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Phase I study of 99mTc-ADAPT6, a scaffold protein-based probe for visualization of HER2 expression in breast cancer
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.ORCID iD: 0000-0002-4695-7858
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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 [en]
HER2, ADAPT6, 99mTc, SPECT, Phase I
National Category
Cancer and Oncology
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-273237OAI: oai:DiVA.org:kth-273237DiVA, id: diva2:1429608
Available from: 2020-05-12 Created: 2020-05-12 Last updated: 2020-05-12
In thesis
1. The ADAPT scaffold as a tool for diagnostic imaging and targeted therapy
Open this publication in new window or tab >>The ADAPT scaffold as a tool for diagnostic imaging and targeted therapy
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
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:nbn:se:kth:diva-273244 (URN)978-91-7873-519-8 (ISBN)
Public defence
2020-06-05, https://kth-se.zoom.us/j/69770628499, Stockholm, 10:00 (English)
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QC 2020-05-12

Available from: 2020-05-12 Created: 2020-05-12 Last updated: 2020-05-12Bibliographically approved

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