Olga Hrytsenko

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 F1s were further characterized. Results. Human insulin was detected in both serum and in the clusters of β cells scattered throughout the Brockmann bodies. Surrounding non-β cells as well as other tissues were negative indicating β 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.

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.

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.

Production of transgenic tilapia homozygous for a humanized insulin gene

Recently we developed transgenic tilapia carrying and expressing a ‘‘humanized’’ tilapia insulin gene (Pohajdak et al. 2004). A mosaic male founder (Wright et al. 2008) was mated to multiple wild-type females and the F1 offspring were screened for the presence of the transgene using PCR. DNA from 13 random transgenic offspring and the founder were also analyzed by Southern hybridization. The genome of all tested fish contained three copies of the humanized gene, suggesting that all three copies were integrated in the same chromosomal locus. Using PCR analysis we determined that inserts are not linked in tandem repeats. Transgenes were expressionally active since [desThr30] human insulin was detected in serum and in clusters of b-cells in F1 offspring (Pohajdak et al. 2004). In the next round of breeding, we crossed two halfsibling transgenic F1’s and obtained 163 offspring. To distinguish homozygous, heterozygous and wildtype fish in this F2 population we estimated relative ratios of total insulin genes (tilapia ? humanized insulin genes) versus tilapia insulin genes using quantitative PCR, similar to our previous approach (Pohajdak et al. 2004). Amplifications were performed on a Rotor Gene 2000 real-time PCR machine using a QuantiFast SYBR Green PCR kit. All data were analyzed using a standard curve method. Considering three integrated transgenes are likely located on the same chromosomal locus, the homozygous: wild-type: heterozygous ratio in the F2 population was expected to be 1:1:2. Indeed, from 163 tested fish we found that 41 fish were homozygous, 39 were nontransgenic and 83 were heterozygous, resulting in an almost perfect Mendelian distribution. Homozygous fish were phenotypically indistinguishable from wild-type and heterozygous fish, suggesting that over-expression of humanized insulin has no obvious influences on fish health. The normal 1:1:2 distribution in the adult transgenic F2 population suggests that the mortality of the homozygous fish at early stages of development was approximately the same as in normal and heterozygous tilapia. Interestingly, even though insulin is a known stimulator of growth and differentiation in fish, we did not observe any morphological differences in the development of transgenic tilapia whose genome contained three or six additional insulin gene copies. Furthermore, crossbreeding of two homozygous siblings resulted in the production of a phenotypically-normal fry population with 100% homozygosity. Thus far, we have noted no O. Hrytsenko B. Pohajdak Department of Biology, Dalhousie University, Halifax, NS B3H 4J1, Canada

Lifelong stable human insulin expression in transgenic tilapia expressing a humanized tilapia insulin gene

We have previously reported the production of transgenic Nile tilapia (Oreochromis niloticus) expressing a humanized tilapia insulin gene created for pancreatic islet xenotransplantation (Pohajdak et al. 2004). The founder was mosaic and his islets did not secret human insulin (Wright et al. 2008). For complex reasons, no further work was done for *5 years (Wright et al. 2011). During this period we accumulated a colony of massive and mostly nonfertile transgenic fish from F1 and F2 generations. Since transient transgene expression was common in early attempts to produce over-expression or novel expression of proteins in large commercially important fish species and since our methodology was similar (except for the use of a tilapia gene ‘‘humanized’’ by site directed mutagenesis), we wondered if these very old fish still produced human insulin. Recently, a breeding program was reestablished and we were able to generate homozygous fish which were too small to bleed for insulin measurements (Hrytsenko et al. 2010). We now have small numbers of larger fish and have compared serum insulin levels in young heterozygous vs homozygous fish. Fasting serum was obtained from three groups of transgenic tilapia as described in Table 1. Serum insulin and glucose levels were measured by Calgary Laboratory Services (Calgary, Canada) using the Abbott Axsym microparticle enzyme immunoassay and Roche Modular P hexokinase methods, respectively. The results are expressed as mean ± SD. Statistical differences between the two groups of young transgenic fish were determined using unpaired t test. Differences were considered significant at 5%. Three observations are made: First, when comparing young heterozygous (Group 2) and young homozygous (Group 3) fish, human insulin levels are significantly higher in the homozygous group (P = 0.00025), but serum glucose levels are not significantly different (P = 0.55161). Second, when compared with historical data (Wright et al. 2000), serum glucose levels in both heterozygous (4.29 mmol/l) and homozygous (4.04 mmol/l) young transgenics are similar to those of young wild-type tilapia housed in the same facility (4.19 mmol/l). Third, human insulin O. Hrytsenko B. Pohajdak Department of Biology, Dalhousie University, Halifax, NS, Canada

Immunohistochemical staining for tilapia and human insulin demonstrates that a tilapia transgenic for humanized insulin is a mosaic

In a previous paper we described the production of transgenic tilapia grown from fertilized eggs microinjected with a tilapia insulin transgene ‘‘humanized’’ by site directed mutagenesis; founder male NT 56 was able to pass this to his F1 offspring, and we were able to measure high levels of circulating humanized insulin in many of these offspring (Pohajdak et al. 2004) and subsequently F2 offspring (unpublished observation). In NT 56’s offspring, the size, shape, and distributions of cells staining for tilapia insulin and human insulin appeared to be identical, suggesting that they are the same cells expressing both peptides—as would be expected. Curiously, we were never able to demonstrate humanized insulin in the serum of NT56, suggesting that production of the transgene was silenced or highly inhibited, possibly due to mosaic integration in the genome. After siring many offspring, NT56 died and was necropsied. As shown in Fig. 1, immunoperoxidase staining of NT 56’s islet tissue for (a) tilapia insulin, using a monoclonal antibody specific for tilapia insulin (Snowdon 2003) and for (b) human insulin (Pohajdak et al. 2004) showed that, unlike his offspring, there were two distinct populations of insulin-positive b-cells. The b-cells that stained for tilapia insulin possessed abundant cytoplasm and were arranged in repetitive units of small tight clusters of multiple b-cells, surrounded by concentric layers of nonb-cells; this pattern is typical for wild-type tilapia (Yang et al. 1999) and for teleost fish in general. In stark contrast, the ‘‘b-cells’’ staining for human insulin were smaller, much less numerous, tended to occur as single cells rather than discrete clusters, and appeared excessively densely granulated (i.e., ‘‘constipated’’ with insulin). Studies with mammalian islets have shown that disruption of normal b-cell interrelationships (i.e., islet architecture), inhibits insulin secretion, so the abnormal architecture of the cells staining for human insulin may account for absence of measurable humanized insulin in NT56’s serum. J. R. Wright Jr (&) Department of Pathology & Laboratory Medicine (Calgary Laboratory Services) and the Julia McFarlane Diabetes Research Centre, Faculty of Medicine, University of Calgary, Room C410, Diagnostic & Scientific Centre, 9, 3535 Research Road NW, Calgary, AB, Canada T2L 2K8 e-mail: jim.wright@cls.ab.ca

Using digital inline holographic microscopy and quantitative phase contrast imaging to assess viability of cultured mammalian cells

Digital inline holographic microscopy was used to record holograms of mammalian cells (HEK293, B16, and E0771) in culture. The holograms have been reconstructed using Octopus software (4Deep inwater imaging) and phase shift maps were unwrapped using the FFT-based phase unwrapping algorithm. The unwrapped phase shifts were used to determine the maximum phase shifts in individual cells. Addition of 0.5 mM H2O2 to cell media produced rapid rounding of cultured cells, followed by cell membrane rupture. The cell morphology changes and cell membrane ruptures were detected in real time and were apparent in the unwrapped phase shift images. The results indicate that quantitative phase contrast imaging produced by the digital inline holographic microscope can be used for the label-free real time automated determination of cell viability and confluence in mammalian cell cultures.

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.

ABSTRACT 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.

Abstract A137: Multi-modal treatment with peptide vaccine, metronomic cyclophosphamide and PD-1 blockade provides effective tumor control through a mechanism of epitope spreading

Future cancer immunotherapies will combine multiple treatments to improve immune responses to cancer through synergistic, multi-modal mechanisms. In Phase 1 and 1b clinical trials, we found that metronomic cyclophosphamide (mCPA; 50 mg BID) enhanced the immunogenicity of a DepoVaxTM (DPX) based cancer vaccine (DPX-Survivac) in ovarian cancer patients. We reproduced these results in preclinical transplantable tumor models which allowed us to study the underlying mechanisms of cyclophosphamide-induced immune modulation, as well as explore additional combinations to enhance the therapeutic effect. In syngeneic murine models, mCPA on alternating weeks (20 mg/kg/day PO) in combination with DPX peptide vaccines provided long-term control of established tumors. Using a HPV-expressing tumor model (C3), we found that the combination of mCPA with vaccination caused selective enrichment of antigen-specific CD8+ T cells resulting in increased immune responses detected by IFN-γ ELISPOT and in vivo cytotoxicity assay, as well as improved protection from tumors. Efficacy of the vaccine and mCPA combination was limited in mice bearing advanced tumors. However, antigen-specific CD8+ T cells could be detected infiltrating the tumor by flow cytometry along with increased expression of PD-1 by RT-qPCR, suggesting that the combination therapy was able to generate strong cytotoxic T cell response but was still subject to tumor induced suppression within the tumor microenvironment. Tumor bearing mice treated with vaccine, mCPA and PD-1 blockade (with anti-PD1 or anti-PDL1) could control and induce regression of established tumors which could not be successfully treated with antibody monotherapy. Ex vivo analysis of tumor infiltrating cells demonstrated that anti-PD1 treatment did not further enhance tumor infiltration with antigen-specific CD8 T cells induced by the vaccine/ mCPA treatment, but did increase the expression of cytotoxic genes such as IFN-γ and granzyme B. This indicates that PD1 blockade may promote increased activity of activated T cells within the tumor microenvironment. We evaluated whether PD-1 blockade could enhance anti-tumor immune responses through epitope spreading by developing a mixed tumor model whereby mice were implanted with a mixture of two different tumor cell lines (C3 and B16-F10) and vaccinated with a C3-specific antigen in combination with mCPA and/or anti-PD1. Splenocytes of mice treated with the tri-therapy produced IFN-γ ELISPOT responses towards both cell lines. This effect could not be detected in tumor bearing mice treated with mCPA/anti-PD1 without vaccine. To confirm these results, we evaluated clonality of tumor infiltrating CD8 T cells using TCRβ sequencing. We conclude that enhanced tumor control mediated by the tri-therapy is mediated by highly active, tumor-specific cytotoxic T lymphocytes produced by a strongly immunogenic vaccine, resulting in epitope spreading that can further facilitate tumor rejection or control. These results provide a rationale for clinical testing of checkpoint blockade therapy in combination with our highly immunogenic combination of mCPA and DPX-Survivac. Citation Format: Genevieve M. Weir, Olga Hrytsenko, Marianne Stanford, Mohan Karkada, Neil Berinstein, Marc Mansour. Multi-modal treatment with peptide vaccine, metronomic cyclophosphamide and PD-1 blockade provides effective tumor control through a mechanism of epitope spreading. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A137.

Abstract 4903: Multimodal therapy with a potent vaccine, metronomic cyclophosphamide and anti-PD-1 enhances immunotherapy of advanced tumors by increasing activation and clonal expansion of tumor infiltrating T cells

Future cancer immunotherapies will combine multiple treatments to improve immune responses to cancer through synergistic, multi-modal mechanisms. In Phase 1 and 1b clinical trials, we found that metronomic cyclophosphamide (mCPA; 50 mg BID) enhanced the immunogenicity of a DepoVaxTM (DPX) based cancer vaccine (DPX-Survivac) in ovarian cancer patients. We reproduced these results in preclinical transplantable tumor models which allowed us to study the underlying mechanisms of cyclophosphamide induced immune modulation, as well as explore additional combinations to enhance the therapeutic effect. Using a HPV-expressing murine tumor model (C3), we found that treatment with mCPA (20 mg/kg/day PO) in combination with a DPX peptide vaccine caused selective enrichment of antigen-specific CD8+ T cells, resulting in increased immune responses detected by IFN-a ELISPOT and in vivo cytotoxicity assay. The combination provided long term-control of tumors when initiated within one week of tumor implantation, however efficacy was limited in mice bearing advanced tumors. Antigen-specific CD8+ T cells could be detected infiltrating advanced tumors by flow cytometry, along with increased expression of PD-1 on the T cells and PD-L1 on the tumor cells, suggesting that the tumor microenvironment (TME) was mediating immune suppression through increased PD-1:PD-L1 signaling. Treatment of tumor bearing mice with vaccine, mCPA and PD-1 blockade (with anti-PD-1 or anti-PD-L1) resulted in tumor control of established tumors which were not successfully treated with antibody monotherapy. Analysis of tumor infiltrating leukocytes by flow cytometry demonstrated that anti-PD-1 treatment did not further enhance tumor infiltration with antigen-specific CD8+ T cells induced by the vaccine/ mCPA treatment. However, RT-qPCR analysis of the tumor detected an increase in expression of cytotoxic T cell gene signatures within the tumor in combination with anti-PD-1 treatment. Clonal analysis was performed of the total TCRâ sequences using gDNA extracted from the tumors. Vaccine and mCPA treatment resulted in selective expansion of clones, as the top 10 clones accounted for 35% of the total TCRâ sequences; tri-therapy including anti-PD-1 significantly enhanced the expansion of T cells within the TME so that the top 10 clones accounted for 46% of the total TCRâ sequences (p Citation Format: Genevieve Weir, Olga Hrytsenko, Tara Quinton, Mohan Karkada, Neil L. Berinstein, Marianne Stanford, Marc Mansour. Multimodal therapy with a potent vaccine, metronomic cyclophosphamide and anti-PD-1 enhances immunotherapy of advanced tumors by increasing activation and clonal expansion of tumor infiltrating T cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4903.