sialyl-Tn Expression Research Articles

Structural insights into Siglec-15 reveal glycosylation dependency for its interaction with T cells through integrin CD11b

Sialic acid-binding Ig-like lectin 15 (Siglec-15) is an immune modulator and emerging cancer immunotherapy target. However, limited understanding of its structure and mechanism of action restrains the development of drug candidates that unleash its full therapeutic potential. In this study, we elucidate the crystal structure of Siglec-15 and its binding epitope via co-crystallization with an anti-Siglec-15 blocking antibody. Using saturation transfer-difference nuclear magnetic resonance (STD-NMR) spectroscopy and molecular dynamics simulations, we reveal Siglec-15 binding mode to α(2,3)- and α(2,6)-linked sialic acids and the cancer-associated sialyl-Tn (STn) glycoform. We demonstrate that binding of Siglec-15 to T cells, which lack STn expression, depends on the presence of α(2,3)- and α(2,6)-linked sialoglycans. Furthermore, we identify the leukocyte integrin CD11b as a Siglec-15 binding partner on human T cells. Collectively, our findings provide an integrated understanding of the structural features of Siglec-15 and emphasize glycosylation as a crucial factor in controlling T cell responses.

Abstract 2966: Exploring the therapeutic potential of a novel Siglec-6 targeted bispecific antibody to selectively kill leukemia cells

Abstract Objectives of the Study: Bispecific antibodies (BsAbs) are a class of immunotherapy drugs that facilitate targeted killing of cancer cells. Here, we report therapeutic potential of a novel Siglec-6/CD3 targeted bsAb against chronic lymphocytic leukemia (CLL) cells. We show here for the first time the functional and therapeutic relevance of Siglec-6 in CLL cell adhesion and migration. Mechanistically, we have shown Siglec-6 mediated cell adhesion and migration through Siglec-6 ligand (sialyl Tn) mediated DOCK8 dependent activation of Cdc42 associated with actin polymerization. To evaluate the therapeutic potential of Siglec-6, we generated a human (hu) Siglec-6 transgenic mouse model and crossed it with the TCL1 CLL mouse model to obtain Siglec-6 x TCL1 mice which develop Siglec-6+ leukemia. A pilot in-vivo study with anti-Siglec-6/CD3 targeted bsAb showed a survival benefit in humanized CD3 (huCD3) mice engrafted with Siglec-6+ leukemic cells. Methods used: Flow cytometry was used to analyze cell surface expression of Siglec-6 and sialyl Tn (sTn). Mass spectrometry analysis was performed to identify Siglec-6 interacting proteins. CRISPR/Cas9 technique was used to generate knock-out (KO) cell lines for mechanistic studies. Phalloidin staining followed by confocal imaging was used to examine actin polymerization. For in-vivo BsAb treatment experiments, huCD3 mice were engrafted with 5 million Siglec-6+ leukemic cells isolated from Siglec-6 x TCL1 mouse splenocytes. Weekly treatment (i.v) was started after mice display 5% circulating leukemia. Results and Conclusion: Compared to Siglec-6+ CLL cells, Siglec-6- CLL cells exhibited significant reduction in adhesion to (~50%) and migration towards (~50%) CLL-BMSCs. Importantly, a Siglec-6 targeted antibody inhibited homing of Siglec-6+ MEC1 cells and primary CLL cells to the spleen and bone marrow in NSG mice (~35%). Mass spectrometry and co-immunoprecipitation analysis in MEC1 cells revealed interaction of Siglec-6 with DOCK8, a guanine nucleotide exchange factor. Stimulation of Siglec-6+ MEC1 cells with sTn resulted in Cdc42 activation and WASP protein recruitment, followed by increased actin polymerization, that were compromised in Siglec-6 or DOCK8 KO MEC1 cells. Siglec-6+ leukemia engrafted huCD3 mice treated with anti-Siglec-6/CD3 BsAb has so far demonstrated a survival benefit with median survival of 18 weeks versus a median survival of 8 weeks in the control group that received a non-targeting BsAb (n=4/group). Significance: We have for the first time shown Siglec-6 dependent recruitment of DOCK8 leading to migration and adhesion of B-CLL cells. An anti-Siglec-6/CD3 BsAb provides a survival benefit against Siglec-6+ leukemia, paving the way for development of Siglec-6 targeted therapeutics to treat CLL. Citation Format: Jessica Nunes, Charlene Mao, Matthew Purcell, Elizabeth Perry, Liwen Zhang, Xiaokui Mo, Matthew Cyr, Meixiao Long, John Byrd, Christoph Rader, Natarajan Muthusamy. Exploring the therapeutic potential of a novel Siglec-6 targeted bispecific antibody to selectively kill leukemia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2966.

The protective role of conjunctival goblet cell mucin sialylation

Gel-forming mucins secreted by conjunctival goblet cells have been implicated in the clearance of allergens, pathogens, and debris. However, their roles remain incompletely understood. Here we show that human and mouse conjunctival goblet cell mucins have Alcian blue-detectable sialic acids, but not sulfates in the steady state. Interestingly, Balb/c mouse strain lacks this sialylation due to a point mutation in a sialyltransferase gene, St6galnac1, which is responsible for sialyl-Tn synthesis. Introduction of intact St6galnac1 to Balb/c restores the sialylation of conjunctival goblet cell mucus. Sialylated mucus efficiently captures and encapsulates the allergen particles in an impenetrable layer, leading to the protection of mice from the development of allergic conjunctivitis. Expression of ST6GALNAC1 and sialyl-Tn is upregulated in humans under conditions with chronic stimuli. These results indicate that the sialylated glycans on the ocular mucins play an essential role in maintaining the conjunctival mucosa by protecting from the incoming foreign bodies such as allergen particles.

ST6GalNAc-I promotes lung cancer metastasis by altering MUC5AC sialylation.

Lung cancer (LC) is the leading cause of cancer‐related mortality. However, the molecular mechanisms associated with the development of metastasis are poorly understood. Understanding the biology of LC metastasis is critical to unveil the molecular mechanisms for designing targeted therapies. We developed two genetically engineered LC mouse models KrasG12D/+; Trp53R172H/+; Ad‐Cre (KPA) and KrasG12D/+; Ad‐Cre (KA). Survival analysis showed significantly (P = 0.0049) shorter survival in KPA tumor‐bearing mice as compared to KA, suggesting the aggressiveness of the model. Our transcriptomic data showed high expression of N‐acetylgalactosaminide alpha‐2, 6‐sialyltransferase 1 (St6galnac‐I) in KPA compared to KA tumors. ST6GalNAc‐I is an O‐glycosyltransferase, which catalyzes the addition of sialic acid to the initiating GalNAc residues forming sialyl Tn (STn) on glycoproteins, such as mucins. Ectopic expression of species‐specific p53 mutants in the syngeneic mouse and human LC cells led to increased cell migration and high expression of ST6GalNAc‐I, STn, and MUC5AC. Immunoprecipitation of MUC5AC in the ectopically expressing p53R175H cells exhibited higher affinity toward STn. In addition, ST6GalNAc‐I knockout (KO) cells also showed decreased migration, possibly due to reduced glycosylation of MUC5AC as observed by low STn on the glycoprotein. Interestingly, ST6GalNAc‐I KO cells injected mice developed less liver metastasis (P = 0.01) compared to controls, while colocalization of MUC5AC and STn was observed in the liver metastatic tissues of control mice. Collectively, our findings support the hypothesis that mutant p53R175H mediates ST6GalNAc‐I expression, leading to the sialyation of MUC5AC, and thus contribute to LC liver metastasis.

Target Score-A Proteomics Data Selection Tool Applied to Esophageal Cancer Identifies GLUT1-Sialyl Tn Glycoforms as Biomarkers of Cancer Aggressiveness.

Esophageal cancer (EC) is a life-threatening disease, demanding the discovery of new biomarkers and molecular targets for precision oncology. Aberrantly glycosylated proteins hold tremendous potential towards this objective. In the current study, a series of esophageal squamous cell carcinomas (ESCC) and EC-derived circulating tumor cells (CTCs) were screened by immunoassays for the sialyl-Tn (STn) antigen, a glycan rarely expressed in healthy tissues and widely observed in aggressive gastrointestinal cancers. An ESCC cell model was glycoengineered to express STn and characterized in relation to cell proliferation and invasion in vitro. STn was found to be widely present in ESCC (70% of tumors) and in CTCs in 20% of patients, being associated with general recurrence and reduced survival. Furthermore, STn expression in ESCC cells increased invasion in vitro, while reducing cancer cells proliferation. In parallel, an ESCC mass spectrometry-based proteomics dataset, obtained from the PRIDE database, was comprehensively interrogated for abnormally glycosylated proteins. Data integration with the Target Score, an algorithm developed in-house, pinpointed the glucose transporter type 1 (GLUT1) as a biomarker of poor prognosis. GLUT1-STn glycoproteoforms were latter identified in tumor tissues in patients facing worst prognosis. Furthermore, healthy human tissues analysis suggested that STn glycosylation provided cancer specificity to GLUT1. In conclusion, STn is a biomarker of worst prognosis in EC and GLUT1-STn glycoforms may be used to increase its specificity on the stratification and targeting of aggressive ESCC forms.

Phenotypic Analysis of Urothelial Exfoliated Cells in Bladder Cancer via Microfluidic Immunoassays: Sialyl-Tn as a Novel Biomarker in Liquid Biopsies.

Bladder cancer is the most common malignancy of the urinary tract, having one of the highest recurrence rates and progression from non-muscle to muscle invasive bladder cancer that commonly leads to metastasis. Cystoscopy and urine cytology are the standard procedures for its detection but have limited clinical sensitivity and specificity. Herein, a microfluidic device, the UriChip, was developed for the enrichment of urothelial exfoliated cells from fresh and frozen urine, based on deformability and size, and the cancer-associated glycan Sialyl-Tn explored as a putative bladder cancer urinary biomarker. Spiking experiments with bladder cancer cell lines showed an isolation efficiency of 53%, while clinical sample analyses revealed retention of cells with various morphologies and sizes. in situ immunoassays demonstrated significantly higher number of Sialyl-Tn-positive cells in fresh and frozen voided urine from bladder cancer patients, compared to healthy individuals. Of note, urothelial exfoliated cells from cryopreserved urine sediments were also successfully isolated by the UriChip, and found to express significantly high levels of Sialyl-Tn. Remarkably, Sialyl-Tn expression is correlated with tumor stage and grade. Overall, our findings demonstrate the potential of UriChip and Sialyl-Tn to detect urothelial bladder cancer cells in follow-up and long-term retrospective studies.

Systematic review and meta-analysis of the clinical survival significance of Sialyl-Tn expression in histological tissues from cancer patients.

BackgroundThe expression of Sialyl-Tn (STn) has been associated with different mechanisms of cancer. Several studies using histological tissue samples from different types of cancer showed differences in the STn expression patterns, but few studies have explored the association between STn expression and decreased overall survival. Therefore, a comprehensive analysis of the clinical significance of STn in cancer patients was performed.MethodsA systematic review and meta-analysis were done of scientific articles published and available from different sources, including MEDLINE/PubMed, Web of Science, Cochrane Library and LILACS. Odds ratio (OR) was represented by the number of deaths during follow-up in positive-STn versus negative-STn patients. OR from each study was pooled and the results were presented in a forest plot.ResultsEleven articles were included in the systematic meta-analysis. The estimated OR of 3.26 (95% CI: 2.12–5.02) was obtained. Furthermore, subgroups studies of gastric cancer showed an OR of 3.74 (95% CI: 2.47–5.66) and other cancer types reported an OR of 2.92 (95% CI: 1.16–7.32).ConclusionsThis study showed that survival time was significantly longer in patients with negative-STn tumors than in those with positive results.

Breast cancer cells expressing cancer-associated sialyl-Tn antigen have less capacity to develop osteolytic lesions in a mouse model of skeletal colonization.

Breast cancer is one of the most prevalent malignancies in women, and approximately 75-80% of patients with advanced breast cancer develop bone metastasis. Expression of the cancer-associated carbohydrate antigen sialyl-Tn (STn) in breast cancer is associated with a poor prognosis; however, involvement of STn in the development of metastatic bone lesions remains unclear. We investigated whether STn expression on breast cancer cells influences intraosseous tumor growth and bone response in mice models of skeletal colonization. STn-positive (STn+) breast cancer cells were generated by stable transfection of an expression vector encoding ST6GaLNAc I into the breast cancer cell line MDA-MB-231. Parental MDA-MB-231 cells not expressing STn antigen were used as STn-negative (STn-) breast cancer cells. Contrary to expectations, STn expression attenuated the development of destructive bone lesions in the in vivo mice models. An in vitro study demonstrated that STn expression impaired adhesion of MDA-MB-231cells to bone marrow stromal cells. This finding in vitro was also confirmed by another breast cancer cell line MCF-7. Cell adhesion to fibronectin and type I collagen was also impaired in STn+ MDA-MB-231 cells compared to that in STn- MDA-MB-231 cells, suggesting integrin dysfunction. Given that the integrin β1 subunit is the main carrier of the STn epitope, it is likely that changes in glycan structure impaired the adhesive capacity of β1 integrin in the bone environment, leading to attenuation of tumor cell engraftment. In conclusion, breast cancer cells expressing STn antigen had less capacity for skeletal colonization, possibly due to impaired adhesive capability.

Truncated O-glycans promote epithelial-to-mesenchymal transition and stemness properties of pancreatic cancer cells.

Aberrant expression of Sialyl‐Tn (STn) antigen correlates with poor prognosis and reduced patient survival. We demonstrated that expression of Tn and STn in pancreatic ductal adenocarcinoma (PDAC) is due to hypermethylation of Core 1 synthase specific molecular chaperone (COSMC) and enhanced the malignant properties of PDAC cells with an unknown mechanism. To explore the mechanism, we have genetically deleted COSMC in PDAC cells to express truncated O‐glycans (SimpleCells, SC) which enhanced cell migration and invasion. Since epithelial‐to‐mesenchymal transition (EMT) play a vital role in metastasis, we have analysed the induction of EMT in SC cells. Expressions of the mesenchymal markers were significantly high in SC cells as compared to WT cells. Equally, we found reduced expressions of the epithelial markers in SC cells. Re‐expression of COSMC in SC cells reversed the induction of EMT. In addition to this, we also observed an increased cancer stem cell population in SC cells. Furthermore, orthotopic implantation of T3M4 SC cells into athymic nude mice resulted in significantly larger tumours and reduced animal survival. Altogether, these results suggest that aberrant expression of truncated O‐glycans in PDAC cells enhances the tumour aggressiveness through the induction of EMT and stemness properties.

Expression of bladder cancer-associated glycans in murine tumor cell lines.

The characterization of murine cell lines is of great importance in order to identify preclinical models that could resemble human diseases. Aberrant glycosylation includes the loss, excessive or novel expression of glycans and the appearance of truncated structures. MB49 and MB49-I are currently the only two murine cell lines available for the development of preclinical bladder cancer models. The glycans Lewis X (LeX), Sialyl lewis X (SLeX) and Sialyl Tn (STn) have previously been associated with aggressiveness, dissemination and poor prognosis in human bladder cancer, additionally N-glycolyl GM3 (NGcGM3) is a neo-antigen expressed in many types of tumors; however, to the best of our knowledge, its expression has not previously been assessed in this type of cancer. Taking into account the relevance of glycans in tumor biology and considering that they can act as targets of therapies and biomarkers, the present study evaluated the expression of LeX, SLeX, STn and NGcGM3 in MB49 and MB49-I cells, in different growth conditions such as monolayer cultures, three-dimensional multicellular spheroids and mouse heterotopic and orthotopic tumors. The expression of LeX was not detected in either cell line, whereas SLeX was expressed in monolayers, spheroids and orthotopic tumors of both cell lines. STn was only identified in MB49 monolayers and spheroids. There are no reports concerning the expression of NGcGM3 in human or murine bladder cancer. In our hands, MB49 and MB49-I expressed this ganglioside in all the growth conditions evaluated. The assessment of its expression in cancer cell lines and patient tumors is of great importance, considering the relevance of this ganglioside in tumor biology. The data obtained by the present study demonstrates that glycan expression may be substantially altered depending on the growth conditions, highlighting the importance of the characterization of murine cancer models. To the best of our knowledge, the present study is the first to examine the expression of cancer-associated glycans, in the two murine cell lines available for the development of preclinical studies in bladder cancer.

Exploring sialyl-Tn expression in microfluidic-isolated circulating tumour cells: A novel biomarker and an analytical tool for precision oncology applications

Circulating tumour cells (CTCs) originating from a primary tumour, lymph nodes and distant metastases hold great potential for liquid biopsies by providing a molecular fingerprint for disease dissemination and its temporal evolution through the course of disease management. CTC enumeration, classically defined on the basis of surface expression of Epithelial Cell Adhesion Molecule (EpCAM) and absence of the pan-leukocyte marker CD45, has been shown to correlate with clinical outcome. However, existing approaches introduce bias into the subsets of captured CTCs, which may exclude biologically and clinically relevant subpopulations. Here we explore the overexpression of the membrane protein O-glycan sialyl-Tn (STn) antigen in advanced bladder and colorectal tumours, but not in blood cells, to propose a novel CTC isolation technology. Using a size-based microfluidic device, we show that the majority (>90%) of CTCs isolated from the blood of patients with metastatic bladder and colorectal cancers express the STn antigen, supporting a link with metastasis. STn+ CTC counts were significantly higher than EpCAM-based detection in colorectal cancer, providing a more efficient cell-surface biomarker for CTC isolation. Exploring this concept, we constructed a glycan affinity-based microfluidic device for selective isolation of STn+ CTCs and propose an enzyme-based strategy for the recovery of viable cancer cells for downstream investigations. Finally, clinically relevant cancer biomarkers (transcripts and mutations) in bladder and colorectal tumours, were identified in cells isolated by microfluidics, confirming their malignant origin and highlighting the potential of this technology in the context of precision oncology.

Humanized anti-Sialyl-Tn antibodies for the treatment of ovarian carcinoma.

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 5625: Myeloid derived suppressor cells (MDSCs) express Sialyl Tn (STn) and are a therapeutic target for anti-STn antibody drug conjugates

Abstract Introduction: Myeloid derived suppressor cells (MDSCs) are functionally defined by their capacity to suppress T cell immunity; therefore inhibiting these cells is of great interest for immuno-oncology applications. STn, the sialylated version of the carbohydrate Tn antigen, is broadly expressed in human cancers such as breast, ovarian, bladder, cervical, colon, and lung and is rarely expressed in normal adult human tissues. The presence of STn in tumors is associated with metastatic disease, poor prognosis, and reduced overall survival. Tumor STn expression is well established and can be leveraged for targeted cancer therapy, however its expression on immuno-suppressive tumor infiltrating lymphocytes such as MDSCs has not been established. Methods: We generated humanized anti-STn mAbs having no cross-reactivity to the asialylated form of STn (Tn) or other glycan antigens. The epitope targeted by these mAbs is the STn glycan itself, not a particular glycopeptide or carrier protein, which offers the broadest potential to bind to multiple STn-glycosylated proteins on cell surfaces. We used these mAbs to quantify specifically the expression of tumor and MDSC STn expression across a set of patient tumor samples. We further treated mice with a humanized anti-STn-MMAE antibody-drug conjugate to evaluate targeted depletion of STn+ MDSCs. In order to evaluate the therapeutic potential of targeting this MDSC phenotype, we further evaluated the immunosuppressive environment of tumors containing STn+ and STn- MDSCs. Results: For the first time, we report the detection of STn on the surface of MDSCs in a growing body of patient tumor samples. Our data in patients as well as xenograft models additionally links STn expression on MDSCs to tumor cell STn expression. We have also demonstrated depletion of STn+ MDSCs after treatment with an anti-STn antibody-drug conjugate in an ovarian carcinoma xenograft model. Conclusions: Our data demonstrated that STn provides a uniquely glycan-specific and potent target for treatment of solid tumors. Anti-STn therapeutics offer the potential to go beyond tumor targeting to also directly target and deplete immune-suppressive MDSCs, thus fostering immune re-engagement and increasing the potential for better patient outcomes. Citation Format: David A. Eavarone, Patricia E. Rao, Jillian M. Prendergast, Adam D. Curtis, Rong Zhang, Lindsay S. Shopland, Jenna N. Stein, Jeff Behrens, Daniel T. Dransfield. Myeloid derived suppressor cells (MDSCs) express Sialyl Tn (STn) and are a therapeutic target for anti-STn antibody drug conjugates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5625.

Abstract 5055: Expression of Tn and SialylTn tumor antigens in intestinal epithelia in APC-mutant mice promotes colorectal carcinogenesis and progression

Abstract Colorectal cancer (CRC) is a leading cause of cancer deaths in the Western world, and several different animal models for CRC have been established to understand its initiation, progression, and metastasis. Mice with mutations in APC (APCm) is widely accepted in the field because APC is the predominant mutated gene in human CRC. Mice carrying APCm only develop multiple intestinal neoplasia, but not carcinoma. Importantly, emerging evidence shows that tumor associated carbohydrate antigens, Tn and sialylTn (STn) appear at an early stage of colon carcinogenesis, and are associated with progression and metastasis. However, the molecular mechanisms underlying the role of Tn/STn antigens in CRC tumorigenesis and progression are unknown. We identified a novel X-linked gene Cosmc (C1GalT1C1) which encodes a molecular chaperone Cosmc regulating O-glycosylation through assisting correct folding of core 1 β3-galactosyltransferase (T-synthase, C1GalT1). T-synthase is the key enzyme that converts Tn antigen to normal core 1 O-glycans on glycoproteins in all animal cells. Cosmc mutations lead to loss of T-synthase activity and resultant expression of Tn/STn antigens in human carcinoma cells and in other diseases. Moreover, in our study, intestinal epithelial cell (IEC)-specific Cosmc-knockout (IEC-Cosmc-KO) mice spontaneously developed invasive tumors mainly in the rectum from 3-12 months of age through complex pathways. To further investigate the role of Tn/STn antigens in CRC formation, progression and metastasis in a context of APC mutations, we generated IEC-Cosmcnull:APC-/+ (double-knockout, DKO) by gene targeting. Totally, 22 DKO mice (13 male and 9 female) and similar numbers of control mouse groups (APC-KO, Cosmc-KO and WT) were generated. Among them, the DKO male and female mice grew slower companied with stress signs and some mortalities around 3-4 months of age. Importantly, both APC-KO and DKO mice had multiple gross tumors in the small intestine, and all DKO mice had more and larger gross tumors in colorectum while only 15% of APC-KO mice developed tumors with smaller sizes in colon. Consistent with our findings, thickened rectum was observed in Cosmc-KO mice. Furthermore, the enlarged inguinal lymph nodes were also found in the DKO mice. Histology confirmed the Tn/STn expression, as well as adenomas and adenocarcinomas in the DKOs, and adenomas in APC-KOs and Cosmc-KO male mice. The distant metastasis of the CRC was not yet observed in these young DKO-mice, since it may require a longer time to develop. These results clearly indicate that the Cosmc deletion resulted in Tn and STn expression, promotes the colorectal carcinogenesis in APC-mutant mice. We are generating more elder DKO and control mice to continue investigating the role of Tn/STn in CRC progression and metastasis by performing detailed molecular analyses. Citation Format: Yuliang Jiang, Su-Ryun Kim, Shweta Kotian, Sean R. Stowell, Guangyu An, Tongzhong Ju. Expression of Tn and SialylTn tumor antigens in intestinal epithelia in APC-mutant mice promotes colorectal carcinogenesis and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5055.

Analysis of sialyl-Lewis x on MUC5AC and MUC1 mucins in pancreatic cancer tissues

Pancreatic adenocarcinoma (PDAC) lacks efficient biomarkers. Mucins are glycoproteins that can carry aberrant glycosylation in cancer. Our objective was to identify cancer-related glycan epitopes on MUC1 and MUC5AC mucins in PDAC as potential biomarkers. We have analysed the tumour-associated carbohydrate antigens sialyl-Lewis x (SLex) and sialyl-Tn (STn) on MUC1 and MUC5AC in PDAC tissues. The selected cohort for this study consisted of twenty-one PDAC tissues positive for SLex antigen and three normal pancreas specimens as controls. STn expression was shown in 76% of the PDAC tissues. MUC1 and MUC5AC were detected in 90% of PDAC tissues. We performed in situ proximity ligation assay combining antibodies against mucins and glycan epitopes to identify specific mucin glycoforms. MUC1-SLex and MUC5AC-SLex were found in 68% and 84% respectively, of the mucin expressing PDAC tissues, while STn hardly colocalized with any of the evaluated mucins. Further analysis by Western blot of MUC5AC and SLex in eight PDAC tissue lysates showed that six out of eight cases were positive for both markers. Moreover, immunoprecipitation of MUC5AC from positive PDAC tissues and subsequent SLex immunodetection confirmed the presence of SLex on MUC5AC. Altogether, MUC5AC-SLex glycoform is present in PDAC and can be regarded as potential biomarker.

ST6GALNAC1 plays important roles in enhancing cancer stem phenotypes of colorectal cancer via the Akt pathway.

Colorectal cancer (CRC) is a mortal disease due to treatment resistance, recurrence and distant metastasis. Emerging evidence has revealed that a small sub-population of cancer cells termed cancer stem cells (CSCs)/ cancer-initiating cells (CICs) is endowed with high levels of tumor-initiating ability, self-renewal ability and differentiation ability and is responsible for treatment resistance, recurrence and distant metastasis. Eradication of CSCs/CICs is essential to improve current treatments. However, the molecular mechanisms by which CSCs/CICs are maintained are still elusive. In this study, we aimed to determine the molecular mechanisms by which colorectal (CR)-CSCs/CICs in are maintained human primary CRC cells. CR-CSCs/CICs were isolated by sphere-culture and the ALDEFLUOR assay, and transcriptome analysis revealed that the gene ST6 N-Acetylgalactosaminide Alpha-2,6-Sialyltransferase 1 (ST6GALNAC1) was expressed at high levels in CR-CSCs/CICs. Overexpression of ST6GALNAC1 enhanced the expression of sialyl-Tn (STn) antigen, which is carried by the CSC marker CD44, and increased the sphere-forming ability and resistance to a chemotherapeutic reagent. The opposite phenomena were observed by gene knockdown using siRNA. Furthermore, the Akt pathway was activated in ST6GANAC1-overexpressed cells, and activation of the pathway was cancelled by gene knockdown of galectin-3. The results indicate that ST6GALNAC1 has a role in the maintenance of CR-CSCs/CICs by activating the Akt pathway in cooperation with galectin-3 and that ST6GalNAC1 (or STn antigen) might be a reasonable molecule for CSC/CIC-targeting therapy.

Sialyl-Tn identifies muscle-invasive bladder cancer basal and luminal subtypes facing decreased survival, being expressed by circulating tumor cells and metastases

ObjectivesTo evaluate the potential of sialyl-Tn (STn), a cancer-associated glycan antigen present in membrane glycoproteins, to improve a recent molecular model for stratification and prognostication of advanced stage bladder tumors based on keratins (KRT14, 5, and 20) expression. In addition, determine the association between STn and disease dissemination based on the evaluation of circulating tumor cells (CTCs) and the metastasis, which is a critical matter to improve patient management. Patients and methodsA retrospective series of 80 muscle-invasive primary bladder tumors and associated metastasis were screened for KRT14, 5, and 20 and STn by real-time polymerase chain reaction and immunohistochemistry. Peripheral blood was collected in a patients’ subset, CTCs were isolated through a size-based microfluidic chip and screened for KRTs and STn. ResultsBasal-like lesions presented worse cancer-specific and disease-free survival compared to luminal tumors. STn antigen inclusion discriminated patients with worst survival in each subgroup (P = 0.047 for luminal; P = 0.027 for basal-like tumors). STn expression in CTCs and distant metastasis was also demonstrated. ConclusionThis work reinforces the potential of the KRT-based model for bladder cancer management and the association of STn with aggressiveness, supporting its inclusion in predictive molecular models toward patient-tailored precision medicine. Moreover, we describe for the first time that CTCs and the metastasis present a basal phenotype and express the STn antigen, highlighting its link with disease dissemination. Future studies should focus on determining the biological and clinical significance of these observations in the context of liquid biopsies. Given the membrane nature of STn, highly specific targeted therapeutics may also be envisaged.

Abstract 36: Novel anti-Sialyl-Tn monoclonal antibodies and antibody-drug conjugates (ADCs) demonstrate tumor specificity in vitro and in vivo antitumor efficacy

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 cell 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 highly selective humanized monoclonal antibodies and antibody drug conjugates (ADCs) targeting TACAs, such as STn. Remarkable sequence homology across all anti-STn mAbs was observed in both heavy and light chains, and hot spots for hypermutation were identified. These antibodies were selected using our glycan microarray that enriches for candidates whose binding is protein-independent, highly selective and demonstrates exceptional target affinity. Lead humanized candidates demonstrated single digit nanomolar EC50s in ELISA/flow cytometric assays, STn selective cell internalization, and STn specific glycan binding on Siamab’s proprietary glycan array. STn binding sites in common tumor lines (ovarian, gastric and breast) were determined per cell and subsequent cytotoxicity assays in these lines demonstrated in vitro efficacy. Tumor microarray experiments revealed membranous staining in cancerous tissues of various indications. Binding studies of anti-STn antibodies to primary human cancer samples by flow cytometry demonstrated that both tumor and Myeloid-Derived Suppressor Cells (MDSC, both myeloid and granulocytic) express STn. In an OVCAR3 xenograft model, 30 days after the last anti-STn ADC dose was given, groups treated (Q7Dx4) exhibited mean tumor volumes below the Day 1 pre-treatment mean tumor volumes (155mm3). Flow cytometric analysis of tumors from these mice demonstrated that anti-STn ADC treatment reduces STn expression on the primary tumor in a dose-dependent manner (Q7Dx4 vs. single dose) compared to the isotype-ADC control. Our data demonstrates that high-affinity, STn-selective mAbs show promise as therapies for solid tumors and could also target MDSCs to promote antitumor immune responses. Citation Format: Jillian M. Prendergast, David A. Eavarone, Patricia E. Rao, Adam D. Curtis, Lindsay S. Shopland, Todd A. Hoffert, Jenna Stein, Jeff Behrens, Daniel T. Dransfield. Novel anti-Sialyl-Tn monoclonal antibodies and antibody-drug conjugates (ADCs) demonstrate tumor specificity in vitro and in vivo antitumor efficacy [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 36. doi:10.1158/1538-7445.AM2017-36

Abstract 3640: Novel humanized anti-Sialyl-Tn, anti-CD3 bispecific antibodies demonstrate tumor and T-cell specificity for immune activation at the tumor site

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

Mucoepidermoid carcinoma-associated expression of MUC5AC, MUC5B and mucin-type carbohydrate antigen sialyl-Tn in the parotid gland

ObjectivesThe aberrant expression of mucins and mucin-type carbohydrates has been described in many types of cancer, including mucoepidermoid carcinoma (MEC), a malignant salivary gland tumor. In this study, we examined the aberrant expression patterns of mucins (MUC1, MUC4, MUC5AC and MUC5B), simple mucin-type carbohydrate antigens (Tn, sialyl-Tn and T) and mature carbohydrate antigens (Lewisa and sulfo-Lewisa antigens) in MEC originating from the parotid gland, which normally does not secrete mucins. DesignWe conducted an immunohistochemical study to investigate the presence of mucins and carbohydrates in 24 MEC samples originating from the parotid gland and in surrounding normal tissue of the same gland in comparison 6 samples of normal salivary glands. The expression levels were compared with respect to the histological grading. Furthermore, 24 MEC samples from non-parotid salivary glands were included. ResultsWe observed loss of topology of membrane-bound MUC1 and MUC4, and de novo expression of MUC5AC, MUC5B and sialyl-Tn in MEC that originated in the parotid gland. Furthermore, mucins MUC1, MUC4 and carbohydrate antigens Tn, sialyl-Tn, T, Lewisa and sulfo-Lewisa were overexpressed in MEC samples compared to surrounding normal salivary gland tissues. MUC1 was expressed in both low- and high grade MECs, whereas MUC4 was not expressed in high grade MECs of the parotid gland. ConclusionDuring the development of MEC in the parotid gland, the genes for gel-forming secretory mucins are switched on. Besides these MEC tissues overexpress short oligosaccharides, suggesting that the glycosylation machinery is altered.