Under conditions of cellular stress, proteins can be post-translationally modified causing them to be recognized by the immune system. One such stress-induced post-translational modification (siPTM) is citrullination, the conversion of arginine residues to citrulline by peptidylarginine deiminase (PAD) enzymes. PAD enzymes are activated by millimolar concentrations of calcium which can occur during apoptosis, leading to precipitation of proteins, their subsequent uptake by B cells and stimulation of antibody responses. Detection of anti-citrullinated protein antibodies (ACPAs) is a diagnostic of rheumatoid arthritis (RA), where immune complexes stimulate inflammation around the joints. More recently, autophagy has been shown to play a role in the presentation of citrullinated peptides on MHC class II molecules to CD4+ helper T cells, suggesting that citrullination may be a way of alerting immune cells to cellular stress. Additionally, inflammation-induced IFNγ and concomitant MHC class II expression on target cells contributes to immune activation. Stressful conditions in the tumor microenvironment induce autophagy in cancer cells as a pro-survival mechanism. Cancer cells also over express PAD enzymes and in light of this the hypothesis that citrullinated peptides stimulate CD4+ T cell responses that would recognize these siPTM's produced during autophagy has been investigated. The induction of potent citrullinated peptide-specific CD4 responses has been shown in both humans and HLA transgenic mouse models. Responses in mouse models resulted in potent anti-tumour responses against tumours expressing either constitutive or IFNγ-inducible MHC class II. The anti-tumour effect relied upon direct recognition of tumours by specific CD4 T cells suggesting that citrullinated peptides are attractive targets for cancer vaccines.

Real-time tumor imaging techniques are increasingly used in oncological surgery, but still need to be supplemented with novel targeted tracers, providing specific tumor tissue detection based on intra-tumoral processes or protein expression. To maximize tumor/non-tumor contrast, targets should be highly and homogenously expressed on tumor tissue only, preferably from the earliest developmental stage onward. Unfortunately, most evaluated tumor-associated proteins appear not to meet all of these criteria. Thus, the quest for ideal targets continues. Aberrant glycosylation of proteins and lipids is a fundamental hallmark of almost all cancer types and contributes to tumor progression. Additionally, overexpression of glycoproteins that carry aberrant glycans, such as mucins and proteoglycans, is observed. Selected tumor-associated glyco-antigens are abundantly expressed and could, thus, be ideal candidates for targeted tumor imaging. Nevertheless, glycan-based tumor imaging is still in its infancy. In this review, we highlight the potential of glycans, and heavily glycosylated proteoglycans and mucins as targets for multimodal tumor imaging by discussing the preclinical and clinical accomplishments within this field. Additionally, we describe the major advantages and limitations of targeting glycans compared to cancer-associated proteins. Lastly, by providing a brief overview of the most attractive tumor-associated glycans and glycosylated proteins in association with their respective tumor types, we set out the way for implementing glycan-based imaging in a clinical practice.

Targeted molecular imaging may overcome current challenges in the preoperative and intraoperative delineation of pancreatic ductal adenocarcinoma (PDAC). Tumor-associated glycans Le^a/c/x, sdi-Le^a, sLe^a, sLe^x, sTn as well as mucin-1 (MUC1) and mucin-5AC (MU5AC) have gained significant interest as targets for PDAC imaging. To evaluate their PDAC molecular imaging potential, biomarker expression was determined using immunohistochemistry on PDAC, (surrounding) chronic pancreatitis (CP), healthy pancreatic, duodenum, positive (LN⁺) and negative lymph node (LN⁻) tissues, and quantified using a semi-automated digital image analysis workflow. Positive expression on PDAC tissues was found on 83% for Le^a/c/x, 94% for sdi-Le^a, 98% for sLe^a, 90% for sLe^x, 88% for sTn, 96% for MUC1 and 67% for MUC5AC, where all were not affected by the application of neoadjuvant therapy. Compared to PDAC, all biomarkers were significantly lower expressed on CP, healthy pancreatic and duodenal tissues, except for sTn and MUC1, which showed a strong expression on duodenum (sTn tumor:duodenum ratio: 0.6, p < 0.0001) and healthy pancreatic tissues (MUC1 tumor:pancreas ratio: 1.0, p > 0.9999), respectively. All biomarkers are suitable targets for correct identification of LN⁺, as well as the distinction of LN⁺ from LN⁻ tissues. To conclude, this study paves the way for the development and evaluation of Le^a/c/x-, sdi-Le^a-, sLe^a-, sLe^x- and MUC5AC-specific tracers for molecular imaging of PDAC imaging and their subsequent introduction into the clinic.

Immunotherapy has been successful in treating many tumour types. The development of additional tumour-antigen binding monoclonal antibodies (mAbs) will help expand the range of immunotherapeutic targets. Lewis histo-blood group and related glycans are overexpressed on many carcinomas, including those of the colon, lung, breast, prostate and ovary, and can therefore be selectively targeted by mAbs. Here we examine the molecular and structural basis for recognition of extended Le^a and Le^xcontaining glycans by a chimeric mAb. Both the murine (FG88.2) IgG3 and a chimeric (ch88.2) IgG1 mAb variants showed reactivity to colorectal cancer cells leading to significantly reduced cell viability. We determined the X-ray structure of the unliganded ch88.2 fragment antigen-binding (Fab) containing two Fabs in the unit cell. A combination of molecular docking, glycan grafting and molecular dynamics simulations predicts two distinct subsites for recognition of Le^a and Le^x trisaccharides. While light chain residues were exclusively used for Le^a binding, recognition of Le^x involved both light and heavy chain residues. An extended groove is predicted to accommodate the Le^a-Le^x hexasaccharide with adjoining subsites for each trisaccharide. The molecular and structural details of the ch88.2 mAb presented here provide insight into its cross-reactivity for various Le^a and Le^xcontaining glycans. Furthermore, the predicted interactions with extended epitopes likely explains the selectivity of this antibody for targeting Lewis-positive tumours.

Small cell lung cancer (SCLC) is an aggressive disease with limited treatment options. Fucosyl-GM1 (FucGM1) is a glycolipid overexpressed in the majority of SCLC tumors but virtually absent from normal healthy tissues. In this study, we validate a FucGM1-targeting T cell–redirecting bispecific (TCB) antibody for the treatment of SCLC. More than 80% of patient-derived xenograft tissues of SCLC expressed FucGM1, whereas only three normal human tissues: pituitary, thymus, and skin expressed low and focal FucGM1. A FucGM1-targeting TCB (SC134-TCB), based on the Fc-silenced humanized SC134 antibody, exhibited nanomolar efficiency in FucGM1 glycolipid and SCLC cell surface binding. SC134-TCB showed potent ex vivo killing of SCLC cell lines with donor-dependent EC50 ranging from 7.2 pmol/L up to 211.0 pmol/L, effectively activating T cells, with picomolar efficiency, coinciding with target-dependent cytokine production such as IFNγ, IL2, and TNFα and robust proliferation of both CD4 and CD8 T cells. The ex vivo SC134-TCB tumor controlling activity translated into an effective in vivo anti-DMS79 tumor therapy, resulting in 100% tumor-free survival in a human peripheral blood mononuclear cell admixed setting and 40% overall survival (55% tumor growth inhibition) with systemically administered human peripheral blood mononuclear cells. Combination treatment with atezolizumab further enhanced survival and tumor growth inhibition (up to 73%). A 10-fold SC134-TCB dose reduction maintained the strong in vivo antitumor impact, translating into 70% overall survival (P < 0.0001). Whole-blood incubation with SC134-TCB, as well as healthy human primary cells analysis, revealed no target-independent cytokine production. SC134-TCB presents an attractive candidate to deliver an effective immunotherapy treatment option for patients with SCLC.