PD-L1 checkpoint blockade delivered by retroviral replicating vector confers anti-tumor efficacy in murine tumor models.

Immune checkpoint inhibitors (CPIs) are associated with a number of immune-related adverse events and low response rates. We provide preclinical evidence for use of a retroviral replicating vector (RRV) selective to cancer cells, to deliver CPI agents that may circumvent such issues and increase efficacy. An RRV, RRV-scFv-PDL1, encoding a secreted single chain variable fragment targeting PD-L1 can effectively compete with PD-1 for PD-L1 occupancy. Cell binding assays showed trans-binding activity on 100% of cells in culture when infection was limited to 5% RRV-scFv-PDL1 infected tumor cells. Further, the ability of scFv PD-L1 to rescue PD-1/PD-L1 mediated immune suppression was demonstrated in a co-culture system consisting of human-derived immune cells and further demonstrated in several syngeneic mouse models including an intracranial tumor model. These tumor models showed that tumors infected with RRV-scFv-PD-L1 conferred robust and durable immune-mediated anti-tumor activity comparable or superior to systemically administered anti-PD-1 or anti PD-L1 monoclonal antibodies. Importantly, the nominal level of scFv-PD-L1 detected in serum is ∼50-150 fold less than reported for systemically administered therapeutic antibodies targeting immune checkpoints. These results support the concept that RRV-scFv-PDL1 CPI strategy may provide an improved safety and efficacy profile compared to systemic monoclonal antibodies of currently approved therapies.

Efficient Therapeutic Protein Expression Using Retroviral Replicating Vector with 2A Peptide in Cancer Models.

Toca 511, a retroviral replicating vector (RRV), uses an internal ribosomal entry site (IRES) to express an optimized yeast cytosine deaminase (yCD2), which converts 5-fluorocytosine to 5-fluorouracil. This configuration is genetically stable in both preclinical mouse models and human clinical trials. However, the use of IRES (∼600 bp) restricts choices of therapeutic transgenes due to limits in RRV genome size. This study replaced IRES with 2A peptides derived from picornaviruses with or without a GSG linker. The data show that GSG-linked 2A (g2A) peptide resulted in higher polyprotein separation efficiency than non-GSG linked 2A peptide. The study also shows that RRV can tolerate insertion of two separate 2A peptides to allow expression of two transgenes without compromising the assembly and function of the virus in addition to insertion of a single 2A peptide to confirm genetic stability with yCD2, green fluorescent protein, and HSV-1 thymidine kinase. In a parallel comparison of the RRV-IRES-yCD2 and RRV-g2A-yCD2 configurations, the study shows the yCD2 protein expressed from RRV-g2A-yCD2 has higher activity, resulting in a higher survival benefit in an intracranial tumor mouse model. These data enable a wider range of potential product candidates that could be developed using the RRV platform.

Retroviral Replicating Vector Delivery of miR-PDL1 Inhibits Immune Checkpoint PDL1 and Enhances Immune Responses In Vitro.

Tumor cells express a number of immunosuppressive molecules that can suppress anti-tumor immune responses. Efficient delivery of small interfering RNAs to treat a wide range of diseases including cancers remains a challenge. Retroviral replicating vectors (RRV) can be used to stably and selectively introduce genetic material into cancer cells. Here, we designed RRV to express shRNA (RRV-shPDL1) or microRNA30-derived shRNA (RRV-miRPDL1) using Pol II or Pol III promoters to downregulate PDL1 in human cancer cells. We also designed RRV expressing cytosine deaminase (yCD2) and miRPDL1 for potential combinatorial therapy. Among various configurations tested, we showed that RRV-miRPDL1 vectors with Pol II or Pol III promoter replicated efficiently and exhibited sustained downregulation of PDL1 protein expression by more than 75% in human cancer cell lines with high expression of PDL1. Immunologic effects of RRV-miRPDL1 were assessed by a trans-suppression lymphocyte assay. In vitro data showed downregulation of PDL1+ tumor cells restored activation of CD8+ T cells and bio-equivalency compared to anti-PDL1 antibody treatment. These results suggest RRV-miRPDL1 may be an alternative therapeutic approach to enhance anti-tumor immunity by overcoming PDL1-induced immune suppression from within cancer cells and this approach may also be applicable to other cancer targets.

Complex Dynamics of Virus Spread from Low Infection Multiplicities: Implications for the Spread of Oncolytic Viruses.

While virus growth dynamics have been well-characterized in several infections, data are typically collected once the virus population becomes easily detectable. Earlier dynamics, however, remain less understood. We recently reported unusual early dynamics in an experimental system using adenovirus infection of human embryonic kidney (293) cells. Under identical experimental conditions, inoculation at low infection multiplicities resulted in either robust spread, or in limited spread that eventually stalled, with both outcomes occurring with approximately equal frequencies. The reasons underlying these observations have not been understood. Here, we present further experimental data showing that inhibition of interferon-induced antiviral states in cells results in a significant increase in the percentage of robust infections that are observed, implicating a race between virus replication and the spread of the anti-viral state as a central mechanism. Analysis of a variety of computational models, however, reveals that this alone cannot explain the simultaneous occurrence of both viral growth outcomes under identical conditions, and that additional biological mechanisms have to be invoked to explain the data. One such mechanism is the ability of the virus to overcome the antiviral state through multiple infection of cells. If this is included in the model, two outcomes of viral spread are found to be simultaneously stable, depending on initial conditions. In stochastic versions of such models, the system can go by chance to either state from identical initial conditions, with the relative frequency of the outcomes depending on the strength of the interferon-based anti-viral response, consistent with the experiments. This demonstrates considerable complexity during the early phase of the infection that can influence the ability of a virus to become successfully established. Implications for the initial dynamics of oncolytic virus spread through tumors are discussed.

Complex spatial dynamics of oncolytic viruses in vitro: mathematical and experimental approaches.

Oncolytic viruses replicate selectively in tumor cells and can serve as targeted treatment agents. While promising results have been observed in clinical trials, consistent success of therapy remains elusive. The dynamics of virus spread through tumor cell populations has been studied both experimentally and computationally. However, a basic understanding of the principles underlying virus spread in spatially structured target cell populations has yet to be obtained. This paper studies such dynamics, using a newly constructed recombinant adenovirus type-5 (Ad5) that expresses enhanced jellyfish green fluorescent protein (EGFP), AdEGFPuci, and grows on human 293 embryonic kidney epithelial cells, allowing us to track cell numbers and spatial patterns over time. The cells are arranged in a two-dimensional setting and allow virus spread to occur only to target cells within the local neighborhood. Despite the simplicity of the setup, complex dynamics are observed. Experiments gave rise to three spatial patterns that we call "hollow ring structure", "filled ring structure", and "disperse pattern". An agent-based, stochastic computational model is used to simulate and interpret the experiments. The model can reproduce the experimentally observed patterns, and identifies key parameters that determine which pattern of virus growth arises. The model is further used to study the long-term outcome of the dynamics for the different growth patterns, and to investigate conditions under which the virus population eliminates the target cells. We find that both the filled ring structure and disperse pattern of initial expansion are indicative of treatment failure, where target cells persist in the long run. The hollow ring structure is associated with either target cell extinction or low-level persistence, both of which can be viewed as treatment success. Interestingly, it is found that equilibrium properties of ordinary differential equations describing the dynamics in local neighborhoods in the agent-based model can predict the outcome of the spatial virus-cell dynamics, which has important practical implications. This analysis provides a first step towards understanding spatial oncolytic virus dynamics, upon which more detailed investigations and further complexity can be built.

Early infection and spread of a conditionally replicating adenovirus under conditions of plaque formation.

Conditionally-replicating adenoviruses (CRAds) and other oncolytic viruses replicate selectively in tumor cells, presenting a potential cancer treatment approach. To optimize application of these viruses, understanding of early spread of these viruses in target cells is important. Here we used a recombinant adenovirus expressing enhanced jellyfish green fluorescent protein (EGFP) in place of the EIA and EIB genes (AdEGFPuci). Infection of susceptible cells (AD-293) under plaque formation conditions (MOI<<1) on gridded culture dishes and daily monitoring allowed visualization of initially infected cells, as well as spread to neighboring cells. We determined key parameters of early infection, including the rate and efficiency of spread from the initially infected cell to other cells. It was noteworthy that a minority of initially infected cells ultimately resulted in plaques. The approaches elucidated here will be useful for determining early infection parameters for CRAds of therapeutic interest.

Jaagsiekte sheep retrovirus biology and oncogenesis.

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of a lung cancer in sheep known as ovine pulmonary adenocarcinoma (OPA). The disease has been identified around the world in several breeds of sheep and goats, and JSRV infection typically has a serious impact on affected flocks. In addition, studies on OPA are an excellent model for human lung carcinogenesis. A unique feature of JSRV is that its envelope (Env) protein functions as an oncogene. The JSRV Env-induced transformation or oncogenesis has been studied in a variety of cell systems and in animal models. Moreover, JSRV studies have provided insights into retroviral genomic RNA export/expression mechanisms. JSRV encodes a trans-acting factor (Rej) within the env gene necessary for the synthesis of Gag protein from unspliced viral RNA. This review summarizes research pertaining to JSRV-induced pathogenesis, Env transformation, and other aspects of JSRV biology.

Jaagsiekte sheep retrovirus encodes a regulatory factor, Rej, required for synthesis of Gag protein.

Retroviruses express Gag and Pol proteins by translation of unspliced genome-length viral RNA. For some retroviruses, transport of unspliced viral RNA to the cytoplasm is mediated by small regulatory proteins such as human immunodeficiency virus Rev, while other retroviruses contain constitutive transport elements in their RNAs that allow transport without splicing. In this study, we found that the betaretrovirus Jaagsiekte sheep retrovirus (JSRV) encodes within the env gene a trans-acting factor (Rej) necessary for the synthesis of Gag protein from unspliced viral RNA. Deletion of env sequences from a JSRV proviral expression plasmid (pTN3) abolished its ability to produce Gag polyprotein in transfected 293T cells, and Gag synthesis could be restored by cotransfection of an env expression plasmid (DeltaGP). Deletion analysis localized the complementing activity (Rej) to the putative Env signal peptide, and a signal peptide expression construct showed Rej activity. Two other betaretroviruses, mouse mammary tumor virus (MMTV) and human endogenous retrovirus type K, encode analogous factors (Rem and Rec, respectively) that are encoded from doubly spliced env mRNAs. Reverse transcriptase-PCR cloning and sequencing identified alternate internal splicing events in the 5' end of JSRV env that could signify analogous doubly spliced Rej mRNAs, and cDNA clones expressing two of them also showed Rej activity. The predicted Rej proteins contain motifs similar to those found in MMTV Rem and other analogous retroviral regulatory proteins. Interestingly, in most cell lines, JSRV expression plasmids with Rej deleted showed normal transport of unspliced JSRV RNA to the cytoplasm; however, in 293T cells Rej modestly enhanced export of unspliced viral RNA (2.8-fold). Metabolic labeling experiments with [(35)S]methionine indicated that JSRV Rej is required for the synthesis of viral Gag polyprotein. Thus, in most cell lines, the predominant function of Rej is to facilitate translation of unspliced viral mRNA.

Identification and mutational analysis of a Rej response element in Jaagsiekte sheep retrovirus RNA.

Jaagsiekte sheep retrovirus (JSRV) is a simple betaretrovirus causing a contagious lung cancer of sheep. JSRV encodes unspliced and spliced viral RNAs, among which unspliced RNA encodes Gag and Pol proteins and a singly spliced mRNA encodes Env protein. In another study we found that JSRV encodes a regulatory protein, Rej, that is responsible for synthesis of Gag polyprotein from unspliced viral RNA. Rej is encoded in the 5' end of env, and it enhances nuclear export or accumulation of cytoplasmic unspliced viral RNA in 293T cells but not in most other cell lines (A. Hofacre, T. Nitta, and H. Fan, J. Virol. 83:12483-12498, 2009). In this study, we found that mutations in the 3' end of env in the context of a cytomegalovirus-driven full-length JSRV expression construct abolished Gag protein synthesis and released viruses in 293T cells. These mutants also showed deficits in accumulation of unspliced viral RNA in the cytoplasm. These mutants defined a Rej-responsive element (RejRE). Inhibition of CRM1 but not Tap function prevented nuclear export/accumulation of cytoplasmic unspliced RNA in 293T cells, similarly to other complex retroviruses that express analogous regulator proteins (e.g., human immunodeficiency virus Rev). Structural modeling of the RejRE with Zuker M-fold indicated a region with a predicted stable secondary structure. Mutational analysis in this region indicated the importance of both secondary structures and primary nucleotide sequences in a central stem-bulge-stem structure. In contrast to 293T cells, mutations in the RejRE did not affect the levels of cytoplasmic unspliced RNA in 293 cells, although the unspliced RNA showed partial degradation, perhaps due to lack of translation. RejRE-containing RNA relocalized Rej protein from the nucleus to the cytoplasm in 293 and rat 208F cells, suggesting binding of Rej to the RejRE.

Multiple domains of the Jaagsiekte sheep retrovirus envelope protein are required for transformation of rodent fibroblasts.

Jaagsiekte sheep retrovirus (JSRV) is an exogenous retrovirus of sheep that induces a contagious lung cancer, ovine pulmonary adenocarcinoma. We previously showed that the gene encoding JSRV envelope protein (Env) appears to function as an oncogene, since it can transform mouse NIH 3T3 cells. The cytoplasmic tail of the Env transmembrane protein (TM) is necessary for the transformation. However, previous experiments did not exclude the involvement of the Env surface protein (SU) in transformation. In this study, we created a series of nested deletion mutants through the SU domain and assessed their ability to transform rodent fibroblasts. All SU deletion mutants downstream of the predicted signal peptide were unable to transform murine NIH 3T3 or rat 208F cells. Transport to the plasma membrane of selected deleted Env proteins was confirmed by confocal immunofluorescence microscopy of hemagglutinin-tagged versions. Additional sequential SU deletion mutants lacking 50-amino-acid (aa) blocks throughout SU also were unable to transform. Furthermore, minimal insertion mutants of two amino acids (Leu/Gln) at various positions in SU also abolished transformation. These data indicate that domains in SU facilitate efficient JSRV transformation. This could reflect a necessity of SU for appropriate configuration of the Env protein or independent activation by SU of a signaling pathway necessary for transformation. Complementation between SU and TM mutants for transformation supported the latter hypothesis. Cotransfection with DeltaGP Y590F (mutant in the TM cytoplasmic tail) with DeltaGP SUDelta103-352 (lacking most of SU) resulted in efficient transformation. The resulting transformants showed evidence for the presence and expression of both mutant plasmids.