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The ADAPT scaffold as a tool for diagnostic imaging and targeted therapy
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.ORCID iD: 0000-0002-4695-7858
2020 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Molecular recognition, or the specific interactions between a protein and its ligand, is central to biology and a key factor for many different clinical and technical applications. Despite antibodies being only one of many different affinity proteins, it has by far been the most successful. However, their large size and complex structure can be limiting in terms of cost and stability. Furthermore, their effector functions can sometimes be undesired or even detrimental. Over the past decades, many alternative affinity proteins have emerged to overcome some of these limitations.

The Albumin Binding Domain (ABD), originally present on the surface of certain bacterial cells, has previously been subjected to combinatorial protein engineering for the generation of ADAPTs (ABD Derived Affinity ProTeins) that bind to different targets. One of these, the ADAPT6, targets HER2 and has shown great promise as a tracer for radionuclide molecular imaging for diagnosis and stratification of HER2 positive patients. The work in this thesis has aimed to optimise the ADAPT6 tracer further and also describes the first-inhuman clinical trial for imaging of HER2-overexpressing breast cancer. The results establish that ADAPT6 is safe and well-tolerated by patients and able to detect primary tumours as well as metastases with very high contrast already 2 hours after injection. However, the high kidney uptake associated with its fast blood clearance prevents further use of ADAPT6 also in a therapeutic setting. By engineering the ADAPT6 to prolong its circulatory half-life and reduce the kidney uptake, this thesis has also aimed to explore the therapeutic potential of this molecule. As a first step towards this goal, the ADAPT6 was genetically fused to an ABD to allow for binding to a patient’s own serum albumin and hence avoid the same extent of renal filtration. Indeed, when evaluated in mice, fusion to ABD increased the retention in circulation by more than 200-fold and exhibited a dramatically decreased renal activity. Treatment of tumour-bearing mice with the ABD-fused ADAPT6 conjugated to a cytotoxic radionuclide significantly prolonged survival by more than two-fold and was not associated with any observable toxicity. Finally, this thesis also describes a novel combinatorial library from which several bispecific ADAPTs have been identified, binding to both albumin and other clinically relevant targets simultaneously. This miniature bispecific scaffold offers an opportunity to combine the benefits associated with small size such as good tissue extravasation and alternative administration routes while still maintaining a sufficient in vivo half-life.

Abstract [sv]

Molekylär igenkänning, eller de specifika interaktionerna mellan ett protein och dess ligand, är ett centralt koncept inom biologin och en nyckelfaktor för många olika kliniska och tekniska tillämpningar. Trots att antikroppar bara är ett av många olika affinitetsproteiner har det varit det tveklöst mest framgångsrika. De har dock vissa begränsningar som beror av deras storlek samt deras komplexa struktur. Dessutom kan deras effektorfunktioner ibland vara oönskade eller till och med medföra negativa effekter. Under de senaste decennierna har därför många alternativa affinitetsproteiner utvecklats.

Proteinet ABD (Albumin Binding Domain), som ursprungligen återfinns på ytan på vissa bakterieceller, har tidigare utsatts för kombinatorisk proteinteknik för att generera ADAPT:er (ABD Derived Affinity ProTeins) som binder till olika målproteiner. En av dessa, ADAPT6, riktar sig mot HER2 och har uppvisat lovande resultat inom molekylär avbildning med hjälp av radionuklider, för diagnos och stratifiering av HER2-positiva patienter. Arbetet i denna avhandling har syftat till att optimera ADAPT6-molekylen ytterligare och beskriver också en första klinisk prövning i människa för avbildning av HER2-överuttryckande bröstcancer. Resultaten visar att ADAPT6 är säker och väl tolererad av patienter och att den kan visualisera primära tumörer såväl som metastaser med mycket hög kontrast redan 2 timmar efter injektion. Dess snabba eliminering från blodet är dock associerad med ett högt upptag i njurarna vilket förhindrar ytterligare användning av ADAPT6 som behandling. Genom att konstruera en ADAPT6-molekyl med förlängd halveringstid har denna avhandling också syftat till att utforska den terapeutiska potentialen för denna molekyl. Som ett första steg mot detta mål fuserades ADAPT6 genetiskt till en ABD-molekyl för att möjliggöra bindning till patientens eget serumalbumin och därmed undvika samma omfattning av filtrering i njurarna. Studier i möss visade att fusion till ABD ökade retentionen i blodet mer än 200 gånger och uppvisade samtidigt en dramatiskt minskad njuraktivitet. Behandling av tumörbärande möss med ABD-fuserad ADAPT6 konjugerad till en cytotoxisk radionuklid förlängde överlevnaden signifikant och var inte associerad med någon toxicitet. Slutligen beskriver denna avhandling också ett nytt kombinatoriskt bibliotek från vilket flera bispecifika ADAPT:er har identifierats, vilka binder till både albumin och andra kliniskt relevanta målprotein samtidigt. Detta minimerade bispecifika protein har potential att åtnjuta de fördelar som är associerade med en liten storlek, såsom ökad vävnadspenetration samt möjligheten till alternativa administrationsvägar, medan det samtidigt kan upprätthålla en tillräcklig halveringstid i blodet.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2020. , p. 91
Series
TRITA-CBH-FOU ; 2020:24
Keywords [en]
Protein G, ABD, ADAPT, affinity proteins, antibody, protein engineering, targeted therapy, nuclear medicine, molecular imaging, HER2, radionuclide therapy
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-273244ISBN: 978-91-7873-519-8 (print)OAI: oai:DiVA.org:kth-273244DiVA, id: diva2:1429650
Public defence
2020-06-05, https://kth-se.zoom.us/j/69770628499, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

QC 2020-05-12

Available from: 2020-05-12 Created: 2020-05-12 Last updated: 2020-05-12Bibliographically approved
List of papers
1. Selection of the optimal macrocyclic chelators for labeling with In-111 and Ga-68 improves contrast of HER2 imaging using engineered scaffold protein ADAPT6
Open this publication in new window or tab >>Selection of the optimal macrocyclic chelators for labeling with In-111 and Ga-68 improves contrast of HER2 imaging using engineered scaffold protein ADAPT6
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2019 (English)In: European journal of pharmaceutics and biopharmaceutics, ISSN 0939-6411, E-ISSN 1873-3441, Vol. 140, p. 109-120Article in journal (Refereed) Published
Abstract [en]

Radionuclide molecular imaging is a promising tool that becomes increasingly important as targeted cancer therapies are developed. To ensure an effective treatment, a molecular stratification of the cancer is a necessity. To accomplish this, visualization of cancer associated molecular abnormalities in vivo by molecular imaging is the method of choice. ADAPTs, a novel type of small protein scaffold, have been utilized to select and develop high affinity binders to different proteinaceous targets. One of these binders, ADAPT6 selectively interacts with human epidermal growth factor 2 (HER2) with low nanomolar affinity and can therefore be used for its in vivo visualization. Molecular design and optimization of labeled anti-HER2 ADAPT has been explored in several earlier studies, showing that small changes in the scaffold affect the biodistribution of the domain. In this study, we evaluate how the biodistribution properties of ADAPT6 is affected by the commonly used maleimido derivatives of the macrocyclic chelators NOTA, NODAGA, DOTA and DOTAGA with the aim to select the best variants for SPECT and PET imaging. The different conjugates were labeled with In-111 for SPECT and Ga-68 for PET. The acquired data show that the combination of a radionuclide and a chelator for its conjugation has a strong influence on the uptake of ADAPT6 in normal tissues and thereby gives a significant variation in tumor-toorgan ratios. Hence, it was concluded that the best variant for SPECT imaging is In-111-(HE)(3)DANS-ADAPT6-GSSC-DOTA while the best variant for PET imaging is Ga-68-(HE)(3)DANS-ADAPT6-GSSC-NODAGA.

Place, publisher, year, edition, pages
ELSEVIER SCIENCE BV, 2019
Keywords
ADAPT, HER2, Radionuclide imaging, Indium-111, Gallium-68, DOTA, NOTA, NODAGA, DOTAGA
Identifiers
urn:nbn:se:kth:diva-272449 (URN)10.1016/j.ejpb.2019.05.008 (DOI)000470947400012 ()31082509 (PubMedID)2-s2.0-85065610735 (Scopus ID)
Note

QC 20200421

Available from: 2020-04-21 Created: 2020-04-21 Last updated: 2020-05-12Bibliographically approved
2. Comparative evaluation of dimeric and monomeric forms of ADAPT scaffold protein for targeting of HER2-expressing tumours
Open this publication in new window or tab >>Comparative evaluation of dimeric and monomeric forms of ADAPT scaffold protein for targeting of HER2-expressing tumours
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2019 (English)In: European journal of pharmaceutics and biopharmaceutics, ISSN 0939-6411, E-ISSN 1873-3441, Vol. 134, p. 37-48Article in journal (Refereed) Published
Abstract [en]

ADAPTs are small engineered non-immunoglobulin scaffold proteins, which have demonstrated very promising features as vectors for radionuclide tumour targeting. Radionuclide imaging of human epidermal growth factor 2 (HER2) expression in vivo might be used for stratification of patients for HER2-targeting therapies. ADAPT6, which specifically binds to HER2, has earlier been shown to have very promising features for in vivo targeting of HER2 expressing tumours. In this study we tested the hypothesis that dimerization of ADAPT6 would increase the apparent affinity to HER2 and accordingly improve tumour targeting. To find an optimal molecular design of dimers, a series of ADAPT dimers with different linkers, -SSSG- (DiADAPT6L1), -(SSSG)(2)- (DiADAPT6L2), and -(SSSG)(3)- (DiADAPT6L3) was evaluated. Dimers in combination with optimal linker lengths demonstrated increased apparent affinity to HER2. The best variants, DiADAPT6L2 and DiADAPT6L3 were site-specifically labelled with In-111 and I-125, and compared with a monomeric ADAPT6 in mice bearing HER2-expressing tumours. Despite higher affinity, both dimers had lower tumour uptake and lower tumour-to-organ ratios compared to the monomer. We conclude that improved affinity of a dimeric form of ADAPT does not compensate the disadvantage of increased size. Therefore, increase of affinity should be obtained by affinity maturation and not by dimerization.

Place, publisher, year, edition, pages
ELSEVIER SCIENCE BV, 2019
Keywords
ADAPT, HER2, Dimer, Radionuclide molecular imaging, Indium-111, Iodine-125
National Category
Pharmaceutical Biotechnology
Identifiers
urn:nbn:se:kth:diva-243967 (URN)10.1016/j.ejpb.2018.11.004 (DOI)000456225000004 ()30408518 (PubMedID)2-s2.0-85056893627 (Scopus ID)
Note

QC 20190301

Available from: 2019-03-01 Created: 2019-03-01 Last updated: 2020-05-12Bibliographically approved
3. Phase I study of 99mTc-ADAPT6, a scaffold protein-based probe for visualization of HER2 expression in breast cancer
Open this publication in new window or tab >>Phase I study of 99mTc-ADAPT6, a scaffold protein-based probe for visualization of HER2 expression in breast cancer
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Radionuclide molecular imaging of human epidermal growth factor (HER2) expression may be helpful to stratify breast and gastroesophageal cancer patients for HER2-targeting therapies. ADAPTs (albumin-binding domain derived affinity proteins) are a new type of small (46-59 amino acids) proteins useful as probes for molecular imaging. The aim of this first in-human study was to evaluate biodistribution, dosimetry, and safety of HER2-specific 99mTc-ADAPT6.

METHODS. Twenty-two patients with HER2-positive (n=11) or HER2-negative (n=11) primary breast cancer were intravenously injected with 385125 MBq. The injected amount of protein was either 500 μg (n=11) or 1000 μg (n=11). Planar scintigraphy followed by SPECT imaging was performed after 2, 4, 6 and 24 h. An additional cohort received a dose of 250 μg, and the planar scintigraphy followed by SPECT imaging was performed after 2 h only.

RESULTS. Injection of 99mTc-ADAPT6 was well tolerated for all doses evaluated in the study, and was not associated with any adverse effects. 99mTc-ADAPT6 cleared rapidly from the blood and the majority of tissues. The normal organs with the highest accumulation were kidney, liver and lung. The effective doses were determined to 0.0090.002 and 0.0100.003 mSv/MBq when injecting protein amounts of 500 and 1000 μg, respectively. Injection of 500 μg resulted in excellent discrimination between HER2-positive and HER2-negative tumors already 2 h after injection (tumor-to-contralateral breast ratio was 3719 vs 52, p < 0.01). The tumor-to-contralateral breast ratios for HER2-positive tumors were significantly (p < 0.5) higher for the injected  mass of 500 μg than for both 250 and 1000 μg. In one patient, the imaging using 99mTc-ADAPT6 revealed three bone metastases, which were not found at the time of diagnosis by CT or 99mTcpyrophosphate bone scan. MRI imaging confirmed this finding.

CONCLUSION. Injections of 99mTc-ADAPT6 are safe and associated with low absorbed and effective doses. A protein dose of 500 μg is preferable for discrimination between tumors with high and low expression of HER2. 99mTc-ADAPT6 is a promising imaging probe for the stratification of patients for HER2-targeting therapy.

Keywords
HER2, ADAPT6, 99mTc, SPECT, Phase I
National Category
Cancer and Oncology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-273237 (URN)
Available from: 2020-05-12 Created: 2020-05-12 Last updated: 2020-05-12
4. Radionuclide Therapy Using ABD-fused ADAPT Scaffold Protein: Proof of Principle
Open this publication in new window or tab >>Radionuclide Therapy Using ABD-fused ADAPT Scaffold Protein: Proof of Principle
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

The molecular recognition characteristics of targeted therapeutics is typically attributed to immunoglobulins. However, recent development of engineered scaffold proteins (ESPs) has provided additional opportunities for the improvement of these targeted therapies. ESPs offer inexpensive production in prokaryotic hosts and high molecular stability as well as convenient approaches to modify the biodistribution. In this study, we have demonstrated successful modification of the biodistribution of a particular ESP known as ADAPT (Albumin-binding domain Derived Affinity ProTein). These ADAPTs are generated through screening of combinatorial libraries based on the rigid scaffold of ABD (Albumin Binding Domain) of protein G. As one of these ADAPTs, ADAPT6 binds to human epidermal growth factor type 2 (HER2) with high affinity. Preclinical and early clinical studies have demonstrated that radiolabeled ADAPT6 can image HER2-expression in tumors with high contrast. However, its rapid glomerular filtration and high renal re-absorption have prevented its use in radionuclide therapy. To modify the biodistribution of ADAPT6 and allow for a therapeutic use, we present here an ADAPT6 genetically fused to ABD. The non-covalent binding of this fusion protein to the host albumin resulted in a 14-fold reduction of renal uptake and appreciable increase of tumor uptake for the best variant, 177Lu-DOTA-ADAPT6-ABD035. Experimental therapy in mice bearing HER2-expressing xenografts demonstrated more than two-fold increase of median survival even after a single injection of 18 MBq 177Lu-DOTA-ADAPT6-ABD035. The injections were not associated with any observable toxicity. Thus, a fusion with ABD and optimization of the molecular design provides ADAPT derivatives with attractive targeting properties for radionuclide therapy.

Keywords
Radionuclide therapy, 177Lu, HER2, half-life extension, ABD, ADAPT, albumin
National Category
Radiology, Nuclear Medicine and Medical Imaging Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-273238 (URN)
Available from: 2020-05-12 Created: 2020-05-12 Last updated: 2020-05-12
5. ADAPT as a single-domain bispecific scaffold capable of albumin-associated haf-life extension for therapeutic applications
Open this publication in new window or tab >>ADAPT as a single-domain bispecific scaffold capable of albumin-associated haf-life extension for therapeutic applications
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Albumin-binding fusion partners are frequently used as a means for the in vivo half-life extension of small therapeutic molecules that would normally be cleared very rapidly from circulation. However, in applications where small size is key, fusion to an additional molecule can be disadvantageous. ABD-Derived Affinity ProTeins (ADAPTs) are a type of scaffold proteins based on one of the albumin binding domains of streptococcal Protein G, with newly introduced binding specificities against numerous targets. Here we engineered this scaffold further and showed that this domain, as small as 6 kDa, can harbor two distinct binding surfaces and utilize them to interact with two targets simultaneously. These novel ADAPTs were developed to bind to both serum albumin as well as another clinically relevant target, thus circumventing the need for an albumin binding fusion partner. To accomplish this, we designed a novel phage display library and used it to successfully select for single-domain bispecific binders towards a panel of targets: TNFa, PSA (Prostate Specific Antigen), CRP (C-reactive protein), renin, angiogenin and insulin. Apart from successfully identifying bispecific binders for all targets, we also demonstrated the formation of the ternary complex of the ADAPT together with albumin and each of the four targets TNFa, PSA, angiogenin and insulin. This simultaneous binding of albumin and other targets presents an opportunity to combine the advantages of small molecules with those of larger ones allowing for lower cost of goods and non-invasive administration routes while still maintaining a sufficient in vivo half-life.

Keywords
Protein engineering, phage display, next generation sequencing, ABD, ADAPT, albumin, half-life extension
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-273241 (URN)
Available from: 2020-05-12 Created: 2020-05-12 Last updated: 2020-05-12

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