WO2010015834A1

WO2010015834A1 - Antibodies specific for misfolded proteins and methods for their production

Info

Publication number: WO2010015834A1
Application number: PCT/GB2009/001944
Authority: WO
Inventors: Mourad Tayebi
Original assignee: The Royal Veterinary College
Priority date: 2008-08-08
Filing date: 2009-08-10
Publication date: 2010-02-11
Also published as: GB0814586D0

Abstract

Methods are provided for producing an antibody specific for a target antigen, in particular, antibodies capable of binding to prion proteins. The methods include adding a target antigen stabilising agent to a sample containing a target antigen, allowing the stabilising agent to stabilise the target antigen, incubating the stabilised target antigen with a conventional antibody immobilised on a solid support to form a complex, wherein the immobilised antibody is specific for the target antigen, immunising a host with the complex, and isolating antibodies specific for the target antigen from the host. Also provided are antibodies produced by the methods together with their use in treatment and diagnosis of disease, e.g. prion diseases.

Description

ANTIBODIES SPECIFIC FOR MISFOLDED PROTEINS AND METHODS FOR

THEIR PRODUCTION

BACKGROUND OF THE INVENTION

The present application relates to novel antibodies and to methods for producing antibodies, in particular antibodies capable of binding to prion proteins.

Transmissible spongiform encephalopathies (TSE) or prion diseases comprise a group of invariably fatal neurodegenerative disorders affecting humans and animals, characterised clinically in humans by a rapidly progressive dementia i.e. memory loss and cognitive deficit and neuropathologically, by spongiform degeneration. Prion diseases such as CJD, BSE, together with Alzheimer's disease and certain forms of Parkinsonism are examples of disorders caused by protein misfolding and in prion disease, an abnormally folded protein (called prions or PrP ), derived from a normal cell surface protein (called PrP ), accumulates in the brain and other organs of affected animals. PrP and PrP are identical when denatured, meaning that the toxicity and infectivity of PrP^Sc is wholly dependent on its conformation, i.e. how it is folded.

The human prion diseases include kuru, Creutzfeldt-Jacob disease (CJD), Gerstmann- Straussler-Scheinker syndrome (GSS), sporadic and familial fatal insomnia (FFI). GSS, FFI and 10-15% of CJD are dominantly inherited disorders associated with specific mutations in the PRNP gene ^l, whereas 85-90% of CJD occur sporadically, and are not associated with mutations in the human prion protein gene . Iatrogenic CJD has resulted from the use of contaminated neurosurgical instruments, corneal grafts, dura matter grafts, and pituitary derived hormone treatments.

In animals, the prototypic naturally occurring prion disease is scrapie, which affects sheep and goats. Scrapie has been recognised for over 200 years ³ and has a worldwide distribution. Other and more recently recognised animal diseases include transmissible mink encephalopathy, chronic wasting disease of mule, deer, and elk ⁴, and bovine spongiform encephalopathy (BSE) ⁵. Prion diseases are characterised by their transmissibility to a wide range of experimental animal species, including mice, hamsters, rats, and demonstrated by the transmission of kuru to chimpanzees ⁶, and CJD and GSS to primates ^{7 8}. Prion diseases can be transmitted by intracranial inoculation for experimental purposes but the appearance in UK cattle in 1986 of BSE, which rapidly evolved into a major epidemic ^9;i0, was widely attributed to transmission of sheep scrapie to cattle via contaminated feed prepared from rendered carcasses ⁹. The more recently described feline spongiform encephalopathy of domestic cats ^u and spongiform encephalopathies of a number of zoo animals, the so-called exotic ungulate encephalopathies ' are also recognised as animal prion diseases, and are thought to have resulted from the same contaminated food given to cattle. A need for testing slaughtered cattle over 30 months of age for bovine spongiform encephalopathy has increased the momentum for developing rapid diagnostic tests. However, perhaps the greatest need is for tests with applicability in the sub-clinical phase of prion disease, requiring a specificity that is currently beyond available immunodiagnostic methods.

Both human and animal prion diseases share common histological features, namely spongiform vacuolation, astrocytosis, and neuronal loss. The spongiform change is not seen by the naked eye and vacuoles measure 1 to 50μm in diameter. Proliferation of astrocytic glial cells is another classical marker of prion diseases. This usually accompanies a loss of nerve cells in the surrounding areas of the brain. Amyloid plaques are deposited in the brains of about 15% of prion disease cases and demonstrate the characteristic red/green birefringence when stained with congo red and exposed to polarised light. Initially these plaques were thought to be indistinguishable from those associated with Alzheimer's disease but immunohistochemical work has shown that they are composed of prion deposits. The deposition of amyloid is especially apparent in GSS, Kuru, and vCJD and is a feature of these diseases.

Classical immunisation techniques applied to raise specific anti-prion antibodies have generally been unsuccessful and until recently, only very few antibodies had been produced that recognised any form of PrP ^{l '} . Scrapie-associated fibrils (SAF) were used as immunogen in order to generate an antibody response in both mice and rabbits. Although purified intact infectious SAF did not elicit detectable natural or experimental immune responses ²¹, a weakly measurable imrmmoreactive antibody reaction was evoked by formic acid-extracted SAF and SDS-solubilised SAF preparations, suggesting that treatment of SAF with detergent or denaturant somehow renders the antigen more immunogenic, perhaps by altering its conformation through a process of denaturation. So-called prion rods were also purified then used to produce anti-PrP antibodies in both rabbits ²² and mice ²³. Similar to the SAF preparations, prion rods also undergo substantial and stringent purification steps ²² which could lead to their denaturation and increased immunogenecity while leading to a remarkable reduction in infectivity.

Both the SAF preparations and the prion rods have given rise to weak immune responses but more importantly the antibodies induced bind weakly or not at all to the abnormal isoform of PrP^c; PrP^Sc, and therefore cannot be used as potent immunogens.

Other immunisation approaches have been used, including synthetic peptide vaccination ^24"29, nucleic acid-based immunisation ^30~32, recombinant PrP ^33~35, dimeric PrP ³⁶, and immunisation with scrapie-infected cells ³⁷, none of which has elicited antibody responses specific for PrP^Sc, except for the 15B3 mAb which apparently binds conformational epitopes on bovine and human PrP^Sc without recognising the same regions in PrP⁰³⁸.

The peptide vaccination approach has proven to be a useful strategy for generating PrP^Sc- selective antibodies ' °, although it is proving crucial to further investigate the structure and mechanisms of formation of PrP^Sc and be able to demonstrate clearly whether these immunogenic motifs are prion disease-associated and not a shared generic epitope with other amyloid-forming diseases .

The nucleic acid-based immunisation strategy ^30>32>42 did not prove to be more potent. Here, mice were immunised with DNA ³ or RNA ⁴³ coding for different human prion proteins, following the synthesis of vectors expressing individual genotypes of either the cellular prion gene (PRNP) or mutant forms of the gene.

Full-length recombinant bovine PrP ³⁸ (rbPrP^23"231), recombinant murine ⁴^⁶ (rmPrP^23'231), and recombinant ovine PrP ⁴⁷ (roPrP^{23 231}) as well as truncated recombinant human PrP ⁴⁸ (rhPrP^91"231) can be expressed in Escherichia coli as an α isoform which can then be folded into β isoforms after reduction of the disulfide bond and lowering the pH to 4.0. The 15B3 ⁴⁹ antibody raised against rbPrP^23"231 appears to be selective for the disease-isoform of PrP, although no further application of this antibody has been reported. Monoclonal antibodies to PrP have been successfully raised by various methods in Prn-p^0/0 mice ⁵⁰ that do not express prion protein ^23>38-^{51 53}. However, generation of monoclonal antibodies that bind specifically the native conformation PrP^Sc, has been difficult to produce.

3F4, an IgG2a kappa chain monoclonal antibody ²¹ was produced by immunising C57BL/6J mice with SAF derived from the hamster 263K prion strain and reacted with hamster but not mouse SAF-derived 263K PrP, indicating strong influence of the host species in determining the antigenic response by the existence of prion epitopes that are species specific.

3F4 was tested with homogenates from normal brain tissues of different species and reacted with both hamster and human PrP in brain preparations but failed to detect PrP in brain tissues from mouse, rat, rabbit, bovine, and sheep. Furthermore, pep-scan analysis of 3F4 has shown that it recognises an epitope present on hamster and human PrP between amino acids 109-113 which is dependent upon the presence of methionines at codon 109 and 112, lacking in other species tested. 3F4 has little or no affinity for native PrP^Sc.

A report from Schenk et al ⁵⁴ was the first clear evidence that immunization of transgenic animals prevented the development of beta-amyloid-plaque formation, and reduced the degree of neuropathological progression in an Alzheimer disease model. Following that report, other groups ^55;56 have demonstrated the effectiveness of immunisation using amyloid-β peptide in reducing neuropathology in transgenic rodents ⁵⁷ and humans ⁵⁸.

Several drugs have been tested for interaction with PrP^Sc and its accumulation, including Polyanions ⁵⁹, Iododoxorubicin, Tetracycline ⁶⁰, Congo red ⁶¹, β-sheet breakers ⁶², Polyene antibiotics ⁶³, Chlorpromazine and Quinacrine ⁶⁴. Only amphotericine analogue MS8209 has had an appreciable effect on the disease course. Otherwise many of these drugs suppress prion replication in vitro, but fail to show significant efficacy in vivo.

Thus, a need exists for improved methods of producing antibodies that are more effective against antigenic targets such as prion proteins. SUMMARY OF THE INVENTION

According to one aspect of the present invention, there is provided a method for producing an antibody specific for a target antigen, the method comprising:-

(i) adding a target antigen stabilising agent to a sample containing a target antigen;

(ii) allowing the stabilising agent to stabilise the target antigen; (iii) incubating the stabilised target antigen with a conventional antibody immobilised on a solid support to form a complex, wherein the immobilised antibody is specific for the target antigen; (iv) immunising a host with the complex; and (v) isolating antibodies specific for the target antigen from the host.

Preferably, the target antigen is a misfolded protein, more preferably a misfolded prion protein, further preferably PrP^Sc or PrP^c. In some embodiments, the target antigen is misfolded native PrP^So.

Preferably, the stabilising agent is selected from Congo red and Thiofiavin T.

Preferably, step (ii) comprises incubating the stabilising agent with the target antigen for up to about 24 hours, preferably up to about 12 hours, further preferably between about 30 minutes and about 5 hours, more preferably about 1 hour and about 3 hours, most preferably about 1 hour or about 2 hours.

Preferably, step (ii) comprises incubating the stabilising agent with the target antigen with continuous rotation. In preferred embodiments, the stabilising agent is incubated with the target antigen at about room temperature.

Preferably, the stablising agent is present at a concentration of between about 50 μg/ml and about 1000 μg/ml, more preferably between about 100 μg/ml and about 500 μg/ml. hi preferred embodiments, the concentration of stabilising agent is increased over time during step (ii), preferably from about 50 μg/ml to about 1000 μg/ml, most preferably from about 100 μg/ml to about 500 μg/ml. In particularly preferred embodiments, the concentration of stabilising agent is increased continually throughout the incubation time of step (ii). In other embodiments, the concentration of stabilising agent is increased in stepped increments over the incubation time.

Preferably, the sample containing the target antigen is a sample of animal tissue, further preferably spleen or a peripheral blood mononuclear cell (PBMC) or part of the CNS, eg. brain.

Preferably, the animal tissue is homogenised prior to addition of the stabilising agent.

Preferably, the target antigen is treated with a proteinase specific for non-target antigen proteins, prior to step (i), (ii) or (iii). hi preferred embodiments the proteinase is proteinase K.

Preferably the proteinase is present at a concentration of between about 10 μg/ml and about 100 μg/ml, more preferably about 50 μg/ml. Preferably, the target antigen is treated with the proteinase for up to about 2 hours, more preferably for up to about 1 hour, most preferably for about 30 minutes. In some embodiments, the target antigen is treated with the proteinase at a temperature of about 37°C.

Preferably, the animal tissue is from an animal infected with a disease and wherein the target antigen is an indicator or a causative agent of that disease.

Preferably, the disease is selected from a disease characterised by protein misfolding, preferably selected from prion diseases, Alzeihmer's Disease, Parkinson's Diseases and Diabetes amyloidosis. Preferably, the prion diseases are selected from CJD, vCJD, BSE, GSS, FFI and Scrapie.

In preferred embodiments, the conventional antibody is immobilised on a solid support by saturating a solid support with the conventional antibody.

hi one embodiment, the conventional antibody is immobilised on the solid support via a second antibody which is immobilised on the solid support and which binds the conventional antibody. Preferably, the host is selected from a mouse, camelid, rabbit, rat, sheep and cow. In preferred embodiments the camelid is selected from an alpaca, camel and llama.

Preferably, prior to immunising the host, the complex is isolated from the sample.

Preferably, the solid support is a bead, further preferably an immunomagnetic bead, more preferably selected from an IgG immunomagnetic bead, IgA immunomagnetic bead, protein G immunomagnetic bead, protein A immunomagnetic bead and streptavidin immunomagnetic bead.

Preferably, the target antigen is immobilised on the solid support by incubating the target antigen with the solid support for up to about 24 hours, preferably for up to about 12 hours, further preferably between about 30 minutes and about 5 hours, more preferably about 1 hour and about 3 hours, most preferably about 1 hour or about 2 hours.

Preferably, the target antigen is incubated with between about 10⁵ and about 10⁷ immunomagnetic particles, more preferably about 10⁶ (25 μl) immunomagnetic particles.

Preferably, the target antigen is incubated with the solid support at between about 0⁰C and about 25°C, preferably between about 2⁰C and about 8°C, more preferably at about 4°C or about room temperature.

According to the invention, there is, therefore, provided a method for producing an anti-PrP^Sc antibody, the method comprising: -

(i) incubating a scrapie infected brain homogenate with Congo Red;

(ii) immobilising Congo Red bound PrP on an anti-PrP immunomagnetic bead to form a PrP/Congo Red immunomagnetic bead complex (iii) immunising a host with the PrP/Congo Red immunomagnetic bead complex; and (iv) isolating antibodies specific for PrP^Sc from the host. Preferably, prior to step (i), the scrapie infected brain homogenate is incubated with proteinase K.

Preferably, the scrapie infected brain homogenate is incubated with about 50μg/ml proteinase K at about 37°C for about 30 minutes.

According to another aspect of the present invention, there is provided an antibody produced by a method of the invention.

Preferably, the antibody is a monoclonal antibody. Preferably, the antibody is an antibody fragment. Preferably, the antibody is conjugated to a growth inhibitory agent. Preferably, the antibody is conjugated to a cytotoxic agent, for example a toxin, antibiotic, lytic enzyme or radioactive isotope.

In some embodiments, the antibody is conjugated to a detectable marker, for example a fluorescent marker.

Another aspect of the present invention relates to a method for the treatment of a disease, the method comprising administering to a patient an effective amount of a composition comprising an antibody of the invention.

Also provided is use of an antibody of the invention in the manufacture of a medicament for the treatment of a disease.

Further provided is an antibody of the invention for use in the treatment of a disease.

Another aspect provides a composition comprising an antibody of the invention for the treatment of a disease.

A further aspect of the present invention relates to a method for diagnosing a disease, the method comprising

(i) contacting a sample obtained from a patient at risk of suffering from the disease with an antibody of the invention; (ii) detecting the presence of an antigen in the sample bound to the antibody; wherein the presence of the antigen indicates that the patient is suffering from the disease or is at risk of suffering from the disease.

hi preferred embodiments, an increased amount of the antigen in the sample versus the amount of the antigen in a control sample indicates that the patient is suffering from or is at risk of suffering from the disease.

Another aspect of the present invention relates to a method for monitoring the progression of a disease, the method comprising

(i) contacting a sample obtained from a patient suffering from the disease with an antibody of the invention;

(ii) detecting the presence of an antigen in the sample bound to the antibody; and

(iii) repeating steps (i) and (ii) at two or more time intervals, wherein an increase in the amount of the antigen bound to the antibody over time is indicative of a progression of the disease and a reduction in the amount of the antigen bound to the antibody over time is indicative of a regression of the disease.

Also provided by the present invention is a kit for the diagnosis, treatment or monitoring of progression of a disease, the kit comprising an antibody of the invention.

Preferably, the disease is selected from a disease characterised by protein misfolding, preferably selected from prion diseases, Alzeihmer's Disease, Parkinson's Diseases and Diabetes amyloidosis. In preferred embodiments, the prion diseases are selected from CJD, vCJD, BSE, GSS, FFI and Scrapie.

The present invention also relates to a pharmaceutical composition comprising the antibody of the present invention as well as a veterinary composition comprising the antibody of the invention.

The route of administration of pharmaceutical compositions according to the invention may be any of those commonly known to those of ordinary skill in the art. For therapy, the antibodies and compositions of the invention can be administered to any patient in accordance with standard techniques. The administration can be by any appropriate mode, including parenterally (e.g., intravenous, intramuscular, intraperitoneal, intra- articular, intrathecal), transdermally, via the pulmonary route, or also, appropriately, by direct infusion with a catheter. The dosage and frequency of administration will depend on the age, sex and condition of the patient, concurrent administration of other drugs, counterindications and other parameters to be taken into account by the clinician. Administration can be local (e.g., local delivery to the lung by pulmonary administration, e.g., intranasal administration) or systemic as indicated. The skilled clinician will be able to determine the appropriate dosing regimen to treat, suppress or prevent disease.

The invention also relates to a drug delivery device comprising the pharmaceutical or veterinary composition of the invention. For example, the drug delivery device can be a parenteral delivery device, intravenous delivery device, intramuscular delivery device, intraperitoneal delivery device, transdermal delivery device, pulmonary delivery device, intraarterial delivery device, intrathecal delivery device, intraarticular delivery device, subcutaneous delivery device, intranasal delivery device, vaginal delivery device, or rectal delivery device. Examples of such delivery devices, include a syringe, a transdermal delivery device (e.g., a patch), a capsule, a tablet, a nebulizer, an inhaler, an atomizer, an aerosolizer, a mister, a dry powder inhaler, a metered dose inhaler, a metered dose sprayer, a metered dose mister, a metered dose atomizer, and a catheter.

In preferred embodiments, the methods and compositions of the invention are for treatment or diagnosis of the disease at an early stage, for example, before symptoms of the disease appear.

In some embodiments, the methods and compositions of the invention are for treatment or diagnosis of the disease at a clinical stage. In this respect, the antibodies of the present invention could be used to reverse early neuropathological changes associated with the diseases, for example by removing or neutralising PrPC reverse neurodegeneration. ⁶⁵'⁶⁶.

Accordingly, as described above, the invention provides compositions (e.g., pharmaceutical and veterinary compositions) comprising an antibody of the invention. The invention also provides a method for treating an individual having a disease or disorder, such as those described herein, comprising administering to said individual a therapeutically effective amount of an antibody of the invention. In some embodiments, the disease or disorder is a neurological disease, such as CJD, etc. The invention also provides for use of an antibody of the invention for the manufacture of a medicament for treatment of a disease or disorder. The invention also relates to use of an antibody as described herein for use in therapy, diagnosis or prophylaxis.

Example embodiments of the present invention will now be described with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a schematic representation of use of Dynabeads M-450 to immunoisolate Congo red-bound prion protein. Immunomagnetic isolation of Congo red-bound prion proteins from brain homogenates (A schematic outline). A & B. After overnight incubation of the sheep anti-mouse IgG coupled 4.5 μm Dynabeads to anti-PrP mAb, the immunocomplex was added to scrapie-infected brain homogenates (pre-incubated with Congo red) and subsequently incubated for 2 hr at room temperature. C & D. The immunocomplex bound to Congo red-bound prion protein was isolated using the MPC-S magnetic device (Dynal), and finally E. This immunocomplex, called PrP/CR-Dynabeads, was used for immunisations of mice and camels;

Figure 2 shows adsorption of Congo red bound-PrP^Sc is proportional to mAb concentration. Decreasing ten-fold dilutions of mAb (starting at 50 mg/ml) were adsorbed to Dynabeads then 1 x 10⁷ of these beads were added to 50ul of a 10% RML brain homogenate bound to Congo red then spiked into an equal volume of 10% Prn-p^0/0 brain homogenate;

Figure 3 shows optimal detection and depletion of Congo red bound-PrP^Sc from homogenised brain. Congo red bound-RML brain homogenate spiked into brain homogenate derived from Prn-p^m mice was proteinase K (PK) treated then incubated with 1 x 10⁷ mAb saturated- Dynabeads (upper panel). In the lower panel, mAb was omitted from the immunocomplex keeping the other conditions identical; Figure 4 shows the effect of medium-term cold storage of the mAb-Dynabeads immunocomplex on depletion of Congo red bound-PrP^Sc. mAb-Dynabeads maintain their ability to capture Congo red bound-PrP^Sc over time;

Figure 5 shows that mAb-Dynabeads bind only a proportion of the Congo red bound-PrP^Sc in a 10% dilution. Congo red bound-RML brain homogenate spiked into brain homogenate derived from Prn-p^m mice in a 1:10 ratio was proteinase K (PK) treated then incubated with decreasing ten- fold dilutions of mAb-Dynabeads (starting from 1 x 10⁷ to 1 x 10⁴). Both bead- bound protein (b) and supernatant (s) were assayed for PrP^Sc levels. Brain homogenate derived from Prn-p^m mice (KO) was also included as negative control;

Figure 6 shows the effect of Dynabeads concentration on Congo red bound-PrP^Sc depletion from various concentrations (1:100, 1:20, and 1/10) of 10% w/v scrapie-infected brain homogenates spiked into Prn-p⁰⁰ brain homogenate. 1 x 10⁴ to 1 x 10⁷ increasing concentration of Dynabeads was incubated with a saturable amount of anti-PrP mAb antibody. Incubation time overnight at 2 - 8°C. Depletion in varying Congo red bound-scrapie brain : Prn-p⁰¹⁰ ratios;

Figure 7 shows the effect of incubation time (2 - 8°C) on depletion/binding of Congo red bound-PrP^Sc using Dynabeads (1 x 10⁷ beads). A 10% Congo red bound-scrapie-infected brain homogenate was incubated with mAb-Dynabreads overnight and samples were analysed at various time points starting from 1 to 24 hours, then amounts of bead-bound and after- wash supernatant PrP^Sc were assessed. PrP^Sc binding to mAb-Dynabeads is time dependent; and

Figure 8 shows treatment of prion-permissive cell lines (ScN2a). Appropriate antibody dilutions of PRIOV and Normal Camel Serum (lμg and 25μg) were added to 1 x 10⁴ ScN2a cells with medium in 6 well plates, and left to incubate for twenty-four hours at 37°C (5% CO₂). The cells were then removed from the wells by gentle scraping using a rubber syringe, and centrifuged at 800 rpm for 5 minutes. The pellet was lysed with 50 μl of lysis buffer, then subsequently proteinase K (PK) treated with lug/ml for 25 minutes at 37⁰C in order to digest normal cellular PrP, and finally treated with ABSF to inhibit the PK. The cells were then stored at -20C until further use. Levels of PrP^Sc in the culture were then analysed by ELISA and Western blot in triplicates for each experiment. Figure 8 shows a clear reduction in PrP^Sc replication when 10,000 ScN2a cells are treated with 1 and 25 μg of PRIO V. Both concentrations show a significant reduction in prion replication (P<0.001) when compared with the NCS control and the No Ab negative control.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to novel antibodies and to methods of their production. Also described are various uses of the novel antibodies.

The methods used in the invention and detailed examples of the invention are set out below:

Within this specification embodiments have been described in a way which enables a clear and concise specification to be written, but it is intended and will be appreciated that embodiments may be variously combined or separated without parting from the invention.

Within this specification, the terms "comprises" and "comprising" are interpreted to mean "includes, among other things". These terms are not intended to be construed as "consists of only".

Within this specification, the term "about" means plus or minus 20%, more preferably plus or minus 10%, even more preferably plus or minus 5%, most preferably plus or minus 2%.

Within this specification, a "functional derivative, variant or analogue thereof is interpreted to mean an antibody preferably having at least about 50%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, the binding affinity of an antibody as described herein. Such binding affinity can readily be assessed using any suitable method, such as those described herein or known in the art.

Within this specification, "antibody or antibody fragment" refers to an antibody (for example IgG, IgM, IgA, IgD or IgE) or fragment (such as a Fab, F(ab')2, Fv, disulphide linked Fv, scFv, closed conformation multispecific antibody, disulphide-linked scFv, diabody) whether derived from any species naturally producing an antibody, or created by recombinant DNA technology; whether isolated from serum, B- cells, hybridomas, transfectomas, yeast or bacteria.

The term "analogue" as used herein referring to a polypeptide means a modified peptide wherein one or more amino acid residues of the peptide have been substituted by other amino acid residues and/or wherein one or more amino acid residues have been deleted from the peptide and/or wherein one or more amino acid residues have been deleted from the peptide and or wherein one or more amino acid residues have been added to the peptide. Such addition or deletion of amino acid residues can take place at the N-terminal of the peptide and/or at the C-terminal of the peptide or they can be within the peptide.

Within this specification, the term "treatment" means treatment of an existing disease and/or prophylactic treatment in order to prevent incidence of a disease.

Within this specification, the term "target antigen" means an antigen to which the antibodies of the invention are desired to bind. In the same way "non-target antigen" means an antigen to which the antibodies of the invention are desired not to bind. However, it will be envisaged that the present invention is not limited to antibodies which bind only target antigens and not non-target antigens. In this respect, some antibodies of the invention are able to bind to both target antigens and non-target antigens, whilst other antibodies of the invention are able to bind selectively to target antigens with limited or no binding to non-target antigens.

Within this specification, the term "stablising agent", means, in particular, an agent that is capable of stablising the conformational structure of a target antigen. Examples include Congo red and Thioflavin T.

Within this specification, the term "host" means, for example, a biological system that is capable of producing antibodies against an antigen introduced into that system. Examples include an animal host, in particular, rabbits, mice, camelids (Alpacas, Llamas, Camels), rats, sheep and cows.

Within this specification, the term "conventional antibody" means an antibody which has been previously found to have the desired binding activity. A conventional antibody could be a commercially available antibody or it could, for example, be an antibody previously discovered during the course of previous unpublished experimental work. Put another way, a conventional antibody is a known or previously identified antibody. In some embodiments a conventional antibody could be an antibody identified by the methods described herein. In such an embodiment, once an antibody has been identified by the methods described herein it will become an antibody known to have the desired binding activity.

Within this specification, the term "animal" includes mammals. The term "mammals" includes humans.

Within this specification, the term "room temperature" means about 21⁰C.

Within this specification, the term "specific" means that the antibody has binding specificity for the antigen in question.

EXAMPLES

Preparation of Congo red-bound native prions

In order to increase conformational differences between PrP^Sc and PrP⁰, Congo red was used to bind PrP^Sc. This was then imrnunoadsorbed to Dynabeads and used to immunise camels and mice.

Congo red is known to bind PrP ^c and is thought to further stabilize protein conformation ⁶⁷. Furthermore, Congo red renders native PrP^Sc resistant to denaturation by boiling in SDS. Neither the properties of PrP^c nor those of predenatured PrP^Sc are changed by the addition of Congo red . In order to study conformational changes induced by Congo red binding to PrP^Sc, scrapie infected brain homogenate was first pre-incubated with an appropriate concentration of Congo red with continous rotation for at least 2h. The mixture was than added to the 35-Dynabeads immunocomplex and incubated for 2h at RT (see preparation of Dynabeads-adsorbed antigen discussed below) Dynabeads^® M-450 sheep anti-mouse IgG

Dynabeads M-450 Sheep anti-Mouse IgG are designed for immunomagnetic separation. Their size is particularly suitable for cell isolation but they are also used for immunoprecipitation of proteins and other purposes. These Dynabeads bind to defined cell surface antigens and other antigens through a mouse primary antibody of the IgG-class. The target cells rosetted with Dynabeads M-450 Sheep anti-Mouse IgG as well as immunoprecipitates can be easily isolated by applying a magnet (Dynal MPC) on the wall of the test tube for 1-2 minutes.

Dynabeads M-450 sheep anti-mouse IgG are monosized, superparamagnetic, polystyrene beads with affinity purified Sheep anti-Mouse IgG covalently bound to the surface. These secondary antibodies bind both heavy and light chain of mouse IgG and IgM light chain. Human cross reactivity is minimal.

Using the Dynabeads protocol (Tayebi et al., MoI Med 2004), optimisation of cell yield and purity is, apart from the chosen antibody, especially dependent on two of the variables involved, the number of beads used and the incubation time. The manufacturer recommends the use of 1-2 x 10⁷ Dynabeads/ml of sample, and a 10-30 minute incubation time.

The affinity and avidity of the primary antibody should be considered as the most important factor in the successful use of Dynabeads M-450 sheep anti-mouse IgG. The reactivity to mouse IgG₁, IgG₂₃ and IgG₂), is very good, while the reactivity to IgG₃ is low due to the fact that this subclass is sparse in normal mouse serum. Concentration of Dynabeads M-450 Sheep anti-Mouse IgG during separation is the single most important parameter for obtaining a good yield, provided that a good primary antibody is used. A concentration of 1- 2 x 10⁷ Dynabeads per ml sample is generally sufficient to reach the plateau in the binding kinetics after 20 min incubation.

Preparation of Dynabeads-adsorbed antigen

Sheep anti-mouse IgG coupled 4.5μm immunomagnetic particles (Dynabeads M450) were used. The Dynabeads M450 were washed 4 times in sterile PBS to remove preservatives, and resuspended with an excess of commercial anti-prion antibody (mAb) to achieve maximal saturation. The Dynabeads/mAb solution was rotated continuously overnight at 4⁰C. After seven washes to remove unbound mAb, the Dynabeads were resuspensed at 10⁷ / ml in sterile PBS, and then stored at 4⁰C until use. IxIO⁶ mAb-coated Dynabeads were incubated with lOOμl of 1% scrapie infected brain homogenate bound to Congo red (as determined by SDS- PAGE) for at least 2 hours at room temperature. The final immuno-isolate was washed 7 times and resuspended in PBS. This immunocomplex, called PrP/CR-Dynabeads was subsequently used for immunisations.

Production of murine (PrioC) and camelid (PrioV) monoclonal antibodies

Anti-PrP/CR-Dynabeads mAbs called PrioC (mouse) and PrioV (camel) mAbs were produced as described above.

Both mice and camels were immunised subcutaneously with PrP/CR-Dynabeads in CFA then in IFA for subsequent boostings. PrP^Sc was first digested with proteinase K (50μg/ml) for 30 minutes at 37⁰C to remove PrP^c prior to binding to Congo red. The complex PrP^Sc/Congo red was then adsorbed to Dynabeads. Mouse Hybridomas (Antibody producing cells) were screened for reactivity to both native PrP^c and PrP^Sc. Positive hybridomas were repeatedly cloned until stable. For camelid antibodies, these were produced using the E coli system.

AU PrioC antibodies produced in mice were of the IgM isotype and PrioV were of the IgG isotype.

Optimisation of commercial anti-prion antibodies

The concentration of the anti-PrP mAb required to saturate the sheep anti-mouse Dynabeads was optimised by varying the binding concentration and then using the mAb-coated beads to immunoprecipitate the same quantity of Congored bound-PrP^Sc from murine scrapie brain homogenates (Fig. 2). These experiments indicated that mAb coupling concentrations > 5 x 10^"2 mg/ml saturated the beads. It was also determined that 2.5 x 10⁶ PrP/CR-Dynabeads loaded onto each lane gave a readily detectable signal in standard western blots. Optimisation of Congo red bound PrP ^c from brain homogenate

Increasing amounts of Congo red bound-PrP ^c were titrated in, in order to achieve maximal specific antigen loading onto Dynabeads saturated with mAb, keeping the concentration of other brain constituents constant by spiking increasing quantities of scrapie brain homogenate into decreasing volumes of homogenates (at equivalent w/v) derived from Prn-p^0/0 mice (Fig. 3). Again Congo red bound-PrP^So binding to the bead surface was highly dependent on the presence of the capturing mAb, although at the highest concentrations used, some nonspecific adsorption to the surface of the Dynabeads was evident. When the supernatant from which PrP had been adsorbed to the Dynabeads was assayed for residual PrP^Sc by western blotting, only partial depletion of the protein was observed under adsorption conditions used, an indication of the inefficacy of PMSF in inactivating the proteinase K.

Effect of medium-term cold storage of the mAb-Dynabeads immunocomplex on depletion of Congo red bound-PrP^Sc

It was investigated whether medium term storage of the immunocomplex mAb-Dynabeads in the laboratory fridge had any direct and detrimental effect on the adsorption of Congo red bound-PrP^Sc from the highest (10%) spiked terminally-sick scrapie brain homogenates into PrP -deficient brain homogenate (Fig. 4)

Surprisingly, after storage at 2-8⁰C, there was little change in the detection of Congo red bound-PrP^Sc after densitometry analysis using Molecular Dynamics STORM 840 phosphoimager (enhanced chemifluorescent substrate (ECF, Amersham) over a period of 60 days.

It was then determined whether the PrP^Sc-Dynabeads were immunogenic in vivo in camels and mice. Optimising the Dynabead concentration needed to achieve maximal Congo red bound- PrP^Sc depletion

To determine the concentration of the relative amount of Dynabeads needed to achieve maximal depletion of Congo red bound-PrP^So molecules, a 10% w/v solution of terminally- sick scrapie brain homogenate bound to Congo red was spiked into Prn-p^m brain homogenate (1:10 ratio) using decreasing amount of beads (ten-fold) ranging from 10⁷ to 10⁴ and was subsequently immunoprecipitated (Fig. 5 & Fig. 6). Both Congo red bound-PrP^Sc adsorbed Dynabeads and the supernatants were assayed. The amount of Congo red bound-PrP^Sc adsorbed to Dynabeads was inversely proportional to the amount detected in the supernatant using mAb. Relative saturation of the Dynabeads was achieved at 1 x 10⁶ beads, then plateaued above this amount. Full depletion of Congo red bound-PrP^Sc was not achieved as shown by the increasing PrP ^c signal in the supernatant when the bead concentration was reduced, and a poor signal has been detected with bead-Congo red bound-PrP^Sc when the beads concentration was below 10⁵.

Using the Molecular Dynamics STORM 840 phosphoimager (enhanced chemifluorescent substrate (ECF, Amersham), a semi-quantitative measure was performed in order to estimate the relative expression levels of Congo red bound-PrP^Sc depleted from a 10% w/v solutions of terminally-sick scrapie brain homogenates that were first mixed in varying ratios (Scrapie : Prn-p^m) and incubated with mAb-Dynabeads, including 1 :100, 1:20, and 1 :10. The measure of the relative depletion of Congo red bound-PrP^Sc was achieved through direct densitometric analysis of the bead- Congo red bound-PrP^Sc after boiling in SDS, rather than substracting it from the post-wash supernatant after the incubation period of the whole irnrnunocomlex as this could give a false measure of the relative amounts present since the concentration of Congo red bound-PrP^Sc could exceed the limit and capacity of the beads to adsorb more proteins.

This has been shown when the highest concentration of the brain homogenate was used (1:10) and compared to other concentrations (1:20 and 1:100) since the same number of beads could deplete more efficiently when lower concentration of the brain homogenate was used. In conclusion, Congo red bound-PrP^Sc binds significantly only when Dynabeads have been precoated with anti-PrP mAb. Moderate depletion of PrP^Sc from brain homogenates reflects the limited capacity for adsorption of the antigen to their surface.

Effect of incubation time on depletion of Congo red bound-PrP^Sc

Congo red bound-PrP^Sc from scrapie-infected brain homogenate spiked into Pm-p^m mice brain homogenate was semi-quantitated using the Molecular Dynamics STORM 840 phosphoimager (enhanced chemifluorescent substrate (ECF, Amersham) over a period of 24 hours (Fig. 7). After adding a 10% preparation of the spiked brain homogenate to the mAb- Dynabeads, samples of the immunocomplex were taken at various time points and depletion versus binding was measured with STORM. With the highest concentration of the spiked brain (10%), it was apparent that the incubation period did not achieve complete depletion, but more Congo red bound-PrP^Sc was shown to bind to the immunocomplex over time. For instance there was a 50% increase in binding over a 24h period. It was also shown that the amount of Congo red bound-PrP^Sc detected in the supernatant, after washing the PrP- Dynabeads immunocomplex, was inversely proportional to bead-Congo red bound-PrP^Sc after SDS boiling.

PrioC mAbs (mouse) raised against native PrP ^C/CR-Dynabeads recognises mouse rPrP

Clones and subclones of hybridomas derived from spleens of Prn-p^0/0 mice hyperimmunised with PrP^s7CR-Dynabeads were screened for reactivity to alpha or beta recombinant protein. Hybridoma clone supernatants, were probed for reactivity with full-length and truncated mouse α or β recombinant prion proteins. The majority of these mAb supernatants reacted strongly to both α and β conformations of rPrP with ELISA. These supernatants were also screened for total IgG and IgM with isotype ELISA.

PrioC mAbs Isotyping

The isotype response for monoclonal antibodies was predominantly of IgM subtype. Epitope mapping of PrioC mAbs

Overlapping 20-mer peptides spanning the mouse PrP sequence 90-230 were used. Depending on the way the immunogen was prepared, supernatants bound different PrP regions.

PrioC mAbs bind native PrP^c/Sc Binding to surface PrP^c on U937 cells

PrioC mAbs were also probed in order to demonstrate whether binding to surface native PrP^c occured. In general PrioC mAbs bound weakly to the surface of U937 cells than either commercial mAb anti-prion, possibly reflecting a lower affinity for PrP .

PrioC mAbs binding to membrane and cytoplasmic PrP^Sc/c with N2a cells

During its synthesis, PrP^c transits through the secretory pathway. The conversion of PrP^c to PrP^Sc is thought to occur after the former reaches the plasma membrane or is reinternalised for degradation ⁶⁹. It was sought to demonstrate cytoplasmic staining of PrP^Sc (and PrP^c) in scrapie infected Neuroblastoma cells (N2a) using PrioC mAbs by immunofluorescence labelling. N2a cells were seeded on glass coverslips and grown to 50% confluence for staining of both external and internal PrP. Prior to immunofluorescence staining and imaging, the infected and non infected N2a cells were first lightly paraformaldehyde fixed and permeabilized with Triton X-100.

An isotype matched IgM control has shown no binding against PrP, in contrast with the positive control, known to bind PrP with high affinity, that decorated the scrapie-infected N2a cells with a ring around the plasma membrane, indicating strong binding to surface PrP^c. PrioC mAbs bound surface PrP on uninfected N2a cells. Since the cells were permeabilized, staining for PrP was also seen inside the cytoplasm, in contrast with mAb positive control where the staining remained at the cell surface forming a ring around it.

Similarly, when PrioC mAbs were used for staining permeabilized Sc N2a cells, binding was also seen in the cytoplasm. Staining was also apparent on the membrane of these Sc N2a. In conclusion, staining for both groups of cells did not show considerable differences of binding by these PrioC mAbs.

PrioC mAbs bind native PrP across species

Normal and scrapie brain homogenate was proteinase K treated and the PrioC mAbs used to immunoprecipitate PrPC and PrPSc from murine, human, ovine, and bovine brain homogenates. Treatment with proteinase K cleaves approximately 67 amino acids from N- terminus of PrP^Sc and completely digests PrP^c. Positive control (biotin-conjugate), which detects all glycoforms and fragments of PrP^c, was used to analyse the immunoprecipitate by western blots after denaturation by boiling in sample buffer for 5 min.

All PrioC mAbs were used to immunoprecipitate PrP from WT and scrapie-infected brains from mice and human vCJD. An anti-mouse IgM, was used as the negative isotype matched control. It was clear that although PrioC mAbs immunoprecipitated both isoforms of PrP from mouse brain homogenates, they recognised vCJD and not control human brain homogenate.

Diagnostic value of PrioC mAbs

A BSE test evaluated by the EC has previously been developed by the inventors that included the measurement of 48 test positive as well as 152 test negative that were all correctly identified (EU website). The BSE test also included dilution series in order to determine the detection limit of the test, and ranged between 10^"1 and 10^"3. All 10^"1, 10^{"1 5} and 10^~2 scored positive. Only 6/8 and 4/12 scored positive for the lO^"2'⁵ and 10^"3 dilutions respectively.

In order to mimic the same format of testing and analysis, PrioC mAb were used. Nine New Zealand control bovine homogenates were also used. Test positive have all scored positive with PrioC mAb (mean reading compared to cut-off). All 10^"1 series scored positive and none of the 10^~2 as well as 10^" dilution series scored positives. See table below for details of the results. Table 1 : Overall results including test positives and negatives as well as dilution series using PrioC mAb

Although the size of the samples was limited and considerably smaller than that used for the BSE EU test, the PrioC mAbs seemed to perform similarly when undiluted samples and dilution series of 10^" were used. In contrast none of the 10^" and 10^" dilution series were detected in this system. In conclusion, the PrioC antibodies raised against native prion proteins have shown promise as diagnostic tools and proved to be very effective in detecting concentrations of PrP^So molecules in the 10^"1 homogenates.

PrioC mAb inhibits propagation of PrP Sc^c i.n chronically infected N2a cells

A range of concentrations of PrioC and control mAbs were added to ScN2a cultures for 4 days (repeated daily treatment). After day five, 25000, 5000, 1000 ScN2a (the 5000 and 1000 cells were diluted into appropriate concentration of uninfected N2a cells to achieve 25000 total number of cells) cells were placed into an ELISPOT plate in quadruplicate. Levels of PrP^Sc in the culture were then analysed with the Scrapie Cell assay. The level of PrP^Sc in cells treated with PrioC mAb, compared with the cells treated with isotype matched negative control IgM was dramatically reduced in a dose-dependent manner.

In contrast, 4-day treatment with control, did not reduce the level of PrP^Sc in ScN2a cultures even at high concentrations.

PrioC inhibition of PrP^Sc level in ScN2a cells was dose-dependent as complete inhibition of PrP^Sc was achieved when a titration of the PrioC, ranging from 27μg/ml to O.l lμg/ml was used on the 25000 ScN2a cell dilution. The 5000 ScN2a cell dilution was also efficiently inhibited by the same titration of antibody, but in contrast with the two cell dilutions, the undiluted 1000 ScN2a cells demonstrated less efficiency in the decrease of PrP^Sc level, particularly below 1 μg/ml.

It should be understood that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the present invention and without diminishing its attendant advantages. It is therefore intended that such changes and modifications are covered by the appended claims.

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Claims

1. A method for producing an antibody specific for a target antigen, the method comprising: -

(ii) allowing the stabilising agent to stabilise the target antigen;

(iii) incubating the stabilised target antigen with a conventional antibody immobilised on a solid support to form a complex, wherein the immobilised antibody is specific for the target antigen;

(iv) immunising a host with the complex; and

(v) isolating antibodies specific for the target antigen from the host.

2. A method according to claim 1 , wherein the target antigen is a misfolded protein.

3. A method according to claim 1 or 2, wherein the target antigen is a prion protein.

4. A method according to claim 3, wherein the prion protein is PrP or PrP .

5. A method according to any preceding claim, wherein the stabilising agent is selected from Congo red and Thioflavin T.

6. A method according to any preceding claim, wherein the sample containing the target antigen is a sample of animal tissue.

7. A method according to claim 6, wherein the animal tissue is selected from spleen, a peripheral blood mononuclear cell (PBMC) and a part of the Central Nervous System (CNS).

8. A method according to claim 6 or 7, wherein the animal tissue is homogenised prior to addition of the stabilising agent.

9. A method according to any of claims 6 to 8, wherein the animal tissue is treated with a proteinase specific for non-target antigen proteins.

10. A method according to any of claims 6 to 9, wherein the animal tissue is from an animal infected with a disease and wherein the target antigen is an indicator of that disease.

11. A method according to claim 10, wherein the disease is characterised by protein misfolding.

12. A method according to claim 10 or 11, wherein the disease is selected from prion diseases, Alzeihmer's Disease, Parkinson's Diseases and Diabetes amyloidosis.

13. A method according to any preceding claim, wherein the host is selected from a mouse, camelid, rabbit, rat, sheep and cow.

14. A method according to any preceding claim, wherein prior to immunising the host, the complex is isolated from the sample.

15. A method according to any preceding claim, wherein the conventional antibody is immobilised on the solid support via a second antibody which is immobilised on the solid support and which binds the conventional antibody.

16. A method according to any preceding claim, wherein the solid support is a bead.

17. A method according to any preceding claim, wherein the solid support is an immunomagnetic bead.

18. A method for producing an anti-PrP^Sc antibody, the method comprising:- (i) incubating a scrapie infected brain homogenate with Congo Red;

(ii) immobilising Congo Red bound PrP on an anti-PrP immunomagnetic bead to form a PrP/Congo Red immunomagnetic bead complex;

(iii) immunising a host with the PrP/Congo Red immunomagentic bead complex; and

(iv) isolating antibodies specific for PrP^Sc from the host.

19. A method according to claim 18, wherein prior to stem (i), the scrapie infected brain homogenate is incubated with proteinase K.

20. An antibody produced by a method according to any preceding claim.

21. An antibody according to claim 20, wherein the antibody is a monoclonal antibody.

22. An antibody according to claim 20 or 21, wherein the antibody is an antibody fragment.

23. An antibody according to any of claims 20 to 22, wherein the antibody is conjugated to a growth inhibitory agent.

24. An antibody according to any of claims 20 to 23, wherein the antibody is conjugated to a cytotoxic agent, for example a toxin, antibiotic, lytic enzyme or radioactive isotope.

25. A method for the treatment of a disease, the method comprising administering to a patient an effective amount of a composition comprising an antibody acccording to any of claims 20 to 24.

26. Use of an antibody according to any of claims 20 to 24 in the manufacture of a medicament for the treatment of a disease.

27. An antibody according to any of claims 20 to 24 for use in the treatment of a disease.

28. A composition comprising an antibody according to any of claims 20 to 24 for the treatment of a disease.

29. A method for diagnosing a disease, the method comprising:-

(i) contacting a sample obtained from a patient at risk of suffering from the disease with an antibody according to any of claims 20 to 24;

(ii) detecting the presence of an antigen in the sample bound to the antibody; wherein the presence of the antigen indicates that the patient is suffering from the disease or is at risk of suffering from the disease.

30. A method according to claim 29, wherein an increased amount of the antigen in the sample versus the amount of the antigen in a control sample indicates that the patient is suffering from or is at risk of suffering from the disease.

31. A method for monitoring the progression of a disease, the method comprising: -

(i) contacting a sample obtained from a patient suffering from the disease with an antibody according to any of claims 20 to 24;

32. A kit for the diagnosis of a disease, the kit comprising an antibody according to any of claims 20 to 24.

33. A method, use, antibody, composition or kit according to any of claims 25 to 32, wherein the disease is characterised by protein misfolding.

34. A method, use, antibody, composition of kit according to any of claims 25 to 33, wherein the disease is selected from prion diseases, Alzeihmer's Disease, Parkinson's Diseases and Diabetes amyloidosis.

35. A pharmaceutical composition comprising an antibody of any of claims 20 to 24.

36. A veterinary composition comprising an antibody of any of claims 20 to 24.