ABSTRACT
The chemoenzymatic remodeled monoclonal antidodies with well-defined glycan structure at the Fc domain display improved biological activities, such as ADCC and ADCP, and are more likely to yield a better safety profile by eliminating the non-human glycans derived from CHO cell culture. We covalently immobilize wild type endoglycosidase S (EndoS), fucosidase, and EndoS2 mutant on magnetic beads through a linker to efficiently generate homogeneous antibody glycoforms without additional purification step to remove endoglycosidase and fucosidase. We also used the biotinylated wild type EndoS2 and EndoS2 mutant in combination with covalently immobilized fucosidase on magnetic beads to allow the sequential removal of endoglycosidases and fucosidase for efficient glyco-engineering and isolation of antibodies without purifying deglycosylated antibody intermediate. Notably, the relatively expensive fucosidase can be recovered to reduce the cost, and the strong affinity of streptavidin to biotin would complete the isolation of biotinylated enzymes. We used Trastuzumab as a model to demonstrate both approaches were reliable for the large-scale production and isolation of antibodies without the residual contamination of endoglycosidase to avoid deglycosylation over storage time.
Subject(s)
Anti-Bacterial Agents/metabolism , Drug Development , Glycoside Hydrolases/metabolism , Trastuzumab/metabolism , alpha-L-Fucosidase/metabolism , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/isolation & purification , Biotinylation , Dose-Response Relationship, Drug , Enzymes, Immobilized/genetics , Enzymes, Immobilized/metabolism , Glycoside Hydrolases/genetics , Magnetic Phenomena , Molecular Structure , Mutation , Structure-Activity Relationship , Trastuzumab/chemistry , Trastuzumab/isolation & purification , alpha-L-Fucosidase/geneticsABSTRACT
Glycosphingolipids (GSLs) bearing the α-galactosyl headgroup and the acyl chain terminated with a phenyl derivative were found to be more potent than α-galactosyl ceramide (αGalCer) to stimulate both murine and human invariant natural killer T (iNKT) cells and to induce an antibody isotope switch to IgG. In this study, we replaced the galactosyl group with glucose (αGlc) and its fluoro-analogs and found that phenyl GSLs with αGlc (C34-Glc) and its fluoro-analog 6F-C34-Glc were stronger than those with αGal in stimulating human iNKT cells but weaker in mice. Their activities have a strong correlation with the binding avidities of the ternary interaction between the iNKT-cell receptor (iNKTCR) and CD1d-GSL complex. It was the iNKTCR rather than CD1d that dictated the species-specific responses. C34-Glc was further used as an adjuvant for a SSEA4-crm-197 vaccine, and after immunization in mice, the vaccine was highly effective against Lewis lung carcinoma.
Subject(s)
Adjuvants, Immunologic/chemistry , Adjuvants, Immunologic/pharmacology , Glycolipids/chemistry , Glycolipids/pharmacology , Lymphocyte Activation/drug effects , Natural Killer T-Cells/drug effects , Animals , Cancer Vaccines/chemistry , Cancer Vaccines/pharmacology , Cell Line , Humans , Mice , Mice, Inbred C57BL , Models, Molecular , Natural Killer T-Cells/immunologyABSTRACT
Antibodies have been developed as therapeutic agents for the treatment of cancer, infection, and inflammation. In addition to binding activity toward the target, antibodies also exhibit effector-mediated activities through the interaction of the Fc glycan and the Fc receptors on immune cells. To identify the optimal glycan structures for individual antibodies with desired activity, we have developed an effective method to modify the Fc-glycan structures to a homogeneous glycoform. In this study, it was found that the biantennary N-glycan structure with two terminal alpha-2,6-linked sialic acids is a common and optimized structure for the enhancement of antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, and antiinflammatory activities.
Subject(s)
Immunoglobulin Fc Fragments/chemistry , Immunoglobulin G/chemistry , Polysaccharides/chemistry , Rituximab/chemistry , Acetylglucosamine/chemistry , Acetylglucosamine/immunology , Animals , Antibodies, Viral/chemistry , Antibodies, Viral/immunology , Antibodies, Viral/therapeutic use , Antibody-Dependent Cell Cytotoxicity , Bacterial Proteins/metabolism , Bacteroides fragilis/enzymology , Cell Line, Tumor , Female , HEK293 Cells , Humans , Immunoglobulin Fc Fragments/immunology , Immunoglobulin G/immunology , Lymphoma, B-Cell/pathology , Mice , Mice, Inbred BALB C , Neuraminidase/metabolism , Orthomyxoviridae Infections/prevention & control , Protein Engineering , Receptors, IgG/immunology , Rituximab/immunology , Sialic Acids/chemistry , Sialic Acids/immunology , Streptococcus pyogenes/enzymology , Structure-Activity Relationship , Trastuzumab/chemistry , Trastuzumab/immunology , alpha-L-Fucosidase/metabolismABSTRACT
Globo H (GH) is a hexasaccharide specifically overexpressed on a variety of cancer cells and therefore, a good candidate for cancer vaccine development. To identify the optimal carrier and adjuvant combination, we chemically synthesized and linked GH to a carrier protein, including keyhole limpet hemocyanion, diphtheria toxoid cross-reactive material (CRM) 197 (DT), tetanus toxoid, and BSA, and combined with an adjuvant, and it was administered to mice for the study of immune response. Glycan microarray analysis of the antiserum obtained indicated that the combination of GH-DT adjuvanted with the α-galactosylceramide C34 has the highest enhancement of anti-GH IgG. Compared with the phase III clinical trial vaccine, GH-keyhole limpet hemocyanion/QS21, the GH-DT/C34 vaccine elicited more IgG antibodies, which are more selective for GH and the GH-related epitopes, stage-specific embryonic antigen 3 (SSEA3) and SSEA4, all of which were specifically overexpressed on breast cancer cells and breast cancer stem cells with SSEA4 at the highest level (>90%). We, therefore, further developed SSEA4-DT/C34 as a vaccine candidate, and after immunization, it was found that the elicited antibodies are also IgG-dominant and very specific for SSEA4.
