Combination of a T cell activating therapy and anti-phosphatidylserine enhances anti-tumour immune responses in a HPV16 E7-expressing C3 tumour model.

DPX is a novel delivery platform that generates targeted CD8 + T cells and drives antigen-specific cytotoxic T cells into tumours. Cancer cells upregulate phosphatidylserine (PS) on the cell surface as a mechanism to induce an immunosuppressive microenvironment. Development of anti-PS targeting antibodies have highlighted the ability of a PS-blockade to enhance tumour control by T cells by releasing immunosuppression. Here, C57BL/6 mice were implanted with HPV16 E7 target-expressing C3 tumours and subjected to low dose intermittent cyclophosphamide (CPA) in combination with DPX-R9F treatment targeting an E7 antigen with and without anti-PS and/or anti-PD-1 targeting antibodies. Immune responses were assessed via IFN-γ ELISPOT assay and the tumour microenvironment was further analyzed using RT-qPCR. We show that the combination of DPX-R9F and PS-targeting antibodies with and without anti-PD-1 demonstrated increased efficacy compared to untreated controls. All treatments containing DPX-R9F led to T cell activation as assessed by IFN-γ ELISPOT. Furthermore, DPX-R9F/anti-PS treatment significantly elevated cytotoxic T cells, macrophages and dendritic cells based on RT-qPCR analysis. Overall, our data indicates that anti-tumour responses are driven through a variety of immune cells within this model and highlights the need to investigate combination therapies which increase tumour immune infiltration, such as anti-phosphotidylserine.

Anti-PD-1 increases the clonality and activity of tumor infiltrating antigen specific T cells induced by a potent immune therapy consisting of vaccine and metronomic cyclophosphamide.

BACKGROUND: Future cancer immunotherapies will combine multiple treatments to generate functional immune responses to cancer antigens through synergistic, multi-modal mechanisms. In this study we explored the combination of three distinct immunotherapies: a class I restricted peptide-based cancer vaccine, metronomic cyclophosphamide (mCPA) and anti-PD-1 treatment in a murine tumor model expressing HPV16 E7 (C3). METHODS: Mice were implanted with C3 tumors subcutaneously. Tumor bearing mice were treated with mCPA (20 mg/kg/day PO) for seven continuous days on alternating weeks, vaccinated with HPV16 E749-57 peptide antigen formulated in the DepoVax (DPX) adjuvanting platform every second week, and administered anti-PD-1 (200 µg/dose IP) after each vaccination. Efficacy was measured by following tumor growth and survival. Immunogenicity was measured by IFN-γ ELISpot of spleen, vaccine draining lymph nodes and tumor draining lymph nodes. Tumor infiltration was measured by flow cytometry for CD8α+ peptide-specific T cells and RT-qPCR for cytotoxic proteins. The clonality of tumor infiltrating T cells was measured by TCRß sequencing using genomic DNA. RESULTS: Untreated C3 tumors had low expression of PD-L1 in vivo and anti-PD-1 therapy alone provided no protection from tumor growth. Treatment with DPX/mCPA could delay tumor growth, and tri-therapy with DPX/mCPA/anti-PD-1 provided long-term control of tumors. We found that treatment with DPX/mCPA/anti-PD-1 enhanced systemic antigen-specific immune responses detected in the spleen as determined by IFN-γ ELISpot compared to those in the DPX/mCPA group, but immune responses in tumor-draining lymph nodes were not increased. Although no increases in antigen-specific CD8α+ TILs could be detected, there was a trend for increased expression of cytotoxic genes within the tumor microenvironment as well as an increase in clonality in mice treated with DPX/mCPA/anti-PD-1 compared to those with anti-PD-1 alone or DPX/mCPA. Using a library of antigen-specific CD8α+ T cell clones, we found that antigen-specific clones were more frequently expanded in the DPX/mCPA/anti-PD-1 treated group. CONCLUSIONS: These results demonstrate how the efficacy of anti-PD-1 may be improved by combination with a potent and targeted T cell activating immune therapy.

Ancestral genomic duplication of the insulin gene in tilapia: An analysis of possible implications for clinical islet xenotransplantation using donor islets from transgenic tilapia expressing a humanized insulin gene.

Tilapia, a teleost fish, have multiple large anatomically discrete islets which are easy to harvest, and when transplanted into diabetic murine recipients, provide normoglycemia and mammalian-like glucose tolerance profiles. Tilapia insulin differs structurally from human insulin which could preclude their use as islet donors for xenotransplantation. Therefore, we produced transgenic tilapia with islets expressing a humanized insulin gene. It is now known that fish genomes may possess an ancestral duplication and so tilapia may have a second insulin gene. Therefore, we cloned, sequenced, and characterized the tilapia insulin 2 transcript and found that its expression is negligible in islets, is not islet-specific, and would not likely need to be silenced in our transgenic fish.

Metronomic cyclophosphamide enhances HPV16E7 peptide vaccine induced antigen-specific and cytotoxic T-cell mediated antitumor immune response.

In clinical trials, metronomic cyclophosphamide (CPA) is increasingly being combined with vaccines to reduce tumor-induced immune suppression. Previous strategies to modulate the immune system during vaccination have involved continuous administration of low dose chemotherapy, studies that have posed unique considerations for clinical trial design. Here, we evaluated metronomic CPA in combination with a peptide vaccine targeting HPV16E7 in an HPV16-induced tumor model, focusing on the cytotoxic T-cell response and timing of low dose metronomic CPA (mCPA) treatment relative to vaccination. Mice bearing C3 tumors were given metronomic CPA on alternating weeks in combination with immunization with a DepoVax vaccine containing HPV16E749-57 peptide antigen every 3 weeks. Only the combination therapy provided significant long-term control of tumor growth. The efficacy of the vaccine was uncompromised if given at the beginning or end of a cycle of metronomic CPA. Metronomic CPA had a pronounced lymphodepletive effect on the vaccine draining lymph node, yet did not reduce the development of antigen-specific CD8+ T cells induced by vaccination. This enrichment correlated with increased cytotoxic activity in the spleen and increased expression of cytotoxic gene signatures in the tumor. Immunity could be passively transferred through CD8+ T cells isolated from tumor-bearing mice treated with the combinatorial treatment regimen. A comprehensive survey of splenocytes indicated that metronomic CPA, in the absence of vaccination, induced transient lymphodepletion marked by a selective expansion of myeloid-derived suppressor cells. These results provide important insights into the multiple mechanisms of metronomic CPA induced immune modulation in the context of a peptide cancer vaccine that may be translated into more effective clinical trial designs.

Cloning and molecular characterization of the glucose transporter 1 in tilapia (Oreochromis niloticus).

Facilitative glucose transporters (GLUTs) are responsible for passively transporting monosaccharides across the plasma membrane. We sequenced and characterized the Nile tilapia (Oreochromis niloticus) GLUT-1 (tGLUT-1) cDNA and genomic DNA. Using rapid amplification of the cDNA ends (RACE), two tGLUT-1 transcripts were detected differing in the length of the 3' untranslated region, 2851 and 4577 bp. Translated tGLUT-1 is a 490 amino acid product, which shares 74% homology with that of humans. Computer analysis of the amino acid sequence predicted 12 transmembrane domains, which are conserved in the GLUT-1 of various species. The tGLUT-1 gene spans more than 11 kb, and similar to the mammalian GLUT-1 genes has a 10 exon, 9 intron organization. Potential promoter regulatory elements have some similarity to those recorded for human, mouse, and rat GLUT-1 genes. Tissue expression studies revealed both GLUT-1 transcripts in liver, Brockmann bodies (BB), heart, small intestine, adipose tissue, white and red muscle, gill, spleen, pituitary gland, and brain. The highest level of expression was detected in tilapia heart, followed by BB, brain, and muscle. Protein based food and glucose had minor or no effects on the level of tGLUT-1 expression in most tissues. The tGLUT-1 mRNA level was significantly induced by glucose and food only in white muscle. Current results suggest that tGLUT-1 is similar to the GLUT-1 of other teleost species and mammals at the genomic, mRNA, and amino acid levels, supporting the concept that tGLUT-1 functions as a ubiquitous basal level glucose transporter.

Regulation of insulin gene expression and insulin production in Nile tilapia (Oreochromis niloticus).

Compared to mammals, little is known about insulin gene expression in fish. Using transient transfection experiments and mammalian insulinoma cell lines we demonstrate that transcription of the Nile tilapia (Oreochromis niloticus) insulin gene is (a) regulated in a beta-cell-specific manner; and (b) not sensitive to the glucose stimulations. Deletion analysis of the 1575 bp 5' insulin gene flanking sequence revealed that cooperative interactions between regulatory elements within the proximal (-1 to -396 bp) and the distal (-396 bp to -1575 bp) promoter regions were necessary for induction of the beta-cell-specific transcription. Effects of glucose and arginine on endogenous insulin secretion, translation, and transcription in isolated tilapia Brockmann bodies were determined using Northern hybridization, Western analysis, and quantitative RT-PCR. Similar to the regulation of mammalian insulin, we found that increases of glucose (1-70 mM) and arginine (0.4-25 mM) induced insulin secretion. However, transcription of the insulin gene was activated only by extremely high concentrations of glucose and arginine added simultaneously. When stimulated for 24 h with low concentrations of both inducers or with either of them added separately, tilapia beta-cells were able to replenish secreted insulin and to maintain insulin stores at a constant level without elevations of the insulin mRNA levels. Since the basal level of insulin mRNA was approximately 3.7-fold higher in tilapia beta-cells than it is in mammalian beta-cells, insulin production in tilapia cells probably relies on an enlarged intracellular insulin mRNA pool and does not require the transcriptional activation of the insulin gene.

Insulin expression in the brain and pituitary cells of tilapia (Oreochromis niloticus).

While the presence of immunoreactive insulin in the central nervous system of many vertebrate species is well known, the origin of brain insulin is still debated. In this study, we applied RT-PCR, quantitative RT-PCR (qRT-PCR), and Northern hybridization to examine expression of the insulin gene in different tissues of an adult teleost fish, the Nile Tilapia (Oreochromis niloticus). We found that the insulin gene is transcribed at a high level in Brockmann bodies (pancreatic islet organs) and at a low level in the brain and pituitary gland. In the brain, insulin transcripts were detected in all areas by qRT-PCR and in situ hybridization. The highest level of insulin mRNA was found in the hypothalamus. The level of insulin transcription in the pituitary gland was 6-fold higher than that in the brain and 4.6-fold higher than that in the hypothalamus. Furthermore, insulin mRNA and immunoreactive insulin-like protein was detected in the pituitary gland using in situ hybridization, immunohistochemistry, and Western blot analysis. Our results indicate that in adult tilapia insulin expression is not restricted to the endocrine pancreatic cells, but also occurs in endocrine cells of the pituitary gland and in the neuronal cells of the brain, suggesting that the brain/pituitary gland might represent extrapancreatic origin of insulin production.

Production of transgenic tilapia with Brockmann bodies secreting [desThrB30] human insulin.

BACKGROUND: Tilapia are commercially important tropical fish which, like many teleosts, have anatomically discrete islet organs called Brockmann bodies. When transplanted into diabetic nude mice, tilapia islets provide long-term normoglycemia and mammalian-like glucose tolerance profiles. METHODS: Using site-directed mutagenesis and linker ligation we have "humanized" the tilapia insulin gene so that it codes for [desThrB30] human insulin while maintaining the tilapia regulatory sequences. Following microinjection into fertilized eggs, we screened DNA isolated from whole fry shortly after hatching by PCR. Positive fish were grown to sexual maturity and mated to wild-types and positive Fl's were further characterized. RESULTS: Human insulin was detected in both serum and in the clusters of beta cells scattered throughout the Brockmann bodies. Surrounding non-beta cells as well as other tissues were negative indicating beta cell specific expression. Purification and sequencing of both A-and B-chains verified that the insulin was properly processed and humanized. CONCLUSIONS: After extensive characterization, transgenic tilapia could become a suitable, inexpensive source of islet tissue that can be easily mass-produced for clinical islet xenotransplantation. Because tilapia islets are exceedingly resistant to hypoxia by mammalian standards, transgenic tilapia islets should be ideal for xenotransplantation using immunoisolation techniques.