Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

• the present invention pertains to novel antibodies directed to the C-terminal part of the PrP Sc isoform of prions.
• the present invention pertains to the use of such antibodies in the diagnosis of bovine spongiform encephalopathy (BSE), the new variant form of Creutzfeld Jacobs Disease (vCJD), sporadic CJD and scrapie.
• BSE bovine spongiform encephalopathy
• vCJD Creutzfeld Jacobs Disease
• sporadic CJD sporadic CJD and scrapie.
• Bovine Spongiform Encephalopathy This disease had been termed Bovine Spongiform Encephalopathy (BSE) and is now widely referred to as "mad cow disease".
• BSE Bovine Spongiform Encephalopathy
• BSE is associated with a transmissible agent, the nature of which is not yet fully understood.
• the agent affects the brain and spinal cord of cattle and lesions are characterized by spongelike changes visible by means of a microscope. This agent is highly stable, and withstands heating to normal cooking temperatures and to even higher temperatures such as those used for pasteurization, sterilization at usual temperatures and time periods, as well as to freezing and drying.
• the BSE agent In cattle naturally infected with BSE, the BSE agent has been found only in brain tissue, in the spinal cord, and in the retina. Additional studies have identified BSE infectivity in the distal ileum, bone marrow, dorsal root ganglion, and trigeminal ganglion of calves that had been fed brain material from BSE-infected animals.
• CJD Creutzfeld- Jacobs disease
• EEG electroencephalographic
• mice inoculated with BSE showed the same pattern of incubation time, clinical signs and brain lesions as mice inoculated with tissues from patients with vCJD from which it was concluded that BSE and vCJD have the same signature or are the same "strain".
• mice One method presently available to determine the presence of the BSE agent in tissues is to inoculate animals, usually mice, with material believed to be infected with BSE. Mouse inoculation studies take, however, a long time of up to 700 days, and failure to identify it in tissues may indicate either true absence of the agent or simply the limited sensitivity of this approach.
• the agent responsible for the development of BSE and other TSE's, such as the vCJD, is smaller than the smallest known virus and has not been completely characterized yet. There are three main theories on the nature of the agent. (1) The agent is a virus with unusual characteristics, (2) the agent is an exclusively host-coded protein ("prion") that is modified to a partially protease-resistant form after infection, and (3) the agent is a small, non-coding regulatory nucleic acid coated with a host-derived protective protein.
• the BSE agent is extremely resistant to heat and to normal sterilization processes. It also does not evoke any detectable immune response or inflammatory reaction in host animals.
• the agent responsible for the disease is distinct from viruses.
• the agent can exist in multiple molecular forms, whereas viruses exist in a single form with distinct ultrastructural morphology.
• these agents are non- immunogenic, in contrast to viruses, which almost always provoke an immune response.
• prions are the cause for the degenerative disease, the only known component thereof is the prion, which is encoded by a chromosomal gene of the individual itself.
• Infectious prions are essentially composed of a protein designated as the "scarpie isoform" of the prion protein, abbreviated as PrP Sc .
• PrP Sc A post-translational process, as yet undefined, obviously generates PrP Sc from the ubiquitous cellular prion protein PrP c .
• PrP Sc and PrP c are encoded by a single copy chromosomal gene and it has been shown that the inoculated prion initiates the production of PrP Sc from the normal host PrP c polypeptide.
• the isoforms are accumulated within the cells in vesicles.
• the isoforms also differ in their conformational structure, exhibited by an increased ⁇ -sheet content which might be a cause for the increased protease resistance of the PrP Sc isoform versus the normal form PrP c .
• PrP Sc isoform which has prevented the manufacture of antibodies specifically recognizing the PrP Sc isoform, while simultaneously avoiding cross-reactivity with the "normal" isoform, PrP c .
• the problem of the present invention therefore resides in providing means, which enables a veterinary and/or physician to specifically detect the presence of agents causing BSE, CJD, vCJD and/or TSE related diseases.
• the present inventors have found that one of the conformational changes, the PrP c undergoes in its transformation to p r ps c j-gsideg j n a modification of the tertiary structure of the C-terminal region of PrP c .
• Fig. 1 shows a picture (10 tines enlarged) of a human brain tissue sample derived from an individuum subject to sporadic CJD treated with the antibody CNCM 1-2476.
• Fig. 2 shows a picture (60 times enlarged) of a human brain tissue sample derived from an individuum subject to sporadic CJD treated with the antibody CNCM 1-2476.
• Fig. 3 shows a picture (60 times enlarged) of a human brain tissue sample derived from an individuum subject to new variant CJD treated with the antibody CNCM 1-2476.
• Fig. 4 shows a picture (20 times enlarged) of a human brain tissue sample derived from an individuum subject to new variant CJD treated with the antibody CNCM 1-2476.
• Fig. 5 shows a picture (20 times enlarged) of a bovine brain tissue sample derived from an animal exhibiting signs of BSE treated with the antibody CNCM 1-2476.
• Fig. 6 shows a picture (10 times enlarged) of a brain tissue sample derived from a goat subject to scrapie treated with the antibody CNCM 1-2476.
• Fig. 7 shows the results of a Dot Blot experiment, wherein various samples of normal brain tissues are exposed to the antibody CNCM 1-2476.
• the peptide of Seq. ID. No. 2 and the conjugate of said peptide and KLH was used.
• the present invention provides for an antibody directed to the C-terminal part of the PrP ⁇ isoform or a part thereof, that is, to a three dimensional conformation of said part of the prion polypeptide exhibited in the "misfolded" form. Since the PrP c form does exhibit a three dimensional conformation in this part of the prion polypeptide obviously different from PrP Sc the present antibodies are capable to selectively bind to the PrP Sc isoform while not binding to the PrP c form. They are therefore capable to distinguish between those isoforms.
• the antibodies are preferably directed to the region comprised by amino acids no.
• the amino acid sequence is amino acid sequence
• Seq. ID No 1 shows a part of C-terminal region of the amino acid sequence of the bovine prion polypeptide that is from amino acid no. 180 to no. 219 as identified in the present invention.
• An antibody as defined here in the present invention shall be capable to differentiate between PrP Sc and PrP c by selectively binding to a three dimensional conformation provided by the C- terminal part of the PrP Sc isoform, which is not present in the PrP c form.
• the antibody may be polyclonal or monoclonal, while due to cross-reactivity reasons monoclonal antibodies are preferred.
• fragments of the antibodies are within the meaning of the present antibodies, in particular those fragment binding to the target, such as the F ab regions or parts thereof. Based on the respective need the skilled person will be able to design the corresponding fragments.
• antibodies is meant to also comprise chimeric antibodies, such as humanized antibodies, wherein the constant region is derived from a human source, while the variable region is derived from animal source, such as mice, so as to decrease a response of the treated individual to the agent.
• Bispecific antibodies are considered in the present invention as well.
• Bispecific antibodies are immuno globulins or fragments thereof wherein the two F ab fragments are directed to different targets each. Thus, one F ab fragment will be directed to the C-terminal part of PrP Sc while the other F ab fragment may be directed to any target, such as a target to used in an assay.
• the antibody may be linked to markers commonly used in the detection of targets, such as a radioactive label, a fluorescent label or a dye.
• the antibody is the antibody produced by the hybridoma cell line CNCM 1-2476, deposited according to the Budapest Treaty with the Pasteur Institute, Paris, France on May 10 th , 2000.
• the above antibodies may be used for the diagnosis and/or treatment of Bovine Spongiform Encephalopathy or Creutzfeld- Jacobs-Disease or variant form thereof or TSE related diseases in mammals, such as e.g. ungulates or humans, in that the antibody selectively binds to the PrP Sc isoform.
• This trait of being capable to use the present antibodies in different species, e.g. human, cow, sheep etc. is a novel and interesting feature of the present antibodies and alleviates the determination methods, such that only one antibody is requierd to investigate all respective individuals.
• a specimen is taken from the individual to be tested, such as a tissue (homogenisate or sections) or a body fluid, e.g. blood, saliva urine or cerebrospinal fluid, and is examined for the presence of the PrP Sc isoform, by contacting the specimen under suitable conditions with the antibody.
• tissue homogenisate or sections
• body fluid e.g. blood, saliva urine or cerebrospinal fluid
• the method for contacting the specimen with the antibody is not subject to any. particular restriction and will be dependent only on the means available and the specimen itself.
• the antibodies may be used e.g. in the methods of ELISA, Dot Blot, Western Blot, immunohistochemistry methods and others, all of which the skilled person is well aquainted with.
• kits which comprises at least one antibody according to the present invention.
• the kit will also comprise buffers, support material and marker agents adapted to the respective assays performed.
• the antibodies of the present invention may also be used in the therapy of prion associated diseases by administering to an affected individual an effective amount of an antibody of the present invention, optionally together with suitable excipients and carriers. Consequently, the present invention will also comprise a pharmaceutical composition containing at least one antibody according to the present invention. Moreover, the present invention also provides for a method for immunizing an individual against the PrP So isoform. In this respect peptides of the C-terminal part found to be different from the normal form (PrP c ) may be used for eliciting an immune response in an individidual, which peptides are preferably derived from the prion sequence amino acid no.
• amino acid sequence is amino acid sequence
• antibodies itself may be used to produce antiidiotypic antibodies, which may be used for immunization. It will be appreciated that for the immunization the skilled person will choose the appropriate time frame between the boosting steps and will select an proper adjuvant if deemed necessary.
• the present antibodies may also be used for raising anti-idiotype antibodies, i.e. antibodies that are directed to the binding region of the antibodies of the present invention.
• anti- idiotypic antibodies represent a mirror-image of the antibodies of the present invention and may be used for the prevention and therapy of the above mentioned diseases.
• the antiidiotypic antibodies are therefore also within the scope of the present invention and may be produced according to methods well known in the art, i.e. by immunizing animals with e.g. the hypervariable region of the antibodies of the present invention and producing polyclonal or monoclonal antibodies against it.
• the antibodies obtained will be selected according to the binding of the present antibodies to said anti-idiotypic antibodies.
• the method for obtaining the antibodies of the present invention comprises the step of immunizing an animal, such as e.g. mice or rabbit with an immunizing amount of a peptide comprising the amino acids of the C-terminal part of the prion polypeptide, preferably a peptide having the following a sequence :
• a peptide is used that had been derived from the above sequence by replacing the Gin residue at location no. 207 of the prion polypeptide by Glu to obtain the following amino acid sequence:
• This sequence has surprisingly proven to elicit an immune response sufficiently strong to be capable to easily raise antibodies, specifically against PrP Sc .
• the surprising result obtained with this peptide may reside in that the native Gin residue may form hydrogen bonds and may such cause insolubility of the peptide, which is not desired when being part of an epitope.
• Glu which is in the sequence of the human PrP, whereas Gin is at the same position in the primary structure of bovine PrP, seemingly does not form such links.
• any of the above peptides for the immunization step harbors the advantage that no knock out mice have to be prepared in before, such as when using the entire prion polypeptide.
• a conjugate of the peptide and a carrier is used, wherein the peptide is bound to the carrier via its N-terminus. It could be shown that this measure obviously provides for a better immune response as compared to binding the peptide to the carrier via the C-terminus.
• spleenocytes After immunizing animals with the antigen spleenocytes are isolated and fused with myeloma cells according to methods and techniques well known in the art. Selection for fused cells is performed using an appropriate medium, which does not support growth of unfused cells, such as the HAT medium. Fused myeloma cells growing in the selection medium are screened for the production of antibodies capable to bind to the peptide used for the immunization step.
• the present invention also pertains to hybridoma cell lines capable of producing the antibodies of the present invention and obtainable according to the above method.
• the lines obtained were found to grow extremely fast, with a doubling time period of about 8 hours. Also the secretion of antibodies was found to be very high as compared to normal hybridoma antibody secretion.
• the hybridoma is CNCM 1-2476 which has been deposited witlf ; the Institute Pasteur, Paris, France on May 10 th , 2000 according to the Budapest Treaty.
• this peptide obviously provide for a three dimensional conformation exhibited exclusively by the PrP Sc isoform and not from the PrP c form.
• this peptide may well be used for immunizing humans or animals, so that the immunized individual may produce an immune response against the "misfolded form" of the prion polypeptide and depleting it from the body in a natural way.
• said polypeptide may also be used for producing drugs against the above mentioned prion associated diseases.
• a 13-amino acid long peptide from the primary structure of the human PrP (human prion protein) has been chosen.
• the peptide is located close to the C-terminal part of the prion protein from amino acid 202 to 214.
• the peptide has been modified by exchanging the Gin residue at location 207 by a Glu residue to result in the following amino acid sequence
• This peptide was bound via its N-terminal part to the carrier protein KLH, keyhole limpet hemocyanin, which represents a novel feature of linking potentially immunogenic peptides to a carrier, since normally the peptide is linked via the C-terminus to the carrier.
• KLH keyhole limpet hemocyanin
• mice were immunized with 0.2 mg KLH-peptide conjugate (prepared according to example 1) per mouse, subcutaneously in Freund's complete adjuvant (CFA) (200 ⁇ l/mouse). After 14 days mice were again immunized with 0.1 mg of KLH-peptide per mouse in Freund's incomplete adjuvant (IF A), intraperitoneally (200 ⁇ l/mouse). After two weeks they were inoculated intraperitoneally with 0.2 mg of KLH-peptide per mouse intraperitoneally in Freund's incomplete adjuvant once more. Blood was taken from the tail vein 10 days after the last inoculation and the presence of antibodies against the conjugate was determined by indirect ELISA. Plates were coated with KLH, KLH-peptide and free peptide. The binding of mouse antibodies was detected by goat anti-mouse antibodies, conjugated with HRP. The immune response of all mice was surprisingly very high.
• CFA Freund's complete adjuvant
• mice On day 4 after the booster injection mice were sacrificed and their spleen was removed. Splenocytes were isolated and fused with mouse myeloma cells NS1 (in ratio 10 : 4) using 50% PEG for 3 minutes according to standard techniques (Liddel, J.E., Cryer, A., A practical guide to monoclonal antibodies. John Wiley&Sons, New York, 1991). Cells were washed and resuspended into 96-well microtiter plates in DMEM (Flow, UK), supplemented with 13% FCS (HyClone, USA) (in the following merely designated DMEM) and with feeder layer of mouse thymocytes. The next day HAT DMEM was added into all wells.
• DMEM Flow, UK
• FCS HyClone, USA
• Plates were washed three times with PBS/Tween20 and goat anti-mouse immunoglobulins, conjugated with HRP (Sigma, USA) were added into the wells at dilution 1 : 5000 in 1 % BSA. After two hours incubation at 37°C plates were washed with PBS Tween 20 and substrate was added to the wells (ABTS H2O2; Sigma, USA). Plates were read at 410 nm. All volumes were 50 ⁇ l.
• Hybridomas from positive wells were transferred into larger volumes (24-well plates) in HT DMEM medium.
• the presence of specific antibodies was determined by indirect ELISA again and selected lines were transferred into tissue culture flasks in DMEM. Finally, selected hybridomas were cloned twice by the limiting dilution method and frozen in liquid nitrogen.
• the lines selected were growing extremely fast in DMEM, i.e. without the need of HT, with a doubling time period of about 8 hours. Also the secretion of antibodies was found to be very high.
• One of the clones obtained was submitted to the Pasteur Institute and received the deposit no CNCM 1-2476.
• the reactivity of the monoclonal antibodies with PrP was tested using the supernatants of the stored clones.
• the monoclonal antibodies were tested by immuno-histochemistry on brain tissue from CJD patients, vCJD " patients and on brains of BSE positive cattle and scrapie infected brains of sheep. For control normal human, bovine and sheep brains were used. The screening was also done by Western blot and dot blot.
• biotinylated goat anti-mouse+rabbit was applied (Ventana, USA) and incubated for 20 minutes at 42°C, followed by an incubation with streptavidin HRP (Ventana) for 20 minutes at 42°C, DAB (diamino benzidine) with Cu for 4 minutes.
• Brain tissue (thalamus or medulla or spinal cord) was homogenized in 10% sucrose, 20 mM HEPES pH 7.5, 2 % sarcosyl and 5 mM EDTA with homogenizer (Potter) (5-times, 0.1 g in 1 ml). Homogenates were centrifuged for 45 minutes at +4°C and 14 000 rpm. The supernatant was frozen at -20°C, whereas the pellet was dissolved in 1 M NaOH. The protein concentration in samples was determined by measuring absorbance at 280 and 260 ran.
• the membranes were then blocked with 5 % milk and incubated with monoclonal antibodies (for CNCM 1-2476 40 ⁇ g/ml) in 1 % milk for 1-2 hours (gentle shaking). After washing of the membranes they were incubated with secondary antibodies, conjugated with HRP (goat anti-mouse, Sigma, USA), diluted 1 : 5000 in 1 % milk (1-2 hours, gentle shaking). Membranes were washed and immune reaction was detected using chemiluminescence kit (ECL, Amersham, USA).
• the monoclonal antibodies provided by the present invention show a high specificity for PrP Sc .
• recognition it was not required to use proteinase K digestion of PrP c prior to the screening method.

Landscapes

• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Medicinal Chemistry (AREA)
• Biomedical Technology (AREA)
• Neurosurgery (AREA)
• Biophysics (AREA)
• Molecular Biology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Biochemistry (AREA)
• Engineering & Computer Science (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Immunology (AREA)
• Genetics & Genomics (AREA)
• Neurology (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Public Health (AREA)
• Hospice & Palliative Care (AREA)
• General Chemical & Material Sciences (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Pharmacology & Pharmacy (AREA)
• Animal Behavior & Ethology (AREA)
• Psychiatry (AREA)
• Veterinary Medicine (AREA)
• Peptides Or Proteins (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Investigating Or Analysing Biological Materials (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Priority Applications (7)

Applications Claiming Priority (2)

Publications (2)

Family

ID=8168816

Family Applications (1)

Country Status (14)

Cited By (2)

Families Citing this family (3)

Family Cites Families (4)

• 2000
  • 2000-05-23 EP EP00111108A patent/EP1158003B2/en not_active Expired - Lifetime
  • 2000-05-23 ES ES00111108T patent/ES2253156T5/es not_active Expired - Lifetime
  • 2000-05-23 DE DE60024787T patent/DE60024787T3/de not_active Expired - Lifetime
  • 2000-05-23 DK DK00111108.7T patent/DK1158003T4/da active
  • 2000-05-23 AT AT00111108T patent/ATE312848T1/de active
• 2001
  • 2001-05-15 PL PL360139A patent/PL208292B1/pl unknown
  • 2001-05-15 CA CA2407905A patent/CA2407905C/en not_active Expired - Fee Related
  • 2001-05-15 WO PCT/IB2001/001666 patent/WO2001090191A2/en not_active Ceased
  • 2001-05-15 KR KR1020027015692A patent/KR100829447B1/ko not_active Expired - Fee Related
  • 2001-05-15 AU AU2001284355A patent/AU2001284355B2/en not_active Ceased
  • 2001-05-15 HU HU0302413A patent/HU229454B1/hu not_active IP Right Cessation
  • 2001-05-15 AU AU8435501A patent/AU8435501A/xx active Pending
  • 2001-05-15 CN CNB01809936XA patent/CN1250572C/zh not_active Expired - Fee Related
  • 2001-05-15 JP JP2001587002A patent/JP5042430B2/ja not_active Expired - Fee Related
  • 2001-05-23 AR ARP010102453A patent/AR029098A1/es active IP Right Grant

Also Published As

Similar Documents

Legal Events