{"id":2635,"date":"2015-08-13T16:50:30","date_gmt":"2015-08-13T15:50:30","guid":{"rendered":"http:\/\/www.blopig.com\/blog\/?p=2635"},"modified":"2015-08-13T16:50:30","modified_gmt":"2015-08-13T15:50:30","slug":"antibody-binding-site-re-design","status":"publish","type":"post","link":"https:\/\/www.blopig.com\/blog\/2015\/08\/antibody-binding-site-re-design\/","title":{"rendered":"Antibody binding site re-design"},"content":{"rendered":"

In this blog post I describe three successful studies on structure based re-design<\/strong> of antibody binding sites, leading to significant improvements of binding affinity<\/strong>.<\/p>\n

In their study Clark et al.<\/em><\/strong>[1] re-designed a binding site of antibody AQC2<\/strong> to improve its binding affinity to the I domain of human integrin VLA1. The authors assessed the effects of the mutations on the binding energy using the CHARMM<\/strong>[2,3] potential with the electrostatic and desolations energies calculated using the ICE software<\/strong>[4]. In total, 83 variants<\/strong> were identified for experimental validation, some of which included multiple mutations. The mutated antibodies were expressed in E. Coli<\/em><\/strong> and the affinity to the antigen was measured. The best mutant included a total of four mutations which improved the affinity by approximately one order of magnitude from 7 nM to 850 pM<\/strong>. The crystal structure<\/strong> of the best mutant was solved to further study the interaction of the mutant with the target.<\/p>\n

\"Figure<\/a>

Figure 1: Comparison of calculated and experimental binding free energies. (Lippow et al., 2007)<\/p><\/div>\n

Lippow et al<\/em><\/strong>.<\/em>[5] studied the interactions of three antibodies \u2013 the anti-epidermal growth factor receptor drug cetuximab<\/strong>[6], the anti-lysozyme antibody D44.1<\/strong> and the anti-lysosyme antibody D1.3<\/strong> with their respective antigens. The energy calculations favoured mutations to large amino acids<\/strong> (such as Phe or Trp) of which most were found to be false positives<\/strong>. More accurate results were obtained using only the electrostatic term<\/strong> of the energy function. The authors improved the binding affinity of D44.1 by one order of magnitude<\/strong> and the affinity of centuximab by 2 orders of magnitude<\/strong>. The antibody D1.3 didn\u2019t show many opportunities for electrostatic improvement<\/strong> and the authors suggest it might be an anomalous<\/strong> antibody.<\/p>\n

Computational methods have recently been used to successfully introduce non-canonical amino acids <\/strong>(NCAA<\/strong>) into the antibody binding site. Xu et al<\/em><\/strong>.<\/em>[7] introduced L-DOPA<\/strong> (L-3,4-dihydroxephenyalanine) into the CDRs of anti-protective antigen scFv antibody M18<\/strong> to crosslink it with its native antigen. The authors used the program Rosetta 3.4<\/strong> to create models of antibody-antigen complex with L-DOPA residues. The distance between L-DOPA and a lysine<\/strong> nucleophile was used as a predictor<\/strong> of crosslinking was. The crosslinking efficiency was quantified as a fraction of antibodies that underwent a mass change, measured using Western blot assays. The measured average efficiency<\/strong> of the mutants was 10%<\/strong> with the maximum<\/strong> efficiency<\/strong> of 52%.<\/strong><\/p>\n

[1]\u00a0\u00a0\u00a0\u00a0\u00a0 Clark LA, Boriack-Sjodin PA, Eldredge J, Fitch C, Friedman B, Hanf KJM, et al. Affinity enhancement of an in vivo matured therapeutic antibody using structure-based computational design. Protein Sci 2006;15:949\u201360. doi:10.1110\/ps.052030506.<\/p>\n

[2]\u00a0\u00a0\u00a0\u00a0\u00a0 Brooks BR, Bruccoleri RE, Olafson DJ, States DJ, Swaminathan S, Karplus M. CHARMM: A Program for Macromolecular Energy, Minimization, and Dynamics Calculations. J Comput Chem 1983;4:187\u2013217.<\/p>\n

[3]\u00a0\u00a0\u00a0\u00a0\u00a0 MacKerel Jr. AD, Brooks III CL, Nilsson L, Roux B, Won Y, Karplus M. CHARMM: The Energy Function and Its Parameterization with an Overview of the Program. In: v. R. Schleyer et al. P, editor. vol. 1, John Wiley & Sons: Chichester; 1998, p. 271\u20137.<\/p>\n

[4]\u00a0\u00a0\u00a0\u00a0\u00a0 Kangas E, Tidor B. Optimizing electrostatic affinity in ligand\u2013receptor binding: Theory, computation, and ligand properties. J Chem Phys 1998;109:7522. doi:10.1063\/1.477375.<\/p>\n

[5]\u00a0\u00a0\u00a0\u00a0\u00a0 Lippow SM, Wittrup KD, Tidor B. Computational design of antibody-affinity improvement beyond in vivo maturation. Nat Biotechnol 2007;25:1171\u20136. doi:10.1038\/nbt1336.<\/p>\n

[6]\u00a0\u00a0\u00a0\u00a0\u00a0 Sato JD, Kawamoto T, Le AD, Mendelsohn J, Polikoff J, Sato GH. Biological effects in vitro of monoclonal antibodies to human epidermal growth factor receptors. Mol Biol Med 1983;1:511\u201329.<\/p>\n

[7]\u00a0\u00a0\u00a0\u00a0\u00a0 Xu J, Tack D, Hughes RA, Ellington AD, Gray JJ. Structure-based non-canonical amino acid design to covalently crosslink an antibody-antigen complex. J Struct Biol 2014;185:215\u201322. doi:10.1016\/j.jsb.2013.05.003.<\/p>\n","protected":false},"excerpt":{"rendered":"

In this blog post I describe three successful studies on structure based re-design of antibody binding sites, leading to significant improvements of binding affinity. In their study Clark et al.[1] re-designed a binding site of antibody AQC2 to improve its binding affinity to the I domain of human integrin VLA1. The authors assessed the effects […]<\/p>\n","protected":false},"author":31,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"nf_dc_page":"","wikipediapreview_detectlinks":true,"_monsterinsights_skip_tracking":false,"_monsterinsights_sitenote_active":false,"_monsterinsights_sitenote_note":"","_monsterinsights_sitenote_category":0,"ngg_post_thumbnail":0,"_jetpack_memberships_contains_paid_content":false,"footnotes":""},"categories":[1],"tags":[],"ppma_author":[493],"class_list":["post-2635","post","type-post","status-publish","format-standard","hentry","category-uncategorized"],"jetpack_featured_media_url":"","jetpack_sharing_enabled":true,"authors":[{"term_id":493,"user_id":31,"is_guest":0,"slug":"jaroslaw","display_name":"jaroslaw nowak","avatar_url":"https:\/\/secure.gravatar.com\/avatar\/d3ddaccc000bffff018a1bdedaebefb1ac9454506ae5203150e7b3efd8dd9a6e?s=96&d=mm&r=g","0":null,"1":"","2":"","3":"","4":"","5":"","6":"","7":"","8":""}],"_links":{"self":[{"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/posts\/2635","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/users\/31"}],"replies":[{"embeddable":true,"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/comments?post=2635"}],"version-history":[{"count":1,"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/posts\/2635\/revisions"}],"predecessor-version":[{"id":2637,"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/posts\/2635\/revisions\/2637"}],"wp:attachment":[{"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/media?parent=2635"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/categories?post=2635"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/tags?post=2635"},{"taxonomy":"author","embeddable":true,"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/ppma_author?post=2635"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}