EP1086134A2

EP1086134A2 - Methods to prepare antibodies which bind to posttranslationally modified amino acids within peptides

Info

Publication number: EP1086134A2
Application number: EP99957063A
Authority: EP
Inventors: Andrew John Garman
Original assignee: AstraZeneca AB
Current assignee: AstraZeneca AB
Priority date: 1998-06-13
Filing date: 1999-06-08
Publication date: 2001-03-28
Also published as: WO1999065946A2; WO1999065946A3

Abstract

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 may be used to identify, localise or measure amino acids within peptides involved in post-translational modifications and, consequently, may be used in screening for inhibitors of enzymes which are involved in such post-translational modifications. In particular 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 threonine is phosphorylated, where such a spectrum or part thereof is useful in screening, particularly in a high throughput manner, for inhibitors of serine or threonine kinase.

Description

METHODS TO PREPARE ANTIBODIES WHICH BIND TO POSTTRANSLAΗONALLY MODIFIED AMINO ACIDS WITHIN PEPTIDES

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

5 may be used to identify, localise or measure amino acids within peptides involved in post- translational modifications and, consequently, may be used in screening for inhibitors of enzymes which are involved in such post-translational modifications. In particular 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. 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. Serine and threonine kinase also represent attractive pharmaceutical targets 0 for a variety of diseases, such as cancer, inflammatory diseases, and immune system disorders. Unfortunately, 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.

For this reason, and to generally facilitate the study of inhibitors of these enzymes in highthroughput assays, a quick and easy method for obtaining antibodies that recognise phosphoserine and phosphothreonine residues would be very valuable. Similar issues also apply to obtaining antibodies that recognise other post-translational modifications to proteins and peptides.

We have surprisingly found that it is possible to obtain useful reagents for the development of assays to enzymes involved in post-translational modification of peptides or proteins by a new approach which involves the use of peptide libraries. Peptide libraries have been used inter alia, for the discovery of novel bioactive peptides (see for example, K. Lam et al., Nature 1991, 354, 82 and R.A. Houghten et al., Nature 1991, 354, 84). To date, peptide libraries have not been employed as immunogens for generating a spectrum of antibodies for use in the development of high throughput assays.

Accordingly we present as a first feature of the invention 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, which method 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.

In essence, 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. When an antibody that will recognise a particular member of the peptide library, or a peptide structurally related to a member of the library, is required, 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. Typically 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. By preparing the antibody spectrum in advance 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. By use of the term "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.

By use of the term "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, 10³, 10⁴, or 10⁵ different peptides. For the avoidance of doubt 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.

By use of the term 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.

By the use of 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.

In order to increase the proportion of antibodies of interest in the antibody spectrum, the peptide library used in the above method is preferably generated to a consensus sequence. For example, 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. Conveniently 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). Alternatively 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. For instance, if 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.

Once prepared, 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. In many cases, 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.

It will be appreciated that the purity, affinity and specificity of the antibody preparation required depends greatly on the use to which the antibody preparation is intended and it will be a routine matter for the scientist skilled in the art to select the most appropriate approach for any particular application.

EXAMPLE 1. Preparation of a phosphoserine peptide library immunogen

The following library was prepared, based essentially on sequences commonly found in proteins containing phosphoserine residues (Meth. Enzymol (1991), 200, 62-81):

AcCAA(K/R)XXpSXF-OH Position: 1 2 3 4 5 6 7 8 9

Where Ac represents an acetyl group on the N terminus 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). Thus, at the positions indicated by "X" in the sequence above, the resin was divided into 19 portions an each reacted with a different protected amino acid and the reactions recombined after coupling. At "K/R", the resin was split into 2 portions, and each reacted with either protected arginine or protected lysine, followed by recombination.

Materials All materials were obtained from Novabiochem and Aldrich. All amino acids were suitably side-chain protected for Fmoc peptide synthesis, as outlined for example in Solid Phase Peptide Synthesis, by E. Atherton and R.C. Sheppard, IRL Press 1989.

Resin preparation The library was synthesised on Fmoc-F-NovaSyn-TGA resin (0.17 mmol/g loading)

(500mg). Resin was washed in N,N-dimethylformamide (DMF) (3 x 5 ml x 5 min.) and subsequently treated with 20% piperidine/DMF (3 x 5 ml x 5 min.) until deprotection was confirmed by the Kaiser test.

Couplings

All residues were coupled using 10 equivalents of the relevant FmocOamino-acid / PyBOP / HOBt and 20 equivalents of diisopropylethylamine (2 x 1.5 hr) in a minimum volume of DMF. Resin was stirred during the coupling cycle. Coupling was repeated until completion was confirmed by the Kaiser test. Capping by acetylation was performed after residues 1,3,5 and 8 using 20% acetic anhydride in DMF (2 x 1.5 hr).

Deprotection

Fmoc deprotection was achieved using 20% piperidine/DMF (3 x 5 ml x 5 min.) followed by thorough washing with DMF/dichloromethane.

Cleavage and purification

Library (150 mg resin) was treated with trifluoroacetic acid

(TFA)/triisopropylsilane/ethanediol/water (92.5/2.5/2.5/2.5) (2 ml) for 3 hr. Resin was washed with excess cleavage mixture (2 x 2 ml). Excess TFA was removed by blowing with nitrogen until there was no residue left. Residue was dissolved in water (2 ml) and washed with excess ether. The aqueous layer was lyophilised and the resulting library stored under nitrogen at < 5°C. EXAMPLE 2. Raising of an immune response to the phosphoserine peptide library

Conjugation

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.

Peptides

Peptide AKT, Biotin-GRPRTSpSFAEG (Sequence ID No.2), was prepared by standard solid phase peptide synthesis procedures. 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). Here pS designates the amino acid phosphoserine.

Immunisations Before immunisation a sample of blood (pre-bleed) was taken from each animal to be used.

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 lOμg 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. After a further hour incubation plates were washed five-times with PBS and the TMB substrate was added. After 30 minutes incubation, the reaction was stopped by the addition of 3M sulphuric acid and the plates were read on an Applied Biosystems plate reader at absorption 450nm. Results were plotted using a four parameter fit; the titre recorded was the dilution of serum which produced half the maximum signal (half titre).

Results mouse Camlib AKT Peptide IKB-diP 1KB no P Peptide Peptide

971 79.7 31.4 2150 141

972 134 2800 40800 292

973 1180 1380 739 12.4

974 727 32000 3040 26

It can be seen that 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

EXAMPLE 3. Preparation of specific antibodies from polyclonal sera by immunoaffinity chromatography

Preparation of specific immuno-affinity column lml (packed volume) of SulfoLink Gel (Pierce) was washed in coupling buffer (50 mM Tris, 5 mM EDTA-Na, pH 8.5), then resuspended in 2mls of the same. The peptide of interest was dilute to lmg per ml in coupling buffer then lml of peptide solution was mixed with the washed SulfoLink gel, in the dark for 1 hour. The gel was washed and lml cysteine at mg/ml was added, to block remaining active groups, and mixed for a further hour. The gel was then washed with 20 volumes of coupling buffer, and was used to pack a small column.

Preparation of specific anti-phosphoserine motif sera.

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.

Results

IKB2P

Column

AKT IKB-diP 1KB no P

Peptide Peptide Peptide

Eluate 40.2 20300 22.1

Flowthrough 1940 80.3 18.3

AKT

Column

AKT IKB-diP 1KB no P

Peptide Peptide Peptide

Eluate 1980 32.3 ND

Flowthrough ND 33600 21.3

ND: not determined

It can be seen that the eluate of each column contain useful antibody activity to the peptide of interest that was immobilised, and that this activity had a significant degree of specificity compared to the other peptides. EXAMPLE 4. Generating monoclonal antibodies to the phosphoserine peptide library

Monoclonal Generation

After death 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.

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. 2x10⁸ ( spleen cells from the above described cell suspension were mixed with

2xl07myeloma cells from the mouse myeloma cell line NSO (available from the European Collection of Animal Cell Cultures under the assession No. 85110503). The tube was centrifuged, and all liquid decanted. To the tube was then slowly added (over 1 minute with constant stirring) 1 ml PEG solution at 37°C. (Boeringer PEG1500) with constant stirring and then stirred for a further minute. The fusion was interrupted by adding Dulbecco's modification of Eagle's medium according to the following scheme: 2ml during the first two minutes, 8ml during the following 4 minutes and another 10ml over 2 minutes. 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.

EXAMPLE 5 Generation of a scFv phage display library to the phosphoserine peptide library

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.

RNA Extraction & cDNA preparation

RNA was extracted from the cell preparation described above by treatment with TRIZOL (Life Technologies) according to the manufacturer's instructions. cDNA was then prepared from this RNA by treatment with reverse transcriptase according to standard methods (Sambrook, Fritsch & Maniatis. Molecular Cloning: A Laboratory Manual 2^nd Edition, Cold Spring Harbor Laboratory Press).

Amplification of the genes encoding the antibody heavy and light chain variable regions Polymerase Chain Reaction was used to amplify the genes encoding the antibody heavy and light chain variable regions from the cDNA described above.

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.

Claims

WO 99/65946 _ j2 . PCT/GB99/01795CLAIMS

1. 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, which method 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.

2. A method as claimed in claim 2 in which the peptide library comprises peptides whose sequences are those found to occur in nature in the vicinity of the post-translationally modified amino acid of interest.

3. A method as claimed in either claim 1 or claim 2 in which the post-translational modification to be recognised is phosphorylation

4. A method as claimed in claim 3 in which the post-translationally modified amino acid is a serine or threonine.

5. Use of an antibody spectrum in the isolation of antibody preparation able to recognise a post-translationally modified amino acid.

6. A method of isolating an antibody preparation from an antibody spectrum generated according to claim 1 which comprises exposing the antibody spectrum to a peptide and isolating the antibody attached to the peptide.

7. Amethod as claimed in claim 6 in which the peptide contains a post-translationally modified amino acid.