WO2000000591A1 - Anticorps monoclonaux humains de l'antigene tumoral uk114, et lymphocytes et hybridomes pour leur production - Google Patents

Anticorps monoclonaux humains de l'antigene tumoral uk114, et lymphocytes et hybridomes pour leur production Download PDF

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Publication number
WO2000000591A1
WO2000000591A1 PCT/EP1999/004333 EP9904333W WO0000591A1 WO 2000000591 A1 WO2000000591 A1 WO 2000000591A1 EP 9904333 W EP9904333 W EP 9904333W WO 0000591 A1 WO0000591 A1 WO 0000591A1
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WO
WIPO (PCT)
Prior art keywords
cells
antibodies
hybridomas
monoclonal antibodies
human
Prior art date
Application number
PCT/EP1999/004333
Other languages
English (en)
Inventor
Alberto Bartorelli
Gianni Bussolati
Ada Funaro
Original Assignee
Zetesis S.P.A.
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 Zetesis S.P.A. filed Critical Zetesis S.P.A.
Priority to EP99931141A priority Critical patent/EP1090107A1/fr
Priority to AU47758/99A priority patent/AU4775899A/en
Priority to CA002331762A priority patent/CA2331762A1/fr
Priority to JP2000557344A priority patent/JP2002519020A/ja
Publication of WO2000000591A1 publication Critical patent/WO2000000591A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man

Definitions

  • the present invention refers to human monoclonal antibodies against the tumor antigen UKl 14, to immortalized lymphocytes and to hybridomas secreting said antibodies.
  • Antibodies specific for the UKl 14 protein have been isolated from the sera of cancer patients and in many cases the specific antibody titer turned out to be significantly increased in patients treated with UKl 01 , the drug containing as the active component the heterologous UKl 14 protein (Bartorelli, A., et al., J. Tumor
  • the antibodies of interest have been analyzed on a panel of tumor cell-lines of different origin and of normal cells so as to evaluate the selective recognition of the tumor-associated antigen.
  • the human antibodies in addition to the purified native protein, all recognized a membrane antigen expressed on gastric tumors (HT-29, Kato III), colon tumors (Colo 684, LoVo), breast tumors (MCF-7, MDA), and on some neuroblastomas (LAN-1, SY5Y) as well as on breast and lung tumors from patients subjected to surgery.
  • a first embodiment of the invention refers therefore to the human monoclonal antibodies directed against the UKl 14 protein.
  • a second embodiment of the invention refers to immortalized lymphocyte cells secreting the monoclonal antibodies against UKl 14.
  • EBV-immortalized cells having the drawbacks of instability and low yield in produced antibody
  • human hybridomas were obtained.
  • Said lines yield generally more stable hybridomas, able to better grow in nude mice in form of ascitic tumor.
  • Three EBV lines selected on the basis of their growing characteristics were fused with the hetero-myeloma K6H6 (Carrol, WL, et al., J. Immunol. Meth. 89:61-69, 1986) obtainable from ATCC and the obtained clones were analyzed for reactivity against the UKl 14 antigen by an ELISA method. The fusion system was very efficient and more than 70% of the obtained clones were reactive.
  • the selected hybridomas were expanded in vitro and cloned by limit dilution.
  • the final result was the selection of three hybridomas: UK#l/2G3hy, UK#l/lAl lhy and UK#l/3D8hy.
  • the first two hybridomas are of IgM-k isotype and the third of IgM- ⁇ .
  • the hybridomas maintained the tumor specificity characteristic of the parental lines, as shown in the following Table:
  • MCF-10 non-malignant breast
  • the reactivity of human monoclonal antibodies on the different tumor lines also showed high differences in terms of number of positive cells and of fluorescence intensity thereby suggesting that different epitopes, recognized by the tree reagents, exist on the target antigen expressed on the membrane, as showed in figure.
  • the three hybridomas have been adapted to grow in vivo in nulnu mice and so are able to grow as ascites tumor, and this allows to increase the specific immunoglobulin concentration of some order of magnitude. Then, the hybridomas have been adapted to grow in synthetic serum-free medium: a main stage to purify antibody eventually destined to clinical use.
  • a further object of the invention refers to human hybridomas that are able to secrete anti UKl 14 human monoclonal antibodies.
  • One of such hybridomas (UK#1/3D8) was deposited at ECACC (European Collection of Cell Cultures) under n° 9706 2409 on June 24 th , 1997.
  • the human antibodies herein described are an optimal clinic/diagnostic tool: first of aspect at present all, they permit to test the human antibody answers to the UKl 01 treatment, at present unknown and different from what is reproducible in the animal models; secondly, the produced reagents could be used in diagnostics as vectors of radioactive isotopes useful in radio-pilot surgery, or in therapy as toxins or drugs vectors.
  • the antibodies of the invention do not cause the undesirable side effects typical of the murine reagents, as they are of human nature.
  • the following example shows in more detail the steps for the preparation of the immortalized cells, of the hybridomas and of the ascitis, as well as the functional characteristics belonging to the lytic power, i) B cell immortalization Human B cells purified from peripheral blood from a carcinoma lung cancer patient successfully treated with UK101 , were enriched by conventional density gradient. The T lymphocytes were removed from the resulting lymphocytic cells by rosetting with sheep erythrocytes. The purified B cells (10 7 ml) were infected by incubation with Epstain Barr virus (EBV) obtained from the B95--8 cell line (10% in the colture medium).
  • EBV Epstain Barr virus
  • lymphoblastoid cell lines three were selected, on the basis of the best growth and secretion characteristics, to be used as partner in the somatic fusion with the myeloma. Since the availability of suitable human myeloma as tumor partners in the cell fusion is very scarce and the products resulting are also very unstable, an hetero-myeloma cell line (human- mouse) provided from ATCC able to fuse with high efficiency and with a better products stability was selected.
  • the used cell line (K6H6/B5) was obtained from the fusion of a murine myeloma with normal human B lymphocytes and selected for being non-secreting and aminopterin-sensitive.
  • the EBV cell lines were fused, in a 2: 1 ratio, with the K6H6/B5 line using polyethylene glycol (pH 7) as promoter agent following a conventional technique. After the fusion the cells were plated on irradiated allogenic lymphocytes constituting the support cells. After 24 hours the selectors were added in the following ratio: hypoxanthine l OO ⁇ M, aminopterin 800nM, thymidine 15 ⁇ M, ouabain 0.1 ⁇ M.
  • KATO III gastric carcinoma
  • MOG-G-UVW astrocytoma
  • HT-29 colon carcinoma selected as tumor target
  • 3,7 MBq of Na 2 [ 51 C R ]0 4 for 1 hour at 37°C.
  • the cells were incubated at 37°C for 1 ,5 hour in the presence of the human antibodies anti-UK114 (ascites diluted 1 : 10) and of 10 ⁇ l of human serum.
  • human serum deprived of C8 C9 factors, or heat-inactivated were used.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne des anticorps monoclonaux humains de l'antigène tumoral UK114 ainsi que leur utilisation à des fins diagnostiques et thérapeutiques.
PCT/EP1999/004333 1998-06-26 1999-06-23 Anticorps monoclonaux humains de l'antigene tumoral uk114, et lymphocytes et hybridomes pour leur production WO2000000591A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP99931141A EP1090107A1 (fr) 1998-06-26 1999-06-23 Anticorps monoclonaux humains de l'antigene tumoral uk114, et lymphocytes et hybridomes pour leur production
AU47758/99A AU4775899A (en) 1998-06-26 1999-06-23 Human monoclonal antibodies against the tumor antigen uk114 and lymphocyte cellsand hybridomas for their production
CA002331762A CA2331762A1 (fr) 1998-06-26 1999-06-23 Anticorps monoclonaux humains de l'antigene tumoral uk114, et lymphocytes et hybridomes pour leur production
JP2000557344A JP2002519020A (ja) 1998-06-26 1999-06-23 腫瘍抗原ｕｋ１１４に対するヒトモノクローナル抗体並びにそれらの産生のためのリンパ性細胞及びハイブリドーマ

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ITMI98A001467 1998-06-26
IT98MI001467 IT1301815B1 (it) 1998-06-26 1998-06-26 Anticorpi monoclonali umani contro l'antigene tumorale uk114 e cellulelinfocitarie e ibridomi per la loro produzione

Publications (1)

Publication Number Publication Date
WO2000000591A1 true WO2000000591A1 (fr) 2000-01-06

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PCT/EP1999/004333 WO2000000591A1 (fr) 1998-06-26 1999-06-23 Anticorps monoclonaux humains de l'antigene tumoral uk114, et lymphocytes et hybridomes pour leur production

Country Status (6)

Country Link
EP (1) EP1090107A1 (fr)
JP (1) JP2002519020A (fr)
AU (1) AU4775899A (fr)
CA (1) CA2331762A1 (fr)
IT (1) IT1301815B1 (fr)
WO (1) WO2000000591A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001064869A2 (fr) * 2000-03-03 2001-09-07 Zetesis S.P.A. Nouvel antigène tumoral

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IT201600127428A1 (it) * 2016-12-16 2018-06-16 Cusani Alberto Bartorelli Nuova proteina ricombinante uk 114 in forma stabile polimerica per uso nella terapia, nella diagnostica e nella prevenzione di neoplasie maligne

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BUSSOLATI, GIANNI (1) ET AL: "Cytolytic and tumor inhibitory antibodies against UK114 protein in the sera of cancer patients.", INTERNATIONAL JOURNAL OF ONCOLOGY, (1997) VOL. 10, NO. 4, PP. 779-785., XP002067972 *
FUNARO A ET AL: "Identification of a 220-kDa membrane tumor-associated antigen by human anti-UK114 monoclonal antibodies selected from the immunoglobulin repertoire of a cancer patient.", EXPERIMENTAL CELL RESEARCH, (1999 MAR 15) 247 (2) 441-50., XP002116994 *
JAMES, K.: "Human monoclonal antibody technology", HANDB. EXP. PHARMACOL. (1994), 113(PHARMACOLOGY OF MONOCLONAL ANTIBODIES) 3-22, XP002116993 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001064869A2 (fr) * 2000-03-03 2001-09-07 Zetesis S.P.A. Nouvel antigène tumoral
WO2001064869A3 (fr) * 2000-03-03 2002-10-10 Zetesis Spa Nouvel antigène tumoral

Also Published As

Publication number Publication date
EP1090107A1 (fr) 2001-04-11
IT1301815B1 (it) 2000-07-07
JP2002519020A (ja) 2002-07-02
ITMI981467A1 (it) 1999-12-26
AU4775899A (en) 2000-01-17
CA2331762A1 (fr) 2000-01-06

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