Selection of antibody single-chain variable fragments with improved carbohydrate binding by phage display

S.J Deng,C.R Mackenzie,J Sadowska,J Michniewicz,N.M Young,D.R Bundle,S.A Narang

doi:10.1016/s0021-9258(17)36914-4

S.J Deng, C.R Mackenzie + Show 5 more

Open Access

https://doi.org/10.1016/s0021-9258(17)36914-4

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Abstract

A single-chain variable fragment (Fv) version of a murine monoclonal antibody, Se155-4, specific for Salmonella serogroup B O-polysaccharide, was used as a model system for testing monovalent phage display as a route for enhancing the relatively low affinities that typify anti-carbohydrate antibodies. Random single-chain Fv mutant libraries generated by chemical and error-prone polymerase chain reaction methods were panned against the serogroup B lipopolysaccharide. Panning of a randomly mutated heavy chain variable domain library indicated selection for improved serogroup B binders and yielded six mutants, five of which showed wild type activity by enzyme immunoassay. Two of these were apparently selected on the basis of better functional single-chain Fv yield in Escherichia coli. A heavy chain mutation (Ile77-->Thr) in one mutant, 3B1, appeared to have a particularly dramatic effect, resulting in yields of approximately 120 mg/liter of functional periplasmic product. The sixth mutant, 4B2, had complementarity determining region 1 (CDR1) and CDR2 mutations and demonstrated 10-fold improved binding, by enzyme immunoassay, relative to the wild type. Extensive analysis of antigen-antibody interactions indicated that the improved binding properties of 4B2 were attributable to a higher association rate constant and interaction with an epitope that is larger than the trisaccharide recognized by the wild type. None of the mutations involved known trisaccharide contact residues; this was consistent with analysis of wild type and mutant single-chain Fvs by titration microcalorimetry. Examination of the structure indicated that two mutations in the heavy chain CDR2 provided improved surface complementarity between the protein and the extended epitope encompassing 2 additional hexose residues. However, introduction of only the CDR2 mutations into the wild type structure failed to confer the improved binding properties of 4B2, indicating an indirect effect by the more distant mutations. Panning of randomly mutated light chain variable domain and full-length single-chain Fv mutant libraries did not yield mutants with improved assembly or binding properties.

Highlights

V, or V, domains and full-length scFv were subjected to mutation by chemical and error-pronePCR methods, and the resulting mutant libraries were panned against serogroup B lipopolysaccharide.Onlythe V, librariesyieldedcloneswith improved binding to serogroup B lipopolysaccharide
T A ~ LIE Amino acid differencesfrom wild type of heavy chain mutants selected from chemical(series 3B) and PCR generated libraries and of site-directed mutants scFv type and clone
({Man-[Abel-Gal-Rhaln)showed that 4B2 binding to these antigens was approximately 10-foldhigher ineach instance, relative to the wild type (Fig. 4)

Summary

Nhe I

P2 trisaccharide methyl glycoside were prepared as described elsewhere.' 30 "C. Following centrifugation, the cells were resuspended in lysis. The With the first round productas template, asecond round introduceda n basic protocol for random chemical mutagenesis was described earlier EagI site, for insertion of the mutant sequence into pSK4, with the (15, 16). Both error-prone PCR and PCR-mediated chemical mutagen- upstream primer 5'-CAAACAGCGGCCGGGTCAGGGTCTAGAATGG-. PCR products were codons between the scFv and gIII sequencebsy ligation of the linearized purified by phenol extraction followed by restriction enzyme digestion. T A ~ LIE Amino acid differencesfrom wild type of heavy chain mutants selected from chemical(series 3B) and PCR (series 4B) generated libraries and of site-directed mutants scFv type and clone

Phage display mutants

RESULTS

DISCUSSION