Danielle Carroll

Genetic Modification of Oncolytic Newcastle Disease Virus for Cancer Therapy

ABSTRACT Clinical development of a mesogenic strain of Newcastle disease virus (NDV) as an oncolytic agent for cancer therapy has been hampered by its select agent status due to its pathogenicity in avian species. Using reverse genetics, we have generated a lead candidate oncolytic NDV based on the mesogenic NDV-73T strain that is no longer classified as a select agent for clinical development. This recombinant NDV has a modification at the fusion protein (F) cleavage site to reduce the efficiency of F protein cleavage and an insertion of a 198-nucleotide sequence into the HN-L intergenic region, resulting in reduced viral gene expression and replication in avian cells but not in mammalian cells. In mammalian cells, except for viral polymerase (L) gene expression, viral gene expression is not negatively impacted or increased by the HN-L intergenic insertion. Furthermore, the virus can be engineered to express a foreign gene while still retaining the ability to grow to high titers in cell culture. The recombinant NDV selectively replicates in and kills tumor cells and is able to drive potent tumor growth inhibition following intratumoral or intravenous administration in a mouse tumor model. The candidate is well positioned for clinical development as an oncolytic virus. IMPORTANCE Avian paramyxovirus type 1, NDV, has been an attractive oncolytic agent for cancer virotherapy. However, this virus can cause epidemic disease in poultry, and concerns about the potential environmental and economic impact of an NDV outbreak have precluded its clinical development. Here we describe generation and characterization of a highly potent oncolytic NDV variant that is unlikely to cause Newcastle disease in its avian host, representing an essential step toward moving NDV forward as an oncolytic agent. Several attenuation mechanisms have been genetically engineered into the recombinant NDV that reduce chicken pathogenicity to a level that is acceptable worldwide without impacting viral production in cell culture. The selective tumor replication of this recombinant NDV, both in vitro and in vivo, along with efficient tumor cell killing makes it an attractive oncolytic virus candidate that may provide clinical benefit to patients.

Phage display and hybridoma generation of antibodies to human CXCR2 yields antibodies with distinct mechanisms and epitopes

Generation of functional antibodies against integral membrane proteins such as the G-protein coupled receptor CXCR2 is technically challenging for several reasons, including limited epitope accessibility, the requirement for a lipid environment to maintain structure and their existence in dynamic conformational states. Antibodies to human CXCR2 were generated by immunization in vivo and by in vitro selection methods. Whole cell immunization of transgenic mice and screening of phage display libraries using CXCR2 magnetic proteoliposomes resulted in the isolation of antibodies with distinct modes of action. The hybridoma-derived antibody fully inhibited IL-8 and Gro-α responses in calcium flux and β-arrestin recruitment assays. The phage-display derived antibodies were allosteric antagonists that showed ligand dependent differences in functional assays. The hybridoma and phage display antibodies did not cross-compete in epitope competition assays and mapping using linear and CLIPS peptides confirmed that they recognized distinct epitopes of human CXCR2. This illustrates the benefits of using parallel antibody isolation approaches with different antigen presentation methods to successfully generate functionally and mechanistically diverse antagonistic antibodies to human CXCR2. The method is likely to be broadly applicable to other complex membrane proteins.

Newcastle disease virus establishes persistent infection in tumor cells in vitro: contribution of the cleavage site of fusion protein and second sialic acid binding site of hemagglutinin-neuraminidase

ABSTRACT Newcastle disease virus (NDV) is an oncolytic virus being developed for the treatment of cancer. Following infection of a human ovarian cancer cell line (OVCAR3) with a recombinant low-pathogenic NDV, persistent infection was established in a subset of tumor cells. Persistently infected (PI) cells exhibited resistance to superinfection with NDV and established an antiviral state, as demonstrated by upregulation of interferon and interferon-induced genes such as myxoma resistance gene 1 (Mx1) and retinoic acid-inducing gene-I (RIG-I). Viruses released from PI cells induced higher cell-to-cell fusion than the parental virus following infection in two tumor cell lines tested, HT1080 and HeLa, and remained attenuated in chickens. Two mutations, one in the fusion (F) protein cleavage site, F117S (F117S), and another in hemagglutinin-neuraminidase (HN), G169R (HN169R), located in the second sialic acid binding region, were responsible for the hyperfusogenic phenotype. F117S improves F protein cleavage efficiency, facilitating cell-to-cell fusion, while HN169R possesses a multifaceted role in contributing to higher fusion, reduced receptor binding, and lower neuraminidase activity, which together result in increased fusion and reduced viral replication. Thus, establishment of persistent infection in vitro involves viral genetic changes that facilitate efficient viral spread from cell to cell as a potential mechanism to escape host antiviral responses. The results of our study also demonstrate a critical role in the viral life cycle for the second receptor binding region of the HN protein, which is conserved in several paramyxoviruses. IMPORTANCE Oncolytic Newcastle disease virus (NDV) could establish persistent infection in a tumor cell line, resulting in a steady antiviral state reflected by constitutively expressed interferon. Viruses isolated from persistently infected cells are highly fusogenic, and this phenotype has been mapped to two mutations, one each in the fusion (F) and hemagglutinin-neuraminidase (HN) proteins. The F117S mutation in the F protein cleavage site improved F protein cleavage efficiency while the HN169R mutation located at the second receptor binding site of the HN protein contributed to a complex phenotype consisting of a modest increase in fusion and cell killing, lower neuraminidase activity, and reduced viral growth. This study highlights the intricate nature of these two mutations in the glycoproteins of NDV in the establishment of persistent infection. The data also shed light on the critical balance between the F and HN proteins required for efficient NDV infection and their role in avian pathogenicity.

Abstract 4556: MEDI5395: An armed oncolytic Newcastle disease virus

Oncolytic viruses are live, replication-competent viruses that infect and/or replicate selectively in tumour cells leading to the destruction of the infected cell. Cell lysis occurs as a natural consequence of the viral life cycle and released virions can infect and kill neighbouring tumour cells leading to an amplified therapeutic effect. Oncolysis has the added benefit of releasing multiple tumour antigens that may further induce an immune-mediated therapeutic response. Newcastle Disease Virus (NDV) is an avian paramyxovirus, which has proven safety and demonstrated efficacy against a variety of preclinical cancer models and in PhI clinical studies as an oncolytic agent, oncolysate or whole cell vaccine. Using reverse genetics, we have generated a recombinant strain of NDV that overcomes environmental and regulatory concerns uncoupling oncolytic potency and avian pathogenicity. Furthermore we have enhanced the immune modulatory properties of NDV by engineering the virus to express granulocyte/macrophage colony-stimulating factor (GM-CSF). We have evaluated the biological characteristics of recNDVGM-CSF (MEDI5395) in vivo and in vitro. MEDI5395 selectively replicates in and kills a wide variety of human and mouse tumour cell lines. Additionally infection of cancer cells with MEDI5395 results in the increased production and secretion of pro-inflammatory cytokines and chemokines which are able to recruit mediators of both the innate and adaptive immune responses. MEDI5395 is a potent activator of the type I interferon response. In vivo, using a range of syngeneic and xenograft models we have demonstrated that NDV treatment has robust anti-tumour activity. In a HT1080 fibrosarcoma xenograft model a single administration (intra-tumoural or systemic) was able to cure 80% of tumour bearing mice. In syngeneic mouse tumour models, which support minimal viral replication MEDI5395 treatment causes significant changes in the local immune suppressive microenvironment and results in long-lasting anti-tumour immune responses. These responses are further enhanced in models that permit greater replication and also when combined with immune checkpoint blockade. The inherent properties of NDV (self-propagation, tumour-selective replication, and immunostimulatory properties) coupled with the ability to genetically engineer NDV to express therapeutic transgenes may provide a multi-modal attack on the tumour, delivering greater benefit to patients. Citation Format: Danielle Carroll, James Harper, Travers Jon, Shannon Burke, Ruth Franks, Christel Navarro, Xing Cheng, Robert Wilkinson, Hong Jin. MEDI5395: An armed oncolytic Newcastle disease virus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4556. doi:10.1158/1538-7445.AM2017-4556

Abstract 2234: Exploring the oncolytic potential of Newcastle Disease Virus

Oncolytic viruses are live, replication-competent viruses that infect and/or replicate selectively in tumour cells leading to the destruction of the infected cell. Cell death occurs as a natural consequence of the viral life cycle and newly produced virions can infect and kill neighbouring tumour cells leading to an amplified therapeutic effect. Oncolysis has the added benefit of potentially producing novel tumour antigens that can induce an immune-mediated therapeutic response. Newcastle Disease Virus (NDV) is an avian paramyxovirus, which has proven safety and demonstrated efficacy against a variety of mammalian cancers in PhI clinical studies. Using reverse genetics, we generated a recombinant strain of NDV that overcomes environmental and regulatory concerns uncoupling oncolytic potency and avian pathogenicity. We have additionally inserted the GM-CSF gene in order to maximize anti-tumour immune response and provide an in situ, patient/tumour- specific vaccine, combined with potent oncolysis. We have evaluated the biological characteristics of recNDVGM-CSF in vivo and in vitro. In vitro recNDVGM-CSF selectively replicates in and kills a wide variety of tumour cell types. In vivo, using a fibrosarcoma model that supports viral replication, a single administration (intra-tumoural or systemic) was able to cure 80% of tumour bearing mice. Additionally, following IV administration recNDVGM-CSF localises to and replicates within the tumour leading to a localised GM-CSF production within the tumour. The inherent properties of NDV (self-propagation, tumour-selective replication, immune activation) coupled with the ability to genetically engineer NDV to express therapeutic transgenes may provide a multi-modal attack on the tumour, delivering greater benefit to patients. Citation Format: James A. Harper, Jon Travers, Ruth Franks, Shannon Burke, Christel Navarro, Cheng Xing, Weijia Wang, Qi Xu, Robert W. Wilkinson, Hong Jin, Danielle Carroll. Exploring the oncolytic potential of Newcastle Disease Virus. [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 2234.

Abstract 4203: Non-invasive imaging of tumor cell death induced by a TRAILR2 agonist

Imaging tumour cell death can give an early indication of treatment efficacy. Tumour necrosis factor (TNF)-related apoptosis-inducing ligand receptor2 (TRAILR2) is a member of the TNF receptor superfamily, which interacts with death receptors (DRs) and induces apoptosis in a broad range of cancer cells but not normal cells[1]. MEDI3039, a newly described agonist of TRAILR2, was used to induce tumour cell death. A NIRF fluorophore-labelled phosphatidylserine (PS)-binding protein (∼15kDa), the C2A domain of Synaptotagmin-I (C2Am-750), which binds to the PS exposed by apoptotic and necrotic cells, was used to image MEDI3039-induced cell death[2]. Non-specific binding was assessed using a site-directed mutant that is inactive in PS binding, which was conjugated to a different fluorophore (iC2Am-680). Binding of C2Am-750 to MEDI3039-treated human colon and breast adenocarcinoma cells in vitro (Colo205 and MDA-Dual respectively) was assessed by flow cytometry and confocal microscopy. Both methods showed that C2Am-750 labelled MEDI3039-treated Colo205 cells, while the inactive iC2Am-680 showed only low non-specific binding. C2Am-750 and iC2Am-680 were also used to monitor the effect of treatment in a Colo205 xenograft model. One cohort of mice (n = 5) received a single dose of MEDI3039 (0.4 mg/kg), while another untreated cohort (n = 5) served as a control group. All mice received a single i.v. injection of a 1:1 mixture of 0.1 μmole/kg C2Am-750 and iC2Am-680 at 16 h after drug treatment, followed by whole body fluorescence imaging (FLI) measurements using an IVIS200 camera at 0 and 3 h post probe injection. FLI measurements in MEDI3039-treated Colo205 tumours showed significant increases in the uptake of C2Am-750 relative to iC2Am-680, both in vivo (4-9 fold, P value NIRF fluorophore-labelled C2Am showed a favourable biodistribution profile, with good tumour penetration and quick clearance of unbound material in vivo. The probe can be used to investigate the efficacy of targeted therapy, or other anti-tumour therapies, at an early stage following treatment. 1. Prasad, S., Kim, J.H., Gupta, S.C. & Aggarwal, B.B. Targeting death receptors for TRAIL by agents designed by Mother Nature. Trends in pharmacological sciences 35, 520-536 (2014). 2. Alam, I.S., Neves, A.A., Witney, T.H., Boren, J. & Brindle, K.M. Comparison of the C2A domain of synaptotagmin-I and annexin-V as probes for detecting cell death. Bioconjugate chemistry 21, 884-891 (2010). Citation Format: Bangwen Xie, Andre Neves, Stefanie R. Mullins, David Tice, Danielle Carroll, Robert W. Wilkinson, Kevin M. Brindle. Non-invasive imaging of tumor cell death induced by a TRAILR2 agonist. [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 4203.

Abstract 427: Host and tumoral CXCR2 signaling contributes to tumor growth

Chemokines are essential mediators of leukocyte migration and inflammation. Additionally they play an important role in tumour growth. The G-protein coupled receptor, CXCR2, and its ligands (CXCL 1,2,3,5,6,7 and IL8) have been shown to promote tumour initiation and growth, chemo-resistance, angiogenesis and immune cell infiltration (neutrophil, myeloid derived suppressor cells (MDSCs) and macrophages) into the tumour microenvironment. However the contribution of host and tumour CXCR2 has not been elucidated. We have developed both murine and human specific anti-CXCR2 antibodies alongside a humanised CXCR2 transgenic mouse to address the contribution of host and tumour CXCR2 signalling to tumour growth and maintenance. In pre-clinical tumours, granulocytic cells (neutrophils and MDSCs) within the tumour increase with size, in parallel with alteration in cell numbers observed within the spleen, peripheral blood and bone marrow. Inhibition of host CXCR2 has a dramatic impact on peripheral neutrophil levels, as well as their ability to become activated. In pre-clinical tumour models, inhibition of CXCR2 results in tumour growth inhibition. Contribution of host or tumour cells to the effect of CXCR2 blockade was model dependent. In the EL4 murine lymphoma model, only inhibition of host/peripheral CXCR2 was able to impact tumour growth. Whereas, in other murine syngeneic models (CT26 and B16), both host and tumour CXCR2 play a role in tumour growth. This was further demonstrated in an NSCLC patient derived xenograft, where the contribution of tumour CXCR2 was stronger than the host. Collectively our data show that CXCR2 inhibition (alone or in combination) has potential to influence growth of a number of tumour types. Future work is focused on understanding the mechanisms underlying the effects of CXCR2 inhibition, which remain key to developing it as an effective anti-cancer therapeutic. Citation Format: Danielle Carroll, James Harper, Karen McDaid, Ruth Franks, Catherine Eberline, Jane Kendrew, Richard Sainson, Judith Anderton, Chris Rossant, Karen Coffman, Ching Ching Leow, Ivan Inigo, Mitchell Reville, Jacintha Shenton, Lesley Young, Simon Barry. Host and tumoral CXCR2 signaling contributes to tumor growth. [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 427. doi:10.1158/1538-7445.AM2015-427