Syed M. Quadri

Caspase inhibition improves ischemia-reperfusion injury after lung transplantation.

Ischemia‐reperfusion injury is associated with cell death in many organ systems. The role of programmed cell death (PCD) pathways and the ultimate clinical relevance of PCD in the context of lung transplantation (LTx) are unknown.

Transbronchial administration of adenoviral-mediated interleukin-10 gene to the donor improves function in a pig lung transplant model

Interleukin-10 (IL-10) gene transfection of donor lungs prior to transplantation is an attractive strategy to reduce ischemia–reperfusion induced lung injury. However, experimental data with gene therapy in large animal models of lung transplantation are generally lacking. We have developed a simple clinically applicable technique for adenoviral-mediated gene delivery of human IL-10 to the lung of large animals that provides homogenous gene expression after 12–24 h of transfection. Using this technique of gene delivery, we have studied the dynamics of adenoviral gene delivery to the lung in the setting of lung transplantation. Although there is a persistent inflammatory response to the adenoviral vector, we achieved significant expression of human IL-10 in lung tissue before lung retrieval to obviate the deleterious impact of the adenoviral vector on the donor lung. The administration of adenoviral-mediated human IL-10 to the donor lung reduced ischemia–reperfusion injury and improved graft function after lung transplantation in this pig lung transplantation model. Transfection of adenoviral-mediated human IL-10 to the donor lung prevented the release of inflammatory cytokines such as IL-6 in lung tissue and plasma. We have demonstrated that IL-10 gene therapy has significant potential to prevent or treat the inflammatory response associated with ischemia–reperfusion injury in lung transplantation. In the future, IL-10 gene therapy could also be used for immunomodulation or tolerance induction.

Yttrium 90-labeled antiferritin followed by high-dose chemotherapy and autologous bone marrow transplantation for poor-prognosis Hodgkin's disease.

PURPOSE This study was undertaken to examine the feasibility of combining radiolabeled antibody therapy with high-dose chemotherapy followed by autologous bone marrow transplantation in patients with poor-prognosis Hodgkins disease. PATIENTS AND METHODS Patients were entered onto this protocol if they had chemotherapy-resistant disease, bulky disease, or extensive prior therapy. Patients received yttrium-labeled antiferritin on day -13, -12, or -11, followed by high-dose cyclophosphamide, carmustine, and etoposide (CBV) on days -6 to -3, and then bone marrow infusion on day 0. RESULTS Twelve patients received both radiolabeled antibody and high-dose chemotherapy followed by autologous transplantation. Two additional patients started the study, but were unable to complete all therapy. Four of 12 patients experienced early transplant-related mortality. Four patients are alive more than 2 years following transplantation and three are free from disease progression at 24+, 25+, and 28+ months following transplantation. The progression-free survival rate at 1 year is estimated to be 21%. Considering the poor prognostic characteristics of these patients, toxicity on this protocol was not necessarily greater than that observed with high-dose chemotherapy alone. CONCLUSION This report demonstrates the feasibility of combining radiolabeled antibody therapy with high-dose chemotherapy and autologous bone marrow transplantation.

Survival of patients with resistant Hodgkin's disease after polyclonal yttrium 90-labeled antiferritin treatment.

PURPOSE A follow-up study was initiated of patients with Hodgkins disease who were treated with yttrium 90-labeled antiferritin. Prescription method, pharmacokinetics, acute and late side effects, and survival were evaluated. METHODS Patients had measurable disease and failed > or = two multiagent chemotherapy regimens previously (N = 44). All patients received 5-mCi indium 111-labeled antiferritin 2 mg intravenously and were scanned repeatedly by gamma camera. In five patients, polyclonal antiferritin (rabbit, pig, or baboon) failed to target the tumor. Thirty-nine patients were injected intravenously with 10-, 20-, 30-, 40-, or 50-mCi yttrium 90-labeled antiferritin 2 to 5 mg. Patients received between one and five cycles. Some patients were supported with 5 x 10(7) autologous bone marrow cells per kilogram. RESULTS Yttrium 90-labeled polyclonal antiferritin does not produce immunologic, pharmacologic, or microbiologic complications in vivo. Bone marrow toxicity is the only side effect observed. Overall response rate is 20 of 39, or 51%. Two patients had stable disease. A significant positive correlation is found between blood radioactivity level 1 hour after radioimmunoconjugate administration and subsequent response of Hodgkins disease. A dosage in millicuries per kilogram provides a higher positive correlation with blood radioactivity levels 1 hour after administration than a dosage in millicuries per square meter of body-surface area or in total millicuries. Fifty percent of patients survive for > or = 6 months. CONCLUSION The low-dose protein used (2 to 5 mg) indicates that the high response rate is due to radiation and not to immunologic effects of the antibody. High-activity administrations followed by bone marrow transplantation are not required for tumor response. The therapeutic ratio of radiolabeled antiferritin is higher than the therapeutic ratio observed in most phase I studies of chemotherapeutic agents. This analysis does not identify a superior mode of treatment for patients with end-stage Hodgkins disease. However, in a heavily pretreated patient population, prolonged survival is observed after relatively inexpensive treatment. Preclinical research with yttrium 90-labeled antiferritin indicates that significant increases in tumor dose can be obtained in the future without an increase in normal tissue toxicity.

Selection of reagents for human radioimmunotherapy.

Promising response rates are noted in patients with refractory Hodgkins disease after radioimmunoglobulin therapy (RIT) with Yttrium-90 labeled polyclonal antiferritin. To explore the most efficacious selection of RIT reagents for use in humans, experimental animal data are reviewed for radiolabeled antiferritin and B72.3. Nude mice with subcutaneously implanted human malignancies provide an excellent primary screen for radiolabeled antibodies under consideration for use in humans. They provide information on the potential of a new reagent to target a human malignancy in vivo. The other determinant of the therapeutic ratio of RIT reagents--normal tissue toxicity--is best analyzed in large animals, such as dogs. Hematologic toxicity is dose limiting in all species and best predicted by a prescription of radiolabeled antibodies in mCi per kilogram body weight and the presence or absence of bone marrow targeting. Per cGy, RIT is more effective in causing BM damage in dogs than in rats. In dogs, bone marrow transplantation with autologous cryopreserved bone marrow cells or G-CSF treatment can accelerate hemopoietic recovery and granulopoiesis, respectively, after RIT. When dose escalation beyond bone marrow toxicity is performed, the liver (dog) or the intestinal tract (rat) become the next dose limiting tissue in dose escalation studies. Significant improvement in RIT results will be achieved when the normal liver uptake of chelated monoclonal antibody in dogs and in human patients can be prevented. The described animal models and continued investigations of RIT in patients with endstage Hodgkins disease will allow for further improvement in the therapeutic ratio of RIT and the applicability of RIT in humans.

Liver Toxicity Induced by Combined External-Beam Irradiation and Radioimmunoglobulin Therapy

High-dose radiation therapy for liver metastases of gastrointestinal malignancies might be improved by combining external-beam irradiation and radioimmunoglobulin therapy. We studied the liver toxicity of the proposed combination in healthy beagle dogs. A total dose of 30 Gy to the whole liver, delivered in 2-Gy fractions over 3 weeks, resulted in mild, temporary veno-occlusive disease (VOD) in three of three dogs. Reversible bone marrow damage was noted after two intravenous injections of 18.5 MBq of yttrium-90-labeled monoclonal antibody ZCE025 per kg body weight in three of three dogs. Administrations of the antibody were separated by 1 week. Three dogs treated by irradiation of the liver with radioimmunoglobulin therapy added during the last 2 weeks of the irradiation showed signs of radiation hepatitis (VOD) starting around 35 days after treatment. One dog had a complete recovery, and two dogs were euthanized in a stage of terminal liver failure around day 90 after treatment. Temporary bone marrow damage was observed after the combined treatment, similar to the bone marrow damage observed after radioimmunoglobulin therapy alone. Earlier studies in the same dog model showed that bone marrow is the dose-limiting organ if radioimmunoglobulin therapy is used alone. The addition of irradiation of the liver to radioimmunoglobulin therapy changes the dose-limiting organ from bone marrow to liver. The radiation hepatitis observed in dogs is very similar to that observed in humans and is reflected in early platelet consumption in the irradiated liver plus late elevations of liver enzymes and VOD in central hepatic veins on histological analysis. Future applications of combined liver irradiation and radioimmunoglobulin therapy in humans should use radioimmunoglobulin therapy agents which show minimal uptake by normal liver.

Antiadrenergic autoimmunity in postural tachycardia syndrome

Abstract Aims Postural tachycardia syndrome (POTS), a common and debilitating cardiovascular disorder, is characterized by an exaggerated heart rate increase during orthostasis and a wide spectrum of adrenergic-related symptoms. To determine the aetiology of POTS, we examined a possible pathophysiological role for autoantibodies against α1-adrenergic (α1AR) and β1/2-adrenergic receptors (β1/2AR). Methods and results Immunoglobulin G (IgG) derived from 17 POTS patients, 7 with recurrent vasovagal syncope (VVS), and 11 normal controls was analysed for its ability to modulate activity and ligand responsiveness of α1AR and β1/2AR in transfected cells and to alter contractility of isolated rat cremaster arterioles in vitro. Immunoglobulin G activation of α1AR and β1/2AR was significantly higher in POTS compared with VVS and controls in cell-based assays. Eight, 11, and 12 of the 17 POTS patients possessed autoantibodies that activated α1AR, β1AR and β2AR, respectively. Pharmacological blockade suppressed IgG-induced activation of α1AR and β1/2AR. Eight of 17 POTS IgG decreased the α1AR responsiveness to phenylephrine and 13 of 17 POTS IgG increased the β1AR responsiveness to isoproterenol irrespective of their ability to directly activate their receptors. Postural tachycardia syndrome IgG contracted rat cremaster arterioles, which was reversed by α1AR blockade. The upright heart rate correlated with IgG-mediated β1AR and α1AR activity but not with β2AR activity. Conclusion These data confirm a strong relationship between adrenergic autoantibodies and POTS. They support the concept that allosteric-mediated shifts in the α1AR and β1AR responsiveness are important in the pathophysiology of postural tachycardia.

Fractionated intravenous administration of 90Y-labeled B72.3 GYK-DTPA immunoconjugate in beagle dogs

B72.3, a monoclonal antibody with reactivity against human adenocarcinomas, was coupled with linker-chelator GYK-DTPA using carbohydrate mediated conjugation chemistry and radiolabeled with yttrium-90. Single and double intravenous injections of radioimmunoconjugate were compared for acute and late normal tissue toxicity in 15 beagle dogs. The second injection was given 4 or 8 days after the first. Pharmacokinetics of the radioimmunoconjugate in blood, bone marrow and urine were similar for first and second injections. Only bone marrow (acute) and liver (late) toxicity were observed. Both liver and bone marrow toxicity were decreased by fractionation of the injections. After double injections, the total equitoxic dose was 15 and 60% higher for bone marrow and liver toxicity, respectively. The mechanisms of normal tissue protection offered by fractionated radioimmunoglobulin therapy (RIT) remain to be defined. Fractionated RIT will have a better therapeutic ratio than single injection RIT, if antitumor effects appear to be less susceptible to fractionation than normal tissues.

Preclinical evaluation of intravenously administered 111in- and 90y-labeled b72.3 immunoconjugate (GYK-DTPA) in beagle dogs

B72.3, a monoclonal antibody with reactivity against human adenocarcinomas was obtained from the Cytogen Corporation in the form of an immunoconjugate coupled with linker-chelator GYK-DTPA by using proprietary carbohydrate directed site specific chemistry. The immunoconjugate was radiolabeled with indium-111 or yttrium-90. A preclinical analysis was performed in 10 normal beagle dogs. The pharmacokinetics of intravenously administered indium- and yttrium-labeled immunoconjugates were compared serially in blood, bone marrow and urine samples. Compared to 90Y less of the 111In label ended up in urine and more was found in blood and bone marrow. Indium-labeled B72.3 GYK-DTPA had relatively higher uptake in most glandular tissues than 111In-labeled antiferritin immunoconjugate. Bone marrow toxicity was the dose limiting side effect after intravenous infusion of 90Y-labeled B72.3 GYK-DTPA. Toxicity was also observed in the liver but not in other organ systems. Recently other investigators obtained similar results with these immunoconjugates in human patients. A preclinical pharmacokinetic analysis of radioimmunoconjugates in beagle dogs provided useful information regarding bone marrow toxicity, liver toxicity and in vivo instability of the immunoconjugate. Data suggest that for future trials in human patients, a more stable chelated immunoconjugate for yttrium is needed to achieve less liver uptake and a better correlation with the 111In-labeled product than the 90Y-labeled B72.3 GYK-DTPA used in this investigation.

Tumour Therapy with Radiolabelled Antibodies

The promise of radiolabelled immunoglobulin therapy (RIT) for selective, patient friendly, cancer treatment has not yet been fulfilled. Only patients with haematological malignancies show sizable response rates after RIT. With solid tumours, intravenous administration of radiolabelled antibodies does not provide sufficient tumour targeting. However, intracompartmental administration may solve this problem, particularly if tumour reactive IgM is used.Clinical progress in RIT depends on understanding the important RIT variables: antigen, antibody, radioisotope, conjugation chemistry, activity escalation, fractionation and protein dose. These are reviewed and a new translational decision tree/flow diagram is presented that can limit analysis to the most important RIT variables for a particular disease. These variables may differ depending on the type and stage of cancer, but the guiding principles in RIT development remain the same: selectivity and accountability. The proper application of these principles leads to the definition of a new series of phase I, II, III studies. These studies are more appropriate for the clinical exploration of RIT and place an emphasis on therapeutic ratio rather than toxicity.