Sialyl Tn Research Articles

A combined computational-experimental approach to define the structural origin of antibody recognition of sialyl-Tn, a tumor-associated carbohydrate antigen

Anti-carbohydrate monoclonal antibodies (mAbs) hold great promise as cancer therapeutics and diagnostics. However, their specificity can be mixed, and detailed characterization is problematic, because antibody-glycan complexes are challenging to crystallize. Here, we developed a generalizable approach employing high-throughput techniques for characterizing the structure and specificity of such mAbs, and applied it to the mAb TKH2 developed against the tumor-associated carbohydrate antigen sialyl-Tn (STn). The mAb specificity was defined by apparent KD values determined by quantitative glycan microarray screening. Key residues in the antibody combining site were identified by site-directed mutagenesis, and the glycan-antigen contact surface was defined using saturation transfer difference NMR (STD-NMR). These features were then employed as metrics for selecting the optimal 3D-model of the antibody-glycan complex, out of thousands plausible options generated by automated docking and molecular dynamics simulation. STn-specificity was further validated by computationally screening of the selected antibody 3D-model against the human sialyl-Tn-glycome. This computational-experimental approach would allow rational design of potent antibodies targeting carbohydrates.

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.

Syntheses and Immunological Evaluation of Self-Adjuvanting Clustered N-Acetyl and N-Propionyl Sialyl-Tn Combined with a T-helper Cell Epitope as Antitumor Vaccine Candidates.

Sialyl-Tn (STn) is a tumor-associated carbohydrate antigen (TACA) rarely observed on healthy tissues. We synthesized two fully synthetic N-acetyl and N-propionyl STn trimer (triSTn) vaccines possessing a T-helper epitope and a TLR2 agonist, since the clustered STn antigens are highly expressed on many cancer cells. Immunization of both vaccines in mice induced the anti-triSTn IgG antibodies, which recognized triSTn-expressing cell lines PANC-1 and HepG2. The N-propionyl triSTn vaccine induced the triSTn-specific IgGs, while IgGs induced by the N-acetyl triSTn vaccine were less specific. These results illustrated that N-propionyl triSTn is a valuable unnatural TACA for anticancer vaccines.

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.

Sialyl Tn Unit with TFA-Labile Protection Realizes Efficient Synthesis of Sialyl Glycoprotein.

Amino acids bearing 4-methylbenzyl (MBn) and 4-methoxybenzyl (MPM)-protected sialic acid were synthesized and used for the 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase synthesis of a glycopeptide. The α-sialyl linkage of the MBn-protected unit was partially cleaved under the final deprotection by trifluoroacetic acid (TFA). In addition, the removal of several MBn groups were incomplete. On the other hand, the MPM-protected unit gave the desired glycopeptide without decomposition of the α-sialyl linkage. Using this unit, peptide thioesters of the tandem repeat unit of MUC1 mucin were synthesized by the N-alkylcysteine (NAC) method and used for the one-pot ligation by the thioester method. As a result, the three tandem repeats of MUC1 carrying sialyl Tn antigens were successfully synthesized.

Correlation of serum sialyl Tn antigen values determined by immunoassay and SRM based method

We previously identified four glycan tumor marker candidates using a HPLC-based method. One candidate was sialyl Tn (STN), NeuAcα2-6-GalNAc. In this study, glycans were prepared from sera by hydrazine treatment followed by fluorescent labeling with aminopyridine. Pyridylaminated-STN levels of 147 gastric cancer, 85 pancreatic cancer and 10 cholangiocarcinoma patients together with 102 normal controls were accurately quantified using HPLC separation followed by selected reaction monitoring (SRM) assay, which used a stable isotope, tetradeuterium-labeled pyridylamino glycan as an internal standard. Additionally, STN values were also quantified using conventional competitive inhibition radioimmunoassay (RIA). The two STN levels determined by RIA and SRM gave a similar distribution pattern in sera. STN levels were increased in sera from cancer patients compared to those from normal controls. Moreover, the STN levels in sera of cancer patients determined by the two different assay procedures showed a good correlation (i.e., correlation coefficient >0.9). Our results suggest it may be better to determine STN levels using SRM instead of RIA.

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

Abstract MIP-071: TARGETING A CHEMORESISTANT OVARIAN CANCER CELL POPULATION VIA THE CARBOHYDRATE ANTIGEN SIALYL TN

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.

A cancer vaccine based on fluorine-modified sialyl-Tn induces robust immune responses in a murine model.

Development of an effective vaccine to target tumor associated carbohydrate antigens, aberrantly expressed on the cell surface of various carcinomas, is an appealing approach toward cancer immunotherapy. However, a major problem of carbohydrate antigens is their poor immunogenicity. Immunization with modified-carbohydrate antigens could improve the immunogenicity and induce cross reaction with the native carbohydrate antigens. In this study, we investigated the antitumor ability of three fluoro-substituted sialyl-Tn (STn) analogues (2, 3, 4) coupled to KLH (keyhole limpet hemocyanin) and studied the mechanism of tumor immunotherapy of the vaccines in a murine model of colon cancer. Vaccination with 4-KLH, in which the two N-acetyl groups of STn are substituted with N-fluoroacetyl groups, could remarkably prolong the survival of tumor-bearing mouse and resulted in a significant reduction in tumor burden of lungs compared with STn-KLH (1-KLH). The vaccine 4-KLH could provoke stronger cytotoxic T lymphocytes immune response, T helper (Th) cell-mediated immune response and an earlier-stage Th1 immune response than 1-KLH, thus breaking immune tolerance and generating a therapeutic response. The 4-KLH vaccine induced strong tumor-specific anti-STn antibodies which could mediate complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity against human tumor cells. Moreover, in the absence of adjuvant, 4-KLH still elicited stronger immune responses than 1-KLH. Our data suggested that 4-KLH is superior in tumor prevention. The strategic hapten fluorination may be a potential approach applicable to the vaccines development for the cancer immunotherapy.

O‐Glycan recognition and function in mice and humans

Glycans function both as direct recognition molecules, where they are bound by a wide variety of glycan‐binding proteins (GBPs), including animal cell receptors, microbial adhesins lectins and toxins, and indirectly glycans function in glycoproteins to facilitate intra‐ and intermolecular interactions that maintain homeostasis and biological integrity. To explore both direct and indirect functions of glycans we have engineered mutations in biosynthetic pathways, most notably in regard to O‐glycan biosynthesis (O‐GalNAc glycans). Normal synthesis of such O‐glycans in animals requires the action of the T‐synthase, a b3‐galactosyltransferase that generates the core 1 O‐glycan Galb1‐3GalNAca1‐Ser/Thr from the Tn antigen precursor GalNAca1‐Ser/Thr. Such normal extended O‐glycans, including those with the core 2 O‐glycan structure and sialyl Lewis x antigen, are recognized by key GBPs, including P‐selectin and L‐selectin. Such specific recognition of O‐glycans by P‐selectin, CLEC2, and other GBPs, directly regulated many cellular interactions and signaling pathways important in development, morphogenesis and organ development. In molecular and biochemical studies we have explored such recognition by generating glycopeptides with specific modifications, including O‐glycans and sulfated tyrosine residues. In terms of additional functions of O‐glycans, we have exploited our discovery that the T‐synthase requires a specific protein chaperone, termed Cosmc, encoded on the X‐chromosome. Cosmc, a resident protein in the endoplasmic reticulum, directly interacts with the newly synthesized T‐synthase to facilitate folding to the active form of the enzyme, that subsequently moves to its functional location in the Golgi apparatus. Thus, Cosmc is the key regulator of Tn antigen expression, and loss of Cosmc, through epigenetic silencing, gene mutations and other pathways, and acquired changes in Cosmc function cause Tn expression in many human tumors and tumor cell lines and in Tn syndrome patients. Targeted deletion of Cosmc leads to loss of normal O‐glycans, expression of the Tn and sialyl Tn antigens, and multiple pathological consequences. In this presentation the loss of Cosmc has been explored in the gastrointestinal tract, where it leads to pathology, chronic inflammation, and changes in the gut microbiome, and in other cell types, such as T cells and B cells, where loss of Cosmc alters cellular maturation and functionalities. Our studies are revealing the multiple ways in which O‐glycans in humans, animals, and other organisms interact within the larger protein‐glycan interactome to regulate biological processes.Support or Funding InformationThis work was supported by NIH grants U01CA168930 and P41GM103694 to R.D.C.

Targeted O‐glycoproteomics explored increased sialylation and identified MUC16 as a poor prognosis biomarker in advanced‐stage bladder tumours

Bladder carcinogenesis and tumour progression is accompanied by profound alterations in protein glycosylation on the cell surface, which may be explored for improving disease management. In a search for prognosis biomarkers and novel therapeutic targets we have screened, using immunohistochemistry, a series of bladder tumours with differing clinicopathology for short‐chain O‐glycans commonly found in glycoproteins of human solid tumours. These included the Tn and T antigens and their sialylated counterparts sialyl‐Tn(STn) and sialyl‐T(ST), which are generally associated with poor prognosis. We have also explored the nature of T antigen sialylation, namely the sialyl‐3‐T(S3T) and sialyl‐6‐T(S6T) sialoforms, based on combinations of enzymatic treatments. We observed a predominance of sialoglycans over neutral glycoforms (Tn and T antigens) in bladder tumours. In particular, the STn antigen was associated with high‐grade disease and muscle invasion, in accordance with our previous observations. The S3T and S6T antigens were detected for the first time in bladder tumours, but not in healthy urothelia, highlighting their cancer‐specific nature. These glycans were also overexpressed in advanced lesions, especially in cases showing muscle invasion. Glycoproteomic analyses of advanced bladder tumours based on enzymatic treatments, Vicia villosa lectin‐affinity chromatography enrichment and nanoLC‐ESI‐MS/MS analysis resulted in the identification of several key cancer‐associated glycoproteins (MUC16, CD44, integrins) carrying altered glycosylation. Of particular interest were MUC16 STn+‐glycoforms, characteristic of ovarian cancers, which were found in a subset of advanced‐stage bladder tumours facing the worst prognosis. In summary, significant alterations in the O‐glycome and O‐glycoproteome of bladder tumours hold promise for the development of novel noninvasive diagnostic tools and targeted therapeutics. Furthermore, abnormal MUC16 glycoforms hold potential as surrogate biomarkers of poor prognosis and unique molecular signatures for designing highly specific targeted therapeutics.

IgG Antibody Response Elicited by a Fully Synthetic Two-Component Carbohydrate-Based Cancer Vaccine Candidate with α-Galactosylceramide as Built-in Adjuvant.

A fully synthetic self-adjuvanting cancer vaccine candidate was constructed through covalent conjugation of invariant natural killer T (iNKT) cell ligand α-galactosylceramide (αGalCer) with sialyl Tn (STn), a representative tumor-associated carbohydrate antigen (TACA). This two-component vaccine STn-αGalCer is devoid of antigenic peptide, featuring the well-defined structure with high simplicity. STn-αGalCer showed remarkable efficacy in inducing antibody class switching from IgM to STn-specific IgG. Subtypes of IgG antibody were primarily IgG1 and IgG3.

Separation of Two Distinct O-Glycoforms of Human IgA1 by Serial Lectin Chromatography Followed by Mass Spectrometry O-Glycan Analysis.

Human immunoglobulin A1 (IgA1), which carries four to six mucin-type O-glycans (O-glycans) on its hinge region (HR), is the most abundant O-glycoprotein in plasma or serum. While normal O-glycans from hematopoietic-originated cells are core 1-based complex structures, many reports showed that the IgA1 from patients with IgA nephropathy (IgAN) carries undergalactosylated or truncated O-glycans such as the Tn antigen and its sialylated version the SialylTn (STn) antigen on the HR. Yet, there is still a debate whether Tn/STn on the HR of IgA1 is specific to the IgA1 from patients with IgAN since these antigens have also been seen in serum IgA1 of healthy individuals. An additional question is whether the O-glycans at all sites on the two HRs of one IgA1 molecule are homogeneous (either all normal or all Tn/STn) or heterogeneous (both normal and Tn/STn O-glycans). To address these questions, we conducted a systematic study on the O-glycans of plasma IgA1 from both IgAN patients and healthy controls using serial HPA and PNA lectin chromatography followed by western blotting and further analysis of O-glycans from HPA-bound and PNA-bound IgA1 fractions by mass spectrometry. Unexpectedly, we found that a variable minor fraction of IgA1 from both IgAN patients and healthy controls had Tn/STn antigens, and that the O-glycoprotein IgA1 molecules from most samples had only two distinct O-glycoforms: one major glycoform with homogeneous normal core 1-based O-glycans and one minor glycoform with homogeneous Tn/STn antigens. These results raised a serious question about the role of Tn/STn antigens on IgA1 in pathogenesis of IgAN, and there is a demand for a practical methodology that any laboratory can utilize to analyze the O-glycans of IgA1. Herein, we describe the methodology we developed in more detail. The method could also be applied to the analysis of any other O-glycosylated proteins.

Hypoxia enhances the malignant nature of bladder cancer cells and concomitantly antagonizes protein O-glycosylation extension.

Invasive bladder tumours express the cell-surface Sialyl-Tn (STn) antigen, which stems from a premature stop in protein O-glycosylation. The STn antigen favours invasion, immune escape, and possibly chemotherapy resistance, making it attractive for target therapeutics. However, the events leading to such deregulation in protein glycosylation are mostly unknown. Since hypoxia is a salient feature of advanced stage tumours, we searched into how it influences bladder cancer cells glycophenotype, with emphasis on STn expression. Therefore, three bladder cancer cell lines with distinct genetic and molecular backgrounds (T24, 5637 and HT1376) were submitted to hypoxia. To disclose HIF-1α-mediated events, experiments were also conducted in the presence of Deferoxamine Mesilate (Dfx), an inhibitor of HIF-1α proteasomal degradation. In both conditions all cell lines overexpressed HIF-1α and its transcriptionally-regulated protein CA-IX. This was accompanied by increased lactate biosynthesis, denoting a shift toward anaerobic metabolism. Concomitantly, T24 and 5637 cells acquired a more motile phenotype, consistent with their more mesenchymal characteristics. Moreover, hypoxia promoted STn antigen overexpression in all cell lines and enhanced the migration and invasion of those presenting more mesenchymal characteristics, in an HIF-1α-dependent manner. These effects were reversed by reoxygenation, demonstrating that oxygen affects O-glycan extension. Glycoproteomics studies highlighted that STn was mainly present in integrins and cadherins, suggesting a possible role for this glycan in adhesion, cell motility and invasion. The association between HIF-1α and STn overexpressions and tumour invasion was further confirmed in bladder cancer patient samples. In conclusion, STn overexpression may, in part, result from a HIF-1α mediated cell-survival strategy to adapt to the hypoxic challenge, favouring cell invasion. In addition, targeting STn-expressing glycoproteins may offer potential to treat tumour hypoxic niches harbouring more malignant cells.

Abstract LB-221: Novel anti-Sialyl-Tn monoclonal antibodies and antibody-drug conjugates (ADCs) demonstrate tumor specificity, unique sequence homology and in vitro and in vivo antitumor efficacy

Abstract Siamab Therapeutics is developing monoclonal antibodies and antibody drug conjugates (ADCs) targeting tumor-associated carbohydrate antigens such as Sialyl-Tn (STn). These antibodies are selected using a proprietary glycan microarray that enriches for candidates whose binding is protein independent, highly specific and demonstrates exceptional target affinity. 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. Siamab has developed over 20 murine mAbs that bind STn with high affinity and specificity. When formatted as ADCs, anti-STn mAb-ADCs target and selectively kill STn expressing cells and also demonstrate efficacy in multiple xenograft studies. Multiple murine mAbs perform well in IHC studies with multiple solid tumor samples and tissue microarrays and demonstrate high tumor specificity. Remarkable sequence homology across all murine mAbs was observed in both heavy and light chains, and hot spots for hypermutation were identified. Humanization was successfully completed using a selection of mAbs and multiple humanized mAbs maintain their target specific binding, affinity and cytotoxicity in vitro. Our data demonstrates that high-affinity, STn-specific mAbs show promise both as therapies for solid tumors and as a potential tool for the development of biomarkers and companion diagnostics enabling selection of patients most likely to respond to therapy. Citation Format: David Eaverone, Jeff Behrens, Jillian Prendergast. Novel anti-Sialyl-Tn monoclonal antibodies and antibody-drug conjugates (ADCs) demonstrate tumor specificity, unique sequence homology and in vitro and in vivo antitumor efficacy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-221.

Interactions of mucins with the Tn or Sialyl Tn cancer antigens including MUC1 are due to GalNAc-GalNAc interactions.

The molecular mechanism(s) underlying the enhanced self-interactions of mucins possessing the Tn (GalNAcα1-Ser/Thr) or STn (NeuNAcα2-6GalNAcα1-Ser/Thr) cancer markers were investigated using optical tweezers (OT). The mucins examined included modified porcine submaxillary mucin containing the Tn epitope (Tn-PSM), ovine submaxillary mucin with the STn epitope (STn-OSM), and recombinant MUC1 analogs with either the Tn and STn epitope. OT experiments in which the mucins were immobilized onto polystyrene beads revealed identical self-interaction characteristics for all mucins. Identical binding strength and energy landscape characteristics were also observed for synthetic polymers displaying multiple GalNAc decorations. Polystyrene beads without immobilized mucins showed no self-interactions and also no interactions with mucin-decorated polystyrene beads. Taken together, the experimental data suggest that in these molecules, the GalNAc residue mediates interactions independent of the anchoring polymer backbone. Furthermore, GalNAc-GalNAc interactions appear to be responsible for self-interactions of mucins decorated with the STn epitope. Hence, Tn-MUC1 and STn-MUC1 undergo self-interactions mediated by the GalNAc residue in both epitopes, suggesting a possible molecular role in cancer. MUC1 possessing the T (Galβ1-3GalNAcα1-Ser/Thr) or ST antigen (NeuNAcα2-3Galβ1-3GalNAcα1-Ser/Thr) failed to show self-interactions. However, in the case of ST-MUC1, self-interactions were observed after subsequent treatment with neuraminidase andβ-galactosidase. This enzymatic treatment is expected to introduce Tn-epitopes and these observations thus further strengthen the conclusion that the observed interactions are mediated by the GalNAc groups.