Aggregation-resistant VHs selected by in vitro evolution tend to have disulfide-bonded loops and acidic isoelectric points.
When panned with a transient heat denaturation approach against target enzymes, a human V(H) (antibody heavy chain variable domain) phage display library yielded V(H)s with composite characteristics of binding, non-aggregation and reversible thermal unfolding. Moreover, selection was characterized by enrichment for V(H)s with (i) an even number of disulfide forming Cys residues in complementarity-determining region (CDR) 1 and CDR3 and (ii) acidic isoelectric points. This parallels naturally occurring camelid and shark single-domain antibodies (sdAbs) which are also characterized by (i) solubility and reversible unfolding, (ii) a high occurrence of disulfide forming Cys in their CDRs, particularly, in CDR1 and CDR3 and (iii) acidic V(H)s as inferred here by a pI distribution analysis, reported here, of pools of human and camelid V(H) and V(H)H (camelid heavy chain antibody V(H)) sequences. Our results, reinforced by previous observations by others, suggest that protein acidification may yet be another mechanism nature has devised to create functional sdAbs and that this concept along with the inclusion of inter-CDR disulfide linkages may be applied to human V(H) domains/libraries for non-aggregation optimization. In addition, calculation of theoretical pIs of V(H)s selected by panning may be used for rapid and precise identification of non-aggregating V(H)s.
Arbabi-Ghahroudi
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M
Institute for Biological Sciences, National Research Council of Canada, Ottawa, ON, Canada K1A 0R6.
To
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Gaudette
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Hirama
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Ding
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MacKenzie
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Tanha
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J
D000818:Animals; D002161:Camelids, New World; D016009:Chi-Square Distribution; D022801:Complementarity Determining Regions / Q000235:genetics; D019020:Directed Molecular Evolution; D004220:Disulfides / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D007526:Isoelectric Point; D019151:Peptide Library; D010957:Plasmids / Q000235:genetics; D011489:Protein Denaturation; D013696:Temperature
D022801:Complementarity Determining Regions; D004220:Disulfides; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D019151:Peptide Library