In vitro antibody affinity maturation targeting germline hotspots.
Affinity-matured antibodies can exhibit increased biological efficacy. Regardless of whether an antibody is isolated from a hybridoma or a human Fv phage library, the antibody affinity for its target may need improvement for therapeutic applications. An increased affinity may allow for a reduced dosage of a therapeutic antibody; toxic side effects may also be reduced. In the immune system, affinity maturation is a process involving somatic hypermutations in B cells. Therefore, germline hotspot residues are most likely to have a major impact on antibody affinity. Here, we describe procedures for germline hotspot mutagenesis with an emphasis on strategies for randomizing hotspots with PCR and phage display, using as an example the anti-CD22 monoclonal antibody.
Ho
Mitchell
M
National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
Pastan
Ira
I
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics; D000915:Antibody Affinity / Q000276:immunology*; D001435:Bacteriophages; D001483:Base Sequence; D018274:Electroporation; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D005434:Flow Cytometry; D005854:Germ Cells / Q000276:immunology*; D008958:Models, Molecular; D008967:Molecular Biology / Q000379:methods*; D008969:Molecular Sequence Data; D019151:Peptide Library; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D000911:Antibodies, Monoclonal; D019151:Peptide Library