Imaging Pulmonary Embolism Using ^99mTc-labeled F（ab）₂ Fragment Of Anti-platelet Membrane Glycoprotein Monoclonal Antibody In Canine Model

Pulmonary embolism, which is associated with significant morbidity and mortality, poses a dilemma for both the clinician and the diagnostician. Although imaging plays a crucial role in the diagnosis of pulmonary embolism （PE） and deep venous thrombosis （DVT）, there has been no single noninvasive diagnostic test that can reliably be used to identify the hematologic status of thrombi. Single photon emission computed tomography （SPECT） is a highly sensitive imaging technique. PE and DVT can be detected by SPECT with Technetium-^99m ( ^99mTc) labeled imaging agents binding to components present predominantly on thromboembolism.We have previously generated McAb SZ51 against P-selectin,which expressed on activated human platelets;and McAb SZ21 against human platelet GPIIIa, which inhibits ADP-induced platelet aggregation in vitro and thrombus formation in vivo. In an attempt to decrease immunogenetic reaction induced by mouse antibody and improve image quality for thrombus detection, we converted SZ21 into mouse/human chimeric antibody （chSZ21）, and prepared its chimeric F（ab）₂ fragment, named chSZ21- F（ab）₂. McAb SZ51 was converted to F（ab）₂ fragment too, named SZ51- F（ab）₂ . The goal of this investigation was to test the feasibility of ^99mTc-SZ51-F（ab）₂ detecting pulmonary embolism and ^99mTc-chSZ21-F（ab）₂ imaging thromboembolism （DVT and PE） in vivo in canine model. MethodsThe mouse/human chimeric antibody （chSZ21） was constructed according the ordinary protocol, to produce chSZ21-F（ab）₂ , chSZ21 were digested overnight with a 1/30 （w/w） ratio of pepsin/IgG at 37℃. The engineered chimeric antibody and its F（ab）₂ fragments were characterized in regard to GPIIb/IIIa antigen binding specificity and anti-platelet activity by flow cytometry assay and ADP-induced platelet aggregation, respectively, in vitro. McAb SZ51 was converted to F（ab）₂ fragment and specific binding of SZ51F（ab）₂ to P-selectin on either human or canine platelets was verified by flow cytometry assay.The thromboembolism model were carried out on beagle canines. In each animal, an 18-gauge catheter was inserted into the distal half of the left or right femoral vein and an 10-mm two-stranded spiral stainless steel coil was placed in the femoral vein at approximately the mid-femur. Another steel embolization coil was inserted into left or right pulmonary artery through catheter in each dog too. 30 minutes after coil placement , X-ray angiographys were obtained to document the pulmonary embolism and the locations of the coils.After modified with 2-iminotholane, SZ51-F（ab）₂ and chSZ21-F（ab）₂ was labeled with ^99mTc using ^99mTc glucoheptonate as an intermediate. ^99mTc-SZ51-F（ab）₂ was used to detect 24-h-old PE and ^99mTc-SZ51-F（ab）₂ was used to image of 24-h-old DVT and PE in a canine model, respectively. Animals were imaged for up to 3 h after injection, heparinized, and sacrificed.ResultSpecific binding of chSZ21-F（ab）₂ to GPIIb/IIIa on either human or canine platelets was verified by flow cytometry assay. The concentration of chSZ21-F（ab）₂ required to inhibit 50% （IC50） of platelet aggregation in platelet-rich plasma was 11.6±7.9 nM and 24.9±18.8 nM for human and canine, respectively. In vivo, focal uptake was observed in planar images as early as 30 min （DVT）, and 60 min （PE） after injection. Lesion uptake of ^99mTc-chSZ21-F（ab）₂ at 3 h post injection was calculated in terms of percentage injected dose per gram （%ID/g） of tissue and averaged 0.076%ID/g PE and 0.083%ID/g DVT. Lesion-to-background ratio averaged 12.8±3.7（PE-to-lung）, 7.2±1.9 （DVT-to-blood）, and 117.0±5.3（DVT-to-muscle）, respectively.After intravenous injection of ^99mTc-SZ51-F（ab）₂, experimental thrombi in dogs could be consistently visualized for 2-3 hours by SPECT . PE had higher uptake of ^99mTc-SZ51-F（ab）₂.DiscussionAdvances in the diagnosis of PE have improved the management of patients with suspected PE, making the diagnostic work-up safer and more accessible. Pulmonary embolism, however, is not reliably detected by current diagnostic methods. Indeed, despite high performances of current imaging modalities like lung scan and contrast-enhanced computed tomographic, the hematologic status of thrombi is still hard to be indentified. However, nuclear imaging by SPECT and positron emission tomography （PET） has the most potential to furnish functional information on cell biologic events which determine the hematologic status of thrombi.PE and DVT are two clinical manifestations of the same pathology. Thus, there is still a role for an alternative, noninvasive imaging study which can detect both PE and DVT on one test. A radiotracer that will bind in high concentration to preexisting deposits of platelets or fibrin and thereby permit imaging of active thrombi within a few hours after injection could be very useful in assessing the need for thrombolysis therapy.The glycoprotein IIb/IIIa receptor （GP IIb/IIIa） is expressed in high density （approximately 80,000 copies/platelet） on the surface of single activated platelet and thus provides a high-capacity target for binding radiotracers. Fibrin remains a significant component of a clot, at all times irrespective of whether the clot is fresh or several-day-old. However, on the other hand, activated platelets-most of which aggregated on a thrombus during its early phase, and a lesser quantity on preformed blood clots-prevent these agents from accumulating enough radioactivity required for visualization of several-day-old clot. For the different components present predominantly between fresh and several-day-old thrombi, theoretically this could distinguish fresh and old thrombi in vivo by using radioactive nuclide labeled anti-GP IIb/IIIa McAb.A limitation of the current study is the slightly high blood pool background of ^99mTc-chSZ21-F（ab）₂ in vivo. In future studies, the improvement in pharmacokinetics and biodistribution of chSZ21 derivatives is likely to facilitate low blood pool background.ConclusionThe chimeric chSZ21-F（ab）₂ has a high affinity for GPIIb/IIIa receptor in activated platelets and was able to localize experimental DVT and PE induced in canine. The PE and DVT were clearly visualized in vivo for up to 24 h after induction. ^99mTc- chSZ21-F（ab）₂ therefore has potential for use as an imaging agent for imaging both fresh thrombi and emboli in a sole test. Our results suggest ^99mTc-SZ51-F（ab）₂ might be a very promising agent for detecting pulmonary embolism.