Abstract Background Ovarian cancer remains the deadliest of the gynecologic cancers in the United States. There have been limited advances in treatment strategies that have seen marked increases in overall survival. Thus, it is essential to continue developing and validating new treatment strategies and markers to identify patients who would benefit from the new strategy. In this report, we sought to further validate applications for a novel humanized anti-Sialyl Tn antibody-drug conjugate (anti-STn-ADC) in ovarian cancer. Methods We aimed to further test a humanized anti-STn-ADC in sialyl-Tn (STn) positive and negative ovarian cancer cell line, patient-derived organoid (PDO), and patient-derived xenograft (PDX) models. Furthermore, we sought to determine whether serum STn levels would reflect STn positivity in the tumor samples enabling us to identify patients that an anti-STn-ADC strategy would best serve. We developed a custom ELISA with high specificity and sensitivity, that was used to assess whether circulating STn levels would correlate with stage, progression-free survival, overall survival, and its value in augmenting CA-125 as a diagnostic. Lastly, we assessed whether the serum levels reflected what was observed via immunohistochemical analysis in a subset of tumor samples. Results Our in vitro experiments further define the specificity of the anti-STn-ADC. The ovarian cancer PDO, and PDX models provide additional support for an anti-STn-ADC-based strategy for targeting ovarian cancer. The custom serum ELISA was informative in potential triaging of patients with elevated levels of STn. However, it was not sensitive enough to add value to existing CA-125 levels for a diagnostic. While the ELISA identified non-serous ovarian tumors with low CA-125 levels, the sample numbers were too small to provide any confidence the STn ELISA would meaningfully add to CA-125 for diagnosis. Conclusions Our preclinical data support the concept that an anti-STn-ADC may be a viable option for treating patients with elevated STn levels. Moreover, our STn-based ELISA could complement IHC in identifying patients with whom an anti-STn-based strategy might be more effective.
Abstract Key words: Sialyl Tn, drug resistance, antibody-drug conjugates, cancer stem cells OBJECTIVES: A successful therapeutic strategy for ovarian cancer will require direct targeting of inherently chemoresistant tumor cells which are comprised in part of cancer stem cells (CSCs) that survive current cytotoxic treatment regimes and drive tumor resurgence. The sialyl-Tn (STn) antigen is a carbohydrate moiety present on tumor cells but rarely seen in normal adult tissue. Importantly, STn has been shown to be present on CSCs in pancreatic, colon, and gastric malignancies. Our objective was to assess the expression of STn and the known CSC marker CD133 in human ovarian cancer (OvCa) cell lines and primary serous carcinomas, and evaluate the ability of STn+ and STn- cells to both grow in an anchorage independent manner and survive standard-of-care cytotoxic therapy. Furthermore, we sought to assess the effect of murine and humanized α -STn antibody-drug conjugates (ADCs) on OvCa cells in vitro and tumor viability in vivo. METHODS: STn and CD133 expression in established OvCa cell lines was analyzed by flow cytometry. STn-CD133-, STn+CD133-, STn-CD133+ and STn+CD133+ cells were purified from OVCAR3 and OVCAR4 by FACS, plated in soft agar, and incubated for 21 days. Colony forming efficiency of each sub-population was calculated. Unsorted cells were treated in vitro with either murine α -STn-monomethyl auristatin E (MMAE) ADC or vehicle control and cell viability was assessed by MTT assay. Subsequently, cells were treated in vitro with α -STn-MMAE, paclitaxel and carboplatin, or appropriate controls, and the profile of cells surviving 72 hours post-treatment was determined by flow cytometric analysis. Finally, OVCAR3-derived mouse xenografts were treated with murine and humanized α-STn-MMAE, unconjugated mAbs alone, and vehicle control. Mice were assessed regularly for tumor growth and cytotoxic effects. RESULTS: In the OvCa cell lines OV90, OVCAR3 and OVCAR4, when grown in traditional 2D culture, STn+ cells comprised 98.4%, 40.0%, and 26.4% of the total cell population, respectively. In each of these cell lines, we readily detected STn+CD133+ sub-populations suggesting that STn is expressed on CD133+ ovarian CSCs. Colony formation assays analyzing FACS-purified STn-CD133-, STn+CD133-, STn-CD133+ and STn+ CD133+ sub-populations suggest that STn expression correlates with anchorage independent growth, a characteristic of cell stemness. Paclitaxel and carboplatin treatment in vitro significantly increased the proportion of STn+ and CD133+ cells, demonstrating the chemoresistant characteristics of these cells. Treatment with the murine α-STn-MMAE ADCs reduced the viability of OvCa cell lines in vitro in a dose-dependent manner. Treatment with murine and humanized α -STn- MMAE antibodies in vivo reduced tumor volumes, whereas vehicle treatment did not impede tumor growth. Interestingly, the unconjugated antibody also had a modest negative impact on tumor volume. CONCLUSION: A novel, highly specific STn antibody identifies the STn antigen in OvCa cell lines and patient samples. STn+ and CD133+ cells demonstrate stem-like characteristics such as anchorage-independent growth and chemoresistance. STn ADCs decreased cell viability in vitro and reduced tumor volumes in vivo, suggesting that specific therapeutic targeting of STn in ovarian tumors may be an effective clinical strategy to eliminate quiescent CSCs. Citation Format: B.R. Rueda, K. Starbuck, D. Eavarone, J. Prendergast, J. Stein, R. Foster, J. Behrens. TARGETING A CHEMORESISTANT OVARIAN CANCER CELL POPULATION VIA THE CARBOHYDRATE ANTIGEN SIALYL TN [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr MIP-071.
The expression of Sialyl-Tn (STn) in tumors is associated with metastatic disease, poor prognosis, and reduced overall survival. STn is expressed on ovarian cancer biomarkers including CA-125 (MUC16) and MUC1, and elevated serum levels of STn in ovarian cancer patients correlate with lower five-year survival rates. In the current study, we humanized novel anti-STn antibodies and demonstrated the retention of nanomolar (nM) target affinity while maintaining STn antigen selectivity. STn antibodies conjugated to Monomethyl Auristatin E (MMAE-ADCs) demonstrated in vitro cytotoxicity specific to STn-expressing ovarian cancer cell lines and tumor growth inhibition in vivo with both ovarian cancer cell line- and patient-derived xenograft models. We further validated the clinical potential of these STn-ADCs through tissue cross-reactivity and cynomolgus monkey toxicity studies. No membrane staining for STn was present in any organs of human or cynomolgus monkey origin, and the toxicity profile was favorable and only revealed MMAE-class associated events with none being attributed to the targeting of STn. The up-regulation of STn in ovarian carcinoma in combination with high affinity and STn-specific selectivity of the mAbs presented herein warrant further investigation for anti-STn antibody-drug conjugates in the clinical setting.
Abstract PD1/PD-L1 and CTLA-4 checkpoint blockade have revolutionized cancer therapy and led to cures in metastatic melanoma, but most patients develop primary and acquired resistance to these therapies. Treating this refractory population requires the discovery of new immune escape mechanisms. Sialic acid–binding immunoglobulin-type lectins (Siglecs) are expressed on the majority of white blood cells of the immune system, play critical roles in immune cell signaling and serve as immune checkpoints to prevent unwanted immune responses. Sialic acid is a ligand for inhibitory Siglecs; hypersialyation is a hallmark of poor prognosis and is believed to help tumors escape from immune surveillance. However, the role of hypersialylation in resistance to immune checkpoint therapies remains unexplored. To study if hypersialylation drives immune escape in melanoma, we profiled the immunosuppressive sialoglycans using Siglec-based high-affinity sialoglycan-binding constructs called ‘HYDRAs'. The current study focuses on understanding Siglec-3, -7 and -9 sialoglycan ligand expression on tumors using the HYDRA-3, -7 and -9 platform, because these Siglecs are the major inhibitory Siglecs on both innate and adaptive immune cells among the fourteen Siglecs in humans. Serial sections from melanoma tumors and healthy tissues were stained with HYDRA-3, -7 or -9 and scored using the semi-qualitative H-score method by a blinded pathologist. HYDRA IHC on healthy and cancerous human tissues demonstrate unique binding patterns with melanomas having high signals for HYDRA-3, -7 and -9. A pre-treatment checkpoint inhibitor therapy cohort (n=53), which contained responders (n=30) and non-responders (n=23) to either aPD1 or aPD1 and aCTLA-4 combination therapy was further studied. Serial sections from each patient was stained with HYDRA-3, -7 or -9 and scored using the semi-qualitative H-score method by our blinded pathologist. Cutoffs were determined in an unbiased manner for each HYDRA individually and each possible HYDRA combination to obtain correlations with patient progression-free and overall survival. A significant tumor H-score cutoff of a combined HYDRA-3 and -7 correlated with poor outcomes. This HYDRA-3 and -7 cutoff did not correlate with other melanoma biomarkers such as BRAF-mutation, liver metastases, PD-L1, nor TILs, suggesting a unique biology independent of these markers. We discovered that melanoma patients with multi-Siglec ligands as profiled by HYDRAs tend to be resistant to PD-1 checkpoint blockade and can be candidates for novel treatments targeting the Siglec-Sialoglycan axis. A larger cohort and longitudinal study are currently underway to examine the Siglec-Sialoglycan axis of immunosuppression in melanoma and late-breaking results will be included in this poster. Citation Format: Adam Petrone, Dennie T. Frederick, Jillian M. Prendergast, James Broderick, Karl Normington, Genevieve Boland, Li Peng. Melanoma patients with multi-Siglec ligands as profiled by HYDRA technology are refractory to PD1 blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 491.
Every cell expresses a molecularly diverse surface glycan coat (glycocalyx) comprising its interface with its cellular environment. In vertebrates, the terminal sugars of the glycocalyx are often sialic acids, 9-carbon backbone anionic sugars implicated in intermolecular and intercellular interactions. The vertebrate brain is particularly enriched in sialic acid-containing glycolipids termed gangliosides. Human congenital disorders of ganglioside biosynthesis result in paraplegia, epilepsy, and intellectual disability. To better understand sialoglycan functions in the nervous system, we studied brain anatomy, histology, biochemistry, and behavior in mice with engineered mutations in St3gal2 and St3gal3, sialyltransferase genes responsible for terminal sialylation of gangliosides and some glycoproteins. St3gal2/3 double-null mice displayed dysmyelination marked by a 40% reduction in major myelin proteins, 30% fewer myelinated axons, a 33% decrease in myelin thickness, and molecular disruptions at nodes of Ranvier. In part, these changes may be due to dysregulation of ganglioside-mediated oligodendroglial precursor cell proliferation. Neuronal markers were also reduced up to 40%, and hippocampal neurons had smaller dendritic arbors. Young adult St3gal2/3 double-null mice displayed impaired motor coordination, disturbed gait, and profound cognitive disability. Comparisons among sialyltransferase mutant mice provide insights into the functional roles of brain gangliosides and sialoglycoproteins consistent with related human congenital disorders.—Yoo, S.-W., Motari, M. G., Susuki, K., Prendergast, J., Mountney, A., Hurtado, A., Schnaar, R. L. Sialylation regulates brain structure and function. FASEB J. 29, 3040-3053 (2015). www.fasebj.org
Abstract Tumor-associated carbohydrate antigens (TACAs) historically have been challenging targets for antibody therapeutics. Sialyl-Tn (STn) is a cancer specific antigen that is expressed on the surface of carcinomas including ovarian, colon, prostate, and pancreatic tumors but is rarely present in normal tissue. STn expression has been linked to innate immune suppression, a chemoresistant phenotype, metastasis, and poor prognosis. Previous attempts to target this antigen in the clinic with synthetic glycan vaccines proved safe but lacked efficacy. We have developed humanized bispecific antibodies targeting STn and CD3 for T-cell recruitment and activation at the tumor site. These bispecific antibodies were selected for optimal tumor targeting using our glycan microarray that enriches for candidates whose binding is protein-independent and glycan specific. STn-selective binding was demonstrated. Current lead candidates exhibited low nanomolar EC50 binding in flow cytometric assays against both STn expressing tumor cells and T cells. Quantification of T-cell activation and T-cell induced tumor killing in vitro provides a basis for the further clinical development of these bispecific antibody candidates. Citation Format: David A. Eavarone, Jillian Prendergast, Patricia E. Rao, Jenna Stein, Jeff Behrens, Daniel T. Dransfield. Novel humanized anti-Sialyl-Tn, anti-CD3 bispecific antibodies demonstrate tumor and T-cell specificity for immune activation at the tumor site [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3640. doi:10.1158/1538-7445.AM2017-3640
Gangliosides are major cell-surface determinants on all vertebrate neurons. Human congenital disorders of ganglioside biosynthesis invariably result in intellectual disability and are often associated with intractable seizures. To probe the mechanisms of ganglioside functions, affinity-captured ganglioside-binding proteins from rat cerebellar granule neurons were identified by quantitative proteomic mass spectrometry. Of the six proteins that bound selectively to the major brain ganglioside GT1b (GT1b:GM1 > 4; p < 10 −4 ), three regulate neurotransmitter receptor trafficking: Thorase (ATPase family AAA domain-containing protein 1), soluble N -ethylmaleimide-sensitive factor (NSF) attachment protein (γ-SNAP), and the transmembrane protein Nicalin. Thorase facilitates endocytosis of GluR2 subunit-containing AMPA-type glutamate receptors (AMPARs) in an ATPase-dependent manner; its deletion in mice results in learning and memory deficits (J. Zhang et al., 2011b). GluR2-containing AMPARs did not bind GT1b, but bound specifically to another ganglioside, GM1. Addition of noncleavable ATP (ATPγS) significantly disrupted ganglioside binding, whereas it enhanced AMPAR association with Thorase, NSF, and Nicalin. Mutant mice lacking GT1b expressed markedly higher brain Thorase, whereas Thorase-null mice expressed higher GT1b. Treatment of cultured hippocampal neurons with sialidase, which cleaves GT1b (and other sialoglycans), resulted in a significant reduction in the size of surface GluR2 puncta. These data support a model in which GM1-bound GluR2-containing AMPARs are functionally segregated from GT1b-bound AMPAR-trafficking complexes. Release of ganglioside binding may enhance GluR2-containing AMPAR association with its trafficking complexes, increasing endocytosis. Disrupting ganglioside biosynthesis may result in reduced synaptic expression of GluR2-contianing AMPARs resulting in intellectual deficits and seizure susceptibility in mice and humans.
