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. 2012 Jun;14(3):336-47.
doi: 10.1007/s11307-011-0500-8.

An Engineered Cysteine-Modified Diabody for Imaging Activated Leukocyte Cell Adhesion Molecule (ALCAM)-positive Tumors

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Free PMC article

An Engineered Cysteine-Modified Diabody for Imaging Activated Leukocyte Cell Adhesion Molecule (ALCAM)-positive Tumors

Katelyn E McCabe et al. Mol Imaging Biol. .
Free PMC article

Abstract

Purpose: The purpose of this study was to generate and evaluate a positron emission tomography (PET) radiotracer targeting activated leukocyte cell adhesion molecule (ALCAM).

Procedures: A human anti-ALCAM single chain variable fragment was reformatted to produce a covalent dimer, termed a cys-diabody (CysDb). Purified CysDb was characterized by gel electrophoresis and size exclusion chromatography, and immunoreactivity was assessed by flow cytometry and immunofluorescence. Targeting and imaging of ALCAM-positive tumors using (64)Cu-DOTA-CysDb were evaluated in mice bearing human pancreatic adenocarcinoma xenografts (HPAF-II or BxPC-3).

Results: CysDb binds specifically to ALCAM-positive cells in vitro with an apparent affinity in the range of 1-3 nM. MicroPET images at 4 h showed specific targeting of positive tumors in vivo, a finding confirmed by biodistribution analysis, with positive/negative tumor ratios of 1.9 ± 0.6 and 2.4 ± 0.6, and positive tumor/blood ratios of 2.5 ± 0.9 and 2.9 ± 0.6 (HPAF-II and BxPC-3, respectively).

Conclusions: Successful imaging with (64)Cu-DOTA-CysDb in animal models suggests further investigation of ALCAM as an imaging biomarker is warranted.

Conflict of interest statement

Conflict of Interest

Anna M. Wu owns stock and is a consultant to ImaginAb, Inc. The other authors declare they have no conflicts of interest.

Figures

Figure 1
Figure 1
a, Determination of ALCAM expression by flow cytometry. Left, HPAF-II; middle, BxPC-3; right, C6. Filled peak, cells only. Solid line, cells incubated with mouse anti-human CD166 monoclonal antibody, followed by FITC-conjugated goat anti-mouse antibody. Dashed line, cells incubated with only FITC-conjugated goat anti-mouse antibody. b, Anti-ALCAM CysDb gene construct showing restriction sites for pEE12 cloning, mammalian leader sequence for protein secretion, 8-aa linker, C-terminal cysteine, and hexahistidine tag. c, Schematic representations of (from left to right) an intact antibody, scFv, unreduced CysDb, and reduced CysDb.
Figure 2
Figure 2
Biochemical characterization of anti-ALCAM CysDb. a, SDS-PAGE analysis of anti-ALCAM CysDb and, for comparison, anti-HER2 CysDb (1, anti-HER2 CysDb; 2, anti-ALCAM CysDb; M, marker; 3, reduced anti-HER2 CysDb; 4, reduced anti-ALCAM CysDb). b, Size exclusion chromatography elution profile of anti-ALCAM CysDb on Superdex 75.
Figure 3
Figure 3
Functional characterization of anti-ALCAM CysDb. a, Evaluation of binding specificity by flow cytometry. Left, HPAF-II; middle, BxPC-3; right, C6. Filled peak, cells only. Solid line, cells incubated with anti-ALCAM CysDb, followed by mouse anti-Penta-His antibody and PE-conjugated goat anti-mouse antibody. Dashed line, cells incubated with only mouse anti-Penta-His antibody and PE-conjugated goat anti-mouse antibody. b, Evaluation of binding to cultured cells by immunofluorescence. Top panels, HPAF-II; middle panels, BxPC-3; bottom panels, C6. Left column, DAPI staining. Middle column, Alexa Fluor 647-anti-ALCAM CysDb staining. Right column, overlay. c, Determination of KD by flow cytometry.
Figure 4
Figure 4
a, MicroPET images of female nude mice bearing HPAF-II (left two panels) or BxPC-3 (right two panels) subcutaneous xenografts in the left shoulder region, and C6 subcutaneous xenografts in the right shoulder region at 4 h post-injection of 64Cu-DOTA-anti-ALCAM CysDb. Upper and lower panels are coronal and transverse slices, respectively. L, liver; K, kidney. b, Ex vivo immunohistochemistry of tumors. Sections of xenografts stained for ALCAM (left, HPAF-II; middle, BxPC-3; right, C6).

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