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Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2013.

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64Cu-1,4,7,10-Tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid-polyethylene glycol 12 anti–tumor-associated glycoprotein 72 diabody

64Cu-PEG12 AVP04-07
, PhD
National Center for Biotechnology Information, NLM, NIH
Corresponding author.

Created: ; Last Update: September 1, 2010.

Chemical name:64Cu-1,4,7,10-Tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid-polyethylene glycol 12 anti–tumor-associated glycoprotein 72 diabody
Abbreviated name:64Cu-PEG12 AVP04-07
Agent Category:Antibodies (diabodies)
Target:Tumor-associated glycoprotein 72 (TAG-72)
Target Category:Antigens
Method of detection:Positron emission tomography (PET)
Source of signal / contrast:64Cu
  • Checkbox In vitro
  • Checkbox Rodents
No structure is available.



The 64Cu-labeled 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA)-polyethylene glycol 12 (PEG12) anti–tumor-associated glycoprotein 72 (TAG-72) diabody conjugate, abbreviated as 64Cu-PEG12 AVP04-07, is a positron emission tomography (PET) tracer that has been developed by Li et al. for imaging tumors by targeting overexpressed TAG-72 antigen (1). Conjugation of monodispersed PEG to antibody fragments has been shown to be an effective approach to reduce kidney retention and increase the tumor uptake of antibody fragments (1). The half-life (t1/2) of 64Cu is 12.7 h.

Antibody fragments such as diabodies and minibodies are ideal for molecular imaging because of their compact size and efficient antigen binding (1-4). However, most radiometal-labeled antibody fragments exhibit high kidney retention, which leads to decreased absolute tumor uptake (5-7). In the case of 64Cu, which has greater tumor retention than radioiodine labels, the advantage is offset by prolonged kidney retention. Use of radioiodine labels results in decreased kidney uptake, but at the expense of lower tumor uptake (1, 8, 9). An approach to avoid the kidney retention issue is to increase the apparent molecular weight of antibody fragments through attachment of PEG (5). This approach has been successfully applied to the engineered antibody fragments against carcinoembryonic antigen (CEA), CD3, and others (5, 10). Increased tumor retention, longer serum t1/2, and decreased kidney uptake have been observed for PEGylated fragments compared with non-PEGylated forms (5, 11). Li et al. reported that attachment of DOTA and PEG3400 to anti-CEA diabody can significantly reduce kidney uptake of the diabody compared with kidney uptake of the parental diabody while preserving high tumor uptake (5, 12). They further generated a PEGylated (PEG3400) anti-TAG-72 diabody (AVP04-07-PEG3400), which reduced kidney retention to levels usually seen with intact immunoglobulin G. Kidney uptake appears to be a function of apparent molecular size (as modified by PEG) and other less defined factors intrinsic to a diabody (1). However, PEG3400 is less than ideal for clinical use because of its inherent polydispersity. Oversubstitution may also inhibit the immunoreactivity of the antibody fragments. In the case of anti-CEA diabody, Li et al. showed that it is necessary to limit the number of PEG3400 particles to approximately one per monomer (5). Li et al. therefore evaluated the effect of monodispersed PEG on the kidney retention and tumor uptake of anti-TAG-72 diabody (1). The investigators synthesized monodispersed PEG (DOTA-PEG12-Cys-VS, where Cys-VS is vinyl sulfone conjugated to the thiol of cysteine) and conjugated this to the amino groups on the anti-TAG-72 diabody (named as PEG12 AVP04-07). Biodistribution studies with 111In-labeled PEG12 AVP04-07 demonstrated low kidney uptake without reduction of the tumor uptake and tumor/blood ratio. Positron emission tomography (PET) imaging with 64Cu-PEG12 AVP04-07 showed high-contrast tumor images in animal models within 24 h after injection (1).



The parental AVP04-07 diabody was generated on the basis of the V-domains from the parent CC49 antibody and the derivative single-chain Fv (scFv) fragments (1, 13). The scFv fragments are orientated as VH–VL (where VH and VL are the variable domains of heavy and light chains, respectively) joined by a G4S linker and have a C-terminal His6 tail. The apparent molecular weight was 52.5 kDa for the AVP04-07 diabody. To construct PEG12 AVP04-07, monodispersed DOTA-PEG12-Cys-VS was first synthesized by coupling N-FMOC-amido-PEG12 acid to Cys-polystyrene Wang resin, followed by reaction with a 10-molar excess of VS. Conjugation of the DOTA-PEG12-Cys-VS to the surface lysines of the diabody was carried out at pH 9.0 at a molar ratio of 50:1 and 20:1, respectively.

Radiometal labeling was performed with 111InCl3 or 64CuCl2 as described in previous reports (1, 12). The radiolabeled materials were purified on Superdex-75 columns. The degree of substitution was estimated to be 1.7 PEGs per monomer for the 20:1 conjugate and 3.0 PEGs per monomer for the 50:1 conjugate. When the conjugates were labeled with 111In, the 20:1 conjugate gave 8.3% incorporation and the 50:1 conjugate gave 92% incorporation. The radiolabeled 50:1 conjugate was used for in vivo studies.

The 111In- and 64Cu-labeled DOTA-Cys-VS-PEG3400-AVP04-07 conjugates (referred to as 111In- and 64Cu-AVP04-07-PEG3400) and DOTA-Cys-VS-PEG27-AVP04-07 conjugates (referred to as 111In-PEG27 AVP04-07 and 64Cu-PEG27 AVP04-07; MICAD chapter on 64Cu-PEG27 AVP04-07) were synthesized similarly. The apparent molecular weight was 105 kDa for the PEG3400 derivatives, but the apparent molecular weight was not described in detail for the PEG12 and PEG27 conjugates. The radiolabeling yields were typically 70%–90% for the PEG conjugates.

In Vitro Studies: Testing in Cells and Tissues


Binding of AVP04-07 diabody to the TAG-72 antigen was determined with bovine submaxillary mucin in a column shift assay (Superdex 200) (1). The AVP04-07 diabody showed >99% immunoreactivity. No data were described for the immunoreactivity of 111In- and 64Cu-labeled conjugates.

Animal Studies



A biodistribution study was performed in athymic nude mice bearing LS-174T xenografts (1). In the case of 111In-AVP04-07-PEG3400, kidney uptake was 8.4% injected dose per gram tissue (ID/g) at 24 h after injection; uptake was 98% ID/g for the non-PEGylated form. The large reduction of kidney uptake with PEGylation was accompanied by an increase in tumor uptake from 22% ID/g (non-PEGylated) to 46% ID/g (PEGylated) at 24 h. The increased tumor retention is evidently due to the prolonged blood clearance of the PEGylated diabody (t1/2β = 36 h) versus the non-PEGylated diabody (t1/2β = 18 h). Biodistribution results for 111In-PEG12 AVP04-07 were nearly equivalent to those for the PEG3400 and PEG27 derivatives. Notably, kidney uptake was initially higher for the PEG12 conjugate (13.4% ID/g) than for the PEG27 conjugate, but fell to lower levels (6.1% ID/g) by 96 h, whereas tumor uptake and blood clearance were nearly identical for the PEG12 and the PEG27 conjugates.

PET imaging demonstrated modest tumor uptake (19.6% ID/g at 46 h) and high kidney uptake (37.4% ID/g at 46 h) for the 64Cu-labeled non-PEGylated AVP04-07 within 2 days after injection. In contrast, 64Cu-PEG12 AVP04-07 showed relatively little kidney uptake (6.7% ID/g at 46 h) over the same period and high tumor uptake (49.3% ID/g at 46 h) as early as 21–22 h after injection. The tumor/blood ratio reached 9:1 at 46 h after injection. The results with the 64Cu-labeled conjugate were similar to those obtained with the 111In-labeled conjugate. The best imaging predicted with imaging figure of merit was at 10 h for the 64Cu-labeled non-PEGylated diabody and at 20 h for the 64Cu-labeled PEG12 version. For the 111In-labeled PEG12 conjugate, imaging was optimal at 30–40 h.

In summary, the PEGylated diabody outperformed the non-PEGylated form in terms of both absolute tumor uptake and tumor/blood ratio for both 111In and 64Cu labels. The results obtained by Li et al. suggested that even PEGs of modest molecular size are efficient in reducing kidney uptake and that the choice of radiometal labels has little effect on the biodistribution. Monodispersed DOTA-PEGylated diabody conjugates are more suitable for imaging of human TAG-72–positive tumors (1).

Other Non-Primate Mammals


No references are currently available.

Non-Human Primates


No references are currently available.

Human Studies


No references are currently available.


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