TY - BOOK
T1 - Generation of Monoclonal Antibodies Targeting Advanced Glycation end Products
T2 - Phage display technology and microarray peptide and protein libraries for antibody development and validation
AU - Wendel, Ulrika Anna Maria
PY - 2018
Y1 - 2018
N2 - This thesis is based on the generation and specificity evaluation of monoclonal antibodies, in the form of single-chain variable fragments (scFv:s) directed against advanced glycation end products (AGE). AGEs are formed in vivo by series of non-enzymatic reactions between protein amines and sugars such as glucose and their degradation products. The final irreversible structures, mostly formed on lysine and arginine side-chain amines, result in altered amino acid charges and a more rigid protein structure caused by crosslinking. These changes can severely affect protein function and give rise to antibody responses and inflammation. AGE is associated with several chronic diseases, such as diabetes, cancer, atherosclerosis and rheumatoid arthritis - possibly as a result of hyperglycemia or an AGE-prone microenvironment. To be able to understand the role of AGE in disease and to detect the modifications that are involved, analysis of specific glycated sites is crucial. However, currently available anti-AGE antibodies are not fully characterized for their binding patterns. Specific antibodies were selected from antibody libraries prepared from spleens of AGE-immunized mice, using phage display technology. ScFv-presenting phages were selected and screened against both full-length in vitro glycated protein collections and peptide libraries produced by solid phase peptide synthesis (SPPS) using glycated lysine building blocks. Selections of binding phages were performed with target epitopes immobilized on magnetic beads or immunotubes. Screening of binding scFv clones was performed by spot-on-spot printing on top of the protein and peptide AGE-libraries immobilized onto microarrays. Characterization of the binding epitopes of the resulting candidate antibodies was performed by extended microarray analysis. Two scFv:s were selected for further evaluation - D1-B2 specific for a carboxymethyllysine (CML) epitope and D12 that binds to methylglyoxal-modified histones. The usability of these two antibodies in biological applications was tested by flow cytometry, immunohistochemistry (IHC), antibody microarray, and immunoprecipitation (IP). Interesting staining patterns were found when incubating the antibodies on tissue microarrays (TMAs) consisting of human pancreatic cancer, and prostate cancer. The AGE antigen libraries were also used for evaluating the specificity of antibodies in mouse model sera. A clear difference in autoantibody reactivity against CML-peptides could be seen in three different rheumatoid arthritis (RA) mouse models. In conclusion, the approach of isolating specific antibodies by phage-display technology together with microarray screening on large synthetic target libraries presents a promising methodology for the generation of new epitope-mapped monoclonal antibodies against AGE. Preliminary IHC results from D1-B2 and D12 open up for further studies of anti-AGE antibodies for use in prognosis and therapeut.
AB - This thesis is based on the generation and specificity evaluation of monoclonal antibodies, in the form of single-chain variable fragments (scFv:s) directed against advanced glycation end products (AGE). AGEs are formed in vivo by series of non-enzymatic reactions between protein amines and sugars such as glucose and their degradation products. The final irreversible structures, mostly formed on lysine and arginine side-chain amines, result in altered amino acid charges and a more rigid protein structure caused by crosslinking. These changes can severely affect protein function and give rise to antibody responses and inflammation. AGE is associated with several chronic diseases, such as diabetes, cancer, atherosclerosis and rheumatoid arthritis - possibly as a result of hyperglycemia or an AGE-prone microenvironment. To be able to understand the role of AGE in disease and to detect the modifications that are involved, analysis of specific glycated sites is crucial. However, currently available anti-AGE antibodies are not fully characterized for their binding patterns. Specific antibodies were selected from antibody libraries prepared from spleens of AGE-immunized mice, using phage display technology. ScFv-presenting phages were selected and screened against both full-length in vitro glycated protein collections and peptide libraries produced by solid phase peptide synthesis (SPPS) using glycated lysine building blocks. Selections of binding phages were performed with target epitopes immobilized on magnetic beads or immunotubes. Screening of binding scFv clones was performed by spot-on-spot printing on top of the protein and peptide AGE-libraries immobilized onto microarrays. Characterization of the binding epitopes of the resulting candidate antibodies was performed by extended microarray analysis. Two scFv:s were selected for further evaluation - D1-B2 specific for a carboxymethyllysine (CML) epitope and D12 that binds to methylglyoxal-modified histones. The usability of these two antibodies in biological applications was tested by flow cytometry, immunohistochemistry (IHC), antibody microarray, and immunoprecipitation (IP). Interesting staining patterns were found when incubating the antibodies on tissue microarrays (TMAs) consisting of human pancreatic cancer, and prostate cancer. The AGE antigen libraries were also used for evaluating the specificity of antibodies in mouse model sera. A clear difference in autoantibody reactivity against CML-peptides could be seen in three different rheumatoid arthritis (RA) mouse models. In conclusion, the approach of isolating specific antibodies by phage-display technology together with microarray screening on large synthetic target libraries presents a promising methodology for the generation of new epitope-mapped monoclonal antibodies against AGE. Preliminary IHC results from D1-B2 and D12 open up for further studies of anti-AGE antibodies for use in prognosis and therapeut.
UR - https://rex.kb.dk/permalink/f/h35n6k/KGL01012062735
M3 - Ph.D. thesis
BT - Generation of Monoclonal Antibodies Targeting Advanced Glycation end Products
PB - Department of Chemistry, Faculty of Science, University of Copenhagen
ER -
