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Human monoclonal antibodies targeting carbonic anhydrase IX for the molecular imaging of hypoxic regions in solid tumours.

Ahlskog JK, Schliemann C, Mårlind J, Qureshi U, Ammar A, Pedley RB, Neri D - Br. J. Cancer (2009)

Bottom Line: These antibodies were able to stain CA IX ex vivo and to target the cognate antigen in vivo.In contrast, in a second animal model (SW1222), distinct staining patterns were observed for pimonidazole and CA IX targeting.We observed a complementary pattern of tumour regions targeted in vivo by the clinical-stage vascular-targeting antibody L19 and the anti-CA IX antibody A3, indicating that a homogenous pattern of in vivo tumour targeting could be achieved by a combination of the two antibodies.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Applied Biosciences, ETH Zürich, Wolfgang-Pauli-Strasse 10, Zurich CH-8093, Switzerland.

ABSTRACT

Background: Hypoxia, which is commonly observed in areas of primary tumours and of metastases, influences response to treatment. However, its characterisation has so far mainly been restricted to the ex vivo analysis of tumour sections using monoclonal antibodies specific to carbonic anhydrase IX (CA IX) or by pimonidazole staining, after the intravenous administration of this 2-nitroimidazole compound in experimental animal models.

Methods: In this study, we describe the generation of high-affinity human monoclonal antibodies (A3 and CC7) specific to human CA IX, using phage technology.

Results: These antibodies were able to stain CA IX ex vivo and to target the cognate antigen in vivo. In one of the two animal models of colorectal cancer studied (LS174T), CA IX imaging closely matched pimonidazole staining, with a preferential staining of tumour areas characterised by little vascularity and low perfusion. In contrast, in a second animal model (SW1222), distinct staining patterns were observed for pimonidazole and CA IX targeting. We observed a complementary pattern of tumour regions targeted in vivo by the clinical-stage vascular-targeting antibody L19 and the anti-CA IX antibody A3, indicating that a homogenous pattern of in vivo tumour targeting could be achieved by a combination of the two antibodies.

Conclusion: The new human anti-CA IX antibodies are expected to be non-immunogenic in patients with cancer and may serve as broadly applicable reagents for the non-invasive imaging of hypoxia and for pharmacodelivery applications.

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Multi-fluorescence microscopy analysis in LS174T xenograft-bearing mice. (A,B) Representative overlays of multiple digital fluorescence images of a LS174T tumour injected with pimonidazole (30 min before killing) and SIP(A3) (6 h before killing) demonstrating perfusion with Hoechst 33342 (1 min before killing) (blue), blood vessel staining (red), pimonidazole binding (green, left panel) and CA IX targeting by SIP(A3) (green, middle panel). (C) ex vivo staining of an adjacent tumour section with a polyclonal anti-CA IX antiserum. (D–F) Higher magnification images of mainly perfused areas of the corresponding tumour sections. (G–I) Higher magnification images of mainly CA IX-positive areas of the corresponding tumour sections. (A–C) Scale bar=500 μm and (D–I) scale bar=100 μm. N indicates necrosis.
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fig6: Multi-fluorescence microscopy analysis in LS174T xenograft-bearing mice. (A,B) Representative overlays of multiple digital fluorescence images of a LS174T tumour injected with pimonidazole (30 min before killing) and SIP(A3) (6 h before killing) demonstrating perfusion with Hoechst 33342 (1 min before killing) (blue), blood vessel staining (red), pimonidazole binding (green, left panel) and CA IX targeting by SIP(A3) (green, middle panel). (C) ex vivo staining of an adjacent tumour section with a polyclonal anti-CA IX antiserum. (D–F) Higher magnification images of mainly perfused areas of the corresponding tumour sections. (G–I) Higher magnification images of mainly CA IX-positive areas of the corresponding tumour sections. (A–C) Scale bar=500 μm and (D–I) scale bar=100 μm. N indicates necrosis.

Mentions: Figure 6 shows representative results of a multi-colour fluorescence microscopy analysis of serial sections from LS174T tumours, following i.v. administration of SIP(A3) (6 h before killing of the mice), of pimonidazole (30 min before killing) and Hoechst 33342 (1 min before killing). The low magnification images (top panels) yield an informative impression of the heterogeneity of these tumours. One can recognise areas with dense vascular structures (red), some of which are well perfused (blue). The structures targeted in vivo by SIP(A3) closely match those stained ex vivo for pimonidazole modification, confirming that these hypoxic areas could be reached by the i.v. administered antibody. Importantly, these areas are superimposable to the structures stained ex vivo with a polyclonal anti-CA IX antiserum, thus indicating that CA IX-positive areas of the tumour could be reached by our reagent. A higher magnification view (bottom panels) shows details of well perfused areas with no detectable CA IX expression, as well as tumour regions efficiently targeted by the A3 antibody. Similar results were obtained with SIP(CC7) (Supplementary Figure).


Human monoclonal antibodies targeting carbonic anhydrase IX for the molecular imaging of hypoxic regions in solid tumours.

Ahlskog JK, Schliemann C, Mårlind J, Qureshi U, Ammar A, Pedley RB, Neri D - Br. J. Cancer (2009)

Multi-fluorescence microscopy analysis in LS174T xenograft-bearing mice. (A,B) Representative overlays of multiple digital fluorescence images of a LS174T tumour injected with pimonidazole (30 min before killing) and SIP(A3) (6 h before killing) demonstrating perfusion with Hoechst 33342 (1 min before killing) (blue), blood vessel staining (red), pimonidazole binding (green, left panel) and CA IX targeting by SIP(A3) (green, middle panel). (C) ex vivo staining of an adjacent tumour section with a polyclonal anti-CA IX antiserum. (D–F) Higher magnification images of mainly perfused areas of the corresponding tumour sections. (G–I) Higher magnification images of mainly CA IX-positive areas of the corresponding tumour sections. (A–C) Scale bar=500 μm and (D–I) scale bar=100 μm. N indicates necrosis.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2736829&req=5

fig6: Multi-fluorescence microscopy analysis in LS174T xenograft-bearing mice. (A,B) Representative overlays of multiple digital fluorescence images of a LS174T tumour injected with pimonidazole (30 min before killing) and SIP(A3) (6 h before killing) demonstrating perfusion with Hoechst 33342 (1 min before killing) (blue), blood vessel staining (red), pimonidazole binding (green, left panel) and CA IX targeting by SIP(A3) (green, middle panel). (C) ex vivo staining of an adjacent tumour section with a polyclonal anti-CA IX antiserum. (D–F) Higher magnification images of mainly perfused areas of the corresponding tumour sections. (G–I) Higher magnification images of mainly CA IX-positive areas of the corresponding tumour sections. (A–C) Scale bar=500 μm and (D–I) scale bar=100 μm. N indicates necrosis.
Mentions: Figure 6 shows representative results of a multi-colour fluorescence microscopy analysis of serial sections from LS174T tumours, following i.v. administration of SIP(A3) (6 h before killing of the mice), of pimonidazole (30 min before killing) and Hoechst 33342 (1 min before killing). The low magnification images (top panels) yield an informative impression of the heterogeneity of these tumours. One can recognise areas with dense vascular structures (red), some of which are well perfused (blue). The structures targeted in vivo by SIP(A3) closely match those stained ex vivo for pimonidazole modification, confirming that these hypoxic areas could be reached by the i.v. administered antibody. Importantly, these areas are superimposable to the structures stained ex vivo with a polyclonal anti-CA IX antiserum, thus indicating that CA IX-positive areas of the tumour could be reached by our reagent. A higher magnification view (bottom panels) shows details of well perfused areas with no detectable CA IX expression, as well as tumour regions efficiently targeted by the A3 antibody. Similar results were obtained with SIP(CC7) (Supplementary Figure).

Bottom Line: These antibodies were able to stain CA IX ex vivo and to target the cognate antigen in vivo.In contrast, in a second animal model (SW1222), distinct staining patterns were observed for pimonidazole and CA IX targeting.We observed a complementary pattern of tumour regions targeted in vivo by the clinical-stage vascular-targeting antibody L19 and the anti-CA IX antibody A3, indicating that a homogenous pattern of in vivo tumour targeting could be achieved by a combination of the two antibodies.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Applied Biosciences, ETH Zürich, Wolfgang-Pauli-Strasse 10, Zurich CH-8093, Switzerland.

ABSTRACT

Background: Hypoxia, which is commonly observed in areas of primary tumours and of metastases, influences response to treatment. However, its characterisation has so far mainly been restricted to the ex vivo analysis of tumour sections using monoclonal antibodies specific to carbonic anhydrase IX (CA IX) or by pimonidazole staining, after the intravenous administration of this 2-nitroimidazole compound in experimental animal models.

Methods: In this study, we describe the generation of high-affinity human monoclonal antibodies (A3 and CC7) specific to human CA IX, using phage technology.

Results: These antibodies were able to stain CA IX ex vivo and to target the cognate antigen in vivo. In one of the two animal models of colorectal cancer studied (LS174T), CA IX imaging closely matched pimonidazole staining, with a preferential staining of tumour areas characterised by little vascularity and low perfusion. In contrast, in a second animal model (SW1222), distinct staining patterns were observed for pimonidazole and CA IX targeting. We observed a complementary pattern of tumour regions targeted in vivo by the clinical-stage vascular-targeting antibody L19 and the anti-CA IX antibody A3, indicating that a homogenous pattern of in vivo tumour targeting could be achieved by a combination of the two antibodies.

Conclusion: The new human anti-CA IX antibodies are expected to be non-immunogenic in patients with cancer and may serve as broadly applicable reagents for the non-invasive imaging of hypoxia and for pharmacodelivery applications.

Show MeSH
Related in: MedlinePlus