Gaurav Malviya

Molecular imaging of rheumatoid arthritis by radiolabelled monoclonal antibodies: new imaging strategies to guide molecular therapies

The closing of the last century opened a wide variety of approaches for inflammation imaging and treatment of patients with rheumatoid arthritis (RA). The introduction of biological therapies for the management of RA started a revolution in the therapeutic armamentarium with the development of several novel monoclonal antibodies (mAbs), which can be murine, chimeric, humanised and fully human antibodies. Monoclonal antibodies specifically bind to their target, which could be adhesion molecules, activation markers, antigens or receptors, to interfere with specific inflammation pathways at the molecular level, leading to immune-modulation of the underlying pathogenic process. These new generation of mAbs can also be radiolabelled by using direct or indirect method, with a variety of nuclides, depending upon the specific diagnostic application. For studying rheumatoid arthritis patients, several monoclonal antibodies and their fragments, including anti-TNF-α, anti-CD20, anti-CD3, anti-CD4 and anti-E-selectin antibody, have been radiolabelled mainly with 99mTc or 111In. Scintigraphy with these radiolabelled antibodies may offer an exciting possibility for the study of RA patients and holds two types of information: (1) it allows better staging of the disease and diagnosis of the state of activity by early detection of inflamed joints that might be difficult to assess; (2) it might provide a possibility to perform ‘evidence-based biological therapy’ of arthritis with a view to assessing whether an antibody will localise in an inflamed joint before using the same unlabelled antibody therapeutically. This might prove particularly important for the selection of patients to be treated since biological therapies can be associated with severe side-effects and are considerably expensive. This article reviews the use of radiolabelled mAbs in the study of RA with particular emphasis on the use of different radiolabelled monoclonal antibodies for therapy decision-making and follow-up.

Synthesis and evaluation of a radioiodinated tracer with specificity for poly(ADP-ribose) polymerase-1 (PARP-1) in vivo

Interest in nuclear imaging of poly(ADP-ribose) polymerase-1 (PARP-1) has grown in recent years due to the ability of PARP-1 to act as a biomarker for glioblastoma and increased clinical use of PARP-1 inhibitors. This study reports the identification of a lead iodinated analog 5 of the clinical PARP-1 inhibitor olaparib as a potential single-photon emission computed tomography (SPECT) imaging agent. Compound 5 was shown to be a potent PARP-1 inhibitor in cell-free and cellular assays, and it exhibited mouse plasma stability but approximately 3-fold greater intrinsic clearance when compared to olaparib. An (123)I-labeled version of 5 was generated using solid state halogen exchange methodology. Ex vivo biodistribution studies of [(123)I]5 in mice bearing subcutaneous glioblastoma xenografts revealed that the tracer had the ability to be retained in tumor tissue and bind to PARP-1 with specificity. These findings support further investigations of [(123)I]5 as a noninvasive PARP-1 SPECT imaging agent.

Radiolabeled Humanized Anti-CD3 Monoclonal Antibody Visilizumab for Imaging Human T-Lymphocytes

Visilizumab is an IgG2 humanized monoclonal antibody (mAb) characterized by non-FcγR binding and specific to the CD3 antigen, expressed on more than 95% of circulating resting T-lymphocytes and on activated T-lymphocytes homing in inflamed tissues. We hypothesized that the use of a radiolabeled anti-CD3 antibody might serve as a diagnostic tool for imaging T-cell traffic and lymphocytic infiltration of tissues and organs affected by autoimmune diseases. Here we describe the results of in vitro and animal experiments with 99mTc-succinimidyl-6-hydrazinonicotinate hydrochloride (SHNH)–visilizumab. Methods: For mAb labeling, we used a 2-step method with a heterobifunctional linker SHNH. Several titrations were performed to obtain the best labeling efficiency. In vitro quality controls included stability assay, cysteine challenge, sodium dodecyl sulfate polyacrylamide gel electrophoresis, binding assay, and immunoreactivity assay. In vivo studies by high-resolution images were performed at 6 and 24 h after the injection of 99mTc-SHNH–visilizumab. These included cell-targeting experiments in BALB/c mice xenografted subcutaneously with an increasing number of HuT78 cells in the leg and displaced with an excess of cold antibody. We also studied irradiated severe combined immunodeficient (SCID) mice reconstituted with human peripheral blood mononuclear cells (hPBMCs) and injected with 99mTc-labeled visilizumab or control mAb. After dynamic imaging for 3 h, major organs were removed, counted, and processed for immunohistologic examination. Results: Visilizumab was labeled with HYNIC with high labeling efficiency (>90%) and high specific activity (SA; 10,360–11,100 MBq/mg), with retained biochemical integrity and in vitro binding activity to CD3-positive cells. The in vivo targeting experiment showed a proportional increase of specific uptake with the number of injected cells, both at 6 and at 24 h, and the in vivo competition study demonstrated more than 60% decreased uptake after an excess of unlabeled antibody. In SCID mice, hPBMCs in different tissues were detected by 99mTc-labeled visilizumab and confirmed by histology. Conclusion: Visilizumab can be efficiently labeled with 99mTc with high efficiency and SA and could be a valuable tool for the study of human T-lymphocyte trafficking and lymphocytic infiltration of tissues and organs.

⁹⁹mTc-anti-TNF-α antibody for the imaging of disease activity in pulmonary sarcoidosis.

Infliximab, a monoclonal antibody directed against tumour necrosis factor (TNF)-α, is used in the treatment of refractory sarcoidosis. However, the clinical response is variable and a tool to select responders beforehand is highly desirable. In this study we evaluated scintigraphy with technetium-99m (99mTc)-labelled infliximab for the imaging of disease activity in patients with pulmonary sarcoidosis. 10 patients were studied using single photon emission computed tomography/computed tomography (CT) 6 h and 20 h after intravenous administration of 370 MBq of 99mTc-infliximab. Correlation analysis was performed between tissue accumulation of 99mTc-infliximab and laboratory parameters (including soluble interleukin-2 receptor and angiotensin-converting enzyme), lung function parameters (including forced expiratory volume in 1 s and the diffusing capacity of the lung for carbon monoxide) and 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET)/CT. Analysis showed selective and variable accumulation of 99mTc-infliximab in the target tissue. Accumulation correlated positively with all four laboratory parameters and negatively with all four lung function parameters, yielding better correlations than serum TNF-α levels or 18F-FDG PET/CT. 99mTc-infliximab accumulation reflects the in situ TNF-α expression in an individual patient and therefore provides valuable information on the presence of the biological target for anti-TNF-α therapy. 99mTc-TNF-α antibody accumulation reflects presence of the biological target for anti-TNF therapy in sarcoidosis http://ow.ly/V92cz

In Vivo Imaging of Natural Killer Cell Trafficking in Tumors

Natural killer cells (NKs) are important effectors of the innate immune system, with marked antitumor activity. Imaging NK trafficking in vivo may be relevant to following up the efficacy of new therapeutic approaches aiming at increasing tumor-infiltrating NKs (TINKs). The specific aims of present study were to efficiently target NKs using a 99mTc-anti-CD56 and to image human NK trafficking in SCID mice bearing human cancer. Methods: The anti-CD56 monoclonal antibody (mAb) was radiolabeled with 99mTc, and in vitro quality controls were performed to test labeling efficiency, stability, and binding affinity to CD56. In vivo biodistribution was determined by injecting 5.5 MBq (104 ng) of radiolabeled antibody in the tail vein of SCID mice, which were then sacrificed at 1, 3, 6, and 24 h after injection. Targeting experiments were performed on 2 groups of SCID mice inoculated subcutaneously with increasing numbers of human NKs in the right thigh (from 2.5 × 106 to 40 × 106) and human granulocytes (CD56−) or anaplastic thyroid cancer (ARO) cells in the contralateral thigh as control. TINK trafficking imaging was achieved by injecting 5.5 MBq of 99mTc-anti-CD56 mAb in SCID mice bearing ARO tumor xenografts in the right thigh, 24 h after being reconstituted with 105, 106, or 107 human NKs. Results: Anti-CD56 mAb was radiolabeled, achieving a radiochemical purity of more than 97% and a specific activity of 3,700 MBq/mg and retaining biochemical integrity and binding activity. In vivo studies revealed physiologic uptake in the liver and kidneys. Targeting experiments confirmed the specificity of labeled antibody to CD56+ cells. Human NK cells injected in CD1 nude mice accumulated in the ARO tumors within 24 h and were imaged as early as 3 h after intravenous administration of 99mTc-anti-CD56. Conclusion: 99mTc-anti-CD56 is a promising tool for in vivo imaging of TINK cell trafficking.

In Vivo Evaluation of TNF-Alpha in the Lungs of Patients Affected by Sarcoidosis

Introduction. Sarcoidosis is a multisystemic granulomatous disorder characterized by multiple noncaseating granulomas involving intrathoracic lymph nodes and lung parenchyma. Recently, the use of anti-tumor necrosis factor alpha (anti-TNFα) agents has been introduced for therapy of chronic and refractory sarcoidosis with controversial results. Infliximab (Remicade) is a chimeric monoclonal antibody (mAb) that recognizes and binds TNFα, neutralizing its biological effects. In the present study,   99mTc labelled infliximab was used to study the expression of TNFα in sarcoid lesions and to evaluate its role as a predictive marker in response to therapy with Remicade. Material and Methods. A total of 10 patients with newly diagnosed sarcoidosis were enrolled together with 10 control patients affected by rheumatoid arthritis. All patients were studied by planar imaging of the chest with   99mTc-infliximab at 6 h and 24 h and total body [18F]-FDG PET/CT. Regions of interest were drawn over the lungs and the right arm and target-to-background ratios were analysed for   99mTc-infliximab. SUVmean and SUVmax were calculated over lungs for FDG. Results and Discussion. Image analysis showed low correlation between T/B ratios and BAL results in patients despite positivity at [18F]-FDG PET. Conclusion. In conclusion, patients with newly diagnosed pulmonary sarcoidosis, with FDG-PET and BAL positivity, showed a negative   99mTc-infliximab scintigraphy.

PET imaging to monitor cancer therapy.

Improved knowledge and understanding of key aspects of cancer has led to the development of novel cancer therapeutics acting through complex pathways and mode of actions. The success of these novel cancer therapeutics is often difficult to predict using standard response criteria based on anatomic changes. Monitoring response to cancer therapy at molecular level using Positron Emission Tomography (PET) has gained popularity in recent years. PET allows longitudinal assessment of specific biological processes rather than just changes in anatomic changes in tumor size. In this review, we provide an overview on application for PET imaging to monitor cancer therapy with emphases on PET of tumor metabolism, cell proliferation, angiogenesis, hypoxia and receptor dynamics.

Radiopharmaceuticals for imaging chronic lymphocytic inflammation

In the last few decades, a number of radiopharmaceuticals for imaging inflammation have been proposed that differ in their specificity and mechanism of uptake in inflamed foci as compared to the traditional inflammation imaging agents. Radiolabelled cytokines represent a reliable tool for the preclinical diagnosis of chronic inflammatory processes, even before anatomical and functional changes occur in affected tissues. Moreover, the introduction of radiolabelled monoclonal antibodies and sophisticated technique like PET/CT now make the field of inflammation imaging highly specific and accurate. In this review, different approaches of the established and experimental radiopharmaceuticals for imaging of chronic inflammation are discussed.

Infection and inflammation imaging

In a recent issue of the journal, Steinhoff K. et al. (2014) stated that Malviya G. et al. have described different tracer uptake in infected joints of the same patient tested with Tc-rituximab (anti-CD20-mAb) and Tc-adalimumab (anti-TNF-α-mAb). This sentence is misleading and requires clarification, in fact we never investigated and reported infection in this patient [1]. Our aimwas to demonstrate that different joints in rheumatoid arthritis patients may have different kind of inflammatory reaction and it is therefore desirable to perform a targeted individualised therapy based on scintigraphic evaluation of joint activity. In particular, I think it is important to correctly describe the terms ‘infection’ and ‘inflammation’. In a recently published letter [2], Signore A. clearly explained this difference and the twowords cannot be used as synonymous. Indeed, we may have an inflammation caused by an infection, but not always we have an infection if there is an inflammation. In case of autoimmune diseases, and Rheumatoid Arthritis in particular, we aim at imaging inflammation, and not infection, since radiopharmaceuticals for infection imaging are different from those used for inflammation imaging [3,4]. Therefore, careful selectionof theseboth terms is required,whileusing them in any specific context, otherwise it might cause misperception.

Isolation and 111In–Oxine Labeling of Murine NK Cells for Assessment of Cell Trafficking in Orthotopic Lung Tumor Model

A noninvasive in vivo imaging method for NK cell trafficking is essential to gain further understanding of the pathogenesis of NK cell mediated immune response to the novel cancer treatment strategies, and to discover the homing sites and physiological distribution of NK cells. Although human NK cells can be labeled for in vivo imaging, little is known about the murine NK cell labeling and its application in animal models. This study describes the isolation and ex vivo radiolabeling of murine NK cells for the evaluation of cell trafficking in an orthotopic model of human lung cancer in mice. Scid-Tg(FCGR3A)Blt transgenic SCID mice were used to isolate NK cells from mouse splenocytes using the CD49b (DX5) MicroBeads positive selection method. The purity and viability of the isolated NK cells were confirmed by FACS analysis. Different labeling buffers and incubation times were evaluated to optimize (111)In-oxine labeling conditions. Functionality of the radiolabeled NK cell was assessed by (51)Cr-release assay. We evaluated physiological distribution of (111)In-oxine labeled murine NK cells in normal SCID mice and biodistribution in irradiated and nonirradiated SCID mice with orthotopic A549 human lung tumor lesions. Imaging findings were confirmed by histology. Results showed that incubation with 0.011 MBq of (111)In-oxine per million murine NK cells in PBS (pH 7.4) for 20 min is the best condition that provides optimum labeling efficiency without affecting cell viability and functionality. Physiological distribution in normal SCID mice demonstrated NK cells homing mainly in the spleen, while (111)In released from NK cells was excreted via kidneys into urine. Biodistribution studies demonstrated a higher lung uptake in orthotopic lung tumor-bearing mice than control mice. In irradiated mice, lung tumor uptake of radiolabeled murine NK cells decreased between 24 h and 72 h postinjection (p.i.), which was accompanied by tumor regression, while in nonirradiated mice, radiolabeled NK cells were retained in the lung tumor lesions up to 72 h p.i. without tumor regression. In tumor-bearing mice that were only irradiated but did not receive radiolabeled murine NK cells, a high tumor burden was observed at 72 h p.i., which indicates that irradiation in combination with murine NK cell allocation, but not irradiation alone, induced a remarkable antitumor effect in the orthotopic A549 lung tumor bearing mouse model. In conclusion, we describe a method to evaluate murine NK cell trafficking and biodistribution, which can be used to determine potential effects of immune-mediated therapeutic agents on NK cell biodistribution.