Generation of a Retargeted Oncolytic Herpes Virus Encoding Adenosine Deaminase for Tumor Adenosine Clearance.

BACKGROUND: Oncolytic viruses are immunotherapeutic agents that can be engineered to encode payloads of interest within the tumor microenvironment to enhance therapeutic efficacy. Their therapeutic potential could be limited by many avenues for immune evasion exerted by the tumor. One such is mediated by adenosine, which induces pleiotropic immunosuppression by inhibiting antitumor immune populations as well as activating tolerogenic stimuli. Adenosine is produced starting from the highly immunostimulatory ATP, which is progressively hydrolyzed to ADP and adenosine by CD39 and CD73. Cancer cells express high levels of CD39 and CD73 ectoenzymes, thus converting immunostimulatory purinergic signal of ATP into an immunosuppressive signal. For this reason, CD39, CD73 and adenosine receptors are currently investigated in clinical trials as targets for metabolic cancer immunotherapy. This is of particular relevance in the context of oncovirotherapy, as immunogenic cell death induced by oncolytic viruses causes the secretion of a high amount of ATP which is available to be quickly converted into adenosine. METHODS: Here, we took advantage of adenosine deaminase enzyme that naturally converts adenosine into the corresponding inosine derivative, devoid of immunoregulatory function. We encoded ADA into an oncolytic targeted herpes virus redirected to human HER2. An engineered ADA with an ectopic signal peptide was also generated to improve enzyme secretion (ADA-SP). RESULTS: Insertion of the expression cassette was not detrimental for viral yield and cancer cell cytotoxicity. The THV_ADA and THV_ADA-SP successfully mediated the secretion of functional ADA enzyme. In in vitro model of human monocytes THP1, this ability of THV_ADA and THV_ADA-SP resulted in the retrieval of eADO-exposed monocytes replication rate, suggesting the proficiency of the viruses in rescuing the immune function. CONCLUSIONS: Encoding ADA into oncolytic viruses revealed promising properties for preclinical exploitation.

Generation of a Novel Mesothelin-Targeted Oncolytic Herpes Virus and Implemented Strategies for Manufacturing.

BACKGROUND: HER2-based retargeted viruses are in advanced phases of preclinical development of breast cancer models. Mesothelin (MSLN) is a cell-surface tumor antigen expressed in different subtypes of breast and non-breast cancer. Its recent identification as a marker of some triple-negative breast tumors renders it an attractive target, presently investigated in clinical trials employing antibody drug conjugates and CAR-T cells. The availability of MSLN-retargeted oncolytic viruses may complement the current immunotherapeutic panel of biological drugs against HER2-negative breast and non-breast tumors. METHODS: A fully virulent, tumor-targeted oncolytic Herpes simplex virus-1 (MSLN-THV) with a selectivity for mesothelin-expressing cancer cells was generated. Recombineering technology was used to replace an essential moiety of the viral glycoprotein D with antibody fragments derived from clinically validated MSLN monoclonal antibodies, and to allow IL12 cargo expression in infected cells. Panels of breast and female reproductive system cell lines were used to verify the oncolytic potential of the viral constructs. A platform for production of the retargeted viruses was developed in HEK 293 cells, providing stable expression of a suitable chimeric receptor. RESULTS: We demonstrated the selectivity of viral infection and cytotoxicity by MSLN-retargeted viruses in a panel of mesothelin-positive cancer cells, originating from breast and female reproductive system tumors. We also developed a second-generation oncolytic MSLN-THV, encoding IL12, to enhance the immunotherapeutic potential of the viral backbone. A non-tumor cell line expressing a chimeric MSLN/Nectin-1 receptor, de-sensitized from antiviral responses by genetic inactivation of the Stimulator of Interferon Genes (STING)-dependent pathway was engineered, to optimize viral yields. CONCLUSIONS: Our proof-of-concept study proposes MSLN-retargeted herpesviruses as potential cancer immunotherapeutics for assessments in preclinical models of MSLN-positive tumors, complementing the available panel of oncolytic viruses to HER2-negative breast tumors.

Retargeted and Multi-cytokine-Armed Herpes Virus Is a Potent Cancer Endovaccine for Local and Systemic Anti-tumor Treatment.

Oncolytic viruses (OVs) are novel anti-tumor agents with the ability to selectively infect and kill tumor cells while sparing normal tissue. Beyond tumor cytolysis, OVs are capable of priming an anti-tumor immune response via lysis and cross-presentation of locally expressed endogenous tumor antigens, acting as an "endovaccine." The effectiveness of OVs, similar to other immunotherapies, can be hampered by an immunosuppressive tumor microenvironment. In this study, we modified a previously generated oncolytic herpes simplex virus (oHSV) retargeted to the human HER2 (hHER2) tumor molecule and encoding murine interleukin-12 (mIL-12), by insertion of a second immunomodulatory molecule, murine granulocyte-macrophage colony-stimulating factor (mGM-CSF), to maximize therapeutic efficacy. We assessed the efficacy of this double-armed virus (R-123) compared to singly expressing GM-CSF and IL-12 oHSVs in tumor-bearing mice. While monotherapies were poorly effective, combination with α-PD1 enhanced the anti-tumor response, with the highest efficacy of 100% response rate achieved by the combination of R-123 and α-PD1. Efficacy was T cell-dependent, and the induced immunity was long lasting and able to reject a second contralateral tumor. Importantly, systemic delivery of R-123 combined with α-PD1 was effective in inhibiting the development of tumor metastasis. As such, this approach could have a significant therapeutic impact paving the way for further development of this platform in cancer immunotherapy.

Integrity of the Antiviral STING-mediated DNA Sensing in Tumor Cells Is Required to Sustain the Immunotherapeutic Efficacy of Herpes Simplex Oncolytic Virus.

The dichotomic contribution of cancer cell lysis and tumor immunogenicity is considered essential for effective oncovirotherapy, suggesting that the innate antiviral immune response is a hurdle for efficacy of oncolytic viruses. However, emerging evidence is resizing this view. By sensing cytosolic DNA, the cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) axis can both counteract viral spread and contribute to the elicitation of adaptive immunity via type I interferon responses. In this paper, we analyzed the tumor-resident function of Sting-mediated DNA sensing in a combined approach of oncovirotherapy and PD-1 immune checkpoint blockade, in an immunocompetent murine model. While supporting increased lytic potential by oncolytic HER2-retargeted HSV-1 in vitro and in vivo, Sting-knockout tumors showed molecular signatures of an immunosuppressive tumor microenvironment. These signatures were correspondingly associated with ineffectiveness of the combination therapy in a model of established tumors. Results suggest that the impairment in antiviral response of Sting-knockout tumors, while favoring viral replication, is not able to elicit an adequate immunotherapeutic effect, due to lack of immunogenic cell death and the inability of Sting-knockout cancer cells to promote anti-tumor adaptive immune responses. Accordingly, we propose that antiviral, tumor-resident Sting provides fundamental contributions to immunotherapeutic efficacy of oncolytic viruses.

Isolation of Two Novel Human Anti-CTLA-4 mAbs with Intriguing Biological Properties on Tumor and NK Cells.

The cytotoxic T lymphocyte-antigen 4 (CTLA-4) has been considered an IC exclusively expressed on T cells, where it counteracts the co-stimulatory CD28 receptor, by competing for its binding to CD-80 and CD-86. We recently found that it is expressed also on tumor and NK cells, suggesting other possible unknown roles of CTLA-4. To shed light on these novel aspects of CTLA-4, we used Ipilimumab, the first FDA approved human antibody targeting CTLA-4, in parallel studies with two novel human mAbs we isolated by using an efficient phage display selection strategy on live activated lymphocytes and purified mouse and human CTLA-4. The selection for cross-reactive mAbs was guaranteed by a high throughput sequencing to identify the sequences commonly enriched by two parallel pannings on human and mouse CTLA-4. Two isolated antibodies were found to bind with high affinity to both human and mouse CTLA-4 and lymphocytes, showing nanomolar or sub-nanomolar Kd values. They were able to kill Treg cells by ADCC, and to activate both human and mouse PBMCs, by strongly increasing cytokines secretion. Interestingly, they activated NK cells, exhibited cytotoxicity against cancer cells by inducing ADCC and inhibited tumor cell growth by affecting CTLA-4 downstream pathways in a similar fashion to CD-80 and CD-86 ligands and differently from Ipilimumab. Moreover, the novel mAbs showed a reduced ability to interfere in the binding of CD-80 ligands to CTLA-4 on T cells with respect to Ipilimumab, suggesting that they could allow for anti-tumor effects without the irAEs associated with the potent antagonistic activity of Ipilimumab.

MHC class II invariant chain-adjuvanted viral vectored vaccines enhances T cell responses in humans.

Strategies to enhance the induction of high magnitude T cell responses through vaccination are urgently needed. Major histocompatibility complex (MHC) class II-associated invariant chain (Ii) plays a critical role in antigen presentation, forming MHC class II peptide complexes for the generation of CD4+ T cell responses. Preclinical studies evaluating the fusion of Ii to antigens encoded in vector delivery systems have shown that this strategy may enhance T cell immune responses to the encoded antigen. We now assess this strategy in humans, using chimpanzee adenovirus 3 and modified vaccinia Ankara vectors encoding human Ii fused to the nonstructural (NS) antigens of hepatitis C virus (HCV) in a heterologous prime/boost regimen. Vaccination was well tolerated and enhanced the peak magnitude, breadth, and proliferative capacity of anti-HCV T cell responses compared to non-Ii vaccines in humans. Very high frequencies of HCV-specific T cells were elicited in humans. Polyfunctional HCV-specific CD8+ and CD4+ responses were induced with up to 30% of CD3+CD8+ cells targeting single HCV epitopes; these were mostly effector memory cells with a high proportion expressing T cell activation and cytolytic markers. No volunteers developed anti-Ii T cell or antibody responses. Using a mouse model and in vitro experiments, we show that Ii fused to NS increases HCV immune responses through enhanced ubiquitination and proteasomal degradation. This strategy could be used to develop more potent HCV vaccines that may contribute to the HCV elimination targets and paves the way for developing class II Ii vaccines against cancer and other infections.

Replicative conditioning of Herpes simplex type 1 virus by Survivin promoter, combined to ERBB2 retargeting, improves tumour cell-restricted oncolysis.

Oncolytic virotherapy is emerging as a promising therapeutic option for solid tumours. Several oncolytic vectors in clinical testing are based on attenuated viruses; thus, efforts are being taken to develop a new repertoire of oncolytic viruses, based on virulent viral genomes. This possibility, however, raises concerns dealing with the safety features of the virulent phenotypes. We generated a double regulated Herpes simplex type-1 virus (HSV-1), in which tumour cell restricted replicative potential was combined to selective entry via ERBB2 receptor retargeting. The transcriptional control of the viral alpha4 gene encoding for the infected cell protein-4 (ICP4) by the cellular Survivin/BIRC5 promoter conferred a tumour cell-restricted replicative potential to a virulent HSV-1 genome. The combination of the additional ERBB2 retargeting further improved the selectivity for tumour cells, conferring to the double regulated virus a very limited ability to infect and propagate in non-cancerous cells. Accordingly, a suitable replicative and cytotoxic potential was maintained in tumour cell lines, allowing the double regulated virus to synergize in vivo with immune checkpoint (anti-PD-1) blockade in immunocompetent mice. Thus, restricting the replicative spectrum and tropism of virulent HSV-1 genomes by combination of conditional replication and retargeting provides an improved safety, does not alter the oncolytic strength, and is exploitable for its therapeutic potential with immune checkpoint blockade in cancer.

Characterizing Hepatitis C Virus-Specific CD4+ T Cells Following Viral-Vectored Vaccination, Directly Acting Antivirals, and Spontaneous Viral Cure.

BACKGROUND AND AIMS: Induction of functional helper CD4+ T cells is the hallmark of a protective immune response against hepatitis C virus (HCV), associated with spontaneous viral clearance. Heterologous prime/boost viral vectored vaccination has demonstrated induction of broad and polyfunctional HCV-specific CD8+ T cells in healthy volunteers; however, much less is known about CD4+ T-cell subsets following vaccination. APPROACH AND RESULTS: We analyzed HCV-specific CD4+ T-cell populations using major histocompatibility complex class II tetramers in volunteers undergoing HCV vaccination with recombinant HCV adenoviral/modified vaccinia Ankara viral vectors. Peptide-specific T-cell responses were tracked over time, and functional (proliferation and cytokine secretion) and phenotypic (cell surface and intranuclear) markers were assessed using flow cytometry. These were compared to CD4+ responses in 10 human leukocyte antigen-matched persons with HCV spontaneous resolution and 21 chronically infected patients treated with directly acting antiviral (DAA) therapy. Vaccination induced tetramer-positive CD4+ T cells that were highest 1-4 weeks after boosting (mean, 0.06%). Similar frequencies were obtained for those tracked following spontaneous resolution of disease (mean, 0.04%). In addition, the cell-surface phenotype (CD28, CD127) memory subset markers and intranuclear transcription factors, as well as functional capacity of peptide-specific CD4+ T-cell responses characterized after vaccination, are comparable to those following spontaneous viral resolution. In contrast, helper responses in chronic infection were infrequently detected and poorly functional and did not consistently recover following HCV cure. CONCLUSIONS: Helper CD4+ T-cell phenotype and function following HCV viral vectored vaccination resembles "protective memory" that is observed following spontaneous clearance of HCV. DAA cure does not promote resurrection of exhausted CD4+ T-cell memory in chronic infection.