An Integrative Approach to Develop and Characterise Antibodies Against the Cancer Associated Antigen Sialyl Lewis A (CA 19-9)

Freitag, A.; Khilji, S. K.; Nedielkov, R.; Kumar, S. M.; Krummhaar, M.; Arndt, J.; Moreira, G. S. G.; Luehle, J.; Goerdeler, F.; Kamphues, C.; Roth, C.; Mroginski, M.-A.; Seeberger, P.; Moeller, H. M.; Moscovitz, O.

doi:10.1101/2025.06.15.659274

bioRxiv · 10.1101/2025.06.15.659274

An Integrative Approach to Develop and Characterise Antibodies Against the Cancer Associated Antigen Sialyl Lewis A (CA 19-9)

Freitag, A.·Khilji, S. K.·Nedielkov, R.·Kumar, S. M.·Krummhaar, M.·Arndt, J.·Moreira, G. S. G.·Luehle, J.·Goerdeler, F.·Kamphues, C.·Roth, C.·Mroginski, M.-A.·Seeberger, P.·Moeller, H. M.·Moscovitz, O.

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Abstract

BackgroundSialyl Lewis A (sLeA), or the CA 19-9 marker, is a tetrasaccharide and a tumour-associated carbohydrate antigen (TACA) overexpressed and abnormally secreted as a serum-borne marker in gastrointestinal malignancies. CA 19-9 is the best validated and only FDA-approved serologic marker clinically used to monitor recurrence, progression, and therapy efficiency in pancreatic ductal adenocarcinoma (PDAC) patients. Due to its altered expression on cancer cells, sLeA is also an attractive target for antibody development. Although recent clinical trials have demonstrated insufficient efficacy of the fully human anti-sLeA 5B1 (MVT-5873) format as a stand-alone drug or an adjuvant therapy in PDAC [1], its safety profile and unique expression in additional malignancies keep CA 19-9 an attractive TACA. Hence, we set out to explore the use of synthetic sLeA to develop novel monoclonal antibodies (mAbs) with improved sLeA recognition and better efficacy. MethodsTwo mAbs targeting sLeA were generated through mice immunisation with synthetic sLeA glycoconjugates, synthetic glycan arrays, and hybridoma technology. We then compared the antigen-binding properties of the newly developed mAbs with the widely used mAb 1116-NS-19- 9 via synthetic glycan arrays, immunohistochemistry (IHC), X-ray crystallography, molecular dynamics (MD) simulation, and Saturation Transfer Difference Nuclear Magnetic Resonance (STD NMR) spectroscopy. ResultsThe newly generated mAbs demonstrated improved affinity and specificity for both synthetic and native sLeA, surpassing the performance of the established mAb 1116-NS-19-9. First, synthetic glycan arrays, surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC) assays confirmed superior antigen-binding properties to synthetic sLeA. In particular, the mAb designated GB11 demonstrated markedly enhanced binding to native sLeA ectopically expressed in B16 melanoma cells. To elucidate the structural origin of GB11s improved antigen binding, we conducted high-resolution mapping of the molecular recognition patterns between sLeA and the different antibodies using X-ray crystallography and STD NMR. These analyses revealed subtle yet critical differences in the glycan engagement and identified key structural features underlying GB11s enhanced recognition of sLeA. MD simulations further supported these observations, indicating distinct orientations of sLeA within the binding pockets of each mAb. ConclusionOur results suggest better recognition of the sLeA antigen by the newly generated GB11 antibody and provide a detailed high-resolution elucidation of the molecular interactions behind it. Our study may provide a novel tool with improved theranostic properties against sLeA-overexpressing malignancies.

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Freitag, A., Khilji, S. K., Nedielkov, R., Kumar, S. M., Krummhaar, M., Arndt, J., Moreira, G. S. G., Luehle, J., Goerdeler, F., Kamphues, C., Roth, C., Mroginski, M.-A., Seeberger, P., Moeller, H. M., Moscovitz, O.. 2025-06-19. An Integrative Approach to Develop and Characterise Antibodies Against the Cancer Associated Antigen Sialyl Lewis A (CA 19-9). https://doi.org/10.1101/2025.06.15.659274

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