Towards a Precision Medicine Approach and In Situ Vaccination against Prostate Cancer by PSMA-Retargeted oHSV

Prostate specific membrane antigen (PSMA) is a specific high frequency cell surface marker of prostate cancers. Theranostic approaches targeting PSMA show no major adverse effects and rule out off-tumor toxicity. A PSMA-retargeted oHSV (R-405) was generated which both infected and was cytotoxic exclusively for PSMA-positive cells, including human prostate cancer LNCaP and 22Rv1 cells, and spared PSMA-negative cells. R-405 in vivo efficacy against LLC1-PSMA and Renca-PSMA tumors consisted of inhibiting primary tumor growth, establishing long-term T immune response, immune heating of the microenvironment, de-repression of the anti-tumor immune phenotype, and sensitization to checkpoint blockade. The in situ vaccination protected from distant challenge tumors, both PSMA-positive and PSMA-negative, implying that it was addressed also to LLC1 tumor antigens. PSMA-retargeted oHSVs are a precision medicine tool worth being additionally investigated in the immunotherapeutic and in situ vaccination landscape against prostate cancers.


Introduction
Prostate cancer (PC) is a frequent cause of cancer among men worldwide, second only to lung tumors, and is the fifth-leading cause of cancer-related death in men [1,2]; it is the most common cancer among men in 84 countries. In 2018, it was diagnosed in 1.2 million patients and caused 359,000 deaths. At diagnosis, prostate cancers fall into three basic groups [3]. In the first group, the tumors present as localized; patients undergo active surveillance, and a fraction of them progress to overt disease. The second group (1/3 of all new cases) includes locally advanced tumors; patients are at significant risk of disease progression and cancer-related death. In the third group, the disease is metastatic at diagnosis. The majority of patients from the second and third groups progress after androgen deprivation (castration) treatment. Prostate cancers are rather slow growing tumors, usually moderately sensitive to immune check-point inhibitors (ICIs) [4] and these attributes made PC a good candidate for dendritic cell (DC)-based immunotherapy. The DC-based autologous vaccine called Sipuleucel-T is an immunotherapeutic approach, which elicits an immune response targeted against castration-resistant prostatic acid phosphatase [5]. It is a personalized treatment made from the patient's blood which works by reprogramming each patient's immune system. Hence, Sipuleucel-T is not available to a large percentage of patients. Therefore, the need for novel immune therapies in prostate cancer remains a high priority [6], and an off-the-shelf derivative would be advantageous. In particular, there is a need for local control of PC, a need to treat any microscopic and confirmed metastases,

R-405 Tropism and Neutralization of Infection by MAbs to PSMA and HSV-1
J cell derivatives, PSMA-positive (SK-OV-3-PSMA, LLC1-PSMA, Renca-PSMA, LNCaP, PC3-PIP, 22Rv1) and PSMA-negative cancer cells (SK-OV-3, LLC1, Renca, PC3) were infected with R-405 virus at an input multiplicity of infection of 10 PFU/cell as titrated in SK-OV-3-PSMA, for 90 min at 37 • C. Pictures were taken 24 h after infection by Nikon Eclipse TS100 fluorescence microscope. For the neutralization assay, replicate monolayers of the PSMA-positive (SK-OV-3-PSMA, LNCaP, PC3-PIP, 22Rv1) and PSMA-negative cancer cells (SK-OV-3) seeded in 96-well plates were preincubated with increasing amounts of MAb D2B or with mouse IgG for 60 min at 37 • C. R-405 (3 PFU/cell) was added to the MAb-containing medium for additional 90 min. Alternatively, R-405 and R-LM5 virions (3 PFU/cell) were pre-incubated with increasing amounts of HSV-1-neutralizing MAb 52S for 1 h at 37 • C, and then allowed to adsorb to cells for 90 min (R-405 to PSMA-positive cancer cells, R-LM5 to SK-OV-3). Viral inoculum was removed, and cells were overlaid with medium containing MAbs for 18 h. The extent of infection was measured by FACS as the percentage of EGFP-positive infected cells. Western blot assay was performed as described [44].

Virus Growth and Cytotoxicity Assay
The number of encapsidated/enveloped infectious and non-infectious viral particles in R-405 stocks was determined as described [42] and expressed as genome copies per ml (gc/mL). R-405 was titrated in the indicated cell lines; plaque number and size were scored after 5 days. The number of viral particles required to obtain a single plaque in the indicated cell lines was calculated as the ratio of gc/mL to the titer values. To measure the extent of virus replication, PSMA-positive (SK-OV-3-PSMA, LLC1-PSMA, Renca-PSMA, LNCaP, PC3-PIP, 22Rv1) and PSMA-negative cancer cells (SK-OV-3, LLC1) were infected at an input multiplicity of 0.1 PFU/cell, as titrated in the corresponding cell line. Unabsorbed virus was inactivated by means of acidic wash (40 mM citric acid, 10 mM KCl, 135 mM NaCl, pH 3.0). Replicate cultures were frozen at the indicated times after infection. The progeny was titrated in SK-OV-3-PSMA cells and plaques were scored 5 days later. For the cytotoxicity assay, PSMA-positive (SK-OV-3-PSMA, LLC1-PSMA, Renca-PSMA, LNCaP, PC3-PIP, 22Rv1) and PSMA-negative cancer cells (SK-OV-3) were seeded in 96 well plates 8 × 10 3 cells/well and infected with R-405 or mock-infected. The input multiplicity of infection was 0.05 PFU/cell as titrated in the corresponding cell line. AlamarBlue (Life Technologies-Thermo Fisher Scientific, Waltham, MA, USA) was added to the culture media (10 µL/well) at the indicated times after infection and incubated for 4 h at 37 • C. Plates were read at 560 nm and 600 nm with GloMax Discover System (Promega Corporation, Madison, WI, USA). For each time point, cell viability was expressed as the percentage of AlamarBlue signal reduction in infected versus uninfected cells, after subtraction of the background value (medium alone).

Detection of Splenocyte Reactivity to Cancer Cells
Freshly explanted spleens were smashed through a 70 µm cell strainer in PBS to isolate splenocytes. Red blood cells in spleens were lysed with ACK buffer, while the splenocytes were resuspended in medium (RPMI 1640 containing 10% heat inactivated FBS, 1% penicillin/streptomycin), counted and seeded in 24 well plates. Suspensions of LLC1-WT and LLC1-PSMA cells were treated with mitomycin 15 µg/mL (Sigma-Aldrich, St. Louis, MO, USA) for 2 h at 37 • C, then washed 3 times with fresh medium. Splenocytes (1 × 10 6 cell/well) were incubated either with 1 × 10 5 LLC1-WT or LLC1-PSMA cells in 0.5 mL medium, and cocultured for 72 h. Media were collected and the amount of secreted IFNγ was quantified by ELISA (IFN-gamma Mouse ELISA Kit, Thermo Fisher Scientific, Waltham, MA, USA).

Detection of Serum Antibodies to Cancer Cells
To detect the serum antibodies to cancer cells, LLC1-WT and LLC1-PSMA cells were trypsinized and resuspended in flow cytometry buffer. For each sample, 0.25 × 10 6 cells were reacted with mouse serum, diluted 1:150, in 96 well plate in ice for 1 h, rinsed with flow cytometry buffer, and finally incubated with anti-mouse APC (1:200, eBioscience). Data were acquired on BD C6 Accuri.

Genetic Engineering of R-405 and Generation of Human and Murine Cell Lines Expressing Human PSMA
We engineered a novel PSMA-retargeted oHSV, called R-405, the backbone of which was similar to that of R-337, the prototype of third generation retargeted oHSVs [24]. R-405 genome is depicted in Figure 1A. The key features are: (i) the insertion of a single chain antibody (scFv) to human PSMA in the glycoprotein gD, along with the deletion of gD aminoacidic (aa) residues 30 and 38; these modifications detarget the virus tropism from natural receptors Nectin1 and herpes virus entry mediator (HVEM) and retarget it to PSMA; (ii) the insertion of the GCN4 peptide in gB for manufacturing purposes; specifically, the GCN4 peptide in gB interacts with an artificial cognate receptor expressed in ad hoc-designed producer cell lines [19,24,45]; the insertion of a single peptide form of murine IL-12 for arming; the single peptide IL-12 consists of the p40 subunit (IL12B) linked to the p35 subunit (IL12A) by means of a 15 amino acid long Glycine-Serine linker; it is produced at higher amounts than the hetero-dimeric IL-12, and causes no adverse effects when expressed intratumorally [24,46,47].
To verify whether R-405 could cause a bona fide infection of PSMA-positive human prostate tumor cells, PSMA-positive LNCaP cells derived from human androgendependent prostate adenocarcinoma lymph node metastasis [48], and PSMA-positive 22Rv1 cells, a human prostate carcinoma epithelial cell line [49] were selected. PC3 cells derived from human PSMA-negative prostate cancer metastasis [50] and PC3-PIP cells which transgenically express PSMA were included. The extent of PSMA expression in the human PC cell lines is shown in Figure 1B.
To express human PSMA in additional human and murine tumor cells, a form of PSMA deleted of the endocytosis motif located in the N-terminal cytoplasmic tail (PSMA∆) was engineered to maximize and stabilize its cell surface expression. The PSMA∆ was expressed in SK-OV-3 cells, a human tumor cell line routinely utilized in our laboratory for the high titer production of HSV stocks, and in the murine tumor LLC1 and Renca cells, syngeneic with C57BL/6 and BALB/c mice, respectively, thus generating LLC1-PSMA∆ and Renca-PSMA∆ (hereinafter called LLC1-PSMA and Renca-PSMA). The extent of PSMA∆ expression is shown in Figure 1C. We previously reported on the construction of the J-PSMA cell line [37]. The parental J cells do not carry a receptor for HSV and cannot be infected by HSV unless they transgenically express Nectin1 or HVEM [36]. Figure 1D shows the specific tropism of R-405 for cells positive for human PSMA. R-405 infected J-PSMA and failed to infect J-Nectin1 or J-HVEM cells; the recombinant virus infected SK-OV-3-PSMA cells, LLC1-PSMA and Renca-PSMA, but not the corresponding wild type PSMA-negative SK-OV-3, LLC1 and Renca cells. Figure 1D additionally shows that R-405 infected the PSMA-positive LNCaP, 22Rv1 and PC3-PIP cells with high efficiency, but failed to infect the PSMA-negative PC3 cells. Altogether the appropriate PSMA-directed tropism was confirmed in all the cell systems utilized, along with the infection of authentic human PSMA-positive prostate cancer cells. It was ascertained whether R-405 infection took place through PSMA by checking whether MAb D2B to PSMA blocked infection [39]. As a positive control, the neutralizing MAb 52S to glycoprotein H (gH) was included [40]. Figure 1E shows that MAb to human PSMA reduced R-405 infection in a dose-dependent manner in human prostate cancer LNCaP and 22Rv1 cells as well as in PC3-PIP and SK-OV-PSMA cells. Conversely, MAb 52S inhibited infection in all cell lines; MAb 52S also inhibited infection of wt SK-OV-3 cells with wt R-LM5. To verify that the chimeric scFv(PSMA)-gD encoded by R-405 is not defective in cell expression and in incorporation in virions, the cell expression of scFv(PSMA)-gD to that of wt-gD encoded by the wt virus R-LM5 was compared using indirect immunofluorescence of infected SK-OV-3-PSMA cells ( Figure 1F). R-405 and R-LM5 virions were purified and it was ascertained that they contained comparable amounts of scFv(PSMA)-gD or wt-gD using western blot with the conformation-independent MAb BD80 against the C-terminus of gD ectodomain (aa 264-274, a region of gD which was not modified in R-405). Figure 1F,G show that there was no major difference between scFv(PSMA)-gD and wt-gD in the extent of cell expression and incorporation in virions.   Figures 1D and 2A). To additionally quantify the differences in the ability of R-405 to infect the above cell lines, replicate amounts of R-405 were plated and the resulting number of plaques were scored. Plating efficiency in Figure 2B shows the decrease in the number of plaques relative to those formed in SK-OV-3-PSMA cells. For each cell line, Figure 2C shows the number of genome copies (gc) required to give rise to a PFU in the indicated cell lines. Parenthetically, the gc/PFU ratio of R-405 in SK-OV-3-PSMA cells (648 gc/PFU) is a figure compatible with those exhibited by retargeted oHSVs [42], and approximately three/four-fold higher than that exhibited by wt-HSV. The gc/PFU values of R-405 in LNCaP, 22Rv1, PC3-PIP e LLC1-PSMA cell lines were 2 orders of magnitude higher than those in SK-OV-3-PSMA and, in Renca-PSMA, the values were 4 orders of magnitude higher. Cumulatively, Figure 2B,C confirmed that the human PC cell lines and the LLC1-PSMA were infected with R-405 at a similar efficiency whereas the infection of Renca-PSMA was 2 orders of magnitude less efficient. R-405 cell-to-cell spread was determined by measuring the plaque size in the PSMA-positive cell lines and was similar for all the cells tested, except for the Renca-PSMA cells, as expected. The ability to form plaques denoted that the R-405 progeny virus had the ability to spread to adjacent PSMA-positive cancer cells. It predicted that, once R-405 was administered to tumors, the progeny virus could spread to adjacent tumor cells within the treated tumors, (Figure 2D,E). Finally, whether R-405 caused the death of PSMA-positive cells was assessed. The cells indicated were infected with R-405, and cytotoxicity was quantified using the AlamarBlue test at 3, 5, and 7 d after infection. Figure 2F shows that R-405 was cytotoxic to all PSMA-positive cells. Altogether R-405 fulfilled several criteria which concordantly indicated that it infected exclusively PSMA-positive cells, replicated in them and was cytotoxic. Importantly, R-405 infected, replicated in, and was cytotoxic to human PSMApositive prostate cancers, and spared human PSMA-negative prostate cancer cells. We concluded that R-405 was suitable for specifically targeting human PSMA-positive PC cells. As far as the murine tumor cells were concerned, they differed significantly one from the other; the LLC1-PSMA expressed PSMA at a higher level (and at a level similar to that in LNCaP and 22Rv1 cells) and sustained R-405 infection at much higher efficiency than Renca-PSMA. With respect to the level of PSMA expression, LLC1-PSMA and Renca-PSMA can be considered to mirror PSMA HIGH and PSMA LOW human PC cells, respectively [33]. Cytotoxicity was determined by AlamarBlue as detailed [21] and expressed at each time point as the percentage of infected relative to uninfected cells. Each column is the average of triplicate samples ± SD.

In Vivo Efficacy of R-405 Monotherapy against LLC1-PSMA Tumors
To provide the first proof-of-principle evidence that R-405 exerts antitumor efficacy against PSMA-positive tumors in syngeneic murine models, we made use of the LLC1-PSMA and Renca-PSMA tumor cells described above, syngeneic with C57BL/6 and BALB/c mice, respectively. Two systems were utilized since it has been well established that tumors vary greatly in their mechanisms of immune response and evasion, also called immune editing. On one extreme are tumors which are very poor in eliciting the immune response; they are designated as immunologically cold, or, in more severe examples, immunologically desert. At the other extreme are tumors which are competent in eliciting an immune response (immune competent or immunologically warm/hot) which they dampen by a variety of immune suppressive mechanisms [51][52][53]. In an accompanying paper in the current Special Issue [54], we reported on the molecular profiling of the immunologically cold LLC1 and the immunologically warm CT26 tumors and the effects of the HER2-retargeted R-337. With respect to the immune evasion strategies, Renca cells are similar to CT26 cells [51]. In addition, properties shown in Figures 1 and 2 indicate that, with respect to the level of PSMA expression, LLC1-PSMA and Renca-PSMA can be considered as mirroring PSMA HIGH and PSMA LOW human PC cells, respectively.
In the first series of in vivo experiments, LLC1-PSMA tumor cells were implanted subcutaneously (s.c.) in syngeneic C57BL/6 mice. The experimental design is shown in Figure 3A. Briefly, the mice received 3 intratumoral (i.t.) injections at 5 d time interval, 5 × 10 7 PFUs for each dose. This dose was chosen based on our previous study regarding the efficacy of the HER2-retargeted oHSV called R-337 against HER2-positive LLC1 tumors [24]. Ex vivo cultures of LLC1-PSMA tumors showed high expression levels of the PSMA transgene (compare Figure 1C to Figure 3B). Figure 3C,D shows that R-405 administration to established LLC1-PSMA tumors caused a complete inhibition of tumor growth and subsequent tumor regression (complete response, CR, in 100% of the mice). Figure 3E shows the highly significant difference in tumor size at d 21 between untreated and R-405-treated mice. The Kaplan-Meyer survival curve mirrored this huge difference ( Figure 3F).
Subsequently, all the R-405-treated mice were simultaneously implanted with two challenge tumors in the right and left flanks, made up of LLC1-PSMA or wt LLC1 (herein LLC1-wt) cells, respectively. Figure 3G-I shows that the mice were fully protected from the LLC1-PSMA challenge. Surprisingly, they were also partly protected from LLC1-wt challenge ( Figure 3J-L), implying that R405 treatment resulted in immune response not only to PSMA but also to LLC1 tumor antigens. In fact, R-405 elicited a long-term T-cell response seen in the splenocytes harvested at sacrifice and measured as an Interferon gamma (IFNγ) release when co-cultured with LLC1 cells. Figure 3M documents the splenocyte reactivity to LLC1-PSMA cells and, to a lesser extent, to LLC1-wt cells. With respect to antibody response, the tumor-bearing mice exhibited reactivity to LLC1-PSMA but not to LLC1-wt cells, consistent with the notion that mice were not tolerant to human PSMA. Surprisingly, the antibody reactivity to PSMA-positive cells was lower in the R-405-treated mice ( Figure 3N). Whether this reflected a lower stimulation of the B-cell compartment consequent to the reduction in tumor volume, peculiar B-T cell circuits, or other mechanisms remains to be investigated. The distant protection and concomitant immune responses induced by the tumor treatment with R-405 exemplified an in situ vaccination, a phenomenon observed with OVs [55][56][57].

Changes to the LLC1-PSMA Tumor Microenvironment Induced by R-405
Collectively, the above data indicated that R-405 monotherapy served as a potent immunotherapeutic treatment against LLC1-based tumors. The purpose of the subsequent experiments was to analyze in some detail the immune response elicited by R-405. The experiment in Figure 3 was replicated ( Figure 4A); the mice were sacrificed 5 d after the end of the virus treatment when the tumor growth curve in the treated mice was declining ( Figure 4B-D). Three series of analyses were conducted, namely characterization of tumorinfiltrating lymphocytes, of intratumoral immune mediators, and of systemic T response and serum antibodies. With respect to infiltrating lymphocytes, R-405-treated tumors exhibited an overall increase in leucocytes (CD45+), including CD8+ cells, and a decrease in the myeloid (CD45+ CD11b+) subpopulation ( Figure 4E-G). The natural killer (NK) cells were decreased in the tumors treated ( Figure 4I), consistent with the increase in MHC-I expression in PSMA-positive tumor cells. However, the fraction of activated NK cells (CD69+, IFNγ+) was increased ( Figure 4H-K); hence, the reduction of NK population in treated tumors could be also the result of activation-induced cell death. The T regulatory cells detected as a CD4+ FoxP3+ population were increased ( Figure 4L), in agreement with previous findings with LLC1-HER2 tumors treated with retargeted-oHSVs [20,24]. A compensatory increase in FoxP3-positive cells during T-cell activation was observed with other OVs, e.g., Newcastle disease virus [58]. A global view of the TME was carried out using RT-PCR analysis which highlighted the high-level expression of the R-405 marker gC glycoprotein and of the R-405-encoded Il12a (Figure 4M,N). The intratumoral mediators of innate response Ifnγ, Cxcl11, and tumor necrosis factor alpha (Tnfα) were increased as was the T-bet transcription factor, which favored Th1 polarization, and the T-regulatory cell FoxP3 factor (Figure 4O-S). Of note, R-405 treatment increased Pd-l1 expression in the tumor cell population ( Figure 4T), raising the possibility that R-405 sensitizes to ICIs. Evidence for long-term adaptive T response rested on high splenocyte reactivity to LLC1-PSMA and LLC1-wt cells in agreement with protection from PSMA positive and from LLC1-wt challenge tumors ( Figure 4U). Moreover, an increase in the CD69 and IFNγ markers in the splenic NK cell population, a feature similar to that in tumor infiltrating immune populations was observed ( Figure 4V,W). Consistent with the decrease in serum reactivity described above (see Figure 3N), a decrease in serum antibodies to LLC1-PSMA was observed ( Figure 4X).

R-405 and Anti-PD-1 Combination Therapy against LLC1-PSMA Tumors
We subsequently asked whether R-405 was sensitized to anti-PD-1 therapy. This experiment required a dose of R-405, which protected approximately 50% of the mice, and not 100% of the mice, as was the case in the experiment in Figure 3. To this end, a decreasing dose experiment was carried out. Mice carrying established LLC1-PSMA tumors were treated twice with doses of R-405 lower than those utilized in Figure 3, ranging from 5 × 10 7 to 2 × 10 5 PFUs ( Figure 5A). The dose/response curves are illustrated in Figure 5B-H. The three highest dosages (down to 5 × 10 6 PFUs) resulted in a CR in 100% of the mice. The lowest dosage 2 × 10 5 PFUs protected only 1 out of six mice. The 3 × 10 6 and 1 × 10 6 PFU dosages induced a CR in 80% and 33% of the mice, respectively.
The schedule of subsequent combination experiments is shown in Figure 5I. Anti-PD-1 antibodies at the dosage utilized herein as monotherapy induced a CR in 1 out of 12 mice ( Figure 5J,K). R-405 was administered twice at the 2 × 10 6 PFU dosage for each injection; it induced a CR in 50% of the mice ( Figure 5L). The combined treatment induced a CR in 5/7 (71%) of the mice ( Figure 5M). The tumor volumes at d 24 and the Kaplan-Meier survival curves showed highly statistically differences in the combination relative to the anti-PD-1 monotherapy or vehicle ( Figure 5N,O). Taken altogether, the results argue in favor of an additive effect.

R-405 Monotherapy and Combination Therapy against Renca-PSMA Tumors
As an immunologically warm model, Renca-PSMA cells implanted s.c. and treated i.t. with R-405 were utilized ( Figure 6A). Upon implantation in mice and tumor growth, the expression of PSMA was maintained in in vivo-grown Renca-PSMA tumors (compare Figure 6B to Figure 1C), which grew at a somehow slower rate than the untreated LLC1-PSMA tumors (compare Figure 3C with Figure 6C). Figure 6D shows that four doses of 1 × 10 8 PFUs each resulted in a CR in 50% of the mice and a partial response (R) in 25% of the mice. The reduction in tumor volume at d 37 and the Kaplan-Meier survival curve showed highly significant differences between the untreated and R-405-treated mice ( Figure 6E,F). The lower efficacy relative to that seen with LLC1-PSMA tumors may be consequent to the low-level expression of PSMA in Renca-PSMA relative to LLC1-PSMA (see Figures 1C, 3B and 6B, in the extent of R-405 replication and cell-spread seen in the two cell types, and possibly of fewer changes to the TME due to lower viral replication. Notably, the highest yield reached in Renca-PSMA and in LLC1-PSMA differed by two orders of magnitude (6 × 10 5 PFU/mL and 8.6 × 10 7 PFU/mL at 72 h, respectively).  The mice which survived the primary tumor were simultaneously implanted with two s.c. challenge tumors made up of Renca-PSMA and Renca-wt cells. The extent of distant protection is reported in Figure 6G-J. Briefly, the mice were fully protected from Renca-PSMA ( Figure 6G,H), and poorly protected from Renca-wt tumor cells ( Figure 6I,J), indicating that, in the Renca-PSMA model, long-term protection was strong against PSMApositive tumors, but weak against the Renca-wt cells.
In the final experiment ( Figure 6K), the effects of anti-PD-1 or anti-CTLA4 antibody monotherapies, and the respective combination therapies ( Figure 6L-Q) were evaluated. Renca tumors are known to be partially sensitive to the anti-CTLA4 monotherapy [59,60]. Monotherapies with the antibodies exerted no-to-modest effects ( Figure 6M,N). At the dosage utilized, R-405 exerted a CR and a PR in 1/6 and 3/6 mice, respectively. The effect of the combination therapy was higher with anti-CTLA4 than with anti-PD-1, and cumulatively affected 100% of the mice (3/5 CR and 2/5 PR), highlighting that the response needed to be determined as a function of tumor specificities. The tumor volumes at d 30 in the six groups of animals is reported in Figure 6R. The difference was highly statistically significant between the untreated mice and those receiving the R-405 monotherapy or the combinations with ICIs. The Kaplan-Meier curve showed a statistically significant advantage for mice receiving the R-405 (monotherapy or combination) relative to the vehicle. Notably, the combined R-405 and anti-CTLA4 therapy improved the survival of mice with respect to the R-405 and anti-CTLA4 monotherapies ( Figure 6S), while R-405 and anti-PD1 combination did not show any advantage relative to R-405 monotherapy.

Discussion
We report on a first proof-of-principle study of the properties and in vivo efficacy of a fully virulent oHSV retargeted to PMSA as an example of precision medicine and in situ vaccination approach against PC. The key features were as follows. (i) The PSMAretargeted IL-12-armed oHSV, called R-405, infected exclusively PSMA-positive cells and spared PSMA-negative cancerous or non-cancerous cells. It infected the cells and caused cytopathic death of the PSMA-positive human prostate cancer LNCaP and 22Rv1 cells, spared the PSMA-negative human prostate cancer PC3 cells, but infected the PC3-PIP cells transgenically expressing PSMA. The PSMA served as the port of entry of the R-405 into such cells. Thus, the human PSMA-positive PC cells were specific targets for R-405. With respect to murine tumor cells, the efficacy of the R-405 infection, cell-to-cell spread, and virus-induced cell death were higher in LLC1-PSMA than in Renca-PSMA cells; these features likely resulted from the intrinsic properties of the two murine tumor cells and, possibly, also from the different extent of PSMA expression. (ii) The in vivo key features of R-405 were its high efficacy against primary tumors (in the LLC1-PSMA model, 100% of mice exhibited a CR when treated with high doses of R-405), and the anti-tumor in situ vaccination effect which resulted in protection against distant challenge tumors. Notable, the immune protection was exerted not only towards the PSMA TAA but, in part, also towards the LLC1 tumor antigens, confirming that the retargeted oHSVs primed for antigen-agnostic antitumor immune response. (iii) The bases of the immune protection were the anti-tumor Th1 polarization of the TME (i.e., increased levels of IL-12, IFNγ, interferon-stimulated genes and TNF), the development of systemic tumor-specific Tlymphocytes (splenocytes), infiltration of the tumor masses by CD8+ and activated NK cells, and the inhibition of the mechanisms of tumor tolerance, exemplified by the depletion of tumor-infiltrating myeloid-derived suppressor cells (MDSCs) and the restoration of MHC-I expression in tumor cells. (iv) R-405 sensitized tumors to a checkpoint blockade-based therapy. The virus treatment inflamed the TME through infection and direct oncolysis, activation of innate and adaptive immunity, and expression of pro-inflammatory cytokines (including the virus-encoded IL-12 and the subsequent IFN cascade). Nevertheless, these modifications also triggered some tumor defenses against immune system, including the increase of PD-L1 expression and the tumor infiltration by Tregs. Both effects were caused by higher IFN levels [54,61] and other mechanisms. The combination therapy of R-405 with ICIs likely neutralized these effects: anti-PD-1 could be able to counteract the increase of PD-L1 expression in LLC1-PSMA tumors and prevent the anergy of the anti-tumor T cells, while anti-CTLA-4 could increase T-cell priming against tumor antigens and/or depleting Treg population in Renca-PSMA tumors. (v) R-405 efficacy was exerted towards two different types of tumors, one immunologically cold, and the other immunologically warm; hence, it appeared to be independent of the tumor immune profile and genotype. The high immunotherapeutic efficacy of R-405 was due in part to the IL-12 payload, known to promote the secretion of IFNγ and additional proinflammatory cytokines which, in turn, recruited and activated the immune effector cells. By means of these mechanisms, IL-12 adjuvanted the immunotherapeutic effects of oncolytic viruses [28]. In fact, a number of OVs are armed with IL-12 [28]. The specific form of mIl-12 encoded by R-405 was a single peptide version, which ensured higher levels of cytokine expression within the R-405treated tumor. R-405 may obviously be improved even more with additional payloads, as appropriate for human PCs. Additional studies should address efficacy in more authentic murine models of PC.
Metastatic castration-resistant prostate cancer remains incurable and fatal, despite the availability of multiple classes of therapy which delay disease progression and prolong life [32]. Beyond the standard of care approaches, the current landscape of treatments against PC includes OV-based treatments, PSMA-targeted therapies, and a personalized vaccine. An overview of these approaches follows: (i) Previous attempts to generate OVs specific for PC cells have included conditionally replicating transcriptionally retargeted adenoviruses (AdVs) carrying key viral genes under prostate specific antigen (PSA) or PSMA enhancer elements [62,63], an alphavirus encoding PSMA and acting as a vaccine [64], a vesicular stomatitis virus (VSV) pseudotyped with measles virus (MeV) glycoprotein, including a PSMAretargeted MeV glycoprotein [65], and an MeV genetically retargeted to PSMA by insertion of scFv in the hemagglutinin gene [66]. The latter virus demonstrated in vivo efficacy against xenotransplants of LNCaP and 22Rv1 human PC cells [66]. Its properties against immunocompetent PSMA-positive murine models and the extent of in vivo off-tumor infections were not reported. The PSMA-MeV had not been additionally investigated in the past 10 years. Hence, how effective it is in eliciting anti-tumor in situ vaccination and in sensitizing to ICIs is not known. AdVs and Reolysin are being tested against PC in ongoing phase 1 or 2 clinical trials, without or in combination with ICIs. (ii) High PSMA expression is frequent (75.21%) in metastatic castration-resistant prostate cancer patients and portends a poor prognosis [31][32][33]. Anti-PSMA antibodies are utilized for diagnostic or combined diagnostic + therapeutic (theranostic) applications, including PSMA-SPECT and the delivery of radiometals. Most of the PSMA-based clinical trials utilize anti-PSMA as a diagnostic or theranostic tool. Of note, a recently closed phase 3 clinical trial regarding the effect of Lutetium-177-PSMA-617 prolonged progression-free survival and overall survival when added to standard care in patients with advanced PSMA-positive metastatic castration-resistant prostate cancer [32].
Notably, a wealth of data convincingly indicated that targeting PSMA results in low toxicity and suggested few off-tumor effects [34,35]. (iii) The importance of PSMA as a target for precision medicine is strengthened by investigations regarding PSMA-targeted chemical antigen receptor T cells (CAR-Ts) [67]. Clinical trials are ongoing. Despite many efforts and rapid developments, the field of CAR-Ts regarding solid tumors still faces difficulties which need to be overcome. Oncolytic viruses, with their ability to attract lymphocytes to TME and to activate them, coupled with high tolerability, could be interesting partners for CAR-Ts. (iv) Very few trials address anti-PC vaccination. Sipuleucel-T is a dendritic cell (DC)based vaccine recommended as first-line therapy for the treatment of metastatic castration-resistant prostate cancer [68]. It must be prepared individually for each patient, starting from the patient's own blood [5,6].
Given the slow progression typical of human PCs, an approach capable of eliciting a long-term adaptive immune response appears to be appropriate. An off-the-shelf therapeutic treatment capable to induce in situ vaccination would respond to this need and would be of practical application and at the reach of a high number of patients. The properties of the PSMA-retargeted oHSVs described in the current proof-of-principle study addressed these challenges. The target patients should include early and advanced stage patients for the purpose of slowing down progression to more advanced stages and to metastatic disease as well as metastatic disease patients. The PSMA-oHSV might ultimately be combined with PSMA T-cell-based therapies to achieve long-term immunoprevention and immunotherapy. We concluded from this proof-of-principle study that PSMA-retargeted oHSVs are worth being additionally investigated as anticancer agents and as candidate therapeutic in situ vaccines against prostate cancer.

Patents
Herpes Simplex Virus (HSV) with modified tropism, uses and process of preparation thereof-WO2009144755 (divisional patent EP2700405) Retargeted