RU2770003C1 - Monoclonal antibody to the c- terminal fragment of anti -muller hormone - Google Patents

Abstract

FIELD: biochemistry.SUBSTANCE: invention relates to the field of biochemistry, in particular to the monoclonal antibody ACMIS-3.EFFECT: invention makes it possible to effectively treat diseases associated with the factor ACMIS-3.3 cl, 5 dwg, 1 tbl, 5 ex

Description

The invention relates to the field of biotechnology, in particular to antibodies against the C-terminal fragment of anti-Müllerian hormone, specifically to monoclonal mouse antibodies against the C-terminal fragment of human anti-Müllerian hormone. The invention can be used in medicine, immunology, biotechnology.

Anti-Müllerian hormone (AMH) is one of the least studied members of the transforming growth factor beta cytokine superfamily. In mammalian embryogenesis, this factor directs the development of the reproductive system according to the male type, and in the postnatal period of life it is involved in the regulation of the balance of sex hormones [Gukasova N.V., Severin S.E. Protein MIS: structure, regulation of expression and molecular mechanism of action // Questions of biological, medical and pharmaceutical chemistry, 2005, 4:3-9]. Of great clinical significance is the determination of the serum level of AMH, which makes it possible to judge the magnitude of the ovarian reserve, the nature of the course of pregnancy in women [La Marca A., Sighinolfi G., Radi D., Argento C., Baraldi E., Artenisio A.C., Stabile G., Volpe A. Anti-Müllerian hormone (AMH) as a predictive marker in assisted reproductive technology (ART) // Human reproduction update, 2009, 16(2):113-130; van Rooij I.A., Broekmans F.J., te Velde E.R., Fauser B.C., Bancsi L.F., de Jong F.H., Themmen A.P. Serum anti-Müllerian hormone levels: a novel measure of ovarian reserve // Human Reproduction, 2002, 17(12):3065-3071] and the presence of normally functioning testicular tissue in the body in men [MacLaughlin D.T., Donahoe P.K. Müllerian inhibiting substance/anti-Müllerian hormone: a potential therapeutic agent for human ovarian and other cancers // Future Oncology, 2010, 6(3):391-405]. In addition, it has been shown that recombinant AMH (rAMH) has antitumor activity against cells that have specific type II AMH receptors (MISRII) on their surface, inducing apoptosis in them [Teixeira J., Maheswaran S., Donahoe P.K. Mullerian inhibiting substance: an instructive developmental hormone with diagnostic and possible therapeutic applications // Endocrine Reviews, 2001, 22(5):657-674].

The human AMH gene is localized on the small arm of the 19th chromosome and consists of 2.75 kb [Cate R.L., Mattaliano R.J., Hession C., Tizard R., Farber N.M., Cheung A., Ninfa E.G., Frey A.Z., Gash D.J. , Chow E.P., Fisher R.A., Bertonis J.M., Torres G., Wallner B.P., Ramachandran K.L., Ragin R.C., Manganaro T.F., Maclaughlin D.T., Donahoe P.K. Isolation of the bovine and human genes for Müllerian inhibiting substance and expression of the human gene in animal cells // Cell, 1986, 45(5):685-698]. It contains 5 exons and 4 introns and encodes the AMH precursor polypeptide (the so-called pro-AMH), consisting of 560 amino acid residues, which, in addition to AMH (25-560), also includes the leader and signal sequences (1-24) [Rey R., Lukas-Croisier C., Lasala C., Bedecarrás P. AMH/MIS: what we know already about the gene, the protein and its regulation // Mol. cell. Endocrinol, 2003, 211(1):21-31]. In the present description, when referring to amino acid positions, we refer to the amino acid sequence of the full-length AMH precursor, which is presented in the UniProt database [http://www.uniprot.org/uniprot/P03971.

The AMH glycoprotein molecule has a mass of about 140 kDa and is a homodimer. It has been shown that AMH in vivo undergoes cleavage into two main fragments, which are N- and C-terminal homodimers with a molecular weight of 115 and 25 kDa, respectively [Cimino I., Casoni F., Liu X., Messina A., Parkash J ., Jamin SP, Catteau-Jonard S., Collier F., Baroncini M., Dewailly D., Pigny P., Prescott M., Campbell R., Herbison AE, Prevot V., Giacobini P. Novel role for anti- Müllerian hormone in the regulation of GnRH neuron excitability and hormone secretion // Nature communications, 2016, 7:10055]. The site of specific proteolysis in each polypeptide chain of the full-length homodimer is located between the amino acid residues Arg451 and Ser452 [Pepinsky RB, Sinclair LK, Chow EP, Mattaliano RJ, Manganaro TF, Donahoe PK, Cate RL Proteolytic processing of mullerian inhibiting substance produces a transforming growth factor-beta -like fragment // Journal of Biological Chemistry, 1988, 263(35):18961-18964]. After such splitting, the AMH molecule enters the active state. At the same time, the C-terminal fragment of AMH (C-AMH), acting as an individual molecule or in a non-covalent complex with an N-terminal fragment, binds to the type II AMH receptor - MISRII - and transmits an activating signal into the cell [di Clemente N., Jamin SP , Lugovskoy A., Carmillo P., Ehrenfels C., Picard JY, Whitty A., Josso N., Pepinsky RB, Cate RL Processing of anti-mullerian hormone regulates receptor activation by a mechanism distinct from TGF-β // Molecular Endocrinology , 2010, 24(11):2193-2206].

Given the possible molecular transformations of the AMH molecule in vivo and in vitro , as well as the diagnostic importance of determining the serum level of AMH for assessing human reproductive potential, it seems important to develop new tools for the detection of AMH derivatives circulating in the blood of patients. In addition, in view of the possibility of using rAMH as an antitumor drug, the development of immunochemical tools for assessing the quality of the resulting rAMH preparations is also very relevant.

Differential detection of AMH and rAMH derivatives in vivo and in vitro can be achieved by obtaining a panel of monoclonal antibodies that recognize various fragments of the AMH molecule and developing new test systems based on them. In addition, various monoclonal antibodies can be used for the synthesis of immunosorbents suitable for isolating individual components from a complex mixture of AMH molecular derivatives, as well as for studying their properties.

From WO2008153433 monoclonal antibodies M1 are known that recognize the C-terminal domain of AMH both in the form of a 25 kDa dimer and a 12.5 kDa monomer.

From US Pat. No. 7,897,350, a composition is known for detecting AMH in mammalian tissue samples, in particular, a F2B12H monoclonal antibody is described that recognizes a proteolysis-resistant human and rat AMH C-terminal fragment under non-reducing conditions.

In the article: Mamsen L.S., Petersen T.S., Jeppesen J.V., Møllgård K., Grøndahl M.L., Larsen A., Ernst E., Oxvig C., Kumar A., Kalra B., Andersen C.Y. Proteolytic processing of anti-Müllerian hormone differs between human fetal tests and adult ovaries // MHR: Basic science of reproductive medicine, 2015, 21(7):571-582 describes monoclonal antibodies AMH-1 and AMH-29 to the C-terminal fragment AMHs that recognize the AMH Val491-Arg502 sequence.

R&D Systems, Inc. produces mouse monoclonal antibodies obtained by immunization with the C-terminal fragment of recombinant human AMH Ala453-Arg560 (Cat# MAB1737).

MyBioSource, Inc. produces mouse monoclonal antibodies obtained by immunization with the C-terminal fragment of recombinant human AMH Ala453-Arg560 (Cat# MBS2001312, Cat# MBS2085832).

Genetex, Inc. produces mouse monoclonal antibodies 5/6 (Cat# GTX42793, Cat# GTX42794) obtained by immunization with a synthetic peptide corresponding to the human AMH Val527-Cys557 sequence.

Abcam launches murine monoclonal antibody MM0475-7H26 (Cat# ab90241) to the C-terminal fragment of human AMH.

The technical problem solved by the authors was to expand the range of antibodies capable of recognizing various epitope regions of the C-terminal fragment of human AMH.

The technical result was achieved by creating monoclonal antibodies ACMIS-1, ACMIS-3, ACMIS-4 and ACMIS-6, which specifically and with high affinity bind to various epitope regions of the C-terminal fragment of human AMH. Moreover, the ACMIS-1 and ACMIS-4 antibodies bind to the SAGATA sequence (Ser452-Ala457 of human AMH), the ACMIS-6 antibody binds to the conformational determinant of human AMH, and the ACMIS-3 antibody recognizes a linear region both in the 70 kD AMH chain and in the 25 kD and 12.5 kD of the C-terminal fragment of AMH and inhibits its binding to the MISRII receptor.

The monoclonal antibody ACMIS-1 contains the variable region of the heavy chain according to Seq ID No: 3 and the variable region of the light chain according to Seq ID No: 4, as well as the hypervariable regions of the variable region of the heavy chain according to Seq ID No: 5-7 and the hypervariable regions of the variable region of the light chains according to Seq ID No: 8-10.

The monoclonal antibody ACMIS-3 contains the variable region of the heavy chain according to Seq ID No:13 and the variable region of the light chain according to Seq ID No:14, as well as the hypervariable regions of the variable region of the heavy chain according to Seq ID No:15-17 and the hypervariable regions of the variable region of the light chains according to Seq ID No:18-20.

The monoclonal antibody ACMIS-4 contains the variable region of the heavy chain according to Seq ID No:23 and the variable region of the light chain according to Seq ID No:24, as well as the hypervariable regions of the variable region of the heavy chain according to Seq ID No:25-27 and the hypervariable regions of the variable region of the light chains according to Seq ID No:28-30.

The monoclonal antibody ACMIS-6 contains the variable region of the heavy chain according to Seq ID No: 33 and the variable region of the light chain according to Seq ID No: 34, as well as the hypervariable regions of the variable region of the heavy chain according to Seq ID No: 35-37 and the hypervariable regions of the variable region of the light chains according to Seq ID No:38-40.

The resulting monoclonal antibodies ACMIS-1 and ACMIS-4 bind specifically and with high efficiency to the N-terminal peptide C-rAMH SAGATA (Ser452-Ala457), but do not bind to C-rAMH in the 70 kDa monomer and do not inhibit C- rAMH with MISRII. The ACMIS-6 antibody binds specifically and with high efficiency to the conformational epitope of C-rAMH and does not bind to the denatured protein, nor does it inhibit the binding of C-rAMH to MISRII. The ACMIS-3 antibody binds to the dimeric and monomeric form of C-rAMH, as well as to the C-terminal region of rAMH in the 70 kDa monomer, in addition, this antibody is able to suppress the binding of C-rAMH to recombinant MISRII.

The technology for obtaining antibodies included the immunization of animals with an antigen, which is a purified C-rAMH, which was obtained according to the method described in the article [Cancer A.Ya., Trofimov A.V., Protasov E.A., Simbirtsev A.S. , Ishchenko A.M. Comparative study of the properties of activated recombinant human anti-Mullerian hormone // Russian Journal of Immunology, 2017, 11(4):755-757; patent for invention RU 2616273]. As antigens for the selection of positive producers of monoclonal antibodies in the production of enzyme-linked immunosorbent assay (ELISA), the same C-rAMH preparation was used as for immunization.

The invention is illustrated by the following drawings:

On FIG. 1A shows the electrophoregram of samples of preparations of the obtained antibodies. A sample of normal mouse IgG immunoglobulins was used as a positive control. M - markers of molecular weights (kDa).

On FIG. 1B, C show photographs of membranes obtained as a result of Western blot analysis of a sample of a mixture of whole and single chain fragmented rAMH (B) and C-rAMH (C). Normal mouse IgG immunoglobulins were used as a negative control. M - markers of molecular weights (kDa).

On FIG. 1D shows the binding curve of antibodies of the ACMIS group with adsorbed C-rAMH.

On FIG. 2 shows the analysis of the specificity of the obtained antibodies to the peptides SAGATA, RSAGATA and AGATA in direct solid-phase ELISA.

On FIG. Figure 3 shows the electropherogram of samples of two eluates obtained during the isolation of rAMH from the culture liquid using the ACMIS-1 immunosorbent. The arrows indicate the bands corresponding to the homodimeric (left) and monomeric (right) forms of C-rAMH. M - markers of molecular weights (kDa).

On FIG. 4. A shows a calibration curve for determining the concentration of C-rAMH in a sandwich ELISA based on ACMIS-3 antibodies and ACMIS-4-Px peroxidase conjugate.

On FIG. 4. B shows the calibration curve for determining the concentration of C-rAMH in a sandwich ELISA based on the MISRII+Fc chimeric protein and the ACMIS-4-Px peroxidase conjugate.

On FIG. 5 shows the binding curve of C-rAMH to the MISRII+Fc chimeric protein in the presence of ACMIS-3 antibodies.

The essence and industrial applicability of the invention are illustrated by the following examples.

Example 1 Preparation of antigens for immunization and screening of hybridomas, immunization of mice and selection of hybridomas.

1.1. Obtaining antigens. For immunization, a highly purified preparation of the C-terminal fragment of recombinant human AMH (C-rAMH), obtained according to a previously developed method [Cancer A.Ya., Trofimov A.V., Protasov E.A., Simbirtsev A.S., Ishchenko A. .M. Comparative study of the properties of activated recombinant human anti-Mullerian hormone // Russian Journal of Immunology, 2017, 11(4):755-757; patent for invention RU 2616273]. Briefly, CHO cells (ATCC, USA) transfected with the human AMH genome (CHO-MIS#26 strain) were grown on CDM4CHO serum-free medium (HyClone, USA). To isolate C-rAMH from the culture liquid, the method of immunoaffinity chromatography was first used on a sorbent prepared on the basis of cyanobromine-activated Sepharose FF (GE, Sweden) and monoclonal antibodies 6E11 [Hudson P.L., Dougas I., Donahoe P.K., Cate R.L., Epstein J. , Pepinsky R.B., MacLaughlin D.T. An immunoassay to detect human mullerian inhibiting substance in males and females during normal development. The Journal of clinical endocrinology and metabolism, 1990, 70(1):16-22] against AMH. After applying the culture liquid to a column equilibrated with phosphate-buffered saline (20 mM, pH 7.4), it was washed with five volumes of phosphate-buffered saline containing 1 M NaCl to remove non-specifically bound molecules, and then eluted with a solution of 0, 1 M glycine pH 2.5 at a flow rate of 1 ml/min. Further, the isolation of C-rAMH from the resulting eluate was carried out by the reverse phase chromatography method using an Agilent 1260 chromatograph equipped with a C18 Jupiter Phenomenex column 4.6x250 mm in size with a pore diameter of 300Å (GE Healthcare, Sweden). The elution was carried out in a gradient system: trifluoroacetic acid - acetonitrile 20-70% for 15 min, at a flow rate of 1.5 ml/min. Peak detection during chromatography was performed at a wavelength of 280 nm.

A preparation containing whole rAMH and a hormone fragmented along one chain was purified by immunoaffinity chromatography on a sorbent prepared on the basis of cyanobrom-activated sepharose FF (GE, Sweden) and monoclonal antibodies 6E11 against AMH. After applying the culture liquid to a column equilibrated with phosphate-buffered saline (20 mM, pH 7.4), it was washed with five volumes of phosphate-buffered saline containing 1 M NaCl to remove non-specifically bound molecules, and then eluted with a solution of 0, 1 M glycine pH 2.5 at a flow rate of 1 ml/min. During chromatography, peaks were detected at a wavelength of 280 nm. [Lorenzo H. K., Teixeira J., Pahlavan N., Laurich V. M., Donahoe P. K., MacLaughlin D. T. New approaches for high-yield purification of Müllerian inhibiting substance improve its bioactivity // Journal of Chromatography B, 2002, 766(1):89- 98; patent for invention RU 2616273].

1.2. Immunization of mice and screening for hybridoma. To obtain monoclonal antibodies, male Balb/c mice were used. Immunization of animals was carried out with purified C-rAMH, obtained according to p. Hybridomas were obtained according to the Milstein-Kohler method, for which animal lymphocytes taken on the 4th day after repeated immunization were mixed with mouse myeloma cells of the Sp2/0 line (at the rate of 50,000 cells per well of a 96-cell plate) in a ratio of 2:1 in the presence of polyethylene glycol with a molecular weight of 1500 Da. Cultivation of cells after fusion was carried out in selective HAT medium (Sigma, United States) in plates with daily mouse peritoneal macrophages. Primary screening of clones was performed 10 days later using direct ELISA. To do this, the antigen (C-rAMH) was immobilized in the wells of the plate by sorption from a solution with a concentration of 1.5 μg/ml in 20 mM borate buffer pH 8.0 for 20 hours at room temperature, then the culture liquid containing the studied antibodies was added, and after 1 hour, a solution (1:4000) of the second antibody, a peroxidase conjugate of goat antibodies against the Fc fragment of mouse IgG immunoglobulins (Sigma, USA), was added.

Cell clones selected from the primary screen, named ACMIS-1, ACMIS-3, ACMIS-4, and ACMIS-6, were re-cloned to test the stability of antibody secretion. For this, clone cells were selected and seeded at the rate of 2 cells per well of a 96-well plate with daily mouse peritoneal macrophages. After 10 days of cultivation, the clones were re-screened to confirm the specificity of the antibodies secreted by them. The cells were then subcultured into culture flasks with an area of 125 cm² with peritoneal mouse macrophages for extension for cryopreservation and/or administration to animals. Further, to generate antibodies, the cells of each hybridoma clone were intraperitoneally injected into mice at the rate of 2 million cells per mouse.

Example 2 Study of the properties of monoclonal antibodies ACMIS-1, ACMIS-3, ACMIS-4 and ACMIS-6.

2.1. Purification of monoclonal antibodies. Antibodies were isolated from ascitic fluids of mice collected 10 days after the introduction of hybridoma cells using protein A affinity chromatography using the MabSelect sorbent (GE, USA) according to the standard protocol according to the instructions. Normal mouse IgG immunoglobulins (Sigma, USA) were used as control antibodies in the assays.

2.2. Study of the molecular properties of antibodies. Denaturing electrophoresis of proteins in polyacrylamide gel was carried out according to the previously described method [Walker J.M. Gradient SDS polyacrylamide gel electrophoresis of proteins. In: Basic protein and peptide protocols. Springer: New York, 1994:35-38]. We used a separating gel with a gradient concentration of acrylamide of 4–20% and a concentrating gel with an acrylamide concentration of 5%. Electrophoresis results were visualized using standard Coomassie G-250 staining. Isotyping of monoclonal antibodies was performed using the Rapid ELISA Mouse Isotyping Kit (ThermoScientific, USA) according to the manufacturer's protocol. The results of isotyping (table 1) and electrophoretic analysis of purified antibodies (Fig. 1. A) showed that all obtained antibodies are IgG immunoglobulins, but belong to different subclasses.

Table 1. Isotypes of obtained antibodies.

Name of the antibody ACMIS-1 ACMIS-3 ACMIS-4 ACMIS-6 Antibody isotype IgG-2a IgG-2a IgG-2b IgG-3

2.3. Study of the specificity of monoclonal antibodies. The specificity of the obtained antibodies was analyzed using a Western blot according to the standard protocol [Mahmood T., Yang P.C. Westernblot: technique, theory, and troubleshooting // North American journal of medical sciences, 2012, 4(9):429]. Both full-length and single-chain fragmented rAMH (Fig. 1. B) and C-rAMH (Fig. 1. C) were used as antigens. For protein transfer, a buffer solution containing 47.9 mM Tris-HCl, 38.6 mM glycine, and 20% methanol and a 0.45 µm nitrocellulose membrane was used. The transfer was carried out for 1 hour at a current of 350 mA, after which the membrane was incubated in a blocking solution (2% BSA in 20 mm phosphate buffer solution, pH 7.4) overnight at +4°C. Then, a solution of primary antibodies (5 μg/ml) in a blocking solution was added; normal mouse immunoglobulins were used as a negative control. After one hour of incubation and two washings, the membrane was also treated for an hour with a solution of a peroxidase conjugate of goat antibodies against mouse IgG immunoglobulins (Sigma, USA) at a concentration of 0.5 µg/ml. To develop the staining, a 0.05% solution of diaminobenzidine (DAB) (Sigma, USA) in phosphate buffer containing 1% dimethyl sulfoxide (Sigma, USA) and 1% hydrogen peroxide (NevaReaktiv, Russia) was used.

The ability of the obtained antibodies to bind to C-rAMH adsorbed on a microtiter plate was shown in direct solid-phase ELISA (Fig. 1. D). When conducting ELISA, capturing antibodies or antigen were absorbed into the wells of the tablet at a concentration of 1.5 μg/ml in 20 mm borate buffer (pH 8.0) overnight at room temperature. At the next stage of the analysis, after washing three times, antigen or antigen-specific antibodies were introduced into the wells, which were incubated for an hour at +37°C. Then, a solution of a peroxidase conjugate of antibodies specific to the previous ELISA component was added to the washed wells at a concentration of 0.5 μg/ml, and incubation was also carried out for an hour at +37°C. Enzyme immunoassay was recorded at a wavelength of 450 nm using a standard method using tetramethylbenzidine and a BioRad Reader Model 680 microplate photometer (BioRad, USA).

2.4. Study of the epitope specificity of monoclonal antibodies. To determine the epitope specificity of the obtained antibodies, we used those obtained by solid-phase synthesis according to the previously proposed protocol [Coin I., Beyermann M., Bienert M. Solid-phase peptide synthesis: from standard procedures to the synthesis of difficult sequences // Nature protocols, 2007, 2 (12):3247] biotinylated peptides with the amino acid sequences SAGATA-(CH ₂ ) ₁₀ -biotin, RSAGATA-(CH ₂ ) ₁₀ -biotin and AGATA-(CH ₂ ) ₁₀ -biotin. For this purpose, peptides containing portions of the amino acid sequence of the N-terminal part of C-rAMH were synthesized and then biotinylated. The SAGATA peptide (Ser452-Ala457) exactly repeats the N-terminal region of C-rAMH, while the RSAGATA (Arg451-Ala457) and AGATA (Ala453-Ala457) peptides differ in that the first of them has one additional amino acid residue Arg (R) before the terminal the Ser (S) residue, while the second peptide lacks this Ser (S) residue. The ACMIS-1 and ACMIS-4 antibodies have been shown to be highly specific for the SAGATA peptide. This is evidenced by the results of direct solid-phase ELISA (Fig. 2), in which streptavidin was adsorbed on a microtiter plate at a concentration of 1.5 μg/ml, then solutions of biotinylated peptides were added: SAGATA, AGATA or RSAGATA (100 ng/ml), then solutions test antibodies (10 mg/ml) and finally an anti-species peroxidase conjugate. At the same time, as can be seen from Fig. 2, there is no specificity of the ACMIS-1 and ACMIS-4 antibodies for the other two peptides. These data indicate that the ACMIS-1 and ACMIS-4 antibodies recognize an epitope that is available during specific proteolysis of the rAMH molecule and can be used to isolate and detect C-rAMH and single-chain fragmented rAMH. The ACMIS-3 and ACMIS-6 antibodies are not specific for the SAGATA, AGATA, and RSAGATA peptides and seem to recognize other epitope regions within C-rAMH. This makes it possible to use them in sandwich ELISA for the detection of C-rAMH or hormone fragmented along one chain as paired antibodies to ACMIS-1 and ACMIS-4.

2.5. Study of the spectrum of rAMH derivatives that bind to monoclonal antibodies. In order to study the spectrum of rAMH derivatives capable of binding to the obtained antibodies, immunosorbents were prepared on their basis, which were then used to isolate rAMH from the culture fluid of producing cells at the stage of preliminary purification of the hormone. Isolation of rAMH preparations from the culture fluid of producer cells was carried out by immunoaffinity chromatography. Immunosorbents were obtained by immobilizing the obtained monoclonal antibodies on cyanobrom-activated Sepharose FF (GE, Sweden). The column equilibrated with phosphate buffer (PB) was washed with five volumes of equilibration buffer after applying the culture liquid free of producer cells. Further, to remove non-specifically bound proteins, one volume of PB containing 1 M NaCl was passed, after which the PB column was again equilibrated and elution was performed with a solution of 0.1 M glycine, pH 2.5, at a flow rate of 1 ml/min. During chromatography, peaks were detected at a wavelength of 280 nm. On FIG. Figure 3 shows the electropherogram of two rAMH preparations obtained by affinity chromatography using the ACMIS-1 immunosorbent. It can be seen that, in addition to C-rAMH, the samples also contain a hormone fragmented along one chain, which can be isolated from these preparations during subsequent purification by immunoaffinity chromatography on sorbents prepared on the basis of antibodies to the N-terminal fragment of rAMH.

3. Using the obtained antibodies to determine the amount of AMH in biological fluids. Two test systems were developed using the obtained antibodies, one of which is designed to determine the concentration of C-rAMH, and the other to test its ability to interact with MISRII.

3.1. ELISA-based test system using monoclonal antibodies specific for various AMH epitopes. In the test system, the assay includes adsorption of ACMIS-3 antibodies at a concentration of 1.5 μg/ml in 20 mM borate buffer (pH 8.0), then the introduction of samples containing C-rAMH, and then the peroxidase conjugate of ACMIS-4-Px antibodies (0.5 µg/ml). On FIG. 4, A shows the calibration curve obtained using the developed test system. The sensitivity of this test system was 30 pg/ml.

3.1. ELISA-based test system using MISRII+Fc. The key component of the test system, which allows testing the ability of C-rAMH to interact with the recombinant MISRII receptor, was the chimeric recombinant MISRII+Fc protein previously obtained in the laboratory. It consists of the extracellular part of the specific AMH receptor - MISRII - and the Fc fragment of human IgG1 immunoglobulin [Cancer A.Ya., Trofimov A.V., Pigareva N.V., Simbirtsev A.S., Ishchenko A.M. Monoclonal antibodies against the human anti-Mullerian hormone receptor as a new tool for the diagnosis and therapy of cancer // Cytokines and inflammation, 2017, 16(3):58-61]. Analysis using this test system at the first stage includes the sorption of I-4 antibodies specific to the Fc fragment of human IgG at a concentration of 1.5 µg/ml in 20 mM borate buffer (pH 8.0). I-4 are capture antibodies for the receptor chimeric construct and orient its MISRII-containing portion towards the ligand. At the next stage, a solution of a chimeric MISRII-containing construct (100 ng/ml) is introduced into the wells of the plate as a specific acceptor for the tested rAMH preparations. After the final incubation of the reaction mixture, the detection of receptor-bound rAMH preparations is carried out using the ACMIS-4-Px antibody peroxidase conjugate (0.5 μg/ml). The calibration curve of the C-rAMH preparation using this test system is shown in Fig. 4, B.

Example 4. Inhibitory activity of monoclonal antibodies against the binding of AMH to a specific MISRII receptor. Further study of the properties of ACMIS-3 monoclonal antibodies showed that they are not only specific for C-rAMH, but also

have the ability to block the interaction of the hormone with MISRII. The use of the test system described in paragraph 3.1. made it possible to show that the intensity of the interaction of C-rAMH with a specific receptor decreases in the presence of ACMIS-3 antibodies (10 μg/ml) (Fig. 5). In the control experiment for incubation with C-rAMH, normal mouse IgG immunoglobulins were used instead of ACMIS-3 antibodies.

Example 5 Synthesis and Sequencing of DNA Encoding Variable Parts of Light and Heavy Chains of Monoclonal Antibodies

5.1. Synthesis and sequencing of DNA encoding the variable portions of the light and heavy chains of monoclonal antibodies ACMIS-1. RNA was isolated from ACMIS-1 hybridoma cells, on the matrix of which, using sets of synthetic primers (Immunogenetics Information System http://www.imgt.org), DNA fragments encoding the variable regions of the heavy (VH) and light (VL) antibody chains were amplified . The resulting DNA fragments were cloned into the pALTA vector (Evrogen) and sequenced from external primers (M13). The sequences of DNA fragments encoding VH and VL are shown in Seq ID No: 1 and Seq ID No: 2, the calculated amino acid sequences of VH and VL are shown in Seq ID No: 3 and Seq ID No: 4. Sequence analysis of the amino acid sequences of the variable regions of the heavy and light chains of the monoclonal antibody ACMIS-1 was performed according to Kabat [Kabat E.A., Wu T.T., Perry H., Gottesman K. And Foeller C. Sequences of Proteins of Immunological Interest, Fifth Edition. NIH Publication, 1991, 91:3242], which made it possible to isolate the CDR regions that determine the complementarity of the antibody to the antigen.

5.2. Synthesis and sequencing of DNA encoding the variable parts of the light and heavy chains of monoclonal antibodies ACMIS-3, ACMIS-4 and ACMIS-6 was carried out as described in paragraph 5.1., while isolating RNA from the cells of the corresponding clone producing monoclonal antibodies.

Based on the above data, the obtained monoclonal antibodies against human C-rAMH can be divided into three independent groups according to specificity. The first group includes antibodies ACMIS-1 and ACMIS-4, which recognize the N-terminal peptide C-rAMH SAGATA (Ser452-Ala457, Fig. 2.), but do not bind to C-rAMH in the 70 kDa monomer (Fig. 1. B) and do not inhibit the binding of C-rAMH to MISRII. The ACMIS-6 antibody can be assigned to the second group, since it is specific to the conformational epitope of C-rAMH and does not bind to the denatured protein (Fig. 1b). The properties of the ACMIS-3 antibody, which can conditionally be attributed to the third group, are of great interest from the point of view of studying the biochemistry of C-rAMH. This antibody is capable of binding not only to the dimeric and monomeric form of C-rAMH, but also to the C-terminal region of rAMH in the 70 kDa monomer (Fig. 1b). Another unique property of the ACMIS-3 antibody is the ability to inhibit the binding of C-rAMH to recombinant MISRII (FIG. 5).

None of the obtained antibodies is capable of recognizing C-rAMH as part of a full-sized hormone molecule. This fact indicates that all obtained antibodies are specific for neodeterminants of only proteolyzed rAMH and its C-terminal fragment.

Thus, the obtained antibodies of the ACMIS group against human C-rAMH can be used to isolate rAMH, as well as to create diagnostic systems for detecting C-AMH in biological fluids and studying the biochemistry of the hormone.

The monoclonal antibodies of the present invention can serve as a basis for the construction of chimeric and humanized antibodies suitable for the creation of drugs.

The Applicant asks to consider the submitted materials of the application "Monoclonal antibody to the C-terminal fragment of anti-Müllerian hormone" in order to issue a patent of the Russian Federation for the invention.

Sequence listing

IP Office :RU

Application number 2021109265

Submission date : 2021-04-05

Name of the invention

Language Name of the invention EN Monoclonal antibody to the C-terminal fragment of anti-Müllerian hormone

Applicant and Inventor :

Applicant's name : Federal State Unitary Enterprise "State Research Institute of Highly Pure Biological Products" of the Federal Medical and Biological Agencies

Language : RU

Name or given name and surname in Latin : Federal State Unitary Enterprise “State Research Institute of Highly Pure Biopreparations” of the Federal Medical and Biological Agency

Sequences

Sequence 11 "DNA encoding the variable region of the heavy chain of the monoclonal antibody ACMIS-3"

Length Molecule type organism Contains fragments of DNA and RNA Missing Sequence 351 DNA Mus muscle Not Not

Characteristics

Feature Key Feature location Qualifiers SOURCE 1..351 MOL_TIPE=genomic DNA
ORGANISM = Mus musculus

--->

Subsequence

caggttcagc tgcagcagtc tggacctgag ctggtgaagc ctggggcttc agtgaagatg 60

tcctgcaagg cttctggata cacattcact gactatgtta taagctgggt gaaggagaga 120

actggacagg gccttgagtg gattggagag attcatcctg gaagtggtag tatttattac 180

aatgagaagt tcaagggcaa ggccacactg actgcagaca aatcctccaa cacagcctac 240

atgcagctca gcagcctgac atctgaggac tctgcggtct atttctgtgc agagacagct 300

cgggctacgt cgggctactg gggccaaggc accactctca cagtctcctc a 351

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Sequence 12 "DNA encoding the light chain variable region of the monoclonal antibody ACMIS-3"

Length Molecule type organism Contains fragments of DNA and RNA Missing Sequence 336 DNA Mus muscle Not Not

Characteristics

Feature Key Feature location Qualifiers SOURCE 1..336 MOL_TIPE=genomic DNA
ORGANISM = Mus musculus

--->

Subsequence

gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60

atctcttgca gatctagtca gagcattgta catagtaatg gaaacaccta tttagaatgg 120

tacctgcaga aaccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180

tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240

agcagagtgg aggctgagga tctgggagtt tattactgct ttcaaggttc acatgttccg 300

ctcacgttcg gtgctgggac caagctggag ctgaaa 336

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Sequence 13 "ACMIS-3 monoclonal antibody heavy chain variable region"

Length Molecule type organism Contains fragments of DNA and RNA Missing Sequence 117 AA Mus muscle Not Not

Characteristics

Feature key Feature location Qualifiers SOURCE 1..117 MOL_TYPE= protein
ORGANISM = Mus musculus

--->

Subsequence

QVQLQQSGPE LVKPGASVKM SCKASGYTFT DYVISWVKER TGQGLEWIGE IHPGSGSIYY 60

NEKEKGKATL TADKSSNTAY MQLSSLTSED SAVYFCAETA RATSGYWGQG TTLTV SS 117

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Sequence 14 "ACMIS-3 Monoclonal Antibody Light Chain Variable Region"

Length Molecule type organism Contains fragments of DNA and RNA Missing Sequence 112 AA Mus muscle Not Not

Characteristics

Feature key Feature location Qualifiers SOURCE 1..112 MOL_TYPE=protein
ORGANISM = Mus musculus

--->

Subsequence

DVLMTQTPLS LPVSLGDQAS ISCRSSQSIV HSNGNTYEW YLQKPGQSPK LLIYKVSNRF 60

SGVPDRFSGS GSGTDFTLKI SRVEAEDLGV YYCFQGSHVP LTFGAGTKLE LK 112

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Sequence 15 "Hypervariable region of the heavy chain of a monoclonal antibody ACMIS-3 (CDRH-1)"

Length Molecule type organism Contains fragments of DNA and RNA Missing Sequence eight AA Mus muscle Not Not

Characteristics

Feature key Feature location Qualifiers SOURCE 1..8 MOL_TYPE=protein
ORGANISM = Mus musculus

--->

Subsequence

GYTFTDYV 8

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Sequence 16 "Hypervariable region of the heavy chain of the monoclonal antibody ACMIS-3 (CDRH-2)"

Length Molecule type organism Contains fragments of DNA and RNA Missing Sequence eight AA Mus muscle Not Not

Characteristics

Feature key Feature location Qualifiers SOURCE 1..8 MOL_TYPE=protein
ORGANISM = Mus musculus

--->

Subsequence

IHPGSGSI 8

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Sequence 17 "Hypervariable region of the heavy chain of the monoclonal antibody ACMIS-3 (CDRH-3)"

Length Molecule type organism Contains fragments of DNA and RNA Missing Sequence ten AA Mus muscle Not Not

Characteristics

Feature Key Feature location Qualifiers SOURCE 1..10 MOL_TYPE=protein
ORGANISM = Mus musculus

--->

Subsequence

AETARATSGY 10

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Sequence 18 "ACMIS-3 Monoclonal Antibody Light Chain Hypervariable Region (CDRL-1)"

Length Molecule type organism Contains fragments of DNA and RNA Missing Sequence eleven AA Mus muscle Not Not

Characteristics

Feature Key Feature location Qualifiers SOURCE 1..11 MOL_TYPE=protein
ORGANISM = Mus musculus

--->

Subsequence

QSIVHSNGNT Y 11

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Sequence 19 "ACMIS-3 Monoclonal Antibody Light Chain Hypervariable Region (CDRL-2)"

Length Molecule type organism Contains fragments of DNA and RNA Missing Sequence 3 AA Mus muscle Not Not

Characteristics

Feature Key Feature location Qualifiers SOURCE 1..3 MOL_TYPE=protein
ORGANISM = Mus musculus

--->

Subsequence

KVS 3

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Sequence 20 "Monoclonal antibody light chain hypervariable region (CDRL-3)"

Length Molecule type organism Contains fragments of DNA and RNA Missing Sequence nine AA Mus muscle Not Not

Characteristics

Feature key Feature location Qualifiers SOURCE 1..9 MOL_TYPE=protein
ORGANISM = Mus musculus

--->

Subsequence

FQGSHVPLT 9

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Claims (3)

1. Monoclonal antibody ACMIS-3 that specifically binds to the C-terminal fragment of human anti-Mullerian hormone and inhibits its binding to the MISRII receptor, containing heavy chain hypervariable regions with sequences of SEQ ID NO: 15-17 and light chain hypervariable regions with sequences of SEQ ID NO: 18-20. 2. The monoclonal antibody of claim 1 having the heavy chain variable region sequence of SEQ ID NO: 13 and the light chain variable region sequence of SEQ ID NO: 14. 3. Monoclonal antibody according to claim 2, encoded by DNA containing the nucleotide sequences according to SEQ ID NO: 11 and SEQ ID NO: 12.

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