WO1994000133A1 - Hormone antimullerienne utilisee pour le traitement de tumeurs et la modulation de l'expression de l'antigene d'histocompatibilite majeure classe i. - Google Patents

Hormone antimullerienne utilisee pour le traitement de tumeurs et la modulation de l'expression de l'antigene d'histocompatibilite majeure classe i. Download PDF

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WO1994000133A1
WO1994000133A1 PCT/US1993/005791 US9305791W WO9400133A1 WO 1994000133 A1 WO1994000133 A1 WO 1994000133A1 US 9305791 W US9305791 W US 9305791W WO 9400133 A1 WO9400133 A1 WO 9400133A1
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mis
tumor
cells
cell
terminal fragment
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Patricia K. Donahoe
Tai Wai Chin
Robert L. Parry
James Epstein
Richard C. Ragin
David T. Maclaughlin
Edward M. Barksdale
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General Hospital Corp
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General Hospital Corp
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/26—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • This application is directed to inhibiting local and metastatic tumor growth by administering to certain tumors an effective amount of M ⁇ llerian Inhibiting Substance or an effecitve amount of the carboxy-terminal fragment
  • This application further concerns inhibiting primary and metastatic tumor growth by transfecting tumor cells with a vector capable of expressing an effective amount of M ⁇ llerian Inhibiting Substance or an effective amount of the carboxy-terminal fragment of MIS. Also, this application is directed to DNA sequences encoding the C-terminal fragment of MIS, vectors containing
  • This application further concerns gene therapy treatments for patients suffering from certain tumors. Also disclosed is a method for modulating class I histocompatibility antigens by administering M ⁇ llerian Inhibiting Substance and Epidermal Growth Factor.
  • M ⁇ llerian Inhibiting Substance is produced by the fetal testis as a 140 kDa glycosylated disulfide-linked homodimer that causes regression of the M ⁇ llerian duct in the male fetus.
  • the protein migrates on gel electrophoresis at an apparent molecular weight of 70 kDa.
  • the protein can be proteolytically cleaved by exogenous plasmin into two distinct fragments that migrate electrophoretically as 57 kDa and 12.5 kDa moieties with cleavage at residue 427 of the intact 535 amino acid monomer (Pepinsky, et al, J. Biol. Chem. 263: 18961-4 (1988)).
  • U.S. Patent No. 4,404, 188 filed July 29, 1981 and entitled "Purified M ⁇ llerian Inhibiting Substance and Method of Purification” describes a process for purifying MIS which comprises treatment with a protein inhibitor, chromatography on ion exchange, chromatography on wheat germ lectin, on concanavalin A and/or on a supported triazinyl dye.
  • U.S. Patent No. 4,487,833, filed on March 1, 1982 and entitled “Method of Preparing Hybridomas and of Purifying Immunogenic Materials” describes a process for separating MIS using immunoaffinity chromatography.
  • the M ⁇ llerian duct develops into the Fallopian tubes, uterus and upper vagina. It is known that MIS causes regression of the
  • MIS has also been shown to play a role in inhibition of oocyte meiosis (Takahashi et al., Mol. Cell. Endocrinol. 47:225- 234 (1986)), testicular descent (Hudson et al., Endocr. Rev. 7:270-283 (1986)), inhibition of fetal lung development (Catlin et al., Am. J. Obstet. Gynecol. 759: 1299-1303 (1988)), inhibition of autophosphorylation of the EGF receptor (Coughlin et al., Mol. Cell. Endocrinol.
  • TILs Tumor-infiltrating-lymphocytes
  • Exogenous genes can be inserted into TILs in vitro and then reinjected into a patient (Rosenberg et al. , Science 233: 1318-21 (1986); Spiess et al , J Notl Cancer Inst 79:1067-75 (1987); Cameron et al, J Exp Med 777:249-63 (1990); Rosenberg et al, N. Engl. J. Med.
  • the major histocompatibility complex is a group of closely linked genes which encode molecules that restrict the specificity of antigen recognition by T lymphocytes.
  • the antigens fall into two classes: class I and class II.
  • the immunological aspects of the present invention are reviewed, for example, by Klein, J., In: Immunology: The Science of Self-Non-Self Discrimination, Wiley-Interscience, NY, pp. 270-309 (1982) and Feldman, M. et al , Scientific American 259:60-85 (1988), which references are herein incorporated by reference.
  • Antigens of class I restrict antigen recognition predominantly by cytotoxic T lymphocytes, whereas antigens of class II restrict recognition by regulatory T cells.
  • Class I MHC molecules are expressed on all cells. In contrast, MHC class II molecules are expressed predominantly, on B lymphocytes.
  • Agents capable of modulating the expression of the MHC class I antigens may be employed in the treatment or prevention of metastatic cancer, immunodeficiency diseases, and organ and tissue rejection.
  • the present invention provides for a method of inhibiting tumor growth comprising administering an effective amount of M ⁇ llerian Inhibiting Substance to a patient, said tumor selected from the group consisting of vulvar epidermoid carcinoma, cervical carcinoma, endometrial adenocarcinoma, ovarian adenocarcinoma, and ocular melanoma.
  • the present inventors have discovered that the M ⁇ llerian duct regression and anti-proliferative bioactivities of MIS reside in its carboxy- terminal domain.
  • the present invention is also directed to a method of inhibiting tumor growth comprising administering an effective amount of the carboxy-terminus of MIS to a patient, said tumor selected from the group consisting of vulvar epidermoid carcinoma, cervical carcinoma, endometrial adenocarcinoma, ovarian adenocarcinoma and ocular melanoma.
  • the present inventors have further discovered that metastatic tumor growth can be inhibited by administering to tumor cells an effective amount of MIS or an effective amount of the C-terminal fragment of MIS.
  • the present invention is further directed to a biologically active pharmaceutical composition, i.e., a composition having M ⁇ llerian duct regression and anti-proliferative activities, containing a pharmaceutically acceptable carrier and the C-terminal fragment of MIS substantially free of the N-terminal fragment.
  • the present invention is further directed to DNA sequences encoding the C-terminal fragment of MIS, vectors containing the DNA sequences and transformed host cells capable of producing the C-terminal fragment.
  • the present invention is further directed to localized tumor treatments comprising activating MIS by cleaving MIS into C- and N-terminal fragments at the tumor site with a proteolytic enzyme.
  • Another goal of this invention is to reduce the concentration of chemotherapeutic agents which are currently used in the treatment of the tumors of this invention.
  • This goal has been achieved by providing for a method of inhibiting tumor growth comprising administering an effective amount of a combination of a chemotherapeutic agent and M ⁇ llerian Inhibiting Substance or an effective amount of a combination of a chemotherapeutic agent and the carboxy-terminal fragment of MIS to a patient, said tumor selected from the group consisting of vulvar epidermoid carcinoma, cervical carcinoma, endometrial adenocarcinoma, ovarian adenocarcinoma, and ocular melanoma.
  • This invention further provides for a pharmaceutical composition
  • a pharmaceutical composition comprising an effective tumor inhibiting amount of proteolytically cleaved M ⁇ llerian Inhibiting Substance, said tumor selected from the group consisting of vulvar epidermoid carcinoma, cervical carcinoma, endometrial adenocarcinoma, ovarian adenocarcinoma, and ocular melanoma, and a pharmaceutically acceptable carrier.
  • the present invention also provides DNA sequences coding for the C- terminal fragment of MIS, recombinant DNA molecules comprising such sequences, hosts comprising such sequences and processes for producing the C-terminal fragment in hosts transformed with those DNA sequences.
  • the present invention is further directed to inhibiting tumor growth comprising transfecting one or more tumor cells with a gene capable of expressing an effective amount of MIS. Moreover, tumor growth can also be inhibited by transfecting one or more tumor cells with a gene capable of expressing an effective amount of the C-terminal fragment of MIS.
  • the present invention further provides a method for inhibiting metastatic tumor growth comprising transfecting one or more tumor cells with a gene capable of expressing an effective amount of MIS. Moreover, metastatic tumor growth can also be inhibited by transfecting one or more tumor cells with a gene capable of expressing an effective amount of the C- terminal fragment of MIS.
  • the present invention also provides gene therapy methods for treating patients with certain tumors.
  • TILs are generated from tumor suspensions cultured in interleukin-2 (IL-2). Cultures of IL-2-stimulated TILs can be transduced in vitro with a gene capable of expressing an effective amount of MIS or an effective amount of the C-terminal fragment of MIS using an appropriate vector.
  • IL-2 interleukin-2
  • the gene-modified autologous TILs will infiltrate into tumors present in the patient. Therefore, the present invention is further directed to a method comprising using TILs as cellular vehicles for in vivo delivery of a gene capable of expressing an effective amount of MIS or an effective amount of the C-terminal fragment of MIS to tumors in a patient.
  • the present invention is also directed to other gene therapy methods for achieving in vivo delivery of a gene capable of expressing an effective amount of MIS or an effective amount of the C-terminal fragment of MIS to tumors in a patient.
  • direct in situ introduction of a gene capable of expressing an effective amount of MIS or an effective amount of the C-terminal fragment of MIS into proliferating tumors can be achieved using retroviral vectors.
  • the present invention further provides methods for modulating MHC class I antigens on cell surfaces using MIS and EGF.
  • MHC Class I antigens appear on the cell surface of all adult cells (Flavell et al, Science 233:437 (1986)). Their orderly yet assynchronous emergence in various embryonic organs and tissues appears to be constrained by growth factors like EGF and augmented by growth inhibitors like MIS. Thus MHC, by responding to growth modulators may act as a chemostat to serve a more basal function during fetal development, in orchestrating the divergent and uneven growth patterns so characteristic of that period.
  • Prolonged survival was enjoyed by fetal and postnatal testis and midgestational renal grafts transplanted beneath the renal capsule of adult congenic mice, confirming previous findings in non-imm ⁇ nosuppressed outbred rats (Foglia R.P. et al, Annals of Surgery 204:402 (1986); Starter M.B. et al , J. Urol. 739:204 (1988))
  • the strategies that enable immature tissues to escape rejection in a graft survival assay were studied by comparing expression of major histocompatibility (MHC) Class 1 and Class II protein and mRNA in each tissue at different ages. In general, graft survival was best when Class I & II expression was low.
  • MHC major histocompatibility
  • Monoclonal antibodies raised against semi-purified bovine M ⁇ llerian Inhibiting Substance (Shima et al, Hybridoma 3:201-214 (1984)) were found to be specific for bovine MIS and not to cross react with high affinity for human, mouse, or rat MIS.
  • the cloning, expression and amplification of the human MIS gene (Cate et al. , Cell 45:685-698 (1986)) enabled purification of sufficient quantities (Pepinsky et al. , J. Biol. Chem. 263: 18961 (1988)) for immunization of mice and rabbits.
  • Mono and polyclonal antibodies were raised to human MIS by conventional techniques.
  • ELISA enzyme linked immunosorbent assay
  • Normative MIS values in humans were established for newborn males and females and for infants, children and adults.
  • the ELISA has been useful in monitoring recombinant vector production of MIS and subsequent purification. It has also been used to investigate MIS half-life and pharmacology in anti- proliferative tumor assays.
  • the ELISA has been used to confirm route of administration and half life in studies of the MIS effect on surfactant production in developing lungs (Catlin et al. , Amer. J. Ob. Gyn 159: 1299 (1988)).
  • MIS was measured in a patient with a sex cord tumor before, during and after multiple operative procedures to excise the undispersed but slow growing tumor.
  • the assay may be used for assessment of: babies with intersex abnormalities and other sexual congenital abnormalities, newborns with Respiratory Distress Syndrome, patients with Sertoli cell neoplasms or other tumors, and patients undergoing In Vitro Fertilization (IVF).
  • Figure 1 shows the results of a semisolid medium (double layer) colony inhibition assay.
  • the left side of the figure shows the effects of the immunoaffinity chromatography method described herein (IAP-MIS) in which colony formation of A431, HT-3, HEC-l-A, NIH:OVCAR-3, andOM431 cells was significantly inhibited (30 nM).
  • the right side of Fig. 1 shows the effect of the salt fraction pre-eluted from the immunoaffinity column (IAP-salt). Stimulation of A431 , OM431 and Hep 3B colony formation was seen when treated with the salt eluted fraction.
  • the symbol "*" indicates p ⁇ 0.05 when compared with control.
  • Figure 2 shows the results of a liquid medium colony inhibition assay.
  • the left side of the figure shows the effect of IAP-MIS in which colony formation of A431 and OM431 cells was significantly inhibited (30 nM).
  • the right side of the figure shows the effect of the salt fraction preeluted from the immunoaffinity column (IAP-salt).
  • the colony formation of A431 and OM431 cells was stimulated by IAP-salt.
  • IAP-MIS and IAP-salt showed no effect on HT-3,and RT4 cells.
  • the symbol "*" indicates p ⁇ O.05 when compared with control.
  • FIG. 3 shows dose dependent inhibition of A431 colony formation by MIS.
  • the left side of the figure shows where MIS purified by dye affinity chromatography (DG-MIS) was tested in increasing doses using the liquid medium colony inhibition assay. Significant inhibition was seen at MIS concentrations of 3.5 and 7.0 nM.
  • the right side of the figure shows that IAP-MIS caused significant inhibition at concentrations of 24, 48 and 96 nM.
  • the symbol "*" indicates p ⁇ 0.05 when compared with controls.
  • Figure 4 shows antibody absorption of DG-MIS.
  • the inhibitory effect of MIS of this degree of purity on A431 colony formation was significantly reduced after absorption with the MIS specific monoclonal antibody (6E11), but not after nonspecific absorption by normal IgG.
  • the symbol "*" indicates p ⁇ O.O5 when compared with MIS alone.
  • Figure 5 shows the effects of various immunopurified fractions on A431 cells in the liquid colony inhibition assay.
  • the IAP-MIS 50 nM
  • the salt eluted fraction stimulated growth.
  • the percent survival of the combination was significantly higher than that of MIS alone.
  • the symbol "*" indicates p ⁇ 0.05 when compared with IAP-MIS alone, although the MIS concentrations were the same.
  • Figure 6 shows the additive effect of cisplatin and DG-MIS.
  • cisplatin and MIS were tested on A431 cells in the liquid medium colony inhibition assay, their effects were additive at cisplatin concentrations of 0.078 and 0.156 mg/ml.
  • the symbol "*" indicates p ⁇ 0.05 when cisplatin plus MIS was compared with cisplatin alone.
  • Figure 7A shows the results of a spheroid assay.
  • DG-MIS (7 nM) inhibited the growth of HT-3 spheroids when compared with control (p ⁇ 0.05).
  • Figure 7B shows the results of a spheroid assay.
  • DG-MIS (7 nM) did not effect the growth of Hep 3B spheroids when compared with control (p ⁇ 0.05).
  • Figure 8A shows MIS serum levels. Alzet pumps loaded with 33 ⁇ g
  • MIS were implanted into CD-I mice. A relatively constant level of MIS was achieved 24 hours after implantation.
  • Figure 8B shows inhibition of tumor growth in vivo by MIS. A431 and OM431 cells were implanted in the subrenal capsule space of CD-I mice. The graft size ratios of both tumors were significantly inhibited in the MIS treated group. The symbol "*" indicates p ⁇ O.O5 when compared with controls.
  • Figure 9 shows dose dependent inhibition of OM431 colony formation by MIS in a liquid colony inhibition assay.
  • the percent survival was 33 % , 29.5 %, 56.6%, 71 %, 109.8%, and 96.8%, respectively, for MIS concentrations of 100.8, 75.6, 50.4, 25.2, 9.8, and 0.98 nM.
  • Significant inhibitions were seen with MIS concentrations of 50.4 (p ⁇ .05), 75.6 (p ⁇ .004), and 100.8 (p ⁇ .006) nM MIS.
  • Figure 10 shows the results of a semisolid medium (double layer) colony inhibition assay. All cell lines were significantly inhibited by rhMIS.
  • the average graft size ratios in the MIS group (n 6) were 2.17 ⁇ 0.26, 2.79 ⁇ 0.33 and 3.91 ⁇ 0.59, respectively. This difference was significant (p ⁇ .02).
  • Figure 11B shows the results of a multicellular spheroid assay of OM482 cells.
  • Figure 12 A shows the results of a subrenal capsule assay of CD-I irradiated mice.
  • Each MIS treated mouse received 48.6 micrograms of MIS over the eight day assay.
  • the growth of the tumors in each MIS group was significantly lower than the control (p ⁇ 0.005).
  • Figure 12B shows the results of a subrenal capsule assay using nude mice.
  • the graft size ratios of the OM431 tumors were significantly greater for the controls (3.02 ⁇ 0.17) compared to the MIS group (1.68 ⁇ 0.09).
  • the MIS treated group received 44.7 micrograms MIS over eight days.
  • the average MIS serum levels of the MIS treated mice on the eighth day of the nude mouse assays were 749 pM.
  • the measured controls had MIS levels of less than 10 pM.
  • the growth of the tumors in each MIS group was significantly lower than the control (p ⁇ 0.001).
  • Figure 12C shows the results of a subrenal capsule assay using nude mice.
  • the graft size ratios of the OM431 tumors were significantly greater for the controls (3.14 ⁇ 0.40) compared to the MIS group (1.69 ⁇ 0.43).
  • the MIS treated group received 130 micrograms MIS over eight days.
  • the average MIS serum levels of the MIS treated mice on the eighth day of the nude mouse assay were 570 pM.
  • the measured controls had MIS levels of less than 10 pM.
  • the growth of the tumors in each MIS group was significantly lower than the control (p ⁇ 0.05).
  • FIG 13 A representative P-100 column chromatogram of a plasmin-cleaved rhMIS preparation. The protein content of each 0.54-ml fraction is given on the ordinate, and the fraction number on the abscissa. The positions of the pools for amino- and carboxy-terminal fragments of rhMIS are indicated by the brackets.
  • FIG 14. This figure shows the electrophoretic separation of rhMIS under reducing conditions before (lane 1, IAP) and after treatment with plasmin (lane 2, Plasmin). Treatment with plasmin results in the characteristic depletion of 70-kDa rhMIS monomer and the appearance of 55-, 34-, and 12.5- to 14-kDa bands. In addition, the protein fragments separated by the P- 100 column are shown. Pool A (lane 3, N-term) contains proteins of 34-55 kDa, while pool B (lane 4, C-term) reveals the presence of essentially only bands of 12.5-14 kDa. The positions of the prestained mol wt markers are given at the left. Figure 15.
  • FIG. 16 The effect of daily addition of rhMIS carboxy-terminus and vehicle control buffer in volumes corresponding to the rhMIS carboxy-terminal domain additions of 5, 10, or 20 ⁇ g.
  • the rhMIS carboxy-terminus significantly inhibits cell proliferation at 20 ⁇ g, and a small dose-dependent buffer effect is noted.
  • Figure 17 shows the amino acid and nucleotide sequences of bovine MIS, C-terminal fragment, having about 109 amino acids.
  • Figure 18 shows the amino acid and nucleotide sequences of human MIS, C-terminal fragment, having about 109 amino acids.
  • FIG. 19 Western analysis of CHO-WT, B9, and L9 cell culture media.
  • MIS was purified from serum-free CHO-WT (lane A), L9 (lane B), and B9 (lane C) media samples by lentil-lectin extraction, and anti-holo-rhMIS primary antibody (MGH-1) was subsequently used for Western analysis.
  • MGH-1 anti-holo-rhMIS primary antibody
  • the predominant 70 and 55 kDa bands in B9 media represent holo- and amino- terminal rhMIS, while the less abundant 70 kDa L9 band corresponds to noncleavable holo-MIS.
  • FIG 20 Northern analysis of CHO-WT, B9, and L9 cells and pulmonary metastases.
  • Total RNA was extracted from CHO-WT, B9, and L9 cells in monolayer culture and from metastases-containing lungs after tail vein injections of SCID mice.
  • MIS mRNA is detectable in both cells and metastases of the B9 and L9 lines, although less abundant in the latter cell lineage. Note the absence of detectable message in CHO-WT cells and metastases. (Exposure time 6 h for B9, CHO-WT; 60 h for L9).
  • FIG. 23 The in vivo growth of CHO-WT, B9, and L9 cell lines was evaluated in a murine subrenal capsule assay. Eight days after implantation beneath the murine renal capsule, the graft size ratios (GSR) were 6.04 ⁇
  • FIG. 24 Pulmonary metastases following tail vein injection of B9, L9, or CHO-WT cells into severe combined immunodeficient mice.
  • FIG. 25 Pulmonary metastases following tail vein injection of OM431 cells into severe combined immunodeficient mice.
  • A) Lungs from animals treated with rhMIS (second row) via an Alzet pump within the peritoneal cavity for 8 days showed a smaller number of surface metastases at six weeks (4.6 ⁇ 2.9) than buffer-treated controls (37 ⁇ 17; p 0.09).
  • B) The cross-sectional area occupied by metastatic OM431 tumors at 6 weeks in the mid-lung of animals treated with rhMIS for 16 days (2.05 ⁇ 0.53%) was significantly less (p 0.01) than the relative area of lung metastases in buffer- treated controls (18.99 ⁇ 5.75 %).
  • Figure 27 shows a comparison of graft size ratio, and histology (architecture and lymphocytic infiltrate) between 14 and 18-day fetal kidney allografts after implantation for 7 days, and between 18-day fetal and 1-day postnatal testis allografts implanted for 10 days.
  • All values represented in (A) are loglO transformed graft size ratios (L X W in final/ L X W in initial), as a measure of individual graft growth, illustrated as range (boxes) and mean (bars). Histologic changes, as demonstrated by loss of architecture (B), and increasing lymphocytic infiltrate (C), were graded using a scale where 1 is best and 5 is worst (Statter M.B.
  • MIS M ⁇ llerian Inhibiting Substance
  • MIS is intended to include compounds and materials which are structurally similar to MIS. Examples of such included substances and materials are salts, derivatives, and aglycone forms of MIS. Additionally, the present invention is intended to include mutant forms of MIS which have substantially the same biological activity as MIS. Examples of such mutant forms would be MIS molecules carrying a deletion, insertion, or alteration in amino acid sequence. MIS may be obtained from any mammalian source or, as indicated above, from non-mammalian sources through the use of recombinant DNA technology, or from the chemical synthesis of the MIS protein. The present inventors have found that MIS is a particularly effective anti-cancer agent due to its anti-proliferative effects on various tumors. In addition, application of MIS to patients has no known unfavorable side effects.
  • carboxy-terminal (C-terminal) fragment of MIS is intended to include compounds and materials structurally similar to the about 12.5 kDa (about 25 kDa under non-reducing conditions) C-terminal fragment of MIS resulting from proteolytic (e.g., plasmin) cleavage at residue 427 of the intact 535 amino acid human MIS monomer.
  • the proteolytic (e.g., plasmin) cleavage site is at residue 443 of the 551 amino acid bovine MIS molecule.
  • “carboxy-terminal (C-terminal) fragment of MIS” is intended to include the about 25 kDa homodimeric C-terminal fragment of MIS.
  • the present inventors have discovered that M ⁇ llerian duct regression and antiproliferative activities reside in the C-terminal domain of MIS.
  • N-terminal fragment of MIS is intended the about 57 kDa fragment resulting from the above-noted cleavage at residue 427 of the intact 535 amino acid human MIS monomer (residue 443 of the 551 amino acid bovine MIS). More prolonged proteolytic exposure results in further proteolysis of the N-terminal fragment of MIS yielding 34- and 22 kDa fragments of the amino-terminal moiety.
  • the C-terminal amino acid and nucleotide sequences for bovine MIS are shown in Figure 17.
  • the C-terminal amino acid and nucleotide sequences for human MIS are shown in Figure 18.
  • a comparison of the amino acid sequence for human and bovine MIS, showing the N- and C-terminal domains is shown in Cate et al, Handbook of Experimental Pharmacology 95/77: 184, edited by M.B. Spoon and A.B. Roberts, Spinger-Verlag Berlin Heidelberg (1990). The contents of Figure 3 of this reference are herein incorporated by reference.
  • the human sequence is shorter in that it misses certain sequences at the N-terminal domain, relative to bovine.
  • the present invention is intended to include mutant forms of the C-terminal fragment of MIS which have substantially the same biological activity as the C-terminal fragment of MIS.
  • mutant forms would be C-terminal fragment of MIS molecules carrying a deletion, insertion, or alteration of amino acid sequence.
  • the C-terminal fragment of MIS can be modified to increase its half-life in vivo.
  • addition of one or more amino acids or other chemical agents to the amino and/or carboxyl end of the C-terminal fragment can be used to increase the fragment's stability.
  • the C-terminal fragment of MIS can be obtained from a mammalian source or through the use of recombinant DNA technology, or from chemical synthesis of the C-terminal polypeptide.
  • the about 25 kDa homodimeric C-terminal fragment of MIS can be generated by proteolytic cleavage of holo rMIS produced from Chinese hamster ovary cells.
  • a gene is said to be a "recombinant" gene if it results from the application of Recombinant DNA Techniques. Examples of recombinant DNA techniques include cloning, mutagenesis, transformation, etc. Recombinant
  • patient is intended to include animal patients. More preferably, “patient” is intended to include mammalian patients, most preferably, human patients.
  • a pharmaceutical preparation for treating the tumors of this invention can be prepared.
  • the use of the method described in U.S. Patent Application No. 07/683,957, filed on April 12, 1991 , and which is fully incorporated by reference is preferred.
  • This preferred method takes advantage of the specificity of antigen-antibody interactions to recover a product having a substantially pure MIS product.
  • this method incorporates the use of immunoaffinity chromatography, but improves upon previously used methods in that the recovered MIS product is substantially free of contaminating enzymes having MIS proteolytic activity or inhibitors of MIS antiproliferative activity.
  • the immunoaffinity chromatography method used herein effectively eliminates contaminating enzymes having MIS proteolytic activity or inhibitors of MIS anti-proliferation activity from an immunoaffinity chromatography matrix by eluting with an effective amount of an alkali metal halide or an alkaline earth metal halide.
  • the MIS is then recovered by eluting with an acid solution having a pH of between about 2.5 and 4.0.
  • This end product is referred to herein as IAP-MIS.
  • the IAP-MIS can be obtained in solution at up to 95 % purity. While the percent purity is comparable to other immunoaffinity purifications processes, the IAP-MIS is substantially free of contaminating proteolytic enzymes or inhibitors of MIS antiproliferative activity.
  • the purified MIS of this invention can be obtained in solution at up to
  • the MIS composition of this invention is substantially free of contaminating proteolytic enzymes or inhibitors of MIS antiproliferative activity.
  • purified MIS is considered to be a MIS composition which is substantially free of contaminating proteolytic enzymes or inhibitors of MIS antiproliferative activity regardless of percent purity.
  • the composition is considered to be substantially free of proteolytic enzymes if gel electrophoresis of the purified MIS product indicates a protein having a molecular weight of 140 kDa or 70 kDa. Gel electrophoresis of such a product will not show time dependent proteolytic fragments which are degradation products of MIS. For example the 57 kDa, 12.5 kDa, 34 kDa and 22 kDa degradation fragments of MIS further described herein will not be readily discernable by standard gel electrophoresis methods in substantially pure holo MIS.
  • the MIS composition will be considered to be substantially free of inhibitors of MIS antiproliferative activity if the MIS blocks proliferation of tumor cells.
  • tumor cells are included herein and in U.S. Patent Application No. 07/683,957. These examples include tumors selected from the group consisting of vulvar epidermoid carcinoma, endometrial adenocarcinoma, cervical carcinoma, endometrial adenocarcinoma, ovarian adenocarcinoma, and other ocular melanoma.
  • the determination of antiproliferative activity of these cells can be achieved by any of the procedures described herein and in U.S. Patent Application No. 07/683,957.
  • the contaminants are separated from the MIS using immunoaffinity chromatography. Separation occurs by eluting the enzymes or inhibitors with an alkali metal halide or an alkaline earth metal halide.
  • an alkali metal halide or an alkaline earth metal halide Such a compound will generally be in solution and an effective amount of halide will be between about 0.1 M and 2.0 M.
  • alkali metals the ions of lithium, sodium and potassium are preferred with sodium being the most preferable.
  • alkaline earth metals the ions of magnesium and calcium are preferred.
  • halides the ions of fluorine, chlorine, bromine and iodine are preferred with chlorine being most preferable.
  • a solution of between about 0.1 and 2.0 M is preferred.
  • concentration of halide can also be varied as elution progresses if desired. This can be accomplished by increasing molar concentration of halide in a stepwise fashion. It is preferred that each step be altered after about 0.1-2.0 bed volumes of solution have contacted the chromatography matrix, although such steps can be further modified as desired.
  • the halide can also be accompanied in solution with an effective amount of a chelating agent.
  • a chelating agent capable of binding metal ions which can inactivate enzymes that require the metal ions for activity.
  • agents include the compounds ethylenediamine tetraacetate (EDTA), and ethylenebis- (oxyethylenenitrilo)tetraacetic acid (EGTA).
  • EDTA ethylenediamine tetraacetate
  • EGTA ethylenebis- (oxyethylenenitrilo)tetraacetic acid
  • Chelants can be effectively added in a range of between 0.1 and 50 mM.
  • the MIS can be recovered by eluting with an acid solution having a pH of between about 2.0 and 4.0.
  • strong acids such as HCl
  • organic acids are preferred because of their relatively mild acid strength.
  • acid amines and imines can be employed as well as monocarboxylic, dicarboxylic and tricarboxylic acids.
  • monocarboxylic acids are acetic, propionic and butyric acid.
  • dicarboxylic acids are succinic, fumaric and malic acid.
  • Preferred as a tricarboxylic acid is citric acid.
  • Preferred among the amines are the acidic amino acids such as aspartic and glutamic acid.
  • the pH of the acid solution can also be incrementally varied as in the application of the halide.
  • MIS 140 kDa homodimer or the 70 kDa subunit of MIS can be included in the pharmaceutical composition.
  • proteolytic enzymes in vivo can proteolytically cleave MIS to its effective form.
  • Such enzymes are represented by the proteolytic compounds described herein.
  • protein fragment is meant to include both synthetic and naturally-occurring amino acid sequences derivable from the naturally occurring amino acid sequence of MIS.
  • the protein is said to be "derivable from the naturally-occurring amino acid sequence of MIS” if it can be obtained by fragmenting the naturally-occurring chosen sequence of MIS, or if it can be synthesized based upon a knowledge of the sequence of the naturally occurring amino acid sequence or of the genetic material (DNA or RNA) which encodes this sequence.
  • proteolytically cleaved refers to an MIS product obtained by treatment with any substance which is capable of cleaving either the homodimer or the 70 kDa subunit of MIS into a protein fragment which inhibits growth of the tumors of this invention.
  • MIS is effective in treating the tumors of this invention when proteolytically cleaved to form protein fragments of about 57 kDa and 12.5 kDa.
  • substances which cleave MIS in this manner include serine proteases, such as plasmin, and endopeptidases.
  • the about 12.5 kDa (about 25 kDa under non-reducing conditions) C- terminal fragment of MIS can be purified from proteolytically cleaved MIS thereby freeing the C-terminal fragment from its association with the N- terminal fragment in the N- and C-terminal non-covalent complex that forms after proteolytic treatment of intact MIS.
  • Suitable purification techniques include column chromatography separation techniques known in the art. For example, the polyacrylamide column technique set forth in Example 3 is particularly suitable for purifying the C-terminal fragment of MIS.
  • the C- terminal fragment of MIS can also be purified by other art-known techniques, provided that the biological activity of the C-terminal fragment is not destroyed during purification. As stated, the antiproliferative activity of MIS resides in its C-terminal domain. Thus, the C-terminal fragment of MIS alone is effective in treating the tumors of this invention.
  • the N-terminal fragment may be present during tumor treatment, but it is not required for inhibition of tumor growth. Cleavage of MIS into N- and C-terminal fragments can occur by exogenous proteolysis or by proteolysis in vivo.
  • MIS is activated to have antiproliferative and M ⁇ llerian duct regression activities upon proteolytic cleavage.
  • MIS can be used as an antiproliferative agent in a site-specific method for treating the tumors of the present invention.
  • the method comprises activating MIS which has been administered to a patient by cleaving the MIS molecule into fragments of about 57 kDa and about 12.5 kDa (about 25 kDa as a homodimer) upon contact with a cleaving substance (e.g., plasmin) located at or provided to the tumor site.
  • a cleaving substance e.g., plasmin
  • substances which cleave MIS in this manner include serine proteases, such as plasmin, and endopeptidases.
  • the cleaving substance can be delivered to the tumor site by perfusion. Methods for perfusing tumors with substances such as plasmin are evident to one having ordinary skill in the art.
  • MIS can be administered to a patient by one or more of the variety of techniques either disclosed herein or known in the art. Post-administration, MIS will remain inactive until activated by cleavage. Since the exogenous cleaving substances are localized at the tumor site, MIS is activated in a site- specific manner at the tumor site. Such a localized tumor treatment can advantageously limit possible toxicity that can be caused by systemic administration of MIS in its activated form.
  • MIS When MIS is administered to a patient in inactive form, without the use of exogenous cleaving substances, endogenous cleaving substances are relied upon to cleave MIS into its activated form.
  • the invention further pertains to polypeptides that, in addition to the chosen sequence, may contain or lack one or more amino acids that may not be present in the naturally-occurring sequence, wherein such polypeptides are functionally similar to or possess antagonist activity to the chosen polypeptide.
  • polypeptides for the present invention are termed "functional derivatives,” provided that they demonstrate activity which is substantially similar to or antagonistic to that of MIS or the C-terminal fragment of MIS.
  • MIS composition or the composition containing the C-terminal fragment of MIS may be in the form of the free amines (on the N-terminus), or acid- addition salts thereof.
  • Common acid solution salts are hydrohalic acid salts, i.e., HBr, HI, or more preferably, HCl.
  • Useful cations are alkali or alkaline earth metallic cations (i.e., Na, K, Li, V ⁇ Ca, V ⁇ Ba, etc.) or amine cations (i.e. , tetraalkylammonium, trialkylammonium, where alkyl can be C,-C, 2 ).
  • the amin ⁇ acid residues of MIS or its C-terminal fragment may be in their protected or unprotected form, using appropriate amino or carboxyl protecting groups.
  • the C-terminal fragment (human or bovine) can be readily produced by the recombinant DNA techniques described in U.S. Patent No. 5,047,336, which is fully incorporated by reference herein. Of particular interest is expression of the C-terminal fragment in E. coli and other bacteria, since the
  • C-terminal fragment is not glycosylated.
  • compositions of this invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby MIS or the C-terminal fragment of MIS or their functional derivatives are combined in admixture with a pharmaceutically acceptable carrier vehicle.
  • Suitable vehicles and their formulation, inclusive of other human proteins, i.e. , human serum albumin, are described for example in Remington's
  • compositions suitable for effective administration will contain an effective amount of MIS or the C-terminal fragment of MIS, or their functional derivatives, together with a suitable amount of carrier vehicle.
  • metastatic tumor growth is also inhibited when exposed to MIS or the C-terminal fragment of MIS.
  • the present invention is further directed to inhibiting primary and metastatic tumor growth by administering to tumor cells an effective amount of MIS or an effective amount of the C-terminal fragment of MIS.
  • the present invention is further directed to a method for inhibiting primary growth of tumors by transfecting tumor cells with a gene capable of expressing an effective amount of MIS or an effective amount of the C- terminal fragment of MIS.
  • a further embodiment of the present invention is directed to a method for inhibiting metastatic tumor growth comprising transfecting tumor cells with a gene capable of expressing an effective amount of MIS or an effective amount of the C-terminal fragment of MIS.
  • vulvar epidermoid carcinomas vulvar epidermoid carcinomas
  • cervical carcinomas endometrial adenocarcinomas
  • ovarian adenocarcinomas ovarian adenocarcinomas
  • ocular melanomas vulvar epidermoid carcinomas
  • cervical carcinomas endometrial adenocarcinomas
  • ovarian adenocarcinomas ovarian adenocarcinomas
  • ocular melanomas ocular melanomas.
  • primary and metastatic growth of prostate, lymphoid, breast, cutaneous and germ cell tumors can also be inhibited by the methods of the present invention.
  • Primary tumor growth and metastases entail different molecular mechanisms.
  • Primary tumor growth involves local proliferation of tumor cells.
  • a tumor cell for a tumor cell to become metastatic, a complex multistep process must occur-i.e., detachment of tumor cells from the local growth, invasion of inter-cellular matrices, penetration of basement membranes of blood vessels, circulation while undergoing homotypic aggregation, extravasation, and induction of angiogenesis in the target organ.
  • Each of these successive steps appears to be mediated by defined molecular mechanisms of the metastatic cell. Therefore, a tumor cell, while exhibiting local tumor growth, may fail to generate metastases if it lacks the molecular machinery required for any one of the sequential metastatic steps.
  • the differences between local (primary) and metastatic-tumor growth are discussed in Eisenbach et al, Cancer Rev. 5:1-18 (1986).
  • various sites may be selected for insertion of the gene coding for MIS or C-terminal fragment of MIS. These sites are usually designated by the restriction endonuclease which cuts them and are well recognized by those of skill in the art.
  • Various methods for inserting DNA sequences into these sites to form recombinant DNA molecules are also well known. These include, for example, dG-dC or dA-dT tailing, direct ligation, synthetic linkers, exonuclease and polymerase- linked repair reactions followed by ligation, or extension of the DNA strand with DNA polymerase and an appropriate single-stranded template followed by ligation.
  • a cloning or expression vehicle useful in this invention need not have a restriction endonuclease site for insertion of the chosen DNA fragment. Instead, the vehicle could be joined to the fragment by alternative means.
  • expression control sequences may also be chosen to effect the expression of the DNA sequences of this invention.
  • These expression control sequences include, for example, the ]ac system, the /3-lactamase system, the trrj system, the tac system, the trc system, the major operator and promoter regions of phase ⁇ , the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast ⁇ -mating factors, promoters for mammalian cells such as the SV40 early promoter, adenovirus late promoter and metallothionine promoter, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses and various combinations thereof.
  • the expression units In mammalian cells, it is additionally possible to amplify the expression units by linking the gene to that for dihydrofolate reductase and applying a selection to host Chinese hamster ovary cells.
  • these DNA sequences are operatively-linked to one or more of the above-described expression control sequences in the expression vector.
  • Such operative linking which may be effected before or after the MIS or C-terminal fragment of MIS DNA sequence is inserted into a cloning vehicle, enables the expression control sequences to control and promote the expression of the DNA sequence.
  • the vector or expression vehicle, and in particular the sites chosen therein for insertion of the selected DNA fragment and the expression control sequence employed in this invention, is determined by a variety of factors, e.g. , number of sites susceptible to a particular restriction enzyme, size of the protein to be expressed, expression characteristics such as start and stop codons relative to the vector sequences, and other factors recognized by those of skill in the art.
  • the choice of a vector, expression control sequence, and insertion site for the MIS or C-terminal fragment of MIS DNA sequence is determined by a balance of these factors, not all selections being equally effective for a given case.
  • the DNA sequences coding for MIS or the C-terminal fragment of MIS that are inserted at the selected site of a cloning or expression vehicle may include nucleotides which are not part of the actual gene coding for MIS or the C-terminal fragment of MIS or may include only a fragment of the actual gene. It is only required that whatever DNA sequence is employed, a transformed host will produce MIS or the C- terminal fragment of MIS.
  • the MIS DNA sequences of this invention may be fused in the same reading frame in an expression vector of this invention to at least a portion of a DNA sequence coding for at least one eukaryotic or prokaryotic signal sequence, or combinations thereof.
  • Such constructions enable the production of, for example, a methionyl or other peptidyl-MIS polypeptide, that is part of this invention.
  • This N-terminal methionine or peptide may either then be cleaved intra- or extra-cellularly by a variety of known processes or the MIS polypeptide with the methionine or peptide attached may be used, uncleaved, in the pharmaceutical compositions and methods of this invention.
  • the cloning vehicle or expression vector containing the MIS or C- terminal fragment of MIS polypeptide coding sequences of this invention is employed in accordance with this invention to transform tumor cells so as to permit expression of an effective amount of MIS or an effective amount of the C-terminal fragment of MIS to inhibit primary or metastatic tumor growth.
  • the MIS polypeptide prepared in accordance with this invention
  • MIS linked to prokaryotic, eukaryotic or combination N-terminal segment to direct excretion, improve stability, improve purification or improve possible cleavage at amino acid residue 443 to release an active C- terminal fragment
  • a precursor of MIS e.g., starting with all or parts of a MIS signal sequence of other eukaryotic or prokaryotic signal sequences
  • a mature MIS polypeptide e.g., a mature MIS polypeptide
  • fmet-MIS polypeptide linked to prokaryotic, eukaryotic or combination N-terminal segment to direct excretion, improve stability, improve purification or improve possible cleavage at amino acid residue 443 to release an active C- terminal fragment
  • the present invention also encompasses substituting codons for those of the MIS or C-terminal fragment of MIS nucleotide sequences. These substituted codons may code for amino acids identical to those coded for by the codons replaced but result in higher yield of the polypeptide. Alternatively, the replacement of one or a combination of codons leading to amino acid replacement or to a longer or shorter polypeptide may alter its properties in a useful way (e.g., increase the stability, increase the solubility or increase the therapeutic activity).
  • the present invention also provides gene therapy methods for treating patients with certain tumors.
  • Tumor-Infiltrating-lymphocytes are prepared from tumor biopsies obtained from patients suffering from tumors by methods known in the art (Rosenberg et al, N. Engl. J. Med. 379:1676-80 (1988); Topalian et al. , J. Immuol 742:3714-25 (1989)).
  • the gene coding for MIS or the C-terminal fragment of MIS can be inserted into an appropriate retroviral vector.
  • the retroviral vector will include a "selection" gene. That is, a gene coding for a product that allows for selection of TILs containing the retrovirus vector with insert.
  • Suitable "selection" genes are those coding for antibiotic resistance, such as the neomycin resistance gene. Other “selection" genes are known in the art.
  • the gene coding for MIS or the C-terminal fragment of MIS is inserted into the N2 retroviral vector which contains a neomycin resistance gene.
  • the retroviral vector with MIS or C-terminal fragment insert can be transfected into an amphotropic packaging cell line.
  • the amphotropic packaging cell lines PA-12 or PA-317 can be used.
  • Suitable retroviral vectors and amphotropic cell lines are described in Miller et al, Mol Cell. Biol. 6:2895-29 (1986); Cornetta et al, J. Virol. Methods 23 :187- 94 (1989); and Anderson et al , Science 226:401-9 (1984).
  • TILs can be cultured in interleukin-2 (IL-2) using art-known techniques.
  • IL-2 interleukin-2
  • a protocol at the National Cancer Institute requires growing the TILs in plastic, gas permeable culture bags (Topalian et al, J. Immunol. Methods 702:127 (1987)). Each bag supports up to 3x10° TIL in a 1.5 liter volume of tissue culture medium containing human serum albumin and IL-2.
  • Knazek et al. J. of Immunol. Methods 727:29-37 (1990) describes an improved method for growing TILs to clinically useful quantities.
  • the Knazek et al method involves growing TILs in hollow fiber cartridges.
  • Cultures of TILs can be transduced with a recombinant retroviral vector containing the MIS or C-terminal fragment of MIS gene insert using art-known techniques. For example, transduction can occur by exposing the TILs to culture supernatant from packaging cell lines transfected with a retroviral vector containing the MIS or C-terminal fragment of MIS gene insert. Transducing cultures of TILs by exposure to culture supernatant from a packaging cell line that produces N2 containing virions is described in Culver et al , Proc. Natl Acad. Sci. USA 88:3155-59 (1991); Kasid et al , Prpc. Natl Acad. Sci.
  • Transduced-TILs can then be selected for in an approprite selection medium.
  • the retroviral vector contains the neomycin transferase gene
  • selection can occur in the neomycin analog G418.
  • TILs containing the retroviral vector will be selected for in the medium.
  • These TILs can then be further grown until the total growth reaches the number of cells ordinarily used for therapy.
  • Current protocols infuse 2-3x10" cells into the patient for therapy. Infusion can occur by any suitable method.
  • the genetically-altered TILs can be re-inserted into the patient intravenously.
  • TILs are known to preferentially localize at the tumor site in vivo. See, for example Culver et al, Proc. Natl. Acad. Sci. USA 88:3155-59 (1991) and Kasid etal, Proc. Natl. Acad. Sci. USA 87:473-7 (1990). Therefore, the present invention provides a method of treating tumors in a patient comprising using TILs as cellular vehicles for transferring a retroviral vector, capable of expressing an effective amount MIS or an effective amount of the C-terminal fragment of MIS, to the tumor site.
  • Another embodiment of the present invention provides a method for direct in situ introduction of a retroviral vector, capable of expressing an effective amount of MIS or an effective amount of the C-terminal fragment of MIS, into proliferating tumors.
  • the gene coding for MIS or the C- terminal fragment of MIS can be inserted into a retroviral vector to form a recombinant construct.
  • this construct can be transfected into an amphotropic packaging cell line using art-known techniques.
  • suitable retroviral vectors and amphotropic cell lines are described in Miller et al, Mol. Cell. Biol. 6:2895-2902 (1986); Cornetta et al, J. Virol.
  • Transfected packaging cell lines are known to continually release the retroviral vector.
  • the transfected packaging cell line can be injected into the tumor mass for direct in situ transfer of the gene coding for MIS or the C-terminal fragment of MIS to the tumor.
  • the transfected packaging cell line can be grafted near or into the tumor to provide a long- lasting source of the retrovirus containing the MIS or C-terminal fragment of MIS gene insert (see Rosenberg et al. , Science 242: 1575-78 (1988) and Wolff et al, PNAS USA 86:9011-9014 (1989)).
  • Rosenberg et al. Science 242: 1575-78 (1988)
  • Wolff et al, PNAS USA 86:9011-9014 (1989) in vivo gene transfer using retroviral vector-producer cells for treating tumors is described in Culver et al , Science 256:1550-52 (1992) and Ram et al., Cancer Research 53:83-88 (19
  • retroviral vectors can also contain one or more or drug susceptibility ("suicide") genes.
  • retrovirus vectors used in the methods of the present invention can further include the gene coding for herpes simplex thymidine kinase (HS-tk). Tumor cells containing the HS-tk gene become sensitive to treatment with ganciclovir (GCV) (Moolten et al , Cancer Res. 46:5276 (1986); Borrelli et al , Proc. Natl. Acad. Sci U.S.A 85: 7572 (1988); Moolten et al, J. Natl Cancer Inst.
  • GCV herpes simplex thymidine kinase
  • the retrovirus vectors of the present invention can include the gene coding for the bacterial enzyme cytosine deaminase.
  • Tumor cells expressing the bacterial enzyme cytosine deaminase convert the ordinarily nontoxic drug 5'-fluorocytosine to the cytotoxic compound 5-fluorouracil, which will kill the tumor cells (Mullen et al , PNAS USA 89:33 (1992)).
  • other drug susceptibility genes can be used. Including a drug susceptibility gene in the vector in addition to the gene coding for MIS or the C-terminal fragment of MIS can increase toxicity to the tumor cells without adversely affecting surrounding normal cells.
  • an “effective amount” of MIS is one which is sufficient to inhibit growth of the tumors of this invention in a human or animal.
  • an “effective amount” of the C-terminal fragment of MIS is one which is sufficient to inhibit growth of the tumors of this invention in a human or animal.
  • inhibition of a tumor implant can be indicated by a decrease in graft size ratio.
  • the graft size ratio is calculated as (L2 x W2 x W2) / (LI x Wl x Wl), wherein LI is the longest diameter of the implant, Wl is the diameter perpendicular to LI, L2 is the longest diameter of the tumor, and W2 is the diameter perpendicular to L2.
  • the volume of the tumor (L2 x W2 x W2) before and after treatment need only be compared.
  • the effective amount may vary depending upon criteria such as the age, weight, physical condition, past medical history, and sensitivity of the recipient.
  • the effective amount will also vary depending on whether administration is oral, intravenous, intramuscular, subcutaneous, local, or by direct application to the tumor. In the case of direct tumor application, it is preferable that a final serum concentration of at least 0.1 nM, preferably about 0.1-1.0 nM, of MIS be achieved.
  • a final serum concentration of at least 0.1 nM, preferably about 0.1-1.0 nM, of the C- terminal fragment of MIS be achieved.
  • Effective individual dosage through the additionally named means of administration can be readily determined by methods well known to those of ordinary skill in the art. For example, using the size ratio calculation as detailed above, one of ordinary skill in the art can determine optimal dosage levels for any means of administration.
  • a serum level of at least 10 ng/ml of MIS In treating a patient with the C-terminal fragment of MIS, it is preferable to achieve a serum level ranging from about 1 ng/ml to about 20 ⁇ g/ml of the C-terminal fragment of MIS.
  • compositions containing MIS or the C-terminal fragment of MIS or their functional derivatives may be administered orally, intravenously, intramuscularly, subcutaneously, or locally. Additional pharmaceutical methods may be employed to control the duration of action. Controlled release preparations may be achieved by the use of polymers to complex or adsorb MIS or the C-terminal fragment of MIS or their functional derivatives.
  • the controlled delivery may be exercised by selecting appropriate macromolecules (for example polyesters, polyamino acids, polyvinyl pyrrolidone, ethylenevinylacetate, methy lcellulose, carboxymethylcellulose, and protamine sulfate) and the concentration of macromolecules as well as the methods of incorporation in order to control release.
  • appropriate macromolecules for example polyesters, polyamino acids, polyvinyl pyrrolidone, ethylenevinylacetate, methy lcellulose, carboxymethylcellulose, and protamine sulfate
  • concentration of macromolecules as well as the methods of incorporation in order to control release.
  • Another possible method to control the duration of action by controlled release preparations is to incorporate MIS or the C-terminal fragment of MIS into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinyl acetate copolymers.
  • MIS or the C-terminal fragment of MIS instead of incorporating MIS or the C-terminal fragment of MIS into these polymeric particles, it is possible to entrap MIS or the C-terminal fragment of MIS in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly(methylmethacrylate) microcapsules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions.
  • compositions which include the proteolytically cleaved MIS protein fragments of this invention can also include chemotherapeutic agents which are known to inhibit tumor growth in a human or animal.
  • the pharmaceutical compositions including proteolytically cleaved MIS protein fragments can include both the N- and C-terminal fragments or the C-terminal fragment alone. When the N-terminal fragment is present in the composition, it may be further cleaved into smaller fragments by prolonged proteolysis.
  • the chemotherapeutic agent included in this composition can be directed to any specific neoplastic disease. Such agents are described in Goodman and Gilman 's The Pharmacological Basis of Therapeutics, 8th Ed., Pergamon Press, New York, N.Y., 1985. It is preferred, however, that the chemotherapeutic agent inhibit growth of the tumors of this invention.
  • the chemotherapeutic agent which is combined with MIS or the C-terminal fragment of MIS will have an additive effect on the treatment of the tumors of this invention.
  • the quantity of chemotherapeutic agent used in treating the tumors of this invention can be reduced from the manufacturer's recommended dose, thereby reducing undesirable side effects.
  • an equivalent effective amount of MIS or the C-terminal fragment of MIS can be added.
  • the use of the term "equivalent effective amount” does not necessarily mean an equivalent weight or volume quantity, but represents the quantity of MIS or the C-terminal fragment of MIS that offers an equal inhibition to tumor growth. This may have to be evaluated on a patient by patient case, but can be determined, for example, by comparing quantities that achieve equal size reduction ratios as defined above.
  • chemotherapeutic agents which can be combined with MIS or the C-terminal fragment of MIS for treatment of the tumors of this invention will be effective between about 0.001 and 10.0 mg/kg body weight of the patient.
  • Administration of the combination of MIS or C-terminal fragment of MIS and chemotherapeutic agent can be accomplished in the same manner as administration of the MIS or C-terminal fragment of MIS alone.
  • chemotherapeutic agents in the pharmaceutical compositions of this invention are nitrogen mustards such as cyclophosphamide, ifosfamide, and melphalan; ethylenimines and methylmelamines such as hexamethylmelamine and thiotepa; pyrimidine analogs such as fluorouracil and fluorodeoxyuridine; vinca alkaloids such as vinblastine; epipodophyllotoxins such as etoposide and teniposide; antibiotics such as actinomycin D, doxorubicin, bleomycin, and mithramycin; biological response modifiers such as interferon a; platinum coordination complexes such as cisplatin and carboplatin; estrogens such as diethylstilbestrol and ethinyl estradiol; antiandrogens such as flutamine; and gonadotropin releasing hormone analogs such as leuprolide.
  • nitrogen mustards such as cyclophosphamide,
  • MHC genes are believed to correlate with the capacity of tumor cells to metastasize (Tanaka, K., et al , Science 228:26 (1985); Eisenbach, L., et al., Cane. Rev. 5: 1-18 (1986), herein incorporated by reference).
  • the metastasis of a tumor cell is a complex multi-step process, involving detachment of tumor cells from a local growth, invasion of inter ⁇ cellular matrices, penetration of basement membranes of blood vessels, circulation while undergoing homotypic aggregation, extravasation, and induction of angiogenesis in the target organ.
  • Tumors which are metastatic have been found to express depressed levels of certain MHC antigens, and in particular, the MHC class I antigen H- 2K. Such metastatic tumors may, however, express normal or elevated levels of a second MHC class I antigen known as H-2D. In contrast, non-metastatic tumors have been found to express normal or elevated levels of the H2-K antigen, but to express depressed levels of the H2-D antigen.
  • cytotoxic T cells In general, such cytotoxic T cells (“CTL”) recognize and destroy foreign or tumor cells through the recognition of a non-MHC cell surface epitope (i.e. a conventional antigen) which is in association with a class I MHC molecule on the surface of the foreign or tumor cell.
  • CTL cytotoxic T cells
  • a non-MHC cell surface epitope i.e. a conventional antigen
  • class I MHC antigen i.e. a conventional antigen
  • the presence of the H-2K class I MHC antigen on a tumor cell is believed to increase the capacity of that cell to be recognized by a CTL - i.e. to increase the cell's immunogenicity (Eisenbach, L. , et al , Cane. Rev.
  • metastatic tumors have been found to manifest abnormally low levels of H-2K MHC class I antigens on their cell surfaces (Eisenbach, L., et al, Int. J. Cane. 32: 113-120 (1983); Eisenbach, L., et al., Int. J. Cane. 34:567-573 (1984); Eisenbach, L., et al , In: Cancer Invasion and Metatasis: Biologic and Therapeutic Aspects (Nicolson, G.L., et al, eds.), Raven Press, NY, pp. 101-121 (1984)); Eisenbach, L., Mod. Trends Hum.
  • Treatment of cells with a- or B-interferon has, however, been found to preferentially increase expression of the H2-D antigen, and thus to increase the metatastic properties of certain tumor cells (Eisenbach, L., 7nt. J. Cane. 32:113-120 (1983)).
  • 7-interferon has been found to preferentially stimulate expression of the H-2K class I MHC antigen, and to therefore decrease the metatastic competence of tumor cells (Eisenbach, L., et al, In: Biochemistry and Molecular Genetics of Tumor Metatasis (Lapis, K., et al , eds.) Boston-Dordrecht-Lancaster: Martinus Nijhoff Publ. 167-184 (1986)).
  • Retinoic acid has been found to be capable of regulating the expression of the H2-D class I MHC antigen, but not of the H2-K class I MHC antigens (Eisenbach, L., et al, Int. J. Cane. 32: 113-120 (1983)). Accordingly, retinoic acid increases the metastatic capacity of tumor cells.
  • the product of the c-fos gene has been found to be capable of regulating the expression of both the H2-K and H2-D antigens (Eisenbach, L., et al, Cane. Rev. 5:1-18 (1986)).
  • H-2K MHC class I antigen has been found to be inversely related to the capacity of a tumor cell to metatasize.
  • H-2K expression is associated with nonmetastatic tumor growth.
  • agents capable of generally increasing MHC class I expression appear to reduce the capacity of tumor cells to proliferate.
  • H-2D MHC class I antigen has been found to be associated with tumor metatasis.
  • An increase in H-2D expression is associated with increased metastatic activity.
  • Embryonic growth and development though explosive, is stringently controlled, responding to exacting genetic and environmental signals, as well as rigorous temporal signals.
  • the embryo evolves in a sea of growth factors and growth inhibitors which play a role in accelerating or delaying disproportionate development. However, little is understood about the mechanisms by which these disparate factors exert their control at that time.
  • MHC Major Histocompatibility Complex
  • EGF Epidermal Growth Factor
  • MIS M ⁇ llerian Inhibiting Substance
  • fetal tissue was sought in a mouse strain containing well established markers for H-2 class I and II loci.
  • Secondary fetal fibroblasts from the C57B1/6 strain were selected, from which one could generate large numbers of cells for RNA extraction.
  • Other, similar mouse strains or cells derived from humans could aternatively be employed.
  • Secondary fetal fibroblasts from the C57B1/6 strain were found to express measurable levels of Class I mRNA.
  • EGF and MIS were tested for their capacity to modulate the expression of the antigens of the MHC and found to be capable of precisely governing the expression of MHC class I mRNA.
  • MHC mRNA expression was found to be up-regulated by the growth inhibitor MIS and down-regulated by the EGF growth factor.
  • MIS ability of MIS to increase the expression of MHC class I mRNA suggests that expression of the MHC class I antigens will be also be increased in the presence of MIS. As discussed above, such an increase in the expression of MHC class I antigen expression is associated with increased immunogenicity, and hence, decreases the capacity of a tumor to metastasize.
  • MIS is capable of increasing the expression of MHC class I mRNA is one aspect of the present invention, and provides a method of controlling the growth of cells during embryonic development and during uncontrolled tumor growth.
  • Human Immunodeficiency Virus HIV is believed to be the causal agent for AIDS (Acquired Immunodeficiency Disease Syndrome).
  • HIV is believed to cause an immunosuppressed state in an individual through its capacity to recognize and selecively inactivate T helper cells.
  • the virus has been found to express antigens whose domains are similar in sequence to those of the MHC class II antigens (Golding, H. et al, ]. Exper. Med. 767:914-923 (1988)).
  • the virus has been hypothesized to confer an immune suppressed condition on an individual by attaching to the T4 (CD4) receptor of helper T lymphocytes, and (since its domains resemble those of a class II MHC molecule) of thereby provoking an immune response against any cell bearing a class II MHC molecule (Ziegler, J.L. et al , Clin. Immunol. Immunopath. 47:305-313 (1986)).
  • T4 CD4 receptor
  • AIDS infection is, therefore, characterized by an impairment of MHC class 1 antigen expression, it frequently results in the establishment of debilitating tumors (such as Kaposi's sarcomas).
  • debilitating tumors such as Kaposi's sarcomas.
  • interferons such as a and 7 to effect expression of MHC class I antigens provides a theoretical basis for their use in treating such tumors in AIDS patients.
  • the use of interferons in the treatment of AIDS is reviewed by Krown, S.E. (In: Developments in Medical Virology: Clinical Aspects of Interferons, (Revel, M., Ed.), Kluwer Academic Pub., Boston, MA, pp. 62-74 (1988)).
  • MIS shares with 7-interferon the capacity to increase the expression of MHC class I mRNA, it may be employed in the same manner as interferon in the treatment of immunodeficiency diseases.
  • individuals who suffer from AIDS or other diseases which result from the prevalence of an immunosuppressed state can be treated with MIS or its functional derivatives or agonists in order to increase their immune response.
  • MIS, its functional derivatives, or its agonists can be used to ameliorate the immune suppressed state of such patients.
  • the MIS of the present invention may be provided in combination (i.e. co-administered) with interferon or other antiviral therapies (such as AZT, etc).
  • interferon or other antiviral therapies such as AZT, etc.
  • Such therapies are reviewed by Clumeck, N., et al. (Amer. J. Med. 85:165-172 (1987)); Jacobs, J.L. (In: Year in Immunology Vol. 3 (Cruse, J.M. et al, Eds.), Karger AG, Basel, pp. 303-309 (1988)) and Sarin, P.S. (Ann. Rev. Pharmacol. Toxicol 28:411-428 (1988)), all of which references are herein incorporated by reference.
  • tissue such as organs, etc.
  • the present invention is directed toward the use of MIS and EGF and their "functional derivatives" in the treatment of metastatic tumors, immunosuppressive and immunodeficiency diseases, and organ and tissue transplant.
  • the MIS or EGF molecule disclosed herein are said to be “substantially free of natural contaminants” if preparations which contain them are substantially free of materials with which these products are normally and naturally found.
  • a “functional derivative” of either MIS or EGF is a compound which posesses a biological activity (either functional or structural) that is substantially similar to a biological activity of MIS or EGF, respectively.
  • the term “functional derivatives” is intended to include the “fragments,” “variants, “ “analogs,” or “chemical derivatives” of a molecule.
  • a “fragment” of a molecule such as either MIS or EGF is meant to refer to any polypeptide subset of the molecule. Fragments of either MIS or EGF which have activity and which are soluble (i.e not membrane bound) are especially preferred.
  • a “variant" of a molecule such as either MIS or EGF is meant to refer to a molecule substantially similar in structure and function to either the entire molecule, or to a fragment thereof. A molecule is said to be "substantially similar” to another molecule if both molecules have substantially similar structures or if both molecules possess a similar biological activity.
  • a molecule such as either MIS or EGF is meant to refer to a molecule substantially similar in function to either the entire molecule or to a fragment thereof.
  • a molecule is said to be a "chemical derivative" of another molecule when it contains additional chemical moieties not normally a part of the molecule. Such moieties may improve the molecule's solubility, absorption, biological half life, etc.
  • the moieties may alternatively decrease the toxicity of the molecule, eliminate or attenuate any undesirable side effect of the molecule, etc. Moieties capable of mediating such effects are disclosed in Remington's Pharmaceutical Sciences (1980).
  • “Toxin-derivatized” molecules constitute a special class of “chemical derivatives.
  • a "toxin-derivatized” molecule is a molecule (such as either MIS or EGF or an antibody to their receptors) which contains a toxin moiety. The binding of such a molecule to a cell brings the toxin moiety into close proximity with the cell and thereby promotes cell death.
  • any suitable toxin moiety may be employed; however, it is preferable to employ toxins such as, for example, the ricin toxin, the diphtheria toxin, radioisotopic toxins, membrane-channel-forming toxins, etc. Procedures for coupling such moieties to a molecule are well known in the art.
  • agonist is intended to refer to an agent which enhances the physiologic response of an organ or organism to the presence of a second agent.
  • an agonist of MIS increases the effectiveness of MIS by increasing an individual's response to the presence of MIS.
  • EGF increases the effectiveness of that agent.
  • Examples of agonists of MIS include antibody (or fragments thereof) to EGF, interferon ( ⁇ , ⁇ , or 7), etc.
  • EGF is an antagonist of MIS.
  • antagonist is intended to refer to an agent which diminishes the physiologic response of an organ or organism to the presence of a second agent.
  • an antagonist of MIS would decrease the effectiveness of MIS by decreasing an individual's response to the presence of MIS.
  • an antagonist of EGF decreases the effectiveness of that agent.
  • An example of an antagonist of EGF is antibody (or fragments thereof) to EGF.
  • MIS, and interferon are examples of antagonists of EGF.
  • MIS or EGF therapeutic advantages of either MIS or EGF (or their functional derivatives, or agonists) may be augmented through the use of functional derivatives of either compound possessing additional amino acid residues added to enhance coupling to carrier or to enhance the activity of the either MIS or EGF.
  • the scope of the present invention is further intended to include functional derivatives of either MIS or EGF which comprise therapeutically active peptide fragments of these molecules. Such fragments may lack certain amino acid residues, or may contain additional amino acid residues, so long as such derivatives exhibit the capacity to affect MHC mRNA expression.
  • MIS and EGF derivatives which may lack (or contain) one or two or three (additional) amino acid residues from either their amino or carboxyl termini.
  • Either MIS or EGF or their functional derivatives or agonists may be obtained either synthetically, through the use of recombinant DNA technology, or by proteolysis.
  • MIS vascular endothelial growth factor
  • EGF EGF-like growth factor
  • two agents are said to be co-administered when they are provided to tissue or to an individual in such close proximity of time that both agents can be observed to exert a detectable effect on the tissue or individual at the same time.
  • a composition is said to be "pharmacologically acceptable” if its administration can be tolerated by a recipient patient.
  • Such an agent is said to be physiologically significant if its presence results in a detectable change in the physiology of a recipient patient.
  • the metastasis-supressive activity of MIS may be provided to a recipient by providing either MIS, or a functional derivative or agonist of MIS. Suppression of metastasis may be achieved through the administration of antibody to EGF or to its cellular receptor, or through the administration of a toxin-derivatized' EGF molecule.
  • MIS can be co-administered with interferon (or other agonists) in order to increase the effectiveness of the therapy.
  • the capacity of EGF to enhance immunogenicity may be provided to a recipient by providing either EGF, or a functional derivative or agonist of EGF. Enhancement of immunogenicity may be achieved through the administration of antibody to MIS or to its cellular receptor, or through the administration of a toxin-derivatized MIS molecule. It is additionally possible to combine the administration of MIS and
  • EGF EGF (or their functional derivatives or agonists) to a patient in order to modulate the effects of these compounds on the expression of the MHC antigens, immunogenicity, or tumor metastasis.
  • other agents, or combinations of agents will also be perceived by those of ordinary skill. Such agents and combinations are intended to be among the equivalents of the present invention.
  • the dosage of administered agent will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition, previous medical history, etc.
  • Either MIS or EGF (or their functional derivatives, agonists, or antagonists) may be administered to patients intravenously, intramuscularly, subcutaneously, enterally, or parenterally.
  • the administration may be by continuous infusion, or by single or multiple boluses.
  • the compounds of the present invention are intended to be provided to recipient subjects in an "effective amount. " An amount of a compound is said to be “effective” if, with regard to the treatment of metastatic tumors, the dosage, route of administration, etc. of the compound is sufficient to suppress or prevent the metastasis of the tumor. Similarly, with respect to the treatment of immunocompromised individuals, an amount of a compound is said to be “effective” if the dosage, route of administration, etc. of the compound is sufficient to alleviate or improve the immune state of the individual.
  • the administration of such compounds may be for either a "prophylactic" or "therapeutic" purpose.
  • the compounds When provided prophylactically to suppress metastasis, the compounds are provided in advance of the detection of any metastasis (for example, at, or shortly after the time of tumor detection).
  • the prophylactic administration of the compounds serve to prevent or attenuate any subsequent metastasis.
  • the compounds When provided therapeutically to suppress metastasis, the compounds are provided at (or shortly after) the detection of an actual metastatic process.
  • the therapeutic administration of the compounds serve to attenuate such actual metastasis.
  • the compounds of the present invention may, thus, be provided either prior to the onset of a metastatic process (so as to suppress an anticipated metastasis) or after the initiation of such a process.
  • the compounds of the present invention may also be provided either prophylactically (i.e. prior to the recognition of such immunosuppressed or immunodeficient state (as to an individual having symptoms of ARC - AIDS Related Condition)), or therapeutically (as to an individual ehibiting symptoms of such an immunocompromized state).
  • EGF EGF
  • its antagonist or its functional derivatives (or any other suitable antagonist of MIS) to prevent or attenuate the rejection of organs or tissue in a transplant recipient
  • such agents can be administered (in any of the manners described above) to the transplant recipient at, or prior to, the time of transplantation.
  • such compounds can be provided to the organ donor at, or after, the time of death or donation.
  • the organ or tissue to be donated will be perfused with the MIS antagonist in order to decrease the expression of MHC class I antigens on the surfaces of the cells of the donated material.
  • Such perfusion may be either systemic (as by introducing the MIS antagonist in a manner so as to cause it to be generally present throughout all or most of the organs or tissue of the donor) or localized (as by bathing, injecting or perfusing such compounds into an isolated organ, tissue or region).
  • an organ or tissue selected for transplantation may be provided with an MIS antagonist (by bathing, injection, or perfusion) after the removal of such material from the donor.
  • the organ may be prepared for storage pending transplantation in a solution or composition containing an MIS antagonist.
  • MIS or EGF molecules of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby these materials, or their functional derivatives, are combined in admixture with a pharmaceutically acceptable carrier vehicle.
  • Suitable vehicles and their formulation, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in Remington's Pharmaceutical Sciences (16th ed. , Osol, A., Ed., Mack, Easton PA (1980)).
  • a pharmaceutically acceptable composition suitable for effective administration such compositions will contain an effective amount of either MIS or EGF molecule, or their functional derivatives, agonists, or antagonists, together with a suitable amount of carrier vehicle.
  • Control release preparations may be achieved through the use of polymers to complex or absorb either MIS or EGF, or their functional derivatives, agonists, or antagonists.
  • the controlled delivery may be exercised by selecting appropriate macromolecules (for example polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine, sulfate) and the concentration of macromolecules as well as the methods of incorporation in order to control release.
  • MIS or EGF molecules or their functional derivatives, agonists, or antagonists, into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers.
  • microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcelluloseorgelatine-microcapsulesandpoly(methylmethacylate) microcapsules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions.
  • coacervation techniques for example, hydroxymethylcelluloseorgelatine-microcapsulesandpoly(methylmethacylate) microcapsules, respectively
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions.
  • compositions of the present invention may be prepared as articles of manufacture, such as "kits. "
  • kits will contain two or more containers which are specially adapted to receive MIS (or EGF) or one of their functional derivatives, and an agonist of MIS (or EGF).
  • dihydrofolate reductase deficient CHO were cotransfected with a linear transcript of both the human
  • MIS and the dihydrofolate reductase genes according to the method of Cate (Cate et al, Cell 45:685-698 (1986)).
  • the transfected CHO cells were amplified in Methotrexate and grown at 37°C in alpha minimal essential medium without ribonucleosides and deoxyribonucleosides, supplemented with 10% bovine MIS-free female fetal calf serum (FCS).
  • FCS bovine MIS-free female fetal calf serum
  • Two different MIS purification protocols were used to provide either partially pure or homogeneous MIS.
  • the immunoaffinity chromatography method used herein was directed toward the recovery of recombinant human MIS (rhMIS).
  • the protein was purified from the conditioned media of Chinese hamster ovary (CHO) cells, transfected with a linear construct of the human rhMIS gene and the DHFR gene, amplified by 30 nM Methotrexate selection, grown to confluence in four liter bioreactors on stainless steel coils as described by Epstein (Epstein et al, In Vitro Cell, and Devel.
  • a 5 ml immunoaffinity column was constructed using approximately 50 mg of a Protein A-Sepharose (BioRad) purified monoclonal anti-human rhMIS antibody [6E11] as described by Hudson (Hudson et al, J. Clin. Endocrinol. Metab. 70: 16-22 (1990)), and covalently attached to Affigel-10 agarose resin (BioRad), per the manufacturer's instructions (approximately 80% coupling efficiency).
  • the column was equilibrated with 100 mis of 20 mM HEPES, pH 7.4, and 200 ml of the concentrated media loaded at 1 column volume/hour at 4°C. After loading, the column was washed with 20 mM HEPES, pH 7.4, until the absorbance at 280 nm returned to baseline (60-100 mis).
  • Elution of rhMIS bound to this column was achieved using 2.0 M sodium thiocyanate (NaSCN); or 1 M Acetic acid, 20 M HEPES, pH 3.0, with or without a pre-elution step containing 0.5 M NaCI, 1 mM EDTA, 0.001 % NP-40, 20 mM HEPES, pH 7.4.
  • NaSCN sodium thiocyanate
  • dilutional effects ranged from 10-50%.
  • MIS concentrations were estimated using an enzyme-linked immunosorbent assay (ELISA) for MIS according to the method of Hudson supra. Protein concentrations were measured by the method of Bradford (Bradford, M.M., Anal. Biochem. 72:248-254 (1976)).
  • Monoclonal antibody was produced by immunizing female A/J mice (Jackson Laboratory, Bar Harbor, ME.) with immunoaffinity purified rhMIS using methods previously described for bovine MIS (Hudson et al, J. Clin. Endocrinol. Metab. 70: 16-22 (1990); Mudgett-Hunter et al, J. Immunol 728: 1327-33 (1982)). Spleen cells producing anti-MIS antibodies were harvested and hybridomas were produced as described by Kohler (Kohler, G., Immunol. Methods 2:285-98 (1981)). A monoclonal line (6E11) was selected and amplified in Dulbecco's modified essential medium supplemented with 15 % FCS. The antibody was precipitated from 6E11 conditioned medium with 50% (NH 4 ),SO 4 , and further purified by protein A-Sepharose CL-4B (Sigma, St. Louis, MO.) chromatography. C. Cell Lines.
  • A431 a cell line derived from a human vulvar epidermoid carcinoma, HT-3 from a human lymph node metastasis of a cervical carcinoma, NIH:OVCAR-3 from a human ovarian adenocarcinoma, HEC- 1-A from a human endometrial adenocarcinoma, RT4 from a human bladder transitional- cell papilloma, and Hep 3B from a human hepatocellular carcinoma were obtained from American Type Culture Collection.
  • OM431 from a human ocular melanoma, was obtained from Dr. James Epstein of Massachusetts General Hospital.
  • the A431, HT-3, NIH:OVCAR-3, OM431 and Hep 3B cells were maintained with alpha minimal essential medium with ribonucleosides and deoxyribonucleosides ( ⁇ -MEM +) supplemented with 10% female FCS and 2 mM L-glutamine.
  • HEC-l-A and RT4 were maintained with McCoy's 5A medium supplemented with 10% female FCS.
  • cells were serially subcultured, then trypsinized at 70-80% confluency to achieve synchrony. This proliferating cell population was then centrifuged at 1500 RPM for 5 minutes and resuspended with 10% female FCS supplemented medium. Cells were counted in a hemocytometer.
  • IAP-MIS and the salt eluted fraction from the immunoaffinity column were tested using A431, HT-3, HEC-IA, NIH:OVCAR-3, OM431, and Hep 3B cells in the conventional double layer agarose colony inhibition assay of Hamburger (Hamburger et al, Science 797:461-463 (1977); Hamburger et al, J. Clin. Invest. 60:846-854 (1977)).
  • the underlayer of the 35 mm culture dishes contained 1 ml of 0.6% agarose (Sigma, St. Louis, MO.) in 10% female FCS supplemented ⁇ -MEM + .
  • the overiayer consisted of 0.3% agarose in 10% female FCS supplemented - MEM + , the cells to be tested (50,000 cells/ml for A431 , HT-3, and 0M431 ; 25,000 cells/ml for HEC-l-A, NIH:OVCAR-3, and Hep 3B), epidermal growth factor (EGF) 10 ng/ml (Sigma, St. Louis, MO.) and one of the following: (a) IAP-MIS (final concentration 30 nM); (b) IAP-salt (final protein concentration 19.3 ⁇ g/ml); or (c) vehicle buffer as a negative control. The dishes were incubated in humid air with 5% CO 2 at 37°C for 10-21 days. Colonies with more than 30 cells were counted with an inverted microscope (Nikon). The results were expressed as percent survival relative to a control group (number of colonies in the test group x 100/number of colonies in the control group).
  • IAP-MIS final concentration 30 nM
  • IAP-salt final protein concentration 19.3 ⁇ g/ml
  • EGF 50 ng/ml was added to A431 and OM431 to suppress monolayer growth but stimulate colony formation according to the method of Lee (Lee et al. Exp. Cell Res. 173: 156- 162 (1987)). The result was expressed as percent survival relative to the vehicle buffer control.
  • DG-MIS was tested in final concentrations of 0.9, 1.8, 3.5, and 7.0 nM using A431 cells (25,000 cells/ml, EGF 50 ng/ml) in the liquid medium colony inhibition assay. IAP-MIS was tested in concentrations of 12, 24, 48, and 96 nM. The results were expressed as percent survival relative to the vehicle buffer control.
  • MIS monoclonal antibody (6E11) and normal rabbit IgG were dialyzed into serum-free ⁇ -MEM + before use. Previous experiments showed maximum absorption of MIS activity at MIS:Ab ratio of 1:3. Therefore, 17.4 ⁇ g of 6E11 was added to 5.6 ⁇ g of DG-MIS. Normal rabbit IgG was diluted with culture medium and added to MIS in the same 1:3 ratio to determine nonspecific absorption. An equivalent amount of protein purified from conditioned medium of untransfected wild type CHO cells served as a negative control when mixed 1:3 with antibody as above. The preparations were mixed at 4°C for 12 hours.
  • Protein A Sepharose 9CL-4B (Sigma, St. Louis, MO.), after being washed in serum free medium, was added and incubated at 4°C for another 12 hours. The mixtures were centrifuged and the supernatants tested in the liquid medium colony inhibition assay using A431 cells (25,000 cells/ml, EGF 50 ng/ml). Percent survival of each group was calculated by comparing the number of colonies in each group to the wild type negative control. Cotreatment of IAP-MIS and the Salt Eluted Fraction (IAP- salt).
  • IAP-MIS was mixed with an equal volume of IAP-salt (protein concentration 0.199 mg/ml) to give a final MIS concentration of 50 nM. It was tested using A431 cells (25,000 cells/ml, EGF 50 ng/ml) in the liquid medium colony inhibition assay and compared to IAP-MIS, IAP-salt, and vehicle buffer. Percent survival was calculated by comparing the number of colonies in each group to that of vehicle buffer negative control.
  • Cisplatin (Bristol Laboratory, Syracuse, NY) was diluted in serum free culture medium to give concentrations of 0, 0.078, 0.156, 0.312 and 0.624 ⁇ g/ml and tested in triplicate with or without DG-MIS (final concentration 7 nM) using A431 cells (25,000 cells/ml, EGF 50 ng/ml) in the liquid medium colony inhibition assay. Vehicle buffer was used as negative control. The percent survival was calculated by comparing the number of colonies grown at each dose to that achieved in the vehicle buffer negative control.
  • Multicellular tumor spheroids of HT-3 and Hep 3B cells were produced by the method described by Yuhas et al. (Yuhas et al, Cancer Res. 37:3639- 3643 (1977)).
  • 10 6 cells of HT-3 or Hep 3B in 10 ml of 10% female FCS supplemented ⁇ -MEM + were plated on the top of 1 % agarose in a 10 cm culture dish and incubated in humid air with 5% CO 2 at 37 °C.
  • Spheroids usually formed in 2-5 days.
  • 0.5 ml of 1 % agarose was added to each well of a 24-well culture plate (Falcon, Oxnard, CA.,#3047) to form a bottom layer before use.
  • A431 and OM431 cells were tested. 10 7 of the cells were centrifuged at 1500 RPM for 5 minutes to form a pellet. 15 ⁇ l of fibrinogen (Sigma, St. Louis, MO., 20 mg/ml dissolved in PBS pH 7.4) was added to the pellet, followed by 8 ⁇ l of thrombin (Sigma, St. Louis, MO., 20 unit/ml dissolved in double-strength Dulbecco' s modified essential medium). The mixture was incubated at 37°C for 15 min. The cell clot thus formed was cut into approximately 100 fragments (1 mm 3 each containing approximately 10 5 cells) in preparation for implantation.
  • MIS was delivered by an Alzet mini-osmotic pump (#2001, Alza, Palo Alto, CA.) placed in the peritoneal cavity at the time of tumor implantation. These pumps have a fill volume of 209 ⁇ 6 ⁇ l and release their contents at a rate of 1.03 ⁇ 0.04 ⁇ l/hr for approximately eight days of delivery time.
  • the pumps were either filled with IAP-MIS (MIS: 159 ⁇ g/ml by ELISA) or with vehicle buffer.
  • a total MIS dose of approximately 33 ⁇ g (230 nM) was given to each mouse in the MIS group over the course of the experiment.
  • Virus and pathogen free female CD-I mice (10 weeks old, average weight 35 g, Charles River Breeding Laboratory, Wilmington, MA.) were given whole body irradiation of 640 rads by a Mark-1 cesium- 137 irradiation 16-24 hours before the experiment (Gajewski et al, Surgical Forum 38:468- 470 (1987)). After inducing anesthesia with an intraperitoneal injection of 0.3 ml of 10% Pentobarbital (Abbott Laboratory, North Chicago, IL), an incision was made in the left flank of the mouse and the left kidney exteriorized. A subcapsular space was developed using a 19-gauge needle trocar.
  • a cell clot was introduced into the space with a segment of 5-0 Nylon suture (approximately 1 mm in length), which was used both to calibrate ocular micrometer measurements and to localize the tumor. Twenty-four mice were implanted with A431 cell clots and 12 mice with OM431 cell clots. The longest diameter (LI) of the implant, the one perpendicular to the longest one (Wl), and the length of the suture were measured with the ocular micrometer of a dissecting microscope. The animals were either treated by IAP-MIS or vehicle buffer delivered by the Alzet pumps placed in the peritoneal cavity.
  • Blood samples at 6, 24, 48, 120 (fifth day) and 192 hours (eighth day) were obtained from selected animals by orbital bleeding and serum MIS levels were measured by ELISA. The animals were sacrificed on the eighth day.
  • the longest diameter (LI) of the tumor, the one perpendicular to the longest one (Wl), and the length of the suture were measured blindly by two independent investigators. After calibration of the measurements, the graft size ratio was represented by (L2 x Wl x W2 / (LI x Wl x Wl). Histologic sections of the kidneys were also obtained and examined. Tumors with cystic change were excluded.
  • the percent survival of the various cell lines after incubation with 30 nM of IAP-MIS was 45 % for A431, 747% for HT-3 , 54% for HEC-l-A, 59% for NIH:OVCAR-3, 34% for OM431, and 114% for Hep 3B.
  • the survival after treatment with MIS in all cell lines except Hep 3B were significantly inhibited by IAP-MIS (p ⁇ 0.05).
  • the growth of Hep 3B was not inhibited by MIS.
  • the percent survival after incubation with IAP-salt were 172% for A431, 93% for HT3, 92% for HEC- 1-A, 120% for OM431, 105 % for NIH:OVCAR-3 and 173% for Hep 133B.
  • the stimulatory effect was significant for A431, OM431 and Hep 3B cells (p ⁇ O.05) (FIG. 1).
  • the percent survival was 55.9 % for A431, 36.8% for OM431 , 100.5 % for HT-3, and 115.8% for RT4 when treated with 30 nM of IAP-MIS.
  • the percent survival was 169.0% for A431, 226.7% for OM431, 101.6% for HT- 3 and 96.5 % for RT4 when treated with IAP-salt (final protein concentration 19.3 ⁇ g/ml).
  • the inhibitory effect of IAP-MIS and the stimulatory effect of IAP-salt were significant for A431 and OM431 colony formation (p ⁇ O.O5) (FIG. 2). Dose Dependent Inhibition of A431 Colony Formation by MIS.
  • the percent survival was 103.4%, 81.4%, 61.3% and 26.5% respectively, for DG-MIS concentrations of 0.9, 1.8, 3.5, and 7.0 nM. Significant inhibitions were seen with DG-MIS concentrations of 3.5 and 7.0 nM (p ⁇ 0.05). The percent survival were 109.7%, 71.1 %, 56.6% , and 33.3% respectively for IAP-MIS concentrations of 12, 24, 48, and 96 nM. Significant inhibitions were seen at IAP-MIS concentrations of 24, 48, and 96 nM (FIG. 3). DG-MIS thus was 10-14 times more potent than IAP-MIS.
  • the percent survival of A431 cells was 51.3% for IAP-MIS alone (50 nM), 116.9% for IAP-salt alone and 81.6% for the combination of these two
  • the percent survival of A431 cells was 100%, 56.5%, 38.7%, 34.3 % and 10.8% respectively for cisplatin concentrations of 0, 0.078, 0.156, 0.312 and 0.624 ⁇ g/ml. With the addition of 7 nM DG-MIS, the percent survival became 39.3%, 32.2%, 23.1 %, 20.4% and 5.4%, respectively, for the same concentrations of cisplatin. The percent survival of cisplatin alone and cisplatin plus MIS was significantly different at the cisplatin concentrations of 0.078 and 0.156 ⁇ g/ml (FIG. 6).
  • the growth of Hep 3B spheroids was faster and uninhibited by MIS (FIG. 7B).
  • the MIS levels in mouse serum were relatively stable from 24 hours to the eighth day after the implantation of the MIS filled Alzet pumps (FIG.
  • the average MIS level of 14 blood samples from day 2 to day 8 was 19.1 ⁇ 2.7 ng/ml (approximately 140 pM).
  • the serum MIS levels of the control mice were undetectable.
  • the growth of the tumors in the MIS group was significantly lower than the control in both cell lines (p ⁇ 0.05) (FIG. 8B).
  • rhMIS Recombinant human MIS
  • DG-MIS serial ion exchange and dye affinity chromatography
  • IAP-MIS immunoaffinity chromatography
  • MIS In evaluating MIS as an anticancer agent, it is important to consider its interaction with available chemotherapies. To evaluate this, MIS and cisplatin were tested alone and in various combinations. Since the inhibitory effects of MIS and cisplatin are additive, cisplatin doses could be lowered when given with MIS; therefore this biological modifier might function as an adjuvant in the multimodality treatment of selected human malignancies.
  • the liquid colony inhibition and the spheroid assays can be used to test repeated doses of either MIS or MIS plus cytotoxic agents. However, asynchronous addition first of MIS, which best effects a proliferative cell population, then the cytotoxic agent, is preferable. DG-MIS, although less purified, consistently showed inhibition of cell growth in vitro. The specificity of the MIS effect was demonstrated by blocking the inhibitory effect with an MIS monoclonal antibody.
  • Wallen et al. (Wallen et al, Cancer Res. 49:2005-2011 (1986)) reported only a minimal antiproliferative activity when an immunopurified MIS preparation was tested against a variety of established cell lines.
  • Example 1 the same poor response with IAP-MIS was observed before the salt elution step was added prior to elution with 1 M acetic acid.
  • the salt fraction eluted from the immunoaffinity column actually showed a growth stimulating effect in some cell lines. Electrophoresis of this salt fraction showed several bands in the region of 14-20 kDa, which are absent in the fraction subsequently eluted by 1 M acetic acid.
  • the subrenal capsule assay was used to test the effect of MIS in vivo.
  • MIS vulvar epidermoid carcinoma cell line
  • A431 a vulvar epidermoid carcinoma cell line
  • OM431 an ocular melanoma line
  • the assay was terminated on day 8, since longer duration led to variable cystic change. Such ' changes can vary the graft size ratio enough to make comparisons unreliable due to imbibition of fluid and cystic changes.
  • dihydrofolate reductase deficient CHO cells were cotransfected with a linear construct of both the human MIS and the dihydrofolate reductase genes as in Example 1.
  • the transfected CHO cells were amplified in Methotrexate and grown at 37 °C in alpha minimal essential medium without ribonucleosides and deoxyribonucleosides, supplemented with 10% bovine MIS-free female fetal calf serum (FCS).
  • FCS bovine MIS-free female fetal calf serum
  • MIS concentrations were estimated using an enzyme-linked immunosorbent assay (ELISA) for MIS, and protein concentrations were measured as in Example 1.
  • ELISA enzyme-linked immunosorbent assay
  • the human ocular melanoma cell lines OM431, OM464, OM467, and
  • OM482 were established in 1984 and kept in liquid nitrogen until early passage ampules were thawed for this study. They were maintained in the alpha modification of Eagle's medium supplemented with ribonucleosides and deoxyribonucleosides ( ⁇ -MEM+) to which was added 10% female FCS and 1 gm/1 L-glutamine. Before study, cells were serially subcultured, then trypsinized at 70-80% confluency. This proliferating cell population was then centrifuged at 1500 RPM for 5 minutes and resuspended with 10% female FCS supplemented medium. Cells were counted in a hemocytometer.
  • the effect of rhMIS was tested using the conventional double layer agarose colony inhibition assay as in Example 1.
  • the underlayer of the 35 mm culture dishes contained 1 ml of 0.6% agarose (Sigma, St. Louis, MO.) in 10% female FCS supplemented ⁇ -MEM + .
  • the overiayer consisted of 0.3% agarose in 10% female FCS supplemented a-MEM + , the cells to be tested (50,000 cells/ml for OM431; 25,000 cells/ml for OM464, OM467 and OM482), epidermal growth factor (EGF) 10 ng/ml (Sigma, St. Louis, MO.) and either rhMIS or vehicle buffer as a negative control.
  • EGF epidermal growth factor
  • the dishes were incubated in humid air with 5 % CO 2 at 37°C for 10-21 days. Colonies with more than 30 cells were counted with an inverted microscope (Nikon). The results are expressed as percent survival relative to a control group (number of colonies in the test group x 100/number of colonies in the control group).
  • Single cell suspensions of OM431 were placed and grown in 24-well culture plates (Falcon, Oxnard, CA.,#3047) at a concentration of 8250 cells per well in 0.5 ml media (c ⁇ -MEM+ with 10% female FCS and 50 ng/ml EGF) as in Example 1. After cell attachment, only those with good single cell dispersion without clumping were used for further study. Agents to be tested were added (50 microliters per well) and were tested in triplicate. The cells were incubated in humid air with 5 % CO 2 at 37°C. Colonies which formed in 5-7 days were stained with Giemsa solution and those with more than 30 cells were counted by eye with an inverted microscope or the counting was automated using a computer based image analyzer.
  • MIS was tested in concentrations of 0.98, 9.8, 25.2, 50.4, 75.6, and 100.8 nM. The results were expressed as percent survival relative to the vehicle buffer control.
  • Multicellular tumor spheroids of OM467 and OM482 cells were produced as described in Example 1.
  • 10 5 cells of OM467 in 1 ml of 10% female FCS supplemented ⁇ -MEM + were plated on top of 1.5 ml of 1 % agarose in a 35x10 culture dish after thorough washing to remove residual trypsin, and incubated in humid air with 5 % CO 2 at 37°C for 2-5 days when spheroids usually formed.
  • 0.5 ml of 1 % agarose was then added to each well of a 24-well culture plate (Falcon, Oxnard, CA.,#3047).
  • OM431 cells grew to graft size of 3.68 ⁇ 0.56; OM482 to 1.97 ⁇ 0.67; OM467 to 1.34 ⁇ 0.5; and OM464 ⁇ 1. Thereafter, 10 7 OM431 cells were centrifuged at 1500 RPM for 5 minutes to form a pellet. 20 ml of fibrinogen (Sigma, St. Louis, MO., 20 mg/ml dissolved in PBS pH 7.4) was added to the pellet, followed by 10 ml of thrombin (Sigma, St. Louis, MO., 20 unit/ml dissolved in double- strength Dulbecco's modified essential medium). The mixture was incubated at 37 °C for 15 min. The cell clot thus formed was cut into approximately 50 fragments (1 mm 3 , each containing approximately 10 5 cells) in preparation for implantation.
  • MIS was delivered by an Alzet mini-osmotic pump (#2001, Alza, Palo Alto, CA.) placed in the peritoneal cavity at the time of tumor implantation. These pumps have a fill volume of approximately 210 ⁇ l and release their contents at a rate of approximately 1 ⁇ l/hr for eight days of delivery time. The pumps were either filled with rhMIS or with vehicle buffer.
  • Virus and pathogen free female CD-I mice (10 weeks old, average weight 35 g, Charles River Breeding Laboratory, Wilmington, MA.) were given whole body irradiation of 640 rads by a Mark-I cesium-137 irradiator 16-24 hours before the experiment as in Example 1.
  • Nude mice (8 weeks old, average weight 24 g, Edwin L. Steele Laboratory, Massachusetts General Hospital, Boston, MA) were also used.
  • Pentobarbital Abbott Laboratory, North Chicago, IL
  • an incision was made in the left flank of the mouse and the left kidney exteriorized. A subcapsular space was developed using a 19- gauge needle trocar.
  • a cell clot was introduced into the space with a segment of 5-0 Nylon suture (approximately 1 mm in length), which was used both to calibrate ocular micrometer measurements and to localize the tumor.
  • Twelve CD-I mice and twenty nude mice were implanted with OM431 cell clots.
  • the longest diameter (LI) of the implant, the diameter pe ⁇ endicular to the longest diameter (Wl), and the length of the suture were measured with the ocular micrometer of a dissecting microscope.
  • the animals were either treated by rhMIS or vehicle buffer delivered by the Alzet pumps placed in the peritoneal cavity. The animals were sacrificed on the eighth day. Blood samples were obtained on the eighth day from the nude mice and serum MIS levels were measured by ELISA.
  • the longest diameter (L2) of the tumor, the diameter pe ⁇ endicular to the longest diameter (W2), and the length of the suture were measured blindly by two independent investigators. After calibration of the measurements, the graft size ratio was represented by (L2 x W2 x W2) / (LI x Wl x Wl). Histologic sections of the kidneys were also obtained and examined. Tumors with cystic change were excluded.
  • Each MIS treated mouse received 48.6 micrograms of MIS over the eight day assay.
  • the graft size ratios of the OM431 tumors were significantly greater, 3.02 ⁇ 0.17 and 3.14 ⁇ 0.40, respectively, for the controls, vs 1.68 ⁇ 0.09 (p ⁇ .001) and 1.69 ⁇ 0.43 (p ⁇ .005) for the MIS group.
  • the MIS treated group received 44.7 micrograms MIS over eight days while in the second nude mouse assay, the MIS group received 130 micrograms.
  • the average MIS serum levels of the MIS treated mice on the eighth day of the nude mouse assays were 749 and 570 pM respectively.
  • the measured controls had MIS levels of less than 10 pM.
  • the growth of the tumors in each MIS group was significantly lower than the control in all three assays (p ⁇ 0.05) (FIGS. 12A-C).
  • Example 2 the effect of recombinant human MIS on three human ocular melanoma cell lines using three different in vitro clonogenic assays were examined.
  • the reliable and reproducible double-layer inhibition assay was used with each cell line. Despite its rather lengthy incubation time (10-21 days), all the cell lines grew well in this assay.
  • OM482, OM43 1 and OM467 were significantly inhibited.
  • the liquid colony inhibition assay was used with the OM431 cell line.
  • OM467 and OM482 failed to grow discrete colonies in this assay. When effective, as with OM431, this assay is rapid (5-7 days) and uses little sample.
  • OM431 showed a clear dose response with inhibition of colony formation with rhMIS concentrations above 25 nM. The percent survival of OM431 correlated well between the two assays. The multicellular spheroid assay was used to recapitulate tumor microregions with cell-cell interactions and nutrient affected growth patterns. OM467 and 482 cell lines formed satisfactory spheroids. OM467 showed significant inhibition while the growth of OM482 was unaffected. OM431 failed to grow spheroids. The availability of all three in vitro assays permits selection of optimal conditions for each tumor.
  • the subrenal capsule assay was used to test the effect of MIS in vivo against OM431, since this line grew large enough to permit comparisons.
  • MIS intraperitoneal constant infusion Alzet pump inserted at the time of tumor implantation, the growth of OM431 was inhibited in vivo. It is important in evaluating this assay that histology be documented for each different tumor, and the experiment completed while the tumors are still solid and before a lymphocytic infiltrate and/or central necrosis occurs.
  • the assay was terminated on day 8 in the irradiated CD-I mice since a longer duration of incubation led to variable histologic changes which can vary the graft size ratio enough to make comparisons unreliable due to imbibition of short lived fluid, inflammatory cell infiltration and cystic changes.
  • nude mice were also used and similar tumor growth inhibition was seen. The same period of time was used when tumors were implanted in the nude mice. No obvious toxicity to animals was observed during the short course of this study. The in vivo effect in this subrenal assay is achieved at picomolar concentrations. In vitro studies, on the other hand, require 10 to 100 fold higher levels, suggesting a failure of cleavage and activation.
  • rhMIS purification by immunoaffinity chromatography of conditioned medium of Chinese hamster ovary cells transfected with the human MIS gene has been described (Pepinsky et al., J. Biol. Chem. 263: 18961-4 (1988)) and recently modified (Ragin, R.C., et al, submitted). Briefly, media were collected every 3-4 days from bioreactor cultures (Epstein et al, In Vitro Cell, and Devel. Biol 25:213-6 (1989)) and stored at -20°C until use. A 5-ml immunoaffinity column was constructed using approximately 50 mg protein-A- Sepharose (Sigma Chemical Co. , St.
  • mice monoclonal antihuman rhMIS antibody Hudson et al, J. Clin. Endocrinol. Metab. 70:16- 22 (1990) covalently attached to Affigel-10 agarose resin (Bio-Rad Laboratories, Richmond, CA).
  • the column was equilibrated with 100 ml 20 mM HEPES, pH 7.4 and 200 ml concentrated medium loaded after filtration through Whatman No. 4 paper (Clifton, NJ) at 1 column vol/h at 4°C. After loading, the column was washed with 20 mM HEPES, pH 7.4, until the absorbance at 280 nm returned to baseline (60 - 100 ml).
  • rhMIS was eluted using 1 M acetic acid in 20 mM HEPES, pH 3.0, after a 1-column vol. preelution wash containing 0.5 m NaCI, 1 mM EDTA, 0.001 % Nonidet P-40 (Sigma Chemical Co.), and 20 mM HEPES, pH 7.4.
  • the acid- eluted immunoaffinity-purified (IAP) fractions were dialyzed overnight against 0.02 M HEPES and 0.001 % Nonidet P-40, pH 7.4.
  • the resulting samples were analyzed for total protein by the Bradford method (Bradford, M.M., Anal. Biochem. 72:248-254 (1976)) and for rhMIS concentrations by an enzyme-linked immunosorbant assay (Hudson et al, J. Clin. Endocrinol. Metab. 70: 16-22 (1990)). They were further examined by polyacrylamide gel electrophoresis (Weber et al., J. Biol. Chem. 244:4406-4412 (1969)), and activity was determined in an in vitro M ⁇ llerian duct regression bioassay and an antiproliferative assay using human A431 vulvar carcinoma cells (see blow).
  • IAP rhMIS (1.1 - 1.5 mg in 2.5 ml 20 mM HEPES buffer, pH 7.4) was incubated with plasmin (EC 3.4.21.7 Sigma Chemical Co.) at a ratio of 20-25:1 rhMIS to plasmin (wt/wt) for 2 hours at room temperature, as previously described (Pepinsky et al., J. Biol. Chem. 263:18961-4 (1988)). The preparation was then placed onto a 21.5 x 16-cm P-100 polyacrylamide column (Bio-Rad Laboratories, Richmond, CA) equilibrated at 4°C with 1.0 M acetic acid in 20 mM HEPES at pH 3.0.
  • plasmin EC 3.4.21.7 Sigma Chemical Co.
  • Protein was eluted in 0.54 ml fractions at a flow rate of approximately 2.0 ml/hour. Ten microliter aliquots were analyzed for protein by the Bradford method (Bradford, M.M., Anal. Biochem. 72:248-254 (1976)). Two peaks of protein, termed A and B, elute from this column. These peaks were pooled separately, frozen in liquid nitrogen, and concentrated by lyophilization in a Savant Speed- Vac apparatus (Hicksville, NY). The resulting pools were dissolved in either 20 mm HEPES, pH 7.4 or 0.3 m sodium phosphate, pH 7.4, so that a final protein concentration of 1 mg/ml was achieved. Elution buffer in volumes similar to those of the pools was also lyophilized and dissolved in buffer, as described above, to serve as controls for the rhMIS bioassays.
  • CMRL 1066 Gibco/Bethesda Research Laboratories, Gaithersburg, MD
  • CMRL 1066 Gibco/Bethesda Research Laboratories, Gaithersburg, MD
  • calf serum agar-coated stainless steel grids above fortified CMRL 1066 (Gibco/Bethesda Research Laboratories, Gaithersburg, MD) medium containing female fetal, and therefore MIS-free, calf serum (Necklaws et al, Endocrinology 778:791-796 (1986) and testosterone at 10 "9 M to enhance the Wolffian duct for direct comparison of the M ⁇ llerian duct in each tissue section.
  • rhMIS protein samples of 0.5 - 8.0 ⁇ g each or buffer controls were added in serum containing CMRL medium after sterile filtration in that solution through a 0.22 ⁇ m Millex GV membrane.
  • the A431 cell line derived from a squamous cell carcinoma of the vulva was maintained in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% MIS-free female calf serum (FCS).
  • DMEM Dulbecco's Modified Eagle's Medium
  • FCS MIS-free female calf serum
  • the cells which have an approximately 22-hour doubling time, were grown to confluency and maintained for an additional 24 hours.
  • A431 cell cultures passaged in this manner are slowed in their transition through the cell cycle and are predominantly found in the G, phase, as confirmed by flow cytometric analysis.
  • the cells were trypsinized, washed with DMEM in 10% FCS, counted, and diluted to a final seeding concentration of approximately 200,000 cells/35-mm culture dish in a total volume of 1.5 ml medium.
  • This medium contains 15 mM HEPES to buffer against the acidification that occurs with the addition of the carboxy-terminus.
  • Various concentrations of carboxy-terminal rhMIS, holo rhMIS, or appropriate buffer controls were added in duplicate, once at the time of plating or three times, at plating and 24 and 48 hours postplating. Controls in DMEM and 10% FCS were examined in triplicate. The cultures were incubated at 37°C in a 95% O 2 - 5 % CO 2 atmosphere. Microscopic inspection and control cell counts were performed every 24 hours for three days.
  • Epitope-specific rabbit polyclonal antibodies were raised to a region of the rhMIS molecule carboxy-terminal to the monobasic consensus cleavage site at position 427 and recently characterized (Pepinsky et al, J. Biol. Chem.
  • This sequence was chosen for its conserved homology among human, bovine, and rat MIS, its difference from other members of the supergene family, and its antigenicity and surface probability, as predicted by the sequence analysis software package of the Genetics Computer Group (University of Wisconsin, version 5), using the criteria outlined by Chou and Fasman (Chou et al, Adv. Enzymol. Relat. Areas. Mol. Biol. 47:45-148 (1978)) and Wolfe et al, Comput. Appl. Biosci. 4:187-191 (1988)).
  • Polyacrylamide gel electrophoresis (Weber et al, J. Biol. Chem. 244:4406-4412 (1969); Laemmli, U.K., Nature 227:680-685 (1970)) was carried out using 15 % homogeneous minigels (1.5 x 80 x 75 mm) overlaid with a 5 % polyacrylamide stacking gel, and 2- to 5- ⁇ g protein samples were run at 110-V and 30-mamp constant current at room temperature. Proteins in the gels were stained with 0.1 % Coomassie brilliant blue R250 (Sigma Chemical Co.) in 50% methanol-10% acetic acid for 1 hour before destaining in 50% methanol - 10% acetic acid. As appropriate, samples were reduced using 0.75 M 2-mercaptoethanol with heating to boiling for 10 minutes before the electrophoresis run. Prestained low molecular weight standards were obtained from Bio-Rad.
  • the blots were incubated with a l:500-fold dilution of rabbit polyclonal anti-rhMIS carboxy- terminal peptide antiserum for 2 hours and washed with 0.05 M Tris-Cl and 0.15 M NaCI before the addition of a 1:1000 dilution of goat amirabbit horseradish peroxidase conjugate (Bio-Rad).
  • Antibody complexes were visualized by the addition of Bio-Rad color reagent (4-chloro-l-naphthol) for 30 minutes, before quenching the reaction with water.
  • FIG. 13 A representative P-100 column elution profile of plasmin-cleaved rhMIS is shown in Figure 13.
  • the later eluting species (pool B) consists chiefly of a single band running just below the 18.5-kDa molecular weight standard in the Coomassie blue-stained polyacrylamide gel of pooled A (N-term) or B (C-term) fractions analyzed after disulfide bond reduction ( Figure 14).
  • the electrophoretic migration patterns generated for intact rhMIS (IAP) and rhMIS incubated with plasmin (Plasmin) are given for comparison.
  • the peptide antibody binds to the intact rhMIS 70-kDa monomer (IAP, lane 1) and the low molecular weight pool B fragments (C-term, lane 3), but not to the 34- to 55-kDa molecular weight species that elute in fraction A from the P- 100 column (N- term, lane 2).
  • the pool B 12- to 14-kDa species is the carboxy-terminal rhMIS.
  • the bioactivity of these isolated pools was tested in the standard MIS 14.5-day-old fetal rat urogenital ridge bioassay.
  • the carboxy-terminal rhMIS domain exhibited a high degree of M ⁇ llerian duct regression activity, as did the intact molecules, although the fragment was approximately 5-fold less active on a molar basis (Table 1).
  • holo and carboxy-terminal rhMIS were not significantly different from one another, but both were different from the amino-terminus at the highest dose (P ⁇ 0.05). Amino-terminal fragments had little or no effect at every dose tested.
  • transforming growth factor-3 a member of the same gene family as MIS
  • Rb a known tumor suppressor gene
  • the rhMIS-C preparations may interfere with cell adhesion, thus reducing the number of cells at the end of the experiment.
  • MIS M ⁇ llerian Inhibiting Substance
  • CHO Chinese hamster ovary
  • M ⁇ llerian Inhibiting Substance is a member of an enlarging gene family that includes the transforming growth factor-/3s (Derynck, R., et al, Nature 376:701-705 (1985)), inhibins (Mason, A.J., et al, Nature 378:659-663 (1985)), activin (Ling, N., et al, Nature 327:779-782 (1986); Vale, W., et al, Nature 327:776-779 (1986)), decapentaplegia protein of Drosophila (Padgett, R.W., et al, Nature 325:81-84 (1987)), Xenopus Vgl (Weeks, D.L., et al, Cell 57:861-867 (1987)), and a series of bone mo ⁇ hogenesis factors (Wozney, J.M., et al, Science 242: 1528-1534 (1988)).
  • MIS is a 140,000 kDa glycoprotein homodimer (Picard, J. Y. , et al, Mol. Cell. Endocrinol. 34:23 (1984); Budzik,
  • bovine MIS partially purified bovine MIS was tested and found to inhibit growth of established M ⁇ llerian-derived tumor cell lines in vitro (Donahoe, P.K., et al, Science 205:913-915 (1979), Fuller, A.F., et al, J. Clin. Endocrinol. Metab. 54: 1051-1055 (1982)) and in vivo (Donahoe, P.K., et al, Ann. Surg. 794:472-480 (1981)), and highly purified bovine MIS inhibited colony growth of a large number of primary M ⁇ llerian-derived cancers obtained directly from patients (Fuller, A.F., et al, Gynecol. Oncol. 22:135-148 (1985)).
  • the human gene for MIS was subsequently isolated and the recombinant protein (rhMIS) expressed in CHO cells (Cate, R.L., et al.Cell 45:685-698 (1986)).
  • rhMIS recombinant protein
  • the purified protein is bioactive, causing regression of the M ⁇ llerian duct of the 14.5 day fetal rat in a semiquantitative organ culture assay (Donahoe, P.K., et al, J. Surg. Res. 23:141-148 (1977))
  • initial studies investigating the antiproliferative effect of this recombinant product showed minimal inhibition (Wallen, J.W., et al, Cancer Res. 49:2005-2011 (1989)).
  • DHFR Dihydrofolate reductase
  • CHO-WT "wild-type" CHO cells
  • B9 a gene for human MIS
  • L9 a mutated gene which produces a noncleavable, inactive form of human MIS
  • OM431 cells transfected with the MIS gene also grew less well in vitro and were substantially inhibited in vivo.
  • This cell line produced approximately 3.0 ⁇ g rhMIS/ml/24 h, as measured by a previously described MIS ELISA (Wallen, J.W., et al, Cancer Res. 49:2005-2011 (1989); Hudson, P.L., et al., J. Clin. Endocrinol. Metab. 70: 16-22 (1990)).
  • B9 cultures contained 30 nM methotrexate in addition.
  • a third CHO cell line, designated L9 was similarly created by transfection of a mutated human MIS gene, which produces a noncleavable protein with arginine #427 changed to threonine (Cate et al. , in Handbook of Experimental Pharmacology, Vol.
  • the OM431 cell line established in vitro from a primary tumor specimen obtained at enucleation (Albert, D.M., et al, Inv. Ophth. Vis. Sci. 25: 1284-1299 (1984)), was confirmed by light and electron microscopy and chromosome analysis to be a human ocular melanoma with Callender (Callender, G.R., Trans. Am. Acad. Ophth. Otol. 36:131 (1931)) epithelial mo ⁇ hology. This cell line was transfected with the pc DNA I/neo. hmis plasmid vector.
  • MIS cDNA (2036 bp) was obtained from the vector, pDl (Cate, R.L., et al, "M ⁇ llerian Inhibiting Substance," in Handbook of Experimental Pharmacology, Vol. 95/11, Peptide Growth Factors and their Receptors II, Sporn and Roberts, eds., Springer- Verlag, New York, pp. 179-210 (1990)), created which had been cut from the pBG312, by incubating for one hour at 37C with Hind III and Not I in buffer C (Promega).
  • This insert was then ligated for 16 hours at 18C to the stable eukaryotic expression vector pcDNA I / NEO (7.1 Kb, Invitrogen) that had previously been cut with the same enzymes.
  • Both the vector and the insert containing vector were run on and cut from 1 % agarose gels, transfected into E. coli using electroporation (2.47 kV, 25uf, 400 ohms) and the cells plated overnight on 1 % agar plates containing 20ug/ml Kanamycin. Colonies were selected and grown overnight in LB broth containing 20ug/ml Kanamycin.
  • Each cell line was carried in the alpha modification of minimal essential media without nucleosides (o;-MEM-), but with added glucose, glutamine, sodium pyruvate, amikacin, and 10% MIS-free female fetal calf serum (FCS).
  • o;-MEM- minimal essential media without nucleosides
  • FCS MIS-free female fetal calf serum
  • Doubling time was calculated by dividing the time in culture (hours) by the number of doublings achieved.
  • Serum-free media was obtained from 72-h monolayer cultures of the transfected and parent CHO and OM431 cell lines for total protein and rhMIS measurement, as well as Western analysis. Protein quantitation was performed by the method of Bradford (Bradford, M.M., et al., Anal. Biochem. 72:248- 254 (1976)), while MIS concentrations were measured by ELISA (Hudson, P.L., et al, J. Clin. Endocrinol. Metab. 70: 16-22 (1990)). MIS was then purified from each media sample for Western analysis (Towbin, H., et al., Proc. Natl. Acad. Sci.
  • MIS expression in vitro and in vivo was performed to evaluate MIS expression in vitro and in vivo.
  • RNA was extracted by a modification of the method of Chirgwin (Chirgwin, J.M., et al, Biochemistry 78:5294-5299 (1979)) using guanidinium thiocyanate/lithium chloride; RNA quantitation was by spectrophotometric analysis and ethidium bromide staining of test gels.
  • Ten ug of total RNA were loaded in each lane of a 1.5% Mo ⁇ holinopropanesulfonic acid-formaldehyde agarose gel, electrophoresed at 5 V/cm, transferred to Biotrans nylon membrane (ICN Biomedicals, Irvine, CA) by capillary action in 25 mM sodium phosphate, and then fixed by UV irradiation.
  • the membrane was prehybridized in plaque screen buffer then hybridized with a random priming (Feinberg, A.P., et al, Anal. Biochem. 737:266-267 (1984)) 3 P-labeled full length human MIS complementary DNA probe as previously described (Kuroda, Lee). Overnight hybridization was performed with 10° cpm/ml in plaque screen buffer containing 0.1 mg/ml tRNA. All hybridizations and washes were done at 65 °C; 75 nM NaCl/7.5 mM Na citrate/0.5% SDS was the most stringent wash. Autoradiographic exposures were for 6 and 60 h.
  • rHMIS Recombinant human MIS was purified using an immunoaffinity chromatography method (Ragin, R.C., et al, Protein Expression and Purification 3(3,1:236-245 (1992)) adapted from previous protocols (Shima, H., et al, Hybridoma 3:201-214 (1984); Pepinsky, R.B., et al, J. Biol. Chem. 263: 18961-18964 (1988)).
  • the conditioned medium of the B9 cell line was loaded onto a 5 ml immunoaffinity column constructed with protein-A-Sepharose (Sigma) purified mouse monoclonal antibody, raised to gel purified rhMIS (Hudson, P.L., et al, J. Clin. Endocrinol. Metab. 70: 16-22 (1990)), which was covalently attached to Affigel-10 agarose resin (BioRad Lab.).
  • the column was washed with 1 column volume of high salt buffer (0.5M NaCI, 1 mM EDTA, 0.001 % nonidet P-40 (NP-40, Sigma), 20 mM HEPES, pH 7.4), prior to elution with 1M acetic acid in 20 mM HEPES, pH 3.0.
  • the MIS containing fractions were immediately neutralized with NaOH, dialyzed overnight versus 20 mM HEPES, 0.001 % NP-40, pH 7.4, and then analyzed for total protein by the method of Bradford (Bradford, M.M., et al, Anal Biochem.
  • the underlayer of 35 mm culture plates consisted of 1 ml of 0.6% agarose (Sigma) in 10% female FCS-supplemented ⁇ -MEM with nucleosides ( ⁇ -MEM+).
  • the overiayer consisted of 0.3% agarose in 10% female FCS supplemented ⁇ -MEM + , the cells (25,000 cells/ml), 10 ng/ml epidermal growth factor (Sigma), and either MIS (28.5 ⁇ g/ml) or buffer alone.
  • Multicellular Tumor Spheroid Assay Multicellular Tumor Spheroid Assay. Multicellular tumor spheroids of CHO-WT, -B9, -L9 cells were produced by the method of Yuhas et al. (Yuhas, J.M., et al, Cancer Res. 37:3639-3643 (1977)).
  • mice Female nude mice (nw) (Pantelouris, E.M., Nature 217:370-371 (1968)) (8 weeks old, average weight 24 g, Edwin L. Steel Laboratory, Massachusetts General Hospital, Boston, MA) were used. All animals were cared for under NIH approved guidelines established by the Massachusetts General Hospital. After inducing anesthesia with an intraperitoneal injection of 0.3 ml of 10% pentobarbital (Abbott Laboratory, North Chicago, IL), a subcapsular space was developed in the left kidney with a 19-gauge needle trocar. A cell clot was then introduced with a 1 mm segment of 5-0 nylon suture, which was used both to calibrate implant measurements and to localize the tumor.
  • pentobarbital Abbott Laboratory, North Chicago, IL
  • the longest diameter (L,) of the implant, the diameter pe ⁇ endicular to the longest diameter (W,), and the length of the suture were measured with the ocular micrometer of a dissecting microscope.
  • Each of the cell lines was implanted into five animals and allowed to grow for eight days, at which time the same measurements were repeated to calculate the graft size ratio (L, x W, x W,) / (L, x W, x W,). Histology was reviewed to assure that the implanted tumor was viable and lacked both an inflammatory infiltrate and central necrosis.
  • SCID severe combined immunodeficient mice
  • OM431 human ocular melanoma cells (untransfected) (10 6 ) were suspended in 0.25 ml media and injected into the tail veins of two groups of SCID mice.
  • ten SCID mice had eight-day Alzet mini osmotic pumps (model #2001, Alza Co ⁇ . Palo Alto, CA.) placed within their peritoneal cavities 24 h prior to the tail vein injections, with half of the pumps containing purified rhMIS (67.8 ⁇ g) and half containing buffer only (200 ⁇ l 20 mM HEPES, pH 7.4).
  • the pumps were removed at nine days. After the animals were sacrificed at six weeks, lungs were prepared and metastases counted as above.
  • Multicellular Tumor Spheroid Assay When tested for its ability to form anchorage independent multicellular tumor spheroids, the B9 cell line produced only loose cellular aggregates. No discrete tumor colonies formed that could be transferred to a 24-well plate. The L9 and CHO-WT lines, on the other hand, produced large spheroids. There was no statistical difference between the relative volumes of spheroids formed by L9 and CHO-WT cells at 9 days.
  • mice injected with OM431 human ocular melanoma cells were treated with exogenous rhMIS.
  • the tumors were much smaller than observed after injection of wild type OM431 cells.
  • Exogenous M ⁇ llerian Inhibiting Substance inhibits the growth of a number of human carcinoma cell lines and primary human tumors both in vitro and in vivo (Donahoe, P.K., et al, Science 205:913-915 (1979); Fuller, A.F., et al, J. Clin. Endocrinol Metab. 54: 1051-1055 (1982); Donahoe, P.K., et al, Ann. Surg. 794:472-480 (1981); Chin, T., et al, Cancer Res.
  • An expression vector carrying the SV40 early promoter and genomic MIS was co-transfected with a plasmid containing an enhancerless mouse DHFR cDNA (Kaufman, R.J., et al, Mol. Cell Biol 2: 1304-1319 (1982)) into a DHFR-deficient CHO cell line (Chasin, L., et al, Proc. Natl Acad. Sci. USA 77:4216-4220 (1980)). Clones were selected and amplified with increasing doses of methotrexate to produce the MIS expressing cell line, B9 (Cate et al , in Handbook of Experimental Pharmacology, Vol. 95/11, Peptide Growth Factors and Their Receptors II, pp.
  • Anchorage independent cell growth has been correlated with cell tumorigenicity and experimental metastatic potential (Mancianti, M.L., et al, Carcinog. Compr. Surv. 77:369-86 (1989); Li, L., et al, J. Natl. Cancer Inst. 87: 1406-1412 (1989); Paraskeva, C, et al, Anticancer Res. 70: 1189-1200 (1990)).
  • the DHFR-deficient CHO-WT cell line used in these experiments grew well as colonies in both agarose and multicellular tumor spheroid assays, reflecting a certain degree of anchorage autonomy.
  • the non-cleavable mutant MIS cell line, L9 grew equally well in these assays, indicating that transfection with the plasmid vectors did not significantly alter in vitro cell growth characteristics. Although we could not achieve comparable transcription ( Figure 20) or translation ( Figure 19) of the non-cleavable mutant, the L9 line provides a reasonable vector control for comparison with B9. Colony growth of the MIS transfected B9 cell line, however, was significantly inhibited in agarose, and B9 cells failed to form multicellular tumor spheroids. Purified rhMIS added exogenously similarly inhibited colony formation of CHO-WT and L9 cells dispersed in agarose, but had no additional inhibitory affect on B9 colony formation. All three cell lines had identical in vitro anchorage dependant doubling times in monolayer culture.
  • the subrenal capsule assay permits the introduction into a nutrient rich environment of a known number of cells whose contained growth can be accurately measured. Cells injected via the tail vein, meanwhile, are given direct access to the circulation.
  • This experimental metastases assay which examines the ability of cells to travel in the circulation and implant at a specific site (Smyth, M.J., et al, Cancer Res. 57:310-317 (1991); Sharkey, R.M., et al, J. Natl. Cancer Inst.
  • Serum MIS levels were not measured during pump delivery to avoid loss of the animals during this lengthy experiment; however, serum levels achieved in recent studies when similar amounts of MIS were delivered via Alzet pumps were 0.5-0.7 nM (Parry, R.L., et al, Cancer Res. 52:1182-6 (1992)).
  • the 400 fold higher concentrations required to produce in vitro effects may be related to more efficient molecular processing, in vivo, to produce a cleaved, biologically active form of the molecule (MacLauglin D.T., et al, Endocrinology 131(1 ):291-296 (1992)) (or that the lung possesses an endogenous enzyme capable of cleaving the MIS).
  • the fact that the metastases of OM431 cells transfected with the MIS gene were markedly suppressed when compared to metastases of wild type untransfected OM431 cells provides important additional evidence for the growth inhibitory effect of MIS on the non M ⁇ llerian ocular melanomas.
  • the biological modifier M ⁇ llerian Inhibiting Substance has the ability to inhibit cell growth and metastases in vitro and in vivo. Its effect can be produced by either the addition of exogenous rhMIS or transfection of the human MIS gene.
  • the availability of cell lines engineered to produce processed, secreted, and biologically active or inactive MIS is currently permitted us to screen for critical "downstream" effects of this hormone, such as changes in cell surface and adhesion molecules, growth factors and their receptors, oncogene and tumor suppressor genes, and essential cell cycle factors.
  • Epidermal Growth Factor tissue culture grade
  • Sigma Chemical Co. Sigma Chemical Co. (St. Louis, Mo.) as a lyophilized powder extracted from the submaxillary glands of male mice (Savage et al, J. Biol. Chem. 247:7609 (1972)). It was reconstituted in sterile pyrogen-free distilled water to a final concentration of 100 ⁇ g/ml, sub-aliquoted and stored at -70 °C.
  • y- interferon was purchased from Amgen Biologicals (Thousand Oaks, Ca.), as a lyophilized powder and dissolved in sterile pyrogen-free water to a final concentration of 1000 units/ml E.
  • Methotrexate selection (Cate et al , Cold Spring Harbor Symposium on Quantitative Biology vol. LI, (1986)) were grown to confluence in four liter bioreactors on stainless steel coils in alpha-Modified Eagle's Medium, supplemented with 10% fetal calf serum, Streptomycin, Gentamicin, L-Glutamine, and Pyruvate. After reaching equilibrium the medium was collected every 3-4 days, then concentrated 20% on a Minitan ultrafilter (Millipore) with a 30,000 d molecular weight cutoff membrane and stored at -70 °C.
  • Minitan ultrafilter Micropore
  • MIS protein as measured by A ⁇
  • ELISA enzyme-linked immunoad sorbant assay
  • the predominant bands at 70 and 55 Kd representing an estimated 30% of the preparation had the predicted amino acid composition and NH 2 terminal sequence of human MIS (Cate et al. , Cell 45:685-698 (1986); Pepinsky et al. , J. Biol. Chem. 263:18961 (1988)).
  • the immunoaffinity purified rMIS preparations (IAP 38 and 50) used in these experiments were found to have protein concentrations of 335 ⁇ g/ml and 399 ⁇ g/ml as determined by Bradford analysis (Bradford, M.M., Anal. Biochem. 72:248-254 (1976)).
  • mice acquired from Jackson labs were mated in a virus free environment; the presence of a vaginal plug was taken as day 0 of pregnancy.
  • Pregnant females were sacrificed and the fetuses harvested under sterile conditions by cesarean section on day 18 of gestation, then decapitated and minced, and the homogenate for each fetus passed through a 6 cc syringe without needle into a sterile 100 ml media bottle with 10 ml of media made 0.25% with trypsin on a stir plate at room temperature for 1 hour.
  • Two pregnant dams with approximately 10 fetuses/dam provide sufficient cells for 20-30 flasks.
  • the trypsin was deactivated by the addition of 10 ml of fetal calf serum and the suspension strained through a sterile funnel containing gauze to remove large particulate matter.
  • the solution was then subdivided into 50 ml conical tubes with 7 ml of media and spun for 5 min. at 2000 9 in a desktop centrifuge, after which the supernatant was discarded and the pellets recovered, pooled and resuspended in 25 ml of media (Dulbecco's modified Eagle's media, 10% fetal calf serum, 0.01 M Hepes, pH 7.4, 10,000 units penicillin and streptomycin). Cells were counted in a hemocytometer and viability determined by trypan blue exclusion.
  • tissue culture flasks were then seeded at a concentration of 4 X 10 6 viable cells (primary fibroblasts) prior to incubation at 37 °C in 5% CO 2 with saturated humidity for 24 hours, when media was changed. The cells were then allowed to grow to confluency (6 days). Each flask was treated with 5 ml of 0.25% trypsin-EDTA for 5 minutes at 37 °C, followed by inactivation with 5 ml of 10% fetal calf serum. Cells were pooled, centrifuged for 5 min.
  • the cells were allowed to continue their growth, and 2 days later various growth factors or lymphokines at the following concentrations were added to each flask, alone or in combination: X-interferon (250 IU/ml, 500 IU/ml and 1000 IU/ml), epidermal growth factor (EGF) 1 ⁇ g/ml, human recombinant M ⁇ llerian Inhibiting Substance (MIS) 1-1.25 ⁇ g/ml, and endotoxin 16 pg/ml. The experiment was terminated 48 hours after addition of the growth factors/lymphokines when viability was again tested with trypan blue.
  • X-interferon 250 IU/ml, 500 IU/ml and 1000 IU/ml
  • EGF epidermal growth factor
  • MIS human recombinant M ⁇ llerian Inhibiting Substance
  • endotoxin 16 pg/ml endotoxin 16 pg/ml
  • Total RNA was prepared by extracting secondary fetal fibroblasts with guanidinium thiocyanate with , 3-mercaptoethanol according to the method of Chirgwin, J.M., et al. (Biochemistry 18:5294 (1979)). The concentration of all RNA samples was determined spectrophotometrically and by electrophoresis on a 2% agarose test gel stained with ethidium bromide to confirm that equivalent amounts of RNA would be loaded on subsequent gels.
  • EDTA EDTA, pH 8.0
  • EDTA EDTA, pH 8.0
  • the gel was washed in 25 mM Na 3 PO 4 , pH 6.3, for 20 minutes 3 times, transferred to Gene Screen (New England Nuclear) overnight in the same buffer, and the RNA then u.v. crosslinked to the filter.
  • bovine serum albumin 0.05 M Tris HCl, pH 7.5, 1 M NaCI,
  • a cDNA probe was selected to analyze for the presence of Class I mRNA.
  • the Class I probe was a nick translated 2.3 kb Bam fragment which contained the 4th exon encoding the 3rd external domain of the H-2K D gene and hybridized to all Class I genes (Weiss E. et al. , EMBO J. 2:453 (1983)).
  • the filters were placed on Kodak X-AR film and the autoradiograms were -Un ⁇
  • MHC myelogenous growth factor
  • Transplantation antigen ubiquitously expressed on the surface of all cells, was first referred as "transplantation antigen", since it was permissive in the recognition of non-self, by participation in the presentation of associated processed antigen for recognition by cytotoxic T-cells.
  • MHC antigens act as a chemostat to precisely modulate growth, with high levels of expression resulting in or correlating with periods of slow or no growth, and low levels of expression resulting in or correlating with proliferative growth or migration.
  • MIS is cleaved at a monobasic consensus cleavage site at amino acid 427 into an N- terminal and a C-terminal fragment which are then held in noncovalent dissociation (Pepinsky et al, J. Biol. Chem. 263:18961 (1988)).
  • MIS Some mechanism for complete dissociation, as seems to be required for TGF-B, may also be required for MIS to exert its biologic activity in the MIS organ culture assay which detects hullerian duct regression (Donahoe et al. , J. Surg. Res. 23: 141 (1977)).
  • the large numbers of mixed mesenchymal and epithelial cells present in the early secondary fetal fibroblast preparation may provide the necessary cleavage enzymes to activate MIS in vitro to produce this response, namely up-regulation of hHC.
  • up-regulation of hHC Of the heterogeneous group of cells comprising secondary fetal fibroblasts, it is not known which proportion of the cell population responds to MIS.
  • growth factors such as insulin, insulin like growth factors, NGF, or FGF; and growth inhibitors such as tumor necrosis factor which is known to up-regulate Class I in other systems; TGF-B and inhibin, with known homology to MIS; and other lymphokines in addition to 7-interferon, may also function in this system.
  • MIS myeloma
  • gonadal steroids are thought to alter immunity, and are known to affect MIS, i.e., estrogen inhibits (Hutson et al, J. Ped. Surg. 7:953-959 (1982)) and testosterone and progesterone enhances MIS (Ikawa et al, Gen. Comn. Endocrinol. 87: 88-102 (1985)) at the receptor level, these steroids may act to modulate the Class I mRNA effect.
  • Gel shift analysis can be done to determine if a specific transacting nuclear protein under the influence of MIS or EGF or ocogene much as myc, fos, or jun binds to the enhancer region of the MHC genes, to further understand factors which control growth in this fetal system.
  • MHC expression is a highly regulated initiator of cell killing which may play an important role in growth control and fetal development by participating in the internal milieu of embryonic tissue remodelling where extensive "programmed cell death" occurs, rather than being limited only to cell killing in response to foreign stimuli.
  • C57BL/6 female mice were mated with C57BL/6 males and the presence of a vaginal plug was taken as day 0 of pregnancy.
  • Pregnant females were sacrificed and fetuses were obtained by Caesarean section on days 14 and 18 of gestation. Some pregnancies went to term and animals were selected at one and six days of age.
  • Kidneys were excised from 14-day and 18-day fetuses, 1-day and 6-day postnatal animals, as well as the mothers. Testes were excised from the 18-day fetuses, 1-day postnatal animals, and adult males. All tissues were flash frozen and stored in liquid nitrogen for RNA determinations.
  • Total RNA was prepared from adult kidney (230 mg, 1.5 kidneys), adult testis (190 mg, 2 testes), and pooled tissue of 6-day postnatal kidney (300 mg, 13 kidneys), 1-day postnatal kidney (300 mg, 37 kidneys), 1-day postnatal testis (160 mg, 400 testes), 18-day fetal kidney (290 mg, 50 kidneys), 18-day fetal testis (160 mg, 410 testes), and 14-day fetal kidney (360 mg, 1000 kidneys), using guanidinium thiocyanate (Chirgwin J.M. et al , Biochemistry 78:5294 (1979)).
  • RNA samples were determined spectrophotometrically and by electrophoresis on a 2 % agarose test gel stained with ethidium bromide to confirm that equivalent amounts of RHA would be loaded on subsequent gels.
  • Samples of total RNA (10 mg) were heated to 55 °C for 15 minutes in 50% deionized formamide, 2.2 M formaldehyde, and 5x running buffer (0.2 M mo ⁇ holinopropanesulfonic acid, pH 7.0, 50 mM sodium acetate, 5 mM EDTA, pH 8.0), placed on ice, then fractionated by electrophoresis (100 V, 4 hrs.) on a 1.0% agarose gel containing 2.2 M formaldehyde in Ix running buffer.
  • the gel was washed in 25 mM Na 3 P0 4 , pH 6.3, for 20 minutes 3 times, transferred to Gene Screen (New England Nuclear) overnight in the same buffer (Maniatis T. et al , In: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, (1982)), and the RNA then UV crosslinked to the filter.
  • bovine serum albumin 0.05 M Tris HCl,
  • Probes were selected to analyze for the presence of Class I, and Class II MHC mRNA.
  • the Class I probe was a nick-translated 2.3 kb BamHI fragment containing the 4th exon encoding the 3rd external domain of the H-2K b gene, and hybridizes to all Class I genes (Weiss, E., et al , EMBO J. 2:453 (1983)).
  • the Class II probe was a nick-translated 1.6 kb Barn-Hind III fragment, containing the 3rd exon encoding the 2nd external domain of the ⁇ - chain of the I- A, ⁇ gene (Larhammar D. et al, Cell 34: 179 (1983)).
  • the filters were placed on Kodak X-AR film and the autoradiograms were developed in 48 hours. Re-hybridization was carried out after washing filters in 0.005 M Tris HCl, pH 8.0, 0.0002 M Na 2 EDTA, 0.05 % sodium pyrophosphate, 0.002 % polyvinyl-pyrrolidone, 0.002 % bovine serum albumin, 0.002% Ficoll, at 65-70 °C for 2-3 hrs with constant agitation. Filters were placed on film for 24 hours to confirm that the previous label was adequately washed off.
  • a functional assay of graft survival in congenic mice was based upon methods of tissue implantation, internal graft measurement, and mo ⁇ hometric and histologic assessment adapted from techniques used previously for developmental (Donahoe P.K., etal. , J. Ped. Surg. 79:863 (1984)), oncologic (Fingert, H., et al , Cane. Res. 47:3824 (1987)), and immunologic (Foglia R.P. et al , Annals of Surgery 204:402 (1986); Foglia, R.P., et al , J. Ped. Surg. 27:608 (1986); Statter M.B. et al , J. Urol.
  • Mouse donor tissue consisted of C57BL/6 adult, 18-day fetal, and 14-day fetal kidney; and adult, 1-day postnatal, and 18-day fetal testis.
  • Excised kidneys or testes were placed in Dulbecco's Modified Eagle's Medium at 4 °C, and divided under a dissecting microscope into 1 mm fragments.
  • Adult recipient B10.A mice were anesthetized and the graft tissue was inserted under the left kidney capsule with a 16-gauge trocar.
  • the long(L) and short(W) axes of the grafts were measured at 10X magnification, at a standard focal distance using a microscope equipped with a calibrated eyepiece, where 10 ocular micrometer units equal 1 mm (Foglia, R.P., et al , Annals of Surgery 204:402 (1986); Foglia, R.P., et al, J. Ped. Surg. 27:608 (1986)).
  • B10.A 18-day and 14-day fetal renal, and adult and 18-day fetal testicular grafts were implanted for 7 days and 10 days beneath the renal capsule of adult recipient B10.A mice, and evaluated for change in size, architecture, and lymphocytic infiltrate.
  • the data for graft size ratio and the bioassays of graft architecture and lymphocytic infiltrate were summarized as mean +. standard deviation.
  • a logarithmic transformation (loglO) was performed on the graft size ratios.
  • An analysis of variance was used to compare the means of the groups for each variable (i.e. graft size ratio logarithmically transformed, graft architecture, and graft infiltrate).
  • Tests of significance of these means involves simul taneous Bonferroni t-tests, in which an overall significance level of 0.05 is preserved by performing each individual test at a level of significance equal to 0.05 divided by the number of determinations made.
  • Two-sample t-tests were used to compare the sizes attained by the respective sets of grafts.
  • the architecture and infiltrate data were tested against the constant value, 1, which was assigned to grafts that demonstrated no evidence of rejection.
  • RNA samples were fractionated by electrophoresis and subsequently transferred to Gene Screen as described above. Filters were prehybridized at 45 °C for 1 hour in hybridization buffer (5 % SDS, 100 mM NaCI, 50 mM Pipes, pH 6.8, 50 mM Na phosphate, 1 mM EDTA). Hybridization was carried out in the same buffer at 45 °C overnight. Filters were washed 2 times in hybridization buffer, then once with 3.2 M tetramethylammonium chloride (TMACL), 1 %
  • Anti sense oligonucleotide probes specific for the respective b and k haplotypes of the Class I H-2K and Class II la genes were chosen to distinguish the contribution of donor and recipient transcripts to the observed signal.
  • the Class I donor specific probe 5'-CCAGAGATCACCTGAATAGT- 3' hybridizes to exon 3 of H-2K ⁇ and the recipient specific probe 5'- CCGTACATCCGTTGGAACGT-3' hybridizes to exon 3 of H-2K k .
  • the Class II donor specific probe 5'-AGCTTGCCAATTGGCCAAAC-3' hybridizes to exon 2 of A ⁇ b, and the recipient specific probe 5'- ATCTTCTCAGTTGAGCAAAC-3' hybridizes to exon 2 of A intuitionk (Benoist, CO. et al , Cell 34: 167 (1983)).
  • Fetal and adult fresh and transplanted tissue were excised, rinsed in OCT compound (Ames Co., Division of Miles Laboratory, Elkhart, IN), and frozen in liquid nitrogen for up to 2 weeks.
  • Eight micron sections cut on a cryostat precooled to -20 °C were mounted on 1 % gelatin coated glass slides then air dried for 30 minutes in preparation for antibody localization of MHC antigen. The sections were fixed in 100% acetone for 4 minutes, washed three times in Tris buffered saline (TBS) for 30 minutes, incubated in 1 % hydrogen peroxide in 50% methanol for 30 minutes to quench the endogenous peroxidase activity, and again washed three times in TBS for 15 minute, all at 4 °C.
  • TBS Tris buffered saline
  • Immunol. 727:2488 (1981)) (Hybritech Inc., San Diego, CA) supplied as 100 ⁇ g of an ammonium sulfate fraction in 0.5 ml PBS and used at an optimal dilution of 1:100.
  • the Class I antibody was a rat monoclonal raised to murine H2 montypic antigen (M 1/42)20 (Hybritech Inc. San Diego, CA) supplied as 100 ⁇ g of an ammonium sulfate fraction in 0.5 ml PBS and used at a dilution of 1:100.
  • Class I and II mRNA levels were much higher in kidney than in testis, and Class I mRNA was higher than Class II mRNA for each tissue. Both started at very low levels in the younger fetus and increased with age. This is observed when total kidney RNA is hybridized to the Class I H-2K b probe.
  • the densitometry levels of adult Class I transcript, when compared to 6-day postnatal, 1-day postnatal, 18-day fetal, and 14-day fetal renal Class I transcripts were approximately 80:50: 18:9:1.
  • Hybridization of the same mRNA to the Class II I-a A also revealed a higher level of expression of Class II transcripts in the adult kidney when compared to that expressed in postnatal and fetal kidney.
  • the relative level of adult Class II transcript, when compared to 6-day postnatal, 1-day postnatal, 18-day fetal, and 14-day fetal renal Class II transcripts were 33: 12:7:6: 1.
  • the hybridization of total testis RNA to the Class I probe showed increasing levels of Class I transcripts through ontogeny, though in all cases the kidney levels were significantly greater than those found for testis of the corresponding developmental stage.
  • the relative levels of adult Class I transcripts when compared to postnatal and 18-day fetal testicular Class I transcripts were 7:2: 1.
  • the level of Class 1 transcripts observed in the adult kidney was about 13x greater than that observed in the adult testis which suggests that the level of Class I transcripts from 18 day kidney was approximately an order of magnitude higher than that of testes from the same stage of development.
  • Hybridization to the Class II probe also revealed much lower levels of Class II transcripts in the adult, postnatal, and fetal testis than those observed in the adult kidney.
  • the adult kidney showed a level of Class II transcripts 16x greater than the level in the adult testis.
  • the relative levels of adult Class II transcripts when compared to 1-day postnatal and 18-day fetal testicular Class II transcripts were 2: 1:1.
  • Syngeneic B10.A grafts implanted beneath the renal capsule of B10.A adult recipients for 7 days, were used to control for the effects of tissue manipulation on graft size ratio and histology.
  • the 14-day and 18-day fetal renal and 18-day fetal testicular syngeneic grafts grew to about the same size as the 14-day and 18-day fetal renal and testicular congenic C57BL/6 grafts implanted beneath the renal capsule of BIO.
  • a adult recipients Histologically, however, the fetal renal and testicular syngeneic grafts show excellent architecture (1 ⁇ 0) and no lymphocytic infiltrate (1 ⁇ 0) with no significant difference noted from the constant value of 1.
  • Hybridization with the donor specific A ⁇ b probe showed levels of class II transcripts in the young implanted kidney and testis grafts that were much greater than the level observed in the adult and fetal renal, and testicular donor tissue prior to implantation.
  • hybridization to the recipient specific Class II probe revealed an induction of A ⁇ k transcripts in the kidney and testis grafts to levels that exceed the level observed in the recipient B10.A renal tissue.
  • grafting of fetal tissue results in a large induction of Class I and Class II mRNA from both the donor graft and the recipient kidney although these fetal grafts are not strongly rejected.
  • the kidney protein as stained by immunohistochemistry did not follow the pattern observed for mRNA.
  • the adult kidney grafted for two days was atrophic but showed an intense amount of diffuse Class I surface protein staining, far in excess of that observed in the recipient adult renal tissue.
  • the Class II protein staining after grafting fetal tissue was minimal, again indicating no induction of Class II protein.
  • Adult kidney grafted for two days showed an intense amount of Class II surface protein, in excess of that seen in the recipient adult renal tissue.
  • testis Class I and II proteins in testes before and after implantation The testis Class I and II protein, as detected by immuno-histochemical staining, was very weak at all stages of development, but appeared to increase slightly throughout ontogeny. The minimal staining seen is localized to the interstitium and not the seminiferous tubules.
  • the testicular tissue after implantation showed a slight increased staining of both Class I and Class II proteins in both the fetal and adult grafts. The staining, however, was very light and never exceeded the low levels seen in the positive control adult renal tissue into which the graft has been implanted. The induction manifested at the mRHA level was not observed in the protein.
  • Adult testis grafts never acheived the intensity of staining seen in adult kidney implants.
  • MHC major histocompatibility complex
  • Testis and kidney were compared to see if the mechanisms of regulation in developing tissues would be tissue specific or universal, with the expectation that some insight might be gained into the sites at which one could intervene therapeutically to regulate the MHC.
  • Using northern analysis we have shown differences between the testis and kidney at all ages in the constituitive expression of Class I and Class II transcripts.
  • Adult mouse kidney has higher levels of Class I and Class II mRNA transcripts than the adult mouse testis.
  • Immature tissue has a lower constituitive level of MHC, and like adult tissue, it can be induced to express high levels of mRNA. Unlike the adult, however, MHC protein is not induced.
  • M ⁇ llerian Inhibiting Substance (MIS) and TGF-b which are important growth modulators in the fetus, can be expected to modulate expression of MHC transcripts or proteins.
  • MIS M ⁇ llerian Inhibiting Substance
  • TGF-b TGF-b
  • fetal and newborn testis like the brain (Gibson, M.J., et al, Science 225:4665 (1984)), is immunopriviledged, not only as a recipient, but as a donor, and thus is useful in clinical transplantation.
  • MIS was purified from conditioned media from a dihydrofolate reductase (DHFR) deficient Chinese hamster ovary (CHO) cell line (Chasin) transfected with a colinear construct of the MIS and DHFR genes (Cate et al. , Cell 45:685-698 (1986); Cate et al , Cold Spring Harbor Symposium on Quantitative Biology vol. LI, (1986)).
  • DHFR dihydrofolate reductase
  • Chosin Chinese hamster ovary
  • the MIS used after purification by carbohydrate affinity was biologically active in an organ culture assay where 1-2 ⁇ g caused regression of the M ⁇ llerian duct.
  • the mice received booster injections of the same dose of MIS mixed 1 : 1 in incomplete Freund's adjuvant (Difco Labs). After ten days the mice were bled from the retrobulbar sinus with Natelson capillary tubes (Monoject Scientific) and the serum anti-MIS antibody titer was assessed semi-quantitatively by El 15A.
  • MIS Conventionally purified MIS was first bound to polyvinyl chloride microtiter plates (Falcon); serially diluted sera from MIS injected and uninjected mice were then added. Colorimetric conversion of 3,3' ,5,5' tetramethyl Benzidine TMB (ICN ImmunoBiologicals) by a goat anti-mouse IgG (H&L) horseradish peroxidase conjugate (New England Nuclear) indicated an antigen specific immune response. Spleen cells from the mouse producing the highest titer of antibody in this screening ELISA assay were selected for fusion.
  • S p 2/0-Ag 14 hypoxanthine guanine deficient mouse myeloma cells were cycled through 20 ⁇ g/ml 8-azaguanine (Sigma) in Dulbecco's Modified Eagle's Medium DMEM containing 4.5 g/L glucose (Hazelton Labs) to assure azaguanine resistance and absence of reversion. After determining a doubling time of 12 hours, the cells were maintained at a concentration of 1 x 10 5 cells/ml and cycled so that the cells were in an exponential phase of growth prior to fusion.
  • Spleen cells 1 x 10 8 from injected mice with the highest titer of anti- MIS antibody were mixed with 2 x 10 2 Sp2/0 myeloma cells and centrifuged at 400 x g for 7 minutes. After aspiration of the supernatant, 1.5 ml of serum free DMEM containing 41 % W:V polyethylene glycol (PEG mw 1450, American Type Culture Collection) was added to the cell pellet. The mixture was pipetted for 1.5 minutes and then centrifuged for 10 minutes at room temperature in serum free DMEM to minimize PEG toxicity.
  • PEG mw 1450 polyethylene glycol
  • the cells were resuspended in HAT-DMEM selective media containing 20% fetal calf serum (Hybridoma screened, Microbiological Associates Bioproducts), 200 mM L- Glutamine, Hypoxanthine 10 "2 M (H), Aminopterin 4 x 1O "5 M (A), Thymidine 1.6 x 10 '3 M (T), and Penicillin/ Streptomycin 2000 U/2mg/ml (all from Sigma).
  • the cells were then transferred to ten 96 well tissue culture plates (Falcon) which had been plated previously with a macrophage feeder layer (10,000 cells/well) obtained from 6 age matched female A/J mice by peritoneal lavage with 5 ml of cold 11.6% sterile sucrose.
  • the plates were placed in a 37 °C incubator in an atmosphere of 6.5 %
  • hybridoma media that gave an absolute optical density value two times that of the media control as assessed by ELISA were considered positive.
  • the hybridoma cells producing anti-MIS antibody were simultaneously expanded to the 24 well level and subcloned by limiting dilution. Mixed populations of hybridomas were frozen during incremental expansion to safe-guard against overgrowth by non-producers. Previous experience indicated improved viability after thawing if the total number of cells had been increased to 1 x 10 5 prior to freezing and storage in liquid nitrogen. Two monoclonals, M10.6 and 6E11, were developed for use in detecting MIS from various sources.
  • Hybridoma cells were amplified in roller bottles in Alpha Modified
  • Antibody was precipitated from ascites, media or blood by treatment with 50% (NH 4 ) 2 SO 4 for 4 hours at 4 °C. After centrifugation at 8000 RPM in a Beckman J2 centrifuge for 20 minutes, the pellet was resuspended in PBS at pH 8.2 in l/50th the original volume and dialyzed. The antibody solution was applied slowly to a 5 ml Protein A Sepharose CL-4B (Sigma) column at 1 column volume/hour. Bound antibody was eluted with 0.1M citrate in PBS pH 3.5, then dialyzed against PBS, pH 7.4 containing 0.03% sodium azide as a preservative.
  • a graded organ culture assay was used to detect MIS.
  • the monoclonal antibody M10.6 or 6E11 or a non-specific antibody was combined at a 3:1 ratio with MIS, at a concentration (2-3 ⁇ g) which caused 3-4 plus regression of the M ⁇ llerian ducts, for an overnight incubation at 4C on a rotary shaker.
  • Each mixture was added, after sterile filtration, to the media beneath the mullerian duct in the organ culture bioassay. Three days later M ⁇ llerian duct regression was assessed histologically.
  • MIS was radiolabelled via the chloramine T Iodination method (Greenwood, et al , Biochem. J. 89: 114 (1963); Hutson et al , J. Ped. 77:953-959 (1982)).
  • Purified antibody, M10.6 or 6E11 was first bound to Falcon polyvinyl chloride microtiter plates. After the plates were blocked with female fetal calf serum, the tagged MIS was added. Determination of the affinity constants was done by measuring bound and unbound radiolabelled MIS.
  • PBS phosphate buffered saline
  • Blocking buffer 5 % female fetal calf serum in PBS, was then added for 2 hours at room temperature.
  • the plates were washed with 4 volumes of PBS, following which unknowns or samples of MIS diluted to provide a standard curve in blocking buffer were applied to the wells and incubated overnight at 4 °C for optimal binding.
  • the wells were then washed with 4 volumes of PBS, and polyclonal antibody (whole serum) at a 1/1000 dilution in blocking buffer was added for 1 hour at room temperature.
  • N terminal fragment was generated by site directed mutagenesis in which a stop codon was introduced at position 427, the site of probable monobasic cleavage.
  • M10.6 recognized the N terminal fragment generated from transfected CHO cells.
  • the C terminal fragment was generated by acid hydrolysis of whole MIS and eluted from an M10.6 affinity column.
  • 6E11 recognized the C terminus at greatly reduced sensitivity but not the N terminal fragment.
  • a mouse anti-rabbit conjugate which had been absorbed against mouse and human serum recognized with specificity and could be used with 6E11 optimally at a 1/5000 dilution. Lengths of incubation times were assessed and abbreviated if possible.
  • the tetramethyl benzidine substrate concentration was constant at 42 mM in DMSO diluted 1/100 in sodium acetate buffer (0.1 M), pH titrated to 4.9 with citric acid followed by the addition of 12 ⁇ moles of 30% H 2 O 2 .
  • Substrate reaction time was exactly 12.5 minutes immediately followed by addition of 2N H 2 SO 4 .
  • the assay can detect picomolar quantities of MIS.
  • TGFB-1 and B-2 have considerable amino acid homology with the C terminal end of MIS and are considered to be representatives of the same gene family.
  • TGF-B, FSH, or LH a similar plate ELISA was done.
  • the analytical method was less sensitive the polyclonal antibody recognized MIS but not TGF-B or the gonadotropins.
  • This ELISA initially established to measure human serum MIS, was first used to monitor the quantity of the MIS protein produced by a Chinese Hamster Ovary cell line which had been transfected with the human MIS gene or various mutant constructs, and to later adapted to assess MIS levels from various clinical sources. These included serum from human umbilical cord blood; serum from premature and normal newborns and those with congenital abnormalities; serum from older normal infants and children and from those with congenital anomalies, as well as serum from normal adults; female serum collected during various stages of the menstrual cycle, ovarian follicular fluid collected during various stages of In Vitro fetilization protocols; and serum of a patient with a rare Sertoli cell MIS producing tumor.
  • the samples were collected and immediately placed on ice during transport to the laboratory where they were stored at -80 °C until assay.
  • the assay permitted a determination of the range of MIS concentrations in newborn male and female sera (9-74 ng/ml in males (average of 27 individuals); 0-2 ng/ml in females (average of 12 individuals)).
  • the assay clearly delineated male cord serum with high MIS levels from female serum in which MIS was barely detectable.
  • serum collected from premature infants in the pediatric ICU showed elevated levels for MIS in the males and undetectable levels in the females.
  • the MIS levels fall gradually after birth in male children and begin to rise slowly to a much lower level in the pre- pubescent female, to reach adult male and female levels of MIS in the 5 ng range.

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Abstract

Cette invention se rapporte au traitement de certaines tumeurs au moyen d'une dose efficace de l'hormone antimüllerienne glycoproteique (MIS). L'invention se rapporte en outre au traitement de certaines tumeurs à l'aide d'une dose efficace du fragment C-terminal de MIS. L'invention se rapporte également à des séquences d'ADN codant le fragment C-terminal de MIS, à des vecteurs contenant la séquence d'ADN et à des cellules hôtes transformées pouvant produire le fragment C-terminal, ainsi qu'au traitement de certaines tumeurs par transfection des cellules tumorales avec un gène codant pour la MIS ou son fragment C-terminal. Des méthodes de thérapie génique permettant d'inhiber la croissance de certaines tumeurs sont également décrites, ainsi qu'une méthode de modulation d'antigènes d'histocompatibilité de Classe I à l'aide de MIS et du facteur de croissance épidermique (EGF).
PCT/US1993/005791 1992-06-19 1993-06-18 Hormone antimullerienne utilisee pour le traitement de tumeurs et la modulation de l'expression de l'antigene d'histocompatibilite majeure classe i. Ceased WO1994000133A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP93916585A EP0646010A4 (fr) 1992-06-19 1993-06-18 Hormone antimullerienne utilisee pour le traitement de tumeurs et la modulation de l'expression de l'antigene d'histocompatibilite majeure classe i.

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US90163792A 1992-06-19 1992-06-19
US07/901,637 1992-06-19
US712593A 1993-01-21 1993-01-21
US08/007,125 1993-01-21

Publications (1)

Publication Number Publication Date
WO1994000133A1 true WO1994000133A1 (fr) 1994-01-06

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PCT/US1993/005791 Ceased WO1994000133A1 (fr) 1992-06-19 1993-06-18 Hormone antimullerienne utilisee pour le traitement de tumeurs et la modulation de l'expression de l'antigene d'histocompatibilite majeure classe i.

Country Status (2)

Country Link
EP (1) EP0646010A4 (fr)
WO (1) WO1994000133A1 (fr)

Cited By (4)

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Publication number Priority date Publication date Assignee Title
US5558241A (en) * 1994-01-06 1996-09-24 Temp Top Container Systems, Inc. Cryotransport chamber
WO2001055212A3 (fr) * 2000-01-27 2002-01-24 Gen Hospital Corp Administration de produits biologiques therapeutiques a partir de matrices tissulaires implantables
EP2676678A1 (fr) 2007-07-17 2013-12-25 The General Hospital Corporation Procédés pour identifier et enrichir des populations de cellules souches cancéreuses ovariennes et cellules souches somatiques et leurs utilisations
US9260759B2 (en) 2007-03-22 2016-02-16 The General Hospital Corporation Pyrazoloanthrone and derivatives thereof for the treatment of cancers expressing MISRII

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WO1989006695A1 (fr) * 1985-10-30 1989-07-27 Biogen, Inc. Dimeres clives de polypeptides semblables a substance d'inhibition mullerienne
WO1992018152A1 (fr) * 1991-04-12 1992-10-29 The General Hospital Corporation Utilisation de l'hormone antimullerienne pour le traitement de certaines tumeurs

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US5011687A (en) * 1984-06-18 1991-04-30 The General Hospital Corporation Purified Mullerian inhibiting substance and process for treating human ovarian cancer cells
ES2096750T3 (es) * 1990-10-31 1997-03-16 Somatix Therapy Corp Vectores retroviricos utiles para la terapia genica.

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WO1989006695A1 (fr) * 1985-10-30 1989-07-27 Biogen, Inc. Dimeres clives de polypeptides semblables a substance d'inhibition mullerienne
WO1992018152A1 (fr) * 1991-04-12 1992-10-29 The General Hospital Corporation Utilisation de l'hormone antimullerienne pour le traitement de certaines tumeurs

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Title
Cancer Research, Volume 49, issued 15 April 1989, WALLEN et al., "Minimal Antiproliferative Effect of Recombinant Mullerian Inhibiting Substance on Gynecological Tumor Cell Lines and Tumor Explants", pages 2005-2011, see page 2010 and 2011. *
Cancer Research, Volume 52, issued 01 March 1992, PARRY et al., "Recombinant Human Mullerian Inhibiting Substance Inhibits Human Ocular Melanoma Cell Lines in Vitro and in Vivo", pages 1182-1186, see entire publication, especially page 1182. *
Journal of Cellular Biochemistry, Supplement 16B, Paper No. G112, issued 25 January 1992, HUDSON et al., "Mullerian Inhibiting Substance (MIS) Slows Epidermoid Carinoma, A431, Cells", see page 123. *
Journal of Immunotherapy, Volume 10, Number 6, issued 1991, ALEXANDER et al., "Adoptively Transferred Tumor-Infiltrating Lymphocytes Can Cure Established Metastatic Tumor in Mice and Persist Long-Term in Vivo as Functional Memory T Lymphocytes", pages 389-397, see entire publication, especially the Abstract. *
Proceeding of the National Academy of Science, Volume 87, issued January 1990, KASID et al., "Human Gene Transfer: Characterization of Human Tumor-Infiltrating Lymphocytes as Vehicles for Retroviral Gene Transfer in Man", pages 473-477, see entire publication, especially the Abstract. *
See also references of EP0646010A4 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5558241A (en) * 1994-01-06 1996-09-24 Temp Top Container Systems, Inc. Cryotransport chamber
WO2001055212A3 (fr) * 2000-01-27 2002-01-24 Gen Hospital Corp Administration de produits biologiques therapeutiques a partir de matrices tissulaires implantables
US6692738B2 (en) 2000-01-27 2004-02-17 The General Hospital Corporation Delivery of therapeutic biologicals from implantable tissue matrices
US7078032B2 (en) 2000-01-27 2006-07-18 The General Hospital Corporation Delivery of therapeutic biologicals from implantable tissue matrices
US9260759B2 (en) 2007-03-22 2016-02-16 The General Hospital Corporation Pyrazoloanthrone and derivatives thereof for the treatment of cancers expressing MISRII
EP2676678A1 (fr) 2007-07-17 2013-12-25 The General Hospital Corporation Procédés pour identifier et enrichir des populations de cellules souches cancéreuses ovariennes et cellules souches somatiques et leurs utilisations
EP2764874A1 (fr) 2007-07-17 2014-08-13 The General Hospital Corporation Procédés pour identifier et enrichir des populations de cellules souches cancéreuses ovariennes et cellules souches somatiques et leurs utilisations

Also Published As

Publication number Publication date
EP0646010A4 (fr) 1997-04-23
EP0646010A1 (fr) 1995-04-05

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