Efficacy of Systemically Administered Retargeted Oncolytic Herpes Simplex Viruses—Clearance and Biodistribution in Naïve and HSV-Preimmune Mice

We investigated the anticancer efficacy, blood clearance, and tissue biodistribution of systemically administered retargeted oncolytic herpes simplex viruses (ReHVs) in HSV-naive and HSV-preimmunized (HSV-IMM) mice. Efficacy was tested against lung tumors formed upon intravenous administration of cancer cells, a model of metastatic disease, and against subcutaneous distant tumors. In naive mice, HER2- and hPSMA-retargeted viruses, both armed with mIL-12, were highly effective, even when administered to mice with well-developed tumors. Efficacy was higher for combination regimens with immune checkpoint inhibitors. A significant amount of infectious virus persisted in the blood for at least 1 h. Viral genomes, or fragments thereof, persisted in the blood and tissues for days. Remarkably, the only sites of viral replication were the lungs of tumor-positive mice and the subcutaneous tumors. No replication was detected in other tissues, strengthening the evidence of the high cancer specificity of ReHVs, a property that renders ReHVs suitable for systemic administration. In HSV-IMM mice, ReHVs administered at late times failed to exert anticancer efficacy, and the circulating virus was rapidly inactivated. Serum stability and in vivo whole blood stability assays highlighted neutralizing antibodies as the main factor in virus inactivation. Efforts to deplete mice of the neutralizing antibodies are ongoing

\u3b1v\u3b23-integrin regulates PD-L1 expression and is involved in cancer immune evasion

Tumors utilize a number of effective strategies, including the programmed death 1/PD ligand 1 (PD-1/PD-L1) axis, to evade immune-mediated control of their growth. PD-L1 expression is mainly induced by IFN receptor signaling or constitutively induced. Integrins are an abundantly expressed class of proteins which play multiple deleterious roles in cancer and exert proangiogenic and prosurvival activities. We asked whether \u3b1v\u3b23-integrin positively regulates PD-L1 expression and the anticancer immune response. We report that \u3b1v\u3b23-integrin regulated constitutive and IFN-induced PD-L1 expression in human and murine cancerous and noncancerous cells. \u3b1v\u3b23-integrin targeted STAT1 through its signaling C tail. The implantation of \u3b23-integrin-depleted tumor cells led to a dramatic decrease in the growth of primary tumors, which exhibited reduced PD-L1 expression and became immunologically hot, with increased IFN\u3b3 content and CD8+ cell infiltration. In addition, the implantation of \u3b23-integrin-depleted tumors elicited an abscopal immunotherapeutic effect measured as protection from the challenge tumor and durable splenocyte and serum reactivity to B16 cell antigens. These modifications to the immunosuppressive microenvironment primed cells for checkpoint (CP) blockade. When combined with anti-PD-1, \u3b23-integrin depletion led to durable therapy and elicited an abscopal immunotherapeutic effect. We conclude that in addition to its previously known roles, \u3b1v\u3b23-integrin serves as a critical component of the cancer immune evasion strategy and can be an effective immunotherapy target

Immunotherapeutic Efficacy of Retargeted oHSVs Designed for Propagation in an Ad Hoc Cell Line

Our laboratory has pursued the generation of cancer-specific oncolytic herpes simplex viruses (oHSVs) which ensure high efficacy while maintaining a high safety profile. Their blueprint included retargeting to a Tumor-Associated Antigen, e.g., HER2, coupled to detargeting from natural receptors to avoid off-target and off-tumor infections and preservation of the full complement of unmodified viral genes. These oHSVs are “fully virulent in their target cancer cells”. The 3rd generation retargeted oHSVs carry two distinct retargeting moieties, which enable infection of a producer cell line and of the target cancer cells, respectively. They can be propagated in an ad hoc Vero cell derivative at about tenfold higher yields than 1st generation recombinants, and more effectively replicate in human cancer cell lines. The R-335 and R-337 prototypes were armed with murine IL-12. Intratumorally-administered R-337 conferred almost complete protection from LLC-1-HER2 primary tumors, unleashed the tumor microenvironment immunosuppression, synergized with the checkpoint blockade and conferred long-term vaccination against distant challenge tumors. In summary, the problem intrinsic to the propagation of retargeted oHSVs—which strictly require cells positive for targeted receptors—was solved in 3rd generation viruses. They are effective as immunotherapeutic agents against primary tumors and as antigen-agnostic vaccines

Immunotherapeutic Efficacy of Retargeted oHSVs Designed for Propagation in an Ad Hoc Cell Line

Our laboratory has pursued the generation of cancer-specific oncolytic herpes simplex viruses (oHSVs) which ensure high efficacy while maintaining a high safety profile. Their blueprint included retargeting to a Tumor-Associated Antigen, e.g., HER2, coupled to detargeting from natural receptors to avoid off-target and off-tumor infections and preservation of the full complement of unmodified viral genes. These oHSVs are “fully virulent in their target cancer cells”. The 3rd generation retargeted oHSVs carry two distinct retargeting moieties, which enable infection of a producer cell line and of the target cancer cells, respectively. They can be propagated in an ad hoc Vero cell derivative at about tenfold higher yields than 1st generation recombinants, and more effectively replicate in human cancer cell lines. The R-335 and R-337 prototypes were armed with murine IL-12. Intratumorally-administered R-337 conferred almost complete protection from LLC-1-HER2 primary tumors, unleashed the tumor microenvironment immunosuppression, synergized with the checkpoint blockade and conferred long-term vaccination against distant challenge tumors. In summary, the problem intrinsic to the propagation of retargeted oHSVs—which strictly require cells positive for targeted receptors—was solved in 3rd generation viruses. They are effective as immunotherapeutic agents against primary tumors and as antigen-agnostic vaccines

A fully-virulent retargeted oncolytic HSV armed with IL-12 elicits local immunity and vaccine therapy towards distant tumors.

Oncolytic herpes simplex viruses (oHSVs) showed efficacy in clinical trials and practice. Most of them gain cancer-specificity from deletions/mutations in genes that counteract the host response, and grow selectively in cancer cells defective in anti-viral response. Because of the deletions/mutations, they are frequently attenuated or over-attenuated. We developed next-generation oHSVs, which carry no deletion/mutation, gain cancer-specificity from specific retargeting to tumor cell receptors-e.g. HER2 (human epidermal growth factor receptor 2)-hence are fully-virulent in the targeted cancer cells. The type of immunotherapy they elicit was not predictable, since non-attenuated HSVs induce and then dampen the innate response, whereas deleted/attenuated viruses fail to contrast it, and since the retargeted oHSVs replicate efficiently in tumor cells, but spare other cells in the tumor. We report on the first efficacy study of HER2-retargeted, fully-virulent oHSVs in immunocompetent mice. Their safety profile was very high. Both the unarmed R-LM113 and the IL-12-armed R-115 inhibited the growth of the primary HER2-Lewis lung carcinoma-1 (HER2-LLC1) tumor, R-115 being constantly more efficacious. All the mice that did not die because of the primary treated tumors, were protected from the growth of contralateral untreated tumors. The long-term survivors were protected from a second contralateral tumor, providing additional evidence for an abscopal immunotherapeutic effect. Analysis of the local response highlighted that particularly R-115 unleashed the immunosuppressive tumor microenvironment, i.e. induced immunomodulatory cytokines, including IFNγ, T-bet which promoted Th1 polarization. Some of the tumor infiltrating cells, e.g. CD4+, CD335+ cells were increased in the tumors of all responders mice, irrespective of which virus was employed, whereas CD8+, Foxp3+, CD141+ were increased and CD11b+ cells were decreased preferentially in R-115-treated mice. The durable response included a breakage of tolerance towards both HER2 and the wt tumor cells, and underscored a systemic immunotherapeutic vaccine response

Efficacy of R-LM113 and R-115 administered early after tumor implantation on the growth of HER2-LLC1 tumors.

<p>(A) Schedule of the treatments. The HER2-transgenic/tolerant mice, implanted with HER2-LLC1 cells, received four loco-regional injections of R-LM113, R-115, or vehicle, at 3–4 days distance, starting at d 3 after tumor implantation. At d 18 or 30, mice received a 1° contralateral challenge tumor. The mice which survived the primary tumor were all resistant to the 1° contralateral challenge tumor; a fraction of them was subsequently analyzed as long survivors (LS) (see, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007209#ppat.1007209.g005" target="_blank">Fig 5</a>). (B-D) Kinetics of tumor growth in mice treated with vehicle (B), R-LM113 (C), R-115 (D). Pooled results from 3 experiments. Statistical significance was calculated using the RM (repeated measures) two way ANOVA-test (until d 21). The figures in panels B-D denote the numbers of tumor free/treated mice (TF), and the mice subsequently analyzed as LS. (E) Volumes of the primary tumors at d 21 after implantation. Statistical significance was calculated using the t-test. (F) Kaplan-Meier survival curves of the three groups of mice. Statistical significance was calculated using the Log-rank (Mantel-Cox) test. Of note, some tumor free mice were sacrificed during the course of the experiment in either arm and were censored. (G) Volumes of 1° contralateral untreated tumors in the R-LM113 and R-115 arms, and in naïve mice, at d 20 and 27 after its implantation. Statistical significance was calculated by means of the t-test. The number of mice in the naïve, R-LM113 and R-115 arms were 15, 4, 8, and 14, 4, 7 at d 20 and 27 after implantation of the contralateral tumor, respectively. The mice decreased in number because of deaths caused by the primary tumor. Four and 6 mice in the R-LM113 and R-115 arms, respectively, survived the primary tumor, received the 1° contralateral tumor and were included in the LS group (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007209#ppat.1007209.g005" target="_blank">Fig 5</a>).</p

Short term intratumoral immune response and quantification of viral genome copy numbers.

<p>A new group of mice treated with R-LM113 or R-115 from d 10 after tumor implantation (same treatment schedule as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007209#ppat.1007209.g004" target="_blank">Fig 4A</a>), was sacrificed 6–7 days after completion of the virus treatment. (A-E) Kinetics of tumor growth for mice treated with vehicle (A), R-LM113 (B, C) or R-115 (D, E). Pooled results from 3 experiments. The virus-treated mice were subdivided in responders (R) and non-responders (NR). The responders showed a regression or slowdown in tumor growth, measured as a reduction in the tumor volume in at least one of the last two measurements before sacrifice (d 2 and d 5 after the last treatment) or increments of the tumor sizes smaller than 50% in the last two measurements. The non responders exhibited a tumor growth similar to that in the vehicle-treated arm. R-LM113 responders (B) and non responders (C). R-115 responders (D) and non responders (E). Statistical significance was calculated using the RM (repeated measures) two way ANOVA-test until d 25. (F) Tumor volumes at d 22. In this and subsequent panels, Black circles, vehicle-treated mice (vehicle). Full green circles, R-LM113 responder mice (R-LM113 R). Open green circles, R-LM113 non-responder mice (R-LM113 NR). Full red circles, R-115 responder mice (R-115 R). Open red circles, R-115 non-responder mice (R-115 NR). (G) Viral genome copy number in tumors, relative to a standard curve prepared by means of purified HSV DNA. Results are expressed as gc/100ng of DNA. (H-O) Tumor infiltrating cells. (Q-X) Splenocytes. (P, Y) PD-L1 expression by CD45+ cell in tumors (P) and in spleens (Y). (F, H-Y) Statistical significance was calculated using the t-test.</p

Transcriptional analysis of tumor specimens and quantification of intratumoral cytokines and molecules.

<p>(A-B) qRT-PCR determination of expression of Ifng and Tbet genes in tumor specimens from mice described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007209#ppat.1007209.g006" target="_blank">Fig 6</a>. Statistical significance was calculated using the t-test. (C-H) Quantification of intratumoral cytokines by Luminex Multiplex Assay in mice described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007209#ppat.1007209.g006" target="_blank">Fig 6</a>. Small tumor specimens were resuspended in non-denaturing lysis buffer and lysed by sonication. Supernatants were assayed by Luminex Multiplex Assay and results were expressed as pg of the analyte/total proteins. Statistical significance was calculated using the t-test. (I) Quantification of intratumoral mIL-12 secretion. R-115- or vehicle-treated mice were sacrificed at d 3, 5 or 7 after completion of the virus treatment (d 21). Tumors were lysed as detailed above, and mIL-12 was quantified by ELISA. Each point represents the average of 4 determinations. Statistical significance was calculated using the two way ANOVA-test.</p

Properties of HER2-expressing murine tumor cells.

<p>(A—F) HER2 expression in HER2-LLC1 (A, B), HER2-B16 (C, D), and SK-OV-3 (E, F) cells, and in the wt LLC1 (A) and B16 (C) cells as controls. HER2 expression was detected by flow cytometry by means of anti-HER2 Ab. (A, C, E) X-axis, fluorescence intensity; y-axis, counts. HER2-LLC1 and HER2-B16, red; LLC1 and B16 wt, blue; the secondary anti-mouse Ab alone, black. The average fluorescence intensities of three independent determinations ± SD were 30420 ± 1155, 8589 ± 334, 43810 ± 1796 for HER2-LLC1, HER2-B16, and SK-OV-3 cells, respectively. (B, D, F) Homogeneity of the HER2-LLC1, HER2-B16 clonal cells, and of SK-OV-3 cells. X-axis, fluorescence intensity; y-axis, side scatter (SSC-A). Figures denote the percentage of cells positive to anti-HER2 Ab. (G) Schematic backbones of R-LM113 and R-115 genomes [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007209#ppat.1007209.ref044" target="_blank">44</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007209#ppat.1007209.ref060" target="_blank">60</a>]. Both R-LM113 and R-115 carry the insertion of BAC (bacterial artificial chromosome) sequences and EGFP (enhanced green fluorescence) gene in the UL3-UL4 intergenic region, the deletion of the aa 6–38 region in gD for detargeting purposes and its replacement with the scFv (single chain antibody) to HER2 for retargeting purposes. In addition, R-115 carries the mIL-12 gene under the hCMV (human cytomegalovirus) promoter in the US1-US2 intergenic region. (H) Relative plating efficiency of R-LM113 and R-115 in SK-OV-3, HER2-LLC1 and HER2-B16 cells, measured as efficiency of plaque formation. Replicate aliquots of R-115 or R-LM113 were plated onto HER2-LLC1, HER2-B16 and SK-OV-3 cell monolayers, in triplicates. Plaques were scored three days later. (I) Yields of R-LM113 and R-115 in SK-OV-3, HER2-LLC1 and HER2-B16 cells. For each cell line replicate cultures were infected with the indicated viruses at 0.1 PFU/cell (as titrated in the respective cell line). Progeny virus was titrated in SK-OV-3 cells. In panels H and I, each column represents the average of triplicates ± SD.</p