Phase I-II study of OBI-888, a humanized monoclonal IgG1 antibody against the tumor-associated carbohydrate antigen Globo H, in patients with advanced solid tumors.

PURPOSE: OBI-888 is a humanized, monoclonal IgG1 antibody specific to the tumor-associated carbohydrate antigen Globo H. We conducted a phase I-II study of OBI-888 in patients with advanced cancer. METHODS: Patients were treated with OBI-888 5, 10, or 20 mg/kg IV weekly in Part A ("3 + 3" design) and 20 mg/kg IV weekly in Part B (Simon's 2-stage design) (1 cycle = 28 days). RESULTS: Overall, 54 patients were treated (Part A, n = 14; Part B, n = 40). OBI-888 was safe and well tolerated across the doses studied, with a low incidence of OBI-888-related treatment emergent adverse events. The maximum tolerated dose of OBI-888 was not reached. No dose-limiting toxicities were noted up to the 20 mg/kg dose level (recommended phase 2 dose). Stable disease (SD) was noted in 28.6% and 20% of Parts A and B, respectively, including three patients with SD for 6+, 7+, and 9 months. Antibody-dependent cellular cytotoxicity (ADCC) was induced after each OBI-888 treatment (average increase, 3.8-fold and 4.7-fold in Parts A and B, respectively), suggesting that ADCC induction is a potential mechanism of action of OBI-888. CONCLUSIONS: OBI-888 was well tolerated. Prolonged SD was noted in three patients. ADCC was induced after each OBI-888 treatment.

Globo H ceramide is an independent prognostic marker for gallbladder cancer.

In recent studies, there has been growing interest in developing cancer therapeutics targeting Globo H ceramide, which is considered as the most prevalent tumor-associated carbohydrate antigen in epithelial cancers. In this study, we aimed to evaluate the expression of Globo H and investigate its prognostic significance in gallbladder cancer (GBC). The tumor specimens and clinical characteristics of GBC patients were collected from the tumor bank and database of Chang Gung Memorial Hospital. Globo H in tumor specimens was detected by immunohistochemistry (IHC) and mass spectrometry analysis. Through data mining, it was discovered that FUT1 and FUT2, which are key enzymes involved in the biosynthesis of Globo H, were significantly up-regulated in human gallbladder cancer (GBC). Consistent with this finding, Globo H expression was detected in 86% (128 out of 149) of GBC specimens using immunohistochemical (IHC) staining. This was the highest frequency among Globo H expressing cancers. Patients with tumors exhibiting higher Globo H expression (H-score ≥ 80) demonstrated significantly shorter disease-free survival (DFS) and overall survival (OS) (P = 0.0001 and P = 0.0004, respectively). In a multivariable Cox regression analysis, elevated Globo H expression was identified as an independent unfavorable predictor for DFS and OS (hazard ratio: 2.29 and 2.32, respectively, P = 0.008 and 0.001) in primary GBC. Globo H is an independent prognostic marker for GBC.

Investigation of the impact of Globo-H expression on the progression of gastric cancer.

Globo-H (GH), a globo-series glycosphingolipid antigen that is synthesized by key enzymes ß1,3-galactosyltransferase V (ß3GalT5), fucosyltransferase (FUT) 1 and 2, is highly expressed on a variety of epithelial cancers rendering it a promising target for cancer immunotherapy. GH-targeting antibody-drug conjugate has been demonstrated an excellent tumor growth inhibition potency in animal models across multiple cancer types including Gastric cancer (GC). This study aims to further investigate the GH roles in GC. Significant correlations were observed between high mRNA expression of GH-synthetic key enzymes and worse overall survival (OS)/post-progression survival for GC patients based on the data from "Kaplan-Meier plotter" database (n=498). The level of GH expression was evaluated in clinical adenocarcinoma samples from 105 patients with GC by immunohistochemistry based on H-score. GH expression (H score ≥ 20; 33.3%) was significantly associated with a poor disease specific survival (DSS) and invasiveness in all samples with P=0.029 and P=0.013, respectively. In addition, it is also associated with shorter DSS and OS in poorly differentiated tumors with P=0.033 and P=0.045, respectively. Particularly, with patients ≥ 65 years of age, GH expression is also significantly associated with the stages (P=0.023), differentiation grade (P=0.038), and invasiveness (P=0.026) of the cancer. Sorted GC NCI-N87 cells with high level of endogenous GH showed higher proliferative activity compared with low-GH-expressing cells based on PCNA expression. Micro-western array analysis on high-GH-expressing GC cells indicated an upregulation in HER2-related signaling proteins including phospho-AKT/P38/JNK and Cyclin D1/Cyclin E1 proteins. Moreover, GH level was shown to be correlated with expression of total HER2 and caveolin-1 in GC cells. Immunoprecipitation study suggested that there are potential interactions among GH, caveolin-1, and HER2. In conclusions, GH level was significantly associated with the worse survival and disease progression in GC patients, especially in older patients. Enhanced cell proliferation activity through interactions among GH, HER2, and caveolin-1 interactions may contribute to GH induced tumor promotion signaling in GC. GH-targeting therapy may be a viable option for the treatment of GC patients.

High expression of embryonic stem cell marker SSEA3 confers poor prognosis and promotes epithelial mesenchymal transition in hepatocellular carcinoma.

BACKGROUND: Malignant cells may arise from dedifferentiation of mature cells and acquire features of the progenitor cells. Definitive endoderm from which liver is derived, expresses glycosphingolipids (GSLs) such as stage-specific embryonic antigen 3 (SSEA3), Globo H, and stage-specific embryonic antigen 4 (SSEA4). Herein, we evaluated the potential prognosis value of the three GSLs and biological functions of SSEA3 in hepatocellular carcinoma (HCC). METHODS: The expression of SSEA3, Globo H, and SSEA4 in tumor tissues obtained from 328 patients with resectable HCC was examined by immunohistochemistry staining. Epithelial mesenchymal transition (EMT) and their related genes were analyzed by transwell assay and qRT-PCR, respectively. RESULTS: Kaplan Meier survival analysis showed significantly shorter relapse-free survival (RFS) for those with higher expression of SSEA3 (p < 0.001), Globo H (p < 0.001), and SSEA4 (p = 0.005) and worse overall survival (OS) for those with high expression of either SSEA3 (p < 0.001) or SSEA4 (p = 0.01). Furthermore, multivariable Cox regression analysis identified the SSEA3 as an independent predictor for RFS (HR: 2.68, 95% CI: 1.93-3.72, p < 0.001) and OS (HR: 2.99, 95% CI: 1.81-4.96, p < 0.001) in HCC. Additionally, SSEA3-ceramide enhanced the EMT of HCC cells, as reflected by its ability to increase migration, invasion and upregulate the expression of CDH2, vimentin, fibronectin, and MMP2, along with ZEB1. Moreover, ZEB1 silencing abrogated the EMT-enhancing effects of SSEA3-ceramide. CONCLUSIONS: Higher expression of SSEA3 was an independent predictor for RFS and OS in HCC and promoted EMT of HCC via upregulation of ZEB1.

Conformational alteration in glycan induces phospholipase Cß1 activation and angiogenesis.

BACKGROUND: In endothelial cells, phospholipase C (PLC) ß1-activated Ca2+ is a crucial second messenger for the signaling pathways governing angiogenesis. PLCß1 is inactivated by complexing with an intracellular protein called translin-associated factor X (TRAX). This study demonstrates specific interactions between Globo H ceramide (GHCer) and TRAX, which highlight a new angiogenic control through PLCß1 activation. METHODS: Globo-series glycosphingolipids (GSLs), including GHCer and stage-specific embryonic antigen-3 ceramide (SSEA3Cer), were analyzed using enzyme-linked immunosorbent assay (ELISA) and Biacore for their binding with TRAX. Angiogenic activities of GSLs in human umbilical vein endothelial cells (HUVECs) were evaluated. Molecular dynamics (MD) simulation was used to study conformations of GSLs and their molecular interactions with TRAX. Fluorescence resonance energy transfer (FRET) analysis of HUVECs by confocal microscopy was used to validate the release of PLCß1 from TRAX. Furthermore, the in vivo angiogenic activity of extracellular vesicles (EVs) containing GHCer was confirmed using subcutaneous Matrigel plug assay in mice. RESULTS: The results of ELISA and Biacore analysis showed a stable complex between recombinant TRAX and synthetic GHCer with KD of 40.9 nM. In contrast, SSEA3Cer lacking a fucose residue of GHCer at the terminal showed ~ 1000-fold decrease in the binding affinity. These results were consistent with their angiogenic activities in HUVECs. The MD simulation indicated that TRAX interacted with the glycan moiety of GHCer at amino acid Q223, Q219, L142, S141, and E216. At equilibrium the stable complex maintained 4.6 ± 1.3 H-bonds. TRAX containing double mutations with Q223A and Q219A lost its ability to interact with GHCer in both MD simulation and Biacore assays. Removal of the terminal fucose from GHCer to become SSEA3Cer resulted in decreased H-bonding to 1.2 ± 1.0 by the MD simulation. Such specific H-bonding was due to the conformational alteration in the whole glycan which was affected by the presence or absence of the fucose moiety. In addition, ELISA, Biacore, and in-cell FRET assays confirmed the competition between GHCer and PLCß1 for binding to TRAX. Furthermore, the Matrigel plug assay showed robust vessel formation in the plug containing tumor-secreted EVs or synthetic GHCer, but not in the plug with SSEA3Cer. The FRET analysis also indicated the disruption of colocalization of TRAX and PLCß1 in cells by GHCer derived from EVs. CONCLUSIONS: Overall, the fucose residue in GHCer dictated the glycan conformation for its complexing with TRAX to release TRAX-sequestered PLCß1, leading to Ca2+ mobilization in endothelial cells and enhancing angiogenesis in tumor microenvironments.

GLORIA: phase III, open-label study of adagloxad simolenin/OBI-821 in patients with high-risk triple-negative breast cancer.

Triple-negative breast cancer (TNBC) has the highest rate of distant metastasis and poorest overall survival among breast cancer subtypes. In a phase II study, adagloxad simolenin (AdaSim), a synthetic Globo H conjugate vaccine administered with adjuvant OBI-821, was shown to induce IgM and IgG anti-Globo H humoral responses in patients with metastatic breast cancer overexpressing the glycosphingolipid Globo H. GLORIA is an ongoing phase III, randomized, open-label clinical trial to evaluate the safety and efficacy of AdaSim and the quality of life (QoL) of patients receiving AdaSim plus standard of care (SOC) versus SOC alone in high-risk, early-stage TNBC. The primary end point is invasive progression-free survival; secondary end points include overall survival, QoL, breast cancer-free interval, distant disease-free survival, safety, and tolerability.

Patients with triple-negative breast cancer generally do very poorly with the current available therapies. A vaccine with a totally different mechanism of action is being investigated in these patients to see how they do with this new therapy. This trial is a very early investigation and is currently ongoing. Clinical Trial Registration: NCT03562637 (ClinicalTrials.gov).

Generation of glycan-specific nanobodies.

The development of antibodies that target specific glycan structures on cancer cells or human pathogens poses a significant challenge due to the immense complexity of naturally occurring glycans. Automated glycan assembly enables the production of structurally homogeneous glycans in amounts that are difficult to derive from natural sources. Nanobodies (Nbs) are the smallest antigen-binding domains of heavy-chain-only antibodies (hcAbs) found in camelids. To date, the development of glycan-specific Nbs using synthetic glycans has not been reported. Here, we use defined synthetic glycans for alpaca immunization to elicit glycan-specific hcAbs, and describe the identification, isolation, and production of a Nb specific for the tumor-associated carbohydrate antigen Globo-H. The Nb binds the terminal fucose of Globo-H and recognizes synthetic Globo-H in solution and native Globo-H on breast cancer cells with high specificity. These results demonstrate the potential of our approach for generating glycan-targeting Nbs to be used in biomedical and biotechnological applications.

Chemoenzymatic Synthesis of Globo-series Glycosphingolipids and Evaluation of Their Immunosuppressive Activities.

Glycosphingolipids (GSLs) play essential roles in many important biological processes, making them attractive synthetic targets. In this paper, a viable chemoenzymatic method is described for the synthesis of globo-series GSLs, namely, Gb4, Gb5, SSEA-4, and Globo H. The strategy uses a chemically synthesized lactoside acceptor equipped with a partial ceramide structure that is uniquely extended by glycosyltransferases in a highly efficient one-pot multiple enzyme (OPME) procedure. A direct and quantitative conversion of Gb4 sphingosine to Globo H sphingosine is achieved by performing two-sequential OPME glycosylations. A reduction and N-acylation protocol allows facile incorporation of various fatty acids into the lipid portions of the GSLs. The chemically well-defined lipid-modified Globo H-GSLs displayed some differences in their immunosuppressive activities, which may benefit the structural modifications of Globo H ceramides in finding new types of immunosuppressive agents. The strategy outlined in this work should be applicable to the rapid access to other complex GSLs.

The role of Globo H and SSEA-4 in the development and progression of cancer, and their potential as therapeutic targets.

Glycans, chains of sugar molecules found conjugated to cell proteins and lipids, contribute to their growth, movement and differentiation. Aberrant glycosylation is a hallmark of several medical conditions including tumorigenesis. Glycosphingolipids (GSLs), consisting of glycans conjugated to a lipid (ceramide) core, are found in the lipid bilayer of eukaryotic cell membranes. GSLs, play an active role in cell processes. Several GSLs are expressed by human embryonic stem cells and have been found to be overexpressed in several types of cancer. In this review, we discuss the data, hypotheses and perspectives related to the GSLs Globo H and SSEA-4.

Dissecting the conformation of glycans and their interactions with proteins.

The use of in silico strategies to develop the structural basis for a rational optimization of glycan-protein interactions remains a great challenge. This problem derives, in part, from the lack of technologies to quantitatively and qualitatively assess the complex assembling between a glycan and the targeted protein molecule. Since there is an unmet need for developing new sugar-targeted therapeutics, many investigators are searching for technology platforms to elucidate various types of molecular interactions within glycan-protein complexes and aid in the development of glycan-targeted therapies. Here we discuss three important technology platforms commonly used in the assessment of the complex assembly of glycosylated biomolecules, such as glycoproteins or glycosphingolipids: Biacore analysis, molecular docking, and molecular dynamics simulations. We will also discuss the structural investigation of glycosylated biomolecules, including conformational changes of glycans and their impact on molecular interactions within the glycan-protein complex. For glycoproteins, secreted protein acidic and rich in cysteine (SPARC), which is associated with various lung disorders, such as chronic obstructive pulmonary disease (COPD) and lung cancer, will be taken as an example showing that the core fucosylation of N-glycan in SPARC regulates protein-binding affinity with extracellular matrix collagen. For glycosphingolipids (GSLs), Globo H ceramide, an important tumor-associated GSL which is being actively investigated as a target for new cancer immunotherapies, will be used to demonstrate how glycan structure plays a significant role in enhancing angiogenesis in tumor microenvironments.

Targeting glycosphingolipids for cancer immunotherapy.

Aberrant expression of glycosphingolipids (GSLs) is a unique feature of cancer and stromal cells in tumor microenvironments. Although the impact of GSLs on tumor progression remains largely unclear, anticancer immunotherapies directed against GSLs are attracting growing attention. Here, we focus on GD2, a disialoganglioside expressed in tumors of neuroectodermal origin, and Globo H ceramide (GHCer), the most prevalent cancer-associated GSL overexpressed in a variety of epithelial cancers. We first summarize recent advances on our understanding of GD2 and GHCer biology and then discuss the clinical development of the first immunotherapeutic agent targeting a glycolipid, the GD2-specific antibody dinutuximab, its approved indications, and new strategies to improve its efficacy for neuroblastoma. Next, we review ongoing clinical trials on Globo H-targeted immunotherapeutics. We end with highlighting how these studies provide sound scientific rationales for targeting GSLs in cancer and may facilitate a rational design of new GSL-targeted anticancer therapeutics.

Signaling pathway of globo-series glycosphingolipids and ß1,3-galactosyltransferase V (ß3GalT5) in breast cancer.

The globo-series glycosphingolipids (GSLs) SSEA3, SSEA4, and Globo-H specifically expressed on cancer cells are found to correlate with tumor progression and metastasis, but the functional roles of these GSLs and the key enzyme ß1,3-galactosyltransferase V (ß3GalT5) that converts Gb4 to SSEA3 remain largely unclear. Here we show that the expression of ß3GalT5 significantly correlates with tumor progression and poor survival in patients, and the globo-series GSLs in breast cancer cells form a complex in membrane lipid raft with caveolin-1 (CAV1) and focal adhesion kinase (FAK) which then interact with AKT and receptor-interacting protein kinase (RIP), respectively. Knockdown of ß3GalT5 disrupts the complex and induces apoptosis through dissociation of RIP from the complex to interact with the Fas death domain (FADD) and trigger the Fas-dependent pathway. This finding provides a link between SSEA3/SSEA4/Globo-H and the FAK/CAV1/AKT/RIP complex in tumor progression and apoptosis and suggests a direction for the treatment of breast cancer, as demonstrated by the combined use of antibodies against Globo-H and SSEA4.

A set of cancer stem cell homing peptides associating with the glycan moieties of glycosphingolipids.

Cancer stem cells (CSCs) are currently believed to be involved in tumor metastasis and relapse. And treatments against CSCs are well concerned issues. Peptides targeting to mouse and human CSCs were screened from an M13 phage display library. The first subset of cancer stem cell homing peptides (CSC HPs), CSC HP-1 to -12, were screened with mouse EMT6 breast cancer stem cells. Among them, CSC HP-1, CSC HP-3, CSC HP-8, CSC HP-9, and CSC HP-10 can bind to mouse CT26 colon CSCs; CSC HP-1, CSC HP-2, CSC HP-3, and CSC HP-8 can bind to mouse Hepa1-6 liver CSCs; as well as CSC HP-1, CSC HP-2, CSC HP-3, CSC HP-8, CSC HP-9, CSC HP-10, and CSC HP-11 can bind to human PANC-1 pancreatic CSCs. The second subset of cancer stem cell homing peptides, CSC HP-hP1 to -hP3, were screened with human PANC-1 pancreatic CSCs. Both CSC HP-hP1 and CSC HP-hP2 were demonstrated able to bind mouse EMT6, CT26 and Hepa1-6 CSCs as well as human colorectal HT29 and lung H1650 CSCs. CSC HP-1 and CSC HP-hP1 could strongly associate with the Globo 4 and Lewis Y glycan epitopes coupled on a microarray chip or Globo 4 and Globo H conjugated on bovine serum albumin. CSC HP-10, CSC HP-11 and CSC HP-hP2 could associate with the disialylated saccharide Neu5Ac-α-2,6-Gal-ß-1,3-(Neu5Ac-α-2,6)-GalNAc coupled on a microarray chip. These results indicate that the CSC HPs may target to the known stem cell glycan markers GbH and Lewis Y as well as the disialylated saccharide.

Globo H expression is associated with driver mutations and PD-L1 expressions in stage I non-small cell lung cancer.

BACKGROUND: Globo H is a tumor-associated carbohydrate antigen exclusively expressed in cancer cells rather than normal tissue. Globo H has been found on many cancers of epithelial origins, and become an attractive target for cancer vaccine. OBJECTIVES: We aimed to study the expression of Globo H in non-small cell lung cancer (NSCLC) patients, and correlated its expression with common driver mutations, clinical outcomes, and status of immune checkpoint, programmed death-ligand 1 (PD-L1). METHODS: The study enrolled 228 patients with surgically resected stage I NSCLC, including 139 patients with adenocarcinoma (ADC) and 89 patients with squamous cell carcinoma (SqCC). Using immunohistochemistry, tumors with moderate to strong membranous staining in ⩾ 1% tumor cells per section were scored as positive Globo H expression. Driver mutations including EGFR, KRAS, BRAF were detected by direct sequencing, while ALK, PI3KCA, FGFR1 and PD-L1 expression was detected by immunohistochemical (IHC) staining. RESULTS: Positive Globo H expression was detected in 88 of the 228 (38.6%) patients. These included 51 of 139 (36.7%) patients with ADC and 37 of 89 (41.6%) patients with SqCC. Positive Globo H expression was significantly associated with EGFR mutation and PD-L1 expression in the ADC group, and PI3KCA overexpression in the SqCC group. The survival analysis showed that Globo H expression was not an independent prognostic factor in stage I NSCLC. CONCLUSIONS: Globo H expression was correlated with specific driver mutations in ADC and SqCC NSCLC tumors, as well as PD-L1 status. Immunotherapy targeting Globo H may have potential application in lung cancer treatment.

Aberrant expression of tumor-associated carbohydrate antigen Globo H in thyroid carcinoma.

BACKGROUND AND OBJECTIVES: The induction of tumor-associated carbohydrate antigen results from altered glycosylation in transformed cells. Globo H is a hexasaccharide glycosphingolipid overexpressed on malignancies of epithelial origin and has become an attractive vaccine target. We aimed to investigate the expression patterns and prognostic value of Globo H in thyroid neoplasms. METHODS: Globo H expression was examined by immunohistochemical analysis using commercial and in-house tissue microarrays. The expression was correlated with clinicopathologic characteristics in papillary thyroid cancer. RESULTS: Normal or benign thyroid lesions were negative for Globo H expression. Globo H was positive in 33% medullary, 24% papillary, 11% undifferentiated, and 8% follicular thyroid cancer. Globo H expression in papillary thyroid cancer was associated with extrathyroidal invasion (P = 0.017), BRAF mutation (P = 0.010), AMES high risk (P = 0.045), and increased ATA risk of recurrence (P = 0.022). CONCLUSIONS: Globo H is specifically expressed in a subset of thyroid malignancies. In papillary thyroid cancer, Globo H expression is associated with invasiveness and BRAF mutation. Immunotherapy targeting Globo H may have potential applications in thyroid cancer. J. Surg. Oncol. 2016;114:853-858. © 2016 2016 Wiley Periodicals, Inc.

Chemical Synthesis of the Tumor-Associated Globo H Antigen.

A derivative of the tumor-associated globo H antigen, a complex hexasaccharide, was synthesized by a convergent and efficient [3+2+1] strategy using various glycosylation methods. All glycosylation reactions afforded good to excellent yields and outstanding stereoselectivity, including the installation of cis α-linked D-galactose and L-fucose. The longest linear sequence for this synthesis was 11 steps from a galactose derivative 11 to give an overall yield of 2.6%. The synthetic target had a free and reactive amino group at the glycan reducing end, facilitating its conjugation with other molecules for various applications.

Novel substrate specificities of two lacto-N-biosidases towards ß-linked galacto-N-biose-containing oligosaccharides of globo H, Gb5, and GA1.

We describe the novel substrate specificities of two independently evolved lacto-N-biosidases (LnbX and LnbB) towards the sugar chains of globo- and ganglio-series glycosphingolipids. LnbX, a non-classified member of the glycoside hydrolase family, isolated from Bifidobacterium longum subsp. longum, was shown to liberate galacto-N-biose (GNB: Galß1-3GalNAc) and 2'-fucosyl GNB (a type-4 trisaccharide) from Gb5 pentasaccharide and globo H hexasaccharide, respectively. LnbB, a member of the glycoside hydrolase family 20 isolated from Bifidobacterium bifidum, was shown to release GNB from Gb5 and GA1 oligosaccharides. This is the first report describing enzymatic release of ß-linked GNB from natural substrates. These unique activities may play a role in modulating the microbial composition in the gut ecosystem, and may serve as new tools for elucidating the functions of sugar chains of glycosphingolipids.

Synthesis and characterization of a novel chemically designed (Globo)3-DTPA-KLH antigen.

In recent years, many experiments have been conducted for the production and evaluation of anticancer glycoconjugated vaccines in developed countries and many achievements have been accomplished with Globo H derivatives. In the current experiment, a new chemically designed triplicate version of (Globo H)3-diethylenetriamine pentaacetic acid (DTPA)-KLH antigen was synthesized and characterized. Immunization with (Globo H)3-DTPA-KLH, a hexasaccharide that is a member of a family of antigenic carbohydrates that are highly expressed in various types of cancers conjugated with DTPA and KLH protein, induced a high level of antibody titer along with an elevated level of IL-4 in mice. Treatment of tumors with the collected sera from immunized mice decreased the tumor size in nude mice as well. None of the immunized mice illustrated any sign of tumor growth after injection of MCF-7 cells compared to the control animals. These findings, based on the newly presented structure of the Globo H antigen, lend exciting and promising evidence for clinical advancement in the development of a therapeutic vaccine in the future.

A Fully Synthetic Self-Adjuvanting Globo H-Based Vaccine Elicited Strong T Cell-Mediated Antitumor Immunity.

Therapeutic cancer vaccines based on the abnormal glycans expressed on cancer cells, such as the globo H antigen, have witnessed great progress in recent years. For example, the keyhole limpet hemocyanin (KLH) conjugate of globo H has been on clinical trials as a cancer vaccine. However, such vaccines have intrinsic problems, such as inconsistence in eliciting T cell-mediated immunity in cancer patients and difficult quality control. To address the issue, a structurally defined fully synthetic glycoconjugate vaccine composed of globo H and monophosphoryl lipid A (MPLA) was developed. The new vaccine was shown to elicit robust IgG1 antibody responses and T cell-dependent immunity, which is desired for anticancer vaccine, and induce significantly faster and stronger immune responses than the globo H-KLH conjugate. Moreover, it was self-adjuvanting, namely, inducing immune responses without the use of an external adjuvant, thus MPLA was not only a vaccine carrier but also a build-in adjuvant. It was also found that antibodies induced by the new vaccine could selectively bind to and mediate strong complement-dependent cytotoxicity to globo H-expressing MCF-7 cancer cell. All of the results have demonstrated that the globo H-MPLA conjugate is a better cancer vaccine than the globo H-KLH conjugate under experimental conditions and is worth further investigation and development.

Affinity of monoclonal antibodies for Globo-series glycans.

Globo-series glycans are human cell-surface carbohydrates that include stem-cell marker SSEA-4 and cancer-cell antigen Globo H. These two hexasaccharides differ only in their terminal saccharide: N-acetylneuraminic acid in SSEA-4 and L-fucose in Globo H. Herein, we evaluated the affinity of the monoclonal antibodies α-SSEA-4 and α-GH for the glycans SSEA-4 and Globo H. Using fluorescence polarization, we find that the two monoclonal antibodies have affinity for their cognate glycan in the low nanomolar range, and have negligible affinity for the non-cognate glycan. Using surface plasmon resonance, we find that each cognate affinity is ∼20-fold greater if the glycan is immobilized on a surface rather than free in solution. We conclude that the terminal saccharide plays a dominant role in the ability of monoclonal antibodies to recognize these Globo-series glycans and that the extraordinary specificity of these antibodies supports their use for identifying and sorting stem-cells (α-SSEA-4) and as an agent in cancer immunotherapy (α-GH).