WO2008153433A1 - Monoclonal antibodies for detection of mullerian inhibiting substance and uses thereof - Google Patents

Abstract

Monoclonal antibodies for the detection and purification of Mullerian Inhibiting Substance.

Description

MONOCLONAL ANTIBODIES FOR THE DETECTION OF MULLERIAN INHIBITING SUBSTANCE AND USES THEREOF

TECHNICAL FIELD This invention relates generally to monoclonal antibodies for the detection and purification of Mullerian Inhibiting Substance.

BACKGROUND

Mullerian Inhibiting Substance (MIS) is a 140 kDa, glycosylated protein that is produced during normal embryogenesis by the Sertoli cells of the embryonic testis, causes the involution of the Mullerian duct, and inhibits female gonadogenesis by producing apoptosis of target gonadal cells. MIS is a member of the TGF-β family, and it causes apoptosis of specific

MIS receptor-bearing cells, while having no effect on cells without receptors.

SUMMARY

MIS monoclonal antibodies can be used in immunoassays and purification of MIS. In one aspect, 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 Blvd., Manassas, VA). The antibody can be selected from the group that includes a chimeric antibody, a humanized monoclonal antibody and an antibody fragment.

In another aspect, 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 Blvd., Manassas, VA). The antibody can be selected from the group that includes a chimeric antibody, a humanized monoclonal antibody and an antibody fragment.

In a further aspect, 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 MgCl₂. 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 MgCI₂.

In another aspect, 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.

In one aspect, 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.

In another aspect, 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.

In a further aspect, 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.

In one aspect, 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.

In a further aspect, 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

Sp2/0 myeloma cells to form mixed hybrid cells, selecting and cloning hybrid cells that produce antibodies that bind the N-terminal domain of MIS, injecting mice with selected hybrid cells that bind the N-terminal domain of MIS, harvesting ascite fluid from injected mice and isolating the monoclonal antibodies from the ascite fluid. In one aspect, a method of inhibiting the spontaneous proteolysis of MIS can include incubating MIS with aprotinin. In a further aspect, a composition can include MIS and aprotinin.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and description below. Other features, objects and advantages of the invention will be apparent from the description and drawings and from the claims.

DESCRIPTION OF DRAWINGS

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 immunoaffϊnity 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 37⁰C. 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 37⁰C.

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

MIS and peak "X" after incubation of HoIoMIS at 4°C for O and 86 days.

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

HoIoMIS under unreduced conditions with different protease inhibitors for 18, 32, and 47 days. 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.

DETAILED DESCRIPTION

Recent evidence suggests cervical and endometrial cancer cells undergo apoptosis with

MIS (Fuller et al., Gynecol Oncol 1984; 17:124-32). 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)). Thus, 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. In addition, 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. Thus, prostatic cancer can be a likely target for the action of MlS.

The ultimate translation of MIS from in vitro studies, to animal trials, and eventually to Phase I clinical trials hinges upon the steady supply of recombinant MIS. However, the production of sufficient quantities of MIS sufficient for clinical use remains to be established.

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 protein expressed by the recombinant cells, as well as those of Sertoli cells, are 70 kD polypeptides which dimerize to form 140 kD disulfide-linked dimers. The purified dimers from Sertoli cells or recombinant cells (e.g., CHO cells transfected with an MIS gene) are active in causing the regression of the rat Mullerian duct in an in vitro standard organ culture assay (Cate et al., Cell, 45: 685-98 (1986)). MIS can be further processed to. produce a C-terminal dimer and an N-terminal dimer. In unreduced conditions, the C-terminal dimer is detected as a 25 kD polypeptide. In reduced conditions, the C-terminal dimer is detected as a 12.5 kD polypeptide. The amino acid sequence for human MIS is provided as NCBI database Accession NP_000470). HoIoMIS refers to intact, glycosylated protein produced by CHO B9 (Lorenzo et al., J. Chromatogr. B, 7656:89- 98 (2001)). MIS is processed and secreted from the embryonic testes as a complex in which the mature region remains non-covalently associated with the prodomain. There is evidence that the two endoproteases, kex2/subtilisin-Iike enzymes, Pc5 and furin, are responsible for cleavage of MIS in vivo (Nachtigal et al., Proc. Natl. Acad. Sci. USA 93:771 1-7716 (1996)). Recombinant MIS protein may be cleaved in vitro with plasmin (Pepinsky et al., J. Biol. Chem., 263: 18961-18964 (1988)). The site of proteolysis is between amino acids Arg 427 and Ser 428 in both in vivo and in vitro proteolysis of MIS (Lee M.M. and Donahoe P. K. Endocrine Rev. 14:152-164 (1993)). 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. For example, 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. For example, a first peptide sequence can represent the N-terminal polypeptide sequence of MIS, and 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

MIS. 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. In general, 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. In a conservative substitution, 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. These families include 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) and 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. Also included as chemical derivatives are those polypeptides which contain one or more naturally-occurring amino acid derivatives of the twenty standard amino acids. For example, 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. IgG, IgD, IgA, IgM and IgE) i.e., a polypeptide that contains an antigen binding site, which specifically binds or immunoreacts with an antigen. 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. In one embodiment, 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.

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 M₅ 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. In one embodiment, the MIS monoclonal antibodies also encompass antibody fragments. Examples of 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. In an antibody fragment that includes one or more heavy chains, 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. For example, 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. Such assays 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.

In one embodiment, MIS monoclonal antibodies can be detectably labeled by linking the antibodies to an enzyme and used in an enzyme immunoassay (EIA). This enzyme, in turn, 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

¹⁵²EU, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriamine pentaacetic acid (EDTA).

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. Examples of particularly useful chemiluminescent labeling compounds include luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt or oxalate ester.

Likewise, a bioluminescent compound can be used to label the antibody. 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. For example, by radioactivity labeling the antibodies, it is possible to detect MIS through the use of a radioimmunoassay (RIA). A good description of RIA can be found in Laboratory Techniques and Biochemistry in Molecular Biology by Work, T. S. et al., ^"North Holland Publishing Company, NY (1978) with particular reference to the chapter entitled "An Introduction to Radioimmune Assay and Related Techniques" by Chard, T., incorporated by reference herein. 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 immunorηetric 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. In a typical immunometric assay, 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.

In a forward sandwich assay, 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.

In another type of sandwich assay, the so-called 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. Ideally 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.

In another embodiment, the MIS monoclonal antibodies can be used in an immunoaffϊnity 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. For example, 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 immunoaffϊnity 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.

Other 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. Alternatively, 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-toyopearl™ or Cibacrom blue 3GA Sepharose™; 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).

Some or all of the foregoing purification steps, in various combinations with immunoaffϊnity chromatography, can also be employed to provide a substantially purified isolated recombinant protein. Preferably, 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. In one embodiment, 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.

In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), 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, acetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; or (10) coloring agents. In the case of capsules, tablets and pills, the 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. In addition to the subject composition, 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.

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.

Alternatively, 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. In addition, 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. Examples of suitable aqueous and nonaqueous carriers which can be employed in the vaccine compositions of the invention 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. 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.

EXAMPLES Materials and Methods Monoclonal Antibodies

The 6El 1 hybridoma as described by Hudson et al., J. Clin. Endocrinol. Metab. 70:16- 22 (1990) was kindly supplied by Massachusetts General Hospital (MGH). The experiments on cultivation of hybridoma cells in flasks for both cryopreservation and for injection to mice were performed. The hybrid cells were generated in mice and the monoclonal anti-human rhMIS (6El 1) antibodies were purified. A new affinity adsorbent based on the 6El 1 antibodies as well as the test-systems for quantitative assay of MIS were developed. 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 10⁶ cells/ampule). RPMI 1640 medium with 20% of FCS was added to the remaining cells and further cultured.

7 days later, a portion (3 x 10⁶) of these cells was cryo-preserved again to increase hybridoma preservation. The remaining cells, at a concentration of 2.5 x 10⁶ cell/mouse, were injected into 20 Balb/c 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.

The ability of hybridoma to produce anti-Holo-MIS monoclonal antibodies was studied in parallel. Culture media supernatants from the wells which contained either 0 (control) or 1 cell clone were screened. To perform screening, the solution of the HoIo-MIS antigen at a concentration of 1 μg/ml in 0.02 M borate buffer, pH 8.0 was introduced to high-sorption plates from Costar, 100 μl/well. The antigen was adsorbed for a night at 20⁰C, then the wells were washed with PBS buffer with 0.05% Tween-20, and the antibodies from the screened wells were added. The conjugate of goat anti-mouse IgG antibodies with horse radish peroxidase was added to the wells an hour after incubation. All single clones were found to be positive for anti- Holo-MIS antibodies. Three clones were transferred to flasks with RPMI 1640 medium conditioned by mouse macrophages for further cryo-preservation and generation of monoclonal antibodies.

7 days after transference of positive clones, a portion (3 x 10⁶) of cells were cryo- preserved once more to increase hybridoma preservation. The remaining cells were injected into 50 Balb/c mice, at 2.5 x 10⁶ cell/mouse. On days 12-14, 5-7 ml/mouse of ascites were taken from euthanized mice. It was demonstrated that the concentration of specific IgG antibodies in ascites came to 2 mg/ml.

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. For this purpose, 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). 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.

Balb/c mice (10 animals) were immunized with 2.5 μg of the purified antigen in 50 μl of PBS and 50 μl 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 μg 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 10⁶ lymphocytes were isolated. These lymphocytes were fused with the myeloma cells at a ratio of 24 x 10⁶ of lymphocytes to 3O x 10⁶ 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.

After 12 days, the clones were screened using the following procedure. The HoIoMIS antigen at a final concentration of 1 μg/ml in 100 μl of PBS/well was adsorbed to the plate and incubated for a night at a room temperature. A 100 μl of the culture medium from the hybridoma was diluted with 100 μl of borate buffer with Tween 20 (0.05%) and incubated with the antigen for an hour at 37°C on a shaker. After washing goat anti-mouse IgG antibodies were added to the wells and incubated for 1 hour, then after washing, o-Phenylenediamine Dihydrochloride (OPD) was added. The reaction was stopped with sulfuric acid, and the optical density of the content was measured with a reader.

Approximately, 1,000 hybrid cells were analyzed by enzyme immunoassay by the level of production of specific anti-Holo-MIS antibodies. The resultant positive hybrids were cloned and additionally assayed by Western blotting using C-terminal domain of HoIo-MIS (kindly supplied by MGH) adsorbed on nitrocellulose strips. As a result, three clones were selected. Two clones were specific to the N-terminal fragment of HoIo-MIS and one clone was specific to the C-terminal fragment. These clones were re-cloned to determine cell segregation and viability. As a result of subsequent screening with the HoIo-MIS antigen, it was demonstrated that one of the two clones specific to the N-terminal domain was segregated. The remaining clones were grown in the amounts sufficient for cryo-preservation and for generation in mice.

To produce ascites, 10 days before injecting, each mouse received i/p 200 μl of pristane. The 1 x 10⁶ 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.

Results

Monoclonal antibodies, Ml, M2, 6El 1 and polyclonal antibodies MGH-6, were compared, in immunoblotting experiments performed after electrophoresis of HoIoMIS in unreduced and reduced conditions. 6El 1 and MGH-6 antibodies were kindly supplied by MGH. HoIoMIS was purified by immunoaffϊnity chromatography using immobilized 6El 1 antibodies. 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).

As evident from Fig. I B₅ under unreduced conditions, 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.

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. To ensure that Ml recognized the C-terminal 25 kD fragment of HoIoMIS specifically, 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. 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.

ELISA Assays

To design test-systems for sandwich ELISA, conjugates of these antibodies with horse radish peroxidase (HRP) were produced. Different pair compositions of the antibodies as well as different series of antibody-antigen interactions were used, in which each of the antibodies served either as a first antibody (also known as capture antibody), or as a second antibody (as known as the labeling or recognizing antibody). In addition, the variant, in which one of the antibodies served as both a capture antibody and a conjugate of the same antibody with HRP - as a recognizing antibody, was tested. The immunoaffinity purified MIS specimen ("IAP"), kindly given by Dr. McLaughlin from MGH, served as a reference sample. ELISA was performed according to the following protocol.

High binding plates were used. 2.0-5.0 mg/ml of each monoclonal antibodies, 6El 1, Ml and M2 were prepared in PBS with 0.1 % ^"Na₃N. The following conjugates were prepared: 6El 1-HRP (0.2 mg/ml, working dilution 1 :400), Ml-HRP (0.2 mg/ml, working dilution 1 :400), M2-HRP (0.2 mg/ml, working dilution 1 :800) are in a stabilizing solution. 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) were prepared. 5 μg of the desired antibody in 100 μl of Buffer A were allowed to adsorbed into each well of the plate for 12-16 hrs and incubated at room temperature in a wet chamber. The plates were washed by rinsing each well 3 times with 150 μl of Buffer B. A 100 μl of Buffer B and 50 μl 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 μl of Buffer B. A 100 μl 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 μl of Buffer B. A 100 μl of tetramethylbenzidine (TMB) was placed into each well of a plate for 15-20 min of incubation at a room temperature to stain the conjugates. The reaction was stopped with 50 μl of IN H₂SO₄. Results were read at 450 nM. The designed test-systems were assayed for sensitivity, i.e. the ability to reliably distinguish between the lower antigen concentration and the background (Table 1).

Table 1 shows the sensitivity of the designed test systems

Results

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.

Using these test systems, sera of randomly selected donors was examined. To determine levels of MIS in these sera, two test systems were used - Ml- M2-HRP and 6El 1- M2-HRP. Ml antibodies to C-terminal MIS _. were used in the M1-M2-HRP test system and 6El 1 antibodies to N-terminal MIS were used in the 6El 1-M2-HRP as recognizing antibodies. 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

As evident from Table 2, MIS concentration in serum determined with different test- systems significantly differed, suggesting that circulating blood MIS is heterogeneous, and this heterogeneity is of unknown origin.

Purification of MIS using Monoclonal Antibodies The potential of the produced antibodies was further studied in the experiments on isolation and purification of MIS secreted by CHO B9 cells.

Using immunoaffinity chromatography with immobilized 6El 1 antibodies, the known method of MIS purification (McLaughlin, Methods Enzymol 198:358-69(1991)) from culture medium of CHO B9 cells cultivated by the method described in Stephen et al., Clinical Cancer Research, 8:2640-2646 (2002), HoIoMIS proteins, immunoaffinity purification (IAP) analogs were isolated. In total, 9 samples of purified HoIoMIS were obtained (C001-C009). See Fig. 3. In each of these purification samples, the protein was determined by double antibody sandwich ELISA assays. 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

6El 1-M2-HRP test-systems in culture supernatants of CHO B9

As evident from Table 3, HoIoMIS concentration determined with M1 -M2-HRP test- system was on the average 15% of that determined with 6El 1 - M2-HRP test-system. The results are similar to the results obtained using human sera.

Using different combinations of Ml, M2, and 6El 1 anti-MIS monoclonal antibodies, other methods of MIS purification were 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. In one set of experiments, a part of the concentrated culture medium was chromatographed with 6El 1- Sepharose to obtain COOl to C009 preparations. See Fig. 3. In another set of experiments, 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 MgCl₂ or 2-3.5M NaCNS ( or KCNS) solutions, pH 7. The eluted protein solutions were immediately dialyzed. Using this protocol, the following preparations of HoIo-MIS were obtained: C003 -1, -2, and -6; C004-1 , -6; C009-1, -6. Chaotropic agents for elution were used to avoid aggregation of proteins during storage.

To compare MIS isolation by different methods, we used supernatant concentrates of the C3, C4, C8 and C9 MIS preparations obtained from 1,000 ml of culture fluid each (see Figure 3). MIS concentrations in the purified samples were estimated by Lowry method. Based on data obtained by Lowry, the reference specimens for each of the test systems were calibrated.

MIS concentrations in all the samples, including the concentrates of culture fluids and the immunoaffinity purified protein solutions 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.

As evident from Table 4, MIS content in the initial samples (C3, C4, C8, and C9) varied from the minimal (15.6 mg/L) in C8 sample to the maximum (25.5 mg/L) in C9. After isolation of MIS with 6El 1-Sepharose, 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

C3, C4, C8, and C9 samples, varied from 75% to 93.6%.

After isolation of MIS with the original M2-Sepharose (samples C003-2-1 , C008-2-1 , and C009-2-1), the protein yield came to 87.3, 89.1, and 83.5%, respectively, i.e., was similar to that after purification with 6El 1-Sepharose. After isolation of MIS with Ml-Sepharose (samples C003-1, C008-1 , and C009-1), the protein yield was 73.0, 67.6, and 61.3%, respectively. The slightly reduced protein yield was due to an incomplete protein adsorption with Ml-Sepharose (step 1). This was confirmed by residual amounts of MIS in the samples eluted after purification with 6El 1- or M2-Sepharose (step 2) (see Figure 3). As evident from Table 4, the amount of MIS purified with Ml-Sepharose was the similar as assayed with different test systems - 6El 1-M2-HRP and M1-M2-HRP. In addition, it was demonstrated that in the samples purified with M2-Sepharose, the Ml -specific MIS content was similar to that in the samples purified with 6El 1-Sepharose (approximately 12%).

Finally, it was demonstrated that purification with 6El 1- or M2-Sepharose allowed production of a total MIS preparation only. Use of consecutive chromatographic steps allowed separation of MIS into the components and study of properties of each of the proteins.

The purified preparations were immediately analyzed by electrophoresis and by iinunoblotting with Ml antibodies. See Figs. 4A and 4B (Lane I, Sample C004-1 under reduced conditions; Lane 2, Sample C004-6 under reduced conditions; Lane 3, Sample C004-1 under non-reduced conditions; Lane 4, Sample C004-6 under non-reduced conditions).

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. In particular, in unreduced conditions there were two components, instead of one, of 140 IcD, as well as the additional component of 55 kD, which was almost absent in the specimen C004-6. In reduced conditions, in the C004-1 sample, the components of 140 kD dissociated cymbatically to those of 70 kD. In addition, 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.

When comparing the intensity of bands on lanes 3 and 4 in Fig. 4A (electrophoresis) and the same lanes in Fig. 4B, one can observe that 140 kD band was more intensive in Fig. 4B. This means that affinity of Ml antibodies to the components of C004-1 was significantly higher than that of C004-6. Notably that Ml antibodies under these experimental conditions did not recognize the 7OkD subunit (reduced conditions) in Fig. 4B, but the 70 kD band markedly appeared after treatment of the nitrocellulose strip with trypsin (see Fig.4C). In Fig. AC, the band's intensity corresponded equally to load in electrophoresis. Lanes 1 , 2, 3, 4 of Fig. 4C correspond to similarly numbered lanes in Fig. 4A.

Analysis of HoloMIS preparations by electrophoresis demonstrated that in unreduced conditions, high-molecular oligomers were present in almost all samples. As electrophoresis was performed with Sodium Dodecyl Sulfate (SDS), a denaturating agent, it was interesting to study HoIo MIS in physiological conditions. So, the specimens C003-1, -2, and -6 were analyzed by gel-permeation chromatography with Superose 12, which allowed protein separation within a range of 1,000 -300,000 D. See Fig. 5 (Chromatogram 1, Gel filtration with Superose 12 of holo MIS purified by immunoaffinity chromatography with Ml column (Sample C003-1); Chromatogram 2, GeI filtration with Superose 12 of holoMIS purifed by immunoaffinity chromatography with Ml and M2 columns (Sample C003-2); Chromatogram 3, Gel filtration with Superose 12 of holoMIS purified by immunoaffinity chromatography with Ml and 6El 1 columns (Sample C003-6); Peak A corresponds to oligomer forms of HoIoMIS (M > 10⁶); Peak B corresponds to dimer of HoIo MIS (M = 3 x 10⁵); Peak C corresponds to holoMIS (M=I .5 x 10⁵)). Chromatography conditions: column 10 x 300 mm, in 50 mM Tris- formiate buffer + 200 mM formiate ammonium, pH 8.0; flow rate 0.8 ml/min, detection 280 nm).

As evident from Fig. 5, in the samples C003-1 and -6, the distribution of HoloMIS among 1 ,000:300:140 kD was 5:28:67 (%), demonstrating that HoloMIS of 140 kD was a major portion. The C003-2 sample had two almost equal components. In this case chromatographic balance was shifted to a fraction with intermediate molecular weight. As C003-2 sample was purified with M2-Sepharose, the data allowed the conclusion that M2 monoclonal antibodies had affinities for molecules of 300 kD.

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, and lanes 6 and 13 correspond to peak C filtrate from C003-6 sample. As evident from Fig. 6, among the C003-1, -2, and -6 samples subjected to electrophoresis in unreduced conditions, only C003-1 contained immunoreactive components of 140 and 25 kD. Notably that IAP preparation (lanes 7 and 14) 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

HoloMIS and was absent on the 70 kD HoloMIS. This was confirmed for all the preparations purified using the above-mentioned procedures. MIS polyclonal antibodies however, recognized and interacted with a 7OkD species of HoloMIS. See Fig. 7 (Lanes 1-7 were subjected to electrophoresis in reduced conditions whereas lanes 8-14 were subjected to electrophoresis 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 MGH₅ 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, and lanes 6 and 13 correspond to peak C filtrate from C003-6 sample). Thus, further experiments were performed to investigate if the neodeterminant was the result of an en2ymatic attack on the intact HoIoMIS. HoIoMIS, under reduced (lanes 1-7 of Fig. 8) and unreduced conditions (lanes 8-14 of Fig. 8) was electrophoresed and transferred onto nitrocellulose sheets. Nitrocellulose sheets with immobilized HoIoMIS were treated with trypsin for 30 mins at 37°C followed by detection with Ml monoclonal antibodies. 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, and lanes 6 and 13 correspond to C003-6 sample from chromatogram 3.

The preparations of HoloMIS purified in compliance with different procedures were demonstrated to differ in molecular structure and assemblies of polypeptide chains and fragments bound either by non-covalent or covalent (S-S) bonds. In order to learn if these preparations differed in bioactivities, the preparations C003-1, -2, -6, were tested for inhibition of proliferation of M0VCAR7 cells. To assay bioactivity of MIS, M0VCAR7 cells at a concentration of 1 x 10³/well in 150 μl of culture medium were seeded over a 96-well plate. A day later, 50 μl of MlS samples, MIS standard or culture medium was added to each well. On days 4-5 of cultivation, 20 μl of (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MMT) solution was added to each well. 4 hours later, 200 μl of culture medium was removed from each well, and 100 μl of acidified isopropanol (0.1N HCl in isopropanol) was added. MMT was eluted by constant stirring of the plate for 30 min. on a shaker. The optical density of the resultant solution was measured at a wave length of 595 nm.

As a result of testing (Fig. 9), the HoloMIS preparation purified with Ml-Sepharose (C003-1), subjected to partial proteolysis, was demonstrated to be most active. 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. As evident from Fig. 9, MIS C003-1 purified with Ml antibodies was most active. Samples C003- 2 and -6 were almost similarly active.

Spontaneous Proteolysis of MIS and inhibition

Two preparations of HoIo-MIS were produced in compliance with classic procedure - immunoaffinity chromatography with 6E1 1-Sepharose (Lorenzo et al., J. Chromatogr. B, 766:89-98 (2001)). The proteins were first precipitated with ammonium sulfate from supernatants of cultivated CHO B9 cells supplied by MGH. The purified preparations were analyzed by SDS-PAAG-electrophoresis. Fig. IO shows electrophoregrams of the COOl sample in reduced conditions (Lanes 1 and 2). In this experiment the protein was analyzed -just after isolation (Lane 1) and 15 days after incubation at 37°C (Lane 2). As evident from Fig. 10, 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.

Upon storage, the sample was subjected to significant changes as demonstrated by the electrophoregram (Lane 2). The maximal intensity corresponds to 57 kD and 12.5 kD subunits which are the N- and C-term domains, respectively. This means that MIS molecule was almost fully splitted into the two key domains. In addition, a less intensive band of 35 kD was noted after storage.

The spontaneous proteolysis of HoIo-MIS was studied in detail. The preparation was kept for a long period in various conditions such as storage at either 4°C or 37°C for several hours or for several months and subsequent analysis by RP-HPLC, electrophoresis and immunoblotting. 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 37⁰C; Chromatogram 5, HoIoMIS after 32 day incubation at 37⁰C). 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 37⁰C

As evident from the data, by day 12, HoIo-MIS was almost fully proteolyzed into C- and N-terminal fragment. Further proteolysis was not observed thus indicating that this proteolysis was a highly specific reaction. At 4°C, the process of spontaneous proteolysis was significantly slower, which was typical for an enzymatic reaction, and was not completed by day 86 (final point of observation). The resultant data is shown in Table 4 and on Fig. 12 in a form of processed chromatograms.

Table 4 shows results of Spontaneous cleavage of holo MIS COOl at 4⁰C

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. Res., 23: 141 (1977); Nachtigal et al., Proc. Natl. Acad. ScL, 93:771 1-7716 (1996); Cigarroa et al. In: Growth Factors, Harwood Acad. Publisher GmbH, UK, 1 :179-191 (1989).

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 37⁰C; Lane 4, HoIo MIS after incubation for 18 days at 37⁰C with PMSF; Lane 5, HoloMIS after incubation for 18 days at 37⁰C with leupeptin; Lane 6, HoloMIS after incubation for 18 days at 37⁰C 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, Pepstatin, aprotinin, and benzamidine; Lane 1 1, Markers). Fig. 14A shows effects of various protease inhibitors on the inhibition of proteolysis of HoloMIS under unreduced conditions. Fig. 14B shows effects of various protease inhibitors on the inhibition of proteolysis of HoloMIS under reduced conditions.

Fig. 14A and B, 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).

The process, of spontaneous proteolysis of HoloMIS and its inhibition was further analyzed by immunoblotting with Ml monoclonal antibodies specific for the C-terminus of MIS See Figs. 15A and B (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°C; Lane 4, HoIo MIS after incubation for 18 days at 37°C with PMSF; Lane 5, HoIoMIS after incubation for 18 days at 37°C with leupeptin; Lane 6, HoIoMIS after incubation for 18 days at 37°C with pepstatin; Lane 7, HoIoMIS after incubation for 18 days at 37°C with aprotinin; Lane 8, HoIoMIS after incubation for 18 days at 37°C with benzamidine; Lane 9, HoIoMIS after incubation for 18 days at 37⁰C with buffer for enzymes; Lane 10, HoIoMIS after incubation for 32 days at 37°C with a cocktail of PMSF, leupeptin, Pepstatin, aprotinin, and benzamidine; Lane 11, Markers). 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.

A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications can be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims:

Claims

WHAT IS CLAIMED IS:

1. An isolated monoclonal antibody wherein the antibody specifically binds to the C- terminal domain of MlS.

2. The monoclonal antibody of claim 1, wherein the antibody further binds to a variant polypeptide having at least 95% homology to the C-terminal domain of MIS.

3. The monoclonal antibody of claim 1, wherein the antibody is produced from the hybridoma cell line deposited with American Type Culture Collection under Accession ^"Number PTA-8390.

4. The monoclonal antibody of claim 1, wherein the antibody is selected from the group consisting of a chimeric antibody, a humanized monoclonal antibody and an antibody fragment.

5. An isolated monoclonal antibody wherein the antibody specifically binds to the N- terminal domain of MIS.

6. The monoclonal antibody of claim 5, wherein the antibody further binds to a variant polypeptide having at least 95% homology to the N-terminal domain of MIS.

7. The monoclonal antibody of claim 5, wherein the antibody is produced from the hybridoma cell line deposited with American Type Culture Collection under Accession Number PT A-8391.

8. The monoclonal antibody of claim 5, wherein the antibody is selected from the group consisting of a chimeric antibody, a humanized monoclonal antibody and an antibody fragment.

9. A method of purifying recombinant MIS from host cells capable of expressing recombinant MIS comprising: binding the recombinant MIS to an antibody, the antibody being a monoclonal antibody that is capable of binding the C-terminal domain of MIS.

10. The method of claim 9, wherein the monoclonal antibody is capable of binding to a variant polypeptide having at least 95% homology to the C-terminal domain of MIS. ύ

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1 1. . The method of claim 9, wherein the monoclonal antibody is conjugated to a chromatography matrix.

12. The method of claim 1 1, wherein the chromatography matrix is Sepharose 4B.

13. The method of claim 9, further comprising recovering the recombinant MIS by eluting with sodium thiocyanate.

14. The method of claim 13, further comprising 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.

15. The method of claim 14, wherein the monoclonal antibody is capable of binding to a variant polypeptide having at least 95% homology to the N-terminal domain of MIS.

16. The method of claim 14, further comprising recovering the recombinant MIS by eluting with MgCl₂.

17. The method of claim 13, further comprising 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.

18. The method of claim 17, further comprising recovering the recombinant MIS by eluting with MgCl₂.

19. A method of purifying recombinant MIS from host cells capable of expressing recombinant MIS comprising: binding the recombinant MIS to an antibody, the antibody being a monoclonal antibody that is capable of binding the N-terminal domain of MIS.

20. The method of claim 19, wherein the monoclonal antibody is capable of binding to a variant polypeptide having at least 95% homology to the N-terminal domain of MIS.

21. A method of quantifying MIS levels comprising detecting MIS in a sample using a sandwich ELISA, wherein the first antibody is 6El 1 monoclonal antibody and the second antibody is labeled with an en2yme.

22. The method of claim 21 , wherein the first antibody is bound to a solid support.

23. The method of claim 21, wherein the second antibody is M2 monoclonal antibody.

24. The method of claim 21 , wherein the second antibody is M 1 monoclonal antibody.

25. The method of claim 21, wherein the enzyme is horseradish peroxidase.

26. A method of quantifying MIS levels comprising 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.

27. The method of claim 26, wherein the first antibody is bound to a solid support.

28. The method of claim 26, wherein the enzyme is horseradish peroxidase.

29. The method of claim 26, wherein the second antibody is 6El 1 monoclonal antibody.

30. The method of claim 26, wherein the second antibody is M2 monoclonal antibody.

31. A method of quantifying MIS levels comprising 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.

32. The method of claim 31 , wherein the first antibody is bound to a solid support.

33. The method of claim 31 , wherein the enzyme is horseradish peroxidase.

34. The method of claim 31, wherein the second antibody is 6El 1 monoclonal antibody.

35. The method of claim 31 , wherein the second antibody is Ml monoclonal antibody.

36. A method of obtaining monoclonal antibodies that specifically bind to the C- terminal domain of MIS comprising: 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.

37. A method of obtaining monoclonal antibodies that specifically bind to the N- terminal domain of MlS comprising: 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 N-terminal domain of MIS; injecting mice with selected hybrid cells that bind the N-terminal domain of MIS; harvesting ascite fluid from injected mice and isolating the monoclonal antibodies from the ascite fluid.

38. A method of inhibiting the spontaneous proteolysis of MIS comprising incubating MIS with aprotinin.

39. A composition comprising MIS and aprotinin.