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Anti-CDCP1 immuno-conjugates for detection and inhibition of ovarian cancer

Identifieur interne : 000828 ( Pmc/Curation ); précédent : 000827; suivant : 000829

Anti-CDCP1 immuno-conjugates for detection and inhibition of ovarian cancer

Auteurs : Brittney S. Harrington [Australie] ; Yaowu He [Australie] ; Tashbib Khan [Australie] ; Simon Puttick [Australie] ; Paul J. Conroy [Australie] ; Thomas Kryza [Australie] ; Tahleesa Cuda [Australie] ; Kamil A. Sokolowski [Australie] ; Brian Wc Tse [Australie] ; Katherine K. Robbins [Australie] ; Buddhika J. Arachchige [Australie] ; Samantha J. Stehbens [Australie] ; Pamela M. Pollock [Australie] ; Sarah Reed [Australie] ; S. John Weroha [États-Unis] ; Paul Haluska [États-Unis] ; Carlos Salomon [Australie] ; Rohan Lourie [Australie] ; Lewis C. Perrin [Australie] ; Ruby H. P. Law [Australie] ; James C. Whisstock [Australie] ; John D. Hooper [Australie]

Source :

RBID : PMC:7019151

Abstract

CUB-domain containing protein 1 (CDCP1) is a cancer associated cell surface protein that amplifies pro-tumorigenic signalling by other receptors including EGFR and HER2. Its potential as a cancer target is supported by studies showing that anti-CDCP1 antibodies inhibit cell migration and survival in vitro, and tumor growth and metastasis in vivo. Here we characterize two anti-CDCP1 antibodies, focusing on immuno-conjugates of one of these as a tool to detect and inhibit ovarian cancer.

Methods: A panel of ovarian cancer cell lines was examined for cell surface expression of CDCP1 and loss of expression induced by anti-CDCP1 antibodies 10D7 and 41-2 using flow cytometry and Western blot analysis. Surface plasmon resonance analysis and examination of truncation mutants was used to analyse the binding properties of the antibodies for CDCP1. Live-cell spinning-disk confocal microscopy of GFP-tagged CDCP1 was used to track internalization and intracellular trafficking of CDCP1/antibody complexes. In vivo, zirconium 89-labelled 10D7 was detected by positron-emission tomography imaging, of an ovarian cancer patient-derived xenograft grown intraperitoneally in mice. The efficacy of cytotoxin-conjugated 10D7 was examined against ovarian cancer cells in vitro and in vivo.

Results: Our data indicate that each antibody binds with high affinity to the extracellular domain of CDCP1 causing rapid internalization of the receptor/antibody complex and degradation of CDCP1 via processes mediated by the kinase Src. Highlighting the potential clinical utility of CDCP1, positron-emission tomography imaging, using zirconium 89-labelled 10D7, was able to detect subcutaneous and intraperitoneal xenograft ovarian cancers in mice, including small (diameter <3 mm) tumor deposits of an ovarian cancer patient-derived xenograft grown intraperitoneally in mice. Furthermore, cytotoxin-conjugated 10D7 was effective at inhibiting growth of CDCP1-expressing ovarian cancer cells in vitro and in vivo.

Conclusions: These data demonstrate that CDCP1 internalizing antibodies have potential for killing and detection of CDCP1 expressing ovarian cancer cells.


Url:
DOI: 10.7150/thno.30736
PubMed: 32104500
PubMed Central: 7019151

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PMC:7019151

Le document en format XML

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<affiliation wicri:level="1">
<nlm:aff id="A1">Mater Research Institute - The University of Queensland, Translational Research Institute, Woolloongabba, QLD 4102, Australia.</nlm:aff>
<country xml:lang="fr" wicri:curation="lc">Australie</country>
<wicri:regionArea>Mater Research Institute - The University of Queensland, Translational Research Institute, Woolloongabba, QLD 4102</wicri:regionArea>
</affiliation>
</author>
<author>
<name sortKey="Sokolowski, Kamil A" sort="Sokolowski, Kamil A" uniqKey="Sokolowski K" first="Kamil A" last="Sokolowski">Kamil A. Sokolowski</name>
<affiliation wicri:level="1">
<nlm:aff id="A6">Preclinical Imaging Facility, Translational Research Institute, Woolloongabba, QLD 4102, Australia.</nlm:aff>
<country xml:lang="fr" wicri:curation="lc">Australie</country>
<wicri:regionArea>Preclinical Imaging Facility, Translational Research Institute, Woolloongabba, QLD 4102</wicri:regionArea>
</affiliation>
</author>
<author>
<name sortKey="Tse, Brian Wc" sort="Tse, Brian Wc" uniqKey="Tse B" first="Brian Wc" last="Tse">Brian Wc Tse</name>
<affiliation wicri:level="1">
<nlm:aff id="A6">Preclinical Imaging Facility, Translational Research Institute, Woolloongabba, QLD 4102, Australia.</nlm:aff>
<country xml:lang="fr" wicri:curation="lc">Australie</country>
<wicri:regionArea>Preclinical Imaging Facility, Translational Research Institute, Woolloongabba, QLD 4102</wicri:regionArea>
</affiliation>
</author>
<author>
<name sortKey="Robbins, Katherine K" sort="Robbins, Katherine K" uniqKey="Robbins K" first="Katherine K." last="Robbins">Katherine K. Robbins</name>
<affiliation wicri:level="1">
<nlm:aff id="A1">Mater Research Institute - The University of Queensland, Translational Research Institute, Woolloongabba, QLD 4102, Australia.</nlm:aff>
<country xml:lang="fr" wicri:curation="lc">Australie</country>
<wicri:regionArea>Mater Research Institute - The University of Queensland, Translational Research Institute, Woolloongabba, QLD 4102</wicri:regionArea>
</affiliation>
</author>
<author>
<name sortKey="Arachchige, Buddhika J" sort="Arachchige, Buddhika J" uniqKey="Arachchige B" first="Buddhika J." last="Arachchige">Buddhika J. Arachchige</name>
<affiliation wicri:level="1">
<nlm:aff id="A7">Centre for Clinical Research, University of Queensland, Herston, Qld 4029, Australia.</nlm:aff>
<country xml:lang="fr" wicri:curation="lc">Australie</country>
<wicri:regionArea>Centre for Clinical Research, University of Queensland, Herston, Qld 4029</wicri:regionArea>
</affiliation>
</author>
<author>
<name sortKey="Stehbens, Samantha J" sort="Stehbens, Samantha J" uniqKey="Stehbens S" first="Samantha J." last="Stehbens">Samantha J. Stehbens</name>
<affiliation wicri:level="1">
<nlm:aff id="A8">Institute of Health and Biomedical Innovation, Queensland University of Technology, Woolloongabba, QLD 4102, Australia.</nlm:aff>
<country xml:lang="fr" wicri:curation="lc">Australie</country>
<wicri:regionArea>Institute of Health and Biomedical Innovation, Queensland University of Technology, Woolloongabba, QLD 4102</wicri:regionArea>
</affiliation>
</author>
<author>
<name sortKey="Pollock, Pamela M" sort="Pollock, Pamela M" uniqKey="Pollock P" first="Pamela M." last="Pollock">Pamela M. Pollock</name>
<affiliation wicri:level="1">
<nlm:aff id="A8">Institute of Health and Biomedical Innovation, Queensland University of Technology, Woolloongabba, QLD 4102, Australia.</nlm:aff>
<country xml:lang="fr" wicri:curation="lc">Australie</country>
<wicri:regionArea>Institute of Health and Biomedical Innovation, Queensland University of Technology, Woolloongabba, QLD 4102</wicri:regionArea>
</affiliation>
</author>
<author>
<name sortKey="Reed, Sarah" sort="Reed, Sarah" uniqKey="Reed S" first="Sarah" last="Reed">Sarah Reed</name>
<affiliation wicri:level="1">
<nlm:aff id="A7">Centre for Clinical Research, University of Queensland, Herston, Qld 4029, Australia.</nlm:aff>
<country xml:lang="fr" wicri:curation="lc">Australie</country>
<wicri:regionArea>Centre for Clinical Research, University of Queensland, Herston, Qld 4029</wicri:regionArea>
</affiliation>
</author>
<author>
<name sortKey="Weroha, S John" sort="Weroha, S John" uniqKey="Weroha S" first="S. John" last="Weroha">S. John Weroha</name>
<affiliation wicri:level="1">
<nlm:aff id="A9">Department of Medical Oncology, Mayo Clinic, Rochester, MN, USA.</nlm:aff>
<country xml:lang="fr" wicri:curation="lc">États-Unis</country>
<wicri:regionArea>Department of Medical Oncology, Mayo Clinic, Rochester, MN</wicri:regionArea>
</affiliation>
</author>
<author>
<name sortKey="Haluska, Paul" sort="Haluska, Paul" uniqKey="Haluska P" first="Paul" last="Haluska">Paul Haluska</name>
<affiliation wicri:level="1">
<nlm:aff id="A9">Department of Medical Oncology, Mayo Clinic, Rochester, MN, USA.</nlm:aff>
<country xml:lang="fr" wicri:curation="lc">États-Unis</country>
<wicri:regionArea>Department of Medical Oncology, Mayo Clinic, Rochester, MN</wicri:regionArea>
</affiliation>
<affiliation wicri:level="1">
<nlm:aff id="A10">Merck Research Laboratories, Rahway, NJ, USA.</nlm:aff>
<country xml:lang="fr" wicri:curation="lc">États-Unis</country>
<wicri:regionArea>Merck Research Laboratories, Rahway, NJ</wicri:regionArea>
</affiliation>
</author>
<author>
<name sortKey="Salomon, Carlos" sort="Salomon, Carlos" uniqKey="Salomon C" first="Carlos" last="Salomon">Carlos Salomon</name>
<affiliation wicri:level="1">
<nlm:aff id="A7">Centre for Clinical Research, University of Queensland, Herston, Qld 4029, Australia.</nlm:aff>
<country xml:lang="fr" wicri:curation="lc">Australie</country>
<wicri:regionArea>Centre for Clinical Research, University of Queensland, Herston, Qld 4029</wicri:regionArea>
</affiliation>
</author>
<author>
<name sortKey="Lourie, Rohan" sort="Lourie, Rohan" uniqKey="Lourie R" first="Rohan" last="Lourie">Rohan Lourie</name>
<affiliation wicri:level="1">
<nlm:aff id="A2">Mater Ovarian Cancer Research Collaborative, Mater Adult Hospital, South Brisbane, QLD 4101, Australia.</nlm:aff>
<country xml:lang="fr" wicri:curation="lc">Australie</country>
<wicri:regionArea>Mater Ovarian Cancer Research Collaborative, Mater Adult Hospital, South Brisbane, QLD 4101</wicri:regionArea>
</affiliation>
<affiliation wicri:level="1">
<nlm:aff id="A11">Mater Health Services, South Brisbane, QLD 4101, Australia.</nlm:aff>
<country xml:lang="fr" wicri:curation="lc">Australie</country>
<wicri:regionArea>Mater Health Services, South Brisbane, QLD 4101</wicri:regionArea>
</affiliation>
</author>
<author>
<name sortKey="Perrin, Lewis C" sort="Perrin, Lewis C" uniqKey="Perrin L" first="Lewis C." last="Perrin">Lewis C. Perrin</name>
<affiliation wicri:level="1">
<nlm:aff id="A2">Mater Ovarian Cancer Research Collaborative, Mater Adult Hospital, South Brisbane, QLD 4101, Australia.</nlm:aff>
<country xml:lang="fr" wicri:curation="lc">Australie</country>
<wicri:regionArea>Mater Ovarian Cancer Research Collaborative, Mater Adult Hospital, South Brisbane, QLD 4101</wicri:regionArea>
</affiliation>
<affiliation wicri:level="1">
<nlm:aff id="A11">Mater Health Services, South Brisbane, QLD 4101, Australia.</nlm:aff>
<country xml:lang="fr" wicri:curation="lc">Australie</country>
<wicri:regionArea>Mater Health Services, South Brisbane, QLD 4101</wicri:regionArea>
</affiliation>
</author>
<author>
<name sortKey="Law, Ruby H P" sort="Law, Ruby H P" uniqKey="Law R" first="Ruby H. P." last="Law">Ruby H. P. Law</name>
<affiliation wicri:level="1">
<nlm:aff id="A5">Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Melbourne, VIC 3800, Australia.</nlm:aff>
<country xml:lang="fr" wicri:curation="lc">Australie</country>
<wicri:regionArea>Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Melbourne, VIC 3800</wicri:regionArea>
</affiliation>
</author>
<author>
<name sortKey="Whisstock, James C" sort="Whisstock, James C" uniqKey="Whisstock J" first="James C." last="Whisstock">James C. Whisstock</name>
<affiliation wicri:level="1">
<nlm:aff id="A5">Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Melbourne, VIC 3800, Australia.</nlm:aff>
<country xml:lang="fr" wicri:curation="lc">Australie</country>
<wicri:regionArea>Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Melbourne, VIC 3800</wicri:regionArea>
</affiliation>
</author>
<author>
<name sortKey="Hooper, John D" sort="Hooper, John D" uniqKey="Hooper J" first="John D" last="Hooper">John D. Hooper</name>
<affiliation wicri:level="1">
<nlm:aff id="A1">Mater Research Institute - The University of Queensland, Translational Research Institute, Woolloongabba, QLD 4102, Australia.</nlm:aff>
<country xml:lang="fr" wicri:curation="lc">Australie</country>
<wicri:regionArea>Mater Research Institute - The University of Queensland, Translational Research Institute, Woolloongabba, QLD 4102</wicri:regionArea>
</affiliation>
<affiliation wicri:level="1">
<nlm:aff id="A2">Mater Ovarian Cancer Research Collaborative, Mater Adult Hospital, South Brisbane, QLD 4101, Australia.</nlm:aff>
<country xml:lang="fr" wicri:curation="lc">Australie</country>
<wicri:regionArea>Mater Ovarian Cancer Research Collaborative, Mater Adult Hospital, South Brisbane, QLD 4101</wicri:regionArea>
</affiliation>
</author>
</analytic>
<series>
<title level="j">Theranostics</title>
<idno type="eISSN">1838-7640</idno>
<imprint>
<date when="2020">2020</date>
</imprint>
</series>
</biblStruct>
</sourceDesc>
</fileDesc>
<profileDesc>
<textClass></textClass>
</profileDesc>
</teiHeader>
<front>
<div type="abstract" xml:lang="en">
<p>CUB-domain containing protein 1 (CDCP1) is a cancer associated cell surface protein that amplifies pro-tumorigenic signalling by other receptors including EGFR and HER2. Its potential as a cancer target is supported by studies showing that anti-CDCP1 antibodies inhibit cell migration and survival
<italic>in vitro</italic>
, and tumor growth and metastasis
<italic>in vivo</italic>
. Here we characterize two anti-CDCP1 antibodies, focusing on immuno-conjugates of one of these as a tool to detect and inhibit ovarian cancer.</p>
<p>
<bold>Methods</bold>
: A panel of ovarian cancer cell lines was examined for cell surface expression of CDCP1 and loss of expression induced by anti-CDCP1 antibodies 10D7 and 41-2 using flow cytometry and Western blot analysis. Surface plasmon resonance analysis and examination of truncation mutants was used to analyse the binding properties of the antibodies for CDCP1. Live-cell spinning-disk confocal microscopy of GFP-tagged CDCP1 was used to track internalization and intracellular trafficking of CDCP1/antibody complexes.
<italic>In vivo</italic>
, zirconium 89-labelled 10D7 was detected by positron-emission tomography imaging, of an ovarian cancer patient-derived xenograft grown intraperitoneally in mice. The efficacy of cytotoxin-conjugated 10D7 was examined against ovarian cancer cells
<italic>in vitro</italic>
and
<italic>in vivo</italic>
.</p>
<p>
<bold>Results</bold>
: Our data indicate that each antibody binds with high affinity to the extracellular domain of CDCP1 causing rapid internalization of the receptor/antibody complex and degradation of CDCP1 via processes mediated by the kinase Src. Highlighting the potential clinical utility of CDCP1, positron-emission tomography imaging, using zirconium 89-labelled 10D7, was able to detect subcutaneous and intraperitoneal xenograft ovarian cancers in mice, including small (diameter <3 mm) tumor deposits of an ovarian cancer patient-derived xenograft grown intraperitoneally in mice. Furthermore, cytotoxin-conjugated 10D7 was effective at inhibiting growth of CDCP1-expressing ovarian cancer cells
<italic>in vitro</italic>
and
<italic>in vivo</italic>
.</p>
<p>
<bold>Conclusions</bold>
: These data demonstrate that CDCP1 internalizing antibodies have potential for killing and detection of CDCP1 expressing ovarian cancer cells.</p>
</div>
</front>
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</TEI>
<pmc article-type="research-article">
<pmc-dir>properties open_access</pmc-dir>
<front>
<journal-meta>
<journal-id journal-id-type="nlm-ta">Theranostics</journal-id>
<journal-id journal-id-type="iso-abbrev">Theranostics</journal-id>
<journal-id journal-id-type="publisher-id">thno</journal-id>
<journal-title-group>
<journal-title>Theranostics</journal-title>
</journal-title-group>
<issn pub-type="epub">1838-7640</issn>
<publisher>
<publisher-name>Ivyspring International Publisher</publisher-name>
<publisher-loc>Sydney</publisher-loc>
</publisher>
</journal-meta>
<article-meta>
<article-id pub-id-type="pmid">32104500</article-id>
<article-id pub-id-type="pmc">7019151</article-id>
<article-id pub-id-type="doi">10.7150/thno.30736</article-id>
<article-id pub-id-type="publisher-id">thnov10p2095</article-id>
<article-categories>
<subj-group subj-group-type="heading">
<subject>Research Paper</subject>
</subj-group>
</article-categories>
<title-group>
<article-title>Anti-CDCP1 immuno-conjugates for detection and inhibition of ovarian cancer</article-title>
</title-group>
<contrib-group>
<contrib contrib-type="author">
<name>
<surname>Harrington</surname>
<given-names>Brittney S.</given-names>
</name>
<xref ref-type="aff" rid="A1">1</xref>
<xref ref-type="aff" rid="A2">2</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>He</surname>
<given-names>Yaowu</given-names>
</name>
<xref ref-type="aff" rid="A1">1</xref>
<xref ref-type="aff" rid="A2">2</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Khan</surname>
<given-names>Tashbib</given-names>
</name>
<xref ref-type="aff" rid="A1">1</xref>
<xref ref-type="aff" rid="A2">2</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Puttick</surname>
<given-names>Simon</given-names>
</name>
<xref ref-type="aff" rid="A3">3</xref>
<xref ref-type="aff" rid="A4">4</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Conroy</surname>
<given-names>Paul J.</given-names>
</name>
<xref ref-type="aff" rid="A5">5</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Kryza</surname>
<given-names>Thomas</given-names>
</name>
<xref ref-type="aff" rid="A1">1</xref>
<xref ref-type="aff" rid="A2">2</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Cuda</surname>
<given-names>Tahleesa</given-names>
</name>
<xref ref-type="aff" rid="A1">1</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Sokolowski</surname>
<given-names>Kamil A</given-names>
</name>
<xref ref-type="aff" rid="A6">6</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Tse</surname>
<given-names>Brian WC</given-names>
</name>
<xref ref-type="aff" rid="A6">6</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Robbins</surname>
<given-names>Katherine K.</given-names>
</name>
<xref ref-type="aff" rid="A1">1</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Arachchige</surname>
<given-names>Buddhika J.</given-names>
</name>
<xref ref-type="aff" rid="A7">7</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Stehbens</surname>
<given-names>Samantha J.</given-names>
</name>
<xref ref-type="aff" rid="A8">8</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Pollock</surname>
<given-names>Pamela M.</given-names>
</name>
<xref ref-type="aff" rid="A8">8</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Reed</surname>
<given-names>Sarah</given-names>
</name>
<xref ref-type="aff" rid="A7">7</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Weroha</surname>
<given-names>S. John</given-names>
</name>
<xref ref-type="aff" rid="A9">9</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Haluska</surname>
<given-names>Paul</given-names>
</name>
<xref ref-type="aff" rid="A9">9</xref>
<xref ref-type="aff" rid="A10">10</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Salomon</surname>
<given-names>Carlos</given-names>
</name>
<xref ref-type="aff" rid="A7">7</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Lourie</surname>
<given-names>Rohan</given-names>
</name>
<xref ref-type="aff" rid="A2">2</xref>
<xref ref-type="aff" rid="A11">11</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Perrin</surname>
<given-names>Lewis C.</given-names>
</name>
<xref ref-type="aff" rid="A2">2</xref>
<xref ref-type="aff" rid="A11">11</xref>
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<name>
<surname>Law</surname>
<given-names>Ruby H. P.</given-names>
</name>
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</contrib>
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<surname>Whisstock</surname>
<given-names>James C.</given-names>
</name>
<xref ref-type="aff" rid="A5">5</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Hooper</surname>
<given-names>John D</given-names>
</name>
<xref ref-type="aff" rid="A1">1</xref>
<xref ref-type="aff" rid="A2">2</xref>
<xref ref-type="corresp" rid="FNA_envelop">✉</xref>
</contrib>
</contrib-group>
<aff id="A1">
<label>1</label>
Mater Research Institute - The University of Queensland, Translational Research Institute, Woolloongabba, QLD 4102, Australia.</aff>
<aff id="A2">
<label>2</label>
Mater Ovarian Cancer Research Collaborative, Mater Adult Hospital, South Brisbane, QLD 4101, Australia.</aff>
<aff id="A3">
<label>3</label>
Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, Australia.</aff>
<aff id="A4">
<label>4</label>
Commonwealth Scientific and Industrial Research Organisation, Probing Biosystems Future Science Platform, Herston, QLD 4029, Australia.</aff>
<aff id="A5">
<label>5</label>
Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Melbourne, VIC 3800, Australia.</aff>
<aff id="A6">
<label>6</label>
Preclinical Imaging Facility, Translational Research Institute, Woolloongabba, QLD 4102, Australia.</aff>
<aff id="A7">
<label>7</label>
Centre for Clinical Research, University of Queensland, Herston, Qld 4029, Australia.</aff>
<aff id="A8">
<label>8</label>
Institute of Health and Biomedical Innovation, Queensland University of Technology, Woolloongabba, QLD 4102, Australia.</aff>
<aff id="A9">
<label>9</label>
Department of Medical Oncology, Mayo Clinic, Rochester, MN, USA.</aff>
<aff id="A10">
<label>10</label>
Merck Research Laboratories, Rahway, NJ, USA.</aff>
<aff id="A11">
<label>11</label>
Mater Health Services, South Brisbane, QLD 4101, Australia.</aff>
<author-notes>
<corresp id="FNA_envelop">✉ Corresponding author: John Hooper, Mater Research Institute - The University of Queensland, Translational Research Institute, Woolloongabba, Qld 4102, Australia; Tel.: +61734437639; E-mail:
<email>john.hooper@mater.uq.edu.au</email>
.</corresp>
<fn fn-type="COI-statement">
<p>Competing Interests: The authors have declared that no competing interest exists.</p>
</fn>
</author-notes>
<pub-date pub-type="collection">
<year>2020</year>
</pub-date>
<pub-date pub-type="epub">
<day>12</day>
<month>1</month>
<year>2020</year>
</pub-date>
<volume>10</volume>
<issue>5</issue>
<fpage>2095</fpage>
<lpage>2114</lpage>
<history>
<date date-type="received">
<day>16</day>
<month>10</month>
<year>2018</year>
</date>
<date date-type="accepted">
<day>13</day>
<month>12</month>
<year>2019</year>
</date>
</history>
<permissions>
<copyright-statement>© The author(s)</copyright-statement>
<copyright-year>2020</copyright-year>
<license license-type="open-access">
<license-p>This is an open access article distributed under the terms of the Creative Commons Attribution License (
<ext-link ext-link-type="uri" xlink:href="https://creativecommons.org/licenses/by/4.0/">https://creativecommons.org/licenses/by/4.0/</ext-link>
). See
<ext-link ext-link-type="uri" xlink:href="http://ivyspring.com/terms">http://ivyspring.com/terms</ext-link>
for full terms and conditions.</license-p>
</license>
</permissions>
<abstract>
<p>CUB-domain containing protein 1 (CDCP1) is a cancer associated cell surface protein that amplifies pro-tumorigenic signalling by other receptors including EGFR and HER2. Its potential as a cancer target is supported by studies showing that anti-CDCP1 antibodies inhibit cell migration and survival
<italic>in vitro</italic>
, and tumor growth and metastasis
<italic>in vivo</italic>
. Here we characterize two anti-CDCP1 antibodies, focusing on immuno-conjugates of one of these as a tool to detect and inhibit ovarian cancer.</p>
<p>
<bold>Methods</bold>
: A panel of ovarian cancer cell lines was examined for cell surface expression of CDCP1 and loss of expression induced by anti-CDCP1 antibodies 10D7 and 41-2 using flow cytometry and Western blot analysis. Surface plasmon resonance analysis and examination of truncation mutants was used to analyse the binding properties of the antibodies for CDCP1. Live-cell spinning-disk confocal microscopy of GFP-tagged CDCP1 was used to track internalization and intracellular trafficking of CDCP1/antibody complexes.
<italic>In vivo</italic>
, zirconium 89-labelled 10D7 was detected by positron-emission tomography imaging, of an ovarian cancer patient-derived xenograft grown intraperitoneally in mice. The efficacy of cytotoxin-conjugated 10D7 was examined against ovarian cancer cells
<italic>in vitro</italic>
and
<italic>in vivo</italic>
.</p>
<p>
<bold>Results</bold>
: Our data indicate that each antibody binds with high affinity to the extracellular domain of CDCP1 causing rapid internalization of the receptor/antibody complex and degradation of CDCP1 via processes mediated by the kinase Src. Highlighting the potential clinical utility of CDCP1, positron-emission tomography imaging, using zirconium 89-labelled 10D7, was able to detect subcutaneous and intraperitoneal xenograft ovarian cancers in mice, including small (diameter <3 mm) tumor deposits of an ovarian cancer patient-derived xenograft grown intraperitoneally in mice. Furthermore, cytotoxin-conjugated 10D7 was effective at inhibiting growth of CDCP1-expressing ovarian cancer cells
<italic>in vitro</italic>
and
<italic>in vivo</italic>
.</p>
<p>
<bold>Conclusions</bold>
: These data demonstrate that CDCP1 internalizing antibodies have potential for killing and detection of CDCP1 expressing ovarian cancer cells.</p>
</abstract>
<kwd-group>
<kwd>CDCP1</kwd>
<kwd>ovarian cancer</kwd>
<kwd>immuno-conjugate</kwd>
<kwd>antibody</kwd>
</kwd-group>
</article-meta>
</front>
<floats-group>
<fig id="F1" position="float">
<label>Figure 1</label>
<caption>
<p>
<bold> Antibody-induced loss of CDCP1 from the cell surface.</bold>
(
<bold>A</bold>
) Western blot analysis of lysates from the indicated cell lines using two mouse monoclonal anti-CDCP1 antibodies, 10D7 and 41-2 (1 µg/ml), a commercial rabbit polyclonal anti-CDCP1 antibody, 4115 (1:2,000 dilution), and an anti-GAPDH antibody (1:10,000). (
<bold>B</bold>
) Flow cytometry analysis of the indicated cell lines for plasma membrane localized CDCP1 using 10D7 and 41-2. Fixed cells were stained with the respective anti-CDCP1 antibody followed by an APC-conjugated anti-mouse IgG, then analysed by flow cytometry. Data are displayed graphically as MFI values corrected for background signal determined from cells stained with only the APC-conjugated anti-mouse IgG. 10D7 and 41-2 identified the same proportion of CDCP1 expressing cells as: HEY 89%; CAOV3 93%; SKOV3 99%; OVTOKO 86%; OVZM6 0%; OVMZ6-CDCP1 75%. (
<bold>C, D</bold>
) Flow cytometry analysis of HEY (c) and OVMZ6-CDCP1 (d) cells treated for 30 minutes at 37°C with 10D7, 41-2 or control IgG
<sub>1</sub>
κ (5µg/ml). Treated cells were fixed and plasma membrane localized CDCP1 detected using fluorescently tagged anti-CDCP1 antibody CD318
<sup>-PE</sup>
. Background signal was assessed by staining treated cells with fluorescently tagged control IgG (IgG
<sup>-PE</sup>
). Data are displayed as MFI values. All data are mean ± SEM from three independent experiments. ***P<0.001.</p>
</caption>
<graphic xlink:href="thnov10p2095g001"></graphic>
</fig>
<fig id="F2" position="float">
<label>Figure 2</label>
<caption>
<p>
<bold> Degradation of CDCP1 induced by internalizing mAbs 41-2 and 10D7.</bold>
(
<bold>A</bold>
) Western blot analysis, using anti-CDCP1 antibody 4115 (1:2,000) and an anti-GAPDH antibody (1:10,000), of lysates from HEY cells treated with isotype matched control IgG (
<italic>lef</italic>
t), 10D7
<italic>(middle)</italic>
or 41-2
<italic>(right)</italic>
for the indicated times. (
<bold>B</bold>
) Graph of fluorescence versus time from HeLa and HeLa-CDCP1 cells treated with 10D7
<sup>pH</sup>
(5µg/ml) (
<italic>left</italic>
), and graph of fluorescence signal from six EOC cell lines following treatment with 10D7
<sup>pH</sup>
or 41-2
<sup>pH</sup>
(5µg/ml) for 8 hours (
<italic>right</italic>
). RFU, relative fluorescence units. (
<bold>C</bold>
) Impact of lysosomal (
<italic>left</italic>
) and proteasomal (
<italic>right</italic>
) inhibition on antibody-induced degradation of CDCP1.
<italic>Top panel</italic>
, Anti-CDCP1 (1:2,000) and -GAPDH (1:10,000) Western blot analysis of HEY cells treated with 10D7 in the presence or absence of the lysosomal inhibitor chloroquine (CLQ; 50 µM), or the proteasomal inhibitor MG132 (20 µM) for the indicated times.
<italic>Bottom panel</italic>
, Graph of the ratio of CDCP1 to GAPDH signal generated from Western blot analyses of lysates from three independent assays assessing the effect of CLQ and MG132 on 10D7-induced degradation of CDCP1. All data represent mean ± SEM from three independent experiments. *P<0.05.</p>
</caption>
<graphic xlink:href="thnov10p2095g002"></graphic>
</fig>
<fig id="F3" position="float">
<label>Figure 3</label>
<caption>
<p>
<bold> 10D7 and 41-2 bind with high affinity to the ECD of CDCP1.</bold>
(
<bold>A</bold>
) Schematic representation of full length CDCP1 (CDCP1
<sup>FL</sup>
) and progressively shorter carboxyl terminal truncations (CDCP1-T358, -S416, -K554, -D665). CUB domains are colored green. (
<bold>B</bold>
) 10D7 and 41-2 (1 µg/ml) Western blot analysis of conditioned media from OVMZ6 cells transiently transfected with a control vector of constructs encoding CDCP1-T358, -S416, -K554, or -D665. (
<bold>C</bold>
) Flow cytometry analysis of HEY cells incubated with: (i) 10D7-QDot
<sup>625</sup>
for 1 h, unlabelled 41-2 for 1 h then 10D7-QDot
<sup>625</sup>
for 1 h, or concurrently with 10D7-QDot
<sup>625</sup>
and 41-2 for 1 h; or (ii) CD318
<sup>-PE</sup>
for 1 h, unlabelled 10D7 or 41-2 for 1 h then CD318
<sup>-PE</sup>
for 1 h, or concurrently with 10D7 or 41-2 and CD318
<sup>-PE</sup>
for 1 h. (
<bold>D</bold>
) Schematic of CDCP1 showing the regions to which antibodies 10D7, 41-2, CD318 and 4115 bind. (
<bold>E</bold>
)
<italic>Top panels</italic>
, Sensorgrams of CDCP1-ECD (concentration range 1.56 to 50 nM) binding to immobilized 10D7
<italic>(left)</italic>
and 41-2
<italic>(right)</italic>
depicting association (increasing signal) and dissociation (reducing signal) over time.
<italic>Bottom panel</italic>
, Table of kinetic parameters. k
<sub>a</sub>
, association rate; k
<sub>d</sub>
, dissociation rate; K
<sub>D</sub>
, affinity constant.</p>
</caption>
<graphic xlink:href="thnov10p2095g003"></graphic>
</fig>
<fig id="F4" position="float">
<label>Figure 4</label>
<caption>
<p>
<bold> 10D7-induces cell surface rapid clustering and lysosomal trafficking of CDCP1.</bold>
(
<bold>A</bold>
) Live-cell confocal microscopy images of HEY-CDCP1
<sup>GFP</sup>
cells treated with 10D7
<sup>pH</sup>
(5µg/ ml). Internalization of CDCP1
<sup>GFP</sup>
and 10D7
<sup>pH</sup>
was observed at 1 frame per second for 600 s. Insets highlight green punctate CDCP1
<sup>GFP</sup>
positive cellular structures at 30 s, and white cellular structures at 600s that are positive for both CDCP1
<sup>GFP</sup>
and 10D7
<sup>pH</sup>
. (
<bold>B</bold>
) Graph of complex formation between IgG
<sup>pH</sup>
and CDCP1
<sup>GFP</sup>
determined as the percentage of IgG
<sup>pH</sup>
signal coincident with CDCP1
<sup>GFP</sup>
signal using ImageJ software analysis. (
<bold>C</bold>
) Images of the plasma membrane and proximal cytoplasmic region of HEY-CDCP1
<sup>GFP</sup>
cells indicating 10D7-induced clustering of CDCP1. In untreated cells CDCP1
<sup>GFP</sup>
is located diffusely on the cell surface. In treated cells, arrowheads highlight rapid 10D7-induced clustering of CDCP1
<sup>GFP</sup>
and its internalization. (
<bold>D</bold>
)
<italic>Left panel</italic>
, Overlay of CDCP1
<sup>GFP</sup>
(green) and 10D7
<sup>pH</sup>
(magenta) signals in HEY-CDCP1
<sup>GFP</sup>
cells after 20 minutes of treatment showing co-localization of within endosomal-like structures.
<italic>Middle panel</italic>
, Black and white image of CDCP1
<sup>GFP</sup>
signal.
<italic>Right panel</italic>
, Black and white image of 10D7
<sup>pH</sup>
signal. (
<bold>E</bold>
) Live-cell confocal microscopy images of HEY-CDCP1
<sup>GFP</sup>
cells treated with IgG7
<sup>pH</sup>
(5µg/ ml). No internalization of CDCP1
<sup>GFP</sup>
was observed within 300 s of treatment with IgG7
<sup>pH</sup>
.</p>
</caption>
<graphic xlink:href="thnov10p2095g004"></graphic>
</fig>
<fig id="F5" position="float">
<label>Figure 5</label>
<caption>
<p>
<bold> CDCP1 is tyrosine phosphorylated during 10D7-induced internalization and degradation.</bold>
(
<bold>A</bold>
) Lysates from HEY cells treated with 10D7 (5µg/ml) for the indicated times were examined by Western blot analysis for CDCP1, p-CDCP1-Y734, Src, p-Src-Y416, and GAPDH. Antibody dilution was 1:2,000 except the anti-GAPDH antibody which was 1:10,000. The graphs display CDCP1 and p-CDCP1-Y734 levels determined by densitometric analysis with data representing mean ± SEM from three independent experiments. (
<bold>B</bold>
) Anti-CDCP1 (1:2,000), -GFP (1:2,000) and -GAPDH (1:10,000) Western blot analysis of fractions collected by cell surface biotinylation of HEY cells expressing CDCP1
<sup>GFP</sup>
, CDCP1
<sup>GFP</sup>
-Y734F, -Y743F or -Y762F. (
<bold>C</bold>
) Analysis of semi-automated computer tracking of CDCP1
<sup>GFP</sup>
and CDCP1
<sup>GFP</sup>
-Y734F, -Y743F and -Y762F in response to 10D7.
<italic>Left</italic>
, representative image of CDCP1
<sup>GFP</sup>
tracks that internalized in response to 10D7
<sup>pH</sup>
in HEY cells. The image is an overlay onto cells of color-coded tracks (violet, tracks that moved the shortest distance; red, the tracks that moved the greatest distance).
<italic>Right</italic>
, Graph of distance moved over 5 min by CDCP1
<sup>GFP</sup>
and CDCP1
<sup>GFP</sup>
-Y734F, -Y743F and -Y762F in response to 10D7 (5 µg/ml). Data are median and range from the 100 tracks with the highest velocity in each experimental group from three independent experiments. ***P<0.001.</p>
</caption>
<graphic xlink:href="thnov10p2095g005"></graphic>
</fig>
<fig id="F6" position="float">
<label>Figure 6</label>
<caption>
<p>
<bold> The Src inhibitor dasatinib blocks 10D7-induced phosphorylation and internalization of CDCP1.</bold>
(
<bold>A</bold>
) HEY cells, treated for 2 h with dasatinib (200 nM), were incubated with 10D7 (5µg/ ml) for the indicated times. Lysates were examined by Western blot analysis for CDCP1 (1:2,000), pCDCP1-Y734 (1:2,000) and GAPDH (1:10,000). (
<bold>B</bold>
) Live-cell confocal microscopy images, acquired at the indicated time points after antibody treatment, of HEY-CDCP1
<sup>GFP</sup>
cells pre-treated with dasatinib (200 nM), then incubated with 10D7
<sup>pH</sup>
.
<italic>Lower panels</italic>
, 10D7
<sup>pH</sup>
signal.
<italic>Middle panels</italic>
, CDCP1
<sup>GFP</sup>
signal.
<italic>Upper panels</italic>
, overlay of 10D7
<sup>pH</sup>
and CDCP1
<sup>GFP</sup>
signals. (
<bold>C</bold>
) Graph of distance moved over 5 min by CDCP1
<sup>GFP</sup>
in response to 10D7 in the presence and absence of dasatinib. Data are median and range from the 100 tracks with the highest velocity in each experimental group from three independent experiments. ***P<0.001.</p>
</caption>
<graphic xlink:href="thnov10p2095g006"></graphic>
</fig>
<fig id="F7" position="float">
<label>Figure 7</label>
<caption>
<p>
<bold> PET-CT imaging of an EOC PDX.</bold>
(
<bold>A</bold>
) Clear cell EOC PDX PH250.
<italic>Left</italic>
, hematoxylin and eosin stained section highlighting clear cell features at 40X with 10X magnification (inset).
<italic>Right</italic>
, Anti-CDCP1 immunohistochemistry (antibody 4115) highlighting strong CDCP1 expression by malignant cells with accentuation of signal on the plasma membrane at 40X and 10X magnification (inset). (
<bold>B</bold>
) Comparison of CDCP1 expression by HEY cells and PDX PH250 cells.
<italic>Left</italic>
, Anti-CDCP1 (antibody 4115; 1:2,000) and GAPDH (1:10,000) Western blot analysis of lysates from HEY cells, a HEY cell xenograft, and a PH250 PDX tumor.
<italic>Right</italic>
, Cell surface CDCP1 receptor number determined by flow cytometry of single cell suspensions of HEY cells and PDX PH250 cells. Receptor numbers per cell are indicated above the flow cytometry peaks. (
<bold>C</bold>
) Representative PET images of mice carrying subcutaneous PH250 PDX tumors on both flanks.
<sup>89</sup>
Zr-10D7 and
<sup>89</sup>
Zr-IgG1κ were injected intravenously three weeks after tumor cell inoculation, and imaging performed 144 h later. White arrowhead, tumor nodules. Yellow arrow,
<sup>89</sup>
Zr-IgG1κ signal accumulated in the spleen. (
<bold>D</bold>
) Quantitative bio-distribution analysis of
<sup>89</sup>
Zr-10D7 and
<sup>89</sup>
Zr-IgG1κ 144 h post injection (n = 4). 10D7 accumulates in tumors to a significantly higher degree than IgG1κ which accumulates in the spleen and liver. ***, P<0.001.</p>
</caption>
<graphic xlink:href="thnov10p2095g007"></graphic>
</fig>
<fig id="F8" position="float">
<label>Figure 8</label>
<caption>
<p>
<bold> 10D7-MMAE selectively inhibits colony formation of CDCP1 expressing but not non-expressing EOC cells.</bold>
(
<bold>A</bold>
) Commassie stained gel of IgG and 10D7, and purified products from reactions of IgG and 10D7 with MMAE. (
<bold>B</bold>
) HEY cells were treated with 10D7-MMAE (5 µg/ml) for the indicated times and lysates examined by Western blot analysis for CDCP1, p-CDCP1-Y734, Src, p-Src-Y416 and GAPDH. Antibody dilution was 1:2,000 except the anti-GAPDH antibody which was 1:10,000. (
<bold>C</bold>
) Representative images of crystal violet stained colonies formed from HEY and OVMZ6-CDCP1 cells after treatment with the indicated concentrations of IgG, IgG-MMAE, 10D7 or 10D7-MMAE. (
<bold>D</bold>
) Graph of crystal violet staining, as a percentage of area (% Area), of colonies formed by HEY, OVMZ6-CDCP1 and OVMZ6 cells after treatment with increasing concentrations of IgG, IgG-MMAE, 10D7 or 10D7-MMAE. Data represent means ± SEM from three independent experiments. ***, P<0.001.</p>
</caption>
<graphic xlink:href="thnov10p2095g008"></graphic>
</fig>
<fig id="F9" position="float">
<label>Figure 9</label>
<caption>
<p>
<bold> 10D7-MMAE reduces tumor burden and increases survival of mice carrying intraperitoneal HEY cell xenografts.</bold>
(
<bold>A</bold>
) After 18 days of HEY cell growth (Day 0 in panel c), tumor burden was assessed in four randomly selected mice by PET-CT imaging using.
<sup>89</sup>
Zr-10D7 was injected intravenously, and imaging performed 144 h later.
<italic>Left</italic>
, anterior-posterior view.
<italic>Right</italic>
, Lateral view. (
<bold>B</bold>
) Bioluminescence imaging. Immediately after PET-CT imaging the remaining mice were administered a single treatment of vehicle, MMAE, 10D7 or 10D7-MMAE. Bioluminsecence imaging was performed 7, 14, 21, 24 and 32 days later. Images from day 24 are shown. (
<bold>C</bold>
) Change in tumor burden quantified by bioluminescent imaging. (
<bold>D</bold>
) Kaplan-Meier survival analysis. Red arrow, day treatments administered.</p>
</caption>
<graphic xlink:href="thnov10p2095g009"></graphic>
</fig>
</floats-group>
</pmc>
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