The present invention relates to immunotherapy. Particularly, the present invention provides a pharmaceutical combination and its applications in regulating tumor microenvironment and cancer immunotherapy.
The new era of cancer treatment in tumor immunology will provide great advancements in the use of immune-oncology (IO) therapy to boost anti-cancer immune response. Immune checkpoint inhibitors (ICIs) are one of the most promising IO therapies that can unleash the power of cytotoxic T lymphocytes (CTLs) to efficiently attack and kill tumors, especially the ICIs targeting PD-1 (Programmed cell death protein 1)/PD-L1 (Programmed death-ligand 1) axis blockade. To date, several ICIs have been developed. However, only about 20% of patients respond to anti-PD-1/anti-PD-L1 antibody monotherapy. About 80% of patients gain no clinical benefit caused by primary and acquired resistance. Resistance to PD-1/PD-L1 blockade is therefore a very important issue to overcome in immunotherapy.
The primary resistance refers to the condition where no responses occur by the PD-1/PD-L1 blockade. In comparing immunotherapy with chemotherapy or targeting therapy, immunotherapy has relatively high rates of primary resistance, and so the clinical benefit is restricted. It is estimated that about 60% of patients receiving immunotherapy have primary resistance. However, acquired resistance refers to the condition where an initial response to PD-1/PD-L1 blockade occurs with the progression of a disease, and a relapse occurs eventually. It is estimated that about 20% of patients receiving immunotherapy have acquired resistance. The low response rates and primary or acquired resistance to PD-1/PD-L1 blockade may be related to the tumor microenvironment (TME) (Annals of Oncology, Volume 27, Issue 8, August 2016, Pages 1492-1504). The TME is a dynamic and complicated composition that controls tumor immune response. The major mechanisms of primary or acquired resistance of PD-1/PD-L1 blockade may include several factors such as TME status, PD-L1 expression, tumor neoantigen expression and presentation, cell signal pathway, immune gene expression, and epigenetic modification.
Numerous combined therapeutic strategies hope to overcome the problem of drug resistance by PD-1/PD-L1 blockade. Many approaches focus on increasing the sensitivity to PD-1/PD-L1 blockade by using anti-PD-1 or anti-PD-L1 antibody in combination with other agents. However, these drug combinations cannot achieve the desired therapeutic benefits, and the efficacy and safety thereof are questionable.
The inventors surprisingly found that tyrosine kinase inhibitors (TKIs) plus histone deacetylase (HDAC) inhibitors significantly improve the anti-cancer efficacy via modulation of TME. Furthermore, the TKIs plus HDAC inhibitors combined with ICIs significantly overcome the primary or acquired resistance by PD-1/PD-L1 blockade, and boost the efficacy of immunotherapy.
In one aspect, the present disclosure provides a method for inhibiting or treating a cancer in a subject through overcoming immune suppression in a tumor microenvironment or stimulating immune response against cancer, comprising administering to the subject 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 a tyrosine kinase inhibitor or a pharmaceutically acceptable salt thereof are formulated in a medicament, or the HDAC inhibitor and a tyrosine kinase inhibitor are each formulated as single medicaments for simultaneous, separate or sequential administration.
In another aspect, the present disclosure provides a pharmaceutical combination for use in a method for inhibiting or treating a cancer in a subject through overcoming immune suppression in a tumor microenvironment or stimulating immune response against cancer, 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 a tyrosine kinase inhibitor or a pharmaceutically acceptable salt thereof are formulated in a medicament, or the HDAC inhibitor and a tyrosine kinase inhibitor are each formulated as single medicaments for simultaneous, separate or sequential administration.
In another aspect, the present disclosure also provides a pharmaceutical 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 a tyrosine kinase inhibitor or a pharmaceutically acceptable salt thereof are formulated in a medicament, or the HDAC inhibitor and a tyrosine kinase inhibitor are each formulated as single medicaments for simultaneous, separate or sequential administration. In some embodiments of the disclosure, the amounts of the HDAC inhibitor and the TKI in the pharmaceutical combination range from about 10% (w/w) to about 70% (w/w) and about 10% (w/w) to about 70% (w/w), respectively. In a further embodiment, the pharmaceutical combination further comprises an immune checkpoint inhibitor. In some embodiments of the disclosure, the amount of immune checkpoint inhibitor in the combination ranges from about 0.5% (w/w) to about 20% (w/w).
In another aspect, the present disclosure provides a use of a combination comprising of a histone deacetylase (HDAC) inhibitor or a pharmaceutically acceptable salt thereof and a tyrosine kinase inhibitor (TKI) or a pharmaceutically acceptable salt thereof in the manufacture of a single medicament or multiple medicaments for inhibiting or treating a cancer in a subject through overcoming immune suppression in tumor microenvironment or stimulating immune response, wherein the HDAC inhibitor or a pharmaceutically acceptable salt thereof and a tyrosine kinase inhibitor or a pharmaceutically acceptable salt thereof are formulated in a medicament, or the HDAC inhibitor and a tyrosine kinase inhibitor are each formulated as single medicaments for simultaneous, separate or sequential administration.
In some embodiments of the disclosure, the amounts of the HDAC inhibitor and the TKI in the combination 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 disclosure provides a method for treating a cancer in a subject through overcoming immune suppression in a tumor microenvironment or stimulating immune response, comprising administering to the subject a combination comprising a histone deacetylase (HDAC) inhibitor or a pharmaceutically acceptable salt thereof, a tyrosine kinase inhibitor (TKI) or a pharmaceutically acceptable salt thereof, in combination with 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 in a medicament or one or two of the HDAC inhibitor or a pharmaceutically acceptable salt thereof, tyrosine kinase inhibitor or a pharmaceutically acceptable salt thereof and immune checkpoint inhibitor are formulated as multiple medicaments for simultaneous, separate or sequential administration.
In another embodiment, the present disclosure provides a pharmaceutical combination for use in a method for treating a cancer in a subject through overcoming immune suppression in a tumor microenvironment or stimulating immune response against cancer, 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, in combination with an immune checkpoint inhibitor; 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 (ICI) are formulated in a medicament or one or two of the HDAC inhibitor or a pharmaceutically acceptable salt thereof, tyrosine kinase inhibitor or a pharmaceutically acceptable salt thereof and immune checkpoint inhibitor are formulated as multiple medicaments for simultaneous, separate or sequential administration.
In another embodiment, the present disclosure provides a use of a combination in the manufacture of a single medicament or multiple medicaments for inhibiting or treating a cancer in a subject through overcoming immune suppression in tumor microenvironment or stimulating 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, in combination with 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 in a medicament or one or two of the HDAC inhibitor or a pharmaceutically acceptable salt thereof, tyrosine kinase inhibitor or a pharmaceutically acceptable salt thereof and immune checkpoint inhibitor are formulated as multiple medicaments for simultaneous, separate or sequential administration.
In one embodiment of the disclosure, the amount of immune checkpoint inhibitor in the combination described herein ranges from about 0.5% (w/w) to about 20% (w/w).
In one embodiment, in the combination described herein, the amounts of the histone deacetylase (HDAC) inhibitor or a pharmaceutically acceptable salt thereof, a tyrosine kinase inhibitor (TKI) or a pharmaceutically acceptable salt thereof and the immune checkpoint inhibitor range from 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 disclosure, the immune checkpoint inhibitor described herein is an anti-cytotoxic T-lymphocyte antigen-4 (CTLA-4) antibody or agent, anti-programmed cell death protein 1 (PD-1) antibody or agent, an anti-programmed death-ligand 1 (PD-L1) antibody or agent, an anti-T-cell immunoglobulin and mucin domain-3 (TIM-3) antibody or agent, anti-B- and T-lymphocyte attenuator (BTLA) antibody or agent, anti-V-domain Ig containing suppressor of T-cell activation (VISTA) antibody or agent, an anti-lymphocyte activation gene-3 (LAG-3) antibody or agent, KIR (killer-cell immunoglobulin-like receptor) inhibitor or antibody, 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 disclosure, the cancer described herein includes, but is not limited to, melanoma, head and neck cancer, merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, colorectal cancer, endometrial carcinoma, cervical cancer, esophageal squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, breast cancer, gastric carcinoma, esophagogastric junction carcinoma, classical Hodgkin lymphoma, Non-Hodgkin lymphoma, urothelial carcinoma, primary mediastinal large B-cell lymphoma, glioblastoma, pancreatic cancer, benign prostate hyperplasia, prostate cancer, ovarian cancer, chronic lymphocytic leukemia, Merkel cell carcinoma, acute myeloid leukemia, gallbladder cancer, cholangiocarcinoma, urinary bladder cancer, and uterine cancer.
In a further embodiment, the caner is an immune checkpoint inhibitor-resistant cancer or a cancer failure to respond to a cancer immunotherapy.
In one embodiment, the subject has not received a cancer therapy. In another embodiment, the subject has received a cancer therapy but failed to the therapy. In some embodiments, the cancer therapy is a radiotherapy, chemotherapy or an immunotherapy. In a further embodiment, the immunotherapy is an anti-PD1 immunotherapy, anti-PD L1 immunotherapy or anti-CTL4 immunotherapy.
In some embodiments of the disclosure, the HDAC inhibitor or a pharmaceutically acceptable salt thereof, as described herein, is a class I-selective HDAC inhibitor or pan-HDAC inhibitor which must inhibit class I HDAC. The examples of the HDAC inhibitor or a pharmaceutically acceptable salt thereof include, but are not limited to, a benzamide class of HDAC inhibitor. Preferably, the HDAC inhibitor is Chidamide, Entinostat, Vorinostat, Romidepsin, Panobinostat, Belinostat, Valproic 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, Givinostat and Dacinostat.
In some embodiments of the disclosure, the TKI or a pharmaceutically acceptable salt thereof, as described herein, is an inhibitor of receptor tyrosine kinases. Preferably, the TKI or a pharmaceutically acceptable salt thereof, as described herein, is an inhibitor of vascular endothelial growth factor receptor (VEGFR). Examples of the TKI or a pharmaceutically acceptable salt thereof include, but are not limited to, Cabozantinib, Regorafenib, Axitinib, Afatinib, Ninetedanib, Crizotinib, Alectinib, Trametinib, Dabrafenib, Sunitinib, Ruxolitinib, Vemurafenib, Sorafenib, Ponatinib, Encorafenib, Brigatinib, Pazopanib, Dasatinib, Imatinib, Lenvatinib, Vandetanib, surufatinib and Sitravatinib.
In some further embodiments, examples of the combination as described herein include, but are not limited to, the following:
In some further embodiments, examples of the combination as described herein include, but are not limited to, the following:
In some embodiments of the disclosure, the method or the combination, as described herein, further comprises administering one or more additional anti-cancer agents.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods and materials are now described. All publications mentioned are incorporated herein by reference.
The term “a” and “an” refers to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. The use of “or” means “and/or,” unless specifically stated otherwise.
As used herein, “subject,” “individual” and “patient” are used interchangeably to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vitro or cultured in vitro are also encompassed.
As used herein, “therapeutically effective amount” means an amount sufficient to treat a subject afflicted with a disease (e.g., a neurodegenerative disease) or to alleviate a symptom or a complication associated with the disease.
As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
As used herein, the term “immunotherapy” refers to the treatment of a subject afflicted with, or at risk of contracting or suffering a recurrence of a disease by a method comprising inducing, enhancing, suppressing or otherwise modifying an immune response.
As used herein, the term “programmed cell death protein 1 (PD-1)” refers to an immunoinhibitory receptor belonging to the CD28 family. PD-1 is expressed predominantly on previously activated T cells in vivo, and binds to two ligands, PD-L1 and PD-L2. The term “PD-1” as used herein includes human PD-1 (hPD-1), variants, isoforms, and species homologs of hPD-1, and analogs having at least one common epitope with hPD-1. The complete hPD-1 sequence can be found under GenBank Accession No. U64863.
As used herein, the term “programmed death-ligand1 (PD-L1)” is one of two cell surface glycoprotein ligands for PD-1 (the other being PD-L2) that downregulate T cell activation and cytokine secretion upon binding to PD-1. The term “PD-L1” as used herein includes human PD-L1 (hPD-L1), variants, isoforms, and species homologs of hPD-L1, and analogs having at least one common epitope with hPD-L1. The complete hPD-L1 sequence can be found under GenBank Accession No. Q9NZQ7.
As used herein, an “antibody” and “antigen-binding fragments thereof” encompass naturally occurring immunoglobulins (e.g., IgM, IgG, IgD, IgA, IgE, etc.) as well as non-naturally occurring immunoglobulins, including, for example, single chain antibodies, chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (e.g., bispecific antibodies), Fab′, F(ab′).sub.2, Fab, Fv, and rIgG. As used herein, an “antigen-binding fragment” is a portion of the full-length antibody that retains the ability to specifically recognize the antigen, as well as various combinations of such portions.
As used herein, the term “cancer” refers to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth results in the formation of malignant tumors that invade neighboring tissues and can also metastasize to distant parts of the body through the lymphatic system or bloodstream. “Cancer” as used herein refers to primary, metastatic and recurrent cancers.
As used herein, the term “combination”, “therapeutic combination” or “pharmaceutical combination”, as used herein, defines either a fixed combination in one dosage unit form or a kit of parts for the combined administration where Compound A and Compound B may be administered independently at the same time or separately within time intervals.
As used herein, the term “pharmaceutically acceptable” is defined herein to refer to those compounds, materials, compositions and/or dosage forms, which are, within the scope of sound medical judgment, suitable for contact with the tissues a subject, e.g., a mammal or human, without excessive toxicity, irritation allergic response and other problem complications commensurate with a reasonable benefit/risk ratio.
As used herein, the term “co-administration” or “combined administration” as used herein is defined to encompass the administration of the selected therapeutic agents to a single patient, and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time.
The present disclosure develops methods and combinations that focus on the regulation of tumor microenvironment components, thereby removing immune suppression in a tumor microenvironment or stimulating an immune system against cancer cells. The tumor microenvironment is an important aspect of cancer biology that contributes to tumor initiation, tumor progression and responses 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, which favors the development 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 been uncovered that can help direct and improve the actions of various cancer therapies, notably immunotherapies that work by potentiating host anti-cancer immune responses. The method and combinations not only provide advantageous effect but also synergistic effect in inhibiting or treating a cancer.
Accordingly, the first aspect of the present disclosure is to provide a method of overcoming immune suppression in a tumor microenvironment or stimulating immune response against cancer naïve or resistant to first-line immune checkpoint inhibitor therapy, comprising administering to a subject a combination of a histone deacetylase inhibitor and a tyrosine kinase inhibitor. In one embodiment, the method comprises administering to the subject a combination of the HDAC inhibitor and the TKI in combination with an immune checkpoint inhibitor. Alternatively, the present disclosure provides a use of a pharmaceutical combination of an HDAC inhibitor and a TKI in the manufacture of a medicament for overcoming immune suppression in a tumor microenvironment or stimulating an immune response against cancer. Alternatively, the present disclosure provides a pharmaceutical combination for overcoming immune suppression in a tumor microenvironment or stimulating immune response against cancer, wherein the pharmaceutical combination comprises an HDAC inhibitor and a TKI. Preferably, the pharmaceutical combination further comprises an immune checkpoint inhibitor.
The second aspect of the present disclosure is to provide a pharmaceutical combination comprising an HDAC inhibitor and a TKI. Preferably, the pharmaceutical combination further comprises an immune checkpoint inhibitor.
In one embodiment, the amounts of the HDAC inhibitor and the TKI in the pharmaceutical combination are 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 pharmaceutical 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 the TKI in the pharmaceutical 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).
An HDAC inhibitor possesses very potent epigenetic modulation properties that significantly improve immune modulation activities. HDACs are classes of enzymes catalyzing removal of an acetyl group from lysine on a histone. Such deacetylation leads the histones to wrap DNA more tightly. HDAC inhibition controls chromatin remodeling resulting in regulation of gene expression. HDACs have been shown to be involved in oncogenic transformation by mediated gene expression that influences the cell cycle progression, proliferation, and apoptosis. HDACs are investigated as possible treatment targets for cancers as well as parasitic, infection (such as AIDS), and inflammatory diseases. Based on their homology of accessory domains to yeast histone deacetylases, the 18 currently known human histone deacetylases are classified into four groups (I-IV). Class I, which includes HDAC1, -2, -3 and -8 is related to yeast RPD3 gene; Class IIA includes HDAC4, -5, -7 and -9; Class IIB including HDAC-6 and -10 is related to yeast Hda1 gene; Class III, also known as the sirtuins, is related to the Sir2 gene and includes SIRT1-7; and Class IV, which contains only HDAC11, has features of both Class I and II.
In one embodiment of the present disclosure, 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 HDACs. In some embodiments, the HDAC inhibitor is a benzamide class of histone deacetylase (HDAC) inhibitors. In some embodiments, the HDAC inhibitor includes, but is not limited to, Chidamide, Entinostat, Vorinostat, Romidepsin, Panobinostat, Belinostat, Valproic 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, Givinostat or Dacinostat. In some embodiments, the HDAC inhibitor is Chidamide, Entinostat, Vorinostat, or Mocetinostat.
Tyrosine kinase (TK) is an enzyme catalyzing transferring a phosphate group from ATP to a tyrosine residue. It functions as a switch in cellular functions such as signal transduction to trigger cell survival, differentiation, proliferation. TKs belong to a large class of enzyme containing receptor tyrosine kinases (RTKs) and non-receptor tyrosine kinases. RTKs are key regulators of cellular processes and are identified to be involved in several pathophysiologies of diseases. So far, twenty subfamilies of RTK 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 (Discoidin domain receptor) in humans. RTK molecules contains two regions, including an extracellular ligand-binding region with a single transmembrane helix, and a cytoplasmic region containing a protein tyrosine kinase domain with additional carboxy-(C-)terminal as well as juxtamembrane regulatory regions. Preferably, the TKI according to the disclosure is an inhibitor of vascular endothelial growth factor receptor (VEGFR) including VEGFR1, VEGFR2, and VEGFR3 to inhibit angiogenesis. More preferably, the TKI is Cabozantinib, Regorafenib, Axitinib, Afatinib, Ninetedanib, Crizotinib, Alectinib, Trametinib, Dabrafenib, Sunitinib, Ruxolitinib, Vemurafenib, Sorafenib, Ponatinib, Encorafenib, Brigatinib, Pazopanib, Dasatinib, Imatinib, Lenvatinib, Vandetanib, Surufatinib or Sitravatinib.
It is believed, though not intended to be restricted by any theory, that multi-targeting kinase inhibitors possess a very potent capacity to modulate TME and boost immune response, especially combined with an immune checkpoint inhibitor such as anti-PD-1 or anti-PD-L1 antibody. It achieves a better therapeutic efficacy outcome than PD-1/PD-L1 blockade monotherapy.
In one embodiment, the immune checkpoint inhibitor can be used in combination with the pharmaceutical combination described herein to stimulate an immune response against cancer cells to treat a cancer. Immune checkpoint inhibitors suitable for use in the present disclosure comprise an antagonist of an inhibitory receptor which inhibits the PD-1, CTLA-4, T cell immunoglobulin-3, B and T lymphocyte attenuator, V-domain Ig suppressor of T cell activation or lymphocyte-activation gene 3 pathway, such as anti-PD-1 antibodies or agents, anti-PD-L1 antibodies or agents, anti-CTLA-4 antibodies or agents, anti-TIM-3 (T cell immunoglobulin-3) antibodies or agents, anti-BTLA (B and T lymphocyte attenuator) antibodies or agents, anti-VISTA (V-domain Ig suppressor of T cell activation) antibodies or agents, anti-LAG-3 (lymphocyte-activation gene 3) antibodies or agents, KIR (killer-cell immunoglobulin-like receptor) antibodies or agents, TIM-3 immunoglobulin domain and mucin domain 3) antibodies or agents, A2AR (adenosine A2A receptor inhibitor, CD276 antibodies or agents, and VCTN1 antibodies or agents. Examples of PD-1 or PD-L1 inhibitors include, without limitation, humanized antibodies blocking human PD-1 such as Pembrolizumab (anti-PD-1 Ab, trade name Keytruda) or Pidilizumab (anti-PD-1 Ab), Bavencio® (anti-PD-L1 Ab, Avelumab), Imfinzi® (anti-PD-L1 Ab, Durvalumab), and Tecentriq® (anti-PD-L1 Ab, Atezolizumab), as well as 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 presentations of soluble PD-1 ligand including small molecular drugs blocking human PD-1/PD-L1 such as BMS-1166, without limitation, PD-L2 Fc fusion protein also known as B7-DC-Ig or AMP-244 and other PD-1 inhibitors presently under investigation and/or development for use in therapy. In addition, immune checkpoint inhibitors may include, without limitation, humanized or fully human antibodies blocking PD-L1 such as Durvalumab and MIH1 and other PD-L1 inhibitors presently under investigation. In some embodiments, the amount of the immune checkpoint inhibitor ranges from about 0.5% (w/w) to about 15% (w/w), about 0.5% (w/w) to about 10% (w/w), about 0.5% (w/w) to about 5% (w/w), about 1.0% (w/w) to about 20% (w/w), about 1.0% (w/w) to about 15% (w/w), about 1.0% (w/w) to about 10% (w/w) or about 1.0% (w/w) to about 5% (w/w).
In one embodiment, the HDAC inhibitor and TKI are administered with the immune checkpoint inhibitor simultaneously or sequentially in either order or in alternation. In some embodiments of the present disclosure, the HDAC inhibitor, the TKI, and the immune checkpoint inhibitor are administered simultaneously.
In a further embodiment, the method further comprises administering one or more additional anti-cancer agents. The additional anti-cancer agent is any anti-cancer agent described herein or known in the art. In one embodiment, the additional anti-cancer agent is a chemotherapy or a platinum-based doublet chemotherapy. In one embodiment, the additional anti-cancer agent is an anti-VEGF antibody or VEGFR small-molecule inhibitor. In other embodiments, the anti-cancer 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 anti-cancer agent is 5-flurouracil (5-FU). In certain embodiments, the additional anti-cancer agent is any other anti-cancer agent known in the art.
The pharmaceutical combination 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 agents for pharmaceutical active substances is well known in the art. For example, the pharmaceutical combinations can be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), lozenges, dragees, capsules, pills, tablets (e.g., those targeted for buccal, sublingual, and systemic absorption), boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; (3) topical application, for example, as a cream, lotion, gel, ointment, or a controlled-release patch or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream, suppository or foam; (5) sublingually; (6) ocularly; (7) transdermally; (8) transmucosally; or (9) nasally.
In a further aspect, the present invention provides a method of treating a cancer in a subject, the method comprising administering a pharmaceutical combination of the invention to the subject.
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 carcinoma, cervical cancer, esophageal squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, breast cancer, gastric carcinoma, esophagogastric junction carcinoma, classical Hodgkin lymphoma, Non-Hodgkin lymphoma, urothelial carcinoma, primary mediastinal large B-cell lymphoma, glioblastoma, pancreatic cancer, benign prostate hyperplasia, prostate cancer, ovarian cancer, chronic lymphocytic leukemia, Merkel cell carcinoma, acute myeloid leukemia, gallbladder cancer, cholangiocarcinoma, urinary bladder cancer, or uterine cancer.
In some embodiments, the pharmaceutical combination of the invention may be provided in a single formulation or medicament. In other embodiments, the pharmaceutical combination of the invention may be provided in separates formulations or medicaments. A pharmaceutical combination may be formulated in a variety of and/or a plurality of forms adapted to one or more preferred routes of administration. Thus, a pharmaceutical combination can be administered via one or more known routes including, for example, oral, parenteral (e.g., intradermal, transcutaneous, subcutaneous, intramuscular, intravenous, intraperitoneal, etc.), or topical (e.g., intranasal, intrapulmonary, intramammary, intravaginal, intrauterine, intradermal, transcutaneous, rectally, etc.). A pharmaceutical combination, or a portion thereof, can be administered to a mucosal surface, such as by administration to, for example, the nasal or respiratory mucosa (e.g., by spray or aerosol). A pharmaceutical combination, or a portion thereof, also can be administered via a sustained or delayed release.
A formulation may be conveniently presented in unit dosage form and may be prepared by methods well known in the art of pharmacy. Methods of preparing a combination with a pharmaceutically acceptable carrier include the step of bringing the pharmaceutical combination of the invention into association with a carrier that constitutes one or more accessory ingredients. In general, a formulation may be prepared by uniformly and/or intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then if necessary, shaping the product into the desired formulations.
The amount of a compound that will be effective in the treatment of a particular disorder or condition, including cancer, will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the progression of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. A preferred dosage will be within the range of about 0.01-1000 mg/kg of body weight, about 0.1 mg/kg to 100 mg/kg, about 1 mg/kg to 100 mg/kg, about 10 mg/kg to 75 mg/kg, about 0.1-1 mg/kg, etc. for the combination or each component of the combination.
The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.
To Overcome the Primary and Acquired Resistance, and HPD to First Line PD-1 Checkpoint Blockade Therapy. Male Balb/c mice bearing subcutaneous CT26 tumors (1×106 cell/mice) were treated with first line of therapy of anti-PD-1 antibody (Purchased from InvivoMab, cat #BE00146) treatment (mean tumor volume: 113 mm3 when treatment began), administered intraperitoneally (i.p.) at 2.5 mg/kg, once every 3 days for 3 doses. When tumors responded to treatment with anti-PD-1 antibody (wherein the tumors were significantly shrunk), then the treatment was continuously given for a further 3 doses (i.e., total 6 doses). If the tumors were shrunk at the beginning of anti-PD-1 antibody treatment (after 3 doses), then they grew gradually as a result of continuous anti-PD-1 antibody treatment (i.e., total 6 doses) due to partially effective in inhibiting tumor growth, and further grew in size to develop acquired resistance. When tumors did not respond at the beginning of treatment of anti-PD-1 antibody (after 3 doses) and met the criteria of 2.5-3 consecutive increases in tumor volume (and tumor volume was <600 mm3), it was considered primary resistance. However, hyperprogressive disease (HPD) has been defined as tumors grew greater than 600 mm3 after 3 doses of first line anti-PD-1 antibody treatment. These mice with primary resistance, acquired resistance and HPD were subsequently reenrolled in a second line of therapy for efficacy study as shown in
Anti-Colorectal Cancer Activity in Animal Models. Animal study was approved and overseen by The Taipei Medical University Institutional Animal Care and Use Committee (TMU IACUC, NO: LAC-2020-0103, LAC-2019-0644). Six- to eight-week-old male Balb/c mice (National Laboratory Animal Center, Taiwan) were used for all animal experiments. CT26 cell line was purchased from ATCC. CT26 tumor cell lines were grown in RPMI-1640 supplemented with 10% (vol/vol) FBS at 37° C., 5% CO2. Tumors were established by s.c. injection of 1×106 CT26 cells with Matrigel (354248, Corning®) into the left flank of mice, and growth determined by measuring two perpendicular diameters. Tumors were allowed to grow for 8-12 days (tumor size about 110-250 mm3) before randomization and treatment. Animals were euthanized when tumors reached more than 3000 mm3 in volume. An anti-IgG antibody (BE0089, Lot #716719J3, Bio X Cell), anti-PD-1 antibody (BE0146, Lot #717918D1, Lot #735019J3, Lot #780120J3, Lot #73501901, 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 i.p. at 2.5 mg/kg twice a week for three weeks. All antibodies were diluted to appropriate concentrations in 100 μL of sterile PBS (pH 7.4, Invitrogen Life Technologies). Axitinib (HY-10065, 30 mg/kg, po daily, MedChemExpress USA), Lenvatinib (HY-10981, 10 mg/kg, po daily, MedChemExpress USA), Olaparib (HY-10162, 50 mg/kg, po daily, MedChemExpress USA), Ibrutinib (HY-10997, 6 mg/kg, po daily, MedChemExpress USA), Cabozantinib (HY-13016, 30 mg/kg, po daily, MedChemExpress USA), Regorafenib (HY-1031, 30 mg/kg, po daily, MedChemExpress USA), RMC-4550 (HY-116009, 30 mg/kg, po daily, MedChemExpress USA), Sitravatinib (HY-16961, 20 mg/kg, po daily, MedChemExpress USA), Entinostat (HY-12163, 20 mg/kg, po q2d, MedChemExpress USA), Vorinostat (HY-10221, 150 mg/kg, po daily, MedChemExpress USA), Chidamide-k30 or Chidamide-HCl salt (50 mg/kg, po daily, produced from GNTbm, Taipei, Taiwan), Celecoxib (50 mg/kg, po daily, capsule/Celebrex®, Pfizer Pharmaceuticals LLC) were given for 16 days. Axitinib, Lenvatinib, Olaparib, Ibrutinib, Cabozantinib, Regorafenib, Entinostat, Vorinostat, RMC-4550, Sitravatinib 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 tumors reached more than 3000 mm3 in volume. The anti-cancer activity was measured from the start of the treatment until the tumor volume reached 3,000 mm3. Tumor volume was calculated as length×width2×0.5. In this study, we defined Complete Response (CR, <0.5 time tumor growth in the tumor bearing mice at three days after the end of treatment); Partial Response (PR, tumor size ≥0.5 time tumor growth, but <1 times tumor growth in the tumor bearing mice at three days after the end of treatment); Stable Disease (SD, tumor size ≥1 time tumor growth, but <5 times tumor growth in the tumor bearing mice at three days after the end of treatment); Progressive Disease (PD, tumor size ≥5 times tumor growth in tumor bearing mice at three days after the end of treatment) for the evaluation of treatment efficacy. The recurrence was defined as when having tumor growth at least 5 fold in mice with CR or PR response after first tumor assessment.
In order to demonstrate that the invented combination can overcome the drug resistance issues after treatment with anti-PD-1 Ab, all mice were treated with anti-PD-1 Ab first.
Tumor Rechallenges in Animal Models. All mice with PR/CR response after treatment were rechallenged with CT26 cells on the contralateral side (please see
Survival Rate in Animal Models. After tumor assessment the tumor volume of the mice was measured once every three or four days (twice/week). The tumor-bearing mice were regarded as dead when the tumor volume reached 3,000 mm3. 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), WIC-11-PE (BM8; BioLegend), CD45 FITC (30-F11; BioLegend). Flow cytometry was performed with a Caliber (BD Biosciences) and the data were analyzed with FACS Diva software (BD Biosciences).
To assess the level of tumor infiltrating lymphocyte in tumors, further assays were performed to analyze the intratumoral CD8+, CD4+, regulatory T-cell (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 the Cabozantinib or Regorafenib treatments with or without Chidamide-k30 plus anti-PD-1 Ab. Briefly, primary tumor tissues were harvested, weighed, and minced into fine fragments. Collagenase IV (Sigma-Aldrich) at 1 mg/mL in HBSS (Invitrogen Life Technologies) was added to each sample at a ratio of 1 mL per 200 mg of tumor tissue. Samples were incubated on an end-over-end shaker for 150 min at 37° C. The resulting tissue homogenates were 0.4-μm filtered and washed three times in PBS (BD Biosciences), and then separated via Percoll gradient to isolate mononuclear cells, and 1×106 cells per sample were used for antibody labeling. CD8+ T-cell level was assessed using previously established phenotypic criteria of CD45+CD3+CD8; Treg cell level was assessed using previously established phenotypic criteria of CD45+CD3+CD25+FoxP3+; PMN-MDSC and M-MDSC cell levels were assessed using previously established phenotypic criteria of CD45+/CD11b+/Ly6G+/Ly6C− and CD45+/CD11b+/Ly6G−/Ly6C+, respectively; TAM cell level was assessed using previously established phenotypic criteria of CD45+CD11b+CHM-11+Ly6C+, and total mononuclear cells were used as a common denominator.
RNA Quantification and Qualification. The drug-resistant mice after first-line anti-PD-1 Ab therapy were randomized and treated with different regimens, and the tumors were excised and collected on day 13 after starting second line treatment. The naïve CT26 tumor-bearing mice were randomized and treated with different regimens, and the tumors were excised and collected on day 9 after starting treatment. All tumor samples were snap-frozen in liquid nitrogen, and samples were then homogenized in Trizol (Invitrogen Life Technologies). RNA Purity and quantification were checked using SimpliNano™-Biochrom Spectrophotometers (Biochrom, MA, USA). RNA degradation and integrity were monitored by Qsep 100 DNA/RNA Analyzer (BiOptic Inc., Taiwan). The results are shown in
Library Preparation for Transcriptome Sequencing. A total amount of 1 μg total RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using KAPA mRNA HyperPrep Kit (KAPA Biosystems, Roche, Basel, Switzerland) following the manufacturer's recommendations, and index codes were added to attribute sequences to each sample. PCR products were purified using KAPA Pure Beads system, and the library quality was assessed on the Qsep 100 DNA/RNA Analyzer (BiOptic Inc., Taiwan).
Bioinformatics. The original data obtained by high-throughput sequencing (Illumina NovaSeq 6000 platform) were transformed into raw sequenced reads by CASAVA base calling and stored in FASTQ format. FastQC and MultiQC were used to check fastq files for quality. The obtained raw paired-end reads were filtered by Trimmomatic (v0.38) to discard low-quality reads, trim adaptor sequences, and eliminate poor-quality bases with the following parameters: LEADING: 3 TRAILING: 3 SLIDINGWINDOW: 4:15 MINLEN: 30. The obtained high-quality data (clean reads) was used for subsequent analysis. Read pairs from each sample were aligned to the reference genome by the HISAT2 software (v2.1.0). FeatureCounts (v1.6.0) was used to count the reads numbers mapped to individual genes. For gene expression, the “Trimmed Mean of M-values” normalization (TMM) was performed DEGseq (v1.36.1) without biological duplicate and the “Relative Log Expression” normalization (RLE) was performed using DESeq2 (v1.22.1) with biological duplicate. Differentially expressed genes (DEGs) analysis of two conditions was performed in R using DEGseq (without biological replicate) and DESeq2 (with biological replicate), which is based on negative binomial distribution and Poisson distribution models, respectively. The resulting p-values were adjusted using the Benjamini and Hochberg's approach for controlling the FDR. GO and KEGG pathway enrichment analysis of DEGs were conducted using clusterProfiler (v3.10.1). Gene set enrichment analysis (GSEA) was performed with 1,000 permutations to identify enriched biological functions and activated pathways from the molecular signatures database (MSigDB). MSigDB is a collection of annotated gene sets for use with GSEA software, including hallmark gene sets, positional gene sets, curated gene sets, motif gene sets, computational gene sets, GO gene sets, oncogenic gene sets, and immunologic gene sets. In addition, Weighted Gene Co-expression Network Analysis (WGCNA) was constructed by the co-expression network based on the correlation coefficient of expression pattern using the WGCNA (v1.64) package in R.
In this example, the mice were treated with second line therapy to mimic the treatment for first line drug resistance occurring in human first line cancer therapy—in which a great portion of human cancer patients receiving first line anti-PD-1 antibody therapy will develop resistance, including primary and acquired resistance or HPD (hyperprogressive disease)—for the evaluation of the anti-cancer potency of second line therapy with tyrosine kinase inhibitors plus HDAC inhibitors combined with 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 drug resistance, the platform with treatment schedule was designed as outlined in
&The second tumor assessment 10 days after the last administration of second line treatment.
# Mice resistant to CT26 re-challenge.
Several reports had indicated that Lenvatinib possessed potent immune modulatory properties that could boost the anti-PD-1 Ab immune response rate in tumor-bearing mice models. We were interested in researching more powerful regimens to regulate the TME for boosting the immune response rate. First, we were to evaluate the Lenvatinib and Lenvatinib combined with anti-PD-1 Ab in CT26-bearing mice model. As shown in
We were very interested to evaluate multiple TKIs combined with anti-PD-1 Ab to boost immune response rate in CT26-bearing mice models. As shown in
Next, we were interested to study whether TKIs plus Chidamide regimen possessed potent regulation of the TME and significant boosting of the immune response in CT26 tumor-bearing mice models. As shown in
In
The potency and the anticancer mechanisms of anti-PD-1 Ab combined with TKIs plus HDACis were further studied in CT26 tumor-bearing mice. As shown in
#Mice resistant to CT26 re-challenge.
&the second tumor assessment 10 days after the last drug administration.
#Mice resistant to CT26 re-challenge.
To determine whether treatment with combination of anti-PD-1 Ab combined with Cabozantinib/Regorafenib or triple combination of anti-PD-1 Ab combined with Cabozantinib/Regorafenib plus Chidamide-k30 affected myeloid-cell and T-cell population in tumors, tumor samples were isolated at day 9 after starting treatment, and immune cells were assessed by flow cytometry (FACS). As shown in
As shown in
As shown in
The anti-cancer activity of anti-PD-1 Ab combined with different TKIs plus Chidamide-k30 was further studied to reassure its potency in CT26 tumor-bearing mice. As shown from
&The second tumor assessment 10 days after the last administration of second line treatment.
#Mice resistant to CT26 re-challenge.
While the present disclosure has been described in conjunction with the specific embodiments set forth above, many alternatives thereto and modifications and variations thereof will be apparent to those of ordinary skill in the art. All such alternatives, modifications and variations are regarded as falling within the scope of the present disclosure.