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Site-specific antibody labeling by covalent photoconjugation of Z domains functionalized for alkyne-azide cycloaddition reactions
KTH, Skolan för bioteknologi (BIO), Proteinteknologi.ORCID-id: 0000-0001-9430-3720
KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
KTH, Skolan för bioteknologi (BIO), Proteinteknologi.ORCID-id: 0000-0002-0695-5188
2015 (engelsk)Inngår i: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 16, nr 17, s. 2522-2529Artikkel i tidsskrift (Annet vitenskapelig) Published
Abstract [en]

Antibodies are extensively used both as research tools and in the clinics for diagnostics and therapy. For many applications the antibodies are labeled, e.g. with detection probes or cytotoxic agents that improve the therapeutic effect. Labeling is typically performed using amine-reactive probes targeting lysine residues accessible on the surface of the protein, resulting in a heterogeneously labeled antibody. One alternative strategy for site-specific labeling is to use the immunoglobulin G (IgG)-binding protein domain Z, which binds to the Fc-region of IgG. By introducing the photoactivable amino acid benzoylphenylalanine (BPA) in the Z domain, a covalent bond can be formed between the Z domain and the antibody by UV irradiation, to produce a site-specifically labeled product. The Z domain with BPA in position 32,  Z32BPA, was synthesized by solid phase peptide synthesis and was further functionalized for alkyne-azide cycloaddition reactions to give alkyne-Z32BPA and azide-Z32BPA for Cu(I)-catalyzed click reaction, and DBCO-Z32BPA for Cu-free strain-promoted click reaction. The reactivity of the functionalized Z domains in Cu(I)-catalyzed and strain-promoted alkyne-azide cycloaddition reactions was analyzed using MALDI-TOF MS and RP-HPLC, showing fast reaction. To further evaluate the concept, the Z32BPA variants were conjugated to the human IgG1 antibody trastuzumab and biotinylated by Cu(I)-catalyzed or strain-promoted alkyne-azide cycloaddition reactions. Western blot analysis of the biotinylated antibodies, using streptavidin-HRP for detection, demonstrated that all Z-antibody conjugates could be site-specifically labeled by the alkyne-azide cycloaddition reactions.

sted, utgiver, år, opplag, sider
Wiley-VCH Verlagsgesellschaft, 2015. Vol. 16, nr 17, s. 2522-2529
Emneord [en]
antibodies, chemoselective conjugation, click chemistry, photoaffinity labeling, solid-phase synthesis
HSV kategori
Identifikatorer
URN: urn:nbn:se:kth:diva-152347DOI: 10.1002/cbic.201500300ISI: 000367719800017PubMedID: 26417902Scopus ID: 2-s2.0-84954401335OAI: oai:DiVA.org:kth-152347DiVA, id: diva2:749741
Merknad

QC 20160201

Tilgjengelig fra: 2014-09-25 Laget: 2014-09-25 Sist oppdatert: 2020-03-09bibliografisk kontrollert
Inngår i avhandling
1. Site-specific labeling of affinity molecules for in vitro and in vivo studies
Åpne denne publikasjonen i ny fane eller vindu >>Site-specific labeling of affinity molecules for in vitro and in vivo studies
2014 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

The thesis is focused on site-specific labeling of affinity molecules for different applications where two types of binding proteins, Affibody molecules and antibodies, have been used. For the purpose of improving the properties of Affibody molecules for in vivo imaging, novel bi-functional chelators for radiolabeling using the radionuclide 111In were evaluated. In a first study, two chelators denoted NOTA and DOTA, respectively, were separately conjugated via maleimide chemistry to a C-terminal cysteine residue in a HER2-binding Affibody molecule (ZHER2:2395). In vivo evaluation using mice with prostate carcinoma cell line xenografts showed that the 111In-NOTA-MMA-ZHER2:2395 tracer exhibited faster clearance from blood than the 111In-DOTA-MMA-ZHER2:2395 counterpart,resulting in improved tumor-to-organ ratios. In a second study the in vivo imaging properties of a third tracer, 111In-NODAGA-MMA-ZHER2:2395, was investigated in tumor-bearing mice. While the tumor uptake was lower than seen for the 111In-DOTA-MMA-ZHER2:2395 tracer, a low uptake in non-targeted organs and a fast clearance from blood resulted in higher tumor-to-organ ratios for 111In-NODAGA-MMA-ZHER2:2395 compared to the DOTA variant.

In a following study, a synthetically produced HER2-targeting affibody variant, denoted ZHER2:S1, was used where NODAGA, NOTA and DOTA chelators instead were conjugated via an amide bond to the N-terminus. In vivo evaluation in mice showed an unfavorable uptake in liver for 111In-NOTA-ZHER2:S1, resulting in a discontinuation. The study showed faster clearance of 111In-NODAGA-ZHER2:S1 from blood, but also an increased uptake in bone in comparison to 111In-DOTA-ZHER2:S1. As bone is a common metastatic site in prostate cancer, the favorable tumor-to-bone ratio for 111In-DOTA-ZHER2:S1 suggests it as the tracer of choice for prostate cancer. Further, the DOTA chelator was also evaluated as conjugated to either N- or C-terminus or to the back of helix 3 via an amide bond, where the in vivo evaluation showed that that C-terminal conjugation resulted in the highest contrast.

Site specificity is also of great importance for labeling antibodies, as conjugation in the antigen-binding regions might influence the affinity. A method for site-specific labeling of antibodies using an IgG-binding domain that becomes covalently attached to the Fc-region of an antibody by photoconjugation was optimized. By investigation of positions most suitable for incorporation of the photoreactive probe, the conjugation efficiencies were increased for antibody subclasses important for both diagnostic and therapeutic applications. In addition, optimized variants were used in combination with an incorporated click-reactive handle for selective labeling of the antibody with a detection molecule.

sted, utgiver, år, opplag, sider
Stockholm: KTH Royal Institute of Technology, 2014. s. 83
Serie
TRITA-BIO-Report, ISSN 1654-2312 ; 2014:14
Emneord
Affibody molecules, molecular imaging, site-specific labeling, solid phase peptide synthesis, IgG-binding domains, photoconjugation.
HSV kategori
Forskningsprogram
Bioteknologi
Identifikatorer
urn:nbn:se:kth:diva-152349 (URN)978-91-7595-252-9 (ISBN)
Disputas
2014-10-17, FR4, Oscar Klein, AlbaNova Universitetscenter, Roslagstullsbacken 21,, Stockholm, 10:00 (engelsk)
Opponent
Veileder
Merknad

QC 20140929

Tilgjengelig fra: 2014-09-29 Laget: 2014-09-25 Sist oppdatert: 2014-10-28bibliografisk kontrollert

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