Tumor-associated Carbohydrate Antigens Research Articles

Synthesis and Thermodynamic Evaluation of Sialyl-Tn MUC1 Glycopeptides Binding to Macrophage Galactose-Type Lectin.

Interactions between the tumor-associated carbohydrate antigens of Mucin 1 (MUC1) and the carbohydrate-binding proteins, lectins, often lead to the creation of a pro-tumor microenvironment favoring tumor initiation, progression, metastasis, and immune evasion. Macrophage galactose binding lectin (MGL) is a C-type lectin receptor found on antigen-presenting cells that facilitates the uptake of carbohydrate antigens for antigen presentation, modulating the immune response homeostasis, autoimmunity, and cancer. Considering the crucial role of tumor-associated forms of MUC1 and MGL in tumor immunology, a thorough understanding of their binding interaction is essential for it to be exploited for cancer vaccine strategies. The synthesis of MUC1 glycopeptide models carrying a single or multiple Tn and/or sialyl-Tn antigen(s) is described. A novel approach for the sialyl-Tn threonine building block suitable for the solid phase peptide synthesis was developed. The thermodynamic profile of the binding interaction between the human MGL and MUC1 glycopeptide models was analyzed using isothermal titration calorimetry. The measured dissociation constants for the sialyl-Tn-bearing peptide epitopes were consistently lower compared to the Tn antigen and ranged from 10 μM for mono- to 100 nM for triglycosylated MUC1 peptide, respectively. All studied interactions, regardless of the glycan's site of attachment or density, exhibited enthalpy-driven thermodynamics.

A Versatile One-Step Enzymatic Strategy for Efficient Imaging and Mapping of Tumor-Associated Tn Antigen.

Tn antigen (CD175), recognized as the precursor monosaccharide (α-GalNAc) of mucin O-glycan, is a well-known tumor-associated carbohydrate antigen (TACA). It has emerged as a potential biomarker for cancer diagnosis and prognosis. However, the role it plays in cancer biology remains elusive due to the absence of a sensitive and selective detection method. In this study, we synthesized two new probes based on a unique uridine-5'-diphospho-α-d-galactose (UDP-Gal) derivative, each functionalized with either a fluorescence or a cleavable biotin tag, to develop an innovative one-step enzymatic labeling strategy, enabling the visualization, enrichment, and site-specific mapping of the Tn antigen with unparalleled sensitivity and specificity. Our versatile strategy has been successfully applied to detect and image Tn antigen across various samples, including the complex cell lysates, live cells, serum, and tissue samples. Compared to the traditional lectin method, this one-step enzymatic method is simpler and more efficient (>10/100-fold in sensitivity). Furthermore, it allowed us to map 454 Tn-glycoproteins and 624 Tn-glycosylation sites from HEK293FTn+ and Jurkat cells. Therefore, our strategy provides an exceptionally promising tool for revealing the biological functions of the Tn antigen and advancing cancer diagnostics.

Positive associations of active immune therapy OBI-833/OBI-821 induced immune responses with clinical outcomes in patients with non-small cell lung cancer.

e14671 Background: OBI-833/OBI-821 (OBI-833) is a novel active immune therapy (cancer vaccine) targeting a tumor-associated carbohydrate antigen, Globo H. Previous reports have shown that OBI-833 can elicit a beneficial immune response in Non-Small Cell Lung Cancer (NSCLC) patients and rendered durable stable disease with some TKI-treated patients. In this study, we further evaluated the effect of OBI-833-induced immune responses on the in-study progression-free survivals (isPFS) during the treatment. Methods: Sera were collected for the evaluations of maximum concentration (Cmax) and exposure (area under the curve: AUC) of anti-Globo H IgM (GH IgM) and IgG (GH IgG), complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and ex-vivo GH IgM induction with PBMC from each subject at baseline. The correlations of OBI-833-induced immune responses with isPFS and best overall response (BOR) were also evaluated. Pearson correlation coefficient analysis was used for all correlation evaluations. Results: Among the 14 OBI-833-treated subjects, 93% and 64% of subjects had significant increase in GH IgM and GH IgG, respectively. Median Cmax GH IgM and Cmax GH IgG were 13.8 µg/mL and 12.4 µg/mL, respectively. While the increase in CDC activities was not significantly correlated with the increase of GH IgM, a weak association between GH IgG and ADCC was observed (r=0.3380, p<0.0001). In addition, a strong association (r=0.84) was observed between Ex-vivo IFN-γ secreting and their Cmax IgM concentration in the OBI-833 Ex-vivo stimulation study. In contrast, weak association (r=0.26) was observed between Ex-vivo GH IgM induction and their Cmax of IgM. Correlations of the immune responses on isPFS were further analyzed. Significant positive correlations were observed for the Cmax and AUC of GH IgM with isPFS (Cmax: r=0.6345, p=0.0148; AUC: r=0.8212, p=0.0003) and the Cmax and AUC of GH IgG with isPFS (Cmax: r=0.7192, p=0.0037; AUC: r=0.6306, p=0.0062). Furthermore, distinct features of GH IgG responses from 2 subjects were identified based on the ADCC-GH IgG scatter plot. Samples from subject 034-006 showed high ADCC activity with relatively low GH IgG concentration, whereas the BOR was at -27%, which has significant negative correlation of tumor response (TR) with GH IgG (r=-0.7381, p=0.0148) and ADCC (r=-0.8565, p=0.0016). In contrast, samples from subject 034-004 showed average ADCC activity and relatively high GH IgG concentration with BOR at -12%, showing a negative correlation of TR with GH IgG (r=-0.8352, p=0.0001) and ADCC (r=-0.5690, p=0.0269). Conclusions: OBI-833 can elicit beneficial immune responses in NSCLC patients. Both GH IgM and GH IgG may contribute to the isPFS and TR of the subjects. Further development of OBI-833 in EGFR-mutated NSCLC patients phase II trial is ongoing.

Donor Preactivation-Based Glycan Assembly: from Manual to Automated Synthesis.

Carbohydrates are called the third chain of life. Carbohydrates participate in many important biochemical functions in living species, and the biological information carried by them is several orders of magnitude larger than that of nucleic acids and proteins. However, due to the intrinsic complexity and heterogeneity of carbohydrate structures, furnishing pure and structurally well-defined glycans for functional studies is a formidable task, especially for homogeneous large-size glycans. To address this issue, we have developed a donor preactivation-based one-pot glycosylation strategy enabling multiple sequential glycosylations in a single reaction vessel.The donor preactivation-based one-pot glycosylation refers to the strategy in which the glycosyl donor is activated in the absence of a glycosyl acceptor to generate a reactive intermediate. Subsequently, the glycosyl acceptor with the same anomeric leaving group is added, leading to a glycosyl coupling reaction, which is then iterated to rapidly achieve the desired glycan in the same reactor. The advantages of this strategy include the following: (1) unique chemoselectivity is obtained after preactivation; (2) it is independent of the reactivity of glycosyl donors; (3) multiple-step glycosylations are enabled without the need for intermediate purification; (4) only stoichiometric building blocks are required without complex protecting group manipulations. Using this protocol, a range of glycans including tumor-associated carbohydrate antigens, various glycosaminoglycans, complex N-glycans, and diverse bacterial glycans have been synthesized manually. Gratifyingly, the synthesis of mycobacterial arabinogalactan containing 92 monosaccharide units has been achieved, which created a precedent in the field of polysaccharide synthesis. Recently, the synthesis of a highly branched arabinogalactan from traditional Chinese medicine featuring 140 monosaccharide units has been also accomplished to evaluate its anti-pancreatic-cancer activity. In the spirit of green and sustainable chemistry, this strategy can also be applied to light-driven glycosylation reactions, where either UV or visible light can be used for the activation of glycosyl donors.Automated synthesis is an advanced approach to the construction of complex glycans. Based on the two preactivation modes (general promoter activation mode and light-induced activation mode), a universal and highly efficient automated solution-phase synthesizer was further developed to drive glycan assembly from manual to automated synthesis. Using this synthesizer, a library of oligosaccharides covering various glycoforms and glycosidic linkages was assembled rapidly, either in a general promoter-activation mode or in a light-induced-activation mode. The automated synthesis of a fully protected fondaparinux pentasaccharide was realized on a gram scale. Furthermore, the automated synthesis of large-size polysaccharides was performed, allowing the assembly of arabinans up to an astonishing 1080-mer using the automated multiplicative synthesis strategy, taking glycan synthesis to a new height far beyond the synthesis of nucleic acids (up to 200-mer) and proteins (up to 472-mer).

Lymph Node Targeting Mediated by Albumin Hitchhiking of Synthetic Tn Glycolipid Leads to Robust In Vivo Antibody Production.

Tn antigen is a tumor-associated carbohydrate antigen, which is present prominently on the tumor cell surfaces and attracts an interest in vaccine development. This work demonstrates that a synthetic Tn antigen carrying glycoconjugate forms a complex with circulating albumin, delivers the antigen to lymph nodes (LNs), and leads to the efficient production of antibodies against the antigen. Synthetic Tn antigen glycoconjugate, possessing DSPE-PEG2000 linker and lipophilic moieties, undergoes micellization in PBS buffer. In the presence of bovine serum albumin (BSA), demicellization of the glycolipid occurs, with a rate constant of 0.18 min-1. In vitro studies show that the glycoconjugate binds preferentially to BSA in the presence of cells. Immunological assessments in mice models reveal the albumin-enabled delivery of the Tn glycoconjugate to antigen-presenting cells in the LNs, specifically leading to a robust humoral immune response. ELISA titers show superior binding, with a saturation dilution of 1:51 200 for Tn glycoconjugate, in comparison to that mediated by the Tn-BSA covalent conjugate with a saturation dilution of 1:6400. Immunohistochemical staining shows delivery of Tn glycoconjugate at the LNs, specifically at the subcapsular sinus and interfollicular areas. The work highlights the potential of albumin-mediated target delivery strategy for cancer immunotherapies.

Abstract 2613: Treatment of solid tumors with antibody drug conjugates targeting aberrant O-glycoproteins

Abstract The lack of antibodies with sufficient cancer selectivity is currently limiting the treatment of solid tumors by immunotherapies. Many solid tumors display aberrant glycosylation that results in the expression of tumor-associated carbohydrate antigens (TACAs) distinct from healthy tissues. Targeting aberrantly glycosylated glycopeptide epitopes provides the cancer selectivity needed for immunotherapy of solid tumors. However, only a few such glycopeptide epitopes have been targeted to date. We have developed a strategy to predict, design, and produce cancer-specific antibodies targeting novel O-glycoprotein epitopes with exquisite cancer specificity. Our mAbs avoid on-target off-tumor toxicity due to the specificity of aberrant glycosylation, and cover a broad range of cancer indications, including breast, lung, colon, pancreatic, and ovarian cancer. Additionally, our antibodies can be used as an IHC companion diagnostic and can be functionalized across a number of modalities, including next-generation CAR-T, T-cell bispecifics, and antibody-drug-conjugates (ADCs).Here, we present two novel antibodies, GO-08 and GO-013, selectively targeting cancer-specific Tn-glycosylated proteins in a site-specific manner with low nanomolar affinities. GO-008 and GO-013 were collectively shown to be highly cancer-selective by immunohistochemistry of sections from a large array of different squamous and adenocarcinomas, including triple-negative breast cancer, lung cancer, gastric cancer, ovarian cancer, pancreatic, and colorectal cancer. The two antibodies were functionalized utilizing ConjuAllTM technology, conjugating the microtubule disrupting payload MMAE via a site-specific beta-glucuronidase cleavable linker. ConjuAllTM functions as a plasma stable, tumor labile linkage, and due to the minimal expression pattern of beta-glucuronidase in healthy tissues, reduces both off- and on-target toxicity due to the minimization of release of free payload in non-tumor tissues. The ADCs based on GO-08 and GO-013 (LCB45A and LCB22A) demonstrated target-specific cytotoxicity invitro and in vivo with tumor elimination in breast and lung PDX models. GO-013 conjugated to MMAE (LCB22A) was well tolerated in cynomolgus monkey toxicity studies. Moreover, the ADCs selectively kill target cells in a human mixed organotypic cancer model without affecting healthy cells, indicating tolerability and safety. The combination of specificity of TACA targeting to differentiate ADC binding from healthy tissue and ConjuAll’s tumor selective payload release promises to enable an excellent anti-cancer therapy. Citation Format: Aaron C. Groen, Nisha Shrestha, Boris Klebanov, Byeongjun Yu, BeomYong Lee, Chul-Woong Chung, Changsik Park, Mikkel K. Aasted, Sally Dabelsteen, Hans H. Wandall. Treatment of solid tumors with antibody drug conjugates targeting aberrant O-glycoproteins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2613.

Abstract 3125: TCX-101, a novel antibody-drug conjugate targeting a tumor-associated carbohydrate antigen for the treatment of solid tumors

Abstract Tumor-associated carbohydrate antigens (TACAs) are short oligosaccharides expressed on the surface of cancer cells and play key roles in cellular communication, immune evasion, apoptosis and metastasis. TACAs are therefore interesting targets for antibody-based therapies in cancer. We have generated an antibody (TCX-101) against a TACA structure with improved binding affinity compared to other monoclonal antibodies (mAbs) against the same target as shown by surface plasmon resonance (~ 10x superior KD), as well as high level of specificity against glycan species related to the target, as accessed by glycan array, flow cytometry, and immunocytochemistry or immunofluorescence. Immunohistochemistry studies on fresh-frozen tissue microarrays showed high expression of the target in various solid tumors (e.g. gastrointestinal, lung, and breast) and only low expression in normal tissues. In vitro, TCX-101 has ~ 4x higher maximum binding, ~ 2x faster binding kinetics, ~ 4x faster cellular internalization and induces ~ 2x higher antibody-dependent cellular cytotoxicity than a reference antibody. Monomethyl auristatin E (MMAE) was used in a drug-antibody ratio (DAR) of 4 to generate an antibody-drug conjugate (ADC). This ADC was effective in in vitro toxicity assays against murine B16 melanoma cells induced to express the target (measurable effect on WT cells), as well as human breast cancer MCF7 WT cells (~ 25x less activity on target-knockout MCF7 cells, Table 1). When tested in vivo, TCX-101 was highly effective in two different xenograft mouse models (B16, MCF7) with more than 95% tumor growth inhibition compared to vehicle, and no signs of toxicity (no weight loss, no histophatological findings). A fully humanized version of TCX-101 with improved binding properties (EC50 6.27 nM vs. 8.13 nM) was generated and will be evaluated in further in vivo experiments. In conclusion, TCX-101 is a promising ADC for the treatment of various solid tumors. IC50 [nM] TCX-101-MMAE 4 DAR B16 KI 7.64 B16 WT no killing observed MCF7 WT 1.97 MCF7 KO 45.97 Citation Format: Fausto G. Gomes, Francesco Muraca, Gustavo M. Schmidt Garcia Moreira, Kathrin Rösch, Maria Bräutigam, Peter Sondermann, Matthias Ocker. TCX-101, a novel antibody-drug conjugate targeting a tumor-associated carbohydrate antigen for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3125.

Highly stereoselective α-glycosylation with GalN3 donors enabled collective synthesis of mucin-related tumor associated carbohydrate antigens.

Mucin-related tumor-associated carbohydrate antigens (TACAs) are important and interesting targets for cancer vaccine therapy. However, efficient access to a library of mucin-related TACAs remains a challenging task. One of the key issues is the challenging construction of α-GalNAc linkages. Here, we report highly stereoselective α-glycosylation with GalN3N-phenyl trifluoroacetimidate donors, which features excellent yields, outstanding stereoselectivities, broad substrate scope and mild reaction conditions. This method is successfully applied to highly stereoselective synthesis of GalN3-α-O-Ser, which served as the common intermediate for collective synthesis of a wide range of TACAs including TN antigen, STN antigen, 2,6 STF antigen, 2,3 STF antigen, glycophorin and cores 1-8 mucin-type O-glycans. In particular, the rationale for this highly stereoselective α-glycosylation is provided for the first time using DFT calculations and mechanistic studies, highlighting the crucial roles of reagent combinations (TMSI and Ph3PO) and the H-bonding directing effect of the N3 group.

Chemical and chemoenzymatic syntheses of sialyl Lewisa tetrasaccharide antigen.

Sialyl Lewisa (sLea), also known as cancer antigen 19-9, is a tumor-associated carbohydrate antigen. In this article, chemical and chemoenzymatic syntheses of a tetrasaccharide glycan 1 structurally derived from sLea are reported. Challenges involved in the chemical synthesis include the highly stereoselective construction of 1,2-cis-α-L-fucoside and α-D-sialoside, as well as the assembly of the 3,4-disubstituted N-acetylglucosamine subunit. Perbenzylated thiofucoside and N-acetyl-5-N,4-O-oxazolidinone protected sialic acid thioglycoside were employed as glycosyl donors, respectively, for the efficient preparation of the desired α-fucoside and α-sialoside. The 3,4-branched glucosamine backbone was established through a 3-O and then 4-O glycosylation sequence in which the 3-hydroxyl group of the glucosamine moiety was glycosylated first and then the 4-hydroxyl. A facile chemoenzymatic approach was also exploited to synthesize the target molecule. The chemically obtained free disaccharide 30 was sequentially sialylated and fucosylated in an enzyme-catalyzed regio- and stereospecific manner to form 1 in high yields. The linker appended 1 can be covalently attached to a carrier protein for further immunological studies.

Unraveling the role of C1GALT1 in abnormal glycosylation and colorectal cancer progression.

C1GALT1 plays a pivotal role in colorectal cancer (CRC) development and progression through its involvement in various molecular mechanisms. This enzyme is central to the O-glycosylation process, producing tumor-associated carbohydrate antigens (TACA) like Tn and sTn, which are linked to cancer metastasis and poor prognosis. The interaction between C1GALT1 and core 3 synthase is crucial for the synthesis of core 3 O-glycans, essential for gastrointestinal health and mucosal barrier integrity. Aberrations in this pathway can lead to CRC development. Furthermore, C1GALT1's function is significantly influenced by its molecular chaperone, Cosmc, which is necessary for the proper folding of T-synthase. Dysregulation in this complex interaction contributes to abnormal O-glycan regulation, facilitating cancer progression. Moreover, C1GALT1 affects downstream signaling pathways and cellular behaviors, such as the epithelial-mesenchymal transition (EMT), by modifying O-glycans on key receptors like FGFR2, enhancing cancer cell invasiveness and metastatic potential. Additionally, the enzyme's relationship with MUC1, a mucin protein with abnormal glycosylation in CRC, highlights its role in cancer cell immune evasion and metastasis. Given these insights, targeting C1GALT1 presents a promising therapeutic strategy for CRC, necessitating further research to develop targeted inhibitors or activators. Future efforts should also explore C1GALT1's potential as a biomarker for early diagnosis, prognosis, and treatment response monitoring in CRC, alongside investigating combination therapies to improve patient outcomes.

Stereoselective Synthesis of Sialyl Lewisa Antigen and the Effective Anticancer Activity of Its Bacteriophage Qβ Conjugate as an Anticancer Vaccine.

Sialyl Lewisa (sLea ), also known as cancer antigen 19-9 (CA19-9), is a tumor-associated carbohydrate antigen. The overexpression of sLea on the surface of a variety of cancer cells makes it an attractive target for anticancer immunotherapy. However, sLea -based anticancer vaccines have been under-explored. To develop a new vaccine, efficient stereoselective synthesis of sLea with an amine-bearing linker was achieved, which was subsequently conjugated with a powerful carrier bacteriophage, Qβ. Mouse immunization with the Qβ-sLea conjugate generated strong and long-lasting anti-sLea IgG antibody responses, which were superior to those induced by the corresponding conjugate of sLea with the benchmark carrier keyhole limpet hemocyanin. Antibodies elicited by Qβ-sLea were highly selective toward the sLea structure, could bind strongly with sLea -expressing cancer cells and human pancreatic cancer tissues, and kill tumor cells through complement-mediated cytotoxicity. Furthermore, vaccination with Qβ-sLea significantly reduced tumor development in a metastatic cancer model in mice, demonstrating tumor protection for the first time by a sLea -based vaccine, thus highlighting the significant potential of sLea as a promising cancer antigen.

Tumor-Associated Extracellular Microvesicles with Fluorine-Modified Carbohydrate Antigens Trigger a Stronger Antitumor Immune Response.

Abnormal glycosylation is a hallmark of tumor development, and tumor-associated carbohydrate antigens are potential immune targets for tumor therapy. Tumor-associated extracellular microvesicles are subcellular vesicles released from cell membranes that have immunogenicity similar to that of precursor cells. However, unmodified tumor-derived microvesicles have weaknesses, such as low immunogenicity, poor biostability, and short half-life in vivo. For the first time, we herein generated extracellular microvesicles containing modified tumor-associated carbohydrate antigens by constructing a cell line with highly expressed antigen-processing enzymes utilizing fluorine-modified monosaccharide substrates via a metabolic oligosaccharide engineering strategy. The microvesicles were applied to tumor immunity, achieving enhanced immunoprophylaxis and immunotherapy effects. Furthermore, the mechanisms of antitumor immunity were explored. Our findings may provide new insights into the adhibition of suitably modified extracellular microvesicles and the development of more effective carbohydrate-based anticancer vaccines.

Tn antigen promotes breast cancer metastasis via impairment of CASC4.

Breast cancer is one of the most serious and deadly cancers in women worldwide, with distant metastases being the leading cause of death. Tn antigen, a tumor-associated carbohydrate antigen, was frequently detected in breast cancer, but its exact role in breast cancer metastasis has not been well elucidated. Here we investigated the impact of Tn antigen expression on breast cancer metastasis and its underlying mechanisms. The expression of Tn antigen was induced in two breast cancer cell lines by deleting T-synthase or Cosmc, both of which are required for normal O-glycosylation. It showed that Tn-expressing cancer cells promoted epithelial-mesenchymal transition (EMT) and metastatic features as compared to Tn(-) control cells both in vitro and in vivo. Mechanistically, we found that cancer susceptibility candidate 4 (CASC4), a heavily O-glycosylated protein, was significantly downregulated in both Tn(+) cells. Overexpression of CASC4 suppressed Tn-induced activation of EMT and cancer metastasis via inhibition of Cdc42 signaling. Furthermore, we confirmed that O-glycosylation is essential for the functional role of CASC4 because defective O-glycosylated CASC4 (mutant CASC4, which lacks nine O-glycosylation sites) exerted marginal metastatic-suppressing effects in comparison with WT CASC4. Collectively, these data suggest that Tn-mediated aberrant O-glycosylation contributes to breast cancer metastasis via impairment of CASC4 expression and function.

A Shiga Toxin B-Subunit-Based Lectibody Boosts T Cell Cytotoxicity towards Gb3-Positive Cancer Cells.

Aberrant glycosylation plays a crucial role in tumour progression and invasiveness. Tumour-associated carbohydrate antigens (TACAs) represent a valuable set of targets for immunotherapeutic approaches. The poor immunogenicity of glycan structures, however, requires a more effective and well-directed way of targeting TACAs on the surface of cancer cells than antibodies. The glycosphingolipid globotriaosylceramide (Gb3) is a well-established TACA present in a multitude of cancer types. Its overexpression has been linked to metastasis, invasiveness, and multidrug resistance. In the present study, we propose to use a dimeric fragment of the Shiga toxin B-subunit (StxB) to selectively target Gb3-positive cancer cells in a StxB-scFv UCHT1 lectibody. The lectibody, comprised of a lectin and the UCHT1 antibody fragment, was produced in E. coli and purified via Ni-NTA affinity chromatography. Specificity of the lectibody towards Gb3-positive cancer cell lines and specificity towards the CD3 receptor on T cells, was assessed using flow cytometry. We evaluated the efficacy of the lectibody in redirecting T cell cytotoxicity towards Gb3-overexpressing cancer cells in luciferase-based cytotoxicity in vitro assays. The StxB-scFv UCHT1 lectibody has proven specific for Gb3 and could induce the killing of up to 80% of Gb3-overexpressing cancer cells in haemorrhagic and solid tumours. The lectibody developed in this study, therefore, highlights the potential that lectibodies and lectins in general have for usage in immunotherapeutic approaches to boost the efficacy of established cancer treatments.

Targeting Solid Cancers with a Cancer-Specific Monoclonal Antibody to Surface Expressed Aberrantly O-glycosylated Proteins.

The lack of antibodies with sufficient cancer selectivity is currently limiting the treatment of solid tumors by immunotherapies. Most current immunotherapeutic targets are tumor-associated antigens that are also found in healthy tissues and often do not display sufficient cancer selectivity to be used as targets for potent antibody-based immunotherapeutic treatments, such as chimeric antigen receptor (CAR) T cells. Many solid tumors, however, display aberrant glycosylation that results in expression of tumor-associated carbohydrate antigens that are distinct from healthy tissues. Targeting aberrantly glycosylated glycopeptide epitopes within existing or novel glycoprotein targets may provide the cancer selectivity needed for immunotherapy of solid tumors. However, to date only a few such glycopeptide epitopes have been targeted. Here, we used O-glycoproteomics data from multiple cell lines to identify a glycopeptide epitope in CD44v6, a cancer-associated CD44 isoform, and developed a cancer-specific mAb, 4C8, through a glycopeptide immunization strategy. 4C8 selectively binds to Tn-glycosylated CD44v6 in a site-specific manner with low nanomolar affinity. 4C8 was shown to be highly cancer specific by IHC of sections from multiple healthy and cancerous tissues. 4C8 CAR T cells demonstrated target-specific cytotoxicity in vitro and significant tumor regression and increased survival in vivo. Importantly, 4C8 CAR T cells were able to selectively kill target cells in a mixed organotypic skin cancer model having abundant CD44v6 expression without affecting healthy keratinocytes, indicating tolerability and safety.

Aberrant Glycosylation as Immune Therapeutic Targets for Solid Tumors.

Glycosylation occurs at all major types of biomolecules, including proteins, lipids, and RNAs to form glycoproteins, glycolipids, and glycoRNAs in mammalian cells, respectively. The carbohydrate moiety, known as glycans on glycoproteins and glycolipids, is diverse in their compositions and structures. Normal cells have their unique array of glycans or glycome which play pivotal roles in many biological processes. The glycan structures in cancer cells, however, are often altered, some having unique structures which are termed as tumor-associated carbohydrate antigens (TACAs). TACAs as tumor biomarkers are glycan epitopes themselves, or glycoconjugates. Some of those TACAs serve as tumor glyco-biomarkers in clinical practice, while others are the immune therapeutic targets for treatment of cancers. A monoclonal antibody (mAb) to GD2, an intermediate of sialic-acid containing glycosphingolipids, is an example of FDA-approved immune therapy for neuroblastoma indication in young adults and many others. Strategies for targeting the aberrant glycans are currently under development, and some have proceeded to clinical trials. In this review, we summarize the currently established and most promising aberrant glycosylation as therapeutic targets for solid tumors.

Supramolecular complex glycoconjugate vaccine generates self-enhancement effects for carbohydrate antigen delivery

Targeting delivery of tumor-associated carbohydrate antigen (TACA)-based vaccine to antigen-presenting cells (APCs) mediated by endogenous antibodies can improve the immunogenicity of TACA. However, an essential requirement of this approach is to generate high titers of endogenous antibodies in vivo through pre-immunization, which complicates the immunization procedure and may cause side effects. Herein, we report a new generation of APC-targeting TACA-based supramolecular complex vaccine, assembled by sialyl Thomsen-nouveau-bovine serum albumin-adamantine (sTn-BSA-Ada) and heptavalent rhamnose (Rha)-modified β-cyclodextrin (β-CD) via host–guest interaction. The complex vaccine retained anti-Rha antibodies recruiting capability and facilitated the APCs uptake of the vaccine via the interaction of the Fc-domain with the Fc receptors on APCs. We demonstrate that direct immunization of complex vaccine elicited anti-Rha and anti-sTn specific immune response synchronously, generating a novel self-enhancement effect that can improve the antigen delivery to APCs in high efficacy. The structure–activity relationship (SAR) study proved that complex vaccine 4 with polyethylene glycol 6 (PEG6) linker in host molecule provoked a robust and specific sTn immune response comparable to the pre-immunization approach. The antisera induced by complex vaccine, either through direct immunization or pre-immunization, exhibited equal potency of cytotoxicity against the sTn expression cancer cells. This study provides a general platform for TACA-based vaccines with self-enhancement effects without the need for pre-immunization.

MUC1 Glycopeptide Vaccine Modified with a GalNAc Glycocluster Targets the Macrophage Galactose C-type Lectin on Dendritic Cells to Elicit an Improved Humoral Response.

Mucin expression and glycosylation patterns on cancer cells differ markedly from healthy cells. Mucin 1 (MUC1) is overexpressed in several solid tumors and presents high levels of aberrant, truncated O-glycans (e.g., Tn antigen). Dendritic cells (DCs) express lectins that bind to these tumor-associated carbohydrate antigens (TACAs) to modulate immune responses. Selectively targeting these receptors with synthetic TACAs is a promising strategy to develop anticancer vaccines and to overcome TACA tolerance. In this work, we prepared, via a solid phase peptide synthesis approach, a modular tripartite vaccine candidate, incorporating a high-affinity glycocluster based on a tetraphenylethylene scaffold, to target the macrophage galactose-type lectin (MGL) on antigen presenting cells. MGL is a C-type lectin receptor that binds Tn antigens and can route them to human leukocyte antigen class II or I, making it an attractive target for anticancer vaccines. Conjugation of the glycocluster to a library of MUC1 glycopeptides bearing the Tn antigen is shown to promote uptake and recognition of the TACA by DCs via MGL. In vivo testing revealed that immunization with the newly designed vaccine construct bearing the GalNAc glycocluster induced a higher titer of anti-Tn-MUC1 antibodies compared to the TACAs alone. Additionally, the antibodies obtained bind a library of tumor-associated saccharide structures on MUC1 and MUC1-positive breast cancer cells. Conjugation of a high-affinity ligand for MGL to tumor-associated MUC1 glycopeptide antigens has a synergistic impact on antibody production.

Galectin-3 and Epithelial MUC1 Mucin-Interactions Supporting Cancer Development.

Aberrant glycosylation of cell surface proteins is a very common feature of many cancers. One of the glycoproteins, which undergoes specific alterations in the glycosylation of tumor cells is epithelial MUC1 mucin, which is highly overexpressed in the malignant state. Such changes lead to the appearance of tumor associated carbohydrate antigens (TACAs) on MUC1, which are rarely seen in healthy cells. One of these structures is the Thomsen-Friedenreich disaccharide Galβ1-3GalNAc (T or TF antigen), which is typical for about 90% of cancers. It was revealed that increased expression of the T antigen has a big impact on promoting cancer progression and metastasis, among others, due to the interaction of this antigen with the β-galactose binding protein galectin-3 (Gal-3). In this review, we summarize current information about the interactions between the T antigen on MUC1 mucin and Gal-3, and their impact on cancer progression and metastasis.

Altered Glycosylation in Progression and Management of Bladder Cancer.

Bladder cancer (BC) is the 10th most common malignancy worldwide, with an estimated 573,000 new cases and 213,000 deaths in 2020. Available therapeutic approaches are still unable to reduce the incidence of BC metastasis and the high mortality rates of BC patients. Therefore, there is a need to deepen our understanding of the molecular mechanisms underlying BC progression to develop new diagnostic and therapeutic tools. One such mechanism is protein glycosylation. Numerous studies reported changes in glycan biosynthesis during neoplastic transformation, resulting in the appearance of the so-called tumor-associated carbohydrate antigens (TACAs) on the cell surface. TACAs affect a wide range of key biological processes, including tumor cell survival and proliferation, invasion and metastasis, induction of chronic inflammation, angiogenesis, immune evasion, and insensitivity to apoptosis. The purpose of this review is to summarize the current information on how altered glycosylation of bladder cancer cells promotes disease progression and to present the potential use of glycans for diagnostic and therapeutic purposes.