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This enables recycling antibodies to achieve comparable therapeutic effect at lower doses than their non\u2010recycling counterparts. The development of such antibodies is typically achieved by histidine doping of their variable regions or by performing\n                    <jats:italic>in vitro<\/jats:italic>\n                    antibody selection campaigns utilizing histidine doped libraries. Both are strategies that may introduce sequence liabilities. Here, we present a methodology that employs a na\u00efve antibody phage display library, consisting of natural variable domains, to discover antibodies that bind \u03b1\u2010cobratoxin from the venom of\n                    <jats:italic>Naja kaouthia<\/jats:italic>\n                    in a pH\u2010dependent manner. As a result, an antibody was discovered that exhibits a 7\u2010fold higher off\u2010rate at pH\u20095.5 than pH 7.4 in bio\u2010layer interferometry experiments. Interestingly, no histidine residues were found in its variable domains, and in addition, the antibody showed pH\u2010dependent binding to a histidine\u2010devoid antigen mutant. As such, the results demonstrate that pH\u2010dependent antigen\u2010antibody binding may not always be driven by histidine residues. By employing molecular dynamics simulations, different protonation states of titratable residues were found, which potentially could be responsible for the observed pH\u2010dependent antigen binding properties of the antibody. Finally, given the typically high diversity of na\u00efve antibody libraries, the methodology presented here can likely be applied to discover recycling antibodies against different targets\n                    <jats:italic>ab initio<\/jats:italic>\n                    without the need for histidine doping.\n                  <\/jats:p>","DOI":"10.1002\/pro.4821","type":"journal-article","created":{"date-parts":[[2023,10,28]],"date-time":"2023-10-28T09:59:03Z","timestamp":1698487143000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":12,"title":["Phage display assisted discovery of a\n                    <scp>pH<\/scp>\n                    \u2010dependent anti\u2010\u03b1\u2010cobratoxin antibody from a natural variable domain library"],"prefix":"10.1002","volume":"32","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7345-8037","authenticated-orcid":false,"given":"Tulika","family":"Tulika","sequence":"first","affiliation":[{"name":"Department of Biotechnology and Biomedicine Technical University of Denmark  Lyngby Denmark"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rasmus W.","family":"Pedersen","sequence":"additional","affiliation":[{"name":"Department of Biotechnology and Biomedicine Technical University of Denmark  Lyngby Denmark"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Charlotte","family":"Rimbault","sequence":"additional","affiliation":[{"name":"Department of Biotechnology and Biomedicine Technical University of Denmark  Lyngby 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