ADC drugs targeting mucin tumor-specific glycotopes

Finding the ideal targeted epitope is key
to developing ADC drugs.
To maximize drug delivery to tumor cells and reduce side effects, ADC-targeted epitopes should be directed at cancer cells rather than normal tissue
.
During cancer progression, the glycosylation pathway is often altered, resulting in new patterns of glycosylation that are selective for cancer cells
.
Mucin is a highly glycosylated protein that is often expressed on tumors and is therefore an ideal presenter of glycoepitopes
.
In this article, we will briefly introduce
the three different types of mucin sugar epitopes and ADC drugs that target mucin tumor-specific sugar epitopes.

Mucin in tumors

Mucins are usually large glycoproteins (200 kDa-200 MDa)
expressed by epithelial cell membranes.
This protein family is characterized by the presence of one or more domains rich in modular proline (Pro), threonine (Thr), serine (Ser) (PTS), where high frequencies of Thr or Ser are partially covalently bound (O-linked glycosylation)
to α-N-acetylgalactosamine (α-GalNAc).
Although the vast majority of mucoproteinglycans belong to the O-linked glycan type, they also contain N-linked glycans
covalently modified by asparagine (Asn).
Glycans typically make up more than 80% of the molecular mass of mucin molecules, and this hydrophilic carbohydrate structure plays a key role
in altering the biophysical properties of cell membranes as well as their topology.

Mucins are often expressed
on the surface of tissue barrier structures, exfoliated (proteolytic cleavage), or secreted.
Their presence maintains the integrity of these barriers, while also limiting contact
with pathogens, toxins and antigens, as well as inflammatory factors that can cause damage and trigger inflammatory responses.
Secreted/exfoliated mucins can help neutralize pathogens, and transmembrane mucins play a role
in sensing the environment and maintaining tissue structure.
Tumorigenesis uses the function of mucins to promote their own growth and survival, enhance motility, limit adhesion, and evade immune surveillance
.
In addition, dysregulation of mucin expression during tumor development may be accompanied by alterations in glycosylation and metabolic mechanisms, resulting in abnormal mucin glycoforms with altered
physical and functional properties.

There are currently four mucins that have been extensively studied in cancer: MUC1, Podxl, MUC16 and MUC5AC
.
MUC1 is an initial member of the mucin family, normally expressed on glandular or luminal epithelial cells of various tissues, and its extended negatively charged sugar branches create selective biophysical barriers with anti-adhesion properties that limit pathogen access
.
MUC1 has proven to be a potential target for ADCs because abnormally glycosylated MUC1 is overexpressed
in most human epithelial carcinoma.

Podxl is a highly glycosylated cell surface salivary protein of the stem cell antigen CD34 family that plays an important role
in cell adhesion and transendothelial migration in normal and cancer tissues.
Its expression is upregulated
in multiple cancer types.
Overexpression of Podxl is associated
with poor prognosis, tumor aggressiveness, and chemotherapy drug resistance.

MUC16, the largest of all known mucins, is a carrier of the CA125 epitope, which is widely used as a serum marker for the detection of ovarian cancer
.

MUC5AC is a secretory gel-forming mucin that normally forms the mucus layer
of the airways.
In cancer, it is upregulated in a range of tumor types, and its expression is mainly in the cytoplasm or on the surface of
tumor cells.

Mucin: Three different types of sugar epitopes

A glycan epitope is a portion
of a carbohydrate recognized by a monoclonal antibody or other glycan-binding protein.
Monoclonal antibodies that bind only glycans typically have much lower affinity than protein-specific monoclonal antibodies, and KD values are typically in the μM range
.
Since glycan epitopes are often tandem on the protein core, the low affinity of glycan-bound monoclonal antibodies can be avoided by producing monoclonal antibodies with two or more recognition repeating glycan epitopes, resulting in the formation of polyvalent complexes
.

Another class of conjugated monoclonal antibodies that exhibit better binding affinity and specificity for tumor-associated carbohydrate antigens recognizes glycopeptide epitopes formed by combinations of sugars and known peptide epitopes
.
They tend to exhibit specificity and affinity comparable to protein antigens
.

The third type of sugar epitope, called the "shielding glycopeptide epitope," is not a true sugar epitope
.
Monoclonal antibodies do not bind directly to sugars, but rather recognize peptide epitopes, and their recognition of peptide sequences is limited
by glycoprotein glycosylation status.
Schematic diagram 1 of these three types of epitopes is shown
.

Figure 1.
Mucin: Three different types of sugar epitopes

ADCs targeting mucin tumor-specific glycotopes

While it is now relatively easy to identify amino acid sequences for regular peptide epitopes, characterization of glycan epitope structures is challenging
due to its complex branched-chain structure.
The exact sugar epitope structure of most mAb/ADC targets is still not very clear
.

• ADCs targeting mucin glycosyls

A prime example of glycosyl-binding antibodies is mouse JAA-F11 IgG3 or humanized hJAA-F11 H2aL2a IgG1, which binds to the T antigen
formed by D-galactose-beta-(1–3)-N-acetyl galactosamine (Gal-β-(1-3)-GalNAc) linked to serine/threonine.
The T antigen is a covert cancerous fetal antigen that is often expressed
by mucin in cancer.
Unlike other monoclonal antibodies that target T antigens, JAA-F11 monoclonal antibodies are highly specific to tumor-associated α-linked T antigens rather than beta-linked structures
expressed on the surface of normal tissues.
JAA-F11 monoclonal antibody is an excellent candidate for the development of ADCs because it rapidly internalizes
after binding to antigens.
When bound to the tubulin inhibitor DM1, hJAA-F11 H2aL2a-DM1 ADC showed strong in vitro cytotoxic activity against triple-negative breast and lung cancer cell lines and significantly reduced tumor growth
in MDA-MB-231 mouse xenograft models.
Given the widespread expression of T antigens in tumors, this ADC has great therapeutic potential
.

Another interesting sugar-chain-binding monoclonal antibody is mouse IgG1 FG129 and its chimeric human IgG1 variant CH129
.
CH129 recognizes sialyl-di-Lewis a polysaccharides with high affinity and two closely related sugar chains expressed in several high molecular weight glycoproteins: sialyl-Lewis a-Lewis^x and Siyl-Lewis ^a
.
CH129-conjugated MMAE (CH129-MMAE) shows excellent in vitro effects
in the pM to nM range.
In the COLO205 xenograft mouse model, CH129-MMAE showed effective tumor growth control and tumor elimination in 7 out of 10 mice at a dose of 5 mg/kg (biweekly
).
CH129 glycotope is expressed in a variety of cancer types, suggesting its broad potential
.
Although this epitope is primarily tumor-specific, FG129 binds weakly to a small subset of cells in gallbladder, ileum, liver, esophagus, pancreas, and thyroid tissue, so the off-target effect during clinical development remains a potential problem
.

• An ADC targeting the MUC1 glycotope

Some MUC1-targeted monoclonal antibodies and single-stranded variable regions (ScFv) have been used as potential cancer treatments, and below we mainly introduce ADC drugs
that target MUC1 glycotopes.

16A is mouse IgG1 targeting the glycoepitope of MUC1
.
This monoclonal antibody binds strongly to the glycopeptide RPAPGS (GalNAc) TAPPAHG, but much weaker to non-glycosylated RPAPGSTAPPAHG (25-fold lower as detected by the ELISA method
).
However, the affinity measured by surface plasmon resonance (SPR) is similar (KD~500-1000 nM).

Abnormal glycosylation on tumors MUC1 has a higher apparent affinity for 16A, which may be due to conformational changes caused by sugar chains that facilitate contact of monoclonal antibodies with their peptide epitopes (i.
e.
, shielding peptide epitopes), or 16A may have intermolecular contact with both the peptide and sugar chain fractions (i.
e.
, glycopeptide epitopes).

16A binds to target epitopes expressed on lung, breast, and gastric cancer tissues and is rapidly internalized, making it an ADC candidate
.
The study found that 16A MMAE ADC, in vitro, can effectively kill lung, breast, pancreas, stomach and ovarian cell lines
.
Furthermore, in a mouse H838 (non-small cell lung cancer) xenograft model, 16A MMAE ADC inhibits tumor growth
in a dose-dependent manner.

Gatipotuzumab, a humanized version of
the mouse IgG1 PankoMab.
This monoclonal antibody was originally intended to maximize differentiation between conformational tumor epitopes (TA-MUC1) and non-glycosylated MUC1 epitopes on carbohydrate-induced MUC1
.
TA-MUC1 epitopes include.
.
.
PDT*RP.
.
.
Amino acids, where T* is GalNAc α1- or similar short, non-sialylated polysaccharides
.
The exact interaction site between PankoMab and its epitope is unknown, but its strong binding to the glycated version of TA-MUC1 and its weak binding to the non-glycated version of the same peptide strongly suggest that the PankoMab epitope is a glycopeptide epitope
。 Due to Gatipotuzumab's ADCC activity, in 2018, Daiichi-Sankyo and Glycotope GmbH entered into an exclusive global licensing agreement to develop ADCs
by combining Daiichi-Sankyo's proprietary ADC technology with Glycotope's gatipotuzumab.

The mouse MuDS6 IgG1 mAb monoclonal antibody recognizes the sialic acid-dependent epitope (sialic acid glycosyl epitope) of MUC1, named "CA6"
.
However, the exact glycan epitope structure remains to be determined, either a glycopeptide epitope or a shielded polypeptide glycotope
.
Humanized DS6 monoclonal antibody is effectively internalized
in an antigen-dependent manner.
The conjugation of humanized DS6 to DM4 to generate SAR566658 ADC induces strong cytotoxicity in vitro and effectively controls tumor growth
in mouse models of multiple human tumor cell lines.
In a Phase I study in patients with CA6-positive metastatic breast cancer (NCT01156870), SAR566658 provided a good safety profile and excellent antitumor activity
.
However, a subsequent phase II study (NCT02984683) in patients with metastatic triple-negative breast cancer initially analyzed its high oculotoxicity , such as keratitis and corneal lesions
.
Although DS6 monoclonal antibodies bind primarily to tumor tissue, they also recognize some normal adult tissues (such as fallopian tubes, alveoli, and urothelium), which may affect their effectiveness
for tumor-specific targeting.

huC242 mAb (Cantuzumab) highly selectively recognizes extracellular CA242 epitopes
present on MUC1 oncogenic antigen (CanAg) sugars.
The exact structure of the epitope has also not been determined, but since it contains sialic acid, it is likely to be one of the three classes of
sugar epitopes described.
Studies have shown that HuC242-DM1 has a potent anti-tumor effect on CanAg-positive COLO205 transplanted tumors, and this ADC can also induce bystander effects, killing proximal antigen-negative tumor cells
.
In a Phase I clinical trial, ImmunoGen partnered with GSK to test the therapeutic potential of huC242-DM1 ADC (SB408075: cantuzumab mertansine) to determine optimal treatment and dose toxicity
.
The activity
of huC242-DM1 was observed in CanAg-strongly expressed tumors.
However, dose-related hepatotoxicity prevented further dose increases and clinical trials were terminated
.
The huC242 DM4 ADC (IMGN242: cantuzumab ravtanstine) conjugated to DM4 resulted in complete tumor regression
in human gastric cancer xenograft mice.
IMGN242 was well
tolerated in a phase I clinical trial (NCT00352131).
In the phase II trial (NCT00620607), a partial response
was observed in 1 of 6 CanAg-positive patients with cancer of the gastric or gastroesophageal junction.
Unfortunately, ocular toxicity
developed in 3 out of 6 patients.

• ADCs targeting other mucin glycotopes

A highly tumor-specific rabbit/human chimeric IgG1 mAb, named PODO447, was recently developed, which binds
to Podxl expressed on tumor cells rather than on normal tissue.
This mAb specifically binds glycopeptide epitopes
composed of O-linked glycans (T-antigens) of the Podxl polypeptide.
PODO447-MMAE ADC induces in vitro cytotoxicity
in various cancer cell lines in an antigen-dependent manner.
In vivo, PODO447 ADC at doses of 2-4 mg/kg resulted in regression
of ovarian and pancreatic cancer tumors.
Phase I trials
are currently underway.

AR9.
6 is a mouse monoclonal antibody that binds to the MUC16 SEA domain 5 conformational epitope
affected by O sugar.
Good targeting and endocytosis of humanized AR9.
6 to tumor cell pairs hints at the therapeutic potential
of this ADC.

NEO-102 (Ensituximab) is a chimeric mouse/human IgG1mAb that recognizes the NPC-1C glycotope of MUC5AC, an abnormally glycosylated epitope that is preferentially expressed
in pancreatic and colorectal cancers.
Thus, NEO-102 can distinguish between native MUC5ACs expressed on normal tissues and variants
of MUC5ACs expressed in tumor tissues.
In an in vivo mouse model, unbound NEO-102 significantly reduced the growth
of CFPAC-1 pancreatic tumor xenograft tumors.
However, in a Phase II clinical trial funded by Precision Biologics, NEO-102 showed moderate antitumor activity
only in patients with refractory metastatic colorectal cancer.
But these patients tolerated unconjugated antibody therapy well, suggesting that coupling NEO-102 to cytotoxins to produce ADCs may safely increase NEO-102's anti-tumor potential
.

Summary

While most ADCs targeting mucin glycotopes have powerful anti-tumor effects in vitro and in animal models, ADCs that have entered clinical trials have so far not performed very well, and some have even shown unexpected side effects
.
Several factors explain these poor results (Figure 2).

First, the shedding or secretion of extracellular mucin domains containing ADC glycotopes can reduce the specific binding of ADCs to their targets, thereby reducing efficacy
.
Therefore, target epitopes entering the circulation should be carefully measured during clinical trials to assess the possibility of
antigen shedding affecting efficacy.
Another factor is the tumor specificity
of the glycan epitope.
Although the ADC-targeted glycotopes described here are primarily expressed in cancer cells, many have lower expression in normal histiocytes subsets, which may lead to significant normal tissue toxicity
.
A third possible limiting factor is that during treatment, tumors may be subjected to selective pressure to alter their glycosyltransferase expression, allowing tumors to escape
.
Therefore, during preclinical models and clinical trials, glycoepitope expression
should be carefully monitored after treatment whenever possible.

Figure 2.
Challenges faced by mucin glycotope ADCs

References

1.
Antibody-Drug Conjugates Targeting Tumor-Specific Mucin Glycoepitopes.

2.
Mucins in cancer: function, prognosis and therapy.