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*Department of Pediatrics, Memorial Sloan-Kettering Cancer Center, New York, NY 10065;
Department of Chemistry, University of Calgary, Calgary, Alberta, Canada; and
Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada
Ab affinity maturation in vivo is always accompanied by negative selection to maintain Ag specificity. In contrast, in vitro affinity maturation can lead to epitope spread, resulting in loss of specificity. Anti-ganglioside-GD2 mAbs are clinically effective against neuroblastoma; pain and neuropathy are major side effects. We used structural relatives of GD2 to define epitope spread during in vitro affinity maturation of an anti-GD2 single-chain variable fragment (scFv) called 5F11-scFv. Clonal dominance identified by polyclonal sequencing was confirmed by analyzing individual clones. Affinity-matured mutations were introduced into scFv-streptavidin for functional studies. Without a negative selector, 19-fold affinity improvement (clone Q, where Q is the symbol for glutamine) was associated with strong cross-reactivity with GM2 and GD1b and moderate cross-reactivity with GD3, resulting in positive immunohistochemical staining of all 13 non-neural normal human tissues, in contrast to none of 13 tissues with parental clone P. With GM2 as a negative selector, clone Y (where Y is the symbol for tyrosine) was generated with only weak cross-reactivity with GD1b, adrenal and thyroid glands, and no staining of other non-neural normal tissues. Even though there was only a 3-fold affinity improvement, clone Y showed significantly higher tumor uptake over parental clone P (134%, p = 0.04), whereas clone Q was inferior (54% of clone P; p = 0.05) as confirmed by tumor-to-normal tissue ratios across 16 organs (41% of clone P; p < 0.0001). Using the less efficient negative selector GD3, a clone mixture (Q, V, and Y, where V is the symbol for valine) emerged. We conclude that epitope spread during affinity maturation can be reduced by negative selection. Furthermore, efficiency of the negative selector depends on its cross-reactive affinity with the matured scFv.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported in part by National Institutes of Health Grant CA106450, Robert Steel Foundation, Hope Street Kids, William H. Goodwin, Alice Goodwin, and the Commonwealth Foundation for Cancer Research, and the Experimental Research Center of Memorial Sloan-Kettering Cancer Center.
2 Address correspondence and reprint requests to Dr. Nai-Kong V. Cheung, Department of Pediatrics, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065. E-mail address: cheungn{at}mskcc.org
3 Abbreviations used in this paper: NB, neuroblastoma; CS, competitive selection; CSN, competitive selection in the presence of a negative selector; DOTA, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; GuHCl, guanidine chloride; IB50, concentration of competitor required to inhibit 50% binding; %ID/g, percentage of injected dose of radiolabeled ligand per gram; IHC, immunohistochemistry; P, parental (clone); SA, streptavidin; scFv, single chain variable fragment; NHS, N-hydroxysuccinimide.
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