Glycosylation is a complex form of post-translational modification that affects more than 50% of cellular proteins and is a key regulator of many eukaryotic processes. Aberrant glycosylation is a common feature of many cancers and plays a crucial role in all stages of tumor development. Glycosylation can regulate the occurrence and progression of tumors and control a variety of physiological and pathological processes, such as cell-cell adhesion, cell-matrix interaction, epithelial-mesenchymal transition, tumor proliferation, invasion, metastasis and angiogenesis. Different aberrant glycosylation mechanisms lead to the formation of tumor-associated carbohydrate antigens (TACA) suitable for selective tumor-targeted therapy. The best characterized TACAs include truncated O-glycans (Tn, TF, and sialyl Tn antigens), gangliosides (GD2, GD3, GM2, GM3, fucosyl-GM1), globosides ( globo-H, SSEA-3, SSEA-4), Lewis antigen and polysialic acid.
For more than two decades, these TACAs have shown potential application in cancer immunotherapy strategies. Glycans can serve as a source for the development of novel clinical biomarkers, providing a range of specific targets for therapeutic intervention. At the same time, TACA can interact with antigen-presenting cells through interactions with glycan-binding receptors (such as SIGLECs, MGL, DC-SIGN), thereby inducing immunosuppressive signals. Therapeutic approaches involving TACA vary, including vaccine-induced active immunity, and genetically engineered monoclonal antibodies. Recently, bispecific antibodies and chimeric antigen receptor T (CAR-T) cells, including those for anti-TACA, have been evaluated clinically and have made impressive progress.
Targeting Glycosphingolipids
Glycosphingolipids (GSLs) are composed of a heterogeneous group of membrane lipids formed by a ceramide backbone covalently linked to a glycan moiety through β-glycosidic bonds. GSL is a universal component of cell membranes, particularly abundant on animal cell surfaces, and plays an important role in molecular signaling, cell adhesion and movement. In vertebrate tissues, they are subdivided into three main series: the globular, ganglionic, and neoarteritis series. The major GSLs characterized by TACA include Globo H, SSEA-3, and SSEA-4, as well as sialic acid-containing glycosphingolipids such as gangliosides GD2, GD3, GM2, fucosyl GM1, and Neu5GCM3. These GSLs can influence cancer progression by controlling cell adhesion, motility and growth, epithelial-to-mesenchymal transition, metastasis, and drug resistance.
- GD2
Ganglioside GD2 is an N-acetylneuraminic acid-containing glycolipid antigen, composed of five monosaccharides, anchored to the lipid bilayer of the plasma membrane through ceramide lipids. The expression level of GD2 in normal tissues is low, but in various types of cancers such as neuroblastoma, small cell lung cancer, melanoma, glioma and sarcoma, the expression of this weak immunogenic antigen can reach 107 molecules per cell. In addition, GD2 has been found to be a potential biomarker for breast cancer stem cells. GD2 can induce tyrosine phosphorylation and activate multiple kinase pathways, leading to enhanced proliferation, migration and invasion capabilities of cancer cells.
Several anti-GD2 monoclonal antibodies have been successfully tested in clinical practice. Dinutuximab (Unituxin™), approved by the U.S. FDA in 2015 for the treatment of high-risk pediatric neuroblastoma, is used in combination with GM-CSF, IL-2 and 13-cis retinoic acid. In addition, BsAbs targeting GD2 have broad prospects in anti-cancer therapy. Some of the BSAbs that bind anti-GD2 and anti-immune cell receptors include 7A4-mAb/FcγRI, 5F11-scFV/CD3, hu3F8-scFV/CD3, and hu3F8-mAb/CD3. Regarding GD2-based tumor vaccines, a bivalent vaccine containing GD2 and GD3 linked to KLH, adjuvanted with OPT-821, was evaluated for the first time in patients with high-risk neuroblastoma in a phase I trial.
- GD3
GD3 is another disialoganglioside that is highly expressed in cancers of neuroectodermal origin. GD3 is considered a marker of neuroectodermal origin in tumors, and several monoclonal antibodies have been developed, such as R24, 2B2, IF4, MG-21, and K641. Multiple clinical trials have been conducted using R24 alone or in combination with other drugs, showing varying clinical response rates. PF-06688992, a human R24-based ADC, is in a Phase I clinical trial in patients with stage III-IV melanoma, with results to be announced. Ecromeximab, another monoclonal antibody with high affinity for GD3, has demonstrated safety in preclinical and phase I studies. However, in a subsequent phase II study of metastatic melanoma, ecromeximab was combined with high-dose IFN-α2, although the overall tolerance was good and the immunogenicity was low, the clinical benefits of this combination were limited. Regarding vaccine strategies, an idiotype monoclonal antibody (BEC2) against well-characterized anti-R24 was developed and demonstrated that BEC2 could mimic GD3 ganglioside and induce anti-GD3 IgG in rabbits.
- Fucosyl-GM1
Ganglioside fucosyl-GM1 (FucGM1) is a tumor-associated antigen that is highly expressed in most human small cell lung cancers but is missing in most normal adult tissues, making FucGM1 an attractive target. Recently, a new nonfucosylated fully human IgG1 antibody (BMS-986012) was developed that is specific for FucGM1. BMS-986012 was evaluated in a Phase I/II clinical trial as a first-line treatment for patients with extensive-stage SCLC. The preliminary results of another phase I/II study evaluating BMS-986012 monotherapy or in combination with nivolumab in patients with relapsed/refractory small cell lung cancer showed that this monoclonal antibody was well tolerated and showed anti-tumor activity in some patients. Finally, an ongoing phase II study is evaluating whether BMS-986012 in combination with carboplatin, etoposide, and nivolumab can improve overall survival in patients with newly diagnosed extensive-stage SCLC.
- GM3
Ganglioside GM3 is widely distributed in almost all types of animal cells and is overexpressed in several human cancers, such as melanoma, lung and brain cancer. Several GM3 (Neu5Gc)-targeting antibodies have been developed, the most characterized being the mouse IgG1 monoclonal antibody 14F7 and its humanized version 14F7hT. 14F7hT showed potent in vivo antitumor activity in solid mouse myeloma models as well as SKOV3 human ovarian cancer and Lewis lung cancer mouse tumors. One strategy for vaccine development is to use the hydrophobic outer membrane protein of Neisseria meningitidis containing GM3 to form small-size proteoliposomes (VSSP) (GlycoVaxGM3-NeuGcGM3/VSSP). The vaccine is reported to be safe and immunogenic. In melanoma patients, the GlycoVaxGM3 vaccine improved overall survival in patients with metastasis after first-line chemotherapy.
- Globo Series
The Globo family of GSLs, such as stage-specific embryonic antigen 3 (SSEA-3), SSEA-4 and globo-H, are specifically expressed on pluripotent stem cells and cancer cells. Among them, the most common cancer-associated antigen is Globo-H ceramide (GHCer), which is overexpressed in several cancers, including breast, gastric, lung, ovarian, endometrial, pancreatic, and prostate cancers. A new antibody-drug conjugate (OBI-999) derived from an anti-Globo-H monoclonal antibody, conjugated to MMAE, has shown good tumor inhibitory effects in different animal models. Two phase I/II clinical trials are currently underway to evaluate the safety, pharmacodynamics and therapeutic activity of anti-Globo-H monoclonal antibody OBI-888 in multiple advanced and metastatic solid tumors, and the safety, pharmacodynamics and therapeutic activity of ADC OBI-999 in advanced solid tumors.
Targeting Mucin O-Glycan Antigens
Simple mucin-type O-glycosylated TACAs, such as Tn antigen (CD175), TF antigen (CD176), and sialyl Tn antigen (STn, CD175s), are very attractive targets for anti-tumor therapy because they are detected in most cancers and usually not present in healthy tissues.
- Tn Antigen
Several monoclonal antibodies that recognize Tn antigens have been generated using different immunization strategies, such as PankoMab, 5E5, KM3413, and 6C5. PankoMab and 5E5 specifically recognize the Tn-MUC1 peptide, while 6C5 specifically recognizes the Tn peptide epitope in dysadherin/FXYD5. PankoMab GEX™ is a fully humanized antibody derived from PankoMab that is glycoengineered to enhance Fc-mediated anti-tumor activity. Phase 1 clinical trials in patients with advanced solid tumors, primarily colon and ovarian cancer, showed the drug was safe and well-tolerated. After promising initial efficacy in patients with ovarian cancer, a phase II study was performed on patients with recurrent ovarian cancer, fallopian tube cancer, or primary peritoneal cancer to evaluate the efficacy of PankoMab GEX TM versus placebo in maintaining response after chemotherapy. However, this study failed to demonstrate improvement in progression-free survival. Another solid tumor Phase I trial evaluated PankoMabGEX™ in combination with an anti-EGFR antibody, demonstrating feasibility and anti-tumor activity in patients with colorectal cancer (CRC) and non-small cell lung cancer.
- Sialyl-Tn Antigen
STn is often co-expressed with Tn, and aberrant expression of STn is associated with dysregulation of the O-glycosylation machinery, including unbalanced expression of Cosmc and STn synthetase (ST6GalNAc-I), leading to more aggressive cancer cell behavior and reduced cell-cell aggregation, increasing tumor growth, extracellular matrix adhesion, migration, invasion and metastasis. A humanized monoclonal antibody (huCC49) targeting sTn has been evaluated in a phase I/II radioimmunotherapy clinical trial. Intraperitoneal radioimmunotherapy with 177Lu-CC49 was well tolerated and appeared to have antitumor activity against intraperitoneal chemotherapy-resistant ovarian cancer. In a phase II clinical trial, radioimmunotherapy of hormone-resistant metastatic prostate cancer with 131I-CC49 combined with IFN enhanced tumor uptake and anti-tumor effects compared with a previous phase II trial of 131I-CC49 alone.
- TF Antigen
TF is present at relatively low levels in many normal tissues, but is found at high levels in cancerous tissues and is associated with invasiveness, tumor growth, and high metastatic potential. JAA-F11 is an IgG3 monoclonal antibody that specifically targets TF-α and has shown tumor growth inhibition and lung metastasis reduction in the breast cancer metastasis model 4T1. A humanized form of JAA-F11 (hJAA-F11) is present in a xenograft model of human breast cancer in SCID mice. Both the naked antibody and the ADC form (hJAA-F11-DM1) inhibit tumor progression in vivo.
Targeting Lewis Antigen
Lewis antigens type I and II are terminally fucosylated carbohydrate structures belonging to the human tissue blood group system. The Lewis antigen and its sialylated form are largely associated with disease progression and dissemination in cancer patients. As cancer-associated antigens, they may become promising targets for new approaches to personalized medicine. Sialyl Lewis A (SLeA) has attracted interest as a therapeutic target, and fully human monoclonal antibodies have been developed, showing improved overall survival in animal models. The antibody, called MVT-5873, demonstrated safety and tolerability on its own when evaluated in pancreatic cancer in combination with gemcitabine and nab-paclitaxel. In addition, CA19-9-positive malignancies are further evaluated in combination with FOLFIRINOX. Another antibody targeting sialic acid LewisA is FG129, and its ADC forms (DM1 and DM4) have shown promising results in preclinical models.
Targeted Polysialic Acid
The glycan modification process attaches monomeric sialic acid entities to the non-reducing end of the glycan tree to form a polysialic acid tail (PSA). Although PSA is almost non-existent in most adult tissues, it is expressed in the progression of some malignant tumors, such as neuroblastoma, breast cancer, laryngeal squamous cell carcinoma, pancreatic cancer, non-small cell lung cancer and small cell lung cancer. The most important physical property of PSA is its anti-adhesion effect, which is beneficial to invasion and metastasis. In addition, the interaction between PSA and inhibitory siglec promotes the immune evasion of tumor cells. At present, several highly specific monoclonal antibodies have been developed: MY.1E12 and MAB735.
The glycan patterns expressed in cancer cells have a significant impact on tumor behavior, and the overexpression of TACA on a variety of cancer cells makes it an attractive target for immunotherapy development. Currently, a variety of TACA-targeting antibodies, ADCs, CAR-T and BsAb have entered the clinical stage, synergizing with other anti-tumor strategies and showing strong therapeutic potential.
References
- Berois, N. et al. Targeting Tumor Glycans for Cancer Therapy: Successes, Limitations, and Perspectives. Cancers(Basel). 2022, 14(3): 645.
- Huang, X.F. et al. Recent advances in tumor associated carbohydrate antigen based chimeric antigenreceptor T cells and bispecific antibodies for anti-cancer immunotherapy. SeminImmunol. 2020, 47: 101390
- Morandi, F. et al. Immunotherapeutic Strategies for Neuroblastoma: Present, Past and Future. Vaccines. 2021, 9: 43.