"Superhumanized" Antibodies: Reduction of Immunogenic Potential by Complementarity-Determining Region Grafting with Human Germline Sequences: Application to an Anti-CD28

Humanized Abs are created by combining, at the genetic level, the complementarity-determining regions of a murine mAb with the framework sequences of a human Ab variable domain. This leads to a functional Ab with reduced immunogenic side effects in human therapy. In this study, we report a new appro...

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Bibliographic Details
Published in:The Journal of immunology (1950) 2002-07, Vol.169 (2), p.1119-1125
Main Authors: Tan, Philip, Mitchell, David A, Buss, Timothy N, Holmes, Margaret A, Anasetti, Claudio, Foote, Jefferson
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Antibodies - chemistry
Antibodies - genetics
Antibodies - pharmacology
Antibodies, Blocking - genetics
Antibodies, Blocking - pharmacology
B7-1 Antigen - metabolism
Binding Sites, Antibody - genetics
CD28 Antigens - genetics
CD28 Antigens - immunology
CD28 Antigens - metabolism
Complementarity Determining Regions - chemistry
Complementarity Determining Regions - genetics
Humans
Immunoglobulin Fab Fragments - biosynthesis
Immunoglobulin Fab Fragments - genetics
Immunoglobulin Heavy Chains - biosynthesis
Immunoglobulin Heavy Chains - chemistry
Immunoglobulin Heavy Chains - genetics
Immunoglobulin Light Chains - biosynthesis
Immunoglobulin Light Chains - chemistry
Immunoglobulin Light Chains - genetics
Immunoglobulin Variable Region - biosynthesis
Immunoglobulin Variable Region - chemistry
Immunoglobulin Variable Region - genetics
Immunosuppressive Agents - pharmacology
Lymphocyte Culture Test, Mixed
Mice
Molecular Sequence Data
Recombinant Fusion Proteins - biosynthesis
Recombinant Fusion Proteins - chemical synthesis
Recombinant Fusion Proteins - chemistry
Sequence Homology, Amino Acid
Species Specificity
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Summary:Humanized Abs are created by combining, at the genetic level, the complementarity-determining regions of a murine mAb with the framework sequences of a human Ab variable domain. This leads to a functional Ab with reduced immunogenic side effects in human therapy. In this study, we report a new approach to humanizing murine mAbs that may reduce immunogenicity even further. This method is applied to humanize the murine anti-human CD28 Ab, 9.3. The canonical structures of the hypervariable loops of murine 9.3 were matched to human genomic V gene sequences whose hypervariable loops had identical or similar canonical structures. Framework sequences for those human V genes were then used, unmodified, with the 9.3 complementarity-determining regions to construct a humanized version of 9.3. The humanized 9.3 and a chimeric 9.3 control were expressed in Escherichia coli as Fab. The humanized Fab showed a moderate loss in avidity in a direct binding ELISA with immobilized CD28-Ig fusion protein (CD28-Ig). Humanized 9.3 blocked ligation of CD28-Ig to cells expressing the CD28 receptor CD80. Lastly, the humanized 9.3 showed biological activity as an immunosuppressant by inhibiting a MLR.
ISSN:0022-1767
1550-6606
DOI:10.4049/jimmunol.169.2.1119