EP1086134A2 - Preparation d'anticorps se liant a des acides amines modifies apres traduction a l'interieur de peptides - Google Patents

Preparation d'anticorps se liant a des acides amines modifies apres traduction a l'interieur de peptides

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Publication number
EP1086134A2
EP1086134A2 EP99957063A EP99957063A EP1086134A2 EP 1086134 A2 EP1086134 A2 EP 1086134A2 EP 99957063 A EP99957063 A EP 99957063A EP 99957063 A EP99957063 A EP 99957063A EP 1086134 A2 EP1086134 A2 EP 1086134A2
Authority
EP
European Patent Office
Prior art keywords
peptide
antibody
post
spectrum
peptides
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Withdrawn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
EP99957063A
Other languages
German (de)
English (en)
Inventor
Andrew John Garman
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.)
AstraZeneca AB
Original Assignee
AstraZeneca AB
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
Priority claimed from GBGB9812772.3A external-priority patent/GB9812772D0/en
Priority claimed from GBGB9819945.8A external-priority patent/GB9819945D0/en
Application filed by AstraZeneca AB filed Critical AstraZeneca AB
Publication of EP1086134A2 publication Critical patent/EP1086134A2/fr
Withdrawn legal-status Critical Current

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Classifications

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

Definitions

  • the invention relates to a process for preparing a spectrum of antibodies by use of a peptide library as an immunogenic substrate.
  • Such antibody spectra are useful in that they
  • the invention relates to preparing a spectrum of antibodies raised using a peptide library in which the majority of peptides contain a consensus sequence for a domain within which a serine or 0 threonine is phosphorylated, where such an spectrum or part therof is useful in screening, particularly in a high throughput manner, for inhibitors of serine or threonine kinase.
  • Protein kinases are key components in the control of many cellular activities and events and, as such, represent attractive pharmaceutical targets.
  • Our understanding of tyrosine kinases has been greatly facilitated by the use of anti-phosphotyrosine antibodies, which are 5 specific enough that they selectively bind to a phophorylated tyrosine itself but not, or at least to a significantly lesser degree, an unphosphorylated tyrosine.
  • anti-phosphotyrosine antibodies which are 5 specific enough that they selectively bind to a phophorylated tyrosine itself but not, or at least to a significantly lesser degree, an unphosphorylated tyrosine.
  • These antibodies have allowed the construction of a number of different types of assay, such as. for example, homogeneous time resolved flourescense (HTRF), for screening compounds as potential inhibitors of tyrosine kinase.
  • HTRF homogeneous time resolved flourescense
  • Serine and threonine kinase also represent attractive pharmaceutical targets 0 for a variety of diseases, such as cancer, inflammatory diseases, and immune system disorders.
  • analogous assays to those used for tyrosine kinase inhibitors can not be prepared for serine or threonine kinases due to the lack of availability of a suitable generic anti-phosphoserine or anti-phosphothreonine antibody which is able to distinguish between the phosphorylated and unphosphorylated forms of serine or threonine, principally because, it 5 is believed, these residues are very small in size (compared to, for example, the much larger phosphotyrosine residue).
  • Antibodies may be raised individually that recognise a specific phosphoserine- or phosphothreonine-containing peptide sequence or protein for example by immunising a suitable animal with the peptide conjugated to a suitable carrier.
  • the antibodies obtained 0 generally recognise a portion of the sequence surrounding the phosphorylated residue, which limits their utility due to their inability to recognise a phosphorylated serine or threonine on a different peptide. Therefore, for each new enzyme and/or substrate found, a new antibody preparation is needed, which is time-consuming. Therefore, generally it has been necessary to rely on the use of radio-isotopes of phosphorous in assay systems of serine or threonine kinase. Such assays are inconvenient in that they require separation steps and require radioactivity handling procedures. This means that they are less well suited for high throughput assays to discover compounds that inhibit these enzymes.
  • a method for the preparation of an antibody spectrum which contains a number of different antibodies of which at least one antibody is able to recognise a post-translationally modified amino acid residue comprises immunisation of at least one animal with a peptide library, wherein the majority of peptides in the peptide library contain the post-translationally modified amino acid of interest, and isolation of the antibody spectrum from each animal.
  • the methods described are used to prepare a spectrum of antibodies or antibody-derived fragments which individually will only recognise a small proportion of individual members of the peptide library.
  • the antibody spectrum may be used to obtain a suitable antibody preparation that can distinguish between this modified, i.e. phosphorylated amino acid, and its unmodified parent, or, alternatively, and more likely, the modified peptide may be itself used to isolate antibodies from the antibody spectrum which are specific for it.
  • an antibody may be isolated from the antibody spectrum raised against the peptide library of the post translationally modified amino acid by the use of immobilised peptide substrate for that particular post translational modifying enzyme.
  • this procedure thereby reduces the time, typically many months by conventional procedures, required to obtain a specific antibody preparation for use in, for example, a new assay, to a matter of days by avoiding the need to raise a specific antibody preparation individually.
  • antibody spectrum we mean a pure or impure preparation of a mixture of either monoclonal or polyclonal antibodies, or fragments thereof, or any other functional preparation derived from an antibody preparation.
  • peptide library we mean a pure or impure preparation of a mixture of peptides, whether peptides derived from natural amino acids or derivatised forms thereof, optionally conjugated to a carrier molecule, for example a carrier protein such as albumin.
  • the library contains at least 10, 150, 10 3 , 10 4 , or 10 5 different peptides.
  • the peptide library may consist of different peptides together in the same sample or may consist of a collection of individual peptide samples, or subsets of the peptide library, which together constitute the peptide library, which are administered to animals and the antibody preparations collected from each animal, or a selection thereof, and pooled to form the antibody spectrum.
  • post-translationally modified amino acid residue we mean an amino acid which undergoes further modification after translation
  • preferred post-translational modifications to which the antibody spectrum is prepared include phosphorylations, in particular of serine and threonine residues.
  • the term “recognise” we mean that at least one antibody in the antibody prepartion has sufficient specificity that it will bind to the amino acid residue of interest or the peptide sequence in preference to the same amino acid residue of interest or peptide sequence when the amino acid residue of interest has or has not been post-translationally modified.
  • the peptide library used in the above method is preferably generated to a consensus sequence.
  • an antibody preparation of general utility for recognising phosphoserine residues in naturally occurring peptide sequences would be obtained by immunising with a library of peptides whose sequences comprised consensus sequence motifs, i.e. sequences that are found in nature to occur in the vicinity of phosphorylated serines. Compilations of such sequences are published from time to time, see for example Meth. Enzymol (1991), 200, 62-81.
  • Isolation of the antibody spectrum from each animal may be by purification of the anti library sera from the animal directly, by isolation of splenocytes from each animal for preparation of monoclonal antibodies according to the methods of Kohler & Milstein (1975) Nature 256, 495, or by any other method known in the art for isolating antibody preparations.
  • Immunisation of animals is in general conventional.
  • the peptide library is conjugated to a suitable carrier, for example ovalbumin, bovine serum albumin, bovine thyroglobulin, keyhole limpet haemacyanin or any other suitable carrier. Conjugation is by conventional methods as described in (Antibodies - A laboratory manual. E Harlowe & D Lane, Cold Spring Harbor Laboratory, 1988).
  • the library is synthesised to allow a single unique point of conjugation which is not part of the consensus sequence motif.
  • the point of conjugation may be a terminal cysteine residue to allow coupling to the carrier which has been activated for reaction with thiol groups, for example by treatment with m- maleimidobenzyl-N-hyrdoxysuccinimide ester (Kitagawa T & Aikawa T (1976) J Biochem 79: 233-236).
  • the peptide may be synthesised with a terminal aminoxy group to allow coupling to the carrier which has been previously activated for reaction with amino groups at low pH.
  • Suitable animals for immunisation include all commonly used laboratory animals. The preferred choice will depend upon the type of antibody being produced.
  • polyclonal antibodies are to be prepared using larger animals, such as rabbits, sheep, goats, donkeys or horses may be preferred. If monoclonal antibodies are to be prepared using mice or rats would be preferred. If phage display is being used any animal for which sufficient antibody coding sequence is known could be used.
  • the antibody spectrum may be preserved for future use by a variety of methods. Particularly preferred are methods that allow the antibody spectrum, or the DNA or RNA encoding for that antibody spectrum, to be preserved for long periods, so that when a need for a new antibody of different specificity arises, a useful antibody preparation may be obtained in a short period of time, i.e. short compared to the time taken to raise an immune response de novo. Such methods include freezing sera, preparation of antibody secreting hybridomas and their preservation in liquid nitrogen, and extraction and cloning of the relevant antibody-encoding genes. The method of the invention also comprises the preparation of polyclonal or monoclonal antibody preparations that have not been selected using the phosphorylated peptide of interest.
  • the polyclonal antibody will contain sufficient numbers of antibody species that recognise the peptide of interest, albeit only partially in many cases, that the preparation may be used directly, either as crude serum, or an IgG preparation or some other preparation. Furthermore, it will be a simple matter to identify those monoclonal antibodies that have broad specificity covering at least a portion of the sequences that may be of interest to the researcher in the future.
  • C represents cysteine (incorporated to facilitate conjugation to carrier)
  • A represents alanine
  • K/R represents either lysine or arginine
  • X represents any of the 20 naturally occurring L amino acids, except cysteine pS represents phosphoserine F represent phenylalanine and OH indicates that the C terminus is free. Sequence ID NO.l.
  • the library was prepared by manual solid phase synthesis using the split and mix methodology of Furka et al. (Abstr. 14 th Int. Congr. Biochem., Prague, Czechoslovakia 1988, 5, 47; Int. J. Pep. Prot. Res. 1991, 37, 487).
  • the resin was divided into 19 portions an each reacted with a different protected amino acid and the reactions recombined after coupling.
  • the resin was split into 2 portions, and each reacted with either protected arginine or protected lysine, followed by recombination.
  • Fmoc deprotection was achieved using 20% piperidine/DMF (3 x 5 ml x 5 min.) followed by thorough washing with DMF/dichloromethane.
  • 2mg of the peptide library was conjugated to 2mg of carrier protein ovalbumin by m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) chemistry, as described by Harlowe and Lane in “Antibodies - A Laboratory Manual", Cold Spring Harbor Laboratory, 1988. After dialysis against PBS, the concentration of the conjugate was adjusted to Img/ml Vaccine preparation
  • the peptide-ovalbumin conjugate solution was mixed with Freund' s Adjuvant to prepare a water in oil emulsion.
  • Peptide AKT Biotin-GRPRTSpSFAEG (Sequence ID No.2)
  • IKB-diP Peptide was CKKERLLDDRHDpSGLDpSMKDE (Sequence ID No.3) and the non-phosphorylated version of this peptide was also prepared ("1KB noP Peptide" Sequence ID No.4).
  • pS designates the amino acid phosphoserine.
  • Rabbits were immunised subcutaneously with 50ug of peptide-ovalbumin conjugate in Freund's Complete Adjuvant on day 0, and subsequently with 50ug of peptide-ovalbumin conjugate in Freund's Incomplete Adjuvant each 4 weeks for a total of 4 injections. A sample of blood was collected 7 days after the final injection.
  • mice were immunised subcutaneously with 20ug of peptide-ovalbumin conjugate in Freund's Complete Adjuvant on day 0, and with 20ug of peptide-ovalbumin conjugate in Freund's Incomplete adjuvant two weeks later, a further two weeks later with 20ug of peptide- ovalbumin conjugate in phosphate buffered saline.
  • a sample of blood was collected 7 days after the final injection. The mice were then allowed to rest between 10 and 20 weeks. Four days before sacrifice each mouse was immunised with lO ⁇ g of conjugate in Phosphate Buffered Saline (PBS) intraveneously. Evaluation of the immune response
  • Peptides of interest (CamLib, AKT, 1KB) were diluted 2ug/ml in carbonate/bicarbonate buffer and used individually to coat micro-titre plates at 1 OOul per well, overnight at 4°C. Before use, each micro-titre plate was washed in PBS and then blocked with 1.5% Milk powder in PBS for one hour at room temperature. Plates were then washed again and dilutions of sera in PBS-Tween were added. After incubation at room temperature for one hour, each plate was washed three times in PBS-Tween, before anti species-horse radish peroxidase was added.
  • the peptide library "Camlib” has elicted a useful level of antibodies to the two peptides of interest, AKT Peptide and IKB-diP Peptide. In the latter case, it can be seen that the response to the unposphorylated peptide 1KB no P Peptide is considerably lower than IKB-diP Peptide
  • Anti library serum (mouse 972) was diluted 1 in 4 in PBS and then applied to the specific immuno-affinity column. The column was then washed with 10ml PBS, before the specific antibodies were recovered by elution with 5 x 1 ml aliquots of 1 OOmM Glycine-HCl pH, 2.5. The eluate was adjusted to neutral pH with 1M Tris-HCl, pH 8.9 and then dialysed against PBS. The titres of both the eluate (which should contain the purified antibody of interest) and the original flowthrough fraction were determined as described in example 2.
  • splenocytes were prepared and fused with NSO cells by standard methods (Kohler & Milstein, Nature (1975) 256, 495), the resulting cells were distributed into 96 well culture dishes and incubated for 2 weeks. The supernatants from the resulting hybridomas were then screened by enzyme immunoassay.
  • mice The spleens from immunised mice were dissociated into a single cell suspension by injecting Dulbecco's modification of Eagle's medium into the spleen sac. 2x10 8 ( spleen cells from the above described cell suspension were mixed with
  • the tube was then centrifuged and resuspended in 30ml DMEM with 10% foetal calf serum. Aliquots of 50ul were distributed to the wells of 6 96-well tissue culture dishes. After 24 hours, 50ul per well DMEM with 10% foetal calf serum and double strength hypoxanthine/aminopterin thymidine (HAT) supplements was added. Each well was fed with 200ul DMEM with 10% foetal calf serum and single strength hypoxanthine/aminopterin/thymidine (HAT) supplements 7 days later.
  • HAT hypoxanthine/aminopterin/thymidine
  • a phage library was prepared from immunised rabbits using methods described by Ridder et al ( Ridder R, Schmitz R, Legay F, Gram H (1995) BIO/TECHNOLOGY 13: 255- 260) - li ce// Preparation
  • Antibody bearing cells were first isolated from the spleen of an immunised rabbit by capture with the IgG fraction from a goat anti-rabbit Ig sera. This cell population was then depleted of cells bearing antibodies specific for the carrier protein component of the immunogen by treatment with immobilised carrier protein.
  • This technique was then used to assemble the heavy and light chain genes into a single fragment in which they are separated by a linker region comprising 45bp encoding for the peptide sequence (GlyGlyGlyGlySer) 3 .
  • This was then cloned into suitable vector such as pCANTAB 5E, (Pharmacia), and the whole construct was used to transform a suitable strain of E.coli such as TGI.
  • the phage library was rescued by subsequent infection with a suitable helper phage such as M13KO7.
  • Phages bearing single chain Fv's (scFvs) specific for particular phospho-serine peptides could then be isolated by panning this library according to standard methods.

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

Abstract

La présente invention concerne un procédé permettant de préparer un spectre d'anticorps en utilisant une échantillothèque de peptides servant de substrat immunogène. De tels spectres d'anticorps sont utiles car ils peuvent s'utiliser pour identifier, localiser ou mesurer des acides aminés dans des peptides impliqués dans les modifications post-traductionnelles, et par conséquent on peut les utiliser pour la recherche systématique d'inhibiteurs d'enzymes impliqués dans de telles modifications post-traductionnelles. Plus particulièrement, l'invention se rapporte à la préparation d'un spectre d'anticorps que l'on a dressé en utilisant une échantillothèque de peptides dans laquelle la majorité des peptides contient une séquence consensus pour un domaine dans laquelle une sérine ou une thréonine est phosphorylée. Il en résulte qu'un tel spectre, ou une partie d'un tel spectre convient particulièrement pour la recherche systématique d'inhibiteurs de sérine ou thréonine kinase, et ce, à relativement grande vitesse.
EP99957063A 1998-06-13 1999-06-08 Preparation d'anticorps se liant a des acides amines modifies apres traduction a l'interieur de peptides Withdrawn EP1086134A2 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
GB9812772 1998-06-13
GBGB9812772.3A GB9812772D0 (en) 1998-06-13 1998-06-13 Assay method
GBGB9819945.8A GB9819945D0 (en) 1998-09-15 1998-09-15 Assay method
GB9819945 1998-09-15
PCT/GB1999/001795 WO1999065946A2 (fr) 1998-06-13 1999-06-08 Preparation d'anticorps se liant a des acides amines modifies apres traduction a l'interieur de peptides

Publications (1)

Publication Number Publication Date
EP1086134A2 true EP1086134A2 (fr) 2001-03-28

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EP99957063A Withdrawn EP1086134A2 (fr) 1998-06-13 1999-06-08 Preparation d'anticorps se liant a des acides amines modifies apres traduction a l'interieur de peptides

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Country Link
EP (1) EP1086134A2 (fr)
WO (1) WO1999065946A2 (fr)

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IE81146B1 (en) * 1987-10-13 2000-05-03 Terrapin Tech Inc Method to produce immunodiagnostic reagents
US5703057A (en) * 1995-04-07 1997-12-30 Board Of Regents The University Of Texas System Expression library immunization

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9965946A3 *

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Publication number Publication date
WO1999065946A2 (fr) 1999-12-23
WO1999065946A3 (fr) 2000-02-03

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