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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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Molecular Imaging and Contrast Agent Database (MICAD) [Internet].

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64Cu-DOTA hu4D5v8 (scFv-CH2-CH3)2

64Cu-DOTA hu4D5v8 scFv-Fc DM
, PhD
National Center for Biotechnology Information, NLM, NIH, Bethesda, MD, vog.hin.mln.ibcn@dacim

Created: ; Last Update: April 3, 2008.

Chemical name:64Cu-DOTA hu4D5v8 (scFv-CH2-CH3)2
Abbreviated name:64Cu-DOTA hu4D5v 8 scFv-Fc DM
Synonym:64Cu-Trastuzumab single-chain Fv-Fc double-mutant antibody fragment
Agent Category:Antibody
Target:Human epidermal growth factor receptor 2 (HER2)
Target Category:Antibody to antigen binding
Method of detection:Positron Emission Tomography (PET)
Source of signal/contrast:64Cu
Activation:No
Studies:
  • Checkbox In vitro
  • Checkbox Rodents
Click on protein, nucleotide (RefSeq), and gene for more information about HER2.

Background

[PubMed]

64Cu-Trastuzumab single-chain Fv-Fc double-mutant antibody fragment (64Cu-DOTA hu4D5v8 scFv-Fc DM), which is formed by the conjugation of 111In with an engineered recombinant humanized monoclonal antibody (MAb) fragment against the antigen of human epidermal growth factor receptor 2 (HER2), has been developed for imaging of human breast cancer (1, 2).

The epidermal growth factor receptors (HER/erbB) with tyrosine kinase activity are involved in transmission of signals controlling normal cell development, growth, differentiation, and survival (2-4). This cell-surface receptor family consists of four distinct members, the epidermal growth factor receptors HER1, HER2, HER3, and HER4. These receptors are found in various combinations in different tissues. There are nine ligands that bind directly to HER1, HER3, or HER4. HER2 can be activated as a result of ligand binding to other HER receptors. The ligandless HER2 receptor is encoded by the human gene HER2/c-erbB2 (HER2/neu). It has been found to be overexpressed in a wide variety of human cancers and in 20-30% of primary breast cancers (5). This overexpression is associated with aggressive tumor growth and metastatic activity. In breast cancer, HER2/neu gene amplification can result in HER2 protein levels in tumor cells that are 10- to 100-fold higher than in normal breast epithelium. HER2 overexpression is considered a negative prognostic indicator for breast cancer.

Radiolabeled MAbs have been developed for both the diagnosis and treatment of tumors (6-8). Anti-HER2 MAbs have been generated for inhibiting the growth of tumor cells that possess activated HER2/neu receptors (9). Murine 4D5 MAb was raised against human breast and ovarian tumor cell lines overexpressing p185HER2. This MAb was found to recognize an extracellular epitope (amino acids 529-627) in the cysteine-rich II domain, which resides very close to the transmembrane region (9). Murine 4D5 MAb was fully humanized by Carter et al. (10) to generate the humAb4D5-8 MAb in an effort to improve the safety and tolerance of the antibody for clinical applications. Trastuzumab is of the IgG1 kappa class and binds to the recombinant HER2 extracellular domain with an affinity constant (Kd) of 0.1 nM. Cardiotoxicity is the most serious complication of trastuzumab use in humans (5, 11). One potential application of a radiolabeled anti-HER2 MAb is the pretreatment imaging of breast cancer patients to predict the cardiotoxicity and therapeutic efficacy of unlabeled trastuzumab.

Molecular imaging with radiolabeled intact antibody is hampered by the relatively slow clearance of the large intact antibody (150 kDa), which contributes to a high background signal (12-14). The blood clearance of antibodies is controlled by the antibody molecular size and the Fc portion (CH2-CH3 region). This problem can be minimized by the use of smaller antibody fragments that are cleared more rapidly through the kidneys. Enzymatically produced fragments such as Fab (55 kDa) and bioengineered fragments (recombinant domain-deleted antibodies) such as single-chain Fv (scFv; 25 kDa) or various minibodies (scFv-Fc DM; 105 kDa) have been shown to be effective in producing higher tumor/blood ratios. The scFv-Fc DM ((scFv-CH2-CH3)2) minibody appears to have certain mutations in the Fc region that mediate Fc receptor interactions and modulate the clearance kinetics of the antibody fragment (12, 13). Various anti-HER2 MAbs have been labeled with 131I, 125I, 111In, 86Y, 90Y, 64Cu, 76Br, or Gd for potential imaging and therapeutic applications (1, 12, 15-20). Labeling with radiometals is generally achieved by indirect labeling with use of a bifunctional chelating agent (21, 22). 64Cu has a half-life (t½) of 12.7 h and decays 17.9% by positron emission, 37.1% by beta emission, and 45% by electron capture (23). These physical properties allow 64Cu to be used for positron emission tomography (PET) and therapy applications.

Synthesis

[PubMed]

Assembly and production of the minibody was similar to the procedure used by Olafsen et al. (24) to prepare the scFv-CH3 dimer. The V genes were isolated and identified from the 10H8.C6.F12 hybridoma cell line. Splice overlap extension PCR was used to create fully synthetic variable genes. Full-length hu4D5v8 VL and VH chain genes were assembled into a scFv with an 18-amino-acid GlySer-rich linker (12). The scFv was then fused to the mammalian expression vector pEE12 to produce the scFv-Fc DM fragment. The anti-p185HER2 scFv-FC DM construct was expressed in NSO murine myeloma cells. The protein was selected in glutamine-deficient medium, screened by ELISA, analyzed by Western blot for size, and purified by dialysis and chromatography. The yield after purification was 27.3 mg/liter.

The purified protein was conjugated to the commercially available 1,4,7,10-tetrazacyclododecane-N,N´,N´´,N´´´-tetraacetic acid (DOTA) by use of the water-soluble N-hydroxysuccinimide (sulfo-NHS) method (25). In this method, N-hydroxysulfosuccinimidyl DOTA (DOTA-OSSu) was prepared by reacting sulfo-NHS and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide with DOTA at 4 °C for 30 min. The fragment hu4D5v8 scFv-Fc DM was then reacted with DOTA-OSSu in 0.1 M Na2HPO4 (pH 7.5) at 4 °C for 24 h with continuous end-over-end mixing. The conjugate was purified by dialysis. The 64Cu labeling was achieved by incubating 290-440 μg of DOTA-hu4D5v8 scFv-Fc DM conjugate with 25.9-111 MBq (0.7-3 mCi) of 64CuCl2 in 0.1 M ammonium citrate (pH 5.5) for 50 min at 43 °C (12). The reaction was stopped by addition of diethylenetriaminepentaacetic acid to 1 mM 64Cu-DOTA-hu4D5v8 scFv-Fc. DM minibody was purified by size-exclusion high-performance chromatography. Immunoreactivity was determined by the cell binding assay with use of the human breast tumor cell line MCF7/HER2. In two radiolabeling experiments, the labeling efficiencies were 100% and 77% with immunoreactivities of 58% and 52%, respectively.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Using competitive binding assay with MCF7/HER2 cells, Olafsen et al. (12) determined that the relative KD of hu4D5v8 scFv-Fc DM was 6.7 nM. In comparison, the KD for intact trastuzumab was estimated to be 2 nM. Immunochemical staining was done in frozen sections of MCF7/HER2 tumors and kidneys from normal nude mouse. The staining patterns of the cell membrane by hu4D5v8 scFv-Fc DM and intact trastuzumab were indistinguishable.

Animal Studies

Rodents

[PubMed]

Olafsen et al. (12) performed in vivo PET imaging of 64Cu-hu4D5v8 scFv-Fc DM in nude mice with s.c. implanted human breast tumor (positive HER2-expressing MCF/HER2; 1,398 ± 4 × 103 receptors/cell (26)) and human breast cancer (low HER2-expressing MD-MBA-231; 115 ± 0.1 × 103 receptors/cell (26)). Each mouse received 4.74-5.18 MBq (128-140 μCi) of 64Cu-hu4D5v8 scFv-Fc DM with a specific activity of 0.37 MBq (1.8 μCi)/μg (38.85 GBq (1.05 Ci)/μmol based on 105 kDa of hu4D5v8 scFv-Fc DM). A whole-body microPET scan was done at 3-4 and 18-21 h after injection. The values for percentage of injected dose (% ID)/g were determined from the images with use of the regions of interest (ROI) technique. After scanning, the animals were sacrificed at 21 h, and tissues were excised for in vitro assay. Imaging showed radioactivity localizations of 64Cu-hu4D5v8 scFv-Fc DM in both MCF/HER2 and MD-MBA-231 tumors at 4 and 21 h. For the MCF/HER2 tumors, the values for the ROI (n = 4) were estimated to be 9.0 ± 2.7 and 11.8 ± 1.0% ID/g (tumor/background ratio = 4.5:1) at 4 and 21 h, respectively. The in vitro assay at 21 h was 12.2 ± 2.4% ID/g. For the MD-MBA-231 tumors, the values for the ROI were 7.7 ± 0.8 and 8.8 ± 1.9% ID/g (tumor/background ratio = 3.4:1) at 4 and 21 h, respectively. The in vitro assay at 21 h was 7.5 ± 0.7% ID/g. The liver and kidneys were the major organs that showed radioactivity uptake. The authors of the study stated that compared with other bioengineered fragments, 64Cu-hu4D5v8 scFv-Fc DM showed improved tumor targeting and reduced kidney uptake.

Other Non-Primate Mammals

[PubMed]

No publication is currently available.

Non-Human Primates

[PubMed]

No publication is currently available.

Human Studies

[PubMed]

No publication is currently available.

NIH Support

NIH Intramural Support. NIH grants CA 43904, CA 48780, CA 86306.

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