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JP-7857367-B2 - Modified Müllerian duct inhibitor (MIS) proteins and their use for the treatment of diseases

JP7857367B2JP 7857367 B2JP7857367 B2JP 7857367B2JP-7857367-B2

Inventors

  • ドナフー パトリシア ケイ．
  • バッバス デメトリオス
  • ペピン デービッド
  • ホアン ミアン ヴァン

Assignees

  • ザ ジェネラル ホスピタル コーポレイション
  • マサチューセッツ・アイ・アンド・イア・インファーマリー

Dates

Publication Date
20260512
Application Date
20240925
Priority Date
20130312

Claims (12)

  1. A recombinant mature Müllerian duct inhibitor (MIS) protein comprising amino acid residues 25-559 of Sequence ID No. 2, wherein the recombinant MIS protein does not contain a FLAG tag.
  2. The recombinant MIS protein according to claim 1, wherein each monomer is a homodimer containing two monomers, each monomer containing residues 25-559 of SEQ ID NO: 2.
  3. A recombinant mature Müllerian inhibitor (MIS) protein comprising an amino acid sequence having at least 98% sequence identity with amino acid residues 26 to 560 of SEQ ID NO: 1, wherein amino acid residue 450 of SEQ ID NO: 1 is changed from Q to R, the FLAG tag is not included, and the recombinant MIS protein exhibits increased cleavage and increased production yield in vitro compared to a wild-type MIS protein having the amino acid sequence of SEQ ID NO: 1 .
  4. The recombinant MIS protein according to claim 3, wherein the recombinant MIS protein is a homodimer comprising two monomers, each monomer containing residues 26 to 560 of SEQ ID NO: 1, and amino acid residue 450 of SEQ ID NO: 1 is changed from Q to R.
  5. A pharmaceutical composition for the treatment of infertility, comprising the recombinant MIS protein described in claim 1 and a pharmaceutically acceptable carrier.
  6. A pharmaceutical composition for the treatment of infertility, comprising the recombinant MIS protein described in claim 2 and a pharmaceutically acceptable carrier.
  7. A pharmaceutical composition for the treatment of infertility, comprising the recombinant MIS protein described in claim 3 and a pharmaceutically acceptable carrier.
  8. A pharmaceutical composition for the treatment of infertility, comprising the recombinant MIS protein described in claim 4 and a pharmaceutically acceptable carrier.
  9. Use of the recombinant MIS protein according to claim 1 in the manufacture of a pharmaceutical product for the treatment of infertility.
  10. Use of the recombinant MIS protein according to claim 2 in the manufacture of a pharmaceutical product for the treatment of infertility.
  11. Use of the recombinant MIS protein according to claim 3 in the manufacture of a pharmaceutical product for the treatment of infertility.
  12. Use of the recombinant MIS protein according to claim 4 in the manufacture of a pharmaceutical product for the treatment of infertility.

Description

This application, which cross-references a related application , claims the benefit under § 119(e) of U.S. Patent Act 61/777,135, filed March 12, 2013, whose contents are incorporated in their entirety by reference. This sequence listing application has been submitted electronically in ASCII format and includes sequence listings incorporated herein by reference in their entirety. An ASCII copy thereof, created on 12 March 2014, is named 030258-076964-PCT_SL.txt and is 28,114 bytes in size. Field of Invention The present invention relates to an improved recombinant human MIS protein having improved cleavage, as well as increased bioactivity and increased potency compared to wild-type human MIS protein. In certain embodiments, the recombinant human MIS protein comprises at least one of the following components: an improved Kex cleavage site for increased cleavage, a FLAG tag, and a non-MIS reader sequence replacing a normal MIS reader sequence. Another aspect of the present invention relates to methods, uses, and kits comprising a recombinant human MIS protein for the treatment of cancer, such as cancer expressing MIS receptor type II (MISRII), or diseases characterized by excess androgens. Government-backed invention was made with government support under authorization number CA17393 granted by the National Institutes of Health (NIH). The government has specific rights to this invention. Background of the Invention: Müllerian inhibitors (MIS), also known as anti-Müllerian hormones (AMH), are 140 kDa disulfide-bonded homodimer glycoprotein members of the large transforming growth factor-β (TGFβ) multigene family of glycoproteins. All proteins in this family are produced as dimeric precursors and undergo post-transcriptional processing for activation requiring cleavage and dissociation to release a bioactive C-terminal fragment. The human MIS gene is located on chromosome 19, and its expression is sexually dimorphic. In males, MIS expression begins in the fetal testes at 9 weeks of gestation and remains at high levels until puberty, when expression levels dramatically decline. In females, MIS is produced only after birth in granulosa cells, at levels similar to adult males, from pre-puberty through menopause, after which expression ceases. In male fetuses, MIS causes regression of the Müllerian duct, the precursor of the Fallopian duct, the uterus, the cervix, and the upper third of the vagina. MIS exerts its biological effects after binding to heterodimers of type I and type II single transmembrane serine/threonine kinase receptors, leading to cross-phosphorylation of the GS-box kinase domain of the type I receptor by the type II receptor. Subsequently, SMAD1, 5, and 8 (but primarily SMAD8) are activated and, together with SMAD4, regulate gene transcription. The only MIS receptor type II (MISRII) gene has been identified in mice, rats, and rabbits, and in humans, its gene is localized on chromosome 12. It is a 65 kD protein detected in embryonic and adult Müllerian duct structures, mammary tissue, prostate tissue, gonads, motor neurons, and the brain. In the fetus, mesothelial cells expressing MISRII in the coelomic epithelium covering the urogenital ridge migrate to and become part of the mesenchymal cells surrounding the Müllerian epithelial cells. Expression is also detected in the gonads and ovarian coelomic epithelium. Type I MIS receptors have been identified in mammals, and depending on the animal species and tissue examined, activin receptor-like kinases (ALK) 2 and 3 are the most promising candidates. In addition to its well-established role in Müllerian duct regression, MIS inhibits the proliferation of diverse human cancer cell lines both in vitro and in vivo. Cell lines exhibiting inhibition are derived from ovarian, cervical, endometrial, prostate, and breast cancers. Even when high concentrations of MIS are maintained systemically in rodents with tumors secreting MIS over long periods or in human patients, no toxicity has been observed in vivo. These findings—relatively limited receptor expression, antiproliferative activity against cancer cells expressing MIS RI and RII, and its apparent non-toxicity—collectively make MIS an ideal reagent for use in combination with existing chemotherapeutic agents for the treatment of ovarian cancer, which are known to develop resistance to these conventional agents. MIS acts via MIS-type II receptor cells and functions as a potential tumor suppressor of ovarian cancer initiation (Teixeira et al, unpublished). MIS may also target the stem/progenitor population 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). MIS (Mild Invasive Surgical Stimulation) shows no apparent toxicity after long-term in vi