Specificity grafting of human antibody frameworks selected from a phage display library: generation of a highly stable humanized anti-CD22 single-chain Fv fragment.
A prerequisite for the enrichment of antibodies screened from phage display libraries is their stable expression on a phage during multiple selection rounds. Thus, if stringent panning procedures are employed, selection is simultaneously driven by antigen affinity, stability and solubility. To take advantage of robust pre-selected scaffolds of such molecules, we grafted single-chain Fv (scFv) antibodies, previously isolated from a human phage display library after multiple rounds of in vitro panning on tumor cells, with the specificity of the clinically established murine monoclonal anti-CD22 antibody RFB4. We show that a panel of grafted scFvs retained the specificity of the murine monoclonal antibody, bound to the target antigen with high affinity (6.4-9.6 nM), and exhibited exceptional biophysical stability with retention of 89-93% of the initial binding activity after 6 days of incubation in human serum at 37 degrees C. Selection of stable human scaffolds with high sequence identity to both the human germline and the rodent frameworks required only a small number of murine residues to be retained within the human frameworks in order to maintain the structural integrity of the antigen binding site. We expect this approach may be applicable for the rapid generation of highly stable humanized antibodies with low immunogenic potential.
National Cancer Institute at Frederick, Frederick, MD 21702, USA.
Jürgen
J
Krauss
Michaela A E
MA
Arndt
Andrew C R
AC
Martin
Huaitian
H
Liu
Susanna M
SM
Rybak
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D015703:Antigens, CD / Q000276:immunology*; D000944:Antigens, Differentiation, B-Lymphocyte / Q000276:immunology*; D001666:Binding Sites, Antibody; D015815:Cell Adhesion Molecules; D002853:Chromatography, Liquid; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D037102:Lectins / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D019151:Peptide Library; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D016415:Sequence Alignment; D044967:Serum; D051918:Sialic Acid Binding Ig-like Lectin 2; D013379:Substrate Specificity; D013816:Thermodynamics
D015703:Antigens, CD; D000944:Antigens, Differentiation, B-Lymphocyte; C497867:CD22 protein, human; C497868:Cd22 protein, mouse; D015815:Cell Adhesion Molecules; D000939:Epitopes; D007135:Immunoglobulin Variable Region; D037102:Lectins; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D051918:Sialic Acid Binding Ig-like Lectin 2