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Imaging Experimental Atherosclerotic Lesions in ApoE Knockout Mice: Enhanced Targeting with Z₂D₃-Anti-DTPA Bispecific Antibody and ^99mTc-Labeled Negatively Charged Polymers

¹ Center for Cardiovascular Targeting, School of Pharmacy, Bouve College of Health Sciences, Northeastern University, Boston, Massachusetts; and ² Division of Cardiology, Department of Medicine, Columbia University Medical Center, New York, New York

View larger version (35K): [in a new window]   FIGURE 1.  (A) Optical density (OD) 280-nm elution profile of Ultrogel-AcA-22 column chromatography for separation of bispecific antibody (peak I [Pk I] = tubes 40–50) from noncross–linked F(ab')2 (peak II [Pk II] = tubes 64–68). (B) Nonreducing SDS–PAGE (6%) of proteins from peaks I and II from Ultrogel AcA-22 column chromatography. MW = molecular weight.

View larger version (18K): [in a new window]   FIGURE 2.  (A) Immunoreactivity of bispecific antibody and Z2D3 F(ab')2 binding to surrogate antigen-coated microtiter wells by ELISA. (B) Immunoreactivity of bispecific antibody and anti-DTPA F(ab')2 binding to DTPA-BSA antigen-coated microtiter wells by ELISA. (C) Demonstration of signal enhancement with bispecific antibody and DTPA-HRP–Suc-PL46 kDa with 5.5 and 9 HRP per polymer relative to Z2D3 F(ab')2 and secondary HRP-conjugated rabbit antihuman IgG antibody (RAH). Negative control is anti-DTPA F(ab')2. OD = optical density.

View larger version (65K): [in a new window]   FIGURE 3.  (A) Ten-micron-thick frozen sections of rabbit atherosclerotic aorta stained with 50 µg/mL (panel a), 10 µg/mL (panel b), 5 µg/mL (panel c), and 1 µg/mL (panel d) of bispecific antibody counterstained with 5.5 HRP polymer, 150 µg/mL Z2D3 F(ab')2 (panel e), or 50 µg/mL (panel f), 10 µg/mL (panel g), 5 µg/mL (panel h), and 1 µg/mL of Z2D3 F(ab')2 (panel i) counterstained with HRP-conjugated secondary antibody. (B) Fluorescent (panels a, c, and e) micrographs show presence or absence of rhodamine fluorescence of surrogate antigen-coated beads with bispecific antibody (panel a), empty beads with bispecific antibody (panel c), or surrogate antigen-coated beads with NSB control (panel e) targeted with rhodamine-labeled DTPA–Suc-PL14.6 kDa. Corresponding light micrographs are shown in panels b, d, and f, respectively.

View larger version (41K): [in a new window]   FIGURE 4.  (A) Anteroposterior images of an ApoE–/– mouse on Western Diet with experimental atherosclerotic lesion in left leg (red arrows) and sham-operated right leg (yellow arrows) injected with NSB control followed by 15 MBq of 99mTc-DTPA–Suc-PL14.6 kDa. The 50-min postpolymer injection image is on left and 2-h image is on right. (B) Anteroposterior images of another ApoE–/– mouse injected with bispecific antibody followed by 99mTc-DTPA–Suc-PL14.6 kDa injection. Top left image was obtained at injection and diagram of various organ activities is in top right. Bottom left image is 30-min image after injection of radiolabeled polymer and bottom right image is 3-h image. Red arrows denote lesion site and yellow arrows denote sham-operated site. (C) Image of another ApoE–/– mouse with experimental atherosclerotic lesion 2 h after injection of 99mTc-DTPA–Suc-PL14.6 kDa. Red arrow = lesion; yellow arrow = sham-operated site.

View larger version (40K): [in a new window]   FIGURE 5.  (A) Localization of radiotracer in left (lesion) and right (sham) femoral arteries determined as %ID/g of total vessel tissue samples injected with NSB control (NSB cont; solid bars) or bispecific antibody (BiSp) (open bars). (B) Immunohistochemical stained frozen sections of left femoral artery lesion with bispecific antibody (panel a), normal right femoral artery from sham-operated region with bispecific antibody (panel b), and left femoral artery (lesion) with NSB control (panel c). (C) Biodistribution of 99mTc-DTPA–Suc-PL14.6 kDa activity in ApoE–/– mice with bispecific antibody (open bars) or NSB control (black bars). Int. = intestine; R = right; L = left.