Thioredoxin system in colorectal cancer: Its role in carcinogenesis, disease progression, and response to treatment.

The thioredoxin system is essential for many physiological processes, including the maintenance of redox signalling pathways. Alterations in the activity, expression and interactions with other signalling pathways can lead to protective or pathophysiological responses. Thioredoxin and thioredoxin reductase, the two main components of this system, are often overexpressed in cancer, including colorectal cancer. This overexpression is often linked with tumour progression and poor outcomes. This review discusses the role of the Trx system in driving colorectal carcinogenesis and disease progression, as well as the challenges of targeting this system. Additionally, the recent advancements in the development of novel and effective thioredoxin inhibitors for colorectal cancer are also explored.

Susceptibility to SARS-CoV-2 omicron following ChAdOx1 nCoV-19 and BNT162b2 versus CoronaVac vaccination.

The emergence of SARS-CoV-2 variants raises concerns of reduced COVID-19 vaccine efficacy. We investigated the humoral immunity in uninfected and previously infected ChAdOx1 nCoV-19, BNT162b2 and CoronaVac vaccinees, who have received complete regimes of vaccines by means of a SARS-CoV-2 surrogate virus blocking test. The ChAdOx1 nCoV-19 (p = 0.0013) and BNT162b2 (p = 0.0005) vaccines induced significant higher blocking activity with longer durability against the Spike (S) protein receptor binding domain (RBD) of wild type SARS-CoV-2 than the CoronaVac vaccine in uninfected vaccinees. Prior infection improved protection in the CoronaVac vaccinees. Subsequent investigation on the breadth of SARS-CoV-2 vaccine-induced antibody blocking responses, revealed that all vaccine platforms cross-protected uninfected vaccinees against all variant of concerns, except Omicron. Prior infection protected the ChAdOx1 nCoV-19 and BNT162b2 vaccinees against Omicron but not CoronaVac vaccinees. Our study suggests that vaccines that induce broader sterilizing immunity are essential to fight against fast-emerging variants.

Molecular and structural basis for Lewis glycan recognition by a cancer-targeting antibody.

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 Lea and Lex containing 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 Lea and Lex trisaccharides. While light chain residues were exclusively used for Lea binding, recognition of Lex involved both light and heavy chain residues. An extended groove is predicted to accommodate the Lea-Lex 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 Lea and Lex containing glycans. Furthermore, the predicted interactions with extended epitopes likely explains the selectivity of this antibody for targeting Lewis-positive tumours.

Glycan-Based Near-infrared Fluorescent (NIRF) Imaging of Gastrointestinal Tumors: a Preclinical Proof-of-Concept In Vivo Study.

PURPOSE: Aberrantly expressed glycans in cancer are of particular interest for tumor targeting. This proof-of-concept in vivo study aims to validate the use of aberrant Lewis glycans as target for antibody-based, real-time imaging of gastrointestinal cancers. PROCEDURES: Immunohistochemical (IHC) staining with monoclonal antibody FG88.2, targeting Lewisa/c/x, was performed on gastrointestinal tumors and their healthy counterparts. Then, FG88.2 and its chimeric human/mouse variant CH88.2 were conjugated with near-infrared fluorescent (NIRF) IRDye 800CW for real-time imaging. Specific binding was evaluated in vitro on human gastrointestinal cancer cell lines with cell-based plate assays, flow cytometry, and immune-fluorescence microscopy. Subsequently, mice bearing human colon and pancreatic subcutaneous tumors were imaged in vivo after intravenous administration of 1 nmol (150 µg) CH88.2-800CW with the clinical Artemis NIRF imaging system using the Pearl Trilogy small animal imager as reference. One week post-injection of the tracer, tumors and organs were resected and tracer uptake was analyzed ex vivo. RESULTS: IHC analysis showed strong FG88.2 staining on colonic, gastric, and pancreatic tumors, while staining on their normal tissue counterparts was limited. Next, human cancer cell lines HT-29 (colon) and BxPC-3 and PANC-1 (both pancreatic) were identified as respectively high, moderate, and low Lewisa/c/x-expressing. Using the clinical NIRF camera system for tumor-bearing mice, a mean tumor-to-background ratio (TBR) of 2.2 ± 0.3 (Pearl: 3.1 ± 0.8) was observed in the HT-29 tumors and a TBR of 1.8 ± 0.3 (Pearl: 1.9 ± 0.5) was achieved in the moderate expression BxPC-3 model. In both models, tumors could be adequately localized and delineated by NIRF for up to 1 week. Ex vivo analysis confirmed full tumor penetration of the tracer and low fluorescence signals in other organs. CONCLUSIONS: Using a novel chimeric Lewisa/c/x-targeting tracer in combination with a clinical NIRF imager, we demonstrate the potential of targeting Lewis glycans for fluorescence-guided surgery of gastrointestinal tumors.

Engineering the Human Fc Region Enables Direct Cell Killing by Cancer Glycan-Targeting Antibodies without the Need for Immune Effector Cells or Complement.

Murine IgG3 glycan-targeting mAb often induces direct cell killing in the absence of immune effector cells or complement via a proinflammatory mechanism resembling oncotic necrosis. This cancer cell killing is due to noncovalent association between Fc regions of neighboring antibodies, resulting in enhanced avidity. Human isotypes do not contain the residues underlying this cooperative binding mode; consequently, the direct cell killing of mouse IgG3 mAb is lost upon chimerization or humanization. Using the Lewisa/c/x -targeting 88mAb, we identified the murine IgG3 residues underlying the direct cell killing and increased avidity via a series of constant region shuffling and subdomain swapping approaches to create improved ("i") chimeric mAb with enhanced tumor killing in vitro and in vivo. Constant region shuffling identified a major CH3 and a minor CH2 contribution, which was further mapped to discontinuous regions among residues 286-306 and 339-378 that, when introduced in 88hIgG1, recapitulated the direct cell killing and avidity of 88mIgG3. Of greater interest was the creation of a sialyl-di-Lewisa-targeting i129G1 mAb via introduction of these selected residues into 129hIgG1, converting it into a direct cell killing mAb with enhanced avidity and significant in vivo tumor control. The human iG1 mAb, termed Avidimabs, retained effector functions, paving the way for the proinflammatory direct cell killing to promote antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity through relief of immunosuppression. Ultimately, Fc engineering of human glycan-targeting IgG1 mAb confers proinflammatory direct cell killing and enhanced avidity, an approach that could be used to improve the avidity of other mAb with therapeutic potential. SIGNIFICANCE: Fc engineering enhances avidity and direct cell killing of cancer-targeting anti-glycan antibodies to create superior clinical candidates for cancer immunotherapy.

The terminal sialic acid of stage-specific embryonic antigen-4 has a crucial role in binding to a cancer-targeting antibody.

Cancer remains a leading cause of morbidity and mortality worldwide, requiring ongoing development of targeted therapeutics such as monoclonal antibodies. Carbohydrates on embryonic cells are often highly expressed in cancer and are therefore attractive targets for antibodies. Stage-specific embryonic antigen-4 (SSEA-4) is one such glycolipid target expressed in many cancers, including breast and ovarian carcinomas. Here, we defined the structural basis for recognition of SSEA-4 by a novel monospecific chimeric antibody (ch28/11). Five X-ray structures of ch28/11 Fab complexes with the SSEA-4 glycan headgroup, determined at 1.5-2.7 Å resolutions, displayed highly similar three-dimensional structures indicating a stable binding mode. The structures also revealed that by adopting a horseshoe-shaped conformation in a deep groove, the glycan headgroup likely sits flat against the membrane to allow the antibody to interact with SSEA-4 on cancer cells. Moreover, we found that the terminal sialic acid of SSEA-4 plays a dominant role in dictating the exquisite specificity of the ch28/11 antibody. This observation was further supported by molecular dynamics simulations of the ch28/11-glycan complex, which show that SSEA-4 is stabilized by its terminal sialic acid, unlike SSEA-3, which lacks this sialic acid modification. These high-resolution views of how a glycolipid interacts with an antibody may help to advance a new class of cancer-targeting immunotherapy.

Monoclonal Antibody Targeting Sialyl-di-Lewisa-Containing Internalizing and Noninternalizing Glycoproteins with Cancer Immunotherapy Development Potential.

Tumor glycans constitute attractive targets for therapeutic antibodies. The sialylated glycocalyx plays a prominent role in cancer progression and immune evasion. Here, we describe the characterization of the mAb, FG129, which targets tumor-associated sialylated glycan, and demonstrate its potential for multimodal cancer therapy. FG129, obtained through BALB/c mouse immunizations with liposomes containing membrane glycan extracts from the colorectal cancer cell line LS180, is an mIgG1κ that targets sialyl-di-Lewisa-containing glycoproteins. FG129, as well as its chimeric human IgG1 variant, CH129, binds with nanomolar functional affinity to a range of colorectal, pancreatic, and gastric cancer cell lines. FG129 targets 74% (135/182) of pancreatic, 50% (46/92) of gastric, 36% (100/281) of colorectal, 27% (89/327) of ovarian, and 21% (42/201) of non-small cell lung cancers, by IHC. In our pancreatic cancer cohort, high FG129 glyco-epitope expression was significantly associated with poor prognosis (P = 0.004). Crucially, the glyco-epitope displays limited normal tissue distribution, with FG129 binding weakly to a small percentage of cells within gallbladder, ileum, liver, esophagus, pancreas, and thyroid tissues. Owing to glyco-epitope internalization, we validated payload delivery by CH129 through monomethyl auristatin E (MMAE) or maytansinoid (DM1 and DM4) conjugation. All three CH129 drug conjugates killed high-binding colorectal and pancreatic cancer cell lines with (sub)nanomolar potency, coinciding with significant in vivo xenograft tumor control by CH129-vcMMAE. CH129, with its restricted normal tissue distribution, avid tumor binding, and efficient payload delivery, is a promising candidate for the treatment of sialyl-di-Lewisa-expressing solid tumors, as an antibody-drug conjugate or as an alternative cancer immunotherapy modality.

Correction to: Combination Treatment with an Antibody-Drug Conjugate (A1mcMMAF) Targeting the Oncofetal Glycoprotein 5T4 and Carboplatin Improves Survival in a Xenograft Model of Ovarian Cancer.

The article Combination Treatment with an Antibody-Drug Conjugate (A1mcMMAF) Targeting the Oncofetal Glycoprotein 5T4 and Carboplatin Improves Survival in a Xenograft Model of Ovarian Cancer, written by Y. Louise Wan, Puja Sapra, James Bolton, Jia Xin Chua, Lindy G. Durrant and Peter L. Stern, was originally published under a CC BY-NC 4.0 license, but has now been made available under a CC BY 4.0 license.

Combination Treatment with an Antibody-Drug Conjugate (A1mcMMAF) Targeting the Oncofetal Glycoprotein 5T4 and Carboplatin Improves Survival in a Xenograft Model of Ovarian Cancer.

BACKGROUND: Recurrence occurs in over 75% of women with epithelial ovarian cancer despite optimal treatment. Selectively killing tumour cells thought to initiate relapse using an antibody-drug conjugate could prolong progression-free survival and offer an improved side-effect profile. A1mcMMAF is an antibody-drug conjugate designed to target cells expressing the tumour-associated antigen 5T4. It has shown to be efficacious in various cell line models and have a greater impact when combined with routine chemotherapeutic regimes. OBJECTIVES: This study aims to explore the potential for the use of a 5T4 antibody-drug conjugate in women with ovarian cancer both as a monotherapy and in combination with platinum-based chemotherapy. METHODS: Immunohistochemical analysis was used to assess 5T4 expression in tumours from patients with ovarian cancer. Effectiveness of A1mcMMAF therapy as a single agent and in combination with carboplatin was assessed in vitro in the ovarian cancer cell line SKOV3 and confirmed in vivo using a serial bioluminescence assay in a SKOV3 xenograft model of ovarian cancer. RESULTS: 5T4 is confirmed as suitably expressed in epithelial ovarian cancers prior to adjuvant therapy and is an independent predictor of poor survival. A1mcMMAF showed specific activity, both in vitro and in vivo, against SKOV3 ovarian cancer cells. When used in combination with carboplatin, in vivo tumour growth was inhibited resulting in prolonged survival in a SKOV3 xenograft model. CONCLUSIONS: These data support further investigation of A1mcMMAF in combination with platinum-based chemotherapy in ovarian and other cancer treatments.

Cancer cell associated glycans as targets for immunotherapy.

Therapeutic anti-glycan antibodies for cancer treatment are limited, in spite of the differential glycophenotype of cancer cells and associated biology. We have generated anti-Le glycan antibodies with clinical potential and multifaceted functionality. Increased understanding of all aspects of glycomic research will ensure the continued development of glycan-targeted immunotherapeutics.

Monoclonal Antibodies Targeting LecLex-Related Glycans with Potent Antitumor Activity.

PURPOSE: To produce antitumor monoclonal antibodies (mAbs) targeting glycans as they are aberrantly expressed in tumors and are coaccessory molecules for key survival pathways. EXPERIMENTAL DESIGN: Two mAbs (FG88.2 and FG88.7) recognizing novel tumor-associated Lewis (Le) glycans were produced by immunizations with plasma membrane lipid extracts of the COLO205 cell line. RESULTS: Glycan array analysis showed that both mAbs bound Le(c)Le(x), di-Le(a), and Le(a)Le(x), as well as Le(a)-containing glycans. These glycans are expressed on both lipids and proteins. Both mAbs showed strong tumor reactivity, binding to 71% (147 of 208) of colorectal, 81% (155 of 192) of pancreatic, 54% (52 of 96) of gastric, 23% (62 of 274) of non-small cell lung, and 31% (66 of 217) of ovarian tumor tissue in combination with a restricted normal tissue distribution. In colorectal cancer, high FG88 glyco-epitope expression was significantly associated with poor survival. The mAbs demonstrated excellent antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), in addition to direct tumor cell killing via a caspase-independent mechanism. Scanning electron microscopy revealed antibody-induced pore formation. In addition, the mAbs internalized, colocalized with lysosomes, and delivered saporin that killed cells with subnanomolar potency. In vivo, the mAbs demonstrated potent antitumor efficacy in a metastatic colorectal tumor model, leading to significant long-term survival. CONCLUSIONS: The mAbs direct and immune-assisted tumor cell killing, pan-tumor reactivity, and potent in vivo antitumor efficacy indicate their potential as therapeutic agents for the treatment of multiple solid tumors. In addition, internalization of saporin conjugates and associated tumor cell killing suggests their potential as antibody drug carriers.