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Despite undertaking thousands of crystallization trials of the humanized 5E5 (h-5E5) Fab in the presence of either the singly or doubly glycosylated peptide antigens corresponding to Tn-MUC1, the Fab is only observed unliganded in the crystal. The conformations of the complementarity-determining regions (CDRs) of the combining site on the h-5E5 Fab do not differ significantly from those reported for liganded murine scFv at 3.0\u2005\u00c5 resolution. While the affinity of the murine 5E5 has previously been reported as\n                    <jats:italic>K<\/jats:italic>\n                    <jats:sub>D<\/jats:sub>\n                    = 1.7\u2005n\n                    <jats:italic>M<\/jats:italic>\n                    for the 24-mer Tn-MUC1 peptide PPAHGVT*SAPDTRPAPGS*T*APPAH prepared by\n                    <jats:italic>in vitro<\/jats:italic>\n                    glycosylation of a synthetic 24-mer MUC1 peptide, the\n                    <jats:italic>K<\/jats:italic>\n                    <jats:sub>D<\/jats:sub>\n                    of the h-5E5 Fab for the shorter doubly glycosylated glycopeptide antigens PAPGS*T*AP and APGS*T*AP was measured here as only 41 and 61\u2005\u00b5\n                    <jats:italic>M<\/jats:italic>\n                    , respectively. Interestingly, the single Fab molecule in the asymmetric unit of space group\n                    <jats:italic>C<\/jats:italic>\n                    2 is observed packed head-to-head with a symmetry-related Fab across a crystallographic twofold axis such that a polypeptide loop from the light chain of each Fab is observed to insert into the antigen-binding pocket of the symmetry-related Fab. While this might suggest that binding of the Tn-MUC1 peptides may have been inhibited by a homophilic association, none was detected. The humanization process has imposed changes in the framework regions of the Fv which may have affected the Vh\u2013Vl interface.\n                  <\/jats:p>","DOI":"10.1107\/s2059798325002554","type":"journal-article","created":{"date-parts":[[2025,3,26]],"date-time":"2025-03-26T11:14:58Z","timestamp":1742987698000},"page":"223-233","update-policy":"https:\/\/doi.org\/10.1107\/cm_01","source":"Crossref","is-referenced-by-count":3,"title":["Structure of the Fab fragment of a humanized 5E5 antibody to a cancer-specific Tn-MUC1 epitope"],"prefix":"10.1107","volume":"81","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1249-8945","authenticated-orcid":true,"given":"Wei","family":"Li","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/04s5mat29","id-type":"ROR","asserted-by":"publisher"}],"department":["Department of Biochemistry and Microbiology"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ulla","family":"Mandel","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/035b05819","id-type":"ROR","asserted-by":"publisher"}],"department":["Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine, Faculty of Health Sciences"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Henk","family":"van Faassen","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/04mte1k06","id-type":"ROR","asserted-by":"publisher"}],"department":["Human Health Therapeutics Research Centre"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Matthew J.","family":"Parker","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/04s5mat29","id-type":"ROR","asserted-by":"publisher"}],"department":["Department of Biochemistry and Microbiology"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9065-7019","authenticated-orcid":true,"given":"Max S. 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