Search

Costimulation via CD137 (4-1BB) enhances antitumor immunity mediated by cytotoxic T lymphocytes. Anti-CD137 agonist antibodies elicit mild liver inflammation in mice, and the maximum tolerated dose of Urelumab, an anti-human CD137 agonist monoclonal antibody, in the clinic was defined by liver inflammation-related side effects. A protease-activated prodrug form of the anti-mouse CD137 agonist antibody 1D8 (1D8 Probody therapeutic, Pb-Tx) was constructed and found to be selectively activated in the tumor microenvironment. This construct, which encompasses a protease-cleavable linker holding in place a peptide that masks the antigen binding site, exerted antitumor effects comparable to the unmodified antibody but did not result in liver inflammation. Moreover, it efficaciously synergized with both PD-1 blockade and adoptive T-cell therapy. Surprisingly, minimal active Pb-Tx reached tumor-draining lymph nodes, and regional lymphadenectomy did not abrogate antitumor efficacy. By contrast, S1P receptor-dependent recirculation of T cells was absolutely required for efficacy. The preferential cleavage of the anti-CD137 Pb-Tx by tumor proteases offers multiple therapeutic opportunities, including neoadjuvant therapy, as shown by experiments in which the Pb-Tx is given prior to surgery to avoid spontaneous metastases., Competing Interests: Competing interest statement: W.M.K., O.V., M.B., B.H., B.I., K.T., J.W., and L.M. are full-time employees of CytomX. A.J.K., E.S., and J.J.E. are full-time employees of BMS. I.M. reports receiving commercial research grants from BMS, Bioncotech, Alligator, Pfizer, Leadartis, Genmab, and Roche; has received speakers bureau honoraria from MSD; and is a consultant or advisory board member for BMS, Roche, Genmab, F-Star, Bioncotech, Bayer, Numab, Pieris, Alligator, and Merck Serono.

The internal ribosome entry site (IRES) in the 5' untranslated region (UTR) of the hepatitis C virus (HCV) genome initiates translation of the viral polyprotein precursor. The unique structure and high sequence conservation of the 5' UTR render the IRES RNA a potential target for the development of selective viral translation inhibitors. Here, we provide an overview of approaches to block HCV IRES function by nucleic acid, peptide, and small molecule ligands. Emphasis will be given to the IRES subdomain IIa, which currently is the most advanced target for small molecule inhibitors of HCV translation. The subdomain IIa behaves as an RNA conformational switch. Selective ligands act as translation inhibitors by locking the conformation of the RNA switch. We review synthetic procedures for inhibitors as well as structural and functional studies of the subdomain IIa target and its ligand complexes.

Thymidylate synthase (TS) is a key enzyme in the biosynthesis of thymidine. The use of TS inhibitors in cancer chemotherapy suffers from resistance development in tumors through upregulation of TS expression. Autoregulatory translation control has been implicated with TS overexpression. TS binding at its own mRNA, which leads to sequestration of the start codon, is abolished when the enzyme forms an inhibitor complex, thereby relieving translation suppression. We have used the protein-binding site from the TS mRNA in the context of a bicistronic expression system to validate targeting the regulatory motif with stabilizing ligands that prevent ribosomal initiation. Stabilization of the RNA by mutations, which were studied as surrogates of ligand binding, suppresses translation of the TS protein. Compounds that stabilize the TS-binding RNA motif and thereby inhibit ribosomal initiation might be used in combination with existing TS enzyme-targeting drugs to overcome resistance development during chemotherapy., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We report a new strategy for cell-type-specific delivery of functional siRNAs into cells. The method involves the noncovalent attachment of siRNAs to ligand-conjugated oligodeoxynucleotides via nucleic acid base-paired interactions. The resulting complexes can be directly applied to cells, leading to specific cellular uptake and gene silencing. The method is simple, economical, and can be easily adapted for other cell surface receptors. Here we show the application of this method for the delivery of siRNAs to folate receptor-expressing cells.

While modern genomic and proteomic technology enables rapid screening of novel proteins and peptides as potential drug candidates, design of delivery systems for these biologics remains challenging especially to achieve site-specific pharmacological actions. This article discusses the issues associated with targeted delivery of protein and peptide drugs at physiochemical, physiological, and intracellular levels with a special focus on cancer therapy.

We previously exploited the frequent overexpression of folate receptors on cancer cells to decorate malignant cell surfaces selectively with folate-hapten conjugates. In antihapten-immunized hosts, this targeted localization of foreign haptens to tumor cells led to rapid accumulation of autologous antihapten IgG, which in turn yielded potent antitumor activity upon stimulation with cytokines (IL-2, IFN-alpha). In an effort to understand the effector mechanisms responsible for tumor regression, we have now investigated the involvement of both humoral and cellular immune components in the tumor destruction process. We report that the dependence of therapeutic efficacy on folate-hapten concentration is bimodal, suggesting that the conjugate must bridge between a cell surface FR and an antihapten IgG in order to mediate killing. Studies with cancer cells in vitro further demonstrate that folate-fluorescein-marked tumor cells are killed primarily by antibody-dependent cellular cytotoxicity and phagocytosis, with no contribution from complement-dependent mechanisms. Investigations of specific immune cell involvement also reveal that asialo-GM1(+)-natural killer cells, macrophages, CD4+ T cells and CD8+ T cells contribute significantly to recognition/removal of the cancer mass, and that elimination of these cell types markedly compromises the therapy. Because the initial antibody-dependent stage of tumor cell killing is shown to lead to a long-term antibody-independent cellular immunity that involves both CD4+ and CD8+ T cells, we propose that F(c) receptor-expressing immune cells not only initiate destruction of the IgG-marked tumor cells, but also participate in presentation of endogenous tumor antigens in a manner that leads to long-term cellular immunity., ((c) 2005 Wiley-Liss, Inc.)

The vitamin, folic acid, has become a useful ligand for targeted cancer therapies because it binds to a tumor-associated antigen known as the folate receptor (FR). By linking folic acid to therapeutic agents, folate-targeted cancer therapies can deliver therapeutic drugs specifically to FR-positive tumor cells. This chapter provides a summary of a specific application of folate-targeted therapies whereby folic acid is exploited to carry an attached hapten (a highly antigenic molecule) to the surfaces of tumor cells for the purpose of rendering the tumors more immunogenic. The basic strategy is to (i) saturate (label) the surface of FR-positive tumor cells with a folate-hapten conjugate against which the cancer-bearing host already has a pre-existing or induced immunity, (ii) allow the surface bound haptens to attract anti-hapten antibodies to the tumor cell surface, and (iii) stimulate Fc receptor-bearing immune cells to mount an antitumor response against the anti-hapten antibody opsonized tumor cells. In immune competent murine tumor models, hapten-marked cancer cells have been shown to be quickly recognized by antibodies and the associated Fc receptor-expressing immune cells dedicated to eliminating antibody-coated target cells. Given the need for cancer cells to escape immune surveillance in order to proliferate and survive in vivo, folate-targeted immunotherapies that mark an otherwise immunologically "invisible" cancer cell as distinctively "non-self" may provide a key strategy for combating malignant disease.

Background and Objective: The arterial thermodilution technique offers the ability to measure cardiac output using only central venous and arterial catheters. However, the technique has been reported to overestimate cardiac output because of a higher loss of cold indicator due to the increased distance between the sites of injection and measurement. In this study, the two techniques were compared with respect to conditions of low cardiac output in which a longer passage time may further increase loss of indicator., Methods: Seventeen anaesthetized dogs were studied during hypovolaemic shock and fluid resuscitation. Cardiac output measurements were carried out simultaneously by arterial and pulmonary artery thermodilution techniques., Results: One-hundred-and-two measurements were performed. The mean cardiac output was 2.28 +/- 1.4Lmin(-1) by the pulmonary arterial technique and 2.29 +/- 1.56Lmin(-1) by the arterial thermodilution technique. The correlation coefficient between the two measurements was 0.95, the precision -0.04 +/- 0.41 Lmin(-1) and the limits of agreement from -0.86 to 0.78Lmin(-1). The agreement was also consistent at low cardiac outputs., Conclusions: The arterial thermodilution technique may serve as a less invasive cardiac output monitor in conditions of severe bleeding and shock.

A quantitative study of carcinoembryonic antigen (CEA) activity was made in plasma and in 25 colorectal tumors, 30 lung tumors, 20 breast tumors, and 20 fibrocystic dysplasias of the breast. The CEA content was detected by enzyme assays (EIA) in solid phase. In colorectal cancer the amount of CEA ranged from 40 to 800 ng/mg protein. In lung cancer, values ranged from 5 to 130 ng/mg protein. Differences in mean content of CEA between normal and cancer yielded the highest significant value of P less than 0.001. In 54 of 95 of cases, high concentrations of CEA in tissues were associated with high values in plasma. In fibrocystic dysplasia of the breast, the CEA concentration levels were lower than in cases of breast carcinoma. The CEA concentration values in fibrocystic dysplasia were associated with the epithelial proliferation and the degree of dysplasia. Therefore, the presence of elevated CEA tissues values may be indicative of patients with higher risk of malignant transformation.

A human lung tumor fetal-associated antigen (LTFA) has been purified from lung tumor tissue by a combination of salt precipitation and ion-exchange chromatography. The purification steps were monitored by double immunodiffusion with the use of a rabbit anti-LTFA-specific antiserum. The isolated protein was tested for its blastogenic properties toward peripheral blood lymphocytes (PBL) by [3H]thymidine incorporation. PBL obtained from 25 healthy individuals, 34 patients with tumors other than lung tumors, 13 patients with lung diseases other than lung tumors, and 51 lung tumor-bearing patients were tested. Only PBL with an in vitro positive response to phytohemagglutinin were employed. Whereas PBL of lung tumor patients showed a significant blastogenic response to the purified antigen in 14 of 22 patients tested (60%), PBL of other patients were completely unreactive (P less than 0.005). The present data suggested that a specific immune response toward an LTFA was present in patients with lung cancers.

The ability of a lung cancer-associated antigen (LCAA) to provoke specific cutaneous delayed-hypersensitivity reactions has been studied on a group of 59 lung cancer patients. Biological activity of LCAA, monitored by skin testing, was demonstrated in 32% (17 of 53) of lung cancer patients, in 48.0% with limited disease, and in 17.2% with extensive disease. All the responders were in the group of normal reactors to standard recall antigens, if three antigens were used (PPDSK-SD, candida). No correlation was found between biological activity of LCAA and level of immunocompetence evaluated by lymphocyte-blastic transformation with PHA and count of rosette E-forming cells. These studies on the capacity to evoke specific DTH reactions in lung-cancer patients will be extended to the use of assays in vitro in the perspective of a more significant evaluation of immunocompetence levels.

49 patients with advanced lung cancer underwent a combined treatment including radiochemoimmunotherapy. Before and at the end of the combined treatment, the immune status of the patients has been evaluated by testing the immunoreactivity to PPD in vivo and the phytohemagglutinin lymphocyte response and the rosette-forming cells in vitro. While the in vitro tests were not significantly modified by immunostimulation, as regards the immunoreactivity to PPD, we observed a positive conversion at the end of the treatment in 11 out of 25 patients who showed a negative reaction before therapy. All the patients who converted from negative to positive showed also a good response to therapy with regression or no progression of the tumor. Survival curves confirmed the prognostic value of PPD reactivity.

Fifty-three inoperable lung cancer patients were treated with radiotherapy combined with immunostimulation with BCG. A good response was obtained in 30 patients (56%); the results were not significantly different than those obtained with a control group of 50 lung cancer patients matched by age, sex and stage of the disease (24 out of 50 equals 46%). These short-term results were compared with the immunologic "status" of patients evaluated before treatment by the parameters monitoring in vivo and in vitro delayed type hypersensitivity. The good response to therapy was documented in a higher percentage of patients with positive skin tests to recall and standard antigens and with normal values of lymphocyte transformation with PHA and Rosette E-forming cells, in comparison with patients with low levels of immunocompetence. An impairment of the cell-mediated immune response was found after combined therapy, presumably due to radiotherapy. BCG was not able to restore the patient's immunocompetence, no effect on host's immune reactivity was demonstrated. As regards humoral immunity, the patients with low levels of IgG before treatment (12/14) showed a good response.

A human lung tumor-associated fetal antigen (LTFA) has been partially isolated and characterized. The antigen that differs in several immunochemical parameters from previously described lung cancer antigens was shared by fetal lung and liver tissue. The neoantigen migrated in immunoelectrophoresis as an alpha2-beta globulin, had an average molecular size of 7S, and was soluble in 50% saturated ammonium sulfate. Whereas LTFA was insensitive to both DNase and RNase treatment, its antigenicity was completely abolished by pronase. The biologic significance of this antigen and its possible clinical use were discussed.

The aim of this study is to compare the levels of carcinoembryonic antigen (CEA) in the gastric juice, stomach mucosa and plasma of gastrectomy patients at risk of developing cancer with those in normal controls and gastric cancer subjects. Blood samples, gastric juices and biopsy material were taken from 52 patients (8 normal, 10 gastric carcinomas, 24 gastroresected according to Billroth II and 10 according to Billroth I). No significant correlation was found between age, sex or smoking habits and CEA levels in plasma, gastric juice or stomach mucosa. A significant correlation between CEA levels in gastric secretion and those in tissue emerged from out data (n = 52; r = 0.67; p less than 0.01). A minor correlation was found between tissue and plasma CEA values (r = 0.34; p less than 0.05). The mean levels of CEA in plasma did not show significant differences between controls and neoplastic risk subjects. The average level of CEA in gastric secretions and in tissue were significantly lower in normal controls than in neoplastic and gastroresected patients; in this latter group, we have observed a correlation between the severity of the histological lesions and the levels of CEA in the biopsy specimens; no correlation was found with the type of operation (Billroth I or Billroth II). The level of CEA in gastric juices and in biopsy material, therefore, appears to be more useful than in plasma in recognizing cancer risk subjects.