OtherBASIC SCIENCE INVESTIGATIONS

Targeting of HER2-Expressing Tumors with a Site-Specifically 99mTc-Labeled Recombinant Affibody Molecule, ZHER2:2395, with C-Terminally Engineered Cysteine

Sara Ahlgren, Helena Wållberg, Thuy A. Tran, Charles Widström, Magnus Hjertman, Lars Abrahmsén, Dietmar Berndorff, Ludger M. Dinkelborg, John E. Cyr, Joachim Feldwisch, Anna Orlova and Vladimir Tolmachev
Journal of Nuclear Medicine May 2009, 50 (5) 781-789; DOI: https://doi.org/10.2967/jnumed.108.056929

Article Figures & Data

Figures

  • FIGURE 1. 
    FIGURE 1. 

    (A) In vitro specificity analysis. One group of culture dishes with SKOV-3 cells was pretreated with saturating amounts of nonlabeled His6-ZHER2:342 before incubation with 99mTc-ZHER2:2395-C. Cell-associated radioactivity was calculated as percentage of total added radioactivity. (B) Cell-associated radioactivity as function of time after interrupted incubation of SKOV-3 cells with 99mTc-ZHER2:2395-C. Cell-associated radioactivity at time zero after interrupted incubation was considered to be 100%. Radioactivity that was removed from cells by treatment with 4 M urea solution in 0.2 M glycine buffer (pH 2.0) was considered to be membrane bound, and remainder was considered to be internalized. Data are presented as mean ± SD (n = 3). Error bars may not be visible because they are smaller than the symbols.

  • FIGURE 2. 
    FIGURE 2. 

    Biodistributions of radiolabeled Affibody molecules 99mTc-H6-ZHER2:342-C, containing His tag, and 99mTc-ZHER2:2395-C, lacking His tag, in NMRI (normal) mice 4 h after injection. Data are expressed as %IA per whole sample for gastrointestinal tract and contents, thyroid, and carcass and as %IA/g for other tissues and are presented as mean ± SD for 4 animals. Saliv = salivary gland.

  • FIGURE 3. 
    FIGURE 3. 

    Specificity of 99mTc-ZHER2:2395-C tumor uptake in SKOV-3 and LS174T xenografts 4 h after injection. To saturate HER2 in tumors, one group of animals for each tumor model was preinjected with 600 μg of nonlabeled His6-ZHER2:342 45 min before injection of radiolabeled conjugate (designated as blocked). All animals were injected with 1 μg of labeled Affibody molecule. Data are expressed as %IA/g and are presented as mean ± SD for 4 animals.

  • FIGURE 4. 
    FIGURE 4. 

    Imaging of HER2 expression in LS174T xenografts in BALB/c nu/nu mice with 99mTc-ZHER2:2395-C. In blocking experiment, HER2 was saturated by preinjection of 600 μg of nonlabeled Affibody molecule. Planar γ-camera images were acquired 1 and 4 h after administration of 99mTc-ZHER2:2395-C. Tumors (right hind leg) were clearly visualized without blocking but were not seen in blocking experiment, indicating specific HER2 binding to 99mTc-ZHER2:2395-C. Animal contours were derived from digital photographs and superimposed over γ-camera images to facilitate interpretation. K = kidney; T = tumor.

  • FIGURE 5. 
    FIGURE 5. 

    Imaging of HER2 expression in SKOV-3 and A431 xenografts in BALB/c nu/nu mice with 99mTc-ZHER2:2395-C. A431 tumors were used as negative controls because they are considered to be non–HER2-expressing tumors. Planar γ-camera images were acquired 1 and 4 h after administration of 99mTc-ZHER2:2395-C in mice with SKOV-3 xenografts and 4 h after administration in mice with A431 xenografts. Tumors (right hind leg) were clearly visualized in SKOV-3 xenografts but were not seen in negative controls, indicating specific HER2 binding to 99mTc-ZHER2:2395-C. Animal contours were derived from digital photographs and superimposed over γ-camera images to facilitate interpretation. pi = after injection.

Tables

  • TABLE 1

    Biodistribution of 99mTc-H6-ZHER2:342-C in SCID Mice Bearing SKOV-3 Tumors

    Uptake (%IA/g) at:
    Tissue1 h2 h4 h
    Blood7 ± 2.02.7 ± 0.51.3 ± 0.4
    Heart3.0 ± 0.51.5 ± 0.40.7 ± 0.3
    Lung7 ± 1.91.8 ± 0.61.5 ± 0.3
    Liver19 ± 1.917 ± 3.619 ± 5
    Spleen2.0 ± 1.02.1 ± 0.22.0 ± 0.2
    Pancreas0.7 ± 0.20.5 ± 0.10.40 ± 0.04
    Kidney63 ± 1289 ± 1073 ± 9
    Stomach1.4 ± 0.61.1 ± 0.10.6 ± 0.1
    Tumor6 ± 1.98.3 ± 1.68.9 ± 3.1
    Skin2.8 ± 0.51.3 ± 0.41.8 ± 0.5
    Muscle0.4 ± 0.10.20 ± 0.030.14 ± 0.03
    Bone1.1 ± 0.21.3 ± 0.30.7 ± 0.2

    • Data are presented as mean ± SD for 3 animals.

  • TABLE 2

    Uptake of 99mTc-ZHER2:2395-C in BALB/c nu/nu Mice Bearing Xenografts of LS174T or SKOV-3 Tumors

    Uptake* in:
    LS174T at:SKOV-3 at:
    Tissue0.5 h1 h4 h6 h4 h
    Blood2.4 ± 0.21.0 ± 0.20.08 ± 0.020.05 ± 0.020.13 ± 0.04
    Lung3.1 ± 0.52.0 ± 0.50.39 ± 0.070.5 ± 0.30.45 ± 0.07
    Liver1.7 ± 0.32.0 ± 0.41.5 ± 0.21.7 ± 0.31.6 ± 0.1
    Spleen1.3 ± 0.21.5 ± 1.30.40 ± 0.060.38 ± 0.060.5 ± 0.2
    Stomach1.9 ± 0.41.3 ± 0.40.37 ± 0.050.36 ± 0.030.44 ± 0.08
    Kidney147 ± 20184 ± 18191 ± 15157 ± 18143 ± 20
    Salivary gland1.1 ± 0.20.7 ± 0.20.28 ± 0.050.28 ± 0.030.35 ± 0.07
    Thyroid0.040 ± 0.0020.026 ± 0.0030.006 ± 0.0010.007 ± 0.0040.005 ± 0.001
    Tumor7.2 ± 2.38.7 ± 1.76.9 ± 2.5†6.6 ± 0.815 ± 3†
    Muscle0.55 ± 0.080.36 ± 0.060.2 ± 0.10.07 ± 0.010.09 ± 0.01
    Intestine2.5 ± 0.41.8 ± 0.51.5 ± 0.32.2 ± 0.52.1 ± 0.3
    Carcass153 ± 2.59.4 ± 1.13.7 ± 0.52.8 ± 0.23.9 ± 0.4

    • ↵* Uptake is reported as %IA/g for all tissues except gastrointestinal tract and its contents, thyroid, and carcass, for which uptake is reported as %IA per whole sample.

    • ↵† Significant difference (P < 0.05) between LS174T xenografts (moderate level of HER2 expression) and SKOV-3 xenografts (high level of HER2 expression).

    • Data are presented as mean ± SD for 4 animals.

  • TABLE 3

    Tumor-to-Organ Ratios for 99mTc-ZHER2:2395-C in BALB/c nu/nu Mice Bearing Xenografts of LS174T or SKOV-3 Tumors

    Tumor-to-organ ratio
    LS174T at:SKOV-3 at:
    Organ0.5 h1 h4 h6 h4 h
    Blood3.1 ± 1.29.1 ± 2.288 ± 24129 ± 35121 ± 24
    Lung2.3 ± 0.64.3 ± 0.517 ± 615 ± 734 ± 5
    Liver4.3 ± 1.24.4 ± 0.64.5 ± 1.44.0 ± 0.49.3 ± 1.5
    Spleen5.8 ± 1.97.8 ± 3.917 ± 417 ± 133 ± 7
    Stomach3.9 ± 1.36.7 ± 1.619 ± 619 ± 335 ± 7
    Kidney0.05 ± 0.010.05 ± 0.010.04 ± 0.010.042 ± 0.0020.11 ± 0.02
    Salivary gland6.4 ± 1.912.0 ± 2.626 ± 1624 ± 444 ± 9
    Muscle13 ± 524 ± 660 ± 3891 ± 22172 ± 24

    • Data are presented as mean ± SD for 4 animals.

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