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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• Micro-Organisms Or Cultivation Processes Thereof (AREA)
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• 1998
  • 1998-06-26 IT IT98MI001467 patent/IT1301815B1/it active IP Right Grant
• 1999
  • 1999-06-23 AU AU47758/99A patent/AU4775899A/en not_active Abandoned
  • 1999-06-23 CA CA002331762A patent/CA2331762A1/en not_active Abandoned
  • 1999-06-23 JP JP2000557344A patent/JP2002519020A/ja active Pending
  • 1999-06-23 WO PCT/EP1999/004333 patent/WO2000000591A1/en not_active Application Discontinuation
  • 1999-06-23 EP EP99931141A patent/EP1090107A1/de not_active Withdrawn

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