Identification of a single tryptophan residue as critical for binding activity in a humanized monoclonal antibody against respiratory syncytial virus.
We have identified a single tryptophan (Trp) residue responsible for loss of binding and biological activity upon ultraviolet (UV) light irradiation in MEDI-493, a humanized monoclonal antibody (MAb) against respiratory syncytial virus (RSV). This finding provides a better understanding of structure-function relationship in a 150-kDa protein. Irradiation of MEDI-493 with UV light resulted in spectral changes typical of Trp photoproducts and in a progressive loss of MEDI-493 binding and biological activity as measured by ELISA, Biacore, and cell-based assays. Mass spectrometric characterization of the proteolytic peptides generated from the UV irradiated MEDI-493 confirmed that most methionine (Met) and a few Trp residues were oxidized to various extents upon exposure to UV light. Among Trp residues, only Trp-105, containing the most solvent-exposed indole moiety in MEDI-493 and residing in a complementary-determining region (CDR) of the heavy chain, was significantly oxidized. When bound to a synthetic antigenic peptide, MEDI-493 showed significant resistance toward binding activity loss during UV irradiation. A second MAb (MEDI-524) with Trp-105 replaced by phenylalanine (Phe) showed a similar pattern of Met oxidation, but no loss of binding and biological activity following irradiation. Treatment of both MAbs with Met- and Trp-specific oxidizing reagents showed that oxidation of Trp-105 correlated with the activity loss, whereas Met oxidation did not affect the activity. These results demonstrate that Trp-105 in MEDI-493 is responsible for the UV light-induced effects.
Wei
Ziping
Z
Department of Analytical Biochemistry, MedImmune, One MedImmune Way, Gaithersburg, Maryland 20878, USA.
Feng
Jinhua
J
Lin
Hung-Yu
HY
Mullapudi
Sombabu
S
Bishop
Eric
E
Tous
Guillermo I
GI
Casas-Finet
Jose
J
Hakki
Fadi
F
Strouse
Robert
R
Schenerman
Mark A
MA
D000595:Amino Acid Sequence / Q000528:radiation effects; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000494:pharmacology* / Q000528:radiation effects; D001665:Binding Sites; D006801:Humans; D010084:Oxidation-Reduction; D010777:Photochemistry; D012136:Respiratory Syncytial Viruses / Q000187:drug effects* / Q000276:immunology*; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000737:chemistry* / Q000528:radiation effects; D014466:Ultraviolet Rays
D000911:Antibodies, Monoclonal; D014364:Tryptophan