EP0814830A1 - Production d'interferon alpha naturel a partir d'un lymphoblastoide - Google Patents

Production d'interferon alpha naturel a partir d'un lymphoblastoide

Info

Publication number
EP0814830A1
EP0814830A1 EP96908839A EP96908839A EP0814830A1 EP 0814830 A1 EP0814830 A1 EP 0814830A1 EP 96908839 A EP96908839 A EP 96908839A EP 96908839 A EP96908839 A EP 96908839A EP 0814830 A1 EP0814830 A1 EP 0814830A1
Authority
EP
European Patent Office
Prior art keywords
interferon
lymphoblastoid
extract
cells
cell culture
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.)
Withdrawn
Application number
EP96908839A
Other languages
German (de)
English (en)
Other versions
EP0814830A4 (fr
Inventor
Peter R. Rothschild
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.)
Rothschild Peter
Original Assignee
Rothschild Peter
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 Rothschild Peter filed Critical Rothschild Peter
Publication of EP0814830A1 publication Critical patent/EP0814830A1/fr
Publication of EP0814830A4 publication Critical patent/EP0814830A4/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/70—Undefined extracts
    • C12N2500/80—Undefined extracts from animals
    • C12N2500/84—Undefined extracts from animals from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/11—Coculture with; Conditioned medium produced by blood or immune system cells

Definitions

  • Interferon Bac ground of the Invention Interferon is found in many vertebrates and seems to be one of the body's main lines of non-specific defense against viral infection. Interferon is a term generically comprehending a group of vertebrate glycoproteins and proteins which are known to have various biological activities, such as antiviral, antiproliferative and immunomodulatory activity. Interferons are all polypeptides consisting of approximately 150 amino acid residues, and has proved tentative successes with its use against hepatitis, herpes simplex, the common cold and even warts. However, one problem with interferon is the burdensome cost which has resulted from the difficulty of getting significant quantities of human interferon by conventional methods.
  • Interferons have generally been named in terms of the species of animal cells producing the substance (e.g., human, murine, bovine, etc.), the type of cell involved (e.g., leukocyte, lymphoblastoid, fibroblast) and, occasionally, the type of inducing material responsible for the interferon production (e.g. , virus, immune). Interferon has been loosely classified by some researchers according to the induction mode as either Type I or Type II, with the former classification comprehending viral and nucleic acid induced interferon, and the latter class including the material produced as a lymphokine through induction by antigens and mitogens.
  • interferons More recently, an orderly nomenclature system for interferon has been devised which classifies interferons into types on the basis of antigenic specificities. Under this newer classification system, the designations alpha, beta and gamma have been used to correspond to previous designations of leukocyte, fibroblast and Type II (immune) interferons, respectively.
  • Alpha and beta interferons are usually acid-stable and correspond to what have been called Type I interferons.
  • Gamma interferons are usually acid-labile and correspond to what have been called Type II interferons.
  • interferon for the treatment of disease in man and animals has been the subject of intense research efforts in many laboratories, both in industry and in educational institutions around the world. In some of the earliest research activities interferon was shown to have antiviral properties and the most successful clinical therapeutical applications to date have been in the treatment of virus-related disease states. More recently it has been found that exogenous interferon is effective for the regression or remission of some metastatic disease states. The literature is replete with reports of research and development efforts directed to defining activities and potential therapeutic uses of interferon. Most of the reports described activities of interferon in vitro or its effects in vivo following parenteral, particularly intramuscular and intradermal administration. There have been some reports of successful topical and intranasal usages.
  • An object of the present invention is to provide a compound which can be used for maintaining a differentiated cell line, specifically, of lymphoblastoid cells capable of producing natural human interferon- alpha.
  • This invention relates to a novel compound and its method of use to maintain a differentiated, responsive lymphoblastoid cell line.
  • the novel compound is an extract of human fetal thymus.
  • the T- lymphocytes are the source of interferon alpha.
  • Lymphoblastoid cells are the "cradle cells" for T- lymphocytes, which are one of the many existing forms of leukocytes. The lymphoblastoid cells are responsible for the interferon alpha endowment of the T- lymphocytes.
  • the production in general, is based on a method originally devised by Cantell in Finland. Essentially his technique consists in making the leukocytes yield their interferon contents by stimulating them with the otherwise innocuous Sendai virus.
  • researchers have been addressing the perspective of growing lymphoblastoid cell cultures in an ideal auxostatic medium with a sterile laboratory environment and subject these to Cantell's method in order to incite them to issue interferon alpha rather than isolating interferon alpha from donated which blood cells.
  • lymphoblastoid cells After a few generations, the lymphoblastoid cells began to lose their differentiation and, once their morphological change was complete, they did not react at all to any stimulus. All the attempts of hampering the loss of cellular differentiation failed. Many methods were tried, from enzyme or chemical influences to rhythmic changes of the medium, without success.
  • the method presented in this invention consists in the addition of a complex of natural substances to the auxostatic medium that contains the lymphoblastoid cell culture to maintain its differentiated state.
  • this substance is an extract made of human fetal thymuses.
  • Human fetal cells are used rather than established human adult cells to allow for a much more vigorous mitosis rate and substantially more economic interferon production.
  • the lymphoblatoid cells do not lose their differentiation, but remain receptive to the stimulation by the Cantell method.
  • these cultures do yield unrestricted volumes of natural interferon alpha that is completely independent of donors. Such production is perfectly predictable, whose volume is only limited by the available space and technological equipment.
  • interferon has been accepted by an international committee assembled to devise a system for the orderly nomenclature of interferons: to qualify as an interferon, a factor must be a protein which exerts non ⁇ specific, antiviral activity at least in homologous cells through cellular metabolic process involving synthesis of both RNA and protein. "Interferon" as used herein in describing the present invention shall be deemed to have that definition and shall contemplate proteins, including glycoproteins, regardless of their cell source.
  • the interferon can be derived from human or animal cell culture, and in accordance with the preferred embodiments is alpha or beta interferon. Proteins having activities similar to natural occurring interferons but with modified amino acid sequences are also contemplated as useful in accordance with this invention.
  • the clinical agents of choice for use in the present invention are human leukocyte interferon and lymphoblastoid interferon, mass produced by procedures involving lymphoblastoid cell cultures, induction of interferon production, via Cantell's method and isolation of interferon from culture media.
  • the fetal thymuses extract is added to the lymphoblastoid cell cultures.
  • the source material is supplied after careful clinical screening of the thymus donors.
  • the thymus cells are extracted under surgical sterility regime, then flashfrozen, and ground up to a 00 level, frozen powder, in a special grinder capable of operating at extremely low temperatures.
  • the powder is then extended on large flat containers in a one millimeter layer and thawed out at room temperature under a vacuum. After the thawing, the dehydrated cells are exposed to an acid gas flow which breaks down their membranes. Then, the open lymphoblastoid (thymus) cells are subjected to ultracentrification with the purpose of separating the DNA contained in their cell nucleuses.
  • the thus obtained substance is then diluted in a physiological, sterile saline solution and added to the auxostatic medium that contains and nourishes the lymphoblastoid cell culture growing in that medium.
  • the extract made of human fetal thymuses is added to the adult lymphoblastoid cell culture at a rate of 2 milliliters per
  • the cells can successfully be used subsequently in the Cantell interferon isolation process.
  • the cells are immortal and continue mitotic divisions as long as the aforementioned fetal thymus extract is added.
  • Human alpha-interferon can be prepared through the following procedures, commonly referred to as the Cantell procedure. The process begins with packs of human leukocytes. The buffy coats in these packs are pooled into centrifuge bottles, and then are diluted with 0.83% ammonium chloride. The mixture is incubated for 15 minutes with intermittent shaking, and is then centrifuged for 20 minutes at 2000 rp . The supernatant is discarded, and the cell pellets are resuspended with a minimal volume of sterile pharmaceutical grade arabic gum (C 5 H 10 O 5 ). The mixture is then diluted with ammonium chloride and centrifuged.
  • the supernatant is again discarded, and the remaining cell pellets are resuspended with a minimal volume of a tissue culture medium such as Minimal Essential Medium (MEM), available from KC Biological.
  • MEM Minimal Essential Medium
  • the cell concentration is determined with a Coulter counter. Interferon induction takes place in glass or plastic bottles.
  • the induction medium contains MEM, 75mM Hepes (available from Calbiochem), 75mM Tricine (available from Sigma Chemical Co.), human gamma serum (18mg/ml), and gentamycin sulfate (from M. A. Bioproducts; 50mcg/ml).
  • the cells are added to the induction vessels at a final concentration of about 5 to 10 million cells per milliliter.
  • the induction vessel is incubated in a 37 degree(s) C. water bath, and alpha-interferon is added as a primer. After two hours, Sendai virus is added to the induction mixture. This causes alpha interferon to be produced in the supernatant by the leukocytes. After a 12- 18 hour incubation time, the induction mixture is centrifuged. The cells are discarded, and the supernatant is then purified. The crude interferon is chilled to 10 degree(s) C. or below in an ice bath. Five molar potassium thiocyanate is added to obtain a final concentration of 0.5M. This solution is stirred for 15 minutes, and then its pH is lowered to 3.3 by adding hydrochloric acid. The mixture is then centrifuged at 2800 rpm for 30 minutes, and the supernatant is discarded.
  • the pellets are then resuspended in 95% ethanol and are stirred for 15 minutes. This suspension is centrifuged at 2800 rpm for 20 minutes, and the pellets are discarded. The mixture is stirred for 10 minutes, and then centrifuged at 280 rpm for 20 minutes. The pellets are discarded. The pH of the supernatant is then adjusted to 8 with sodium hydroxide. This solution is stirred for 10 minutes, followed by centrifugation at 2800 rpm for 20 minutes. The supernatant is discarded, and the pellets are resuspended with 0.5M potassium thiocyanate in a 0.1M sodium phosphate buffer. This suspension is stirred at 4 degree(s) C.
  • the suspension is centrifuged at 2800 rpm for 20 minutes, and the pellets are discarded.
  • the pH of the supernatant is adjusted to 5.3 with hydrochloric acid.
  • the pH of the supernatant is adjusted to 2.8 with hydrochloric acid, followed by further stirring for 20 minutes. This mixture is centrifuged at 2800 rpm, and the resulting pellet is purified human alpha- interferon.
  • Interferon Preparations are commercially available from Hoffmann-LaRoche, Burroughs-Wellcome and Schering- Plough.
  • the fetal thymus extract could be used with these methods as well.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Cell Biology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention a pour objet un extrait de thymus foetal humain. Cet extrait, une fois ajouté aux cultures cellulaires d'un lymphoblastoïde, permet aux cellules de rester dans un état différencié et de répondre aux stimuli, ce qui favorise la production d'interféron alpha par les cellules du lymphoblastoïde.
EP96908839A 1995-03-15 1996-03-15 Production d'interferon alpha naturel a partir d'un lymphoblastoide Withdrawn EP0814830A4 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US404667 1989-09-07
US40466795A 1995-03-15 1995-03-15
PCT/US1996/003613 WO1996028184A1 (fr) 1995-03-15 1996-03-15 Production d'interferon alpha naturel a partir d'un lymphoblastoide

Publications (2)

Publication Number Publication Date
EP0814830A1 true EP0814830A1 (fr) 1998-01-07
EP0814830A4 EP0814830A4 (fr) 2001-12-12

Family

ID=23600547

Family Applications (1)

Application Number Title Priority Date Filing Date
EP96908839A Withdrawn EP0814830A4 (fr) 1995-03-15 1996-03-15 Production d'interferon alpha naturel a partir d'un lymphoblastoide

Country Status (4)

Country Link
EP (1) EP0814830A4 (fr)
CN (1) CN1148342A (fr)
AU (1) AU5254596A (fr)
WO (1) WO1996028184A1 (fr)

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
No further relevant documents disclosed *
See also references of WO9628184A1 *

Also Published As

Publication number Publication date
WO1996028184A1 (fr) 1996-09-19
EP0814830A4 (fr) 2001-12-12
AU5254596A (en) 1996-10-02
CN1148342A (zh) 1997-04-23

Similar Documents

Publication Publication Date Title
Chomyn [29] Platelet-mediated transformation of human mitochondrial DNA-less cells
Galbraith et al. Transferrin binding by human lymphoblastoid cell lines and other transformed cells
EP0302894B1 (fr) Procede de culture de leucocytes
US20120027678A1 (en) Method of using stem cells to aid in diagnosis
EP0240348B1 (fr) Agent et procédé pour l'élimination d'acides nucléiques et/ou d'endotoxine
Fernandez-Botran et al. Methods in cellular immunology
Kitamura et al. Differentiation and transdifferentiation of mast cells; a unique member of the hematopoietic cell family
CN1122716C (zh) 体外细胞培养和移植试剂盒
KR860000898B1 (ko) 인체 융모막성선 자극호르몬(Human Chorionic Gonadotropin)의 제조방법
JPS6154040B2 (fr)
US5759856A (en) Cell culture system
WO1996028184A1 (fr) Production d'interferon alpha naturel a partir d'un lymphoblastoide
EP0254593A2 (fr) Préparation et applications d'interféron gamma
US4452893A (en) Cell growth medium supplement
Wilt The concept of messenger RNA and cytodifferentiation
JP2003511389A (ja) 調節／アンフォールディング型のエズリンペプチド
US4124448A (en) Process for the large scale production of human growth hormone by serial secondary suspension culture
KR970002165B1 (ko) γ-인터페론의 제조방법과 그 용도
Chang et al. Spontaneous Cytotoxicity by Monocyte‐Enriched Subpopulations of Human Peripheral Blood Mononuclear Cells against Human or Mouse Anchorage‐Dependent Tumour Cell Lines: Contribution of NK‐like Cells
US7521234B2 (en) Mammalian immortalized liver cell
SU745948A1 (ru) Штамм перевиваемой клеточной линии фибробластов эмбриона овцы фэо, продуцирующий бычий лейкозный вирус (блв)
JP2566761B2 (ja) 骨髄単球系細胞
JPS5823793A (ja) ヒトｔ細胞増殖因子の製造方法
USRE30753E (en) Process for the large scale production of human growth hormone by serial secondary suspension culture
EP0945463B1 (fr) Procédé de préparation d'interféron [alpha] leucocytaire humain

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19971009

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

A4 Supplementary search report drawn up and despatched

Effective date: 20011026

AK Designated contracting states

Kind code of ref document: A4

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20010401