Development of a Novel Long-Lived ImmunoPET Tracer for Monitoring Lymphoma Therapy in a Humanized Transgenic Mouse Model

Molecular Imaging Program at Stanford (MIPS), Department of Radiology and.
Bioconjugate Chemistry (Impact Factor: 4.82). 05/2012; 23(6). DOI: 10.1021/bc300039r
Source: PubMed

ABSTRACT Positron emission tomography (PET) is an attractive imaging tool to localize and quantify tracer biodistribution. ImmunoPET with an intact mAb typically requires two to four days to achieve optimized tumor-to-normal ratios. Thus, a positron emitter with a half-life of two to four days such as zirconium-89 [(89)Zr] (t(1/2): 78.4 h) is ideal. We have developed an antibody-based, long-lived immunoPET tracer (89)Zr-Desferrioxamine-p-SCN (Df-Bz-NCS)-rituximab (Zr-iPET) to image tumor for longer durations in a humanized CD20-expressing transgenic mouse model. To optimize the radiolabeling efficiency of (89)Zr with Df-Bz-rituximab, multiple radiolabelings were performed. Radiochemical yield, purity, immunoreactivity, and stability assays were carried out to characterize the Zr-iPET for chemical and biological integrity. This tracer was used to image transgenic mice that express the human CD20 on their B cells (huCD20TM). Each huCD20TM mouse received a 7.4 MBq/dose. One group (n = 3) received a 2 mg/kg predose (blocking) of cold rituximab 2 h prior to (89)Zr-iPET; the other group (n = 3) had no predose (nonblocking). Small animal PET/CT was used to image mice at 1, 4, 24, 48, 72, and 120 h. Quality assurance of the (89)Zr-iPET demonstrated NCS-Bz-Df: antibody ratio (c/a: 1.5 ± 0.31), specific activity (0.44-1.64 TBq/mol), radiochemical yield (>70%), and purity (>98%). The Zr-iPET immunoreactivity was >80%. At 120 h, Zr-iPET uptake (% ID/g) as mean ± STD for blocking and nonblocking groups in spleen was 3.2 ± 0.1% and 83.3 ± 2.0% (p value <0.0013.). Liver uptake was 1.32 ± 0.05% and 0.61 ± 0.001% (p value <0.0128) for blocking and nonblocking, respectively. The small animal PET/CT image shows the spleen specific uptake of Zr-iPET in mice at 120 h after tracer injection. Compared to the liver, the spleen specific uptake of Zr-iPET is very high due to the expression of huCD20. We optimized the radiolabeling efficiency of (89)Zr with Df-Bz-rituximab. These radioimmunoconjugate lots were stable up to 5 days in serum in vitro. The present study showed that (89)Zr is well-suited for mAbs to image cancer over an extended period of time (up to 5 days).

  • [Show abstract] [Hide abstract]
    ABSTRACT: Due to its long half-life (78 h) and decay properties (77% electron capture, 23% β(+), Emax = 897 keV, Eav = 397 keV, Eγ = 909 keV, Iγ = 100%) (89)Zr is an appealing radionuclide for immunoPET imaging with whole IgG antibodies. Derivatives of the siderophore desferrioxamine-B (H3DFO) are the most widely used bifunctional chelators for coordination of (89)Zr(4+) because the radiolabeling of the resulting immunoconjugates is rapid under mild conditions. (89)Zr-DFO complexes are reportedly stable in vitro but there is evidence that (89)Zr(4+) is released in vivo, and subsequently taken up by the skeleton. We have evaluated a novel tripodal tris(hydroxypyridinone) chelator, H3CP256 and its bifunctional maleimide derivative, H3YM103, for coordination of Zr(4+) and compared the NMR spectra, and the (89)Zr(4+) radiolabeling, antibody conjugation, serum stability and in vivo distribution of radiolabelled immunoconjugates with those of H3DFO and its analogues. H3CP256 coordinates (89)Zr(4+) at carrier-free concentrations forming [(89)Zr(CP256)](+). Both H3DFO and H3CP256 were efficiently radiolabelled using [(89)Zr(C2O4)4](4-) at ambient temperature in quantitative yield at pH 6-7 at millimolar concentrations of chelator. Competition experiments demonstrate that (89)Zr(4+) dissociates from [(89)Zr(DFO)](+) in the presence of one equivalent of H3CP256 (relative to H3DFO) at pH 6-7, resulting largely in [(89)Zr(CP256)](+). To assess the stability of H3DFO and H3YM103 immunoconjugates radiolabelled with (89)Zr, maleimide derivatives of the chelators were conjugated to the monoclonal antibody trastuzumab via reduced cysteine side chains. Both immunoconjugates were labelled with (89)Zr(4+) in >98% yield at high specific activities and the labeled immunoconjugates were stable in serum with respect to dissociation of the radiometal. In vivo studies in mice indicate that (89)Zr(4+) dissociates from YM103-trastuzumab with significant amounts of activity becoming associated with bones and joints (25.88 ± 0.58% ID g(-1) 7 days post-injection). In contrast, <8% ID g(-1) of (89)Zr activity becomes associated with bone in animals administered (89)Zr-DFO-trastuzumab over the course of 7 days. The tris(hydroxypyridinone) chelator, H3CP256, coordinates (89)Zr(4+) rapidly under mild conditions, but the (89)Zr-labelled immunoconjugate, (89)Zr-YM103-trastuzumab was observed to release appreciable amounts of (89)Zr(4+)in vivo, demonstrating inferior stability when compared with (89)Zr-DFO-trastuzumab. The significantly lower in vivo stability is likely to be a result of lower kinetic stability of the Zr(4+) tris(hydroxypyridinone complex) relative to that of DFO and its derivatives.
    Dalton Transactions 10/2014; DOI:10.1039/C4DT02978J · 4.10 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The field of tumor immunology has seen an explosion of renewed interest over the last decade. With the FDA approval of new immunotherapies for prostate cancer and melanoma, as well as several exciting new drugs in clinical trials, tumor immunology is becoming an increasingly important topic in preclinical studies and patient care. However, the current methods for assessing the immune status of a patient and tumor are limited, which has led to the development of novel molecular imaging methods for assessing tumor immunology. From cell tracking for cellular therapeutics to assessing the tumor immune microenvironment, these imaging methods have the potential to further preclinical understanding of immunotherapies and potentially translate into clinically useful tests to predict and assess therapeutic response of these exciting new agents. In this review, we first discuss the recent advances in cancer immunotherapy, followed by a detailed review of the current state of molecular imaging for tumor immunology. Finally, we discuss opportunities for further development and innovation in this rapidly growing field.
    Advances in Cancer Research 01/2014; 124:257-96. DOI:10.1016/B978-0-12-411638-2.00008-2 · 4.26 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Purpose We evaluated the dosimetry of [89Zr]rituximab, an anti-CD20 immunoPET tracer to image B cell non-Hodgkin’s lymphoma (NHL) using a humanized transgenic mouse model that expresses human CD20 transgenic mice (huCD20TM). Procedures Rituximab was conjugated to desferrioxamine (Df) for radiolabeling of Zirconium-89. [89Zr]rituximab (2.8 ± 0.2 MBq) was tail vein-injected into huCD20T mice. Positron emission tomography (PET)/CT imaging was performed on the two groups of mice (blocking = 2 mg/kg pre-dose of rituximab and non-blocking; n = 5) at eight time points (1, 4, 24, 48, 72, 96, 120, and 168 h) post injection. Results The novel [89Zr]rituximab PET tracer had good immunoreactivity, was stable in human serum, and was able to specifically target human CD20 in mice. The human equivalents of highest dose (mean ± SD) organs with and without pre-dose are liver (345 ± 284 μSv/MBq) and spleen (1165 ± 149 μSv/MBq), respectively. Conclusions Dosimetry of the human patient whole-body dose was found to be 145 MBq per annum, and the patient dose-limiting organ will be the liver (with rituximab pre-dose blocking) and spleen for non-blocking. The [89Zr]rituximab (t½ = 78.4 h) imaging of B cell NHL patients could permit the observation of targeting lesions in NHL patients over an extended period due to longer half-life as compared to the [64Cu] rituximab (t½ = 12.7 h).
    Molecular Imaging & Biology 12/2014; DOI:10.1007/s11307-014-0810-8 · 2.87 Impact Factor