TWI874649B - A pharmaceutical combination and method for overcoming immune suppression or stimulating immune response against cancer - Google Patents

Abstract

The invention relates to a method of overcoming immune suppression in tumor microenvironment or stimulating immune response against cancer, comprising administering to a subject a combination of a histone deacetylase (HDAC) inhibitor and a tyrosine kinase inhibitor (TKI).

Description

A drug combination and method for overcoming immunosuppression or stimulating anti-cancer immune response

The present invention relates to immunotherapy. Specifically, the present invention provides a drug combination and its application in regulating tumor microenvironment and cancer immunotherapy.

The new era of cancer treatment in tumor immunology will provide great progress in the use of immuno-oncology (IO) therapy to enhance anti-cancer immune response. Immune checkpoint inhibitors (ICIs) are one of the most promising IO therapies, which can release the ability of cytotoxic T lymphocytes (CTLs) to effectively attack and kill tumors, especially ICIs that target the PD-1 (programmed cell death protein 1)/PD-L1 (programmed death ligand 1) axis blockade. To date, several STIs have been developed. However, only about 20% of patients respond to anti-PD-1/anti-PD-L1 antibody monotherapy. About 80% of patients do not obtain clinical benefits caused by primary and acquired resistance. Therefore, resistance to PD1/PD-L1 blockade is a very important problem to overcome with immunotherapy.

Primary resistance refers to the absence of a response to PD-1/PD-L1 blockade. Immunotherapy has a relatively high rate of primary resistance compared to immunotherapy and chemotherapy or targeted therapies, and therefore the clinical benefit is limited. It is estimated that approximately 60% of patients receiving immunotherapy have primary resistance. However, acquired resistance refers to the absence of an initial response to PD-1/PD-L1 blockade as the disease progresses and eventually relapses. It is estimated that approximately 20% of patients receiving immunotherapy have acquired resistance. Low response rates and primary or acquired resistance to PD1/PD-L1 blockade can be associated with the tumor microenvironment ( TME) ( Annals of Oncology , Vol. 27, No. 8 , August 2016 , pp . 1492-1504 ) . The TME is a dynamic and complex complex that controls tumor immune responses. The main mechanisms of primary or acquired resistance to PD-1/PD-L1 blockade may include several factors, such as TME status, PD-L1 expression, tumor neoantigen expression and presentation, cell signaling pathways, immune gene expression, and epigenetic modifications.

Many combination therapy strategies hope to overcome the resistance problem through PD-1/PD-L1 blockade. Many approaches focus on increasing sensitivity to PD1/PD-L1 blockade by using anti-PD-1 antibodies or anti-PD-L1 antibodies in combination with other agents. However, these drug combinations fail to achieve the desired therapeutic benefit, and their efficacy and safety are questionable.

The inventors unexpectedly discovered that tyrosine kinase inhibitors (TKIs) plus histone deacetylase (HDAC) inhibitors significantly improve anticancer efficacy by modulating TME. In addition, TKI plus HDAC inhibitors combined with ICIs significantly overcome primary or acquired resistance through PD1/PD-L1 blockade and improve the efficacy of immunotherapy.

In one aspect, the present invention provides a method for inhibiting or treating cancer in an individual by overcoming immunosuppression or stimulating an anti-cancer immune response in the tumor microenvironment, comprising administering to the individual a combination comprising a histone deacetylase (HDAC) inhibitor or a pharmaceutically acceptable salt thereof and a tyrosine kinase inhibitor (TKI) or a pharmaceutically acceptable salt thereof; wherein the HDAC inhibitor or a pharmaceutically acceptable salt thereof and the tyrosine kinase inhibitor or a pharmaceutically acceptable salt thereof are formulated as one agent, or the HDAC inhibitor and the tyrosine kinase inhibitor are each formulated as a single agent for simultaneous, separate or sequential administration.

In another aspect, the present invention provides a drug combination for use in a method for inhibiting or treating cancer in an individual by overcoming immunosuppression in the tumor microenvironment or stimulating an anti-cancer immune response, wherein the combination comprises a histone deacetylase (HDAC) inhibitor or a pharmaceutically acceptable salt thereof and a tyrosine kinase inhibitor (TKI) or a pharmaceutically acceptable salt thereof; wherein the HDAC inhibitor or a pharmaceutically acceptable salt thereof and the tyrosine kinase inhibitor or a pharmaceutically acceptable salt thereof are formulated as one medicament, or the HDAC inhibitor and the tyrosine kinase inhibitor are each formulated as a single medicament for simultaneous, separate or sequential administration.

In another aspect, the present invention also provides a drug combination comprising a histone deacetylase (HDAC) inhibitor or a pharmaceutically acceptable salt thereof and a tyrosine kinase inhibitor (TKI) or a pharmaceutically acceptable salt thereof; wherein the HDAC inhibitor or a pharmaceutically acceptable salt thereof and the tyrosine kinase inhibitor or a pharmaceutically acceptable salt thereof are formulated as one agent, or the HDAC inhibitor and the tyrosine kinase inhibitor are each formulated as a single agent for simultaneous, separate or sequential administration. In some embodiments of the present invention, the amount of the HDAC inhibitor and the TKI in the drug combination is in the range of about 10% (w/w) to about 70% (w/w) and about 10% (w/w) to about 70% (w/w), respectively. In another embodiment, the pharmaceutical composition further comprises an immune checkpoint inhibitor. In some embodiments of the present invention, the amount of the immune checkpoint inhibitor in the composition is in the range of about 0.5% (w/w) to about 20% (w/w).

In another aspect, the present invention provides a use of a combination comprising a histone deacetylase (HDAC) inhibitor or a pharmaceutically acceptable salt thereof and a tyrosine kinase inhibitor (TKI) or a pharmaceutically acceptable salt thereof for the manufacture of a single agent or multiple agents for inhibiting or treating cancer in an individual by overcoming immunosuppression or stimulating an immune response in the tumor microenvironment, wherein the HDAC inhibitor or a pharmaceutically acceptable salt thereof and the tyrosine kinase inhibitor or a pharmaceutically acceptable salt thereof are formulated as one agent, or the HDAC inhibitor and the tyrosine kinase inhibitor are each formulated as a single agent for simultaneous, separate or sequential administration.

In some embodiments of the invention, the amounts of HDAC inhibitor and TKI in the combinations described herein range from about 10% (w/w) to about 70% (w/w) and about 10% (w/w) to about 70% (w/w), respectively.

In one embodiment, the present invention provides a method for treating cancer in an individual by overcoming immunosuppression or stimulating an immune response in a tumor microenvironment, comprising administering to the individual a combination of a histone deacetylase (HDAC) inhibitor or a pharmaceutically acceptable salt thereof, a tyrosine kinase inhibitor (TKI) or a pharmaceutically acceptable salt thereof, and an immune checkpoint inhibitor (ICI); wherein the histone deacetylase (HDAC) inhibitor is The HDAC inhibitor or a pharmaceutically acceptable salt thereof, the tyrosine kinase inhibitor (TKI) or a pharmaceutically acceptable salt thereof, and the immune checkpoint inhibitor are formulated as one agent, or one or both of the HDAC inhibitor or a pharmaceutically acceptable salt thereof, the tyrosine kinase inhibitor or a pharmaceutically acceptable salt thereof, and the immune checkpoint inhibitor are formulated as multiple agents for simultaneous, separate or sequential administration.

In another embodiment, the present invention provides a drug combination for use in a method for treating cancer in an individual by overcoming immunosuppression in the tumor microenvironment or stimulating an anti-cancer immune response, wherein the combination comprises a histone deacetylase (HDAC) inhibitor or a pharmaceutically acceptable salt thereof, a tyrosine kinase inhibitor (TKI) or a pharmaceutically acceptable salt thereof, and an immune checkpoint inhibitor; wherein the histone deacetylase (HDAC) inhibitor An enzyme (HDAC) inhibitor or a pharmaceutically acceptable salt thereof, a tyrosine kinase inhibitor (TKI) or a pharmaceutically acceptable salt thereof, and an immune checkpoint inhibitor (ICI) are formulated as one agent, or one or both of the HDAC inhibitor or a pharmaceutically acceptable salt thereof, the tyrosine kinase inhibitor or a pharmaceutically acceptable salt thereof, and the immune checkpoint inhibitor are formulated as multiple agents for simultaneous, separate or sequential administration.

In another embodiment, the present invention provides a combination for use in the manufacture of a single agent or multiple agents for inhibiting or treating cancer in an individual by overcoming immunosuppression or stimulating immune response in the tumor microenvironment, wherein the combination comprises a histone deacetylase (HDAC) inhibitor or a pharmaceutically acceptable salt thereof, a tyrosine kinase inhibitor (TKI) or a pharmaceutically acceptable salt thereof, and an immune checkpoint inhibitor (ICI); The histone deacetylase (HDAC) inhibitor or a pharmaceutically acceptable salt thereof, the tyrosine kinase inhibitor (TKI) or a pharmaceutically acceptable salt thereof, and the immune checkpoint inhibitor are formulated as one agent, or one or both of the HDAC inhibitor or a pharmaceutically acceptable salt thereof, the tyrosine kinase inhibitor or a pharmaceutically acceptable salt thereof, and the immune checkpoint inhibitor are formulated as a plurality of agents for simultaneous, separate or sequential administration.

In one embodiment of the invention, the amount of immune checkpoint inhibitor in the combination described herein is in the range of about 0.5% (w/w) to about 20% (w/w).

In one embodiment, in the combinations described herein, the amounts of the histone deacetylase (HDAC) inhibitor or a pharmaceutically acceptable salt thereof, the tyrosine kinase inhibitor (TKI) or a pharmaceutically acceptable salt thereof, and the immune checkpoint inhibitor are in the range of about 10% (w/w) to about 70% (w/w), about 10% (w/w) to about 70% (w/w), and about 0.5% (w/w) to about 20% (w/w), respectively.

In some embodiments of the present invention, the immune checkpoint inhibitors described herein are anti-cytotoxic T lymphocyte antigen 4 (CTLA-4) antibodies or reagents, anti-programmed cell death protein 1 (PD-1) antibodies or reagents, anti-programmed death-ligand 1 (PD-L1) antibodies or reagents, anti-T cell immunoglobulin and mucin domain 3 (TIM-3) antibodies or reagents, anti-B lymphocyte and T lymphocyte attenuation factor (BTLA) antibodies or reagents, anti-V domain Ig containing T cell activation inhibitor (VISTA) antibodies or reagents, anti-lymphocyte activation gene 3 (LAG-3) antibodies or reagents, KIR (killer cell immunoglobulin-like receptor) inhibitors or antibodies, A2AR (adenosine A2A receptor) inhibitor, CD276 inhibitor or antibody or VTCN1 inhibitor or antibody. More preferably, the immune checkpoint inhibitor is pembrolizumab, lambrolizumab, pidilizumab, nivolumab, durvalumab, avelumab or atezolizumab.

In some embodiments of the present invention, the cancers described herein include, but are not limited to, melanoma, head and neck cancer, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, colorectal cancer, endometrial cancer, cervical cancer, esophageal squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, breast cancer, gastric cancer, esophageal gastric junction cancer, classical Hodgkin lymphoma, non-Hodgkin lymphoma, urothelial carcinoma, primary septal large B-cell lymphoma, neuroglioblastoma, pancreatic cancer, benign prostatic hyperplasia, prostate cancer, ovarian cancer, chronic lymphocytic leukemia, Merkel cell carcinoma, carcinoma), acute myeloid leukemia, gallbladder cancer, bile duct cancer, bladder cancer, and uterine cancer.

In another embodiment, the cancer is an immune checkpoint inhibitor-resistant cancer or a cancer that has failed to respond to cancer immunotherapy.

In one embodiment, the individual has not received cancer therapy. In another embodiment, the individual has received cancer therapy but failed to cure. In some embodiments, the cancer therapy is radiation therapy, chemotherapy, or immunotherapy. In another embodiment, the immunotherapy is anti-PD1 immunotherapy, anti-PD L1 immunotherapy, or anti-CTL4 immunotherapy.

In some embodiments of the present invention, the HDAC inhibitor or a pharmaceutically acceptable salt thereof as described herein is a class I selective HDAC inhibitor or a pan-HDAC inhibitor that must inhibit class I HDAC. Examples of HDAC inhibitors or pharmaceutically acceptable salts thereof include (but are not limited to) benzamide HDAC inhibitors. Preferably, the HDAC inhibitor is Chidamide, Entinostat, Vorinostat, Romidepsin, Panobinostat, Belinostat, Valproic Acid, or the like. acid), Mocetinostat, Abexinostat, Pracinostat, Resminostat, Givinostat, Quisinostat, Domatinostat, Quisnostat, CUDC-101, CUDC-907, Pracinostat, Citarinostat, Droxinostat, Abexinostat, Ricolinostat, Tacedinaline, Fimepinostat, Tubacin, Resminostat, ACY-738, Tinostamustine, Tubastatin A A), Givinostat and Dacinostat.

In some embodiments of the present invention, the TKI as described herein or a pharmaceutically acceptable salt thereof is an inhibitor of receptor tyrosine kinase. Preferably, the TKI as described herein or a pharmaceutically acceptable salt thereof is an inhibitor of vascular endothelial growth factor receptor (VEGFR). Examples of TKI or a pharmaceutically acceptable salt thereof include (but are not limited to) Cabozantinib, Regorafenib, Axitinib, Afatinib, Ninetedanib, Crizotinib, Alectinib, Trametinib, Dabrafenib, Sunitinib, Ruxolitinib, ib), Vemurafenib, Sorafenib, Ponatinib, Encorafenib, Brigatinib, Pazopanib, Dasatinib, Imatinib, Lenvatinib, Vandetanib, Surufatinib, and Sitravatinib.

In some other embodiments, examples of the combination described herein include (but are not limited to) the following: (i) anti-CTLA4 antibody, anti-PD1 or anti-PD L1 antibody (ICI), regorafenib, cabozantinib, ibrutinib, axitinib or a pharmaceutically acceptable salt thereof (TKI) and cedamide or cedamide-k30 or a pharmaceutically acceptable salt thereof (HDAC); (ii) anti-CTLA-4 anti-PD1 or anti-PD L1 antibody (ICI), regorafenib, cabozantinib, ibrutinib, axitinib or a pharmaceutically acceptable salt thereof (TKI) and cedamide-HCl salt (HDAC).

In some other embodiments, examples of the combination described herein include (but are not limited to) the following: (1) anti-CTLA-4 antibody (ICI) + regorafenib (TKI) + cedamide or cedamide-k30 (HDAC); (2) anti-CTLA-4 antibody (ICI) + cabozantinib (TKI) + cedamide or cedamide-k30 (HDAC); and (3) anti-CTLA-4 antibody (ICI) + cabozantinib (TKI) + cedamide-HCl salt (HDAC).

In some embodiments of the invention, the methods or combinations described herein further comprise administering one or more additional anti-cancer agents.

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which the present invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are now described. All publications mentioned are incorporated herein by reference.

The terms "a" and "an" refer to one or more than one (ie, at least one) of the grammatical objects of the article. By way of example, "element" means one element or more than one element. Unless otherwise stated, the use of "or" means "and/or."

As used herein, the terms "individual", "person" or "patient" are used interchangeably herein and refer to vertebrates, preferably mammals, and more preferably humans. Mammals include (but are not limited to) mice, monkeys, humans, farm animals, sports animals and pets. It also covers tissues, cells and their progeny of biological entities obtained in vitro or cultured in vitro.

As used herein, "therapeutically effective amount" means an amount sufficient to treat a subject suffering from a disease (e.g., a neurodegenerative disease) or to alleviate symptoms or complications associated with the disease.

As used herein, the terms "treat," "treating," "treatment," and similar terms refer to the reduction or improvement of a disorder and/or symptoms associated therewith. It should be understood that treating a disorder or condition does not require, although not preclude, complete elimination of the disorder, condition, or symptoms associated therewith.

As used herein, the term "immunotherapy" refers to the treatment of an individual suffering from a disease or at risk of infection or recurrence of a disease by methods involving inducing, enhancing, suppressing or otherwise modifying an immune response.

As used herein, the term "programmed cell death protein 1 (PD-1)" refers to an immunosuppressive receptor belonging to the CD28 family. PD-1 is primarily expressed on previously activated T cells in vivo and binds to two ligands, PD-L1 and PD-L2. As used herein, the term "PD-1" includes human PD-1 (hPD-1), variants, isoforms and species homologs of hPD-1, and analogs having at least one common antigenic determinant of hPD-1. The complete hPD-1 sequence can be found under GenBank accession number U64863.

As used herein, the term "programmed death-ligand 1 (PD-L1)" is one of the two cell surface glycoprotein ligands of PD-1 (the other is PD-L2), which downregulates T cell activation and interleukin secretion after binding to PD-1. As used herein, the term "PD-L1" includes human PD-L1 (hPD-L1), variants, isoforms and species homologs of hPD-L1, and analogs that share at least one common antigenic determinant with hPD-L1. The complete hPD-L1 sequence can be found in Genbank Accession No. Q9NZQ7.

As used herein, "antibodies" and "antigen-binding fragments" thereof encompass naturally occurring immunoglobulins (e.g., IgM, IgG, IgD, IgA, IgE, etc.) and non-naturally occurring immunoglobulins, including, for example, single-chain antibodies, chimeric antibodies (e.g., humanized mouse antibodies), heteroconjugate antibodies (e.g., bispecific antibodies), Fab', F(ab').sub.2, Fab, Fv, and rIgG. As used herein, "antigen-binding fragments" are portions of full-length antibodies that retain the ability to specifically recognize antigens, as well as various combinations of such portions.

As used herein, the term "cancer" refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth lead to the formation of malignant tumors that invade neighboring tissues and can also metastasize to distant parts of the body via the lymphatic system or bloodstream. As used herein, "cancer" refers to primary, metastatic, and recurrent cancers.

As used herein, the terms "combination", "therapeutic combination" or "pharmaceutical combination" as used herein define a fixed combination in one unit dosage form or a kit of parts for combined administration, wherein Compound A and Compound B can be administered simultaneously or separately, independently, or at intervals in time.

As used herein, the term "pharmaceutically acceptable" is defined herein to refer to those compounds, materials, compositions and/or dosage forms that are suitable for contact with the tissues of subjects (e.g., mammals or humans) without excessive toxicity, irritation, allergic reactions and other problematic complications commensurate with a reasonable benefit/risk ratio, within the scope of sound medical judgment.

As used herein, the term "co-administration" or "administration in combination" is defined to encompass administration of selected therapeutic agents to a single patient, and is intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or administered at the same time.

The present invention develops methods and compositions that focus on modulating tumor microenvironment components to remove immunosuppression or stimulate the immune system against cancer cells in the tumor microenvironment. The tumor microenvironment is an important aspect of cancer biology that contributes to tumor initiation, tumor progression, and response to therapy. The tumor microenvironment is composed of a heterogeneous cell population that includes malignant cells and cells that support tumor proliferation, invasion, and metastatic potential through extensive crosstalk. Tumor cells often induce an immunosuppressive microenvironment that favors the generation of immunosuppressive populations of immune cells, such as myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs). Therefore, targets within the tumor microenvironment have not been revealed that can help guide and improve the effects of various cancer therapies, especially immunotherapies, that work by enhancing host anti-cancer immune responses. The methods and combinations provide not only advantageous effects but also synchronous effects in inhibiting or treating cancer.

Therefore, the first aspect of the present invention provides a method for overcoming immunosuppression in the tumor microenvironment or stimulating an anti-cancer natural immune response or resistance to a first-line immune checkpoint inhibitor therapy, which comprises administering a combination of a histone deacetylase inhibitor and a tyrosine kinase inhibitor to an individual. In one embodiment, the method comprises administering a combination of an HDAC inhibitor and a TKI in combination with an immune checkpoint inhibitor to an individual. Alternatively, the present invention provides a drug combination of an HDAC inhibitor and a TKI in the manufacture of a medicament for overcoming immunosuppression in the tumor microenvironment or stimulating an anti-cancer immune response. Alternatively, the present invention provides a drug combination for overcoming immunosuppression in the tumor microenvironment or stimulating an anti-cancer immune response, wherein the drug combination comprises an HDAC inhibitor and a TKI. Preferably, the drug combination further comprises an immune checkpoint inhibitor.

A second aspect of the present invention is to provide a drug combination comprising an HDAC inhibitor and a TKI. Preferably, the drug combination further comprises an immune checkpoint inhibitor.

In one embodiment, the amount of HDAC inhibitor and TKI in the drug combination ranges from about 10% (w/w) to about 70% (w/w) and about 10% (w/w) to about 70% (w/w), respectively.

In some embodiments, the amount of the HDAC inhibitor in the drug combination ranges from about 20% (w/w) to about 70% (w/w), about 30% (w/w) to about 70% (w/w), about 40% (w/w) to about 70% (w/w), about 20% (w/w) to about 60% (w/w), about 30% (w/w) to about 60% (w/w), about 40% (w/w) to about 60% (w/w), or about 35% (w/w) to about 60% (w/w).

In some embodiments, the amount of TKI in the drug combination ranges from about 20% (w/w) to about 70% (w/w), about 30% (w/w) to about 70% (w/w), about 40% (w/w) to about 70% (w/w), about 20% (w/w) to about 60% w/w), about 30% (w/w) to about 60% w/w), about 40% (w/w) to about 60% (w/w), or about 35% (w/w) to about 60% (w/w).

HDAC inhibitors have extremely potent epigenetic modulatory properties that significantly improve immunomodulatory activity. HDACs are a class of enzymes that catalyze the removal of acetyl groups from lysines on histones. This deacetylation allows histones to more tightly package DNA. HDAC inhibition controls chromatin remodeling that leads to regulation of gene expression. HDACs have been shown to be involved in oncogenic transformation by mediating gene expression that affects cell cycle progression, proliferation, and apoptosis. HDACs are being studied as possible therapeutic targets for cancer as well as parasitic, infectious (such as AIDS), and inflammatory diseases. The 18 currently known human histone deacetylases are classified into four groups (I to IV) based on the homology of their auxiliary domains to yeast histone deacetylases. Class I, which includes HDAC1, HDAC2, HDAC3, and HDAC8, is related to the yeast RPD3 gene; class IIA includes HDAC4, HDAC5, HDAC7, and HDAC9; class IIB, which includes HDAC6 and HDAC10, is related to the yeast Hda1 gene; class III, also called deacetylases, is related to the irS2 gene and includes SIRT1-7; and class IV, which contains only HDAC11, has characteristics of both class I and class II.

In one embodiment of the present invention, the HDAC inhibitor is an inhibitor of class I HDAC or class II HDAC. Preferably, the HDAC inhibitor is a selective inhibitor of class I HDAC. In some embodiments, the HDAC inhibitor is a histone deacetylase (HDAC) inhibitor of the benzamide class. In some embodiments, HDAC inhibitors include, but are not limited to, cedamide, entinostat, vorinostat, romidepsin, panobinostat, belinostat, valproic acid, mosetinol, abexinostat, prestostat, resminostat, givistostat, quinostat, dumanostat, quistostat, CUDC-101, CUDC-907, prestostat, sitamostat, zositamostat, abexinostat, recostat, tekdinol, feminostat, tubasin, resminostat, ACY-738, tenostin, tubastatin A, givistostat and danostat. In some embodiments, the HDAC inhibitor is cedamide, entinostat, vorinostat or mosetinol.

Tyrosine kinases (TKs) are enzymes that catalyze the transfer of a phosphate group from ATP to a tyrosine residue. They act as switches in cellular functions, such as signal transduction that triggers cell survival, differentiation, and proliferation. TKs belong to a class of enzymes that contain receptor tyrosine kinases (RTKs) and non-receptor tyrosine kinases. RTKs are key regulators of cellular processes and have been identified as being involved in several pathophysiological diseases. To date, twenty subfamilies of RTKs have been identified, such as EGFR (epidermal growth factor receptor), FGFR (fibroblast growth factor receptor), VEGFR (vascular endothelial growth factor receptor), RETR (RET receptor), EPHR (Eph receptor), and DDR (discoideum domain receptor) in humans. RTK molecules contain two regions, including an extracellular ligand binding region with a single transmembrane helix, and a cytoplasmic region containing a protein tyrosine kinase domain with an additional carboxyl-(C-) terminus and a juxtamembrane regulatory region. Preferably, the TKI according to the present invention is a vascular endothelial growth factor receptor (VEGFR) inhibitor, including VEGFR1, VEGFR2 and VEGFR3 to inhibit angiogenesis. More preferably, the TKI is cabozantinib, regorafenib, axitinib, afatinib, nintedanib, crizotinib, alectinib, trametinib, dabrafenib, sunitinib, ruxolitinib, vemurafenib, sorafenib, ponatinib, conafenib, brigatinib, pazopanib, dasatinib, imatinib, lenvatinib, vandetanib, surufatinib or setrinib.

Although not intending to be bound by any theory, it is believed that multi-targeted kinase inhibitors have the ability to modulate TME and enhance immune responses very effectively, especially in combination with immune checkpoint inhibitors (such as anti-PD-1 or anti-PD-L1 antibodies), which achieve better therapeutic efficacy results than PD-1/PD-L1 blockade monotherapy.

In one embodiment, immune checkpoint inhibitors can be used in combination with the drug combinations described herein to stimulate anti-cancer cellular immune responses and treat cancer. Immune checkpoint inhibitors suitable for the present invention include inhibitory receptor antagonists that inhibit PD-1, CTLA-4, T cell immunoglobulin 3, B and T lymphocyte depletion factor, T cell activation or lymphocyte activation gene 3 pathway V domain Ig inhibitors, such as anti-PD-1 antibodies or reagents, anti-PD-L1 antibodies or reagents, anti-CTLA-4 antibodies or reagents, anti-TIM-3 (T cell immunoglobulin-3) antibodies or reagents, anti-BTLA (B and T lymphocyte depletion factor) antibodies or reagents, anti-VISTA (V domain Ig inhibitor of T cell activation) antibodies or reagents, anti-LAG-3 (lymphocyte activation gene 3) antibodies or reagents, KIR (killer cell immunoglobulin-like receptor) antibody or reagent, TIM-3 (T cell immunoglobulin domain and mucin domain 3) antibody or reagent, A2AR (adenosine A2A receptor) inhibitor, CD276 antibody or reagent and VCTN1 antibody or reagent. Examples of PD-1 or PD-L1 inhibitors include, but are not limited to, humanized antibodies that block human PD-1, such as pembrolizumab (anti-PD-1 Ab, trade name ^Keytruda® ) or pilizumab (anti-PD-1 Ab), ^Bavencio® (anti-PD-L1 Ab, avelumab), ^Imfinzi® (anti-PD-L1 Ab, durvalumab) and ^Tecentriq® (anti-PD-L1 Ab, atezolizumab), and fully human antibodies, such as nivolumab (anti-PD-1 Ab, trade name ^Opdivo® ) and cemiplimab-rwlc (anti-PD-1 Ab, trade name ^Libtayo® ). Other PD-1 inhibitors may include small molecule drugs that express soluble PD-1 ligands, including those that block human PD1/PD-L1, such as BMS-1166, but not limited to PD-L2 Fc fusion protein, also known as B7-DC-Ig or AMP-244 and other PD-1 inhibitors currently in research and/or development for therapy. In addition, immune checkpoint inhibitors may include (but are not limited to) humanized or fully human antibodies that block PD-L1 (such as durvalumab and MIH1) and other PD-L1 inhibitors currently under investigation. In some embodiments, the amount of the immune checkpoint inhibitor ranges from about 0.5% (w/w) to about 15% (w/w), 0.5% (w/w) to about 10% (w/w), 0.5% (w/w) to about 5% (w/w), 1.0% (w/w) to about 20% (w/w), 1.0% (w/w) to about 15% (w/w), 1.0% (w/w) to about 10% (w/w), or 1.0% (w/w) to about 5% (w/w).

In one embodiment, the HDAC inhibitor and the TKI and immune checkpoint inhibitor are administered simultaneously or sequentially in any order or alternating. In some embodiments of the present invention, the HDAC inhibitor, TKI and immune checkpoint inhibitor are administered simultaneously.

In another embodiment, the method further comprises administering one or more additional anticancer agents. The additional anticancer agent is any anticancer agent described herein or known in the art. In one embodiment, the additional anticancer agent is chemotherapy or platinum-based dual chemotherapy. In one embodiment, the additional anticancer agent is an anti-VEGF antibody or a VEGFR small molecule inhibitor. In other embodiments, the anticancer agent is a platinum agent (e.g., cisplatin, carboplatin), a mitotic inhibitor (e.g., paclitaxel, albumin-bound paclitaxel, docetaxel, taxotere, docecad), a fluorinated vinca alkaloid (e.g., vinflunine, javlor), vinorelbine, vinblastine, etoposide, or pemetrexed gemcitabin. In one embodiment, the additional anticancer agent is 5-fluorouracil (5-FU). In certain embodiments, the additional anticancer agent is any other anticancer agent known in the art.

The pharmaceutical compositions of the present invention may be formulated with a "carrier". As used herein, "carrier" includes any solvent, dispersion medium, vehicle, coating, diluent, antibacterial and/or antifungal agent, isotonic agent, absorption delaying agent, buffer, carrier solution, suspension, colloid and the like. The use of such media and/or such reagents for pharmaceutical active substances is well known in the art. For example, the pharmaceutical composition can be specifically formulated for administration in solid or liquid form, including forms suitable for: (1) oral administration, such as drenches (aqueous or non-aqueous solutions or suspensions), buccal tablets, dragees, capsules, pills, tablets (e.g., tablets targeted for buccal, sublingual, and systemic absorption), boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, such as by administration of, for example, The drug may be administered subcutaneously, intramuscularly, intravenously, or epidurally as a sterile solution or suspension or as a sustained-release formulation; (3) topically, for example, to the skin in the form of a cream, lotion, gel, ointment, or controlled-release patch or spray; (4) intravaginally or intrarectally, for example, in the form of a pessary, cream, suppository, or foam; (5) sublingually; (6) ocularly; (7) transdermally; (8) transmucosally; or (9) nasally.

In another aspect, the present invention provides a method for treating cancer in an individual, the method comprising administering to the individual a pharmaceutical combination of the present invention.

In some embodiments, the cancer includes, but is not limited to, melanoma, head and neck cancer, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, colorectal cancer, endometrial cancer, cervical cancer, esophageal squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, breast cancer, gastric cancer, esophageal gastric junction cancer, classical Hodgkin's lymphoma, non-Hodgkin's lymphoma, urothelial carcinoma, primary septal large B-cell lymphoma, neuroglioblastoma, pancreatic cancer, benign prostatic hyperplasia, prostate cancer, ovarian cancer, chronic lymphocytic leukemia, Merkel cell carcinoma, acute myeloid leukemia, gallbladder cancer, bile duct cancer, bladder cancer, or uterine cancer.

In some embodiments, the drug combination of the present invention can be provided in a single formulation or dosage form. In other embodiments, the drug combination of the present invention can be provided in separate formulations or dosage forms. The drug combination can be formulated in a variety of forms and/or multiple forms suitable for one or more preferred routes of administration. Therefore, the drug combination can be administered via one or more known routes, including, for example, oral, parenteral (e.g., intradermal, transdermal, subcutaneous, intramuscular, intravenous, intraperitoneal, etc.) or topical (e.g., intranasal, intrapulmonary, intramammary, intravaginal, intrauterine, intradermal, transdermal, rectal, etc.). The drug combination or a portion thereof can be administered to a mucosal surface, such as by administration to, for example, the nasal mucosa or respiratory mucosa (e.g., by spray or aerosol). The drug combination or a portion thereof can also be administered via sustained or delayed release.

The formulation may be presented in unit dosage form and may be prepared by any method known in the art of pharmacy. The method of preparing the combination with a pharmaceutically acceptable carrier includes the step of combining the drug combination of the present invention with a carrier constituting one or more accessory ingredients. In general, the formulation can be prepared by uniformly and/or intimately combining the active compound with a liquid carrier, a finely powdered solid carrier, or both, and then, if necessary, shaping the product into the desired formulation.

The amount of compound effective for treating a particular disease or condition, including cancer, will depend on the nature of the disease or condition and can be determined by standard clinical techniques. In addition, in vitro assays may be used to help identify optimal dosage ranges as appropriate. The exact dosage to be used in the formulation will also depend on the route of administration and the progression of the disease or condition, and should be determined according to the judgment of the physician and the circumstances of each patient. For the combination or each component of the combination, the preferred dosage will be in the range of 0.01 to 1000 mg/kg body weight, 0.1 mg/kg to 100 mg/kg, 1 mg/kg to 100 mg/kg, 10 mg/kg to 75 mg/kg, 0.1 to 1 mg/kg, etc.

The present invention is illustrated by the following examples. It should be understood that the specific examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the present invention as described herein. Example Materials and Methods

To overcome primary and acquired resistance and HPD to first-line PD - 1 checkpoint blockade therapy. Male Balb/c mice (1×10 ⁶ cells/mouse) bearing subcutaneous CT26 tumors were treated with anti-PD-1 antibody (purchased from InvivoMab, cat#BE00146) as first-line therapy (mean tumor volume: 113 mm ³ at the start of treatment) at 2.5 mg/kg intraperitoneally (ip) once every 3 days for 3 doses. When the tumor responded to treatment with anti-PD-1 antibody (with significant tumor shrinkage), treatment was continued for 3 doses (i.e., 6 doses in total). If the tumor shrinks at the start of anti-PD-1 antibody treatment (after 3 doses), it gradually grows due to continuous anti-PD-1 antibody treatment (i.e., 6 doses in total) due to partially effective inhibition of tumor growth, and further increases in size to produce acquired resistance. When the tumor does not respond at the start of anti-PD-1 antibody treatment (after 3 doses) and meets the criteria of 2.5 to 3 consecutive increases in tumor size (and tumor size <600 mm ³ ), it is considered primary resistance. However, hyperprogressive disease (HPD) is defined as tumor growth greater than 600 mm ³ after 3 doses of first-line anti-PD-1 antibody treatment. These mice with primary resistance, acquired resistance and HPD were then re-selected for efficacy studies in the second-line therapy, as shown in Figure 1 ( A ) . The second-line therapy was as follows. Anti-IgG antibody (purchased from InvivoMab, cat#BE0089, Bio X Cell), anti-PD-1 antibody (purchased from InvivoMab, cat#BE0146, Bio X Cell) and anti-CTLA-4 antibody (purchased from InvivoMab, cat#BE0164, Bio X Cell) as controls were intraperitoneally administered at 2.5 mg/kg, once every 3 days for 6 doses. Anti-CTLA-4 Ab (2.5 mg/kg) + Chidamide-HCl (50 mg/kg) + Celecoxib (50 mg/kg), anti-CTLA-4 Ab (2.5 mg/kg) + Regorafenib (30 mg/kg) + Chidamide-k30 (50 mg/kg), and anti-CTLA-4 Ab (2.5 mg/kg) + Cabozantinib (30 mg/kg) + Chidamide-k30 (50 mg/kg) were administered orally daily for 16 times. Tumor diameter was measured every 2 to 3 days, and tumor volume was calculated using a caliper (in mm ³ ). Anticancer activity was measured starting with self-treatment until the tumor volume reached 3,000 mm ^3. Tumor volume was calculated as length × width2 × ^0.5 .

Anti-colorectal cancer activity in animal models. Animal studies were approved and supervised by the Taipei Medical University Institutional Animal Care and Use Committee (TMU IACUC, No.: LAC-2020-0103, LAC-2019-0644). For all animal experiments, six- to eight-week-old male BALB/c mice (National Laboratory Animal Center, Taiwan) were used. CT26 cell line was purchased from ATCC. CT26 tumor cell line was grown in RPMI-1640 supplemented with 10% (v/v) _FBS at 37°C, 5% CO2. Tumors were established by subcutaneously injecting 1×10 ⁶ CT26 cells with Matrigel (354248, Corning®) into the left flank of mice, and growth was determined by measuring two perpendicular diameters. Tumors were allowed to grow for 8 to 12 days (tumor size approximately 110 to 250 mm ³ ) before randomization and treatment. Animals were euthanized when tumor volume reached more than 3000 mm ³ . Anti-IgG antibody (BE0089, Lot #716719J3, Bio X Cell), anti-PD-1 antibody (BE0146, Lot #717918D1, Lot #735019J3, Lot #780120J3, Lot #735019O1, Bio X Cell), anti-PD-L1 antibody (BE0101, Lot #720619F1, Bio X Cell) and anti-CTLA-4 antibody (BE0164, Lot #702418A2B, Bio X Cell) were administered intraperitoneally at 2.5 mg/kg twice a week for three weeks. All antibodies were diluted to the appropriate concentration in 100 μL of sterile PBS (pH 7.4, Invitrogen Life Technologies). Axitinib (HY-10065, 30 mg/kg, oral daily, US MedChemExpress), lenvatinib (HY-10981, 10 mg/kg, oral daily, US MedChemExpress), olaparib (HY-10162, 50 mg/kg, oral daily, US MedChemExpress), ibrutinib (HY-10997, 6 mg/kg, oral daily, US MedChemExpress), cabozantinib (HY-13016, 30 mg/kg, oral daily, US MedChemExpress), regorafenib (HY-1031, 30 mg/kg, oral daily, US MedChemExpress), RMC-4550 (HY-116009, 30 mg/kg, oral daily, US MedChemExpress), sestretinib (HY-16961, 20 mg/kg, oral daily, MedChemExpress, USA), entinostat (HY-12163, 20 mg/kg, oral q2d, MedChemExpress, USA), vorinostat (HY-10221, 150 mg/kg, oral daily, MedChemExpress, USA), chidamide-k30 or chidamide-HCl salt (50 mg/kg, oral daily, produced by GNTbm, Taipei, Taiwan), celecoxib (50 mg/kg, oral daily, capsule/Celebrex ^® , Pfizer Pharmaceuticals LLC) for 16 days. Axitinib, lenvatinib, olaparib, ibrutinib, cabozantinib, regorafenib, entinostat, vorinostat, RMC-4550, selutinib, and celecoxib were dissolved in DMSO and diluted in PBS before administration. Chidamide-k30 and chidamide-HCl salt were dissolved in water. Animals were euthanized when tumor volume reached more than 3,000 mm ^3. Anticancer activity was measured starting with self-treatment until tumor volume reached 3,000 mm ³ . Tumor volume was calculated as length × ^width2 × 0.5. In this study, we defined complete response (CR, tumor growth <0.5-fold in tumor-bearing mice three days after the end of treatment); partial response (PR, tumor size ≥0.5-fold tumor growth, but <1-fold tumor growth in tumor-bearing mice three days after the end of treatment); stable disease (SD, tumor size ≥1-fold tumor growth, but <5-fold tumor growth in tumor-bearing mice three days after the end of treatment); progressive disease (PD, tumor size ≥5-fold tumor growth in tumor-bearing mice three days after the end of treatment) to evaluate treatment efficacy. Relapse was defined as tumor growth of at least 5-fold in mice with CR or PR responses after the first tumor assessment.

In order to demonstrate that the combination of the present invention can overcome the drug resistance problem after treatment with anti-PD-1 Ab, all mice were first treated with anti-PD-1 Ab.

Tumor Rechallenge in Animal Models. All mice with a PR/CR response after treatment were rechallenged with CT26 cells on the contralateral side (see Table 6 ( F ) ). Rechallenge with CT26 was performed 34±2 days after the initial tumor assessment (day 27±2), which was 7 days (day 41±2), in which 5×10 ⁶ CT26 cells/mouse were injected. After rechallenge with CT26 cells, tumors were allowed to grow for 7 days (day 41±2) to determine the baseline as 1 fold. After another 10 days (day 51±2), tumor growth was evaluated for rechallenge. A response is considered a recurrence/relapse induced by rechallenge if two of the following criteria are met: first, tumor size more than doubles compared to baseline on day 41±2; second, tumor volume on day 51±2 exceeds 300 mm ³ . Relapse occurs when immunity is not fully activated. Inhibition of tumor growth means that immunity is activated.

Survival rate in animal model. After tumor assessment, the tumor volume of mice was measured every three or four days (twice a week). When the tumor volume reached 3,000 mm ³ , the tumor-bearing mice were considered dead. All treatment groups were recorded and analyzed.

Flow cytometry . The following antibodies and reagents were used for flow cytometry: CD8a PerCP-Cy5.5 (53-6.7; BioLegend), CD4 PE (GK 1.5; BioLegend), CD25 PerCP-Cy5.5 (PC61; BioLegend), Foxp3 PE (MF14; BioLegend), CD3 APC (17A2; BioLegend), CD11b APC (M1/70; BioLegend), Ly-6C PerCP-Cy5.5 (HK 1.4; BioLegend), Ly-6G PE (1A8; BioLegend), MHC-ll-PE (BM8; BioLegend), CD45 FITC (30-F11; BioLegend). Flow cytometry was performed with a caliper (BD Biosciences), and data were analyzed with FACS Diva software (BD Biosciences).

To assess the level of tumor-infiltrating lymphocytes in tumors, further analysis was performed to analyze intratumoral CD8 ⁺ , CD4 ⁺ , regulatory T cells (Treg), PMN-MDSC, M-MDSC, TAM populations. Tumor-infiltrating lymphocytes were first purified from tumor samples excised from mice on day 12 after initiation of cabozantinib or regorafenib treatment in the presence or absence of cedabendin-k30 plus anti-PD-1 Ab. Briefly, primary tumor tissue was harvested, weighed, and minced into small fragments. HBSS (Invitrogen Life Technologies) containing 1 mg/mL collagenase IV (Sigma-Aldrich) was added to each sample at a ratio of 1 mL per 200 mg of tumor tissue. The samples were incubated on an endover end shaker at 37°C for 150 min. The resulting tissue homogenate was filtered through 0.4 μm and washed three times in PBS (BD Biosciences), separated by Percoll gradient to isolate mononuclear cells, and 1×10 ⁶ cells per sample were used for antibody labeling. CD8 ⁺ T cells were assessed using the previously established phenotypic criteria of CD45 ⁺ CD3 ⁺ CD8; Treg cell levels were assessed using the previously established phenotypic criteria of CD45 ⁺ CD3 ⁺ CD25 ⁺ FoxP3 ⁺ ; PMN-MDSC and M-MDSC cell levels were assessed using the previously established phenotypic criteria of CD45 ⁺ /CD11b ⁺ /Ly6G ⁺ / ^Ly6C- and CD45 ⁺ /CD11b ⁺ / ^Ly6G- /Ly6C ⁺ , respectively; TAM cell levels were assessed using the previously established phenotypic criteria of CD45 ⁺ CD11b ⁺ CHM-ll ⁺ Ly6C ⁺ , and total monocytes were used as the common denominator.

RNA quantification and qualitative analysis. Drug-resistant mice after first-line anti-PD-1 Ab therapy were randomly divided into groups and treated with different regimens, and the tumors were removed and collected on the 13th day after the start of second-line treatment. Mice naturally carrying CT26 tumors were randomly divided into groups and treated with different regimens, and the tumors were removed and collected on the 9th day after the start of treatment. All tumor samples were quickly frozen in liquid nitrogen, and the samples were then homogenized in Trizol (Invitrogen Life Technologies). RNA purity and quantification were checked using a SimpliNano™-Biochrom spectrophotometer (Biochrom, MA, USA). RNA degradation and integrity were monitored by a Qsep 100 DNA/RNA analyzer (BiOptic Inc., Taiwan). The results are shown in Figures 13 and 14 .

Library preparation for transcriptome sequencing. A total amount of 1 μg of total RNA per sample was used as input material for RNA sample preparation. Sequencing libraries were generated using the KAPA mRNA High Prep Kit (KAPA Biosystems, Roche, Basel, Switzerland) following the manufacturer's recommendations, and index codes were added to characterize sequences to each sample. PCR products were purified using the KAPA Pure Bead System, and library quality was assessed on a Qsep 100 DNA/RNA Analyzer (BiOptic Inc., Taiwan).

Bioinformatics . The raw data obtained by high-throughput sequencing (Illumina NovaSeq 6000 platform) were converted into raw sequencing reads by CASAVA base identification and stored in FASTQ format. The quality of fastq files was checked using FastQC and MultiQC. The raw paired-end reads obtained were filtered by Trimmomatic (v0.38) to discard low-quality reads, trim the junction sequences, and eliminate poor-quality bases with the following parameters: LEADING: 3 TRAILING: 3 SLIDINGWINDOW: 4: 15 MINLEN: 30. The obtained high-quality data (clear reads) were used for subsequent analysis. Read pairs from each sample were aligned to the reference genome using HISAT2 software (v2.1.0). The number of reads mapped to individual genes was counted using FeatureCounts (v1.6.0). For gene expression, Trimmed Mean (TMM) normalization was performed using DEGseq (v1.36.1) without biological replicates and Relative Log Expression (RLE) normalization was performed using DESeq2 (v1.22.1) with biological replicates. Analysis of differentially expressed genes (DEGs) between the two conditions was performed in R using DEGseq (without biological replicates) and DESeq2 (with biological replicates), based on negative binomial and Poisson distribution models, respectively. The obtained p-values were adjusted using the method of Benjamini and Hochberg to control the FDR. GO and KEGG pathway enrichment analysis of DEGs was performed using clusterProfiler (v3.10.1). Genome enrichment analysis (GSEA) was performed with 1,000 permutations to identify enriched biological functions and activation pathways from the molecular signature database (MSigDB). MSigDB is a collection of annotated genes used with the GSEA software, which includes marker genes, positional genes, validated genes, motif genes, computational genes, GO genes, oncogenes, and immune genes. In addition, weighted gene co-expression network analysis (WGCNA) was constructed using the co-expression network package based on the correlation coefficient of the expression pattern using WGCNA (v1.64) in R.

Examples 1 : To overcome the carrying CT26 In mice, tyrosine kinase inhibitors HDAC Inhibitors and Antibodies CTLA - 4 Antibodies from first-line PD - 1 Ab Resistance to treatment

In this example, mice were treated with second-line therapy to mimic the treatment of first-line resistance that occurs in human first-line cancer therapy, where most human cancer patients receiving first-line anti-PD-1 antibody therapy will develop resistance, including primary and acquired resistance or hyperprogressive disease (HPD) - used to evaluate the anti-cancer efficacy of second-line therapy using a combination of a tyrosine kinase inhibitor plus an HDAC inhibitor and an anti-CTLA-4 antibody when first-line anti-PD-1 antibody therapy has failed. To evaluate the effectiveness of different treatments for first-line anti-PD-1 antibody resistance, a platform with a treatment schedule was designed, as outlined in Figure 1 ( A ) . As shown in FIG1 , first, 110 mice were treated with anti-PD-1 antibody as first-line treatment, and 10 mice were treated with anti-IgG antibody as negative control. 18 of the 110 mice achieved a response by first-line anti-PD-1 antibody treatment, with an objective response rate (ORR) of 16.4% (18/110). As shown in FIG1 ( B ) and ( C ) , the tumor volume of mice responding to first-line anti-PD-1 Ab treatment was significantly suppressed compared with the anti-IgG Ab group in the mouse model bearing CT26 tumors. Eighty-five of the 110 mice showed primary resistance to first-line anti-PD-1 antibody treatment, with an incidence of 77.3% (85/110). In addition, acquired resistance to first-line anti-PD-1 antibody treatment gradually developed and eventually relapsed at an incidence of 28% (7/25), as shown in Table 1. These results suggest that primary resistance to first-line anti-PD-1 antibody treatment is a very challenging issue for cancer immunotherapy. We were interested in whether TKI plus HDAC inhibitors could improve ICI sensitivity by modulating the TME. For the study, tumors were allowed to grow for 8 days (tumor size averaged approximately 113 mm 3 ) before first-line treatment with anti-PD-1 antibody (2.5 mg/ ^kg ; Lot#735019O1) administered intraperitoneally for three consecutive doses (once every 3 days). In mice that responded to first-line anti-PD-1 antibody treatment, tumors were suppressed or maintained, that is, tumors continued to shrink and achieved CR or PR responses, with an ORR of 16.4%. However, when tumors met treatment failure criteria: (1) after three doses of first-line anti-PD-1 antibody treatment, tumor volume continued to increase by 2.5 to 3 times on day 16 (average tumor size was 396.8 mm ³ ), and (2) tumor volume was <600 mm ³ , mice were defined as developing primary resistance and were reselected for second-line treatment. These mice with primary resistance to anti-PD-1 Ab therapy were further randomized. Regarding HPD mice (incidence approximately 10%, 11/110), defined as having hyperprogressive disease with tumor volume >600 mm ³ , the mean tumor volume was 754.7 mm ³ , as shown in Figure 1 ( A ) . There were nine different treatment groups (n=9 to 10 mice/group) as indicated. Mice with primary resistance were randomly divided into five different second-line treatment groups, including anti-IgG Ab (2.5 mg/kg; Lot #716719J3), anti-CTLA-4 Ab (2.5 mg/kg; Lot #702418A2B), the combination of anti-CTLA-4 Ab and chidamide-HCl salt (50 mg/kg) plus celecoxib as a positive control, the combination of anti-CTLA-4 Ab and regorafenib (30 mg/kg) plus chidamide-k30 (50 mg/kg), and the combination of anti-CTLA-4 Ab and cabozantinib (30 mg/kg) plus chidamide-k30 (50 mg/kg). Anti-CTLA-4 antibody was administered intraperitoneally (ip) for six consecutive doses (once every 3 days). Chidamide-k30, chidamide-HCl salt, regorafenib and cabozantinib were given by oral administration once a day for 16 days. As shown in Figures 2 ( A ) and 2 ( B ) , the combination of anti-CTLA-4 Ab and regorafenib plus chidamide-k30, and the combination of anti-CTLA-4 Ab and cabozantinib plus chidamide-k30 inhibited tumor growth more effectively than the positive control of anti-CTLA-4 Ab and chidamide-HCl salt plus celecoxib group and anti-CTLA-4 Ab group alone. These results indicate that the combination of anti-CTLA-4 Ab and regorafenib or cabozantinib plus chidamide-k30 has a very effective modulation of TME activity to overcome primary resistance to first-line treatment with anti-PD-1 Ab. This result also shows that the combination of anti-CTLA-4 Ab and cedabenb-k30 plus regorafenib or cabozantinib is more effective than the combination of anti-CTLA-4 Ab and cedabenb-HCl salt plus celecoxib to overcome primary resistance. As shown in Figure 2 ( C ) , the weight of mice in each treatment group did not decrease significantly. As shown in Figures 3 ( A ) to 3 ( C ) , HPD mice were treated with a combination of anti-CTLA-4 Ab (2.5 mg/kg) and cabozantinib (30 mg/kg) plus cedabenb-HCl salt (50 mg/kg) for 16 days, and large tumors unexpectedly showed growth inhibition and some tumors were controlled by continuous shrinkage under this treatment regimen. The results show that the combination of anti-CTLA-4 Ab and cedabenb-HCl salt plus cabozantinib is an extremely effective regimen for restoring HPD. Our previous studies have demonstrated that cedabenb-HCl salt modulates the TME more effectively than cedabenb-k30. This result also shows that the faster tumor growth in HPD mice compared to primary resistant mice requires treatment with a combination of anti-CTLA-4 Ab and cabozantinib plus cedabenb-HCl salt to significantly inhibit tumor growth through immune regulation in the TME, thereby enhancing the immune response. The individual results of each drug-resistant mouse are also shown in Figure 4. In the anti-IgG Ab group, 5 mice achieved PD with rapid tumor growth. Treatment with anti-CTLA-4 Ab achieved 5 SD mice and 2 PD mice (compared to anti-IgG Ab). Treatment with anti-CTLA-4 Ab and the combination of chidamide-HCl salt plus cenecoxib as a positive control showed that 3 mice achieved CR, 4 mice achieved SD, and 1 mouse achieved PD (response rate 37.5%). However, treatment with the combination of anti-CTLA-4 Ab and regorafenib plus chidamide-k30 showed that 5 mice achieved CR, 1 mouse achieved PR, and only 2 mice achieved SD (response rate 62.5%). When it was a combination of anti-CTLA-4 Ab and cabozantinib plus chidamide-k30, the results showed that 3 mice achieved CR, 1 mouse achieved PR, and 3 mice achieved SD (response rate 57.1%). However, treatment of HPD mice with a combination of anti-CTLA-4 Ab and cabozantinib plus cedamide-HCl salt showed that 2 mice achieved CR and 9 mice achieved SD (response rate 18.1%). Although the immune response rate of HPD mice was very high, most tumors were inhibited from growing after treatment with this second-line regimen. This result is interesting because HPD tumors are extremely difficult to effectively reduce and inhibit. Regarding the incidence of acquired resistance, it was observed that the initial first-line anti-PD-1 Ab treatment had an effective response in mice carrying CT26, but after continuous treatment with anti-PD-1 Ab, it did not effectively inhibit tumor growth. This phenomenon is defined as acquired resistance to anti-PD-1 Ab treatment. We focused on evaluating the therapeutic effect of the combination of anti-PTLA-4 Ab and regorafenib plus cedamide-k30 in mice with acquired resistance to anti-PD-1 Ab, as shown in Figure 5. Treatment with the combination of anti-CTLA-4 Ab and regorafenib plus cedamide-k30 effectively inhibited tumor growth in mice with primary resistance to anti-PD-1 Ab (as shown in Figure 5 ( A ) and Figure 2 ( A ) ), however, the same regimen showed significant inhibition of tumor growth in mice with acquired resistance to anti-PD-1 Ab treatment, as shown in Figure 5 ( B ) , which showed that 1 mouse achieved CR and 6 mice achieved SD (response rate 14.1%), as shown in Figure 5 ( C ) . In addition, we were interested in evaluating the survival of mice with primary, acquired resistance or HPD to anti-PD-1 Ab treatment. As shown in Figure 5 ( D ) , for mice with primary resistance, the combination of anti-CTLA-4 Ab (2.5 mg/kg) and regorafenib (30 mg/kg) plus cedabenb-k30 (50 mg/kg) achieved an overall survival rate of 87.5%; the combination of anti-CTLA-4 Ab (2.5 mg/kg) and cabozantinib (30 mg/kg) plus cedabenb-k30 (50 mg/kg) achieved an overall survival rate of 71.4%; and the combination of anti-CTLA-4 Ab (2.5 mg/kg) and cedabenb-HCl salt (50 mg/kg) plus celecoxib (50 mg/kg) as a positive control achieved an overall survival rate of 37.5%. This result shows that both groups are extremely powerful in prolonging survival compared to the positive control group. Regarding HPD mice, treatment with the combination of anti-CTLA-4 Ab (2.5 mg/kg) and cabozantinib (30 mg/kg) plus cedabenb-HCl salt (50 mg/kg) achieved an excellent overall survival rate of 45.4%. This result is noteworthy because HPD mice only achieved an ORR of 18.1%, but achieved an overall survival rate of 45.4% due to significant inhibition of tumor growth. The results suggest that the regimen may have had a strong modulatory ability of the TME, resulting in continuous tumor suppression that will be maintained even one month after the end of treatment. Similar results were also seen in the treatment group of the combination of anti-CTLA-4 Ab and regorafenib/cabozantinib plus cedabenamide-k30, where the overall survival rate was better than the ORR. As shown in Figure 5 ( E ) , mice with acquired resistance achieved an overall survival rate of 57.1% after treatment with a combination of anti-CTLA-4 Ab (2.5 mg/kg) and regorafenib (30 mg/kg) plus cedabenamide-k30 (50 mg/kg). This is similar to the survival rate of HPD mice. These results fully demonstrate that the combination of anti-CTLA-4 Ab and regorafenib/tozatinib plus cedabenamide-HCl salt/cedabenamide-k30 has extremely effective activity to modulate the TME and significantly improve the immune response to overcome resistance to first-line anti-PD-1 Ab treatment. As shown in Table 2 , the initial ORR assessment was performed 3 days after the last drug administration, however, due to the observation of continuous tumor shrinkage, a second ORR assessment was additionally scheduled 10 days after the last drug administration. In mice with primary resistance to first-line anti-PD-1 Ab therapy, treatment with the combination of anti-CTLA-4 Ab and regorafenib plus cedamide-k30 significantly increased the ORR from 62.5% to 87.5%, and significantly increased the CR from 5 to 7 mice in the second assessment. Similar phenomena were also shown in the combination group of anti-CTLA-4 Ab and cabozantinib plus cedamide-k30: it significantly increased the ORR from 57.1% to 100% and significantly improved the CR/PR from 4 to 7 mice in the second evaluation in mice with primary resistance to first-line anti-PD-1 Ab therapy. Regarding HPD mice, treatment with the combination of anti-CTLA-4 Ab and cabozantinib plus cedamide-HCl salt significantly increased the ORR from 18.1% to 45.4% in the second evaluation. Finally, for mice with acquired resistance to first-line anti-PD-1 Ab therapy, treatment with the combination of anti-CTLA-4 Ab and regorafenib plus cedamide-k30 also showed increased anti-cancer activity in the second evaluation, with an ORR of 14.2% to 28.5%. Taken together, all these results indicate that the regimen of anti-CTLA-4 Ab in combination with regorafenib/cabozantinib plus chidamide-HCl salt/chidamide-k30 is highly potent in overcoming primary, acquired resistance, and HPD to first-line anti-PD-1 Ab treatment.

Table 1. One hundred and twenty male Balb/c mice bearing subcutaneous CT26 tumors were treated with anti-PD-1 and anti-IgG (as negative control) antibodies (2.5 mg/kg) as a line of therapy every 3 days for 3 doses. Number of mice Whether there is a response to first-line anti- PD - 1 antibody therapy Types of resistance to first-line anti -PD - 1 antibody therapy 10 Treatment with anti-IgG antibody (as negative control) N/A 18 yes Response ^* 7 Initial response followed by significant tumor growth Acquired resistance ^** 85 no Primary resistance ^*** * : Reaction rate ( CR % plus PR % ) : 18/110 , 16.4 % ; ** : Acquired resistance rate : 7/25 , 28.0 % ; *** : Primary resistivity : 85/110 , 77.3 % . ​ ​ ​ ​ ​ ​ ​

Table 2. Response rates after treatment with different second - line regimens in CT26-bearing mice with primary, acquired resistance, or HPD to first-line anti-PD-1 Ab treatment. Resistance plan Initial tumor volume ( mm ³ ) ORR (%) PD SD PR CR ORR (%) ^& PD ^& SD ^& PR ^& CR ^& Survival rate (%) Relapse * Immunity ^# Primary resistance to first-line anti-PD-1 Ab therapy Anti-IgG Ab as control 396 0% 4 1 0 0 0% 5 0 0 0 0% (0/5) - - Anti-CTLA-4 Ab 0% 0 7 0 0 0% 2 5 0 0 0% (0/7) - - Anti-CTLA-4 Ab + Chidamide-HCl + Celecoxib 37.5% 1 4 0 3 37.5% 2 3 0 3 37.5% (3/8) 0% (0/3) 100% (3/3) Anti-CTLA-4 Ab+Regorafenib+Chidamide-k30 62.5% 0 2 1 5 87.5% 0 1 0 7 87.5% (7/8) 0% (0/6) 100% (6/6) Anti-CTLA-4 Ab+cabozantinib+chidamide-k30 57.1% 0 3 1 3 100% 0 0 3 4 71.4% (5/7) 0% (0/4) 100% (4/4) Hyperprogressive disease (HPD) in response to first-line anti-PD-1 Ab therapy Anti-CTLA-4 Ab + Cabozantinib + Chidamide-HCl salt 669 18.1% 0 9 0 2 45.4% 3 3 3 2 45.4% (5/11) 0% (0/2) 100% (2/2) Acquired resistance to first-line anti-PD-1 Ab therapy Anti-CTLA-4 Ab+Regorafenib+Chidamide-k30 477 14.2% 0 6 0 1 28.5% 0 5 1 1 57.1% (4/7) 0% (0/1) - *: Relapse/recurrence was defined as tumor growth of at least 5-fold in mice with CR or PR response after the first tumor assessment. &: Second tumor assessment 10 days after the last dose of second-line therapy #: Mice resistant to CT26 re-challenge. -: Not tested Response assessment criteria: Fold change in tumor size compared to baseline PD: x ≧5; SD: 1 ≦ x ＜ 5; PR: 0.5 ≦ x ＜ 1; CR: x ＜ 0.5

Examples 2 : For research purposes CT26 In mice TKI and resistance PD - 1 Ab The anti-cancer effect of the combination.

Several reports indicate that lenvatinib has potent immunomodulatory properties that can increase the immune response rate of anti-PD-1 Ab in tumor-bearing mouse models. We are interested in new searches for more powerful regimens to modulate the TME to increase the immune response rate. First, we evaluated lenvatinib and the combination of lenvatinib and anti-PD-1 Ab in a mouse model bearing CT26. As shown in Figures 6 ( A ) and 6 ( B ) , the combination of lenvatinib (10 mg/kg) with or without anti-PD-1 Ab (2.5 mg/kg) significantly inhibited tumor growth than anti-PD-1 Ab (2.5 mg/kg) alone. Individual tumor assessments showed that 1 mouse achieved CR, 1 mouse achieved PR, 4 mice achieved SD, and 5 mice achieved PD (response rate 18%) in the anti-PD-1 Ab group. The lenvatinib group showed that 1 mouse achieved CR, 2 mice achieved PR, 5 mice achieved SD, and 2 mice achieved PD (response rate 30%). The combination group of anti-PD-1 Ab and lenvatinib showed that 2 mice achieved CR, 7 mice achieved SD, and 3 mice achieved PD (response rate 17%). Although the immune response rate seemed to be lower in the combination group of anti-PD-1 Ab and lenvatinib, 8 mice showed significant inhibition of tumor growth, as shown in Figure 6 ( B ) . Compared with the anti-IgG or anti-PD-1 Ab group as shown in Figure 6 ( C ) , only the body weight in the lenvatinib and lenvatinib and anti-PD-1 Ab combination treatment groups was slightly reduced. In addition, the survival rate was analyzed as shown in Figure 6 ( D ) . After tumor implantation, mice bearing CT26 tumors were euthanized when the tumor volume reached 3000 mm ^3. Compared with the anti-PD-1 Ab group, the combination of anti-PD-1 Ab and lenvatinib or the regimen of lenvatinib alone was more effective in prolonging survival. As shown in Figure 6 (E), the recurrence rate showed that the combination of anti-PD-1 Ab and lenvatinib had more and stronger ability to activate the immune system to avoid recurrence than lenvatinib or anti-PD-1 Ab treatment alone. Next, we are interested in evaluating the immunity stimulated by different treatments in the re-challenge experiment as outlined in Figure 6 ( F ) . Mice with CR or PR entered a 7-day washout phase (until day 34±2) without any further treatment. Subsequently, rechallenge was performed by inoculating the same type of cancer cells (CT26; 5×10 ⁶ ) in the opposite flank for an additional 7 days (Day 41±2), and the tumor volume was then determined as baseline (1-fold). Rechallenge tumors were allowed to grow for 10 days and then assessed to evaluate tumor growth (Day 51±2). Immunization was defined as negative when it met two conditions: tumor volume exceeded 300 mm ³ , or tumor size exceeded 2-fold compared to baseline. If the immune memory is activated after treatment, the immunity is active and specific for the recognition of cancer cells with the same antigen, and the growth of the inoculated tumor during the re-attack period will be inhibited, so the immune definition is positive. If the immune memory is not induced or not fully activated, resulting in the growth of the inoculated tumor during the re-attack (tumor recurrence), the immunity will be defined as negative. As shown in Figure 6 ( G ) , in the anti-PD-1 Ab group, 2 mice that achieved PR and CR showed 0% tumor recurrence after re-attack. The results show that these PR and CR mice achieved 100% overall immune memory. In the combination group of lenvatinib and anti-PD-1 Ab, 2 mice that achieved CR showed 0% tumor recurrence after re-attack. It also showed 100% global immune memory in these mice with CR or PR. In the lenvatinib only group, the 3 mice that achieved CR/PR showed 33% tumor recurrence after re-challenge. It showed 67% global immune memory. Re-challenge experiments were used to confirm whether these regimens stimulate the ability of the immune system to activate immune memory directly or indirectly.

Examples 3 : For research purposes CT26 Tyrosine kinase inhibitors in mice ( TKI ) and resistance PD - 1 Ab The anti-cancer effect of the combination.

We are very interested in evaluating multiple TKIs in combination with anti-PD-1 Ab in a mouse model bearing CT26 to improve the immune response rate. As shown in Figure 7 ( A ) , cabozantinib, ibrutinib, axitinib, and olaparib, poly ADP-ribose polymerase inhibitors (PARPi) were combined with anti-PD-1 Ab. Compared with the treatment of anti-PD-1 Ab combined with ibrutinib, axitinib, and olaparib, the combination of cabozantinib and anti-PD-1 Ab significantly inhibited tumor growth. As shown in Figure 7 ( B ) , the individual tumor sizes (fold change) and ORR indicated that the anti-PD-1 antibody (2.5 mg/kg) group achieved 1 CR, 1 SD, and 7 PD, with an ORR (objective response rate) of 11%; the combination of cabozantinib (30 mg/kg) and anti-PD-1 Ab (2.5 mg/kg) achieved 1 CR, 7 SD, and 1 PD, with an ORR of 11%; the combination of axitinib (12.5 mg/kg) and anti-PD-1 Ab (2.5 mg/kg) achieved 1 CR, 2 SD, and 6 PD, with an ORR of 11%; the combination of ibrutinib (6 mg/kg) and anti-PD-1 Ab (2.5 mg/kg) achieved 1 CR, 1 SD, and 7 PD, with an ORR of 11%; and the combination of olaparib (50 mg/kg) and anti-PD-1 Ab (2.5 In this study, the combination of cabozantinib (30 mg/kg) and anti-PD-1 Ab (2.5 mg/kg) achieved 1 SD and 8 PD, with an ORR of 0%. In this study, the combination of cabozantinib (30 mg/kg) and anti-PD-1 Ab (2.5 mg/kg) achieved the best antitumor effect relative to TME control. Next, we are interested in evaluating whether the combination of anti-PD-1 Ab with cabozantinib plus COX-2 inhibitors or HDAC inhibitors can improve the immune response rate. The possibility of adding COX-2 inhibitors or HDAC inhibitors that inhibit PGE2 synthesis to the regimen combined with cabozantinib anti-PD-1 Ab may improve anticancer activity. As shown in Figure 8 ( A ) , the combination of anti-PD-1 antibody (2.5 mg/kg) with cabozantinib (30 mg/kg) plus celecoxib (50 mg/kg) or cedabenb-k30 (50 mg/kg) achieved excellent effects in inhibiting tumor growth compared with anti-PD-1 Ab treatment alone. Celecoxib is a selective COX-2 inhibitor, and cedabenb is a benzamide-based HDAC inhibitor that selectively inhibits HDAC1, HDAC2, HDAC3, and HDAC10. As shown in Figure 8 ( B ) , the individual tumor sizes (fold change) and ORR indicated that the anti-PD-1 antibody (2.5 mg/kg) group achieved 1 CR, 1 PR, 2 SD, and 4 PD, with an ORR of 25%; the combination group of anti-PD-1 Ab (2.5 mg/kg) and cabozantinib (30 mg/kg) plus celecoxib (50 mg/kg) achieved 4 CR and 3 SD, with an ORR of 57%; the combination group of anti-PD-1 Ab (2.5 mg/kg) and cabozantinib (30 mg/kg) plus cedabenb-k30 (50 mg/kg) achieved 6 CR and 1 PR, with an ORR of 100%. These data show that the combination of anti-PD-1 Ab (2.5 mg/kg) and cabozantinib (30 mg/kg) plus cedabenb-k30 (50 mg/kg) achieved better ORR relative to TME control compared to the combination of anti-PD-1 Ab (2.5 mg/kg) and cabozantinib (30 mg/kg) plus celecoxib (50 mg/kg). This is the first time that the combination of TKI plus HDAC inhibitor and ICI has been found to significantly improve the immune response rate, which can be attributed to the regulation of TME. As shown in Figure 8 ( C ) , there was no significant loss of weight in mice treated with different regimens. After implantation, mice bearing CT26 tumors were euthanized when the tumor volume reached 3000 ^mm3 . As shown in Figure 8 ( D ) , the combination of anti-PD-1 Ab and cabozantinib plus cedabenin-k30 was extremely powerful in extending survival compared with other groups, achieving 100% survival rate on day 60. This result shows that cabozantinib plus cedabenin-k30 can have strong immunomodulatory activity in TME. Subsequently, the recurrence rate in each group was evaluated as shown in Figure 8 ( E ) . The results show that there was no recurrence in the combination group of anti-PD-1 Ab, anti-PD-1 Ab and cabozantinib plus cedabenin-k30 or celecoxib. In the re-attack experiment, as outlined in Figure 6 ( F ) , the immunity activated by treatment with anti-PD-1 Ab, anti-PD-1 Ab and cabozantinib plus cedabenin-k30 or celecoxib was studied. As shown in Figure 8 ( F ) , no tumor growth occurred after re-attack. The results showed that these PR and CR mice achieved 100% overall immune memory to recognize the inoculated CT26 cells during the re-challenge period. This shows that the combination of anti-PD-1 Ab with cabozantinib plus cedabenb-k30 or celecoxib is very effective in activating immune memory activity to prevent relapse.

Examples 4 : For research purposes CT26 Tyrosine kinase inhibitors in mice ( TKI ) Chidamide - k30 The anti-cancer effect of the combination.

Next, we were interested in studying whether the combination of TKI plus cedamide has a strong modulation of the TME and a significant improvement in immune response in a mouse model bearing CT26 tumors. As shown in Figure 9 ( A ) , several TKIs were combined with cedamide-k30 to test their ability to inhibit tumor growth. The combination of cedamide-k30 with lenvatinib or axitinib inhibited tumor growth more effectively than lenvatinib, axitinib, and cedamide-k30 treatment alone. As shown in Figure 9 ( B ) , the individual tumor sizes (fold change) and ORR indicated that the chidamide-k30 (50 mg/kg) group achieved 2 CR, 4 SD, and 2 PD, with an ORR of 25%; the lenvatinib (10 mg/kg) group achieved 1 PR, 5 SD, and 2 PD, with an ORR of 12.5%; the axitinib (30 mg/kg) group achieved 3 CR, 1 SD, and 4 PD, with an ORR of 37.5%; the combination of lenvatinib (10 mg/kg) and chidamide-k30 (50 mg/kg) achieved 3 CR, 3 SD, and 2 PD, with an ORR of 37.5%; the combination of axitinib (30 mg/kg) and chidamide-k30 (50 mg/kg) achieved 4 CR, 3 SD, and 1 PD, with an ORR of 50%. It seems that the combination of cedabenamide-k30 and lenvatinib or axitinib has an additive effect on the regulation of TME, which is shown in the immune response rate of mice carrying CT26 tumors. As shown in Figure 9 ( C ) , there was no significant loss of mouse weight in different treatments. After implantation, when the tumor volume reached 3000 mm ³ , mice carrying CT26 tumors were euthanized. As shown in Figure 9 ( D ) , the combination of cedabenamide-k30 and axitinib or lenvatinib showed an increase in survival rate compared with lenvatinib or cedabenamide-k30 treatment alone. The recurrence rate was also evaluated as shown in Figure 9 ( E ) . The results show that the combination of TKI and cedabenamide-k30 can have a more powerful activity to activate the immune system to avoid recurrence. To further prove the theory, we have studied other TKIs in combination with cedamide-k30 in a mouse model bearing CT26 tumors. Regorafenib and cabozantinib are two potent oral multi-kinase inhibitors tested. Cabozantinib is a potent multi-tyrosine kinase inhibitor used to inhibit c-MET, VEGFR1, VEGFR2, VEGFR3, AXL and RET. Regorafenib is an oral multi-kinase inhibitor used to inhibit VEGFR1, VEGFR2, VEGFR3, TIE-2, RET, KIT and PDGFR. As shown in Figure 9 ( F ) , the combination of cedamide-k30 with regorafenib or cabozantinib was extremely effective in inhibiting tumor growth compared to treatment with cedamide-k30, regorafenib and cabozantinib alone. As shown in Figure 9(G) , the individual tumor sizes (fold change) and ORR indicated that the chidamide-k30 (50 mg/kg) group achieved 2 CR, 4 SD, and 2 PD, with an ORR of 25%; the regorafenib (30 mg/kg) group achieved 2 PR, 4 SD, and 2 PD, with an ORR of 25%; the cabozantinib (30 mg/kg) group achieved 3 CR, 2 SD, and 2 PD, with an ORR of 43%; the combination group of regorafenib (30 mg/kg) and chidamide-k30 (50 mg/kg) achieved 7 CR and 1 SD, with an ORR of 87.5%; and the combination group of cabozantinib (30 mg/kg) and chidamide-k30 (50 mg/kg) achieved 5 CR, 1 PR, and 2 SD, with an ORR of 75%. It seems that the combination of cedamide-k30 and regorafenib has a more effective synergistic effect on the regulation of TME in mice bearing CT26 tumors than the combination of cedamide-k30 and cabozantinib, and thus improves the immune response rate. In summary, these data show that the combination of cedamide-k30 and regorafenib or cabozantinib is more effective in regulating TME and significantly enhances the immune response rate in mice bearing CT26 tumors compared to the combination treatment of cedamide-k30 and axitinib or lenvatinib. As shown in Figure 9 ( H ) , the weight of mice did not change significantly. The survival rate of the combination group of cedamide-k30 and regorafenib or cabozantinib was evaluated. As shown in Figure 9 ( I ) , the combination of cedamide-k30 and regorafenib is extremely effective in prolonging survival compared to the combination of cedamide-k30 and cabozantinib. The data revealed that the combination of cedamide-k30 with regorafenib or cabozantinib increased the immune response rate and survival rate without combining with ICI. This regimen that improves the combination of cedamide-k30 with regorafenib or cabozantinib has the potential to activate CTL or NK cells to kill tumor cells, without any combination with ICI. To further confirm the anti-cancer efficacy of these two combinations, recurrence was monitored. As shown in FIG9 ( J ) , based on the observation that no relapse occurred in 7 mice that achieved CR, the combination of cedamide-k30 and regorafenib was very effective in activating the immune system/anti-cancer activity; however, in the combination group of cedamide-k30 and cabozantinib and the single treatment group, some mice that achieved CR had relapses. Rechallenge experiments (summarized as shown in FIG6 ( F ) ) indicate that the regimen of the combination of cedamide-k30 and cabozantinib can more effectively induce the generation of memory T cells than the regimen of the combination of cedamide-k30 and regorafenib, as shown in FIG9 ( K ) . Based on the results of the relapse and rechallenge experiments, it seems highly likely that the 7 mice that achieved CR after treatment with the combination of chidamide-k30 and regorafenib did not relapse due to complete tumor irradiation, however, some mice (2/7) subsequently failed to develop or only partially activated immune memory, resulting in growth of the inoculated tumor during the rechallenge period.

Examples 5 : For research purposes CT26 Mouse anti- PD - 1 Ab With cabozantinib or regorafenib plus chidamide - k30 Anticancer activity of the combination.

In Figure 9 , our results show that the combination of cedamide-k30 and cabozantinib or regorafenib has a very effective inhibition of tumor growth activity. Next, we are interested in studying the regimen of adding anti-PD-1 Ab to the combination of cabozantinib or regorafenib plus cedamide-k30 in a mouse model carrying CT26 tumors. As shown in Figure 10 ( A ) , the regimen of anti-PD-1 Ab in combination with cabozantinib plus cedamide-k30 is more effective in inhibiting tumor growth than the regimen of anti-PD-1 Ab in combination with cabozantinib or the regimen of anti-PD-1 Ab in combination with cedamide-k30 plus celecoxib as a positive control (the positive control has previously been shown to be a very promising combination for treatment with immunotherapy). This result suggests that cabozantinib may be a more effective drug than celecoxib in the triple combination. Based on the data, cabozantinib may better control the TME than celecoxib. The individual tumor sizes (fold change) and ORR shown in FIG10(B) indicate that the anti-PD-1 Ab (2.5 mg/kg) group achieved 1 CR, 3 SD, and 5 PD, with an ORR of 11%; the combination group of anti-PD-1 Ab (2.5 mg/kg) and cedabenb-k30 (50 mg/kg) plus celecoxib (50 mg/kg) (as a positive control) achieved 5 CR, 1 SD, and 3 PD, with an ORR of 56%; the combination group of anti-PD-1 Ab (2.5 mg/kg) and cabozantinib (30 mg/kg) achieved 3 CR, 1 PR, 3 SD, and 2 PD, with an ORR of 44.0%; the combination group of anti-PD-1 Ab (2.5 mg/kg) and cabozantinib (30 mg/kg) plus cedabenb-k30 (50 mg/kg) achieved 5 CR and 4 SD, with an ORR of 56%. It appears that the two groups of anti-PD-1 Ab and cabozantinib plus cedabenamide-k30 combination groups and the positive control group achieved an ORR of 56%. However, it appears that the tumors of each mouse treated with the combination of anti-PD-1 Ab and cabozantinib plus cedabenamide-k30 inhibited growth more significantly than the tumors of mice treated with the combination of anti-PD-1 Ab and cedabenamide-k30 plus cenecoxib (no tumors were inhibited in three mice, as shown in Figure 10 ( B ) ). As shown in Figure 10 ( C ) , treatment with the combination of anti-PD-1 Ab and cabozantinib plus cedabenamide-k30 initially caused weight loss, but the weight of the mice eventually recovered after continuous treatment. The survival rate of the combination group of anti-PD-1 Ab and cabozantinib plus cedabenamide-k30 was evaluated compared with the combination group of anti-PD-1 Ab and cabozantinib. As shown in Figure 10 ( D ) , the combination of anti-PD-1 Ab and cabozantinib plus cedabenamide-k30 was more effective in prolonging survival than the combination of anti-PD-1 Ab and cabozantinib. After implantation, mice carrying CT26 tumors were euthanized when the tumor volume reached 3000 ^mm3 . This result also shows that cedabenamide is an extremely important component for improving the combination of anti-PD-1 Ab and cabozantinib to significantly improve the ORR and survival rate in mice carrying CT26 tumors. Also based on the evaluation of the relapse rate as shown in Figure 10 ( E ) , the combination of anti-PD-1 Ab and cabozantinib plus cedabenb-k30 was more effective in avoiding relapse than the combination of anti-PD-1 Ab and cabozantinib. This result also shows that the combination of anti-PD-1 Ab and cabozantinib plus cedabenb-k30 fully activates the immune system to monitor cancer cells and avoid relapse. Next, we are interested in evaluating the triple combination of anti-PD-1 Ab and regorafenib plus cedabenb-k30. As shown in Figure 10 ( F ) , the combination of anti-PD-1 Ab (2.5 mg/kg) and regorafenib (30 mg/kg) plus cedamide-k30 (50 mg/kg) was more effective in inhibiting tumor growth than the combination of anti-PD-1 Ab (2.5 mg/kg) and regorafenib (30 mg/kg). The group of anti-PD-1 Ab (2.5 mg/kg) and cedamide-k30 (50 mg/kg) plus celecoxib (50 mg/kg) was combined as a positive control. This result also indicates that cedamide is a key component to improve the combination of anti-PD-1 Ab and regorafenib to significantly increase the immune response rate for inhibiting tumor growth. As shown in Figure 10 ( G ) , the individual tumor sizes (fold change) and ORR indicated that the anti-PD-1 Ab (2.5 mg/kg) group achieved 1 CR, 3 SD, and 5 PD, with an ORR of 11%; the combination group of anti-PD-1 Ab (2.5 mg/kg) and chidamide-k30 (50 mg/kg) plus celecoxib (50 mg/kg) (as a positive control) achieved 5 CR, 1 SD, and 3 PD, with an ORR of 56%; the combination group of anti-PD-1 Ab (2.5 mg/kg) and cabozantinib (30 mg/kg) achieved 5 SD and 4 PD, with an ORR of 0%; the combination group of anti-PD-1 Ab (2.5 mg/kg) and regorafenib (30 mg/kg) plus chidamide-k30 (50 mg/kg) achieved 3 CR and 6 SD, with an ORR of 33%. Although the ORR in the combination group of anti-PD-1 Ab (2.5 mg/kg) and regorafenib (30 mg/kg) plus cedamide-k30 (50 mg/kg) was lower than that in the combination group of anti-PD-1 Ab and cedamide-k30 plus celecoxib positive control, tumor growth in each mouse was significantly inhibited compared to the positive control group in which tumors were not inhibited in three of the mice, as shown in Figure 10 (G ). As shown in Figure 10 (H), treatment with the combination of anti-PD-1 Ab and regorafenib plus cedamide-k30 initially caused weight loss, but the weight of the mice eventually recovered after continuous treatment. The survival rate of the combination of anti-PD-1 Ab and regorafenib plus cedamide-k30 was evaluated compared with the combination of anti-PD-1 Ab and regorafenib. As shown in Figure 10 ( I ) , the combination of anti-PD-1 Ab and regorafenib plus cedamide-k30 was more effective in prolonging survival than the combination of anti-PD-1 Ab and regorafenib. This result once again proves that cedamide is an extremely important component that contributes to the combination of anti-PD-1 Ab and regorafenib to significantly improve the survival rate in mice carrying CT26 tumors. What surprised us was that the ORR of the combination of anti-PD-1 Ab and regorafenib plus cedamide-k30 was only 33%, but the overall survival rate was as high as 77%. Clearly, the regimen has a strong modulation of tumor immune activity, and despite the cessation of drug administration, it continues to shrink tumors. Similar results can be found in mice resistant to first-line therapy with anti-PD-1 Ab, which were then treated with second-line therapy with a combination of anti-PD-1 Ab and regorafenib/cabozantinib plus cedamide-k30, as shown in Figures 4 and 5 ( D ) . Relapse rates were assessed as shown in Figure 10 (J). None of the mice in the combination group of anti-PD-1 Ab and regorafenib plus cedamide-k30 experienced a relapse. Rechallenge experiments were performed and the results are shown in Figure 10 ( K ) . The combination of anti-PD-1 Ab and regorafenib plus cedamide-k30 and the combination of anti-PD-1 Ab and cabozantinib plus cedamide-k30 both have extremely effective immune activation to prevent the growth of inoculated tumors through the re-attack process. In addition, we also found that the overall immunity rate in mice with CR or PR was lower after treatment with the combination of anti-PD-1 Ab and cabozantinib. Based on the results of immune activation, a combination of anti-PD-1 Ab and multikinase inhibitors (such as regorafenib or cabozantinib plus cedamide-k30) is proposed to activate specific immune memory and thus carry out strong anti-cancer activity. It is beneficial to control the TME to improve tumor immune response rate and avoid recurrence. Chidamide, a subtype-selective HDAC1, HDAC2, HDAC3 and HDAC10 inhibitor and a potent epigenetic immunomodulator, has been approved for the treatment of R/R PTCL (relapsed/refractory peripheral T-cell lymphoma) and ER ⁺ /Her- ^2- breast cancer by China NMPA.

Examples 6 : For research in carrying CT26 In mice ICI and tyrosine kinase inhibitors ( TKI ) Add histone deacetylase inhibitor ( HDACi ) The anticancer activity of the combination.

The efficacy and anti-cancer mechanism of the combination of anti-PD-1 Ab and TKI plus HDAC were further investigated in mice bearing CT26 tumors. As shown in FIG11 ( A ) , we evaluated the combination of anti-PD-1 Ab and regorafenib plus different HDAC inhibitors such as cedabenb (inhibits HDAC1, HDAC2, HDAC3 and HDAC10), vorinostat (SAHA, pan-HDAC inhibitor) and entinostat (inhibits HDAC1, HDAC2 and HDAC3). The results showed that the combination of anti-PD-1 Ab (2.5 mg/kg) and regorafenib (30 mg/kg) plus cedabenb-k30 (50 mg/kg) had a more potent inhibition of tumor growth than the combination of anti-PD-1 Ab and vorinostat or entinostat. The individual tumor sizes (fold change) and ORR shown in FIG11 (B) indicated that the anti-PD-1 Ab (2.5 mg/kg) group achieved 4 SD and 6 PD, with an ORR (objective response rate) of 0%; the combination of anti-PD-1 Ab (2.5 mg/kg) and regorafenib (30 mg/kg) plus cedabenb-k30 (50 mg/kg) achieved 2 CR, 1 PR, and 7 PD, with an ORR of 30%; the combination of anti-PD-1 Ab (2.5 mg/kg) and regorafenib (30 mg/kg) plus vorinostat (150 mg/kg) achieved 3 CR, 1 PR, 2 SD, and 4 PD, with an ORR of 40%; the combination of anti-PD-1 Ab (2.5 mg/kg) and regorafenib (30 mg/kg) plus entinostat (20 mg/kg) achieved 3 CR, 1 PR, 1 SD, and 5 PD, with an ORR of 40%. The ORR of the combination group of 2.5 mg/kg) plus cedabenb-k30 (50 mg/kg) was lower than that of the combination group of anti-PD-1 Ab and regorafenib plus vorinostat (ORR of 40%) or entinostat (ORR of 40%) (ORR of 30%), but compared with the combination group of anti-PD-1 Ab and regorafenib plus vorinostat or entinostat (4 PD and 5 PD, respectively), the combination group of anti-PD-1 Ab (2.5 mg/kg) and regorafenib (30 mg/kg) plus cedabenb-k30 (50 mg/kg) significantly inhibited tumor growth in each mouse (no mouse obtained PD, as shown in Figure 11 ( B ) ). As shown in Figure 11 ( C ) , anti-PD-1 The combination of Ab and regorafenib plus cedamide-k30 or entinostat initially reduced body weight, but then the mice eventually recovered their weight. As shown in Figure 11 ( D ) , the overall survival rate was as follows: the combination of anti-PD-1 Ab and regorafenib plus cedamide-k30 > the combination of anti-PD-1 Ab and regorafenib plus entinostat > the combination of anti-PD-1 Ab and regorafenib plus vorinostat > the anti-PD-1 Ab group. Notably, no mice that achieved ORR had relapse (right panel of Figure 11 ( D ) ). Next, the triple combination with cabozantinib was tested. As shown in Figure 11 ( E ) , the combination group of cabozantinib (30 mg/kg) and cedabenbine-k30 (50 mg/kg) as a control (also studied in Figure 9) showed more effective tumor growth inhibition than the combination group of anti-PD-1 Ab (2.5 mg/kg) and cabozantinib (30 mg/kg) plus cedabenbine-k30 (50 mg/kg). However, the regimen of anti-PD-1 Ab (2.5 mg/kg) in combination with cabozantinib (30 mg/kg) plus cedabenb-k30 (50 mg/kg) or entinostat (20 mg/kg) was more effective in inhibiting tumor growth than the regimen of anti-PD-1 Ab (2.5 mg/kg) in combination with cabozantinib (30 mg/kg) plus vorinostat (150 mg/kg) or anti-PD-1 Ab (2.5 mg/kg) treatment alone. As shown in Figure 11 ( F ) , the individual tumor sizes (fold change) and ORR indicated that the anti-PD-1 Ab (2.5 mg/kg) group achieved 4 SD and 6 PD, with an ORR (objective response rate) of 0%; the combination of anti-PD-1 Ab (2.5 mg/kg) and cabozantinib (30 mg/kg) plus cedabenb-k30 (50 mg/kg) achieved 4 CR, 5 SD and 1 PD, with an ORR of 40%; the combination of anti-PD-1 Ab (2.5 mg/kg) and cabozantinib (30 mg/kg) plus vorinostat (150 mg/kg) achieved 1 CR, 2 PR, 4 SD and 3 PD, with an ORR of 30%; the combination of anti-PD-1 Ab (2.5 mg/kg) and cabozantinib (30 mg/kg) plus entinostat (20 mg/kg) achieved 3 CR, 2 PR, 3 SD and 1 PD, with an ORR of 56%. The combination of cabozantinib (30 mg/kg) and chidamide-k30 (50 mg/kg) achieved 6 CR, 3 SD and 1 PD, with an ORR of 60%, which showed similar results compared with Figure 9 ( G ) . Although the ORR of the combination group of anti-PD-1 Ab (2.5 mg/kg) and cabozantinib (30 mg/kg) plus cedabenb-k30 (50 mg/kg) was lower than that of the combination group of anti-PD-1 Ab and cabozantinib plus entinostat (ORR of 56%) (ORR of 40%), the combination group of anti-PD-1 Ab (2.5 mg/kg) and cabozantinib (30 mg/kg) plus cedabenb-k30 (50 mg/kg) or entinostat (20 mg/kg) significantly inhibited tumor growth in each mouse compared with the combination group of anti-PD-1 Ab and cabozantinib plus vorinostat or anti-PD-1 Ab treatment alone (3 PD and 6 PD, respectively) (only one mouse developed PD, as shown in Figure 11 ( F ) ). As shown in Figure 11 ( G ) , only the combination group of cabozantinib and cedamide-k30 initially reduced body weight, but the mice eventually recovered body weight. As shown in Figure 11 (H), the overall survival rate was as follows: the combination group of anti-PD-1 Ab and cabozantinib plus cedamide-k30 > the combination group of anti-PD-1 Ab and cabozantinib plus entinostat > the combination group of anti-PD-1 Ab and cabozantinib plus vorinostat > the anti-PD-1 Ab group. Despite the higher ORR, the combination group of cabozantinib (30 mg/kg) and cedamide-k30 (50 mg/kg) had the same survival rate as the combination group of anti-PD-1 Ab and cabozantinib plus cedamide-k30. This result again shows that the combination of anti-PD-1 Ab and cabozantinib plus cedamide-k30 has a very effective immunomodulatory activity despite drug cessation. There was no relapse in the combination of anti-PD-1 Ab and cabozantinib plus cedamide-k30 and cabozantinib (30 mg/kg) and cedamide-k30 (50 mg/kg). Next, we are interested in evaluating tumor growth inhibition and the activity of different ICI combinations, such as the combination of anti-PD-1/anti-PD-L1/anti-CTLA-4 Ab and regorafenib/cabozantinib plus cedamide-k30, in mice bearing CT26 tumors. As shown in Figure 11 ( I ) , the combination of anti-PD-L1 Ab (2.5 mg/kg) with regorafenib (30 mg/kg) plus cedabenb-k30 (50 mg/kg) was more effective in inhibiting tumor growth than the combination of anti-CTLA-4 Ab (2.5 mg/kg) or anti-PD-1 Ab (2.5 mg/kg) with regorafenib (30 mg/kg) plus cedabenb-k30 (50 mg/kg). As shown in Figure 11 ( J ) , the individual tumor sizes (fold change) and ORR indicated that the anti-PD-1 Ab (2.5 mg/kg) group achieved 4 SD and 6 PD, with an ORR of 0%; the combination group of anti-PD-1 Ab (2.5 mg/kg) and regorafenib (30 mg/kg) plus cedabenb-k30 (50 mg/kg) achieved 2 CR, 1 PR and 7 PD, with an ORR of 30%; the combination group of anti-PD-L1 Ab (2.5 mg/kg) and regorafenib (30 mg/kg) plus cedabenb-k30 (50 mg/kg) achieved 5 CR, 3 PR and 1 SD, with an ORR of 89%; the combination group of anti-CTLA-4 Ab (2.5 mg/kg) and regorafenib (30 mg/kg) plus cedabenb-k30 (50 mg/kg) achieved 4 CR, 2 PR and 4 SD, with an ORR of 60%. The activity of inhibiting tumor growth is as follows: the combination of anti-PD-L1 Ab and regorafenib plus cedabenbine-k30> the combination of anti-CTLA-4 Ab and regorafenib plus cedabenbine-k30> the regimen> the combination of anti-PD-1 Ab and regorafenib plus cedabenbine-k30> the anti-PD-1 Ab regimen. As shown in Figure 11 ( K ) , the combination group of anti-PD-L1 Ab and regorafenib plus cedabenbine-k30 initially severely reduced body weight, but then the weight of the mice eventually recovered. The combination group of anti-CTLA-4/anti-PD-1 Ab and regorafenib plus cedabenbine-k30 initially had a slight decrease in body weight and the weight of the mice eventually recovered. These results indicate that the combination of anti-PD-L1 Ab and regorafenib plus cedamide-k30 has more effective tumor growth inhibition activity, but may have stronger toxicity and may require further evaluation of other treatment regimens. As shown in Figure 11 ( L ) , we confirmed whether the combination of different ICIs and regorafenib plus cedamide-k30 has different overall survival rates. The overall survival rates are as follows: the combination of anti-CTLA-4 Ab and regorafenib plus cedamide-k30> the combination of anti-PD-L1 Ab and regorafenib plus cedamide-k30> the combination of anti-PD-1 Ab and regorafenib plus cedamide-k30> the anti-PD-1 Ab group. The ORR of the two drug combinations (the combination of anti-CTLA-4 Ab and regorafenib plus cedabenbine-k30 and the combination of anti-PD-1 Ab and regorafenib plus cedabenbine-k30) was lower than the overall survival rate, which may be related to the anti-cancer mechanism of such regimens in effectively regulating the TME. However, only one mouse had a relapse in the combination group of anti-PD-L1 Ab and regorafenib plus cedabenbine-k30. This also shows that these regimens are very effective in activating the immune system to avoid relapse. As shown in Figure 11 ( M ) , the combination of anti-CTLA-4 Ab (2.5 mg/kg) and cabozantinib (30 mg/kg) plus cedabenbine-k30 (50 mg/kg) inhibited tumor growth more effectively than other treatments. Individual tumor sizes (fold change) and ORRs as shown in Figure 11 ( N ) indicate that the combination of anti-PD-1 Ab (2.5 mg/kg) achieved 4 SD and 6 PD, with an ORR of 0%; the combination of cabozantinib (30 mg/kg) and cedabenbine-k30 (50 mg/kg) achieved 6 CR, 3 SD, and 1 PD, with an ORR of 60%; the combination of anti-PD-1 Ab (2.5 mg/kg) and cabozantinib (30 mg/kg) plus cedabenbine-k30 (50 mg/kg) achieved 4 CR, 5 SD, and 1 PD, with an ORR of 40%; the combination of anti-PD-L1 Ab (2.5 mg/kg) and cabozantinib (30 mg/kg) plus cedabenbine-k30 (50 mg/kg) achieved 3 CR, 3 PR, and 4 SD, with an ORR of 60%; the combination of anti-CTLA-4 Ab (2.5 mg/kg) and cabozantinib (30 mg/kg) plus cedamide-k30 (50 mg/kg) achieved 8 CR, 1 PR and 1 SD, with an ORR of 90%. The activity of inhibiting tumor growth is as follows: the combination of anti-CTLA-4 Ab and cabozantinib plus cedamide-k30> the combination of cabozantinib and cedamide-k30> the combination of anti-PD-L1 Ab and cabozantinib plus cedamide-k30> the combination of anti-PD-1 Ab and cabozantinib plus cedamide-k30. As shown in Figure 11 ( O ) , the combination of anti-PD-1 Ab and cabozantinib plus cedamide-k30 alone generally maintained body weight. However, the combination group of anti-PD-L1/anti-CTLA-4 Ab and cabozantinib plus cedamide-k30 or the combination group of cabozantinib and cedamide-k30 initially had a severe decrease in weight, but then the mice eventually recovered their weight. As shown in Figure 11 ( P ) , the overall survival rate was as follows: the combination group of anti-CTLA-4 Ab and cabozantinib plus cedamide-k30> the combination group of anti-PD-1 Ab and cabozantinib plus cedamide-k30=the combination group of cabozantinib plus cedamide-k30>the anti-PD-1 Ab group. Only one mouse had a relapse in the combination group of anti-PD-L1 Ab and cabozantinib plus cedamide. This result also shows that these regimens are very effective in activating the immune system to avoid relapse. Next, we are interested in comparing the regimens of anti-PD-1 Ab in combination with different TKIs plus cedabenb-k30. As shown in Figure 11 ( Q ) , the regimen of anti-PD-1 Ab in combination with regorafenib or cabozantinib plus cedabenb-k30 inhibits tumor growth more effectively than other regimens. RMC-4550 is a well-known effective SHP-2 inhibitor and reports show that it is effective in improving ORR when combined with anti-PD-1 Ab in animal models. However, our data show that RMC-4550 does not have the ability to increase anti-cancer activity compared to regorafenib or cabozantinib in mice carrying CT26 tumors when combined with anti-PD-1 Ab plus cedabenb-k30. Individual tumor sizes (fold change) and ORRs as shown in Figure 11 ( R ) indicate that the anti-PD-1 Ab (2.5 mg/kg) group achieved 4 SD and 6 PD, with an ORR of 0%; the combination group of anti-PD-1 Ab (2.5 mg/kg) and regorafenib (30 mg/kg) plus cedabenbine-k30 (50 mg/kg) achieved 2 CR, 1 PR, and 7 PD, with an ORR of 30%; the combination group of anti-PD-L1 Ab (2.5 mg/kg) and cabozantinib (30 mg/kg) plus cedabenbine-k30 (50 mg/kg) achieved 4 CR, 5 SD, and 1 PD, with an ORR of 40%; the combination group of anti-PD-1 Ab (2.5 mg/kg) and RMC-4550 (30 mg/kg) plus cedabenbine-k30 (50 mg/kg) achieved 1 CR, 1 PR, 2 SD and 6 PD, with an ORR of 20%. The activity of inhibiting tumor growth is as follows: the combination of anti-PD-L1 Ab and cabozantinib plus cedabenbine-k30> the combination of anti-PD-1 Ab and regorafenib plus cedabenbine-k30> the combination of anti-PD-1 Ab and RMC-4550 plus cedabenbine-k30. The weight of mice is shown in Figure 11 ( S ) . As shown in Figure 11 ( T ) , the overall survival rate is as follows: the combination group of anti-PD-1 Ab and regorafenib plus cedabenbine-k30 = the combination group of anti-PD-1 Ab and cabozantinib plus cedabenbine-k30> the combination group of anti-PD-1 Ab and RMC-4550 plus cedabenbine-k30> the anti-PD-1 Ab group. Only the combination group of anti-PD-1 Ab and RMC-4550 plus cedabenb-k30 had relapses. As shown in Table 3 , normal ORR evaluation was performed 3 days after the last drug administration. However, as shown in Table 4 , it was unexpectedly found that tumors in mice bearing CT26 tumors continued to shrink after stopping drug administration, so a second ORR evaluation was performed 10 days after the last drug administration. In the second ORR evaluation, the ORR of the combination group of anti-PD-1 Ab (2.5 mg/kg) and regorafenib (30 mg/kg) plus cedabenb-k30 (50 mg/kg) was significantly enhanced by 30% to 60%. The 3 mice that initially achieved SD continued to have tumor shrinkage and achieved 1 PR and 2 CR, because the drug effect continued to activate CTL and NK in the mouse immune system, resulting in 4 CR, 2 PR, 3 SD and 1 PD results in the second evaluation. Similar phenomena also existed in the combination group of anti-PD-1 Ab (2.5 mg/kg) and regorafenib (30 mg/kg) plus entinostat (20 mg/kg), and the ORR increased from 40% to 50%. In addition, it was found that in the combination group of anti-CTLA-4 Ab (2.5 mg/kg) and regorafenib (30 mg/kg) plus cedabenb-k30 (50 mg/kg), the ORR increased from 60% to 80% and more mice achieved CR. Although the ORR of anti-PD-L1 Ab (2.5 mg/kg) and the combination group of regorafenib (30 mg/kg) plus cedabenb-k30 (50 mg/kg) did not change, more mice became CR (5 to 8). In the combination group of anti-PD-1 Ab (2.5 mg/kg) and cabozantinib (30 mg/kg) plus cedabenb-k30 (50 mg/kg), the ORR was significantly enhanced by 40% to 60%. Although the ORR of the combination group of anti-PD-1 Ab (2.5 mg/kg) and cabozantinib (30 mg/kg) plus entinostat (20 mg/kg) did not change, more mice became CR (3 to 5). Similar phenomena also exist in the combination group of anti-PD-L1/anti-CTLA-4 Ab (2.5 mg/kg) and cabozantinib (30 mg/kg) plus cedabenb-k30 (50 mg/kg), with CR increasing from 3 to 5 and 8 to 9, respectively. These data indicate that the combination of anti-PD-1/anti-PD-L1/anti-CTLA-4 Ab and regorafenib or cabozantinib plus cedabenb-k30 or entinostat has extremely potent activity on the activation of CTL or NK. This is an important finding that the inherent anti-cancer efficacy of ICI and the combination of TKI plus HDACi in mice bearing CT26 tumors is revealed by observing the improved anti-cancer efficacy in mice with initial SD or PR as a delayed development of enhanced immunity. Finally, immunity was further studied by re-challenge experiments (as shown in the upper right row of Table 4 ). The study showed that almost all regimens had significant activity in immune stimulation and thus effectively inhibited the proliferation of CT26 cancer cells inoculated by re-challenge. However, only two regimens (the combination of anti-PD-1 Ab with cabozantinib plus entinostat and the combination of anti-PD-1 Ab with RMC-4550 plus cedabenb) showed tumor growth.

Table 3. Efficacy of HDAC inhibitors plus tyrosine kinase inhibitors with or without ICI in a mouse model bearing CT26 tumors. plan Initial tumor volume ( mm ³ ) ORR (%) PD SD PR CR Survival rate (%) Relapse * (Relapse / recurrence ) Immune # ( attack again ) Figure 6 Anti-PD-1 Ab 190 18% 5 4 1 1 18% (2/11) 100% (2/2) 100% (2/2) Lenvatinib 30% 2 5 2 1 10% (1/10) 67% (2/3) 67% (2/3) Anti-PD-1 Ab + Lenvatinib 17% 3 7 0 2 25% (3/12) 0% (0/2) 100% (2/2) Figure 7 Anti-PD-1 Ab 299 11% 7 1 0 1 Anti-PD-1 Ab + cabozantinib 11% 1 7 0 1 Anti-PD-1 Ab + axitinib 11% 6 2 0 1 Anti-PD-1 Ab + Ibrutinib 11% 7 1 0 1 Anti-PD-1 Ab + Olaparib 0% 8 1 0 0 Figure 8 Anti-PD-1 Ab 177 25% 4 2 1 1 38% (3/8) 0% (0/2) 100% (2/2) Anti-PD-1 Ab + cabozantinib + celecoxib 57% 0 3 0 4 71% (5/7) 0% (0/4) 100% (4/4) Anti-PD-1 Ab+cabozantinib+chidamide-k30 100% 0 0 1 6 100% (7/7) 0% (0/7) 100% (7/7) Figure 9 Chidamide-k30 251 25% 2 4 0 2 25% (2/8) 0% (0/2) Lenvatinib 13% 2 5 1 0 0% (0/8) 100% (1/1) Lenvatinib + Chidamide-k30 38% 2 3 0 3 38% (3/8) 0% (0/3) Axitinib 38% 4 1 0 3 38% (3/8) 0% (0/3) Axitinib + Chidamide-k30 50% 1 3 0 4 50% (4/8) 0% (0/4) Regorafenib 25% 2 4 0 2 13% (1/8) 50% (1/2) 50% (1/2) Regorafenib + Chidamide-k30 88% 0 1 0 7 88% (7/8) 0% (0/7) 71% (5/7) Cabozantinib 43% 2 2 0 3 14% (1/7) 67% (2/3) 100% (3/3) Cabozantinib + Chidamide-k30 75% 0 2 1 5 75% (6/8) 17% (1/6) 100% (6/6) Figure 10 Anti-PD-1 Ab 218 11% 5 3 0 1 22% (2/9) 0% (0/1) 100% (1/1) Anti-PD-1 Ab + cabozantinib 44% 2 3 1 3 33% (3/9) 50% (2/4) 67% (2/3) Anti-PD-1 Ab + Regorafenib 0% 4 5 0 0 0% (0/9) Anti-PD-1 Ab+Chidamide-k30+Cenecoxib 56% 3 1 0 5 44% (4/9) 20% (1/5) 100% (5/5) Anti-PD-1 Ab+cabozantinib+chidamide-k30 56% 0 4 0 5 56% (5/9) 0% (0/5) 100% (5/5) Anti-PD-1 Ab+Regorafenib+Chidamide-k30 33% 0 6 0 3 78% (7/9) 0% (0/3) 100% (3/3) *: Relapse/recurrence is defined as tumor growth of at least 5-fold in mice with CR or PR response after the first tumor assessment. #: Mice resistant to CT26 rechallenge. Response assessment criteria: Fold change in tumor size compared to baseline PD: x≧5; SD: 1≦x<5; PR: 0.5≦x<1; CR: x<0.5

Table 4. Anticancer activity of ICI and the combination of TKI plus HDAC inhibitor in the CT26 tumor - bearing mouse model. plan Initial tumor volume (mm ³ ) ORR (%) PD SD PR CR ORR (%) ^& PD ^& SD ^& PR ^& CR ^& Survival rate (%) Relapse / recurrence ​ ​ ​ Immune # ( attack again ) Figure 11A , B , C and D Anti-PD-1 Ab 243 0% 6 4 0 0 0% 8 2 0 0 0% (0/10) Anti-PD-1 Ab+Regorafenib+Chidamide-k30 30% 0 7 1 2 60% 1 3 2 4 60% (6/10) 0% (0/3) 100% (3/3) Anti-PD-1 Ab+regorafenib+vorinostat 40% 4 2 1 3 40% 4 2 0 4 40% (4/10) 0% (0/4) 100% (4/4) Anti-PD-1 Ab+Regorafenib+Entinostat 40% 5 1 1 3 50% 5 0 0 5 50% (5/10) 0% (0/4) 100% (4/4) Figure 11E , F , G and H Anti-PD-1 Ab 0% 6 4 0 0 0% 8 2 0 0 0% (0/10) Cabozantinib + Chidamide-k30 60% 1 3 0 6 60% 3 1 0 6 60% (6/10) 0% (0/6) 100% (6/6) Anti-PD-1 Ab+Regorafenib+Chidamide-k30 30% 0 7 1 2 60% 1 3 2 4 60% (6/10) 0% (0/3) 100% (3/3) Anti-PD-L1 Ab+Regorafenib+Chidamide-k30 89% 0 1 3 5 89% 0 1 0 8 78% (7/9) 13% (1/8) 100% (8/8) Anti-CTLA-4 Ab+Regorafenib+Chidamide-k30 60% 0 4 2 4 80% 1 1 1 7 90% (9/10) 0% (0/6) 100% (6/6) Figure 11I , J , K and L Anti-PD-1 Ab 0% 6 4 0 0 0% 8 2 0 0 0% (0/10) Anti-PD-1 Ab+cabozantinib+chidamide-k30 40% 1 5 0 4 60% 3 1 1 5 60% (6/10) 0% (0/4) 100% (4/4) Anti-PD-1 Ab + cabozantinib + vorinostat 30% 3 4 2 1 30% 6 1 2 1 30% (3/10) 33% (1/3) 100% (3/3) Anti-PD-1 Ab+cabozantinib+entinostat 56% 1 3 2 3 56% 2 2 0 5 33% (3/9) 40% (2/5) 60% (3/5) Figure 11M , N , O and P Anti-PD-1 Ab 0% 6 4 0 0 0% 8 2 0 0 0% (0/10) Cabozantinib + Chidamide-k30 60% 1 3 0 6 60% 3 1 0 6 60% (6/10) 0% (0/6) 100% (6/6) Anti-PD-1 Ab+cabozantinib+chidamide-k30 40% 1 5 0 4 60% 3 1 1 5 60% (6/10) 0% (0/4) 100% (4/4) Anti-PD-L1 Ab+cabozantinib+chidamide-k30 60% 0 4 3 3 60% 2 2 1 5 60% (6/10) 17% (1/6) 100% (6/6) Anti-CTLA-4 Ab+cabozantinib+chidamide-k30 90% 0 1 1 8 90% 0 1 0 9 90% (9/10) 0% (0/9) 100% (9/9) Figure 11Q , R , S and T Anti-PD-1 Ab 0% 6 4 0 0 0% 8 2 0 0 0% (0/10) Anti-PD-1 Ab+Regorafenib+Chidamide-k30 30% 0 7 1 2 60% 1 3 2 4 60% (6/10) 0% (0/3) 100% (3/3) Anti-PD-1 Ab+cabozantinib+chidamide-k30 40% 1 5 0 4 60% 3 1 1 5 60% (6/10) 0% (0/4) 100% (4/4) Anti-PD-1 Ab+RMC-4550+Chidamide-k30 20% 6 2 1 1 10% 7 2 0 1 10% (1/10) 50% (1/2) 50% (1/2) *: Relapse/recurrence was defined as tumor growth of at least 5-fold in mice with CR or PR response after the first tumor assessment. &: Second tumor assessment 10 days after the last drug administration #: Mice resistant to CT26 rechallenge. Response assessment criteria: Fold change in tumor size compared to baseline PD: x≧5; SD: 1≦x<5; PR: 0.5≦x<1; CR: x<0.5

Examples 7 : In Carry CT26 In mice with tumors, with or without chidamide - k30 Combination of resistance PD - 1 Ab Comparison of tumor cell populations between cabozantinib or regorafenib

To determine whether treatment with anti-PD-1 Ab in combination with cabozantinib/regorafenib or the triple combination of anti-PD-1 Ab and cabozantinib/regorafenib plus cedamide-k30 affects myeloid and T cell populations in tumors, tumor samples were isolated on day 9 after the start of treatment and immune cells were assessed by flow cytometry (FACS ). As shown in FIG12 , CD4 ⁺ T cells and Tregs were significantly altered after treatment with anti-PD-1 Ab with or without cedamide-k30 and cabozantinib or regorafenib, as shown in FIG12 ( A ) . Flow cytometry results showed that the combination of anti-PD-1 Ab and cabozantinib or regorafenib was effective in reducing Treg cells, which is beneficial for activating immunity in tumors in response to tumor expansion. In addition, the regimen of only the combination of PD-1 Ab + regorafenib + cedamide-k30 caused a significant increase in CD8 ⁺ T cell infiltration. Regarding MDSCs, it is known that these bone marrow-derived immature cells are often elevated in tumor-bearing hosts and have potent immunosuppressive activity. Compared with other regimens, treatment with the combination of anti-PD-1 Ab and regorafenib plus cedabenb-k30 significantly reduced PMN-MDSC cells and tumor-associated macrophages (TAMs) in the tumor, as shown in Figure 12 ( B ) , indicating that this triple combination is more likely to result in an increase in tumor-infiltrating lymphocytes (TILs) by inducing the depletion of immunosuppressive cells PMN-MDSC cells and TAMs.

Examples 8 : Adjustable carry CT26 Tumor in mice TME Gene expression in , Chidamide - k30 With cabozantinib / Regorafenib plus CTLA - 4 Ab Combination to overcome the first-line resistance PD - 1 Ab Resistance to treatment.

As shown in Figure 13 , the expression of genes that were modulated by treatment with different second-line regimens to overcome resistance to first-line anti-PD-1 Ab treatment was analyzed. As shown in Figure 13 ( A ) , the results showed that the regimen of anti-CTLA-4 Ab in combination with chidamide-k30 plus cabozantinib or regorafenib was more effective in inducing interferon gamma-related gene expression than the combination of chidamide-HCl salt and celecoxib with or without anti-CTLA-4 Ab therapy and anti-CTLA-4 Ab alone. Similar results also showed that the combination of anti-CTLA-4 Ab and chidamide-k30 plus cabozantinib or regorafenib significantly enhanced the expression of interferon-β-related genes compared with the combination of chidamide-HCl salt and celecoxib with or without anti-CTLA-4 Ab and anti-CTLA-4 Ab alone, as shown in Figure 13 ( B ) . The expression of genes related to T cell-mediated cytotoxicity was analyzed, as shown in Figure 13 ( C ) . The combination of anti-CTLA-4 Ab and chidamide-k30 plus cabozantinib or regorafenib was more effective in inducing the expression of genes related to T cell-mediated cytotoxicity than the combination of chidamide-HCl salt and celecoxib or anti-CTLA-4 Ab alone. However, the expression of genes related to angiogenic activity was significantly downregulated in the combination of anti-CTLA-4 Ab and chidamide-k30 plus regorafenib, as shown in Figure 13 ( D ) . Taken together, all the modulations of gene expression described above suggest that in order to effectively overcome resistance caused by first-line anti-PD-1 Ab treatment, TME regulation involves gene expression affected by cells including immune cells in CT26 tumors.

Examples 9 : Chidamide is used to significantly regulate the carrying CT26 Tumor in mice TME The gene expressed in PD - 1 Ab With regorafenib / Cabozantinib plus chidamide - k30 The key component in the combined solution.

As shown in FIG14 , gene expression analysis of CT26 tumors showed induction of a plethora of immune-related pathways by cedamide. As shown in FIG14 ( A ) , a comparison of the regimen of anti-PD-1 Ab alone and the combination of anti-PD-1 Ab and cabozantinib showed that the regimen of the combination of anti-PD-1 Ab and cabozantinib was more effective in increasing the expression level of genes associated with the activity of trend factors. In addition, we unexpectedly found that the regimen of the combination of anti-PD-1 Ab and cabozantinib plus cedamide-k30 was more effective in enhancing the expression of genes associated with the activity of trend factors compared with the regimen of the combination of anti-PD-1 Ab or anti-PD-1 Ab and cabozantinib. Similar results were also shown for the regimen of the combination of anti-PD-1 Ab and regorafenib plus cedamide-k30. The expression of immune response-related genes was analyzed as shown in Figure 14 ( B ) . The combination of anti-PD-1 Ab and cabozantinib plus cedabenbine-k30 was more effective in increasing the expression of immune response-related genes compared with the combination of anti-PD-1 Ab alone or anti-PD-1 Ab and cabozantinib. Similar results were also shown for the combination of anti-PD-1 Ab and regorafenib plus cedabenbine-k30. Next, we analyzed the expression of genes associated with the marker interferon gamma response as shown in Figure 14 ( C ) . The combination of anti-PD-1 Ab and cabozantinib plus cedabenbine-k30 was more effective in increasing the expression of genes associated with the marker interferon gamma response compared with the combination of anti-PD-1 Ab alone or anti-PD-1 Ab and cabozantinib. Similar results also showed that the combination of anti-PD-1 Ab and regorafenib plus cedamide-k30 was more effective in increasing the expression of genes associated with the interferon gamma response than the combination of anti-PD-1 Ab alone or anti-PD-1 Ab and regorafenib. The effect on downregulation of gene expression was analyzed. As shown in Figure 14 ( D ) , the expression of genes related to transmembrane receptor protein tyrosine kinase activity was more significantly downregulated in the combination group of anti-PD-1 Ab and regorafenib plus cedamide-k30 compared with the combination group of anti-PD-1 Ab alone or anti-PD-1 Ab and regorafenib. As shown in Figure 14 ( E ) , angiogenic activity-related gene expression was significantly downregulated in the combination group of anti-PD-1 Ab and regorafenib plus cedamide-k30 compared to the combination group of anti-PD-1 Ab alone or anti-PD-1 Ab and regorafenib. Significant modulation of gene expression was observed on day 9 after starting treatment with the combination including cedamide-k30, resulting in upregulation of genes related to trendinogen activity, immune response, and marker interferon gamma. These results suggest that the combination of anti-PD-1 Ab and regorafenib/cabozantinib plus cedamide-k30 can complement and increase the efficacy of immunotherapy in the mouse model bearing CT26 tumors. However, when compared with the combination groups of anti-PD-1 Ab alone or anti-PD-1 Ab and regorafenib, down-regulation of gene expression related to transmembrane receptor protein tyrosine kinase activity and angiogenesis activity was significant in the combination group of anti-PD-1 Ab and regorafenib plus cedamide-k30. Taken together, our data suggest and support that the regimen of ICI and TKI plus HDACi combination has potent modulatory activity in the TME to increase the immune response rate of mice bearing CT26 tumors.

Examples 10 : To confirm again, CT26 In mice ICI and tyrosine kinase inhibitors ( TKI ) Add histone deacetylase inhibitor ( HDACi ) The anticancer activity of the combination.

The anticancer activity of the combination of anti-PD-1 Ab and different TKIs plus cedabenamide-k30 was further studied to reconfirm its efficacy in mice bearing CT26 tumors. As shown in Figures 15 ( A ) to 15 ( D ) , the combination of anti-PD-1 Ab and regorafenib plus cedabenamide-k30 was evaluated. The results in Figure 15 ( A ) show that the combination of anti-PD-1 Ab (2.5 mg/kg) and regorafenib (30 mg/kg) plus cedabenamide-k30 (50 mg/kg) has a more potent inhibition of tumor growth than the combination of anti-PD-1 Ab and regorafenib. As shown in Figure 15 ( B ) , the individual tumor sizes (fold change) and ORR indicated that the anti-PD-1 Ab group achieved 8 PD, with an ORR (objective response rate) of 0%; the combination group of anti-PD-1 Ab and regorafenib achieved 5 SD and 1 PD, with an ORR of 0%; the combination group of anti-PD-1 Ab and regorafenib plus cedamide-k30 achieved 3 CR and 4 SD, with an ORR of 43%. The data showed that the combination of anti-PD-1 Ab and regorafenib plus cedamide-k30 had more effective anti-tumor activity than the combination of anti-PD-1 Ab and regorafenib. As shown in Figure 15 ( C ) , the combination group of anti-PD-1 Ab and regorafenib and the combination group of anti-PD-1 Ab and regorafenib plus cedamide-k30 initially reduced body weight, but then the mice eventually recovered their weight. The overall survival rate is shown in Figure 15 ( D ) . The combination group of anti-PD-1 Ab and regorafenib plus cedabenb-k30> the combination group of anti-PD-1 Ab and regorafenib> the anti-PD-1 Ab group. The combination group of anti-PD-1 Ab and regorafenib plus cedabenb-k30 did not relapse. The results also showed that the combination group of anti-PD-1 Ab and regorafenib plus cedabenb-k30 was extremely effective in activating the immune system to avoid relapse. As shown in Figures 15 ( E ) to 15 ( H ) , the combination of anti-PD-1 Ab and cabozantinib plus cedabenb-k30 was evaluated. The results in Figure 15 ( E ) show that the combination of anti-PD-1 Ab and cabozantinib plus cedabenb-k30 has more effective tumor growth inhibition than the combination of anti-PD-1 Ab and cabozantinib. As shown in FIG15 ( F ) , the individual tumor sizes (fold change) and ORR indicated that the anti-PD-1 Ab group achieved 8 PD, with an ORR (objective response rate) of 0%; the combination group of anti-PD-1 Ab and cabozantinib achieved 1 CR, 6 SD and 1 PD, with an ORR of 13%; the combination group of anti-PD-1 Ab and cabozantinib plus cedabenamide-k30 achieved 3 CR, 1 PR and 4 SD, with an ORR of 50%. The data showed that the combination of anti-PD-1 Ab and cabozantinib plus cedabenamide-k30 had more effective anti-tumor activity than the combination of anti-PD-1 Ab and cabozantinib. As shown in FIG15 ( G ) , the weight of the mice did not change significantly. As shown in Figure 15 ( H ) , the combination of anti-PD-1 Ab and cabozantinib plus cedabenamide-k30 prolonged survival compared to other groups, reaching a 63% survival rate on day 54. Next, the recurrence rate in each group was evaluated. The results showed that there was no recurrence in the combination group of anti-PD-1 Ab and cabozantinib and the combination group of anti-PD-1 Ab and cabozantinib plus cedabenamide-k30. Then, we confirmed whether the combination of TKI and HDACi has more effective tumor growth inhibition activity than in the presence of ICI. As shown in Figures 15 ( I ) to 15 ( L ) , the combination of different TKIs and cedabenamide-k30 was evaluated. As shown in Figure 15 ( I ) , the combination of regorafenib and cedabenin-k30 and the combination of situtinib and cedabenin-k30 showed excellent antitumor activity. As shown in Figure 15 ( J ) , the individual tumor size (fold change) and ORR indicated that the combination of regorafenib and cedabenin-k30 achieved 6 CR, 1 PR and 1 SD, with an ORR of 88%; the combination of cabozantinib and cedabenin-k30 achieved 1 CR and 7 SD, with an ORR of 13%; the combination of situtinib and cedabenin-k30 achieved 6 CR and 1 SD, with an ORR of 86%. As shown in Figure 15 ( K ) , the weight of mice did not change significantly. As shown in Figure 15 (L), the combination of regorafenib and cedamide-k30 significantly prolonged the survival rate compared with the other groups, reaching 100% survival rate on day 54. This data shows that the combination of regorafenib and cedamide-k30 has effective immunomodulatory activity. The results of the recurrence rate show that there is no recurrence in the combination group of different TKIs and cedamide-k30. The results of the second tumor assessment and immunity/immunomemory are shown in Table 5 , which have confirmed the conclusions described in Examples 4 and 6 ( Figure 9 and Table 4 ). Treatment with a regimen of regorafenib combined with chidamide-k30 in the absence of anti-PD-1 Ab showed incomplete or partial immunity in some mice with complete tumor irradiation, and in the presence of anti-PD-1, showed delayed development of immunity with enhanced anti-cancer activity observed in a second tumor assessment.

Table 5. Efficacy of HDAC inhibitors plus tyrosine kinase inhibitors with or without ICI in a mouse model bearing CT26 tumors. plan Initial tumor volume ( mm ³ ) ORR (%) PD SD PR CR ORR (%) ^& PD ^& SD ^& PR ^& CR ^& Survival rate (%) Relapse / recurrence ​ ​ ​ Immune # ( attack again ) Figure 15A , B , C and D 227 Anti-PD-1 Ab 0% 8 0 0 0 0% 8 0 0 0 0% Anti-PD-1 Ab + Regorafenib 0% 1 5 0 0 0% 6 0 0 0 0% Anti-PD-1 Ab+Regorafenib+Chidamide-k30 43% 0 4 0 3 86% 1 0 1 5 86% 0% (0/3) 100% (3/3) Figure 15E , F , G and H Anti-PD-1 Ab 0% 8 0 0 0 0% 8 0 0 0 0% Anti-PD-1 Ab + cabozantinib 13% 1 6 0 1 13% 5 2 0 1 25% 0% (0/1) 100% (1/1) Anti-PD-1 Ab+cabozantinib+chidamide-k30 50% 0 4 1 3 50% 1 3 0 4 63% 0% (0/4) 100% (4/4) Figure 11I , J , K and L Anti-PD-1 Ab 0% 8 0 0 0 0% 8 0 0 0 0% Regorafenib + Chidamide-k30 88% 0 1 1 6 88% 0 1 0 7 100% 0% (0/7) 86% (6/7) Cabozantinib + Chidamide-k30 13% 0 7 0 1 25% 3 3 1 1 25% 0% (0/1) 100% (1/1) Selutinib + Chidamide-k30 86% 0 1 0 6 86% 1 0 0 6 86% 0% (0/6) 100% (6/6) *: Relapse/recurrence was defined as tumor growth of at least 5-fold in mice with CR or PR response after the first tumor assessment. &: Second tumor assessment 10 days after the last dose of second-line therapy #: Mice resistant to CT26 re-challenge. Response assessment criteria: Fold change in tumor size compared to baseline PD: x≧5; SD: 1≦x<5; PR: 0.5≦x<1; CR: x<0.5

Although the present invention has been described in conjunction with the specific embodiments described above, many alternatives and modifications and variations thereof will be apparent to those of ordinary skill in the art. All such alternatives, modifications and variations are considered to be within the scope of the present invention.

Figure 1 (A) to (C) shows the continuous treatment course and response results of first-line anti-PD-1 Ab treatment. Male Balb/c mice (1×10 ⁶ cells/mouse) bearing subcutaneous CT26 tumors were treated with first-line anti-PD-1 Ab therapy (mean tumor volume: 113 mm ³ when treatment started). Mice were intraperitoneally (ip) administered 2.5 mg/kg of anti-PD-1 Ab or IgG once every 3 days for 3 doses. When mice responded to anti-PD-1 Ab with tumor reduction, they were given three more doses. (A) Continuous treatment schedule, (B) Tumor size (mm ³ ) from mice responding to first-line anti-PD-1 Ab treatment compared to control group treated 6 times with anti-IgG antibody, (C) Fold change of tumors in (B).

Figure 2 (A) to (C) show the results of second-line treatment in mice with primary resistance to anti-PD-1 antibodies. In first-line anti-PD-1 antibody therapy, mice were defined as primary resistant if the tumor showed a 2.5- to 3-fold continuous increase in tumor volume and the volume was <600 mm ^3. These mice were then re-selected and divided into five groups in second-line treatment for efficacy studies. In second-line treatment, anti-IgG antibody served as a control and anti-IgG and anti-CTLA-4 antibodies were administered intraperitoneally (ip) at 2.5 mg/kg once every 3 days for 6 dosings. The combinations in second-line treatment were: anti-CTLA-4 antibody (2.5 mg/kg) and cedabenb-HCl salt (50 mg/kg) plus celecoxib (50 mg/kg); anti-CTLA-4 antibody (2.5 mg/kg) and regorafenib (30 mg/kg) plus cedabenb-k30 (50 mg/kg); and anti-CTLA-4 antibody (2.5 mg/kg) and cabozantinib (30 mg/kg) plus cedabenb-k30 (50 mg/kg). The combinations were administered orally once daily for 16 times. (A) Tumor size (mm ³ ), (B) Tumor fold change, (C) Mouse ^weight3 .

Figures 3(A) to (C) show the results of second-line treatment in mice with hyperprogressive disease (HPD) tumors during anti-PD-1 antibody therapy. After three doses of first-line anti-PD-1 antibody, mice were defined as having hyperprogressive disease (HPD) if the tumor volume was >600 mm ^3. These mice were then selected for second-line treatment for efficacy studies. The combination in the second-line treatment was a combination of anti-CTLA-4 antibody (2.5 mg/kg) and cabozantinib (30 mg/kg) plus cedamide-HCl salt (50 mg/kg). The combination was administered intraperitoneally and orally. The antibody was administered intraperitoneally once every 3 days for 6 doses, and the combination of cabozantinib and cedamide-HCl salt was administered once a day for 16 days. (A) Tumor fold change, (B) Mouse weight, (C) The combination of anti-CTLA-4 antibody and cabozantinib plus cedabenbine-HCl achieved 2 CR, 9 SD, with an ORR of 18.1%.

Figures 4(A) to (F) show the results of individual tumor volumes in second-line treatment of anti-PD-1 Ab primary resistant mice (as shown in Figures 2 and 3). (A) As a control, the anti-IgG antibody group achieved 5 PD, with an ORR of 0%. (B) The anti-CTLA-4 antibody group achieved 5 SD and 2 PD, with an ORR of 0%. (C) The combination of anti-CTLA-4 antibody and cedabenb-HCl salt plus celecoxib group achieved 3 CR, 4 SD and 1 PD, with an ORR of 37.5%. (D) The combination of anti-CTLA-4 antibody and regorafenib plus cedabenb-k30 group achieved 5 CR and 1 PR, 2 SD, with an ORR of 62.5%. (E) The combination of anti-CTLA-4 antibody and cabozantinib plus cedabenbine-k30 group achieved 3 CR and 1 PR, 3 SD, with an ORR of 57.1%. (F) In HPD mice, treatment with the combination of anti-CTLA-4 antibody and cabozantinib plus cedabenbine-HCl salt achieved 2 CR, 9 SD, with an ORR of 18.1%.

Figures 5(A) to (E) show the results of second-line treatment for acquired resistance to first-line anti-PD-1 antibody treatment. Mice were treated with first-line treatment of 2.5 mg/kg of anti-PD-1 antibody intraperitoneally once every 3 days for 6 doses. Mice were defined as having acquired resistance (relapse) to anti-PD-1 antibody if the tumor volume increased more than 2-fold at 2- to 3-fold treatment and then through all 6 treatments. These mice were then treated with second-line treatment of anti-CTLA-4 antibody with a combination of regorafenib plus cedamide-k30 for efficacy studies. (A) For comparison, tumor size in different treatments from Figure 2(A) is shown. (B) Treatment duration and tumor size for first-line and second-line treatment of mice with acquired anti-PD-1 antibody resistance. (C) Individual tumor volumes in mice with acquired anti-PD-1 antibody resistance after second-line treatment with an anti-EGFR-4 antibody in combination with regorafenib plus cedabenbine-k30 regimen. Treatment achieved 1 CR and 6 SD, with an ORR of 14.1%. (D) Overall survival of mice with primary resistance to anti-PD-1 antibody after second-line treatment. (E) Overall survival of mice with acquired resistance to anti-PD1 antibody after second-line treatment.

Figure 6 (A) to (G) show the results of antitumor effects and immunological assessments of lenvatinib treatment alone or in combination with anti-PD-1 Ab in a mouse model bearing CT26 tumors. Balb/c mice bearing CT26 tumors were treated with different treatment modes as indicated. Anti-IgG antibody, IgG control (2.5 mg/kg); anti-PD-1 monoclonal antibody, PD-1 (2.5 mg/kg); lenvatinib (10 mg/kg); combination of anti-PD-1 Ab (2.5 mg/kg) and lenvatinib (10 mg/kg). (A) Total tumor volume. (B) Individual tumor volume. (C) Mouse weight. (D) Animal survival rate. (E) Relapse rate. (F) Experimental design of rechallenge schedule. (G) Mice bearing recurrent CT26 tumors (i.e., achieving a CR or PR response) were re-challenged with CT26 cells on day 34 and assessed for relapse on day 51. Relapse induced by rechallenge was defined as a tumor increase of at least 2-fold compared to the tumor size on day 41 and a tumor volume exceeding 300 mm ³ . The relapse rate after CT26 cell re-challenge is shown in each group. After tumor implantation, mice bearing CT26 tumors were treated as indicated and euthanized when the tumor volume was 3000 mm ³ . Data are presented as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, one-way ANOVA with Tukey's test. *, compared with IgG; #, compared with PD-1.

Figure 7 (A) and (B) show the results of the efficacy comparison of cabozantinib, ibrutinib, axitinib, olaparib, and cedamide-k30 in combination with anti-PD-1 antibodies. Balb/c mice bearing CT26 tumors were treated with different treatment modes as indicated. Anti-IgG antibody, IgG control (2.5 mg/kg); anti-PD-1 monoclonal antibody, PD-1 (2.5 mg/kg); cedamide-k30 (50 mg/kg); celecoxib (50 mg/kg); cabozantinib (30 mg/kg); ibrutinib (6 mg/kg); axitinib (12.5 mg/kg); olaparib (50 mg/kg). (A) Total tumor volume and mouse weight. (B) Individual tumor volume. After tumor implantation, mice bearing CT26 tumors were treated as indicated and euthanized when the tumor volume reached 3000 mm ^3. Data are presented as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, one-way ANOVA with Tukey's test. *, compared with IgG; #, compared with PD-1.

Figure 8 (A) to (F) show the results of the treatment response and immune assessment of cabozantinib plus celecoxib or cedamide-k30 in combination with anti-PD-1 antibodies in mice bearing CT26 tumors. Balb/c mice bearing CT26 tumors were treated with different treatment modes as indicated. Anti-IgG antibody, IgG control (2.5 mg/kg); anti-PD-1 monoclonal antibody, PD-1 (2.5 mg/kg); cabozantinib (30 mg/kg); cedamide-k30 (50 mg/kg); celecoxib (50 mg/kg). (A) Total tumor volume. (B) Individual tumor volume. (C) Mouse weight. (D) Animal survival rate. (E) Relapse rate. (F) Mice carrying recurrent CT26 tumors were re-challenged with CT26 cells and evaluated as described in Figure 6(G). The relapse rate after CT26 cell re-challenge is shown in each group. After tumor implantation, mice carrying CT26 tumors were treated as indicated and euthanized when the tumor volume was 3000 ^mm3 . Data are given as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, one-way ANOVA using Tukey's test. *, compared with IgG; #, compared with PD-1.

Figure 9 (A) to (K) show the results of the treatment response and immunological assessment of the combination of different tyrosine kinase inhibitors and Chidamide-k30 in mice bearing CT26 tumors. Balb/c mice bearing CT26 tumors were treated with different treatment modes as indicated. Vehicle, 5% DMSO; Lenvatinib (10 mg/kg); Axitinib (30 mg/kg); Regorafenib (30 mg/kg); Cabozantinib (30 mg/kg); and Chidamide-k30 (50 mg/kg). (A) Total tumor volume for lenvatinib and axitinib alone or in combination with chidamide-k30 treatment; (B) individual tumor volume; (C) mouse weight; (D) animal survival; and (E) relapse rate. (F) Total tumor volume for regorafenib and cabozantinib alone or in combination with chidamide-k30 treatment; (G) individual tumor volume, (H) mouse weight, (I) animal survival; and (J) relapse rate. (K) Mice carrying recurrent CT26 tumors were re-challenged with CT26 cells and evaluated as described in Figure 6(G). Relapse rates after CT26 cell re-challenge are shown in each group. After tumor implantation, mice bearing CT26 tumors were treated as indicated and euthanized when the tumor volume reached 3000 mm ^3. Data are presented as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, one-way ANOVA with Tukey's test. *, compared with IgG; #, compared with PD-1. The p value for overall survival was determined using the log-rank (Mantel-Cox) test, comparing each group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 10 (A) to (K) show the results of the treatment response and immune assessment of cabozantinib and regorafenib in combination with chidamide-k30 plus anti-PD-1 antibody in mice bearing CT26 tumors. Balb/c mice bearing CT26 tumors were treated with different treatment modes as indicated. Anti-IgG antibody, IgG control (2.5 mg/kg); anti-PD-1 monoclonal antibody, PD-1 (2.5 mg/kg); cabozantinib (30 mg/kg); regorafenib (30 mg/kg); chidamide-k30 (50 mg/kg). The combination with cabozantinib is shown as (A) total tumor volume, (B) individual tumor volume, (C) mouse weight, (D) animal survival rate, and (E) recurrence rate. However, the combination with regorafenib is shown in (F) total tumor volume, (G) individual tumor volume, (H) mouse weight, (I) animal survival, and (J) relapse rate. (K) Mice carrying recurrent CT26 tumors were re-challenged with CT26 cells and evaluated as described in Figure 6(G). The relapse rate after CT26 cell re-challenge is shown in each group. After tumor implantation, mice carrying CT26 tumors were treated as indicated and euthanized when the tumor volume was 3000 ^mm3 . Data are presented as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, one-way ANOVA with Tukey's test. *, compared with IgG; #, compared with PD-1. The p value of overall survival was determined using the log-rank (Mantel-Cox) test, comparing each two groups, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 11 (A) to (T) show the results of the treatment response of ICI and the combination of TKI plus HDAC inhibitor (HDACi) in CT26-bearing mice. Balb/c mice bearing CT26 tumors were treated with different treatment modes as indicated. IgG, anti-IgG Ab as control (2.5 mg/kg); anti-PD-1 monoclonal antibody PD-1 (2.5 mg/kg); anti-PD-L1 monoclonal antibody (2.5 mg/kg), PD-L1 (2.5 mg/kg); anti-CTLA-4 monoclonal antibody (2.5 mg/kg), CTLA-4 (2.5 mg/kg); regorafenib (30 mg/kg); cabozantinib (30 mg/kg); cedabendin-k30 (50 mg/kg); vorinostat (150 mg/kg); entinostat (20 mg/kg); RMC-4550 (30 mg/kg). First, the combination of anti-PD-1 Ab and regorafenib plus different HDAC inhibitors was evaluated in mice bearing CT26 tumors. (A) Total tumor volume, (B) individual tumor volume, (C) mouse weight, (D) animal survival and relapse rate were recorded. Second, the combination of anti-PD-1 Ab and cabozantinib plus different HDAC inhibitors was evaluated in mice bearing CT26. (E) Total tumor volume, (F) individual tumor volume, (G) mouse weight, (H) animal survival and relapse rate were recorded. Third, the combination of different ICIs and regorafenib plus cedabenbine-k30 was evaluated in mice bearing CT26. (I) Total tumor volume, (J) individual tumor volume, (K) mouse weight, (L) animal survival and relapse rate. Fourth, the combination of different ICIs with cabozantinib plus cedabenb-k30 was evaluated in mice bearing CT26. (M) Total tumor volume, (N) individual tumor volume, (O) mouse weight, (P) animal survival and recurrence rate were recorded. Fifth, the combination of anti-PD-1 Ab and different TKIs plus cedabenb-k30 was evaluated in mice bearing CT26. (Q) Total tumor volume, (R) individual tumor volume, (S) mouse weight, (T) animal survival and recurrence rate were recorded. After tumor implantation, mice bearing CT26 tumors were treated as indicated and euthanized when the tumor volume was 3000 mm ³ . Data are presented as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, one-way ANOVA with Tukey's test. *, compared with IgG; #, compared with PD-1. The p value of overall survival was determined using the log-rank (Mantel-Cox) test, comparing each group, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Figure 12 (A) and (B) show the results of immune cell population analysis of lymphocytes and bone marrow-derived MDSC in CT26-bearing mouse tumors. Mice bearing CT26 tumors were treated with anti-IgG Ab, IgG (2.5 mg/kg) as a control; anti-PD-1 monoclonal antibody, PD-1 (2.5 mg/kg); cabozantinib (30 mg/kg); regorafenib (30 mg/kg); and cedabendin-k30 (50 mg/kg). Mice bearing CT26 tumors were treated with different treatment modes as indicated. Tumor samples were isolated on day 9 after treatment to analyze cell populations in the tumors. (A) Tumor size and flow cytometry results of CD3, CD4, CD8 and Treg cell populations in tumors for each treatment group. Results are shown as mean + SD. *p < 0.05 vs. anti-IgG Ab, (n = 8 to 12). (B) Flow cytometry results of bone marrow-derived CD11b, PMN-MDSC, M-MDSC and tumor macrophage populations in tumors. Results are shown as mean + SD. *p < 0.05 vs. anti-IgG Ab. (n = 8 to 12).

Figure 13 (A) to (D) show overcoming resistance to first-line anti-PD-1 Ab therapy by modulating gene expression in the TME of mice bearing CT26 tumors with a combination of cedamide-HCl/cedamide-k30 and cabozantinib/regorafenib plus anti-CTLA-4 Ab. Tumors were analyzed 13 days after starting second-line treatment for gene expression by RNA-seq. Heatmaps of gene expression and scores associated with (A) interferon gamma, (B) interferon beta, (C) T cell-mediated cytotoxicity, and (D) angiogenic activity. NES: normalized enrichment score; FDR: false discovery rate. The signature score was calculated based on the mean log2 (TPM) of its individual member genes; P value: Mann-Whitney test, two-tailed. TPM, transcripts per million; DGE, differential genetic expression.

Figure 14 (A) to (E) shows that anti-PD-1 Ab and regorafenib/cabozantinib plus cedamide-k30 regimens, with cedamide as the key component, significantly modulate gene expression in the TME of mice bearing CT26 tumors. Tumors were analyzed 9 days after the start of treatment for gene expression by RNA-seq. Heatmaps of gene expression associated with (A) trendinator activity, (B) immune response, (C) interferon gamma, (D) transmembrane receptor protein tyrosine kinase activity, and (E) angiogenic activity are shown with scores. NES: normalized enrichment score; FDR: false discovery rate. The signature score was calculated based on the mean log2 (TPM) of its individual member genes; P value: Mann-Whitney test, two-tailed. TPM, transcripts per million; DGE, differential genetic expression.

Figure 15 (A) to (L) show the results of the treatment response of the combination of TKI plus HDAC inhibitor (Cedamide-k30) with or without anti-PD-1 antibody in CT26-bearing mice. Balb/c mice bearing CT26 tumors were treated with different treatment modes as indicated. IgG, anti-IgG Ab as control (2.5 mg/kg); vehicle, 5% DMSO; PD-1, anti-PD-1 monoclonal antibody (2.5 mg/kg); regorafenib (30 mg/kg); cabozantinib (30 mg/kg); Cedamide-k30 (50 mg/kg); setrutinib (20 mg/kg); celecoxib (50 mg/kg). First, the combination of anti-PD-1 Ab and regorafenib with or without cedamide-k30 was evaluated in mice bearing CT26 tumors. (A) Total tumor volume, (B) Individual tumor volume, (C) Mouse weight, (D) Animal survival and recurrence rate were recorded. Second, the combination of anti-PD-1 Ab and cabozantinib with or without cedamide-k30 was evaluated in mice bearing CT26. (E) Total tumor volume, (F) Individual tumor volume, (G) Mouse weight, (H) Animal survival and recurrence rate were recorded. Third, the combination of different TKIs with cedamide-k30 was evaluated in mice bearing CT26. (I) Total tumor volume, (J) individual tumor volume, (K) mouse weight, (L) animal survival and relapse rate. After tumor implantation, mice bearing CT26 tumors were treated as indicated and euthanized when the tumor volume reached 3000 mm ^3. Data are presented as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, one-way ANOVA with Tukey's test. *, compared with IgG; #, compared with PD-1. The p value of overall survival was determined using the log-rank (Mantel-Cox) test to compare the groups, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Claims (13)

A use of a combination comprising a histone deacetylase (HDAC) inhibitor or a pharmaceutically acceptable salt thereof and a tyrosine kinase inhibitor (TKI) or a pharmaceutically acceptable salt thereof for manufacturing a single agent or multiple agents for inhibiting or treating cancer in an individual by overcoming immunosuppression or stimulating immune response in a tumor microenvironment, wherein the HDAC inhibitor or a pharmaceutically acceptable salt thereof and the tyrosine kinase inhibitor or a pharmaceutically acceptable salt thereof are formulated as one agent, or the H The DAC inhibitor and the tyrosine kinase inhibitor are each formulated as a single agent for simultaneous, separate or sequential administration; wherein the HDAC inhibitor or a pharmaceutically acceptable salt thereof is chidamide, entinostat, or vorinostat or a pharmaceutically acceptable salt thereof; and the TKI or a pharmaceutically acceptable salt thereof is regorafenib or a pharmaceutically acceptable salt thereof. A combination for the manufacture of a single agent or multiple agents for inhibiting or treating cancer in an individual by overcoming immunosuppression or stimulating immune response in the tumor microenvironment, wherein the combination comprises a combination of a histone deacetylase (HDAC) inhibitor or a pharmaceutically acceptable salt thereof, a tyrosine kinase inhibitor (TKI) or a pharmaceutically acceptable salt thereof, and an immune checkpoint inhibitor (ICI); wherein the histone deacetylase (HDAC) inhibitor or a pharmaceutically acceptable salt thereof, the tyrosine kinase inhibitor (TKI) or a pharmaceutically acceptable salt thereof, and the immune checkpoint inhibitor are formulated as one agent, or the HDAC inhibitor or a pharmaceutically acceptable salt thereof, the tyrosine kinase inhibitor or a pharmaceutically acceptable salt thereof, and the immune checkpoint inhibitor are formulated as one agent. One or both of the inhibitors are formulated as a plurality of agents for simultaneous, separate or sequential administration; wherein the HDAC inhibitor or a pharmaceutically acceptable salt thereof is chidamide, entinostat, or vorinostat or a pharmaceutically acceptable salt thereof and the TKI or a pharmaceutically acceptable salt thereof is regorafenib or a pharmaceutically acceptable salt thereof; or the HDAC inhibitor or a pharmaceutically acceptable salt thereof is The salt is chidamide or a pharmaceutically acceptable salt thereof and the TKI or a pharmaceutically acceptable salt thereof is cabozantinib or a pharmaceutically acceptable salt thereof; and the immune checkpoint inhibitor is an anti-cytotoxic T-lymphocyte antigen 4 (CTLA-4) antibody, an anti-programmed cell death protein 1 (PD-1) antibody, or an anti-programmed death-ligand 1 (PD-L1) antibody. The use of claim 1 or 2, wherein the cancer is melanoma, head and neck cancer, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, colorectal cancer, endometrial cancer, cervical cancer, esophageal squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, breast cancer, gastric cancer, esophageal gastric junction cancer, classical Hodgkin lymphoma, non-Hodgkin lymphoma lymphoma), urothelial carcinoma, primary septal large B-cell lymphoma, neuroglioblastoma, pancreatic cancer, benign prostatic hyperplasia, prostate cancer, ovarian cancer, chronic lymphocytic leukemia, Merkel cell carcinoma, acute myeloid leukemia, gallbladder cancer, bile duct cancer, bladder cancer, or uterine cancer. The use of claim 1 or 2, wherein the cancer is an immune checkpoint inhibitor-resistant cancer or a cancer that is unresponsive to cancer immunotherapy. For use as claimed in item 1 or 2, the individual has not received cancer treatment. The use as claimed in claim 5, wherein the individual has received cancer treatment but the treatment has failed. For use as claimed in claim 2, wherein the immune checkpoint inhibitor is pembrolizumab, lambrolizumab, pidilizumab, nivolumab, durvalumab, avelumab or atezolizumab. The use of claim 1 or 2, wherein the amount of the HDAC inhibitor and the TKI in the combination is in the range of about 10% (w/w) to about 70% (w/w) and about 10% (w/w) to about 70% (w/w), respectively. The use of claim 2, wherein the amount of the immune checkpoint inhibitor in the combination is in the range of about 0.5% (w/w) to about 20% (w/w). The use of claim 1 or 2, wherein the combination further comprises one or more additional anticancer agents. A drug combination, comprising the following drug combination, the drug combination comprising a histone deacetylase (HDAC) inhibitor or a pharmaceutically acceptable salt thereof and a tyrosine kinase inhibitor (TKI) or a pharmaceutically acceptable salt thereof; wherein the HDAC inhibitor or a pharmaceutically acceptable salt thereof and the tyrosine kinase inhibitor or a pharmaceutically acceptable salt thereof are formulated as one drug, or the HDAC inhibitor and the tyrosine kinase inhibitor are each formulated for simultaneous, separate or sequential administration. A single drug for sequential administration; wherein the amount of the HDAC inhibitor and the TKI in the drug combination is in the range of about 10% (w/w) to about 70% (w/w) and about 10% (w/w) to about 70% (w/w), respectively; wherein the HDAC inhibitor or a pharmaceutically acceptable salt thereof is chidamide, entinostat, or vorinostat or a pharmaceutically acceptable salt thereof; and the TKI or a pharmaceutically acceptable salt thereof is regorafenib or a pharmaceutically acceptable salt thereof. A drug combination, wherein the drug combination comprises a histone deacetylase (HDAC) inhibitor or a pharmaceutically acceptable salt thereof, a tyrosine kinase inhibitor (TKI) or a pharmaceutically acceptable salt thereof, and an immune checkpoint inhibitor, wherein the HDAC inhibitor or a pharmaceutically acceptable salt thereof and the tyrosine kinase inhibitor or a pharmaceutically acceptable salt thereof are formulated as one medicament, or the HDAC inhibitor and the tyrosine kinase inhibitor are each formulated as a single medicament for simultaneous, separate or sequential administration; wherein the amount of the HDAC inhibitor and the TKI in the drug combination is about 10% (w/w) to about 70% (w/w) and about 10% (w/w) to about 70% (w/w), respectively. /w); wherein the HDAC inhibitor or its pharmaceutically acceptable salt is cedamide, entinostat, or vorinostat or its pharmaceutically acceptable salt and the TKI or its pharmaceutically acceptable salt is regorafenib or its pharmaceutically acceptable salt; or the HDAC inhibitor or its pharmaceutically acceptable salt is cedamide or its pharmaceutically acceptable salt and the TKI or its pharmaceutically acceptable salt is cabozantinib or its pharmaceutically acceptable salt; and the immune checkpoint inhibitor is an anti-cytotoxic T-lymphocyte antigen 4 (CTLA-4) antibody, an anti-programmed cell death protein 1 (PD-1) antibody, or an anti-programmed death-ligand 1 (PD-L1) antibody. A drug combination as claimed in claim 12, wherein the immune checkpoint inhibitor in the drug combination is in the range of about 0.5% (w/w) to about 20% (w/w).