WO2021110121A1 - Hdac6选择性抑制剂及其组合疗法 - Google Patents

Hdac6选择性抑制剂及其组合疗法 Download PDF

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WO2021110121A1
WO2021110121A1 PCT/CN2020/133799 CN2020133799W WO2021110121A1 WO 2021110121 A1 WO2021110121 A1 WO 2021110121A1 CN 2020133799 W CN2020133799 W CN 2020133799W WO 2021110121 A1 WO2021110121 A1 WO 2021110121A1
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antibody
seq
preparation
combination
amino acid
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French (fr)
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李振虎
李富
任兆翔
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基石药业（苏州）有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D211/80—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members
    • C07D211/84—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen directly attached to ring carbon atoms
    • C07D211/86—Oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
    • C07D307/04—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D309/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings
    • C07D309/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
    • C07D309/04—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having no double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings

Definitions

  • the present invention relates to a treatment method for treating a condition of a subject that benefits from modulating immune function, which comprises administering to the subject a pharmaceutically effective amount of an HDAC6 selective inhibitor.
  • the present invention also relates to a combination therapy, which comprises administering to the subject a pharmaceutically effective amount of an HDAC6 selective inhibitor and one or more therapeutic agents.
  • the present invention also provides a therapeutic combination comprising: (1) a selective inhibitor of HDAC6, and (2) one or more therapeutic agents; it can be used to separately, simultaneously or sequentially administer to individuals in need for treatment Or prevent the subject's conditions that benefit from modulating immune function.
  • the present invention also relates to a composition, medicament or preparation or kit, which comprises: (1) a selective inhibitor of HDAC6; (2) one or more therapeutic agents.
  • MM Multiple myeloma
  • BM bone marrow
  • Proteasome inhibitors include Bortezomib, Carfilzomib, Ixazomib and Marizomib, which can block the degradation of polyubiquitinated misfolded proteins and induce ER stress and triggering the apoptosis of MM cells are the basis for the treatment of multiple myeloma.
  • the disease can become resistant through undefined mechanisms.
  • HDACs histone deacetylase
  • pan-HDAC Pan-HDAC
  • class I HDAC inhibitors inhibit both class I (HDAC 1, 2, 3, and 8) and class IIb (HDAC 6 and 10) enzymes
  • class I HDAC inhibitors only inhibit class I enzymes. Since then, class I HDAC inhibitors have been tested and have shown efficacy, but they still have hematological toxicity and dose-limiting adverse reactions.
  • Several studies have reported that the acute toxicity of non-selective HDACi is mainly caused by the inhibition of Class I HDACs. Therefore, the toxicity of pan-HDAC inhibitors or class I HDAC inhibitors is still an obstacle to successful clinical use.
  • HDAC6 plays a role in regulating the function of aggregates and affects the acetylation status of several proteins including ⁇ -tubulin, which is very important for the regulation of microtubule stability and function. It has been reported that HDAC6 is overexpressed in B-cell and T-cell lymphoma, and its inhibitory effect through selective HDAC6 inhibitors has been demonstrated to be active in preclinical models of lymphoma and MM.
  • Immunotherapy is a method of treating a subject's condition that excessively benefits from modulating immune function.
  • cancer immunotherapy In the development of various types of cancer immunotherapy, the main obstacle encountered by scientists and clinicians is to break the tolerance to self-antigens (cancer) in order to trigger a strong anti-tumor response, which leads to tumor regression. Unlike the development of traditional tumor-targeted small and macromolecular drugs, cancer immunotherapy targets cells of the immune system, which have the potential to generate effector cell memory banks to induce longer-lasting effects and minimize recurrence.
  • PD-1 receptor and PD-1 ligands 1 and 2 play an indispensable role in immune regulation.
  • PD-1 expressed on activated T cells is activated by PD-L1 (also known as B7-H 1) and activated by PD-L2 expressed by stromal cells, tumor cells, or both, thereby triggering T cell death and localization Immunosuppression provides an environment of immune tolerance for tumor development and growth. In contrast, inhibiting this interaction in non-clinical animal models can enhance local T cell responses and mediate anti-tumor activity.
  • the present invention relates to a treatment method for the treatment of a subject's condition that benefits from modulating immune function; the present invention also relates to a combination therapy; the present invention also provides a therapeutic combination, which can be used separately, simultaneously or sequentially Is administered to an individual to treat or prevent a subject's conditions that benefit from modulating immune function; the present invention also relates to a composition, drug or formulation or kit.
  • selective HDAC6 inhibitors are expected to prevent the severe toxicity observed in pan-HDAC inhibitors and provide a broader therapeutic window for the clinic.
  • selective HDAC6 inhibitors have unique characteristics in the treatment of cancer, especially HDAC inhibition: they have a dual function of synergistic effect with proteasome inhibitors and increase the anti-tumor function of immune checkpoints by activating myeloid cells.
  • the combination of a selective HDAC6 inhibitor and one or more therapeutic agents synergistically enhances the anti-tumor function, especially the anti-multiple myeloma function.
  • the HDAC6 inhibitors that can be used in the combination of the present invention have been described generally and specifically in the published PCT patent application WO2018/130155, which is incorporated herein by reference.
  • the present invention relates to a method for a condition of a subject that benefits from modulating immune function, which comprises administering to the subject a pharmaceutically effective amount of an HDAC6 selective inhibitor.
  • the present invention also relates to a method for a condition of a subject that benefits from modulating immune function, which comprises administering to the subject a pharmaceutically effective amount of a selective HDAC6 inhibitor and one or more therapeutic agents.
  • the present invention also provides a therapeutic combination comprising: (1) a selective HDAC6 inhibitor, and (2) one or more therapeutic agents; it can be used to separately, simultaneously or sequentially administer to individuals in need for treatment Or prevent the subject's conditions that benefit from modulating immune function.
  • the present invention relates to a composition, medicament or formulation comprising a selective HDAC6 inhibitor, which is used in combination with one or more therapeutic agents for the treatment of subjects who benefit from modulating immune function disease.
  • the present invention also relates to a composition, medicament or preparation, which comprises: (1) a selective HDAC6 inhibitor; (2) one or more therapeutic agents.
  • the present invention also provides a kit comprising a first container, a second container and a package insert, wherein the first container contains at least one dose of the drug, the drug contains a selective HDAC6 inhibitor, and the second container contains at least one dose
  • the drug contains a second therapeutic agent.
  • the present invention also relates to the use of a combination of a selective HDAC6 inhibitor and one or more therapeutic agents in the preparation of a medicament for the treatment of a condition in a subject that benefits from modulating immune function.
  • the present invention relates to the use of a selective HDAC6 inhibitor in the preparation of a medicament for the treatment of a condition in a subject that benefits from modulating immune function, the selective HDAC6 inhibitor and one or more Therapeutic agent combination.
  • the present invention relates to the use of one or more therapeutic agents in the manufacture of a medicament for the treatment of a condition in a subject that benefits from modulating immune function, the one or more therapeutic agents and Selective HDAC6 inhibitor combination.
  • the selective HDAC6 inhibitor and one or more therapeutic agents are administered to the subject simultaneously, separately or sequentially.
  • the selective HDAC6 inhibitor is a compound represented by formula (I) and formula (II), or a pharmaceutically acceptable compound thereof Salt or enantiomer, diastereomer, racemate, solvate, hydrate, polymorph, prodrug or active metabolite,
  • E 1 and E 2 are selected from -O-, -CH 2 -and -CH 2 -CH 2 -;
  • R 2 is selected from: H, F, Cl, Br, I;
  • R is selected from: F, Cl, Br, I;
  • n is selected from: 0 or 1.
  • the selective HDAC6 inhibitor is a compound of formula A or a pharmaceutically acceptable salt or enantiomer, non- Enantiomers, racemates, solvates, hydrates, polymorphs, prodrugs or active metabolites:
  • the one or more therapeutic agents are PD-1 axis binding antagonists or proteasome inhibitors
  • the PD-1 axis binding antagonist is selected from PD-1 binding antagonists, PD-L1 binding antagonists and PD-L2 binding antagonists; more preferably, the PD-1 axis binding antagonist is an antibody or its antigen binding Fragment; especially preferably, the antibody is an anti-PD-1 antibody; most preferably, the antibody is a monoclonal antibody;
  • the proteasome inhibitor is bortezomib, carfilzomib, ixazomib or marezomib.
  • the antibody or antigen-binding fragment thereof binds to an epitope of PD-1, and the epitope comprises: SEQ ID NO: 24 amino acids at positions 128, 129, 130, 131, and 132 and at least one amino acid at positions 35, 64, 82, and 83;
  • the antibody or antigen-binding fragment thereof binds to an epitope of human PD-1 and murine PD-1, wherein the epitope includes SEQ ID NO: 128, 129, 130, 131, and 132 on 24 Site amino acid.
  • the antibody or antigen-binding fragment thereof comprises an amino acid sequence selected from SEQ ID NOs: 1, 2,
  • the sequences in the group consisting of 3, 4, 5, 6, 7, 8 and 9 have at least 70%, 80%, 90% or 95% homology, wherein the antibody specifically binds to PD-1; preferably Yes, the antibody or antigen-binding fragment thereof comprises an amino acid sequence, and the amino acid sequence is selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, and 9.
  • use, combination, composition, drug, preparation or kit the antibody or antigen-binding fragment thereof comprises:
  • the heavy chain variable region which has an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2
  • sequence in or a sequence that has at least 70%, 80%, 90% or 95% homology with it.
  • the light chain variable region which has an amino acid sequence selected from SEQ ID NOs: 3, 4, 5, 6, 7, 8 and 9
  • the antibody specifically binds PD-1.
  • the antibody or antigen-binding fragment thereof comprises:
  • the antibody or antigen-binding fragment thereof comprises a complementarity determining region (CDR), which has an amino acid sequence selected from SEQ ID NOs: A sequence in the group consisting of 10-23, wherein the antibody specifically binds PD-1;
  • CDR complementarity determining region
  • the antibody comprises:
  • the heavy chain variable region CDR1 includes an amino acid sequence selected from SEQ ID NO: 10,
  • Heavy chain variable region CDR2 the sequence includes an amino acid sequence selected from SEQ ID NO: 11,
  • the heavy chain variable region CDR3 contains an amino acid sequence selected from SEQ ID NO: 13,
  • the light chain variable region CDR1 includes an amino acid sequence selected from SEQ ID NO: 14,
  • the light chain variable region CDR2 includes an amino acid sequence selected from SEQ ID NO: 19,
  • the light chain variable region CDR3 includes an amino acid sequence selected from SEQ ID NO: 21.
  • the selective HDAC6 inhibitor is a compound of formula A or an enantiomer, diastereomer, or exoisomer.
  • the one or more therapeutic agents are antibodies or antigen-binding fragments thereof, which are antibody 2E5, comprising:
  • the antibody specifically binds PD-1.
  • the selective HDAC6 inhibitor is a compound of the following formula or an enantiomer, a diastereomer, or an exoisomer Racemates, solvates, hydrates, polymorphs, prodrugs or active metabolites:
  • the one or more therapeutic agents are bortezomib, carfilzomib, ixazomib or marezomib.
  • Figure 1 shows the chemical structure of the compound of formula A and ACY-1215.
  • Figure 2 shows the effect of the combination of a compound of formula A and ixazomib on tumor growth in a MM.1S xenograft model.
  • Figure 3 shows the expression levels of Ac- ⁇ -tubulin and Ac-H3K9 proteins in MM.1S tumor tissue.
  • Figure 4 shows the effect of the combination of a compound of formula A and antibody 2E5 on tumor growth in a CT26 homologous model.
  • Figure 5 shows the PBMC-mediated killing effect of the compound of formula A on HCT116 cells, MCF-7 cells, NCI-H292 or HuH-7 cells.
  • Figure 6 shows the RNA transcriptome analysis performed by PCA analysis. The results show that the gene expression pattern of Compound A is different from other HDAC inhibitors.
  • Figure 7 shows RNA transcriptome analysis using GO enrichment analysis. The results show that the compound of formula A specifically modulates immune cell functions in cell lines with different indications.
  • Figure 8 shows RNA transcriptome analysis using GO enrichment analysis. The results show that the different expressed genes induced by the compound of formula A can be enriched in the myeloid cell differentiation pathway of Daudi and HT-29 cells.
  • Figure 9 shows that the compound of formula A activates myeloid cells and increases the M1/M2 ratio.
  • Combination refers to the simultaneous or sequential administration of the drugs or therapeutic agents of the present invention.
  • the HDAC6 described herein can be administered simultaneously or sequentially in separate unit dosage forms with another therapeutic agent, such as an anti-PD-1 antibody or antigen-binding fragment thereof, or simultaneously administered in a single unit dosage form together with another therapeutic agent.
  • the drug or therapeutic agent that can be used in the "combination of the invention” includes the free base of the compound and all pharmaceutically acceptable salts of the compound.
  • pharmaceutically effective amount or “clinically effective amount” or “therapeutically effective amount” refers to an amount sufficient to provide an observable improvement in the symptoms and symptoms observed on the clinical baseline of the disorder.
  • pharmaceutically acceptable is defined herein as those suitable for contact with the tissues of individuals (such as mammals or humans) within the scope of reasonable medical judgment without excessive toxicity, irritation of allergic reactions and other complications and problems. Compounds, materials, compositions and/or dosage forms with a reasonable benefit/risk ratio commensurate with it.
  • administering or “administration” when applied to animals, humans, experimental subjects, cells, tissues, organs or biological fluids refers to exogenous drugs, therapeutic agents, diagnostic agents or compositions and subjects, Contact of cells, tissues, organs or biological fluids.
  • co-administration or “combination administration” as used herein is defined to encompass the administration of selected therapeutic agents to a single patient, and is meant to include treatment regimens in which the drugs are not necessarily administered via the same route or simultaneously.
  • treatment includes treatments that alleviate, alleviate or reduce at least one symptom of an individual or affect the delay in disease progression.
  • the treatment may be to eliminate one or several symptoms of the disorder or completely eradicate the disorder (e.g., cancer).
  • the term “treatment” also means preventing, delaying the onset (ie, the time before the clinical manifestations of the disease) and/or reducing the risk of the development or deterioration of the disease.
  • protection refers to preventing, delaying or treating (or all as appropriate) the development or persistence or exacerbation of an individual's disease.
  • Subjects to be administered include, but are not limited to: humans (ie, men or women of any age group, for example, pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young Adults, middle-aged adults or older adults)) and/or non-human animals, for example, mammals, for example, primates (for example, cynomolgus monkeys, rhesus monkeys), cows, pigs, horses, sheep , Goats, rodents, cats and/or dogs.
  • the subject is a human.
  • the subject is a non-human animal.
  • the terms "human", “patient” and “subject” are used interchangeably herein.
  • cell proliferative disorder and “proliferative disorder” refer to disorders related to a certain degree of abnormal cell proliferation.
  • the cell proliferative disorder refers to cancer.
  • the cell proliferative disorder is a tumor.
  • Tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer cancer
  • cancer cancer
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  • cancer cancer
  • cancer and “cancerous” refer to or describe a physiological condition in mammals that is usually characterized by unregulated cell growth.
  • examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies.
  • cancers include, but are not limited to, squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal cancer , Hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral cancer, liver tumor, breast cancer, colon cancer, Rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, superficial spreading melanoma, Malignant lentigines melanoma, acral melanoma, nodular melanoma, multiple myeloma, and
  • cancers suitable for treatment by the antibodies of the invention include breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, glioblastoma, non-Hodgkin’s lymphoma (NHL) , Renal cell carcinoma, prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, ovarian cancer, mesothelioma and multiple myeloma.
  • the cancer is selected from: small cell lung cancer, glioblastoma, neuroblastoma, melanoma, breast cancer, gastric cancer, colorectal cancer (CRC), and hepatocellular carcinoma.
  • the cancer is selected from: non-small cell lung cancer, colorectal cancer, glioblastoma, and breast cancer, including those metastatic forms of cancer.
  • programmed death 1 As used herein, the terms “programmed death 1”, “programmed cell death 1”, “protein PD-1”, “PD-1”, “PD1”, “PDCD1”, “hPD-1” and “ “hPD-F” can be used interchangeably, and includes variants, isotypes, species homologs of human PD-1, and analogs having at least one common epitope of PD-1.
  • an “antibody” is an immunoglobulin molecule capable of specifically binding to a target such as carbohydrates, polynucleotides, lipids, and polypeptides via at least one antigen recognition site located in the variable region of the immunoglobulin molecule.
  • the term encompasses not only complete polyclonal or monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab')2, Fv), single chain (scFv) and domain antibodies (including Such as shark and camel antibodies) and fusion proteins containing antibodies, and any other modified configurations of immunoglobulin molecules containing antigen recognition sites.
  • Antibodies include any class of antibodies, such as IgG, IgA, or IgM (or subclasses thereof), and the antibody need not be of any specific class.
  • immunoglobulins can be classified into different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgAl, and IgA2.
  • the heavy chain constant regions corresponding to different types of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
  • antibody includes intact antibodies and any antigen-binding fragments (ie, “antigen-binding portions") or single chains thereof.
  • Antibody refers to a protein comprising at least two heavy chains (H) and two light chains (L) connected to each other by disulfide bonds, or an antigen-binding portion thereof.
  • Each heavy chain is composed of a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region.
  • the heavy chain constant region is composed of three domains, CH1, CH2 and CH3.
  • Each light chain consists of a light chain variable region (abbreviated as VL herein) and a light chain constant region.
  • the light chain constant region consists of a domain CL.
  • VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDR), interspersed with more conservative regions called framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminal to the carboxy terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with antigens.
  • antibody refers to an immunoglobulin or its fragments or derivatives thereof, and includes any polypeptide of the antigen binding site it contains, regardless of whether it is produced in vitro or in vivo.
  • the term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, non-specific, humanized, single-stranded, chimeric, synthetic, recombinant, hybrid, Mutant, grafted antibodies.
  • antibody also includes antibody fragments such as Fab, F(ab')2, FV, scFv, Fd, dAb and other antibody fragments that retain the antigen-binding function, that is, can specifically bind to PD-1. Normally, such fragments will include antigen-binding fragments.
  • antigen-binding fragment or "antigen-binding portion" of an antibody as used herein refers to one or more fragments of an intact antibody that retain the ability to specifically bind to a given antigen.
  • the antigen-binding function of an antibody can be performed by fragments of intact antibodies.
  • binding fragments encompassed by the term "antigen-binding fragment” of the antibody include Fab; Fab'; F(ab')2; Fd fragment composed of VH domain and CH1 domain; VL structure composed of one arm of antibody Fv fragment composed of domain and VH domain; single domain antibody (dAb) fragment (Ward et al., Nature 341:544-546, 1989) and isolated complementarity determining region (CDR).
  • antigen-binding fragment refers to an antibody molecule that contains amino acids responsible for the binding between a specific antibody and an antigen.
  • the antigen-binding fragment only binds a part of the antigen. That is, the part of the antigen molecule responsible for the specific interaction with the antigen-binding fragment is called "epitope” or "antigenic determinant”.
  • An antigen-binding fragment usually includes an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), however, it does not necessarily include both.
  • VL antibody light chain variable region
  • VH antibody heavy chain variable region
  • a so-called Fd antibody fragment consists of only the VH domain, but still retains some of the antigen-binding functions of a complete antibody.
  • epitope is defined as an antigenic determinant, which specifically binds/recognizes the binding fragment.
  • the binding fragment can specifically bind/react with a unique conformation or continuous epitope for the target structure, such as human PD-1 and murine PD-1 (mouse or rat).
  • the conformational or discontinuous epitope is characterized in that the polypeptide antigen is two or more discrete amino acid residues separated in the primary sequence, but when the polypeptide is folded into a natural protein/antigen, it is gathered together on the surface of the molecule. Two or more discrete amino acid residues of an epitope are present in independent parts of one or more polypeptide chains.
  • epitope that binds to PD-1 refers to an antibody that specifically binds to a specific epitope of PD-1, which can be defined by a linear amino acid sequence or a partial three-dimensional structure of PD-1. Binding means that the affinity for the antibody in the part of PD-1 is significantly greater than its affinity for other related polypeptides.
  • substantially greater affinity refers to a measurable increase in the affinity for PD-1 compared to the affinity of other related polypeptides.
  • the affinity for a specific part of PD- is at least 1.5 times, 2 times, 5 times, 10 times, 100 times, 103 times, 104 times, 105 times, 106 times or greater than other proteins.
  • the binding affinity is determined by enzyme-linked immunosorbent assay (ELISA), or by fluorescence activated cell sorting (FACS) analysis or surface plasmon resonance (SPR). More preferably, the binding specificity is obtained by fluorescence activated cell sorting (FACS) analysis.
  • ELISA enzyme-linked immunosorbent assay
  • FACS fluorescence activated cell sorting
  • SPR surface plasmon resonance
  • “Homology” refers to the sequence similarity between two polypeptide sequences when they are optimally aligned. When the position in both of the two compared sequences is occupied by the same amino acid monomer subunit, for example, if the position in the light chain CDR of two different Abs is occupied by alanine, the two Abs are at that position. Homologous. The percent homology is the number of homology positions shared by the two sequences divided by the total number of positions compared X 100. For example, if 8 out of 10 positions in two sequences match or are homologous when the sequences are optimally aligned, then the two sequences are 80% homologous. Generally, the comparison is made when two sequences are aligned to produce the maximum percent homology. For example, the comparison can be performed by the BLAST algorithm, where the parameters of the algorithm are selected to produce the largest match between each sequence over the full length of each reference sequence.
  • pharmaceutically acceptable salt refers to a salt of the compound of the present invention, which is prepared from a compound with specific substituents discovered in the present invention and a relatively non-toxic acid or base.
  • a base addition salt can be obtained by contacting the neutral form of the compound with a sufficient amount of base in a pure solution or a suitable inert solvent.
  • Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salt or similar salts.
  • the acid addition salt can be obtained by contacting the neutral form of the compound with a sufficient amount of acid in a pure solution or a suitable inert solvent.
  • Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, hydrogen carbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, Hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts, the organic acid includes, for example, acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, Similar acids such as fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid and methanesulfonic acid; also include salts of amino acids (such as arginine, etc.) , And salts of organic acids such as glucuronic acid (see Berge et al., "
  • the salt is contacted with a base or acid in a conventional manner, and the parent compound is separated, thereby regenerating the neutral form of the compound.
  • the parent form of the compound differs from its various salt forms in certain physical properties, such as solubility in polar solvents.
  • “Pharmaceutically acceptable salt” as used herein is a derivative of the compound of the present invention, wherein the parent compound is modified by forming a salt with an acid or a salt with a base.
  • pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of bases (such as amines), alkali metal or organic salts of acids (such as carboxylic acids), and the like.
  • Pharmaceutically acceptable salts include conventional non-toxic salts or quaternary ammonium salts of parent compounds, such as salts formed by non-toxic inorganic or organic acids. Conventional non-toxic salts include, but are not limited to, those derived from inorganic acids and organic acids.
  • the inorganic or organic acids are selected from 2-acetoxybenzoic acid, 2-hydroxyethanesulfonic acid, acetic acid, ascorbic acid, Benzenesulfonic acid, benzoic acid, bicarbonate, carbonic acid, citric acid, edetic acid, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, glucoheptose, gluconic acid, glutamic acid, glycolic acid, Hydrobromic acid, hydrochloric acid, hydroiodide, hydroxyl, hydroxylnaphthalene, isethionic acid, lactic acid, lactose, dodecylsulfonic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, nitric acid, oxalic acid, Pamoic acid, pantothenic acid, phenylacetic acid, phosphoric acid, polygalacturonic acid, propionic acid, salicylic acid,
  • the pharmaceutically acceptable salt of the present invention can be synthesized from the parent compound containing acid or base by conventional chemical methods. Generally, such salts are prepared by reacting these compounds in free acid or base form with a stoichiometric amount of appropriate base or acid in water or an organic solvent or a mixture of both. Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.
  • the compounds provided by the present invention also exist in prodrug forms.
  • the prodrugs of the compounds described herein easily undergo chemical changes under physiological conditions to transform into the compounds of the invention.
  • prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in the in vivo environment.
  • Certain compounds of the present invention may exist in unsolvated or solvated forms, including hydrated forms.
  • the solvated form is equivalent to the unsolvated form, and both are included in the scope of the present invention.
  • Certain compounds of the present invention may have asymmetric carbon atoms (optical centers) or double bonds. Racemates, diastereomers, geometric isomers and individual isomers are all included in the scope of the present invention.
  • enantiomer or “optical isomer” refers to stereoisomers that are mirror images of each other.
  • cis-trans isomer or “geometric isomer” is caused by the inability to rotate freely because of double bonds or single bonds of ring-forming carbon atoms.
  • diastereomer refers to a stereoisomer in which the molecule has two or more chiral centers and the relationship between the molecules is non-mirror-image relationship.
  • the compound of the present invention may be specific.
  • tautomer or “tautomeric form” means that at room temperature, the isomers of different functional groups are in dynamic equilibrium and can be transformed into each other quickly. If tautomers are possible (such as in solution), the chemical equilibrium of tautomers can be reached.
  • proton tautomer also called prototropic tautomer
  • proton migration such as keto-enol isomerization and imine-ene Amine isomerization.
  • Valence isomers include some recombination of bonding electrons to carry out mutual transformations.
  • keto-enol tautomerization is the tautomerism between two tautomers of pentane-2,4-dione and 4-hydroxypent-3-en-2-one.
  • the term “enriched in one isomer”, “enriched in isomers”, “enriched in one enantiomer” or “enriched in enantiomers” refers to one of the isomers or pairs of
  • the content of the enantiomer is less than 100%, and the content of the isomer or enantiomer is greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or 96% or greater, or 97% or greater, or 98% or greater, or 99% or greater, or 99.5% or greater, or 99.6% or greater, or 99.7% or greater, or 99.8% or greater, or greater than or equal 99.9%.
  • the term “isomer excess” or “enantiomeric excess” refers to the difference between the relative percentages of two isomers or two enantiomers. For example, if the content of one isomer or enantiomer is 90%, and the content of the other isomer or enantiomer is 10%, the isomer or enantiomer excess (ee value) is 80% .
  • optically active (R)- and (S)-isomers and D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If you want to obtain an enantiomer of a compound of the present invention, it can be prepared by asymmetric synthesis or derivatization with chiral auxiliary agents, in which the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide pure The desired enantiomer.
  • the molecule when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), it forms a diastereomeric salt with an appropriate optically active acid or base, and then passes through a conventional method known in the art The diastereoisomers are resolved, and then the pure enantiomers are recovered.
  • the separation of enantiomers and diastereomers is usually accomplished through the use of chromatography, which uses a chiral stationary phase and is optionally combined with chemical derivatization (for example, the formation of amino groups from amines). Formate).
  • the compound of the present invention may contain unnatural proportions of atomic isotopes on one or more of the atoms constituting the compound.
  • compounds can be labeled with radioisotopes, such as tritium ( 3 H), iodine-125 ( 125 I), or C-14 ( 14 C).
  • deuterium can be substituted for hydrogen to form deuterated drugs.
  • the bond formed by deuterium and carbon is stronger than the bond formed by ordinary hydrogen and carbon.
  • deuterated drugs can reduce toxic side effects and increase drug stability. , Enhance the efficacy, extend the biological half-life of drugs and other advantages. All changes in the isotopic composition of the compounds of the present invention, whether radioactive or not, are included in the scope of the present invention.
  • treatment includes the effect that occurs when a subject suffers from a specific disease, disorder, or condition, which reduces the severity of the disease, disorder, or condition, or delays or slows the disease, disorder Or the development of a condition ("therapeutic treatment"), and also includes effects that occur before the subject begins to suffer from a specific disease, disorder, or condition ("prophylactic treatment").
  • the HDAC inhibitor described herein may be any HDAC inhibitor. Therefore, HDAC inhibitors can be selective or non-selective for specific types of histone deacetylases.
  • the HDAC inhibitor is a selective HDAC inhibitor. More preferably, the HDAC inhibitor is an HDAC6 inhibitor.
  • the HDAC6 inhibitors that can be used in the combination of the present invention have been described generally and specifically in the published PCT patent application WO2018/130155, which is incorporated herein by reference.
  • the HDAC6 inhibitor described herein is a compound represented by formula (I) and formula (II), or a pharmaceutically acceptable compound thereof Salt or enantiomer, diastereomer, racemate, solvate, hydrate, polymorph, prodrug or active metabolite,
  • E 1 and E 2 are selected from -O-, -CH 2 -and -CH 2 -CH 2 -;
  • R 2 is selected from: H, F, Cl, Br, I;
  • R is selected from: F, Cl, Br, I;
  • n is selected from: 0 or 1.
  • the selective HDAC6 inhibitor is the following compound or its pharmaceutically acceptable salt or enantiomer, diastereomer, racemate, solvate, hydrate, polymorph, Prodrugs or active metabolites:
  • the selective HDAC6 inhibitor is a compound of the following formula A, or a pharmaceutically acceptable salt or enantiomer, diastereomer, racemate, solvate, or hydrate thereof , Polymorphs, prodrugs or active metabolites:
  • the PD-1 axis binding antagonist described herein is selected from the group consisting of PD-1 binding antagonist, PD-L1 binding antagonist and PD -L2 binding antagonist; more preferably, the PD-1 axis binding antagonist is an antibody or an antigen-binding fragment thereof; especially preferably, the antibody is an anti-PD-1 antibody; most preferably, the antibody is a monoclonal antibody.
  • the antibodies or antigen-binding fragments thereof that can be used in the combination of the present invention are generally and specifically described in WO2018/53709 and CN107840887, which application is incorporated herein by reference.
  • the anti-PD-1 antibody or antigen-binding fragment thereof binds to an epitope of PD-1, the epitope comprising: SEQ ID NO: No. 128, 129, 130, 131 on 24 And the amino acid at position 132 and at least one amino acid at positions 35, 64, 82, and 83.
  • the anti-PD-1 antibody or antigen-binding fragment thereof binds to an epitope of human PD-1 and murine PD-1, wherein the epitope includes SEQ ID NO: 24 Amino acids at positions 128, 129, 130, 131 and 132.
  • the aforementioned antibody or antigen-binding fragment thereof has at least one of the following properties:
  • the above antibody or antigen-binding fragment thereof comprises an amino acid sequence which is selected from SEQ ID NOs: 1, 2
  • the sequences in the group consisting of 3, 4, 5, 6, 7, 8 and 9 have at least 70%, 80%, 90% or 95% homology, wherein the antibody specifically binds to PD-1.
  • the above-mentioned antibody or antigen-binding fragment thereof comprises an amino acid sequence selected from SEQ ID NOs: 1, 2, Sequences in the group consisting of 3, 4, 5, 6, 7, 8, and 9, wherein the antibody specifically binds to PD-1.
  • use, combination, composition, drug, preparation or kit, the above-mentioned antibody, or antigen-binding fragment thereof comprises:
  • the heavy chain variable region whose amino acid sequence has at least 70%, 80%, 90% or 95% homology with a sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2; as well as
  • the light chain variable region whose amino acid sequence is at least 70%, 80%, 90% or 95% of the sequence selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8 and 9 % Homology,
  • the antibody specifically binds PD-1.
  • use, combination, composition, drug, preparation or kit, the above-mentioned antibody or antigen-binding fragment thereof comprises:
  • the antibody specifically binds PD-1.
  • the antibody comprises:
  • the antibody specifically binds PD-1.
  • the antibody comprises:
  • the antibody specifically binds PD-1.
  • the antibody comprises:
  • the antibody specifically binds PD-1.
  • the antibody comprises:
  • the antibody specifically binds PD-1.
  • the antibody comprises:
  • the antibody specifically binds PD-1.
  • the antibody comprises:
  • the antibody specifically binds PD-1.
  • the antibody comprises:
  • the antibody specifically binds PD-1.
  • the antibody comprises:
  • the antibody specifically binds PD-1.
  • the antibody comprises:
  • the antibody specifically binds PD-1.
  • the antibody comprises:
  • the antibody specifically binds PD-1.
  • the above-mentioned antibody or antigen-binding fragment thereof comprises a complementarity determining region (CDR), which
  • amino acid sequence is selected from the sequence in the group consisting of SEQ ID Nos: 10-23,
  • the antibody specifically binds PD-1.
  • the above-mentioned antibody or antigen-binding fragment thereof comprises: CDR1, CDR2 and
  • the CDR3 sequence of the heavy chain variable region contains a sequence selected from the group consisting of SEQ ID NO: 12 and SEQ ID NO: 13.
  • the antibody specifically binds PD-1.
  • the CDR3 sequence of the light chain variable region of the above antibody preferably comprises a sequence selected from the group consisting of SEQ ID NOs: 20, 21, 22, and 23
  • the CDR2 sequence of the heavy chain variable region of the above antibody preferably comprises a sequence selected from the group consisting of SEQ ID NO: 11
  • the CDR2 sequence of the light chain variable region of the above-mentioned antibody preferably comprises a sequence selected from the group consisting of SEQ ID NO: 19.
  • the CDR1 sequence of the heavy chain variable region of the above antibody preferably comprises a sequence selected from the group consisting of SEQ ID NO: 10
  • the CDR1 sequence of the light chain variable region of the above antibody preferably comprises a sequence selected from SEQ ID NOs: 14, 15, 16, 17 and 18.
  • the antibody or antigen-binding fragment thereof comprises:
  • a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences
  • a light chain variable region comprising CDR1, CDR2 and CDR3 sequences, wherein
  • the heavy chain variable region CDR1 includes an amino acid sequence selected from SEQ ID NO: 10,
  • Heavy chain variable region CDR2 the sequence includes an amino acid sequence selected from SEQ ID NO: 11,
  • Heavy chain variable region CDR3 the sequence contains the amino acid sequence selected from the group consisting of SEQ ID NO: 12 and SEQ ID NO: 13,
  • the light chain variable region CDR1 includes an amino acid sequence selected from the group consisting of SEQ ID NO: 14-18,
  • the light chain variable region CDR2 includes an amino acid sequence selected from SEQ ID NO: 19,
  • the sequence includes an amino acid sequence selected from the group consisting of SEQ ID NO: 20-23,
  • the antibody specifically binds PD-1.
  • the antibody comprises:
  • the heavy chain variable region CDR1 includes an amino acid sequence selected from SEQ ID NO: 10,
  • Heavy chain variable region CDR2 the sequence includes an amino acid sequence selected from SEQ ID NO: 11,
  • the heavy chain variable region CDR3 contains an amino acid sequence selected from SEQ ID NO: 12,
  • the light chain variable region CDR1 includes an amino acid sequence selected from SEQ ID NO: 14,
  • the light chain variable region CDR2 includes an amino acid sequence selected from SEQ ID NO: 19,
  • CDR3 of the light chain variable region includes an amino acid sequence selected from SEQ ID NO: 20,
  • the antibody specifically binds PD-1.
  • the antibody comprises:
  • the heavy chain variable region CDR1 includes an amino acid sequence selected from SEQ ID NO: 10,
  • Heavy chain variable region CDR2 the sequence includes an amino acid sequence selected from SEQ ID NO: 11,
  • the heavy chain variable region CDR3 contains an amino acid sequence selected from SEQ ID NO: 13,
  • the light chain variable region CDR1 includes an amino acid sequence selected from SEQ ID NO: 14,
  • the light chain variable region CDR2 includes an amino acid sequence selected from SEQ ID NO: 19,
  • the light chain variable region CDR3 includes an amino acid sequence selected from SEQ ID NO: 21,
  • the antibody specifically binds PD-1.
  • the antibody comprises:
  • the heavy chain variable region CDR1 includes an amino acid sequence selected from SEQ ID NO: 10,
  • Heavy chain variable region CDR2 the sequence includes an amino acid sequence selected from SEQ ID NO: 11,
  • the heavy chain variable region CDR3 contains an amino acid sequence selected from SEQ ID NO: 13,
  • the light chain variable region CDR1 includes an amino acid sequence selected from SEQ ID NO: 15,
  • the light chain variable region CDR2 includes an amino acid sequence selected from SEQ ID NO: 19,
  • the light chain variable region CDR3 includes an amino acid sequence selected from SEQ ID NO: 21,
  • the antibody specifically binds PD-1.
  • the antibody comprises:
  • the heavy chain variable region CDR1 includes an amino acid sequence selected from SEQ ID NO: 10,
  • Heavy chain variable region CDR2 the sequence includes an amino acid sequence selected from SEQ ID NO: 11,
  • the heavy chain variable region CDR3 contains an amino acid sequence selected from SEQ ID NO: 13,
  • the light chain variable region CDR1 includes an amino acid sequence selected from SEQ ID NO: 16,
  • the light chain variable region CDR2 includes an amino acid sequence selected from SEQ ID NO: 19,
  • the light chain variable region CDR3 includes an amino acid sequence selected from SEQ ID NO: 21,
  • the antibody specifically binds PD-1.
  • the antibody comprises:
  • the heavy chain variable region CDR1 includes an amino acid sequence selected from SEQ ID NO: 10,
  • Heavy chain variable region CDR2 the sequence includes an amino acid sequence selected from SEQ ID NO: 11,
  • the heavy chain variable region CDR3 contains an amino acid sequence selected from SEQ ID NO: 12,
  • the light chain variable region CDR1 includes an amino acid sequence selected from SEQ ID NO: 17,
  • the light chain variable region CDR2 includes an amino acid sequence selected from SEQ ID NO: 19,
  • the light chain variable region CDR3 includes an amino acid sequence selected from SEQ ID NO: 21,
  • the antibody specifically binds PD-1.
  • the antibody comprises:
  • the heavy chain variable region CDR1 includes an amino acid sequence selected from SEQ ID NO: 10,
  • Heavy chain variable region CDR2 the sequence includes an amino acid sequence selected from SEQ ID NO: 11,
  • the heavy chain variable region CDR3 contains an amino acid sequence selected from SEQ ID NO: 12,
  • the light chain variable region CDR1 includes an amino acid sequence selected from SEQ ID NO: 16,
  • the light chain variable region CDR2 includes an amino acid sequence selected from SEQ ID NO: 19,
  • the light chain variable region CDR3 includes an amino acid sequence selected from SEQ ID NO: 21,
  • the antibody specifically binds PD-1.
  • the antibody comprises:
  • the heavy chain variable region CDR1 includes an amino acid sequence selected from SEQ ID NO: 10,
  • Heavy chain variable region CDR2 the sequence includes an amino acid sequence selected from SEQ ID NO: 11,
  • the heavy chain variable region CDR3 contains an amino acid sequence selected from SEQ ID NO: 13,
  • the light chain variable region CDR1 includes an amino acid sequence selected from SEQ ID NO: 17,
  • the light chain variable region CDR2 includes an amino acid sequence selected from SEQ ID NO: 19,
  • the light chain variable region CDR3 includes an amino acid sequence selected from SEQ ID NO: 21,
  • the antibody specifically binds PD-1.
  • the antibody comprises:
  • the heavy chain variable region CDR1 includes an amino acid sequence selected from SEQ ID NO: 10,
  • Heavy chain variable region CDR2 the sequence includes an amino acid sequence selected from SEQ ID NO: 11,
  • the heavy chain variable region CDR3 contains an amino acid sequence selected from SEQ ID NO: 13,
  • the light chain variable region CDR1 includes an amino acid sequence selected from SEQ ID NO: 17,
  • the light chain variable region CDR2 includes an amino acid sequence selected from SEQ ID NO: 19,
  • the light chain variable region CDR3 includes an amino acid sequence selected from SEQ ID NO: 22,
  • the antibody specifically binds PD-1.
  • the antibody comprises:
  • the heavy chain variable region CDR1 includes an amino acid sequence selected from SEQ ID NO: 10,
  • Heavy chain variable region CDR2 the sequence includes an amino acid sequence selected from SEQ ID NO: 11,
  • the heavy chain variable region CDR3 contains an amino acid sequence selected from SEQ ID NO: 12,
  • the light chain variable region CDR1 includes an amino acid sequence selected from SEQ ID NO: 18,
  • the light chain variable region CDR2 includes an amino acid sequence selected from SEQ ID NO: 19,
  • CDR3 of the light chain variable region includes an amino acid sequence selected from SEQ ID NO: 23,
  • the antibody specifically binds PD-1.
  • the antibody comprises:
  • the heavy chain variable region CDR1 includes an amino acid sequence selected from SEQ ID NO: 10,
  • Heavy chain variable region CDR2 the sequence includes an amino acid sequence selected from SEQ ID NO: 11,
  • the heavy chain variable region CDR3 contains an amino acid sequence selected from SEQ ID NO: 12,
  • the light chain variable region CDR1 includes an amino acid sequence selected from SEQ ID NO: 18,
  • the light chain variable region CDR2 includes an amino acid sequence selected from SEQ ID NO: 19,
  • CDR3 of the light chain variable region includes an amino acid sequence selected from SEQ ID NO: 20,
  • the antibody specifically binds PD-1.
  • the antibody is a chimeric antibody or a humanized antibody or a human antibody.
  • the antibody exhibits at least one of the following properties:
  • the KD that binds to human PD-1 is 2.15E-10M or less, and the KD that binds to mouse PD-1 is 1.67E-08M or less;
  • the above proteasome inhibitor is bortezomib, carfilzomib, ixazomib or marezomib, preferably ixazomib Meter.
  • the present invention relates to a treatment method for treating a condition of a subject that benefits from modulating immune function, which comprises administering to the subject a pharmaceutically effective amount of an HDAC6 selective inhibitor.
  • the present invention also relates to a combination therapy, which comprises administering to the subject a pharmaceutically effective amount of an HDAC6 selective inhibitor and one or more therapeutic agents.
  • the present invention also provides a therapeutic combination comprising: (1) a selective inhibitor of HDAC6, and (2) one or more therapeutic agents; it can be used to separately, simultaneously or sequentially administer to individuals in need for treatment Or prevent the subject's conditions that benefit from modulating immune function.
  • the present invention relates to a method for treating a condition in a subject that benefits from modulating immune function, which comprises administering to the subject a pharmaceutically effective amount of a selective HDAC6 inhibitor and one or more treatments Agent.
  • the present invention also relates to the use of a combination of a selective HDAC6 inhibitor and one or more therapeutic agents in the preparation of a medicament for the treatment of a condition in a subject that benefits from modulating immune function.
  • the present invention relates to the use of a selective HDAC6 inhibitor in the preparation of a medicament for the treatment of a condition in a subject benefiting from modulating immune function, the selective HDAC6 inhibitor and one or more therapeutic agents combination.
  • the present invention relates to the use of one or more therapeutic agents in the preparation of a medicament for the treatment of a condition in a subject benefiting from modulating immune function, the one or more therapeutic agents and the selectivity HDAC6 inhibitor combination.
  • the selective HDAC6 inhibitor and one or more therapeutic agents are administered to the subject simultaneously, separately or sequentially.
  • the selective HDAC6 inhibitor is a compound represented by formula (I) and formula (II), or a pharmaceutically acceptable salt or enantiomer or diastereomer Constructs, racemates, solvates, hydrates, polymorphs, prodrugs or active metabolites,
  • E 1 and E 2 are selected from -O-, -CH 2 -and -CH 2 -CH 2 -;
  • R2 is selected from: H, F, Cl, Br, I;
  • R is selected from: F, Cl, Br, I;
  • n is selected from: 0 or 1.
  • the selective HDAC6 inhibitor is the following compound or its pharmaceutically acceptable salt or enantiomer, diastereomer, racemate, solvate, hydrate, polymorph, Prodrugs or active metabolites:
  • the selective HDAC6 inhibitor is a compound of formula A or a pharmaceutically acceptable salt or enantiomer, diastereomer, racemate, solvate, hydrate, poly Crystal form, prodrug or active metabolite:
  • the one or more therapeutic agents are (1) PD-1 axis binding antagonists or (2) proteasome inhibitors.
  • the proteasome inhibitor is bortezomib, carfilzomib, ixazomib or marezomib.
  • the above-mentioned PD-1 axis binding antagonist is selected from PD-1 binding antagonists; preferably, the PD-1 axis binding antagonist is an antibody or an antigen-binding fragment thereof; particularly preferably, the antibody is an anti-PD -1 antibody; most preferably, the antibody is a monoclonal antibody.
  • Any PD-1 axis binding antagonist described herein can be used in the method or use of the present invention.
  • the antibody or antigen-binding fragment thereof binds to an epitope of PD-1, and the epitope comprises: SEQ ID NO: 24 amino acids at positions 128, 129, 130, 131 and 132 and At least one amino acid in positions 35, 64, 82, and 83;
  • the antibody or antigen-binding fragment thereof binds to an epitope of human PD-1 and murine PD-1, wherein the epitope includes SEQ ID NO: 128, 129, 130, 131, and 132 on 24 Site amino acid.
  • the antibody or antigen-binding fragment thereof comprises an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, and 9.
  • the sequences in the group have at least 70%, 80%, 90% or 95% homology, wherein the antibody specifically binds to PD-1; preferably, the antibody or antigen-binding fragment thereof contains an amino acid sequence, so The amino acid sequence is selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, and 9.
  • the antibody or antigen-binding fragment thereof comprises:
  • the heavy chain variable region which has an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2, or has at least 70%, 80%, 90%, or 95% identity with it. Source sequence; and
  • the light chain variable region which has an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8 and 9, or at least 70%, 80%, 90% of the sequence. % Or 95% sequence homology,
  • the antibody specifically binds PD-1.
  • the antibody or antigen-binding fragment thereof comprises a specific sequence of the heavy chain and light chain selected from the antibody described in Table 1.
  • the antibody or antigen-binding fragment thereof comprises:
  • the antibody or antigen-binding fragment thereof comprises a complementarity determining region (CDR), which has an amino acid sequence selected from the group consisting of SEQ ID NOs: 10-23, wherein the antibody is specific Binding PD-1;
  • CDR complementarity determining region
  • the antibody or antigen-binding fragment thereof comprises a complementarity determining region (CDR), which comprises a CDR sequence selected from Table 2;
  • CDR complementarity determining region
  • the antibody comprises:
  • the heavy chain variable region CDR1 includes an amino acid sequence selected from SEQ ID NO: 10,
  • Heavy chain variable region CDR2 the sequence includes an amino acid sequence selected from SEQ ID NO: 11,
  • the heavy chain variable region CDR3 contains an amino acid sequence selected from SEQ ID NO: 13,
  • the light chain variable region CDR1 includes an amino acid sequence selected from SEQ ID NO: 14,
  • the light chain variable region CDR2 includes an amino acid sequence selected from SEQ ID NO: 19,
  • the light chain variable region CDR3 includes an amino acid sequence selected from SEQ ID NO: 21.
  • the one or more therapeutic agents are proteasome inhibitors, preferably bortezomib, carfilzomib, ixazomib or marezomib.
  • the selective HDAC6 inhibitor is compound A of the following formula or enantiomers, diastereomers, racemates, solvates, hydrates, polymorphs, prodrugs Or active metabolite:
  • the one or more therapeutic agents are antibody 2E5 or antigen-binding fragments thereof, comprising:
  • the antibody specifically binds PD-1.
  • the selective HDAC6 inhibitor is compound A of the following formula or enantiomers, diastereomers, racemates, solvates, hydrates, polymorphs, prodrugs Or active metabolite:
  • the one or more therapeutic agents are proteasome inhibitors, preferably bortezomib, carfilzomib, ixazomib or marezomib.
  • the methods or uses of the present invention can be used to treat or prevent immune disorders or disorders of subjects that benefit from modulating immune function, such as cancer.
  • the method or use of the present invention is also used to inhibit the growth of tumor cells in a subject.
  • the above-mentioned tumor cells are selected from multiple myeloma (MM), melanoma, kidney cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer. Head cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer and rectal cancer.
  • the cancer is a solid tumor or blood cancer, such as non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, Esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycotic fungus, Merkel cell Cancer and other hematological malignancies, such as classical Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell/histiocytosis-rich B-cell lymphoma, EBV positive and negative PTLD, and EBV Related diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T cell lymphoma
  • CHL Hodgkin lymph
  • the methods described herein can also be used to treat conditions that require enhanced immunogenicity, such as increasing tumor immunogenicity for the treatment of cancer.
  • a method for enhancing immune function in a subject suffering from cancer which comprises administering to the subject an effective amount of a selective HDAC6 inhibitor and an anti-PD-1 antibody or antigen-binding fragment thereof.
  • Also provided herein is a method for treating or delaying the development of cancer in a subject, comprising administering to the individual an effective amount of a selective HDAC6 inhibitor and an anti-PD-1 antibody or antigen-binding fragment thereof; or administering an effective amount of a selective HDAC6 Inhibitors and bortezomib, carfilzomib, ixazomib or marezomib.
  • the subject has been treated with the selective HDAC6 inhibitor before the combination treatment with the selective HDAC6 inhibitor and the anti-PD-1 antibody or antigen-binding fragment thereof.
  • the subject has a cancer that is resistant to HDAC6 inhibitors (e.g., drug-resistant multiple myeloma).
  • resistance to HDAC6 inhibitors includes recurrence of cancer or refractory cancer. Recurrence may refer to the recurrence of cancer in the original site or a new site after treatment.
  • resistance to HDAC6 inhibitors includes the progression of cancer during treatment with the HDAC6 inhibitor.
  • resistance to HDAC6 inhibitors includes cancers that do not respond to treatment. Cancer may develop resistance at the beginning of treatment or become resistant during treatment. In some embodiments, the cancer is early or late.
  • any of the methods described herein can be tested in various models known in the art (e.g., a combination therapy comprising administering an effective amount of a selective HDAC6 inhibitor and a combination of an anti-PD-1 antibody or antigen-binding fragment thereof) , Such as clinical or preclinical clinical models.
  • An effective amount of the above-mentioned selective HDAC6 inhibitor (e.g. Compound A) and anti-PD-1 antibody or its antigen-binding fragment (e.g. antibody 2E5) can be administered; or a selective HDAC6 inhibitor (e.g. Compound A) and protease Body inhibitors (for example, bortezomib, carfilzomib, ixazomib, or marezomib) to prevent or treat diseases.
  • a selective HDAC6 inhibitor e.g. Compound A
  • protease Body inhibitors for example, bortezomib, carfilzomib, ixazomib, or marezomib
  • the appropriate dosage of the above-mentioned selective HDAC6 inhibitor, anti-PD-1 antibody or its antigen-binding fragment and proteasome inhibitor can be selected according to the type of disease to be treated, the selective HDAC6 inhibitor, anti-PD-1 antibody or its The type of antigen-binding fragment and proteasome inhibitor, the severity and course of the disease, the severity of the treatment, the individual's clinical symptoms, the individual's clinical history and response to treatment are determined.
  • the combination therapy with the above-mentioned selective HDAC6 inhibitor and anti-PD-1 antibody or antigen-binding fragment thereof, or the combination therapy with the selective HDAC6 inhibitor and proteasome inhibitor is synergistic, Therefore, relative to the effective dose of a selective HDAC6 inhibitor or anti-PD-1 antibody (or an antigen-binding fragment thereof) or a proteasome inhibitor alone, the effective dose in the combination is reduced.
  • the therapeutically effective amount of the PD-1 antibody or antigen-binding fragment thereof provided herein to humans is about 0.0001 mg/kg to about 100 mg/kg (e.g., about 0.01 mg/kg, about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg) of the patient's body weight, whether it is administered once or multiple times.
  • the antibody or antigen-binding fragment thereof used is about 0.01 mg/kg, about 0.1 mg/kg, about 0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg, about 0.6 mg/kg.
  • the antibody or antigen-binding fragment thereof is administered at a dose of /kg.
  • the PD-1 antibody or antigen-binding fragment thereof is administered in a unit dose of about 1 mg to about 100 mg, preferably in a unit dose of about 5 mg, about 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, and 50 mg.
  • the appropriate dose of the selective HDAC6 inhibitor (such as Compound A) described above should be the amount of the compound that is the lowest dose effective to produce a therapeutic effect.
  • Such effective doses will generally depend on the aforementioned factors.
  • the dose range of the compound of the present invention to a patient is about 0.0001 to about 100 mg per kilogram of body weight, preferably about 1 mg/kg, about 2 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg .
  • the dosage may be between about 10 to 2000 mg; alternatively, the dosage may be about 100 to 1000 mg per day, or about 200 to about 600 mg per day.
  • the effective daily dose of the active compound may be one, two, three, four or more sub-doses administered in unit dosage form at appropriate intervals throughout the day.
  • the unit dose is about 10 to 1000 mg, more preferably about 100 to 1000 mg, about 150 to about 600 mg, about 200 to about 500 mg, or about 300 to about 400 mg, particularly preferably about 10 mg, about 50 mg, about 100 mg, about 150 mg , About 200mg, about 250mg, about 300mg, about 350mg, about 400mg, about 450mg, about 500mg.
  • the appropriate dose of the protease inhibitors described above should be the amount of the compound that is the lowest dose effective to produce a therapeutic effect.
  • Such effective doses will generally depend on the aforementioned factors.
  • the dose range of the compound of the present invention to a patient is about 0.0001 to about 100 mg per kilogram of body weight, preferably about 0.01 mg/kg, about 0.1 mg/kg, about 0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg.
  • the dosage can be between about 0.1 to 1000 mg.
  • the dosage may be about 1 to 500 mg, about 5 to 200 mg, about 10 to 50 mg, or preferably about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40mg, 45mg, or 50mg.
  • the effective daily dose of the active compound can be administered in unit dosage form in one, two, three, four or more sub-doses at appropriate intervals throughout the day.
  • the unit dose is about 1 to 500 mg, about 5 to 200 m, about 10 to 50 mg, or preferably about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, About 30 mg, about 35 mg, about 40 mg, about 45 mg, or about 50 mg.
  • the administered dose can be changed during the course of treatment.
  • the initial administered dose may be higher than the subsequent administered dose.
  • the dose administered may vary during the course of treatment.
  • the above-mentioned selective HDAC6 inhibitors or one or more therapeutic agents can be administered at specific doses at multiple intervals, for example, once a day, twice a day or more; once a week, twice a week or more; once a month, Two or more times; once a week, once every two weeks, once every three weeks, once a month or once every two months, or less.
  • the dose administered can vary with the course of treatment. For example, in certain embodiments, the initial dose administered may be higher than subsequent doses.
  • the dose administered is adjusted during the course of treatment based on the response of the subject to be treated.
  • the dosage regimen can be adjusted to achieve an optimal response (e.g., therapeutic response). For example, a single dose or multiple divided doses can be administered over a period of time.
  • the combination therapy of the present invention includes administration of the selective HDAC6 inhibitor described above and one or more therapeutic agents (e.g., an anti-PD-1 antibody or an antigen-binding fragment thereof or a proteasome inhibitor).
  • the above-mentioned selective HDAC6 inhibitors and one or more therapeutic agents can be administered in any suitable manner known in the art.
  • the above-mentioned selective HDAC6 inhibitor and one or more therapeutic agents can be administered sequentially (at different times) or simultaneously. Medicine (at the same time).
  • the selective HDAC6 inhibitor and one or more therapeutic agents are in the form of separate preparations; in some embodiments, the selective HDAC6 inhibitor and one or more therapeutic agents (for example, an anti-PD-1 antibody or an antigen-binding fragment thereof or a proteasome inhibitor) are present in the same composition or formulation.
  • the above-mentioned selective HDAC6 inhibitor and one or more therapeutic agents can be administered through the same route of administration or different routes of administration.
  • Administration e.g., anti-PD-1 antibody or its antigen-binding fragment or proteasome inhibitor
  • the PD-1 antibody or antigen-binding fragment thereof is implanted, by inhalation, intrathecal, intraventricular or intranasal intravenous, intramuscular, subcutaneous, topical, oral, transdermal, intraperitoneal, or intraorbital , Oral administration, preferably intravenous administration.
  • the selective HDAC6 inhibitors described above are administered by implantation, inhalation, intrathecal, intraventricular or intranasal intravenous, intramuscular, subcutaneous, topical, oral, transdermal, intraperitoneal, or intraorbital , Oral administration, preferably oral administration.
  • each therapeutic agent in the combination therapy of the present invention can be simultaneously (i.e., in the same drug), simultaneously (i.e., in separate drugs administered one after the other in any order), or in any Sequential administration.
  • the therapeutic agents in the combination therapy are in different dosage forms (one drug is a tablet or capsule, the other drug is a sterile liquid) and/or are administered according to different dosing schedules (such as chemotherapy drugs), they are given sequentially Medicine is particularly useful.
  • Biotherapeutics that are administered at least once a day, and less frequently, for example, once a week, once every two weeks, or once every three weeks.
  • the therapeutic agent for example, anti-PD-1 antibody or its antigen-binding fragment or proteasome inhibitor
  • the selective HDAC6 inhibitor provide a synergistic combination, and the ratio of the combination range is 1: It is preferably between 200 and 200:1 parts by weight, which can be about 1:100, about 1:95, about 1:90, about 1:85, about 1:80, about 1:75, about 1:70, About 1:65, about 1:60, about 1:55, about 1:50, about 1:45, about 1:40, about 1:35, about 1:30, about 1:25, about 1:20, About 1:15, about 1:10, about 1:8, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2 or about 1:1, about 2:1 About 3:1, about 4:1, about 5:1, about 6:1, about 10:1, about 20:1, about 30:1, about 40:1, about 50:1, about 60:1, About 70:1, about 80:1, about 90:1, about 100:1.
  • the method may further include additional therapies.
  • Additional therapy can be radiation therapy, surgery (e.g., mastectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or the foregoing The combination. Additional therapy can take the form of adjuvant or neoadjuvant therapy.
  • the additional therapy is the administration of small molecule enzyme inhibitors or anti-metastatic agents.
  • the additional therapy is the administration of a side effect limiting agent (e.g., an agent intended to reduce the occurrence and/or severity of side effects of the treatment, such as an anti-nausea agent, etc.).
  • the additional therapy is radiation therapy.
  • the additional therapy is surgery.
  • the additional therapy is a combination of radiation therapy and surgery.
  • the additional therapy is gamma radiation.
  • the combination therapy of the present invention can be used to remove tumors before or after surgery, and can be used before, during or after radiotherapy.
  • the combination therapy of the present invention is administered to patients who have not been previously treated with biotherapeutics or chemotherapeutics.
  • the combination therapy is administered to patients who have failed to achieve a sustained response after previous treatment with a biotherapeutic agent or chemotherapeutic agent (ie, experienced treatment).
  • the present invention provides a composition comprising a selective HDAC6 inhibitor as described above (e.g. Compound A) and an anti-PD-1 antibody or antigen-binding fragment thereof (e.g., antibody 2E5) and optionally a pharmaceutically acceptable The excipient or carrier.
  • a composition comprising the selective HDAC6 inhibitor (e.g. Compound A) as described above and a proteasome inhibitor (e.g. bortezomib, carfilzomib, ixazomib or marezomib) ) And optional pharmaceutically acceptable excipients or carriers.
  • the pharmaceutically acceptable excipients or carriers used in the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquid, gel or solid carriers, aqueous media, non-aqueous media, antimicrobial materials, penetrating materials, Buffers, antioxidants, anesthetics, suspending/dispersing agents, chelating agents, diluents, adjuvants, agents or non-toxic auxiliary substances, other components known in the art or various combinations of the above.
  • Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickeners, colorants, emulsifiers or stabilizers, such as sugar and Cyclodextrin.
  • Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, mercaptoglycolic acid, sorbitol, butyl fennel Ether, butylated hydroxytoluene and/or propyl gallate.
  • pharmaceutically acceptable excipients or carriers may include, for example, aqueous vehicles, such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's Injections, non-aqueous vehicles such as fixed oils.
  • aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's Injections
  • non-aqueous vehicles such as fixed oils.
  • Plant origin cottonseed oil, corn oil, sesame oil or peanut oil, antibacterial or antibacterial concentration of antibacterial agent, isotonic agent (such as sodium chloride or glucose), buffer (such as phosphate or citrate buffer), antibacterial agent Oxidizing agents (such as sodium bisulfate), local anesthetics as procaine hydrochloride, suspending agents and dispersing agents, such as sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose or polyvinylpyrrolidone, emulsifiers, such as polysorbate 80 (TWEEN-80), chelating agent or chelating agent, such as EDTA (ethylenediamine tetraacetic acid) or EGTA (ethylene glycol tetraacetic acid), ethanol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid Or lactic acid.
  • isotonic agent such as sodium chloride or glucose
  • buffer such as phosphate or citrate buffer
  • Antimicrobial agents that can be used as carriers can be added to the pharmaceutical composition in a multi-dose container that includes phenol or cresol, mercury, benzyl alcohol, chlorobutanol, methylparaben and propylparaben Formate, thimerosal, benzalkonium chloride and benzethonium chloride.
  • Suitable excipients may include, for example, water, saline, dextrose, glycerol, or ethanol.
  • Suitable non-toxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, or such as sodium acetate, sorbitan monolaurate, triethanolamine oleate or cyclic Dextrin reagent.
  • the pharmaceutical composition can be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained-release preparation or powder.
  • Oral preparations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, magnesium carbonate and the like.
  • the pharmaceutical composition is formulated as an injectable composition.
  • the pharmaceutical composition for injection can be prepared in any conventional form, such as a liquid solvent, suspension, emulsifier or solid form suitable for the production of liquid solvent, suspension or emulsifier.
  • the present invention also provides a medicament, which comprises the selective HDAC6 inhibitor as described above and an anti-PD-1 antibody or an antigen-binding fragment thereof and optionally a pharmaceutically acceptable excipient or carrier.
  • the present invention also provides a medicament comprising the selective HDAC6 inhibitor and proteasome inhibitor (bortezomib, carfilzomib, ixazomib or marezomib) as described above and optionally a pharmaceutical Acceptable excipients or carriers.
  • the drug comprising the above-mentioned selective HDAC6 inhibitor and anti-PD-1 antibody or antigen-binding fragment thereof can be provided in the form of a liquid formulation or by reconstitution with sterile water and water for injection before use. Prepared as a dry powder.
  • the selective HDAC6 inhibitors and anti-PD-1 antibodies or antigen-binding fragments thereof described herein can be provided as a product or a kit, which includes a first container and a second container and a package insert.
  • the first container contains at least one dose of the drug containing the PD-1 axis binding antagonist
  • the second container contains at least one dose of the drug containing fexofitinib
  • the product or kit further includes a package insert containing a selective HDAC6 inhibitor and an anti-PD-1 antibody or antigen-binding fragment thereof (or the selective HDAC6 inhibitor and Instructions for the combination of bortezomib, carfilzomib, ixazomib, or marezomib) to treat or delay the progression of an individual's cancer or enhance the immune function of an individual with cancer.
  • the first and second containers may be composed of the same or different shapes (e.g., vials, syringes, and bottles) and/or materials (e.g., plastic or glass).
  • the article or kit may also contain other materials that can be used to administer the drug, such as diluents, filters, IV bags and tubes, needles, and syringes.
  • the preparation also includes one or more other agents (e.g., chemotherapeutic agents and anti-tumor agents).
  • Suitable containers for one or more reagents include, for example, bottles, vials, bags, and syringes.
  • the selective HDAC6 inhibitor is compound A
  • the anti-PD-1 antibody or antigen-binding fragment thereof is antibody 2E5, which comprises: a) a heavy chain variable region whose amino acid sequence is selected from SEQ ID NO : The sequence shown in 2; and b) the light chain variable region, the amino acid sequence of which is selected from the sequence shown in SEQ ID NO: 3.
  • a selective HDAC6 inhibitor e.g. Compound A
  • an anti-PD-1 antibody or antigen-binding fragment thereof e.g., antibody 2E5
  • a proteasome inhibitor boron Tiezomib, Carfilzomib, Ishazomib or Marezomib
  • the dosage of PD-1 antibody or its antigen-binding fragment is about 1 mg to about 100 mg in a unit dose, preferably about 5 mg, about 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg; selective HDAC6
  • the dose of the inhibitor is about 10 to 1000 mg, more preferably about 100 to 1000 mg, about 150 to about 600 mg, about 200 to about 500 mg, or about 300 to about 400 mg, particularly preferably about 10 mg, about 50 mg, or about 100 mg , About 150mg, about 200mg, about 250mg, about 300mg, about 350mg, about 400mg, about 450mg, about 500mg; or proteasome inhibitor (bortezomib, carfilzomib, ixazomib or marezomib) dose About 1 to 500 mg, about 5 to 200 mg, about 10 to 50 mg, or preferably about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg,
  • one or more therapeutic agents for example, anti-PD-1 antibodies or antigen-binding fragments thereof or proteasome inhibitors
  • HDAC6 inhibitors provide a synergistic combination, which The ratio of the combination range is between 1:200 to 200:1 parts by weight, preferably, it can be about 1:100, about 1:95, about 1:90, about 1:85, about 1:80, or about 1.
  • the compound of formula A and ACY-1215 were prepared by Wuxi Biologics (Shanghai, China).
  • HT-29 and NCI-H292 were purchased from ATCC.
  • Tumor cells were cultured in McCoy's 5A (Thermo Fisher Scientific, 16600082) or RPMI 1640 (Thermo Fisher Scientific, 110905098), supplemented with 10% FBS (Thermo Fisher Scientific, 10099141) and 1% penicillin-streptomycin (Thermo Fisher Scientific, 110905098). Fisher Scientific, 15140122).
  • ACY-1215 in assay buffer [50mM HEPES, pH 7.4, 100mM KCl, 0.001% Tween-20, 0.05% BSA, and 20 ⁇ M tris(2-carboxyethyl)phosphine] and then dilute to the final concentration of 6 Times.
  • the HDAC enzyme was diluted to 1.5 times the final concentration in the assay buffer and pre-incubated with ACY-1215 for 10 minutes.
  • the amount of FTS (HDAC1, HDAC2, HDAC3, and HDAC6) or MAZ-1675 (HDAC4, HDAC5, HDAC7, HDAC8, and HDAC9) used by each enzyme is equal to the Michaelis constant (Km), which is determined by a titration curve.
  • MM.1S purchased from ATCC
  • CT26 purchased from ATCC
  • MM.1S was subcutaneously inoculated with MM.1S (5x10 6 ) tumor cells in 0.2ml PBS supplemented with Matrigel (1:1) or CT26 (3x10 5 ) tumor cells in 0.2ml PBS on the right posterior side for Tumor development.
  • Treatment starts when the average tumor size in the MM.1S model reaches 150mm 3 and the average tumor size in the CT26 model reaches 70mm 3.
  • the date of randomization is marked as day 1.
  • Use a caliper to measure the tumor volume in two dimensions, 3 times a week (MM.1S) or twice (CT26), and use the following formula to express the volume in mm 3 : V (L x W x W)/2, where V Is the tumor volume, L is the tumor length (the longest tumor dimension) and W is the tumor width (the longest tumor dimension perpendicular to L).
  • the tumor volume and body weight were measured twice a week.
  • TGI tumor growth inhibition
  • TGI(%) [1-(Ti-T0)/(Vi-V0)] ⁇ 100
  • Ti is the average tumor volume of the treatment group in a given day
  • T0 is the average tumor volume of the treatment group on the day of grouping
  • Vi is the average tumor volume of the vehicle control group on the same day as Ti
  • V0 is the average tumor volume of the vehicle group on the day of grouping.
  • Coefficient of drug interaction (CDI) was used to analyze the synergistic inhibitory effect of the drug combination.
  • CDI ⁇ 1 indicates synergy
  • CDI ⁇ 0.7 indicates obvious synergy
  • CDI>1 indicates antagonism
  • immobilizable reactive dye 700 (BD Biosciences, 564997) for live/dead cell identification. Perform cell surface staining at 4°C for 30 minutes in the dark.
  • Antibodies used include PE mouse anti-human CD11c (BD Biosciences, 555392), BB515 mouse anti-human CD86 (BD Biosciences, 564544), PE mouse anti-human CD274 (BD Biosciences, 557924). All flow cytometry analyses were performed using Cytoflex (Beckman Coulter), and FlowJo software (BD Biosciences) was used for analysis.
  • the tumor tissue was collected from the mouse model, and then quickly frozen in a freezer at -80°C after two washings with ice PBS.
  • the samples were lysed with RIPA lysis buffer (Sigma, R0278) containing protease inhibitor cocktail (Roche, 04693124001) and phosphatase inhibitor cocktail 2 (Sigma, P5726). Grind the tumor at full speed with Tissuelyser LT for 5 minutes, and place the tissue lysate on ice for 30 minutes.
  • the protein concentration was determined by Pierce TM BCA protein analysis kit (Thermo Scientific, 23225).
  • the sample was denatured in the sample buffer, and then the same amount of protein was separated on a 4-12% Bis-Tris gel (Thermo Scientific, NP0336BOX) according to the molecular weight, and transferred to a polyvinylidene fluoride membrane.
  • the membrane was blocked in 5% skim milk in TBST for 1 hour, and probed overnight at 4°C with the indicated primary antibody: Ac- ⁇ -tubulin antibody (Cell Signaling Technology, #5335); anti-acetyl Basal histone H3 (Lys9) antibody (Millipore, #07-352); GAPDH (Cell Signaling Technology, 5174).
  • the blot was washed three times with TBST for about 15 minutes, and then incubated with the following secondary antibodies for 1 hour: goat anti-rabbit secondary antibody (Thermo, 31462); goat anti-mouse secondary antibody (Santa, F2816). After washing 3 times with TBST, the HRP substrate in the West Femto Highest Sensitivity Kit (Thermo Scientific, 34096) was added to the membrane, and the chemiluminescence was detected with Tanon 5200multi.
  • the data obtained in the cell study was evaluated by the unpaired Student's t test. After Levene test, tumor weight or tumor volume data were analyzed by independent T test. P ⁇ 0.05 indicates a significant difference.
  • the above results indicate that the compound of formula A is a highly selective and selective HDAC6 inhibitor with high HDAC6/HDAC1 selectivity.
  • ACY-1215 data is obtained from the literature (Santo, L., et al., Preclinical activity, pharmacodynamic, and pharmacokinetic properties of a selective HDAC6inhibitor, ACY-1215, in combination with bortezomib in multiple myeloma.Blood, 2012.119(11): p .2579-89.).
  • the plasma of the compound of formula A in the plasma of CB-17SCID mice, SD rats and beagle dogs was determined by high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) .
  • Table 4 shows the PK parameters of the compound of formula A in rat and dog plasma after a single intravenous (i.v.) or oral (p.o.) dose.
  • the PK data (Table 4) at the lowest tested p.o. dose determined relatively low and high oral bioavailability in rats (19.9%) and dogs (75.0%), respectively.
  • the compound of formula A showed a tissue distribution with a medium volume of distribution (Vss) value in rats (2.81 L/kg) and dogs (1.40 L/kg).
  • the clearance rate in rats is high (89.5 mL/min/kg) and the clearance rate in dogs is moderate (16.8 mL/min/kg).
  • the apparent terminal half-life (T1/2) of rats is 0.482h and that of dogs is 1.29h (Table 4). In conclusion, no abnormal data was found in the PK analysis of the compound of formula A.
  • the 1S human myeloma cancer xenograft model evaluates the in vivo efficacy of the compound of formula A as a monotherapy or in combination with ixazomib.
  • the data shows that the monotherapy of the compound of formula A can produce significant tumor growth inhibitory effect, and the combination of the compound of formula A and ixazomib can synergistically improve the efficacy compared with each single drug ( Figure 2).
  • MM.1S tumor cells were subcutaneously implanted into female CB-17SCID mice. After randomization, each group of 7 mice, the mice were treated with ixazomib (4mg/kg), formula A compound (75mg/kg) monotherapy or a combination of ixazomib and formula A compound (4mg/ kg+75mg/kg) treatment.
  • the tumor volume (A) and body weight (B) were recorded three times a week.
  • the tumor volume and TGI on day 41 after vaccination are as follows: the TGI of ixazomib (4mg/kg) monotherapy is 68.65%; the TGI of formula A compound (75mg/kg) monotherapy The TGI of the combination therapy of ixazomib and the compound of formula A (4mg/kg+75mg/kg) was 92.58%. All data represent the mean ⁇ SEM. Statistical analysis was performed by independent t-test, which was tested by Levene test. ** P ⁇ 0.01 compared with the vehicle group; *** P ⁇ 0.001 compared with the vehicle group; ### P ⁇ 0.001 compared with the compound of formula A-75mg/kg group.
  • MM.1S myeloma tumor tissues of the designated treatment group were collected within 1, 4, or 24 hours after the last administration.
  • the MM.1S myeloma tumor tissue was dissected for immunoblotting to detect the expression levels of Ac- ⁇ -tubulin and Ac-H3K9.
  • Western blot analysis was performed using corresponding antibodies.
  • B and C are the quantitative data of Ac- ⁇ -tubulin and Ac-H3K9, respectively. Values are expressed as mean ⁇ SEM.
  • the statistical analysis of B and C was performed by one-way ANOVA analysis followed by Newman-Keuls test.
  • the statistical analysis of c and d was performed by one-way ANOVA analysis, followed by Dunnett's test. Compared with vehicle group: * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001.
  • the compound of formula A improves the anti-tumor activity of anti-PD-1 immune checkpoint blockers
  • CT26 tumor cells were subcutaneously implanted into female Balb/c mice. After randomization (8 mice per group), the mice were treated with antibody 2E5 (1mg/kg, ip, BIW ⁇ 5 times), compound of formula A (30 or 75 mg/kg, po, BID ⁇ 17 days) monotherapy Or use the compound of formula A and antibody 2E5 in combination with a dose consisting of monotherapy.
  • the tumor volume (A) and body weight (B) were recorded twice a week. All data represent the mean ⁇ SEM.
  • Statistical analysis was performed by independent t-test, which was tested by Levene test. * P ⁇ 0.05 compared with the vehicle group; ** P ⁇ 0.01 compared with the vehicle group; ## P ⁇ 0.01 compared with the compound of formula A-30mg/kg group.
  • the anti-PD-1 antibody ie, antibody 2E5
  • TGI tumor growth inhibition
  • BID tumor growth inhibition
  • monotherapy with compound of formula A inhibited tumor growth by 9.85% and 31.28%.
  • the compound of formula A is used in combination with an anti-PD-1 monoclonal antibody (ie, antibody 2E5) at 30 mg/kg and 75 mg/kg twice a day
  • the TGI increases to 69.87% and 66.77%, respectively.
  • 3 out of 8 mice treated with the compound of formula A (30 mg/kg) in combination with antibody 2E5 showed complete regression.
  • the combination of a compound of formula A and an anti-PD-1 immune checkpoint blocker synergistically improves tumor growth inhibition in the CT26 model.
  • Cytotoxic T cells play a vital role in cancer immunotherapy.
  • HuH-7 cells purchased from the Cell Bank of the Chinese Academy of Sciences
  • Test substance compound of formula A, stock solution: 10mM.
  • PBMCs Frozen PBMC (code-named TBD) from healthy donors, ordered from the supplier Stemexppress.
  • the cryotube Immerse the cryotube in a 37°C water bath and shake it gently to quickly thaw the frozen PBMC cells.
  • the cells were then transferred to 10 mL assay medium (RPMI-1640+10% FBS), centrifuged at 1500 rpm for 10 minutes, and the supernatant was aspirated.
  • the cells were resuspended in an appropriate volume of assay medium containing 1 ⁇ g/mL CD3 and 1 ⁇ g/mL CD28 Ab to make the cell density 1.5 ⁇ 10 6 cells/mL, and incubated in an incubator at 37° C. for 24 h.
  • the activated PBMC was collected in a 50 mL test tube, centrifuged at 1500 rpm for 10 minutes, and the supernatant was aspirated. Resuspend with an appropriate volume of assay medium and adjust to an appropriate concentration with the same medium. Add activated PBMC to make the E:T ratio (ratio of effector cells to target cells) 10:1. Then 0.1 ⁇ M, 0.5 ⁇ M, 1 ⁇ M, or 2 ⁇ M of the formula A compound was added to the co-cultured HCT116 and PBMC, and cultured in an incubator at 37° C. for 48 hours.
  • the detection steps for MCF-7 cells, NCI-H292 cells and HuH-7 cells are the same as the detection steps for HCT116 cells described above.
  • the compound of formula A showed significantly enhanced PBMC-mediated tumor killing efficacy.
  • RNA transcriptome analysis shows the different gene expression patterns of the compound of formula A and other HDAC inhibitors
  • Example 1 we found that the combination of the compound of formula A and the anti-PD-1 antibody (ie, antibody 2E5) has synergistically enhanced anti-tumor efficacy in the CT26 mouse model.
  • the anti-PD-1 antibody ie, antibody 2E5
  • the anti-PD-1 antibody ie, antibody 2E5
  • five indications cell lines were tested with formula A compounds or other HDAC inhibitors, including Daudi (lymphoma), MM.
  • HDAC inhibitors include Vorinostat (Pan-HDAC inhibitor), Entinostat (Class I HDAC inhibitor) and ACY-1215 (selective HDAC6 inhibitor) ( Figure 1).
  • HDAC inhibitors including Vorinostat (Pan-HDAC inhibitor), Entinostat (Class I HDAC inhibitor), and ACY-1215 (selective HDAC6 inhibitor). Extract RNA and perform RNA sequencing. Then PCA analysis was performed. A loop of 3 dots represents a group of repeats 3 times.
  • the principal component analysis (PCA) data shows that, unlike other HDAC inhibitors, the compound of formula A induces a unique gene transcription pattern.
  • the transcription pattern induced by the compound of formula A is significantly different from other HDAC inhibitors, which indicates that the compound of formula A acts through a different pathway from other HDAC inhibitors and has new functions and unique mechanisms of action.
  • the compound of formula A specifically regulates immune cell function in cell lines with different indications
  • Fig. 8 Based on the genes specifically changed in the compound of formula A, we further carried out gene ontology analysis related to myeloid cells.
  • the experimental results of Fig. 8 show that the different expressed genes induced by the compound of formula A can be enriched in the myeloid cell differentiation pathway of Daudi and HT-29 cells (Fig. 8), which indicates that, unlike other HDAC inhibitors, the compound of formula A is specific sexually regulate the function of myeloid cells. Therefore, it is found that the transcription pattern induced by the compound of formula A is significantly different from other HDAC inhibitors, which indicates that the compound of formula A has a new function and a unique mechanism of action.
  • the compound of formula A uniquely activates myeloid cells
  • the effect of the compound of formula A on the differentiation pathway of myeloid cells and leukocytes indicates that the compound of formula A increases DC maturation and affects the M1/M2 balance.
  • To evaluate the effect on DC cells we treated HT-29 and NCI-H292 cells with the compound of formula A and ACY-1215, and then cultured them with immature DC derived from myeloid.
  • HT-29 and NCI-H292 cells were pretreated with a compound of formula A or ACY-1215 at a specified concentration for 24 hours, and then co-cultured with myeloid-derived immature DC for 24 hours.
  • the CD86 expression of CD11c + DC was evaluated by flow cytometry (A and B in Figure 9).
  • M1 and M2 polarized macrophages showed anti-tumor and tumor-promoting functions, respectively.
  • M1 and M2 from myeloid origin were treated with the compound of formula A and ACY-1215 in vitro, respectively ( Figure 9C).
  • M1 and M2 derived from myeloid samples were treated with the compound of formula A or ACY-1215 at the specified concentration for 6 days.
  • Monitor cell viability by staining with Fixable Viability Stain 700.
  • Data are expressed as mean ⁇ SEM. ***P ⁇ 0.001, **P ⁇ 0.01 and *P ⁇ 0.05, used for the comparison between the dose group of each compound of formula A and the DMSO treatment group.
  • Statistical data was obtained by unpaired t test.
  • the results show that compared with the ACY-1215 treatment group, the tumor cells treated with the compound of formula A can significantly enhance the maturation of CD11c + DC by inducing higher expression of the activation markers CD86 (AB in Figure 9) and MHCII (data not shown) .
  • the experimental results of Fig. 9C show that when treated with the compound of formula A, the M1/M2 ratio increased, but in the ACY-1215 treatment group, the M1/M2 ratio did not change. Therefore, the experimental results show that the compound of formula A, but not ACY-1215, can activate DC maturation and change the balance of macrophages, thereby having anti-tumor function.

Abstract

用于治疗受益于调节免疫功能的受试者病症的治疗方法，其包括向所述受试者施用药学有效量的HDAC6选择性抑制剂。还涉及一种组合疗法，其包括向所述受试者施用药学有效量的HDAC6选择性抑制剂和一种或多种治疗剂。还提供了一种治疗组合，其包含：(1)HDAC6选择性抑制剂，和(2)一种或多种治疗剂；其可用于分别、同时或顺序向有需要的个体施用以治疗或预防受益于调节免疫功能的受试者病症。还涉及一种组合物、药物或者制剂或者试剂盒，其包含：(1)HDAC6选择性抑制剂；(2)一种或多种治疗剂。

Description

HDAC6选择性抑制剂及其组合疗法 技术领域
本发明涉及用于治疗受益于调节免疫功能的受试者病症的治疗方法，其包括向所述受试者施用药学有效量的HDAC6选择性抑制剂。本发明还涉及一种组合疗法，其包括向所述受试者施用药学有效量的HDAC6选择性抑制剂和一种或多种治疗剂。本发明还提供了一种治疗组合，其包含：(1)HDAC6选择性抑制剂，和(2)一种或多种治疗剂；其可用于分别、同时或顺序向有需要的个体施用以治疗或预防受益于调节免疫功能的受试者病症。本发明还涉及一种组合物、药物或者制剂或者试剂盒，其包含：(1)HDAC6选择性抑制剂；(2)一种或多种治疗剂。
背景技术
多发性骨髓瘤(MM)是一种B细胞恶性肿瘤，其特征为与免疫球蛋白高产量相关的骨髓(BM)浆细胞的增殖。尽管可以进行治疗，但MM被认为无法治愈，所有骨髓瘤患者的5年总生存率仅为42％。蛋白酶体抑制剂包括硼替佐米(Bortezomib)、卡非佐米(Carfilzomib)、伊沙佐米(Ixazomib)和马瑞佐米(Marizomib)，它们可阻断多泛素化错误折叠的蛋白质的降解，诱导ER应激并触发MM细胞的凋亡，是多发性骨髓瘤治疗的治疗基础。但是，该疾病可通过未明确定义的机制而变得耐药。在这些候选药物中，组蛋白去乙酰化酶(HDACs)抑制剂已在临床上显示出有希望的功效。HDAC抑制剂大致分为两类，泛HDAC(Pan-HDAC)抑制剂和I类HDAC抑制剂。泛HDAC抑制剂同时抑制I类(HDAC 1、2、3和8)和IIb类(HDAC 6和10)酶，I类HDAC抑制剂仅抑制I类酶。此后，已对I类HDAC抑制剂进行了测试并显示出功效，但其仍具有血液学毒性和剂量限制的不良反应。几项研究报告称非选择性HDACi的急性毒性主要是通过I类HDAC抑制引起的。因此，泛HDAC抑制剂或I类HDAC抑制剂的毒性问题仍然是临床成功使用的障碍。
HDAC6作为分子伴侣，在调节聚集体功能中起作用，并影响包括α-微管蛋白(tubulin)在内的几种蛋白质的乙酰化状态，这对于微管稳定性和功能的调节十分重要。据报道，在B细胞和T细胞淋巴瘤中HDAC6过表达，且其通过选择性HDAC6抑制剂的抑制作用已在淋巴瘤和MM的临床前模型中 证明具有活性。免疫疗法是治疗过度受益于调节免疫功能的受试者病症的一种方法。在各种类型的癌症免疫疗法的开发中，科学家和临床医生遇到的主要障碍是打破对自身抗原(癌症)的耐受性，以便引发强有力的抗肿瘤反应，从而导致肿瘤消退。与传统的靶向肿瘤的小分子和大分子药物的开发不同，癌症免疫疗法靶向免疫系统的细胞，这些细胞具有产生效应细胞记忆库的潜力，以诱导更持久的作用并使复发最小化。
程序性死亡1(PD-1)受体和PD-1配体1和2(分别为PD-L1和PD-L2)在免疫调节中起着不可或缺的作用。在激活的T细胞上表达的PD-1被PD-L1(也称为B7-H 1)激活，并由基质细胞、肿瘤细胞或两者表达的PD-L2激活，从而引发T细胞死亡和局部免疫抑制为肿瘤的发展和生长提供免疫耐受的环境。相反，在非临床动物模型中抑制这种相互作用可以增强局部T细胞反应并介导抗肿瘤活性。
尽管在癌症的治疗方面有许多最新进展，但是仍然需要对遭受癌症影响的个体进行更有效和/或增强的治疗。本发明涉及用于治疗受益于调节免疫功能的受试者病症的治疗方法；本发明还涉及一种组合疗法；本发明还提供了一种治疗组合，其可用于分别、同时或顺序向有需要的个体施用以治疗或预防受益于调节免疫功能的受试者病症；本发明还涉及一种组合物、药物或者制剂或者试剂盒。
发明概述
针对以上技术问题，我们发现，选择性HDAC6抑制剂有望预防在泛HDAC抑制剂中观察到的严重毒性，并为临床提供更广阔的治疗窗口。具体而言，我们发现选择性HDAC6抑制剂在治疗癌症方面，尤其是HDAC抑制方面具有独特的特征：具有与蛋白酶体抑制剂协同作用和通过活化髓样细胞增加免疫检查点抗肿瘤功能的双重功能。我们还发现，选择性HDAC6抑制剂和一种或多种治疗剂的组合协同增强了抗肿瘤功能，尤其是抗多发性骨髓瘤的功能。可用于本发明组合中的HDAC6抑制剂已在公开的PCT专利申请WO2018/130155中一般性地和具体地描述，该申请以引用方式并入本文。
本发明涉及一种用于受益于调节免疫功能的受试者病症的方法，其包括向所述受试者施用药学有效量的HDAC6选择性抑制剂。
本发明还涉及一种用于受益于调节免疫功能的受试者病症的方法，其包 括向所述受试者施用药学有效量的选择性HDAC6抑制剂和一种或多种治疗剂。
本发明还提供了一种治疗组合，其包含：(1)选择性HDAC6抑制剂，和(2)一种或多种治疗剂；其可用于分别、同时或顺序向有需要的个体施用以治疗或预防受益于调节免疫功能的受试者病症。在一个实施方案中，本发明涉及一种包含选择性HDAC6抑制剂的组合物、药物或制剂，其与一种或多种治疗剂组合使用，用于治疗治疗受益于调节免疫功能的受试者病症。在一个实施方案中，本发明还涉及一种组合物、药物或者制剂，其包含：(1)选择性HDAC6抑制剂；(2)一种或多种治疗剂。
本发明还提供了一种试剂盒，其包括第一容器、第二容器和包装插页，其中第一容器包含至少一个剂量的药物，该药物包含选择性HDAC6抑制剂，第二容器包含至少一个剂量的药物，该药物包含第二种治疗剂。
本发明还涉及选择性HDAC6抑制剂和一种或多种治疗剂的组合在制备用于治疗用于受益于调节免疫功能的受试者病症的药物中的用途。在一个实施方案中，本发明涉及选择性HDAC6抑制剂在制备用于治疗用于受益于调节免疫功能的受试者病症的药物中的用途，所述选择性HDAC6抑制剂和一种或多种治疗剂组合。在另一个实施方案中，本发明涉及一种或多种治疗剂在制备用于治疗用于受益于调节免疫功能的受试者病症的药物中的用途，所述一种或多种治疗剂和选择性HDAC6抑制剂组合。
在上述方法、用途、组合、组合物、药物、制剂或试剂盒的实施方案中，所述选择性HDAC6抑制剂与一种或多种治疗剂同时、分别或顺序向该受试者施用。
在上述方法、用途、组合、组合物、药物、制剂或试剂盒的实施方案中，所述选择性HDAC6抑制剂为式(I)和式(II)所示化合物，或其药学上可接受的盐或对映异构体、非对映异构体、外消旋体、溶剂合物、水合物、多晶型、前药或活性代谢物，
Figure PCTCN2020133799-appb-000001
其中，
L1选自：单键、-NH-、-C(＝O)-NH-；
E 1、E 2选自-O-、-CH 2-和-CH 2-CH 2-；
R 2选自：H、F、Cl、Br、I；
R选自：F、Cl、Br、I；
n选自：0或1。
在上述方法、用途、组合、组合物、药物、制剂或试剂盒的实施方案中，所述选择性HDAC6抑制剂为式A的化合物或其药学上可接受的盐或对映异构体、非对映异构体、外消旋体、溶剂合物、水合物、多晶型、前药或活性代谢物:
Figure PCTCN2020133799-appb-000002
在上述方法、用途、组合、组合物、药物、制剂或试剂盒的实施方案中，所述一种或多种治疗剂为PD-1轴结合拮抗剂或者蛋白酶体抑制剂；
优选的，PD-1轴结合拮抗剂选自PD-1结合拮抗剂，PD-L1结合拮抗剂和PD-L2结合拮抗剂；更优选的，PD-1轴结合拮抗剂是抗体或其抗原结合片段；尤其优选地，该抗体是抗PD-1抗体；最优选地，该抗体是单克隆抗体；
优选的，所述蛋白酶体抑制剂为硼替佐米、卡非佐米、伊沙佐米或者马瑞佐米。
在上述方法、用途、组合、组合物、药物、制剂或试剂盒的实施方案中，所述抗体或其抗原结合片段结合于PD-1的一个表位，所述表位包含：SEQ ID NO﹕24上第128、129、130、131和132位点氨基酸和第35、64、82、83位中至少一个氨基酸；
优选的，所述抗体或其抗原结合片段结合于人PD-1和鼠PD-1的一个表位，其中，所述表位包括SEQ ID NO﹕24上第128、129、130、131和132位点氨基酸。
在上述方法、用途、组合、组合物、药物、制剂或试剂盒的实施方案中，所述抗体或其抗原结合片段包含一个氨基酸序列，所述氨基酸序列与选自由 SEQ ID NOs﹕1、2、3、4、5、6、7、8和9所组成的组中的序列具有至少70％、80％、90％或95％的同源性，其中所述抗体特异性结合PD-1；优选的，所述抗体或其抗原结合片段包含一个氨基酸序列，所述氨基酸序列选自由SEQ ID NOs﹕1、2、3、4、5、6、7、8和9所组成的组中的序列。
在上述方法、用途、组合、组合物、药物、制剂或试剂盒的实施方案中，所述抗体或其抗原结合片段包含：
a)重链可变区，其具有的氨基酸序列选自由SEQ ID NO﹕1和SEQ ID NO﹕2所组成的组
中的序列或与其具有至少70％、80％、90％或95％的同源性的序列；以及
b)轻链可变区，其具有的氨基酸序列选自由SEQ ID NOs﹕3、4、5、6、7、8和9所组成的
组中的序列或与其具有至少70％、80％、90％或95％的同源性的序列，
其中所述抗体特异性结合PD-1。
更优选的，所述抗体或其抗原结合片段包含：
a)重链可变区，其氨基酸序列选自SEQ ID NO﹕2所示的序列；以及
b)轻链可变区，其氨基酸序列选自SEQ ID NO﹕3所示的序列。
在上述的方法、用途、组合、组合物、药物、制剂或试剂盒的实施方案中，所述抗体或其抗原结合片段包含互补决定区(CDR)，其具有的氨基酸序列选自由SEQ ID NOs﹕10-23所组成的组中的序列，其中所述抗体特异性结合PD-1；
优选的，所述抗体包含：
a)重链可变区CDR1，序列包含选自SEQ ID NO﹕10所示的氨基酸序列，
b)重链可变区CDR2，序列包含选自SEQ ID NO﹕11所示的氨基酸序列，
c)重链可变区CDR3，序列包含选自SEQ ID NO﹕13所示的氨基酸序列，
d)轻链可变区CDR1，序列包含选自SEQ ID NO﹕14所示的氨基酸序列，
e)轻链可变区CDR2，序列包含选自SEQ ID NO﹕19所示的氨基酸序列，
f)轻链可变区CDR3，序列包含选自SEQ ID NO﹕21所示的氨基酸序列。
在上述方法、用途、组合、组合物、药物、制剂或试剂盒的一个实施方案中，所述选择性HDAC6抑制剂为式A化合物或对映异构体、非对映异构 体、外消旋体、溶剂合物、水合物、多晶型、前药或活性代谢物:
Figure PCTCN2020133799-appb-000003
式A；
所述一种或多种治疗剂为抗体或其抗原结合片段，其为抗体2E5，包含：
a)重链可变区，其氨基酸序列选自SEQ ID NO﹕2所示的序列；以及
b)轻链可变区，其氨基酸序列选自SEQ ID NO﹕3所示的序列，
其中所述抗体特异性结合PD-1。
在上述的方法、用途、组合、组合物、药物、制剂或试剂盒的一个实施方案中，所述选择性HDAC6抑制剂为下式化合物或对映异构体、非对映异构体、外消旋体、溶剂合物、水合物、多晶型、前药或活性代谢物:
Figure PCTCN2020133799-appb-000004
式A
并且，所述一种或多种治疗剂为硼替佐米、卡非佐米、伊沙佐米或者马瑞佐米。
附图说明
图1为式A化合物和ACY-1215的化学结构。
图2显示式A化合物与伊沙佐米组合对MM.1S异种移植模型的肿瘤生长的影响。
图3为MM.1S肿瘤组织的Ac-α-微管蛋白和Ac-H3K9蛋白表达水平。
图4显示式A化合物与抗体2E5组合对CT26同系模型的肿瘤生长的影响。
图5显示式A化合物的PBMC介导的HCT116细胞、MCF-7细胞、NCI-H292或HuH-7细胞杀伤效果。
图6显示用PCA分析进行RNA转录组分析，结果表明A化合物与其他HDAC抑制剂不同的基因表达模式。
图7显示用GO富集分析进行RNA转录组分析，结果表明式A化合物特异性调节不同适应症细胞系中的免疫细胞功能。
图8显示用GO富集分析进行RNA转录组分析，结果表明式A化合物诱导的不同表达基因可以在Daudi和HT-29细胞的髓样细胞分化途径中富集。
图9显示式A化合物激活了髓样细胞并增加了M1/M2比率。
发明详述
定义
为使本发明可更容易理解，在下文中具体地定义某些技术和科学术语。除非在本文件中其他地方具体地定义，否则本文所使用的所有其他技术和科学术语均具有本领域普通技术人员通常所理解的含义。
术语“约”或“大概”应具有在指定值或范围的10％内，优选在5％内的含义。
除非在本文中另外说明或与上下文明显矛盾，否则如本文(包括随附的权利要求书)所用，词语的单数形式诸如“一(a/an)”和“该(the)”应理解为涵盖单数和复数形式。
“组合”“治疗组合”或“药物组合”以及相关术语是指同时或依次给药本发明的药物或治疗剂。例如，本文所述的HDAC6可以与另一治疗剂例如抗PD-1抗体或其抗原结合片段以分开的单位剂型同时或依次给药，或与另一治疗剂一起呈单一单位剂型同时给药。
除非另外说明或通过原文明确指出，否则关于可用于“本发明组合”中的药物或者治疗剂包含化合物的游离碱和化合物的所有药学上可接受的盐。
术语“药学有效量”或“临床有效量”或“治疗有效量”是指足以提供在病症的临床基线上观察得到的症候和症状方面具有可观察到的改进的量。
术语“药学上可接受的”在本文中定义为那些在合理医学判断范围内适合接触个体(例如哺乳动物或人)的组织而不会产生过量毒性、刺激过敏性反应和其他并发症问题且具有与之相称的合理的效益/风险比的化合物、材料、组合物和/或剂型。
“施用”或“给药”在其应用于动物、人类、实验受试者、细胞、组织、 器官或生物流体时是指外源性药物、治疗剂、诊断剂或组合物与受试者、细胞、组织、器官或生物流体的接触。
本文中所使用的术语“共同施用”或“组合施用”定义为涵盖向单个患者施用所选治疗剂，且意指包括药物不一定通过相同途径施用或同时施用的治疗方案。
本文中所使用的术语“治疗”包括缓解、减轻或降低个体的至少一种症状或影响疾病进展延迟的治疗。例如，治疗可以是消除病症的一或数个症状或完全根除病症(例如癌症)。在本发明的含义中，术语“治疗”也表示阻止、延迟发病(即出现疾病的临床表现前的时间)和/或降低疾病发展或恶化的风险。术语“保护”在本文中指预防、延迟或治疗(或适当时全部)个体疾病的发展或持续或加剧。
给药的“受试者”包括但不限于：人(即，任何年龄组的男性或女性，例如，儿科受试者(例如，婴儿、儿童、青少年)或成人受试者(例如，年轻的成人、中年的成人或年长的成人))和/或非人的动物，例如，哺乳动物，例如，灵长类(例如，食蟹猴、恒河猴)、牛、猪、马、绵羊、山羊、啮齿动物、猫和/或狗。在一些实施方案中，受试者是人。在一些实施方案中，受试者是非人动物。本文可互换使用术语“人”、“患者”和“受试者”。
术语“细胞增殖性病症”和“增殖性病症”指与一定程度的异常细胞增殖有关的病症。在一个实施方案中，细胞增殖性病症指癌症。在一个实施方案中，细胞增殖性病症是肿瘤。
“肿瘤”在用于本文时指所有赘生性(neoplastic)细胞生长和增殖，无论是恶性的还是良性的，及所有癌前(pre-cancerous)和癌性细胞和组织。术语“癌症”、“癌性”、“细胞增殖性病症”、“增殖性病症”和“肿瘤”在本文中提到时并不互相排斥。
术语“癌症”和“癌性”指向或描述哺乳动物中特征通常为细胞生长不受调节的生理疾患。癌症的例子包括但不限于癌、淋巴瘤、母细胞瘤、肉瘤和白血病或淋巴样恶性肿瘤。此类癌症的更具体例子包括但不限于鳞状细胞癌(例如上皮鳞状细胞癌)、肺癌(包括小细胞肺癌、非小细胞肺癌、肺的腺癌、和肺的鳞癌)、腹膜癌、肝细胞癌、胃癌(包括胃肠癌和胃肠基质癌)、胰腺癌、成胶质细胞瘤、宫颈癌、卵巢癌、肝癌、膀胱癌、尿道癌、肝瘤、乳腺癌、结肠癌、直肠癌、结肠直肠癌、子宫内膜癌或子宫癌、唾液腺癌、肾癌、前 列腺癌、外阴癌、甲状腺癌、肝癌、肛门癌、阴茎癌、黑素瘤、浅表扩散性黑素瘤、恶性雀斑样痣黑素瘤、肢端黑素瘤、结节性黑素瘤、多发性骨髓瘤和B细胞淋巴瘤(包括低级/滤泡性非何杰金氏淋巴瘤(NHL)、小淋巴细胞性(SL)NHL、中级/滤泡性NHL、中级弥漫性NHL、高级成免疫细胞性NHL、高级成淋巴细胞性NHL、高级小无核裂细胞性NHL、贮积病(bulky disease)NHL、套细胞淋巴瘤、AIDS相关淋巴瘤、和瓦尔登斯特伦氏(Waldenstrom)巨球蛋白血症)、慢性淋巴细胞性白血病(CLL)、急性成淋巴细胞性白血病(ALL)、毛细胞性白血病、慢性成髓细胞性白血病、和移植后淋巴增殖性病症(PTLD)，以及与瘢痣病(phakomatoses)、水肿(诸如与脑瘤有关的)和梅格斯氏(Meigs)综合征有关的异常血管增殖、脑瘤和脑癌，以及头颈癌，及相关转移。在某些实施方案中，适合于通过本发明的抗体来治疗的癌症包括乳腺癌、结肠直肠癌、直肠癌、非小细胞肺癌、成胶质细胞瘤、非何杰金氏淋巴瘤(NHL)、肾细胞癌、前列腺癌、肝癌、胰腺癌、软组织肉瘤、卡波西(Kaposi)氏肉瘤、类癌癌(carcinoid carcinoma)、头颈癌、卵巢癌、间皮瘤和多发性骨髓瘤。在一些实施方案中，癌症选自：小细胞肺癌、成胶质细胞瘤、成神经细胞瘤、黑素瘤、乳腺癌、胃癌、结肠直肠癌(CRC)和肝细胞癌。还有、在一些实施方案中、癌症选自：非小细胞肺癌、结肠直肠癌、成胶质细胞瘤和乳腺癌，包括那些癌症的转移性形式。
如本文中所使用，术语“程序性死亡1”、“程序性细胞死亡1”、“蛋白PD-1”、“PD-1”、“PD1”、“PDCD1”、“hPD-1”和“hPD-F”可互换使用，并且包括变体、同种型、人PD-1的物种同源物和具有PD-1的至少一个共同表位的类似物。
“抗体”是能够经由至少一个位于免疫球蛋白分子的可变区中的抗原识别位点特异性结合至靶标诸如碳水化合物、多核苷酸、脂质、多肽的免疫球蛋白分子。如本文中所使用，该术语不仅涵盖完整多克隆或单克隆抗体，而且涵盖其片段(诸如Fab、Fab’、F(ab’)2、Fv)、单链(scFv)和结构域抗体(包括例如鲨鱼和骆驼抗体)以及包含抗体的融合蛋白，和包含抗原识别位点的免疫球蛋白分子的任何其他修饰的构型。抗体包括任何类别的抗体，诸如IgG、IgA或IgM(或其亚类)，且该抗体不必为任何特定类别。根据其重链的恒定区的抗体氨基酸序列，免疫球蛋白可被归为不同类别。存在五种主要类别的免疫球蛋白：IgA、IgD、IgE、IgG和IgM，且这些中的几种可进一步分成 亚类(同种型)，例如IgGl、IgG2、IgG3、IgG4、IgAl和IgA2。对应于不同种类的免疫球蛋白的重链恒定区分别称为α、δ、ε、γ和μ。不同类别的免疫球蛋白的亚单位结构和三维构型是众所周知的。
如本文中所使用，术语“抗体”包括完整抗体和任何抗原结合片段(即“抗原结合部分”)或其单链。“抗体”是指包含至少两条重链(H)和两条轻链(L)并通过二硫键相互连接的，或其抗原结合部分的蛋白质。每条重链由重链可变区(本文缩写为VH)和重链恒定区组成。重链恒定区由三个结构域，CH1、CH2和CH3组成。每条轻链由轻链可变区(本文缩写为VL)和轻链恒定区的。轻链恒定区由一个结构域CL组成。VH和VL区可以进一步细分成高变区，称为互补决定区(CDR)，与更保守的称为构架区(FR)的区域散布。每个VH和VL由三个CDR和四个FR组成，从氨基末端到羧基末端以下面的顺序排列：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。重链和轻链的可变区包含与抗原相互作用的结合结构域。
术语“抗体”，在本申请中所用的是指免疫球蛋白或其片段或它们的衍生物，并且包括其包含的抗原结合位点的任何多肽，而不管其是否是在体外或体内产生。该术语包括，但不限于，多克隆、单克隆、单特异性的、多特异性的、非特异性的、人源化、单链的、嵌合的、合成的、重组的、杂合的、突变的、嫁接的抗体。术语“抗体”还包括抗体片段例如Fab、F(ab')2、FV、scFv、Fd、dAb和其它保留抗原结合功能的抗体片段，即，能够与PD-1的特异性结合。通常情况下，这样的片段将包括抗原结合片段。
如本文所使用的术语抗体的“抗原结合片段”或“抗原结合部分”是指保留与给定抗原特异性结合的能力的完整抗体的一个或多个片段。抗体的抗原结合功能可由完整抗体的片段执行。涵盖在术语抗体的“抗原结合片段”内的结合片段的实例包括Fab；Fab’；F(ab’)2；由VH结构域和CH1结构域组成的Fd片段；由抗体的单臂的VL结构域和VH结构域组成的Fv片段；单结构域抗体(dAb)片段(Ward等人，Nature 341:544-546,1989)和分离的互补决定区(CDR)。术语“抗原结合片段”、“抗原结合结构域”和“结合片段”是指一种抗体分子，其包含负责具体的抗体和抗原之间的结合的氨基酸。例如，其中的抗原是大的，抗原结合片段只结合抗原的一部分。即抗原分子中负责与抗原结合片段特异性相互作用的部分被称为“表位”或“抗原决定簇”。
抗原结合片段通常包括抗体轻链可变区(VL)和抗体重链可变区(VH)，然 而，它不一定必须包括两者。例如，一个所谓的Fd抗体片段仅由VH结构域组成，但仍保留了完整抗体的一些抗原结合功能。
上述术语“表位”定义为抗原决定簇，其特异性结合/识别结合片段。结合片段可以特异性与针对靶结构独特的构象或连续表位进行结合/反应，例如人类PD-1和鼠PD-1(小鼠或大鼠)。构象或不连续表位的特征在于多肽抗原在一级序列中是分离的两个或多个离散的氨基酸残基，但多肽折叠成天然蛋白/抗原时是一起聚集在在分子的表面上的。表位的两个或多个离散的氨基酸残基存在于一个或多个多肽链的独立部分。当多肽链折叠成三维结构，这些残基聚集在分子表面以构成表位。与此相反，由两个或多个离散的氨基酸残基组成的连续或线性表位，其存在于多肽链的单个线性区段。
术语“结合PD-1的表位”是指抗体特异性结合PD-1的特定表位，其可通过直链氨基酸序列或PD-1的部分三维结构来定义结合。结合是指，对于PD-1的部分中的抗体的亲和力比其对其他相关多肽的亲和力显着更大。术语“基本上更大的亲和力”是指与其他相关多肽的亲和力相比，在对PD-1的部分的亲和性呈可测量的增加。优选地，对PD-的特定部分的亲和力相比其他蛋白质至少是1.5倍、2倍、5倍、10倍、100倍、103倍、104倍、105倍、106倍或更大。优选地，结合亲和力是通过酶联免疫吸附测定(ELISA)，或通过荧光激活细胞分选(FACS)分析或表面等离子体共振(SPR)测定的。更优选地，结合特异性由荧光激活细胞分选(FACS)分析得到。
“同源性”是指当两个多肽序列最佳比对时在两个序列之间的序列相似性。当两个比较的序列的两者中的位置由相同氨基酸单体亚单位占据时，例如如果两个不同Ab的轻链CDR中的位置由丙氨酸占据时，贝两个Ab在该位置处同源。同源性百分比是由两个序列共有的同源性位置的数目除以所比较的位置的总数目X 100。例如，如果当序列最佳比对时，两个序列中的10个位置中的8个位置匹配或同源，则该两个序列是80％同源的。通常，在两个序列被比对以产生最大同源性百分比时进行比较。例如，比较可通过BLAST算法进行，其中该算法的参数被选择以在各参考序列的完整长度上的各序列之间产生最大匹配。
术语“药学上可接受的盐”是指本发明化合物的盐，由本发明发现的具有特定取代基的化合物与相对无毒的酸或碱制备。当本发明的化合物中含有相对酸性的功能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的碱 与这类化合物的中性形式接触的方式获得碱加成盐。药学上可接受的碱加成盐包括钠、钾、钙、铵、有机胺或镁盐或类似的盐。当本发明的化合物中含有相对碱性的官能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的酸与这类化合物的中性形式接触的方式获得酸加成盐。药学上可接受的酸加成盐的实例包括无机酸盐，所述无机酸包括例如盐酸、氢溴酸、硝酸、碳酸、碳酸氢根、磷酸、磷酸一氢根、磷酸二氢根、硫酸、硫酸氢根、氢碘酸、亚磷酸等；以及有机酸盐，所述有机酸包括如乙酸、丙酸、异丁酸、马来酸、丙二酸、苯甲酸、琥珀酸、辛二酸、反丁烯二酸、乳酸、扁桃酸、邻苯二甲酸、苯磺酸、对甲苯磺酸、柠檬酸、酒石酸和甲磺酸等类似的酸；还包括氨基酸(如精氨酸等)的盐，以及如葡糖醛酸等有机酸的盐(参见Berge等人.,"Pharmaceutical Salts",Journal of Pharmaceutical Science 66:1-19(1977))。本发明的某些特定的化合物含有碱性和酸性的官能团，从而可以被转换成任一碱或酸加成盐。
优选地，以常规方式使盐与碱或酸接触，再分离母体化合物，由此再生化合物的中性形式。化合物的母体形式与其各种盐的形式的不同之处在于某些物理性质，例如在极性溶剂中的溶解度不同。
本文所用的“药学上可接受的盐”属于本发明化合物的衍生物，其中，通过与酸成盐或与碱成盐的方式修饰所述母体化合物。药学上可接受的盐的实例包括但不限于：碱(比如胺)的无机酸或有机酸盐、酸(比如羧酸)的碱金属或有机盐等等。药学上可接受的盐包括常规的无毒性的盐或母体化合物的季铵盐，例如无毒的无机酸或有机酸所形成的盐。常规的无毒性的盐包括但不限于那些衍生自无机酸和有机酸的盐，所述的无机酸或有机酸选自2-乙酰氧基苯甲酸、2-羟基乙磺酸、乙酸、抗坏血酸、苯磺酸、苯甲酸、碳酸氢根、碳酸、柠檬酸、依地酸、乙烷二磺酸、乙烷磺酸、富马酸、葡庚糖、葡糖酸、谷氨酸、乙醇酸、氢溴酸、盐酸、氢碘酸盐、羟基、羟萘、羟乙磺酸、乳酸、乳糖、十二烷基磺酸、马来酸、苹果酸、扁桃酸、甲烷磺酸、硝酸、草酸、双羟萘酸、泛酸、苯乙酸、磷酸、多聚半乳糖醛、丙酸、水杨酸、硬脂酸、亚乙酸、琥珀酸、氨基磺酸、对氨基苯磺酸、硫酸、单宁、酒石酸和对甲苯磺酸。
本发明的药学上可接受的盐可由含有酸根或碱基的母体化合物通过常规化学方法合成。一般情况下，这样的盐的制备方法是：在水或有机溶剂或 两者的混合物中，经由游离酸或碱形式的这些化合物与化学计量的适当的碱或酸反应来制备。一般地，优选醚、乙酸乙酯、乙醇、异丙醇或乙腈等非水介质。
除了盐的形式，本发明所提供的化合物还存在前药形式。本文所描述的化合物的前药容易地在生理条件下发生化学变化从而转化成本发明的化合物。此外，前体药物可以在体内环境中通过化学或生化方法被转换到本发明的化合物。
本发明的某些化合物可以以非溶剂化形式或者溶剂化形式存在，包括水合物形式。一般而言，溶剂化形式与非溶剂化的形式相当，都包含在本发明的范围之内。
本发明的某些化合物可以具有不对称碳原子(光学中心)或双键。外消旋体、非对映异构体、几何异构体和单个的异构体都包括在本发明的范围之内。
除非另有说明，术语“对映异构体”或者“旋光异构体”是指互为镜像关系的立体异构体。
除非另有说明，术语“顺反异构体”或者“几何异构体”系由因双键或者成环碳原子单键不能自由旋转而引起。
除非另有说明，术语“非对映异构体”是指分子具有两个或多个手性中心，并且分子间为非镜像的关系的立体异构体。
除非另有说明，“(D)”或者“(+)”表示右旋，“(L)”或者“(-)”表示左旋，“(DL)”或者“(±)”表示外消旋。
本发明的化合物可以存在特定的。除非另有说明，术语“互变异构体”或“互变异构体形式”是指在室温下，不同官能团异构体处于动态平衡，并能很快的相互转化。若互变异构体是可能的(如在溶液中)，则可以达到互变异构体的化学平衡。例如，质子互变异构体(proton tautomer)(也称质子转移互变异构体(prototropic tautomer))包括通过质子迁移来进行的互相转化，如酮-烯醇异构化和亚胺-烯胺异构化。价键异构体(valence tautomer)包括一些成键电子的重组来进行的相互转化。其中酮-烯醇互变异构化的具体实例是戊烷-2,4-二酮与4-羟基戊-3-烯-2-酮两个互变异构体之间的互变。
除非另有说明，术语“富含一种异构体”、“异构体富集”、“富含一种对映体”或者“对映体富集”指其中一种异构体或对映体的含量小于100％，并且，该异构体或对映体的含量大于等于60％，或者大于等于70％，或者大于等于 80％，或者大于等于90％，或者大于等于95％，或者大于等于96％，或者大于等于97％，或者大于等于98％，或者大于等于99％，或者大于等于99.5％，或者大于等于99.6％，或者大于等于99.7％，或者大于等于99.8％，或者大于等于99.9％。
除非另有说明，术语“异构体过量”或“对映体过量”指两种异构体或两种对映体相对百分数之间的差值。例如，其中一种异构体或对映体的含量为90％，另一种异构体或对映体的含量为10％，则异构体或对映体过量(ee值)为80％。
可以通过的手性合成或手性试剂或者其他常规技术制备光学活性的(R)-和(S)-异构体以及D和L异构体。如果想得到本发明某化合物的一种对映体，可以通过不对称合成或者具有手性助剂的衍生作用来制备，其中将所得非对映体混合物分离，并且辅助基团裂开以提供纯的所需对映异构体。或者，当分子中含有碱性官能团(如氨基)或酸性官能团(如羧基)时，与适当的光学活性的酸或碱形成非对映异构体的盐，然后通过本领域所公知的常规方法进行非对映异构体拆分，然后回收得到纯的对映体。此外，对映异构体和非对映异构体的分离通常是通过使用色谱法完成的，所述色谱法采用手性固定相，并任选地与化学衍生法相结合(例如由胺生成氨基甲酸盐)。
本发明的化合物可以在一个或多个构成该化合物的原子上包含非天然比例的原子同位素。例如，可用放射性同位素标记化合物，比如氚( 3H)，碘-125( 125I)或C-14( 14C)。又例如，可用重氢取代氢形成氘代药物，氘与碳构成的键比普通氢与碳构成的键更坚固，相比于未氘化药物，氘代药物有降低毒副作用、增加药物稳定性、增强疗效、延长药物生物半衰期等优势。本发明的化合物的所有同位素组成的变换，无论放射性与否，都包括在本发明的范围之内。
“疾病”、“障碍”和“病症”在本文中可互换地使用。
除非另作说明，否则，本文使用的术语“治疗”包括受试者患有具体疾病、障碍或病症时所发生的作用，它降低疾病、障碍或病症的严重程度，或延迟或减缓疾病、障碍或病症的发展(“治疗性治疗”)，还包括受试者开始患有具体疾病、障碍或病症之前发生的作用(“预防性治疗”)。
组蛋白脱乙酰基酶(HDAC)抑制剂
在上述方法、用途、组合、组合物、药物、制剂或试剂盒的实施方案中，本文所述的HDAC抑制剂可为任何HDAC抑制剂。因此，HDAC抑制剂对于特定类型的组蛋白脱乙酰基酶可为选择性或非选择性的。优选地，HDAC抑制剂是选择性HDAC抑制剂。更优选地，HDAC抑制剂是HDAC6抑制剂。可用于本发明组合中的HDAC6抑制剂已在公开的PCT专利申请WO2018/130155中一般性地和具体地描述，该申请以引用方式并入本文。
在上述方法、用途、组合、组合物、药物、制剂或试剂盒的实施方案中，本文所述的HDAC6抑制剂为式(I)和式(II)所示化合物，或其药学上可接受的盐或对映异构体、非对映异构体、外消旋体、溶剂合物、水合物、多晶型、前药或活性代谢物，
Figure PCTCN2020133799-appb-000005
其中，
L1选自：单键、-NH-、-C(＝O)-NH-；
E 1、E 2选自-O-、-CH 2-和-CH 2-CH 2-；
R 2选自：H、F、Cl、Br、I；
R选自：F、Cl、Br、I；
n选自：0或1。
优选地，选择性HDAC6抑制剂为下列化合物或或其药学上可接受的盐或对映异构体、非对映异构体、外消旋体、溶剂合物、水合物、多晶型、前药或活性代谢物：
Figure PCTCN2020133799-appb-000006
最优选地，所述选择性HDAC6抑制剂为如下式A化合物，或其药学上可接受的盐或对映异构体、非对映异构体、外消旋体、溶剂合物、水合物、多晶型、前药或活性代谢物:
Figure PCTCN2020133799-appb-000007
PD-1轴结合拮抗剂
在上述方法、用途、组合、组合物、药物、制剂或试剂盒的实施方案中，本文所述的PD-1轴结合拮抗剂选自PD-1结合拮抗剂，PD-L1结合拮抗剂和PD-L2结合拮抗剂；更优选的，PD-1轴结合拮抗剂是抗体或其抗原结合片段；尤其优选地，该抗体是抗PD-1抗体；最优选地，该抗体是单克隆抗体。可用于本发明组合中的抗体或其抗原结合片段在WO2018/53709、CN107840887中一般性地和具体地描述，该申请以引用方式并入本文。
在一个实施方案中，所述抗PD-1抗体或其抗原结合片段，其结合于PD-1的一个表位，所述表位包含：SEQ ID NO﹕24上第128、129、130、131和 132位点氨基酸和第35、64、82、83位中至少一个氨基酸。
在一个实施方案中，所述抗PD-1抗体或其抗原结合片段，其结合于人PD-1和鼠PD-1的一个表位，其中，所述表位包括SEQ ID NO﹕24上第128、129、130、131和132位点氨基酸。
在一个实施方案中，如上文所述的抗体或其抗原结合片段，其中鼠PD-1是小鼠或大鼠PD-1。
在一些实施方案中，如上文所述的抗体或其抗原结合片段，其中所述抗体
a)结合于人PD-1，KD为2.15E-10M以下；并且
b)结合于鼠PD-1，KD为1.67E-08M以下。
在一些实施方案中，上述抗体或其抗原结合片段具有下列性质中的至少一种：
a)结合于人PD-1，KD为4.32E-10M至2.15E-10M，并且结合于小鼠PD-1，KD为5.39E-08M至1.67E-08M；
b)实质上不结合于人CD28、CTLA-4；
c)增加T细胞的增殖；
d)增加干扰素-γ的产生；或
e)增加白细胞介素-2的分泌。
在上述方法、用途、组合、组合物、药物、制剂或试剂盒的实施方案中，上述抗体或其抗原结合片段，其包含一个氨基酸序列，所述氨基酸序列与选自由SEQ ID NOs﹕1、2、3、4、5、6、7、8和9所组成的组中的序列具有至少70％、80％、90％或95％的同源性，其中所述抗体特异性结合PD-1。
在上述方法、用途、组合、组合物、药物、制剂或试剂盒的实施方案中，上述抗体或其抗原结合片段，其包含一个氨基酸序列，所述氨基酸序列选自由SEQ ID NOs﹕1、2、3、4、5、6、7、8和9所组成的组中的序列，其中所述抗体特异性结合PD-1。
在上述方法、用途、组合、组合物、药物、制剂或试剂盒的实施方案中，上述抗体，或其抗原结合片段，包含：
a)重链可变区，其氨基酸序列与选自由SEQ ID NO﹕1和SEQ ID NO﹕2所组成的组中的序列具有至少70％、80％、90％或95％的同源性；以及
b)轻链可变区，其氨基酸序列与选自由SEQ ID NOs﹕3、4、5、6、7、 8和9所组成的组中的序列具有至少70％、80％、90％或95％的同源性，
其中所述抗体特异性结合PD-1。
在上述方法、用途、组合、组合物、药物、制剂或试剂盒的实施方案中，上述抗体或其抗原结合片段，包含：
a)重链可变区，其氨基酸序列选自由SEQ ID NO﹕1和SEQ ID NO﹕2组成的组中的序列；以及
b)轻链可变区，其氨基酸序列选自由SEQ ID NOs﹕3、4、5、6、7、8和9所组成的组中的序列，
其中所述抗体特异性结合PD-1。
在一些具体实施方案中，所述抗体包含：
a)重链可变区，其氨基酸序列选自SEQ ID NO﹕1所示的序列；以及
b)轻链可变区，其氨基酸序列选自SEQ ID NO﹕3所示的序列，
其中所述抗体特异性结合PD-1。
或在一些具体实施方案中，所述抗体包含：
a)重链可变区，其氨基酸序列选自SEQ ID NO﹕2所示的序列；以及
b)轻链可变区，其氨基酸序列选自SEQ ID NO﹕3所示的序列，
其中所述抗体特异性结合PD-1。
或在一些具体实施方案中，所述抗体其包含：
a)重链可变区，其氨基酸序列选自SEQ ID NO﹕2所示的序列；以及
b)轻链可变区，其氨基酸序列选自SEQ ID NO﹕4所示的序列，
其中所述抗体特异性结合PD-1。
或在一些具体实施方案中，所述抗体包含：
a)重链可变区，其氨基酸序列选自SEQ ID NO﹕2所示的序列；以及b)轻链可变区，其氨基酸序列选自SEQ ID NO﹕5所示的序列，
其中所述抗体特异性结合PD-1。
或在一些具体实施方案中，所述抗体包含：
a)重链可变区，其氨基酸序列选自SEQ ID NO﹕1所示的序列；以及
b)轻链可变区，其氨基酸序列选自SEQ ID NO﹕6所示的序列，
其中所述抗体特异性结合PD-1。
或在一些具体实施方案中，所述抗体包含：
a)重链可变区，其氨基酸序列选自SEQ ID NO﹕1所示的序列；以及
b)轻链可变区，其氨基酸序列选自SEQ ID NO﹕5所示的序列，
其中所述抗体特异性结合PD-1。
或在一些具体实施方案中，所述抗体包含：
a)重链可变区，其氨基酸序列选自SEQ ID NO﹕2所示的序列；以及
b)轻链可变区，其氨基酸序列选自SEQ ID NO﹕6所示的序列，
其中所述抗体特异性结合PD-1。
或在一些具体实施方案中，所述抗体包含：
a)重链可变区，其氨基酸序列选自SEQ ID NO﹕2所示的序列；以及
b)轻链可变区，其氨基酸序列选自SEQ ID NO﹕7所示的序列，
其中所述抗体特异性结合PD-1。
或在一些具体实施方案中，所述抗体包含：
a)重链可变区，其氨基酸序列选自SEQ ID NO﹕1所示的序列；以及
b)轻链可变区，其氨基酸序列选自SEQ ID NO﹕8所示的序列，
其中所述抗体特异性结合PD-1。
或在一些具体实施方案中，所述抗体包含：
a)重链可变区，其氨基酸序列选自SEQ ID NO﹕2所示的序列；以及
b)轻链可变区，其氨基酸序列选自SEQ ID NO﹕9所示的序列，
其中所述抗体特异性结合PD-1。
具体序列详见表1和序列表信息：
表1抗体的重链、轻链具体序列
Figure PCTCN2020133799-appb-000008
Figure PCTCN2020133799-appb-000009
另一方面，在上述方法、用途、组合、组合物、药物、制剂或试剂盒的实施方案中，上述抗体或其抗原结合片段，包含互补决定区(CDR)，其
氨基酸序列选自由SEQ ID NOs﹕10-23所组成的组中的序列，
其中所述抗体特异性结合PD-1。
另一方面，在上述方法、用途、组合、组合物、药物、制剂或试剂盒的实施方案中，上述抗体或其抗原结合片段，其包含：包含CDR1，CDR2和
CDR3序列的重链可变区；以及包含CDR1，CDR2和CDR3序列的轻链可变区，
其中重链可变区CDR3序列包含选自由SEQ ID NO﹕12和SEQ ID NO﹕13所组成的组
中的氨基酸序列及其保守性修饰，
其中所述抗体特异性结合PD-1。
上述抗体的轻链可变区CDR3序列优选包含选自由SEQ ID NOs﹕20、21、22和23所组
成的组中的氨基酸序列及其保守性修饰。
上述抗体的重链可变区CDR2序列优选包含选自由SEQ ID NO﹕11所组成的组中的
氨基酸序列及其保守性修饰。
上述抗体的轻链可变区CDR2序列优选包含选自由SEQ ID NO﹕19所组成的组中的
氨基酸序列及其保守性修饰。
上述抗体的重链可变区CDR1序列优选包含选自由SEQ ID NO﹕10所组成的组中的
氨基酸序列及其保守性修饰。
上述抗体的轻链可变区CDR1序列优选包含选自由SEQ ID NOs﹕14、15、16、17和18
所组成的组中的氨基酸序列及其保守性修饰。
在一些具体实施方案中，所述抗体或其抗原结合片段包含：
包含CDR1、CDR2和CDR3序列的重链可变区；以及
包含CDR1、CDR2和CDR3序列的轻链可变区，其中
a)重链可变区CDR1，序列包含选自SEQ ID NO﹕10所示的氨基酸序列，
b)重链可变区CDR2，序列包含选自SEQ ID NO﹕11所示的氨基酸序列，
c)重链可变区CDR3，序列包含选自由SEQ ID NO﹕12和SEQ ID NO﹕13所组成的组中所示的氨基酸序列，
d)轻链可变区CDR1，序列包含选自由SEQ ID NO﹕14-18所组成的组中所示的氨基酸序列，
e)轻链可变区CDR2，序列包含选自SEQ ID NO﹕19所示的氨基酸序列，
f)轻链可变区CDR3，序列包含选自由SEQ ID NO﹕20-23所组成的组中所示的氨基酸序列，
其中所述抗体特异性结合PD-1。
在一些具体实施方案中，所述抗体包含：
a)重链可变区CDR1，序列包含选自SEQ ID NO﹕10所示的氨基酸序列，
b)重链可变区CDR2，序列包含选自SEQ ID NO﹕11所示的氨基酸序列，
c)重链可变区CDR3，序列包含选自SEQ ID NO﹕12所示的氨基酸序列，
d)轻链可变区CDR1，序列包含选自SEQ ID NO﹕14所示的氨基酸序列，
e)轻链可变区CDR2，序列包含选自SEQ ID NO﹕19所示的氨基酸序列，
f)轻链可变区CDR3，序列包含选自SEQ ID NO﹕20所示的氨基酸序列，
其中所述抗体特异性结合PD-1。
在一些具体实施方案中，所述抗体包含：
a)重链可变区CDR1，序列包含选自SEQ ID NO﹕10所示的氨基酸序列，
b)重链可变区CDR2，序列包含选自SEQ ID NO﹕11所示的氨基酸序列，
c)重链可变区CDR3，序列包含选自SEQ ID NO﹕13所示的氨基酸序列，
d)轻链可变区CDR1，序列包含选自SEQ ID NO﹕14所示的氨基酸序列，
e)轻链可变区CDR2，序列包含选自SEQ ID NO﹕19所示的氨基酸序列，
f)轻链可变区CDR3，序列包含选自SEQ ID NO﹕21所示的氨基酸序列，
其中所述抗体特异性结合PD-1。
在一些具体实施方案中，所述抗体包含：
a)重链可变区CDR1，序列包含选自SEQ ID NO﹕10所示的氨基酸序列，
b)重链可变区CDR2，序列包含选自SEQ ID NO﹕11所示的氨基酸序列，
c)重链可变区CDR3，序列包含选自SEQ ID NO﹕13所示的氨基酸序列，
d)轻链可变区CDR1，序列包含选自SEQ ID NO﹕15所示的氨基酸序列，
e)轻链可变区CDR2，序列包含选自SEQ ID NO﹕19所示的氨基酸序列，
f)轻链可变区CDR3，序列包含选自SEQ ID NO﹕21所示的氨基酸序列，
其中所述抗体特异性结合PD-1。
在一些具体实施方案中，所述抗体包含：
a)重链可变区CDR1，序列包含选自SEQ ID NO﹕10所示的氨基酸序列，
b)重链可变区CDR2，序列包含选自SEQ ID NO﹕11所示的氨基酸序列，
c)重链可变区CDR3，序列包含选自SEQ ID NO﹕13所示的氨基酸序列，
d)轻链可变区CDR1，序列包含选自SEQ ID NO﹕16所示的氨基酸序列，
e)轻链可变区CDR2，序列包含选自SEQ ID NO﹕19所示的氨基酸序列，
f)轻链可变区CDR3，序列包含选自SEQ ID NO﹕21所示的氨基酸序列，
其中所述抗体特异性结合PD-1。
在一些具体实施方案中，所述抗体包含：
a)重链可变区CDR1，序列包含选自SEQ ID NO﹕10所示的氨基酸序列，
b)重链可变区CDR2，序列包含选自SEQ ID NO﹕11所示的氨基酸序列，
c)重链可变区CDR3，序列包含选自SEQ ID NO﹕12所示的氨基酸序列，
d)轻链可变区CDR1，序列包含选自SEQ ID NO﹕17所示的氨基酸序列，
e)轻链可变区CDR2，序列包含选自SEQ ID NO﹕19所示的氨基酸序列，
f)轻链可变区CDR3，序列包含选自SEQ ID NO﹕21所示的氨基酸序列，
其中所述抗体特异性结合PD-1。
在一些具体实施方案中，所述抗体包含：
a)重链可变区CDR1，序列包含选自SEQ ID NO﹕10所示的氨基酸序列，
b)重链可变区CDR2，序列包含选自SEQ ID NO﹕11所示的氨基酸序列，
c)重链可变区CDR3，序列包含选自SEQ ID NO﹕12所示的氨基酸序列，
d)轻链可变区CDR1，序列包含选自SEQ ID NO﹕16所示的氨基酸序列，
e)轻链可变区CDR2，序列包含选自SEQ ID NO﹕19所示的氨基酸序列，
f)轻链可变区CDR3，序列包含选自SEQ ID NO﹕21所示的氨基酸序列，
其中所述抗体特异性结合PD-1。
在一些具体实施方案中，所述抗体包含：
a)重链可变区CDR1，序列包含选自SEQ ID NO﹕10所示的氨基酸序列，
b)重链可变区CDR2，序列包含选自SEQ ID NO﹕11所示的氨基酸序列，
c)重链可变区CDR3，序列包含选自SEQ ID NO﹕13所示的氨基酸序列，
d)轻链可变区CDR1，序列包含选自SEQ ID NO﹕17所示的氨基酸序列，
e)轻链可变区CDR2，序列包含选自SEQ ID NO﹕19所示的氨基酸序列，
f)轻链可变区CDR3，序列包含选自SEQ ID NO﹕21所示的氨基酸序列，
其中所述抗体特异性结合PD-1。
在一些具体实施方案中，所述抗体包含：
a)重链可变区CDR1，序列包含选自SEQ ID NO﹕10所示的氨基酸序列，
b)重链可变区CDR2，序列包含选自SEQ ID NO﹕11所示的氨基酸序列，
c)重链可变区CDR3，序列包含选自SEQ ID NO﹕13所示的氨基酸序列，
d)轻链可变区CDR1，序列包含选自SEQ ID NO﹕17所示的氨基酸序列，
e)轻链可变区CDR2，序列包含选自SEQ ID NO﹕19所示的氨基酸序列，
f)轻链可变区CDR3，序列包含选自SEQ ID NO﹕22所示的氨基酸序列，
其中所述抗体特异性结合PD-1。
在一些具体实施方案中，所述抗体包含：
a)重链可变区CDR1，序列包含选自SEQ ID NO﹕10所示的氨基酸序列，
b)重链可变区CDR2，序列包含选自SEQ ID NO﹕11所示的氨基酸序列，
c)重链可变区CDR3，序列包含选自SEQ ID NO﹕12所示的氨基酸序列，
d)轻链可变区CDR1，序列包含选自SEQ ID NO﹕18所示的氨基酸序列，
e)轻链可变区CDR2，序列包含选自SEQ ID NO﹕19所示的氨基酸序列，
f)轻链可变区CDR3，序列包含选自SEQ ID NO﹕23所示的氨基酸序列，
其中所述抗体特异性结合PD-1。
在一些具体实施方案中，所述抗体包含：
a)重链可变区CDR1，序列包含选自SEQ ID NO﹕10所示的氨基酸序列，
b)重链可变区CDR2，序列包含选自SEQ ID NO﹕11所示的氨基酸序列，
c)重链可变区CDR3，序列包含选自SEQ ID NO﹕12所示的氨基酸序列，
d)轻链可变区CDR1，序列包含选自SEQ ID NO﹕18所示的氨基酸序列，
e)轻链可变区CDR2，序列包含选自SEQ ID NO﹕19所示的氨基酸序列，
f)轻链可变区CDR3，序列包含选自SEQ ID NO﹕20所示的氨基酸序列，
其中所述抗体特异性结合PD-1。
具体CDR序列详见表2信息：
表2的重链、轻链具体序列
Figure PCTCN2020133799-appb-000010
在一些实施方案中，所述抗体是嵌合抗体或人源化抗体或人抗体。
在一些实施方案中，其中所述抗体显示下列性质中的至少一种：
a)结合人PD-1的KD为2.15E-10M以下，并且结合小鼠PD-1的KD为1.67E-08M以下；
b)实质上不结合人CD28、CTLA-4；
c)增加T细胞增殖；
d)增加干扰素-γ的产生；或
e)增加白细胞介素-2的分泌。
蛋白酶体抑制剂
在上述方法、用途、组合、组合物、药物、制剂或试剂盒的实施方案中，上述蛋白酶体抑制剂为硼替佐米、卡非佐米、伊沙佐米或者马瑞佐米，优选为伊沙佐米。
治疗方法或用途
本发明涉及用于治疗受益于调节免疫功能的受试者病症的治疗方法，其包括向所述受试者施用药学有效量的HDAC6选择性抑制剂。本发明还涉及一种组合疗法，其包括向所述受试者施用药学有效量的HDAC6选择性抑制剂和一种或多种治疗剂。本发明还提供了一种治疗组合，其包含：(1)HDAC6选择性抑制剂，和(2)一种或多种治疗剂；其可用于分别、同时或顺序向有需要的个体施用以治疗或预防受益于调节免疫功能的受试者病症。
在一个方面，本发明涉及一种用于治疗受益于调节免疫功能的受试者病症的方法，其包括向所述受试者施用药学有效量的选择性HDAC6抑制剂和一种或多种治疗剂。本发明还涉及选择性HDAC6抑制剂和一种或多种治疗剂的组合在制备用于治疗受益于调节免疫功能的受试者病症的药物中的用途。在一个实施方案中，本发明涉及选择性HDAC6抑制剂在制备用于治疗受益于调节免疫功能的受试者病症的药物中的用途，所述选择性HDAC6抑制剂和一种或多种治疗剂组合。在另一个实施方案中，本发明涉及一种或多种治疗剂在制备用于治疗受益于调节免疫功能的受试者病症的药物中的用途，所述一种或多种治疗剂和选择性HDAC6抑制剂组合。
在上述方法或用途的实施方案中，所述选择性HDAC6抑制剂与一种或多种治疗剂同时、分别或顺序向该受试者施用。
在上述方法或用途的实施方案中，所述选择性HDAC6抑制剂为式(I)和式(II)所示化合物，或其药学上可接受的盐或对映异构体、非对映异构体、外消旋体、溶剂合物、水合物、多晶型、前药或活性代谢物，
Figure PCTCN2020133799-appb-000011
其中，
L1选自：单键、-NH-、-C(＝O)-NH-；
E 1、E 2选自-O-、-CH 2-和-CH 2-CH 2-；
R2选自：H、F、Cl、Br、I；
R选自：F、Cl、Br、I；
n选自：0或1。
优选地，选择性HDAC6抑制剂为下列化合物或或其药学上可接受的盐或对映异构体、非对映异构体、外消旋体、溶剂合物、水合物、多晶型、前药或活性代谢物：
Figure PCTCN2020133799-appb-000012
优选的，所述选择性HDAC6抑制剂为式A的化合物或其药学上可接受的盐或对映异构体、非对映异构体、外消旋体、溶剂合物、水合物、多晶型、前药或活性代谢物:
Figure PCTCN2020133799-appb-000013
在上述方法或用途的实施方案中，所述一种或多种治疗剂为(1)PD-1轴结合拮抗剂或者(2)蛋白酶体抑制剂。
在一个实施方案中，所述蛋白酶体抑制剂为硼替佐米、卡非佐米、伊沙佐米或者马瑞佐米。
在一个实施方案中，上述PD-1轴结合拮抗剂选自PD-1结合拮抗剂；优选的，PD-1轴结合拮抗剂是抗体或其抗原结合片段；尤其优选地，该抗体是抗PD-1抗体；最优选地，该抗体是单克隆抗体。
本文所述的任何PD-1轴结合拮抗剂均可用于本发明的方法或用途。
在一个实施方案中，所述抗体或其抗原结合片段结合于PD-1的一个表位，所述表位包含：SEQ ID NO﹕24上第128、129、130、131和132位点氨基酸和第35、64、82、83位中至少一个氨基酸；
优选的，所述抗体或其抗原结合片段结合于人PD-1和鼠PD-1的一个表位，其中，所述表位包括SEQ ID NO﹕24上第128、129、130、131和132位点氨基酸。
在一个实施方案中，所述抗体或其抗原结合片段包含一个氨基酸序列，所述氨基酸序列与选自由SEQ ID NOs﹕1、2、3、4、5、6、7、8和9所组成的组中的序列具有至少70％、80％、90％或95％的同源性，其中所述抗体特异性结合PD-1；优选的，所述抗体或其抗原结合片段包含一个氨基酸序列，所述氨基酸序列选自由SEQ ID NOs﹕1、2、3、4、5、6、7、8和9所组成的组中的序列。
在一个实施方案中，所述抗体或其抗原结合片段包含：
a)重链可变区，其具有的氨基酸序列选自由SEQ ID NO﹕1和SEQ ID NO﹕2所组成的组中的序列或与其具有至少70％、80％、90％或95％的同源性的序列；以及
b)轻链可变区，其具有的氨基酸序列选自由SEQ ID NOs﹕3、4、5、6、 7、8和9所组成的组中的序列或与其具有至少70％、80％、90％或95％的同源性的序列，
其中所述抗体特异性结合PD-1。
优选的，所述抗体或其抗原结合片段包含选自表1中所述的抗体的重链、轻链具体序列。
更优选的，所述抗体或其抗原结合片段包含：
a)重链可变区，其氨基酸序列选自SEQ ID NO﹕2所示的序列；以及
b)轻链可变区，其氨基酸序列选自SEQ ID NO﹕3所示的序列。
在一个实施方案中，所述抗体或其抗原结合片段包含互补决定区(CDR)，其具有的氨基酸序列选自由SEQ ID NOs﹕10-23所组成的组中的序列，其中所述抗体特异性结合PD-1；
优选的，所述抗体或其抗原结合片段包含互补决定区(CDR)，其包含选自表2的CDR序列；
更优选的，所述抗体包含：
a)重链可变区CDR1，序列包含选自SEQ ID NO﹕10所示的氨基酸序列，
b)重链可变区CDR2，序列包含选自SEQ ID NO﹕11所示的氨基酸序列，
c)重链可变区CDR3，序列包含选自SEQ ID NO﹕13所示的氨基酸序列，
d)轻链可变区CDR1，序列包含选自SEQ ID NO﹕14所示的氨基酸序列，
e)轻链可变区CDR2，序列包含选自SEQ ID NO﹕19所示的氨基酸序列，
f)轻链可变区CDR3，序列包含选自SEQ ID NO﹕21所示的氨基酸序列。
在一个实施方案中，所述一种或多种治疗剂为蛋白酶体抑制剂，优选为硼替佐米、卡非佐米、伊沙佐米或者马瑞佐米。
在一个实施方案中，所述选择性HDAC6抑制剂为下式化合物A或对映异构体、非对映异构体、外消旋体、溶剂合物、水合物、多晶型、前药或活性代谢物:
Figure PCTCN2020133799-appb-000014
所述一种或多种治疗剂为抗体2E5或其抗原结合片段，包含：
a)重链可变区，其氨基酸序列选自SEQ ID NO﹕2所示的序列；以及
b)轻链可变区，其氨基酸序列选自SEQ ID NO﹕3所示的序列，
其中所述抗体特异性结合PD-1。
在一个实施方案中，所述选择性HDAC6抑制剂为下式化合物A或对映异构体、非对映异构体、外消旋体、溶剂合物、水合物、多晶型、前药或活性代谢物:
Figure PCTCN2020133799-appb-000015
并且，所述一种或多种治疗剂为蛋白酶体抑制剂，优选为硼替佐米、卡非佐米、伊沙佐米或者马瑞佐米。
适应症和受试者
本发明的方法或用途可用于治疗治疗或预防免疫病症或受益于调节免疫功能的受试者病症，例如癌症。本发明的方法或用途还用于抑制受试者中肿瘤细胞的生长。在一个实施方案中，上述肿瘤细胞选自多发性骨髓瘤(MM)、黑素瘤、肾癌、前列腺癌、乳腺癌、结肠癌、肺癌、骨癌、胰腺癌、皮肤癌、头部或颈部癌、皮肤或眼内恶性黑素瘤、子宫癌、卵巢癌和直肠癌。
在上述的一些实施方案中，所述癌症为实体瘤或血液癌，如非小细胞肺癌、小细胞肺癌、肾细胞癌、结肠直肠癌、卵巢癌、乳腺癌、胰腺癌、胃癌、膀胱癌、食道癌、间皮瘤、黑素瘤、头颈癌、甲状腺癌、肉瘤、前列腺癌、胶质母细胞瘤、宫颈癌、胸腺癌、白血病、淋巴瘤、骨髓瘤、真菌病真菌，默克尔细胞癌和其他血液系统恶性肿瘤，例如经典霍奇金淋巴瘤(CHL)、原发性纵隔大B细胞淋巴瘤、T-细胞/组织细胞丰富的B细胞淋巴瘤、EBV阳性和阴性PTLD、以及EBV相关的弥漫性大B细胞淋巴瘤(DLBCL)、浆母细胞淋巴瘤、结外NK/T细胞淋巴瘤、鼻咽癌和HHV8-相关的原发性渗出性 淋巴瘤、霍奇金淋巴瘤、中枢神经系统肿瘤(CNS)、例如原发性中枢神经系统淋巴瘤、脊髓轴肿瘤、脑干神经胶质瘤；优选的，所述癌症选自黑素瘤、肾癌、前列腺癌、乳腺癌、结肠癌、肺癌、骨癌、胰腺癌、皮肤癌、头部或颈部癌、皮肤或眼内恶性黑素瘤、子宫癌、卵巢癌和直肠癌。
本文描述的方法还可用于治疗需要增强免疫原性的病症，例如增加用于治疗癌症的肿瘤免疫原性。本文还提供了在患有癌症的受试者中增强免疫功能的方法，包括向该受试者施用有效量的选择性HDAC6抑制剂和抗PD-1抗体或其抗原结合片段。
本文还提供了用于治疗或延缓受试者癌症发展的方法，包括向该个体施用有效量的选择性HDAC6抑制剂和抗PD-1抗体或其抗原结合片段；或者施用有效量的选择性HDAC6抑制剂和硼替佐米、卡非佐米、伊沙佐米或者马瑞佐米。
在上述实施方案中，在用选择性HDAC6抑制剂和抗PD-1抗体或其抗原结合片段联合治疗之前，已经用选择性HDAC6抑制剂治疗了受试者。
在一些实施方案中，受试者患有对HDAC6抑制剂具有抗性的癌症(例如耐药性多发性骨髓瘤)。在一些实施方案中，对HDAC6抑制剂的抗性包括癌症或难治性癌症的复发。复发可能是指治疗后在原始部位或新部位再次出现癌症。在一些实施方案中，对HDAC6抑制剂抗性包括在用对HDAC6抑制剂治疗期间癌症的进展。在一些实施方案中，对HDAC6抑制剂的抗性包括对治疗无反应的癌症。癌症可能在治疗开始时产生耐药性，或者在治疗期间变得耐药。在一些实施方案中，癌症处于早期或晚期。
可以在本领域已知的各种模型中测试本文所述的任何方法的功效(例如，包括给予有效量的选择性HDAC6抑制剂和抗PD-1抗体或其抗原结合片段的组合的联合治疗)，例如临床或临床前临床模型。
剂量水平和剂量方案
可以施用有效量的上文中所述的选择性HDAC6抑制剂(例如化合物A)和抗PD-1抗体或其抗原结合片段(例如抗体2E5)；或者选择性HDAC6抑制剂(例如化合物A)和蛋白酶体抑制剂(例如，硼替佐米、卡非佐米、伊沙佐米或者马瑞佐米)以预防或治疗疾病。上文中所述的选择性HDAC6抑制剂、抗PD-1抗体或其抗原结合片段以及蛋白酶体抑制剂的适当剂量可以根据待治 疗的疾病类型，选择性HDAC6抑制剂、抗PD-1抗体或其抗原结合片段以及蛋白酶体抑制剂的类型，疾病的严重程度和病程，治疗的严重程度、个体的临床症状，个体的临床病史和对治疗的反应来确定。在一些实施方案中，用上文中所述的选择性HDAC6抑制剂和抗PD-1抗体或其抗原结合片段的联合治疗、或者选择性HDAC6抑制剂和蛋白酶体抑制剂的联合治疗是协同的，因此相对于单独的选择性HDAC6抑制剂或抗PD-1抗体(或其抗原结合片段)或者蛋白酶体抑制剂的有效剂量而言，降低了组合中的有效剂量。
一般而言，本文提供给人的PD-1抗体或其抗原结合片段的治疗有效量为约0.0001mg/kg至约100mg/kg(例如，约0.01mg/kg、约0.1mg/kg、约0.5mg/kg、约1mg/kg)的患者体重，无论是一次还是多次给药。在一些实施方案中，所使用的抗体或其抗原结合片段是约0.01mg/kg、约0.1mg/kg、约0.3mg/kg、约0.4mg/kg、约0.5mg/kg、约0.6mg/kg、约0.7mg/kg、约0.8mg/kg、约0.9mg/kg、约1mg/kg、约2mg/kg、约5mg/kg、约10mg/kg、约15mg/kg、约20mg/kg、25mg/kg、约30mg/kg、约35mg/kg、约40mg/kg、约45mg/kg、约50mg/kg、约55mg/kg、约60mg/kg、约65mg/kg、约70mg/kg、约75mg/kg、约80mg/kg、约85mg/kg、约90mg/kg、约95mg/kg或约100mg/kg。在一些实施方案中，以约50mg/kg或更少、10mg/kg或更少、5mg/kg或更少、1mg/kg或更少、0.5mg/kg或更少、或小于或等于0.1mg/kg的剂量施用抗体或其抗原结合片段。
例如，PD-1抗体或其抗原结合片段以约1mg至约100mg的单位剂量，优选以约5mg、约10mg、15mg、20mg、25mg、30mg、35mg、40mg、45mg、50mg的单位剂量施用。
一般而言，上文中所述的选择性HDAC6抑制剂(例如化合物A)的合适的剂量应为有效产生治疗作用的最低剂量的化合物的量。这样的有效剂量通常将取决于上述因素。通常，本发明化合物对患者的剂量范围为每千克体重约0.0001至约100mg，优选约1mg/kg、约2mg/kg、约5mg/kg、约10mg/kg、约15mg/kg、约20mg/kg。例如，剂量可以在约10至2000mg之间；或者，剂量可以是每天约100至1000mg，或每天约200至约600mg。如果需要，活性化合物的有效日剂量可以一，二，三，四或更多个亚剂量在一天中的适当间隔分别以单位剂型的形式给药。优选的，单位剂量为约10至1000mg、更优选约100至1000mg，约150至约600mg、约200至约500mg、或约300 至约400mg、尤其优选约10mg、约50mg、约100mg、约150mg、约200mg、约250mg、约300mg、约350mg、约400mg、约450mg、约500mg。
一般而言，上文中所述的蛋白酶抑制剂(例如，硼替佐米、卡非佐米、伊沙佐米或者马瑞佐米)的合适的剂量应为有效产生治疗作用的最低剂量的化合物的量。这样的有效剂量通常将取决于上述因素。通常，本发明化合物对患者的剂量范围为每千克体重约0.0001至约100mg，优选约约0.01mg/kg、约0.1mg/kg、约0.3mg/kg、约0.4mg/kg、约0.5mg/kg、约0.6mg/kg、约0.7mg/kg、约0.8mg/kg、约0.9mg/kg、约1mg/kg、约2mg/kg、约5mg/kg、约10mg/kg、约15mg/kg、约20mg/kg。例如，剂量可以在约0.1至1000mg之间。或者，剂量可以是约1至500mg，约5至200mg，约10至50mg，或优选约1mg、约2mg、约3mg、约4mg、约5mg、约10mg、15mg、20mg、25mg、30mg、35mg、40mg、45mg、或者50mg。如果需要，活性化合物的有效日剂量可以一、二、三、四或更多个亚剂量在一天中的适当间隔分别以单位剂型的形式给药。优选的，单位剂量为约1至500mg、约5至200m、约10至50mg，或优选约1mg、约2mg、约3mg、约4mg、约5mg、约10mg、约15mg、约20mg、约25mg、约30mg、约35mg、约40mg、约45mg、或者约50mg。
在一些实施方案中，施用剂量可以在治疗过程中改变。例如，在一些实施方案中，初始施用剂量可以高于随后的施用剂量。在一些实施方案中，取决于受试者的反应，给药剂量可以在治疗过程中变化。
上述选择性HDAC6抑制剂或者一种多种治疗剂可以多次间隔施用特定剂量，例如每天一次，每天两次或更多次；每周一次，每周两次或更多次；每月一次，两次或更多次；每周一次，每两周一次，每三周一次，每月一次或每两个月一次，或者更少。在一些实施方案中，施用的剂量可以随治疗过程而变化。例如，在某些实施方案中，施用的初始剂量可以高于随后的剂量。在某些实施方案中，根据治疗对象的反应，在治疗过程中调节给药剂量。可以调整剂量方案以达到最佳应答(例如治疗应答)。例如，可以在一段时间内施用单剂量或多次分剂量施用。
在一些实施方案中，本发明的联合疗法包括施用上文中所述的选择性HDAC6抑制剂和一种或多种治疗剂(例如抗PD-1抗体或其抗原结合片段或者蛋白酶体抑制剂)。上文中所述的选择性HDAC6抑制剂和一种或多种治 疗剂(例如抗PD-1抗体或其抗原结合片段或者蛋白酶体抑制剂)可以以本领域已知的任何合适方式施用。例如，上文中所述的选择性HDAC6抑制剂和一种或多种治疗剂(例如抗PD-1抗体或其抗原结合片段或者蛋白酶体抑制剂)可以依次给药(在不同时间)或同时给药(同时)。在一些实施方案中，选择性HDAC6抑制剂和一种或多种治疗剂(例如抗PD-1抗体或其抗原结合片段或者蛋白酶体抑制剂)以单独的制剂形式存在；在一些实施方案中，选择性HDAC6抑制剂和一种或多种治疗剂(例如抗PD-1抗体或其抗原结合片段或者蛋白酶体抑制剂)存在于同一组合物或者制剂中。
上文中所述的选择性HDAC6抑制剂和一种或多种治疗剂(例如抗PD-1抗体或其抗原结合片段或者蛋白酶体抑制剂)可以通过相同的给药途径或不同的给药途径来给药。在一些实施方案中，PD-1抗体或其抗原结合片段通过植入、通过吸入、鞘内、心室内或鼻内静脉内、肌内、皮下、局部、口服、经皮、腹膜内、眶内、口服给药，优选静脉内给药。在一些实施方案中，上文中所述的选择性HDAC6抑制剂通过植入、吸入、鞘内、心室内或鼻内静脉内、肌内、皮下、局部、口服、经皮、腹膜内、眶内、口服给药，优选口服给药。
在一些实施方案中，本发明的联合疗法中的每种治疗剂可以同时(即，在相同药物中)，同时(即，在以任何顺序一个接一个地给药的单独药物中)或以任何顺序顺序给药。当组合疗法中的治疗剂为不同剂型(一种药物为片剂或胶囊剂，另一种药物为无菌液体)和/或按不同的给药方案(例如化疗药物)给药时，顺序给药特别有用。至少每天一次给药，以及不那么频繁地给药的生物治疗剂，例如每周一次，每两周一次或每三周一次。
在一另方面，本发明的联合疗法中的治疗剂(例如抗PD-1抗体或其抗原结合片段或者蛋白酶体抑制剂)与选择性HDAC6抑制剂提供协同组合，其组合范围比例介于1:200到200:1重量份数之间优选的，可为约1:100、约1:95、约1:90、约1:85、约1:80、约1:75、约1:70、约1:65、约1:60、约1:55、约1:50、约1:45、约1:40、约1:35、约1:30、约1:25、约1:20、约1:15、约1:10、约1:8、约1:6、约1:5、约1:4、约1:3、约1:2或约1:1、约2:1、约3:1、约4:1、约5:1、约6:1、约10:1、约20:1、约30:1、约40:1、约50:1、约60:1、约70:1、约80:1、约90:1、约100:1。
在一些实施方案中、所述方法可以进一步包括另外的疗法。附加疗法可 以是放射疗法、手术(例如、乳房切除术和乳房切除术)、化学疗法、基因疗法、DNA疗法、病毒疗法、RNA疗法、免疫疗法、骨髓移植、纳米疗法、单克隆抗体疗法或前述的组合。附加疗法可以采取辅助或新辅助疗法的形式。在一些实施方案中，另外的疗法是小分子酶抑制剂或抗转移剂的施用。在一些实施方案中，另外的疗法是施用副作用限制剂(例如，旨在减轻治疗副作用的发生和/或严重性的试剂，例如抗恶心剂等)。在一些实施方案中，另外的疗法是放射疗法。在一些实施方案中，该另外的疗法是手术。在一些实施例中，附加疗法是放射疗法和手术的组合。在一些实施方案中，另外的疗法是伽马辐射。
本发明的联合疗法可以在手术之前或之后用于去除肿瘤，并且可以在放射疗法之前，之中或之后使用。在一些实施方案中，将本发明的联合疗法施用于先前未曾用生物治疗剂或化学治疗剂治疗过的患者。在其他实施方案中，将组合疗法施用于在用生物治疗剂或化学治疗剂(即经历过治疗)的先前治疗后未能实现持续应答的患者。
药物组合物、制剂或者试剂盒
本发明提供了一种组合物，其包含如上文所述的选择性HDAC6抑制剂(例如化合物A)和抗PD-1抗体或其抗原结合片段(例如抗体2E5)以及任选的药学上可接受的赋形剂或载体。本发明还提供了一种组合物，其包含如上文所述的选择性HDAC6抑制剂(例如化合物A)和蛋白酶体抑制剂(例如硼替佐米、卡非佐米、伊沙佐米或者马瑞佐米)以及任选的药学上可接受的赋形剂或载体。
用于本文公开的药物组合物中的药学上可接受的赋形剂或载体可包括例如药学上可接受的液体、凝胶或固体载体、水性介质、非水性介质、抗微生物材料、渗透材料、缓冲剂、抗氧化剂、麻醉剂、悬浮/分散剂、螯合剂、稀释剂、佐剂、剂或无毒辅助物质，本领域已知的其他组分或上述的各种组合。
合适的组分可以包括例如抗氧化剂、填充剂、粘合剂、崩解剂、缓冲剂、防腐剂、润滑剂、增香剂、增稠剂、着色剂、乳化剂或稳定剂，例如糖和环糊精。合适的抗氧化剂可以包括例如甲硫氨酸、抗坏血酸、EDTA、硫代硫酸钠、铂、过氧化氢酶、柠檬酸、半胱氨酸、巯基甘油、巯基乙醇酸、脱水 山梨醇、丁基茴香醚、丁基化羟基甲苯和/或没食子酸丙酯。
此外，药学上可接受的赋形剂或载体可包括例如水性媒介物，例如氯化钠注射剂、林格氏注射剂、等渗右旋糖注射剂、无菌水注射剂或右旋糖和乳酸林格氏剂注射剂，非水媒介物例如固定油。植物来源、棉籽油、玉米油、芝麻油或花生油、抑菌或抑菌浓度的抗菌剂、等渗剂(例如氯化钠或葡萄糖)、缓冲剂(例如磷酸盐或柠檬酸盐缓冲剂)、抗氧化剂(例如硫酸氢钠)、局部麻醉剂作为盐酸普鲁卡因、悬浮剂和分散剂，例如羧甲基纤维素钠，羟丙基甲基纤维素或聚乙烯吡咯烷酮、乳化剂，例如聚山梨酯80(TWEEN-80)，螯合剂或螯合剂，例如EDTA(乙二胺四乙酸)或EGTA(乙二醇四乙酸)、乙醇、聚乙二醇、丙二醇、钠氢氧化物、盐酸、柠檬酸或乳酸。可用作载体的抗微生物剂可以在多剂量容器中添加到药物组合物中，该容器包括苯酚或甲酚、汞、苯甲醇、氯丁醇、对羟基苯甲酸甲酯和丙基对羟基苯甲酸酯、硫柳汞、苯扎氯铵和苄索氯铵。合适的赋形剂可包括例如水、盐水、右旋糖、甘油或乙醇。合适的无毒辅助物质可以包括，例如，润湿剂或乳化剂、pH缓冲剂、稳定剂、溶解度增强剂、或诸如乙酸钠、脱水山梨糖醇单月桂酸酯、三乙醇胺油酸酯或环糊精的试剂。
药物组合物可以是液体溶液、悬浮液、乳剂、丸剂、胶囊剂、片剂、缓释制剂或粉剂。口服制剂可以包括标准载体，例如药用级的甘露醇、乳糖、淀粉、硬脂酸镁、聚乙烯吡咯烷酮、糖精钠、纤维素、碳酸镁等。
在一些实施方案中，药物组合物被配制成可注射的组合物。可以以任何常规形式制备注射用药物组合物，例如液体溶剂、悬浮液、乳化剂或适合于生产液体溶剂，悬浮液或乳化剂的固体形式。
本发明还提供了一种药物，其包含如上文所述的选择性HDAC6抑制剂和抗PD-1抗体或其抗原结合片段以及任选的药学上可接受的赋形剂或载体。本发明还提供了一种药物，其包含如上文所述的选择性HDAC6抑制剂和蛋白酶体抑制剂(硼替佐米、卡非佐米、伊沙佐米或者马瑞佐米)以及任选的药学上可接受的赋形剂或载体。
在一些实施方案中，包含上文所述的选择性HDAC6抑制剂和抗PD-1抗体或其抗原结合片段的药物可以液体制剂形式提供或通过在使用前用无菌水用注射用水重构冻干粉末来制备。
本文所述的选择性HDAC6抑制剂和抗PD-1抗体或其抗原结合片段(或 者上文所述的选择性HDAC6抑制剂和硼替佐米、卡非佐米、伊沙佐米或者马瑞佐米)可以作为制品或试剂盒提供，其包括第一容器和第二容器以及包装说明书。第一容器包含至少一个剂量的包含PD-1轴结合拮抗剂的药物，第二容器包含至少一个剂量的包含非索非替尼的药物，以及包装插页或标签，其中包含用于治疗患者的说明书，癌症使用药物。在一些实施方案中，制品或试剂盒还包括包装插页，该包装插页包含用于选择性HDAC6抑制剂和抗PD-1抗体或其抗原结合片段(或者上文所述的选择性HDAC6抑制剂和硼替佐米、卡非佐米、伊沙佐米或者马瑞佐米)联用以治疗或延迟个体癌症的进展或增强患有癌症的个体的免疫功能的说明书。第一和第二容器可以由相同或不同的形状(例如，小瓶、注射器和瓶子)和/或材料(例如，塑料或玻璃)组成。
制品或药盒还可包含可用于施用药物的其他材料，例如稀释剂、过滤器、静脉输液袋和输液管、针头和注射器。在一些实施方案中，制品还包括一种或多种其他试剂(例如，化学治疗剂和抗肿瘤剂)。用于一种或多种试剂的合适容器包括，例如，瓶、小瓶、袋子和注射器。
本文所述的任何选择性HDAC6抑制剂、抗PD-1抗体或其抗原结合片段或者蛋白酶体抑制剂均可用于本发明的药物组合物，药物和试剂盒中。例如，在一些实施方案中，选择性HDAC6抑制剂是化合物A，抗PD-1抗体或其抗原结合片段是抗体2E5，其包含：a)重链可变区，其氨基酸序列选自SEQ ID NO﹕2所示的序列；以及b)轻链可变区，其氨基酸序列选自SEQ ID NO﹕3所示的序列。
在一些实施方案中，在上述组合物、制剂或者试剂盒中，选择性HDAC6抑制剂(例如化合物A)和抗PD-1抗体或其抗原结合片段(例如抗体2E5)以及蛋白酶体抑制剂(硼替佐米、卡非佐米、伊沙佐米或者马瑞佐米)具有本文中所描述的剂量和组合比例。例如，PD-1抗体或其抗原结合片段的剂量为约1mg至约100mg的单位剂量，优选以约5mg、约10mg、15mg、20mg、25mg、30mg、35mg、40mg、45mg、50mg；选择性HDAC6抑制剂(例如化合物A)的剂量为约10至1000mg，更优选约100至1000mg、约150至约600mg、约200至约500mg或约300至约400mg，尤其优选约10mg、约50mg、约100mg、约150mg、约200mg、约250mg、约300mg、约350mg、约400mg、约450mg、约500mg；或者蛋白酶体抑制剂(硼替佐米、卡非佐米、伊 沙佐米或者马瑞佐米)的剂量为约1至500mg，约5至200mg，约10至50mg，或优选约1mg、约2mg、约3mg、约4mg、约5mg、约10mg、约15mg、约20mg、约25mg、约30mg、约35mg、约40mg、约45mg、或者约50mg。
在一另方面，在上述组合物、制剂或者试剂盒中，一种或多种治疗剂(例如抗PD-1抗体或其抗原结合片段或者蛋白酶体抑制剂)与HDAC6抑制剂提供协同组合，其组合范围比例介于1:200到200:1重量份数之间，优选的，可为约1:100、约1:95、约1:90、约1:85、约1:80、约1:75、约1:70、约1:65、约1:60、约1:55、约1:50、约1:45、约1:40、约1:35、约1:30、约1:25、约1:20、约1:15、约1:10、约1:8、约1:6、约1:5、约1:4、约1:3、约1:2或约1:1、约2:1、约3:1、约4:1、约5:1、约6:1、约10:1、约20:1、约30:1、约40:1、约50:1、约60:1、约70:1、约80:1、约90:1、约100:1。
根据本文包含的教导，本发明的这些和其他方面，包括下面列出的示例性具体实施方案，将是显而易见的。该说明书被认为足以使本领域技术人员能够实施本发明。根据前面的描述，除了本文中示出和描述的之外，本发明的各种修改对于本领域技术人员而言将变得显而易见，并且落入所附权利要求的范围内。本文引用的所有出版物，专利和专利申请出于所有目的通过引用整体并入本文。
具体实施方式
实施例
缩略语表
缩略语 定义
p.o. 灌胃给药
QD 每天一次
QW 每周一次
Rb 视网膜母细胞瘤
s.c. 皮下注射
i.v. 静脉给药
SEM 标准误差
SERD 选择性雌激素受体下调剂
TGI 肿瘤生长抑制
实施例1
试剂和细胞系
式A化合物和ACY-1215由Wuxi Biologics(中国，上海)制备。HT-29和NCI-H292购自ATCC。肿瘤细胞分别培养于McCoy's 5A(Thermo Fisher Scientific,16600082)或RPMI 1640(Thermo Fisher Scientific,110905098)，该培养基补充有10％FBS(Thermo Fisher Scientific,10099141)和1％青霉素-链霉素(Thermo Fisher Scientific,15140122)。
HDAC酶测定
溶于测定缓冲液[50mM HEPES,pH 7.4,100mM KCl,0.001％Tween-20,0.05％BSA，以及20μM三(2-羧乙基)膦]中溶解ACY-1215并随后稀释至最终浓度的6倍。在添加底物之前，将HDAC酶在测定缓冲液中稀释至最终浓度的1.5倍，并与ACY-1215预温育10分钟。每种酶使用的FTS(HDAC1、HDAC2、HDAC3和HDAC6)或MAZ-1675(HDAC4、HDAC5、HDAC7、HDAC8和HDAC9)的量等于米氏常数(Km)，其通过滴定曲线确定。用0.3M测序级胰蛋白酶(Sigma-Aldrich)将FTS或MAZ-1675在测定缓冲液中稀释至最终浓度的6倍。将底物/胰蛋白酶混合物添加到酶/化合物混合物中，并将板摇动60秒，然后放入SpectraMax M5微量滴定板读数器中。在肽底物中的赖氨酸侧链脱乙酰基化后，在30分钟内监测酶促反应中7-氨基-4-甲氧基香豆素的释放，并计算反应的线性速率。由Cerep进行HDAC11、sirtuin1和sirtuin2测定。
体内抗肿瘤活性
研究中与动物操作、护理和处理有关的所有程序均按照国际实验动物评估和认可委员会(AAALAC)的指导原则进行。在接种肿瘤之前，收集处于指数生长期的MM.1S(购自ATCC)和CT26(购自ATCC)并通过细胞计数器定量。每只小鼠在右后侧皮下接种补充有基质胶(1：1)的0.2ml PBS中的MM.1S(5x10 6)肿瘤细胞或0.2ml PBS中的CT26(3x10 5)肿瘤细胞，用于肿瘤发展。当MM.1S模型中的平均肿瘤尺寸达到150mm 3且CT26模型中的平均肿瘤尺寸达到70mm 3时开始治疗。随机化日期被标记为第1天。使用卡尺在两个维度测量肿瘤体积，每周3次(MM.1S)或两次(CT26)，并使用以下 公式以mm 3表示体积：V＝(L x W x W)/2，其中V是肿瘤体积，L是肿瘤长度(最长的肿瘤维度)且W是肿瘤宽度(垂直于L的最长的肿瘤维度)。每周两次测量肿瘤体积和体重。使用以下公式计算每组的肿瘤生长抑制(TGI)：TGI(％)＝[1-(Ti-T0)/(Vi-V0)]×100；Ti是给定日中治疗组的平均肿瘤体积，T0是分组当天治疗组的平均肿瘤体积，Vi是在与Ti同一天的媒介物对照组平均肿瘤体积，且V0是分组当天媒介物组的平均肿瘤体积。使用药物相互作用系数(CDI)分析药物组合的协同抑制作用。CDI按以下公式计算：CDI＝AB/(A×B)。AB是2种药物联合治疗组与对照组的肿瘤体积的比值，且A或B是单一药物组与对照组的肿瘤体积的比值。因此，CDI<1表示协同作用，CDI<0.7表示明显的协同作用，CDI＝1表示可加性，CDI>1表示拮抗作用。
流式细胞仪
根据制造商的规程，使用可固定活性染料700(BD Biosciences,564997)进行活/死细胞鉴别。避光在4℃下进行细胞表面染色30分钟。使用的抗体包括PE小鼠抗人类CD11c(BD Biosciences,555392)、BB515小鼠抗人类CD86(BD Biosciences,564544)、PE小鼠抗人类CD274(BD Biosciences,557924)。所有流式细胞仪分析均使用Cytoflex(Beckman Coulter)进行，并使用FlowJo软件(BD Biosciences)进行分析。
蛋白质印迹法
经过指定的处理后，从小鼠模型中收集肿瘤组织，并在两次冰PBS洗涤后于-80℃的冷冻器中速冻。用含有蛋白酶抑制剂混合物(Roche,04693124001)和磷酸酶抑制剂混合物2(Sigma，P5726)的RIPA裂解缓冲液(Sigma，R0278)裂解样品。用Tissuelyser LT全速研磨肿瘤5分钟，并将组织裂解物在冰上放置30分钟。通过Pierce TMBCA蛋白质分析试剂盒确定蛋白质浓度(Thermo Scientific,23225)。将样品在样品缓冲液中变性，然后根据分子量在4-12％Bis-Tris凝胶(Thermo Scientific，NP0336BOX)上分离等量的蛋白质，并转移到聚偏二氟乙烯膜上。于室温，在TBST的5％脱脂牛奶中将膜封闭1小时，并在4℃下用所示的一抗探测过夜：Ac-α-微管蛋白抗体(Cell Signaling Technology,#5335)；抗乙酰基组蛋白H3(Lys9)抗体(Millipore, #07-352)；GAPDH(Cell Signaling Technology,5174)。用TBST将印迹洗涤三次约15分钟，然后与以下二抗一起温育1小时：山羊抗兔二抗(Thermo,31462)；山羊抗小鼠二抗(Santa,F2816)。TBST洗涤3次后，将West Femto最高灵敏度试剂盒(Thermo Scientific，34096)中的HRP底物添加到膜上，并用Tanon 5200multi检测化学发光。
统计分析
细胞研究中获得的数据通过未配对的Student t检验进行评估。在Levene检验之后，通过独立T检验分析了肿瘤重量或肿瘤体积数据。P<0.05表明存在显著差异。
实施例1-1
式A化合物在HDAC抑制方面具有独特的特征
为了评估式A化合物对HDAC6和其他HDAC家族成员的抑制能力和选择性，使用了无细胞HDAC酶测定。对比ACY-1215(Ricolinostat,选择性HDAC6抑制剂)，示出了式A化合物的结构(图1)。体外研究表明式A化合物是具有不同HDAC抑制特征的HDAC6选择性抑制剂(表3)。结果表明式A化合物抑制HDAC6活性(IC 50＝0.90nM)和HDAC1活性(IC 50＝73.64nM,HDAC1/HDAC6选择性倍数＝81.82)的功效与ACY-1215至少相当(HDAC6IC 50＝2.19nM,HDAC1IC 50＝63.99nM,HDAC1/HDAC6选择性倍数＝29.21)。除了HDAC6和HDAC1，还检查了式A化合物对HDAC家族的抑制作用的选择性(Reaction Biology Corporation,PA,US)。结果显示式A化合物抑制HDAC2活性(IC 50＝2973nM)、HDAC3活性(IC 50＝1990nM)、HDAC4活性(IC 50＝919nM)、HDAC5活性(IC 50＝442nM)、HDAC7活性(IC 50＝254nM)、HDAC8活性(IC 50＝137nM)、HDAC9活性(IC 50＝773nM)、HDAC10活性(IC 50＝2890nM)、HDAC11活性(IC 50＝812nM)(表3)。以上结果表明，式A化合物是具有高度HDAC6/HDAC1选择性的高选择性选择性HDAC6抑制剂。
表3.式A化合物对HDAC家族酶活性的抑制作用
Figure PCTCN2020133799-appb-000016
ACY-1215数据获取自文献(Santo,L.,et al.,Preclinical activity,pharmacodynamic,and pharmacokinetic properties of a selective HDAC6inhibitor,ACY-1215,in combination with bortezomib in multiple myeloma.Blood,2012.119(11):p.2579-89.)。
实施例1-2
式A化合物药代动力学分析
对于式A化合物的药代动力学研究，通过高效液相色谱-串联质谱法(LC-MS/MS)测定CB-17SCID小鼠，SD大鼠和小猎犬(beagle dog)血浆中的式A化合物。表4显示单次静脉内(i.v.)或口服(p.o.)剂量后大鼠和狗血浆中式A化合物的PK参数。在最低测试的p.o.剂量下的PK数据(表4)，分别在大鼠(19.9％)和狗(75.0％)中确定相对较低和较高的口服生物利用度。在i.v.给药后，式A化合物在大鼠(2.81L/kg)和狗(1.40L/kg)中显示出具有中等分布体积(Vss)值的组织分布。大鼠中的清除率很高(89.5mL/min/kg)而狗中的清除率中等(16.8mL/min/kg)。大鼠的表观终末半衰期(T1/2)为0.482h，狗为1.29h(表4)。总之，在式A化合物PK分析中没有发现异常数据。
表4.式A化合物药代动力学特征
Figure PCTCN2020133799-appb-000017
Figure PCTCN2020133799-appb-000018
p.o.和i.v.溶液的组成
大鼠:30％(v/v)PEG400/70％(v/v)(5％(v/v)Kolliphor EL于水中))
狗:30％(v/v)PEG400/70％(v/v)(5％(v/v)Kolliphor EL于水中)
**AUC 最后:对大鼠为AUC 0-48小时,i.v.且AUC 0-48小时,p.o.，且对狗为AUC 0-24小时
实施例1-3
式A化合物和伊沙佐米的组合在人多发性骨髓瘤模型中协同增强对肿瘤细胞的增殖抑制
由于HDAC6选择性抑制剂的单独使用或与蛋白酶体抑制剂伊沙佐米的组合使用在多发性骨髓瘤患者中显示出良好的抗增殖潜力，我们着手在雌性CB-17SCID小鼠中使用MM.1S人骨髓瘤癌症异种移植模型评估式A化合物作为单一疗法或与伊沙佐米组合的体内疗效。数据显示式A化合物单一疗法可产生明显的肿瘤生长抑制作用，且与各单一药物相比，式A化合物与伊沙佐米的组合可协同提高疗效(图2)。
如图2所示，将MM.1S肿瘤细胞皮下植入雌性CB-17SCID小鼠中。随机分组后，每组7只小鼠，小鼠在指定时间用伊沙佐米(4mg/kg)、式A化合物(75mg/kg)单一疗法或伊沙佐米和式A化合物组合(4mg/kg+75mg/kg)处理。每周记录肿瘤体积(A)和体重(B)三次。对于肿瘤体积(A)，接种后第41天的肿瘤体积和TGI如下所示：伊沙佐米(4mg/kg)单一疗法的TGI为68.65％；式A化合物(75mg/kg)单一疗法的TGI为51.25％；伊沙佐米和式A化合物(4mg/kg+75mg/kg)组合疗法的TGI为92.58％。所有数据代表平均值±SEM。统计分析通过独立的t检验进行，该检验通过Levene检验进行检验。 **与媒介物组相比P＜0.01； ***与媒介物组相比P＜0.001； ###与式A化合物-75mg/kg组相比P＜0.001。
MM.1S肿瘤的Ac-α-微管蛋白和Ac-H3K9蛋白表达水平也用蛋白质印迹法检测(图3)。在式A化合物单药治疗组和组合治疗组中，式A化合物的最终剂量后1小时和4小时，观察到α-微管蛋白乙酰化(HDAC6的底物)显 著增加和H3K9乙酰化(HDAC1、2和3的底物)小幅增加。这些数据表明式A化合物选择性影响HDAC6下游信号传导，并小幅调节I类HDAC下游信号传导。总体而言，肿瘤样本分析的结果表明，式A化合物是一种抗MM肿瘤生长的口服有效选择性HDAC6抑制剂，其抑制α-微管蛋白和H3K9去乙酰化作用。
如图3所示，在进行图2的体内式A化合物抗肿瘤功效研究之后，在最后一次给药后的1、4或24小时内收集了指定治疗组的MM.1S骨髓瘤肿瘤组织。解剖MM.1S骨髓瘤肿瘤组织以进行免疫印迹测定，以检测Ac-α-微管蛋白和Ac-H3K9表达水平。使用相应抗体进行蛋白质印迹分析。B和C分别为Ac-α-微管蛋白和Ac-H3K9的定量数据。数值表示为平均值±SEM。B和C的统计分析是通过单因素ANOVA分析，然后进行Newman-Keuls检验进行的。对c和d的统计分析是通过单因素ANOVA分析进行的，然后进行邓奈特检验(Dunnett’s test)。与媒介物组相比： *P<0.05, **P<0.01, ***P<0.001。
实施例1-4
式A化合物改善抗PD-1免疫检查点阻断剂的抗肿瘤活性
为了探索式A化合物和PD-1阻断剂在实体瘤中的组合潜力，我们在CT26鼠结肠癌同系小鼠模型中评估式A化合物与抗体2E5组合的抗肿瘤效果，其中抗体2E5是人源化抗PD-1抗体(图4)。
如图4所示，将CT26肿瘤细胞皮下植入雌性Balb/c小鼠中。随机分组后(每组8只小鼠)，小鼠用抗体2E5(1mg/kg,ip,BIW×5次)、式A化合物(30或75mg/kg,po,BID x 17天)单一疗法处理或用式A化合物与抗体2E5组合以单一疗法组成的剂量处理。每周记录两次肿瘤体积(A)和体重(B)。所有数据代表平均值±SEM。统计分析通过独立的t检验进行，该检验通过Levene检验进行检验。 *与媒介物组相比P＜0.05； **与媒介物组相比P＜0.01； ##与式A化合物-30mg/kg组相比P＜0.01。
结果表明，抗PD-1抗体(即，抗体2E5)的单一疗法产生了43.85％的肿瘤生长抑制(TGI)；在分别以30mg/kg和75mg/kg每天两次(BID)的剂量水平给药17天后，式A化合物的单一疗法抑制9.85％和31.28％的肿瘤生长。当式A化合物与抗PD-1单克隆抗体(即，抗体2E5)组合使用时，以30mg/kg 和75mg/kg每天两次给药，TGI分别增至69.87％和66.77％。用式A化合物(30mg/kg)与抗体2E5组合治疗的8只小鼠中有3只显示出完全消退。当式A化合物的剂量分别为30mg/kg和75mg/kg时，CDI值分别为0.623和0.883。综上所述，式A化合物和抗PD-1免疫检查点阻断剂的组合协同改善CT26模型中的肿瘤生长抑制。
实施例2
式A化合物的PBMC介导的HCT116细胞、MCF-7细胞、NCI-H292或HuH-7细胞杀伤效果
实验设计
细胞毒性T细胞在癌症免疫治疗中起着至关重要的作用。本研究的目的是在HCT116细胞模型中，评价在式A化合物的PBMC介导的肿瘤细胞杀伤的效果(效应细胞和靶细胞比(E:T)：PBMCs：HCT116＝10:1)。
材料与方法
细胞和试剂：
· HCT116细胞(购自中国科学院细胞库)
· MCF-7细胞(购自ATCC)
· NCI-H292细胞(购自ATCC)
· HuH-7细胞(购自中国科学院细胞库)
· 无菌PBS(Hyclone，CAT#SH30256.01)
· FBS(Gibco CAT#100991-148)
· 胰蛋白酶0.25％EDTA(Gibco，CAT#25200-072)
· CFSE(eBioscience，CAT#65-0850-84)
· 测定培养基：RPMI1640+10％FBS
· 纯化的NA/LE小鼠抗人CD3(BD,Cat.No.：555336)
· 纯化的NA/LE小鼠抗人类CD28(BD,Cat.No.：555725)
· Fixable Viability Stain 700(BD,Cat.No.:564997)。
测试物：式A化合物，储备液：10mM。
PBMCs：来自健康供体的冷冻PBMC(代号为TBD)，从供应商Stemexppress订购。
检测步骤
第1天：细胞接种
在McCoy的5A+10％FBS+1％PS+3％NaHCO 3培养基中解离并收集HCT116细胞，重悬于适当体积的PBS中，并将细胞密度调整为5-20×10 6细胞/mL。在PBS(预热至室温)中制备2X CFSE溶液(5μM)，并向肿瘤细胞溶液中加入等体积的5μM的CFSE染料溶液，立即混合均匀，然后在黑暗中于37℃孵育5-10分钟。通过添加4-5体积的冰冷测定介质(含有≥10％的血清)停止标记，并在冰上孵育5分钟。然后用预热的测定培养基洗涤细胞两次，并计数活细胞。将细胞重悬于5-6mL测定培养基中并调节至所需细胞密度，在24孔板中接种1×10 5细胞/孔。
第1天：PBMC复苏和活化
将冷冻管浸入37℃的水浴中并轻轻摇动来快速融化冷冻的PBMC细胞。然后将细胞转移到10mL测定培养基(RPMI-1640+10％FBS)中，以1500rpm离心10分钟，吸出上清液。将细胞重悬于适当体积的含有1μg/mL CD3和1μg/mL CD28 Ab的测定培养基中，使细胞密度为1.5×10 6细胞/mL，并在37℃的培养箱中孵育24h。
第2天：细胞杀伤试验
将活化的PBMC收集到50mL试管中，以1500rpm离心10分钟，吸出上清液。用适当体积的测定培养基重悬，并用相同的培养基调节至合适的浓度，加入活化的PBMC，使E:T比(效应细胞和靶细胞比)为10:1。然后将0.1μM、0.5μM、1μM或2μM式A化合物加入共培养的HCT116和PBMC，并在37℃的培养箱中培养48小时。
第4天：流式细胞仪分析
将细胞用胰蛋白酶解离后，在室温下用L/D染色15分钟后，在流式细胞仪上运行样品。然后使用FlowJo对数据进行分析，并记录CFSE +肿瘤细胞的L/D +％。
MCF-7细胞、NCI-H292细胞和HuH-7细胞的检测步骤与上文所述针对HCT116细胞的检测步骤相同。
实验结果
根据图5可知，在MCF-7、NCI-H292、HUH-7或HCT116细胞中，式A化合物表现出明显增强的PBMC介导的肿瘤杀伤效力。
实施例3
实施例3-1
RNA转录组分析显示了式A化合物与其他HDAC抑制剂不同的基因表达模式
在实施例1中，我们发现式A化合物和抗PD-1抗体(即，抗体2E5)的组合在CT26小鼠模型中具有协同增强的抗肿瘤功效。为了探索式A化合物在特定适应症中促进I/O治疗(肿瘤免疫疗法)的机制，用式A化合物或其他HDAC抑制剂检测了5种适应症细胞系，包括Daudi(淋巴瘤)，MM.1S(多发黑色素瘤)，NCI-H292(肺癌)，MCF-7(乳腺癌)和HT-29(结肠癌)，所述其他HDAC抑制剂包括Vorinostat(Pan-HDAC抑制剂)，Entinostat(I类HDAC抑制剂)和ACY-1215(选择性HDAC6抑制剂)(图1)。
用DMSO、式A化合物或其他HDAC抑制剂处理肿瘤细胞系，包括Vorinostat(Pan-HDAC抑制剂)、Entinostat(I类HDAC抑制剂)和ACY-1215(选择性HDAC6抑制剂)。提取RNA并进行RNA测序。然后进行了PCA分析。一个循环3个点代表一组重复3次。
实验结果
根据图6可知，主成分分析(PCA)数据显示，与其他HDAC抑制剂不同，式A化合物诱导了独特的基因转录模式。由式A化合物诱导的转录模式与其他HDAC抑制剂显着不同，这表明了式A化合物通过与其他HDAC抑制剂不同的途径起作用，具有新功能和独特作用机理。
实施例3-2
式A化合物特异性调节不同适应症细胞系中的免疫细胞功能
为了进一步分析那些特异变化的基因的功能，我们基于式A化合物特异变化的基因在5个适应症细胞系中进行了基因本体(GO)分析(图7)。
用DMSO、式A化合物或ACY-1215处理肿瘤细胞系。提取RNA并进行RNA测序。在式A化合物组中发生显著特异性变化的DEG(不同的表达基因)用于GO分析。显示了十大富集途径。
实验结果
图7的实验结果表明，式A化合物诱导的不同表达基因可以丰富Daudi细胞中T细胞增殖的正向调控。
基于式A化合物特异变化的基因，我们进一步进行了髓样细胞相关的基因本体分析。图8的实验结果表明，式A化合物诱导的不同表达基因可以在Daudi和HT-29细胞的髓样细胞分化途径中富集(图8)，这表明与其他HDAC抑制剂不同，式A化合物特异性调节髓样细胞的功能。因此，发现由式A化合物诱导的转录模式与其他HDAC抑制剂显着不同，这表明式A化合物的具有新功能和独特作用机理。
实施例3-3
式A化合物独特地激活髓样细胞
式A化合物对髓样细胞和髓样白细胞分化途径的影响表明式A化合物增加DC成熟并影响M1/M2平衡。为了评估对DC细胞的作用，我们用式A化合物和ACY-1215处理了HT-29和NCI-H292细胞，然后用源自髓样的未成熟DC培养它们。
具体而言，将HT-29和NCI-H292细胞用指定浓度的式A化合物或ACY-1215预处理24小时，然后与源自髓样的未成熟DC共培养24小时。通过流式细胞术评估CD11c +DC的CD86表达(图9中A和B)。
另一方面，M1和M2极化的巨噬细胞分别显示抗肿瘤和促肿瘤功能。为了探索式A化合物对M1/M2巨噬细胞平衡的影响，分别在体外用式A化合物和ACY-1215处理了髓样来源的M1和M2(图9C)。具体而言，用指定浓度的式A化合物或ACY-1215处理源自髓样的M1和M2 6天。通过用Fixable Viability Stain 700染色来监测细胞生存力。通过流式细胞术计数M1和M2细胞数，并计算M1/M2比。数据表示为平均值±SEM。***P<0.001，**P<0.01和*P<0.05，用于各式A化合物剂量组与DMSO治疗组之间的比较。通过非配对t检验获得统计数据。
实验结果
结果表明，与ACY-1215处理组相比，式A化合物处理的肿瘤细胞可通 过诱导活化标记CD86(图9的A-B)和MHCII(数据未显示)的更高表达来明显增强CD11c +DC的成熟。另一方面，图9C的实验结果表明，当用式A化合物治疗时，M1/M2比增加了，但是在ACY-1215治疗组中，M1/M2比没有改变。因此，实验结果表明式A化合物，而非ACY-1215，可激活DC成熟并改变巨噬细胞平衡，从而具有抗肿瘤功能。

Claims (43)

  1. 一种用于治疗受益于调节免疫功能的受试者病症的方法，其包括向所述受试者施用药学有效量的选择性HDAC6抑制剂。
  2. 一种用于治疗受益于调节免疫功能的受试者病症的方法，其包括向所述受试者施用药学有效量的选择性HDAC6抑制剂和一种或多种治疗剂。
  3. 选择性HDAC6抑制剂和一种或多种治疗剂的组合在制备用于治疗受益于调节免疫功能的受试者病症的药物中的用途。
  4. 选择性HDAC6抑制剂在制备用于治疗受益于调节免疫功能的受试者病症的药物中的用途，所述选择性HDAC6抑制剂和一种或多种治疗剂组合。
  5. 一种或多种治疗剂在制备用于治疗受益于调节免疫功能的受试者病症的药物中的用途，所述一种或多种治疗剂和选择性HDAC6抑制剂组合。
  6. 选择性HDAC6抑制剂和一种或多种治疗剂的组合，其用于治疗受益于调节免疫功能的受试者病症，包括向所述受试者施用药学有效量的选择性HDAC6抑制剂和一种或多种治疗剂。
  7. 包含选择性HDAC6抑制剂药物组合物、药物、制剂或试剂盒，所述选择性HDAC6抑制剂与一种或多种治疗剂组合使用，用于治疗治疗受益于调节免疫功能的受试者病症。
  8. 一种组合物，药物，制剂或试剂盒，其包含选择性HDAC6抑制剂，其与一种或多种治疗剂组合使用，用于治疗治疗受益于调节免疫功能的受试者病症。
  9. 一种组合物，药物，制剂或试剂盒，其包含选择性HDAC6抑制剂、一种或多种治疗剂和一种或多种药学上可接受的载体。
  10. 权利要求1-9中任一项的方法、用途、组合、组合物、药物、制剂或试剂盒，其中所述选择性HDAC6抑制剂与一种或多种治疗剂同时、分别或顺序向该受试者施用。
  11. 权利要求1-10中任一项的方法、用途、组合、组合物、药物、制剂或试剂盒，其中所述选择性HDAC6抑制剂为式(I)和式(II)所示化合物，其药学上可接受的盐或其异构体，
    Figure PCTCN2020133799-appb-100001
    其中，
    L1选自：单键、-NH-、-C(＝O)-NH-；
    E 1、E 2各自独立地选自-O-、-CH 2-和-CH 2-CH 2-；
    R 2选自：H、F、Cl、Br、I；
    R选自：F、Cl、Br、I；
    n选自：0或1。
  12. 根据1-10中任一项的方法、用途、组合、组合物、药物、制剂或试剂盒，其中所述选择性HDAC6抑制剂为:
    Figure PCTCN2020133799-appb-100002
  13. 根据权利要求1-10中任一项的方法、用途、组合、组合物、药物、制剂或试剂盒，其中所述选择性HDAC6抑制剂为如下化合物A:
    Figure PCTCN2020133799-appb-100003
  14. 根据权利要求1-13中任一项的方法、用途、组合、组合物、药物、制剂或试剂盒，其中所述一种或多种治疗剂为PD-1轴结合拮抗剂或者蛋白酶体抑制剂；
    优选的，PD-1轴结合拮抗剂选自PD-1结合拮抗剂，PD-L1结合拮抗剂和PD-L2结合拮抗剂；更优选的，PD-1轴结合拮抗剂是抗体或其抗原结合片段；尤其优选地，该抗体是抗PD-1抗体；最优选地，该抗体是单克隆抗体；
    优选的，所述蛋白酶体抑制剂为硼替佐米、卡非佐米、伊沙佐米或者马瑞佐米。
  15. 根据权利要求1-14中任一项的方法、用途、组合、组合物、药物、制剂或试剂盒，其中所述抗体或其抗原结合片段结合于PD-1的一个表位，所述表位包含：SEQ ID NO﹕24上第128、129、130、131和132位点氨基酸和第35、64、82、83位中至少一个氨基酸；
    优选的，所述抗体或其抗原结合片段结合于人PD-1和鼠PD-1的一个表位，其中，所述表位包括SEQ ID NO﹕24上第128、129、130、131和132位点氨基酸；
    其中鼠PD-1是小鼠或大鼠PD-1。
  16. 根据权利要求1-15的方法、用途、组合、组合物、药物、制剂或试剂盒，其中所述抗体
    a)结合于人PD-1，KD为2.15E-10M以下；并且
    b)结合于鼠PD-1，KD为1.67E-08M以下。
  17. 根据权利要求1-16的方法、用途、组合、组合物、药物、制剂或试剂盒，其中所述抗体具有下列性质中的至少一种：
    a)结合于人PD-1，KD为4.32E-10M至2.15E-10M，并且结合于小鼠PD-1，KD为5.39E-08M至
    1.67E-08M；
    b)实质上不结合于人CD28、CTLA-4；
    c)增加T细胞的增殖；
    d)增加干扰素-γ的产生；或
    e)增加白细胞介素-2的分泌。
  18. 根据权利要求1-17的方法、用途、组合、组合物、药物、制剂或试剂盒，其中所述抗体或其抗原结合片段包含一个氨基酸序列，所述氨基酸序列与选自由SEQ ID NOs﹕1、2、3、4、5、6、7、8和9所组成的组中的序列具有至少70％、80％、90％或95％的同源性，其中所述抗体特异性结合PD-1。
  19. 根据权利要求1-18的方法、用途、组合、组合物、药物、制剂或试剂盒，其中所述抗体或其抗原结合片段包含一个氨基酸序列，所述氨基酸序列选自由SEQ ID NOs﹕1、2、3、4、5、6、7、8和9所组成的组中的序列，其中所述抗体特异性结合PD-1。
  20. 根据权利要求1-19的方法、用途、组合、组合物、药物、制剂或试剂盒，其中所述抗体或其抗原结合片段包含：
    a)重链可变区，其具有的氨基酸序列与选自由SEQ ID NO﹕1和SEQ ID NO﹕2所组成的组中的序列具有至少70％、80％、90％或95％的同源性；以及
    b)轻链可变区，其具有的氨基酸序列与选自由SEQ ID NOs﹕3、4、5、6、7、8和9所组成的组中的序列具有至少70％、80％、90％或95％的同源性，
    其中所述抗体特异性结合PD-1。
  21. 根据权利要求1-20的方法、用途、组合、组合物、药物、制剂或试剂盒，其中所述抗体或其抗原结合片段包含：
    a)重链可变区，其具有的氨基酸序列选自由SEQ ID NO﹕1和SEQ ID NO﹕2组成的组中所述序列；以及
    b)轻链可变区，其具有的氨基酸序列选自由SEQ ID NOs﹕3、4、5、6、7、8和9所组成的组中的序列，其中所述抗体特异性结合PD-1。
  22. 根据权利要求1-21的方法、用途、组合、组合物、药物、制剂或试剂盒，其中所述抗体或其抗原结合片段包含：
    a)重链可变区，其氨基酸序列选自SEQ ID NO﹕2所示的序列；以及
    b)轻链可变区，其氨基酸序列选自SEQ ID NO﹕3所示的序列，
    其中所述抗体特异性结合PD-1。
  23. 根据权利要求1-22的方法、用途、组合、组合物、药物、制剂或试剂盒，其中所述抗体或其抗原结合片段包含互补决定区(CDR)，其具有的氨基酸序列选自由SEQ ID NOs﹕10-23所组成的组中的序列，其中所述抗体特异性结合PD-1。
  24. 根据权利要求1-23的方法、用途、组合、组合物、药物、制剂或试剂盒，其中所述抗体或其抗原结合片段包含：
    包含CDR1、CDR2和CDR3序列的重链可变区；以及
    包含CDR1、CDR2和CDR3序列的轻链可变区，
    其中重链可变区CDR3序列包含选自由SEQ ID NO﹕12和SEQ ID NO﹕13所组成的组中的氨基酸序列及其保守性修饰，其中所述抗体特异性结合PD-1。
  25. 根据权利要求1-24的方法、用途、组合、组合物、药物、制剂或试剂盒，其中所述抗体轻链可变区CDR3序列包含选自由SEQ ID NOs﹕20、21、22和23所组成的组中的氨基酸序列及其保守性修饰。
  26. 根据权利要求1-25的方法、用途、组合、组合物、药物、制剂或试剂盒，其中所述抗体重链可变区CDR2序列包含选自由SEQ ID NO﹕11所组成的组中的氨基酸序列及其保守性修饰。
  27. 根据权利要求1-26的方法、用途、组合、组合物、药物、制剂或试剂盒，其中所述抗体轻链可变区CDR2序列包含选自由SEQ ID NO﹕19所组成的组中的氨基酸序列及其保守性修饰。
  28. 根据权利要求1-27的方法、用途、组合、组合物、药物、制剂或试剂盒，其中所述抗体重链可变区CDR1序列包含选自由SEQ ID NO﹕10所组成的组中的氨基酸序列及其保守性修饰。
  29. 根据权利要求1-26方法、用途、组合、组合物、药物、制剂或试剂盒，其中所述抗体轻链可变区CDR1序列包含选自由SEQ ID NOs﹕14、15、16、17和18所组成的组中的氨基酸序列及其保守性修饰。
  30. 根据权利要求1-29的方法、用途、组合、组合物、药物、制剂或试 剂盒，其中所述抗体包含：
    a)重链可变区CDR1，序列包含选自SEQ ID NO﹕10所示的氨基酸序列，
    b)重链可变区CDR2，序列包含选自SEQ ID NO﹕11所示的氨基酸序列，
    c)重链可变区CDR3，序列包含选自SEQ ID NO﹕13所示的氨基酸序列，
    d)轻链可变区CDR1，序列包含选自SEQ ID NO﹕14所示的氨基酸序列，
    e)轻链可变区CDR2，序列包含选自SEQ ID NO﹕19所示的氨基酸序列，
    f)轻链可变区CDR3，序列包含选自SEQ ID NO﹕21所示的氨基酸序列，
    其中所述抗体特异性结合PD-1。
  31. 根据权利要求1-30的方法、用途、组合、组合物、药物、制剂或试剂盒，其中所述抗体是嵌合抗体或人源化抗体或人抗体或哺乳动物抗体。
  32. 根据权利要求1-31的方法、用途、组合、组合物、药物、制剂或试剂盒，其中所述抗体或抗原结合其片段是骆驼化的单链结构域抗体、双抗体、scFv、scFv二聚体、BsFv、dsFv、(dsFv)2、dsFv-dsFv'、Fv片段、Fab、Fab'、F(ab')2、ds双功能抗体(dsdiabody)、纳米抗体、结构域抗体或二价结构域抗体。
  33. 根据权利要求1-32任一项的方法、用途、组合、组合物、药物、制剂或试剂盒，其中所述抗体或抗原结合其片段还包含免疫球蛋白恒定区。
  34. 根据权利要求1-33中任一项的方法、用途、组合、组合物、药物、制剂或试剂盒，其中：
    所述选择性HDAC6抑制剂为如下式A化合物:
    Figure PCTCN2020133799-appb-100004
    化合物A；
    所述一种或多种治疗剂为抗体或其抗原结合片段，其为抗体2E5，包含：
    a)重链可变区，其氨基酸序列选自SEQ ID NO﹕2所示的序列；以及
    b)轻链可变区，其氨基酸序列选自SEQ ID NO﹕3所示的序列，
    其中所述抗体特异性结合PD-1。
  35. 根据权利要求1-13中任一项的方法、用途、组合、组合物、药物、 制剂或试剂盒，其中，所述选择性HDAC6抑制剂为如下化合物A：
    Figure PCTCN2020133799-appb-100005
    化合物A
    所述一种或多种治疗剂为硼替佐米、卡非佐米、伊沙佐米或者马瑞佐米。
  36. 根据权利要求1-35中任一项所述的任一项的方法、用途、组合、组合物、药物、制剂或试剂盒，其中，所述选择性HDAC6抑制剂以约0.0001至约100mg/kg，优选约1mg/kg、约2mg/kg、约5mg/kg、约10mg/kg、约15mg/kg、约20mg/kg给药；
    优选的，所述选择性HDAC6抑制剂单位剂量为约10至1000mg，更优选约100至1000mg，约200至约600mg，或约300至约500mg，尤其优选约10mg、约50mg、约100mg、约150mg、约200mg、约250mg、约300mg、约350mg、约400mg、约450mg、约500mg。
  37. 根据权利要求36所述的任一项的方法、用途、组合、组合物、药物、制剂或试剂盒，其中，所述PD-1轴结合拮抗剂以约0.01mg/kg至约100mg/kg，例如，约0.1mg/kg-10mg/kg、约0.5mg/kg-5mg/kg、或约1mg/kg-约2mg/kg给药；
    优选的，所述PD-1轴结合拮抗剂的单位剂量为约1mg至约100mg的单位剂量，优选为约5mg-50mg、约10mg-40mg、15mg-30mg、或20mg-25mg。
  38. 根据权利要求36所述的任一项的方法、用途、组合、组合物、药物、制剂或试剂盒，其中，所述蛋白酶体抑制剂以约0.0001至约100mg mg/kg，优选约0.01mg/kg-约1mg/kg、约0.1mg/kg-约0.5mg/kg、约1mg/kg-20mg/kg、约2mg/kg-约15mg/kg、约5mg/kg-约10mg/kg的剂量给药；
    优选的，所述蛋白酶体抑制剂的单位剂量为约1至500mg，约5至200mg，约10至50mg，约2mg至约30mg、约10mg至约20mg。
  39. 根据权利要求1-38中任一项所述的任一项的方法、用途、组合、组合物、药物、制剂或试剂盒，其中，所述选择性HDAC6抑制剂以约100至1000mg的单位剂量，优选约200至约600mg，或约300至约500mg的单位 剂量配制成制剂，尤其是口服制剂；所述PD-1轴结合拮抗剂以约0.01mg/kg至约100mg/kg的单位剂量，优选以约5mg-50mg的单位剂量配制成制剂，尤其是注射剂；或者所述蛋白酶体抑制剂以约1至500mg，优选约5至50mg的单位剂量配制成制剂，尤其是注射剂。
  40. 根据权利要求1-39中任一项所述的任一项的方法、用途、组合、组合物、药物、制剂或试剂盒，其中，所述选择性HDAC6抑制剂以口服给药；所述PD-1轴结合拮抗剂通过静脉内输注给药；所述蛋白酶体抑制剂通过口服给药；
    优选的，在治疗周期中，所述选择性HDAC6抑制剂每天给药1次、2次或者更多次；所述PD-1轴结合拮抗剂每个月给药1次、每两周给药1次、每周给药1次、2次或者更多次、每天给药1次、2次或者更多次；所述蛋白酶体抑制剂每个月给药1次、每两周给药1次、每周给药1次、2次或者更多次、每天给药1次、2次或者更多次；
    更优选的，所述HDAC6抑制剂每天给药1次；所述PD-1轴结合拮抗剂每两周给药1次；或者所述蛋白酶体抑制剂每两周给药1次。
  41. 根据权利要求1-39中任一项所述的方法、用途、组合、组合物、药物、制剂或试剂盒，其中，所述受试者已经接受了至少一轮先前的治疗。
  42. 根据权利要求1-38中任一项所述的方法、用途、组合、组合物、药物、制剂或试剂盒，其中，先前的治疗是手术切除，移植，局部治疗，全身治疗和/或最佳支持治疗。
  43. 根据权利要求1-38中任一项所述的方法、用途、组合、组合物、药物、制剂或试剂盒，其中，其中所述受益于调节免疫功能的受试者的病症为癌症，具体地，所述癌症为实体瘤或血液癌，如非小细胞肺癌、小细胞肺癌、肾细胞癌、结肠直肠癌、卵巢癌、乳腺癌、胰腺癌、胃癌、膀胱癌、食道癌、间皮瘤、黑素瘤、头颈癌、甲状腺癌、肉瘤、前列腺癌、胶质母细胞瘤、宫颈癌、胸腺癌、白血病、淋巴瘤、骨髓瘤、真菌病真菌、默克尔细胞癌和其他血液系统恶性肿瘤，例如经典霍奇金淋巴瘤(CHL)、原发性纵隔大B细胞淋巴瘤、T-细胞/组织细胞丰富的B细胞淋巴瘤、EBV阳性和-阴性PTLD、以及EBV相关的弥漫性大B细胞淋巴瘤(DLBCL)、浆母细胞淋巴瘤、结外NK/T细胞淋巴瘤、鼻咽癌和HHV8-相关的原发性渗出性淋巴瘤、霍奇金淋巴瘤、中枢神经系统肿瘤(CNS)，例如原发性中枢神经系统淋巴瘤、脊髓 轴肿瘤、脑干神经胶质瘤、多发性骨髓瘤；
    优选的，所述癌症选自多发性骨髓瘤或乳腺癌。
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011106632A1 (en) * 2010-02-26 2011-09-01 Millennium Pharmaceuticals, Inc. Substituted hydroxamic acids and uses thereof
CN103429574A (zh) * 2010-11-16 2013-12-04 阿塞蒂隆制药公司 作为蛋白质去乙酰化酶抑制剂的嘧啶羟基酰胺化合物和其使用方法
WO2015007870A1 (en) * 2013-07-18 2015-01-22 Institut De Recherche Pour Le Developpement (I.R.D.) New compounds for the treatment and/or prevention of parasitic diseases and method of production of thereof
WO2015100363A1 (en) * 2013-12-23 2015-07-02 The Trustees Of Columbia University In The City Of New York Selective hdac6 inhibitors
WO2018130155A1 (zh) * 2017-01-10 2018-07-19 南京明德新药研发股份有限公司 Hdac6选择性抑制剂及其制备方法和应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011106632A1 (en) * 2010-02-26 2011-09-01 Millennium Pharmaceuticals, Inc. Substituted hydroxamic acids and uses thereof
CN103429574A (zh) * 2010-11-16 2013-12-04 阿塞蒂隆制药公司 作为蛋白质去乙酰化酶抑制剂的嘧啶羟基酰胺化合物和其使用方法
WO2015007870A1 (en) * 2013-07-18 2015-01-22 Institut De Recherche Pour Le Developpement (I.R.D.) New compounds for the treatment and/or prevention of parasitic diseases and method of production of thereof
WO2015100363A1 (en) * 2013-12-23 2015-07-02 The Trustees Of Columbia University In The City Of New York Selective hdac6 inhibitors
WO2018130155A1 (zh) * 2017-01-10 2018-07-19 南京明德新药研发股份有限公司 Hdac6选择性抑制剂及其制备方法和应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
A RAY, D S DAS, Y SONG, T HIDESHIMA, Y-T TAI, D CHAUHAN, K C ANDERSON: "Combination of a novel HDAC6 inhibitor ACY-241 and anti-PD-L1 antibody enhances anti-tumor immunity and cytotoxicity in multiple myeloma", LEUKEMIA, STOCKTON PRESS, LONDON, vol. 32, no. 3, 1 March 2018 (2018-03-01), London, pages 843 - 846, XP055507573, ISSN: 0887-6924, DOI: 10.1038/leu.2017.322 *

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