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Targeting molecules for diagnostics of Head and Neck squamous cell carcinoma
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery.
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Description
Abstract [en]

To personalize treatment for cancer, correct staging of the primary tumor, nodal disease and metastatic disease is of essence. By targeting tumor specific receptors with radiolabeled antibodies, specificity and accuracy of imaging may be improved. Radio-immunodiagnostics can potentially detect small volume disease, occult metastasis and recurrent cancer in treated tissue. This thesis focuses on evaluation of radio-immunoconjugates directed towards CD44v6, which is a surface receptor overexpressed in many head and neck squamous cell carcinomas. At the outset, the monoclonal chimeric antibody cMab U36 and its cleavage products Fab’ and F(ab’)2 were labeled with 125I and assessed in vitro and in vivo (paper I). The best distribution pattern and tumor to organ ratio was achieved with F(ab’)2. Due to the immunological responses humans can develop towards chimeric antibodies, they are not optimal for clinical use, and subsequently fully human antibody fragments were developed. AbD15179, which is a monovalent fragment, was labeled with 111In and 125I and evaluated in vitro and in mice bearing CD44v6-expressing tumors. Tumor to organ ratios were improved compared to cMab U36 derived fragments, and 111In-AbD15179 displayed a more favorable distribution compared to 125I-AbD15179 (Paper II). A bivalent Fab-dHXL, AbD19384 derived from AbD15179, was then constructed and labeled with 125I and evaluated in cell- and biodistribution studies. Furthermore, an imaging study in a small animal PET was performed with 124I-AbD19384 (Paper III). Uptake in kidneys was reduced and liver uptake increased compared to AbD15179 reflecting the larger molecule. The high CD44v6 expressing tumor was clearly visualized with maximum uptake at 48 hours post injection.In paper IV human single chain fragments towards CD44v6v were selected, and the top candidates A11 and H12 were further evaluated in vitro and in vivo. Single chain fragments are small molecules exhibiting fast clearance and high affinity to the target. The study proved this by demonstrating superior tumor to blood ratios of radiolabeled A11 and H12 compared to previously studied molecules. 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2017. , 54 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1319
Keyword [en]
HNSCC, CD44v6, radio-immunodiagnosis, immuno-PET, tumor targeting, antibodies, antibody fragments
National Category
Otorhinolaryngology
Research subject
Oto-Rhino-Laryngology
Identifiers
URN: urn:nbn:se:uu:diva-315210ISBN: 978-91-554-9864-1 (print)OAI: oai:DiVA.org:uu-315210DiVA: diva2:1083264
Public defence
2017-05-12, Skoogsalen, Ing. 78-79 Akademiska sjukhuset, Uppsala, 09:00 (Swedish)
Opponent
Supervisors
Available from: 2017-04-20 Created: 2017-03-20 Last updated: 2017-04-20
List of papers
1. A novel CD44v6 targeting antibody fragment with improved tumor-to-blood ratio
Open this publication in new window or tab >>A novel CD44v6 targeting antibody fragment with improved tumor-to-blood ratio
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2012 (English)In: International Journal of Oncology, ISSN 1019-6439, Vol. 40, no 5, 1525-1532 p.Article in journal (Refereed) Published
Abstract [en]

The chimeric monoclonal antibody U36 (cMAb U36) recognizes the CD44v6 antigen. Its potential as a radioimmunotargeting agent, as well as its safety, has been shown in previous studies in head and neck cancer patients. However, intact MAbs have long circulation time in the blood and tumor targeting may also be hampered due to the slow and incomplete diffusion into solid tumors. In comparison, smaller monovalent Fab' and divalent F(ab')2 fragments are expected to exhibit shorter circulating half-lives, better tumor penetration and are thus more likely to yield better imaging results. In this study, novel F(ab')2 and Fab' fragments from cMAb U36 were radiolabeled with 125I and the characteristics of the conjugates in vitro were examined. The biodistribution of the conjugates were then evaluated in nude mice bearing CD44v6-expressing xenograft tumors. Furthermore, the penetration depth and distribution in tumor tissue was assessed by autoradiography in selected tumor samples. The in vitro experiments showed that the conjugates were stable and had intact affinity to CD44v6. The biodistribution study demonstrated superior tumor-to-blood ratio for the novel cMAb U36 fragment 125I-F(ab')2 compared with both the intact MAb and the monovalent fragment form. Autoradiography also revealed better tumor penetration for 125I-F(ab')2. This study demonstrates that the use of antibody fragments may improve radioimmunotargeting and possibly improve the management of head and neck malignancies.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Otorhinolaryngology
Research subject
Oto-Rhino-Laryngology
Identifiers
urn:nbn:se:uu:diva-170945 (URN)10.3892/ijo.2012.1352 (DOI)000302273400023 ()22307465 (PubMedID)
Available from: 2012-03-14 Created: 2012-03-14 Last updated: 2017-03-20Bibliographically approved
2. In vivo characterization of the novel CD44v6-targeting Fab fragment AbD15179 for molecular imaging of squamous cell carcinoma: a dual-isotope study
Open this publication in new window or tab >>In vivo characterization of the novel CD44v6-targeting Fab fragment AbD15179 for molecular imaging of squamous cell carcinoma: a dual-isotope study
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2014 (English)In: EJNMMI Research, ISSN 2191-219X, E-ISSN 2191-219X, Vol. 4, 11Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: Patients with squamous cell carcinoma in the head and neck region (HNSCC) offer a diagnostic challenge due to difficulties to detect small tumours and metastases. Imaging methods available are not sufficient, and radio-immunodiagnostics could increase specificity and sensitivity of diagnostics. The objective of this study was to evaluate, for the first time, the in vivo properties of the radiolabelled CD44v6-targeting fragment AbD15179 and to assess its utility as a targeting agent for radio-immunodiagnostics of CD44v6-expressing tumours.

METHODS: The fully human CD44v6-targeting Fab fragment AbD15179 was labelled with 111In or 125I, as models for radionuclides suitable for imaging with SPECT or PET. Species specificity, antigen specificity and internalization properties were first assessed in vitro. In vivo specificity and biodistribution were then evaluated in tumour-bearing mice using a dual-tumour and dual-isotope setup.

RESULTS: Both species-specific and antigen-specific binding of the conjugates were demonstrated in vitro, with no detectable internalization. The in vivo studies demonstrated specific tumour binding and favourable tumour targeting properties for both conjugates, albeit with higher tumour uptake, slower tumour dissociation, higher tumour-to-blood ratio and higher CD44v6 sensitivity for the 111In-labelled fragment. In contrast, the 125I-Fab demonstrated more favourable tumour-to-organ ratios for liver, spleen and kidneys.

CONCLUSIONS: We conclude that AbD15179 efficiently targets CD44v6-expressing squamous cell carcinoma xenografts, and particularly, the 111In-Fab displayed high and specific tumour uptake. CD44v6 emerges as a suitable target for radio-immunodiagnostics, and a fully human antibody fragment such as AbD15179 can enable further clinical imaging studies.

Keyword
Radio-immunodiagnostics, Antibody fragment, CD44v6, Molecular imaging, Fab, I-125, In-111
National Category
Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:uu:diva-221094 (URN)10.1186/2191-219X-4-11 (DOI)000357859200001 ()24598405 (PubMedID)
Available from: 2014-03-25 Created: 2014-03-25 Last updated: 2017-03-20Bibliographically approved
3. Evaluation of a novel type of imaging probe based on a recombinant bivalent mini-antibody construct for detection of CD44v6-expressing squamous cell carcinoma
Open this publication in new window or tab >>Evaluation of a novel type of imaging probe based on a recombinant bivalent mini-antibody construct for detection of CD44v6-expressing squamous cell carcinoma
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2015 (English)In: International journal of oncology, ISSN 1791-2423, Vol. 48, no 2, 461-470 p.Article in journal (Refereed) Published
Abstract [en]

We have developed the CD44v6-targeting human bivalent antibody fragment AbD19384, an engineered recombinant human bivalent Fab antibody formed via dimerization of dHLX (synthetic double helix loop helix motif) domains, for potential use in antibody-based molecular imaging of squamous cell carcinoma in the head and neck region. This is a unique construct that has, to the best of our knowledge, never been assessed for molecular imaging in vivo before. The objective of the present study was to evaluate for the first time the in vitro and in vivo binding properties of radio-iodinated AbD19384, and to assess its utility as a targeting agent for molecular imaging of CD44v6-expressing tumors. Antigen specificity and binding properties were assessed in vitro. In vivo specificity and biodistribution of 125I-AbD19384 were next evaluated in tumor-bearing mice using a dual-tumor setup. Finally, AbD19384 was labeled with 124I, and its imaging properties were assessed by small animal PET/CT in tumor bearing mice, and compared with 2-deoxy-2-[18F]fluoro-D-glucose (18F-FDG). In vitro studies demonstrated CD44v6-specific binding with slow off-rate for AbD19384. A favorable biodistribution profile was seen in vivo, with tumor-specific uptake. Small animal PET/CT images of 124I-AbD19384 supported the results through clearly visible high CD44v6-expressing tumors and faintly visible low expressing tumors, with superior imaging properties compared to 18F-FDG. Tumor-to-blood ratios increased with time for the conjugate (assessed up to 72 h p.i.), although 48 h p.i. proved best for imaging. Biodistribution and small-animal PET studies demonstrated that the recombinant Fab-dHLX construct AbD19384 is a promising tracer for imaging of CD44v6 antigen expression in vivo, with the future aim to be used for individualized diagnosis and early detection of squamous cell carcinomas in the head and neck region. Furthermore, this proof-of-concept research established the feasibility of using recombinant Fab-dHLX constructs for in vivo imaging of tumor biomarkers.

Keyword
molecular imaging; CD44v6; Fab-dHLX; F(ab')(2); recombinant antibodies; immuno-PET; head and neck squamous cell carcinoma
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-270259 (URN)10.3892/ijo.2015.3290 (DOI)000366897500003 ()26676731 (PubMedID)
Available from: 2015-12-22 Created: 2015-12-22 Last updated: 2017-03-20Bibliographically approved
4. Generation and evaluation of antibody agents for molecular imaging of CD44v6 expressing cancers
Open this publication in new window or tab >>Generation and evaluation of antibody agents for molecular imaging of CD44v6 expressing cancers
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(English)Manuscript (preprint) (Other academic)
Keyword
scFv, recombinant antibody formats, CD44v6, squamous cell carcinoma, molecular imaging
National Category
Other Basic Medicine
Identifiers
urn:nbn:se:uu:diva-316722 (URN)
Available from: 2017-03-14 Created: 2017-03-14 Last updated: 2017-03-20

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