WO2020042941A1 - 抗her2抗体药物偶联物在治疗尿路上皮癌中的用途 - Google Patents

抗her2抗体药物偶联物在治疗尿路上皮癌中的用途 Download PDF

Info

Publication number
WO2020042941A1
WO2020042941A1 PCT/CN2019/101283 CN2019101283W WO2020042941A1 WO 2020042941 A1 WO2020042941 A1 WO 2020042941A1 CN 2019101283 W CN2019101283 W CN 2019101283W WO 2020042941 A1 WO2020042941 A1 WO 2020042941A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
her2
use according
variable region
chain variable
Prior art date
Application number
PCT/CN2019/101283
Other languages
English (en)
French (fr)
Inventor
房健民
Original Assignee
荣昌生物制药烟台有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to JP2020529589A priority Critical patent/JP7105304B2/ja
Priority to KR1020207029858A priority patent/KR102520974B1/ko
Priority to KR1020237012001A priority patent/KR20230051318A/ko
Priority to AU2019330680A priority patent/AU2019330680A1/en
Application filed by 荣昌生物制药烟台有限公司 filed Critical 荣昌生物制药烟台有限公司
Priority to EP19855043.6A priority patent/EP3845252A4/en
Priority to RU2020117761A priority patent/RU2750817C1/ru
Priority to US16/652,413 priority patent/US20200289663A1/en
Priority to CA3077260A priority patent/CA3077260C/en
Priority to SG11202009130XA priority patent/SG11202009130XA/en
Priority to KR1020247006234A priority patent/KR20240033092A/ko
Priority to CN201980002467.0A priority patent/CN111655295B/zh
Priority to BR112020010856A priority patent/BR112020010856A8/pt
Publication of WO2020042941A1 publication Critical patent/WO2020042941A1/zh
Priority to JP2022111260A priority patent/JP7470154B2/ja
Priority to AU2022252734A priority patent/AU2022252734A1/en

Links

Images

Classifications

    • 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
    • A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
    • 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
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • 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
    • A61K31/5365—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 ortho- or peri-condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/05—Dipeptides
    • 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
    • A61K47/6847—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 hormone or a hormone-releasing or -inhibiting factor
    • 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
    • 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
    • A61K47/6861—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 kidney or bladder cancer cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0021—Intradermal administration, e.g. through microneedle arrays, needleless injectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0043—Nose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/02—Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the invention relates to the use of an anti-HER2 (human epidermal growth factor receptor 2, human epidermal growth factor receptor 2) antibody drug conjugate in the treatment of urothelial cancer.
  • HER2 human epidermal growth factor receptor 2, human epidermal growth factor receptor 2
  • Urinary epithelial cell carcinoma (Urothelial carcinoma, UC; also known as transitional cell carcinoma, TCC) is a type of cancer that is usually found in the urinary system: kidney, bladder and accessory organs. It is the most common type of bladder cancer and cancer of the ureter, urethra and umbilical duct. It is the second most common type of kidney cancer, accounting for 5-10% of all primary renal malignancies.
  • Urethral epithelium (also called transitional epithelium) is the lining of the bladder, the ureters and the inside of the urethra, and the renal pelvis (the part of the kidney where urine is collected). It consists of urothelial cells or transition cells. These cells can become cancerous cells, known as urothelial carcinoma (or transitional cell carcinoma).
  • urothelial cancer can be non-invasive (only in the lining of the bladder) or invasive (growing into other layers of the bladder wall). Among them, non-invasive urothelial cancer is only in the endometrium of the bladder and does not grow deeper in the bladder wall. At the time of diagnosis, tumors in 50% -60% of patients with urothelial carcinoma are non-invasive.
  • non-invasive urothelial cancer examples include: non-invasive flat urothelial cancer (also known as carcinoma in situ); non-invasive papillary urothelial cancer, high-grade; non-invasive papillary urothelial cancer Low-grade malignancy; non-invasive papillary urothelial tumors with low malignant potential are less likely to develop aggressive cancer.
  • non-invasive flat urothelial cancer also known as carcinoma in situ
  • non-invasive papillary urothelial cancer high-grade
  • non-invasive papillary urothelial cancer Low-grade malignancy
  • non-invasive papillary urothelial tumors with low malignant potential are less likely to develop aggressive cancer.
  • invasive urothelial cancer grows from the endometrium of the bladder to deeper layers of the bladder wall, such as connective tissue (known as the lamina propria) and muscle layers (called the muscle layer).
  • connective tissue known as the lamina propria
  • muscle layers called the muscle layer.
  • tumors in 40% -50% of patients with urothelial carcinoma are invasive.
  • urothelial cancer can start from any part of the urinary tract, including but not limited to the renal pelvis, ureter, bladder or urethra.
  • the current first-line therapy is: combination therapy of gemcitabine and cisplatin; and radiotherapy is targeted at
  • the effect of urothelial cancer is not ideal, and it is generally used as an adjuvant therapy; when treating cancer in the renal pelvis / ureter epithelium, BCG injection therapy (catheter injection of M. tuberculosis) can be used.
  • Urinary epithelial carcinoma has multiple centers and is prone to recurrence.
  • total bladder resection is preferred, and strict regular review is required after surgery. Therefore, treatment is difficult and the recurrence rate is high.
  • mitomycin a chemotherapeutic agent
  • the administration of mitomycin (a chemotherapeutic agent) to the bladder as a one-time dose early in the postoperative period (within 24 hours) or as a six-dose regimen a few weeks after surgery is also an option for some patients.
  • Cisplatin-based chemotherapy is still the gold standard for patients with metastatic UC.
  • the overall response rate (ORR) of cisplatin-based chemotherapy is 60-70%, and the overall survival (OS) is 14-15 months. 5 years The survival rate is 13-15%. After relapse, platinum-based chemotherapy has an ORR of about 15% and a median OS of about 7 months.
  • Changchun flunin has been approved in Europe for the treatment of advanced urinary epithelium or metastatic TCC (Bellmunt, J. et al., J Clin Oncol. 27 (27): 4454-4461 (2009)).
  • Several agents have shown modest activity as tested by single agent therapy, and median survival is 5 to 10 months (Yafi, F.A. et al., Current Oncol. 18 (1): e25-e34 (2011)).
  • Docetaxel is administered as a remission option to patients with transitional cell carcinoma (NCCN 2014), and the medical community in the United States and Canada recognizes docetaxel treatment based on evidence from a phase 2 study Protocol for advanced disease (WO2016 / 064649A1).
  • Atezolizumab is the first anti-PD-L1 cancer immunotherapy drug approved for marketing in 2016.
  • Atezolizumab for locally advanced or metastatic urethral bladder cancer stage III
  • OS overall survival
  • mUC metastatic urothelial carcinoma
  • the median overall survival of the atezolizumab group was 11.1 months and that of the chemotherapy group was 10.6 months; the confirmed objective response rates of the evaluable patients in this population were 23% and At 22%, the median response duration was 15.9 months versus 8.3 months.
  • the median progression-free survival of this population was 2.4 months (control: 4.2 months).
  • the 12-month overall survival data were 39.2% for atezolizumab and 32.4% for chemotherapy;
  • the median overall survival of atezolizumab was 8.3 months, taxane was 7.5 months, and vinflunine was 9.2 months.
  • Nivolumab was approved by the FDA in 2017 for patients with locally advanced or metastatic urothelial cancer.
  • Nivolumab is an anti-PD-1 monoclonal antibody.
  • Clinical data show that Nivolumab's objective response rate ( The ORR was 19.6%, with a median overall survival rate of 8.7 months.
  • the most common serious adverse events include: urinary tract infection, sepsis, diarrhea, small bowel obstruction, and general health deterioration.
  • the most common adverse reactions include fatigue, muscle and bone pain, nausea, and loss of appetite.
  • Nivolumab treatment was discontinued in 17% of patients due to adverse reactions, and drug administration was delayed due to adverse reactions in 46% of patients. During clinical development, 3 patients died of treatment-related deaths due to pneumonia or cardiovascular failure.
  • the main problem of the current clinical stage of PD-L1 / PD-1 immunotherapy drugs is the poor treatment effect, which is mainly reflected in the treatment data such as objective response rate (ORR) and median overall survival.
  • ORR objective response rate
  • Another major problem is the relatively high proportion of serious adverse reactions. For example, Nivolumab caused 3 deaths in related clinical trials.
  • FGFR fibroblast growth factor receptor
  • FGFR FGFR
  • FGFR is only suitable for patients with urothelial cancer who have certain FGFR gene mutations, while FGFR is only in 15% to 20% of metastatic urothelial cancer and 40% to 70% % Non-muscle invasive bladder cancer overexpression (2018 ASCO Annual Meeting, Response Found Found Advanced Urothelial Carcinoma With FGFR Inhibitor).
  • the invention discloses a method for treating urothelial cancer.
  • the method comprises injecting an effective amount of an antibody drug conjugate (ADC) into a patient, wherein the ADC is an anti-HER2 antibody coupled to a cytotoxic molecule.
  • ADC antibody drug conjugate
  • the cytotoxic molecule includes, but is not limited to, a tubulin inhibitor or a DNA damaging agent.
  • the tubulin inhibitor includes, but is not limited to, dolastatin and auristatin cytotoxic molecules, maytansine cytotoxic molecules;
  • the DNA damaging agent includes, but is not limited to, card Calicheamicin, duocarmycin, and azithromycin derivative PBD (pyrrolobenzodiazepine, pyrrolobenzodiazepine) ), Camptothecin derivatives SN-38.
  • the auristatin-like cytokine molecule includes, but is not limited to, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and auristatin F (auristatin F; AF) or their puppet organisms
  • the maytansinoid cytotoxic molecules include, but are not limited to, DM1, DM3, DM4 or their puppet organisms (advanced molecules of antibody-drug conjugates, advances in research, Hu Xinyue et al , China Pharmaceutical Biotechnology, December 2017, Vol. 12, No. 6) (Research progress of maytansinoid antibody drug conjugates, Zhou Lei, et al., Chinese Journal of New Drugs, Vol. 25, No.
  • the cytotoxic molecule may also be amanitins, anthracyclines, baccatins, camptothecins, cemadotins, colchicine ( colchicines, colcimids, combetastatins, cryptophycins, decodermolides, docetaxel, doxorubicin, echinomycin (echinomycins), eleutherobins, epothilones, estramustines, lexitropsins, maytansines, methotrexate ), Netropsins, puromycins, rhizoxins, taxanes, tubulysins, or vinca alkaloids.
  • the cytotoxic molecule is not limited to the above-mentioned categories, and includes all drugs available for ADC.
  • an antibody drug conjugate for use in preparing a medicament for treating bladder cancer;
  • the antibody drug conjugate comprises an antibody or a functional fragment thereof that binds HER2,
  • the antibody comprises a heavy chain variable region and a light chain variable region, wherein (i) the heavy chain variable region includes three CDR regions, wherein the amino acid sequences of the CDR regions each have a sequence such as SEQ ID NO: 1 The amino acid sequences shown in Figures 2, 2 and 3; and (ii) the light chain variable region comprises three CDR regions, wherein the amino acid sequences of the CDR regions have the sequences shown in SEQ ID NOs: 4, 5 and 6, respectively. Amino acid sequence.
  • the antibody may also be an antibody that competitively binds to the same or similar epitope as the CDR-defined antibody described above.
  • the term "antibody” may include a full-length antibody or an antibody fragment that binds, is reactively related, or complexes with HER2.
  • An antibody can be any protein, protein-like molecule, or polypeptide that binds, complexes, or reacts with a portion of a population of cells seeking therapeutic modification.
  • the antibody may be a chimeric antibody or a functionally active fragment thereof, a humanized antibody or a functionally active fragment thereof, a human antibody or a functionally active fragment thereof.
  • the antibody can also be an antibody or a functionally active fragment of another species other than the above, for example: a mouse antibody or a functionally active fragment thereof, a rat antibody or a functionally active fragment thereof, a sheep antibody or a functionally active fragment thereof, a rabbit antibody or Functionally active fragment.
  • the antibody may be a polyclonal antibody or a monoclonal antibody. In some embodiments, the antibody may be a bispecific antibody. In addition, the antibody may be a functionally active fragment, a derivative of the antibody, or the like.
  • “Functional” means that the fragments, derivatives, or analogs can identify the same antigen, and antibodies that recognize fragments, derivatives, or analogs derived from the antigen, such as but not limited to: F (ab ') 2 , Fab, Fab ', Fv fragments and heavy and light chain dimers of antibodies, or any minimal fragments thereof, like Fvs or single chain antibodies (SCAs).
  • the antibody may be a fusion protein of the antibody.
  • Antibodies may also include analogs and derivatives that are modified or unmodified (ie, covalently linked through any molecule), so long as such covalent linkages allow the antibody to retain its antigen-binding immunospecificity.
  • Examples include, but are not limited to, analogs and derivatives of antibodies, including further modifications such as: glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protection / blocker groups, proteases Cleavage, attachment to cellular antibody units or other proteins, etc. Any number of chemical modifications can be achieved using known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis in the presence of tunicamycin, and the like.
  • analogs or derivatives may include one or more unnatural amino acids.
  • the antibody may have modifications (eg, substitutions, deletions, or additions) in amino acid residues that interact with the Fc receptor.
  • the antibody-drug conjugate has a structure of the general formula Ab- (LU) n, where Ab represents the antibody or a functional fragment thereof, L represents a linker; U represents a coupled cytotoxic molecule, n An integer from 1 to 8 representing the number of molecules of the therapeutic agent bound to each antibody.
  • the linker is connected to the antibody or a functional fragment thereof through a thiol group and / or an amino group, and the cytotoxic molecule is coupled to the antibody in a fixed or non-targeted manner.
  • the linker of the present invention may be selected from the following:
  • the linker of the present invention is preferably maleimido-hexanoyl-valine-citrulline-p-aminobenzyloxy (Maleimido-Caproyl-Valine-Citrulline-p-Aminobenzyloxy, mc-vc-pAB) and horse Maleimidocaproyl (mc).
  • the linker of the present invention may also be selected from a triglycine peptide linker, which is a new linker for ADC drug conjugates developed in recent years.
  • a triglycine peptide linker which is a new linker for ADC drug conjugates developed in recent years.
  • ADCs Antibody-Drug Conjugates
  • you can also choose glucuronide linker (gluc -tubulysin) (Patrick J. Burke et al, Glucuronide-linked antibody-tubulysin conjugates display activity in MDR + and heterogeneous tumor models, Molecular Cancer Therapeutics, 2018).
  • the antibody or functional fragment thereof is derived from an antibody secreted by a hybridoma deposited at the General Microbiology Center of the China Microbial Species Collection Management Committee under the accession number CGMCC No. 8102 on August 22, 2013; address: No. 3, No. 1 Courtyard, Beichen West Road, Chaoyang District, Beijing;
  • the antibody is a humanized antibody
  • the antibody is an antibody secreted by CHO cells deposited with the China Type Culture Collection under the accession number CCTCC C2013170 on November 6, 2013. Address: Wuhan University, Wuhan, Hubei
  • the name of the antibody drug conjugate used is RC48-mc-vc-pAB-MMAE, which conforms to the structure of the general formula Ab- (LU) n, and RC48 (a humanized anti-HER2 monoclonal (Antibodies) are coupled to MMAE through the linker mc-vc-pAB, and the number of couplings ranges from 1-8, including coupling 1, 2, 3, 4, 5, 6, 7, 8 One or a combination of antibody drug conjugates with varying numbers of 1-8 MMAEs.
  • the urothelial cancer is locally advanced urothelial cancer, locally advanced or metastatic urothelial cancer, HER2 (human epidermal growth factor receptor 2, also called ErbB-2) , C-erbB2 or HER2 / neu) positive urothelial carcinoma, HER2-positive locally advanced or metastatic urothelial carcinoma.
  • HER2 human epidermal growth factor receptor 2, also called ErbB-2
  • C-erbB2 or HER2 / neu positive urothelial carcinoma
  • HER2-positive locally advanced or metastatic urothelial carcinoma HER2-positive locally advanced or metastatic urothelial carcinoma.
  • the medicament described in the present invention may be administered intranasally, subcutaneously, intradermally, intramuscularly, or intravenously.
  • the medicament also includes a pharmaceutically acceptable carrier; the medicament is preferably a lyophilizer or a liquid preparation; the carrier includes a stabilizer, a protective agent, a buffer solution, a lyophilized protective agent, an active protective agent, a surfactant, One or more of an adsorption carrier and an absorption enhancer.
  • Figure 1 SDS-PAGE image of purified human recombinant protein HER2-ECD, stained by Coomassie blue. The loading amount was 10 ⁇ g per well.
  • Figure 2 SDS-PAGE analysis chart of cRC48 (chimeric antibody) and RC48 (humanized antibody). The amount of antibody per well was 2 ⁇ g.
  • Figure 3 shows the binding affinity of HER2-ECD of the humanized antibody RC48 determined by ELISA experiments, and the binding affinity constant Kd is calculated.
  • Herceptin and cRC48 served as controls.
  • FIG. 4A shows the ability of the anti-HER2 humanized antibody RC48 to bind to HER2 + cells SK-BR3, BT474, and HER2-cell MDA-MB468 by flow cytometry.
  • Figure 4B Shows the ability of anti-HER2 antibodies to bind to BT474 cell surface antigen at different antibody concentrations by flow cytometry.
  • Anti-HER2 antibodies include Herceptin, cRC48, RC48. A total of 5 ⁇ 10 4 cells were analyzed.
  • Figure 5 Shows that RC48 only shows specific binding affinity for HER2, but does not bind to EGFR, HER3, HER4.
  • a cDNA fragment encoding HER2-ECD (amino acids Thr23 to Thr652, GenBank accession number M11730) was cloned into a pcDNA3 (Invitrogen) expression vector by PCR.
  • cDNA of the HER2-ECD coding region is obtained from HER2 + SKBR3 cell line (ATCC number: HTB-30) by RT-PCR method (the kit uses ImProm-IITM Reverse Transcription System Reverse Transcription System).
  • the primers are: P1: 5'CG GGATCCTG CCACCAGCTGTGCGCC (SEQ ID NO: 7), P2: 5 ' GCT CTAGA TCAGTTGATGGGGCAAGGCT (SEQ ID NO: 8), the underlined sequences are the introduced BamHI and XbaI restriction sites, respectively, to reverse
  • the recorded HER2-ECD cDNA was used as a template for PCR amplification with the above primers.
  • the amplification conditions were: denaturation at 94 ° C for 30s, annealing at 60 ° C for 30s, extension at 72 ° C for 1 minute, 30 cycles, and extension at 72 ° C for 10 minutes.
  • PCR fragment was then recovered, digested with BamHI and XbaI enzyme (NEB), and ligated to the pcDNA3 vector.
  • a polyhistidine tag was added to the C-terminus of HER2-ECD to facilitate purification.
  • HEK293 cells ATCC, USA
  • His-labeled soluble protein HER2-ECD was purified from the cell culture medium by Ni-NTA affinity chromatography (Qiagen). SDS-PAGE and Coomassie brilliant blue staining showed that the purified HER2-ECD protein was more than 95% homogeneous. The results are shown in Figure 1.
  • Soluble HER2-ECD appears as a monomer with a relative molecular weight of about 75 kDa, which is slightly larger than the calculated molecular weight (71 kDa), indicating that the protein is glycosylated in HEK293 cells.
  • the purified HER2-ECD protein was further concentrated and transferred to a sterile pH 7.4 PBS buffer for subsequent in vivo and in vitro analysis.
  • mice were immunized using the HER2-ECD prepared above as an antigen to prepare monoclonal antibodies. Immunization, hybridoma cell fusion, and preliminary screening were performed according to standard procedures (Reference: WHO Technical Report Series, No. 822, 1992 Annex 3).
  • the serum of the mice was detected by an enzyme-linked immunosorbent assay (ELISA), and the spleens of the two mice with the highest anti-HER2 antibody titers were removed, and then fused with myeloma cells P3X63Ag8 (ATCC CRL-1580).
  • the fused cells were diluted into 10 96-well plates, and preliminary screening was performed by ELISA method according to the binding ability with HER2-ECD.
  • Nunc Maxisorb 96-well plates were coated with HER2-ECD (0.2-1 ⁇ g / ml), and then incubated with gradient dilution of mouse serum or hybridoma supernatant (100 ⁇ L).
  • the mouse-derived anti-HER2 antibody was detected with a horseradish peroxidase-conjugated goat F (ab ') 2 anti-mouse IgG and Fc-specific secondary antibody (Invitrogen).
  • the supernatants of 400 hybridoma cell lines were screened by ELISA, and 36 of them showed strong HER2-ECD binding.
  • Ten hybridoma cells with the strongest HER2 binding ability were selected, and subcloned hybridoma cell lines were screened by the limiting dilution method.
  • BD FACS Calibur flow cytometry
  • hybridoma cell line mRC48 (mouse-derived IgG1k) was identified through sequence analysis, which has strong HER2 binding capacity.
  • the hybridoma cell mRC48 was deposited on August 22, 2013 under the deposit number No. 8102 at the General Microbial Center of the China Microbial Strain Collection Management Committee (the date of conversion to deposit under the Budapest Treaty was October 29, 2013).
  • variable regions of the heavy and light chains of the above-mentioned hybridoma cell clones mRC48 were rapidly amplified using the commercial kit SMARTTM RACE cDNA Amplification Kit (Clontech) and sequenced according to the instructions.
  • the primers in the kit were used.
  • Add the reverse transcriptase SMARTScribeTM Reverse Transcriptase follow the steps provided in the kit to perform reverse transcription to obtain the RACE-Ready first-strand cDNA, and then perform two rounds of PCR, the first round of PCR to obtain the cDNA as a template.
  • the UPM is a 5 'end primer and the 3' end is mRC48-VL-1 / mRC48-VH-1.
  • the PCR reaction conditions were: pre-denaturation at 94 ° C for 5 minutes; 25 amplification cycles (denaturation at 94 ° C for 30s, annealing at 68 ° C for 30s, extension at 72 ° C for 2 minutes); and final extension at 72 ° C for 10 minutes.
  • the product of the first round of PCR was used as the template.
  • the NUP provided in the kit was the 5′-end primer and the 3′-end primer was mRC48-VL-2 / mRC48-VH-2.
  • the PCR reaction conditions were: Pre-denaturation at 94 ° C for 5min; 25 amplification cycles (denaturation at 94 ° C for 30s, annealing at 68 ° C for 30s, extension at 72 ° C for 2min); extension at 72 ° C for 10min. In this way, the variable regions of the heavy and light chains of the aforementioned hybridoma cell clone mRC48 were obtained.
  • the specific primer sequences are as follows:
  • mRC48-VL-1 5’-GTTGGTGCAGCATCAGCCCGTT-3 ’(SEQ ID NO.9)
  • mRC48-VL-2 5’-GTTCACTGCCATCAATCTTCCAC-3 ’(SEQ ID NO.10)
  • mRC48-VH-1 5’-GCCAGTGGATAGACAGATGG-3 ’(SEQ ID NO.11)
  • mRC48-VH-2 5’-AGGTCACTGTCACTGGCTCAG-3 ’(SEQ ID NO.12)
  • the PCR product was purified by agarose gel electrophoresis and subcloned into the pCR2.1TOPO cloning vector (Invitrogen). Ten independent cloned plasmid DNAs were obtained by PCR, and then sequenced with M13 forward and reverse primers. DNA sequence analysis showed that all 10 clones had cDNA encoding the same VH or VL polypeptide.
  • the amino acid sequence of the complementarity determining region (CDR) is defined by the Kabat coding table and is listed in Table 1. Sequence comparison analysis showed that the CDRs of anti-HER2 mRC48 were significantly different from known HER2 antibodies including Herceptin (trastuzumab).
  • VH Light chain
  • VL CDR1 DYYIH (SEQ ID No.1) KASQDVGTAVA (SEQ ID NO.4)
  • CDR2 RVNPDHGDSYYNQKFKD SEQ ID No. 2
  • WASIRHT SEQ ID NO.5
  • CDR3 ARNYLFDHW SEQ ID No. 3
  • HQFATYT SEQ ID NO.6
  • Humanized anti-HER2 monoclonal antibody mRC48 was humanized by transplanting the light or heavy chain CDRs into the human IgG1 ⁇ framework region.
  • the light chain variable region (RC48-VL) of the humanized RC48 antibody and the heavy chain variable region (RC48-VH) of the humanized RC48 antibody were designed to combine into a humanized anti-HER2 antibody: RC48.
  • the overall sequence of RC48-VH is 84% similar to the human IgG1VH gene.
  • the RC48 antibody comprises a light chain variable region RC48-VL and a heavy chain variable region RC48-VH.
  • Humanized anti-HER2 monoclonal antibody RC48 was obtained by CDR grafting.
  • the variable region of the heavy or light chain was directly synthesized by Nanjing Kingsray Biotechnology Co., Ltd.
  • the synthetic variable region included the Kozak consensus sequence and the start code Daughter, heavy or light chain signal peptide, human-derived framework region and murine CDR, variable region and human IgG1k constant region are linked into a complete fragment by overlapping extension PCR method.
  • the primers for overlap extension PCR are:
  • Heavy chain VH1 5 CGCGGATCC GCCGCCACCATGGGATGGAGCT3 ′ (SEQ ID NO: 13)
  • VH2 5GATGGGCCCTTGGTGCTAGCGGAGCTCACTGTCACCAGTGTT3 (SEQ ID NO: 14)
  • VL1 5 CGCGGATCC GCCGCCACCATGGACATGAGGGT 3 (SEQ ID NO: 17)
  • VL2 5 GATGGTGCAGCCACAGTACGCTTTATCTCAACTTTTGT
  • CL2 5 CCGGAATTCACACTCTCCCCTGTTGAAGC 3 (SEQ ID NO: 20)
  • variable region and VH2 are: denaturation at 94 ° C for 30s, annealing at 60 ° C for 30s, extension at 72 ° C for 1 minute, 30 cycles, and extension at 72 ° C for 10 minutes.
  • the amplification conditions were: denaturation at 94 ° C for 30s, annealing at 60 ° C for 30s, extension at 72 ° C for 2 minutes, 30 cycles, and extension at 72 ° C for 10 minutes.
  • variable region of the light chain was first amplified using the synthetic variable region as a template, VL1 and VL2 as primers, the human IgG1 ⁇ light chain constant region as a template, and CL1 and CL2 as primers, respectively.
  • the constant region and amplification conditions are: denaturation at 94 ° C for 30s, annealing at 60 ° C for 30s, extension at 72 ° C for 1 minute, 30 cycles, and extension at 72 ° C for 10 minutes.
  • the amplification conditions were: denaturation at 94 ° C for 30s, annealing at 60 ° C for 30s, extension at 72 ° C for 2 minutes, 30 cycles, and extension at 72 ° C for 10 minutes.
  • a humanized anti-HER2 monoclonal antibody RC48 was obtained, in which RC48 contained human IgG1 ⁇ heavy chain constant region and heavy chain variable region RC48-VH, and human IgG1 ⁇ light chain constant region and light chain variable region RC48-VL.
  • the human-mouse chimeric antibody cRC48 was also obtained by the same method.
  • the variable region of the mouse and the constant region of human IgG1k were ligated into a complete fragment by overlapping extension PCR.
  • Chimeric anti-HER2 RC48 (referred to as cRC48) is composed of a mouse-human chimeric cRC48 heavy and light chain. RC48 includes the humanized heavy chain RC48-VH and the humanized light chain RC48-VL. Both cRC48 and RC48 can be expressed.
  • the antibodies were collected from CHO cell supernatants, purified by Protein A, and analyzed by SDS-PAGE under reducing and non-reducing conditions (see Figure 2).
  • CHO cells secreting RC48 antibody as described above i.e., CHO cells transfected with human IgG1 ⁇ heavy chain constant region and heavy chain variable region RC48-VH, and human IgG1 ⁇ light chain constant region and light chain variable region RC48-VL
  • RC48 antibody as described above (i.e., CHO cells transfected with human IgG1 ⁇ heavy chain constant region and heavy chain variable region RC48-VH, and human IgG1 ⁇ light chain constant region and light chain variable region RC48-VL) It was deposited at the China Type Culture Collection on November 6, 2013 under the accession number C2013170.
  • HER2-binding affinity constant (Kd) of cRC48 (chimeric antibody) and RC48 antibody (humanized antibody) was measured by ELISA method.
  • the specific method can be seen in Example 1, that is, the 96-well plate was coated with soluble HER2-ECD protein, and then Incubate with diluted antibodies (Herceptin and chimeric cRC48 as controls) and use HRP-conjugated sheep F (ab ') 2 anti-human IgG Fc- for HER2-ECD-related antibodies (all forms of human IgG1 ⁇ ) Specific secondary antibodies were detected (invitrogen).
  • Flow cytometry was used to detect the binding of endogenously expressed HER2 of human breast cancer cells to the humanized anti-HER2 antibody RC48.
  • the results are shown in Figure 3. 6 ⁇ g of control human IgG, Herceptin, cRC48, and RC48 were incubated with human breast cancer cells SK-BR-3, BT474, and HER2-cell MDA-MB468 (2 ⁇ 10 7 cells) in two HER2 + cell lines. Incubate for 30-45 minutes.
  • Herceptin, cRC48, and RC48 were tested by ELISA.
  • the ELISA method is described in Example 1.
  • the 96-well plates were coated with the antigens EGFR, HER2, HER3, and HER4, and the loading amount of each well was 20ng.
  • Linked sheep F (ab ′) 2 anti-mouse IgG and Fc-specific secondary antibody were tested. The results are shown in Figure 5.
  • Monoclonal antibody against RC48 was obtained from CHO cell culture solution by Protein A, and the purity was over 95% by SDS-PAGE electrophoresis and SEC analysis.
  • the obtained antibody protein was dialyzed into a PBS buffer solution with a 30KD membrane package, concentrated, and the concentration was calibrated with an ultraviolet absorbance spectrophotometer for subsequent coupling reaction.
  • PBS buffer solution was used to prepare reducing agent and protecting agent as follows: 1 ⁇ 20mmol / L TCEP (Tris-2-carboxyethyl-phosphine), 1 ⁇ 20mmol / L DTPA (Diethylene triamine pentacetate) acid.
  • TCEP Tris-2-carboxyethyl-phosphine
  • DTPA Diethylene triamine pentacetate
  • Different ratios can be added in a certain concentration range, mixed with a certain concentration of monoclonal antibodies (such as: 5-30mg / ml) according to a certain volume ratio (such as 1: 1), and the molar ratio of TCEP to the final concentration of the antibody is 0.5-6.0: 1. Stir the reaction at 25 ° C for 2h.
  • the concentration of free sulfhydryl groups was detected by DTNB method at 412nm, and the number of free thiol groups was calculated by the molar ratio with the antibody. TCEP reduction is reproducible, and the number of free thiol groups can reach 1.0-8.0 after reduction.
  • the ELISA plate was coated with the recombinant protein HER2-ECD (concentration: 0.5 mg / ml), and overnight at 2 ° C-8 ° C. The plate washer washes 3 times. 3% BSA-PBST solution was blocked, 37 degrees 2h. The plate washer washes 3 times. Adding sample: Dilute the standard line with PBST solution from 1000ng / ml to 11 points, 100 ⁇ l / well, 37 ° C for 2h. The plate washer washes 3 times. The secondary antibody (goat anti-human IgG-Fc-HRP) was diluted 5000-fold with PBST solution. Add TMB color development solution to develop color, and develop color in the dark at room temperature for 8-10 minutes. The experiment was terminated with 2M H2SO4, and the microplate reader was read at 450 / 655nm. The results are shown in Table 4.
  • Example 7 Efficacy and safety of a single drug in the treatment of HER2-positive locally advanced or metastatic urothelial cancer
  • the purpose of this experiment was to evaluate the efficacy and safety of single-agent therapy for HER2-positive locally advanced or metastatic urothelial carcinoma.
  • the antibody-drug conjugate used was RC48-mc-vc-pAB-MMAE.
  • RC48 was coupled to MMAE through the linker mc-vc-pAB, and the number of couplings varied from 1-8.
  • the selection criteria for the subjects are:
  • Age 18 years (minimum age) to 80 years (maximum age)
  • the selection criteria are:
  • Subjects who have been diagnosed with locally advanced or metastatic disease that cannot be surgically removed have disease progression or intolerance after receiving at least first-line systemic chemotherapy;
  • ECOG physical status is 0 or 1 point
  • Contraceptives or condoms should be surgically sterilized, postmenopausal, or agree to use at least one medically approved contraceptive (such as an intrauterine device) during the study treatment period and within 6 months after the end of the study treatment period , Contraceptives or condoms); male subjects should agree to use at least one medically approved contraceptive (such as condoms, abstinence, etc.) during the study treatment period and within 6 months after the end of the study treatment period;
  • HIV test result is positive
  • This study will include subjects who have previously received at least first-line systemic chemotherapy failure or intolerance of locally advanced or metastatic urothelial cancer, and have measurable lesions, general conditions and good organ function. All subjects submitted tumor tissue pathological sections to the central laboratory for confirmation of positive HER2 expression, and the positive definition was defined as immunohistochemistry (IHC) of 2+ or 3+ (regardless of the results of fluorescence in situ hybridization [FISH] detection).
  • IHC immunohistochemistry
  • TRAEs treatment-related adverse reactions
  • ALT 50.0%, grade 1-2
  • sensory 50.0%, Grade 1-2
  • white blood cell count 50.0%, grade 1-2
  • SAEs drug-related serious adverse events
  • Patient 01001 Female, 57 years old, with multiple lung metastases after right pelvic cancer surgery. Pathological diagnosis of urothelial carcinoma, HER2, IHC, 3+;
  • FIG. 7 it can be seen that, after 6 weeks of treatment, the tumor lesion located beside the right psoas muscle has shrunk from 56 ⁇ 48mm to 33 ⁇ 22mm, a reduction of 72.9%.
  • Patient 01007 male, 63 years old, postoperative bladder cancer, right pelvic cancer, lung metastasis, liver metastasis, cervical lymph node metastasis, mediastinal metastasis, multiple bone metastases.
  • FIG. 8 it can be seen that tumor lesions located in the left upper lobe, liver metastasis, and mediastinal lymph nodes undergo a 6-week treatment, and the tumor area at the lesions has been significantly reduced.
  • the left upper lobe was reduced from 45 ⁇ 36mm to 28 ⁇ 22mm, a decrease of 61.9%
  • liver metastasis was reduced from 38 ⁇ 32mm to 25 ⁇ 21mm, a decrease of 56.8%
  • mediastinal lymph nodes were reduced from 29 ⁇ 15mm to 18 ⁇ 7mm, a 71.03% decrease.
  • the anti-HER2 monoclonal antibody-MMAE coupling agent drug of the present invention has a very significant therapeutic effect; by comparison with the currently marketed drugs, it can also be seen that it is significantly better than the current one that has been approved by the European Union.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Oncology (AREA)
  • Cell Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Urology & Nephrology (AREA)
  • Dermatology (AREA)
  • Endocrinology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Otolaryngology (AREA)
  • Toxicology (AREA)
  • Hematology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicinal Preparation (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

涉及抗HER2抗体药物偶联物(ADC)在制备用于治疗尿路上皮癌的药物中的用途。所述的药物对于尿路上皮癌特别是局部晚期或转移性尿路上皮癌患者安全有效，能有效延长患者生存期。

Description

抗HER2抗体药物偶联物在治疗尿路上皮癌中的用途 技术领域
本发明涉及一种抗HER2(人类表皮生长因子受体2，human epidermal growth factor receptor 2)抗体药物偶联物在治疗尿路上皮癌中的用途。
背景技术
尿路上皮细胞癌(Urothelial carcinoma，UC；或称移行细胞癌，Transitional cell carcinoma，TCC)是一种癌症类型，其通常存在于泌尿系统：肾、膀胱和附属器官中。其为膀胱癌和输尿管、尿道和脐尿管癌症的最常见类型。其为第二最常见的肾癌类型，占所有原发性肾脏恶性肿瘤的5-10％。
尿路上皮(也称为过渡性上皮)是膀胱，输尿管和尿道内侧以及肾盂(尿液收集的肾脏部分)内衬。它由尿路上皮细胞或过渡细胞组成。这些细胞可以变成癌细胞，即称为尿路上皮癌(或移行细胞癌)。
根据癌变细胞的侵入性，尿路上皮癌可以是非侵入性的(仅在膀胱的衬里中)或侵入性的(生长到膀胱壁的其他层中)。其中，非侵入性尿路上皮癌仅在膀胱内膜中并且未在膀胱壁中更深地生长。在诊断时，50％-60％的尿路上皮癌患者的肿瘤为非侵入性的。非侵入性尿路上皮癌的类型包括：非侵袭性扁平尿路上皮癌(也称为原位癌)；非侵袭性乳头状尿路上皮癌，高级别；非侵袭性乳头状尿路上皮癌，低度恶性；具有低恶性潜能的非侵袭性乳头状尿路上皮肿瘤发展成侵袭性癌症的可能性很小。
相比之下，侵入性尿路上皮癌从膀胱内膜生长到膀胱壁的较深层，例如结缔组织(称为固有层)和肌肉层(称为肌层)。在诊断时，40％-50％的尿路上皮癌患者的肿瘤为侵袭性的。
理论上，尿路上皮癌可以从泌尿道的任何部位开始，包括但不限于肾盂、输尿管、膀胱或尿道。
当相关肿瘤细胞未转移时，手术切除治疗为首选的治疗方案，对于肿瘤已经转移的患者，一般需要采取抗癌药物治疗，目前的一线疗法是：吉西他滨和顺铂的联合疗法；而放射疗法针对尿路上皮癌的效果并不理想，一般用作辅助治疗的手段；当治疗肾盂/输尿管上皮中的癌时，可以使用BCG注射疗法(导管注射牛结核杆菌)。
尿路上皮癌具有多中心，易复发的特点，对于肿瘤累及肌层的患者首选膀胱全切，且术后还需严格规律复查，因此，治疗难度大，复发率高。(李学松，王刚，张骞编.泌尿外科病例精粹，北京大学医学出版社，2017)。在术后早期(24小时内)作为一次性剂量或在外科手术后几周作为六剂量方案将丝裂霉素(一种化疗药物)给予至膀胱中也是部分患者可选择的治疗方案。目前基于顺铂的化疗仍然是转移性UC患者的治疗金标准，基于顺铂的化疗的总体反应率(ORR)为60-70％，总生存期(OS)为14-15个月，5年生存率为13-15％，复发后的患者基于铂的化疗，ORR约为15％，中位OS约为7个月。
在欧洲已批准长春氟宁用于治疗泌尿上皮晚期或转移性TCC(Bellmunt,J.等人，J Clin Oncol.27(27)：4454-4461(2009))。经单一药剂疗法测试，若干药剂已显示适中活性，且中值存活期为5至10个月(Yafi,F.A.等人，Current Oncol.18(1)：e25-e34(2011))。在转移性情况中，多西他赛(Docetaxel)作为缓解性选择向移行细胞癌患者投与(NCCN 2014)，且美国及加拿大(Canada)医学界基于来自2期研究的证据认可多西他赛治疗晚期疾病的方案(WO2016/064649A1)。
近年来，治疗尿路上皮癌的新药主要有：1.罗氏的Atezolizumab是2016年批准上市的首个抗PD-L1癌症免疫治疗药物，根据最新的Atezolizumab治疗局部晚期或转移性尿道膀胱癌三期临床试验(IMvigor211)试验结果表明：Atezolizumab未满足III期IMvigor211试验的主要临床终点，即改善局部晚期或转移性尿路上皮癌(mUC)患者二线治疗的总生存期(OS)，在共234例患者(atezolizumab组116例，化疗组118例)中，atezolizumab组的中位总生存期为11.1个月，化疗组为10.6个月；该人群中可评估患者的确诊客观反应率分别为23％和22％，中位反应持续时间为15.9个月对8.3个月。
该人群的中位无进展生存期为2.4个月(对照为4.2个月)，在意向治疗人群的探索性分析中，12个月的总生存率数据：atezolizumab为39.2％，化疗为32.4％；atezolizumab的中位总生存期为8.3个月，紫杉烷为7.5个月，长春氟宁为9.2个月。(The ASCO Post,IMvigor211 Trial:Atezolizumab vs Chemotherapy in Platinum-Treated Advanced Urothelial Carcinoma,Matthew Stenger,9/17/2018)
此外，由于美国数据监测委员会观测到使用Atezolizumab单一疗法与基于铂的化疗疗法相比，发现了PD-L1的低表达肿瘤患者的存活率下降的问 题，因此在2018年6月，美国FDA发出了限制Atezolizumab(Tecentriq)在适合含顺铂化疗的局部晚期或转移性尿路上皮癌患者中使用的要求。要求在使用Atezolizumab前需先行检测PD-L1表达水平。这进一步表明了Atezolizumab应用于治疗尿路上皮癌的局限性。
2.百时美施贵宝公司的Nivolumab于2017年被FDA批准用于局部晚期或转移性尿路上皮癌患者，Nivolumab是一种抗PD-1单克隆抗体，临床数据显示，Nivolumab的客观缓解率(ORR)为19.6％，中位数总生存率为8.7个月，最常见严重不良事件包括：尿路感染，败血症，腹泻，小肠梗阻，以及总体健康状况退化。最常见的不良反应包括疲劳，肌与骨骼疼痛，恶心，以及食欲减退。17％患者因不良反应停止Nivolumab治疗，46％患者因不良反应延迟给药。在临床开展中，出现了3例患者由于肺炎或心血管衰竭导致与治疗相关的死亡。
综合以上内容来看，目前临床阶段的PD-L1/PD-1免疫治疗药物的主要问题是治疗效果不佳，主要体现在客观缓解率(ORR)和中位数总生存期等治疗数据方面不理想，另一个主要问题是严重不良反应占比相对较高，例如：Nivolumab在相关临床试验中造成3例死亡病例。
3.强生公司旗下的杨森公司的erdafitinib在2018年被FDA授予突破性药物资格，用于治疗接受化疗后病情进展且肿瘤中存在特定成纤维细胞生长因子(FGFR)基因改变的局部晚期或转移性尿路上皮癌患者的治疗，该药物属于一种成纤维细胞生长因子受体(FGFR)酪氨酸激酶抑制剂；其二期临床研究(BLC2001，NCT02365597)的结果显示，erdafitinib治疗的总缓解率为40％(完全缓解率3％，部分缓解率37％)，中位无进展生存期为5.5个月，中位总生存期13.8个月，在总计99例患者中，7例患者因治疗相关不良事件停止治疗(https://www.jnj.com/media-center/press-releases/erdafitinib-phase-2-study-results-show-promise-in-the-treatment-of-metastati c-urothelial-cancer)。由于该药的治疗靶点是FGFR，因此，其仅适用于具有某些FGFR基因突变的尿路上皮癌患者，而FGFR仅在15％至20％的转移性尿路上皮癌和40％至70％的非肌层浸润性膀胱癌过表达(2018 ASCO Annual Meeting，Responses Found in Advanced Urothelial Carcinoma With FGFR Inhibitor)。
从目前的治疗来看，晚期尿路上皮癌恶性程度高，预后差，特别是常规化疗失败后，治疗手段有限，免疫治疗仅能使部分患者获益，而且可用的免疫治疗抑制剂数量也极为有限，客观缓解率不高，治疗副作用较大，或者使 用存在特定基因要求；目前患者能够选择的治疗药物并不多，因此，仍需要开发治疗效果更为显著、适用更广的药物，以满足临床的迫切需求。
发明内容
本发明公开了一种治疗尿路上皮癌的方法，所述方法包括向患者注射有效量的抗体药物偶联物(ADC)，所述ADC为抗HER2抗体偶联细胞毒分子。所述细胞毒分子包括但不限于微管蛋白抑制剂或DNA损伤剂。所述微管蛋白抑制剂包括但不限于海兔毒素(dolastatin)及奥瑞他汀(auristatin)类细胞毒分子，美登素(maytansine)类细胞毒分子；所述DNA损伤剂包括但不限于卡奇霉素(calicheamicin)类、倍癌霉素(duocarmycin)类、安曲霉素类衍生物PBD(pyrrolobenzodiazepine，吡咯并苯并二氮杂
Figure PCTCN2019101283-appb-000001
)、喜树碱类衍生物SN-38。所述奥瑞他汀(auristatin)类细胞素分子包括但不限于单甲基澳瑞他汀E(monomethyl auristatin E；MMAE)、单甲基澳瑞他汀F(monomethyl auristatin F；MMAF)、和澳瑞他汀F(auristatin F；AF)或它们的洐生物，所述美登素类细胞毒分子包括但不限于DM1、DM3、DM4或它们的洐生物(抗体药物偶联物的弹头分子研究进展，胡馨月等，中国医药生物技术，2017年12月，第12卷第6期)(美登素类抗体药物偶联物研究进展，周磊等，中国新药杂志2016年第25卷第22期，第2521-2530页)。所述细胞毒分子还可以是瓢菌素(amanitins)、蒽环类物(anthracyclines)、浆果赤霉素(baccatins)、喜树碱(camptothecins)、西马多丁(cemadotins)、秋水仙碱(colchicines)、秋水仙胺(colcimids)、考布他汀(combretastatins)、隐菲辛(cryptophycins)、圆皮海绵内酯(discodermolides)、多烯紫杉醇(docetaxel)、阿霉素(doxorubicin)、棘霉素(echinomycins)、艾榴塞洛素(eleutherobins)、埃博霉素(epothilones)、雌莫司汀(estramustines)、偏端霉素(lexitropsins)、美登素(maytansines)、氨甲蝶呤(methotrexate)、纺锤菌素(netropsins)、嘌呤霉素(puromycins)、根瘤菌素(rhizoxins)、紫杉烷(taxanes)、微管蛋白裂解素(tubulysins)、或长春花生物碱(vinca alkaloids)。所述细胞毒分子不限于上述类别，包括所有可用于ADC的药物。
本发明的另一个方面是提供了一种抗体药物偶联物(ADC)在制备用于治疗膀胱癌的药物中的用途；所述抗体药物偶联物包含结合HER2的抗体或其功能性片段，其中所述抗体包含重链可变区和轻链可变区，其中(i)所述重链可变区包含三个CDR区，其中所述CDR区的氨基酸序列分别具有如SEQ ID NO：1、2和3所示的氨基酸序列；和(ii)所述轻链可变区包含三个CDR区，其中所述CDR区的氨基酸序列分别具有如SEQ ID NO：4、5和6 所示的氨基酸序列。所述抗体还可以是与上述CDR限定的抗体竞争性结合相同或相近表位的抗体。
本发明中，术语“抗体”可以包括全长抗体或与HER2结合、反应性相关、或复合的抗体片段。抗体可以是任何蛋白质、类蛋白质分子、或多肽，其与寻求治疗性修饰的细胞群的一部分结合、复合、或反应。所述抗体可为嵌合抗体或其功能活性片段、人源化抗体或其功能活性片段、人类抗体或其功能活性片段。亦可为上述物种以外的其他物种的抗体或其其功能活性片段，例如：小鼠抗体或其功能活性片段、大鼠抗体或其功能活性片段、羊抗体或其功能活性片段、兔子抗体或其功能活性片段。抗体可为多克隆抗体或单克隆抗体。于一些实施例中，抗体可为双特异性抗体。此外，抗体也可为功能性活性片段、抗体的衍生物或类似物。“功能性”代表所述片段、衍生物或类似物可辨认相同的抗原，辨认衍生自抗原的片段、衍生物或类似物的抗体，例如但不限于：F(ab’) 2、Fab、Fab’、Fv片段及抗体的重链及轻链二聚体、或其任何最小片段像是Fvs或单链抗体(SCAs)。此外，抗体可为抗体的融合蛋白。抗体也可包括经修饰或未经修饰(亦即，通过任何分子的共价连接)的类似物和衍生物，只要这样的共价连接允许抗体保留其抗原结合免疫特异性。例如但不限于：抗体的类似物和衍生物，包括经过进一步修饰，例如：糖基化、乙酰化、聚乙二醇化、磷酸化、酰胺化、通过已知的保护/阻隔基衍生化、蛋白酶切割、连接至细胞抗体单元或其他蛋白质等。可利用已知技术实现任何大量的化学修饰，包括但不限于特异性化学切割、乙酰化、甲酰化、衣霉素存在下的代谢合成等。此外，类似物或衍生物可包括一种或多种非天然氨基酸。在一些实施例中，抗体可在与Fc受体作用的氨基酸残基中具有修饰(例如：取代、删除、或增加)。另一方面，所述抗体药物偶联物具有通式Ab-(L-U)n的结构，其中Ab表示所述的抗体或其功能性片段，L表示接头；U表示偶联的细胞毒分子，n为1至8的整数，代表每一抗体上结合的治疗剂的分子数量。
另一方面，所述接头与所述抗体或其功能性片段通过巯基和/或氨基连接，所述细胞毒分子定点或非定点偶联所述抗体。本发明的接头可以选自以下列表中的接头：
Figure PCTCN2019101283-appb-000002
本发明的接头优选马来酰亚胺基-己酰基-缬氨酸-瓜氨酸-p-氨基苄氧基(Maleimido-Caproyl-Valine-Citrulline-p-Aminobenzyloxy,mc-vc-pAB)和马来酰亚胺基己酰基(Maleimidocaproyl，mc)。
本发明的接头也可选择三甘氨酸肽接头(triglycyl peptide linker)，该接头是近年来开发的用于ADC药物偶联物的新接头。(Rajeeva Singh et al,A New Triglycyl Peptide Linker for Antibody–Drug Conjugates(ADCs)with Improved Targeted Killing of Cancer Cells,MCT-16-002,Published June 2016.)，此外，也可以选择葡萄糖醛酸接头(glucuronide-tubulysin)(Patrick J.Burke et al,Glucuronide-linked antibody-tubulysin conjugates display activity in MDR+and heterogeneous tumor models,Molecular Cancer Therapeutics,2018)。
[根据细则26改正08.10.2019]　
另一方面，其中所述抗体或其功能性片段衍生自在2013年08月22日以保藏编号CGMCC No.8102保藏于中国微生物菌种保藏管理委员会普通微生物中心的杂交瘤所分泌的抗体；地址：北京市朝阳区北辰西路1号院3号；
[根据细则26改正08.10.2019]　
另一方面，其中所述抗体是人源化抗体，优选地所述抗体是在2013年11月06日以保藏编号CCTCC C2013170保藏于中国典型培养物保藏中心的CHO细胞所分泌的抗体。地址：湖北 武汉 武汉大学
在一个实施例中，所使用的抗体药物偶联物名称为RC48-mc-vc-pAB –MMAE，符合通式Ab-(L-U)n的结构，RC48(一种人源化的抗HER2单克隆抗体)通过连接子mc-vc-pAB偶联MMAE，偶联数量为1-8个不等，包括偶联1个，2个，3个，4个，5个，6个，7个，8个或为偶联1-8个MMAE数量不等的抗体药物偶联物的组合。
本发明中，所述尿路上皮癌为无法接受手术切除的局部进展尿路上皮癌、局部晚期或转移性尿路上皮癌、HER2(人类表皮生长因子受体2，也称之为ErbB-2、c-erbB2或HER2/neu)阳性尿路上皮癌、HER2阳性局部晚期或转移性尿路上皮癌。
本发明中所述药物可通过鼻内、皮下、皮内、肌内或静脉内施用。
所述药物还包括药学上可接受的载体；所述药物优选为冻干剂或液体制剂；所述载体包括稳定剂、保护剂、缓冲液、冻干保护剂、活性保护剂、表面活性剂、吸附载体、吸收促进剂中的一种或多种。
附图说明
图1：纯化人重组蛋白HER2-ECD的SDS-PAGE图，经考马斯亮蓝染色。每孔上样量为10μg。
图2：表示cRC48(嵌合抗体)，RC48(人源化抗体)的SDS-PAGE分析图，抗体每孔上样量为2μg。
图3：显示了通过ELISA实验测定的人源化抗体RC48的HER2-ECD的结合亲和力，并计算出结合亲和力常数Kd。本实验中Herceptin和cRC48作为对照。
图4A:表示用流式细胞术分析抗HER2人源化抗体RC48结合HER2+细胞SK-BR3，BT474，HER2-细胞MDA-MB468的能力。
图4B:显示流式细胞术分析不同抗体浓度下抗HER2抗体结合BT474细胞表面抗原的能力。抗HER2抗体包括Herceptin，cRC48，RC48。共分析了5×10 4个细胞。
图5：表示RC48只对HER2表现出特异性结合亲和力，而对EGFR，HER3，HER4没有结合。
图6.受试者编号01001疗效示意图
患者基本情况：女，57岁，右肾盂癌术后，多发肺转移。病理诊断尿路上皮癌，HER2 IHC 3+；
图7.受试者编号01003疗效示意图
患者基本情况：女，45岁，右肾盂癌术后，腹腔淋巴结转移。病理诊断尿路上皮癌，HER2 IHC 3+。
图8.受试者编号01007疗效示意图
患者基本情况：男，63岁，膀胱癌术后，右肾盂癌术后，肺转移，肝转移，颈部淋巴结转移，纵隔转移，多发骨转移。病理诊断尿路上皮癌,，HER2 IHC 3+；
具体实施方式
实施例1.鼠源单克隆抗体mRC48的制备与序列分析
1、HER2抗原的表达和纯化
编码HER2-ECD(氨基酸Thr23至Thr652，GenBank登录号为M11730)的cDNA片段通过PCR克隆到pcDNA3(Invitrogen公司)表达载体上。
具体方法为：通过RT-PCR方法从HER2+SKBR3细胞株(ATCC号：HTB-30)中获得HER2-ECD编码区的cDNA(试剂盒采用Promega公司的ImProm-IITM Reverse Transcription System逆转录系统)。
引物为：P1：5’CG GGATCCTGCCACCAGCTGTGCGCC(SEQ ID NO：7)，P2：5’GCT CTAGA TCAGTTGATGGGGCAAGGCT(SEQ ID NO：8)，下划线序列分别为引入的BamHI、XbaI酶切位点，以反转录的HER2-ECD的cDNA为模板用上述引物进行PCR扩增，扩增条件是：94℃变性30s，60℃退火30s，72℃延伸1分钟，循环30次，最后72℃延伸10分钟。然后将PCR片段回收，用BamHI和XbaI酶(NEB)进行酶切，与pcDNA3载体连接。HER2-ECD的C末端加入了一个多聚组氨酸标签以方便纯化。用构建的DNA表达载体转染HEK293细胞(美国ATCC)，利用Ni-NTA亲和层析(Qiagen)从细胞培养液中纯化His-标记的可溶性蛋白HER2-ECD。SDS-PAGE及考马斯亮蓝染色表明纯化的HER2-ECD蛋白具有95％以上的同质性，结果见图1。可溶性的HER2-ECD以单体形式出现，相对分子量约为75kDa，比计算的分子量(71kDa)稍大，表明蛋白在HEK293细胞中进行了糖基化。将纯化得到的HER2-ECD蛋白进一步浓缩，并转移到无菌pH7.4的PBS缓冲液中，用于随后的体内和体外分析。
2、杂交瘤细胞的生产与筛选
使用上述制备的HER2-ECD作为抗原免疫小鼠，制备单克隆抗体。免 疫反应、杂交瘤细胞融合和初筛都按照标准步骤(参考文献：WHO Technical Report Series，No.822，1992 Annex 3)进行。0.25ml HER2-ECD蛋白(50-100μg)和0.25ml弗氏完全佐剂(Difco Lab)等体积混合均匀后免疫4只Balb/c小鼠(购自上海斯莱克实验动物有限责任公司)，间隔2周后进行第2次注射，用弗氏不完全佐剂(Difco Lab)，抗原量为25-50μg/0.5ml/只小鼠，间隔3周后进行第3次注射，注射剂量同第2次，第3次注射后10天取血。用酶联免疫吸附试验(ELISA)检测小鼠的血清，将血清中抗HER2抗体滴度最大的2只小鼠的脾脏取出，然后与骨髓瘤细胞P3X63Ag8(ATCC CRL-1580)融合。将融合细胞稀释到10块96孔板上，根据与HER2-ECD的结合能力用ELISA方法进行初筛。在典型的ELISA实验中，用HER2-ECD(0.2-1μg/ml)包被Nunc Maxisorb 96孔板，然后用梯度稀释的小鼠血清或杂交瘤上清液(100μL)孵育。鼠源的抗HER2抗体用辣根过氧化物酶偶联的山羊F(ab′)2抗鼠IgG Fc特异的二抗(Invitrogen公司)进行检测。
用ELISA法对400个杂交瘤细胞株的上清液进行筛选，其中36个表现出强的HER2-ECD结合力。选择HER2结合能力最强的十株杂交瘤细胞，通过有限稀释方法筛选亚克隆杂交瘤细胞株。通过悬浮亚克隆杂交瘤细胞株培养，蛋白纯化，用ELISA确定HER2的结合亲和力，用流式细胞仪(BD FACS Calibur)来进一步测试上述抗体对自然表达在人乳腺癌细胞株表面的HER2的结合能力(更详细的描述见实施例4)。最终通过序列分析确定了一株杂交瘤细胞系mRC48(鼠源IgG1k)，它具有强的HER2结合能力。所述杂交瘤细胞mRC48在2013年08月22日以保藏编号No.8102保藏于中国微生物菌种保藏管理委员会普通微生物中心(转为按照布达佩斯条约之保藏的日期为2013年10月29日)。
3、抗HER2杂交瘤细胞克隆mRC48的序列分析
上述杂交瘤细胞克隆mRC48重链和轻链的可变区根据说明书利用商业试剂盒SMARTTM RACE cDNA Amplification Kit(Clontech公司)快速扩增5’末端进行测序。
用RNApure Tissue Kit(北京康威世纪生物科技有限公司)从杂交瘤细胞中提取总RNA，使用SMARTTM RACE cDNA Amplification Kit进行总RNA的反转录，以总RNA为模板，利用试剂盒中的引物，加入反转录酶SMARTScribeTM Reverse Transcriptase，按照试剂盒提供的步骤进行反转录获得RACE-Ready第一链cDNA，然后进行两轮PCR，第一轮PCR以获得的cDNA为模板，试剂盒中提供的UPM为5’端引物，3’端引物为 mRC48-VL-1/mRC48-VH-1。PCR反应条件为：94℃预变性5min；25个扩增循环(94℃变性30s，68℃退火30s，72℃延伸2min)；最后72℃延伸10min。
第二轮PCR，以第一轮PCR的产物为模板，以试剂盒中提供的NUP为5’端引物，3’端引物为mRC48-VL-2/mRC48-VH-2，PCR反应条件为：94℃预变性5min；25个扩增循环(94℃变性30s，68℃退火30s，72℃延伸2min)；72℃延伸10min。这样既获得上述杂交瘤细胞克隆mRC48重链和轻链的可变区。
具体引物序列如下：
mRC48-VL-1:5’-GTTGGTGCAGCATCAGCCCGTT-3’(SEQ ID NO.9)
mRC48-VL-2:5’-GTTCACTGCCATCAATCTTCCAC-3’(SEQ ID NO.10)
mRC48-VH-1:5’-GCCAGTGGATAGACAGATGG-3’(SEQ ID NO.11)
mRC48-VH-2:5’-AGGTCACTGTCACTGGCTCAG-3’(SEQ ID NO.12)
PCR产物通过琼脂糖凝胶电泳进行纯化，并亚克隆到pCR2.1TOPO克隆载体上(Invitrogen公司)。通过PCR，获得10个独立克隆的质粒DNA，进而用M13正向和反向引物测序。DNA序列分析表明，这10个克隆都具有编码相同VH或VL多肽的cDNA。互补决定区(CDR)的氨基酸序列通过Kabat编码表定义，并在表1中列出。序列比较分析表明，抗HER2 mRC48的CDR与已知的HER2抗体包括Herceptin(曲妥珠单抗)有显著区别。
表1.抗HER2单克隆抗体mRC48 CDR的氨基酸序列
  重链(VH) 轻链(VL)
CDR1 DYYIH(SEQ ID NO.1) KASQDVGTAVA(SEQ ID NO.4)
CDR2 RVNPDHGDSYYNQKFKD(SEQ ID NO.2) WASIRHT(SEQ ID NO.5)
CDR3 ARNYLFDHW(SEQ ID NO.3) HQFATYT(SEQ ID NO.6)
实施例2.抗HER2单克隆抗体mRC48的人源化(源引自CN105008398A中相应方法)
通过移植轻链或重链CDR到人的IgG1κ框架区来人源化鼠抗HER2单克隆抗体mRC48。
设计了人源化RC48抗体的轻链可变区(RC48-VL)，以及人源化RC48抗体的重链可变区(RC48-VH)，从而组合成为人源化抗HER2抗体：RC48。RC48-VH的整体序列与人IgG1VH基因的相似性分别84％。RC48抗体包含轻链可变区RC48-VL和重链可变区RC48-VH。
人源化抗HER2单克隆抗体RC48通过CDR移植的方法获得，重链或轻链可变区是由南京金斯瑞生物科技有限公司直接合成，合成的可变区包括Kozak共有序列、起始密码子，重链或轻链信号肽，人源框架区和鼠源CDR，可变区与人IgG1k恒定区是通过重叠延伸PCR的方法连接成完整片段。
重叠延伸PCR的引物是：
重链VH1：5 CGCGGATCC GCCGCCACCATGGGATGGAGCT3′(SEQ ID NO：13)
VH2：5GATGGGCCCTTGGTGCTAGCGGAGCTCACTGTCACCAGTGTT3(SEQ ID NO：14)
CH1：5 GCTAGCACCAAGGGCCCATC 3(SEQ ID NO：15)
CH2：5 CCGGAATTCTTTACCGGGAGACAGGGAGA 3(SEQ ID NO：16)
轻链：VL1：5 CGCGGATCC GCCGCCACCATGGACATGAGGGT 3(SEQ ID NO：17)
VL2：5 GATGGTGCAGCCACAGTACGCTTTATCTCAACTTTTG T 3
AC3(SEQ ID NO：18)
CL1：5 CGTACTGTGGCTGCACCAT 3(SEQ ID NO：19)
CL2：5 CCGGAATTCACACTCTCCCCTGTTGAAGC 3(SEQ ID NO：20)
对于重链的扩增，首先以合成的可变区为模板，以VH1和VH2为引物，人IgG1κ重链恒定区为模板，以CH1和CH2为引物，分别扩增重链的可变区、恒定区，扩增条件均是：94℃变性30s，60℃退火30s，72℃延伸1分钟，循环30次，最后72℃延伸10分钟。再以两次的PCR产物为模板，VH1和CH2为引物，扩增RC48的重链序列。扩增条件均是：94℃变性30s，60℃退火30s，72℃延伸2分钟，循环30次，最后72℃延伸10分钟。
对于轻链的扩增，首先以合成的可变区为模板，以VL1和VL2为引物，以人IgG1κ轻链恒定区为模板，以CL1和CL2为引物，分别扩增轻链的可变区、恒定区，扩增条件均是：94℃变性30s，60℃退火30s，72℃延伸1分钟，循环30次，最后72℃延伸10分钟。再以两次的PCR产物为模板，VL1和CL2为引物，扩增轻链序列。扩增条件均是：94℃变性30s，60℃退火30s，72℃延伸2分钟，循环30次，最后72℃延伸10分钟。
因此，得到了人源化抗HER2单克隆抗体RC48，其中RC48包含人IgG1κ重链恒定区和重链可变区RC48-VH，以及人IgG1κ轻链恒定区和轻链可变区RC48-VL。
人-鼠嵌合抗体cRC48也是通过相同的方法获得，将鼠的可变区和人IgG1k恒定区通过重叠延伸PCR的方法连接成完整片段。
将上述扩增得到的各片段分别亚克隆到表达载体pcDNA3.0上。将构建的不同质粒转染到悬浮CHO细胞(Invitrogen)中，以产生不同的重组抗体，并利用Protein A进行纯化和后续的特征分析。嵌合的抗-HER2 RC48(被称为cRC48)是由鼠-人嵌合cRC48重链和轻链组成。RC48包括人源化重链RC48-VH和人源化轻链RC48-VL。cRC48和RC48都能够表达，从CHO细胞上清液中收集所述抗体，经Protein A进行纯化，在还原与非还原条件下用SDS-PAGE分析(见图2)。如上所述分泌RC48抗体的CHO细胞(即转染了人IgG1κ重链恒定区和重链可变区RC48-VH，以及人IgG1κ轻链恒定区和轻链可变区RC48-VL的CHO细胞)在2013年11月06日以保藏编号C2013170保藏于中国典型培养物保藏中心。
实施例3.抗HER2 RC48抗体的表征分析
用ELISA法测定cRC48(嵌合抗体)和RC48抗体(人源化抗体)的HER2-结合亲和力常数(Kd)，具体方法可见实施例1，即以可溶性HER2-ECD蛋白包被96孔板，之后与稀释的抗体(Herceptin和嵌合cRC48作为对照)一起孵育，对于与HER2-ECD相关的抗体(所有形式的人IgG1κ)用HRP-偶联的羊F(ab’)2抗-人IgG Fc-特异性二抗进行检测(invitrogen)。绘制结合曲线并进一步用单一位点特异性结合非线性方程(Journal of Immunological Methods，270：155-162，2002)，为每一个抗HER2抗体计算表面结合亲和力常数(Kd)值(图3中展示的是由3次独立的ELISA实验得出的一条典型HER2-结合曲线)。ELISA试验结果见图3。
从3次独立的测定试验可知，与cRC48(平均亲和常数77pM)和Herceptin(平均亲和常数97pM)相比，RC48(人源化抗体)有显著提高的HER2-ECD结合亲和力，平均亲和常数为44pM，结果见表2。
表2.本发明的抗体与Herceptin的平均亲和常数比较
样品 平均亲和常数
Herceptin 97pM
cRC48 77pM
RC48 44pM
实施例4.RC48(人源化抗体)与HER2的结合力
1)RC48抗体与HER2的结合亲和力试验
利用流式细胞仪来检测人乳腺癌细胞内源表达的HER2与人源化抗HER2抗体RC48的结合力，结果见图3。用6μg对照组人IgG、Herceptin、cRC48、RC48分别与两种HER2+细胞系人乳腺癌细胞SK-BR-3、BT474以及HER2-细胞MDA-MB468(2×10 7个细胞)共同孵育，冰上孵育30-45分钟。用4ml冷的PBS充分洗涤2次之后，细胞表面结合的抗体通过偶联R-PE的山羊抗人IgG Fc(15μl，25μg/mL)特异性二抗检测，然后利用流式细胞仪(BDFACSCalibur)进行分析。对照组人IgG1没有检测出与上述三种癌症细胞的结合。相比而言，Herceptin、cRC48、RC48与两种HER2阳性细胞强 烈结合，但与HER2阴性细胞不结合，这说明这种结合是HER2特异性的(如图4a所示)。通过比较同种对照组的平均荧光强度发现，与Herceptin和cRC48相比，RC48表现出更高的结合亲和力。通过滴定抗HER2抗体的浓度，以及流式细胞术中分析的细胞数目，得出基于细胞的抗HER2的抗体与细胞表面HER2的结合曲线，结果见图4b。人源化的抗HER2抗体RC48表现出明显的结合亲和力，与BT474细胞表面HER2结合亲和力Kd为4nM，Herceptin、cRC48分别为10nM和5nM，结果见表3：
样品 结合亲和力Kd
Herceptin 10nM
cRC48 5nM
RC48 4nM
2)抗原结合特异性试验
用ELISA方法测定Herceptin、cRC48、RC48结合不同的表面抗原EGFR、HER2、HER3、HER4的能力。ELISA方法见实施例1。分别用抗原EGFR、HER2、HER3、HER4包被96孔板，每孔上样量为20ng，用不同的抗HER2抗体进行孵育，即Herceptin、cRC48、RC48抗体，然后用辣根过氧化物酶偶联的羊F(ab′)2抗鼠IgG Fc特异的二抗(Invitrogen公司)进行检测。结果见图5，结果表明Herceptin、cRC48、RC48抗体与EGFR、HER3、HER4几乎无结合力，而与HER2有强的结合力，说明Herceptin、RC48对HER2的结合有高度的特异性。
实施例5.抗体偶联物的制备
1)单克隆抗体RC48的纯化
经Protein A从CHO细胞培养液中捕获得到RC48的单克隆抗体，SDS-PAGE电泳及SEC分析纯度达到95％以上。用30KD膜包将所获抗体蛋白超滤透析到PBS缓冲液中，浓缩，用紫外吸光光度计标定浓度，用于后续的偶联反应。
2)单克隆抗体RC48与药物分子的偶联
用PBS缓冲液分别配制还原剂和保护剂如下：1～20mmol/L  TCEP(Tris-2-carboxyethyl-phosphine)、1～20mmol/L DTPA(Diethylene triamine pentacetate acid)母液，还原剂用量根据所需偶联率不同可在一定浓度范围内添加，与一定浓度单克隆抗体(如：5～30mg/ml)按照一定体积比(如1∶1)混合，TCEP与抗体的终浓度摩尔比0.5～6.0∶1，于25℃搅拌反应2h。用DTNB法于412nm检测自由巯基浓度，与抗体的摩尔比计算自由巯基个数。TCEP还原可重复性好，还原后自由巯基数可以达到1.0-8.0。
TCEP还原后抗体可直接进行后续偶联。配制一定浓度(10mM)药物(vc-MMAE、vc-MMAF、mc-MMAF)(购自上海皓元化学科技有限公司)溶于25％的DMSO(dimethyl sulfoxide，二甲亚砜)，按照药物与巯基的摩尔比0.3～2.8∶1缓慢加药，于25℃搅拌反应2h。用DTNB法于412nm检测自由巯基浓度(接近0)，Sephadex G-25纯化除去残余未反应药物以及DMSO等游离小分子，SDS-PAGE电泳、反相高效液相(R-HPLC)疏水高效液相(HIC-HPLC)法检测偶联情况。
实施例6.抗体药物偶联物亲和力的测定
ELISA法测亲和力
用重组蛋白HER2-ECD(浓度为0.5mg/ml)包被ELISA板，2℃-8℃过夜。洗板机洗板3次。3％BSA-PBST溶液封闭，37度2h。洗板机洗板3次。加样：用PBST溶液稀释标线从1000ng/ml起梯度稀释11个点，100μl/孔，37度2h。洗板机洗板3次。用PBST溶液稀释二抗(山羊抗人IgG-Fc-HRP)5000倍。加入TMB显色液显色，室温避光显色8-10分钟。用2M H2SO4终止试验，酶标仪450/655nm处读数。结果见表4。
表4.抗体药物偶联物与T-DM1亲和力比较(VC为mc-vc-pAB缩写)
Figure PCTCN2019101283-appb-000003
由结果可知，RC48-VC-MMAE(即RC48-mc-vc-pAB–MMAE)、RC48-VC-MMAF、RC48-MC-MMAF的HER2-ECD亲和力与T-DM1相当。
实施例7.单药治疗HER2阳性局部晚期或转移性尿路上皮癌的有效性和安全性实验
本实验的目的是评价单药治疗HER2阳性局部晚期或转移性尿路上皮癌的有效性和安全性。所使用的抗体药物偶联物为RC48-mc-vc-pAB–MMAE，RC48通过连接子mc-vc-pAB偶联MMAE，偶联数量为1-8个不等。
其中对于受试者的选择标准为：
年龄：18岁(最小年龄)至80岁(最大年龄)
其中入选标准为：
1.自愿同意参与研究并签署知情同意书；
2.男性或女性，年龄大于等于18岁且小于60岁；
3.预期生存期大于等于12周；
4.具有病理学确诊的无法接受手术完全切除的局部进展或转移性膀胱尿路上皮癌；
5.受试者确诊为无法手术切除的局部进展或转移性疾病后接受过至少一线全身化疗后出现疾病进展或不能耐受；
6.至少具有RECIST 1.1标准规定的可测量病灶；
7.中心实验室确认的HER2表达阳性；
8.ECOG体力状况0或1分；
9.足够的心、骨髓、肝、肾功能；
10.对于女性受试者：应为手术绝育、绝经后的患者，或同意在研究治疗期间和研究治疗期结束后6个月内至少采用一种经医学认可的避孕措施(如宫内节育器，避孕药或避孕套)；男性受试者应同意在研究治疗期间和研究治疗期结束后6个月内至少采用一种经医学认可的避孕措施(如避孕套、禁欲等)；
11.愿意且能够遵从试验和随访程序安排。
同时，排除标准为：
1.已知对重组人源化抗HER2单抗-MMAE偶联剂药物及其组分过敏者；
2.研究治疗开始前4周内接受过其他抗肿瘤治疗；
3.既往接受过重组人源化抗HER2单抗-MMAE偶联剂药物；
4.研究给药开始前4周内进行过大型手术且未完全恢复；
5.研究给药开始前4周内接种过活疫苗或计划在研究期间接受任何疫苗；
6.其他可能影响方案依从性或干扰结果解释的严重的、无法控制的伴随疾病；
7.研究给药开始前5年内患有其他恶性肿瘤；
8.患有中枢神经系统转移和/或癌性脑膜炎；
9.有在过去2年内需要系统治疗的活动性自身免疫性疾病；
10.既往接受过异体造血干细胞移植或实体器官移植；
11.伴有临床症状或需要对症处理的大量胸水或腹水；
12.妊娠或哺乳期妇女；
13.HIV检测结果阳性；
14.活动性乙型肝炎或丙型肝炎患者；
15.活动性结核病史；
16.患有任何其它疾病，代谢异常，体格检查异常或实验室检查异常，根据研究者判断，有理由怀疑患者具有不适合使用研究药物的某种疾病或状态，或者将会影响研究结果的解读，或者使患者处于高风险的情况；
17.估计患者参加本临床研究的依从性不足。
具体实验方法：
本研究将纳入既往接受过至少一线全身化疗失败或不能耐受的局部晚期或转移性尿路上皮癌受试者，且具有可测量病灶，一般情况及器官功能良好。所有受试者送检肿瘤组织病理切片至中心实验室检测确认HER2表达阳性，其阳性定义为免疫组化(IHC)为2+或3+(无论荧光原位杂交[FISH]检测结果)。
本研究中HER2免疫组化(IHC)的结果判定依据中国《乳腺癌HER2检测指南(2014版)》中的HER2判读标准。具体如表5所示。
表5中国《乳腺癌HER2检测指南(2014版)》中的HER2判读标准。
Figure PCTCN2019101283-appb-000004
符合所有研究人群标准的受试者接受RC48-ADC治疗(2.0mg/kg，静脉滴注，每2周一次)，每6周进行疗效评价，直至发生疾病进展、不可耐受毒性或主动退出。研究的主要终点指标为独立评估的客观缓解率(ORR)，次要研究终点为无进展生存期、总生存期以及治疗的安全性。
研究结果：
该研究于2017年12月启动，截至2018年7月31日，共入组18例受试者接受治疗，其中男性15例，女性3例，中位年龄63岁。原发病灶包括膀胱(50.0％)、肾盂(27.8％)和输尿管(22.2％)，主要的转移部位为肺、肝以及淋巴结转移。16例(88.9％)既往接受过一线铂类治疗。受试者肿瘤HER2表达经中心实验室确认的免疫组化(IHC)结果为11例(61.1％)IHC2+，7例(38.9％)IHC3+。
18例受试者中有13例受试者进行了疗效评估，部分缓解(PR)10例(4例已确认PR(连续两次评效为PR称为确认PR)。根据RECIST标准“当每一个受试者符合部分或者完全缓解标准而且在随后的时间点(一般是四周后)再次做疗效确认后才能认为是完全或者部分缓解”，即受试者连续两次评效为PR，称为确认PR。其他6例还未到再次疗效确认时间点，目前仅完 成一次确认)，客观缓解率(ORR)为76.9％(10/13)，疾病控制率(DCR)(13例疗效评估者中10例PR，2例疾病稳定(SD))为92.3％(12/13)。目前受试者最长治疗时间超过7个月。获得疾病缓解的受试者中，7例(53.8％)接受过紫杉类药物治疗，4例(30.8％)接受过PD-1/PD-L1治疗。
18例受试者中，有16例接受了安全性评估，安全性评估中最常见的治疗相关不良反应(TRAEs)为ALT升高(50.0％,1-2级)、感觉减退(50.0％,1-2级)和白细胞计数降低(50.0％,1-2级)；≥3级的TRAE为中性粒细胞计数下降(12.5％，3级)。无药物相关严重不良事件(SAE)发生。
下方为部分病例直观治疗效果描述：
1.患者01001：女，57岁，右肾盂癌术后，多发肺转移。病理诊断尿路上皮癌，HER2 IHC 3+；
根据图6可以看出：经过长达12周的治疗，其位于右肺下叶的肿瘤病灶经过6周、12周的治疗后，肿瘤病灶从54×31mm缩小到37×23mm，缩小了49％。
2.患者01003，女，45岁，右肾盂癌术后，腹腔淋巴结转移。病理诊断尿路上皮癌，HER2 IHC 3+；
根据图7可以看出：通过6周的治疗，其位于右腰大肌旁的肿瘤病灶从56×48mm缩小到33×22mm，缩小了72.9％。
3.患者01007，男，63岁，膀胱癌术后，右肾盂癌术后，肺转移，肝转移，颈部淋巴结转移，纵隔转移，多发骨转移。病理诊断尿路上皮癌,，HER2 IHC 3+；
根据图8可以看出：位于左上肺叶、肝转移、纵隔淋巴结的肿瘤病灶经过为期6周的治疗，病灶处的肿瘤面积均有明显的缩小。左上肺叶从45×36mm缩小到28×22mm，减少61.9％，肝转移处从38×32mm缩小到25×21mm，减少56.8％，纵隔淋巴结从29×15mm缩小到18×7mm，减少71.03％。
以上临床数据及直观病理检测均表明本发明的抗HER2单抗-MMAE偶联剂药物具有非常显著的治疗效果；通过与目前的上市药物对比，也可以看出：其明显优于目前已经被欧盟和美国批准的同类药物，例如：罗氏的Atezolizumab三期临床数据显示ORR仅为23％，百时美施贵宝公司的Nivolumab的ORR仅为19.6％，而杨森公司的erdafitinib仅针对具有某些FGFR基因突变的尿路上皮癌患者，反观本发明的重组人源化抗HER2单抗 -MMAE偶联剂药物的ORR为76.9％，疾病控制率(DCR)为92.3％，明显优于目前上市的同类药物，并且副作用也明显小于同类药物，未发生任何严重不良反应(SAE)，针对的患者更为广泛，为急需治疗的尿路上皮癌患者提供了另一种选择；由此可见，本发明的抗HER2抗体偶联物在治疗尿路上皮癌中有着极佳的应用前景，能够有效改善，甚至逆转患者的疾病发展进程，取得了预料不到的技术效果。

Claims (18)

  1. 一种抗体药物偶联物(ADC)在制备用于治疗尿路上皮癌的药物中的用途，所述抗体药物偶联物为抗HER2抗体或其功能性片段偶联细胞毒分子。
  2. 权利要求1所述的用途，所述细胞毒分子包括但不限于微管蛋白抑制剂或DNA损伤剂。
  3. 权利要求2所述的用途，所述微管蛋白抑制剂包括但不限于海兔毒素(dolastatin)及奥瑞他汀(auristatin)类细胞毒分子，美登素(maytansine)类细胞毒分子；所述DNA损伤剂包括但不限于卡奇霉素类(calicheamicin)、倍癌霉素(duocarmycin)类、安曲霉素类衍生物PBD、喜树碱类衍生物SN-38。
  4. 权利要求1所述的用途，所述细胞毒分子包括但不限于瓢菌素(amanitins)、蒽环类物(anthracyclines)、浆果赤霉素(baccatins)、喜树碱(camptothecins)、西马多丁(cemadotins)、秋水仙碱(colchicines)、秋水仙胺(colcimids)、考布他汀(combretastatins)、隐菲辛(cryptophycins)、圆皮海绵内酯(discodermolides)、多烯紫杉醇(docetaxel)、阿霉素(doxorubicin)、棘霉素(echinomycins)、艾榴塞洛素(eleutherobins)、埃博霉素(epothilones)、雌莫司汀(estramustines)、偏端霉素(lexitropsins)、美登素(maytansines)、氨甲蝶呤(methotrexate)、纺锤菌素(netropsins)、嘌呤霉素(puromycins)、根瘤菌素(rhizoxins)、紫杉烷(taxanes)、微管蛋白裂解素(tubulysins)、或长春花生物碱(vinca alkaloids)。
  5. 权利要求3所述的用途，所述奥瑞他汀(auristatin)类细胞素分子包括但不限于MMAE或MMAF或它们的洐生物，所述美登素类细胞毒分子包括但不限于DM1、DM4或它们的洐生物。
  6. 根据前述任一权利要求所述的用途，所述抗体或其功能性片段与以下CDR序列限定的抗体竞争性结合相同表位；所述CDR序列限定的抗体包含重链可变区和轻链可变区，其中
    (i)所述重链可变区包含三个CDR区，其中所述CDR区的氨基酸序列分别具有如SEQ ID NO：1、2和3所示的氨基酸序列；和
    (ii)所述轻链可变区包含三个CDR区，其中所述CDR区的氨基酸序列分别具有如SEQ ID NO：4、5和6所示的氨基酸序列。
  7. 根据权利要求1-5中任一项的用途，所述抗体或其功能性片段包含重链可变区和轻链可变区，其中
    (i)所述重链可变区包含三个CDR区，其中所述CDR区的氨基酸序列分别具有如SEQ ID NO：1、2和3所示的氨基酸序列；和
    (ii)所述轻链可变区包含三个CDR区，其中所述CDR区的氨基酸序列分别具有如SEQ ID NO：4、5和6所示的氨基酸序列。
  8. 根据前述任一权利要求所述的用途，所述抗体药物偶联物具有通式Ab-(L-U)n的结构，其中Ab表示所述的抗体或其功能性片段，L表示接头；U表示偶联的细胞毒分子，n为1至8的整数，代表每一抗体上结合的治疗剂的分子数量。
  9. 权利要求8所述的用途，所述偶联的细胞毒分子包括但不限于DM1、DM3、DM4、MMAE、MMAF、AF(auristatin F)或它们的洐生物。
  10. 根据前述任一权利要求所述的用途，所述接头与所述抗体或其功能性片段通过巯基和/或氨基连接。
  11. 权利要求10所述的用途，所述接头选自马来酰亚胺基-己酰基-缬氨酸-瓜氨酸-p-氨基苄氧基(Maleimido-Caproyl-Valine-Citrulline-p-Aminobenzyloxy,mc-vc-pAB)、马来酰亚胺基己酰基(Maleimidocaproyl，mc)、三甘氨酸肽接头(triglycyl peptide linker)、3-马来酰亚胺-丙酸(3-maleimido-propionic acid)、Mal-di-EG-OPFP(perfluorophenyl 3-(2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)ethoxy)ethoxy)propanoate)、Mal-di-EG-Osu(2,5-dioxopyrrolidin-1-yl 3-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)propanoate)、Mal-Tri-EG-OSu(2,5-dioxopyrrolidin-1-yl 3-(2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)ethoxypro panoate)、Mal-Tetra-EG-OSu(2,5-dioxopyrrolidin-1-yl 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-oxo-7,10,13,16-tetraoxa-4-azan onadecan-19-oate)、Br-di-EG-OSu(2,5-dioxopyrrolidin-1-yl 3(2-(2-(2-bromoacetamido)ethoxy)ethoxy)propanoate)、Py-ds-prp-OSu(2-5-dioxopyrrolidin-1-yl 3-(pyridine-2-yldisulfanyl)propanoate)、Py-ds-Prp-OPEP(perfluorophenyl 3-(pyridine-2-yldisulfanyl)propanoate)、Py-ds-dmBut-OSu(2,5-dioxopyrrolidin-1-yl 4-methyl-4-(pyridine-2-yldisulfanyl)、Py-ds-dmBut-OPF(perfluorophenyl  4-methyl-4-(pyridine-2-yldisulfanyl)pentanoate)、SMCC(N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate)、MBS(3-maleimidobenzoic acid N-hydroxysuccinimide ester)、SATA(S-(N-succinimidyl)thioacetate)、SPDP((N-succinimidyl 3-(2-pyridyldithio)propionate)、SMPT((N-succinimidyloxy carbonyl)-1-methyl-1-(2-pyridyldithio)toluene)。
  12. 根据前述任一权利要求所述的用途，所述细胞毒分子定点或非定点偶联所述抗体。
  13. 根据前述任一权利要求所述的用途，其中所述抗体或其功能性片段为鼠源、嵌合或人源化的。
  14. 根据前述任一权利要求所述的用途，其中所述抗体或其功能性片段衍生自在2013年08月22日以保藏编号CGMCC No.8102保藏于中国微生物菌种保藏管理委员会普通微生物中心的杂交瘤所分泌的抗体。
  15. 根据前述任一权利要求所述的用途，其中所述抗体是人源化抗体，优选地所述抗体是在2013年11月06日以保藏编号CCTCC C2013170保藏于中国典型培养物保藏中心的CHO细胞所分泌的抗体。
  16. 根据前述任一项权利要求所述的用途，所述尿路上皮癌为无法接受手术切除的局部进展尿路上皮癌、局部晚期或转移性尿路上皮癌、HER2阳性尿路上皮癌、HER2阳性局部晚期或转移性尿路上皮癌。
  17. 根据前述任一项权利要求所述的用途，所述药物还包括药学上可接受的载体；所述药物优选为冻干剂或液体制剂；所述载体包括稳定剂、保护剂、缓冲液、冻干保护剂、活性保护剂、表面活性剂、吸附载体、吸收促进剂中的一种或多种。
  18. 根据前述任一项权利要求所述的用途，所述药物可通过鼻内、皮下、皮内、肌内或静脉内施用。
PCT/CN2019/101283 2018-08-29 2019-08-19 抗her2抗体药物偶联物在治疗尿路上皮癌中的用途 WO2020042941A1 (zh)

Priority Applications (14)

Application Number Priority Date Filing Date Title
RU2020117761A RU2750817C1 (ru) 2018-08-29 2019-08-19 Применение конъюгата анти-her2 антитело-лекарственное средство при лечении уротелиальной карциномы
KR1020237012001A KR20230051318A (ko) 2018-08-29 2019-08-19 요로상피 암종의 치료에서 항―ｈｅｒ２ 항체―약물 접합체의 용도
AU2019330680A AU2019330680A1 (en) 2018-08-29 2019-08-19 Use of anti-HER2 antibody-drug conjugate in treating urothelial carcinoma
CA3077260A CA3077260C (en) 2018-08-29 2019-08-19 Use of anti-her2 antibody-drug conjugate in treating urothelial carcinoma
EP19855043.6A EP3845252A4 (en) 2018-08-29 2019-08-19 USE OF AN ANTI-HER2-DRUG ANTIBODY CONJUGATE IN THE TREATMENT OF UROTHELIAL CARCINOMA
KR1020207029858A KR102520974B1 (ko) 2018-08-29 2019-08-19 요로상피 암종의 치료에서 항―ｈｅｒ２ 항체―약물 접합체의 용도
US16/652,413 US20200289663A1 (en) 2018-08-29 2019-08-19 Use of anti-her2 antibody-drug conjugate in treating urothelial carcinoma
JP2020529589A JP7105304B2 (ja) 2018-08-29 2019-08-19 尿路上皮がんの治療における抗ｈｅｒ２抗体－薬物コンジュゲートの使用
SG11202009130XA SG11202009130XA (en) 2018-08-29 2019-08-19 Use of anti-her2 antibody-drug conjugate in treating urothelial carcinoma
KR1020247006234A KR20240033092A (ko) 2018-08-29 2019-08-19 요로상피 암종의 치료에서 항―ｈｅｒ２ 항체―약물 접합체의 용도
CN201980002467.0A CN111655295B (zh) 2018-08-29 2019-08-19 抗her2抗体药物偶联物在治疗尿路上皮癌中的用途
BR112020010856A BR112020010856A8 (pt) 2018-08-29 2019-08-19 Uso de conjugado de anticorpo anti-her2-fármaco no tratamento de carcinoma urotelial
JP2022111260A JP7470154B2 (ja) 2018-08-29 2022-07-11 尿路上皮がんの治療における抗ｈｅｒ２抗体－薬物コンジュゲートの使用
AU2022252734A AU2022252734A1 (en) 2018-08-29 2022-10-11 Use of anti-HER2 antibody-drug conjugate in treating urothelial carcinoma

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201810998055.4 2018-08-29
CN201810998055 2018-08-29

Publications (1)

Publication Number Publication Date
WO2020042941A1 true WO2020042941A1 (zh) 2020-03-05

Family

ID=69643195

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/101283 WO2020042941A1 (zh) 2018-08-29 2019-08-19 抗her2抗体药物偶联物在治疗尿路上皮癌中的用途

Country Status (12)

Country Link
US (1) US20200289663A1 (zh)
EP (1) EP3845252A4 (zh)
JP (2) JP7105304B2 (zh)
KR (3) KR20230051318A (zh)
CN (1) CN111655295B (zh)
AU (2) AU2019330680A1 (zh)
BR (1) BR112020010856A8 (zh)
CA (1) CA3077260C (zh)
RU (1) RU2750817C1 (zh)
SG (1) SG11202009130XA (zh)
TW (1) TWI767139B (zh)
WO (1) WO2020042941A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022242692A1 (en) * 2021-05-21 2022-11-24 Remegen Co., Ltd. Use of antibody-drug conjugate in combination with immune checkpoint inhibitor in treatment of urothelial cancer

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20230051318A (ko) * 2018-08-29 2023-04-17 레메젠 코, 리미티드 요로상피 암종의 치료에서 항―ｈｅｒ２ 항체―약물 접합체의 용도
IL308508A (en) * 2021-05-24 2024-01-01 Remegen Co Ltd Use of a HER2-targeted antibody-drug conjugate for the treatment of low-HER2-expressing breast cancer
CN115925954A (zh) * 2022-12-28 2023-04-07 广州誉衡生物科技有限公司 抗-pd-1抗体及其在制备治疗尿路上皮癌患者的药物中的用途

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103319599A (zh) * 2013-05-27 2013-09-25 上海交联药物研发有限公司 一种抗人ErbB2抗体—美登木素偶联物及其应用
WO2015074528A1 (zh) * 2013-11-19 2015-05-28 烟台荣昌生物工程有限公司 抗her2抗体及其缀合物
WO2016064649A1 (en) 2014-10-24 2016-04-28 Eli Lilly And Company Therapy for urothelial carcinoma
US20170209594A1 (en) * 2015-06-25 2017-07-27 Immunomedics, Inc. Synergistic effect of anti-trop-2 antibody-drug conjugate in combination therapy for triple-negative breast cancer when used with microtubule inhibitors or parp inhibitors
CN107029244A (zh) * 2016-02-04 2017-08-11 浙江昭华生物医药有限公司 抗her2抗体-药物偶联物及其应用
WO2018102589A2 (en) * 2016-11-30 2018-06-07 Atyr Pharma, Inc. Anti-hrs antibodies and combination therapies for treating cancers

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150080559A1 (en) * 2011-05-27 2015-03-19 Ambrx, Inc. Compositions Containing, Methods Involving, and Uses of Non-Natural Amino Acid Linked Dolastatin Derivatives
US10407743B2 (en) * 2014-05-22 2019-09-10 Synthon Biopharmaceuticals B.V. Site-specific conjugation of linker drugs to antibodies and resulting ADCs
MX2017014736A (es) * 2015-05-29 2018-03-23 Genentech Inc Metodos terapeuticos y diagnosticos para cancer.
CN107789631B (zh) * 2017-11-03 2021-03-16 合肥瀚科迈博生物技术有限公司 抗人ErbB2双表位抗体-药物偶联物及其应用
KR20230051318A (ko) * 2018-08-29 2023-04-17 레메젠 코, 리미티드 요로상피 암종의 치료에서 항―ｈｅｒ２ 항체―약물 접합체의 용도

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103319599A (zh) * 2013-05-27 2013-09-25 上海交联药物研发有限公司 一种抗人ErbB2抗体—美登木素偶联物及其应用
WO2015074528A1 (zh) * 2013-11-19 2015-05-28 烟台荣昌生物工程有限公司 抗her2抗体及其缀合物
WO2016064649A1 (en) 2014-10-24 2016-04-28 Eli Lilly And Company Therapy for urothelial carcinoma
US20170209594A1 (en) * 2015-06-25 2017-07-27 Immunomedics, Inc. Synergistic effect of anti-trop-2 antibody-drug conjugate in combination therapy for triple-negative breast cancer when used with microtubule inhibitors or parp inhibitors
CN107029244A (zh) * 2016-02-04 2017-08-11 浙江昭华生物医药有限公司 抗her2抗体-药物偶联物及其应用
WO2018102589A2 (en) * 2016-11-30 2018-06-07 Atyr Pharma, Inc. Anti-hrs antibodies and combination therapies for treating cancers

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
"Essence of Urology Cases", 2017, PEKING UNIVERSITY MEDICAL PRESS
ANONYMOUS: "[ASCO 2019] China's original research ADC drug appeared on the international stage, bringing a breakthrough for the treatment of HER2 positive urothelial cancer", 5 June 2019 (2019-06-05), pages 1 - 2, XP055680652, Retrieved from the Internet <URL:http://www.sohu.com/a/318774405_333130> *
BELLMUNT, J. ET AL., J CLIN. ONCOL., vol. 27, no. 27, 2009, pages 4454 - 4461
HU XINYUE ET AL., CHIN MED BIOTECHNOL, vol. 12, no. 6, December 2017 (2017-12-01)
JOURNAL OF IMMUNOLOGICAL METHODS, vol. 270, 2002, pages 155 - 162
LI MEI-REN ET AL : "Gene amplification and protein expression of the human epidermal growth factor receptor 2 in Urothelial Carcinoma of bladder", JOURNAL OF GANNAN MEDICAL UNIVERSITY, vol. 35, no. 4, 31 August 2015 (2015-08-31), Gannan Yixueyuan CN, pages 514 - 517, 528, XP009519603, ISSN: 1001-5779 *
PATRICK J. BURKE ET AL.: "Glucuronide-linked antibody-tubulysin conjugates display activity in MDR+ and heterogeneous tumor models", MOLECULAR CANCER THERAPEUTICS, 2018
RAJEEVA SINGH ET AL.: "ANew Triglycyl Peptide Linker for Antibody-Drug Conjugates (ADCs) with Improved Targeted Killing of Cancer Cells", MCT-16-002, June 2016 (2016-06-01)
VLACHOSTERGIOS, PANAGIOTIS J. ET AL.: "Antibody-Drug Conjugates in Bladder Cancer", BLADDER CANCER, vol. 4, no. 3, 30 July 2018 (2018-07-30), pages 247 - 259, XP055680273, DOI: 10.3233/BLC-180169 *
YAFI, F.A. ET AL., CURRENT ONCOL., vol. 18, no. 1, 2011, pages e25 - e34
ZHOU LEI ET AL.: "Research progress of maytansinoid antibody drug conjugates", CHINESE JOURNAL OF NEW DRUGS, vol. 25, no. 22, pages 2521 - 2530

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022242692A1 (en) * 2021-05-21 2022-11-24 Remegen Co., Ltd. Use of antibody-drug conjugate in combination with immune checkpoint inhibitor in treatment of urothelial cancer

Also Published As

Publication number Publication date
KR20240033092A (ko) 2024-03-12
EP3845252A1 (en) 2021-07-07
CA3077260A1 (en) 2020-03-05
EP3845252A4 (en) 2022-06-15
TW202023626A (zh) 2020-07-01
RU2750817C1 (ru) 2021-07-05
CN111655295A (zh) 2020-09-11
AU2019330680A1 (en) 2020-04-23
TWI767139B (zh) 2022-06-11
JP7105304B2 (ja) 2022-07-22
BR112020010856A8 (pt) 2023-03-07
JP2022130744A (ja) 2022-09-06
KR20230051318A (ko) 2023-04-17
JP2021504427A (ja) 2021-02-15
TW202310877A (zh) 2023-03-16
BR112020010856A2 (pt) 2021-03-09
AU2022252734A1 (en) 2022-11-03
KR102520974B1 (ko) 2023-04-17
SG11202009130XA (en) 2020-10-29
CA3077260C (en) 2023-03-07
JP7470154B2 (ja) 2024-04-17
CN111655295B (zh) 2024-05-28
US20200289663A1 (en) 2020-09-17
KR20200134268A (ko) 2020-12-01

Similar Documents

Publication Publication Date Title
JP6728264B2 (ja) 抗ｈｅｒ２抗体及びその結合体
WO2020042941A1 (zh) 抗her2抗体药物偶联物在治疗尿路上皮癌中的用途
CA3160557A1 (en) Treatment of cancer
TWI843105B (zh) 抗her2 抗體藥物偶聯物在治療尿路上皮癌中的用途
JP2024086799A (ja) 尿路上皮がんの治療における抗ｈｅｒ２抗体－薬物コンジュゲートの使用

Legal Events

Date Code Title Description
ENP Entry into the national phase

Ref document number: 3077260

Country of ref document: CA

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19855043

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2019330680

Country of ref document: AU

Date of ref document: 20190819

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2020529589

Country of ref document: JP

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 20207029858

Country of ref document: KR

Kind code of ref document: A

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112020010856

Country of ref document: BR

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 112020010856

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20200529

ENP Entry into the national phase

Ref document number: 2019855043

Country of ref document: EP

Effective date: 20210329