Reducing Epitope Spread during Affinity Maturation of an Anti-Ganglioside GD2 Antibody

Abstract

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.

Human derived dimerization tag enhances tumor killing potency of a T-cell engaging bispecific antibody

“…Subsequently, 2 cysteine mutations, residue S44C on the heavy chain and residue A100C on the light chain, were introduced by site-directed mutagenesis (Stratagene, CA) to stabilize the 5F11 scFv. Additionally, one affinity maturation mutation P104Y 19 was also incorporated into the 5F11 scFv. The final sequence of the GD2xCD3 BsAb was as follows : QVQLQQSGPELVKPGASVKISCKTSGYKFTEYTMHW VKQSHGKCLEWIGGINPNNGGTNYNQKFKGKATLTV DKSSSTAYMELRSLTSEDSAVYYCARDTTVPYAYWGQG TTVTVSSGGGGSGGGGSGGGGSDIELTQSPAIMSASPG EKVTMTCSASSSISYMHWYQQKPGTSPKRWIYDTSKLAS SVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQRSSYPLT FGCGTKLEIKRASTKGPGGGGSGGGGSGGGGSQVQLV QSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPG KGLEWIGYINPSRGYTNYNQKFKDRFTISRDNSKNTAFL QMDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVS SGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTIT CSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRF SGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQG TKLQITR. To construct the GD2xCD3-HDD BsAb, the following sequences were added to the C-terminus of the GD2xCD3 BsAb: IgG3 hinge (TPLGDTTHTSG), HNF1a dimerization domain (MVSKLSQLQTELLAALLESGLSKEALIQALGE), and a spacer (GSGGAP).…”

“…Gangliosides were directly immobilized onto the CM5 sensor chip via hydrophobic interaction, as previously described. 19 The active surface contained a 1:1 mixture of GD2 and GM1, and the reference surface contained only GM1. BsAb samples were diluted in HBS-EP buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% v/v Surfactant P20) at varying concentrations and injected over the sensor surface at a flow rate of 30 mL/min over 1-2 min.…”

“…More recently, several laboratories have reported the presence of GD2 on breast cancer stem cells, 14,15 neuroectodermal 16 and mesenchymal stem cells. 17,18 The anti-GD2£anti-CD3 tsc-BsAb (GD2xCD3) were composed of single polypeptide chains containing the scFv of anti-GD2 monoclonal antibody 5F11 [19][20][21] and the humanized scFv of the anti-CD3 antibody OKT3 22 without and with the HNF1a dimerization domain (HDD) (see Fig. 1A and 1B).…”

Alteration of Electrostatic Surface Potential Enhances Affinity and Tumor Killing Properties of Anti-ganglioside GD2 Monoclonal Antibody hu3F8

“…Affinity maturation of carbohydrate-specific antibodies without the loss of specificity has been attempted by incorporating limited point mutations (6,26). A hierarchy of cross-reactivity usually accompanies increased binding affinity (6), rendering affinity maturation of anti-carbohydrate antibodies more complicated.…”

“…GD3, GD2, GD1b, GT1b, and GQ1b are classified as belonging to the b-series gangliosides (5), whereas GM2 and GT2 are in the a-and c-series, respectively. Because of epitope similarity to GD2 in the synthetic pathways, GD3, GM2, and GD1b are potential cross-reactive epitopes during anti-GD2 antibody development (6).…”