Recognition of Dimeric Lewis X by Anti-Dimeric Lex Antibody SH2

Sinthuja Jegatheeswaran; Ari Asnani; Adam Forman; Jenifer L Hendel; Christopher J Moore; Ali Nejatie; An Wang; Jo-Wen Wang; France-Isabelle Auzanneau

doi:10.3390/vaccines8030538

Recognition of Dimeric Lewis X by Anti-Dimeric Le^x Antibody SH2

Vaccines (Basel). 2020 Sep 17;8(3):538. doi: 10.3390/vaccines8030538.

Authors

Sinthuja Jegatheeswaran^{1

2}, Ari Asnani^{1

3}, Adam Forman^{1

4}, Jenifer L Hendel^{1

5}, Christopher J Moore^{1

6}, Ali Nejatie^{1

7}, An Wang^{1

8}, Jo-Wen Wang^{1

9}, France-Isabelle Auzanneau¹

Affiliations

¹ Department of Chemistry, University of Guelph, Guelph, ON N1G 2W1, Canada.
² Immunology Department, University of Toronto, 1 King's College Circle, Toronto, ON M5S-1A8, Canada.
³ Department of Chemistry, Universitas Jenderal Soedirman, Purwokerto, Jawa Tengah 53123, Indonesia.
⁴ Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, ON M5S-3H6, Canada.
⁵ Research and Development, Ludger Ltd., Culham Science Centre, Abingdon, Oxfordshire OX14-3EB, UK.
⁶ Quality Control, SteriMax Inc., 2770 Portland Dr, Oakville, ON L6H-6R4, Canada.
⁷ Department of Chemistry, Simon Fraser University, Burnaby, BC V5A1S6, Canada.
⁸ SGS-CSTC Standards Technical Services Co., Ltd. 4/F, 4th Building, 889 Yishan Road, Xuhui District, Shanghai 200233, China.
⁹ IQVIA, QuintilesIMS, Clinical Research, 10188 Telesis Ct #400, San Diego, CA 92121, USA.

Abstract

The carbohydrate antigen dimeric Lewis X (DimLe^x), which accumulates in colonic and liver adenocarcinomas, is a valuable target to develop anti-cancer therapeutics. Using the native DimLe^x antigen as a vaccine would elicit an autoimmune response against the Le^x antigen found on normal, healthy cells. Thus, we aim to study the immunogenic potential of DimLe^x and search internal epitopes displayed by DimLe^x that remain to be recognized by anti-DimLe^x monoclonal antibodies (mAbs) but no longer possess epitopes recognized by anti-Le^x mAbs. In this context, we attempted to map the epitope recognized by anti-DimLe^x mAb SH2 by titrations and competitive inhibition experiments using oligosaccharide fragments of DimLe^x as well as Le^x analogues. We compare our results with that reported for anti-Le^x mAb SH1 and anti-polymeric Le^x mAbs 1G5F6 and 291-2G3-A. While SH1 recognizes an epitope localized to the non-reducing end Le^x trisaccharide, SH2, 1G5F6, and 291-2G3-A have greater affinity for DimLe^x conjugates than for Le^x conjugates. We show, however, that the Le^x trisaccharide is still an important recognition element for SH2, which (like 1G5F6 and 291-2G3-A) makes contacts with all three sugar units of Le^x. In contrast to mAb SH1, anti-polymeric Le^x mAbs make contact with the GlcNAc acetamido group, suggesting that epitopes extend further from the non-reducing end Le^x. Results with SH2 show that this epitope is only recognized when DimLe^x is presented by glycoconjugates. We have reported that DimLe^x adopts two conformations around the β-d-GlcNAc-(1→3)-d-Gal bond connecting the Le^x trisaccharides. We propose that only one of these conformations is recognized by SH2 and that this conformation is favored when the hexasaccharide is presented as part of a glycoconjugate such as DimLe^x-bovine serum albumin (DimLe^x-BSA). Proper presentation of the oligosaccharide candidate via conjugation to a protein or lipid is essential for the design of an anti-cancer vaccine or immunotherapeutic based on DimLe^x.

Keywords: ELISA; SH2; conformational epitope; dimeric Lewis X; tumor-associated carbohydrate antigens.

Grants and funding

RG03820/Natural Sciences and Engineering Research Council of Canada