Vascular attack by 5,6-dimethylxanthenone-4-acetic acid combined with B7.1 (CD80)-mediated immunotherapy overcomes immune resistance and leads to the eradication of large tumors and multiple tumor foci

The promise of cancer immunotherapy is that it will not only eradicate primary tumors but will generate systemic antitumor immunity capable of destroying distant metastases. A major problem that must first be surmounted relates to the immune resistance of large tumors. Here we reveal that immune resistance can be overcome by combining immunotherapy with a concerted attack on the tumor vasculature. The functionally related antitumor drugs 5,6-dimethylxanthenone-4-acetic acid (DMXAA) and flavone acetic acid (FAA), which cause tumor vasculature collapse and tumor necrosis, were used to attack the tumor vasculature, whereas the T-cell costimulator B7.1 (CD80), which costimulates T-cell proliferation via the CD28 pathway, was used to stimulate antitumor immunity. The injection of cDNA (60–180 µg) encoding B7.1 into large EL-4 tumors (0.8 cm in diameter) established in C57BL/6 mice, followed 24 h later by i.p. administration of either DMXAA (25 mg/kg) or FAA (300 mg/kg), resulted in complete tumor eradication within 2–6 weeks. In contrast, monotherapies were ineffective. Both vascular attack and B7.1 immunotherapy led to up-regulation of heat shock protein 70 on stressed and dying tumor cells, potentially augmenting immunotherapy. Remarkably, large tumors took on the appearance of a wound that rapidly ameliorated, leaving perfectly healed skin. Combined therapy was mediated by CD8+ T cells and natural killer cells, accompanied by heightened and prolonged antitumor cytolytic activity (P < 0.001), and by a marked increase in tumor cell apoptosis. Cured animals completely rejected a challenge of 1 x 107 parental EL-4 tumor cells but not a challenge of 1 x 104 Lewis lung carcinoma cells, demonstrating that antitumor immunity was tumor specific. Adoptive transfer of 2 x 108 splenocytes from treated mice into recipients bearing established (0.8 cm in diameter) tumors resulted in rapid and complete tumor rejection within 3 weeks. Although DMXAA and B7.1 monotherapies are complicated by a narrow range of effective doses, combined therapy was less dosage dependent. Thus, a broad range of amounts of B7.1 cDNA were effective in combination with 25 mg/kg DMXAA. In contrast, DMXAA, which has a very narrow range of high active doses, was effective at a low dose (18 mg/kg) when administered with a large amount (180 µg) of B7.1 cDNA. Importantly, combinational therapy generated heightened antitumor immunity, such that gene transfer of B7.1 into one tumor, followed by systemic DMXAA treatment, led to the complete rejection of multiple untreated tumor nodules established in the opposing flank. These findings have important implications for the future direction and utility of cancer immunotherapies aimed at harnessing patients’ immune responses to their own tumors.

Anti-inflammatory immunotherapy for multiple sclerosis/experimental autoimmune encephalomyelitis (EAE) disease

Multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), are inflammatory diseases of the central nervous system (CNS) characterized by localized areas with demyelination. Disease is believed to be an autoimmune disorder mediated by activated immune cells such as T- and B-lymphocytes and macrophages/microglia. Lymphocytes are primed in the peripheral tissues by antigens, and clonally expanded cells infiltrate the CNS. They produce large amounts of inflammatory cytokines, nitric oxide (NO) that lead to demyelination and axonal degeneration. Although several studies have shown that oligodendrocytes (OLGs), the myelin-forming glial cells in the CNS, are sensitive to cell death stimuli, such as cytotoxic cytokines, anti-myelin antibodies, NO, and oxidative stress, in vitro, the mechanisms underlying injury to the OLGs in MS/EAE remain unclear. The central role of glutamate receptors in mediating excitotoxic neuronal death in stroke, epilepsy, trauma and MS has been well established. Glutamate is the major excitatory amino acid transmitter within the CNS and it's signaling is mediated by a number of postsynaptic ionotropic and metabotropic receptors. Inflammation can be blocked with anti-cell adhesion molecules MAb, simultaneously protected oligodendrocytes and neurons against glutamate-mediated damage with the AMPA/kainate antagonist NBQX, and the NMDA receptor antagonist GPE, could thus be effective therapies for multiple sclerosis.

The Australian multiple sclerosis (MS) immunotherapy study: A prospective, multicentre study of drug utilisation using the MSBase platform

To prospectively characterise treatment persistence and predictors of treatment discontinuation in an Australian relapsing-remitting multiple sclerosis (RRMS) population. Tertiary MS treatment centres participating in the MSBase registry prospectively assessed treatment utilisation, persistence, predictors of treatment discontinuation and switch rates. Multivariable survival analyses were used to compare treatment persistence between drugs and to identify predictors of treatment discontinuation. 1113 RRMS patients were studied. Patients persisted on their first disease-modifying therapy (DMT) for a median of 2.5 years. Treatment persistence on GA was shorter than on all IFNβ products (p<0.03). Younger age at treatment initiation and higher EDSS were predictive of DMT discontinuation. Patients persisted on subsequent DMTs, for 2.3 years. Patients receiving natalizumab (NAT) as a subsequent DMT persisted longer on treatment than those on IFNβ or GA (p<0.000). The primary reason for treatment discontinuation for any drug class was poor tolerability. Annualised switch or cessation rates were 9.5–12.5% for individual IFNβ products, 11.6% for GA and 4.4% for NAT. This multicentre MS cohort study is the first to directly compare treatment persistence on IFNβ and GA to NAT. We report that treatment persistence in our Australian RRMS population is short, although patients receiving IFNβ as a first DMT persisted longer on treatment than those on GA. Additionally, patients receiving NAT as a subsequent DMT were more likely to persist on treatment than those switched to IFNβ or GA. EDSS and age at DMT initiation were predictive of DMT discontinuation. Treatment intolerance was the principal reason for treatment cessation.

Hypoallergenic variants of the major latex allergen Hev b 6.01 retaining human T lymphocyte reactivity

Hev b 6.01 is a major allergen of natural rubber latex with sensitization of 70–86% of latex glove-allergic subjects. Recently, we mapped the immunodominant T cell sites of Hev b 6.01 to the highly IgE-reactive hevein (Hev b 6.02) domain. Hev b 6.01 contains 14 cysteine residues with multiple disulphide bridges stabilizing tertiary conformation. With the goal of a standardized specific immunotherapy we developed hypoallergenic Hev b 6.01 mutants by site-directed mutagenesis of selected cysteine residues (3, 12, 17, and 41) within the Hev b 6.02 domain. Peptides corresponding to the Hev b 6.02 domain of two of the mutants were also synthesized. These mutants and peptide variants showed markedly decreased or ablated latex-allergic patient serum IgE binding by immunoblotting and ELISA. Basophil activation testing confirmed markedly decreased activation with successive cysteine substitutions of the mutants and complete abrogation with the Hev b 6.02 (Cys 3, 12, 17, 41 Ala) peptide. Retention of T cell reactivity is crucial for effective specific immunotherapy and all mutants and peptide variants maintained their latex-specific T cell reactivity. The ablated allergenicity but retained T cell reactivity of the Hev b 6.02 (Cys 3, 12, 17, 41 Ala) peptide suggests this peptide is a suitable candidate for inclusion in a latex immunotherapy preparation.

Effects of survivin antagonists of growth of established tumors and B7-1 immunogene therapy

Background: Survivin, a member of the inhibitor of apoptosis (IAP) protein family, is detectable in most types of cancer, and its presence is associated with a poor prognosis. We determined the effects of gene-based therapies that inhibit survivin function in a mouse tumor model. Methods: Using five to six mice per treatment group, we injected tumors derived from mouse EL-4 thymic lymphoma cells with plasmids encoding antisense survivin, a dominant-negative mutant survivin, and the T-cell costimulator B7-1. Expression of endogenous survivin and the proteins encoded by the injected plasmids were examined by immunohistochemical staining of tumor sections and by western blot and flow cytometry analyses of isolated tumor cells. Tumor growth, the generation of antitumor cytotoxic T-lymphocyte (CTL) activity, apoptosis, and the contribution of leukocyte subsets to antitumor activity were measured. All statistical tests were two-sided. Results: Large (1.0-cm diameter) tumors had approximately 10-fold more survivin than small (0.2-cm diameter) tumors. At 28 days after injection, antisense and dominant-negative mutant survivin plasmids statistically significantly inhibited the growth of both small (P = .006 and P = .0018, respectively) and large (P<.001 for both plasmids) EL-4 tumors compared with tumors injected with empty plasmid. The growth of large tumors was further inhibited by intratumoral injection with antisense survivin and B7-1 (P = .004); thus, inhibition of survivin expression renders large tumors susceptible to B7-1-mediated immunotherapy. Mice whose tumors were completely eradicated by injection of B7-1 remained tumor free for 26 days after re-injection with EL-4 cells (when the experiment ended). Compared with tumors injected with empty plasmid, tumors injected with survivin-based plasmids had increased apoptosis, and animals bearing such tumors generated more antitumor CTLs. Conclusion: Intratumoral injection of plasmids that block survivin expression and stimulate the generation of tumor-specific CTLs may be beneficial for the treatment of large lymphomas.

An in vivo cytotoxicity threshold for influenza A virus-specific effector and memory CD8+ T cells

Influenza A virus infection of C57BL/6 (B6) mice is characterized by prominent CD8 T cell responses to H2Db complexed with peptides from the viral nucleoprotein (NP366, ASNENMETM) and acid polymerase (PA224, SSLENFRAYV). An in vivo cytotoxicity assay that depends on the adoptive transfer of peptide-pulsed, syngeneic targets was used in this study to quantitate the cytotoxic potential of DbNP366- and DbPA224-specific acute and memory CD8 T cells following primary or secondary virus challenge. Both T cell populations displayed equivalent levels of in vivo effector function when comparable numbers were transferred into naive B6 hosts. Cytotoxic activity following primary infection clearly correlated with the frequency of tetramer-stained CD8 T cells. This relationship looked, however, to be less direct following secondary exposure, partly because the numbers of CD8DbNP366 T cells were greatly in excess. However, calculating the in vivo E:T ratios indicated that in vivo lysis, like many other biological functions, is threshold dependent. Furthermore, the capacity to eliminate peptide-pulsed targets was independent of the differentiation state (i.e., primary or secondary effectors) and was comparable for the two T cell specificities that were analyzed. These experiments provide insights that may be of value for adoptive immunotherapy, where careful consideration of both the activation state and the number of effector cells is required.

Targeting hepatitis B virus and human papillomavirus induced carcinogenesis : novel patented therapeutics

Viral infections leading to carcinogenesis tops the risk factors list for the development of human cancer. The decades of research has provided ample scientific evidence that directly links 10-15% of the worldwide incidence of human cancers to the infections with seven human viruses. Moreover, the insights gained into the molecular pathogenetic and immune mechanisms of hepatitis B virus (HBV) and human papillomavirus (HPV) viral transmission to tumour progression, and the identification of their viral surface antigens as well as oncoproteins have provided the scientific community with opportunities to target these virus infections through the development of prophylactic vaccines and antiviral therapeutics. The preventive vaccination programmes targeting HBV and high risk HPV infections, linked to hepatocellular carcinoma (HCC) and cervical cancer respectively have been recently reported to alter age-old cancer patterns on an international scale. In this review, with an emphasis on HBV and HPV mediated carcinogenesis because of the similarities and differences in their global incidence patterns, viral transmission, mortality, molecular pathogenesis and prevention, we focus on the development of recently identified HBV and HPV targeting innovative strategies resulting in several patents and patent applications.

Depression's multiple comorbidities explained by (neuro)inflammatory and oxidative & nitrosative stress pathways

There is now evidence that depression, as characterized by melancholic symptoms, anxiety, and fatigue and somatic (F&S) symptoms, is the clinical expression of peripheral cell-mediated activation, inflammation and induction of oxidative and nitrosative stress (IO&NS) pathways and of central microglial activation, decreased neurogenesis and increased apoptosis. This review gives an explanation for the multiple “co-morbidities” between depression and a large variety of a) brain disorders related to neurodegeneration, e.g. Alzheimer’s, Parkinson’s and Huntington’s disease, multiple sclerosis and stroke; b) medical disorders, such as cardiovascular disorder, chronic fatigue syndrome, chronic obstructive pulmonary disease, rheumatoid arthritis, psoriasis, systemic lupus erythematosus, inflammatory bowel disease, irritable bowel syndrome, leaky gut, diabetes type 1 and 2, obesity and the metabolic syndrome, and HIV infection; and c) conditions, such as hemodialysis, interferon-α-based immunotherapy, the postnatal period and psychosocial stressors. The common denominator of all those disorders/conditions is the presence of microglial activation and/or activation of peripheral IO&NS pathways. There is evidence that shared peripheral and / or central IO&NS pathways underpin the pathophysiology of depression and the previously mentioned disorders and that activation of these IO&NS pathways contributes to shared risk. The IO&NS pathways function as a smoke sensor that detect threats in the peripheral and central parts of the body and signal these threats as melancholic, anxiety, and fatigue and somatic (F&S) symptoms. The presence of concomitant depression is strongly associated with a lower quality of life and increased morbidity and mortality in medical disorders. This may be explained since depression contributes to increased (neuro)inflammatory burden and may therefore drive the inflammatory and degenerative progression. It is concluded that the activation of peripheral and / or central IO&NS pathways may explain the co-occurrence of depression with the above disorders. This shows that depression belongs to the spectrum of inflammatory and degenerative disorders.

Role of the EpCAM (CD326) in prostate cancer metastasis and progression

Despite significant advances in surgery, radiotherapy and chemotherapy to treat prostate cancer (CaP), many patients die of secondary disease (metastases). Current therapeutic approaches are limited, and there is no cure for metastatic castration-resistant prostate cancer (CRPC). Epithelial cell adhesion molecule (EpCAM, also known as CD326) is a transmembrane glycoprotein that is highly expressed in rapidly proliferating carcinomas and plays an important role in the prevention of cell–cell adhesion, cell signalling, migration, proliferation and differentiation. Stably and highly expressed EpCAM has been found in primary CaP tissues, effusions and CaP metastases, making it an ideal candidate of tumour-associated antigen to detect metastasis of CaP cells in the circulation as well as a promising therapeutic target to control metastatic CRPC disease. In this review, we discuss the implications of the newly identified roles of EpCAM in terms of its diagnostic and metastatic relevance to CaP. We also summarize EpCAM expression in human CaP and EpCAM-mediated signalling pathways in cancer metastasis. Finally, emerging and innovative approaches to the management of the disease and expanding potential therapeutic applications of EpCAM for targeted strategies in future CaP therapy will be explored.

The use of sensitive chemical antibodies for diagnosis: detection of low levels of Epcam in breast cancer

EpCAM is expressed at low levels in a variety of normal human epithelial tissues, but is overexpressed in 70–90% of carcinomas. From a clinico-pathological point of view, this has both prognostic and therapeutic significance. EpCAM was first suggested as a therapeutic target for the treatment of epithelial cancers in the 1990s. However, following several immunotherapy trials, the results have been mixed. It has been suggested that this is due, at least in part, to an unknown level of EpCAM expression in the tumors being targeted. Thus, selection of patients who would benefit from EpCAM immunotherapy by determining EpCAM status in the tumor biopsies is currently undergoing vigorous evaluation. However, current EpCAM antibodies are not robust enough to be able to detect EpCAM expression in all pathological tissues.

Here we report a newly developed EpCAM RNA aptamer, also known as a chemical antibody, which is not only specific but also more sensitive than current antibodies for the detection of EpCAM in formalin-fixed paraffin-embedded primary breast cancers. This new aptamer, together with our previously described aptamer, showed no non- specific staining or cross-reactivity with tissues that do not express EpCAM. They were able to reliably detect target proteins in breast cancer xenograft where an anti-EpCAM antibody (323/A3) showed limited or no reactivity. Our results demonstrated a more robust detection of EpCAM using RNA aptamers over antibodies in clinical samples with chromogenic staining. This shows the potential of aptamers in the future of histopathological diagnosis and as a tool to guide targeted immunotherapy.

Production and characterization of hypoallergenic chicken eggs

This project aimed to develop novel therapeutics and methods to tackle egg allergy. In this study, recombinant variants of egg allergens were produced that may be used in future immunotherapy. Genetic engineering was also used to develop new strategies to produce non-allergenic chicken eggs.

Coassembled nanostructured bioscaffold reduces the expression of proinflammatory cytokines to induce apoptosis in epithelial cancer cells

The local inflammatory environment of the cell promotes the growth of epithelial cancers. Therefore, controlling inflammation locally using a material in a sustained, non-steroidal fashion can effectively kill malignant cells without significant damage to surrounding healthy cells. A promising class of materials for such applications are the nanostructured scaffolds formed by epitope containing minimalist self-assembled peptides (SAPs), as they are bioactive on a cellular length scale, whilst presenting as an easily handled hydrogel. Here, we show that the assembly process distributes an anti-inflammatory polysaccharide, fuccoidan, localised to the nanofibers to function as an anti-inflammatory biomaterial for cancer therapy. We show that it supports healthy cells, whilst inducing apoptosis in cancerous endothelial cells, as demonstrated by the downregulation of the proinflammatory gene and protein expression pathways associated with epithelial cancer progression. Our findings highlight an innovative material approach with potential applications as local epithelial cancer immunotherapy and drug delivery vehicles.

Production and immunological analysis of IgE reactive recombinant egg white allergens expressed in Escherichia coli.

IgE-mediated allergy to chicken egg affects a large number of children and adults worldwide. The current management strategy for egg allergy is strict avoidance, however this is impractical due to the presence of eggs in a range of foods and pharmaceutical products including vaccines. Strict avoidance also poses nutritional disadvantages due to high nutritional value of eggs. Allergen specific immunotherapy is being pursued as a curative treatment, in which an allergic individual is gradually exposed to the allergen to induce tolerance. Use of recombinant proteins for immunotherapy has been beneficial due to the purity of the recombinant proteins compared to natural proteins. In this study, we produced IgE reactive recombinant egg white proteins that can be used for future immunotherapy. Using E. coli as an expression system, we successfully produced recombinant versions of Gal d 1, 2 and 3, that were IgE reactive when tested against a pool of egg allergic patients' sera. The IgE reactivity indicates that these recombinant proteins are capable of eliciting an immune response, thus being potential candidates for immunotherapy. We have, for the first time, attempted to produce recombinant versions of all 4 major egg white allergens in E. coli, and successfully produced 3, with only Gal d 4 showing loss of IgE reactivity in the recombinant version. The results suggest that egg allergy in Australian populations may mainly be due to IgE reactivity to Gal d 3 and 4, while Gal d 1 shows higher IgE reactivity. This is the first report of a collective and comparative immunological analysis of all 4 egg white allergens. The significance of this study is the potential use of the IgE reactive recombinant egg white proteins in immunotherapy to treat egg allergic patients.

Hypoallergenic variant of the major egg white allergen Gal d 1 produced by disruption of cysteine bridges

Gal d 1 (Ovomucoid) is the most allergenic protein in the chicken egg white. Hypoallergenic variants of this allergen can be used in immunotherapy as an egg allergy treatment approach. We hypothesized that disruption of selected cysteine-cysteine disulfide bridges by mutagenesis will allow us the production of a hypoallergenic variant of the protein.