WO2015131822A1 - Antibody-drug conjugate pertuzumab-mcc-dm1, composition of pertuzumab-mcc-dm1 and trastuzumab and uses thereof - Google Patents

Antibody-drug conjugate pertuzumab-mcc-dm1, composition of pertuzumab-mcc-dm1 and trastuzumab and uses thereof Download PDF

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WO2015131822A1
WO2015131822A1 PCT/CN2015/073629 CN2015073629W WO2015131822A1 WO 2015131822 A1 WO2015131822 A1 WO 2015131822A1 CN 2015073629 W CN2015073629 W CN 2015073629W WO 2015131822 A1 WO2015131822 A1 WO 2015131822A1
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cancer
antibody
pertuzumab
trastuzumab
her2
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French (fr)
Chinese (zh)
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沈竞康
孟韬
马兰萍
张永良
彭红丽
王昕�
王英
陈驎
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中国科学院上海药物研究所
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5355—Non-condensed oxazines and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6855—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from breast cancer cell
    • 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
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes

Definitions

  • the present invention relates to antibody-drug conjugates and methods for treating hyperproliferative disorders therewith, and in particular to an antibody-drug conjugate Pertuzumab-MCC-DM1 (P-DM1) and its dimerization with a therapeutically effective amount of HER2 A composition of the inhibitor antibody Trastuzumab, and a method of treating a hyperproliferative disorder using the antibody-drug conjugate or composition.
  • P-DM1 Pertuzumab-MCC-DM1
  • HER2 is a receptor tyrosine kinase that binds to the surface of cell membranes and is encoded by the proto-oncogene HER2/neu. It belongs to the epidermal growth factor receptor family, which includes HER1 (erbB 1, EGFR), HER2 (erbB2, NEU), HER3 (erbB3), and HER4 (erbB4). HER2 is involved in signaling pathways that lead to cell growth and differentiation. Overexpression of HER2 was observed in approximately 25% to 30% of human breast cancer. Studies have shown that breast cancer is more invasive and more aggressive when HER2 protein is overexpressed. Moreover, HER2-positive tumors grow and spread faster than tumors that are not HER2-positive. HER2-positive breast cancer is reported to be 2.5 times more recurrent than non-HER2 positive breast cancer.
  • trastuzumab is a humanized monoclonal antibody directed against the Her-2/neu proto-oncogene product that selectively binds to region IV of the HER2 (or HER2/neu) receptor. Trastuzumab inhibits tumor cell growth by binding to the HER2 protein.
  • trastuzumab (trade name: Herceptin) was approved by the US Food and Drug Administration (FDA) for breast cancer treatment with positive Her-2 expression.
  • Herceptin provides significantly better results for patients with HER2-positive tumors than chemotherapy alone, nearly 50% of Her-2 positive patients are insensitive to trastuzumab treatment (Singer CF, Predicting the efficacy) Of Trastuzumab-based therapy in breast cancer: current standards and future strategies, Biochim Biophys Acta, 2008, 1786(2): 105-113), and most Her2-positive patients after one year of trastuzumab treatment, It will be resistant to it. Therefore, there is still a clinical need to develop new cancer therapies for HER2 for patients who are unresponsive or underreactive to Herceptin treatment, have tumors that overexpress HER2 or other diseases associated with HER2 expression.
  • Antibody-drug conjugate uses monoclonal antibodies to specifically recognize the characteristics of specific antigens on tumor cells, and coupled with small molecule chemotherapy drugs, can achieve accurate anti-antibiotics Tumor material delivery to tumor target cell release.
  • ADC Antibody-drug conjugate
  • T-DM1 (trastuzumab conjugated with maytansin DM1) was approved by the FDA for the treatment of HER2-positive metastatic breast cancer.
  • T-DM1 showed potent anti-tumor activity in the Trastuzumab-sensitive and Trastuzumab-resistant tumor cell lines overexpressing HER2, and in human xenograft models of human breast cancer.
  • T-DM1 The mechanism of action of T-DM1 showed that in addition to the action of small molecule DM1, it also includes the action of Trastuzumab itself and the role of ADCC (Trastuzumab Emtansine: A Unique Antibody-Drug Conjugate in Development for Human Epidermal Growth Factor Receptor 2-Positive Cancer, Clin Cancer Res, 2011, 17 (20), 6437-6447).
  • Pertuzumab is also a monoclonal antibody that acts on HER dimerization.
  • Pertuzumab differs from TTrastuzumab in the epitope binding region of the light and heavy chains, Pertuzumab binds to an epitope within subdomain 2 of HER2, and the epitope of TTrastuzumab is located at subdomain 4. Pertuzumab works by blocking the binding of HER2 to other HER family members, including HER1 (epidermal growth factor receptor; EGFR), HER3, and HER4.
  • HER1 epipidermal growth factor receptor
  • Pertuzumab inhibits ligand-initiated intracellular signaling. Inhibition of these signaling pathways can lead to growth arrest and apoptosis, respectively.
  • T-DM1 and Pertuzumab showed stronger anti-tumor activity than single agents in cell and animal models (Dual targeting of HER2-positive cancer with Trastuzumab-emtansine (T-DM1) and pertuzumab: critical role for neuregulin blockade In anti-tumor response to combination therapy, Clin Cancer Res, Published Online First on October 4, 2013).
  • T-DM1 Trastuzumab-emtansine
  • pertuzumab critical role for neuregulin blockade In anti-tumor response to combination therapy
  • P-DM1 Pertuzumab-MCC-DM1
  • a composition thereof in combination with a therapeutically effective amount of the HER2 dimerization inhibitor antibody Trastuzumab.
  • the present invention provides an antibody-drug conjugate Pertuzumab-MCC-DM1 (P-DM1) having the following structure:
  • mAb refers to pertuzumab
  • Pertuzumab is linked to maytansinoid DM1 via a bifunctional linker reagent SMCC, wherein n represents a drug-antibody ratio, and the range thereof is preferably an integer between 1 and 8. Value, wherein 1, 2, 3, 4, 5, 6, 7, or 8 drug moieties are covalently bound to the antibody Pertuzumab.
  • the invention also provides a therapeutic composition for treating a hyperproliferative disease or condition, wherein the therapeutic composition comprises a therapeutically effective amount of P-DMl and a therapeutically effective amount of a HER2 dimerization inhibitor antibody.
  • the HER2 dimerization inhibitor antibody is preferably trastuzumab
  • the therapeutically effective amount of P-DMl and the therapeutically effective amount of a HER2 dimerization inhibitor antibody can be administered in combination as a formulation or alternately.
  • the invention also provides methods of using the therapeutic agent combination to treat an extracellular and/or in situ treatment of a mammalian cell, an organism's hyperproliferative disease or condition.
  • Another aspect of the invention also provides a method of using the therapeutic composition of the invention to treat a hyperproliferative disease or condition in a mammal or a human, such as a cancer, including a disease modulated by HER2 or KDR9 (VEGF receptor 1) method.
  • the invention also provides a method of treating a hyperproliferative disease or condition comprising administering to a mammal or human in need of such treatment a therapeutically effective amount of a P-DM1 and HER2 dimerization inhibitor antibody agent.
  • P-DM1 and HER2 dimerization inhibitor antibody agents are administered in the form of a combined formulation
  • the P-DM1 and HER2 dimerization inhibitor antibody agents are administered separately (alternately, sequentially) as a combination of therapeutic agents; further, the P-DM1 and HER2 dimerization inhibitors
  • the antibody agent is administered to a patient having a hyperproliferative disorder at intervals of about 3 weeks or once a week;
  • the mammal is a HER2-positive patient; further, the HER2-positive patient has received Trastuzumab and other chemotherapeutic agents.
  • Another aspect of the invention provides the antibody-drug conjugate P-DM1 and therapeutic composition of the invention for use in the manufacture of a hyperproliferative disease or disorder, such as cancer, for use in treating a mammal or a human Use in drugs regulated by HER2).
  • the hyperproliferative disorder of the present invention is cancer; further, the hyperproliferative disorder is a cancer expressing HER2; further, the cancer is breast cancer, gastric cancer, ovarian cancer, cervical cancer, prostate cancer, Testicular cancer, genitourinary tract cancer, non-small cell lung cancer, colon cancer, bone cancer, pancreatic cancer, colon-rectal cancer, liver cancer and cholangiocarcinoma, skin cancer, esophageal cancer, laryngeal cancer, glioblastoma, neuroblast Tumor, adenocarcinoma, oral cancer, thyroid cancer, lung cancer, epidermoid carcinoma, large cell carcinoma, small cell carcinoma, kidney cancer, small intestine cancer, colon cancer, rectal cancer, nasopharyngeal cancer, bladder cancer, melanoma, lip cancer , hair cell cancer, follicular carcinoma, brain and central nervous system cancer, Hodgkin's lymphoma or leukemia, preferably breast cancer.
  • the cancer is breast cancer,
  • Another aspect of the invention also provides a method for predicting an effective pharmaceutical combination for in vivo efficacy by qualitative and quantitative analysis of efficacy data from in vitro cell proliferation and in vivo tumor xenograft experiments, wherein said combination comprises P-DM1 and HER2 dimerization Inhibitor antibody.
  • the present invention provides a method for determining a compound to be used in combination for cancer treatment, comprising: a) treating a tumor cell line in vitro with a combination of therapeutic agents of Pertuzumab-MCC-DM1 and a HER2 dimerization inhibitor antibody, and b) Synergistic or non-synergistic effects are measured whereby a synergistic combination of therapeutic agents is determined for cancer treatment.
  • the present invention provides a method for determining a compound to be used in combination for cancer treatment, comprising: (a) administering Pertuzumab-MCC-DM1 and HER2 dimerization to HER2-amplified breast cancer cells or gastric cancer cells. A combination of therapeutic agents of the inhibitor antibody, and (b) measuring inhibition of cell proliferation, thereby determining a synergistic therapeutic combination for cancer treatment.
  • the combination index (CI) was determined by a combination of P-DM1 and Trastuzumab in a fixed concentration ratio, or the combination index (CI) was determined by a combination of a fixed concentration of P-DM1 and various concentrations of Trastuzumab.
  • a CI value of less than 0.9 indicates synergy; a CI value between 0.9 and 1.1 indicates additive; and a CI value greater than 1.1 indicates antagonistic.
  • the HER2-amplified breast cancer cells are BT-474, SK-BR-3; and the HER2-amplified gastric cancer cells are NCI-N87.
  • the antibody-drug conjugate of the present invention (P-DM1 can be prepared by the following method:
  • the pertituzate monoclonal antibody was replaced by a desalting chromatography (SephadexTM G25 desalting column) into a reaction buffer (potassium phosphate/NaCl/EDTA), and the antibody was concentrated to prepare a pertuzumab antibody;
  • the MCC-DM1 mother liquid prepared in the step (2) is added to the pertuzumab prepared in the step (1) to carry out a conjugation reaction.
  • the antibody was initially titrated with a number of excess MCC-DM1 to determine the desired DM1:mAb ratio. Then adding 8-10 times excess molar ratio of SMCC-DM1 mother liquor for reaction;
  • the coupling reaction mixture was purified by gel filtration using a sodium succinate/NaCl buffer using a gel filtration column (Superdex 200), and a peak sample was collected according to UV280 ultraviolet absorption value, or ultrafiltered several times.
  • treating refers to both therapeutic treatment and prophylactic or preventative measures, wherein the goal is to prevent or slow down (mitigate) unwanted physiological changes or conditions, such as the growth of high proliferative conditions such as cancer, formation. Or spread.
  • advantageous or desirable clinical outcomes include, but are not limited to, alleviating symptoms, attenuating the extent of the disease, stabilizing the disease state (ie, not worsening), delaying or slowing the progression of the disease, improving or reducing the disease state, and rehabilitation (whether part Still complete), whether detectable or undetectable.
  • Treatment can also mean prolonging survival as compared to expected survival without treatment.
  • Subjects in need of treatment include subjects who have long had a disease or condition as well as subjects who are predisposed to having the disease or condition or who are to be prevented from developing a condition or condition.
  • terapéuticaally effective amount refers to an amount of a compound of the invention that (i) treats a particular disease, condition, or condition described herein, (ii) alleviates, ameliorates, or eliminates one or more of a particular disease, condition, or condition. a symptom, or (iii) preventing or delaying the onset of one or more symptoms of a particular disease, condition, or condition.
  • a therapeutically effective amount of the drug can reduce the number of cancer cells; reduce the volume of the tumor; inhibit (ie, slow down, preferably stop) the infiltration of the cancer cells into the surrounding organs; inhibition (ie, a certain degree of slowing, preferably stop) a tumor metastasis; a degree of inhibition of tumor growth; and/or a degree of alleviation of one or more symptoms associated with cancer.
  • the drug inhibits cancer cell growth and/or kills existing cancer cells, it can be cytostatic and/or cytotoxic.
  • efficacy can be measured, for example, by evaluating time to disease progression (TTP) and/or assay response rate (RR).
  • High proliferative disorders refer to tumors, cancer, and neoplastic tissues, including pre-malignant and non-neoplastic stages, but also include psoriasis, endometriosis, polyps, and fibroadenomas.
  • cancer and “cancerous” refer to or describe a physiological condition in a mammal that is typically characterized by unregulated cell growth.
  • a “tumor” includes one or more cancerous cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma and leukemia or lymphoid malignancies.
  • squamous cell carcinoma eg, epithelial squamous cell carcinoma
  • lung cancer including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma
  • peritoneal cancer hepatocellular carcinoma
  • gastric cancer including Gastrointestinal cancer
  • rectal cancer colorectal cancer, endometrial cancer or uterine cancer
  • salivary gland cancer kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, and head and neck cancer.
  • pharmaceutically acceptable indicates that the substance or composition must be chemically and/or toxicologically compatible with the other ingredients that make up the formulation and/or the mammal to which it is treated.
  • synergistic refers to a combination of therapeutic agents that are more effective than the additive effect of two or more single agents.
  • the determination of the synergistic interaction between the antibodies of the P-DM1 and HER2 dimerization inhibitors can be based on the results obtained from the assays described herein.
  • Combination therapies provide "synergistic” and prove to be “synergistic”, ie, the effect achieved when the active ingredients are used together is greater than the sum of the effects resulting from the separate use.
  • Synergistic effects can be obtained when the active ingredients are as follows: (1) co-formulation and simultaneous administration or delivery in a combined unit dose formulation; (2) alternating delivery as separate formulations; or (3) providing some other regimen.
  • a synergistic effect can be obtained when the compounds are administered sequentially or while being administered (eg, by different injections in separate syringes).
  • an effective amount of each active ingredient is administered sequentially (i.e., continuously in time) during alternation therapy.
  • a combination of Pertuzumab and a small molecule chemotherapeutic drug, a combination of a P-DM1 and a HER2 dimerization inhibitor antibody exhibits a synergistic effect in inhibiting the growth of cancer cells in vitro and in vivo, and can achieve a HER2-mediated A better therapeutic effect of a disease such as cancer.
  • the antibody-drug conjugate P-DM1 of the present invention and its combination with the antibody trastuzumab significantly enhance the therapeutic effect, providing a more effective method for treating a HER2-mediated condition such as cancer.
  • Figure 1 shows the inhibition of cell proliferation in vitro by BT-474 cells in vitro after treatment with various concentrations of Pertuzumab-MCC-DM1 (P-DM1), Trastuzumab, and fixed concentration ratio (1:1) of P-DM1 and Trastuzumab alone.
  • Figure 2 shows the inhibition of cell proliferation of BT-474 cells in vitro by various fixed concentrations of Pertuzumab-MCC-DM1 (P-DM1) in combination with Trastuzumab, and various concentrations of Trastuzumab alone for 5 days.
  • P-DM1 Pertuzumab-MCC-DM1
  • FIG 3 shows the inhibition of cell proliferation in vitro by NCI-N87 cells in vitro after treatment with various concentrations of Pertuzumab-MCC-DM1 (P-DM1), Trastuzumab, and fixed concentration ratio (1:10) of P-DM1 and Trastuzumab alone.
  • FIG 4 shows the inhibition of cell proliferation of NCI-N87 cells in vitro by various fixed concentrations of Pertuzumab-MCC-DM1 (P-DM1) in combination with Trastuzumab, and treatment with various concentrations of Trastuzumab alone for 5 days.
  • P-DM1 Pertuzumab-MCC-DM1
  • Figure 5 shows the inhibition of cell proliferation in vitro by SK-BR-3 cells in vitro after treatment with various concentrations of Pertuzumab-MCC-DM1 (P-DM1), Trastuzumab, and various fixed concentrations of P-DM1 in combination with Trastuzumab.
  • P-DM1 Pertuzumab-MCC-DM1
  • Figure 6 shows the inhibition of cell proliferation in vitro by Calcu-3 cells after 5 days of treatment with various concentrations of Pertuzumab-MCC-DM1 (P-DM1), and (B) shows various fixed concentrations of P-DM1 in combination with Trastuzumab. Inhibition of cell proliferation in vitro by Calu-3 cells after 5 days of treatment with various concentrations of Trastuzumab (+ vehicle) alone.
  • Figure 7 shows the inhibition of cell proliferation in vitro by DU-145 cells treated with various concentrations of Pertuzumab-MCC-DM1 (P-DM1) for 3 days, and (B) shows various fixed concentrations of P-DM1 in combination with Trastuzumab.
  • P-DM1 Pertuzumab-MCC-DM1
  • Fig. 8(A) shows the inhibition of cell proliferation of SKOV-3 cells in vitro after treatment with various concentrations of Pertuzumab-MCC-DM1 (P-DM1) for 3 days, and (B) shows various fixed concentrations of P-DM1 in combination with Trastuzumab.
  • P-DM1 Pertuzumab-MCC-DM1
  • Figure 9 is a graph showing the change in mean tumor volume over time in Herceptin-resistant human breast cancer BT-474/T721 nude mice xenografts as follows: (1) solvent control; (2) P-DM1 3 mg/kg (3) P-DM1 10 mg/kg; (4) P-DM1 3 mg/kg + Trastuzumab 10 mg/kg.
  • the desalted single antigen solution was replaced by a desalting chromatography (SephadexTM G25 desalting column) into a reaction buffer (50 mM potassium phosphate/50 mM NaCl/2 mM EDTA, pH 7.5), and the antibody concentration was concentrated to 5 mg/ml to prepare.
  • a desalting chromatography SephadexTM G25 desalting column
  • a reaction buffer 50 mM potassium phosphate/50 mM NaCl/2 mM EDTA, pH 7.5
  • the MCC-DM1 was weighed and fully dissolved with dimethylacetamide (DMA) to prepare a 10 mg/ml MCC-DM1 mother liquor;
  • DMA dimethylacetamide
  • the MCC-DM1 mother liquid configured in the step (2) is added to the pertuzumab prepared in the step (1) to carry out a conjugation reaction.
  • the antibody was initially titrated with a number of excess MCC-DM1 to determine the desired DM1:mAb ratio, which is typically a 6-10 fold molar excess for human antibodies.
  • the DMA in the reaction is within 5% v/v.
  • the reaction temperature is 25 ° C and the reaction time is from 1.5 to about 20 hours.
  • the reactions were divided into three groups:
  • the coupling reaction mixture was gel-filtered using a gel filtration column (Superdex 200) with a sodium succinate/150 mM NaCl buffer of pH 5.0, and a peak sample was collected according to the UV280 ultraviolet absorption value, or ultrafiltered several times.
  • the Ab antibody molecule in the final conjugate is measured by measuring the absorbance of the conjugate at 252 and 280 nm and using the known extinction coefficient of DM1 and antibody at these two wavelengths. The number of DM1 molecules (calculated as Equation 1).
  • the coupling rate of the product was calculated by mass spectrometry and compared. The data is shown in Table 1.
  • determining the coupling ratio means measuring the amount of a small molecule drug attached to each monoclonal antibody molecule, that is, the molar ratio of the drug to the antibody in the conjugate. It can be determined by two methods: ultraviolet spectrophotometry and mass spectrometry.
  • Ultraviolet spectrophotometry Determination of the coupling value (DAR) by measuring the UV absorbance of the conjugate at 252 nM and 280 nM, and calculating by Lamberbeer's law; mass spectrometry: for the antibody-maytansinoid
  • DAR coupling value
  • mass spectrometry for the antibody-maytansinoid
  • UV absorption values were determined by UV spectrophotometry at 252 nM and 280 nM, and antibody and drug concentrations were calculated by Lambert Beer's law; the concentration of the conjugate described in the present invention is single The sum of the anti-concentration and the maytansinoid (DM1) drug attached to the mAb.
  • DM1 maytansinoid
  • the experimental materials used in the following experiments were derived from: DMEM medium, F12K medium, RPMI 1640 medium, 0.25% trypsin-EDTA, fetal bovine serum, 100 x sodium pyruvate, 100 x streptomycin, available from Gibco.
  • Sulforhodamine B (SRB) was purchased from Sigma.
  • BT-474 breast cancer cells and NCI-N87 gastric cancer cells are from Kunming Cell Bank of Chinese Academy of Sciences
  • SK-BR-3 breast cancer cells are from Shanghai Bogu Biotechnology Co., Ltd.
  • Calu-3 lung cancer cells are from Shanghai Institute of Life Sciences, Chinese Academy of Sciences.
  • the library, SKOV-3 ovarian cancer cells and DU-145 prostate cancer cells were obtained from the American Type Culture Collection (ATCC). All other reagents were of analytical grade.
  • 96-well flat-bottom polystyrene Corning, Cat. No. 3599). Synergy 2 microplate reader (Bio-Tek).
  • SRB sulforhodamine B
  • SRB is a pink anionic dye which is easily soluble in water and can specifically bind to basic amino acids of proteins in cells under acidic conditions. It produces an absorption peak at a wavelength of 510 nm. The absorbance is linearly positively correlated with the amount of cells. Therefore, it can be used for quantitative detection of the number of cells.
  • the cell lines selected in this example were: BT-474, SK-BR-3 breast cancer cells, NCI-N87 gastric cancer cells, Calu-3 lung cancer cells, SKOV-3 ovarian cancer cells, and DU-145 prostate cancer cells.
  • BT-474, SK-BR-3, NCI-N87 cells in 1640 medium containing 10% fetal bovine serum, Calu-3, SKOV-3, DU-145 cells in DMEM medium containing 10% fetal bovine serum The cells were cultured to a logarithmic growth phase at 37 ° C in a 5% CO 2 incubator, and the cells in the logarithmic growth phase were seeded at a density of 2 x 103 to 9 ⁇ 103 cells per well to a 96-well culture plate.
  • Inhibition rate (%) (A control - A administration) / A control ⁇ 100%.
  • the inhibition of drug 1 (D) 1 in combination with drug 2 (D) 2 was X%, and (Dx) 1 and (Dx) 2 were concentrations at which X% was inhibited when drug 1 and drug 2 were used alone. For each compound, the % growth value at each concentration measured by the SRB assay was used.
  • Pertuzumab-MCC-DM1 (P-DM1) alone or in combination with Trastuzumab was used to study the proliferation of a variety of HER2-overexpressing tumor cell lines in vitro, and also to non-HER2 overexpressing tumor cell lines. Study on the proliferation of cell culture in vitro.
  • the data were analyzed using the Chou & Talalay method at a fixed concentration ratio of the P-DM1 and Trastuzumab groups.
  • the combination index (CI) was determined by combining the combined index (CI), or a combination of various concentrations of Trastuzumab at a fixed concentration of P-DM1.
  • a CI value of less than 0.9 indicates synergy; a CI value between 0.9 and 1.1 indicates additive; and a CI value greater than 1.1 indicates antagonistic.
  • P-DM1 alone produced anti-tumor cell proliferation activity, as shown in Figure 1.
  • the combination of P-DM1 and Trastuzumab in a fixed concentration ratio (1:1) for HER2 overexpressing BT-474 cells resulted in synergistic anti-cells. Proliferative activity.
  • Mouse xenograft studies showed similar results, with P-DM1 in combination with Trastuzumab resulting in greatly enhanced anti-tumor efficacy compared to treatment with a single agent (Figure 9).
  • HER2 overexpressing NCI-N87 cells in combination with Tastuzumab (P-DM1: Tastuzumab 1:10) using a fixed ratio of P-DM1 also resulted in synergistic anti-cell proliferative activity, except for the highest (10 ⁇ g/ A combination of mL P-DM1 and 100 ⁇ g/mL Trastuzumab) or lowest (0.0256 ng/mL P-DM1 and 0.256 ng/mL Trastuzumab) concentrations.
  • Example 3 In vivo anti-tumor efficacy assay:
  • the efficacy of the combination of the invention can be measured in vivo by implanting an allograft or xenograft of cancer cells in a rodent and treating the tumor with the combination.
  • Test mice were treated with drugs or controls and monitored for weeks or longer to measure time to tumor doubling, log cell killing, and tumor suppression.
  • BALB/cA-nude nude mice 6-7 weeks old, purchased from Shanghai Slack Laboratory Animals LLC. Certificate No.: SCXK (Shanghai) 2012-0002. Feeding environment: SPF level.
  • the nude mice were subcutaneously inoculated with human breast cancer BT-474/T721 cells, and after the tumors were grown to 100-200 mm 3 , the animals were randomly grouped (D0).
  • the dosage and administration schedule are shown in Table 2.
  • the tumor volume was measured twice a week, the rats were weighed, and data were recorded.
  • the tumor volume (V) is calculated as:
  • V 1/2 ⁇ a ⁇ b2
  • a and b represent length and width, respectively.
  • T/C (%) (TT 0 ) / (CC 0 ) ⁇ 100, where T and C are the tumor volumes at the end of the experiment; T 0 and C 0 are the tumor volumes at the beginning of the experiment.
  • D0 first administration time
  • IV intravenous administration
  • Figure 9 shows the efficacy of Herceptin-resistant human breast cancer BT-474/T721 nude mice after administration as follows: (1) solvent control; (2) P-DM1 3 mg/kg; (3) P- DM1 10 mg/kg; (4) P-DM1 3 mg/kg + Trastuzumab 10 mg/kg.
  • the results showed that P-DM1 (3, 10 mg/kg, IV, once a week for 3 times) significantly inhibited the growth of subcutaneous xenografts in BT-474/T721 nude mice, with tumor inhibition rates of 72% and 97%, respectively.

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Abstract

Provided are antibody-drug conjugate Pertuzumab-MCC-DM1 (that is, P-DM1) and a treating agent composition comprising the P-DM1 and an HER2 dimerisation inhibitor antibody. Also provided are uses of the P-DM1 and the treating agent composition in treating highly proliferative diseases.

Description

抗体-药物偶联物Pertuzumab-MCC-DM1、其与Trastuzumab的组合物及其应用Antibody-drug conjugate Pertuzumab-MCC-DM1, its composition with Trastuzumab and application thereof 技术领域Technical field
本发明涉及抗体-药物偶联物以及用其治疗高增殖性病症的方法，具体涉及一种抗体-药物偶联物Pertuzumab-MCC-DM1(P-DM1)及其与治疗有效量的HER2二聚化抑制剂抗体Trastuzumab的组合物，以及使用所述抗体-药物偶联物或组合物治疗高增殖性病症的方法。The present invention relates to antibody-drug conjugates and methods for treating hyperproliferative disorders therewith, and in particular to an antibody-drug conjugate Pertuzumab-MCC-DM1 (P-DM1) and its dimerization with a therapeutically effective amount of HER2 A composition of the inhibitor antibody Trastuzumab, and a method of treating a hyperproliferative disorder using the antibody-drug conjugate or composition.
背景技术Background technique
HER2(ErbB2)是结合在细胞膜表面的受体酪氨酸激酶，由原癌基因HER2/neu编码。属于表皮生长因子受体家族，该家族包括HER1(erbB 1，EGFR)、HER2(erbB2，NEU)、HER3(erbB3)及HER4(erbB4)。HER2参与导致细胞生长和分化的信号传导途径。在大约25％-30％的人乳腺癌中观察到有HER2的过表达。研究显示当HER2蛋白质过表达时乳癌浸润性强，更具有侵袭性。而且HER2阳性肿瘤比不呈HER2阳性的肿瘤生长和蔓延得更快。据报道HER2阳性乳癌是非HER2阳性乳癌复发率的2.5倍。HER2 (ErbB2) is a receptor tyrosine kinase that binds to the surface of cell membranes and is encoded by the proto-oncogene HER2/neu. It belongs to the epidermal growth factor receptor family, which includes HER1 (erbB 1, EGFR), HER2 (erbB2, NEU), HER3 (erbB3), and HER4 (erbB4). HER2 is involved in signaling pathways that lead to cell growth and differentiation. Overexpression of HER2 was observed in approximately 25% to 30% of human breast cancer. Studies have shown that breast cancer is more invasive and more aggressive when HER2 protein is overexpressed. Moreover, HER2-positive tumors grow and spread faster than tumors that are not HER2-positive. HER2-positive breast cancer is reported to be 2.5 times more recurrent than non-HER2 positive breast cancer.
曲妥珠单抗(Trastuzumab)是一种针对Her-2/neu原癌基因产物的人源化单克隆抗体，其选择性地结合在HER2(或HER2/neu)受体的区域IV。曲妥珠单抗通过与HER2蛋白质结合抑制肿瘤细胞生长。1998年，曲妥珠单抗(商品名：赫赛汀，Herceptin)获得美国食品及药物管理局(Food and Drug Administration，FDA)批准上市，用于Her-2表达阳性的乳腺癌治疗。虽然Herceptin的开发给具有HER2阳性肿瘤的患者提供了比单独的化疗显著要好的结果，但另有将近50％的Her-2阳性患者对曲妥珠单抗治疗不敏感(Singer CF，Predicting the efficacy of Trastuzumab-based therapy in breast cancer：current standards and future strategies，Biochim Biophys Acta，2008，1786(2)：105-113)，并且大部分Her2阳性患者在经过1年的曲妥珠单抗治疗后，即会对其产生耐药性。因此，临床上仍需要为对Herceptin治疗不响应或响应不足，具有过表达HER2的肿瘤或其它与HER2表达有关的疾病的患者开发新的针对HER2的癌症疗法。Trastuzumab is a humanized monoclonal antibody directed against the Her-2/neu proto-oncogene product that selectively binds to region IV of the HER2 (or HER2/neu) receptor. Trastuzumab inhibits tumor cell growth by binding to the HER2 protein. In 1998, trastuzumab (trade name: Herceptin) was approved by the US Food and Drug Administration (FDA) for breast cancer treatment with positive Her-2 expression. Although the development of Herceptin provides significantly better results for patients with HER2-positive tumors than chemotherapy alone, nearly 50% of Her-2 positive patients are insensitive to trastuzumab treatment (Singer CF, Predicting the efficacy) Of Trastuzumab-based therapy in breast cancer: current standards and future strategies, Biochim Biophys Acta, 2008, 1786(2): 105-113), and most Her2-positive patients after one year of trastuzumab treatment, It will be resistant to it. Therefore, there is still a clinical need to develop new cancer therapies for HER2 for patients who are unresponsive or underreactive to Herceptin treatment, have tumors that overexpress HER2 or other diseases associated with HER2 expression.
抗体-药物偶联物(antibody-drug conjugate，ADC)利用单克隆抗体特异性识别肿瘤细胞上特定抗原的特点，偶联小分子化疗药物，可实现精准地将抗 肿瘤物质递送到肿瘤靶细胞释放。2013年3月，Roche公司的ADC药物T-DM1(曲妥珠单抗Trastuzumab与美登素DM1偶联)获得FDA的认可用于HER2阳性转移性乳腺癌治疗。T-DM1在过表达HER2的Trastuzumab敏感性和Trastuzumab抗性肿瘤细胞系，和人乳腺癌的异种移植物模型中均显示出有力的抗肿瘤活性。T-DM1的作用机理研究显示除了小分子DM1的作用外，还包括Trastuzumab本身的作用和ADCC作用(Trastuzumab Emtansine：A Unique Antibody-Drug Conjugate in Development for Human Epidermal Growth Factor Receptor 2-Positive Cancer，Clin Cancer Res，2011，17(20)，6437-6447)。帕妥珠单抗Pertuzumab亦是一个作用于HER二聚化的单克隆抗体。通过结合HER2，阻滞了HER2与其它HER受体的杂二聚，导致有丝分裂活化蛋白激酶和PI3K通路受阻，进而出现肿瘤细胞生长停滞和凋亡，从而减缓了肿瘤的生长。Pertuzumab与TTrastuzumab的区别在于轻链和重链的表位结合区，Pertuzumab结合在HER2的亚结构域2内的表位，而TTrastuzumab的表位则位于亚结构域4。Pertuzumab通过阻断HER2与其它HER家族成员，包括HER1(表皮生长因子受体；EGFR)，HER3，和HER4的结合来起作用的。这种结合是在存在配体下及经MAP激酶和PI3激酶发信号所需要的。因此，Pertuzumab可抑制配体启动的细胞内信号传导。抑制这些信号传导途径分别能导致生长阻滞和调亡。将T-DM1与Pertuzumab联用在细胞及动物模型上显示出较单个试剂更强的抗肿瘤活性(Dual targeting of HER2-positive cancer with Trastuzumab-emtansine(T-DM1)and pertuzumab：critical role for neuregulin blockade in anti-tumor response to combination therapy，Clin Cancer Res，Published Online First on October 4，2013)。但目前尚无关于将Pertuzumab与小分子化疗药物小分子化疗药物偶联，来治疗HER2介导的疾病的相关报道。Antibody-drug conjugate (ADC) uses monoclonal antibodies to specifically recognize the characteristics of specific antigens on tumor cells, and coupled with small molecule chemotherapy drugs, can achieve accurate anti-antibiotics Tumor material delivery to tumor target cell release. In March 2013, Roche's ADC drug T-DM1 (trastuzumab conjugated with maytansin DM1) was approved by the FDA for the treatment of HER2-positive metastatic breast cancer. T-DM1 showed potent anti-tumor activity in the Trastuzumab-sensitive and Trastuzumab-resistant tumor cell lines overexpressing HER2, and in human xenograft models of human breast cancer. The mechanism of action of T-DM1 showed that in addition to the action of small molecule DM1, it also includes the action of Trastuzumab itself and the role of ADCC (Trastuzumab Emtansine: A Unique Antibody-Drug Conjugate in Development for Human Epidermal Growth Factor Receptor 2-Positive Cancer, Clin Cancer Res, 2011, 17 (20), 6437-6447). Pertuzumab is also a monoclonal antibody that acts on HER dimerization. By binding to HER2, heterodimerization of HER2 with other HER receptors is blocked, leading to mitosis-activated protein kinase and PI3K pathways, resulting in tumor cell growth arrest and apoptosis, thereby slowing tumor growth. Pertuzumab differs from TTrastuzumab in the epitope binding region of the light and heavy chains, Pertuzumab binds to an epitope within subdomain 2 of HER2, and the epitope of TTrastuzumab is located at subdomain 4. Pertuzumab works by blocking the binding of HER2 to other HER family members, including HER1 (epidermal growth factor receptor; EGFR), HER3, and HER4. This binding is required in the presence of a ligand and signaling via MAP kinase and PI3 kinase. Thus, Pertuzumab inhibits ligand-initiated intracellular signaling. Inhibition of these signaling pathways can lead to growth arrest and apoptosis, respectively. The combination of T-DM1 and Pertuzumab showed stronger anti-tumor activity than single agents in cell and animal models (Dual targeting of HER2-positive cancer with Trastuzumab-emtansine (T-DM1) and pertuzumab: critical role for neuregulin blockade In anti-tumor response to combination therapy, Clin Cancer Res, Published Online First on October 4, 2013). However, there are no reports on the coupling of Pertuzumab with small molecule chemotherapy drugs for small molecule chemotherapy to treat HER2-mediated diseases.
发明内容Summary of the invention
为了克服上述技术问题，本发明的目的在于提供一种抗体-药物偶联物Pertuzumab-MCC-DM1(P-DM1)及其与治疗有效量的HER2二聚化抑制剂抗体Trastuzumab的组合物。In order to overcome the above technical problems, it is an object of the present invention to provide an antibody-drug conjugate Pertuzumab-MCC-DM1 (P-DM1) and a composition thereof in combination with a therapeutically effective amount of the HER2 dimerization inhibitor antibody Trastuzumab.
为了达到上述发明目的，本发明采用如下技术方案：In order to achieve the above object, the present invention adopts the following technical solutions:
本发明提供一种具有如下结构的抗体-药物偶联物Pertuzumab-MCC-DM1(P-DM1)： The present invention provides an antibody-drug conjugate Pertuzumab-MCC-DM1 (P-DM1) having the following structure:
Figure PCTCN2015073629-appb-000001
Figure PCTCN2015073629-appb-000001
其中，mAb指帕妥珠单抗Pertuzumab，Pertuzumab经双功能连接体试剂SMCC与美登素生物碱DM1连接，上式中n表示药物-抗体比，且其范围优选为1至8之间的整数值，其中，1、2、3、4、5、6、7或8个药物部分共价结合在抗体Pertuzumab上。Wherein, mAb refers to pertuzumab, Pertuzumab is linked to maytansinoid DM1 via a bifunctional linker reagent SMCC, wherein n represents a drug-antibody ratio, and the range thereof is preferably an integer between 1 and 8. Value, wherein 1, 2, 3, 4, 5, 6, 7, or 8 drug moieties are covalently bound to the antibody Pertuzumab.
本发明还提供一种用于治疗高增殖性疾病或病症的治疗剂组合物，其中，所述治疗剂组合物包含治疗有效量的P-DM1和治疗有效量的HER2二聚化抑制剂抗体。The invention also provides a therapeutic composition for treating a hyperproliferative disease or condition, wherein the therapeutic composition comprises a therapeutically effective amount of P-DMl and a therapeutically effective amount of a HER2 dimerization inhibitor antibody.
进一步地，所述HER2二聚化抑制剂抗体优选为曲妥珠单抗(Trastuzumab)；Further, the HER2 dimerization inhibitor antibody is preferably trastuzumab;
进一步地，所述治疗有效量的P-DM1和所述治疗有效量的HER2二聚化抑制剂抗体可以组合成配制剂的形式施用或交替地施用。Further, the therapeutically effective amount of P-DMl and the therapeutically effective amount of a HER2 dimerization inhibitor antibody can be administered in combination as a formulation or alternately.
本发明还提供使用所述治疗剂组合物体外和/或原位治疗哺乳动物细胞、生物体高增殖性疾病或病症的方法。本发明的另一方面还提供一种使用本发明的治疗剂组合物来治疗哺乳动物或人高增殖性疾病或病症(诸如癌症，包括受HER2或KDR9(VEGF受体1)调控的疾病)的方法。The invention also provides methods of using the therapeutic agent combination to treat an extracellular and/or in situ treatment of a mammalian cell, an organism's hyperproliferative disease or condition. Another aspect of the invention also provides a method of using the therapeutic composition of the invention to treat a hyperproliferative disease or condition in a mammal or a human, such as a cancer, including a disease modulated by HER2 or KDR9 (VEGF receptor 1) method.
本发明还提供一种治疗高增殖性疾病或病症的方法，包括对需要此类治疗的哺乳动物或人施用治疗有效量的P-DM1和HER2二聚化抑制剂抗体剂。The invention also provides a method of treating a hyperproliferative disease or condition comprising administering to a mammal or human in need of such treatment a therapeutically effective amount of a P-DM1 and HER2 dimerization inhibitor antibody agent.
进一步地，所述P-DM1和HER2二聚化抑制剂抗体剂是以组合配制剂形式施用的；Further, the P-DM1 and HER2 dimerization inhibitor antibody agents are administered in the form of a combined formulation;
进一步地，所述P-DM1和HER2二聚化抑制剂抗体剂作为治疗剂组合交替地(交替，序贯给药)分开施用；更进一步地，所述P-DM1和HER2二聚化抑制剂抗体剂是以约3周间隔或每周1次间隔对具有高增殖性病症的病人施用的；Further, the P-DM1 and HER2 dimerization inhibitor antibody agents are administered separately (alternately, sequentially) as a combination of therapeutic agents; further, the P-DM1 and HER2 dimerization inhibitors The antibody agent is administered to a patient having a hyperproliferative disorder at intervals of about 3 weeks or once a week;
进一步地，所述哺乳动物是HER2阳性患者；更进一步地，所述HER2阳性患者已经接受Trastuzumab和其它化疗试剂疗法。Further, the mammal is a HER2-positive patient; further, the HER2-positive patient has received Trastuzumab and other chemotherapeutic agents.
本发明的另一个方面还提供本发明所述的抗体-药物偶联物P-DM1和治疗剂组合物在制备用于治疗哺乳动物或人高增殖性疾病或病症(诸如癌症，包括 受HER2调控)的药物中的用途。Another aspect of the invention provides the antibody-drug conjugate P-DM1 and therapeutic composition of the invention for use in the manufacture of a hyperproliferative disease or disorder, such as cancer, for use in treating a mammal or a human Use in drugs regulated by HER2).
其中，本发明所述高增殖性病症是癌症；进一步地，所述高增殖性病症是表达HER2的癌症；更进一步地，所述癌症是乳腺癌、胃癌、卵巢癌、宫颈癌、前列腺癌、睾丸癌、生殖泌尿道癌、非小细胞肺癌、结肠癌、骨癌、胰腺癌、结肠-直肠癌、肝癌和胆管癌、皮肤癌、食管癌、喉癌、成胶质细胞瘤、成神经细胞瘤、腺癌、口腔癌、甲状腺癌、肺癌、表皮样癌、大细胞癌、小细胞癌、肾癌、小肠癌、大肠癌、直肠癌、鼻咽癌、膀胱癌、黑素瘤、唇癌、毛细胞癌、滤泡性癌、脑和中枢神经系统癌、霍奇金淋巴癌或白血病，优选为乳腺癌。Wherein the hyperproliferative disorder of the present invention is cancer; further, the hyperproliferative disorder is a cancer expressing HER2; further, the cancer is breast cancer, gastric cancer, ovarian cancer, cervical cancer, prostate cancer, Testicular cancer, genitourinary tract cancer, non-small cell lung cancer, colon cancer, bone cancer, pancreatic cancer, colon-rectal cancer, liver cancer and cholangiocarcinoma, skin cancer, esophageal cancer, laryngeal cancer, glioblastoma, neuroblast Tumor, adenocarcinoma, oral cancer, thyroid cancer, lung cancer, epidermoid carcinoma, large cell carcinoma, small cell carcinoma, kidney cancer, small intestine cancer, colon cancer, rectal cancer, nasopharyngeal cancer, bladder cancer, melanoma, lip cancer , hair cell cancer, follicular carcinoma, brain and central nervous system cancer, Hodgkin's lymphoma or leukemia, preferably breast cancer.
本发明的另一个方面还提供通过定性和定量分析来自体外细胞增殖和体内肿瘤异种移植物实验的功效数据为体内功效预测有效药物组合的方法，其中所述组合包括P-DM1和HER2二聚化抑制剂抗体。Another aspect of the invention also provides a method for predicting an effective pharmaceutical combination for in vivo efficacy by qualitative and quantitative analysis of efficacy data from in vitro cell proliferation and in vivo tumor xenograft experiments, wherein said combination comprises P-DM1 and HER2 dimerization Inhibitor antibody.
本发明提供一种用于为癌症治疗确定要组合使用的化合物的方法，包括：a)用Pertuzumab-MCC-DM1和HER2二聚化抑制剂抗体的治疗剂组合处理体外肿瘤细胞系，并b)测量协同或非协同效应，由此为癌症治疗确定协同治疗剂组合。The present invention provides a method for determining a compound to be used in combination for cancer treatment, comprising: a) treating a tumor cell line in vitro with a combination of therapeutic agents of Pertuzumab-MCC-DM1 and a HER2 dimerization inhibitor antibody, and b) Synergistic or non-synergistic effects are measured whereby a synergistic combination of therapeutic agents is determined for cancer treatment.
进一步地，本发明还提供一种用于为癌症治疗确定要组合使用的化合物的方法，包括：(a)对HER2扩增的乳腺癌细胞或胃癌细胞施用Pertuzumab-MCC-DM1和HER2二聚化抑制剂抗体的治疗剂组合，和(b)测量对细胞增殖的抑制，由此为癌症治疗确定协同治疗剂组合。其中，以固定浓度比例的P-DM1和Trastuzumab组合测定组合指数(CI)，或以固定浓度的P-DM1联合各种浓度的Trastuzumab组合测定组合指数(CI)。CI值小于0.9指示协同性；CI值介于0.9-1.1之间指示加和性；而CI值大于1.1指示拮抗性。Further, the present invention provides a method for determining a compound to be used in combination for cancer treatment, comprising: (a) administering Pertuzumab-MCC-DM1 and HER2 dimerization to HER2-amplified breast cancer cells or gastric cancer cells. A combination of therapeutic agents of the inhibitor antibody, and (b) measuring inhibition of cell proliferation, thereby determining a synergistic therapeutic combination for cancer treatment. Among them, the combination index (CI) was determined by a combination of P-DM1 and Trastuzumab in a fixed concentration ratio, or the combination index (CI) was determined by a combination of a fixed concentration of P-DM1 and various concentrations of Trastuzumab. A CI value of less than 0.9 indicates synergy; a CI value between 0.9 and 1.1 indicates additive; and a CI value greater than 1.1 indicates antagonistic.
其中，所述HER2扩增的乳腺癌细胞是BT-474、SK-BR-3；所述HER2扩增的胃癌细胞是NCI-N87。Wherein, the HER2-amplified breast cancer cells are BT-474, SK-BR-3; and the HER2-amplified gastric cancer cells are NCI-N87.
本发明的抗体-药物偶联物(P-DM1可以通过如下方法来制备：The antibody-drug conjugate of the present invention (P-DM1 can be prepared by the following method:
1)抗体的缓冲液交换1) Buffer exchange of antibodies
利用脱盐层析(SephadexTM G25脱盐柱)方式置换帕妥珠单抗原液至反应缓冲液(磷酸钾盐/NaCl/EDTA)中，并浓缩抗体，制备帕妥珠单抗抗体；The pertituzate monoclonal antibody was replaced by a desalting chromatography (SephadexTM G25 desalting column) into a reaction buffer (potassium phosphate/NaCl/EDTA), and the antibody was concentrated to prepare a pertuzumab antibody;
2)MCC-DM1母液的制备2) Preparation of MCC-DM1 mother liquor
称取MCC-DM1，用二甲基乙酰胺(DMA)充分溶解制备母液； Weigh MCC-DM1 and prepare the mother liquor by fully dissolving with dimethylacetamide (DMA);
3)偶联反应3) Coupling reaction
在步骤(1)所制备的帕妥珠单抗中加入步骤(2)所配制的MCC-DM1母液进行缀合反应。最初用若干过量的MCC-DM1滴定抗体以测定希望的DM1∶mAb比例。然后加入8-10倍过量摩尔比的SMCC-DM1母液进行反应；The MCC-DM1 mother liquid prepared in the step (2) is added to the pertuzumab prepared in the step (1) to carry out a conjugation reaction. The antibody was initially titrated with a number of excess MCC-DM1 to determine the desired DM1:mAb ratio. Then adding 8-10 times excess molar ratio of SMCC-DM1 mother liquor for reaction;
4)纯化4) Purification
采用凝胶过滤柱(Superdex 200)将偶联反应混合物用琥珀酸钠/NaCl缓冲液凝胶过滤纯化，根据UV280紫外吸收值收集出峰样品，或超滤数遍。The coupling reaction mixture was purified by gel filtration using a sodium succinate/NaCl buffer using a gel filtration column (Superdex 200), and a peak sample was collected according to UV280 ultraviolet absorption value, or ultrafiltered several times.
在本发明中，作出如下定义：In the present invention, the following definitions are made:
术语“治疗”或“处理”指治疗性处理及预防性或防范性措施二者，其中目标是预防或减缓(减轻)不想要的生理学变化或病症，诸如高增殖性状况诸如癌症的生长，形成或传播。为了本发明，有利或期望的临床结果包括但不限于：缓解症状，削弱疾病的程度，疾病状态稳定(即不恶化)，延迟或减缓疾病发展，改善或减轻疾病状态，及康复(无论是部分的还是完全的)，无论是可检测的还是不可检测的。“治疗”或“处理”还可以指与不接受治疗的预期存活相比延长存活。需要治疗的受试者包括早就患有疾病或病症的受试者以及倾向于患上疾病或病症的受试者或者要预防状况或病症的受试者。The term "treating" or "treating" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the goal is to prevent or slow down (mitigate) unwanted physiological changes or conditions, such as the growth of high proliferative conditions such as cancer, formation. Or spread. For the purposes of the present invention, advantageous or desirable clinical outcomes include, but are not limited to, alleviating symptoms, attenuating the extent of the disease, stabilizing the disease state (ie, not worsening), delaying or slowing the progression of the disease, improving or reducing the disease state, and rehabilitation (whether part Still complete), whether detectable or undetectable. "Treatment" or "treatment" can also mean prolonging survival as compared to expected survival without treatment. Subjects in need of treatment include subjects who have long had a disease or condition as well as subjects who are predisposed to having the disease or condition or who are to be prevented from developing a condition or condition.
术语“治疗有效量”指本发明化合物的量，其(i)治疗本文所述特定疾病，状况，或病症，(ii)减轻，改善，或消除特定疾病，状况，或病症的一种或多种症状，或(iii)预防或延迟特定疾病，状况，或病症的一种或多种症状的发作。在癌症的情况中，药物的治疗有效量可减少癌细胞数；缩小肿瘤体积；抑制(即一定程度的减缓，优选停止)癌细胞浸润到周围器官中；抑制(即一定程度的减缓，优选停止)肿瘤转移；一定程度的抑制肿瘤生长；和/或一定程度的减轻与癌症有关的一种或多种症状。就药物可抑制癌细胞生长和/或杀死现有癌细胞的程度而言，它可以是抑制细胞的和/或毒害细胞的。对于癌症疗法，可以例如通过评价距疾病发展的时间(TTP)和/或测定响应率(RR)来测量功效。The term "therapeutically effective amount" refers to an amount of a compound of the invention that (i) treats a particular disease, condition, or condition described herein, (ii) alleviates, ameliorates, or eliminates one or more of a particular disease, condition, or condition. a symptom, or (iii) preventing or delaying the onset of one or more symptoms of a particular disease, condition, or condition. In the case of cancer, a therapeutically effective amount of the drug can reduce the number of cancer cells; reduce the volume of the tumor; inhibit (ie, slow down, preferably stop) the infiltration of the cancer cells into the surrounding organs; inhibition (ie, a certain degree of slowing, preferably stop) a tumor metastasis; a degree of inhibition of tumor growth; and/or a degree of alleviation of one or more symptoms associated with cancer. To the extent that the drug inhibits cancer cell growth and/or kills existing cancer cells, it can be cytostatic and/or cytotoxic. For cancer therapy, efficacy can be measured, for example, by evaluating time to disease progression (TTP) and/or assay response rate (RR).
“高增殖性病症”指肿瘤，癌症，和新生物组织，包括恶变前和非新生物阶段，而且还包括银屑病，子宫内膜异位症，息肉和纤维腺瘤。"High proliferative disorders" refer to tumors, cancer, and neoplastic tissues, including pre-malignant and non-neoplastic stages, but also include psoriasis, endometriosis, polyps, and fibroadenomas.
术语“癌症”和“癌性”指向或描述哺乳动物中特征通常为细胞生长不受调节的生理状况。“肿瘤”包括一个或多个癌性细胞。癌症的例子包括但不限于癌，淋巴瘤，母细胞瘤，肉瘤和白血病或淋巴样恶性肿瘤。此类癌症的更 具体例子包括鳞状细胞癌(例如上皮鳞状细胞癌)，肺癌(包括小细胞肺癌，非小细胞肺癌，肺的腺癌，和肺的鳞癌)，腹膜癌，肝细胞癌，胃癌(包括胃肠癌)，膜腺癌，成胶质细胞瘤，宫颈癌，卵巢癌，肝癌，膀肮癌，肝瘤，乳腺癌，结肠癌，直肠癌，结肠直肠癌，子宫内膜癌或子宫癌，唾液腺癌，肾癌，前列腺癌，外阴癌，甲状腺癌，肝癌，肛门癌，阴茎癌，以及头颈癌。The terms "cancer" and "cancerous" refer to or describe a physiological condition in a mammal that is typically characterized by unregulated cell growth. A "tumor" includes one or more cancerous cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma and leukemia or lymphoid malignancies. More of this type of cancer Specific examples include squamous cell carcinoma (eg, epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, and gastric cancer (including Gastrointestinal cancer), membrane adenocarcinoma, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver tumor, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer , salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, and head and neck cancer.
术语“药学可接受的”指明该物质或组合物必须是在化学和/或毒理学方面与构成配制剂的其它成分和/或用它治疗的哺乳动物相容的。The term "pharmaceutically acceptable" indicates that the substance or composition must be chemically and/or toxicologically compatible with the other ingredients that make up the formulation and/or the mammal to which it is treated.
本文中所使用的术语“协同的”指比两种或更多种单一药剂的加和效果更有效的治疗剂组合。P-DM1和HER2二聚化抑制剂的抗体之间的协同相互作用的确定可以基于自本文所述测定法获得的结果。The term "synergistic" as used herein refers to a combination of therapeutic agents that are more effective than the additive effect of two or more single agents. The determination of the synergistic interaction between the antibodies of the P-DM1 and HER2 dimerization inhibitors can be based on the results obtained from the assays described herein.
组合疗法可提供“协同性”和证明是“协同的”，即一起使用活性成分时所实现的效果大于分开使用所致效果之和。当活性成分如下所述时可获得协同效应：(1)以组合的单位剂量配制剂共配制和同时施用或递送；(2)作为分开的配制剂交替递送；或(3)提供一些其它方案。当在交替疗法中递送时，当化合物序贯施用或边送时(例如通过分开的注射器中的不同注射)可获得协同效应。一般而言，在交替疗法期间，序贯地(即时间上连续地)施用有效剂量的每种活性成分。Combination therapies provide "synergistic" and prove to be "synergistic", ie, the effect achieved when the active ingredients are used together is greater than the sum of the effects resulting from the separate use. Synergistic effects can be obtained when the active ingredients are as follows: (1) co-formulation and simultaneous administration or delivery in a combined unit dose formulation; (2) alternating delivery as separate formulations; or (3) providing some other regimen. When delivered in alternation therapy, a synergistic effect can be obtained when the compounds are administered sequentially or while being administered (eg, by different injections in separate syringes). In general, an effective amount of each active ingredient is administered sequentially (i.e., continuously in time) during alternation therapy.
有益效果：Beneficial effects:
在本发明的中，将Pertuzumab与小分子化疗药物偶联，P-DM1和HER2二聚化抑制剂抗体的组合物在体外和在体内在抑制癌细胞生长中显示协同效应，可以达到对HER2介导的病症如癌症的较好治疗效果。本发明所述的抗体-药物偶联物P-DM1及其与抗体曲妥珠单抗的组合显著地提高了治疗效果，提供了治疗基于HER2介导的病症如癌症更有效的方法。In the present invention, a combination of Pertuzumab and a small molecule chemotherapeutic drug, a combination of a P-DM1 and a HER2 dimerization inhibitor antibody exhibits a synergistic effect in inhibiting the growth of cancer cells in vitro and in vivo, and can achieve a HER2-mediated A better therapeutic effect of a disease such as cancer. The antibody-drug conjugate P-DM1 of the present invention and its combination with the antibody trastuzumab significantly enhance the therapeutic effect, providing a more effective method for treating a HER2-mediated condition such as cancer.
本发明的另外的优点和新颖的特征部分在下面的说明中列出，部分在检查下面的说明书后对于本领域技术人员是显而易见的或可以通过本发明的实践而学会。凭借所附权利要求中特别指出的手段/工具、组合、组合物和方法可以认识和获得本发明的优点。Additional advantages and novel features of the invention are set forth in the description which follows. The advantages of the invention will be realized and attained by the <RTIgt;
附图说明DRAWINGS
图1显示单独各种浓度的Pertuzumab-MCC-DM1(P-DM1)、Trastuzumab以及固定浓度比例(1∶1)的P-DM1和Trastuzumab组合处理5天后对BT-474细胞体外细胞增殖抑制作用。 Figure 1 shows the inhibition of cell proliferation in vitro by BT-474 cells in vitro after treatment with various concentrations of Pertuzumab-MCC-DM1 (P-DM1), Trastuzumab, and fixed concentration ratio (1:1) of P-DM1 and Trastuzumab alone.
图2显示各种固定浓度的Pertuzumab-MCC-DM1(P-DM1)联合Trastuzumab，和单独的各种浓度的Trastuzumab处理5天后对BT-474细胞体外细胞增殖抑制作用。Figure 2 shows the inhibition of cell proliferation of BT-474 cells in vitro by various fixed concentrations of Pertuzumab-MCC-DM1 (P-DM1) in combination with Trastuzumab, and various concentrations of Trastuzumab alone for 5 days.
图3显示单独各种浓度的Pertuzumab-MCC-DM1(P-DM1)、Trastuzumab以及固定浓度比例(1∶10)的P-DM1和Trastuzumab组合处理5天后对NCI-N87细胞体外细胞增殖抑制作用。Figure 3 shows the inhibition of cell proliferation in vitro by NCI-N87 cells in vitro after treatment with various concentrations of Pertuzumab-MCC-DM1 (P-DM1), Trastuzumab, and fixed concentration ratio (1:10) of P-DM1 and Trastuzumab alone.
图4显示各种固定浓度的Pertuzumab-MCC-DM1(P-DM1)联合Trastuzumab，和单独的各种浓度的Trastuzumab处理5天后对NCI-N87细胞体外细胞增殖抑制作用。Figure 4 shows the inhibition of cell proliferation of NCI-N87 cells in vitro by various fixed concentrations of Pertuzumab-MCC-DM1 (P-DM1) in combination with Trastuzumab, and treatment with various concentrations of Trastuzumab alone for 5 days.
图5显示单独各种浓度的Pertuzumab-MCC-DM1(P-DM1)、Trastuzumab以及各种固定浓度的P-DM1联合Trastuzumab组合处理5天后对SK-BR-3细胞体外细胞增殖抑制作用。Figure 5 shows the inhibition of cell proliferation in vitro by SK-BR-3 cells in vitro after treatment with various concentrations of Pertuzumab-MCC-DM1 (P-DM1), Trastuzumab, and various fixed concentrations of P-DM1 in combination with Trastuzumab.
图6(A)显示单独各种浓度的Pertuzumab-MCC-DM1(P-DM1)处理5天后对Calcu-3细胞体外细胞增殖抑制作用，(B)显示各种固定浓度的P-DMl联合Trastuzumab，和单独的各种浓度的Trastuzumab(+溶媒)处理5天后对Calu-3细胞体外细胞增殖抑制作用。Figure 6 (A) shows the inhibition of cell proliferation in vitro by Calcu-3 cells after 5 days of treatment with various concentrations of Pertuzumab-MCC-DM1 (P-DM1), and (B) shows various fixed concentrations of P-DM1 in combination with Trastuzumab. Inhibition of cell proliferation in vitro by Calu-3 cells after 5 days of treatment with various concentrations of Trastuzumab (+ vehicle) alone.
图7(A)显示单独各种浓度的Pertuzumab-MCC-DM1(P-DM1)处理3天后对DU-145细胞体外细胞增殖抑制作用，(B)显示各种固定浓度的P-DM1联合Trastuzumab，和单独的各种浓度的Trastuzumab(+溶媒)处理3天后对DU-145细胞体外细胞增殖抑制作用。Figure 7 (A) shows the inhibition of cell proliferation in vitro by DU-145 cells treated with various concentrations of Pertuzumab-MCC-DM1 (P-DM1) for 3 days, and (B) shows various fixed concentrations of P-DM1 in combination with Trastuzumab. In vitro cell proliferation inhibition of DU-145 cells after 3 days of treatment with various concentrations of Trastuzumab (+ vehicle).
图8(A)显示单独各种浓度的Pertuzumab-MCC-DM1(P-DM1)处理3天后对SKOV-3细胞体外细胞增殖抑制作用，(B)显示各种固定浓度的P-DM1联合Trastuzumab，和单独的各种浓度的Trastuzumab(+溶媒)处理3天后对SKOV-3细胞体外细胞增殖抑制作用。Fig. 8(A) shows the inhibition of cell proliferation of SKOV-3 cells in vitro after treatment with various concentrations of Pertuzumab-MCC-DM1 (P-DM1) for 3 days, and (B) shows various fixed concentrations of P-DM1 in combination with Trastuzumab. In vitro cell proliferation inhibition of SKOV-3 cells after 3 days of treatment with various concentrations of Trastuzumab (+ vehicle) alone.
图9显示如下给药后对Herceptin-抗药性人乳腺癌BT-474/T721裸小鼠移植瘤的肿瘤体积均值随时间变化的图：(1)溶剂对照；(2)P-DM1 3mg/kg；(3)P-DM1 10mg/kg；(4)P-DM1 3mg/kg+Trastuzumab 10mg/kg。Figure 9 is a graph showing the change in mean tumor volume over time in Herceptin-resistant human breast cancer BT-474/T721 nude mice xenografts as follows: (1) solvent control; (2) P-DM1 3 mg/kg (3) P-DM1 10 mg/kg; (4) P-DM1 3 mg/kg + Trastuzumab 10 mg/kg.
具体实施方式detailed description
在此详细地述及本发明的某些实施方案，所附结构和公式中例示了它们的例子。虽然将会连同所列举的实施方案来描述本发明，应当理解它们并非意图将本发明限于那些实施方案。相反，本发明旨在涵盖所有备选方案、修改和等 同方案，其可以包括在权利要求所限定的本发明范围内。本领域技术人员会领会与本文中所描述的方法和材料相似的或等同的许多方法和材料可用于实施本发明。本发明绝非限于所描述的方法和材料。倘若所收入的文献、专利和相似的材料与本申请不同或矛盾，包括但不限于所定义的术语，术语的用法，所描述的技术，诸如此类，以本申请为准。Certain embodiments of the invention are described in detail herein, examples of which are illustrated in the accompanying structures and formulas. While the invention will be described in conjunction with the illustrated embodiments, it is understood that the invention is not intended to limit the invention. Instead, the present invention is intended to cover all alternatives, modifications, and the like. The same is intended to be included within the scope of the invention as defined by the appended claims. Those skilled in the art will appreciate that many methods and materials similar or equivalent to those described herein can be used in the practice of the invention. The invention is in no way limited to the methods and materials described. In the event that the documents, patents, and similar materials that are included are different or contradictory to the present application, including but not limited to the terms defined, the usage of the terms, the techniques described, and the like, the present application controls.
实施例Example
为了例示本发明，包括下列实施例。然而，应当理解，这些实施例不限制本发明，而只是意图提示实施本发明的方法。In order to illustrate the invention, the following examples are included. However, it should be understood that these examples do not limit the invention, but are merely intended to suggest a method of practicing the invention.
实施例1：Pertuzumab-MCC-DM1(P-DM1)的制备Example 1: Preparation of Pertuzumab-MCC-DM1 (P-DM1)
制备步骤如下：The preparation steps are as follows:
1)抗体的缓冲液交换1) Buffer exchange of antibodies
利用脱盐层析(SephadexTM G25脱盐柱)方式置换帕妥珠单抗原液至反应缓冲液(50mM磷酸钾盐/50mM NaCl/2mM EDTA，pH7.5)中，并浓缩抗体浓度至5mg/ml，制备帕妥珠单抗抗体；The desalted single antigen solution was replaced by a desalting chromatography (SephadexTM G25 desalting column) into a reaction buffer (50 mM potassium phosphate/50 mM NaCl/2 mM EDTA, pH 7.5), and the antibody concentration was concentrated to 5 mg/ml to prepare. Pertuzumab antibody;
2)MCC-DM1母液的制备2) Preparation of MCC-DM1 mother liquor
称取MCC-DM1，用二甲基乙酰胺(DMA)充分溶解制备10mg/ml的MCC-DM1母液；The MCC-DM1 was weighed and fully dissolved with dimethylacetamide (DMA) to prepare a 10 mg/ml MCC-DM1 mother liquor;
3)偶联反应3) Coupling reaction
在步骤(1)所制备的帕妥珠单抗中加入步骤(2)所配置的MCC-DM1母液进行缀合反应。最初用若干过量的MCC-DM1滴定抗体以测定希望的DM1∶mAb比例，对于人抗体通常这个范围是6-10倍摩尔过量。反应中DMA在5％v/v以内。反应温度为25℃，反应时间为1.5至约20小时。反应分为三组进行：The MCC-DM1 mother liquid configured in the step (2) is added to the pertuzumab prepared in the step (1) to carry out a conjugation reaction. The antibody was initially titrated with a number of excess MCC-DM1 to determine the desired DM1:mAb ratio, which is typically a 6-10 fold molar excess for human antibodies. The DMA in the reaction is within 5% v/v. The reaction temperature is 25 ° C and the reaction time is from 1.5 to about 20 hours. The reactions were divided into three groups:
a)反应中加入8倍过量摩尔比的SMCC-DM1母液；a) adding 8 times excess molar ratio of SMCC-DM1 mother liquor to the reaction;
b)反应中加入9倍过量摩尔比的SMCC-DM1母液；b) adding a 9-fold excess molar ratio of the SMCC-DM1 mother liquor to the reaction;
c)反应中加入10倍过量摩尔比的SMCC-DM1母液；c) adding a 10-fold excess molar ratio of SMCC-DM1 mother liquor to the reaction;
4)纯化4) Purification
采用凝胶过滤柱(Superdex 200)将偶联反应混合物用pH 5.0的琥珀酸钠/150mM NaCl缓冲液凝胶过滤纯化，根据UV280紫外吸收值收集出峰样品，或超滤数遍。通过测定缀合物在252和280nm的吸光度并使用DM1和抗体在这两个波长的已知消光系数来测量最终缀合物中的每个Ab抗体分子的 DM1分子的数目(计算公式如式1)。同时使用质谱分析法计算产品的偶联率，并进行对比。数据见表1。The coupling reaction mixture was gel-filtered using a gel filtration column (Superdex 200) with a sodium succinate/150 mM NaCl buffer of pH 5.0, and a peak sample was collected according to the UV280 ultraviolet absorption value, or ultrafiltered several times. The Ab antibody molecule in the final conjugate is measured by measuring the absorbance of the conjugate at 252 and 280 nm and using the known extinction coefficient of DM1 and antibody at these two wavelengths. The number of DM1 molecules (calculated as Equation 1). At the same time, the coupling rate of the product was calculated by mass spectrometry and compared. The data is shown in Table 1.
式1 Formula 1
Figure PCTCN2015073629-appb-000002
Figure PCTCN2015073629-appb-000002
表1投料比对偶联率的影响Table 1 Effect of feed ratio on coupling rate
Figure PCTCN2015073629-appb-000003
Figure PCTCN2015073629-appb-000003
本实验中偶联率的测定方法：测定偶联率是指测定每个单抗分子上连接有小分子药物的数量，即偶联物中药物与抗体的摩尔比。可采用紫外分光光度法和质谱分析法两种方法测定。紫外分光光度法：通过测定偶联物在252nM和280nM处的紫外吸收值，并通过朗伯比尔定律进行计算得出偶联率(DAR)；质谱分析法：对抗体-美登素生物碱偶联物进行ESI-MS分析，可以获得连接不用个数药物的峰型图，根据各个峰的面积进行计算得出偶联率。通过两种方法获得的偶联率具有很好的一致性。The method for determining the coupling rate in this experiment: determining the coupling ratio means measuring the amount of a small molecule drug attached to each monoclonal antibody molecule, that is, the molar ratio of the drug to the antibody in the conjugate. It can be determined by two methods: ultraviolet spectrophotometry and mass spectrometry. Ultraviolet spectrophotometry: Determination of the coupling value (DAR) by measuring the UV absorbance of the conjugate at 252 nM and 280 nM, and calculating by Lamberbeer's law; mass spectrometry: for the antibody-maytansinoid The ESI-MS analysis of the conjugates allows the peak pattern of the connected drug to be obtained, and the coupling ratio is calculated based on the area of each peak. The coupling rates obtained by the two methods are very consistent.
本实验中偶联物浓度测定：采用紫外分光光度法，测定在252nM和280nM处的紫外吸收值，通过朗伯比尔定律计算抗体和药物的浓度；本发明中所述的偶联物浓度是单抗浓度与连接到单抗上的美登素生物碱(DM1)药物的总和。Conjugate concentration determination in this experiment: UV absorption values were determined by UV spectrophotometry at 252 nM and 280 nM, and antibody and drug concentrations were calculated by Lambert Beer's law; the concentration of the conjugate described in the present invention is single The sum of the anti-concentration and the maytansinoid (DM1) drug attached to the mAb.
实施例2：体外细胞增殖测定生物活性实验Example 2: In vitro cell proliferation assay Bioactivity assay
以下实验中所用的实验材料来源于：DMEM培养基、F12K培养基、RPMI1640培养基、0.25％胰蛋白酶-EDTA、胎牛血清、100×丙酮酸钠、100×青链霉素购自Gibco公司。磺酰罗丹明B(Sulforhodamine B，SRB)购自Sigma公司。BT-474乳腺癌细胞和NCI-N87胃癌细胞来自中国科学院昆明细胞库，SK-BR-3乳腺癌细胞来自上海博谷生物科技有限公司，Calu-3肺癌细胞来自中国科学院上海生命科学研究院细胞库，SKOV-3卵巢癌细胞和DU-145前列腺癌细胞来自美国典型培养物保藏中心(ATCC)。其他试剂均是分析纯。96孔平底聚苯乙烯(Corning，产品目录号3599)。Synergy 2酶标仪(Bio-Tek)。The experimental materials used in the following experiments were derived from: DMEM medium, F12K medium, RPMI 1640 medium, 0.25% trypsin-EDTA, fetal bovine serum, 100 x sodium pyruvate, 100 x streptomycin, available from Gibco. Sulforhodamine B (SRB) was purchased from Sigma. BT-474 breast cancer cells and NCI-N87 gastric cancer cells are from Kunming Cell Bank of Chinese Academy of Sciences, SK-BR-3 breast cancer cells are from Shanghai Bogu Biotechnology Co., Ltd., Calu-3 lung cancer cells are from Shanghai Institute of Life Sciences, Chinese Academy of Sciences. The library, SKOV-3 ovarian cancer cells and DU-145 prostate cancer cells were obtained from the American Type Culture Collection (ATCC). All other reagents were of analytical grade. 96-well flat-bottom polystyrene (Corning, Cat. No. 3599). Synergy 2 microplate reader (Bio-Tek).
在本实施例中，研究了将Pertuzumab-MCC-DM1(P-DM1)单独或与 Trastuzumab组合对肿瘤细胞系增殖的作用。In this example, the study of Pertuzumab-MCC-DM1 (P-DM1) alone or with Effect of Trastuzumab combination on proliferation of tumor cell lines.
本实施例使用磺酰罗丹明B(SRB)比色法来评价药物组合的抗增殖作用。SRB是一种粉红色阴离子染料，易溶于水，在酸性条件下可特异性地与细胞内组成蛋白质的碱性氨基酸结合；在510nm波长下产生吸收峰，吸光值与细胞量成线性正相关，故可用作细胞数的定量检测。This example uses the sulforhodamine B (SRB) colorimetric method to evaluate the anti-proliferative effect of the drug combination. SRB is a pink anionic dye which is easily soluble in water and can specifically bind to basic amino acids of proteins in cells under acidic conditions. It produces an absorption peak at a wavelength of 510 nm. The absorbance is linearly positively correlated with the amount of cells. Therefore, it can be used for quantitative detection of the number of cells.
本实施例选择的细胞系有：BT-474、SK-BR-3乳腺癌细胞、NCI-N87胃癌细胞、Calu-3肺癌细胞、SKOV-3卵巢癌细胞和DU-145前列腺癌细胞。The cell lines selected in this example were: BT-474, SK-BR-3 breast cancer cells, NCI-N87 gastric cancer cells, Calu-3 lung cancer cells, SKOV-3 ovarian cancer cells, and DU-145 prostate cancer cells.
BT-474、SK-BR-3、NCI-N87细胞在含10％胎牛血清的1640培养基中，Calu-3、SKOV-3、DU-145细胞在含10％胎牛血清的DMEM培养基中，于37℃，5％CO2培养箱中培养至对数生长期，将处于对数生长期的上述细胞分别以2×103～9×103个细胞每孔的密度接种至96孔培养板，每孔100μL，培养24小时后，加入不同浓度的药物分别作用3天或5天，分别以3、4或5倍稀释制备9个浓度，每个浓度设复孔，以使得所有组合药物处理组在同一块板，并设相应浓度的溶媒对照及无细胞培养基孔。作用结束后，贴壁细胞倾去培养液，加入4℃预冷的三氯乙酸溶液(30％，w/v)100μl，于4℃固定1小时，随后用去离子水冲洗5遍，室温干燥后，每孔加入0.4％(w/v)的SRB染液(Sigma，1％冰乙酸配制)100μL，室温下孵育染色30min后，用1％冰乙酸冲洗4次，去除未结合的染料，室温晾干。每孔加入10mM Tris溶液100μL，室温下孵育染色15min后，用1％冰乙酸冲洗五次洗去未结合的SRB，室温干燥后，每孔加入10mM Tris缓冲液(pH＝10.5)溶解与细胞蛋白结合的染料，采用Synergy 2酶标仪(Bio-Tek)波长510nm和690nm处测定光吸收值(OD值)，并得到A＝OD510-OD690。BT-474, SK-BR-3, NCI-N87 cells in 1640 medium containing 10% fetal bovine serum, Calu-3, SKOV-3, DU-145 cells in DMEM medium containing 10% fetal bovine serum The cells were cultured to a logarithmic growth phase at 37 ° C in a 5% CO 2 incubator, and the cells in the logarithmic growth phase were seeded at a density of 2 x 103 to 9 × 103 cells per well to a 96-well culture plate. After 100 μL per well, after 24 hours of culture, different concentrations of the drug were added for 3 or 5 days respectively, and 9 concentrations were prepared by dilution at 3, 4 or 5 times, respectively, and each well was set to make all the combined drug treatment groups. In the same plate, set the corresponding concentration of vehicle control and cell-free medium wells. After the end of the action, the adherent cells were decanted, and 100 μl of a pre-cooled trichloroacetic acid solution (30%, w/v) at 4 ° C was added and fixed at 4 ° C for 1 hour, followed by washing 5 times with deionized water and drying at room temperature. After that, add 100 μL of 0.4% (w/v) SRB stain (Sigma, 1% glacial acetic acid) per well, incubate for 30 min at room temperature, and then rinse 4 times with 1% glacial acetic acid to remove unbound dye. Dry. Add 100 μL of 10 mM Tris solution to each well, incubate for 15 min at room temperature, wash the unbound SRB with 5 times of 1% glacial acetic acid, and dry at room temperature. Add 10 mM Tris buffer (pH=10.5) to each well to dissolve the cell protein. The bound dye was measured for light absorption (OD value) at a wavelength of 510 nm and 690 nm using a Synergy 2 plate reader (Bio-Tek), and A = OD 510 - OD 690 was obtained.
抑制率(％)＝(A对照-A给药)/A对照×100％。Inhibition rate (%) = (A control - A administration) / A control × 100%.
用CalcuSyn软件程序使用Chou和Talalay组合法，通过等效图解方程CI＝(D)1/(Dx)1+(D)2/(Dx)2确定组合指数(CI)。药物1(D)1与药物2(D)2组合的抑制X％，(Dx)1和(Dx)2是抑制X％时的单独使用药物1和药物2时的浓度。对于各化合物而言，使用通过SRB测定法测得的各浓度下的生长％值。The combination index (CI) was determined by the Cau and Talalay combination method using the CalcuSyn software program by the equivalent graphical equation CI = (D) 1 / (Dx) 1 + (D) 2 / (Dx) 2 . The inhibition of drug 1 (D) 1 in combination with drug 2 (D) 2 was X%, and (Dx) 1 and (Dx) 2 were concentrations at which X% was inhibited when drug 1 and drug 2 were used alone. For each compound, the % growth value at each concentration measured by the SRB assay was used.
本实验使用Pertuzumab-MCC-DM1(P-DM1)单独或联合Trastuzumab对多种HER2过表达的肿瘤细胞系进行了体外细胞培养增殖作用的研究，同时也对非HER2过表达的肿瘤细胞系进行了体外细胞培养增殖作用的研究。使用Chou&Talalay方法分析了数据，以固定浓度比例的P-DM1和Trastuzumab组 合测定组合指数(CI)，或以固定浓度的P-DM1联合各种浓度的Trastuzumab组合测定组合指数(CI)。CI值小于0.9指示协同性；CI值介于0.9-1.1之间指示加和性；而CI值大于1.1指示拮抗性。P-DM1单独处理均能产生抗肿瘤细胞增殖活性，如图1所示，对于HER2过表达的BT-474细胞使用固定浓度比例(1∶1)的P-DM1与Trastuzumab组合导致协同的抗细胞增殖活性。小鼠异种移植物研究显示了相似的结果，P-DM1联合Trastuzumab导致与用单独药剂治疗相比大大增强的抗肿瘤功效(图9)。如图3所示，HER2过表达的NCI-N87细胞使用固定浓度比例的P-DM1与Tastuzumab联合(P-DM1：Tastuzumab为1∶10)也导致协同的抗细胞增殖活性，除了最高(10μg/mL P-DM1和100μg/mL Trastuzumab)或最低(0.0256ng/mL P-DM1和0.256ng/mL Trastuzumab)浓度的组合。如图3-5所示，在HER2过表达的NCI-N87、SK-BR-3细胞中，各种浓度的Trastuzumab与多个固定浓度的P-DM1组合均导致协同的抗细胞增殖活性。如图2所示，在BT-474细胞中，各种浓度的Trastuzumab与多个固定浓度的P-DM1组合均导致协同的抗细胞增殖活性，除了与高浓度的Trastuzumab(25或100μg/mL)的组合。如图6所示，对HER2过表达的Calu-3细胞，固定浓度的P-DM1(0.5或5μg/mL)与一定浓度范围的Trastuzumab(0.1～6.5μg/mL)组合导致协同的抗细胞增殖活性。如图7所示，对非HER2过表达的DU-145细胞，各种浓度的Trastuzumab与固定浓度的P-DM1(7或10u g/mL)组合导致协同的抗细胞增殖活性。如图8所示，对非HER2过表达的SKOV-3细胞，单独的P-DM1产生抑制肿瘤细胞增殖作用；固定浓度的P-DM1与Trastuzumab组合不比单独的P-DM1更有效。In this experiment, Pertuzumab-MCC-DM1 (P-DM1) alone or in combination with Trastuzumab was used to study the proliferation of a variety of HER2-overexpressing tumor cell lines in vitro, and also to non-HER2 overexpressing tumor cell lines. Study on the proliferation of cell culture in vitro. The data were analyzed using the Chou & Talalay method at a fixed concentration ratio of the P-DM1 and Trastuzumab groups. The combination index (CI) was determined by combining the combined index (CI), or a combination of various concentrations of Trastuzumab at a fixed concentration of P-DM1. A CI value of less than 0.9 indicates synergy; a CI value between 0.9 and 1.1 indicates additive; and a CI value greater than 1.1 indicates antagonistic. P-DM1 alone produced anti-tumor cell proliferation activity, as shown in Figure 1. The combination of P-DM1 and Trastuzumab in a fixed concentration ratio (1:1) for HER2 overexpressing BT-474 cells resulted in synergistic anti-cells. Proliferative activity. Mouse xenograft studies showed similar results, with P-DM1 in combination with Trastuzumab resulting in greatly enhanced anti-tumor efficacy compared to treatment with a single agent (Figure 9). As shown in Figure 3, HER2 overexpressing NCI-N87 cells in combination with Tastuzumab (P-DM1: Tastuzumab 1:10) using a fixed ratio of P-DM1 also resulted in synergistic anti-cell proliferative activity, except for the highest (10 μg/ A combination of mL P-DM1 and 100 μg/mL Trastuzumab) or lowest (0.0256 ng/mL P-DM1 and 0.256 ng/mL Trastuzumab) concentrations. As shown in Figures 3-5, in HER2 overexpressing NCI-N87, SK-BR-3 cells, various concentrations of Trastuzumab combined with multiple fixed concentrations of P-DM1 resulted in synergistic anti-cell proliferation activity. As shown in Figure 2, in BT-474 cells, various concentrations of Trastuzumab combined with multiple fixed concentrations of P-DM1 resulted in synergistic anti-cell proliferative activity, except with high concentrations of Trastuzumab (25 or 100 μg/mL). The combination. As shown in Figure 6, a fixed concentration of P-DM1 (0.5 or 5 μg/mL) in combination with a certain concentration range of Trastuzumab (0.1-6.5 μg/mL) resulted in synergistic anti-cell proliferation in HER2 overexpressing Calu-3 cells. active. As shown in Figure 7, for non-HER2 overexpressing DU-145 cells, various concentrations of Trastuzumab in combination with a fixed concentration of P-DMl (7 or 10 u g/mL) resulted in synergistic anti-cell proliferative activity. As shown in Figure 8, for non-HER2 overexpressing SKOV-3 cells, P-DM1 alone produced inhibition of tumor cell proliferation; a fixed concentration of P-DM1 in combination with Trastuzumab was no more effective than P-DM1 alone.
实施例3：体内抗肿瘤功效测定实验：Example 3: In vivo anti-tumor efficacy assay:
可以在体内测量本发明的组合的功效，即在啮齿类动物中植入癌细胞的同种异体移植物或异种移植物，并用所述组合处理肿瘤。将受试小鼠用药物或对照处理，并监测数周或更长时间以测量到达肿瘤倍增的时间，对数细胞杀伤，和肿瘤抑制。The efficacy of the combination of the invention can be measured in vivo by implanting an allograft or xenograft of cancer cells in a rodent and treating the tumor with the combination. Test mice were treated with drugs or controls and monitored for weeks or longer to measure time to tumor doubling, log cell killing, and tumor suppression.
1)实验动物1) Experimental animals
BALB/cA-nude裸小鼠，6-7周，雄性，购自上海斯莱克实验动物有限责任公司。合格证号：SCXK(沪)2012-0002。饲养环境：SPF级。BALB/cA-nude nude mice, 6-7 weeks old, purchased from Shanghai Slack Laboratory Animals LLC. Certificate No.: SCXK (Shanghai) 2012-0002. Feeding environment: SPF level.
2)实验步骤2) Experimental steps
裸小鼠皮下接种人乳腺癌BT-474/T721细胞，待肿瘤生长至100-200mm3 后，将动物随机分组(D0)。给药剂量和给药方案见表2。每周测2次瘤体积，称鼠重，记录数据。肿瘤体积(V)计算公式为：The nude mice were subcutaneously inoculated with human breast cancer BT-474/T721 cells, and after the tumors were grown to 100-200 mm 3 , the animals were randomly grouped (D0). The dosage and administration schedule are shown in Table 2. The tumor volume was measured twice a week, the rats were weighed, and data were recorded. The tumor volume (V) is calculated as:
V＝1/2×a×b2其中a、b分别表示长、宽。V = 1/2 × a × b2 where a and b represent length and width, respectively.
T/C(％)＝(T-T0)/(C-C0)×100，其中T、C为实验结束时的肿瘤体积；T0、C0为实验开始时的肿瘤体积。T/C (%) = (TT 0 ) / (CC 0 ) × 100, where T and C are the tumor volumes at the end of the experiment; T 0 and C 0 are the tumor volumes at the beginning of the experiment.
表2给药剂量和给药方案Table 2 Dosage and dosing schedule
Figure PCTCN2015073629-appb-000004
Figure PCTCN2015073629-appb-000004
D0：第一次给药时间；IV：静脉给药；D0: first administration time; IV: intravenous administration;
实验开始时小鼠数目：对照组n＝10，治疗组n＝6。Number of mice at the start of the experiment: n=10 in the control group and n=6 in the treatment group.
3)实验结果3) Experimental results
图9显示了如下给药后对Herceptin-抗药性人乳腺癌BT-474/T721裸小鼠移植瘤的疗效：(1)溶剂对照；(2)P-DM1 3mg/kg；(3)P-DM1 10mg/kg；(4)P-DM1 3mg/kg+Trastuzumab 10mg/kg。结果显示，P-DM1(3、10mg/kg，IV，每周1次，共3次)明显抑制BT-474/T721裸小鼠皮下移植瘤的生长，抑瘤率分别为72％和97％，其中高剂量组有1/6肿瘤部分消退；Trasutuzumab(10mg/kg，IV，每周1次，共3次)明显增效P-DM1的抗肿瘤作用，使抑瘤率从单用P-DM1 3mg/kg时的72％提高到127％，并有5/6肿瘤部分消退，增效时毒性没有明显增加。因此，P-DM1(3、10mg/kg，IV，每周1次，共3次)剂量依赖性地明显抑制Herceptin-抗药人乳腺癌BT-474/T721裸小鼠皮下移植瘤的生长，引起肿瘤部分消退；Trastuzumab(10mg/kg，IV，每周1次，共3次)明显增效P-DM1的抗肿瘤作用。Figure 9 shows the efficacy of Herceptin-resistant human breast cancer BT-474/T721 nude mice after administration as follows: (1) solvent control; (2) P-DM1 3 mg/kg; (3) P- DM1 10 mg/kg; (4) P-DM1 3 mg/kg + Trastuzumab 10 mg/kg. The results showed that P-DM1 (3, 10 mg/kg, IV, once a week for 3 times) significantly inhibited the growth of subcutaneous xenografts in BT-474/T721 nude mice, with tumor inhibition rates of 72% and 97%, respectively. Among them, 1/6 tumor partial regression in the high-dose group; Trasutuzumab (10 mg/kg, IV, once a week for 3 times) significantly enhanced the anti-tumor effect of P-DM1, and the tumor inhibition rate was from P-only. 72% of DM1 3mg/kg increased to 127%, and 5/6 tumors partially resolved, and there was no significant increase in toxicity when synergistic. Therefore, P-DM1 (3, 10 mg/kg, IV, once a week for 3 times) dose-dependently inhibited the growth of subcutaneous xenografts in Herceptin-resistant human breast cancer BT-474/T721 nude mice. Tumor partial regression; Trastuzumab (10 mg / kg, IV, once a week, a total of 3 times) significantly enhanced the anti-tumor effect of P-DM1.
综上所述，本发明所述的抗体-药物偶联物P-DM1及其与抗体曲妥珠单抗的组合显著地提高了治疗效果，本发明的方法提供了治疗基于HER2介导的病症如癌症更有效的方法。 In summary, the antibody-drug conjugate P-DM1 of the present invention and its combination with the antibody trastuzumab significantly improve the therapeutic effect, and the method of the present invention provides treatment for a HER2-mediated disorder. A more effective method such as cancer.

Claims (10)

  1. 一种具有如下结构的抗体-药物偶联物Pertuzumab-MCC-DM，即P-DM1：An antibody-drug conjugate Pertuzumab-MCC-DM having the following structure, namely P-DM1:
    Figure PCTCN2015073629-appb-100001
    Figure PCTCN2015073629-appb-100001
    其中，mAb指帕妥珠单抗Pertuzumab，Pertuzumab经双功能连接体试剂SMCC与美登素生物碱DM1连接，上式中n表示药物-抗体比。Among them, mAb refers to pertuzumab, Pertuzumab is linked to maytansinoid DM1 via the bifunctional linker reagent SMCC, where n represents the drug-antibody ratio.
  2. 根据权利要求1所述的抗体-药物偶联物P-DM1，其特征在于，所述药物-抗体比n的范围为1至8之间的整数值，其中，1、2、3、4、5、6、7或8个药物部分共价结合在抗体Pertuzumab上。The antibody-drug conjugate P-DM1 according to claim 1, wherein the drug-antibody ratio n ranges from an integer value between 1 and 8, wherein 1, 2, 3, 4, 5, 6, 7 or 8 drug moieties are covalently bound to the antibody Pertuzumab.
  3. 一种用于制备治疗高增殖性疾病或病症的药物的治疗剂组合物，其特征在于，所述治疗剂组合物包含治疗有效量的根据权利要求1或2所述的抗体-药物偶联物P-DM1和治疗有效量的HER2二聚化抑制剂抗体。A therapeutic composition for the preparation of a medicament for the treatment of a hyperproliferative disease or condition, characterized in that the therapeutic composition comprises a therapeutically effective amount of the antibody-drug conjugate according to claim 1 or P-DM1 and a therapeutically effective amount of a HER2 dimerization inhibitor antibody.
  4. 根据权利要求3所述的治疗剂组合物，其特征在于，所述HER2二聚化抑制剂抗体为曲妥珠单抗Trastuzumab。The therapeutic composition according to claim 3, wherein the HER2 dimerization inhibitor antibody is trastuzumab Trastuzumab.
  5. 根据权利要求3或4所述的治疗剂组合物，其特征在于，所述治疗有效量的抗体-药物偶联物P-DM1和所述治疗有效量的HER2二聚化抑制剂抗体可以组合成配制剂的形式施用或交替地施用。The therapeutic composition according to claim 3 or 4, wherein the therapeutically effective amount of the antibody-drug conjugate P-DM1 and the therapeutically effective amount of the HER2 dimerization inhibitor antibody can be combined into The form of the formulation is administered or alternately administered.
  6. 根据权利要求3或4所述的治疗剂组合物，其特征在于，所述高增殖性病症是癌症。A therapeutic composition according to claim 3 or 4, wherein the hyperproliferative disorder is cancer.
  7. 根据权利要求3或4所述的治疗剂组合物，其特征在于，所述高增殖性病症是表达HER2的癌症。The therapeutic composition according to claim 3 or 4, wherein the hyperproliferative disorder is a cancer that expresses HER2.
  8. 根据权利要求7所述的治疗剂组合物，其特征在于，所述癌症是乳腺癌、胃癌、卵巢癌、宫颈癌、前列腺癌、睾丸癌、生殖泌尿道癌、非小细胞肺癌、结肠癌、骨癌、胰腺癌、结肠-直肠癌、肝癌和胆管癌、皮肤癌、食管癌、喉癌、成胶质细胞瘤、成神经细胞瘤、腺癌、口腔癌、甲状腺癌、肺癌、表皮样癌、大细胞癌、小细胞癌、肾癌、小肠癌、大肠癌、直肠癌、鼻咽癌、膀胱癌、黑素瘤、唇癌、毛细胞癌、滤泡性癌、脑和中枢神经系统癌、霍奇金淋 巴癌或白血病。The therapeutic composition according to claim 7, wherein the cancer is breast cancer, gastric cancer, ovarian cancer, cervical cancer, prostate cancer, testicular cancer, genitourinary tract cancer, non-small cell lung cancer, colon cancer, Bone cancer, pancreatic cancer, colon-rectal cancer, liver cancer and cholangiocarcinoma, skin cancer, esophageal cancer, laryngeal cancer, glioblastoma, neuroblastoma, adenocarcinoma, oral cancer, thyroid cancer, lung cancer, epidermoid carcinoma , large cell carcinoma, small cell carcinoma, kidney cancer, small intestine cancer, colorectal cancer, rectal cancer, nasopharyngeal cancer, bladder cancer, melanoma, lip cancer, hair cell cancer, follicular carcinoma, brain and central nervous system cancer Hodgkin Ba or cancer.
  9. 根据权利要求8所述的治疗剂组合物，其特征在于，所述癌症是乳腺癌或胃癌。The therapeutic composition according to claim 8, wherein the cancer is breast cancer or gastric cancer.
  10. 权利要求1或2所述的P-DM1在制备用于治疗高增殖性疾病或病症的药物中的应用。 Use of P-DM1 according to claim 1 or 2 for the manufacture of a medicament for the treatment of a hyperproliferative disease or condition.
PCT/CN2015/073629 2014-03-05 2015-03-04 Antibody-drug conjugate pertuzumab-mcc-dm1, composition of pertuzumab-mcc-dm1 and trastuzumab and uses thereof WO2015131822A1 (en)

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