TY - BOOK
T1 - Identification and evaluation of nextgeneration PTM-specific antibodies
T2 - A microarray-based approach
AU - Persson, Nina Emilia
PY - 2017
Y1 - 2017
N2 - Over the past 30 years, antibody-based therapies have been established as important tools fortreatment of cancer, autoimmune diseases, inflammatory diseases, organ transplantations andinfectious diseases. There are today over 47 FDA approved monoclonal antibody products fordiagnostics and therapy. Including antibodies target several different categories of antigens suchas proteins, glycoproteins and glycolipids. Glycoproteins have become highlighted in cancerresearch since they are frequently involved in the initiation and spreading of cancer. One form ofglycosylation of proteins is the O-glycosylation. Incomplete O-glycan synthesis in cancer lead totruncated carbohydrate structures like the Tn-antigen (GalNac-Ser/Thr) or the STn-antigen(Neu5Acα2-6GalNAc-O-Ser/Thr). This type of aberrant glycosylation is very frequently foundin cancers and correlate with metastatic behaviour and bad prognosis for the patients. Severalmonoclonal antibodies have been generated against the Tn- and STn-antigens, but none has yetreached approval for therapeutic or diagnostic use. Indicating the need for a new generation ofantibodies against this type aberrant glycosylation.The two major techniques used for the production of monoclonal antibodies are somatic cellhybridization (hybridoma technology) and phage display. For both techniques, efforts have beenmade to develop new screening assays, but early identification of fine specific of antibodiesremains a bottleneck in the overall procedure of antibody generation. An optimal selectionprocess needs to exclude cross-reactive or suboptimal binders at an early stage to avoid problemsin the downstream evaluation process.In this thesis, a microarray screening assay is described. This assay combines an antibody and anantigen microarray for high content screening of multiple single-chain fragment variable (scFv)clones. Two different analyses are performed on the same microarray. There is no need for anypurification or enrichment before screening. In the first analysis, the ability of the individualscFv clone to bind to the soluble form of the antigens is evaluated. Favouring selection for scFvwith higher affinity rather than avidity. In the second analysis the binding of scFv to denselypresentedantigens enable the identification of clones with lower affinity, resulting in an overallhigher hit rate. This workflow allows for early selection of high affinity binders againstpredefined peptide and glycopeptide targets.Using phage display and the microarray screening assay a new anti-Tn, G2-D11 was identified.This antibody differs in specificity compared to other anti-Tn antibodies. It is specific for twoadjacent Tn-antigens and compared to other Tn-antigen binding antibodies the influenced by therest of the peptide backbone is limited. Indicating a high degree of carbohydrate epitopedominance and independence of the peptide backbone. With an impressive affinity (KDapp=1.3 x10-8 M) and no cross-reactivity to either STn epitope or blood group A antigens, the G2-D11differ from Tn-antigen binding antibodies reported in the literature. A cohort of 80 tissuesections of gastric carcinoma were all stained positive in immunohistochemistry with G2-D11.With these characteristics, G2-D11 might be a promising candidate for a general anti-Tn reagentto be used in research, diagnosis or therapy.In subsequent project, a new antibody library was constructed from the heavy chain variabledomain of G2-D11 and random light chains domains from immunized mice. Using this antibodylibrary and our screening assay, we could select a number of antibodies specific for the Tnantigenon a peptide from the CD43 protein and not reactive to either the Tn antigen on adifferent peptide or to the corresponding non-modified CD43 peptide. This illustrates theusefulness of antibody engineering and the strength of our antibody development concept foridentification of new carbohydrate binding antibodies.
AB - Over the past 30 years, antibody-based therapies have been established as important tools fortreatment of cancer, autoimmune diseases, inflammatory diseases, organ transplantations andinfectious diseases. There are today over 47 FDA approved monoclonal antibody products fordiagnostics and therapy. Including antibodies target several different categories of antigens suchas proteins, glycoproteins and glycolipids. Glycoproteins have become highlighted in cancerresearch since they are frequently involved in the initiation and spreading of cancer. One form ofglycosylation of proteins is the O-glycosylation. Incomplete O-glycan synthesis in cancer lead totruncated carbohydrate structures like the Tn-antigen (GalNac-Ser/Thr) or the STn-antigen(Neu5Acα2-6GalNAc-O-Ser/Thr). This type of aberrant glycosylation is very frequently foundin cancers and correlate with metastatic behaviour and bad prognosis for the patients. Severalmonoclonal antibodies have been generated against the Tn- and STn-antigens, but none has yetreached approval for therapeutic or diagnostic use. Indicating the need for a new generation ofantibodies against this type aberrant glycosylation.The two major techniques used for the production of monoclonal antibodies are somatic cellhybridization (hybridoma technology) and phage display. For both techniques, efforts have beenmade to develop new screening assays, but early identification of fine specific of antibodiesremains a bottleneck in the overall procedure of antibody generation. An optimal selectionprocess needs to exclude cross-reactive or suboptimal binders at an early stage to avoid problemsin the downstream evaluation process.In this thesis, a microarray screening assay is described. This assay combines an antibody and anantigen microarray for high content screening of multiple single-chain fragment variable (scFv)clones. Two different analyses are performed on the same microarray. There is no need for anypurification or enrichment before screening. In the first analysis, the ability of the individualscFv clone to bind to the soluble form of the antigens is evaluated. Favouring selection for scFvwith higher affinity rather than avidity. In the second analysis the binding of scFv to denselypresentedantigens enable the identification of clones with lower affinity, resulting in an overallhigher hit rate. This workflow allows for early selection of high affinity binders againstpredefined peptide and glycopeptide targets.Using phage display and the microarray screening assay a new anti-Tn, G2-D11 was identified.This antibody differs in specificity compared to other anti-Tn antibodies. It is specific for twoadjacent Tn-antigens and compared to other Tn-antigen binding antibodies the influenced by therest of the peptide backbone is limited. Indicating a high degree of carbohydrate epitopedominance and independence of the peptide backbone. With an impressive affinity (KDapp=1.3 x10-8 M) and no cross-reactivity to either STn epitope or blood group A antigens, the G2-D11differ from Tn-antigen binding antibodies reported in the literature. A cohort of 80 tissuesections of gastric carcinoma were all stained positive in immunohistochemistry with G2-D11.With these characteristics, G2-D11 might be a promising candidate for a general anti-Tn reagentto be used in research, diagnosis or therapy.In subsequent project, a new antibody library was constructed from the heavy chain variabledomain of G2-D11 and random light chains domains from immunized mice. Using this antibodylibrary and our screening assay, we could select a number of antibodies specific for the Tnantigenon a peptide from the CD43 protein and not reactive to either the Tn antigen on adifferent peptide or to the corresponding non-modified CD43 peptide. This illustrates theusefulness of antibody engineering and the strength of our antibody development concept foridentification of new carbohydrate binding antibodies.
UR - https://rex.kb.dk/primo-explore/fulldisplay?docid=KGL01010613080&context=L&vid=NUI&search_scope=KGL&tab=default_tab&lang=da_DK
M3 - Ph.D. thesis
BT - Identification and evaluation of nextgeneration PTM-specific antibodies
PB - Department of Chemistry, Faculty of Science, University of Copenhagen
ER -
