Modified Müllerian duct inhibitor (MIS) proteins and their use for the treatment of diseases

Abstract

To provide modified recombinant human Mullerian Inhibiting Substance (MIS) proteins which have improved cleavage, increased bioactivity, and increased potency, as compared to wild-type human MIS proteins.SOLUTION: Provided is a recombinant MIS protein comprising a combination of a modification of a non-MIS leader sequence or a functional fragment thereof in place of the MIS leader sequence in a particular amino acid sequence and a modification of at least one amino acid in a particular amino acid sequence, to increase cleavage as compared to in the absence of a modification, the recombinant MIS protein having increased cleavage and increased yield of production in vitro as compared to wild-type MIS protein.SELECTED DRAWING: Figure 1B

Description

Technical Field

[0001] Cross-reference of related applications This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 61 / 777,135, filed Mar. 12, 2013, the entire content of which is incorporated herein by reference.

[0002] Sequence List This application is electronically submitted in ASCII format and includes a Sequence Listing, which is incorporated herein by reference in its entirety. The ASCII copy, created on Mar. 12, 2014, is named 030258-076964-PCT_SL.txt and is 28,114 bytes in size.

[0003] Field of Invention The present invention relates to an improved cleavage and an improved recombinant human MIS protein having increased biological activity and increased potency compared to the wild-type human MIS protein. In certain embodiments, the recombinant human MIS protein comprises at least one of the following components: an improved Kex cleavage site for increasing cleavage, a FLAG tag, and a non-MIS leader sequence replacing the normal MIS leader sequence. Another aspect of the present invention relates to methods, uses, and kits comprising a recombinant human MIS protein for the treatment of cancer, such as cancer expressing MIS receptor type II (MISRII), or for the treatment of diseases characterized by excessive androgen.

[0004] Government support This invention was made with government support under Grant No. CA17393 awarded by the National Institutes of Health (NIH). The government has certain rights in this invention.

Background Art

[0005] Background of the Invention Müllerian inhibitors (MIS), also known as anti-Müllerian hormone (AMH), are 140 kDa disulfide-bonded homodimer glycoprotein members of the large transforming growth factor-β (TGFβ) multigene family of glycoproteins. All proteins in this family are produced as dimeric precursors and undergo post-transcriptional processing for activation requiring cleavage and dissociation to release a bioactive C-terminal fragment.

[0006] The human MIS gene is located on chromosome 19, and its expression is sexually dimorphic. In males, MIS expression begins in the fetal testes at 9 weeks of gestation and remains at high levels until puberty, when expression levels dramatically decline. In females, MIS is produced only after birth in granulosa cells, at levels similar to adult males, from pre-puberty through menopause, after which expression ceases. In male fetuses, MIS causes regression of the Müllerian duct, the precursor of the Fallopian duct, the uterus, the cervix, and the upper third of the vagina.

[0007] MIS exerts its biological effects after binding to heterodimers of type I and type II single transmembrane serine-threonine kinase receptors, leading to cross-phosphorylation of the GS-box kinase domain of the type I receptor by the type II receptor. Subsequently, SMAD1, 5, and 8 (but primarily SMAD8) are activated and, together with SMAD4, regulate gene transcription. The only MIS receptor type II (MISRII) gene has been identified in mice, rats, and rabbits, and in humans, its gene is localized on chromosome 12. It is a 65 kD protein detected in embryonic and adult Müllerian duct structures, mammary tissue, prostate tissue, gonads, motor neurons, and the brain. In the fetus, mesothelial cells expressing MISRII in the coelomic epithelium covering the urogenital ridge migrate to and become part of the mesenchymal cells surrounding the Müllerian epithelial cells. Expression is also detected in the gonads and ovarian coelomic epithelium. Type I MIS receptors have been identified in mammals, and depending on the animal species and tissue examined, activin receptor-like kinases (ALK) 2 and 3 are the most promising candidates.

[0008] In addition to its well-established role in Müllerian duct regression, MIS inhibits the proliferation of a variety of human cancer cell lines in vitro and in vivo. Cell lines exhibiting inhibition are derived from ovarian, cervical, endometrial, prostate, and breast cancers. Even when high concentrations of MIS are maintained systemically in rodents with tumors secreting MIS over long periods or in human patients, no toxicity has been observed in vivo. These findings—relatively limited receptor expression, antiproliferative activity against cancer cells expressing MIS RI and RII, and its apparent non-toxicity—collectively make MIS an ideal reagent for use in combination with existing chemotherapeutic agents for the treatment of ovarian cancer, which are known to develop resistance to these conventional agents.

[0009] MIS acts via MIS-type II receptor cells and functions as a potential tumor suppressor of ovarian cancer initiation (Teixeira et al, unpublished). MIS can also target stem / progenitor populations of ovarian cancer cell lines as a receptor-mediated event (Meirelles et al, 2012; Wei et al, 2010). MIS can be used for the treatment of cancer, for example, by expressing MISRII. MISRII is expressed in the majority of epithelial ovarian cancers (Masiakos et al. 1999; Bakkum-Gamez et al. 2008; Song et al. 2009).

[0010] MIS has no apparent toxicity after long-term in vivo treatment and inhibits the growth of various cancers in vitro and in vivo (Pieretti-Vanmarcke et al. 2006b). Epithelial ovarian cancers replicate the histology of the embryonic Müllerian duct and its diverse subtypes (Scully 1977); for example, serous cystadenocarcinoma resembles the embryonic Fallopian duct, endometrioid carcinoma resembles the endometrium, and mucinous carcinoma resembles the cervix. In addition, MIS functions synergistically or supplementally with cancer drugs commonly used to control tumor growth (Pieretti-Vanmarcke et al. 2006a).

[0011] It has been previously reported that chemotherapeutic agents are typically multidrug-resistant and / or resistant to chemotherapeutic agents in ovarian cancer stem cells. In particular, numerous studies have reported that ovarian cancer and cell lines are heterogeneous with ovarian cancer stem cell populations that are resistant to chemotherapeutic agents but responsive to MIS. MIS specifically targets ovarian cancer side population cells, as well as populations of CD44+, CD24+, EpCam+, and E-cadherin-negative cells, which have stem / progenitor cell characteristics that are largely responsive to chemotherapeutic agents in current clinical use for ovarian cancer (Wei et al, 2010). In particular, MIS has been shown to inhibit ovarian cancer cells both in vitro and in vivo, and may specifically target and inhibit the proliferation of ovarian cancer progenitor cell populations enriched with CD44+, CD24+, EpCAM+, and E-cadherin-cell surface markers. To adapt clinical trials of MIS in ovarian cancer patients, the production of recombinant human MIS must be optimized to increase yield and purity.

[0012] However, the refinements obtained from the purification of native and wild-type MIS are complex, and their yields are low. Furthermore, the cleavage required to produce the active fragment of MIS is inefficient. Human MIS proteins are produced from a preproprotein containing a leader sequence. The leader sequence (amino acids 1-25 of SEQ ID NO: 1) is cleaved, and the remaining preprotein (often called "holo-human MIS") is cleaved post-translationally, yielding N-terminal and C-terminal domains. These covalently bonded N-terminal and C-terminal domains form monomers, and two identical monomers (containing the N and C-terminal domains) together form a homodimer. Holo-human MIS is most promisingly cleaved into its N and C-terminal domains by furin or the related prohormone convertase PC5, which is expressed in the gonads. The cleavage is primarily cleaved by R, which has a serine +1 site, leaving a single nucleotide at the MIS cleavage site. -4 XXR -1 This occurs at a kex-like site characterized by [specific feature]. The purified C-terminal domain is a biologically active component, and cleavage is required for its biological activity. A second cleavage site, whose importance is unknown, has been observed less frequently at residues 229-230 (corresponding to amino acid residues 254-255 of SEQ ID NO: 1). Non-cleavable mutants of MIS are not biologically active, and mutations in the human gene that truncate the carboxy-terminal domain lead to persistent Müllerian syndrome. The role of the amino-terminal domain in vivo may be to assist protein folding and to facilitate the delivery of the C-terminal peptide to its receptor. In one study (Cate, Pepinsky, et al.), the addition of an N-terminal peptide was shown to enhance the biological activity of the C-terminal component in vitro, although the mechanism was unclear. Cleavage of recombinant MIS expressed by CHO cells is incomplete, thus requiring cleavage with an exogenous serine protease, such as plasmin, to enhance its biological activity.

[0013] Therefore, there is a need for more efficient methods to produce high concentrations of human MIS proteins for use as therapeutic biological agents. [Overview of the project]

[0014] The present invention relates to a modified recombinant human MIS protein having improved cleavage, increased bioactivity, and increased efficacy compared to a wild-type human MIS protein. The recombinant human MIS protein comprises, with or without internal labeling or tagging to facilitate its purification, a combination of the following components to improve the yield of the bioactive protein: a modified Kex cleavage site for increased cleavage, and a non-MIS reader sequence replacing the normal MIS reader sequence.

[0015] Therefore, the inventors hereby modified the native human MIS amino acid sequence to perform the following combinations: (i) modifying the first cleavage site to increase cleavage, thereby increasing the efficacy and bioactivity of the MIS without the insertion of a tag to facilitate purification; and (ii) modifying the endogenous leader sequence of the MIS to increase the yield of the bioactive protein. Surprisingly, the addition of the leader sequence combined with the modified first cleavage site significantly increased both the yield of the protein produced and the amount of cleavage from the first cleavage site of the recombinant MIS protein. Furthermore, there is an unmet need for the study of other bindings, which would be extremely important both for having a bioactive MIS form that can be labeled for use as a receptor, and for patient selection for treatment, and for addressing the molecular mechanism of MIS interaction in tissues with various receptors. In addition, its labeled ligand would be essential for determining whether other receptors or other binding proteins are present in various tissues. Here, the inventors demonstrate the production of internally epitope-tagged MIS that retains complete biological activity in a Müllerian duct regression assay. In one embodiment, the tag is a "FLAG" tag for the availability of high-quality reagents used for its detection and purification.

[0016] Herein, we demonstrate that the substitution of the MIS reader sequence with that of human serum albumin (HSA), combined with the modification of the first endogenous cleavage site from RAQR / S (SEQ ID NO: 26) to RARR / S (SEQ ID NO: 27), results in greater expression, increased C-terminal cleavage, and reduced unwanted potential internal cleavage when produced in CHO cells.

[0017] In another embodiment, recombinant human MIS is treated with a more efficient cleavage site at the carboxyl terminus of the N-terminal domain, thereby eliminating the need for exogenous cleavage. This recombinant MIS protein can be used both therapeutically and as a probe molecule without an identification tag.

[0018] Importantly, modification of the endogenous leader sequence to a different leader sequence, such as the human serum albumin (HSA) leader sequence, increases the production of MIS proteins. Surprisingly, the inventors demonstrate that the combination of the leader sequence and the modified cleavage site increases cleavage from the first cleavage site from 37% to over 80%. This was unexpected because an increase in protein production typically saturates available or endogenous cleavage enzymes, and therefore an increase in protein yield is accompanied by a decrease in post-translational processing, including cleavage.

[0019] Accordingly, the present invention relates to a method for treating cancer, such as cancer expressing MIS receptor II (MISRII), using recombinant human MIS protein (e.g., polypeptide, or nucleic acid encoding recombinant human MIS protein), or a functional fragment thereof, or derivatives or variants thereof.

[0020] Accordingly, one aspect of the present invention relates to a recombinant Müllerian inhibitor (MIS) protein comprising a combination of a non-MIS leader sequence or a functional fragment thereof that replaces the MIS leader sequence of amino acids 1-25 of SEQ ID NO: 1, and a modification of at least one amino acid between residues 448-452 of SEQ ID NO: 1, in order to increase cleavage compared to an unmodified protein. The recombinant MIS protein has increased cleavage and increased production yield in vitro compared to a wild-type MIS protein corresponding to the amino acid residues of SEQ ID NO: 1. In certain embodiments, the recombinant MIS protein lacks a leader sequence. In these embodiments, the recombinant MIS protein can be produced from a preproprotein comprising a non-MIS leader sequence or a functional fragment thereof that replaces the MIS leader sequence of amino acids 1-25 of SEQ ID NO: 1, where the leader sequence is cleaved during production. In certain embodiments, the recombinant MIS protein comprises a Tag protein.

[0021] In certain embodiments, the non-MIS reader sequence is an albumin reader sequence or a functional fragment thereof, for example, a human serum albumin (HSA) reader sequence or a functional fragment thereof. In certain embodiments, the HSA reader sequence includes the amino acid sequence of SEQ ID NO: 6 or a variant that is at least 80% homologous thereto, or a functional fragment thereof, for example, an HSA sequence or variant that is at least 80% homologous thereto, containing at least 10 amino acids, or at least about 11 amino acids, or at least 15 amino acids of SEQ ID NO: 6. In certain embodiments, the fragment of the HSA reader sequence is The selection is made from the group consisting of TIFF0007857367000001.tif19164.

[0022] In certain embodiments, the non-MIS reader sequence is an immunoglobulin signal peptide (IgSP-tPA) fused to a tissue-type plasminogen activator propeptide, a mouse immunoglobulin signal peptide (IgSP), or an MPIF-1 signal sequence. TIFF0007857367000002.tif5128, stanniocalcin signal sequence TIFF0007857367000003.tif5128, invertase signal sequence TIFF0007857367000004.tif5128, yeast mating factor alpha signal sequence (K. lactis killer toxin leader sequence), hybrid signal sequence TIFF0007857367000005.tif5128, HSA / MFα-1 hybrid signal sequence TIFF0007857367000006.tif5128, K. lactis killer / MFα-1 fusion leader sequence TIFF0007857367000007.tif5128, immunoglobulin Ig signal sequence TIFF0007857367000008.tif5128, Fibulin B precursor signal sequence TIFF0007857367000009.tif5128, clusterin precursor signal sequence TIFF0007857367000010.tif5128, and insulin-like growth factor binding protein 4 signal sequence TIFF0007857367000011.tif5128, or is selected from the group consisting of functional fragments thereof.

[0023] In certain embodiments, the modification of amino acid 450 of SEQ ID NO: 1 from Q to R increases cleavage from the first cleavage site compared to the amount of cleavage of those without such modification. In certain embodiments, the recombinant MIS protein further comprises a modification of amino acid 452 of SEQ ID NO: 1 from S to R, which increases cleavage compared to those without such modification.

[0024] In certain embodiments, the recombinant MIS proteins disclosed herein comprise a FLAG tag, e.g., amino acid The tag comprises TIFF0007857367000012.tif4128 or a functional derivative or variant thereof. In certain embodiments, the tag, for example, the FLAG tag, is located after amino acid residue 452 of SEQ ID NO: 1 and before amino acid residue 453 of SEQ ID NO: 1. In certain embodiments, the tag is located at the N-terminus of the C-terminal domain of MIS. In certain embodiments, the tag is 50 amino acids or less in length, for example, about 50, about 40, about 30, about 20, or about 10 amino acids or less, or about 7 amino acids in length.

[0025] In certain embodiments, the recombinant MIS proteins disclosed herein comprise the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3, or a functional fragment thereof. These may be encoded by the nucleic acid sequences of SEQ ID NO: 4 and SEQ ID NO: 5, respectively.

[0026] Another aspect of the present invention relates to a pharmaceutical composition comprising a recombinant MIS protein, as discussed herein, and a pharmaceutically acceptable carrier.

[0027] Another aspect of the present invention relates to a polynucleotide encoding a recombinant MIS protein discussed herein, where, for example, the polynucleotide corresponds to SEQ ID NO: 4 or SEQ ID NO: 5, or a nucleotide having at least 95% sequence identity with the nucleic acid sequence SEQ ID NO: 4 or SEQ ID NO: 5, respectively. Another aspect of the technique described herein relates to a vector comprising a polynucleotide of SEQ ID NO: 4 or SEQ ID NO: 5, or a nucleotide having at least 95% sequence identity with the nucleic acid sequence SEQ ID NO: 4 or SEQ ID NO: 5, respectively. In certain embodiments, the vector is a viral vector or an expression vector, e.g., pcDNA3.1, or an alternative vector for Escherichia coli (E. coli) or a bacteriophage. In certain embodiments, the viral vector is selected from the group consisting of adenovirus vectors, poxvirus vectors, and lentivirus vectors. In certain embodiments, the viral vector is an adeno-associated virus (AAV), e.g., recombinant AAV serotype 9 (rAAV9).

[0028] In certain embodiments, the vector comprises a nucleic acid sequence encoding a recombinant MIS protein or a fragment thereof, having at least 95% sequence identity with the nucleic acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5, wherein the nucleic acid sequence is conjugated to a tissue or cell-type specific promoter. In certain embodiments, a host cell containing such a vector is also included in the present invention.

[0029] In certain embodiments, a vector comprising the polynucleotides discussed herein may express recombinant MIS proteins at a constant level over a required period of time.

[0030] Another aspect of the present invention relates to human MIS proteins produced by post-translational processing of recombinant human MIS proteins discussed herein.

[0031] Another aspect of the technology discussed herein relates to a pharmaceutical composition comprising the vector and pharmaceutically acceptable carrier discussed herein. Another aspect of the technology discussed herein relates to a purified preparation or substantially purified human MIS protein produced from the recombinant human MIS protein discussed herein.

[0032] Another aspect of the technology discussed herein is a method for treating a subject having cancer, comprising the step of administering a composition comprising a recombinant MIS protein, wherein the recombinant MIS protein, with or without an internal tag protein, comprises a modification of at least one amino acid between residues 448-452 of SEQ ID NO: 1 to increase cleavage compared to an unmodified protein. In certain embodiments, the recombinant MIS protein has increased cleavage and increased production yield in vitro compared to a wild-type MIS protein corresponding to the amino acid residue of SEQ ID NO: 1.

[0033] In certain embodiments, recombinant human MIS proteins (e.g., polypeptides and / or nucleic acids encoding recombinant human MIS proteins), or functional fragments, derivatives, or variants thereof, can be used to treat cancer. In certain embodiments, a recombinant human MIS protein that can be used to treat cancer comprises amino acid residues 25-559 of SEQ ID NO: 2 or its functional fragment. In certain embodiments, a recombinant human MIS protein that can be used to treat cancer comprises amino acid residues 25-567 of SEQ ID NO: 3 or its functional fragment. In certain embodiments, cancer is MIS-responsive II (MISRII) cancer, or cancer expressing MISRII, for example, ovarian cancer, or includes ovarian cancer cells, vulvar epithelial cancer cells, cervical cancer cells, endometrial adenocarcinoma cells, and ovarian adenocarcinoma. In certain embodiments, cancer includes, but is not limited to, breast cancer, lung cancer, head and neck cancer, bladder cancer, stomach cancer, nervous system cancer, osteosarcoma, bone marrow cancer, brain tumor, colon cancer, esophageal cancer, endometrial cancer, gastrointestinal cancer, gingival cancer, kidney cancer, liver cancer, nasopharyngeal cancer, ovarian cancer, prostate cancer, pancreatic cancer, skin cancer, stomach cancer, testicular cancer, tongue cancer, melanoma, ocular melanoma, or uterine cancer.

[0034] In certain embodiments disclosed herein, the administration of recombinant MIS protein is performed before, during, or after the administration of further agents or cancer treatment.

[0035] In certain embodiments, Müllerian duct inhibitor (MIS) receptors are measured in biological samples obtained from a subject, such as cancer or tumor tissue samples, or in cancer cells or tumor cells, such as biopsy tissue samples.

[0036] In certain embodiments, the cancer is a chemotherapy-resistant or multidrug-resistant cancer, for example, a cancer resistant to paclitaxel, cisplatin, rapamycin, pyrazoloanthrone, or doxorubicin.

[0037] In certain embodiments, recombinant MIS proteins can be administered via any of the following routes, for example, intravenous, intracutaneous, intramuscular, intraarterial, intrafocal, intradermal, or subcutaneous, or by aerosol administration. In certain embodiments, the administration is therapeutic or prophylactic. In all embodiments disclosed herein, the subject is a mammal, such as a human.

[0038] In certain embodiments, at least one further agent is administered to the subject in combination with (e.g., before, during, or after) the administration of recombinant human MIS. This is, for example, a therapeutic or chemotherapeutic agent, for example, paclitaxel, cisplatin, doxorubicin, rapamycin, pyrazoloanthrone, for example, but not limited to, anthra(1,9-cd)pyrazole-6(2H)-one (SP600125), or N1-methyl-1,9-pyrazoloanthrone (M-SP600125), or functional derivatives or functional analogs thereof. In certain embodiments, the chemotherapeutic agent is a radiotherapeutic agent.

[0039] Another aspect of the technology disclosed herein relates to a method for reducing the dose of a chemotherapeutic agent for the treatment of cancer. The method comprises the step of administering to a subject a therapeutically effective dose of a recombinant MIS protein, wherein the recombinant MIS protein comprises a Q-to-R modification of amino acid 450 of SEQ ID NO: 1, and the therapeutically effective dose of the chemotherapeutic agent in the presence of the recombinant MIS protein is lower than the therapeutically effective dose of the chemotherapeutic agent alone. In certain embodiments, the recombinant MIS protein optionally includes a tag protein.

[0040] Another aspect of the technology disclosed herein relates to the use of recombinant MIS proteins for the manufacture of drugs for the treatment of cancer. The recombinant MIS protein comprises a Q-to-R modification of amino acid 450 of SEQ ID NO: 1, and cancer expresses a Müllerian duct inhibitor (MIS) receptor.

[0041] Another aspect of the present invention relates to a packaging material and a product comprising a pharmaceutical composition comprising a recombinant MIS protein disclosed herein. The packaging material includes a label indicating that the pharmaceutical composition can be administered in an effective dose for a sufficient period of time to treat or reduce the risk of cancer expressing Müllerian inhibitor (MIS) receptors.

[0042] Another aspect of the technology disclosed herein relates to a method for treating a subject with cancer. The method includes the step of evaluating the expression and / or activity of Müllerian duct inhibitor receptor II (MISRII) in a biological sample obtained from the subject. A clinician considers whether the result indicates the presence of MISRII expression and / or activity, and the clinician directs the subject to treatment with a pharmaceutical composition comprising the recombinant MIS protein disclosed herein.

[0043] Another aspect of the technology disclosed herein relates to the use of recombinant MIS proteins to reduce plasma serum levels of one or more androgens in subjects requiring reduction. The recombinant MIS protein comprises a Q-to-R modification of amino acid 450 of SEQ ID NO: 1 and optionally includes a tag, such as a Flag tag. In a particular embodiment, a recombinant human MIS protein that can be used to reduce androgen levels comprises amino acid residues 25-559 of SEQ ID NO: 2 or its functional fragment.

[0044] In certain embodiments, one or more androgens are testosterone. In certain embodiments, the required subjects have benign prostatic hyperplasia, or prostate cancer, or polycystic ovary disease, and / or precocious puberty. In other embodiments, the required subjects have, but are not limited to, benign prostatic hyperplasia (BPH), prostate cancer, testicular cancer, androgen-dependent acne, male pattern baldness, precocious puberty, hyperandrogenicity, hirsutism, masculinization, polycystic ovary syndrome (POCS), hyperandrogenicity (HA), and insulin resistance (IR), as well as diseases or disorders selected from the group including acanthosis nigricans (AN) (HIAR-AN) syndrome, ovarian follicular cell proliferation, follicular maturation inhibition, atresia, anovulation, dysmenorrhea, dysfunctional uterine bleeding, infertility, and androgen-producing tumors.

[0045] Another aspect of the technology disclosed herein is a method for treating an androgen-dependent disease or disorder, comprising the step of administering to a subject an effective amount of a pharmaceutical composition comprising a recombinant MIS protein disclosed herein, or a preparation of MIS protein from the cleavage of a recombinant MIS protein disclosed herein. The pharmaceutical composition reduces the level of at least one androgen in the subject's plasma serum, causing a reduction in at least one symptom of an androgen-dependent disease or disorder.

[0046] Another aspect of the technology disclosed herein relates to a method for reducing plasma levels of one or more androgens in a subject. The method comprises the step of administering an effective amount of recombinant MIS protein, wherein the recombinant MIS protein comprises a Q-to-R modification of amino acid 450 of SEQ ID NO: 1, the recombinant MIS protein optionally comprises a tag, and the recombinant MIS protein reduces plasma serum levels of one or more androgens in a subject.

[0047] In certain embodiments, the subjects have diseases or disorders characterized by androgen dependence, for example, but are not limited to, benign prostatic hyperplasia (BPH), prostate cancer, testicular cancer, androgen-dependent acne, male pattern baldness, precocious puberty, hyperandrogenemia, hirsutism, masculinization, polycystic ovary syndrome (POCS), hyperandrogenemia (HA), and insulin resistance (IR), as well as acanthosis nigricans (AN) (HIAR-AN) syndrome, ovarian follicular cell proliferation, follicular maturation inhibition, atresia, anovulation, dysmenorrhea, dysfunctional uterine bleeding, infertility, and androgen-producing tumors.

[0048] Another aspect of the technology disclosed herein relates to a kit comprising a recombinant MIS protein disclosed herein, or a preparation of an MIS protein produced by post-translational processing of a recombinant MIS protein disclosed herein, and a pharmaceutically acceptable carrier. In certain embodiments, the kit may optionally include instructions for the use of the recombinant MIS protein for the treatment of cancer or androgen-dependent diseases. [Invention 1001] A recombinant Müllerian inhibitor (MIS) protein comprising a combination of a non-MIS reader sequence or a functional fragment thereof replacing the MIS reader sequence of amino acids 1-25 of SEQ ID NO: 1, and a modification of at least one amino acid between residues 447-451 of SEQ ID NO: 1, wherein the recombinant MIS protein has increased cleavage and increased production yield in vitro compared to a wild-type MIS protein corresponding to the amino acid residues of SEQ ID NO: 1. [Invention 1002] The recombinant MIS protein of the present invention 1001, wherein the recombinant MIS protein further comprises a tag protein. [Invention 1003] The recombinant MIS protein of the present invention 1001, wherein the recombinant MIS protein comprises at least a non-MIS reader sequence or a functional fragment thereof in place of the MIS reader sequence of amino acids 1-25 of SEQ ID NO: 1, and a modification of at least one amino acid between residues 447-451 of SEQ ID NO: 1, in order to increase cleavage compared to the unmodified MIS protein. [Invention 1004] The recombinant MIS protein of the present invention 1001, wherein the non-MIS reader sequence is an albumin reader sequence or a functional fragment thereof. [Invention 1005] The recombinant MIS protein of the present invention 1004, wherein the albumin reader sequence is a human serum albumin (HSA) reader sequence or a functional fragment thereof. [Invention 1006] The recombinant MIS protein of the present invention 1005, wherein the HSA reader sequence comprises the amino acid sequence of SEQ ID NO: 6 or a variant that is at least 80% homologous thereto. [Invention 1007] The recombinant MIS protein of the present invention 1005, wherein the HSA reader sequence fragment comprises at least 10 amino acids of SEQ ID NO: 6 or a variant that is at least 80% homologous thereto. [Invention 1008] The recombinant MIS protein of the present invention 1005, wherein the HSA reader sequence comprises at least 15 amino acids of SEQ ID NO: 6 or a variant that is at least 80% homologous thereto. [Invention 1009] The recombinant MIS protein of the present invention 1005, wherein the HSA reader sequence comprises at least 11 amino acids of SEQ ID NO: 6 or a variant that is at least 80% homologous thereto. [Invention 1010] The HSA reader sequence fragment is Recombinant MIS protein of the present invention 1005, selected from the group consisting of TIFF0007857367000013.tif19149. [Invention 1011] The aforementioned non-MIS reader sequence is fused to a tissue-type plasminogen activator propeptide in an immunoglobulin signal peptide (IgSP-tPA), a mouse immunoglobulin signal peptide (IgSP), and an MPIF-1 signal sequence. TIFF0007857367000014.tif5128, stanniocalcin signal sequence TIFF0007857367000015.tif5128, invertase signaling sequence TIFF0007857367000016.tif5128, yeast junction factor alpha signal sequence (K. lactis killer toxin reader sequence), hybrid signal sequence TIFF0007857367000017.tif5128, HSA / MFα-1 hybrid signal sequence TIFF0007857367000018.tif5128, K. lactis killer / MFα-1 fusion leader sequence TIFF0007857367000019.tif5128, immunoglobulin Ig signaling sequence TIFF0007857367000020.tif5128, fibrin B precursor signal sequence TIFF0007857367000021.tif5128, clathelin precursor signal sequence TIFF0007857367000022.tif5128, and insulin-like growth factor-binding protein 4 signal sequence A recombinant MIS protein according to the present invention 1001, selected from the group consisting of TIFF0007857367000023.tif5128, or fragments thereof. [Invention 1012] The recombinant MIS protein of the present invention 1001, comprising a modification of amino acid 449 of sequence number 1 from Q to R to increase cleavage compared to the unmodified protein. [Invention 1013] The recombinant MIS protein of the present invention 1001 further comprises a modification of amino acid 451 of SEQ ID NO: 1 from S to R to increase cleavage compared to the unmodified protein. [Invention 1014] The recombinant MIS protein of the present invention 1001, wherein the aforementioned tag is a FLAG tag. [Invention 1015] The FLAG tag is an amino acid sequence A recombinant MIS protein according to the present invention 1014, comprising TIFF0007857367000024.tif5128, or a functional fragment or variant thereof. [Invention 1016] The recombinant MIS protein of the present invention 1014, wherein the FLAG tag is located after amino acid residue 451 of SEQ ID NO: 1 and before amino acid residue 452 of SEQ ID NO: 1. [Invention 1017] The recombinant MIS protein of the present invention 1014, wherein the FLAG tag is located between amino acid residues 451-452 of sequence number 1. [Invention 1018] A recombinant MIS protein according to the present invention 1001, comprising the amino acid sequence of SEQ ID NO: 2 or a functional fragment thereof. [Invention 1019] A recombinant MIS protein according to the present invention 1001, comprising the amino acid sequence of SEQ ID NO: 3 or a functional fragment thereof. [Invention 1020] The recombinant MIS protein of the present invention 1018, encoded by the nucleic acid sequence of Sequence ID: 4. [Invention 1021] The recombinant MIS protein of the present invention 1019, encoded by the nucleic acid sequence of Sequence ID: 5. [Invention 1022] A pharmaceutical composition comprising any recombinant MIS protein according to invention 1001 to 1021 and a pharmaceutically acceptable carrier. [Invention 1023] A polynucleotide encoding any of the recombinant MIS proteins described in invention 1001 to 1021. [Invention 1024] The polynucleotide of the present invention 1023, wherein the nucleotide corresponds to sequence number 4, or a nucleotide having at least 95% sequence identity with the nucleic acid sequence of sequence number 4. [Invention 1025] The polynucleotide of the present invention 1023, wherein the nucleotide corresponds to sequence number 5, or a nucleotide having at least 95% sequence identity with the nucleic acid sequence of sequence number 5. [Invention 1026] A vector comprising any of the polynucleotides 1023 to 1025 of the present invention. [Invention 1027] The vector according to the present invention 1026, wherein the vector is a viral vector or an expression vector. [Invention 1028] The vector of the present invention 1027, wherein the expression vector is pcDNA3.1 or a cDNA or genomic vector for bacteria (e.g., Escherichia coli) or bacteriophages. [Invention 1029] The vector of the present invention 1027, wherein the viral vector is selected from the group consisting of adenovirus vectors, poxvirus vectors, and lentivirus vectors. [Invention 1030] A vector according to any one of the invention 1026 to 1029, wherein the nucleic acid sequence encodes a recombinant MIS protein or a functional fragment thereof having at least 95% sequence identity with the nucleic acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5, and the nucleic acid sequence is ligated to act on a tissue or cell type-specific promoter. [Invention 1031] Human MIS protein produced by post-translational processing of recombinant human MIS protein according to Invention 1001. [Invention 1032] A host cell containing any of the vectors described in invention 1026 to 1030. [Invention 1033] A pharmaceutical composition comprising a vector according to any of invention 1026 to 1030 and a pharmaceutically acceptable carrier. [Invention 1034] A purified preparation of human MIS protein produced from any recombinant human MIS protein according to invention 1001 to 1021. [Invention 1035] A method for treating a subject having cancer, the method comprising the step of administering a composition comprising a recombinant MIS protein, wherein the recombinant MIS protein comprises a combination of a non-MIS reader sequence or a functional fragment thereof replacing the MIS reader sequence of amino acids 1-25 of SEQ ID NO: 1, and a modification of at least one amino acid between residues 447-451 of SEQ ID NO: 1, in order to increase cleavage compared to an unmodified MIS protein, and the recombinant MIS protein has increased cleavage and increased production yield in vitro compared to a wild-type MIS protein corresponding to the amino acid residues of SEQ ID NO: 1. [Invention 1036] The method of the present invention 1035, wherein the recombinant MIS protein further comprises a tag protein. [Invention 1037] The method of the present invention 1035, wherein the recombinant MIS protein comprises at least a non-MIS reader sequence or a functional fragment thereof in place of the MIS reader sequence of amino acids 1-25 of SEQ ID NO: 1, and a modification of at least one amino acid between residues 447-451 of SEQ ID NO: 1, in order to increase cleavage compared to the unmodified protein. [Invention 1038] The method of the present invention 1035, wherein the non-MIS reader sequence is an albumin reader sequence or a functional fragment thereof. [Invention 1039] The method of the present invention 1038, wherein the albumin reader sequence is a human serum albumin (HSA) reader sequence or a functional fragment thereof. [Invention 1040] The method of the present invention 1035, wherein the recombinant MIS protein comprises a modification of amino acid 449 of sequence number 1 from Q to R to increase cleavage compared to the unmodified protein. [Invention 1041] The method of the present invention 1035, wherein the tag is a FLAG tag containing the amino acid sequence of sequence number 8 or a functional fragment thereof. [Invention 1042] The method of the present invention 1035, wherein the cancer is MIS-responsive II cancer. [Invention 1043] The method of the present invention 1035, wherein the cancer is ovarian cancer. [Invention 1044] The method of the present invention 1035, wherein the cancer is chemotherapy-resistant or multidrug-resistant. [Invention 1045] The method of the present invention 1035, wherein the administration of the recombinant MIS protein is performed before, during, or after the administration of an additional agent or cancer treatment. [Invention 1046] The method of the present invention 1035, wherein the cancer expresses Müllerian duct inhibitor receptor II (MISRII). [Invention 1047] The method of the present invention 1046, wherein the expression of the Müllerian duct inhibitor (MIS) receptor is measured in a biological sample obtained from the subject. [Invention 1048] The method of the present invention 1047, wherein the biological sample is a cancer or tumor tissue sample or cancer cells or tumor cells. [Invention 1049] The method of the present invention 1047, wherein the biological sample is a biopsy tissue sample. [Invention 1050] The method of the present invention 1035, wherein the cancer is ovarian cancer cells, vulvar epithelial cancer cells, cervical cancer cells, endometrial adenocarcinoma cells, and ovarian adenocarcinoma cells. [Invention 1051] The method of the present invention 1035, wherein the cancer is selected from the group consisting of breast cancer, lung cancer, head and neck cancer, bladder cancer, stomach cancer, nervous system cancer, osteosarcoma, bone marrow cancer, brain tumor, colon cancer, esophageal cancer, endometrial cancer, gastrointestinal cancer, gingival cancer, kidney cancer, liver cancer, nasopharyngeal cancer, ovarian cancer, prostate cancer, skin cancer, stomach cancer, testicular cancer, tongue cancer, melanoma, ocular melanoma, or uterine cancer. [Invention 1052] The method of the present invention 1044, wherein the cancer is a paclitaxel or doxorubicin-resistant cancer. [Invention 1053] The method of the present invention 1035, wherein the administration is intravenous, intracutaneous, intramuscular, intraarterial, intralesional, transdermal, or subcutaneous, or by aerosol administration. [Invention 1054] The method of the present invention 1035, wherein the administration is a prophylactic administration. [Invention 1055] The method of the present invention 1035, wherein the administration is therapeutic. [Invention 1056] The method of the present invention 1035, wherein the subject is a mammal. [Invention 1057] The method of the present invention 1056, wherein the subject is a human. [Invention 1058] The method of the present invention 1035, wherein at least one additional agent is administered to the subject in combination with (for example, before, during, or after) the administration of the recombinant human MIS. [Invention 1059] The method of the present invention 1058, wherein the additional agent is a therapeutic agent or a chemotherapy agent. [Invention 1060] The method of the present invention 1059, wherein the chemotherapeutic agent is selected from the group consisting of paclitaxel, cisplatin, doxorubicin, rapamycin, and pyrazoloanthrone. [Invention 1061] The method of the present invention 1059, wherein the chemotherapeutic agent is a radiotherapy agent. [Invention 1062] The method of the present invention 1059, wherein the chemotherapeutic agent is pyrazoloanthrone. [Invention 1063] The method of the present invention 1062, wherein the pyrazoloanthrone is anthra(1,9-cd)pyrazole-6(2H)-one (SP600125) or a functional derivative or functional analog thereof. [Invention 1064] A method for reducing the dose of a chemotherapeutic agent for the treatment of cancer, the method comprising the step of administering a therapeutically effective dose of a recombinant MIS protein to a target, wherein the recombinant MIS protein comprises a combination of a modification of amino acid 449 of SEQ ID NO: 1 from Q to R and a non-MIS reader sequence or a functional fragment thereof replacing the MIS reader sequence of amino acids 1-25 of SEQ ID NO: 1, and the therapeutically effective dose of the chemotherapeutic agent in the presence of the recombinant MIS protein is less than the therapeutically effective dose of the chemotherapeutic agent alone. [Invention 1065] The method of the present invention 1064, wherein the recombinant MIS protein further comprises a tag protein. [Invention 1066] Use of recombinant MIS protein for the manufacture of a drug for treating cancer, wherein the recombinant MIS protein comprises a combination of a modification from Q to R at amino acid 449 of SEQ ID NO: 1 and a non-MIS reader sequence or a functional fragment thereof in place of the MIS reader sequence of amino acids 1-25 of SEQ ID NO: 1, and the cancer expresses a Müllerian duct inhibitor (MIS) receptor. [Invention 1067] Use of the invention 1066, wherein the recombinant MIS protein further comprises a tag protein. [Invention 1068] Use of the present invention 1066, wherein the Müllerian duct inhibitor (MIS) receptor is an MIS type II receptor or its homolog or functional fragment. [Invention 1069] A manufactured product comprising a packaging material and a pharmaceutical composition comprising any recombinant MIS protein of the present invention 1001 to 1021, wherein the packaging material includes a label indicating that the pharmaceutical composition can be administered in a sufficient dose for a sufficient period of time to treat or reduce the risk of cancer expressing a Müllerian duct inhibitor (MIS) receptor. [Invention 1070] A method for treating a subject suffering from cancer, the method comprising the step of evaluating the expression and / or activity of Müllerian duct inhibitor receptor II (MISRII) in a biological sample obtained from the subject, wherein a clinician reviews the results, and if the results indicate the presence of MISRII expression and / or activity, the clinician instructs the subject to treat with a pharmaceutical composition of the present invention 1022 or 1033. [Invention 1071] The method of the present invention 1070, wherein the biological sample is a tissue sample. [Invention 1072] The method of the present invention 1071, wherein the tissue sample is a cancer or tumor tissue sample or cancer cells or tumor cells. [Invention 1073] The method of the present invention 1071, wherein the biological sample is a biopsy tissue sample. [Invention 1074] The method of the present invention 1070, wherein the cancer is ovarian cancer cells, vulvar epithelial cancer cells, cervical cancer cells, endometrial adenocarcinoma cells, and ovarian adenocarcinoma cells. [Invention 1075] The method of the present invention 1070, wherein the cancer is breast cancer, lung cancer, head and neck cancer, bladder cancer, stomach cancer, nervous system cancer, osteosarcoma, bone marrow cancer, brain tumor, colon cancer, esophageal cancer, endometrial cancer, gastrointestinal cancer, gingival cancer, kidney cancer, liver cancer, nasopharyngeal cancer, ovarian cancer, prostate cancer, skin cancer, stomach cancer, testicular cancer, tongue cancer, melanoma, ocular melanoma, or uterine cancer. [Invention 1076] The use of a recombinant MIS protein to reduce plasma serum levels of one or more androgens in a target subject, wherein the recombinant MIS protein comprises a combination of a Q-to-R modification of amino acid 449 of SEQ ID NO: 1 and a non-MIS reader sequence or a functional fragment thereof replacing the MIS reader sequence of amino acids 1-25 of SEQ ID NO: 1. [Invention 1077] Use of the invention 1076, wherein the recombinant MIS protein further comprises a tag protein. [Invention 1078] Use of the present invention 1076, wherein one or more androgens are testosterone. [Invention 1079] The use of the aforementioned subject is one having benign prostatic hyperplasia. [Invention 1080] The use of the present invention 1076 is for a subject who has prostate cancer. [Invention 1081] Use of the present invention 1076 in which the subject requiring the use has polycystic ovary disease and / or precocious puberty. [Invention 1082] Use of Invention 1076 in which the subject requiring the use has a disease or disorder selected from the group consisting of benign prostatic hyperplasia (BPH), prostate cancer, testicular cancer, androgen-dependent acne, male pattern baldness, precocious puberty, hyperandrogenemia, hirsutism, masculinization, polycystic ovary syndrome (POCS), hyperandrogenemia (HA), and insulin resistance (IR), as well as acanthosis nigricans (AN) (HIAR-AN) syndrome, ovarian follicular cell proliferation, suppression of follicular maturation, atresia, anovulation, dysmenorrhea, dysfunctional uterine bleeding, infertility, and androgen-producing tumors. [Invention 1083] A method for treating an androgen-dependent disease or disorder, the method comprising the step of administering an effective amount of a pharmaceutical composition of the present invention 1022 or 1033 to a subject, wherein the pharmaceutical composition reduces the level of at least one androgen in the plasma serum of the subject, thereby causing a reduction in at least one symptom of an androgen-dependent disease or disorder. [Invention 1084] A method for reducing plasma levels of one or more androgens in a subject, the method comprising the step of administering an effective amount of recombinant MIS protein, wherein the recombinant MIS protein comprises a combination of a modification of amino acid 449 of SEQ ID NO: 1 from Q to R and a non-MIS reader sequence or a functional fragment thereof in place of the MIS reader sequence of amino acids 1-25 of SEQ ID NO: 1, wherein the recombinant MIS protein reduces plasma serum levels of one or more androgens in a subject. [Invention 1085] The method of the present invention 1084, wherein the recombinant MIS protein further comprises a tag protein. [Invention 1086] The method of the present invention 1084, wherein the subject has a disease or disorder characterized by androgen dependence. [Invention 1087] The method of any of items 1084 to 1086 of the present invention, wherein the disease or disorder is selected from the group consisting of benign prostatic hyperplasia (BPH), prostate cancer, testicular cancer, androgen-dependent acne, male pattern baldness, precocious puberty, hyperandrogenemia, hirsutism, masculinization, polycystic ovary syndrome (POCS), hyperandrogenemia (HA), and insulin resistance (IR), as well as acanthosis nigricans (AN) (HIAR-AN) syndrome, ovarian follicular cell proliferation, suppression of follicular maturation, atresia, anovulation, dysmenorrhea, dysfunctional uterine bleeding, infertility, and androgen-producing tumors. [Invention 1088] A kit comprising any recombinant MIS protein according to invention 1001 to 1021, a pharmaceutically acceptable carrier, and a recombinant MIS protein according to invention 1001 to 1021. [Invention 1089] The kit of the present invention 1088 optionally further comprises instructions for the use of recombinant MIS proteins for the treatment of cancer or androgen-dependent diseases. [Brief explanation of the drawing]

[0049] [Figure 1A]Figures 1A and 1B are schematic diagrams showing the design of a novel recombinant MIS component with an albumin leader sequence. Figure 1A shows that the leader sequences of MIS (25 amino acids) and albumin (24 amino acids) have 20% identity and 5 conserved amino acids. [Figure 1B] Figures 1A and 1B are schematic diagrams showing the designs of novel recombinant MIS components having an albumin leader sequence. Figure 1B is a schematic diagram showing the designs of RF (improved cleavage site + flag tag), LRF (leader sequence + improved cleavage site + flag tag), and LR (leader sequence + improved cleavage site) components, including the arrangement of a flag tag (F), an improved cleavage site (R), and an albumin leader (L). [Figure 2] This shows MIS production and cleavage in CHOK1 clones stably transfected with recombinant human LR-MIS and LRF-MIS components. Western blotting of 4% reduced SDS gel of culture supernatant 72 hours after use of an anti-MIS goat polyclonal antibody targeting the c-terminus of MIS. Purified RF-MIS, CHO93 medium, and B9 medium are used as positive controls. [Figure 3A] Figures 3A–3B show purified recombinant MIS analyzed by Western blotting of reduced SDS gel to assess the amount of cleavage. Figure 3A shows purified recombinant RF-MIS, LRF-MIS, and WT-MIS being compared using antibodies against the N-terminus that can recognize the holoMIS monomer, the cleaved N-terminus, and the potential cleavage product including the N-terminal portion. [Figure 3B] Figures 3A–3B show purified recombinant MIS analyzed by Western blotting of reduced SDS gel to assess the amount of cleavage. Figure 3B shows the detection of purified recombinant RF-MIS, LRF-MIS, and WT-MIS using antibodies against the C-terminus that can recognize the holoMIS monomer, the cleaved C-terminus, and the potential cleavage product including the C-terminus portion. [Figure 4A]Figures 4A–4B show a comparison of WT, RF, and LRF recombinant MIS at 5 μg / ml (35 μM) in Müllerian duct regression biopsy. Recombinant human MIS products were incubated with fetal rat urogenital ridges for 72 hours. Figure 4A shows representative sections from both the treated ridge and the untreated contralateral control ridge being compared for ridge-Müllerian duct regression. [Figure 4B] Figures 4A-4B show a comparison of WT, RF, and LRF recombinant MIS at 5 μg / ml (35 μM) in Müllerian duct regression biopsy. Recombinant human MIS products were incubated with fetal rat urogenital ridges for 72 hours. Figure 4B is a histogram showing the frequency distribution of these scores in Figure 4A. (LRF-MIS N=6, RF-MIS N=39). W: Wolffian duct; M: Müllerian duct. [Figure 5A] Figures 5A-5B show the amino acids of the wild-type MIS protein (SEQ ID NO: 1), with corresponding amino acid residues using the conventional amino acid labeling nomenclature (where the first numbered amino acid begins after the leader sequence). Figure 5A shows the amino acid sequence of the wild-type MIS protein SEQ ID NO: 1, with the leader sequence (thick line) and the first and second cleavage sites highlighted. The corresponding amino acids, numbered using the conventional numbering method, are shown in brackets. [Figure 5B] Figures 5A-5B show the amino acids of the wild-type MIS protein (Sequence ID: 1), with corresponding amino acid residues using the conventional nomenclature of amino acid labeling (where the first numbered amino acid begins after the leader sequence). Figure 5B shows a table of features on the amino acid residues on Sequence ID: 1 corresponding to the amino acid residues using the usual MIS nomenclature (where the first numbered amino acid begins after the leader sequence). Figure 5B discloses "RAQR / S" as Sequence ID: 26. [Modes for carrying out the invention]

[0050] Detailed description of the invention The present invention relates to an improved recombinant human MIS protein having at least one of the following features: improved cleavage, increased bioactivity, and increased potency, and which can be produced in higher yields compared to wild-type human MIS proteins. Herein, the recombinant human MIS protein comprises a combination of an improved Kex cleavage site for increased cleavage and a non-MIS reader sequence replacing a normal MIS reader sequence, thereby improving the yield of the bioactive protein. In certain embodiments, the improved MIS may or may not have an internal label or tag to facilitate its purification.

[0051] Therefore, in this instance, the inventors modified the native human sequence to induce endogenous cleavage and thereby increase the efficacy of MIS. The inventors also optionally inserted tags to facilitate its purification.

[0052] The inventors further modified the recombinant human MIS protein to include a non-MIS leader sequence instead of the 25-amino acid MIS leader sequence of SEQ ID NO: 1 (amino acids 1-25). In certain embodiments, the leader sequence comprises an albumin leader sequence, e.g., a human serum albumin sequence (HSA) or a functional fragment or variant thereof. In certain embodiments, the leader sequence comprises the 24 amino acids of SEQ ID NO: 6 or a functional fragment thereof, replacing amino acid residues 1-25 of SEQ ID NO: 1. Surprisingly, this addition further increased the cleavage of the recombinant MIS protein. This combination led to a higher yield of a more homogeneous product, accompanied by increased potency due to the increased cleavage. This combination of modifications produces recombinant human MIS variants that could be adapted to further binding studies, which would be crucial for both patient selection for treatment and addressing the molecular mechanism of MIS interactions in tissues with various receptors. Furthermore, the labeled ligand will be essential for determining whether other receptors or other binding proteins are present in various tissues. In this specification, we demonstrate the production of internally epitope-tagged MIS that retains full biological activity in Müllerian duct regression assays. In one embodiment, the tag is a "FLAG" tag due to the availability of high-quality reagents for its detection and purification.

[0053] As discussed herein, the present invention provides a method for treating various conditions by administering an effective amount of recombinant human MIS proteins and their functional fragments and derivatives disclosed herein to a target of interest. Conditions that can be treated with the compounds of the present invention or pharmaceutically active compositions thereof include any condition that is treated or has reduced symptoms by administration of human MIS, or by activation of MIS signaling or activation of MISRII, thereby benefiting from the administration of recombinant human MIS proteins and their functional fragments and derivatives. Representative conditions in this regard include, but are not limited to, cancers expressing MIS receptors, such as cancers expressing MISRII, such as, but are not limited to, cancers of the ovaries, cervix, and endometrium. Other conditions that can be treated with MIS, or in which activation of MIS signaling has reduced symptoms, include proliferative disorders, such as cancer, or abnormally high androgen levels, such as polycystic ovary disease, precocious puberty, and other hyperandrogen disorders, such as testotoxicosis, or any androgen-dependent tumor, such as prostate cancer.

[0054] definition For convenience, specific terms used throughout this application (including in the specification, examples, and claims) are summarized here. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this invention belongs.

[0055] The terms “Müllerian inhibitor” and “MIS” are used interchangeably herein and refer to compounds and materials structurally similar to MIS, also known as anti-Müllerian hormone or AMH. “MIS” or “Müllerian inhibitor” means a polypeptide having an amino acid sequence that is at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identical to amino acid residues 26–560 of SEQ ID NO: 1. The present invention intends to include mutants of recombinant human MIS having substantially the same or greater biological activity as wild-type MIS. Examples of such mutant MIS molecules have deletions, insertions, or alterations in the amino acid sequence of wild-type MIS (e.g., amino acid residues 26–560 of SEQ ID NO: 1). Other forms of the substances contained include, for example, salts, functional derivatives, and aglycone forms of wild-type MIS and recombinant human MIS. Furthermore, recombinant human MIS proteins can be obtained using recombinant DNA technology or from the chemical synthesis of MIS proteins. For citation purposes only, the wild-type human MIS nucleic acid corresponds to RefSeq No: NM_000479, which is incorporated herein by reference.

[0056] The terms “Müllerian duct inhibitor type II receptor” or “MISRII” are used interchangeably herein and refer to the type II receptor for MIS. The term “MISRII” is intended to encompass all MIS receptors and functional derivatives of MISRII that are substantially homologous to MISRII. MISRII is also known as AMHR2. For reference purposes only, the nucleic acid sequence of human MISRII corresponds to NM_020547 and GenBank No: AF172932, which is incorporated herein by reference.

[0057] The term “wild-type” refers to the naturally occurring polynucleotide sequence or a portion thereof that codes for a protein, or the protein sequence or a portion thereof, respectively, as it normally exists in vivo. Therefore, as disclosed herein, the wild-type amino acid sequence for the preproprotein of human MIS corresponds to Sequence ID No. 1, where amino acid residues 1–25 correspond to the leader sequence. The proprotein of MIS contains amino acid residues 26–560 of Sequence ID No. 1 (e.g., lacking the 1–25 leader sequence), which is post-translationally processed by cleavage as disclosed herein to form a bioactive MIS homodimer.

[0058] As used herein, the term "soluble MIS polypeptide" refers to an MIS polypeptide that does not contain at least some or all of the amino acids that allow for functional binding to a membrane.

[0059] "A polynucleotide encoding MIS" means a polynucleotide encoding a polypeptide that has at least approximately 60%, at least approximately 70%, at least approximately 80%, at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99% sequence identity with any of the amino acid sequences corresponding to amino acid residues 26-560 of SEQ ID NO: 1.

[0060] The term "mutation" refers to any change in the genetic material of an organism, in particular a change in the wild-type polynucleotide sequence (i.e., deletion, substitution, addition, or alteration), or any change in the wild-type protein sequence. The term "variant" is used interchangeably with "mutant." While changes in the genetic material are often expected to result in changes in protein function, the terms "mutant" and "variant" refer to changes in the wild-type protein sequence, regardless of whether the change alters protein function (e.g., increase, decrease, or addition of a new function) or whether the change has an effect on protein function (e.g., the mutation or alteration is silent). The term "mutation" is used interchangeably with "polymorphism" herein.

[0061] As used herein, the terms “agent” or “compound” refer to a chemical or biological product, or a combination of chemical or biological products, administered to a subject to treat, prevent, or control a disease or condition. A chemical or biological product may preferably, but not necessarily, be a low molecular weight compound, but may also be a larger compound, or any organic or inorganic molecule, such as modified and unmodified nucleic acids, such as antisense nucleic acids, RNAi, such as siRNA or siRNA, peptides, peptide mimes, receptors, ligands, and antibodies, aptamers, polypeptides, nucleic acid analogs, or variants thereof. For example, nucleic acids, amino acids, or carbohydrate oligomers include, but are not limited to, proteins, oligonucleotides, ribozymes, DNAzymes, glycoproteins, siRNA, lipoproteins, aptamers, and their variants and combinations.

[0062] The term “nucleic acid” is publicly known in the art. As used herein, “nucleic acid” generally refers to molecules (i.e., strands) of DNA, RNA, or their derivatives or analogues that include nucleic acid bases. Nucleic acid bases include, for example, the spontaneously occurring purine or pyrimidine bases found in DNA (adenine “A”, guanine “g”, thymine “T”, or cytosine “C”) or RNA (A, G, uracil “U”, or C). The term “nucleic acid” encompasses the terms “oligonucleotide” and “polynucleotide” as subgenres of the term “nucleic acid.” The term “oligonucleotide” refers to a molecule with a length of about 3 to about 100 nucleic acid bases. The term “polynucleotide” refers to at least one molecule with a length greater than about 100 nucleic acid bases. The term “nucleic acid” also means polynucleotides, such as deoxyribonucleotides (DNA), and, where appropriate, ribonucleotides (RNA). The term should be understood to include, as equivalents, analogs (similarities) of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiments described, single-stranded (sense or antisense) and double-stranded polynucleotides. The terms “polynucleotide sequence” and “nucleotide sequence” are also interchangeable herein.

[0063] As used herein, the term “gene” means a nucleic acid containing an open reading frame that codes for a polypeptide, including both exons and (optionally) intron sequences. “Genes” also means the coding sequence of a gene product, as well as the non-coding regions of a gene product, including the 5'UTR and 3'UTR regions, introns, and promoters of the gene product. These definitions generally refer to single-stranded molecules, but in certain embodiments, they may also include additional strands that are partially, substantially, or completely complementary to a single-stranded molecule. Thus, nucleic acids may include double-stranded molecules, or double-stranded molecules containing one or more complementary strands or “complements” of a particular sequence containing a molecule. As used herein, single-stranded nucleic acids may be indicated by the prefix “ss”, double-stranded nucleic acids by the prefix “ds”, and triple-stranded nucleic acids by the prefix “is”. The term “gene” means a segment of DNA associated with the production of a polypeptide chain, which includes the regions before and after the coding region, as well as intervention sequences (introns) between individual coding segments (exons). A promoter is a region of a nucleic acid sequence that controls the initiation and rate of transcription. It may contain elements to which regulatory proteins and molecules can bind to initiate specific transcriptions of the nucleic acid sequence, such as RNA polymerase and other transcription factors. The term "enhancer" refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence. Enhancers can function in either direction and may be upstream or downstream of the promoter.

[0064] As used herein, the term “gene product” includes RNA (e.g., mRNA) transcribed from a gene, or polypeptides encoded by or translated from RNA.

[0065] The terms “polypeptide” and “protein” are interchangeable and refer to polymers of amino acid residues, not limited to their minimum length. Peptides, oligopeptides, dimers, and polymers, etc., composed of linearly arranged amino acids linked by peptide bonds, are included in the definition, regardless of whether they are produced biologically, recombinetally, or synthetically, or whether they are composed of spontaneously occurring or non-spontaneous amino acids. Both full-length proteins and their fragments are included in the definition. The term also includes concurrent (e.g., cleavage of the leader sequence of amino acids 1-25 of SEQ ID NO: 1) and post-translational modifications of polypeptides, such as disulfide bond formation, glycosylation, acetylation, phosphorylation, proteolytic cleavage (e.g., cleavage by furin or metalloproteinases and prohormone converters (PCs)), etc. Furthermore, for the purposes of this invention, “polypeptide” includes proteins with modifications to the native sequence, such as deletions, additions, and substitutions (generally, those that are virtually conserved as known to those skilled in the art), as long as the protein maintains the desired activity. These modifications may be intentional, for example, through site-directed mutagenesis, or accidental, for example, through mutations in the host producing the protein, or through errors in PCR amplification or other recombinant DNA methods. Polypeptides or proteins are composed of linearly arranged amino acids linked by peptide bonds, but in contrast to peptides, they have well-defined conformations. Unlike polypeptides, proteins generally consist of chains of 50 or more amino acids. For the purposes of this invention, the term “peptide” as used herein typically refers to a sequence of amino acids consisting of a single chain of D- or L-amino acids, or a mixture of D- or L-amino acids, linked by peptide bonds. Generally, peptides contain at least two amino acid residues and are less than about 50 amino acid lengths.

[0066] The incorporation of non-natural amino acids into peptides (or components of other compositions excluding protease recognition sequences), including synthetic non-native amino acids, substituted amino acids, or one or more D-amino acids, is required in certain situations. D-amino acid-containing peptides exhibit increased stability in vitro or in vivo compared to L-amino acid-containing forms. This makes the construction of D-amino acid-incorporating peptides particularly useful when greater in vivo or intracellular stability is required or needed. More specifically, D-peptides are resistant to endogenous peptidases and proteases, thereby providing superior oral transepithelial and transdermal delivery of conjugated drugs and conjugates, improved bioavailability of membrane-sustained complexes (discussed further below), and extended intravascular and stromal lifespan when such properties are required. The use of D-isomer peptides can also enhance the transdermal and oral transepithelial delivery of conjugated drugs and other cargo molecules. Furthermore, D-peptides can be efficiently processed for major histocompatibility complex class II restriction presentation to T helper cells, and are therefore less likely to induce humoral immune responses throughout the organism. Therefore, peptide conjugates can be constructed, for example, using D-isomers of cell-penetrating peptide sequences, L-isomers of cleavage sites, and D-isomers of therapeutic peptides. In certain embodiments, recombinant human MIS proteins consist of D- or L-amino acid residues. The use of naturally occurring L-amino acids has the advantage that their degradation products are relatively non-toxic to cells or organisms.

[0067] In further embodiments, recombinant human MIS proteins or fragments or derivatives thereof may be retro-inverso peptides. A retro-inverso peptide is a peptide in which the orientation of the bound peptide is reversed at at least one position, i.e., the amino- and carboxy-terminuses are reversed relative to the amino acid side chains. This allows the retro-inverso analog to have opposite ends and opposite direction of peptide bonds while appropriately maintaining a side-chain topology similar to that of the native peptide sequence. Retro-inverso peptides may contain L-amino acids, D-amino acids, or a mixture of L-amino acids and D-amino acids, where all amino acids may be D-isomers. A partial retro-inverso peptide analog is a polypeptide in which only a portion of the sequence is reversed and substituted with enantiomer amino acid residues. Since the retro-inverted portion of such an analog has opposite amino and carboxy-terminuses, the amino acid residues adjacent to the retro-inverted portion are substituted with side-chain-similar α-substituted geminal-diaminomethanes and malonates, respectively. The retro-inverso form of cell-penetrating peptides has been found to function efficiently in transposing across membranes, similar to its native form. The synthesis of retro-inversopeptide analogs is described in Bonelli, F. et al., Int J Pept Protein Res. 24(6):553-6 (1984); Verdini, A and Viscomi, GC, J. Chem. Soc. Perkin Trans. 1:697-701 (1985); and U.S. Patent No. 6,261,569, which are incorporated herein by reference in their entirety. Methods for the solid-phase synthesis of partial retro-inversopeptide analogs are also described (EP 97994-B), which are also incorporated herein by reference in their entirety.

[0068] As used herein, the terms "peptide," "polypeptide," or "fragment" of a molecule refer to a subset of adjacent polypeptides of that molecule. As used herein, the term "protein fragment" includes both synthetically derived and naturally occurring amino acid sequences derived from the naturally occurring amino acid sequence of MIS (SEQ ID NO: 1). A protein is said to be "derived from the naturally occurring amino acid sequence of a recombinant human MIS protein" if it can be obtained by fragmenting a recombinant human MIS protein, or if it can be synthesized based on knowledge of the naturally occurring amino acid sequence or the genetic material (DNA or RNA) encoding this sequence. Thus, "fragment" of a molecule means referring to a subset of any polypeptide of that molecule. In certain embodiments, a functional fragment of a recombinant human MIS includes at least a C-terminal domain and at least an N-terminal domain. In certain embodiments, a functional fragment includes a portion of the C-terminal and / or N-terminal domain of a recombinant human MIS protein (e.g., a fragment). Recombinant human MIS protein fragments that have greater activity than, or at least greater than, the wild-type MIS protein of Sequence ID No. 1 disclosed herein, and are soluble, are also included for use in the present invention.

[0069] A fragment of recombinant human MIS protein, for example, a functional fragment of SEQ ID NO: 2 or 3 useful in the methods disclosed herein, has at least 30% of the activity of the polypeptide SEQ ID NO: 2 or 3 in vivo to induce Müllerian regression in the Müllerian regression bioassay disclosed herein, for example, in the examples. In other words, a functional fragment of recombinant human MIS protein is a fragment that can bind to and activate MISRII, or induce at least 30% of the same activity as SEQ ID NO: 2 or 3, either alone or as a fusion protein, to induce Müllerian regression in the Müllerian regression bioassay disclosed herein (Figure 4). The fragments disclosed herein may be soluble (i.e., not membrane-bound). A “fragment” may be at least about 6, at least about 9, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, at least about 100, at least about 250, at least about 300 nucleic acids or amino acids, or any integer between these. Typical fragments include C-terminal truncations, N-terminal truncations, or truncations of both C and N terminals (e.g., deletions of at least 1, at least 2, at least 3, at least 4, at least 5, at least 8, at least 10, at least 15, at least 20, at least 25, at least 40, at least 50, at least 75, at least 100, or more amino acids, removed from the N-terminus, C-terminus, or both). Those skilled in the art can produce such fragments by simple deletion analysis. Such fragments of SEQ ID NO: 2 or 3 could be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, or more than 10 amino acids, e.g., 15, 30, 50, 100, or more than 100 amino acids, removed from the respective N-terminus and / or C-terminus of SEQ ID NO: 2 or 3.Those skilled in the art can easily identify the smallest peptide fragment of SEQ ID NO: 2 or 3 useful for the methods and compositions disclosed herein, or the fusion proteins disclosed herein, by sequentially deleting the N and / or C-terminal amino acids from SEQ ID NO: 2 or 3, or sequentially deleting the N-terminal and C-terminal amino acids from recombinant human MIS protein, and evaluating the function of the resulting peptide fragments, either individually or when cleaved. Those skilled in the art can form a functional fragment with multiple smaller fragments. These can be joined by crosslinking peptide linkers. Those skilled in the art can immediately select linkers to maintain wild-type conformation. Those skilled in the art can easily evaluate the function of recombinant human MIS protein compared to the recombinant human MIS protein corresponding to SEQ ID NO: 2 or 3, as disclosed herein for activating MISRII, or in Müllerian duct regression bioassays disclosed herein. If, using such an in vivo assay, a fragment of recombinant human MIS protein has at least 30% of the biological activity of the recombinant human MIS protein corresponding to SEQ ID NO: 2 or 3 disclosed herein, then the fragment is considered a valid recombinant human MIS protein fragment and can be used in the compositions and methods disclosed herein. In certain embodiments, the fragment of SEQ ID NO: 2 or 3 may have less than 200, less than 150, less than 100, less than 50, or less than 20 amino acids of SEQ ID NO: 2 or 3. In certain embodiments, the fragment of SEQ ID NO: 2 or 3 may have a peptide length of less than 100. However, as stated above, the fragment must be at least about 6, at least about 9, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, at least about 100, at least about 250, at least about 500 nucleic acids or amino acids, or any integer between these.

[0070] As used herein, the term “derivative” refers to peptides that have been chemically modified by, for example, ubiquitination, labeling, pegylation (derivativeation with polyethylene glycol), or the addition of other molecules. A molecule is also a derivative of another molecule if it contains additional chemical components that are not part of the molecule's normal structure. Such components can improve the solubility, absorption, biological half-life, etc., of the molecule. Alternatively, they can reduce the toxicity of the molecule, eliminate or reduce unwanted side effects, etc. Components that can mediate such effects are disclosed in Remington's Pharmaceutical Sciences, 18th edition, AR Gennaro, Ed., MackPubl., Easton, PA (1990).

[0071] When used in conjunction with “derivative,” “variant,” or “fragment,” the term “functional” means a polypeptide having biological activity (either functional or structural) substantially similar to the biological activity of the polypeptide that is its functional derivative, variant, or fragment. The term functional derivative is intended to include molecular fragments, analogs, or chemical derivatives. “Substantially similar” in this context means that the biological activity, for example, the activation of MISRII, is at least 25%, at least 35%, or at least 50% of the activity of the cited polypeptide, for example, the corresponding wild-type MIS polypeptide or recombinant human MIS protein, and preferably at least 60%, 70%, 80%, 90%, 95%, 100%, or more (i.e., the variant or derivative has greater activity than the wild-type), for example, 110%, 120%, or more. In other words, a functional fragment that is "substantially similar" to a recombinant human MIS protein in this context means that at least 25%, at least 35%, or at least 50% of the relevant or required biological activity of the corresponding recombinant human MIS protein is maintained.In an example of a functional fragment or peptide of recombinant human MIS protein disclosed herein (e.g., SEQ ID NO: 2 or 3), the functional fragment of SEQ ID NO: 2 or 3 is a protein or peptide containing a portion of SEQ ID NO: 2 or 3 that retains activity, preferably to activate MISRII or induce Müllerian regression in the Müllerian regression bioassay disclosed herein, with at least 25%, at least 35%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, or more compared to the full-length SEQ ID NO: 2 or 3 (i.e., the variant or derivative has greater activity than the wild-type MIS of SEQ ID NO: 1 or the recombinant human MIS protein of SEQ ID NO: 2 or 3), for example, at least 110%, at least 120%, or more. As another example, a fragment of MIS (e.g., amino acids 26-560 of SEQ ID NO: 1) would be a protein or peptide containing a portion of amino acids 26-560 of SEQ ID NO: 1 that retains activity for Müllerian duct regression. Preferably, the fragment of amino acids 26-560 of SEQ ID NO: 1 retains at least 25%, at least 35%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, or more (i.e., the variant or derivative has greater activity than the wild type), for example, at least 110%, at least 120%, or more, compared to the full-length amino acids 26-560 of SEQ ID NO: 1, in order to induce Müllerian duct regression in the Müllerian duct regression bioassay disclosed herein in the examples.As another example, a fragment of the HSA reader sequence of Sequence ID No. 6 may be a protein or peptide containing a portion of Sequence ID No. 6 that, as measured by an assay such as that disclosed in its entirety by reference in U.S. Patent No. 5,759,802, which is incorporated herein by reference, has at least 25%, at least 35%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, or more than (i.e., the variant or derivative has greater activity than the wild-type HSA sequence), for example, at least 110%, at least 120%, or more activity compared to the full-length HSA sequence of Sequence ID No. 6.

[0072] The terms “functional derivative” and “mimicking,” “biologically active variant,” or “biologically active fragment” refer to compounds that are interchangeable and possess biological activity (either functional or structural) substantially similar to the biological activity of the substance or molecule that is its functional derivative (e.g., recombinant human MIS protein). The term “functional derivative” is intended to include molecular fragments, variants, analogs, or chemical derivatives.

[0073] The term “functional derivative” is intended to include “fragments,” “variants,” “analogs,” or “chemical derivatives” of molecules. Molecules are said to be “substantially similar” to another molecule if both molecules have substantially similar structures or if both molecules have similar biological activity. Thus, if two molecules have similar activity, they are considered variants, because this term is used herein even if the structure of one molecule is not found in the other, or even if the amino acid residue sequences are not identical. “Analogs” of recombinant human MIS proteins mean molecules that are substantially similar in function to the whole molecule or a fragment thereof. As used herein, a molecule is said to be a “chemical derivative” of another molecule if it contains further chemical components that are not part of the usual molecule. Such components can improve the solubility, absorption, biological half-life, etc., of the molecule. Components can also reduce the toxicity of the molecule, or eliminate or reduce unwanted side effects of the molecule. Components capable of mediating such effects are disclosed in Remington's Pharmaceutical Sciences, 18th edition, AR Gennaro, Ed., MackPubl., Easton, PA(1990).

[0074] In recombinant human MIS proteins, a “variant” means a molecule that is substantially similar in structure and function to the whole molecule or any of its fragments. Therefore, as used herein, the term “variant” means a peptide or nucleic acid that differs from a naturally occurring polypeptide or nucleic acid by the deletion, addition, substitution, or side-chain modification of one or more amino acids or nucleic acids, but still retains one or more specific functions or biological activities of the naturally occurring molecule. Amino acid substitutions include modifications in which an amino acid is replaced by a different naturally occurring or unconventional amino acid residue. Such substitutions can be classified as “conserved” if an amino acid residue in a polypeptide is replaced by another naturally occurring amino acid having similar properties in terms of polarity, side-chain functionality, or size. Substitutions encompassed by the present invention may also be “unconserved” if an amino acid residue present in a peptide is replaced by an amino acid with different properties, for example, a naturally occurring amino acid from a different group (e.g., substitution of a charged or hydrophobic amino acid with alanine), or if a naturally occurring amino acid is replaced by an unconventional amino acid. In certain embodiments, amino acid substitutions are conserved. When using the term "variant" in reference to a polynucleotide or polypeptide, it includes polynucleotides or polypeptides that may be modified in their primary, secondary, or tertiary structure compared to each of the referenced polynucleotides or polypeptides (for example, compared to the wild-type polynucleotide or polypeptide). A "variant" of a recombinant human MIS protein is a molecule that is substantially similar in structure and function, i.e., its function is its ability to activate MISRII.

[0075] For example, variants of recombinant human MIS proteins may include different mutations or modifications from the cited amino acids in SEQ ID NO: 2 or 3. In certain embodiments, a variant of SEQ ID NO: 2 or 3 is a fragment of SEQ ID NO: 2 or 3 disclosed herein. In certain embodiments, a variant may be a different isoform of SEQ ID NO: 2 or 3 and may include different isomeric amino acids. A variant may be a spontaneously occurring, synthetic, recombinant, or chemically modified polynucleotide or polypeptide isolated or produced using methods known in the art. A variant may include conserved or non-conserved amino acid changes, as described below. Polynucleotide changes may result in amino acid substitutions, additions, deletions, fusions, and truncations in the polypeptide encoded by the cited sequence. A variant may include amino acid insertions, deletions, or substitutions, including, but not limited to, insertions of ornithine, which do not normally occur in human proteins, in the peptide sequence on which the variant is based.

[0076] In describing polypeptides, the term "conservative substitution" refers to a change in the amino acid composition of a polypeptide that does not substantially alter its activity. For example, a conservative substitution is the substitution of an amino acid residue with a different amino acid residue that has similar chemical properties. Conservative amino acid substitutions include the substitution of leucine with isoleucine or valine, the substitution of aspartic acid with glutamic acid, or the substitution of threonine with serine. A "conservative amino acid substitution" is made by substituting one amino acid with another that has similar structural and / or chemical properties. For example, the substitution of leucine with isoleucine or valine, the substitution of aspartic acid with glutamic acid, or the substitution of threonine with serine. Thus, “conservative substitutions” of a particular amino acid sequence refer to substitutions of these amino acids that are not important to polypeptide activity, or substitutions of important amino acids with other amino acids that have similar properties (e.g., acidic, basic, positive or negative charge, polar or nonpolar, etc.) that do not reduce the peptide activity (i.e., the ability of a peptide to reduce T-reg cells and / or inflammatory cytokines, as disclosed herein). Tables of conserved amino acids that provide functionally similar amino acids are known in the art. For example, each of the following six groups contains amino acids that are conserved substitutions with respect to each other: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W) (see also Creighton, Proteins, WH Freeman and Company (1984)).In certain embodiments, individual substitutions, deletions, or additions, such as altering, adding, or deleting a single amino acid or a small proportion of amino acids, can also be considered “conservative substitutions” if the alteration does not reduce the activity of the MIS (i.e., the ability of a recombinant human MIS protein or variant to induce Müllerian regression in vivo, which can be determined using the Müllerian regression bioassay disclosed herein). Insertions or substitutions are typically in the range of about 1 to 5 amino acids. The selection of conservative amino acids can be based on the position of the amino acid to be substituted in the peptide, for example, whether the amino acid is outside the peptide and exposed to the solvent, or inside the peptide and not exposed to the solvent.

[0077] In another embodiment, a person skilled in the art can select an amino acid that will substitute for an existing amino acid based on its position, i.e., its exposure to the solvent (i.e., whether the amino acid is exposed to the solvent or is on the outer surface of the peptide or polypeptide compared to an amino acid located internally that is not exposed to the solvent). Such selections of conserved amino acid substitutions are known in the art and are disclosed, for example, in Dordo et al, J. Mol Biol, 1999, 217, 721-739, and Taylor et al, J. Theor. Biol. 119(1986);205-218, and S. French and B. Robson, J. Mol. Evol. 19(1983)171. Thus, a person skilled in the art can select a conserved amino acid substitution suitable for an amino acid on the outside of a protein or peptide (i.e., an amino acid exposed to the solvent). For example, but not limited to these, the following substitutions can be used: Y with F, T with S or K, P with A, E with D or Q, N with D or G, R with K, G with N or A, T with S or K, D with N or E, I with L or V, F with Y, S with T or A, R with K, G with N or A, K with R, and A with S, K or P.

[0078] In another embodiment, those skilled in the art may select conserved amino acid substitutions suitable for the inner amino acids of a protein or peptide. For example, suitable conserved substitutions can be used for the inner amino acids of a protein or peptide (i.e., amino acids that are not exposed to the solvent). For example, but not limited to, the following conserved substitutions can be used: Y with F, T with A or S, I with L or V, W with Y, M with L, N with D, G with A, T with A or S, D with N, I with L or V, F with Y or L, S with A or T, and A with S, G, T or V. In certain embodiments, non-conserved amino acid substitutions are also included in the range of variants. A variant of a recombinant human MIS protein, for example, a variant of SEQ ID NO: 2 or 3, means any molecule that is substantially similar in structure and function to either the whole molecule or any fragment thereof of SEQ ID NO: 2 or 3.

[0079] The terms “homology,” “identity,” and “similarity” refer to the degree of sequence similarity between two peptides or between optimally aligned nucleic acid molecules. Homologousity and identity can be determined, respectively, by comparing positions in each sequence that can be aligned for comparison purposes. For example, this is based on the use of standard homology software, e.g., BLAST, version 2.2.14, at default positions. If equivalent positions in the compared sequences are occupied by the same base or amino acid, the molecules are identical at that position. If equivalent positions are occupied by similar amino acids (e.g., similar in three-dimensional structure and / or electrical properties, e.g., conserved amino acid substitutions), the molecules can be referred to as homologous (similar) at that position. Expressions of homology / similarity or identity as percentages mean a function of the number of similar or identical amino acids at positions shared by the compared sequences, respectively. Sequences that are “unrelated” or “non-homologous” have less than 40% identity. Preferably, they have less than 25% identity with the sequences disclosed herein.

[0080] As used herein, the term “sequence identity” means that two polynucleotide or amino acid sequences are identical on a comparison window (i.e., identical nucleotide-wise or residue-wise). The term “percentage of sequence identity” is calculated by comparing two optimally aligned sequences on a comparison window, determining the number of positions in both sequences where the same nucleic acid base (e.g., A, T, C, GU, or I) or residue is present, calculating the number of matching positions, dividing the number of matching positions within the comparison window (i.e., window size) by the total number of positions, and multiplying the result by 100 to obtain the percentage of sequence identity.

[0081] As used herein, the term “substantially identical” refers to a polynucleotide or amino acid sequence that contains at least 85% sequence identity, preferably at least 90–95%, and more commonly at least 99% sequence identity, compared to the referenced sequence, on a comparison window at least 18 nucleotides (6 amino acids), and more frequently at 24–48 nucleotides (8–16 amino acids). Here, the percentage of sequence identity is calculated by comparing the referenced sequence to a sequence that may contain deletions or additions of 20% or less of the entire referenced sequence on the comparison window. The referenced sequence may be a subset of a larger sequence. When used to describe polypeptides, the term “similarity” is determined by comparing the amino acid sequence and conserved amino acid substitutions of one polypeptide to the sequence of a second polypeptide.

[0082] As used herein, the terms “homolog” or “homogenetic” are interchangeable and, when used to describe polynucleotides or polypeptides, indicate that two polynucleotides or polypeptides, or a given sequence thereof, are identical in at least 70% of their nucleotides, typically about 75–99%, more preferably at least about 98–99%, with appropriate nucleotide insertions or deletions, or amino acid insertions or deletions, when arbitrarily aligned and compared, for example, using BLAST, version 2.2.14 with default parameters. As used herein, the terms “homolog” or “homogenetic” also mean homology in terms of structure and / or function. With respect to sequence homology, sequences are homologous if they are identical in at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or at least 99%. Determining homologous genes or peptides of the present invention can be easily done by those skilled in the art.

[0083] The term "substantially homologous" refers to sequences that are identical by at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. Homologous sequences may be the same functional genes in different species. Determining homologs of the genes or peptides of the present invention can be easily done by those skilled in the art.

[0084] When two molecules have substantially similar structures or similar biological activities, for example, when both molecules can activate MISRII, the molecules are said to be "substantially similar." Thus, if two molecules have similar activities (i.e., a variant of recombinant human MIS protein that can activate MISRII similar to the MIS protein corresponding to SEQ ID NO: 1 or the recombinant human MIS protein corresponding to SEQ ID NO: 2 or 3), they are considered variants and are included for the uses disclosed herein, even if the structure of one molecule is not found in the other or the amino acid residue sequences are not identical. In particular, when the term “substantially similar” is used to define a recombinant human MIS protein that includes a functional variant of a recombinant human MIS protein compared to the recombinant human MIS protein encoded by SEQ ID NO: 2 or 3, it means that a particular sequence of interest, for example, a variant or derivative sequence of a recombinant human MIS protein, differs from the sequence of the natural (or wild-type) MIS of SEQ ID NO: 1 or the recombinant human MIS protein (i.e., encoded by SEQ ID NO: 2 or 3) by one or more substitutions, deletions, or additions, although its net effect retains at least some of the biological activity found in the recombinant human MIS protein disclosed herein. Alternatively, nucleic acid and amino acid sequences that have less similarity to the recombinant human MIS protein but possess comparable biological activity are considered equivalent. In polynucleotide sequence determination, all subject polynucleotide sequences that can encode substantially similar amino acid sequences are considered substantially similar to the cited polynucleotide sequence, regardless of differences in codon sequences.A nucleotide sequence is "substantially similar" to a specific nucleic acid sequence of Sequence ID: 4 or 5 disclosed herein if (a) the nucleotide sequence hybridizes to the coding region of a native MIS nucleic acid, (b) the nucleotide sequence can hybridize to the nucleotide sequence of a recombinant human MIS protein encoded by Sequence ID: 4 or 5 under moderate stringent conditions and has similar biological activity to the recombinant human MIS protein, or (c) it is a degenerate nucleotide sequence as a result of genetic coding with respect to the nucleotide sequence defined in (a) or (b). A substantially similar protein would typically have more than 80% similarity to the corresponding sequence of the native protein.

[0085] In the context of peptide sequences, the term "substantially similar" indicates that a polypeptide contains a sequence that has at least about 60% sequence identity to the reference sequence, or 70%, 80%, or 85% sequence identity to the reference sequence, or most preferably 90% identity, on a comparison window of about 10 to 20 amino acid residues. In the context of amino acid sequences, "substantially similar" further includes conserved amino acid substitutions. This means that a polypeptide is substantially similar to a second polypeptide if, for example, two peptides differ by one or more conserved substitutions.

[0086] In one embodiment, the term “human homolog” refers to a DNA sequence having at least about 55% homology to the full-length nucleotide sequence of a recombinant MIS protein gene, such as one encoded by the genome of a human or animal, for example, a mouse or a transgenic animal. In one embodiment, the term “human homolog” for a protein identified as being associated with a recombinant human MIS protein refers to an amino acid sequence having 40% homology to the full-length amino acid sequence of a protein identified as being associated with a recombinant human MIS protein, such as one encoded by the genome of a transgenic animal of the present invention. More preferably, the homology is at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 75%, more preferably at least about 80%, more preferably at least about 85%, more preferably at least about 90%, and more preferably at least about 95%. As described above, the homology is at least about 50% to 100%, and in all intervals in between (i.e., 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, etc.). Determining human homologs of the genes of the present invention can be easily done by those skilled in the art.

[0087] In describing polypeptides, the term "conservative substitution" refers to a change in the amino acid composition of a polypeptide that does not substantially alter its activity. Therefore, a "conservative substitution" of a particular amino acid sequence refers to substitutions of amino acids that are not important to polypeptide activity, or substitutions of important amino acids that do not substantially alter activity, but instead use other amino acids with similar properties (e.g., acidic, basic, positive or negative charge, polar or nonpolar). Tables of conservative amino acids providing functionally similar amino acids are publicly known in the art. For example, each of the following six groups contains amino acids that are conserved substitutions for each other: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W) (see also Creighton, Proteins, WH Freeman and Company (1984)). Furthermore, individual substitutions, deletions, or additions that alter, add, or delete a single amino acid or a small proportion of amino acids in the encoded sequence are also considered "conserved substitutions."

[0088] As used herein, the term "non-conservative" means the substitution of an amino acid with a different amino acid residue having different chemical properties. Non-conservative substitutions include, but are not limited to, the substitution of aspartic acid (D) with glycine (G); the substitution of asparagine (N) with lysine (K); or the substitution of alanine (A) with arginine (R).

[0089] For sequence comparison, typically one sequence serves as the reference sequence against which the test sequence is compared. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, subsequence coordinates are specified as needed, and a sequence algorithm program is specified. The sequence comparison algorithm then calculates the percentage of identity between the test sequence and the reference sequence based on the specified program parameters.

[0090] The optimal arrangement (alignment) of sequences for comparison can be achieved, for example, by Smith and Waterman's local homology algorithm (Adv. Appl. Math. 2:482 (1981) incorporated herein by reference), by Needleman and Wunsch's homology alignment algorithm (J. Mol. Biol. 48:443-53 (1970) incorporated herein by reference), by Pearson and Lipman's similarity search method (Proc. Natl. Acad. Sci. USA 85:2444-48 (1988) incorporated herein by reference), by running these algorithms on a computer (e.g., GAP, BESTFIT, FASTA, and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (General, Ausubel et al. (eds.), Current Protocols in This can be done according to Molecular Biology, 4th ed., John Wiley and Sons, New York (1999).

[0091] One example of a useful algorithm is PILEUP. PILEUP produces multiple sequence alignments from a group of related sequences using progressive pairwise alignment to show the degree of sequence identity. It also plots a genealogy or dendrogram showing the clustering relationships used to produce the alignments. PILEUP employs a simplification of the progressive alignment method of Feng and Doolittle (J. Mol. Evol. 25:351-60 (1987) incorporated herein by reference). The method used is similar to that described by Higgins and Sharp (Comput. Appl. Biosci. 5:151-53 (1989) incorporated herein by reference). The program can align up to 300 sequences, each with a maximum length of 5,000 nucleotides or amino acids. The multiple alignment procedure begins with alignment of two of the most similar sequences, creating a cluster of the two aligned sequences. Next, this cluster is aligned to the next most relevant or aligned sequence cluster. Two clusters of sequences are aligned by a simple extension of the alignment of the two individual sequences in pairs. The final alignment is achieved by a series of progressive pair alignments. The program is executed by specifying specific sequences and their amino acid or nucleotide arrangements for the region of sequence comparison, and by specifying program parameters. For example, a cited sequence can be compared to other test sequences to determine the percentage of sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps.

[0092] Another example of an algorithm suitable for determining the proportion of sequence identity and sequence similarity is the BLAST algorithm by Altschul et al. (see J. Mol. Biol. 215:403-410 (1990), incorporated herein by reference). (See also Zhang et al., Nucleic Acid Res. 26:3986-90 (1998); Altschul et al., Nucleic Acid Res. 25:3389-402 (1997), incorporated herein by reference). Software for performing BLAST analysis is available on the National Center for Biotechnology Information website. This algorithm relates to the initial identification of high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that fit or satisfy a certain positive threshold score T when aligned with words of the same length in the database sequence. T is also called the neighbor word score threshold (Altschul et al. (1990), op. cit.). These initial neighbor word hits serve as seeds to initiate a search to find longer HSPs containing them. Word hits are then extended in both directions along their respective sequences as long as the cumulative alignment score increases. The extension of word hits in each direction is interrupted if: the cumulative alignment score drops by an amount X from its maximum achieved value; the cumulative score becomes zero or lower due to the accumulation of one or more negative score residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.The BLAST program uses, by default, a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-9 (1992), incorporated herein by reference), alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0093] In addition to calculating the degree of sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-77 (1993), incorporated herein by reference). One criterion of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a cited sequence if the smallest sum probability of the test nucleic acid being compared to the cited nucleic acid is less than approximately 0.1, more typically less than approximately 0.01, and most typically less than approximately 0.001.

[0094] The terms "insertion" or "deletion" typically refer to a range of approximately 1 to 5 amino acids. Acceptable variations can be experimentally determined by synthesizing peptides while systematically creating nucleotide insertions, deletions, or substitutions in the sequence using recombinant DNA technology.

[0095] In the context of peptides, the term "substitution" refers to a change in an amino acid, such as the substitution of an amino acid with a different substance, for example, another amino acid or amino acid component. Substitutions can be conservative or non-conservative.

[0096] A “molecular analog,” such as recombinant human MIS protein, e.g., SEQ ID NO: 2 or 3, refers to a molecule that is functionally similar to the whole molecule or any of its fragments. The term “analog” is intended to also include allelic, species, and induced variants. Analogies typically differ from naturally occurring peptides at one or more positions, often in terms of conserved substitutions. Analogies typically exhibit at least 80% or 90% sequence identity with the native peptide. Certain analogs also include modifications of non-native amino acids or N- or C-terminal amino acids. Examples of non-native amino acids, but not limited to, include acedisubstituted amino acids, N-alkyl amino acids, lactate, 4-hydroxyproline, γ-carboxyglutamine, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, and δ-N-methylarginine. As described below, fragments and analogs can be screened for prophylactic or therapeutic efficacy in transgenic animal models.

[0097] "Covalent bonding" refers to a linkage, either directly or indirectly (for example, via a linker), through a covalent chemical bond.

[0098] As used herein, the term “fusion protein” refers to a recombinant protein comprising two or more proteins. A fusion protein can be produced, for example, by ligating a nucleic acid sequence encoding one protein to a nucleic acid encoding another protein so as to constitute a single open reading frame that can be translated intracellularly into a single polypeptide containing all the intended proteins. The order of protein arrangement is diverse. As a non-limiting example, a nucleic acid sequence encoding a recombinant human MIS fusion protein is obtained from a nucleotide sequence encoding a recombinant human MIS protein or a functional derivative, fragment, or variant, fused within a frame to either the 5' or 3' end of a gene encoding a first fusion partner, e.g., an IgG1 Fc fragment. In this method, based on gene expression, the recombinant human MIS protein or its functional derivative, fragment, or variant is functionally expressed and fused to the N-terminus or C-terminus of IgG1 Fc. In certain embodiments, the polypeptide probe is modified so that the functionality of the recombinant human MIS protein or its functional derivative, fragment, or variant is not substantially affected in terms of biological activity by fusion to the first fusion partner, e.g., IgG1 Fc.

[0099] "Specifically binding" or "specific binding" means a compound or antibody that recognizes and binds to the required polypeptide, but does not substantially recognize or bind to other molecules in the sample, such as a biological sample naturally containing the polypeptide of the present invention.

[0100] "Substantially pure" means nucleic acids or other molecules that have been isolated from naturally occurring components. Typically, a polypeptide is substantially pure if it contains at least about 60% by weight, at least about 70% by weight, at least about 80% by weight, at least about 90% by weight, at least about 95% by weight, or at least about 99% by weight, without naturally occurring proteins and spontaneously occurring organic molecules. For example, substantially pure polypeptides can be obtained by extraction from natural sources, by expression of recombinant nucleic acids in cells that do not normally express that protein, or by chemical synthesis.

[0101] "Enhanced proteolytic stability" means a reduction in the rate or degree of proteolytic degradation of the peptide sequence by at least about 2%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% compared to a control sequence under the same conditions (e.g., in vivo or in vitro system, e.g., in cells or cell lysates). Peptides with enhanced proteolytic stability may include any of the following modifications, e.g., insertions, deletions, or point mutagenesis that remove or eliminate sites involved in proteolytic cleavage at specific sites. The sites of proteolytic cleavage can be identified based on known target sequences or using computer software (e.g., software described in Gasteiger et al., Protein Identification and Analysis Tools on the ExPASy Server. In John M. Walker, ed. The Proteomics Protocols Handbook, Humana Press (2005)). Alternatively, the proteolytic sites can be experimentally determined, for example, by Western blotting of the protein after expression or incubation in a cell line or cell lysate, and sequencing of the identified fragments to determine the next cleavage site.

[0102] As used herein to describe nucleic acid molecules, the term “recombinant” means a genome, cDNA, virus, semi-synthetic, and / or synthetic source that is not related in any way to all or part of the naturally associated polynucleotides in terms of their origin or manipulation. As used herein with respect to proteins or polypeptides, the term “recombinant” means a polypeptide produced by the expression of recombinant polynucleotides. As used with respect to host cells, the term “recombinant” means a host cell into which recombinant polynucleotides have been introduced. Recombinant is also used herein to mean a material (e.g., a cell, nucleic acid, protein, or vector) that has been modified by the introduction of a heterologous material (e.g., a cell, nucleic acid, protein, or vector).

[0103] The terms “subject” and “individual” are used interchangeably herein and refer to an animal, such as a human, to which treatment is administered, including prophylactic treatment, with the pharmaceutical compositions according to the present invention. The term “subject” as used herein refers to both human and non-human animals. The terms “non-human animal” and “non-human mammal” are used interchangeably herein and include all vertebrates, such as mammals, such as non-human primates (especially higher primates), sheep, dogs, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, and non-mammals, such as birds, amphibians, reptiles, etc. In one embodiment, the subject is a human. In another embodiment, the subject is an animal substitute as an experimental animal or disease model. The term does not imply a specific age or sex. This includes living and neonatal subjects, as well as fetuses, regardless of whether they are male or female. Examples of subjects include humans, dogs, cats, cattle, goats, and mice. The term "subjects" is intended to include transgenic species. The term "subjects" also includes mammals, such as humans, to which treatment, e.g., therapeutic and / or prophylactic treatment, is administered with compositions comprising recombinant human MIS proteins disclosed herein.

[0104] The term “tissue” is intended to include complete cells, blood, blood preparations such as plasma and serum, bone, joints, muscles, smooth muscle, and organs.

[0105] The terms “disease” or “disorder” are interchangeable herein and mean any change in any of several conditions of the body or organs that interrupts or impairs the performance of a function and / or causes symptoms, such as discomfort, inadequacy, pain, or death, in a person or a person who comes into contact with a person. Disease or disorder includes distemper, ailing, ailment, amlady, disorder, sickness, illness, and complaint, inderdisposion, affection.

[0106] The terms “malignant (tumor)” and “cancer” are used interchangeably herein and refer to diseases characterized by uncontrolled, abnormal proliferation of cells. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system. The terms are also intended to include diseases of any organ or tissue in mammals characterized by an inadequately controlled or uncontrolled increase of normal or abnormal cells in its tissue, and its effects on the body as a whole. Diseases of cancer included in the scope of the definition include benign neoplasms, dysplasias, hyperplasias, and metastatic growths or other changes, such as vitiligo, which often precedes the development of cancer.

[0107] As used herein, the term “tumor” refers to a population of transformed cells characterized by the inclusion of vascular structures of at least some vascular origin. Transformed cells are characterized by rapid and continuous uncontrolled cell proliferation of neoplasms, even after the stimulus to initiate new growth has ceased. The term “tumor” is more broadly used to include tumor parenchymal cells and supporting stroma, including vascular vessels that infiltrate the tumor parenchymal cell population. Tumors are generally malignant tumors, i.e., cancers with the ability to metastasize (i.e., metastatic tumors), but tumors can also be non-malignant (i.e., non-metastatic tumors). Tumors are cancerous, a deadly natural process, a neoplastic disease. Cancer cells are highly undifferentiated and exhibit invasive and metastatic characteristics.

[0108] As used herein, the terms “metastatic” or “metastatic tumor” refer to a secondary tumor that arises from cells that branch off and grow elsewhere in the body from a primary tumor, and, if the primary tumor is a solid tumor, from cells that have been separated and transported. As used herein, a primary tumor refers to a tumor that is organized in the location or organ in which it exists and has not metastasized from another location to that location. As used herein, a “malignant tumor” refers to a tumor that is invasive and metastatic and exhibits a high degree of anaplasia. Anaplasia is the reversal of cells to an immature or less differentiated form and occurs in most malignant tumors.

[0109] As used herein, the term "treatment-resistant cancer" means a cancer present in a subject that is resistant to or resistant to at least two different anticancer agents, such as chemotherapy agents, and typically, as defined herein, means that the subject is being treated with at least two different anticancer agents that do not provide an effective treatment.

[0110] The term "sensitization" is used interchangeably herein and means making cells susceptible or responsive to other secondary agents, such as other prodrugs, or other environmental effects, such as radiation.

[0111] As used herein, the terms “treatment” or “procedure” include both therapeutic and prophylactic or preventive measures, the purpose of which is to prevent or slow the development of a disease, for example, to reduce the development of a tumor, the spread of cancer, or at least one effect or symptom of an inappropriate proliferation or population of cells, such as a condition, disease, or disorder associated with cancer. Treatment is “effective,” as defined herein, generally if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of a disease is reduced or halted. That is, “treatment” includes not only improvement of symptoms or markers but also a measurable reduction in one or more symptoms or measurable markers of a disease or disorder (e.g., cancer), and / or halt to at least a slowing of the progression or worsening of symptoms that would be expected in the absence of treatment. Measurable reduction includes a statistically significant decrease in any of the measurable markers or symptoms. Beneficial or desired clinical outcomes include, but are not limited to, relief of one or more symptoms, whether detectable or undetectable; a reduction in the severity of the disease; a state of stabilization (i.e., not exacerbation) of the disease; a delay or slowing of disease progression; relief or reduction of the disease state; and remission (whether partial or complete), whether detectable or undetectable. “Treatment” may also mean an extended survival time compared to the survival time predicted without treatment. Persons requiring treatment include those already diagnosed with cancer and those at risk of developing secondary tumors due to metastasis. In certain embodiments, treatment may be a prophylactic measure.

[0112] As used herein, the term “effective dose” means the amount of recombinant human MIS protein disclosed herein to alleviate at least one or more symptoms of a disease or disorder, and relates to a sufficient amount of the pharmaceutical composition to produce the desired effect. As used herein, the term “therapeutic effective dose,” for example, the therapeutic effective dose of a pharmaceutical composition containing at least one recombinant human MIS protein disclosed herein, means a sufficient amount of the composition to treat a disease in a reasonable benefit / risk ratio applicable to any medical treatment. Therefore, the term “therapeutic effective dose” means the amount of the composition disclosed herein that is sufficient to produce a therapeutically or prophylactically significant reduction in symptoms or clinical markers associated with cancer or a cancer-mediated condition.

[0113] A therapeutically or prophylactically significant reduction in symptoms is defined as an increase or decrease in the measured parameter compared to a control or untreated subject, for example, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 125%, at least about 150%, or more. The measured or measurable parameter is a clinically detectable marker of the disease, such as an increase or decrease in the level of a biological marker, as well as a parameter or marker for the disease or disorder related to a clinically acceptable scale of symptoms. However, the overall daily dosage of any composition or formulation disclosed herein will be determined by the attending physician within the bounds of sound medical judgment. The exact amount required will vary depending on factors such as the type of disease to be treated.

[0114] With respect to the treatment of subjects with cancer with a pharmaceutical composition comprising at least one recombinant human MIS protein disclosed herein, the term “therapeutic effective dose” means a safe and sufficient amount to prevent or delay tumor development and further proliferation or metastasis in a cancer patient. This amount can treat or quell cancer, slow the progression of cancer, slow or inhibit tumor growth, slow or inhibit tumor metastasis, slow or inhibit the establishment of secondary tumors at metastatic sites, or inhibit the formation of new tumor metastases. The effective dose for treating cancer depends on the tumor to be treated, the severity of the tumor, the level of drug resistance of the tumor, the species to be treated, the age and general condition of the subject, the mode of administration, etc. Therefore, it is not possible to specify an exact “effective dose.” However, for any given case, an appropriate “effective dose” can be determined by a person skilled in the art using only conventional experiments. The efficacy of a treatment can be determined by a person skilled in the art; for example, efficacy can be evaluated in animal models of cancer and tumors. For example, the administration of any treatment or composition or formulation that leads to the treatment of a rodent having cancer and a reduction in at least one symptom of cancer, such as a reduction in tumor size or a slowing or cessation of the rate of tumor growth, indicates an effective treatment. In embodiments in which a composition is used to treat cancer, the efficacy of the composition can be determined using an experimental animal model of cancer, such as a wild-type mouse or rat, or preferably a transplant of tumor cells. When using an experimental animal model, the efficacy of the treatment is demonstrated if the reduction in cancer symptoms, such as a reduction in tumor size or a slowing or cessation of the rate of tumor growth, occurs faster in treated animals than in untreated animals. “Faster” means, for example, that the reduction in tumor size is at least 5% faster, more preferably, for example, 1 day faster, 2 days faster, 3 days faster, or faster.

[0115] As used herein, the term “treatment” when referring to cancer treatment means a reduction in cancer symptoms and / or biochemical markers. For example, a significant reduction in at least one cancer biochemical marker would be considered an effective treatment. Examples of such cancer biochemical markers include CD44, telomerase, TGF-α, TGF-β, erbB-2, erbB-3, MUC1, MUC2, CK20, PSA, CA125, and FOBT. A reduction in the rate of cancer cell proliferation by at least about 10% would also be considered an effective treatment by the methods disclosed herein. As another example, a reduction in cancer symptoms, such as a slowdown in the rate of cancer growth by at least about 10%, or a cessation of tumor size increase, or a reduction in tumor size by at least about 10%, or a reduction in tumor spread (i.e., tumor metastasis) by at least about 10%, would also be considered an effective treatment by the methods disclosed herein. In certain embodiments, it is desirable, but not required, for the therapeutic agent to actually kill the tumor.

[0116] The term “prophylactic effective dose” refers to the amount of recombinant human MIS protein or its functional fragment or variant that is effective in the required dosage and organs to achieve the desired prophylactic outcome, for example, to prevent the development of cancer in subjects at risk of cancer progression. Typically, a prophylactic dose of recombinant human MIS protein or its functional fragment or variant is administered to subjects in the pre- or early stages of cancer, or to subjects with a genetic predisposition to cancer, such as, but not limited to, subjects with gene mutations that increase the likelihood of developing ovarian cancer. In certain embodiments, the prophylactic effective dose is less than the therapeutic effective dose. The prophylactic effective dose of recombinant human MIS protein or its functional fragment or variant is also the amount in which the beneficial effects of any of the compounds outweigh the toxic or adverse effects.

[0117] As used herein, the terms “prevention” and “prevention” mean avoiding or delaying the signs of a disease or disorder, such as an autoimmune disease, or one or more symptoms or measurable markers. Delaying the signs of a symptom or marker is a delay in the time it takes for such symptoms or markers to appear in a control or untreated subject who has a similar potential or susceptibility to the progression of the disease or disorder. The terms “prevention” and “prevention” include not only avoiding or preventing the symptoms or markers of a disease, but also reducing the severity or degree of any one of the symptoms or markers of a disease, to the extent of these symptoms or markers in a control or individual who has a similar potential or susceptibility to the progression of the disease or disorder, or to the extent of symptoms or markers that are likely to occur based on historical or statistical standards of the population affected by the disease or disorder. "Severity reduction" means a reduction of at least 10% in the severity or degree of symptoms or measurable disease markers compared to the control or reference standard, e.g., at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% (i.e., no symptoms or measurable markers).

[0118] As used herein, the terms “administration” and “introduction” are interchangeable herein and refer to the placement of the metabolic regulator agent of the present invention to a subject by a method or route that results in at least partial localization of the recombinant human MIS protein at the required site. The compounds of the present invention can be administered by any suitable route that results in effective treatment in the subject. In certain embodiments, for the treatment of cancer, the recombinant human MIS protein can be placed directly at or near the site of a tumor, or administered systemically.

[0119] "Composition" or "Pharmaceutical Composition" means a composition that is interchangeably used herein and typically comprises excipients, such as conventionally used in the art and pharmaceutically acceptable carriers suitable for administration to cells. Cells may be the part of a target, for example, for therapeutic, diagnostic, or preventive purposes. Cells may also be cultured and may be, for example, cells as part of an assay for screening potential pharmaceutical compositions, and cells may also be parts of a transgenic animal for research purposes. A composition may also be a cell culture in which a polypeptide or polynucleotide encoding the metabolic regulator of the present invention is present in the cells or cell medium. Furthermore, compositions for topical (e.g., oral mucosa, respiratory mucosa) and / or oral administration may form solutions, suspensions, tablets, pills, capsules, sustained-release formulations, mouthwashes, or powders, which are well known in the art and described herein. Furthermore, compositions for topical (e.g., oral mucosa, respiratory mucosa) and / or oral administration may form liquids, suspensions, tablets, pills, capsules, sustained-release formulations. A composition may comprise stabilizers and preservatives. Examples of carriers, stabilizers, and adjuvants are disclosed in University of the Sciences in Philadelphia (2005) Remington: The Science and Practice of Pharmacy with Facts and Comparisons, 21st Ed.

[0120] As used herein, the terms “parenteral administration” and “administered parenterally” mean a mode of administration, usually by infusion, other than intra-intestinal and local administration, and include, but are not limited to, intravenous, intramuscular, intra-arterial, intra-shearing, intraventricular, intracapsular, intraorbital, intracardiac, intracutaneous, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intrathecal, intraventricular, intraspinal, and intrasternal injections and infusions. As used herein, the terms “systemic administration,” “systemically administered,” “peripheral administration,” and “peripherally administered” mean the administration of recombinant human MIS proteins such that it enters the animal system and thereby enters metabolism and other similar systems, for example, subcutaneous administration.

[0121] The term "pharmaceutically acceptable" is used herein to mean that these compounds, materials, compositions, and / or dosage forms are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, within the bounds of sound medical judgment, and that they are balanced by a reasonable benefit / risk ratio.

[0122] As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, related to maintaining the activity of the agent of interest and carrying or transporting it from one organ or body part to another organ or body part. In addition to being “pharmaceutically acceptable” as defined herein, each carrier must be “acceptable” in the sense that it is compatible with the other components of the formulation. A pharmaceutical formulation contains the compound of the present invention in combination with one or more pharmaceutically acceptable components. The carrier may be in the form of a solid, semi-solid, or liquid diluent, cream, or capsule. These pharmaceutical preparations are further subjects of the present invention. Typically, the amount of the active compound is 0.1 to 95% by weight of the preparation, preferably 0.2 to 20% by weight in the preparation for parenteral use, and preferably 1 to 50% by weight in the preparation for oral administration. For clinical use of the method of the present invention, the targeted delivery composition of the present invention is formulated into a pharmaceutical composition or pharmaceutical preparation for parenteral administration, for example, intravenously, mucosally, for example, intranasally, intraintestinally, for example orally, topically, for example transdermally, orally, for example via corneal dissection, or other forms of administration.

[0123] As used herein, the term “oncogene” refers to a nucleic acid sequence that encodes a mutated and / or overexpressed form of a polypeptide of a normal gene, thereby relieving the cell of its normal growth limitations in a dominant manner, thereby conferring tumorigeneic ability to the cell, either alone or in conjunction with other changes. Examples of “oncogenes” include gp40 (v-fms); p21 (ras); p55 (v-myc); p65 (gag-jun); pp60 (v-src); v-abl; v-erb; v-erba; v-fos, etc. Proto-oncogenes refer to the normal expression of nucleic acids that express the normal cellular equivalent of an oncogene, and these genes are typically those that are usually associated with signaling or regulation of cell growth.

[0124] As used herein, the term “regeneration” means the regrowth of a population of cells, an organ, or a tissue, which in certain embodiments is after disease or injury.

[0125] The term “vector” refers to a nucleic acid capable of transporting another ligated nucleic acid. A plasmid is a species of the genus encompassed by “vector.” Typically, the term “vector” refers to a nucleic acid sequence containing the origin of replication, as well as other substances for replication and / or maintenance in a host cell. A vector capable of directing the expression of an actionably ligated gene and / or nucleic acid sequence is referred to herein as an “expression vector.” Generally, useful expression vectors are often in the form of a “plasmid,” which, in its vector form, does not bind to a chromosome and typically refers to a circular double-stranded DNA loop containing substances or encoded DNA for stabilization or transient expression. Other expression vectors that can be used in the methods disclosed herein include, for example, plasmids, episomes, bacterial artificial chromosomes, yeast artificial chromosomes, bacteriophages, or viral vectors, which can be integrated into the host genome and autopneumogenerated within a particular cell. Vectors can be DNA or RNA. Other forms of expression vectors known to those skilled in the art that provide equivalent function may also be used, such as self-replicating extrachromosomal vectors or vectors that are incorporated into the host genome. Preferred vectors are those capable of autonomous replication and / or expression of ligated nucleic acids. Vectors capable of directing the expression of actionably ligated genes are referred to herein as “expression vectors.” Expression vectors can induce stable or transient expression of DNA. A typical expression vector for use in the present invention is pcDNA3.1.

[0126] The term "viral vector" refers to a virus-related vector used as a virus or as a carrier of nucleic acid components into cells. Components can be incorporated and packaged into non-replicating, deficient viral genomes, such as adenoviruses, adeno-associated viruses (AAVs), or herpes simplex virus (HSV), or others, such as retroviral and lentiviral vectors, for infection or transduction into cells. The vector may or may not be incorporated into a cellular genome. Components may optionally contain viral sequences for transfection. Alternatively, components can be incorporated into vectors capable of episomal replication, such as EPV and EBV vectors.

[0127] As used herein, the terms “promoter,” “promoter region,” or “promoter element” are interchangeable and refer to a segment of a nucleic acid sequence, typically but not limited to DNA, RNA, or analogues thereof, that controls the transcription of an actionably linked nucleic acid sequence. A promoter region includes specific sequences sufficient for RNA polymerase recognition, binding, and transcription initiation. This portion of the promoter region is referred to as the promoter. Furthermore, the promoter region includes sequences that regulate RNA polymerase recognition, binding, and transcription initiation. These sequences may be cis-acting or trans-acting. Depending on the nature of the control, the promoter may be constitutive or regulated.

[0128] The term “regulatory sequence” is used interchangeably with “regulatory element” herein and refers to a segment of nucleic acid, typically but not limited to DNA or RNA or analogues thereof, that regulates the transcription of an actionably linked nucleic acid sequence, thereby functioning as a transcription modulator. Regulatory sequences regulate the expression of actionably linked genes and / or nucleic acid sequences. Regulatory sequences often include “regulatory factors,” which are nucleic acid sequences that are transcription-binding domains and are recognized by the nucleic acid-binding domains of transcription proteins and / or transcription factors, repressors, or enhancers. Typically, regulatory sequences include, but not limited to, transcription promoters, inducible promoters, and transcription factors, optional action sequences for controlling transcription, sequences encoding appropriate mRNA-ribosome binding sites, and sequences for controlling the termination of transcription and / or translation. Regulatory sequences may be single regulatory sequences, multiple regulatory sequences, or modified regulatory sequences or fragments thereof. A modified regulatory sequence is a regulatory sequence in which the nucleic acid sequence has been altered or modified by certain means, such as, but not limited to, mutation, methylation, etc.

[0129] As used herein, the term “operatably linked” refers to the functional relationship of a nucleic acid sequence to regulatory sequences of nucleotides, such as promoters, enhancers, transcription and translation stop sites, and other signal sequences. For example, operational linkage of a nucleic acid sequence, typically DNA, to a regulatory sequence or promoter region refers to a physical and functional relationship between DNA and a regulatory sequence or promoter such that the transcription of the DNA is initiated from the regulatory sequence or promoter by an RNA polymerase that specifically recognizes, binds to, and transcribes that DNA. To optimize expression and / or in vitro transcription, it may be necessary to modify the regulatory sequences for the expression of nucleic acids or DNA in the cell type in which they are expressed. The demand or need for such modifications can be determined empirically. Enhancers do not need to be located in close proximity to the coding sequence they enhance. Furthermore, a gene transcribed from a promoter that is trans-regulated by a factor transcribed by a second promoter can be said to be operationally linked to the second promoter. In such a case, the transcription of the first gene is said to be operationally linked to the first promoter and also operationally linked to the second promoter.

[0130] As used herein, the term “biological sample” also means cells or populations of cells, or specific amounts of tissue or fluid from an object. Often, a sample is removed from an object, but the term “biological sample” can also refer to cells or tissues that are analyzed in vivo, i.e., not removed from an object. Often, “biological sample” would include cells from an object, but the term can also refer to non-cellular biological samples that can be used to measure protein phosphorylation levels, such as non-cellular fractions of blood, saliva, or urine. In certain embodiments, “biological sample” or “tissue sample” refers to a sample of tissue or fluid isolated from an individual, and includes, but is not limited to, blood, plasma, serum, tumor biopsy, urine, feces, sputum, cerebrospinal fluid, pleural fluid, nipple aspirate, lymph, external skin sections, airways, intestines, urogenital tract, tears, saliva, lotion, cells (including, but not limited to, blood cells), tumors, organs, and samples of in vitro cell culture components. In certain embodiments, the biological sample is from excision, bronchoscopic biopsy, or core-needle biopsy of primary, secondary, or metastatic tumors, or from cell blocks from pleural fluid. Furthermore, fine-needle aspiration biological samples are also useful. In certain embodiments, the biological sample is primary ascites cells. The sample may be fresh, frozen, fixed, or optionally paraffin-embedded, or may be subjected to other tissue preservation methods, such as methods for preserving the phosphorylation state of polypeptides in the biological sample. The biological sample may also mean a sample of biological tissue or fluid containing proteins or cells. Such samples include, but are not limited to, tissues isolated from a subject or animal. The biological sample may also include tissue sections, e.g., biopsy and autopsy samples, frozen sections taken for histological purposes, blood, plasma, serum, sputum, stool, tears, mucus, hair, and skin. The biological sample also includes explants and primary and / or transformed cell cultures obtained from patient tissue.Biological samples can be obtained by removing a sample of cells from a subject, but can also be achieved by using previously isolated cells (e.g., those isolated by another person at a different time and / or for another purpose), or by performing the method of the present invention in vivo. Archival tissues, e.g., those with a treatment or outcome history, can also be used. Biological samples include, but are not limited to, tissue biopsies, scrapes (e.g., buccal friction pieces), whole blood, plasma, serum, urine, saliva, cell cultures, or cerebrospinal fluid. Biological samples also include tissue biopsies and cell cultures. Biological samples can be obtained by removing a sample of cells from a subject, but can also be achieved by using previously isolated cells (e.g., those isolated by another person), or by performing the method of the present invention in vivo. Such samples include, but are not limited to, whole blood, cultured cells, primary cell preparations, sputum, amniotic fluid, tissue or microneedle biopsy samples, and ascites. In certain embodiments, the biological sample is taken from a human patient, and in other embodiments, the biological sample is taken from any mammal, such as a rodent, an animal model of a disease, a commercial animal, a companion animal, a dog, a cat, a sheep, a cow, and a pig. The biological sample may be pretreated as necessary for storage or preservation by diluting or concentrating it with a suitable buffer solution as necessary. At physiological pH, any of several standard aqueous buffer solutions may be used with one of various buffers, such as phosphoric acid, Tris, etc. The biological sample may be stored for use in a specific environment before being used in the assays disclosed herein. Such storage may be at +4°C, or frozen, for example, at -20°C or -80°C, provided that a cryopreservative suitable for maintaining cell viability when the cells are thawed is used.

[0131] As used herein, the terms “reduction,” “decrease,” or “decrease” generally mean a reduction of only a statistically significant amount relative to the citation. However, to avoid doubt, as defined herein, “reduction” means a statistically significant reduction of at least 10% compared to the citation level, e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or greater than that, less than 100%, or 100% (i.e., a level of non-existence compared to the cited sample), or any reduction of 10–100% compared to the citation level. As used in the context of levels of gene expression or activity, the terms “decrease” or “inhibition” mean a reduction in protein or nucleic acid levels or activity in cells, cell extracts, or cell supernatants. For example, such a reduction may occur due to a decrease in RNA stability, transcription, or translation, an increase in proteolysis, or RNA interference. Preferably, this reduction is at least about 5%, at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 80%, or at least about 90% of the level of expression or activity under control conditions.

[0132] As used herein, the term “low” generally means lower by a statistically significant amount. To avoid ambiguity, “low” means a statistically significant level that is at least 10% lower than the citation level, e.g., at least 20% lower than the citation level, at least 30% lower than the citation level, at least 40% lower than the citation level, at least 50% lower than the citation level, at least 60% lower than the citation level, at least 70% lower than the citation level, at least 80% lower than the citation level, at least 90% lower than the citation level, less than 100% lower than the citation level, and 100% lower (i.e., the level of non-existence compared to the cited sample).

[0133] As used herein, the terms “increase” or “increase” generally mean an increase of a statistically significant amount. To avoid doubt, as defined herein, “increase” means a statistically significant increase of at least 10% compared to the citation level, for example, an increase of at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or more compared to the citation level, for example, an increase of at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times or more compared to the citation level. As used in the context of gene or protein expression or activity, the term “increase” means a positive change in protein or nucleic acid levels or activity in a cell, cell extract, or cell supernatant. For example, such an increase may result from an increase in RNA stability, transcription, or translation, or a decrease in proteolysis. Preferably, this increase is at least 5%, at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 80%, at least about 100%, at least about 200%, or at least about 500% or more, above the level of expression or activity under control conditions.

[0134] As used herein, the term “high” generally means a statistically significant amount higher than the citation. To avoid ambiguity, “high” means a statistically significant level that is at least 10% higher than the citation level, for example, at least 20% higher, at least 30% higher, at least 40% higher, at least 50% higher, at least 60% higher, at least 70% higher, at least 80% higher, at least 90% higher, at least 100% higher, at least twice as high, at least three times higher, at least four times higher, at least five times higher, at least ten times higher, or more.

[0135] The articles “a” and “an” are used herein to refer to one or more (i.e., at least one) of the grammatical purposes of the article. For example, “element” means one element or more than one element.

[0136] Except in the examples, or unless otherwise indicated, all figures expressing quantities of components or reaction conditions used herein should be understood in all cases as being modified by the term “approximately.” When used in conjunction with percentages, the term “approximately” may mean ±1%. The present invention is further illustrated in detail by the following examples, but the scope of the invention should not be limited thereto.

[0137] It should be understood that the present invention is not limited to the specific methods, protocols, and reagents described herein, but can be diverse. The terms used herein are for the purpose of describing specific examples and are not intended to limit the scope of the invention as defined solely by the claims. Other features and advantages of the present invention will become apparent from the following detailed description, drawings, and claims. Müllerian duct inhibitors (MIS)

[0138] While not intended to be a theoretical link, Müllerian repressors (MISs) are a component of the TGFβ multiple gene family of glycoproteins. All proteins in this family are produced as dimeric precursors, undergoing post-transcriptional processing for activation that requires cleavage and dissociation to release a bioactive C-terminal fragment. MISs are 140kDa dimers composed of identical 70kDa disulfide-bonded monomers, each consisting of a 57kDa N-terminal domain and a 12.5kDa carboxyl terminus (C-terminus). Thus, MISs contain two identical monomers (hence the term "homodimer"), each monomer containing two non-covalently held domains: the N-terminal and C-terminal domains. The purified C-terminal domain is the bioactive component and requires cleavage for activity. The N-terminal domain assists in protein folding in vivo, facilitating delivery of the C-terminal peptide to its receptor, e.g., MISRI and MISRII. Non-cleaved mutants of MIS are biologically inactive.

[0139] The carboxyl-terminal active domain exhibits amino acid homology with members of the TGFb family, such as TGF-B 1, 2, and 3, inhibin, activin, and osteomorphonomastic proteins, as well as members of growth and differentiation factors (GDFs). The structure of the MIS carboxyl-terminal domain is supported by seven cysteines involved in both intramolecular and intermolecular disulfide crosslinks, which derive its structural stability, as evidenced by its homology to the three-dimensional structure of TGFb, as revealed by molecular modeling (Lorenzo, Donahoe, et al., unpublished).

[0140] Like other TGFb members, MIS is cleaved by plasmin to form its amino-terminal and carboxyl-terminal domains. This proteolytic process is required for its physiological activity and occurs at sites similar to the dinucleotide cleavage sites found in the TGFb sequence. The resulting products are closely associated in a non-covalent complex that dissociates at low pH. Therefore, technically complex and time-consuming protocols involving plasmin treatment and molecular size exclusion chromatography are required to enhance or complete the separation of the carboxyl terminus from the amino terminus.

[0141] MIS contains two major cleavage sites that are sensitive to plasmin. The first single-nucleotide site is located at amino acid positions 426-427 of human wild-type MIS (corresponding to amino acids 451-452 of Sequence ID No. 1 as herein). Cleavage at this site, which releases the active carboxy-terminal domain of MIS, resembles the consensus-Frin cleavage site. The second cleavage site, identified by amino-terminal sequencing of the MIS fragment (also indicated as "R / S"), is located at residues 229-230 of the amino acid-terminal domain of wild-type MIS (corresponding to amino acids 254-255 of Sequence ID No. 1 as herein). This site contains R / S but does not follow the consensus-Arg-X-(Arg / Lys)-Arg for Furin cleavage. Separation of the purified carboxy-terminus from amino-terminal MIS after digestion with exogenous plasmin has previously been performed using molecular-size exclusion chromatography under acidic conditions. This technique requires extreme care to control MIS digestion. This is because prolonged incubation of MIS in plasmin produces the carboxy-terminal MIS domain and other 22 and 34 kDa fragments due to cleavage of both the first and second sites, making separation from each other extremely difficult by size exclusion. Since all fragments formed after plasmin digestion, except for the carboxy-terminal domain, are glycosylated, wheat germ lectin affinity can be used instead of size chromatography to purify the carboxy-terminal domain of MIS. After plasmin cleavage, the resulting fragments can be subjected to a wheat germ lectin column at pH 3.5 to dissociate the amino-terminal and carboxy-terminal domains, as disclosed in Lorenzo et al., J. Chromatography, (2001), 776; 89-98, which is incorporated in whole herein by reference.

[0142] Therefore, to overcome some of the problems related to avoiding the production of MIS fragments, for example, carboxy-terminal MIS domain + 22 and 34 kDa fragments by cleavage of both the first and second sites, the inventors modified the first cleavage site at amino acid positions 426-427 of human wild-type MIS (corresponding to amino acids 451-452 of Sequence ID No. 1 herein). To facilitate the purification of the C-terminal domain without requiring complex methods using wheat germ lectin affinity or size chromatography columns, the inventors included a tag at the N-terminus of the C-terminal domain, which is the most flexible C-terminal domain.

[0143] Furthermore, the wild-type MIS protein is produced as a prohormone containing an N-terminal leader sequence corresponding to amino acid residues 1-25 of SEQ ID NO: 1. Processing of the mature hormone MIS protein may involve cleavage and removal of the leader sequence (e.g., amino acids 1-25 of SEQ ID NO: 1), cleavage of the MIS protein at a first site to produce N-terminal and C-terminal domains, and formation of these domains to monomers, which are disulfides linked by intrachain and interchain disulfide bonds to the same monomer to form a bioactive homodimer MIS protein. Leader Array

[0144] While not intended to lead to theory, leader sequences improve the expression and / or secretion of the polypeptide in question in host cells and are useful for recombinant protein production. Generally, an efficient method for screening proteins required by genetic engineering procedures is known to be to express a fusion protein containing the required protein (e.g., MIS) and a prepropeptide (signal peptide + propeptide) in host cells, then to cleave (process) it intracellularly with host enzymes, and then to secrete it extracellularly. However, according to this method, the fusion protein must be cleaved twice by host enzymes to become a mature protein, leading to a decrease in the yield of mature proteins and contamination of the mature proteins with residual fusion proteins.

[0145] Therefore, secreted proteins are first expressed in the cell in a precursor form containing a leader sequence that ensures they enter the secretory pathway. Such leader sequences, also called signal peptides, direct the expressed product across the endoplasmic reticulum (ER) membrane. Signal peptides are generally cleaved by signal peptidases during translocation to the ER. After entering the secretory pathway, the protein is transported to the Golgi apparatus. From the Golgi, the protein can follow different pathways leading to compartments such as cellular vacuoles or the cell membrane, or it can be transported out of the cell to be secreted into an external medium (Pfeffer and Rothman (1987) Ann. Rev. Biochem. 56:829-852).

[0146] For the industrial production of secreted proteins, the proteins to be produced must be efficiently secreted from host cells or host organisms. The signal peptide can be, for example, the native signal peptide of the protein to be produced, a heterologous signal peptide, or a hybrid of native and heterologous signal peptides. However, there are several problems with the use of currently known signal peptides. One problem that often arises when producing human proteins from non-human host cells or organisms is that the native signal peptide does not ensure efficient translocation and / or cleavage of the signal peptide. This leads to a low rate of protein secretion and / or secretion of mature proteins exhibiting N-terminal elongation due to inaccurate cleavage of the signal peptide. Therefore, the selection of the signal peptide is crucial for the industrial production of proteins.

[0147] In the addition of leader sequences that direct protein secretion, the precursor form may include supplemental leader sequences that are cleaved during maturation. These supplemental leader peptides, called propeptides, typically follow the signal peptide. Substantially, all peptide hormones, numerous bioactive proteins (e.g., growth factors, receptors, and cell adhesion molecules, and MIS, etc.), as well as many bacterial toxins and viral envelope glycoproteins, contain propeptides that are post-translationally cleaved to mature and produce bioactive proteins (Seidah and Chretien (1999) Brain Res. 848:45-62).

[0148] Peptides are further cleaved by enzymes called proprotein convertases. Mammalian proprotein convertases include, for example, subtilisine convertase, PCSK1, PCSK2, and furin. Furin is widely expressed and located in the trans-Golgi network. Furin activates numerous proprotein substrates in the secretory pathway compartment (Thomas (2002) Nat Rev Mol Cell Biol. 3:753-766). More specifically, furin localizes to the trans-Golgi network, a late Golgi structure responsible for sorting secretory pathway proteins to their final destinations, including cellular structures, endosomes, lysosomes, and secretory granules. The cleavage site of furin has been extensively studied. The cleavage site is located after the carboxyl-terminus arginine of the consensus sequence RXL / RR (where X can represent any amino acid (Nakayama (1997) Biochem. J 327:625-635)). The cleavage efficiency increases when X is lysine, valine, isoleucine, or alanine (Watanabe et al (1992) J Biol. Chem. 267:8270-8274).

[0149] In certain embodiments, the recombinant human MIS protein includes a modified leader sequence in place of the wild-type leader sequence of the MIS protein SEQ ID NO: 1. In certain embodiments, the native leader sequence of amino acid residues 1-25 of SEQ ID NO: 1 is replaced with a non-MIS leader sequence, for example, an albumin leader sequence or a functional fragment thereof, in certain embodiments, the non-MIS leader sequence is a human serum albumin sequence (HSA), for example, the leader sequence corresponding to SEQ ID NO: 6 encoded by the nucleic acid corresponding to SEQ ID NO: 7.

[0150] In certain embodiments, the HAS sequence is a functional fragment of SEQ ID NO: 6, for example, at least 23, at least 22, at least 21, at least 20, at least 19, at least 18, at least 17, at least 16, at least 15, at least 14, at least 13, at least 12, at least 11, at least 10, or less than 10 consecutive or discontinuous amino acids of SEQ ID NO: 6. Modified versions of the HAS reader sequence are also included in the use of the present invention and are disclosed in U.S. Patent No. 5,759,802, which is incorporated entirely herein by reference. In certain embodiments, a functional fragment of the HSA reader sequence is disclosed in European Patent No. EP2277889, which is incorporated entirely by reference. TIFF0007857367000025.tif5128 or its variants. HSA signal sequence ( The prepro region of TIFF0007857367000026.tif5128) is a fragment, for example, the prepro region of the HSA signal sequence ( TIFF0007857367000027.tif5128) or its variants, for example Includes TIFF0007857367000028.tif5128.

[0151] In certain embodiments, the leader sequence is identical to the amino acid residues of SEQ ID NO: 6 by at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0152] The HAS reader sequence used herein resulted in an expected increase in the yield of recombinant human MIS protein (both higher concentrations and higher production) (see Figures 2 and 3). However, the presence of the HSA reader sequence resulted in a surprising and unexpected increase in cleavage from a first cleavage site (corresponding to the cleavage sites 451 / 452 of SEQ ID NO: 1 (or 426 / 427 in the conventional amino acid nomenclature of wild-type MIS protein)) (see Figures 2 and 3). This increased yield and increased cleavage were surprising because, with increasing yield (and therefore more protein produced by the cell), a decrease in cleavage would be expected as the activity of the available cleaving enzymes saturates and expands too much. However, instead, the exact opposite result occurred: cleavage from the first cleavage site increased with increasing protein production.

[0153] Other leader sequences are also included for use in recombinant human MIS proteins disclosed herein, for example, to substitute amino acids 1-25 of Sequence ID No. 1. Such leader sequences are known to those skilled in the art and include leader sequences containing an immunoglobulin signal peptide (IgSP-tPA) fused to a tissue-type plasminogen activator propeptide, as disclosed in US 2007 / 0141666, which is incorporated herein by reference in its entirety. Numerous other signal peptides are used for the production of secreted proteins. One of them is the mouse immunoglobulin signal peptide (IgSP, EMBL Accession No. M13331). IgSP was first identified in 1983 by Loh et al. (Cell. 33:85-93). IgSP is known to induce excellent expression in mammalian cells. For example, European Patent No. 0382762 discloses a method for producing horseradish peroxidase by constructing a fusion peptide between IgSP and horseradish peroxidase.

[0154] Other leader sequences, though not limited to these, include the MPIF-1 signal sequence (for example, amino acids 1-21 of GenBank Accession number AAB51134). TIFF0007857367000029.tif5128; stanniocalcin signal sequence TIFF0007857367000030.tif5128; Invertase signaling sequence TIFF0007857367000031.tif5128; Yeast conjugation factor alpha signaling sequence (e.g., K. lactis killer toxin reader sequence); Hybrid signaling sequence TIFF0007857367000032.tif5128; HSA / MFα-1 hybrid signal sequence (also known as HSA / kex2) TIFF0007857367000033.tif5128; K. lactis killer / MFα-1 fusion leader sequence TIFF0007857367000034.tif5128; Immunoglobulin Ig signaling sequence TIFF0007857367000035.tif5128 Fibrin B precursor signal sequence TIFF0007857367000036.tif5128; Clasterin precursor signal sequence TIFF0007857367000037.tif5128; and insulin-like growth factor binding protein 4 signal sequence Includes TIFF0007857367000038.tif5128.

[0155] When it is required to produce recombinant MIS in a bacterial system, the leader sequence may include a bacterial leader sequence, such as those disclosed in U.S. Application 2011 / 0020868. Several other secretory signals for use in expressing recombinant polypeptides or proteins are described. For example, U.S. Patent Nos. 5,914,254, 4,963,495, European Patent No. 0177343, U.S. Patent No. 5,082,783, PCT Publication No. WO 89 / 10971, U.S. Patent Nos. 6,156,552, 6,495,357; 6,509,181; 6,524,827; 6,528,298; 6,558,939; 6,608,018; 6,617,143, U.S. Patent Nos. 5,595,898; 5,698,435; and 6,204,023, U.S. Patent No. 6,258,560, PCT Publication No. WO 01 / 21662, WO These are patent applications 02 / 068660, U.S. Patent Publication No. 2003 / 0044906, U.S. Patent No. 5,641,671, and European Patent No. EP 0 121 352. Modified severed area

[0156] As discussed herein, the preparation of MIS proteins for preclinical use is complex and inefficient. Human MIS proteins are produced from a preproprotein containing a leader sequence. The leader sequence (amino acids 1-25 of SEQ ID NO: 1) is cleaved, and the remaining protein corresponding to amino acids 26-560 of SEQ ID NO: 1 (often called "holo-human MIS") is further cleaved post-translation to produce N-terminal and C-terminal domains. These N-terminal and C-terminal domains form monomers, and two identical monomers (containing the N and C-terminal domains) together form a homodimer. Holo-human MIS is cleaved into its N and C-terminal domains by furin or related prohormone convertase PC5, most promisingly expressed in the gonads. The cleavage first occurs at a kex-like site characterized by R-4 XXR-1, which has serine at a +1 site that is monobasic but makes the MIS cleavage site more furin / hex-consensus. The purified C-terminal domain is the biologically active component and cleavage is required for its biological activity. A second cleavage site, whose importance is not yet known, is observed less frequently at residues 229–230 (corresponding to amino acids 254–255 of SEQ ID NO: 1). Non-cleaving mutants of MIS are not biologically active, and mutants in the human gene with truncated carboxyl-terminal domain lead to persistent Müllerian syndrome. Cleavage of the MIS protein recombinantly expressed by CHO cells is incomplete and inefficient, thus requiring cleavage with exogenous serine proteases, such as plasmin, to enhance its biological activity.

[0157] Here, the inventors modified the kex-like site, characterized by R-4 XXR-1 with R at the -2 position, to make the single-basic MIS cleavage site more like a consensus Kex / Furin recognition site. In particular, in one embodiment, the recombinant human MIS protein is produced from a proprotein in which the amino acid residue at position 450 of SEQ ID NO:1 is changed from Q (glutamine or Gln) to R (arginine or Arg). This mutation can be denoted as amino acid Q450R in SEQ ID NO:1. This corresponds to a change in amino acid residue 425 of the MIS (Q425R), numbered in conventional protein numbering where the first numbered amino acid begins after the leader sequence.

[0158] The amino acid sequence modification of Q450R in SEQ ID NO: 1 allows for the production of highly purified cleaved preparations of fully bioactive human MIS proteins.

[0159] In another embodiment, the first cleavage site in the MIS protein, for example, the single nucleotide site located at amino acid positions 426-427 of human wild-type MIS (corresponding to amino acids 451-452 of Sequence ID No. 1 as herein), can be modified to an amino acid recognition site recognized by a different cleavage enzyme. For example, the first cleavage site in the MIS protein, for example, the single nucleotide site located at amino acid positions 426-427, can be modified to an amino acid sequence recognized by a protease or peptidase, for example, prohormone converters (PCs), or other cleavage agents expressed by cells and found in surrounding tissues, or produced by microorganisms that can establish infection in mammals. The enzymatically cleavable peptide may, but is not required, contain one or more amino acids in addition to the amino acid recognition sequence. The additional amino acids can be added to the amino terminus, carboxy terminus, or both the amino and carboxy terminus of the recognition sequence. For example, in an automated peptide synthesizer, means for adding amino acids to an amino acid sequence, and means for detecting peptide cleavage, for example by chromatographic analysis of the amino acid products of such cleavage, are known to those skilled in the art of the present invention.

[0160] Prohormone convertases constitute a family of serine proteases structurally related to bacterial subtilisin and yeast kexin. Several eukaryotic members of this family are currently known. Prohormone convertases (PCs) most often cleave precursor polypeptides at specific basic residues after a selected pair of basic residues, producing bioactive peptides and proteins. Many members of the insulin family of proteins (e.g., insulin, Igf-1) are substrates for PCs. Tags to enhance purification

[0161] In certain embodiments, the recombinant human MIS protein includes at least one internal label or "tag." In certain embodiments, the tag may be, for example, c-myc, polyhistidine, or a FLAG tag. In certain embodiments, the tag is a FLAG tag, for example, the FLAG tag of SEQ ID NO: 8. The FLAG tag may be encoded by the nucleic acid of SEQ ID NO: 9.

[0162] In certain embodiments, the tag on the recombinant human MIS protein is located internally at the carboxyl terminus immediately downstream from the cleavage site. It is the most flexible part of the C-terminus, the "finger tip" of the C-terminus, as it is not involved in receptor binding and specificity determination (Papakostas et al, 2010, Lorenzo et al, 2002). In certain embodiments, labeling at this site is most likely to preserve biological activity. In certain embodiments, the tag, e.g., the FLAG tag, is located after the first cleavage site, for example, after amino acid 450 of SEQ ID NO: 1 (corresponding to amino acid residue 425 in conventional protein nomenclature). In certain embodiments, the tag is located between amino acids 452 and 453 of SEQ ID NO: 1 (corresponding to amino acid residues 427 and 428 under the usual amino acid nomenclature of the MIS protein).

[0163] In another embodiment, the tag or label is located at any position between amino acids 450 and 560 of SEQ ID NO: 1. In a particular embodiment, the tag is inserted two amino acid residues after the modified amino acid at position 450 of SEQ ID NO: 1. However, the N-terminal tag position of the C-terminal domain of the MIS is preferred because the C-terminal position of the C-terminal domain inactivates the C-terminal domain as a whole, significantly reducing the biological activity of the MIS protein.

[0164] In certain embodiments, the recombinant MIS protein contains more than one tag, e.g., at least two, at least three, at least four, or more than four tags. In certain embodiments, the tags are sequential (e.g., successively); in certain embodiments, they are dispersed (e.g., intermittently) in the recombinant human MIS protein. Preferably, the tags do not interfere with or substantially affect the biological activity of the recombinant MIS protein function in binding to and activation of MISRII. In certain embodiments, if the recombinant MIS protein contains more than one tag, the tags are the same tag. In other embodiments, if the recombinant MIS protein contains more than one tag, the tags are different tags; for example, the recombinant MIS protein may contain a FLAG tag and a histidine tag. The small size of the Flag tag allows it to be contained within the flexible, unbound N-terminal domain at the C-terminus. Therefore, in certain embodiments, any tag known to those skilled in the art can be used instead of the Flag tag, for example, tags of about 5-10 amino acids, about 10-15 amino acids, about 15-20 amino acids, about 20-30 amino acids, or about 30-50 amino acids. In certain embodiments, tags longer than 50 amino acids are not recommended because they may sterically interfere with the flexible N-terminus of the C-terminal domain, thereby inhibiting the biological activity of the recombinant MIS protein.

[0165] In certain embodiments, tag-labeled recombinant human MIS proteins, such as those labeled with FLAG, e.g., the LRF recombinant human MIS protein disclosed herein (see Figure 1), can be eluted in a single step to produce highly purified, efficiently cleaved preparations with full biological activity. When scaled up, this purification of recombinant human MIS proteins would be suitable for clinical application. Furthermore, it would be useful in various binding assays in both clinical and experimental settings. Internal labeling of MIS during translation is more effective than labeling after protein purification because iodization or biotinylation significantly reduces MIS biological activity. Surprisingly, the inventors discovered that LRF recombinant human MIS protein components exhibit higher biological activity than wild-type MIS. Insertion of the FLAG tag sequence has several other distinct advantages. Firstly, its unique amino acid domain is not present in any other gene (except mouse brain phosphatase), making high-FLAG antibodies highly specific. Secondly, protein elution with 3xFLAG peptide is specific to FLAG MIS and not specific to other proteins that bind nonspecifically to agarose beads.

[0166] Surprisingly, compared to native MIS or previously prepared unlabeled RAQR / R (SEQ ID NO: 28) MIS, FLAG-labeled, cleavage-optimized recombinant human MIS (e.g., RF recombinant human MIS or RAQR / S (SEQ ID NO: 27) FLAG MIS) were bioactive, while FLAG-labeled, uncleavage-optimized recombinant human MIS (e.g., RAQR / R (SEQ ID NO: 28) FLAG MIS) were not. The presence of the acidic FLAG tag near the cleavage site may impair the degree of cleavage, thus leading to a loss of activity. As a result, the inventors did not anticipate the enhancement of cleavage with the addition of the Flag tag. Furthermore, holo-RAQR / R FLAG MIS ("RAQR / R" as disclosed in SEQ ID NO: 28) preparations in CHO (or HEK) cells are not bioactive. This is because, in contrast to what was reported by Kurian (Cancer Res., 1995. 1;343-349) when the component lacks the FLAG tag, endogenous processing does not occur at the RAQR / R (SEQ ID NO: 28) cleavage site. On the other hand, the retention of serine at position 428 and the conversion of the single-nucleotide site to a double-nucleotide site (corresponding to Q>R at amino acid position 425 using conventional protein nomenclature, or Q>R at position 450 of SEQ ID NO: 1) make the endogenous cleavage site more efficient and highly specific. Furthermore, tags such as FLAG MIS are powerful tools for binding studies and can be used to immunoprecipitate endogenous MISRII without crosslinking. Therefore, in certain embodiments, labeled recombinant human MIS proteins, e.g., MIS with an internal FLAG, are useful in an efficient method for producing a highly pure and biologically active internally labeled form of MIS, which can be used for MIS binding studies and for tracking in pharmacokinetic studies, and for scale-up for preclinical and clinical use. Variants of human recombinant MIS proteins

[0167] In certain embodiments, the recombinant human MIS protein disclosed herein may have modifications to the core MIS protein sequence, for example, modifications within amino acid residues 26-560 of SEQ ID NO: 1 (including the modification of amino acid residue 450 from Q to R in SEQ ID NO: 1) and / or the insertion of a tag at the beginning of the C-terminal domain. Such variants are considered homologous to the wild-type MIS protein.

[0168] As used herein, the term "polypeptide" refers to a polymer of amino acids and its equivalents, and does not refer to a specific length of the product. Thus, peptides, oligopeptides, and proteins are included in the definition of polypeptide. A derivative is a polypeptide having a conserved amino acid substitution compared to another sequence. A derivative may further include other modifications of the protein, such as glycosylation, acetylation, or phosphorylation.

[0169] In certain embodiments, a recombinant human MIS protein is at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% similar to a homologous recombinant human MIS protein. As used herein, “similarity” or “percentage of similarity” in the context of two polypeptide sequences means two or more sequences or subsequences that are identical or have a particular percentage of identical amino acid residues or their conserved substitutions when compared and aligned for maximum match using one of the following sequence comparison algorithms or by visual inspection. For example, if the first amino acid sequence is identical to or conservatively substituted to the second amino acid sequence by at least 50%, 60%, 70%, 75%, 80%, 90%, or 95% when compared to an alignment of polypeptides aligned by a computer similarity program known in the art, as described below, then the first amino acid sequence can be considered similar to the second amino acid sequence.

[0170] Homogenies, functional derivatives, and functional fragments of MIS Sequence ID No. 1 are also included in the use of the present invention and can be identified, for example, by expression of MIS from an expression library (e.g., Sambrook et al. (2001). Molecular cloning: a laboratory manual, 3rd ed. (Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press); see also Ausubel et al., op. cit.). A mutated endogenous gene sequence can be called a heterologous transgene. For example, a transgene encoding a mutation in MIS not known in the spontaneously occurring genome is a heterologous transgene with respect to mouse and non-mouse, e.g., human species. MIS proteins, e.g., disclosed in U.S. Patents 5,427,780, 5,359,033, and 5,661,126 (all incorporated herein by reference).

[0171] Variations in the primary structure of the core human MIS protein sequence (e.g., alteration of amino acid 26-560 of SEQ ID NO: 1 (including alteration of amino acid residue 450 from Q to R in SEQ ID NO: 1), and / or insertion of a tag at the beginning of the N-terminus of the C-terminal domain), or its functional fragments or homologs, are included for use in the present invention and include, for example, deletions, additions, and substitutions. Substitutions may be conserved or non-conserved. Differences between recombinant human MIS proteins and variants generally involve preserving desired properties, mitigating or removing unwanted properties, and adding desired or new properties. For example, variants of recombinant human MIS proteins may have superior activity compared to wild-type MIS proteins.

[0172] The core human MIS protein sequence of the recombinant human MIS protein disclosed herein (e.g., amino acid residues 26-560 of SEQ ID NO: 1) can be immediately manipulated to alter the amino acid sequence of the protein. Genes encoding the MIS protein or its functional fragment, homolog, or variant can be manipulated by various known techniques for in vitro mutagenesis, in particular, to produce variants of the spontaneously occurring human protein or its fragment, referred herein as variants or mutains, and can be used in accordance with the present invention. Other modifications to recombinant human MIS proteins

[0173] Recombinant human MIS proteins useful in the present invention can also be modified at their amino termini, for example, to increase their hydrophilicity. Increased hydrophobicity enhances the exposure of the peptide on the surface of the lipid-based carrier into which the parent peptide-lipid conjugate is incorporated. Polar groups suitable for binding to peptides to increase their hydrophilicity are known, but are not limited to, acetyl ("Ac"), 3-cyclohexylalanyl ("Cha"), and acetyl-serine ("Ac"), for example. The compounds include Ser"), acetyl-seryl-serine ("Ac-Ser-Ser-"), succinyl ("Suc"), succinyl-serine ("Suc-Ser"), succinyl-seryl-serine ("Suc-Ser-Ser"), methoxysuccinyl ("MeO-Suc"), methoxysuccinyl-serine ("MeO-Suc-Ser"), methoxysuccinyl-seryl-serine ("MeO-Suc-Ser-Ser"), and seryl-serine ("Ser-Ser-") groups, polyethylene glycol ("PEG"), polyacrylamide, polyacrylomorpholine, polyvinylpyrrolidone, polyhydroxy groups, and carboxysaccharides, such as lactobionic, N-acetylneuraminic acid, and sialic acid groups. The carboxyl groups of these groups are to be linked to the N-terminus of the peptide via amide bonds. Currently, the preferred N-terminal modification is the methoxy-succinyl modification.

[0174] In certain embodiments, recombinant human MIS proteins can be fused with one or more fusion partners. In certain embodiments, one of the fusion partners is an Fc protein (e.g., mouse Fc or human Fc). The fusion protein may further include a second fusion partner, such as a purification or detection tag, a protein that can be detected directly or indirectly (e.g., green fluorescent protein, hemagglutinin, or alkaline phosphatase), a DNA-binding domain (e.g., GAL4 or LexA), a gene-activating domain (e.g., GAL4 or VP16), a purification tag, or a secretion signal peptide (e.g., preprotrypsin signal peptide).

[0175] In one embodiment, the recombinant human MIS protein fusion protein useful in the methods and compositions disclosed herein may comprise a human Fc protein or a functional fragment thereof. Therefore, in one embodiment, the recombinant human MIS protein fusion protein useful in the methods and compositions disclosed herein comprises a human Fc molecule as the first fusion partner, where the Fc fragment may be SEQ ID NO: 10 or a functional variant or functional derivative thereof. SEQ ID NO: 10 is as follows: TIFF0007857367000039.tif32155

[0176] Variations and modifications of recombinant human MIS proteins and vectors can provide means for increasing or decreasing recombinant human MIS protein expression and targeting it. For example, recombinant human MIS proteins can be ligated to molecularly targeted molecules to target cancer cells or ovarian cells, creating cancer-specific or ovarian-specific recombinant human MIS proteins, respectively.

[0177] In one embodiment, recombinant human MIS protein is fused to a second fusion partner, for example, a carrier molecule to enhance bioavailability. Such carriers are known in the art and include poly(alkyl)glycols, such as polyethylene glycol (PEG). Fusion to serum albumin can also increase the serum half-life of the therapeutic peptide.

[0178] In certain embodiments, recombinant human MIS proteins may also be fused to a second fusion partner, such as a polypeptide that targets the product at a desired position, or to a tag, for example, to facilitate purification as needed. In certain embodiments, the tag and fusion partner may be designed to be cleavable as needed. Another modification of particular consideration is binding to a polymer, such as covalent bonding. In one embodiment, polymers, such as polyethylene glycol (PEG) or methoxypolyethylene glycol (mPEG), can increase the in vivo half-life of the protein to which they are bound. Methods for PEGylating polypeptide agents are known to those skilled in the art, and consideration is given to, for example, how large a PEG polymer to use.

[0179] In certain embodiments, recombinant human MIS proteins or their functional fragments are modified to achieve an appropriate circulating half-life that affects drug administration, drug delivery, and efficacy. Many approaches have been taken to increase the half-life of biotherapeutic drugs. Small proteins less than 60 kD are rapidly cleared by the kidneys and therefore do not reach their targets. This means that higher doses are required to achieve efficacy. Modifications to recombinant human MIS proteins and their fragments included in the methods of the present invention for increasing the half-life of proteins in circulation include PEGylation; conjugation or gene fusion with proteins, e.g., transferrin (WO06096515A2), albumin, growth hormone (US2003104578AA); conjugation with cellulose (Levy and Shoseyov, 2002); conjugation or fusion with Fc fragments; glycosylation and mutagenesis approaches (Carter, 2006), which are incorporated herein by reference in whole.

[0180] In PEGylation, polyethylene glycol (PEG) is conjugated to recombinant human MIS proteins or fragments, which can be, for example, plasma proteins, antibodies, or antibody fragments. The first studies on the effects of antibody PEGylation were conducted in the 1980s. Conjugation can be carried out enzymatically or chemically and is well established in the art (Chapman, 2002; Veronese and Pasut, 2005). With PEGylation, the overall size increases, reducing the opportunity for renal filtration. PEGylation further protects against proteolytic degradation and slows clearance from the blood. Furthermore, it has been reported that PEGylation can reduce immunogenicity and increase solubility. The improvement in pharmacokinetics by PEG addition is due to several different mechanisms: increased molecular size, protection from proteolytic degradation, reduced antigenicity, and masking of specific sequences from cellular receptors. In the case of antibody fragments (Fab), PEGylation has been shown to achieve a 20-fold increase in plasma half-life (Chapman, 2002).

[0181] To date, several approved PEGylated drugs exist, such as PEG-interferon alpha-2b (PEG-INTRON), designated in 2000, and alpha-2a (Pegasys), designated in 2002. A PEGylated antibody fragment against TNF-alpha, called Cimzia or Certolizumab Pegol, was submitted for FDA approval in 2007 for the treatment of Crohn's disease and was approved on April 22, 2008. The limitation of PEGylation is the difficulty in synthesizing long monodisperse species, especially when PEG chains exceeding 1000 kD are required. For many applications, monodisperse PEGs with chain lengths exceeding 10000 kD are used, resulting in a population of conjugates with PEG chains of different lengths, which requires extensive analysis to ensure equivalent batches with each production run. Different PEG chain lengths can result in different biological activities and therefore different pharmacokinetics. Another limitation of PEGylation is the reduction in affinity or activity, as observed with alpha-interferon Pegasys, which has only 7% of its native antiviral activity but exhibits improved pharmacokinetics due to an increased plasma half-life.

[0182] In certain embodiments, recombinant human MIS proteins or fragments thereof are conjugated to long-lived proteins, such as albumin, which is 67 kD and has a plasma half-life of 19 days in humans (Dennis et al., 2002). Albumin is the most abundant protein in plasma and is involved in plasma pH regulation, but it also functions as a carrier of substances in plasma. In the case of CD4, the increase in plasma half-life has been achieved after fusing it with human serum albumin (Yeh et al., 1992). Other examples of fusion proteins include insulin, human growth hormone, transferrin, and cytokines (see Ali et al., 1999; Duttaroy et al., 2005; Melder et al., 2005; Osborn et al., 2002a; Osborn et al., 2002b; Sung et al., 2003, as well as US2003104578A1, WO06096515A2, and WO07047504A2, which are incorporated herein by reference in their entirety).

[0183] The effects of glycosylation on plasma half-life and protein activity have been extensively studied. In the case of tissue plasminogen activator (tPA), the addition of a new glycosylation site reduced plasma clearance and improved potency (Keyt et al., 1994). Glycoengineering has been successfully applied to several proteins and immunoglobulins (Elliott et al., 2003; Raju and Scallon, 2007; Sinclair and Elliott, 2005; Umana et al., 1999). Furthermore, glycosylation affects the stability of immunoglobulins (Mimura et al., 2000; Raju and Scallon, 2006).

[0184] In certain embodiments, recombinant human MIS proteins or fragments thereof can be fused to Fc fragments of IgG (Ashkenazi and Chamow, 1997). The Fc fusion approach has been utilized, for example, in Trap Technology developed by Regeneron (e.g., IL1 trap and VEGF trap). The use of albumin to extend the half-life of peptides is described in US2004001827A1. Positive effects of albumin have also been reported for Fab fragments and scFv-HSA fusion proteins (Smith et al., 2001). The extended serum half-life of albumin has been demonstrated to be due to a recirculation process mediated by FcRn (Anderson et al., 2006; Chaudhury et al., 2003; Smith et al., 2001).

[0185] In certain embodiments, the recombinant human MIS protein is conjugated to a biotinylated Fc protein, such as the one disclosed in U.S. Patent Application No. 2010 / 0209424, which is incorporated in its entirety herein by reference.

[0186] As used herein, the terms “conjugate” or “conjugation” refer to the joining of two or more substances to form one substance. For example, the methods of the present invention provide a conjugation of a recombinant human MIS protein (i.e., SEQ ID NO: 2 or 3 or its fragments, derivatives, or variants) linked to another substance, such as a component, for example, an Ig carrier particle, for example, IgG1 Fc, which stabilizes the recombinant human MIS protein. The joining can be achieved by linkers, chemical modifications, peptide linkers, chemical linkers, covalent or non-covalent bonding, or protein fusion, or by any means known to those skilled in the art. The joining may be permanent or reversible. In certain embodiments, several linkers may be included to take advantage of the desired properties of each linker and each protein in the conjugate. Flexible linkers and linkers that increase the solubility of the conjugate are considered, either alone or in conjunction with other linkers disclosed herein. Peptide linkers can be linked to one or more proteins in a conjugate by expressing DNA encoding the linker. The linker may be acid-cleaved, photocleaved, and heat-sensitive. Methods for conjugation are known to those skilled in the art and are incorporated for use in the present invention.

[0187] According to the present invention, a recombinant human MIS protein (i.e., SEQ ID NO: 2 or 3, or a fragment, derivative, or variant thereof) can be conjugated to a first fusion partner by any suitable means known in the art (see, for example, U.S. Patent Publications 4,625,014, 5,057,301 and 5,514,363, which are incorporated herein by reference). For example, a recombinant human MIS protein can be covalently conjugated to IgG1 Fc directly or via one or more linkers. In one embodiment, the recombinant human MIS protein disclosed herein is conjugated directly to a first fusion partner (e.g., Fc), and in another embodiment, the recombinant human MIS protein disclosed herein can be conjugated to a first fusion partner (e.g., IgG1 Fc) via a linker, for example, a transport-enhancing linker.

[0188] A wide variety of methods for conjugating recombinant human MIS proteins disclosed herein with a first fusion partner (e.g., Fc) are known in the art. Such methods are described, for example, in Hermanson (1996, Bioconjugate Techniques, Academic Press), U.S. Patents US 6,180,084 and US 6,264,914, which are incorporated herein by reference in their entirety, and include, for example, methods used to ligate haptens to carriers conventionally used in applicable immunology (see Harlow and Lane, 1988, "Antibodies: A laboratory manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). In certain cases, recombinant human MIS proteins have been shown to lose potency or functionality upon conjugation, depending, for example, on the conjugation procedure or chemical group used. However, by considering a wide variety of conjugation methods, those skilled in the art can find conjugation methods that do not affect, or have the least effect on, the potency or functionality of a substance, such as recombinant human MIS proteins to be conjugated.

[0189] Suitable methods for the conjugation of recombinant human MIS proteins disclosed herein with a first fusion partner (e.g., Fc) include, for example, carbodiimide conjugation (Bauminger and Wilchek, 1980, Meth. Enzymol. 70: 151-159). Alternatively, components can be linked to a targeting agent, as described by Nagy et al., Proc. Natl. Acad. Sci. USA 93:7269-7273 (1996), and Nagy et al., Proc. Natl. Acad. Sci. USA 95:1794-1799 (1998), which are incorporated herein in their entirety by reference. Other methods for conjugation that can be used include, for example, sodium periodate oxidation, as well as reductive alkylation and glutaraldehyde crosslinking of the following suitable reactants.

[0190] Those skilled in the art can use a variety of methods to conjugate recombinant human MIS proteins disclosed herein to a first fusion partner (e.g., Fc), for example, but not limited to, aminocaproic acid horseradish peroxidase (HRP), or heterobifunctional crosslinkers, such as carbonyl-reactive and sulfhydryl-reactive crosslinkers. Heterobifunctional crosslinkers typically contain two reactive groups capable of binding to two different functional targets on proteins and other macromolecules in a two or three-step process, which can limit the degree of polymerization often associated with the use of homobifunctional crosslinkers. Such multi-step protocols can provide greater control over the size of the conjugate and the molar ratio of its components.

[0191] The term “linker” or “crosslinking agent” refers to any means of linking two or more substances, for example, a recombinant human MIS protein disclosed herein, to a first fusion partner (e.g., Fc). The linker may be a covalent or non-covalent linker. An example of a covalent linker includes a linker component covalently bonded to one or more proteins to be linked. The linker may also be a non-covalent linker, for example, an organometallic bond via a metal center, such as a platinum atom. A variety of functionalities can be used for covalent bonding. For example, amide groups, for example, carboxylic acid derivatives, ethers, esters, for example, organic and inorganic esters, urethanes, urea, etc. To provide a linkage, effector molecules and / or probes can be modified by oxidation, hydroxylation, substitution, reduction, etc., to provide a site for linkage. Modifications that do not significantly reduce the function of the recombinant human MIS protein disclosed herein or the first fusion partner (e.g., Fc) will be considered preferable. targeting

[0192] In certain embodiments, recombinant human MIS proteins, or functional fragments or homologs thereof, for use in the methods and compositions disclosed herein, can be targeted to cancer or ovarian cells via a targeting ligand. A targeting ligand is a molecule, such as a small molecule, protein, or fragment thereof, that binds with high affinity and specifically to a target, such as a cell surface marker on a pre-selected cell, such as a cell surface protein, or to a receptor that is present to a greater extent on the pre-selected cell target than on any other body tissue. Therefore, in certain embodiments, recombinant human MIS proteins for use in the methods and compositions disclosed herein can be fused to Fc and / or optionally to a targeting molecule. In certain embodiments, a nucleic acid molecule encoding a targeting ligand can be fused to a nucleotide encoding a recombinant human MIS protein, or a fragment, homolog, or variant thereof. Another example of a targeting ligand is a group of cadherin domains from human cadherins. Targeting ligand components bound to recombinant human MIS proteins include spontaneously occurring, recombinant, or manipulated ligands or fragments thereof that can bind to pre-selected target cells.

[0193] Further examples of targeted ligands, but not limited to these, include antibodies and parts thereof that specifically bind to pre-selected surface proteins with high affinity. “High affinity” means an equilibrium dissociation constant of at least molar concentration, as determined by assay methods known in the art, such as BiaCore analysis. In one embodiment, the targeted ligand may also include one or more immunoglobulin-binding domains isolated from antibodies produced against a given tissue-specific surface protein or target tissue-specific receptor. As used herein, “immunoglobulin” or “antibody” means, in the context of this invention, a mammalian peptide or fragment thereof, including humans, that includes a framework region from an immunoglobulin gene that specifically binds to and recognizes an antigen, such as a tissue-specific surface protein, a tissue-specific receptor, or a part thereof. If a targeted fusion polypeptide intended as a mammalian therapeutic agent is to be used, the immunoglobulin-binding region should be obtained from the corresponding mammalian immunoglobulin. If a targeted fusion polypeptide is intended for non-therapeutic use, such as diagnostics and ELISA, the immunoglobulin-binding region can be obtained from either human or non-human sources, such as mice. Human immunoglobulin genes or gene fragments include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant regions, as well as numerous immunoglobulin variable region genes. The light chain can be classified as either kappa or lambda. The heavy chain can be classified as gamma, mu, alpha, delta, or epsilon, which define the classes of immunoglobulins: IgG, IgM, IgA, IgD, and IgE, respectively. Within each IgG class, different isomers exist (e.g., IgG1, IgG2, etc.). Typically, the antigen-binding region of an antibody will be the most important factor in determining binding specificity and affinity.

[0194] The typical immunoglobulin (antibody) structural unit of human IgG contains a tetramer. Each tetramer consists of a pair of two identical polypeptide chains, each pair having one light chain (approximately 25 kD) and one heavy chain (approximately 50-70 kD). The N-terminus of each chain defines a variable region of approximately 100-110 or more amino acids, which is the main cause of antigen recognition. The terms “variable light chain” (VL) and “variable heavy chain” (VH) refer to these light and heavy chains, respectively. Antibodies exist as complete immunoglobulins or as several well-characterized fragments produced by digestion with various peptidases. For example, pepsin digests antibodies under disulfide crosslinking in the hinge region to produce F(ab)'2, a dimer of Fab, which is itself a light chain linked to VH-CH by disulfide bonds. F(ab)'2 can be reduced under mild conditions to break the disulfide crosslinks in the hinge region, thereby converting the F(ab)'2 dimer into a Fab' monomer. The Fab' monomer is essentially Fab with a portion of the hinge region. While a variety of antibody fragments are defined with respect to the digestion of a complete antibody, those skilled in the art will recognize that such fragments can be newly synthesized chemically or by recombinant DNA technology. Thus, the terms immunoglobulin or antibody as used herein also include antibody fragments produced by modification of a complete antibody, or newly synthesized using recombinant DNA technology (e.g., single-stranded Fv(scFv)), or identified using a phage display library (e.g., see McCafferty et al. (1990) Nature 348:552-554). Furthermore, the fusion polypeptides of the present invention include the variable region of the heavy chain (VH) or light chain (VL) of an immunoglobulin, as well as a tissue-specific surface protein and its target receptor binding portion. A method for producing such variable regions is described in Reiter, et al. (1999) J. Mol. Biol. 290:685-698.

[0195] Methods for preparing antibodies are known in the art (see, for example, Kohler & Milstein (1975) Nature 256:495-497; Harlow & Lane (1988) Antibodies: a Laboratory Manual, Cold Spring Harbor Lab., Cold Spring Harbor, NY). The genes encoding the heavy and light chains of the antibody in question can be cloned from cells. For example, the gene encoding a monoclonal antibody can be cloned from a hybridoma and used to produce recombinant monoclonal antibodies. Gene libraries encoding the heavy and light chains of monoclonal antibodies can also be prepared from hybridomas or plasma cells. Random combinations of heavy and light chain gene products create a large pool of antibodies with different antigen specificities. Techniques for producing single-chain or recombinant antibodies (U.S. Patent No. 4,946,778; U.S. Patent No. 4,816,567) can be adapted to produce fusion polypeptides and antibodies used in the methods of the present invention. Furthermore, transgenic mice or other organisms, such as other mammals, can be used to express human or humanized antibodies. Alternatively, phage display technology can be used to identify antibodies, antibody fragments, such as variable domains, and Fab fragments of heteromers that specifically bind to a given antigen.

[0196] Screening and selection of preferred immunoglobulins (e.g., antibodies) can be carried out by a variety of methods known in the art. An initial screening for the presence of tissue-specific or target receptor-specific monoclonal antibodies can be carried out, for example, via ELISA-based methods or the use of phage display. A second screening is preferably performed to identify and select monoclonal antibodies required for use in the construction of the tissue-specific fusion polypeptide of the present invention. The second screening can be carried out by any suitable method known in the art. One method called "Biosensor Modification-Assisted Profiling" ("BiaMAP") (U.S. Patent Application Publication 2004 / 101920) allows for the rapid identification of hybridoma clones that produce monoclonal antibodies with the required characteristics. More specifically, monoclonal antibodies are classified into different epitope-associated groups based on the evaluation of antibody-antigen interactions. Purification of recombinant human MIS proteins

[0197] The recombinant human MIS proteins disclosed herein, as well as their functional fragments and derivatives, can be obtained by any suitable method. For example, polypeptides can be produced using conventional recombinant nucleic acid technologies, such as DNA or RNA, preferably DNA. Guidance and information on methods and materials for polypeptide production using recombinant DNA technology can be found in numerous publications and reference manuals. See, for example, Sambrook et al, 1989, Molecular Cloning - A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press; Ausubel et al. (eds.), 1994, Current Protocols in Molecular Biology, John Wiley & Sons, Inc.; Innis et al. (eds.), 1990 PCR Protocols, Academic Press.

[0198] Alternatively, recombinant human MIS proteins or their functional fragments can be obtained directly by chemical synthesis, for example, by using commercially available peptide synthesizers according to the vendor's instructions. Methods and materials for the chemical synthesis of polypeptides are known in the art. See, for example, Merrifield, 1963, "Solid Phase Synthesis," J. Am. Chem. Soc. 83:2149-2154.

[0199] In certain embodiments, recombinant human MIS protein, or its functional fragment, derivative, or variant, can be expressed intracellularly after the introduction of protein-encoding DNA, such as the nucleic acid encoding the human MIS protein, or its homolog or functional fragment, either in a conventional expression vector disclosed herein, or by a catheter, or by cells transformed with nucleic acid ex vivo and transplanted into a target. Assay for determining the activity of recombinant human MIS protein

[0200] In one embodiment, the biological activity of the human recombinant MIS protein disclosed herein can be assayed using an organ culture assay system. The assay system used is a Müllerian duct suppression organ culture assay, as described in Donahoe et al, J. Surg. Res., 23, 141-148, 1977. The urogenital ridge was excised from 14-day-old female rat fetuses and transferred to an organ culture dish (Falcon, 3010). The specimens were placed on a stainless steel grid coated with a thin layer of 2% agar and incubated for 72 hours at 37°C in 5% CO2 and 95% air on 2 mL of culture medium [CMRL 1066 containing 10% fetal bovine serum and 1% penicillin (10,000 units / ml)] or a 1:1 mixture of culture medium and supernatant or gradient fraction to be tested. The incubated tissue was then coated with a mixture of 2% agar and albumin at 44°C, fixed in buffered formaldehyde, dehydrated in ethanol, washed in xylene, and embedded in paraffin. For viewing under a light microscope, 8-micrometer serial sections were stained with hematoxylin and eosin. Sections from the head end of the Müllerian tube were assigned code numbers and graded for regression on a scale from O to V (Donahoe et al, Biol. Reprod., 15, 329-334, 1976). For each assay, five slides were read with 6–8 sections per slide. The activity grade was listed as the integer closest to the mean. The test group for the fractionation procedure shows at least 10 assays. If the mean was between two numbers, both numbers were listed. Grade 0 indicates no regression. Müllerian ducts, lined with columnar epithelial cells whose nuclei are oriented basally, have a broad, patent lumen. Grade I is minimal regression. The duct is slightly smaller, and the surrounding mesenchyme is either aggregated around the duct, as seen in plastic sections, or a transparent region is present around the duct, as seen in paraffin sections. Grade II refers to mild regression. The duct is even smaller, and the aggregation of mesenchyme or the transparent region around the duct is more pronounced.Shorter epithelial cell nuclei lose their basal orientation. Grade III is moderate regression. The ducts are extremely small and disordered. The apex of the urogenital ridge is inadequately developed distal to Wolffian ducts. Grade IV is severe regression. The ducts are replaced by cellular vortices. Grade V refers to complete regression. No ductal remnants are observable. Positive tissue controls using fetal testes and negative tissue controls of Müllerian ducts incubated alone or with muscle were included in each experiment. Müllerian ducts were exposed to extracts from non-testicular tissue, inactive testicular fractions, or saline used as biochemical controls. Aliquots of all fractions were dialyzed against distilled water, freeze-dried, and protein components were measured. Delivery of recombinant human MIS proteins

[0201] Methods known in the art for the therapeutic delivery of recombinant human MIS proteins and / or nucleic acids encoding them can be used in a subject to treat a disease or disorder, such as cancer. These methods include, for example, cell transfection, gene therapy, direct administration on a delivery vehicle or pharmaceutically acceptable carrier, or indirect delivery by providing recombinant cells containing nucleic acids encoding the targeted fusion polypeptide of the present invention.

[0202] In certain embodiments, recombinant human MIS protein is cleaved in vitro to form a bioactive halo-dimer of MIS containing two identical monomers, each consisting of an N-terminal domain and a C-terminal domain, and then administered to a subject.

[0203] A variety of delivery systems are known and can be used to administer recombinant human MIS proteins to a target (before or after cleavage into their bioactive form). These include, for example, liposome encapsulation, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), and the construction of nucleic acids as part of retroviruses or other vectors. Methods of delivery may be enteral or parenteral, but may include intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, pulmonary, intranasal, intraocular, epidural, and oral routes. Recombinant human MIS proteins can be administered by any conventional route, for example, by injection or bolus injection, by absorption via the epithelium or cutaneous mucosa (e.g., oral mucosa, rectal and intestinal mucosa), and may be administered together with other bioactive agents. Administration may be systemic or topical. Furthermore, it may be required to introduce the pharmaceutical composition containing recombinant human MIS protein into the central nervous system via one of the appropriate routes, such as intracerebroventricular and intrasacral injection, either before or after cleavage to its bioactive form. Intracerebroventricular injection can be facilitated, for example, by an intracerebroventricular catheter connected to a reservoir, such as an Ommaya reservoir. Lung administration can also be used, for example, by the use of an inhaler or nebulizer and by formulation with an aerosolizing agent. Methods for treating proliferative disorders and cancer

[0204] One aspect of the present invention provides a method for treating cancer, such as cancer expressing MISRII, in a subject. Thus, one aspect of the present invention broadly relates to a method for treating a proliferative disease or disorder. Here, the proliferative disease or disorder is related to cells expressing MIS receptor, such as cells expressing MISRII. In certain embodiments, the proliferative disease or disorder is cancer. Here, the cancer or cancer cells express at least one MIS receptor. For example, cancer or cancer cells expressing MISRII. The method of the present invention includes administering to a subject having a proliferative disorder an effective amount of a recombinant human MIS protein or a functional fragment thereof as disclosed herein. Here, the cells related to the proliferative disorder express at least one MIS receptor. For example, the cells express MISRII. For example, an effective amount of the recombinant human MIS protein or a functional fragment thereof as disclosed herein is administered to a subject having cancer expressing at least one MIS receptor, such as MISRII. Thereby, by using the method of the present invention, it is possible to intervene in a proliferative disease, such as cancer, relieve symptoms, and in certain cases, cure the disease. In certain embodiments, the recombinant human MIS protein that can be used for treating a proliferative disease and cancer includes the amino acid sequence residues 25 to 559 of SEQ ID NO: 2 or a fragment thereof.

[0205] Examples of such diseases where the proliferation of cells expressing at least one MIS receptor, such as MISRII, is the cause of the disease are cancer, such as cervical cancer and ovarian cancer. In certain embodiments, the cancer expressing at least one MIS receptor, such as MISRII, is cancer cells. In certain embodiments, such cancer cells expressing at least one MIS receptor, such as MISRII, are, for example, but not limited to, ovarian cancer cells, vulvar epidermoid cancer cells, cervical cancer cells, endometrial adenocarcinoma cells, ovarian adenocarcinoma.

[0206] In another embodiment, cancers expressing at least one MIS receptor, such as MISRII, include, but are not limited to, breast cancer, lung cancer, head and neck cancer, bladder cancer, gastric cancer, nervous system cancers, osteosarcoma, myeloid cancer, brain tumors, colon cancer, esophageal cancer, endometrial cancer, gastrointestinal cancer, genitourinary cancer, gastric cancer, lymphoma, melanoma, glioma, bladder cancer, pancreatic cancer, gingival cancer, kidney cancer, retinal cancer, liver cancer, nasopharyngeal cancer, ovarian cancer, oral cancer, bladder cancer, hematological neoplasms, follicular lymphoma, cervical cancer, multiple myeloma, osteosarcoma, thyroid cancer, prostate cancer, rectal cancer, prostate cancer, skin cancer, gastric cancer, testicular cancer, tongue cancer, or uterine cancer.

[0207] In another embodiment, the invention relates to the use of a recombinant human MIS protein, or a functional fragment, derivative, or variant thereof, disclosed herein, for the treatment of any disorder, wherein administration of the MIS protein or nucleic acid encoding the MIS protein, or activation of MISRII, is all or part of a treatment regimen.

[0208] In certain embodiments, the cancer is an MIS-responsive cancer, such as, but not limited to, ovarian cancer and cervical cancer. In certain embodiments, the cancer is a cancer expressing MISRII, such as, but not limited to, ovarian cancer and cervical cancer. In certain embodiments, the disorder is a disorder associated with an androgen excess state, such as that disclosed in U.S. Patent No. 6,673,352, which is hereby incorporated by reference in its entirety. In certain embodiments, the methods of the invention are used for the treatment of prostate cancer, polycystic ovary disorder, benign prostatic hyperplasia, and precocious puberty.

[0209] In certain embodiments, the cancer is a chemotherapy-resistant or multi-drug resistant cancer, such as when the cancer is resistant to paclitaxel, cisplatin, rapamycin, pyrazoloanthrone, or doxorubicin.

[0210] In relevant embodiments, the tissue to be treated is tumor tissue expressing at least one MIS receptor, for example, MISRII of the subject. For example, tumor tissue is cancer such as, but is not limited to, solid tumors, metastases, skin cancer, breast cancer, ovarian cancer, cervical cancer, hemangioma, or angiofibroma. Typical solid tumor tissues treatable with the pharmaceutical compositions of the present invention include, but is not limited to, tumors of tissues such as the lungs, pancreas, chest, intestines, larynx, and ovaries. In a particular embodiment, the solid tumor tissue treatable by the method of the invention is the thyroid gland, and the type of cancer is medullary thyroid carcinoma.

[0211] In related embodiments, the present invention considers the implementation of a method of administering a composition comprising the recombinant human MIS protein disclosed herein, or a functional fragment thereof, in combination with other therapies, such as conventional chemotherapy for solid tumors, and to suppress the establishment of metastasis. For example, chemotherapeutic agents used in chemotherapy include, but are not limited to, paclitaxel, cisplatin, doxorubicin, rapamycin, pyrazoloanthrone, for example, but not limited to, anthra(1,9-cd)pyrazole-6(2H)-one (SP600125) or N1-methyl-1,9-pyrazoloanthrone (M-SP600125), or functional derivatives or analogs thereof. In certain embodiments, the chemotherapeutic agent is a radiotherapeutic agent. Administration of the compounds disclosed herein is typically performed before, concurrently with, and / or after chemotherapy. However, inhibiting cell proliferation after a chemotherapy regimen is also within the scope of the present invention, at a time when tumor tissue would respond to toxic attack by inducing angiogenesis to restore blood and nutrient supply to the tumor tissue. Furthermore, if a subject is identified as being at risk of developing cancer, for example, if the subject is positive for cancer cell or tumor biomarkers, the pharmaceutical compositions of the present invention for the treatment of proliferative disorders, such as cancer, can be administered prophylactically and / or before tumor development. Insofar as the method is applied to inhibit cell proliferation, the method can also be applied to inhibit tumor tissue growth, inhibit tumor metastasis formation, and regression of established tumors.

[0212] In certain embodiments, the expression of Müllerian inhibitor (MIS) receptors is measured in biological samples obtained from a subject, such as cancer or tumor tissue samples or cancer cells or tumor cells, such as biopsy tissue samples.

[0213] The presence of MISRII in cells in a fluid such as blood may indicate the presence of cancer. The presence of MISRII in a fluid or at a site not near the tumor may indicate metastasis. In certain embodiments, the compounds of the present invention are administered to a subject, and in certain embodiments, the compounds of the present invention are administered to a subject in a pharmaceutical composition comprising one or more therapeutic agents.

[0214] The methods of the present invention disclosed herein provide parenteral and oral administration of recombinant human MIS proteins, or functional fragments thereof, or derivatives disclosed herein, in combination with other pharmaceutical compositions, to subjects in need of such treatment. Parenteral administration includes, but is not limited to, intravenous (IV), intramuscular (IM), subcutaneous (SC), intraperitoneal (IP), intranasal, and inhalation routes. In the methods of the present invention, the recombinant human MIS proteins, or functional fragments thereof, or analogs disclosed herein are preferably administered orally. IV, IM, SC, and IP administrations may be by bolus or infusion and may be by sustained-release implantable devices, such as, but not limited to, pumps, sustained-release formulations, and mechanical devices. The formulation, route, and method of administration, as well as the dosage, will depend on the disorder to be treated and the medical history of the subject. Generally, the dosage administered by subcutaneous infusion will be greater than the equivalent dosage for treatment given intravenously or intramuscularly. Preferably, the dosage of the compound of the present invention will be about 0.1 mg to about 250 mg. In certain embodiments, the dosage of the compound of the present invention will be about 1 mg to about 60 mg.

[0215] The present invention provides a method for treating cancer expressing at least one MIS receptor, e.g., MISRII, which is useful for treating proliferation-related diseases or disorders associated with cancer expressing at least one MIS receptor, e.g., MISRII, comprising the step of contacting tissue where proliferation is occurring or at risk of occurring with a therapeutically effective amount of a recombinant human MIS protein or a functional fragment thereof disclosed herein.

[0216] In certain embodiments, and in many embodiments, the subjects treated by the methods of the present invention are human subjects. However, it should be understood that the principles of the present invention demonstrate that the invention is effective for all mammals. In this context, mammals are understood to include any mammalian species for which treatment of cancer or growth-related disorders is required, particularly agricultural and domestic mammalian species, as well as transgenic animals. use

[0217] In another embodiment, the present invention provides a method for treating a variety of conditions by administering an effective amount of the recombinant human MIS protein or a functional fragment of the present invention to a subject in need. Conditions that can be treated with the compounds of the present invention or pharmaceutical compositions containing the same include any conditions whose symptoms are treated or alleviated by the administration of MIS or activation of MIS signaling or MISRII, thereby benefiting from the administration of recombinant human MIS protein or a functional derivative thereof. Representative conditions in this regard include, but are not limited to, cancers expressing MIS receptors, such as cancers expressing MISRII, such as, but are not limited to, ovarian cancer, cervical cancer, and endometrial cancer. Other conditions that can be treated with MIS, or whose symptoms are alleviated by activation of MIS signaling, include, for example, rheumatoid arthritis, proliferative disorders such as cancer, treatment of prostate cancer, polycystic ovarian disease, benign prostatic hyperplasia, and precocious puberty, and other hyperandrogen disorders such as orchiditis.

[0218] Accordingly, the present invention relates to the use of recombinant human MIS protein or a functional derivative thereof for the treatment of any of the following disorders: administration of the MIS protein or nucleic acid encoding the MIS protein or a functional derivative thereof, or activation of MISRII, which constitutes the whole or part of the treatment plan. In certain embodiments, the recombinant human MIS protein that can be used comprises amino acid residues 25-559 of SEQ ID NO: 2 or its functional fragment.

[0219] In certain embodiments, the method of the present invention relates to the use of recombinant human MIS protein or a functional derivative thereof in combination with other therapeutic agents, such as chemotherapeutic agents. Here, the chemotherapeutic agent, such as paclitaxel or MIS, can be used at lower doses, thereby reducing side effects. Use of recombinant human MIS proteins or their functional derivatives or analogs for the treatment of hyperandrogen conditions

[0220] In another embodiment, recombinant human MIS protein or its functional derivatives or analogs can be used to treat disorders associated with excessive androgen production in a subject. The inventors have previously demonstrated that administration of MIS protein and / or MIS nucleic acid reduces androgen levels in a subject, as disclosed in U.S. Patent No. 6,673,352 and U.S. Patent Application No. 10 / 683,346, which are incorporated herein by reference in their entirety. Transgenic mice overexpressing MIS have also been shown to have reduced serum testosterone concentrations, indicating that MIS administration reduces serum testosterone levels (Sriraman et al., J Androl. 2001, 22(5):750-8 and Trbovich et al., PNAS, 2001 Mar 13;98(6):3393-7). MIS has also been shown to suppress both androgen-stimulated cellular proliferation and androgen-dependent survival. MIS controls prostate growth by suppressing testicular testosterone synthesis and directly regulates androgen-inducible gene expression and growth in the prostate at the cellular level (Trann et al, Mol Endocrinol. 2006, 20(10):2382-91).

[0221] Androgens stimulate or regulate the development and maintenance of masculine characteristics in vertebrates by binding to androgen receptors. Also known as androgen hormones or testoids, androgens are precursors to estrogen, a female hormone. The most well-known androgen is testosterone.

[0222] While not intended to be a theoretical link, excessive androgen production by the adrenal glands and / or ovaries can lead to androgenic excess, resulting from increased sensitivity of local tissues to circulating androgens. Androgenic excess can affect different tissues and organ systems, leading to overt masculinization of clinical conditions ranging from acne to hirsutism.

[0223] Hyperandrogenism, referring to the overproduction and secretion of androgens and their precursors, is a common, and sometimes serious, endocrine disorder in women of reproductive age. Excess androgens and their precursors originate from the adrenal glands and ovaries in varying proportions, manifesting a range of effects depending on the amount of excess androgen. Clinical manifestations range from hirsutism (excessive, male-pattern hair growth, sometimes accompanied by acne) to masculinization (clitoral enlargement, temporary baldness, voice deepening, or increased muscle mass).

[0224] Hyperandrogenism can occur as part of the symptoms of a wide range of diseases. These include polycystic ovary syndrome (PCOS), which is a variable combination of hirsutism, infertility, obesity, insulin resistance, and polycystic ovary syndrome; HAIR-AN syndrome (hyperandrogenism, insulin resistance, and acanthosis nigricans); ovarian membranous cell proliferation (HAIR-AN with luteinized follicular cell tumors in the ovarian stroma); and other signs of high intraovarian androgen concentrations (e.g., follicular maturation restriction, atresia, anovulation, dysmenorrhea, dysfunctional uterine bleeding, infertility); androgen-producing tumors (masculinizing ovaries or adrenal tumors).

[0225] Hirsutism is excessive visible hair growth characterized by an increase in the number and length of terminal hairs in androgen-sensitive areas. Race, family, genetics, and ethnicity all influence the occurrence of hirsutism. Hirsutism is difficult to quantify, the entire body needs to be examined, and its discovery must be carefully documented, with particular attention directed to the jaw, lips, sideburns, chest and sternum, midline between the umbilicus and pubis, and thighs.

[0226] Ferriman and Gallwey published a grading scale for staging hirsutism, which is generally known to those skilled in the art. This system is the most widely used and evaluates body areas for hirsutism from "none" to "severe" with scores of 0 - 4 respectively. Scores of 8 and above are consistent with a diagnosis of hirsutism. This scale is another way to objectively evaluate excessive hair without reference to hair thickness. The scoring system helps in quantifying hirsutism and in evaluating treatment response. Even scores above 8 are defined by the patient. From a clinical perspective, the patient can determine whether he or she notices a difference. Photographs are useful for documentation and for following the course of treatment.

[0227] Virilization is relatively rare. It occurs with marked hyperandrogenemia and is characterized by transient balding, breast atrophy, androgenic muscle development, clitoral hypertrophy, amenorrhea, deepening of the voice, and marked hirsutism.

[0228] Current medical treatments for women are directed at the adrenal glands, ovaries, and androgen receptors. Glucocorticoid therapy is directed at the adrenal glands but is limited in specific cases by suppression without the requirement for cortisol synthesis. GnRH therapy is directed at the ovaries but is expensive and its long-term effects are unknown. Additionally, therapy using oral contraceptives may be inappropriate as most contain progestins along with androgenic activity.

[0229] Since abnormal androgen production is associated with the pathways of many diseases and / or disorders for which there are no acceptable treatments, there is a need to find small molecules that inhibit the production of gonadotropins and / or androgens in mammals for treatment and / or prevention.

[0230] Therefore, in one embodiment, recombinant human MIS protein or its functional derivative or analog can be used to treat disorders associated with excessive androgen production in a subject. In a particular embodiment, the recombinant human MIS protein that can be used comprises amino acid residues 25-559 of SEQ ID NO: 2 or its functional fragment.

[0231] The term "androgen" is used herein to mean a steroid that promotes the development of male characteristics, and includes steroid derivatives of androstanes, such as testosterone, androstanedione, and their analogues.

[0232] As used herein, a disease condition or disorder characterized by "androgen dependence" is a disease condition exacerbated by, or caused by, insufficient, excessive, inappropriate, or uncontrolled androgen production. Examples of such diseases in men, but not limited to, include, BPH, metastatic prostate cancer, testicular cancer, androgen-dependent acne, male pattern baldness, and precocious puberty in boys. Examples of such diseases in women, but not limited to, include, hyperandrogenemia, hirsutism, masculinization, POCS, HAIR-AN syndrome, follicular cell proliferation, follicular maturation inhibition, atresia, anovulation, dysmenorrhea, dysfunctional uterine bleeding, infertility, and androgen-producing cells.

[0233] As used herein, “androgen inhibitor” means an effective dose of pyrazoloanthrone or its functional derivatives or analogs, such as SP600125, as defined herein. It will reduce androgen in vivo levels to normal or below normal when administered to a subject for the prevention or treatment of a disease condition exacerbated or caused by excessive or uncontrolled androgen production.

[0234] In certain embodiments, recombinant human MIS proteins or their functional derivatives or analogs disclosed herein can be used to treat prostate cancer. The effects of androgens on prostate cancer are known, and there are treatments for prostate cancer by androgen deficiency, including androgen blockade and inhibition of androgen synthesis (Huggins et al., Archs. Surg., Vol. 43, pp. 209-223 (1941); J. Steroid Biochem. Molec. Biol., Vol. 37, pp. 349-362 (1990)). Furthermore, steroid hormones are widely used as contraceptives. Antispermatogenic agents are male contraceptives that inhibit spermatogenesis, the process that leads to mature sperm. Agents that interfere with this process include androgens and antiandrogens. Since the antiandrogenic effects of recombinant human MIS proteins or their functional derivatives or analogs disclosed herein are reversible, recombinant human MIS proteins can also be used as male contraceptives (Korolkovas, A., Essentials Of Medicinal Chemistry, Second Edition, pp.1032 (1988)).

[0235] In certain embodiments, other agents may be used in combination with a pharmaceutical composition comprising recombinant human MIS proteins or their functional derivatives or analogs disclosed herein for the treatment of excess androgens in a subject. In certain embodiments, the agent functions to reduce serum androgen levels and block peripheral androgenic activity. Examples of such agents, but not limited to, include suppression and modulated estrogen therapy of ovarian androgens by administration of estrogens and / or progestins (i.e., birth control pills) or GnRH agonists, glucocorticoids (e.g., dexamethasone, prednisolone), antiandrogens (e.g., spironolactone, flutamide, cyproterone acetate), 5α-reductase inhibitors (e.g., finasteride), bromocriptine, and suppression of adrenal androgens by administration of insulin-sensitive agents (e.g., metformin, thiazolidinedione).

[0236] Subjects who are eligible to accept treatment with recombinant human MIS proteins or their functional derivatives or analogs by the methods disclosed herein are identified subjects with diseases or disorders associated with excessive androgen levels, for example, disorders, for example, but not limited to, BPH, prostate cancer, benign prostatic hyperplasia, testicular cancer, androgen-dependent acne, male pattern baldness, precocious puberty, hyperandrogenemia, hirsutism, masculinization, POCS, HIAR-AN syndrome, ovarian follicular cell proliferation, follicular maturation inhibition, atresia, anovulation, dysmenorrhea, dysfunctional uterine bleeding, infertility, and androgen-producing tumors.

[0237] In certain embodiments, subjects who are receptive to treatment with recombinant human MIS proteins or their functional derivatives or analogs by the methods disclosed herein are subjects having congenical adrenal hyperplasma. This can be generally identified by those skilled in the art. CAH is most typically an autosomal recessive disorder characterized by a deficiency or functional deficit of the enzyme 21-hydrolase. Alternatively, subjects having CAH may have a loss and / or reduction of function of the enzyme 11α-hydrolase and / or the enzyme 3α-hydroxysteroid dehydrogenase. When these enzymes are absent or function at low levels, the body is unable to produce adequate amounts of the adrenal steroid hormones, cortisol and aldosterone. Adrenal hyperplasma is followed by high levels of ACTH, which stimulates the over-secretion of androgen precursors for cortisol and aldosterone synthesis. CAH may manifest in utero or develop postnatally. At birth, pseudohermaphroditism may be present.

[0238] 21-Hydroxylase deficiency is the most common autosomal recessive disorder (more common than cystic fibrosis) and manifests itself with high levels of 17-hydroxyprogesterone. 11α-Hydroxylase deficiency is characterized by high levels of 11-deoxycortisol (compound S) and high levels of deoxycorticosterone (DOC), a mineralocorticoid. Hypertension and hypokalemia may be prominent features of 11α-Hydroxylase deficiency. Another form of CH, 3α-Hydroxysteroid dehydrogenase deficiency, results in high levels of pregnenolone, 17-hydroxypregnenolone, and DHEA. This condition is fatal if undetected, as corticosteroids are not synthesized.

[0239] Partial deficiencies in the aforementioned enzymes that manifest after puberty lead to high levels of adrenal steroids via the same mechanism. These elevations are less pronounced than those associated with congenital conditions, and this condition is referred to as non-classical (adult-onset or late-onset) CAH. Therefore, in certain embodiments, patients with non-classical (adult-onset or late-onset) CAH are eligible for treatment with recombinant human MIS protein or its functional derivatives or analogs by the methods disclosed herein.

[0240] In certain embodiments, subjects who are receptive to treatment with recombinant human MIS protein or its functional derivatives or analogs by the methods disclosed herein are female subjects with testosterone levels of approximately 2.0 ng / mL (200 ng / dL, 8.92 nmol / L) or higher, or testosterone levels at least approximately 2.5 times the upper limit of the standard range. In certain embodiments, such subjects have Sertoli-Leydig cell tumors, hilar cell tumors, and lipoid cell (adrenal) tumors, which are the most common. Sertoli-Leydig cell tumors reach a size that is palpable at the time of clinical diagnosis, while hilar cell tumors and lipoid cell tumors are difficult to detect by any means due to their small size.

[0241] In certain embodiments, subjects who are receptive to treatment with recombinant human MIS protein or its functional derivatives or analogs by the methods disclosed herein are subjects having adrenal tumors (adenomas, calcinomas) that secrete elevated levels of androgens. In such embodiments, subjects who are receptive to treatment by the methods disclosed herein can be identified by having DHEAS levels of approximately 7 μg / mL (18 μmol / L) or higher.

[0242] Other subjects for whom the methods of treating excessive androgenic conditions disclosed herein may be accepted include, for example, classical or non-classical (late-onset) CAH, Cushing's syndrome (subjects with Cushing's syndrome secrete elevated androgens), hyperandrogenemia, insulin resistance, and acanthosis nigricans. In certain embodiments, other subjects for whom the methods of treating excessive androgenic conditions disclosed herein may be accepted include, for example, patients with mild androgen disorders, for example, but not limited to, ovulatory PCOS (ovulatory hyperandrogen subjects with polycystic ovaries on ultrasound), idiopathic hyperandrogenemia (subjects with ovulatory antiandrogen but with normal ovaries on ultrasound), and idiopathic hirsutism (subjects with the androgen phenotype of normal androgens).

[0243] Reference testosterone and DHEAS levels are widely known to those skilled in the art and are disclosed in Guay et al, International Journal of Impotence Research (2004) 16, 112-120, which are incorporated herein by reference in their entirety. In summary, normal androgen levels for women aged 20–49 years are in the following ranges: DHEAS; approximately 195.6–140.4 ug / dl; serum testosterone approximately 51.5–33.7 ng / dl and free testosterone 1.51–1.03 pg / ml. Accordingly, subjects who are eligible to accept treatment with pyrazoloanthrone or its functional derivatives or analogs by the methods disclosed herein have an increase of at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, or more in DHEAS (195.6 μg / dl), serum testosterone (51.5 ng / dl), or free testosterone (1.51 pg / ml) compared to the highest range of normal values ​​for DHEAS, serum testosterone, or free testosterone. In certain embodiments, subjects who are eligible to receive treatment with pyrazoloanthrone or a functional derivative or analog thereof by the method disclosed herein have an increase of at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 10 times, or more in DHEAS (195.6 μg / dl), serum testosterone (51.5 ng / dl), or free testosterone (1.51 pg / ml) compared to the highest range of normal values ​​for DHEAS, serum testosterone, or free testosterone.

[0244] DHEAS can be measured by those skilled in the art using kits from Diagnostic Products Corporation of Los Angeles, California, USA. Cross-reactivity has been previously measured with DHEAS as 100%: 0.121% (androstenedione), 15% (9-hydroxyandrostenedione), 0.046% (estrone-3 sulfate), 0.55% (androsterone sulfate), 0.5% (DHEA), and all other tested steroids are at negligible levels. Free testosterone can be measured by those skilled in the art using Coat a Count Kits of Diagnostic Products Corporation, Los Angeles, California, USA. Cross-reactivity has been previously measured: 0.41% (dihydrotestosterone), 0.01% (androstenedione), 0.10% (methyltestosterone), and 0.01% (all other tested steroids). Overall serum testosterone levels can be measured by those skilled in the art using the Immunochem serum testosterone kit of ICN Biomedicals Inc., Diagnostic Division of Costa Mesa, California, USA.

[0245] Assays for measuring serum pregnenolone and 17-hydroxypregnenolone can be performed by those skilled in the art using kits from Quest Laboratory in Tarzana, California, USA. The free androgen index (FAI) can be calculated using the following formula: (Total testosterone ng / dl x 0.0347) / (SHBG nmol / l) x 100 = FAI. Administration of pharmaceutical compositions

[0246] Recombinant human MIS proteins or their derivatives or functional fragments may be administered by any route known in the art or described herein, for example, orally, parenterally (e.g., intravenously or intramuscularly), intraperitoneally, rectally, cutaneously, nasally, vaginally, by inhalation, cutaneously (patch), or orally. Recombinant human MIS proteins or their derivatives or functional fragments may be administered in any of the following dosages or dosing regimens.

[0247] With respect to the therapeutic methods of the present invention, the administration of recombinant human MIS protein or polynucleotides encoding such recombinant human MIS protein or functional fragments thereof is not intended to be limited to a specific form of administration, dosage, or frequency of administration. The present invention considers all forms of administration, e.g., intramuscular, intravenous, intraperitoneal, intravesical, intraarterial, intrafocal, subcutaneous, or any other route sufficient to provide a dosage suitable for treating autoimmune or immune-related diseases as disclosed herein. An effective dose of recombinant human MIS protein, e.g., a therapeutically effective dose, may be administered to a patient in a single dose or multiple doses. When multiple doses are administered, the doses may be separated from each other for only, for example, 1 hour, 3 hours, 6 hours, 8 hours, 1 day, 2 days, 1 week, 2 weeks, or 1 month. For example, a composition containing recombinant human MIS protein may be administered for, for example, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20 weeks, or more than that. For any particular subject, a specific medication plan should be adjusted over time according to the individual needs and the expert judgment of the person managing or directing the administration of the composition. For example, if a lower dose does not provide sufficient therapeutic activity, the dose of the therapeutic agent can be increased.

[0248] The attending physician will ultimately determine the appropriate dosage and administration plan, but effective doses of recombinant human MIS protein or its derivatives or functional fragments can be administered in doses of 0.0001, 0.01, 0.01, 0.1, 1, 5, 10, 25, 50, 100, 500, or 1,000 mg / kg. Effective doses can be estimated from dose-response curves obtained from in vitro or animal model test bioassays or systems. In certain embodiments, the dose of recombinant human MIS protein is approximately 1 pg / kg to 10 mg / kg (patient body weight), although lower or higher doses may also be administered.

[0249] In certain embodiments, the reference range for recombinant human MIS dosages is evaluated from the US citation group and is described in Antimullerian Hormone (AMH), Serum from Mayo Medical Laboratories. Retrieved April 2012. In certain embodiments, female subjects may be administered recombinant human MIS in the following dosages: <5 ng / mL (younger women than 24 months), <10 ng / mL (women 24 months to 12 years), 1 to 10 ng / mL (women 13 to 45 years), and >1 ng / mL (women over 45 years). In certain embodiments, male subjects may be administered recombinant human MIS in the following dosages: 15 to 500 ng / mL (younger men than 24 months), 7 to 240 ng / mL (men 24 months to 12 years), and >1 ng / mL (men over 12 years). If the person being measured is vitamin D deficient, the MIS measurement may become less accurate.

[0250] Furthermore, since additive, synergistic, or competitive effects have been demonstrated with MIS and rapamycin, AzadC, doxorubicin, cisplatin, and paclitaxel, the recombinant human MIS disclosed herein may be administered in combination with selective targeted therapy to achieve greater activity against ovarian cancer than, for example, the use of recombinant human MIS or chemotherapeutic agents alone.

[0251] Dosages for specific patients or subjects can be determined by those skilled in the art using conventional considerations (e.g., by appropriate conventional pharmaceutical protocols). A physician, for example, may initially prescribe a relatively low dose and then increase the dose until an appropriate response is obtained. The dose administered to a patient is, depending on the indication, sufficient to produce a beneficial therapeutic response over time or, for example, to reduce symptoms or other appropriate activity. Dosage is determined by the efficacy of the particular formulation, and the activity, stability, or serum half-life of the recombinant human MIS protein or its functional derivative or fragment disclosed herein, as well as the patient's condition, the autoimmune disease to be treated, and the patient's body weight and surface area to be treated. The size of the dose may also be determined by the presence, nature, and extent of any adverse side effects associated with the administration of a particular vector, formulation, etc., in a particular subject. Therapeutic compositions comprising recombinant human MIS protein or its functional derivatives or functional fragments are optionally tested in one or more suitable in vitro and / or in vivo animal models of disease, e.g., as disclosed herein in the Examples, to confirm efficient tissue metabolism and evaluate dosage, according to methods known in the art. In particular, dosage may be determined first in the relevant assay by therapeutic versus non-therapeutic activity, stability, or other appropriate criteria (e.g., comparison of treated vs. untreated cells or animal models). The formulation is administered in a ratio determined by the LD50 of the formulation and / or by observations of adverse effects of any of the recombinant human MIS protein or its functional derivatives or functional fragments at various concentrations, such as those applicable to the majority of patients or overall health. Administration may be in single or divided doses.

[0252] In determining the effective dose of recombinant human MIS protein or its functional derivative or functional fragment to be administered in the treatment or prevention of disease, physicians assess circulating plasma levels, drug toxicity, and disease progression. The selected dose level will depend on a variety of factors, such as the activity of the specific compound of the present invention used or its ester, salt, or amide; the route of administration; the time of administration; the rate of elimination of the specific compound used; the duration of treatment; the compounds and / or materials used in combination with the specific compound used; age, sex, weight, condition, overall health, and the patient's previous medical history, as well as factors known in the medical field.

[0253] In certain embodiments, the recombinant human MIS proteins disclosed herein may be administered in doses that are in line with best medical practice, taking into account the individual patient's clinical conditions, site and method of administration, administration schedule, patient's age, sex, weight, and other factors known to the healthcare provider.

[0254] The dosage regimen for compositions comprising recombinant human MIS proteins or functional fragments or variants disclosed herein can be adjusted to provide the optimal required response (e.g., therapeutic or prophylactic response). For example, a single bolus may be administered, divided doses may be administered over time, or the dosage may be proportionally reduced or increased as indicated by the urgency of the treatment situation. For ease of administration and uniformity of administration, it is particularly advantageous to prepare parenteral compositions in dosage unit form.

[0255] Furthermore, the actual dose levels of recombinant human MIS proteins in pharmaceutical compositions may vary to obtain an amount of the active ingredient that is not toxic to the subject and is effective in achieving the required therapeutic response, composition, and mode of administration for a particular subject. Pharmaceutical compositions comprising recombinant human MIS proteins or functional fragments or variants disclosed herein may be "therapeutic effective doses" and / or "preventive effective doses." Generally, an appropriate daily dose of a composition comprising recombinant human MIS proteins or functional fragments or variants disclosed herein would be the amount of recombinant human MIS protein that is the lowest dose effective in obtaining a therapeutic effect, such as the reduction of symptoms of proliferative disorders or cancer disclosed herein. Such an effective dose will generally depend on the factors described above.

[0256] If necessary, an effective daily dose of a composition comprising the recombinant human MIS protein or a functional fragment or variant disclosed herein may be administered separately in unit doses at appropriate intervals throughout the day, in two, three, four, five, six, or more sub-doses.

[0257] The dosage level administered to the subject may remain constant for a required period, for example, at least one week, at least two weeks, at least three weeks, at least one month, at least two months, at least three months, at least six months, at least one year, or at least five years. Alternatively, the dosage level administered to the subject may vary depending on the progression of the condition being treated.

[0258] Dosage values ​​may vary depending on the type and severity of cancer to be alleviated. It should be further understood that for any particular subject, a specific dosing regimen should be adjusted over time according to the individual's needs and the expert judgment of the person administering and supervising the administration of the composition, and that the dosing ranges described herein are typical examples only and are not intended to limit the scope and implementation of the invention.

[0259] The efficacy and toxicity of a compound can be determined in cell culture or experimental animals by standard pharmaceutical procedures, such as ED50 (effective in 50% of the population) and LD50 (lethal in 50% of the population). The ratio of toxicity to therapeutic effect is called the therapeutic index, which can be expressed as the LD50 / ED50 ratio. Pharmaceutical compositions exhibiting a large therapeutic index are preferred. Suitable experimental models that can be used may be the use of the Müllerian duct regression bioassay disclosed herein in the examples, or in vivo cancer models widely known to those skilled in the art. In vivo cancer models are discussed in Frese et al., "Maximizing mouse cancer models," Nat Rev Cancer. 2007 Sep;7(9):645-58, and Santos et al., Genetically modified mouse models in cancer studies. Clin Transl Oncol. 2008 Dec;10(12):794-803, and "Cancer stem cells in mouse models of cancer," 6th Annual MDI Stem Cell Symposium, MDI Biological Lab, Salisbury Cove, ME, August 10-11, 2007, which are incorporated herein by reference in their entirety.

[0260] For example, the therapeutically effective dose can be initially evaluated in a cell culture assay or in an animal model, typically in mice, rabbits, dogs, or pigs. Animal models are also used to achieve the required concentration range and route of administration. This information can then be used to determine useful doses and routes for administration in other subjects. Generally, the therapeutically effective dose depends on the required therapeutic effect; for example, the therapeutically effective dose of recombinant human MIS protein can be evaluated in a mouse model of cancer or using the Müllerian duct regression bioassay disclosed herein in Examples and Figure 4.

[0261] A physician or veterinarian skilled in the art can immediately determine and prescribe the effective amount of the required pharmaceutical composition. For example, a physician or veterinarian may disclose the dosage of the compound of the present invention used in the pharmaceutical composition at a level lower than the amount required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. It should also be noted that humans are generally treated for longer periods than mice or other experimental animals illustrated herein. The duration of treatment is proportional to the course of the disease and the duration of the drug's effectiveness. The dosage may be a single dose or multiple doses over a period of several days, but a single dose is preferred.

[0262] In certain embodiments, recombinant human MIS proteins (e.g., proteins, or nucleic acids encoding recombinant human MIS proteins or fragments thereof) can be administered to humans and other animals for therapeutic purposes by any suitable route of administration, e.g., orally, nasally, e.g., spray, rectally, vaginally, parenterally, intracapsularly, and topically, in the form of powders, ointments, or drops, e.g., orally and sublingually.

[0263] After preparation in a pharmaceutically acceptable carrier at the required dose, the pharmaceutical composition comprising the recombinant human MIS protein or its functional fragment or variant disclosed herein can be administered to a subject. The pharmaceutical composition comprising the recombinant human MIS protein or its functional fragment or variant can be administered to a subject by any suitable means. Generally, suitable means of administration include, but are not limited to, topical, oral, parenteral (e.g., intravenous, subcutaneous, or intramuscular), rectal, intravesical, vaginal, intraperitoneal, ocular, or nasal routes.

[0264] In certain embodiments, it may be required to administer a pharmaceutical composition containing recombinant human MIS protein to an area of ​​treatment. This can be achieved, for example, by local injection during surgery, by local application, for example, by injection, by catheter, or by implant, where the implant may be made of porous, non-porous, or gelatinous material, including membranes, e.g., sialastic membranes, fibers, or commercially available skin substitutes. In certain embodiments, the recombinant human MIS protein disclosed herein may be applied to muscles using topical creams, patches, intramuscular injections, etc.

[0265] In certain embodiments, recombinant human MIS proteins may be administered to a subject orally (e.g., in capsules, suspensions, or tablets) or parenterally. Conventional methods for oral administration include administering recombinant human MIS proteins in any one of the following forms: tablets, suspensions, solutions, emulsions, capsules, powders, syrups, etc. Well-known techniques for delivering recombinant human MIS proteins orally or intravenously while maintaining their biological activity are preferred. Parenteral administration may include, for example, intramuscular, intravenous, intra-articular, intra-arterial, intra-shelter, subcutaneous, or intraperitoneal administration. Recombinant human MIS proteins may also be administered orally, transdermally, topically, by inhalation (intrabronchial, intranasal, oral inhalation, or intranasal drops), or rectally. Administration may be topical or systemic, as indicated. Agents, such as nucleic acids encoding recombinant human MIS proteins or functional fragments thereof, may also be delivered using vectors, such as viral vectors, by methods known to those skilled in the art.

[0266] When compositions comprising recombinant human MIS proteins or functional fragments or variants disclosed herein are administered parenterally, they are generally prepared in a form that can be injected in a unit dose (e.g., a solution, suspension, or emulsion). Suitable pharmaceutical formulations for injection include sterile aqueous solutions or dispersions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. The carrier may be a solvent or dispersion medium, for example, water, ethanol, polyols (e.g., glycols, propylene glycols, liquid polyethylene glycols), suitable mixtures thereof, and vegetable oils.

[0267] As used herein, the term “dosage unit” refers to a physically distinct unit suitable as a dosage for a mammalian subject to be treated. Herein, each unit contains a predetermined amount of active compound calculated to produce the required therapeutic effect in relation to the required pharmaceutical carrier. Details for the dosage unit forms of the present invention are determined by, or directly depend upon, (a) the specific characteristics of the recombinant human MIS protein or its functional fragment or variant disclosed herein, and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of synthesizing recombinant human MIS proteins into activators for the treatment of susceptibility in an individual.

[0268] A pharmaceutically acceptable composition comprising a recombinant human MIS protein or a functional fragment or variant thereof disclosed herein can be suspended in an aqueous vehicle and introduced via a conventional subcutaneous injection needle or using an injection pump. Pharmaceutical composition

[0269] In certain embodiments, recombinant human MIS proteins or functional fragments or variants disclosed herein can be prepared by any suitable means, for example, in a sterile injectable solution. This can be prepared, for example, by incorporating the recombinant human MIS protein into a required amount of a suitable solvent together with various other components as needed.

[0270] Pharmaceutical formulations of compositions comprising recombinant human MIS proteins or functional fragments or variants disclosed herein can be administered to a patient in an injectable formulation comprising any suitable carrier, such as a variety of vehicles, adjuvants, additives, and diluents. Alternatively, the compounds utilized in the present invention can be administered parenterally in the form of sustained-release subcutaneous implants or targeted delivery systems, such as monoclonal antibodies, vectored delivery, ion electrophoresis, polymer matrices, liposomes, and microspheres. Examples of delivery systems useful for the present invention include those provided for U.S. Patent Nos. 5,225,182; 5,169,383; 5,167,616; 4,959,217; 4,925,678; 4,487,603; 4,486,194; 4,447,233; 4,447,224; 4,439,196 and 4,475,196. Other such implants, delivery systems, and modules are known to those skilled in the art.

[0271] Adequate fluidity can be maintained, for example, by coating, such as the use of lecithin; by maintaining the required size in the case of dispersions; and by the use of surfactants. Non-aqueous vehicles, such as cottonseed oil, sesame oil, olive oil, soybean oil, corn oil, sunflower oil, or peanut oil, and esters, such as isopropyl myristate, can also be used as solvent systems for compound compositions. Furthermore, various additives that enhance the stability, sterility, and isotonicity of the composition, such as antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of microbial action can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid. In many cases, the inclusion of isotonic agents, such as sugars and sodium chloride, will be required. Extended absorption of injectable pharmaceutical forms can be achieved by the use of absorption-delaying agents, such as aluminum monostearate and gelatin. However, according to the present invention, any vehicle, diluent, or additive used must be compatible with the compound.

[0272] In another embodiment, compositions comprising recombinant human MIS proteins or functional fragments or variants thereof disclosed herein may include lipid-based formulations. Any known lipid-based drug delivery system can be used in carrying out the present invention. For example, multivesicular liposomes, multilayer liposomes, and monolayer liposomes can all be used, insofar as a sustained-release ratio of the encapsulated active compound can be established. Methods for creating a multivesicular liposome drug delivery system with controlled release are described in PCT publications WO 9703652, WO 9513796, and WO 9423697, which are incorporated herein by reference in their entirety.

[0273] The composition of synthetic membrane vesicles is typically a combination of phospholipids, usually with steroids, particularly cholesterol. Other phospholipids or other lipids may also be used. Examples of lipids useful for synthetic membrane vesicle production include phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Preferred embodiments include, for example, phosphatidylcholine, dipalmitoylphosphatidylcholine, distearylphosphatidylcholine, dioleylphosphatidylcholine, dipalmitoylphosphatidylglycerol, and dioleylphosphatidiglycerol.

[0274] In the preparation of lipid-based vesicles containing recombinant human MIS proteins or their functional fragments or variants, variables such as the efficacy of the active compound encapsulation, the labiality of the active compound, the uniformity and size of the resulting vesicle population, the active compound-to-lipid ratio, permeability, the instability of the preparation, and the pharmaceutically acceptable formulation should be considered.

[0275] In another embodiment, recombinant human MIS proteins can be delivered in vesicles, particularly liposomes (see Langer (1990) Science 249:1527-1533). In yet another embodiment, recombinant human MIS proteins can be delivered in a controlled release system. In one embodiment, a pump can be used (see Langer (1990), cited above). In another embodiment, a polymer material can be used (see Howard et al. (1989) J. Neurosurg. 71:105). In another embodiment, the activator of the present invention is a nucleic acid encoding a recombinant human MIS protein, and the nucleic acid can be administered in vivo to promote the expression of the protein encoded therein by constructing it as part of a suitable nucleic acid expression vector so that it becomes intracellular, for example by using a retrovirus (see, for example, U.S. Patent No. 4,980,286), or by direct injection, or by using microparticle irradiation (see, for example, Genetic Onc, Biolistic, Dupont), or by coating it with a lipid or cell surface receptor or transfectant, or by ligating it to a homobox-like peptide known to enter the nucleus (see, for example, Joliot et al., 1991, Proc. Natl. Acad. Sci. USA 88:1864-1868), and administering it.

[0276] Prior to introduction, compositions comprising recombinant human MIS proteins or functional fragments or variants disclosed herein can be sterilized by any of the many available techniques in the art, for example, by gamma irradiation or electron beam sterilization.

[0277] In another embodiment of the present invention, the recombinant human MIS protein or its functional fragment or variant disclosed herein may be administered and / or formulated in conjunction with (e.g., in combination with) any other therapeutic agent. For the purpose of administration, the recombinant human MIS protein or its functional fragment or variant disclosed herein may preferably be formulated as a pharmaceutically acceptable composition. The pharmaceutically acceptable composition of the present invention comprises the compound of the present invention and a pharmaceutically acceptable carrier, wherein the compound is present in the composition in an amount useful for treating the condition in question. Appropriate concentrations and doses can be readily determined by those skilled in the art.

[0278] Pharmaceutically acceptable carriers are known to those skilled in the art. For compositions prepared as liquid solutions, acceptable carriers include salts and sterile water, and may optionally include antioxidants, buffers, bacteriostatic agents, and other additives. The compositions may also be prepared as pills, capsules, particles, or tablets, which, in addition to the compounds of the present invention, include diluents, dispersants and surfactants, binders, and lubricants. Those skilled in the art may further prepare the compounds of the present invention in appropriate forms and in accordance with acceptable practices, such as those disclosed in Remington's Pharmaceutical Sciences, Gennaro, Ed., Mack Publishing Co., Easton, Pa. 1990.

[0279] The compositions of the present invention may take any of the following forms, but are not limited to, solutions, suspensions, dispersions, ointments (including oral ointments), creams, pastes, gels, powders (toothpaste), toothpaste, lozenges, ointments, chewing gum, mouth sprays, sachets, mouthwashes, aerosols, tablets, capsules, and transdermal patches, comprising one or more resolvins and / or protectins of the present invention, or analogs thereof.

[0280] Preparations of compositions comprising recombinant human MIS proteins or functional fragments or variants disclosed herein can be prepared by several means known to those skilled in the art. In certain embodiments, a formulation can be prepared for administration as an aerosol formulation by combining, for example, (i) a sufficient amount of recombinant human MIS protein or functional fragment or variant disclosed herein to provide effective doses for multiple therapies, (ii) an effective amount of water to stabilize each of the formulations, (iii) a sufficient amount of spray to spray multiple doses from an aerosol canister, and (iv) any further optional component, such as ethanol as a cosolvent, to disperse the compound. These components can be dispersed by shaking or by ultrasonic energy using a conventional mixer or homogenizer. Bulk formulations can be transferred to smaller individual aerosol vials using valve-valve transfer, pressure injection, or a conventional cold-fill method. It is not required to enable stabilizers used in suspended aerosol formulations in the spray. Substances that are not sufficiently soluble can be coated onto drug particles in an appropriate amount, and then these coated particles can be incorporated into the formulation as described above.

[0281] In certain embodiments, compositions comprising recombinant human MIS proteins disclosed herein can be administered to a subject as a pharmaceutical composition together with a pharmaceutically acceptable carrier. In certain embodiments, these pharmaceutical compositions optionally further comprise one or more additional therapeutic agents. In certain embodiments, the additional therapeutic agent is an autoimmune disease agent, such as an immunosuppressant. In certain embodiments, the additional therapeutic agent is a corticosteroid. In certain embodiments, the additional therapeutic agent is Prednisone, methylprednisolone, Kenalog, Medrol Oral, Medrol (Pak) Oral, Depo-Medrol Inj, prednisolone Oral, Solu-Medrol Inj, hydrocortisone Oral, Cortef Oral, Solu-Medrol IV, cortisone Oral, Celestone Soluspan Inj, Orapred ODT Oral, Orapred Oral, Prelone Oral, methylprednisolone acetate Inj , Prednisone Intensol Oral, betamethasone acet & sod phos Inj, Veripred, Celestone Oral, methylprednisolone sodium succ IV, methylprednisolone sodium succ Inj, Millipred Oral, Solu-Medrol (PF) Inj, Solu-Cortef Inj, Aristospan Intra-Articular Inj, hydrocortisone sod succinate Inj, prednisolone sodium phosphate Oral, methylprednisolone sod suc(PF) IV, Solu-Medrol (PF) IV, triamcinolone hexacetonide Inj, A-Hydrocort Inj, A-Methapred Inj, Millipred DP Oral, Flo-Pred Oral,The following are selected from the group consisting of Aristospan Intralesional Inj, betamethasone Oral, methylprednisolone sod succ (PF) Inj, hydrocortisone sod succ (PF) Inj, Solu-Cortef (PF) Inj, prednisolone acetate Oral, dexamethasone in 0.9% NaCl IV, Rayos, and levothyroxine. Of course, such therapeutic agents are known to those skilled in the art and can be readily substituted by them. These should not be interpreted as exclusive or limiting.

[0282] Wetting agents, emulsifiers, and lubricants, such as sodium lauryl sulfate and sodium stearate, as well as colorants, dissociating agents, coating agents, sweeteners, flavors, fragrances, preservatives, and antioxidants may also be present in this composition. Examples of pharmaceutically acceptable antioxidants include water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; oil-soluble antioxidants, such as ascorbic acid palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, etc.; and metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0283] The formulations of the present invention include those suitable for intravenous, oral, nasal, topical, transdermal, buccal, sublingual, rectal, vaginal, and / or parenteral administration. The formulations can conveniently be provided in unit dose forms and can be prepared by any method known to those skilled in the art of pharmaceuticals. The amount of active ingredient that can be combined with a carrier material to produce a single dose form will generally be the amount of compound that produces a therapeutically effective dose. Generally, from 100 percent, this amount will be in the range of about 1 percent to about 99 percent of the active ingredient, preferably about 5 percent to about 70 percent, and most preferably about 10 percent to about 30 percent.

[0284] Formulations of the present invention suitable for oral administration may be in the form of capsules, pills, tablets, lozenges (flavored, usually using sucrose and acacia or tragacanth), powders, particles, aqueous or non-aqueous liquid solutions or suspensions, oil-in-water or water-in-oil liquid emulsions, elixirs or syrups, or flavored tablets (inactive, e.g., using gelatin and glycerin, or sucrose and acacia), and / or mouthwash, each containing a predetermined amount of the compound of the present invention as the active ingredient. The compound of the present invention may also be administered as a bolus, lick, or paste.

[0285] In the solid dosage form of the present invention for oral administration (capsules, tablets, pills, dragées, powders, particles, etc.), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, e.g., sodium citrate, dicalcium phosphate, and / or the following components: fillers or bulking agents, e.g., starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; binders, e.g., carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; humectants, e.g., glycerol Solids, disintegrants such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; solution decolorizers such as paraffin; absorption accelerators such as quaternary ammonium compounds; wetting agents such as cetyl alcohol and glycerol monostearate; absorbents such as kaolin and bentonite clay; lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof; and colorants. In the case of capsules, tablets, and pills, the pharmaceutical composition may also include cyclic agents. Similar types of solid compositions can also be used as fillers in soft and hard-filled gelatin capsules with excipients such as lactose or milk, and high molecular weight polyethylene glycol, etc.

[0286] Tablets can be made by compression or molding, with one or more auxiliary components as optional. Compressed tablets can be prepared using a binder (e.g., gelatin or hydroxypropyl methylcellulose), a lubricant, an inert diluent, a preservative, a disintegrant (e.g., sodium glycolate starch or cross-linked carboxymethylcellulose), a surface-active or dispersant. Molded tablets can be made by molding a powdered compound moistened with an inert liquid diluent using appropriate machinery.

[0287] Tablets and other solid dosage forms of the pharmaceutical compositions of the present invention, such as dragées, capsules, pills, and particles, may optionally be prepared with or with coatings and shells, such as enteric coatings and other coatings known in pharmaceutical formulation techniques. They may also be formulated to provide sustained or controlled release of the active ingredient therein, for example, using various proportions of hydroxypropyl methylcellulose, other polymer matrices, liposomes, and / or microspheres to provide the desired release profile. They may be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating a sterilizer into the form of a sterile solid composition that can be dissolved in sterile water, or by several other immediately-injectable sterile media. These compositions may also optionally contain opacifying agents, and they may be compositions that release the active ingredient alone or, preferably, in a delayed manner, in a specific part of the gastrointestinal tract. Examples of embedding compositions that can be used include polymer substances and waxes. The active ingredient may also be in microencapsulated form, which, if appropriate, contains one or more of the above-described excipients.

[0288] Liquid administration forms for oral administration of the compounds of the present invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs.

[0289] In addition to the active ingredient, the liquid dosage form may include inert diluents commonly used in the art, such as water or solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, as well as mixtures thereof. Besides inert diluents, the oral composition may also include adjuvants, such as humectants, emulsifiers and suspending agents, sweeteners, flavorings, colorants, fragrances, and preservatives.

[0290] In addition to the active compound, the suspension may contain suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methhydroxyl, bentonite, agar and tragacanth, and mixtures thereof.

[0291] In certain cases, compositions comprising recombinant human MIS proteins or functional fragments or variants thereof disclosed herein may be suitable formulations for rectal or vaginal administration, for example, suppositories, which can be prepared by mixing one or more compounds of the present invention with one or more suitable non-irritating excipients or carriers, such as cocoa butter, polyethylene glycol, suppository wax, or salicylates, which are solid at room temperature but liquid at body temperature, thereby releasing the active compounds. Suitable carriers and formulations for such administrations are known in the art.

[0292] For topical or transdermal administration of the recombinant human MIS protein of the present invention, for example, for intramuscular administration, the dosage forms include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The recombinant human MIS protein or its functional fragment or variant disclosed herein can be mixed under sterile conditions with a pharmaceutically acceptable carrier and any required preservative, buffer, or propellant.

[0293] Ointments, pastes, creams, and gels may, in addition to the active compounds of the present invention, contain excipients such as animal and vegetable oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silicic acid, talc, and zinc oxide, as well as mixtures thereof. Powders and sprays may, in addition to the compounds of the present invention, contain excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate, and polyamide powders, or mixtures thereof. Sprays may further contain conventional propellants such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons such as butane and propane.

[0294] Transdermal patches offer the further advantage of providing controlled delivery of the recombinant human MIS protein of the present invention to the body. Such a dosage form can be made by dissolving or dispersing the compound in a suitable medium. Absorption enhancers can also be used to increase the flow of the compound across the skin. The rate of such flow can be controlled by providing a rate-controlled membrane or by dispersing the active compound in a polymer matrix or gel.

[0295] A pharmaceutical composition of the present invention suitable for parenteral administration comprises one or more compounds of the present invention in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions, or emulsions, or sterile powders that can be reconstituted into sterile injectable solutions or dispersions immediately before use. It may contain antioxidants, buffers, bacteriostatic agents, solutes to be isotonic with the blood of the recipient to whom the formulation is intended, or suspensions or thickeners.

[0296] Suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate). Appropriate fluidity can be maintained, for example, by the use of coating materials (e.g., lecithin), by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0297] These compositions may also include adjuvants, such as preservatives, humectants, emulsifiers, and dispersants. Prevention of microbial action can be ensured by including various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid. Isotonic agents, such as sugars and sodium chloride, may also be required in the composition. Furthermore, extended absorption of the injectable pharmaceutical form can be achieved by including absorption-delaying agents, such as aluminum monostearate and gelatin.

[0298] In certain cases, it is required to slow the absorption of a drug from subcutaneous or intramuscular injection in order to prolong its effect. This can be achieved by using aqueous suspensions of crystalline or amorphous materials with poor water solubility. The rate of drug absorption depends on its rate of dissolution, which may depend on the crystal size and morphology. Alternatively, delayed absorption of parenterally administered drug forms can be achieved by dissolving and suspending the drug in an oil vehicle.

[0299] Injectable depot formulations are created by forming a microencapsulation matrix of the target compound in a biodegradable polymer, such as polylactide-polyglycolide. The rate of drug release can be controlled depending on the ratio of the drug to the polymer and the properties of the specific polymer used. Other examples of biodegradable polymers include poly(orthoesters) and poly(anhydride). Depot injectable formulations can also be prepared by encapsulating the drug in liposomes or microemulsions compatible with body tissues.

[0300] In certain embodiments, recombinant human MIS proteins or their functional fragments or variants disclosed herein may be isolated and / or purified, or substantially purified, by one or more purification methods described herein or known to those skilled in the art. Generally, the purity is at least 90%, particularly 95%, and often exceeds 99%. In certain embodiments, spontaneously occurring compounds are excluded from the general description of the broader genus.

[0301] In certain embodiments, the composition comprises at least one recombinant human MIS protein in combination with a pharmaceutically acceptable carrier. Specific examples of materials that can function as pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate, ethyl laurylate, and agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic salt solutions; Ringer's solution; ethyl alcohol; phosphate buffer; and other non-toxic compatible substances used in pharmaceutical formulations.

[0302] In certain embodiments, compositions comprising recombinant human MIS proteins or functional fragments or variants thereof disclosed herein may contain one or more acidic functional groups that can form pharmaceutically acceptable salts with pharmaceutically acceptable bases. The terms “pharmaceutically acceptable salts, esters, amides, and prodrugs” as used herein refer to these carboxylate salts, amino acid addition salts, esters, amides, and prodrugs of the compounds of the present invention. They are within the bounds of sound medical judgment, suitable for use in contact with patient tissue, effective for the intended use of the compounds of the present invention, and balanced by a reasonable benefit / risk ratio, without excessive toxicity, irritation, allergic reactions, etc. The term “salt” refers to relatively non-toxic inorganic and organic acid addition salts of the compounds of the present invention.

[0303] These salts can be prepared in situ during the final isolation and purification of the compound, or by reacting the purified compound in free salt form separately with a suitable organic or inorganic acid and isolating the salt formed thereby. These may include cations based on alkali metals and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium, etc., as well as non-toxic ammonium, quaternary ammonium, and amine cations, such as, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, etc. (see, for example, Berge SM, et al., "Pharmaceutical Salts," J. Pharm. Sci., 1977;66:1-19, which is incorporated herein in its entirety by reference).

[0304] The term “pharmaceutically acceptable ester” refers to the relatively non-toxic esterified products of the compounds of the present invention. These esters can be prepared in situ during the final isolation and purification of the compound, or by reacting the purified compound in its free acid form or hydroxyl form separately with a suitable esterifying agent. Carboxylic acids can be converted to esters via treatment with alcohol in the presence of a catalyst. The term is further intended to include lower hydrocarbon groups that can be solvated under physiological conditions, such as alkyl esters, methyl, ethyl, and propyl esters.

[0305] As used herein, “pharmaceutically acceptable salt or prodrug” is a salt or prodrug that is free from excessive toxicity, irritation, allergic reactions, etc., is balanced by a reasonable benefit / risk ratio, is effective for its intended use, is suitable for use in contact with patient tissue, and is within the bounds of sound medical judgment. These compounds include, if possible, the amphoteric form of the r compound of the present invention.

[0306] The term “salt” refers to relatively non-toxic inorganic and organic acid addition salts of the compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compound, or by reacting the purified compound in free base form separately with a suitable organic or inorganic acid and isolating the salt formed thereby. These may include cations based on alkali metals and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium, etc., as well as non-toxic ammonium, quaternary ammonium, and amine cations, such as, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, etc. (see, for example, Berge SM, et al., “Pharmaceutical Salts,” J. Pharm. Sci., 1977;66:1-19, which is incorporated herein in whole by reference).

[0307] The term “prodrug” refers to a compound or agent that is rapidly converted in vivo to produce an active recombinant human MIS protein, such as a biologically active or functionally active MIS protein, or a nucleic acid (e.g., mRNA, DNA, MOD-RNA) encoding a functionally active MIS protein. In certain embodiments, recombinant human MIS protein prodrugs may be activated by hydrolysis in the blood, for example, through cleavage of a leader sequence, or through cleavage of a first cleavage site that produces the N-terminal and C-terminal domains for the production of a biologically active MIS protein, similar to how insulin is activated from a proprotein to an active insulin protein. A detailed discussion is provided in T. Higachi and V. Stella, “Pro-drugs as Novel Delivery Systems,” Vol. 14 of the ACS Symposium Series, and in Bioreversible Carriers in: Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987 (both incorporated herein in their entirety by reference). As used herein, a prodrug is a compound that is metabolized or converted into a biologically, pharmaceutically, or therapeutically active form upon in vivo administration. Prodrugs may be designed to alter the metabolic stability or transport properties of recombinant human MIS proteins in order to block side effects or toxicity, or to alter other characteristics or properties of recombinant human MIS proteins.

[0308] Based on knowledge of the in vivo pharmacokinetic processes of MIS and drug metabolism or post-translational protein processing, once pharmaceutically active compounds are identified, those skilled in the art can generally design recombinant human MIS protein prodrugs that can be activated in vivo to increase the level of bioactive MIS protein in a subject (see, for example, Nogrady (1985) Medicinal Chemistry: A Biochemical Approach, Oxford University Press, NY, pages 388–392). Conventional procedures for the selection and preparation of appropriate prodrugs are described, for example, in "Design of Prodrugs," ed. H. Bundgaard, Elsevier, 1985. Suitable examples of prodrugs include methyl, ethyl, and glycerol esters of the corresponding acids.

[0309] As discussed herein, in certain embodiments, compositions comprising recombinant human MIS proteins or functional fragments or variants thereof disclosed herein can be conjugated or covalently bound to targeting agents to increase their tissue specificity and targeting to cells, such as muscle cells. Targeting agents may include, but are not limited to, antibodies, cytokines, and receptor ligands, as discussed in the “Targeting” section. In certain embodiments, the targeting agent is overexpressed on the cells to be targeted, such as muscle cells, compared to non-muscle cells.

[0310] Regardless of the chosen route of administration, the compounds and / or pharmaceutically acceptable compositions of the present invention, which can be used in a suitable hydrate form, are prepared into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art. gene therapy

[0311] In certain embodiments, nucleic acids encoding recombinant human MIS proteins or functional fragments thereof disclosed herein can be appropriately administered as vectors, such as viral vectors.

[0312] In certain embodiments, nucleic acids encoding recombinant human MIS proteins can be effectively used in gene therapy. For example, generally refer to U.S. Patent No. 5,399,346, which is incorporated in whole herein by reference. The general principle is to introduce its polynucleotides into target cells in a patient, where they are transcribed into proteins.

[0313] Cellular transfer can be facilitated by appropriate techniques known in the art, for example, by providing polynucleotides in the form of a suitable vector, or by encapsulating polynucleotides in liposomes.

[0314] The required aspect of gene therapy is the provision of polynucleotides in a manner that replicates within cells and enhances and prolongs the desired effect. This allows the polynucleotides to be ligated to an appropriate promoter, such as the native promoter of the corresponding gene, a heterologous promoter endogenously active in liver, nerve, bone, muscle, skin, joint, or chondrocytes, or a heterologous promoter that can be induced by an appropriate agent.

[0315] Expression vectors compatible with eukaryotic cells, preferably compatible with vertebrate cells, can be used to create recombinant human MIS proteins, or their functional derivatives, functional variants, or functional fragments, as disclosed herein. Eukaryotic cell expression vectors are known in the art and are available from several commercial sources. Typically, such vectors are provided to contain a convenient recognition site for insertion of the desired DNA segment. These vectors may be viral vectors, such as adenoviruses, adeno-associated viruses, poxviruses, such as orthopox (vaccinia and attenuated vaccinia), avipox, lentiviruses, and murine leukemia virus.

[0316] Alternatively, in certain embodiments, plasmid expression vectors can be used. Plasmid expression vectors include, but are not limited to, the following: pcDNA3.1, pET vector (Novagen(r)), pGEX vector (GE Life Sciences), and pMAL vector (New England labs. Inc.) (for protein expression in E. coli host cells, e.g., BL21, BL21(DE3) and AD494(DE3)pLysS, Rosetta(DE3), and Origami(DE3)) (Novagen(r)); Strong CMV promoter-based pcDNA3.1 (Invitrogen(tm) Inc.) and pCIneo vector (Promega) (for expression in mammalian cell lines, e.g., CHO, COS, HEK-293, Jurkat, and MCF-7); and replication-incompetent adenovirus vectors pAdeno X, pAd5F35, pLP-Adeno-X-CMV (Clontech(r)). pAd / CMV / V5-DEST, pAd-DEST vector (Invitrogen (tm) Inc.) (for adenovirus-mediated gene transfer and expression in mammalian cells); pLNCX2, pLXSN, and pLAPSN retrovirus (retroviral vectors from Clonetech for use in the Retro-X (tm) system for retrovirus-mediated gene transfer and expression in mammalian cells); pLenti4 / V5-DEST(tm), pLenti6 / V5-DEST(tm), and pLenti6.2 / V5-GW / lacZ (INVITROGEN (tm) Inc.) (for lentivirus-mediated gene transfer and expression in mammalian cells); adeno-associated virus expression vectors, e.g., pAAV-MCS and pAAV-IRES-hrGFP (for adeno-associated virus-mediated gene transfer and expression in mammalian cells); BACpak6 baculovirus (Clontech (r)) and pFastBac(tm) HT ( Invitrogen (tm) Inc.) (for expression in Spodoptera frugiperda 9 (Sf9) and Sf11 insect cell lines); pMT / BiP / V5-His (Invitrogen (tm) Inc.) (for expression in Drosophila Schneider S2 cells); Pichia expression vectors pPICZα, pPICZ, pFLDα and pFLD (Invitrogen (tm) Inc.) (for expression in Pichia pastoris), vectors pMETα and pMET (for expression in P. methanolica); pYES2 / GS and pYD1 (Invitrogen (tm) Inc.) vector (for expression in the yeast Saccharomyces cerevisiae). Recent advances in large-scale heterologous protein expression in Chlamydomonas reinhardtii are described in Griesbeck C. et. al. 2006 Mol. Biotechnol. 34:213-33 and Fuhrmann M. 2004, Methods Mol Med. 94:191-5. Exogenous heterologous coding sequences are inserted into the nuclear, chloroplast, and mitochondrial genomes via heterologous recombination. The chloroplast expression vector p64, containing aminoglycoside adenyltransferase (aadA), the most versatile chloroplast selection marker conferring resistance to spectinomycin or streptomycin, can be used to express exogenous proteins in chloroplasts. The biolistic gene gun method is used to introduce vectors into algae. Upon transfer to chloroplasts, the exogenous DNA is released from the gene gun particle and integrated into the chloroplast genome via homologous recombination.

[0317] The viral vector systems that can be used in the present invention are not limited to, but include: (a) adenovirus vectors; (b) retrovirus vectors; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV 40 vectors; (f) polyomavirus vectors; (g) papillomavirus vectors; (h) picornavirus vectors; (i) poxvirus vectors, e.g., orthopox, e.g., vaccinia virus vector or avipox, e.g., canarypox or fowlpox; and (j) helper-dependent or gutless adenoviruses. In preferred embodiments, the vector is an adenovirus. Replication-deficient viruses may also be released.

[0318] The vector may or may not be incorporated into the cell genome. Its components may, if necessary, include a viral sequence for transfection. Alternatively, the components may be incorporated into an episomal replication-capable vector, such as EPV and EBV vectors.

[0319] The nucleic acid expression components for recombinant human MIS proteins disclosed herein, such as DNA, MOD-RNA, or RNAa, can generally be ligated to regulatory sequences, such as promoters or enhancers, to ensure the expression of the component in target cells. Further details of the vectors and other components are described below.

[0320] Typical regulatory sequences, though not limited to these, include transcription promoters, inducible promoters, and transcription elements, optional actuarial sequences for controlling transcription, sequences encoding appropriate mRNA-ribosome binding sites, and sequences for controlling the termination of transcription and / or translation. The term “regulatory elements” includes nucleic acids that induce or control the transcription of protein-coding sequences to which they are actuarially linked, such as start signals, enhancers, and promoters. In certain examples, the transcription of recombinant genes is under the control of a promoter sequence (or other transcriptional regulatory sequence) that controls the expression of the recombinant gene in the cell type in which expression is intended. It will also be understood that recombinant genes may be under the control of the same or different transcriptional regulatory sequences as those that control the transcription of naturally occurring proteins. In certain examples, promoter sequences are recognized by the cell’s synthetic mechanism, or introduced synthetic mechanism, which is required to initiate the transcription of a particular gene.

[0321] A regulatory sequence may be a single regulatory sequence, multiple regulatory sequences, modified regulatory sequences, or fragments thereof. A modified regulatory sequence is a regulatory sequence whose nucleic acid has been altered or modified by specific means, such as, but not limited to, mutation, methylation, etc. Regulatory sequences useful in the methods disclosed herein are promoter elements sufficient to make promoter-dependent gene expression controllable for cell type-specific, tissue-specific, or induceable by an external signal or agent (e.g., an enhancer or repressor). Such elements may be located within the 5' or 3' region of a native gene, or within an intron.

[0322] As used herein, the term “tissue-specific promoter” means a nucleic acid sequence that functions as a promoter, that is, controls the expression of a selected nucleic acid sequence ligated to a promoter, and selectively influences the expression of a selected nucleic acid sequence in specific cells of a tissue, such as ovarian-derived cells.

[0323] The term "constitutively active promoter" refers to a promoter of a gene that is always expressed within a given cell. Typical promoters for use in mammalian cells include cytomegalovirus (CMV), while those for use in prokaryotic cells include bacteriophage T7 and T3 promoters, among others. The term "inducible promoter" refers to a promoter of a gene that can be expressed in response to a given signal, such as the addition or removal of an agent. Non-exclusive examples of inducible promoters include "tet-on" and "tet-off" promoters, or promoters that are controlled in specific tissue types.

[0324] In certain embodiments, viral vectors containing nucleic acid sequences encoding the recombinant human MIS protein or a functional fragment thereof, such as DNA, MOD-RNA, or RNAa, can be used. For example, retroviral vectors can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for the precise packaging of the viral genome and its integration into host cell DNA. The nucleic acid sequences encoding the recombinant human MIS protein are cloned into one or more vectors, which facilitate the delivery of the gene to the patient. More details on retroviral vectors can be found in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of retroviral vectors for delivering the mdrl gene to hematopoietic stem cells to make the stem cells more resistant to chemotherapy. Other references detailing the use of retroviral vectors in gene therapy include Cloves et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993).

[0325] The production of recombinant retroviral vectors containing the gene in question is typically carried out in two steps. First, a sequence encoding a recombinant human MIS protein, or a functional derivative, functional variant, or functional fragment thereof, can be inserted alone or fused with -Fc into a retroviral vector containing metabolic regulators (including promoter and / or enhancer elements, which may be provided by viral terminal repeat sequences (LTRs) or by internal promoters / enhancers and associated splicing signals), sequences necessary for the efficient expression of infectious virions of viral RNA (e.g., packaging signals (Psi), tRNA primer binding sites (-PBS), 3' regulatory sequences required for reverse transcription (+PBS), and sequences required for efficient packaging into viral LTRs). The LTRs contain sequences required for the association of viral genomic RNA, reverse transcriptase, and integrase functions, as well as sequences related to packaging the expression of genomic RNA within the viral particle.

[0326] Following the preparation of recombinant retroviral vectors, the vector DNA is introduced into a packaging cell system. The packaging cell system provides the viral proteins required in trans for packaging the viral genomic RNA into viral particles having the desired host range (e.g., viral coding core (gag), polymerase (pol), and envelope (env) proteins). The host range is partially controlled by the type of envelope gene product expressed on the surface of the viral particle. The packaging cell system may express ecotropic, amphotropic, or xenotropic envelope gene products. Alternatively, the packaging cell system may lack the sequence encoding the viral envelope (env) protein. In this case, the packaging cell system can package the viral genome into particles lacking membrane-associated proteins (e.g., the env protein). To produce viral particles containing membrane-associated proteins that allow the virus to enter cells, a packaging cell line containing a retroviral sequence can be transfected with a sequence encoding a membrane-associated protein (for example, the G protein of varicella stomatitis virus (VSV)). The transfected packaging cell line can then produce viral particles containing the membrane-associated proteins expressed by that transfected packaging cell line. These viral particles, containing viral genomic RNA derived from one virus and capsided by the envelope protein of another virus, are called pseudotyped viral particles.

[0327] Adenoviruses are another viral vector that can be used in gene therapy. Adenoviruses are particularly attractive vehicles for delivering genes to respiratory epithelium. Adenoviruses spontaneously infect respiratory epithelium and cause mild disease. Other targets for adenovirus-based delivery systems are the liver, central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being able to infect non-divided cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) provides a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrates the use of adenovirus vectors for delivering genes to rhesus monkey respiratory epithelium. Other preferred viral vectors include poxviruses, such as vaccinia viruses, such as attenuated vaccinia, such as Modified Virus Ankara (MVA) or NYVAC, AviPox, such as fowlpox or canarypox. Other examples of adenovirus use in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT Publication WO94 / 12649; and Wang, et al., Gene Therapy 2:775-783 (1995). In another embodiment, a lentiviral vector, such as the HIV-based vector described in U.S. Patent Nos. 6,143,520; 5,665,557; and 5,981,276, which are incorporated herein by reference in their entirety. In a particular embodiment, a viral vector, such as an adeno-associated virus (AAV) vector, is used.Typical AAV vectors are described in Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Patent No. 5,436,146, incorporated herein by reference; Gao et al., Gene Therapy 2005, 5, 285-297; Vandenberghe et al., Gene Therapy 2009, 16, 311-319; Gao et al., PNAS 2002, 99, 11854-11859; Gao et al., PNAS 2003, 100, 6081-6086; Gao et al., J. of Virology 2004, 78, 6381-6388; Molecular Cloning: A Laboratory Manual (4th edition) ed. by M. Green and Disclosed in J. Sambrook. In certain embodiments, the AAV vectors are AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.10, and AAV2.5. It should be noted that the selection of a particular type of AAV vector may depend on the target tissue.

[0328] In certain embodiments, if a recombinant human MIS protein encoded by a viral vector is endogenously expressed in a subject, the expression level of the recombinant human MIS protein disclosed herein may remain constant over a required period, for example, at least one week, at least two weeks, at least three weeks, at least one month, at least two months, at least three months, at least six months, at least one year, or at least five years. In certain embodiments, the expression of the recombinant human MIS protein disclosed herein may be maintained over a required period at or beyond a therapeutically effective level.

[0329] Another approach to gene therapy involves transferring genes to cells in tissue culture by methods such as electroporation, lipofection, calcium phosphate transfection, or viral infection. Typically, the transfer method involves transferring a selection marker to the cells. The cells are then picked up and placed under selection to isolate those expressing the transferred gene. These cells are then delivered to the patient.

[0330] U.S. Patent No. 5,676,954 (in its entirety by reference) reports on the injection of genetic material complexed with a cationic liposome carrier into mice. U.S. Patent Nos. 4,897,355, 4,946,787, 5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859, 5,703,055, and International Publication No. WO 94 / 9469 (in its entirety by reference) provide cationic lipids for use in transfecting DNA into cells and mammals. U.S. Patent Nos. 5,589,466, 5,693,622, 5,580,859, 5,703,055, and International Publication No. WO 94 / 9469 (in whole incorporated herein by reference) provide a method for delivering DNA-cationic lipid complexes to mammals. Such cationic lipid complexes or nanoparticles can also be used to deliver proteins.

[0331] Genes or nucleic acid sequen...

Claims

1. A recombinant mature Müllerian duct inhibitor (MIS) protein comprising amino acid residues 25-559 of Sequence ID No. 2, wherein the recombinant MIS protein does not contain a FLAG tag.

2. The recombinant MIS protein according to claim 1, wherein each monomer is a homodimer comprising two monomers, each monomer containing residues 25-559 of SEQ ID NO:

2.

3. A recombinant mature Müllerian inhibitor (MIS) protein comprising an amino acid sequence having at least 98% sequence identity with amino acid residues 26-560 of SEQ ID NO: 1, wherein amino acid residue 450 of SEQ ID NO: 1 is changed from Q to R, the FLAG tag is absent, and the recombinant MIS protein exhibits increased cleavage and increased production yield in vitro compared to a wild-type MIS protein having the amino acid sequence of SEQ ID NO:

1.

4. The recombinant MIS protein according to claim 3, wherein the recombinant MIS protein is a homodimer comprising two monomers, each monomer containing residues 26-560 of SEQ ID NO: 1, and amino acid residue 450 of SEQ ID NO: 1 is changed from Q to R.

5. A pharmaceutical composition for the treatment of infertility, comprising the recombinant MIS protein described in claim 1 and a pharmaceutically acceptable carrier.

6. A pharmaceutical composition for the treatment of infertility, comprising the recombinant MIS protein according to claim 2 and a pharmaceutically acceptable carrier.

7. A pharmaceutical composition for the treatment of infertility, comprising the recombinant MIS protein according to claim 3 and a pharmaceutically acceptable carrier.

8. A pharmaceutical composition for the treatment of infertility, comprising the recombinant MIS protein according to claim 4 and a pharmaceutically acceptable carrier.

9. Use of the recombinant MIS protein according to claim 1 in the manufacture of a pharmaceutical product for the treatment of infertility.

10. Use of the recombinant MIS protein according to claim 2 in the manufacture of a pharmaceutical product for the treatment of infertility.

11. Use of the recombinant MIS protein according to claim 3 in the manufacture of a pharmaceutical product for the treatment of infertility.

12. Use of the recombinant MIS protein according to claim 4 in the manufacture of a pharmaceutical product for the treatment of infertility.

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