Mucins are typically large glycoproteins (200 kDa–200 MDa) expressed by epithelial cell membranes [16]. This protein family is characterized by the presence of one or more modular mucin proline (Pro), threonine (Thr), serine (Ser) (PTS)-rich domains with a high frequency of Thr or Ser amino acid residues covalently conjugated with an $\alpha$-N-acetylgalactosamine ($\alpha$-GalNAc) moiety (O-linked glycosylation) [17, 18]. The presence of the Pro interrupts the alpha-helix architecture allowing for an extended secondary structure with accessible Ser/Thr sites for glycan addition and, perhaps, display of glycans emanating radially from the protein core [19]. Although the vast majority of mucin glycans are of this O-linked glycan type, most also contain N-linked glycans through additional covalent modification of asparagine (Asn) residues [15]. Glycans often comprise more than 80% of the molecular mass of mucin molecules and this hydrophilic carbohydrate structure is thought to play key roles in modifying the biophysical properties of cellular membranes as well as alter their topology [16, 20]. Following their complex synthesis in the cis and trans Golgi, mucins can either be secreted or anchored into the plasma membrane via protein transmembrane domains or posttranslational attachment of lipids [15].

Mucins are frequently expressed, shed (proteolytic cleavage) or secreted on the apical surface of barrier structures of tissues, especially epithelial barriers contacting the extra-tissue environment (mucosal surface epithelium) and endothelial layers contacting the circulation [18]. Their presence maintains the integrity of these barriers while also limiting contact with pathogens, toxins and antigens and inflammatory agents that may cause damage and trigger inflammatory responses. Secreted/shed mucins can help neutralize pathogens and transmembrane mucins play a role in sensing the environment and maintaining tissue architecture. Tumorigenesis exploits the function of mucins (stochastically) to promote their own growth and survival; enhance motility; limit adhesion; and evade immune surveillance [18]. In addition, dysregulated expression of mucins during tumor progression may be accompanied by changes to glycosylation and metabolic machinery that result in aberrant mucin glycoforms with altered physical and functional properties including immune evasion [18, 21]. In this review, we will selectively focus on four mucins that have been explored extensively for their role in cancer: transmembrane mucins Mucin 1 (MUC1), podocalyxin (Podxl), and MUC16 and secreted mucin MUC5AC (Table 1, Ref. [22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43]).

MUC1 is the founding member of large family of mucins normally expressed on glandular or luminal epithelial cells in a variety of tissues, where its extended negatively charged sugar branches create a selective biophysical barrier with anti-adhesive properties, limiting pathogen accessibility and preventing colonization of mucosal surfaces [23]. MUC1 has proved to be an interesting target for ADC development as aberrantly glycosylated MUC1 is overexpressed in most human epithelial cancers [25]. Podxl is a highly glycosylated cell surface sialomucin of the CD34 family of stem cell antigens and plays important roles in cell adhesion and transendothelial migration in normal and cancer tissues [44, 45]. Its expression is upregulated by a wide variety of cancer types and Podxl overexpression is consistently linked to poor prognosis, more aggressive tumor progression, unfavorable treatment outcomes, and possibly chemoresistance [32, 33, 34]. In keeping with its normal distribution on hematopoietic and early embryonic tissue stem cell populations, Podxl appears to be expressed by a highly mobile subset of tumor initiating cells. Furthermore, inactivation of the PODXL gene or dampening of its expression in cell lines cripples their ability to form tumors in xenografts and dampens their “tumorsphere” forming potential in vitro [46, 47]. MUC16, a MUC1 relative, is the largest of all known mucins and is the carrier of the Cancer Antigen 125 (CA125) epitope, which is widely used as a serum marker for the detection of ovarian cancer [35, 48, 49]. MUC5AC is a secreted, gel-forming, mucin that normally composes the airway mucus layer [50, 51]. In cancer, its expression is upregulated by an array of tumor types and its expression is predominantly cytoplasmic or at the apical pole of tumor cells [41].

O-linked glycosylation of mucins is initiated with the monosaccharide GalNAc $\alpha$-linked primarily to Ser/Thr residues. This O-linked GalNAc is then further extended by addition of different monosaccharides catalyzed by 30 or more distinct glycosyltransferases (GTF) [52]. In contrast to N-linked glycosylation which occurs co-translationally in the endoplasmic reticulum (ER), O-linked glycan post-translational modifications are initiated in the Golgi apparatus and occur after most key protein folding events have taken place [52]. Several mechanisms can contribute to the alterations in O-linked GalNAc glycosylation observed in cancer including changes in expression or localization of GTFs, altered expression or activity of glycosidases (enzymes responsible for the hydrolysis of glycosidic linkages), changes in pH of the Golgi apparatus, modifications in the identity and concentration of UDP-sugar donors, and finally, large alterations in the abundance of the target mucin substrates (apomucins) themselves [53, 54]. These alterations produce three classes of TACAs: (1) oncofetal antigens that are rare in normal adult tissue but commonly expressed in fetal tissue; (2) neoantigens expressed only in tumor cells; and (3) altered levels of normal antigens [54]. Oncofetal antigens and neoantigens are ideal targets for ADCs as they are typically tumor-restricted in adults. These oncofetal and neoantigen TACAs can be either truncated O-linked glycans that reveal hidden or cryptic antigens (Tn-, sTn- or T-antigens) or altered terminal glycans (Lewis system carbohydrates, for example) (Fig. 1) [54, 55]. While the focus of this review is on mucin glycoepitopes, other tumor-expressed glycoproteins also display TACAs. One good example is the carcinoembryonic antigen (CEA) or CECAM5, which is a glycoprotein member of the immunoglobulin family and one of the first described tumor antigens [56]. Although originally thought to be an embryo-restricted marker that is (re)expressed on tumors, CEA is also found on normal mucosal tissue in adults [57]. However, its glycosylation profile is altered on cancer cells with increased expression of Lewis X and Lewis Y motifs [58, 59].

Fig. 1.

Glycan structures of tumor-associated carbohydrate antigens. (Left) Examples of extended glycans expressed on normal cells. (Middle) Examples of common truncated O-linked glycans. (Right) Examples of altered terminal O-linked glycans. Created with BioRender.com.

By definition, a glycoepitope refers to a carbohydrate moiety that is recognized by a mAb or other glycan-binding protein [60]. While mAbs that only bind monosaccharide units (glycan-binding mAbs) exist, their affinity is usually much lower than protein-specific mAbs with equilibrium dissociation constant (K${}_{\mathrm{\text{D}}}$) values in the micromolar ($\mu$M) range compared to nanomolar (nM) range, respectively [61, 62]. Since glycan epitopes are frequently repeated on a protein core, especially in the case of mucins with multiple tandem glycosylation targets, low affinity of glycan-binding mAb can be circumvented by generating mAbs with two or more binding sites that recognize repeating glycan-epitope to form a multivalent complex [63]. This is reflected by their early expression as deca-valent IgM and their observed class switching bias toward IgG${}_3$ in mice and IgG${}_2$ in humans—mAb isotypes that tend to self-associate through their constant regions to form multivalent networks [61, 64, 65]. As an alternative approach, mucins may accommodate bi-paratopic mAb that recognize two different nonoverlapping epitopes on the same antigen to improve glycan-binding mAb avidity. Bi-paratopic ADCs have superior internalization profiles compared to monospecific and bispecific counterparts (reviewed in ref [66]).

Another class of TACA-binding mAbs that, in general, tend to exhibit improved binding affinities and specificity are those mAbs that recognize a glycoepitope formed by combinations of a glycan and a defined peptide epitope (i.e., glycopeptide glycoepitopes) [67, 68]. These tend to show specificity and affinities more typically associated with protein antigens. A third type of glycoepitope that we have named “shielded-peptide glycoepitopes” are not true glycoepitopes per se as the mAb does not directly bind the glycan, but instead recognize polypeptide epitopes whose accessibility to the peptide sequence is restricted by the altered glycosylation status of the glycoprotein expressed on normal cells. A schematic representation of these three classes of epitopes is presented in Fig. 2.

Fig. 2.

Schematic representation of glycan-only, glycopeptide and shielded-peptide glycoepitopes. Created with BioRender.com.

While the identification of the amino acid sequence of a regular peptide epitope is now relatively easy, the characterization of a glycoepitope structures remains complex and challenging due to their complex and branched chain structure. For this reason, the exact glycoepitope structure of most mAb/ADC targets discussed in this review is still unknown. Nevertheless, we have included mAbs/ADCs with sufficient epitope mapping data to suggest that their epitope falls within one of three categories described in Fig. 2.

A prime example of a glycan-binding mAb is the murine JAA-F11 IgG${}_3$ or humanized hJAA-F11 H2aL2a IgG${}_1$ that binds the T-antigen (sometimes call a Thomsen-Friedenreich (TF)-antigen) formed by the disaccharide, D-galactose-beta-(1–3)-N-acetyl galactosamine (Gal-$\beta$-(1–3)-GalNAc), alpha ($\alpha$)-linked to Ser/Thr peptide residues [69, 71] (Fig. 1, Table 2). The T-antigen is a cryptic oncofetal antigen frequently expressed by mucins in cancer [107]. In contrast to other T-antigen targeting mAbs, the JAA-F11 mAbs are highly specific for the alpha-linked tumor-associated T-antigen and not the beta-linked structure expressed on the surface of normal tissues [69, 71]. JAA-F11 mAbs are excellent candidates for ADC development as they are rapidly internalized upon binding their ligand [71, 108]. Indeed, when conjugated to the microtubulin inhibitor DM1 (N2${}^{\prime }$-deacetyl-N2${}^{\prime }$-(3-mercapto-1-oxopropyl)-maytansine), the hJAA-F11 H2aL2a-DM1 ADC demonstrated in vitro cytotoxic activity against various triple negative human breast cancer and lung cancer cell lines and significantly reduced MDA-MB-231 tumor growth in a mouse xenograft model [71]. This ADC has the potential to treat a significant number of patients, given the widespread expression of the T-antigen (up to 80% positive breast tumors) and its expression by a variety of cancer types including breast, lung, prostate, colon, bladder, and ovarian cancers [69, 70]. Although the ability to kill xenografted tumors appears quite promising, this effect required an intense dose regimen (15 mg/kg 3 times a week for the first week followed by weekly injections for 5 weeks) and the study did not include a non-targeting ADC raising concerns of the specific efficacy of antibody-mediated tumor killing in this study [71].

Another interesting glycan-binding mAb is the mouse IgG${}_1$ FG129 and its chimeric human IgG${}_1$ variant, CH129 (Table 2). These recognize, with high affinity (KinExA K${}_{\mathrm{\text{D}}}$ ~ 21–59 nM [72]), terminal sialyl-di-Lewis${}^{\text{a}}$ (s-di-Le${}^{\text{a}}$) glycans as well as the two closely related glycans, sialyl-Lewis${}^{\text{a}}$-Lewis${}^{\text{x}}$ (sLe${}^{\text{a}}$/sLe${}^{\text{x}}$) and sialyl-Lewis${}^{\text{a}}$ (sLe${}^{\text{a}}$) expressed on several high molecular weight glycoproteins (likely mucins) (Fig. 1) [72]. In vitro studies with colorectal and pancreatic cancer cell lines demonstrate that CH129 mAb binding to the cell surface of tumors drives epitope internalization. Conjugation with three different linker-payloads (monomethyl auristatin E (MMAE) or two maytansinoids (DM1 and DM4)) leads to higher cytotoxicity compared to their respective non-targeting rituximab (RTX) control ADCs. CH129 coupled to MMAE (CH129-MMAE) showed an impressive half-maximal effective concentration (EC${}_{50}$) in the picomolar (pM)-to-nM range and its non-targeting control RTX-MMAE showed no cytotoxicity. Using an in vivo evaluation in a COLO205 xenograft mouse model, CH129–MMAE conjugate exhibits potent control of tumor growth and elimination of tumors in 7 of 10 mice for the duration of the study with a therapeutic dose of 5 mg/kg (biweekly) [72]. The CH129 glycoepitope is expressed on a variety of cancer types suggesting a broad potential for therapeutic interventions. While this epitope is mostly tumor-specific, FG129 binds weakly to a small percentage of cells within gallbladder, ileum, liver, esophagus, pancreas, and thyroid tissues and therefore off-target effects during clinical development remain a potential concern [72].

Several MUC1-directed mAbs and single-chain variable fragments (scFv) have been generated as potential cancer therapeutics for CAR-T, radioimmunotherapy (RIT) and mAb therapy that may have applications as ADCs (reviewed in ref [109]). These mAbs, as a collection, target glycan or peptide epitopes as well as glycoepitopes [109]. Below we highlight examples of established MUC1-directed ADC candidates that target glycoepitopes.

16A is a murine IgG${}_1$ that targets a glycoepitope of MUC1 [73, 74]. This mAb strongly binds to the glycopeptide RPAPGS(GalNAc)TAPPAHG, an aberrantly glycosylated tandem repeat region of MUC1 but displays much weaker binding (25-fold lower) to the non-glycosylated RPAPGSTAPPAHG peptide (relative affinity determined by ELISA) [73]. Interestingly, the affinity of 16A for the isolated peptide and glycosylated peptide measured by surface plasmon resonance (SPR) are comparable (SPR K${}_D$~ 500–1000 nM) [73]. The much higher apparent affinity for aberrantly glycosylated MUC1 on live cells and tumors might be due to conformational changes induced by the glycan that facilitates access of the mAb to its peptide epitope (i.e., a shielded-peptide glycoepitope) or, alternatively, 16A may engage in intermolecular contacts simultaneously with the peptide and the glycan moiety (i.e., glycopeptide epitope). The 16A mAb binds to target epitopes expressed on lung, breast (including triple-negative breast cancer) and gastric cancer tissues and is rapidly internalized supporting its further development as a therapeutic ADC candidate [74]. To test its potential, Pan et al. [74] generated the 16A-MMAE ADC and demonstrated that it potently kills lung, breast, pancreatic, gastric, and ovarian cell lines in vitro. Furthermore, using the adenocarcinoma H838 (non-small cell lung cancer) in a mouse xenograft model, they demonstrated that the 16A-MMAE ADC inhibits tumor growth in a dose dependent manner (minimum therapeutic dose ~5 mg/kg) [74]. However, the omission of a non-targeting ADC as a control in these experiments somewhat limits the ability to evaluate conclusions of its “on”, versus “off” target efficacy [74].

Gatipotuzumab, formerly known as PankoMab-GEX, is the humanized version of the mouse IgG${}_1$ PankoMab. This mAb was initially generated to maximally discriminate between the carbohydrate-induced conformational tumor epitope on MUC1 (TA-MUC1) and the non-glycosylated MUC1 epitope [79]. The TA-MUC1 epitope includes the …PDT*RP… amino acid, where T* is O-glycosylated with GalNAc$\alpha$1- or a similar short, non-sialylated, glycan such as Gal$\beta$-(1–3)-GalNAc$\alpha$1- (core-1/T-antigen) [110]. The exact interaction sites between PankoMab and its epitope are unknown, but its strong binding with the glycosylated version of the TA-MUC1 peptide and weak binding with the non-glycosylated version of the same peptide strongly suggest that PankoMab epitope is a glycopeptide epitope [79]. Antibody affinity estimates range from ~1 to 7 nM (Scatchard K${}_{\mathrm{\text{D}}}$) for tumor cell lines [79]. Due to its antibody dependent cellular cytotoxicity (ADCC) activity [79], Gatipotuzumab (unconjugated form) was tested in Phase I clinical trials (NCT01222624) in patients with advanced TA-MUC1-positive carcinomas and Phase II trial (NCT01899599) in patients with TA-MUC1-positive ovarian tumors. While the mAb was well tolerated in ovarian cancer patients, unfortunately, gatipotuzumab did not improve disease outcomes compared to the placebo [81, 83]. The potential of gatipotuzumab to function as an ADC, however, has not been fully explored. Its rapid internalization and its capacity to induce toxin-mediated antigen-specific tumor cell killing in vitro suggest it is a good candidate for further research [79]. Interestingly, in 2018, Daiichi-Sankyo (Japan) and Glycotope GmbH (Germany) entered into an exclusive worldwide licensing agreement to develop an ADC by combining Daiichi-Sankyo’s proprietary ADC technology with Glycotope’s gatipotuzumab [111].

The murine MuDS6 IgG${}_1$ mAb was generated by Smith et al. in 1999 [112] in a screen to produce a mAb that reacts with an antigen that is tumor cell surface expressed but with limited expression on normal tissue. The resulting mAb was later humanized as huDS6 IgG${}_1$ mAb [113]. The DS6 mAbs recognize a sialic acid-dependent epitope (sialoglycotope) of MUC1 designated “CA6” [84, 86]. However, the exact glycoepitope structure remains to be determined and could be either a glycopeptide epitope or a shielded-peptide glycoepitope. The huDS6 mAb is efficiently internalized in an antigen dependent manner and has been conjugated to the cytotoxic maytansinoid derivative DM4 to generate SAR566658 (huDS6-SPDB-DM4 ADC) [86, 114]. This ADC induces targeted in vitro cytotoxicity and effectively controls tumor growth in murine xenograft tumor models using a variety of human tumor cell lines. Importantly, the efficacy of SAR566658 is associated with high CA6 expression in tumor targets [86]. In a Phase I study in CA6-positive patients with metastatic breast cancer (NCT01156870), SAR566658 provided a favorable safety profile and encouraging antitumor activity [87, 115]. However, a subsequent Phase II study in metastatic triple-negative breast cancer patients (NCT02984683) was discontinued following preliminary analyses showing that the benefit/risk balance was not favorable due to a higher-than-expected incidence of ophthalmologic events (e.g., keratitis and keratopathy) [86, 88]. While DS6 mAbs predominantly bind to tumor tissues, they also recognized some normal adult tissues (e.g., fallopian tube, pulmonary alveoli and urothelium) and these could compromise its utility for tumor-specific targeting [84].

The C242 mouse IgG${}_1$ mAb or its humanized version huC242 mAb (cantuzumab) recognize, with high selectivity, the extracellular CA242 epitope present on the cancer antigen (CanAg) glycoform of MUC1 [91, 116, 117]. The exact structure of the epitope, too, has not been determined but, because it contains a sialic acid, it is likely one of the three classes of glycoepitopes described in this review [116]. First demonstrated in 1996 by Liu et al. [92], the murine C242 mAb combined with the maytansinoid DM1 (C242-DM1) can effectively eradicate CanAg-expressing human tumor xenografts in mice in an antigen-specific manner. The capacity of its humanized version (huC242-DM1) to eliminate CanAg-positive COLO205 xenograft tumors was later confirmed and it was also demonstrated that this ADC can induce an interesting bystander effect and kill the proximally located antigen-negative tumor cells [93]. The therapeutic potential of the huC242-DM1 ADC (SB-408075: cantuzumab mertansine) was tested by ImmunoGen (USA) in partnership with GlaxoSmithKline (UK) in a Phase 1 clinical trial to determine the optimal treatment schedule and the limiting dose toxicity [95, 96, 97]. Patients with CanAg-expressing solid malignancies were included in these trials and early signs of activity of huC242–DM1 in tumors with strong intensity of CanAg was observed. However, dose-related hepatoxicity halted further dose escalation and therapeutic trials and development was discontinued [118]. Evaluation of a derivative of huC242 conjugated to DM4 (IMGN242: cantuzumab ravtanstine) caused complete tumor regression in mice bearing human gastric carcinoma xenografts [94]. IMGN242 was well tolerated in a Phase I clinical trial (NCT00352131) [119]. In a Phase II trial (NCT00620607) [120], in 6 patients with CanAg-positive gastric or gastroesophageal junction cancer, a partial response was observed in 1 patient. Unfortunately, 3 out of 6 patients developed ocular toxicities that were assessed to be study drug related [99] and the huC242-DM4 program was later discontinued [118].

While MUC1 glycoepitopes are interesting targets for the development of ADCs, the few targeting mAbs that reached the clinical trial stage have, to date, failed to be approved for cancer treatment. Possible explanations for these disappointing results will be discussed at the end of this review.

Abnormal mucin-glycosylation patterns in cancer are not restricted to MUC1. We recently developed a highly tumor specific rabbit/human chimeric IgG${}_1$ mAb, named PODO447 that reacts with Podxl expressed on tumor cells but not normal tissue [47]. This mAb specifically binds a glycopeptide epitope consisting of the core 1 O-linked glycan (T-antigen) in the context of the Podxl polypeptide but does not recognize general core 1 glycans decorating other proteins [100]. The PODO447 affinity for human tumor cell lines is well into the sub-nanomolar range (KinExA K${}_{\mathrm{\text{D}}}$ 8 to 190 pM) [121]. PODO447 is effectively internalized in cancer cells and, when conjugated to a protease-cleavable linker with MMAE (vedotin), PODO447-ADC induces in vitro cytotoxicity in variety of cancer cell lines in an antigen-dependent manner [47]. In vivo, PODO447-ADC treatment leads to regression of xenografted human ovarian and pancreatic tumors with a therapeutic dose of 2–4 mg/kg [100]. Further development of this promising ADC awaits Phase I and Phase II trials.

AR9.6 is a murine mAb that binds a conformational epitope of the SEA domain 5 of MUC16 that is influenced by O-linked glycosylation [101]. Interestingly, binding of unconjugated AR9.6 with MUC16 leads to a significant reduction of tumor growth in a pancreatic tumor model by blocking MUC16-dependent activation of ErbB (EGF) receptors on the cancer cell surface and downstream attenuation of oncogenic AKT and GSK3$\beta$ signaling [101]. This mAb has also been conjugated to a near-infrared fluorescent dye and radiolabeled with [${}^{89}$Zr]Zr${}^{4+}$ to enable PET-imaging. In both cases, these AR9.6 conjugations lead to MUC16-dependent tumor targeting in different in vivo cancer models [102, 122]. Good target-mediated internalization of a humanized AR9.6 by tumor cells suggests promising ADC therapeutic potential for this mAb [102, 122].

Finally, NEO-102 (ensituximab) is a chimeric mouse/human IgG${}_1$ mAb that recognizes the NPC-1C glycoepitope of MUC5AC and is described as an aberrantly glycosylated epitope preferentially expressed in pancreatic and colorectal cancers. NEO-102 can therefore discriminate between the native MUC5AC expressed on normal tissue and a variant of MUC5AC expressed by tumors [103, 123]. The exact structure of the epitope remains to be identified, but its dependence on a tumor-specific glycosylation profile suggests it is one of the three classes of glycoepitope described in this review. In an in vivo mouse model, unconjugated NEO-102 significantly reduced human pancreatic CFPAC-1 tumor xenograft growth [103]. However, in a Phase II clinical trial [106] funded by Precision Biologics Inc (USA), NEO-102 only demonstrated modest antitumor activity in patients with refractory metastatic colorectal cancer [105]. Interestingly, the unconjugated mAb treatment was well-tolerated by these patients suggesting that linking NEO-102 to a cytotoxin to generate an ADC could safely increase the antitumor potential of NEO-102 [104, 105].