Modified mullerian inhibiting substance (MIS) proteins and uses thereof for 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 TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 61 / 777,135, filed March 12, 2013, the contents of which are incorporated by reference in their entirety.

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

[0003] FIELD OF THEINVENTION The present invention relates to an improved recombinant human MIS protein with improved cleavage, increased biological activity and increased potency compared to wild-type human MIS protein. In a particular embodiment, the recombinant human MIS protein comprises at least one of the following components: an improved Kex cleavage site for increased cleavage, a FLAG tag, and a non-MIS leader sequence instead of the normal MIS leader sequence. Another embodiment of the present invention relates to methods, uses, and kits comprising recombinant human MIS protein for the treatment of cancer, such as cancers expressing MIS receptor type II (MISRII), or for the treatment of diseases characterized by androgen excess.

[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 technology]

[0005] 2. Background of the Invention Müllerian inhibitory substance (MIS), also known as anti-Müllerian hormone (AMH), is a 140 kDa disulfide-linked homodimeric glycoprotein member of the large transforming growth factor-β (TGFβ) multigene family of glycoproteins. The proteins of this family are all produced as dimeric precursors, and post-transcriptional processing for activation requires cleavage and dissociation to release the biologically active 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 the 9th week of gestation and continues at high levels until puberty, when expression levels drop dramatically. In females, MIS is produced only after birth in granulosa cells at levels similar to those in adult males from prepuberty through menopause, after which expression ceases. In male fetuses, MIS causes regression of the Müllerian ducts, precursors of the fallopian tubes, the uterus, the cervix, and the upper third of the vagina.

[0007] MIS exerts its biological effects after binding to a heterodimer 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. SMAD1, 5, and 8 (but mainly SMAD8) are then activated, which together with SMAD4 regulate gene transcription. Only one MIS receptor type II (MISRII) gene has been identified in mice, rats, and rabbits, and in humans, the gene is localized to chromosome 12. It is a 65 kD protein that has been detected in embryonic and adult Müllerian structures, breast tissue, prostate tissue, gonads, motor neurons, and 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, with activin receptor-like kinases (ALK) 2 and 3 being the most likely candidates, depending on the animal species and tissue examined.

[0008] In addition to its well-established role in regression of the Mullerian duct, MIS inhibits the growth of a variety of human cancer cell lines in vitro and in vivo. Cell lines showing inhibition are from ovarian, cervical, endometrial, prostate, and breast cancers. No toxicity has been observed in vivo, even when high concentrations of MIS are maintained systemically in rodents or human patients with tumors secreting MIS for long periods of time. These findings of relatively restricted receptor expression, antiproliferative activity against cancer cells expressing MIS RI and RII, and its apparent non-toxicity, taken together, make MIS an ideal reagent for use in combination with existing chemotherapeutic drugs for the treatment of ovarian cancer, which are known to become resistant to these conventional agents.

[0009] MIS acts through MIS type II receptor cells and functions as a potential tumor suppressor in ovarian cancer initiation (Teixeira et al, unpublished). MIS may 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 cancers, for example, those 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 also inhibits the growth of various cancers in vitro and in vivo without apparent toxicity after long-term treatment in vivo (Pieretti-Vanmarcke et al. 2006b). Epithelial ovarian cancer recapitulates the archetypal histology of the embryonic Mullerian duct and its various subtypes (Scully 1977), e.g., serous cystadenocarcinoma resembles the embryonic fallopian tube, endometrioid carcinoma resembles the endometrium, and mucinous carcinoma resembles the cervix. MIS also functions synergistically or additively with cancer drugs commonly used to control tumor growth (Pieretti-Vanmarcke et al. 2006a).

[0011] Chemotherapeutic agents have previously been reported to select for ovarian cancer stem cells, which are typically multidrug resistant and / or resistant to chemotherapeutic agents. In particular, there are numerous findings reporting that ovarian cancers 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, which have stem / progenitor cell characteristics that are poorly responsive to chemotherapeutic agents in current clinical use for ovarian cancer, as well as populations of CD44+, CD24+, EpCam+, and E-cadherin-negative cells (Wei et al, 2010). In particular, MIS has been shown to inhibit ovarian cancer cells both in vitro and in vivo, and can specifically target and inhibit the proliferation of ovarian cancer progenitor cell populations enriched by CD44+, CD24+, EpCAM+, and E-cadherin-cell surface markers. To accommodate clinical trials of MIS in ovarian cancer patients, production of recombinant human MIS must be optimized to increase yield and purity.

[0012] However, preparations obtained from purification of native and wild-type MIS are complex and the yields are low. Moreover, the cleavage required to produce active fragments of MIS is also inefficient. Human MIS protein is produced from a preproprotein that contains a leader sequence. The leader sequence (amino acids 1-25 of SEQ ID NO:1) is cleaved, and the remaining preprotein (often referred to as "holo-human MIS") is post-translationally cleaved to yield the N- and C-terminal domains. These covalently linked N- and C-terminal domains form a monomer, and two identical monomers (containing the N- and C-terminal domains) together form a homodimer. Holo-human MIS is cleaved into the N- and C-terminal domains, most likely by furin or a related prohormone convertase PC5, which is expressed in the gonads. Cleavage occurs primarily at the R-terminal end with a serine at the +1 site, making the MIS cleavage site monobasic. -4 XXR -1 The purified C-terminal domain is the biologically active component, and cleavage is required for biological activity. A second cleavage site, the significance of which 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 duct syndrome. The role of the amino-terminal domain in vivo may be to assist in folding of the protein and to facilitate 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 biological activity of the C-terminal component in vitro, but the mechanism was unclear. Cleavage of recombinant MIS expressed by CHO cells was incomplete, which makes cleavage with exogenous serine proteases, such as plasmin, required to enhance biological activity.

[0013] Therefore, there is a need for more efficient methods for producing high concentrations of human MIS protein for use as a therapeutic biological agent. Summary of the Invention

[0014] The present invention relates to a modified recombinant human MIS protein having improved cleavage, increased biological activity, and increased potency compared to wild-type human MIS protein, which contains a combination of the following components: a modified Kex cleavage site for increased cleavage, and a non-MIS leader sequence in place of the normal MIS leader sequence, with or without an internal label or tag to facilitate its purification, to improve yields of biologically active protein.

[0015] Thus, herein, the inventors have made changes to the native human MIS amino acid sequence to combine: (i) modifying the first cleavage site to increase cleavage, thereby increasing the potency and biological activity of MIS without the insertion of a tag to facilitate purification, and (ii) modifying the endogenous leader sequence of MIS to increase the yield of biologically active protein. Surprisingly, the addition of the leader sequence in combination with the modified first cleavage site significantly increased both the yield of protein produced and the amount of cleavage from the first cleavage site of the recombinant MIS protein. Furthermore, there is an unmet need to have a form of biologically active MIS that can be labeled for use with receptors, as well as other binding studies that would be crucial for both selecting patients for therapy and addressing questions of molecular mechanisms regarding the interaction of MIS in tissues with various receptors. In addition, the labeled ligand would be essential to determine whether other receptors or other binding proteins are present in various tissues. Here, we demonstrate the production of an internally epitope-tagged MIS that retains full biological activity in a Müllerian regression assay. In one embodiment, the tag is a "FLAG" tag due to the availability of high quality reagents used for its detection and purification.

[0016] Here we demonstrate that replacement of the MIS leader sequence with that of human serum albumin (HSA), combined with alteration 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 engineered with a more efficient cleavage site at the carboxy terminus of the N-terminal domain, thereby eliminating the need for exogenous cleavage. This recombinant MIS protein can be used both as a therapeutic and as a probe molecule without an identification tag.

[0018] Importantly, alteration of the endogenous leader sequence to another leader sequence, such as the human serum albumin (HSA) leader sequence, increases production of the MIS protein. Surprisingly, the inventors demonstrate that the combination of a leader sequence with an altered cleavage site increases cleavage from the first cleavage site from 37% to more than 80%. This was unexpected since increased protein production typically saturates available or endogenous cleavage enzymes, and thus increased protein yield is accompanied by decreased post-translational processing, including cleavage.

[0019] Thus, the present invention relates to methods of using recombinant human MIS protein (e.g., a polypeptide or a nucleic acid encoding a recombinant human MIS protein), or a functional fragment, or a derivative or variant thereof, to treat cancer, e.g., cancers expressing MIS receptor II (MISRII).

[0020] Thus, one aspect of the invention relates to a recombinant Müllerian inhibitory substance (MIS) protein comprising a combination of a non-MIS leader sequence or functional fragment thereof replacing the MIS leader sequence of amino acids 1-25 of SEQ ID NO:1 and at least one amino acid modification between residues 448-452 of SEQ ID NO:1 to increase cleavage compared to the absence of modification. The recombinant MIS protein has increased cleavage in vitro and increased production yield compared to the 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 functional fragment thereof replacing the MIS leader sequence of amino acids 1-25 of SEQ ID NO:1, wherein 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 leader sequence is an albumin leader sequence or a functional fragment thereof, such as a human serum albumin (HSA) leader sequence or a functional fragment thereof. In certain embodiments, the HSA leader sequence comprises the amino acid sequence of SEQ ID NO:6 or a variant at least 80% homologous thereto, or a functional fragment thereof, such as an HSA sequence comprising at least 10 amino acids, or at least about 11 amino acids, or at least 15 amino acids of SEQ ID NO:6 or a variant at least 80% homologous thereto. In certain embodiments, a fragment of the HSA leader sequence is TIFF2024178382000002.tif19164.

[0022] In certain embodiments, the non-MIS leader sequence is an immunoglobulin signal peptide fused to a tissue-type plasminogen activator propeptide (IgSP-tPA), a mouse immunoglobulin signal peptide (IgSP), an MPIF-1 signal sequence, TIFF2024178382000003.tif5128, stanniocalcin signal sequence TIFF2024178382000004.tif5128, invertase signal sequence TIFF2024178382000005.tif5128, Yeast mating factor alpha signal sequence (K. lactis killer toxin leader sequence), hybrid signal sequence TIFF2024178382000006.tif5128, HSA / MFα-1 hybrid signal sequence TIFF2024178382000007.tif5128, K. lactis killer / MFα-1 fusion leader sequence TIFF2024178382000008.tif5128, immunoglobulin Ig signal sequence TIFF2024178382000009.tif5128, fibulin B precursor signal sequence TIFF2024178382000010.tif5128, clusterin precursor signal sequence TIFF2024178382000011.tif5128, and insulin-like growth factor binding protein 4 signal sequence TIFF2024178382000012.tif5128, or a functional fragment thereof.

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

[0024] In certain embodiments, the recombinant MIS proteins disclosed herein include a FLAG tag, e.g., the amino acid TIFF2024178382000013.tif4128 or a functional derivative or variant thereof. In certain embodiments, the tag, e.g., a 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 N-terminal to the C-terminal domain of MIS. In certain embodiments, the tag is 50 amino acids or less, e.g., about 50, about 40, about 30, about 20, or about 10 amino acids or less in length, or about 7 amino acids in length.

[0025] In certain embodiments, the recombinant MIS protein disclosed herein comprises the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3, or a functional fragment thereof, which can 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 pharma- ceutically acceptable carrier.

[0027] Another aspect of the present invention relates to a polynucleotide encoding a recombinant MIS protein as 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 of SEQ ID NO:4 or SEQ ID NO:5, respectively. Another aspect of the technology 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 of SEQ ID NO:4 or SEQ ID NO:5, respectively. In certain embodiments, the vector is a viral vector or an expression vector, such as pcDNA3.1, or an alternative vector for E. coli or bacteriophage. In certain embodiments, the viral vector is selected from the group consisting of an adenoviral vector, a poxviral vector, and a lentiviral vector. In certain embodiments, the viral vector is an adeno-associated virus (AAV), such as 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 operably linked to a tissue or cell type specific promoter. In certain embodiments, host cells comprising such vectors are also included in the present invention.

[0029] In certain embodiments, vectors containing the polynucleotides discussed herein are capable of expressing recombinant MIS protein at constant levels for a required period of time.

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

[0031] Another aspect of the technology discussed herein relates to a pharmaceutical composition comprising the vector discussed herein and a pharma- ceutically acceptable carrier.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 relates to a method for treating a subject having cancer comprising administering a composition comprising a recombinant MIS protein, wherein the recombinant MIS protein comprises at least one amino acid modification between residues 448-452 of SEQ ID NO:1, with or without an internal tag protein, to increase cleavage relative to the absence of the modification. In certain embodiments, the recombinant MIS protein has increased cleavage and increased production yield in vitro relative to the wild-type MIS protein corresponding to the amino acid residues of SEQ ID NO:1.

[0033] In certain embodiments, the recombinant human MIS protein disclosed herein (e.g., a polypeptide and / or a nucleic acid encoding a recombinant human MIS protein), or a functional fragment or derivative or variant thereof, can be used to treat cancer. In certain embodiments, the recombinant human MIS protein that can be used to treat cancer comprises amino acid residues 25-559 of SEQ ID NO:2, or a functional fragment thereof. In certain embodiments, the recombinant human MIS protein that can be used to treat cancer comprises amino acid residues 25-567 of SEQ ID NO:3, or a functional fragment thereof. In certain embodiments, the cancer is a MIS Responsive II (MISRII) cancer, or where the cancer expresses MISRII, e.g., ovarian cancer, or includes ovarian cancer cells, vulvar epithelial cancer cells, cervical cancer cells, endometrial adenocarcinoma cells, and ovarian adenocarcinoma. In certain embodiments, the cancer includes, but is not limited to, breast cancer, lung cancer, head and neck cancer, bladder cancer, gastric cancer, cancer of the nervous system, osteosarcoma, bone marrow cancer, brain cancer, colon cancer, esophageal cancer, endometrial cancer, gastrointestinal cancer, gum cancer, renal 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, administration of the recombinant MIS protein occurs before, during, or after administration of an additional agent or cancer treatment.

[0035] In certain embodiments, Müllerian inhibitory substance (MIS) receptor is measured in a biological sample obtained from the subject, such as a cancer or tumor tissue sample or cancer or tumor cells, such as a biopsy tissue sample.

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

[0037] In certain embodiments, the recombinant MIS protein can be administered via any route, for example, intravenously, intradermally, intramuscularly, intraarterially, intralesionally, intradermally, or subcutaneously, or by aerosol administration. In certain embodiments, the administration is therapeutic or prophylactic. In all aspects disclosed herein, the subject is a mammal, for example a human.

[0038] In certain embodiments, at least one additional 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, the chemotherapeutic agent is paclitaxel, cisplatin, doxorubicin, rapamycin, pyrazoloanthrone, for example, but not limited to, anthra(1,9-cd)pyrazol-6(2H)-one (SP600125), or N1-methyl-1,9-pyrazoloanthrone (M-SP600125), or functional derivatives or functional analogues 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 dosage of a chemotherapeutic agent for the treatment of cancer, comprising administering to a subject a therapeutically effective amount of a recombinant MIS protein, wherein the recombinant MIS protein comprises a Q to R modification at amino acid 450 of SEQ ID NO:1, and the therapeutically effective amount of the chemotherapeutic agent in the presence of the recombinant MIS protein is lower than the therapeutically effective amount of the chemotherapeutic agent alone.In certain embodiments, the recombinant MIS protein optionally comprises a tag protein.

[0040] Another aspect of the technology disclosed herein relates to the use of a recombinant MIS protein for the manufacture of a medicament for treating a cancer, the recombinant MIS protein comprising a Q to R modification at amino acid 450 of SEQ ID NO:1, wherein the cancer expresses the Mullerian inhibitory substance (MIS) receptor.

[0041] Another aspect of the present invention relates to an article of manufacture comprising packaging material and a pharmaceutical composition comprising a recombinant MIS protein as disclosed herein, the packaging material comprising a label indicating that the pharmaceutical composition can be administered in an effective dosage and for a sufficient period of time to treat or reduce the risk of cancer that expresses the Müllerian inhibitory substance (MIS) receptor.

[0042] Another aspect of the technology disclosed herein relates to a method of treating a subject suffering from cancer, comprising assessing expression and / or activity of Mullerian inhibitory substance receptor II (MISRII) in a biological sample obtained from the subject. A clinician considers whether the results indicate the presence of MISRII expression and / or activity, and the clinician prescribes treatment of the subject with a pharmaceutical composition comprising a recombinant MIS protein disclosed herein.

[0043] Another aspect of the technology disclosed herein relates to the use of a recombinant MIS protein to reduce plasma serum levels of one or more androgens in a subject in need thereof, the recombinant MIS protein comprising a Q to R modification at amino acid 450 of SEQ ID NO:1, and optionally comprising a tag, e.g., 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 a functional fragment thereof.

[0044] In certain embodiments, the one or more androgens are testosterone.In certain embodiments, the subject in need has benign prostatic hyperplasia or prostate cancer, or polycystic ovarian disease, and / or precocious puberty.In another embodiment, the subject in need has a disease or disorder selected from the group including, but not limited to, benign prostatic hyperplasia (BPH), prostate cancer, testicular cancer, androgen-dependent acne, male pattern baldness, precocious puberty, hyperandrogenemia, hirsutism, virilization, polycystic ovarian syndrome (POCS), hyperandrogenemia (HA), and insulin resistance (IR), and acanthosis nigricans (AN) (HIAR-AN) syndrome, ovarian theca cell hyperplasia, follicular maturation arrest, atresia, anovulation, dysmenorrhea, dysfunctional uterine bleeding, infertility, and androgen-producing tumors.

[0045] Another aspect of the technology disclosed herein relates to a method for treating a disease or disorder characterized by androgen dependency, comprising administering to a subject an effective amount of a pharmaceutical composition comprising a recombinant MIS protein as disclosed herein, or a preparation of MIS protein from cleavage of the recombinant MIS protein as disclosed herein, which pharmaceutical composition reduces the level of at least one androgen in the plasma serum of the subject, causing a reduction in at least one symptom of the disease or disorder characterized by androgen dependency.

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

[0047] In certain embodiments, the subject has a disease or disorder characterized by androgen dependence, such as, but not limited to, a disease or disorder selected from the group including benign prostatic hyperplasia (BPH), prostate cancer, testicular cancer, androgen-dependent acne, male pattern baldness, precocious puberty, hyperandrogenemia, hirsutism, virilization, polycystic ovary syndrome (POCS), hyperandrogenism (HA), and insulin resistance (IR), as well as acanthosis nigricans (AN) (HIAR-AN) syndrome, ovarian theca hyperplasia, follicular maturation arrest, 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 MIS protein produced by post-translational processing of the recombinant MIS protein disclosed herein, and a pharma- ceutically acceptable carrier. In certain embodiments, the kit may optionally include instructions for using the recombinant MIS protein for the treatment of cancer or androgen-dependent disease. [The present invention 1001] A recombinant Mullerian inhibitory substance (MIS) protein comprising a combination of a non-MIS leader sequence or a functional fragment thereof in place of the MIS leader sequence of amino acids 1 to 25 of SEQ ID NO:1 and at least one amino acid modification between residues 447 to 451 of SEQ ID NO:1 to increase cleavage compared to the absence of the modification, wherein the recombinant MIS protein has increased cleavage and increased production yield in vitro compared to the wild-type MIS protein corresponding to the amino acid residues of SEQ ID NO:1. [The present invention 1002] The recombinant MIS protein of the present invention, wherein said recombinant MIS protein further comprises a tag protein. [The present invention 1003] 1001. A recombinant MIS protein according to the present invention, comprising at least a non-MIS leader sequence or a functional fragment thereof in place of the MIS leader sequence of amino acids 1 to 25 of SEQ ID NO:1, and at least one amino acid modification between residues 447 to 451 of SEQ ID NO:1, to increase cleavage compared to the unmodified form. [The present invention 1004] The recombinant MIS protein of the present invention, wherein said non-MIS leader sequence is an albumin leader sequence or a functional fragment thereof. [The present invention 1005] The recombinant MIS protein of the present invention, wherein said albumin leader sequence is a human serum albumin (HSA) leader sequence or a functional fragment thereof. [The present invention 1006] 1005. The recombinant MIS protein of the present invention, wherein said HSA leader sequence comprises the amino acid sequence of SEQ ID NO:6 or a variant thereof which is at least 80% homologous thereto. [The present invention 1007] 1005. The recombinant MIS protein of the present invention, wherein said fragment of the HSA leader sequence comprises at least 10 amino acids of SEQ ID NO:6 or a variant thereof that is at least 80% homologous thereto. [The present invention 1008] 1005. The recombinant MIS protein of the present invention, wherein said HSA leader sequence comprises at least 15 amino acids of SEQ ID NO:6 or a variant thereof that is at least 80% homologous thereto. [The present invention 1009] 1005. The recombinant MIS protein of the present invention, wherein said HSA leader sequence comprises at least 11 amino acids of SEQ ID NO:6 or a variant thereof that is at least 80% homologous thereto. [The present invention 1010] The fragment of the HSA leader sequence is The recombinant MIS protein of the present invention 1005 is selected from the group consisting of TIFF2024178382000014.tif19149. [The present invention 1011] The non-MIS leader sequence is an immunoglobulin signal peptide fused to a tissue-type plasminogen activator propeptide (IgSP-tPA), a murine immunoglobulin signal peptide (IgSP), or an MPIF-1 signal sequence. TIFF2024178382000015.tif5128, stanniocalcin signal sequence TIFF2024178382000016.tif5128, invertase signal sequence TIFF2024178382000017.tif5128, Yeast mating factor alpha signal sequence (K. lactis killer toxin leader sequence), hybrid signal sequence TIFF2024178382000018.tif5128, HSA / MFα-1 hybrid signal sequence TIFF2024178382000019.tif5128, K. lactis killer / MFα-1 fusion leader sequence TIFF2024178382000020.tif5128, immunoglobulin Ig signal sequence TIFF2024178382000021.tif5128, fibrin precursor B signal sequence TIFF2024178382000022.tif5128, clusterin precursor signal sequence TIFF2024178382000023.tif5128, and insulin-like growth factor binding protein 4 signal sequence TIFF2024178382000024.tif5128, or a fragment thereof. [The present invention 1012] 1001. A recombinant MIS protein of the present invention comprising a modification of amino acid 449 of SEQ ID NO:1 from Q to R to increase cleavage compared to without said modification. [The present invention 1013] 1001. The recombinant MIS protein of the present invention, further comprising an modification of amino acid 451 of SEQ ID NO:1 from S to R to increase cleavage relative to the absence of said modification. [The present invention 1014] The recombinant MIS protein of the present invention, wherein said tag is a FLAG tag. [The present invention 1015] The FLAG tag has the amino acid sequence TIFF2024178382000025.tif5128, or a functional fragment or variant thereof. [The present invention 1016] The recombinant MIS protein of the present invention 1014, wherein said 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. [The present invention 1017] The recombinant MIS protein of the present invention, wherein said FLAG tag is located between amino acid residues 451-452 of SEQ ID NO:1. [The present invention 1018] 1001. A recombinant MIS protein of the present invention, comprising the amino acid sequence of SEQ ID NO:2 or a functional fragment thereof. [The present invention 1019] 1001. A recombinant MIS protein of the present invention, comprising the amino acid sequence of SEQ ID NO:3 or a functional fragment thereof. [The present invention 1020] A recombinant MIS protein of the present invention, encoded by the nucleic acid sequence of SEQ ID NO:4. [The present invention 1021] A recombinant MIS protein of the present invention, encoded by the nucleic acid sequence of SEQ ID NO:5. [The present invention 1022] A pharmaceutical composition comprising any one of the recombinant MIS proteins of the present inventions 1001 to 1021 and a pharma- ceutically acceptable carrier. [The present invention 1023] A polynucleotide encoding any one of the recombinant MIS proteins of the present inventions 1001 to 1021. [The present invention 1024] The polynucleotide of the present invention, wherein said nucleotides correspond to SEQ ID NO:4 or a nucleotide having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:4. [The present invention 1025] The polynucleotide of the present invention, wherein the nucleotides correspond to SEQ ID NO:5 or a nucleotide having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:5. [The present invention 1026] A vector comprising any one of the polynucleotides of the present invention 1023 to 1025. [The present invention 1027] The vector of claim 1026, wherein the vector is a viral vector or an expression vector. [The present invention 1028] The vector of the present invention 1027, wherein said expression vector is pcDNA3.1 or a cDNA or genomic vector for a bacterium (eg, E. coli) or a bacteriophage. [The present invention 1029] The vector of the present invention, wherein the viral vector is selected from the group consisting of an adenoviral vector, a poxvirus vector, and a lentivirus vector. [The present invention 1030] The vector of any of claims 1026 to 1029, wherein the nucleic acid sequence encodes a recombinant MIS protein or a functional fragment thereof having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:4 or SEQ ID NO:5, and the nucleic acid sequence is operably linked to a tissue or cell type specific promoter. [The present invention 1031] A human MIS protein produced by post-translational processing of the recombinant human MIS protein of the present invention 1001. [The present invention 1032] A host cell comprising any one of the vectors of the present invention 1026 to 1030. [The present invention 1033] A pharmaceutical composition comprising any one of the vectors of the present inventions 1026 to 1030 and a pharma- ceutically acceptable carrier. [The present invention 1034] A purified preparation of human MIS protein produced from a recombinant human MIS protein of any of the present inventions 1001 to 1021. [The present invention 1035] 1. A method for treating a subject having cancer, the method comprising administering a composition comprising a recombinant MIS protein, wherein the recombinant MIS protein comprises a combination of a non-MIS leader sequence or a functional fragment thereof in place of the MIS leader sequence of amino acids 1-25 of SEQ ID NO:1 and at least one amino acid modification between residues 447-451 of SEQ ID NO:1 to increase cleavage compared to the absence of the modification, and 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. [The present invention 1036] The method of claim 1035, wherein the recombinant MIS protein further comprises a tag protein. [The present invention 1037] The method of claim 1035, wherein the recombinant MIS protein comprises at least a non-MIS leader sequence or a functional fragment thereof in place of the MIS leader sequence of amino acids 1 to 25 of SEQ ID NO:1, and at least one amino acid modification between residues 447 to 451 of SEQ ID NO:1 to increase cleavage compared to the absence of the modification. [The present invention 1038] The method of claim 1035, wherein said non-MIS leader sequence is an albumin leader sequence or a functional fragment thereof. [The present invention 1039] The method of claim 1038, wherein said albumin leader sequence is a human serum albumin (HSA) leader sequence or a functional fragment thereof. [The present invention 1040] 1035. The method of claim 1035, wherein said recombinant MIS protein comprises a modification of amino acid 449 of SEQ ID NO:1 from Q to R to increase cleavage compared to the absence of said modification. [The present invention 1041] The method of claim 1035, wherein said tag is a FLAG tag comprising the amino acid sequence of SEQ ID NO:8 or a functional fragment thereof. [The present invention 1042] The method of claim 1035, wherein said cancer is a MIS-responsive II cancer. [The present invention 1043] The method of claim 1035, wherein the cancer is ovarian cancer. [The present invention 1044] The method of claim 1035, wherein said cancer is a chemotherapy-resistant or multidrug-resistant cancer. [The present invention 1045] The method of claim 1035, wherein the administration of said recombinant MIS protein occurs before, during or after the administration of an additional agent or cancer treatment. [The present invention 1046] The method of claim 1035, wherein said cancer expresses Mullerian inhibitory substance receptor II (MISRII). [The present invention 1047] The method of claim 1046, wherein expression of said Mullerian inhibiting substance (MIS) receptor is measured in a biological sample obtained from said subject. [The present invention 1048] The method of claim 1047, wherein said biological sample is a cancer or tumor tissue sample or a cancer or tumor cell. [The present invention 1049] The method of claim 1047, wherein said biological sample is a biopsy tissue sample. [The present invention 1050] The method of claim 1035, wherein said cancer is an ovarian cancer cell, a vulvar epithelial cancer cell, a cervical cancer cell, an endometrial adenocarcinoma cell, or an ovarian adenocarcinoma cell. [The present 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, cancer of the nervous system, osteosarcoma, bone marrow cancer, brain cancer, colon cancer, esophageal cancer, endometrial cancer, gastrointestinal cancer, gingival cancer, renal cancer, liver cancer, nasopharyngeal cancer, ovarian cancer, prostate cancer, skin cancer, stomach cancer, testicular cancer, tongue cancer, melanoma, ocular melanoma, or uterine cancer. [The present invention 1052] The method of claim 1044, wherein said cancer is a paclitaxel or doxorubicin resistant cancer. [The present invention 1053] The method of claim 1035, wherein said administration is intravenous, intradermal, intramuscular, intraarterial, intralesional, transdermal, or subcutaneous, or by aerosol administration. [The present invention 1054] The method of claim 1035, wherein said administering is prophylactic administering. [The present invention 1055] The method of claim 1035, wherein said administering is therapeutic administering. [The present invention 1056] The method of claim 1035, wherein the subject is a mammal. [The present invention 1057] The method of claim 1056, wherein the subject is a human. [The present invention 1058] The method of claim 1035, wherein at least one additional agent is administered to the subject in combination with (eg, before, during, or after) the administration of said recombinant human MIS. [The present invention 1059] The method of claim 1058, wherein said additional agent is a therapeutic or chemotherapeutic agent. [The present invention 1060] The method of claim 1059, wherein said chemotherapeutic agent is selected from the group consisting of paclitaxel, cisplatin, doxorubicin, rapamycin, and pyrazoloanthrone. [The present invention 1061] The method of claim 1059, wherein said chemotherapeutic agent is a radiotherapeutic agent. [The present invention 1062] The method of claim 1059, wherein said chemotherapeutic agent is a pyrazoloanthrone. [The present invention 1063] The method of claim 1062, wherein said pyrazoloanthrone is anthra(1,9-cd)pyrazol-6(2H)-one (SP600125) or a functional derivative or analog thereof. [The present invention 1064] 1. A method for reducing the dosage of a chemotherapeutic agent for the treatment of cancer, the method comprising administering to a subject a therapeutically effective amount of a recombinant MIS protein comprising a combination of a Q to R modification at amino acid 449 of SEQ ID NO:1 and a non-MIS leader sequence, or a functional fragment thereof, in place of the MIS leader sequence at amino acids 1-25 of SEQ ID NO:1, and wherein a therapeutically effective amount of the chemotherapeutic agent in the presence of the recombinant MIS protein is less than a therapeutically effective amount of the chemotherapeutic agent alone. [The present invention 1065] The method of claim 1064, wherein said recombinant MIS protein further comprises a tag protein. [The present invention 1066] 1. Use of a recombinant MIS protein for the manufacture of a medicament for treating cancer, wherein the recombinant MIS protein comprises a combination of a Q to R modification at amino acid 449 of SEQ ID NO:1 and a non-MIS leader sequence or a functional fragment thereof in place of the MIS leader sequence at amino acids 1 to 25 of SEQ ID NO:1, and wherein the cancer expresses a Mullerian inhibitory substance (MIS) receptor. [The present invention 1067] The use of the present invention 1066, wherein said recombinant MIS protein further comprises a tag protein. [The present invention 1068] The use of the present invention 1066, wherein said Mullerian inhibiting substance (MIS) receptor is a MIS type II receptor or a homologue or functional fragment thereof. [The present invention 1069] An article of manufacture comprising packaging material and a pharmaceutical composition comprising any one of the recombinant MIS proteins of the present inventions 1001 to 1021, the packaging material comprising a label indicating that the pharmaceutical composition can be administered in a sufficient dosage and for a sufficient period of time to treat or reduce the risk of a cancer expressing the Mullerian inhibitory substance (MIS) receptor. [The present invention 1070] A method for treating a subject suffering from cancer, the method comprising a step of assessing expression and / or activity of Mullerian inhibitory substance receptor II (MISRII) in a biological sample obtained from the subject, wherein a clinician examines the results and, if the results indicate the presence of MISRII expression and / or activity, the clinician prescribes treatment to the subject with a pharmaceutical composition of the present invention 1022 or 1033. [The present invention 1071] The method of claim 1070, wherein said biological sample is a tissue sample. [The present invention 1072] The method of claim 1071, wherein said tissue sample is a cancer or tumor tissue sample or a cancer or tumor cell. [The present invention 1073] The method of claim 1071, wherein said biological sample is a biopsy tissue sample. [The present invention 1074] The method of claim 1070, wherein said cancer is an ovarian cancer cell, a vulvar epithelial cancer cell, a cervical cancer cell, an endometrial adenocarcinoma cell, or an ovarian adenocarcinoma cell. [The present invention 1075] The method of claim 1070, wherein the cancer is breast cancer, lung cancer, head and neck cancer, bladder cancer, stomach cancer, cancer of the nervous system, osteosarcoma, bone marrow cancer, brain cancer, 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. [The present invention 1076] 1. Use of a recombinant MIS protein for reducing plasma serum levels of one or more androgens in a subject in need thereof, the recombinant MIS protein comprising a combination of a Q to R modification at amino acid 449 of SEQ ID NO:1 and a non-MIS leader sequence or a functional fragment thereof in place of the MIS leader sequence at amino acids 1 to 25 of SEQ ID NO:1. [The present invention 1077] The use of the present invention 1076, wherein said recombinant MIS protein further comprises a tag protein. [The present invention 1078] The use of the present invention 1076, wherein the one or more androgens is testosterone. [The present invention 1079] The use of invention 1076, wherein said subject in need thereof has benign prostatic hyperplasia. [The present invention 1080] The use of invention 1076, wherein said subject in need thereof has prostate cancer. [The present invention 1081] The use of invention 1076, wherein said subject in need thereof has polycystic ovary disease, and / or precocious puberty. [The present invention 1082] The use of the present invention 1076, wherein the subject in need thereof 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, virilization, polycystic ovary syndrome (POCS), hyperandrogenism (HA), and insulin resistance (IR), and acanthosis nigricans (AN) (HIAR-AN) syndrome, ovarian theca cell hyperplasia, follicular maturation arrest, atresia, anovulation, dysmenorrhea, dysfunctional uterine bleeding, infertility, and androgen-producing tumors. [The present invention 1083] A method for treating a disease or disorder characterized by androgen dependence, the method comprising administering to a subject an effective amount of a pharmaceutical composition of the present invention 1022 or 1033, wherein the pharmaceutical composition reduces the level of at least one androgen in the plasma serum of the subject, causing a reduction in at least one symptom of the disease or disorder characterized by androgen dependence. [The present invention 1084] 1. A method for reducing plasma levels of one or more androgens in a subject, the method comprising administering an effective amount of a recombinant MIS protein, wherein the recombinant MIS protein comprises a combination of a Q to R modification at amino acid 449 of SEQ ID NO:1 and a non-MIS leader sequence or a functional fragment thereof in place of the MIS leader sequence at amino acids 1-25 of SEQ ID NO:1, wherein the recombinant MIS protein reduces plasma serum levels of one or more androgens in the subject. [The present invention 1085] The method of claim 1084, wherein the recombinant MIS protein further comprises a tag protein. [The present invention 1086] The method of claim 1084, wherein the subject has a disease or disorder characterized by androgen dependence. [The present invention 1087] Any of the methods of the present invention 1084 to 1086, 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, virilization, polycystic ovary syndrome (POCS), hyperandrogenism (HA), and insulin resistance (IR), and acanthosis nigricans (AN) (HIAR-AN) syndrome, ovarian theca cell hyperplasia, follicular maturation arrest, atresia, anovulation, dysmenorrhea, dysfunctional uterine bleeding, infertility, and androgen-producing tumors. [The present invention 1088] A kit comprising any one of the recombinant MIS proteins of the present inventions 1001 to 1021 and a pharma- ceutically acceptable carrier. [The present invention 1089] The kit of the present invention 1088, optionally further comprising instructions for use of the recombinant MIS protein for the treatment of cancer or the treatment of an androgen-dependent disease. [Brief description of the drawings]

[0049] [Figure 1A]1A-1B are schematic diagrams showing the design of the new recombinant MIS construct with the albumin leader sequence. Figure 1A shows that the leader sequences of MIS (25 amino acids) and albumin (24 amino acids) share 20% identity and 5 conserved amino acids. [Figure 1B] 1A-1B are schematic diagrams showing the design of new recombinant MIS constructs with albumin leader sequences. Figure 1B is a schematic diagram showing the design of RF (improved cleavage site + Flag tag), LRF (leader sequence + improved cleavage site + Flag tag), and LR (leader sequence + improved cleavage site) constructs, including the arrangement of Flag tag (F), improved cleavage site (R), and albumin leader (L). [Diagram 2] MIS production and cleavage in CHOK1 clones stably transfected with recombinant human LR-MIS and LRF-MIS constructs. Western blot of 4% reducing SDS gel of medium supernatant after 72 hours in culture with anti-MIS goat polyclonal antibody targeting the c-terminus of MIS. Purified RF-MIS, CHO93 medium, and B9 medium as positive controls. [Figure 3A] Figures 3A-3B show purified recombinant MIS analyzed by Western blot of reducing SDS gels to assess the amount of cleavage. Figure 3A shows purified recombinant RF-MIS, LRF-MIS, and WT-MIS are compared using an antibody against the N-terminus that can recognize the holoMIS monomer, the cleaved N-terminus, and potential cleavage products including portions of the N-terminus. [Figure 3B] Figures 3A-3B show purified recombinant MIS analyzed by Western blot of reducing SDS gels to assess the amount of cleavage. Figure 3B shows detection of purified recombinant RF-MIS, LRF-MIS, and WT-MIS with an antibody against the C-terminus that can recognize potential cleavage products including holo-MIS monomer, the cleaved C-terminus, and parts of the C-terminus. [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 biopsies. Recombinant human MIS products were incubated with fetal rat urogenital ridges for 72 hours. Figure 4A shows representative sections from both treated and untreated contralateral control ridges 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 biopsies. 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 the corresponding amino acid residues using the conventional nomenclature of amino acid labeling (starting with the first numbered amino acid after the leader sequence). Figure 5A shows the amino acid sequence of the wild-type MIS protein of SEQ ID NO:1, highlighting the leader sequence (bold line) and the first and second cleavage sites. The corresponding amino acids numbered using the conventional numbering system are shown in brackets. [Figure 5B] Figures 5A-5B show the amino acid sequence of the wild type MIS protein (SEQ ID NO:1) with the corresponding amino acid residues using conventional nomenclature for amino acid labeling (starting with the first numbered amino acid after the leader sequence). Figure 5B shows a table showing the features on the amino acid residues on SEQ ID NO:1 that correspond to the amino acid residues using conventional nomenclature for MIS (starting with the first numbered amino acid after the leader sequence). Figure 5B discloses "RAQR / S" as SEQ ID NO:26. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0050] Detailed Description of the Invention The present invention relates to an improved recombinant human MIS protein that has at least one of the following features: improved cleavage, increased biological activity, increased potency, and can be produced in higher yields compared to wild-type human MIS protein, where the recombinant human MIS protein includes a combination of the following features: an improved Kex cleavage site for increased cleavage, and a non-MIS leader sequence in place of the normal MIS leader sequence, improving the yield of biologically active protein. In certain embodiments, the improved MIS is with or without an internal label or tag to facilitate its purification.

[0051] Thus, herein we have made changes to the native human sequence to increase the endogenous cleavage and thus the potency of MIS, and, optionally, we have also inserted a tag to facilitate its purification.

[0052] The inventors further improved the recombinant human MIS protein to include a non-MIS leader sequence in place of the 25 amino acid MIS leader sequence from amino acids 1-25 of SEQ ID NO:1. In certain embodiments, the leader sequence includes an albumin leader sequence, e.g., human serum albumin sequence (HSA), or a functional fragment or variant thereof. In certain embodiments, the leader sequence includes 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. This addition surprisingly further increased cleavage of the recombinant MIS protein. This combination led to higher yields of a more homogeneous product, with increased potency due to increased cleavage. This combination of changes creates a recombinant human MIS variant that can meet the previously unmet need to have a form of bioactive MIS labeled for use with receptors, as well as other binding studies that would be crucial for both patient selection for therapy and for addressing questions of molecular mechanisms regarding the interaction of MIS in tissues with various receptors. Furthermore, a labeled ligand would be essential to determine whether additional receptors or other binding proteins are present in various tissues. Herein, we demonstrate the production of an internally epitope-tagged MIS that retains full biological activity in the Mullerian duct regression assay. 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 the recombinant human MIS protein and its functional fragments and derivatives disclosed herein to a subject in need thereof. Conditions that can be treated by the compounds of the present invention or pharmaceutical compositions containing them include any condition that can be treated or that can be reduced by administering human MIS, or by activating MIS signaling or activating MISRII, thereby benefiting from the administration of recombinant human MIS protein and its functional fragments and derivatives. Representative conditions in this regard include, for example, but are not limited to, cancers that express MIS receptors, such as cancers that express MISRII, such as, but are not limited to, ovarian, cervical, and endometrial cancers. Another condition that can be treated with MIS or where activation of MIS signaling reduces symptoms is a proliferative disease, such as cancer, or an abnormally high androgen stage, such as polycystic ovarian disease, precocious puberty, and other hyperandrogen disorders, such as testotoxicosis, or any androgen-dependent tumor, such as prostate cancer.

[0054] definition For convenience, certain terms used throughout this application (including the specification, examples, and claims) are collected here. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0055] The terms "Mullerian inhibitor" and "MIS" are used interchangeably herein and are also known as anti-Mullerian hormone or AMH, and refer to compounds and materials structurally similar to MIS. "MIS" or "Mullerian inhibitor" refers to 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 is intended to include mutant forms of recombinant human MIS that have 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 included material are, for example, salts, functional derivatives, and aglycone forms of wild-type MIS and recombinant human MIS.Furthermore, human recombinant MIS protein can be obtained using recombinant DNA technology or from chemical synthesis of MIS protein.For reference purposes only, wild-type human MIS nucleic acid corresponds to RefSeq No: NM_000479, which is incorporated herein by reference.

[0056] The term "Mullerian inhibitory substance type II receptor" or "MISRII" is used interchangeably herein and refers to the type II receptor for MIS. The term "MISRII" is intended to encompass all MIS receptors that are substantially homologous to MISRII and functional derivatives of MISRII. MISRII is also known alternatively 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 a naturally occurring polynucleotide sequence encoding a protein, or a portion thereof, or a protein sequence, or a portion thereof, respectively, as it normally exists in vivo. Thus, as disclosed herein, the wild type amino acid sequence for the preproprotein of human MIS corresponds to SEQ ID NO:1, of which amino acid residues 1-25 correspond to the leader sequence. The proprotein of MIS comprises amino acid residues 26-560 of SEQ ID NO:1 (e.g., lacking the 1-25 leader sequence), which are subsequently post-translationally processed by cleavage as disclosed herein to form the biologically active MIS homodimer.

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

[0059] A "polynucleotide encoding MIS" refers to a polynucleotide encoding a polypeptide having 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% sequence identity to any of the amino acid sequences corresponding to amino acid residues 26 to 560 of SEQ ID NO:1.

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

[0061] The term "agent" or "compound" as used herein refers to a chemical or biological product, or a combination of chemicals or biological products, administered to a subject to treat, prevent, or control a disease or condition. The chemical or biological product is preferably, but not necessarily, 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, peptidomimetics, receptors, ligands, and antibodies, aptamers, polypeptides, nucleic acid analogs, or variants thereof. For example, oligomers of nucleic acids, amino acids, or carbohydrates, including, but not limited to, proteins, oligonucleotides, ribozymes, DNAzymes, glycoproteins, siRNAs, lipoproteins, aptamers, and variants and combinations thereof.

[0062] The term "nucleic acid" is known in the art. As used herein, "nucleic acid" generally refers to a molecule (i.e., a strand) of DNA, RNA, or a derivative or analog thereof, that contains a nucleobase. Nucleic acid bases include, for example, naturally 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", each as a subgenus of the term "nucleic acid". The term "oligonucleotide" refers to a molecule of about 3 to about 100 nucleobases in length. The term "polynucleotide" refers to at least one molecule that is greater than about 100 nucleobases in length. The term "nucleic acid" also refers to polynucleotides, such as deoxyribonucleotides (DNA), and, where appropriate, ribonucleotides (RNA). The term should be understood to include, as equivalents, either RNA or DNA analogs (analogs) made from nucleotide analogs, and, as applicable to the described embodiments, single-stranded (sense or antisense) and double-stranded polynucleotides. The terms "polynucleotide sequence" and "nucleotide sequence" are also used interchangeably herein.

[0063] As used herein, the term "gene" refers to a nucleic acid that includes an open reading frame that encodes a polypeptide, including both exon and (optionally) intron sequences. "Gene" refers to the coding sequence of a gene product, as well as the non-coding regions of the 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 will also encompass additional strands that are partially, substantially, or completely complementary to the single-stranded molecule. Thus, a nucleic acid may encompass a double-stranded molecule that includes one or more complementary strands or "complements" of a particular sequence that includes the molecule. As used herein, a single-stranded nucleic acid may be denoted by the prefix "ss," a double-stranded nucleic acid by the prefix "ds," and a triple-stranded nucleic acid by the prefix "is." The term "gene" refers to a segment of DNA involved in the production of a polypeptide chain, including the regions before and after the coding region, as well as the intervening sequences (introns) between the 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, such as RNA polymerase and other transcription factors, to which regulatory proteins and molecules may bind to initiate specific transcription of a nucleic acid sequence. The term "enhancer" refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence. Enhancers may function in either direction and may be upstream or downstream of a promoter.

[0064] As used herein, the term "gene product" is used to include RNA transcribed from a gene (eg, mRNA) or a polypeptide encoded by a gene or translated from RNA.

[0065] The terms "polypeptide" and "protein" are used interchangeably and refer to a polymer of amino acid residues and are not limited to a minimum length. Peptides, oligopeptides, dimers, and multimers, etc., composed of a linear sequence of amino acids linked by peptide bonds, whether produced biologically, recombinantly, or synthetically, and whether composed of naturally occurring or non-naturally occurring amino acids, are also included in the definition. Both full-length proteins and fragments thereof are included in the definition. The term also includes co-translational (e.g., cleavage of the leader sequence at amino acids 1-25 of SEQ ID NO:1) and post-translational modifications of the polypeptide, such as disulfide bond formation, glycosylation, acetylation, phosphorylation, proteolytic cleavage (e.g., cleavage by furin or metalloproteases and prohormone convertases (PC)), etc. Additionally, for purposes of the present invention, "polypeptide" encompasses proteins containing modifications to the native sequence, such as deletions, additions, and substitutions (generally conservative in nature, as known to those of skill in the art), so long as the protein maintains the required activity. These modifications may be deliberate, for example by site-directed mutagenesis, or may be accidental, for example by mutation of the host that produces the protein, or through errors in PCR amplification or other recombinant DNA methods. Polypeptides or proteins are composed of a linear sequence of amino acids linked by peptide bonds, but in contrast to peptides, have a well-defined conformation. Proteins, unlike polypeptides, generally consist of a chain of 50 or more amino acids. For the purposes of the present invention, the term "peptide" as used herein refers to a sequence of amino acids typically composed 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 acids in length.

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

[0067] In yet further embodiments, the recombinant human MIS protein or a fragment or derivative thereof may be a retro-inverso peptide. A retro-inverso peptide refers to a peptide with a reversed orientation of the peptide bonds at at least one position, i.e., a reversed amino- and carboxy-terminus relative to the amino acid side chains. Thus, a retro-inverso analog has opposite termini and opposite orientation of peptide bonds while maintaining the same side chain topology as the native peptide sequence. A retro-inverso peptide may contain L- or D-amino acids, or a mixture of L- 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 replaced with the opposite, enantiomeric amino acid residue. The retro-inversed portion of such an analog has opposite amino and carboxy termini, so that the amino acid residues adjacent to the retro-inversed portion are replaced with side chain-analogous α-substituted geminal-diaminomethane and malonate, respectively. The retro-inverso forms of cell-penetrating peptides have been found to function as efficiently as the native forms in translocating across membranes. The synthesis of retro-inverso peptide 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-inverso peptide analogs have been described (EP 97994-B), which are also incorporated herein by reference in their entirety.

[0068] The term "fragment" of a peptide, polypeptide, or molecule as used herein refers to any contiguous polypeptide subset of that molecule. The term "protein fragment" as used herein includes both synthetic and naturally occurring amino acid sequences derivable 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 recombinant human MIS protein" if it can be obtained by fragmenting recombinant human MIS protein or if it can be synthesized based on the knowledge of the naturally occurring amino acid sequence or the genetic material (DNA or RNA) that codes for this sequence. Thus, a "fragment" of a molecule is meant to refer to any polypeptide subset of that molecule. In certain embodiments, a functional fragment of recombinant human MIS includes at least the C-terminal domain and at least the N-terminal domain. In certain embodiments, a functional fragment includes a portion of the C-terminus and / or a portion of the N-terminal domain (e.g., a fragment) of recombinant human MIS protein. Fragments of recombinant human MIS protein that have greater activity than, or at least the activity of, the wild-type MIS protein of SEQ ID NO:1 disclosed herein and that are soluble are also encompassed 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 of SEQ ID NO:2 or 3 in vivo, for example to cause Müllerian duct regression in the Müllerian duct regression bioassay disclosed herein in the Examples. In other words, a functional fragment of recombinant human MIS protein is a fragment that can cause at least 30% of the same activity as SEQ ID NO:2 or 3, alone or as a fusion protein, to bind and activate MISRII or cause Müllerian duct regression in the Müllerian duct regression bioassay disclosed herein (Figure 4). The fragments disclosed herein can be soluble (i.e., not membrane bound). A "fragment" can 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 therebetween. Exemplary fragments include C-terminal truncations, N-terminal truncations, or both C- and N-terminal truncations (e.g., deletion 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). One skilled in the art can generate such fragments by simple deletion analysis. Such fragments of SEQ ID NO:2 or 3 can 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 N-terminus and / or C-terminus of SEQ ID NO:2 or 3, respectively.One skilled in the art can easily identify the smallest peptide fragment of SEQ ID NO:2 or 3 that is useful in the methods and compositions disclosed herein, or in the fusion proteins disclosed herein, by sequentially deleting N- and / or C-terminal amino acids from SEQ ID NO:2 or 3, or sequentially deleting N- and C-terminal amino acids from recombinant human MIS protein, and evaluating the function of the resulting peptide fragment, either alone or when truncated. One skilled in the art can form a functional fragment with multiple smaller fragments. These can be linked by crosslinking peptide linkers. One skilled in the art can readily select a linker to maintain the wild-type conformation. One skilled in the art can easily evaluate the function of recombinant human MIS protein as disclosed herein to activate MISRII, or in the Mullerian duct regression bioassay disclosed herein, compared to the recombinant human MIS protein corresponding to SEQ ID NO:2 or 3. Using such in vivo assays, if a fragment of a recombinant human MIS protein has at least 30% of the biological activity of a 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, a fragment of SEQ ID NO:2 or 3 can be less than 200, or less than 150, or less than 100, or less than 50, or less than 20 amino acids of SEQ ID NO:2 or 3. In certain embodiments, a fragment of SEQ ID NO:2 or 3 is less than 100 peptides in length. However, as noted above, a fragment should 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 therebetween.

[0070] The term "derivative" as used herein refers to a peptide that has been chemically modified, for example, but not limited to, by ubiquitination, labeling, pegylation (derivatization with polyethylene glycol), or the addition of other molecules. A molecule is also a derivative of another molecule when it contains additional chemical moieties that are not normally part of the molecule. Such moieties can improve the solubility, absorption, biological half-life, etc. of the molecule. The moieties can alternatively reduce the toxicity of the molecule, eliminate or reduce unwanted side effects of the molecule, etc. Moieties that can mediate such effects are disclosed in Remington's Pharmaceutical Sciences, 18th edition, AR Gennaro, Ed., MackPubl., Easton, PA (1990).

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

[0072] The terms "functional derivative" and "mimetic" or "biologically active variant" or "biologically active fragment" are used interchangeably and refer to a compound that has a biological activity (either functional or structural) that is substantially similar to the biological activity of a substance or molecule (e.g., recombinant human MIS protein) that is its functional derivative. The term functional derivative is intended to include fragments, variants, analogs, or chemical derivatives of a molecule.

[0073] The term "functional derivative" is intended to include "fragments," "variants," "analogs," or "chemical derivatives" of a molecule. A molecule is said to be "substantially similar" to another molecule if both molecules have a substantially similar structure or if both molecules have a similar biological activity. Thus, if two molecules have similar activity, they are considered to be variants, since the term is used herein even if the structure of one of the molecules is not found in the other, or even if the sequence of amino acid residues is not identical. An "analog" of a recombinant human MIS protein is meant to refer to a molecule that is substantially similar in function to the entire molecule or to a fragment thereof. As used herein, a molecule is said to be a "chemical derivative" of another molecule when it contains additional chemical moieties that are not normally part of the molecule. Such moieties can improve the solubility, absorption, biological half-life, etc. of the molecule. The moieties can alternatively reduce the toxicity of the molecule, eliminate or attenuate unwanted side effects of the molecule, etc. Ingredients capable of mediating such effects are disclosed in Remington's Pharmaceutical Sciences, 18th edition, AR Gennaro, Ed., MackPubl., Easton, PA (1990).

[0074] A "variant" of recombinant human MIS protein is meant to refer to a molecule that is substantially similar in structure and function to either the entire molecule or a fragment thereof. Thus, the term "variant" as described herein refers to a peptide or nucleic acid that differs from a naturally occurring polypeptide or nucleic acid by one or more amino acid or nucleic acid deletions, additions, substitutions, or side chain modifications, 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 with a different naturally occurring or non-conventional amino acid residue. Such substitutions can be classified as "conservative" when an amino acid residue contained in a polypeptide is replaced with another naturally occurring amino acid of similar properties in terms of either polarity, side chain functionality, or size. Substitutions encompassed by the present invention can also be "non-conservative" when an amino acid residue present in the peptide is replaced with an amino acid having different properties, for example, a naturally occurring amino acid from a different group (e.g., replacement of a charged or hydrophobic amino acid with alanine), or when a naturally occurring amino acid is replaced with a non-conventional amino acid. In certain embodiments, the amino acid substitutions are conservative. The term variant, when used in reference to a polynucleotide or polypeptide, includes polynucleotides or polypeptides that may be altered in primary, secondary, or tertiary structure compared to the referenced polynucleotide or polypeptide, respectively (e.g., compared to a wild-type polynucleotide or polypeptide). A "variant" of recombinant human MIS protein refers to a molecule that is substantially similar in structure and function, i.e., function is the ability to activate MISRII.

[0075] For example, variants of recombinant human MIS protein may contain mutations or modifications that differ from the recited amino acids in SEQ ID NO:2 or 3. In certain embodiments, variants of SEQ ID NO:2 or 3 are fragments of SEQ ID NO:2 or 3 disclosed herein. In certain embodiments, variants may be different isoforms of SEQ ID NO:2 or 3 and may contain different isomeric amino acids. Variants may be naturally occurring, synthetic, recombinant, or chemically modified polynucleotides or polypeptides isolated or produced using methods known in the art. Variants may contain conservative or non-conservative 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 recited sequence. Variants may contain insertions and substitutions of amino acids and other molecules that do not normally occur in the peptide sequence on which the variant is based, for example, amino acid insertions, deletions, or substitutions, including but not limited to the insertion of ornithine, which does not normally occur in human proteins.

[0076] The term "conservative substitution" when describing a polypeptide refers to a change in the amino acid composition of a polypeptide that does not substantially change the activity of the polypeptide. For example, conservative substitution refers to the replacement of an amino acid residue with a different amino acid residue that has similar chemical properties. Conservative amino acid substitutions include the replacement of leucine with isoleucine or valine, the replacement of aspartic acid with glutamic acid, or the replacement of threonine with serine. A "conservative amino acid substitution" is made by replacing one amino acid with another that has similar structural and / or chemical properties. For example, the replacement of leucine with isoleucine or valine, the replacement of aspartic acid with glutamic acid, or the replacement of threonine with serine. Thus, a "conservative substitution" of a particular amino acid sequence refers to the substitution of those amino acids that are not important for the polypeptide activity, or the substitution of an amino acid with another amino acid that has similar properties (e.g., acidic, basic, positive or negative charge, polar or non-polar, etc.) such that even the substitution of an important amino acid does not decrease the activity of the peptide (i.e., the ability of the peptide to reduce T-reg cells and / or reduce inflammatory cytokines as disclosed herein). Tables of conservative amino acids providing functionally similar amino acids are known in the art. For example, each of the following six groups contains amino acids that are conservative substitutions for one another: 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 that change, add, or delete a single amino acid or a small percentage of amino acids can also be considered "conservative substitutions" if the change does not reduce the activity of MIS (i.e., the ability of a recombinant human MIS protein or variant to cause Müllerian duct regression in vivo, which can be determined using the Müllerian duct regression bioassay disclosed herein). Insertions or substitutions are typically in the range of about 1-5 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 on the exterior of the peptide and exposed to solvent, or on the interior and not exposed to solvent.

[0077] In another embodiment, one skilled in the art can select an amino acid that will replace an existing amino acid based on the position of the existing amino acid, 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 inside that is not exposed to the solvent). Such conservative amino acid substitution selection is known in the art and is 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, one skilled in the art can select a conservative amino acid substitution that is suitable for the amino acid on the outside of a protein or peptide (i.e., the amino acid exposed to the solvent). For example, but not limited to, the following substitutions may 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, A with S, K or P.

[0078] In another embodiment, one skilled in the art can also select suitable conservative amino acid substitutions for amino acids in the interior of a protein or peptide. For example, suitable conservative substitutions can be used for amino acids in the interior of a protein or peptide (i.e., amino acids not exposed to solvent). For example, but not limited to, the following conservative 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-conservative amino acid substitutions are also included in the scope of variants. A variant of recombinant human MIS protein, for example, a variant of SEQ ID NO: 2 or 3, refers to any molecule substantially similar in structure and function to the entire molecule of SEQ ID NO: 2 or 3, or any of its fragments.

[0079] The terms "homology", "identity" and "similarity" refer to the degree of sequence similarity between two peptides or optimally aligned nucleic acid molecules. Homology and identity can each be determined by comparing positions in each sequence that can be aligned for comparison purposes. For example, it is based on the use of standard homology software, such as BLAST, version 2.2.14, at default positions. If the equivalent position in the compared sequences is occupied by the same base or amino acid, the molecules are identical at that position. If the equivalent position is occupied by a similar amino acid (e.g., similar in steric structure and / or electrical properties, e.g., conservative amino acid substitution), the molecules can be referred to as being homologous (similar) at that position. Expression as a percentage of homology / similarity or identity means a function of the number of similar or identical amino acids, respectively, at the positions shared by the compared sequences. A sequence that is "unrelated" or "non-homologous" has less than 40% identity. Preferably, it is less than 25% identical to the sequences disclosed herein.

[0080] As used herein, the term "sequence identity" means that two polynucleotide or amino acid sequences are identical (i.e., nucleotide-by-nucleotide or residue-by-residue) over a comparison window. The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions in both sequences that have identical nucleic acid bases (e.g., A, T. C, GU or I) or residues, calculating the number of matched positions, dividing the number of matched positions within the comparison window (i.e., the window size) by the total number of positions, and multiplying the result by 100 to calculate the percentage of sequence identity.

[0081] As used herein, the term "substantially identical" refers to a polynucleotide or amino acid sequence characteristic that the polynucleotide or amino acid sequence comprises a sequence having at least 85% sequence identity, preferably at least 90-95% sequence identity, and more usually at least 99% sequence identity, relative to the reference sequence over a comparison window of at least 18 nucleotide (6 amino acid) positions, and frequently over 24-48 nucleotide (8-16 amino acid) positions, where the percent sequence identity is calculated by comparing the reference sequence to a sequence that may contain 20% or less of the entire deletion or addition of the reference sequence over the comparison window. The reference sequence may be a subset of a larger sequence. The term "similarity" when used to describe polypeptides is determined by comparing the amino acid sequence and conservative amino acid substitutions of one polypeptide to the sequence of a second polypeptide.

[0082] As used herein, the terms "homolog" or "homologs" are used interchangeably and, when used to describe polynucleotides or polypeptides, indicate that two polynucleotides or polypeptides, or designated sequences thereof, are identical, with appropriate nucleotide insertions or deletions, or amino acid insertions or deletions, in at least 70% of the nucleotides, usually about 75-99% of the nucleotides, and more preferably at least about 98-99% of the nucleotides, when optionally aligned and compared, for example, using BLAST, version 2.2.14 with default parameters. As used herein, the terms "homolog" or "homologous" also refer to homology with respect to structure and / or function. With respect to sequence homology, sequences are homologous if they are 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% identical. Determining homologs of genes or peptides of the present invention can be readily performed by one of skill in the art.

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

[0084] Molecules are said to be "substantially similar" if they have a substantially similar structure or if they have a similar biological activity, for example, if they can activate MISRII. Thus, if two molecules have a similar activity (i.e., a variant of a 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 to be variants and are included for the purposes disclosed herein, even if the structure of one of the molecules is not found in the other or the sequence of amino acid residues is not identical. Thus, if two molecules have a similar biological activity, they are considered to be variants, as the term is used herein, even if the structure of one of the molecules is not found in the other or the sequence of amino acid residues is not identical. In particular, the term "substantially similar" when used to define recombinant human MIS proteins, including functional variants of recombinant human MIS proteins compared to recombinant human MIS proteins encoded by SEQ ID NO:2 or 3, means that a particular subject sequence, e.g., a variant or derivative sequence of recombinant human MIS protein, differs from the sequence of the native (or wild-type) MIS of SEQ ID NO:1 or recombinant human MIS protein (i.e., encoded by SEQ ID NO:2 or 3) by one or more substitutions, deletions, or additions, the net effect of which is to retain 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 have comparable biological activity are considered to be equivalent. In determining polynucleotide sequences, all subject polynucleotide sequences that can code for substantially similar amino acid sequences are considered to be substantially similar to the reference polynucleotide sequence, regardless of differences in codon sequence.A nucleotide sequence is "substantially similar" to a particular nucleic acid sequence of SEQ ID NO:4 or 5 disclosed herein if (a) the nucleotide sequence hybridizes to the coding region of a naturally occurring MIS nucleic acid, (b) the nucleotide sequence is capable of hybridizing under moderately stringent conditions to the nucleotide sequence of recombinant human MIS protein encoded by SEQ ID NO:4 or 5 and has biological activity similar to recombinant human MIS protein, or (c) is a nucleotide sequence that is degenerate as a result of the genetic code to the nucleotide sequence defined in (a) or (b). A substantially similar protein will typically have greater than about 80% similarity to the corresponding sequence of the native protein.

[0085] The term "substantially similar" as used in the context of peptide sequences indicates that the polypeptide comprises a sequence having 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, over a comparison window of about 10-20 amino acid residues. In the context of amino acid sequences, "substantially similar" further includes conservative substitutions of amino acids. Thus, a polypeptide is substantially similar to a second polypeptide, for example, where the two peptides differ by one or more conservative 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 the sequence of the recombinant MIS protein gene as encoded by the genome of a human or animal, e.g., a mouse or transgenic animal. In one embodiment, the term "human homolog" for a protein identified as related to the recombinant human MIS protein refers to an amino acid sequence having 40% homology to the full-length amino acid sequence of the protein identified as related to the recombinant human MIS protein as encoded by the genome of a transgenic animal of the 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%, more preferably at least about 95%. As stated above, the homology is at least about 50% to 100%, and all intervals therebetween (i.e., 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, etc.). Determination of human homologs of the genes of the present invention can be readily performed by one of skill in the art.

[0087] The term "conservative substitution" when describing a polypeptide refers to a change in the amino acid composition of the polypeptide that does not substantially change the activity of the polypeptide. Thus, a "conservative substitution" of a particular amino acid sequence refers to the substitution of those amino acids that are not important for the polypeptide activity, or the substitution of an amino acid with another amino acid that has similar properties (e.g., acidic, basic, positive or negative charge, polar or non-polar, etc.) such that even the substitution of an important amino acid does not substantially change the activity. Tables of conservative amino acids providing functionally similar amino acids are known in the art. For example, each of the following six groups contains amino acids that are conservative substitutions for one another: 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 addition, individual substitutions, deletions, or additions that change, add, or delete a single amino acid or a small percentage of amino acids in an encoded sequence are also "conservative substitutions."

[0088] As used herein, the term "non-conservative" refers to 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 a reference sequence to which a test sequence is compared. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and a sequence algorithm program is designated. The sequence comparison algorithm then calculates the percentage of identity of the test sequence to the reference sequence, based on the designated program parameters.

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

[0091] One example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive pairwise alignment to show the percentage of sequence identity. It also plots a tree or dendogram showing the clustering relationships used to create the alignment. PILEUP uses 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 a pairwise alignment of the two most similar sequences, creating a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of the two individual sequences. The final alignment is achieved by a series of progressive pairwise alignments. The program is run by specifying particular sequences and their amino acid or nucleotide positions for regions of sequence comparison and by specifying the program parameters. For example, a reference 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 percentage of sequence identity and sequence similarity is the BLAST algorithm by Altschul et al. (see J. Mol. Biol. 215:403-410 (1990), which is 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), which are incorporated herein by reference). Software for performing BLAST analysis is available at the Internet website of the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or satisfy some positive threshold score T when aligned with words of the same length in the database sequence. T is also referred to as the neighborhood word score threshold (Altschul et al. (1990), supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. The extension of the word hits in each direction is discontinued when: the cumulative alignment score falls by an amount X from its maximum achieved value; the cumulative score becomes 0 or lower due to the accumulation of one or more negative-scoring 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 as defaults a wordlength (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 percentage of sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-77 (1993), incorporated herein by reference). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides a measure of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered to be similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid with the reference nucleic acid is less than about 0.1, more typically less than about 0.01, and most typically less than about 0.001.

[0094] The term "insertion" or "deletion" typically ranges from about 1 to 5 amino acids. Permitted variations can be determined experimentally by synthetically producing the peptide while systematically creating nucleotide insertions, deletions, or substitutions in the sequence using recombinant DNA technology.

[0095] The term "substitution" when referring to a peptide refers to a change in an amino acid to a different substance, e.g., another amino acid or amino acid component. Substitutions can be conservative or non-conservative substitutions.

[0096] An "analog" of a molecule, such as a recombinant human MIS protein, e.g., SEQ ID NO: 2 or 3, refers to a molecule similar in function to either the entire molecule or a fragment thereof. The term "analog" is intended to include allelic, species, and induced variants. Analogs typically differ from naturally occurring peptides at one or a few positions, often in terms of conservative substitutions. Analogs typically exhibit at least 80% or 90% sequence identity with the native peptide. Particular analogs also include unnatural amino acids or modifications of the N- or C-terminal amino acids. Examples of unnatural amino acids include, but are not limited to, 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, δ-N-methylarginine. Fragments and analogs can be screened for prophylactic or therapeutic efficacy in transgenic animal models, as described below.

[0097] "Covalently bonded" means linked directly or indirectly (eg, via a linker) by a covalent chemical bond.

[0098] The term "fusion protein" as used herein refers to a recombinant protein of two or more proteins. Fusion proteins can be produced, for example, by linking 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 in a cell into a single polypeptide with all the intended proteins. The order of arrangement of the proteins can be varied. 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 thereof, fused in frame to either the 5' or 3' end of a first fusion partner, for example, a gene encoding an IgG1 Fc fragment. In this way, upon expression of the gene, the recombinant human MIS protein or a functional derivative, fragment, or variant thereof is functionally expressed and fused to the N-terminus or C-terminus of IgG1 Fc. In certain embodiments, the modification of the polypeptide probe is performed such that the functionality of the recombinant human MIS protein or a functional derivative, fragment, or variant thereof is not substantially affected in terms of biological activity by fusion to the first fusion partner, for example, IgG1 Fc.

[0099] "Specifically binds" or "specific binding" refers to a compound or antibody that recognizes and binds to a desired polypeptide but does not substantially recognize or bind to other molecules in a sample, e.g., a biological sample that naturally contains the polypeptide of the invention.

[0100] "Substantially pure" refers to a nucleic acid or other molecule that has been separated from components that naturally accompany it. Typically, a polypeptide is substantially pure if it is at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% by weight free from naturally associated proteins and naturally occurring organic molecules. For example, a substantially pure polypeptide can be obtained by extraction from a natural source, by expression of a recombinant nucleic acid in a cell that does not normally express that protein, or by chemical synthesis.

[0101] "Enhanced proteolytic stability" refers to a reduction in the rate or extent of proteolysis of a 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 an in vitro system, such as a cell or cell lysate). Peptides with enhanced proteolytic stability can include any modification, such as insertions, deletions, or point mutagenesis that delete or remove sites involved in proteolytic cleavage at specific sites. Sites of proteolytic cleavage can be identified based on known target sequences or using computer software (e.g., the 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, proteolytic sites can be determined experimentally, e.g., by Western blot on the protein after expression or incubation in cell lines or cell lysates, and sequencing of identified fragments to determine the site of subsequent cleavage.

[0102] The term "recombinant" as used herein to describe a nucleic acid molecule means of genomic, cDNA, viral, semisynthetic, and / or synthetic origin that is not related in whole or in part to a polynucleotide with which it is naturally associated in terms of its origin or manipulation. The term recombinant as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide. The term recombinant as used with respect to a host cell means a host cell into which a recombinant polynucleotide has been introduced. Recombinant is also used herein to refer to material (e.g., a cell, nucleic acid, protein, or vector) that has been altered by the introduction of 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, e.g., a human, to which a treatment, including prophylactic treatment, with a pharmaceutical composition according to the present invention is applied. As used herein, the term "subject" refers to a human and a non-human animal. The terms "non-human animal" and "non-human mammal" are used interchangeably herein and include all vertebrates, e.g., mammals, e.g., non-human primates (especially higher primates), sheep, dogs, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits, cows, and non-mammals, e.g., birds, amphibians, reptiles, and the like. In one embodiment, the subject is a human. In another embodiment, the subject is an animal surrogate, e.g., an experimental animal or a disease model. The term does not refer to a particular age or sex. This includes living and newborn subjects, as well as fetuses, whether male or female. Examples of subjects include humans, dogs, cats, cows, goats, and mice. The term subject is intended to include transgenic species. The term subject also includes mammals, e.g., humans, to which treatment, e.g., therapeutic and / or prophylactic treatment, with compositions comprising recombinant human MIS proteins disclosed herein is directed.

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

[0105] The terms "disease" or "disorder" are used interchangeably herein and refer to any change in some state of the body or organs that interrupts or impedes the performance of functions and / or causes symptoms, such as discomfort, disability, suffering, or death, in the afflicted person or in people in contact with the person. Disease or disorder includes distemper, ailing, ailment, amlady, disorder, sickness, illness, complaint, inderdisposion, affection.

[0106] The terms "malignancy (tumor)" and "cancer" are used interchangeably herein to refer to a disease characterized by the uncontrolled, abnormal growth of cells. Cancer cells can spread locally or via the bloodstream and lymphatic system to other parts of the body. The term is also intended to include any disease of an organ or tissue in a mammal characterized by a poorly controlled or uncontrolled increase in normal or abnormal cells in that tissue, and its effects on the body as a whole. Cancer diseases included within the definition include benign neoplasia, dysplasia, hyperplasia, as well as metastatic growth or other changes, such as leukoplakia, which often precede the development of cancer.

[0107] As used herein, the term "tumor" refers to a population of transformed cells characterized, at least in part, by the inclusion of angiogenic vasculature. Transformed cells are characterized by rapid and continuous neoplastic uncontrolled cell proliferation, even after the stimuli to initiate new proliferation have ceased. The term "tumor" is used broadly to include tumor parenchymal cells and supporting stroma, including angiogenic blood vessels that infiltrate the tumor parenchymal cell population. Tumors are generally malignant tumors, i.e., cancers that have the ability to metastasize (i.e., metastatic tumors), although tumors can also be non-malignant (i.e., non-metastatic tumors). Tumors are a hallmark of cancer, a natural process that is fatal, a neoplastic disease. Cancer cells exhibit invasive and metastatic properties and are highly anaplastic.

[0108] As used herein, the term "metastatic" or "metastatic tumor" refers to a secondary tumor that grows from a primary tumor elsewhere in the body, arising from cells that have detached and been transported from the primary tumor if the primary tumor is a solid tumor. As used herein, a primary tumor refers to a tumor that is organized in the location or organ in which it resides and has not metastasized to that location from another location. As used herein, a "malignant tumor" is one that has the properties of invasion and metastasis and exhibits a high degree of anaplasia. Anaplasia is the reversion of cells to an immature or less differentiated form and occurs in most malignant tumors.

[0109] As used herein, the term "treatment-resistant cancer" refers to a cancer present in a subject that is resistant or refractory to at least two different anti-cancer agents, e.g., chemotherapeutic agents, typically meaning that the subject has been treated with at least two different anti-cancer agents that do not provide an effective treatment, as the term is defined herein.

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

[0111] As used herein, the term "treatment" or "treatment" refers to both therapeutic treatment and prophylactic or preventative treatment, where the purpose is to prevent or slow the development of a disease, e.g., to reduce at least one effect or symptom of a condition, disease, or disorder associated with tumor development, cancer spread, or inappropriate proliferation or cell population, e.g., cancer. Treatment, as defined herein, is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if the progression of a disease is reduced or stopped. That is, "treatment" includes not only the 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 at least a halt in the slowing of the progression or worsening of symptoms that would be expected in the absence of treatment. A measurable reduction includes any statistically significant decline in a measurable marker or symptom. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, whether detectable or undetectable, reduction in the extent of disease, stabilization (i.e., not worsening) of disease, delay or slowing of disease progression, alleviation or mitigation of disease state, and remission (whether partial or total). "Treatment" can also mean prolonged survival compared to expected survival in the absence of treatment. Those in need of treatment include those who have already been diagnosed with cancer and those who may develop secondary tumors through metastasis. In certain embodiments, treatment can be a preventative measure.

[0112] The term "effective amount" as used herein refers to an 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 a pharmaceutical composition to provide the required effect. As used herein, the term "therapeutically effective amount", e.g., a therapeutically effective amount of a pharmaceutical composition comprising at least one recombinant human MIS protein disclosed herein, refers to a sufficient amount of the composition to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment. Thus, the term "therapeutically effective amount" refers to an amount of a composition disclosed herein that is sufficient to cause 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, 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 in a measured parameter compared to a control or untreated subject. Measured or measurable parameters are clinically detectable markers of disease, such as an increase or decrease in the level of a biological marker, as well as parameters related to a clinically accepted scale of symptoms or markers for a disease or disorder. However, the overall daily dosage of the compositions or formulations disclosed herein will be determined by the attending physician within the scope 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 a subject having cancer with a pharmaceutical composition comprising at least one recombinant human MIS protein disclosed herein, the term "therapeutically effective amount" refers to an amount that is safe and sufficient to prevent or delay the development and further growth or spread of metastases of tumors in a cancer patient. This amount can treat or quell cancer, slow the course of cancer progression, 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 amount for the treatment of cancer depends on the tumor to be treated, the severity of the tumor, the drug resistance level of the tumor, the species to be treated, the age and general condition of the subject, the mode of administration, and the like. This makes it impossible to specify an exact "effective amount." However, for any given case, an appropriate "effective amount" can be determined by one of skill in the art using only routine experimentation. The efficacy of the treatment can be determined by one of skill in the art, for example, efficacy can be evaluated in animal models of cancer and tumors. For example, treatment of rodents with cancer and any treatment or administration of a composition or formulation that leads to a reduction in at least one symptom of cancer, such as a reduction in tumor size or a slowing or cessation of tumor growth, indicates an effective treatment. In embodiments in which the 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 tumor cell transplant. When using an experimental animal model, the efficacy of the treatment is demonstrated if a reduction in the symptom of cancer, such as a reduction in tumor size or a slowing or cessation of tumor growth, occurs faster in treated animals versus untreated animals. "Shorter" 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 is used to refer to the reduction of symptoms and / or biochemical markers of cancer. For example, a significant reduction of at least one biochemical marker of cancer would be considered an effective treatment. Examples of such biochemical markers of cancer include CD44, telomerase, TGF-α, TGF-β, erbB-2, erbB-3, MUC1, MUC2, CK20, PSA, CA125 and FOBT. A reduction in the rate of proliferation of cancer cells by at least about 10% would also be considered an effective treatment by the methods disclosed in the present invention. As another example, a reduction in symptoms of cancer, such as a slowing down of the rate of cancer proliferation by at least about 10%, or a halt in the increase of tumor size, or a reduction in tumor size by at least about 10%, or a reduction in the spread of tumors (i.e., tumor metastasis) by at least about 10%, would also be considered an effective treatment by the methods disclosed in the present invention. In certain embodiments, it is preferred, but not required, that the therapeutic agent actually kill the tumor.

[0116] The term "prophylactically effective amount" refers to an amount of recombinant human MIS protein or its functional fragment or variant that is effective in the required dosage and organ to achieve the required prophylactic result, for example, to prevent the development of cancer in a subject at risk of developing cancer. Typically, a prophylactic dose of recombinant human MIS protein or its functional fragment or variant is administered to a subject at a pre- or early stage of cancer, or to a subject with a genetic predisposition to cancer, for example, but not limited to, a subject with a genetic mutation that increases the subject's chance of developing ovarian cancer. In certain embodiments, a prophylactically effective amount is less than a therapeutically effective amount. A prophylactically effective amount of recombinant human MIS protein or its functional fragment or variant is also an amount in which any toxic or harmful effects of the compound are outweighed by the beneficial effects.

[0117] As used herein, the terms "prophylaxis" and "prevention" refer to the avoidance or delay of one or more symptoms or measurable markers of a disease or disorder, such as an autoimmune disease. The delay of symptoms or markers is the delay relative to the time when such symptoms or markers appear in a control or untreated subject with a similar likelihood or susceptibility to develop a disease or disorder. The terms "prevention" and "prophylaxis" include not only the avoidance or prevention of disease symptoms or markers, but also the reduction of the severity or extent of any one of the symptoms or markers of a disease, relative to these symptoms or markers in a control or individual with a similar likelihood or susceptibility to develop a disease or disorder, or relative to symptoms or markers that are likely to occur based on historical or statistical standards of a population affected by a disease or disorder. "Reduction in severity" means a reduction in the severity or extent of a symptom or measurable disease marker by at least 10%, e.g., at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% (i.e., the absence of symptoms or measurable markers) relative to a control or reference standard.

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

[0119] "Composition" or "pharmaceutical composition" are used interchangeably herein and refer to a composition that typically includes an excipient, e.g., a pharma- ceutically acceptable carrier, conventional in the art and suitable for administration to a cell. The cell may be part of a subject, e.g., for therapeutic, diagnostic, or prophylactic purposes. The cell may also be cultured, e.g., as part of an assay to screen potential pharmaceutical compositions, or the cell may be part of a transgenic animal for research purposes. The composition may also be a cell culture, in which a polypeptide or polynucleotide encoding a metabolic regulator of the present invention is present in the cell or in the cell medium. Additionally, 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, as are well known in the art and described herein. Additionally, compositions for topical (e.g., oral mucosa, respiratory mucosa) and / or oral administration may form solutions, suspensions, tablets, pills, capsules, sustained release formulations. The composition may include 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" refer to modes of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intrathecal, intraventricular, intraspinal, and intrasternal injection and infusion. As used herein, the terms "systemic administration", "administered systemically", "peripheral administration", and "administered peripherally" refer to administration of recombinant human MIS protein such that it enters the animal's system and thereby enters the metabolism and other similar systems, e.g., subcutaneous administration.

[0121] The term "pharmacologically acceptable" is used herein to mean that those compounds, materials, compositions, and / or dosage forms are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio.

[0122] The term "pharmaceutical acceptable carrier" as used herein means a pharma-ceutically acceptable material, composition, or vehicle, e.g., a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, associated with carrying, carrying, or transporting the activity of an agent of interest from one organ or body part to another. In addition to being "pharmaceutical acceptable" as defined herein, each carrier must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation. A pharmaceutical formulation comprises a compound of the invention in combination with one or more pharma-ceutically acceptable ingredients. The carrier may be in the form of a solid, semi-solid, or liquid diluent, cream, or capsule. These pharmaceutical preparations are further objects of the present invention. Usually, the amount of active compound is 0.1-95% by weight of the preparation, preferably 0.2-20% by weight in the preparation for parenteral use, and preferably 1-50% by weight in the preparation for oral administration. For clinical use of the methods of the invention, the targeted delivery compositions of the invention are formulated into pharmaceutical compositions or formulations for parenteral administration, e.g., intravenous, mucosal, e.g., intranasal, enteral, e.g., oral, topical, e.g., transdermal, ophthalmic, e.g., via corneal scarification, or other modes of administration.

[0123] As used herein, the term "oncogene" refers to a nucleic acid sequence that encodes a polypeptide that is a mutated and / or overexpressed version of a normal gene that relieves cells from the normal constraints of proliferation in a dominant manner, thereby conferring tumorigenicity to the cell, either alone or in combination with other alterations. 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, and the like. Protooncogene refers to the normal expression of a nucleic acid that expresses the normal cellular equivalent of an oncogene, typically a gene that is normally associated with signaling or control of cell proliferation.

[0124] As used herein, the term "regeneration" refers to the regrowth of a cell population, organ, or tissue, in certain embodiments, following disease or trauma.

[0125] The term "vector" refers to a nucleic acid capable of transporting another nucleic acid to which it is linked. Plasmids are species of the genus encompassed by "vector". The term "vector" typically refers to a nucleic acid sequence that includes an origin of replication and other materials for replication and / or maintenance in a host cell. A vector capable of directing the expression of an operably linked gene and / or nucleic acid sequence is referred to herein as an "expression vector". In general, useful expression vectors are often in the form of "plasmids", which in their vector form are not bound to chromosomes and typically refer to circular double-stranded DNA loops that contain materials or encoded DNA for stabilization or transient expression. Other expression vectors can be used in the methods disclosed herein, such as, but not limited to, plasmids, episomes, bacterial artificial chromosomes, yeast artificial chromosomes, bacteriophages, or viral vectors, and such vectors can be integrated into the genome of a host and replicate automatically in a particular cell. Vectors can be DNA or RNA. Other forms of expression vectors known to those skilled in the art that serve equivalent functions can also be used, such as self-replicating extrachromosomal vectors or vectors that integrate into the host genome. Preferred vectors are those that can replicate autonomously and / or express linked nucleic acids. Vectors that can direct the expression of operably linked genes are referred to herein as "expression vectors." Expression vectors can cause stable or transient expression of DNA. An exemplary expression vector for use in the present invention is pcDNA3.1.

[0126] The term "viral vector" refers to the use of virus or virus-associated vectors as carriers of nucleic acid constructs into cells. The constructs can be incorporated and packaged into non-replicative defective viral genomes, such as adenovirus, adeno-associated virus (AAV), or herpes simplex virus (HSV), or other, such as retrovirus and lentivirus vectors, for infecting or transducing cells. The vectors may or may not be integrated into the cell genome. The constructs may optionally include viral sequences for transfection. Alternatively, the constructs can be incorporated into vectors capable of episomal replication, such as EPV and EBV vectors.

[0127] As used herein, the interchangeably used terms "promoter", "promoter region", or "promoter element" refer to a segment of a nucleic acid sequence, typically but not limited to DNA or RNA or their analogs, that controls the transcription of an operably linked nucleic acid sequence. The promoter region contains specific sequences that are 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 contains sequences that regulate RNA polymerase recognition, binding, and transcription initiation. These sequences may be cis-acting or responsive to trans-acting factors. Promoters may be constitutive or regulated, depending on the nature of the regulation.

[0128] The term "regulatory sequence" is used interchangeably herein with "regulatory element" and refers to a segment of nucleic acid, typically but not limited to DNA or RNA or analogs thereof, that regulates the transcription of an operably linked nucleic acid sequence, thereby functioning as a transcriptional modulator. Regulatory sequences regulate the expression of operably linked genes and / or nucleic acid sequences. Regulatory sequences often include "regulatory elements," which are nucleic acid sequences that are transcription binding domains and are recognized by transcription proteins and / or nucleic acid binding domains of transcription factors, repressors, or enhancers. Typically, regulatory sequences include, but are not limited to, transcription promoters, inducible promoters, and transcription factors, optional operative sequences for controlling transcription, sequences encoding appropriate mRNA ribosomal binding sites, and sequences for controlling the termination of transcription and / or translation. Regulatory sequences can be a single regulatory sequence or multiple regulatory sequences, or modified regulatory sequences or fragments thereof. Modified regulatory sequences are regulatory sequences in which the nucleic acid sequence has been changed or modified by a particular means, such as but not limited to, mutation, methylation, etc.

[0129] The term "operably linked" as used herein refers to the functional relationship of a nucleic acid sequence with nucleotide regulatory sequences, such as promoters, enhancers, transcription and translation termination sites, and other signal sequences. For example, operably linked nucleic acid sequences, typically DNA, to regulatory sequences or promoter regions refers to the physical and functional relationship between the DNA and the regulatory sequences or promoter such that transcription of the DNA is initiated from the regulatory sequences or promoter by an RNA polymerase that specifically recognizes, binds to, and transcribes the DNA. To optimize expression and / or in vitro transcription, it may be necessary to refine the regulatory sequences for expression of the nucleic acid or DNA in the cell type in which it is expressed. The need or necessity for such refinements can be determined empirically. Enhancers need not be located in close proximity to the coding sequences that 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 operably linked to the second promoter. In such a case, transcription of the first gene is said to be operably linked to the first promoter and also operably linked to the second promoter.

[0130] As used herein, the term "biological sample" also refers to a cell or a population of cells, or a specific amount of tissue or fluid from a subject. Often, a sample has been removed from a subject, but the term "biological sample" can also refer to cells or tissues that are analyzed in vivo, i.e., not removed from a subject. Often, a "biological sample" will include cells from a subject, but the term can also refer to non-cellular biological samples, such as non-cellular fractions of blood, saliva, or urine, that can be used to measure protein phosphorylation levels. In certain embodiments, a "biological sample" or "tissue sample" refers to a sample of tissue or fluid isolated from an individual, including, but not limited to, blood, plasma, serum, tumor biopsy, urine, stool, sputum, cerebrospinal fluid, pleural fluid, nipple aspirate, lymph, external sections of skin, respiratory tract, intestinal tract, genitourinary tract, tears, saliva, milk, cells (including but not limited to blood cells), tumors, organs, and in vitro cell culture components. In certain embodiments, the biological sample is from a resection, a bronchoscopic biopsy, or a core needle biopsy of a primary, secondary, or metastatic tumor, or a cell block from pleural fluid. In addition, fine needle aspirate biological samples are also useful. In certain embodiments, the biological sample is a primary ascites cell. 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. Biological samples may also refer to samples of biological tissues or fluids, including proteins or cells. Such samples include, but are not limited to, tissues isolated from a subject or animal. Biological samples may also include sections of tissues, such as biopsy and autopsy samples, frozen sections taken for histological purposes, blood, plasma, serum, sputum, stool, tears, mucus, hair, and skin. Biological samples also include explants obtained from patient tissues and primary and / or transformed cell cultures.A biological sample can be provided by removing a sample of cells from a subject, but can also be achieved by using previously isolated cells (e.g., isolated by another person at a different time and / or for a different purpose) or by performing the methods of the invention in vivo. Archival tissues, e.g., those with treatment or outcome history, can also be used. Biological samples include, but are not limited to, tissue biopsies, scrapes (e.g., buccal scrapes), whole blood, plasma, serum, urine, saliva, cell cultures, or cerebrospinal fluid. Biological samples also include tissue biopsies, 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., isolated by another person) or by performing the methods of the invention in vivo. Such samples include, but are not limited to, whole blood, cultured cells, primary cell preparations, sputum, amniotic fluid, tissue or fine needle biopsy samples, ascites, among others. 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 rodents, animal models of disease, commercial animals, companion animals, dogs, cats, sheep, cattle, and pigs. The biological sample can be pretreated as needed for storage or preservation by diluting or concentrating with an appropriate buffer solution as needed. Any of several standard aqueous buffer solutions can be used, using one of a variety of buffers, such as phosphate, Tris, etc., at physiological pH. The biological sample can be stored in a particular environment for use prior to use in the assays disclosed herein. Such storage can be at +4°C or frozen, such as at -20°C or -80°C, as long as an appropriate cryopreservative is used to maintain cell viability when the cells are thawed.

[0131] The terms "reduction", "decrease" or "reduction" as used herein generally refer to a decrease by a statistically significant amount relative to the reference. However, for the avoidance of doubt, as defined herein, "reduction" refers to a statistically significant decrease of at least 10% relative to the reference 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 more, a decrease of less than or equal to 100% (i.e., a level of absence relative to the reference sample), or a decrease anywhere from 10-100% relative to the reference level. The terms "reduction" or "inhibition" as used in the context of the level of expression or activity of a gene refer to a decrease in protein or nucleic acid levels or activity in a cell, cell extract, or cell supernatant. For example, such a decrease may occur due to a decrease in RNA stability, transcription, or translation, an increase in protein degradation, or RNA interference. Preferably, the 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] The term "low" as used herein generally means lower by a statistically significant amount. For the avoidance of doubt, "low" means a statistically significant level at least 10% lower than the cited level, for example, a level at least 20% lower than the cited level, a level at least 30% lower than the cited level, a level at least 40% lower than the cited level, a level at least 50% lower than the cited level, a level at least 60% lower than the cited level, a level at least 70% lower than the cited level, a level at least 80% lower than the cited level, a level at least 90% lower than the cited level, less than 100% of the cited level, and a level 100% lower (i.e., a level of absence compared to the cited sample).

[0133] The term "increase" or "increase" as used herein generally refers to an increase of a statistically significant amount. For the avoidance of doubt, as defined herein, "increase" refers to a statistically significant increase of at least 10% compared to the cited level, such as 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 cited level, such as an increase of at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold or more compared to the cited level. The term "increase" as used in the context of gene or protein expression or activity refers to a positive change in protein or nucleic acid level or activity in a cell, cell extract, or cell supernatant. For example, such an increase can occur due to an increase in RNA stability, transcription, or translation, or a decrease in protein degradation. Preferably, the 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 over the level of expression or activity under control conditions.

[0134] As used herein, the term "high" generally means higher by a statistically significant amount relative to the reference. For the avoidance of doubt, "high" means a statistically significant level that is at least 10% higher than the reference level, e.g., 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 2-fold higher, at least 3-fold higher, at least 4-fold higher, at least 5-fold higher, at least 10-fold higher, or more, relative to the reference level.

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

[0136] Other than in the examples, or unless otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood to be modified in all instances by the term "about." The term "about," when used in conjunction with percentages, can mean ±1%. The present invention is further described in detail by the following examples, which should not be construed to limit the scope of the present invention.

[0137] It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc. described herein, as these may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Other features and advantages of the present invention will become apparent from the following detailed description, drawings, and claims. Mullerian Inhibitory Substance (MIS)

[0138] Without intending to be bound by theory, Müllerian inhibitory substance (MIS) is a member of the TGFβ multigene family of glycoproteins. The proteins of this gene family are all produced as dimeric precursors, which undergo post-transcriptional processing for activation that requires cleavage and dissociation to release the biologically active C-terminal fragment. MIS is a 140 kDa dimer of identical 70 kDa disulfide-linked monomers, each composed of a 57 kDa N-terminal domain and a 12.5 kDa carboxy terminus (C-terminus). MIS thus contains two identical monomers (hence referred to as "homodimers"), each of which contains two non-covalently held domains, the N-terminal and the C-terminal domain. The purified C-terminal domain is the biologically active component, and cleavage is required for activity. The N-terminal domain aids in folding of the protein in vivo and facilitates delivery of the C-terminal peptide to its receptors, e.g., MISRI and MISRII. Non-truncating mutants of MIS are biologically inactive.

[0139] The carboxy-terminal activation domain shares amino acid homology with members of the TGFb family, such as TGF-B 1, 2, and 3, inhibins, activins, and bone morphogenetic proteins, as well as members of the growth and differentiation factors (GDFs). The structure of the MIS carboxy-terminal domain is supported by seven cysteines involved in both intra- and intermolecular disulfide bridges that confer structural stability, as revealed by homology to the three-dimensional structure of TGFb using molecular modeling (Lorenzo, Donahoe, et al., unpublished).

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

[0141] MIS contains two major cleavage sites that are sensitive to plasmin. The first monobasic site is located at amino acid positions 426-427 of human wild-type MIS (corresponding to amino acids 451-452 of SEQ ID NO:1 herein). Cleavage at this site, which releases the active carboxy-terminal domain of MIS, resembles a consensus furin cleavage site. The second cleavage site (also designated "R / S"), identified by amino-terminal sequencing of the MIS fragment, is located at residues 229-230 of the amino-terminal domain of wild-type MIS (corresponding to amino acids 254-255 of SEQ ID NO:1 herein). This site, although it contains an R / S, does not follow the consensus Arg-X-(Arg / Lys)-Arg for furin cleavage. Separation of the purified carboxy-terminus from the amino-terminal MIS following digestion with exogenous plasmin has previously employed molecular size exclusion chromatography under acidic conditions. This technique requires extreme care to control MIS digestion. Because prolonged incubation of MIS in plasmin produces the carboxy-terminal MIS domain and other fragments of 22 and 34 kDa due to cleavage at both the first and second sites, which are extremely difficult to separate from each other 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 applied to a wheat germ lectin column at pH 3.5 to dissociate the amino- and carboxy-terminal domains, as disclosed in Lorenzo et al., J. Chromatography, (2001), 776; 89-98, which is incorporated herein by reference in its entirety.

[0142] Therefore, to overcome some of the problems associated with avoiding the production of fragments of MIS, e.g., the carboxy-terminal MIS domain +22 and 34 kDa fragments by cleavage at both the first and second sites, the inventors engineered the first cleavage site at amino acid positions 426-427 of human wild-type MIS (corresponding to amino acids 451-452 of SEQ ID NO:1 herein).To aid in the purification of the C-terminal domain without the need for complex methods using wheat germ lectin affinity or size chromatography columns, the most flexible C-terminal domain, the inventors included a tag at the N-terminus of the C-terminal domain.

[0143] Additionally, wild-type MIS protein is produced as a prohormone that includes an N-terminal leader sequence, corresponding to amino acid residues 1-25 of SEQ ID NO: 1. Processing of the mature hormone MIS protein can 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 into monomers that are disulfide linked by intrachain and interchain disulfide bonds to identical monomers to form the biologically active homodimeric MIS protein. Leader sequence

[0144] Without intending to be bound by theory, leader sequences improve the expression and / or secretion of a polypeptide of interest in a host cell and are useful for recombinant production of proteins. In general, an efficient method for screening a protein required by genetic engineering procedures is known in which a fusion protein containing a required protein (e.g., MIS) and a prepropeptide (signal peptide+propeptide) is expressed in a host cell, then cleaved (processed) intracellularly by the host enzyme, and then secreted extracellularly. However, according to this method, the fusion protein must be cleaved twice by the host enzyme to become a mature protein, which causes a decrease in the yield of the mature protein and contamination of the mature protein with the residual fusion protein.

[0145] Thus, secreted proteins are first expressed in cells in precursor forms that contain leader sequences that ensure their entry into the secretory pathway. Such leader sequences, also called signal peptides, direct the expressed product across the membrane of the endoplasmic reticulum (ER). Signal peptides are generally cleaved by signal peptidases during translocation into the ER. After entering the secretory pathway, proteins are transported to the Golgi apparatus. From the Golgi, the protein can follow different pathways that lead to compartments such as the cell vacuole or the cell membrane, or it can be sent out of the cell to be secreted into the external medium (Pfeffer and Rothman (1987) Ann. Rev. Biochem. 56:829-852).

[0146] For industrial production of secreted proteins, the protein to be produced needs to be efficiently secreted from the host cell or host organism. 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 occurs 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 that exhibit N-terminal extensions due to incorrect cleavage of the signal peptide. This makes the selection of the signal peptide crucial for industrial production of proteins.

[0147] In addition of leader sequences that direct the secretion of the protein, precursor forms may contain additional leader sequences that are cleaved during maturation. These additional leader peptides, called propeptides, usually follow the signal peptide. Virtually all peptide hormones, many biologically active 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 produce the mature and biologically active protein (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, subtilisin convertase, PCSK1, PCSK2, and furin. Furin is widely expressed and located in the trans-Golgi network. Furin activates a number of proprotein substrates in secretory pathway compartments (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 cytoplasmic structures, endosomes, lysosomes, and secretory granules. The site at which furin cleaves has been extensively studied. The cleavage site is located after the carboxy-terminal arginine of the consensus sequence RXL / RR, where X can represent any amino acid (Nakayama (1997) Biochem. J 327:625-635). Cleavage efficiency is increased 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 comprises an altered leader sequence instead of the wild-type leader sequence of the MIS protein of 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, such as, but not limited to, an albumin leader sequence or a functional fragment thereof, in certain embodiments, the non-MIS leader sequence is a human serum albumin sequence (HSA), such as the leader sequence corresponding to SEQ ID NO: 6 encoded by a nucleic acid corresponding to SEQ ID NO: 7.

[0150] In certain embodiments, the HAS sequence is a functional fragment of SEQ ID NO:6, e.g., 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 contiguous or non-contiguous amino acids of SEQ ID NO:6. Modified forms of the HAS leader sequence are also included for use in the present invention and are disclosed in U.S. Patent No. 5,759,802, which is incorporated herein by reference in its entirety. In certain embodiments, a functional fragment of the HSA leader sequence is a functional fragment of the HSA leader sequence as disclosed in European Patent EP 2277889, which is incorporated herein by reference in its entirety. TIFF2024178382000026.tif5128 or a variant thereof. The prepro region of TIFF2024178382000027.tif5128) is a fragment, e.g., the prepro region of the HSA signal sequence ( TIFF2024178382000028.tif5128) or variants thereof, e.g. Includes TIFF2024178382000029.tif5128.

[0151] In certain embodiments, the leader sequence is 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% identical to the amino acid residues of SEQ ID NO:6.

[0152] The HAS leader sequence used herein caused the expected increase in yield (both higher concentration and higher production) of recombinant human MIS protein (see Figures 2 and 3). However, the presence of the HSA leader sequence caused a surprising and unexpected increase in cleavage from the first cleavage site (corresponding to the cleavage site at 451 / 452 in SEQ ID NO:1 (or 426 / 427 in conventional amino acid nomenclature for the wild-type MIS protein) (see Figures 2 and 3). This increased yield and increased cleavage was surprising because with increased yield (and therefore more protein produced by the cell), one would expect a decrease in cleavage as the activity of the available cleavage enzyme becomes saturated and overextended. However, this was not the case, and in fact just the opposite result occurred: increased cleavage from the first cleavage site with increased protein production.

[0153] Other leader sequences are also included for use in the recombinant human MIS protein disclosed herein, for example to replace amino acids 1-25 of SEQ ID NO:1. Such leader sequences are known to those of skill in the art and include leader sequences comprising an immunoglobulin signal peptide fused to a tissue-type plasminogen activator propeptide (IgSP-tPA), as disclosed in US 2007 / 0141666, which is incorporated herein by reference in its entirety. A number of other signal peptides are used for the production of secreted proteins, one of which is the murine immunoglobulin signal peptide (IgSP, EMBL Accession No. M13331). IgSP was first identified by Loh et al. in 1983 (Cell. 33:85-93). IgSP is known to cause good expression in mammalian cells. For example, EP 0382762 discloses a method for producing horseradish peroxidase by constructing a fusion peptide between IgSP and horseradish peroxidase.

[0154] Other leader sequences include, but are not limited to, the MPIF-1 signal sequence (e.g., amino acids 1-21 of GenBank Accession number AAB51134). TIFF2024178382000030.tif5128; stanniocalcin signal sequence TIFF2024178382000031.tif5128; Invertase signal sequence TIFF2024178382000032.tif5128; yeast mating factor alpha signal sequence (e.g., K. lactis killer toxin leader sequence); hybrid signal sequence TIFF2024178382000033.tif5128; HSA / MFα-1 hybrid signal sequence (also known as HSA / kex2) TIFF2024178382000034.tif5128; K. lactis killer / MFα-1 fusion leader sequence TIFF2024178382000035.tif5128; Immunoglobulin Ig signal sequence TIFF2024178382000036.tif5128Fibrin B precursor signal sequence TIFF2024178382000037.tif5128; Clusterin precursor signal sequence TIFF2024178382000038.tif5128; and insulin-like growth factor binding protein 4 signal sequence Includes TIFF2024178382000039.tif5128.

[0155] Where it is desired to produce recombinant MIS in a bacterial system, the leader sequence may comprise a bacterial leader sequence, such as that disclosed in US Application 2011 / 0020868. Several other secretion signals have been described for use in expressing recombinant polypeptides or proteins. See, for example, U.S. Pat. No. 5,914,254, U.S. Pat. No. 4,963,495, European Patent No. 0 177 343, U.S. Pat. No. 5,082,783, PCT Publication No. WO 89 / 10971, U.S. Pat. No. 6,156,552, U.S. Pat. Nos. 6,495,357; 6,509,181; 6,524,827; 6,528,298; 6,558,939; 6,608,018; 6,617,143, U.S. Pat. Nos. 5,595,898; 5,698,435; and 6,204,023, U.S. Pat. No. 6,258,560, PCT Publication Nos. WO 01 / 21662, WO No. 02 / 068660, and U.S. Application Publication No. 2003 / 0044906, U.S. Patent No. 5,641,671, and European Patent No. EP 0 121 352. Modified cleavage site

[0156] As discussed herein, preparation of MIS protein for preclinical use is complicated and inefficient. Human MIS protein is produced from a preproprotein that includes a leader sequence. The leader sequence (amino acids 1-25 of SEQ ID NO:1) is excised, and the remaining protein, corresponding to amino acids 26-560 of SEQ ID NO:1 (often referred to as "holo-human MIS"), is further post-translationally cleaved to yield N- and C-terminal domains. These N- and C-terminal domains form monomers, and two identical monomers (containing the N- and C-terminal domains) together form homodimers. Holo-human MIS is cleaved into N- and C-terminal domains, most likely by furin or a related prohormone convertase PC5, expressed in the gonads. Cleavage occurs initially at a kex-like site characterized by an R-4 XXR-1 with a serine at the +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 biological activity. A second cleavage site, the significance of which is unknown, is observed less frequently at residues 229-230 (corresponding to amino acids 254-255 of SEQ ID NO:1). Non-cleavable mutants of MIS are not biologically active, and mutants in the human gene that truncate the carboxy-terminal domain lead to persistent Müllerian duct syndrome. Cleavage of recombinantly expressed MIS protein by CHO cells is incomplete and inefficient, which requires cleavage with exogenous serine proteases, such as plasmin, to enhance biological activity.

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

[0158] This alteration of the amino acid sequence of Q450R of SEQ ID NO:1 allows for the production of highly purified truncated preparations of human MIS protein with full biological activity.

[0159] In another embodiment, the first cleavage site in the MIS protein, e.g., the monobasic site located at amino acid positions 426-427 of human wild-type MIS (corresponding herein to amino acids 451-452 of SEQ ID NO:1), 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, e.g., the monobasic site located at amino acid positions 426-427, can be modified to an amino acid sequence recognized by proteases or peptidases, e.g., prohormone convertases (PC's), or other cleavage agents expressed by cells and found in surrounding tissues, or produced by microorganisms capable of establishing an infection in a mammal. The enzyme-cleavable peptide can, but is not required to, include one or more amino acids in addition to the amino acid recognition sequence. The additional amino acids can be added to the amino terminus, the carboxy terminus, or both the amino and carboxy termini of the recognition sequence. Means for adding amino acids to an amino acid sequence, for example in an automated peptide synthesizer, and means for detecting cleavage of the peptide, for example by chromatographic analysis for the amino acid products of such cleavage, are known to those of skill in the art to which the present invention pertains.

[0160] Prohormone convertases constitute a family of serine proteases structurally related to bacterial subtilisins and yeast kexins. Several eukaryotic members of this family are now known. Prohormone convertases (PC's) cleave precursor polypeptides at specific basic residues, most often after selected pairs of basic residues, to generate biologically active peptides and proteins. Many members of the insulin family of proteins (e.g., insulin, Igf-1) are substrates for PC's. Tags to enhance purification

[0161] In certain embodiments, the recombinant human MIS protein comprises at least one internal label or "tag". In certain embodiments, the tag can 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 can be encoded by the nucleic acid of SEQ ID NO:9.

[0162] In certain embodiments, the tag on the recombinant human MIS protein is internal at the carboxy terminus immediately downstream from the cleavage site. It is the most flexible part of the C-terminus, the "finger tip" of the C-terminus, since it is not relevant for determining receptor binding and specificity (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, e.g., after amino acid 450 of SEQ ID NO:1 (corresponding to amino acid residue 425 under 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 conventional amino acid nomenclature of the MIS protein).

[0163] In another embodiment, the tag or label is located anywhere 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, location of the tag N-terminal to the C-terminal domain of MIS is preferred, since location C-terminal to the C-terminal domain renders the C-terminal domain totally inactive and greatly reduces the biological activity of the MIS protein.

[0164] In certain embodiments, the recombinant MIS protein comprises more than one tag, for example, at least two, at least three, at least four, or more than four tags. In certain embodiments, the tags are consecutive (for example, one after the other), in certain embodiments, they are dispersed (for example, intermittent) 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 activating MISRII. In certain embodiments, when the recombinant MIS protein comprises more than one tag, the tags are the same tag. In another embodiment, when the recombinant MIS protein comprises more than one tag, the tags are different tags, for example, the recombinant MIS protein may comprise a FLAG tag and a histidine tag. The small size of the Flag tag allows it to be included in the flexible non-binding N-terminal domain of the C-terminus. Therefore, in certain embodiments, any tag known to those skilled in the art can be used in place of the Flag tag, for example, a tag 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, recombinant human MIS protein tagged with a tag, e.g., FLAG tagged, e.g., the LRF recombinant human MIS protein disclosed herein (see FIG. 1), can be eluted in a single step to produce a highly purified, efficiently cleaved preparation with full biological activity. When scaled up, this purification of recombinant human MIS protein would be suitable for clinical applications. Furthermore, it would be useful for various binding assays, both clinical and experimental. Internal labeling of MIS during translation is more effective than labeling after purification of the protein, because iodination or biotinylation greatly reduces MIS biological activity. Surprisingly, the inventors discovered that the LRF recombinant human MIS protein construct is more biologically active than wild-type MIS. The insertion of the FLAG tag sequence has several other distinct advantages. First, its unique amino acid domain is not present in any other gene (except mouse brain phosphatase), making the anti-FLAG antibody extremely specific. Second, elution of proteins with 3xFLAG peptide is specific for FLAG MIS and not for other proteins that bind non-specifically to the agarose beads.

[0166] Surprisingly, when compared to native MIS or previously prepared unlabeled RAQR / R (SEQ ID NO:28) MIS, FLAG-tagged, cleavage-optimized recombinant human MIS (e.g., RF recombinant human MIS or RARR / S (SEQ ID NO:27) FLAG MIS) was bioactive, whereas FLAG-tagged, non-cleavage-optimized recombinant human MIS (e.g., RAQR / R (SEQ ID NO:28) FLAG MIS) was not. The presence of an acidic FLAG tag near the cleavage site may impair the extent of cleavage, thus causing a loss of activity. Thus, the inventors did not expect an enhancement of cleavage with the addition of the Flag tag. Furthermore, holo-RAQR / R FLAG MIS ("RAQR / R" disclosed in SEQ ID NO:28) preparations in CHO (or HEK) cells are not bioactive. Because no endogenous processing occurs at the RAQR / R (SEQ ID NO:28) cleavage site, in contrast to what was reported by Kurian (Cancer Res., 1995. 1;343-349) when that construct lacked the FLAG tag. On the other hand, retention of the serine at position 428 and conversion of the monobasic site to dibasic (corresponding to Q>R at amino acid position 425, or Q>R at position 450, SEQ ID NO:1, using conventional protein nomenclature) makes 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. Thus, in certain embodiments, labeled recombinant human MIS protein, e.g., MIS with an internal FLAG, is useful in an efficient method for producing highly pure and biologically active internally labeled forms of MIS that can be used for MIS binding studies and for follow-up in pharmacokinetic studies, and for scale-up for preclinical and clinical use. Variants of human recombinant MIS protein

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

[0168] As used herein, the term "polypeptide" refers to a polymer of amino acids and its equivalents, without referring to a specific length of the product. Thus, peptides, oligopeptides, and proteins are included in the definition of a polypeptide. A derivative is a polypeptide that has conservative amino acid substitutions compared to another sequence. Derivatives further include other modifications of the protein, such as glycosylation, acetylation, phosphorylation, and the like.

[0169] In certain embodiments, the 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 refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or conservative substitutions thereof that are the same, when compared and aligned for maximum correspondence as measured using one of the sequence comparison algorithms below or by visual inspection. For example, a first amino acid sequence can be considered similar to a second amino acid sequence if the first amino acid sequence is at least 50%, 60%, 70%, 75%, 80%, 90%, or 95% identical to or conservatively substituted for the second amino acid sequence when compared to an equal number of amino acids contained in the first sequence, or when compared to an alignment of polypeptides aligned by computer similarity programs known in the art, such as those described below.

[0170] Homologs and functional derivatives and fragments of MIS of SEQ ID NO:1 are also included for use in the present invention and can be identified, for example, by expression of MIS from an expression library (see, 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., supra). A mutated endogenous gene sequence can be referred to as a heterologous transgene. For example, a transgene encoding a mutation in MIS that is not known in the naturally occurring genome is a heterologous transgene with respect to murine and non-murine, e.g., human, species. MIS proteins, such as those disclosed in U.S. Pat. Nos. 5,427,780, 5,359,033, and 5,661,126, which are incorporated herein by reference in their entireties.

[0171] Variations in the primary structure of the core human MIS protein sequence (e.g., insertion of a tag at amino acids 26-560 of SEQ ID NO:1 (including modification of amino acid residue 450 from Q to R of SEQ ID NO:1), and / or at the beginning of the N-terminus of the C-terminal domain), or functional fragments or homologs thereof, are encompassed for use in the present invention and include, for example, deletions, additions, and substitutions. Substitutions may be conservative or non-conservative. Differences between the recombinant human MIS protein and the variants generally preserve desired properties, reduce or eliminate undesired properties, and add desired or new properties. For example, a variant of the recombinant human MIS protein may have superior activity compared to the wild-type MIS protein.

[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 readily engineered to alter the amino acid sequence of the protein. Genes encoding MIS proteins or functional fragments, homologs, or variants thereof can be engineered by a variety of known techniques for in vitro mutagenesis, among others, to produce variants of the naturally occurring human protein or fragments thereof, referred to herein as variants or muteins, and used in accordance with the present invention. Other modifications to the recombinant human MIS protein

[0173] Recombinant human MIS proteins useful in the present invention can also be modified, for example, at their amino termini to increase their hydrophilicity. Increasing hydrophobicity enhances exposure of the peptide on the surface of a lipid-based carrier in which the parent peptide-lipid conjugate is incorporated. Polar groups suitable for attachment to peptides to increase their hydrophilicity are known and include, for example, but are not limited to, acetyl ("Ac"), 3-cyclohexylalanyl ("Cha"), acetyl-serine ("Ac"). 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 carboxy sugars, such as lactobionic, N-acetylneuraminic acid, and sialic acid groups. The carboxy group of these groups may be linked to the N-terminus of the peptide via an amide bond. Currently, the preferred N-terminal modification is the methoxy-succinyl modification.

[0174] In certain embodiments, the recombinant human MIS protein can be fused to 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 can further include a second fusion partner, such as a purification or detection tag, such as a directly or indirectly detectable protein (e.g., green fluorescent protein, hemagglutinin, or alkaline phosphatase), a DNA binding domain (e.g., GAL4 or LexA), a gene activation 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 include a human Fc protein or a functional fragment thereof. Thus, in one embodiment, the recombinant human MIS protein fusion protein useful in the methods and compositions disclosed herein includes a human Fc molecule as a first fusion partner, where the Fc fragment may be SEQ ID NO:10 or a functional variant or derivative thereof. SEQ ID NO:10 is as follows: TIFF2024178382000040.tif32155

[0176] Variations and modifications to recombinant human MIS protein and vectors can be used to increase or decrease recombinant human MIS protein expression and provide a means for targeting. For example, recombinant human MIS protein can be linked to molecular targeting molecules to target cancer cells or ovarian cells, making recombinant human MIS protein cancer-specific or ovarian tissue-specific, respectively.

[0177] In one embodiment, the recombinant human MIS protein is fused to a second fusion partner, e.g., a carrier molecule to enhance bioavailability. Such carriers are known in the art and include poly(alkyl)glycols, e.g., 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 protein can also be fused to a second fusion partner, such as a polypeptide that targets the product to a desired location, or a tag that facilitates purification, for example, if necessary. In certain embodiments, the tag and fusion partner can be designed to be cleavable, if necessary. Another modification that is particularly contemplated is conjugation, for example, covalent attachment, to a polymer. In one aspect, polymers, such as polyethylene glycol (PEG) or methoxypolyethylene glycol (mPEG), can increase the in vivo half-life of the protein to which they are attached. Methods for PEGylation of polypeptide agents are known to those skilled in the art, and take into consideration, for example, how large a PEG polymer to use.

[0179] In certain embodiments, recombinant human MIS protein or its functional fragments are modified to achieve a suitable circulating half-life, which affects dosing, drug administration and efficacy. Many approaches have been taken to increase the half-life of biological therapeutics. Small proteins under 60 kD are quickly cleared by the kidney and therefore do not reach their target. This means that high dosages are required to achieve efficacy. Modifications to recombinant human MIS protein and its fragments included in the method of the present invention to increase the half-life of proteins in circulation include PEGylation; conjugation or gene fusion with proteins such as 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 their entirety.

[0180] In the case of PEGylation, polyethylene glycol (PEG) is attached to a recombinant human MIS protein or fragment, which can be, for example, a plasma protein, an antibody, or an antibody fragment. The first studies on the effect of PEGylation of antibodies were performed in the 1980s. Conjugation can be performed enzymatically or chemically and is well established in the art (Chapman, 2002; Veronese and Pasut, 2005). PEGylation increases the overall size, decreasing the chance of renal filtration. PEGylation also protects against proteolytic degradation and slows clearance from the blood. In addition, it has been reported that PEGylation can reduce immunogenicity and increase solubility. The improvement of pharmacokinetics by the addition of PEG is due to several different mechanisms: increasing the size of the molecule, protection from proteolytic degradation, reducing antigenicity, and masking certain sequences from cellular receptors. In the case of antibody fragments (Fab), a 20-fold increase in plasma half-life has been achieved with PEGylation (Chapman, 2002).

[0181] To date, there are several approved PEGylated drugs, 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. A limitation of PEGylation is the difficulty of synthesizing long monodisperse species, especially when PEG chains greater than 1000 kD are required. For many applications, monodisperse PEGs with chain lengths greater than 10,000 kD are used, resulting in a population of conjugates with PEG chains of different lengths, which requires extensive analysis to ensure equivalent batches per production. Different lengths of PEG chains may result in different biological activities and therefore different pharmacokinetics. Another limitation of PEGylation is reduced affinity or activity, as has been observed with the alpha-interferon Pegasys, which has only 7% of the native antiviral activity but improved pharmacokinetics due to increased plasma half-life.

[0182] In a particular embodiment, the recombinant human MIS protein or a fragment thereof is conjugated to a long-lived protein, 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 control, but also functions as a carrier of substances in plasma. In the case of CD4, an increase in plasma half-life has been achieved after fusing it to human serum albumin (Yeh et al., 1992). Further examples of fusion proteins are 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 by reference in their entireties).

[0183] The effect of glycosylation on plasma half-life and protein activity has been extensively studied. In the case of tissue plasminogen activator (tPA), the addition of new glycosylation sites 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 protein or a fragment thereof can be fused to the Fc fragment of IgG (Ashkenazi and Chamow, 1997). The Fc fusion approach is utilized, for example, in the Trap Technology (e.g., IL1 trap and VEGF trap) developed by Regeneron. The use of albumin to extend the half-life of peptides is described in US2004001827A1. The positive effect of albumin has also been reported for Fab fragments and scFv-HSA fusion proteins (Smith et al., 2001). It has been demonstrated that the extended serum half-life of albumin is due to the recycling process mediated by FcRn (Anderson et al., 2006; Chaudhury et al., 2003; Smith et al., 2001).

[0185] In a specific embodiment, recombinant human MIS protein is conjugated to a biotinylated Fc protein, as disclosed in US Application No. 2010 / 0209424, which is incorporated herein by reference in its entirety.

[0186] As used herein, the term "conjugate" or "conjugation" refers to the binding of two or more substances to form one substance. For example, the method of the present invention provides for the conjugation of a recombinant human MIS protein (i.e., SEQ ID NO: 2 or 3, or a fragment, derivative, or variant thereof) with another substance, such as a first fusion partner that stabilizes the recombinant human MIS protein, such as an Ig carrier particle, such as IgG1 Fc. The binding can be by linker, chemical modification, peptide linker, chemical linker, covalent or non-covalent bond, or protein fusion, or by any means known to those skilled in the art. The linkage can be permanent or reversible. In certain embodiments, several linkers can 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 alone or together with other linkers disclosed herein. Peptide linkers can be linked by expressing DNA that codes for the linker to one or more proteins in the conjugate.Linkers can be acid-cleavable, photocleavable, and heat-sensitive linkers.Methods for conjugation are known to those skilled in the art and are included for use in the present invention.

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

[0188] A wide variety of methods are known in the art for conjugating the recombinant human MIS protein disclosed herein with a first fusion partner (e.g., Fc). Such methods are described, for example, in Hermanson (1996, Bioconjugate Techniques, Academic Press), U.S. Patent No. 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 link haptens to carriers as conventionally used in applied immunology (see Harlow and Lane, 1988, "Antibodies: A laboratory manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). It is recognized that in certain cases, recombinant human MIS protein may lose efficacy or functionality upon conjugation, for example, depending on the conjugation procedure or chemical group utilized therein. However, given the wide variety of methods for conjugation, one skilled in the art can find a conjugation method that does not affect, or has the least effect on, the efficacy or functionality of the substance, such as recombinant human MIS protein, to be conjugated.

[0189] Suitable methods for conjugation of recombinant human MIS protein 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, a component 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 by reference in their entirety. Another method for conjugation that can be used is, for example, sodium periodate oxidation, and reductive alkylation and glutaraldehyde crosslinking following the appropriate reaction.

[0190] Those skilled in the art can use a variety of methods to conjugate the recombinant human MIS protein disclosed herein to a first fusion partner (e.g., Fc), such as, 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 that can bind two different functional targets to proteins and other macromolecules in a two- or three-step process that can limit the degree of polymerization often associated with the use of homobifunctional crosslinkers. Such multi-step protocols can provide great control over the size of the conjugate and the molar ratio of the constituents.

[0191] The term "linker" or "crosslinker" refers to any means of linking two or more substances, such as the recombinant human MIS protein disclosed herein, with a first fusion partner (e.g., Fc). The linker may be a covalent or non-covalent linker. Examples of covalent linkers include a covalent bond or a linker moiety covalently attached to the protein or proteins to be linked. The linker may be a non-covalent linker, such as an organometallic bond via a metal center, such as a platinum atom. A variety of functionalities can be used for covalent linkage, such as amide groups, such as carboxylic acid derivatives, ethers, esters, such as organic and inorganic esters, urethanes, ureas, etc. To provide for binding, the effector molecule and / or probe can be modified by oxidation, hydroxylation, substitution, reduction, etc. to provide a site for binding. It will be appreciated that modifications that do not significantly reduce the function of the recombinant human MIS protein disclosed herein or the first fusion partner (e.g., Fc) are preferred. targeting

[0192] In certain embodiments, the recombinant human MIS protein for use in the methods and compositions disclosed herein, or its functional fragment or homologue, can be targeted to cancer or ovarian cells via a targeting ligand. A targeting ligand is a molecule, e.g., a small molecule, protein or fragment thereof, that specifically binds with high affinity to a target, e.g., a cell surface marker on a preselected cell, e.g., a cell surface protein, e.g., a receptor that is present to a greater extent on the preselected cell target than on any other body tissue. Thus, in certain embodiments, the recombinant human MIS protein for use in the methods and compositions disclosed herein can be fused to Fc and / or optionally to a targeting molecule. In certain embodiments, the nucleic acid molecule encoding the targeting ligand can be fused to a nucleotide that encodes the recombinant human MIS protein, or a fragment, homologue, or variant thereof. Another example of a targeting ligand is a group of cadherin domains from human cadherin. The targeting ligand construct bound to the recombinant human MIS protein includes naturally occurring, recombinant, or engineered ligands or fragments thereof that can bind to preselected target cells.

[0193] Further examples of targeting ligands include, but are not limited to, antibodies and portions thereof that specifically bind to preselected surface proteins with high affinity. "High affinity" means an equilibrium dissociation constant of at least molar, as determined by assay methods known in the art, such as BiaCore analysis. In one embodiment, the targeting ligand may also include one or more immunoglobulin binding domains isolated from antibodies raised against a given tissue-specific surface protein or target tissue-specific receptor. The term "immunoglobulin" or "antibody" as used herein refers to a mammalian, including human, peptide or fragment thereof that specifically binds to and recognizes an antigen, which in the present case is a tissue-specific surface protein, tissue-specific receptor, or portion thereof, including a framework region from an immunoglobulin gene. If the intended targeting fusion polypeptide will be used as a mammalian therapeutic, the immunoglobulin binding region should be derived from the corresponding mammalian immunoglobulin. If the targeting fusion polypeptide is intended for non-therapeutic use, e.g., diagnostics and ELISA, the immunoglobulin binding region can be derived from either human or non-human, e.g., mouse. Human immunoglobulin genes or gene fragments include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant regions, as well as a myriad of immunoglobulin variable region genes. Light chains can be classified as either kappa or lambda. Heavy chains can be classified as gamma, mu, alpha, delta, or epsilon, which define the immunoglobulin classes 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 most important in determining the specificity and affinity of binding.

[0194] A typical immunoglobulin (antibody) structural unit of human IgG comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one light chain (about 25 kD) and one heavy chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100-110 or more amino acids that are primarily responsible for antigen recognition. The terms "variable light chain" (VL) and "variable heavy chain" (VH) refer to these light and heavy chains, respectively. Antibodies exist as intact immunoglobulins or as several well-characterized fragments produced by digestion with various peptidases. For example, pepsin digests antibodies under the disulfide bridge in the hinge region to produce F(ab)'2, a dimer of Fab, which is itself a light chain linked to VH-CH by a disulfide bond. F(ab)'2 can be reduced under mild conditions to break the disulfide bridges in the hinge region, thereby converting the F(ab)'2 dimer into a Fab' monomer. The Fab' monomer is essentially a Fab with a portion of the hinge region. Although various antibody fragments are defined with respect to the digestion of the complete antibody, one skilled in the art will recognize that such fragments can be synthesized de novo, either chemically or by using recombinant DNA technology. Thus, the term immunoglobulin or antibody as used herein also includes antibody fragments produced by the modification of a complete antibody or those synthesized de novo using recombinant DNA technology (e.g., single chain Fv (scFv)), or those identified using a phage display library (see, e.g., McCafferty et al. (1990) Nature 348:552-554). In addition, the fusion polypeptide of the present invention includes the variable regions of the immunoglobulin heavy (VH) or light (VL) chains, as well as tissue-specific surface proteins and their target receptor-binding portions. Methods for producing such variable regions are described in Reiter, et al. (1999) J. Mol. Biol. 290:685-698.

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

[0196] Screening and selection of preferred immunoglobulins (e.g., antibodies) can be performed by a variety of methods known in the art. Initial screening for the presence of tissue-specific or target receptor-specific monoclonal antibodies can be performed, for example, via the use of ELISA-based methods or phage display. A secondary screen is preferably performed to identify and select the desired monoclonal antibodies for use in constructing the tissue-specific fusion polypeptides of the invention. The secondary screening can be performed 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 desired characteristics. More specifically, monoclonal antibodies are classified into different epitope-related groups based on evaluation of antibody-antigen interactions. Purification of recombinant human MIS protein

[0197] The recombinant human MIS protein disclosed herein, as well as its functional fragments and derivatives, can be obtained by any suitable method. For example, the polypeptide can be produced using conventional recombinant nucleic acid technology, such as DNA or RNA, preferably DNA. Guidance and information on methods and materials for producing polypeptides using recombinant DNA technology can be found in numerous articles and reference manuals. For example, see 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 protein or a functional fragment thereof can be obtained directly by chemical synthesis, for example, by using a commercially available peptide synthesizer according to the vendor's instructions. Methods and materials for 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 a functional fragment, derivative, or variant thereof, can be expressed intracellularly following introduction of DNA encoding the protein, e.g., a nucleic acid encoding the human MIS protein, or a homolog or functional fragment thereof, e.g., in a conventional expression vector disclosed herein, or by a catheter, or by cells transformed with the nucleic acid ex vivo and implanted into a subject. 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 the Mullerian Inhibition Organ Culture Assay, described in Donahoe et al, J. Surg. Res., 23, 141-148, 1977. Urogenital ridges were excised from 14-day-old female rat fetuses and transferred to organ culture dishes (Falcon, 3010). The specimens were placed on stainless steel grids coated with a thin layer of 2% agar and incubated at 37°C in 5% CO2 and 95% air for 72 hours on 2 mL of culture medium [CMRL 1066 containing 10% fetal bovine serum, 1% penicillin (10,000 units / ml)] or a 1:1 mixture of culture medium and supernatant or gradient fraction to be tested. The incubated tissues were 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. Serial sections of 8 micrometers were stained with hematoxylin and eosin for viewing under light microscopy. Sections from the cephalic end of the Mullerian duct were assigned a code number and graded for regression on a scale of 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. Activity grades are listed as the nearest integer to the average. Test groups for the fractionation procedure represent at least 10 assays. If the average was between two numbers, both numbers are listed. Grade 0 indicates no regression. The Müllerian ducts, lined with columnar epithelial cells whose nuclei have a basal orientation, have wide patent lumens. Grade I is minimal regression. The ducts are a little smaller and the surrounding mesenchyme is either condensed around the ducts as seen in plastic sections or there are clear areas around the ducts as seen in paraffin sections. Grade II refers to mild regression. The ducts are smaller and the condensation of mesenchyme or clear areas around the ducts are more evident.The nuclei of the shorter epithelial cells lose their orientation at their base. Grade III is moderate regression. The ducts are extremely small and disorganized. The tip of the urogenital ridge is poorly developed distal to the Wolffian duct. Grade IV is severe regression. The ducts are replaced by whorls of cells. Grade V refers to complete regression. No remnants of the ducts can be observed. Positive tissue controls using fetal testes and negative tissue controls in which Müllerian ducts were incubated alone or with muscle were included in each experiment. Müllerian ducts were exposed to extracts from non-testis tissue, to inactive testis fractions, or to saline, which served as biochemical controls. Aliquots of all fractions were dialyzed against distilled water, lyophilized, and protein content was measured. Delivery of recombinant human MIS protein

[0201] Methods known in the art for therapeutic delivery of recombinant human MIS protein and / or nucleic acids encoding same can be used to treat a disease or disorder, such as cancer, in a subject, such as cell transfection, gene therapy, direct administration in a delivery vehicle or pharma- ceutically acceptable carrier, indirect delivery by providing recombinant cells containing a nucleic acid encoding a targeted fusion polypeptide of the invention.

[0202] In a specific embodiment, recombinant human MIS protein is cleaved in vitro to form a biologically active halo-dimer of MIS, which contains two identical monomers, each consisting of the N-terminal and C-terminal domains, and then administered to a subject.

[0203] A variety of delivery systems are known and can be used to administer recombinant human MIS protein to a subject (before or after it has been cleaved into its biologically active form), such as encapsulation in liposomes, 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), construction of the nucleic acid as part of a retrovirus or other vector, etc. The method of introduction can be enteral or parenteral, including, but not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, pulmonary, intranasal, intraocular, epidural, and oral routes. The recombinant human MIS protein can be administered by any conventional route, such as by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other biologically active agents. Administration can be systemic or local. Furthermore, it may be required to introduce the pharmaceutical composition containing the recombinant human MIS protein into the central nervous system, either before or after cleavage into its biologically active form, by any suitable route, for example, intraventricular and intrathecal injection. Intraventricular injection can be facilitated, for example, by an intraventricular catheter connected to a reservoir, for example, an Ommaya reservoir. Pulmonary administration can also be used, for example, by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. Methods for Treating Proliferative Diseases and Cancer - Patent application

[0204] One aspect of the invention provides a method for treating cancer, e.g., a cancer expressing MISRII, in a subject. Accordingly, one aspect of the invention relates generally to a method for treating a proliferative disease or disorder, wherein the proliferative disease or disorder is associated with cells expressing a MIS receptor, e.g., cells expressing MISRII. In a particular embodiment, the proliferative disease or disorder is a cancer, wherein the cancer or cancer cells express at least one MIS receptor, e.g., a cancer or cancer cell expressing MISRII. The method of the invention includes administering an effective amount of a recombinant human MIS protein or functional fragment thereof as disclosed herein to a subject having a proliferative disorder, wherein the cells associated with the proliferative disorder express at least one MIS receptor. For example, the cells express MISRII. For example, an effective amount of a recombinant human MIS protein or functional fragment thereof as disclosed herein is administered to a subject having a cancer expressing at least one MIS receptor, e.g., MISRII. Thus, the methods of the invention can be used to intervene in proliferative diseases, such as cancer, alleviate symptoms, and in certain cases, cure the disease. In certain embodiments, a recombinant human MIS protein that can be used to treat proliferative diseases and cancer comprises amino acid sequence residues 25-559 of SEQ ID NO:2, or a fragment thereof.

[0205] Examples of such diseases in which the proliferation of cells expressing at least one MIS receptor, such as MISRII, is the cause of the disease are cancers, such as cervical cancer and ovarian cancer. In certain embodiments, the cancer expressing at least one MIS receptor, such as MISRII, is a cancer cell. 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, and ovarian adenocarcinoma.

[0206] In another embodiment, the cancer expressing at least one MIS receptor, e.g., MISRII, is, for example, but not limited to, breast cancer, lung cancer, head and neck cancer, bladder cancer, stomach cancer, cancer of the nervous system, osteosarcoma, bone marrow cancer, brain tumor, colon cancer, esophageal cancer, endometrial cancer, gastrointestinal cancer, genitourinary cancer, stomach cancer, lymphoma, melanoma, glioma, bladder cancer, pancreatic cancer, gum 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, stomach cancer, testicular cancer, tongue cancer, or uterine cancer.

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

[0208] In certain embodiments, the cancer is MIS-responsive cancer, such as, but not limited to, ovarian cancer and cervical cancer.In certain embodiments, the cancer is MISRII-expressing cancer, such as, but not limited to, ovarian cancer and cervical cancer.In certain embodiments, the disorder is an excess androgen state-related disorder, such as, for example, as disclosed in U.S. Patent No. 6,673,352, which is incorporated herein by reference in its entirety.In certain embodiments, the method of the present invention is used to treat prostate cancer, polycystic ovarian disorder, benign prostatic hyperplasia, and precocious puberty.

[0209] In certain embodiments, the cancer is a chemotherapy-resistant or multidrug-resistant cancer, for example, where the cancer is a paclitaxel-, cisplatin-, rapamycin-, pyrazoloanthrone-, or doxorubicin-resistant cancer.

[0210] In a related embodiment, the tissue to be treated is a tumor tissue expressing at least one MIS receptor, e.g., MISRII in the subject. For example, the tumor tissue is a cancer, such as, but not limited to, a solid tumor, a metastasis, a skin cancer, a breast cancer, an ovarian cancer, a cervical cancer, a hemangioma, or an angiofibroma. Exemplary solid tumor tissue treatable by the pharmaceutical composition of the present invention includes, but is not limited to, tumors of tissues such as lung, pancreas, breast, intestine, larynx, ovary, etc. 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 contemplates the practice of administering a composition comprising a recombinant human MIS protein, or a functional fragment thereof, as disclosed herein, in combination with other therapies, such as conventional chemotherapy for solid tumors, and to inhibit the establishment of metastases. For example, the chemotherapeutic agents used in chemotherapy include, but are not limited to, paclitaxel, cisplatin, doxorubicin, rapamycin, pyrazoloanthrone, such as, but not limited to, anthra(1,9-cd)pyrazol-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 radiotherapy agent. The administration of the compounds disclosed herein is typically performed before, during, and / or after chemotherapy. However, it is also within the scope of the present invention to inhibit cell proliferation after a chemotherapy regimen, when the tumor tissue will respond to the toxic attack by inducing angiogenesis to restore blood and nutrient supply to the tumor tissue. Furthermore, if a subject has been identified as being at risk for developing cancer, for example, a subject who is positive for a biomarker for cancer cells or tumors, the pharmaceutical compositions of the present invention for treating a proliferative disease, for example, cancer, can be administered prophylactically and / or prior to tumor development. Insofar as the method is applied to inhibiting cell proliferation, it can also be applied to inhibiting tumor tissue growth, inhibiting tumor metastasis formation, and regressing established tumors.

[0212] In certain embodiments, expression of a Mullerian inhibitory substance (MIS) receptor is measured in a biological sample obtained from the subject, such as a cancer or tumor tissue sample or cancer or tumor cells, such as a biopsy tissue sample.

[0213] The presence of MISRII in cells in fluids such as blood may indicate the presence of cancer. The presence of MISRII in fluids or in sites that are not near tumors 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 that includes one or more therapeutic agents.

[0214] The methods of the invention disclosed herein provide for parenteral and oral administration of the recombinant human MIS protein disclosed herein, or functional fragments or derivatives thereof, in combination with other pharmaceutical compositions, to a subject 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 invention, the recombinant human MIS protein disclosed herein, or functional fragments or analogs thereof, is preferably administered orally. IV, IM, SC, and IP administration may be by bolus or infusion, and may be by sustained release implantable devices, including, 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. In general, the dosage administered by subcutaneous injection will be higher than the equivalent dosage of the treatment given intravenously or intramuscularly. Preferably, the dosage of the compound of the invention will be administered at a dosage of about 0.1 mg to about 250 mg. In certain embodiments, the dosage of the compound of the invention will be from about 1 mg to about 60 mg.

[0215] The methods of the present invention for treating cancers expressing at least one MIS receptor, e.g., MISRII, are useful for treating a proliferation-related disease or disorder associated with a cancer expressing at least one MIS receptor, e.g., MISRII, comprising contacting a tissue in which proliferation is occurring or at risk of occurring with a therapeutically effective amount of a recombinant human MIS protein, or functional fragment thereof, as disclosed herein.

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

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

[0218] Thus, the present invention relates to the use of recombinant human MIS protein or a functional derivative thereof for the treatment of any disorder, where administration of MIS protein or a nucleic acid encoding MIS protein or a functional derivative thereof or activation of MISRII is the whole or part of a treatment regimen. 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 a functional fragment thereof.

[0219] In a particular embodiment, the methods of the invention relate to the use of recombinant human MIS protein or a functional derivative thereof with other therapeutic agents, e.g., chemotherapeutic agents, where the chemotherapeutic agents, e.g., paclitaxel or MIS, can be used in lower dosages, thereby reducing side effects. Use of recombinant human MIS protein or functional derivatives or analogs thereof for the treatment of hyperandrogenic conditions - Patents.com

[0220] In another embodiment, recombinant human MIS protein or its functional derivative or analogue can be used for the treatment of disorders related to excess androgen production in a subject.The present inventors have previously demonstrated that administration of MIS protein and / or MIS nucleic acid reduces the level of androgen in a subject and reduces the serum level of androgen 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 that overexpress MIS have also been shown to have reduced serum testosterone concentrations, and administration of MIS 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 inhibit both androgen-stimulated proliferation and androgen-dependent survival of cells; MIS controls prostate growth by suppressing testicular testosterone synthesis and also directly regulates androgen-induced 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 control the development and maintenance of male characteristics in vertebrates by binding to the androgen receptor. Androgens, also known as androgenic hormones or testoids, are precursors of the female hormones estrogen. The primary and best known androgen is testosterone.

[0222] Without intending to be bound by theory, excess androgen production by the adrenal glands and / or ovaries may result in androgen excess, which may result from increased sensitivity of local tissues to circulating androgens. Androgen excess can affect different tissues and organ systems, resulting in overt virilization of clinical conditions ranging from acne to hirsutism.

[0223] Hyperandrogenism, which refers to the excessive production and secretion of androgens and their precursors, is a common and sometimes severe endocrine disorder in women of reproductive age. Excess androgens and precursors arise from the adrenal glands and ovaries in different proportions and manifest different effects depending on the amount of excess androgen. Clinical signs range from hirsutism (excessive male-pattern hair growth, sometimes accompanied by acne) to virilization (clitoromegaly, temporary balding, deepening of the voice, or increased musculature).

[0224] Hyperandrogenism occurs as part of a wide range of disease manifestations, including polycystic ovary syndrome (PCOS), a variable combination of hirsutism, infertility, obesity, insulin resistance, and polycystic ovaries; HAIR-AN syndrome (hyperandrogenism, insulin resistance, and acanthosis nigricans); theca cell hyperplasia (HAIR-AN with luteinizing theca cell tumors in the ovarian stroma); and other manifestations of high intraovarian androgen concentrations (e.g., arrested follicular maturation, atresia, anovulation, dysmenorrhea, dysfunctional uterine bleeding, infertility); and androgen-producing tumors (virilizing ovarian or adrenal tumors).

[0225] Hirsutism is excessive, recognizable hair growth characterized by an increase in the number and length of terminal hairs in androgen-sensitive areas. Race, ancestry, genetics, and ethnicity all influence the occurrence of hirsutism. Hirsutism is difficult to quantify; the entire body must be examined and findings must be carefully recorded; particular attention should be given to the chin, lips, sideburns, chest and sternum, midline between the navel and pubic bone, and thighs.

[0226] Ferriman and Gallwey have published a grading scale for grading hirsutism, which is commonly known to those skilled in the art. This system is the most widely used and evaluates body areas for hirsutism from "none" to "intense" with scores of 0 to 4, respectively. Scores of 8 and above are consistent with a diagnosis of hirsutism. This scale is another way of objectively assessing excess hair, without relying on hair thickness. The scoring system is a helpful aid in quantifying hirsutism and in assessing treatment response. Even a score above 8 gives the patient a definition. From a clinical perspective, the patient can decide whether he or she notices a difference. Photographs are useful for documentation and to follow the course of treatment.

[0227] Virilization is relatively uncommon. It occurs in the setting of marked hyperandrogenism and is characterized by temporary baldness, breast atrophy, androgenic muscular development, clitoral enlargement, 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 in certain cases is limited by the unwanted suppression of cortisol synthesis. GnRH therapy is directed at the ovaries, but is expensive and its long-term effects are unknown. Furthermore, therapy using oral contraceptives may be inappropriate since most contain progestins with androgenic activity.

[0229] Because abnormal production of androgens is associated with the pathways of many diseases and / or disorders for which no acceptable treatment exists, 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] Thus, in one embodiment, a recombinant human MIS protein or a functional derivative or analog thereof can be used for the treatment of a disorder associated with excess 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 a functional fragment thereof.

[0231] The term "androgen" means a steroid that promotes the development of male characteristics, and is used herein to include steroid derivatives of androstane, such as testosterone, androstanedione, and analogs.

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

[0233] As used herein, "androgen inhibition" refers to an effective amount of a pyrazoloanthrone or a functional derivative or analog thereof, as defined herein, such as SP600125, that will reduce in vivo levels of androgens to normal or below normal when administered to a subject for the prevention or treatment of a disease state exacerbated by or caused by excessive or uncontrolled androgen production.

[0234] In certain embodiments, the recombinant human MIS protein or its functional derivative or analogue 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 deprivation, 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)). In addition, 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 effect of the recombinant human MIS protein or its functional derivatives or analogs disclosed herein is reversible, the recombinant human MIS protein can also be used as a male contraceptive (Korolkovas, A., Essentials Of Medicinal Chemistry, Second Edition, pp.1032 (1988)).

[0235] In certain embodiments, other agents can be used in combination with pharmaceutical compositions comprising recombinant human MIS protein or functional derivatives or analogs thereof disclosed herein for the treatment of excess androgens in a subject. In certain embodiments, the agents function to reduce serum-free androgen levels and block peripheral androgen action. Examples of such agents include, but are not limited to, suppression of ovarian androgens and moderate estrogen therapy by administration of estrogens and / or progestins (i.e., birth control pills) or GnRH agonists, suppression of adrenal androgens by administration of glucocorticoids (e.g., dexamethasone, prednisolone), antiandrogens (e.g., spironolactone, flutamide, cyproterone acetate), 5α-reductase inhibitors (e.g., finasteride), bromocriptine, and insulin-sensitizing agents (e.g., metformin, thiazolidinediones).

[0236] Subjects amenable to treatment with a recombinant human MIS protein or a functional derivative or analog thereof according to the methods disclosed herein are those who have been identified as having a disease or disorder associated with excess androgen levels, e.g., a disorder, e.g., a disease or disorder selected from the group including, but not limited to, BPH, prostate cancer, benign prostatic hyperplasia, testicular cancer, androgen-dependent acne, male pattern baldness, precocious puberty, hyperandrogenemia, hirsutism, virilization, POCS, HIAR-AN syndrome, ovarian theca cell hyperplasia, follicular maturation arrest, atresia, anovulation, dysmenorrhea, dysfunctional uterine bleeding, infertility, and androgen-producing tumors.

[0237] In certain embodiments, subjects amenable to treatment with recombinant human MIS protein or its functional derivatives or analogs according to the methods disclosed herein are subjects with congenic adrenal hyperplasma, which can be generally identified by those skilled in the art. CAH is most typically an autosomal recessive disorder in which the enzyme 21-hydrolase is absent or functionally deficient. Alternatively, subjects with CAH may have loss and / or reduction of function of the 11α-hydrolase enzyme and / or the 3α-hydroxy-steroid dehydrogenase enzyme. When these enzymes are absent or function at low levels, the body cannot make adequate amounts of the adrenal steroid hormones, cortisol and aldosterone. High levels of ACTH ensue, which stimulates adrenal hyperplasia and hypersecretion 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. 11a-Hydroxylase deficiency is characterized by high levels of 11-deoxy-cortisol (compound S), which results in high levels of deoxycorticosterone (DOC), a mineralocorticoid. Hypertension and hypokalemia can be prominent features of 11a-hydroxylase deficiency. Another form of CH, 3a-hydroxy-steroid dehydrogenase deficiency, results in high levels of pregnenolone, 17-hydroxy-pregnenolone, and DHEA. This condition is fatal if undetected, as corticosteroids are not synthesized.

[0239] Partial deficiency of the above enzyme manifests after puberty and causes high levels of adrenal steroids through the same mechanism. The elevation is not as significant as that associated with congenital conditions, and this condition is called non-classical (adult or late onset) CAH. Therefore, in certain embodiments, the subject that is amenable to treatment with recombinant human MIS protein or its functional derivative or analog according to the methods disclosed herein is a subject with non-classical (adult or late onset) CAH.

[0240] In certain embodiments, subjects amenable to treatment with recombinant human MIS protein or its functional derivative or analogue by the methods disclosed herein are female subjects with testosterone levels of about 2.0 ng / mL (200 ng / dL, 8.92 nmol / L) or higher, or testosterone levels at least about 2.5 times the upper limit of the normal range. In certain embodiments, such subjects have Sertoli-Leydig cell tumors, hilar cell tumors, and lipoid cell (para-adrenal) tumors, which are the most common. Sertoli-Leydig cell tumors reach a palpable size 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 amenable to treatment with recombinant human MIS protein or a functional derivative or analog thereof according to the methods disclosed herein are subjects with tumors (adenomas, carcinomas) of the adrenal gland that secrete elevated levels of androgens. In such embodiments, subjects amenable to treatment according to the methods disclosed herein can be identified by having DHEAS levels of about 7 μg / mL (18 μmol / L) or greater.

[0242] Other subjects amenable to the methods of treatment of excess androgenic conditions disclosed herein 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 amenable to the methods of treatment of excess androgenic conditions disclosed herein include, for example, patients with mild androgenic disorders, such as, but not limited to, ovulatory PCOS (ovulatory hyperandrogenic subjects with polycystic ovaries on ultrasound), idiopathic hyperandrogenicism (ovulatory antiandrogenic subjects, but with normal ovaries on ultrasound), and idiopathic hirsutism (subjects with normal androgenic androgenic phenotype).

[0243] Reference testosterone and DHEAS levels are well known to those skilled in the art and are disclosed in Guay et al, International Journal of Impotence Research (2004) 16, 112-120, which is incorporated herein by reference in its entirety. In summary, normal androgen levels for women aged 20-49 years are in the following ranges: DHEAS; about 195.6-140.4 ug / dl; serum testosterone about 51.5-33.7 ng / dl and free testosterone 1.51-1.03 pg / ml. Thus, a subject amenable to treatment with a pyrazoloanthrone or a functional derivative or analog thereof according to the methods disclosed herein will 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, serum testosterone, or free testosterone compared to the highest range of normal values ​​for DHEAS (195.6 μg / dl), serum testosterone (51.5 ng / dl), or free testosterone (1.51 pg / ml). In certain embodiments, a subject amenable to treatment with a pyrazoloanthrone or a functional derivative or analog thereof according to the methods disclosed herein has at least about a 2-fold, at least about a 3-fold, at least about a 4-fold, at least about a 5-fold, at least about a 10-fold, or more increase in DHEAS, serum testosterone, or free testosterone compared to the highest range of normal values ​​for DHEAS (195.6 μg / dl), serum testosterone (51.5 ng / dl), or free testosterone (1.51 pg / ml).

[0244] DHEAS can be measured by one 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% (anrostenedione), 15% (9-hydroxyandrostenedione), 0.046% (estrone-3 sulfate), 0.55% (androsterone sulfate), 0.5% (DHEA), and negligible for all other tested steroids. Free testosterone can be measured by one skilled in the art using Coat a Count Kits of Diagnostic Products Corporation, Los Angeles, California, USA. Cross-reactivity has previously been measured: 0.41% (dihydrotestosterone), 0.01% (androstenedione), 0.10% (methyltestosterone), and 0.01% (all other tested steroids). Total serum testosterone levels can be measured by one 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 one of skill 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) x100 = FAI. Administration of the Pharmaceutical Composition

[0246] The recombinant human MIS protein or a derivative or functional fragment thereof can 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, skin (patch), or ocularly. The recombinant human MIS protein or a derivative or functional fragment thereof can be administered in any dosage or dosing regimen.

[0247] With respect to the therapeutic methods of the present invention, it is not intended that the administration of recombinant human MIS protein or polynucleotides encoding such recombinant human MIS protein or functional fragments thereof be limited to a particular form of administration, dosage, or frequency of administration. The present invention contemplates all forms of administration, for example, intramuscular, intravenous, intraperitoneal, intravesicular, intraarterial, intralesional, subcutaneous, or any other route sufficient to provide a suitable dosage to treat an autoimmune disease or immune-related disease disclosed herein. An effective amount, for example, a therapeutically effective amount, of recombinant human MIS protein can be administered to a patient in a single dose or multiple doses. When multiple doses are administered, the doses can be separated from each other by, 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 comprising a recombinant human MIS protein agent can be administered for, for example, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20 weeks, or more. For any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, for example, dosages of therapeutic agents can be increased if lower dosages do not provide sufficient therapeutic activity.

[0248] Although the attending physician will ultimately determine the appropriate amount and administration schedule, an effective amount of recombinant human MIS protein or a derivative or functional fragment thereof can be provided at a dosage of 0.0001, 0.01, 0.01 0.1, 1, 5, 10, 25, 50, 100, 500, or 1,000 mg / kg. Effective dosages can be extrapolated from dose-response curves obtained from in vitro or animal model test bioassays or systems. In certain embodiments, the dosage of recombinant human MIS protein is about 1 pg / kg to 10 mg / kg (of patient body weight), although lower or higher dosages can also be administered.

[0249] In certain embodiments, reference ranges for the dosage of recombinant human MIS are evaluated from the US Citation Group and described in Antimullerian Hormone (AMH), Serum from Mayo Medical Laboratories. Retrieved April 2012. In certain embodiments, female subjects can be administered the following dosages of recombinant human MIS: less than 5 ng / mL (younger women younger than 24 months), less than 10 ng / mL (women aged 24 months to 12 years), 1 to 10 ng / mL (women aged 13 to 45 years), and greater than 1 ng / mL (women over 45 years). In certain embodiments, male subjects can be administered the following dosages of recombinant human MIS: 15 to 500 ng / mL (males younger than 24 months), 7 to 240 ng / mL (males aged 24 months to 12 years), 0.7 to 20 ng / mL and greater than 1 ng / mL (males over 12 years). MIS measurements may be less accurate if the person being measured is vitamin D deficient.

[0250] Furthermore, because additivity, synergy, or competition has been demonstrated with MIS and rapamycin, AzadC, doxorubicin, cisplatin, and paclitaxel, the recombinant human MIS disclosed herein can be administered in combination with selective targeted therapies, for example, to achieve greater activity against ovarian cancer than the use of recombinant human MIS or the chemotherapeutic agent alone.

[0251] Dosages for a particular patient or subject can be determined by those skilled in the art using conventional considerations (e.g., by appropriate conventional pharmaceutical protocols). A physician can, for example, prescribe a relatively low dosage initially and then increase the dosage until an appropriate response is obtained. The dosage administered to a patient is sufficient to provide a beneficial therapeutic response in the patient over time, or, for example, to reduce symptoms or other appropriate activity, depending on the indication. The dosage is determined by the potency 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 condition of the patient, the autoimmune disease to be treated, and the weight and surface area of ​​the patient to be treated. The size of the dose is also determined by the existence, nature, and extent of any adverse side effects associated with the administration of a particular vector, formulation, etc. in a particular subject. The therapeutic composition comprising recombinant human MIS protein or its functional derivative or functional fragment is optionally tested in one or more suitable in vitro and / or in vivo animal models of disease to confirm efficient tissue metabolism and to assess dosage, according to methods known in the art, for example, in the Mullerian regression bioassay disclosed herein in the Examples. Known to those skilled in the art. In particular, dosage may be determined first by therapeutic vs. non-therapeutic activity, stability, or other suitable criteria (e.g., comparison of treated vs. untreated cells or animal models) in relevant assays. The formulation is administered at a ratio determined by the LD50 of the relevant formulation and / or the observation of side effects of recombinant human MIS protein or its functional derivative or functional fragment at various concentrations, as applicable to the majority or overall health of the patient. Administration may be in single or divided doses.

[0252] In determining the effective amount of recombinant human MIS protein or its functional derivative or functional fragment to be administered in the treatment or prevention of a disease, the physician will evaluate circulating plasma levels, formulation toxicity, and disease progression. The selected dosage level will depend on a variety of factors, such as the activity of the particular compound of the invention or its esters, salts, or amides used, the route of administration, the time of administration, the rate of excretion of the particular compound used, the duration of treatment, the compounds and / or materials used in combination with the particular compound used, the age, sex, weight, condition, general health, and previous medical history of the patient being treated, as well as factors known in the medical arts.

[0253] In certain embodiments, the recombinant human MIS proteins disclosed herein can be administered at dosages in accordance with good medical practice, taking into account the clinical condition of the individual patient, the site and method of administration, the schedule of administration, the age, sex, weight of the patient, and other factors known to medical practitioners.

[0254] Dosage regimens of compositions comprising recombinant human MIS protein or functional fragments or variants thereof disclosed herein can be adjusted to provide the optimum desired response (e.g., therapeutic or prophylactic response). For example, a single bolus can be administered, several divided doses can be administered over time, or the dosage can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in dosage unit form.

[0255] Furthermore, the actual dosage level of the recombinant human MIS protein in the pharmaceutical composition may be varied to obtain an amount of active ingredient that is not toxic to the subject and is effective to achieve the desired therapeutic response, composition, and mode of administration for a particular subject. The pharmaceutical composition comprising the recombinant human MIS protein or its functional fragment or variant disclosed herein may be a "therapeutically effective amount" and / or a "prophylactically effective amount". In general, the appropriate daily dosage of the composition comprising the recombinant human MIS protein or its functional fragment or variant disclosed herein will be the amount of recombinant human MIS protein that is the lowest dosage effective to obtain a therapeutic effect, such as reduction of the symptoms of a proliferative disorder or cancer disclosed herein. Such an effective amount will generally depend on the factors described above.

[0256] If desired, an effective daily dosage of a composition comprising a recombinant human MIS protein or a functional fragment or variant thereof disclosed herein can be administered in two, three, four, five, six or more separate sub-doses at appropriate intervals throughout the day, optionally in unit dosage forms.

[0257] The dosage level administered to a subject can be constant over the required period of time, e.g., at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 1 year, or at least 5 years, or alternatively, the dosage level administered to a subject can vary depending on the progression of the condition being treated.

[0258] Dosage values ​​may vary depending on the type and severity of the cancer to be alleviated. It should be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual needs and the professional judgment of the person administering and supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope and practice of the invention.

[0259] The efficacy and toxicity of a compound can be determined in cell cultures or experimental animals by standard pharmaceutical procedures, such as ED50 (a dose effective in 50% of the population) and LD50 (a dose lethal in 50% of the population). The dose ratio of toxicity to therapeutic effect is called the therapeutic index, which can be expressed as the ratio LD50 / ED50. Pharmaceutical compositions that exhibit large therapeutic indices are preferred. Suitable experimental models that can be used can be the use of the Mullerian regression bioassay disclosed herein in the Examples, or in vivo cancer models that are well 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 by reference in their entireties.

[0260] For example, a therapeutically effective amount can be first evaluated in cell culture assays or in an animal model, usually a mouse, rabbit, dog, or pig. An animal model can also be used to achieve the required concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in other subjects. In general, a therapeutically effective amount depends on the therapeutic effect required; for example, a therapeutically effective amount of recombinant human MIS protein can be evaluated in a mouse model of cancer or using the Mullerian Duct Regression bioassay disclosed herein in the Examples and Figure 4.

[0261] A physician or veterinarian skilled in the art can readily determine and prescribe the effective amount of pharmaceutical composition required. For example, a physician or veterinarian can disclose the dosage of the compound of the present invention used in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. It is also noted that humans are generally treated longer than mice or other experimental animals exemplified herein. The treatment has a length proportional to the length of the disease process and the effectiveness of the drug. The dosage can 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 protein (e.g., a protein or a nucleic acid encoding a recombinant human MIS protein or a fragment thereof) can be administered to humans and other animals for treatment by any suitable route of administration, such as orally, nasally, e.g., by spray, rectally, vaginally, parenterally, intravesically, and topically, by powder, ointment, or drops, e.g., buccal and sublingual.

[0263] After formulation in a pharma- ceutical acceptable carrier at the required dosage, 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 using any suitable means. In general, suitable means of administration include, but are not limited to, topical, oral, parenteral (e.g., intravenous, subcutaneous, or intramuscular), rectal, intravesical, intravaginal, intraperitoneal, ocular, or nasal routes.

[0264] In certain embodiments, it may be required to administer a pharmaceutical composition comprising recombinant human MIS protein to the area in need of treatment. This may be accomplished, for example, but not limited to, by local injection during surgery, by local application, for example, by injection, by catheter, or by implant, where the implant may be made of a porous, non-porous, or gelatinous material, including membranes, for example, sialastic membranes, fibers, or commercially available skin substitutes. In certain embodiments, the recombinant human MIS protein disclosed herein may be applied to muscle using topical creams, patches, intramuscular injections, and the like.

[0265] In certain embodiments, the recombinant human MIS protein can be administered to a subject orally (e.g., in a capsule, suspension, or tablet) or by parenteral administration. Conventional methods for oral administration include administering the recombinant human MIS protein in any one of the following: tablets, suspensions, solutions, emulsions, capsules, powders, syrups, and the like are available. Well-known techniques for delivering recombinant human MIS protein orally or intravenously and retaining biological activity are preferred. Parenteral administration can include, for example, intramuscular, intravenous, intraarticular, intraarterial, intrathecal, subcutaneous, or intraperitoneal administration. The recombinant human MIS protein can also be administered orally, transdermally, topically, by inhalation (intrabronchial, intranasal, oral inhalation, or intranasal drops), or rectally. Administration can be local or systemic as indicated. An agent, e.g., a nucleic acid encoding a recombinant human MIS protein or a functional fragment thereof, can also be delivered using a vector, e.g., a viral vector, by methods known to those skilled in the art.

[0266] When a composition comprising a recombinant human MIS protein or a functional fragment or variant thereof disclosed herein is administered parenterally, it will generally be formulated in a unit dosage injectable form (e.g., solution, suspension, emulsion). Pharmaceutical preparations suitable for injection include sterile aqueous solutions or dispersions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. The carrier can be a solvent or dispersion medium, including, for example, water, ethanol, polyol (e.g., glycol, propylene glycol, liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.

[0267] The term "dosage unit" form as used herein refers to a physically discrete unit suitable as a unitary dosage for a mammalian subject to be treated, where each unit contains a predetermined amount of active compound calculated to produce a desired therapeutic effect in association with a required pharmaceutical carrier. The details for the dosage unit forms of the present invention are determined by or directly depend on (a) the unique 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 technical field of synthesizing recombinant human MIS protein into an active agent for treating a susceptibility in an individual.

[0268] A pharma- ceutically 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 hypodermic needle or using an infusion pump. Pharmaceutical Compositions

[0269] In certain embodiments, the recombinant human MIS protein or functional fragments or variants thereof disclosed herein can be formulated in any suitable manner, for example, in a sterile injectable solution, which can be prepared, for example, by incorporating the recombinant human MIS protein in the required amount of an appropriate solvent with various other ingredients, as needed.

[0270] Pharmaceutical formulations of compositions comprising the recombinant human MIS protein or functional fragments or variants thereof disclosed herein can be administered to patients in any compatible carrier, such as injectable formulations containing 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, iontophoresis, polymer matrices, liposomes, and microspheres. Examples of delivery systems useful in the present invention include those provided in 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] Proper fluidity can be maintained, for example, by the use of a coating, such as 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. In addition, 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, it will be necessary to include isotonic agents, such as sugars, sodium chloride, and the like. Prolonged absorption of injectable pharmaceutical forms can be brought about by the use of agents that delay absorption, such as aluminum monostearate and gelatin. According to the present invention, however, any vehicle, diluent, or additive used would have to be compatible with the compound.

[0272] In another embodiment, the composition comprising the recombinant human MIS protein or its functional fragment or variant disclosed herein may comprise a lipid-based formulation. Any of the well-known lipid-based drug delivery systems can be used to practice the present invention. For example, multivesicular liposomes, multilamellar liposomes, and unilamellar liposomes can all be used as long as they can establish a sustained release rate of the encapsulated active compound. Methods for making controlled release multivesicular liposome drug delivery systems 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 usually a combination of phospholipids, usually with steroids, especially cholesterol.Other phospholipids or other lipids can also be used.Examples of lipids useful for synthetic membrane vesicle production include phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides, in preferred embodiments, for example, phosphatidylcholine, dipalmitoylphosphatidylcholine, distearylphosphatidylcholine, dioleylphosphatidylcholine, dipalmitoylphosphatidylglycerol, and dioleylphosphatidiglycerol.

[0274] In preparing lipid-based vesicles containing recombinant human MIS protein or a functional fragment or variant thereof, variables such as efficacy of active compound encapsulation, lability of the active compound, uniformity and size of the resulting vesicle population, active compound to lipid ratio, permeability, instability of the preparation, and pharmaceutical acceptability of the formulation should be considered.

[0275] In another embodiment, recombinant human MIS protein can be delivered in a vesicle, in particular a liposome (see Langer (1990) Science 249:1527-1533). In yet another embodiment, recombinant human MIS protein can be delivered in a controlled release system. In one embodiment, a pump can be used (see Langer (1990) supra). In another embodiment, a polymeric material can be used (see Howard et al. (1989) J. Neurosurg. 71:105). In another embodiment, where the active agent of the invention is a nucleic acid encoding a recombinant human MIS protein, the nucleic acid can be administered in vivo to promote expression of the protein encoded therein by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, for example, by use of a retrovirus (see, e.g., U.S. Patent No. 4,980,286), or by direct injection, or by use of particle bombardment (e.g., gene gun, Biolistic, Dupont), or by coating with lipids or cell surface receptors or transfecting agents, or by administering it linked to a homobox-like peptide known to enter the nucleus (see, e.g., Joliot et al., 1991, Proc. Natl. Acad. Sci. USA 88:1864-1868).

[0276] Prior to introduction, a composition comprising a recombinant human MIS protein or a functional fragment or variant thereof disclosed herein can be sterilized by any of a number of techniques available 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 can 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 is preferably formulated as a pharmaceutical composition. The pharmaceutical composition of the present invention comprises a compound of the present invention and a pharma- ceutical acceptable carrier, wherein the compound is present in the composition in an amount useful for treating the condition in question. Appropriate concentrations and dosages can be readily determined by those skilled in the art.

[0278] Pharmaceutically acceptable carriers are known to those skilled in the art. For compositions formulated as liquid solutions, acceptable carriers include saline and sterilized water, and may optionally include antioxidants, buffers, bacteriostatic agents, and other additives. Compositions can also be formulated as pills, capsules, granules, 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 can further formulate the compounds of the present invention in an appropriate manner and according to accepted practices, for example, as disclosed in Remington's Pharmaceutical Sciences, Gennaro, Ed., Mack Publishing Co., Easton, Pa. 1990.

[0279] The compositions of the present invention can be in any form, including but not limited to solutions, suspensions, dispersions, ointments (including oral ointments), creams, pastes, gels, powders (toothpastes), toothpastes, lozenges, salves, chewing gums, mouth sprays, pastilles, sachets, mouthwashes, aerosols, tablets, capsules, transdermal patches, which contain one or more resolvins and / or protectins, or analogs thereof, of the present invention.

[0280] Formulations of compositions comprising recombinant human MIS protein or functional fragments or variants thereof disclosed herein can be prepared by several means known to those skilled in the art. In certain embodiments, the formulations can be prepared for administration as aerosol formulations, for example, by combining (i) a sufficient amount of recombinant human MIS protein or functional fragments or variants thereof disclosed herein to provide multiple therapeutically effective doses, (ii) the addition of water in an amount effective to stabilize each of the formulations, (iii) a propellant in an amount sufficient to spray multiple doses from an aerosol canister, and (iv) any additional optional components, such as ethanol as a co-solvent, to disperse the compound. The components can be dispersed using a conventional mixer or homogenizer, by shaking, or by ultrasonic energy. The bulk formulation can be transferred to smaller individual aerosol vials using valve-valve displacement methods, pressure-fed injection, or by conventional cold-fill methods. It is not required that the stabilizers used in suspension aerosol formulations be made available in the propellant. Those that are not sufficiently soluble can be coated onto the drug particles in an appropriate amount and the coated particles can then be incorporated into the formulation as described above.

[0281] In certain embodiments, compositions comprising the recombinant human MIS protein disclosed herein can be administered to a subject as a pharmaceutical composition together with a pharma- ceutically 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 drug, 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,Aristospan Intralesional Inj, betamethasone oral, methylprednisolone sod suc (PF) Inj, hydrocortisone sod succ (PF) Inj, Solu-Cortef (PF) Inj, prednisolone acetate oral, dexamethasone in 0.9% NaCl IV, Rayos, levothyroxine. Of course, such therapeutic agents are known to those of skill in the art and can be readily substituted by those of skill in the art. They should not be construed as being exclusive or limiting.

[0282] Wetting agents, emulsifiers, and lubricants, such as sodium lauryl sulfate and sodium stearate, as well as coloring agents, dissociating agents, coating agents, sweeteners, flavorings, fragrances, preservatives, and antioxidants may also be present in the composition. Examples of pharma-ceutically acceptable antioxidants include water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc., oil-soluble antioxidants, such as ascorbyl 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 may conveniently be provided in unit dosage form and may be prepared by any method known to those skilled in the art of pharmacy. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutically effective amount. Generally, out of one hundred percent, this amount will range from about 1 percent to about 99 percent of the active ingredient, preferably from about 5 percent to about 70 percent, and most preferably from 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, typically with sucrose and acacia or tragacanth), powder, granules, or aqueous or non-aqueous liquid solutions or suspensions, oil-in-water or water-in-oil liquid emulsions, elixirs or syrups, or pastilles (inert, e.g., with gelatin and glycerin, or sucrose and acacia), and / or mouthwashes, etc., each containing a predetermined amount of a compound of the present invention as an active ingredient. The compounds of the present invention may also be administered as a bolus, electuary, or paste.

[0285] In the solid dosage forms of the present invention for oral administration (capsules, tablets, pills, dragees, powders, granules, etc.), the active ingredient is mixed with one or more pharma- ceutically acceptable carriers, such as sodium citrate, dicalcium phosphate, and / or the following ingredients: fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; humectants, such as glycerol; In the case of capsules, tablets, and pills, the pharmaceutical composition may also include cyclic agents.Similar types of solid compositions may also be used as fillers in soft and hard-filled gelatin capsules, using excipients such as lactose or milk, and high molecular weight polyethylene glycols, etc.

[0286] A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binders (e.g., gelatin or hydroxypropylmethylcellulose), lubricants, inert diluents, preservatives, disintegrants (e.g., sodium starch glycolate or cross-linked carboxymethylcellulose), surface-active or dispersing agents. Molded tablets may be made by molding, in a suitable machine, the powdered compound moistened with an inert liquid diluent.

[0287] Tablets and other solid dosage forms of the pharmaceutical composition of the present invention, such as dragees, capsules, pills, and particles, can be optionally provided with or prepared with coatings and shells, such as enteric coatings and other coatings known in the pharmaceutical formulation art. They can also be formulated to provide slow or controlled release of the active ingredient therein, for example, using various proportions of hydroxypropylmethylcellulose, other polymer matrices, liposomes, and / or microspheres to provide the required release profile. They can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions can also optionally contain opacifying agents, and they can be compositions that release the active ingredient alone or preferably in a certain part of the gastrointestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.

[0288] Liquid dosage forms for oral administration of the compounds of the invention include pharma- ceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs.

[0289] In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or solvents, solubilizing and emulsifying agents, 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, corn, germ, olive, castor oil, and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof. In addition to the inert diluents, oral compositions may also contain adjuvants, such as wetting agents, emulsifying and suspending agents, sweeteners, flavoring agents, coloring agents, perfumes, and preservatives.

[0290] Suspensions may contain, in addition to the active compounds, suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.

[0291] In certain examples, compositions comprising a recombinant human MIS protein or functional fragment or variant thereof disclosed herein may be in a formulation suitable for rectal or vaginal administration, e.g., a formulation suitable for 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, including, for example, cocoa butter, polyethylene glycol, a suppository wax, or a salicylate, that are solid at room temperature but liquid at body temperature, thereby releasing the active compound. Suitable carriers and formulations for such administration are known in the art.

[0292] Dosage forms for topical or transdermal, e.g., intramuscular, administration of the recombinant human MIS protein of the present invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The recombinant human MIS protein disclosed herein or a functional fragment or variant thereof can be mixed under sterile conditions with a pharma- ceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.

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

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

[0295] Pharmaceutical compositions of the present invention suitable for parenteral administration contain one or more compounds of the present invention in combination with one or more pharma- ceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, or sterile powders that can be reconstituted immediately before use into sterile injectable solutions or dispersions, which may contain antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[0296] Examples of 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 such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0297] These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be necessary to include isotonic agents, for example, sugars, sodium chloride, and the like, in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption, for example, aluminum monostearate and gelatin.

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

[0299] Injectable depot forms are made by forming microencapsulated matrices of the subject compounds in biodegradable polymers, such as polylactide-polyglycolide. The rate of drug release can be controlled depending on the ratio of drug to polymer and the nature of the particular polymer used. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

[0300] In certain embodiments, the recombinant human MIS protein or functional fragment or variant thereof 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 more than 99%. In certain embodiments, naturally occurring compounds are excluded from the broader genus general description.

[0301] In certain embodiments, the composition comprises at least one recombinant human MIS protein in combination with a pharma- ceutically acceptable carrier. Specific examples of materials which can function as pharma- ceutically 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 wax, 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 laurate, agar, buffers such as magnesium hydroxide and aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, phosphate buffers, and other non-toxic compatible substances used in pharmaceutical formulations.

[0302] In certain embodiments, compositions comprising the recombinant human MIS protein or functional fragments or variants thereof disclosed herein may contain one or more acidic functional groups, which can form pharma- ceutically acceptable salts with pharma- ceutically acceptable bases. As used herein, the term "pharma- ceutically acceptable salts, esters, amides, and prodrugs" refers to those carboxylate salts, amino acid addition salts, esters, amides, and prodrugs of the compounds of the invention that are within the scope of sound medical judgment and are suitable for use in contact with the tissues of a patient, effective for the intended use of the compounds of the invention, without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit / risk ratio. The term "salts" refers to the relatively non-toxic inorganic and organic acid addition salts of the compounds of the invention.

[0303] These salts can be prepared in situ during the final isolation and purification of the compound, or by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed. These can 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 by reference in its entirety).

[0304] The term "pharmaceutical acceptable esters" 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 separately reacting the purified compound in free acid form or hydroxyl with a suitable esterifying agent. Carboxylic acids can be converted to esters via treatment with an 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, a "pharmaceutically acceptable salt or prodrug" is a salt or prodrug that is effective for the intended use, commensurate with a reasonable benefit / risk ratio, without undue toxicity, irritation, allergic response, etc., and that is suitable for use in contact with the tissues of a patient, within the scope of sound medical judgment. These compounds include zwitterionic forms of the r compounds of the invention, where possible.

[0306] The term "salts" 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 separately reacting the purified compound in free base form with a suitable organic or inorganic acid and isolating the salt thus formed. These can 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 by reference in its entirety).

[0307] The term "prodrug" refers to a compound or agent that is rapidly converted in vivo to produce an active recombinant human MIS protein, e.g., 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, the recombinant human MIS protein prodrug can be activated by hydrolysis in blood, e.g., through cleavage of the leader sequence or the first cleavage site that generates the N-terminal and C-terminal domains for the production of a biologically active MIS protein, similar to the activation of insulin from a proprotein to an active insulin protein. A detailed discussion is provided in T. Higashi 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 of which are incorporated herein by reference in their entirety). As used herein, a prodrug is a compound that, upon in vivo administration, is metabolized or converted into a biologically, pharma- ceutical or therapeutically active form of the compound. Prodrugs may be designed to alter the metabolic stability or transport properties of recombinant human MIS protein to block side effects or toxicity, or to alter other characteristics or properties of recombinant human MIS protein.

[0308] Based on knowledge of the pharmacokinetic process of MIS in vivo and drug metabolism or post-translational protein processing, once a pharmacologic active compound is identified, one skilled in the art can generally design a recombinant human MIS protein prodrug that can be activated in vivo to increase the level of biologically active MIS protein in a subject (see, for example, Nogrady (1985) Medicinal Chemistry A Biochemical Approach, Oxford University Press, NY, pages 388-392). Routine procedures for the selection and preparation of suitable 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 protein or functional fragments or variants thereof disclosed herein can be conjugated or covalently linked to a targeting agent to increase their tissue specificity and targeting to cells, e.g., muscle cells. Targeting agents can include, for example, 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, e.g., muscle cells, compared to non-muscle cells.

[0310] Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrate form, and / or pharmaceutical compositions of the present invention, are formulated into pharma- ceutically acceptable dosage forms by conventional methods known to those skilled in the art. Gene therapy

[0311] In certain embodiments, a nucleic acid encoding a recombinant human MIS protein or functional fragment thereof disclosed herein may be suitably administered as a vector, for example a viral vector.

[0312] In certain embodiments, nucleic acids encoding recombinant human MIS protein can be effectively used in gene therapy treatments. See, for example, generally, U.S. Patent No. 5,399,346, which is incorporated herein by reference in its entirety. The general principle is to introduce the polynucleotide into target cells in the patient, where it is transcribed into the protein.

[0313] Transfer into the cell can be facilitated by any suitable technique known in the art, for example by providing the polynucleotide in the form of a suitable vector, or by encapsulating the polynucleotide in a liposome.

[0314] The desired aspect of gene therapy is to provide a polynucleotide in such a way that it will replicate in cells and enhance and prolong the desired effect, whereby the polynucleotide is operably linked to a suitable promoter, e.g., the native promoter of the corresponding gene, a heterologous promoter that is endogenously active in liver, nerve, bone, muscle, skin, joint, or cartilage cells, or a heterologous promoter that can be induced by a suitable agent.

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

[0316] Alternatively, in certain embodiments, a plasmid expression vector can be used. Plasmid expression vectors include, but are not limited to, pcDNA3.1, pET vectors (Novagen®), pGEX vectors (GE Life Sciences), and pMAL vectors (New England labs. Inc.) (for protein expression in E. coli host cells, such as BL21, BL21(DE3) and AD494(DE3)pLysS, Rosetta (DE3), and Origami(DE3) (Novagen®); the strong CMV promoter-based pcDNA3.1 (Invitrogen® Inc.) and pCIneo vectors (Promega) (for expression in mammalian cell lines, such as CHO, COS, HEK-293, Jurkat, and MCF-7); the replication-incompetent adenovirus vectors pAdeno X, pAd5F35, pLP-Adeno-X-CMV (Clontech®), pAd / CMV / V5-DEST, pAd-DEST vectors (Invitrogen (tm) Inc.) (for adenovirus-mediated gene transfer and expression in mammalian cells); pLNCX2, pLXSN, and pLAPSN retroviruses (retroviral vectors for use with the Retro-X (tm) system from Clontech for retroviral-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, such as 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™ Inc.) (for expression in Spodoptera frugiperda 9 (Sf9) and Sf11 insect cell lines); pMT / BiP / V5-His (Invitrogen™ Inc.) (for expression in Drosophila Schneider S2 cells); Pichia expression vectors pPICZα, pPICZ, pFLDα and pFLD (Invitrogen™ Inc.) (for expression in Pichia pastoris), vectors pMETα and pMET (for expression in P. methanolica); pYES2 / GS and pYD1 (Invitrogen™ Inc.) vectors (for expression in the yeast Saccharomyces cerevisiae). Recent progress in large-scale expression heterologous proteins in Chlamydomonas reinhardtii is 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 by heterologous recombination. The chloroplast expression vector p64, which carries the most versatile chloroplast selectable marker aminoglycoside adenyltransferase (aadA), conferring resistance to spectinomycin or streptomycin, can be used to express foreign proteins in chloroplasts. Biolistic gene gun methods are used to introduce the vector into algae. Upon transfer into chloroplasts, the foreign DNA is released from the gene gun particles and integrated into the chloroplast genome by homologous recombination.

[0317] The viral vector systems that can be used in the present invention include, but are not limited to, (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, such as orthopox, e.g., vaccinia virus vectors or avipox, e.g., canarypox, or fowlpox, and (j) helper-dependent or gutless adenoviruses. In a preferred embodiment, the vector is an adenovirus. Replication-defective viruses can also be free.

[0318] The vector may or may not integrate into the cellular genome. The construct may optionally include viral sequences for transfection. Alternatively, the construct may be integrated into a vector capable of episomal replication, such as EPV and EBV vectors.

[0319] Constructs, e.g., DNA, MOD-RNA or RNAa, for expression of a nucleic acid encoding a recombinant human MIS protein disclosed herein can generally be operably linked to regulatory sequences, e.g., promoters, enhancers, etc., to ensure expression of the construct in a target cell. Other details of vectors and other constructs are described in more detail below.

[0320] Exemplary regulatory sequences include, but are not limited to, transcriptional promoters, inducible promoters, and transcriptional elements, optional operative sequences for controlling transcription, sequences encoding suitable mRNA ribosomal binding sites, 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 operably linked, such as initiation signals, enhancers, and promoters. In certain instances, transcription of a recombinant gene is under the control of a promoter sequence (or other transcriptional control sequence) that controls expression of the recombinant gene in a cell type in which expression is intended. It will also be understood that a recombinant gene can be under the control of transcriptional control sequences that are the same as or different from those sequences that control the transcription of naturally occurring proteins. In certain instances, a promoter sequence is recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate transcription of a particular gene.

[0321] The regulatory sequence may be a single regulatory sequence, or multiple regulatory sequences, or modified regulatory sequences, or fragments thereof. Modified regulatory sequences are regulatory sequences whose nucleic acids have been altered or modified by specific means, including but not limited to, mutation, methylation, etc. Regulatory sequences useful in the methods disclosed herein are promoter elements that are sufficient to render promoter-dependent gene expression controllable for cell type-specific, tissue-specific, or inducible by external signals or agents (e.g., enhancers or repressors). Such elements may be located within the 5' or 3' regions of the native gene, or within introns.

[0322] As used herein, the term "tissue-specific promoter" refers to a nucleic acid sequence that functions as a promoter, i.e., controls the expression of a selected nucleic acid sequence operably linked to it, and selectively affects expression of the selected nucleic acid sequence in particular cells of a tissue, e.g., cells of ovarian origin.

[0323] The term "constitutively active promoter" refers to a promoter of a gene that is constantly expressed in a given cell. Exemplary promoters for use in mammalian cells include cytomegalovirus (CMV), those for use in prokaryotic cells include bacteriophage T7 and T3 promoters, and the like. The term "inducible promoter" refers to a promoter of a gene that can be expressed in response to a given signal, e.g., the addition or removal of an agent. Non-limiting examples of inducible promoters are "tet-on" and "tet-off" promoters, or promoters that are regulated in a particular tissue type.

[0324] In certain embodiments, viral vectors containing a nucleic acid sequence, e.g., DNA, MOD-RNA or RNAa, encoding the recombinant human MIS protein or a functional fragment thereof disclosed herein 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 necessary constructs for correct packaging of the viral genome and integration into the host cell DNA. The nucleic acid sequence encoding the recombinant human MIS protein is cloned into one or more vectors, which facilitates delivery of the gene to the patient. More details about retroviral vectors can be found in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of retroviral vectors to deliver 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 are Clowes 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 carrying genes of interest is typically carried out in two steps. First, a sequence encoding a recombinant human MIS protein, or a functional derivative, variant, or fragment thereof, alone or fused to -Fc, can be inserted into a retroviral vector that contains metabolic regulators (including promoter and / or enhancer elements, which may be provided by viral long terminal repeats (LTRs) or by internal promoters / enhancers and associated splicing signals), sequences required for efficient expression of viral RNA into infectious virions (e.g., packaging signal (Psi), tRNA primer binding site (-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 associated with packaging the expression of genomic RNA into viral particles.

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

[0327] Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are particularly attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia and cause a 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-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) provide a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrate the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Another preferred viral vector is a poxvirus, such as a vaccinia virus, such as an attenuated vaccinia, such as Modified Virus Ankara (MVA) or NYVAC, an avipox, such as fowlpox or canarypox. Other examples of the use of adenovirus 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, lentiviral vectors are used, such as HIV-based vectors described in U.S. Patent Nos. 6,143,520; 5,665,557; and 5,981,276, the entireties of which are incorporated by reference herein. In certain embodiments, viral vectors are used, such as adeno-associated virus (AAV) vectors.Exemplary AAV vectors are described in Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Patent No. 5,436,146, which is incorporated by reference in its entirety; 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 The AAV vector is disclosed in J. Sambrook. In certain embodiments, the AAV vector is AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.10, 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, when 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 can be constant for a required period of time, e.g., at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 1 year, or at least 5 years. In certain embodiments, the expression of the recombinant human MIS protein disclosed herein can be maintained at or above a therapeutically effective level for a required period of time.

[0329] Another approach to gene therapy involves transferring genes to cells in tissue culture by methods such as electroporation, lipofection, calcium phosphate transfe...

Claims

1. A recombinant Mullerian inhibitory substance (MIS) protein comprising amino acid residues 25 to 559 of SEQ ID NO:2, said recombinant MIS protein not containing a FLAG tag.

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

2.

3. A recombinant Mullerian inhibiting substance (MIS) protein comprising an amino acid sequence having at least 98% sequence identity to amino acid residues 25 to 560 of SEQ ID NO:1, wherein amino acid residue 450 of SEQ ID NO:1 is changed from Q to R, and the recombinant MIS protein does not contain a FLAG tag.

4. The recombinant MIS protein described in claim 3, wherein the recombinant MIS protein is a homodimer comprising two monomers, each monomer containing residues 25 to 560 of SEQ ID NO:1, and wherein 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 of claim 1 and a pharma- ceutically acceptable carrier.

6. A pharmaceutical composition for the treatment of infertility, comprising the recombinant MIS protein of claim 2 and a pharma- ceutically acceptable carrier.

7. A pharmaceutical composition for the treatment of infertility, comprising the recombinant MIS protein of claim 3 and a pharma- ceutically acceptable carrier.

8. A pharmaceutical composition for treating infertility, comprising the recombinant MIS protein of claim 4 and a pharma- ceutically acceptable carrier.

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

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

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

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

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