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64Cu-Labeled, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-chelated, and cysteine-modified anti-activated leukocyte cell adhesion molecule diabody

64Cu-DOTA-CysDb
, PhD
National Center for Biotechnology Information, NLM, NIH

Created: ; Last Update: November 28, 2012.

Chemical name:64Cu-Labeled, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-chelated, and cysteine-modified anti-activated leukocyte cell adhesion molecule diabody
Abbreviated name:64Cu-DOTA-CysDb
Synonym:
Agent Category:Antibodies (diabody)
Target:Activated leukocyte cell adhesion molecule (ALCAM/CD166)
Target Category:Adhesion molecules
Method of detection:Positron emission tomography
Source of signal / contrast:64Cu
Activation:No
Studies:
  • Checkbox In vitro
  • Checkbox Rodents
No structure available.

Background

[PubMed]

The 64Cu-labeled, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-chelated, and cysteine-modified anti-activated leukocyte cell adhesion molecule (ALCAM) diabody (CysDb), abbreviated as 64Cu-DOTA-CysDb, was synthesized by McCabe et al. for use with positron emission tomography (PET) of tumors expressing ALCAM (1).

Antibodies have long been considered to be the most attractive agents for molecular imaging because of their high specificity and binding affinity (2, 3). However, the usefulness of full antibodies as imaging agents is limited by their long circulation time in blood (several days to weeks), which requires a long time to optimally accumulate in tumors (1–2 days) (2). Because of their large molecular size (150 kDa), antibodies also exhibit poor tumor-penetrating ability, which leads to poor signal/noise ratio. To improve antibody pharmacokinetics without compromising the affinity and specificity, a promising solution is to reduce the antibody size or alter the Fc receptor-binding domain with protein engineering (2, 3). Indeed, various antibody fragments have been generated, such as single-chain variable fragment (scFv; 25–30 kDa), Fab (~50 kDa) and F(ab')2 (~110 kDa) fragments, bivalent scFv (tandem scFv and diabody, 50–60 kDa), and scFv-fusion proteins (80 kDa for minibody and 105 kDa for scFv-Fc) (2, 4). These fragments exhibit good tumor penetration, fast clearance kinetics, high affinity, and high tumor/blood ratio, which are desirable for imaging agents. However, several issues with the use of antibody fragments as imaging agents remain to be solved, one of which is the diverse effects of labeling chemistry on the binding affinity of antibody fragments (5, 6).

To reduce the diverse effects of labeling chemistry, various strategies have been designed. For example, Vaidyanathan and Zalutsky prepared 18F-labeled antibody fragments using N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB), which reacts with the ε-amino group of surface-exposed lysine residues on proteins (7). Labeling with this approach showed no loss of the antibody fragment affinity. Olafsen et al. further improved efficiency and speed of [18F]SFB production by adapting the synthesis to a three-step, one-pot, microwave-assisted method, followed by purification using either a single cartridge or high-performance liquid chromatography (HPLC) (7, 8). Alternatively, McCabe et al. first conjugated the diabody to DOTA, followed by 64Cu-radiolabeling through DOTA (1). All these strategies have been reported to be efficient and have fewer diverse effects on the binding affinity of antibody fragments. This chapter summarizes the data obtained with 64Cu-DOTA-CysDb. In another chapter, the data obtained with [18F]FB-Cys-Db are summarized.

Synthesis

[PubMed]

The anti-ALCAM CysDb construct was developed via polymerase chain reaction (PCR) amplification of the VH and VL sequences of the anti-ALCAM antibody, followed by cloning of the PCR fragments into the mammalian expression vector pEE12 with insertion of a GlySer-rich, eight amino acid linker between the two single-chain variable fragments (scFv) and with attachment of a cysteine and hexahistidine tag at the C-terminus (1). The expression vector was then transfected into NSo mouse myeloma cells for production of the CysDb. The anti-ALCAM CysDb was purified from NSo terminal cultures with a yield of ~10 mg/L supernatant. SDS-PAGE analysis showed that, under non-reducing conditions, the anti-ALCAM CysDb migration was consistent with its predicted dimeric molecular weight of ~50 kDa, and under reducing conditions the migration was consistent with its predicted monomeric molecular weight. The lack of additional bands on the SDS-PAGE gel and the single peak on the chromatogram indicated high purity of the final products. Size-exclusion chromatography confirmed production of the scFv dimer.

For radiolabeling, the anti-ALCAM CysDb was first conjugated with DOTA by incubating the diabody with DOTA at a molar ratio of 1:200 for 12–16 h at 4°C (1). Size-exclusion chromatography showed that the DOTA-conjugated CysDb (DOTA-CysDb) eluted slightly earlier (22.4 min) than the unconjugated CysDb (23.8 min), and the large peak area corresponding to the conjugate indicated high conjugation efficiency (the ratio of DOTA to CysDb in the final product was not reported). 64Cu-Labeling was achieved by incubating 14.8–18.5 MBq (0.4–0.5 mCi) 64Cu with 200–300 μg DOTA-CysDb for 50 min at 43°C. The 64Cu-labeling efficiency was 82 ± 11% (n = 4). The radiochemical purity and specific activity of 64Cu-DOTA-CysDb were not reported.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

McCabe et al. analyzed the ALCAM expression in two human pancreatic adenocarcinoma cell lines HPAF-II and BxPC-3, and one rat glioma cell line C6 using the mouse anti-human ALCAM monoclonal antibody and flow cytometry (1). The results showed that HPAF-II and BxPC-3 were positive for cell surface ALCAM, while C6 was negative. The specific antibody-binding capacity (ABC) for HPAF-II and BxPC-3 cells was between 2.5 × 105 and 3.0 × 105, respectively (n = 2, detailed data not shown). ABC is a term that provides quantitative information about the number of antibodies binding to the surface molecules on individual cells.

The specific binding of CysDb with ALCAM-positive HPAF-II and BxPC-3 cells was also analyzed with flow cytometry. The affinity, expressed as the equilibrium binding constant, was determined to be in the range of 1–3 nM. The nonspecific binding was minimal. Under fluorescent microscopy and with flow cytometry, the Alexa Fluor 647–labeled CysDb retained the immunoreactivity of the native CysDb and bound specifically to ALCAM-positive cells. The binding affinity of 64Cu-DOTA-CysDb was not reported.

Animal Studies

Rodents

[PubMed]

MicroPET was performed at 4 h and 21 h, respectively, after tail vein injection of 3.7–6.1 MBq (100–165 μCi, 85–95 Ci/mmol) 64Cu-DOTA-CysDb in each nude mouse that had an ALCAM-positive (HPAF-II or BxPC-3) and an ALCAM-negative (C6) subcutaneous tumor (n = 4–5 mice/group) (1). The ALCAM-positive but not the ALCAM-negative tumors were clearly detected at 4 h after injection. Very high signal was observed in the kidneys and liver. Ex vivo gamma counting of tumors and organs harvested at 21 h after injection confirmed specific targeting of the probe, with positive tumor uptakes of 1.8 ± 0.5% and 2.5 ± 0.5% injected dose per gram tissue (ID/g) for HPAF-II and BxPC-3, respectively (P = 0.08). The C6-negative tumor uptake (1.0 ± 0.1% ID/g) was comparable to that seen in the blood (0.7 ± 0.1% ID/g for HPAF-II tumor-bearing mice (P < 0.01) and 0.9 ± 0.1% ID/g for BxPC-3 tumor-bearing mice (P > 0.1)) (Table 1). Uptake differences between ALCAM-positive and ALCAM-negative tumors were statistically significant (P < 0.01 for both HPAF-II versus C6 and BxPC-3 versus C6). The positive/negative tumor uptake ratios were 1.9 ± 0.6 and 2.4± 0.6 for HPAF-II/C6 and BxPC-3/C6, respectively. The positive tumor/blood radioactivity ratios were 2.5 ± 0.9 and 2.9 ± 0.6 for HPAF-II/blood and BxPC-3/blood, respectively. Consistent biodistribution data were obtained in non-tumor-bearing Alcam/− mice (n = 2) at 4 h after injection of 64Cu-DOTA-CysDb (Table 1), suggesting that accumulation of the probe in the liver and kidneys is not due to ALCAM expression.

Table 1. Biodistribution of 64Cu-DOTA-CysDb in mice bearing HPAF-II or BxPC-3 and C6 tumors and in Alcam-knockout mice

TissuePercent of ID/g (mean ± SD)
HPAF-II (n = 5)BxPC-3 (n = 4)Alcam-knockout (n = 2)
21 h21 h4 h
Liver6.6 ± 0.56.8 ± 1.45.4 ± 0.1
Spleen1.7 ± 0.21.8 ± 0.32.2 ± 0.7
Kidneys42 ± 5.040 ± 0.890 ± 3.5
Lung1.8 ± 0.52.3 ± 0.32.1 ± 0.3
Blood0.7 ± 0.10.9 ± 0.11.0 ± 0.3
Positive tumor (T)1.8 ± 0.52.5 ± 0.5
Negative tumor (C6)1.0 ± 0.11.0 ± 0.1
T/blood2.5 ± 0.92.9 ± 0.6
T/C61.9 ± 0.62.4 ± 0.6

The radioactive uptake was also measured directly from the PET images acquired at 4 h and 21 h (1). Images were normalized to the activity of the injected dose and animal body weight. Total image units were converted into total activity using a conversion factor determined from a 64Cu phantom, and the activity was decay-corrected back to the time of dose calibration and injection to calculate the % ID/g for each region of interest. The uptake values in positive tumors at 21 h after injection were determined to be 1.6 ± 0.2% ID/g and 1.7 ± 0.4% ID/g for HPAF-II and BxPC-3, respectively. These values were lower than the corresponding ex vivo biodistribution values (P < 0.05), but the disparity was consistent. The uptake values at 4 h were slightly lower (P < 0.01), with 1.5 ± 0.2% ID/g for HPAF-II and 1.4 ± 0.5% ID/g for BxPC-3, but the tumor/background ratios (4.6 ± 1.1 and 5.1 ± 1.7 for HPAF-II at 4 h and 21 h, respectively; 5.4 ± 3.3 and 4.7 ± 3.2 for BxPC-3 at 4 h and 21 h, respectively) were not significantly different at the two time points (P > 0.5 for both HPAF-II and BxPC-3).

Immunohistochemistry of the harvested tumors confirmed that the HPAF-II and BxPC-3 tumors were positive for ALCAM, while the C6 tumors were negative, consistent with the results determined for each cell line in vitro (1).

Other Non-Primate Mammals

[PubMed]

No references are currently available.

Non-Human Primates

[PubMed]

No references are currently available.

Human Studies

[PubMed]

No references are currently available.

References

1.
McCabe K.E., Liu B., Marks J.D., Tomlinson J.S., Wu H., Wu A.M. An engineered cysteine-modified diabody for imaging activated leukocyte cell adhesion molecule (ALCAM)-positive tumors. Mol Imaging Biol. 2012;14(3):336–47. [PMC free article: PMC3227780] [PubMed: 21630083]
2.
Olafsen T. andWu, A.M. Antibody vectors for imaging. Semin Nucl Med. 2010;40(3):167–81. [PMC free article: PMC2853948] [PubMed: 20350626]
3.
Kaur S., Venktaraman G., Jain M., Senapati S., Garg P.K., Batra S.K. Recent trends in antibody-based oncologic imaging. Cancer Lett. 2012;315(2):97–111. [PMC free article: PMC3249014] [PubMed: 22104729]
4.
Girgis M.D., Kenanova V., Olafsen T., McCabe K.E., Wu A.M., Tomlinson J.S. Anti-CA19-9 diabody as a PET imaging probe for pancreas cancer. J Surg Res. 2011;170(2):169–78. [PubMed: 21601881]
5.
Sirk S.J., Olafsen T., Barat B., Bauer K.B., Wu A.M. Site-specific, thiol-mediated conjugation of fluorescent probes to cysteine-modified diabodies targeting CD20 or HER2. Bioconjug Chem. 2008;19(12):2527–34. [PMC free article: PMC2668938] [PubMed: 19053310]
6.
Shen B.Q., Xu K., Liu L., Raab H., Bhakta S., Kenrick M., Parsons-Reponte K.L., Tien J., Yu S.F., Mai E., Li D., Tibbitts J., Baudys J., Saad O.M., Scales S.J., McDonald P.J., Hass P.E., Eigenbrot C., Nguyen T., Solis W.A., Fuji R.N., Flagella K.M., Patel D., Spencer S.D., Khawli L.A., Ebens A., Wong W.L., Vandlen R., Kaur S., Sliwkowski M.X., Scheller R.H., Polakis P., Junutula J.R. Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates. Nat Biotechnol. 2012;30(2):184–9. [PubMed: 22267010]
7.
Vaidyanathan G. andZalutsky, M.R. Labeling proteins with fluorine-18 using N-succinimidyl 4-[18F]fluorobenzoate. Int J Rad Appl Instrum B. 1992;19(3):275–81. [PubMed: 1629016]
8.
Olafsen T., Sirk S.J., Olma S., Shen C.K., Wu A.M. ImmunoPET using engineered antibody fragments: fluorine-18 labeled diabodies for same-day imaging. Tumour Biol. 2012;33(3):669–77. [PubMed: 22392499]
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