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==Development==
==Development==
As with other antibody mimetics, the idea behind developing the Affibody molecule was to apply a combinatorial [[protein engineering]] approach on a small and robust protein scaffold. The aim was to generate new binders capable of specific binding to different target proteins, while retaining the favorable folding and stability properties, and ease of bacterial expression of the parent molecule.<ref>{{cite journal|doi=10.1093/protein/8.6.601|last1=Nord|first1=K|last2=Nilsson|first2=J|last3=Nilsson|first3=B|last4=Uhlén|first4=M|last5=Nygren|first5=P-A|year=1995|title=A combinatorial library of an α-helical bacterial receptor domain|journal=Prot. Eng.|volume=8|issue=6|pages=601–608|pmid=8532685}}</ref><ref>{{cite journal|doi=10.1038/nbt0897-772|last1=Nord|first1=K|last2=Gunneriusson|first2=E|last3=Ringdahl|first3=J|last4=Ståhl|first4=S|last5=Uhlén|first5=M|last6=Nygren|first6=P-A|year=1997|title=Binding proteins selected from combinatorial libraries of an α-helical bacterial receptor domain|journal=Nature Biotechnology|volume=15|issue=8|pages=772–777|pmid=9255793}}</ref>
As with other antibody mimetics, the idea behind developing the Affibody molecule was to apply a combinatorial [[protein engineering]] approach on a small and robust protein scaffold. The aim was to generate new binders capable of specific binding to different target proteins with almost good affinity, while retaining the favorable folding and stability properties, and ease of bacterial expression of the parent molecule.<ref>{{cite journal|doi=10.1093/protein/8.6.601|last1=Nord|first1=K|last2=Nilsson|first2=J|last3=Nilsson|first3=B|last4=Uhlén|first4=M|last5=Nygren|first5=P-A|year=1995|title=A combinatorial library of an α-helical bacterial receptor domain|journal=Prot. Eng.|volume=8|issue=6|pages=601–608|pmid=8532685}}</ref><ref>{{cite journal|doi=10.1038/nbt0897-772|last1=Nord|first1=K|last2=Gunneriusson|first2=E|last3=Ringdahl|first3=J|last4=Ståhl|first4=S|last5=Uhlén|first5=M|last6=Nygren|first6=P-A|year=1997|title=Binding proteins selected from combinatorial libraries of an α-helical bacterial receptor domain|journal=Nature Biotechnology|volume=15|issue=8|pages=772–777|pmid=9255793}}</ref>


The original Affibody protein scaffold was designed based on the Z domain (the [[immunoglobulin G]] binding domain) of [[protein A]]. In contrast to [[Antibody|antibodies]], Affibody molecules are composed of [[Alpha helix|alpha helices]] and lack [[disulfide]] bridges. The parent three-helix bundle structure is currently the fastest folding protein structure known.<ref>{{cite journal|doi=10.1110/ps.03541304|last1=Arora|first1=P|last2=Oas|first2=T|last3=Myers|first3=J|year=2004|title=Fast and faster: A designed variant of the B-domain of protein A folds in 3 μsec|journal=Protein Sci.|volume=13|issue=4|pages=847–853|pmid=15044721|pmc=2280057}}</ref>
The original Affibody protein scaffold was designed based on the Z domain (the [[immunoglobulin G]] binding domain) of [[protein A]]. In contrast to [[Antibody|antibodies]], Affibody molecules are composed of [[Alpha helix|alpha helices]] and lack [[disulfide]] bridges. The parent three-helix bundle structure is currently the fastest folding protein structure known.<ref>{{cite journal|doi=10.1110/ps.03541304|last1=Arora|first1=P|last2=Oas|first2=T|last3=Myers|first3=J|year=2004|title=Fast and faster: A designed variant of the B-domain of protein A folds in 3 μsec|journal=Protein Sci.|volume=13|issue=4|pages=847–853|pmid=15044721|pmc=2280057}}</ref>
Line 15: Line 15:
Affibody molecules are based on a three-helix bundle domain, which can be expressed in soluble and [[proteolytic]]ally stable forms in various host cells on its own or via fusion with other protein partners.<ref>{{cite journal|last1=Ståhl|first1=S|last2=Nygren|first2=P-A|year=1997|title=The use of gene fusions to protein A and protein G in immunology and biotechnology|journal=Pathol. Biol.|location=Paris|volume=45|issue=1|pages=66–76|pmid=9097850}}</ref>
Affibody molecules are based on a three-helix bundle domain, which can be expressed in soluble and [[proteolytic]]ally stable forms in various host cells on its own or via fusion with other protein partners.<ref>{{cite journal|last1=Ståhl|first1=S|last2=Nygren|first2=P-A|year=1997|title=The use of gene fusions to protein A and protein G in immunology and biotechnology|journal=Pathol. Biol.|location=Paris|volume=45|issue=1|pages=66–76|pmid=9097850}}</ref>


They tolerate modification and are independently folding when incorporated into [[fusion protein]]s. Head-to-tail fusions of Affibody molecules of the same specificity have proven to give [[avidity]] effects in target binding, and head-to-tail fusion of Affibody molecules of different specificities makes it possible to get bi- or multi-specific affinity proteins. Fusions with other proteins can also be created genetically<ref>{{cite journal|doi=10.1016/S0022-1759(01)00563-4|last1=Rönnmark|first1=J|last2=Hansson|first2=M|last3=Nguyen|first3=T|last4=Uhlén|first4=M|last5=Robert|first5=A|last6=Ståhl|first6=S|last7=Nygren|first7=P-A|year=2002|title=Construction and characterization of affibody-Fc chimeras produced in Escherichia coli|journal=J. Immunol. Methods|volume=261|issue=1–2|pages=199–211|pmid=11861078}}</ref><ref>{{cite journal|doi=10.1016/j.jim.2003.06.001|last1=Rönnmark|first1=J|last2=Kampf|first2=C|last3=Asplund|first3=A|last4=Höiden-Guthénberg|first4=I|last5=Wester|first5=K|last6=Pontén|first6=F|last7=Uhlén|first7=M|last8=Nygren|first8=P-A|year=2003|title=Affibody-beta-galactosidase immunoconjugates produced as soluble fusion proteins in the Escherichia coli cytosol|journal=J. Immunol. Methods|volume=281|issue=1–2|pages=149–160|pmid=14580889}}</ref> or by spontaneous isopeptide bond formation.<ref>{{cite journal|doi=10.1073/pnas.1519214113|last1=Veggiani|first1=G|last2=Nakamura|first2=T|last3=Brenner|first3=M|last4=Gayet|first4=R|last5=Yan|first5=J|last6=Robinson|first6=C|last7=Howarth|first7=M|year=2016|title=Programmable polyproteams built using twin peptide superglues|journal=PNAS|volume=113|issue=5|pages=1202–1207|pmid= 26787909}}</ref> A site for site-specific conjugation is facilitated by introduction of a single cysteine at a desired position.
They tolerate modification and are independently folding when incorporated into [[fusion protein]]s. Head-to-tail fusions of Affibody molecules of the same specificity have proven to give [[avidity]] effects in target binding, and head-to-tail fusion of Affibody molecules of different specificities makes it possible to get bi- or multi-specific affinity proteins. Fusions with other proteins can also be created genetically<ref>{{cite journal|doi=10.1016/S0022-1759(01)00563-4|last1=Rönnmark|first1=J|last2=Hansson|first2=M|last3=Nguyen|first3=T|last4=Uhlén|first4=M|last5=Robert|first5=A|last6=Ståhl|first6=S|last7=Nygren|first7=P-A|year=2002|title=Construction and characterization of affibody-Fc chimeras produced in Escherichia coli|journal=J. Immunol. Methods|volume=261|issue=1–2|pages=199–211|pmid=11861078}}</ref><ref>{{cite journal|doi=10.1016/j.jim.2003.06.001|last1=Rönnmark|first1=J|last2=Kampf|first2=C|last3=Asplund|first3=A|last4=Höiden-Guthénberg|first4=I|last5=Wester|first5=K|last6=Pontén|first6=F|last7=Uhlén|first7=M|last8=Nygren|first8=P-A|year=2003|title=Affibody-beta-galactosidase immunoconjugates produced as soluble fusion proteins in the Escherichia coli cytosol|journal=J. Immunol. Methods|volume=281|issue=1–2|pages=149–160|pmid=14580889}}</ref> or by spontaneous isopeptide bond formation.<ref>{{cite journal|doi=10.1073/pnas.1519214113|last1=Veggiani|first1=G|last2=Nakamura|first2=T|last3=Brenner|first3=M|last4=Gayet|first4=R|last5=Yan|first5=J|last6=Robinson|first6=C|last7=Howarth|first7=M|year=2016|title=Programmable polyproteams built using twin peptide superglues|journal=PNAS|volume=113|issue=5|pages=1202–1207|pmid= 26787909}}</ref> A site for site-specific conjugation is facilitated by introduction of a single cysteine at a desired position, therefore this engineered protein can be use to conjugate to radionuclides such as Technetium-99m and Indium-111 to visualize receptor-overexpressing tumors.<ref>{{Cite journal|last=Altai M, Wållberg H, Orlova A, Rosestedt M, Hosseinimehr SJ, Tolmachev V, Ståhl S.|first=|date=2012|title=Order of amino acids in C-terminal cysteine-containing peptide-based chelators influences cellular processing and biodistribution of 99mTc-labeled recombinant Affibody molecules|url=https://link.springer.com/article/10.1007/s00726-011-0927-x|journal=Amino Acids|volume=42(5)|pages=1975-1985|doi=10.1007/s00726-011-0927-x|pmid=21573874|via=}}</ref><ref>{{Cite journal|last=Tolmachev V1, Friedman M, Sandström M, Eriksson TL, Rosik D, Hodik M, Ståhl S, Frejd FY, Orlova A.|first=|date=2009|title=Affibody molecules for epidermal growth factor receptor targeting in vivo: aspects of dimerization and labeling chemistry|url=http://jnm.snmjournals.org/content/50/2/274.abstract|journal=J Nucl Med|volume=50(2)|pages=274-283|doi=10.2967/jnumed.108.055525.|pmid=19164241|via=}}</ref>


A number of different Affibody molecules have been produced by [[chemical synthesis]]. Since they do not contain [[cysteine]]s or disulfide bridges, they fold spontaneously and reversibly into the correct three-dimensional structures when the [[protection group]]s are removed after synthesis.<ref name="Nord 2001">{{cite journal|last1=Nord|first1=K|last2=Nord|first2=O|last3=Uhlén|first3=M|last4=Kelley|first4=B|last5=Ljungqvist|first5=C|last6=Nygren|first6=P-A|year=2001|title=Recombinant human factor VIII-specific affinity ligands selected from phage-displayed combinatorial libraries of protein A|journal=Eur. J. Biochem.|volume=268|issue=15|pages=1–10|pmid=11488921|doi=10.1046/j.1432-1327.2001.02344.x}}</ref><ref>{{cite journal|last1=Engfeldt|first1=T|last2=Renberg|first2=B|last3=Brumer|first3=H|last4=Nygren|first4=P-A|last5=Karlström|first5=EA|year=2005|title=Chemical synthesis of triple-labelled three-helix bundle binding proteins for specific fluorescent detection of unlabelled protein|journal=Chem. BioChem.|volume=6|issue=6|pages=1043–1050|pmid=15880677|doi=10.1002/cbic.200400388}}</ref> In some studies, temperatures above the melting temperature have been used, with retained binding properties following return to ambient conditions.<ref name="Finnzymes">{{cite web|publisher=Finnzymes |title=Phusion Hot Start High-Fidelity DNA Polymerase |url=http://www.finnzymes.fi/pcr/phusion_hot_start_high_fidelity_dna_polymerase.html |deadurl=yes |archiveurl=https://web.archive.org/web/20090328110438/http://www.finnzymes.fi/pcr/phusion_hot_start_high_fidelity_dna_polymerase.html |archivedate=2009-03-28 }}</ref> Cross-linked variants have been produced as well.
A number of different Affibody molecules have been produced by [[chemical synthesis]]. Since they do not contain [[cysteine]]s or disulfide bridges, they fold spontaneously and reversibly into the correct three-dimensional structures when the [[protection group]]s are removed after synthesis.<ref name="Nord 2001">{{cite journal|last1=Nord|first1=K|last2=Nord|first2=O|last3=Uhlén|first3=M|last4=Kelley|first4=B|last5=Ljungqvist|first5=C|last6=Nygren|first6=P-A|year=2001|title=Recombinant human factor VIII-specific affinity ligands selected from phage-displayed combinatorial libraries of protein A|journal=Eur. J. Biochem.|volume=268|issue=15|pages=1–10|pmid=11488921|doi=10.1046/j.1432-1327.2001.02344.x}}</ref><ref>{{cite journal|last1=Engfeldt|first1=T|last2=Renberg|first2=B|last3=Brumer|first3=H|last4=Nygren|first4=P-A|last5=Karlström|first5=EA|year=2005|title=Chemical synthesis of triple-labelled three-helix bundle binding proteins for specific fluorescent detection of unlabelled protein|journal=Chem. BioChem.|volume=6|issue=6|pages=1043–1050|pmid=15880677|doi=10.1002/cbic.200400388}}</ref> In some studies, temperatures above the melting temperature have been used, with retained binding properties following return to ambient conditions.<ref name="Finnzymes">{{cite web|publisher=Finnzymes |title=Phusion Hot Start High-Fidelity DNA Polymerase |url=http://www.finnzymes.fi/pcr/phusion_hot_start_high_fidelity_dna_polymerase.html |deadurl=yes |archiveurl=https://web.archive.org/web/20090328110438/http://www.finnzymes.fi/pcr/phusion_hot_start_high_fidelity_dna_polymerase.html |archivedate=2009-03-28 }}</ref> Cross-linked variants have been produced as well.

Revision as of 13:18, 11 September 2017

Affibody molecules are small, robust and easily proteins engineered to bind to a large number of target proteins or peptides with high affinity, imitating monoclonal antibodies, and are therefore a member of the family of antibody mimetics. Affibody molecules are used in biochemical research and are being developed as potential new biopharmaceutical drugs.[1] These molecules can be used for molecular recognition in diagnostic and therapeutic applications.[2][3]

Development

As with other antibody mimetics, the idea behind developing the Affibody molecule was to apply a combinatorial protein engineering approach on a small and robust protein scaffold. The aim was to generate new binders capable of specific binding to different target proteins with almost good affinity, while retaining the favorable folding and stability properties, and ease of bacterial expression of the parent molecule.[4][5]

The original Affibody protein scaffold was designed based on the Z domain (the immunoglobulin G binding domain) of protein A. In contrast to antibodies, Affibody molecules are composed of alpha helices and lack disulfide bridges. The parent three-helix bundle structure is currently the fastest folding protein structure known.[6]

Affibody molecules with unique binding properties are acquired by randomization of 13 amino acids located in two alpha-helices involved in the binding activity of the parent protein domain. Lately, amino acids outside of the binding surface have been substituted in the scaffold to create a surface entirely different from the ancestral protein A domain.

Specific Affibody molecules binding a desired target protein can be “fished out” from pools (libraries) containing billions of different variants, using phage display.

Production

Affibody molecules are based on a three-helix bundle domain, which can be expressed in soluble and proteolytically stable forms in various host cells on its own or via fusion with other protein partners.[7]

They tolerate modification and are independently folding when incorporated into fusion proteins. Head-to-tail fusions of Affibody molecules of the same specificity have proven to give avidity effects in target binding, and head-to-tail fusion of Affibody molecules of different specificities makes it possible to get bi- or multi-specific affinity proteins. Fusions with other proteins can also be created genetically[8][9] or by spontaneous isopeptide bond formation.[10] A site for site-specific conjugation is facilitated by introduction of a single cysteine at a desired position, therefore this engineered protein can be use to conjugate to radionuclides such as Technetium-99m and Indium-111 to visualize receptor-overexpressing tumors.[11][12]

A number of different Affibody molecules have been produced by chemical synthesis. Since they do not contain cysteines or disulfide bridges, they fold spontaneously and reversibly into the correct three-dimensional structures when the protection groups are removed after synthesis.[13][14] In some studies, temperatures above the melting temperature have been used, with retained binding properties following return to ambient conditions.[15] Cross-linked variants have been produced as well.

Properties

An Affibody molecule consists of three alpha helices with 58 amino acids and has a molar mass of about 6 kDa. A monoclonal antibody, for comparison, is 150 kDa, and a single-domain antibody, the smallest type of antigen-binding antibody fragment, 12–15 kDa.

Affibody molecules have been shown to withstand high temperatures (90 °C) or acidic and alkaline conditions (pH 2.5 or pH 11, respectively).[16][17][18]

Binders with an affinity of down to sub-nanomolar have been obtained from naïve library selections, and binders with picomolar affinity have been obtained following affinity maturation.[19] Affibodies conjugated to weak electrophiles bind their targets covalently.[20]Combination of small size, high affinity and specificity makes Affibody molecules promising ligands for both molecular imaging and therapeutical applications. [21][22]

Applications

Affibody molecules can be used for protein purification,[13] enzyme inhibition,[15] research reagents for protein capture and detection,[23][24] diagnostic imaging[19] and targeted therapy.[25] The HER2/neu specific Affibody ABY-025 is in clinical development for tumor diagnosis.[26][27] Recently, anti-ZEGFR Affibody ZEGFR:2377 was used to visualize ZEGR expressing tumor in mice xenograft successfully.[28]

References

  1. ^ Frejd FY, Kim KT (2017). "Affibody molecules as engineered protein drugs". Exp Mol Med. 49(3): e306. doi:10.1038/emm.2017.35. PMID 28336959.
  2. ^ Garousi J1, Andersson K, Mitran B, Pichl ML, Ståhl S, Orlova A, Löfblom J, Tolmachev V (2016). "PET imaging of epidermal growth factor receptor expression in tumours using 89Zr-labelled ZEGFR:2377 Affibody molecules". Int J Oncol. 48(4): 1325–1332. doi:10.3892/ijo.2016.3369. PMID 26847636 – via SPANDIDOS PUBLICATIONS.{{cite journal}}: CS1 maint: multiple names: authors list (link) CS1 maint: numeric names: authors list (link)
  3. ^ Sörensen J1, Sandberg D, Sandström M, Wennborg A, Feldwisch J, Tolmachev V, Åström G, Lubberink M, Garske-Román U, Carlsson J, Lindman H. (2014). "First-in-human molecular imaging of HER2 expression in breast cancer metastases using the 111In-ABY-025 affibody molecule". J Nucl Med. 55(5): 730–735. doi:10.2967/jnumed.113.131243. PMID 24665085.{{cite journal}}: CS1 maint: multiple names: authors list (link) CS1 maint: numeric names: authors list (link)
  4. ^ Nord, K; Nilsson, J; Nilsson, B; Uhlén, M; Nygren, P-A (1995). "A combinatorial library of an α-helical bacterial receptor domain". Prot. Eng. 8 (6): 601–608. doi:10.1093/protein/8.6.601. PMID 8532685.
  5. ^ Nord, K; Gunneriusson, E; Ringdahl, J; Ståhl, S; Uhlén, M; Nygren, P-A (1997). "Binding proteins selected from combinatorial libraries of an α-helical bacterial receptor domain". Nature Biotechnology. 15 (8): 772–777. doi:10.1038/nbt0897-772. PMID 9255793.
  6. ^ Arora, P; Oas, T; Myers, J (2004). "Fast and faster: A designed variant of the B-domain of protein A folds in 3 μsec". Protein Sci. 13 (4): 847–853. doi:10.1110/ps.03541304. PMC 2280057. PMID 15044721.
  7. ^ Ståhl, S; Nygren, P-A (1997). "The use of gene fusions to protein A and protein G in immunology and biotechnology". Pathol. Biol. 45 (1). Paris: 66–76. PMID 9097850.
  8. ^ Rönnmark, J; Hansson, M; Nguyen, T; Uhlén, M; Robert, A; Ståhl, S; Nygren, P-A (2002). "Construction and characterization of affibody-Fc chimeras produced in Escherichia coli". J. Immunol. Methods. 261 (1–2): 199–211. doi:10.1016/S0022-1759(01)00563-4. PMID 11861078.
  9. ^ Rönnmark, J; Kampf, C; Asplund, A; Höiden-Guthénberg, I; Wester, K; Pontén, F; Uhlén, M; Nygren, P-A (2003). "Affibody-beta-galactosidase immunoconjugates produced as soluble fusion proteins in the Escherichia coli cytosol". J. Immunol. Methods. 281 (1–2): 149–160. doi:10.1016/j.jim.2003.06.001. PMID 14580889.
  10. ^ Veggiani, G; Nakamura, T; Brenner, M; Gayet, R; Yan, J; Robinson, C; Howarth, M (2016). "Programmable polyproteams built using twin peptide superglues". PNAS. 113 (5): 1202–1207. doi:10.1073/pnas.1519214113. PMID 26787909.
  11. ^ Altai M, Wållberg H, Orlova A, Rosestedt M, Hosseinimehr SJ, Tolmachev V, Ståhl S. (2012). "Order of amino acids in C-terminal cysteine-containing peptide-based chelators influences cellular processing and biodistribution of 99mTc-labeled recombinant Affibody molecules". Amino Acids. 42(5): 1975–1985. doi:10.1007/s00726-011-0927-x. PMID 21573874.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  12. ^ Tolmachev V1, Friedman M, Sandström M, Eriksson TL, Rosik D, Hodik M, Ståhl S, Frejd FY, Orlova A. (2009). "Affibody molecules for epidermal growth factor receptor targeting in vivo: aspects of dimerization and labeling chemistry". J Nucl Med. 50(2): 274–283. doi:10.2967/jnumed.108.055525.. PMID 19164241. {{cite journal}}: Check |doi= value (help)CS1 maint: multiple names: authors list (link) CS1 maint: numeric names: authors list (link)
  13. ^ a b Nord, K; Nord, O; Uhlén, M; Kelley, B; Ljungqvist, C; Nygren, P-A (2001). "Recombinant human factor VIII-specific affinity ligands selected from phage-displayed combinatorial libraries of protein A". Eur. J. Biochem. 268 (15): 1–10. doi:10.1046/j.1432-1327.2001.02344.x. PMID 11488921.
  14. ^ Engfeldt, T; Renberg, B; Brumer, H; Nygren, P-A; Karlström, EA (2005). "Chemical synthesis of triple-labelled three-helix bundle binding proteins for specific fluorescent detection of unlabelled protein". Chem. BioChem. 6 (6): 1043–1050. doi:10.1002/cbic.200400388. PMID 15880677.
  15. ^ a b "Phusion Hot Start High-Fidelity DNA Polymerase". Finnzymes. Archived from the original on 2009-03-28. {{cite web}}: Unknown parameter |deadurl= ignored (|url-status= suggested) (help)
  16. ^ Ahlgren, S; Wållberg, H; Tran, TA; Widström, C; Hjertman, M; Abrahmsén, L; Berndorff, D; Dinkelborg, LM; Cyr, JE (2009). "Targeting of HER2-expressing tumors with a site-specifically 99mTc-labeled recombinant affibody molecule, ZHER2:2395, with C-terminally engineered cysteine". J. Nucl. Med. 50 (5): 781–789. doi:10.2967/jnumed.108.056929. PMID 19372467. {{cite journal}}: Unknown parameter |displayauthors= ignored (|display-authors= suggested) (help)
  17. ^ Orlova, A; Rosik, D; Sandström, M; Lundqvist, H.; Einarsson, L; Tolmachev, V (2007). "Evaluation of [(111/114m)In]CHX-A"-DTPA-ZHER2:342, an affibody ligand conjugate for targeting of HER2-expressing malignant tumors". Q. J. Nucl. Med. Mol. Imaging. 51 (4): 314–23. PMID 17464277.
  18. ^ Tran, T; Engfeldt, T; Orlova, A; Sandström, M; Feldwisch, J; Abrahmsén, L; Wennborg, A; Tolmachev, V; Karlström, AE (2007). "(99m)Tc-maEEE-Z(HER2:342), an Affibody molecule-based tracer for the detection of HER2 expression in malignant tumors". Bioconjug. Chem. 18 (6): 1956–64. doi:10.1021/bc7002617. PMID 17944527. {{cite journal}}: Unknown parameter |displayauthors= ignored (|display-authors= suggested) (help)
  19. ^ a b Orlova, A; Magnusson, M; Eriksson, TL; Nilsson, M; Larsson, B; Höidén-Guthenberg, I; Widström, C; Carlsson, J; Tolmachev, V (2006). "Tumor imaging using a picomolar affinity HER2 binding affibody molecule". Cancer Res. 66 (8): 4339–48. doi:10.1158/0008-5472.CAN-05-3521. PMID 16618759. {{cite journal}}: Unknown parameter |displayauthors= ignored (|display-authors= suggested) (help)
  20. ^ Holm, L; Moody, P; Howarth, M (2009). "Electrophilic Affibodies Forming Covalent Bonds to Protein Targets". The Journal of Biological Chemistry. 284 (47): 32906–13. doi:10.1074/jbc.M109.034322. PMC 2781706. PMID 19759009.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  21. ^ Lofblom J, Feldwisch J, Tolmachev V, Carlsson J, Stahl S and Frejd FY (2010). "Affibody molecules: engineered proteins for therapeutic, diagnostic and biotechnological applications". FEBS Lett. 584(12): 2670–2680. doi:10.1016/j.febslet.2010.04.014. PMID 20388508 – via Elsevier, Science Direct.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  22. ^ Tolmachev V, Orlova A, Nilsson FY, Feldwisch J, Wennborg A and Abrahmsen L (2007). "Affibody molecules: potential for in vivo imaging of molecular targets for cancer therapy". Expert Opin Biol Ther. 7(4): 555–568. doi:10.1517/14712598.7.4.555. PMID 17373906.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  23. ^ Renberg, B; Nordin, J; Merca, A; Uhlén, M; Feldwisch, J; Nygren, P-A; Karlström, AE (2007). "Affibody molecules in protein capture microarrays: evaluation of multidomain ligands and different detection formats". J. Proteome Res. 6 (1): 171–179. doi:10.1021/pr060316r. PMID 17203961.
  24. ^ Lundberg, E; Höidén-Guthenberg, I; Larsson, B; Uhlén, M; Gräslund, T (2007). "Site-specifically conjugated anti-HER2 Affibody molecules as one-step reagents for target expression analyses on cells and xenograft samples". J. Immunol. Methods. 319 (1–2): 53–63. doi:10.1016/j.jim.2006.10.013. PMID 17196217.
  25. ^ Tolmachev, V; Orlova, A; Pehrson, R; Galli, J; Baastrup, B; Andersson, K; Sandström, M; Rosik, D; Carlsson, J (2007). "Radionuclide therapy of HER2-positive microxenografts using a 177Lu-labeled HER2-specific Affibody molecule". Cancer Res. 67 (6): 2773–82. doi:10.1158/0008-5472.CAN-06-1630. PMID 17363599. {{cite journal}}: Unknown parameter |displayauthors= ignored (|display-authors= suggested) (help)
  26. ^ Gebauer, M; Skerra, A (2009). "Engineered protein scaffolds as next-generation antibody therapeutics". Current Opinion in Chemical Biology. 13 (3): 245–55. doi:10.1016/j.cbpa.2009.04.627. PMID 19501012.
  27. ^ Sörensen J, Velikyan I, Sandberg D, Wennborg A, Feldwisch J, Tolmachev V, Orlova A, Sandström M, Lubberink M, Olofsson H, Carlsson J, Lindman H (2016). "Measuring HER2-Receptor Expression In Metastatic Breast Cancer Using [68Ga]ABY-025 Affibody PET/CT". Theranostics. 6(2): 262–271. doi:10.7150/thno.13502.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  28. ^ Andersson KG, Oroujeni M, Garousi J, Mitran B, Ståhl S, Orlova A, Löfblom J, Tolmachev V. (2016). "Feasibility of imaging of epidermal growth factor receptor expression with ZEGFR:2377 affibody molecule labeled with 99mTc using a peptide-based cysteine-containing chelator". Int J Oncol. 49(6): 2285–2293. doi:10.3892/ijo.2016.3721. PMID 27748899.{{cite journal}}: CS1 maint: multiple names: authors list (link)
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