Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
  • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
  • A61K51/1045—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IG], 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • 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 mammalian immune system has a matchless ability to produce molecules with specificity and avidity for a particular spatial and polar structure, as may be found with sequences of amino acids and sugars. For a long period of time, one was dependent upon producing antibodies employing the immune system jln vivo. The resulting polyclonal antibodies demonstrated high specificity for a specific antigen, but could not discriminate between various sites on the antigen and, furthermore, were a mixture of antibodies of varying specificity and avidity. thus, one observed the averaging over the entire composition and not the properties of a specific antibody.
• human hybridomas which produce antibodies allogenic to a human host, particularly for _in. vivo applications, human hybridomas remain of great interest. In other instances, even with the difficulties encountered with human-human crosses, the human hybridoma may be preferable to a heterogeneic cross, where the resulting hubridoma may lose the genetic information for the monoclonal antibodies after a number of passages.
• Monoclonal antibodies for these purposes desirably are specific for a particular type of cancer or subset of cancers, rather than being specific for a particular host cancer cell. it is therefore desirable to develop monoclonal antibodies which can be used in the diagnosis and treatment of human cancers.
• Lymphocytes derived from a neoplastic human host are immortalized by fusion with human fusion partners to provide human x human hybridomas secreting monoclonal antibodies specific for a neoplastic cell.
• monoclonal antibodies specific for solid tumor cells such as cervical cancer cells are provided for use in diagnostics and therapy.
• Human monoclonal antibodies specific for neoplastic cells from soiid tumors are obtained from human x human fusions employing B lymphocytes, e.g., from lymph nodes draining a solid tumor. Particularly, lymph nodes are selected which appear to be active based on necrosis of tumor cells in the vicinity of the lymph node in an immunocompetent host.
• the draining lymph node(s) may be isolated in conjunction with a variety of human tissue, e.g., cervix, mammary, colon, lungs, prostate, skin, etc. Of particular interest are lymph nodes from the spinal area.
• the fusion partner may be any convenient immortalized B-cell, which does not secrete immunoglobulins, individual chains or fragments thereof, can be selected against, as with HAT medium, and desirably has a high fusion efficiency.
• Illustrative fusion partners are UC729-6, J-4 (SKO-007) , and GM1500 6TG-A12.
• the fusions may be performed as described in the literature employing PEG1500 as fusogen, plating the cells in HAT medium in a plurality of wells and then screening supernatants in the viable cell wells for antibodies of interest. Wells positive for reactivity are then cloned by limiting dilution and expanded.
• CLNH5 and CLNH11 are obtained by fusion between the fusion partner UC729-6 with lymphocytes from lymph node cells of a patient having cervical cancer.
• UC729-6 is on deposit at the A.T.C.C. with Accession No. CRL 8061. UC729-6 was deposited for patent purposes in conjunction with the filing of application Serial No. 247,652.
• the lymphocytes employed for fusion were from a draining lymph node from the spinal area and peripheral blood lymphocytes from a patient having cervical carcinoma.
• the fusion is performed by combining the patient's lymphocytes from the lymph node with the fusion partner UC729-6 at a ratio of about 2:1 in a solution of about 35% polyethylene glycol in HEPES buffered RPMI 1640.
• the mixture of cells is then suspended in appropriate selective medium, particularly HAT medium containing about 10% fetal bovine serum, placed in wells at about 10 cells per well and a sufficient time permitted for the cells to grow.
• the selective medium is replaced from time to time.
• Wells from the above fusion provided clones specifically reactive with the cervical cancer cells of the host patient which were designated CLNH5 and 11. These wells provided human IgM and IgG monoclonal antibodies, respectively, which react wit antigen found on a variety of cervical carcinomas and other tumor cell lines, e.g., small cell carcinoma of the lung, but not with normal tissues and normal cell lines, which were tested.
• the hybridomas and monoclonal antibodies can find use in a variety of ways, particularly as sources of genetic material, as reagents, and as precursors to products which find use as reagents.
• the subject hybridomas may be used as a source for genetic material.
• the subject hybridomas may be fused with other fusion partners to provide novel hybridomas having the same secretory capabilities as CLNH5 and 11 to provide antibodies having the same specificity.
• Such fusions may result in the production of antibodies having different heavy chains so as to provide the other classes or subclasses of antibodies, e.g., G, A or M.
• the hybridoma may also be used as a source of DNA, which by hybrid DNA technology, the genes may be excised, introduced into a lymphoma for production of the mature antibodies.
• the monoclonal antibodies can be used in a variety of ways, both in vivo and ⁇ n vitro diagnosis, as well as in therapy.
• Labels may include radionuclides, enzymes, fluorescers, toxins or the cytotoxic fragment of toxins, particles, metals, metalloids, etc.
• the antibodies may be incorporated in liposo e membranes or modified with lipids, so as to be incorporated in such membranes.
• the antibodies by themselves or labeled may be used in _in vitro diagnosis for measuring the presence of antigens associated with a neoplasm such as cervical cancer, for _in vivo diagnosis for introduction into a host, e.g., intravenously, in a physiologically acceptable carrier, e.g., PBS, or may be introduced for therapeutic purposes in the same manner.
• the antibodies by themselves or labeled may also be used for treating a neoplasm in human host such as cervical carcinoma, pro.state tumor, colon carcinoma,
• the antibodies of this invention are easily soluble in physiological saline, and therefore can be injected intravenously or intramuscularly as a saline solution 5 or a drip. Furthermore, the antibodies of the invention can be used in the form of an ointment or suppository.
• Fab fragments F(ab')p fragments, or Fv fragments may suffice.
• Lymph nodes were teased with nugent forceps in RPMI 1640 media and isolated lymphocytes were
• Lymphocytes were counted and mixed at a ratio of 2:1 with the human lymphoblastoid B cell line UC729-6 (Handley and Royston. 1982, in Hybridomas in Cancer
• each well contains a 0.6mm hole over which is placed a 6mm diameter glass fiber filter.
• Surface tension prevents fluid volumes less than lOOyl from draining through the hole until a vacuum is applied. When vacuum is applied, fluid is drawn through the filter and out the drain hole leaving particulate matter trapped on the filter.
• 50 ⁇ l of hybridoma supernatant were incubated 30 min at room temperature. Filters were then washed and incubated with 50 ⁇ l of a horseradish peroxidase conjugated goat anti-human Ig for an additional 30 min.
• Table 1 outlines the results of the fusion attempting to produce anti-SCCC (squamous cell carcinoma of cervix) human MoAbs.
• Hybridomas CLNH5 and CLNHll were cloned and expanded when found to react with the cervical carcinoma cell lines, CaSki and Hela.
• SCCC The relative amounts of human MoAb bound to each of the cell lines listed was measured by EIA.
• Antibody (IgM) secreted by CLNH5 shows positive reactivity with carcinomas of the cervix (CaSki, Hela) , lung (T293, Calu-1, and SK-MES-1) , melanoma (SK-MEL-28) , and prostate (LnCap) and was 9 negative for normal fibroblasts, T lymphocytes and peripheral blood lymphocytes.
• Antibody (IgG) secreted by CLNHll shows positive reactivity with carcinomas of the cervix (CaSki, Hela) , prostate (PC-3) , breast (ZR-76-1) , colon (COLO-205) and melanoma (G-361) and was negative for normal fibroblasts (WI * 38 and MRC-9) , T. lymphocytes and peripheral blood lymphocytes.
• Hybridoma CLNH5 -(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-a)-2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
• IgM binds to human cervical carcinoma cells and the other carcinomas mentioned above.
• Hybridoma CLNH5 can be proliferated in HAT medium (medium containing hypoxanthine, amethopterin and thymidine) .
• Hybridoma CLNHll -
• IgG binds to human cervical carcinoma cells, and the other carcinomas mentioned above.
• the relative DNA content (the ratio to the DNA content of normal human lymphocytes) was determined by a method which comprises dyeing the hybridoma and then separating and analyzing it by a cytofluorometer.
• the subject monoclonal antibodies are useful for the diagnosing, imaging and potentially for treating cervical carcinoma as well as other reactive tumors. Because of the specificity of the monoclonal antibodies over a rnage of cervical carcinomas from different hosts, the subject antibodies can be used in different hosts, rather than solely with the host source of the antigen. Because the subject antibodies are human, they are less likely to produce a significant immune response when employed in jLn vivo diagnosis or therapy.

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• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Immunology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• General Health & Medical Sciences (AREA)
• Organic Chemistry (AREA)
• Medicinal Chemistry (AREA)
• Biochemistry (AREA)
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• Epidemiology (AREA)
• Animal Behavior & Ethology (AREA)
• Oncology (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Cell Biology (AREA)
• Physics & Mathematics (AREA)
• Pharmacology & Pharmacy (AREA)
• Genetics & Genomics (AREA)
• Molecular Biology (AREA)
• Optics & Photonics (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Peptides Or Proteins (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Medicines Containing Material From Animals Or Micro-Organisms (AREA)

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Cited By (19)

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Citations (2)

• 1982
  • 1982-05-21 JP JP57084843A patent/JPS58201994A/ja active Granted
• 1983
  • 1983-05-19 SK SK3566-83A patent/SK279249B6/sk unknown
  • 1983-05-19 CZ CS833566A patent/CZ280677B6/cs not_active IP Right Cessation
  • 1983-05-20 DE DE3382764T patent/DE3382764T2/de not_active Expired - Lifetime
  • 1983-05-20 AU AU17729/83A patent/AU560595B2/en not_active Expired
  • 1983-05-20 EP EP83902157A patent/EP0109441B1/en not_active Expired - Lifetime
  • 1983-05-20 JP JP58502264A patent/JPS60501359A/ja active Pending
  • 1983-05-20 AT AT83902157T patent/ATE114189T1/de not_active IP Right Cessation
  • 1983-05-20 HU HU832697A patent/HU190908B/hu not_active IP Right Cessation
  • 1983-05-20 WO PCT/US1983/000781 patent/WO1983004313A1/en not_active Ceased
  • 1983-05-23 ZA ZA833686A patent/ZA833686B/xx unknown
  • 1983-05-31 IN IN683/CAL/83A patent/IN157982B/en unknown
• 1984
  • 1984-01-19 FI FI840219A patent/FI86077C/fi not_active IP Right Cessation
  • 1984-01-20 DK DK025084A patent/DK164510C/da not_active IP Right Cessation

Patent Citations (2)

Non-Patent Citations (5)

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