WO2020259667A1 - Use of combination of tlr agonist and anti-ox40 antibody or antigen binding fragment thereof in preparation of medicament for treating tumors - Google Patents

Abstract

Disclosed is the use of a combination of a TLR agonist and an anti-OX40 antibody or antigen binding fragment thereof in the preparation of the medicament for treating tumors. Specifically, the TLR agonist is the compound represented by formula (I) or a pharmaceutically acceptable salt thereof.

Description

Application of TLR agonist combined with anti-OX40 antibody or antigen-binding fragment thereof in preparing medicine for treating tumor Technical field

The present disclosure relates to the use of a TLR agonist combined with an anti-OX40 antibody or an antigen-binding fragment thereof in the preparation of a medicine for preventing or treating tumors.

Background technique

Today's human society has long faced severe health challenges. However, traditional therapies such as surgery, chemotherapy and radiotherapy often have little effect in the treatment of solid tumors that have spread. Tumor immunotherapy is a continuing hot spot in the field of tumor treatment. Recent studies have shown that enhancing the function of anti-tumor T cells can be used to fight cancer. There is a large amount of evidence that tumor cells “evade” the immune system by inducing active immune tolerance mediated mainly by regulatory T lymphocytes (Treg; Quezda et al. Immunol Rev 2011; 241:104-118). Therefore, the balance between effector T lymphocytes (Teff) and tolerogenic Treg is essential for effective anti-tumor immunotherapy. Therefore, an effective anti-tumor immune response can be obtained by enhancing the effector function of tumor-specific Teff and/or by reducing the inhibitory function of tumor-specific Treg. The CD134 (OX40) receptor has been shown to be a key receptor that mediates these responses (Sugamura, K, Ishii, N, Weinberg, A. Therapeutic targeting of the effector T-cell co-stimulatory molecule OX40. Nature Rev Imm 2004; 4:420-431).

Toll-like receptors (TLRs) are an important class of protein molecules involved in innate immunity. TLRs are monomeric transmembrane non-catalytic receptors, usually expressed in sentinel cells such as macrophages and dendritic cells, and can recognize structurally conserved molecules produced by microorganisms. Once these microorganisms break through physical barriers such as the skin or intestinal mucosa, they will be recognized by TLRs and then activate immune cell responses. (Mahla, RS. et al., Front Immunol. 4:248 (2013)). The ability of the immune system to widely recognize pathogenic microorganisms is partly due to the widespread existence of Toll-like immune receptors.

There are at least 10 different TLRs in mammals. Some ligands for these receptors and the corresponding signal cascades have been identified. Many diseases and obstacles are related to abnormalities of TLRs, such as melanoma, non-small cell lung cancer, hepatocellular carcinoma, basal cell carcinoma (basalcellcarcinoma), renal cell carcinoma, myeloma, allergic rhinitis, asthma, chronic obstructive pneumonia (COPD) ), ulcerative colitis, liver fibrosis, HBV, Flaviviridae virus, HCV, HPV, RSV, SARS, HIV or influenza virus infection.

TLR7 is a member of the subgroup of TLRs (TLRs 3, 7, 8 and 9) and is limited to the endosomal compartment of cells that specialize in detecting non-hex nucleic acids. TLR7 plays a key role in antiviral defense by recognizing ssRNA (Diebold S.S. et al., Science, 2004:303, 1529-1531; and Lund J.M. et al., PNAS, 2004: 101, 5598-5603). Currently, the TLR7 inhibitors that have been marketed are mainly used for topical administration, such as imiquimod for the treatment of condyloma acuminatum.

WO2018095426A provides a TLR7 agonist, the structure is as follows:

OX40 (also known as CD134) is a member of the tumor necrosis factor receptor (TNFR) superfamily. It is a glycoprotein with a molecular weight of about 50kDa expressed on the cell surface. The extracellular ligand binding domain of OX40 is composed of 4 cysteine-rich domains (CRD). The natural ligand of OX40 is OX40L (CD252), and 3 OX40 correspond to the ligands that bind to the trimer to form an OX40-OX40L complex.

OX40 is mainly expressed on activated T cells, and OX40 is a secondary co-stimulatory molecule, which is expressed after 24-72 hours after activation. The OX40 ligand OX40L is mainly expressed on activated antigen presenting cells. T lymphocytes expressing OX40 have been confirmed to exist in the draining lymph nodes of various human malignant tumors and cancer patients. In a mouse model of severe combined immunodeficiency (SCID), the interaction of OX40 and OX40L binding domains can enhance anti-tumor immunity, resulting in various human malignant tumor cell lines, such as lymphoma, prostate cancer, colon cancer and breast cancer tumors Growth inhibition.

At present, many international pharmaceutical companies are developing monoclonal antibodies against OX40, which specifically stimulate immune activation, improve the patient's own immune system response to tumors, and achieve the purpose of killing tumor cells. Related patents such as WO2013038191, WO2015153513, WO2016179517, WO2017096182 and so on. So far, the anti-OX40 antibodies developed by companies such as AstraZeneca and BMS have been in phase II clinical trials, and related products of companies such as Genentech and GSK are also in clinical trials.

WO2017021912A discloses a combination of an antigen binding protein that binds to OX40 and a TRL7/8 modulator or a TRL4 modulator. Specific examples disclose the experimental data of the anti-OX40 antibody OX86 and TRL4 agonist in the CT-26 model, However, the combination of OX40 antibody and TRL7/8 modulator is not effective.

Summary of the invention

The present disclosure provides a use of a combination of a TLR agonist and an anti-OX40 antibody or an antigen-binding fragment thereof in the preparation of a drug for preventing or treating tumors.

The TLR agonists provided in the present disclosure can be selected from TLR1 agonists, TLR2 agonists, TLR3 agonists, TLR4 agonists, TLR5 agonists, TLR6 agonists, TLR7 agonists, TLR8 agonists, TLR9 agonists, preferably TLR7 agonists or TLR9 agonists, most preferably TLR7 agonists.

The TLR agonists provided by the present disclosure can be selected from Heplisav, resiquimod, SD-101, Dynavax, DV-281, imiquimod, cobitolimod, entolimod, lemonolimod, Poly-ICLC, Grass MATA MPL, G-100, AST-008, GSK-1795091 , Tilsotolimod, KMRC-011, CMB-305, rintatolimod, AZD-1419, influenza-PAL, SAR-439794, MIS-416, MGN-1601, GSK-2245035, VTX-1463, motolimod, GS-9688, LHC-165 , BDB-001, PGV-001, AV-7909, DSP-0509, DPX-E7, RG-7854, telratolimod, vesatolimod, poly-ICLC adjuvanted vaccines, MVAME-03, Riboxxim, G-305, PUL-042, litenimod , DRibbles vaccine, TMX-202, ISA-201, PEPA-10, CpG-ODN(K3), AL-034, Vaxart, CBLB-612, Pseudomonas aeruginosa, IR-103, VAX-161, VAX-125, MEDI19197 or The compound represented by formula (I) or its complex or pharmaceutically acceptable salt thereof, preferably the TLR7 agonist imiquimod, GSK-2245035, LHC-165, DSP-0509, RG-7854, vesatolimod, TMX-202, AL-034 or The compound represented by formula (I) or its complex or its pharmaceutically acceptable salt,

The anti-OX40 antibody or antigen-binding fragment thereof described in the present disclosure is selected from MEDI6469, PF-04518600, KHK4083, BMS986178, MEDI0562, MOXR0916, MEDI6383, INCAGN01949.

The anti-OX40 antibody described in the present disclosure specifically binds to human OX40, and contains the CDRs shown below: heavy chain HCDR1, HCDR2, HCDR3 shown in the amino acid sequence of SEQ ID NO: 1, 2, and 3, or with SEQ ID The HCDR1, HCDR2, and HCDR3 shown in NO: 1, 2, and 3 respectively have HCDR variants with 3, 2 or 1 amino acid difference; and the light chain LCDR1 shown in the amino acid sequence of SEQ ID NO: 6, 7, and 8 respectively , LCDR2, LCDR3 or LCDR1, LCDR2, LCDR3 shown in SEQ ID NO: 6, 7, 8 with 3, 2 or 1 amino acid difference, respectively, preferably containing SEQ ID NO: 3, 4, 5 The heavy chain HCDR1, HCDR2, HCDR3 shown in the amino acid sequence and the light chain LCDR1, LCDR2, LCDR3 shown in the amino acid sequence of SEQ ID NO: 6, 7, and 8, respectively.

The above-mentioned CDR sequences are shown in the following table:

name	sequence	Numbering
HCDR1	RYSVH	SEQID NO: 3
HCDR2	MIWDGGNTDYNSALKS	SEQID NO: 4
HCDR3	NPLYFSYAMDY	SEQID NO: 5
LCDR1	RASQDISNYLN	SEQID NO: 6
LCDR2	YTSRLQS	SEQID NO: 7
LCDR3	QQVNTFPFT	SEQID NO: 8

In some embodiments, the CDR (including 3 heavy chain CDRs and 3 light chain CDRs) of the monoclonal antibody or antigen-binding fragment has 3, 2 or 1 amino acid difference CDR variants are screened by affinity maturation methods. Obtained CDR variants with 3, 2 or 1 amino acid difference.

In a preferred embodiment, the heavy chain HCDR2 variant of the OX40 antibody is shown in the amino acid sequence of SEQID NO: 12.

name	sequence	Numbering
HCDR2 V1	MIWDGGNTDYNAALKS	SEQID NO: 12

The anti-OX40 antibody described in the present disclosure is a murine antibody, a chimeric antibody or a humanized antibody, preferably a humanized antibody.

In one embodiment, the humanized antibody comprises the heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 11 or has at least 95% sequence identity with the heavy chain variable region, preferably comprising the amino acid sequence shown in SEQ ID NO: 11 The variable region of the heavy chain shown in the sequence.

In one embodiment, the anti-OX40 antibody or antigen-binding fragment thereof comprises: the light chain variable region shown in the amino acid sequence of SEQ ID NO: 10 or having at least 95% sequence identity with it, preferably comprising the light chain variable region shown in SEQ ID NO: 10 ID NO: The variable region of the light chain shown in the 10 amino acid sequence.

In one embodiment, the humanized antibody includes the heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 11 and the light chain variable region shown in the amino acid sequence of SEQ ID NO: 10.

In one embodiment, the anti-OX40 antibody includes a constant region; preferably, the antibody is a chimeric antibody or a humanized antibody, and the heavy chain constant region of the antibody is derived from human IgG1, IgG2, and IgG3 or IgG4 or its mutant sequence. The light chain constant region is derived from human κ, λ chain or its mutant sequence. Preferably, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO: 13 or has at least 95% sequence thereof Identity, the amino acid sequence of the constant region of the light chain is shown in SEQ ID NO: 14 or has at least 95% sequence identity with it, and most preferably the amino acid sequence of the constant region of the heavy chain is shown in SEQ ID NO: 13, The amino acid sequence of the light chain constant region is shown in SEQ ID NO: 14.

IgG1 heavy chain constant region:

Constant region of kappa light chain:

In a preferred embodiment, the anti-OX40 antibody comprises: the heavy chain amino acid sequence is shown in SEQ ID NO: 15 or has at least 85% sequence identity with it and the light chain amino acid sequence is shown in SEQ ID NO: 16 or It has at least 85% sequence identity; preferably, the heavy chain amino acid sequence is shown in SEQ ID NO: 15 and the light chain amino acid sequence is shown in SEQ ID NO: 16.

Heavy chain:

Light chain:

In some embodiments, TLR agonists can be used with a uniform dose or a weight-based dose. In other embodiments, the TLR agonist is administered as a uniform dose. In some embodiments, the TLR agonist is administered as a weight-based dose. In order to administer a TLR agonist, the dosage may be within the following range of 0.1-1000 μg/kg, 0.5-500 μg/kg, 1-500 μg/kg; specifically, it may be 0.10 μg/kg, 0.20 μg/kg, 0.30 μg/kg, 0.40μg/kg, 0.50μg/kg, 0.60μg/kg, 0.70μg/kg, 0.80μg/kg, 0.90μg/kg, 1.00μg/kg, 5μg/kg, 10μg/kg, 15μg/kg, 20μg/kg , 25μg/kg, 30μg/kg, 35μg/kg, 40μg/kg, 45μg/kg, 50μg/kg, 55μg/kg, 60μg/kg, 65μg/kg, 70μg/kg, 75μg/kg, 80μg/kg, 85μg /kg, 90μg/kg, 95μg/kg, 100μg/kg, 105μg/kg, 110μg/kg, 115μg/kg, 120μg/kg, 125μg/kg, 130μg/kg, 135μg/kg, 140μg/kg, 145μg/kg , 150μg/kg, 155μg/kg, 160μg/kg, 165μg/kg, 170μg/kg, 175μg/kg, 180μg/kg, 185μg/kg, 190μg/kg, 195μg/kg, 200μg/kg, 205μg/kg, 210μg /kg, 215μg/kg, 220μg/kg, 225μg/kg, 230μg/kg, 235μg/kg, 240μg/kg, 245μg/kg, 250μg/kg, 255μg/kg, 260μg/kg, 265μg/kg, 270μg/kg , 275μg/kg, 280μg/kg, 285μg/kg, 290μg/kg, 295μg/kg, 300μg/kg, 305μg/kg, 310μg/kg, 315μg/kg, 320μg/kg, 325μg/kg, 330μg/kg, 335μg /kg, 340μg/kg, 345μg/kg, 350μg/kg, 355μg/kg, 360μg/kg, 365μg/kg, 370μg/kg, 375μg/kg, 380μg/kg, 385μg/kg, 390μg/kg, 395μg/kg , 400μg/kg, 405μg/kg, 410μg/kg, 415μg/kg, 420μg/kg, 425μg/kg, 430μg/kg, 435μg/kg, 440μg/kg, 445μg/kg, 450μg/kg, 455μg/kg, 460μg /kg, 465μg/kg, 470μg/kg, 475μg/k g, 480μg/kg, 485μg/kg, 490μg/kg, 495μg/kg, 500μg/kg; or for the administration of TLR agonists, the dosage may be within the following range of 0.001-100mg, 0.01-50mg, 0.1-20mg, 0.1 -10mg, 0.1-5mg, for example: 0.001mg, 0.01mg, 0.02mg, 0.03mg, 0.04mg, 0.05mg, 0.05, 0.06mg, 0.07mg, 0.08mg, 0.09mg, 0.10mg, 0.15mg, 0.20mg, 0.25mg, 0.30mg, 0.35mg, 0.40mg, 0.45mg, 0.50mg, 0.55mg, 0.60mg, 0.65mg, 0.70mg, 0.75mg, 0.80mg, 0.85mg, 0.90mg, 0.95mg, 1.00mg, 1.05mg , 1.10mg, 1.15mg, 1.20mg, 1.25mg, 1.30mg, 1.35mg, 1.40mg, 1.45mg, 1.50mg, 1.55mg, 1.60mg, 1.65mg, 1.70mg, 1.75mg, 1.80mg, 1.85mg, 1.90 mg, 1.95mg, 2.00mg, 2.05mg, 2.10mg, 2.15mg, 2.20mg, 2.25mg, 2.30mg, 2.35mg, 2.40mg, 2.45mg, 2.50mg, 2.55mg, 2.60mg, 2.65mg, 2.70mg, 2.75mg, 2.80mg, 2.85mg, 2.90mg, 2.95mg, 3.00mg, 3.05mg, 3.10mg, 3.15mg, 3.20mg, 3.25mg, 3.30mg, 3.35mg, 3.40mg, 3.45mg, 3.50mg, 3.55mg , 3.60mg, 3.65mg, 3.70mg, 3.75mg, 3.80mg, 3.85mg, 3.90mg, 3.95mg, 4.00mg, 4.05mg, 4.10mg, 4.15mg, 4.20mg, 4.25mg, 4.30mg, 4.35mg, 4.40 mg, 4.45mg, 4.50mg, 4.55mg, 4.60mg, 4.65mg, 4.70mg, 4.75mg, 4.80mg, 4.85mg, 4.90mg, 4.95mg, 5.00mg, 5.50mg, 6.00mg, 6.50mg, 7.00mg, 7.50mg, 8.00mg, 8.50mg, 9.00mg, 9.50mg, 10.00mg, 10.50mg, 11.00mg, 11.50mg, 12.00mg, 12.50m g, 13.00mg, 13.50mg, 14.00mg, 14.50mg, 15.00mg, 15.50mg, 16.00mg, 16.50mg, 17.00mg, 17.50mg, 18.00mg, 18.50mg, 19.00mg, 19.50mg, 20.00mg, 20.50mg, 21.00mg, 21.50mg, 22.00mg, 22.50mg, 23.00mg, 23.50mg, 24.00mg, 24.50mg, 25.00mg, 25.50mg, 26.00mg, 26.50mg, 27.00mg, 27.50mg, 28.00mg, 28.50mg, 29.00mg , 29.50mg, 30.00mg, 30.50mg, 31.00mg, 31.50mg, 32.00mg, 32.50mg, 33.00mg, 33.50mg, 34.00mg, 34.50mg, 35.00mg, 35.50mg, 36.00mg, 36.50mg, 37.00mg, 37.50 mg, 38.00mg, 38.50mg, 39.00mg, 39.50mg, 40.00mg, 40.50mg, 41.00mg, 41.50mg, 42.00mg, 42.50mg, 43.00mg, 43.50mg, 44.00mg, 44.50mg, 45.00mg, 45.50mg, 46.00mg, 46.50mg, 47.00mg, 47.50mg, 48.00mg, 48.50mg, 49.00mg, 49.50mg, 50.00mg.

In an alternative embodiment, the dose of the TLR agonist is selected from 0.10 mg, 0.15 mg, 0.20 mg, 0.25 mg, 0.30 mg, 0.35 mg, 0.40 mg, 0.45 mg, 0.50 mg, 0.55 mg, 0.60 mg, 0.65 mg, 0.70mg, 0.75mg, 0.80mg, 0.85mg, 0.90mg, 0.95mg, 1.0mg, 2.0mg, 3.0mg, 4.0mg, 5.0mg, 6.0mg, 7.0mg, 8.0mg, 9.0mg, 10mg, 11mg, 12mg , 13mg, 14mg, 15mg, 16mg, 17mg, 18mg, 19mg, 20mg, 25mg, 30mg, 35mg, 40mg, 45mg, 50mg.

In an alternative embodiment, the dose of the TLR agonist is selected from 0.5 mg, 1.0 mg, 2.0 mg, 3.0 mg, 4.0 mg, 5.0 mg, 6.0 mg, 7.0 mg, 8.0 mg, 9.0 mg, 10 mg, 11 mg, 12 mg, 13mg, 14mg, 15mg.

In an alternative embodiment, the dose of the TLR agonist is selected from 0.5 mg, 1 mg, 2.0 mg, 3 mg, 4.0 mg, 6.0 mg, 8 mg, 9 mg, and 12 mg.

In the present disclosure, the frequency of administration of TLR agonists is 3 times a day, 2 times a day, once a day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, weekly Once, once every 2 weeks, once every 3 weeks, once every 4 weeks; preferably once a week, once every 2 weeks.

In an alternative embodiment, the frequency of administration of the TLR agonist is once a week.

In an alternative embodiment, the frequency of TLR agonist administration is once every 2 weeks.

In an alternative embodiment, the frequency of administration of the TLR agonist is once every 3 days.

In certain embodiments, the combined use provided by the present disclosure can be used with a uniform dose or a weight-based dose. In some embodiments, the anti-OX40 antibody or antigen binding portion thereof is administered as a uniform dose. In some embodiments, the anti-OX40 antibody or antigen binding portion thereof is administered as a weight-based dose. In order to administer the anti-OX40 antibody, the dosage may be in the following range: 0.01-10.0 mg/kg, 0.1-5 mg/kg, 0.1-2 mg/kg, for example, the dosage may be 0.01 mg/kg, 0.02 mg/kg, 0.03 mg /kg, 0.04mg/kg, 0.05mg/kg, 0.06mg/kg, 0.07mg/kg, 0.08mg/kg, 0.09mg/kg, 0.1mg/kg, 0.2mg/kg, 0.3mg/kg, 0.4mg /kg, 0.5mg/kg, 0.6mg/kg, 0.7mg/kg, 0.8mg/kg, 0.9mg/kg, 1.0mg/kg, 1.2mg/kg, 1.4mg/kg, 1.6mg/kg, 1.8mg /kg, 2.0mg/kg, 2.2mg/kg, 2.4mg/kg, 2.6mg/kg, 2.8mg/kg, 3.0mg/kg, 3.2mg/kg, 3.4mg/kg, 3.6mg/kg, 3.8mg /kg, 4.0mg/kg, 4.2mg/kg, 4.4mg/kg, 4.6mg/kg, 4.8mg/kg, 5.0mg/kg, 5.2mg/kg, 5.4mg/kg, 5.6mg/kg, 5.8mg /kg, 6.0mg/kg, 6.2mg/kg, 6.4mg/kg, 6.6mg/kg, 6.8mg/kg, 7.0mg/kg, 7.2mg/kg, 7.4mg/kg, 7.6mg/kg, 7.8mg /kg, 8.0mg/kg, 8.2mg/kg, 8.4mg/kg, 8.6mg/kg, 8.8mg/kg, 9.0mg/kg, 9.2mg/kg, 9.4mg/kg, 9.6mg/kg, 9.8mg /kg, 10.0mg/kg; in order to administer the OX40 antibody, the dose can also be within the following range 1-1000mg, 1-500mg, 1-300mg, 1-200mg, specifically 1mg, 2mg, 3mg, 4mg, 5mg, 6mg, 7mg, 8mg, 9mg, 10mg, 11mg, 12mg, 13mg, 14mg, 15mg, 16mg, 17mg, 18mg, 19mg, 20mg, 21mg, 22mg, 23mg, 24mg, 25mg, 26mg, 27mg, 28mg, 29mg, 30mg, 31mg, 32mg, 33mg, 34mg, 35mg, 36mg, 37mg, 38mg, 39mg, 40mg, 41mg, 42mg, 43mg, 44mg, 45mg, 46mg, 47mg, 48mg, 49mg, 50mg, 50mg, 60mg, 70mg, 80mg, 90mg, 100mg, 110mg, 120mg, 1 30mg, 140mg, 150mg, 160mg, 170mg, 180mg, 190mg, 200mg, 200mg, 225mg, 250mg, 275mg, 300mg, 325mg, 350mg, 375mg, 400mg, 425mg, 450mg, 475mg, 500mg.

In an alternative embodiment, the dose of the anti-OX40 antibody is selected from 0.03 mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8mg/kg, 0.9mg/kg, 1.0mg/kg, 1.2mg/kg, 1.4mg/kg, 1.6mg/kg, 1.8mg/kg, 2.0mg/kg, 2.2mg/kg, 2.4 mg/kg, 2.6mg/kg, 2.8mg/kg, 3.0mg/kg, 3.2mg/kg, 3.4mg/kg, 3.6mg/kg, 3.8mg/kg, 4.0mg/kg, 4.2mg/kg, 4.4 mg/kg, 4.6mg/kg, 4.8mg/kg, 5.0mg/kg.

In an alternative embodiment, the anti-OX40 antibody dose is selected from 0.03 mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 1 mg/kg, 3 mg/kg , 5mg/kg.

In an alternative embodiment, the anti-OX40 antibody dose is selected from 0.03 mg/kg.

In an alternative embodiment, the anti-OX40 antibody dose is selected from 0.1 mg/kg.

In an alternative embodiment, the anti-OX40 antibody dose is selected from 0.3 mg/kg.

In an alternative embodiment, the anti-OX40 antibody dose is selected from 1 mg/kg.

In an alternative embodiment, the anti-OX40 antibody dose is selected from 3 mg/kg.

In an alternative embodiment, the anti-OX40 antibody dose is selected from 5 mg/kg.

The frequency of administration of anti-OX-40 antibody in the present disclosure is 3 times a week, 2 times a week, once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once a month, every 3 times. -6 months once, preferably 3 times a week or once every 4 weeks, most preferably once every 3 weeks.

The tumor described in the present disclosure is selected from squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma Cancer, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer , Endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, soft tissue sarcoma, neuroblastoma, penile cancer, melanoma, superficial spreading melanoma Tumors, lentigo melanoma, acral melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma, chronic lymphocytic leukemia (CLL), non-Hodgkin’s lymphoma (NHL) ), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, post-transplant lymphoproliferative disorder (PTLD), and phakomatoses, edema (such as associated with brain tumors) The abnormal blood vessel proliferation, brain tumor, brain cancer, and head and neck cancer related to Meigs syndrome, preferably colon cancer and non-Hodgkin’s lymphoma.

The present disclosure provides a use of a compound represented by formula (I) or a complex or a pharmaceutically acceptable salt thereof in combination with the above-mentioned anti-OX40 antibody or antigen-binding fragment in the preparation of a medicine for preventing or treating tumors. The tumor is preferably colon cancer.

The pharmaceutically acceptable salts of TLR agonists in the present disclosure can be hydrochloride, phosphate, hydrogen phosphate, sulfate, hydrogen sulfate, sulfite, acetate, oxalate, malonate, valeric acid Salt, glutamate, oleate, palmitate, stearate, laurate, borate, p-toluenesulfonate, methanesulfonate, isethionate, maleate , Malate, tartrate, benzoate, pamoate, salicylate, vanillate, mandelate, succinate, gluconate, lactobionate or lauryl sulfonate, etc. , Maleate and hydrochloride are preferred, and dihydrochloride is most preferred.

The present disclosure provides a method for treating tumors, comprising administering to a patient a therapeutically effective amount of the above-mentioned TLR agonist and a therapeutically effective amount of the above-mentioned anti-OX40 antibody or antigen-binding fragment.

The present disclosure provides a method for treating colon cancer, which comprises administering to a patient a therapeutically effective amount of compound dihydrochloride represented by formula (I) and a therapeutically effective amount of the OX40 antibody or antigen-binding fragment.

The administration route of the anti-OX40 antibody or antigen-binding fragment in the present disclosure includes intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral administration routes, such as by injection or infusion. The "parenteral administration" refers to administration modes other than enteral and local administration by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, Intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, intralesional, And electroporation in vivo. In certain embodiments, the anti-OX40 antibody or antigen-binding fragment is administered by a non-parenteral route, in certain embodiments, orally. Other non-parenteral routes include topical, epidermal or mucosal routes of administration, for example, intranasal, vaginal, rectal, sublingual, or topical.

The route of administration of TLR agonists in the present disclosure may be the same as or different from the above-mentioned immune checkpoint inhibitors, specifically including oral, nasal, topical, intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, intralesional or other gastric Parenteral administration route, the administration route of the TLR agonist in the present disclosure is preferably intratumoral administration in the lesion.

The present disclosure provides a method for treating tumors, comprising administering to a patient a therapeutically effective amount of the above-mentioned TLR agonist and a therapeutically effective amount of the above-mentioned anti-OX40 antibody or antigen-binding fragment.

The present disclosure provides a method for treating tumors, which includes intratumorally administering to a patient a therapeutically effective amount of the above-mentioned TLR agonist and tumor or intravenously administering to the patient a therapeutically effective amount of the above-mentioned anti-OX40 antibody or antigen-binding fragment.

The present disclosure provides a method for treating tumors, which comprises intratumorally administering a therapeutically effective amount of compound dihydrochloride of formula (I) to a patient and intravenously administering a therapeutically effective amount of the above-mentioned OX40 antibody or antigen-binding fragment to the patient.

The anti-OX40 antibody or antigen-binding fragment of the present disclosure can be constituted in a composition, for example, a pharmaceutical composition containing an antibody and a pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" as used herein includes any and all solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic agents and absorption delaying agents that are physiologically compatible. In one embodiment, the carrier for the antibody-containing composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, intraperitoneal, spinal or epidermal administration (e.g., by injection or infusion), the pharmaceutical composition of the present disclosure may include One or more pharmaceutically acceptable salts, antioxidants, aqueous and non-aqueous carriers, and/or adjuvants such as preservatives, wetting agents, emulsifiers and dispersants.

The combination of the TLR agonist provided by the present disclosure and the anti-OX40 antibody or the antigen-binding fragment thereof has unexpected remote effects.

the term

In order to make the present disclosure easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have meanings commonly understood by those of ordinary skill in the art to which this disclosure belongs.

The use of the term "uniform dose" refers to the dose administered to a patient regardless of the patient's weight or body surface area (BSA). For example, a 60 kg person and a 100 kg person will receive the same dose of antibody (e.g., 240 mg of anti-OX40 antibody).

The term "weight-based dose" mentioned herein refers to the dose calculated based on the weight of the patient and administered to the patient. For example: 10.0mg/kg means that 10.0mg per kg is administered based on the body weight of the subject.

The "combination" described in the present disclosure is a mode of administration, which means that at least an anti-OX40 antibody and a TLR agonist are administered within a certain period of time, and both drugs show pharmacological effects. The time limit may be within one administration cycle, preferably within 4 weeks, within 3 weeks, within 2 weeks, within 1 week, or within 24 hours. The anti-OX40 antibody and TLR agonist can be administered simultaneously or sequentially. This period includes treatments in which the anti-OX40 antibody and the TLR agonist are administered via the same route of administration or different routes of administration.

The terms "anti-OX40 antibody" and "antibody that binds to OX40" refer to antibodies that can bind to OX40 with sufficient affinity so that the antibody can be used as a diagnostic and/or therapeutic agent for targeting OX40.

The term "antibody" described in the present disclosure refers to an immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the immunoglobulin heavy chain are different, and their antigenicity is also different. According to this, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are μ chain, δ chain, and γ chain respectively. , Α chain and ε chain. The same type of Ig can be divided into different subclasses according to the difference in the amino acid composition of the hinge region and the number and position of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chain is divided into κ chain or λ chain by the difference of the constant region. Each of the five Ig types can have κ chain or λ chain. The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (such as bispecific antibodies), and antibody fragments, as long as they exhibit The desired antigen binding activity can be obtained.

In the present disclosure, the antibody light chain variable region described in the present disclosure may further comprise a light chain constant region, and the light chain constant region comprises human or murine κ, λ chains or variants thereof, preferably κ constant Area.

In the present disclosure, the antibody heavy chain variable region of the present disclosure may further comprise a heavy chain constant region, and the heavy chain constant region comprises human or murine IgG1, IgG2, IgG3, IgG4 or variants thereof, The IgG1 constant region is preferred.

The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly and is the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant region (C region). The variable region includes 3 hypervariable regions (HVR) and 4 framework regions (FR) with relatively conservative sequences. Three hypervariable regions determine the specificity of the antibody, also known as complementarity determining regions (CDR). Each light chain variable region (VL) and heavy chain variable region (VH) is composed of 3 CDR regions and 4 FR regions. The sequence from the amino terminal to the carboxy terminal is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2 and HCDR3. The number and position of the CDR amino acid residues of the LCVR region and the HCVR region of the antibody or antigen-binding fragment described in the present disclosure comply with the known Kabat numbering rules.

The antibodies of the present disclosure include murine antibodies, chimeric antibodies, and humanized antibodies, preferably humanized antibodies.

The term "antibody framework (FR)" as used herein refers to a part of the variable domain VL or VH, which serves as a scaffold for the antigen binding loop (CDR) of the variable domain. Essentially, it is a variable domain without CDRs.

The term "amino acid difference" refers to the difference between a polypeptide and its variants, in a certain or certain amino acid positions on the polypeptide fragment, wherein the variants can be replaced, inserted or inserted into certain positions or positions on the polypeptide. Obtained by missing amino acids.

The "mutated sequence" mentioned in the present disclosure means that the nucleotide sequence and amino acid sequence of the present disclosure are different from the nucleotide sequence and amino acid sequence of the present disclosure under the condition that the nucleotide sequence and amino acid sequence of the present disclosure are modified by mutation, such as insertion or deletion. Nucleotide sequence and amino acid sequence in percent sequence identity. The sequence identity described in the present disclosure may be at least 85%, 90% or 95%, non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%. Sequence comparison and determination of the percent identity between the two sequences can be performed through the default settings of the BLASTN/BLASTP algorithm available on the National Center For Biotechnology Institute website.

"Homology" and "identity" herein refer to the sequence similarity between two polynucleotide sequences or between two polypeptides. When positions in two comparison sequences are occupied by the same base or amino acid monomer subunit, for example, if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position . The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100. For example, in the best sequence alignment, if there are 6 matches or homology in 10 positions in the two sequences, then the two sequences are 60% homologous; if there are 95 matches in 100 positions in the two sequences Or homology, then the two sequences are 95% homologous. Generally speaking, the comparison is made when two sequences are aligned to obtain the maximum percent homology.

Description of the drawings

Figure 1. Growth curve of tumor tissue on the right side after treatment;

Figure 2. Growth curve of left tumor tissue after treatment;

Figure 3. Body weight change curve of tumor-bearing mice during treatment with test compound.

Detailed ways

The following examples are used to further describe the disclosure, but these examples do not limit the scope of the disclosure.

Example 1. Preparation of anti-OX40 antibody (drug A)

1. Preparation of antibodies

The experimental methods without specific conditions in the examples of the present disclosure usually follow conventional conditions, such as Cold Spring Harbor's antibody technology experimental manual, molecular cloning manual; or according to the conditions recommended by the raw material or commodity manufacturer. Reagents without specific sources are conventional reagents purchased on the market.

The anti-human OX40 monoclonal antibody library is generated by immunizing mice. Experimental mice of BalB/C and A/J strains (Comparative Medicine Center of Yangzhou University, Animal Production License Number: SCXK (苏)2017-007), female, 10 weeks old.

The immune antigen is human OX40 recombinant protein with Fc tag (OX40-Fc: OX40 Leu 29-Ala 216 (Accession#NP_003318) fused with Fc), purchased from Acro Biosystems, catalog number #OX40-H5255, expressed in HEK293 as usual Method purification. Emulsify OX40-Fc with Freund's adjuvant: Freund's complete adjuvant (sigma-aldrich, F5881-10ML) for the first immunization, and Freund's incomplete adjuvant (sigma-aldrich, F5506-10ML) for the remaining booster immunization. The ratio of antigen to adjuvant is 1:1, and 25μg protein/200μl/mouse is injected per immunization. See Table 1 below for details.

Table 1.

Day 1	The first immunization, complete Freund's adjuvant.
Day 21	The second immunization, incomplete Freund's adjuvant.
Day 35	The third immunization, incomplete Freund's adjuvant.
Day 42	Blood sampling and serum titer testing
Day 49	The fourth immunization, incomplete Freund's adjuvant.
Day 56	Blood sampling and serum titer testing

The mouse serum was detected by the following ELISA method to determine the antibody titer of the mouse serum and the neutralizing activity of blocking OX40/OX40L binding. The mice with strong serum titer, affinity and ligand binding blocking ability were selected for a final immunization and then sacrificed. Take spleen cells and SP2/0 myeloma cells (

CRL-1581 ^TM ) fusion and plating to obtain hybridomas, the target hybridomas are selected by the following indirect ELISA, capture ELISA, and cell-based functional screening, and monoclonal antibodies are established by the limiting dilution method.

The 19 established strains of OX40 mouse monoclonal antibodies were produced by serum-free expression, and purified mouse monoclonal antibodies were obtained by protein A affinity chromatography technology. Through indirect ELISA, capture ELISA and cell functional activity screening of hybridoma cells secreting activated anti-OX40 antibody. The brief steps of functional screening are as follows: culture GS-H2/OX40 stable cell line (purchased from genscript, cat#M00608). Prepare diluted test antibody and OX40L (Sino Biological, 13127-H04H) solution and add them to GS-H2/OX40 cells in the logarithmic growth phase. After culture, the cell supernatant is collected and the IL-8 content in the supernatant is determined ( Use human IL-8 kit, cisbio, cat#62IL8PEB).

According to cell functional activity, 10 strains with high activity were selected for gene cloning and sequencing. The total cell RNA can be obtained by conventional RNA extraction technology, and then the PCR product of the variable region of the monoclonal antibody can be obtained by reverse transcription polymerase chain reaction (RT-PCR). The PCR products were separated and recovered by agarose gel, then cloned into gene vector and transformed into E. coli. Several transformed colonies were randomly selected, and the variable regions of monoclonal antibodies were amplified by PCR for gene sequencing. The corresponding sequence of the obtained exemplary murine monoclonal antibody is shown below.

The heavy and light variable region sequences of the murine monoclonal antibody m2G3 are as follows:

m2G3 heavy chain variable region:

m2G3 light chain variable region:

The sequence of the CDR region of the mouse monoclonal antibody m2G3 is shown in Table 2:

Table 2. CDR region sequence of mouse monoclonal antibody m2G3

name	sequence	Numbering
HCDR1	RYSVH	SEQID NO: 3
HCDR2	MIWDGGNTDYNSALKS	SEQID NO: 4
HCDR3	NPLYFSYAMDY	SEQID NO: 5
LCDR1	RASQDISNYLN	SEQID NO: 6
LCDR2	YTSRLQS	SEQID NO: 7
LCDR3	QQVNTFPFT	SEQID NO: 8

2. ELISA identification and screening methods of antibodies

2.1 Indirect ELISA method:

Dilute 20× coating buffer to 1× with deionized water, and prepare antigen human OX40-His (Acro biosytems, OXL-H52Q8) with 1× coating solution (carbonate buffer) to make the final concentration 2μg /mL, add 100μL per well, and incubate at 4℃ overnight or 37℃ for 2h. The plate was washed once with PBST, 200μL of blocking solution (PBST containing 5% skim milk) was added to each well, incubated at 37°C for 2h, and the plate was washed 4 times with PBST. Add 100μL of the diluted primary antibody to each well (the concentration of the antibody to be tested is 5 times diluted from 10000ng/ml, 7 gradients, namely 10000ng/ml, 2000ng/ml, 400ng/ml, 80ng/ml, 16ng/ml, 3.2 ng/ml, 0.64ng/ml, blank wells are pure diluent (2.5% skimmed milk in PBST), incubate at 37°C for 40 minutes. Wash the plate 4 times with PBST, dilute the enzyme-labeled secondary antibody (HRP-labeled goat anti-mouse IgG, purchased from Jackson Immunoresearch, Cat#115036071 or HRP-labeled goat anti-human IgG, purchased from Jackson Immunoresearch, Cat#109036098) with PBST buffer, Add 100μL to each well and incubate at 37°C for 40min. Wash the plate 4 times with PBST, add 100μL TMB chromogenic solution to each well, incubate at room temperature and dark for 3-15 minutes, add 50μl stop solution (1M sulfuric acid) to each well. Set the parameters of the microplate reader, read the OD value at 450-630nm, and save the experimental data.

2.2 Capture ELISA:

Dilute 20×PBS buffer with deionized water to 1×, prepare GAM secondary antibody (Jackson Immunoresearch, 115-006-071) with 1×PBS to make the final concentration 2μg/mL, add 100μL to each well, 4℃ Incubate overnight or 37°C for 2h; wash the plate once with PBST, add 200μL blocking solution (PBST containing 5% skim milk) to each well, incubate at 37°C for 2h, wash the plate with PBST 4 times. Add 100μL of the diluted primary antibody to each well (the concentration of the antibody to be tested is 5 times diluted from 10000ng/ml, 7 gradients, namely 10000ng/ml, 2000ng/ml, 400ng/ml, 80ng/ml, 16ng/ml, 3.2 ng/ml, 0.64ng/ml, blank wells are pure diluent (2.5% skimmed milk in PBST), incubate at 37°C for 40 minutes. Wash the plate 4 times with PBST, dilute human OX40-FC-biotin (Acro biosystem, OX0-H5255, labeled Biotin) with 2.5% skim milk in PBST, add 100 μl to each well, incubate at 37°C for 40 min, wash the plate 4 times with PBST. Add 100μL TMB color developing solution to each well, incubate at room temperature for 3-15 minutes, add 50μl stop solution (1M sulfuric acid) to each well, set the microplate reader parameters, read the OD value at 450-630nm, and save the experimental data.

2.3 Ligand binding blocking ELISA:

Dilute 20× coating buffer to 1× with deionized water, and prepare antigen OX40L-His (Acro biosytems, OXL-H52Q8) with 1× coating solution (carbonate buffer) to make the final concentration 2μg/ mL, add 100μL to each well and incubate at 4°C overnight or incubate at 37°C for 2h; wash the plate once with PBST; add 200μL blocking solution (PBST containing 5% skim milk) to each well and incubate at 37°C for 2h; wash the plate with PBST 4 times; use The pre-prepared 200ng/ml human OX40-Fc solution (prepared in 2.5% skimmed milk) was serially diluted with mouse serum/antibody, and then pre-incubated at room temperature for 40 minutes, then added to the blocked OX40L plate, 100μL/ Incubate wells for 40 minutes; wash the plate with PBST 4 times; dilute HRP-labeled goat anti-human secondary antibody (GAH-HRP, Jackson Immunoresearch, 109-035-006) with PBST buffer, add 100 μl to each well, incubate at 37°C for 40 minutes; PBST Wash the plate 4 times; add 100μL TMB color developing solution to each well, and incubate at room temperature for 3-15 minutes; add 50μl stop solution (1M sulfuric acid) to each well, set the microplate reader parameters, read the OD value at 450-630nm, and save Experimental data.

3. Construction and expression of anti-OX40 recombinant chimeric antibody

The variable region of the heavy chain (VH) of the murine antibody m2G3 in the present disclosure plus the constant region of the human immunoglobulin heavy chain, and the variable region of the light chain (VL) plus the constant region of the human immunoglobulin Kappa light chain, respectively Cloned into a eukaryotic expression vector and transfected cells to produce mouse-human chimeric antibodies. The heavy chain vector is designed as follows: signal peptide + heavy chain variable region sequence + human IgG1 constant region sequence. The light chain vector is designed as follows: signal peptide + light chain variable region sequence + human Kappa constant region sequence. The above sequences were inserted into pCEP4 vector (Thermofisher, V04450). After the vector plasmid is obtained, the plasmid is drawn out, and the plasmid is sent to sequencing for verification. The qualified plasmid was transfected into human 293F cells with PEI and cultured continuously, and the 293F cells were cultured with serum-free medium (Shanghai Optima Biotech, OPM-293CD03) to the logarithmic growth phase for cell transfection. Dissolve 21.4μg of chimeric antibody light chain plasmid and 23.6μg of chimeric antibody heavy chain plasmid in 10ml

Mix well in I Reduced Serum Medium (GIBCO, 31985-070), then add 200μg PEI, mix well, incubate at room temperature for 15min, and add to 50mL cells. Cell culture conditions: 5% CO ₂ , 37°C, 125 rpm/min. During the culture period, feed was added on the 1st and 3rd day until the cell viability was less than 70%, and the cell supernatant was collected and centrifuged. Load the cell culture fluid after centrifugation and filtration on the antibody purification affinity column, wash the column with phosphate buffer, elution with glycine hydrochloric acid buffer (pH 2.7 0.1M Gly-HCl), neutralize with 1M Tris hydrochloric acid pH 9.0, and After dialysis with phosphate buffer, purified chimeric antibody Ch2G3 was finally obtained.

4. In vitro binding affinity and kinetic experiments

The mouse antibody and chimeric antibody were tested for the affinity of human OX40 (the method steps are the same as the ELISA identification and screening method of 2 antibodies). Among them, m2G3-NC and ch2G3-NC are negative controls. The results show that the chimeric antibody Ch2G3 has high affinity with human OX40.

5. Anti-OX40 antibody in vitro cell reporter gene experiment

Coat the well plate with anti-CD3 antibody (Chempartner, A05-001), place overnight at 4 degrees, and wash 3 times with PBS; harvest Jurkat-NF-Kb luc-hOX40 cells (ATCC, TIB-152 (stable cell line from Shanghai Ruizhi) Chemical construction)) and Raji cells (ATCC, CCL-86), resuspend and mix the two cells. Add 50μl/well of diluted antibody to be tested and 50μl/well of the mixed two kinds of cells to the cell plate, and incubate the cell plate in a 37°C 5% CO ₂ incubator for 5 hours. Add 100 μl one-Glo TM luciferase reagent (Promega, Cat#E6120) to each well, and incubate at room temperature for more than 3 minutes. Test the luminescence signal on the computer and record the RLU reading. The EC50 (nM) of the Ch2G3 antibody is 0.6371, showing that the chimeric antibody Ch2G3 effectively activates the reporter gene.

6. Mouse antibody humanization experiment

In order to reduce the possible immunogenicity, the murine antibody was humanized. The heavy chain variable region (VH) and the light chain variable region (VL) of the chimeric antibody were respectively used for site-specific amino acid mutations in the FR (framework region) region. According to different combinations of amino acid mutations, different humanized antibody weights were designed. Humanized antibodies can be produced by transfecting cells with plasmids that combine different light and heavy chains. The brief description is as follows. First design the expression vector: the heavy chain vector is designed as follows: signal peptide + mutant heavy chain variable region sequence + human IgG1 constant region sequence. The light chain vector is designed as follows: signal peptide + mutated light chain variable region sequence + human Kappa constant region sequence. The above sequences were inserted into pCEP4 vector (Thermofisher, V04450). Ask a third-party gene synthesis company to synthesize the expression vector according to the above design, and after obtaining the vector plasmid, the plasmid will be large-scaled and sent to sequence verification. The qualified plasmid was transfected into human 293F cells with PEI and cultured continuously, and the 293F cells were cultured with serum-free medium (Shanghai Optima Biotech, OPM-293CD03) to the logarithmic growth phase for cell transfection. Dissolve 21.4μg of humanized antibody light chain plasmid and 23.6μl of humanized antibody heavy chain plasmid in 10ml

Mix well in I Reduced Serum Medium (GIBCO, 31985-070), then add 200μg PEI, mix well, incubate at room temperature for 15min, and add to 50mL cells. Cell culture conditions: 5% CO ₂ , 37°C, 125 rpm/min. During the culture period, feed was added on the 1st and 3rd day until the cell viability was less than 70%, and the cell supernatant was collected and centrifuged. Load the cell culture fluid after centrifugation and filtration on the antibody purification affinity column, wash the column with phosphate buffer, elution with glycine hydrochloride buffer (pH 2.7 0.1M Gly-HCl), neutralize with 1M Tris hydrochloric acid pH 9.0 And phosphate buffer dialysis to obtain purified humanized antibody finally.

The sequence of the humanized variable region is as follows:

>hu2G3 VH1

>hu2G3 VL1

Note: The sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the italic in the sequence is the FR sequence, and the underline is the CDR sequence.

In order to obtain a better humanized 2G3 antibody, the amino acid sequence of hu2G3VH1 was mutated, and the heavy chain variable region sequence after mutation is as follows:

hu2G3 VH1.1

The sequence of HCDR2 obtained after mutation is as follows:

Table 3. HCDR2 sequence obtained after mutation

name	sequence	Numbering
HCDR2 V1	MIWDGGNTDYNAALKS	SEQID NO: 12

The heavy chain variable region hu2G3 VH1.1 and the light chain variable region hu2G3 VL1 are combined to form a new optimized humanized antibody, see Table 4.

Table 4. New humanized 2G3 antibodies obtained after mutation

Antibody	VH	VL
2G3	hu2G3 VH1.1	hu2G3 VL1

The aforementioned humanized antibody was subjected to affinity evaluation (refer to the capture ELISA in Example 2), and the experimental results showed that the humanized molecule can bind to OX40.

The light chain variable region and the light chain constant region sequence (for example: shown in SEQ ID NO: 14) are combined to form the final light chain sequence, and each heavy chain variable region and the heavy chain constant region (for example, SEQ ID NO: 13) combined to form the final heavy chain sequence. The specific light and heavy chain constant regions are not limited to the constant regions of the antibodies of the present disclosure, and other light and heavy chain constant regions and their mutants known in the art can also be used to increase the performance of the antibody. An exemplary constant region is as follows:

IgG1 heavy chain constant region:

Constant region of kappa light chain:

The full-length amino acid sequence of an exemplary 2G3 antibody is as follows:

2G3 heavy chain:

2G3 light chain:

7. In vitro binding affinity and kinetic test of humanized antibody

The Biacore method is a recognized method for objectively detecting mutual affinity and kinetics of proteins. We use Biacore T200 (GE) to analyze the OX40 antibody to be tested in the present disclosure to characterize the affinity and binding kinetics.

Using the kit provided by Biacore, the recombinant anti-OX40 antibody to be tested of the present disclosure was covalently connected to the CM5 (GE) chip using the NHS standard amino coupling method. Then, a series of concentration gradients of human OX40-His protein (Yiqiao Shenzhou #10481-H08H) diluted in the same buffer were injected into each cycle at a flow rate of 30 μL/min. After injection, all were regenerated in the kit Reagent regeneration. Track the antigen-antibody binding kinetics for 3 minutes and track the dissociation kinetics for 10 minutes. Using GE's BIAevaluation software to analyze the obtained data with a 1:1 (Langmuir) binding model, the chimeric antibody ka (kon), kd (koff) and K _D values determined by this method are shown in Table 5 below.

Table 5. In vitro binding affinity results of humanized antibodies

Antibody to be tested	ka(1/Ms)	kd(1/s)	K D (M)
2G3	4.371E+05	2.023E-03	4.628E-09

The results show that 2G3 can effectively bind OX40.

8. Antibodies block OX40 and OX40L binding ELISA test

After covering the plate with the ligand OX40 ligand (Acrobiosystem, OXL-H52Q8), add the antibody to be tested in a gradient dilution (antibody is diluted in a solution containing human Bio-OX40-FC (Acrobiosystem, OX40-H5255, labeled Biotin) and pre-incubated for 40 minutes After adding the plate), after incubating for 40 minutes, wash the plate. Then add SA-HRP (Jackson Immunoresearch, 016-030-084), after incubating for 40 minutes, add the color developing solution and stop solution, and measure the OD value. The results are shown in Table 6 below.

Table 6. Experimental results of antibody blocking the binding of OX40 and OX40L

Antibody	2G3
IC50(nM)	1.88

The results show that 2G3 can block the binding of OX40 to OX40L.

Humanized antibody activity data

Coat the plate with anti-CD3 antibody (Chempartner, A05-001), place overnight at 4°C, and wash 3 times with PBS; harvest Jurkat-NF-Kb luc-hOX40 cells (ATCC, TIB-152 (stabilized cell line by Ruizhi Chemical Company) Build) and Raji cells (ATCC, CCL-86), resuspend and mix the two cells. Add 50μl/well of the diluted antibody to be tested, and 50μl/well of the mixed two cells to the plates, and place the cell plate at 37 degrees Incubate for 5 hours in a 5% CO ₂ incubator. Add one-Glo TM luciferase reagent (Promega, Cat#E6120) to each well and incubate for more than 3 minutes at room temperature. Test the luminescence signal on the computer and record the RLU reading. The results are shown below. Table, the results show that 2G3 can effectively activate the reporter gene.

Table 7. Experimental results of humanized antibody activation of reporter gene

Antibody	2G3
EC50(nM)	2.294

10. In vitro cell function test

Isolate CD4+ memory T cells, add the antibody to be tested to a 96-well plate coated with anti-CD3 antibody (Chempartner, A05-001), incubate at 37°C for 72 hours, take the supernatant to detect IFN-γ, the result is shown in Figure 2. . GPX4 and 2G3 can significantly enhance the release of IFN-γ, and 2G3 can achieve the maximum stimulation effect at 10ng/mL.

11. Anti-OX40 antibody inhibits tumor cell growth

B-hTNFRSF4 (OX40) humanized mouse (B-hTNFRSF4 (OX40) humanized mouse, Biocytogen Jiangsu Gene Biotechnology Co., Ltd.), female, 17-20g, 6-7 weeks. Collect logarithmic growth phase MC38 tumor cells 7 (purchased from Nanjing Yinhe Biomedicine Co., Ltd.), adjust the cell concentration to 5×10 ⁶ /mL with PBS buffer, and inoculate 0.1 mL of cell suspension on the flanks of OX40 mice. Observe the mice after inoculation and monitor the growth of tumors. On the 7th day after inoculation, the average tumor volume on the flank of the tumor-bearing mice reached 102.5mm ^3. Grouping and drug administration observations were made according to the tumor size. The specific grouping information is as follows:

Table 8. Mice grouping and dosage table

G	Dosage (mg/kg)	Route of administration	Dosing frequency
Vehicle(IgG1)	3	i.p.	Q3D×6
GPX4	3	i.p.	Q3D×6
2G3	0.3	i.p.	Q3D×6
2G3	1	i.p.	Q3D×6
2G3	3	i.p.	Q3D×6

To detect the growth inhibitory effect of (OX40) humanized mouse MC38 colon cancer cell transplantation tumor. Measurement of tumor volume and weight of tumor-bearing mice: using vernier calipers to measure twice a week, the calculation formula of tumor volume is V = 0.5axb ² , a and b represent the long diameter and wide diameter of the tumor respectively; tumor growth transplanted tumor TGI (%) = [1-T/C]×100. The body weight of all tumor-bearing mice was measured twice a week.

On the 20th day after the treatment, the average tumor volume of the Vehicle (IgG1) control group reached 1732.593 mm ³ , and the average tumor volume of the mice in the low-dose administration group (0.3 mg/kg) of the test compound 2G3 reached 930.37 mm ³ . The average tumor volume of tumor-bearing mice in the middle-dose administration group (2G3 1mg/kg) and the high-dose administration group (2G3 3mg/kg) were 303.49mm ³ and 155.79mm ³ , respectively. The middle and high-dose groups inhibited tumor growth compared with the control group The difference was obvious (**P<0.01), and showed a preliminary dose-dependent relationship. The tumor growth inhibition rate reached 49%, 88% and 97.0%, respectively. The average tumor volume of GPX4 3mg/kg tumor-bearing mice was 362.47mm ³ , which was significantly different from the Vehicle group. It also showed significant tumor growth inhibition (*P<0.05), and its tumor growth inhibition rate reached 84. %

The experiment was ended on the 20th day after administration. The results are shown in Table 13 and Figure 3. All the treated mice were euthanized, and the subcutaneous transplanted tumor mass of the tumor-bearing mice was stripped and weighed. The vehicle group had an average tumor mass weight of 1.568g, and the test compound 2G3 in the low-dose group (0.3mg/kg), medium-dose group (1mg/kg) and high-dose group (3mg/kg), the average tumor weight was 0.926, respectively g, 0.251g and 0.181g, among which the high-dose group and the control group are significantly different, and the tumor growth inhibition effect is obvious (**P<0.01). At the same time, the average tumor weight of GPX4 3mg/kg in the administration group was 0.372g, which was significantly different from that in the Vehicle group, and also showed a significant inhibitory effect on the growth of MC38 tumor cells (**P<0.01).

During the experiment, there was no obvious abnormality in the body weight of the treated tumor-bearing mice, and no obvious abnormal behavior and other manifestations were observed during the drug treatment.

Table 9. Anti-tumor effect in mice

Example 2. Preclinical evaluation of anti-OX40 antibody (drug A) and compound dihydrochloride (drug B) represented by formula (I) and single or combined drugs on the growth of hOX40 transgenic mice MC38 colon cancer xenografts .

1. Experimental materials

1) Tumor cells

MC38 tumor cells were purchased from Nanjing Yinhe Biomedical Co., Ltd. The cells were cultured in RPMI1640 medium containing 10% fetal bovine serum, and the cells were digested and passaged with EDTA-containing trypsin as usual, passaged twice a week, and placed in a 37°C, 5% CO ₂ incubator for continued cultivation. Tumor cells in the logarithmic growth phase are used for the establishment of transplanted tumor models in vivo.

2) Experimental animals

75 humanized B-hTNFRSF4 (OX40) mice, female, 4-5 weeks, weighing 14-19g, provided by Biocytogen Jiangsu Gene Biotechnology Co., Ltd.

Laboratory animal breeding

All the mice were raised in an SPF animal room IVC constant temperature and pressure system, where the temperature was 20-26°C, the humidity was 40-70%, and the light cycle was 12 hours bright and 12 hours dark. 6 mice were raised in each cage, the size of the cage was 325mmx210mmx180mm, and the litter in the cage was changed twice a week.

3) Test sample

Drug A: The preparation method is as in Example 1, specifically 2G3.

Drug B:

Preparation method: Dissolve the compound represented by formula (I) (40 mg, 0.105 mmol) in 0.5 mL of a mixed solvent of isopropanol and tetrahydrofuran (V/V=1:1), stir to completely dissolve, heat up to 50°C, and add dropwise A 4M hydrogen chloride solution in isopropanol (0.055 mL, 0.22 mmol) was cooled to room temperature and stirred for 16 hours, and a white solid was precipitated. The reaction solution was filtered, the filter cake was collected, and dried in vacuum to obtain drug B.

Mouse TLR9 agonist: ODN-1826, purchased from Suzhou Hongxun Biotechnology Co., Ltd.

Table 10.

4) Other reagents and experimental instruments

Table 11.

article	Brand	article	Brand
RPMI 1640medium	HeClone	PBS	Sigma
FBS	GIBCO	Inverted microscope	OLYMPUS
PenicillinG	SangonBiotech	CO 2 incubator	Thermo
Streptomycin sulfate	SangonBiotech	Balance	Acclulab
0.25% Trypsin-EDTA	SangonBiotech	Analytical Balances	Denver Instrument
Trypan Blue	GIBCO	ABSOLUTE Digimatic	Mitutoyo corp

2. Experimental method

Collect the P9 generation of logarithmic growth phase MC38 tumor cells, adjust the cell concentration to 5× ^{10 6} /mL with PBS buffer, and inoculate 0.1 mL of cell suspension on the left and right bilateral flank of OX40 mice. Observe the mice after inoculation and monitor the growth of tumors. On the 9th day after inoculation, the average tumor volume on the right flanks of tumor-bearing mice reached 123.7mm ^3. Grouping and drug administration observations were made according to the size of the tumor on the right side. The specific grouping information is as follows Table 12.

Table 12. Administration and treatment methods

Note: N: Number of animals used;

i.p.,: intraperitoneal injection;

i.t.,: intratumoral injection (intratumoral injection only on the right side);

Q3D: Dosing once every three days.

Administration volume: intratumoral injection volume is 0.1mL/mouse, intraperitoneal injection is adjusted according to the weight of tumor-bearing mice (0.1mL/10g).

The first and fourth groups of this experiment ended on the 18th day after grouping, and the second, third, and fifth to tenth groups ended on the 21st day after grouping.

The first group is the vehicle group.

The configuration of the test compound is shown in Table 13.

Table 13.

Test index

The main purpose is to detect the growth inhibitory effect of (OX40) humanized mouse MC38 colon cancer cell transplantation tumor. Tumor volume and weight measurement of tumor-bearing mice: using vernier calipers to measure twice a week, the tumor volume calculation formula is V=0.5axb ² , a and b represent the long diameter and wide diameter of the tumor respectively;

RTV T/C: Relative Tumor Volume RTV (Relative Tumor Volume)=V _t /V _o , V _o is the tumor volume of each mouse in the group at the beginning of the administration, and V _t is the measurement after each administration Tumor volume.

T/C(%)=TRTV/CRTV 100%

TRTV: average relative tumor volume RTV of tumor-bearing mice in the test drug group; CRTV: average relative tumor volume of mice in the control group

Tumor growth transplant tumor TGI (%) = [1-T/C]×100

The body weight of all tumor-bearing mice was measured twice a week. At the same time, calculate the rate of change in weight gain of mice after administration: BWL(%)=(BW _i -BW ₀ )/BW ₀ ×100, BW _i is the average body weight after the start of administration, and BW ₀ is the average at the first administration body weight.

All data are expressed in Mean±SEM, and the statistical analysis software SPSS20.0 is used to test the data for normal distribution and homogeneity of variance. According to the test results, one-way analysis of variance or nonparametric test Kruskal-Wallis method is selected. *P<0.05 will be considered as a statistically significant difference.

2. Experimental results

Table 14. Shows the tumor volume on the right side of the mouse after treatment

The tumor volume on the right side of the mouse after treatment (Mean±SEM) n=6

Note: *P＜0.05, **P＜0.01, compared with the vehicle group.

Table 15. Shows the tumor volume on the left side of the mouse after treatment

Mouse left tumor volume after treatment (Mean±SEM) n=6

Note: *P＜0.05, **P＜0.01, compared with the vehicle group.

The tumor growth inhibitory effects of drug A and drug B alone or in combination on the hOX40 transgenic mouse MC38 colon cancer xenograft model are shown in Table 16 (right side).

Table 16.

Note: a. Data is expressed as "average value ± standard error";

b. *P<0.05, **P<0.01, compared with the vehicle group;

c. Use one-way analysis of variance (one-way ANOVA) for significant differences between treatment groups.

The tumor growth inhibitory effects of drug A and drug B alone or in combination on the hOX40 transgenic mouse MC38 colon cancer xenograft model are shown in Table 17 (left side).

Table 17.

Note: a. Data is expressed as "average value ± standard error";

b. *P<0.05, **P<0.01, compared with the vehicle group;

c. Use one-way analysis of variance (one-way ANOVA) for significant differences between treatment groups.

Table 18. Tumor mass (g) (Mean±SEM) n=6

4. Experiment summary and discussion

This experiment tested the inhibitory effects of anti-OX40 antibody (drug A) and TLR7 agonist (drug B) alone or in combination on the growth of MC38 colon cancer xenografts in hOX40 transgenic mice. On the 18th day after administration, the average tumor volume of the vehicle control group reached 2793.66mm ³ (right side) and 2794.89mm ³ (left side). At the same time, drug B in the single-drug group was 25μg/mouse, and the average tumor volume of mice in the treatment group It also reached 2767.03mm ³ (right side) and 2175.03mm ³ (left side). The two groups of tumor-bearing mice were euthanized according to the experimental protocol and animal welfare requirements.

On the same day, the average tumor volume on the right side of the tumor-bearing mice in group 2, group 3, group 5, group 6, group 7, group 8, 9 and group 10 was 649.96mm ³ , 768.28mm ³ , 1013.28mm ³ , 824.27mm ³ , 334.23mm ³ , 536.72mm ³ , 231.48mm ³ and 239.03mm ³ , which are significantly different from the Vehicle group, and have a significant inhibitory effect on tumor growth (*P<0.05,* *P<0.01) (see Figure 1, Table 14, Table 16), the tumor growth inhibition rate reached 76.83%, 73.75%, 63.42%, 70.48%, 88.03%, 81.63%, 92.0% and 91.69% (see Table 16).

The average tumor volume on the left side of the tumor-bearing mice were 1084.84mm ³ , 917.22mm ³ , 1470.54mm ³ , 947.32mm ³ , 490.11mm ³ , 644.32mm ³ , 498.02mm ³ and 349.66mm ³ , respectively, and the Vehicle group The difference is significant, and it has a significant effect of inhibiting tumor growth (*P<0.05, **P<0.01), and its tumor growth inhibition rate reached 48.48%, 69.09%, 43.51%, 62.62%, 76.66%, 77.28%, respectively , 79.44% and 90.22% (see Figure 2, Table 15, Table 17).

In the course of the experiment, the 3rd group 1# and 5th group 48# tumor-bearing mice due to weight loss, the 4th group 23# tumor-bearing mice due to poor state, respectively on the 7th day, 10th day and 18th day after grouping There was death on the day, and the 1# tumor-bearing mouse was dissected and found that there was obvious foreign body in the abdominal cavity of hematoma tissue and tumor metastasis. The weight changes of the remaining tumor-bearing mice with other treatments were not significantly abnormal, and there were no obvious abnormal behaviors and other manifestations during the drug treatment. Vehicle group 20# and 32# died on the 18th day after grouping due to the heavy tumor burden (total tumor volume>3000mm ³ ).

Claims (16)

1. The use of a TLR agonist combined with an anti-OX40 antibody or an antigen-binding fragment thereof in the preparation of a medicine for preventing or treating tumors.
2. The use according to claim 1, wherein the TLR agonist is selected from the group consisting of TLR1 agonist, TLR2 agonist, TLR3 agonist, TLR4 agonist, TLR5 agonist, TLR6 agonist, TLR7 agonist, TLR8 agonist, A TLR9 agonist, preferably a TLR7 agonist or a TLR9 agonist, and most preferably a TLR7 agonist.
3. The use according to claim 2, wherein the TLR agonist is selected from Heplisav, SD-101, resiquimod, Dynavax, DV-281, imiquimod, cobitolimod, entolimod, lefitolimod, Poly-ICLC, Grass MATA MPL, G-100 , AST-008, GSK-1795091, tilsotolimod, KMRC-011, CMB-305, rintatolimod, AZD-1419, influenza-PAL, SAR-439794, MIS-416, MGN-1601, GSK-2245035, VTX-1463, motolimod , GS-9688, LHC-165, BDB-001, PGV-001, AV-7909, DSP-0509, DPX-E7, RG-7854, telratolimod, vesatolimod, poly-ICLC adjuvanted vaccines, MVAME-03, Riboxxim, G -305, PUL-042, litenimod, DRibbles vaccine, TMX-202, ISA-201, PEPA-10, AL-034, CpG-ODN (K3), Vaxart, CBLB-612, Pseudomonas aeruginosa, IR-103, VAX- 161, VAX-125, MEDI19197 or the compound represented by formula (I) or its complex or pharmaceutically acceptable salt thereof, preferably TLR7 agonist: imiquimod, GSK-2245035, LHC-165, DSP-0509, RG-7854, vesatolimod , TMX-202, AL-034 or the compound represented by formula (I) or its complex or its pharmaceutically acceptable salt, most preferably the compound represented by formula (I) or its complex or its pharmaceutically acceptable salt

   
4. The use according to any one of claims 1 to 3, wherein the anti-OX40 antibody comprises the CDRs shown below: heavy chain HCDR1, HCDR2, HCDR3 shown in SEQ ID NO: 3, 4, and 5 amino acid sequences, or SEQ ID NOs: HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 3, 4, and 5 are HCDR variants with 3, 2 or 1 amino acid differences, respectively; Chain LCDR1, LCDR2, LCDR3 or LCDR1, LCDR2 or LCDR3 shown in SEQ ID NO: 6, 7 and 8 with 3, 2 or 1 amino acid difference, respectively, preferably containing SEQ ID NO: 3, The heavy chain HCDR1, HCDR2, HCDR3 shown in the amino acid sequence of 4 and 5 and the light chain LCDR1, LCDR2 and LCDR3 shown in the amino acid sequence of SEQ ID NO: 6, 7, and 8, respectively.
5. The use according to claim 4, characterized in that the heavy chain HCDR2 variant of the anti-OX40 antibody is shown in the amino acid sequence of SEQID NO: 12.
6. The use according to any one of claims 4-5, characterized in that the anti-OX40 antibody is a murine antibody, a chimeric antibody or a humanized antibody, preferably a humanized antibody.
7. The use according to claim 6, characterized in that the humanized antibody comprises a heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 11 or having at least 95% sequence identity with it, preferably comprising SEQ ID NO: 11 heavy chain variable region shown in the amino acid sequence.
8. The use according to any one of claims 6-7, characterized in that the anti-OX40 antibody or antigen-binding fragment thereof contains the amino acid sequence shown in SEQ ID NO: 10 or has at least 95% sequence identity with the light The chain variable region preferably includes the light chain variable region shown in the amino acid sequence of SEQ ID NO: 10.
9. The use according to claim 8, characterized in that the humanized antibody comprises the heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 11 and the light chain variable region shown in the amino acid sequence of SEQ ID NO: 10 Area.
10. The use according to any one of claims 6-9, characterized in that the anti-OX40 antibody comprises a constant region, and the heavy chain constant region of the antibody is derived from human IgG1, IgG2, IgG3 or IgG4 or its mutant sequence The light chain constant region is derived from human kappa, lambda chains or their mutant sequences. Preferably, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO: 13 or has at least 95% sequence identity with it. The amino acid sequence of the constant region is shown in SEQ ID NO: 14 or has at least 95% sequence identity with it. Most preferably, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO: 13, and the light chain constant region is The amino acid sequence is shown in SEQ ID NO: 14.
11. The use according to any one of claims 4-10, characterized in that the anti-OX40 antibody comprises: a heavy chain amino acid sequence as shown in SEQ ID NO: 15 or at least 85% sequence identity and light chain amino acid sequence with it It is shown in SEQ ID NO: 16 or has at least 85% sequence identity with it; preferably, the heavy chain amino acid sequence is shown in SEQ ID NO: 15 and the light chain amino acid sequence is shown in SEQ ID NO: 16.
12. The use according to any one of claims 1 to 3, characterized in that the anti-OX40 antibody or its antigen-binding fragment is selected from MEDI6469, PF-04518600, KHK4083, BMS986178, MEDI0562, MOXR0916, MEDI6383, INCAGN01949.
13. The use according to any one of claims 1-12, wherein the tumor is selected from squamous cell carcinoma, lung cancer, peritoneal cancer, hepatocellular carcinoma, gastric cancer, pancreatic cancer, glioblastoma, cervical cancer , Ovarian cancer, liver cancer, bladder cancer, urethral cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, Anal cancer, soft tissue sarcoma, neuroblastoma, penile cancer, melanoma, superficial spreading melanoma, lentigines melanoma, acral melanoma, nodular melanoma, multiple bone marrow Tumor, B-cell lymphoma, chronic lymphocytic leukemia, non-Hodgkin’s lymphoma, acute lymphoblastic leukemia, hairy cell leukemia, chronic myeloblastic leukemia, post-transplant lymphoproliferative disorders, brain tumors, brain Cancer and head and neck cancer, preferably colon cancer and non-Hodgkin's lymphoma.
14. The use according to any one of claims 3-13, characterized in that the TLR7 agonist is the dihydrochloride of the compound represented by formula (I).
15. The anti-OX40 antibody or antigen-binding fragment thereof according to any one of claims 4-14, wherein the dosage is selected from 0.01-10.0 mg/kg or 1-1000 mg, and the frequency of the medicine is 3 times a week, weekly Twice, once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once a month, once every 3-6 months, preferably 3 times a week or once every 4 weeks.
16. The use according to claim 15, characterized in that the dosage of the TLR agonist is selected from 0.1-1000 μg/kg or 0.001-100 mg, and the administration frequency is 3 times a day, 2 times a day, once a day, every 2 days Once, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, preferably once a week, every 2 Once a week.

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