Identification of a Single Tryptophan Residue as Critical for Binding Activity in a Humanized Monoclonal Antibody against Respiratory Syncytial Virus

Abstract

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.

Effect of pH and Light on Aggregation and Conformation of an IgG1 mAb

“…38 A similar loss of bioactivity with the loss of Trp in the CDR was also observed for an IgG1 mAb by Wei et al 43 Methionine oxidation to methionine sulfoxide in the Fc was also noted by Liu et al 42 and Lam et al 41 33 In those studies the mAb was photoirradiated with light at 254 nm rather than the broad spectrum light noted in the previous studies. They identified a series of dithiohemiacetal and thioether cross-links formed following photoirradiation and suggest the involvement of thiyl radicals in the processes.…”

“…30 For comparison, the light-induced cleavage of glutathione disulfide led predominantly to thiyl radicals. 30 It is well documented that radical mediated damage to proteins may lead to a variety of potentially negative consequences such as fragmentation 23,38 , aggregation 25,38,39 , oxidation [40][41][42][43] , changes to surface hydrophobicity, conformational changes 39 and even denaturation. 38,39 It may also ultimately result in the loss of activity or simply generate new reactive species propagating the radical reactions.…”

“…25,38,[41][42][43] Common to all of these studies was photodegradation resulting in the oxidation of tryptophan 38,43 and methionine 38,41,42 with differences noted in specific regions of each antibody. Qi et al observed substantial oxidation of light chain Trp94 in the CDR resulting in multiple oxidation products along with secondary oxidation of Met261, Met437 and His294 of the heavy chain.…”

Applications of immunochemistry in human health: advances in vaccinology and antibody design (IUPAC Technical Report)

“…The efficacy of therapeutic antibodies is often reduced by chemical degradations such as deamidation, isomerization, succinimide formation, methionine and tryptophan oxidation, and cysteinylation of unpaired cysteine in the CDR region [330][331][332][333][334], and antibodies with such tendencies should be, therefore, avoided as clinical candidate molecules. Among various degradation pathways, asparagine deamidation and isomerization in the CDRs seems to play a major role.…”

Assessing monoclonal antibody product quality attribute criticality through clinical studies

“…Deamidation at Asn-33 and oxidation at Trp-105 in the light chain and heavy chains, respectively, of two therapeutic mAbs represent examples of this class of attributes. 14,15 Alternatively, a PQA may affect efficacy by changing the in vivo concentration by impacting clearance rates. Attributes that have been shown to impact FcRn binding, e.g., oxidation of Met-252 and -428, are candidates that may impact efficacy in this indirect fashion.…”