These preclinical data indicate that SGM-101 is an attractive candidate for FGS of CEA-expressing tumors and is currently assessed in clinical trials.
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.
340 Background: Carcinoembryonic antigen (CEA) is a widely known tumor marker that is clearly expressed in gastrointestinal tract cancer. We utilized a CEA-specific chimeric antibody conjugated to a near infrared (NIR) fluorophore to facilitate CEA-targeted fluorescence image–guided surgery (FGS) of gastric cancer. The anti-CEA antibody, SGM-101 is conjugated with NIR dye BM-105, which has an absorbance band centered at 705 nm. Methods: RNA sequencing data of 34 gastric cancer cell lines from Cancer Cell Line Encyclopedia were screened and validated by qPCR and western blotting. Flow cytometry and confocal microscopy were performed by SGM-101, Alexa Fluor-680, Isotype-101 and Isotype-680 to quantify fluorescence intensity. SGM-101(n = 5) or Isotype-101(n = 2) was injected to mouse xenografts through a tail vein which had been subcutaneously implanted with MKN-45, SNU-16, and SNU-668. IVIS Spectrum quantified radiant efficiency of fluorescence in the region of interest at serial time points. The extracted tumor in peak time was analyzed by confocal imaging for microdistribution. In addition, 85As2mLuc were injected intraperitoneally in 6-week-old female BALB/c-nu mice for peritoneal carcinomatosis. Bioluminescence/fluorescence imaging was performed with IVIS Spectrum at peak time and analyzed via Living Image. Histologic evaluations were processed with H&E and Immunohistochemistry (IHC) data by a pathologist. Results: RNA expression of ceacam5 and protein expression of CEA in gastric cell lines was measured by RNA sequencing, qPCR, and western blotting. CEA expression patterns displays similar with fluorescence intensity patterns which were quantified through flow cytometry and immunocytochemistry show that CEA localized in membranes. In subcutaneously implanted model, the radiant efficiency of each group shows that the accumulation of SGM-101 has significantly higher fluorescence signal in the high CEA expressing group (MKN-45) and medium expressing group (SNU-16) while no fluorescence signal was observed in the CEA negative group (SNU-668) via IVIS Spectrum. Biodistribution of SGM-101 indicates that the maximum peak accumulation point was 48 hours after tail vein injection. Frozen tissue which was extracted at peak detection time shows micro-distribution of SGM-101 and expression of extracted tissue CEA expression was validated with IHC by pathological analysis. In the peritoneal carcinomatosis model, the imaging of fluorescence detection patterns corresponds with bioluminescence imaging and histological evaluation. Conclusions: CEA expression corresponded with intensity of in vitro fluorescence immunodetection and a tumor area accumulation in gastric cancer xenografts by SGM-101. This study indicates that NIR tumor specific imaging can be a feasible tool for image-guided surgery.
Anti-Müllerian 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 receptor-like 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 (COV434-AMHRII, SKOV3-AMHRII, 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 COV434-AMHRII and SKOV3-AMHRII cells. These biological effects were induced via ALK3 recruitment by AMHRII, as ALK3-AMHRII 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 IgG1-like bispecific antibodies (BsAbs) against AMHRII and ALK2, and against AMHRII and ALK3 were designed and evaluated. In vivo, COV434-AMHRII tumor cell xenograft growth was significantly reduced in all BsAb-treated groups compared with that in the vehicle group (P=0.018 for BsAb 12G4-3D7; P=0.001 for all other BsAbs). However, the growth of COV434-AMHRII tumor cell xenografts was slower in mice treated with the anti-AMRII-ALK2 BsAb 12G4-2F9 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 anti-AMHRII/anti-AMHRI BsAbs.
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