WO2020063858A1 - Use of combination of tlr agonist and immune checkpoint inhibitor in preparation of drugs for treating tumors - Google Patents

Abstract

Use of a combination of a TLR agonist and an immune checkpoint inhibitor in preparation of drugs for treating tumors. Specifically, the TLR agonist is a compound of formula (I) and a pharmacologically acceptable salt thereof, and the immune checkpoint inhibitor is selected from a group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.

Description

Application of TLR agonist and immune checkpoint inhibitor in preparing medicine for treating tumor Technical field

The present disclosure relates to the use of a TLR agonist combined with an immune checkpoint inhibitor in the manufacture of a medicament for treating tumors.

Background technique

Protein programmed death 1 (PD-1) is an inhibitory member of the CD28 receptor family, which also includes CD28, CTLA-4, ICOS, and BTLA. PD-1 is expressed on activated B cells, T cells, and myeloid cells. The original members of the family, CD28 and ICOS, were found based on the functional effects of increasing the proliferation of T cells after the addition of monoclonal antibodies. PD-1 was found by screening for differential expression in apoptotic cells. At present, Nivolumab by Ono and Pembrolizumab by Merck have been successfully approved for marketing for the treatment of unresectable or metastatic melanoma, non-small cell lung cancer, advanced renal cell carcinoma, Hodgkin lymphoma, recurrent or metastatic Squamous cell carcinoma.

Toll-like receptors (TLRs) are an important class of protein molecules involved in innate immunity. TLRs are monomeric non-catalytic receptors that are commonly 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 are recognized by TLRs, which in turn activates immune cell responses. (Mahla, RS. Et al., Front Immunol. 4: 248 (2013)). The ability of the immune system to recognize pathogenic microorganisms is partly due to the widespread presence of Toll-like immune receptors.

There are at least 10 different TLRs in mammals. Ligands and corresponding signal cascades for some of these receptors have been identified. Many diseases and disorders are related to abnormal TLRs, such as melanoma, non-small cell lung cancer, hepatocellular 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, etc.

TLR7 is a member of the TLRs ( TLRs 3, 7, 8, and 9) subgroup and is limited to the endosome compartment of cells that specifically detect non-nucleic acids. TLR7 plays a key role in antiviral defense by identifying ssRNAs (Diebold S.S. et al., Science, 2004: 303, 1529-1531; and Lund J.M. et al., PNAS, 2004: 101, 5598-5603). Currently, TLR7 inhibitors on the market are mainly used for surface administration, for example, imiquimod is used to treat genital warts.

WO2018095426A provides a TLR7 agonist with the following structure:

FatoSato-Kaneko et al. Found that combined therapy with intratumoral injection of TLR agonist and PD-1 blocker can activate tumor-associated macrophages (TAM) and induce tumor-specific adaptive immune response, in recurrent or metastatic Inhibition of primary tumors and prevention of metastasis in a head and neck squamous cell carcinoma (HNSCC) model (JCI Insight. 2017 Sep 21; 2 (18): e93397.).

Yosuke Ota et al. Found that intravenous injection of TLR7 agonist DSP-0509 in combination with anti-PD-1 antibody has a synergistic effect on antitumor immune response (AACR 2018 Proceedings: Abstracts 4726).

Naoto Nishii et al. Found that the combination of the TLR7 agonist resiquimod with PD-L1 antibody in the mouse head and neck squamous cell carcinoma model SCCVII and colon cancer model Colon26 concluded that low-dose requimod could be used as PD-1 / PD- A companion drug for L1 blockade therapy (Oncotarget, 2018, Vol. 9, (No. 17), pp: 13301-13312).

MEDIMMUNE has carried out clinical trials on the combination of TLR7 agonist MEDI9197 and durvalumab in the treatment of solid tumors or T-cell lymphoma, and no results have been found (NCT02556463).

Summary of the Invention

The present disclosure provides a use of a TLR agonist and an immune checkpoint inhibitor in the manufacture of a medicament for treating a tumor.

The TLR agonist provided in the present disclosure may be a compound represented by formula (I) or a complex thereof or a pharmaceutically acceptable salt thereof,

The pharmaceutically acceptable salts of TLR agonists in the present disclosure may be hydrochloride, phosphate, hydrogen phosphate, sulfate, hydrogen sulfate, sulfite, acetate, oxalate, malonate, valerate Salt, glutamate, oleate, palmitate, stearate, laurate, borate, p-toluenesulfonate, mesylate, isethionate, maleate , Malate, tartrate, benzoate, paraben, salicylate, vanillate, mandelate, succinate, gluconate, lactobionate or laurylsulfonate, etc. Of these, maleate and hydrochloride are preferred, and dihydrochloride is most preferred.

The immune checkpoint inhibitors described in this disclosure may be selected from the group consisting of a programmed death-1 (PD-1) inhibitor, a programmed death ligand-1 (PD-L1) inhibitor, or a cytotoxic-T-lymphocyte-related Protein-4 (CTLA-4) inhibitor.

In some embodiments, the PD-1 receptor inhibitor described in the present disclosure is an antibody or an antigen-binding fragment thereof that specifically binds PD-1 and inhibits PD-1 activity; the PD-L1 inhibitor is An antibody or antigen-binding fragment thereof capable of specifically binding PD-L1 and inhibiting PD-L1 activity; the CTLA-4 inhibitor is an antibody or antigen-binding thereof capable of specifically binding CTLA-4 and inhibiting CTLA-4 activity Fragment.

The antibody or antigen-binding fragment thereof that specifically binds PD-1 and inhibits PD-1 activity described in the present disclosure is an anti-PD-1 antibody or an antigen-binding fragment thereof.

The antibodies or antigen-binding fragments thereof that can specifically bind PD-L1 and inhibit the activity of PD-L1 described in the present disclosure are anti-PD-L1 antibodies or antigen-binding fragments thereof.

The antibody or antigen-binding fragment thereof that specifically binds CTLA-4 and inhibits CTLA-4 activity described in the present disclosure is an anti-CTLA-4 antibody or antigen-binding fragment thereof.

In some embodiments, the light chain variable region of the PD-1 antibody described in the present disclosure comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 respectively.

The heavy chain variable region of the PD-1 antibody comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

The CDR sequences described above are shown in the following table:

name	sequence	Numbering
HCDR1	SYMMS	SEQID NO: 1
HCDR2	TISGGGANTYYPDSVKG	SEQID NO: 2
HCDR3	QLYYFDY	SEQID NO: 3
LCDR1	LASQTIGTWLT	SEQID NO: 4
LCDR2	TATSLAD	SEQID NO: 5
LCDR3	QQVYSIPWT	SEQID NO: 6

Preferably, the PD-1 antibody is a humanized antibody or a fragment thereof.

In an alternative embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof described in the present disclosure is selected from the group consisting of Fab, Fab'-SH, Fv, scFv, and (Fab ') 2 fragments of antibody fragments.

Immunoglobulins can be derived from any generally known isotype, including but not limited to IgA, secreted IgA, IgG, and IgM. IgG subclasses are also well known to those skilled in the art and include, but are not limited to, IgG1, IgG2, IgG3, and IgG4. "Isotype" refers to an Ab species or subclass (eg, IgM or IgG1) encoded by a heavy chain constant region gene. In some alternative embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof described in the present disclosure comprises a heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4 isotype, preferably comprising the IgG1 or IgG4 isotype Heavy chain constant region.

In other alternative embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a light chain constant region of a light chain constant region of kappa or lambda.

Further, the humanized antibody light chain variable region sequence is preferably a sequence as shown in SEQ ID NO: 10 or a variant thereof, and the variant preferably has a 0-10 amino acid change in the light chain variable region, more Preferably it is an amino acid change of A43S; the sequence of the humanized antibody heavy chain variable region is the sequence shown in SEQ ID NO: 9 or a variant thereof, and the variant preferably has 0-10 in the heavy chain variable region The amino acid change is more preferably an amino acid change of G44R.

The sequences of the heavy and light chains of the aforementioned humanized antibodies are as follows:

Heavy chain variable region

Light chain variable region

Preferably, the humanized antibody light chain sequence is the sequence shown in SEQ ID NO: 8 or a variant thereof; the variant preferably has an amino acid change of 0-10 in the variable region of the light chain, more preferably A43S amino acid changes; the humanized antibody heavy chain sequence is the sequence shown in SEQ ID NO: 7 or a variant thereof, the variant preferably has a 0-10 amino acid change in the heavy chain variable region, more An amino acid change of G44R is preferred.

In another embodiment, the light chain sequence of the humanized antibody is the sequence shown in SEQ ID NO: 8, and the heavy chain sequence is the sequence shown in SEQ ID NO: 7.

The sequences of the heavy and light chains of the humanized antibody are as follows:

Heavy chain

Light chain

In certain embodiments, the TLR agonist is a compound represented by formula (I) or a complex thereof or a pharmaceutically acceptable salt thereof, and the immune checkpoint inhibitor is selected from an anti-PD-1 antibody whose heavy and light chain The sequences are shown in SEQ ID NO: 7 and SEQ ID NO: 8 in the present disclosure.

In certain embodiments, the heavy chain variable region of the anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 11-13, respectively, and the anti-PD-L1 antibody Or the light chain variable region of the antigen-binding fragment comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 14-16, respectively;

details as follows:

HCDR1 is selected from:

SYWMH ID: 11

HCDR2 is selected from:

RIGPNSGFTSYNEKFKN SEQ ID ID: 12

HCDR3 is selected from:

GGSSYDYFDY SEQ ID ID: 13

LCDR1 is selected from:

RASESVSIHGTHLMH SEQ ID ID: 14

LCDR2 is selected from:

AASNLES ID: 15

LCDR3 is selected from:

QQSFEDPLT: SEQ ID NO: 16.

In certain embodiments, the PD-L1 antibody or antigen-binding fragment comprises an amino acid sequence: SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity light chain variable region CDR sequences, and amino acids Sequence: SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13 have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98%, or 99% sequence identity heavy chain variable region CDR sequences.

In certain embodiments, the PD-L1 antibody or antigen-binding fragment may be selected from a murine antibody, a chimeric antibody, a humanized antibody, a human antibody, preferably a humanized antibody.

In certain embodiments, the PD-L1 antibody or antigen-binding fragment comprises and the amino acid sequence SEQ ID NO: 17 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity heavy chain variable region sequences, and the amino acid sequence SEQ ID NO: 18 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity light chain variable region sequences.

In certain embodiments, the PD-L1 antibody or antigen-binding fragment further comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, preferably a human IgG2 or IgG4 heavy chain constant region More preferably, the IgG4 heavy chain constant region comprises F234A and L235A mutations; the humanized antibody light chain further comprises a constant region of a human-derived kappa, lambda chain, or a variant thereof.

In certain embodiments, the PD-L1 antibody or antigen-binding fragment comprises and the amino acid sequence SEQ ID NO: 19 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity heavy chain sequence, and the amino acid sequence SEQ ID NO: 21 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity light chain sequences.

In some embodiments, the heavy chain sequence of the PD-L1 antibody or antigen-binding fragment is SEQ ID NO: 19, and the light chain sequence is SEQ ID NO: 21.

Note: italics in the sequence are FR sequences; underlined are CDR sequences.

Heavy chain sequence

Heavy chain sequence encoding gene sequence

Light chain sequence

Light chain sequence encoding gene sequence:

In some embodiments, the anti-CTLA-4 antibody or antigen-binding fragment thereof provided in the present disclosure is selected from the group consisting of ipilimumab, vermelimumab, AGEN-1884, CS-1002, XmAb-20717, REGN-4659, BCD-145, MEDI-5752 , AK-104, MK-1308, BMS-986249, BMS-986218, PF-06753512, preferably ipilimumab, vermelimumab.

The combination of a TLR agonist and an immune checkpoint inhibitor provided in the present disclosure can adjust the dosage regimen to provide the optimal desired response, for example, maximum therapeutic response and / or minimal adverse effects.

In some embodiments, the dose of the TLR agonist may be in the following range: 0.0001-20.0 mg / kg, optionally 0.001-10.0 mg / kg, optionally 0.001-5.0 mg / kg, optionally 0.001- 1.0mg / kg.

In an alternative embodiment, the dose of the TLR agonist is 0.0010 mg / kg, 0.0015 mg / kg, 0.0020 mg / kg, 0.0025 mg / kg, 0.0030 mg / kg, 0.0035 mg / kg, 0.0040 mg / kg, 0.0045 mg / kg, 0.0050mg / kg, 0.0055mg / kg, 0.0060mg / kg, 0.0065mg / kg, 0.0070mg / kg, 0.0075mg / kg, 0.0080mg / kg, 0.0085mg / kg, 0.0090mg / kg, 0.0095mg / kg, 0.0100mg / kg, 0.0105mg / kg, 0.0110mg / kg, 0.0115mg / kg, 0.0120mg / kg, 0.0125mg / kg, 0.0130mg / kg, 0.0135mg / kg, 0.0140mg / kg, 0.0145mg / kg, 0.0150mg / kg, 0.0155mg / kg, 0.0160mg / kg, 0.0165mg / kg, 0.0170mg / kg, 0.0175mg / kg, 0.0180mg / kg, 0.0185mg / kg, 0.0190mg / kg, 0.0195mg / kg, 0.0200mg / kg, 0.0205mg / kg, 0.0210mg / kg, 0.0215mg / kg, 0.0220mg / kg, 0.0225mg / kg, 0.0230mg / kg, 0.0235mg / kg, 0.0240mg / kg, 0.0245mg / kg, 0.0250mg / kg, 0.0255mg / kg, 0.0260mg / kg, 0.0265mg / kg, 0.0270mg / kg, 0.0275mg / kg, 0.0280mg / kg, 0.0285mg / kg, 0.0290mg / kg, 0.0295mg / kg, 0.0300mg / kg, 0.0305mg / kg, 0.0310mg / kg, 0.0315mg / kg, 0.0320mg / kg, 0.0325mg / kg, 0.0330mg / kg, 0.0335mg / kg, 0.0340mg / kg, 0.0345mg / kg , 0.0350 mg / kg, 0.0355mg / kg, 0.0360mg / kg, 0.0365mg / kg, 0.0370mg / kg, 0.0375mg / kg, 0.0380mg / kg, 0.0385mg / kg, 0.0390mg / kg, 0.0395mg / kg , 0.0400mg / kg, 0.0405mg / kg, 0.0410mg / kg, 0.0415mg / kg, 0.0420mg / kg, 0.0425mg / kg, 0.0430mg / kg, 0.0435mg / kg, 0.0440mg / kg, 0.0445mg / kg , 0.0450mg / kg, 0.0455mg / kg, 0.0460mg / kg, 0.0465mg / kg, 0.0470mg / kg, 0.0475mg / kg, 0.0480mg / kg, 0.0485mg / kg, 0.0490mg / kg, 0.0495mg / kg , 0.0500mg / kg, 0.0505mg / kg, 0.0510mg / kg, 0.0515mg / kg, 0.0520mg / kg, 0.0525mg / kg, 0.0530mg / kg, 0.0535mg / kg, 0.0540mg / kg, 0.0545mg / kg , 0.0550mg / kg, 0.0555mg / kg, 0.0560mg / kg, 0.0565mg / kg, 0.0570mg / kg, 0.0575mg / kg, 0.0580mg / kg, 0.0585mg / kg, 0.0590mg / kg, 0.0595mg / kg , 0.0600mg / kg, 0.0605mg / kg, 0.0610mg / kg, 0.0615mg / kg, 0.0620mg / kg, 0.0625mg / kg, 0.0630mg / kg, 0.0635mg / kg, 0.0640mg / kg, 0.0645mg / kg , 0.0650mg / kg, 0.0655mg / kg, 0.0660mg / kg, 0.0665mg / kg, 0.0670mg / kg, 0.0675mg / kg, 0.0680mg / kg, 0.0685mg / kg, 0.0690mg / kg, 0.0695mg / kg 0.0700mg / kg, 0. 0705mg / kg, 0.0710mg / kg, 0.0715mg / kg, 0.0720mg / kg, 0.0725mg / kg, 0.0730mg / kg, 0.0735mg / kg, 0.0740mg / kg, 0.0745mg / kg, 0.0750mg / kg, 0.0755 mg / kg, 0.0760mg / kg, 0.0765mg / kg, 0.0770mg / kg, 0.0775mg / kg, 0.0780mg / kg, 0.0785mg / kg, 0.0790mg / kg, 0.0795mg / kg, 0.0800mg / kg, 0.0805 mg / kg, 0.0810mg / kg, 0.0815mg / kg, 0.0820mg / kg, 0.0825mg / kg, 0.0830mg / kg, 0.0835mg / kg, 0.0840mg / kg, 0.0845mg / kg, 0.0850mg / kg, 0.0855 mg / kg, 0.0860mg / kg, 0.0865mg / kg, 0.0870mg / kg, 0.0875mg / kg, 0.0880mg / kg, 0.0885mg / kg, 0.0890mg / kg, 0.0895mg / kg, 0.0900mg / kg, 0.0905 mg / kg, 0.0910mg / kg, 0.0915mg / kg, 0.0920mg / kg, 0.0925mg / kg, 0.0930mg / kg, 0.0935mg / kg, 0.0940mg / kg, 0.0945mg / kg, 0.0950mg / kg, 0.0955 mg / kg, 0.0960mg / kg, 0.0965mg / kg, 0.0970mg / kg, 0.0975mg / kg, 0.0980mg / kg, 0.0985mg / kg, 0.0990mg / kg, 0.0995mg / kg, 0.1000mg / kg, 0.1005 mg / kg, 0.1010mg / kg, 0.1015mg / kg, 0.1020mg / kg, 0.1025mg / kg, 0.1030mg / kg, 0.1035mg / kg, 0.1040mg / kg, 0.1045mg / kg, 0.1050mg / kg, 0.1055 mg / kg, 0.1060mg / kg, 0.1065mg / kg, 0.1070mg / kg, 0.1075mg / kg, 0.1080mg / kg, 0.1085mg / kg, 0.1090mg / kg, 0.1095mg / kg, 0.1100mg / kg, 0.1105mg / kg, 0.1110mg / kg, 0.1115mg / kg, 0.1120mg / kg, 0.1125mg / kg, 0.1130mg / kg, 0.1135mg / kg, 0.1140mg / kg, 0.1145mg / kg, 0.1150mg / kg, 0.1155mg / kg, 0.1160mg / kg, 0.1165mg / kg, 0.1170mg / kg, 0.1175mg / kg, 0.1180mg / kg, 0.1185mg / kg, 0.1190mg / kg, 0.1195mg / kg, 0.1200mg / kg, 0.1205mg / kg, 0.1210mg / kg, 0.1215mg / kg, 0.1220mg / kg, 0.1225mg / kg, 0.1230mg / kg, 0.1235mg / kg, 0.1240mg / kg, 0.1245mg / kg, 0.1250mg / kg, 0.1255mg / kg, 0.1260mg / kg, 0.1265mg / kg, 0.1270mg / kg, 0.1275mg / kg, 0.1280mg / kg, 0.1285mg / kg, 0.1290mg / kg, 0.1295mg / kg, 0.1300mg / kg, 0.1305mg / kg, 0.1310mg / kg, 0.1315mg / kg, 0.1320mg / kg, 0.1325mg / kg, 0.1330mg / kg, 0.1335mg / kg, 0.1340 mg / kg, 0.1345mg / kg, 0.1350mg / kg, 0.1355mg / kg, 0.1360mg / kg, 0.1365mg / kg, 0.1370mg / kg, 0.1375mg / kg, 0.1380mg / kg, 0.1385mg / kg, 0.1390mg / kg, 0.1395mg / kg, 0.1400mg / kg, 0.1405mg / kg, 0.1410mg / kg, 0.1415mg / kg 0.1420mg / kg, 0.1425mg / kg, 0.1430mg / kg, 0.1435mg / kg, 0.1440mg / kg, 0.1445mg / kg, 0.1450mg / kg, 0.1455mg / kg, 0.1460mg / kg, 0.1465mg / kg, 0.1470 mg / kg, 0.1475 mg / kg, 0.1480 mg / kg, 0.1485 mg / kg, 0.1490 mg / kg, 0.1495 mg / kg, 0.1500 mg / kg.

In some embodiments, the administered dose of the TLR agonist is selected from the group consisting of 0.01-200 mg, 0.05-100 mg, and 0.10-50 mg, and specifically, 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07mg, 0.08mg, 0.09mg, 0.10mg, 0.11mg, 0.12mg, 0.13mg, 0.14mg, 0.15mg, 0.16mg, 0.17mg, 0.18mg, 0.19mg, 0.20mg, 0.21mg, 0.22mg, 0.23mg , 0.24mg, 0.25mg, 0.26mg, 0.27mg, 0.28mg, 0.29mg, 0.30mg, 0.31mg, 0.32mg, 0.33mg, 0.34mg, 0.35mg, 0.36mg, 0.37mg, 0.38mg, 0.39mg, 0.40 mg, 0.41mg, 0.42mg, 0.43mg, 0.44mg, 0.45mg, 0.46mg, 0.47mg, 0.48mg, 0.49mg, 0.50mg, 0.51mg, 0.52mg, 0.53mg, 0.54mg, 0.55mg, 0.56mg, 0.57mg, 0.58mg, 0.59mg, 0.60mg, 0.61mg, 0.62mg, 0.63mg, 0.64mg, 0.65mg, 0.66mg, 0.67mg, 0.68mg, 0.69mg, 0.70mg, 0.71mg, 0.72mg, 0.73mg , 0.74mg, 0.75mg, 0.76mg, 0.77mg, 0.78mg, 0.79mg, 0.80mg, 0.81mg, 0.82mg, 0.83mg, 0.84mg, 0.85mg, 0.86mg, 0.87mg, 0.88mg, 0.89mg, 0.90 mg, 0.91mg, 0.92mg, 0.93mg, 0.94mg, 0.95mg, 0.96mg, 0.97mg, 0.98mg, 0. 99mg, 1.00mg, 1.01mg, 1.02mg, 1.03mg, 1.04mg, 1.05mg, 1.06mg, 1.07mg, 1.08mg, 1.09mg, 1.10mg, 1.11mg, 1.12mg, 1.13mg, 1.14mg, 1.15mg, 1.16mg, 1.17mg, 1.18mg, 1.19mg, 1.20mg, 1.21mg, 1.22mg, 1.23mg, 1.24mg, 1.25mg, 1.26mg, 1.27mg, 1.28mg, 1.29mg, 1.30mg, 1.31mg, 1.32mg , 1.33mg, 1.34mg, 1.35mg, 1.36mg, 1.37mg, 1.38mg, 1.39mg, 1.40mg, 1.41mg, 1.42mg, 1.43mg, 1.44mg, 1.45mg, 1.46mg, 1.47mg, 1.48mg, 1.49 mg, 1.50mg, 1.51mg, 1.52mg, 1.53mg, 1.54mg, 1.55mg, 1.56mg, 1.57mg, 1.58mg, 1.59mg, 1.60mg, 1.61mg, 1.62mg, 1.63mg, 1.64mg, 1.65mg, 1.66mg, 1.67mg, 1.68mg, 1.69mg, 1.70mg, 1.71mg, 1.72mg, 1.73mg, 1.74mg, 1.75mg, 1.76mg, 1.77mg, 1.78mg, 1.79mg, 1.80mg, 1.81mg, 1.82mg , 1.83mg, 1.84mg, 1.85mg, 1.86mg, 1.87mg, 1.88mg, 1.89mg, 1.90mg, 1.91mg, 1.92mg, 1.93mg, 1.94mg, 1.95mg, 1.96mg, 1.97mg, 1.98mg, 1.99 mg, 2.00mg, 2.00mg, 2.50mg, 3.00mg, 3.50mg, 4.00mg, 4.50mg, 5.00mg, 5.50mg, 6.00mg, 6.5 0mg, 7.00mg, 7.50mg, 8.00mg, 8.50mg, 9.00mg, 9.50mg, 10.00mg, 10.50mg, 11.00mg, 11.50mg, 12.00mg, 12.50mg, 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, 22.00 mg, 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, 31.50 mg, 32.00mg, 32.50mg, 33.00mg, 33.50mg, 34.00mg, 34.50mg, 35.00mg, 35.50mg, 36.00mg, 36.50mg, 37.00mg, 37.50mg, 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 administered dose of the TLR agonist is selected from the group consisting of 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 TLR agonist is administered in a dose selected from the group consisting of 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, 12mg, 13mg, 14mg, 15mg.

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

The frequency of TLR agonist administration in the present disclosure is 3 times per day, 2 times per day, 1 time per day, 1 time every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, every Once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, preferably once every 3 days, once a week or once every 2 weeks.

In an alternative embodiment, the TLR agonist is administered once a week.

In an alternative embodiment, the TLR agonist is administered every two weeks.

In an alternative embodiment, the TLR agonist is administered every three days.

In certain embodiments of the present disclosure, the dose of the anti-PD-1 antibody or antigen-binding fragment thereof may be in the following range: 0.1-10.0 mg / kg, 0.1-5 mg / kg, 1-5 mg / kg, 2-5 mg / kg, for example, the dosage can be 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, 2.4mg / 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, 8.6 mg / kg, 8.8 mg / kg, 9.0 mg / kg, 9.2 mg / kg, 9.4 mg / kg, 9.6 mg / kg, 9.8 mg / kg, 10.0 mg / kg.

In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof may also be in the range of 1-1000mg, 80-800mg, 80-700mg, 80-600mg, 80-500mg, 80-400mg, 80- 300mg, 100-300mg or 200-300mg. For example, the administered dose may be 1.0 mg, 1.2 mg, 1.4 mg, 1.6 mg, 1.8 mg, 2.0 mg, 2.2 mg, 2.4 mg, 2.6 mg, 2.8 mg, 3.0 mg, 3.2 mg, 3.4 mg, 3.6 mg, 3.8 mg, 4.0mg, 4.2mg, 4.4mg, 4.6mg, 4.8mg, 5.0mg, 5.2mg, 5.4mg, 5.6mg, 5.8mg, 6.0mg, 6.2mg, 6.4mg, 6.6mg, 6.8mg, 7.0mg, 7.2mg, 7.4mg, 7.6mg, 7.8mg, 8.0mg, 8.2mg, 8.4mg, 8.6mg, 8.8mg, 9.0mg, 9.2mg, 9.4mg, 9.6mg, 9.8mg, 10.0mg, 15mg, 20mg, 25mg , 30mg, 35mg, 40mg, 45mg, 50mg, 55mg, 60mg, 65mg, 70mg, 75mg, 80mg, 85mg, 90mg, 95mg, 100mg, 105mg, 110mg, 115mg, 120mg, 125mg, 130mg, 135mg, 140mg, 145mg, 150mg , 155mg, 160mg, 165mg, 170mg, 175mg, 180mg, 185mg, 190mg, 195mg, 200mg, 205mg, 210mg, 215mg, 220mg, 225mg, 230mg, 235mg, 240mg, 245mg, 250mg, 255mg, 260mg, 265mg, 270mg, 275mg , 280mg, 285mg, 290mg, 295mg, 300mg, 305mg, 310mg, 315mg, 320mg, 325mg, 330mg, 335mg, 340mg, 345mg, 350mg, 355mg, 360mg, 365mg, 370mg, 375mg, 380mg, 385mg, 390mg, 395mg, 40 0mg, 405mg, 410mg, 415mg, 420mg, 425mg, 430mg, 435mg, 440mg, 445mg, 450mg, 455mg, 460mg, 465mg, 470mg, 475mg, 480mg, 485mg, 490mg, 495mg, 500mg, 505mg, 510mg, 515mg, 520mg, 525mg, 530mg, 535mg, 540mg, 545mg, 550mg, 555mg, 560mg, 565mg, 570mg, 575mg, 580mg, 585mg, 590mg, 595mg, 600mg.

In an alternative embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof is administered at a dose of 80 mg, 100 mg, 160 mg, 200 mg, 240 mg, 300 mg, 320 mg, 400 mg, 500 mg, 600 mg, 700 mg, or 800 mg.

In an alternative embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof is administered at a dose of 200 mg.

In an optional embodiment of the present disclosure, the frequency of administration of the immune checkpoint inhibitor is once a week, once every two weeks, once every three weeks, once every four weeks, once a month, or every three to six times. Dosing is performed monthly or more frequently.

In an alternative embodiment, the frequency of dosing the anti-PD-1 antibody or antigen-binding fragment thereof is once every 2 weeks or once every 3 weeks.

In some embodiments, the dose of the PD-L1 antibody or antigen-binding fragment thereof is selected from 1-50 mg / kg, preferably from 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7mg / kg, 8mg / kg, 9mg / kg, 10mg / kg, 11mg / kg, 12mg / kg, 13mg / kg, 14mg / kg, 15mg / kg, 16mg / kg, 17mg / kg, 18mg / kg , 19mg / kg, 20mg / kg, 21mg / kg, 22mg / kg, 23mg / kg, 24mg / kg, 25mg / kg, 26mg / kg, 27mg / kg, 28mg / kg, 29mg / kg, 30mg / kg, 31mg / kg, 32mg / kg, 33mg / kg, 34mg / kg, 35mg / kg, 36mg / kg, 37mg / kg, 38mg / kg, 39mg / kg, 40mg / kg, 42mg / kg, 45mg / kg, 47mg / kg 50 mg / kg, more preferably 1 mg / kg, 3 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg.

In an alternative embodiment, the PD-L1 antibody or antigen-binding fragment thereof has a dose of 50-3000 mg, preferably from 50 mg, 60 mg, 70 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 375 mg, 400mg, 425mg, 450mg, 475mg, 500mg, 550mg, 600mg, 650mg, 700mg, 750mg, 800mg, 850mg, 900mg, 950mg, 1000mg, 1050mg, 1100mg, 1150mg, 1200mg, 1250mg, 1300mg, 1350mg, 1400mg, 1450mg, 1500mg, 1500mg, 1550mg, 1600mg, 1650mg, 1700mg, 1750mg, 1800mg, 1850mg, 1900mg, 1950mg, 2000mg, 2050mg, 2100mg, 2150mg, 2200mg, 2250mg, 2300mg, 2350mg, 2400mg, 2450mg, 2500mg, 2550mg, 2600mg, 2650mg, 2700mg, 2750mg, 2750mg 2800mg, 2850mg, 2900mg, 2950mg, 3000mg.

In an alternative embodiment, the frequency of administration of the PD-L1 antibody or antigen-binding fragment may be once a week, every 2 times, every 3 weeks, every 4 weeks, preferably every 2 weeks or every 3 weeks. once.

In an alternative embodiment, the administered dose of the TLR agonist is selected from the group consisting of 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, 0.70 mg, 0.75 mg, 0.80 mg, 0.85mg, 0.90mg, 0.95mg, 1.00mg, 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, the frequency of administration is once every 3 days, once a week or once every 2 weeks; the immune checkpoint inhibitor is selected from anti-PD -1 antibody or antigen-binding fragment thereof or anti-PD-L1 antibody or antigen-binding fragment thereof, said anti-PD-1 antibody or antigen-binding fragment thereof is administered at a dose of 0.1-10.0 mg / kg or 1-1000 mg, and the frequency of administration Once every 2 weeks or once every 3 weeks, the anti-PD-L1 antibody or antigen-binding fragment thereof is administered at a dose selected from 1-50 mg / kg or 50-3000 mg, and the administration frequency is once every 2 weeks or every 3 weeks Once a week.

In an alternative embodiment, the TLR agonist is administered in a dose selected from the group consisting of 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, 12mg, 13mg, 14mg, 15mg, 16mg, 17mg, 18mg, 19mg, 20mg, the frequency of administration is once every 3 days, once a week or once every 2 weeks; the immune checkpoint inhibitor is selected from anti-PD-1 antibodies or The antigen-binding fragment or the anti-PD-L1 antibody or the antigen-binding fragment thereof, the anti-PD-1 antibody or the antigen-binding fragment thereof is administered at a dose of 0.1-10.0 mg / kg or 1-1000 mg, and the administration frequency is every 2 weeks Once or every 3 weeks, the anti-PD-L1 antibody or antigen-binding fragment thereof is administered at a dose selected from 1-50 mg / kg or 50-3000 mg, and the administration frequency is once every two weeks or once every three weeks.

In an alternative embodiment, the TLR agonist is administered in a dose selected from the group consisting of 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, 12mg, 13mg, 14mg, 15mg, 16mg, 17mg, 18mg, 19mg, 20mg, the frequency of administration is once every 3 days, once a week or once every 2 weeks; the immune checkpoint inhibitor is selected from anti-PD-1 antibodies or The antigen-binding fragment or anti-PD-L1 antibody or the antigen-binding fragment thereof, the anti-PD-1 antibody or the antigen-binding fragment thereof is administered at a dose of 80 mg, 100 mg, 160 mg, 200 mg, 240 mg, 300 mg, 320 mg, 400 mg, 500 mg, 600mg, 700mg or 800mg, the frequency of administration is once every 2 weeks or once every 3 weeks, the anti-PD-L1 antibody or antigen-binding fragment thereof is administered at a dose selected from 1-50 mg / kg or 50-3000 mg, administered The frequency is once every 2 weeks or once every 3 weeks.

In an alternative embodiment, the TLR agonist is administered in a dose selected from the group consisting of 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, 12mg, 13mg, 14mg, 15mg, 16mg, 17mg, 18mg, 19mg, 20mg, the frequency of administration is once every 3 days, once a week or once every 2 weeks; the immune checkpoint inhibitor is selected from anti-PD-1 antibodies or An antigen-binding fragment or an anti-PD-L1 antibody or an antigen-binding fragment thereof, the anti-PD-1 antibody or an antigen-binding fragment thereof is administered at a dose of 200 mg, and the administration frequency is once every 2 weeks or once every 3 weeks, said The anti-PD-L1 antibody or antigen-binding fragment thereof is administered at a dose selected from 1-50 mg / kg or 50-3000 mg, and the administration frequency is once every 2 weeks or once every 3 weeks.

In an alternative embodiment, the dose of the TLR agonist is selected from 0.5 mg, 1.0 mg, 2.0 mg, and 3.0 mg, and the frequency of administration is once a week or once every two weeks; the immune checkpoint inhibitor is selected from anti-PD -1 antibody or antigen-binding fragment thereof or anti-PD-L1 antibody or antigen-binding fragment thereof, the anti-PD-1 antibody or antigen-binding fragment thereof is administered at a dose of 200 mg, and the administration frequency is once every 2 weeks or every 3 weeks Once, the dose of the anti-PD-L1 antibody or antigen-binding fragment thereof is selected from 1-50 mg / kg or 50-3000 mg, and the frequency of administration is once every 2 weeks or once every 3 weeks.

In an alternative embodiment, the administration dose of the TLR agonist is selected from the group consisting of 2.0 mg, 4.0 mg, 6.0 mg, 8.0 mg, 9 mg, and 12 mg, and the administration frequency is once every 3 days or once a week; immune checkpoint inhibition The agent is selected from an anti-PD-1 antibody or an antigen-binding fragment thereof or an anti-PD-L1 antibody or an antigen-binding fragment thereof, the anti-PD-1 antibody or the antigen-binding fragment thereof is administered at a dose of 200 mg, and the administration frequency is every 2 weeks Once or every 3 weeks, the anti-PD-L1 antibody or antigen-binding fragment thereof is administered at a dose selected from 1-50 mg / kg or 50-3000 mg, and the administration frequency is once every two weeks or once every three weeks.

In an alternative embodiment, the TLR agonist is administered at a dose of 0.50 mg, and the administration frequency is once a week or once every two weeks; the immune checkpoint inhibitor is selected from an anti-PD-1 antibody or an antigen-binding fragment thereof or an anti-PD-1 antibody PD-L1 antibody or antigen-binding fragment thereof, the anti-PD-1 antibody or antigen-binding fragment thereof is administered at a dose of 200 mg, and the administration frequency is once every 2 weeks or once every 3 weeks, and the anti-PD-L1 antibody or The dose of the antigen-binding fragment is selected from 1-50 mg / kg or 50-3000 mg, and the frequency of administration is once every two weeks or once every three weeks. .

In an alternative embodiment, the TLR agonist is administered at a dose of 1 mg, and the frequency of administration is once a week or once every two weeks; the immune checkpoint inhibitor is selected from an anti-PD-1 antibody or an antigen-binding fragment thereof or an anti-PD -L1 antibody or antigen-binding fragment thereof, the anti-PD-1 antibody or antigen-binding fragment thereof is administered at a dose of 200 mg, and the administration frequency is once every 2 weeks or once every 3 weeks, and the anti-PD-L1 antibody or its The dose of the antigen-binding fragment is selected from 1-50 mg / kg or 50-3000 mg, and the frequency of administration is once every 2 weeks or once every 3 weeks.

In an alternative embodiment, the TLR agonist is administered at a dose of 2 mg, and the administration frequency is once a week or once every two weeks; the immune checkpoint inhibitor is selected from an anti-PD-1 antibody or an antigen-binding fragment thereof or an anti-PD -L1 antibody or antigen-binding fragment thereof, the anti-PD-1 antibody or antigen-binding fragment thereof is administered at a dose of 200 mg, and the administration frequency is once every 2 weeks or once every 3 weeks, and the anti-PD-L1 antibody or its The dose of the antigen-binding fragment is selected from 1-50 mg / kg or 50-3000 mg, and the frequency of administration is once every 2 weeks or once every 3 weeks.

In an alternative embodiment, the TLR agonist is administered at a dose of 3 mg, and the administration frequency is once a week or once every two weeks; the immune checkpoint inhibitor is selected from an anti-PD-1 antibody or an antigen-binding fragment thereof or an anti-PD -L1 antibody or antigen-binding fragment thereof, the anti-PD-1 antibody or antigen-binding fragment thereof is administered at a dose of 200 mg, and the administration frequency is once every 2 weeks or once every 3 weeks, and the anti-PD-L1 antibody or its The dose of the antigen-binding fragment is selected from 1-50 mg / kg or 50-3000 mg, and the frequency of administration is once every 2 weeks or once every 3 weeks.

In an alternative embodiment, the TLR agonist is administered at a dose of 0.50 mg, and the administration frequency is every 2 weeks; the immune checkpoint inhibitor is selected from an anti-PD-1 antibody or an antigen-binding fragment thereof or an anti-PD-L1 antibody Or its antigen-binding fragment, the anti-PD-1 antibody or its antigen-binding fragment is administered at a dose of 200 mg, and the frequency of administration is once every 2 weeks or every 3 weeks, the anti-PD-L1 antibody or its antigen-binding fragment The dosage is selected from 1-50 mg / kg or 50-3000 mg, and the frequency of administration is once every 2 weeks or once every 3 weeks.

In an alternative embodiment, the TLR agonist is administered at a dose of 1 mg and the frequency of administration is every two weeks; the immune checkpoint inhibitor is selected from an anti-PD-1 antibody or an antigen-binding fragment thereof or an anti-PD-L1 antibody or The anti-PD-1 antibody or the antigen-binding fragment thereof is administered at a dose of 200 mg and the frequency of administration is once every 2 weeks or every 3 weeks. The administration dose is selected from 1-50 mg / kg or 50-3000 mg, and the administration frequency is once every 2 weeks or once every 3 weeks.

In an alternative embodiment, the TLR agonist is administered at a dose of 2 mg, and the administration frequency is every 2 weeks; the immune checkpoint inhibitor is selected from an anti-PD-1 antibody or an antigen-binding fragment thereof or an anti-PD-L1 antibody or The anti-PD-1 antibody or the antigen-binding fragment thereof is administered at a dose of 200 mg and the frequency of administration is once every 2 weeks or every 3 weeks. The administration dose is selected from 1-50 mg / kg or 50-3000 mg, and the administration frequency is once every 2 weeks or once every 3 weeks.

In an alternative embodiment, the TLR agonist is administered at a dose of 3 mg and the frequency of administration is every 2 weeks; the immune checkpoint inhibitor is selected from an anti-PD-1 antibody or an antigen-binding fragment thereof or an anti-PD-L1 antibody or The anti-PD-1 antibody or the antigen-binding fragment thereof is administered at a dose of 200 mg and the frequency of administration is once every 2 weeks or every 3 weeks. The administration dose is selected from 1-50 mg / kg or 50-3000 mg, and the administration frequency is once every 2 weeks or once every 3 weeks.

In an alternative embodiment, the TLR agonist is administered at a dose of 2.0 mg, and the administration frequency is once every 3 days or once a week; the immune checkpoint inhibitor is selected from an anti-PD-1 antibody or an antigen-binding fragment thereof or Anti-PD-L1 antibody or antigen-binding fragment thereof, the anti-PD-1 antibody or antigen-binding fragment thereof is administered at a dose of 200 mg, and the administration frequency is once every 2 weeks or once every 3 weeks, the anti-PD-L1 antibody Or the antigen-binding fragment thereof is administered at a dose selected from 1-50 mg / kg or 50-3000 mg, and the administration frequency is once every 2 weeks or once every 3 weeks.

In an alternative embodiment, the TLR agonist is administered at a dose of 4.0 mg, and the administration frequency is once every 3 days or once a week; the immune checkpoint inhibitor is selected from an anti-PD-1 antibody or an antigen-binding fragment thereof or Anti-PD-L1 antibody or antigen-binding fragment thereof, the anti-PD-1 antibody or antigen-binding fragment thereof is administered at a dose of 200 mg, and the administration frequency is once every 2 weeks or once every 3 weeks, the anti-PD-L1 antibody Or the antigen-binding fragment thereof is administered at a dose selected from 1-50 mg / kg or 50-3000 mg, and the administration frequency is once every 2 weeks or once every 3 weeks.

In an alternative embodiment, the TLR agonist is administered at a dose of 6.0 mg, and the administration frequency is once every 3 days or once a week; the immune checkpoint inhibitor is selected from an anti-PD-1 antibody or an antigen-binding fragment thereof or Anti-PD-L1 antibody or antigen-binding fragment thereof, the anti-PD-1 antibody or antigen-binding fragment thereof is administered at a dose of 200 mg, and the administration frequency is once every 2 weeks or once every 3 weeks, and the anti-PD-L1 antibody Or the antigen-binding fragment thereof is administered at a dose selected from 1-50 mg / kg or 50-3000 mg, and the administration frequency is once every 2 weeks or once every 3 weeks.

In an alternative embodiment, the TLR agonist is administered at a dose of 8.0 mg, and the administration frequency is once every 3 days or once a week; the immune checkpoint inhibitor is selected from an anti-PD-1 antibody or an antigen-binding fragment thereof or Anti-PD-L1 antibody or antigen-binding fragment thereof, the anti-PD-1 antibody or antigen-binding fragment thereof is administered at a dose of 200 mg, and the administration frequency is once every 2 weeks or once every 3 weeks, the anti-PD-L1 antibody Or the antigen-binding fragment thereof is administered at a dose selected from 1-50 mg / kg or 50-3000 mg, and the administration frequency is once every 2 weeks or once every 3 weeks.

In an alternative embodiment, the dose of the TLR agonist is 9 mg, and the frequency of administration is once every 3 days or once a week; the immune checkpoint inhibitor is selected from an anti-PD-1 antibody or an antigen-binding fragment thereof or an anti-PD-1 antibody. PD-L1 antibody or antigen-binding fragment thereof, the anti-PD-1 antibody or antigen-binding fragment thereof is administered at a dose of 200 mg, and the administration frequency is once every 2 weeks or once every 3 weeks, and the anti-PD-L1 antibody or The dose of the antigen-binding fragment is selected from 1-50 mg / kg or 50-3000 mg, and the frequency of administration is once every two weeks or once every three weeks.

In an alternative embodiment, the TLR agonist is administered at a dose of 12 mg, and the frequency of administration is once every 3 days or once a week; the immune checkpoint inhibitor is selected from an anti-PD-1 antibody or an antigen-binding fragment thereof or an anti-PD-1 antibody. PD-L1 antibody or antigen-binding fragment thereof, the anti-PD-1 antibody or antigen-binding fragment thereof is administered at a dose of 200 mg, and the administration frequency is once every 2 weeks or once every 3 weeks, and the anti-PD-L1 antibody or The dose of the antigen-binding fragment is selected from 1-50 mg / kg or 50-3000 mg, and the frequency of administration is once every two weeks or once every three weeks.

In certain embodiments, any combination therapy disclosed in this disclosure lasts for at least 3 weeks, or at least 1 month, or at least 3 months, or at least 6 months, or 9 months, or at least 1 year, or at least 18 months, or at least 24 months, or at least 3 years, or at least 5 years.

The tumor described in the present disclosure is selected from lung cancer, non-small cell lung cancer, hepatocellular carcinoma, basal cell carcinoma, renal cell carcinoma, myeloma, rectal cancer, colon cancer, breast cancer, gastric cancer, bone cancer, pancreatic cancer, skin cancer , Head or neck cancer, skin or eye malignant melanoma, uterine cancer, ovarian cancer, anal cancer, gastric cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vaginal cancer, He Jiejin 'S disease, non-Hodgkin's lymphoma, esophageal cancer, small bowel cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, acute myeloid cells Leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) Neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T Any combination of the tumor cells and lymphoma, preferably hepatocellular carcinoma, colorectal cancer, colon cancer, non-small cell lung cancer.

In an alternative embodiment, the tumor in the use in the present disclosure is hepatocellular carcinoma, intrahepatic bile duct cell carcinoma, bile duct pancreatic carcinoma, and mixed liver cancer.

In an alternative embodiment, the tumor in the use in the present disclosure is lung cancer.

In an alternative embodiment, the tumor in the use in the present disclosure is non-small cell lung cancer, and the non-small cell lung cancer described in the present disclosure is selected from squamous cell carcinoma and non-squamous cell carcinoma, preferably non-phosphorous cell carcinoma .

In an alternative embodiment, the tumor in the use in the present disclosure is rectal cancer, colon cancer.

The combined route of administration of the present disclosure includes, but is not limited to, oral, parenteral, and transdermal administration. The parenteral administration includes, but is not limited to, intravenous, subcutaneous, and intramuscular injection.

The immune checkpoint inhibitors of the present disclosure can be incorporated into a composition, for example, a pharmaceutical composition containing an antibody and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In one embodiment, the carrier for the antibody-containing composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, abdominal, spinal or epidermal administration (e.g., by injection or infusion), and 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, emulsifying agents, and dispersing agents.

In a preferred embodiment of the present disclosure, the PD-1 antibody or antigen-binding fragment thereof is administered by injection, for example, subcutaneously or intravenously or intraperitoneally. Before injection, the PD-1 antibody or antigen-binding fragment thereof is required. Formulated in injectable form. An injectable form of an anti-PD-1 antibody or an antigen-binding fragment thereof in a particularly preferred embodiment of the present disclosure is an injection solution or a lyophilized powder injection, which comprises a PD-1 antibody or an antigen-binding fragment thereof, a buffer, a stabilizer, Optionally also contains a surfactant. The buffer may be selected from one or more of acetate, citrate, succinate, and phosphate. The stabilizer may be selected from sugars or amino acids, preferably disaccharides such as sucrose, lactose, trehalose, maltose. The surfactant is selected from polyoxyethylene hydrogenated castor oil, glycerin fatty acid ester, polyoxyethylene sorbitan fatty acid ester, preferably the polyoxyethylene sorbitan fatty acid ester is polysorbate 20, 40, 60, or 80 Of these, polysorbate 20 is most preferred. The most preferred injectable form of the PD-1 antibody or antigen-binding fragment thereof comprises the PD-1 antibody or antigen-binding fragment thereof, an acetate buffer, trehalose, and polysorbate 20.

The administration route of the TLR agonist in the present disclosure may be the same as or different from the above immune checkpoint inhibitors, and specifically includes oral administration, nasal cavity, topical, intravenous, intramuscular, subcutaneous, intraperitoneal, intralesional, and spinal Or other parenteral routes of administration.

In order to facilitate administration, the TLR agonist needs to be configured into a composition. When the TLR agonist is administered orally, the carrier of the composition includes, but is not limited to, a filler, a lubricant, a disintegrant, a binder, and the like.

In some embodiments, the TLR agonist is administered to a patient by intralesional (specifically, intratumoral) injection, or by inhalation. The carrier of the composition includes, but is not limited to, buffer Agent, osmotic pressure regulator.

In an alternative embodiment of the present disclosure, the TLR agonist is administered at a dose of 0.50 mg, the frequency of administration is once a week or once every two weeks, and the tumor is administered intratumorally; the immune checkpoint inhibitor is selected from an anti-PD-1 antibody Or an antigen-binding fragment thereof or an anti-PD-L1 antibody or an antigen-binding fragment thereof, the anti-PD-1 antibody or the antigen-binding fragment thereof is administered at a dose of 200 mg, and the administration frequency is once every two weeks or once every three weeks. The anti-PD-L1 antibody or antigen-binding fragment thereof is administered at a dose selected from 1-50 mg / kg or 50-3000 mg, and the administration frequency is once every two weeks or once every three weeks.

In an alternative embodiment, the TLR agonist is administered at a dose of 1 mg, the frequency of administration is once a week or once every two weeks, and the tumor is administered intratumorally; the immune checkpoint inhibitor is selected from an anti-PD-1 antibody or an antigen thereof A binding fragment or an anti-PD-L1 antibody or an antigen-binding fragment thereof, the anti-PD-1 antibody or an antigen-binding fragment thereof is administered at a dose of 200 mg, and the administration frequency is once every 2 weeks or once every 3 weeks, and the anti-PD -L1 antibody or antigen-binding fragment thereof is administered at a dose selected from 1-50 mg / kg or 50-3000 mg, and the administration frequency is once every 2 weeks or once every 3 weeks.

In an alternative embodiment, the TLR agonist is administered at a dose of 2 mg, the frequency of administration is once a week or once every two weeks, and the tumor is administered intratumorally; the immune checkpoint inhibitor is selected from an anti-PD-1 antibody or an antigen thereof A binding fragment or an anti-PD-L1 antibody or an antigen-binding fragment thereof, the anti-PD-1 antibody or an antigen-binding fragment thereof is administered at a dose of 200 mg, and the administration frequency is once every 2 weeks or once every 3 weeks, and the anti-PD -L1 antibody or antigen-binding fragment thereof is administered at a dose selected from 1-50 mg / kg or 50-3000 mg, and the administration frequency is once every 2 weeks or once every 3 weeks.

In an alternative embodiment, the TLR agonist is administered at a dose of 3 mg, the frequency of administration is once a week or once every two weeks, and the tumor is administered intratumorally; the immune checkpoint inhibitor is selected from an anti-PD-1 antibody or an antigen thereof A binding fragment or an anti-PD-L1 antibody or an antigen-binding fragment thereof, the anti-PD-1 antibody or an antigen-binding fragment thereof is administered at a dose of 200 mg, and the administration frequency is once every 2 weeks or once every 3 weeks, and the anti-PD -L1 antibody or antigen-binding fragment thereof is administered at a dose selected from 1-50 mg / kg or 50-3000 mg, and the administration frequency is once every 2 weeks or once every 3 weeks.

In an alternative embodiment, the TLR agonist is administered at a dose of 0.50 mg, and the administration frequency is once every 2 weeks, and is administered intratumorally; the immune checkpoint inhibitor is selected from an anti-PD-1 antibody or an antigen-binding fragment thereof or Anti-PD-L1 antibody or antigen-binding fragment thereof, the anti-PD-1 antibody or antigen-binding fragment thereof is administered at a dose of 200 mg, and the administration frequency is once every 2 weeks or once every 3 weeks, the anti-PD-L1 antibody Or the antigen-binding fragment thereof is administered at a dose selected from 1-50 mg / kg or 50-3000 mg, and the administration frequency is once every 2 weeks or once every 3 weeks.

In an alternative embodiment, the TLR agonist is administered at a dose of 1 mg, and the frequency of administration is once every two weeks, and is administered intratumorally; the immune checkpoint inhibitor is selected from an anti-PD-1 antibody or an antigen-binding fragment thereof or an anti-PD-1 antibody. PD-L1 antibody or antigen-binding fragment thereof, the anti-PD-1 antibody or antigen-binding fragment thereof is administered at a dose of 200 mg, and the administration frequency is once every 2 weeks or once every 3 weeks, and the anti-PD-L1 antibody or The dose of the antigen-binding fragment is selected from 1-50 mg / kg or 50-3000 mg, and the frequency of administration is once every two weeks or once every three weeks.

In an alternative embodiment, the TLR agonist is administered at a dose of 2 mg, and the frequency of administration is every 2 weeks, and the tumor is administered intratumorally; the immune checkpoint inhibitor is selected from an anti-PD-1 antibody or an antigen-binding fragment thereof or an anti-PD-1 antibody. PD-L1 antibody or antigen-binding fragment thereof, the anti-PD-1 antibody or antigen-binding fragment thereof is administered at a dose of 200 mg, and the administration frequency is once every 2 weeks or once every 3 weeks, and the anti-PD-L1 antibody or The dose of the antigen-binding fragment is selected from 1-50 mg / kg or 50-3000 mg, and the frequency of administration is once every two weeks or once every three weeks.

In an alternative embodiment, the TLR agonist is administered at a dose of 3 mg, and the administration frequency is once every 2 weeks, and is administered intratumorally; the immune checkpoint inhibitor is selected from an anti-PD-1 antibody or an antigen-binding fragment thereof or an anti-PD-1 antibody PD-L1 antibody or antigen-binding fragment thereof, the anti-PD-1 antibody or antigen-binding fragment thereof is administered at a dose of 200 mg, and the administration frequency is once every 2 weeks or once every 3 weeks, and the anti-PD-L1 antibody or The dose of the antigen-binding fragment is selected from 1-50 mg / kg or 50-3000 mg, and the frequency of administration is once every 2 weeks or once every 3 weeks.

In an alternative embodiment, the TLR agonist is administered at a dose of 2.0 mg, the frequency of administration is once every 3 days or once a week, and the oral administration is performed; the immune checkpoint inhibitor is selected from an anti-PD-1 antibody or its An antigen-binding fragment or an anti-PD-L1 antibody or an antigen-binding fragment thereof, the anti-PD-1 antibody or an antigen-binding fragment thereof is administered at a dose of 200 mg, and the administration frequency is once every 2 weeks or once every 3 weeks, and the antibody The dose of PD-L1 antibody or antigen-binding fragment thereof is selected from 1-50 mg / kg or 50-3000 mg, and the frequency of administration is once every two weeks or once every three weeks.

In an alternative embodiment, the TLR agonist is administered at a dose of 4.0 mg, the frequency of administration is once every 3 days or once a week, and the oral administration is performed; the immune checkpoint inhibitor is selected from an anti-PD-1 antibody or its An antigen-binding fragment or an anti-PD-L1 antibody or an antigen-binding fragment thereof, the anti-PD-1 antibody or an antigen-binding fragment thereof is administered at a dose of 200 mg, and the administration frequency is once every 2 weeks or once every 3 weeks, and the antibody The dose of PD-L1 antibody or antigen-binding fragment thereof is selected from 1-50 mg / kg or 50-3000 mg, and the frequency of administration is once every two weeks or once every three weeks.

In an alternative embodiment, the TLR agonist is administered at a dose of 6.0 mg, the frequency of administration is once every 3 days or once a week, and the oral administration is performed; the immune checkpoint inhibitor is selected from an anti-PD-1 antibody or its An antigen-binding fragment or an anti-PD-L1 antibody or an antigen-binding fragment thereof, the anti-PD-1 antibody or an antigen-binding fragment thereof is administered at a dose of 200 mg, and the administration frequency is once every 2 weeks or once every 3 weeks, and the antibody The dose of PD-L1 antibody or antigen-binding fragment thereof is selected from 1-50 mg / kg or 50-3000 mg, and the frequency of administration is once every two weeks or once every three weeks.

In an alternative embodiment, the TLR agonist is administered at a dose of 8.0 mg, the frequency of administration is once every 3 days or once a week, and the oral administration is performed; the immune checkpoint inhibitor is selected from an anti-PD-1 antibody or its An antigen-binding fragment or an anti-PD-L1 antibody or an antigen-binding fragment thereof, the anti-PD-1 antibody or an antigen-binding fragment thereof is administered at a dose of 200 mg, and the administration frequency is once every 2 weeks or once every 3 weeks, and the antibody The dose of PD-L1 antibody or antigen-binding fragment thereof is selected from 1-50 mg / kg or 50-3000 mg, and the frequency of administration is once every two weeks or once every three weeks.

In an alternative embodiment, the TLR agonist is administered at a dose of 9 mg, the frequency of administration is once every 3 days or once a week, and the oral administration is performed; the immune checkpoint inhibitor is selected from an anti-PD-1 antibody or an antigen thereof A binding fragment or an anti-PD-L1 antibody or an antigen-binding fragment thereof, the anti-PD-1 antibody or an antigen-binding fragment thereof is administered at a dose of 200 mg, and the administration frequency is once every 2 weeks or once every 3 weeks, and the anti-PD -L1 antibody or antigen-binding fragment thereof is administered at a dose selected from 1-50 mg / kg or 50-3000 mg, and the administration frequency is once every 2 weeks or once every 3 weeks.

In an alternative embodiment, the TLR agonist is administered at a dose of 12 mg, the frequency of administration is once every 3 days or once a week, and orally administered; the immune checkpoint inhibitor is selected from an anti-PD-1 antibody or an antigen thereof A binding fragment or an anti-PD-L1 antibody or an antigen-binding fragment thereof, the anti-PD-1 antibody or an antigen-binding fragment thereof is administered at a dose of 200 mg, and the administration frequency is once every 2 weeks or once every 3 weeks, and the anti-PD -L1 antibody or antigen-binding fragment thereof is administered at a dose selected from 1-50 mg / kg or 50-3000 mg, and the administration frequency is once every 2 weeks or once every 3 weeks.

In an alternative embodiment, the TLR agonist is administered orally, intratumorally, or by inhalation of a pharmaceutical composition containing the TLR agonist.

The present disclosure provides a TLR agonist-containing pharmaceutical composition, wherein the compound represented by formula (I) or a complex thereof or a pharmaceutically acceptable salt thereof has a pH of 3.0-7.0, specifically 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, and the preferred pH range is 4.5-6.0.

The TLR agonist-containing pharmaceutical composition provided by the present disclosure, wherein the buffering agent may include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, Tartrate, fumarate, glycylglycine and other organic acid salts.

Specifically, a buffering agent prevents a change in pH in a solution in the form of a "buffer", and a "buffer" refers to a buffer that is resistant to changes in pH by the action of its acid-base conjugate component. Examples of the buffer for controlling the pH in an appropriate range include acetate buffer, succinate buffer, gluconate buffer, histidine buffer, oxalate buffer, lactate buffer, phosphate Salt buffer, citrate buffer, tartrate, buffer, fumarate buffer, glycylglycine buffer and other organic acid buffers.

A "citrate buffer" is a buffer that includes citrate ions. Examples of the citrate buffer include citric acid-sodium citrate, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-magnesium citrate, and the like. A preferred citrate buffer is citric acid-sodium citrate.

A "succinate buffer" is a buffer that includes succinate ions. Examples of the succinate buffer include succinate-sodium succinate, succinate-potassium succinate, succinate-calcium succinate and the like. A preferred succinate buffer is succinate-sodium succinate.

A "phosphate buffer" is a buffer that includes phosphate ions. Examples of the phosphate buffer include disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, and the like. A preferred phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate.

"Acetate buffer" is a buffer that includes acetate ions. Examples of the acetate buffer include acetate-sodium acetate, histidine acetate, acetate-potassium acetate, calcium acetate, acetate-magnesium acetate, and the like. The preferred acetate buffer is acetate-sodium acetate.

In an alternative embodiment, the buffer is selected from acetate buffer, citric acid-citric acid buffer, preferably from citric acid-sodium citrate buffer.

In an alternative embodiment, the concentration of the buffer in the TLR agonist-containing pharmaceutical composition is 1-150 mM (mmol), and may be 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45mM, 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, 80mM, 85mM, 90mM, 95mM, 100mM, 105mM, 110mM, 115mM, 120mM, 125mM, 130mM, 135mM, 140mM, 145mM or 150mM, preferably 10-80mM, Most preferred is 20 mM.

Further, the osmotic pressure of the TLR agonist-containing pharmaceutical composition is 150-500 mOsmol / kg, preferably 200-400 mOsmol / kg, most preferably 280-320 mOsmol / kg, and specifically 280 mOsmol / kg, 281 mOsmol / kg, 282 mOsmol / kg, 283mOsmol / kg, 284mOsmol / kg, 285mOsmol / kg, 286mOsmol / kg, 287mOsmol / kg, 288mOsmol / kg, 289mOsmol / kg, 290mOsmol / kg, 291mOsmol / kg, 292mOsmol / kg, 293mOsmol / kg, 294mOsmol / kg , 295mOsmol / kg, 296mOsmol / kg, 297mOsmol / kg, 298mOsmol / kg, 299mOsmol / kg, 300mOsmol / kg, 301mOsmol / kg, 302mOsmol / kg, 303mOsmol / kg, 304mOsmol / kg, 305mOsmol / kg, 306mOsmol / kg / kg, 308mOsmol / kg, 309mOsmol / kg, 310mOsmol / kg, 311mOsmol / kg, 312mOsmol / kg, 313mOsmol / kg, 314mOsmol / kg, 315mOsmol / kg, 316mOsmol / kg, 317mOsmol / kg, 318mOsmol / kg, 319mOsmol / kg Or 320mOsmol / kg.

In an alternative embodiment, the TLR agonist-containing pharmaceutical composition further comprises an osmotic pressure regulator, which is preferably selected from sodium chloride, glucose, sorbitol, glycerol, PEG, propylene glycol, and the like The content of one or more osmotic pressure regulators is between 0.001 and 100 mg / ml.

In a preferred embodiment, the osmotic pressure regulator is sodium chloride, and the concentration of sodium chloride is about 0.1-100 mg / ml. Non-limiting examples include 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, and 1.5 mg. / mL, 2.0mg / mL, 2.5mg / mL, 3.0mg / mL, 3.5mg / mL, 4.0mg / mL, 4.5mg / mL, 5.0mg / mL, 5.5mg / mL, 6.0mg / mL, 6.5mg / mL, 7.0mg / mL, 7.5mg / mL, 8.0mg / mL, 8.5mg / mL, 9.0mg / mL, 9.5mg / mL, 10.0mg / mL, preferably 4-10mg / ml.

In an alternative embodiment, the concentration of the TLR agonist in the TLR agonist-containing pharmaceutical composition is 0.01-10 mg / mL, preferably 0.1-10 mg / mL, specifically 0.1 mg / mL, 0.2 mg / mL, 0.3mg / mL, 0.4mg / mL, 0.5mg / mL, 0.6mg / mL, 0.7mg / mL, 0.8mg / mL, 0.9mg / mL or 1.0mg / ml, 1.1mg / mL, 1.2mg / mL, 1.3mg / mL, 1.4mg / mL, 1.5mg / mL, 1.6mg / mL, 1.7mg / mL, 1.8mg / mL, 1.9mg / mL, 2.0mg / mL, 2.1mg / mL, 2.2mg / mL, 2.3mg / mL, 2.4mg / mL, 2.5mg / mL, 2.6mg / mL, 2.7mg / mL, 2.8mg / mL, 2.9mg / mL, 3.0mg / mL, 3.1mg / mL, 3.2mg / mL, 3.3mg / mL, 3.4mg / mL, 3.5mg / mL, 3.6mg / mL, 3.7mg / mL, 3.8mg / mL, 3.9mg / mL, 4.0mg / mL, 4.1mg / mL, 4.2mg / mL, 4.3 mg / mL, 4.4 mg / mL, 4.5 mg / mL, 4.6 mg / mL, 4.7 mg / mL, 4.8 mg / mL, 4.9 mg / mL, 5.0 mg / mL.

In one embodiment, the TLR agonist-containing pharmaceutical composition in the present disclosure comprises: 0.01-10 mg / ml of a compound represented by formula (I) or a complex thereof or a pharmaceutically acceptable salt thereof, the pH of the pharmaceutical composition About 3.0-7.0.

In one embodiment, the TLR agonist-containing pharmaceutical composition in the present disclosure comprises: a) 0.01-10 mg / ml of a compound represented by formula (I) or a complex thereof or a pharmaceutically acceptable salt thereof, b) 1-150 mM Citric acid-sodium citrate buffer, the pH of the pharmaceutical composition is about 3.0-7.0.

In one embodiment, the TLR agonist-containing pharmaceutical composition in the present disclosure comprises: a) 0.01-10 mg / ml of a compound represented by formula (I) or a complex thereof or a pharmaceutically acceptable salt thereof, b) 1-150 mM Citric acid-sodium citrate buffer, c) 0.1-100 mg / ml sodium chloride, and the pH of the pharmaceutical composition is about 3.0-7.0.

In one embodiment, the TLR agonist-containing pharmaceutical composition in the present disclosure comprises: a) 0.1-10 mg / ml of a compound represented by formula (I) or a complex thereof or a pharmaceutically acceptable salt thereof, and 10-80 mM lemon Acid-sodium citrate buffer, pH range 4.5-6.0.

The present disclosure also provides a method for preparing the aforementioned TLR agonist-containing pharmaceutical composition. The method includes a step of mixing a TLR agonist and a buffer liquid phase, and further, a step of adding sodium chloride.

In some embodiments, the aforementioned TLR agonist-containing pharmaceutical composition is stable at 25 ° C for at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months.

In some embodiments, the aforementioned TLR agonist-containing pharmaceutical composition is stable at 60 ° C for at least 10 days, at least 20 days, or at least 30 days.

The present disclosure also relates to a pharmaceutical composition comprising a TLR agonist, an immune checkpoint inhibitor, and one or more pharmaceutically acceptable carriers, excipients, and diluents. The pharmaceutical composition can be made into any pharmaceutically acceptable dosage form. For example, they can be formulated as tablets, capsules, pills, granules, solutions, suspensions, syrups, injections (including injections, sterile powders for injections and concentrated solutions for injections), suppositories, inhalants or sprays Agent.

The present disclosure also provides a pharmaceutical packaging box in which a pharmaceutical composition of a TLR agonist and an immune checkpoint inhibitor according to the present invention is packaged.

The present disclosure combines the administration of a TLR agonist and an immune checkpoint inhibitor, thereby enhancing antitumor activity and improving the therapeutic effect of tumors.

In the present disclosure, a method for treating a tumor is provided, which comprises administering to a patient a therapeutically effective amount of the above-mentioned TLR agonist and an immune checkpoint inhibitor.

Unless interpreted to the contrary, terms in this disclosure have the following meanings:

The term "frequency of administration" as used herein refers to the frequency of the administered dose of a drug disclosed herein at a given time. The frequency of dosing can be indicated as the number of doses per given time, for example, once a week, once every two weeks.

The “combination” described in the present disclosure is a mode of administration, which means that at least one dose of an immune checkpoint inhibitor and a TLR agonist is administered within a certain period of time, and both of the drugs show a pharmacological effect. The time period may be within a dosing cycle, preferably within 4 weeks, within 3 weeks, within 2 weeks, within 1 week, or within 24 hours. Immune checkpoint inhibitors and TLR agonists can be administered simultaneously or sequentially. This term includes treatment in which immune checkpoint inhibitors and TLR agonists are administered by the same route or different routes of administration.

The "humanized antibody" in the present disclosure, also known as CDR-grafted antibody, refers to the transplantation of mouse CDR sequences into the human antibody variable region framework, that is, different types Antibodies produced in human germline antibody framework sequences. It can overcome the strong antibody variable antibody response induced by the chimeric antibody because it carries a large amount of mouse protein components. Such framework sequences can be obtained from a public DNA database including germline antibody gene sequences or published references. For example, germline DNA sequences of human heavy chain and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet www.mrccpe.com.ac.uk/vbase), and in Kabat, EA, etc. People, 1991 Sequences of Proteins of Immunological Interest, 5th edition. In a preferred embodiment of the present disclosure, the CDR sequence of the PD-1 humanized antibody is selected from the group consisting of SEQ ID NO: 1,2,3,4,5,6.

The “antigen-binding fragment” described in the present disclosure refers to a Fab fragment, Fab ′ fragment, F (ab ′) 2 fragment, and Fv fragment sFv fragment that binds to human PD-1; The antibody is selected from one or more CDR regions of SEQ ID NO: 1 to SEQ ID NO: 6. The Fv fragment contains the variable region of the heavy chain and light chain of the antibody, but has no constant region and has the smallest antibody fragment with all antigen-binding sites. Generally, Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming the structure required for antigen binding. The variable regions of two antibodies can also be linked into a single polypeptide chain with different linkers, called single chain antibodies (single chain antibodies) or single chain Fv (sFv). The term "binding to PD-1" in this disclosure refers to the ability to interact with human PD-1. The term "antigen-binding site" in the present disclosure refers to a three-dimensional spatial site on the antigen that is discontinuous and is recognized by the antibody or antigen-binding fragment in the present disclosure.

"Immune checkpoint inhibitors" as described in this disclosure do not include TLR agonists.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. The weight change curve of Hepa1-6 orthotopic transplantation model tumor-bearing mice in the treatment of drug A and drug B;

Figure 2. Hepa1-6 orthotopic transplantation model tumor-bearing mice in the treatment of drug A and drug B weight change curve;

Figure 3. Hepa1-6 orthotopic transplantation model tumor-bearing mice serum AFP concentration in tumor-bearing mice during drug A and drug B treatment;

Figure 4. Tumor weight of Hepa1-6 orthotopic transplantation model tumor-bearing mice in the treatment of drug A and drug B.

Figure 5. Body weight of mice treated with an LLC lung cancer animal model of hPD-1 humanized transgenic mice;

Figure 6. Curves of body weight changes of mice with the human lung cancer transgenic mouse model of hPD-1 after treatment with an LLC lung cancer animal model;

Figure 7. Tumor weight data after the end of an LLC lung cancer animal model experiment with hPD-1 humanized transgenic mice.

Figure 8. Efficacy of intratumoral administration of drug A or combination with PD-L1 monoclonal antibody drug C on subcutaneously transplanted tumors of MC38 / H-11 mice (administration end, Mean ± SEM, n = 10);

Figure 9. Efficacy of intratumoral administration of drug A alone or in combination with PD-L1 monoclonal antibody drug C on subcutaneously transplanted tumors in MC38 / H-11 mice (non-administration end, n = 10);

Figure 10. Effect of drug A, drug C single and combination of two drugs on body weight of tumor-bearing nude mice (Mean ± SD; n = 10).

detailed description

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

Example 1 The pre-clinical verification of the same compound dihydrochloride (drug A) as shown in the test formula (I) in female PD-1 HuGEMM mice was the same as that of in situ vaccination with PD-1 antibody (drug B). Effect of Source Transplantation Model Hepa1-6

Experimental material

1) Animal

Strain: PD-1HuGEMM mouse; Supplier: Nanjing Yinhe Biological Medicine Co., Ltd. Age: 6-9 weeks (start of administration); Gender: Female; Number of animals: 115

2) Test and control drugs

Drug A Dihydrochloride Compound of Formula (I)

Preparation method: A compound represented by formula (I) (40 mg, 0.105 mmol, compound represented by formula (I) is prepared by a method disclosed in WO2018095426A) is dissolved in a mixed solvent of 0.5 mL of isopropanol and tetrahydrofuran (V / V = 1: 1) ), Stir to dissolve completely, raise the temperature to 50 ° C., dropwise add a 4M solution of hydrogen chloride in isopropanol (0.055 mL, 0.22 mmol), cool to room temperature and stir for 16 hours, and precipitate a white solid. The reaction solution was filtered, and the filter cake was collected and dried under vacuum to obtain Drug A.

Drug B

Anti-PD-1 antibody, PD-1 antibody disclosed in WO2017054646A, the sequences of heavy and light chains are shown in SEQ ID NO: 7, SEQ ID NO: 8, 200 mg, lyophilized powder injection.

IgG4: negative control, supplier: Shanghai Hengrui Pharmaceutical Co., Ltd.

3) ELISA kit for mouse AFP detection: the manufacturer is R & D

4) Model

The PD-1HuGEMM model is a transgenic mouse model that expresses human and mouse PD-1 genes in C57BL / 6 background mice. The homology model Hepa1-6 was used for the efficacy experiment.

2. Experimental method

A logarithmic tumor cell line Hepa1-6 was used to inoculate the right scapula of 4-7 week-old HuPD-1 human female mice. When subcutaneously inoculated Hepa1-6 seed tumors grow to 700-1000 mm ³ , the seed tumors are dissected out, cut to a diameter of 2-3 mm, and inoculated into mouse livers in situ. Four days after the inoculation, 63 mice in good condition were selected and randomly divided into 9 groups of 7 mice each. The day of the grouping was defined as Day0, and administration was started on Day0 (dosing treatment was started on the day of grouping). Dosing started on day 0 and ended on day 21. The nine dose groups, administration routes, and administration frequencies are as described in Table 1.

Tumor weighing: The animals in the experiment measured the weight of the mice twice a week. After the experiment, the tumor mass was dissected, the tumor weight was measured, and the T / C weight percentage was calculated. T weight and C weight respectively represent the administration group and the vehicle control. The tumor weight of the group. Data was collected using StudyDirector (version 3.1.399.19, vendor Studylog System, Inc) software.

AFP test: Serum samples were collected on day 21 (at the end of the experiment) of group administration for AFP test. The day of group dosing was defined as Day 0. (Approximately 100 μL of whole blood was collected per mouse, and 30-40 μL of serum samples were isolated).

Statistical analysis was performed using SPSS software based on tumor weight data at the end of the trial. All data comparisons were analyzed using Ttest. All data were analyzed using SPSS 18.0 and / or GraphPad Prism 5.0. All experimental results are expressed as "mean ± standard error". A P value of less than 0.05 is considered a significant difference.

Table 1.

Note: N: the number of animals in each group; i.p .: intraperitoneal injection; p.o .: oral gavage; QD: once a day; Q3D: once every 3 days;

Table 2. Test drug and control drug preparation methods

Note: The storage conditions are 4-8 ° C, and the type of administration is to adjust the dosage according to body weight (administration volume = 10 μL / g)

3. Experimental results

1) The weights of tumor-bearing mice in each treatment group and solvent-treated group at different time points are shown in Tables 3 and 4.

Weight of tumor-bearing mice in each treatment group and solvent treatment group Table 3

Weight of tumor-bearing mice in each treatment group and solvent treatment group Table 4

Note: Data are expressed as "mean ± standard error"

The weight change curve and weight change curve of tumor-bearing mice in each treatment group and solvent-treated group are shown in Fig. 1 and Fig. 2, respectively, from the first group to the ninth group.

Percentage change in body weight of tumor-bearing mice in group 1, group 2, group 3, group 4, group 5, group 6, group 7, group 8, and group 9 on day 21 after group treatment They are 5.72%, 9.53%, 11.73%, 10.56%, 8.35%, 10.83%, 8.15%, 6.63% and 8.59%.

In this experiment, a model of orthotopic liver transplantation was selected. After the tumor grew to a certain degree, it was very easy to invade, causing small granular tumors in the viscera of the mouse (such as the mesentery, medial peritoneum, etc.), resulting in Mice developed acute discomfort and caused death. In the experiment, the mice were well tolerant to the drug, and the weight loss of the mice did not exceed 10%.

2) The AFP concentration data and statistical analysis of tumor-bearing mice in each treatment group and solvent treatment group are shown in Table 5.

AFP concentration data and statistical analysis of data in tumor-bearing mice Table 5

Notes: a. Data are expressed as "mean ± standard error".

b. Using independent sample T-test to analyze the serum AFP value.

Hepa1-6 orthotopic transplantation model tumor-bearing mice in drug A and drug B treatment of serum AFP concentration in tumor-bearing mice is shown in Figure 3, in order from group 1 to group 9.

On the 21st day after the group treatment, compared with the control group, the test drugs Group 2, Group 3, Group 4, Group 5, Group 6, and Group 7 did not have statistically significant anti-hepatogen. The role of Hepa1-6 tumor growth in the allogeneic transplantation model (P> 0.05). However, compared with the vehicle-treated group, groups 8 and 9 of the high-dose combination treatment group have statistically significant anti-hepatic orthotopic transplantation model Hepa1-6 tumor growth effect (P <0.05) .

3) Tumor weight data and statistical analysis of tumor-bearing mice in each treatment group and solvent treatment group Table 6

Tumor weight data and statistical analysis of tumor-bearing mice Table 6

Notes: a. Data are expressed as "mean ± standard error";

b. Using independent sample T-test to analyze tumor weight.

The tumor weights of Hepa1-6 orthotopic transplantation model tumor-bearing mice in the treatment of drug A and drug B are shown in Figure 4, which are group 1 to group 9, respectively.

On the 21st day after the group treatment, compared with the control group, the test drugs Group 2, Group 3, Group 4, Group 5, Group 6, and Group 7 did not have statistically significant anti-hepatogen. The role of Hepa1-6 tumor growth in the allogeneic transplantation model (P> 0.05). The high-dose combination treatment groups 8 and 9 had statistically significant anti-hepatic orthotopic transplantation model Hepa1-6 tumor growth compared with the vehicle treatment group (P <0.05). .

Combined with the above experimental results, it was proved that the intraperitoneal injection of drug B at 3 mg / kg combined with oral drug A at 1 mg / kg and 3 mg / kg could significantly inhibit the tumor growth of orthotopic liver transplantation model Hepa1-6. Oral drug A combined with intraperitoneal injection of drug B has significant synergistic antitumor effect.

Example 2: Detecting the compound dihydrochloride (drug A) of formula (I) at different dosages in combination with PD-1 antibody (drug B) alone or in situ against human lung cancer of human PD Evaluation of the inhibitory effect of the growth of transplanted tumors, in which drug A was administered by nebulization.

1.Experimental materials

1) tumor cells

LLC tumor cells were cultured in RPMI1640 medium containing 10% fetal bovine serum. Cells were digested and passaged with trypsin containing EDTA as usual, passaged twice a week, placed in a 37 ° C, 5% CO ₂ incubator and continued to grow . Tumor cells in logarithmic growth phase were used to establish a model of xenograft tumors in vivo.

2) Experimental animals

Sixty B6 / JNju-pdcd1em2Cin345 / Nju humanized PD-1 transgenic mice, female, 4-8 weeks, provided by Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

3) Test sample

Drug A Dihydrochloride Compound of Formula (I)

Dissolve the compound represented by formula (I) (40 mg, 0.105 mmol, prepared by the method disclosed in WO2018095426) in 0.5 mL of a mixed solvent of isopropanol and tetrahydrofuran (V / V = 1: 1), The solution was stirred and completely dissolved. The temperature was raised to 50 ° C., and a 4M solution of hydrogen chloride in isopropanol (0.055 mL, 0.22 mmol) was added dropwise, and the mixture was cooled to room temperature and stirred for 16 hours to precipitate a white solid. The reaction solution was filtered, and the filter cake was collected and dried under vacuum to obtain Drug A.

Drug A is configured according to the prescription in Table 7.

Table 7

Material name	Composition of each medicine prescription
Drug A	1mg
Citric acid	2.101mg
Sodium chloride	7.50mg
Sodium hydroxide	N / A
Water for Injection	1ml

Drug B

For the PD-1 antibody disclosed in WO2017054646A, the sequences of the heavy and light chains are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively;

IgG4: negative control, supplier: Shanghai Hengrui Pharmaceutical Co., Ltd.

4) Experimental equipment

The small animal atomized drug delivery system is Sansbio SA-YLS-8B.

2. Experimental steps and methods

1) LLC mouse lung cancer orthotopic tumor cell inoculation

After digestion with trypsin, LLC tumor cells in logarithmic growth phase were collected, P10, the cell concentration was adjusted to 8 × 10 ⁶ / mL with PBS buffer, and 0.025 mL of LLC cell suspension was injected into lung tissue with an insulin injection needle. The injected mice need to be anesthetized, and all mice are weighed before the cell injection operation and recorded as the initial weight. The mice to be operated were fully anesthetized with a mixture of isoflurane and oxygen. The animals were placed in the left lateral position at the time of injection. All mice were pelted before surgery, sterilized with iodophor and alcohol cotton balls, and then operated. The skin and muscle tissue were cut at the third and fourth ribs on the left side of the animal, and the left lung lobe of the animal was found under a dissecting microscope. The cells were injected into the left lung lobe with an insulin needle, and the wound was sutured with 5-0, and the mice were placed in the cage after recovery of activity. All surgical instruments were autoclaved in advance. The day of surgery was PG-D0. On the 6th day after the inoculation, mice with a poor state of rejection and underweight were selected and grouped according to body weight and observed.

The specific grouping information is shown in Table 8 below:

Table 8. Administration routes, doses and groups of pharmacodynamic experiments of LLCPD animal models of hPD-1 humanized transgenic mice

Remarks: i.p .: intraperitoneal administration; QD: once a day; Q3D once every 3 days.

2) Test drug configuration

Table 9.

3) Evaluation indicators

The main purpose is to test the inhibitory effect of test compounds on hPD-1 humanized mouse LLC lung cancer cell transplantation tumors. Measurement of tumor volume and weight of tumor-bearing mice: At the end of the experiment, tumor masses were dissected out and the tumor weight was measured.

All mice were euthanized on the 15th day after the start of the experiment, and the experiment was ended.

The data were expressed by Mean ± SEM. The statistical analysis software Graphgad Prism was used to test the data for normal distribution and homogeneity of variance. One-way analysis of variance was selected based on the test results. * P <0.05 will be considered a statistically significant difference.

3. Experimental results

FIG. 5 shows the body weight of an animal model of LLC lung cancer treated with a humanized transgenic mouse of hPD-1;

FIG. 6 is a curve of body weight changes of an animal model of LLC lung cancer of a humanized transgenic mouse of hPD-1 after treatment;

Figure 7 shows tumor weight data after the end of an LLC lung cancer animal model experiment of hPD-1 humanized transgenic mice (* P <0.05, ** P <0.01, compared to the solvent group).

Table 10. Tumor mass (g) (Mean ± SEM) n = 8

Group	Tumor mass (g) a	P value
1	0.354 ± 0.067	-
2	0.091 ± 0.036 **	0.0014
3	0.135 ± 0.043 **	0.0084
4	0.12 ± 0.08 **	0.0038
5	0.082 ± 0.052 ***	0.001
6	0.031 ± 0.018 ***	0.001

Notes: a. Data are expressed as "mean ± standard error";

b. ** P <0.01, *** P≤0.001, compared with the solvent group.

This experiment examined the inhibitory effect of test compound drug A combined with drug B at different doses on the growth of LLC orthotopic transplantation tumors of lung cancer cells.

During the experiment, the test compound drug A was administered by nebulization, and the control group was nebulized and administered the same volume of the drug A placebo solution. Body weight measurements were performed twice a week during the dosing period. The data and details are shown in Figure 5. The average tumor mass in the solvent control group was 0.354g, and the tumor weights in the other administration groups were 0.091g (Drug A 1mL nebulized, QD), 0.135g (Drug A 3mL nebulized, QD), 0.12 g (drug B 3mg / kg IP, Q3D), 0.082g (drug A 1mL, nebulization, QD + drug B 3mg / kg, ip, Q3D) and 0.031g (drug A 3mL, nebulization, QD + drug B 3mg / kg , Ip, Q3D), each treatment group showed a significant inhibitory effect on the in situ growth of LLC tumor cell mice lungs, which was significantly different from the solvent control group (** P <0.01, *** P≤0.001 ). (See Table 10)

Example 3 Evaluation of a compound represented by formula (I) dihydrochloride (drug A) as a single drug intratumorally or in combination with PD-L1 monoclonal antibody (drug C) for MC38 / H-11 mouse subcutaneously transplanted tumor Efficacy.

1.Experimental materials

1) tumor cells

MC-38 / H-11 cells are mouse colon cancer MC-38 cells that have been knocked out of mouse endogenous PD-L1 by CRISPR / Cas9 technology, transfected and express human PD-L1 monoclonal cells. Therefore, MC- 38 / H-11 cells only highly expressed human PD-L1 protein. MC-38 / H-11 was adhered to a 10-cm petri dish and cultured under the condition of adding RPMI1640 medium with 10% fetal calf serum and penicillin and streptomycin in a 37 ° C incubator with 5% CO2 air to cultivate. Passage three times a week. When the cells are growing exponentially, trypsinize, collect, count, and inoculate cells.

2) Experimental animals

Fifty C57BL / 6 mice, 6-7 weeks, ♀, purchased from Shanghai Lingchang Biological Technology Co., Ltd. Production license number: SCXK (Shanghai) 2013-0018; animal certificate number 2013001829559. Rearing environment: SPF level.

3) Test drug

Drug A

Dissolve the compound represented by formula (I) (40 mg, 0.105 mmol, prepared by the method disclosed in WO2018095426) in 0.5 mL of a mixed solvent of isopropanol and tetrahydrofuran (V / V = 1: 1), The solution was stirred and completely dissolved. The temperature was raised to 50 ° C., and a 4M solution of hydrogen chloride in isopropanol (0.055 mL, 0.22 mmol) was added dropwise, and the mixture was cooled to room temperature and stirred for 16 hours to precipitate a white solid. The reaction solution was filtered, and the filter cake was collected and dried under vacuum to obtain Drug A.

Drug A is configured according to the prescription in Table 11.

Table 11

Material name	Composition of each medicine prescription
Drug A	1mg
Citric acid	4.208mg
Sodium chloride	7.50mg
Sodium hydroxide	N / A
Water for Injection	1ml

Drug C

The anti-PD-L1 antibody has a heavy chain sequence of SEQ ID NO: 19 and a light chain sequence of SEQ ID NO: 21.

2.Experimental methods and steps

Mice were inoculated bilaterally under the left and right armpits. Each mouse was inoculated with 1.3 × 10 ⁶ MC38 / H-11 cells. After the tumors reached 80-150 mm ³ , they were grouped according to tumor size. 50 μg of drug A (it) was administered into the right tumor of nude mice in the drug A group and the combined group, and the injection volume was 50 μL, administered twice a week (BIW) for a total of 4 times; the drug group C and the combined drug group were naked The mice were injected intraperitoneally with 10 mg / kg of drug C and administered once every two days (QOD) for a total of 6 times; the control group was intraperitoneally administered with 50 μL of physiological saline and intraperitoneally injected with 10 mL / kg physiological saline. See Table 12 for specific dosages and dosing schedules.

Table 12.

D0: time of first administration; i.p .: intraperitoneal administration; i.t .: intratumoral injection; QOD: administration once every 2 days; BIW: twice weekly.

The experimental index is to examine the effect of the drug on tumor growth, and the specific index is T / C% or tumor inhibition rate TGI (%).

The tumor diameter is measured twice a week with a vernier caliper, and the tumor volume (V) calculation formula is:

V = 1/2 × a × b ² , where a and b represent length and width, respectively.

T / C (%) = (TT ₀ ) / (CC ₀ ) × 100 where T and C are tumor volumes at the end of the experiment; T ₀ and C ₀ are tumor volumes at the beginning of the experiment.

Tumor inhibition rate (TGI) (%) = 100-T / C (%).

When the tumor regresses, the tumor suppression rate (TGI) (%) = 100- (TT ₀ ) / T ₀ × 100

If the tumor is smaller than the initial volume, that is, T <T ₀ or C <C ₀ , it is defined as tumor partial regression (PR); if the tumor completely disappears, it is defined as tumor complete regression (CR).

The tumor volume or tumor weight of the two groups was compared using a two-tailed Student's test, and P <0.05 was defined as a statistically significant difference.

3. Experimental results

As shown in Table 13 and Figure 8: On the administration side (proximal), 50 μg / drug A (group 2) and 10 mg / kg drug C (group 3) significantly inhibited MC38 / H-11 nude mice subcutaneously. The growth rate of transplanted tumors was 32.1% and 34.4%, respectively. The combination of drug A and drug C (group 4) also significantly inhibited the growth of subcutaneously transplanted tumors in MC38 / H-11 nude mice, with a tumor suppression rate of 58.8%. Compared with the single drug of drug C (group 3), the combined group (group 4) has a significant synergistic antitumor effect, and compared with the single drug of drug A (group 2), it has a synergistic antitumor effect (P = 0.067).

Table 13. Efficacy of intratumoral administration of drug A alone or in combination with drug B on subcutaneously transplanted tumors of MC38 / H-11 mice (drug A administration side (proximal) data, Mean ± SEM, n = 10)

D0: time of first administration, * P <0.05, ** P <0.01, *** P <0.001 vs. solvent group; ^# P <0.05 vs. drug C 10mg / kg; ^$ P = 0.067 vs. drug A 50μg / only.

As shown in Table 14 and Figure 9: On the non-dose side (distal end), 50 μg / drug A (group 2) and 10 mg / kg drug C (group 3) significantly inhibited MC38 / H-11 nude mice The growth rate of subcutaneous transplanted tumors was 41.8% and 34.7%, respectively. The combination of drug A and drug C (group 4) also significantly inhibited the growth of subcutaneously transplanted tumors in nude mice of MC38 / H-11, with a tumor suppression rate of 48.3%, and there was a synergistic trend with the respective single drug ratios.

Table 14. Efficacy of drug A intratumorally administered alone or in combination with drug B on MC38 / H-11 mice subcutaneously transplanted tumors (data of non-administration side (distal) of drug A, Mean ± SEM, n = 10 )

D0: time of first administration; * P <0.05, ** P <0.01 vs. Vehicle.

As shown in Figure 10, 50 μg / drug A (group 2) and 10 mg / kg of drug C single (group 3) and combination (group 4) mice gained normal weight, and the control group (group 1) Compared with that, there was no significant difference. It is suggested that tumor-bearing mice can tolerate drug A and drug C alone or in combination, and there is no significant change in weight gain.

Example 4

Mix the dihydrochloride of the compound represented by formula (I) with water for injection, adjust the pH with acetic acid-sodium acetate, sodium dihydrogen phosphate-disodium hydrogen phosphate, stir and dissolve it for future use, and examine the condition of 60 ℃ under different pH Under the stability, the data are shown in Tables 15 and 16:

Table 15

Table 16

It can be known from the experimental results that the change in pH affects the stability of the compound represented by formula (I) dihydrochloride in the solution. When the pH value is between 3.0 and 4.0, the total impurities significantly increase after standing at 60 ° C for 20 days. When the pH of the injection solution is between 4.5 and 6.0, the stability is good, which is beneficial to the long-term storage of the composition.

Example 5,

The dihydrochloride and the buffer solution of the compound represented by formula (I) are arranged according to the prescribed amount in Table 17, and after stirring and dissolving, the pH is adjusted, and then set aside, and the stability at 60 ° C is investigated. The data are shown in Table 18:

Table 17

Table 18

From the experimental results, it can be known that the dihydrochloride of the compound represented by formula (I) has good stability in an acetate and citrate buffer system with a pH value of 5.0.

Example 6. Screening of citrate concentration

According to the formulation of the compound dihydrochloride of formula (I) shown in Table 19, the stability at 60 ° C was investigated. The data is shown in Table 20:

Table 19

Table 20

From the experimental results, it can be seen that the dihydrochloride of the compound shown in (I) has good stability and the pH value is stable between 4.5 and 5.5 in the acetate buffer solution of each concentration, and the compound shown in (I) The dihydrochloride has good stability in the buffer salt concentration of 10mM to 80mM.

Example 7,

The prescription formulation of the dihydrochloride compound represented by formula (I) was arranged according to Table 21, and the stability at 60 ° C was examined. The results are shown in Table 22.

Table 21

Table 22

From the experimental results, it can be known that the stability of the pharmaceutical composition is good when osmotic pressure regulators are added, but in order to avoid or reduce the stimulus of administration, it is necessary to add osmotic pressure regulators to maintain the isotonicity of the pharmaceutical composition, taking into account the clinical use In the case of administration of blood sodium to patients, sodium chloride is preferred as an osmotic regulator.

Example 8,

A solution of the compound dihydrochloric acid represented by formula (I) was prepared according to the prescribed amount in Table 23, sterilized, and divided. Place it at a temperature of 60 ° C ± 2 ° C to check the stability. The specific data is shown in Table 24.

Table 23

Active ingredient	5mg / ml
Citrate concentration	40mM
Sodium chloride	7.5mg / ml
pH value	5.5

Table 24

Zh

0 month

10 days

30 days

Total Miscellaneous	1.2%	1.4	1.4
content	98.9%	98.9%	100.2%
pH value	5.50	5.49	5.48

Claims (23)

1. Use of a TLR agonist combined with an immune checkpoint inhibitor in the manufacture of a medicament for treating tumors.
2. The use according to claim 1, wherein the TLR agonist is a compound represented by formula (I) or a complex thereof or a pharmaceutically acceptable salt thereof

   
3. The use according to claim 2, wherein the immune checkpoint inhibitor is selected from a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.
4. The use according to claim 3, wherein the PD-1 inhibitor is an anti-PD-1 antibody or an antigen-binding fragment thereof.
5. The use according to claim 4, wherein the light chain variable region of the anti-PD-1 antibody comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 respectively. The heavy chain variable region of the PD-1 antibody comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively.
6. The use according to claim 5, wherein the anti-PD-1 antibody or antigen-binding fragment thereof is selected from a humanized antibody or a fragment thereof.
7. The use according to claim 5 or 6, wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 isotype, preferably comprising an IgG1 or IgG4 isotype Heavy chain constant region.
8. The use according to claim 6, wherein the light chain variable region sequence of the humanized antibody is a sequence shown in SEQ ID NO: 10 or a variant thereof, and the heavy chain variable region of the humanized antibody The sequence is the sequence shown in SEQ ID NO: 9 or a variant thereof.
9. The use according to claim 6, the light chain sequence of the humanized antibody is the sequence shown as SEQ ID NO: 8 or a variant thereof; the heavy chain sequence of the humanized antibody is as SEQ ID NO : 7 or a variant thereof.
10. The use according to claim 6, wherein the humanized antibody light chain sequence is a sequence shown in SEQ ID NO: 8, and the heavy chain sequence is a sequence shown in SEQ ID NO: 7.
11. The use according to claim 3, wherein the PD-L1 inhibitor is an anti-PD-L1 antibody or an antigen-binding fragment thereof.
12. The use according to claim 11, wherein the heavy chain variable region of the anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 11-13, respectively, and the anti-PD- The light chain variable region of the L1 antibody or antigen-binding fragment comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 14-16, respectively.
13. The extract according to claim 12, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region shown in SEQ ID NO: 17 or a variant thereof, and Light chain variable regions or variants thereof.
14. The use according to claim 13, characterized in that the PD-L1 antibody or antigen-binding fragment further comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, preferably human IgG2 or The IgG4 heavy chain constant region, more preferably contains the IgG4 heavy chain constant region that introduces F234A and L235A mutations; the humanized antibody light chain further comprises a human-derived kappa, lambda chain or variant thereof constant region.
15. The use according to claim 14, wherein the PD-L1 antibody or antigen-binding fragment comprises a heavy chain as shown in SEQ ID NO: 19 and a light chain as shown in SEQ ID NO: 21.
16. The use according to claim 12, wherein the PD-L1 antibody or antigen-binding fragment is selected from a murine antibody, a chimeric antibody, a humanized antibody, a human antibody, preferably a humanized antibody.
17. The use according to any one of claims 1-6, the tumor is selected from the group consisting of lung cancer, non-small cell lung cancer, hepatocellular carcinoma, basal cell carcinoma, renal cell carcinoma, myeloma, rectal cancer, colon cancer, breast cancer, Gastric cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or eye malignant melanoma, uterine cancer, ovarian cancer, anal cancer, gastric cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer , Vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small bowel cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penis Cancer, chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer , Renal pelvis cancer, central nervous system neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermis Like cancer, squamous cell carcinoma, and T cell lymphoma, preferably hepatocellular carcinoma, rectal cancer, colon cancer, and non-small cell lung cancer.
18. The use according to any one of claims 2-17, wherein the TLR agonist is a dihydrochloride of a compound represented by formula (I).
19. The use according to any one of claims 1-18, the TLR agonist is administered at a dose selected from 0.0001-20.0mg / kg or 0.01-200mg, and the frequency of administration is 3 times a day, 2 times a day, 1 day Times, once every 2 days, 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 every 3 Once a day, once a week or once every two weeks.
20. The use according to any one of claims 4-10, 17-19, wherein the anti-PD-1 antibody or antigen-binding fragment thereof is administered at a dose selected from 0.1-10.0 mg / kg or 1-1000 mg, and the frequency of administration Once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once a month, once every 3-6 months, preferably once every 2 weeks or once every 3 weeks.
21. The use according to any one of claims 11-16, 17-19, the dose of the anti-PD-L1 antibody or antigen-binding fragment thereof is selected from 1-50 mg / kg or 50-3000 mg, and the frequency of administration is once a week , Once every 2 weeks, once every 3 weeks, once every 4 weeks, preferably once every 2 weeks or once every 3 weeks.
22. The use according to any one of claims 1 to 21, wherein the TLR agonist is administered orally, intratumorally or by inhalation.
23. A pharmaceutical composition containing a compound represented by formula (I) or a complex thereof or a pharmaceutically acceptable salt thereof, said

    

    The pH of the pharmaceutical composition is 3.0-7.0, preferably 4.5-6.0.

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