WO2022007947A1 - 抗cd47抗体或其抗原结合片段和dna甲基化转移酶抑制剂的组合及其用途 - Google Patents

抗cd47抗体或其抗原结合片段和dna甲基化转移酶抑制剂的组合及其用途 Download PDF

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WO2022007947A1
WO2022007947A1 PCT/CN2021/105470 CN2021105470W WO2022007947A1 WO 2022007947 A1 WO2022007947 A1 WO 2022007947A1 CN 2021105470 W CN2021105470 W CN 2021105470W WO 2022007947 A1 WO2022007947 A1 WO 2022007947A1
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antibody
administration
pharmaceutical combination
dose
azacitidine
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PCT/CN2021/105470
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English (en)
French (fr)
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伍伟伟
陈炳良
左洪莉
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信达生物制药(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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins

Definitions

  • the present invention relates to the field of medicine. Specifically, the present invention relates to an anti-tumor drug combination and use thereof, in particular to a drug combination comprising an anti-CD47 antibody or an antigen-binding fragment thereof and a DNA methyltransferase inhibitor and its use in the prevention and/or treatment of tumors and/or use in cancer.
  • AML Acute myeloid leukemia
  • AML is a group of highly heterogeneous diseases characterized by abnormal clonal proliferation of hematopoietic stem cells, and is the most common type of acute leukemia in adults.
  • An article in The Lancet Hematology in 2018 summarized the epidemiological data of AML in 184 regions around the world and showed that the incidence of AML was the highest in Australia, about 4.8/100,000 people, of which the incidence in China was about 2.6/10 million people.
  • According to the latest 2020 Cancer Journal estimates there will be approximately 19,940 new cases of AML in the United States in 2020, and approximately 11,180 deaths, with a male-to-female ratio of approximately 1.4:1.
  • hypomethylating agents including: azacitidine or decitabine
  • Venetoclax for the treatment of AML at home and abroad.
  • MDS Myelodysplastic syndromes
  • AML Acute myeloid leukemia
  • SEER epidemiological data show that the annual incidence of MDS is about 4.9/100,000 people, and the incidence increases significantly with age.
  • the incidence of MDS is only 0.1/100,000 in children, adolescents and young adults under 40 years old, but as high as 30.2/100,000 and 59.8/100,000 in people aged 70-79 and over 80 years old, respectively.
  • MDS is divided into two categories according to the International Prognostic Scoring System (IPSS, IPSS-R): lower risk group and higher risk group.
  • IPSS International Prognostic Scoring System
  • the goal of treatment is to improve the peripheral blood cell count and the quality of life of the patient, and the treatment methods are mainly supportive treatment such as watchful waiting or promoting hematopoiesis; while the higher-risk group (IPSS intermediate-risk 2 and high-risk and IPSS-R intermediate-risk, IPSS-R intermediate-risk, High-risk and very high-risk), the treatment goal is to remove the malignant clone of MDS and prolong the survival of patients.
  • the main treatment options are the treatment of demethylating agents (Hypomethylating agents, HMA), chemotherapy, hematopoietic stem cell transplantation and supportive care.
  • HMA is the most commonly used drug for high-risk MDS patients. Although HMA monotherapy can prolong survival, only 40-50% of patients respond to HMA drug therapy, and this effect is often not durable, and eventually occurs within 2 years. Relapse, and once relapsed, the median survival of these patients is only 4.3 months.
  • the inventors have surprisingly found that the drug combination of the present application can act synergistically and/or improve the incidence and/or severity of adverse events (AEs) in therapy against cancer, especially AML and MDS Prophylactic and/or therapeutic effects.
  • AEs adverse events
  • the present invention provides the following embodiments:
  • the present invention provides a pharmaceutical combination comprising (i) an anti-CD47 antibody or antigen-binding fragment thereof; and (ii) a DNA methyltransferase inhibitor,
  • the anti-CD47 antibody or its antigen-binding fragment comprises 6 CDRs, wherein HCDR1, HCDR2 and HCDR3 comprise as shown in GSISSYYWS (SEQ ID NO:1), YIYYSGSTNYNPSLKS (SEQ ID NO:2) and ARGKTGSAA (SEQ ID NO:3)
  • the sequence or consists of the amino acid sequence wherein the HCDR1 is located at H27-H35 in the Kabat numbering system, and the HCDR3 is located at H93-H102 in the Kabat numbering system, and the HCDR2, LCDR1, LCDR2 and LCDR3 are determined by the Kabat definition rules;
  • DNA methyltransferase inhibitor is selected from azacitidine or a pharmaceutically acceptable salt thereof.
  • the anti-CD47 antibody or antigen-binding fragment thereof in the pharmaceutical combination of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence of SEQ ID NO:7 or its A sequence having at least 90%, 95%, 98% or 99% identity, and the light chain variable region comprises or has at least 90%, 95%, 98% or 99% identity to the sequence of SEQ ID NO: 8 sequence.
  • the anti-CD47 antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises or is at least 90%, 95%, 98% or 99% identical to the sequence of SEQ ID NO:9 and the light chain comprises the sequence of SEQ ID NO: 10 or a sequence indicated by at least 90%, 95%, 98% or 99% identity thereto.
  • the present invention also provides the following embodiments:
  • the single administration dose of (i) is selected from 1-50 mg/kg, for example 1 mg/kg, 2 mg/kg, 3 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg /kg, 35 mg/kg, 40 mg/kg, 45 mg/kg or 50 mg/kg; preferably 1 mg/kg, 30 mg/kg or 45 mg/kg; or (i) a single administration dose selected from about 60-3000 mg, For example about 60 mg, 120 mg, 180 mg, 300 mg, 600 mg, 900 mg, 1200 mg, 1500 mg, 1800 mg, 2100 mg, 2400 mg, 2700 mg or 3000 mg, preferably about 60 mg, 1800 mg, 2700 mg; and
  • the single administration dose of (ii) is selected from the group consisting of 70-80 mg/m 2 , such as 70 mg/m 2 , 75 mg/m 2 or 80 mg/m 2 ; preferably 75 mg/m 2 ; or the single administration dose of (ii) is selected from about 120-140 mg, such as about 120 mg, 130 mg, 140 mg, preferably about 130 mg.
  • parenteral administration preferably intravenous administration
  • (ii) is for parenteral administration, preferably subcutaneous injection.
  • each dosing cycle being two weeks, three weeks, four weeks, one month, two months, three months , four months, five months, half a year or more, preferably two, three or four weeks, wherein (i) and (ii) may be administered separately, simultaneously or sequentially.
  • a method for preventing or treating cancer comprising administering to a patient in need thereof an effective amount of the pharmaceutical combination according to any one of the preceding embodiments, the cancer being a solid tumor and a hematological tumor, the The blood tumor is AML or MDS.
  • kits of parts comprising a pharmaceutical combination according to any one of the preceding embodiments, preferably the kit comprises one or more single pharmaceutical dosage units.
  • the single pharmaceutical dosage unit comprises about 60-3000 mg, preferably about 60 mg, 120 mg, 180 mg, 1800 mg or 2700 mg, more preferably about 60 mg or 1800 mg of the aforementioned anti-CD47 antibody or its antigen binding agent fragments; and
  • One or more single pharmaceutical dosage units comprising a dose of about 120-140 mg, such as 120 mg, 130 mg, 140 mg, preferably about 130 mg of azacitidine or a pharmaceutically acceptable salt thereof .
  • the drug combination of the anti-CD47 antibody or its antigen-binding fragment of the present invention and a DNA methyltransferase inhibitor can provide better anti-tumor efficacy.
  • the anti-CD47 antibody or antigen-binding fragment thereof in the pharmaceutical combination is the fully human monoclonal anti-CD47 antibody or antigen-binding fragment thereof ADI-26630 disclosed in WO2019042285A1, a DNA methyltransferase inhibitor of the present invention for azacitidine.
  • the anti-tumor efficacy obtained by the pharmaceutical combination of the present invention is significantly better than the anti-tumor efficacy obtained by single-drug administration of the anti-CD47 antibody or its antigen-binding fragment or DNA methyltransferase inhibitor.
  • Figure 1 shows the induction of CD47 expression by azacitidine in MV-4-11 cells.
  • Figure 2 shows the induction of CD47 expression by azacitidine in HL60 cells.
  • Figure 3 shows the effect of anti-CD47 antibody, azacitidine alone and in combination on tumor growth in MV-4-11 tumor-bearing mice.
  • Figure 4 shows the effects of anti-CD47 antibody, azacitidine alone and in combination on the body weight of MV-4-11 tumor-bearing mice.
  • Figure 5 shows the effect of anti-CD47 antibody, azacitidine single drug and combination (the anti-CD47 antibody was administered on the first day after the end of azacitidine administration) on the tumor volume of MV-4-11 tumor-bearing mice .
  • Figure 6 shows the effect of anti-CD47 antibody, azacitidine single drug and combination (the anti-CD47 antibody was administered on the third day after the end of azacitidine administration) on the tumor volume of MV-4-11 tumor-bearing mice .
  • Figure 7 shows the effect of anti-CD47 antibody, azacitidine single drug and combination (the anti-CD47 antibody was administered on the fifth day after the end of azacitidine administration) on the tumor volume of MV-4-11 tumor-bearing mice .
  • Figure 8 shows the effects of anti-CD47 antibody, azacitidine alone and in combination on tumor growth in HL60 tumor-bearing mice.
  • Figure 9 shows the effect of anti-CD47 antibody, azacitidine single drug and combination on body weight of HL60 tumor-bearing mice.
  • Figure 10 shows the effect of anti-CD47 antibody, azacitidine single agent and combination (the anti-CD47 antibody was administered on the first day after the end of azacitidine administration) on the tumor volume of HL60 tumor-bearing mice.
  • Figure 11 shows the effect of anti-CD47 antibody combined with azacitidine administration at different times on tumor volume in HL60 tumor-bearing mice.
  • the term “comprising” or “comprising” means the inclusion of stated elements, integers or steps, but not the exclusion of any other elements, integers or steps.
  • the term “comprising” or “comprising” is used, unless otherwise indicated, it also encompasses situations consisting of the recited elements, integers or steps.
  • reference to an antibody variable region that "comprises” a particular sequence is also intended to encompass antibody variable regions that consist of that particular sequence.
  • the terms "complete antibody” and “intact antibody” are used interchangeably herein to refer to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds.
  • Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region consists of three domains, CH1, CH2 and CH3.
  • Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region consists of one domain, CL.
  • variable region refers to the domain of an antibody heavy chain or light chain that is involved in antibody binding to an antigen.
  • the variable domains of the heavy and light chains of native antibodies generally have similar structures, with each variable domain comprising four conserved framework regions (FRs) and three complementarity determining regions.
  • FRs conserved framework regions
  • a single VH or VL domain may be sufficient to confer antigen binding specificity.
  • VH or VL domains from antibodies that bind to a particular antigen can be used to isolate antibodies that bind the antigen to screen libraries of complementary VL or VH domains, respectively. See, eg, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).
  • CDR regions or “CDRs” or “hypervariable regions” (used interchangeably herein with hypervariable regions “HVR”), are the variable domains of antibodies that are hypervariable in sequence and Structurally defined loops ("hypervariable loops") and/or regions containing antigen-contacting residues ("antigen contact points") are formed.
  • the CDRs are mainly responsible for binding to antigenic epitopes.
  • the CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2 and CDR3, numbered sequentially from the N-terminus.
  • the CDRs located within the variable domains of antibody heavy chains are referred to as HCDR1, HCDR2 and HCDR3, while the CDRs located within the variable domains of antibody light chains are referred to as LCDR1, LCDR2 and LCDR3.
  • the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment systems, including Example: Chothia based on the three-dimensional structure of antibodies and topology of CDR loops (Chothia et al.
  • the sequences are aligned for optimal comparison purposes (e.g., between the first and second amino acid sequences or nucleic acid sequences for optimal alignment. Gaps are introduced in one or both or non-homologous sequences can be discarded for comparison purposes).
  • the length of the reference sequences aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60% and even more preferably at least 70%, 80% , 90%, 100% of the reference sequence length.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position.
  • Sequence comparisons and calculation of percent identity between two sequences can be accomplished using mathematical algorithms.
  • the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm (at http://www.gcg.com) is used that has been integrated into the GAP program of the GCG software package available), using the Blossum 62 matrix or the PAM250 matrix and gap weights 16, 14, 12, 10, 8, 6, or 4 and length weights 1, 2, 3, 4, 5, or 6, to determine the distance between two amino acid sequences percent identity.
  • the GAP program in the GCG software package (available at http://www.gcg.com) is used, using the NWSgapdna.CMP matrix and gap weights 40, 50, 60, 70 or 80 and A length weight of 1, 2, 3, 4, 5, or 6 determines the percent identity between two nucleotide sequences.
  • a particularly preferred set of parameters (and one that should be used unless otherwise specified) is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.
  • antigen-binding fragment is a portion or segment of an intact or complete antibody having fewer amino acid residues than an intact or complete antibody, which is capable of binding an antigen or competing with an intact antibody (ie, with the intact antibody from which the antigen-binding fragment is derived) bind antigen.
  • Antigen-binding fragments can be prepared by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies.
  • Antigen binding fragments include, but are not limited to, Fab, Fab', F(ab') 2 , Fv, single chain Fv, diabody, single domain antibody (sdAb).
  • the Fab fragment is a monovalent fragment consisting of VL, VH, CL and CH1 domains, eg, Fab fragments can be obtained by papain digestion of complete antibodies.
  • digestion of complete antibodies by pepsin below the disulfide bond in the hinge region produces F(ab') 2 , which is a dimer of Fab', a bivalent antibody fragment.
  • F(ab') 2 can be reduced under neutral conditions by breaking the disulfide bond in the hinge region, thereby converting the F(ab') 2 dimer to a Fab' monomer.
  • a Fab' monomer is basically a Fab fragment with a hinge region (for a more detailed description of other antibody fragments see: Fundamental Immunology, ed.
  • the Fv fragment consists of the VL and VH domains of the antibody one-arm. Additionally, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, using recombinant methods, they can be linked by a synthetic linker peptide that enables the production of the two domains as a single protein chain, described in The VL and VH domains of a single protein chain are paired to form a single-chain Fv.
  • the antibody fragments can be obtained by chemical methods, recombinant DNA methods or protease digestion.
  • monoclonal antibody refers to a preparation of an antibody molecule having a single amino acid composition, not to the method by which it is produced.
  • Monoclonal antibodies or antigen-binding fragments thereof can be produced, for example, by hybridoma techniques, recombinant techniques, phage display techniques, synthetic techniques such as CDR grafting, or a combination of these or other techniques known in the art.
  • the term “specifically binds” or “binding” when used in reference to antigens and antibodies means that the antibody forms a complex with an antigen that is relatively stable under physiological conditions.
  • Methods for determining whether an antibody specifically binds to an antigen include, for example, surface plasmon resonance assays, MSD assays (Estep, P. et al., High throughput solution-based measurement of antibody-antigen affinity). and epitope binning, MAbs, 2013.5(2):p.270-278), ForteBio affinity assay (Estep, P et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning.MAbs, 2013.5(2): p.270-8), etc.
  • anti-CD47 antibody refers to an antibody that is capable of specifically binding to the CD47 protein or its fragment, and block or inhibit the binding of CD47 to SIRP ⁇ on the surface of macrophages, so that the antibody can be used as a preventive and/or therapeutic agent targeting CD47.
  • non-fixed combination means that the active ingredients (eg, (i) an anti-CD47 antibody or antigen-binding fragment thereof, and (ii) a DNA methyltransferase inhibitor (eg, azacitidine)) are concomitantly as separate entities , without a specific time limit, or at the same or different time intervals, sequentially administered to a subject, wherein such administration provides prophylactically or therapeutically effective levels of the two antibodies in the subject.
  • active ingredients eg, (i) an anti-CD47 antibody or antigen-binding fragment thereof, and (ii) a DNA methyltransferase inhibitor (eg, azacitidine)
  • a DNA methyltransferase inhibitor eg, azacitidine
  • the anti-CD47 antibody or antigen-binding fragment thereof and a DNA methyltransferase inhibitor (eg, azacitidine) used in the drug combination are administered at levels not exceeding those used alone.
  • the term "fixed combination" means the drug of the invention.
  • the two antibodies in the combination are administered to the subject simultaneously in the form of a single entity.
  • the administration dose and/or time interval of the two antibodies in the pharmaceutical combination of the present invention are selected so that the combined use of each antibody can be used in the treatment of The disease or condition produces a greater effect than any one antibody alone can achieve.
  • Each antibody may be in a separate formulation, which may or may not be the same.
  • administration refers to the physical introduction of each active ingredient in the pharmaceutical combination of the present invention into a subject using any of a variety of methods and delivery systems known to those skilled in the art.
  • Routes of administration for each antibody in the pharmaceutical combinations of the present invention include oral, intravenous (eg, infusion (also known as drip) or injection), intramuscular, subcutaneous, intraperitoneal, spinal, topical, or other parenteral routes of administration.
  • parenteral administration refers to modes of administration other than parenteral and topical administration, including, without limitation, intramuscular, intraarterial, intravenous, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital , intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, and in vivo electroporation.
  • each antibody in the pharmaceutical combination of the present invention can be formulated into capsules, tablets, injections (including infusion or injection), syrups, sprays, lozenges, liposomes or suppositories and the like.
  • the term "effective amount” refers to an amount effective to achieve the desired prophylactic and/or therapeutic result, at the required dosage and for the required period of time.
  • the prophylactically and/or therapeutically effective amount may vary depending on a variety of factors, such as the disease state, the age, sex, and weight of the subject.
  • a therapeutically effective amount is any amount in which the toxic or detrimental effects are outweighed by the therapeutically beneficial effects.
  • a “therapeutically effective amount” preferably inhibits a measurable parameter (eg, tumor growth rate) by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and still more, relative to an untreated subject. Preferably at least about 80%.
  • prophylactically effective amount refers to an amount effective to achieve the desired prophylactic result, at the required dose and for the required period of time. Typically, a prophylactically effective amount is less than a therapeutically effective amount because the prophylactic dose is administered in a subject prior to or at an earlier stage of the disease.
  • dose is the amount of a drug that elicits a prophylactic and/or therapeutic effect. Dosages are related to the amount of drug in free form unless otherwise stated. If the drug is in the form of a pharmaceutically acceptable salt, the amount of drug is increased proportionally to the amount of drug in free form. For example, the dosage will be stated on the product packaging or product information sheet. For example, the administered dose is calculated in milligrams per kilogram (mg/kg) based on the patient's weight or in milligrams per square meter of body surface area (mg/m2) based on the patient's height and weight.
  • mg/kg milligrams per kilogram
  • mg/m2 milligrams per square meter of body surface area
  • single drug dosage unit refers to a single drug dosage form comprising an anti-CD47 antibody or antigen-binding fragment thereof of the invention and/or comprising a multi-receptor tyrosine kinase inhibitor of the invention for a single administration to a patient single-dose dosage form.
  • the single pharmaceutical dosage form may be a parenterally administered dosage form, such as a vial, ampule, prefilled needle or prefilled syringe for injection, containing the drug in solution or lyophilized powder, or parenterally.
  • Dosage forms such as tablets, capsules, lozenges, powders, suspensions, etc. for oral administration.
  • inhibitor refers to the reduction of certain parameters by a given molecule (eg (i) an anti-CD47 antibody or antigen-binding fragment thereof; (ii) a DNA methyltransferase inhibitor.
  • a given molecule eg (i) an anti-CD47 antibody or antigen-binding fragment thereof; (ii) a DNA methyltransferase inhibitor.
  • the term includes an inhibition of at least 5% , 10%, 20%, 30%, 40% or more activity. Thus, inhibition need not be 100%.
  • treating in reference to a disease refers to alleviating the disease (ie, slowing or arresting or reducing the development of the disease or at least one clinical symptom thereof), preventing or delaying the onset or development or progression of the disease.
  • prevention includes the inhibition or delay of the onset or frequency of a disease or disorder or its symptoms, which generally refers to the administration of a drug prior to the onset of a sign or symptom, particularly in a subject at risk .
  • subject refers to mammals and non-mammals. Mammal refers to any member of the mammalian species, which includes, but is not limited to: humans; non-human primates, cows, horses, sheep, pigs, rabbits, dogs, cats, and the like. The term “subject” does not limit a particular age or gender. In some embodiments, the subject is a human.
  • AE adverse event
  • AE is any unfavorable and often unexpected or unwanted sign (including abnormal laboratory findings), symptom or disease associated with the use of a medical treatment.
  • adverse events can be associated with activation of the immune system in response to treatment or expansion of immune system cells (eg, T cells) in response to treatment.
  • the medical treatment can have one or more associated AEs, and each AE can have the same or different levels of severity.
  • OS all survival
  • progression-free survival is the time from a patient's first use of an investigational drug until disease progression or death from any cause.
  • the pharmaceutical combination of the present invention comprises (i) an anti-CD47 antibody or antigen-binding fragment thereof; and (ii) a DNA methyltransferase inhibitor, preferably the DNA methyltransferase inhibitor is azacitidine.
  • the anti-CD47 antibody or antigen-binding fragment thereof in the pharmaceutical combination of the present invention comprises 3 CDRs in the heavy chain variable region amino acid sequence shown in SEQ ID NO:7 and 3 CDRs in the amino acid sequence shown in SEQ ID NO:8 Three CDRs in the amino acid sequence of the light chain variable region.
  • the anti-CD47 antibody or antigen-binding fragment thereof in the pharmaceutical combination of the present invention comprises 6 CDRs, wherein HCDR1, HCDR2 and HCDR3 comprise, for example, GSISSYYWS (SEQ ID NO: 1), YIYYSGSTNYNPSLKS (SEQ ID NO: 2) and the amino acid sequence shown in ARGKTGSAA (SEQ ID NO: 3) or consisting of the amino acid sequence, and wherein LCDR1, LCDR2 and LCDR3 comprise such as RASQGISRWLA (SEQ ID NO: 4), AASSLQS (SEQ ID NO: 5) and the amino acid sequence shown in QQTVSFPIT (SEQ ID NO: 6) or consist of the amino acid sequence; wherein the HCDR1 is located at H27-H35 in the Kabat numbering system, HCDR3 is located at H93-H102 in the Kabat numbering system, and the HCDR2 , LCDR1, LCDR2 and LCDR3 are determined by the Kabat definition
  • the anti-CD47 antibody or antigen-binding fragment thereof comprises a heavy chain variable region VH and a light chain variable region VL, wherein the heavy chain variable region comprises or has at least 90% of the sequence of SEQ ID NO:7,95 %, 98% or 99% identical sequence, and the light chain variable region comprises or has at least 90%, 95%, 98% or 99% identity to the sequence of SEQ ID NO: 8.
  • the anti-CD47 antibody or antigen-binding fragment thereof comprises heavy and light chain sequences, wherein the heavy chain comprises or is at least 90%, 95%, 98% or 99% identical to the sequence of SEQ ID NO:9 and the light chain comprises or is at least 90%, 95%, 98% or 99% identical to the sequence of SEQ ID NO: 10.
  • the anti-CD47 antibody is the anti-CD47 antibody ADI-26630 disclosed in WO2019042285A1.
  • the present invention provides the aforementioned pharmaceutical combinations of the present invention for use in preventing and/or treating the severity of at least one symptom or sign of cancer or inhibiting cancer cell growth in a subject.
  • the present invention provides methods of preventing or treating cancer comprising administering to a subject in need thereof an effective amount of a pharmaceutical combination of the present invention.
  • the effective amount includes a prophylactically effective amount and a therapeutically effective amount.
  • the present invention provides the use of the aforementioned pharmaceutical combination of the present invention in the preparation of a medicament for preventing or treating cancer.
  • Cancers described herein include solid tumors and hematological malignancies, eg, acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS).
  • AML acute myeloid leukemia
  • MDS myelodysplastic syndromes
  • the anti-CD47 antibody or antigen-binding fragment thereof in the pharmaceutical combination of the present invention may be administered to a subject in need thereof in one or more doses, wherein in the case of multiple doses, 1 week, 2 weeks after the previous dose , 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks to administer the next dose.
  • a dose of anti-CD47 antibody or antigen-binding fragment thereof may be selected from 1-50 mg/kg of subject body weight (eg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg /kg, 25mg/kg, 30mg/kg, 35mg/kg, 40mg/kg, 45mg/kg or 50mg/kg, preferably 1mg/kg, 30mg/kg or 45mg/kg); or, in the pharmaceutical combination of the present invention
  • a single administration dose of an anti-CD47 antibody or antigen-binding fragment thereof may be selected from about 60-3000 mg, for example about 60 mg, 120 mg, 180 mg, 300 mg, 600 mg, 900 mg, 1200 mg, 1500 mg, 1800 mg, 2100 mg, 2400 mg, 2700 mg, or 3000 mg, Preferably about 60 mg, 1800 mg, 2700 mg.
  • the anti-CD47 antibody or antigen-binding fragment thereof in the pharmaceutical combination of the present invention is administered to the subject first at a low dose (eg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 5 mg/kg), and then at a high dose (eg, 10 mg).
  • a low dose eg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 5 mg/kg
  • a high dose eg, 10 mg
  • a single administered dose of the antibody or antigen-binding fragment thereof is administered to a subject at a low dose (eg, 60 mg, 120 mg, 180 mg, 300 mg), followed by a high dose (eg, 600 mg, 600 mg, 900 mg, 1200 mg, 1500 mg, 1800 mg, 2100 mg, 2400 mg, 2700 mg or 3000 mg) administered to the subject.
  • a low dose eg, 60 mg, 120 mg, 180 mg, 300 mg
  • a high dose eg, 600 mg, 600 mg, 900 mg, 1200 mg, 1500 mg, 1800 mg, 2100 mg, 2400 mg, 2700 mg or 3000 mg
  • the DNA methyltransferase inhibitor (eg, azacitidine) in the pharmaceutical combination of the present invention may be administered to a subject in need thereof in one or more doses, wherein in the case of multiple doses, the previous Dosing is followed by administration about once a day, once every two days, once every three days, once every four days, or on a regimen of once a day for one week and every three weeks for one week off.
  • Dose of a DNA methylation inhibitor e.g., azacitidine
  • a DNA methylation inhibitor may be selected 70-80mg / m 2 body surface area per square meter of the subject (e.g.
  • a single administration dose of a DNA methyltransferase inhibitor (eg, azacitidine) in the pharmaceutical combination of the present invention is about 120-140 mg, eg, about 120 mg, 130 mg, 140 mg, preferably about 130 mg.
  • the pharmaceutical combination of the present invention may be in any dosage form known to those skilled in the art, such as tablets, capsules, granules, syrups, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions , solutions, syrups, aerosols, ointments, creams and injections, etc.
  • the anti-CD47 antibody or its antigen-binding fragment and the DNA methyltransferase inhibitor eg, azacitidine
  • the dosage forms may be different or the same.
  • the anti-CD47 antibody or antigen-binding fragment thereof in the pharmaceutical combination of the present invention is in a dosage form for intravenous administration, eg, an intravenous injection.
  • the DNA methyltransferase inhibitor (eg, azacitidine) in the pharmaceutical combination of the present invention is a subcutaneously administered dosage form, such as a subcutaneous injection.
  • the pharmaceutical combination of the present invention may be a pharmaceutical dosage unit, eg, a single pharmaceutical dosage unit.
  • the administration period of the pharmaceutical combination of the present invention can be two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, half a year or longer, preferably two weeks, three months Weeks or four weeks, wherein the anti-CD47 antibody or antigen-binding fragment thereof is administered 2, 3, or 4 times per cycle, and/or DNA methyltransferase inhibition is administered 5, 6, or 7 times per cycle agent, preferably the DNA methyltransferase inhibitor is administered continuously from 1 to 7 days.
  • the anti-CD47 antibody or antigen-binding fragment thereof and the DNA methyltransferase inhibitor in the pharmaceutical combination of the present invention may be administered separately, simultaneously or sequentially.
  • the pharmaceutical combination of the present invention is administered in a four-week cycle, wherein the anti-CD47 antibody or antigen-binding fragment thereof is administered at a dose of 1 mg/kg on the first day of the first week, and the first day of the second week and thereafter.
  • a dose of 30 mg/kg is given daily, preferably intravenously, and a DNA methyltransferase inhibitor (eg, azacitidine) is given at a dose of 75 mg/m 2 daily, preferably subcutaneously, during the first week of each cycle.
  • a DNA methyltransferase inhibitor eg, azacitidine
  • the pharmaceutical combination of the present invention results in an increased, preferably synergistically increased response to tumors compared to monotherapy administered with an anti-CD47 antibody or antigen-binding fragment thereof or monotherapy administered with a DNA methyltransferase inhibitor such as azacitidine. growth inhibition.
  • the pharmaceutical combination of the present invention results in decreased tumor growth compared to administration of monotherapy of an anti-CD47 antibody or antigen-binding fragment thereof or monotherapy of a DNA methyltransferase inhibitor (eg, azacitidine). Inhibit at least about 60%, about 70%, about 80%, about 90%, about 100%.
  • administration of the pharmaceutical combinations of the present invention results in increased tumor regression, tumor shrinkage, and/or disappearance.
  • the pharmaceutical combination of the present invention prevents tumor recurrence and/or increases the duration of survival in a subject, eg, increases the duration of survival by more than 15 days, more than 1 month, more than 3 months, more than 6 months, More than 12 months, more than 18 months, more than 24 months, more than 36 months, or more than 48 months.
  • the pharmaceutical combinations of the present invention increase progression-free survival or overall survival.
  • the pharmaceutical combinations of the present invention can reduce adverse events, eg, hematologic toxicities, non-hematologic toxicities, or other toxicities, resulting from administration of each single agent.
  • Another object of the present invention is to provide a kit comprising a pharmaceutical combination of the present invention and instructions for using the pharmaceutical combination, preferably the kit comprises one or more single pharmaceutical dosage units.
  • kit of parts of the present invention contains in the same package:
  • One or more single pharmaceutical dosage units comprising a dose of about 60-3000 mg, preferably about 60 mg, 120 mg, 180 mg, 1800 mg or 2700 mg, more preferably about 60 mg or 1800 mg as before the anti-CD47 antibody or antigen-binding fragment thereof;
  • One or more single drug dosage units comprising a dose of about 120-140 mg, such as about 120 mg, 130 mg, 140 mg, preferably about 130 mg, of azacitidine which is pharmaceutically acceptable Salt.
  • An anti-CD47 antibody is an antibody that blocks or inhibits the interaction of CD47 ligand SIRP ⁇ with CD47 by binding to CD47, such as any of the anti-CD47 monoclonal antibodies disclosed in Patent Publication No. WO2019042285A1.
  • the entire contents of this PCT application are hereby incorporated by reference for the purposes of this application.
  • This example exemplifies the use of the anti-CD47 antibody ADI-26630 disclosed in WO2019042285A1. It should be noted that those skilled in the art can also use other anti-CD47 antibodies, including but not limited to other anti-CD47 antibodies disclosed in WO2019042285A1, in the pharmaceutical combination of the present invention.
  • amino acid sequences of the CDR regions, light and heavy chain variable regions, light and heavy chains of the anti-CD47 antibody ADI-26630 are given in the "Detailed Description" of the present application.
  • the anti-CD47 antibody in the pharmaceutical combination of the present invention can be produced by any antibody preparation technique known to those skilled in the art.
  • antibody expression and purification see Patent Publication No. WO2019042285A1.
  • MV-4-11 cells ATCC, CRL-9591TM
  • HL60 Shanghai Cell Bank, Chinese Academy of Sciences, TcHu23
  • serially diluted azacitidine the drug source was the same as in Example 2, the highest concentration was 500ug
  • human CD47 protein expression was detected by flow cytometry at 24 hours.
  • Example 3 Establishment of MV-4-11 (human myelomonocytic leukemia cells) tumor-bearing NOD-SCID mouse model and the anti-tumor effect of the drug combination of the present invention
  • NOD-SCID mice (15-18 g, 35-41 days) were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. The grade is SPF, the number is 60, and the certificate number is 1100112011009957. Experiments were carried out after acclimation and rearing for 3 days after arrival. The subcutaneous dose of MV-4-11 (ATCC, CRL-9591TM) was 6 million per mouse. Cells were grouped according to tumor volume on day 6 after subcutaneous inoculation.
  • mice A total of 60 mice were divided into 8 groups according to tumor volume, with 7 mice in each group.
  • 3Anti-CD47 antibody-0.1mg/kg (the anti-CD47 antibody was administered on the first day after the end of azacitidine administration)
  • the anti-CD47 antibody administration time point in the single drug group was the same as that of anti-CD47 antibody + azacitidine -0.1+3mg/kg (anti-CD47 antibody was administered on the first day after the end of azacitidine administration) group, anti-CD47 antibody+azacitidine-0.1+3mg/kg (anti-CD47 antibody was administered in azacitidine
  • the anti-CD47 antibody in the group started to be administered on the first day after the end of the administration of azacitidine, and was administered 3 times in total;
  • the anti-CD47 antibody administration time point in the single drug group was the same as that of anti-CD47 antibody+azacitidine -0.1+3mg/kg (the anti-CD47 antibody was administered on the third day after the end of azacitidine administration) group, the anti-CD47 antibody+azacitidine-0.1+3mg/kg (the anti-CD47 antibody was administered in azacitidine
  • the anti-CD47 antibody in the group started to be administered on the third day after the end of the administration of azacitidine, and was administered 3 times in total;
  • Anti-CD47 antibody - 0.1 mg/kg (anti-CD47 antibody was administered on the fifth day after the end of azacitidine administration)
  • the time point of anti-CD47 antibody administration in the single drug group was the same as that of anti-CD47 antibody + azacitidine -0.1+3mg/kg (the anti-CD47 antibody was administered on the fifth day after the end of azacitidine administration) group, the anti-CD47 antibody+azacitidine-0.1+3mg/kg (the anti-CD47 antibody was administered in azacitidine
  • the anti-CD47 antibody in the group started to be administered on the fifth day after the end of the administration of azacitidine, and was administered 3 times in total;
  • the mode of administration was IP (intraperitoneal injection).
  • TGI Tumor inhibition rate
  • Tvolcontrol–Tvoltreated the terminal tumor volume after administration in the control group – the final volume of the tumor in the administration group after administration;
  • Tvolcontrol–Tvolpredose terminal tumor volume after administration in the control group.
  • TGI Tumor Inhibition Rate
  • Figure 3 The effect of tumor volume of tumor-bearing mice in each group over time is shown in Figure 3, and the body weight change rate of tumor-bearing mice in each group is shown in Figure 4.
  • Figure 5, Figure 6 and Figure 7 show the effect of azacitidine combined administration at different times on tumor volume in tumor-bearing mice over time.
  • the single-drug anti-CD47 antibody was -0.1 mg/kg (the anti-CD47 antibody was administered with azacitidine).
  • the administration started on the first day after the end of the administration) group, the anti-CD47 antibody-0.1 mg/kg (the anti-CD47 antibody was administered on the third day after the end of azacitidine administration) group, the anti-CD47 antibody-0.1 mg/kg ( The tumor inhibition rates of the anti-CD47 antibody group started on the fifth day after the azacitidine administration were 53%, 26%, and 42%, respectively.
  • the tumor inhibition rate of single-agent azacitidine-3 mg/kg was 51%.
  • Co-administration of anti-CD47 antibody and azacitidine Anti-CD47 antibody + azacitidine-0.1 + 3 mg/kg (anti-CD47 antibody started on the first day after the end of azacitidine administration) group, anti-CD47 antibody +Azacitidine-0.1+3mg/kg (anti-CD47 antibody started on the third day after azacitidine administration) group, anti-CD47 antibody+azacitidine-0.1+3mg/kg (anti-CD47 The anti-CD47 antibody combined with azacitidine had a significantly higher tumor inhibition rate than the anti-CD47 antibody combined with azacitidine.
  • Anti-CD47 antibody single-drug and azacitidine single-drug groups have good tumor-inhibiting effects, and there are statistical differences (the statistical method adopts two-way analysis of variance, in Figure 5, Figure 6 and Figure 7, ** represents P value is less than 0.01, **** represents P value is less than 0.0001), indicating that anti-CD47 antibody and azacitidine have a synergistic effect in anti-tumor, and have a good tumor inhibitory effect.
  • Example 4 Establishment of HL60 (human myeloid leukemia) tumor-bearing NOD-SCID mouse model and the antitumor effect of the drug combination of the present invention
  • NOD-SCID mice (15-18 g, 35-41 days) were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. The grade is SPF, the quantity is 70, and the certificate number is 1100112011010881. Experiments were carried out after acclimation and rearing for 3 days after arrival.
  • HL60 (Shanghai Cell Bank, Chinese Academy of Sciences, TcHu23) was subcutaneously inoculated at 5 million per mouse. Cells were grouped according to tumor volume on day 7 after subcutaneous inoculation.
  • mice A total of 75 mice were divided into 6 groups according to tumor volume, with 6 mice in each group.
  • the frequency of drug administration in the anti-CD47 antibody group is shown in Table 4. The drug was administered from the 7th day, and was administered 3 times in total;
  • the administration frequency of azacitidine is once a day, starting from the 7th day, for 5 consecutive administrations;
  • Anti-CD47 antibody+azacitidine-0.1+3mg/kg group anti-CD47 antibody on the third day after the end of azacitidine administration
  • the drug was administered on the same day, and the drug was administered 3 times in total;
  • the mode of administration was IP (intraperitoneal injection).
  • TGI Tumor inhibition rate
  • Tvolcontrol–Tvoltreated the terminal tumor volume after administration in the control group – the final volume of the tumor in the administration group after administration;
  • Tvolcontrol–Tvolpredose terminal tumor volume after administration in the control group.
  • TGI Tumor Inhibition Rate
  • the tumor inhibition rate of the single-drug anti-CD47 antibody-0.2 mg/kg group was 63%.
  • the tumor inhibition rate in the single-agent azacitidine-3 mg/kg group was 65%.
  • Anti-CD47 antibody+azacitidine-0.2+3mg/kg group (anti-CD47 antibody and azacitidine were administered at the same time on the first day), anti-CD47 antibody+azacitidine-0.2+3mg/kg (anti-CD47 antibody Administered on the first day after the end of azacitidine administration) group, anti-CD47 antibody + azacitidine-0.2 + 3 mg/kg (anti-CD47 antibody started on the third day after the end of azacitidine administration.
  • the tumor inhibition rates of the drug) group were 85%, 98%, and 91%, respectively.
  • the tumor inhibition rate after the combined administration of anti-CD47 antibody and azacitidine was significantly higher than that of the anti-CD47 antibody single drug and azacitidine single drug group, which had a good tumor inhibition effect, and there was a statistical difference (statistical method used Two-way analysis of variance, in Figure 10 and Figure 11, **** represents a P value less than 0.0001), indicating that anti-CD47 antibody and azacitidine have a synergistic effect in anti-tumor, and have a good tumor inhibitory effect.
  • mice in the anti-CD47 antibody + azacitidine-0.2 mg/kg + 3 mg/kg were treated with azacitidine continuously.
  • Body weight decreased during administration, and gradually recovered after administration.
  • the other groups of mice did not show significant weight loss after administration, and the mice behaved normally.
  • Anti-CD47 antibody produced by Innovent Biopharmaceutical (Suzhou) Co., Ltd., specification 1ml/100mg or 5ml/500mg.
  • Azacitidine produced by Baxter Oncology GmbH, specification 100mg.
  • Relapsed/refractory AML defined as one of the following:
  • Recurrence within 12 months after consolidation or intensive therapy after reaching CR (recurrence is defined as: leukemia cells reappeared in peripheral blood or blast cells in bone marrow> 5% after reaching CR, and the above symptoms have been excluded after consolidation chemotherapy. bone marrow regeneration and other reasons);
  • Newly diagnosed elderly AML intolerant to standard induction chemotherapy defined as meeting the following conditions:
  • Age ⁇ 18 years old, gender is not limited.
  • ECOG PS Eastern Cooperative Oncology Group Performance Status
  • Renal function Serum creatinine (Creatinine, Cr) ⁇ 1.5 ⁇ ULN or endogenous creatinine clearance rate (Creatinine clearance rate, CCr) ⁇ 60ml/min (calculated by CKD-EPI formula, see Annex 5 for the specific calculation formula);
  • Coagulation function International Normalized Ratio (INR) ⁇ 1.5, and Prothrombin time (PT) or Activated partial thromboplastin time (APTT) ⁇ 1.5 ⁇ ULN.
  • Female subjects of childbearing age or male subjects whose partners are women of childbearing age should take effective contraceptive measures throughout the treatment period and 6 months after the treatment period.
  • IPSS-R prognostic score > 3 MDS patients with intermediate and high risk (IPSS-R prognostic score > 3) diagnosed by bone marrow aspiration or biopsy;
  • Liver function Serum total bilirubin (TBIL) ⁇ 1.5 ⁇ Upper Limit of Normal value (ULN); Alanine transaminase (ALT) and aspartate aminotransferase Enzyme (Aspartate amino transferase, AST) ⁇ 2.5 ⁇ ULN.
  • Albumin Albumin (Albumin, ALB)> 30g/L. Subjects with Gilbert syndrome require TBIL ⁇ 3 ⁇ ULN.
  • Renal function Serum creatinine (Creatinine, Cr) ⁇ 1.5 ⁇ ULN or endogenous creatinine clearance rate (Creatinine clearance rate, CCr) ⁇ 50ml/min (calculated by CKD-EPI formula, see Annex 5 for the specific calculation formula).
  • Coagulation function International Normalized Ratio (INR) ⁇ 1.5, and Prothrombin time (PT) or Activated partial thromboplastin time (APTT) ⁇ 1.5 ⁇ ULN;
  • the specific dosage regimen is: anti-CD47 antibody: intravenous injection of 1.0 mg/kg on the first day of the first week, and intravenous injection of 30 mg/kg on the first day of the second week and thereafter.
  • Azacitidine 75 mg/m 2 , administered by subcutaneous injection every day from day 1 to day 7, every 4 weeks as a cycle.

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Abstract

提供了抗CD47抗体或其抗原结合片段和DNA甲基化转移酶抑制剂的药物组合、包含所述药物组合的药物组合物或成套药盒、以及使用所述药物组合、药物组合物或成套药盒预防和/或治疗肿瘤和/或癌症的方法。抗CD47抗体或其抗原结合片段联合DNA甲基化转移酶抑制剂具有优异的抗肿瘤药效，尤其在AML和MDS适应症发挥协同抗肿瘤作用。

Description

抗CD47抗体或其抗原结合片段和DNA甲基化转移酶抑制剂的组合及其用途
本申请要求申请日为2020/7/10的中国专利申请2020106622282的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明涉及医药学领域。具体地，本发明涉及一种抗肿瘤药物组合及其用途，尤其涉及一种包含抗CD47抗体或其抗原结合片段和DNA甲基化转移酶抑制剂的药物组合及其在预防和/或治疗肿瘤和/或癌症中的用途。
背景技术
急性髓系白血病(Acute myeloid leukemia,AML)是一组造血干细胞克隆性增殖异常的高度异质性疾病，是成人急性白血病中最常见的类型。2018年柳叶刀血液学杂志上一篇文章汇总了全球184个地区的AML流行病学数据显示：AML发病率以澳大利亚最高，约为4.8/10万人，其中中国发病率约为2.6/10万人。根据2020年最新的癌症杂志估计，2020年美国新发AML人数约19,940人，死亡人数约11180人，男女比例约为1.4：1。近年来，国内外先后有批准的去甲基化药物(hypomethylating agents,HMA)(包括：阿扎胞苷或地西他滨)联合维奈克拉(Venetoclax)用于治疗AML。尽管这些药物的联合提高了患者的预后，但半数以上的AML患者最终仍将面临复发，一旦复发，这些患者的中位生存期往往不超过1年。
骨髓增生异常综合征(myelodysplastic syndromes,MDS)是一类起源于造血干细胞的异质性髓系克隆性疾病，其特点是髓系发育异常，表现为无效造血、难治性血细胞减少，高风险向急性髓系白血病(acute myeloid leukemia,AML)转化。最新的SEER流行病学数据显示，MDS每年的发病率约为4.9/10万人，且随着年龄增高，发病率显著增高。MDS的发病率在＜40岁的儿童、青少年及年轻人群中仅为0.1/10万，但在70～79岁和80岁以上的人群中分别高达30.2/10万及59.8/10万。MDS依据国际预后积分系统(IPSS，IPSS-R)分为两大类：较低危组与较高危组，较低危组通常包含IPSS低危和中危l以及IPSS-R极低危、低危组，治疗目标是改善患者外周血血细胞计数和提高患者生活质量，治疗方法以观察等待或促造血等支持治疗为主；而较高危组(IPSS中危2和高危以及IPSS-R中危、高危和极高危)，治疗目标是清除MDS恶性克隆和延长患者生存期，主要的治疗选择有去甲基化药物(Hypomethylating agents,HMA)的治疗、化 疗、造血干细胞移植及支持治疗。HMA是较高危MDS患者最常用的治疗药物尽管HMA单药治疗可以使生存期延长，但仅有40～50％的患者对HMA药物治疗有效，而且这种疗效往往不持久，最终会在2年内复发，一旦复发，这些患者的中位生存期只有4.3个月。
因此，开发安全有效的HMA的用药方案是目前AML和MDS适应症在临床亟需解决的问题。
发明内容
本发明人令人惊讶地发现，本申请的药物组合能够以协同作用和/或改善治疗中的不良事件(AE)发生率和/或严重程度的方式对癌症、尤其是对AML和MDS起到预防和/或治疗的效果。
具体而言，本发明提供了以下实施方案：
第一方面，本发明提供了一种药物组合，其包含(i)抗CD47抗体或其抗原结合片段；和(ii)DNA甲基化转移酶抑制剂，
其中抗CD47抗体或其抗原结合片段包含6个CDR，其中HCDR1、HCDR2和HCDR3包含如GSISSYYWS(SEQ ID NO:1)、YIYYSGSTNYNPSLKS(SEQ ID NO:2)和ARGKTGSAA(SEQ ID NO:3)所示的氨基酸序列或由所述氨基酸序列组成，且其中LCDR1、LCDR2和LCDR3包含如RASQGISRWLA(SEQ ID NO:4)、AASSLQS(SEQ ID NO:5)和QQTVSFPIT(SEQ ID NO:6)所示的氨基酸序列或由所述氨基酸序列组成；其中所述HCDR1在Kabat编号系统中位于H27-H35，HCDR3在Kabat编号系统中位于H93-H102，所述HCDR2、LCDR1、LCDR2和LCDR3由Kabat定义规则确定；
其中DNA甲基化转移酶抑制剂选自阿扎胞苷或其药学上可接受的盐。
在一个实施方案中，本发明的药物组合中的抗CD47抗体或其抗原结合片段包含重链可变区和轻链可变区，其中重链可变区包含SEQ ID NO:7的序列或与其具有至少90％,95％,98％或99％同一性的序列，且轻链可变区包含SEQ ID NO:8的序列或与其具有至少90％,95％,98％或99％同一性的序列。
在一个实施方案中，所述抗CD47抗体或其抗原结合片段包含重链和轻链，其中重链包含SEQ ID NO:9的序列或与其具有至少90％,95％,98％或99％同一性的序列，且轻链包含SEQ ID NO：10的序列或与其具有至少90％,95％,98％或99％同一性所示的序列。
另一方面，本发明还提供以下实施方式：
1.根据前述实施方案中的任一项的药物组合，其中
(i)的单次施用剂量选自1-50mg/kg，例如1mg/kg、2mg/kg、3mg/kg、5mg/kg、10mg/kg、15mg/kg、20mg/kg、25mg/kg、30mg/kg、35mg/kg、40mg/kg、45mg/kg或50mg/kg；优选地为1mg/kg、30mg/kg或45mg/kg；或(i)的单次施用剂量选自约60-3000mg，例如约60mg、120mg、180mg、300mg、600mg、900mg、1200mg、1500mg、1800mg、2100mg、2400mg、2700mg或3000mg，优选地为约60mg、1800mg、2700mg；和
(ii)的单次施用剂量选自70-80mg/m 2，例如70mg/m 2、75mg/m 2或80mg/m 2；优选地为75mg/m 2；或(ii)的单次施用剂量选自约120-140mg，例如约120mg、130mg、140mg，优选地为约130mg。
2.根据前述实施方案中任一项的药物组合，其中
(i)先给予1mg/kg的单次施用剂量，再给予30mg/kg的单次施用剂量；和
(ii)给予75mg/m 2的单次施用剂量。
3.根据前述实施方案中任一项的药物组合，其中
(i)为胃肠外给药，优选为静脉注射给药；和
(ii)为胃肠外给药，优选为皮下注射给药。
4.根据前述实施方案中任一项的药物组合，所述药物组合是按给药周期施用的，每个给药周期为二周、三周、四周、一个月、两个月、三个月、四个月、五个月、半年或更长时间，优选为二周、三周或四周，其中(i)和(ii)可以分开、同时或依次施用。
5.根据实施方案4所述的药物组合，其中
在每个周期施用2次、3次或4次(i)，和
在每个周期施用5次、6次或7次(ii)，优选为第1至7天连续施用(ii)。
6.根据前述实施方案5所述的药物组合，其中四周为一个周期施用所述药物组合，其中(i)第一周的第一天给予剂量1mg/kg，第二周及以后每周的第一天给予剂量30mg/kg，优选静脉注射，且(ii)每个周期第一周每天给予剂量75mg/m 2，优选皮下注射。
7.根据前述实施方案中任一项所述的药物组合，其用于预防或治疗癌症，其中所述癌症为实体瘤和血液肿瘤，所述血液肿瘤为AML或MDS。
8.根据前述实施方案中任一项所述的药物组合在制备用于预防或治疗癌症的药物中的用途，所述癌症为实体瘤和血液肿瘤，所述血液肿瘤为AML或MDS。
9.用于预防或治疗癌症的方法，所述方法包括向有需要的患者施用有效量的根据前述实施方案中任一项所述的药物组合，所述癌症为实体瘤和血液肿瘤，所述血液肿瘤为AML或MDS。
10.成套药盒，其包含根据前述实施方案中任一项所述的药物组合，优选地所述药盒包含一个或多个单次药物剂量单元。
11.根据实施方案10所述的成套药盒，其中:
(1)所述单次药物剂量单元包含约60-3000mg，优选地约60mg、120mg、180mg、1800mg或2700mg，更优选地约60mg或1800mg剂量的如前所述的抗CD47抗体或其抗原结合片段；和
(2)一个或多个单次药物剂量单元，所述单次药物剂量单元包含约120-140mg，例如120mg、130mg、140mg，优选为约130mg剂量的阿扎胞苷其药学上可接受的盐。
12.根据实施方案10或11所述的成套药盒，其还包含用于指示所述药物组合的使用方法的说明书。
本发明的抗CD47抗体或其抗原结合片段与DNA甲基化转移酶抑制剂的药物组合能够提供更好的抗肿瘤药效。在一个实施方案中，药物组合中的抗CD47抗体或其抗原结合片段是WO2019042285A1中公开的全人单克隆抗CD47抗体或其抗原结合片段ADI-26630，本发明的DNA甲基化转移酶抑制剂为阿扎胞苷。本发明的药物组合取得的抗肿瘤药效显著优于抗CD47抗体或其抗原结合片段或DNA甲基化转移酶抑制剂以单药施用取得的抗肿瘤药效。
附图说明
结合以下附图一起阅读时，将更好地理解以下详细描述的本发明的优选实施方案。出于说明本发明的目的，图中显示了目前优选的实施方案。然而，应当理解本发明不限于图中所示实施方案的精确安排和手段。
图1表示阿扎胞苷在MV-4-11细胞中诱导CD47表达情况。
图2表示阿扎胞苷在HL60细胞中诱导CD47表达情况。
图3表示抗CD47抗体、阿扎胞苷单药及联合后对MV-4-11荷瘤小鼠肿瘤生长的影响。
图4表示抗CD47抗体、阿扎胞苷单药及联合后对MV-4-11荷瘤小鼠体重的影响。
图5表示抗CD47抗体、阿扎胞苷单药及联合后(抗CD47抗体于阿扎胞苷给药结束后第一天开始给药)对MV-4-11荷瘤小鼠的肿瘤体积影响。
图6表示抗CD47抗体、阿扎胞苷单药及联合后(抗CD47抗体于阿扎胞苷给药结束后第三天开始给药)对MV-4-11荷瘤小鼠的肿瘤体积影响。
图7表示抗CD47抗体、阿扎胞苷单药及联合后(抗CD47抗体于阿扎胞苷给药结束后第五天开始给药)对MV-4-11荷瘤小鼠的肿瘤体积影响。
图8表示抗CD47抗体、阿扎胞苷单药及联合后对HL60荷瘤小鼠肿瘤生长的影响。
图9表示抗CD47抗体、阿扎胞苷单药及联合后对HL60荷瘤小鼠体重的影响。
图10表示抗CD47抗体、阿扎胞苷单药及联合后(抗CD47抗体于阿扎胞苷给药结束后第一天开始给药)对HL60荷瘤小鼠肿瘤体积的影响。
图11表示抗CD47抗体联合阿扎胞苷不同时间给药对HL60荷瘤小鼠肿瘤体积的影响。
具体实施方式
在详细描述本发明之前，应了解，本发明不受限于本说明书中的特定方法及实验条件，因为所述方法以及条件是可以改变的。另外，本文所用术语仅是供说明特定实施方案之用，而不意欲为限制性的。
I.定义
除非另有定义，否则本文中使用的所有技术和科学术语均具有与本领域一般技术人员通常所理解的含义相同的含义。为了本发明的目的，下文定义了以下术语。
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小10％的下限和比指定数字数值大10％的上限的范围内的数字数值。
术语“和/或”当用于连接两个或多个可选项时，应理解为意指可选项中的任一项或可选项中的任意两项或更多项。
如本文中所用，术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。在本文中，当使用术语“包含”或“包括”时，除非另有指明，否则也涵盖由所述及的要素、整数或步骤组成的情形。例如，当提及“包含”某个具体序列的抗体可变区时，也旨在涵盖由该具体序列组成的抗体可变区。
术语“完全抗体”和“完整抗体”在本文中可互换地用来指包含由二硫键相互连接的至少两条重链(H)和两条轻链(L)的糖蛋白。每条重链由重链可变区(本文中缩写为VH)和重链恒定区组成。重链恒定区由3个结构域CH1、CH2和CH3组成。每条轻链由轻链可变区(本文中缩写为VL)和轻链恒定区组成。轻链恒定区由一个结构域CL组成。
术语“可变区”或“可变结构域”是指参与抗体与抗原结合的抗体重链的结构域或轻链的结构域。天然抗体的重链和轻链的可变结构域通常具有相似的结构，其中每个可变结构域包含四个保守的构架区(FR)和三个互补决定区。(参见，例如，Kindt等Kuby Immunology，6th ed.,W.H.Freeman and Co.91页(2007))。单个VH或VL结构域可 以足以给予抗原结合特异性。此外，可以使用来自与特定抗原结合的抗体的VH或VL结构域来分离结合所述抗原的抗体，以分别筛选互补VL或VH结构域的文库。参见，例如，Portolano等，J.Immunol.150：880-887(1993)；Clarkson等，Nature 352：624-628(1991)。
“互补决定区”或“CDR区”或“CDR”或“高变区”(在本文中与超变区“HVR”可以互换使用)，是抗体可变结构域中在序列上高变并且形成在结构上确定的环(“超变环”)和/或含有抗原接触残基(“抗原接触点”)的区域。CDR主要负责与抗原表位结合。重链和轻链的CDR通常被称作CDR1、CDR2和CDR3，从N-端开始顺序编号。位于抗体重链可变结构域内的CDR被称作HCDR1、HCDR2和HCDR3，而位于抗体轻链可变结构域内的CDR被称作LCDR1、LCDR2和LCDR3。在一个给定的轻链可变区或重链可变区氨基酸序列中，各CDR的精确氨基酸序列边界可以使用许多公知的抗体CDR指派系统的任一种或其组合确定，所述指派系统包括例如：基于抗体的三维结构和CDR环的拓扑学的Chothia(Chothia等人.(1989)Nature 342:877-883，Al-Lazikani等人,“Standard conformations for the canonical structures of immunoglobulins”,Journal of Molecular Biology,273,927-948(1997))，基于抗体序列可变性的Kabat(Kabat等人,Sequences of Proteins of Immunological Interest,第4版,U.S.Department of Health and Human Services,National Institutes of Health(1987)),AbM(University of Bath)，Contact(University College London)，国际ImMunoGeneTics database(IMGT)(万维网imgt.cines.fr/)，以及基于利用大量晶体结构的近邻传播聚类(affinity propagation clustering)的North CDR定义。
为确定两个氨基酸序列或两个核酸序列的同一性百分数，将所述序列出于最佳比较目的比对(例如，可以为了最佳比对而在第一和第二氨基酸序列或核酸序列之一或二者中引入空位或可以为比较目的而抛弃非同源序列)。在一个优选实施方案中，为比较目的，所比对的参考序列的长度是至少30％、优选地至少40％、更优选地至少50％、60％和甚至更优选地至少70％、80％、90％、100％的参考序列长度。随后比较在对应氨基酸位置或核苷酸位置处的氨基酸残基或核苷酸。当第一序列中的位置由第二序列中对应位置处的相同氨基酸残基或核苷酸占据时，则所述分子在这个位置处是相同的。
可以利用数学算法实现两个序列间的序列比较和同一性百分数的计算。在一个优选实施方案中，使用已经集成至GCG软件包的GAP程序中的Needlema和Wunsch((1970)J.Mol.Biol.48:444-453)算法(在http://www.gcg.com可获得)，使用Blossum 62矩阵或PAM250矩阵和空位权重16、14、12、10、8、6或4和长度权重1、2、3、4、5或6，确定两个氨基酸序列之间的同一性百分数。在又一个优选的实施方案中，使用GCG软件包中的GAP程序(在http://www.gcg.com可获得)，使用NWSgapdna.CMP矩阵和空位权 重40、50、60、70或80和长度权重1、2、3、4、5或6，确定两个核苷酸序列之间的同一性百分数。特别优选的参数集合(和除非另外说明否则应当使用的一个参数集合)是采用空位罚分12、空位延伸罚分4和移码空位罚分5的Blossum 62评分矩阵。
还可以使用PAM120加权余数表、空位长度罚分12，空位罚分4，利用已经并入ALIGN程序(2.0版)的E.Meyers和W.Miller算法,((1989)CABIOS,4:11-17)确定两个氨基酸序列或核苷酸序列之间的同一性百分数。
术语“抗原结合片段”是比完整或完全抗体的氨基酸残基数要少的完整或完全抗体的一部分或一段，其能结合抗原或与完整抗体(即与抗原结合片段所来源的完整抗体)竞争结合抗原。可以通过重组DNA技术、或通过酶或化学切割完整的抗体制备抗原结合片段。抗原结合片段包括但不限于Fab、Fab’、F(ab’) 2、Fv、单链Fv、双体抗体(diabody)、单结构域抗体(sdAb)。所述Fab片段是一种由VL、VH、CL和CH1结构域组成的单价片段，例如，通过木瓜蛋白酶消化完全抗体能够获得Fab片段。此外，通过胃蛋白酶在铰链区的二硫键下面消化完全抗体产生F(ab') 2，其为Fab’的二聚体，是二价的抗体片段。F(ab') 2可以在中性条件下通过破坏铰链区中的二硫键而被还原，由此将F(ab') 2二聚体转化为Fab'单体。Fab'单体基本上是具有铰链区的Fab片段(其它抗体片段的更详细的描述请参见：基础免疫学(Fundamental Immunology),W.E.Paul编辑,Raven Press,N.Y.(1993))。所述Fv片段由抗体单臂的VL和VH结构域组成。另外，虽然Fv片段的两个结构域VL和VH由独立的基因编码，但是使用重组方法，可以将它们通过能够使这两个结构域作为单条蛋白链产生的合成性连接肽连接，在所述单条蛋白链中VL区和VH区配对以形成单链Fv。可以通过化学方法、重组DNA方法或蛋白酶消化法获得所述抗体片段。
术语“单克隆抗体”指具有单一氨基酸组成的抗体分子的制备物，而不指其产生的方法。单克隆抗体或其抗原结合片段可以例如通过杂交瘤技术、重组技术、噬菌体展示技术、合成技术例如CDR嫁接、或此类或其它本领域已知的技术的组合来产生。
当谈及抗原和抗体时使用的术语“特异性结合”或“结合”意指抗体与生理条件下相对稳定的抗原形成复合物。用于确定抗体是否与抗原特异性结合的方法是本领域熟知的并且例如包括表面等离振子共振测定法、MSD测定法(Estep,P.等人,High throughput solution-based measurement of antibody-antigen affinity and epitope binning,MAbs,2013.5(2):p.270-278)、ForteBio亲和力测定法(Estep,P等人，High throughput solution Based measurement of antibody-antigen affinity and epitope binning.MAbs,2013.5(2):p.270-8)等。
术语“抗CD47抗体”、“抗CD47”、“CD47抗体”或“结合CD47的抗体”可互换地使用，是指这样的抗体，所述抗体能够以足够的亲和力特异性结合CD47蛋白或其片段，并 阻断或抑制CD47与巨噬细胞表面SIRPα的结合，以致所述抗体可以用作靶向CD47的预防剂和/或治疗剂。
术语“药物组合”是指非固定组合产品或固定组合产品，包括但不限于药盒、药物组合物。术语“非固定组合”意指活性成分(例如，(i)抗CD47抗体或其抗原结合片段、以及(ii)DNA甲基化转移酶抑制剂(例如阿扎胞苷)以分开的实体被同时、无特定时间限制或以相同或不同的时间间隔、依次地施用于受试者，其中这类施用在受试者体内提供预防或治疗有效水平的所述两种抗体。在一些实施方案中，药物组合中使用的抗CD47抗体或其抗原结合片段和DNA甲基化转移酶抑制剂(例如阿扎胞苷)以不超过它们单独使用时的水平施用。术语“固定组合”意指本发明药物组合中的两种抗体以单个实体的形式被同时施用于受试者。优选对本发明药物组合中的两种抗体的施用剂量和/或时间间隔进行选择，从而使各抗体的联合使用能够在治疗疾病或病症时产生大于单独使用任何一种抗体所能达到的效果。各抗体可以各自呈单独的制剂形式，其制剂形式可以相同也可以不同。
术语“施用”指用本领域技术人员已知的多种方法和递送系统中的任一种将本发明的药物组合中的各活性成分物理导入至受试者。本发明的药物组合中的各抗体的施用途径包括口服、静脉内(例如输注(又称滴注)或注射)、肌内、皮下、腹膜内、脊髓、局部或其他胃肠外施用途径。本文所用的短语“胃肠外施用”指胃肠和局部施用之外的施用方式，非限制性地包括肌内、动脉内、静脉内，鞘内、淋巴内、病灶内、囊内、眶内、心内、皮内、腹膜内、经气管、皮下、表皮下、关节内、囊下、蛛网膜下、脊柱内、硬膜外和胸骨内注射和输注，以及体内电穿孔。相应地，本发明的药物组合中的各抗体可以被配制成胶囊剂、片剂、注射剂(包括输液或注射液)、糖浆、喷雾剂、锭剂、脂质体或栓剂等。
术语“有效量”指以需要的剂量并持续需要的时间段，有效实现所需预防和/或治疗结果的量。可以根据多种因素如疾病状态、受试者的年龄、性别和重量等变动预防和/或治疗有效量。治疗有效量是任何有毒或有害作用不及治疗有益作用的量。相对于未治疗的受试者，“治疗有效量”优选地抑制可度量参数(例如肿瘤生长率)至少约20％、更优选地至少约40％、甚至更优选地至少约60％和仍更优选地至少约80％。可以在预示人肿瘤中的功效的动物模型系统中评价本发明的药物组合抑制可度量参数(例如，肿瘤体积)的能力。“预防有效量”指以需要的剂量并持续需要的时间段，有效实现所需预防结果的量。通常，由于预防性剂量在受试者中在疾病较早阶段之前或在疾病较早阶段使用，故预防有效量小于治疗有效量。
术语“剂量”是引发预防和/或治疗效果的药物的量。除非另有说明，否则剂量与游离 形式的药物的量有关。如果药物是可药用盐形式，药物的量与游离形式的药物的量相比成比例地增加。例如，剂量将在产品包装或产品信息单中声明。例如给药剂量根据患者的体重按每千克毫克数(mg/kg)计算或根据患者的身高体重按每平方米体表面积毫克数(mg/㎡)计算。
术语“单次药物剂量单元”是指用于单次施用于患者的包含本发明的抗CD47抗体或其抗原结合片段的单次药物剂型和/或包含本发明的多受体酪氨酸激酶抑制剂的单次药物剂型。所述单次药物剂型可以是经胃肠外施用的剂型，例如注射用的小瓶、安瓿、预充针或预充式注射器，其中含有药物的溶液或冻干粉，或者是经胃肠内施用的剂型，例如口服施用的片剂、胶囊、锭剂、散剂、混悬剂等。
术语“抑制”是指给定分子(例如(i)抗CD47抗体或其抗原结合片段；(ii)DNA甲基化转移酶抑制剂使得某些参数的降低。例如，该术语包括抑制至少5％、10％、20％、30％、40％或更多的活性。因此，抑制不必是100％。
在谈及疾病时，术语“治疗”是指减轻所述疾病(即，减缓或阻止或减少所述疾病或其至少一个临床症状的发展)、防止或延迟所述疾病的发作或发展或进展。
术语“预防”包括对疾病或病症或其症状的发生或发生频率的抑制或推迟，其通常是指在病征或症状发生前，特别是在具有风险受试者的病征或症状发生前的药物施用。
术语“受试者”指哺乳动物和非哺乳动物。哺乳动物指哺乳类的任何成员，其包括但不限于：人；非人灵长类动物，牛、马、羊、猪、兔、狗和猫等。术语“受试者”并不限定特定的年龄或性别。在一些实施方案中，受试者是人。
术语“不良事件”(AE)是与医学治疗的使用相关的任何不利且通常非预期或不想要的病征(包括异常实验室发现)、症状或疾病。例如，不良事件可以与免疫系统响应治疗而激活或免疫系统细胞(例如T细胞)响应治疗而扩增相关。医学治疗可以具有一种或多种相关AE，且各AE可以具有相同或不同水平的严重度。
术语“总生存”或“OS”为患者从首次使用所研究药物至任何原因导致其死亡的时间。
术语“无进展生存”或“PFS”为患者首次使用所研究药物到出现疾病进展或任何原因导致死亡的时间。
II.本发明的药物组合
本发明的药物组合包含(i)抗CD47抗体或其抗原结合片段；和(ii)DNA甲基化转移酶抑制剂，优选地所述DNA甲基化转移酶抑制剂为阿扎胞苷。
在一个实施方案中，本发明药物组合中的抗CD47抗体或其抗原结合片段包含SEQ ID NO:7所示的重链可变区氨基酸序列中的3个CDR和SEQ ID NO:8所示的轻链可变 区氨基酸序列中的3个CDR。
又在一个实施方案中，本发明的药物组合中的抗CD47抗体或其抗原结合片段包含6个CDR，其中HCDR1、HCDR2和HCDR3包含如GSISSYYWS(SEQ ID NO:1)、YIYYSGSTNYNPSLKS(SEQ ID NO:2)和ARGKTGSAA(SEQ ID NO:3)所示的氨基酸序列或由所述氨基酸序列组成，且其中LCDR1、LCDR2和LCDR3包含如RASQGISRWLA(SEQ ID NO:4)、AASSLQS(SEQ ID NO:5)和QQTVSFPIT(SEQ ID NO:6)所示的氨基酸序列或由所述氨基酸序列组成；其中所述HCDR1在Kabat编号系统中位于H27-H35，HCDR3在Kabat编号系统中位于H93-H102，所述HCDR2、LCDR1、LCDR2和LCDR3由Kabat定义规则确定。
优选地，所述抗CD47抗体或其抗原结合片段包含重链可变区VH和轻链可变区VL，其中重链可变区包含SEQ ID NO:7的序列或与其具有至少90％,95％,98％或99％同一性的序列，且轻链可变区包含SEQ ID NO:8的序列或与其具有至少90％,95％,98％或99％同一性的序列。
优选地，所述抗CD47抗体或其抗原结合片段包含重链和轻链序列，其中重链包含SEQ ID NO:9的序列或与之具有至少90％,95％,98％或99％同一性的序列，且轻链包含SEQ ID NO：10的序列或与之具有至少90％,95％,98％或99％同一性的序列。
例示的抗CD47抗体重链可变区：
Figure PCTCN2021105470-appb-000001
例示的抗CD47抗体的轻链可变区：
Figure PCTCN2021105470-appb-000002
例示的抗CD47抗体的重链：
Figure PCTCN2021105470-appb-000003
Figure PCTCN2021105470-appb-000004
例示的抗CD47抗体的轻链：
Figure PCTCN2021105470-appb-000005
优选地，所述抗CD47抗体或是WO2019042285A1中公开的抗CD47抗体ADI-26630。
III.本发明的药物组合的用途和使用本发明的药物组合的治疗方法
本发明提供了前述本发明的药物组合，其用于预防和/或治疗受试者中癌症的至少一种症状或指征的严重性或抑制癌细胞生长。
本发明提供了预防或治疗癌症的方法，其包括向有需要的受试者施用有效量的本发明的药物组合。所述有效量包括预防有效量和治疗有效量。
本发明提供了前述本发明的药物组合在制备用于预防或治疗癌症的药物中的用途。
本发明所述癌症包括实体瘤和血液学恶性肿瘤，例如，急性骨髓性白血病(AML)和骨髓增生异常综合征(MDS)。
本发明药物组合中的抗CD47抗体或其抗原结合片段可以以一个或多个剂量施用于有需要的受试者，其中在施用多个剂量的情况下，在前一剂量之后1周、2周、3周、4周、5周、6周、7周、8周、9周或10周施用下一个剂量。抗CD47抗体或其抗原结合片段的一个剂量可以选自1-50mg/kg受试者体重(例如1mg/kg、2mg/kg、3mg/kg、5mg/kg、10mg/kg、15mg/kg、20mg/kg、25mg/kg、30mg/kg、35mg/kg、40mg/kg、45mg/kg或50mg/kg，优选地为1mg/kg、30mg/kg或45mg/kg)；或者，本发明药物组合中的抗CD47抗体或其抗原结合片段的单次施用剂量可以选自约60-3000mg，例如约60mg、120mg、180mg、300mg、600mg、900mg、1200mg、1500mg、1800mg、2100mg、2400mg、2700mg或3000mg，优选地为约60mg、1800mg、2700mg。
本发明药物组合中的抗CD47抗体或其抗原结合片段先以一个低剂量(例如1mg/kg、2mg/kg、3mg/kg、5mg/kg)施用受试者，再以一个高剂量(例如10mg/kg、15mg/kg、20mg/kg、25mg/kg、30mg/kg、35mg/kg、40mg/kg、45mg/kg或50mg/kg)施用受试者；或者，本发明药物组合中的抗CD47抗体或其抗原结合片段的单次施用剂量先以低剂量(例如60mg、120mg、180mg、300mg)施用受试者，再以高剂量(例如600mg、600mg、900mg、1200mg、1500mg、1800mg、2100mg、2400mg、2700mg或3000mg) 施用受试者。本发明药物组合中的DNA甲基化转移酶抑制剂(例如阿扎胞苷)可以以一个或多个剂量施用于有需要的受试者，其中在施用多个剂量的情况下，在前一剂量之后约一天一次、每两天一次、每三天一次或每四天一次施用，或以一天一次施用一周且每三周停一周的方案施用。DNA甲基化转移酶抑制剂(例如阿扎胞苷)的一个剂量可以选自70-80mg/m 2受试者每平方米体表面积(例如70mg/m 2、75mg/m 2或80mg/m 2，优选地为75mg/m 2)；或者，本发明药物组合中的DNA甲基化转移酶抑制剂(例如阿扎胞苷)的单次施用剂量约120-140mg，例如约120mg、130mg、140mg，优选地为约130mg。
本发明的药物组合可以是本领域技术人员已知的任何剂型，例如片剂、胶囊剂、颗粒剂、糖浆剂、粉末、锭剂、药囊、扁囊剂、酏剂、混悬剂、乳剂、溶液、糖浆剂、气雾剂、软膏剂、乳膏剂和注射剂等。其中，抗CD47抗体或其抗原结合片段与DNA甲基化转移酶抑制剂(例如阿扎胞苷)可以各自呈单独的剂型，其剂型可以是不同或相同的。
在一个实施方案中，本发明的药物组合中的抗CD47抗体或其抗原结合片段是静脉内施用剂型，例如静脉内注射剂。本发明的药物组合中的DNA甲基化转移酶抑制剂(例如阿扎胞苷)是皮下施用剂型，例如皮下注射剂。
本发明的药物组合可以是药物剂量单元、例如单次药物剂量单元。
本发明的药物组合的给药周期可以为二周、三周、四周、一个月、两个月、三个月、四个月、五个月、半年或更长时间，优选为二周、三周或四周，其中在每个周期施用2次、3次或4次抗CD47抗体或其抗原结合片段，和/或在每个周期施用5次、6次或7次DNA甲基化转移酶抑制剂，优选为第1至7天连续施用DNA甲基化转移酶抑制剂。
本发明的药物组合中的抗CD47抗体或其抗原结合片段和DNA甲基化转移酶抑制剂可以分开、同时或依次施用。
在一些实施方案中，本发明的药物组合以四周为一个周期施用，其中抗CD47抗体或其抗原结合片段第一周的第一天给予剂量1mg/kg，第二周及以后每周的第一天给予剂量30mg/kg，优选静脉注射，且DNA甲基化转移酶抑制剂(例如阿扎胞苷)每个周期第一周每天给予剂量75mg/m 2，优选皮下注射。
与施用抗CD47抗体或其抗原结合片段的单一疗法或施用DNA甲基化转移酶抑制剂(例如阿扎胞苷)的单一疗法相比，本发明的药物组合导致增加、优选协同地增加对肿瘤生长的抑制作用。在一些实施方案中，与施用抗CD47抗体或其抗原结合片段的单一疗法或DNA甲基化转移酶抑制剂(例如阿扎胞苷)的单一疗法相比，本发明的药物组合导致肿瘤生长被抑制至少约60％、约70％、约80％、约90％、约100％。在一些实施方 案中，本发明的药物组合的施用导致增加肿瘤消退、肿瘤缩小和/或消失。在一些实施方案中，与未治疗的受试者或与使用抗CD47抗体或其抗原结合片段的单一疗法或使用DNA甲基化转移酶抑制剂(例如阿扎胞苷)的单一疗法相比，本发明的药物组合预防受试者的肿瘤复发和/或增加生存持续时间，例如，将生存持续时间增加多于15天、多于1个月、多于3个月、多于6个月、多于12个月、多于18个月、多于24个月、多于36个月、或多于48个月。在一些实施方案中，本发明的药物组合可增加无进展生存或总体生存。
在一些实施方案中，本发明的药物组合可以减少由施用各单药导致的不良事件，例如，血液学毒性反应、非血液学毒性反应或其他毒性反应。
IV.本发明的药盒
本发明的另一个目的是提供一种成套药盒，其包含本发明的药物组合以及使用所述药物组合的说明书，优选地所述药盒包含一个或多个单次药物剂量单元。
在一个实施方案中，本发明的成套药盒在同一包装内包含：
(1)一个或多个单次药物剂量单元，所述单次药物剂量单元包含约60-3000mg，优选地约60mg、120mg、180mg、1800mg或2700mg，更优选地约60mg或1800mg剂量的如前所述的抗CD47抗体或其抗原结合片段；和
(2)一个或多个单次药物剂量单元，所述单次药物剂量单元包含约120-140mg，例如约120mg、130mg、140mg，优选为约130mg剂量的阿扎胞苷其药学上可接受的盐。
本发明所述的各个实施方案/技术方案以及各个实施方案/技术方案中的特征应当被理解为可以任意进行相互组合，这些相互组合得到的各个方案均包括在本发明的范围内，就如同在本文中具体地且逐一地列出了这些相互组合而得到的方案一样，除非上下文清楚地显示并非如此。
描述以下实施例以辅助对本发明的理解。不意在且不应当以任何方式将实施例解释成对本发明的保护范围的限制。
实施例
通过以下实施例向本领域普通技术人员提供如何制备和使用本发明的方法和组合物的完整公开和描述，并且不旨在限制本发明所涵盖的范围。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
以下结合具体实施例，对本发明作进一步说明。应理解，以下实施例仅用于说明本 发明而非用于限定本发明的范围。
实施例1.抗CD47抗体的生产和纯化
抗CD47抗体是通过结合CD47，从而阻断或抑制CD47配体SIRPα与CD47相互作用的抗体，例如专利公开号WO2019042285A1中公开的任一抗CD47单克隆抗体。为了本申请的目的，该PCT申请的全部内容特此并入本文作为参考。本实施例例示使用的是WO2019042285A1中公开的抗CD47抗体ADI-26630。需要说明的是，本领域技术人员也可以使用其他抗CD47抗体，包括但不限于WO2019042285A1中公开的其他抗CD47抗体用于本发明的药物组合中。
抗CD47抗体ADI-26630的CDR区、轻链可变区和重链可变区、轻链和重链的氨基酸序列在本申请的“发明详述”中给出。
本发明的药物组合中的抗CD47抗体可以通过本领域技术人员公知的任一抗体制备技术来生成。抗体表达纯化参见专利公开号WO2019042285A1。
实施例2体外实验探索阿扎胞苷是否诱导肿瘤细胞上调表达CD47
将MV-4-11细胞(ATCC，CRL-9591TM)和HL60(细胞中科院上海细胞库，TcHu23)铺于96孔板，加入梯度稀释的阿扎胞苷(药物来源同实施例2，最高浓度500ug/ml，3倍梯度稀释)后，于24小时通过流式检测人CD47蛋白表达。
通过阿扎胞苷刺激24小时候，细胞表面的CD47表达水平呈现上升趋势，且随着阿扎胞苷浓度梯度上升(见图1、图2)。这一现象在MV-4-11和HL60两种细胞都有观测到。说明在使用AZA后上调了肿瘤细胞表面的CD47表达，CD47的上调使AZA给药后抑制巨噬细胞吞噬肿瘤细胞，揭示了AZA出现耐药的机理。为了解除这一耐药机理，需要联合其他药物解除抑制作用，通过两者的协同作用，更好地发挥阿扎胞苷的药效。下述实施例3和4将阿扎胞苷和本发明的抗CD47抗体联用后，揭示了抗CD47抗体可以解除这一抑制，在下述两个适应症模型中验证了联合后的可以发挥显著抗肿瘤作用。
实施例3.MV-4-11(人髓性单核细胞白血病细胞)荷瘤的NOD-SCID小鼠模型的建立和本发明的药物组合的抗肿瘤作用
实验动物及来源
NOD-SCID小鼠(15-18g，35-41天)购自北京维通利华实验动物技术有限公司。等级SPF级，数量为60只，合格证编号为1100112011009957。到达后驯化饲养3天后进 行实验。MV-4-11(ATCC，CRL-9591TM)皮下接种量为6个million每只小鼠。细胞皮下接种后第6天根据肿瘤体积进行分组。
表1实验药物及来源
Figure PCTCN2021105470-appb-000006
表2给药方案及过程
Figure PCTCN2021105470-appb-000007
给药：
共60只小鼠，根据瘤体积分组，共分为8组，每组7只。
①h-IgG-4mg/kg组给药频率见表2，从第6天开始给药，共给药3次；
②阿扎胞苷给药频率为一天一次，从第6天开始给药，连续给药5次；
③抗CD47抗体-0.1mg/kg(抗CD47抗体于阿扎胞苷给药结束后第一天开始给药)单药组中的抗CD47抗体给药时间点同抗CD47抗体+阿扎胞苷-0.1+3mg/kg(抗CD47抗体于阿扎胞苷给药结束后第一天开始给药)组，抗CD47抗体+阿扎胞苷-0.1+3mg/kg(抗CD47抗体于阿扎胞苷给药结束后第一天开始给药)组中的抗CD47抗体于阿扎胞苷给药结束后第一天开始给药，共给药3次；
④抗CD47抗体-0.1mg/kg(抗CD47抗体于阿扎胞苷给药结束后第三天开始给药)单药组中的抗CD47抗体给药时间点同抗CD47抗体+阿扎胞苷-0.1+3mg/kg(抗CD47抗体于阿扎胞苷给药结束后第三天开始给药)组，抗CD47抗体+阿扎胞苷-0.1+3mg/kg(抗CD47抗体于阿扎胞苷给药结束后第三天开始给药)组中的抗CD47抗体于阿扎胞苷给药结束后第三天开始给药，共给药3次；
⑤抗CD47抗体-0.1mg/kg(抗CD47抗体于阿扎胞苷给药结束后第五天开始给药)单药组中的抗CD47抗体给药时间点同抗CD47抗体+阿扎胞苷-0.1+3mg/kg(抗CD47抗体于阿扎胞苷给药结束后第五天开始给药)组，抗CD47抗体+阿扎胞苷-0.1+3mg/kg(抗CD47抗体于阿扎胞苷给药结束后第五天开始给药)组中的抗CD47抗体于阿扎胞苷给药结束后第五天开始给药，共给药3次；
给药方式均为IP(腹腔注射)。
实验结果和结论：
肿瘤体积测定：
采用游标卡尺测定肿瘤的最大长轴(L)和最大宽轴(W)，肿瘤体积按如下公式计算：V＝L×W 2/2。
相对肿瘤抑制率计算：
肿瘤抑制率TGI(％)：100％(Tvolcontrol–Tvoltreated)/(Tvolcontrol–Tvolpredose)
Tvolcontrol–Tvoltreated：对照组给药后肿瘤终末体积–给药组给药后肿瘤终末体积；
Tvolcontrol–Tvolpredose：对照组给药后肿瘤终末体积–对照组给药前肿瘤体积。
表3.肿瘤抑制率(TGI)和肿瘤消失数目
Figure PCTCN2021105470-appb-000008
Figure PCTCN2021105470-appb-000009
各组荷瘤小鼠肿瘤体积随时间的影响如图3示，各组荷瘤小鼠的体重变化率如图4所示，抗CD47抗体单药、阿扎胞苷单药或者抗CD47抗体与阿扎胞苷不同时间联合用药对荷瘤小鼠肿瘤体积随时间的影响如图5、图6和图7所示。
由表3可知，给药结束后，相对于h-IgG组，在MV-4-11接种后第23天，单药抗CD47抗体-0.1mg/kg(抗CD47抗体于阿扎胞苷给药结束后第一天开始给药)组、抗CD47抗体-0.1mg/kg(抗CD47抗体于阿扎胞苷给药结束后第三天开始给药)组、抗CD47抗体-0.1mg/kg(抗CD47抗体于阿扎胞苷给药结束后第五天开始给药)组的肿瘤抑制率分别为53％、26％、42％。单药阿扎胞苷-3mg/kg的肿瘤抑制率为51％。抗CD47抗体与阿扎胞苷联合给药抗CD47抗体+阿扎胞苷-0.1+3mg/kg(抗CD47抗体于阿扎胞苷给药结束后第一天开始给药)组、抗CD47抗体+阿扎胞苷-0.1+3mg/kg(抗CD47抗体于阿扎胞苷给药结束后第三天开始给药)组、抗CD47抗体+阿扎胞苷-0.1+3mg/kg(抗CD47抗体于阿扎胞苷给药结束后第五天开始给药)组的肿瘤抑制率分别为78％、67％、77％，抗CD47抗体与阿扎胞苷联合时的肿瘤抑制率明显高于抗CD47抗体单药以及阿扎胞苷单药组，具有很好的抑瘤效果，且具有统计学差异(统计方法采用双因素方差分析，在图5、图6和图7中，**代表P值小于0.01，****代表P值小于0.0001)，表明抗CD47抗体和阿扎胞苷在抗肿瘤方面具有协同作用，具有很好的抑瘤效果。
另外，由图4可知，抗CD47抗体单药、阿扎胞苷单药或者抗CD47抗体与阿扎胞苷联合用药在临床前小鼠模型中均未见到明显毒副作用，小鼠体重正常，未出现明显下降。
由图3、图5、图6、图7和表3可以看出，抗CD47抗体具有一定的抗肿瘤药效，与阿扎胞苷联用后，解除了阿扎胞苷的耐药性，显著增强其抗肿瘤作用，说明抗CD47抗体和阿扎胞苷联用具有协同效应。
实施例4.HL60(人骨髓白血病)荷瘤的NOD-SCID小鼠模型的建立和本发明的药物组合的抗肿瘤作用
实验动物及来源
NOD-SCID小鼠(15-18g，35-41天)购自北京维通利华实验动物技术有限公司。等级SPF级，数量为70只，合格证编号为1100112011010881。到达后驯化饲养3天后进行实验。HL60(中科院上海细胞库，TcHu23)皮下接种量为5个million每只小鼠。细胞皮下接种后第7天根据肿瘤体积进行分组。
实验药物及来源
同实施例3
表4给药方案及过程
Figure PCTCN2021105470-appb-000010
给药：
共75只小鼠，根据瘤体积分组，共分为6组，每组6只。
①h-IgG-4mg/kg组给药频率均见表4，从第7天开始给药，共给药3次；
②抗CD47抗体组的药物给药频率见表4，从第7天开始给药，共给药3次；
③阿扎胞苷给药频率为一天一次，从第7天开始给药，连续给药5次；
④抗CD47抗体+阿扎胞苷-0.2mg/kg+3mg/kg组(抗CD47抗体与阿扎胞苷第一天同时给药)组的抗CD47抗体于阿扎胞苷给药第一天同时给药，共给药3次；
⑤抗CD47抗体+阿扎胞苷-0.2mg/kg+3mg/kg(抗CD47抗体于阿扎胞苷给药结束后第一天开始给药)组的抗CD47抗体于阿扎胞苷给药结束后第1天开始给药，共给药3次；
抗CD47抗体+阿扎胞苷-0.1+3mg/kg(抗CD47抗体于阿扎胞苷给药结束后第三天开始给药)组的抗CD47抗体于阿扎胞苷给药结束后第3天开始给药，共给药3次；
给药方式均为IP(腹腔注射)。
实验结果及结论
肿瘤体积测定：
采用游标卡尺测定肿瘤的最大长轴(L)和最大宽轴(W)，肿瘤体积按如下公式计算：V＝L×W 2/2。
相对肿瘤抑制率计算：
肿瘤抑制率TGI(％)：100％×(Tvolcontrol–Tvoltreated)/(Tvolcontrol–Tvolpredose)
Tvolcontrol–Tvoltreated：对照组给药后肿瘤终末体积–给药组给药后肿瘤终末体积；
Tvolcontrol–Tvolpredose：对照组给药后肿瘤终末体积–对照组给药前肿瘤体积。
表5.肿瘤抑制率(TGI)和肿瘤消失数目
Figure PCTCN2021105470-appb-000011
各组荷瘤小鼠肿瘤体积随时间的影响如图8所示，各组荷瘤小鼠的体重变化率如图9所示，抗CD47抗体与阿扎胞苷第一天同时给药和不同时间联合给药对荷瘤小鼠肿瘤体积随时间的影响如图10、图11所示。
由表5可知，给药结束后，相对于h-IgG组，在HL60接种后第25天，单药抗CD47抗体-0.2mg/kg组的肿瘤抑制率为63％。单药阿扎胞苷-3mg/kg组的肿瘤抑制率为65％。抗CD47抗体+阿扎胞苷-0.2+3mg/kg组(抗CD47抗体与阿扎胞苷第一天同时给药)、抗CD47抗体+阿扎胞苷-0.2+3mg/kg(抗CD47抗体于阿扎胞苷给药结束后第一天开始给 药)组、抗CD47抗体+阿扎胞苷-0.2+3mg/kg(抗CD47抗体于阿扎胞苷给药结束后第三天开始给药)组的肿瘤抑制率分别为85％、98％、91％。
抗CD47抗体和阿扎胞苷联合给药后的肿瘤抑制率明显高于抗CD47抗体单药以及阿扎胞苷单药组，具有很好的抑瘤效果，且具有统计学差异(统计方法采用双因素方差分析，在图10、图11中，****代表P值小于0.0001)，表明抗CD47抗体和阿扎胞苷在抗肿瘤方面具有协同作用，具有很好的抑瘤效果。
本实验中，抗CD47抗体+阿扎胞苷-0.2mg/kg+3mg/kg(抗CD47抗体于阿扎胞苷给药结束后第三天开始给药)组小鼠在阿扎胞苷连续给药过程中体重下降，给药结束后体重逐渐恢复。其他各组小鼠在给药后未出现明显的体重下降情况，小鼠行为正常。
由图8、图10和图11可以看出，抗CD47抗体具有一定的抗肿瘤药效，与阿扎胞苷联用后，解除了阿扎胞苷的耐药性，显著增强其抗肿瘤作用，说明抗CD47抗体和阿扎胞苷联用具有协同效应。
实施例5
1.受试药物
抗CD47抗体：信达生物制药(苏州)有限公司生产，规格1ml/100mg或5ml/500mg。
阿扎胞苷：Baxter Oncology GmbH公司生产，规格100mg。
AML适应症入组标准：
1.根据2016年WHO AML诊断标准确诊的AML患者(除外M3型及BCR-ABL阳性AML)。
2.复发/难治性或初诊不耐受标准诱导化疗的老年AML患者。
·复发/难治性AML，定义为满足下列条件之一：
1)初治病例经过标准方案诱导化疗2疗程未达到完全缓解(CR)；
2)曾达到CR后经过巩固或强化治疗，12个月内复发者(复发的定义为：达到CR后外周血再次出现白血病细胞或骨髓中原始细胞＞5％，且上述表现已除外巩固化疗后骨髓再生等其他原因)；
3)曾达到CR后经过巩固或强化治疗12个月后复发但经过标准方
案再诱导未达到CR者；
4)累计2次或多次复发者；
5)自体造血干细胞移植后复发；
·初诊不耐受标准诱导化疗的老年AML，定义为同时满足以下条件：
1)≥60周岁，因体能状态或器官功能不耐受标准的诱导化疗(如DA、IA、MA方案等)；
2)未接受过抗白血病化疗及其他免疫治疗(羟基脲、白细胞分离术除外)；
3)骨髓或外周血中原始细胞≥20％；或原始细胞＜20％但同时克隆性重现性细胞遗传学异常如t(8；21)(q22；q22)、inv(16)(p13；q22)或t(16；16)(p13；q22)。
3.年龄≥18周岁，性别不限。
4.根据美国东部肿瘤协作组体力状态评分(Eastern Cooperative Oncology Group Performance Status,ECOG PS)为0～2分。
5.具有充分的器官功能，定义如下：
1)血常规：入组前白细胞计数(White blood cell,WBC)需＜25×109/L。
2)肝功能：血清总胆红素(Total bilirubin,TBIL)≤1.5×正常上限
(Upper Limit of Normal value,ULN)；丙氨酸氨基转移酶(Alanine transaminase,ALT)和天门冬氨酸氨基转移酶(Aspartate aminotransferase,AST)≤2.5×ULN。白蛋白(albumin,ALB)＞30g/L。Gilbert综合征受试者要求TBIL≤3×ULN。
3)肾功能：血清肌酐(Creatinine,Cr)≤1.5×ULN或内生肌酐清除率(Creatinine clearance rate,CCr)≥60ml/min(用CKD-EPI公式计算，具体计算公式见附件5)；
4)凝血功能：国际标准化比率(International Normalized Ratio,INR)≤1.5，且凝血酶原时间(Prothrombin time,PT)或活化部分凝血活酶时间(Activated partial thromboplastin time,APTT)≤1.5×ULN。
6.预期生存时间≥12周。
7.育龄期女性受试者或伴侣为育龄期妇女的男性受试者，需在整个治疗期及治疗期后6个月采取有效的避孕措施。
8.能够耐受骨髓穿刺及活检，并按方案要求的时间点接受该检查。
9.签署书面知情同意书，而且能够遵守方案规定的访视及相关程序。
MDS适应症入组标准：
1.根据2016WHO诊断标准及IPSS-R预后评分，经骨髓穿刺或活检确诊的中高危(IPSS-R预后评分＞3)的MDS患者；
2.年龄≥18周岁；
3.根据美国东部肿瘤协作组体力状态评分(Eastern Cooperative Oncology Group  Performance Status,ECOG PS，见附件4)为0～2分；
4.不适合或拒绝接受强化疗及异基因造血干细胞移植(因年龄、合并症、没有合适供体等因素)；
5.未接受过去甲基化治疗或化疗的初诊MDS患者(允许输血、造血生长因子及补充造血原料治疗)；
6.具有充分的器官功能，定义如下：
1)肝功能：血清总胆红素(Total bilirubin,TBIL)≤1.5×正常上限(Upper Limit of Normal value,ULN)；丙氨酸氨基转移酶(Alanine transaminase,ALT)和天门冬氨酸氨基转移酶(Aspartate amino transferase,AST)≤2.5×ULN。白蛋白(Albumin,ALB)＞30g/L。Gilbert综合征受试者要求TBIL≤3×ULN。
2)肾功能：血清肌酐(Creatinine,Cr)≤1.5×ULN或内生肌酐清除率(Creatinine clearance rate,CCr)≥50ml/min(用CKD-EPI公式计算，具体计算公式见附件5)。
3)凝血功能：国际标准化比率(International Normalized Ratio,INR)≤1.5，且凝血酶原时间(Prothrombin time,PT)或活化部分凝血活酶时间(Activated partial thromboplastin time,APTT)≤1.5×ULN；
7.预期生存时间≥12周；
8.育龄期女性受试者或伴侣为育龄期妇女的男性受试者，需在整个治疗期及治疗期后6个月采取有效的避孕措施；
9.能够耐受在筛选期及检测时间点进行骨髓穿刺及活检检查；
10.签署书面知情同意书，而且能够遵守方案规定的访视及相关程序。
2.给药方法及过程
具体给药方案为：抗CD47抗体：第1周的第一天静脉注射1.0mg/kg，第2周及以后每周的第一天静脉注射30mg/kg。阿扎胞苷：75mg/m 2，第1至第7天每天皮下注射给药，每4周为一个周期。
3.实验结果
本研究的联合用药在AML和MDS适应症上显示出安全性和有效性。
尽管已经出于说明本发明的目的显示了某些代表性实施方案和细节，但是本领域技术人员显而易见的是可以对它们进行多种变化和修改而不脱离主题发明的范围。在这个方面，本发明范围仅由以下权利要求限定。

Claims (15)

  1. 药物组合，其包含(i)抗CD47抗体或其抗原结合片段；和(ii)DNA甲基化转移酶抑制剂或其药学上可接受的盐，其中所述抗CD47抗体或其抗原结合片段包含如下三个重链CDR和三个轻链CDR：
    -GSISSYYWS(SEQ ID NO:1)的重链VH CDR1；
    -YIYYSGSTNYNPSLKS(SEQ ID NO:2)的重链VH CDR2；
    -ARGKTGSAA(SEQ ID NO:3)的重链VH CDR3；
    -RASQGISRWLA(SEQ ID NO:4)的轻链VL CDR1；
    -AASSLQS(SEQ ID NO:5)的轻链VL CDR2；和
    -QQTVSFPIT(SEQ ID NO:6)的轻链VL CDR3；
    所述DNA甲基化转移酶抑制剂选自阿扎胞苷或其药学上可接受的盐。
  2. 如权利要求1所述的药物组合，其中所述抗CD47抗体或其抗原结合片段包含重链可变区和轻链可变区，其中重链可变区包含SEQ ID NO:7的序列或与其具有至少90％,95％,98％或99％同一性的序列，且轻链可变区包含SEQ ID NO:8的序列或与其具有至少90％,95％,98％或99％同一性的序列。
  3. 如权利要求1所述的药物组合，其中所述抗CD47抗体或其抗原结合片段包含重链氨基酸序列和轻链氨基酸序列，其中重链包含SEQ ID NO:9或与之具有至少90％,95％,98％或99％同一性的重链序列，且轻链包含SEQ ID NO:10或与之具有至少90％,95％,98％或99％同一性的轻链序列。
  4. 如权利要求1-3中任一项所述的药物组合，其中
    (i)的单次施用剂量选自约1-50mg/kg，例如约1mg/kg、2mg/kg、3mg/kg、5mg/kg、10mg/kg、15mg/kg、20mg/kg、25mg/kg、30mg/kg、35mg/kg、40mg/kg、45mg/kg或50mg/kg；优选地为约1mg/kg、30mg/kg或45mg/kg；或(i)的单次施用剂量选自约60-3000mg，例如约60mg、120mg、180mg、300mg、600mg、900mg、1200mg、1500mg、1800mg、2100mg、2400mg、2700mg或3000mg，优选地为约60mg、1800mg、2700mg；和
    (ii)的单次施用剂量选自约70-80mg/m 2，例如约70mg/m 2、75mg/m 2或80mg/m 2；优选地为约75mg/m 2；或(ii)的单次施用剂量选自约120-140mg，例如约120mg、130mg、140mg，优选地为约130mg。
  5. 如前述权利要求中任一项的药物组合，其中
    (i)先给予1mg/kg的单次施用剂量，再给予30mg/kg的单次施用剂量；
    (ii)给予75mg/m 2的单次施用剂量。
  6. 如前述权利要求中任一项的药物组合，其中
    (i)为胃肠外给药，优选为静脉注射给药；和
    (ii)为胃肠外给药，优选为皮下注射给药。
  7. 如前述权利要求中任一项的药物组合，其中所述药物组合的给药周期可以为二周、三周、四周、一个月、两个月、三个月、四个月、五个月、半年或更长时间，任选地，优选为二周、三周或四周，其中(i)和(ii)可以分开、同时或依次施用。
  8. 如权利要求7所述的药物组合，其中
    在每个周期施用2次、3次或4次(i)，和
    在每个周期施用5次、6次或7次(ii)，优选为第1至7天连续施用(ii)。
  9. 如前述权利要求8所述的药物组合，其中以四周为一个周期施用所述药物组合，其中(i)第一周的第一天给予剂量1mg/kg，第二周及以后每周的第一天给予剂量30mg/kg，优选静脉注射，且(ii)每个周期第一周每天给予剂量75mg/m 2，优选皮下注射。
  10. 如前述权利要求中任一项所述的药物组合，其用于预防或治疗癌症，所述癌症为实体瘤和血液肿瘤，所述血液肿瘤为AML或MDS。
  11. 如前述权利要求中任一项所述的药物组合在制备用于预防或治疗癌症的药物中的用途，所述癌症为实体瘤和血液肿瘤，所述血液肿瘤为AML或MDS。
  12. 用于预防或治疗癌症的方法，所述方法包括向有需要的患者施用有效量的如前述权利要求中任一项所述的药物组合，所述癌症为实体瘤和血液肿瘤，所述血液肿瘤为AML或MDS。
  13. 成套药盒，其包含如前述权利要求1-10中任一项所述的药物组合，优选地所述药盒包含一个或多个单次药物剂量单元。
  14. 如权利要求13所述的成套药盒，其中：
    (1)一个或多个单次药物剂量单元，所述单次药物剂量单元包含约60-3000mg，优选地约60mg、120mg、180mg、1800mg或2700mg，更优选地约60mg或1800mg剂量的权利要求1-3中的抗CD47抗体或其抗原结合片段；和
    (2)一个或多个单次药物剂量单元，所述单次药物剂量单元包含约120-140mg，例如约120mg、130mg、140mg，优选为约130mg剂量的阿扎胞苷其药学上可接受的盐。
  15. 如权利要求13或14所述的成套药盒，其还包含用于指示如前述权利要求中任一项所述的药物组合的使用方法的说明书。
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