Safe and Effective Sarcoma Therapy through Bispecific Targeting of EGFR and uPAR.

Sarcomas differ from carcinomas in their mesenchymal origin. Therapeutic advancements have come slowly, so alternative drugs and models are urgently needed. These studies report a new drug for sarcomas that simultaneously targets both tumor and tumor neovasculature. eBAT is a bispecific angiotoxin consisting of truncated, deimmunized Pseudomonas exotoxin fused to EGF and the amino terminal fragment of urokinase. Here, we study the drug in an in vivo "ontarget" companion dog trial as eBAT effectively kills canine hemangiosarcoma and human sarcoma cells in vitro We reasoned the model has value due to the common occurrence of spontaneous sarcomas in dogs and a limited lifespan allowing for rapid accrual and data collection. Splenectomized dogs with minimal residual disease were given one cycle of eBAT followed by adjuvant doxorubicin in an adaptive dose-finding, phase I-II study of 23 dogs with spontaneous, stage I-II, splenic hemangiosarcoma. eBAT improved 6-month survival from <40% in a comparison population to approximately 70% in dogs treated at a biologically active dose (50 µg/kg). Six dogs were long-term survivors, living >450 days. eBAT abated expected toxicity associated with EGFR targeting, a finding supported by mouse studies. Urokinase plasminogen activator receptor and EGFR are targets for human sarcomas, so thorough evaluation is crucial for validation of the dog model. Thus, we validated these markers for human sarcoma targeting in the study of 212 human and 97 canine sarcoma samples. Our results support further translation of eBAT for human patients with sarcomas and perhaps other EGFR-expressing malignancies. Mol Cancer Ther; 16(5); 956-65. ©2017 AACR.

Potent antirheumatic activity of a new DNA vaccine targeted to B7-2/CD28 costimulatory signaling pathway in autoimmune arthritis.

Rheumatoid arthritis is a proinflammatory autoimmune disease attributed to failure of both CD4(+)CD25(+) regulatory T (Tr) and CD8(+)CD28(-) suppressor T (Ts) cells to control autoreactive CD4(+)CD28(+) Th1 (Th1) and autoantibody-producing B cells. Here we show a single intramuscular injection of our novel targeted DNA vaccine encoding Pseudomonas exotoxin A and costimulatory molecule B7-2 without autoantigens in a collagen-induced arthritis model simultaneously increased Tr and Ts cells and selectively decreased autoreactive Th1 cells. The vaccine induced a shift from Th1 to Th2 and Th3 cellular and cytokine profiles and a decrease in CD4(+)/CD8(+) cell ratios. Importantly, the vaccine showed potent antirheumatic activity by clinical and other examinations such as X-ray, histopathology, and anti-type II collagen IgG levels and was comparable to methotrexate, the current "gold standard" treatment. As an effective stimulator of both Tr and Ts cells and a specific suppressor of autoreactive Th1 cells, this vaccine is a promising therapeutic approach for rheumatoid arthritis.

Neuroradiographic changes following convection-enhanced delivery of the recombinant cytotoxin interleukin 13-PE38QQR for recurrent malignant glioma.

OBJECT: Convection-enhanced delivery (CED) is a novel method for delivering therapeutic agents to infiltrative brain tumor cells. For agents administered by CED, changes on magnetic resonance (MR) imaging directly resulting from catheter placement, infusion, and the therapeutic compound may confound any interpretation of tumor progression. As part of an ongoing multiinstitutional Phase I study, 14 patients with recurrent malignant glioma underwent CED of interleukin (IL) 13-PE38QQR, a recombinant cytotoxin consisting of human IL-13 conjugated with a truncated Pseudomonas exotoxin. Serial neuroradiographic changes were assessed in this cohort of patients. METHODS: Patients were treated in two groups: Group 1 patients received IL13-PE38QQR before and after tumor resection; Group 2 patients received infusion only after tumor resection. Preoperative and postinfusion MR images were obtained prospectively at specified regular intervals. Changes were noted along catheter tracks on postresection MR images obtained in all patients. A simple grading system was developed to describe these changes. When MR imaging changes appeared to be related to IL1 3-PE38QQR, patients were followed up without instituting new antitumor therapy. CONCLUSIONS: As CED of therapeutic agents becomes more common, clinicians and investigators must become aware of associated neuroimaging changes that should be incorporated into toxicity assessment. We have developed a simple grading system to facilitate communication about these changes among investigators. Biological imaging modalities that could possibly distinguish these changes from recurrent tumor should be evaluated. In this study the authors demonstrate the challenges in determining efficacy when surrogate end points such as time to tumor progression as defined by new or progressive contrast enhancement on MR imaging are used with this treatment modality.

Progress report of a Phase I study of the intracerebral microinfusion of a recombinant chimeric protein composed of transforming growth factor (TGF)-alpha and a mutated form of the Pseudomonas exotoxin termed PE-38 (TP-38) for the treatment of malignant brain tumors.

TP-38 is a recombinant chimeric targeted toxin composed of the EGFR binding ligand TGF-alpha and a genetically engineered form of the Pseudomonas exotoxin, PE-38. After in vitro and in vivo animal studies that showed specific activity and defined the maximum tolerated dose (MTD), we investigated this agent in a Phase I trial. The primary objective of this study was to define the MTD and dose limiting toxicity of TP-38 delivered by convection-enhanced delivery in patients with recurrent malignant brain tumors. Twenty patients were enrolled in the study and doses were escalated from 25 ng/mL to 100 with a 40 mL infusion volume delivered by two catheters. One patient developed Grade IV fatigue at the 100 ng/mL dose, but the MTD has not been established. The overall median survival after TP-38 for all patients was 23 weeks whereas for those without radiographic evidence of residual disease at the time of therapy, the median survival was 31.9 weeks. Overall, 3 of 15 patients, with residual disease at the time of therapy, have demonstrated radiographic responses and one patient with a complete response and has survived greater than 83 weeks.

Interleukin-13 receptor-directed cytotoxin for malignant glioma therapy: from bench to bedside.

Central nervous system malignant neoplasias, in particular, glioblastoma multiforme (GBM) have defied all current therapeutic modalities. New therapies involving tumor targeting approach are being explored. This approach relies on the identification of unique or over-expressed cell surface receptors or antigens on tumor cells. In that regard, we have identified receptor for an immune regulatory cytokine, interleukin-13 (IL-13), which is over-expressed on human malignant glioma cell lines and primary tumor cell cultures. To target IL-13 receptors (IL-13R) for cancer therapy, we have developed a recombinant fusion protein composed of IL-13 and a mutated form of Pseudomonas exotoxin (IL13-PE38QQR or IL-13 cytotoxin). The IL-13 cytotoxin was found to be highly selective and potent in killing human GBM cells in vitro while normal cells including immune cells, endothelial cells and normal brain cells were generally spared the cytotoxic effect of IL-13 cytotoxin. This is because these cells either expressed none or expressed low levels of IL-13R. Consistent with in vitro cytotoxic activity, IL-13 cytotoxin mediated remarkable anti-tumor activity to human glioma in animal xenograft models. The direct injection of IL-13 cytotoxin into subcutaneous human GBM tumors grown in nude mice produced complete and durable regression of established tumors. Intravenous and intraperitoneal administration of IL-13 cytotoxin also reduced tumor burden significantly with fewer complete responders. All animals tolerated therapy well with minimal toxicity to vital organs. Pre-clinical safety and toxicity studies were performed in mice, rats and monkeys. Systemic administration of IL-13 cytotoxin appeared to be well tolerated at high doses (up to 50 microg/kg). Intrabrain parenchyma administration of IL-13 cytotoxin at doses up to 100 microg/ml was very well tolerated without any evidence of gross or microscopic necrosis, whereas at 500 microg/ml dose, localized necrosis was observed in normal rat brain. Based on these encouraging pre-clinical studies, three Phase I/II clinical trials in adults with malignant glioma have been initiated. The first clinical trial involves convection-enhanced delivery (CED) of IL-13 cytotoxin into recurrent malignant glioma. This route of IL-13 cytotoxin administration appears to be fairly well tolerated with no neurotoxicity. The second clinical trial involves infusion of IL-13 cytotoxin by CED following tumor resection. The initial stage of the second study assessed histologic effect of drug administered prior to resection. In third one, IL-13 cytotoxin is infused by CED followed by tumor resection. All three clinical trials are currently ongoing.

IL-13 fusion cytotoxin ameliorates chronic fungal-induced allergic airway disease in mice.

IL-13 has emerged as a major contributor to allergic and asthmatic responses, and as such it represents an attractive target in these diseases. In this study, IL-13-responsive cells in the lung were targeted via the intranasal administration of IL-13-PE38QQR (IL-13-PE), comprised of human IL-13 and a derivative of Pseudomonas exotoxin, to Aspergillus fumigatus-sensitized mice challenged with A. fumigatus spores, or conidia. Mice received 50, 100, or 200 ng of IL-13-PE or diluent alone (i.e., control group) on alternate days from day 14 to day 28 after the conidia challenge. The control group of mice exhibited significant airway hyperreactivity, goblet cell hyperplasia, and peribronchial fibrosis at day 28 after conidia. Although the two lower doses of IL-13-PE had limited therapeutic effects in mice with fungal-induced allergic airway disease, the highest dose of IL-13-PE tested significantly reduced all features of airway disease compared with the control group. Whole lung mRNA expression of IL-4Ralpha and IL-13Ralpha1 was markedly reduced, whereas bronchoalveolar lavage and whole lung levels of IFN-gamma were significantly elevated in mice treated with 200 ng of IL-13-PE compared with the control group. This study demonstrates that a therapy designed to target IL-13-responsive cells in the lung ameliorates established fungal-induced allergic airway disease in mice.

Purging of mammary carcinoma cells during ex vivo culture of CD34+ hematopoietic progenitor cells with recombinant immunotoxins.

Tumor cells have been found in autologous hematopoietic cell transplants used after high-dose chemotherapy. To specifically eliminate contaminating mammary tumor cells during ex vivo expansion of CD34+ hematopoietic progenitor cells, we used recombinant immunotoxins (ITs) directed against cell-surface antigens expressed on mammary carcinoma cells. ITs were expressed from fusion cDNAs combining a single-chain antibody fragment (scFv) directed against the Erb-B2 or epidermal growth factor (EGF) receptors with a truncated Pseudomonas exotoxin A fragment devoid of its cell-binding domain. CD34+ hematopoietic progenitor cells did not express Erb-B2 and EGF receptors as detected by Western blotting. Ex vivo expansion of total hematopoietic cells or of colony-forming cells from CD34+ progenitors in the presence of stem-cell factor (SCF), interleukin-1 (IL-1), IL-3, IL-6, and erythropoietin (Epo) was not affected when ITs were added to the cultures. In contrast, MDA-MB 453 and MCF-7 mammary carcinoma cells were depleted in a dose- and time-dependent manner by more than 3 log in coculture with CD34+ cells over a period of 7 days in the presence of 100 to 1,000 ng/mL of anti-Erb-B2 IT. This included elimination of the subpopulations with regrowth potential. Similarly, addition of either anti-Erb-B2 or anti-EGF receptor ITs to primary breast cancer cells isolated from patients with metastatic disease resulted in elimination of cytokeratin-positive cells in seven of seven samples. ITs are highly efficient and convenient to use for the depletion of mammary tumor cells during ex vivo expansion of hematopoietic progenitor-cell autografts.

Group A streptococcal pyrogenic exotoxin: pyrogenicity, alteration of blood-brain barrier, and separation of sites for pyrogenicity and enhancement of lethal endotoxin shock.

Group A streptococcal pyrogenic exotoxin type C (SPE C) was shown to produce fever by crossing the blood-brain barrier. The toxin directly stimulated the hypothalamic fever response control center, thus bypassing a requirement for endogenous pyrogen release. SPE C was detected in the cerebrospinal fluids of toxin-treated rabbits by pyrogen tests and a hemagglutination inhibition assay. The toxin altered the permeability of the blood-brain barrier to endotoxin, Streptococcus pneumoniae, and Haemophilus influenzae as well as to itself. SPE C did not alter the in vivo differential and total counts of peripheral blood leukocytes and did not elicit endogenous pyrogen release from leukocytes in vitro. In vivo, peripheral blood platelet counts remained unchanged after SPE treatment. Cycloheximide pretreatment of rabbits did not inhibit fever production by SP C. In contrast to the hypothermia observed in mice treated with endotoxin intravenously susceptibility to lethal endotoxin shock. The abilities of SPE C to produce fever and enhance lethal shock were shown to be separate functions of the molecule; fever results from stimulation of the hypothalamus, and enhancement appears not to involve the central nervous system.