WO2021175326A1 - CTB006与Ponatinib联合应用 - Google Patents

CTB006与Ponatinib联合应用 Download PDF

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WO2021175326A1
WO2021175326A1 PCT/CN2021/079403 CN2021079403W WO2021175326A1 WO 2021175326 A1 WO2021175326 A1 WO 2021175326A1 CN 2021079403 W CN2021079403 W CN 2021079403W WO 2021175326 A1 WO2021175326 A1 WO 2021175326A1
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trail
ctb006
antibody
ponatinib
cancer
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PCT/CN2021/079403
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French (fr)
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钟晓燕
曹峰琦
李哲
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北京先通生物医药技术有限公司
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Priority to CN202180019028.8A priority Critical patent/CN115243719A/zh
Priority to EP21763694.3A priority patent/EP4115902A4/en
Priority to JP2022579146A priority patent/JP2023518132A/ja
Priority to US17/909,658 priority patent/US20230112450A1/en
Publication of WO2021175326A1 publication Critical patent/WO2021175326A1/zh

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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/50—Pyridazines; Hydrogenated pyridazines
    • A61K31/5025—Pyridazines; Hydrogenated pyridazines ortho- or peri-condensed with heterocyclic ring systems
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/191—Tumor necrosis factors [TNF], e.g. lymphotoxin [LT], i.e. TNF-beta
    • 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
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • 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
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen

Definitions

  • This article relates to an anti-tumor drug combination, in particular to an anti-tumor drug combination including TRAIL-R2 agonistic antibody (such as CTB006 antibody) and Ponatinib.
  • TRAIL-R2 agonistic antibody such as CTB006 antibody
  • Ponatinib This article also relates to tumor treatment methods using TRAIL-R2 agonistic antibodies and panatinib in combination.
  • TRAIL Tumor necrosis factor-related apoptosis-inducing ligand
  • Apo2L tumor necrosis factor
  • TNF tumor necrosis factor
  • TRAIL is expressed in immune system cells such as NK cells, T cells, macrophages and dendritic cells. Mature TRAIL exists in membrane-bound or soluble form, and the soluble form is the extracellular active part of membrane-bound TRAIL that is hydrolyzed by protease. These two forms of TRAIL can form trimers and can induce apoptosis of target cells by interacting with TRAIL receptors (TRAIL-R) on target cells.
  • TRAIL-R TRAIL receptors
  • TRAIL-R1 or DR4
  • TRAIL-R2 or DR5
  • DcR1 TRAIL-R3
  • TRAIL-R4 DcR2
  • OPG Osteoprotegerin
  • All five receptors of TRAIL have significant homology in their extracellular ligand binding domains.
  • the intracellular fragments of TRAIL-R1 and TRAIL-R2 contain a conserved functional domain, the so-called "death domain", which is responsible for transducing apoptosis signals.
  • Ponatinib hydrochloride is a multi-target kinase inhibitor, produced and sold by ARIAD Pharmaceuticals under the trade name Its clinical indications are limited to hematological tumors, for example, certain types of chronic myelogenous leukemia (CML) and acute lymphocytic leukemia (ALL).
  • CML chronic myelogenous leukemia
  • ALL acute lymphocytic leukemia
  • an anti-tumor drug combination which includes an apoptosis inducer and a multi-target kinase inhibitor.
  • the apoptosis-inducing agent is a TRAIL-R2 binding agent.
  • the TRAIL-R2 binding agent is a TRAIL-R2 agonist.
  • the TRAIL-R2 agonist is TRAIL-R2 natural ligand TRAIL or TRAIL-R2 agonist antibody.
  • the TRAIL-R2 agonist antibody is B273, kanalimumab, CTB006 antibody, Hexabody-DR5/DR5, or CTB003 antibody.
  • the multi-target kinase inhibitor is Ponatinib or a pharmaceutically acceptable salt thereof.
  • the multi-target kinase inhibitor is panatinib hydrochloride.
  • the tumor expresses TRAIL-R2 on the cell surface.
  • the tumor is breast cancer, pancreatic cancer, colorectal cancer, gastric cancer, lung cancer, liver cancer, esophageal cancer, or ovarian cancer.
  • the apoptosis-inducing agent and the multi-target kinase inhibitor are present in the same or different pharmaceutical compositions or pharmaceutical kits.
  • the dosage form of the apoptosis-inducing agent is suitable for intravenous administration, and the dosage form of the multi-target kinase inhibitor is suitable for oral administration.
  • a method of treating tumors in a subject which comprises simultaneously, sequentially or separately applying a therapeutically effective amount of an apoptosis inducer and a multi-target kinase inhibitor to the subject ⁇ Administration.
  • the apoptosis-inducing agent is a TRAIL-R2 binding agent.
  • the TRAIL-R2 binding agent is a TRAIL-R2 agonist.
  • the TRAIL-R2 agonist is TRAIL-R2 natural ligand TRAIL or TRAIL-R2 agonist antibody.
  • the TRAIL-R2 agonist antibody is B273, kanalimumab, CTB006 antibody, Hexabody-DR5/DR5, or CTB003 antibody.
  • the multi-target kinase inhibitor is panatinib or a pharmaceutically acceptable salt thereof.
  • the multi-target kinase inhibitor is panatinib hydrochloride.
  • the tumor expresses TRAIL-R2 on the cell surface.
  • the tumor is breast cancer, pancreatic cancer, colorectal cancer, gastric cancer, lung cancer, liver cancer, esophageal cancer, or ovarian cancer.
  • the apoptosis-inducing agent is administered by intravenous injection, and the multi-target kinase inhibitor is administered orally.
  • this article provides the application of apoptosis-inducing agents in the preparation of drugs that are administered in combination with multi-target kinase inhibitors to treat tumors.
  • the apoptosis-inducing agent is a TRAIL-R2 binding agent.
  • the TRAIL-R2 binding agent is a TRAIL-R2 agonist.
  • the TRAIL-R2 agonist is TRAIL-R2 natural ligand TRAIL or TRAIL-R2 agonist antibody.
  • the TRAIL-R2 agonist antibody is B273, kanalimumab, CTB006 antibody, Hexabody-DR5/DR5, or CTB003 antibody.
  • the kinase inhibitor inhibits activation of the MEK/ERK signaling pathway.
  • the multi-target kinase inhibitor is panatinib or a pharmaceutically acceptable salt thereof.
  • the multi-target kinase inhibitor is panatinib hydrochloride.
  • the tumor expresses TRAIL-R2 on the cell surface.
  • the tumor is breast cancer, pancreatic cancer, colorectal cancer, gastric cancer, lung cancer, liver cancer, esophageal cancer, or ovarian cancer.
  • this document provides a method for inducing tumor cell apoptosis, including contacting the tumor cell with an apoptosis inducer and a multi-target kinase inhibitor.
  • the apoptosis inducer is CTB006 antibody
  • the multi-target kinase inhibitor is panatinib or its hydrochloride.
  • provided herein is a method for enhancing an anti-tumor effect in a subject who is intolerant to panatinib or a pharmaceutically acceptable salt thereof, which comprises administering the CTB006 antibody to the subject.
  • Figure 1 shows the tumor-killing effect of CTB006 antibody combined with Ponatinib on MCF-7 cells.
  • Figure 2 shows the killing effect of CTB006 antibody combined with Ponatinib on S2Lm7AA cells.
  • Figure 3 shows the killing effect of CTB006 antibody combined with Ponatinib on S2VP10 cells.
  • Figure 4 shows the killing effect of CTB006 antibody combined with Ponatinib on HT29 cells.
  • Figure 5 shows the killing effect of CTB006 antibody combined with Ponatinib on SGC-7901 cells.
  • Figure 6 shows the killing effect of CTB006 antibody combined with Ponatinib on N87 cells.
  • Figure 7 shows the killing effect of CTB006 antibody combined with Ponatinib on A549 cells.
  • Figure 8 shows the killing effect of CTB006 antibody combined with Ponatinib on SK-HEP-1 cells.
  • Figure 9 shows the killing effect of CTB006 antibody combined with Ponatinib on Huh-1 cells.
  • Figure 10 shows the killing effect of CTB006 antibody combined with Ponatinib on TE1 cells.
  • Figure 11 shows the expression of DR5 in primary ovarian cancer ascites tumor cells.
  • Figure 12 shows the killing effect of CTB006 antibody and Ponatinib on primary tumor cells S1 (A) and S2 (B).
  • Figure 13 shows the efficacy of CTB006 combined with Ponatinib on pancreatic cancer transplantation model MIA-Paca-2.
  • A Tumor volume change
  • B Survival curve.
  • Figure 14 shows the effect of CTB006 combined with Ponatinib on the PDX xenograft model CS237.
  • Figure 15 shows the effect of CTB006 combined with Ponatinib on the PDX xenograft model CS263.
  • A Tumor volume change
  • B Survival curve.
  • Figure 16 shows the effect of CTB006 combined with Ponatinib on the PDX xenograft model CS264.
  • Figure 17 shows the effect of CTB006 combined with Ponatinib on the PDX xenograft model CS225.
  • Figure 18 shows the effect of CTB006 combined with Ponatinib on the PDX xenograft model CS266.
  • Figure 19 shows the effect of CTB006 combined with Ponatinib on the PDX xenograft model GAS033.
  • Figure 20 shows the killing effect of different types of DR5 agonists in combination with Ponatinib on A549 cells.
  • Figure 21 shows the killing effect of different types of DR5 agonists in combination with Ponatinib on S2VP10 cells.
  • Figure 22 shows the killing effect of different types of DR5 agonists in combination with Ponatinib on MCF7 cells.
  • Figure 23 shows the activation of different tumor cell apoptosis and proliferation signaling pathways under different drug treatment conditions (colon cancer cell SW1116 (A), gastric cancer cell 7901 (B), colorectal cancer HT29 (C), pancreatic cancer S2Lm7AA (D) )).
  • Lane-1 Cell control
  • Lane-2 1000ng/ml CTB006 treatment group
  • Lane-3 1 ⁇ M Ponatinib treatment group
  • Lane-4 Combination group (1000ng/ml CTB006 and 1 ⁇ M Pontatinib).
  • apoptosis inducer refers to various chemical reagents (such as small molecule drugs), biological reagents (such as polypeptides, antibodies), or even physical agents that can promote apoptosis of cells (such as tumor cells) in vitro or in vivo. Treatment (such as radiation), etc.
  • agonistic antibody refers to an antibody that can bind to its corresponding antigen and trigger the biological activity of the antigen.
  • the antigen is TRAIL-R2, and when the agonistic antibody binds to it, it can trigger the apoptotic activity of its intracellular death domain.
  • kinase inhibitor refers to a chemical or biological agent capable of inhibiting, attenuating, or eliminating kinase phosphorylation of its substrate protein.
  • multi-target kinase inhibitor refers to a kinase inhibitor capable of acting on multiple kinases. For example, when administered in vivo, multi-target kinase inhibitors can inhibit multiple signaling pathways.
  • multi-target kinase inhibitors such as Lapatinib (targets are EGFR and HER2), Bosutilib (targets are SRC and ABL), Imatinib (targets are BCR-ABL, C-KIT and PDGFR), Pontatinib (targets are BCR-ABL, C-KIT and PDGFR), The points are ABL, PDGFR, VEGFR, Src, etc.), and so on.
  • the multi-target kinase inhibitor is Ponatinib or a pharmaceutically acceptable salt thereof.
  • CTB006 or "CTB006 antibody” refers to an agonistic antibody that targets TRAIL-R2.
  • CGMCC Common Microorganism Collection and Management Center
  • the antibody has the heavy chain CDR amino acid sequence SYFIH (SEQ ID NO: 1), WIYPGNVNTKYSEKFKG (SEQ ID NO: 2) and GEAGYFD (SEQ ID NO: 3), and the light chain CDR amino acid sequence KASQDVSTAVA (SEQ ID NO: 4), WASTRHT (SEQ ID NO: 5) and QQHYRTPW (SEQ ID NO: 6).
  • SYFIH SEQ ID NO: 1
  • WIYPGNVNTKYSEKFKG SEQ ID NO: 2
  • GEAGYFD SEQ ID NO: 3
  • KASQDVSTAVA SEQ ID NO: 4
  • WASTRHT SEQ ID NO: 5
  • QQHYRTPW SEQ ID NO: 6
  • the synergy between CTB006 and Ponatinib is determined by the coefficient of drug interaction (CDI).
  • the nature of the action is antagonistic.
  • subject refers to an animal (preferably a human) suffering from or suspected of having a certain disease, or, when predicting susceptibility, the “subject” may also include healthy individuals. This term can often be used interchangeably with “patient”, “patient”, “test subject”, “treatment subject” and so on.
  • pharmaceutically acceptable salt refers to a relatively non-toxic inorganic acid or organic acid addition salt of a drug.
  • the nature of the salt is generally not critical, provided that it is pharmaceutically acceptable.
  • pharmaceutically acceptable salts refer to inorganic or organic acid addition salts that are basically harmless to animals or humans, such as hydrochloride, hydrobromide, phosphate, sulfate, formate, acetate, Ascorbate, benzenesulfonate, benzoate, cinnamate, citrate, fumarate, glutamate, glycolate, lactate, maleate, methanesulfonate, Salicylate, stearate, succinate, tartrate, etc.
  • Such salts can be formed by methods well known to those skilled in the art.
  • the CTB006 antibody is administered in combination with Ponatinib. In other embodiments, the CTB006 antibody is administered in combination with a pharmaceutically acceptable salt of Ponatinib (e.g., hydrochloride).
  • a pharmaceutically acceptable salt of Ponatinib e.g., hydrochloride
  • terapéuticaally effective amount refers to the amount of the active compound or pharmaceutical composition sufficient to cause the biological or medical response desired by the clinician in the subject.
  • the “therapeutically effective amount” can usually be determined by those skilled in the art according to factors such as the route of administration, the subject's weight, age, and the condition of the disease. For example, a typical daily dosage may range from 0.01 mg to 100 mg of active ingredient per kg body weight.
  • a drug combination containing an apoptosis inducer and a multi-target kinase inhibitor is provided.
  • drug combination not only means that the apoptosis inducer and the multi-target kinase inhibitor can coexist in the same pharmaceutical preparation, but also means that the apoptosis inducer and the multi-target kinase inhibitor can be used as separate agents.
  • the pharmaceutical preparations are present in the same pharmaceutical kit, or even the apoptosis inducer and the multi-target kinase inhibitor can be present as separate pharmaceutical preparations in different pharmaceutical kits. In fact, as long as the apoptosis-inducing agent and the multi-target kinase inhibitor can exist in the subject at a certain time by administration, this kind of administration method adopts this "drug combination".
  • Apoptosis inducers and/or multi-target kinase inhibitors are usually formulated with pharmaceutically acceptable carriers to form pharmaceutical preparations or pharmaceutical compositions.
  • pharmaceutically acceptable carrier refers to solid or liquid diluents, fillers, antioxidants, stabilizers and other substances that can be safely administered to animals or humans. These substances are suitable for humans and/or humans. It is administered to animals without excessive side effects, and at the same time, it is suitable for maintaining the vitality of the drugs or active agents located therein.
  • Suitable administration routes include, for example, oral, intravenous infusion, intramuscular injection, subcutaneous injection, subperitoneal, rectal, sublingual, or inhalation, transdermal, etc. routes.
  • apoptosis inducers and/or multi-target kinase inhibitors can be formulated with these pharmaceutically acceptable carriers into any clinically acceptable dosage form, such as tablets, granules, powders, capsules, injections, Suppositories, drops, topical ointments, ointments, medicated oils, or sprays, etc.
  • apoptosis inducers and multi-target kinase inhibitors there are provided methods for treating tumors (or cancers) by administering a combination of apoptosis inducers and multi-target kinase inhibitors.
  • co-administration as used herein, for example, for the combination of CTB006 antibody and Ponatinib, it includes CTB006 antibody and Ponatinib administered separately, for example, CTB006 antibody is administered before or after Ponatinib administration, and also includes Ponatinib and CTB006
  • the antibodies are administered simultaneously in the same pharmaceutical formulation or in the form of separate pharmaceutical formulations. In embodiments of sequential administration, usually Ponatinib and CTB006 antibody coexist in the subject at least part of the time.
  • Ponatinib and CTB006 antibody may have a synergistic effect, for example, the amount of one of the drugs is lower than its therapeutically effective amount when administered alone, or preferably, the amount of both drugs is lower than The therapeutically effective dose when used alone.
  • Some embodiments of the present invention are based at least in part on the inventors' discovery that the combined administration of an apoptosis inducer and a multi-target kinase inhibitor produces a significant synergistic effect.
  • the synergy can also be reflected in: 1) A new therapeutic effect is produced, for example, it can be treated with the apoptosis inducer or the multi-target kinase inhibitor treatment. Responsive tumors; 2) reduced side effects of treatment; 3) extended dosing interval; 4) shortened treatment time, and so on.
  • the anti-tumor drug combinations and tumor treatment methods provided herein utilize the synergistic effects of apoptosis inducers and multi-target kinase inhibitors to enhance tumor treatment effects.
  • the use of an apoptosis-inducing agent can enhance the tumor growth inhibitory effect of kinase inhibitors (for example, Ponatinib).
  • the use of apoptosis-inducing agents can reduce the treatment time of kinase inhibitors (such as Ponatinib), thereby improving patient tolerance.
  • Example 1 Combination of CTB006 and Ponatinib can significantly enhance tumor cell apoptosis
  • CDI coefficient of drug interaction
  • the experimental method is as follows: In a 96-well plate, add 3000 tumor cells in logarithmic growth phase and different concentrations of CTB006 antibody and Ponatinib (hydrochloride form, CAS: 1114544-31-8) (Yaodu) in each well. After culturing for 24 hours at 37°C, 5% CO 2 , the survival of each tumor cell was observed by the ATPlite method.
  • CTB006 and Ponatinib alone had no killing effect on S2VP10.
  • the combination of the two has a killing effect.
  • the CDI value of CTB006 antibody at different concentrations combined with 500 nM Ponatinib was less than 1; when Ponatinib at different concentrations was used in combination with 1000 ng/ml CTB006, the CDI value was less than 1 (see Table 3), indicating that the CTB006 antibody is combined with
  • the combined effect of Ponatinib on pancreatic cancer S2VP10 cells is synergistic (see Figure 3).
  • the CDI value of CTB006 antibody at different concentrations in combination with 500 nM Ponatinib was less than 1.
  • the CDI value was less than 1 (see Table 4), indicating that CTB006 antibody and Ponatinib
  • the combined effect of Ponatinib on colorectal cancer HT29 cells is synergistic (see Figure 4).
  • the CDI value of CTB006 antibody at different concentrations combined with 500 nM Ponatinib was less than 1; when Ponatinib at different concentrations was used in combination with 1000 ng/ml CTB006, the CDI value was less than 1 (see Table 5), indicating that CTB006 antibody is combined with The combined effect of Ponatinib on gastric cancer SGC-7901 cells is synergistic (see Figure 5).
  • the CDI value of CTB006 antibody in combination with 500 nM of Ponatinib at different concentrations was less than 1.
  • the CDI value was less than 1 (see Table 6), indicating that the CTB006 antibody is combined with The combined effect of Ponatinib on gastric cancer N87 cells is synergistic (see Figure 6).
  • the CDI value of CTB006 antibody in combination with 500 nM of Ponatinib at different concentrations was less than 1.
  • the CDI value was less than 1 (see Table 7), indicating that CTB006 antibody is combined with The combined effect of Ponatinib on lung cancer A549 cells is synergistic (see Figure 7).
  • the CDI value of CTB006 antibody at different concentrations combined with 500 nM Ponatinib was less than 1; when Ponatinib at different concentrations was combined with 500 ng/ml CTB006, the CDI value was less than 1 (see Table 8), indicating that CTB006 antibody is combined with The combined effect of Ponatinib combination on liver cancer SK-HEP-1 cells is synergistic (see Figure 8).
  • the CDI value of CTB006 antibody in combination with 500 nM of Ponatinib at different concentrations was less than 1; the CDI value of CTB006 in combination with Ponatinib at different concentrations and 1000 ng/ml was less than 1 (see Table 9), indicating that CTB006 antibody and Ponatinib
  • the combined effect of Ponatinib on Huh-1 cells of liver cancer is synergistic (see Figure 9).
  • the CDI value of CTB006 antibody in combination with 500 nM of Ponatinib at different concentrations was less than 1; the CDI value of CTB006 in combination with Ponatinib at different concentrations and 1000 ng/ml was less than 1 (see Table 10), indicating that the CTB006 antibody and Ponatinib
  • the combined effect of Ponatinib on TE1 cells of esophageal cancer is synergistic (see Figure 10).
  • Figure 11 shows the DR5 expression of primary tumor cells S1 and S2 isolated from ascites from two ovarian cancer patients. Through flow cytometry, it is found that both S1 and S2 express DR5, but the abundance difference is large, and the expression level of S2 is four times that of S1.
  • CTB006 and Ponatinib have limited killing effects on both S1 and S2 when used as a single agent.
  • Different concentrations of CTB006 antibody and 100nM Ponatinib used in combination the tumor cells were significantly reduced, and their CDI values were less than 1 (see Tables 11 and 12), indicating that the combination of CTB006 antibody and Ponatinib synergistically enhanced the effect of the combination of CTB006 antibody and Ponatinib on primary tumor cells of ovarian cancer ascites. Killing effect.
  • a number of tumor animal model tests were selected, including pancreatic cancer cell MIA-PACA-2CDX model and multiple PDX models, covering colorectal cancer (CS237, CS263, CS264), lung cancer (CS225) ), and gastric cancer (CS266, GAS033) multiple cancers.
  • P ⁇ 0.05 indicates a significant statistical difference, marked with *; P ⁇ 0.01 indicates a very significant statistical difference, marked with **.
  • a CDX model was established by subcutaneously transplanting MIA-Paca-2 into BALB/c nude nude mice (Vitonic). The tumor volume was about 0.1cm 3 and the administration was started. After reaching 1cm 3 , the animals were ethically euthanized and the survival time was recorded. The administration group was given CTB006 (10mg/kg, iv, qw), Ponatinib (30mg/kg, po, qd) or a combination of both, and the control group was given equal volume of saline. In this experiment, the administration was stopped after 22 days of continuous administration, the control group was euthanized, and the other groups continued to observe until the corresponding endpoint.
  • Figure 13A shows the results.
  • both the CTB006 single administration group and the combined administration group showed good tumor growth inhibition effects, and there was no significant difference between the two.
  • Figure 13B shows the survival curve of each group of animals.
  • the median survival time of the control group was 22 days.
  • the median survival time of CTB006, Ponatinib and CTB006 combined with Ponatinib were 43 days, 34.5 days and 54.5 days, respectively.
  • the combination medication showed significant survival benefits.
  • CTB006 may enhance the efficacy of patients who are intolerant to Ponatinib.
  • TGI tumor inhibition rates
  • G2 group CTB006 single-drug group
  • G3 group Ponatinib single-drug group
  • G4 group CTB006 combined with ponatinib.
  • TGI tumor inhibition rate
  • the survival curve was made based on the time when the average tumor volume reached 1.0cm 3 in each group.
  • the median survival time of the control group was 39 days, the CTB006 single-drug group, and the Ponatinib single-drug group CTB006 and Ponatinib combined group were 48 days, 56 days, 78 days. Days ( Figure 15B).
  • TGI tumor inhibition rates
  • TGI tumor inhibition rate
  • TGI tumor inhibition rate
  • DR5 natural ligand TRAIL Yiqiao Shenzhou
  • DR5 agonistic monoclonal antibody B273 Daiichi Sankyo
  • kanalimumab AMG655, Amgen
  • second-generation multimeric DR5 agonistic antibody Hexabody-DR5/DR5 Genmab, GEN1029
  • DR5 bispecific antibody DR5/DR4 (CTB003, Sinotau, see patent application PCT/CN07/001453).
  • CTB003, Sinotau see patent application PCT/CN07/001453
  • Table 13 The CDI of different types of DR5 agonists and Ponatinib on A549 cells
  • pancreatic cancer cell S2VP10 different types of DR5 agonists and Ponatinib alone at the corresponding concentration have no obvious killing effect on S2VP10. When the same dosage is used in combination, it has obvious killing effect.
  • the CDI values are all less than 1 (see Table 14), indicating that the combined effect of different types of DR5 agonists and Ponatinib on pancreatic cancer S2VP10 cells is synergistic (see Figure 21).
  • DR5 agonists such as TRAIL, Hexabody-DR5/DR5 and DR5/DR4
  • ponatinib alone at the corresponding concentration
  • B273 and AMG655 have obvious killing effect on MCF7 when used alone
  • Ponatinib is ineffective when used alone.
  • the CDI values are all less than 1 (see Table 15), indicating that the combined effect of different types of DR5 agonists and Ponatinib on breast cancer MCF7 cells is synergistic (see Figure 22).
  • Lanes 1-4 in Figure 23 are the control group, treated with CTB006, treated with Ponatinib, and treated with CTB006+Ponatinib, respectively.
  • Caspase-3blot showed that in the four cell lines, Ponatinib single-drug treatment did not induce caspase cleavage. No matter CTB006 was single-drug or combined with Ponatinib, there was significant cleavage, indicating that the cells are in apoptotic state. After CTB006+Ponatinib co-treatment, the cutting of caspase-3 was enhanced compared with CTB006 single-drug treatment.
  • the combination of CTB006 and Ponatinib can activate the Caspase signaling pathway, promote caspase cleavage, and activate the endogenous and exogenous apoptosis pathways of tumor cells; at the same time, the combination can enhance the inhibition of MEK and ERK phosphorylation, and inhibit the MEK/ERK signaling pathway. The activation thereby inhibits the growth of tumor cells.

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Abstract

本文提供了抗肿瘤药物组合，其包括TRAIL-R2激动型抗体(如CTB006)和帕纳替尼(Ponatinib)或其盐酸盐。本文还提供了肿瘤治疗方法，其包括以治疗有效量的TRAIL-R2激动型抗体和帕纳替尼或其盐酸盐向患者给药。这二者的协同作用有助于增强肿瘤治疗效果。

Description

CTB006与Ponatinib联合应用 技术领域
本文涉及抗肿瘤药物组合，尤其涉及包括TRAIL-R2激动型抗体(如CTB006抗体)和帕纳替尼(Ponatinib)的抗肿瘤药物组合。本文还涉及联合使用TRAIL-R2激动型抗体和帕纳替尼的肿瘤治疗方法。
背景技术
肿瘤坏死因子相关的凋亡诱导配体(tumor necrosis factor-related apoptosis-inducing ligand，TRAIL)，在文献中也称为Apo2L或TNFSF10，于上世纪90年代被鉴定，属于肿瘤坏死因子(TNF)超家族。TRAIL在免疫系统细胞如NK细胞、T细胞、巨噬细胞和树突状细胞中表达。成熟的TRAIL以膜结合形式或可溶性形式存在，其中可溶性形式为膜结合TRAIL被蛋白酶水解而得到的胞外活性部分。这两种形式的TRAIL可形成三聚体并能够通过与靶细胞上的TRAIL受体(TRAIL-R)相互作用而诱导靶细胞凋亡。在人类已鉴定了五种与TRAIL有结合活性的受体。其中的两种TRAIL-R1(或称为DR4)和TRAIL-R2(或称为DR5)能够转导细胞凋亡信号，而其他三种TRAIL-R3(DcR1)、TRAIL-R4(DcR2)和护骨蛋白(osteoprotegerin，OPG)不转导细胞凋亡信号。TRAIL的所有五种受体在它们的细胞外配体结合结构域中有显著的同源性。TRAIL-R1和TRAIL-R2的细胞内片段含有保守的功能结构域，即所谓的“死亡结构域”，其负责转导细胞凋亡信号。已发现直接将重组人TRAIL用于诱导人肿瘤细胞凋亡具有生物半衰期短和稳定差等问题。一些研究机构和生物公司开发了不同的TRAIL-R激动型单克隆抗体，试图替代TRAIL以增强抗肿瘤效果，但临床有效性依然有限。
帕纳替尼(Ponatinib)盐酸盐是一种多靶点激酶抑制剂，由ARIAD Pharmaceuticals公司生产销售，商品名为
Figure PCTCN2021079403-appb-000001
其临床适应症仅局限于血液肿瘤，例如，某些类型的慢性髓性白血病(CML)和急性淋巴细胞白血病(ALL)。
发明内容
在一方面，本文提供了抗肿瘤药物组合，其包括细胞凋亡诱导剂和多靶点激酶抑制剂。
在一些实施方案中，所述细胞凋亡诱导剂为TRAIL-R2结合剂。
在一些实施方案中，所述TRAIL-R2结合剂为TRAIL-R2激动剂。
在一些实施方案中，所述TRAIL-R2激动剂为TRAIL-R2天然配体TRAIL或TRAIL-R2激动型抗体。
在一些实施方案中，所述TRAIL-R2激动型抗体为B273、可那木单抗、CTB006抗体、Hexabody-DR5/DR5、或CTB003抗体。
在一些实施方案中，所述多靶点激酶抑制剂为帕纳替尼(Ponatinib)或其药学上可接受的盐。
在一些实施方案中，所述多靶点激酶抑制剂为帕纳替尼盐酸盐。
在一些实施方案中，所述肿瘤在细胞表面表达TRAIL-R2。
在一些实施方案中，所述肿瘤为乳腺癌、胰腺癌、结直肠癌、胃癌、肺癌、肝癌、食管癌、或卵巢癌。
在一些实施方案中，所述细胞凋亡诱导剂和多靶点激酶抑制剂存在于同一或不同的药物组合物或药物试剂盒中。
在一些实施方案中，所述细胞凋亡诱导剂的剂型适合于静脉给药，所述多靶点激酶抑制剂的剂型适合于口服给药。
另一方面，本文提供了在受试者中治疗肿瘤的方法，其包括同时地、依序地或分别地以治疗有效量的细胞凋亡诱导剂和多靶点激酶抑制剂向所述受试者给药。
在一些实施方案中，所述细胞凋亡诱导剂为TRAIL-R2结合剂。
在一些实施方案中，所述TRAIL-R2结合剂为TRAIL-R2激动剂。
在一些实施方案中，所述TRAIL-R2激动剂为TRAIL-R2天然配体TRAIL或TRAIL-R2激动型抗体。
在一些实施方案中，所述TRAIL-R2激动型抗体为B273、可那木单抗、CTB006抗体、Hexabody-DR5/DR5、或CTB003抗体。
在一些实施方案中，所述多靶点激酶抑制剂为帕纳替尼或其药学上可接受的盐。
在一些实施方案中，所述多靶点激酶抑制剂为帕纳替尼盐酸盐。
在一些实施方案中，所述肿瘤在细胞表面表达TRAIL-R2。
在一些实施方案中，所述肿瘤为乳腺癌、胰腺癌、结直肠癌、胃癌、肺癌、肝癌、食管癌、或卵巢癌。
在一些实施方案中，所述细胞凋亡诱导剂通过静脉注射给药，所述多靶点激酶抑制剂通过口服给药。
另一方面，本文提供了细胞凋亡诱导剂在制备与多靶点激酶抑制剂联合给药以治疗肿瘤的药物中的应用。
在一些实施方案中，所述细胞凋亡诱导剂为TRAIL-R2结合剂。
在一些实施方案中，所述TRAIL-R2结合剂为TRAIL-R2激动剂。
在一些实施方案中，所述TRAIL-R2激动剂为TRAIL-R2天然配体TRAIL或TRAIL-R2激动型抗体。
在一些实施方案中，所述TRAIL-R2激动型抗体为B273、可那木单抗、CTB006抗体、Hexabody-DR5/DR5、或CTB003抗体。
在一些实施方案中，所述激酶抑制剂抑制MEK/ERK信号通路的活化。
在一些实施方案中，所述多靶点激酶抑制剂为帕纳替尼或其药学上可接受的盐。
在一些实施方案中，所述多靶点激酶抑制剂为帕纳替尼盐酸盐。
在一些实施方案中，所述肿瘤在细胞表面表达TRAIL-R2。
在一些实施方案中，所述肿瘤为乳腺癌、胰腺癌、结直肠癌、胃癌、肺癌、肝癌、食管癌、或卵巢癌。
另一方面，本文提供了诱导肿瘤细胞凋亡的方法，包括使所述肿瘤细胞与细胞凋亡诱导剂和多靶点激酶抑制剂接触。
在一些实施方案中，所述细胞凋亡诱导剂为CTB006抗体，所述多靶点激酶抑制剂为帕纳替尼或其盐酸盐。
另一方面，本文提供了在对帕纳替尼或其药学上可接受的盐不耐受的受试者中增强抗肿瘤效果的方法，其包括以CTB006抗体向所述受试者给药。
附图说明
图1显示了CTB006抗体与Ponatinib联合对MCF-7细胞的肿瘤杀伤作用。
图2显示了CTB006抗体与Ponatinib联合对S2Lm7AA细胞的杀伤作用。
图3显示了CTB006抗体与Ponatinib联合对S2VP10细胞的杀伤作用。
图4显示了CTB006抗体与Ponatinib联合对HT29细胞的杀伤作用。
图5显示了CTB006抗体与Ponatinib联合对SGC-7901细胞的杀伤作用。
图6显示了CTB006抗体与Ponatinib联合对N87细胞的杀伤作用。
图7显示了CTB006抗体与Ponatinib联合对A549细胞的杀伤作用。
图8显示了CTB006抗体与Ponatinib联合对SK-HEP-1细胞的杀伤作用。
图9显示了CTB006抗体与Ponatinib联合对Huh-1细胞的杀伤作用。
图10显示了CTB006抗体与Ponatinib联合对TE1细胞的杀伤作用。
图11显示了卵巢癌腹水原代肿瘤细胞的DR5表达情况。
图12显示了CTB006抗体与Ponatinib对原代肿瘤细胞S1(A)和S2(B)的杀伤作用。
图13显示了CTB006与Ponatinib联合对胰腺癌移植瘤模型MIA-Paca-2的疗效。(A)肿瘤体积变化，(B)生存曲线。
图14显示了CTB006与Ponatinib联合对PDX移植瘤模型CS237的作用。
图15显示了CTB006与Ponatinib联合对PDX移植瘤模型CS263的作用。(A)肿瘤体积变化，(B)生存曲线。
图16显示了CTB006与Ponatinib联合对PDX移植瘤模型CS264的作用。
图17显示了CTB006与Ponatinib联合对PDX移植瘤模型CS225的作用。
图18显示了CTB006与Ponatinib联合对PDX移植瘤模型CS266的作用。
图19显示了CTB006与Ponatinib联合对PDX移植瘤模型GAS033的作用。
图20显示了不同类型的DR5激动剂与Ponatinib联合对A549细胞的杀伤作用。
图21显示了不同类型的DR5激动剂与Ponatinib联合对S2VP10细胞的杀伤作用。
图22显示了不同类型的DR5激动剂与Ponatinib联合对MCF7细胞的杀伤作用。
图23显示了不同药物处理作用条件下不同肿瘤细胞凋亡及增殖信号通路激活情况(结肠癌细胞SW1116(A)，胃癌细胞7901(B)，结直肠癌HT29(C)，胰腺癌S2Lm7AA(D))。Lane-1：细胞对照；Lane-2：1000ng/ml CTB006处理组；Lane-3：1μM Ponatinib处理组；Lane-4：合用组(1000ng/ml CTB006和1μM Ponatinib)。
具体实施方式
除非另有说明，本文使用的所有技术和科学术语具有本领域普通技术人员所通常理解的含义。
术语“细胞凋亡诱导剂”指能够在体外或体内促进细胞(如肿瘤细胞)凋亡(apoptosis)的各种化学试剂(如小分子药物)、生物试剂(如多肽，抗体)、或者甚至物理处理(如辐射)等。
术语“激动型抗体”指能够与其对应的抗原结合并触发该抗原的生物学活性的抗体。在一些实施方案中，该抗原为TRAIL-R2，该激动型抗体与其结合时可触发其胞内死亡结构域的凋亡活性。
术语“激酶抑制剂”指能够抑制、减弱、或消除激酶磷酸化其底物蛋白的化学或生物试剂。相应地，术语“多靶点激酶抑制剂”指能够作用于多种激酶的激酶抑制剂。例如，当体内给药时，多靶点激酶抑制剂可抑制多个信号通路。已知的多靶点激酶抑制剂例如有Lapatinib(靶点为EGFR和HER2)、Bosutilib(靶点为SRC和ABL)、Imatinib(靶点为BCR-ABL、C-KIT和PDGFR)，Ponatinib(靶点为ABL、PDGFR、VEGFR、Src等)，等等。在一些实施方案中，该多靶点激酶抑制剂为Ponatinib或其药学上可接受的盐。
术语“CTB006”或“CTB006抗体”指一种靶向TRAIL-R2的激动型抗体。产生CTB006抗体的鼠-鼠杂交瘤细胞已于2006年4月11日保藏于中国普通微生物菌种保藏管理中心(CGMCC)，保藏号为1691，参见PCT申请公开WO2016/074245(通过引用将其全文并入本文)。该抗体具有重链CDR氨基酸序列SYFIH(SEQ ID NO：1)、WIYPGNVNTKYSEKFKG(SEQ ID NO：2)和GEAGYFD(SEQ ID NO：3)，以及轻链CDR氨基酸序列KASQDVSTAVA(SEQ ID NO：4)、WASTRHT(SEQ ID NO：5)和QQHYRTPW(SEQ ID NO：6)。该PCT申请还描述了该抗体的轻链和重链氨基酸序列以及人源化嵌合抗体序列。本领域技术人员应理解，在已知抗体CDR序列情况下，可以对该抗体其它部分序列的氨基酸或甚至该CDR中少数氨基酸进行替换而不改变或基 本上不改变其结合活性。另外，可以预见的，与该抗体具有相同表位特异性的其它抗体也应具有相应的促凋亡活性。因此，可以考虑将这些抗体变体用于替换本文描述的CTB006抗体与所述激酶机制联合使用。这些抗体变体也包括在本发明的范围内。下文实施例中提及CTB006时，采用的是其人源化嵌合抗体。
术语“协同作用”或“协同增效作用”指在两种或更多种试剂、实体、因子或物质之间产生的作用大于其各自作用的总和的效果。在一些实施方案中，例如CTB006与Ponatinib之间的协同作用是通过药物相互作用系数(coefficient of drug interaction，即CDI)来确定的。在一些实施方案中，CDI按下列公式计算：CDI＝AB/A×B，其中AB是两药联合组与对照组检测值的比值(例如联合用药组的存活率)，A或B是各药单独使用组与对照组检测值的比值(例如单独用药组的存活率)。如果CDI<1，可以认为两药作用性质为协同，CDI<0.7时可以认为两药协同作用非常显著；如果CDI＝1，认为两药作用性质为相加；如果CDI>1，则认为两药作用性质为拮抗。
术语“受试者”指患有或者怀疑患有某种疾病的动物(优选人)，或者，在预测易感性时“受试者”也可包括健康个体。该术语通常可以和与“患者”、“病人”、“检测对象”、“治疗对象”等互换使用。
术语“药学上可接受的盐”指某药物的相对无毒的无机酸或有机酸加成盐。盐的性质通常并不关键，前提是它是药学上可接受的。这里“药物上可接受的盐”指对于动物或人体基本无害的无机或有机酸加成盐，例如盐酸盐、氢溴酸盐、磷酸盐、硫酸盐、甲酸盐、乙酸盐、抗坏血酸盐、苯磺酸盐、苯甲酸盐、肉桂酸盐、柠檬酸盐、富马酸盐、谷氨酸盐、羟乙酸盐、乳酸盐、马来酸盐、甲磺酸盐、水杨酸盐、硬脂酸盐、琥珀酸盐、酒石酸盐等。这类盐可以通过本领域技术人员所熟知的方法形成。
在一些实施方案中，CTB006抗体与Ponatinib联合给药。在另一些实施方案中，CTB006抗体与Ponatinib的药学上可接受的盐(例如盐酸盐)联合给药。
术语“治疗有效量”指足以在受试者体内引起临床医师所期望的生物学或医学反应的活性化合物或药物组合物的量。“治疗有效量”可通常由本领域技术人员根据给药途径、受试者的体重、年龄、病情等因素而确定。例如，典型的日剂量范围可以为每kg体重0.01mg至100mg活性成分。
在本发明的一些方面，提供了含有细胞凋亡诱导剂和多靶点激酶抑制剂的药物组合。这里使用术语“药物组合”，不仅意味着细胞凋亡诱导剂和多靶点激酶抑制剂可以共存于同一药物制剂中，还意味着细胞凋亡诱导剂和多靶点激酶抑制剂可以作为分开的药物制剂存在于同一药物试剂盒中，或甚至细胞凋亡诱导剂和多靶点激酶抑制剂可以作为分开的药物制剂存在于不同的药物试剂盒中。实际上，只要通过给药使得细胞凋亡诱导剂和多靶点激酶抑制剂能够在某个时间同时存在于受试者体内，则这种给药方式就采用了这种“药物组合”。
细胞凋亡诱导剂和/或多靶点激酶抑制剂通常与药物上可接受的载体一起配制，形成药物制剂或药物组合物。这里“药物上可接受的载体”指对于动物体或人体而言，可以安全地给药的固体或液体稀释剂、填充剂、抗氧化剂、稳定剂等物质，这些物质适合于对人和/或动物给药而无过度的不良副反应，同时适合于维持位于其中的药物或活性剂的活力。依照给药途径的不同，可以采用本领域众所周知的各种不同的载体，包括，但不限于糖类、淀粉、纤维素及其衍生物、麦芽糖、明胶、滑石、硫酸钙、植物油(例如蓖麻油)、合成油、多元醇、藻酸、磷酸缓冲液、乳化剂、等渗盐水、和/或无热原水等。适合使用的给药途径例如包括口服、静脉内输注、肌肉内注射、皮下注射、腹膜下、直肠、舌下，或经吸入、透皮等途径。相应地，细胞凋亡诱导剂和/或多靶点激酶抑制剂可以与这些药物上可接受的载体一起配制为任何临床上可接受的剂型，例如片剂、颗粒、粉末、胶囊、注射制剂、栓剂、滴剂、外贴药膏、软膏、药油、或喷雾剂，等等。
在本发明的一些方面，提供了以细胞凋亡诱导剂和多靶点激酶抑制剂联合给药以治疗肿瘤(或癌症)的方法。这里所用的术语“联合给药”，例如对于CTB006抗体和Ponatinib组合而言，其包括CTB006抗体与Ponatinib顺序地分别给药，例如CTB006抗体在Ponatinib给药之前或之后给药，还包括Ponatinib与CTB006抗体在同一药物制剂中或以分开的药物制剂形式同时给药。在顺序给药的实施方案中，通常Ponatinib与CTB006抗体至少在部分时间上共存于受试者体内。在联合给药的一些实施方案中，Ponatinib与CTB006抗体可以具有协同作用，例如其中的一种药物的用量低于其单独用药时的治疗有效量，或者优选地，两种药物的用量均低于其单独用药时的治疗有效量。
本发明的一些实施方案至少部分地基于发明人发现以细胞凋亡诱导剂和多靶点激酶抑制剂联合给药会产生明显的协同作用。除了上文已阐述的通过CDI证实协同作用之外，协同作用还可以体现在：1)产生了新的治疗效果，例如可以治疗对该细胞凋亡诱导剂或多靶点激酶抑制剂治疗均无应答的肿瘤；2)减少了治疗副作用；3)延长了给药时间间隔；4)缩短了治疗时间，等等。
本文提供的抗肿瘤药物组合和肿瘤治疗方法利用细胞凋亡诱导剂和多靶点激酶抑制剂的协同作用来增强肿瘤治疗效果。在一些实施方案中，细胞凋亡诱导剂(例如CTB006抗体)的使用可增强激酶抑制剂(例如Ponatinib)的肿瘤生长抑制作用。在一些实施方案中，细胞凋亡诱导剂(例如CTB006抗体)的使用可减少激酶抑制剂(例如Ponatinib)的治疗时间，从而改善患者耐受性。
以下通过具体实施例来进一步阐述本发明。
实施例1 CTB006与Ponatinib合用可显著增强肿瘤细胞的凋亡
两药合用的效果分别通过肿瘤细胞株和癌症病人来源的原代肿瘤细胞验证。
1.1 CTB006与Ponatinib联合用药可以协同杀伤多种肿瘤细胞系
CTB006与Ponatinib联合用药的体外药效学，通过对多个癌种肿瘤细胞株进行体外联合杀伤作用进行验证。
CTB006抗体与Ponatinib两药相互作用我们采用药物相互作用系数(coefficient of drug interaction,CDI) [1,2]评价CTB006抗体与化疗药物的联合作用。CDI按下列公式计算：CDI＝AB/A×B；AB是两药联合组与对照组检测值的比值(联合用药组的存活率)，A或B是各药单独使用组与对照组检测值的比值(单独用药组的存活率)。如CDI<1，证明两药作用性质为协同，CDI<0.7时为两药协同作用非常显著；如CDI＝1，则两药作用性质为相加；如CDI>1，则两药作用性质为拮抗。
实验方法如下：于96孔板，每孔加入3000处于对数生长期的肿瘤细胞以及不同浓度的CTB006抗体和Ponatinib(盐酸盐形式，CAS：1114544-31-8)(药渡)。37℃，5％CO 2，培养24h后，用ATPlite方法观察各肿瘤细胞的存活情况。
1.1.1 CTB006与Ponatinib合用可以协同杀伤乳腺癌细胞MCF-7
结果如图1所示，CTB006与Ponatinib单独使用对MCF-7有一定程度的杀伤作用。两者联合使用，杀伤效果更为明显。不同浓度的CTB006抗体与500nM的Ponatinib联合用药时，其CDI值均小于1；不同浓度的Ponatinib与1000ng/ml的CTB006联合用药时，其CDI值均小于1(见表1)，表明CTB006抗体与Ponatinib联合用药对乳腺癌MCF-7细胞的联合效果为协同增效作用(见图1)。
表1 CDI表明CTB006和Ponatinib协同杀伤MCF-7细胞
Figure PCTCN2021079403-appb-000002
*协同作用(CDI<1)；**显著协同作用(CDI<0.7)
1.1.2 CTB006与Ponatinib合用可以协同杀伤胰腺癌细胞S2Lm7AA
在胰腺癌细胞S2Lm7AA实验中，CTB006与Ponatinib单独使用对S2Lm7AA没有杀伤作用。两者联合使用，有杀伤效果。不同浓度的CTB006抗体与500nM的Ponatinib联合用药时，其CDI值均小于1；不同浓度的Ponatinib与1000ng/ml的CTB006联合用药时，其CDI值均小于1(见表2)，表明CTB006抗体与Ponatinib联合用药对胰腺癌S2Lm7AA细胞的联合效果为协同增效作用(见图2)。
表2 CTB006抗体和Ponatinib对S2Lm7AA细胞两药作用的存活率及CDI
Figure PCTCN2021079403-appb-000003
*协同作用(CDI<1)；**显著协同作用(CDI<0.7)
1.1.3 CTB006与Ponatinib合用可以协同杀伤胰腺癌细胞S2VP10
在胰腺癌细胞S2VP10实验中，CTB006和Ponatinib单独使用对S2VP10没有杀伤作用。两者联合使用，有杀伤效果。不同浓度的CTB006抗体与500nM的Ponatinib联合用药时，其CDI值均小于1；不同浓度的Ponatinib与1000ng/ml的CTB006联合用药时，其CDI值均小于1(见表3)，表明CTB006抗体与Ponatinib联合用药对胰腺癌S2VP10细胞的联合效果为协同增效作用(见图3)。
表3 CTB006抗体与Ponatinib对S2VP10细胞两药作用的存活率及CDI
Figure PCTCN2021079403-appb-000004
Figure PCTCN2021079403-appb-000005
*表示协同作用(CDI<1)；**表示协同作用非常显著(CDI<0.7)
1.1.4 CTB006与Ponatinib合用可以协同杀伤结直肠癌细胞HT29
不同浓度的CTB006抗体与500nM的Ponatinib联合用药时，其CDI值均小于1；不同浓度的Ponatinib与1000ng/ml的CTB006联合用药时，其CDI值均小于1(见表4)，表明CTB006抗体与Ponatinib联合用药对结直肠癌HT29细胞的联合效果为协同增效作用(见图4)。
表4 CTB006抗体与Ponatinib对HT29细胞两药作用的存活率及CDI
Figure PCTCN2021079403-appb-000006
*表示协同作用(CDI<1)；**表示协同作用非常显著(CDI<0.7)
1.1.5 CTB006与Ponatinib合用可以协同杀伤胃癌细胞SGC-7901
不同浓度的CTB006抗体与500nM的Ponatinib联合用药时，其CDI值均小于1；不同浓度的Ponatinib与1000ng/ml的CTB006联合用药时，其CDI值均小于1(见表5)，表明CTB006抗体与Ponatinib联合用药对胃癌SGC-7901细胞的联合效果为协同增效作用(见图5)。
表5 CTB006抗体与Ponatinib对SGC-7901细胞两药作用的存活率及CDI
Figure PCTCN2021079403-appb-000007
Figure PCTCN2021079403-appb-000008
*表示协同作用(CDI<1)；**表示协同作用非常显著(CDI<0.7)
1.1.6 CTB006与Ponatinib合用可以协同杀伤胃癌细胞N87
不同浓度的CTB006抗体与500nM的Ponatinib联合用药时，其CDI值均小于1；不同浓度的Ponatinib与1000ng/ml的CTB006联合用药时，其CDI值均小于1(见表6)，表明CTB006抗体与Ponatinib联合用药对胃癌N87细胞的联合效果为协同增效作用(见图6)。
表6 CTB006抗体与Ponatinib对N87细胞两药作用的存活率及CDI
Figure PCTCN2021079403-appb-000009
*表示协同作用(CDI<1)；**表示协同作用非常显著(CDI<0.7)
1.1.7 CTB006与Ponatinib合用可以协同杀伤肺癌细胞A549
不同浓度的CTB006抗体与500nM的Ponatinib联合用药时，其CDI值均小于1；不同浓度的Ponatinib与1000ng/ml的CTB006联合用药时，其CDI值均小于1(见表 7)，表明CTB006抗体与Ponatinib联合用药对肺癌A549细胞的联合效果为协同增效作用(见图7)。
表7 CTB006抗体与Ponatinib对A549细胞两药作用的存活率及CDI
Figure PCTCN2021079403-appb-000010
*表示协同作用(CDI<1)；**表示协同作用非常显著(CDI<0.7)
1.1.8 CTB006与Ponatinib合用可以协同杀伤肝癌细胞SK-HEP-1
不同浓度的CTB006抗体与500nM的Ponatinib联合用药时，其CDI值均小于1；不同浓度的Ponatinib与500ng/ml的CTB006联合用药时，其CDI值均小于1(见表8)，表明CTB006抗体与Ponatinib联合用药对肝癌SK-HEP-1细胞的联合效果为协同增效作用(见图8)。
表8 CTB006抗体与Ponatinib对SK-HEP-1细胞两药作用的存活率及CDI
Figure PCTCN2021079403-appb-000011
*表示协同作用(CDI<1)；**表示协同作用非常显著(CDI<0.7)
1.1.9 CTB006与Ponatinib合用可以协同杀伤肝癌细胞Huh-1
不同浓度的CTB006抗体与500nM的Ponatinib联合用药时，其CDI值均小于1；不同浓度的Ponatinib与1000ng/ml的CTB006联合用药时，其CDI值均小于1(见表9)，表明CTB006抗体与Ponatinib联合用药对肝癌Huh-1细胞的联合效果为协同增效作用(见图9)。
表9 CTB006抗体与Ponatinib对Huh-1细胞两药作用的存活率及CDI
Figure PCTCN2021079403-appb-000012
*表示协同作用(CDI<1)；**表示协同作用非常显著(CDI<0.7)
1.1.10 CTB006与Ponatinib合用可以协同杀伤食管癌细胞TE1
不同浓度的CTB006抗体与500nM的Ponatinib联合用药时，其CDI值均小于1；不同浓度的Ponatinib与1000ng/ml的CTB006联合用药时，其CDI值均小于1(见表10)，表明CTB006抗体与Ponatinib联合用药对食管癌TE1细胞的联合效果为协同增效作用(见图10)。
表10 CTB006抗体与Ponatinib对TE1细胞两药作用的存活率及CDI
Figure PCTCN2021079403-appb-000013
Figure PCTCN2021079403-appb-000014
*表示协同作用(CDI<1)；**表示协同作用非常显著(CDI<0.7)
1.2 CTB006与Ponatinib合用可显著增强对卵巢癌腹水原代肿瘤细胞的杀伤作用
富集卵巢癌肿瘤病人腹水中肿瘤细胞。不同药物处理下，观察肿瘤细胞的生长状况。
图11所示为两例卵巢癌病人腹水分离所得的原代肿瘤细胞S1和S2的DR5表达情况。通过流式检测发现，S1与S2均表达DR5，但丰度差异较大，S2表达量为S1的四倍。
如图12A和12B所示，CTB006与Ponatinib单药使用时，对S1和S2的杀伤效果均有限。不同浓度的CTB006抗体与100nM的Ponatinib联合使用，肿瘤细胞明显减少，其CDI值均小于1(见表11、12)，表明CTB006抗体与Ponatinib联合用药协同增强了对卵巢癌腹水原代肿瘤细胞的杀伤效果。
表11 CTB006抗体与Ponatinib对原代肿瘤细胞S1两药作用的存活率及CDI
Figure PCTCN2021079403-appb-000015
*表示协同作用(CDI<1)；**表示协同作用非常显著(CDI<0.7)
表12 CTB006抗体与Ponatinib对原代肿瘤细胞S2两药作用的存活率及CDI
Figure PCTCN2021079403-appb-000016
Figure PCTCN2021079403-appb-000017
*表示协同作用(CDI<1)；**表示协同作用非常显著(CDI<0.7)
实施例2 CTB006与Ponatinib合用的体内药效学实验数据
为检测CTB006与Ponatinib合用的体内药效，选择了多个肿瘤动物模型测试，包括胰腺癌细胞MIA-PACA-2CDX模型和多个PDX模型，覆盖肠癌(CS237,CS263,CS264)、肺癌(CS225)、和胃癌(CS266,GAS033)多个癌肿。数学统计中，采用ANOVA分析，P<0.05表示具有显著统计学差异，用*标注；P<0.01表示具有非常显著统计学差异，用**标注。
2.1 CTB006与Ponatinib合用可显著延长胰腺癌CDX模型停药后的肿瘤生长
于BALB/c nude裸鼠(维通利华)皮下移植MIA-Paca-2建立CDX模型，肿瘤体积约0.1cm 3开始给药，达到1cm 3后，动物按伦理安乐死并记录生存时间。给药组分别给以CTB006(10mg/kg,iv,qw)，Ponatinib(30mg/kg,p.o,qd)单药或两者合用，对照组给以等体积生理盐水。本实验连续给药22天后停止给药，对照组安乐死，其余组持续观察至相应终点。
结果如图13A所示，在给药期间，CTB006单独给药组与联合给药组均显示出良好的抑制肿瘤生长的作用，两者未有显著差异。停药之后，CTB006与Ponatinib联合治疗组比CTB006单独治疗组有更加有效的抑制肿瘤生长的效果(IndependentT-test，p＝0.003，Day 47)。图13B为各组动物的生存曲线，对照组中位生存时间22天，CTB006，Ponatinib和CTB006与Ponatinib联合治疗组中位生存期分别为43天，34.5天和54.5天。联合用药表现出明显的生存获益。
上述结果说明CTB006与Ponatinib合用可显著延长CDX肿瘤模型停药后的生长，显示出更加有效的治疗效果。CTB006或可能增强对Ponatinib不耐受病人的疗效。
2.2 CTB006与Ponatinib合用可显著抑制PDX肿瘤抑制瘤模型的生长
于裸鼠或NOD/SCID鼠皮下建立病人来源的移植瘤模型，分别给以CTB006(10mg/kg,iv,qd)，Ponatinib(30mg/kg,p.o,qd)单药或两者合用，对照组给以等体积生理盐水。观察各组动物肿瘤生长状况，评估药物疗效。
结果显示CTB006与Ponatinib合用相比于单独用药，可显著抑制多个瘤种PDX模型肿瘤生长，在停药之后这种效应依旧存在，两药之间体现出加合甚至协同的效应。
2.2.1对结直肠癌PDX模型CS237的药效
结果如图14所示，在给药后第52天，CTB006单药组，Ponatinib单药组，CTB006与Ponatinib联合治疗组的肿瘤抑制率(TGI)分别为25.16％、43.8％、和70％。说明CTB006与Ponatinib联合治疗组有更好的治疗效果。
2.2.2对结直肠癌PDX模型CS263的作用
在直肠癌PDX模型CS263模型中，在给药后第44天，阴性对照组平均肿瘤体积达到1.5cm 3，按伦理安乐死，其余各治疗组停止给药，继续观察后续效果。
给药后第44天，G2组：CTB006单药组，G3组：Ponatinib单药组，G4组：CTB006与Ponatinib合用组的肿瘤抑制率(TGI)分别为27.8％，45.3％，80.8％，CTB006与Ponatinib合用比单药组表现出更好的治疗效果(图15A)(G2vsG4，P＝0.05；G3vsG4，P＝0.02；ANOVADunettT 3)。
在停止给药后的继续观察过程中，发现Ponatinib单药组的肿瘤生长速率明显加快，而联合用药组的肿瘤生长速率略有加快，较单药治疗组，仍较为缓和。以各组平均肿瘤体积达到1.0cm 3的时间为限做存活曲线，对照组的中位生存期39天,CTB006单药组，Ponatinib单药组CTB006与Ponatinib合用组为48天、56天、78天(图15B)。
上述结果说明CTB006与Ponatinib联合治疗组有更好的治疗PDX移植瘤模型CS263的效果。
2.2.3对结直肠癌PDX模型CS264的作用
结果如图16所示，在给药后第32天，5-Fu阳性对照组，CTB006单药组，Ponatinib单药组CTB006与Ponatinib合用组的肿瘤抑制率(TGI)分别为31.2％，-16％，32.4％，80.9％。CTB006单用在该模型中没有显示治疗效果，但与Ponatinib合用明显增强了Ponatinib的治疗效果。
上述结果说明CTB006与Ponatinib联合治疗组有更好的治疗PDX移植瘤模型CS264的效果。
2.2.4对肺癌PDX模型CS225的作用
结果如图17所示，在给药后第28天，Taxol阳性对照组，CTB006单药组，Ponatinib单药组CTB006与Ponatinib合用组的肿瘤抑制率(TGI)分别为50％，14％，50％,32.4％，77％。
上述结果说明CTB006与Ponatinib联合治疗组有更好的治疗PDX移植瘤模型CS225的效果。
2.2.5对胃癌PDX模型CS266的作用
结果如图18所示，在给药后第31天，阴性对照组与CTB006单独治疗组平均肿瘤体积超过达到1cm 3时，处死阴性对照组、5-Fu阳性对照组，Ponatinib单独治疗组与联合治疗组停止给药，继续观察。
在第31天，5-Fu阳性对照组，CTB006单药组，Ponatinib单药组CTB006与Ponatinib合用组的肿瘤抑制率(TGI)分别为57.5％,22％,87.7％,94.6％。
在停止给药后的继续观察过程中，发现Ponatinib单独治疗组的肿瘤生长速率明显加快，抑瘤效果很快丧失，而联合治疗组的肿瘤生长速率未发生明显改变，能够继续维持抑制肿瘤生长。
上述结果说明CTB006与Ponatinib联合治疗组有更好的治疗PDX移植瘤模型CS266的效果。
2.2.6对胃癌PDX模型GAS033的作用
结果如图19所示，在给药后第16天，CTB006单药组，Ponatinib单药组CTB006与Ponatinib合用组的肿瘤抑制率(TGI)分别为-3.0％,37％,61％。
上述结果说明CTB006与Ponatinib联合治疗组有更好的治疗PDX移植瘤模型GAS033的效果。
实施例3.Ponatinib与其他DR5激动剂的联合用药
为了验证Ponatinib与CTB006的协同增效作用是否适用于所有的DR5激动剂，根据文献及相关专利数据，表达并纯化了相关类型的其他DR5激动剂，包括：DR5天然配体TRAIL(义翘神州)；DR5激动型单抗B273(第一三共)，可那木单抗(AMG655，安进)；第二代多聚型DR5激动型抗体Hexabody-DR5/DR5(
Figure PCTCN2021079403-appb-000018
Genmab，GEN1029)；DR5双特异性抗体：DR5/DR4(CTB003，Sinotau，参见专利申请PCT/CN07/001453)。体外多种细胞验证不同类型的DR5激动剂与Ponatinib的联合作用。方法如上所述。
3.1不同类型的DR5激动剂与Ponatinib合用可以协同杀伤肺癌细胞A549
在肺癌细胞A549实验中，不同类型的DR5激动剂与Ponatinib在相应浓度下单独使用对A549没有杀伤作用。同等剂量联合使用时，有明显的杀伤效果。其CDI值均小于1(见表13)，表明不同类型的DR5激动剂与Ponatinib联合用药对肺癌A549细胞的联合效果为协同增效作用(见图20)。
表13不同类型的DR5激动剂与Ponatinib对A549细胞两药作用的CDI
Figure PCTCN2021079403-appb-000019
Figure PCTCN2021079403-appb-000020
3.2不同类型的DR5激动剂与Ponatinib合用可以协同杀伤胰腺癌细胞S2VP10
在胰腺癌细胞S2VP10实验中，不同类型的DR5激动剂与Ponatinib在相应浓度下单独使用对S2VP10杀伤作用不明显。同等剂量联合使用时，有明显的杀伤效果。其CDI值均小于1(见表14)，表明不同类型的DR5激动剂与Ponatinib联合用药对胰腺癌S2VP10细胞的联合效果为协同增效作用(见图21)。
表14不同类型的DR5激动剂与Ponatinib对S2VP10细胞两药作用的CDI
Figure PCTCN2021079403-appb-000021
3.3不同类型的DR5激动剂与Ponatinib合用可以协同杀伤乳腺癌细胞MCF7
在乳腺癌细胞MCF7实验中，大部分类型的DR5激动剂(如TRAIL，Hexabody-DR5/DR5及DR5/DR4)与Ponatinib在相应浓度下单独使用对MCF7杀伤作用不明显。同等剂量联合使用时，有明显的杀伤效果。另有B273与AMG655单独用药时对MCF7有明显的杀伤作用，而Ponatinib单独使用无效，当两者联合使用时，其杀伤效果更为显著。其CDI值均小于1(见表15)，表明不同类型的DR5激动剂与Ponatinib联合用药对乳腺癌MCF7细胞的联合效果为协同增效作用(见图22)。
表15不同类型的DR5激动剂与Ponatinib对MCF7细胞两药作用的CDI
Figure PCTCN2021079403-appb-000022
实施例4.Ponatinib与CTB006合用可能的机理
为了探究Ponatinib与CTB006之间协同作用的分子机制，研究两者合用是否可以激活细胞凋亡通路，四株肿瘤细胞结肠癌细胞SW1116(A)，胃癌细胞7901(B)，结直肠癌HT29(C)，胰腺癌S2Lm7AA(D)，经相应药物处理过夜，取细胞裂解液做Western blot检测Caspase剪切测定细胞凋亡信号通路的激活情况，以及MEK/ERK信号通路的磷酸化水平反映细胞增生情况。
图23中Lane 1-4分别为对照组，CTB006处理，Ponatinib处理，和CTB006+Ponatinib共同处理。caspase-3blot显示，在四株细胞里，Ponatinib单药处理并不诱导caspase的切割，CTB006无论是单药还是与Ponatinib合用，均有明显的剪切，表明细胞处于凋亡状态。CTB006+Ponatinib共同处理后，caspase-3的切割均比CTB006单药处理增强。
在7901(B)，HT29(C)，S2Lm7AA(D)三株细胞里，caspase-8，9剪切上，结果相似，Ponatinib单药处理并不诱导caspase-8，9的切割，CTB006无论是单药还是与Ponatinib合用，均有明显的剪切，表明细胞内外源的凋亡通路均已激活。而7901和S2Lm7AA这两株细胞在内源性凋亡的标记物caspase-9的剪切上，CTB006+Ponatinib共同处理后也比CTB006单药处理略为增强。
在MEK/ERK通路上，在四株细胞中，阴性对照组及CTB006单药组p-MEK，p-ERK均没有显著变化，Ponatinib单独用药组p-MEK，p-ERK均明显下调，而合用组p-MEK和p-ERK下降更为显著。
综上所述，CTB006与Ponatinib联合用药可激活Caspase信号通路，促进caspase剪切，激活肿瘤细胞的内外源凋亡通路；同时联合用药可以增强抑制MEK，ERK的磷酸化，抑制MEK/ERK信号通路的活化从而抑制肿瘤细胞生长。
参考文献：
1.汤秀红，秦叔逵，陈惠英，等.三氧化二砷与顺铂合用抗人肝癌细胞株QGY-7701的实验研究[J].肿瘤防治研究，2002，29(5)：362-364.
2.廖志勇，张胜华，甄永苏.Geldanamycin增强顺铂的抗肿瘤作用[J].癌症，2000,19(8)：731-734.

Claims (34)

  1. 抗肿瘤药物组合，包括细胞凋亡诱导剂和多靶点激酶抑制剂。
  2. 如权利要求1所述的抗肿瘤药物组合，其中所述细胞凋亡诱导剂为TRAIL-R2结合剂。
  3. 如权利要求2所述的抗肿瘤药物组合，其中所述TRAIL-R2结合剂为TRAIL-R2激动剂。
  4. 如权利要求3所述的抗肿瘤药物组合，其中所述TRAIL-R2激动剂为TRAIL-R2天然配体TRAIL或TRAIL-R2激动型抗体。
  5. 如权利要求4所述的抗肿瘤药物组合，其中所述TRAIL-R2激动型抗体为B273、可那木单抗、CTB006抗体、Hexabody-DR5/DR5、或CTB003抗体。
  6. 如前述权利要求任一项所述的抗肿瘤药物组合，其中所述多靶点激酶抑制剂为帕纳替尼(Ponatinib)或其药学上可接受的盐。
  7. 如前述权利要求任一项所述的抗肿瘤药物组合，其中所述多靶点激酶抑制剂为帕纳替尼盐酸盐。
  8. 如前述权利要求任一项所述的抗肿瘤药物组合，其中所述肿瘤在细胞表面表达TRAIL-R2。
  9. 如前述权利要求任一项所述的抗肿瘤药物组合，其中所述肿瘤为乳腺癌、胰腺癌、结直肠癌、胃癌、肺癌、肝癌、食管癌、或卵巢癌。
  10. 如前述权利要求任一项所述的抗肿瘤药物组合，其中所述细胞凋亡诱导剂和多靶点激酶抑制剂存在于同一或不同的药物组合物或药物试剂盒中。
  11. 如前述权利要求任一项所述的抗肿瘤药物组合，其中所述细胞凋亡诱导剂的剂型适合于静脉给药，所述多靶点激酶抑制剂的剂型适合于口服给药。
  12. 在受试者中治疗肿瘤的方法，包括同时地、依序地或分别地以治疗有效量的细胞凋亡诱导剂和多靶点激酶抑制剂向所述受试者给药。
  13. 如权利要求12所述的方法，其中所述细胞凋亡诱导剂为TRAIL-R2结合剂。
  14. 如权利要求13所述的方法，其中所述TRAIL-R2结合剂为TRAIL-R2激动剂。
  15. 如权利要求14所述的方法，其中所述TRAIL-R2激动剂为TRAIL-R2天然配体TRAIL或TRAIL-R2激动型抗体。
  16. 如权利要求15所述的方法，其中所述TRAIL-R2激动型抗体为B273、可那木单抗、CTB006抗体、Hexabody-DR5/DR5、或CTB003抗体。
  17. 如权利要求12-16任一项所述的方法，其中所述多靶点激酶抑制剂为帕纳替尼或其药学上可接受的盐。
  18. 如权利要求12-17任一项所述的方法，其中所述多靶点激酶抑制剂为帕纳替尼盐酸盐。
  19. 如权利要求12-18任一项所述的方法，其中所述肿瘤在细胞表面表达TRAIL-R2。
  20. 如权利要求12-19任一项所述的方法，其中所述肿瘤为乳腺癌、胰腺癌、结直肠癌、胃癌、肺癌、肝癌、食管癌、或卵巢癌。
  21. 如权利要求12-20任一项所述的方法，其中所述细胞凋亡诱导剂通过静脉注射给药，所述多靶点激酶抑制剂通过口服给药。
  22. 细胞凋亡诱导剂在制备与多靶点激酶抑制剂联合给药以治疗肿瘤的药物中的应用。
  23. 如权利要求22所述的应用，其中所述细胞凋亡诱导剂为TRAIL-R2结合剂。
  24. 如权利要求23所述的应用，其中所述TRAIL-R2结合剂为TRAIL-R2激动剂。
  25. 如权利要求24所述的应用，其中所述TRAIL-R2激动剂为TRAIL-R2天然配体TRAIL或TRAIL-R2激动型抗体。
  26. 如权利要求25所述的应用，其中所述TRAIL-R2激动型抗体为B273、可那木单抗、CTB006抗体、Hexabody-DR5/DR5、或CTB003抗体。
  27. 如权利要求22-26任一项所述的应用，其中所述多靶点激酶抑制剂抑制MEK/ERK信号通路的活化。
  28. 如权利要求22-27任一项所述的应用，其中所述多靶点激酶抑制剂为帕纳替尼或其药学上可接受的盐。
  29. 如权利要求22-28任一项所述的应用，其中所述多靶点激酶抑制剂为帕纳替尼盐酸盐。
  30. 如权利要求22-29任一项所述的应用，其中所述肿瘤在细胞表面表达TRAIL-R2。
  31. 如权利要求22-30任一项所述的应用，其中所述肿瘤为乳腺癌、胰腺癌、结直肠癌、胃癌、肺癌、肝癌、食管癌、或卵巢癌。
  32. 诱导肿瘤细胞凋亡的方法，包括使所述肿瘤细胞与细胞凋亡诱导剂和多靶点激酶抑制剂接触。
  33. 如权利要求32所述的方法，其中所述细胞凋亡诱导剂为CTB006抗体，所述多靶点激酶抑制剂为帕纳替尼或其盐酸盐。
  34. 在对帕纳替尼或其药学上可接受的盐不耐受的受试者中增强抗肿瘤效果的方法，包括以CTB006抗体向所述受试者给药。
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