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Generation and engineering of ABD-derived affinity proteins for clinical applications
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology. (Sophia Hober)ORCID iD: 0000-0001-5908-4315
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proteins that specifically recognize and bind to other molecules or structures are important tools in industrial and medical applications. Binding proteins engineered from small stable scaffold proteins have been utilized for several purposes due to their favorable biophysical properties, tolerance to mutagenesis, efficient tissue penetration and ease of production. The 46 amino acid long albumin-binding domain (ABD) derived from the bacterial receptor Protein G is a promising scaffold that has been explored in this thesis. The scaffold was subjected to combinatorial protein engineering for generation of ABD-derived binding proteins with novel specificities. Furthermore, the medical potential of engineered ABD- derived affinity proteins (ADAPTs) was evaluated in a series of pre-clinical studies.

In the first studies, ADAPTs suitability as tracers for radionuclide molecular imaging was evaluated. Factors influencing biodistribution and tumor targeting properties were assessed in mice models bearing HER2 positive xenografts. All tested ADAPT constructs demonstrated high and specific targeting of HER2-expressing tumor cells as well as fast clearance from circulation. The results also showed that the size and character of the N- terminus affected the biodistribution profile of ADAPTs. Moreover, the targeting properties of ADAPTs proved to be highly influenced by the residualizing properties of the attached radionuclide label. Taken together, the results provided the first evidence that tumor imaging can be performed using ADAPTs and the favorable pharmacokinetic profiles in the studied mice models suggest that the scaffold is a promising candidate for clinical applications.

In the last study, a platform for generation of stable ABD-derived affinity proteins with novel binding specificities was established using a multi-step approach combining directed evolution and rational protein design. A broad combinatorial protein library with 20 randomized positions in ABD was designed and binders against three distinct targets were selected using phage display. Characterization of the selected binders provided information regarding optimal positions to randomize in a final library. In addition, the isolated binders were subjected to mutagenesis in certain surface exposed positions and mutations that provided increased stability were introduced into the original scaffold. Finally, a more focused combinatorial protein library consisting of 11 randomized positions was designed and constructed. The library was validated by selections against the same set of targets as for the first, broad library. The isolation of highly stable affinity ligands confirms that the library can be used for generation of diverse and stable affinity molecules.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2018. , p. 84
Series
TRITA-CBH-FOU ; 2018:7
Keywords [en]
ABD, ADAPT, affinity proteins, protein engineering, radionuclide molecular imaging, HER2
National Category
Pharmaceutical Biotechnology
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-224256ISBN: 978-91-7729-715-4 (print)OAI: oai:DiVA.org:kth-224256DiVA, id: diva2:1190660
Public defence
2018-04-13, FR4 Oskar Kleins Auditorium, Roslagstullsbacken 21, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

QC 20180315

Available from: 2018-03-15 Created: 2018-03-15 Last updated: 2018-03-15Bibliographically approved
List of papers
1. ADAPT, a Novel Scaffold Protein-Based Probe for Radionuclide Imaging of Molecular Targets That Are Expressed in Disseminated Cancers
Open this publication in new window or tab >>ADAPT, a Novel Scaffold Protein-Based Probe for Radionuclide Imaging of Molecular Targets That Are Expressed in Disseminated Cancers
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2015 (English)In: Cancer Research, ISSN 0008-5472, E-ISSN 1538-7445, Vol. 75, no 20, p. 4364-4371Article in journal (Refereed) Published
Abstract [en]

Small engineered scaffold proteins have attracted attention as probes for radionuclide-based molecular imaging. One class of these imaging probes, termed ABD-Derived Affinity Proteins (ADAPT), has been created using the albumin-binding domain (ABD) of streptococcal protein G as a stable protein scaffold. In this study, we report the development of a clinical lead probe termed ADAPT6 that binds HER2, an oncoprotein overexpressed in many breast cancers that serves as a theranostic biomarker for several approved targeting therapies. Surface-exposed amino acids of ABD were randomized to create a combinatorial library enabling selection of high-affinity binders to various proteins. Furthermore, ABD was engineered to enable rapid purification, to eradicate its binding to albumin, and to enable rapid blood clearance. Incorporation of a unique cysteine allowed site-specific conjugation to a maleimido derivative of a DOTA chelator, enabling radionuclide labeling, In-111 for SPECT imaging and Ga-68 for PET imaging. Pharmacologic studies in mice demonstrated that the fully engineered molecule In-111/Ga-68-DOTA(HE) 3-ADAPT6 was specifically bound and taken up by HER2-expressing tumors, with a high tumor-to-normal tissue ratio in xenograft models of human cancer. Unbound tracer underwent rapid renal clearance followed by high renal reabsorption. HER2-expressing xenografts were visualized by gamma-camera or PET at 1 hour after infusion. PET experiments demonstrated feasibility for discrimination of xenografts with high or low HER2 expression. Our results offer a preclinical proof of concept for the use of ADAPT probes for noninvasive in vivo imaging.

Place, publisher, year, edition, pages
American Association for Cancer Research Inc., 2015
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:kth:diva-180154 (URN)10.1158/0008-5472.CAN-14-3497 (DOI)000365601900013 ()26297736 (PubMedID)2-s2.0-84945567447 (Scopus ID)
Note

QC 20160113

Available from: 2016-01-13 Created: 2016-01-07 Last updated: 2018-03-15Bibliographically approved
2. Influence of Histidine-Containing Tags on the Biodistribution of ADAPT Scaffold Proteins.
Open this publication in new window or tab >>Influence of Histidine-Containing Tags on the Biodistribution of ADAPT Scaffold Proteins.
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2016 (English)In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 27, no 3, p. 716-726Article in journal (Refereed) Published
Abstract [en]

Engineered scaffold proteins (ESP) are high-affinity binders that can be used as probes for radionuclide imaging. Histidine-containing tags enable both efficient purification of ESP and radiolabeling with (99m)Tc(CO)3. Earlier studies demonstrated that the use of a histidine-glutamate-histidine-glutamate-histidine-glutamate (HE)3-tag instead of the commonly used hexahistidine (H6)-tag reduces hepatic uptake of radiolabeled ESP and short peptides. Here, we investigated the influence of histidine-containing tags on the biodistribution of a novel type of ESP, ADAPTs. A series of anti-HER2 ADAPT probes having H6- or (HE)3-tags in the N-termini were prepared. The constructs, (HE)3-ADAPT6 and H6-ADAPT6, were labeled with two different nuclides, (99m)Tc or (111)In. The labeling with (99m)Tc(CO)3 utilized the histidine-containing tags, while (111)In was attached through a maleimido derivative of DOTA conjugated to the N-terminus. For (111)In-labeled ADAPTs, the use of (HE)3 provided a significantly (p < 0.05) lower hepatic uptake at 1 h after injection, but there was no significant difference in hepatic uptake of (111)In-(HE)3-ADAPT6 and H6-ADAPT6 at later time points. Interestingly, in the case of (99m)Tc, (99m)Tc(CO)3-H6-ADAPT6 provided significantly (p < 0.05) lower uptake in a number of normal tissues and was more suitable as an imaging probe. Thus, the influence of histidine-containing tags on the biodistribution of the novel ADAPT scaffold proteins was different compared to its influence on other ESPs studied so far. Apparently, the effect of a histidine-containing tag on the biodistribution is highly dependent on the scaffold composition of the ESP.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2016
National Category
Biological Sciences Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-184209 (URN)10.1021/acs.bioconjchem.5b00677 (DOI)000372478600026 ()26781756 (PubMedID)2-s2.0-84962209236 (Scopus ID)
Funder
Swedish Cancer Society, CAN 2015/350Swedish Research Council, 2015-02353 621-2012-5088
Note

QC 20160405

Available from: 2016-03-30 Created: 2016-03-30 Last updated: 2018-03-15Bibliographically approved
3. Influence of the N -Terminal Composition on Targeting Properties of Radiometal-Labeled Anti-HER2 Scaffold Protein ADAPT6
Open this publication in new window or tab >>Influence of the N -Terminal Composition on Targeting Properties of Radiometal-Labeled Anti-HER2 Scaffold Protein ADAPT6
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2016 (English)In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 27, no 11, p. 2678-2688Article in journal (Refereed) Published
Abstract [en]

Radionuclide-imaging-based stratification of patients to targeted therapies makes cancer treatment more personalized and therefore more efficient. Albumin-binding domain derived affinity proteins (ADAPTs) constitute a novel group of imaging probes based on the scaffold of an albumin binding domain (ABD). To evaluate how different compositions of the N-terminal sequence of ADAPTS influence their biodistribution, a series of human epidermal growth factor receptor type 2 (HER2)-binding ADAPT6 derivatives with different N-terminal sequences were created: GCH(6)DANS (2), GC(HE)(3)DANS (3), GCDEAVDANS (4), and GCVD.ANS(5). These were compared with the parental variant: GCSS(HE)(3)DEAVDANS (1). All variants were site-specifically conjugated with a maleimido-derivative of a DOTA chelator and labeled with In-III. Binding to HER2-expressing cells in vitro, in vivo biodistribution as well as targeting properties of the new variants were compared with properties of the In-III-labeled parental ADAPT variant 1 (In-III-DOTA-1). The composition of the N-terminal sequence had an apparent influence on biodistribution of ADAPT6 in mice. The use of a hexahistidine tag in (InD)-In-III-OTA-2 was associated with elevated hepatic uptake compared to the (HE)(3)-containing counterpart, In-III-DOTA-3. All new variants without a hexahistidine tag demonstrated lower uptake in blood, lung, spleen, and muscle compared to uptake in the parental variant. The best new variants, In-III-DOTA-3 and In-III-DOTA-5, provided tumor uptakes of 14.6 +/- 2.4 and 12.5 +/- 1.3% ID/g at 4 h after injection, respectively. The tumor uptake of In-III-DOTA-3 was significantly higher than the uptake of the parental In-III-DOTA-1 (9.1 +/- 2.0% ID/g). The tumor-to-blood ratios of 395 +/- 75 and 419 +/- 91 at 4 h after injection were obtained for In-III-DOTA-5 and (IIII)n-DOTA-3, respectively. In conclusion, the N-terminal sequence composition affects the biodistribution and targeting properties of ADAPT-based imaging probes, and its optimization may improve imaging contrast.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2016
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-199556 (URN)10.1021/acs.bioconjchem.6b00465 (DOI)000388430700011 ()2-s2.0-84996523484 (Scopus ID)
Note

QC 20170113

Available from: 2017-01-13 Created: 2017-01-09 Last updated: 2018-03-15Bibliographically approved
4. Radionuclide Tumor Targeting Using ADAPT Scaffold Proteins: Aspects of Label Positioning and Residualizing Properties of the Label
Open this publication in new window or tab >>Radionuclide Tumor Targeting Using ADAPT Scaffold Proteins: Aspects of Label Positioning and Residualizing Properties of the Label
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2018 (English)In: Journal of Nuclear Medicine, ISSN 0161-5505, E-ISSN 1535-5667, Vol. 59, no 1, p. 93-99Article in journal (Refereed) Published
Abstract [en]

Visualization of cancer-associated alterations of molecular phenotype using radionuclide imaging is a noninvasive approach to stratifying patients for targeted therapies. The engineered albumin-binding domain-derived affinity protein (ADAPT) is a promising tracer for radionuclide molecular imaging because of its small size (6.5 kDa), which satisfies the precondition for efficient tumor penetration and rapid clearance. Previous studies demonstrated that the human epidermal growth factor receptor type 2 (HER2)-targeting ADAPT6 labeled with radiometals at the N terminus is able to image HER2 expression in xenografts a few hours after injection. The aim of this study was to evaluate whether the use of a non-residualizing label or placement of the labels at the C terminus would further improve the targeting properties of ADAPT6. Methods: Two constructs, Cys(2)-ADAPT6 and Cys(59)-ADAPT6, having the (HE)(3)DANS sequence at the N terminus were produced and site-specifically labeled using In-111-DOTA or I-125-iodo-((4-hydroxyphenyl) ethyl) maleimide (HPEM). The conjugates were compared in vitro and in vivo. HER2-targeting properties and biodistribution were evaluated in BALB/C nu/nu mice bearing ovarian carcinoma cell (SKOV-3) xenografts. Results: Specific HER2 binding and high affinity were preserved after labeling. Both Cys(2)-ADAPT6 and Cys59-ADAPT6 were internalized slowly by HER2-expressing cancer cells. Depending on the label position, uptake at 4 h after injection varied from 10% to 22% of the injected dose per gram of tumor tissue. Regardless of terminus position, the I-125-HPEM label provided more than 140-fold lower renal uptake than the In-111-DOTA label at 4 after injection. The tumor-to-organ ratios were, in contrast, higher for both of the (111)InDOTA- labeled ADAPT variants in other organs. Tumor-to-blood ratios for In-111-labeled Cys(2)-ADAPT6 and Cys(59)-ADAPT6 did not differ significantly (250-280), but In-111-DOTA-Cys(59)-ADAPT6 provided significantly higher tumor-to-lung, tumor-to-liver, tumor-to-spleen, and tumor-to-muscle ratios. Radioiodinated variants had similar tumor-to-organ ratios, but I-125-HPEM-Cys(59)-ADAPT6 had significantly higher tumor uptake and a higher tumor-to-kidney ratio. Conclusion: Residualizing properties of the label strongly influence the targeting properties of ADAPT6. The position of the radiolabel influences targeting as well, although to a lesser extent. Placement of a label at the C terminus yields the best biodistribution features for both radiometal and radiohalogen labels. Low renal retention of the radioiodine label creates a precondition for radionuclide therapy using I-131-labeled HPEM-Cys(59)-ADAPT6.

Place, publisher, year, edition, pages
Society of Nuclear Medicine, 2018
Keywords
molecular biology, ADAPT, HER2, biodistribution, molecular imaging, terminal sequence
National Category
Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:kth:diva-221362 (URN)10.2967/jnumed.117.197202 (DOI)000419116200021 ()28864631 (PubMedID)2-s2.0-85040078184 (Scopus ID)
Funder
Swedish Cancer SocietySwedish Research Council
Note

QC 20180117

Available from: 2018-01-17 Created: 2018-01-17 Last updated: 2018-03-15Bibliographically approved
5. Comparative evaluation of tumor targeting using the anti-HER2 ADAPT scaffold protein labeled at the C-terminus with indium-111 or technetium-99m
Open this publication in new window or tab >>Comparative evaluation of tumor targeting using the anti-HER2 ADAPT scaffold protein labeled at the C-terminus with indium-111 or technetium-99m
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2017 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 14780Article in journal (Refereed) Published
Abstract [en]

ABD-Derived Affinity Proteins (ADAPTs) is a novel class of engineered scaffold proteins derived from an albumin-binding domain of protein G. The use of ADAPT6 derivatives as targeting moiety have provided excellent preclinical radionuclide imaging of human epidermal growth factor 2 (HER2) tumor xenografts. Previous studies have demonstrated that selection of nuclide and chelator for its conjugation has an appreciable effect on imaging properties of scaffold proteins. In this study we performed a comparative evaluation of the anti-HER2 ADAPT having an aspartate-glutamate-alanine-valine-aspartate-alanine-asparagine-serine (DEAVDANS) N-terminal sequence and labeled at C-terminus with (99)mTc using a cysteine-containing peptide based chelator, glycine-serine-serine-cysteine (GSSC), and a similar variant labeled with In-111 using a maleimido derivative of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator. Both (99)mTc-DEAVDANS-ADAPT6-GSSC and In-111-DEAVDANS-ADAPT6-GSSC-DOTA accumulated specifically in HER2-expressing SKOV3 xenografts. The tumor uptake of both variants did not differ significantly and average values were in the range of 19-21% ID/g. However, there was an appreciable variation in uptake of conjugates in normal tissues that resulted in a notable difference in the tumor-to-organ ratios. The In-111-DOTA label provided 2-6 fold higher tumor-to-organ ratios than (99)mTc-GSSC and is therefore the preferable label for ADAPTs.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 2017
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:kth:diva-217934 (URN)10.1038/s41598-017-15366-w (DOI)000414569900003 ()2-s2.0-85033458225 (Scopus ID)
Note

QC 20171121

Available from: 2017-11-21 Created: 2017-11-21 Last updated: 2018-03-15Bibliographically approved
6. Design, construction and characterization of an ABD-based library with improved stability
Open this publication in new window or tab >>Design, construction and characterization of an ABD-based library with improved stability
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Recombinant affinity proteins binding specifically to other molecules are important tools for many clinical and industrial applications. Small robust protein scaffolds have proven to be well suited as frameworks for generation of novel affinity binders due to their stability. Here we used the albumin-binding domain (ABD) of protein G from Streptococcus G148 as scaffold to design a new combinatorial library capable of generating stable binders to various target proteins with high affinity and specificity. To create a robust framework able to generate highly stable binders, mutations in the non-binding region were evaluated and residues providing increased stability were introduced into the scaffold. By combining rational design with combinatorial protein engineering we also evaluated the surface exposed amino acids at the albumin-binding interface and identified 11 residues suitable for randomization. The potency of the novel scaffold library was assessed by screening for binders using phage display against three distinct targets; complement factor 4, (C4), insulin and interleukin-6 (IL-6). Binders in the nanomolar range with melting temperature above 57°C were selected for all three targets. Notably, the identified IL-6 binders were characterized by extreme thermal stability with variants demonstrating organized structures even at 90°C. This demonstrates that stable binders with distinct specificities can be generated and thus proves the high potential of the library.

Keywords
ABD, alternative scaffold, library design, protein engineering, directed evolution, rational design, in vitro selection, phage display
National Category
Other Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-224254 (URN)
Note

QC 20180315

Available from: 2018-03-15 Created: 2018-03-15 Last updated: 2018-03-15Bibliographically approved

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