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Haylock, Anna-Karin
Publications (10 of 11) Show all publications
Mortensen, A., Spiegelberg, D., Haylock, A.-K., Lundqvist, H. & Nestor, M. (2018). Preclinical evaluation of a novel engineered recombinant human anti-CD44v6 antibody for potential use in radio-immunotherapy. International Journal of Oncology, 52(6), 1875-1885
Open this publication in new window or tab >>Preclinical evaluation of a novel engineered recombinant human anti-CD44v6 antibody for potential use in radio-immunotherapy
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2018 (English)In: International Journal of Oncology, ISSN 1019-6439, Vol. 52, no 6, p. 1875-1885Article in journal (Refereed) Published
Abstract [en]

CD44v6 is overexpressed in a variety of cancers, rendering it a promising target for radio-immunotherapy (RIT). In this study, we have characterized a novel engineered recombinant monoclonal anti-CD44v6 antibody, AbN44v6, and assessed its potential for use in RIT using either Lu-177 or I-131 as therapeutic radionuclides. In vitro affinity and specificity assays characterized the binding of the antibody labeled with Lu-177, I-125 or I-131. The therapeutic effects of Lu-177-AbN44v6 and I-131-AbN44v6 were investigated using two in vitro 3D tumor models with different CD44v6 expression. Finally, the normal tissue biodistribution and dosimetry for Lu-177-AbN44v6 and I-125-AbN44v6/I-131-AbN44v6 were assessed in vivo using a mouse model. All AbN44v6 radioconjugates demonstrated CD44v6-specific binding in vitro. In the in vitro 3D tumor models, dose-dependent therapeutic effects were observed with both Lu-177-AbN44v6 and I-131-AbN44v6, with a greater significant therapeutic effect observed on the cells with a higher CD44v6 expression. Biodistribution experiments demonstrated a greater uptake of Lu-177-AbN44v6 in the liver, spleen and bone, compared to I-125-AbN44v6, whereas I-125-AbN44v6 demonstrated a longer circulation time. In dosimetric calculations, the critical organs for Lu-177-AbN44v6 were the liver and spleen, whereas the kidneys and red marrow were considered the critical organs for I-131-AbN44v6. The effective dose was in the order of 0.1 mSv/MBq for both labels. In conclusion, AbN44v6 bound specifically and with high affinity to CD44v6. Furthermore, in vitro RIT demonstrated growth inhibition in a CD44v6-specific activity-dependent manner for both radioconjugates, demonstrating that both Lu-177-AbN44v6 and I-131-AbN44v6 may be promising RIT candidates. Furthermore, biodistribution and dosimetric analysis supported the applicability of both conjugates for RIT. The CD44v6-specific therapeutic effects observed with radiolabeled AbN44v6 in the 3D tumor models in vitro, combined with the beneficial dosimetry in vivo, render AbN44v6 a potential candidate for RIT.

Place, publisher, year, edition, pages
SPANDIDOS PUBL LTD, 2018
Keywords
radio-immunotherapy, 3D tumor models, dosimetry, biodistribution, Lu-177, I-131
National Category
Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:uu:diva-356443 (URN)10.3892/ijo.2018.4364 (DOI)000432241200010 ()29658563 (PubMedID)
Funder
Swedish Research CouncilSwedish Cancer Society
Available from: 2018-07-31 Created: 2018-07-31 Last updated: 2018-07-31Bibliographically approved
Haylock, A.-K., Nilvebrant, J., Mortensen, A., Velikyan, I., Nestor, M. & Falk, R. (2017). Generation and evaluation of antibody agents for molecular imaging of CD44v6-expressing cancers. OncoTarget, 8(39), 65152-65170
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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2017 (English)In: OncoTarget, ISSN 1949-2553, E-ISSN 1949-2553, Vol. 8, no 39, p. 65152-65170Article in journal (Refereed) Published
Abstract [en]

Aim: The aim of this study was to generate and characterize scFv antibodies directed to human CD44v6, as well as to radiolabel and evaluate top candidates in vitro and in vivo for their potential use in CD44v6-targeted molecular imaging in cancer patients.

Materials and methods: Phage display selections were used to isolate CD44v6-specific scFvs. A chain shuffling strategy was employed for affinity maturation based on a set of CD44v6-specific first-generation clones. Two second-generation scFv clones were then chosen for labeling with 111In or 125I and assessed for CD44v6-specific binding on cultured tumor cells. In vivo uptake and distribution was evaluated in tumor-bearing mice using a dual tumor model. Finally, a proof-of-concept small animal PET-CT study was performed on one of the candidates labeled with 124I.

Results: Two affinity-matured clones, CD44v6-scFv-A11 and CD44v6-scFv-H12, displayed promising binding kinetics. Seven out of eight radiolabeled conjugates demonstrated CD44v6-specific binding. In vivo studies on selected candidates demonstrated very advantageous tumor-to-organ ratios, in particular for iodinated conjugates, where 125I-labeled scFvs exhibited favorable kinetics and tumor-to-blood ratios above five already at 24 hours p. i.. The small animal PET-CT study using 124I-labeled CD44v6-scFv-H12 was in line with the biodistribution data, clearly visualizing the high CD44v6-expressing tumor.

Conclusion: The single chain fragments, CD44v6-scFv-A11 and CD44v6-scFv-H12 specifically bind to CD44v6, and the radiolabeled counterparts provide high tumor-to-blood ratios and fast clearance from organs and blood. We conclude that radioiodinated CD44v6-scFv-A11 and CD44v6-scFv-H12 possess features highly suitable for stringent molecular imaging.

Keywords
scFv, recombinant antibody formats, CD44v6, squamous cell carcinoma, molecular imaging
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Cancer and Oncology Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:uu:diva-335192 (URN)10.18632/oncotarget.17996 (DOI)000410291200039 ()29029420 (PubMedID)
Funder
Swedish Cancer Society, CAN 2015/1080, CAN 2015/385Swedish Research Council, 2013-30876-104113-30, 637-2013-468Swedish Society for Medical Research (SSMF)Knut and Alice Wallenberg Foundation, 2008.0133
Note

Marika Nestor and Ronny Falk shared senior authorship.

Available from: 2017-12-08 Created: 2017-12-08 Last updated: 2017-12-08Bibliographically approved
Haylock, A.-K. (2017). Targeting molecules for diagnostics of Head and Neck squamous cell carcinoma. (Doctoral dissertation). Uppsala: Acta Universitatis Upsaliensis
Open this publication in new window or tab >>Targeting molecules for diagnostics of Head and Neck squamous cell carcinoma
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
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. p. 54
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1319
Keywords
HNSCC, CD44v6, radio-immunodiagnosis, immuno-PET, tumor targeting, antibodies, antibody fragments
National Category
Otorhinolaryngology
Research subject
Oto-Rhino-Laryngology
Identifiers
urn:nbn:se:uu:diva-315210 (URN)978-91-554-9864-1 (ISBN)
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
Spiegelberg, D., Stenberg, J., Haylock, A.-K. & Nestor, M. (2016). A real-time in vitro assay as a potential predictor of in vivo tumor imaging properties. Nuclear Medicine and Biology, 43(1), 12-18
Open this publication in new window or tab >>A real-time in vitro assay as a potential predictor of in vivo tumor imaging properties
2016 (English)In: Nuclear Medicine and Biology, ISSN 0969-8051, E-ISSN 1872-9614, Vol. 43, no 1, p. 12-18Article in journal (Refereed) Published
Abstract [en]

Introduction: Selective tumor targeting strategies based on cell surface molecules enable new personalized diagnosis and treatments, potentially lowering adverse effects and increasing efficacy. Radio-immunotargeting generally relies on a molecule binding to a cancer-specific target. It is therefore important to understand the properties of molecular interactions in their working environment and how to translate these properties measured in vitro into the in vivo molecular imaging situation. Methods: Time resolved interaction analysis in vitro was compared with a corresponding in vivo xenograft mouse model. The antibody fragment AbD15179 was labeled with I-125 or In-111, and analyzed on cell lines with differing CD44v6 expression in vitro, and in a dual tumor xenograft model derived from the same cell lines. In vitro LigandTracer measurements were analyzed with TraceDrawer and Interaction Map. Conjugate sensitivity, kinetics, and signal-to-background ratios were assessed for both tumor cells in vitro and xenograft tumors in vivo. Results: In vitro results revealed a general biphasic appearance of a high- and a low-affinity interaction event. The In-111-labeled fragment displayed the largest proportion of the high-affinity interaction with increased sensitivity and retention compared to I-125-Fab. In vivo results were in agreement with in vitro data, with increased retention, higher sensitivity and better contrast for the In-111-labeled fragment compared to I-125. Conclusions: Time resolved binding characteristics measured in vitro largely matched the in vivo performance for the conjugates, which is promising for future studies. In vitro time-resolved LigandTracer assays are efficient, rapid, and in this study shown to be able to predict in vivo outcomes. Advances in Knowledge and Implications for Patient Care: Further studies are needed to confirm these findings, but the method is promising considering the ethical need to reduce the use of laboratory animals, as well as reducing costs for the development of tumor targeting compounds in the future.

Keywords
Radio-immunotargeting, Radio-immunodiagnostics, Molecular imaging, AbD15179, CD44v6, HNSCC
National Category
Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:uu:diva-274920 (URN)10.1016/j.nucmedbio.2015.09.004 (DOI)000367419400003 ()26702782 (PubMedID)
Available from: 2016-01-27 Created: 2016-01-26 Last updated: 2017-11-30Bibliographically approved
Spiegelberg, D., Stenberg, J., Haylock, A.-K. & Nestor, M. (2015). A real-time in vitro assay as a potential predictor of in vivo tumour imaging properties. Paper presented at 28th Annual Congress of the European-Association-of-Nuclear-Medicine (EANM), OCT 10-14, 2015, Hamburg, GERMANY. European Journal of Nuclear Medicine and Molecular Imaging, 42(S1), S306-S307
Open this publication in new window or tab >>A real-time in vitro assay as a potential predictor of in vivo tumour imaging properties
2015 (English)In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 42, no S1, p. S306-S307Article in journal, Meeting abstract (Other academic) Published
National Category
Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:uu:diva-269147 (URN)000363013202115 ()
Conference
28th Annual Congress of the European-Association-of-Nuclear-Medicine (EANM), OCT 10-14, 2015, Hamburg, GERMANY
Note

Meeting Abstract: PW057

Available from: 2015-12-18 Created: 2015-12-14 Last updated: 2017-12-01Bibliographically approved
Haylock, A.-K., Spiegelberg, D., Mortensen, A. C., Selvaraju, R. K., Nilvebrant, J., Eriksson, O., . . . Nestor, M. V. (2015). Evaluation of a novel type of imaging probe based on a recombinant bivalent mini-antibody construct for detection of CD44v6-expressing squamous cell carcinoma. International journal of oncology, 48(2), 461-470
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, p. 461-470Article 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.

Keywords
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: 2017-11-15Bibliographically approved
Spiegelberg, D., Mortensen, A. C., Haylock, A.-K., Selvaraju, R. K., Stenerlöw, B. & Nestor, M. (2014). Evaluation of biomarkers for imaging and radio-immunotherapy in combination with HSP90 inhibition in squamous cell carcinomas. Paper presented at Annual Congress of the European-Association-of-Nuclear-Medicine (EANM), OCT 18-22, 2014, Gothenburg, SWEDEN. European Journal of Nuclear Medicine and Molecular Imaging, 41(S2), S638-S638, Article ID P972.
Open this publication in new window or tab >>Evaluation of biomarkers for imaging and radio-immunotherapy in combination with HSP90 inhibition in squamous cell carcinomas
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2014 (English)In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 41, no S2, p. S638-S638, article id P972Article in journal, Meeting abstract (Other academic) Published
National Category
Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:uu:diva-247704 (URN)000348841902154 ()
Conference
Annual Congress of the European-Association-of-Nuclear-Medicine (EANM), OCT 18-22, 2014, Gothenburg, SWEDEN
Available from: 2015-03-31 Created: 2015-03-23 Last updated: 2017-12-04Bibliographically approved
Mortensen, A. C., Spegelberg, D., Haylock, A.-K., Selvaraju, R. K., Tolmachev, V. & Nestor, M. (2014). In vivo characterization of a novel engineered CD44v6-targeting bivalent antibody fragment AbD19384 for molecular imaging of squamous cell carcinoma. Paper presented at Annual Congress of the European-Association-of-Nuclear-Medicine (EANM), OCT 18-22, 2014, Gothenburg, SWEDEN. European Journal of Nuclear Medicine and Molecular Imaging, 41(S2), S447-S447, Article ID P309.
Open this publication in new window or tab >>In vivo characterization of a novel engineered CD44v6-targeting bivalent antibody fragment AbD19384 for molecular imaging of squamous cell carcinoma
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2014 (English)In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 41, no S2, p. S447-S447, article id P309Article in journal, Meeting abstract (Other academic) Published
National Category
Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:uu:diva-247698 (URN)000348841901302 ()
Conference
Annual Congress of the European-Association-of-Nuclear-Medicine (EANM), OCT 18-22, 2014, Gothenburg, SWEDEN
Available from: 2015-04-02 Created: 2015-03-23 Last updated: 2017-12-04Bibliographically approved
Haylock, A.-K., Spiegelberg, D., Nilvebrant, J., Sandström, K. & Nestor, M. (2014). In vivo characterization of the novel CD44v6-targeting Fab fragment AbD15179 for molecular imaging of squamous cell carcinoma: a dual-isotope study. EJNMMI Research, 4, Article ID 11.
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, article id 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.

Keywords
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: 2017-12-05Bibliographically approved
Spiegelberg, D., Haylock, A.-K. & Nestor, M. (2013). In vivo characterization of CD44v6-targeting Fab fragments for molecular imaging of squamous cell carcinoma: a dual isotope study. Paper presented at Annual Congress of the European-Association-of-Nuclear-Medicine (EANM); OCT 19-23, 2013; Lyon, FRANCE. European Journal of Nuclear Medicine and Molecular Imaging, 40(Suppl. 2), S185-S185
Open this publication in new window or tab >>In vivo characterization of CD44v6-targeting Fab fragments for molecular imaging of squamous cell carcinoma: a dual isotope study
2013 (English)In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 40, no Suppl. 2, p. S185-S185Article in journal, Meeting abstract (Other academic) Published
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-211599 (URN)000325853400291 ()
Conference
Annual Congress of the European-Association-of-Nuclear-Medicine (EANM); OCT 19-23, 2013; Lyon, FRANCE
Available from: 2013-12-03 Created: 2013-11-27 Last updated: 2017-12-06Bibliographically approved
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