[go: up one dir, main page]

WO2011006001A1 - Modèle animal pour l'évaluation de thérapies d'adjuvant du cancer - Google Patents

Modèle animal pour l'évaluation de thérapies d'adjuvant du cancer Download PDF

Info

Publication number
WO2011006001A1
WO2011006001A1 PCT/US2010/041420 US2010041420W WO2011006001A1 WO 2011006001 A1 WO2011006001 A1 WO 2011006001A1 US 2010041420 W US2010041420 W US 2010041420W WO 2011006001 A1 WO2011006001 A1 WO 2011006001A1
Authority
WO
WIPO (PCT)
Prior art keywords
tumor
animal
agent
cancer
lymph node
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/US2010/041420
Other languages
English (en)
Inventor
Anil D. Bagri
Maresa Caunt
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Genentech Inc
Original Assignee
Genentech Inc
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
Application filed by Genentech Inc filed Critical Genentech Inc
Publication of WO2011006001A1 publication Critical patent/WO2011006001A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0331—Animal model for proliferative diseases
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0393—Animal model comprising a reporter system for screening tests

Definitions

  • the present invention relates to methods and compositions useful for identifying and testing compounds for use in treating adjuvant cancer.
  • Adjuvant therapy refers to treatment administered to newly diagnosed cancer patients following removal of all detectable disease, typically by surgery (Mano et al., (2008) Clinical Colorectal Cancer 7: 178-183).
  • high recurrence rates in many indications suggests micro-metastases or residual tumor often remain behind.
  • the aim of adjuvant therapy is to target residual disease and thereby improve disease free and overall survival, and to potentially cure the patient's cancer.
  • Adjuvant chemotherapy and radiotherapy are currently approved following surgery for colon, lung, breast, prostate, pancreatic and certain other cancers. While relatively safe and efficacious, some patients continue to progress and these treatments are associated with a number of toxicities. Thus, additional therapies for adjuvant cancer are desirable.
  • preclinical tumor models simulate treatment in a first-line setting rather than treatment following disease progression, or in the adjuvant setting.
  • human cancer cell lines are injected into immune-deficient mice, which are then treated with test agents, and evaluated for size of the primary tumor and not metastatic burden or survival (Sausville and Burger, (2006) Cancer Res 66:3351-3354). Minor modifications to these models allow them to better reflect the biology relevant to adjuvant disease.
  • the primary tumor is allowed to establish followed by either surgical excision or chemo-ablation with an aggressive treatment regimen.
  • Another preclinical model is the "Micro-Metastases Model,” which involves the use of selected xenografts or syngeneic tumors that reliably and reproducibly metastasize to a particular organ, typically lung, liver, bone or brain. The implanted tumor is allowed to grow and metastasize prior to surgical excision of the primary mass.
  • Some of the first pre-clinical adjuvant chemotherapy studies were performed in the mid-1970s using the transplantable murine colon 26 tumor model. Colon 26 tumor fragments implanted subcutaneously in mice provide an example of this model (Corbett, T.H. et al., (1975) Cancer Res 35:2434-2439; Schabel, F.M. et al., Cancer 40:558-568).
  • Orthotopic or Genetic Engineered Mouse (GEM) models can also be used for adjuvant studies. Tumors are either implanted into the orthotopic site from which they originated or arise de novo in a specific organ. This approach better models the impact of the local microenvironment and stromal factors on tumor growth and metastasis. Although orthotopic implantation can give rise to robust primary tumor growth, these primary tumors do not develop according to the natural disease progression process of de novo tumors. GEM models of cancer faithfully recapitulate several aspects of the corresponding human disease, and have the potential to accurately model various disease settings for testing novel therapeutic agents (Singh, M. et al, (2006) Clin Cancer Res 12:5312-28; Hanahan, D.
  • transgenic mice typically express oncogenes or exhibit deletion of tumor suppressors in an organ-specific contest through the use of tissue-selective promoters. While the inclusion of surgical procedures would make this an attractive model of adjuvant cancer, in some instances the low rate of metastases and the technical challenges of surgery can limit its practical use.
  • the present invention is based, in part, on the discovery of a novel animal model of adjuvant cancer.
  • Most pre-clinical models of cancer simulate treatment in the first-line setting rather than adjuvant disease. Metastases and primary disease show significantly different properties, thus, animal models of adjuvant cancer are desirable to enable pre-clinically testing of agents useful for treating patients in the adjuvant setting.
  • the invention provides a method of making an animal model of adjuvant disease, comprising the steps of implanting a tumor cell into a tissue or organ of an animal, wherein the tumor cell establishes a tumor; allowing the tumor to metastasize to a sentinel lymph node; and removing the tumor from the animal.
  • the invention also provides a method of making an animal model of adjuvant disease, comprising the steps of providing an animal comprising a tumor in a tissue or organ of the animal; allowing the tumor to metastasize to a sentinel lymph node; and removing the tumor from the animal.
  • the invention provides an animal model for adjuvant disease, wherein the animal comprises a lymph node comprising tumor cells, wherein the tumor cells originated from a tumor in a tissue or organ for which the lymph node is a sentinel lymph node, and wherein the tumor has been removed from the animal.
  • the tumor cells metastasize to a distant site.
  • the invention further provides a method of identifying an agent for treating adjuvant disease, comprising the steps of implanting a tumor cell into a tissue or organ of an animal, wherein the tumor cell establishes a tumor; allowing the tumor to metastasize to a sentinel lymph node; removing the tumor from the animal; and administering a test agent to the animal, wherein the test agent is an agent for treating adjuvant disease if the test agent reduces or prevents metastasis of tumor cells in the sentinel lymph node to a distant site.
  • the invention also provides a method of identifying an agent for treating adjuvant disease, comprising the step of
  • test agent is an agent for treating adjuvant disease if the test agent reduces or prevents metastasis of tumor cells in the sentinel lymph node to a distant site.
  • the test agent may be a polypeptide, antibody or small molecule compound.
  • the invention provides a method of testing efficacy of an agent for treating adjuvant disease, comprising the steps of implanting a tumor cell into a tissue or organ of an animal, wherein the tumor cell establishes a tumor; allowing the tumor to metastasize to a sentinel lymph node; removing the tumor from the animal;
  • a method of testing efficacy of an agent for treating adjuvant disease comprising the steps of administering the agent to the animal generated using the methods of the present invention; and analyzing the animal for metastasis of tumor cells in the sentinel lymph node to a distant site, wherein the agent is efficacious for treating adjuvant disease if it reduces or prevents said metastasis.
  • the animal may be a mouse.
  • the tissue or organ may be any tissue or organ where the tissue or organ contains numerous blood and lymph vessels, is associated with one or a few unambiguous lymph nodes, and where the tumor cells in the tissue or organ are easily removed by surgery.
  • the tissue or organ is an ear, tail, or hindfoot.
  • the distant site may be any tissue or organ in the animal including, but not limited to, lung, liver, brain, spleen, skin or bone.
  • Figure Ia is schematic diagram depicting the generation of a mouse model of adjuvant disease using the methods of the invention. Briefly, at day 0, tumor cells are implanted into the ear of the mouse. At about day 12, the tumor has metastasized to the sentinel lymph node (SLN) and the primary tumor in the ear is removed.
  • Figure Ib is a graph showing the percent of such animals that have tumor cells in the SLN over time.
  • Figure Ic is a graph showing the percent of such animals that have tumor cells in the lung.
  • Figure 2 is a schematic diagram outlining how agents are tested for their efficacy as treatment for adjuvant cancer using the mouse model of adjuvant disease using the methods of the invention.
  • cancer refers to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Included in this definition are benign and malignant cancers as well as dormant tumors or micrometastatses. Examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
  • cancers include squamous cell cancer, lung cancer (including small- cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer, as well as B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL;
  • B-cell lymphoma including low grade/follicular non-Hodgkin's lymphoma (NHL
  • Macroglobulinemia may be used to treat chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblasts leukemia; and post- transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs' syndrome.
  • CLL chronic lymphocytic leukemia
  • ALL acute lymphoblastic leukemia
  • Hairy cell leukemia a chronic myeloblasts leukemia
  • PTLD post- transplant lymphoproliferative disorder
  • Tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer is meant the spread of cancer from its primary site to other places in the body. Cancer cells can break away from a primary tumor, penetrate into lymphatic and blood vessels, circulate through the bloodstream, and grow in a distant focus (metastasize) in normal tissues elsewhere in the body.
  • Metastasis can be local or distant. Metastasis is a sequential process, contingent on tumor cells breaking off from the primary tumor, traveling through the bloodstream, and stopping at a distant site. At the new site, the cells establish a blood supply and can grow to form a life-threatening mass. Both stimulatory and inhibitory molecular pathways within the tumor cell regulate this behavior, and interactions between the tumor cell and host cells in the distant site are also significant.
  • micrometastasis is meant a small number of cells that have spread from the primary tumor to other parts of the body. Micrometastasis may or may not be detected in a screening or diagnostic test.
  • Cancer recurrence herein refers to a return of cancer following treatment, and includes return of cancer in the primary organ, as well as distant recurrence, where the cancer returns outside of the primary organ.
  • Adjuvant disease or “adjuvant cancer” as used herein refers to cancer that develops after removal of all detectable cancer. In some embodiments the cancer is removed by surgery.
  • adjuvant therapy refers to therapy given after definitive surgery, where no evidence of residual disease can be detected, so as to reduce the risk of disease recurrence.
  • the goal of adjuvant therapy is to prevent or delay recurrence of the cancer, and therefore to reduce the chance of cancer-related death.
  • Definitive surgery is used as that term is used within the medical community. Definitive surgery includes, for example, procedures, surgical or otherwise, that result in removal or resection of the tumor, including those that result in the removal or resection of all grossly visible tumor. Definitive surgery includes, for example, complete or curative resection or complete gross resection of the tumor. Definitive surgery includes procedures that occurs in one or more stages, and includes, for example, multi-stage surgical procedures where one or more surgical or other procedures are performed prior to resection of the tumor. Definitive surgery includes procedures to remove or resect the tumor including involved organs, parts of organs and tissues, as well as surrounding organs, such as lymph nodes, parts of organs, or tissues.
  • maintenance therapy is meant a therapeutic regimen that is given to reduce the likelihood of disease recurrence or progression.
  • Maintenance therapy can be provided for any length of time, including extended time periods up to the life-span of the subject. Maintenance therapy can be provided after initial therapy or in conjunction with initial or additional therapies. Dosages used for maintenance therapy can vary and can include diminished dosages as compared to dosages used for other types of therapy.
  • concurrent administration includes a dosing regimen when the administration of one or more agent(s) continues after discontinuing the
  • “monotherapy” is meant a therapeutic regimen that includes only a single therapeutic agent for the treatment of the cancer or tumor during the course of the treatment period.
  • an "agent for treating adjuvant disease” or an “agent for treating adjuvant cancer” refers to a small molecular weight substance, a polynucleotide, a polypeptide, an isolated protein, a recombinant protein, an antibody, or conjugates or fusion proteins thereof, that ameliorates or treats one or more symptoms of adjuvant cancer.
  • anti-cancer therapy refers to a therapy useful in treating cancer.
  • anti-cancer therapeutic agents include, but are not limited to, e.g., surgery, chemotherapeutic agents, growth inhibitory agents, cytotoxic agents, agents used in radiation therapy, anti-angiogenesis agents, apoptotic agents, anti- tubulin agents, and other agents to treat cancer, such as anti-HER-2 antibodies, anti- CD20 antibodies, an epidermal growth factor receptor (EGFR) antagonist (e.g., a tyrosine kinase inhibitor), HER1/EGFR inhibitor (e.g., erlotinib (Tarceva ® ), platelet derived growth factor inhibitors (e.g., Gleevec (Imatinib Mesylate)), a COX-2 inhibitor (e.g., celecoxib), interferons, cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the following targets ErbB2,
  • EGFR epiderma
  • an "anti-angiogenesis agent” or “angiogenesis inhibitor” refers to a small molecular weight substance, a polynucleotide, a polypeptide, an isolated protein, a recombinant protein, an antibody, or conjugates or fusion proteins thereof, that inhibits angiogenesis, vasculogenesis, or undesirable vascular permeability, either directly or indirectly. It should be understood that the anti-angiogenesis agent includes those agents that bind and block the angiogenic activity of the angiogenic factor or its receptor.
  • an anti-angiogenesis agent is an antibody or other antagonist to an angiogenic agent as defined above, e.g., antibodies to VEGF-A or to the VEGF-A receptor (e.g., KDR receptor or FIt-I receptor), anti-PDGFR inhibitors such as GleevecTM (Imatinib Mesylate).
  • Anti-angiogensis agents also include native angiogenesis inhibitors, e.g., angiostatin, endostatin, etc. See, e.g., Klagsbrun and D'Amore, Annu. Rev.
  • cytotoxic agent refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells.
  • the term is intended to include radioactive isotopes (e.g. At 211 , 1 131 , 1 125 , Y 90 , Re 186 , Re 188 ,
  • chemotherapeutic agents such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof.
  • a "chemotherapeutic agent” is a chemical compound useful in the treatment of cancer.
  • chemotherapeutic agents include is a chemical compound useful in the treatment of cancer.
  • chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and
  • methylamelamines including altretamine, triethylenemelamine,
  • trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC- 1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBl-TMl); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,
  • cholophosphamide estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e. g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall (see, e.g., Agnew, Chem Intl. Ed.
  • dynemicin including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®
  • doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2- pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potf ⁇ romycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analog
  • dromostanolone propionate epitiostanol, mepitiostane, testolactone
  • anti-adrenals such as aminoglutethimide, mitotane, trilostane
  • folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;
  • amsacrine bestrabucil
  • bisantrene edatraxate
  • defofamine demecolcine
  • diaziquone elfornithine
  • elliptinium acetate an epothilone; etoglucid; gallium nitrate;
  • hydroxyurea lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin;
  • phenamet pirarubicin; losoxantrone; podophyllinic acid; 2- ethylhydrazide;
  • procarbazine PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spiro germanium; tenuazonic acid; triaziquone; 2,2',2"- trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol;
  • mitolactol pipobroman; gacytosine; arabinoside ("Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL® paclitaxel (Bristol- Myers Squibb Oncology, Princeton, N.
  • ABRAXANE ® Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel American Pharmaceutical Partners, Schaumberg, Illinois
  • TAXOTERE® doxetaxel Rhone- Poulenc Rorer, Antony, France
  • chloranbucil GEMZAR® gemcitabine
  • 6-thioguanine mercaptopurine
  • methotrexate platinum analogs such as cisplatin, oxaliplatin and carboplatin
  • vinblastine platinum
  • platinum etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; NAVELB INE® vinorelbine
  • VP- 16 etoposide
  • novantrone novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including the treatment regimen of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); inhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva ® )) and VEGF-A that reduce cell proliferation and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • DMFO difluorometlhylornith
  • anti-hormonal agents that act to regulate or inhibit hormone action on tumors
  • SERMs selective estrogen receptor modulators
  • tamoxifen including NOLVADEX® tamoxifen
  • raloxifene including NOLVADEX® tamoxifen
  • droloxifene 4-hydroxytamoxifen
  • trioxifene keoxifene
  • LYl 17018, onapristone and FARESTON- toremifene
  • aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole, RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole
  • anti- androgens such as flutamide
  • ANGIOZYME® ribozyme and a HER2 expression inhibitor
  • vaccines such as gene therapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine
  • PROLEUKIN® rIL-2 PROLEUKIN® rIL-2
  • LURTOTECAN® topoisomerase 1 inhibitor ABARELIX® rmRH
  • pharmaceutically acceptable salts, acids or derivatives of any of the above are examples of any of the above.
  • cytokine is a generic term for proteins released by one cell population which act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones.
  • cytokines include growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); epidermal growth factor; hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-alpha and -beta; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-alpha; platelet-growth factor; transforming growth factors (TGFs) such as TGF-alpha and TGF-beta; insulin-like growth factor-I and -II; erythropoie
  • a “growth inhibitory agent” when used herein refers to a compound or composition which inhibits growth of a cell in vitro and/or in vivo.
  • the growth inhibitory agent may be one which significantly reduces the percentage of cells in S phase.
  • growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase), such as agents that induce Gl arrest and M-phase arrest.
  • Classical M-phase blockers include the vincas (vincristine and vinblastine), TAXOL®, and topo II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.
  • DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found in The Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycle regulation, oncogenes, and antineoplastic drugs” by Murakami et al. (WB Saunders: Philadelphia, 1995), especially p. 13.
  • prodrug refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active parent form. See, e.g., Wilman, "Prodrugs in Cancer Chemotherapy” Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Harbor (1986) and Stella et al., "Prodrugs: A Chemical Approach to
  • the prodrugs of this invention include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, ⁇ -lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide- containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs which can be converted into the more active cytotoxic free drug.
  • cytotoxic drugs that can be derivatized into a prodrug form for use in this invention include, but are not limited to, those chemotherapeutic agents described above.
  • radiation therapy is meant the use of directed gamma rays or beta rays to induce sufficient damage to a cell so as to limit its ability to function normally or to destroy the cell altogether. It will be appreciated that there will be many ways known in the art to determine the dosage and duration of treatment.
  • Typical treatments are given as a one time administration and typical dosages range from 10 to 200 units (Grays) per day.
  • Reduce or inhibit is meant the ability to cause an overall decrease preferably of 20% or greater, more preferably of 50% or greater, and most preferably of 75%, 85%, 90%, 95%, or greater.
  • Reduce or inhibit can refer to the symptoms of the disorder being treated, including the presence or size of metastases or micrometastases,
  • intravenous infusion refers to introduction of a drug into the vein of an animal or human patient over a period of time greater than approximately 5 minutes, preferably between approximately 30 to 90 minutes, although, according to the invention, intravenous infusion is alternatively
  • intravenous bolus or “intravenous push” refers to drug administration into a vein of an animal or human such that the body receives the drug in approximately 15 minutes or less, preferably 5 minutes or less.
  • subcutaneous administration refers to introduction of a drug under the skin of an animal or human patient, preferable within a pocket between the skin and underlying tissue, by relatively slow, sustained delivery from a drug receptacle.
  • the pocket may be created by pinching or drawing the skin up and away from underlying tissue.
  • subcutaneous infusion refers to introduction of a drug under the skin of an animal or human patient, preferably within a pocket between the skin and underlying tissue, by relatively slow, sustained delivery from a drug receptacle for a period of time including, but not limited to, 30 minutes or less, or 90 minutes or less.
  • the infusion may be made by subcutaneous implantation of a drug delivery pump implanted under the skin of the animal or human patient, wherein the pump delivers a predetermined amount of drug for a predetermined period of time, such as 30 minutes, 90 minutes, or a time period spanning the length of the treatment regimen.
  • subcutaneous bolus refers to drug administration beneath the skin of an animal or human patient, where bolus drug delivery is preferably less than approximately 15 minutes, more preferably less than 5 minutes, and most preferably less than 60 seconds. Administration is preferably within a pocket between the skin and underlying tissue, where the pocket is created, for example, by pinching or drawing the skin up and away from underlying tissue.
  • the term "therapeutically effective amount” refers to an amount of a drug effective to treat a disease or disorder in a mammal.
  • the therapeutically effective amount of the drug may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the disorder.
  • the therapeutically effective amount of the drug may reduce the number or proliferation of micrometastases; reduce or prevent the growth of a dormant tumor; or reduce or prevent the recurrence of a tumor after treatment or removal (e.g., using an anti-cancer therapy such as surgery, radiation therapy, or chemotherapy).
  • an anti-cancer therapy such as surgery, radiation therapy, or chemotherapy.
  • the drug may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic.
  • efficacy in vivo can, for example, be measured by assessing the duration of survival, disease free survival (DFS), time to disease progression (TTP), duration of progression free survival (PFS), the response rates (RR), duration of response, time in remission, and/or quality of life.
  • DFS disease free survival
  • TTP time to disease progression
  • PFS duration of progression free survival
  • RR response rates
  • the effective amount may improve disease free survival (DFS), improve overall survival (OS), decrease likelihood of recurrence, extend time to recurrence, extend time to distant recurrence (i.e., recurrence outside of the primary site), cure cancer, improve symptoms of cancer (e.g., as gauged using a cancer specific survey), reduce appearance of second primary cancer, etc.
  • DFS disease free survival
  • OS overall survival
  • Currence i.e., recurrence outside of the primary site
  • cure cancer improve symptoms of cancer (e.g., as gauged using a cancer specific survey), reduce appearance of second primary cancer, etc.
  • Treatment refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented, including those in which the occurrence or recurrence of cancer is to be prevented.
  • an antibody can be chimeric, human, humanized, and/or affinity matured.
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible mutations, e.g., naturally occurring mutations, that may be present in minor amounts. Thus, the modifier “monoclonal” indicates the character of the antibody as not being a mixture of discrete antibodies.
  • such a monoclonal antibody typically includes an antibody comprising a polypeptide sequence that binds a target, wherein the target-binding polypeptide sequence was obtained by a process that includes the selection of a single target binding polypeptide sequence from a plurality of polypeptide sequences.
  • the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, or recombinant DNA clones.
  • a selected target binding sequence can be further altered, for example, to improve affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence is also a monoclonal antibody of this invention.
  • polyclonal antibody In contrast to polyclonal antibody
  • each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.
  • monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins.
  • the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14 (3): 253-260 (1995), Harlow et al, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-CeIl Hybridomas 563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g., U.S. Patent No.
  • phage-display technologies see, e.g., Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. MoI. Biol. 222: 581-597 (1992); Sidhu et al., J. MoI. Biol. 338(2): 299-310 (2004); Lee et al., J. MoI. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al, J. Immunol.
  • Methods 284(1-2): 119-132(2004), and technologies for producing human or human-like antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993); Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Patent Nos.
  • the monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see, e.g., U.S. Patent No. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA 81 :6851-6855 (1984)).
  • Chimeric antibodies include PRIMATIZED® antibodies wherein the antigen-binding region of the antibody is derived from an antibody produced by, e.g., immunizing macaque monkeys with the antigen of interest.
  • multivalent antibody denotes an antibody comprising three or more antigen binding sites.
  • the multivalent antibody is engineered to have the three or more antigen binding sites and is generally not a native sequence IgM or IgA antibody.
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
  • a humanized antibody is a human
  • immunoglobulin in which residues from a HVR of the recipient are replaced by residues from a HVR of a non-human species (donor antibody) such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and/or capacity.
  • donor antibody such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and/or capacity.
  • FR residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications may be made to further refine antibody performance.
  • a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the FRs are those of a human immunoglobulin sequence.
  • the humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • a "human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
  • Human antibodies can be produced using various techniques known in the art, including phage-display libraries. Hoogenboom and Winter, J. MoI. Biol, 227:381 (1991); Marks et al., J. MoI. Biol, 222:581 (1991). Also available for the preparation of human monoclonal antibodies are methods described in Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.
  • Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., immunized xenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding XENOMOUSETM technology). See also, for example, Li et al, Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodies generated via a human B-cell hybridoma technology.
  • Antibody fragments comprise a portion of an intact antibody, preferably comprising the antigen binding region thereof.
  • Examples of antibody fragments include Fab, Fab', F(ab') 2 , and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecif ⁇ c antibodies formed from antibody fragments.
  • Fully fragments of the antibodies of the invention comprise a portion of an intact antibody, generally including the antigen-binding or variable region of the intact antibody or the Fc region of an antibody that retains FcR binding capability.
  • antibody fragments include linear antibodies, single-chain antibody molecules, and multispecif ⁇ c antibodies formed from antibody fragments.
  • an “intact antibody” is one comprising heavy and light variable domains as well as an Fc region.
  • Fv is the minimum antibody fragment which contains a complete antigen-binding site.
  • a two-chain Fv species consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association.
  • one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a "dimeric" structure analogous to that in a two-chain Fv species. It is in this configuration that the three HVRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer.
  • the Fab fragment contains the heavy- and light-chain variable domains and also contains the constant domain of the light chain and the first constant domain (CHl) of the heavy chain.
  • Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHl domain including one or more cysteines from the antibody hinge region.
  • Fab '-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group.
  • F(ab') 2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
  • Fc region herein is used to define a C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions.
  • the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl- terminus thereof.
  • the C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Accordingly, a composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue.
  • a “blocking” antibody or an “antagonist” antibody is one that inhibits or reduces biological activity of the antigen it binds.
  • Preferred blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.
  • An "agonist antibody”, as used herein, is an antibody that mimics at least one of the functional activities of a polypeptide of interest.
  • An "antigen" is a predetermined antigen to which an antibody can selectively bind.
  • the target antigen may be polypeptide, carbohydrate, nucleic acid, lipid, hapten, or another naturally occurring or synthetic compound.
  • the target antigen is a polypeptide.
  • An "acceptor human framework” for the purposes herein is a framework comprising the amino acid sequence of a VL or VH framework derived from a human immunoglobulin framework, or from a human consensus framework.
  • An acceptor human framework "derived from" a human immunoglobulin framework or human consensus framework may comprise the same amino acid sequence thereof, or may contain pre-existing amino acid sequence changes.
  • pre-existing amino acid changes preferably no more than five, and more preferably four or less, and still more preferably three or less, pre-existing amino acid changes are present.
  • pre-existing amino acid changes are present in a VH, preferably those changes are only at three, two, or one of positions 71, 73, and 78; for instance, the histidine residues at those positions may be alanine residues.
  • the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.
  • the phrase "substantially similar” or “substantially the same,” as used herein, denotes a sufficiently high degree of similarity between two numeric values such that one of skill in the art would consider the difference between the two values to be of little or no biological and/or statistical significance within the context of the biological characteristic measured by said values.
  • the difference between said two values is preferably less than about 50%, more preferably less than about 40%, still more preferably less than about 30%, even more preferably less than about 20%, and most preferably less than about 10% as a function of the value for the
  • the difference between said two values is preferably greater than about 10%, more preferably greater than about 20%, still more preferably greater than about 30%, even more preferably greater than about 40%, and most preferably greater than about 50% as a function of the value for the
  • Detection includes any means of detecting, including direct and indirect detection.
  • An "animal” is any non-human vertebrate. In certain aspects,
  • the vertebrate is a non-human mammal.
  • Mammals include, but are not limited to, farm animals (including sheep and cows), cats, dogs, horses, primates, rabbits, and rodents (including mice and rats).
  • An "isolated" polypeptide or “isolated” antibody is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
  • the polypeptide will be purified (1) to greater than 95% by weight of polypeptide as determined by the Lowry method, or more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue, or silver stain.
  • Isolated polypeptide includes the polypeptide in situ within recombinant cells since at least one component of the polypeptide's natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step.
  • label when used herein refers to a compound or composition which is conjugated or fused directly or indirectly to a reagent such as a nucleic acid probe or an antibody and facilitates detection of the reagent to which it is conjugated or fused.
  • the label may itself be detectable (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable.
  • a “native sequence” polypeptide comprises a polypeptide having the same amino acid sequence as a polypeptide derived from nature.
  • a native sequence polypeptide can have the amino acid sequence of naturally occurring polypeptide from any mammal.
  • Such native sequence polypeptide can be isolated from nature or can be produced by recombinant or synthetic means.
  • the term "native sequence” polypeptide specifically encompasses naturally occurring truncated or secreted forms of the polypeptide (e.g., an extracellular domain sequence), naturally occurring variant forms (e.g., alternatively spliced forms) and naturally occurring allelic variants of the polypeptide.
  • a polypeptide "variant” means a biologically active polypeptide having at least about 80% amino acid sequence identity with the native sequence polypeptide.
  • variants include, for instance, polypeptides wherein one or more amino acid residues are added, or deleted, at the N- or C-terminus of the polypeptide.
  • a variant will have at least about 80% amino acid sequence identity, more preferably at least about 90% amino acid sequence identity, and even more preferably at least about 95% amino acid sequence identity with the native sequence polypeptide.
  • amino acid modification refers to a change in the amino acid sequence of a predetermined amino acid sequence.
  • exemplary modifications include an amino acid substitution, insertion, and/or deletion.
  • the preferred amino acid modification herein is a substitution.
  • amino acid modification at refers to the substitution or deletion of the specified residue, or the insertion of at least one amino acid residue adjacent to the specified residue.
  • insertion adjacent to a specified residue is meant insertion within one to two residues thereof. The insertion may be N-terminal or C-terminal to the specified residue.
  • amino acid substitution refers to the replacement of at least one existing amino acid residue in a predetermined amino acid sequence with another different “replacement” amino acid residue.
  • the replacement residue or residues may be "naturally occurring amino acid residues" (i.e., encoded by the genetic code) and selected from the group consisting of: alanine (Ala); arginine (Arg); asparagine (Asn); aspartic acid (Asp); cysteine (Cys); glutamine (GIn); glutamic acid (GIu); glycine (GIy); histidine (His); isoleucine (He): leucine (Leu); lysine (Lys); methionine (Met); phenylalanine (Phe); proline (Pro); serine (Ser); threonine (Thr); tryptophan (Trp); tyrosine (Tyr); and valine (VaI).
  • the replacement residue is not cysteine.
  • substitution with one or more non-naturally occurring amino acid residues is also encompassed by the definition of an amino acid substitution herein.
  • a "non-naturally occurring amino acid residue” refers to a residue, other than those naturally occurring amino acid residues listed above, which is able to covalently bind adjacent amino acid residues(s) in a polypeptide chain. Examples of non-naturally occurring amino acid residues include norleucine, ornithine, norvaline, homoserine, and other amino acid residue analogues such as those described in Ellman et ah, Meth. Enzym. 202:301-336 (1991).
  • substitutions are meant to refer to amino acid substitutions that substitute functionally equivalent amino acids.
  • Conservative amino acid changes result in silent changes in the amino acid sequence of the resulting polypeptide.
  • one or more amino acids of a similar polarity act as functional equivalents and result in a silent alteration within the amino acid sequence of the polypeptide.
  • substitutions within a group may be considered conservative with respect to structure and function.
  • the skilled artisan will recognize that the role of a particular residue is determined by its context within the three-dimensional structure of the molecule in which it occurs. For example, Cys residues may occur in the oxidized (disulfide) form, which is less polar than the reduced (thiol) form.
  • the long aliphatic portion of the Arg side chain may constitute a critical feature of its structural or functional role, and this may be best conserved by substitution of a nonpolar, rather than another basic, residue.
  • side chains containing aromatic groups Trp, Tyr, and Phe
  • substitution of one of these side chains with a member of the acidic or uncharged polar group may be conservative with respect to structure and function.
  • Residues such as Pro, GIy, and Cys can have direct effects on the main-chain conformation, and often may not be substituted without structural distortions.
  • amino acid insertion refers to the incorporation of at least one amino acid into a predetermined amino acid sequence. While the insertion will usually consist of the insertion of one or two amino acid residues, the present application contemplates larger "peptide insertions", e.g., insertion of about three to about five or even up to about ten amino acid residues.
  • the inserted residue(s) may be naturally occurring or non-naturally occurring as disclosed above.
  • amino acid deletion refers to the removal of at least one amino acid residue from a predetermined amino acid sequence.
  • Amino acids may be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)):
  • Carriers as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the
  • physiologically acceptable carrier is an aqueous pH-buffered solution.
  • physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low-molecular-weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt- forming counterions such as sodium; and/or nonionic surfactants such as TWEENTM, polyethylene glycol (PEG), and PLURONICSTM.
  • buffers such as phosphate, citrate, and other organic acids
  • antioxidants including ascorbic acid
  • the present invention features, in part, a novel animal model of adjuvant cancer that allows more reliable evaluation of agents impacting the spread and growth of metastatic cells in distant organs.
  • the invention provides a method of making an animal model of adjuvant disease, comprising the steps of implanting a tumor cell into a tissue or organ of an animal, wherein the tumor cell establishes a tumor; allowing the tumor to metastasize to a sentinel lymph node; and removing the tumor from the animal.
  • This animal model of adjuvant disease may also be generated by providing an animal comprising a tumor in a tissue or organ of the animal, allowing the tumor to metastasize to a sentinel lymph node, and removing the tumor from the animal.
  • the tumor metastasizes from the sentinel lymph nodes to a distant site. Due to an increasing number of murine tumor cell lines, and the fact that such lines can also be derived from genetic mouse models, this technique is likely to be used more broadly for testing new agents in the adjuvant setting.
  • a murine tumor cell line is implanted on the ear pinnae of syngeneic immune-competent mice. Implantation of tumors into mice with an intact immune system allows for improved modeling of the role of tumor infiltrates, and immune and inflammatory cells, in regulating tumor cell growth, angiongenesis, lymphangiogenesis and metastases. The tumors are allowed to establish and disseminate spontaneously, first to the sentinel lymph node and subsequently to distant sites such as the lung. Surgical removal of the primary tumor via ear clipping can be performed in a relatively high-throughput manner.
  • the methods of the invention may be performed with any non-human animal, including non-human mammals.
  • Mammals include, but are not limited to, farm animals (including sheep and cows), monkeys, cats, dogs, horses, primates, rabbits, and rodents (including mice and rats).
  • the animal is a monkey, rabbit, rat or mouse.
  • tumor cells or tumor fragments may be implanted into the tissue or organ of the animal wherein the tumor cells or tumor fragments are allowed to establish a tumor in the tissue or organ.
  • Such methods may include, for example, transplanting murine tumors in syngeneic hosts and xenografts of human tumors grown in immunodeficient animals. See, e.g., Fiebig et al., Human tumor xenografts and explants. In: Teicher BA, editor. Animal models in cancer research. Totowa (NJ): Humana Press, Inc. 113-37 (2001).
  • the tumor may arise in the tissue or organ by genetically modifying the animal.
  • the animal may be genetically modified to express an oncogene or exhibit deletion of tumor suppressors in an organ-specific or tissue-specific context through the use of organ or tissue-selective promoters and/or other inducible promoters.
  • Methods of making such genetically modified animals are well know in the art (Hanahan D. and Folkman J (1996) Cell 86:353-364).
  • Certain methods of the invention contemplate implanting or otherwise generating a tumor in a tissue or organ of an animal, wherein the tumor metastasizes to a sentinel lymph node.
  • the tissue or organ may be any tissue or organ that (1) has a rich blood and lymphatic vasculature, (2) is associated with one or a few unambiguous lymph nodes, and (3) contains the tumor such that the tumor is easily removed.
  • the murine ear has stereotypic blood and lymphatic vascular networks, providing an ideal bed for tumor establishment and metastasis by hematogenous or lymphatic routes.
  • the murine ear is also associated with a single sentinel lymph node and can be removed quite readily by clipping.
  • exemplary tissues or organs in the mouse that are useful for performing the methods of the invention include the tail, which is associated with the inguinal lymph node, the ventral iliac lymph node and the caudal auxiliary lymph node; and the hindfoot, which is associated with the popliteal lymph node.
  • Lymph nodes associated with specific tissues and organs are known in the art. See, e.g., Harrell, M.I. et al. (2008) J Immunol Methods 332: 170-174.
  • the tumor in the tissue or organ is removed after it metastasizes to a sentinel lymph node.
  • the tumor may be removed from the animal using various methods known in the art, including without limitation, cytotoxic chemotherapy, irradiation, anti-tumor targeted therapy or a toxin specific to a transgenic tumor cell.
  • the tumor is removed surgically.
  • tumor cells from the sentinel lymph node metastasize to a distant site may be any organ or tissue in the animal, including without limitation, lung, liver, spleen, brain, skin and bone.
  • the distant site may be any organ or tissue in the animal, including without limitation, lung, liver, spleen, brain, skin and bone.
  • a mouse with a tumor in an ear has the ear removed after the tumor is allowed to metastasize to a sentinel lymph node. After removal of the ear, tumor cells from the sentinel lymph node metastasize to the lung.
  • the tumor metastasizes from the ear to the sentinel lymph node by about 12 days after initial implantation of tumor cells in the ear ( Figure Ib).
  • the animals show no sign of any detectable metastases to the lung.
  • These animals exhibit the presence of tumor cells in the lung after about 20 days post- implantation of the tumor cells into the ear. Similar methods may be carried out using a different organ or tissue as the distant site for metastasis.
  • the invention also provides the animals made by the methods of the invention. Accordingly, the invention provides an animal model for adjuvant disease, wherein the animal comprises a lymph node comprising tumor cells, wherein the tumor cells originated from a tumor in a tissue or organ for which the lymph node is a sentinel lymph node, and wherein the tumor has been removed from the animal. In certain embodiments the tumor cells metastasize to a distant site.
  • the animal models of adjuvant cancer provided by the present invention are useful in a number of different applications.
  • the animal models are useful for identifying agents for treating adjuvant cancer.
  • the invention provides a method of identifying an agent for treating adjuvant disease, comprising the steps of implanting a tumor cell into a tissue or organ of an animal, wherein the tumor cell establishes a tumor; allowing the tumor to metastasize to a sentinel lymph node; removing the tumor from the animal; and administering a test agent to the animal, wherein the test agent is an agent for treating adjuvant disease if the test agent reduces or prevents metastasis of tumor cells in the sentinel lymph node to a distant site.
  • the invention also provides a method of identifying an agent for treating adjuvant disease, comprising the step of
  • test agent is an agent for treating adjuvant disease if the test agent reduces or prevents metastasis of tumor cells in the sentinel lymph node to a distant site.
  • the test agent may be a small molecule compound, a
  • polynucleotide a polypeptide, an isolated protein, a recombinant protein, an antibody or conjugates or fusion proteins thereof.
  • the animal models of the invention may also be used for testing efficacy of an agent for treating adjuvant cancer.
  • the invention provides a method of testing efficacy of an agent for treating adjuvant disease, comprising the steps of implanting a tumor cell into a tissue or organ of an animal, wherein the tumor cell establishes a tumor; allowing the tumor to metastasize to a sentinel lymph node; removing the tumor from the animal; administering the agent to the animal; and analyzing the animal for metastasis of tumor cells in the sentinel lymph node to a distant site, wherein the agent is efficacious for treating adjuvant disease if it reduces or prevents said metastasis.
  • Also provided is a method of testing efficacy of an agent for treating adjuvant disease comprising the steps of administering the agent to an animal generated using the methods of the present invention; and analyzing the animal for metastasis of tumor cells in the sentinel lymph node to a distant site, wherein the agent is efficacious for treating adjuvant disease if it reduces or prevents said metastasis
  • Methods of the invention wherein agents for treating adjuvant cancer are identified or tested may also comprise administering other anti-cancer therapy in combination with the test agent to determine, for example, efficacy of the combination therapy.
  • anti-cancer therapies are detailed above and include, without limitation, chemotherapeutic agents, growth inhibitory agents, cytotoxic agents, radiation therapy, anti-angiogenesis agents, apoptotic agents, etc.
  • the test agent and the anti-cancer therapy may be administered to the animal simultaneously or sequentially.
  • a test agent may be co-administered to an animal of the invention with one or more anti-cancer therapy or other therapeutic agent.
  • the test agent may be administered first followed by one or more anticancer therapies or other therapeutic agent. Administration of one or more anticancer therapy or other therapeutic agent first is also contemplated.
  • C ⁇ LacZ cells (ATCC) were grown in DMEM, 10% FBS and maintained at 37 0 C in a 5% CO 2 95% humidity incubator. Cells were trypsinized, counted and resuspended in PBS at 100x10 6 cells/ml. BaIb-C nude mice were anesthetized under isofluorane and l ⁇ l cell suspension of C ⁇ LacZ cells was injected intradermally into the tip of an ear in each animal using a lOul Hamilton syringe with a 0.5" point 2 needle (Hamilton).
  • Animals were analyzed for tumor cell presence in the sentinel lymph node on each day noted above, as well as tumor cell presence in the blood and lungs.
  • Tumors cells were detected by using an enzymatic reaction to detect the trangenically expressed ⁇ -galactosidase using a commercially available kit (Pierce Biotechnology Cat# 75707) as described in the manufacturer's instructions. Briefly, the tissues to be analyzed were collected, lysed with M-PER mammalian protein extraction reagent (Pierce Biotechnology) and homogenized. 25ul of lysate was evaluated using a ⁇ -galactosidase assay kit (Pierce). A cell suspension of C6LacZ cells was used as a positive control. Using serial dilutions of C6LacZ cells, the sensitivity of this technique was evaluated. This technique was able to detect as few as 10 LacZ positive cells in a lymph node.
  • Results using this technique to detect tumor cells show that C6LACZ tumor cells are initially trapped in the SLN at day 3 and 100% of SLN have detectable tumor cells by day 12 (Figure Ib). Presence of tumor cells in the blood was observed at day 12. Lungs were also assessed both visually for gross metastases as well as by the technique described above (homogenized and assayed in the ⁇ - galactosidase test) to detect mico-metastases. No animals had tumor cells in the lungs prior to day 18 ( Figure Ic). This allowed us to define the time frame at which metastasis to the sentinel lymph node had occurred, but prior to metastasis to the lung.
  • Surgical excision of the tumor was performed by the following method: Animals were anesthetized under isofluorane, the ear was swabbed with iodine and ethanol and the tumor was removed. Pressure was applied to the ear using sterile gauze followed by silver nitrate to cauterize any bleeding, followed by 5% topical lidocaine. Animals were randomized, 20 per group, and treatment initiated to allow for evaluation of adjuvant therapeutic regimens. At day 26, 14 days after treatment initiation, animals were sacrificed and lymph nodes, blood and lungs collected for analysis. Visual counts of lungs were carried out.
  • mice were implanted with cells as described above and 24 hours later the primary tumor removed. These mice showed no signs of metastasis in the SNL, blood or lung even after mice had been allow to recover for 4 weeks.
  • Example 2 Method of identifying or testing an agent using the adjuvant cancer model
  • C ⁇ LacZ cells are implanted into the ear pinnae as described above.
  • the day the cells are implanted is denoted day 0. 100% of animals are noted to have visually evident tumor lesions at the site of metastasis (i.e., the sentinel lymph node) by day 12.
  • Tumors are then surgically removed as described above and animals are randomly sorted into 2 groups.
  • Treatment with a control antibody (anti-ragweed) or a test agent is initiated at the appropriate dose, e.g., 25mg/kg twice weekly.
  • animals from both groups are sacrificed, lymph node metastases are confirmed, and lungs are harvested and evaluated for the presence of metastases. Metastatic nodule number and size are also analyzed for each animal. See Figure 2.
  • Agents that are able to inhibit metastases from the lymph node to the lung are identified as agents useful for treating adjuvant cancer.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Environmental Sciences (AREA)
  • Cell Biology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Zoology (AREA)
  • Animal Husbandry (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention porte sur des procédés et sur des compositions utiles pour identifier et tester des agents pour une thérapie d'adjuvant du cancer.
PCT/US2010/041420 2009-07-09 2010-07-08 Modèle animal pour l'évaluation de thérapies d'adjuvant du cancer Ceased WO2011006001A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US22428509P 2009-07-09 2009-07-09
US61/224,285 2009-07-09

Publications (1)

Publication Number Publication Date
WO2011006001A1 true WO2011006001A1 (fr) 2011-01-13

Family

ID=42359524

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2010/041420 Ceased WO2011006001A1 (fr) 2009-07-09 2010-07-08 Modèle animal pour l'évaluation de thérapies d'adjuvant du cancer

Country Status (1)

Country Link
WO (1) WO2011006001A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102750861A (zh) * 2012-06-23 2012-10-24 关真民 胃癌种植性转移演示模型
WO2015013432A1 (fr) * 2013-07-23 2015-01-29 Genentech, Inc. Modèle de cancer colorectal

Citations (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
WO1991010741A1 (fr) 1990-01-12 1991-07-25 Cell Genesys, Inc. Generation d'anticorps xenogeniques
US5545806A (en) 1990-08-29 1996-08-13 Genpharm International, Inc. Ransgenic non-human animals for producing heterologous antibodies
US5545807A (en) 1988-10-12 1996-08-13 The Babraham Institute Production of antibodies from transgenic animals
US5569825A (en) 1990-08-29 1996-10-29 Genpharm International Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
WO1996033735A1 (fr) 1995-04-27 1996-10-31 Abgenix, Inc. Anticorps humains derives d'une xenosouris immunisee
WO1996034096A1 (fr) 1995-04-28 1996-10-31 Abgenix, Inc. Anticorps humains derives de xeno-souris immunisees
US5625126A (en) 1990-08-29 1997-04-29 Genpharm International, Inc. Transgenic non-human animals for producing heterologous antibodies
US5633425A (en) 1990-08-29 1997-05-27 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5661016A (en) 1990-08-29 1997-08-26 Genpharm International Inc. Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
WO1998024893A2 (fr) 1996-12-03 1998-06-11 Abgenix, Inc. MAMMIFERES TRANSGENIQUES POSSEDANT DES LOCI DE GENES D'IMMUNOGLOBULINE D'ORIGINE HUMAINE, DOTES DE REGIONS VH ET Vλ, ET ANTICORPS PRODUITS A PARTIR DE TELS MAMMIFERES
US6075181A (en) 1990-01-12 2000-06-13 Abgenix, Inc. Human antibodies derived from immunized xenomice
US6150584A (en) 1990-01-12 2000-11-21 Abgenix, Inc. Human antibodies derived from immunized xenomice
US6982321B2 (en) 1986-03-27 2006-01-03 Medical Research Council Altered antibodies
US7087409B2 (en) 1997-12-05 2006-08-08 The Scripps Research Institute Humanization of murine antibody

Patent Citations (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US6982321B2 (en) 1986-03-27 2006-01-03 Medical Research Council Altered antibodies
US5545807A (en) 1988-10-12 1996-08-13 The Babraham Institute Production of antibodies from transgenic animals
WO1991010741A1 (fr) 1990-01-12 1991-07-25 Cell Genesys, Inc. Generation d'anticorps xenogeniques
US6150584A (en) 1990-01-12 2000-11-21 Abgenix, Inc. Human antibodies derived from immunized xenomice
US6075181A (en) 1990-01-12 2000-06-13 Abgenix, Inc. Human antibodies derived from immunized xenomice
US5661016A (en) 1990-08-29 1997-08-26 Genpharm International Inc. Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
US5625126A (en) 1990-08-29 1997-04-29 Genpharm International, Inc. Transgenic non-human animals for producing heterologous antibodies
US5633425A (en) 1990-08-29 1997-05-27 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5569825A (en) 1990-08-29 1996-10-29 Genpharm International Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
US5545806A (en) 1990-08-29 1996-08-13 Genpharm International, Inc. Ransgenic non-human animals for producing heterologous antibodies
WO1996033735A1 (fr) 1995-04-27 1996-10-31 Abgenix, Inc. Anticorps humains derives d'une xenosouris immunisee
WO1996034096A1 (fr) 1995-04-28 1996-10-31 Abgenix, Inc. Anticorps humains derives de xeno-souris immunisees
WO1998024893A2 (fr) 1996-12-03 1998-06-11 Abgenix, Inc. MAMMIFERES TRANSGENIQUES POSSEDANT DES LOCI DE GENES D'IMMUNOGLOBULINE D'ORIGINE HUMAINE, DOTES DE REGIONS VH ET Vλ, ET ANTICORPS PRODUITS A PARTIR DE TELS MAMMIFERES
US7087409B2 (en) 1997-12-05 2006-08-08 The Scripps Research Institute Humanization of murine antibody

Non-Patent Citations (50)

* Cited by examiner, † Cited by third party
Title
615TH MEETING BELFAST, 1986
AGNEW, CHEM INTL. ED. ENGL., vol. 33, 1994, pages 183 - 186
BRUGGEMANN ET AL., YEAR IN IMMUNOL., vol. 7, 1993, pages 33
CORBETT, T.H. ET AL., CANCER RES, vol. 35, 1975, pages 2434 - 2439
EGBERTS J.H. ET AL.: "Dexamethasone reduces tumor recurrence and metastasis after pancreatic tumor resection in SCID mice.", CANCER BIOL. THER., vol. 7, no. 7, July 2008 (2008-07-01), pages 1044 - 1050, XP002595691 *
FELLOUSE, PROC. NATL. ACAD. SCI. USA, vol. 101, no. 34, 2004, pages 12467 - 12472
FERRARA; ALITALO, NATURE MEDICINE, vol. 5, 1999, pages 1359 - 1364
FIDLER, I.J., NAT. REV. CANCER, vol. 3, 2003, pages 453 - 458
FIDLER, I.J., SEMIN CANCER BIOL, vol. 12, 2002, pages 89 - 96
FISHWILD ET AL., NATURE BIOTECHNOL., vol. 14, 1996, pages 845 - 851
HANAHAN, D. ET AL., GENES & DEVELOPMENT, vol. 21, 2007, pages 2258 - 2270
HARRELL, M.I. ET AL., J IMMUNOL METHODS, vol. 332, 2008, pages 170 - 174
HARRIS, BIOCHEM. SOC. TRANSACTIONS, vol. 23, 1995, pages 1035 - 1038
HONGO ET AL., HYBRIDOMA, vol. 14, no. 3, 1995, pages 253 - 260
HOOGENBOOM; WINTER, J. MOL. BIOL., vol. 227, 1991, pages 381
HURLE; GROSS, CURR. OP. BIOTECH., vol. 5, 1994, pages 428 - 433
JAKOBOVITS ET AL., NATURE, vol. 362, 1993, pages 255 - 258
JAKOBOVITS ET AL., PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 2551
JONCS ET AL., NATURE, vol. 321, 1986, pages 522 - 525
KLAGSBRUN; D'AMORE, ANNU. REV. PHYSIOL., vol. 53, 1991, pages 217 - 39
KOHLER; MILSTEIN, NATURE, vol. 256, 1975, pages 495 - 97
KUREBAYASHI J. ET AL.: "Postsurgical oral administration of Uracil and Tegafur inhibits progression of micrometastasis of human breast cancer cells in nude mice", CLINICAL CANCER RES., vol. 3, 1997, pages 653 - 659, XP002595690 *
LEE ET AL., J. IMMUNOL. METHODS, vol. 284, no. 1-2, 2004, pages 119 - 132
LEE ET AL., J. MOL. BIOL., vol. 340, no. 5, 2004, pages 1073 - 1093
LONBERG, NATURE, vol. 368, 1994, pages 856 - 859
LONBERG; HUSZAR, INTERN. REV. IMMUNOL., vol. 13, 1995, pages 65 - 93
MANO ET AL., CLINICAL COLORECTAL CANCER, vol. 7, 2008, pages 178 - 183
MARKS ET AL., BIOLTECHNOLOGY, vol. 10, 1992, pages 779 - 783
MARKS ET AL., J. MOL. BIOL., vol. 222, 1991, pages 581
MORRISON ET AL., PROC. NATL. ACAD. SCI. USA, vol. 81, 1984, pages 6851 - 6855
MORRISON, NATURE, vol. 368, 1994, pages 812 - 813
NEUBERGER, NATURE BIOTECHNOL., vol. 14, 1996, pages 826
PODSYPANINA, K. ET AL., SCIENCE, vol. 32, 2008, pages 1841 - 4
PRESTA, CURR. OP. STRUCT. BIOL., vol. 2, 1992, pages 593 - 596
RIECHMANN ET AL., NATURE, vol. 332, 1988, pages 323 - 329
SATO., INT. J. CLIN. ONCOL., vol. 8, 2003, pages 200 - 206
SAUSVILLE; BURGER, CANCER RES, vol. 66, 2006, pages 3351 - 3354
SCHABEL, F.M. ET AL., CANCER, vol. 40, pages 558 - 568
SIDHU ET AL., J. MOL. BIOL., vol. 338, no. 2, 2004, pages 299 - 310
SINGH, M. ET AL., CLIN CANCER RES, vol. 12, 2006, pages 5312 - 28
STREIT; DETMAR, ONCOGENE, vol. 22, 2003, pages 3172 - 3179
THIERY, J.P, NAT. REV. CANCER, vol. 2, 2002, pages 442 - 454
THURMAN G.B. ET AL.: "Acute inflammatory changes in subcutaneous microtumors in the ears of mice induced by intravenous CM101 (GBS toxin)", J. CANCER RES. CLIN. ONCOL., vol. 122, 1996, pages 549 - 553, XP008125274 *
TONINI ET AL., ONCOGENE, vol. 22, 2003, pages 6549 - 6556
TORGENSON M J ET AL: "Natural History of Pancreatic Cancer Recurrence Following ''Curative'' Resection in Athymic Mice", JOURNAL OF SURGICAL RESEARCH, vol. 149, no. 1, 1 September 2008 (2008-09-01), ACADEMIC PRESS INC., SAN DIEGO, CA, US, pages 57 - 61, XP023905484, ISSN: 0022-4804, [retrieved on 20080814], DOI: 10.1016/J.JSS.2007.08.024 *
VAN DYKE, T. ET AL., CELL, vol. 108, 2002, pages 135 - 144
VASWANI; HAMILTON, ANN. ALLERGY, ASTHMA & IMMUNOL., vol. 1, 1998, pages 105 - 115
WEINBERG, R.A., CANCER CELL, vol. 14, 2008, pages 283 - 4
WILMAN: "Prodrugs in Cancer Chemotherapy", BIOCHEMICAL SOCIETY TRANSACTIONS, vol. 14, pages 375 - 382
YOKOYAMA H. ET AL.: "Biological significance of isolated tumor cells and micrometastasis in lymph nodes evaluated using a green fluorescent protein-tagged human gastric cancer cell line.", HUM. CANCER BIOL., vol. 12, no. 2, 15 January 2006 (2006-01-15), pages 361 - 368, XP002595689 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102750861A (zh) * 2012-06-23 2012-10-24 关真民 胃癌种植性转移演示模型
WO2015013432A1 (fr) * 2013-07-23 2015-01-29 Genentech, Inc. Modèle de cancer colorectal
JP2016525358A (ja) * 2013-07-23 2016-08-25 ジェネンテック， インコーポレイテッド 結腸直腸がんのモデル

Similar Documents

Publication Publication Date Title
EP3038647B1 (fr) Polythérapie pour le traitement du glioblastome
TWI577695B (zh) 用於輔助及先導性輔助療法之血管內皮生長因子（ｖｅｇｆ）-特異性拮抗劑及早期腫瘤之治療
CN104271601B (zh) 使用pd-1轴结合拮抗剂和vegf拮抗剂治疗癌症的方法
RU2519669C2 (ru) Лечение антителами против vegf
TWI612971B (zh) 用於乳癌治療之抗-血管新生療法
EP3355902B1 (fr) Combinaison d'un antagoniste de la liaison de l'axe pd-1 et d'un inhibiteur de alk dans le traitement du cancer alk-négatif
CN102498129B (zh) 使用bv8拮抗剂或g-csf拮抗剂抑制肿瘤转移
CN102458467A (zh) 辅助癌症疗法
TWI451875B (zh) 用於治療先前治療過之乳癌之抗－血管新生療法
WO2011006001A1 (fr) Modèle animal pour l'évaluation de thérapies d'adjuvant du cancer
CN103476430A (zh) 抗egfl7抗体用于治疗的给药
HK1144290A (en) Treatment with anti-vegf antibodies
HK1144202A (en) Treatment with anti-vegf antibodies
HK1200857B (zh) 使用pd-1轴结合拮抗剂和vegf拮抗剂治疗癌症的方法

Legal Events

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

Ref document number: 10732603

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 10732603

Country of ref document: EP

Kind code of ref document: A1