Immune-competent Animals Research Articles

Orally bioavailable glutamine antagonist prodrug JHU-083 penetrates mouse brain and suppresses the growth of MYC-driven medulloblastoma

A subset of poor-prognosis medulloblastoma has genomic amplification of MYC. MYC regulates glutamine metabolism in multiple cellular contexts. We modified the glutamine analog 6-diazo-5-oxo-l-norleucine (DON) to mask its carboxylate and amine functionalities, creating a prodrug termed JHU-083 with increased oral bioavailability. We hypothesized that this prodrug would kill MYC-expressing medulloblastoma. JHU-083 treatment caused decreased growth and increased apoptosis in human MYC-expressing medulloblastoma cell lines. We generated a mouse MYC-driven medulloblastoma model by transforming C57BL/6 mouse cerebellar stem and progenitor cells. When implanted into the brains of C57BL/6 mice, these cells formed large cell/anaplastic tumors that resembled aggressive medulloblastoma. A cell line derived from this model was sensitive to JHU-083 in vitro. Oral administration of JHU-038 led to the accumulation of micromolar concentrations of DON in the mouse brain. JHU-083 treatment significantly increased the survival of immune-competent animals bearing orthotopic tumors formed by the mouse cerebellar stem cell model as well as immune-deficient animals bearing orthotopic tumors formed by a human MYC-amplified medulloblastoma cell line. These data provide pre-clinical justification for the ongoing development and testing of orally bioavailable DON prodrugs for use in medulloblastoma patients.

CD8+CD122+PD-1+ Tregs Synergize With Costimulatory Blockade of CD40/CD154, but Not B7/CD28, to Prolong Murine Allograft Survival.

A transplanted organ is always rejected in the absence of any immunosuppressive treatment due to vigorous alloimmunity. However, continuously global immunosuppression with a conventional immunosuppressant may result in severe side effects, including nephrotoxicity, tumors and infections. Tregs have been widely used to inhibit allograft rejection, especially in animal models. However, it's well accepted that administration of Tregs alone is not satisfactory in immune-competent wild-type animals. Therefore, it's imperative to promote Treg therapies under the cover of other approaches, including costimulatory blockade. In the present study, we demonstrated that administration of in vitro-expanded CD8+CD122+PD-1+ Tregs synergized with costimulatory blockade of CD40/CD154, but not B7/CD28, to prolong skin allograft survival in wild-type mice and to reduce cellular infiltration in skin allografts as well. Treg treatment and blockade of CD40/CD154, but not B7/CD28, also exhibited an additive effect on suppression of T cell proliferation in vitro and pro-inflammatory cytokine expression in skin allografts. Importantly, blocking B7/CD28, but not CD40/CD154, costimulation decreased the number of transferred CD8+CD122+PD-1+ Tregs and their expression of IL-10 in recipient mice. Furthermore, it's B7/CD28, but not CD40/CD154, costimulatory blockade that dramatically reduced IL-10 production by CD8+CD122+PD-1+ Tregs in vitro, suggesting that B7/CD28, but not CD40/CD154, costimulation is critical for their production of IL-10. Indeed, infusion of IL-10-deficient CD8+CD122+PD-1+ Tregs failed to synergize with anti-CD154 Ab treatment to further prolong allograft survival. Our data may explain why blocking B7/CD28 costimulatory pathway does not boost IL-10-dependent Treg suppression of alloimmunity. Thus, these findings could be implicated in clinical organ transplantation.

A CD117-Amanitin Antibody Drug Conjugate (ADC) Effectively Depletes Human and Non-Human Primate Hematopoietic Stem and Progenitor Cells (HSPCs): targeted Non-Genotoxic Conditioning for Bone Marrow Transplant

Current regimens for patient preparation, or conditioning, prior to bone marrow transplantation (BMT) limit the use of this curative procedure due to regimen-related mortality and morbidities. To address this, we developed a CD117-ADC to specifically deplete recipient HSPCs. CD117 is an ideal target for an ADC-based approach to patient preparation as it is selectively expressed on HSPCs. In addition, CD117 is frequently overexpressed on in acute myelogenous leukemia (AML). Thus, a CD117-ADC may enable curative treatment while potentially improving disease control prior to and following transplant. We developed a potent CD117 antibody conjugated to amanitin (AM), an RNA polymerase inhibitor, that depletes primary human CD34+ cells in vitro with an IC50 of 21pM (Fig. 1A). The ADC was designed to have a fast half-life, allowing the HSPC ablating ADC to clear prior to stem cell transplant. Humanized NSG mice treated with the anti-CD117-AM had >95% depletion of human HSPCs in bone marrow following a single dose of 0.1 mg/kg (Fig. 1B). The specificity of anti-CD117-AM was confirmed by the persistence of peripheral human lymphocytes, indicating the ability to maintain adaptive immunity. In addition, a single dose of anti-CD117-AM extended survival of NSG mice challenged with CD117+ patient derived xenografts (PDX), demonstrating potent anti-tumor effects. We treated NHPs with the cross-reactive anti-CD117-AM to determine if the ADC could deplete HSPCs in immune-competent animals. Dose-dependent decreases in phenotypic HSPCs and colony-forming units (CFUs) were observed in the bone marrow 7 days after dosing, with >95% HSPC depletion after a single dose of 0.3 mg/kg (Fig. 1C, 1D). Depletion was target- and AM-dependent, as the antibody alone and isotype-AM had no effect. In the periphery a transient decrease in reticulocytes was observed at day 4 with a neutrophil nadir at day 18. WBC and lymphocytes were within normal range throughout. Transient off-target effects consisted of reversible thrombocytopenia (Day 4-8) and reversible and mild liver enzyme levels (Day 4-10). Fractionated dosing achieved similar efficacy with reduced off-target effects. As expected, the fast half-life anti-CD117-AM was rapidly cleared (t1/215-18 h), providing appropriate pharmacokinetics for BMT. In summary, anti-CD117-AM can selectively deplete HSPCs in humanized NSG mice and NHP. In addition, the ADC exhibited potent anti-tumor activity in AML PDX models. This strategy could preserve the adaptive immune system with delayed onset of neutrophil nadir, potentially shortening the period of neutropenia during BMT. Targeted non-genotoxic conditioning with ADCs could provide a significant improvement to current approaches to patient preparation with an acceptable safety profile prior to BMT and gene therapies.

Chimeric tumor modeling reveals role of partial PDL1 expression in resistance to virally induced immunotherapy

Expression of PDL1 on the surface of tumor cells can blunt the efficacy of many cancer immunotherapies. For example, our lab has previously shown that tumors derived from malignant cells incapable of expressing PDL1 are highly susceptible to immunotherapy induced by oncolytic virus treatment while tumors derived from PDL1 capable cells are highly resistant. In patient biopsies, however, expression of PDL1 on malignant cells is often not uniform with some cells expressing PDL1 while others do not. Importantly, how this partial PDL1 positivity influences the outcomes of immunotherapy remains largely unknown. In the current work, we expand on our previous findings by generating partially PDL1 positive tumors in immune competent animals and asking what percentage of tumor cells must express PDL1 for a tumor to become functionally resistant to oncolytic treatment. Our results indicate that the responsiveness of partially PDL1+ tumors correlates linearly with the percentage of PDL1 capable cells present at the initiation of treatment. Additionally, we observe that tumors which relapse after treatment display a significant increase in the numbers of PDL1 capable cells present suggesting that specific editing of mixed tumors might play a role in disease relapse. These data indicate that varying levels of PDL1 expression can play a significant role in the outcomes of oncolytic immunotherapy and challenges the concept that tumors should be viewed as simply PDL1+ or PDL1−.

LDH-A regulates the tumor microenvironment via HIF-signaling and modulates the immune response.

Previous studies show that LDH-A knockdown reduces orthotopic 4T1 breast tumor lactate and delays tumor growth and the development of metastases in nude mice. Here, we report significant changes in the tumor microenvironment (TME) and a more robust anti-tumor response in immune competent BALB/c mice. 4T1 murine breast cancer cells were transfected with shRNA plasmids directed against LDH-A (KD) or a scrambled control plasmid (NC). Cells were also transduced with dual luciferase-based reporter systems to monitor HIF-1 activity and the development of metastases by bioluminescence imaging, using HRE-sensitive and constitutive promoters, respectively. The growth and metastatic profile of orthotopic 4T1 tumors developed from these cell lines were compared and a primary tumor resection model was studied to simulate the clinical management of breast cancer. Primary tumor growth, metastasis formation and TME phenotype were significantly different in LDH-A KD tumors compared with controls. In LDH-A KD cells, HIF-1 activity, hexokinase 1 and 2 expression and VEGF secretion were reduced. Differences in the TME included lower HIF-1α expression that correlated with lower vascularity and pimonidazole staining, higher infiltration of CD3+ and CD4+ T cells and less infiltration of TAMs. These changes resulted in a greater delay in metastases formation and 40% long-term survivors (>20 weeks) in the LDH-A KD cohort following surgical resection of the primary tumor. We show for the first time that LDH-depletion inhibits the formation of metastases and prolongs survival of mice through changes in tumor microenvironment that modulate the immune response. We attribute these effects to diminished HIF-1 activity, vascularization, necrosis formation and immune suppression in immune competent animals. Gene-expression analyses from four human breast cancer datasets are consistent with these results, and further demonstrate the link between glycolysis and immune suppression in breast cancer.

LB1572 Two rat models of psoriasis for drug development

Psoriasis is the most common chronic autoimmune skin condition, impacting roughly 125 million people worldwide. The pathogenesis is not wholly understood, though an assumed complex interplay between environmental factors, immune dysregulation and genetic susceptibility. Data suggests that an environmental/pathogenic insult to the skin precipitates the activation of immune cells in the skin leading to a dysregulated pro-inflammatory response. This response triggers secretion of IL-12 and IL-23, causing migration and differentiation of Th17 and Th1 cells. These cells release cytokines such as IL-22 and IL-17A/F driving proliferation of keratinocytes and epithelial cells. It has become increasingly important to model this disease, in immune competent animals such as rats. To this end, MD Biosciences has optimized two psoriasis models in the rat. IMQ mimics a pathogenic insult to the skin, eliciting a robust Th17 immune response leading to plaque formation. IMQ is applied to the shaved backs and ears of naïve rats, and disease progression and skin thickening is monitored. Diseased animals exhibit erythema and plaque formation shortly after the start of the study and progress through termination accompanied by increased biomarkers, IL-22, IL17A and IL-17F. IL-23 is the main pathogenic cytokine in the initiation of psoriasis disease progression and has become a lead target for the clinical treatment. Exogenous IL-23 can be delivered directly to the dermis of rats to induce a downstream cascade of inflammatory biomarker upregulation, erythema and epidermal hyperplasia, similar to what is seen in human psoriatic plaques. The IMQ- and IL-23-induced psoriasis models are robust pre-clinical models to help develop therapeutics, as both models mimic specific disease pathologies. These models can be used to assess efficacy of candidate drugs for pathogenic pathway targeting such as Th17 and IL-23/IL-17. The versatility of the models and the benefits of the rat as a host organism make these models ideal for pre-clinical drug development for psoriasis or other Th17/IL-17 dependent diseases.

Antibody and cytokine response to Cystoisospora suis infections in immune-competent young pigs

BackgroundTo date, investigations on the immune response to Cystoisospora suis infections focused on suckling piglets, the age group clinically most affected. Actively immunizing piglets is unfeasible due to their immature immune system and the typically early infection in the first days after birth. Therefore, understanding and possibly enhancing the immune response of immune-competent animals is the prerequisite to develop a passive immunization strategy for piglets which currently rely on very limited treatment options.MethodsTo investigate antibody and cytokine responses of immune-competent animals and the impact of the oral immunization protocol on their immune response, growers with unknown previous exposure to C. suis (10–11 weeks-old) were infected one or three times with different doses (600 and 6000 or 200 and 2000, respectively) of C. suis oocysts, and compared to uninfected controls. Oocyst excretion was evaluated, and blood and intestinal mucus antibody titers were determined by IFAT. Systemic production of Th1, Th2, inflammatory and regulatory cytokines was determined in different immune compartments at mRNA and (after stimulation with a recombinant merozoite-protein) at protein level by PCR and multiplex fluorescent immunoassay, respectively.ResultsInfection generated significantly increased serum IgA and IgG levels against C. suis sporozoites and merozoites, irrespective of infection mode, with IgG against merozoites showing the strongest increase. No clinical signs and only occasional excretion were observed. The systemic cytokine response to C. suis was only weak. Nonetheless, in white blood cells, IL-4, IL-6 and IL-10 mRNA-levels significantly increased after infection, whereas IFN-ɣ, IL-2 and TGF-β expression tended to decrease. In mesenteric lymph nodes (MLN), IL-10 and TNF-α levels were elevated while splenic cytokine expression was unaltered upon infection. Stimulated MLN-derived lymphocytes from infected pigs produced slightly more IL-12 and less IFN-α than controls.ConclusionsAn infection and a subsequent systemic immune response can be induced in immune-competent animals by all evaluated infection models and growers can be used as models to mimic sow immunizations. The immune response to C. suis, although mild and with considerable variation in cytokine expression, was characterized by a Th2-associated and regulatory cytokine profile and antibody production. However, none of the parameters clearly stood out as a potential marker associated with protection. Antibody titers were significantly positively related with oocyst excretion and might thus serve as correlates for parasite replication or severity of infection.

Development of experimental sporothrichosis in a murine model with yeast and mycelial forms of Sporothrix schenkii

Sporotrichosis is a subcutaneous disease that affects both men and a variety of animals caused by the dimorphic fungus Sporothrix schenckii . The aims of this study were to observe differences in virulence between inocula containing the yeast (Y) or mycelial (M) forms, to compare the reactions induced by the two inocula injected in different anatomical sites, and to verify the possibility of horizontal transmission of the disease by contact between infected and healthy animals. All animals were observed during a period of nine weeks. Mice were inoculated subcutaneously in the paw pads and in the regions of the hip and shoulder joints. Another group of non-inoculated healthy mice was kept in direct contact with mice developing the subcutaneous lesions. Animals of groups Y and M developed lesions due to infections and clinical symptoms very characteristic of sporothrichosis. Lesions were more prominent and with longer duration when occurring in the pads than in any other site of inoculation. Non-inoculated mice remained healthy as it occurred to animals in the control group. The development of typical clinical symptoms for sporothrichosis in all inoculated groups suggests that no difference exists in virulence between the yeast and mycelial forms of the same isolated of S. schenckii . Moreover, the paw pads appear to be the site of choice for the inoculation for experimental sporotrichosis in the murine model. In addition, this study also showed that the contact with sick animals and contaminated materials is insufficient to transmit the infection to immune competent animals with intact skin.

Ultrasound targeted gene therapy in a mouse model of prostate cancer: Evaluation of immune response.

120 Background: Gene transfer to malignant sites using human adenoviruses (hAd) has been limited because of their immunogenicity. Murine cells often lack some of the receptors needed for hAd infection; therefore, are generally non-permissive for hAd infection and replication, which limits translational studies of adenoviral gene transfer techniques. We developed a gene transfer method, which uses a combination of lipid-encapsulated perfluorocarbon microbubbles (MBs) and ultrasound (US) to shield and deliver hAds to a specific tissue bypassing the requirement of the coxsackie and adenovirus receptor (CAR). Methods: Transduction efficiency and GFP protein expression of hAd.GFP was assessed by flow cytometry and fluorescence microscopy in murine TRAMP-C2 and human DU145 prostate cancer cells. Innate and acquired immunity response was determined by ELISA and CTL assay in C57BL/6 mice bearing TRAMP-C2 syngeneic tumor grafts following injections of MBs-Ad.GFP complexes in the presence or absence of ultrasound. Results: We observed that the murine prostate cancer cells TRAMP-C2 were transduced less efficiently by hAd.GFP than the human DU145 cells. We showed in vitro that the transduction rate was increased significantly in both TRAMP-C2 and DU145 prostate cancer cells when delivering the Ad particles by a combination of MBs and US. Moreover, we observed expression of the GFP transgene in both cell lines at 48 hours and 72 hours. Lack of activation of the innate and acquired immunity was observed in vivo by quantifying IL-6 and TNF-α cytokines, and by assaying neutralizing IgG antibodies and CTLs activity, following intratumoral or intravenous injections of MBs-Ad.GFP complexes in the presence or absence of ultrasound. Conclusions: This study demonstrates the feasibility of using the TRAMP-C2 murine model of prostate adenocarcinoma to translate our ultrasound-mediated MB-Ad delivery system from the bench to the clinic. Our data provides evidence that the TRAMP-C2 prostate cancer graft model is a suitable system to study in immune competent animals the capacity of lipid-encapsulated perfluorocarbon MBs and US, to shield and deliver hAds to a site-specific tissue bypassing the requirement of specific receptors.

Novel Conditioning Regimens for Blood Stem Cell Transplantation

Hematopoietic stem cell (HSC) therapies rely on chemotherapy and/or radiation to overcome host barriers to permit cell engraftment and provide life-long donor derived hematopoiesis. In conventional allogeneic transplantation these modalities function to both suppress immune rejection and create sufficient niche space to allow HSC to engraft. For gene-corrected or gene-modified cells that are derived from autologous patient sources, access to the HSC niche constitutes the primary barrier. Non-genotoxic approaches are being developed with the goal to target molecules expressed on hematopoietic stem and progenitors. We have studied the use of monoclonal antibodies (mAbs) that target the molecule CD117, also known as cKit, to deplete endogenous stem cells and safely allow engraftment of purified HSC. CD117 is a cell surface molecule expressed at high levels on HSC and early myeloid progenitors as well as other non-hematopoietic cell types. It is a receptor tyrosine kinase that upon binding to its ligand, stem cell factor (SCF), propagates intracellular signaling pathways that regulate activities including cell survival, proliferation and differentiation. Studies in immune deficient mice showed that a single dose of anti-CD117 mAb transiently depletes HSC, creating a therapeutic window for donor HSC to engraft1. We have developed for use in clinical transplantation an anti-human CD117 (anti-hCD117) mAb. This antibody significantly inhibits in vitro proliferation of human HSC and effectively depletes human HSC in mice xenografted with human cord blood cells2, and is effective in depleting HSC and progenitors from the bone marrow of non-human primates (NHP). Although CD117 is expressed on non-hematopoietic cells including mast cells, melanocytes, and cells in the gastrointestinal tract, antibody administration in animals and in healthy human volunteers, show little to no off-target toxicity. We are currently testing in a Phase I clinical trial the anti-hCD117 mAb as the sole conditioning agent for the transplantation of children with severe combined immunodeficiency (SCID). We have observed that the effect of the mouse anti-CD117 mAb is more potent in immune deficient mice compared to immune sufficient wildtype mice as evidenced by markedly inferior HSC engraftment results in wildtype recipients. Thus, we are exploring next generation approaches to achieve deeper and more extended depletion of HSC and progenitors in immune competent animals. In vivo HSC clearance by the mouse anti-CD117 mAb is dependent on Fc-mediated antibody effector functions. We therefore tested combination therapy with anti-CD117 mAb plus biologic agents that inhibit CD47, a myeloid-specific immune checkpoint, with the goal to promote phagocytosis of target cells that bind the anti-CD117 mAb. The combined treatment led to elimination of >99% of host HSCs and robust multilineage blood reconstitution following HSC transplantation3. These studies show that a conditioning approach using anti-CD117 antibodies is feasible, and has the potential to replace genotoxic agents with targeted and safer biologic agents. 1. Czechowicz A, Kraft D, Weissman IL, et al, Efficient transplantation via antibody-based clearance of hematopoietic stem cell niches. Science. 2007; 318: 1296. 2. Logan AC, Czechowicz A, Kelley BV, et al. Anti-CD117 (c-Kit) monoclonal antibodies deplete human hematopoietic stem cells and facilitate their replacement in humanized NOD/SCID/IL2Rg-/- mice: A non-toxic conditioning regimen for allotransplantation. Blood . 2012; 120: 4099. 3. Chhabra A, Ring AM, Weiskopf K, et al.Hematopoietic stem cell transplantation in immunocompetent hosts without radiation or chemotherapy. Sci Transl Med . 2016; 8: 351RA105. Disclosures No relevant conflicts of interest to declare.

Sub-clonal analysis of the murine C1498 acute myeloid leukaemia cell line reveals genomic and immunogenic diversity

BackgroundIn acute myeloid leukaemia (AML)-affected patients, the presence of heterogeneous sub-clones at diagnosis has been shown to be responsible for minimal residual disease and relapses. The role played by the immune system in this leukaemic sub-clonal hierarchy and maintenance remains unknown. As leukaemic sub-clone immunogenicity could not be evaluated in human AML xenograft models, we assessed the sub-clonal diversity of the murine C1498 AML cell line and the immunogenicity of its sub-clones in immune-competent syngeneic mice. MethodologyThe murine C1498 cell line was cultured in vitro and sub-clonal cells were generated after limiting dilution. The genomic profiles of 6 different sub-clones were analysed by comparative genomic hybridization arrays (CGH). The sub-clones were then injected into immune-deficient and – competent syngeneic mice. The immunogenicities of the sub-clones was evaluated through 1) assessment of mouse survival, 2) determination of leukaemic cell infiltration into organs by flow cytometry and the expression of a fluorescent reporter gene, 3) assessment of the CTL response ex vivo and 4) detection of residual leukaemic cells in the organs via amplification of the genomic reporter gene by real-time PCR (qPCR). ResultsGenomic analyses revealed heterogeneity among the parental cell line and its derived sub-clones. When injected individually into immune-deficient mice, all sub-clones induced cases of AML with different kinetics. However, when administered into immune-competent animals, some sub-clones triggered AML in which no mice survived, whereas others elicited reduced lethality rates. The AML-surviving mice presented efficient anti-leukaemia CTL activity ex vivo and eliminated the leukaemic cells in vivo. ConclusionWe showed that C1498 cell sub-clones presented genomic heterogeneity and differential immunogenicity resulting either in immune escape or elimination. Such findings could have potent implications for new immunotherapeutic strategies in patients with AML.

Genomic analysis of Isometamidium Chloride resistance in Trypanosoma congolense

Isometamidium Chloride (ISM) is one of the principal drugs used to counteract Trypanosoma congolense infection in livestock, both as a prophylactic as well as a curative treatment. However, numerous cases of ISM resistance have been reported in different African regions, representing a significant constraint in the battle against Animal African Trypanosomiasis.In order to identify genetic signatures associated with ISM resistance in T. congolense, the sensitive strain MSOROM7 was selected for induction of ISM resistance in a murine host. Administered ISM concentrations in immune-suppressed mice were gradually increased from 0.001 mg/kg to 1 mg/kg, the maximal dose used in livestock. As a result, three independent MSOROM7 lines acquired full resistance to this concentration after five months of induction, and retained this full resistant phenotype following a six months period without drug pressure. In contrast, parasites did not acquire ISM resistance in immune-competent animals, even after more than two years under ISM pressure, suggesting that the development of full ISM resistance is strongly enhanced when the host immune response is compromised. Genomic analyses comparing the ISM resistant lines with the parental sensitive line identified shifts in read depth at heterozygous loci in genes coding for different transporters and transmembrane products, and several of these shifts were also found within natural ISM resistant isolates. These findings suggested that the transport and accumulation of ISM inside the resistant parasites may be modified, which was confirmed by flow cytometry and ex vivo ISM uptake assays that showed a decrease in the accumulation of ISM in the resistant parasites.

Immune-mediated effects targeting hepatitis C virus in a syngeneic replicon cell transplantation mouse model

ObjectiveHCV is characterised by its ability to establish chronic infection in hepatocytes and to replicate in the presence of an inflammation. We mimicked this situation in vivo in immune-competent mice...

Humanized mouse model for assessing the human immune response to xenogeneic and allogeneic decellularized biomaterials.

Current assessment of biomaterial biocompatibility is typically implemented in wild type rodent models. Unfortunately, different characteristics of the immune systems in rodents versus humans limit the capability of these models to mimic the human immune response to naturally derived biomaterials. Here we investigated the utility of humanized mice as an improved model for testing naturally derived biomaterials. Two injectable hydrogels derived from decellularized porcine or human cadaveric myocardium were compared. Three days and one week after subcutaneous injection, the hydrogels were analyzed for early and mid-phase immune responses, respectively. Immune cells in the humanized mouse model, particularly T-helper cells, responded distinctly between the xenogeneic and allogeneic biomaterials. The allogeneic extracellular matrix derived hydrogels elicited significantly reduced total, human specific, and CD4+ T-helper cell infiltration in humanized mice compared to xenogeneic extracellular matrix hydrogels, which was not recapitulated in wild type mice. T-helper cells, in response to the allogeneic hydrogel material, were also less polarized towards a pro-remodeling Th2 phenotype compared to xenogeneic extracellular matrix hydrogels in humanized mice. In both models, both biomaterials induced the infiltration of macrophages polarized towards a M2 phenotype and T-helper cells polarized towards a Th2 phenotype. In conclusion, these studies showed the importance of testing naturally derived biomaterials in immune competent animals and the potential of utilizing this humanized mouse model for further studying human immune cell responses to biomaterials in an invivo environment.

Contrasting Views on the Role of Mesenchymal Stromal/Stem Cells in Tumour Growth: A Systematic Review of Experimental Design.

The effect of mesenchymal stromal/stem cells (MSCs) on tumour growth remains controversial. Experimental evidence supports both an inhibitory and a stimulatory effect. We have assessed factors responsible for the contrasting effects of MSCs on tumour growth by doing a meta-analysis of existing literature between 2000 and May 2017. We assessed 183 original research articles comprising 338 experiments. We considered (a) in vivo and in vitro experiments, (b) whether in vivo studies were syngeneic or xenogeneic, and (c) if animals were immune competent or deficient. Furthermore, the sources and types of cancer cells and MSCs were considered together with modes of cancer induction and MSC administration. 56% of all 338 experiments reported that MSCs promote tumour growth. 78% and 79% of all experiments sourced human MSCs and cancer cells, respectively. MSCs were used in their naïve and engineered form in 86% and 14% of experiments, respectively, the latter to produce factors that could alter either their activity or that of the tumour. 53% of all experiments were conducted in vitro with 60% exposing cancer cells to MSCs via coculture. Of all in vivo experiments, 79% were xenogeneic and 63% were conducted in immune-competent animals. Tumour growth was inhibited in 80% of experiments that used umbilical cord-derived MSCs, whereas tumour growth was promoted in 64% and 57% of experiments that used bone marrow- and adipose tissue-derived MSCs, respectively. This contrasting effect of MSCs on tumour growth observed under different experimental conditions may reflect differences in experimental design. This analysis calls for careful consideration of experimental design given the large number of MSC clinical trials currently underway.

Abstract 2361: Novel animal models for prediction of cancer vaccine response

Abstract Predicting cancer vaccine response is a challenge that must be addressed in immune competent animals that recapitulate human disease and human response to therapy. Furthermore, genetically heterogeneous humans respond differently to vaccination due, in part, to their varying MHC haplotypes and polymorphisms including those found in costimulatory or checkpoint genes. We previously showed that human HER2 transgenic (Tg) mice of different genetic backgrounds have distinct response to HER2 vaccines in the order of BALB > (BALBxB6)F1 > (B6xDR3)F1 > B6 (Radkevich-Brown 2010). We anticipate even greater variance among human populations, and seek genetically diverse animal models to project human cancer vaccine response. Domestic felines develop mammary tumors with similar pathology and etiology as human cancer, and expression of EGFR, HER2 and HER3 genes that are highly homologous to their human counterparts. Intramuscular electrovaccination of healthy outbred domestic shorthair cats with heterologous (xenogeneic) or single point-mutated self HER2 DNA induces HER2-specific T cells in ∼30% of vaccinated cats (Gibson 2015). Humoral immune responses were induced by both vaccine constructs, with point-mutated feline HER2 vaccine producing antibodies to unique epitopes on feline mammary carcinoma (FMC). The latter inhibits FMC growth in vitro and delays tumor growth in SCID mice. Vaccination with wild type feline HER2 DNA fails to induce specific immunity and even hinders feline HER2 immune activation by other vaccines. Studies in domestic cats provide novel insight on the range and nature of vaccine response in healthy cats which can also be tested in feline cancer patients. Strategic modification of minimal residues may be most effective in inducing reactivity to cancer associated self antigens. To address genetic regulation of vaccine response, diversity outbred (DO) mice offer a pragmatic option. These mice were generated by cross-breeding 8 inbred mouse strains to ensure broad genetic diversity among the mice (Svenson 2012). The human HER2 gene is introduced by breeding DO mice with our human HER2 transgenic (Tg) mice. F1 mice represent a genetically heterogeneous population that expresses human HER2 as a self antigen. Specific genes impacting vaccine response will be identified by genome-wide quantitative trait locus (QTL) scan to associate haplotype or single nucleotide variants with vaccine response. Findings in these mice may lead to the identification of critical genes that regulate cancer vaccine response. Candidate vaccines identified in DO mice can be further validated in cats and expanded to human trials. With these novel animal models, cancer vaccines can be designed and tested with greater precision, and patients with reactive genotypes may be identified before vaccination. (Supported by CA76340) Citation Format: Heather Gibson, Richard F. Jones, Joyce D. Reyes, Wei-Zen Wei. Novel animal models for prediction of cancer vaccine response. [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 2361.

Quantitative in vivo optical tomography of cancer progression & vasculature development in adult zebrafish.

We describe a novel approach to study tumour progression and vasculature development in vivo via global 3-D fluorescence imaging of live non-pigmented adult zebrafish utilising angularly multiplexed optical projection tomography with compressive sensing (CS-OPT). This “mesoscopic” imaging method bridges a gap between established ~μm resolution 3-D fluorescence microscopy techniques and ~mm-resolved whole body planar imaging and diffuse tomography. Implementing angular multiplexing with CS-OPT, we demonstrate the in vivo global imaging of an inducible fluorescently labelled genetic model of liver cancer in adult non-pigmented zebrafish that also present fluorescently labelled vasculature. In this disease model, addition of a chemical inducer (doxycycline) drives expression of eGFP tagged oncogenic K-RASV12 in the liver of immune competent animals. We show that our novel in vivo global imaging methodology enables non-invasive quantitative imaging of the development of tumour and vasculature throughout the progression of the disease, which we have validated against established methods of pathology including immunohistochemistry. We have also demonstrated its potential for longitudinal imaging through a study of vascular development in the same zebrafish from early embryo to adulthood. We believe that this instrument, together with its associated analysis and data management tools, constitute a new platform for in vivo cancer studies and drug discovery in zebrafish disease models.

A Novel Model of Metastatic Conjunctival Melanoma in Immune-Competent Mice.

Conjunctival melanoma (CM) is an ocular surface tumor that can lead to fatal metastases. Patients developing, tumor-associated lymphangiogenesis have a significantly increased risk of metastatic disease, because tumor spread primarily occurs via lymphatic vessels to the draining lymph node. Here, we describe a novel immune-competent mouse model of CM that displays tumor-associated lymphangiogenesis with development of metastatic tumors. C57BL/6N mice received C57BL/6N-derived dermal melanoma cells (hepatocyte growth factor [HGF] cyclin dependent kinase-4 [Cdk4]+) or B16F10 via subconjunctival injection. A clinical score quantified primary tumor growth and metastases were identified by macroscopic examination of the draining lymph nodes, lung, and spleen. Confirmation of tumors and metastases was achieved by immunohistochemical staining for markers of pigmented cells (tyrosinase related protein-2 [TRP2]) and S-100, and of cell proliferation (Ki67). The intra- and peritumoral CD31+ blood and lymphatic vessel endothelium hyaluronan receptor-1 (LYVE-1)+ lymphatic vessels were quantified immunohistochemically. All mice rapidly developed aggressive TRP2+, S100+, and Ki67+ CM. Metastatic tumors were found in the lymph node (9%) and lung (6%) of HGF-Cdk4(R24C)-treated mice and in the spleen (8%) and lung (17%) of B16F10-treated mice. The amount of peri- and intratumoral blood vessels was significantly increased compared with lymphatic vessels. This CM model in immune-competent animals offers new possibilities to study the pathobiology of tumor growth, invasion, and mechanisms of metastatic tumor spread, and provides a robust model to explore new immune-based and antilymphangiogenic treatment modalities of this malignancy.

Abstract 3216: Immunogenicity and genomic profiling reveal sub-clonal diversity of a murine acute myeloid leukemia (AML) cell line

Abstract In acute myeloid leukemia (AML), clonal heterogeneity at diagnosis and the presence of minor sub-clones were shown to be responsible for minimal residual disease and relapses in patients. Several studies have proposed that their persistence could be due to their dormant state (i.e. quiescence and/or control of their growth by immune cells). As contribution of immunity to clonal persistence could not be evaluated in xenografts models, we assessed for the clonal diversity of a murine AML cell line. Genomic analyses revealed heterogeneity among the parental cell line and its derived clones. Injected individually into immune-deficient mice, all clones induced AML with different kinetics. However, when administered into immune-competent animals, some clones triggered AML with no mice survival, whereas others elicited reduced lethality rates. The leukemia-surviving mice presented an efficient anti-tumoral CTL activity and reduced lymphopenia. Investigating for residual leukemic cells in the organs of these mice, elimination rather than persistence was observed. Thus, we showed that genetic heterogeneity among the clones led to differential immunogenicity resulting either in their immune escape or elimination. Such findings could have potent implications for new immunotherapeutic strategies in patients. Citation Format: Carine Brinster, Virginie DRISS, Bruno Quesnel, Martin Figeac, Frédéric Leprêtre, Céline Villenet, Isabelle Briche. Immunogenicity and genomic profiling reveal sub-clonal diversity of a murine acute myeloid leukemia (AML) cell line. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3216. doi:10.1158/1538-7445.AM2015-3216

Manipulating adenovirus hexon hypervariable loops dictates immune neutralisation and coagulation factor X-dependent cell interaction in vitro and in vivo.

Adenoviruses are common pathogens, mostly targeting ocular, gastrointestinal and respiratory cells, but in some cases infection disseminates, presenting in severe clinical outcomes. Upon dissemination and contact with blood, coagulation factor X (FX) interacts directly with the adenovirus type 5 (Ad5) hexon. FX can act as a bridge to bind heparan sulphate proteoglycans, leading to substantial Ad5 hepatocyte uptake. FX “coating” also protects the virus from host IgM and complement-mediated neutralisation. However, the contribution of FX in determining Ad liver transduction whilst simultaneously shielding the virus from immune attack remains unclear. In this study, we demonstrate that the FX protection mechanism is not conserved amongst Ad types, and identify the hexon hypervariable regions (HVR) of Ad5 as the capsid proteins targeted by this host defense pathway. Using genetic and pharmacological approaches, we manipulate Ad5 HVR interactions to interrogate the interplay between viral cell transduction and immune neutralisation. We show that FX and inhibitory serum components can co-compete and virus neutralisation is influenced by both the location and extent of modifications to the Ad5 HVRs. We engineered Ad5-derived HVRs into the rare, native non FX-binding Ad26 to create Ad26.HVR5C. This enabled the virus to interact with FX at high affinity, as quantified by surface plasmon resonance, FX-mediated cell binding and transduction assays. Concomitantly, Ad26.HVR5C was also sensitised to immune attack in the absence of FX, a direct consequence of the engineered HVRs from Ad5. In both immune competent and deficient animals, Ad26.HVR5C hepatic gene transfer was mediated by FX following intravenous delivery. This study gives mechanistic insight into the pivotal role of the Ad5 HVRs in conferring sensitivity to virus neutralisation by IgM and classical complement-mediated attack. Furthermore, through this gain-of-function approach we demonstrate the dual functionality of FX in protecting Ad26.HVR5C against innate immune factors whilst determining liver targeting.