Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Patent
1993-06-23
1994-10-25
Hill, Jr., Robert J.
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
435 691, 435 694, 530300, 530351, C12P 2106, C12Q 100, C07K 300, C07K 1500
Patent
active
053590333
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD OF INVENTION
This invention relates to cleaved dimers of Mullerian inhibiting substance-like polypeptides. More particularly, this invention relates to such dimers, methods of producing them and methods of using them in the treatment of cancer and tumors, especially those of the female genital tract. The dimers of this invention are also useful in compositions and methods for contraception.
BACKGROUND OF THE INVENTION
Mullerian Inhibiting Substance (MIS) is a glycoprotein produced by the Sertoli cells of the embryonic testis. It is a non-steroidal factor that causes regression of the Mullerian duct, the anlagen of the internal female reproductive tract [Jost, Rec. Prog. Horm. Res., 8, 379-418 (1953)]. MIS, in addition to its important role in development, has been shown to be cytotoxic to the human ovarian tumor cell line HOC-21, both in vitro and in vivo (in a nude mouse model) [Donahoe et al., Science, 205, 913-15 (1979); Fuller et al., J. Clin. Endocrinol. Metab., 54, 1051-55 (1982); Donahoe et al., Ann. Surg., 194, 472-80 (1981)].
Both human MIS and bovine MIS have been cloned and expressed in various bacterial and animal host cells using both genomic and cDNA sequences. The products of such recombinant transformed cells, as well as those of Sertoli cells, are 70K polypeptides which dimerize to form 140K disulfide-linked dimers. The purified dimers from Sertoli cells or recombinant cells (e.g., CHO cells transfected with an MIS gene) are active in vitro in causing regression of the rat Mullerian duct in a standard organ culture assay [Cate et al., Cell, 45, pp. 685-98 (1986)].
SUMMARY OF THE INVENTION
The present invention relates to dimers of MIS-like polypeptides (bovine, human or other mammal) which may be processed to produce a C-terminal dimer and a N-terminal dimer. These dimers may remain non-covalently associated with each other and are active in a standard organ culture assay for biological activity of an MIS protein [Cate et al., supra]. The associated dimers, which may also be separated from each other by boiling, acidification or treatment with a detergent such as deoxycholate, are useful separately or in combination in the treatment of cancer, especially cancer of the female genital tract. The dimers of this invention are also useful in compositions and methods for contraception.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic description of one process for producing N-terminal and C-terminal dimers of this invention from a dimer of a human MIS.
FIG. 2 depicts the genomic DNA of a human MIS. It also depicts the amino acid sequence of immature and mature human MIS--those sequences are interrupted by four introns at the DNA level. In this figure, the amino acids are represented by single letter codes as follows:
______________________________________ Phe: F Leu: L Ile: I Met: M
Val: V Ser: S Pro: P Thr: T
Ala: A Tyr: Y His: H Gln: Q
Asn: N Lys: K Asp: D Glu: E
Cys: C Trp: W Arg: R Gly: G
______________________________________
FIG. 3 depicts the DNA and amino acid sequences of immature and mature bovine MIS. It is a genomic/cDNA composite sequence.
FIG. 4 is a SDS-PAGE of plasmin digested MIS which shows that plasmin cleavage of MIS produces an N-terminal (110 kDa) and C-terminal (25 kDa) dimer.
FIG. 5 is a SDS-PAGE which shows that deoxycholate dissociates the non-covalent complex between the N- and C-terminal dimers.
FIG. 6 is a schematic outline of the construction of plasmid pD1.
FIGS. 7A-7D are schematic outlines of the construction of plasmid pJ103.
FIG. 8 depicts the nucleotide sequence of oligomers MIS100, MIS103, MIS104, MIS105, MIS106.
FIG. 9 is a SDS-PAGE analysis of purified mutant 103 produced by L9C16 CHO cells. Wild type MIS or mutant 103 were analyzed under reducing and non-reducing conditions, before and after digestion with plasmin.
FIG. 10 is a schematic outline of the construction of plasmid pJ100.
FIG. 11 is a SDS-PAGE analysis of purified N-terminal dimer produced by L7118 CHO cells.
FIG. 12 depicts regression of the Muller
REFERENCES:
patent: 4404188 (1983-09-01), Donahoe et al.
Cate Richard L.
Pepinsky R. Blake
Biogen Inc.
Elrifi Ivor R.
Haley, Jr. James F.
Hill Jr. Robert J.
Wang Gian P.
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