Pharmaceutical composition containing TLR agonist and multiple antibodies, combined formulation, pharmaceutical use, and method of treatment

Abstract

Provided are a pharmaceutical composition containing a TLR agonist and multiple antibodies, a combined formulation, pharmaceutical use, and a method of treatment. The TLR agonist and two or more antibodies for treating cancer are used in combination. Each active pharmaceutical ingredient has a different anti-tumor immune activation effect, and the combination exhibits a synergistic effect. Compared with the monotherapies, the combination features improved efficacy and a reduced dose, thereby reducing the toxicity, the possibility of adverse events, and the possibility of acquired immune resistance.

Description

A pharmaceutical composition, combination formulation, pharmaceutical use, and treatment methods containing a TLR agonist and multiple antibodies.

[0001] Cross-references to related applications

[0002] This application claims priority to Chinese Patent Application No. CN 202510125118.5, filed on January 26, 2025, entitled "A pharmaceutical composition, combination formulation, and pharmaceutical use and treatment method containing a TLR agonist and multiple antibodies", and Chinese Patent Application No. CN 202510919201.X, filed on July 3, 2025, entitled "A pharmaceutical composition, combination formulation, and pharmaceutical use and treatment method containing a TLR agonist and multiple antibodies", the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] This application belongs to the field of oncology technology, and specifically relates to a pharmaceutical composition, combination formulation, pharmaceutical use, and treatment method containing a TLR agonist and multiple antibodies. Background Technology

[0004] Immunotherapy and targeted therapy have become important tools in clinical cancer treatment. Immunotherapy works by activating or enhancing the body's own immune system, enabling it to recognize and attack tumor cells, thereby achieving a therapeutic effect. Current clinical approaches to cancer immunotherapy rely on the systemic administration of high doses of one or two biological immunomodulators. These drugs aim to restore pre-set conditions of tumor-induced immunosuppression while stimulating adaptive immune responses against tumor-specific antigens (TSA) and tumor-associated antigens (TAA).

[0005] Toll-like receptors (TLRs) are present on many cells of the immune system and participate in innate immune responses. The TLR receptor family includes six transmembrane TLRs (TLR-1, 2, 4, 5, 6, and 10) and four endosomal TLRs (TLR-3, 7, 8, and 9). Each PAMP (pathogen-associated molecular pattern) is recognized by a different TLR: lipopolysaccharide (TLR4), lipopeptide (TLR2 and TLR6 or TLR1), flagellin (TLR5), single-stranded RNA (TLR7 / 8), double-stranded RNA (TLR3), and DNA containing a CpG motif (TLR9).

[0006] Several TLR agonists for cancer treatment are currently under development, including TLR9 agonists such as CpG oligodeoxynucleotide (CpG ODN); TLR7 / 8 agonists such as retsimod (R848), BDB001, BDB018, and NKTR-262; TLR8 agonists such as VTX-2337 (Motolimod); TLR7 agonists such as imiquimod, RO7119929, SHR2150, TQ-A3334, and LHC165; TLR5 agonists such as CBLB502 (also known as Entolimod); TLR4 agonists such as GLA-SE (glucopyranosyl lipid A stable emulsion) and GSK1795091; and TLR3 agonists such as rintatolimod and poly-ICLC. Clinical trials have already investigated the ability of various TLR agonists to promote anti-tumor immunity. Antitumor responses are largely attributed to their ability to stimulate APCs (such as dendritic cells (DCs)) and thereby activate tumor-specific T cell responses.

[0007] CpG ODN is a synthetically produced oligodeoxynucleotide (ODN) containing unmethylated cytosine-phosphate-guanine dinucleotide (CpG). Upon recognition by the individual's immune system, CpG ODN exhibits strong immunostimulatory activity, eliciting both innate and adaptive immune responses. CpG ODN can activate plasmacytoid dendritic cells (pDCs), causing them to secrete type I interferon (IFN), and can also promote the expression of co-stimulatory molecules such as CD80 and CD86 on DCs and tumors expressing TLR9. This can lead to the secretion of various cytokines / chemokines and activate natural killer (NK) cells, TH1 cells, and cytotoxic T lymphocytes (CTLs). Encouraging results have been achieved in phase I and II clinical trials using CpG ODN as an adjuvant for tumor vaccines or in combination therapy with chemotherapy for cancer treatment.

[0008] Targeted therapy involves intervening in specific molecules or signaling pathways on tumor cells by targeting their molecular targets, using drugs or other treatments to directly interfere with or block the growth and spread of tumor cells, thereby killing or inhibiting them. Targeted therapy primarily utilizes over 100 antibodies approved in recent years. Generally, therapeutic antibodies kill tumor cells through three mechanisms: (1) Direct antibody action, which involves blocking or activating ligand / receptor signaling activity, inducing apoptosis, and delivering drugs or cytotoxic agents. Antibody receptor activation can directly kill tumor cells. Antibodies can also mediate tumor cell killing through receptor antagonistic activity. (2) Immune-mediated cell killing mechanisms, including complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), T cell function regulation, etc. Immune-mediated tumor cell killing can be accomplished through the following mechanisms: induction of phagocytosis, activation of complement, antibody-dependent cell-mediated cytotoxicity, targeting of tumors by genetically modified T cells via single-chain variable fragments (scFv), activation of T cells through antibody-mediated antigen cross-presentation by dendritic cells, and inhibition of T cell inhibitory receptors. (3) The specific effects of antibodies on the tumor vascular system and matrix induce ablation of vascular and stromal cells by capturing vascular receptor antagonists or ligands.

[0009] Therapeutic monoclonal antibody drugs have advanced the research and development of anticancer drugs, and significant benefits have been obtained from their use. However, some issues still need further research and resolution, such as antibody immunogenicity; a significant proportion of patients do not respond to treatment or become refractory; and more than half of the antibodies fail to achieve long-term effective inhibition and killing of tumor cells. Furthermore, the use of high systemic doses of monoclonal antibodies is accompanied by a high frequency of serious adverse events.

[0010] To address the limitations of the aforementioned therapies in cancer treatment, targeted therapy and immunotherapy have begun to evolve towards combination therapy, and more combination therapy regimens are expected to emerge in the future to further improve treatment efficacy. Some companies have conducted beneficial explorations in combination therapy with cancer immunotherapy. For example, the I / II clinical trial ILLUMINATE-204 (Clinicaltrials.gov, NCT02644967), involving the intratumoral TLR9 agonist IMO-2125 (Tilsotolimod) combined with the CTLA4 antibody ipilimumab for the treatment of PD-1 inhibitor-refractory advanced melanoma, showed that this combination therapy was generally well-tolerated and effective in PD-1 inhibitor-refractory advanced melanoma, including durable responses (Cara L. Haymaker1, Robert H. Andtbacka, Douglas B. Johnson, etc., 2020 Virtual ESMO Congress). However, the pivotal registrational phase 3 clinical trial ILLUMINATE-301 (Clinicaltrials.gov, NCT03445533), in combination with the CTLA4 antibody ipilimumab for the treatment of PD-1 refractory advanced melanoma, failed to meet the primary endpoint of ORR.

[0011] In conclusion, new approaches to cancer treatment are still needed in this field to improve treatment outcomes. Summary of the Invention

[0012] While current cancer immunotherapy methods offer benefits to a limited number of patients, their adherence and applicability are limited by acquired resistance and a high rate of adverse immune events. Furthermore, while concurrent administration of multiple antibodies significantly enhances antitumor efficacy, it can also trigger a range of adverse reactions, leading to poor tolerability and making treatment unsustainable.

[0013] To address the aforementioned problems in the prior art, this application provides a pharmaceutical composition, combination formulation, pharmaceutical use, and treatment method containing a TLR agonist and multiple antibodies, overcoming many limitations of current cancer immunotherapy. This application combines multiple cancer-treating antibodies with simultaneous administration of a TLR agonist, exhibiting a synergistic effect. Compared to therapies relying on a single drug, this enhances efficacy, reduces dosage, thereby lowering the likelihood of toxicity and adverse events, and reducing the possibility of acquired immune resistance.

[0014] In a first aspect, this application provides a pharmaceutical composition comprising a TLR agonist and multiple antibodies, wherein:

[0015] (a) One TLR agonist;

[0016] (b) Two or more antibodies for treating cancer;

[0017] (c) Pharmaceutically acceptable carriers or excipients.

[0018] In some embodiments, the pharmaceutical composition does not contain other active ingredients.

[0019] In a second aspect, this application provides a combination formulation containing a TLR agonist and multiple antibodies, comprising:

[0020] (a) One TLR agonist;

[0021] (b) Two or more antibodies for treating cancer.

[0022] In some embodiments, the combination formulation does not contain other active ingredients.

[0023] In some embodiments, in the combination formulation, each antibody in (b) is formulated as a separate pharmaceutical composition; or at least two antibodies are formulated together as a pharmaceutical composition (e.g., when there are three antibodies in (b), two are formulated together as a pharmaceutical composition and the other is formulated as a separate pharmaceutical composition); or all antibodies are formulated as a single pharmaceutical composition.

[0024] In some embodiments, in the combined formulation, (a) and (b) are each formulated as separate pharmaceutical compositions; or a portion of (a) and (b) are formulated together as a pharmaceutical composition, and the remainder of (b) is formulated as a separate pharmaceutical composition.

[0025] In some embodiments, the pharmaceutical composition contains a pharmaceutically acceptable carrier or excipient.

[0026] The pharmaceutical compositions described in the first aspect and the combination formulations described in the second aspect are further described in detail below.

[0027] In some embodiments, the TLR agonist is selected from TLR1 agonists, TLR2 agonists, TLR3 agonists, TLR4 agonists, TLR5 agonists, TLR6 agonists, TLR7 agonists, TLR8 agonists, TLR7 / 8 dual agonists, TLR9 agonists, and TLR10 agonists. Preferably, the TLR agonist is selected from TLR3 agonists, TLR4 agonists, TLR5 agonists, TLR6 agonists, TLR7 agonists, TLR8 agonists, TLR7 / 8 dual agonists, and TLR9 agonists. More preferably, the TLR agonist is selected from TLR7 agonists, TLR8 agonists, TLR7 / 8 dual agonists, and TLR9 agonists.

[0028] In some embodiments, the TLR agonist is selected from CpG ODN, R848, BDB001, Poly I:C, BDB018, NKTR-262, VTX-2337 (Motolimod), Imiquimod, RO7119929, SHR2150, TQ-A3334, LHC165, CBLB502 (also known as Entolimod), GLA-SE (glucopyranosyl lipid A stable emulsion), GSK1795091, Rintatolimod, poly-ICLC, and pharmaceutically acceptable salts thereof. The CpG ODN includes types A, B, and C.

[0029] In some embodiments, the TLR9 agonist is CpG ODN or a pharmaceutically acceptable salt thereof, including type A, type B, and type C. Preferably, the TLR9 agonist is type C CpG ODN or a pharmaceutically acceptable salt thereof.

[0030] In some embodiments, the CpG ODN includes chemical modifications. In some embodiments, the CpG ODN includes modification with one or more phosphate ester groups. In some embodiments, the modification with one or more phosphate ester groups is a thiophosphate bond. In some embodiments, the phosphate backbone of the CpG ODN is fully modified with thiophosphate.

[0031] In some embodiments, the CpG ODN is CpG HP007, CpG HP021, CpG HP026, CpG 684, SD-101, G10 CpG ODN, CpG 1018, CpG 7909, IMO-2125, and pharmaceutically acceptable salts thereof.

[0032] In some embodiments, the TLR9 agonist is a pharmaceutically acceptable salt of CpG HP007. In some embodiments, the TLR7 / 8 dual agonist is R848 or a pharmaceutically acceptable salt thereof. The TLR3 agonist is Poly I:C or a pharmaceutically acceptable salt thereof.

[0033] In some embodiments, (a) is a TLR9 agonist; or a TLR7 / 8 agonist; or a TLR3 agonist; preferably, (a) is CpG HP007 or a pharmaceutically acceptable salt thereof; or R848 or a pharmaceutically acceptable salt thereof; or Poly I:C or a pharmaceutically acceptable salt thereof.

[0034] In some embodiments, the cancer-treating antibody targets PD-1, CTLA-4, TIGIT, PD-L1, PD-L2, OX40, 4-1BB, ICOS, LAG3, TIM3, IDO, HER2, CD20, CD22, CD27, CD40, CD40L, GITR, EGFR, CD2, CD19, CD33, CD37, CD38, CD44, CD45, CD47, CD52, CD56, CD70, CD79, 5T4, AGS-5, AGS-16, angiopoietin 2, B7.1, and B7. 2. B7DC, B7H1, B7H2, B7H3, BT-062, BTLA, CAIX, carcinoembryonic antigen, Cripto, ED-B, ErbB1, ErbB2, ErbB3, ErbB4, EGFL7, EpCAM, EphA2, EphA3, EphB2, FAP, fibronectin, folate receptor, ganglioside GM3, GD2, glucocorticoid-induced tumor necrosis factor receptor GITR, gp100, gpA33, GPNMB, ICOS, IGF1R, integrin αν, integrin ανβ, KIR, Lewis Y, mesothelin, c-MET, MN carbonic anhydrase IX, MUC1, MUC16, cohesin-4, NKGD2, NOTCH, OX40L, PSCA, PSMA, RANKL, ROR1, ROR2, SLC44A4, STING, multiligand proteoglycan-1, TACI, TAG-72, tendinin, TIM3, TRAILR1, TRAILR2, VEGFR-1, VEGFR-2, VEGFR-3, SSTR, SSTR2, CXCR4, NTR, GRPR. Preferably, the antibody for treating cancer targets one, two, or more (e.g., three) of PD-1, CTLA-4, TIGIT, OX40, and CD40. More preferably, the antibody for treating cancer or a combination thereof targets one, two, or more (e.g., three) of PD-1, CTLA-4, TIGIT, and CD40.

[0035] In some embodiments, the antibody used to treat cancer is a monoclonal antibody, a bispecific antibody, a trispecific antibody, an antibody-drug conjugate (ADC), or a radionuclide conjugate (RDC).

[0036] In some embodiments, (b) is an anti-CD40 antibody and an anti-CTLA4 antibody; or an anti-PD-1 antibody, an anti-CTLA4 antibody and an anti-TIGIT antibody; or an anti-PD1 antibody, an anti-CTLA4 antibody and an anti-CD40 antibody; or an anti-OX40 antibody, an anti-CTLA4 antibody and an anti-TIGIT antibody.

[0037] In some embodiments, the anti-PD-1 antibody is a monoclonal antibody selected from Keytruda, Opdivo, cimiprimab, toripalimab, sintilimab, camrelizumab, patelimab, sugelimab, penapralimab, cepalimumab, and slulimab; the anti-CTLA4 antibody is a monoclonal antibody selected from ipilimumab and tremelimumab; and the anti-TIGIT antibody is a monoclonal antibody selected from domvanalimab, belrestotug, and tiragol. The following are listed: umab, ospermumab, JS-006, vibostolimab, Lancastotug, Renvistobart, Etigilimab, HB-0030, PM-1021, AB-308, COM-902, M6223, ASP-8374, BAT-6005, Tamgiliblimab; the anti-CD40 antibody is a monoclonal antibody selected from SEA-CD40, Sotigalimab, BSI-038, and Anti-CD40. ChiLob7 / 4 (BioNTech SE), 2141-V11, CDX-1140, YH-003, Selicrelumab, Mitazalimab, ILB-2101, ABBV-927, LVGN-7409; the anti-OX40 antibody is a monoclonal antibody selected from BGB-A445, INBRX-106, YH-002, INCAGN-1949, MEDI-6469, GEN-1055, BAT-6026, IBI-101, ABBV-368, MEDI-0562, HFB-3010.

[0038] In some embodiments, the bispecific antibody is selected from: epalolimbab / tovoretalimab (CTLA4 inhibitor / PD1 inhibitor), canducilin / tovoretalimab (CTLA4 inhibitor / PD1 inhibitor), Volrustomig (CTLA4 inhibitor / PD1 inhibitor), Danvilostomig (CTLA4 inhibitor / PD1 inhibitor), Lorigerlimab (CTLA4 inhibitor / PD1 inhibitor), Vudalimab (CTLA4 inhibitor / PD1 inhibitor), SHS-010 (CTLA4 inhibitor / PD1 inhibitor), REMD-511 (CTLA4 inhibitor / PD1 inhibitor), YH-008 (CD40 inhibitor / PD1 inhibitor), FS-120 (4-1BB agonist / OX40 agonist), YH-006 (CTLA4 inhibitor / OX40 agonist), ATOR-1015 (CTLA4 inhibitor / OX40 agonist), APVO-603 (4-1BB agonist / OX40 agonist). The following are selected as specific antibodies: CD40 agonist, GEN-1042 (4-1BB agonist / CD40 agonist), IMB-071703 (4-1BB agonist / CD40 agonist), SM2256 (4-1BB agonist / CD40 agonist), Rilvegostomig (PD1 inhibitor / TIGIT inhibitor), ZG-005 (PD1 inhibitor / TIGIT inhibitor), REMD-532 (PD1 inhibitor / TIGIT inhibitor), IBI-321 (PD1 inhibitor / TIGIT inhibitor), BC008-1A (PD1 inhibitor / TIGIT inhibitor), ZGGS15 (LAG3 inhibitor / TIGIT inhibitor); and trispecific antibodies selected from GBD209 (CTLA4 inhibitor / PD1 inhibitor / TIGIT inhibitor), FG-T903 (CD112R antagonist / PD1 inhibitor / TIGIT inhibitor), and HC010 (hcbiopharma) (CTLA4 inhibitor / PD1 inhibitor / VEGF inhibitor).

[0039] In some embodiments, the ADC drug is selected from: Mylotarg, Besponsa, Kadcyla, Polivy, Lumoxiti, Enhertu, Adcetris, Padcev, Tivdak, Blenrep, Trodelvy, Akalux, Zynlonta, Adrenaline, Luconsartuzumab, and Elahere.

[0040] In some embodiments, the radionuclide conjugate drug RDC is selected from: Pluvicto and Lutathera.

[0041] In some embodiments, the pharmaceutical composition or combination formulation comprises:

[0042] (a) CpG HP007 or a pharmaceutically acceptable salt thereof; or R848 or a pharmaceutically acceptable salt thereof; or Poly I:C or a pharmaceutically acceptable salt thereof;

[0043] (b) Anti-CD40 antibody and anti-CTLA4 antibody; or anti-PD-1 antibody, anti-CTLA4 antibody and anti-TIGIT antibody; or anti-PD1 antibody, anti-CTLA4 antibody and anti-CD40 antibody; or anti-OX40 antibody, anti-CTLA4 antibody and anti-TIGIT antibody. The antibody is preferably a monoclonal antibody.

[0044] In some embodiments, the pharmaceutical composition or combination formulation comprises:

[0045] (a) CpG HP007 or a pharmaceutically acceptable salt thereof;

[0046] (b) Anti-CD40 antibody and anti-CTLA4 antibody; or anti-PD1 antibody, anti-CTLA4 antibody and anti-CD40 antibody; or anti-OX40 antibody, anti-CTLA4 antibody and anti-TIGIT antibody. The antibody is preferably a monoclonal antibody.

[0047] In some embodiments, the pharmaceutical composition or combination formulation comprises:

[0048] (a) R848 or a pharmaceutically acceptable salt thereof;

[0049] (b) Anti-CD40 antibody and anti-CTLA4 antibody; or anti-PD1 antibody, anti-CTLA4 antibody and anti-CD40 antibody. The antibody is preferably a monoclonal antibody.

[0050] In some embodiments, the pharmaceutical composition or combination formulation comprises:

[0051] (a) Poly I:C or a pharmaceutically acceptable salt thereof;

[0052] (b) Anti-CD40 antibody and anti-CTLA4 antibody; or anti-PD1 antibody, anti-CTLA4 antibody and anti-CD40 antibody. The antibody is preferably a monoclonal antibody.

[0053] In some embodiments, the mass ratio of (a):(b) in the pharmaceutical composition or formulation is (1-1000):(2-6000), preferably (2-500):(4-3000), more preferably (10-200):(20-3000). Most preferably (10-100):(40-2000).

[0054] In one set of embodiments, the pharmaceutical composition or combination formulation contains a TLR9 agonist (such as CpG). When present, the amount is 1-1000 parts, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000 parts, preferably 2-500 parts, more preferably 10-200 parts, and most preferably 40-100 parts; TLR7 / 8 dual agonists (such as R When present, 1-1000 parts of 848 or its pharmaceutically acceptable salt are used, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000 parts, preferably 2-500 parts, more preferably 10-200 parts, and most preferably 10-100 parts; TLR3 agonists (such as Poly) When I:C or its pharmaceutically acceptable salt is present, it is in the form of 1-1000 parts, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000 parts, preferably 2-5 parts. 00 parts, more preferably 10-200 parts, most preferably 10-100 parts; when anti-CTLA4 antibody is present, it is 1-2000 parts, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200.

[0055] 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 parts, preferably 2-1000 parts, more preferably 10-500 parts, and most preferably 40-400 parts; when anti-CD40 antibody is present, it is 1-500 parts, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500 parts, preferably 2-200 parts, more preferably 10- 100 parts, optimally 30-100 parts; when anti-PD1 antibody is present, the quantity is 1-2000 parts, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700. 1800, 1900, 2000 parts, preferably 2-1500 parts, more preferably 10-1000 parts, and most preferably 100-1000 parts; when anti-OX40 antibody is present, it is 1-500 parts, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500 parts, preferably 2-200 parts, more preferably 10-200 parts, and most preferably 20-50 parts; anti-TIG When IT antibodies are present, the quantity is 1-1000 parts, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000 parts, preferably 2-500 parts, more preferably 10-500 parts, and most preferably 100-200 parts.

[0056] In some embodiments, the pharmaceutical composition or combination formulation comprises (a) 1-1000 parts of CpG HP007 or a pharmaceutically acceptable salt thereof; or 1-1000 parts of R848 or a pharmaceutically acceptable salt thereof; or 1-1000 parts of Poly I:C or a pharmaceutically acceptable salt thereof.

[0057] (b) 1-500 doses of anti-CD40 antibody and 1-2000 doses of anti-CTLA4 antibody; or 1-2000 doses of anti-PD-1 antibody, 1-2000 doses of anti-CTLA4 antibody and 1-1000 doses of anti-TIGIT antibody; or 1-2000 doses of anti-PD1 antibody, 1-2000 doses of anti-CTLA4 antibody and 1-500 doses of anti-CD40 antibody; or 1-500 doses of anti-OX40 antibody, 1-2000 doses of anti-CTLA4 antibody and 1-1000 doses of anti-TIGIT antibody.

[0058] In some embodiments, the pharmaceutical composition or combination formulation comprises (a) 2-500 parts of CpG HP007 or a pharmaceutically acceptable salt thereof; or 2-500 parts of R848 or a pharmaceutically acceptable salt thereof; or 2-500 parts of Poly I:C or a pharmaceutically acceptable salt thereof.

[0059] (b) 2-200 doses of anti-CD40 antibody and 2-1000 doses of anti-CTLA4 antibody; or 2-1500 doses of anti-PD-1 antibody, 2-1000 doses of anti-CTLA4 antibody and 2-500 doses of anti-TIGIT antibody; or 2-1500 doses of anti-PD1 antibody, 2-1000 doses of anti-CTLA4 antibody and 2-200 doses of anti-CD40 antibody; or 2-200 doses of anti-OX40 antibody, 2-1000 doses of anti-CTLA4 antibody and 2-500 doses of anti-TIGIT antibody.

[0060] In some embodiments, the pharmaceutical composition or combination formulation comprises (a) 10-200 parts of CpG HP007 or a pharmaceutically acceptable salt thereof; or 10-200 parts of R848 or a pharmaceutically acceptable salt thereof; or 10-200 parts of Poly I:C or a pharmaceutically acceptable salt thereof.

[0061] (b) 10-100 doses of anti-CD40 antibody and 10-500 doses of anti-CTLA4 antibody; or 10-1000 doses of anti-PD-1 antibody, 10-500 doses of anti-CTLA4 antibody and 10-500 doses of anti-TIGIT antibody; or 10-1000 doses of anti-PD1 antibody, 10-500 doses of anti-CTLA4 antibody and 10-100 doses of anti-CD40 antibody; or 10-200 doses of anti-OX40 antibody, 10-500 doses of anti-CTLA4 antibody and 10-500 doses of anti-TIGIT antibody.

[0062] In some embodiments, the pharmaceutical composition or combination formulation comprises (a) 40-100 parts of CpG HP007 or a pharmaceutically acceptable salt thereof; or 10-100 parts of R848 or a pharmaceutically acceptable salt thereof; or 10-100 parts of Poly I:C or a pharmaceutically acceptable salt thereof.

[0063] (b) 30-100 doses of anti-CD40 antibody and 40-400 doses of anti-CTLA4 antibody; or 100-1000 doses of anti-PD-1 antibody, 40-400 doses of anti-CTLA4 antibody and 100-200 doses of anti-TIGIT antibody; or 100-1000 doses of anti-PD1 antibody, 40-400 doses of anti-CTLA4 antibody and 30-100 doses of anti-CD40 antibody; or 20-50 doses of anti-OX40 antibody, 40-400 doses of anti-CTLA4 antibody and 100-200 doses of anti-TIGIT antibody.

[0064] In some embodiments, the pharmaceutical composition or combination formulation comprises (a) 40 or 100 parts of CpG HP007 or a pharmaceutically acceptable salt thereof; or 10, 30 or 100 parts of R848 or a pharmaceutically acceptable salt thereof; or 10, 30 or 100 parts of Poly I:C or a pharmaceutically acceptable salt thereof.

[0065] (b) 30, 50, or 100 doses of anti-CD40 antibody and 40, 200, or 400 doses of anti-CTLA4 antibody; or 100, 500, or 1000 doses of anti-PD-1 antibody, 40, 200, or 400 doses of anti-CTLA4 antibody and 167 doses of anti-TIGIT antibody; or 100, 500, or 1000 doses of anti-PD1 antibody, 40, 200, or 400 doses of anti-CTLA4 antibody and 30, 50, or 100 doses of anti-CD40 antibody; or 40 doses of anti-OX40 antibody, 40, 200, or 400 doses of anti-CTLA4 antibody and 167 doses of anti-TIGIT antibody.

[0066] In some embodiments, the pharmaceutical composition or combination formulation comprises, (a) 40-100 parts of CpG HP007 or a pharmaceutically acceptable salt thereof;

[0067] (b) 30-100 doses of anti-CD40 antibody and 40-400 doses of anti-CTLA4 antibody; or 100-1000 doses of anti-PD1 antibody, 40-400 doses of anti-CTLA4 antibody and 30-100 doses of anti-CD40 antibody; or 20-50 doses of anti-OX40 antibody, 40-400 doses of anti-CTLA4 antibody and 100-200 doses of anti-TIGIT antibody.

[0068] In some embodiments, the pharmaceutical composition or combination formulation comprises, (a) 40-100 parts of CpG HP007 or a pharmaceutically acceptable salt thereof;

[0069] (b) 50-100 doses of anti-CD40 antibody and 200-400 doses of anti-CTLA4 antibody; or 1000 doses of anti-PD1 antibody, 200-400 doses of anti-CTLA4 antibody and 50-100 doses of anti-CD40 antibody; or 40 doses of anti-OX40 antibody, 200-400 doses of anti-CTLA4 antibody and 167 doses of anti-TIGIT antibody.

[0070] In some embodiments, the pharmaceutical composition or combination formulation comprises: (a) 100 parts of CpG HP007 or a pharmaceutically acceptable salt thereof; (b) 50 parts of anti-CD40 antibody and 200 parts of anti-CTLA4 antibody; or (a) 100 parts of CpG HP007 or a pharmaceutically acceptable salt thereof; (b) 40 parts of anti-OX40 antibody, 200 parts of anti-CTLA4 antibody and 167 parts of anti-TIGIT antibody; or (a) 40 parts of CpG HP007 or a pharmaceutically acceptable salt thereof; (b) 1000 parts of anti-PD1 antibody, 400 parts of anti-CTLA4 antibody and 100 parts of anti-CD40 antibody.

[0071] In some embodiments, the pharmaceutical composition or combination formulation comprises, (a) 10-100 parts of R848 or a pharmaceutically acceptable salt thereof; or 10-100 parts of Poly I:C or a pharmaceutically acceptable salt thereof;

[0072] (b) 30-100 doses of anti-CD40 antibody and 40-400 doses of anti-CTLA4 antibody; or 100-1000 doses of anti-PD1 antibody, 40-400 doses of anti-CTLA4 antibody and 30-100 doses of anti-CD40 antibody.

[0073] In some embodiments, the pharmaceutical composition or combination formulation comprises: (a) 30 parts of R848 or a pharmaceutically acceptable salt thereof; (b) 50 parts of anti-CD40 antibody and 200 parts of anti-CTLA4 antibody; or (a) 30 parts of R848 or a pharmaceutically acceptable salt thereof; (b) 50 parts of anti-CD40 antibody, 200 parts of anti-CTLA4 antibody and 500 parts of anti-PD1 antibody; or (a) 10 parts of R848 or a pharmaceutically acceptable salt thereof; (b) 30 parts of anti-CD40 antibody, 40 parts of anti-CTLA4 antibody and 100 parts of anti-PD1 antibody; or (a) 100 parts of R848 or a pharmaceutically acceptable salt thereof; (b) 100 parts of anti-CD40 antibody, 400 parts of anti-CTLA4 antibody and 1000 parts of anti-PD1 antibody.

[0074] In some embodiments, the pharmaceutical composition or combination formulation comprises: (a) 30 parts of Poly I:C or a pharmaceutically acceptable salt thereof; (b) 50 parts of anti-CD40 antibody and 200 parts of anti-CTLA4 antibody; or (a) 30 parts of Poly I:C or a pharmaceutically acceptable salt thereof; (b) 50 parts of anti-CD40 antibody, 200 parts of anti-CTLA4 antibody and 500 parts of anti-PD1 antibody; or (a) 10 parts of Poly I:C or a pharmaceutically acceptable salt thereof; (b) 30 parts of anti-CD40 antibody, 40 parts of anti-CTLA4 antibody and 100 parts of anti-PD1 antibody; or (a) 100 parts of Poly I:C or a pharmaceutically acceptable salt thereof; (b) 100 parts of anti-CD40 antibody, 400 parts of anti-CTLA4 antibody and 1000 parts of anti-PD1 antibody.

[0075] In a third aspect, the pharmaceutical composition described in the first aspect and the combination formulation described in the second aspect of this application are used for the prevention and / or treatment of cancer.

[0076] In a fourth aspect, this application provides pharmaceutical use of the pharmaceutical composition described in the first aspect and the combination formulation described in the second aspect for the preparation of a medicament for the prevention and / or treatment of cancer.

[0077] In a fifth aspect, this application provides a method for preventing and / or treating cancer, comprising the steps of administering to a subject an effective amount of the pharmaceutical composition described in the first aspect or the combination preparation described in the second aspect.

[0078] In some embodiments, the cancer is a solid tumor, including but not limited to colorectal cancer, melanoma, breast cancer, lung cancer, stomach cancer, head and neck tumors, prostate cancer, liver cancer, nasopharyngeal carcinoma, esophageal cancer, and cervical cancer.

[0079] In some embodiments, the method for preventing and / or treating cancer includes the step of administering an effective amount of the pharmaceutical composition described in the first aspect to a subject.

[0080] In some embodiments, the pharmaceutical composition can be administered to the subject parenterally, including but not limited to intratumoral injection (e.g., multiple intratumoral injection), subcutaneous injection, intravenous injection, intramuscular injection, nasal inhalation, or administration via an interventional device. Preferably, the pharmaceutical composition is administered to the subject via intratumoral injection. Intratumoral administration significantly reduces the dosage of each active ingredient, thereby increasing the breadth of pharmacological action to enhance antitumor efficacy, reducing toxicity, and decreasing the likelihood of acquired immune resistance.

[0081] In some embodiments, the method for preventing and / or treating cancer includes the step of administering an effective amount of the combination preparation described in the first aspect to a subject.

[0082] In some embodiments, the combination formulation can be administered to the subject via parenteral administration, including but not limited to intratumoral injection (e.g., multi-point intratumoral injection), subcutaneous injection, intravenous injection, intramuscular injection, nasal inhalation, or administration via an interventional device; the combination formulation can be administered to the subject simultaneously, sequentially, or at intervals.

[0083] In some embodiments, the routes of administration of (a) and (b) in the combined formulation are the same or different. Preferably, (a) and (b) are administered via the same route, such as intratumoral injection or subcutaneous injection; preferably, (a) and (b) are administered via different routes, such as intratumoral injection or intravenous injection, for example, (a) is administered to the subject via intratumoral injection; (b) is administered to the subject via intravenous injection; preferably, a portion of (a) and (b) are administered via the same route, while the remaining portion of (b) is administered via different routes, such as a portion of (a) and (b) being administered to the subject via intratumoral injection, and the remaining portion of (b) being administered to the subject via intravenous injection.

[0084] In some embodiments, in the combined formulation, (a) and (b) are mixed and administered to the subject simultaneously, such as via intratumoral injection or subcutaneous injection. (a) and (b) of the combined formulation are administered to the subject simultaneously via different administration methods, such as via intratumoral injection, intravenous injection, or subcutaneous injection, respectively. Alternatively, a portion of (a) and (b) are mixed and administered to the subject simultaneously via intratumoral injection, while the remaining portion of (b) is administered to the subject simultaneously via intravenous injection.

[0085] In some embodiments, (a) and (b) of the combination formulation may be administered to the subject sequentially and at intervals via the same or different administration methods, preferably via different administration methods; more preferably, via intratumoral injection, intravenous injection, or subcutaneous injection, respectively. For example, (a) and (b) may be administered to the subject via intratumoral injection and intravenous injection, respectively; or a portion of (a) and (b) may be administered to the subject simultaneously via intratumoral injection, and the remaining portion of (b) may be administered to the subject simultaneously via intravenous injection.

[0086] In some embodiments, the different antibodies in (b) of the combined formulation are administered via the same or different routes. Preferably, the different antibodies in (b) are administered via the same route, by intratumoral injection, intravenous injection, or subcutaneous injection. Preferably, the different antibodies in (b) are administered via different routes, by intratumoral injection or intravenous injection, respectively.

[0087] In some embodiments, in the combination formulation, the antibodies in (b) are mixed and administered to the subject simultaneously, or the antibodies are administered to the subject as a single pharmaceutical composition; such as by intravenous injection, intratumoral injection, or subcutaneous injection; or a portion of the antibodies are administered to the subject by intratumoral injection, and the remaining portion of the antibodies are administered to the subject simultaneously by intravenous injection.

[0088] In some embodiments, in the combined formulation, the antibodies in (b) can be administered to the subject sequentially and at intervals via the same or different administration methods, preferably via the same administration method; more preferably via intravenous injection, intratumoral injection or subcutaneous injection; preferably, a portion of the antibodies is administered to the subject via intratumoral injection and the remaining portion is administered to the subject via intravenous injection.

[0089] In some embodiments, the combined formulation,

[0090] (a) CpG HP007 or a pharmaceutically acceptable salt thereof; or R848 or a pharmaceutically acceptable salt thereof; or Poly I:C or a pharmaceutically acceptable salt thereof; by intratumoral injection;

[0091] (b) The antibody is an anti-CD40 antibody and an anti-CTLA4 antibody, administered intratumorally with the anti-CD40 antibody and intravenously with the anti-CTLA4 antibody; or an anti-PD-1 antibody, an anti-CTLA4 antibody, and an anti-TIGIT antibody, administered intravenously; or an anti-PD1 antibody, an anti-CTLA4 antibody, and an anti-CD40 antibody, wherein the anti-PD1 antibody and anti-CTLA4 antibody are administered intravenously and the anti-CD40 antibody is administered intratumorally; or an anti-OX40 antibody, an anti-CTLA4 antibody, and an anti-TIGIT antibody, wherein the anti-OX40 antibody is administered intratumorally and the anti-CTLA4 antibody and anti-TIGIT antibody are administered intravenously. The antibody is preferably a monoclonal antibody.

[0092] In some implementations, the administration or application can be performed by a single or multiple administrations; preferably, administration can be performed by 1 to 12 administrations.

[0093] In some embodiments, in the pharmaceutical composition or combination formulation, (a) has a single dose of 0.01-10 mpk and (b) has a single dose of 0.02-60 mpk; preferably, (a) has a single dose of 0.02-5 mpk and (b) has a single dose of 0.04-30 mpk. More preferably, (a) has a single dose of 0.1-2 mpk and (b) has a single dose of 0.2-30 mpk. Most preferably, (a) has a single dose of 0.1-1 mpk and (b) has a single dose of 0.4-20 mpk.

[0094] In some embodiments, when a TLR9 agonist (such as CpG ODN or a pharmaceutically acceptable salt thereof) is present in the pharmaceutical composition or combination formulation, the single-dose administration is 0.01-10 mpk, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0. 70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3, 4, 5, 6, 7, 8, 9, 10 mpk, preferably 0.02-5 mpk, more preferably 0.1-2 mpk, most preferably 0.4-1 mpk; TLR7 / 8 dual agonist (such as R When 848 or its pharmaceutically acceptable salts are present, the single dose is 0.01-10 mpk, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0. 70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3, 4, 5, 6, 7, 8, 9, 10 mpk, preferably 0.02-5 mpk, more preferably 0.1-2 mpk, most preferably 0.1-1 mpk; TLR3 agonists (such as Poly When I:C or its pharmaceutically acceptable salts are present, the single dose is 0.01-10 mpk, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.0, 1.1, 1.2, 1.3, 1.4. The dosages are 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3, 4, 5, 6, 7, 8, 9, 10 mpk, preferably 0.02-5 mpk, more preferably 0.1-2 mpk, and most preferably 0.1-1 mpk; when anti-CTLA4 antibody is present, the single dose is 0.01-20 mpk, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 mpk, preferably 0.02-10 mpk; more preferably 0.1-5 mpk, most preferably 0.4-4 mpk; when anti-CD40 antibody is present, its single dose is 0.01-5 mpk, such as 0.01, 0. 0.2, 0.03, 0.04, 0.05, 0.1, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.0, 2, 3, 4, 5 mpk, preferably 0.02-2 mpk; more preferably 0.1-1 mpk, most preferably 0.3-1 mpk; when anti-PD1 antibody is present, its single dose is 0.01-20 mpk, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.15, 0. 20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 mpk, preferably 0.02-15 mpk, more preferably 0.1-10 mpk, most preferably 1-10 mpk; when anti-OX40 antibody is present, its single-dose administration is... The dosage is 0.01-5 mpk, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.0, 2, 3, 4, 5 mpk, preferably 0.02-2 mpk; more preferably 0.1-2 mpk, most preferably 0.2-0.5 mpk; when anti-TIGIT antibody is present, the single dose is 0.01-10 mpk, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.0.7, 0.08, 0.09, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1, 1.5, 1.67, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10 mpk, preferably 0.02-5 mpk, more preferably 0.1-5 mpk, and most preferably 1-2 mpk.

[0095] In some embodiments, the pharmaceutical composition or combination formulation comprises (a) CpG HP007 or a pharmaceutically acceptable salt thereof, administered at a single dose of 0.01-10 mpk; or R848 or a pharmaceutically acceptable salt thereof, administered at a single dose of 0.01-10 mpk; or Poly I:C or a pharmaceutically acceptable salt thereof, administered at a single dose of 0.01-10 mpk.

[0096] (b) Anti-CD40 antibody, single dose of 0.01-5 mpk, and anti-CTLA4 antibody, single dose of 0.01-20 mpk; or anti-PD-1 antibody, single dose of 0.01-20 mpk, anti-CTLA4 antibody, single dose of 0.01-20 mpk, and anti-TIGIT antibody, single dose of 0.01-10 mpk; or anti-PD1 antibody, single dose of 0.01-20 mpk, anti-CTLA4 antibody, single dose of 0.01-20 mpk, and anti-CD40 antibody, single dose of 0.01-5 mpk; or anti-OX40 antibody, single dose of 0.01-5 mpk, anti-CTLA4 antibody, single dose of 0.01-20 mpk, and anti-TIGIT antibody, single dose of 0.01-10 mpk.

[0097] In some embodiments, the pharmaceutical composition or combination formulation comprises (a) CpG HP007 or a pharmaceutically acceptable salt thereof, administered at a single dose of 0.02-5 mpk; or R848 or a pharmaceutically acceptable salt thereof, administered at a single dose of 0.02-5 mpk; or Poly I:C or a pharmaceutically acceptable salt thereof, administered at a single dose of 0.02-5 mpk;

[0098] (b) Anti-CD40 antibody, single dose of 0.02-2 mpk, and anti-CTLA4 antibody, single dose of 0.02-10 mpk; or anti-PD-1 antibody, single dose of 0.02-15 mpk, anti-CTLA4 antibody, single dose of 0.02-10 mpk, and anti-TIGIT antibody, single dose of 0.02-5 mpk; or anti-PD1 antibody, single dose of 0.02-15 mpk, anti-CTLA4 antibody, single dose of 0.02-10 mpk, and anti-CD40 antibody, single dose of 0.02-2 mpk; or anti-OX40 antibody, single dose of 0.02-2 mpk, anti-CTLA4 antibody, single dose of 0.02-10 mpk, and anti-TIGIT antibody, single dose of 0.02-5 mpk.

[0099] In some embodiments, the pharmaceutical composition or combination formulation comprises (a) CpG HP007 or a pharmaceutically acceptable salt thereof, administered at a single dose of 0.1-2 mpk; or R848 or a pharmaceutically acceptable salt thereof, administered at a single dose of 0.1-2 mpk; or Poly I:C or a pharmaceutically acceptable salt thereof, administered at a single dose of 0.1-2 mpk.

[0100] (b) Anti-CD40 antibody, single dose of 0.1-1 mpk, and anti-CTLA4 antibody, single dose of 0.1-5 mpk; or anti-PD-1 antibody, single dose of 0.1-10 mpk, anti-CTLA4 antibody, single dose of 0.1-5 mpk, and anti-TIGIT antibody, single dose of 0.1-5 mpk; or anti-PD1 antibody, single dose of 0.1-10 mpk, anti-CTLA4 antibody, single dose of 0.1-5 mpk, and anti-CD40 antibody, single dose of 0.1-1 mpk; or anti-OX40 antibody, single dose of 0.1-2 mpk, anti-CTLA4 antibody, single dose of 0.1-5 mpk, and anti-TIGIT antibody, single dose of 0.1-5 mpk.

[0101] In some embodiments, the pharmaceutical composition or combination formulation comprises (a) CpG HP007 or a pharmaceutically acceptable salt thereof, administered at a single dose of 0.4-1 mpk; or R848 or a pharmaceutically acceptable salt thereof, administered at a single dose of 0.1-1 mpk; or Poly I:C or a pharmaceutically acceptable salt thereof, administered at a single dose of 0.1-1 mpk.

[0102] (b) Anti-CD40 antibody, single dose of 0.3-1 mpk, and anti-CTLA4 antibody, single dose of 0.4-4 mpk; or anti-PD-1 antibody, single dose of 1-10 mpk, anti-CTLA4 antibody, single dose of 0.4-4 mpk, and anti-TIGIT antibody, single dose of 1-2 mpk; or anti-PD1 antibody, single dose of 1-10 mpk, anti-CTLA4 antibody, single dose of 0.4-4 mpk, and anti-CD40 antibody, single dose of 0.3-1 mpk; or anti-OX40 antibody, single dose of 0.2-0.5 mpk, anti-CTLA4 antibody, single dose of 0.4-4 mpk, and anti-TIGIT antibody, single dose of 1-2 mpk.

[0103] In some embodiments, the pharmaceutical composition or combination formulation comprises (a) CpG HP007 or a pharmaceutically acceptable salt thereof, administered at a single dose of 0.4 or 1 mpk; or R848 or a pharmaceutically acceptable salt thereof, administered at a single dose of 0.1, 0.3 or 1 mpk; or Poly I:C or a pharmaceutically acceptable salt thereof, administered at a single dose of 0.1, 0.3 or 1 mpk;

[0104] (b) Anti-CD40 antibody, administered at a single dose of 0.3, 0.5, or 1 mpk, and anti-CTLA4 antibody, administered at a single dose of 0.4, 2, or 4 mpk; or anti-PD-1 antibody, administered at a single dose of 1, 5, or 10 mpk, anti-CTLA4 antibody, administered at a single dose of 0.4, 2, or 4 mpk, and anti-TIGIT antibody, administered at a single dose of 1.67 mpk; or anti-PD1 antibody, administered at a single dose of 1, 5, or 10 mpk, anti-CTLA4 antibody, administered at a single dose of 0.4, 2, or 4 mpk, and anti-CD40 antibody, administered at a single dose of 0.3, 0.5, or 1 mpk; or anti-OX40 antibody, administered at a single dose of 0.4 mpk, anti-CTLA4 antibody, administered at a single dose of 0.4, 2, or 4 mpk, and anti-TIGIT antibody, administered at a single dose of 1.67 mpk.

[0105] In some embodiments, the pharmaceutical composition or combination formulation comprises (a) CpG HP007 or a pharmaceutically acceptable salt thereof, administered at a single dose of 0.4-1 mpk;

[0106] (b) Anti-CD40 antibody, single dose of 0.3-1 mpk, and anti-CTLA4 antibody, single dose of 0.4-4 mpk; or anti-PD-1 antibody, single dose of 1-10 mpk, anti-CTLA4 antibody, single dose of 0.4-4 mpk, and anti-CD40 antibody, single dose of 0.3-1 mpk; or anti-OX40 antibody, single dose of 0.2-0.5 mpk, anti-CTLA4 antibody, single dose of 0.4-4 mpk, and anti-TIGIT antibody, single dose of 1-2 mpk.

[0107] In some embodiments, the pharmaceutical composition or combination formulation comprises (a) CpG HP007 or a pharmaceutically acceptable salt thereof, administered at a single dose of 0.4-1 mpk;

[0108] (b) Anti-CD40 antibody, single dose of 0.5-1 mpk, and anti-CTLA4 antibody, single dose of 2-4 mpk; or anti-PD-1 antibody, single dose of 10 mpk, anti-CTLA4 antibody, single dose of 2-4 mpk, and anti-CD40 antibody, single dose of 0.5-1 mpk; or anti-OX40 antibody, single dose of 0.4 mpk, anti-CTLA4 antibody, single dose of 2-4 mpk, and anti-TIGIT antibody, single dose of 1.67 mpk.

[0109] In some embodiments, the pharmaceutical composition or combination formulation comprises: (a) CpG HP007 or a pharmaceutically acceptable salt thereof, administered at a single dose of 1 mpk; (b) an anti-CD40 antibody, administered at a single dose of 0.5 mpk, and an anti-CTLA4 antibody, administered at a single dose of 2 mpk; or (a) CpG HP007 or a pharmaceutically acceptable salt thereof, administered at a single dose of 1 mpk; (b) an anti-OX40 antibody, administered at a single dose of 0.4 mpk, an anti-CTLA4 antibody, administered at a single dose of 2 mpk, and an anti-TIGIT antibody, administered at a single dose of 1.67 mpk; or (a) CpG HP007 or a pharmaceutically acceptable salt thereof, administered at a single dose of 0.4 mpk; (b) an anti-PD-1 antibody, administered at a single dose of 10 mpk, an anti-CTLA4 antibody, administered at a single dose of 4 mpk, and an anti-CD40 antibody, administered at a single dose of 1 mpk.

[0110] In some embodiments, the pharmaceutical composition or combination formulation comprises (a) R848 or a pharmaceutically acceptable salt thereof, administered at a single dose of 0.1-1 mpk; or Poly I:C or a pharmaceutically acceptable salt thereof, administered at a single dose of 0.1-1 mpk.

[0111] (b) anti-CD40 antibody, with a single dose of 0.3-1 mpk, and anti-CTLA4 antibody, with a single dose of 0.4-4 mpk; or anti-PD-1 antibody, with a single dose of 1-10 mpk, anti-CTLA4 antibody, with a single dose of 0.4-4 mpk, and anti-CD40 antibody, with a single dose of 0.3-1 mpk.

[0112] In some embodiments, the pharmaceutical composition or combination formulation comprises (a) R848 or a pharmaceutically acceptable salt thereof, administered at a single dose of 0.3 mpk; (b) an anti-CD40 antibody, administered at a single dose of 0.5 mpk, and an anti-CTLA4 antibody, administered at a single dose of 2 mpk; or (a) R848 or a pharmaceutically acceptable salt thereof, administered at a single dose of 0.3 mpk; (b) an anti-PD-1 antibody, administered at a single dose of 5 mpk, an anti-CTLA4 antibody, administered at a single dose of 2 mpk, and an anti-CD40 antibody, administered at a single dose of 0.5 mpk. Alternatively, (a) R848 or a pharmaceutically acceptable salt thereof, administered at a single dose of 0.1 mpk; (b) anti-PD-1 antibody, administered at a single dose of 1 mpk, anti-CTLA4 antibody, administered at a single dose of 0.4 mpk, and anti-CD40 antibody, administered at a single dose of 0.3 mpk; or (a) R848 or a pharmaceutically acceptable salt thereof, administered at a single dose of 1 mpk; (b) anti-PD-1 antibody, administered at a single dose of 10 mpk, anti-CTLA4 antibody, administered at a single dose of 4 mpk, and anti-CD40 antibody, administered at a single dose of 1 mpk.

[0113] In some embodiments, the pharmaceutical composition or combination formulation comprises (a) Poly I:C or a pharmaceutically acceptable salt thereof, administered at a single dose of 0.3 mpk; (b) an anti-CD40 antibody, administered at a single dose of 0.5 mpk, and an anti-CTLA4 antibody, administered at a single dose of 2 mpk; or (a) Poly I:C or a pharmaceutically acceptable salt thereof, administered at a single dose of 0.3 mpk; (b) an anti-PD-1 antibody, administered at a single dose of 5 mpk, an anti-CTLA4 antibody, administered at a single dose of 2 mpk, and an anti-CD40 antibody, administered at a single dose of 0.5 mpk; or (a) Poly I:C or a pharmaceutically acceptable salt thereof, administered at a single dose of 0.1 mpk; (b) an anti-PD-1 antibody, administered at a single dose of 1 mpk, an anti-CTLA4 antibody, administered at a single dose of 0.4 mpk, and an anti-CD40 antibody, administered at a single dose of 0.3 mpk; or (a) Poly I:C or a pharmaceutically acceptable salt thereof, administered at a single dose of 1 mpk; (b) anti-PD-1 antibody, administered at a single dose of 10 mpk, anti-CTLA4 antibody, administered at a single dose of 4 mpk, and anti-CD40 antibody, administered at a single dose of 1 mpk.

[0114] In some implementations, the subject is a mammal.

[0115] In some implementations, the subjects are humans, dogs, or cats.

[0116] In some embodiments, the methods for preventing and / or treating cancer may be used in combination with surgery, radiotherapy, chemotherapy, and / or electromagnetic therapy.

[0117] Compared with the prior art, this application has the following beneficial effects:

[0118] This application combines different active pharmaceutical ingredients, TLR agonists, and various cancer-treating antibodies, each of which has different anti-tumor immune-activating effects and a synergistic effect.

[0119] This application, by combining different active pharmaceutical ingredients, can improve efficacy compared to therapies that rely on a single drug, and can reduce dosage, thereby reducing the likelihood of toxicity and adverse events, and reducing the possibility of acquired drug resistance.

[0120] TLR agonists have synergistic and toxicity-reducing effects. They can be used in conjunction with immunotherapy. When combined with multiple antibodies, they not only enhance anti-tumor effects but also reduce the systemic dosage of antibodies. Intratumoral administration can further significantly reduce the dosage, thereby increasing the breadth of pharmacological effects to enhance anti-tumor efficacy, reducing the likelihood of toxicity and adverse events, and improving tolerability.

[0121] This application improves therapeutic efficacy by using lower doses of the active pharmaceutical ingredient and increasing the number of therapeutic drugs with synergistic mechanisms, while reducing the frequency and severity of adverse events and the possibility of acquired resistance. Attached Figure Description

[0122] Figure 1 shows the tumor volume in mice after administration of drugs in groups G1, G2, G5, G6, G10, G12, G15, and G22 in Table 1 of Example 2. It illustrates the tumor-suppressing effect of CpG C it combined with anti-mCD40 it+anti-mCTLA-4 iv, or combined with anti-mCD40 it+anti-mCTLA-4 / anti-mPD-1 iv, or combined with anti-mOX40 it+anti-mCTLA-4 / anti-mTIGIT iv.

[0123] Figure 2 shows the tumor volume in mice after administration of Poly I:Cit in groups G1, G4, G5, G6, G7, G10, G11, G14, G16, G17, and G18 in Table 1 of Example 2. It shows the tumor-suppressing effect of Poly I:Cit combined with anti-mCD40it+anti-mCTLA-4 iv, or combined with anti-mCD40it+anti-mCTLA-4 / anti-mPD-1 iv at different doses.

[0124] Figure 3 shows the tumor volume in mice after administration of drugs in groups G1, G3, G5, G6, G7, G10, G11, G13, G19, G20, and G21 in Table 1 of Example 2. It shows the tumor-suppressing effect of R848 it combined with anti-mCD40 it + anti-mCTLA-4 iv, or combined with anti-mCD40 it + anti-mCTLA-4 / anti-mPD-1 iv at different doses. Detailed Implementation

[0125] Reference will now be made to detailed embodiments of this application, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of some embodiments may be used in other embodiments to produce further embodiments.

[0126] Unless otherwise stated, all terms used to disclose this invention (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Further guidance is provided below for a better understanding of the teachings of this invention. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0127] the term

[0128] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0129] The terms “containing,” “comprising,” and “including” as used in this invention are synonyms and are inclusive or open-ended, not excluding additional, uncited members, elements, or method steps.

[0130] The term "two or more" as used in this invention refers to more than two types, such as three or four types.

[0131] In this invention, the numerical range represented by endpoints includes all numerical values ​​and fractions contained within that range, as well as the endpoints mentioned.

[0132] This invention relates to concentration values, which include fluctuations within a certain range. For example, fluctuations are allowed within a corresponding precision range. For instance, 2% may fluctuate within ±0.1%. For larger values ​​or values ​​that do not require overly precise control, even greater fluctuations are permitted. For example, 100mM may fluctuate within ranges of ±1%, ±2%, ±5%, etc. Regarding molecular weight, fluctuations of ±10% are allowed.

[0133] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0134] TLR agonists:

[0135] Toll-like receptors (TLRs) are a family of receptors that recognize microbial-derived molecular structures (pathogen-associated molecular models). Immune cells expressing TLRs are activated by binding to these pathogen-associated molecular models. TLRs are a class of naturally occurring pattern recognition receptors, highly expressed in both humans and mice, and their family includes a series of receptors such as TLR1-TLR10. TLRs can recognize many substances derived from pathogenic microorganisms, which are all TLR agonists. Activation of TLR ligands can activate downstream signaling pathways, triggering the release of various bioactive factors. TLR ligands can be used as adjuvants or immune activators in cancer therapy.

[0136] TLR1 and TLR2 agonists:

[0137] TLR1 molecules are expressed on the cell membrane and typically form heterodimers with TLR2 to recognize ligands of bacterial origin, such as lipoproteins and peptidoglycans, as well as fungal origin, such as yeast polysaccharides. Exemplary TLR1 and TLR2 agonists include, but are not limited to, synthetically produced bacterial lipoproteins (BLP) and Pam3CSK4.

[0138] TLR3 agonists:

[0139] TLR3 molecules are expressed on the endosomal membrane and recognize viral-derived double-stranded RNA. Exemplary TLR3 agonists include, but are not limited to, Poly I:C, Rintatolimod, and poly-ICLC.

[0140] Poly I:C is a synthetic double-stranded ribonucleic acid (BSA) and an interferon inducer with antiviral and immunomodulatory functions.

[0141] TLR4 agonists:

[0142] TLR4 is a unique molecule in the Toll receptor family, capable of being expressed on both the cell membrane and the endosomal membrane. Furthermore, TLR4 is the only Toll-like receptor that can simultaneously recruit both MyD88 and TRIF adaptor proteins. Exemplary TLR4 agonists include, but are not limited to, monophosphate lipid A (MPL) and Glucopyranosyl Lipid-A (GLA).

[0143] TLR5 agonists:

[0144] TLR5 is expressed on the cell membrane and recruits downstream MyD88 to promote the expression of inflammatory cytokines. TLR5 primarily recognizes flagellin from commensal bacteria and exogenous microorganisms. Exemplary TLR4 agonists include, but are not limited to, flagellin, CBLB502 (also known as Entolimod), etc.

[0145] TLR6 agonists:

[0146] Similar to TLR1, TLR6 is expressed on the cell membrane and forms a heterodimer with TLR2 to exert its immune function. Its downstream recruited adaptor protein is MyD88, which primarily activates the NF-κB signaling pathway. TLR6 recognizes various ligands, including bacterial acyl lipoproteins, heat shock proteins, and HMGB1, as well as synthetic lipoproteins FSL-1 and Pam2CSK4.

[0147] TLR7 agonists:

[0148] TLR7 molecules are expressed on the endosomal membrane and form heterodimers with TLR8 to recognize viral-derived single-stranded RNA and synthetically produced ligands such as CpG-A and Poly G3, promoting type I interferon expression. Exemplary TLR7 agonists include, but are not limited to, imiquimod and loxoribin.

[0149] TLR8 agonists:

[0150] TLR8 is expressed on the endosome membrane, and its recognized ligands and downstream signals are consistent with those of TLR7. Only human TLR8 recognizes single-stranded RNA; mouse TLR8 cannot. Exemplary TLR8 agonists include, but are not limited to, VTX-2337.

[0151] TLR9 agonists:

[0152] TLR9 is an intracellular pattern recognition receptor found in the endosomal compartments of mouse plasmacytoid dendritic cells, macrophages, natural killer cells, and other APCs. In humans, TLR9 is primarily expressed in B cells and pDCs. The physiological ligand of TLR9 is a bacterial dsDNA fragment with an unmethylated CpG dinucleotide. TLR9 agonists are artificial oligonucleotides with an unmethylated CpG motif. They can induce signaling cascades, leading to transcriptional programs that result in inflammatory processes, enhanced killing of cancer cells, and the generation of adaptive immune responses.

[0153] CpG ODN:

[0154] CpG ODN refers to CpG oligodeoxynucleotides, which are at least about ten nucleotides in length and include one unmethylated CpG. CpG ODN is single-stranded. The entire CpG ODN can be unmethylated or partially unmethylated. CpG ODN includes D-type (also known as A-type), K-type (also known as B-type), C-type, and P-type ODN.

[0155] Commonly used CpG ODNs include CpG HP007 (SEQ ID NO.1: 5'-tcgcg aacgt tcgcc gcgta cgtacgcgg-3'), CpG HP021 (SEQ ID NO.2: 5'-tcgca acgtt gcctt cgaag g-3'), CpG HP026 (SEQ ID NO.3: 5'-tcgcg acgtt cgccg acgtt cgta-3'), CpG 684 (SEQ ID NO.4: 5'-tcgac gttcg tcgtt cgtcg ttc-3'), SD-101 (SEQ ID NO.5: 5'-tcgaa cgttc gaacg ttcga acgtt cgaat-3'), G10 CpG ODN (SEQ ID NO.6: 5'-ggggg ggggg gacga tcgtc ggggg ggggg-3'), CpG 1018 (SEQ ID NO.7: 5'-tgact gtgaa cgttc gagatga-3'), CpG 7909 (SEQ ID NO.8: 5'-tcgtc gtttt gtcgt tttgt cgtt-3'), IMO-2125 (SEQ ID NO.9: 5'-TCG*AACG*TTCG*-XG*CTTG*CAAG*CT-5', where G* represents 2'-deoxy-7-denitro-guanosine, and X is the glycerol linker).

[0156] Cancer and tumor:

[0157] Cancer and tumor are interchangeable terms that express the same meaning. Cancer refers to a pathological condition in the body characterized by uncontrolled cell proliferation. Cancer cells grow uncontrollably, can invade surrounding tissues and metastasize, endangering an individual's life. Examples of cancer include, but are not limited to: malignant epithelial tumors, lymphomas, blastomas, and leukemia. More specific examples of cancer include solid tumors and hematologic malignancies, including but not limited to: lung (small cell and non-small cell) cancer, breast cancer, prostate cancer, carcinoid tumors, bladder cancer, stomach cancer, pancreatic cancer, liver cancer (hepatocellular carcinoma), hepatoblastoma, colorectal cancer, head and neck cancer, nasopharyngeal carcinoma, squamous cell carcinoma, esophageal cancer, ovarian cancer, uterine cancer, endometrial cancer, cervical cancer, mesothelioma, melanoma, sarcoma, osteosarcoma, liposarcoma, thyroid cancer, desmoidoma, acute myeloid leukemia (AML), and chronic myeloid leukemia (CML).

[0158] Antibody:

[0159] Antibodies include polyclonal antibodies, monoclonal antibodies, and antibody fragments. "Antibody fragments" include the antigen-binding fragments of these antibodies, including Fab, F(ab')2, Fd, Fv, Fab'-SH, scFv, bispecific antibodies, and the smallest antibody recognition unit, as well as single-chain derivatives of these antibodies and fragments, such as scFv-Fc. Antibody types can be selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD. Furthermore, antibodies include naturally occurring antibodies and non-naturally occurring antibodies, including, for example, chimeric, bifunctional, humanized antibodies and human antibodies, as well as related synthetic isoforms. The term "antibody" is used interchangeably with "immunoglobulin."

[0160] PD-1:

[0161] PD-1 is expressed on T cells activated after TCR-mediated antigen stimulation, as well as on B cells, NK cells, and monocytes. The function of PD-1 is to control T cell activation in peripheral tissues to prevent tissue damage caused by persistent inflammation. Prolonged or high-level antigen exposure (e.g., in advanced cancer) leads to PD-1-mediated T cell exhaustion. During T cell sensitization, IFNγ induces PD-L1 in APCs as a counterregulatory mechanism after T cell activation. Under TCR activation, PD-1 ligation with PD-L1 leads to phosphorylation of PD1 by Lek tyrosine kinase, which subsequently recruits SHP2 phosphatase to the intracellular domain of PD-1, thereby dephosphorylating and inactivating TCR and CD28, thus blocking downstream activation signals from stimulation and co-stimulation during T cell sensitization. Similarly, in the effector phase of tumors, PD-L1 is induced by IFNγ and expressed by tumor cells. PD-L1 attaches to PD1+ Teff cells (PD1+ Teff cells recognize MHC-I-presented tumor antigens via TCR), recruits SHP2, thereby inactivating TCR signaling, reducing cytokine production, and inducing T cell apoptosis or PD1+TIL depletion and dysfunction, thus preventing Teff cell infiltration into tumors. Blocking PD1 with antagonistic mAbs works by restoring the immune function of depleted T cells in the periphery and can enhance tumor killing through various immune mechanisms, including directly inhibiting T cell apoptosis, leading to the restoration of T cell effector function, enhancing APC-mediated T cell activation, downregulating Treg-mediated suppression, downregulating immunosuppressive cytokines IL-10 and TGFβ, and upregulating IL-2 and IFN-γ, while simultaneously resisting PD-L1-induced expression in tumors. Clinical evidence supports a model that blocking the PD1 signaling axis is most effective for tumors that have already elicited an endogenous T cell response but are suppressed by the binding of PD-1 to ligands PD-L1 and PD-L2. However, the response to αPD1 mAb in PD-L1(-) tumors suggests that a pre-existing immune response is not always an absolute necessity for tumor rejection after αPD1 treatment.

[0162] CTLA-4:

[0163] As T cell activation progresses, counter-regulatory mechanisms are induced, preventing the activation and survival of T cells with excessively weak or strong TCR / MHC / Ag interactions. One of the inducing signals expressed on Teff cells is CTLA-4, which is induced when TCR / MHC / Ag interactions are too strong or prolonged. CTLA-4 has functions beyond CD28, thereby blocking the co-stimulatory signaling provided by CD80 / 86. This limits the polyclonal nature of TCR variants and the selection, proliferation, and survival of T cell clones. Furthermore, CTLA-4 is constitutively highly expressed by Tregs, which can also inhibit CD80 / 86 co-stimulatory signaling by directly blocking interactions with CD28 and by trans-endocytosis through membrane flaps of CD80 / 86-containing DCs. Increased T cell stimulation leads to increased secretion of Th1 cytokines such as TNFα, IL2, and IFNylate, as well as increased T cell proliferation (↑CD3 / Ki67). Therefore, blocking CTLA4 with αCTLA mAb will increase the co-stimulatory signal provided by CD28 to continue, ultimately enabling T cell clones with strong TCR / MHC / Ag affinity to survive and proliferate, and increasing TCR variant diversity.

[0164] TIGIT:

[0165] TIGIT, a relatively new molecule discovered recently, belongs to the class of cell surface proteins with immunosuppressive functions. Its full name is T cell immune receptor with Ig and ITIM domains, also known as WUCAM, Vstm3, or VSIG9. It is an inhibitory receptor belonging to the Ig superfamily and possesses many characteristic structures of immunoregulatory proteins, such as extracellular immunoglobulin domains, type I transmembrane structures, and intracellular immune receptor tyrosine inhibitory / activating motifs (ITIM / ITAM).

[0166] CD40:

[0167] CD40 (Bp50) is a surface antigen associated with T cell and B cell function. Its precursor contains 297 amino acids and is a type I transmembrane glycoprotein composed of an N-terminal signal peptide (20 amino acids), an extracellular domain (193 amino acids), a transmembrane domain (22 amino acids), and a cytoplasmic domain (62 amino acids). The extracellular domain contains four CDRs with a total of 22 cysteine ​​residues. Within the 71 amino acids of the hinge domain, there are 9 serine residues and 7 threonine residues, which are potential O-glycosylation sites, as well as 2 potential N-glycosylation sites. Therefore, CD40 has a high degree of glycosylation. The molecular weight estimated from the amino acid sequence is 28 kDa, but the molecular weight after glycosylation is 40-50 kDa. CD40 is expressed on B cells, thymic epithelial cells, activated monocytes / macrophages, dendritic cells, hematopoietic progenitor cells, epithelial cells, endothelial cells, and some tumor cell lines, such as the HepG2 liver cancer cell line and the HS294T melanoma cell line. PMA, anti-IgM antibodies, anti-CD20 antibodies, and IFN-γ can all stimulate CD40 expression on B cells, and IFN-γ can also stimulate CD40 expression on tumor cells. CD40 signal transduction mainly regulates the activity of non-receptor tyrosine protein kinases, such as Lyn, Fyn, and Syk, and can also activate PI3K, phospholipase Cg2, Rel / NF-κB transcription factors, and induce Bcl-xL, Cdk4, and Cdk6 proteins.

[0168] OX40:

[0169] OX40 (also known as CD134) is an important T cell costimulatory molecule and a member of the tumor necrosis factor receptor (TNF) superfamily, primarily expressed on activated T cells. The ligand OX40L (also known as CD252) binds to three molecules of OX40 protein in a trimeric form, forming a hexamer complex. This complex activates downstream signaling pathways such as NF-κB, PI3K, and AKT, thereby regulating genes involved in T cell division and survival, promoting the transcription of cytokine genes, and enhancing the expression of cytokine receptors—all crucial for cell survival.

[0170] Effective dosage:

[0171] An effective dose refers to the amount of medication that is effective in treating or preventing the recurrence of a condition or its signs or symptoms. A single dose may not produce the full therapeutic effect; a series of doses may be required for the full therapeutic effect. Therefore, an effective therapeutic dose can be administered in single or multiple applications of a single unit. The dosage depends on the individual being treated, the individual's immune system's ability to synthesize antibodies, and the expected level of protection. The exact amount of active ingredient required depends on the physician's judgment and varies from person to person.

[0172] Anti-tumor effects:

[0173] In this invention, anti-tumor effects refer to the inhibition of tumor cell growth, reduction of tumor cell number, tumor shrinkage, tumor disappearance, improvement of individual clinical symptoms, slowing of tumor growth, extension of individual life, improvement of individual quality of life, and suppression of tumor metastasis and recurrence. Anti-tumor effects include both the treatment of tumors and the prevention of tumor occurrence and recurrence.

[0174] Pharmaceutically acceptable salts:

[0175] Pharmaceutically acceptable salts are those that, within the bounds of reasonable medical judgment, are suitable for contact with the tissues of mammals, particularly humans, without excessive toxicity, irritation, allergic reactions, etc., and whose benefits / risks are proportionate to a reasonable ratio. If the compound is basic, pharmaceutically acceptable salts include salts prepared from inorganic acids as well as salts prepared from organic acids. If the compound is acidic, pharmaceutically acceptable salts include salts prepared from inorganic bases and / or organic bases.

[0176] Pharmaceutically acceptable salts of exemplary CpG oligonucleotides include: ammonium salts, alkali metal salts such as sodium, lithium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, zinc salts, salts formed with organic bases (e.g., organic amines), such as N-Me-D-reduced glucosamine, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride, choline, tromethamine, dicyclohexylamine, tert-butylamine, and salts formed with amino acids such as arginine, lysine, etc.

[0177] Pharmaceutically acceptable carriers or excipients:

[0178] Pharmaceutically acceptable means any substance permitted by the relevant government regulatory authority as acceptable for human or livestock use. A carrier refers to one or more solid or liquid fillers, diluents, or encapsulating substances, which may be organic, inorganic, natural, or synthetic. This includes various solutions, diluents, solvents, dispersants, liposomes, emulsifiers, antibacterial agents, antifungal agents, isotonic and de-absorption agents, and other carriers suitable for use in the pharmaceutical combinations of this invention. Excipients include, for example, solvents, fillers, buffers, tension modifiers, and preservatives. In some embodiments, the pharmaceutical composition may contain one or more excipients acting as solvents, fillers, buffers, and tension modifiers. The choice of pharmaceutically acceptable carriers and excipients depends on the manner of application of the pharmaceutical combinations provided by this invention.

[0179] Subjects:

[0180] In this invention, "subject" and "patient" refer to mammals. Exemplary subjects include humans, dogs, and cats.

[0181] The embodiments of the present invention will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this invention, or follow experimental manuals or conventional conditions in the art, or other experimental methods known in the art, or follow the conditions recommended by the manufacturer.

[0182] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0183] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0184] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0185] Example 1: Tumor suppression experiment in a mouse model of colon cancer (MC38)

[0186] Experimental materials:

[0187] PBS was purchased from Solarbio, catalog number P1010. Unless otherwise specified, the concentration of all PBS solutions was 0.01M and the pH value was 7.2-7.4.

[0188] TLR receptor agonists:

[0189] TLR9 agonist CpG C1: 5'-tcgcgaacgttcgccgcgtacgtacgcgg-3', SEQ ID NO.: 1, (CpG HP007), was provided by Huapu Biotechnology (Hebei) Co., Ltd.

[0190] TLR7 / 8 agonist R848 (Resiquimod), RCE, catalog number: 113202.

[0191] TLR3 agonist Poly I:C, Sigma, catalog number P1350-25MG.

[0192] Antibody:

[0193] Anti-mPD-1 (clone number: RMP1-14), BioXcell, catalog number: 861023J2.

[0194] Anti-mCTLA4 (clone number: 9D9), BioXcell, catalog number: 809522M2.

[0195] Anti-mTIGIT (clone number: 1B4), Absolute, catalog number: Ab01258-1.1 / T2434B03.

[0196] Anti-mCD40 (clone number: FGK4.5 / FGK45), BioXcell, catalog number: BE0016-2.

[0197] Anti-mOX40 (clone number: OX-36), BioXcell, catalog number: BE0031.

[0198] Mice:

[0199] Female C57BL / 6N mice aged 6-7 weeks, Beijing Vital River Biotechnology Co., Ltd.

[0200] Cell lines:

[0201] MC38 tumor cells. MC38 tumor cells were cultured in DMEM medium containing 10% fetal bovine serum. MC38 cells in the exponential growth phase were collected and resuspended in PBS to a suitable concentration for subcutaneous inoculation in C57BL / 6 mice.

[0202] Experimental methods:

[0203] The experimental mice were subcutaneously injected with 1×10⁻⁶ spores on their right back. 6 MC38 cells were resuspended in PBS (0.1 mL / cell), and tumor growth was observed regularly until the tumor reached an average volume of 150 mm². 3 Mice were randomly assigned to groups based on tumor size and body weight for drug administration. Treatment was administered every 3 days for 5 consecutive days. Before administration, all animals were weighed, and tumor volume was measured using calipers. Tumor volume was estimated using the following formula: Volume = Length × Width 2 ×0.5. All data are expressed as averages in mm. 3 +SEM. The day of grouping is designated as Day 0 (D0), and the administration begins on Day 1 (D1) according to the experimental design.

[0204] Calculate the relative tumor volume increase ratio T / C (%), T / C (%) = (mean of relative tumor volume in the treatment group / mean of relative tumor volume in the control group) × 100, and then calculate the tumor growth inhibition rate (TGI, %) = (1-T / C) × 100.

[0205] During the experiment, mice whose body weight decreased by more than 20% compared to the first day of administration, or mice deemed unsuitable by a veterinarian, were euthanized. Total tumor volume exceeding 2000 mm² was also considered. 3 The mice will be euthanized.

[0206] Six mice were in each experimental group. They were administered either systemically (iv) at a volume of 10 μL / g or intratumorally (it) at a volume of 50 μL / mouse. The administration cycle was Q3D for a total of 5 administrations.

[0207] Example 2: The therapeutic effects of different TLR agonists combined with different antibodies in a murine MC38 colon cancer model

[0208] The animals are grouped as shown in Table 1:

[0209] Table 1

[0210] [Corrected according to Rule 91, 27.01.2026] The experimental results are shown in Table 2 and Figures 1 to 3.

[0211] Table 2: Antitumor effects of different treatment groups for D13 Note: First, Bartlett's test was performed to check the consistency of variance among the groups. P < 0.05 indicates that the data does not conform to a normal distribution. Therefore, the Kruskal-Wallis test was used to check whether there are groups with different medians, and Conover's non-parametric all-pairs comparison test was run for all possible pairwise comparisons.

[0212] Figures 1 to 3 show the tumor volume in mice after administration of the drugs listed in Table 1 for groups G1, G2, G5, G6, G10, G12, G15, G22; groups G1, G4, G5, G6, G7, G10, G11, G14, G16, G17, G18; and groups G1, G3, G5, G6, G7, G10, G11, G13, G19, G20, G21. Figure 1 shows the tumor-suppressing effects of CpG C it combined with anti-mCD40 it + anti-mCTLA-4 iv, or combined with anti-mCD40 it + anti-mCTLA-4 / anti-mPD-1 iv, or combined with anti-mOX40 it + anti-mCTLA-4 / anti-mTIGIT iv. Figure 2 shows the antitumor effects of Poly I:C it combined with anti-mCD40 it + anti-mCTLA-4 iv, or combined with anti-mCD40 it + anti-mCTLA-4 / anti-mPD-1 iv at different doses. Figure 3 shows the antitumor effects of R848 it combined with anti-mCD40 it + anti-mCTLA-4 iv, or combined with anti-mCD40 it + anti-mCTLA-4 / anti-mPD-1 iv at different doses.

[0213] As shown in Figure 1 and Table 2, compared with the negative control (PBS) group, the TLR9 agonist CpGC alone showed a certain antitumor effect (G2, TGI = 62.7%); the anti-mCD40 it + anti-mCTLA-4 iv group showed a considerable tumor-suppressive effect (G10, TGI = 61.9%); when CpGC was administered in combination with two antibodies, it did not further enhance the antitumor effect of these two antibodies (G12, TGI = 65.6%). When CpGC was administered in combination with three antibodies, the antitumor effect was further enhanced (G15, TGI = 85.1%; G22, TGI = 75.2%).

[0214] As shown in Figure 2 and Table 2, compared with the negative control (PBS) group, the TLR3 agonist Poly I:C alone had no antitumor effect (G4, TGI = 0.8%); the anti-mCD40 it + anti-mCTLA-4 iv group had a strong antitumor effect (G10, TGI = 61.9%); when Poly I:C was administered in combination with the two antibodies, it did not further enhance the antitumor effect of the two antibodies (G14, TGI = 64.5%). However, when Poly I:C was used in combination with the three antibodies, the antitumor effect was further enhanced, showing a synergistic antitumor effect. Furthermore, the antitumor effect showed a dose-dependent relationship with the dosage of Poly I:C and each antibody. Within the dosage range of this experiment, the medium dose showed the best antitumor effect (G16, TGI = 68.5%; G17, TGI = 89.1%; G18 = 84.4%). Moreover, the antitumor effect of the low-dose group of Poly I:C + three antibodies (G16, TGI = 68.5%) was comparable to that of the group that only received the medium dose of the three antibodies (G11, TGI = 71.0%). This indicates that Poly I:C can significantly reduce the dosage of the antibodies without weakening the antitumor effect.

[0215] As shown in Figure 3 and Table 2, compared with the negative control (PBS) group, administration of the TLR7 / 8 agonist R848 alone showed a certain antitumor effect (G3, TGI = 41.7%); administration of anti-mCD40 it + anti-mCTLA-4 iv showed a strong antitumor effect (G10, TGI = 61.9%); when R848 was combined with two antibodies, it did not further enhance the antitumor effect of these two antibodies (G13, TGI = 56.8%). However, when R848 was used in combination with three antibodies, the antitumor effect was further enhanced, showing a synergistic antitumor effect. Furthermore, the antitumor effect showed a dose-dependent relationship with the dosage of R848 and each antibody. Within the dosage range of this experiment, medium and high doses showed comparable antitumor effects (G19, TGI = 71.3%; G20, TGI = 93.0%; G21, TGI = 91.8%). Moreover, the antitumor effect of the low-dose group of R848 + the three antibodies (G19, TGI = 71.3%) was comparable to that of the group that only received medium doses of the three antibodies (G11, TGI = 71.0%). This indicates that R848 can significantly reduce the dosage of the antibodies without weakening the antitumor effect.

[0216] In summary, the combined administration of different TLR agonists and three antibodies demonstrated excellent antitumor effects. Furthermore, the tumor-suppressive effect showed a dose-dependent relationship with the dosage of both the TLR receptor agonist and the antibody. Moreover, the TLR receptor agonist significantly reduced the antibody dosage while maintaining excellent antitumor activity. Additionally, altering the type of TLR receptor agonist or antibody could also result in superior antitumor effects.

[0217] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A pharmaceutical composition comprising a TLR agonist and multiple antibodies, characterized in that, Include: (a) One TLR agonist; (b) Two or more antibodies for treating cancer; (c) Pharmaceutically acceptable carriers or excipients.

2. The pharmaceutical composition according to claim 1, characterized in that, The pharmaceutical composition does not contain any other active ingredients.

3. A combination formulation containing a TLR agonist and multiple antibodies, characterized in that, It includes: (a) One TLR agonist; (b) Two or more antibodies for treating cancer.

4. The combination formulation according to claim 3, characterized in that, The combined formulation does not contain any other active ingredients.

5. The pharmaceutical composition according to claim 1 or 2, or the combination formulation according to any one of claims 3-4, characterized in that, The TLR agonist is selected from TLR3 agonists, TLR4 agonists, TLR5 agonists, TLR7 agonists, TLR8 agonists, TLR7 / 8 dual agonists, and TLR9 agonists.

6. The pharmaceutical composition or combination formulation according to claim 5, characterized in that, The TLR9 agonist is selected from CpG ODN and pharmaceutically acceptable salts thereof, preferably from CpG HP007, CpG HP021, CpG HP026, CpG 684, SD-101, G10 CpG ODN, CpG 1018, CpG 7909, IMO-2125 and pharmaceutically acceptable salts thereof; The TLR7 / 8 dual agonist is R848 or a pharmaceutically acceptable salt thereof; The TLR3 agonist is Poly I:C or a pharmaceutically acceptable salt thereof.

7. The pharmaceutical composition or combination formulation according to claim 5 or 6, characterized in that, (a) is a TLR9 agonist; or a TLR7 / 8 agonist; or a TLR3 agonist; Preferably, (a) is CpG HP007 or a pharmaceutically acceptable salt thereof; or R848 or a pharmaceutically acceptable salt thereof; or Poly I:C or a pharmaceutically acceptable salt thereof.

8. The pharmaceutical composition or combination formulation according to any one of claims 1-7, characterized in that, The antibody used to treat cancer targets one, two, or more of PD-1, CTLA-4, TIGIT, OX40, and CD40.

9. The pharmaceutical composition or combination formulation according to any one of claims 1-8, characterized in that, The antibodies used to treat cancer are monoclonal antibodies, bispecific antibodies, trispecific antibodies, antibody-drug conjugates (ADCs), or radiopharmaceutical-radioactive conjugates (RDCs).

10. The pharmaceutical composition or combination formulation according to claim 8 or 9, characterized in that, (b) is anti-CD40 antibody and anti-CTLA4 antibody; or anti-PD-1 antibody, anti-CTLA4 antibody and anti-TIGIT antibody; or anti-PD1 antibody, anti-CTLA4 antibody and anti-CD40 antibody; or anti-OX40 antibody, anti-CTLA4 antibody and anti-TIGIT antibody.

11. The pharmaceutical composition or combination formulation according to claim 10, characterized in that, Include: (a) CpG HP007 or a pharmaceutically acceptable salt thereof; or R848 or a pharmaceutically acceptable salt thereof; or Poly I:C or a pharmaceutically acceptable salt thereof; (b) Anti-CD40 antibody and anti-CTLA4 antibody; or anti-PD-1 antibody, anti-CTLA4 antibody and anti-TIGIT antibody; or anti-PD1 antibody, anti-CTLA4 antibody and anti-CD40 antibody; or anti-OX40 antibody, anti-CTLA4 antibody and anti-TIGIT antibody.

12. The pharmaceutical composition or combination formulation according to any one of claims 1-11, characterized in that, In the pharmaceutical composition or formulation, the mass ratio of (a):(b) is (1-1000):(2-6000), preferably (2-500):(4-3000), more preferably (10-200):(20-3000). Most preferably (10-100):(40-2000).

13. The pharmaceutical composition or combination formulation according to claim 12, characterized in that, Contains (a) 1-1000 parts of CpG HP007 or a pharmaceutically acceptable salt thereof; or 1-1000 parts of R848 or a pharmaceutically acceptable salt thereof; or 1-1000 parts of Poly I:C or a pharmaceutically acceptable salt thereof; (b) 1-500 doses of anti-CD40 antibody and 1-2000 doses of anti-CTLA4 antibody; or 1-2000 doses of anti-PD-1 antibody, 1-2000 doses of anti-CTLA4 antibody, and 1-1000 doses of anti-TIGIT antibody; or 1-2000 doses of anti-PD1 antibody, 1-2000 doses of anti-CTLA4 antibody, and 1-500 doses of anti-CD40 antibody; or 1-500 doses of anti-OX40 antibody, 1-2000 doses of anti-CTLA4 antibody, and 1-1000 doses of anti-TIGIT antibody; Preferably, it comprises: (a) 40-100 parts of CpG HP007 or a pharmaceutically acceptable salt thereof; or 10-100 parts of R848 or a pharmaceutically acceptable salt thereof; or 10-100 parts of Poly I:C or a pharmaceutically acceptable salt thereof; (b) 30-100 doses of anti-CD40 antibody and 40-400 doses of anti-CTLA4 antibody; or 100-1000 doses of anti-PD-1 antibody, 40-400 doses of anti-CTLA4 antibody and 100-200 doses of anti-TIGIT antibody; or 100-1000 doses of anti-PD1 antibody, 40-400 doses of anti-CTLA4 antibody and 30-100 doses of anti-CD40 antibody; or 20-50 doses of anti-OX40 antibody, 40-400 doses of anti-CTLA4 antibody and 100-200 doses of anti-TIGIT antibody.

14. The pharmaceutical composition or combination formulation according to claim 13, characterized in that, Contains 40-100 parts of (a) CpG HP007 or a pharmaceutically acceptable salt thereof; (b) 50-100 doses of anti-CD40 antibody and 200-400 doses of anti-CTLA4 antibody; or 1000 doses of anti-PD1 antibody, 200-400 doses of anti-CTLA4 antibody and 50-100 doses of anti-CD40 antibody; or 40 doses of anti-OX40 antibody, 200-400 doses of anti-CTLA4 antibody and 167 doses of anti-TIGIT antibody; Preferably, it comprises: (a) 100 parts of CpG HP007 or a pharmaceutically acceptable salt thereof; (b) 50 parts of anti-CD40 antibody and 200 parts of anti-CTLA4 antibody; or (a) 100 parts of CpG HP007 or a pharmaceutically acceptable salt thereof; (b) 40 parts of anti-OX40 antibody, 200 parts of anti-CTLA4 antibody and 167 parts of anti-TIGIT antibody; or (a) 40 parts of CpG HP007 or a pharmaceutically acceptable salt thereof; (b) 1000 parts of anti-PD1 antibody, 400 parts of anti-CTLA4 antibody and 100 parts of anti-CD40 antibody.

15. The pharmaceutical composition or combination formulation according to claim 13, characterized in that, Contains 10-100 parts of (a) R848 or a pharmaceutically acceptable salt thereof; or 10-100 parts of Poly I:C or a pharmaceutically acceptable salt thereof; (b) 30-100 doses of anti-CD40 antibody and 40-400 doses of anti-CTLA4 antibody; or 100-1000 doses of anti-PD1 antibody, 40-400 doses of anti-CTLA4 antibody and 30-100 doses of anti-CD40 antibody. Preferably, it comprises: (a) 30 parts of R848 or a pharmaceutically acceptable salt thereof, or 30 parts of Poly I:C or a pharmaceutically acceptable salt thereof; (b) 50 parts of anti-CD40 antibody and 200 parts of anti-CTLA4 antibody; or (a) 30 parts of R848 or a pharmaceutically acceptable salt thereof, or 30 parts of Poly I:C or a pharmaceutically acceptable salt thereof; (b) 50 parts of anti-CD40 antibody, 200 parts of anti-CTLA4 antibody and 500 parts of anti-PD1 antibody; or (a) 10 parts of R848 or a pharmaceutically acceptable salt thereof, or 10 parts of Poly I:C or a pharmaceutically acceptable salt thereof; (b) 30 parts of anti-CD40 antibody, 40 parts of anti-CTLA4 antibody and 100 parts of anti-PD1 antibody; or (a) 100 parts of R848 or a pharmaceutically acceptable salt thereof, or Poly I:C or a pharmaceutically acceptable salt thereof. (a) 100 parts of I:C or its pharmaceutically acceptable salt; (b) 100 parts of anti-CD40 antibody, 400 parts of anti-CTLA4 antibody, and 1000 parts of anti-PD1 antibody.

16. The pharmaceutical composition or combination preparation according to any one of claims 1-15, for the prevention and / or treatment of cancer; preferably, the cancer is a solid tumor; more preferably, the cancer is selected from colorectal cancer, melanoma, breast cancer, lung cancer, gastric cancer, head and neck tumors, prostate cancer, liver cancer, nasopharyngeal carcinoma, esophageal cancer, and cervical cancer.

17. The pharmaceutical use of the pharmaceutical composition or combination preparation according to any one of claims 1-15, for the preparation of a medicament for the prevention and / or treatment of cancer; preferably, the cancer is a solid tumor; more preferably, the cancer is selected from colorectal cancer, melanoma, breast cancer, lung cancer, gastric cancer, head and neck tumors, prostate cancer, liver cancer, nasopharyngeal carcinoma, esophageal cancer, and cervical cancer.

18. A method for preventing and / or treating cancer, characterized in that, The method includes the following steps: administering to a subject an effective amount of the pharmaceutical composition or combination formulation according to any one of claims 1-15; preferably, the cancer is a solid tumor; more preferably, the cancer is selected from colorectal cancer, melanoma, breast cancer, lung cancer, gastric cancer, head and neck tumors, prostate cancer, liver cancer, nasopharyngeal carcinoma, esophageal cancer, and cervical cancer.

19. The method according to claim 18, characterized in that, The pharmaceutical composition or combination formulation is administered to the subject via parenteral administration, preferably via intratumoral injection, subcutaneous injection, intravenous injection, intramuscular injection, nasal inhalation, or via an interventional device.

20. The method according to claim 19, characterized in that, The combined formulation can be administered to the subject simultaneously, sequentially, or at intervals.

21. The method according to claim 19 or 20, characterized in that, In the combined formulation, (a) and (b) may be administered via the same or different routes; Preferably, (a) and (b) are administered via the same route, either by intratumoral injection or subcutaneous injection; or (a) and (b) are administered via different routes, with (a) administered to the subject via intratumoral injection. (b) administered to the subject via intravenous injection; Alternatively, a portion of (a) and (b) may be administered via the same route, while the remaining portion of (b) may be administered via a different route, with a portion of (a) and (b) administered to the subject via intratumoral injection and the remaining portion of (b) administered to the subject via intravenous injection.

22. The method according to claim 20 or 21, characterized in that, In the combined formulation, (a) and (b) are mixed and administered to the subject simultaneously; or (a) and (b) are administered to the subject simultaneously via different administration methods; preferably, (a) and (b) are mixed and administered to the subject simultaneously via intratumoral injection or subcutaneous injection, or (a) and (b) are administered to the subject simultaneously via different administration methods selected from intratumoral injection and intravenous injection; or a portion of (a) and (b) are mixed and administered to the subject simultaneously via intratumoral injection, and the remaining portion of (b) is administered to the subject simultaneously via intravenous injection.

23. The method according to claim 20 or 21, characterized in that, The combination formulation (a) and (b) can be administered to the subject sequentially or at intervals via the same or different administration methods. Preferably, the subject is administered via intratumoral injection or intravenous injection. More preferably, (a) and (b) are administered to the subject via intratumoral injection and intravenous injection, respectively. Alternatively, a portion of (a) and (b) are administered to the subject via intratumoral injection simultaneously, and the remaining portion of (b) is administered to the subject via intravenous injection simultaneously.

24. The method according to claim 19 or 20, characterized in that, In the combined formulation, (b) the different antibodies are administered via the same or different routes; Preferably, in (b), the different antibodies are administered via the same route, such as intratumoral injection, intravenous injection, or subcutaneous injection; or in (b), the different antibodies are administered via different routes, such as a portion of the antibodies in (b) being administered to the subject via intratumoral injection, and the remaining portion in (b) being administered to the subject via intravenous injection.

25. The method according to claim 24, characterized in that, In the combined formulation, the antibodies in (b) are mixed and administered to the subject simultaneously, or the antibodies are administered to the subject as a single drug composition, preferably by intravenous injection, intratumoral injection, or subcutaneous injection; or a portion of the antibodies are administered to the subject by intratumoral injection, and the remaining portion of the antibodies are administered to the subject by intravenous injection.

26. The method according to claim 24, characterized in that, In the combined formulation, the antibodies in (b) can be administered to the subject sequentially and at intervals via the same or different administration methods. Preferably, the antibodies are administered via the same method, such as intratumoral injection, intravenous injection, or subcutaneous injection; or a portion of the antibodies are administered to the subject via intratumoral injection, and the remaining portion is administered to the subject via intravenous injection.

27. The method according to any one of claims 22-26, characterized in that, In the aforementioned combination formulation (a) CpG HP007 or a pharmaceutically acceptable salt thereof; or R848 or a pharmaceutically acceptable salt thereof; or Poly I:C or a pharmaceutically acceptable salt thereof; by intratumoral injection; (b) Anti-CD40 antibody and anti-CTLA4 antibody, with anti-CD40 antibody injected intratumorally and anti-CTLA4 antibody injected intravenously; or anti-PD-1 antibody, anti-CTLA4 antibody and anti-TIGIT antibody, injected intravenously; or anti-PD1 antibody, anti-CTLA4 antibody and anti-CD40 antibody, wherein anti-PD1 antibody and anti-CTLA4 antibody are injected intravenously and anti-CD40 antibody is injected intratumorally; or anti-OX40 antibody, anti-CTLA4 antibody and anti-TIGIT antibody, wherein anti-OX40 antibody is injected intratumorally and anti-CTLA4 antibody and anti-TIGIT antibody are injected intravenously.

28. The method according to any one of claims 18-27, characterized in that, Giving can be done in one instance or multiple instances.

29. The method according to any one of claims 18-28, characterized in that, In the pharmaceutical composition or combination formulation, (a) has a single dose of 0.01-10 mpk and (b) has a single dose of 0.02-60 mpk; preferably, (a) has a single dose of 0.02-5 mpk and (b) has a single dose of 0.04-30 mpk; more preferably, (a) has a single dose of 0.1-2 mpk and (b) has a single dose of 0.2-30 mpk; most preferably, (a) has a single dose of 0.1-1 mpk and (b) has a single dose of 0.4-20 mpk.

30. The method according to claim 29, characterized in that, The pharmaceutical composition or combination formulation comprises, (a) CpG HP007 or a pharmaceutically acceptable salt thereof, administered at a single dose of 0.01-10 mpk; or R848 or a pharmaceutically acceptable salt thereof, administered at a single dose of 0.01-10 mpk; or Poly I:C or a pharmaceutically acceptable salt thereof, administered at a single dose of 0.01-10 mpk; (b) anti-CD40 antibody, single dose of 0.01-5 mpk, and anti-CTLA4 antibody, single dose of 0.01-20 mpk; or anti-PD-1 antibody, single dose of 0.01-20 mpk, anti-CTLA4 antibody, single dose of 0.01-20 mpk, and anti-TIGIT antibody, single dose of 0.01-10 mpk; or anti-PD1 antibody, single dose of 0.01-20 mpk, anti-CTLA4 antibody, single dose of 0.01-20 mpk, and anti-CD40 antibody, single dose of 0.01-5 mpk; or anti-OX40 antibody, single dose of 0.01-5 mpk, anti-CTLA4 antibody, single dose of 0.01-20 mpk, and anti-TIGIT antibody, single dose of 0.01-10 mpk; Preferably, the pharmaceutical composition or combination formulation comprises (a) CpG HP007 or a pharmaceutically acceptable salt thereof, administered at a single dose of 0.4-1 mpk; or R848 or a pharmaceutically acceptable salt thereof, administered at a single dose of 0.1-1 mpk; or Poly I:C or a pharmaceutically acceptable salt thereof, administered at a single dose of 0.1-1 mpk. (b) Anti-CD40 antibody, single dose of 0.3-1 mpk, and anti-CTLA4 antibody, single dose of 0.4-4 mpk; or anti-PD-1 antibody, single dose of 1-10 mpk, anti-CTLA4 antibody, single dose of 0.4-4 mpk, and anti-TIGIT antibody, single dose of 1-2 mpk; or anti-PD1 antibody, single dose of 1-10 mpk, anti-CTLA4 antibody, single dose of 0.4-4 mpk, and anti-CD40 antibody, single dose of 0.3-1 mpk; or anti-OX40 antibody, single dose of 0.2-0.5 mpk, anti-CTLA4 antibody, single dose of 0.4-4 mpk, and anti-TIGIT antibody, single dose of 1-2 mpk.

31. The method according to claim 30, characterized in that, The drug or combination formulation comprises (a) CpG HP007 or a pharmaceutically acceptable salt thereof, administered in a single dose of 0.4-1 mpk; (b) Anti-CD40 antibody, single dose of 0.5-1 mpk, and anti-CTLA4 antibody, single dose of 2-4 mpk; or anti-PD-1 antibody, single dose of 10 mpk, anti-CTLA4 antibody, single dose of 2-4 mpk, and anti-CD40 antibody, single dose of 0.5-1 mpk; or anti-OX40 antibody, single dose of 0.4 mpk, anti-CTLA4 antibody, single dose of 2-4 mpk, and anti-TIGIT antibody, single dose of 1.67 mpk; Preferably, the pharmaceutical composition or combination formulation comprises: (a) CpG HP007 or a pharmaceutically acceptable salt thereof, administered at a single dose of 1 mpk; (b) an anti-CD40 antibody, administered at a single dose of 0.5 mpk, and an anti-CTLA4 antibody, administered at a single dose of 2 mpk; or (a) CpG HP007 or a pharmaceutically acceptable salt thereof, administered at a single dose of 1 mpk; (b) an anti-OX40 antibody, administered at a single dose of 0.4 mpk, an anti-CTLA4 antibody, administered at a single dose of 2 mpk, and an anti-TIGIT antibody, administered at a single dose of 1.67 mpk; or (a) CpG HP007 or a pharmaceutically acceptable salt thereof, administered at a single dose of 0.4 mpk; (b) an anti-PD-1 antibody, administered at a single dose of 10 mpk, an anti-CTLA4 antibody, administered at a single dose of 4 mpk, and an anti-CD40 antibody, administered at a single dose of 1 mpk.

32. The method according to claim 30, characterized in that, The drug or combination formulation comprises (a) R848 or a pharmaceutically acceptable salt thereof, administered at a single dose of 0.1-1 mpk; or Poly I:C or a pharmaceutically acceptable salt thereof, administered at a single dose of 0.1-1 mpk; (b) anti-CD40 antibody, with a single dose of 0.3-1 mpk, and anti-CTLA4 antibody, with a single dose of 0.4-4 mpk; or anti-PD-1 antibody, with a single dose of 1-10 mpk, anti-CTLA4 antibody, with a single dose of 0.4-4 mpk, and anti-CD40 antibody, with a single dose of 0.3-1 mpk; Preferably, the pharmaceutical composition or combination formulation comprises (a) R848 or a pharmaceutically acceptable salt thereof, or Poly I:C or a pharmaceutically acceptable salt thereof, administered at a single dose of 0.3 mpk; (b) an anti-CD40 antibody, administered at a single dose of 0.5 mpk, and an anti-CTLA4 antibody, administered at a single dose of 2 mpk; or (a) R848 or a pharmaceutically acceptable salt thereof, or Poly I:C or a pharmaceutically acceptable salt thereof, administered at a single dose of 0.3 mpk; (b) an anti-PD-1 antibody, administered at a single dose of 5 mpk, an anti-CTLA4 antibody, administered at a single dose of 2 mpk, and an anti-CD40 antibody, administered at a single dose of 0.5 mpk; or (a) R848 or a pharmaceutically acceptable salt thereof, or Poly I:C or a pharmaceutically acceptable salt thereof, administered at a single dose of 0.3 mpk; or (b) an anti-PD-1 antibody, administered at a single dose of 5 mpk, an anti-CTLA4 antibody, administered at a single dose of 2 mpk, and an anti-CD40 antibody, administered at a single dose of 0.5 mpk; or (a) R848 or a pharmaceutically acceptable salt thereof, or Poly I:C or a pharmaceutically acceptable salt thereof, administered at a single dose of 0.3 mpk. (a) I:C or a pharmaceutically acceptable salt thereof, administered at a single dose of 0.1 mpk; (b) anti-PD-1 antibody, administered at a single dose of 1 mpk, anti-CTLA4 antibody, administered at a single dose of 0.4 mpk, and anti-CD40 antibody, administered at a single dose of 0.3 mpk; or (a) R848 or a pharmaceutically acceptable salt thereof, or Poly I:C or a pharmaceutically acceptable salt thereof, administered at a single dose of 1 mpk; (b) anti-PD-1 antibody, administered at a single dose of 10 mpk, anti-CTLA4 antibody, administered at a single dose of 4 mpk, and anti-CD40 antibody, administered at a single dose of 1 mpk.

33. The method according to claims 18-32, characterized in that, The subjects are mammals; preferably, the subjects are humans, dogs, or cats.

34. The method according to claims 18-33, characterized in that, Used in combination with surgery, radiotherapy, chemotherapy and / or electromagnetic therapy.

Images