WO2002034776A2 - Epitopes de pai-1 - Google Patents

Epitopes de pai-1 Download PDF

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Publication number
WO2002034776A2
WO2002034776A2 PCT/BE2001/000186 BE0100186W WO0234776A2 WO 2002034776 A2 WO2002034776 A2 WO 2002034776A2 BE 0100186 W BE0100186 W BE 0100186W WO 0234776 A2 WO0234776 A2 WO 0234776A2
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WIPO (PCT)
Prior art keywords
pai
antibody
peptide
fragment
protein
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PCT/BE2001/000186
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English (en)
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WO2002034776A3 (fr
Inventor
Paul Declerck
Jean-Marie Stassen
Ann-Pascal Bijnens
Ann Gils
Thu Hoa Ngo
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K.U.Leuven Research And Development
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Priority claimed from DE2000153251 external-priority patent/DE10053251A1/de
Priority claimed from DE2001110235 external-priority patent/DE10110235A1/de
Application filed by K.U.Leuven Research And Development filed Critical K.U.Leuven Research And Development
Priority to AU2002216847A priority Critical patent/AU2002216847A1/en
Publication of WO2002034776A2 publication Critical patent/WO2002034776A2/fr
Publication of WO2002034776A3 publication Critical patent/WO2002034776A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8121—Serpins
    • C07K14/8132—Plasminogen activator inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention belongs to the field of cardiovascular and thromboembolic diseases or cancer and related diseases.
  • the invention relates to proteins, peptides comprising specified epitopes of plasminogen activator inhibitor type-1 (PAI-1) and nucleic acids encoding said proteins or peptides.
  • PAI-1 plasminogen activator inhibitor type-1
  • the invention further relates to methods of screening for substances capable of inhibiting said PAI-1, substances identifiable with said method and pharmaceutical compositions comprising said substances.
  • PAI-1 plasminogen activator inhibitor type-1
  • PAI-1 converts spontaneously into an inactive, latent conformation that can be partially reactivated (31).
  • a non-inhibitory substrate conformation has been identified (15-17). Elucidation of the possible diversity of the binding regions of various inhibitory monoclonal antibodies may lead to an understanding of the various molecular mechanisms involved and will support the rational design of PAI-1 neutralizing drugs.
  • the invention belongs to the field of cardiovascular and thromboembolic diseases or cancer and related diseases.
  • the invention relates to proteins, peptides comprising specified epitopes of plasminogen activator inhibitor type-1 (PAI-1) and nucleic acids encoding said proteins or peptides.
  • PAI-1 plasminogen activator inhibitor type-1
  • the invention further relates to methods of screening for substances capable of inhibiting said PAI-1, substances identifiable with said method and pharmaceutical compositions comprising said substances.
  • the invention further relates to antibodies or fragments or derivatives thereof capable of inhibiting said PAI-1, said antibodies, fragments or derivatives thereof identifiable with said method and pharmaceutical compositions comprising said antibodies, fragments or derivatives.
  • Figure 1 Effect of monoclonal antibodies on the rate of displacement of PAB from tPA (A) and uPA (B) by free PAI-1 (solid line), and its complexes with MA-44E4 (squares), MA-42A2F6 (triangles) and MA-56A7C10 (reversed triangles). All reactions were carried out in 0.1 M Hepes/NaOH pH 7.4 buffer with 0.1 M NaCl at 25°C. Stopped flow traces show reactions of PAI-1 (1.3 ⁇ M) with 0.15 ⁇ M tPA, 400 ⁇ M PAB (A) and with 0.08 ⁇ M uPA, 100 ⁇ M PAB (B).
  • PAI- 1/antibody complexes were preformed by incubation of 1.6 ⁇ M PAI-1 with 1.25- 1.4 molar excess of antibody. PAI-1 /antibody complexes reacted with 1.1 ⁇ M tPA, 180 ⁇ M PAB (A) and with 0.22 ⁇ M uPA, 100 ⁇ M PAB (B).
  • Loop 1 (residues 185-200) and loop 2 (residues 242-246) are represented in green and the reactive site loop is represented in orange.
  • the residues of the epitope of MA-44E4 are indicated as yellow spheres and the residues of the epitope of MA- 42A2F6 and/or MA-56A7C10 as blue spheres.
  • the figures of PAI-1 in the active and latent conformation are generated by the Molscript programma, using the coordinates of active (17) and latent PAI-1 (19), respectively.
  • PAI-1 plasminogen activator inhibitor-1
  • the proteins or peptides according to the invention comprise human/mammalian or preferably human/rat PAI-1 chimeric proteins as exemplified in example 1.
  • the proteins or peptides according to the invention preferably are of mammalian origin, such as rodents (e.g. rats, hamster, mice) or cattle, horse, pig and most preferably human.1
  • peptide shorter than the indicated amino acid length e.g. from amino acids 1 to 16.
  • functional derivative as used herein is a peptide or protein with a modification of e.g. a point mutation, such as amino acid deletion, insertion or replacement resulting in essentially unaltered immunological properties such as antibody binding.
  • the invention further relates to a protein or peptide comprising the epitope of PAI-1 characterized by the amino acids 185-187 of PAI-1: His Arg Arg or a fragment or a functional derivative thereof.
  • the invention further relates to a protein or peptide comprising the epitope of PAI-1 characterized by the amino acids 242-244 of PAI-1
  • Glu Lys Glu or a fragment or a functional derivative thereof.
  • the invention further relates to a protein or peptide comprising the epitope of PAI-1 characterized by the amino acids 350-356 of PAI-1
  • the invention further relates to a protein or peptide comprising the epitope of PAI-1 characterized by the amino acids 350, 355, and 356 of PAI-1
  • the invention further relates to a peptide according to the invention, characterized in that it consists of the amino acids His Arg Arg (amino acids 185 to 187 of PAI-1).
  • the invention further relates to a peptide according to the invention, characterized in that it consists of the amino acids Glu Lys Glu (amino acids 242-244 of PAT).
  • the invention further relates to a peptide according to the invention, characterized in that it consists of the amino acids Glu Glu He He Met Asp Arg (amino acids 350-356 of PAI-1).
  • the invention further relates to a peptide according to the invention, characterized in that it consists of the amino acids Glu Asp Arg (amino acids 350, 355, and 356 of PAI-1).
  • the invention further relates to a protein or peptide comprising two or more of said epitopes of PAI-1 as described supra or a functional derivative thereof.
  • Said protein or peptide may comprise said two or more epitopes of PAI-1 in a way that they are consecutively linked together or may comprise a spacer which may be a different protein or peptide or a spacer which is a chemical molecule different from a protein.
  • Human PAI-1 itself comprises or consists of the amino acid sequence Nal His His Pro Pro Ser Tyr Nal Ala His Leu Ala Ser Asp Phe Gly Nal Arg Nal Phe Gin Gin Nal Ala Gin Ala Ser Lys Asp Arg Asn Nal Nal Phe Ser Pro Tyr Gly Nal Ala Ser Val Leu Ala Met Leu Gin Leu Thr Thr Gly Gly Glu Thr Gin Gin Gin He Gin Ala Ala Met Gly Phe Lys He Asp Asp Lys Gly Met Ala Pro Ala Leu Arg His Leu Tyr Lys Glu Leu Met Gly Pro Trp Asn Lys Asp Glu He Ser Thr Thr Asp Ala He Phe Nal Gin Arg Asp Leu Lys Leu Nal Gin Gly Phe Met Pro His Phe Phe Arg Leu Phe Arg Ser Thr Nal Lys Gin Nal Asp Phe Ser Glu Nal Glu Arg Ala Arg Phe He He He He He He He He He He He He He He He
  • nucleic acid encoding any one of the proteins or peptides according to the invention as described supra.
  • a suitable cell or cell line preferably an eukaryotic cell or a cell line, to be transformed with nucleic acid constructs to express said peptide or protein according to the invention may be any cell or cell line known to the expert in the field.
  • Examples of such cells or cell lines useful for producing the transformed cell lines of the invention include mammalian cells or cell lines (e.g.
  • HEK human embryonic kidney
  • BHK BHK
  • CHO Ltk "
  • f ⁇ broblasts myelomas
  • neuroblastomas hybridomas
  • oocytes embryonic stem cells
  • insect cell lines e.g., using baculovirus vectors such as pPbac or pMbac (Stratagene, La Jolla, USA)
  • yeast e.g., Pichia pastoris or using yeast expression vectors such as pYESHIS (Invitrogen, San Diego, USA)
  • fungi fungi.
  • Another important embodiment of the invention is a method of screening for substances capable of inhibiting PAI-1 characterized in that a) a peptide or a protein according to the invention as described supra or entire PAI-1 is incubated with a test compound in a suited buffer b) tPA or a fragment thereof and a monoclonal antibody specific for the peptide or protein as described supra or PAI-1 and a monoclonal antibody specific for tPA are detectably labelled with a reporter c) unbound antibodies are washed out d) the amount of reporter is measured and e) the value obtained is compared to the value obtained in the absence of the test compound.
  • Said method is exemplified in a non-limiting manner in example 2.
  • reporter genes encoding reporters to be used in a method according to the invention include, but are not limited to E. coli ⁇ -galactosidase ( ⁇ -gal, Luban and Goff, 1995; Curr Opin Biotechnol 6, 59-64), xanthine-guanine phosphoribosyl transferase (Chu and Berg, 1985; Nucleic Acids Res 13, 2921-2930), galactokinase (Schumperli et al., 1982; Proc Natl Acad Sci USA 79, 257-261), interleukin-2 (Cullen, 1986; Cell 46, 973-982), thymidine kinase (Searle et al., 1985; Mol Cell Biol 5, 1480-1489), alkaline phosphatase (Toh et al., 1989; Eur J Biochem 182, 231-237; Henthorn et al., 1988; Proc Natl Acad Sci USA 85, 6342
  • reporter genes such as reporter enzymes
  • bioassays can be carried out for biologically active proteins such as interleukin-2.
  • Enzyme assays can be performed when the reporter gene product is a reporter enzyme such as alkaline phosphatase or ⁇ -galactosidase.
  • various types of immunoassays such as competitive immunoassays, direct immunoassays and indirect immunoassays may be used.
  • Such immunoassays involve the formation of immune complexes containing the reporter gene product and a measurable "reporter" or a "label".
  • reporter includes moieties that can be detected directly, such as fluorochromes and radiolabels, and moieties such as enzymes that must be reacted or derivatized to be detected.
  • Another more preferred embodiment of the invention is a method according to the invention, wherein the peptide or protein according to the invention as described supra or entire PAI-1 is labelled in such a way or with a suitable label to differentiate binding of said peptide or protein or PAI-1 to tPA from non-binding or reduced binding.
  • Another more preferred embodiment of the invention is a method according to the invention, wherein said antibody specific for said peptide or protein according to the invention (described supra) or entire PAI-1 is labelled with Europium kryptate (see example 2).
  • Another important embodiment of the invention is a method according to the invention, wherein said antibody specific for tPA or a fragment thereof is labelled with XL665 (see example 2).
  • Another more preferred embodiment of the invention is a method according to the invention, wherein said method is a high throughput screening (HTS) or a ultra high throughput screening (UHTS).
  • HTS relates to an experimental setup wherein a large number of compounds is tested simultaneously.
  • said HTS setup may be carried out in microplates, may be partially or fully automated and may be linked to electronic devices such as computers for data storage, analysis, and interpretation using bioinformatics.
  • said automation may involve robots capable of handling large numbers of microplates and capable of carrying out several thousand tests per day.
  • a test compound which shows a desired inhibitory function in a cell- free system will also be tested in a cell-based system using a cell line expressing a protein or peptide according to the present invention.
  • the term HTS also comprises ultra high throughput screening formats (UHTS).
  • said UHTS formats may be carried out using 384- or 1536-well microplates, sub-microliter or sub-nanoliter pipettors, improved plate readers and procedures to deal with evaporation.
  • HTS methods are described e.g. in US 5876946 A or US 5902732 A.
  • the expert in the field can adapt the method described below to a HTS or UHTS format without the need of carrying out an inventive step.
  • Another preferred aspect of the present invention relates to the use of a protein or peptide according to the invention as described supra for the identification or design of an inhibitor of PAI-1.
  • Another preferred aspect of the present invention relates to the use of an antibody specific for a protein or peptide according to the invention or specific to a different epitope of PAI-1 in a method according to the invention.
  • Such antibodies to be used in a method according to the invention are disclosed as non-limiting examples in example 1 and 2.
  • These PAI-1 - specific antibody proteins according to the invention consist of the variable regions of both chains which are held together by the adjacent constant region. These may be formed by protease digestion, e.g. with papain, from conventional antibodies, but similar Fab fragments may also be produced in the mean time by genetic engineering.
  • Fv fragments fragment of the variable part
  • an PAI-1 -specific antibody molecule according to the invention is such an Fv fragment. Since these Fv-fragments lack the covalent bonding of the two chains by the cysteines of the constant chains, the Fv fragments are often stabilised.
  • variable regions of the heavy and of the light chain by a short peptide fragment, e.g. of 10 to 30 amino acids, preferably 15 amino acids.
  • a single peptide strand is obtained consisting of NH and NL, linked by a peptide linker.
  • An antibody protein of this kind is known as a single-chain-Fv (scFv).
  • scFv-antibody proteins of this kind known from the prior art are described in Huston et al. (1988, P ⁇ AS 16: 5879-5883). Therefore, in another preferred embodiment the method according to the invention is characterised in that the human or humanized antibody protein is a single-chain-Fv protein (scFv).
  • scFv as a multimeric derivative. This is intended to lead, in particular, to recombinant antibodies with improved pharmacokinetic and biodistribution properties as well as with increased binding avidity.
  • scFv were prepared as fusion proteins with multimerisation domains.
  • the multimerisation domains may be, e.g. the CH3 region of an IgG or coiled coil structure (helix structures) such as Leucin-zipper domains.
  • helix structures such as Leucin-zipper domains.
  • the interaction between the NH/NL regions of the scFv are used for the multimerisation (e.g. di-, tri- and pentabodies).
  • the method according to the invention is characterised in that the human or humanized antibody protein is an PAI-1 -specific diabody antibody fragment.
  • diabody the skilled person means a bivalent homodimeric scFv derivative (Hu et al, 1996, P ⁇ AS 16: 5879-5883).
  • the shortening of the Linker in an scFv molecule to 5- 10 amino acids leads to the formation of homodimers in which an inter-chain NH/VL-superimposition takes place.
  • Diabodies may additionally be stabilised by the incorporation of disulphide bridges. Examples of diabody-antibody proteins from the prior art can be found in Perisic et al. (1994, Structure 2: 1217-1226).
  • minibody means a bivalent, homodimeric scFv derivative. It consists of a fusion protein which contains the CH3 region of an immunoglobulin, preferably IgG, most preferably IgGl as the dimerisation region which is connected to the scFv via a Hinge region (e.g. also from IgGl) and a Linker region.
  • Hinge region e.g. also from IgGl
  • Linker region e.g. also from IgGl
  • the disulphide bridges in the Hinge region are mostly formed in higher cells and not in prokaryotes.
  • the method according to the invention is characterised in that the human or humanized antibody protein is a PAI-1 -specific minibody antibody fragment. Examples of minibody-antibody proteins from the prior art can be found in Hu et al. (1996, Cancer Res.
  • triabody By triabody the skilled person means a: trivalent homotrimeric scFv derivative (Kortt et al. 1997 Protein Engineering 10: 423-433). ScFv derivatives wherein NH-NL are fused directly without a linker sequence lead to the formation of trimers.
  • miniantibodies which have a bi-, tri- or tetravalent structure and are derived from scFv.
  • the multimerisation is carried out by di-, tri- or tetrameric coiled coil structures (Pack et al., 1993 Biotechnology 11:, 1271-1277; Lovejoy et al. 1993 Science 259: 1288-1293; Pack et al, 1995 J. Mol. Biol. 246: 28-34).
  • Yet another important embodiment is a substance identifiable with a method according to the invention characterized in that it is capable of specifically inhibiting PAI-1.
  • Said substance can be an antagonist or modulator of PAI-1.
  • said substance is an antibody or a functional fragment or derivative thereof.
  • Another important embodiment is the use of a substance, i.e. an antagonist or a modulator of PAI-1 according to the invention for the manufacture of a medicament in the treatment of thromboembolic and cardiovascular diseases, preferably stroke, or cancer and related diseases.
  • diseases include, but are not limited to global and focal ischaemia, ischaemic and haemorrhagic stroke, global cerebral ischaemia with heart stoppage, post-ischaemic neurocytotoxic conditions, hypoglycaemia, diabetic polyneuropathy, hypoxia, anoxia, brain edema, brain pressure (elevated intracranial pressure), hypertonia, hypotonia, cardiac infarction, breast cancer, prostate cancer, bladder cancer, liver cancer and cancer of the gastrointestinal tract.
  • diseases include, but are not limited to global and focal ischaemia, ischaemic and haemorrhagic stroke, global cerebral ischaemia with heart stoppage, post-ischaemic neurocytotoxic conditions, hypoglycaemia, diabetic polyneuropathy, hypoxia, anoxia
  • the invention also relates to a pharmaceutical composition characterized in that it comprises a substance according to the invention and optionally pharmaceutically acceptable carriers or excipients.
  • a pharmaceutically acceptable carrier can contain physiologically acceptable compounds that act, for example, to stabilize or to increase the absorption of a PAI-1 antagonist or modulator.
  • physiologically acceptable compounds include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients (see also e.g. Remington's Pharmaceutical Sciences (1990), 18th ed. Mack Publ., Easton.).
  • a pharmaceutically acceptable carrier including a physiologically acceptable compound, depends, for example, on the route of administration of the composition.
  • PfuTwboTM DNA polymerase was purchased from Stratagene (La Jolla, CA). Synthetic oligonucleotides (for PCR and DNA sequencing) were synthesized by Amersham Pharmacia Biotech (Uppsala, Sweden).
  • the vectors pIGE20-humanPAI-l and pIGE20-PAl-l-stab were constructed as described before (26;27).
  • Luria Broth (LB) growth medium was purchased from Life Technologies (Ghent, Belgium). Tissue-type plasminogen activator (Actilyse ® ) was a kind gift from Boehringer Ingelheim (Brussels, Belgium). Recombinant human PAI-1, porcine PAI-1, rat PAI-1, murine PAI-1 and PAI-1-stab were expressed in Escherichia coli and were produced essentially as described (26- 29). Most chemical reagents including dithiotreitol and the proteinase inhibitors leupeptin, phenylmethanesulfonylfluoride, pepstatin, benzamidine hydrochloride and antipain were from Sigma (St.Louis,USA).
  • SP Sepharose ® Fast Flow and Heparin- Sepharose ® CL 6B were purchased from Amersham Pharmacia Biotech (Uppsala, Sweden).
  • DNA manipulation techniques were carried out according to standard procedures and following the instructions of the manufacturers. Plasmid DNA was isolated using Nucleobond ® cartridges (Machery-Nagel, D ⁇ ren, Germany), DNA fragments were purified using the QIAquick ® Gel Extraction kit (QIAGEN GmbH, Hilden, Germany) and PCR was performed using the Gene Amp ® 2400 (Perkin-Elmer). DNA was sequenced with the Autoread Sequencing ® kit and the Automated Laser Fluorescent ALF ® apparatus (both from Amersham Pharmacia Biotech).
  • mutants were created using a method based on the QuickChangeTM site-directed mutagenesis kit from Stratagene (La Jolla, CA). Therefore, pIGE20-PAI-l-wt (26) and pIGE20-PAI-l-stab (27) were used as template to introduce mutations in PAI-1 -wt and in PAI-1 -stab, respectively.
  • PCR was performed using 2.5 units of PfuTurboTM DNA polymerase, 50 ng of template, 125 ng of each primer, 0.2 nmol of each dNTP in 50 ⁇ l buffer containing 10 mM KC1, 10 mM(NH 4 ) 2 SO 4 , 20 mM Tris-HCl (pH 8.8), 2 mM MgSO 4 , 0.1% Triton X-100 and 100 ⁇ g/ml nuclease-free bovine serum albumin. After an initial DNA denaturation step (95 °C, 30 s), 16 PCR cycles were performed (95 °C, 30 s; 55 °C, 60s; 68 °C, 12 min). Subsequently, DNA was subjected to a Dpnl digestion prior to transformation of DHl ⁇ Escherichia coli. For all mutants, large-scale DNA preparations were made and the PAI-1 encoding region was sequenced entirely.
  • PAI-1 -wt and PAI-1 mutants were expressed in MCI 061 Escherichia coli cells using a method described before (26).
  • competent MC1061 cells were cotransformed with pAcI and either pIGE20-PAI-l-wt or one of the pIGE20-PAI-l mutant constructs.
  • Clonal isolates were grown and PAI-1 expression was induced by increasing the temperature to 42 °C. After 3 h cells were harvested and disrupted by pressure. The cell lysate was cleared by ultracentrifugation and the PAI-1 containing supernatant was collected and immediately subjected to purification as described previously (30).
  • PAI-1 containing elution fractions were examined for their inhibitory activity towards t-PA by SDS-PAGE. Fractions containing the highest proportion of functionally active PAI-1 were pooled and used for further experiments.
  • Quantitation of the formed reaction products was done by densitometric scanning of the gels using the Imagemaster ® system (Amersham Pharmacia Biotech (Uppsala, Sweden) (26).
  • PAI-1 activity was quantitated (31) after pre-incubation of human PAI-1 with monoclonal antibody. Briefly, 100 ⁇ l PAI-1 (50 ng/ml with an activity of 80% of the theoretical maximal value) was incubated with 100 ⁇ l of serial two-fold dilutions of the antibodies (MA-
  • PAI-1 PAI-1.
  • the reaction mixture was allowed to react two hours at room temperature. 50 ⁇ l samples of this reaction mixture were incubated with 50 ⁇ l of t-PA (20 lU/ml) at 37°C for 15 min in wells of a microtiter plate. Then, 100 ⁇ l of a solution containing plasminogen (1 ⁇ M), CNBr- digested fibrinogeh (1 ⁇ M) and S-2403 (0.6 mM) was added. The residual PA activity was measured by recording the absorbance change at 405 nm. 100 % PAI-1 activity was defined as the PAI-1 activity observed in the absence of monoclonal antibody. The percentage inhibition by the monoclonal antibody was then calculated from the residual PAI-1 activity measured in the presence of the monoclonal antibody.
  • Quantitation of the formed reaction products was done by densitometric scanning of the gels using the Imagemaster ® system (Amersham Pharmacia Biotech (Uppsala, Sweden) (26).
  • Affinity constants for the binding between monoclonal antibodies and PAI-l-wt and PAI-1 mutants were determined using the BIAcoreTM3000 analytical system equipped with the CM5 sensor chip (BIAcore AB) as described previously (32).
  • the monoclonal antibodies were coupled covalently to 2000 resonance units (using a concentration of 10 ⁇ g/ml in 10 mM acetate buffer, pH 4.5) using the automatic Wizard mode.
  • PAI-1 variants diluted in 0.01 M Hepes, 0.15 M NaCl, 3 mM EDTA, 0,005 % Tween 20, pH 7.4 to a final PAI-1 antigen concentration ranging from 18 to 1200 nM were injected at a flow rate of 20 ⁇ l/min (injection volume is 40 ⁇ l).
  • injection volume is 40 ⁇ l.
  • the chip was regenerated using 10 ⁇ l of a 15 mM HC1 solution.
  • the analyses of the association and dissociation phases were made with the software of the BIAcoreTM 3000 (Langmuir binding, local or global fit).
  • PAI-l-wt Isolation of the latent and substrate conformation of PAI-l-wt was performed as described previously (14). In brief, active PAI-l-wt was converted into the latent form by incubation at 37 °C for 24 h. To separate the latent from the substrate conformation, the inactivated sample was applied to immobilized 1-PA-S478A: the non-binding fraction (i.e. the latent conformation) was collected and bound PAI-1 (i.e. the substrate conformation) was eluted using a 0.1 M acetate buffer containing 2 mM gluthation and 1.5 M NaCl, pH 5.5.
  • PAB p-aminobenzamidine
  • tPA a fluorescent probe inhibitor of serine proteinases
  • PAI-1 (1.6 ⁇ M) or its complex with each monoclonal antibody, when reacted with the PAB/proteinase complex, causes a decrease in fluorescence emission of the PAB due to its displacement from the active site of the enzyme (34).
  • the reactions were carried out using an SX-18MN microvolume stopped-flow reaction analyzer (Applied Photophysics Ltd) equipped with a fluorescence detector or an SLM 8000 spectrofluorimeter for slower reactions. Excitation wavelengths were 320 and 330 mm for uPA and tPA, respectively.
  • MA-44E4, MA-42A2F6 and MA-56A7C10 inhibited PAI-1 activity in a dose-dependent manner revealing that MA-56A7C10 is the most potent inhibitor (i.e. 80 ⁇ 5 %> inhibition using a two-fold molar excess of MA vs 21 ⁇ 6 % and 15 ⁇ 6 % for MA- 42A2F6 and MA-44E4, respectively).
  • PAI-1 inhibition exceeding 85 % is observed for all three antibodies.
  • MA-44E4 binds human PAI-1 with a K A of 4 x 10 8 M "1 , but lacks affinity (i.e. K A at least 400- fold less) for porcine, murine and rat PAI-1 (Table II, A).
  • the K A values of MA-42A2F6 and MA-56A7C10 for human PAI-1 are 3.8 x 10 8 M _1 and 1.3 x 10 9 M "1 , respectively (Table II, B).
  • the affinity constants of both antibodies for porcine PAI-1 are reduced 6- and 9-fold, respectively, and no binding to murine or rat PAI-1 was observed.
  • Glu 350 In the stretch of residues in human PAI-1 C-terminal from position 327 (24), only one charged amino acid in human PAI-1, Glu 350 , differs from the corresponding residues in murine and rat PAI-1, whereas it is conserved in porcine PAI-1. Therefore, we hypothesized that Glu is involved in the interaction of PAI-1 with MA-42A2F6 and MA-56A7C10.
  • PAI-1-H185A His 185 to Ala
  • PAI-1-R187A Arg 187 to Ala
  • PAI-1-H190A His 190 to Ala
  • PAI-1-K191A Lys 191 to Ala
  • PAI-1-E242A Lys 242 to Ala
  • PAI-1-K243A Lys 243 to Ala
  • PAI-1-E244A Glu 244 to Ala
  • PAI-1-D355A Asp 355 to Ala and PAI-1-R356A, Arg 356 to Ala
  • PAI-1-H185A-R186A PAI-1- R186A-R187A
  • PAI-1-H190A-K191A PAI-1-D355A-R356A
  • two triple mutants PAI-1 - H185A-R186A-R187A
  • PAI-1 -E242A-K243A-E244A PAI-1 -E242A-K243A-E244A
  • PAI-1 - E242A-K243A-E244A-E350A quadruple mutant
  • MA-44E4 reacts with active, latent and substrate conformations of human PAI-1 with comparable affinity (Table III).
  • affinity constants indicate that both antibodies react preferentially with PAI-1 in the active conformation.
  • the affinity of the latter antibodies for the substrate conformation is reduced 9- and 5-fold, respectively and the affinity for the latent form is decreased 56- and 25-fold, respectively.
  • the affinity constants for the stable variant is similar to that for human PAI-1. Characterization of the PAI-1 mutants
  • Table IN summarizes the conformational distribution of all PAI-1 mutants studied, in comparison with PAI-l-wt.
  • inhibitory activity towards t-PA and the ratios between the various conformations were similar as for PAI-l-wt, i.e. 68 % to 85% occurred in the active form, 9.4 % to 22 % in the latent form and 3.0 % to 12 % in the substrate conformation.
  • alanine mutations at positions 187, 190, 191, 193, 355 and 356 caused a reduction in PAI-1 activity ( ⁇ 52 %), together with an increase of non-reactive PAI-1 (latency > 41 %).
  • MA-44E4 exhibited an affinity of 4 x 10 8 M "1 towards PAI-l-wt.
  • the affinity for PAI-1-D181 A (Table N) was nearly identical to that for PAI-l-wt, indicating that this residue is not involved in the interaction with MA-44E4.
  • MA-44E4 did not bind to PAI-1-H185A-R186A- R187A (p ⁇ 0.0001 vs PAI-l-wt, Table N).
  • the data in Table N demonstrate that the affinity of MA-44E4 is completely lost by any combination of two adjacent alanine mutations between residues 185 and 187 (p ⁇ 0.0001 vs PAI-l-wt).
  • the affinities of MA-42A2F6 and MA-56A7C10 for PAI-l-wt were 4 x 10 8 M "1 and 1 x 10 9 M " l . From the data shown in Table NI it can be deduced that the affinity of MA-42A2F6 and MA- 56A7C10 for PAI-1-E350A and PAI-1-E242A-K243A-E244A is 10-fold reduced (p ⁇ 0.005 vs PAI-l-wt), indicating that these residues contribute to the epitope of both antibodies.
  • the affinity of MA-42A2F6 and MA-56A7C10 for the mutants PAI-1-H185A-R186A-R187A, PAI- 1-H190A-K191A, PAI-1-D193A and PAI-1-D355A-R356A is at least 3-fold reduced compared to that of PAI-l-wt.
  • the affinity data for these mutants can not be unambiguously interpreted because of their increased latency.
  • the data further confirm the possible role of residue Glu 350 (p ⁇ 0.0001 vs PAI-1- stab). Because of the latency of PAI-l-stab-H190A-K191A and PAI-l-stab-D355A-R356A, the ⁇ . ⁇ . 11 ⁇ ⁇ -- _ _ • -- --j LI ⁇ -• -j i r ⁇ - _190 T __-.191 i .-355 , .- j /- _- antibodies was determined for a set of PAI-1 variants in which a single residue was substituted with alanine.
  • the proposed epitope localization indicates that monoclonal antibody binding might impair the PAI-1 /proteinase complex formation through interference with the residues involved in the formation of the initial, reversible complex.
  • the effect of the monoclonal antibodies on the rate of PAI-1/proteinase complex formation was studied.
  • the dependencies of k obs on PAI-1 concentration were linear for both proteinases (0-2.5 ⁇ M PAI-1), and the values of ki for the reactions with tPA and uPA (4.5 ⁇ 0.3 and 1.9 ⁇ 0.3 xlO 7 M ' V 1 , respectively) indicate that the initial complex formation is diffusion limited.
  • k 0 b S for the displacement of PAB by PAI-1 was 35 ⁇ 2 and 2.2 ⁇ 0.4 s "1 for tPA and uPA, respectively (Fig. 1).
  • the presence of monoclonal antibodies induced a significant decrease in the rate of the PAB displacement (Fig. 1).
  • the values of k 0bs were decreased by 500-900 and 4,500- 6,500 fold with MA-44E4 and MA-42A2F6 for uPA and tPA, respectively, and the reaction was completely prevented (k 0bS ⁇ 10 "4 s "1 ) by MA-56A7C10.
  • the considerable reduction of the rate constant for displacement of PAB is due to a considerable decrease in the reactivity of proteinases to PAI-1 /Mab complexes compared with free PAI-1.
  • the epitope of CB5B10 was localized in ⁇ -helix E and the turn connecting helix E and strand si A (52).
  • Epitope mapping of the inhibitory anti-rat PAI-1 MA-124K1 revealed the major contribution of residues Glu 212 and Glu 220 localized on strand slB and s2B (53).
  • loop 1 might be a target to modulate PAI-1 activity (22).
  • the current localization of the epitope for MA-44E4 provides the first proof that targeting this particular region results in the inactivation of PAI-1. The latter hypothesis is therefore likely to form the molecular basis for the inhibitory effect of MA-44E4.
  • MA-56A7C10 virtually completely blocks PAB displacement from the proteinase active site providing convincing evidence for the hypothesized molecular mechanism of inactivation in combination with the particular epitope localization.
  • MA-42A2F6 has a considerable effect but not as pronounced as MA-56A7C10. This difference could be due either to the lower affinity ( ⁇ 3.4 fold) or to differences in the relative contribution of residues 242-244 and the residues in the distal hinge region.
  • the observed effect of MA-44E4 is most likely due to an, indirect, steric hindrance.
  • the 384- well plates were purchased from Canberra Packard, Dreieich, Germany (Optiplate for HTRF; Cat.-No. 6005256).
  • the anti-PAI-1 monoclonal antibody MA-45C12, the anti-tPA monoclonal antibody MA-51H8 and the human wild-type PAI-1 (2.35 mg/ml; 60% activity; 1.1 Mio U/ml) were purchased from Prof. Declerck, University of Leuven.
  • the tPA (580 000 U/mg protein; 1 mg/ml) was from the Department of Biopharmaceutical Production, however, is also commercially available. All other materials were of highest grade commercially available.
  • the labeling of MA-45C12 with XL665 at a molar ratio of approx. 1.5 XL/Ab and the labeling of MA-51H8 with europium kryptate at a molar ratio of approx. 5-6 K/Ab were done at CIS bio international (Bagnol sur Ceze, France).
  • test compound in assay buffer final concentration 5 ⁇ g/ml; 1 % DMSO
  • PAI-1 3 U/well
  • HSA 0.025% HSA
  • HSA 0.01 %> 10 ⁇ l PAI-1 (3 U/well) in assay buffer supplemented with 0.025% HSA; final concentration of HSA 0.01 %>
  • the plates are then incubated at room temperature for 30 minutes. After the incubation period 20 ⁇ l detection mix for active PAI-1 are added and the plates incubated for another 30 minutes. After this second incubation period 25 ⁇ l KF (1400 mM) are added to each well.
  • the time-resolved fluorescence of the wells is then measured in a Packard Discovery fluorescence reader (Ex 335 nm; Em 620 nm and 665 nm) (Canberra Packard, Dreieich, Germany). The signal is stable for at least 10 hours. The ratio of the two emission signals is then calculated (Em665*10.000/Em620).
  • the long-living signal at 620 nm is derived from the europium kryptate label.
  • the long-living signal at 665 nm is derived from XL665 label and the fluorescence resonance energy transfer (FRET) which happens when both labels are in close proximity. FRET happens only when active PAI-1 binds to tPA which is present in the detection mix.
  • Assay buffer 50 mM Tris, 100 mM NaCl, 0.01% Tween 80, adjusted to pH 7.5 with HC1 Detection mix for active PAI-1 :
  • Each assay microtiter plate contains wells with vehicle controls (f. c. 1%- DMSO in assay buffer) as reference for the total active PAI-1 (100% CTL; high values) and wells with vehicle controls and assay buffer/HSA instead of PAI-1 as controls for non-active PAI-1 (0% CTL; low values).
  • vehicle controls f. c. 1%- DMSO in assay buffer
  • SAA vehicle controls and assay buffer/HSA instead of PAI-1 as controls for non-active PAI-1 (0% CTL; low values).
  • Each assay microtiter plate contains also wells with 10 ⁇ M BIBT2809BS as reference inhibition and should result in a value of about 30 % CTL.
  • the analysis of the data is performed by the calculation of the percentage of the ratio 665/620 in the presence of the test compound compared to the ratio 665/620 of the high control values: (ratio(sample)-ratio(low))* 100/(ratio(high)-ratio(low)) .
  • An inhibitor of the target protein will give values between 100 % CTL (no inhibition) and 0% CTL (complete inhibition). Values of more than 100 % CTL are normally related to compound- specific physico-chemical properties (e.g. solubility, absorption, fluorescence) or indirect biochemical effects such as allosteric regulation.
  • PAI-1 K A (1/M) Sequence alignment between AA and AA in PAI-1 (1)
  • Murine NB E MGPVr ⁇ K-lp-ISTMAIFVQRDLE VQGFMPHFFK FfiTMVKQVDFS- ⁇ t
  • the amino acids are represented using one letter codes.
  • the charged amino acids in human PAI-1 are underlined.
  • the residues in porcine, rat and murine PAI-1 that differ from corresponding position in human PAI-1 are underlined. Charged residues hypothesized to explain the differential species reactivity are indicated.
  • PAI-1 Active PAI-1 4.2 ⁇ 1.210 s 3.8 ⁇ 0.9x10" 1.3 ⁇ 0.510 y Latent PAI-1 1.3 ⁇ 0.110 8 6.8 ⁇ 1.610 6 5.2 ⁇ 0.210 7 Substrate PAI-1 1.6 + 0.110 8 4.4 ⁇ 1.510 7 2J ⁇ 1.410 8 PAI-1 -stab 5.7 + 0.110 8 3.9 ⁇ 0.110 8 6.2 ⁇ 0.110 8
  • PAI-1 -variant MA-42A2F6 MA-56A7C10

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Abstract

Cette invention concerne le domaine des maladies cardiovasculaires et thromboemboliques et des cancers et maladies associée. L'invention concerne des protéines, des peptides comportant des épitopes spécifiés de l'inhibiteur de type 1 de l'activateur du plasminogène (PAI-1), et des acides nucléiques codant ces protéines ou peptides. L'invention traite également de méthodes de criblage de substances pouvant inhiber ledit PAI-1, de substances pouvant être identifiées au moyen de ladite méthode et de compositions pharmaceutiques comportant lesdites substances.
PCT/BE2001/000186 2000-10-26 2001-10-25 Epitopes de pai-1 WO2002034776A2 (fr)

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Cited By (9)

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WO2005094863A1 (fr) * 2004-03-30 2005-10-13 Den Kgl.Veterinær-Og Landbohøjskole Ameliorations dans le traitement du cancer et prediction efficace de traitement du cancer par le blocage et la detection d'inhibiteurs de la protease
WO2005094810A2 (fr) * 2004-03-05 2005-10-13 Karo Bio Ab Nouvelles compositions pharmaceutiques
US7374886B2 (en) 1999-04-09 2008-05-20 Rigshospitalet Tissue inhibitor of matrix metalloproteinases type-1 (TIMP-1) as a cancer marker and postoperative marker for minimal residual disease or recurrent disease in patients with a prior history of cancer
WO2009013753A1 (fr) * 2007-07-24 2009-01-29 Thrombotech Ltd. Peptides issus de l'inhibiteur 1 de l'activateur du plasminogène et leurs utilisations
US20090105157A1 (en) * 2006-05-30 2009-04-23 Obschestvo Ogranichennoi Otvetstennostyu Sia Peptides Peptide substance stimulating regeneration of central nervous system neurons, pharmaceutical composition on its base, and the method of its application
US7807379B2 (en) 1999-04-09 2010-10-05 Rigshospitalet Tissue inhibitor of matrix metalloproteinases type-1 (TIMP-1) as a cancer marker
WO2015125904A1 (fr) * 2014-02-21 2015-08-27 アステラス製薬株式会社 Nouvel anticorps anti-pai-1 humain
US9845363B2 (en) 2013-08-13 2017-12-19 Sanofi Antibodies to plasminogen activator inhibitor-1 (PAI-1) and uses thereof
EP3620472A1 (fr) 2013-08-13 2020-03-11 Sanofi Anticorps dirigés contre l'inhibiteur des activateurs du plasminogène de type 1 (pai-1) et leurs utilisations

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BIJNENS ANN-PASCALE ET AL: "The distal hinge of the reactive site loop and its proximity. A target to modulate plasminogen activator inhibitor-1 activity." JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 276, no. 48, 30 November 2001 (2001-11-30), pages 44912-44918, XP002217300 November 30, 2001 ISSN: 0021-9258 *
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US7807379B2 (en) 1999-04-09 2010-10-05 Rigshospitalet Tissue inhibitor of matrix metalloproteinases type-1 (TIMP-1) as a cancer marker
US7374886B2 (en) 1999-04-09 2008-05-20 Rigshospitalet Tissue inhibitor of matrix metalloproteinases type-1 (TIMP-1) as a cancer marker and postoperative marker for minimal residual disease or recurrent disease in patients with a prior history of cancer
WO2005094810A2 (fr) * 2004-03-05 2005-10-13 Karo Bio Ab Nouvelles compositions pharmaceutiques
WO2005094810A3 (fr) * 2004-03-05 2006-01-12 Karobio Ab Nouvelles compositions pharmaceutiques
WO2005094863A1 (fr) * 2004-03-30 2005-10-13 Den Kgl.Veterinær-Og Landbohøjskole Ameliorations dans le traitement du cancer et prediction efficace de traitement du cancer par le blocage et la detection d'inhibiteurs de la protease
US8524674B2 (en) 2006-05-30 2013-09-03 Obschestvo S Ogranichennoi Otvetstvennostyu “SIA Peptides” Method of improving the conditioned reflex habit, the muscle tonus, or the motion coordination of a patient after suffering trauma to the brain cortex
US20090105157A1 (en) * 2006-05-30 2009-04-23 Obschestvo Ogranichennoi Otvetstennostyu Sia Peptides Peptide substance stimulating regeneration of central nervous system neurons, pharmaceutical composition on its base, and the method of its application
JP2010534238A (ja) * 2007-07-24 2010-11-04 スロムボーテック リミテッド プラスミノーゲン活性化因子阻害物質１由来のペプチドおよびその使用
EP2468289A1 (fr) * 2007-07-24 2012-06-27 Thrombotech Ltd. Peptides dérivés d'un inhibiteur-1 d'activateur de plasminigène et utilisations correspondantes
US8507436B2 (en) 2007-07-24 2013-08-13 D-Pharm Ltd. Peptides derived from plasminogen activator inhibitor-1 and uses thereof
WO2009013753A1 (fr) * 2007-07-24 2009-01-29 Thrombotech Ltd. Peptides issus de l'inhibiteur 1 de l'activateur du plasminogène et leurs utilisations
US9845363B2 (en) 2013-08-13 2017-12-19 Sanofi Antibodies to plasminogen activator inhibitor-1 (PAI-1) and uses thereof
JP7223069B2 (ja) 2013-08-13 2023-02-15 サノフイ プラスミノーゲン活性化因子阻害剤－１（ｐａｉ－１）に対する抗体及びその使用
JP2021155440A (ja) * 2013-08-13 2021-10-07 サノフイSanofi プラスミノーゲン活性化因子阻害剤−１（ｐａｉ−１）に対する抗体及びその使用
EP3620472A1 (fr) 2013-08-13 2020-03-11 Sanofi Anticorps dirigés contre l'inhibiteur des activateurs du plasminogène de type 1 (pai-1) et leurs utilisations
CN106029884A (zh) * 2014-02-21 2016-10-12 安斯泰来制药株式会社 新型抗人pai-1抗体
US9803024B2 (en) 2014-02-21 2017-10-31 Astellas Pharma Inc. Anti-human PAI-1 antibody
JPWO2015125904A1 (ja) * 2014-02-21 2017-03-30 アステラス製薬株式会社 新規抗ヒトｐａｉ−１抗体
KR20160124840A (ko) 2014-02-21 2016-10-28 아스텔라스세이야쿠 가부시키가이샤 신규 항인간 pai-1 항체
WO2015125904A1 (fr) * 2014-02-21 2015-08-27 アステラス製薬株式会社 Nouvel anticorps anti-pai-1 humain

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