Siglec-7, an immune checkpoint receptor, has emerged as a promising target for cancer immunotherapy due to its involvement in the regulation of immune and inflammatory responses. However, while its participation in immunoediting and immune evasion is well established, understanding its biological context, relevant ligands, and associated signalling pathways remains limited. Understanding these aspects is crucial for the development of effective immunotherapies targeting Siglec-7. In this study, three expression constructs of Siglec-7 were designed, expressed, and characterised, including an analysis of the oligomeric state of its extracellular domain. The N-terminal V-set Ig carbohydrate recognition domain was also produced in an isotopically double-labelled (13C,15N) mammalian cell growth medium. Two stable constructs suitable for biophysical and structural studies were identified. These findings reveal the noncovalent dimerisation of Siglec-7, offering new insights into its possible ligand interactions, signal transduction mechanisms, or receptor/ligand clustering. The dimerisation of Siglec-7 may be essential to achieve multivalent, high-avidity interactions with glycoconjugates, which may result in enhanced or alternative signalling processes within the NK cell immune synapse. In addition, a detailed protocol for generating double-labelled Siglec-7 in HEK293 cells, which may apply to other proteins under similar conditions, was described. These findings contribute to a better understanding of the biophysical and structural properties of Siglec-7 and are key to the design of more precise and effective cancer immunotherapies targeting Siglec-7.

Department of Biochemistry Faculty of Science Charles University Hlavova 2030 12840 Prague Czech Republic

Department of Chemical Sciences University of Naples Federico 2 Via Cintia 4 80126 Napoli Italy

Department of Chemical Sciences University of Naples Federico 2 Via Cintia 4 80126 Napoli Italy; CEINGE Biotecnologie Avanzate Franco Salvatore Via Gaetano Salvatore 486 80145 Napoli Italy

Department of Chemical Sciences University of Naples Federico 2 Via Cintia 4 80126 Napoli Italy; CEINGE Biotecnologie Avanzate Franco Salvatore Via Gaetano Salvatore 486 80145 Napoli Italy; Department of Chemistry School of Science Osaka University 1 1 Machikaneyama Toyonaka 560 0043 Osaka Japan

EMBL Hamburg Unit c o DESY Notkestrasse 85 22607 Hamburg Germany

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