Background: Despite advances in therapy of Hodgkin's lymphoma (HL), a proportion of patients will not respond or relapse. The authors had previously identified CD25, IL-2R alpha, as a target for systemic radioimmunotherapy of HL since most normal cells do not express CD25, but it is expressed by a minority of Hodgkin/Reed-Sternberg (HRS) cells and most Tregs rosetting around HRS cells. Study Design and Treatment: This was a single institution, nonrandomized, open-label phase I/II trial of radiolabeled Y-90-daclizumab, an anti-CD25 monoclonal antibody, BEAM (carmustine, etoposide, cytarabine, and melphalan) conditioning treatment followed by autologous hematopoietic stem cell transplant (ASCT). Four patients with refractory and relapsed HL were treated in this trial with 3 patients receiving a single dose of 564.6-574.6 MBq Y-90-daclizumab and the fourth patient receiving two doses of 580.9-566.1 MBq Y-90-daclizumab followed by high-dose chemotherapy and ASCT. Results: All 4 evaluable patients treated with Y-90-daclizumab obtained complete responses (CRs) that are ongoing 4.5-7 years following their stem cell transplant. The spectrum and severity of adverse events were mild and more importantly none of the patients, including several with multiple therapies before this treatment, developed the myelodysplastic syndrome. Discussion: Targeting by daclizumab was not directed primarily at tumor cells, but rather the nonmalignant CD25-expressing T cells adjacent to the HRS cells and Y-90-daclizumab provided strong enough beta emissions to kill CD25-negative tumor cells at a distance by a crossfire effect. Furthermore, the strong beta irradiation killed normal cells in the tumor microenvironment. Conclusions: Y-90-daclizumab (anti-CD25), high-dose BEAM chemotherapy and ASCT was well tolerated and yielded sustained complete remissions in all 4 patients with recurrent HL patients who completed their treatment. Significance: Despite advances, a proportion of patients with HL will not have a CR to their initial treatment, and some with CRs will relapse. They demonstrated that the addition of Y-90-daclizumab into the preconditioning regimen for refractory and relapsed HL patients with high-dose BEAM chemotherapy and ASCT provided sustained CRs in the 4 patients studied. Two of these patients were highly refractory to multiple prior treatments with bulky disease at entry into this study, including 1 patient who never entered a remission and had failed 6 different therapeutic regimens. Despite the small number of patients treated in this study, the sustained clinical benefit in these patients indicates a highly effective treatment. The daclizumab was directed primarily not at HRS cells themselves but toward nonmalignant T cells rosetting around malignant cells. Y-90 provided strong beta emissions that killed antigen nonexpressing tumor cells at a distance by a crossfire effect. Furthermore, the strong beta radiation killed normal cells in the tumor microenvironment that nurtured the malignant cells in the lymphomatous mass. The present study supports expanded analysis of Y-90-daclizumab as part of the regimen of ASCT in patients with refractory and relapsed HL.

The peripheral blood represents only a small fraction of the total number of lymphocytes in the body. To develop a more thorough understanding of T cell dynamics, including the effects of SIV/SHIV/HIV infection on immune cell depletion and immune reconstitution following combination antiretroviral therapy (cART), one needs to utilize approaches that allow direct visualization of lymphoid tissues. In the present study, noninvasive in vivo imaging of the CD4+ T cell pool has revealed that the timing of the CD4+ T cell pool reconstitution following initiation of ART in SIV-infected nonhuman primates (NHPs) appears seemingly stochastic among clusters of lymph nodes within the same host. At 4 weeks following initiation or interruption of cART, the changes observed in peripheral blood (PB) are primarily related to changes in the whole-body CD4 pool rather than changes in lymphocyte trafficking. Lymph node CD4 pools in long-term antiretroviral-treated and plasma viral load-suppressed hosts appear suboptimally reconstituted compared with healthy controls, while splenic CD4 pools appear similar between the 2 groups.

We investigated the effect of shed antigen mesothelin on the tumor uptake of amatuximab, a therapeutic anti-mesothelin mAb clinically tested in mesothelioma patients. The B3 mAb targeting a nonshed antigen was also analyzed for comparison. The mouse model implanted with A431/H9 tumor, which expresses both shed mesothelin and nonshed Lewis-Y antigen, provided an ideal system to compare the biodistribution and PET imaging profiles of the two mAbs. Our study demonstrated that the tumor and organ uptakes of 89Zr-B3 were dose-independent when 3 doses, 2, 15, and 60 μg B3, were compared at 24 h after injection. In contrast, tumor and organ uptakes of 89Zr-amatuximab were dose-dependent, whereby a high dose (60 μg) was needed to achieve tumor targeting comparable to the low dose (2 μg) of 89Zr-B3, suggesting that shed mesothelin may affect amatuximab tumor targeting as well as serum half-life. The autoradiography analysis showed that the distribution of 89Zr-B3 was nonuniform with the radioactivity primarily localized at the tumor periphery independent of the B3 dose. However, the autoradiography analysis for 89Zr-amatuximab showed dose-dependent distribution profiles of the radiolabel; at 10 μg dose, the radiolabel penetrated toward the tumor core with its activity comparable to that at the tumor periphery, whereas at 60 μg dose, the distribution profile became similar to those of 89Zr-B3. These results suggest that shed antigen in blood may act as a decoy requiring higher doses of mAb to improve serum half-life as well as tumor targeting. Systemic mAb concentration should be at a severalfold molar excess to the shed Ag in blood to overcome the hepatic processing of mAb-Ag complexes. On the other hand, mAb concentration should remain lower than the shed Ag concentration in the tumor ECS to maximize tumor penetration by passing binding site barriers.

Expression of the chemokine receptor CXCR4 by many cancers correlates with aggressive clinical behavior. As part of the initial studies in a project whose goal was to quantify CXCR4 expression on cancers non-invasively, we examined CXCR4 expression in cancer samples by immunohistochemistry using a validated anti-CXCR4 antibody. Among solid tumors, we found expression of CXCR4 on significant percentages of major types of kidney, lung, and pancreatic adenocarcinomas, and, notably, on metastases of clear cell renal cell carcinoma and squamous cell carcinoma of the lung. We found particularly high expression of CXCR4 on adrenocortical cancer (ACC) metastases. Microarrays of ACC metastases revealed correlations between expression of CXCR4 and other chemokine system genes, particularly CXCR7/ACKR3, which encodes an atypical chemokine receptor that shares a ligand, CXCL12, with CXCR4. A first-in-human study using 64Cu-plerixafor for PET in an ACC patient prior to resection of metastases showed heterogeneity among metastatic nodules and good correlations among PET SUVs, CXCR4 staining, and CXCR4 mRNA. Additionally, we were able to show that CXCR4 expression correlated with the rates of growth of the pulmonary lesions in this patient. Further studies are needed to understand better the role of CXCR4 in ACC and whether targeting it may be beneficial. In this regard, non-invasive methods for assessing CXCR4 expression, such as PET using 64Cu-plerixafor, should be important investigative tools.

Rapidly increasing number of therapeutic antibodies are being repurposed to imaging probes for noninvasive diagnosis, as well as monitoring during treatment or disease recurrence. Though antibody-based imaging involves tracer doses (~3 log lower than therapeutic doses), and immune responses are severely reduced in patients with impaired immunity, formation of anti-tracer antibodies (ATA) has been observed hampering further diagnostic monitoring. Here, we explored the potential to develop humoral responses to intravenously administered tracer dose of a monoclonal antibody F(ab΄)2 fragment, and associated with host related immune measures in 49 rhesus macaques categorized into healthy (uninfected controls), SIV-progressors, SIV non-progressors, or total body irradiated (TBI). Antibody fragment administered in tracer amount (~100μg) induced immune responses with significantly lower odds in SIV-progressors or TBI macaques (P<0.005) as compared to healthy animals. Peripheral blood (PB) CD4+ cell counts, but not CD20+ cell levels, were associated with significantly higher risk of developing a humoral response (P<0.001). Doubling the PB CD4+ counts is associated with an odds ratio of developing an immune response of 1.73. Among SIV-infected animals, CD4+ cell count was a stronger predictor of immune response than plasma SIV-RNA levels. Both SIV-progressors and TBI macaques showed higher odds of responses with increasing CD4+ counts, however when compared to healthy or SIV non-progressors with similar CD4+ count, they were still functionally incompetent in generating a response (P<0.01). Moreover, presence of ATA in systemic circulation altered the in vivo biodistribution by increasing hepatic uptake and decreasing plasma radiotracer clearance, with minimal to no binding detected in targeted tissues.

Introduction: Tumors over-expressing the human epithelial receptor 2 (HER2) or exhibiting amplification or mutation of its proto-oncogene have a poorer prognosis.Using trastuzumab and/or other HER2 targeted therapies can increase overall survival in patients with HER2(+) tumors making it critical to accurately identify patients who may benefit.We report on a Phase 0 study of the imaging agent, 111 In-CHX-A"-DTPA trastuzumab, in patients with known HER2 status to evaluate its safety and biodistribution and to obtain preliminary data regarding its ability to provide an accurate, whole-body, non-invasive means to determine HER2 status.Methods: 111 In-CHX-A"-DTPA trastuzumab was radiolabeled on-site and slowly infused into 11 patients who underwent single (n=5) or multiple (n=6) ɣ-camera (n=6) and/or SPECT (n=8) imaging sessions. Results:No safety issues were identified.Visual and semi-quantitative imaging data were concordant with tissue HER2 expression profiling in all but 1 patient.The biodistribution showed intense peak liver activity at the initial imaging timepoint (3.3h) and a single-phase clearance fit of the average time-activity curve (TAC) estimated t 1/2 =46.9h (R 2 =0.97; 95%CI 41.8 to 53h).This was followed by high gastrointestinal (GI) tract activity peaking by 52h.Linear regression predicted GI clearance by 201.2h (R 2 =0.96; 95%CI 188.5to 216.9h).Blood pool had lower activity with its maximum on the initial images.Non-linear regression fit projected a t 1/2 =34.2h (R 2 =0.96; 95%CI 25.3 to 46.3h).Assuming linear whole-body clearance, linear regression projected complete elimination (x-intercept) at 256.5hr (R 2 =0.96; 95%CI 186.1 to 489.2h). Conclusion:111 In-CHX-A"-DTPA trastuzumab can be safely imaged in humans.The biodistribution allowed for visual and semiquantitative analysis with results concordant with tissue expression profiling in 10 of 11 patients.Advances in Knowledge and Implications for Patient Care Using readily available components and on-site radiolabeling 111 In-CHX-A"-DTPA trastuzumab SPECT imaging may provide an economical, non-invasive means to detect HER2 over-expression.

ObjectivesThe aim of this study was to investigate the pharmacokinetics of Cu-64-DOTA (1,4,7,10-azacyclododecane-N,N ',N '',N '''-tetraacetic acid), a positron surrogate analog of the late gadolinium (Gd)-enhancement cardiac magnetic resonance agent, Gd-DOTA, in a rat model of chronic myocardial infarction (MI) and its microdistribution in the cardiac fibrosis by autoradiography.MethodsDOTA was labeled with Cu-64-acetate. CD rats (n=5) with MI by left anterior descending coronary artery ligation and normal rats (n=6) were injected intravenously with Cu-64-DOTA (18.5 MBq, 0.02 mmol DOTA/kg). Dynamic PET imaging was performed for 60 min after injection. F-18-Fluorodeoxyglucose ([F-18]-FDG) PET imaging was performed to identify the viable myocardium. For the region of interest analysis, the Cu-64-DOTA PET image was coregistered to the [F-18]-FDG PET image. To validate the PET images, slices of heart samples from the base to the apex were analyzed using autoradiography and by histological staining with Masson's trichrome.ResultsCu-64-DOTA was rapidly taken up in the infarct area. The time-activity curves demonstrated that Cu-64-DOTA concentrations in the blood, fibrotic tissue, and perfusion-rich organs peaked within a minute post injection; thereafter, it was rapidly washed out in parallel with blood clearance and excreted through the renal system. The blood clearance curve was biphasic, with a distribution half-life of less than 3 min and an elimination half-life of similar to 21.8 min. The elimination half-life of Cu-64-DOTA from the focal fibrotic tissue (similar to 22.4 min) and the remote myocardium (similar to 20.1 min) was similar to the blood elimination half-life. Consequently, the uptake ratios of focal fibrosis-to-blood and remote myocardium-to-blood remained stable for the time period between 10 and 60 min. The corresponding ratios obtained from images acquired from 30 to 60 min were 1.09 and 0.59, respectively, indicating that the concentration of Cu-64-DOTA in the focal fibrosis was 1.85 (1.09/0.59) times greater than that in the remote myocardium. Thus, this finding indicates that the extracellular volume fraction was 1.85 times greater in the focal fibrosis than in the remote myocardium. The accumulation of Cu-64-DOTA in fibrotic tissue was further supported by autoradiography and histology images. The autoradiography images of Cu-64-DOTA in the fibrotic tissues were qualitatively superimposed over the histology images of the fibrotic tissues. The histology images of the infarct areas were characterized by a heterogeneous distribution of thin bands of fibrotic collagen, myocytes, and expanded extracellular space.ConclusionCu-64-DOTA is a useful surrogate positron analog of Gd-DOTA, enabling quantitative measurement of the uptake values in fibrotic tissues by dynamic PET imaging and calculation of the extracellular volume fractions of the fibrotic tissues. At a microscopic level, the distribution of Cu-64-DOTA is nonuniform, corresponding to the heterogeneous distribution of expanded extracellular space in the setting of MI.

ObjectivesThe aim of the study is to evaluate the pharmacokinetics and microbiodistribution of Cu-64-labeled collagen-binding peptides.MethodsThe affinity constant (K-D), association (k(a)), and dissociation rate constant (k(d)) for the peptide collagelin or its analog (named CRPA) binding to collagen were measured by biolayer interferometric analysis. Rats (n=4-5) with myocardial infarction or normal were injected intravenously with the Cu-64-labeled peptides or Cu-64-DOTA as a control. Dynamic PET imaging was performed for 60min at 7-8 weeks after infarct. Fluorine-18 fluorodeoxyglucose PET imaging was performed to identify the viable myocardium. To validate the PET images, slices of heart samples from the base to the apex were analyzed using autoradiography and histology.ResultThe peptides bound to collagen with a K-D of approximate to 0.9 mu mol/l. The Cu-64-peptides and Cu-64-DOTA accumulated in the infarct area (confirmed by autoradiography and histology images) within 1min of injection and were excreted rapidly through the renal system. The blood clearance curves were biphasic with elimination half-lives of 21.92.4, 26.2 +/- 4.6, and 21.2 +/- 2.1min for Cu-64-CRPA, Cu-64-collagelin, and the control Cu-64-DOTA, respectively. The clearance half-lives from the focal fibrotic tissue (24.1 +/- 1.5, 25.6 +/- 8.0, and 21.4 +/- 1.3min, respectively) and remote myocardium (20.8 +/- 0.7, 21.0 +/- 5.5, and 19.1 +/- 2.4min, respectively) were not significantly different. The uptake ratios of infarct-to-remote myocardium (1.93 +/- 0.18, 2.15 +/- 0.38, and 1.88 +/- 0.08, respectively) for Cu-64-CRPA, Cu-64-collagelin, and Cu-64-DOTA remained stable for the time period between 10 and 60min.ConclusionThe distribution of the Cu-64-collagelin probes corresponds to the heterogeneous distribution of expanded extracellular space in the setting of myocardial infarction. The overall washout rate from the fibrous tissue was determined by the slow washout rate (t(1/2)20min) of the peptides from the extracellular space to the vasculature, not by the dissociation rate (t(1/2)<2min) of the Cu-64-peptides from collagen.

A disulfide-bridged cyclic RGD peptide, named iRGD (internalizing RGD, c(CRGDK/RGPD/EC)), is known to facilitate tumor targeting as well as tissue penetration. After the RGD motif-induced targeting on αv integrins expressed near tumor tissue, iRGD encounters proteolytic cleavage to expose the CendR motif that promotes penetration into cancer cells via the interaction with neuropilin-1. Based on these proteolytic cleavage and internalization mechanism, we designed an iRGD-based monolithic imaging probe that integrates multiple functions (cancer-specific targeting, internalization and fluorescence activation) within a small peptide framework. To provide the capability of activatable fluorescence signaling, we conjugated a fluorescent dye to the N-terminal of iRGD, which was linked to the internalizing sequence (CendR motif), and a quencher to the opposite C-terminal. It turned out that fluorescence activation of the dye/quencher-conjugated monolithic peptide probe requires dual (reductive and proteolytic) cleavages on both disulfide and amide bond of iRGD peptide. Furthermore, the cleavage of the iRGD peptide leading to fluorescence recovery was indeed operative depending on the tumor-related angiogenic receptors (αvβ3 integrin and neuropilin-1) in vitro as well as in vivo. Compared to an ‘always fluorescent’ iRGD control probe without quencher conjugation, the dye/quencher-conjugated activatable monolithic peptide probe visualized tumor regions more precisely with lower background noise after intravenous injection, owing to the multifunctional responses specific to tumor microenvironment. All these results, along with minimal in vitro and in vivo toxicity profiles, suggest potential of the iRGD-based activatable monolithic peptide probe as a promising imaging agent for precise tumor diagnosis.

Glypican-3 (GPC3) represents an attractive target for hepatocellular carcinoma (HCC) therapy because it is highly expressed in HCC but not in adult normal tissue. Recently, high affinity anti-GPC3 antibodies have been developed; however, full antibodies may not penetrate evenly into tumor parenchyma, reducing their effectiveness. In this study, we compared a whole IgG antibody, anti-GPC3 YP7, with an anti-GPC3 human heavy chain antibody, HN3, with regard to their relative therapeutic effects. Both YP7 and HN3 bound to GPC3-positive A431/G1 cells and were internalized by the cells by in vitro evaluation with (125)I- and (111)In-radiolabeling antibodies. In vivo biodistribution and tumor accumulation was performed with (111)In-labeled antibodies, and intratumoral microdistribution was evaluated using fluorescently labeled antibodies (IR700). HN3 showed similar high tumor accumulation but superior homogeneity within the tumor compared with YP7. Using the same IR700 conjugated antibodies photoimmunotherapy (PIT) was performed in vitro and in a tumor-bearing mouse model in vivo. PIT with IR700-HN3 and IR700-YP7 demonstrated that comparable results could be achieved despite of low reaccumulation 24 h after the first NIR light exposure. These results indicated that a heavy-chain antibody, HN3, showed more favorable characteristics than YP7, a conventional IgG, as a therapeutic antibody platform for designing molecularly targeted agents against HCC.

Amatuximab is a chimeric high-affinity monoclonal IgG1/k antibody targeting mesothelin that is being developed for treatment of mesothelin-expressing cancers. Considering the ongoing clinical development of amatuximab in these cancers, our objective was to characterize the biodistribution, and dosimetry of 111Indium (111In) radiolabelled amatuximab in mesothelin-expressing cancers. Between October 2011 and February 2013, six patients including four with malignant mesothelioma and two with pancreatic adenocarcinoma underwent Single Photon Emission Computed Tomography-Computed Tomography (SPECT/CT) imaging following administration of 111In amatuximab. SPECT/CT images were obtained at 2-4 hours, 24-48 hours and 96-168 hours after radiotracer injection. In all patients, tumor to background ratios (TBR) consistently met or exceeded an uptake of 1.2 (range 1.2-62.0) which is considered the minimum TBR that can be visualized. TBRs were higher in tumors of patients with mesothelioma than pancreatic adenocarcinoma. 111In-amatuximab uptake was noted in both primary tumors and metastatic sites. The radiotracer dose was generally well-tolerated and demonstrated physiologic uptake in the heart, liver, kidneys and spleen. This is the first study to show tumor localization of an anti-mesothelin antibody in humans. Our results show that 111In-amatuximab was well tolerated with a favorable dosimetry profile. It localizes to mesothelin expressing cancers with a higher uptake in mesothelioma than pancreatic cancer.

Ionizing irradiation is used routinely to induce myeloablation and immunosuppression. However, it has not been possible to evaluate the extent of ablation without invasive biopsy. For lymphoid recovery, peripheral blood (PB) lymphocytes (PBLs) have been used for analysis, but they represent <2% of cells in lymphoid tissues (LTs). Using a combination of single-photon emission computed tomography imaging and a radiotracer ((99m)Tc-labeled rhesus immunoglobulin G1 anti-CD4R1 (Fab')2), we sequentially imaged CD4(+) cell recovery in rhesus macaques following total body irradiation (TBI) and reinfusion of vector-transduced, autologous CD34(+) cells. Our results present for the first time a sequential, real-time, noninvasive method to evaluate CD4(+) cell recovery. Importantly, despite myeloablation of circulating leukocytes following TBI, total depletion of CD4(+) lymphocytes in LTs such as the spleen is not achieved. The impact of TBI on LTs and PBLs is discordant, in which as few as 32.4% of CD4(+) cells were depleted from the spleen. In addition, despite full lymphocyte recovery in the spleen and PB, lymph nodes have suboptimal recovery. This highlights concerns about residual disease, endogenous contributions to recovery, and residual LT damage following ionizing irradiation. Such methodologies also have direct application to immunosuppressive therapy and other immunosuppressive disorders, such as those associated with viral monitoring.

Objectives: To investigate the effect of the injection dose of MORAb-009 (amatuximab, an anti-mesothelin monoclonal antibody), the tumor size and the level of shed mesothelin on the uptake of the antibody in mesothelin-positive tumor and organs by biodistribution (BD) and positron emission tomography (PET) imaging studies.Methods: 2-S-(4-Isothiocyanatobenzyl)-1,4,7-triazacydononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) was conjugated to amatuximab and labeled with (CuCl2)-Cu-64 in 0.25 M acetate buffer, pH 4.2. The resulting Cu-64-NOTA-amatuximab was purified with a PD 10 column. To investigate the dose effect or the effect of tumor size, the BD was performed in groups of nude mice (n = 5) with mesothelin-expressing A431/H9 tumors (range, 80-300 mm(3)) one day after iv injection of Cu-64-NOTA-amatuximab (10 mu Ci) containing a total amatuximab dose of 2, 30, or 60 mu g. The BD and PET imaging were also investigated 3, 24 and 48 h after injecting a total dose of 30 mu g (10 mu Ci for BD), and 2 or 60 mu g (300 mu Ci for PET), respectively.Results: Comparing the results of the BDs from three different injection doses, the major difference was shown in the uptake (%ID/g) of the radiolabel in tumor, liver and blood. The tumor uptake and blood retention from 30 and 60 mu g doses were greater than those from 2 mu g dose, whereas the liver uptake was smaller. The BD studies also demonstrated a positive correlation between tumor size (or the level of shed mesothelin in blood) and liver uptake. However, there was a negative correlation between tumor size (or the shed mesothelin level) and tumor uptake and between tumor size and blood retention. These findings were confirmed by the PET imaging study, which clearly visualized the tumor uptake with the radiolabel concentrated in the tumor core and produced a tumor to liver ratio of 1.2 at 24 h post-injection with 60 mu g amatuximab, whereas the injection of 2 mu g amatuximab produced a tumor to liver ratio of 0.4 at 24 h post-injection.Conclusion: Our studies using a nude mouse model of A431/H9 tumor demonstrated that the injection of a high amatuximab dose (30 to 60 mu g) could provide a beneficial effect in maximizing tumor uptake while maintaining minimum liver and spleen uptakes of the radiolabel, and in facilitating its penetration into the tumor core. Published by Elsevier Inc.

AIM:Effectiveness of Glypican-3 (GPC3)-targeted photoimmunotherapy (PIT) combined with the nanoparticle albumin-bound paclitaxel (nab-paclitaxel) for hepatocellular carcinoma was evaluated.MATERIALS & METHODS:GPC3 expressing A431/G1 cells were incubated with a phthalocyanine-derivative, IRDye700DX (IR700), conjugated to an anti-GPC3 antibody, IR700-YP7 and exposed to near-infrared light. Therapeutic experiments combining GPC3-targeted PIT with nab-paclitaxel were performed in A431/G1 tumor-bearing mice.RESULTS:IR700-YP7 bound to A431/G1 cells and induced rapid target-specific necrotic cell death by near-infrared light exposure in vitro. IR700-YP7 accumulated in A431/G1 tumors. Tumor growth was inhibited by PIT compared with nontreated control. Additionally, PIT dramatically increased nab-paclitaxel delivery and enhanced the therapeutic effect.CONCLUSION:PIT targeting GPC3 combined with nab-paclitaxel is a promising method for treating hepatocellular carcinoma.

Near-infrared (NIR) fluorophores have several advantages over visible-light fluorophores, including superior tissue penetration and lower autofluorescence. We recently accessed a new class of readily synthesized NIR cyanines containing a novel C4'-O-alkyl linker, which provides both high chemical stability and excellent optical properties. In this study, we provide the first in vivo analysis of this new class of compounds, represented by the tetrasulfonate FNIR-774 (Frederick NIR 774). Monoclonal antibody (mAb) conjugates of FNIR-774 were compared to conjugates of the commercially available dye (IRDye800CW (IR800)), one of the most widely used NIR fluorophores for clinical translation. Both dyes were conjugated to panitumumab (pan) or cetuximab (cet) with ratios of 1:2 or 1:5. Conjugates of both dyes demonstrated similar quenching capacity, stability, and brightness in target cells in vitro. In contrast, in vivo imaging in mice showed different pharmacokinetics between pan-FNIR-774 (1:5) and pan-IR800 (1:5), or cet-FNIR-774 (1:5) and cet-IR800 (1:5). Particularly at the higher labeling density, mAb-FNIR-774 conjugates showed superior specific accumulation in tumors compared with mAb-IR800 conjugates. Thus, FNIR-774 conjugates showed superior in vivo pharmacokinetics compared with IR800 conjugates, independent of the mAb. These results suggest that FNIR-774 is a promising fluorescent probe for NIR optical imaging.

1472 Objectives To investigate the pharmacokinetics of 64Cu-DOTA, a positron surrogate analog of late Gd-enhancement cardiac magnetic resonance agent, Gd-DOTA in a rat model of chronic myocardial infarction (MI) and its micro-distribution in the cardiac fibrosis by autoradiography and histology. Methods DOTA was labeled with 64Cu-acetate. CD rats (n=3-5) with MI by LAD ligation and 2 normal rats were injected iv with the 64Cu-DOTA (0.5 mCi with 0.02 mmol DOTA/Kg). The PET imaging was performed for 60 min after injection at 7 and 14 week post MI. 18F-FDG PET imaging was performed to identify the viable myocardium. In addition, 64Cu-acetate PET was performed for both MI and normal rat. For the VOI analysis, the 64Cu-DOTA PET image was co-registered to the 18F-FDG PET image. To validate the PET images, slices of heart samples from base to apex were analyzed using autoradiography and histological staining with Masson’s Trichrome. Results The dynamic PET imaging showed that the presence of the radioactivity reflected the blood pool signal with high levels of radioactivity in perfusion-rich tissues including lungs, cardiac fibrosis, myocardium and liver. However, 64Cu-DOTA was retained longer in fibrotic tissue than the normal tissues, and excreted rapidly via the renal system. And SUVs (30-60 min) in fibrosis were 1.53 ± 0.25 and 1.54 ± 0.12 at 7 and 14 week post-MI, respectively. The fibrosis/remote myocardium ratios were 2.53 ± 0.18 and 2.17 ± 0.25 at 7 and 14 week post-MI, respectively and the corresponding fibrosis/blood ratios were 1.53 ± 0.21 and 1.37 ± 0.13, respectively. For 64Cu-acetate, there was no accumulation in myocardium. The autoradiography and histology analysis indicate that 64Cu-DOTA accumulated on collagen rich area of cardiac fibrosis. Conclusions 64Cu-DOTA is retained longer in collagen rich area in the extracellular space and may be useful as a surrogate positron analog of Gd-DOTA to quantify its absolute uptake in cardiac fibrosis. Research Support Clinical Center, NIH and NCI Contract No. HHSN261200800001E

Despite significant advances in the treatment of Hodgkin's lymphoma (HL), a significant proportion of patients will not respond or will subsequently relapse. We identified CD25, the IL-2 receptor alpha subunit, as a favorable target for systemic radioimmunotherapy of HL. The scientific basis for the clinical trial was that, although most normal cells with exception of Treg cells do not express CD25, it is expressed by a minority of Reed-Sternberg cells and by most polyclonal T cells rosetting around Reed-Sternberg cells. Forty-six patients with refractory and relapsed HL were evaluated with up to seven i.v. infusions of the radiolabeled anti-CD25 antibody (90)Y-daclizumab. (90)Y provides strong β emissions that kill tumor cells at a distance by a crossfire effect. In 46 evaluable HL patients treated with (90)Y-daclizumab there were 14 complete responses and nine partial responses; 14 patients had stable disease, and nine progressed. Responses were observed both in patients whose Reed-Sternberg cells expressed CD25 and in those whose neoplastic cells were CD25(-) provided that associated rosetting T cells expressed CD25. As assessed using phosphorylated H2AX (γ-H2AX) as a bioindicator of the effects of radiation exposure, predominantly nonmalignant cells in the tumor microenvironment manifested DNA damage, as reflected by increased expression of γ-H2AX. Toxicities were transient bone-marrow suppression and myelodysplastic syndrome in six patients who had not been evaluated with bone-marrow karyotype analyses before therapy. In conclusion, repeated (90)Y-daclizumab infusions directed predominantly toward nonmalignant T cells rosetting around Reed-Sternberg cells provided meaningful therapy for select HL patients.