Novel anti-Sialyl-Tn monoclonal antibodies and antibody-drug conjugates demonstrate tumor specificity and anti-tumor activity.

Targeted therapeutics that can differentiate between normal and malignant tumor cells represent the ideal standard for the development of a successful anti-cancer strategy. The Sialyl-Thomsen-nouveau antigen (STn or Sialyl-Tn, also known as CD175s) is rarely seen in normal adult tissues, but it is abundantly expressed in many types of human epithelial cancers. We have identified novel antibodies that specifically target with high affinity the STn glycan independent of its carrier protein, affording the potential to recognize a wider array of cancer-specific sialylated proteins. A panel of murine monoclonal anti-STn therapeutic antibodies were generated and their binding specificity and efficacy were characterized in vitro and in in vivo murine cancer models. A subset of these antibodies were conjugated to monomethyl auristatin E (MMAE) to generate antibody-drug conjugates (ADCs). These ADCs demonstrated in vitro efficacy in STn-expressing cell lines and significant tumor growth inhibition in STn-expressing tumor xenograft cancer models with no evidence of overt toxicity.

Human xeno-autoantibodies against a non-human sialic acid serve as novel serum biomarkers and immunotherapeutics in cancer.

Human carcinomas can metabolically incorporate and present the dietary non-human sialic acid Neu5Gc, which differs from the human sialic acid N-acetylneuraminic acid (Neu5Ac) by 1 oxygen atom. Tumor-associated Neu5Gc can interact with low levels of circulating anti-Neu5Gc antibodies, thereby facilitating tumor progression via chronic inflammation in a human-like Neu5Gc-deficient mouse model. Here we show that human anti-Neu5Gc antibodies can be affinity-purified in substantial amounts from clinically approved intravenous IgG (IVIG) and used at higher concentrations to suppress growth of the same Neu5Gc-expressing tumors. Hypothesizing that this polyclonal spectrum of human anti-Neu5Gc antibodies also includes potential cancer biomarkers, we then characterize them in cancer and noncancer patients' sera, using a novel sialoglycan microarray presenting multiple Neu5Gc-glycans and control Neu5Ac-glycans. Antibodies against Neu5Gcα2-6GalNAcα1-O-Ser/Thr (GcSTn) were found to be more prominent in patients with carcinomas than with other diseases. This unusual epitope arises from dietary Neu5Gc incorporation into the carcinoma marker Sialyl-Tn, and is the first example of such a novel mechanism for biomarker generation. Finally, human serum or purified antibodies rich in anti-GcSTn-reactivity kill GcSTn-expressing human tumors via complement-dependent cytotoxicity or antibody-dependent cellular cytotoxicity. Such xeno-autoantibodies and xeno-autoantigens have potential for novel diagnostics, prognostics, and therapeutics in human carcinomas.

Sensitive and specific detection of the non-human sialic Acid N-glycolylneuraminic acid in human tissues and biotherapeutic products.

BACKGROUND: Humans are genetically defective in synthesizing the common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc), but can metabolically incorporate it from dietary sources (particularly red meat and milk) into glycoproteins and glycolipids of human tumors, fetuses and some normal tissues. Metabolic incorporation of Neu5Gc from animal-derived cells and medium components also results in variable contamination of molecules and cells intended for human therapies. These Neu5Gc-incorporation phenomena are practically significant, because normal humans can have high levels of circulating anti-Neu5Gc antibodies. Thus, there is need for the sensitive and specific detection of Neu5Gc in human tissues and biotherapeutic products. Unlike monoclonal antibodies that recognize Neu5Gc only in the context of underlying structures, chicken immunoglobulin Y (IgY) polyclonal antibodies can recognize Neu5Gc in broader contexts. However, prior preparations of such antibodies (including our own) suffered from some non-specificity, as well as some cross-reactivity with the human sialic acid N-acetylneuraminic acid (Neu5Ac). METHODOLOGY/PRINCIPAL FINDINGS: We have developed a novel affinity method utilizing sequential columns of immobilized human and chimpanzee serum sialoglycoproteins, followed by specific elution from the latter column by free Neu5Gc. The resulting mono-specific antibody shows no staining in tissues or cells from mice with a human-like defect in Neu5Gc production. It allows sensitive and specific detection of Neu5Gc in all underlying glycan structural contexts studied, and is applicable to immunohistochemical, enzyme-linked immunosorbent assay (ELISA), Western blot and flow cytometry analyses. Non-immune chicken IgY is used as a reliable negative control. We show that these approaches allow sensitive detection of Neu5Gc in human tissue samples and in some biotherapeutic products, and finally show an example of how Neu5Gc might be eliminated from such products, by using a human cell line grown under defined conditions. CONCLUSIONS: We report a reliable antibody-based method for highly sensitive and specific detection of the non-human sialic acid Neu5Gc in human tissues and biotherapeutic products that has not been previously described.

Evidence for dynamic interplay of different oligomeric states of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase by biophysical methods.

The bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) is a key enzyme for the biosynthesis of sialic acids, the terminal sugars of glycoconjugates associated with a variety of physiological and pathological processes such as cell adhesion, development, inflammation and cancer. In this study, we characterized rat GNE by different biophysical methods, analytical ultracentrifugation, dynamic light-scattering and size-exclusion chromatography, all revealing the native hydrodynamic behavior and molar mass of the protein. We show that GNE is able to reversibly self-associate into different oligomeric states including monomers, dimers and tetramers. Additionally, it forms non-specific aggregates of high molecular mass, which cannot be unequivocally assigned a distinct size. Our results also indicate that ligands of the epimerase domain of the bifunctional enzyme, namely UDP-N-acetylglucosamine and CMP-N-acetylneuraminic acid, stabilize the protein against aggregation and are capable of modulating the quaternary structure of the protein. The presence of UDP-N-acetylglucosamine strongly favors the tetrameric state, which therefore likely represents the active state of the enzyme in cells.

Identification of human L-fucose kinase amino acid sequence.

Fucose is a major component of complex carbohydrates. L-Fucose kinase (fucokinase) takes part in the salvage pathway for reutilization of fucose from the degradation of oligosaccharides. The amino acid sequence of human fucokinase was derived from a cDNA encoding a protein of hitherto unidentified function. Human fucokinase polypeptide chain consists of 990 amino acids with a predicted molecular mass of 107 kDa. The C-terminal part of its amino acid sequence showed sequence motifs typical for sugar kinases. Fucokinase full-length protein and a deletion mutant lacking the first 363 amino acids of the N-terminus were expressed in Escherichia coli BL21 cells. Both proteins displayed fucokinase activity. These results reveal that the discovered cDNA encodes the fucokinase protein and they confirm that a functional kinase domain is located in the C-terminal part of the enzyme.