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Site-specific labeling of affinity molecules for in vitro and in vivo studies
KTH, School of Biotechnology (BIO), Protein Technology.ORCID iD: 0000-0001-9430-3720
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
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.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2014. , 83 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2014:14
Keyword [en]
Affibody molecules, molecular imaging, site-specific labeling, solid phase peptide synthesis, IgG-binding domains, photoconjugation.
National Category
Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-152349ISBN: 978-91-7595-252-9 (print)OAI: oai:DiVA.org:kth-152349DiVA: diva2:749756
Public defence
2014-10-17, FR4, Oscar Klein, AlbaNova Universitetscenter, Roslagstullsbacken 21,, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

QC 20140929

Available from: 2014-09-29 Created: 2014-09-25 Last updated: 2014-10-28Bibliographically approved
List of papers
1. Evaluation of a Maleimido Derivative of NOTA for Site-Specific Labeling of Affibody Molecules
Open this publication in new window or tab >>Evaluation of a Maleimido Derivative of NOTA for Site-Specific Labeling of Affibody Molecules
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2011 (English)In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 22, no 5, 894-902 p.Article in journal (Refereed) Published
Abstract [en]

Radionuclide molecular imaging has the potential to improve cancer treatment by selection of patients for targeted therapy. Affibody molecules are a class of small (7 kDa) high-affinity targeting proteins with appreciable potential as molecular imaging probes. The NOTA chelator forms stable complexes with a number of radionuclides suitable for SPECT or PET imaging. A maleimidoethylmonoamide NOTA (MMA-NOTA) has been prepared for site-specific labeling of Affibody molecules having a unique C-terminal cysteine. Coupling of the MMA-NOTA to the anti-HER2 Affibody molecule Z(HER2:239S) resulted in a conjugate with an affinity (dissociation constant) to HER2 of 72 pM. Labeling of [MMA-NOTA-Cys(61)]-Z(HER2:239S) with In-111 gave a yield of >95% after 20 min at 60 degrees C. In vitro cell tests demonstrated specific binding of [In-111-MMA-NOTA-Cys(61)]-Z(HER2:239S) to HER2-expressing cell lines. In mice bearing prostate cancer DU-145 xenografts, the tumor uptake of [In-111-MMA-NOTA-Cys(61)]-Z(HER2:239S) was 8.2 +/- 0.9% IA/g and the tumor-to-blood ratio was 31 +/- 1 (4 h postinjection). DU-145 xenografts were clearly visualized by a gamma camera. Direct in vivo comparison of [In-111-MMA-NOTA-Cys(61)]-Z(HER2:239S) and [In-111-MMA-DOTA-Cys(61)]-Z(HER2:239S) demonstrated that both conjugates provided equal radioactivity uptake in tumors, but the tumor-to-organ ratios were better for [In-111-MMA-NOTA-Cys(61)]-Z(HER2:239S) due to more efficient clearance from normal tissues. In conclusion, coupling of MMA-NOTA to a cysteine-containing Affibody molecule resulted in a site-specifically labeled conjugate, which retains high affinity, can be efficiently labeled, and allows for high-contrast imaging.

National Category
Biochemistry and Molecular Biology Biochemistry and Molecular Biology Organic Chemistry
Identifiers
urn:nbn:se:kth:diva-34392 (URN)10.1021/bc100470x (DOI)000290691600007 ()2-s2.0-79959574104 (Scopus ID)
Note
QC 20110607Available from: 2011-06-07 Created: 2011-06-07 Last updated: 2017-12-11Bibliographically approved
2. Preclinical evaluation of anti-HER2 Affibody molecules site-specifically labeled with In-111 using a maleimido derivative of NODAGA
Open this publication in new window or tab >>Preclinical evaluation of anti-HER2 Affibody molecules site-specifically labeled with In-111 using a maleimido derivative of NODAGA
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2012 (English)In: Nuclear Medicine and Biology, ISSN 0969-8051, E-ISSN 1872-9614, Vol. 39, no 4, 518-529 p.Article in journal (Refereed) Published
Abstract [en]

Introduction: Affibody molecules have demonstrated potential for radionuclide molecular imaging. The aim of this study was to synthesize and evaluate a maleimido derivative of the 1,4,7-triazacyclononane-l-glutaric acid-4,7-diacetic acid (NODAGA) for site-specific labeling of anti-HER2 Affibody molecule. Methods: The maleimidoethylmonoamide NODAGA (MMA-NODAGA) was synthesized and conjugated to Z(HER2:2395) Affibody molecule having a C-terminal cysteine. Labeling efficiency, binding specificity to and cell internalization by HER2-expressing cells of [In-111-MMA-NODAGA-Cys(61)]-Z(HER2:2395) were studied. Biodistribution of [In-111-MMA-NODAGA-Cys(61)]-Z(HER2:2395) and [In-111-MMA-DOTA-Cys(61)]-Z(HER2:2395) was compared in mice. Results: The affinity of [MMA-NODAGA-Cys(61)]-Z(HER2:2395) binding to HER2 was 67 pM. The In-1111-labeling yield was 99.6%+/- 0.5% after 30 min at 60 degrees C. [In-111-MMA-NODAGA-Cys(61)]-Z(HER2:2395) bound specifically to HER2-expressing cells in vitro and in vivo. Tumor uptake of [In-111-MMA-NODAGA-Cys(61)]-ZHER(2:2395) in mice bearing DU-145 xenografts (4.7%+/- 0.8% ID/g) was lower than uptake of [In-111-MMA-DOTA-Cys(61)]-Z(HER2:2395) (7.5%+/- 1.6% ID/g). However, tumor-to-organ ratios were higher for [In-111-MMA-NODAGA-Cys(61)]-Z(HER2:2395) due to higher clearance rate from normal tissues. Conclusions: MMA-NODAGA is a promising chelator for site-specific labeling of targeting proteins containing unpaired cysteine. Appreciable influence of chelators on targeting properties of Affibody molecules was demonstrated.

Keyword
HER2, Affibody molecules, Radionuclides, Receptor targeting, Molecular imaging, Indium-111, Maleimido-NODAGA
National Category
Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:kth:diva-96444 (URN)10.1016/j.nucmedbio.2011.10.013 (DOI)000303790600008 ()2-s2.0-84860342736 (Scopus ID)
Funder
Swedish Research Council
Note
QC 20120605Available from: 2012-06-05 Created: 2012-06-04 Last updated: 2017-12-07Bibliographically approved
3. Comparative evaluation of synthetic anti-HER2 Affibody molecules site-specifically labelled with In-111 using N-terminal DOTA, NOTA and NODAGA chelators in mice bearing prostate cancer xenografts
Open this publication in new window or tab >>Comparative evaluation of synthetic anti-HER2 Affibody molecules site-specifically labelled with In-111 using N-terminal DOTA, NOTA and NODAGA chelators in mice bearing prostate cancer xenografts
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2012 (English)In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 39, no 3, 481-492 p.Article in journal (Refereed) Published
Abstract [en]

Purpose In disseminated prostate cancer, expression of human epidermal growth factor receptor type 2 (HER2) is one of the pathways to androgen independence. Radionuclide molecular imaging of HER2 expression in disseminated prostate cancer might identify patients for HER2-targeted therapy. Affibody molecules are small (7 kDa) targeting proteins with high potential as tracers for radionuclide imaging. The goal of this study was to develop an optimal Affibody-based tracer for visualization of HER2 expression in prostate cancer. Methods A synthetic variant of the anti-HER2 Z(HER2:342) Affibody molecule, Z(HER2:S1), was N-terminally conjugated with the chelators DOTA, NOTA and NODAGA. The conjugated proteins were biophysically characterized by electrospray ionization mass spectroscopy (ESI-MS), circular dichroism (CD) spectroscopy and surface plasmon resonance (SPR)-based biosensor analysis. After labelling with In-111, the biodistribution was assessed in normal mice and the two most promising conjugates were further evaluated for tumour targeting in mice bearing DU-145 prostate cancer xenografts. Results The HER2-binding equilibrium dissociation constants were 130, 140 and 90 pM for DOTA-Z(HER2:S1), NOTA-Z(HER2:S1) and NODAGA-Z(HER2:S1), respectively. A comparative study of In-111-labelled DOTA-Z(HER2:S1), NOTA-Z(HER2:S1) and NODAGA-Z(HER2:S1) in normal mice demonstrated a substantial influence of the chelators on the biodistribution properties of the conjugates. In-111-NODAGA-Z(HER2:S1) had the most rapid clearance from blood and healthy tissues. In-111-NOTA-Z(HER2:S1) showed high hepatic uptake and was excluded from further evaluation. In-111-DOTA-Z(HER2:S1) and In-111-NODAGAZHER2: S1 demonstrated specific uptake in DU-145 prostate cancer xenografts in nude mice. The tumour uptake of In-111-NODAGA-Z(HER2:S1), 5.6 +/- 0.4% ID/g, was significantly lower than the uptake of In-111-DOTA-Z(HER2:S1), 7.4 +/- 0.5% ID/g, presumably because of lower bioavailability due to more rapid clearance. In-111-NODAGA-Z(HER2:S1) provided higher tumour-to-blood ratio, but somewhat lower tumour-to-liver, tumour-to-spleen and tumour-to-bone ratios. Conclusion Since distant prostate cancer metastases are situated in bone or bone marrow, the higher tumour-to-bone ratio is the most important. This renders In-111-DOTA-Z(HER2:S1) a preferable agent for imaging of HER2 expression in disseminated prostate cancer.

Keyword
HER2 expression, Affibody molecule, Radionuclide molecular imaging, Prostate cancer, Macrocyclic chelators
National Category
Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:kth:diva-93940 (URN)10.1007/s00259-011-1992-9 (DOI)000302287200015 ()2-s2.0-84859635393 (Scopus ID)
Funder
Swedish Research Council
Note
QC 20120503Available from: 2012-05-03 Created: 2012-05-03 Last updated: 2017-12-07Bibliographically approved
4. Influence of DOTA Chelator Position on Biodistribution and Targeting Properties of In-111-Labeled Synthetic Anti-HER2 Affibody Molecules
Open this publication in new window or tab >>Influence of DOTA Chelator Position on Biodistribution and Targeting Properties of In-111-Labeled Synthetic Anti-HER2 Affibody Molecules
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2012 (English)In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 23, no 8, 1661-1670 p.Article in journal (Refereed) Published
Abstract [en]

Affibody molecules are a class of affinity proteins. Their small size (7 kDa) in combination with the high (subnanomolar) affinity for a number of cancer-associated molecular targets makes them suitable for molecular imaging. Earlier studies demonstrated that the selection of radionuclide and chelator may substantially influence the tumor-targeting properties of affibody molecules. Moreover, the placement of chelators for labeling of affibody molecules with Tc-99m at different positions in affibody molecules influenced both blood clearance rate and uptake in healthy tissues. This introduces an opportunity to improve the contrast of affibody-mediated imaging. In this comparative study, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was conjugated to the synthetic affibody molecule Z(HER2:S1) at three different positions: DOTA-A1-Z(HER2:S1) (N-terminus), DOTA-K58-Z(HER2:S1) (C-terminus), and DOTA-K50-Z(HER2:S1) (middle of helix 3). The affinity for HER2 differed slightly among the variants and the K-D values were determined to be 133 pM, 107 pM and 94 pM for DOTA-A1-Z(HER2:S1), DOTA-K50-Z(HER2:S1), and DOTA-K58-Z(HER2:S1), respectively. Z(HER2:S1) K50-DOTA showed a slightly lower melting point (57 degrees C) compared to DOTA-A1-Z(HER2:S1) (64 degrees C) and DOTA-K5S-Z(HER2:S1) (62 degrees C), but all variants showed good refolding properties after heat treatment All conjugates were successfully labeled with In-III resulting in a radiochemical yield of 99% with preserved binding capacity. In vitro specificity studies using SKOV-3 and LS174T cell lines showed that the binding of the radiolabeled compounds was HER2 receptor mediated, which also was verified in vivo using BALB/C nu/nu mice with LS174T and Ramos lymphoma xenografts. The three conjugates all showed specific uptake in L5174T xenografts in nude mice, where DOTA-A1-Z(HER2:S1) and DOTA-K58-Z(HER2:S1) showed the highest uptake. Overall, DOTA-K58-Z(HER2:S1) provided the highest tumor-to-blood ratio, which is important for a high contrast imaging. In conclusion, the positioning of the DOTA chelator influences the cellular processing and the biodistribution pattern of radiolabeled affibody molecules, creating preconditions for imaging optimization.

Keyword
Prostate-Cancer Xenografts, Metastatic Breast-Cancer, Monoclonal-Antibody, Binding-Proteins, Imaging Agents, Ovarian-Cancer, In-Vivo, Receptor, Her2, Overexpression
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-102343 (URN)10.1021/bc3002369 (DOI)000307487300016 ()2-s2.0-84865145661 (Scopus ID)
Funder
Swedish Research Council
Note

QC 20120914

Available from: 2012-09-14 Created: 2012-09-14 Last updated: 2017-12-07Bibliographically approved
5. Site-Specific Photoconjugation of Antibodies Using Chemically Synthesized IgG-Binding Domains
Open this publication in new window or tab >>Site-Specific Photoconjugation of Antibodies Using Chemically Synthesized IgG-Binding Domains
2014 (English)In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 25, no 3, 481-488 p.Article in journal (Refereed) Published
Abstract [en]

Site-specific labeling of antibodies can be performed using the immunoglobulin-binding Z domain, derived from staphylococcal protein A (SpA), which has a well-characterized binding site in the Pc region of antibodies. By introducing a photoactivable probe in the Z domain, a covalent bond can be formed between the Z domain and the antibody by irradiation with UV light. The aim of this study was to improve the conjugation yield for labeling of different subclasses of IgG having different sequence composition, using a photoactivated Z domain variant. Four different variants of the Z domain (Z5BPA, Z5BBA, Z32BPA, and Z32BBA) were synthesized to investigate the influence of the position of the photoactivable probe and the presence of a flexible linker between the probe and the protein. For two of the variants, the photoreactive benzophenone group was introduced as part of an amino acid side chain by incorporation of the unnatural amino acid benzoylphenylalanine (BPA) during peptide synthesis. For the other two variants, the photoreactive benzophenone group was attached via a flexible linker by coupling of benzoylbenzoic acid (BBA) to the e-amino group of a selectively deprotected lysine residue. Photoconjugation experiments using human IgG1, mouse IgG I, and mouse IgG2A demonstrated efficient conjugation for all antibodies. It was shown that differences in linker length had a large impact on the conjugation efficiency for labeling of mouse IgG1, whereas the positioning of the photoactivable probe in the sequence of the protein had a larger effect for mouse IgG2A. Conjugation to human IgG1 was only to a minor extent affected by position or linker length. For each subclass of antibody, the best variant tested using a standard conjugation protocol resulted in conjugation efficiencies of 41-66%, which corresponds to on average approximately one Z domain attached to each antibody. As a combination of the two best performing variants, Z5BBA and Z32BPA, a Z domain variant with two photoactivable probes (Z5BBA32BPA) was also synthesized with the aim of targeting a wider panel of antibody subclasses and species. This new reagent could efficiently couple to all antibody subclasses that were targeted by the single benzophenone-labeled Z domain variants, with conjugation efficiencies of 26-41%.

Keyword
Photo-Cross-Linking, Protein-A, Staphylococcus-Aureus, Crystal-Structure, Fragment, Complex, Conjugation, Resolution, Proximity, Regions
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-144547 (URN)10.1021/bc400440u (DOI)000333435900004 ()2-s2.0-84896512087 (Scopus ID)
Note

QC 20140428

Available from: 2014-04-28 Created: 2014-04-24 Last updated: 2017-12-05Bibliographically approved
6. Site-specific antibody labeling by covalent photoconjugation of Z domains functionalized for alkyne-azide cycloaddition reactions
Open this publication in new window or tab >>Site-specific antibody labeling by covalent photoconjugation of Z domains functionalized for alkyne-azide cycloaddition reactions
2015 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 16, no 17, 2522-2529 p.Article in journal (Other academic) 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.

Place, publisher, year, edition, pages
Wiley-VCH Verlagsgesellschaft, 2015
Keyword
antibodies, chemoselective conjugation, click chemistry, photoaffinity labeling, solid-phase synthesis
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-152347 (URN)10.1002/cbic.201500300 (DOI)000367719800017 ()2-s2.0-84954401335 (Scopus ID)
Note

QC 20160201

Available from: 2014-09-25 Created: 2014-09-25 Last updated: 2017-12-05Bibliographically approved

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