WO1989004670A1 - Procede de production d'anticorps - Google Patents

Procede de production d'anticorps Download PDF

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Publication number
WO1989004670A1
WO1989004670A1 PCT/GB1988/001030 GB8801030W WO8904670A1 WO 1989004670 A1 WO1989004670 A1 WO 1989004670A1 GB 8801030 W GB8801030 W GB 8801030W WO 8904670 A1 WO8904670 A1 WO 8904670A1
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WO
WIPO (PCT)
Prior art keywords
material according
immunogen
cell
lipid
silica
Prior art date
Application number
PCT/GB1988/001030
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English (en)
Inventor
Bruce Fletcher Clark
Original Assignee
Imperial Cancer Research Technology Ltd
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 Imperial Cancer Research Technology Ltd filed Critical Imperial Cancer Research Technology Ltd
Publication of WO1989004670A1 publication Critical patent/WO1989004670A1/fr

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Classifications

    • 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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants

Definitions

  • the present invention relates to processes and materials for producing antibodies, including monoclonal antibodies, against lipid antigens or haptens of low immunogenicity.
  • This method is suitable for generating antibody-producing cells for use in production of hybridoma cell lines secreting monoclonal antibodies against the original immunogen and has resulted in yields of 10 - 15% of hybridomas producing monoclonal antibodies against glycolipids compared with ⁇ 0.1% of specific antibody secreting hybridomas obtained by conventional immunisation using glycolipid coated on the surface of acid-treated S.Minnesota bacteria.
  • an immunogenic material comprising a lipid immunogen and silica, preferably in the form of an injectable composition.
  • the silica will be finely divided and preferably it is of a grade useful in thin layer chromatography such as silica gel 60.
  • lipids for instance certain glycolipids are capable of raising an immune response when administered alone (and may be regarded as antigens) whereas others probably do not. However it is believed that substantially all lipids will raise an immune response when presented in association with silica in accordance with the present invention and the term "lipid immunogen" therefore encompasses all such lipids.
  • the glycolipids fall into two sub-classes; gangliosides and glycosphingolipids. The invention may be applied advantageously to materials from either of these classes.
  • the immunogenic material according to the present invention comprises a major proportion of silica and a minor proportion of the lipid immunogen, preferably the weight ratio of silica to lipid immunogen is at least 5:1 more preferably 10:1 and most preferably at least 50:1.
  • the immunogenic material according to the present invention should contain sufficient lipid immunogen to elicit an immune response and the quantity of silica should not be so great as to prohibit satisfactory adminisrtration of the immunogenic material to the host.
  • these considerations define a practical upper limit to the weight ratio of silica to lipid immunogen in the immunogenic material. In most situations however this ratio will be less than 1000:1, preferably 500:1 or less.
  • the immunogenic material according to the present invention may also comprise suitable excipients and carriers such as are conventionally used in immunisation, for instance water for injections, buffers, preservatives and antioxidants.
  • suitable excipients and carriers such as are conventionally used in immunisation, for instance water for injections, buffers, preservatives and antioxidants.
  • the immunogen material is provided in unit- or multi-dosage form ready for administration or as a lyophilized solid suitable for reconstitution by addition. of water for injection to provide a unit- or multi-dose quantity of an injectable formulation.
  • the present invention also provides a method for immunising a host against a lipid immunogen or for producing antibodies against a lipid immunogen which comprises administering at least one dose, for instance two or three doses of an immunogenic material according to the invention to the host. Where more than one dose is administered the interval between doses is selected to enhance the desired immune response and would normally be in the range of several days to several weeks but, particularly in the case of third and subsequent doses, the interval may be as long as several months. Polyclonal antibodies against the lipid immunogen can be obtained form the body fluids, of such immunised hosts.
  • the immunogenic materials of the present invention are in the production of monoclonal antibodies against the original immunogen.
  • Monoclonal antibodies (MAb's) were first described by Kohler and Milstein [Nature, 256, 495-497, (1975)] and have since found widespread use in research and are increasingly used in therapy and diagnosis.
  • the now almost routine process for obtaining MAb's against a particular immunogen involves innoculating animals with the immunogen, obtaining antibody-producing cells from the innoculated animal and immortalising these cells or instance by fusion with immortal cells, usually myeloma cells, derived from the same or a different species, to form an antibody-producing hybridoma cell line.
  • the present immunogenic materials are particularly suitable for use in producing monoclonal antibodies against glycolipids because of the good immune response obtained and the relatively large number of antibody-producing cells which may be obtained from the innoculated animal.
  • the present invention provides a process for producing a cell capable of secreting antibodies against a lipid immunogen which process comprises innoculating an animal with an immunogenic material as hereinbefore defined and removing antibody secreting cells from the animal.
  • the present invention is useful in obtaining cells which produce antibodies against disease associated lipid immunogens and may also be applied to production of cells which produce antibodies which have no effect in protecting the host against any disease and no therapeutic effect i.e. antibodies against non-disease non-therapeutic antigens and haptens.
  • the immunogenic material is administered by the intrasplenic route.
  • the administration is repeated after a period of from several days to several weeks and, when necessary, further innoculations may be made after similar intervals. The most appropriate interval will depend upon the particular lipid immunogen and the type of immune response which it is desired to elicit.
  • cells which secrete IgG antibodies are produced by several innoculations given over a relatively long period.
  • cells which secrete IgM antibodies are produced by several innoculations given in quick succession, for instance at intervals of one or a few days.
  • the invention provides a cell capable of secreting antibodies against a lipid immunogen which cell has been taken from an animal innoculated with an immunogenic material as hereinbefore defined.
  • the invention also provides, in a further aspect, a process for producing a immortal cell capable of secreting antibodies against a lipid immunogen which process comprises immortalising an antibody-secreting cell, the antibody-secreting cell having been taken from an animal innoculated with an immunogenic material as hereinbefore defined.
  • the term "immortal cell” means cell capable of repeated cell division ad infinitum under appropriate culture conditions.
  • the present invention further provides an immortal hybrid cell capable of secreting antibodies against a lipid immunogen which cell is an immortalised antibody-producing cell capable of secreting antibodies against the lipid immunogen taken from an animal innoculated with an immunogenic material as hereinbefore defined, or is a descendant of such a cell.
  • the present invention also provides a process for producing monoclonal antibodies against a lipid immunogen comprising culturing an immortal cell as hereinbefore defined or a descendant of such a cell and recovering the monoclonal antibodies.
  • Immortalisation is preferrably achieved by fusion with an immortal cell as previously described; in this case immortalisation affords an immortal hybrid cell capable of secreting antibody against the lipid immunogen. Immortalisation is usually followed by screening for appropriate antibody secreting cells.
  • the invention provides monoclonal antibodies against a lipid immunogen which antibodies are obtained from a culture of an immortal hybrid cell as hereinbefore defined or a descendant of such a cell.
  • Polyclonal and especially monoclonal antibodies against lipid immunogens are useful in passive immunisation, as diagnostic agents, as agents for localisation of tumours (when the lipid is one associated with tumour cells but not with normal cells) in which case the antibody will be coupled to a detectable label such as a radioisotope, and as therapeutic agents targetted to reach particular cells within the body.
  • the antibody is directed against a lipid associated with tumour cells but not normal cells and is coupled to cytostatic or cytotoxic agent.
  • Antibodies against lipids associated with tumour but not normal cells can be obtained by making a solvent extract of normal cells and a similar extract of tumour cells from the same tissue, removing particulate matter and separating the lipids by thin layer chromatography on silica, extracts of normal and tumour cells being run in parallel. After visualisation the location of bands within the tumour extract lane which do not correspond with bands in the normal lane is determined. Silica and lipid from such bands is removed from the chromatogram and used as an immunogenic material to raised antibodies as previously described.
  • the invention also provides diagnostic immunoassay methods comprising contacting a sample suspected to contain a lipid immunogen with an antibody according to the invention against the immunogen, and kits for performing such assays comprising the antibody and at least one other reagent.
  • diagnostic immunoassay methods comprising contacting a sample suspected to contain a lipid immunogen with an antibody according to the invention against the immunogen, and kits for performing such assays comprising the antibody and at least one other reagent.
  • the skilled man will be familiar with the various types of immunoassay and with the available methods for detecting any antibody - immunogen interaction.
  • Figure 1 shows thin layer chromatograms on Merck precoated HPTLC aluminium sheets of lipid extracts of normal (N) and tumour (T) colonic mucosae. Each 1cm wide lane contained an amount of lipid extract equivalent to 20mg wet weight of tissue. The chromatograms were developed with chloroform/methanol containing 0.2% KCl (60:35:8, by vol.). GSL bands were revealed using the orcinol-ferric chloride reagent (Sigma Chemical Company). Region A of an unstained chromatogram was removed and used to immunise mice.
  • Foetal brain (1gm) homogenised in distilled water (1ml) was extracted with twenty volumes chloroform:methanol (1:1) and the solvent extract dried under a stream of nitrogen at 70°C.
  • the dried extract was partitioned (30 volumes/g tissue) in di-isopropyl-ether/1-butanol /50mMol aqueous sodium chloride (6:4:5 by volume) (3).
  • Low molecular weight contaminants were removed by 300 ⁇ 15mm Sephadex G-50 gel filtration.
  • the void volume was lyophilized and the gangliosides redissolved in 1.0ml chloroformxmethanol (1:1) such that the extract form 1.0g wet weight tissue was contained in 1.0ml of solvent mixture.
  • Gangliosides in the extracts were then fractionated by thin layer chromatography on aluminium backed silica gel plates (Art: 5553; E. Merck, Darmstadt, West Germany) which were developed in chloroform/methanol/0.2% aqueous calcium chloride (60:40:9 by volume). Aliquots of extract equivalent to 16mg of wet weight foetal brain were run in 0.5x6.0cm lanes with parallel duplicates.
  • GMl Monosialoganglioside
  • GDI disialoganglioside
  • GDI disialoganglioside
  • Gangliosides on parallel chromatograms were visualised with orcinol ferric chloride reagent (Sigma Chemical Co.). Using visualised glycolipids as markers, silica was scraped from the corresponding chromatograms from a point 0.1cm. from the origin to 1.0cm below the solvent front, thereby excluding residual material.
  • silica and associated glycolipids were suspended in phosphate buffered saline (lOmg wet weight equivalents of foetal brain/ml) by sonication in a Dawe sonicleaner (Ultrasonics Ltd., USA) at maximum setting until homogeneous by visual examination.
  • mice Young adult female Balb-C mice were anaesthetized and their spleens exposed through incisions in the body wall. Fifty microlitres of silica/ganglioside suspension was injected into the exposed spleen. The spleen was replaced in the peritoneal cavity and the incision closed with sutures. The procedure was repeated either one week or three months later.
  • RPMI Roswell Park Memorial Institute medium 1640
  • oxaloacetic acid 1.0x10 M
  • sodium pyruvate 0.5x10 M
  • insulin 0.2 units/ml
  • the cell suspension was distributed (lOOul/well) for growth in 8 ⁇ 96 well culture plates (Costar, Cambridge, MA02139, USA), containing mouse peritoneal macrophages as feeder cells (about 2.5 ⁇ 10 4 peritoneal exudate cells per well were dispersed in 100ul aliquots of RPMI), foetal calf serum
  • the second antibody preparation was twice absorbed with pig liver powder (Sigma Chemical Co.) before use.
  • Monoclonal antibodies 127-11 and U13A were used as reference antibodies on foetal brain and neuroblastoma sections respectively.
  • Antibody free medium RPMI + 20% foetal calf serum
  • Slides were examined with a Zeiss fluorescent microscope.
  • HPTLC high performance thin layer chromatography
  • Silica (about 15mg) was removed from a region of an unstained chromatogram of a colon extract where parallel stained chromatograms that revealed differences in glycolipid composition between normal and tumor tissue (Fig. 1). Silica plus associated glycolipids were resuspended in 10mM sodium phosphate buffer, pH7.2, 0.17M NaCl (10mg wet weight equivalent of tissue/ml) by using sonication until homogeneous by visual inspection. Mice were immunised by the intrasplenic route as described by Thorpe et al. (18); 2 injections of the suspension (50 ⁇ l each) were given 2 weeks apart. Production of hybridomas
  • the fused cells were dispersed in 75ml of medium containing foetal calf serum (20%), hyposanthine (2.0 ⁇ 10 -4 M) , thymidine (3.2 ⁇ 10 -5 M), aminopterin (8.0 ⁇ 10 -7 M), oxaloacetic acid (1.0 ⁇ 10 -3 M), sodium pyruvate (0.5 ⁇ 10 -1 M) and insulin (0.2 units/ml), and were distributed (100 ⁇ l/well) into 8 x 96 well culture plates (Costar,
  • mice peritoneal exudate cells 2.5 ⁇ 10 4 /well attached to the plastic well as feeder cells.
  • the latter cells were added in 100 ⁇ l of complete medium and were incubated in the cell for 24h prior to adding the fused cells.
  • Suprenatant medium was removed for antibody screening from those wells which contained sufficient hybridoma cells to cover at least half the bottom of the well.
  • the medium was replaced with fresh medium containing half the previous concentrations of hypoxanthine and thymidine and no aminopterin.
  • the culture supematant ⁇ were screened for antibodies on frozen sections of tissue by using indirect immunofluorescence (24).

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

La substance immunigène décrite comprend un lipide immunigène et de la silice.
PCT/GB1988/001030 1987-11-27 1988-11-25 Procede de production d'anticorps WO1989004670A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB878727834A GB8727834D0 (en) 1987-11-27 1987-11-27 Process for producing antibodies
GB8727834 1987-11-27

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WO1989004670A1 true WO1989004670A1 (fr) 1989-06-01

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001049277A2 (fr) * 1999-12-29 2001-07-12 William Leslie Porter Modulation d'une reponse immune

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE1942161A1 (de) * 1968-08-20 1970-02-26 Miles Lab Trockenes,in Wasser dispergierbares Aluminiumhydroxidgel mit darauf absorbierter Antikoerper produzierender oder immunisierender Substanz
DE1945251A1 (de) * 1969-09-06 1971-03-11 Karl Dr Med Theurer Verfahren zur Beeinflussung der quantitativen Antikoerperbildung bei der aktiven Immunisierung und der Desensibilisierung

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE1942161A1 (de) * 1968-08-20 1970-02-26 Miles Lab Trockenes,in Wasser dispergierbares Aluminiumhydroxidgel mit darauf absorbierter Antikoerper produzierender oder immunisierender Substanz
DE1945251A1 (de) * 1969-09-06 1971-03-11 Karl Dr Med Theurer Verfahren zur Beeinflussung der quantitativen Antikoerperbildung bei der aktiven Immunisierung und der Desensibilisierung

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Chemical Abstracts, vol. 81, no. 1, 8 July 1974 (Columbus, Ohio, US) D. Mancino et el.: "Adjuvant activity of silicon-containing substances. I. Effect of an amorphous silica on the production of antibodies against bovine Y -globulin in guinea pigs", see page 187 *
Chemical Abstracts, vol. 96, no. 23, 7 June 1982 (Columbus, Ohio, US) Kurisaki, Junichi et al.: "Antibody responses of mouse IgE to bovine B -lactoglobulin" see page 498 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001049277A2 (fr) * 1999-12-29 2001-07-12 William Leslie Porter Modulation d'une reponse immune
WO2001049277A3 (fr) * 1999-12-29 2002-11-28 William Leslie Porter Modulation d'une reponse immune

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