Development of a Novel Long-Lived ImmunoPET Tracer for Monitoring

May 23, 2012 - Positron emission tomography (PET) is an attractive imaging tool to localize and quantify tracer biodistribution. ImmunoPET with an int...
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Development of a Novel Long-Lived ImmunoPET Tracer for Monitoring Lymphoma Therapy in a Humanized Transgenic Mouse Model Arutselvan Natarajan, Frezghi Habte, and Sanjiv S. Gambhir*,§ Molecular Imaging Program at Stanford (MIPS), Department of Radiology and § Bioengineering, Department of Materials Science and Engineering, Bio-X Program, Stanford University, Stanford, California, United States S Supporting Information *

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 [89Zr] (t1/2: 78.4 h) is ideal. We have developed an antibody-based, long-lived immunoPET tracer 89ZrDesferrioxamine-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 89Zr 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 89Zr-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 89Zr-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 95 >95 84.2 ± 2.8

dx.doi.org/10.1021/bc300039r | Bioconjugate Chem. 2012, 23, 1221−1229

Bioconjugate Chemistry

Article

cells. The average immunoreactive fraction of 89Zr-Df-Bzrituximab from each lot was 0.85 ± 0.07 (n = 6). The control experiment was performed with CD20 negative Jurkat cells, and CD20 positive Ramos cells preblocked with 100-fold excess of cold rituximab over 89Zr-rituximab. The immunoreactivity of the tracer was 84.2 ± 2.8% (Mean ± STD) compared to the binding effect on same cells but preblocked with cold rituximab and Jurkat cells were 5 ± 1.6% (Mean ± STD) and 4 ± 2.1% (Mean ± STD) respectively, indicating the specific reactivity of the tracer. Small Animal PET and CT Imaging. To evaluate antibody-based PET tracer targeting anti-CD20 B-cell lymphoma, we used a huCD20TM model that mimics a human B-cell lymphoma tumor and provides a clearance pattern such that rituximab recognizes CD-20 antigens on Bcells in human lymphoma patients. The in vivo targeting ability of 89Zr-Df-Bz-rituximab in huCD20TM was demonstrated at various time points post tail-vein injection of radiopharmaceutical, with and without blocking of CD20 antigens by cold rituximab. Each mouse was imaged at various time points (1, 4, 24, 48, 72, 96, and 120 h) after tracer injection of 7−8 MBq of 89 Zr-Df-Bz-rituximab (2−3 μg of rituximab). Figures 3 and 4 show the immunoPET images of huCD20TM using the 89Zr-Df-Bz-rituximab tracer. Figure 3A PET images were obtained at 4, 24, 48, 72, 96, and 120 h after injection of tracer. At each time point, PET/CT images were obtained from the control huCD20TM (Figure 3 top row: blocking = preblocked with 100-fold excess cold rituximab over tracer mass 2 h prior to tracer injection) and experimental huCD20TM (Figure 3 bottom row: nonblocking = tracer alone). Figure 3B images shown are huCD20 images of just CT and co-registered with PET/CT for organ delineation. Organs marked as follows: H = heart, L = liver, and the spleen is marked with a yellow arrow. From these images, it is evident that 89Zr-Df-Bz-rituximab had uptake primarily in the spleen, a major site for B cells, which express CD20 antigen. Figure 4 shows immunoPET images of huCD20TM at the maximum intensity projection of 89Zr-Df-Bz-rituximab after 5 days post injection of the tracer. The spleen and liver uptake (%ID/g) is shown in Figure 5 at 120 h post injection compared with the predose group (human CD20 antigens were preblocked with cold rituximab). The radiopharmaceutical uptake (%ID/g) in the preblocked (n = 3) and unblocked (n = 3) groups is 3.19 ± 0.14, and 83.3 ± 1.99 for spleen; 1.32 ± 0.05, and 0.61 ± 0.01 for liver, respectively.

Figure 1. Purity and serum stability of the 89Zr-Df-rituximab. (A) Radio-HPLC chromatogram PET tracer shows the purity of the tracer. The PET tracer was eluted by SEC 3000 column at a flow rate of 1 mL/min using PBS. Radioactivity of each of the 1 mL fractions was measured by a gamma counter and the data was used to plot the chromatogram. (B) Serum stability of 89Zr-Df-rituximab assayed by cellulose acetate electrophoresis (CAE) at 45 min: The results of serum stability study of PET tracer tested by CAE are shown. CAE was performed for 45 min with barbital buffer (0.05 M, pH 8.6) at room temperature. Radioconjugate (50 μg) was mixed per mL of human serum and kept at 37 °C for 3 days. At various time points (0, 24, 72, and 120 h), 2−10 μL samples were drawn and tested for stability on CAE. Radioactivity peaks in the figure correspond to radioconjugate in the serum at days 1−5, demonstrating that radioconjugates are at identical migration distance.

serum and greater than 98% of the immunoconjugate was intact for up to 5 days. Cell Binding Study. Figure 2 shows the binding fraction of the 89Zr-Df-Bz-rituximab tested with CD20 positive Ramos

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DISCUSSION To monitor NHL therapy, we have developed a novel immunoPET tracer using 89Zr radionuclide that chelated to desferrioxamine (Df) linked to rituximab. This work was conducted in an effort to eventually translate the methods developed here for human lymphoma patient management through PET imaging. In this report, we present in vitro characterization and preclinical evaluation of 89Zr-Df-rituximab tracer in a huCD20TM mouse model, which expresses the human CD20. Previously, we developed a 64Cu-DOTArituximab immunoPET tracer22 that received FDA approval for a physician-sponsored IND (#104995) for safety and clearance and is currently being tested in a clinical trial. We are also interested in developing immunoPET tracers for radioimmunotherapy monitoring with longer half-life radionuclides for comparable half-life of antibody (2−5 days). To match the half-life of antibody, currently very few long half-life PET

Figure 2. Live cell binding assay for the determination of the immunoreactive fraction of an 89Zr-rituximab tracer using Ramos cells. The assay was set up using six concentrations of Ramos cells in 1:2 dilutions from 3.3 to 0.1 million cells/mL, and the final concentrations of tracer was 10 ng/mL. Cells were used at various concentrations to infinite antigen excess (1/y-intercept). A plot of (X axis) total/bound activity against (Y axis) 1/normalized cell concentration (mL/million) provided immunoreactive fraction (0.85 ± 0.07). X axis of graph represents cell concentrations (mL/million) 0.3, 0.6, 1.2, 2.4, 4.8, and 9.62, the corresponding immunoreactive fractions (mean ± SD) represent Y axis are 1.25 ± 0.11, 1.35 ± 0.12, 1.5 ± 0.18, 1.95 ± 0.12, 2.78 ± 0.24, and 4.16 ± 0.11, respectively. The data represented in the graph are corrected with subtraction of immunoreactive fractions on preblocked cells [nonspecific binding (mean ± SD) = 0.008 ± 0.001] with cold rituximab prior to tracer application. 1225

dx.doi.org/10.1021/bc300039r | Bioconjugate Chem. 2012, 23, 1221−1229

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Figure 3. ImmunoPET images of 89Zr-Df-rituximab on huCD20TM model. The representative images were scanned (5−20 min) at 4−120 h after tracer injection (7.4 MBq/2−3 μg of rituximab). Panels A,B: top (blocked = Predose; control) and bottom (nonblocked = no predose; experiment) rows are huCD20TM coronal images of preblocked and unblocked, respectively. Panel A represents a mouse that received 4 mg/kg cold rituximab 2 h prior to tracer injection and panel B represents a mouse that received the PET tracer only. Panel C shows the CT and PET/CT images at 120 h after tracer injection for organ identification. Spleen is indicated by the yellow arrow. The other major organs are as marked (H = heart and L = liver). The color bar shows tracer %ID/g.

Figure 4. ImmunoPET images of huCD20TM at 120 h post injection with maximum intensity projections(MIP) shows mouse with and without predose of rituximab. The 89Zr-Df-rituximab uptake by spleen is very high compared to preblocked mouse by cold rituximab. ImmunoPET tracer was stable even after 120 h, and no PET signals were observed in bone marrow. Figure 5. Quantitative analysis of organ uptake estimated from the ROI of the images. Data shown are tracer uptake values at 120 h post injection by liver (gray bar) and spleen (black bar) of huCD20 mice with and without predose groups. Y axis is %ID/gram and X axis shows comparison of control and experiment groups. Tracer uptake by the liver is similar for both study groups, and the spleen uptake for the group without predose is >50 times higher than that with the predose group, confirming the specificity of the tracer. The data represent the mean ± STD of 3 mice per group.

radionuclides are available for use in clinical studies; i.e., 124I and 89Zr. Although iodination (124I) of antibodies for PET is simple, it has widespread limitations of lower resolution and enzymatic deiodination.32,33 Recently, 89Zr-immunoPET tracers are being increasingly used in clinical studies.34,35 This is because the low production cost, purity, stability, and extended half-life period of the 89Zr radionuclides make it a perfect choice to link to an antibody for targetable PET tracers. On the other hand, GMP-grade Df-BzNCS bifunctional chelator is also commercially available for 89 Zr labeling for clinical studies. The Df-Bz-NCS ligand has already shown good biological performance when used in protein conjugation of radionuclides such as 68Ga, 66Ga, 225Ac, 177 Lu, and lead radionuclides.36,37 Furthermore, currently both Df-Bz-NCS conjugated antibodies and 89Zr-labeled antibodies are in clinical trials after FDA approval. Although few studies have shown that DTPA can be used for the chelation of 89Zr ions, it shows demetalation in vivo and hence Df continues to

remain the best chelator for 89Zr.38−40 Thus, in our study, DfBz-NCS was utilized to prepare Df-Bz-rituximab according to published procedures.25,41 In the present report, we optimized the reaction conditions for conjugation of Df and radiolabeling of 89Zr to chelate for high radiochemical yield and specific activity specifically for the rituximab antibody. The conjugation optimization yielded maximum 2.6 chelates per antibody (Table 1) at pH 9.0, with 10-fold molar excess ratio after 45 min; although chelate 1226

dx.doi.org/10.1021/bc300039r | Bioconjugate Chem. 2012, 23, 1221−1229

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times. Thus, compared to other published studies our tracer with high specific activity showed very high contrast images over extended period of time. This was reflected in Figures 3B and 4 at 120 h where the immunoPET demonstrated very high contrast images for spleen of nonblocked-huCD20TM, compared to blocked-huCD20TM. ROI quantitative analysis of 89Zr-Df-rituximab uptake in spleen (Figure 5), of nonblocked-huCD20TM, was approximately 25-fold higher %ID/g than blocked-huCD20TM. Thus, our study data observed from the high specific activity tracer may be useful for other mAbs and mAb-fragments for high imaging contrast and for imaging for extended periods of time. Zirconium is known to bind plasma proteins44 and is later deposited in mineral bone.45,46 However, in our study we did not see bone uptake of the radionuclide originating from the breakdown of the 89Zr-antibody or any nonspecific association of 89Zr to antibody, which could then easily transchelate to plasma proteins compared to 89Zr bound to Df. The results reported here are consistent with previous investigations and confirm that 89Zr is a suitable radionuclide for labeling full, intact antibodies (150 kDa).36 The 89Zr has the potential to solve many problems associated with various PET isotopes such as (a) PET isotopes of 64Cu and 86Y having short half-life, (b) 64Cu, 68Ga, and 124I demonstrated to have high uptake in background tissue, (c) 124I are known for low in vivo stability (for any internalizing antibody−antigen constructs) and poor dosimetry. Contrary to the above-mentioned isotopes, 89Zrlabeled mAbs can be used for both localizing tumors and measuring the long-term effects (5 days) of drug treatment from a single radiotracer injection. Such measurements cannot be achieved by using 64Cu,124I, and 99mTc radiolabeled rituximab.23,24,47 The image quality and chelate chemistry of 89 Zr-Df-mab immunoPET tracers were well-demonstrated by various PET images.36,41,48 For example, studies pertaining to 89 Zr-Df-trastuzumab represent one of the most promising radiotracers for noninvasive immunoPET measurements of HER2/neu expression in vivo.48

ratio per antibody was dictated by the number of lysine molecules per antibody, in our case the c/a is comparable to the results reported elsewhere using different antibody.41 Although we have achieved 2.6 c/a, we realize that too many lysine group modifications may alter the biodistribution from the parent molecule.42,43 Hence, we restrict our chelate conjugation to not more than 2.0 per antibody for the use of preclinical studies. Similarly, the radiolabeling optimization yielded maximum 77.0 ± 2.16 (mean ± STD) at pH 7, and after purification, the final product purity was >98% monomeric (Figure 1A and Table 2). The 89Zr-Df-Bz-rituximab conjugate showed good in vitro stability for up to 5 days at 37 °C in 0.9% saline, 50 mM DTPA, and human serum (Figure 1B). Our results confirmed with previous reports elsewhere that the Df-Bz-SCN linked immunoconjugates were stable over a 6 day period in serum at 37 °C.36,41 After 5 days, less than 1.5% activity appeared at 5 cm in CAE at 45 min, which may correspond to 89Zrtranschelated protein in serum, possibly due to demetalation from the 89Zr-Df-rituximab (Figure 1B). Figure 2 shows the estimation of immunoreactive fraction of 89Zr-Df-Bz-rituximab used in the preclinical studies. The immunoreactive fraction of the 89Zr-Df-rituximab was measured by specific in vitro cellular association assays using CD20-positive Ramos cells prior to each preclinical study. The average immunoreactive fraction of 89 Zr-Df-rituximab (Figure 2) was found to be 0.85 (n = 6). Control experiments were performed using the CD20-negative Jurkat cells (no CD20 expression), and Ramos cells preblocked with 100-fold excess of cold unmodified rituximab 1 h prior to tracer application showed