l-fucose, a sugary regulator of antitumor immunity and immunotherapies

Abstract

l-fucose is a dietary sugar that is used by cells in a process called fucosylation to post-translationally modify and regulate protein behavior and function. As fucosylation plays essential cellular functions in normal organ and immune developmental and homeostasis, it is perhaps not surprising that it has been found to be perturbed in a number of pathophysiological contexts, including cancer. Increasing studies over the years have highlighted key roles that altered fucosylation can play in cancer cell-intrinsic as well as paracrine signaling and interactions. In particular, studies have demonstrated that fucosylation impact tumor:immunological interactions and significantly enhance or attenuate antitumor immunity. Importantly, fucosylation appears to be a posttranslational modification that can be therapeutically targeted, as manipulating the molecular underpinnings of fucosylation has been shown to be sufficient to impair or block tumor progression and to modulate antitumor immunity. Moreover, the fucosylation of anticancer agents, such as therapeutic antibodies, has been shown to critically impact their efficacy. In this review, we summarize the underappreciated roles that fucosylation plays in cancer and immune cells, as well as the fucosylation of therapeutic antibodies or the manipulation of fucosylation and their implications as new therapeutic modalities for cancer.

1 ∣. INTRODUCTION

The monosaccharide l-fucose (also known as 6-deoxy-l-galactose) is a dietary sugar that can play important roles in cellular biology. Although similar to hexose sugars such as glucose, l-fucose is structurally distinct in its l-configuration and lack of a hydroxyl group on the carbon at the 6-position (C-6). Also distinct is the way in which it is used by cells as a substrate that is conjugated as a posttranslational modifier onto proteoglycans (proteins subject to glycosylation, also known as glycoproteins). This process, called fucosylation, can regulate crucial aspects of protein behavior and function, and thus, cellular biology. Although l-fucose and fucosylation have been established to be essential for normal immune and organ development processes, precise underlying molecular mechanisms have largely not been delineated. Increasing numbers of studies have begun to elucidate and highlight fucosylation-regulated mechanisms that govern key aspects of pathogenic biology in cancer. In particular, numerous emerging studies highlight important pathophysiological roles and functions of l-fucose and fucosylation in tumor immunology. Importantly, unlike other posttranslational modifications (e.g., phosphorylation), the systemic augmentation or suppression of fucosylation using dietary l-fucose or small molecule inhibitors, respectively, has been shown to yield significant therapeutic potential. However, the precise extents of antitumor (or protumor) effects elicited by modulation of fucosylation appear to be tumor context-specific and remain to be explored and refined. Here, we review important findings regarding the roles of fucosylation in tumor biology and tumor immunology and highlight therapeutic insights and potential opportunities for much needed honing and development of fucosylation-centered modalities.

2 ∣. FUCOSYLATION: REGULATION OF SUBSTRATE AVAILABILITY AND STRUCTURE-FUNCTION

The process by which cells uptake, guanidine diphosphate (GDP)-couple, and conjugate l-fucose as a posttranslational modifier of proteins is called fucosylation. This process is regulated at the substrate availability and downstream structure–function levels (Figure 1).

FIGURE 1.

l-fucose (red triangle) uptaken into cells is phosphorylated by FUK and GDP-coupled (light green circle) by GFPP, yielding GDP-fucose, which is transported into the ER/Golgi via SLC35C1/2 transporters, where it is conjugated onto proteins by 13 ER/Golgi-resident fucosyltransferases (FUTs). GDP-fucose can also be supplied in the cell by conversion from GDP-mannose (dark green circle) by the GMD/FX enzymes. FUK, GFPP, and SLC35C1/2 regulate global GDP-fucose availability (i.e., functional amplitude of fucosylation), whereas the FUTs are structure–function determining, and in the context of tumors, delineate tumor-promoting versus tumor-suppressing functions. ER, endoplasmic reticulum; FUK, fucokinase; FS, GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase; GDP, guanidine diphosphate; GFPP, GDP-fucose-pyrophosphorylase, GMD, GDP-mannose 4,6-dehydratase. The figure was created with BioRender.com

The first step of fucosylation comprises the sourcing of cellular l-fucose, from either endogenous or exogenous sources. Endogenous free l-fucose is derived from intracellular recycling via hydrolysis and liberation during the recycling of proteoglycans in lysosomes.^1,2 In contrast, exogenous (dietary) l-fucose is found in a variety of plants. It has been detected in the cell walls of potato (Solanum tuberosum),³ cassava tuber (Manihot utilissima),⁴ kiwi (Actinidia arguta),⁵ and Thale cress (Arabidopsis thaliana)⁶; in seeds including soybeans (Glycine max),⁷ Winged beans (Psophocarpus tetragonolobus),⁸ and Rapeseed (Brassica napus)⁹ in mucilage of young leaves and stems of “Junsai” (Brasenia schreberi).¹⁰ Notably, l-fucose has been reported to be enriched at significantly higher amounts in seaweed in the form of sulfated l-fucose polymers known as fucoidan. Fucoidan, which has been subject to extensive commercialization as a nutraceutical supplement for an array of potential health-promoting purposes,¹¹ is found in the intercellular mucilage of several species of seaweed. Seaweeds that have been used as sources of fucoidan have included Ecklonia kurome,^12-14 Laminaria angustata var longissimi,¹⁵ Fucus vesiculosus,¹⁶ Kjellmaniella crassifolia,¹⁷ and Fucus serratus.¹⁸

Regardless of the source, l-fucose must be phosphorylated and then coupled to GDP, producing GDP-fucose, for subsequent conjugation onto proteins. Intracellular levels of GDP-fucose are maintained by two distinct pathways, the salvage and the de novo synthetic pathways. Via the salvage pathway, free l-fucose in the cytosol is phosphorylated by fucokinase (FUK), and the intermediate, fucose-1-phosphate, is further converted to GDP-fucose by GDP-fucose-pyrophosphorylase (GFPP).^19,20 The salvage pathway is responsible for compensating and replenishing GDP-fucose to sustain physiological functions when the de novo pathway is defective.²¹

The de novo synthetic pathway is a highly conserved process by which intracellular GDP-mannose is converted into GDP-fucose (representing another endogenous source of l-fucose-derived substrate). In the cytoplasm, GDP-fucose is converted from GDP-mannose by GDP-mannose 4,6-dehydratase (GMD) and GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase (FX) sequentially.^22,23 This mechanism has been reported to produce up to 80%–90% of cellular GDP-fucose under “normal” (i.e., nondisease/pathological) circumstances,^24,25 although the relative contribution of either de novo or salvage pathways to total intracellular GDP-fucose levels likely varies by pathophysiological and cell type-specific context. Together, by regulating fucosylation at the substrate level, the de novo synthetic and the salvage pathways regulate the functional amplitude of cellular fucosylation.

Once GDP-fucose is transported into the lumen of Golgi apparatus and endoplasmic reticulum (ER) via the SLC35C1 and SLC35C2 transporters,²⁶ it serves as a substrate that is conjugated onto proteins on N-glycans by 11 fucosyltransferases (FUTs) in the Golgi^27,28 and/or on O-glycans by two protein O-fucosyltransferases (POFUTs) in the ER.^29,30 The 13 different FUTs catalyze a diverse range of fucose-conjugated structures. There are two well-established types of N-linked fucosylated structures, core fucosylation (α-1,6-linkage) catalyzed by FUT8 and terminal fucosylation (α-1,2/3/4-linkage) catalyzed by the other FUTs (FUT1–7 and FUT9–11), among which FUT1 and FUT2 are responsible for the synthesis of α-1,2 linkages, while FUT3–7/9–11 are involved in the generation of α-1,3/4 linkages.^27,31,32 The diverse topologies of fucosylation are structure–function-regulating and therefore can result in diverse functional consequences. In cancer, for example, the profile of FUT expression delineates whether fucosylation elicits tumor-promoting or tumor-suppressing effects.

3 ∣. ABERRANT FUCOSYLATION IN CANCER

Protein fucosylation is crucial for maintaining the “normal” cell and tissue physiology and functions, including, for example, cellular adhesion, organ and immune development, and fertilization (Table 1). Suppression of global substrate-level fucosylation in mice (by FX knockout, which abrogates the de novo synthetic pathway) results in severe neutrophilia, myeloproliferation, and the absence of leukocyte selectin ligand expression.²³ In melanoma, the downregulation of FUK also significantly enhances motility while altering tumor immunological profiles.³³ Aberrant fucosylation resulting from abnormal or pathologically deregulated expression of specific FUTs has been reported to result in several pathological processes, such as inflammatory conditions, malignancy, and tumor metastasis (Table 1). In the context of cancer, our understanding of the specific mechanistic roles of FUTs has been generally limited, although increasing studies showcase the wide range of contributions that FUTs make to the pathogenesis of cancer.

TABLE 1.

Roles of fucosylation in normal cellular/organ development and biology versus cancer

Organ/tissue	Normal cell/organ biology	FUTs	References (PMID)	Cancer	FUTs	References (PMID)
Peripheral blood					FUT4	31097000
			24838610		POFUT1	30344988
	Leukocyte trafficking and homing	FUT1/2	8752218	Myeloid leukemia		20645416
		FUT4/7	8822932		FUT8	27967290
			11485743	Lymphoma	-	23493549
				MM	-	30844485
Breast	-	-	-	Breast cancer	FUT1	27560716
						27375041
					FUT4	23887626
						26701615
						28692034
					FUT8	28982386
Lung	Pulmonary development	FUT8	16236725	NSCLC	FUT4	32629386
						31104223
					FUT7	28860805
					FUT8	26443198
						23267084
Liver	Hepatic development	FUT2	28056490	Hepatocellular carcinoma	FUT4/6/8	24232099
					FUT7	11935309
						15666088
					FUT8	23352314
Brain	Synapse formation and neurite outgrowth	FUT1/2	15686343	Glioblastoma		29669750
					FUT8	25727145
Gastrointestinal system						33323540
	Host–microbe interactions	FUT2	17673542	Gastric cancer	FUT3/5	21978830
	Gut microbiome maintaining	FUT3	25263220		FUT8	24732908
			25274297	Colorectal cancer	FUT1	17459061
			28988527		FUT3/5/6	24253505
						28771224
					POFUT1	30250219
						9000578
Skin	Keratinocyte migration (differentiation and epidermis renewal)	FUT1	23625752	Melanoma	FUT1	29924834
						17897065
					FUT4	25672851
						31961483
					FUT8	28609658
Prostate	Origination of Lewis/Core-type Fuco-PSAs	FUT3/8	27494861	Prostate cancer	FUT3/6/7	24178760
					FUT8	24906821
Pancreatic	Lewis b, Lewis y, and H-type antigens	FUT1/2	10728712	Pancreatic cancer	FUT1	10728712
					FUT3	11058871
						29963165
Oral/head and neck	-	-	-	Oral squamous cell carcinoma	FUT1	23242548
					FUT3/6	25428916
Ovary						29130097
					FUT1	20003467
	-	-	-	Ovarian cancer		27240592
					FUT8	11093814

Abbreviations: FUT, fucosyltransferase; MM, multiple myeloma; NSCLC, non-small cell lung cancer; POFUT, protein O-fucosyltransferase; PSA, prostate-specific antigen.

The most extensively characterized aberration in FUT function in cancer is FUT8-mediated core fucosylation. FUT8 is frequently upregulated in human tumors and is associated with worse clinical outcomes in cancer patients.^34,35 The crucial role of FUT8 in tumorigenesis has been clearly demonstrated in studies of breast, liver, lung cancer, and melanoma showing that depletion of FUT8 effectively blocks tumor growth and metastasis.^36-39 Based on its protumorigenic function, extensive studies have highlighted corefucosylated proteins, suggesting their clinical utility as potential diagnostic and prognostic biomarkers in a wide range of cancer types (Table 2). In addition to core fucosylation, increasing studies report that α-1,3/4 fucosylation branching catalyzed by FUTs 3–7 and 9–11 is also upregulated in tumors, eliciting tumor-promoting functions when further assessed in cell-based assays and in vivo models (Table 1). Specifically, Lewis antigen structures comprising α-1,2/3/4-linkage fucosylation, which are known to decorate tumor cell membrane proteins, have been demonstrated to promote cellular adhesion and extravasation in the process of metastasis through binding to E/P-selectin expressed on endothelial cells.⁴⁰ Consistent with this notion, FUTs that mediate this type of fucosylation topology, such as FUT4, are altered in cancers and promote tumorigenic events such as epithelial–mesenchymal transition.⁴¹ Conversely, α-1,2 terminal fucosylation mediated by FUT1–2 was reported to exhibit tumor-suppressive effects in melanoma, oral/head and neck, gastric, and hepatocellular carcinoma, whereas it promotes tumor progression in bladder, breast, epidermoid, ovarian, and prostate tumors. Our understanding of how FUTs regulate cancer development and progression is an actively growing area, it is becoming increasingly clear that their contributions are determined by specific expression profiles in different cancer types, which will dictate the functional balance of tumor-promoting versus tumor-suppressing forms of fucosylation.

TABLE 2.

Fucosylated glycoproteins as diagnostic/prognostic markers in cancer

Fucosylated biomarker	Type of cancer	FUTs	References (PMID)
EGFR	Lung cancer	FUT8	18754874
			21709263
PSA	Prostate cancer	FUT8	20861084
			17432893
AGP	Lung cancer	FUT6	31601857
	Esophagus, stomach, lung, breast, liver, pancreas, colon and rectum carcinomas	α(1,3) linkage	23509786
			27295180
			15536618
AFP	Hepatocellular carcinoma	FUT8	2415789
			31451706
			24899827
A1AT	Lung cancer	FUT8	25347993
	Hepatocellular carcinoma	α(1,2/3/4) linkage	20811639
	Ovarian cancer		3265332
HP	Pancreatic cancer	FUT8	16385567
		α(1,3/4) linkage	18646007
	Colorectal Cancer		22006099
	Gastric cancer		28052004
	NSCLC		24018448
	Prostate cancer		24802742
	Ovarian cancer		1511415
			1451094
	Breast cancer		18218651
	Hepatocellular carcinoma		24807840
			26503433
PON1	Hepatocellular carcinoma	FUT8	25702281
			22751611
	SCLC		24085812
			30619732
GM1	SCLC	FUT1/2	16880505
			9334808
E-cadherin	Lung cancer	FUT8	22634079
	Colorectal Cancer		19302290
CD147	OSCC	FUT8	23078850
	ESCC		32474852
CA19-9	Pancreatic cancer	FUT3	31221853
CP	Hepatocellular carcinoma	FUT8	24799124
	Pancreatic cancer	Sialyl-Lewis X	25595436
Fetuin-A	Cholangiocarcinoma	α(1,3/6) linkage	28561948
L1CAM	Melanoma	FUT8	28609658

Abbreviations: A1AT, α-1-antitrypsin; AFP, α-fetoprotein; AGP, α1-acid glycoprotein; CP, ceruloplasmin; EGFR, epidermal growth factor receptor; ESCC, esophageal squamous cell carcinoma; FUTs, fucosyltransferases; GM1, monosialotetrahexosylganglioside; HP, haptoglobin; L1CAM, L1 cell adhesion molecule; NSCLC, non-small cell lung cancer; OSCC, oral squamous cell carcinoma; PON, paraoxonase 1; PSA, prostate-specific antigen; SCLC, small cell lung cancer.

4 ∣. ROLES OF FUCOSYLATION AT THE TUMOR:IMMUNE INTERFACE

Importantly, whereas studies on the roles of FUTs and fucosylation in cancer have largely focused on tumor cell autonomous signaling, fucosylation plays key functional roles in other cells within the tumor microenvironment—particularly immune cells—that are significant determinants of tumor progression and therapeutic responses. Immunotherapies are among the most effective emerging treatment modalities for many tumor types. However, across all cancers, their efficacy is hampered by a number of underlying reasons ranging from the paucity of tumor-infiltrating immune cells to the lack of target protein expression to immunosuppressed tumor microenvironment. These therapeutic hurdles are areas of active study aimed at increasing immunotherapy efficacy. Fucosylation can mechanistically regulate aspects of immune cell biology that are highly relevant to the mechanisms underlying multiple types of immunotherapies. However, this remains an emerging area and our increased understanding of how fucosylation regulates tumor:immune interactions and immunology is expected to help delineate how fucosylation might itself be leveraged as a therapeutic approach to help improve immunotherapeutic efficacy and clinical outcomes.

Interactions between tumor and immune cells are fundamentally governed by the maturation and differentiation of individual immune cell types—processes that are stringently orchestrated to ensure specific, timely, and durable innate and adaptive immune responses. To precisely control such aspects of immune cell biology, the cells implement diverse posttranslational modifications to rapidly and precisely establish spatiotemporal control of key proteins that can regulate and alter these processes. As stated above, one such modifier is the addition of l-fucose to proteins through the process of fucosylation. Unlike other posttranslational modifications including phosphorylation, ubiquitination, degradation, or proteolytic processing, which have been subject to extensive study in the context of tumor microenvironments and their potential impact on immunotherapy efficacy, fucosylation is another important form of posttranslational modification, the contributions of which have generally been underappreciated. Previous studies have highlighted a number of important roles that fucosylation plays in normal immune cell biology. These functions range from myeloid and lymphocyte development (e.g., thymic B- and T-cell maturation) to immune cell signaling and behaviors in several key immune cell lineages, demonstrating the broad regulatory impact of this modification in the immune system (Table 3).⁴²

TABLE 3.

Roles of fucosylation in immune cell biology

Immune cell lineage	Role of fucosylation	FUTs	References (PMID)
Immature B cell	Mediates BCR development via u heavy chain fucosylation	FUT8	22084235
			20622883
Hematopoietic stem cell	Pushes development toward leukocyte lineages via fucosylation of Notch	POFUT1	15653671
			15692013
			11524432
			21464368
Mature B cell	Promotes signaling of BCR	POFUT1	22269039
			17714731
			1688813
			16888140
Monocyte	Adhesion and migration of monocytes to areas of infection	FUT1/2	24665114
			10075584
Macrophages	Shifts polarization towards M1 phenotype	FUT1/2	24838610
Developing lymphocyte	Signals proper developmental patterns of T and B cells in the thymus. Suppresses B cells in the thymus	POFUT1	21464368
Neutrophils	E- and P-selectin-mediated adhesion and extravasation	FUT1/2	31530991
			11133780

Abbreviations: BCR, B-cell receptor; FUT, fucosyltransferase; POFUT, protein O-fucosyltransferase.

In addition to its contributions to normal immune cell development, the pathological deregulation of fucosylation is implemented in a number of immunological diseases. One well-established example is leukocyte adhesion deficiency ll (LAD-ll).^43-46 Patients suffering from LAD-ll possess mutations in the GDP-fucose transporter gene SLC35C1, which results in the attenuation of cellular protein fucosylation rates.^45,47 Although LAD-ll may manifest through several phenotypes, the leukocyte compartment is significantly impaired as these cells lose their ability to bind to vessel walls, leading to uncontrolled rolling of leukocytes and failure of leukocyte extravasation.⁴⁸ Patients with LAD-ll present with chronic bacterial infections due to the lack of immune infiltration that would normally deal with such infections.⁴⁹ Notably, the administration of l-fucose to children suffering from the disorder is sufficient to at least partially mitigate symptoms of the disease.⁵⁰ Another example of a fucosylation-related immune disease is rheumatoid arthritis (RA), a pathology that is characterized by joint pain resulting from immune-mediated destruction of cartilage.⁵⁰ Intriguingly, several studies have reported that RA patients generally exhibit reduced serum levels of l-fucose and fucosylated proteoglycans compared with healthy individuals, despite overexpression of all FUTs except FUT8 and FUT13 (also POFUT2), suggesting potentially altered substrate-level perturbations in fucosylation that play key roles in the generation and/or maintenance of immune tolerance in lymphocytes.^28,42,51-55

Whereas fucosylation has been demonstrated to play pivotal pathogenic roles in the progression of immunological diseases, less is known about the immunological roles of l-fucose and fucosylation in cancer pathogenesis. Moreover, in the context of tumor immunity, studies on the roles of fucosylation have predominantly focused on T cells.

To date, a number of studies—focusing largely on FUT8, which mediates tumor-promoting core fucosylation in tumor cells—have reported both tumor-promoting and tumor-suppressing effects of fucosylation in the context of tumor immunity. Several studies have highlighted the significant roles of fucosylation in the regulation of T-cell activity. FUT8 knockout mice exhibit impaired development and increased apoptosis of thymic T cells, indicating the requirement of core fucosylation for the development and viability.⁵⁶ Further, fucosylation of the CD4⁺ T-cell receptor (TCR), is required for full activation of CD4⁺ T cells.^57,58 A study by Fujii et al.⁵⁸ identified FUT8 as a FUT that fucosylates TCR, critically mediating CD4⁺ T activation; knockout of FUT8 significantly attenuates CD4⁺ T-cell activation. Consistent with the notion of pathological T-cell activation by aberrant fucosylation, core fucosylation of CD4⁺ TCR was reported to be increased in tissues of patients with irritable bowel syndrome and systemic lupus erythematosus.^57,58 Collectively, these findings indicate that FUT8-mediated core fucosylation is crucial for general T-cell development, viability, and CD4⁺ T-cell activation—and importantly, that aberrant TCR fucosylation is associated with the pathogenesis of specific immunological diseases.

However, core fucosylation has also been reported by Okada et al.⁵⁹ to conversely induce CD8⁺ T-cell exhaustion by stabilizing cell surface PD1. Moreover, Huang et al.⁶⁰ found that FUT8-mediated core fucosylation stabilizes cell surface B7H3 on tumor cells, enforcing multipronged immune checkpoint signaling to suppress T-cell activity in breast tumors. These findings illustrate the complex and divergent roles of fucosylation within the tumor immune microenvironment, including potentially differential effects on CD4⁺ versus CD8⁺ T cells, as well as on tumor and other stromal cells within the tumor microenvironment. Consistent with these possibilities, FUT8 has previously been reported to significantly impact both immune tolerance and T-cell autoreactivity.

Although the aforementioned studies focused exclusively on core fucosylation mediated by FUT8, it is important to recognize that the 12 other FUTs mediate a range of diverse fucosylated glycan structures. As the individual FUTs can elicit divergent effects in cancer cell-autonomous signaling and biology (Table 1), the precise expression profiles of the individual FUTs specific to individual immune cell subtypes is expected to similarly elicit different immunological effects mediated by the different fucosylated glycan structures. Consistent with these possibilities, in contrast to the CD8⁺ T-cell exhaustion mediated by FUT8, FUT7-mediated noncore fucosylation enhances CD8⁺ T-cell activity in models of leukemia, breast cancer, and melanoma.⁶¹

Thus, rather than a binary on/off signal, the impact of fucosylation on T cells (and other immune cells in the tumor microenvironment) likely exists as a multifaceted balance of regulation. Future studies are needed to address how subtle changes in the fucosylation landscape in immune cells can significantly change immunological outcomes.

5 ∣. AFUCOSYLATED THERAPEUTIC ANTIBODIES FOR THE TREATMENT OF CANCER

In addition to impacting the biology of tumor and immune cells, fucosylation of therapeutic molecules such as antibodies has been reported to impact efficacy, and fucosylation itself has been modulated as an experimental anticancer approach.

Therapeutic antibodies are a highly effective class of anticancer therapy that has been demonstrated to suppress tumor growth and spread via a number of mechanisms including direct inhibition of key signaling pathways by blocking ligand:receptor binding or receptor dimerization/activation, and immune-mediated antitumor activity, including (i) induction of complement-mediated toxicity, (ii) phagocytosis of tumor cells, and (iii) antibody-dependent cytotoxicity (ADCC) ^62,63

Importantly, the direct glycosylation of antibodies can vary depending on the cells in which they are produced and can significantly influence a given antibody's ability to recruit immune effector cells and to trigger target neutralization and cytotoxicity.⁶⁴ For example, the differential glycosylation of CAMPATH-1H, a humanized antibody used for the treatment of leukemia, lymphoma, and RA, was found to associate with differential extents of ADCC, depending on the cell line that the antibody was produced in [65]. Initial studies of therapeutic antibodies against human immunodeficiency virus (HIV) revealed that differential glycosylation of the Fc regions is a significant determinant of their therapeutic efficacy. Specifically, profiling of natural Fc region glycosylation variants (a.k.a., glycoforms) of anti-HIV antibodies revealed that galactosylation or fucosylation of the Fc region is associated with significantly enhanced or reduced anti-viral activity and HIV control, respectively.^66,67 The removal of core-linked fucose from Fc has been recognized as a method to improve the immune-mediated activity of therapeutic antibodies. Initial structural modeling studies suggested that differential glycosylation at a conserved Asn297 residue on the IgG Fc region impacts the structural conformation of the FC receptor:FC interaction that mediates ADCC responses.⁶⁸ Further studies revealed that FC fucosylation significantly enhances FC:FC receptor interaction in an FC receptor-dependent manner, as among FCγ receptor isoforms, FCγ receptor IIIa binds with 50-fold increased affinity to afucosylated IgG1 FC compared with other FCγ receptor isoforms including IIA and IIB. Importantly, the increased binding affinity also synergistically increased ADCC.⁶⁹ Indeed, depletion of l-fucose from IgG1 enhances the binding of IgG1 to FCγ receptor IIIa.⁷⁰ Notably, N-glycosylation of FCγ receptor IIIa at Asn162 was found to be required for enhanced binding affinity to afucosylated IgG FC, indicating that differential glycosylation and fucosylation of Fc and cognate receptors can significantly impact binding affinity and consequent immunological effects.⁷¹

Numerous efforts have been undertaken to produce afucosylated antibodies to improve immune-mediated therapeutic efficacy.⁷² To reduced fucosylation on antibodies, glycoengineering efforts have targeted fucosylation mechanisms in the cells used to produce the antibodies. Efforts targeting GDP-fucose substrate-level production/availability have included the knockout of GMD/FX enzymes (which mediate de novo synthesis of GDP-fucose from GDP-mannose) or SLC35C1 (Golgi transporter of l-fucose).^73-76 Downstream structure–function-targeted efforts have included knockdown or knockout of FUT8.^77,78 In each case, the targeted perturbation in cellular fucosylation resulted in increased production of antibodies exhibiting reduced fucosylation. However, these approaches can be limited by generally incomplete abrogation of fucosylation, resulting in increased ratios of afucosylated:fucosylated antibodies produced.

A number of afucosylated antibodies have been tested in clinical trials for the treatment of various cancers, and a small number are commercially produced (Table 4).

TABLE 4.

Afucosylated anticancer antibodies

Commercial name	Target	Pathology	Commercial/clinical trial phase	References (PMID)
ARGX-111	c-MET	Multiple cancers	Phase 1 (completed): NCT02055066	29733746
Bemarituzumab	FGFR2b	Gastric cancer, gastroesophageal junction adenocarcinoma	Phases 1, 2, and 3: NCT05111626; NCT05052801; NCT02213289; NCT03694522; NCT03343301	29733746
		Transitional cell carcinoma of the bladder	Phase 1: NCT02318329	29733746
BMS-986218	CTLA4	Prostate cancer	Early Phase 1: NCT04301414	29733746
		ALC; advanced malignant solid neoplasm; primary and metastatic liver cancer; metastatic lung carcinoma; metastatic malignant solid neoplasm	Phases 1 and 2: NCT04785287	29733746
Cusatuzumab	CD70	AML	Phase 2: NCT04023526	29733746
Gatipotuzumab/PankoMab-GEX	Glycoepitope of Muc1 (TA-Muc1	Ovarian epithelial cancer, primary peritoneal cancer recurrent fallopian tube cancer	Phase 2 (completed): NCT01899599	29733746
		Solid tumors	Phase 1 (completed)	29733746
GSK2849330	HER3	Multiple cancers, neoplasms	Phase 1: NCT01222624; NCT01966445; NCT02345174	29733746
Inebilizumab	CD19	Advanced B-cell malignancies; blood cancer; relapsed/refractory aggressive BCL; CLL; DLBCL	Phases 1 and 2 (completed): NCT01453205	29733746
Lumretuzumab	HER3	Breast cancer; neoplasms	Phase 1 (completed): NCT01918254; NCT01482377	29733746
MEDI-570	ICOS (CD278)	Angioimmunoblastic T-cell lymphoma; T-cell lymphoma follicular variant	Phase 1: NCT02520791	29733746
Mogamulizumab	CC chemokine receptor 4 (CCR4)	CTL; relapsed or refractory CCR4-positive adult T-cell leukemia-lymphoma	Commercially available (POTELIGEO): NCT04745234	29733746
		DLBCL; HCC; recurrent and/refractory Hodgkin lymphoma; recurrent/refractory NHL	Phases 1 and 2: NCT03309878	29733746
		Stage IB–IIB CTL	Phase 2: NCT04128072	29733746
Obinutuzumab (a.k.a., GA101 or Gazyva)	CDC20	CLL, NHL	Commercially available (GA101): NCT04908228; NCT03059251	29733746
		BCL	Trial Phases 1, 2, and 3	25143487; 23835718; 22431570; 24401022
SEA-BCMA	B-cell maturation antigen	Relapsed or refractory multiple myeloma	Phase 1: NCT03582033	29733746
SEA-CD40	CD40	Hodgkin's disease; melanoma; NSCLC	Phase 2: NCT04993677	29733746
Tomuzotuximab/CetuGEX	EGFR	Head and neck squamous cell carcinoma	Phase 2: NCT02052960	29733746
		Solid tumor	Phase 1	29733746
TrasGex	HER2	Solid tumor	Phase 1	29733746
TRX518	Glucocorticoid-induced TNF receptor	Unresectable Stage III or Stage IV MM or other solid tumor malignancies	Phase 1 (completed): NCT01239134	29733746
Ublituximab	CD20	BCL; CLL; marginal zone lymphoma; NHL; primary central nervous system lymphoma; SLL; Waldenstrom's macroglobulinemia	Phases 1 and 2: NCT01647971; NCT01744912l	29733746
		CLL	Phase 2: NCT02656303	29733746
		CLL, Mantle cell lymphoma	Phase 1: NCT02013128	29733746
		CLL; NHL	Phase 2: NCT03207256	29733746
		DLBCL; follicular lymphoma; marginal zone lymphoma; mediastinal large BCL; SLL	Phase 1: NCT04806035	29733746
		Follicular lymphoma, SLL	Phase 2: NCT03828448	29733746

Abbreviations: ALC, advanced lung carcinoma; AML, acute myeloid leukemia; BCL, B-cell lymphoma; CLL, chronic lymphocytic leukemia; CTL, cutaneous T-cell lymphoma; DLBCL, diffuse large B-cell lymphoma; HCC, hepatocellular carcinoma; MM, malignant melanoma; NHL, non-Hodgkin's lymphoma; NSCLC, non-small cell lung cancer; SLL, small lymphocytic lymphoma.

6 ∣. TARGETING FUCOSYLATION IN CANCER

In parallel to the development of afucosylated antibodies with enhanced therapeutic potential for anticancer activities, systemic inhibition of protein fucosylation has also been an area of active study as a potential anticancer therapeutic modality. A few small molecule inhibitors of fucosylation have been studied and developed in recent years that inhibit fucosylation at the substrate level (FUK/GMD/FX) and at the downstream FUT level.^79,80

Substrate-level fucosylation inhibitors have been developed based on fluorinated analogs of l-fucose, which can principally be uptaken into cells and converted into inhibitor analogs of GDP-fucose that bind tightly to and inhibit FUTs.^79,81 The most studied inhibitor analog is 2-fluoro-fucose, 2FF.⁸² This inhibitor has been preclinically tested for therapeutic effects in the context of a number of different cancer types including liver and breast cancers.^83,84 Recent studies have developed higher potency inhibitors of fucosylation based on 2FF. These include, for example, unnatural α-anomers and endogenous α-anomers of 2-fluoro-fucose-1-phosphate (A2FF1P and B2FF1P, respectively), which exhibit 4–7-fold increased potency for inhibiting fucosylation compared with 2FF.⁸⁵ In parallel, fluorinated versions of GDP-mannose-1-phosphate, Fucotrim I/II, have recently been developed to suppress fucosylation via de novo synthetic pathway conversion to fluorinated GDP-fucose-1-phosphate.⁸⁶ Fluorinated rhamnosides were also recently developed and reported to robustly inhibit fucosylation.⁸⁵

In preclinical breast cancer models, 2FF inhibited tumor growth and increased the antitumor capability of immune cells.⁸⁷ These data promoted a phase I clinical trial for solid tumors, which resulted in one complete response in head and neck squamous cell carcinoma and stable disease in 36% of patients. However, the clinical trial was discontinued due to significant rates of thromboembolic events.⁸⁸ Given the pleiotropic roles of the 13 FUTs across the different cell and tissue types, it is not necessarily surprising that systemic and chronic inhibition of fucosylation without FUT-specific targeting would result in deleterious adverse events and co-morbidities. Thus, although these 2FF and 2FF analogs represent potent substrate-level inhibitors of fucosylation and important tools for studying fucosylation, their implementation as therapeutic agents need to be carefully and contextually considered given the potential side effects.

Based on the potential and unintended deleterious effects elicited by systemic inhibition of fucosylation, FUT-targeted efforts have been anticipated to provide more tumor-specific suppression. Given previously reported protumorigenic roles of FUT8 in driving tumor cell-autonomous signaling (e.g., increase metastatic capacity), FUT8, in particular, has been the subject of study as an antitumor target.⁸⁹ Other inhibitors with narrower FUT-specific affinities have recently been developed, as derivatives of GDP-fucose, and have been reported to exhibit enhanced inhibitory specificity for FUTs including FUT6 and FUT8.^79,89,90 It is important to acknowledge, however, the divergent roles that individual FUTs have been reported to play—divergence for which it might be detrimental to inhibit their functions. For example, FUT8 has been reported to be required for T-cell development, viability, and activation.^56-58 However, it has also been reported to induce CD8⁺ T-cell exhaustion⁵⁶ and to stabilize tumor cell immune checkpoint proteins, enforcing immune checkpoint inhibition of T-cell activity.⁶⁰ Thus, specific targeting of FUT8 (and other individual FUTs) might inadvertently abrogate the balance of antitumor immunity and tumor autonomous signaling, promoting tumor progression. Given the pleiotropic roles of FUT8 (and other individual FUTs), further studies are required to delineate the key determinants of response (i.e., FUT expression, sex, age, type, and stage of cancer) and are expected to guide the clinical utility of FUT inhibitors.

In contrast to the efforts aimed at inhibiting fucosylation for the treatment of cancers, increasing fucosylation has also been reported to elicit therapeutically potent antitumor effects with the potential to augment immunotherapies. As mentioned above, clinical-grade l-fucose has been implemented as an experimental treatment for leukocyte adhesion deficiency-II (LAD-II), a rare genetic disorder in children driven by mutations in the de novo GDP-fucose synthetic pathway that abrogates fucosylation in leukocyte precursors, impairing their ability to extravasate from the vasculature.⁹¹ Oral administration of l-fucose, which increases fucosylation via the fucose salvage pathway, is sufficient to compensate for cellular fucosylation that is suppressed by de novo pathway mutations in LAD-II patients, at least partially reverting symptoms of this pathology, demonstrating the potential clinical utility and the safety of l-fucose supplementation.⁵⁰

In the context of cancer, preclinical in vivo mouse models of breast cancer, Ehrlich carcinoma, and melanoma have demonstrated antitumorigenic effects of l-fucose.^33,92,93 In the melanoma models performed by our laboratory, oral l-fucose results in net decreases in tumor growth, suggesting that despite divergent and potentially tumor-promoting functions of individual FUTs, the systemic dosing of l-fucose tips the functional balance of fucosylation toward tumor suppression. Notably, the fucosylation-triggered tumor suppression might be immune-mediated, as overall immune infiltration of melanomas was increased by l-fucose supplementation.³³ Consistent with this notion, the ex vivo treatment of CD8⁺ T cells with exogenous recombinant FUT7 before adoptive cell transfer into melanomabearing mice resulted in increased T-cell fucosylation associated with enhanced cytotoxic activities in vitro and melanoma tumor suppression in vivo.⁶¹ Studies elucidating how l-fucose triggers antitumor immunity are forthcoming. Increasing our knowledge of (1) what is being fucosylated and (2) how l-fucose affects specific immune cell populations in the tumor microenvironment is expected to further delineate the rationale and contexts for the clinical utility of l-fucose.

In addition to supplementation with l-fucose in preclinical cancer models, a seaweed-derived l-fucose-rich extract called fucoidan has exhibited antitumorigenic properties in vivo and in vitro.^94-101 In the context of immunotherapy, this is particularly exciting as fucoidan has been shown to enhance the efficacy of immune checkpoint blockade.¹⁰²

7 ∣. CONCLUSION

In conclusion, growing bodies of study have highlighted l-fucose and fucosylation as key components in cancer biology and in tumor immunology. Although it is clear that fucosylation plays crucial roles in the regulation of immune cell biology, particularly in the context of the tumor immune microenvironment, how fucosylation can be most effectively modulated to suppress tumors remains unclear. Specifically, there is a need to increase our understanding of how to leverage fucosylation to (i) suppress tumor autonomous signaling, (ii) enhance antitumor immune biology, and (iii) enhance immunotherapeutic molecules (e.g., afucosylated therapeutic anti-bodies) and modalities (e.g., coadministration of l-fucose vs. fucosylation inhibitors with other antitumor therapeutics). Further studies are expected to help deconvolute the complexities of tumor:immune contexts that will therapeutically benefit from modulating fucosylation and provide contextual frameworks in which increasing or inhibiting fucosylation will enhance the efficacies of specific immunotherapeutic modalities.

DATA AVAILABILITY STATEMENT

This manuscript did not generate any new data; it is a review of previously published scientific studies for which the data are available on a study-by-study basis.