WO1988000240A1 - Preparation d'anticorps monoclonaux - Google Patents

Preparation d'anticorps monoclonaux Download PDF

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Publication number
WO1988000240A1
WO1988000240A1 PCT/GB1987/000461 GB8700461W WO8800240A1 WO 1988000240 A1 WO1988000240 A1 WO 1988000240A1 GB 8700461 W GB8700461 W GB 8700461W WO 8800240 A1 WO8800240 A1 WO 8800240A1
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WO
WIPO (PCT)
Prior art keywords
complex
wells
monoclonal antibody
ligand
cells
Prior art date
Application number
PCT/GB1987/000461
Other languages
English (en)
Inventor
Colin Henry Self
Original Assignee
Antibody Technology Limited
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 Antibody Technology Limited filed Critical Antibody Technology Limited
Priority to GB08804802A priority Critical patent/GB2199833A/en
Publication of WO1988000240A1 publication Critical patent/WO1988000240A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins

Definitions

  • This invention relates to the preparation of monoclonal antibodies. More specifically this inventi relates to the preparation of secondary monoclonal antibodies against a complex of a ligand and a binding protein against such ligand which secondary monoclonal antibody is not an antibody against the ligand or agains its binding protein.
  • European Patent Application No. 85901495.3 (PCT/GB/85/120 and W085/4422) describes a novel class of antibodies. Said European Patent Application is incorporated herein. Because of the special and unusua nature of those antibodies it has been found that a larg number of animals have to be immunised and used to produ a large number of hybridomas to give rise to those antibodies. It would be an advantage if a method could be used which reduces the time involved in such procedur Such an improved method has been discovered and is one also applicable to antibodies against other complexes. It is particularly surprising that antibodies able to bind complexes so specifically are obtainable by rapid methods.
  • the present invention provides a method of producing a secondary monoclonal antibody against a complex of a ligand and a binding protein against said ligand which secondary monoclonal antibody- is not an antibody against the ligand or against its binding protein which method comprises introducing the complex directly to immunocompetent cells, thereafter fusing said immunocompetent cells with neoplastic perpetuating cells and selecting and growing hybridomas capable of producing said secondary monoclonal antibody and recovering said secondary monoclonal antibody produced thereby.
  • the method of this invention may be adapt to the preparation of a secondary monoclonal antibody against a complex of a ligand whicu is large or small
  • small molecule means a molecule of molecular weight less than 5000. Such small molecules most aptl has a molecular weight of less than 2000 and preferably have a molecular weight of less than 1200.
  • the binding protein used in this invention may be an antibody or other proteinaceous material such as an enzyme or specific binding protein such as a steroid binding protein or a vitimin binding protein.
  • Preferab 5 the binding protein used is an antibody.
  • Such antibodies may be referred to as "primary" antibodi
  • this invention provid a method of producing a secondary monoclonal antibody 10 against a complex of a small molecule and a primary monoclonal antibody against said small molecule which secondary monoclonal antibody is not an antibody against the ligand or against its primary monoclonal antibody wh method comprises introducing the complex directly to 5 immunocompetent cells, thereafter fusing said immunocompetent cells with neoplastic perpetuating cells and selecting and growing hybridomas capable of producin said secondary monoclonal antibody and recovering said monoclonal antibody produced thereby.
  • An advantage of the method of this invention is that incubation of the complex by the immunocompetent cells needs to take place for only a relatively short time as opposed to the longer periods generally required if the complex is introduced to an animal in convention manner (ie to the whole animal by a systemic mode of administration, such as intramuscular injection, as opposed to direct administration of the complex to the immunocompetent cells) ' .
  • Good titres of the desired monoclonal antibodies can be achieved by the method of this invention.
  • the direct administration of the complex to the immunocompetent cells may take place in vitro (for exam to a suspension of cells such as lymphocytes) but I presently prefer to introduce the complex to the immunocompetent cells in vivo, for example by administr into the spleen (that is intrasplenic immunisation) . I have found this lattermethod to yield particularly suitable results.
  • Intrasplenic immunisation may be effected by injection of a solution (or suspension) of complex directly into the spleen of an animal such as a mouse.
  • the immunisation may be repeated.
  • Such a second (or subsequent) administration may be directly to the immunocompetent cells or (when in vivo) systemically by conventional methods such as intramuscul injection or intraperitoneal injection.
  • this invention provides a method as hereinbefore described wherein the complex is introduced directly to the immunocompetent cells (normal intrasplenically) and then later the animal is immunised systemically.
  • the time between the direct and conventional immunisation may be, for example, 7-21 day or more aptly 10-15_lays.
  • the systemic immunisation may precede the direct immunisation.
  • the time between conventional and direct methods may be, for example, 7-2_days or more aptly 10-15 days.
  • the complex used for immunisation may be prepared by the general methods of my earlier European Patent Application but I have found it particularly convenient to simply mix generally with stirring the components in distilled water or a suitable buffer such as Tris pH 7.4 buffer. This simple mixing can be for as short as a few minutes with more soluble materials but longer perio may be required with less soluble materials. Solubilis agents such as Tweens can be used In low levels in conventional manner if desired.
  • the ligand may be pres in excess if desired (subject to any toxic effect of a toxic ligand) and may also be administered in addition t the complex. Less preferably the complex of ligand and binding protein may be one in which the two are covalent linked.
  • immunocompotent cell such as lymphoc
  • lymphoc any suitable immunocompotent cell such as lymphoc
  • the cells may be fused with neoplastic perpetuating cells such as myeloma cells in conventional manner for producing hybri cell lines for monoclonal antibody preparation. I prefe to use mouse cells but cells of other animals may be employed if desired, for example rat, human, sheep, bird or the like.
  • the growth and selection of clones and isolation of secondary antibody may be performed as in my earlier European Patent Application No. 85901495.3.
  • the antibodies may be employed as described in that applicat
  • the favoured classes of small molecule to be employed are as described in my earlier European Patent Application.
  • Larger molecules that may be employed are preferably naturally occuring materials such as those useful as diagnostic markers, for example enzymes.
  • Any suitable method of recovering the desired monoclonal antibodies may be employed and many such methods are known in the art, also for example my earlier European Patent Application.
  • Clones were seen after three to four days and they were screened for the production of antibody to complexes of the primary anti with oestrone sulphate as follows: 96-well ELISA- microtitre plates (Nunc, Denmark) were- coated with prim anti-oestrone sulphate antibody (2ug of protein A purifi material per well in 200ul of 50mM bicarbonate buffer pH 9.6 incubated in the wells overnight at RT) . The wells were then glazed with 200 ul of the same buffer containi 0.2% casein by incubation for lhr at 37C. The wells were then washed four times with 50mM Tris pH 7.4 containing 0.02% Tween 20.
  • a series of wells then received 150ul per well of a solution of lOOug/ml oestro sulphate in 50mM Tris buffer pH 7.4. Another series of the wells received 150ul of the buffer alone. Both series were then incubated for 1 hr at 37C after which 50ul of culture fluid from each hybridoma clone was adde to an oestrone sulphate containing wells and to an oestrone s ⁇ lphate free well and the mixtures incubated a 37c for 1 hr. The solutions were shaken from the wells and the wells washed four times with 200 ul 50 mM Tris p 7.4 containing 0.02% Tween 20.
  • Mouse monoclonal antibo remaining bound to the wells was- then detected by the addition of 200 ul of a donkey anti-mouse IgG and IgM polyclonal antibody labelled with alkaline phosphatase (Guildhay Antisera Ltd, Guildford, Surrey) which had bee purified free of activity against rabbit IgG by immunoabsorption on a column of cyanogen bromide activated sepharose linked to rabbit IgG and then dilute to 1:1000 of the original concentration in 50mM Tris pH 7.4. The plates were incubated for 1 hr at room temperature and washed four times with 50mM Tris pH 7.4.
  • a Balb/c mouse was taken anaesthetized in halothan its spleen exposed and immunised by three direct subcapsular injections with a total of 50ug freeze-dried protein
  • a purified mouse anti-oestradiol monoclonal antibody dissolved in 100 ul of distilled water containi 5 ug of oestradiol and incubated at RT for 30 minutes.
  • mice Four days later the mouse was killed by cervical disloca the spleen removed. Single cell preparations were made and the cells fused with mouse myeloma line NSO followin the procedure described in Galfre, G., Howe, S.C., Milst c, Butcher, G.W. and Howard, J.C. (1977). Antibodies to major histocompatability antigens produced by hybrid cell lines, Nature, 266, 550-552 but using RPMI medium containing 10% Clex - tissue culture medium supplement obtained from Dextran Products Limited, Scarborough, Ontario, Canada. Also the RPMI HAT medium (Flow
  • the wells were then washed four times with 50mM Tris pH 7.4 containing 0.02% Tween 20.
  • a series of wells then received 150 ul per well of a solution of 50 ug/ml oestradiol in 50mM Tris buffer pH 7.4.
  • Another series of the wells received 150 ul of the buffer alone. Both series were then incubated for 30 minutes at room temperature (22C) after which 50 ul of culture fluid from each hybridoma clone was added to an oestradiol containing wells and to an oestradiol free well and the mixtures incubated for a further 30 minutes at room temperature.
  • the plates were incubated for lhr at room temperature and washed four times with 50mM Tris pH 7.4. Each well then received 200 ul of lOmM para-nitrophenol phosphate (Sigma Chemical Co Ltd, Lond in 50mM bicarbonate buffer pH 10.3 containing 3.3mM MgC12 and the paltes were incubated at room temperature in the dark.
  • the alkaline phosphatase activity was determined by measurement of the absorption at 405nm and clear differences were evident after incubation for one hour and were recorded.
  • Example 1 The method of Example 1 may be repeated using a monoclonal antibody against oestrone sulphate.
  • Example 3 The method of Example 3 may be repeated for progesterone instead of oestrone sulphate.
  • a Balb/c mouse was taken anaesthetized with halotha its spleen exposed and immunised by three direct subcaps injections with a total of 50ug freeze-dried protein
  • a Purified mouse anti-progesterone monoclonal antibody dissolved in 100 ul of phosphate buffered saline containing 50 ug of progesterone and incubated at RT fo 2 hours. Four days later the mouse was killed by cervical dislocation the spleen removed.
  • Single cell preparations were made from the spleen and the cells fused with mouse myeloma line NSO following the convent procedure described in Galfre, G., Howe, SC, Milstein, C, Butcher, GW and Howard, JC (1977) Antibodies to majo histocompatability antigens produced by hybrid cell line Nature, 266, 550-552 but using RPMI medium containing 10 Clex - tissue culture medium supplement obtained from Dextron Products Limited, Scarborough, Ontario, Canada. Also the RPMI HAT medium (Flow Laboratories Ltd, Irvine, Scotland) was added immediately after the fusion had been performed.
  • the culture fluids in group 4 did not contain primary antibody against progesterone as follows Micro-Elisa well strips (Dynatech Im ulon II) were coated with anti-Mouse IgM antibody (u-chain specific) (Sigma Chemical Co Cat. No. M1147) by leaving 100 ul of 50 mM Bicarbonate buffer pH 9.6 containing 1 ug of th antibody in each well overnight at room temperature. The wells were then glazed by leaving 100 ul of 0.2% casein in the same buffer in them for a further hour at room temperature. They were then washed four times wit TT. 50 ul of each culture fluid was put into individua wells followed by 50 ul of 50 mM Tris pH 7.4.
  • Example 1 The method of Example 1 was repeated using Ag8 myeloma cells instead of NSO myelomacells.
  • Balb/c mice are taken and immunised by intro- peritoneal injection of lOOug of progesterone mixed in 80 ul of complete Freund's adjuvant and emulsified with 80 ul of phosphate buffered saline containing 100 ug of a primary monoclonal antibody against progesterone. Two weeks later they receive another similar immunisati but with incomplete Freund's adjuvant. Two weeks afte this they receive their last immunisation by means of a direct subcapsular intrasplenic injection of 100 ug of the antibody mixed with 100 ug of progesterone in 100 u phosphate buffered saline.
  • hybridomas Three days after this their spleens are removed and used to make hybridomas as in the previous examples with NSO myeloma cells.
  • the hybridomas are screened for the production of IgG class antibody against the complex of the primary monoclonal and progesterone as follows:
  • Quadruplicate wells then receive 50 ul of culture fluid to be screened and 50 ul of 50 mM Tris pH 7.4 and are incubated for 45 minutes at room temperature. Unbound material is then shaken out and the wells washed four times with 50 mM Tris pH 7.4 containing 0.02% Tween 20. Each well then
  • the quadruplicate wells are split into pairs and to one set is added 50 ul of a saturated solution of progesterone made by heating an excess of progesterone in 50mM Tris buffer pH 7.4 at 56C, cooling to room temperature and separating the saturated solution. The other set receiv 50 ul of the buffer alone. All of the wells then receiv 50 ul of a 1:1000 dilution of a conjugate of the primary anti-progesterone antibody previously conjugated to alkaline phosphatase by the method of Valler, A., D.E.
  • Micro-Elisa well strips (Dynatech Im ulon II) are coated with anti-Mouse IgG antibody (Sigma Chemical Co Cat. No. M8624) by leaving 100 ul of 50 mM bicarbonate buffer pH 9.6 containing 1 ug of the antibody in each well overnight at room temperature. The wells are then glazed by leaving 100 ul of 0.2% casein in the same buff in them for a further hour at room temperature. They are then washed four times with TT. 50 ul of each culture fluid was put into individual wells followed by 50 ul of 50 mM Tris pH 7.4. The wells are incubated for one hour at room temperature and then washed a further four times with TT.
  • hybridomas producing the culture fluids giving rise to culture fluids of group 4 are grown up the antibody they produced purified and used in assays with the primary antibody for progesterone determination.
  • hybridomas are screened both for the production of IgM antibody as before (employing microtit plates coated with anti-Mouse IgM - Sigma Chemical Co Cat. No. M1147) and also for IgG as before (employing microtitre plates coated with anti-Mouse IgG - Sigma Chemical Co Cat. No. M8642).
  • Example 8 The method of Example 8 was repeated but the dire immunisation was carried out 14 days before the first immunisation by intra peritoneal injection (and not after the last intra peritoneal injection).
  • Example 8 is repeated but with additional injections of intra peritoneal doses of 100 ug progeste in 100 ul incomplete Freund's adjuvant between each of the injections in that previous Example.

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

Abstract

Procédé de production d'un anticorps monoclonal secondaire contre un complexe d'un ligand et une protéine de liaison contre ledit ligand, ledit anticorps monoclonal secondaire n'étant pas un anticorps contre le ligand ou contre sa protéine de liaison. Ce procédé consiste à introduire le complexe directement dans des cellules immunocompétentes, à provoquer ensuite la fusion desdites cellules immunocompétentes avec des cellules néoplastiques se perpétuant, et à sélectionner et cultiver des hybridomes pouvant produire ledit anticorps monoclonal secondaire, et à extraire ledit anticorps monoclonal secondaire ainsi produit.
PCT/GB1987/000461 1986-07-02 1987-07-01 Preparation d'anticorps monoclonaux WO1988000240A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
GB08804802A GB2199833A (en) 1986-07-02 1987-07-01 Preparation of monoclonal antibodies

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB868616174A GB8616174D0 (en) 1986-07-02 1986-07-02 Monoclonal antibodies
GB8616174 1986-07-02

Publications (1)

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WO1988000240A1 true WO1988000240A1 (fr) 1988-01-14

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Application Number Title Priority Date Filing Date
PCT/GB1987/000461 WO1988000240A1 (fr) 1986-07-02 1987-07-01 Preparation d'anticorps monoclonaux

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AU (1) AU7649187A (fr)
GB (2) GB8616174D0 (fr)
WO (1) WO1988000240A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0264219A2 (fr) * 1986-10-09 1988-04-20 Syntex (U.S.A.) Inc. Récepteurs pour des complexes immunologiques, leurs procédés de préparation et méthodes d'essais les utilisant
WO1991019001A1 (fr) * 1990-05-25 1991-12-12 British Technology Group Ltd Diagnostic immunologique de la polyarthrite rhumatoide
WO1993011153A1 (fr) * 1991-11-25 1993-06-10 Peptide Therapeutics Limited Peptides et anticorps utilises dans le traitement de la polyarthrite rhumatoide
US5837686A (en) * 1991-11-25 1998-11-17 Peptide Therapeutics Limited Peptides and antibodies for treatment of rheumatoid arthritis
EP0918218A2 (fr) * 1997-11-21 1999-05-26 Daiichi Pure Chemicals Co. Ltd. Méthode pour l'immunoessai
US8389296B2 (en) 2007-04-23 2013-03-05 Selective Antibodies Limited Assay devices and methods and components for use therein

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU656181B2 (en) * 1991-05-03 1995-01-27 Pasteur Sanofi Diagnostics Heterobifunctional antibodies possessing dual catalytic and specific antigen binding properties and methods using them

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1985004422A1 (fr) * 1984-03-30 1985-10-10 Cambridge Patent Developments Ltd. Anticorps, fabrication et utilisation

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1985004422A1 (fr) * 1984-03-30 1985-10-10 Cambridge Patent Developments Ltd. Anticorps, fabrication et utilisation

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Biological Abstracts, Volume 76, No. 6, 1983, (Philadelphia, PA, US), P.L. WITTE et al.: "Improved Efficiency of Hybridoma Ascites Production by Intrasplenic Inoculation in Mice", see page 4524, Abstract 41616, & J. Natl. Cancer Inst. 70(3): 575-577, 1983 *
Biological Abstracts, Volume 78, No. 7, 1984, (Philadelphia, PA., US), M. SPITZ et al.: "Intrasplenic Primary Immunization for the Production of Monoclonal Antibodies", see page 5803, Abstract 51560, & J. Immunol. Methodes 70(1): 39-44, 1984 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0264219A2 (fr) * 1986-10-09 1988-04-20 Syntex (U.S.A.) Inc. Récepteurs pour des complexes immunologiques, leurs procédés de préparation et méthodes d'essais les utilisant
EP0264219A3 (en) * 1986-10-09 1990-04-11 Syntex (U.S.A.) Inc. Receptors for immune complexes, their methods of production, compositions containing them, assay methods and assay kits using them
US6326159B1 (en) 1986-10-09 2001-12-04 Dade Behring Marburg Gmbh Receptors for immune complexes
WO1991019001A1 (fr) * 1990-05-25 1991-12-12 British Technology Group Ltd Diagnostic immunologique de la polyarthrite rhumatoide
GB2246780A (en) * 1990-05-25 1992-02-12 Nat Res Dev Antibody reagents specific for IgA- alpha 1 antitrypsin complex indicative of rheumatoid arthritis
GB2246780B (en) * 1990-05-25 1994-04-27 Nat Res Dev Immunodiagnostic assay for rheumatoid arthritis
US5827668A (en) * 1990-05-25 1998-10-27 Peptide Therapeutics Limited Immunodiagnostic assay for rheumatoid arthritis
WO1993011153A1 (fr) * 1991-11-25 1993-06-10 Peptide Therapeutics Limited Peptides et anticorps utilises dans le traitement de la polyarthrite rhumatoide
US5837686A (en) * 1991-11-25 1998-11-17 Peptide Therapeutics Limited Peptides and antibodies for treatment of rheumatoid arthritis
EP0918218A2 (fr) * 1997-11-21 1999-05-26 Daiichi Pure Chemicals Co. Ltd. Méthode pour l'immunoessai
EP0918218A3 (fr) * 1997-11-21 2000-01-05 Daiichi Pure Chemicals Co. Ltd. Méthode pour l'immunoessai
US8389296B2 (en) 2007-04-23 2013-03-05 Selective Antibodies Limited Assay devices and methods and components for use therein

Also Published As

Publication number Publication date
GB8616174D0 (en) 1986-08-06
GB8804802D0 (en) 1988-03-30
AU7649187A (en) 1988-01-29
GB2199833A (en) 1988-07-20

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