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

Abstract

The invention provides an anti-TLR7 antibody or an antigen binding fragment and a pharmaceutical composition thereof, the antibody or the antigen binding fragment thereof can be specifically bound with a human or monkey TLR7 antigen and is not bound with mouse TLR7, has remarkable TLR7 antigen binding activity, and can effectively inhibit various inflammatory cytokines generated by TLR7 activation. The anti-TLR7 antibody or the antigen binding fragment thereof can be used as a single agent or a drug combination and can be used for treating and / or preventing diseases related to TLR7 target pathology, including immune inflammation related diseases, allergic diseases, infectious diseases or cancers and the like.

Description

Technical Field

[0001] This invention belongs to the fields of immunology and molecular biology, and in particular to an anti-TLR7 antibody or its antigen-binding fragment, pharmaceutical composition and its application. Background Technology

[0002] Toll-like receptors (TLRs) belong to the pattern recognition receptor (PRR) family. They activate downstream signaling pathways by rapidly recognizing pathogen-associated molecular patterns (PAMPs) of invading microorganisms, triggering an immune response. TLR7 is an innate immune RNA sensor expressed in B cells, dendritic cells, and monocytes / macrophages, primarily in intracellular vesicles such as the endoplasmic reticulum, endosomes, and lysosomes. It is a transmembrane signal transduction receptor that responds not only to single-stranded RNA (ssRNA) from pathogens but also to its own ssRNA. TLR7 can induce a series of signal transductions by recognizing single-stranded RNA, leading to the release of cytokines such as TNF-α (tumor necrosis factor-α), IL-1 (interleukin-1), IL-6 (interleukin-6), IL-12 (interleukin-12), and INF-α (interferon-α).

[0003] Recent studies have shown that Toll-like receptor 7 (TLR7) is closely associated with autoimmune diseases such as systemic lupus erythematosus (SLE) (References: 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, immune complexes can activate TLR7 in plasmacytoid dendritic cells (pDCs), leading to the secretion of high levels of IFN-α and exacerbating clinical symptoms (Reference: Pathogenesis of systemiclupus erythematosus: risks, mechanisms and therapeutic targets. Ann Rheum Dis. 2023 Aug; 82(8):999-1014.). Furthermore, TLR7 plays a crucial role in B cell differentiation. TLR7 in B cells drives SLE patients to form autoreactive antibody-secreting cells through various pathways. For example, in extrafollicular reactions, TLR7 can promote the differentiation of resting naive B cells into activated naive B cells, which then differentiate into DN2 B cells, and further differentiate into antibody-secreting cells, producing pathogenic autoantibodies that promote and worsen SLE symptoms. In summary, overactivation of TLR7 severely affects the occurrence, development, and prognosis of SLE (References: 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] Systemic lupus erythematosus (SLE) is a chronic, multi-systemic autoimmune disease characterized by a high concentration of autoantibodies in the blood and damage to multiple organs. Symptoms are complex and diverse, encompassing almost all organ systems. Common manifestations include skin rashes, arthritis, kidney involvement, and neurological involvement. The mortality rate is 2.6 times higher than in the general population (Reference: Systemic lupus erythematosus. Nat Rev Dis Primers 2016, 2, 16039.). The etiology of SLE is complex, involving multiple factors including genetics, environment, immunity, and endocrine disorders. SLE patients exhibit abnormally increased apoptosis and defective clearance of late-stage apoptotic debris, leading to increased exposure to autoantigens. Innate and adaptive immune cells react abnormally to autoantigens, producing various autoantibodies and forming autoimmune complexes that deposit in tissues. This results in complement pathway activation, neutrophil and monocyte aggregation, and proliferation of autoreactive lymphocytes, ultimately causing damage to various organs throughout the body.

[0005] Currently, the main clinical treatments for SLE involve downregulating the overactive autoimmune system using glucocorticoids, antimalarial drugs, and immunosuppressants. However, despite these treatments, drug-resistant patients can still develop life-threatening conditions, such as lupus nephritis. Furthermore, the use of glucocorticoids is limited due to various side effects. In addition, SLE is currently incurable and requires long-term medication to control symptoms; therefore, there is a need for new treatments with fewer side effects and better efficacy.

[0006] In April 2022, researchers from the Australian National University and other research institutions published their findings in Nature. In this study, the authors, through whole-genome sequencing and whole-exome sequencing, for the first time identified the Y264H single-gene mutation in TLR7 as one of the causes of SLE and induces severe SLE. This discovery provides a clinical basis for developing TLR7-targeted therapies (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, numerous studies have been conducted on the treatment of SLE by targeting the TLR7 signaling pathway. For example, dihydroartemisinin alleviates the SLE phenotype by inducing dendritic cell apoptosis through inhibiting the TLR7 / 9-MyD88-IRAKs signaling pathway. Kensuke Miyake's research team screened the anti-mouse TLR7 monoclonal antibody A94B10. In a mouse animal model, the study found that this antibody can reduce IgG deposition and autoantibody production in the glomeruli, inhibit the release of inflammatory factors from immune cells, eliminate the increase of lupus-associated monocytes, 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.). Taking all factors into consideration, targeting and inhibiting TLR7 shows promise as a potential treatment strategy for SLE, and even for many common autoimmune diseases including dry eye, rheumatoid arthritis, and multiple sclerosis. Furthermore, TLR7 is closely related to the occurrence, development, and prognosis of numerous diseases; therefore, other potential indications for TLR7 antagonist antibodies 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 autoimmune 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 TLR7agonist 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-related anemia (see reference: Chronic TLR7 and TLR9 signaling drive anemia via differentiation of specialized hemophagocytes[J]. Science, 2019, 363(6423):eaao5213.).

[0007] Therefore, it is necessary to develop a novel anti-TLR7 antibody to provide a treatment and / or preventative agent for immune-inflammatory diseases, allergic diseases, infectious diseases, and cancer. Summary of the Invention

[0008] To overcome the deficiencies of the prior art, this application provides an anti-TLR7 antibody or its antigen-binding fragment that specifically binds to TLR7, wherein the anti-TLR7 antibody or its antigen-binding fragment can inhibit cytokines induced by TLR7 agonists, as well as a corresponding preparation method and use of the anti-TLR7 antibody or its antigen-binding fragment.

[0009] In a first aspect, the present invention provides an anti-TLR7 antibody or an antigen-binding fragment thereof, said anti-TLR7 antibody or antigen-binding fragment comprising 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 any of the amino acid sequences shown in GYX1FTX2YX3 (SEQ ID NO: 70), GFTFSX4YX5 (SEQ ID NO: 71), GFSLTX6X7G (SEQ ID NO: 176), ARX8QIX9HGWNEGVNDY (SEQ ID NO: 177), and SEQ ID NO: 39-50, or an amino acid sequence having at least 80% identity with any of the amino acid sequences shown in GYX1FTX2YX3 (SEQ ID NO: 70), GFTFSX4YX5 (SEQ ID NO: 71), GFSLTX6X7G (SEQ ID NO: 176), ARX8QIX9HGWNEGVNDY (SEQ ID NO: 177), and SEQ ID NO: 39-50;

[0011] The amino acid sequence of the light chain variable region contains ENIX. 10 SY (SEQ ID NO: 178), QX 11HFGIPWT (SEQ ID NO: 179), any of the amino acid sequences shown in SEQ ID NO: 51, 53-68, 169-170, or ENIX 10 SY (SEQ ID NO: 178), QX 11 HX 12 The amino acid sequences shown in GIPWT (SEQ ID NO: 179), SEQ ID NO: 51, 53-68, 169-170 have at least 80% amino acid identity.

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

[0013] The amino acid sequence of CDR-H1 includes any of the amino acid sequences shown in GYX1FTX2YX3 (SEQ ID NO: 70), GFTFSX4YX5 (SEQ ID NO: 71), and GFSLTX6X7G (SEQ ID NO: 176), or an amino acid sequence that has at least 80% identity with any of the amino acid sequences shown in GYX1FTX2YX3 (SEQ ID NO: 70), GFTFSX4YX5 (SEQ ID NO: 71), and GFSLTX6X7G (SEQ ID NO: 176);

[0014] The amino acid sequence of CDR-H2 includes any of the amino acid sequences shown in SEQ ID NO: 39-44, or an amino acid sequence that has at least 80% identity with any of the amino acid sequences shown in SEQ ID NO: 39-44.

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

[0016] The amino acid sequence of CDR-L1 includes SEQ ID NO: 51, 53-56, 169-170, and ENIX. 10 The amino acid sequence shown in SY (SEQ ID NO: 178), or any of the amino acid sequences shown in SEQ ID NO: 51, 53-56, 169-170, ENIX 10The amino acid sequences shown in any of SY (SEQ ID NO: 178) have at least 80% amino acid sequence identity;

[0017] The amino acid sequence of CDR-L2 includes any of the amino acid sequences shown in SEQ ID NO: 57-62, or an amino acid sequence that has at least 80% identity with any of the amino acid sequences shown in SEQ ID NO: 57-62.

[0018] The amino acid sequence of CDR-L3 includes SEQ ID NO: 63-68, QX 11 The amino acid sequence shown in HFGIPWT (SEQ ID NO: 179), or the sequence of SEQ ID NO: 63-68, QX 11 The amino acid sequences shown in any of the HFGIPWT (SEQ ID NO: 179) have at least 80% amino acid sequence identity.

[0019] In SEQ ID NO: 70-71, 176-179, X can be any natural amino acid residue, such as 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), and valine (V).

[0020] In one specific embodiment of the present invention, X1 in SEQ ID NO: 70 represents T or I; X2 represents E, T or K; and X3 represents P, F or W.

[0021] In one specific embodiment of the present invention, X4 in SEQ ID NO: 71 represents S or D; X5 represents T or G.

[0022] In one specific embodiment of the present invention, X6 in SEQ ID NO: 176 represents G, Y, S, T, D, H, W, Q, E or N; X7 represents Y or H.

[0023] In one specific embodiment of the present invention, X8 in SEQ ID NO: 177 represents V or A; X9 represents Y or R.

[0024] In one specific embodiment of the present invention, X in SEQ ID NO: 178 10 Represents Y, R, K, D, N, Q, E, H, or S.

[0025] In one specific embodiment of the present invention, X in SEQ ID NO: 179 11 Represents H or S.

[0026] Preferably, the CDR-H1 comprises any of the amino acid sequences shown in SEQ ID NO: 33-38, 148-157 or an amino acid sequence having at least 80% identity with any of the amino acid sequences shown in SEQ ID NO: 33-38, 148-157.

[0027] Preferably, the CDR-H3 comprises any of the amino acid sequences shown in SEQ ID NO: 45-50, 158-160 or an amino acid sequence having at least 80% identity with any of the amino acid sequences shown in SEQ ID NO: 45-50, 158-160.

[0028] Preferably, the CDR-L1 comprises any of the amino acid sequences shown in SEQ ID NO: 51-56, 161-170 or an amino acid sequence having at least 80% identity with any of the amino acid sequences shown in SEQ ID NO: 51-56, 161-170.

[0029] Preferably, the CDR-L3 comprises any of the amino acid sequences shown in SEQ ID NO: 63-69, 171 or an amino acid sequence having at least 80% identity with any of the amino acid sequences shown in SEQ ID NO: 63-69, 171.

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

[0031] Table 1. Amino acid sequences of the antibody's CDR

[0032]

[0033] Table 2 shows the amino acid sequences of the antibody's CDR (the unshown portions are identical to those of 34G12-h458).

[0034]

[0035]

[0036] In this application, the amino acid division of the antibody CDR region adopts the IMGT numbering system.

[0037] The structure of the anti-TLR7 antibody or its antigen-binding fragment includes nanobodies, chimeric antibodies, Fab fragments, Fab' fragments, Fd fragments, Fv fragments, bispecific antibodies, multispecific antibodies, dAb fragments, F(ab')2 fragments, single-chain antibody fragments (scFv), or linear antibodies.

[0038] The anti-TLR7 antibody or its antigen-binding fragment may be a humanized antibody.

[0039] Preferably, the anti-TLR7 antibody or its antigen-binding fragment has been modified, the modification including humanization, and the modification site is located in the CDR region, frame region and / or constant region of the antibody.

[0040] Preferably, the anti-TLR7 antibody or its antigen-binding fragment specifically binds to human TLR7 or monkey TLR7 and does not bind to mouse TLR7; and / or inhibits the function of human TLR7 or monkey TLR7.

[0041] Preferably, the heavy chain variable region comprises any 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 with any of the amino acid sequences shown in SEQ ID NO: 5-11, 172, 174, 180, 182.

[0042] Preferably, the light chain variable region comprises any 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 with any of the amino acid sequences shown in SEQ ID NO: 12-18, 173, 175, 181, 183.

[0043] In one specific embodiment, the heavy chain variable region further includes humanized sequences on SEQ ID NO: 8, 11.

[0044] In one specific embodiment, the humanization modification site is located in the frame region, and the frame region of the heavy chain variable region contains any of the amino acid sequences shown in SEQ ID NO: 86-109 and 191-209.

[0045] In one specific embodiment, the light chain variable region further includes humanized sequences on SEQ ID NO: 16, 18.

[0046] In one specific embodiment, the humanization modification site is located in the frame region, and the frame region of the light chain variable region contains any of the amino acid sequences shown in SEQ ID NO: 110-123, 184-190, and 210-220.

[0047] In one specific embodiment, the heavy chain variable region further includes sequences mutated on SEQ ID NO: 180, 182.

[0048] In one specific embodiment, the light chain variable region further includes sequences mutated on SEQ ID NO: 181, 183.

[0049] In one specific embodiment, the heavy chain variable region further includes sequences mutated on SEQ ID NO: 172, 174.

[0050] In one specific embodiment, the light chain variable region further includes sequences mutated on SEQ ID NO: 173, 175.

[0051] Preferably, the mutation region includes the CDR region and / or the frame region.

[0052] Preferably, the CDR mutation sites in the heavy chain variable region include amino acids at positions 31, 32, 98, 101, 104, and 109; and the frame region mutation sites include amino acids at positions 20, 48, 37, 60-63, 67, 71, 76, and 78.

[0053] Preferably, the CDR region mutation sites of the light chain variable region include amino acids 27-28, 30, and 90; the frame region mutation sites include amino acids 43 and 100.

[0054] The mutations in the heavy chain variable region and the light chain variable region are shown in Table 11-16.

[0055] Preferably, the light chain constant region includes Igκ (kappa) or Igλ (lambda).

[0056] Preferably, the heavy chain constant region includes IgG1, IgG2, IgG3, IgG4, IgM, IgA1, or IgA2.

[0057] In one specific embodiment, the light chain constant region includes Igκ, and the light chain constant region includes the amino acid sequence shown in SEQ ID NO: 221.

[0058] In one specific embodiment, the heavy chain constant region includes IgG1.

[0059] Preferably, the IgG1 further comprises an LALA mutation, and the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 222.

[0060] Preferably, the amino acid sequence of the anti-TLR7 antibody or its antigen-binding fragment comprises any amino acid sequence in SEQ ID NO: 72-85, or has at least 80% identity with any amino acid sequence in SEQ ID NO: 72-85.

[0061] In one specific embodiment of the present invention, the heavy chain amino acid sequence of the anti-TLR7 antibody or its antigen-binding fragment includes any of the amino acid sequences shown in SEQ ID NO: 72-78.

[0062] In one specific embodiment of the present invention, the light chain amino acid sequence of the anti-TLR7 antibody or its antigen-binding fragment includes any of the amino acid sequences shown in SEQ ID NO: 79-85.

[0063] The anti-TLR7 antibody or its antigen-binding fragment can be obtained using existing conventional techniques, such as chemical synthesis or expression using eukaryotic or prokaryotic expression systems.

[0064] A second aspect of the present invention provides an application of the above-described anti-TLR7 antibody or its antigen-binding fragment thereof, the application comprising:

[0065] Application of A in the preparation of fusion constructs, wherein the fusion constructs include the above-mentioned anti-TLR7 antibody or its antigen-binding fragment and other bioactive effector molecules, wherein the other bioactive effector molecules include antibodies or their antigen-binding fragments or other functional components targeting other targets besides any of the above-mentioned anti-TLR7 antibodies or their antigen-binding fragments;

[0066] Application of B in detecting TLR7 expression;

[0067] Application of C in the preparation of TLR7 antagonists or inhibitors.

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

[0069] A third aspect of the present invention provides a fusion construct comprising the above-described anti-TLR7 antibody or its antigen-binding fragment.

[0070] Preferably, the fusion construct further comprises other bioactive effector molecules, including antibodies or antigen-binding fragments of other targets besides the anti-TLR7 antibody or its antigen-binding fragment described above, or other functional components.

[0071] Preferably, the functional components include, but are not limited to, one or more of the following: serum albumin, cytokines, transferrin, scaffold proteins, oligopeptides, oligopeptide polymers, polypeptides, polypeptide polymers, polysaccharides, fatty acid chains, avidin, biotin, streptavidin, toxins, drugs, nucleic acids, radionuclides and their markers, PEG or Fc fragments.

[0072] The structure of the anti-TLR7 antibody or its antigen-binding fragment is a nanobody, chimeric antibody, Fab fragment, Fab' fragment, Fd fragment, Fv fragment, bispecific antibody, multispecific antibody, dAb fragment, F(ab')2 fragment, single-chain antibody, or linear antibody.

[0073] Preferably, the fusion construct comprises one or more of the aforementioned anti-TLR7 antibodies or their antigen-binding fragments.

[0074] Preferably, the fusion construct contains one or more other bioactive molecules, which may be the same or different bioactive molecules.

[0075] The anti-TLR7 antibody or its antigen-binding fragment is directly or indirectly linked to other biological effector molecules.

[0076] Preferably, the indirect connection can be a connection via a connector, a functional structural domain, and / or a connector sub-connector for coupling.

[0077] The connector is selected from linker peptides, oligopeptides, oligopeptide polymers, polypeptides, polypeptide polymers, PEG, nucleic acids, polysaccharides, fatty acid chains, biotin, streptavidin, or avidin.

[0078] The functional domain is one or more of the following: Fc fragment, serum albumin, cytokines, transferrin, or scaffold protein.

[0079] The connectors used for coupling include functional group connectors.

[0080] The functional linker includes reactive groups such as thiol, amino, hydroxyl and / or carboxyl groups, which can covalently couple anti-TLR7 antibody or its antigen-binding fragment to a bioactive effector molecule.

[0081] Preferably, the direct or indirect connection includes direct or indirect connection to the N-terminus, C-terminus, and / or internal residues of the anti-TLR7 antibody or its antigen-binding fragment and / or other target antibodies or their antigen-binding fragments.

[0082] The connection sequence of the anti-TLR7 antibody or its antigen-binding fragment, and other target antibodies contained in the fusion construct can be that the N-terminus, C-terminus and / or internal residues of one antibody are linked to the N-terminus, C-terminus and / or internal residues of another antibody.

[0083] In a fourth aspect, the present invention provides a nucleic acid encoding the aforementioned anti-TLR7 antibody or antigen-binding fragment or the aforementioned fusion construct. For example, the nucleic acid comprises DNA and / or mRNA.

[0084] In some embodiments, the nucleic acid is DNA that encodes the aforementioned anti-TLR7 antibody or antigen-binding fragment or the aforementioned fusion construct.

[0085] Preferably, the nucleotide sequence encoding the heavy chain variable region of the anti-TLR7 antibody or its antigen-binding fragment comprises any nucleotide sequence in SEQ ID NO: 19-25 or its degenerate sequence, or has at least 80% identity with any nucleotide sequence in SEQ ID NO: 19-25 and has the function of encoding an anti-TLR7 antibody or its antigen-binding fragment.

[0086] Preferably, the nucleotide sequence encoding the light chain variable region of the anti-TLR7 antibody or its antigen-binding fragment comprises any nucleotide sequence or its degenerate sequence in SEQ ID NO: 26-32, or a nucleotide sequence having at least 80% identity with any nucleotide sequence in SEQ ID NO: 26-32 and having the function of encoding an anti-TLR7 antibody or its antigen-binding fragment.

[0087] Preferably, the nucleotide sequence of the heavy chain encoding the anti-TLR7 antibody or its antigen-binding fragment comprises any nucleotide sequence or its degenerate sequence in SEQ ID NO: 130, 132, 134, 136, 138, 140, 142, or a nucleotide sequence having at least 80% identity with any nucleotide sequence in SEQ ID NO: 130, 132, 134, 136, 138, 140, 142 and having the function of encoding the anti-TLR7 antibody or its antigen-binding fragment.

[0088] Preferably, the nucleotide sequence of the light chain encoding the anti-TLR7 antibody or its antigen-binding fragment comprises any nucleotide sequence or its degenerate sequence in SEQ ID NO: 131, 133, 135, 137, 139, 141, 143, or a nucleotide sequence having at least 80% identity with any nucleotide sequence in SEQ ID NO: 131, 133, 135, 137, 139, 141, 143 and having the function of encoding the anti-TLR7 antibody or its antigen-binding fragment.

[0089] In a fifth aspect, the present invention provides a carrier comprising the above-described nucleic acid.

[0090] The vector described can be expressed in prokaryotic or eukaryotic cells.

[0091] For example, expression vectors can be introduced into host cells through transient or stable transfection.

[0092] In a sixth aspect, the present invention provides a host cell comprising the aforementioned nucleic acid or the aforementioned vector.

[0093] The host cell can be a eukaryotic cell or a prokaryotic cell.

[0094] Eukaryotic cells include animal and plant cells, such as T cells, yeast cells, HEK293 cells, or CHO cells, etc.

[0095] Prokaryotic cells, such as Escherichia coli.

[0096] A seventh aspect of the present invention provides a method for preparing a host cell, the method comprising introducing the above-mentioned nucleic acid or vector into a host cell.

[0097] In an eighth aspect, the present invention provides a method for preparing an anti-TLR7 antibody or its antigen-binding fragment or fusion construct thereof, the method comprising culturing the host cells described above and expressing the anti-TLR7 antibody or its antigen-binding fragment or the fusion construct thereof.

[0098] In a ninth aspect, the present invention provides a method for preparing an anti-TLR7 antibody or an antigen-binding fragment thereof, the method comprising synthesizing the anti-TLR7 antibody or the antigen-binding fragment thereof by chemical synthesis.

[0099] In a tenth aspect, the present invention provides a product for treating, preventing, and / or diagnosing diseases related to TLR7, said product comprising any of the following:

[0100] A) The above-mentioned anti-TLR7 antibody or its antigen-binding fragment;

[0101] B) The aforementioned fusion construct;

[0102] C) The aforementioned nucleic acids;

[0103] D) The aforementioned carrier; or,

[0104] E) The aforementioned host cells.

[0105] Preferably, the product may be a diagnostic kit, a drug, or a diagnostic chip, etc.

[0106] More preferably, the TLR7-related diseases include, but are not limited to, immune-inflammatory diseases, allergic diseases, infectious diseases, or cancer, etc.

[0107] The drug may be an antibody-drug conjugate (ADC), which comprises the anti-TLR7 antibody described in this invention or its antigen-binding fragment or fusion construct, and other drugs covalently bound thereto.

[0108] In an eleventh aspect of the present invention, the use of A)-E) above in the preparation of products for treating and / or preventing TLR7-related diseases, or in the preparation of diagnostic products or tracers for TLR7-related diseases.

[0109] Preferably, the TLR7-related diseases include, but are not limited to, immune-inflammatory diseases, allergic diseases, infectious diseases, or cancer, etc.

[0110] The products mentioned can be pharmaceuticals, etc.

[0111] The drug may be an antibody-drug conjugate (ADC), which comprises the anti-TLR7 antibody described in this invention or its antigen-binding fragment or fusion construct, and other drugs covalently bound thereto.

[0112] The diagnostic product may be a diagnostic kit or a diagnostic chip.

[0113] In a twelfth aspect, the present invention provides a method for detecting TLR7, the method comprising binding a sample to be tested with the aforementioned anti-TLR7 antibody or its antigen-binding fragment, and then detecting the content of the complex formed by TLR7 and the anti-TLR7 antibody or its antigen-binding fragment.

[0114] The detection method described herein is to detect the presence or content of TLR7. Here, "presence" refers to a qualitative analysis indicating its presence or absence, and "content" can be the expression level or protein concentration, etc.

[0115] In a thirteenth aspect of the present invention, a method for diagnosing TLR7-related diseases is provided, the method comprising taking a sample, combining the sample with a diagnostic product for the aforementioned disease, and detecting the content of a complex formed by TLR7 and an anti-TLR7 antibody or its antigen-binding fragment.

[0116] Preferably, the TLR7-related diseases include, but are not limited to, immune-inflammatory diseases, allergic diseases, infectious diseases, or cancer, etc.

[0117] In a fourteenth aspect, the present invention provides a method for treating and / or preventing TLR7-related diseases, the method comprising administering the aforementioned product for treating and / or preventing the disease to an individual.

[0118] Preferably, the TLR7-related diseases include, but are not limited to, immune-inflammatory diseases, allergic diseases, infectious diseases, or cancer, etc.

[0119] The “anti-TLR7 antibody or its antigen-binding fragment” in this invention exemplifies the CDR sequence obtained by partitioning according to the IMGT method. In addition, various other numbering methods can be used to partition the CDR region, including but not limited to Kabat, Chothia, AbM, Contact, etc.

[0120] The terms “comprising” or “including” in this invention are open-ended descriptions that include the specified ingredients or steps described, as well as other specified ingredients or steps that do not materially affect them.

[0121] The "immunoinflammatory diseases" in this invention include, but are not limited to, connective tissue diseases, musculoskeletal diseases (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.), hematologic diseases (autoimmune hemolytic anemia, aplastic anemia, idiopathic thrombocytopenic purpura, etc.), digestive diseases (Crohn's disease, ulcerative colitis, ileitis, etc.), hepatobiliary and pancreatic diseases and endocrine diseases (autoimmune hepatitis, viral hepatitis, alcoholic hepatitis, non-alcoholic steatohepatitis, primary sclerosing cholangitis, primary biliary cirrhosis, Sjögren's syndrome, type 1 diabetes, autoimmune thyroiditis, Basel-Dürer's disease, Hashimoto's disease, etc.), and respiratory diseases (chronic obstructive pulmonary disease, cystic fibrosis, interstitial lung disease, etc.). This includes diseases related to immune-mediated inflammation such as pneumonia, neurological disorders (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, arteriosclerosis, etc.), skin and epidermal system (psoriasis, pemphigus, vitiligo, contact dermatitis, eczema, etc.), renal system (glomerulonephritis, diabetic nephropathy, IgA nephropathy, purpuric nephritis, nephropathy, interstitial cystitis, etc.), endocrine system (type 1 diabetes, autoimmune thyroiditis, Basel-Duhr's disease, Hashimoto's disease, etc.), systemic inflammation (Behçet's disease, antiphospholipid antibody syndrome, IgG4-related diseases, sepsis, hemorrhage, hypersensitivity reactions, transplant rejection, shock symptoms caused by cancer chemotherapy, etc.), etc.

[0122] The "allergic diseases" in this invention include, but are not limited to, atopic dermatitis, asthma, allergic reactions, pseudo-allergic reactions, food allergies, rhinitis, otitis media, drug reactions, insect sting reactions, plant reactions, latex allergies, conjunctivitis, urticaria, etc.

[0123] In this invention, "infectious disease" includes, but is not limited to, diseases caused by infection with viruses (single-stranded RNA viruses, double-stranded RNA viruses, single-stranded DNA viruses, double-stranded DNA viruses, etc.), bacteria (Gram-negative bacteria, Gram-positive bacteria, acid-fast bacteria, actinomycetes, spirochetes, spirilla, rickettsiae, chlamydia, mycoplasma, etc.), fungi (dermatophytes, Candida, Cryptococcus, Aspergillus, Pneumocystis, Malassezia, etc.), parasites (filariasis, trematodes, tapeworms, trematodes, Echinococcus tapeworm, Entamoeba histolytica, fleas, lice, mites, roundworms, pinworms, etc.).

[0124] The term "cancer" in this invention includes, but is not limited to, lymphoma, leukemia, breast cancer, lung cancer, and skin cancer.

[0125] As used herein, the term "fusion construct" defines the fusion of an antibody or antigen-binding fragment thereof with another compound. The fusion construct may comprise one or more antibodies or antigen-binding fragments thereof, which may be the same or different. The fusion construct may also comprise one or more other compounds, which may also be the same or different. The compounds may be protein compounds or non-protein compounds. When the compound is a protein compound or the fusion construct comprises only multiple antibodies or antigen-binding fragments thereof, the fusion construct may also be called a fusion protein. When the compound is fused with an antibody or antigen-binding fragment thereof in a conjugated form, the fusion construct may also be called a conjugate.

[0126] The "medicine" described in this invention can be used to treat humans or non-human animals, such as non-human mammals. The medicine may contain pharmaceutically acceptable carriers, excipients, or salts commonly found in the art.

[0127] The drug can be administered via any suitable route of administration, such as gastrointestinal (e.g., oral) or non-gastrointestinal (e.g., intravenous, intramuscular, subcutaneous, intradermal, intra-organ, intranasal, intraocular, intravenous, intracerebral, intrathecal, transdermal, rectal, etc.).

[0128] The drug can be in any suitable dosage form, such as a gastrointestinal or 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 inhalers, lotions, ointments, plasters, pastes, patches, eye drops, nasal drops, sublingual tablets, suppositories, aerosols, effervescent tablets, pellets, gels, etc. All dosage forms of the drug can be prepared according to conventional pharmaceutical manufacturing methods.

[0129] The term "pharmaceutically acceptable" as used in this invention refers to the biological activity and characteristics of the active substances in the applied product that neither significantly stimulate the organism nor inhibit it.

[0130] The “…method” described in this invention can be used for the purpose of diagnosing, treating and / or preventing diseases, or for the purpose of diagnosing, treating and / or preventing non-diseases.

[0131] The "antigen-binding fragment" described in this invention is a portion of an antibody that retains the specific binding activity of the antibody; that is, any part of the antibody is capable of specifically binding to an epitope on the antibody's target molecule. It includes, for example, Fab, Fab', F(ab')2, Fv, Fd, and variants of these fragments. For example, the heavy and / or light chains of the antibody, the variable regions of the heavy and / or light chains of the antibody, or single or more CDRs from the heavy or light chains of the antibody.

[0132] Nanobodies or single-domain antibodies refer to the variable domain of the antibody heavy chain (VHH), which has independent antigen-binding activity.

[0133] Chimeric antibodies are antibodies in which a portion of the heavy chain and / or light chain originates from a specific source or species, while the remainder of the heavy chain and / or light chain originates from different sources or species.

[0134] Single-chain antibodies are antibodies composed of variable regions of the heavy chain and variable regions of the light chain linked by a short peptide (linker) of 15 to 20 amino acids.

[0135] Fab is a monovalent segment composed of VL, VH, CL and CH1 domains.

[0136] Fab' is a Fab fragment with one or more cysteine ​​residues at the C-terminus of the CH1 domain.

[0137] F(ab')2 is a divalent segment containing two Fab segments connected by disulfide bonds in the hinge region.

[0138] Fd is an Fd fragment composed of VH and CH1 domains.

[0139] Fv is a fragment composed of the VL and VH domains of the antibody single arm.

[0140] dAb fragments are antibody fragments composed of VH domains.

[0141] The "linear antibody" described in this invention comprises one or more pairs of antibody fragments tandemly connected together. The antibody fragments may be Fd segments (VH-CH1), single-chain antibodies (scFv), antibody fragments (Fab), or single-domain antibodies (VHH). These fragments are tandemly connected by linker peptides to form a continuous antibody structure.

[0142] Wherein, VH represents the variable region of the heavy chain, VL represents the variable region of the light chain, CH represents the constant region of the heavy chain, and CL represents the constant region of the light chain.

[0143] The term "comprising" or "including" as used in this invention is an open-ended expression. When used to describe the sequence of a protein or nucleic acid, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the same or similar activity as the original sequence.

[0144] The "homology" or "identity" referred to in this invention means that, in the use of protein sequences or nucleotide sequences, those skilled in the art can adjust the sequences according to actual work needs, and the used sequences, compared with sequences obtained by prior art, have (including but not limited to) homology / identity of 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%, or 99.9%.

[0145] The "humanized antibody" described in this invention refers to an antibody whose framework region or the entire antibody is encoded by a human antibody gene.

[0146] The "individual" referred to in this invention can be a human or a non-human mammal. The non-human mammal 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, pigs, cattle, sheep, horses, donkeys, foxes, raccoon dogs, minks, camels, dogs, cats, rabbits, mice (e.g., rats, mice, guinea pigs, hamsters, gerbils, chinchillas, squirrels) or monkeys, etc.

[0147] The term "treatment" as used in this invention refers to slowing down, interrupting, preventing, controlling, stopping, alleviating, reducing, or reversing a sign, symptom, disorder, condition, or progression or severity of a disease after it has begun to develop, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders.

[0148] The term "prevention" as used in this invention refers to a method implemented to prevent or delay the occurrence of a disease, condition, or symptom in the body.

[0149] In this invention, "34G12" is the same as "34G12B8".

[0150] This invention provides a novel anti-TLR7 antibody or its antigen-binding fragment. A mouse anti-human TLR7 antibody is obtained through screening. Further screening and modification, such as humanizing the obtained mouse anti-human TLR7 antibody and further modifying the CDR, framework, and variable region of the humanized antibody, yields an antibody that specifically binds to different variants of human TLR7 and monkey TLR. This antibody exhibits good antigen-binding activity against TLR7 and can effectively inhibit various inflammatory cytokines produced by TLR7 activation, making it suitable for the treatment and / or prevention of TLR7-related diseases. Attached Figure Description

[0151] Figure 1 The results are obtained by flow cytometry analysis of the binding selectivity of chimeric anti-human TLR7 antibodies (83A7-c, 182A1H9-c, 247C6A2-c, 7C6A2-c, 13G1F4-c, 34G12B8-c, 17D1D2-c, 13H17L-c) to the antigen (human TLR7: variant 1).

[0152] Figure 2 The results are obtained by flow cytometry analysis of the selectivity of chimeric anti-human TLR7 antibodies (83A7-c, 182A1H9-c, 247C6A2-c, 7C6A2-c, 13G1F4-c, 34G12B8-c, 17D1D2-c, 13H17L-c) to the antigen (cynomolgus monkey TLR7).

[0153] Figure 3 The results are obtained by flow cytometry analysis of the selectivity of chimeric anti-human TLR7 antibodies (83A7-c, 247C6A2-c, 13G1F4-c, 17D1D2-c, 13H17L-c, 7C6A2-c, 34G12B8-c) to antigens (mouse TLR7, HEK293T).

[0154] Figure 4 The flow cytometry results show the selective binding ability of chimeric anti-human TLR7 antibodies (247C6A2-c, 7C6A2-c, 13G1F4-c, 34G12B8-c, 17D1D2-c, 13H17L-c) to the antigen (human TLR7: variant 2). The graph represents the specific binding to the antigen.

[0155] Figure 5 Flow cytometry analysis results of the binding selectivity of chimeric anti-human TLR7 antibodies (83A7-c, 182A1H9-c, 247C6A2-c, 13G1F4-c, 17D1D2-c, 13H17L-c, 7C6A2-c, 34G12B8-c) to antigens (human TLR7: variant 1, human Unc93B1, HEK293T).

[0156] Figure 6 The results are obtained by flow cytometry analysis of the binding selectivity of chimeric anti-human TLR7 antibodies (247C6A2-c, 13H17L-c) to antigens (human TLR7: variant 2, human Unc93B1, HEK293T).

[0157] Figure 7 Flow cytometry analysis results 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).

[0158] Figure 8 The graphs show the effects of different concentrations of chimeric anti-human TLR7 antibodies (247C6A2-c, 17D1D2-c, 13H17L-c, and 34G12B8-c) on IL-6 production in human PBMCs treated with GS-9620. Figure A shows the effect of different concentrations of chimeric anti-human TLR7 antibodies (17D1D2-c and 13H17L-c) on IL-6 production in human PBMCs treated with GS-9620; Figure B shows the effect of different concentrations of chimeric anti-human TLR7 antibodies (17D1D2-c, 13H17L-c, and 34G12B8-c) on IL-6 production in human PBMCs treated with GS-9620; Figure C shows the effect of different concentrations of chimeric anti-human TLR7 antibodies (247C6A2-c and 13H17L-c) on IL-6 production in human PBMCs treated with GS-9620.

[0159] Figure 9 The graphs show the effects of different concentrations of chimeric anti-human TLR7 antibodies (247C6A2-c, 17D1D2-c, 13H17L-c, and 34G12B8-c) on IFN-α production in human PBMCs treated with GS-9620. Figure A shows the effect of different concentrations of chimeric anti-human TLR7 antibodies (17D1D2-c and 34G12B8-c) on IFN-α production in human PBMCs treated with GS-9620; Figure B shows the effect of different concentrations of chimeric anti-human TLR7 antibodies (17D1D2-c and 13H17L-c) on IFN-α production in human PBMCs treated with GS-9620; and Figure C shows the effect of different concentrations of chimeric anti-human TLR7 antibodies (247C6A2-c and 13H17L-c) on IFN-α production in human PBMCs treated with GS-9620.

[0160] Figure 10The effect of adding different concentrations of chimeric anti-human TLR7 antibodies 13G1F4-c, 17D1D2-c, 13H17L-c, and 34G12B8-c on IL-6 production in DSR-6434-treated human PBMCs is shown in the figure.

[0161] Figure 11 The effect of 13H17L-c, a chimeric anti-human TLR7 antibody, on IFN-α production in DSR-6434-treated human PBMCs is shown in the figure.

[0162] Figure 12 The effect of adding different concentrations of chimeric anti-human TLR7 antibodies 13H17L-c, 17D1D2-c, 34G12B8-c, and 13G1F4-c on TNF-α production in DSR-6434-treated human PBMCs is shown in the figure.

[0163] Figure 13 The effect of chimeric anti-human TLR7 antibodies 13H17L-c, 34G12B8-c, and 13G1F4-c on IP-10 production in DSR-6434-treated human PBMCs is shown in the figure.

[0164] Figure 14 Flow cytometry analysis results of the binding selectivity of 13H17L humanized antibodies (13H17L-h1—13H17L-h22) to antigens (human TLR7: variant 2, human Unc93B1, HEK293T).

[0165] Figure 15 Flow cytometry analysis results of the binding selectivity of 34G12B8 humanized antibodies (34G12-h1, 2, 3, 4, 5, 7) to antigens (human TLR7: variant 1, human Unc93B1, HEK293T).

[0166] Figure 16 Flow cytometry analysis results of the binding selectivity of 34G12B8 humanized antibodies (34G12-h8, 9, 11, 13, 14, 15, 16, 17, 18, 19) to antigens (human TLR7: variant 1, human Unc93B1, HEK293T).

[0167] Figure 17 Flow cytometry analysis results of the binding selectivity of 34G12B8 humanized antibodies (34G12-h9, 11, 13, 14, 15, 16, 17, 19) to antigens (cynomolgus monkey TLR7, cynomolgus monkey Unc93B1, HEK293T).

[0168] Figure 18The graph shows the effect of 13H17L humanized antibody (13H17L-h1—13H17L-h22) on IL-6 production in human PBMCs treated with GS-9620. In the graph, A shows the effect of 13H17L-h3, 4, 5, 7, 9, and 10; B shows the effect of 13H17L-h11 to 13H17L-h17 and 13H17L-h19 to 13H17L-h22.

[0169] Figure 19 The figure shows the effect of the 34G12B8 humanized antibody (34G12-h1—34G12-h19) on IL-6 production in human PBMCs treated with GS-9620.

[0170] Figure 20 The figure shows the effect of the 34G12B8 humanized antibody (34G12-h1—34G12-h19) on TNFα production in human PBMCs treated with GS-9620.

[0171] Figure 21 The effect of humanized TLR7 antibody on IL-6 in CD34+ humanized mice.

[0172] Figure 22 Flow cytometry analysis results of the binding selectivity of anti-human TLR7 affinity maturation antibodies (34G12-h542, 34G12-h458) to antigens (human TLR7: variant 1, human Unc93B1, HEK293T).

[0173] Figure 23 Flow cytometry analysis results of the binding selectivity of anti-human TLR7 affinity maturation antibodies (h587, h588, h589, h542) to antigens (human TLR7: variant 1, human Unc93B1, HEK293T).

[0174] Figure 24 Effects of anti-human TLR7 affinity maturation antibodies (34G12-h458, 34G12-h488, 34G12-h487, 34G12-h489, 34G12-h485, 34G12-h486) on the production of IL-6 and TNF-α in human PBMCs treated with GS-9620.

[0175] Figure 25Effects of anti-human TLR7 affinity maturation 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 the production of IL-6 and TNF-α in GS-9620-treated human PBMCs.

[0176] Figure 26 Effects of anti-human TLR7 affinity maturation 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 the production of IL-6 and TNF-α in GS-9620-treated human PBMCs.

[0177] Figure 27 Effects of anti-human TLR7 affinity maturation antibodies (34G12-h458, 34G12-h556, 34G12-h557) on downstream signaling in HEK293-TLR7 reporter cells treated with GS-9620.

[0178] Figure 28 Effects of anti-human TLR7 affinity maturation antibodies (34G12-h542, 34G12-h587, 34G12-h588, 34G12-h589) on downstream signaling in HEK293-TLR7 reporter cells treated with GS-9620.

[0179] Figure 29 The effect of anti-human TLR7 affinity maturation antibody (34G12-h458) on the proportion of plasma cell formation in SLE-PBMCs was detected by flow cytometry.

[0180] Figure 30 The effect of anti-human TLR7 affinity maturation antibody (34G12-h458) on the proportion of plasma cell formation in HV (healthy population)-PBMCs was detected by flow cytometry.

[0181] Figure 31 The effect of anti-human TLR7 affinity maturation antibody (34G12-h542) on the proportion of plasma cell formation in HV-PBMCs (healthy individuals) was detected by flow cytometry.

[0182] Figure 32The effect of anti-human TLR7 affinity maturation antibody (34G12-h458) on IL-6 in CD34+ humanized mice.

[0183] Figure 33 Detection of endosomes and Ba / F3 cell lines containing human Unc93B1-G4S-Flag-T2A-human TLR7. Detailed Implementation

[0184] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions in the embodiments of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0185] Unless otherwise specified, all materials, reagents, instruments, etc. used in the following examples are commercially available.

[0186] Example 1: Preparation of mouse anti-human TLR7 antibody

[0187] 1-1: Immunity

[0188] 1-1-1 Construction of Ba / F3 cell line overexpressing human Unc93B1-G4S-Flag-T2A-human TLR7

[0189] use Max DNA Polymerase (Takara) was used to integrate the human Unc93B1-G4S-Flag-T2A-human TLR7 gene into the 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 the human TLR7 gene is shown in SEQ ID NO: 2.

[0190] The transposon vector and transposon helper plasmid were introduced into the Ba / F3 cell line (ATCC) using the transfection reagent Lip3000 (Thermo) to establish a Ba / F3 cell line overexpressing human Unc93B1-G4S-Flag-T2A-human TLR7.

[0191] 1-1-2 Endosome Extraction

[0192] The Ba / F3 cell line overexpressing human Unc93B1-G4S-Flag-T2A-human TLR7, established in step 1-1-1, was thoroughly homogenized on ice under aseptic conditions using a Dounce homogenizer. Sucrose gradient centrifugation samples were prepared using sucrose buffer of different concentrations, and endosomes were separated by ultracentrifugation. The crude endosome extract was collected and purified again by ultracentrifugation to obtain the target endosome.

[0193] The obtained endosomes and the above-mentioned cell lines were tested. The results are as follows: Figure 33 As shown.

[0194] 1-1-3 Immunization of Mice

[0195] Divided into three groups,

[0196] Group 1: Ba / F3 cell lines overexpressing human Unc93B1-G4S-Flag-T2A-human TLR7 established in 1-1-1, or endosomes purified in 1-1-2, were combined with adjuvants. Gold Adjuvant (sigma) was mixed and used as an antigen, and administered to Balbc or SJL mice once a week via the soles of the feet, tail, and intraperitoneal cavity.

[0197] Group 2: Ba / F3 cell lines overexpressing human Unc93B1-G4S-Flag-T2A-human TLR7 established in 1-1-1, or endosomes purified in 1-1-2, were combined with adjuvants. Gold Adjuvant (sigma) and CpG / Alhydrogel The adjuvant 2% (Invivogen) mixture was used as an antigen. Balbc or SJL mice were immunized once every two weeks for the first immunization, and the remaining immunizations were administered once a week to the soles of the feet, tail, and intraperitoneal cavity.

[0198] Group 3: Ba / F3 cell lines overexpressing human Unc93B1-G4S-Flag-T2A-human TLR7 established in 1-1-1, or endosomes purified in 1-1-2, were combined with Freund's solution and CpG / Alhydrogel as adjuvants. The adjuvant 2% (Invivogen) mixture was used as an antigen. Balbc mice were immunized once every two weeks for the first immunization, and the remaining immunizations were administered once a week to the soles of the feet, tail, and intraperitoneal cavity.

[0199] For the three groups mentioned above, during the 8th immunization, Ba / F3 cell lines overexpressing human Unc93B1-G4S-Flag-T2A-human TLR7, suspended in 1×PBS, or purified endosomes were administered intraperitoneally.

[0200] The spleen was removed on the 5th day after the last immunization and used to make hybridomas.

[0201] 1-2 Preparation of hybridoma

[0202] Immunized spleen cells and Sp2 / 0 cell lines (from [Company Name]) were mixed and fused using a BTX electrofusion instrument. Following cell fusion, the cells were cultured in DMEM (manufactured by [Company Name]) containing HAT (Sigma) medium (containing 10% FBS (Gibco)) for hybridoma screening. Hybridoma colonies were collected, thus creating monoclonal hybridomas.

[0203] 1-3 Screening for human TLR7 binding antibodies using cell-based ELISA

[0204] 1-3-1 Construction of HEK293T cell line overexpressing human TLR7

[0205] use The human TLR7 gene (SEQ ID NO: 2) was integrated into the lentiviral vector pCDH (BioWind) using Max DNA Polymerase (Takara). The lentiviral vector was then introduced into the HEK293-derived packaging cell line Lenti-X 293T (Sigma) using the transfection reagent EZ Trans (manufactured by Heyuan Liji). The culture supernatant was collected 48 hours later as a viral suspension. This viral suspension was added to HEK293T cells to establish a HEK293T cell line overexpressing human TLR7.

[0206] 1-3-2 Screening for human TLR7 binding antibodies using cell-based ELISA

[0207] Hybridomas that formed colonies under a microscope were collected and their culture supernatants were used for screening. HEK293T cell lines overexpressing human TLR7, constructed in a 1-3-1 manner, were added to an ELISA plate. After three days of culture, the culture medium was removed, and the cells were fixed and perforated with 0.1% Triton X-100 (Solepro). Skim milk was then added for blocking. The culture supernatant was added to the ELISA plate for color development, and the hybridomas that produced anti-hTLR7 antibodies were preliminarily screened using an ELISA reader.

[0208] 1-4 Screening for human TLR7 binding antibodies using flow cytometry analysis

[0209] 1-4-1 Construction of HEK293T cell line overexpressing human Unc93B1-Flag

[0210] use The human Unc93B1 (SEQ ID NO: 1)-Flag gene was integrated into the lentiviral vector pCDH (BioWind) using Max DNA Polymerase (Takara). The lentiviral vector was then introduced into the HEK293-derived packaging cell line Lenti-X 293T (Sigma) using the transfection reagent EZ Trans (manufactured by Heyuan Liji). The culture supernatant was collected after 48 hours as a viral suspension. This viral suspension was added to HEK293T cells to establish a HEK293T cell line overexpressing human Unc93B1-Flag. 1-4-2 Construction of HEK293T cell line overexpressing human Unc93B1-Flag / human TLR7

[0211] use The human Unc93B1 (SEQ ID NO: 1)-Flag gene was integrated into the lentiviral vector pCDH (BioWind) using Max DNA Polymerase (Takara). The lentiviral vector was then introduced into the HEK293-derived packaging cell line Lenti-X 293T (Sigma) using the transfection reagent EZ Trans (manufactured by Heyuan Liji). The culture supernatant was collected 48 hours later as the viral suspension. This viral suspension was added to HEK293T cells overexpressing human TLR7 to establish a HEK293T cell line overexpressing human Unc93B1-Flag / human TLR7.

[0212] 1-4-3 Construction of HEK293T cell line overexpressing human TLR7 (variant 2)

[0213] use The human TLR7 (variant 2, SEQ ID NO: 144) gene was integrated into the lentiviral vector pCDH (BioWind) using Max DNA Polymerase (Takara). The lentiviral vector was then introduced into the HEK293-derived packaging cell line Lenti-X 293T (Sigma) using the transfection reagent EZ Trans (manufactured by Heyuan Liji). The culture supernatant was recovered after 48 hours to obtain the viral suspension. This viral suspension was added to HEK293T cells to establish a HEK293T cell line overexpressing human TLR7 (variant 2).

[0214] Construction of HEK293T cell line overexpressing human Unc93B1 (SEQ ID NO: 1)-Flag / human TLR7 (variant 2) 1-4-4

[0215] use The human Unc93B1 (SEQ ID NO: 1)-Flag gene was integrated into the lentiviral vector pCDH (BioWind) using Max DNA Polymerase (Takara). The lentiviral vector was then introduced into the HEK293-derived packaging cell line Lenti-X 293T (Sigma) using the transfection reagent EZ Trans (manufactured by Heyuan Liji). The culture supernatant was recovered after 48 hours as a viral suspension. This viral suspension was added to HEK293T cells overexpressing human TLR7 (variant 2) to establish a HEK293T cell line overexpressing human Unc93B1-Flag / human TLR7 (variant 2).

[0216] Construction of Ba / F3 cell lines overexpressing human Unc93B1 (SEQ ID NO: 1)-Flag / human TLR7 (variant 2) 1-4-5

[0217] use Max DNA Polymerase (Takara) was used to integrate the human Unc93B1 (SEQ ID NO: 1)-Flag gene into the lentiviral vector pCDH (BioWind). The human TLR7 (variant 2, SEQ ID NO: 144) gene was integrated into the lentiviral vector pCDH (BioWind) using Max DNA Polymerase (Takara). The lentiviral vector was then introduced into the HEK293-derived packaging cell line Lenti-X 293T (Sigma) using the transfection reagent EZTrans (manufactured by Heyuan Liji). The culture supernatant was recovered after 48 hours as a viral suspension. This viral suspension was added to Ba / F3 cells to establish a Ba / F3 cell line overexpressing human Unc93B1-Flag / human TLR7 (variant 2).

[0218] 1-4-6 Screening for human TLR7 binding antibodies by flow cytometry analysis

[0219] Hybridoma culture supernatants producing human TLR7 antibodies were initially screened using a 1-3-2 cell-based ELISA. HEK293T cell lines overexpressing human TLR7 and those not expressing it at all, and / or HEK293T cell lines overexpressing human Unc93B1-Flag / human TLR7 and HEK293T cell lines overexpressing human Unc93B1-Flag, were stained in their culture supernatants and analyzed by flow cytometry (Beckman, DxFLEX) to screen for hybridomas producing anti-hTLR7 antibodies. Seven hybridomas producing mouse anti-human TLR7 antibodies were selected and named 83A7, 182A1H9, 247C6A2, 13G1F4, 17D1D2, 7C6A2, and 34G12B8.

[0220] In this application specification, the antibodies produced by hybridomas 83A7, 182A1H9, 247C6A2, 13G1F4, 17D1D2, 7C6A2, and 34G12B8 are named 83A7 antibody, 182A1H9 antibody, 247C6A2 antibody, 13G1F4 antibody, 17D1D2 antibody, 7C6A2 antibody, and 34G12B8 antibody, respectively.

[0221] Example 2: Determination of the nucleotide and amino acid sequences of cDNA encoding the variable region of mouse anti-human TLR7 antibody 2-1 cDNA synthesis

[0222] Total RNA was recovered from hybridomas containing 83A7, 182A1H9, 247C6A2, 13G1F4, 17D1D2, 7C6A2, and 34G12B8 using TRIzol Reagent (Ambion). Then, PrimeScript was used to analyze the RNA. TM 1st Strand cDNA SynthesisKit (Takara) synthesized cDNA.

[0223] 2-2 Amplification and Sequence Determination of Variable Region Gene Fragments of Mouse Immunoglobulin Heavy and Light Chains

[0224] Antibodies VH and VL were amplified using universal antibody light and heavy chain primers. The PCR products were cloned into pUC19-T, and colony sequencing was performed to interpret the nucleotide sequences encoding the variable regions of the antibodies.

[0225] 2-2-1 Mouse anti-human TLR7 antibody (83A7)

[0226] The amino acid sequence encoded by the nucleotide sequence of the cDNA encoding the heavy chain variable region of the determined 83A7 antibody begins at SEQ ID NO: 5 in the sequence listing.

[0227] The amino acid sequence encoded by the nucleotide sequence of the cDNA encoding the light chain variable region of the determined 83A7 antibody begins at SEQ ID NO: 12 in the sequence listing.

[0228] 2-2-2 Mouse anti-human TLR7 antibody (182A1H9)

[0229] The amino acid sequence encoded by the nucleotide sequence of the cDNA encoding the heavy chain variable region of the determined antibody 182A1H9 begins with SEQ ID NO: 6 in the sequence listing.

[0230] The amino acid sequence encoded by the nucleotide sequence of the cDNA encoding the light chain variable region of the determined antibody 182A1H9 begins with SEQ ID NO: 13 in the sequence listing.

[0231] 2-2-3 Mouse anti-human TLR7 antibody (247C6A2)

[0232] The amino acid sequence encoded by the nucleotide sequence of the cDNA encoding the heavy chain variable region of the determined antibody 247C6A2 begins with SEQ ID NO: 7 in the sequence listing.

[0233] The amino acid sequence encoded by the nucleotide sequence of the cDNA encoding the light chain variable region of the determined antibody 247C6A2 begins with SEQ ID NO: 14 in the sequence listing.

[0234] 2-2-4 mouse anti-human TLR7 antibody (13G1F4)

[0235] The amino acid sequence encoded by the nucleotide sequence of the cDNA encoding the heavy chain variable region of the determined 13G1F4 antibody begins at SEQ ID NO: 8 in the sequence listing.

[0236] The amino acid sequence encoded by the nucleotide sequence of the cDNA encoding the light chain variable region of the determined 13G1F4 antibody begins at SEQ ID NO: 15 in the sequence listing.

[0237] 2-2-5 mouse anti-human TLR7 antibody (17D1D2)

[0238] The amino acid sequence encoded by the nucleotide sequence of the cDNA encoding the heavy chain variable region of the determined 17D1D2 antibody begins at SEQ ID NO: 9 in the sequence listing.

[0239] The amino acid sequence encoded by the nucleotide sequence of the cDNA encoding the light chain variable region of the determined 17D1D2 antibody begins at SEQ ID NO: 16 in the sequence listing.

[0240] 2-2-6 mouse anti-human TLR7 antibody (7C6A2)

[0241] The amino acid sequence encoded by the nucleotide sequence of the cDNA encoding the heavy chain variable region of the determined 7C6A2 antibody begins at SEQ ID NO: 10 in the sequence listing.

[0242] The amino acid sequence encoded by the nucleotide sequence of the cDNA encoding the light chain variable region of the determined 7C6A2 antibody begins at SEQ ID NO: 17 in the sequence listing.

[0243] 2-2-7 mouse anti-human TLR7 antibody (34G12B8)

[0244] The amino acid sequence encoded by the nucleotide sequence of the cDNA encoding the heavy chain variable region of the determined 34G12B8 antibody begins at SEQ ID NO: 11 in the sequence listing.

[0245] The amino acid sequence encoded by the nucleotide sequence of the cDNA encoding the light chain variable region of the determined 34G12B8 antibody begins at SEQ ID NO: 18 in the sequence listing.

[0246] Example 3: Preparation of chimeric anti-human TLR7 antibody

[0247] Construction of a chimeric anti-TLR7 antibody expression vector

[0248] 3-1-1 Construction of the vector pcDNA3.4-LK for expressing chimeric light chains

[0249] The approximately 6 kb fragment obtained by digesting plasmid pcDNA3.4 with restriction endonucleases BamHI and HindIII, and the DNA fragment containing the DNA sequence encoding the human light chain signal sequence and the human kappa chain constant region (SEQ ID NO: 128) were ligated with T4 ligase (Takara) to construct pcDNA3.4-LK.

[0250] 3-1-2 Construction of the chimeric IgG1 heavy chain expression vector pCDNA3.4-G1

[0251] The DNA fragment containing the light chain signal sequence and the human kappa chain constant region of plasmid pcDNA3.4-LK, which was digested with restriction endonucleases BamHI and HindIII, and the DNA fragment containing the DNA sequence encoding the human heavy chain signal sequence and the human IgG1-LALA constant region (SEQ ID NO: 129) were ligated with T4 ligase (Takara) to construct pcDNA3.4-G1.

[0252] 3-1-3 Construction of 83A7 chimeric anti-human TLR7 antibody expression vector

[0253] A DNA fragment encoding the nucleotide sequence of the 83A7-c antibody heavy chain was synthesized, wherein the nucleotide sequence encodes the variable region of the 83A7-c antibody heavy chain, represented by nucleotides 58-405 bp of the nucleotide sequence shown in SEQ ID NO: 130. The synthesized DNA fragment was inserted into pcDNA3.4-G1 using assembly mix (China-US Taihe), with the insertion position after the signal sequence and before the constant region nucleotide sequence, thereby constructing an expression vector for the 83A7-c antibody heavy chain. The 83A7-c antibody heavy chain contains the signal sequence and has the amino acid sequence of SEQ ID NO: 72.

[0254] A DNA fragment (SEQ ID NO: 131) containing the DNA sequence encoding the 83A7-c antibody light chain was synthesized. Using pcDNA3.4-LK prepared in Example 3-1-1, an 83A7-c antibody light chain expression vector was constructed using the same method as described above. The 83A7-c antibody light chain contains a signal sequence and has the amino acid sequence of SEQ ID NO: 79.

[0255] 3-1-4 Construction of 182A1H9 chimeric anti-human TLR7 antibody expression vector

[0256] A DNA fragment containing the nucleotide sequence encoding the 182A1H9-c heavy chain was synthesized, wherein the nucleotide sequence encodes the heavy chain variable region of the 182A1H9-c antibody, represented by nucleotides 58-426 bp of the nucleotide sequence shown in SEQ ID NO: 132. A 182A1H9-c heavy chain expression vector was constructed using the same method as in Example 3-1-3. The 182A1H9-c heavy chain contains a signal sequence and has the amino acid sequence of SEQ ID NO: 73.

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

[0258] 3-1-5 Construction of a chimeric anti-human TLR7 antibody expression vector based on 247C6A2

[0259] A DNA fragment containing a heavy chain nucleotide sequence encoding the 247C6A2-c antibody was synthesized, wherein the nucleotide sequence encodes the heavy chain variable region of the 247C6A2-c antibody, as shown in SEQ ID NO: 134, nucleotide sequence 58-420 bp. A 247C6A2-c heavy chain expression vector was constructed using the same method as in Example 3-1-3. The 247C6A2-c heavy chain contains a signal sequence having the amino acid sequence of SEQ ID NO: 74.

[0260] A DNA fragment (SEQ ID NO: 135) containing a DNA sequence encoding the 247C6A2-c light chain was synthesized. Using pcDNA3.4-LK prepared in Example 3-1-1, a 247C6A2-c light chain expression vector was constructed using the same method as in Example 3-1-3. The 247C6A2-c light chain contains a signal sequence and has the amino acid sequence of SEQ ID NO: 81.

[0261] 3-1-6 Construction of 13G1F4 chimeric anti-human TLR7 antibody expression vector

[0262] A DNA fragment containing the nucleotide sequence encoding the 13G1F4-c heavy chain was synthesized, wherein the nucleotide sequence encodes the heavy chain variable region of the 13G1F4-c antibody, represented by nucleotides 58-405 bp of the nucleotide sequence shown in SEQ ID NO: 136. A 13G1F4-c heavy chain expression vector was constructed using the same method as in Example 3-1-3. The 13G1F4-c heavy chain contains a signal sequence and has the amino acid sequence of SEQ ID NO: 75.

[0263] A DNA fragment (SEQ ID NO: 137) containing a DNA sequence encoding the 13G1F4-c light chain was synthesized. Using pcDNA3.4-LK prepared in Example 3-1-1, a 13G1F4-c light chain expression vector was constructed using the same method as in Example 3-1-3. The 13G1F4-c light chain contains a signal sequence and has the amino acid sequence of SEQ ID NO: 82.

[0264] 3-1-7 Construction of a 17D1D2 chimeric anti-human TLR7 antibody expression vector

[0265] A DNA fragment containing the nucleotide sequence encoding the 17D1D2-c heavy chain was synthesized, wherein the nucleotide sequence encodes the heavy chain variable region of the 17D1D2-c antibody, represented by nucleotides 58-405 bp of the nucleotide sequence shown in SEQ ID NO: 138. A 17D1D2-c heavy chain expression vector was constructed using the same method as in Example 3-1-3. The 17D1D2-c heavy chain contains a signal sequence and has the amino acid sequence of SEQ ID NO: 76.

[0266] A DNA fragment (SEQ ID NO: 139) containing a DNA sequence encoding the 17D1D2-c light chain was synthesized. Using pcDNA3.4-LK prepared in Example 3-1-1, a 17D1D2-c light chain expression vector was constructed using the same method as in Example 3-1-3. The 17D1D2-c light chain contains a signal sequence and has the amino acid sequence of SEQ ID NO: 83.

[0267] Construction of 3-1-8 7C6A2 chimeric anti-human TLR7 antibody expression vector

[0268] A DNA fragment containing the nucleotide sequence encoding the 7C6A2-c heavy chain was synthesized, wherein the nucleotide sequence encodes the heavy chain variable region of the 7C6A2-c antibody, represented by nucleotides 58-411 bp of the nucleotide sequence shown in SEQ ID NO: 140. A 7C6A2-c heavy chain expression vector was constructed using the same method as in Example 3-1-3. The 7C6A2-c heavy chain contains a signal sequence and has the amino acid sequence of SEQ ID NO: 77.

[0269] A DNA fragment (SEQ ID NO: 141) containing a DNA sequence encoding the 7C6A2-c light chain was synthesized. Using pcDNA3.4-LK prepared in Example 3-1-1, a 7C6A2-c light chain expression vector was constructed using the same method as in Example 3-1-3. The 7C6A2-c light chain contains a signal sequence and has the amino acid sequence of SEQ ID NO: 84.

[0270] Construction of 34G12B8 chimeric anti-human TLR7 antibody expression vector (3-1-9)

[0271] A DNA fragment containing the nucleotide sequence encoding the 34G12B8-c heavy chain was synthesized, wherein the nucleotide sequence encodes the heavy chain variable region of the 34G12B8-c antibody, represented by nucleotides 58-423 bp of the nucleotide sequence shown in SEQ ID NO: 142. A 34G12B8-c heavy chain expression vector was constructed using the same method as in Example 3-1-3. The 34G12B8-c heavy chain contains a signal sequence and has the amino acid sequence of SEQ ID NO: 78.

[0272] A DNA fragment (SEQ ID NO: 143) containing the DNA sequence encoding the 34G12B8-c light chain was synthesized. Using pcDNA3.4-LK prepared in Example 3-1-1, a 34G12B8-c light chain expression vector was constructed using the same method as in Example 3-1-3. The 34G12B8-c light chain contains a signal sequence and has the amino acid sequence of SEQ ID NO: 85.

[0273] 3-2 Preparation of chimeric anti-human TLR7 antibody

[0274] 3-2-1 Preparation of chimeric 83A7 antibody

[0275] For Expi293 cells (Gibco), passage and culture were performed according to the manual. 3 × 10⁶ cells in the logarithmic growth phase were... 8 One Expi293 cell (Gibco) was seeded in a 250 ml shake flask (nest) and treated with Expi293... TM Expression Medium (Gibco) diluted and adjusted to 3×10 6 Cells / ml. Add 50 μg of the heavy chain expression vector constructed in step 3-1-3 and 50 μg of the light chain expression vector constructed in step 3-1-3 to 5 ml of Opti-MEM medium (Gibco) and mix well. Add 100 μg of polyethyleneimine (Bioscience) to 5 ml of Opti-MEM medium (Gibco) and mix well. After mixing the vector and polyethyleneimine, let stand for 15 minutes before adding to Expi293 cells. Incubate at 37°C and 7% CO2 with shaking for 4 days. Filter the resulting supernatant using a vacuum filter cup (Nest) to produce the chimeric 83A7-c antibody.

[0276] Preparation of 3-2-2 chimeric 182A1H9 antibody

[0277] Using the heavy chain expression vector and light chain expression vector constructed in 3-1-4 above, a chimeric 182A1H9-c antibody was manufactured by the same method as in 3-2-1 above.

[0278] Preparation of 3-2-3 chimeric 247C6A2 antibody

[0279] Using the heavy chain expression vector and light chain expression vector constructed in 3-1-5 above, a chimeric 247C6A2-c antibody was manufactured by the same method as in 3-2-1 above.

[0280] Preparation of 3-2-4 chimeric 13G1F4 antibody

[0281] Using the heavy chain expression vector and light chain expression vector constructed in 3-1-6 above, a chimeric 13G1F4-c antibody was manufactured by the same method as in 3-2-1 above.

[0282] Preparation of 3-2-5 chimeric 17D1D2 antibody

[0283] Using the heavy chain expression vector and light chain expression vector constructed in 3-1-7 above, a chimeric 17D1D2-c antibody was manufactured by the same method as in 3-2-1 above.

[0284] Preparation of 3-2-6 chimeric 13H17L antibody

[0285] Using the heavy chain expression vector constructed in 3-1-6 above and the light chain expression vector constructed in 3-1-7 above, a chimeric 13H17L-c antibody was manufactured by the method described in 3-2-1 above (the 13H17L antibody was obtained by combining the heavy chain of the 13G1F4 antibody and the light chain of the 17D1D2 antibody).

[0286] Preparation of 3-2-7 chimeric 7C6A2 antibody

[0287] Using the heavy chain expression vector and light chain expression vector constructed in 3-1-8 above, a chimeric 7C6A2-c antibody was manufactured by the same method as in 3-2-1 above.

[0288] Preparation of 3-2-8 chimeric 34G12B8 antibody

[0289] Using the heavy chain expression vector and light chain expression vector constructed in 3-1-9 above, a chimeric 34G12B8-c antibody was manufactured by the same method as in 3-2-1 above.

[0290] Purification of 3-3 chimeric anti-human TLR7 antibody

[0291] The target antibody was purified from the culture supernatants obtained in Examples 3-2 using a series of steps of Protein A affinity chromatography. The culture supernatant was loaded onto a chromatography column filled with Protein A (Yisheng) and equilibrated with binding buffer (Sangon Biotech). The column was then washed twice with 5 column volumes of binding buffer. Elution buffer (Sangon Biotech) was then used for equilibration with 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 membrane to further concentrate the antibody. Finally, the purified sample was obtained by filtration using a 0.22 μM disposable syringe filter (PALL).

[0292] Example 4: In vitro evaluation of chimeric anti-human TLR7 antibody

[0293] 4-1 Evaluation of the binding selectivity of chimeric anti-human TLR7 antibodies

[0294] 4-1-1 Establishment of HEK293T cell line overexpressing mouse TLR7

[0295] use The mouse TLR7 gene (SEQ ID NO: 126) was integrated into the lentiviral vector pCDH (BioWind) using Max DNA Polymerase (Takara). The lentiviral vector was then introduced into the HEK293-derived packaging cell line Lenti-X 293T (Sigma) using the transfection reagent EZ Trans (manufactured by Heyuan Liji). The culture supernatant was collected 48 hours later as a viral suspension. This viral suspension was added to HEK293T cells to establish a HEK293T cell line overexpressing mouse TLR7.

[0296] 4-1-2 Establishment of HEK293T cell line overexpressing cynomolgus monkey TLR7

[0297] use The cynomolgus monkey TLR7 gene (SEQ ID NO: 127) was integrated into the lentiviral vector pCDH (BioWind) using Max DNA Polymerase (Takara). The lentiviral vector was then introduced into the HEK293-derived packaging cell line Lenti-X 293T (Sigma) using the transfection reagent EZ Trans (manufactured by Heyuan Liji). The culture supernatant was recovered after 48 hours to obtain the viral suspension. This viral suspension was added to HEK293T cells to establish a HEK293T cell line overexpressing cynomolgus monkey TLR7.

[0298] 4-1-3 Establishment of HEK293T cell line overexpressing cynomolgus monkey TLR7 / cynomolgus monkey Unc93B1

[0299] use Max DNA Polymerase (Takara) was used to integrate the cynomolgus monkey Unc93B1 gene (SEQ ID NO: 145) into the lentiviral vector pCDH (BioWind). The lentiviral vector was introduced into the HEK293-derived packaging cell line Lenti-X 293T (Sigma) using the transfection reagent EZ Trans (manufactured by Heyuan Liji). The culture supernatant was recovered after 48 hours as a viral suspension. This viral suspension was added to HEK293T cells overexpressing cynomolgus monkey TLR7 to establish a HEK293T cell line overexpressing cynomolgus monkey TLR7 / Unc93B1.

[0300] 4-1-4 Evaluation of the binding selectivity of chimeric anti-human TLR7 antibodies using flow cytometry

[0301] The following cell lines were used: HEK293T cell lines overexpressing human TLR7 (established in 1-3-1), HEK293T cell lines overexpressing mouse TLR7 (established in 4-1-1), HEK293T cell lines overexpressing cynomolgus monkey TLR7 (established in 4-1-2), HEK293T cell lines overexpressing cynomolgus monkey TLR7 / cynomolgus monkey Unc93B1 (established in 4-1-3), and HEK293T cell lines overexpressing human TLR7 (variant 2) (established in 1-4-3). Cell lines were permeabilized with 1× Biolegend permeabilizing agent. The following antibody concentrations were used: chimeric 83A7 antibody, 182A1H9 antibody, 247C6A2 antibody, 13G1F4 antibody, 17D1D2 antibody, 13H17L antibody, 7C6A2 antibody, and 34G12B8 antibody obtained in Example 3, and Goat anti-human IgG was used as a secondary antibody. H&L-FITC (Abcam) staining. The antibody binding selectivity was evaluated using flow cytometry, and mean fluorescence intensity (MFI) was compared.

[0302] The results showed that antibodies 83A7, 182A1H9, 247C6A2, 13G1F4, 17D1D2, 13H17L, 7C6A2, and 34G12B8 specifically bound to human TLR7. Figure 1 );

[0303] Antibodies 83A7, 182A1H9, 247C6A2, 13G1F4, 17D1D2, 13H17L, 7C6A2, and 34G12B8 specifically bind to TLR7 in cynomolgus monkeys. Figure 2 The series of anti-human TLR7 antibodies screened in this study, such as 83A7 antibody, 247C6A2 antibody, 13G1F4 antibody, 17D1D2 antibody, 13H17L antibody, 7C6A2 antibody, and 34G12B8 antibody, do not bind to mouse TLR7. Figure 3 The results showed that the antibody did not specifically bind to the antigen; 83A7 antibody, 182A1H9 antibody, 247C6A2 antibody, 13G1F4 antibody, 17D1D2 antibody, 7C6A2 antibody, 34G12B8 antibody, and 13H17L antibody all specifically bind to human TLR7-variant 2. Figure 4 Its EC 50 See Table 3

[0304] Table 3EC 50 Results Display

[0305]

[0306] Note: N / A indicates maladaptation.

[0307] 4-2 The antigen-binding activity of chimeric anti-human TLR7 antibody was obtained using flow cytometry.

[0308] HEK293T cells overexpressing human Unc93B1-Flag / human TLR7 were permeabilized with 1× Biolegend membrane permeabilization solution. Cells were then stained with a series of antibody dilutions using the chimeric 83A7, 182A1H9, 247C6A2, 13G1F4, 17D1D2, 13H17L, 7C6A2, and 34G12B8 antibodies obtained in Example 3, and with Goat anti-human IgG H&L-FITC (Abcam) as a secondary antibody. Antibody binding activity was evaluated by flow cytometry, and MFI was compared. The results are as follows: Figure 5 As shown, the binding activities from strongest to weakest are: 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), and chimeric 182A1H9 antibody (182A1H9-c), among which EC 50:13H17L-c 0.4608nM, 17D1D2-c 0.5407nM, 13G1F4-c 0.4263nM, 247C6A2-c 0.5754nM, 34G12B8-c 0.5021nM, 83A7-c 0.9462nM, 7C6A2-c 33.71nM.

[0309] HEK293T cells overexpressing human Unc93B1-Flag / human TLR7 (variant 2) were permeabilized with 1× Biolegend permeabilization solution and stained with serially diluted antibody concentrations of the chimeric 247C6A2 and 13H17L antibodies obtained in Example 3, and Goat anti-human IgG H&L-FITC (Abcam) as a secondary antibody. MFI was analyzed by flow cytometry to evaluate antibody binding activity. Figure 6 As shown, the results indicate that 247C6A2-c and 13H17L-c specifically bind to this cell.

[0310] Ba / F3 cell lines overexpressing human Unc93B1-G4S-Flag-T2A-human TLR7 were permeabilized with 1× Biolegend permeabilization agent. Cells were then stained with serially diluted chimeric 83A7, 182A1H9, and 247C6A2 antibodies obtained in Example 3, and with Goat anti-human IgG H&L-FITC (Abcam) as a secondary antibody. Antibody binding activity was evaluated using flow cytometry, and MFI was compared. Figure 7 As shown, the results indicate that the binding activity, from strongest to weakest, is as follows: chimeric 247C6A2 antibody (247C6A2-c), chimeric 83A7 antibody (83A7-c), and chimeric 182A1H9 antibody (182A1H9-c), among which EC... 50 :247C6A2-c 0.6793nM, 83A7-c 2.234nM. Its EC 50 See Table 4.

[0311] Table 4EC 50 Results Display

[0312]

[0313] Note: N / A indicates maladaptation.

[0314] 4-3 Inhibitory effect of chimeric anti-human TLR7 antibody on cytokine production

[0315] Human PBMCs were purchased from Shanghai Aoneng Biotechnology Co., Ltd. in frozen form and thawed according to the instructions before use. The solution was adjusted to 2×10⁻⁶ using RPMI 1640 (Gibco) containing 10% FBS (Excell), 1mM sodium pyruvate (Gibco), 0.1mM MEM-Non-Essential Amino Acids (Gibco), 50mM 2-mercaptoethanol (Gibco), 50U / ml penicillin, and 50U / ml streptomycin (Procell). 6 100 μl of PBMCs at a concentration of cells / ml were seeded into each well of a 96-well cell culture plate. Different concentrations of chimeric anti-human TLR7 antibodies (247C6A2, 13G1F4, 17D1D2, 13H17L, 34G12B8, or human IgG1-LALA control antibody) were added to 80 μl / well for pretreatment at 37°C for 6 hours. Then, 0.5 μM or 1 μM TLR7 agonist GS-9620 (MCE) was added to 20 μl / well, and the plates were thoroughly mixed and incubated at 37°C and 5% CO2 for approximately 20 hours. After thorough mixing, the plates were centrifuged at 1500 rpm for 5 minutes. The concentration of IL-6 in the supernatant was determined by sandwich ELISA (Sinobio); the concentration of IFN-α in the supernatant was determined by sandwich ELISA (Mabtech).

[0316] Figure 8 This study demonstrates that chimeric anti-human TLR7 antibodies inhibit IL-6 production in human PBMCs treated with GS-9620. 247C6A2-c, 13H17L-c, 17D1D2-c, and 34G12B8-c all inhibited IL-6 production in human PBMCs. On the other hand, no inhibition was observed with the human IgG1-LALA control antibody even at a concentration of 5 μg / ml.

[0317] Figure 9 The results showed that the chimeric anti-human TLR7 antibody inhibited IFN-α production in human PBMCs treated with GS-9620. 247C6A2-c, 13H17L-c, 17D1D2-c, and 34G12B8-c all inhibited IFN-α production in human PBMCs. On the other hand, no significant inhibition was observed with the human IgG1-LALA control antibody even at a concentration of 5 μg / ml.

[0318] Human PBMCs were purchased from Shanghai Aoneng Biotechnology Co., Ltd. in frozen form and thawed according to the instructions before use. The solution was adjusted to 2×10⁻⁶ using RPMI 1640 (Gibco) containing 10% FBS (Excell), 1mM sodium pyruvate (Gibco), 0.1mM MEM-Non-Essential Amino Acids (Gibco), 50mM 2-mercaptoethanol (Gibco), 50U / ml penicillin, and 50U / ml streptomycin (Procell). 6 100 μl of PBMCs at a concentration of cells / ml were seeded into each 96-well cell culture plate. Different concentrations of chimeric anti-human TLR7 antibodies (13G1F4, 17D1D2, 13H17L, 34G12B8, or human IgG1-LALA control antibody) were added to each well at 80 μl / well. The plates were then pretreated at 37°C for 6 hours. Then, 20 μl / well was added to... Figure 10-13 Different concentrations of DSR-6434 (MCE) were incubated at 37°C and 5% CO2 for approximately 20 hours after thorough mixing. After thorough mixing, the plates were centrifuged at 1500 rpm for 5 minutes. The concentrations of IL-6, TNF-α, and IP-10 in the supernatant were determined using a sandwich ELISA (Sinobio), a sandwich ELISA (Proteintech), and a sandwich ELISA (Proteintech).

[0319] Figure 10 This study demonstrates that chimeric anti-human TLR7 antibodies inhibit IL-6 production in human PBMCs treated with DSR-6434. 13H17L-c, 17D1D2-c, 13G1F4-c, and 34G12B8-c can inhibit IL-6 production in human PBMCs. On the other hand, no inhibition was observed with the human IgG1-LALA control antibody even at a concentration of 5 μg / ml.

[0320] Figure 11 The results showed that the chimeric anti-human TLR7 antibody inhibited IFN-α production in human PBMCs treated with DSR-6434. 13H17L-c also inhibited IFN-α production in human PBMCs. On the other hand, no inhibition was observed with the human IgG1-LALA control antibody at a concentration of 5 μg / ml.

[0321] Figure 12This study demonstrated that chimeric anti-human TLR7 antibodies inhibited TNF-α production in human PBMCs treated with DSR-6434. 13H17L-c, 17D1D2-c, 13G1F4-c, and 34G12B8-c all inhibited TNF-α production in human PBMCs. On the other hand, no inhibition was observed with the human IgG1-LALA control antibody even at a concentration of 5 μg / ml.

[0322] Figure 13 The chimeric anti-human TLR7 antibody was shown to inhibit IP-10 production in human PBMCs treated with DSR-6434. 13H17L-c, 13G1F4-c, and 34G12B8-c also inhibited IP-10 production in human PBMCs. On the other hand, no inhibition was observed with the human IgG1-LALA control antibody even at a concentration of 1 μg / ml.

[0323] Example 5: Manufacturing of humanized anti-human TLR7 antibody

[0324] 5-1 Humanization Design of Anti-human TLR7 Antibody

[0325] Humanization design of 5-1-1 antibodies

[0326] Structural modeling of the variable region of murine antibodies was performed using The Bioluminate module within the company was used for structural modeling. Based on the RCSB database and according to the resolution of structural resolution and the similarity criteria of the light and heavy chain framework sequences, 4AEH (PDBID) was selected for structural modeling of the variable region of the murine antibody. The templates with the highest homology for 13H17L were IGHV1-2*02 (VH) and IGKV1-27*01 (VL); for 34G12B8, the templates with the highest homology were IGHV4-38-2*02 (VH) and IGKV1-39*01 (VL). Humanization was performed using the CDR grafting method. Receptors with high homology were selected from the human germline database based on the shared sequences defined by IMGT numbers. Donor residues on the receptor were selected and transferred based on information such as the Vernier region, canonical structure, and key amino acid residues at the light and heavy chain interface identified by the Bioluminate module. This was done to maintain the original antibody affinity and function while providing stability. Candidate derivatized sequences were designed using a back mutations strategy, with 10 sequences for 13H17L and 19 sequences for 34G12, for later experiments to test affinity and immunogenicity.

[0327] Humanization of the heavy chain variable region of the 5-1-1-2 13H17L antibody

[0328] (1) The designed heavy chain variable region was named 13H17L-GH1-H0~13H17L-GH1-H10. The amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 in this sequence are the same as those of CDR-H1, CDR-H2 and CDR-H3 of the 13G1F4 antibody.

[0329] Humanization of the light chain variable region of the 5-1-1-3 13H17L antibody

[0330] (2) The designed light chain variable region was named 13H17L-GL1-L0~13H17L-GL1-L9. In this sequence, CDR-L1, CDR-L2 and CDR-L3 are the same as the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 of the 17D1D2 antibody.

[0331] 5-1-1-4 Humanization of the heavy chain variable region of the 34G12B8 antibody

[0332] (3) The designed heavy chain variable region is named 34G12-GH1-H0~34G12-GH1-H12. The amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 in this sequence are the same as those of CDR-H1, CDR-H2 and CDR-H3 of the 34G12B8 antibody.

[0333] Humanization of the variable region of the light chain of the 5-1-1-5 34G12B8 antibody.

[0334] (4) The designed light chain variable region is named 34G12-GL1-L0~34G12-GL1-L3. In this sequence, CDR-L1, CDR-L2 and CDR-L3 are the same as the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 of the 34G12B8 antibody.

[0335] 5-2 Designing Humanized Antibodies by Combining Heavy and Light Chains

[0336] 5-2-1 Design of Humanized Antibody 13H17L Anti-TLR7

[0337] The description describes the combination of light and heavy chains into antibodies, with names and corresponding light and heavy chain names as follows:

[0338] Table 5. Design combinations of humanized antibody 13H17L anti-TLR7 antibodies:

[0339]

[0340]

[0341] 5-2-2 Design of humanized antibody 34G12 anti-TLR7 antibody

[0342] Table 6. Design combinations of humanized antibody 34G12 anti-TLR7 antibodies:

[0343] Antibody name Light chain variable region Heavy chain variable region 34G12-h1 34G12-GL1-L0 34G12-GH1-H0 34G12-h2 34G12-GL1-L0 34G12-GH1-H1 34G12-h3 34G12-GL1-L1 34G12-GH1-H0 34G12-h4 34G12-GL1-L1 34G12-GH1-H1 34G12-h5 34G12-GL1-L2 34G12-GH1-H1 34G12-h6 34G12-GL1-L2 34G12-GH1-H2 34G12-h7 34G12-GL1-L2 34G12-GH1-H3 34G12-h8 34G12-GL1-L2 34G12-GH1-H4 34G12-h9 34G12-GL1-L2 34G12-GH1-H5 34G12-h10 34G12-GL1-L2 34G12-GH1-H6 34G12-h11 34G12-GL1-L2 34G12-GH1-H7 34G12-h12 34G12-GL1-L2 34G12-GH1-H8 34G12-h13 34G12-GL1-L3 34G12-GH1-H8 34G12-h14 34G12-GL1-L3 34G12-GH1-H9 34G12-h15 34G12-GL1-L3 34G12-GH1-H10 34G12-h16 34G12-GL1-L3 34G12-GH1-H11 34G12-h17 34G12-GL1-L3 34G12-GH1-H12 34G12-h18 34G12-GL1-L3 34G12B8-mouse-VH 34G12-h19 34G12B8-mouse-VL 34G12-GH1-H12

[0344] Table 7 Humanization Demonstration of the Framework Area

[0345]

[0346]

[0347] Table 8 Humanization Demonstration of the Framework Area

[0348]

[0349]

[0350] 5-3 Preparation of Humanized Anti-Human TLR7 Antibody

[0351] 5-3-1 Construction of the 13H17L humanized antibody heavy chain expression vector

[0352] A DNA fragment containing a sequence encoding the variable region of the 13H17L-GH1-H0 heavy chain was synthesized. Using Assemblymix (China-US Taihe), the synthesized DNA fragment was inserted into pcDNA3.4-G1, with the insertion position after the signal sequence and before the constant region nucleotide sequence, thus constructing the 13H17L-GH1-H0-huIgG1-LALA heavy chain expression vector. The 13H17L-GH1-H0-huIgG1-LALA heavy chain contains the signal sequence.

[0353] The heavy chain expression vectors 13H17L-GH1-H1 to 13H17L-GH1-H10 were also constructed using the same method described above.

[0354] 5-3-2 Construction of the 13H17L humanized antibody light chain expression vector

[0355] A DNA fragment containing a DNA sequence encoding the variable region of the 13H17L-GL1-L0 light chain was synthesized. Using pcDNA3.4-LK prepared in Example 3-1-1, the 13H17L-GL1-L0-kappa expression vector was constructed using the same method as described above. The light chain of 13H17L-GL1-L0-kappa contains a signal sequence.

[0356] Light chain expression vectors such as 13H17L-GL1-L1 to 13H17L-GL1-L9 were also constructed using the same method.

[0357] 5-3-3 Construction of 34G12 humanized antibody heavy chain expression vector

[0358] A DNA fragment containing a sequence encoding the variable region of the 34G12-GH1-H0 heavy chain was synthesized. Using Assemblymix (China-US Taihe), the synthesized DNA fragment was inserted into pcDNA3.4-G1, with the insertion position after the signal sequence and before the constant region nucleotide sequence, thus constructing the 34G12-GH1-H0-huIgG1-LALA heavy chain expression vector. The 34G12-GH1-H0-huIgG1-LALA heavy chain contains the signal sequence.

[0359] The heavy chain expression vectors of 34G12-GH1-H1 to 34G12-GH1-H12 were also constructed using the same method described above.

[0360] 5-3-4 Construction of 34G12 humanized antibody light chain expression vector

[0361] A DNA fragment containing a DNA sequence encoding the variable region of the 34G12-GL1-L0 light chain was synthesized. Using pcDNA3.4-LK prepared in Example 3-1-1, the 34G12-GL1-L0-kappa expression vector was constructed using the same method as described above. The light chain of 34G12-GL1-L0-kappa contains a signal sequence.

[0362] Light chain expression vectors such as 34G12-GL1-L1 to 34G12-GL1-L3 were also constructed using the same method.

[0363] 5-3-5 Preparation of humanized antibodies

[0364] 5-3-5-1 Manufacturing of Humanized Antibodies

[0365] The heavy and light chains designed in Example 5-2 were combined, and each humanized antibody was manufactured using the expression vectors constructed in 5-3-1 to 5-3-4 in the same manner as in Example 3-2-1.

[0366] 5-3-5-2 Purification of Humanized Antibodies

[0367] The target antibody was purified from the culture supernatants obtained in Example 5-3-5-1 using a series of steps of Protein A affinity chromatography. The culture supernatant was loaded onto a chromatography column filled with Protein A (Yisheng) and equilibrated with binding buffer (Sangon Biotech). The column was then washed twice with 5 column volumes of binding buffer. Elution was then performed with elution buffer (Sangon Biotech) at 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 membrane to further concentrate the antibody. Finally, the purified sample was obtained by filtration using a 0.22 μM disposable syringe filter (PALL).

[0368] Example 6: In vitro activity of humanized anti-human TLR7 antibody

[0369] Evaluation of the binding selectivity of humanized anti-human TLR7 antibodies (6-1)

[0370] 6-1-1 Evaluation of the binding selectivity of chimeric anti-human TLR7 antibodies using flow cytometry

[0371] HEK293T cells overexpressing human Unc93B1-Flag / human TLR7, established in step 1-4-2, were permeabilized with 1× Biolegend. Cells were then serially diluted with humanized 13H17L or 34G12 antibodies obtained in Example 5 and stained with Goat anti-human IgG H&L-FITC (Abcam) as a secondary antibody. The binding selectivity of the antibodies was evaluated using flow cytometry, and mean fluorescence intensity (MFI) was compared.

[0372] HEK293T cells overexpressing human TLR7, established in step 1-3-1, were permeabilized with 1× Biolegend and stained with serially diluted humanized 13H17L or 34G12 antibodies obtained in Example 5, and Goat antihuman IgG H&L-FITC (Abcam) as a secondary antibody. The binding selectivity of the antibodies was evaluated by flow cytometry, and mean fluorescence intensity (MFI) was compared.

[0373] HEK293T cells overexpressing human Unc93B1-Flag / human TLR7 (variant 2) established in step 1-4-4 were permeabilized with 1× Biolegend and stained with serially diluted humanized 13H17L antibody obtained in Example 5 and Goat anti-human IgG H&L-FITC (Abcam) as a secondary antibody. The binding selectivity of the antibodies was evaluated by flow cytometry, and mean fluorescence intensity (MFI) was compared.

[0374] The HEK293T cell line overexpressing cynomolgus macaque TLR7 / Unc93B1, established in 4-1-3, was permeabilized with 1× Biolegend. Cells were then stained with a series of diluted humanized 34G12 antibody concentrations obtained in Example 5 and with Goat anti-human IgG H&L-FITC (Abcam) as a secondary antibody. The antibody binding selectivity was evaluated using flow cytometry, and mean fluorescence intensity (MFI) was compared.

[0375] 1) The 13H17L humanized antibody (13H17L-h1—13H17L h22) specifically binds to HEK293T cells overexpressing human Unc93B1-Flag / human TLR7 (variant 2), and the results are as follows: Figure 14 As shown.

[0376] 2) The 34G12 humanized antibody (34G12-h1—34G12-h19) specifically binds to HEK293T cells overexpressing human Unc93B1-Flag / human TLR7. The results are as follows: Figure 15-16 As shown.

[0377] 3) The 34G12 humanized antibody (34G12-h1—34G12-h19) specifically binds to HEK293T cells overexpressing monkey Unc93B1-Flag / monkey TLR7. The results are as follows: Figure 17 As shown.

[0378] 4) Its EC 50 See Table 9

[0379] Table 9EC 50 Results Display

[0380]

[0381]

[0382] 6-2 Inhibitory effect of humanized anti-human TLR7 antibody on cytokine production

[0383] Human PBMCs were purchased from Shanghai Aoneng Biotechnology Co., Ltd. in frozen form and thawed according to the instructions before use. The solution was adjusted to 2×10⁻⁶ using RPMI 1640 (Gibco) containing 10% FBS (Excell), 1mM sodium pyruvate (Gibco), 0.1mM MEM-Non-Essential Amino Acids (Gibco), 50mM 2-mercaptoethanol (Gibco), 50U / ml penicillin, and 50U / ml streptomycin (Procell). 6 100 μl of PBMCs at a concentration of cells / ml were seeded into each 96-well cell culture plate. Different concentrations of humanized anti-human TLR7 antibody (13H17L antibody, 34G12 antibody, or human IgG1-LALA control antibody) were added to 80 μl / well, and the plates were pretreated at 37°C for 6 hours. Then, 1 μM GS-9620 (MCE) or 3 nM DSR-6434 (MCE) was added to 20 μl / well, and the plates were thoroughly mixed and incubated at 37°C and 5% CO2 for approximately 20 hours. After thorough mixing, the plates were centrifuged at 1500 rpm for 5 minutes. The concentration of IL-6 in the supernatant was measured using a sandwich ELISA (Sinobio); the concentration of TNF-α in the supernatant was also measured using a sandwich ELISA (Sinobio).

[0384] 1) such as Figure 18 The humanized 13H17L antibodies (13H17L-h1—13H17L-h22) effectively inhibited IL-6 production in human PBMCs treated with GS-9620. On the other hand, no inhibition was observed with the human IgG1-LALA control antibody.

[0385] 2) such as Figures 19-20 The 34G12 humanized antibody (34G12-h1—34G12-h19) effectively inhibited the production of IL-6 and TNFα in human PBMCs treated with GS-9620. On the other hand, no inhibition was observed with the human IgG1-LALA control antibody.

[0386] Example 7: In vivo activity of humanized anti-human TLR7 antibody

[0387] Validation of the activity of 7-1 humanized anti-human TLR7 antibody in CD34+ humanized mice

[0388] 7-1-1CD34+ humanized mouse modeling

[0389] Four-week-old female NSG-SGM3 mice were irradiated with 100 cGy. After irradiation, the mice were returned to new cages and injected via the tail vein with 100 μL of hu-CD34+ cells (1*10e5 / mouse) within 24 hours. Bactrim water was continuously administered post-irradiation. Blood samples were collected after cell seeding for flow cytometry analysis to determine PBMC reconstitution efficiency, as needed for the experimental procedures.

[0390] 7-1-2 Validation of the activity of humanized anti-human TLR7 antibody in CD34+ humanized mice

[0391] The experiment involved in vivo injection of antibodies and TLR7 agonists. Mice were first divided into groups: 9-week-old (5 weeks after human CD34+ cell injection) female NSG-SGM3 mice were divided into 5 groups of 2-4 mice each. As described in Table 10, mice in group G3 received a tail vein injection of 10 mg / kg of 34G12-h14 antibody. Overnight (18 hours), group G1 received PBS, while groups G2 and G3 received a tail vein injection of 0.01 mg / kg of the TLR7 agonist DSR-6434. Two hours later, 100 μl of blood was collected from each mouse, centrifuged at 5000 rpm for 5 minutes, and the supernatant was collected. Serum IL-6 was detected using an ELISA kit (human IL-6 kit), with the mouse serum diluted 4-fold before use.

[0392] Table 10 Grouping Information

[0393]

[0394] Experimental results are as follows Figure 21 As shown, the hybridoma-selected TLR7 antibody effectively inhibited the release of the inflammatory cytokine IL-6 induced by the TLR7 agonist DSR-6434 in CD34+ mice, preliminarily verifying that the hybridoma-selected TLR7 antibody is active in vivo.

[0395] Example 8: Preparation of Anti-human TLR7 Affinity Maturation Antibody

[0396] Affinity maturation design of 8-1 antibodies

[0397] The three-dimensional structure of antibody-antigen complexes was simulated using AlphaFold2 or its derivative models. By analyzing the molecular interactions at the binding interface, key amino acid residues that potentially affect binding affinity were identified. A single-point mutant library was constructed by rationally designing these key amino acid residues. Selective binding ability screening was performed using overexpression cell lines, and positive mutants with higher affinity and improved function were selected through in vitro functional experiments. These selected positive single-point mutations were combined to construct multiple mutants. Affinity and function verification of these multiple mutants was performed, ultimately obtaining the target antibody sequence with optimized performance. 8-1-2 Affinity maturation of the variable region of the 34G12 antibody heavy chain.

[0398] Affinity maturation of the light and heavy chain variable regions of 8-1-2-1 34G12-h8 and 34G12-h14 antibodies

[0399] Based on binding and functional experiments, it was determined that humanized antibodies 34G12-h8 and 34G12-h14 would require further optimization. After multiple rounds of single-point mutation verification for affinity maturation, it was determined that the LC mutation H90S and the HC mutation A98V+N104W+M109N+Y101R+G31Y would significantly improve affinity. The parent antibody for affinity maturation based on h14 was designated 34G12-h458 (shown in Tables 11-12 and 15), and the parent antibody for affinity maturation based on h8 was designated 34G12-h542 (shown in Tables 13-14 and 16). Further affinity maturation was then performed on these two parent antibodies.

[0400] Affinity maturation of the light and heavy chain variable regions of 8-1-2-2 34G12-h458 and 34G12-h542 antibodies

[0401] As shown in Tables 11-16, several variants with significantly enhanced affinity were obtained by site-directed mutagenesis of the parental antibodies 34G12-h458 and 34G12-h542. The mutation sites in Tables 11-16 are described with reference to the light and heavy chain sequences of the parent antibody; for example, 34G12-h458 references the sequence of 34G12-h14, and 34G12-h477 references the sequence of 34G12-h458. The heavy chain variable region sequence of 34G12-h458 is shown in SEQ ID NO: 172, and the light chain variable region sequence is shown in SEQ ID NO: 173. The heavy chain variable region sequence of 34G12-h8 is shown in SEQ ID NO: 180, and the light chain variable region sequence is shown in SEQ ID NO: 181. The heavy chain variable region sequence of 34G12-h542 is shown in SEQ ID NO: 174, and the light chain variable region sequence is shown in SEQ ID NO: 175. The heavy chain variable region sequence of 34G12-h14 is shown in SEQ ID NO: 182, and the light chain variable region sequence is shown in SEQ ID NO: 183.

[0402] 1) CDR mutations based on h458: 34G12-h477, 34G12-h483, 34G12-h493-34G12-h500, and 34G12-h544 are affinity maturation antibodies that undergo single-point or combined mutations based on 34G12-h458, with the aim of improving affinity or drug-likeness.

[0403] 2) CDR mutations 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, and 34G12-h601 are affinity maturation antibodies that have undergone single-point or combined mutations based on 34G12-h542, with the aim of improving antibody affinity or drug-likeness.

[0404] 3) FR mutations based on h458: 34G12-h512, 34G12-h513, 34G12-h522-34G12-h528, and 34G12-h541 are point mutations based on 34G12-h458, which aim to improve antibody affinity or drug-likeness. 34G12-h529-34G12-h535 are reversion mutations based on 34G12-h458, which aim to reduce reversion mutation sites and improve drug-likeness.

[0405] 4) FR mutations based on h542: 34G12-h545, 34G12-h546, 34G12-h548, 34G12-h554, 34G12-h555, 34G12-h576, and 34G12-h583 are point mutations based on 34G12-h542, the purpose of which is to improve antibody affinity or drug-likeness. 34G12-h577-34G12-h580 and 34G12-h583-34G12-h586 are reversion mutations based on 34G12-h542, the purpose of which is to reduce reversion mutation sites and improve drug-likeness.

[0406] 5) CDR+FR mutations based on h458: 34G12-h465, 34G12-h480, 34G12-h482, 34G12-h484-34G12-h489, and 34G12-h491 are point mutations performed on the basis of 34G12-h458, with the aim of improving affinity or drug-likeness.

[0407] 6) CDR+FR mutations based on h542: 34G12-h559, 34G12-h564-34G12-h567, 34G12-h598, and 34G12-h600 are point mutations performed on the basis of 34G12-h542, with the aim of improving affinity or drug-likeness.

[0408] The binding affinity of the mature antibody mutants is similar to or higher than that of the parent antibody, and both can inhibit the production of IL-6 and TNF-α in human PBMCs treated with GS-9620. Exemplary results can be found in Examples 9-10.

[0409] Table 11. Explanation of CDR region mutations in 34G12-h458

[0410]

[0411] Table 12. Explanation of CDR region mutations in the 34G12-h458 affinity maturation-related mutation sequence.

[0412]

[0413] Table 13, Explanation of CDR region mutations in 34G12-h542

[0414]

[0415]

[0416] Table 14. Explanation of CDR region mutations in the 34G12-h542 affinity maturation-related mutation sequence.

[0417]

[0418]

[0419] Table 15. Explanation of frame region mutations in 34G12-h458 and related mutation sequences.

[0420]

[0421] Table 16. Explanation of frame region mutations in 34G12-h542 and related mutation sequences.

[0422]

[0423]

[0424] Preparation of 8-2 Anti-human TLR7 Affinity Maturation Antibody

[0425] 8-2-1 Construction of 34G12 affinity maturation antibody light and heavy chain expression vector

[0426] This invention employs site-directed mutagenesis technology, designing specific primers targeting the coding sequence of the variable region on a plasmid template containing the antibody gene. Single or multiple amino acid site mutations are introduced via high-fidelity PCR amplification to obtain mature mutants with antibody affinity. After the PCR product is digested with DpnI enzyme to remove the template plasmid, it is transformed into competent cells for cloning and screening, ultimately successfully constructing a series of point mutant plasmids.

[0427] 8-2-2 Preparation of anti-human TLR7 affinity maturation antibody

[0428] 8-2-2-1 Manufacturing of Anti-human TLR7 Affinity Maturation Antibody

[0429] The heavy and light chains designed in Example 8-1 were combined, and anti-human TLR7 affinity maturation antibodies were manufactured using the expression vectors constructed in Example 8-2-1 through the same method as in Example 3-2-1.

[0430] Purification of anti-human TLR7 affinity maturation antibody (8-2-2-2)

[0431] The target antibody was purified from the culture supernatants obtained in Example 8-2-2-1 using a series of steps of Protein A affinity chromatography. The culture supernatant was loaded onto a chromatography column filled with Protein A (Yisheng) and equilibrated with binding buffer (Sangon Biotech). The column was then washed twice with 5 column volumes of binding buffer. Elution was then performed with elution buffer (Sangon Biotech) at 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 membrane to further concentrate the antibody. Finally, the purified sample was obtained by filtration using a 0.22 μM disposable syringe filter (PALL).

[0432] Example 9: In vitro activity of anti-human TLR7 affinity maturation antibody

[0433] 9-1 Evaluation of the binding selectivity of anti-human TLR7 affinity maturation antibodies using flow cytometry

[0434] HEK293T cells overexpressing human Unc93B1-Flag / human TLR7, established in step 1-4-2, were permeabilized with 1× Biolegend permeabilization agent. Cells were then stained with serially diluted anti-human TLR7 affinity maturation antibody obtained in Example 8 and Goat anti-human IgG H&L-FITC (Abcam) as a secondary antibody. The binding selectivity of the antibodies was evaluated using flow cytometry, and mean fluorescence intensity (MFI) was compared.

[0435] Exemplary results are as follows Figure 22-23 As shown, all anti-human TLR7 affinity maturation antibodies specifically bound to this cell. After affinity maturation, the maximum fluorescence value (Emax) of 34G12-h458 and 34G12-h542 antibodies binding to the target antigen significantly increased, and their EC50 values ​​also increased. 50 The Emax value is shown in Table 17.

[0436] Table 17 EC 50 Display of Emax value results

[0437] Ab 34G12-h542 34G12-h458 34G12-h8 34G12-h14 <![CDATA[EC 50 (nM)]]> 0.8463 0.8749 0.505 0.4803 Emax 47992.35 43851.9 29441.55 30522.65 Ab 34G12-h587 34G12-h588 34G12-h589 34G12-h542 <![CDATA[EC 50 (nM)]]> 0.6753 0.6838 0.739 0.5853 Emax 69932.4 61286.6 49015.5 48727.3

[0438] 9-2 Inhibitory effect of anti-human TLR7 affinity maturation antibody on cytokine production

[0439] Human PBMCs were purchased from Shanghai Aoneng Biotechnology Co., Ltd. in frozen form and thawed according to the instructions before use. Three healthy donor PBMCs were used in this study to validate antibody efficacy. The antibody efficacy was adjusted to 2×10⁻⁶ 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). 6 100 μl of PBMCs at a concentration of cells / ml were seeded into each 96-well cell culture plate. Different concentrations of all antibodies used as maturation antibodies against human TLR7 affinity or human IgG1-LALA control antibody (Baiying) were added to each well at 80 μl / well for pretreatment at 37°C for 6 hours. Then, 1 μM GS-9620 (MCE) was added to each well at 20 μl / well, and the plates were thoroughly mixed and incubated at 37°C and 5% CO2 for approximately 20 hours. After thorough mixing, the plates were centrifuged at 1500 rpm for 5 minutes. The concentration of IL-6 in the supernatant was determined by sandwich ELISA (Sinobio); the concentration of TNF-α in the supernatant was also determined by sandwich ELISA (Sinobio).

[0440] like Figure 24-26 The anti-human TLR7 affinity maturation antibodies shown inhibited IL-6 production in human PBMCs from three different donors treated with GS-9620. All anti-human TLR7 affinity maturation antibodies inhibited IL-6 production in human PBMCs. On the other hand, no significant inhibition was observed with the human IgG1-LALA control antibody.

[0441] As shown in the figure Figure 24-26 The anti-human TLR7 affinity maturation antibodies shown inhibited TNF-α production in human PBMCs from three different donors treated with GS-9620. All anti-human TLR7 affinity maturation antibodies inhibited TNF-α production in human PBMCs. On the other hand, no significant inhibition was observed with the human IgG1-LALA control antibody.

[0442] 9-3 Anti-human TLR7 affinity maturation antibody inhibits HEK-Blue TM Expression of downstream signaling of the hTLR7 reporter gene in cells

[0443] HEK-Blue TMThe hTLR7 reporter cell line was purchased from Invivogen. Cells were resuscitated according to the manufacturer's instructions before experiments. Cells were cultured in DMEM (GibcoBASIC DMEM, High Glucose) containing 10% FBS (Hyclone), 100 U / ml penicillin, and 100 U / ml streptomycin (Procell). After the cells reached optimal condition, they were harvested and the cell density was adjusted to 2.5E+06 / ml using HEK-Blue assay medium. 160 μL of cell suspension was added to each well of a 96-well plate. 20 μL of HEK-Blue assay medium was added to each well after diluting the antibody (starting concentration 6 μg / mL, diluted 3.16 times in 8 steps, 2 replicates per well). The plates were incubated for 4 hours. 20 μL of GS-9620 (final concentration 1 μM, diluted with HEK-Blue assay medium) was added to each well. 20 μL of HEK-Blue assay medium was used as a negative control. Transfer the 96-well plate to a 37°C, 5% CO2 incubator and incubate for 12-16 hours. Use a microplate reader to detect the OD value at a wavelength of 620 nm.

[0444] The results are as follows Figure 27-28 As shown, 1 μM GS-9620 stimulates HEK-Blue TM After hTLR7 reporter cells were added, the addition of an anti-human TLR7 affinity maturation antibody significantly inhibited the expression of its downstream signals.

[0445] 9-4 Anti-human TLR7 affinity maturation antibody inhibits plasma cell formation

[0446] Healthy human PBMCs were purchased in frozen form from Shanghai Aoneng Biotechnology Co., Ltd., and SLE patient PBMCs were purchased in frozen form from Shanghai Junxing Biotechnology Co., Ltd. After thawing according to the instructions, they were used. The concentration of RPMI 1640 (Gibco), containing 10% FBS (Hyclone), 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 1.25 × 10⁻⁶. 6 800 μl of PBMCs at a concentration of 1 cell / ml were seeded into each 24-well cell culture plate. Different concentrations of anti-human TLR7 affinity maturation antibody were added to each well at 100 μl. Then, different concentrations of GS-9620 (MCE) were added to each well at 100 μl. After thorough mixing, the cells were cultured at 37°C and 5% CO2 for approximately 6 days. Cells were then collected and plasma cell IgD levels were analyzed by flow cytometry. - CD27 h CD38 hThe proportion of ).

[0447] like Figures 29-31 As shown, 1 μM GS-9620 stimulation of PBMCs can detect the formation of plasma cells. The addition of 5 μg / ml anti-human TLR7 affinity maturation antibody can significantly reduce the proportion of plasma cells, but hIgG1-LALA does not reduce the proportion of plasma cells, indicating that anti-human TLR7 affinity maturation antibody can significantly inhibit the formation of plasma cells.

[0448] Example 10: In vivo activity of anti-human TLR7 affinity maturation antibody

[0449] Validation of the activity of 10-1 anti-human TLR7 affinity maturation antibody in CD34+ humanized mice

[0450] 10-1-1CD34+ humanized mice

[0451] Female genO-BRGSF-HIS4 mice were purchased from Shanghai Kinuowei Biotechnology Co., Ltd. for this experiment.

[0452] Validation of the activity of 10-1-2 anti-human TLR7 affinity maturation antibody in CD34+ humanized mice

[0453] Mice were randomly divided into two groups. On the first day, group 1 mice were untreated, while group 2 mice were injected with 5 mg / kg of 34G12-h458 antibody via the tail vein. After 18 hours, group 1 mice were injected with PBS, and group 2 mice were injected with 0.01 mg / kg of TLR7 agonist DSR-6434 via the tail vein. Two hours later, 100 μl of blood was collected from each mouse, centrifuged at 5000 rpm for 5 min, and the supernatant was collected. The mouse serum IL-6 was detected using an ELISA kit (human IL-6 kit), and the mouse serum was used after being diluted 4 times.

[0454] like Figure 32 As shown, the anti-human TLR7 affinity maturation antibody 34G12-h458 effectively inhibited the release of the inflammatory cytokine IL-6 induced by the TLR7 agonist DSR-6434 in CD34+ mice, preliminarily verifying that the anti-human TLR7 affinity maturation antibody 34G12-h458 is active in vivo.

[0455] Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made to the embodiments of the present invention by those skilled in the art without departing from the spirit and essence of the invention, and such modifications or substitutions should all be within the scope of the present invention. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should also be covered within the protection scope of the present invention.

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 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; and / or 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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