Top

Molecular Imaging and Biology

Published in:

01-06-2012 | Research Article

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

Authors: Katelyn E. McCabe, Bin Liu, James D. Marks, James S. Tomlinson, Hong Wu, Anna M. Wu

Published in: Molecular Imaging and Biology | Issue 3/2012

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 ⁶⁴Cu-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 ⁶⁴Cu-DOTA-CysDb in animal models suggests further investigation of ALCAM as an imaging biomarker is warranted.

Wu AM (2009) Antibodies and antimatter: the resurgence of immuno-PET. J Nucl Med 50:2–5PubMedCrossRef

Wu AM, Olafsen T (2008) Antibodies for molecular imaging of cancer. Cancer J 14:191–197PubMedCrossRef

Wu AM, Senter PD (2005) Arming antibodies: prospects and challenges for immunoconjugates. Nat Biotechnol 23:1137–1146PubMedCrossRef

Cai W, Olafsen T, Zhang X et al (2007) PET imaging of colorectal cancer in xenograft-bearing mice by use of an 18F-labeled T84.66 anti-carcinoembryonic antigen diabody. J Nucl Med 48:304–310PubMedCrossRef

Olafsen T, Cheung CW, Yazaki PJ et al (2004) Covalent disulfide-linked anti-CEA diabody allows site-specific conjugation and radiolabeling for tumor targeting applications. Protein Eng Des Sel 17:21–27PubMedCrossRef

Sirk SJ, Olafsen T, Barat B, Bauer KB, Wu AM (2008) Site-specific, thiol-mediated conjugation of fluorescent probes to cysteine-modified diabodies targeting CD20 or HER2. Bioconjug Chem 19:2527–2534PubMedCrossRef

King JA, Ofori-Acquah SF, Stevens T, Al-Mehdi AB, Fodstad O, Jiang WG (2004) Activated leukocyte cell adhesion molecule in breast cancer: prognostic indicator. Breast Cancer Res 6:R478–R487PubMedCrossRef

Burkhardt M, Mayordomo E, Winzer KJ et al (2006) Cytoplasmic overexpression of ALCAM is prognostic of disease progression in breast cancer. J Clin Pathol 59:403–409PubMedCrossRef

Kulasingam V, Zheng Y, Soosaipillai A, Leon AE, Gion M, Diamandis EP (2009) Activated leukocyte cell adhesion molecule: a novel biomarker for breast cancer. Int J Cancer 125:9–14PubMedCrossRef

10.

Weichert W, Knosel T, Bellach J, Dietel M, Kristiansen G (2004) ALCAM/CD166 is overexpressed in colorectal carcinoma and correlates with shortened patient survival. J Clin Pathol 57:1160–1164PubMedCrossRef

11.

Sawhney M, Matta A, Macha MA et al (2009) Cytoplasmic accumulation of activated leukocyte cell adhesion molecule is a predictor of disease progression and reduced survival in oral cancer patients. Int J Cancer 124:2098–2105PubMedCrossRef

12.

Mezzanzanica D, Fabbi M, Bagnoli M et al (2008) Subcellular localization of activated leukocyte cell adhesion molecule is a molecular predictor of survival in ovarian carcinoma patients. Clin Cancer Res 14:1726–1733PubMedCrossRef

13.

Chen R, Yi EC, Donohoe S et al (2005) Pancreatic cancer proteome: the proteins that underlie invasion, metastasis, and immunologic escape. Gastroenterology 129:1187–1197PubMedCrossRef

14.

Kristiansen G, Pilarsky C, Wissmann C et al (2003) ALCAM/CD166 is up-regulated in low-grade prostate cancer and progressively lost in high-grade lesions. Prostate 54:34–43PubMedCrossRef

15.

Kristiansen G, Pilarsky C, Wissmann C et al (2005) Expression profiling of microdissected matched prostate cancer samples reveals CD166/MEMD and CD24 as new prognostic markers for patient survival. J Pathol 205:359–376PubMedCrossRef

16.

Patel DD, Wee SF, Whichard LP et al (1995) Identification and characterization of a 100-kD ligand for CD6 on human thymic epithelial cells. J Exp Med 181:1563–1568PubMedCrossRef

17.

Bowen MA, Patel DD, Li X et al (1995) Cloning, mapping, and characterization of activated leukocyte-cell adhesion molecule (ALCAM), a CD6 ligand. J Exp Med 181:2213–2220PubMedCrossRef

18.

van Kempen LC, Meier F, Egeblad M et al (2004) Truncation of activated leukocyte cell adhesion molecule: a gateway to melanoma metastasis. J Invest Dermatol 122:1293–1301PubMedCrossRef

19.

van Kilsdonk JW, Wilting RH, Bergers M et al (2008) Attenuation of melanoma invasion by a secreted variant of activated leukocyte cell adhesion molecule. Cancer Res 68:3671–3679PubMedCrossRef

20.

Liu B, Conrad F, Cooperberg MR, Kirpotin DB, Marks JD (2004) Mapping tumor epitope space by direct selection of single-chain Fv antibody libraries on prostate cancer cells. Cancer Res 64:704–710PubMedCrossRef

21.

Liu B, Conrad F, Roth A, Drummond DC, Simko JP, Marks JD (2007) Recombinant full-length human IgG1s targeting hormone-refractory prostate cancer. J Mol Med 85:1113–1123PubMedCrossRef

22.

Roth A, Drummond DC, Conrad F et al (2007) Anti-CD166 single chain antibody-mediated intracellular delivery of liposomal drugs to prostate cancer cells. Mol Cancer Ther 6:2737–2746PubMedCrossRef

23.

Galfre G, Milstein C (1981) Preparation of monoclonal antibodies: strategies and procedures. Methods Enzymol 73:3–46PubMedCrossRef

24.

Olafsen T, Gu Z, Sherman MA et al (2007) Targeting, imaging, and therapy using a humanized antiprostate stem cell antigen (PSCA) antibody. J Immunother 30:396–405PubMedCrossRef

25.

Bebbington CR, Renner G, Thomson S, King D, Abrams D, Yarranton GT (1992) High-level expression of a recombinant antibody from myeloma cells using a glutamine synthetase gene as an amplifiable selectable marker. Biotechnology (NY) 10:169–175CrossRef

26.

Olafsen T, Kenanova VE, Sundaresan G et al (2005) Optimizing radiolabeled engineered anti-p185HER2 antibody fragments for in vivo imaging. Cancer Res 65:5907–5916PubMedCrossRef

27.

Loening AM, Gambhir SS (2003) AMIDE: a free software tool for multimodality medical image analysis. Mol Imaging 2:131–137PubMedCrossRef

28.

Nelson AL, Dhimolea E, Reichert JM (2010) Development trends for human monoclonal antibody therapeutics. Nat Rev Drug Discov 9:767–774PubMedCrossRef

29.

Leyton JV, Olafsen T, Sherman MA et al (2009) Engineered humanized diabodies for microPET imaging of prostate stem cell antigen-expressing tumors. Protein Eng Des Sel 22:209–216PubMedCrossRef

30.

Schmidt MM, Wittrup KD (2009) A modeling analysis of the effects of molecular size and binding affinity on tumor targeting. Mol Cancer Ther 8:2861–2871PubMedCrossRef

31.

Eder M, Knackmuss S, Le Gall F et al (2010) (68)Ga-labelled recombinant antibody variants for immuno-PET imaging of solid tumours. Eur J Nucl Med Mol Imaging 37:1397–1407PubMedCrossRef

32.

Schneider DW, Heitner T, Alicke B et al (2009) In vivo biodistribution, PET imaging, and tumor accumulation of 86Y- and 111In-antimindin/RG-1, engineered antibody fragments in LNCaP tumor-bearing nude mice. J Nucl Med 50:435–443PubMedCrossRef

33.

Anderson CJ, Ferdani R (2009) Copper-64 radiopharmaceuticals for PET imaging of cancer: advances in preclinical and clinical research. Cancer Biother Radiopharm 24:379–393PubMedCrossRef

34.

Shokeen M, Anderson CJ (2009) Molecular imaging of cancer with copper-64 radiopharmaceuticals and positron emission tomography (PET). Acc Chem Res 42:832–841PubMedCrossRef

35.

Yazaki PJ, Wu AM, Tsai SW et al (2001) Tumor targeting of radiometal labeled anti-CEA recombinant T84.66 diabody and t84.66 minibody: comparison to radioiodinated fragments. Bioconjug Chem 12:220–228PubMedCrossRef

36.

Ahlgren S, Wallberg H, Tran TA et al (2009) Targeting of HER2-expressing tumors with a site-specifically 99mTc-labeled recombinant affibody molecule, ZHER2:2395, with C-terminally engineered cysteine. J Nucl Med 50:781–789PubMedCrossRef

37.

Arano Y (1998) Strategies to reduce renal radioactivity levels of antibody fragments. Q J Nucl Med 42:262–270PubMed

38.

Piazza T, Cha E, Bongarzone I et al (2005) Internalization and recycling of ALCAM/CD166 detected by a fully human single-chain recombinant antibody. J Cell Sci 118:1515–1525PubMedCrossRef

39.

Liu K, Lepin EJ, Wang M-W, Guo F, Lin W-Y, Chen Y-C, Sirk SJ, Olma S, Phelps ME, Zhao X-Z, Tseng H-R, van Dam RM, Wu AM, Shen CK-F (2011) Microfluidic-based ¹⁸F labeling of biomolecules for immuno-positron emission tomography. Molecular Imaging 10:168–176. doi:10.2310/7290.2010.00043 PubMed

40.

Horner MJ RL, Krapcho M, Neyman N, Aminou R, Howlader N, Altekruse SF, Feuer EJ, Huang L, Mariotto A, Miller BA, Lewis DR, Eisner MP, Stinchcomb DG, Edwards BK (eds) SEER Cancer Statistics Review, 1975–2006, National Cancer Institute. Bethesda, MD, based on November 2008 SEER data submission, posted to the SEER web site, 2009. Available at http://seer.cancer.gov/csr/1975_2006/

41.

Parsons CM, Sutcliffe JL, Bold RJ (2008) Preoperative evaluation of pancreatic adenocarcinoma. J Hepatobiliary Pancreat Surg 15:429–435PubMedCrossRef

42.

Foss CA, Fox JJ, Feldmann G et al (2007) Radiolabeled anti-claudin 4 and anti-prostate stem cell antigen: initial imaging in experimental models of pancreatic cancer. Mol Imaging 6:131–139PubMed

43.

Hausner SH, Abbey CK, Bold RJ et al (2009) Targeted in vivo imaging of integrin alphavbeta6 with an improved radiotracer and its relevance in a pancreatic tumor model. Cancer Res 69:5843–5850PubMedCrossRef

44.

Strickland LA, Ross J, Williams S et al (2009) Preclinical evaluation of carcinoembryonic cell adhesion molecule (CEACAM) 6 as potential therapy target for pancreatic adenocarcinoma. J Pathol 218:380–390PubMedCrossRef

45.

Vervoort L, Burvenich I, Staelens S et al (2010) Preclinical evaluation of monoclonal antibody 14C5 for targeting pancreatic cancer. Cancer Biother Radiopharm 25:193–205PubMedCrossRef

46.

Mariani G, Molea N, Bacciardi D et al (1995) Initial tumor targeting, biodistribution, and pharmacokinetic evaluation of the monoclonal antibody PAM4 in patients with pancreatic cancer. Cancer Res 55:5911s–5915sPubMed

47.

Kahlert C, Weber H, Mogler C et al (2009) Increased expression of ALCAM/CD166 in pancreatic cancer is an independent prognostic marker for poor survival and early tumour relapse. Br J Cancer 101:457–464PubMedCrossRef

48.

Wiiger MT, Gehrken HB, Fodstad O, Maelandsmo GM, Andersson Y (2010) A novel human recombinant single-chain antibody targeting CD166/ALCAM inhibits cancer cell invasion in vitro and in vivo tumour growth. Cancer Immunol Immunother 59:1665–1674PubMedCrossRef

Title: An Engineered Cysteine-Modified Diabody for Imaging Activated Leukocyte Cell Adhesion Molecule (ALCAM)-Positive Tumors
Authors: Katelyn E. McCabe
Bin Liu
James D. Marks
James S. Tomlinson
Hong Wu
Anna M. Wu
Publication date: 01-06-2012
Publisher: Springer-Verlag
Published in: Molecular Imaging and Biology / Issue 3/2012
Print ISSN: 1536-1632
Electronic ISSN: 1860-2002
DOI: https://doi.org/10.1007/s11307-011-0500-8

At a glance: The STEP trials

Springer Medicine

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

Abstract

Purpose

Procedures

Results

Conclusions

At a glance: The STEP trials

Springer Medicine

Abstract

Purpose

Procedures

Results

Conclusions

Please log in to get access to this content

Other articles of this Issue 3/2012

Correlation of the Ga-68-Bombesin Analog Ga-68-BZH3 with Receptors Expression in Gliomas as Measured by Quantitative Dynamic Positron Emission Tomography (dPET) and Gene Arrays

Spectral Unmixing Imaging of Wavelength-Responsive Fluorescent Probes: An Application for the Real-Time Report of Amyloid Beta Species in Alzheimer’s Disease

Dual-Labeling Strategies for Nuclear and Fluorescence Molecular Imaging: A Review and Analysis

An Evaluation of 2-deoxy-2-[18F]Fluoro-D-Glucose and 3′-deoxy-3′-[18F]-Fluorothymidine Uptake in Human Tumor Xenograft Models

Multispectral Optoacoustic Tomography of Matrix Metalloproteinase Activity in Vulnerable Human Carotid Plaques

Microvessel Density But Not Neoangiogenesis Is Associated with 18F-FDG Uptake in Human Atherosclerotic Carotid Plaques