Anti-TLR7 antibody or antigen-binding fragment thereof, pharmaceutical composition and use thereof

Abstract

Provided in the present invention are an anti-TLR7 antibody or an antigen-binding fragment thereof, and a pharmaceutical composition thereof. The antibody or the antigen-binding fragment thereof can specifically bind to a human or simian TLR7 antigen and does not bind to murine TLR7, exhibits significant TLR7 antigen-binding activity, and can effectively inhibit various inflammatory cytokines produced upon TLR7 activation. The anti-TLR7 antibody or the antigen-binding fragment thereof can be used, either as a monotherapy or in combination with other drugs, for treating and / or preventing diseases pathologically associated with the TLR7 target, including immune inflammation-related diseases, allergic diseases, infectious diseases, cancers, etc.

Description

Anti-tlr7 antibody or antigen-binding fragment thereof, pharmaceutical composition and application thereof TECHNICAL FIELD

[0001] The present application belongs to the field of immunology and molecular biology, in particular to an anti-TLR7 antibody or antigen-binding fragment thereof, a pharmaceutical composition and application thereof. BACKGROUND

[0002] Toll like receptors (TLRs) belong to the family of pattern recognition receptors (PRRs), which activate downstream signaling pathways by rapidly recognizing pathogen associated molecular patterns (PAMPs) of invading microorganisms to trigger immune responses of the body. TLR7 is a kind of innate immune RNA sensor, which is expressed in B cells, dendritic cells and monocytes / macrophages and mainly expressed in intracellular vesicles such as endoplasmic reticulum, endosome and lysosome, and is a kind of transmembrane signal transduction receptor. This receptor not only reacts to single-stranded RNA (ssRNA) from pathogens, but also reacts to ssRNA from the body itself. TLR7 can cause a series of signal transduction by recognizing single-stranded RNA, leading to the release of cytokines such as TNF-α (tumor necrosis factor alpha), IL-1 (interleukin 1), IL-6 (interleukin 6), IL-12 (interleukin 12), INF-α (interferon type I alpha).

[0003] Recent studies have shown that Toll-like receptor 7 (TLR7) is closely related to autoimmune diseases such as Systemic Lupus Erythematosus (SLE) (Ref. TLR7 gain-of-function genetic variation causes human lupus. Nature 2022, 605, 349-356. and Toll-Like Receptors (TLRs): Structure, Functions, Signaling, and Role of Their Polymorphisms in Colorectal Cancer Susceptibility. Biomed Res Int. 2021: 1157023.). In SLE disease, immune complexes can activate TLR7 in plasmacytoid dendritic cells (pDCs), which in turn secrete high levels of IFN-a, exacerbating the clinical condition (Ref. Pathogenesis of systemic lupus erythematosus: risks, mechanisms and therapeutic targets. Ann Rheum Dis. 2023 Aug; 82(8): 999-1014.). In addition, TLR7 plays an important role in B cell differentiation, and TLR7 in B cells drives the formation of autoreactive antibody-secreting cells in SLE patients through different pathways, for example, in the extracellular follicular response, TLR7 can promote the differentiation of resting naive B cells into activated naive B cells, and then into DN2 B cells, and then into antibody-secreting cells, producing pathogenic autoantibodies, promoting and exacerbating SLE symptoms. In summary, the overactivation of TLR7 seriously affects the occurrence, development and prognosis of SLE (Ref. TLR7 drives human lupus. Nat Immunol. 2022, 23, 817. and Toll-Like Receptors (TLRs): Structure, Functions, Signaling, and Role of Their Polymorphisms in Colorectal Cancer Susceptibility. Biomed Res Int. 2021: 1157023.).

[0004] [Corrected according to Rule 26 24.10.2025] SLE is a chronic multisystem autoimmune disease characterized by the presence of large amounts of autoantibodies in the blood of patients and multiple organ damage. The symptoms are very complex and diverse, and clinical manifestations almost cover all organ systems. Common manifestations include skin rash, arthritis, kidney involvement, and nervous system involvement, and the mortality rate is increased by 2.6 times compared with ordinary people (reference Systemic lupus erythematosus. Nat Rev Dis Primers 2016, 2, 16039.). The etiology of SLE is complex, and is related to genetics, environment, immunity, and endocrine and other factors. SLE is prone to occur in women of childbearing age, and the prevalence in China is about 31-70 / 10 million. At present, the number of SLE patients in China exceeds 1 million, and about 8 million people worldwide, and the prevalence of SLE diagnosis is increasing (reference Global epidemiology of systemic lupus erythematosus. Nat Rev Rheumatol. 2021, 17, 515-532). SLE patients have abnormal increase in cell apoptosis, and defects in clearance of late apoptotic debris, leading to increased opportunities for exposure to self-antigens, abnormal reaction of innate and adaptive immune cells to self-antigens, production of various autoantibodies and deposition of autoimmune complexes in tissues, resulting in activation of the complement pathway, aggregation of neutrophils and monocytes, and proliferation of autoreactive lymphocytes, ultimately causing damage to various organs throughout the body.

[0005] The main treatment for SLE in clinical practice is to down-regulate the over-activated autoimmune system by glucocorticoids, antimalarial drugs and immunosuppressive drugs. However, despite such treatment, drug-resistant patients still have life-threatening manifestations, such as lupus nephritis. In addition, the use of glucocorticoids is limited due to various side effects. In addition, SLE cannot be cured at present, and long-term medication is required to control symptoms, so a new type of therapeutic drug with fewer side effects and better efficacy is needed.

[0006] In April 2022, researchers from the Australian National University and other research institutions published their research results in the journal Nature. In this study, the authors first identified the Y264H single gene mutation of TLR7 as one of the causes of SLE and induced severe SLE through whole genome sequencing and whole exome sequencing. This finding provides clinical evidence for the development of TLR7-targeted treatment methods (Reference: Toll-Like Receptors (TLRs): Structure, Functions, Signaling, and Role of Their Polymorphisms in Colorectal Cancer Susceptibility. Biomed Res Int. 2021: 1157023). In recent years, there have been numerous studies on targeting the TLR7 signaling pathway to treat SLE. For example, dihydroartemisinin induces dendritic cell apoptosis by inhibiting the TLR7 / 9-MyD88-IRAKs signaling pathway, alleviating SLE phenotype; Kensuke Miyake's research team screened the anti-mouse TLR7 monoclonal antibody A94B10, and found that in mouse animal models, this antibody can reduce IgG deposition in glomeruli and autoantibody production, inhibit the release of immune cell inflammatory factors, eliminate the increase of monocytes associated with lupus, protect NZBWF1 mice from lupus nephritis and prolong their survival (Reference: Anti-TLR7 antibody protects against lupus nephritis in NZBWF1 mice by targeting B cells and patrolling monocytes. Frontiers in Immunology, 2021, 12: 777197.). In summary, targeting and inhibiting TLR7 is expected to become a potential treatment strategy for SLE and even multiple common autoimmune diseases, including dry eye, rheumatoid arthritis, and multiple sclerosis. In addition, TLR7 is closely related to the occurrence, development, and prognosis of numerous diseases, so other potential indications of TLR7 antagonistic antibodies also include antiphospholipid syndrome (APS) (Reference: Lipid presentation by the protein C receptor links coagulation with autoimmunity. Science, 2021, 371, eabc0956.), metabolic diseases such as obesity, diabetes, and even self-immune diseases exacerbated by obesity (Reference Lupus autoimmunity and metabolic parameters are exacerbated upon high fat diet-induced obesity due to TLR7 signaling. Frontiers in immunology, 2019, 10:2015.; Increased adipose tissue expression of Toll-like receptor (TLR)-7 in obese individuals: significance in metabolic disease. Journal of Glycomics & Lipidomics, 2015, 5(4): 1.; Toll-like receptor 7 (TLR7) is expressed in adipocytes and the pharmacological TLR7 agonist imiquimod and adipocyte-derived cell-free nucleic acids (cfDNA) regulate adipocyte function. International Journal of Molecular Sciences, 2022, 23(15): 8475.; T-bet+ B cells accumulate in adipose tissue and exacerbate metabolic disorder during obesity. Cell metabolism, 2022, 34(8): 1121-1136.e6.; Nucleic acid-targeting pathways promote inflammation in obesity-related insulin resistance. Cell reports, 2016, 16(3): 717-730.chronic obstructive pulmonary disease (COPD) (Reference: TLR7 promotes smoke-induced experimental lung damage through the activity of mast cell tryptase. Nature Communications, 2023, 14(1): 7349.), macrophage activation syndrome (MAS), and severe malaria anemia (Reference: Chronic TLR7 and TLR9 signaling drives anemia via differentiation of specialized hemophagocytes [J]. Science, 2019, 363(6423): eaao5213.), etc.

[0007] Therefore, it is necessary to develop a new anti-TLR7 antibody, and to provide a therapeutic and / or prophylactic agent for immune inflammatory-related diseases, allergic diseases, infectious diseases, and cancers. SUMMARY

[0008] To overcome the defects of the prior art, the present application provides an anti-TLR7 antibody or an antigen-binding fragment thereof having specific binding to TLR7, which can inhibit cytokines induced by TLR7 agonists, and a corresponding preparation method and use of the anti-TLR7 antibody or the antigen-binding fragment thereof.

[0009] In a first aspect of the present application, an anti-TLR7 antibody or an antigen-binding fragment thereof is provided, which comprises a heavy chain variable region and / or a light chain variable region, wherein,

[0010] the amino acid sequence of the heavy chain variable region comprises GYX1FTX2YX3(SEQ ID NO: 70), GFTFSX4YX5(SEQ ID NO: 71), GFSLTX6X7G (SEQ ID NO: 176), ARX8QIX9HGWNEGVNDY (SEQ ID NO: 177), the amino acid sequence of any one of SEQ ID NOs: 39-50, or an amino acid sequence having at least 80% identity to the amino acid sequence of any one of GYX1FTX2YX3(SEQ ID NO: 70), GFTFSX4YX5(SEQ ID NO: 71), GFSLTX6X7G (SEQ ID NO: 176), ARX8QIX9HGWNEGVNDY (SEQ ID NO: 177), SEQ ID NOs: 39-50;

[0011] the amino acid sequence of the light chain variable region comprises ENIX 10 SY (SEQ ID NO: 178), QX 11 HFGIPWT (SEQ ID NO: 179), SEQ ID NOs: 51, 53-68, 169-170, or an amino acid sequence having at least 80% identity to the amino acid sequence of any one of ENIX 10 SY (SEQ ID NO: 178), QX 11 HX 12 GIPWT (SEQ ID NO: 179), SEQ ID NOs: 51, 53-68, 169-170.

[0012] Preferably, the anti-TLR7 antibody or antigen-binding fragment thereof comprises CDR-H1, CDR-H2, and CDR-H3 of a heavy chain variable region; and / or, CDR-L1, CDR-L2, and CDR-L3 of a light chain variable region.

[0013] the amino acid sequence of CDR-H1 comprises GYX1FTX2YX3(SEQ ID NO: 70), GFTFSX4YX5(SEQ ID NO: 71), GFSLTX6X7G (SEQ ID NO: 176), or an amino acid sequence having at least 80% identity to the amino acid sequence of any one of GYX1FTX2YX3(SEQ ID NO: 70), GFTFSX4YX5(SEQ ID NO: 71), GFSLTX6X7G (SEQ ID NO: 176);

[0014] The amino acid sequence of CDR-H2 comprises any one of the amino acid sequences set forth in SEQ ID NOs: 39-44, or an amino acid sequence that has at least 80% identity to any one of the amino acid sequences set forth in SEQ ID NOs: 39-44;

[0015] The amino acid sequence of CDR-H3 comprises any one of the amino acid sequences set forth in SEQ ID NOs: 45-50, ARX8QIX9HGWNEGVNDY (SEQ ID NO: 177), or an amino acid sequence that has at least 80% identity to any one of the amino acid sequences set forth in SEQ ID NOs: 45-50, ARX8QIX9HGWNEGVNDY (SEQ ID NO: 177).

[0016] The amino acid sequence of CDR-L1 comprises any one of the amino acid sequences set forth in SEQ ID NOs: 51, 53-56, 169-170, ENIX 10 SY (SEQ ID NO: 178), or an amino acid sequence that has at least 80% identity to any one of the amino acid sequences set forth in SEQ ID NOs: 51, 53-56, 169-170, ENIX 10 SY (SEQ ID NO: 178);

[0017] The amino acid sequence of CDR-L2 comprises any one of the amino acid sequences set forth in SEQ ID NOs: 57-62, or an amino acid sequence that has at least 80% identity to any one of the amino acid sequences set forth in SEQ ID NOs: 57-62;

[0018] The amino acid sequence of CDR-L3 comprises any one of the amino acid sequences set forth in SEQ ID NOs: 63-68, QX 11 HFGIPWT (SEQ ID NO: 179), or an amino acid sequence that has at least 80% identity to any one of the amino acid sequences set forth in SEQ ID NOs: 63-68, QX 11 HFGIPWT (SEQ ID NO: 179).

[0019] wherein X in SEQ ID NOs: 70-71, 176-179 can be any natural amino acid residue, for example, alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), threonine (T), proline (P), serine (S), tryptophan (W), tyrosine (Y), valine (V).

[0020] In an embodiment of the application, X1in SEQ ID NO: 70 represents T or I; X2represents E, T or K; X3represents P, F or W.

[0021] In an embodiment of the application, X4in SEQ ID NO: 71 represents S or D; X5represents T or G.

[0022] In an embodiment of the application, X6in SEQ ID NO: 176 represents G, Y, S, T, D, H, W, Q, E or N; X7represents Y or H.

[0023] In an embodiment of the application, X8in SEQ ID NO: 177 represents V or A; X9represents Y or R.

[0024] In an embodiment of the application, X10in SEQ ID NO: 178 represents Y, R, K, D, N, Q, E, H or S. 10

[0025] In an embodiment of the application, X11in SEQ ID NO: 179 represents H or S. 11

[0026] Preferably, the CDR-H1 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 33-38, 148-157, or an amino acid sequence that has at least 80% identity to an amino acid sequence as set forth in any one of SEQ ID NOs: 33-38, 148-157.

[0027] Preferably, the CDR-H3 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 45-50, 158-160, or an amino acid sequence that has at least 80% identity to an amino acid sequence as set forth in any one of SEQ ID NOs: 45-50, 158-160.

[0028] Preferably, the CDR-L1 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 51-56, 161-170, or an amino acid sequence that has at least 80% identity to an amino acid sequence as set forth in any one of SEQ ID NOs: 51-56, 161-170.

[0029] Preferably, the CDR-L3 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 63-69, 171, or an amino acid sequence that has at least 80% identity to an amino acid sequence as set forth in any one of SEQ ID NOs: 63-69, 171.

[0030] ​​In one embodiment of the present application, the amino acid sequence of the anti-TLR7 antibody or antigen-binding fragment thereof comprises any one of the following groups (see Tables 1 and 2 for details):

[0031] Table 1 Amino acid sequences of CDRs of antibodies

[0032] Table 2 Amino acid sequences of CDRs of antibodies (the part not shown is the same as 34G12-h458)

[0033] The division of the amino acids of the CDR regions of the antibodies in the present application uses the IMGT numbering system.

[0034] The structure of the anti-TLR7 antibody or antigen-binding fragment thereof includes a nanobody, a chimeric antibody, a Fab fragment, a Fab' fragment, a Fd fragment, a Fv fragment, a bispecific antibody, a multispecific antibody, a dAb fragment, a F(ab')2 fragment, a single chain antibody (scFv), or a linear antibody.

[0035] The anti-TLR7 antibody or antigen-binding fragment thereof can be a humanized antibody.

[0036] Preferably, the anti-TLR7 antibody or antigen-binding fragment thereof is modified, and the modification includes humanization, and the modification site is located in the CDR region, the framework region, and / or the constant region of the antibody.

[0037] Preferably, the anti-TLR7 antibody or antigen-binding fragment thereof specifically binds to human TLR7 or monkey TLR7 and does not bind to murine TLR7; and / or, inhibits the function of human TLR7 or monkey TLR7.

[0038] Preferably, the heavy chain variable region comprises the amino acid sequence shown in any one of SEQ ID NOs: 5-11, 172, 174, 180, and 182, or an amino acid sequence having at least 80% identity to the amino acid sequence shown in any one of SEQ ID NOs: 5-11, 172, 174, 180, and 182.

[0039] Preferably, the light chain variable region comprises the amino acid sequence shown in any one of SEQ ID NOs: 12-18, 173, 175, 181, and 183, or an amino acid sequence having at least 80% identity to the amino acid sequence shown in any one of SEQ ID NOs: 12-18, 173, 175, 181, and 183.

[0040] In one embodiment, the heavy chain variable region further comprises a sequence that is humanized at SEQ ID NO: 8, 11.

[0041] In one embodiment, the humanization engineering site is located in the framework region, and the framework region of the heavy chain variable region comprises an amino acid sequence as set forth in any one of SEQ ID NO: 86-109, 191-209.

[0042] In one embodiment, the light chain variable region further comprises a sequence that is humanized at SEQ ID NO: 16, 18.

[0043] In one embodiment, the humanization engineering site is located in the framework region, and the framework region of the light chain variable region comprises an amino acid sequence as set forth in any one of SEQ ID NO: 110-123, 184-190, 210-220.

[0044] In one embodiment, the heavy chain variable region further comprises a sequence that is mutated at SEQ ID NO: 180, 182.

[0045] In one embodiment, the light chain variable region further comprises a sequence that is mutated at SEQ ID NO: 181, 183.

[0046] In one embodiment, the heavy chain variable region further comprises a sequence that is mutated at SEQ ID NO: 172, 174.

[0047] In one embodiment, the light chain variable region further comprises a sequence that is mutated at SEQ ID NO: 173, 175.

[0048] Preferably, the mutation region comprises a CDR region and / or a framework region.

[0049] Preferably, the CDR region mutation site of the heavy chain variable region comprises amino acids at positions 31, 32, 98, 101, 104, 109; and the framework region mutation site comprises amino acids at positions 20, 48, 37, 60-63, 67, 71, 76, 78.

[0050] Preferably, the CDR region mutation site of the light chain variable region comprises amino acids at positions 27-28, 30, 90; and the framework region mutation site comprises amino acids at positions 43, 100.

[0051] The mutation of the heavy chain variable region and the light chain variable region is shown in Tables 11-16.

[0052] Preferably, the light chain constant region comprises Ig kappa (kappa) or Ig lambda (lambda).

[0053] Preferably, the heavy chain constant region comprises IgG1, IgG2, IgG3, IgG4, IgM, IgA1 or IgA2.

[0054] In one embodiment, the light chain constant region comprises Ig kappa, and the light chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 221.

[0055] In one embodiment, the heavy chain constant region comprises IgG1.

[0056] Preferably, the IgG1 further comprises LALA mutation, and the heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 222.

[0057] Preferably, the amino acid sequence of the anti-TLR7 antibody or antigen binding fragment thereof comprises any one of SEQ ID NOs: 72-85, or has at least 80% identity to any one of SEQ ID NOs: 72-85.

[0058] In one embodiment of the present application, the heavy chain amino acid sequence of the anti-TLR7 antibody or antigen binding fragment thereof comprises any one of SEQ ID NOs: 72-78.

[0059] In one embodiment of the present application, the light chain amino acid sequence of the anti-TLR7 antibody or antigen binding fragment thereof comprises any one of SEQ ID NOs: 79-85.

[0060] The anti-TLR7 antibody or antigen binding fragment thereof can be obtained by using conventional techniques. For example, chemical synthesis or expression using eukaryotic or prokaryotic expression system.

[0061] In a second aspect of the present application, there is provided a use of the anti-TLR7 antibody or antigen binding fragment thereof as described above, which use comprises:

[0062] A use in preparing a fusion construct comprising the anti-TLR7 antibody or antigen binding fragment thereof as described above and other biologically active effector molecules, which other biologically active effector molecules comprise antibodies or antigen binding fragments thereof or other functional components targeting other targets other than any of the anti-TLR7 antibodies or antigen binding fragments thereof as described above;

[0063] B use in detecting TLR7 expression;

[0064] Use of C in the preparation of a TLR7 antagonist or inhibitor.

[0065] Preferably, the other functional components include, but are not limited to, one or more of serum albumin, cytokines, transferrin, scaffold proteins, oligopeptides, oligopeptide polymers, polypeptides, polypeptide polymers, polysaccharides, fatty chains, avidin, biotin, streptavidin, toxins, drugs, nucleic acids, radionuclides and labels thereof, PEG or Fc fragments.

[0066] In a third aspect of the present application, a fusion construct is provided, which comprises the anti-TLR7 antibody or antigen-binding fragment thereof as described above.

[0067] Preferably, the fusion construct further comprises other bioactive effector molecules, which include antibodies or antigen-binding fragments thereof against other targets or other functional components in addition to the anti-TLR7 antibody or antigen-binding fragment thereof as described above.

[0068] Preferably, the functional components include, but are not limited to, one or more of serum albumin, cytokines, transferrin, scaffold proteins, oligopeptides, oligopeptide polymers, polypeptides, polypeptide polymers, polysaccharides, fatty chains, avidin, biotin, streptavidin, toxins, drugs, nucleic acids, radionuclides and labels thereof, PEG or Fc fragments.

[0069] The anti-TLR7 antibody or antigen-binding fragment thereof has a structure of a nanobody, a chimeric antibody, a Fab fragment, a Fab' fragment, a Fd fragment, a Fv fragment, a bispecific antibody, a multispecific antibody, a dAb fragment, a F(ab')2 fragment, a single-chain antibody or a linear antibody.

[0070] Preferably, the fusion construct comprises one or more of the anti-TLR7 antibody or antigen-binding fragment thereof.

[0071] Preferably, the fusion construct comprises one or more of other bioactive effector molecules, which can be the same or different bioactive effector molecules.

[0072] The anti-TLR7 antibody or antigen-binding fragment thereof is directly or indirectly connected to the other bioactive effector molecules.

[0073] Preferably, the indirect connection can be through a linker, a functional domain and / or a linker for conjugation.

[0074] The linker is selected from a connecting peptide, an oligopeptide, an oligopeptide polymer, a polypeptide, a polypeptide polymer, PEG, a nucleic acid, a polysaccharide, a fatty chain, biotin, streptavidin or avidin.

[0075] The functional domain is a combination of one or more of Fc fragment, serum albumin, cytokine, transferrin or scaffold protein.

[0076] The linker for conjugation includes a functional group linker.

[0077] The functional group linker includes a thiol, amino, hydroxyl and / or carboxyl reactive group, which allows covalent conjugation between the anti-TLR7 antibody or antigen-binding fragment thereof and the biologically active effector molecule.

[0078] Preferably, the direct or indirect linkage includes direct or indirect linkage to the N-terminus, C-terminus and / or internal residues of the anti-TLR7 antibody or antigen-binding fragment thereof and / or other target antibodies or antigen-binding fragments thereof.

[0079] The linkage order of the anti-TLR7 antibody or antigen-binding fragment thereof, other target antibodies in the fusion construct can be linkage of the N-terminus, C-terminus and / or internal residues of one antibody to the N-terminus, C-terminus and / or internal residues of another antibody.

[0080] In a fourth aspect, the present application provides a nucleic acid encoding the anti-TLR7 antibody or antigen-binding fragment described above or the fusion construct described above. For example, the nucleic acid includes DNA and / or mRNA.

[0081] In some embodiments, the nucleic acid is DNA encoding the anti-TLR7 antibody or antigen-binding fragment described above or the fusion construct described above.

[0082] Preferably, the nucleotide sequence encoding the heavy chain variable region of the anti-TLR7 antibody or antigen-binding fragment thereof comprises any one of the nucleotide sequences in SEQ ID NO: 19-25 or a degenerate sequence thereof, or a nucleotide sequence having at least 80% identity to any one of the nucleotide sequences in SEQ ID NO: 19-25 and having the function of encoding the anti-TLR7 antibody or antigen-binding fragment thereof.

[0083] Preferably, the nucleotide sequence encoding the light chain variable region of the anti-TLR7 antibody or antigen-binding fragment thereof comprises any one of the nucleotide sequences in SEQ ID NO: 26-32 or a degenerate sequence thereof, or a nucleotide sequence having at least 80% identity to any one of the nucleotide sequences in SEQ ID NO: 26-32 and having the function of encoding the anti-TLR7 antibody or antigen-binding fragment thereof.

[0084] Preferably, the nucleotide sequence encoding the heavy chain of the anti-TLR7 antibody or antigen-binding fragment thereof comprises any one of SEQ ID NO: 130, 132, 134, 136, 138, 140, 142, or a degenerate sequence thereof, or a nucleotide sequence having at least 80% identity to any one of SEQ ID NO: 130, 132, 134, 136, 138, 140, 142, and having a function of encoding the anti-TLR7 antibody or antigen-binding fragment thereof.

[0085] Preferably, the nucleotide sequence encoding the light chain of the anti-TLR7 antibody or antigen-binding fragment thereof comprises any one of SEQ ID NO: 131, 133, 135, 137, 139, 141, 143, or a degenerate sequence thereof, or a nucleotide sequence having at least 80% identity to any one of SEQ ID NO: 131, 133, 135, 137, 139, 141, 143, and having a function of encoding the anti-TLR7 antibody or antigen-binding fragment thereof.

[0086] In a fifth aspect of the present application, a vector is provided, wherein the vector comprises the nucleic acid as described above.

[0087] The vector can express in a prokaryotic or eukaryotic cell.

[0088] For example, the expression vector can be introduced into a host cell by a method of transient transfection or stable transfection.

[0089] In a sixth aspect of the present application, a host cell is provided, wherein the host cell comprises the nucleic acid as described above or the vector as described above.

[0090] The host cell can be a eukaryotic cell or a prokaryotic cell.

[0091] The eukaryotic cell includes an animal or plant cell, such as a T cell, a yeast cell, a HEK293 cell, a CHO cell, and the like.

[0092] The prokaryotic cell is, for example, an Escherichia coli.

[0093] In a seventh aspect of the present application, a method for preparing a host cell is provided, wherein the method comprises introducing the nucleic acid or the vector as described above into a host cell.

[0094] In an eighth aspect of the present application, a method for preparing an anti-TLR7 antibody or antigen-binding fragment thereof or a fusion construct is provided, wherein the method comprises culturing the host cell as described above to express the anti-TLR7 antibody or antigen-binding fragment thereof or the fusion construct.

[0095] In a ninth aspect, the present application provides a method for preparing an anti-TLR7 antibody or an antigen-binding fragment thereof, comprising synthesizing the anti-TLR7 antibody or the antigen-binding fragment thereof by a chemical synthesis method.

[0096] In a tenth aspect, the present application provides a product for treating, preventing and / or diagnosing a TLR7-related disease, comprising any one of the following:

[0097] A) the anti-TLR7 antibody or the antigen-binding fragment thereof described above;

[0098] B) the fusion construct described above;

[0099] C) the nucleic acid described above;

[0100] D) the vector described above; or,

[0101] E) the host cell described above.

[0102] Preferably, the product can be a diagnostic kit or a drug or a diagnostic chip, etc.

[0103] More preferably, the TLR7-related disease includes, but is not limited to, an immune inflammation-related disease, an allergic disease, an infectious disease or a cancer, etc.

[0104] The drug can be an antibody-drug conjugate (ADC) comprising the anti-TLR7 antibody or the antigen-binding fragment thereof or the fusion construct described in the present application, and other drugs covalently bound thereto.

[0105] In an eleventh aspect, the present application provides the use of A)-E) described above in the preparation of a product for treating and / or preventing a TLR7-related disease, or in the preparation of a diagnostic product or a tracer for a TLR7-related disease.

[0106] Preferably, the TLR7-related disease includes, but is not limited to, an immune inflammation-related disease, an allergic disease, an infectious disease or a cancer, etc.

[0107] The product can be a drug, etc.

[0108] The drug can be an antibody-drug conjugate (ADC) comprising the anti-TLR7 antibody or the antigen-binding fragment thereof or the fusion construct described in the present application, and other drugs covalently bound thereto.

[0109] The diagnostic product can be a diagnostic kit or a diagnostic chip.

[0110] In a twelfth aspect, the present application provides a method for detecting TLR7, which comprises combining a sample to be detected with the anti-TLR7 antibody or antigen-binding fragment thereof described above, and then detecting the content of the complex formed by TLR7 and the anti-TLR7 antibody or antigen-binding fragment thereof.

[0111] The detection method is for detecting the presence or content of TLR7. The presence indicates qualitative analysis of the presence or absence, and the content can be expression level or protein concentration, etc.

[0112] In a thirteenth aspect, the present application provides a method for diagnosing a TLR7-related disease, which comprises sampling, combining the sample with the diagnostic product for the disease described above, and detecting the content of the complex formed by TLR7 and the anti-TLR7 antibody or antigen-binding fragment thereof.

[0113] Preferably, the TLR7-related disease includes but is not limited to immune inflammation-related diseases, allergic diseases, infectious diseases, or cancers, etc.

[0114] In a fourteenth aspect, the present application provides a method for treating and / or preventing a TLR7-related disease, which comprises administering the disease treatment and / or prevention product described above to an individual.

[0115] Preferably, the TLR7-related disease includes but is not limited to immune inflammation-related diseases, allergic diseases, infectious diseases, or cancers, etc.

[0116] The "anti-TLR7 antibody or antigen-binding fragment thereof" in the present application exemplarily shows the CDR sequences obtained according to the IMGT method, and in addition, other numbering methods can be used for dividing the CDR region, including but not limited to Kabat, Chothia, AbM, Contact, etc.

[0117] The term "comprising" or "including" in the present application is an open description, which contains the specified components or steps described, and other specified components or steps that do not materially affect.

[0118] The "immune inflammation-related disease" in the present application includes, but is not limited to, connective tissue, musculoskeletal system (systemic lupus erythematosus, rheumatoid arthritis, juvenile idiopathic arthritis, adult Still's disease, ankylosing spondylitis, systemic scleroderma, polymyositis, dermatomyositis, psoriatic arthritis, osteoarthritis, mixed connective tissue disease, muscular dystrophy, etc.), blood system (autoimmune hemolytic anemia, aplastic anemia, idiopathic thrombocytopenic purpura, etc.), digestive system (Crohn's disease, ulcerative colitis, ileitis, etc.), liver, gallbladder, pancreas and endocrine system (autoimmune hepatitis, viral hepatitis, alcoholic hepatitis, non-alcoholic fatty liver disease, primary sclerosing cholangitis, primary biliary cirrhosis, Sjogren's syndrome, type 1 diabetes, autoimmune thyroiditis, Behcet's disease, Hashimoto's disease, etc.), respiratory system (chronic obstructive pulmonary disease, cystic fibrosis, interstitial pneumonia, etc.), brain and nervous system (multiple sclerosis, myasthenia gravis, meningitis, encephalomyelitis, autoimmune encephalitis, etc.), visual system (uveitis, trachoma, endophthalmitis, etc.), cardiovascular system (vasculitis syndrome, granulomatous polyangiitis, Wegener's granulomatosis, myocarditis, ischemic heart disease, atherosclerosis, etc.), skin and epidermis system (psoriasis, eczema, vitiligo, contact dermatitis, eczema, etc.), kidney system (glomerulonephritis, diabetic nephropathy, IgA nephropathy, purpura nephritis, nephropathy, interstitial cystitis, etc.), endocrine system (type 1 diabetes, autoimmune thyroiditis, Behcet's disease, Hashimoto's disease, etc.), and other immune inflammation-related diseases, systemic inflammation (Behcet's disease, anti-phospholipid antibody syndrome, IgG4-related disease, sepsis, hemorrhage, hypersensitivity, transplant rejection, shock symptoms due to cancer chemotherapy, etc.), and the like.

[0119] The "allergic disease" in the present application includes, but is not limited to, atopic dermatitis, asthma, anaphylaxis, anaphylactoid reaction, food allergy, rhinitis, otitis media, drug reaction, insect sting reaction, plant reaction, latex allergy, conjunctivitis, urticaria, and the like.

[0120] The "infection" in the present application includes, but is not limited to, diseases caused by infection with viruses (single-stranded RNA virus, double-stranded RNA virus, single-stranded DNA virus, double-stranded DNA virus, etc.), bacteria (Gram-negative bacteria, Gram-positive bacteria, acid-fast bacteria, actinomycetes, spirochetes, spirochetes, rickettsia, chlamydia, mycoplasma, etc.), fungi (trichophyton, candida, cryptococcus, aspergillus, pneumocystis, malassezia, etc.), parasites (filariasis, trematode, tapeworm, distome, echinococcus, amoeba, flea, louse, mite, roundworm, pinworm, etc.), and the like.

[0121] The "cancer" in the present application includes, but is not limited to, lymphoma, leukemia, breast cancer, lung cancer, skin cancer, and the like.

[0122] The "fusion construct" used herein defines a fusion of the antibody or antigen-binding fragment thereof of the present application with another compound. The fusion construct can comprise one or more antibodies or antigen-binding fragments thereof, which can be the same or different. The fusion construct can comprise one or more additional compounds, which can also be the same or different. The compound can be a proteinaceous compound or a non-proteinaceous compound. In the case where the compound is a proteinaceous compound or the fusion construct comprises only antibodies or antigen-binding fragments thereof, the fusion construct can also be referred to as a fusion protein. In the case where the compound is fused to the antibody or antigen-binding fragment thereof in a conjugated form, the fusion construct can also be referred to as a conjugate.

[0123] The "drug" of the present application can be used for the treatment of a human or a non-human animal, such as a non-human mammal. The drug can comprise a pharmaceutically acceptable carrier, adjuvant or salt commonly known in the art.

[0124] The drug can be administered by any suitable route, such as a gastrointestinal route (e.g., oral) or a non-gastrointestinal route (e.g., intravenous, intramuscular, subcutaneous, intradermal, intraorgan, intranasal, intraocular, instillation, intracerebral, intrathecal, transdermal, intrarectal, etc.).

[0125] The drug can be in any suitable dosage form, such as a gastrointestinal dosage form or a non-gastrointestinal dosage form, preferably including but not limited to tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, microemulsions, suspensions, injections, sprays, aerosols, powder sprays, lotions, ointments, plasters, pastes, patches, eye drops, nose drops, sublingual tablets, suppositories, aerosols, effervescent tablets, dripping pills, gels, and the like. The various dosage forms of the drug can be produced according to conventional methods in the pharmaceutical field.

[0126] The "pharmaceutically acceptable" of the present application means neither significantly stimulating the organism nor inhibiting the biological activity and properties of the active substance of the product to be administered.

[0127] The "… method" of the present application can be for the diagnosis, treatment and / or prevention of a disease or for the diagnosis, treatment and / or prevention of a non-disease.

[0128] The "antigen-binding fragment" of the present application is a part of an antibody that retains the specific binding activity of the antibody, i.e., any part of an antibody is capable of specifically binding to an epitope on the target molecule of the antibody. It includes, for example, Fab, Fab', F(ab')2, Fv, Fd, and variants of these fragments. For example, the heavy chain and / or light chain of an antibody, the heavy chain variable region and / or the light chain variable region of an antibody, or a single or more than two CDRs from the heavy chain or light chain of an antibody. Among them,

[0129] Nanobody or single-domain antibody refers to the variable domain of heavy chain (VHH) of an antibody, which has independent antigen binding activity.

[0130] Chimeric antibody refers to an antibody in which part of the heavy chain and / or light chain is derived from a specific source or species, while the rest of the heavy chain and / or light chain is derived from a different source or species.

[0131] Single-chain antibody is an antibody formed by connecting the variable domain of heavy chain and the variable domain of light chain through a short peptide linker of 15-20 amino acids.

[0132] Fab, i.e., a monovalent fragment composed of VL, VH, CL and CH1 domains.

[0133] Fab', i.e., a Fab fragment with one or more cysteine residues at the C-terminus of the CH1 domain.

[0134] F(ab')2, i.e., a bivalent fragment containing two Fab fragments connected by a disulfide bond in the hinge region.

[0135] Fd, an Fd fragment composed of VH and CH1 domains.

[0136] Fv, an Fv fragment composed of VL and VH domains of a single arm of an antibody.

[0137] dAb fragment, an antibody fragment composed of a VH domain.

[0138] The "linear antibody" of the present application includes one or more pairs of tandem antibody fragments, which can be Fd segments (VH-CH1), single-chain antibodies (scFv), antibody fragments (Fab), or single-domain antibodies (VHH), which are connected together by a linker peptide to form a continuous antibody structure.

[0139] Among them, VH represents the variable domain of heavy chain, VL represents the variable domain of light chain, CH represents the constant domain of heavy chain, and CL represents the constant domain of light chain.

[0140] The term "comprise" or "comprising" as used herein is open-ended and means that the protein or nucleic acid can consist of the recited sequence, or can have additional amino acids or nucleotides at either or both ends of the protein or nucleic acid, but still have the same or similar activity as the original sequence.

[0141] The term "homology" or "identity" as used herein means that the sequence can be adjusted by the person skilled in the art according to the actual work, and the sequence has (including but not limited to) for example 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% homology / identity compared with the sequence obtained by the prior art.

[0142] The term "humanized antibody" as used herein means that the framework region of the antibody or all of the antibody is encoded by human antibody genes.

[0143] The term "individual" as used herein can be a human or a non-human mammal, which can be a wild animal, a zoo animal, an economic animal, a pet, a laboratory animal, etc. Preferably, the non-human mammal includes but is not limited to a pig, a cow, a sheep, a horse, an ass, a fox, a raccoon dog, a mink, a camel, a dog, a cat, a rabbit, a mouse (for example, a rat, a mouse, a guinea pig, a hamster, a gerbil, a chinchilla, a squirrel), or a monkey, etc.

[0144] The term "treatment" as used herein means to slow down, interrupt, stop, control, reduce, or reverse the progression or severity of a sign, a symptom, a disorder, a condition, or a disease after the disease has started to develop, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders.

[0145] The term "prevention" as used herein means a way to prevent or delay the occurrence of a disease or a condition or a symptom in the body.

[0146] In the present application, "34G12" is the same as "34G12B8".

[0147] The application provides a new anti-TLR7 antibody or antigen binding fragment thereof, a mouse anti-human TLR7 antibody is obtained by screening, the mouse anti-human TLR7 antibody is obtained by further screening and modification, such as humanization of the mouse anti-human TLR7 antibody, further modification of the CDR, framework and variable region of the humanized antibody, and the antibody obtained can specifically bind to different variants of human TLR7 and monkey TLR, has good antigen binding activity of the anti-TLR7 antibody, can effectively inhibit various inflammatory cytokines generated due to TLR7 activation, and is used for treating and / or preventing TLR7 related diseases. BRIEF DESCRIPTION OF DRAWINGS

[0148] Figure 1 is a flow cytometry analysis result of the binding selection ability of chimeric anti-human TLR7 antibodies (83A7-c, 182A1H9-c, 247C6A2-c, 7C6A2-c, 13G1F4-c, 34G12B8-c, 17D1D2-c, 13H17L-c) and antigens (human TLR7: variant 1).

[0149] Figure 2 is a flow cytometry analysis result of the binding selection ability of chimeric anti-human TLR7 antibodies (83A7-c, 182A1H9-c, 247C6A2-c, 7C6A2-c, 13G1F4-c, 34G12B8-c, 17D1D2-c, 13H17L-c) and antigens (cynomolgus monkey TLR7).

[0150] Figure 3 is a flow cytometry analysis result of the binding selection ability of chimeric anti-human TLR7 antibodies (83A7-c, 247C6A2-c, 13G1F4-c, 17D1D2-c, 13H17L-c, 7C6A2-c, 34G12B8-c) and antigens (mouse TLR7, HEK293T).

[0151] Figure 4 is a flow cytometry analysis result of the binding selection ability of chimeric anti-human TLR7 antibodies (247C6A2-c, 7C6A2-c, 13G1F4-c, 34G12B8-c, 17D1D2-c, 13H17L-c) and antigens (human TLR7: variant 2), which is a graph showing specific binding to the antigen.

[0152] Figure 5 is a flow cytometry analysis result of the binding selection ability of chimeric anti-human TLR7 antibodies (83A7-c, 182A1H9-c, 247C6A2-c, 13G1F4-c, 17D1D2-c, 13H17L-c, 7C6A2-c, 34G12B8-c) and antigens (human TLR7: variant 1, human Unc93B1, HEK293T).

[0153] Figure 6 is a flow cytometry analysis result of the binding selectivity of chimeric anti-human TLR7 antibodies (247C6A2-c, 13H17L-c) to antigens (human TLR7: variant 2, human Unc93B1, HEK293T).

[0154] Figure 7 is a flow cytometry analysis result of the binding selectivity of chimeric anti-human TLR7 antibodies (83A7-c, 182A1H9-c, 247C6A2-c) to antigens (human TLR7: variant 1, human Unc93B1, Ba / F3).

[0155] Figure 8 is a graph showing the effect of different concentrations of chimeric anti-human TLR7 antibodies 247C6A2-c, 17D1D2-c, 13H17L-c, 34G12B8-c on the production of IL-6 by human PBMC treated with GS-9620, wherein A is a graph showing the effect of different concentrations of chimeric anti-human TLR7 antibodies 17D1D2-c, 13H17L-c on the production of IL-6 by human PBMC treated with GS-9620; B is a graph showing the effect of different concentrations of chimeric anti-human TLR7 antibodies 17D1D2-c, 13H17L-c, 34G12B8-c on the production of IL-6 by human PBMC treated with GS-9620; C is a graph showing the effect of different concentrations of chimeric anti-human TLR7 antibodies 247C6A2-c, 13H17L-c on the production of IL-6 by human PBMC treated with GS-9620.

[0156] Figure 9 is a graph showing the effect of different concentrations of chimeric anti-human TLR7 antibodies 247C6A2-c, 17D1D2-c, 13H17L-c, 34G12B8-c on the production of IFN-a by human PBMC treated with GS-9620, wherein A is a graph showing the effect of different concentrations of chimeric anti-human TLR7 antibodies 17D1D2-c, 34G12B8-c on the production of IFN-a by human PBMC treated with GS-9620; B is a graph showing the effect of different concentrations of chimeric anti-human TLR7 antibodies 17D1D2-c, 13H17L-c on the production of IFN-a by human PBMC treated with GS-9620; C is a graph showing the effect of different concentrations of chimeric anti-human TLR7 antibodies 247C6A2-c, 13H17L-c on the production of IFN-a by human PBMC treated with GS-9620.

[0157] Figure 10 is a graph showing the effect of different concentrations of chimeric anti-human TLR7 antibodies 13G1F4-c, 17D1D2-c, 13H17L-c, 34G12B8-c on the production of IL-6 by human PBMC treated with DSR-6434.

[0158] Figure 11 is a graph showing the effect of 13H17L-c, a chimeric anti-human TLR7 antibody, on DSR-6434-treated human PBMCs producing IFN-a.

[0159] Figure 12 is a graph showing the effect of 13H17L-c, 17D1D2-c, 34G12B8-c, 13G1F4-c, chimeric anti-human TLR7 antibodies, on DSR-6434-treated human PBMCs producing TNF-a at different concentrations.

[0160] Figure 13 is a graph showing the effect of 13H17L-c, 34G12B8-c, 13G1F4-c, chimeric anti-human TLR7 antibodies, on DSR-6434-treated human PBMCs producing IP-10.

[0161] Figure 14 is a flow cytometry analysis result of the binding selection ability of 13H17L humanized antibodies (13H17L-h1-13H17L-h22) to antigens (human TLR7: variant 2, human Unc93B1, HEK293T).

[0162] Figure 15 is a flow cytometry analysis result of the binding selection ability of 34G12B8 humanized antibodies (34G12-h1, 2, 3, 4, 5, 7) to antigens (human TLR7: variant 1, human Unc93B1, HEK293T).

[0163] Figure 16 is a flow cytometry analysis result of the binding selection ability of 34G12B8 humanized antibodies (34G12-h8, 9, 11, 13, 14, 15, 16, 17, 18, 19) to antigens (human TLR7: variant 1, human Unc93B1, HEK293T).

[0164] Figure 17 is a flow cytometry analysis result of the binding selection ability of 34G12B8 humanized antibodies (34G12-h9, 11, 13, 14, 15, 16, 17, 19) to antigens (cynomolgus monkey TLR7, cynomolgus monkey Unc93B1, HEK293T).

[0165] Figure 18 is a graph showing the effect of 13H17L humanized antibodies (13H17L-h1-13H17L-h22) on GS-9620-treated human PBMCs producing IL-6, wherein A is a graph showing the effect of 13H17L-h3, 4, 5, 7, 9, 10, and B is a graph showing the effect of 13H17L-h11-13H17L-h17, 13H17L-h19-13H17L-h22.

[0166] Figure 19 is a graph of the effect of 34G12B8 humanized antibodies (34G12-h1 - 34G12-h19) on IL-6 production by GS-9620 treated human PBMC.

[0167] Figure 20 is a graph of the effect of 34G12B8 humanized antibodies (34G12-h1 - 34G12-h19) on TNF production by GS-9620 treated human PBMC.

[0168] Figure 21 is a graph of the effect of humanized TLR7 antibodies on IL-6 in CD34+ humanized mice.

[0169] Figure 22 is a flow cytometry analysis of the binding selectivity of anti-human TLR7 affinity matured antibodies (34G12-h542, 34G12-h458) to antigens (human TLR7: variant 1, human Unc93B1, HEK293T).

[0170] Figure 23 is a flow cytometry analysis of the binding selectivity of anti-human TLR7 affinity matured antibodies (h587, h588, h589, h542) to antigens (human TLR7: variant 1, human Unc93B1, HEK293T).

[0171] Figure 24 is the effect of anti-human TLR7 affinity matured antibodies (34G12-h458, 34G12-h488, 34G12-h487, 34G12-h489, 34G12-h485, 34G12-h486) on IL-6 and TNF-α production by GS-9620 treated human PBMC.

[0172] Figure 25 is the effect of anti-human TLR7 affinity matured antibodies (34G12-h542, 34G12-h568, 34G12-h569, 34G12-h576, 34G12-h577, 34G12-h578, 34G12-h579, 34G12-h580, 34G12-h581, 34G12-h583, 34G12-h584, 34G12-h585, 34G12-h586) on IL-6 and TNF-α production by GS-9620 treated human PBMC.

[0173] Figure 26 Effect of anti-human TLR7 affinity matured antibodies (34G12-h542, 34G12-h546, 34G12-h548, 34G12-h552, 34G12-h559, 34G12-h560, 34G12-h561, 34G12-h562, 34G12-h563, 34G12-h564, 34G12-h565, 34G12-h566, 34G12-h567) on IL-6 and TNF-a production from GS-9620 treated human PBMCs.

[0174] Figure 27 Effect of anti-human TLR7 affinity matured antibodies (34G12-h458, 34G12-h556, 34G12-h557) on downstream signaling in GS-9620 treated HEK293-TLR7 reporter cells.

[0175] Figure 28 Effect of anti-human TLR7 affinity matured antibodies (34G12-h542, 34G12-h587, 34G12-h588, 34G12-h589) on downstream signaling in GS-9620 treated HEK293-TLR7 reporter cells.

[0176] Figure 29 Flow cytometry detection of the effect of anti-human TLR7 affinity matured antibodies (34G12-h458) on the proportion of plasma cell formation in SLE-PBMCs.

[0177] Figure 30 Flow cytometry detection of the effect of anti-human TLR7 affinity matured antibodies (34G12-h458) on the proportion of plasma cell formation in HV (healthy human population)-PBMCs.

[0178] Figure 31 Flow cytometry detection of the effect of anti-human TLR7 affinity matured antibodies (34G12-h542) on the proportion of plasma cell formation in HV-PBMCs (healthy human population).

[0179] Figure 32 Graph of the effect of anti-human TLR7 affinity matured antibodies (34G12-h458) on IL-6 in CD34+ humanized mice.

[0180] Figure 33 Endosome and Ba / F3 cell line assay for human Unc93B1-G4S-Flag-T2A-human TLR7. DETAILED DESCRIPTION

[0181] In order to make the person skilled in the art better understand the technical solutions in the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor should belong to the scope of protection of the present application.

[0182] The materials, reagents, instruments and the like used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0183] Example 1: Preparation of mouse anti-human TLR7 antibody

[0184] 1-1: Immunization

[0185] 1-1-1 Construction of Ba / F3 cell strain overexpressing human Unc93B1-G4S-Flag-T2A-human TLR7

[0186] Using Max DNA Polymerase (Takara) to integrate human Unc93B1-G4S-Flag-T2A-human TLR7 gene into transposon vector pPB-EF1α-MCS-puro (purchased from Yunzhou Biotechnology Co., Ltd.). The nucleotide sequence of Unc93B1 is shown in SEQ ID NO: 1, and the nucleotide sequence of human TLR7 gene is shown in SEQ ID NO: 2.

[0187] The transposon vector and transposon auxiliary plasmid were used to gene introduce Ba / F3 cell strain (ATCC) using transfection reagent Lip3000 (Thermo), thereby establishing Ba / F3 cell strain overexpressing human Unc93B1-G4S-Flag-T2A-human TLR7.

[0188] 1-1-2 Endosome extraction

[0189] The Ba / F3 cell strain overexpressing human Unc93B1-G4S-Flag-T2A-human TLR7 established in 1-1-1 was ground under sterile conditions using a Dounce homogenizer on ice. Different concentrations of sucrose buffer were used to prepare sucrose gradient centrifugation samples, and endosomes were separated by ultracentrifuge. After collecting the endosome crude extract, it was purified again by ultracentrifuge to obtain the target endosome.

[0190] The obtained endosome and the above cell strain were detected. The obtained endosome and the above cell strain were detected, and the results are shown in Figure 33.

[0191] 1-1-3 Immunization of mice

[0192] Divided into three groups,

[0193] First group: Ba / F3 cell line overexpressing human Unc93B1-G4S-Flag-T2A-human TLR7 established in 1-1-1 or endosome purified in 1-1-2 were mixed with Gold Adjuvant (sigma) as an adjuvant as an antigen, and administered to the footpad, tail, and intraperitoneally of Balbc or SJL mice once a week in total; Gold Adjuvant(sigma) mixed as an adjuvant as an antigen, and administered to the footpad, tail, and intraperitoneally of Balbc or SJL mice once a week in total;

[0194] Second group: Ba / F3 cell line overexpressing human Unc93B1-G4S-Flag-T2A-human TLR7 established in 1-1-1 or endosome purified in 1-1-2 were mixed with Gold Adjuvant (sigma) and CpG / adjuvant 2%(Invivogen) mixed as an adjuvant as an antigen, and administered to the footpad, tail, and intraperitoneally of Balbc or SJL mice once a week in total; adjuvant 2%(Invivogen) mixed as an adjuvant as an antigen, and administered to the footpad, tail, and intraperitoneally of Balbc or SJL mice once a week in total;

[0195] Third group: Ba / F3 cell line overexpressing human Unc93B1-G4S-Flag-T2A-human TLR7 established in 1-1-1 or endosome purified in 1-1-2 were mixed with Freund's and CpG / adjuvant 2%(Invivogen) mixed as an adjuvant as an antigen, and administered to the footpad, tail, and intraperitoneally of Balbc or SJL mice once a week in total.

[0196] In the above three groups, at the 8th immunization, Ba / F3 cell line overexpressing human Unc93B1-G4S-Flag-T2A-human TLR7 or purified endosome suspended in 1x PBS was administered intraperitoneally.

[0197] Spleen was removed 5 days after the last immunization, and used to prepare hybridoma.

[0198] 1-2 Preparation of hybridoma

[0199] Spleen cells after immunization and Sp2 / 0 cell line (company) were mixed, and cell fusion was performed using BTX electrofusion apparatus. After cell fusion, to screen hybridoma, culture solution (containing 10% FBS (Gibco)) containing HAT (Sigma) was used in DMEM (derived from biological company) to culture, and the resulting hybridoma colony was recovered to thereby prepare monoclonal hybridoma.

[0200] 1-3 Screening of human TLR7 binding antibody by cell-based ELISA

[0201] 1-3-1 Construction of HEK293T cell line overexpressing human TLR7

[0202] Using Max DNA Polymerase (Takara) to integrate human TLR7 gene (SEQ ID NO: 2) into lentiviral vector pCDH (Bio-Rad). The lentiviral vector was introduced into HEK293-derived packaging cell line Lenti-X 293T (Sigma) using transfection reagent EZ Trans (Geneseed Biotech Co., Ltd.). The culture supernatant was recovered after 48 hours and used as a virus suspension. The virus suspension was added to HEK293T cells to establish a HEK293T cell line overexpressing human TLR7.

[0203] 1-3-2 Screening of human TLR7-binding antibodies by cell-based ELISA

[0204] Hybridomas that formed colonies under a microscope were collected for screening, and the HEK293T cell line overexpressing human TLR7 constructed in 1-3-1 was added to the enzyme-labeled plate. After three days of culture, the culture medium was removed, and after fixation, the membrane was broken with 0.1% Triton X-100 (Solebo Co., Ltd.). Non-fat milk was added for blocking, and the culture supernatant was added to the enzyme-labeled plate for color development. Enzyme-labeled instrument was used for analysis to preliminarily screen hybridomas producing anti-hTLR7 antibodies.

[0205] 1-4 Screening of human TLR7-binding antibodies by flow cytometry analysis

[0206] 1-4-1 Construction of HEK293T cell line overexpressing human Unc93B1-Flag

[0207] Using Max DNA Polymerase (Takara) to integrate human Unc93B1 (SEQ ID NO: 1)-Flag gene into lentiviral vector pCDH (Bio-Rad). The lentiviral vector was introduced into HEK293-derived packaging cell line Lenti-X 293T (Sigma) using transfection reagent EZ Trans (Geneseed Biotech Co., Ltd.). The culture supernatant was recovered after 48 hours and used as a virus suspension. The virus suspension was added to HEK293T cells to establish a HEK293T cell line overexpressing human Unc93B1-Flag.

[0208] Using Max DNA Polymerase (Takara) was used to integrate the human Unc93B1 (SEQ ID NO: 1)-Flag gene into the lentiviral vector pCDH (Bio-Rad). The lentiviral vector was introduced into the HEK293-derived packaging cell line Lenti-X 293T (Sigma) using the transfection reagent EZ Trans (Wako Pure Chemical Industries, Ltd.). After 48 hours, the culture supernatant was recovered as a virus suspension. The virus suspension was added to HEK293T cells overexpressing human TLR7, thereby establishing a HEK293T cell line overexpressing human Unc93B1-Flag / human TLR7.

[0209] 1-4-3 Construction of a HEK293T cell line overexpressing human TLR7 (variant 2)

[0210] Using Max DNA Polymerase (Takara) was used to integrate the human TLR7 (variant 2, SEQ ID NO: 144) gene into the lentiviral vector pCDH (Bio-Rad). The lentiviral vector was introduced into the HEK293-derived packaging cell line Lenti-X 293T (Sigma) using the transfection reagent EZ Trans (Wako Pure Chemical Industries, Ltd.). After 48 hours, the culture supernatant was recovered as a virus suspension. The virus suspension was added to HEK293T cells, thereby establishing a HEK293T cell line overexpressing human TLR7 (variant 2).

[0211] 1-4-4 Construction of a HEK293T cell line overexpressing human Unc93B1 (SEQ ID NO: 1)-Flag / human TLR7 (variant 2)

[0212] Using Max DNA Polymerase (Takara) was used to integrate the human Unc93B1 (SEQ ID NO: 1)-Flag gene into the lentiviral vector pCDH (Bio-Rad). The lentiviral vector was introduced into the HEK293-derived packaging cell line Lenti-X 293T (Sigma) using the transfection reagent EZ Trans (Wako Pure Chemical Industries, Ltd.). After 48 hours, the culture supernatant was recovered as a virus suspension. The virus suspension was added to HEK293T cells overexpressing human TLR7 (variant 2), thereby establishing a HEK293T cell line overexpressing human Unc93B1-Flag / human TLR7 (variant 2).

[0213] 1-4-5 Construction of a Ba / F3 cell line overexpressing human Unc93B1 (SEQ ID NO: 1)-Flag / human TLR7 (variant 2)

[0214] Using Max DNA Polymerase (Takara) to integrate the human Unc93B1 (SEQ ID NO: 1)-Flag gene into the lentiviral vector pCDH (Bio-Rad). The lentiviral vector was introduced into the HEK293-derived packaging cell line Lenti-X 293T (Sigma) using the transfection reagent EZ Trans (Bio-Rad). After 48 hours, the culture supernatant was recovered as a virus suspension. The virus suspension was added to Ba / F3 cells to establish a Ba / F3 cell line overexpressing human Unc93B1-Flag / human TLR7 (variant 2). Max DNA Polymerase (Takara) to integrate the human TLR7 (variant 2, SEQ ID NO: 144) gene into the lentiviral vector pCDH (Bio-Rad). The lentiviral vector was introduced into the HEK293-derived packaging cell line Lenti-X 293T (Sigma) using the transfection reagent EZ Trans (Bio-Rad). After 48 hours, the culture supernatant was recovered as a virus suspension. The virus suspension was added to Ba / F3 cells to establish a Ba / F3 cell line overexpressing human Unc93B1-Flag / human TLR7 (variant 2).

[0215] 1-4-6 Screening of human TLR7-binding antibodies by flow cytometry analysis

[0216] The hybridoma culture supernatant producing human TLR7 antibodies was preliminarily screened in the cell-based ELISA of 1-3-2 for screening. For the HEK293T cell line overexpressing human TLR7 and the HEK293T cells not expressing at all and / or the HEK293T cell line overexpressing human Unc93B1-Flag / human TLR7 and the HEK293T cell line overexpressing human Unc93B1-Flag subjected to cell membrane permeation treatment with 1x wash buffer permeabilizing agent (Biolegend), the culture supernatant was stained separately, and the analysis by flow cytometry (Beckman, DxFLEX) was performed to screen the hybridoma producing anti-hTLR7 antibodies. As a result, seven hybridomas producing mouse anti-human TLR7 antibodies were selected, and were designated as 83A7, 182A1H9, 247C6A2, 13G1F4, 17D1D2, 7C6A2, 34G12B8, respectively.

[0217] In the present specification, the antibodies produced by the hybridomas 83A7, 182A1H9, 247C6A2, 13G1F4, 17D1D2, 7C6A2, 34G12B8 are designated as 83A7 antibody, 182A1H9 antibody, 247C6A2 antibody, 13G1F4 antibody, 17D1D2 antibody, 7C6A2 antibody, 34G12B8 antibody, respectively.

[0218] Example 2: Determination of the nucleotide sequence and amino acid sequence of the cDNA encoding the variable region of the mouse anti-human TLR7 antibody

[0219] Total RNA was recovered from the hybridomas of 83A7, 182A1H9, 247C6A2, 13G1F4, 17D1D2, 7C6A2, 34G12B8 using TRIzol Reagent (Ambion), and then cDNA was synthesized using PrimeScript TM 1st Strand cDNA Synthesis Kit (Takara).

[0220] 2-2 Amplification and sequence determination of variable region gene segments of mouse immunoglobulin heavy and light chains

[0221] Antibody VH and VL were amplified using universal antibody light and heavy chain primers. The PCR products were cloned into pUC19-T, colony sequenced, and the nucleotide sequences encoding the variable regions of the antibodies were interpreted.

[0222] 2-2-1 Mouse anti-human TLR7 antibody (83A7)

[0223] The amino acid sequence encoded by the determined nucleotide sequence of the cDNA encoding the heavy chain variable region of the 83A7 antibody begins with SEQ ID NO: 5 of the Sequence Listing.

[0224] The amino acid sequence encoded by the determined nucleotide sequence of the cDNA encoding the light chain variable region of the 83A7 antibody begins with SEQ ID NO: 12 of the Sequence Listing.

[0225] 2-2-2 Mouse anti-human TLR7 antibody (182A1H9)

[0226] The amino acid sequence encoded by the determined nucleotide sequence of the cDNA encoding the heavy chain variable region of the 182A1H9 antibody begins with SEQ ID NO: 6 of the Sequence Listing.

[0227] The amino acid sequence encoded by the determined nucleotide sequence of the cDNA encoding the light chain variable region of the 182A1H9 antibody begins with SEQ ID NO: 13 of the Sequence Listing.

[0228] 2-2-3 Mouse anti-human TLR7 antibody (247C6A2)

[0229] The amino acid sequence encoded by the determined nucleotide sequence of the cDNA encoding the heavy chain variable region of the 247C6A2 antibody begins with SEQ ID NO: 7 of the Sequence Listing.

[0230] The amino acid sequence encoded by the determined nucleotide sequence of the cDNA encoding the light chain variable region of the 247C6A2 antibody begins with SEQ ID NO: 14 of the Sequence Listing.

[0231] 2-2-4 Mouse anti-human TLR7 antibody (13G1F4)

[0232] The amino acid sequence encoded by the determined nucleotide sequence of the cDNA encoding the heavy chain variable region of the 13G1F4 antibody begins at SEQ ID NO: 8 of the Sequence Listing.

[0233] The amino acid sequence encoded by the determined nucleotide sequence of the cDNA encoding the light chain variable region of the 13G1F4 antibody begins at SEQ ID NO: 15 of the Sequence Listing.

[0234] 2-2-5 Mouse anti-human TLR7 antibody (17D1D2)

[0235] The amino acid sequence encoded by the determined nucleotide sequence of the cDNA encoding the heavy chain variable region of the 17D1D2 antibody begins at SEQ ID NO: 9 of the Sequence Listing.

[0236] The amino acid sequence encoded by the determined nucleotide sequence of the cDNA encoding the light chain variable region of the 17D1D2 antibody begins at SEQ ID NO: 16 of the Sequence Listing.

[0237] 2-2-6 Mouse anti-human TLR7 antibody (7C6A2)

[0238] The amino acid sequence encoded by the determined nucleotide sequence of the cDNA encoding the heavy chain variable region of the 7C6A2 antibody begins at SEQ ID NO: 10 of the Sequence Listing.

[0239] The amino acid sequence encoded by the determined nucleotide sequence of the cDNA encoding the light chain variable region of the 7C6A2 antibody begins at SEQ ID NO: 17 of the Sequence Listing.

[0240] 2-2-7 Mouse anti-human TLR7 antibody (34G12B8)

[0241] The amino acid sequence encoded by the determined nucleotide sequence of the cDNA encoding the heavy chain variable region of the 34G12B8 antibody begins at SEQ ID NO: 11 of the Sequence Listing.

[0242] The amino acid sequence encoded by the determined nucleotide sequence of the cDNA encoding the light chain variable region of the 34G12B8 antibody begins at SEQ ID NO: 18 of the Sequence Listing.

[0243] Example 3: Preparation of chimeric anti-human TLR7 antibodies

[0244] 3-1 Construction of expression vectors for chimeric anti-TLR7 antibodies

[0245] 3-1-1 Construction of a vector pcDNA3.4-LK expressing a chimeric light chain

[0246] A DNA fragment (SEQ ID NO: 128) containing a DNA sequence encoding a human light chain signal sequence and a human kappa chain constant region was ligated to the approximately 6 kb fragment obtained by digesting the plasmid pcDNA3.4 with the restriction enzymes BamH I and Hind III using T4 ligase (Takara), thereby constructing pcDNA3.4-LK.

[0247] 3-1-2 Construction of a chimeric IgG1 type heavy chain expression vector pCDNA3.4-G1

[0248] A DNA fragment in which the DNA sequence encoding a human heavy chain signal sequence and a human IgG1-LALA constant region was ligated to the DNA fragment in which the light chain signal sequence and the human kappa chain constant region were removed by digesting the plasmid pcDNA3.4-LK with the restriction enzymes BamH I and Hind III using T4 ligase (Takara), thereby constructing pcDNA3.4-G1.

[0249] 3-1-3 Construction of an 83A7 chimeric anti-human TLR7 antibody expression vector

[0250] A DNA fragment containing a DNA sequence encoding the heavy chain of the 83A7-c antibody was synthesized, and the synthesized DNA fragment was inserted into pcDNA3.4-G1 using assembly mix (Stratagene) at a position after the signal sequence and before the constant region nucleotide sequence, thereby constructing a heavy chain expression vector for the 83A7-c antibody. The heavy chain of the 83A7-c antibody contains a signal sequence and has the amino acid sequence of SEQ ID NO: 72.

[0251] A DNA fragment containing a DNA sequence encoding the light chain of the 83A7-c antibody was synthesized (SEQ ID NO: 131). Using pcDNA3.4-LK prepared in Example 3-1-1, a light chain expression vector for the 83A7-c antibody was constructed by the same method as described above. The light chain of the 83A7-c antibody contains a signal sequence and has the amino acid sequence of SEQ ID NO: 79.

[0252] 3-1-4 Construction of a 182A1H9 chimeric anti-human TLR7 antibody expression vector

[0253] A DNA fragment containing a DNA sequence encoding the heavy chain of the 182A1H9-c antibody was synthesized (SEQ ID NO: 133). Using the pcDNA3.4-LK prepared in Example 3-1-1, a 182A1H9-c light chain expression vector was constructed by the same method as in Example 3-1-3. The light chain of 182A1H9-c contains a signal sequence and has the amino acid sequence of SEQ ID NO: 80.

[0254] A DNA fragment containing a DNA sequence encoding the heavy chain of the 182A1H9-c antibody was synthesized (SEQ ID NO: 133). Using the pcDNA3.4-LK prepared in Example 3-1-1, a 182A1H9-c light chain expression vector was constructed by the same method as in Example 3-1-3. The light chain of 182A1H9-c contains a signal sequence and has the amino acid sequence of SEQ ID NO: 80.

[0255] 3-1-5 Construction of 247C6A2 chimeric anti-human TLR7 antibody expression vector

[0256] A DNA fragment containing a DNA sequence encoding the heavy chain of the 182A1H9-c antibody was synthesized (SEQ ID NO: 133). Using the pcDNA3.4-LK prepared in Example 3-1-1, a 182A1H9-c light chain expression vector was constructed by the same method as in Example 3-1-3. The light chain of 182A1H9-c contains a signal sequence and has the amino acid sequence of SEQ ID NO: 80.

[0257] A DNA fragment containing a DNA sequence encoding the heavy chain of the 182A1H9-c antibody was synthesized (SEQ ID NO: 133). Using the pcDNA3.4-LK prepared in Example 3-1-1, a 182A1H9-c light chain expression vector was constructed by the same method as in Example 3-1-3. The light chain of 182A1H9-c contains a signal sequence and has the amino acid sequence of SEQ ID NO: 80.

[0258] 3-1-6 Construction of 13G1F4 chimeric anti-human TLR7 antibody expression vector

[0259] A DNA fragment containing a DNA sequence encoding the 13G1F4-c heavy chain was synthesized (SEQ ID NO: 137). Using the pcDNA3.4-LK prepared in Example 3-1-1, a 13G1F4-c light chain expression vector was constructed by the same method as in Example 3-1-3. The light chain of 13G1F4-c contains a signal sequence, and has the amino acid sequence of SEQ ID NO: 82.

[0260] A DNA fragment containing a DNA sequence encoding the 13G1F4-c heavy chain was synthesized (SEQ ID NO: 137). Using the pcDNA3.4-LK prepared in Example 3-1-1, a 13G1F4-c light chain expression vector was constructed by the same method as in Example 3-1-3. The light chain of 13G1F4-c contains a signal sequence, and has the amino acid sequence of SEQ ID NO: 82.

[0261] 3-1-7 Construction of 17D1D2 chimeric anti-human TLR7 antibody expression vector

[0262] A DNA fragment containing a DNA sequence encoding the 17D1D2-c heavy chain was synthesized (SEQ ID NO: 139). Using the pcDNA3.4-LK prepared in Example 3-1-1, a 17D1D2-c light chain expression vector was constructed by the same method as in Example 3-1-3. The light chain of 17D1D2-c contains a signal sequence, and has the amino acid sequence of SEQ ID NO: 83.

[0263] A DNA fragment containing a DNA sequence encoding the 17D1D2-c heavy chain was synthesized (SEQ ID NO: 139). Using the pcDNA3.4-LK prepared in Example 3-1-1, a 17D1D2-c light chain expression vector was constructed by the same method as in Example 3-1-3. The light chain of 17D1D2-c contains a signal sequence, and has the amino acid sequence of SEQ ID NO: 83.

[0264] 3-1-8 Construction of 7C6A2 chimeric anti-human TLR7 antibody expression vector

[0265] A DNA fragment containing a DNA sequence encoding the 7C6A2-c heavy chain was synthesized (SEQ ID NO: 139). Using the pcDNA3.4-LK prepared in Example 3-1-1, a 17D1D2-c light chain expression vector was constructed by the same method as in Example 3-1-3. The light chain of 17D1D2-c contains a signal sequence, and has the amino acid sequence of SEQ ID NO: 83.

[0266] A DNA fragment containing a DNA sequence encoding the 7C6A2-c light chain was synthesized (SEQ ID NO: 141). Using the pcDNA3.4-LK prepared in Example 3-1-1, a 7C6A2-c light chain expression vector was constructed by the same method as in Example 3-1-3. The light chain of 7C6A2-c contains a signal sequence, and has an amino acid sequence of SEQ ID NO: 84.

[0267] 3-1-9 Construction of 34G12B8 Chimeric Anti-human TLR7 antibody expression vector

[0268] A DNA fragment containing a DNA sequence encoding the 34G12B8-c heavy chain nucleotide sequence encoding the heavy chain variable region of the 34G12B8-c antibody shown in the nucleotide sequence of SEQ ID NO: 142 from 58 to 423 bp was synthesized. A 34G12B8-c heavy chain expression vector was constructed by the same method as in Example 3-1-3. The heavy chain of 34G12B8-c contains a signal sequence, and has an amino acid sequence of SEQ ID NO: 78.

[0269] A DNA fragment containing a DNA sequence encoding the 34G12B8-c light chain was synthesized (SEQ ID NO: 143). Using the pcDNA3.4-LK prepared in Example 3-1-1, a 34G12B8-c light chain expression vector was constructed by the same method as in Example 3-1-3. The light chain of 34G12B8-c contains a signal sequence, and has an amino acid sequence of SEQ ID NO: 85.

[0270] 3-2 Preparation of chimeric anti-human TLR7 antibody

[0271] 3-2-1 Preparation of chimeric 83A7 antibody

[0272] For Expi293 cells (Gibco), passaging and culturing were performed according to the manual. 3 x 10 8 Expi293 cells (Gibco) in the logarithmic growth phase were inoculated in a 250 ml flask (nest) and diluted with Expi293 TM Expression Medium (Gibco) to adjust to 3 x 10 6Cells / ml. To 5 ml of Opti-MEM medium (Gibco), 50 μg of the heavy chain expression vector constructed in the above 3-1-3 and 50 μg of the light chain expression vector constructed in the above 3-1-3 were added, and mixed. To 5 ml of Opti-MEM medium (Gibco), 100 μg of polyethylenimine (Bioscience) was added, and mixed. After mixing the vector and polyethylenimine, it was added to Expi293 cells after being left still for 15 minutes. It was cultured for 4 days in a 37°C, 7% CO2 incubator, and the obtained supernatant was filtered with a vacuum suction filter cup (Nest), thereby manufacturing the chimeric 83A7-c antibody.

[0273] 3-2-2 Preparation of chimeric 182A1H9 antibody

[0274] Using the heavy chain expression vector and the light chain expression vector constructed in the above 3-1-4, the chimeric 182A1H9-c antibody was manufactured by the method of the above 3-2-1.

[0275] 3-2-3 Preparation of chimeric 247C6A2 antibody

[0276] Using the heavy chain expression vector and the light chain expression vector constructed in the above 3-1-5, the chimeric 247C6A2-c antibody was manufactured by the method of the above 3-2-1.

[0277] 3-2-4 Preparation of chimeric 13G1F4 antibody

[0278] Using the heavy chain expression vector and the light chain expression vector constructed in the above 3-1-6, the chimeric 13G1F4-c antibody was manufactured by the method of the above 3-2-1.

[0279] 3-2-5 Preparation of chimeric 17D1D2 antibody

[0280] Using the heavy chain expression vector and the light chain expression vector constructed in the above 3-1-7, the chimeric 17D1D2-c antibody was manufactured by the method of the above 3-2-1.

[0281] 3-2-6 Preparation of chimeric 13H17L antibody

[0282] Using the heavy chain expression vector constructed in the above 3-1-6 and the light chain expression vector constructed in the above 3-1-7, the chimeric 13H17L-c antibody (13H17L antibody is obtained by expressing a combination of the heavy chain of the 13G1F4 antibody and the light chain of the 17D1D2 antibody) was manufactured by the method of the above 3-2-1.

[0283] 3-2-7 Preparation of chimeric 7C6A2 antibody

[0284] Using the heavy chain expression vector and the light chain expression vector constructed in the above 3-1-8, a chimeric 7C6A2-c antibody was produced by the method described in the above 3-2-1.

[0285] 3-2-8 Production of chimeric 34G12B8 antibody

[0286] Using the heavy chain expression vector and the light chain expression vector constructed in the above 3-1-9, a chimeric 34G12B8-c antibody was produced by the method described in the above 3-2-1.

[0287] 3-3 Purification of chimeric anti-human TLR7 antibody

[0288] The target antibody was purified from each of the culture supernatants obtained in Example 3-2 by a series of procedures of Protein A affinity chromatography. After the culture supernatant was loaded onto a chromatography column packed with Protein A (GE Healthcare) equilibrated with binding buffer (Sangon Biotech), the chromatography column was washed twice with 5 column volumes of binding buffer. Then, elution was performed with elution buffer (Sangon Biotech), and the equilibration buffer was 1M Tris-HCl, pH 7.4 (Sangon Biotech). The antibody buffer was replaced with PBS using an Amicon Ultra-15 centrifugal filter equipped with an Ultracel-30 filter membrane, and the antibody was further concentrated. Finally, the purified sample was prepared by filtration using a 0.22μM disposable needle filter (PALL).

[0289] Example 4: In vitro evaluation of chimeric anti-human TLR7 antibody

[0290] 4-1 Evaluation of binding selection ability of chimeric anti-human TLR7 antibody

[0291] 4-1-1 Establishment of HEK293T cell strain overexpressing mouse TLR7

[0292] Using Max DNA Polymerase (Takara) to integrate the mouse TLR7 gene (SEQ ID NO: 126) into the lentiviral vector pCDH (System Biosciences). The lentiviral vector was introduced into the HEK293-derived packaging cell line Lenti-X 293T (Sigma) using the transfection reagent EZ Trans (Genechem). After 48 hours, the culture supernatant was recovered as a virus suspension. The virus suspension was added to HEK293T cells to establish a HEK293T cell strain overexpressing mouse TLR7.

[0293] 4-1-2 Establishment of HEK293T cell strain overexpressing cynomolgus monkey TLR7

[0294] Using Max DNA Polymerase (Takara) to integrate the cynomolgus monkey TLR7 gene (SEQ ID NO: 127) into the lentivirus vector pCDH (Bio-Rad). The lentivirus vector was introduced into the HEK293-derived packaging cell line Lenti-X 293T (Sigma) using the transfection reagent EZ Trans (Wako Pure Chemical Industries, Ltd.). After 48 hours, the culture supernatant was recovered as a virus suspension. The virus suspension was added to HEK293T cells to establish a HEK293T cell line overexpressing cynomolgus monkey TLR7.

[0295] 4-1-3 Establishment of a HEK293T cell line overexpressing cynomolgus monkey TLR7 / cynomolgus monkey Unc93B1

[0296] Using Max DNA Polymerase (Takara) to integrate the cynomolgus monkey Unc93B1 gene (SEQ ID NO: 145) into the lentivirus vector pCDH (Bio-Rad). The lentivirus vector was introduced into the HEK293-derived packaging cell line Lenti-X 293T (Sigma) using the transfection reagent EZ Trans (Wako Pure Chemical Industries, Ltd.). After 48 hours, the culture supernatant was recovered as a virus suspension. The virus suspension was added to the HEK293T cells overexpressing cynomolgus monkey TLR7 to establish a HEK293T cell line overexpressing cynomolgus monkey TLR7 / cynomolgus monkey Unc93B1.

[0297] 4-1-4 Evaluation of the binding selectivity of chimeric anti-human TLR7 antibodies using a flow cytometer

[0298] The HEK293T cell strain overexpressing human TLR7 established in 1-3-1, the HEK293T cell strain overexpressing mouse TLR7 established in 4-1-1, the HEK293T overexpressing cynomolgus monkey TLR7 established in 4-1-2, the HEK293T overexpressing cynomolgus monkey TLR7 / cynomolgus monkey Unc93B1 established in 4-1-3, the HEK293T cell strain overexpressing human TLR7 (variant 2) established in 1-4-3 were subjected to cell membrane permeation treatment with 1x permeabilization buffer (Biolegend), and stained with the chimeric 83A7 antibody, 182A1H9 antibody, 247C6A2 antibody, 13G1F4 antibody, 17D1D2 antibody, 13H17L antibody, 7C6A2 antibody, 34G12B8 antibody obtained in Example 3 at an antibody concentration dilution series, and goat anti-human IgG H&L-FITC (Abeam) as a secondary antibody. For evaluation of the binding selectivity of the antibodies, analysis was performed using a flow cytometer and the mean fluorescence intensity (MFI) was compared.

[0299] As a result, the 83A7 antibody, 182A1H9 antibody, 247C6A2 antibody, 13G1F4 antibody, 17D1D2 antibody, 13H17L antibody, 7C6A2 antibody, 34G12B8 antibody specifically bound to human TLR7 (FIG. 1);

[0300] The 83A7 antibody, 182A1H9 antibody, 247C6A2 antibody, 13G1F4 antibody, 17D1D2 antibody, 13H17L antibody, 7C6A2 antibody, 34G12B8 antibody specifically bound to cynomolgus monkey TLR7 (FIG. 2); the series of anti-human TLR7 antibodies screened herein, such as the 83A7 antibody, 247C6A2 antibody, 13G1F4 antibody, 17D1D2 antibody, 13H17L antibody, 7C6A2 antibody, 34G12B8 antibody did not bind to mouse TLR7 (FIG. 3, the results show that the antibodies do not specifically bind to the antigen); the 83A7 antibody, 182A1H9 antibody, 247C6A2 antibody, 13G1F4 antibody, 17D1D2 antibody, 7C6A2 antibody, 34G12B8 antibody, 13H17L antibody specifically bound to human TLR7-variant 2 (FIG. 4). The EC 50 See Table 3

[0301] Table 3 EC 50 The results show

[0302] Note: N / A means not applicable

[0303] 4-2 Antigen binding activity of chimeric anti-human TLR7 antibodies using a flow cytometer

[0304] The HEK293T cell strain overexpressing human Unc93B1-Flag / human TLR7 was subjected to cell membrane permeation treatment with 1x Permeabilizing Solution (Biolegend), and stained with the chimeric 83A7 antibody, 182A1H9 antibody, 247C6A2 antibody, 13G1F4 antibody, 17D1D2 antibody, 13H17L antibody, 7C6A2 antibody, 34G12B8 antibody obtained in Example 3 at an antibody concentration dilution series and Goat anti human IgG H&L-FITC (Abeam) as a secondary antibody. For evaluation of the binding activity of the antibodies, analysis was performed using a flow cytometer and MFI was compared. The results are shown in FIG. 5, and the binding activity was in order from strong to weak as follows: chimeric 13G1F4 antibody (13G1F4-c), chimeric 13H17L antibody (13H17L-c), chimeric 34G12B8 antibody (34G12B8-c), chimeric 17D1D2 antibody (17D1D2-c), chimeric 247C6A2 antibody (247C6A2-c), chimeric 83A7 antibody (83A7-c), chimeric 7C6A2 antibody (7C6A2-c), chimeric 182A1H9 antibody (182A1H9-c), wherein EC 50 : 13H17L-c 0.4608 nM, 17D1D2-c 0.5407 nM, 13G1F4-c 0.4263 nM, 247C6A2-c 0.5754 nM, 34G12B8-c 0.5021 nM, 83A7-c 0.9462 nM, 7C6A2-c 33.71 nM.

[0305] The HEK293T cell strain overexpressing human Unc93B1-Flag / human TLR7 (variant 2) was subjected to cell membrane permeation treatment with 1x Permeabilizing Solution (Biolegend), and stained with the chimeric 247C6A2, 13H17L antibodies obtained in Example 3 at an antibody concentration dilution series and Goat anti human IgG H&L-FITC (Abeam) as a secondary antibody. For evaluation of the binding activity of the antibodies, MFI was analyzed using a flow cytometer. As shown in FIG. 6, the results are that 247C6A2-c, 13H17L-c specifically bind to the cells.

[0306] The Ba / F3 cell strain overexpressing human Unc93B1-G4S-Flag-T2A-human TLR7 was treated with 1x transmembrane reagent (Biolegend) for cell membrane permeation, and stained with the chimeric 83A7 antibody, 182A1H9 antibody, 247C6A2 antibody obtained in Example 3, antibody concentration dilution series, and goat anti-human IgG H&L-FITC (Abeam) as a secondary antibody. For evaluation of the binding activity of the antibodies, analysis was performed using a flow cytometer and MFI was compared. As shown in FIG. 7, the results were that the binding activity was in order of chimeric 247C6A2 antibody (247C6A2-c), chimeric 83A7 antibody (83A7-c), and chimeric 182A1H9 antibody (182A1H9-c) from strong to weak, and the EC 50 : 247C6A2-c 0.6793 nM, 83A7-c 2.234 nM. The EC 50 See Table 4.

[0307] Table 4 EC 50 The results show

[0308] Note: N / A represents not applicable

[0309] 4-3 Inhibitory effect of chimeric anti-human TLR7 antibodies on cytokine production

[0310] Human PBMCs were purchased from Shanghai Aobio Biotech Co., Ltd. in a frozen form, and used after thawing according to the instructions. The RPMI 1640 (Gibco) containing 10% FBS (Excell), 1 mM sodium pyruvate (Gibco), 0.1 mM MEM-Non-Essential Amino Acids (Gibco), 50 mM 2-mercaptoethanol (Gibco), 50 U / ml penicillin, and 50 U / ml streptomycin (Procell) was adjusted to 2x10 6PBMC at a concentration of 2 x 105cells / ml were each inoculated into 100 μl in a 96-well cell culture plate, 80 μl / well of different concentrations of 247C6A2 antibody, 13G1F4 antibody, 17D1D2 antibody, 13H17L antibody, 34G12B8 antibody or human IgG1-LALA control antibody (Biolink) as chimeric anti-human TLR7 antibody was added, and pre-treatment was performed for 6 hours in a 37°C incubator. Then, 0.5 μM or 1 μM of TLR7 agonist GS-9620 (MCE) was added in 20 μl / well, after thorough stirring, and incubation was performed at 37°C, 5% CO2for about 20 hours. After thorough stirring of the plate, centrifugation was performed at 1500 rpm for 5 minutes, and the concentration of IL-6 contained in the supernatant was determined by sandwich ELISA (Sinobio); the concentration of IFN-α contained in the supernatant was determined by sandwich ELISA (Mabtech).

[0311] Figure 8 shows that chimeric anti-human TLR7 antibodies inhibit the production of IL-6 of human PBMC treated with GS-9620. 247C6A2-c, 13H17L-c, 17D1D2-c, 34G12B8-c can inhibit the production of IL-6 of human PBMC. On the other hand, no inhibition was observed even at a concentration of 5 μg / ml of human IgG1-LALA control antibody.

[0312] Figure 9 shows that chimeric anti-human TLR7 antibodies inhibit the production of IFN-α of human PBMC treated with GS-9620. 247C6A2-c, 13H17L-c, 17D1D2-c, 34G12B8-c can inhibit the production of IFN-α of human PBMC. On the other hand, no significant inhibition was observed even at a concentration of 5 μg / ml of human IgG1-LALA control antibody.

[0313] Human PBMC were purchased from Shanghai Aobio Biotech Co., Ltd. in frozen form, and used after thawing according to the instructions. RPMI 1640 (Gibco) containing 10% FBS (Excell), 1 mM sodium pyruvate (Gibco), 0.1 mM MEM-Non-Essential Amino Acids (Gibco), 50 mM 2-mercaptoethanol (Gibco), 50 U / ml penicillin and 50 U / ml streptomycin (Procell) was adjusted to 2 x 105cells / ml, and 100 μl was inoculated into each well of a 96-well cell culture plate. 80 μl / well of different concentrations of 247C6A2 antibody, 13G1F4 antibody, 17D1D2 antibody, 13H17L antibody, 34G12B8 antibody or human IgG1-LALA control antibody (Biolink) as chimeric anti-human TLR7 antibody was added, and pre-treatment was performed for 6 hours in a 37°C incubator. Then, 0.5 μM or 1 μM of TLR7 agonist GS-9620 (MCE) was added in 20 μl / well, after thorough stirring, and incubation was performed at 37°C, 5% CO2for about 20 hours. After thorough stirring of the plate, centrifugation was performed at 1500 rpm for 5 minutes, and the concentration of IL-6 contained in the supernatant was determined by sandwich ELISA (Sinobio); the concentration of IFN-α contained in the supernatant was determined by sandwich ELISA (Mabtech). 6PBMC at a concentration of 1 x 106cells / ml were each inoculated into 100 μl in a 96-well cell culture plate, 80 μl / well of 13G1F4 antibody, 17D1D2 antibody, 13H17L antibody, 34G12B8 antibody, or human IgG1-LALA control antibody (Biyong) as a chimeric anti-human TLR7 antibody was added at different concentrations, and pre-treatment was performed in a 37°C incubator for 6 hours. Then, 20 μl / well of DSR-6434 (MCE) at different concentrations as shown in FIGS. 10-13 was added, and after thorough stirring, incubation was performed at 37°C, 5% CO2for about 20 hours. After thorough stirring of the plate, centrifugation was performed at 1500 rpm for 5 minutes, and the concentration of IL-6 contained in the supernatant was determined by sandwich ELISA (Sinobio), the concentration of TNF-α contained in the supernatant was determined by sandwich ELISA (Proteintech), and the concentration of IP-10 contained in the supernatant was determined by sandwich ELISA (Proteintech).

[0314] FIG. 10 shows that the chimeric anti-human TLR7 antibody inhibits the production of IL-6 of human PBMC treated with DSR-6434. 13H17L-c, 17D1D2-c, 13G1F4-c, 34G12B8-c can inhibit the production of IL-6 of human PBMC. On the other hand, no inhibition was observed even at a concentration of 5 μg / ml of human IgG1-LALA control antibody.

[0315] FIG. 11 shows that the chimeric anti-human TLR7 antibody inhibits the production of IFN-α of human PBMC treated with DSR-6434. 13H17L-c can inhibit the production of IFN-α of human PBMC. On the other hand, no inhibition was observed even at a concentration of 5 μg / ml of human IgG1-LALA control antibody.

[0316] FIG. 12 shows that the chimeric anti-human TLR7 antibody inhibits the production of TNF-α of human PBMC treated with DSR-6434. 13H17L-c, 17D1D2-c, 13G1F4-c, 34G12B8-c can inhibit the production of TNF-α of human PBMC. On the other hand, no inhibition was observed even at a concentration of 5 μg / ml of human IgG1-LALA control antibody.

[0317] FIG. 13 shows that the chimeric anti-human TLR7 antibody inhibits the production of IP-10 of human PBMC treated with DSR-6434. 13H17L-c, 13G1F4-c, 34G12B8-c can inhibit the production of IP-10 of human PBMC. On the other hand, no inhibition was observed even at a concentration of 1 μg / ml of human IgG1-LALA control antibody.

[0318] Example 5: Production of humanized anti-human TLR7 antibody

[0319] 5-1 Design of humanization of anti-human TLR7 antibody

[0320] 5-1-1 Design of humanization of 13H17L antibody

[0321] Structural modeling of murine antibody variable region Structural modeling of murine antibody variable region was performed by Bioluminate module in Schrödinger®. Based on RCSB database, and according to the resolution of structure resolution and the similarity criteria of light and heavy chain framework sequences, 4AEH (PDB ID) was selected for structural modeling of murine antibody variable region. The highest homology template VH of 13H17L was IGHV1-2*02, and the VL was IGKV1-27*01; the highest homology template VH of 34G12B8 was IGHV4-38-2*02, and the VL was IGKV1-39*01. Humanization was performed by CDR grafting method. According to the consensus sequence defined by IMGT numbering, the homologous acceptor was selected in the human germline database. Based on the information of Vernier zone, Canonical structure and key amino acid residues of light and heavy chain interface identified by Bioluminate module, the donor residues on the acceptor were selected and moved in. In order to maintain the original antibody affinity and function, and at the same time provide stability. Back mutations strategy was used to design candidate humanization sequences, 10 for 13H17L and 19 for 34G12, which were used for later affinity and immunogenicity tests.

[0322] 5-1-1-2 Humanization of heavy chain variable region of 13H17L antibody

[0323] (1) The designed heavy chain variable region was named 13H17L-GH1-H0~13H17L-GH1-H10, and the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 in the sequence were the same as those of CDR-H1, CDR-H2 and CDR-H3 of 13G1F4 antibody.

[0324] 5-1-1-3 Humanization of light chain variable region of 13H17L antibody

[0325] (2) The designed light chain variable region was named 13H17L-GL1-L0~13H17L-GL1-L9, and the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 in the sequence were the same as those of CDR-L1, CDR-L2 and CDR-L3 of 17D1D2 antibody.

[0326] 5-1-1-4 Humanization of heavy chain variable region of 34G12B8 antibody

[0327] (3) The designed heavy chain variable region is named 34G12-GH1-H0~34G12-GH1-H12, and the CDR-H1, CDR-H2 and CDR-H3 in the sequence are the same as the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 of the 34G12B8 antibody.

[0328] 5-1-1-5 Humanization of the light chain variable region of the 34G12B8 antibody.

[0329] (4) The designed light chain variable region is named 34G12-GL1-L0~34G12-GL1-L3, and the CDR-L1, CDR-L2 and CDR-L3 in the sequence are the same as the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 of the 34G12B8 antibody.

[0330] 5-2 Design of humanized antibodies by combining heavy chains and light chains

[0331] 5-2-1 Design of humanized antibody 13H17L anti-TLR7 antibody

[0332] The light chain and the heavy chain are combined into an antibody, and the name is as follows:

[0333] Table 5 Design combination of humanized antibody 13H17L anti-TLR7 antibody

[0334] 5-2-2 Design of humanized antibody 34G12 anti-TLR7 antibody

[0335] Table 6 Design combination of humanized antibody 34G12 anti-TLR7 antibody

[0336] Table 7 Framework region humanization display

[0337] Table 8 Framework region humanization display

[0338] 5-3 Preparation of humanized anti-human TLR7 antibody

[0339] 5-3-1 Construction of 13H17L humanized antibody heavy chain expression vector

[0340] A DNA fragment containing a DNA sequence encoding the variable region of the heavy chain of 13H17L-GH1-H0 was synthesized. The synthesized DNA fragment was inserted into pcDNA3.4-G1 using assembly mix (Stratagene) after the signal sequence and before the constant region nucleotide sequence, thereby constructing a heavy chain expression vector for 13H17L-GH1-H0-huIgGl-LALA. The heavy chain of 13H17L-GH1-H0-huIgGl-LALA contains a signal sequence.

[0341] Heavy chain expression vectors for 13H17L-GH1-H1 to 13H17L-GH1-H10 were also constructed by the same method.

[0342] 5-3-2 Construction of a light chain expression vector for a humanized 13H17L antibody

[0343] A DNA fragment containing a DNA sequence encoding the variable region of the light chain of 13H17L-GL1-L0 was synthesized. A light chain expression vector for 13H17L-GL1-L0-kappa was constructed by the same method as described above using pcDNA3.4-LK prepared in Example 3-1-1. The light chain of 13H17L-GL1-L0-kappa contains a signal sequence.

[0344] Light chain expression vectors for 13H17L-GL1-L1 to 13H17L-GL1-L9 were also constructed by the same method.

[0345] 5-3-3 Construction of a heavy chain expression vector for a humanized 34G12 antibody

[0346] A DNA fragment containing a DNA sequence encoding the variable region of the heavy chain of 34G12-GH1-H0 was synthesized. A heavy chain expression vector for 34G12-GH1-H0-huIgGl-LALA was constructed by inserting the synthesized DNA fragment into pcDNA3.4-G1 using assembly mix (Stratagene) after the signal sequence and before the constant region nucleotide sequence. The heavy chain of 34G12-GH1-H0-huIgGl-LALA contains a signal sequence.

[0347] Heavy chain expression vectors for 34G12-GH1-H1 to 34G12-GH1-H12 were also constructed by the same method.

[0348] 5-3-4 Construction of a light chain expression vector for a humanized 34G12 antibody

[0349] A DNA fragment containing a DNA sequence encoding the variable region of the light chain of 34G12-GL1-L0 was synthesized. Using the pcDNA3.4-LK prepared in Example 3-1-1, a 34G12-GL1-L0-kappa expression vector was constructed by the same method as described above. The light chain of 34G12-GL1-L0-kappa contains a signal sequence.

[0350] Light chain expression vectors for 34G12-GL1-L1 to 34G12-GL1-L3 were also constructed by the same method as described above.

[0351] 5-3-5 Production of humanized antibodies

[0352] 5-3-5-1 Production of humanized antibodies

[0353] Each of the humanized antibodies was produced by the same method as in Example 3-2-1 using each of the expression vectors constructed in 5-3-1 to 5-3-4, by combining the heavy chain designed in Example 5-2 with the light chain.

[0354] 5-3-5-2 Purification of humanized antibodies

[0355] Each of the target antibodies was purified from each of the culture supernatants obtained in Example 5-3-5-1 by a series of procedures of Protein A affinity chromatography. After loading the culture supernatant onto a chromatography column packed with Protein A (GE Healthcare) equilibrated with binding buffer (Genview), the column was washed twice with 5 column volumes of binding buffer. Then, elution was performed with elution buffer (Genview), and the equilibration buffer was 1M Tris-HCl, pH 7.4 (Genview). The antibody buffer was replaced with PBS using an Amicon Ultra-15 centrifugal filter equipped with an Ultracel-30 filter membrane, and the antibody was further concentrated. Finally, the purified sample was prepared by filtration using a 0.22 μM disposable needle filter (PALL).

[0356] Example 6: In vitro activity of humanized anti-human TLR7 antibodies

[0357] 6-1 Evaluation of binding selection ability of humanized anti-human TLR7 antibodies

[0358] 6-1-1 Evaluation of binding selection ability of chimeric anti-human TLR7 antibodies using a flow cytometer

[0359] HEK293T cells overexpressing human Unc93B1-Flag / human TLR7 established in 1-4-2 were treated with 1x Transmembrane Permeabilization Buffer (Biolegend) for cell membrane permeabilization, stained with a concentration dilution series of the humanized 13H17L or 34G12 antibodies obtained in Example 5 and Goat anti human IgG H&L-FITC (Abeam) as secondary antibody. For evaluation of the binding selectivity of the antibodies, the analysis was performed by flow cytometry and the mean fluorescence intensity (MFI) was compared.

[0360] HEK293T cells overexpressing human TLR7 established in 1-3-1 were treated with 1x Transmembrane Permeabilization Buffer (Biolegend) for cell membrane permeabilization, stained with a concentration dilution series of the humanized 13H17L or 34G12 antibodies obtained in Example 5 and Goat anti human IgG H&L-FITC (Abeam) as secondary antibody. For evaluation of the binding selectivity of the antibodies, the analysis was performed by flow cytometry and the mean fluorescence intensity (MFI) was compared.

[0361] HEK293T cells overexpressing human Unc93B1-Flag / human TLR7 (variant 2) established in 1-4-4 were treated with 1x Transmembrane Permeabilization Buffer (Biolegend) for cell membrane permeabilization, stained with a concentration dilution series of the humanized 13H17L antibodies obtained in Example 5 and Goat anti human IgG H&L-FITC (Abeam) as secondary antibody. For evaluation of the binding selectivity of the antibodies, the analysis was performed by flow cytometry and the mean fluorescence intensity (MFI) was compared.

[0362] HEK293T cell strain overexpressing cynomolgus TLR7 / cynomolgus Unc93B1 established in 4-1-3 were treated with 1x Transmembrane Permeabilization Buffer (Biolegend) for cell membrane permeabilization, stained with a concentration dilution series of the humanized 34G12 antibodies obtained in Example 5 and Goat anti human IgG H&L-FITC (Abeam) as secondary antibody. For evaluation of the binding selectivity of the antibodies, the analysis was performed by flow cytometry and the mean fluorescence intensity (MFI) was compared.

[0363] 1) 13H17L humanized antibodies (13H17L-h1 - 13H17L h22) specifically bind to HEK293T cells overexpressing human Unc93B1-Flag / human TLR7 (variant 2) with the results shown in Figure 14.

[0364] 2) 34G12 humanized antibodies (34G12-h1 - 34G12-h19) specifically bind to HEK293T cells overexpressing human Unc93B1-Flag / human TLR7. The results are shown in Figures 15-16.

[0365] 3) 34G12 humanized antibodies (34G12-h1 - 34G12-h19) specifically bind to HEK293T cells overexpressing monkey Unc93B1-Flag / monkey TLR7. The results are shown in Figure 17.

[0366] 4) EC 50 See Table 9

[0367] Table 9 EC 50 Results show

[0368] 6-2 Cytokine production inhibition effect of humanized anti-human TLR7 antibodies

[0369] Human PBMCs were purchased from Shanghai Aobio Biotech Co., Ltd. in frozen form, and used after thawing according to the instructions. PBMCs were adjusted to a concentration of 2 x 10 6 Each 100 μl of PBMCs adjusted to a concentration of 2 x 10

[0370] 1) As shown in Figure 18, 13H17L humanized antibodies (13H17L-h1 - 13H17L-h22) effectively inhibited the production of IL-6 by human PBMCs treated with GS-9620. On the other hand, no inhibition was observed with the human IgG1-LALA control antibody.

[0371] 2) As shown in Figures 19-20, 34G12 humanized antibodies (34G12-h1 - 34G12-h19) effectively inhibited IL-6 and TNFa production by GS-9620 treated human PBMC. In contrast, no inhibition was observed with the human IgGl -LALA control antibody.

[0372] Example 7: In vivo activity of humanized anti-human TLR7 antibodies

[0373] 7-1 Validation of humanized anti-human TLR7 antibody activity in vivo in CD34+ humanized mice

[0374] 7-1-1 Modeling of CD34+ humanized mice

[0375] 4-week old NSG-SGM3 female mice were whole body irradiated at 100 cGy. After irradiation, mice were returned to a new cage and injected with hu-CD34+ cells (umbilical cord blood derived) 100 uL CD34+ cells (1*10e5 per mouse) via tail vein within 24 hours post irradiation and Bactrim water was given continuously post irradiation. Blood was taken for flow cytometry to check PBMC reconstitution efficiency post cell inoculation as per experimental requirement.

[0376] 7-1-2 Validation of humanized anti-human TLR7 antibody activity in vivo in CD34+ humanized mice

[0377] Experiments were performed with in vivo injection of antibody + in vivo injection of TLR7 agonist. First, mice were grouped: 9-week old (human CD34+ cell injected 5w) female NSG-SGM3 mice were divided into 5 groups, 2-4 mice per group; G3 mice were injected with 10 mg / kg of 34G12-h14 antibody via tail vein as described in Table 10; after overnight (18h), G1 group was injected with PBS and G2, G3 groups were injected with 0.01 mg / kg TLR7 agonist DSR-6434 via tail vein; 2 hours later, 100 μl of blood was taken from all mice, centrifuged at 5000 rpm for 5 min, and the supernatant was collected; ELISA kit was used to detect mouse serum IL-6 (human IL-6 kit), and mouse serum was diluted 4-fold before use.

[0378] Table 10 Grouping situation

[0379] The experimental results are shown in Figure 21. Hybridoma screening TLR7 antibodies effectively inhibited the release of inflammatory factor IL-6 induced by TLR7 agonist DSR-6434 in CD34+ mice, which preliminarily verified that hybridoma screening TLR7 antibodies have activity in vivo.

[0380] Example 8: Manufacture of affinity matured antibodies against human TLR7 antibodies

[0381] Affinity maturation design of 8-1 antibody

[0382] By simulating the three-dimensional structure of the antibody-antigen complex using AlphaFold2 or its derivative models, key amino acid residues with potential impact on binding affinity are identified by analyzing the molecular interactions between the binding interface. Rational design is performed on the key amino acid residues to construct a single-point mutant library. The positive mutants with higher affinity and improved function are screened through the selection of binding ability screening by overexpression system cells and combined with in vitro functional experiments. The positive single-point mutations screened are combined to construct multiple mutants. The multiple mutants are verified for affinity and function, and finally the antibody sequence with optimized performance is obtained.

[0383] 8-1-2 Affinity maturation of 34G12 antibody heavy chain variable region

[0384] 8-1-2-1 Affinity maturation of 34G12-h8, 34G12-h14 antibody light and heavy chain variable region

[0385] According to the binding and functional experiments, it is determined that the humanized antibodies 34G12-h8 and 34G12-h14 are further optimized, and after multiple rounds of verification of single-point mutations by affinity maturation, it is determined that the LC mutation H90S and the HC mutation A98V+N104W+M109N+Y101R+G31Y will greatly improve the affinity. The parent subjected to affinity maturation on the basis of h14 is called 34G12-h458 (shown in Tables 11-12, 15), and the parent subjected to affinity maturation on the basis of h8 becomes 34G12-h542 (shown in Tables 13-14, 16). On the basis of these two parents, affinity maturation is further performed.

[0386] As shown in Tables 11-16, by site-directed mutagenesis of the parent antibodies 34G12-h458 and 34G12-h542, a plurality of variants with significantly enhanced affinity were obtained. The mutation position description in Table 11-16 refers to the light and heavy chain sequence of the parent, such as 34G12-h458 referring to the sequence of 34G12-h14, and 34G12-h477 referring to the sequence of 34G12-h458. The heavy chain variable region sequence of 34G12-h458 is shown as SEQ ID NO: 172, and the light chain variable region sequence is shown as SEQ ID NO: 173, the heavy chain variable region sequence of 34G12-h8 is shown as SEQ ID NO: 180, and the light chain variable region sequence is shown as SEQ ID NO: 181, the heavy chain variable region sequence of 34G12-h542 is shown as SEQ ID NO: 174, and the light chain variable region sequence is shown as SEQ ID NO: 175, the heavy chain variable region sequence of 34G12-h14 is shown as SEQ ID NO: 182, and the light chain variable region sequence is shown as SEQ ID NO: 183.

[0387] 1) CDR mutation based on h458: 34G12-h477, 34G12-h483, 34G12-h493-34G12-h500, 34G12-h544 are affinity maturation antibodies based on single point or combination mutation of 34G12-h458, aiming to improve affinity or improve drugability.

[0388] 2) CDR mutation based on h542: 34G12-h552, 34G12-h560, 34G12-h561-34G12-h563, 34G12-h568, 34G12-h569, 34G12-h581, 34G12-h587-34G12-h589, 34G12-h591-34G12-h599, 34G12-h601 are affinity maturation antibodies based on single point or combination mutation of 34G12-h542, aiming to improve antibody affinity or improve drugability.

[0389] 3) FR mutation based on h458: 34G12-h512, 34G12-h513, 34G12-h522-34G12-h528, 34G12-h541 are point mutations based on 34G12-h458, aiming to improve antibody affinity or improve drugability, 34G12-h529-34G12-h535 are back mutations based on 34G12-h458, aiming to reduce back mutation sites and improve drugability.

[0390] 4) FR mutation based on h542: 34G12-h545, 34G12-h546, 34G12-h548, 34G12-h554, 34G12-h555, 34G12-h576, 34G12-h583 are point mutations based on 34G12-h542, aiming to improve the affinity of the antibody or improve the drugability, 34G12-h577-34G12-h580, 34G12-h583-34G12-h586 are back mutations based on 34G12-h542, aiming to reduce the back mutation sites and improve the drugability.

[0391] 5) CDR+FR mutation based on h458: 34G12-h465, 34G12-h480, 34G12-h482, 34G12-h484-34G12-h489, 34G12-h491 are point mutations based on 34G12-h458, aiming to improve the affinity or improve the drugability.

[0392] 6) CDR+FR mutation based on h542: 34G12-h559, 34G12-h564-34G12-h567, 34G12-h598, 34G12-h600 are point mutations based on 34G12-h542, aiming to improve the affinity or improve the drugability.

[0393] The binding ability of the affinity matured antibody mutants is similar to or higher than that of the parent, and they can all inhibit the production of IL-6 and TNF-α of human PBMC treated by GS-9620. Exemplary results can be found in Examples 9-10.

[0394] Table 11. Mutation explanation of CDR region of 34G12-h458

[0395] Table 12. Mutation explanation of CDR region of 34G12-h458 affinity maturation related mutant sequence

[0396] Table 13. Mutation explanation of CDR region of 34G12-h542

[0397] Table 14. Mutation explanation of CDR region of 34G12-h542 affinity maturation related mutant sequence

[0398] Table 15. Mutation explanation of framework region of 34G12-h458 and related mutant sequence

[0399] Table 16 Frame region mutation explanation of 34G12-h542 and related mutant sequences

[0400] 8-2 Preparation of affinity matured antibodies against human TLR7

[0401] 8-2-1 Construction of light and heavy chain expression vectors of 34G12 affinity matured antibodies

[0402] The present application uses site-directed mutagenesis technology to design specific primers for the target variable region coding sequence on the plasmid template containing the antibody gene. Single or multiple amino acid site mutations are introduced by high-fidelity PCR amplification to obtain antibody affinity maturation mutants. After the PCR product is digested with Dpnl to remove the template plasmid, it is transformed into competent cells for cloning and screening, and finally a series of point mutant plasmids are successfully constructed.

[0403] 8-2-2 Preparation of affinity matured antibodies against human TLR7

[0404] 8-2-2-1 Preparation of affinity matured antibodies against human TLR7

[0405] The heavy chain and light chain designed in Example 8-1 are combined, and the affinity matured antibodies against human TLR7 are prepared by the same method as in Example 3-2-1 using the expression vectors constructed in 8-2-1.

[0406] 8-2-2-2 Purification of affinity matured antibodies against human TLR7

[0407] From the culture supernatant obtained in Example 8-2-2-1, the target antibody is purified by a series of procedures of Protein A affinity chromatography. After loading the culture supernatant into a chromatography column packed with Protein A (Yixing) equilibrated with binding buffer (Shenguo), the chromatography column is washed twice with 5 times the column volume of binding buffer. Then elution is performed with elution buffer (Shenguo), and the equilibration buffer is 1M Tris-HCl, pH 7.4 (Shenguo). The antibody buffer is replaced with PBS using an Amicon Ultra-15 centrifugal filter equipped with an Ultracel-30 filter membrane, and the antibody is further concentrated. Finally, a 0.22μM disposable needle filter (PALL) is used for filtration to prepare the purified sample.

[0408] Example 9: In vitro activity of affinity matured antibodies against human TLR7

[0409] 9-1 Evaluation of the binding selection ability of affinity matured antibodies against human TLR7 using flow cytometry

[0410] HEK293T cells overexpressing human Unc93B1-Flag / human TLR7 established in 1-4-2 were subjected to cell membrane permeation treatment with 1x transfection agent (Biolegend), and were stained with the anti-human TLR7 affinity matured antibodies obtained in Example 8 at a concentration dilution series and goat anti-human IgG H&L-FITC (Abeam) as a secondary antibody. For evaluation of the binding selectivity of the antibodies, analysis was performed by flow cytometry and the mean fluorescence intensity (MFI) was compared.

[0411] Exemplary results are shown in Figures 22-23, all of the anti-human TLR7 affinity matured antibodies specifically bound to the cells, and the Emax values of the binding of 34G12-h458 and 34G12-h542 to the target antigen were significantly increased after affinity maturation, and the EC50 values of the two antibodies were also significantly reduced. 50 and Emax values are shown in Table 17

[0412] Table 17 EC 50 and Emax values results

[0413] 9-2 Cytokine production inhibition effect of anti-human TLR7 affinity matured antibodies

[0414] Human PBMCs were purchased from Shanghai Aobio Biotech Co., Ltd. in frozen form, and were used after thawing according to the instructions. In this study, three healthy donor PBMCs were used for antibody efficacy verification, and the PBMCs were adjusted to a concentration of 2x10 6 Cells / ml with RPMI 1640 (Gibco) containing 10% FBS (Excell), 1 mM sodium pyruvate (Gibco), 0.1 mM MEM-Non-Essential Amino Acids (Gibco), 50 mM 2-mercaptoethanol (Gibco), 50 U / ml penicillin and 50 U / ml streptomycin (Procell) to 100 μl / well, and 80 μl / well of different concentrations of all antibodies as anti-human TLR7 affinity matured antibodies or human IgG1-LALA control antibody (Bing) were added for 6 hours of pre-treatment in a 37°C incubator. Then, 1 μM GS-9620 (MCE) was added at 20 μl / well, and after thorough mixing, the cells were cultured at 37°C, 5% CO2 for about 20 hours. After thoroughly mixing the plate, centrifugation was performed at 1500 rpm for 5 minutes, and the concentration of IL-6 contained in the supernatant was determined by sandwich ELISA (Sinobio); the concentration of TNF-α contained in the supernatant was determined by sandwich ELISA (Sinobio).

[0415] Anti-human TLR7 affinity matured antibodies inhibited IL-6 production from three different donor human PBMCs treated with GS-9620 as shown in Figures 24-26. All anti-human TLR7 affinity matured antibodies inhibited IL-6 production from human PBMCs. On the other hand, no significant inhibition was observed with the human IgGl-LALA control antibody.

[0416] Anti-human TLR7 affinity matured antibodies inhibited TNF-a production from three different donor human PBMCs treated with GS-9620 as shown in Figures 24-26. All anti-human TLR7 affinity matured antibodies inhibited TNF-a production from human PBMCs. On the other hand, no significant inhibition was observed with the human IgGl-LALA control antibody.

[0417] 9-3 Anti-human TLR7 affinity matured antibodies inhibited downstream signaling of HEK-Blue TM hTLR7 reporter cells

[0418] HEK-Blue TM HEK-Blue hTLR7 reporter cell line was purchased from Invivogen and experiments were performed after the cells were recovered according to the instructions. The cells were continuously cultured in DMEM (Gibco BASIC DMEM, High Glucose) medium containing 10% FBS (Hyclone), 100 U / ml penicillin and 100 U / ml streptomycin (Procell). The cells were cultured to good condition, and after the cells were collected, the cell density was adjusted to 2.5E+06 / ml using HEK-Blue detection medium, 160 μL of cell suspension was added to a 96-well plate, 20 μL of HEK-Blue detection medium was added after the antibody was diluted (the starting concentration of the antibody was 6 μg / mL, and it was diluted by 3.16 times for 8 gradients, and 2 replicate wells were detected), and it was incubated in an incubator for 4 h. 20 μL of GS-9620 (final concentration 1 μM, diluted with HEK-Blue detection medium) was added to each well. 20 μL of HEK-Blue detection medium was used as a negative control. The 96-well plate was moved to a 37°C, 5% CO2 incubator and incubated for 12-16 h. An enzyme labeler was used to detect the OD value at 620 nm.

[0419] The results are shown in Figures 27-28. After HEK-Blue TM hTLR7 reporter cells were stimulated with 1 μM GS-9620, the addition of anti-human TLR7 affinity matured antibodies significantly inhibited the expression of downstream signaling.

[0420] 9-4 Anti-human TLR7 affinity matured antibodies inhibited plasmacytoid cell formation

[0421] PBMC of healthy human were purchased from Shanghai Aobio Biotech Co., Ltd. in frozen form, and PBMC of SLE patients were purchased from Shanghai Junxing Biotechnology Co., Ltd. in frozen form, which were used after thawing according to the instructions. PBMC were adjusted to a concentration of 1.25 x 10 6 Cells were seeded at 800 μl / well in 24-well cell culture plates with PBMC of healthy human at a concentration of 1.25 x 10 - CD27 h CD38 h ) were collected and detected by flow cytometry.

[0422] As shown in Figures 29-31, 1 μM GS-9620 stimulated PBMC cells to detect the formation of plasma cells, and the addition of 5 μg / ml of anti-human TLR7 affinity matured antibody could significantly reduce the proportion of plasma cells, but hlgGl-LALA did not reduce the proportion of plasma cells, indicating that the anti-human TLR7 affinity matured antibody could significantly inhibit the formation of plasma cells.

[0423] Example 10: In vivo activity of anti-human TLR7 affinity matured antibody

[0424] 10-1 Anti-human TLR7 affinity matured antibody in vivo activity verification in CD34+ humanized mice

[0425] 10-1-1 CD34+ humanized mice

[0426] GenO-BRGSF-HIS4 female mice were purchased from Shanghai Keyeui Biological Technology Co., Ltd. for this experiment.

[0427] 10-1-2 Anti-human TLR7 affinity matured antibody in vivo activity verification in CD34+ humanized mice

[0428] The mice were divided into two groups, on the first day, group 1 mice were not treated, group 2 mice were injected with 5mg / kg of 34G12-h458 antibody via tail vein; overnight (18h) group 1 was injected with PBS, group 2 was injected with 0.01mg / kg TLR7 agonist DSR-6434 via tail vein; 2 hours later, 100ul of blood was taken from each mouse, centrifuged at 5000rpm for 5min, and the supernatant was taken; ELISA kit was used to detect mouse serum IL-6 (human IL-6 kit), and the mouse serum was diluted 4 times before use.

[0429] As shown in Figure 32, the anti-human TLR7 affinity matured antibody 34G12-h458 effectively inhibited the release of the inflammatory factor IL-6 induced by the TLR7 agonist DSR-6434 in CD34+ mice in vivo, which preliminarily verified that the anti-human TLR7 affinity matured antibody 34G12-h458 has activity in vivo.

[0430] Although the present application has been described in detail with reference to the preferred embodiments, the present application is not limited to the preferred embodiments. Various equivalent modifications or replacements can be made to the embodiments of the present application by those skilled in the art without departing from the spirit and essence of the present application, and these modifications or replacements should be within the scope of the present application. Any person skilled in the art can easily think of changes or replacements within the technical scope disclosed by the present application, and these changes or replacements should be within the protection scope of the present application.

Claims

1. An anti-TLR7 antibody or antigen-binding fragment thereof, characterized in that, The anti-TLR7 antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and / or a light chain variable region, wherein The amino acid sequence of the heavy chain variable region comprises GYX1FTX2YX3 (SEQ ID NO: 70), GFTFSX4YX5 (SEQ ID NO: 71), GFSLTX6X7G (SEQ ID NO: 176), ARX8QIX9HGWNEGVNDY (SEQ ID NO: 177), the amino acid sequence represented by any one of SEQ ID NOs: 39-50, or an amino acid sequence having at least 80% identity to the amino acid sequence represented by any one of GYX1FTX2YX3 (SEQ ID NO: 70), GFTFSX4YX5 (SEQ ID NO: 71), GFSLTX6X7G (SEQ ID NO: 176), ARX8QIX9HGWNEGVNDY (SEQ ID NO: 177), and SEQ ID NOs: 39-50; The amino acid sequence of the heavy chain variable region comprises ENIX 10 SY (SEQ ID NO: 178), QX 11 HFGIPWT (SEQ ID NO: 179), any one of SEQ ID NOs: 51, 53-68, 169-170, or an amino acid sequence having at least 80% identity to ENIX 10 SY (SEQ ID NO: 178), QX 11 HFGIPWT (SEQ ID NO: 179), any one of SEQ ID NOs: 51, 53-68, 169-170, or an amino acid sequence having at least 80% identity to ENIX The X 1-11 may be any natural amino acid residue.

2. The anti-TLR7 antibody or the antigen-binding fragment thereof according to claim 1, wherein X1in SEQ ID NO: 70 represents T or I; X2represents E, T or K; and X3represents P, F or W; X4in SEQ ID NO: 71 represents S or D; and X5represents T or G; X6in SEQ ID NO: 176 represents G, Y, S, T, D, H, W, Q, E or N; and X7represents Y or H; X8in SEQ ID NO: 177 represents V or A; and X9represents Y or R; X in SEQ ID NO: 178 10 represents Y, R, K, D, N, Q, E, H, or S; X in SEQ ID NO: 179 11 represents H or S.

3. The anti-TLR7 antibody or the antigen-binding fragment thereof according to claim 1 or 2, wherein The CDR-H1 comprises the amino acid sequence represented by any one of SEQ ID NOs: 33-38 and 148-157, or an amino acid sequence having at least 80% identity to the amino acid sequence represented by any one of SEQ ID NOs: 33-38 and 148-157; The amino acid sequence of the CDR-H2 comprises the amino acid sequence represented by any one of SEQ ID NOs: 39-44, or an amino acid sequence having at least 80% identity to the amino acid sequence represented by any one of SEQ ID NOs: 39-44; The CDR-H3 comprises the amino acid sequence represented by any one of SEQ ID NOs: 45-50 and 158-160, or an amino acid sequence having at least 80% identity to the amino acid sequence represented by any one of SEQ ID NOs: 45-50 and 158-160; The CDR-L1 comprises the amino acid sequence represented by any one of SEQ ID NOs: 51-56 and 161-170, or an amino acid sequence having at least 80% identity to the amino acid sequence represented by any one of SEQ ID NOs: 51-56 and 161-170; The amino acid sequence of the CDR-L2 comprises any one of the amino acid sequences shown in SEQ ID NO: 57-62, or an amino acid sequence having at least 80% identity to any one of the amino acid sequences shown in SEQ ID NO: 57-62; The CDR-L3 comprises any one of the amino acid sequences shown in SEQ ID NO: 63-69, 171, or an amino acid sequence having at least 80% identity to any one of the amino acid sequences shown in SEQ ID NO: 63-69, 171.

4. The anti-TLR7 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that, The anti-TLR7 antibody or antigen-binding fragment thereof is engineered, and the engineering includes humanization, and the engineering site is located in the CDR region, the framework region and / or the constant region of the antibody.

5. The anti-TLR7 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, characterized in that, The anti-TLR7 antibody or antigen-binding fragment thereof includes a nanobody, a chimeric antibody, a Fab fragment, a Fab' fragment, a Fd fragment, a Fv fragment, a bispecific antibody, a multispecific antibody, a dAb fragment, a F(ab')2 fragment, a single-chain antibody or a linear antibody.

6. The anti-TLR7 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, characterized in that, The anti-TLR7 antibody or antigen-binding fragment thereof specifically binds to human TLR7 or monkey TLR7 and does not bind to murine TLR7; and / or, inhibits the function of human TLR7 or monkey TLR7.

7. The anti-TLR7 antibody or antigen-binding fragment thereof according to any one of claims 1-6, characterized in that, The heavy chain variable region comprises any one of the amino acid sequences shown in SEQ ID NO: 5-11, 172, 174, 180, 182, or an amino acid sequence having at least 80% identity to any one of the amino acid sequences shown in SEQ ID NO: 5-11, 172, 174, 180, 182; The light chain variable region comprises any one of the amino acid sequences shown in SEQ ID NO: 12-18, 173, 175, 181, 183, or an amino acid sequence having at least 80% identity to any one of the amino acid sequences shown in SEQ ID NO: 12-18, 173, 175, 181, 183.

8. The anti-TLR7 antibody or antigen-binding fragment thereof according to any one of claims 1-7, characterized in that, The amino acid sequence of the anti-TLR7 antibody or antigen-binding fragment thereof comprises any one of the amino acid sequences shown in SEQ ID NO: 72-85, or an amino acid sequence having at least 80% identity to any one of the amino acid sequences shown in SEQ ID NO: 72-85.

9. Use of an anti-TLR7 antibody or antigen binding fragment thereof according to any one of claims 1 to 8, characterized in that, The application includes: A use in preparing a fusion construct comprising the anti-TLR7 antibody or antigen-binding fragment thereof of any one of claims 1-8 and other biologically active effector molecules, which include antibodies or antigen-binding fragments thereof of other targets or other functional components other than the anti-TLR7 antibody or antigen-binding fragment thereof of any one of claims 1-8; B use in detecting TLR7 expression; C use in preparing a TLR7 antagonist or inhibitor.

10. Use according to claim 9, characterized in that, The other functional components include one or two or more combinations of serum albumin, a cytokine, transferrin, a scaffold protein, an oligopeptide, an oligopeptide polymer, a polypeptide, a polypeptide polymer, a polysaccharide, a fatty chain, avidin, biotin, streptavidin, a toxin, a drug, a nucleic acid, a radionuclide and a label thereof, PEG or an Fc fragment.

11. A fusion construct, characterized in that, The fusion construct comprises the anti-TLR7 antibody or antigen-binding fragment thereof according to any one of claims 1-8.

12. The fusion construct of claim 11, wherein, The fusion construct further comprises other bioactive effector molecules, which include antibodies or antigen-binding fragments thereof or other functional components of other targets other than the anti-TLR7 antibody or antigen-binding fragment thereof according to any one of claims 1-8, Preferably, the other functional components include, but are not limited to, one or more of serum albumin, cytokines, transferrin, scaffold proteins, oligopeptides, oligopeptide polymers, polypeptides, polypeptide polymers, polysaccharides, fatty chains, avidin, biotin, streptavidin, toxins, drugs, nucleic acids, radionuclides and markers thereof, PEG or Fc fragments, or a combination of two or more thereof.

13. A nucleic acid, characterized in that, The nucleic acid encodes the anti-TLR7 antibody or antigen-binding fragment thereof according to any one of claims 1-8 or the fusion construct according to any one of claims 11-12.

14. A vector, characterized in that, The vector comprises the nucleic acid according to claim 13.

15. A host cell, characterized in that, The host cell comprises the nucleic acid according to claim 13 or the vector according to claim 14.

16. A method of producing an anti-TLR7 antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 or a fusion construct according to any one of claims 11 to 12, characterized in that, The preparation method comprises culturing the host cell according to claim 15 to express the anti-TLR7 antibody or antigen-binding fragment thereof or the fusion construct.

17. A method of producing an anti-TLR7 antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, characterized in that, The preparation method comprises synthesizing the anti-TLR7 antibody or antigen-binding fragment thereof by chemical synthesis.

18. A product for the treatment, prevention and / or diagnosis of a disease related to TLR7, characterized in that it comprises a compound according to any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof. The product for treating, preventing and / or diagnosing a TLR7-related disease comprises any one of the following: A) the anti-TLR7 antibody or antigen-binding fragment thereof according to any one of claims 1-8; B) the fusion construct according to any one of claims 11-12; C) the nucleic acid according to claim 13; D) the vector according to claim 14; or E) the host cell according to claim 15.

19. Use of the anti-TLR7 antibody or antigen-binding fragment thereof according to any one of claims 1-8, the fusion construct according to any one of claims 11-12, the nucleic acid according to claim 13, the vector according to claim 14, or the host cell according to claim 15 in the preparation of a product for treating and / or preventing a TLR7-related disease, or in the preparation of a diagnostic product or tracer for a TLR7-related disease.

20. The use according to claim 19, characterized in that, The TLR7-related disease includes immune inflammation-related diseases, allergic diseases, infectious diseases or cancers.

21. A method of detecting TLR7, comprising contacting a sample with an antibody of claim 1 and detecting the presence of a complex between the antibody and TLR7. The detection method comprises combining a sample to be detected with the anti-TLR7 antibody or antigen-binding fragment thereof according to any one of claims 1-8, and then detecting the content of the complex formed by TLR7 and the anti-TLR7 antibody or antigen-binding fragment thereof.

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