WO2015131822A1 - Conjugué anticorps-médicament pertuzumab-mcc-dm1, composition de pertuzumab-mcc-dm1 et de trastuzumab et utilisations associées - Google Patents

Conjugué anticorps-médicament pertuzumab-mcc-dm1, composition de pertuzumab-mcc-dm1 et de trastuzumab et utilisations associées 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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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
    • A61K47/68033—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a maytansine
    • 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

L'invention concerne un conjugué anticorps-médicament, le Pertuzumab-MCC-DM1 (à savoir le P-DM1), ainsi qu'une composition d'agents thérapeutiques contenant le P-DM1 et un anticorps inhibiteur de la dimérisation du HER2. L'invention concerne également des utilisations du P-DM1 et de la composition d'agents thérapeutiques dans le traitement de maladies hautement prolifératives.
PCT/CN2015/073629 2014-03-05 2015-03-04 Conjugué anticorps-médicament pertuzumab-mcc-dm1, composition de pertuzumab-mcc-dm1 et de trastuzumab et utilisations associées WO2015131822A1 (fr)

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