Anti-Müllerian hormone concentration regulates activin receptor-like kinase-2/3 expression levels with opposing effects on ovarian cancer cell survival.

Anti­Müllerian hormone (AMH) type II receptor (AMHRII) and the AMH/AMHRII signaling pathway are potential therapeutic targets in ovarian carcinoma. Conversely, the role of the three AMH type I receptors (AMHRIs), namely activin receptor­like kinase (ALK)2, ALK3 and ALK6, in ovarian cancer remains to be clarified. To determine the respective roles of these three AMHRIs, the present study used four ovarian cancer cell lines (COV434­AMHRII, SKOV3­AMHRII, OVCAR8, KGN) and primary cells isolated from tumor ascites from patients with ovarian cancer. The results demonstrated that ALK2 and ALK3 may be the two main AMHRIs involved in AMH signaling at physiological endogenous and supraphysiological exogenous AMH concentrations, respectively. Supraphysiological AMH concentrations (25 nM recombinant AMH) were associated with apoptosis in all four cell lines and decreased clonogenic survival in COV434­AMHRII and SKOV3­AMHRII cells. These biological effects were induced via ALK3 recruitment by AMHRII, as ALK3­AMHRII dimerization was favored at increasing AMH concentrations. By contrast, ALK2 was associated with AMHRII at physiological endogenous concentrations of AMH (10 pM). Based on these results, tetravalent IgG1­like bispecific antibodies (BsAbs) against AMHRII and ALK2, and against AMHRII and ALK3 were designed and evaluated. In vivo, COV434­AMHRII tumor cell xenograft growth was significantly reduced in all BsAb­treated groups compared with that in the vehicle group (P=0.018 for BsAb 12G4­3D7; P=0.001 for all other BsAbs). However, the growth of COV434­AMHRII tumor cell xenografts was slower in mice treated with the anti­AMRII­ALK2 BsAb 12G4­2F9 compared with that in animals that received a control BsAb that targeted AMHRII and CD5 (P=0.048). These results provide new insights into type I receptor specificity in AMH signaling pathways and may lead to an innovative therapeutic approach to modulate AMH signaling using anti­AMHRII/anti­AMHRI BsAbs.

Anti-Müllerian hormone (AMH) autocrine signaling promotes survival and proliferation of ovarian cancer cells.

In ovarian carcinoma, anti-Müllerian hormone (AMH) type II receptor (AMHRII) and the AMH/AMHRII signaling pathway are potential therapeutic targets. Here, AMH dose-dependent effect on signaling and proliferation was analyzed in four ovarian cancer cell lines, including sex cord stromal/granulosa cell tumors and high grade serous adenocarcinomas (COV434-AMHRII, SKOV3-AMHRII, OVCAR8 and KGN). As previously shown, incubation with exogenous AMH at concentrations above the physiological range (12.5-25 nM) decreased cell viability. Conversely, physiological concentrations of endogenous AMH improved cancer cell viability. Partial AMH depletion by siRNAs was sufficient to reduce cell viability in all four cell lines, by 20% (OVCAR8 cells) to 40% (COV434-AMHRII cells). In the presence of AMH concentrations within the physiological range (5 to 15 pM), the newly developed anti-AMH B10 antibody decreased by 25% (OVCAR8) to 50% (KGN) cell viability at concentrations ranging between 3 and 333 nM. At 70 nM, B10 reduced clonogenic survival by 57.5%, 57.1%, 64.7% and 37.5% in COV434-AMHRII, SKOV3-AMHRII, OVCAR8 and KGN cells, respectively. In the four cell lines, B10 reduced AKT phosphorylation, and increased PARP and caspase 3 cleavage. These results were confirmed in ovarian cancer cells isolated from patients' ascites, demonstrating the translational potential of these results. Furthermore, B10 reduced COV434-MISRII tumor growth in vivo and significantly enhanced the median survival time compared with vehicle (69 vs 60 days; p = 0.0173). Our data provide evidence for a novel pro-survival autocrine role of AMH in the context of ovarian cancer, which was targeted therapeutically using an anti-AMH antibody to successfully repress tumor growth.

Toxicity and pharmacokinetic profile of SGM-101, a fluorescent anti-CEA chimeric antibody for fluorescence imaging of tumors in patients.

The real-time improvement of the intraoperative discrimination between different tissue types (particularly between tumor and adjacent normal tissue) using intraoperative imaging represents a considerable advance for oncology surgeons. However, the development of imaging agents is much slower than that of drug therapies, although surgery represents one of the few curative treatments for many solid tumors. SGM-101 is a recently described, innovative antibody conjugate in which the near-infrared fluorochrome BM-104 is covalently linked to a chimeric monoclonal antibody against carcinoembryonic antigen (CEA). SGM-101 was developed with the goal of providing oncology surgeons with an intraoperative imaging tool that allows the visualization of CEA-overexpressing tumors. This antigen is overexpressed in a wide range of human carcinomas, such as colorectal, gastric, pancreatic, non-small cell lung and breast carcinomas. Here we characterized SGM-101 safety prior to its clinical testing for real-time cancer mapping by oncology surgeons. Safety pharmacology and toxicology studies were performed after intravenous injection of SGM-101 in Wistar rats and in Beagle dogs. SGM-101 metabolism and pharmacokinetics were analyzed in rats and mice. Finally, the potential toxicity of the BM-104 dye and SGM-101 cross-reactivity were assessed in a panel of 42 human tissues. Our pre-clinical toxicology, pharmacology and pharmacokinetic results demonstrated the absence of significant adverse effects of both SGM-101 and BM-104 at doses well above the anticipated maximal human exposure. Taken together, the results of the pharmacology, pharmacokinetic and toxicology studies support the development of SGM-101 as a potentially useful and safe tumor-specific imaging tool that might improve the complete tumor resection rate.

SGM-101: An innovative near-infrared dye-antibody conjugate that targets CEA for fluorescence-guided surgery.

PURPOSE: Fluorescence-guided surgery (FGS) provides surgeons with new opportunities to improve real-time cancer nodule detection and tumor margin visualization. Currently, the most important challenge in this field is the development of fluorescent dyes that specifically target tumors. We developed, characterized and evaluated SGM-101, an innovative antibody-dye conjugate in which the fluorochrome BM104, which has an absorbance band centered at 700 nm, is coupled to a chimeric monoclonal antibody (mAb) against carcinoembryonic antigen (CEA). METHODS: The dye to mAb ratio, binding to CEA and photobleaching of SGM-101 were determined. FGS was performed and results analyzed using different mouse models of human digestive tumors. RESULTS: SGM-101 allowed the detection of tumor nodules in three different colon cancer models: LS174T human colorectal adenocarcinoma cell-induced peritoneal carcinomatosis (PC) and liver metastases, and orthotopic grafts of HT29 human colorectal adenocarcinoma cells. In the PC model, submillimeter-sized nodules were detected during SGM-101-based FGS and SGM-101 predictive positive values ranged from 99.04% to 90.24% for tumor nodules >10 mg and nodules <1 mg, respectively. Similarly, in the orthotopic model of pancreatic cancer using BxPC3 (pancreas adenocarcinoma) cells, SGM-101 could clearly delineate tumors in vivo with a tumor-to-background ratio of 3.5, and penetrated in tumor nodules, as demonstrated by histological analysis. Free BM105 dye (BM104 with an activated ester for conjugation to the antibody) and an irrelevant conjugate did not induce any NIR fluorescence. CONCLUSION: These preclinical data indicate that SGM-101 is an attractive candidate for FGS of CEA-expressing tumors and is currently assessed in clinical trials.