The generation and characterisation of anti-glycan monoclonal antibodies

Glycomic profiling of tumour tissues consistently shows alterations in N- and O-glycosylation profiles of glycoproteins and glycolipids compared to healthy tissues, with important functional implications for cancer cell biology. Despite the attractiveness of the targets, there are very few mAbs recognising glycans as they induce low affinity IgM responses. The aim of this study was to develop a novel immunisation protocol, with the objective of increasing the immunogenicity of tumour-associated carbohydrate or glycolipid antigens, leading to the generation of novel anti-glycan monoclonal antibodies (mAbs) with potent cytotoxicity against cancers. Mice were immunised with, tumour cell plasma membrane glycolipid extracts incorporated into liposomes in the presence or absence of alpha-galactosylceramide (α-GalCer) and/or anti-CD40 mAb and/or with engineered cells, which express specific glycolipids. Three IgG3 murine mAbs (FG88.2, FG88.7 and FG2811.72) with subnanomolar potency were generated, suggesting that the immunisation protocols succeeded in increasing the immunogenicity of glycolipid antigens which promoted antibody class switching and affinity maturation. FG88.2 and FG88.7 mAbs recognised the novel Lewis a/c/x (Lea/c/x) glycans expressed on glycolipids and glycoproteins whereas FG2811.72 is an ultraspecific mAb targeting SSEA-4 glycolipid. Immunohistochemical analysis showed that FG88.2 glyco-epitopes were overexpressed on a broad range of tumour types with limited cross reactivity with normal human tissues. Characterisation of effector functions of FG88 and FG2811.72 mAbs demonstrated that they were capable of inducing potent antibody dependent cellular cytotoxicity (ADCC; EC50 FG88 mAbs: 10-9 M; EC50 FG2811.72 mAb: 10-10 M) and complement dependent cytotoxicity (CDC; EC50 FG88 mAbs: 10-9 M; EC50 FG2811.72 mAb: 10-9 M). In addition, FG88 mAbs were capable of inducing direct tumour cell death via oncosis which induces pore formation and lysis of tumour cells. Preliminary results showed that FG2811.72 mAb inhibited tumour cell growth in the absence of immune cells and complement proteins, but the mechanism of tumour cell growth inhibition remains unexplored. The excellent in vitro cytotoxicity of FG88 mAbs translated into potent anti-tumour efficacy and significant survival improvement in a colorectal hepatic metastasis xenograft model in which mAb treatment was initiated 10 days after tumour initiation and development of liver metastasis. No other studies, to date, have shown tumour eradication of 10-day established tumours and liver metastases. Traditional cancer chemotherapy is often accompanied with severe side effects to cancer patients. Antibody-drug conjugates (ADCs) are composed of a therapeutic mAb chemically linked to a potent cytotoxic drug. By linking mAbs to cytotoxic drugs, ADCs selectively target tumour cells, thus improving the therapeutic index of chemotherapeutic agents. ADCs work via the internalisation of antibody-antigen complexes into cancer cells, followed by the release of the cytotoxic drugs in the lysosomes which then causes cancer cell death. FG88 and FG2811.72 mAbs deliver Zaporin conjugates into tumour cells with subnanomolar potency, suggesting they have the potential to be used as ADCs. Murine mAbs have limited clinical utility due to their short half-life in serum, their suboptimal in stimulation of Fc effector functions and their high immunogenicity in humans. Therefore chimeric FG88.2 (CH88.2; IgG1) mAb was generated. Although CH88.2 mAb maintained antigen specificity and affinity, it had reduced CDC activity and importantly, it lost its ability to induce direct tumour cell death. In this study, the E345R mutation [a single amino acid substitution at position 345 (Glu345→ Arg)] was introduced into CH88.2 to generate a mutant version of CH88.2 named ECH88.2 mAb. The ECH88.2 mAb showed restored direct cell killing ability and the CDC activity was improved. In addition, ECH88.2 demonstrated excellent ADCC activity. Lewis a/c/x (Lea/c/x) glycans were expressed by both glycolipids and glycoproteins. Studies on the role of glycolipids/glycoproteins in inducing direct tumour cell death suggested that it was primarily mediated by glycolipids. In contrast, both glycolipids and glycoproteins were involved in ADCC, CDC and mAb internalisation into tumour cells. In summary, the aim of this study was achieved by generating three novel anti-glycan mAbs which bind to tumour associated carbohydrate antigens with high affinity. Their strong tumour specificity and limited cross reactivity to normal tissues, as well as their potent cytotoxicity effects, suggest that they should have great potential as therapeutic anti-cancer mAbs. Furthermore, their ability to internalise into tumour cells suggests that they may have potential as drug carriers. Importantly, this study demonstrates a novel approach: by introducing the E345R mutation in the Fc region of the chimeric mAbs it promotes non-covalent interaction in the presence of antigen leading to increased cell surface binding, improved CDC activity and restoration of the direct killing ability of the mouse mAbs

Monoclonal Antibodies Against Tumour-Associated Carbohydrate Antigens

Glycomic profiling of tumour tissues consistently shows alterations in N- and O-glycosylation profiles of glycoproteins and glycolipids compared to healthy tissues, with important functional implications for cancer cell biology. The overexpression of tumour-associated carbohydrate antigens (TACAs), as a result of aberrant glycosylation in tumours, is usually correlated with poor prognosis and survival of cancer patients. In tumours, TACAs are associated with worse tumour progression than the deletion and inactivation of tumour suppressor genes. The findings of TACAs acting are not merely tumour markers but also constitute part of the machinery in inducing cancer metastasis and invasiveness further strengthen the scientific rationales for immunotherapy targeting TACAs. Despite the attractiveness of the TACAs, there are very few anti-glycan monoclonal antibodies (mAbs), as glycans usually induce low-affinity IgM responses. This chapter provides an overview of TACAs, direct killing anti-glycan mAbs, and introduces two murine mAbs (FG88 mAbs) that recognise Lewis carbohydrate antigens overexpressed on tumour glycoconjugates with high functional affinity. Although the production of anti-glycan mAbs against cancers is not new, the production of high-affinity IgG anti-glycan mAbs is novel. FG88 mAbs definitely have great potential in cancer therapy and serve as valuable tools in glycobiology research

Effect of Metal Chelators on γ-Secretase Indicates That Calcium and Magnesium Ions Facilitate Cleavage of Alzheimer Amyloid Precursor Substrate

Gamma-secretase is involved in the production of Aβ amyloid peptides. It cleaves the transmembrane domain of the amyloid precursor protein (APP) at alternative sites to produce Aβ and the APP intracellular domain (AICD). Metal ions play an important role in Aβ aggregation and metabolism, thus metal chelators and ligands represent potential therapeutic agents for AD treatment. A direct effect of metal chelators on γ-secretase has not yet been investigated. The authors used an in vitro  γ-secretase assay consisting of cleavage of APP C100-3XFLAG by endogenous γ-secretase from rodent brains and human neuroblastoma SH-SY5Y, and detected AICD production by western blotting. Adding metalloprotease inhibitors to the reaction showed that clioquinol, phosphoramidon, and zinc metalloprotease inhibitors had no significant effect on γ-secretase activity. In contrast, phenanthroline, EDTA, and EGTA markedly decreased γ-secretase activity that could be restored by adding back calcium and magnesium ions. Mg2+ stabilized a 1,000 kDa presenilin 1 complex through blue native gel electrophoresis and size-exclusion chromatography. Data suggest that Ca2+ and Mg2+ stabilize γ-secretase and enhance its activity

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

Comparing the CASI-4R and the PGBI-10 M for Differentiating Bipolar Spectrum Disorders from Other Outpatient Diagnoses in Youth

We compared 2 rating scales with different manic symptom items on diagnostic accuracy for detecting pediatric bipolar spectrum disorder (BPSDs) in outpatient mental health clinics. Participants were 681 parents/guardians of eligible children (465 male, mean age = 9.34) who completed the Parent General Behavior Inventory-10-item Mania (PGBI-10M) and mania subscale of the Child and Adolescent Symptom Inventory-Revised (CASI-4R). Diagnoses were based on KSADS interviews with parent and youth. Receiver operating characteristic (ROC) analyses and diagnostic likelihood ratios (DLRs) determined discriminative validity and provided clinical utility, respectively. Logistic regressions tested for incremental validity in the CASI-4R mania subscale and PGBI-10M in predicting youth BPSD status above and beyond demographic and common diagnostic comorbidities. Both CASI-4R and PGBI-10M scales significantly distinguished BPSD (N=160) from other disorders (CASI-4R: Area under curve (AUC) = .80, p .05). Diagnostic likelihood ratios indicated low scores on either scale (CASI: 0–5; PGBI-10M: 0–6) cut BPSD odds to 1/5 of those with high scores (CASI DLR− = 0.17; PGBI-10M DLR− = 0.18). High scores on either scale (CASI: 14+; PGBI-10M: 20+) increased BPSD odds about fourfold (CASI DLR+ = 4.53; PGBI-10M DLR+ = 3.97). Logistic regressions indicated the CASI-4R mania subscale and PGBI-10M each provided incremental validity in predicting youth BPSD status. The CASI-4R is at least as valid as the PGBI-10M to help identify BPSDs, and can be considered as part of an assessment battery to screen for pediatric BPSDs

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

Destabilization of β Cell FIT2 by saturated fatty acids alter lipid droplet numbers and contribute to ER stress and diabetes

Western-type diets are linked to obesity and diabetes partly because of their high–saturated fatty acid (SFA) content. We found that SFAs, but not unsaturated fatty acids (USFAs), reduced lipid droplets (LDs) within pancreatic β cells. Mechanistically, SFAs, but not USFAs, reduced LD formation by inducing S-acylation and proteasomal, mediated degradation of fat storage–inducing transmembrane protein 2 (FIT2), an endoplasmic reticulum (ER) resident protein important for LD formation. Targeted ablation of FIT2 reduced β cell LD numbers, lowered β cell ATP levels, reduced Ca(2+) signaling, dampened vesicle exocytosis, down-regulated β cell transcription factors, up-regulated unfolded protein response genes, and finally, exacerbated diet-induced diabetes in mice. Subsequent mass spectrometry studies revealed increased C16:0 ceramide accumulation in islets of diet-induced diabetes mice lacking β cell FIT2. Inhibition of ceramide synthases ameliorated the enhanced ER stress and improved insulin secretion. FIT2 was reduced in mouse diabetic islets, and separately, overexpression of FIT2 increased the number of intracellular LDs and rescued SFA-induced ER stress and apoptosis, thereby highlighting the protective role of FIT2 and LDs against β cell lipotoxicity

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

©2020 American Association for Cancer Research. 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

Identification of Genes with Allelic Imbalance on 6p Associated with Nasopharyngeal Carcinoma in Southern Chinese

Nasopharyngeal carcinoma (NPC) is a malignancy of epithelial origin. The etiology of NPC is complex and includes multiple genetic and environmental factors. We employed case-control analysis to study the association of chromosome 6p regions with NPC. In total, 360 subjects and 360 healthy controls were included, and 233 single nucleotide polymorphisms (SNPs) on 6p were examined. Significant single-marker associations were found for SNPs rs2267633 (p = 4.49×10−5), rs2076483 (most significant, p = 3.36×10−5), and rs29230 (p = 1.43×10−4). The highly associated genes were the gamma-amino butyric acid B receptor 1 (GABBR1), human leukocyte antigen (HLA-A), and HLA complex group 9 (HCG9). Haplotypic associations were found for haplotypes AAA (located within GABBR1, p-value  = 6.46×10−5) and TT (located within HLA-A, p = 0.0014). Further investigation of the homozygous genotype frequencies between cases and controls suggested that micro-deletion regions occur in GABBR1 and neural precursor cell expressed developmentally down-regulated 9 (NEDD9). Quantitative real-time polymerase chain reaction (qPCR) using 11 pairs of NPC biopsy samples confirmed the significant decline in GABBR1 and NEDD9 mRNA expression in the cancer tissues compared to the adjacent non-tumor tissue (p<0.05). Our study demonstrates that multiple chromosome 6p susceptibility loci contribute to the risk of NPC, possibly though GABBR1 and NEDD9 loss of function

A piggyBac transposon-based mutagenesis system for the fission yeast Schizosaccharomyces pombe

The TTAA-specific transposon piggyBac (PB), originally isolated from the cabbage looper moth, Trichoplusia ni, has been utilized as an insertional mutagenesis tool in various eukaryotic organisms. Here, we show that PB transposes in the fission yeast Schizosaccharomyces pombe and leaves almost no footprints. We developed a PB-based mutagenesis system for S. pombe by constructing a strain with a selectable transposon excision marker and an integrated transposase gene. PB transposition in this strain has low chromosomal distribution bias as shown by deep sequencing-based insertion site mapping. Using this system, we obtained loss-of-function alleles of klp5 and klp6, and a gain-of-function allele of dam1 from a screen for mutants resistant to the microtubule-destabilizing drug thiabendazole. From another screen for cdc25-22 suppressors, we obtained multiple alleles of wee1 as expected. The success of these two screens demonstrated the usefulness of this PB-mediated mutagenesis tool for fission yeast