An albumin leader sequence coupled with a cleavage site modification enhances the yield of recombinant C-terminal Mullerian Inhibiting Substance

Abstract

Mullerian Inhibiting Substance (MIS) has been shown to inhibit ovarian cancer cells both in-vitro and in-vivo. Furthermore, recent evidence suggests that MIS may effectively target a putative ovarian cancer progenitor cell population enriched by a panel of CD44+, CD24+, Ep-CAM+, and E-cadherin-cell surface markers. In order to accommodate clinical testing of MIS in ovarian cancer patients, the production of recombinant human MIS must be optimized to increase yield and purity. Here we show that, compared to wild type, the substitution of the MIS leader sequence to that of human serum albumin, combined with a modification of the endogenous cleavage site from RAQR/S to a furin/kex2 RARR/S consensus site results in high expression, increased C-terminus cleavage and a reduction in unwanted cryptic internal cleavage products when produced in CHO cells. Purified MIS containing these alterations retains its capacity to induce regression of the Mullerian duct in fetal rat embryonic urogenital ridge assays.

AMH/MIS as a contraceptive that protects the ovarian reserve during chemotherapy

“…We confirmed that Misr2 was expressed by granulosa cells at all early stages of folliculogenesis, including primordial follicles ( (Fig. S2 B and C), which can be detected by an ELISA specific to the human protein (2,21). The circulating level of rhMIS was remarkably stable over the 60 d of the experiment (Fig.…”

“…The ELISA to measure human MIS was performed as previously reported (21). Briefly, a 96-well plate was coated with mouse monoclonal anti-human recombinant MIS antibody (clone 6E11) overnight at 4°C and was blocked with 1% BSA/PBS and Tween 20 (PBST) (Jackson ImmunoResearch) for 2 h at room temperature.…”

“…RhMIS protein was purified as previously described (21). Briefly, a CHO-K1 clone stably transfected with the LR-MIS vector was grown in HYPERflasks (Corning), and serum-free conditioned medium collected after 72 h of incubation was concentrated 10-20× using a tangential flow over a size-exclusion membrane.…”

“…To address these concerns, we created a cDNA construct (21) in which we replaced the Leader sequence with that of albumin and introduced a Q425R substitution in the C-terminal cleavage site to produce an MIS protein analog (LR-MIS, which we will refer to simply as "rhMIS"), with increased activating cleavage and secretion. The LR-MIS transgene also was used to produce an optimized adeno-associated virus serotype 9 (AAV9) gene therapy vector compatible with in vivo use, which we will refer to as "AAV9-MIS" (22).…”

“…The availability of a biologically active rhMIS protein that can be produced and purified to high yields using chinese hamster ovary (CHO) cells (21) allowed us to explore higher and longer dosing in vivo, which previously had been impractical with the lower-cleavage wildtype protein (39) or impossible using commercial C-terminal recombinant MIS protein (R&D Systems, Inc.), which we found to be devoid of activity. For example, incubation of fetal [embryonic day (E)14.5] female rat urogenital ridges with 5 μg/mL of rhMIS for 72 h in ex vivo cultures causes nearly complete regression of the Müllerian duct (Fig.…”