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55 Identification of the O-glycan epitope targeted by an anti-human carcinoma monoclonal antibody (mAb) NEO-201
  1. Massimo Fantini1,
  2. Anjum Zaki1,
  3. Sharon Mavroukakis1,
  4. Christina Annunziata2,
  5. Philip Arlen1,
  6. Kwong Tsang1 and
  7. Kwong Tsang1
  1. 1Precision Biologics, Inc., Bethesda, MD, USA
  2. 2National Cancer Institute, Bethesda, MD, USA


Background NEO-201 is a humanized IgG1 mAb reactive against multiple human carcinomas, but not normal epithelial tissues. NEO-201 can mediate antitumor activity through multiple mechanisms such as antibody-dependent cellular cytotoxicity, complement dependent cytotoxicity, and blockade of the CEACAM5/CEACAM1 immune checkpoint inhibitory pathway. In addition to solid tumors, flow cytometry analysis has demonstrated that NEO-201 binds to 98.9% of CD15+ granulocytes, human regulatory T cells as well as various human hematological neoplastic cell lines. However, NEO-201 does not bind to other immune subsets and to the majority of CD4+ T cells. Furthermore, we have demonstrated that NEO-201 binds to mammalian expressed rhCEACAM6 but not bacterial expressed rhCEACAM6. These findings suggest that NEO-201 binds to glycans linked to specific proteins. Glycosylation is an important post-translation modification of protein and is affected by oncogenesis. Aberrant O-glycans may serve as potential targets to improve the monitoring and treatment of cancers. Based on this information, this study was designed to focus on the identification of the O-glycan binding epitope of NEO-201.

Methods An O-glycan array consisting of 94 O-glycans was used to identify the O-glycans targeted by NEO-201. O-glycan profiles were elucidated from human pancreatic cancer cell line (CFPAC-1), human hematological neoplastic cell lines (HL60, U937, K562) and human neutrophils. Different truncated C-terminus of CEACAM6 and CEACAM5 gene constructs were designed and the truncated CEACAM6 and CEACAM5 proteins were expressed in mammalian expression system to identify the NEO-201 binding region in CEACAM6 and CEACEAM5.

Results The O-glycan array analysis shows that NEO-201 interacts with O-glycans 01, 02 (Tn antigens), 06 (Core 1), 023 (Core 2), 026 (Core 3) and 039 (Core 4). The Core-1 binding interaction was the strongest of any observed. The O-glycan profiling studies demonstrated that CFPAC-1 and human neutrophils express mostly the Core 1 profile. HL-60 expresses mainly the extended Core 1 profile, U937 expresses mainly the extended Core 1 and Core 2 profiles and K562 expresses only the Core 2 profile. Flow cytometry analysis demonstrated that NEO-201 binds to CFPAC-1, human neutrophils, HL60 and U937 cells but not to K562. We also proved that in both CEACAM5 and CEACAM6 NEO-201 binds to regions containing threonine. GalNAc residue can be added onto threonine to form O-glycans.

Conclusions This study demonstrated that NEO-201 binds strongly to Core 1 and/or extended Core 1 O-glycans and confirms our finding that NEO-201 binds only mammalian expressed rhCEACAM6 express O-glycans.

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