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An Albumin-Binding Domain As a Scaffold for Bispecific Affinity Proteins

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An Albumin-Binding Domain As a Scaffold for Bispecific Affinity Proteins An albumin-binding domain as a scaffold for bispecific affinity proteins J o h a n n i lv e B r a n T Doctoral Thesis in Biotechnology Stockholm, Sweden 2012 An albumin-binding domain as a scaffold An albumin-binding domain as a scaffold for bispecific affinity proteins for bispecific affinity proteins JOHAN NILVEBRANT JOHAN NILVEBRANT Royal Institute of Technology Royal Institute of Technology School of Biotechnology School of Biotechnology Stockholm 2012 Stockholm 2012 © Johan Nilvebrant © Johan Nilvebrant Stockholm 2012 Stockholm 2012 Royal Institute of Technology Royal Institute of Technology School of Biotechnology School of Biotechnology AlbaNova University Center AlbaNova University Center SE-106 91 Stockholm SE-106 91 Stockholm Sweden Sweden Printed by E-Print Printed by E-Print Oxtorgsgatan 9 Oxtorgsgatan 9 SE-111 57 Stockholm SE-111 57 Stockholm Sweden Sweden ISBN 978-91-7501-569-9 ISBN 978-91-7501-569-9 TRITA-BIO Report 2012:21 TRITA-BIO Report 2012:21 ISSN 1654-2312 ISSN 1654-2312 iii iii Johan Nilvebrant (2012): An albumin-binding domain as a scaffold for bispecific affinity Johan Nilvebrant (2012): An albumin-binding domain as a scaffold for bispecific affinity proteins. proteins. Division of Proteomics, School of Biotechnology, Royal Institute of Technology (KTH), Stockholm, Division of Proteomics, School of Biotechnology, Royal Institute of Technology (KTH), Stockholm, Sweden. Sweden. Abstract Abstract Protein engineering and in vitro selection systems are powerful methods to generate binding proteins. Protein engineering and in vitro selection systems are powerful methods to generate binding proteins. In nature, antibodies are the primary affinity proteins and their usefulness has led to a widespread use In nature, antibodies are the primary affinity proteins and their usefulness has led to a widespread use both in basic and applied research. By means of combinatorial protein engineering and protein library both in basic and applied research. By means of combinatorial protein engineering and protein library technology, smaller antibody fragments or alternative non-immunoglobulin protein scaffolds can be technology, smaller antibody fragments or alternative non-immunoglobulin protein scaffolds can be engineered for various functions based on molecular recognition. In this thesis, a 46 amino acid small engineered for various functions based on molecular recognition. In this thesis, a 46 amino acid small albumin-binding domain derived from streptococcal protein G was evaluated as a scaffold for the albumin-binding domain derived from streptococcal protein G was evaluated as a scaffold for the generation of affinity proteins. Using protein engineering, the albumin binding has been generation of affinity proteins. Using protein engineering, the albumin binding has been complemented with a new binding interface localized to the opposite surface of this three-helical complemented with a new binding interface localized to the opposite surface of this three-helical bundle domain. By using in vitro selection from a combinatorial library, bispecific protein domains bundle domain. By using in vitro selection from a combinatorial library, bispecific protein domains with ability to recognize several different target proteins were generated. In paper I, a bispecific with ability to recognize several different target proteins were generated. In paper I, a bispecific albumin-binding domain was selected by phage display and utilized as a purification tag for highly albumin-binding domain was selected by phage display and utilized as a purification tag for highly efficient affinity purification of fusion proteins. The results in paper II show how protein engineering, efficient affinity purification of fusion proteins. The results in paper II show how protein engineering, in vitro display and multi-parameter fluorescence-activated cell sorting can be used to accomplish the in vitro display and multi-parameter fluorescence-activated cell sorting can be used to accomplish the challenging task of incorporating two high affinity binding-sites, for albumin and tumor necrosis challenging task of incorporating two high affinity binding-sites, for albumin and tumor necrosis factor-alpha, into this new bispecific protein scaffold. Moreover, the native ability of this domain to factor-alpha, into this new bispecific protein scaffold. Moreover, the native ability of this domain to bind serum albumin provides a useful characteristic that can be used to extend the plasma half-lives bind serum albumin provides a useful characteristic that can be used to extend the plasma half-lives of proteins fused to it or potentially of the domain itself. When combined with a second targeting of proteins fused to it or potentially of the domain itself. When combined with a second targeting ability, a new molecular format with potential use in therapeutic applications is provided. The ability, a new molecular format with potential use in therapeutic applications is provided. The engineered binding proteins generated against the epidermal growth factor receptors 2 and 3 in papers engineered binding proteins generated against the epidermal growth factor receptors 2 and 3 in papers III and IV are aimed in this direction. Over-expression of these receptors is associated with the III and IV are aimed in this direction. Over-expression of these receptors is associated with the development and progression of various cancers, and both are well-validated targets for therapy. development and progression of various cancers, and both are well-validated targets for therapy. Small bispecific binding proteins based on the albumin-binding domain could potentially contribute Small bispecific binding proteins based on the albumin-binding domain could potentially contribute to this field. The new alternative protein scaffold described in this thesis is one of the smallest to this field. The new alternative protein scaffold described in this thesis is one of the smallest structured affinity proteins reported. The bispecific nature, with an inherent ability of the same structured affinity proteins reported. The bispecific nature, with an inherent ability of the same domain to bind to serum albumin, is unique for this scaffold. These non-immunoglobulin binding domain to bind to serum albumin, is unique for this scaffold. These non-immunoglobulin binding proteins may provide several advantages as compared to antibodies in several applications, proteins may provide several advantages as compared to antibodies in several applications, particularly when a small size and an extended half-life are of key importance. particularly when a small size and an extended half-life are of key importance. Keywords: albumin-binding domain, bispecific, albumin, affinity protein, phage display, Keywords: albumin-binding domain, bispecific, albumin, affinity protein, phage display, staphylococcal display, orthogonal affinity purification, TNF-α, ErbB2, ErbB3 staphylococcal display, orthogonal affinity purification, TNF-α, ErbB2, ErbB3 © Johan Nilvebrant 2012 © Johan Nilvebrant 2012 iv iv Papers in this thesis Papers in this thesis This thesis is based on the following papers, which are referred to in the text by their This thesis is based on the following papers, which are referred to in the text by their Roman numerals (I-IV). They are included in the appendix. Roman numerals (I-IV). They are included in the appendix. I Alm, T., Yderland, L., Nilvebrant, J., Halldin, A., Hober, S. (2010). A small I Alm, T., Yderland, L., Nilvebrant, J., Halldin, A., Hober, S. (2010). A small bispecific protein selected for orthogonal affinity purification, Biotechnol. J., 5: bispecific protein selected for orthogonal affinity purification, Biotechnol. J., 5: 605-617 605-617 II Nilvebrant, J., Alm, T., Hober, S., Löfblom, J. (2011). Engineering bispecificity II Nilvebrant, J., Alm, T., Hober, S., Löfblom, J. (2011). Engineering bispecificity into a single albumin-binding domain, PLoS ONE, 6(10): e25791 into a single albumin-binding domain, PLoS ONE, 6(10): e25791 III Nilvebrant, J., Åstrand, M., Georgieva, M., Björnmalm, M., Löfblom, J., Hober, III Nilvebrant, J., Åstrand, M., Georgieva, M., Björnmalm, M., Löfblom, J., Hober, S. Engineering of bispecific affinity proteins with nanomolar affinity for both S. Engineering of bispecific affinity proteins with nanomolar affinity for both ErbB2 and albumin, manuscript ErbB2 and albumin, manuscript IV Nilvebrant*, J., Åstrand*, M., Löfblom, J., Hober, S. Development and IV Nilvebrant*, J., Åstrand*, M., Löfblom, J., Hober, S. Development and characterization of small bispecific three-helical ErbB3/albumin-binding domains characterization of small bispecific three-helical ErbB3/albumin-binding domains aimed at therapeutic applications, manuscript aimed at therapeutic applications, manuscript * Authors contributed equally * Authors contributed equally All papers and figures derived from them have been reproduced with permission from All papers and figures derived from them have been reproduced with permission from copyright holders. copyright holders. v v Papers not included in the thesis Papers not included in the thesis Boström*, T., Nilvebrant* J., Hober, S. (2012). Purification systems based on bacterial Boström*, T., Nilvebrant* J., Hober, S. (2012). Purification systems based on bacterial surface proteins. Protein Purification, R. Ahmad (Ed.), ISBN: 978-953-307-831-1, InTech surface proteins. Protein Purification, R. Ahmad (Ed.), ISBN: 978-953-307-831-1, InTech
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