Human framework adaptation of a mouse anti-human IL-13 antibody.
Humanization of a potent neutralizing mouse anti-human IL-13 antibody (m836) using a method called human framework adaptation (HFA) is reported. HFA consists of two steps: human framework selection (HFS) and specificity-determining residue optimization (SDRO). The HFS step involved generation of a library of m836 antigen binding sites combined with diverse human germline framework regions (FRs), which were selected based on structural and sequence similarities between mouse variable domains and a repertoire of human antibody germline genes. SDRO consisted of diversifying specificity-determining residues and selecting variants with improved affinity using phage display. HFS of m836 resulted in a 5-fold loss of affinity, whereas SDRO increased the affinity up to 100-fold compared to the HFS antibody. Crystal structures of Fabs in complex with IL-13 were obtained for m836, the HFS variant chosen for SDRO, and one of the highest-affinity SDRO variants. Analysis of the structures revealed that major conformational changes in FR-H1 and FR-H3 occurred after FR replacement, but none of them had an evident direct impact on residues in contact with IL-13. Instead, subtle changes affected the V(L)/V(H) (variable-light domain/variable-heavy domain) interface and were likely responsible for the 5-fold decreased affinity. After SDRO, increased affinity resulted mainly from rearrangements in hydrogen-bonding pattern at the antibody/antigen interface. Comparison with m836 putative germline genes suggested interesting analogies between natural affinity maturation and the engineering process that led to the potent HFA anti-human IL-13 antibody.
Centocor R&D, Inc., 3210 Merryfield Row, San Diego, CA 92121, USA.
Johan
J
Fransson
Alexey
A
Teplyakov
Gopalan
G
Raghunathan
Ellen
E
Chi
Wendy
W
Cordier
Thai
T
Dinh
Yiqing
Y
Feng
Jill
J
Giles-Komar
Gary
G
Gilliland
Bridget
B
Lollo
Thomas J
TJ
Malia
Walter
W
Nishioka
Galina
G
Obmolova
Shanrong
S
Zhao
Yonghong
Y
Zhao
Ronald V
RV
Swanson
Juan C
JC
Almagro
D000595:Amino Acid Sequence; D000818:Animals; D057134:Antibodies, Neutralizing / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D000937:Antigen-Antibody Reactions; D001665:Binding Sites; D018360:Crystallography, X-Ray; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D018793:Interleukin-13 / Q000037:antagonists & inhibitors* / 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
D057134:Antibodies, Neutralizing; D007135:Immunoglobulin Variable Region; D018793:Interleukin-13; D019151:Peptide Library; D011993:Recombinant Fusion Proteins