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WO2008153433A1 - Anticorps monoclonaux pour la détection de la substance inhibitrice des canaux de müller et leurs utilisations - Google Patents

Anticorps monoclonaux pour la détection de la substance inhibitrice des canaux de müller et leurs utilisations Download PDF

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Publication number
WO2008153433A1
WO2008153433A1 PCT/RU2007/000313 RU2007000313W WO2008153433A1 WO 2008153433 A1 WO2008153433 A1 WO 2008153433A1 RU 2007000313 W RU2007000313 W RU 2007000313W WO 2008153433 A1 WO2008153433 A1 WO 2008153433A1
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Prior art keywords
mis
antibody
monoclonal antibody
terminal domain
antibodies
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PCT/RU2007/000313
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English (en)
Inventor
Alexander Viktorovich Trofimov
Alexander Mitrofanovich Ischenko
Lidiya Alexandrovna Kuzmina
Alexander Vladimirovich Zhakhov
Sergey Vladimirovich Rodin
Sergey Vasilyevich Martyushin
Evgeny Alexandrovich Protasov
Alexander Vladimirovich Petrov
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State Research Institute Of Highly Pure Biopreparations (Srihpb)
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Priority to PCT/RU2007/000313 priority Critical patent/WO2008153433A1/fr
Publication of WO2008153433A1 publication Critical patent/WO2008153433A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • 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
    • C07K14/8114—Kunitz type inhibitors
    • C07K14/8117—Bovine/basic pancreatic trypsin inhibitor (BPTI, aprotinin)

Definitions

  • TECHNICAL FIELD This invention relates generally to monoclonal antibodies for the detection and purification of Mullerian Inhibiting Substance.
  • MIS Mullerian Inhibiting Substance
  • MIS receptor-bearing cells while having no effect on cells without receptors.
  • an isolated monoclonal antibody includes an antibody that specifically binds to the C-terminal domain of MIS.
  • the antibody can further bind to a variant polypeptide having at least 95% homology to the C-terminal domain of MIS.
  • the antibody can be produced from the hybridoma cell line deposited with American Type Culture Collection under Accession Number PTA-8390 (deposited with the American Type Culture Collection, 10801 University Boulevard., Manassas, VA).
  • the antibody can be selected from the group that includes a chimeric antibody, a humanized monoclonal antibody and an antibody fragment.
  • an isolated monoclonal antibody includes an antibody that specifically binds to the N-terminal domain of MIS.
  • the antibody can further bind to a variant polypeptide having at least 95% homology to the N-terminal domain of MIS.
  • the antibody can be produced from the hybridoma cell line deposited with American Type Culture Collection under Accession Number PTA-8391 (deposited with the American Type Culture Collection, 10801 University Boulevard., Manassas, VA).
  • the antibody can be selected from the group that includes a chimeric antibody, a humanized monoclonal antibody and an antibody fragment.
  • a method of purifying recombinant MIS from host cells capable of expressing recombinant MIS can include binding recombinant MIS to an antibody, the antibody being a monoclonal antibody that is capable of binding the C-terminal domain of MIS.
  • the monoclonal antibody can bind to a variant polypeptide having at least 95% homology to the C- terminal domain of MIS.
  • the monoclonal antibody can be conjugated to a chromatography matrix.
  • the chromatography matrix can be Sepharose 4B.
  • the method of purifying recombinant MIS can include recovering the recombinant MIS by eluting with sodium thiocyanate.
  • the method can further include binding the recombinant MIS to a second antibody, the antibody being a monoclonal antibody that is capable of binding the " N-terminal domain of MIS.
  • the monoclonal antibody can bind to a variant polypeptide having at least 95% homology to the N-terminal domain of MIS.
  • the method can further include recovering the recombinant MIS by eluting with MgCl 2 .
  • the method can further include binding the recombinant MIS to a second antibody, the antibody being a monoclonal antibody produced from a hybridoma cell line 6El 1 and capable of binding the full length MIS.
  • the method can include recovering the recombinant MIS by eluting with MgCI 2 .
  • a method of purifying recombinant MIS from host cells capable of expressing recombinant MIS can include binding the recombinant MIS to an antibody, the antibody being a monoclonal antibody that is capable of binding the N-terminal domain of MIS.
  • the monoclonal antibody can bind to a variant polypeptide having at least 95% homology to the C-terminal domain of MIS.
  • a method of quantifying MIS levels can include detecting MIS in a sample using a sandwich ELISA, wherein the first antibody can be 6El 1 monoclonal antibody and the second antibody can be labeled with an enzyme.
  • the first antibody can be bound to a solid support.
  • the second antibody can be the M2 monoclonal antibody or the M 1 monoclonal antibody.
  • the enzyme can be horseradish peroxidase.
  • a method of quantifying MIS levels can include detecting MIS in a sample using a sandwich ELISA, wherein the first antibody is Ml monoclonal antibody and the second antibody is labeled with an enzyme.
  • the first antibody can be bound to a solid support.
  • the enzyme can be horseradish peroxidase.
  • the second antibody can be the 6El 1 monoclonal antibody or the M2 monoclonal antibody.
  • a method of quantifying MIS levels can include detecting MIS in a sample using a sandwich ELISA, wherein the first antibody is M2 monoclonal antibody and the second antibody is labeled with an enzyme.
  • the first antibody can be bound to a solid support.
  • the enzyme can be horseradish peroxidase.
  • the second antibody can be the 6El 1 monoclonal antibody or the Ml monoclonal antibody.
  • a method of obtaining monoclonal antibodies that specifically bind to the C-terminal domain of MIS can include sensitizing mice with an immunizing amount of recombinant MIS, obtaining lymphocytes from the mice and fusing the lymphocytes with Sp2/0 myeloma cells to form mixed hybrid cells, selecting and cloning hybrid cells that produce antibodies that bind the C-terminal domain of MIS, injecting mice with selected hybrid cells that bind the C-terminal domain of MIS, harvesting ascite fluid from injected mice and isolating the monoclonal antibodies from the ascite fluid.
  • a method of obtaining monoclonal antibodies that specifically bind to the N-terminal domain of MIS can include sensitizing mice with an immunizing amount of recombinant MIS, obtaining lymphocytes from the mice and fusing the lymphocytes with
  • a method of inhibiting the spontaneous proteolysis of MIS can include incubating MIS with aprotinin.
  • a composition can include MIS and aprotinin.
  • Fig. IA is an immunoblot showing holoMIS detected with Ml, M2, 6El 1 and MGH-6 antibodies in reduced conditions.
  • Fig. IB is an immunoblot showing holoMIS detected with Ml, M2, 6El 1 and MGH-6 antibodies in unreduced conditions.
  • Fig. 2A is an immunoblot showing holoMIS detected with Ml antibodies before and after treatment with plasmin in reduced conditions.
  • Fig. 2B is an immunoblot showing holoMIS detected with Ml antibodies before and after treatment with plasmin in unreduced conditions.
  • Fig. 3 is a diagram showing different protocols for the purification of MIS.
  • Fig. 4A is a gradient gel (4-20%) showing holoMIS after immnoaffinity chromatography.
  • Fig. 4B is an immunoblot showing holoMIS detected with Ml antibodies.
  • Fig. 4C is an immnunoblot showing holoMIS detected with Ml antibodies after treatment with trypsin
  • Fig. 5 is a chromatogram of HoloMIS after purification by immunoaffinity chromatography.
  • Fig. 6 is an immunoblot analyzing HoloMIS after purification by immunoaffinity chromatography. MIS was detected using Ml monoclonal antibodies.
  • Fig. 7 is an immunoblot analyzing HoIoMIS after purification by immunoaff ⁇ nity chromatography. MIS was detected using rabbit anti-MIS polyclonal antibodies.
  • Fig. 8 is an immunoblot analyzing HoIoMIS after purification by immunoaffinity chromatography. Purified MIS was immobilized on the nitrocellulose sheet and incubated with trypsin for 30 minutes at 37 0 C. MIS was detected using Ml monoclonal antibodies.
  • Fig. 9 is a graph showing MIS activity following purification by immunoaffinity chromatography with Ml monoclonal antibodies, M2 monoclonal antibodies or 6El 1 monoclonal antibodies. MIS biological activity was assayed using M0VCAR7 cells.
  • Fig. 10 is gel showing purified MIS in reduced conditions, immediately after purification and 15 days after incubation at 37 0 C.
  • Fig. 1 IA is a chromatogram of HoIoMIS after incubation at 37°C for 1, 4, 12, and 32 days.
  • Fig. 1 IB is a bar graph showing the levels of C-terminal MIS, HoIoMIS and N-terminal MIS and peak "X" after incubation of HoIoMIS at 37°C for O, 12, 18, 32, and 47 days.
  • Fig. 12 is a bar graph showing the levels of C-terminal MIS, HoIoMIS and " N-terminal
  • Fig. 13 is a bar graph showing the levels of C-terminal MIS, HoIoMIS and N-terminal MIS and peak "X" after incubation of HoIoMIS with different protease inhibitors for O, 12 and 13 days.
  • Fig. 14A is gradient gel (4-20%) showing inhibition of spontaneous cleavage of
  • Fig. 14B is gradient gel (4-20%) showing inhibition of spontaneous cleavage of HoIoMIS under reduced conditions with different protease inhibitors, for 18, 32, and 47 days.
  • Fig. 15A is an immunoblot showing inhibition of spontaneous cleavage of HoIoMIS under unreduced conditions with different protease inhibitors for 18, 32, and 47 days. MIS was detected using Ml monoclonal antibodies.
  • Fig. 15B is an immunoblot showing inhibition of spontaneous cleavage of HoIoMIS under reduced conditions with different protease inhibitors for 18, 32, and 47 days. MIS was detected using Ml monoclonal antibodies.
  • MIS Magnetic et al., Gynecol Oncol 1984; 17:124-312.
  • the MIS Type II receptor is expressed in human breast cancer cell lines, normal human and rat breast, human fibroadenomas and carcinomas and also in a cell line derived from normal breast tissue (Segev et al., J Biol Chem 276:26799-8068 (2001)).
  • breast tissue can be a likely target for the action of MIS.
  • MIS is also present in the prostate and MIS inhibits growth of a human prostate cancer cell line in vitro.
  • MIS suppresses testosterone production (Teixeira et al., Endocrinology 140:4732-8 (1999), Trbovich et al., Proc Natl Acad Sci USA 13:3393-7 (2001), Teixeira et al., Proc Natl Acad Sci USA 96: 13831 -8 (1999). Therefore, MIS can exert a double effect on prostatic cancer by inhibition of tumor growth and by lowering testosterone levels.
  • prostatic cancer can be a likely target for the action of MlS.
  • MIS Both human MIS and bovine MIS have been cloned and expressed in various bacterial and animal host cells using both genomic and cDNA sequences.
  • the purified dimers from Sertoli cells or recombinant cells e.g., CHO cells transfected with an MIS gene
  • MIS can be further processed to.
  • MIS human MIS
  • MIS is processed and secreted from the embryonic testes as a complex in which the mature region remains non-covalently associated with the prodomain.
  • MIS can encompass any fragments, variants, analogs, agonists, chemical derivatives, functional derivatives or functional fragments of a MIS polypeptide.
  • MIS can be identified by its structure, including its polypeptide sequence.
  • the structure can be described by a single polypeptide sequence representing the full polypeptide sequence of MIS, or the structure can be described by a partial sequence, corresponding to a fragment of MIS.
  • a plurality of partial sequences can be combined to make a larger partial sequence or the full sequence.
  • a first peptide sequence can represent the N-terminal polypeptide sequence of MIS
  • a second sequence can represent the C-terminal polypeptide sequence of MIS.
  • a fragment of MIS is a fragment of the MIS protein which has an amino acid sequence which is unique to MIS.
  • the fragment can be as few as 6 amino acids, although it can be 7, 8, 9, 10, 1 1, 12, 13, 14, 15 or more amino acids.
  • a variant of a MIS polypeptide can include a molecule which is substantially similar to
  • the variant of a MIS polypeptide can have comparable biological activity to the MIS polypeptide, the C-terminal domain of MIS or the N-terminal domain of MIS.
  • Variants of MIS can have for example, 60% or greater homology, 70% or more, 80% or more, 90% or more or 95% or more homology, with either the full length polypeptide of MIS or the C-terminal domain of MIS or the N-terminal domain of MIS.
  • Variant peptides may be covalently prepared by direct chemical synthesis of the variant peptide, using methods well known in the art.
  • Variants of MIS may further include, for example, deletions, insertions or substitutions of residues within the amino acid sequence. Any combination of deletion, insertion, and substitution may also be made to arrive at the final construct, provided that the final construct possesses the desired activity.
  • an amino acid residue of the polypeptide can be replaced by another amino acid residue in a conservative substitution. Examples of conservative substitutions include, for example, the substitution of one non-polar (i.e., hydrophobic) residue such as isoleucine, valine, leucine or methionine for another non-polar residue; the substitution of one polar (i.e.
  • hydrophilic residue for another polar residue such as a substitution between arginine and lysine, between glutamine and asparagine, or between glycine and serine; the substitution of one basic residue such as lysine, arginine or histidine for another basic residue; or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another acidic residue.
  • an amino acid residue can be replaced with an amino acid residue having a chemically similar side chain. Families of amino acid residues having side chains with chemical similarity have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • a conservative substitution may also include the use of a chemically derivatized residue in place of a non-derivatized residue.
  • a chemical derivative residue is a residue chemically derivatized by reaction of a functional group of the residue. Examples of such chemical derivatives include, but are not limited to, those molecules in which free amino
  • SUBSTITUTE SHEEf (RULE 26) groups have been derivatized to form, for example, amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups.
  • Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters, or other types of esters or hydrazides.
  • Free hydroxyl groups may be derivatized to form O-acyl or O- alkyl derivatives.
  • chemical derivatives are those polypeptides which contain one or more naturally-occurring amino acid derivatives of the twenty standard amino acids.
  • 4-hydroxyproline may be substituted for proline; 5-hydroxylsine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine.
  • An amino acid residue of the polypeptide can be replaced by another amino acid residue in a non-conservative substitution. In some cases, a non-conservative substitution will not alter the relevant properties of the polypeptide.
  • the relevant properties can be, without limitation, ability to bind to an antibody that recognizes MIS such as full length MIS, the C- terminal domain of MIS and/or the N-terminal domain of MIS or other biological activity.
  • An antibody is an immunoglobulin and any antigen-binding portion of an immunoglobulin (e.g.
  • Antibodies can include at least one heavy (H) chain and at least one light (L) chain inter-connected by at least one disulfide bond.
  • V H is the heavy chain variable region of an antibody.
  • V L is the light chain variable region of an antibody.
  • antibodies can include monoclonal antibodies.
  • a monoclonal antibody is an antibody produced by a single clone of hybridoma cells.
  • Techniques for generating monoclonal antibodies include, but are not limited to, the hybridoma technique (see Kohler & Milstein (1975) Nature 256:495-497), the trioma technique, the human B-cell hybridoma technique (see Kozbor et al. (1983) Immunol. Today 4:72), the EBV hybridoma technique (see Cole, et al., 1985 In: Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) and phage display.
  • MIS antibodies To produce MIS antibodies, host animals can be injected with MIS polypeptides or fragments thereof. Rats and mice, especially mice, are preferred for obtaining monoclonal antibodies. Host animals can be injected with the C-terminal domain of HoIoMIS or the N- terminal domain of HoIoMIS. Host animals can also be injected with MIS polypeptides of overlapping sequence across a desired area of the MIS protein. For example, peptide antigens can be designed in tandem order of linear amino acid sequence of a protein, or staggered in linear sequence of the protein. In addition, antibodies to three dimensional epitopes, i.e., non linear epitopes, can also be prepared, based on, for example, the crystallographic data of proteins.
  • Hosts can also be injected with peptides of different lengths encompassing a desired target sequence.
  • Antibodies obtained from that injection can be screened against the short antigens of MIS.
  • Antibodies prepared against an MIS peptide can be tested for activity against that peptide as well as the full length MIS protein.
  • Antibodies prepared against a C- or N- terminal domain of MIS can be tested for activity against that domain.
  • Suitable antibodies can have affinities of at least about 10 "6 M 5 10 "7 M, 10 "8 M, 10 ⁇ 9 M, 10 ' '° M, 10 "11 M or 10 "12 M toward the MIS peptide, the C-terminal domain of MIS, the N-terminal domain of MIS and/or the full length MIS protein:
  • Monoclonal antibodies can be produced in cells which produce antibodies and those cells used to generate monoclonal antibodies by using standard fusion techniques for forming hybridoma cells. See G. Kohler, et al., Nature, 256:456 (1975). Typically this involves fusing an antibody-producing cell with an immortal cell line such as a myeloma cell to produce the hybrid cell. Alternatively, monoclonal antibodies can be produced from cells by the method of Huse, et al., Science, 256:1275 (1989). Monoclonal antibodies can also be prepared according to U.S. Patent No. 4,487,833, hereby incorporated by reference.
  • Purification methods can include salt precipitation (for example, with ammonium sulfate), ion exchange chromatography (for example, on a cationic or anionic exchange column run at neutral pH and eluted with step gradients of increasing ionic strength), gel filtration chromatography (including gel filtration HPLC), and chromatography on affinity resins such as protein A, protein G, hydroxyapatite, and anti-antibody.
  • Antibodies can also be purified on affinity columns according to methods known in the art. Other methods of antibody purification are described for example, in Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y.
  • Monoclonal antibodies can be further manipulated or modified to generate chimeric or humanized antibodies.
  • Chimeric antibodies are encoded by immunoglobulin genes that have been genetically engineered so that the light and heavy chain genes are composed of immunoglobulin gene segments belonging to different species. For example, substantial portions of the variable (V) segments of the genes from a mouse monoclonal antibody, e.g., obtained as described herein, can be joined to substantial portions of human constant (C) segments. Such a chimeric antibody is likely to be less antigenic to a human than a mouse monoclonal antibody.
  • the MIS monoclonal antibodies also encompass antibody fragments.
  • antibody fragments include Fab, Fab', Fab'-SH, F(ab') 2 , and Fv fragments, diabodies and any antibody fragment that has a primary structure consisting of one uninterrupted sequence of contiguous amino acid residues, including without limitation: single-chain Fv (scFv) molecules, single chain polypeptides containing only one light chain variable domain, or a fragment thereof that contains the three CDRs of the light chain variable domain, without an associated heavy chain moiety and single chain polypeptides containing only one heavy chain variable region, or a fragment thereof containing the three CDRs of the heavy chain variable region, without an associated light chain moiety; and multispecific or multivalent structures formed from antibody fragments.
  • scFv single-chain Fv
  • the heavy chain(s) can contain any constant domain sequence (e.g. CHl in the IgG isotype) found in a non-Fc region of an intact antibody, and/or can contain any hinge region sequence found in an intact antibody, and/or can contain a leucine zipper sequence fused to or situated in the hinge region sequence or the constant domain sequence of the heavy chain(s).
  • Suitable leucine zipper sequences include the jun and fos leucine zippers taught by Kostelney et al., J. Immunol, 148: 1547- 1553 (1992) and the GCN4 leucine zipper described in U.S. Pat. No. 6,468,532.
  • Fab and F(ab') 2 fragments lack the Fc fragment of intact antibody and are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab') 2 fragments).
  • a level of MIS in a sample can be measured qualitatively or quantitatively using an assay, for example, in an immunochromatographic format.
  • a qualitative assay can be distinguish between the presence or absence of MIS, or can distinguish between categories of MIS levels in a sample, such as absent, low concentration, medium concentration or high concentration, or combinations thereof.
  • a quantitative assay can provide a numerical measure of MIS in a sample.
  • the assay can include contacting MIS with an antibody that recognizes MIS, detecting MIS by mass spectrometry, assaying a sample including cells for expression (e.g., of mRNA or polypeptide) of the MIS gene by the cells, or a combination of measurements.
  • the assay can include contacting a sample with an antibody that recognizes MIS and a mass spectrometry measurement.
  • MIS can be detected in plasma or other bodily fluids which can be obtained from a mammalian body, such as interstitial fluid, urine, whole blood, saliva, serum, lymph, gastric juices, bile, sweat, tear fluid and brain and spinal fluids. Bodily fluids can be processed (e.g. serum) or unprocessed.
  • the mammalian subject can be a human.
  • the levels of MIS can be measured using an immunoassay, i.e. by contacting the sample with an antibody that binds specifically to the marker and measuring any binding that has occurred between the antibody and at least one species of MIS in the sample.
  • immunoassays can be competitive and non-competitive assay systems and levels of MIS can be measured using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), sandwich immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays and protein A immunoassays.
  • MIS monoclonal antibodies can be detectably labeled by linking the antibodies to an enzyme and used in an enzyme immunoassay (EIA).
  • EIA enzyme immunoassay
  • This enzyme when later exposed to an appropriate substrate, will react with the substrate in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or by visual means.
  • Enzymes which can be used detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
  • the detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the enzyme. Detection can also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards. It is also possible to label an anti-MIS monoclonal antibody with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can be then be detected due to fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine. The antibody can also be detectably labeled using fluorescence emitting metals such as
  • EDTA diethylenetriamine pentaacetic acid
  • the antibody also can be detectably labeled by coupling it to a chemiluminescent compound.
  • the presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • particularly useful chemiluminescent labeling compounds include luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt or oxalate ester.
  • Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.
  • Detection can be accomplished using any of a variety of other immunoassays.
  • a radioimmunoassay RIA
  • the radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography.
  • An antibody molecule can also be adapted for use in an immunor ⁇ etric assay, also known as a two-site or sandwich assay or sandwich ELISA assay.
  • the sandwich ELISA requires two antibodies that bind to epitopes that do not overlap on the antigen. This can be accomplished with two monoclonal antibodies that recognize discrete sites.
  • a quantity of unlabeled antibody (or fragment of antibody) is bound to a solid support or carrier and a quantity of detectably labeled soluble antibody is added to permit detection and/or quantitation of the ternary complex formed between solid-phase antibody, antigen, and labeled antibody.
  • one antibody (the capture antibody) is purified and bound to a solid phase typically attached to the bottom of a plate well. Antigen is then added and allowed to complex with the bound antibody. Unbound products are then removed with a wash, and a labeled second antibody (the detection antibody) is allowed to bind to the antigen, thus completing the sandwich. The assay is then quantitated by measuring the amount of labeled second antibody bound to the matrix, through the use of a colorimetric substrate.
  • Major advantages of this technique are that the antigen does not need to be purified prior to use, and that these assays are very specific. However, one disadvantage is that not all antibodies can be used. Monoclonal antibody combinations must be qualified as matched pairs, meaning that they can recognize separate epitopes on the antigen so they do not hinder each other's binding.
  • simultaneous and reverse assays are used.
  • a simultaneous assay involves a single incubation step as the antibody bound to the solid support or carrier and labeled second antibody are both added to the sample being tested at the same time. After the incubation is completed, the solid support or carrier is washed to remove the residue of fluid sample and uncomplexed labeled antibody. The presence of labeled antibody associated with the solid support or carrier is then determined as it would be in a conventional forward sandwich assay.
  • ELISA procedures utilize substrates that produce soluble products.
  • the enzyme substrates should be stable, safe and inexpensive.
  • Popular enzymes are those that convert a colorless substrate to a colored product, e.g., p-nitrophenyl phosphate (pNPP), which is converted to the yellow p-nitrophenol by alkaline phosphatase.
  • Substrates used with peroxidase can include 2,2'-azo-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine (OPD) and 3,3'5,5'- tetramethylbenzidine base (TMB), which yield green, orange and blue colors, respectively.
  • the MIS monoclonal antibodies can be used in an immunoaff ⁇ nity chromatography method to obtain purified MIS protein using methods known to those skilled in the art.
  • MIS can include the C-terminal domain of MIS or the N-terminal domain of MIS.
  • the recombinant MIS protein can be concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Following the concentration step, the concentrate can be applied to immunoaff ⁇ nity chromatography.
  • Antibodies to the full length MIS, C -terminal domain of MIS or N-terminal domain of MIS can be used in the purification of MIS in accordance with known methods.
  • the affinity columns utilizing monoclonal antibodies to the MIS full length, C-terminal domain of MIS or N-terminal domain of MIS can be used in various combinations to obtain purified MIS.
  • Suitable purification methods include an anion exchange resin (e.g. a matrix or substrate having pendant diethylaminoethyl (DEAE) or polyetheyleneimine (PEI) groups).
  • the matrices can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification.
  • a cation exchange step can be employed.
  • Suitable cation exchangers include various insoluble matrices such as sulfopropyl or carboxymethyl groups.
  • the purification of the MIS protein can also include one or more column steps over such affinity resins as concanavalin A-agarose, heparin-toyopearlTM or Cibacrom blue 3GA SepharoseTM; or by hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether.
  • MIS can be purified using High Performance Liquid Chromatography (HPLC).
  • the isolated human MIS is purified so that it is substantially free of other proteins.
  • Isolated or purified human MIS can be formulated into a suitable composition.
  • the composition can include a stabilizer that inhibits the spontaneous proteolysis of MIS.
  • the stabilizer can include protease inhibitors such as aprotinin.
  • Purified MIS can further be formulated and introduced through oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous or intranasal routes. Oral delivery of MIS can supplement injections of purified MIS.
  • Purified MIS can include the C-terminal domain of MIS or the N-terminal domain of MIS.
  • Pharmaceutical formulations suitable for oral administration can be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia), each containing a predetermined amount of a subject composition thereof as an active ingredient.
  • Compositions of the present invention can also be administered as a bolus, electuary, or paste.
  • the subject composition can be mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, acety
  • compositions can also include buffering agents.
  • Solid compositions of a similar type can also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
  • a tablet can be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets can be prepared using binder (for example, gelatin or hydroxypropyl methyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface- active or dispersing agent.
  • Molded tablets can be made by molding in a suitable machine a mixture of the subject composition moistened with an inert liquid diluent. Tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, can optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms can contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers
  • Suspensions in addition to the subject composition, can contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, macrocrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, macrocrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • MIS can be administered parenterally as injections (intravenous, intramuscular or subcutaneous).
  • the pharmaceutical can optionally contain one or more adjuvants. Any suitable adjuvant can be used, such as aluminum hydroxide, aluminum phosphate, plant and animal oils, and the like, with the amount of adjuvant depending on the nature of the particular adjuvant employed.
  • the pharmaceutical composition can also contain at least one stabilizer, such as carbohydrates such as sorbitol, mannitol, starch, sucrose, dextrin, and glucose, as well as proteins such as albumin or casein, and buffers such as alkali metal phosphates and the like.
  • the pharmaceutical formulation that are suitable for parenteral administration can be formulated into pharmaceutically-acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powder which can be reconstituted into sterile injectable solutions or dispersions just prior to use, and can further contain antioxidants, buffers, bacteriostats, solutes (which render the formulation isotonic with the blood of the intended recipient) or suspending or thickening agents.
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • the formulated MIS pharmaceutical compositions can be stored in a sterile glass container sealed with a rubber stopper through which liquids can be injected and compositions withdrawn by syringe.
  • Hybridoma cells were grown in cultivation flasks on RPMI 1640 + 20% of fetal calf serum (FCS) conditioned with mouse macrophages to saturate the medium with growth factors. A portion of cells were cloned in parallel in 96-well culture plates containing the same culture media by method of serial dilutions, counting 2 cells/well. In a logarithmic phase of growth, a portion of cells from a flask was cryo-preserved in an ampule (3 x 10 6 cells/ampule). RPMI 1640 medium with 20% of FCS was added to the remaining cells and further cultured.
  • FCS fetal calf serum
  • mice 7 days later, a portion (3 x 10 6 ) of these cells was cryo-preserved again to increase hybridoma preservation.
  • the remaining cells at a concentration of 2.5 x 10 6 cell/mouse, were injected into 20 Balb/c mice.
  • mice Three days before injection, mice were subjected to i/p injections of 0.5 ml of pristane. Ascites were formed in mice on day 14 after injection, and the ascite fluid taken from euthanized mice.
  • Monoclonal anti-human rhMIS (6El 1) antibodies were isolated from ascite fluids by ammonium sulfate precipitation. The final concentration of ammonium sulfate was 45%. The precipitate was washed by centrifugation and dissolved in phosphate buffer. The antibodies were purified by chromatography with Protein A-Sepharose. From data of electrophoresis, the resultant fraction contained 95% of IgG antibodies. 900 mg of the antibodies was raised.
  • the raised monoclonal antibodies were characterized by the ability to interact with HoIo-MIS protein.
  • the purified antibodies were immobilized on Sepharose 4B.
  • Sepharose 4B was activated in two stages, first, with epichlorohydrin, and then with di- azide of adipic acid. The unreacted active groups of the carrier were blocked with triethylamine.
  • 6El 1 antibodies were immobilized at 4°C at stirring. The concentration of immobilized protein was estimated by a difference between the initial amount of protein and that after immobilization, using BCA-assay Kit (Sigma).
  • the produced 6El 1 Sepharose adsorbent was used for isolation of HoIo-MIS from culture fluid of CHO B9 cells (obtained from MGH).
  • rHoIo-MIS Purified rHoIo-MIS at a concentration of 0.5 mg/ml, 90-95% pure (from data of electrophoresis) was produced. In this preparation a protein fragment of either 25 kD (in unreduced conditions) or 12.5 kD (in reduced conditions) was identified. These parameters corresponded to the C-terminal domain of HoIo-MIS, as confirmed by data of Western blotting with rabbit 249 antibodies (MGH). Mice were immunized with the purified antigen to raise novel clones of antibodies for use in MIS isolation and purification and for design of high-efficient test-systems.
  • MGH Western blotting with rabbit 249 antibodies
  • Balb/c mice (10 animals) were immunized with 2.5 ⁇ g of the purified antigen in 50 ⁇ l of PBS and 50 ⁇ l of complete Freund's adjuvant (CFA). One half of this volume was injected into a mouse back pad and another half into the crest. On day 28, after immunization (short schedule) one mouse was boosted by i/v injection of 2.5 ⁇ g of the purified antigen. After four days, the groin lymph nodes were taken from this mouse to isolate lymphoid cells. A total of 24 x 10 6 lymphocytes were isolated.
  • CFA complete Freund's adjuvant
  • lymphocytes were fused with the myeloma cells at a ratio of 24 x 10 6 of lymphocytes to 3O x 10 6 of Sp2/0 myeloma cells.
  • Cell hybridization was performed in PEG/DMSO by method of serial dilutions. The resultant hybrids were seeded over five 96-well culture plates on RPMI 1640 medium with 20% of FCS.
  • the HoIoMIS antigen at a final concentration of 1 ⁇ g/ml in 100 ⁇ l of PBS/well was adsorbed to the plate and incubated for a night at a room temperature.
  • a 100 ⁇ l of the culture medium from the hybridoma was diluted with 100 ⁇ l of borate buffer with Tween 20 (0.05%) and incubated with the antigen for an hour at 37°C on a shaker.
  • OPD o-Phenylenediamine Dihydrochloride
  • each mouse received i/p 200 ⁇ l of pristane.
  • the 1 x 10 6 cell/mouse of the cloned cells were injected to abdominal cavity of a mouse. Two weeks later, ascites were harvested from the euthanized mice.
  • the specific IgG antibodies were isolated with Protein-A-Sepharose. These antibodies were designated as M-I, specific to the C-terminal domain, and M-2, specific to the N-terminal domain of MIS.
  • the antibodies were analyzed by immunoblotting using the C-terminal fragment of HoIo-MIS (C- term-25), kindly supplied by MGH.
  • Figs. IA and B are immunoblots of HoIoMIS detected with Ml antibodies (lane 1), M2 antibodies (lane 2), 6El 1 antibodies (lane 3) and MGH-6 antibodies (lane 4) respectively in reduced (Fig. IA) and unreduced conditions (Fig. I B).
  • the Ml antibodies recognized 140 kD HoIoMIS and the 25 kD C-terminal fragment of HoIoMIS.
  • the remaining antibodies, M2, 6El 1 and MGH-6 recognized high-molecular weight species of HoIoMIS at 140-150 kD and other higher molecular weight species corresponding to dimers, oligomers and MIS aggregates as well as the 110 kD N-terminal fragment of HoIoMIS.
  • Fig. IA As evident from Fig. IA, under reduced conditions, the 6El 1 and M2 antibodies did not recognize any MIS fragment.
  • the MGH-6 antibodies recognized the HoIo MIS fragment of 70 kD and the 57 kD fragment of the N-terminal domain.
  • the Ml antibodies recognized the 12.5 kD protein, the C-term monomer, but did not recognize 70 kD subunit of HoIoMIS.
  • purified HoIoMIS was treated with plasmin according to the method described in Pepinsky et al., J. Biol. Chem., 263:18961-18964 (1988) and detected by immunoblotting with Ml antibodies (Fig. 2A and B). Figs.
  • FIG. 2A and B are immunoblots of holoMIS detected with Ml antibodies before plasmin treatment (lane 1) and after plasmin treatment (lane 2) in reduced (Fig. 2A) and unreduced conditions (Fig. 2B).
  • the Ml antibodies recognized the fragment of 25 kD (C- terminal HoloMIS dimer) as well as the 12.5 kD monomer. Therefore, Ml antibodies are specific to the C-term 25 kD fragment, namely, to the linear determinant.
  • Double antibody sandwich ELISA assays performed using various combinations of 6EI 1, M2, M I antibodies allowed the conclusion that 6El 1 and M2 antibodies recognized only the N-terminal fragment of HoIo MIS molecule but did not compete with each other suggesting they were specific to different fragment of the molecule.
  • HRP horse radish peroxidase
  • Buffer A Buffer for antibody binding: 0.02M borate buffer, pH8.0, 0.15M NaCl
  • Buffer B 0.02M borate buffer, pH8.0, 0.15M NaCl, 0.05% Tween-20
  • Buffer C PBS, pH7,4, 0.05% Tween-20, 5% Fetal calf serum
  • Buffer D 0.02M borate buffer, pH 8.0, 0.15M NaCl, 0.05% Tween-20, 0.5 mg/ml BSA
  • the plates were washed by rinsing each well 3 times with 150 ⁇ l of Buffer B.
  • a 100 ⁇ l of Buffer B and 50 ⁇ l of tested MIS sample in Buffer C were placed into relevant wells for a 2 hr incubation at 37°C in a wet chamber with shaking.
  • the plates were then washed by rinsing each well 3 times with 150 ⁇ l of Buffer B.
  • a 100 ⁇ l of the relevant conjugate in Buffer D was placed into relevant wells for 1 hr incubation at 37°C in a wet chamber with shaking.
  • the plates were washed after conjugate binding by rinsing each well 3 times with 150 ⁇ l of Buffer B.
  • TMB tetramethylbenzidine
  • Table 1 shows the sensitivity of the designed test systems
  • Test system nos. 3 and 9 were the most sensitive ELISA assays as these test systems were able to detect MIS concentrations at 20 to 40 pg/ml.
  • MIS protein purified by immunoaffinity chromatography (IAP) was used as a reference sample in both test-systems. Data of MIS concentrations in the sera determined by ELISA with these test-systems are demonstrated in Table 2. Table 2 shows MIS concentrations in donor sera as determined by M 1 - M2-HRP and 6El 1 - M2-HRP test-systems
  • test-systems Two different test-systems were used - the first test system utilized Ml antibodies to C-terminal MIS and the second test system utilized 6El 1 antibodies to N- terminal MIS as capture antibodies. M2 antibodies conjugated to HRP were used as recognizing antibodies in both test-systems. r Table 3 shows the relative content of HoIoMIS (%) estimated with M I - M2-HRP and
  • MIS purification was examined (Fig. 3). For these experiments, 9 independent cycles of CHO B9 cells were grown. In these experiments the volume of culture fluid for MIS isolation varied from 2 to I l L. The culture media was centrifuged and approximately 25-fold concentrated by ultrafiltration with hollow fibers. Ultrafiltration was used instead of precipitation with ammonium sulfate, as 10-15% of protein was lost after precipitation compared to 1-3% lost after ultrafiltration.
  • HoIoMIS was isolated and purified in a set of parallel experiments.
  • a part of the concentrated culture medium was chromatographed with 6El 1- Sepharose to obtain COOl to C009 preparations. See Fig. 3.
  • the remaining culture medium was first purified with Ml-Sepharose followed by either M2- or 6E1 1 -Sepharose. Elution was performed with either 2-3.5 M MgCl 2 or 2-3.5M NaCNS ( or KCNS) solutions, pH 7. The eluted protein solutions were immediately dialyzed.
  • MIS concentrations in all the samples were assayed by ELISA using test system nos. 3 and 9 indicated in Table 1. This data is shown in Table 4 (columns 2 and 3, respectively). In addition, Table 4 shows the calculated data of M 1 -specific MIS portion in the tested samples.
  • Table 4 shows MIS concentrations in the samples prior and after immunoaffinity purification as well as a specific content (%) of Ml -specific MIS in different samples.
  • MIS content in the initial samples varied from the minimal (15.6 mg/L) in C8 sample to the maximum (25.5 mg/L) in C9.
  • the protein yield determined as a relation between MIS content in the purified samples C003, C004, C008, and C009 and the calculated MIS content in
  • Fig.4A shows that HoIoMIS purified with Ml-Sepharose (Sample C004-1) significantly differed from that purified with 6E 1 1 -Sepharose (Sample C004-6).
  • the former protein had a more complicated molecular spectrum.
  • the components of 140 kD dissociated cymbatically to those of 70 kD.
  • the component of 57 kD increased, and a monomer of 12.5 kD appeared.
  • Fig. 4B is an immunoblot showing holoMIS detected with MI monoclonal antibodies. Lanes 1 , 2, 3, 4 of Fig. 4B correspond to similarly numbered lanes in Fig. 4A. Immunoblotting data further demonstrated that in reduced conditions, C004-1 sample was significantly enriched with the C-term 12.5 kD fragment as well as with HoloMIS subpopulation containing neodeterminant.
  • Fig. 6 is an immunoblot analyzing HoIo MIS after purification by immunoaffinity chromatography and gel filtration. Lanes 1-7 were subjected to electrophoresis in reduced conditions whereas lanes 8-14 were subjected to electropheresis in unreduced conditions.
  • Lanes 1 and 8 correspond to Peak B filtrate from C003-1 sample
  • lanes 3 and 10 correspond to Peak B filtrate from C003-2 sample
  • lanes 5 and 12 correspond to Peak B filtrate from C003-6 sample
  • lanes 7 and 14 correspond to IAP sample obtained from MGH
  • lanes 2 and 9 correspond to Peak C filtrate from sample C003-1
  • lanes 4 and 1 1 correspond to peak C filtrate from C003-2 sample
  • lanes 6 and 13 correspond to peak C filtrate from C003-6 sample.
  • IAP preparation contained HoloMIS significantly subjected to processing with the exposed neodeterminant specific to M l antibodies. It is remarkable that the neodeterminant was exhibited only in an intact molecule of
  • Lanes 1 and 8 correspond to Peak B filtrate from C003-1 sample
  • lanes 3 and 10 correspond to Peak B filtrate from C003-2 sample
  • lanes 5 and 12 correspond to Peak B filtrate from C003-6 sample
  • lanes 7 and 14 correspond to IAP sample obtained from MGH 5
  • lanes 2 and 9 correspond to Peak C filtrate from sample C003-1
  • lanes 4 and 1 1 correspond to peak C filtrate from C003-2 sample
  • lanes 6 and 13 correspond to peak C filtrate from C003-6 sample).
  • Fig. 8 shows that the treatment of immobilized HoIoMlS with trypsin resulted in the appearance of a neodeterminant recognized by Ml antibodies.
  • Immobilized holoMIS was obtained from gel filtration (shown as chromatograms 1 , 2 and 3 in Fig. 5).
  • Lanes 1 and 8 correspond to C003-1 sample from chromatogram 2
  • lanes 3 and 10 correspond to C003-2 sample from chromatogram 2
  • lanes 5 and 12 correspond to C003-6 from chromatogram 2
  • lanes 7 and 14 correspond to IAP sample obtained from MGH
  • lanes 2 and 9 correspond to C003-1 sample from chromatogram 3
  • lanes 4 and 11 correspond to C003-2 from chromatogram 3
  • lanes 6 and 13 correspond to C003-6 sample from chromatogram 3.
  • MMT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
  • FIG. 9 shows 3 curves which correspond to activities of 3 proteins - C003-1, -2, and -6 which were purified by immunoaffinity chromatography with Ml -, M2- and 6E1 1-Sephrose columns respectively.
  • MIS C003-1 purified with Ml antibodies was most active.
  • Samples C003- 2 and -6 were almost similarly active.
  • Fig. IO shows electrophoregrams of the COOl sample in reduced conditions (Lanes 1 and 2).
  • the protein was analyzed -just after isolation (Lane 1) and 15 days after incubation at 37°C (Lane 2).
  • the freshly isolated sample was characterized by a 70 kD band, corresponding to a full-length MIS subunit, and by other less intensive bands of 57 and 12.5 kD corresponding to N- and C- terminal domains subunits, respectively.
  • Fig. 1 1 shows chromatograms of the protein subjected to incubation at 37°C. (Chromatogram 1, before incubation, but approximately 1 week storage at 4°C; Chromatogram 2, HoIoMIS after 1 day incubation at 37°C; Chromatogram 3, HoIoMIS after 4 day incubation at 37°C; Chromatogram 4, HoIoMIS after 12 day incubation at 37 0 C; Chromatogram 5, HoIoMIS after 32 day incubation at 37 0 C).
  • Fig. 1 1 demonstrates that the component with lesser time of retention (about 7.5 min) was increased, while the one with time of retention about 1 1 min, was moved to the right, to the hydrophobic region, as time passed, and became asymmetric. From ELISA data and electrophoresis data, peak A in Fig. 1 1 corresponded to the C-terminal fragment of HoIoMIS and peak B corresponded to the N-terminal fragment of HoIoMIS. In addition, a minor peak (about 9 min of retention) was seen. This peak was designated peak X and has not yet been identified. The chromatograms were processed and presented in a form of a table (Table 3) and a diagram (Fig. 11 B).
  • Table 3 shows the results of spontaneous cleavage of holo MIS COO 1 at 37 0 C
  • Table 4 shows results of Spontaneous cleavage of holo MIS COOl at 4 0 C
  • the C-terminal fragment of HoIoMIS was isolated and purified.
  • the lyophilized protein was produced by purification with RP-HPLC in a gradient of trifluoroacetic acid (pH 2) and acetonitrile.
  • N-terminal analysis demonstrated that the produced protein contained the sequence Ser-Ala-GIy-Ala-Thr..., similar to that of the C-term fragment obtained when cleaved by the kex2/subtilisin-like endoprotease.
  • the C-terminal fragment of HoIoMIS was demonstrated to possess bioactivity in the test of degeneration of the Muller's ducts isolated from rat embryos using the method described in Donahoe et al. J. Surg.
  • protease inhibitors We further studied the influence of protease inhibitors on the spontaneous proteolysis of HoIoMIS.
  • the inhibitors of serine proteases, cysteinases, and aspartases such as phenylmethylsulfamide fluoride (PMSF), benzamidine, aprotinin, leupeptin, pepstatin, and a cocktail of inhibitors were studied.
  • the effects of the inhibitors were analyzed with RP- HPLC, electrophoresis and immunoblotting.
  • the data of the processed chromatograms of HoIoMIS subjected to incubation at 37°C in the presence of inhibitors are shown in Table 5 and on Fig. 13.
  • Fig. 13 demonstrates that the cocktail protease inhibitors effectively inhibited spontaneous cleavage of HoIo-MIS.
  • Table 5 shows the inhibition of holoMIS spontaneous cleavage
  • Fig. 14 demonstrates the effects of various protease inhibitors on the inhibition of spontaneous proteolysis of HoloMIS (Lane 1 , HoloMIS after storage at 4°C for not more than 7 days; Lane 2, HoloMIS after incubation for 47 days at 37°C; Lane 3, HoloMIS after incubation for 18 days at 37 0 C; Lane 4, HoIo MIS after incubation for 18 days at 37 0 C with PMSF; Lane 5, HoloMIS after incubation for 18 days at 37 0 C with leupeptin; Lane 6, HoloMIS after incubation for 18 days at 37 0 C with pepstatin; Lane 7, HoloMIS after incubation for 18 days at 37°C with aprotinin; Lane 8, HoloMIS after incubation for 18 days at 37°C with benzamidine; Lane 9, HoloMIS after incubation for 18 days at 37°C with buffer for enzymes; Lane 10, HoloMIS after incubation for 32 days at 37°C with a cocktail of PMSF, leupeptin, Pep
  • lane 2 demonstrates that there is increased proteolysis of HoloMIS into C-terminal fragments of 25 kD in unreduced conditions (Fig. 14A, lane 2) or 12.5 kD in reduced conditions (Fig. 14B, lane 2).
  • Fig. 14B shows that the intensity of the band corresponding to the 7OkD species was decreased whereas the intensities of the bands corresponding to the 55 and 12.5 kD species increased. This suggested that spontaneous proteolysis of HoloMIS had occurred.
  • Complete inhibition of spontaneous proteolysis was demonstrated for protease inhibitor aprotinin (Figs.l2A and B, lanes 7), and for a cocktail of enzymes (Figs. 14A and B, lanes 10), and partial inhibition of proteolysis was shown for PMSF, ieupeptin, and benzamidine (Figs.14A and B, lanes 4, 5, and 8).
  • Fig. 15A is an immunoblot of inhibition of spontaneous cleavage of holoMIS under unreduced conditions.
  • Fig. 15B is an immunoblot of inhibition of spontaneous cleavage of holoMIS under reduced conditions.
  • Figs. 15A and B confirm the specificity of spontaneous proteolysis and inhibition effects.

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Abstract

L'invention porte sur des anticorps monoclonaux pour la détection et la purification de la substance inhibitrice des canaux de Müller.
PCT/RU2007/000313 2007-06-09 2007-06-09 Anticorps monoclonaux pour la détection de la substance inhibitrice des canaux de müller et leurs utilisations WO2008153433A1 (fr)

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ITRM20130455A1 (it) * 2013-08-05 2015-02-06 Alfonso Baldi Ligandi dell'ormone anti-mulleriano
WO2017050974A1 (fr) * 2015-09-25 2017-03-30 INSERM (Institut National de la Santé et de la Recherche Médicale) Anticorps de neutralisation de l'hormone anti-müllérienne (l'amh) et leurs utilisations
CN106674348A (zh) * 2017-01-06 2017-05-17 刘玲 抗苗勒管激素抗体及其制备方法
EP3257867A1 (fr) * 2016-06-17 2017-12-20 Biomérieux Procédé de préparation d'anticorps anti-amh et leurs utilisations
RU2764795C1 (ru) * 2021-04-05 2022-01-21 Федеральное государственное унитарное предприятие «Государственный научно-исследовательский институт особо чистых биопрепаратов» Федерального медико-биологического агентства Моноклональное антитело к С-концевому фрагменту антимюллерова гормона
EP3885363A4 (fr) * 2018-11-20 2022-08-24 Xiamen Innodx Biotech Co., Ltd Anticorps spécifique pour amh et ses applications

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US9861711B2 (en) 2013-08-05 2018-01-09 Pietro Giulio Signorile Labeled ligands of anti-Mullerian hormone for diagnosis of endometriosis
WO2015019269A1 (fr) * 2013-08-05 2015-02-12 Signorile Pietro Giulio Ligands marqués d'hormone antimüllérienne pour le diagnostic de l'endométriose
ITRM20130455A1 (it) * 2013-08-05 2015-02-06 Alfonso Baldi Ligandi dell'ormone anti-mulleriano
WO2017050974A1 (fr) * 2015-09-25 2017-03-30 INSERM (Institut National de la Santé et de la Recherche Médicale) Anticorps de neutralisation de l'hormone anti-müllérienne (l'amh) et leurs utilisations
US10774142B2 (en) 2015-09-25 2020-09-15 Inserm (Institut National De La Sante Et De La Recherche Medicale) Anti-mullerian hormone (AMH) neutralizing antibodies and uses thereof
RU2764198C2 (ru) * 2016-06-17 2022-01-14 Биомерье Способ получения антител против амн и их применение
WO2017216334A1 (fr) * 2016-06-17 2017-12-21 bioMérieux Procédé de préparation d'anticorps anti-amh et leurs utilisations
CN109563163A (zh) * 2016-06-17 2019-04-02 生物梅里埃公司 用于制备抗-amh抗体的方法及其用途
EP3257867A1 (fr) * 2016-06-17 2017-12-20 Biomérieux Procédé de préparation d'anticorps anti-amh et leurs utilisations
US11225518B2 (en) 2016-06-17 2022-01-18 bioMérieux Method for preparing anti-AMH antibodies and uses of same
CN106674348A (zh) * 2017-01-06 2017-05-17 刘玲 抗苗勒管激素抗体及其制备方法
EP3885363A4 (fr) * 2018-11-20 2022-08-24 Xiamen Innodx Biotech Co., Ltd Anticorps spécifique pour amh et ses applications
RU2764795C1 (ru) * 2021-04-05 2022-01-21 Федеральное государственное унитарное предприятие «Государственный научно-исследовательский институт особо чистых биопрепаратов» Федерального медико-биологического агентства Моноклональное антитело к С-концевому фрагменту антимюллерова гормона

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