Mullerian Inhibiting Substance (MIS) causes regression of female reproductive tract structures in the male fetus. To investigate whether MIS can be a therapeutic for ovarian cancer, which originates from the Mullerian duct derived coelomic epithelium, binding of MIS to both ovarian cancer cell lines and primary ascites cells from patients with stage III or IV cystadenocarcinoma was demonstrated. These cells express the MIS type II receptor mRNA and respond to MIS in growth inhibition assays. These experiments paralleled efforts to produce MIS that would be suitable for clinical trial in humans because of concern over mouse immunogens in the immunoaffinity purification of MIS and the preponderance of high molecular weight aggregates and multiple products of the primary and secondary cleavage sites of holo MIS. In order to address those concerns, preliminary studies were done that indicate conditioned serum free media from MIS transfected mammalian cells could be used to advantage to produce a homogeneous preparation of MIS by size exclusion chromatography with an FPLC system. Peptides with high affinity can also be used in purification, and tobacco sources for rhMIS will be compared to mammalian sources. Thus, the first Specific Aim is to purify homogenous MIS using these approaches and to scale up production for use in clinical trials. The second Specific Aim is to crystallize MIS or its derivative in order to begin the process of characterizing the secondary structure of MIS and understand the molecular interactions both between the amino and carboxy terminal MIS domains and between the C terminal domain and its receptor. The third Specific Aim is to determine if the MIS type II receptor is mutated in patients with ovarian cancer by sequence analysis and correlate absence of functional receptor with the occurance of ovarian cancer. The fourth Specific Aim is to study the tissue specific and developmental expression of the MIS type II receptor using antibodies developed during the past grant and others to be developed during the course of the proposed grant. A homogeneous MIS product whose structure is defined by crystallography, which can be scaled up for human use in clinical trials against human ovarian cancer, can then be used in parallel with MIS type II receptor molecular probes and antibodies to select tumors appropriate for such therapies.
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