A Prospective, Cohort Study of SITOIGANAP to Treat Glioblastoma When Given in Combination With Granulocyte-Macrophage Colony-Stimulating Factor/Cyclophosphamide/Bevacizumab/Nivolumab or Granulocyte-Macrophage Colony-Stimulating Factor/Cyclophosphamide/Bevacizumab/Pembrolizumab in Patients Who Failed Prior Treatment With Surgical Resection, Radiation, and Temozolomide.

Background: Glioblastoma (GBM) is the most common primary, malignant brain tumor in adults and has a poor prognosis. The median progression-free survival (mPFS) of newly diagnosed GBM is approximately 6 months. The recurrence rate approaches 100%, and the case-fatality ratio approaches one. Half the patients die within 8 months of recurrence, and 5-year survival is less than 10%. Advances in treatment options are urgently needed. We report on the efficacy and safety of a therapeutic vaccine (SITOIGANAP: Epitopoietic Research Corporation) administered to 21 patients with recurrent GBM (rGBM) under a Right-to-Try/Expanded Access program. SITOIGANAP is composed of both autologous and allogeneic tumor cells and lysates. Methods: Twenty-one patients with rGBM received SITOIGANAP on 28-day cycles in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF), cyclophosphamide, bevacizumab, and an anti-programmed cell death protein-1 (anti-PD-1) monoclonal antibody (either nivolumab or pembrolizumab). Results: The mPFS was 9.14 months, and the median overall survival (mOS) was 19.63 months from protocol entry. Currently, 14 patients (67%) are at least 6 months past their first SITOIGANAP cycle; 10 patients (48%) have received at least six cycles and have a mOS of 30.64 months and 1-year survival of 90%. The enrollment and end-of-study CD3+/CD4+ T-lymphocyte counts strongly correlate with OS. Conclusions: The addition of SITOIGANAP/GM-CSF/cyclophosphamide to bevacizumab and an anti-PD-1 monoclonal antibody resulted in a significant survival benefit compared to historic control values in rGBM with minimal toxicity compared to current therapy.

Therapeutic Immunization against Glioblastoma.

Glioblastoma is the most common form of brain cancer in adults that produces severe damage to the brain leading to a very poor survival prognosis. The standard of care for glioblastoma is usually surgery, as well as radiotherapy followed by systemic temozolomide chemotherapy, resulting in a median survival time of about 12 to 15 months. Despite these therapeutic efforts, the tumor returns in the vast majority of patients. When relapsing, statistics suggest an imminent death dependent on the size of the tumor, the Karnofsky Performance Status, and the tumor localization. Following the standard of care, the administration of Bevacizumab, inhibiting the growth of the tumor vasculature, is an approved medicinal treatment option approved in the United States, but not in the European Union, as well as the recently approved alternating electric fields (AEFs) generator NovoTTF/Optune. However, it is clear that regardless of the current treatment regimens, glioma patients continue to have dismal prognosis and novel treatments are urgently needed. Here, we describe different approaches of recently developed therapeutic glioma brain cancer vaccines, which stimulate the patient's immune system to recognize tumor-associated antigens (TAA) on cancer cells, aiming to instruct the immune system to eventually attack and destroy the brain tumor cells, with minimal bystander damage to normal brain cells. These distinct immunotherapies may target particular glioma TAAs which are molecularly defined, but they may also target broad patient-derived tumor antigen preparations intentionally evoking a very broad polyclonal antitumor immune stimulation.

First clinical results of a personalized immunotherapeutic vaccine against recurrent, incompletely resected, treatment-resistant glioblastoma multiforme (GBM) tumors, based on combined allo- and auto-immune tumor reactivity.

Glioblastoma multiforme (GBM) patients have a poor prognosis. After tumor recurrence statistics suggest an imminent death within 1-4.5 months. Supportive preclinical data, from a rat model, provided the rational for a prototype clinical vaccine preparation, named Gliovac (or ERC 1671) composed of autologous antigens, derived from the patient's surgically removed tumor tissue, which is administered together with allogeneic antigens from glioma tissue resected from other GBM patients. We now report the first results of the Gliovac treatment for treatment-resistant GBM patients. Nine (9) recurrent GBM patients, after standard of care treatment, including surgery radio- and chemotherapy temozolomide, and for US patients, also bevacizumab (Avastin™), were treated under a compassionate use/hospital exemption protocol. Gliovac was given intradermally, together with human GM-CSF (Leukine(®)), and preceded by a regimen of regulatory T cell-depleting, low-dose cyclophosphamide. Gliovac administration in patients that have failed standard of care therapies showed minimal toxicity and enhanced overall survival (OS). Six-month (26 weeks) survival for the nine Gliovac patients was 100% versus 33% in control group. At week 40, the published overall survival was 10% if recurrent, reoperated patients were not treated. In the Gliovac treated group, the survival at 40 weeks was 77%. Our data suggest that Gliovac has low toxicity and a promising efficacy. A phase II trial has recently been initiated in recurrent, bevacizumab naïve GBM patients (NCT01903330).

Oil-based emulsion vaccine adjuvants.

Vaccine adjuvants are critical components in experimental and licensed vaccines used in human and veterinary medicine. When aiming to evoke an immune response to a purified antigen, the administration of antigen alone is often insufficient, unless the antigen contains microbial structures or has a natural particulate structure. In most cases, the rationale to use an adjuvant is obvious to the experimental immunologist or the professional vaccinologist, who is familiar with the nature of the antigen, and the aim of the vaccine to elicit a specific antibody response and/or a specific type of T cell response. In this unit, we describe protocols to formulate antigens with oil-based emulsions. Such emulsions represent a major prototype adjuvant category that is frequently used in experimental preclinical vaccines, as well as veterinary and human vaccines.

Development of immune memory to glial brain tumors after tumor regression induced by immunotherapeutic Toll-like receptor 7/8 activation.

The efficacy of immunotherapeutic TLR7/8 activation by resiquimod (R848) was evaluated in vivo, in the CNS-1 rat glioma model syngeneic to Lewis rats. The immune treatment was compared with cytotoxic cyclophosphamide chemotherapy, and as well, was compared with the combination cytotoxic and immunotherapeutic treatments. We found that parenteral treatment with the TLR7/8 agonist, resiquimod, eventually induced complete tumor regression of CNS-1 glioblastoma tumors in Lewis rats. Cyclophosphamide (CY) treatment also resulted in dramatic CNS-1 remission, while the combined treatment showed similar antitumor effects. The resiquimod efficacy appeared not to be associated with direct injury to CNS-1 growth, while CY proved to exert tumoricidal cytotoxicity to the tumor cells. Rats that were cured by treatment with the innate immune response modifier resiquimod proved to be fully immune to secondary CNS-1 tumor rechallenge. They all remained tumor-free and survived. In contrast, rats that controlled CNS-1 tumor growth as a result of CY treatment did not develop immune memory, as demonstrated by their failure to reject a secondary CNS-1 tumor challenge; they showed a concomittant outgrowth of the primary tumor upon secondary tumor exposure. Rechallenge of rats that initially contained tumor growth by combination chemo-immunotherapy also failed to reject secondary tumor challenge, indicating that the cytotoxic effect of the CY likely extended to the endogenous memory immune cells as well as to the tumor. These data demonstrate strong therapeutic antitumor efficacy for the immune response modifier resiquimod leading to immunological memory, and suggest that CY treatment, although effective as chemotherapeutic agent, may be deleterious to maintenance of long-term antitumor immune memory. These data also highlight the importance of the sequence in which a multi-modal therapy is administered.

Exploring the Therapeutic Efficacy of Glioma Vaccines Based on Allo- and Syngeneic Antigens and Distinct Immunological Costimulation Activators.

The efficacy of a various immunotherapeutic immunisation strategies for malignant glioma brain cancer was evaluated in the syngeneic CNS-1 Lewis rat glioma model. A prototype glioma cancer vaccine, which was composed of multivalent antigens derived from allogeneic and syngeneic cells and lysates, formed the prototype preparation of antigens. These antigens reflect the autologous antigens derived from the patient's surgically removed tumor tissue, as well as allogeneic antigens form glioma tumor tissue surgically removed from donor patients. This antigen mixture provides a broad spectrum of tumor associated antigens (TAA) and helps to prevent escape of tumor immune surveillance when given as a vaccine. This antigen preparation was administered in a therapeutic setting with distinct single or multiple co-stimulation-favouring immunostimulants and evaluated for inhibition of tumor growth. Our prototype vaccine was able to arrest progression of tumor growth when co-delivered in a specific regimen together with the costimulating multi-TLR agonist, Bacille Calmette Guerin (BCG) and interleukin-2, or with the Toll-Like receptor (TLR) 7/8 activator resiquimod.

Trends in vaccine adjuvants.

Adjuvants are essential components of most clinically used vaccines. This is because the majority of nonliving vaccines are relatively poor inducers of adaptive immunity unless effective adjuvants are co-administered. Aluminum salts (alum) have been used as adjuvants with great success for almost a century and have been particularly effective at promoting protective humoral immunity. However, alum is not optimally effective for diseases where cell-mediated immunity is required for protection. Furthermore, adjuvants including oil-in-water emulsions have shown improved efficacy for avian influenza protection suggesting that even for diseases where humoral immunity can confer protection, there is scope for developing improved adjuvants. There have been major developments in antigen discovery over the past decade, which has accelerated the vaccine development process for new indications and this demands a new generation of adjuvants that can drive and specifically direct the desired immune responses. A number of systems are under investigation that combine different types of adjuvants into specific formulations with greater activity. Additionally, targeting of vaccines to specific immune cells shows great promise. In the case of cancer and chronic infectious diseases, it may be difficult to develop effective vaccines without blocking immune regulatory pathways, which impede cell-mediated responses. However, increased understanding of immunology and particularly the innate immune system is informing vaccine adjuvant research and consequently driving the development of novel and specifically directed vaccine adjuvant strategies. In this article we address the importance of adjuvants in vaccine development, the known mode of action of specific adjuvants and recent developments in this important field.

A novel soluble immune-type receptor (SITR) in teleost fish: carp SITR is involved in the nitric oxide-mediated response to a protozoan parasite.

BACKGROUND: The innate immune system relies upon a wide range of germ-line encoded receptors including a large number of immunoglobulin superfamily (IgSF) receptors. Different Ig-like immune receptor families have been reported in mammals, birds, amphibians and fish. Most innate immune receptors of the IgSF are type I transmembrane proteins containing one or more extracellular Ig-like domains and their regulation of effector functions is mediated intracellularly by distinct stimulatory or inhibitory pathways. METHODOLOGY/PRINCIPAL FINDINGS: Carp SITR was found in a substracted cDNA repertoire from carp macrophages, enriched for genes up-regulated in response to the protozoan parasite Trypanoplasma borreli. Carp SITR is a type I protein with two extracellular Ig domains in a unique organisation of a N-proximal V/C2 (or I-) type and a C-proximal V-type Ig domain, devoid of a transmembrane domain or any intracytoplasmic signalling motif. The carp SITR C-proximal V-type Ig domain, in particular, has a close sequence similarity and conserved structural characteristics to the mammalian CD300 molecules. By generating an anti-SITR antibody we could show that SITR protein expression was restricted to cells of the myeloid lineage. Carp SITR is abundantly expressed in macrophages and is secreted upon in vitro stimulation with the protozoan parasite T. borreli. Secretion of SITR protein during in vivo T. borreli infection suggests a role for this IgSF receptor in the host response to this protozoan parasite. Overexpression of carp SITR in mouse macrophages and knock-down of SITR protein expression in carp macrophages, using morpholino antisense technology, provided evidence for the involvement of carp SITR in the parasite-induced NO production. CONCLUSION/SIGNIFICANCE: We report the structural and functional characterization of a novel soluble immune-type receptor (SITR) in a teleost fish and propose a role for carp SITR in the NO-mediated response to a protozoan parasite.

Therapeutic vaccination against malignant gliomas based on allorecognition and syngeneic tumor antigens: proof of principle in two strains of rat.

In the present study we investigated whether allogeneic glioma cells can be utilized to evoke prophylactic or therapeutic immune-mediated elimination of syngeneic glioma in two rat strains. Fisher 344 and Sprague-Dawley (SD) rats were injected with two syngeneic glioma cell lines, 9L and C6, respectively, resulting in progressive tumor growth. 9L is syngeneic to the Fisher 344 and allogeneic to the SD rats, while C6 cells are syngeneic to SD rats and allogeneic to Fisher 344 rats. Both rat strains were subcutaneously injected with their respective allogeneic tumor cells, which proved unable to grow progressively. The allogeneic cells were either rejected immediately in SD rats or within 25 days in Fisher rats, after limited tumor outgrowth. Both rat strains were subsequently challenged with their respective syngeneic glioma tumor cells and once more 10 days later with a fivefold higher dose. SD rats, even after reinjection with five times the original dosage of C6 cells, remained tumor free for at least 360 days. Similarly, Fisher rats, after initially rejecting allogeneic tumors, failed to develop syngeneic tumors. To determine anti-tumor immunity against established glioma tumors under more demanding therapeutic conditions, rats were first injected subcutaneously with their respective syngeneic tumor and vaccinated once or repeatedly (at 5-day intervals) with a mixture of the allogeneic or xenogeneic cells, with or without a lysate from the same syngeneic tumor, which served as a therapeutic vaccine preparations. The control group received either no treatment or syngeneic instead of allogeneic cells. In both strains of rats, we demonstrated that the therapeutically vaccinated groups were able to significantly reduce tumor growth, while complete rejection of tumors was noted in the SD rats. Immunization with syngeneic tumor cells alone failed to evoke anti-tumor immunity. We conclude that therapeutic immunization with a combination of allogeneic cells and syngeneic lysates induces rejection of malignant gliomas and offers a protective effect against challenge with syngeneic tumor cells. This immunization approach may prove useful as a post-surgery adjuvant therapy in future cancer treatment protocols, or even as a stand-alone therapeutic tumor vaccination.

Molecular immunophenotyping of lungs and spleens in naive and vaccinated chickens early after pulmonary avian influenza A (H9N2) virus infection.

In a respiratory-infection-model with the avian influenza A H9N2 virus we studied lung and splenic immune reactions in chickens using a recently developed 5K chicken immuno-microarray. Groups of chickens were either mock-immunized (referred to as non-immune), vaccinated with inactivated viral antigen only (immune) or with viral antigen in a water-in-oil (W/O) immunopotentiator (immune potentiated). Three weeks after vaccination all animals were given a respiratory infection. Immune potentiated birds developed inhibitory antiviral antibodies, showed minimal lung histopathology and no detectable viral sequences, while non-immune animals showed microscopic immunopathology and detectable virus. Immune birds, receiving antigen in saline only, showed minimal microscopic histopathology, and intermediate levels of virus detection. These classical features in the different groups were mirrored by overlapping or specific mRNA gene expression profiles in lungs and spleen using microarray analysis. To our knowledge this is the first study demonstrating pneumonia-associated lung pathology of the low pathogenic avian influenza H9N2 virus. Our data provide insights into the molecular interaction of this virus with its natural host when naive or primed by vaccination.

Structure- and oil type-based efficacy of emulsion adjuvants.

Oil-based emulsions are well-known immunopotentiators for inactivated, "killed" vaccines. We addressed the relationship between emulsion structure and levels of in vivo antibody formation to inactivated New Castle Disease virus (NDV) and Infectious Bronchitis virus (IBV) as antigens in 3-week-old chickens. The use of a polymeric emulsifier allowed for direct comparison of three types of emulsions, water-in-oil (W/O), oil-in-water (O/W) and W/O-in-water (W/O/W), while maintaining an identical content of components for each vehicle. They were prepared with either non-metabolizable, mineral oil or metabolizable, Miglyol 840. In addition, we assessed the inherent release capacity of each emulsion variant in vitro. Remarkably, we noted that W/O-type emulsions induced the best immune responses, while they released no antigen during 3 weeks. In general, mineral oil vaccines showed superior efficacy compared to Miglyol 840-based vaccines.

Structure-activity relations of water-in-oil vaccine formulations and induced antigen-specific antibody responses.

Water-in-oil (W/O) emulsions are known as most effective adjuvants to generate high and durable antibody responses to vaccine antigens following a single immunization. However, their structural requirements remain poorly understood. Here we addressed the significance of certain pharmaceutical characteristics including water/oil ratios--ranging from 60/40 to 30/70 (w/w(%))--droplet size and type of oil, i.e. non-metabolizable (mineral oil) versus metabolizable (Miglyol 840). Stability of emulsions was accomplished by the use of a polymeric emulsifier. Distinct W/O emulsions were formulated with inactivated (i) infectious bronchitis virus (iIBV) and Newcastle disease virus (iNDV), and evaluated in immunized chickens for magnitude and duration of in vivo antiviral antibody formation and local reactions. A high mineral oil content proved most effective for antibody response formation. In general, a larger droplet size evoked higher antibody responses for both oil types. Inoculum residues proved lower using biodegradable Miglyol, when compared to mineral oil, for all emulsion variants. Especially water-to-oil ratio and droplet size may provide useful parameters for improving (antiviral) antibody production by W/O emulsions.

Identification and molecular cloning of functional chicken IL-12.

By a combination of large-scale sequencing, bioinformatics, and traditional molecular biology, we identified the long-searched-for cDNA sequences encoding the homologues of the chicken IL-12p35 and IL-12p40 chains. These molecules are the first discovered nonmammalian IL-12 subunits. The homologies of the chicken IL-12p35 and IL-12p40 proteins to the corresponding known subunits of various species, i.e., humans, sheep, horse, cat, bovine, mouse, and woodchuck, ranged between 21 and 42%, respectively. The expression of IL-12 subunits was observed in lymphoid cells and proved to be dependent on the cell type and stimulus, while expression was not detected in stimulated primary chicken embryo fibroblast cells. Following transient expression of both molecules in COS-7 cells, we confirmed the necessity of heterodimerization into IL-12p70 to yield bioactivity as was also shown for its mammalian counterparts. The chicken IL-12p70 molecule, generated either by transient coexpression of monomeric IL-12p35 and monomeric IL-12p40 or as a fusion protein (as in a fusion linker construct), induced IFN-gamma synthesis and proliferative activity of freshly exposed chicken splenocytes. The high degree of functional similarity between chicken IL-12 and IL-12 of higher mammalian vertebrates, despite their poor sequence homology, illustrates the conservation and vital importance of the IL-12 molecule since the evolutionary dichotomy of birds and mammals >300 million years ago. In this article, we describe the first nonmammalian IL-12 molecule and show that this chicken IL-12 molecule is bioactive.

Characterization of the first nonmammalian T2 cytokine gene cluster: the cluster contains functional single-copy genes for IL-3, IL-4, IL-13, and GM-CSF, a gene for IL-5 that appears to be a pseudogene, and a gene encoding another cytokinelike transcript, KK34.

A genomics approach based on the conservation of synteny was used to develop a bacterial artificial chromosome (BAC) contig across the chicken T2 cytokine gene cluster. Sequencing of representative BACs showed that the chicken genome encodes genes for the homologs of mammalian interleukin-3 (IL-3), IL-4, IL-5, IL-13, and granulocyte-macrophage colony-stimulating factor (GM-CSF). These sequences represent the first T2 cytokines found outside of mammals, and their location demonstrates that the T2 cluster is ancient (at least 300 million years old). Four of these genes (IL-3, IL-4, IL-13, and GM-CSF) are expressed at the mRNA level and can be expressed as recombinant protein. In contrast to the other four genes, the chicken IL-5 (ChIL-5) gene we sequenced lacks a recognizable promoter and regulatory sequences in the predicted 3'-untranslated region (3'-UTR). Further, there is no evidence for its expression at the mRNA level. We, therefore, hypothesize that it is a pseudogene. Genomic analysis revealed that a recently characterized cytokinelike transcript, KK34, not identified in our initial analysis of the BAC sequence, is also encoded in this cluster. This gene may represent a duplication of an ancestral IL-5 gene and may encode the functional homolog of IL-5 in the chicken.