Chimerization of the Anti-Viral CD8⁺ T Cell Response with A Broad Anti-Tumor T Cell Response Reverses Inhibition of Checkpoint Blockade Therapy by Oncolytic Virotherapy

Abstract

Although immune checkpoint inhibition (ICI) has produced profound survival benefits in a broad variety of tumors, a proportion of patients do not respond. Treatment failure is in part due to immune suppressive tumor microenvironments (TME), which is particularly true of hepatocellular carcinoma (HCC). Since oncolytic viruses (OV) can generate a highly immune-infiltrated, inflammatory TME, we developed a vesicular stomatitis virus expressing interferon-ß (VSV-IFNß) as a viro-immunotherapy against HCC. Since HCC standard of care atezolizumab/bevacizumab incorporates ICI, we tested the hypothesis that pro-inflammatory VSV-IFNß would recruit, prime, and activate anti-tumor T cells, whose activity anti-PD-L1 ICI would potentiate. However, in a partially anti-PD-L1-responsive model of HCC, addition of VSV-IFNß abolished anti-PD-L1 therapy. Cytometry by Time of Flight showed that VSV-IFNß expanded dominant anti-viral effector CD8 T cells with concomitant, relative disappearance of anti-tumor T cell populations which are the target of anti-PD-L1. However, by expressing a range of HCC tumor antigens within VSV, the potent anti-viral response became amalgamated with an anti-tumor T cell response generating highly significant cures compared to anti-PD-L1 ICI alone. Our data provide a cautionary message for the use of highly immunogenic viruses as tumor-specific immune-therapeutics by showing that dominant anti-viral T cell responses can inhibit sub-dominant anti-tumor T cell responses. However, by chimerizing anti-viral and anti-tumor T cell responses through encoding tumor antigens within the virus, oncolytic virotherapy can be purposed for very effective immune driven tumor clearance and can generate anti-tumor T cell populations upon which immune checkpoint blockade can effectively work.

INTRODUCTION

Hepatocellular Carcinoma (HCC) represents the most common presentation of cancer within the liver and the 6th most common cancer worldwide^{1, 2,3}. Although immune checkpoint inhibition (ICI) therapy has produced significant survival benefits in a broad range of tumor types, a proportion of patients with putatively ICI-sensitive tumors, such as hepatocellular carcinoma, either do not respond or experience diminishing returns from ongoing therapy. The anti-PD-L1 monoclonal antibody atezolizumab in combination with the anti-vascular endothelial growth factor (VEGF) antibody bevacizumab has recently been approved as front-line therapy for HCC⁴. In the phase III imbrave150 study, this combination reduced the risk of death by 56% and the risk of disease progression by 40% compared to the treatment control arm sorafenib. However, although atezolizumab/bevacizumab has clinical activity, response rates reach about 30% and the majority of patients ultimately succumb to the disease⁵.

A major factor contributing to treatment failure in HCC is believed to be the immune suppression mediated by the tumor microenvironment (TME)^{6, 7, 8, 9}. In this respect, oncolytic viruses (OV) infect and selectively replicate within tumor cells, leading to selective cancer cell lysis^{10, 11, 12, 13}. Pro-inflammatory viral oncolysis also modifies the TME and helps to reverse immune-sparse, or -suppressive, microenvironments into highly infiltrated inflammatory immune environments^{14, 15, 16}. To exploit the inflammatory aspects of OV therapy, we have developed the Vesicular Stomatitis Virus (VSV) as an oncolytic and immunotherapeutic agent against different tumor types with the rationale that intratumoral (IT) delivery of VSV would disrupt the immune-suppressive TME and convert it into a virus-inflamed, immune infiltrated tumor site^{15, 17, 18, 19, 20, 21}. To increase both safety and immune-stimulating potency, we also expressed the IFN-ß gene from the virus^{21, 22, 23}. Following IND-enabling toxicology studies^{24, 25}, we conducted a first-in-human clinical study of VSV expressing human IFN-ß (VSV-hIFNβ) using IT injection under ultrasound guidance in patients with liver tumors [NCT01628640]. Fifteen patients were treated with a dose range of 10⁵ to 1.8×10⁷ median tissue culture infectious dose (TCID₅₀) and the maximum tolerated dose was 3×10⁶ TCID₅₀. Preliminary evidence of efficacy included one partial response by Response Evaluation Criteria in Solid Tumors (RECIST) v1.1 criteria and two patients with stable disease (manuscript in preparation), making it clear that further improvements to the therapeutic regimen are required.

Since current standard of care HCC therapy atezolizumab/bevacizumab incorporates ICI, in the current study we sought to develop our initial clinical approach by combining virotherapy with ICI. Therefore, here we tested the hypothesis that the heavily pro-inflammatory activity of vesicular stomatitis virus expressing interferon-ß (VSV-IFNß) virotherapy would convert the immune suppressive TME of HCC to an immune stimulatory environment. In turn, this would recruit/prime/activate potentially anti-tumor T cells, the activity of which anti-PD-L1 ICI therapy would be able to potentiate further, thereby generating superior anti-tumor therapy to either therapy alone^{26, 27, 28, 29, 30, 31, 32}. This hypothesis is consistent with the model that inflammatory killing of tumor cells through viral oncolysis will recruit antigen presenting cells (APC) to the tumor site, release high loads of tumor associated antigens (TAA), and facilitate presentation of these TAA to CD8 and CD4 T cells in the draining lymph nodes, thereby generating systemically active anti-tumor T cell immunity^{26, 27, 28, 29, 30, 31, 32}.

To test this hypothesis, we used a slow developing model of HCC, which resembles the etiology of human HCC³³, in which a Sleeping Beauty transposon integrates ß-catenin and hMet oncogenes into the livers of neonatal mice resulting in multi-focal tumor formation (SB-HCC) over 100–150 days. Tumor bearing mice were partially susceptible to anti-PD-L1 therapy (~ 50% long term cures), thereby mimicking at least one aspect of the human disease and current therapy. To our surprise, rather than observing even additive benefit to therapy, and irrespective of the relative timings of both treatments, the addition of VSV-IFNß to anti-PD-L1 therapy largely abolished the therapeutic effects of anti-PD-L1 ICI alone. We show that VSV-IFNß treatment generated a highly significant expansion of one, or a few, dominant anti-viral effector CD8⁺ T cell populations with the concomitant relative disappearance of those putative anti-tumor T cell populations which are the target of anti-PD-L1 treatment. Based on our previous studies showing that inclusion of a tumor antigen within VSV induces CD8⁺ T cell dependent anti-tumor therapy directed specifically against the virally encoded antigen^{19, 34, 35, 36}, we hypothesized that it would be possible to amalgamate the potent anti-viral response with an anti-tumor T cell response by expressing immunologically relevant tumor associated antigens from within the virus. Here we show that by expressing a multitude of putative, uncharacterized, tumor antigens within VSV using a cDNA library derived from three different SB-HCC explants (VSV-SB-HCC1,2,3) we observed highly significant improvements compared to anti-PD-L1 ICI alone - even in the absence of added anti-PD-L1 treatment.

Our data provide a cautionary message for the use of highly immunogenic viruses as tumor-specific immune-therapeutics. We show that oncolytic virotherapy can induce anti-viral T cell responses which can actively inhibit, or obscure, weak anti-tumor T cell responses leading, potentially, to decreased anti-tumor therapy. However, by chimerizing anti-viral and anti-tumor T cell responses through encoding tumor antigens within the virus, at least a proportion of the anti-viral T cell response is co-opted into an anti-tumor response. In this way, oncolytic virotherapy can be purposed for very effective immune based tumor clearance and can generate anti-tumor T cell populations upon which immune checkpoint blockade can effectively work.

RESULTS

The hMet and S45Y ß-catenin Sleeping Beauty transposon system induces multi-focal immune suppressive HCC.

Concomitant expression of activated hMet and ß-catenin leads to multi-focal liver tumors in murine models following hydrodynamic tail vein injection³³, thereby mimicking an expression signature seen in a proportion of HCC patients^{33, 37, 38}. We confirmed that the hMet + S45Y ß-catenin/Sleeping Beauty transposon plasmid system consistently induced multi-focal tumors in the livers of C57Bl/6 mice (Figs. 1A–B). Histologic signs of liver tumor development were observed as early as 4 weeks following hydrodynamic tail vein injection, initially with well-differentiated morphology but minimal immune infiltration. By 7 weeks post injection, livers were densely population by tumors, with large pseudo-cysts, fat deposits, and signs of extramedullary hematopoiesis and shortly thereafter inevitably reached a terminal endpoint (Figs. 1A–B).

Figures 1. The hMet + S45Y ß-Catenin Sleeping Beauty transposon system induces liver tumors which are infiltrated by PD-1⁺ CD8 T cells.

Following hydrodynamic injection of hMet + S45Y ß-Catenin plasmids, animals were sacrificed at 4 and 7 weeks for pathologic studies. A. Livers (animals A1 and A4) demonstrated appreciable neoplasia with a well-differentiated morphology and minimal immune infiltration by week 4. This progressed by week 7 (animals C1 and C4) with neoplastic cells overtaking the majority of the tumor, with large pseudo-cysts, fat deposits, and signs of extramedullary hematopoiesis (more prominent in C4). B. Livers from a control mouse and from a mouse 7 weeks after hMet + S45Y ß-Catenin hydrodynamic tail-vein injection. C-D. Following hydrodynamic injection of hMet + S45Y ß-Catenin, animals were sacrificed at day 22 and day 29 and analyzed for CD8+ (C) and CD4+ (D) T cells by flow cytometry. D. Similar to C, tumor-infiltrating CD4 T cells were evaluated. E-F. Proportions of CD8+ (E) and CD4+ (F) T cells expressing Tim3 and/or PD1⁺ in livers of tumor bearing or tumor naïve mice at days 22 and day 29. G. Proportions of PD-L1⁺ CD86⁺ dendritic cells (DCs) in mice 22 and 29 days after hMet + S45Y ß-Catenin injection compared to tumor naïve mice. H. Hematoxylin and eosin staining combined with PD-L1 immunohistochemistry of the livers of FVB mice at weeks 4, 5, 6, and 7 after hMet + S45Y ß-Catenin hydrodynamic tail-vein injection. 10x magnification. Significance for C-D determined by ordinary one-way ANOVA. Significance for E-G was determined by 2-way ANOVA with multiple comparisons using Tukey’s multiple comparisons test. Statistical significance was set with * indicating a p value less than 0.05, ** < 0.01, *** < 0.001, and **** < 0.0001.

Whilst CD8⁺ T cell infiltration into livers was 10-fold higher in tumor-bearing mice than in non-tumor bearing control animals at both days 22 and 29 post hydrodynamic injection, CD4⁺ T cell infiltration trended towards increased levels but did not reach significance (Figs. 1C–D). The majority of liver infiltrating CD8⁺ and CD4⁺ T cells in non-tumor bearing animals had minimal expression of either Tim-3 or PD-1, although there was a small but detectable population of Tim-3⁺/PD-1⁺ CD4⁺ T cells (Figs. 1E–F). In contrast, the proportion of Tim-3⁺/PD-1⁺ CD8⁺ T cells in tumor bearing livers at both days 22 and 29 was significantly increased (≥ 50% of the total CD8⁺ T cell population) compared to the control non-tumor bearing mice, suggesting that presence of tumor induced a profound state of CD8⁺ T cell dysfunction/exhaustion (Fig. 1E). This change was not observed in the liver infiltrating CD4⁺ T cell population, where trends towards increased proportions of Tim-3⁺/PD-1⁺ CD4⁺ T cells did not reach significance compared to non-tumor bearing mice (Fig. 1F). Consistent with development of an immune suppressive TME within hMet + S45Y ß-catenin liver (SB-HCC) tumors, expression of PD-L1 on liver infiltrating CD11c⁺/MHCII⁺ dendritic cells was significantly increased in tumor bearing compared to non-tumor bearing mice (Fig. 1G). Finally, macroscopic visualization of tumor bearing livers showed high levels of PD-L1 expression associated with the multi-focal tumor lesions which increased progressively with tumor development from 4 through 7 weeks post hydrodynamic injection (Fig. 1H). Taken together, these data indicate that SB-HCC tumors develop a highly immune suppressive TME within 20–30 days of hydrodynamic plasmid injection, thereby re-capitulating this aspect of human HCC in which a highly immunosuppressive TME³⁹ is associated with exhausted CD8⁺ T cells with reduced anti-tumor cytotoxicity^{40, 41, 42}.

SB-HCC is responsive to CD8⁺ T cell-mediated checkpoint blockade with anti-PD-L1.

Consistent Consistent with SB-HCC liver tumors developing a highly immune suppressive, PD-L1 dependent TME analogous to the human disease, treatment with anti-PD-LI ICI led to cures of up to 50% of treated mice by day 150 post hydrodynamic injection (Fig. 2A). Therapy with anti-PD-L1 ICI was completely dependent upon CD8⁺ T cells (Fig. 2A) but not on CD4⁺ T cells (Fig. 2B). Over two separate experiments, depletion of NK cells showed a trend towards increased efficacy of anti-PD-L1 therapy compared to non-depleted mice, although this did not reach significance (Fig. 2C). While not definitive, this may suggest that NK-mediated immune suppression plays a role in HCC development in this model.

Figure 2. Responsiveness of SB-HCC to CD8-mediated immune checkpoint blockade is abolished by VSV virotherapy.

Animals in all arms underwent hydrodynamic injection of hMet + S45Y ß-Catenin at day 0. N=67 animals (A-C). A-C. Control IgG or anti-PD-L1 ICI therapy was initiated at day 21 for 6 doses (days 21,23,25,28,30,31) in non-depleted mice or in mice depleted of CD8 (A), CD4 (B) or NK cells (C) as shown (8 total doses). Survival with time is shown. D. Mice hydrodynamically injected with hMet + S45Y ß-Catenin at day 0 were treated with PBS or VSV-mIFNß on days 21, 23 and 25 (3 doses, 10⁸ pfu/dose), followed by control IgG isotype or anti-PD-L1 on days 28, 30, 32, 35, 37, 39 (6 doses, 200μg per dose). Survival with time is shown. E. Mice hydrodynamically injected with hMet + S45Y ß-Catenin at day 0 were treated with control IgG isotype or anti-PD-L1 on days 21, 23, 25, 28, 30, 33 (6 doses, 200μg per dose) and with PBS or VSV-mIFNß on days 40, 42 and 44 (3 doses, 10⁸ pfu/dose). Survival with time is shown. Significance was determined through survival curve comparison testing using a log-rank Mantel-Cox test. Statistical significance was set with * indicating a p value less than 0.05, ** < 0.01, *** < 0.001, and **** < 0.0001.

Combination VSV-IFNß oncolytic virotherapy with anti-PD-L1 ICI abolishes the therapy of ICI alone.

Our initial hypothesis was that early IT treatment of HCC with VSV-IFNß would convert the immune-suppressive TME into a pro-inflammatory environment. In turn, this would liberate HCC tumor associated antigens (HCC_TAA) leading to the priming of anti-HCC_TAA T cells upon which immune checkpoint therapy could work. Contrary to this hypothesis, we observed that early IT treatment with VSV-IFNß (day 21–25 for three doses) prior to anti-PD-L1 ICI (day 28–39 for 6 doses) completely abolished the survival benefit generated by ICI alone and was no better than either virus alone or control treatment alone (Fig. 2D). When this sequencing was reversed and IT virus was administered later (day 40–44) than anti-PD-L1 ICI (day 21–33), the virus still inhibited the effects of anti-PD-L1 therapy alone (Fig. 2E), although there was a (non-significant) trend to an improvement of the combination (virus + ICI) therapy compared to the virus alone. Similar inhibition of the therapeutic effects of anti-PD-L1 therapy by IT virus were observed with other schedules of administration including when virus and ICI overlapped (Supplemental Fig. 1). Taken together, these data show that the addition of a highly immunogenic oncolytic virus to a weak, anti-PD-L1-sensitive anti-tumor immune response acted consistently to inhibit the therapeutic anti-HCC_TAA immune response.

An immuno-dominant anti-viral rapid effector CD8⁺ T cell population replaces sub-dominant putative anti-HCC_TAA T cells.

We observed that, when IT virus was given early either before, or co-incidentally, with anti-PD-L1 ICI, the therapeutic effects of ICI therapy alone were lost (Fig. 2D, Supplemental Fig. 1), with the survival curves beginning to separate about 10 days after the last treatment with anti-PD-L1. Therefore, we used Cytometry by Time of Flight (CyTOF) analysis using t-distributed Stochastic Neighbor Embedding (tSNE) with Rphenograph, 22 populations to analyze tumor infiltrating lymphocyte populations (TIL) (Fig. 3A). Using differential immune marker expression, we defined a total of 9 distinct immune populations and reanalyzed through tSNE with FlowSOM, 9 populations (Fig. 3B). When comparing TIL populations between naïve and HCC tumor bearing mice, the presence of developing HCC tumor in the liver induced expansion of a CD8⁺ T cell population with markers of terminal effector cells, as defined by high Tim-3 (CD366) and PD-1 expression (Fig. 3A), at day 38 post hydro-dynamic injection (Fig. 3C–D). This population, which we identify as ‘Exhausted CD8 T Cells’, correlates to cluster 17 in our 22-population tSNE analysis (Fig. 3A). This tumor-expanded population also expressed high levels of Lymphocyte Activation Gene-3 (LAG3, CD223), T-cell immunoglobulin and mucin domain-3 (TIM3), T cell immunoreceptor with Ig and ITIM domains (TIGIT), programmed cell death protein 1 (PD-1) and CD39 which are also associated with terminal effector and exhausted CD8⁺ T cells^{43, 44, 45}. These data validate the use of SB-HCC as a model for human HCC, as the TME of HCC is enriched with exhausted, PD-1 expressing CD8⁺ T cells that represent the main subset of TIL and display anti-tumor cytotoxic activity in HCC46, 47, 48.

Figure 3. IT treatment with VSV-mIFNß generates a dominant anti-viral CD8 T cell response which replaces the anti-tumor T cell response.

A. Treatment regimen. Following hydrodynamic injection of hMet + S45Y ß-Catenin on day 0, animals were treated with anti-PD-L1 on day 21 for 6 doses concurrently with VSV-mIFNß for 3 doses. All animals were sacrificed on day 38. Livers were processed into single-cell suspensions and analyzed by the Mayo CyTOF core facility. N=16, 4 animals per group, groups were pooled for Rphenograph tSNE analysis, 22 groups. Scatterplot and mean heat map shown. B. Following initial analysis (A), 9 distinct immune populations were identified and pooled data was re-analyzed using FlowSOM tSNE analysis. ‘Exhausted CD8 T cells’ were identified by expression of CD8, Tim-3, LAG-3, CD39, and PD-1 expression, ‘B cells’ by CD38 and CD19; ‘CD4 T cells’ by CD4; ‘Memory CD4 T Cells’ by CD4, CCR7, and CD62L; ‘Memory CD8 T Cells’ by CD8, CCR7, and CD62L; ‘NK Cells’ by NK1.1; ‘NKT Cells’ by NK1.1 and CD8; and ‘Anti-viral CD8 T Cells’ by CD8, GranzB, and PD-L1. C. Pooled analysis of lymphocytes within livers of tumor-free mice, first 10,000 events. D. Pooled analysis of lymphocytes within livers of SB-HCC-bearing mice, first 10,000 events. E. Pooled analysis of lymphocytes within livers of SB-HCC bearing mice treated with anti-PD-L1, first 10,000 events. F. Pooled analysis of lymphocytes within livers of SB-HCC-bearing mice treated with anti-PD-L1 and VSV-mIFNß, first 10,000 events.

Treatment of HCC-bearing mice with ICI alone (days 21–34) induced detectable changes in the landscape of CD8⁺ T cell populations, with a particularly significant further proportional increase in the ‘Exhausted CD8 T Cells’ (Cluster 17) population which is characterized by activation/inhibitory markers (TIM-3, TIGIT, LAG-3, CD39, and PD-1) (Fig. 3E). We hypothesize this population to be anti-HCC_TAA CD8⁺ T cells upon which ICI therapy was operative.

In contrast, treatment of HCC-bearing mice with ICI (days 21–34) and VSV-IFNß early (days 21–25) - under which conditions the therapeutic effects of anti-PD-L1 ICI therapy were abolished by addition of virus (Supplemental Fig. 1) – induced a predominant population of putative viral specific CD8⁺ T cells (Clusters 18, 19, 20, 22, Fig. 3A; ‘Anti-viral CD8 T Cells,’ Fig. 3B&F) which proportionately downregulated most of all the other CD8⁺ T cell populations, including the putative anti-HCC_TAA CD8⁺ T cells (cluster 17, Fig. 3A; ‘Exhausted CD8 T Cells,’ Fig. 3B&F). This population was characterized by relatively high levels of expression of Granzyme B, interleukin 7 receptor (IL-7R, CD127), leukocyte antigen B associated transcript 3 (Bat3), and PD-L1 as well as relatively low levels of CD69 compared to the terminally differentiated putative anti-tumor CD8⁺ T cell population – an overall profile consistent with the presence of anti-viral effector CD8⁺ T cells.

Taken together, these data strongly argue that the abolition of therapeutic benefit achieved with ICI alone by addition of virus to ICI therapy was due to a rapid induction/expansion of anti-viral effector CD8⁺ T cells at the expense of anti-tumor, PD-1 expressing terminal effector CD8⁺ T cells. Moreover, the repolarization of the effector response of CD8⁺ T cells away from anti-HCC_TAA CD8⁺ T cells towards a population of anti-viral effector CD8⁺ T cells would allow the concomitantly administered anti-PD-L1 ICI to focus upon the anti-viral, rather than anti-HCC_TAA CD8⁺ T cells – thereby re-invigorating the anti-viral, rather than anti-tumor, T cell response.

Directing the anti-viral response to become an anti-tumor response.

Our previous studies have shown that inclusion of a tumor antigen within VSV induces CD8⁺ T cell dependent anti-tumor therapy directed specifically against the virally encoded antigen as a result of the potent immune stimulating adjuvant properties of infection with VSV^{19, 34, 35, 36}. Therefore, we hypothesized that it would be possible to amalgamate the potent anti-viral response (Fig. 3) with an anti-tumor T cell response by expressing immunologically relevant tumor associated antigens from within the virus. To test this in the hMet + S45Yß-catenin / Sleeping Beauty transposon system, we initially used the model tumor antigen OVALBUMIN (OVA) against which we could following anti-tumor T cell responses using SIINFEKL tetramer analysis. Following hydrodynamic injection of the hMet + S45Yß-catenin + pOVA / Sleeping Beauty transposon plasmid system, in which the model antigen OVA was co-expressed in the HCC tumors, anti-OVA T cells were detected in mice with hMet + S45Yß-catenin + OVA tumors from both tumor and spleen but not in naïve, non-tumor-bearing animals (Figs. 4A–B). Even though in this case OVA is a foreign non-tolerized antigen, these data were consistent with the generation of anti-HCC_TAA T cells in these tumors as suggested from Figs. 3B–E. Furthermore, addition of anti-PD-L1 to tumor bearing mice significantly enhanced the anti-OVA T cell response approximately threefold in both tumor and spleen, confirming that anti-PD-L1 ICI was able to expand anti-HCC_TAA CD8⁺ T cell responses in vivo (Figs. 4A–B).

Figure 4. Expression of a HCC_TAA within VSV overcomes the loss of anti-TAA CD8+ T cells by VSV virotherapy.

A-B. Following hydrodynamic injection of HCC-OVA (day 0), mice were treated with anti-PD-L1 (day 6 for 6 doses). On day 23 livers (A) and spleens (B) were processed to single cell suspensions and analyzed by flow cytometry for SIINFEKL tetramer positive CD8+ T cells. (n=12, 4 per group, representative flow data shown). C-E. Following hydrodynamic injection of HCC-OVA (day 0), mice were treated with anti-PD-L1 (day 6 for 6 doses) followed by VSV-GFP or VSV-IFNß (3 doses, days 18,20,22). On day 23 livers (D) and spleens (E) were analyzed by flow cytometry for SIINFEKL tetramer positive CD8+ T cells. (n=28, 4 animals per group). F-H. Following hydrodynamic injection of HCC-OVA (day 0), mice were treated with anti-PD-L1 (day 23, 6 doses) followed by VSV expressing ovalbumin (VSV-OVA) for 3 doses (day 90, 92, 94). On day 98 livers and spleens were analyzed by flow cytometry for SIINFEKL tetramer positive CD8+ T cells. (n=12, 4 per group. Representative flow data shown as well as pooled ratio of SIINFEKL-tetramer⁺ to VSV-N⁺ CD8 T cells). Significance was determined through ordinary one-way ANOVA with Tukey’s multiple comparison tests. Statistical significance was set with * indicating a p value less than 0.05, ** < 0.01, *** < 0.001, and **** < 0.0001.

Administration of ICI confirmed the anti-PD-L1 enhanced expansion of anti-OVA T cells in both liver and spleen (Figs. 4C–E). However, consistent with the effective replacement of anti-tumor T cells with anti-viral T cells seen in Fig. 3, the addition of either early (Fig. 3) or late (Figs. 4C–D) VSV (VSV-GFP or VSV-IFNß) to HCC-OVA tumor-bearing mice ablated almost entirely the anti-HCC_TAA(OVA) T cell response in the liver, whether or not anti-PD-LI ICI was also administered (Figs. 4C–D). The diminution of the anti-HCC_TAA(OVA) T cell response by addition of VSV-GFP or VSV-IFNß was less complete in the spleen (Figs. 4C&E) suggesting that the inflammatory anti-viral response is most dominant within the liver TME.

Therefore, we tested the hypothesis that it would be possible to amalgamate the anti-viral response with an anti-tumor response by expressing the HCC_TAA(OVA) from within VSV. When ICI was given in the absence of virus to allow for maximal expansion of the anti-tumor T cell response, treatment with VSV-OVA alone at a late stage (Fig. 4F) generated significantly greater numbers of anti-OVA T cells in both liver and spleen compared to the levels spontaneously generated in response to growth of HCC-OVA tumors alone (Figs. 4G&H compared to Figs. 4A&B). In addition, treatment with early anti-PD-L1 ICI combined with late VSV-OVA significantly expanded the levels of anti-HCC_TAA(OVA) CD8⁺ T cells to high levels as a percentage of total CD8⁺ T cells in both liver and spleen (Figs. 4G&H). Interestingly, the expansion of anti-HCC_TAA(OVA) CD8⁺ T cells by the addition of anti-PD-L1 ICI was significantly greater than the relative expansion of anti-VSV T cells (measured by tetramer directed against the immune-dominant VSV-N_{52 – 59} peptide of the VSV N protein) (Figs. 4G&H).

Taken together these data show that by encoding a putative HCC_TAA within the oncolytic VSV highly significant numbers of anti-HCC_TAA T cells were generated in vivo which were substrates for expansion by anti-PD-L1 ICI therapy and which expanded preferentially over at least one component of the immune-dominant anti-viral response.

Selection of putative Sleeping Beauty TAA through prediction of high MHC Class I binding epitopes.

Although it was encouraging that encoding a TAA within VSV could generate ICI-expandable anti-tumor T cells, the experiments of Fig. 4 used the immunogenic model OVA antigen to which the C57Bl/6 mice were not tolerized. Therefore, to expand the concept of VSV-TAA to therapeutic efficacy against real putative HCC_TAA, RNAseq analysis of SB-HCC tumors was used to identify the top ten genes whose expression was upregulated in tumors compared to normal liver (Fig. 5A). Predicted binding affinities of peptide epitopes from these genes to the relevant H2K^b and H2D^b MHC Class I molecules of C57Bl/6 mice identified several strong binding epitopes from the Lcn2, Lect2, Smagp, Nsdh1 and Plrg1 genes (Fig. 5B–C), suggesting that over-expression of these genes in Sleeping Beauty HCC may expose potential neo-antigens for endogenous CD8⁺ T cell priming/recognition. To test the ability of the three highest over-expressed genes (Lcn2, Lect2, Smagp) encoded separately within VSV to break tolerance to these potential HCC_TAA, VSV-IFNß co-expressing the relevant genes were tested as vaccinating agents in vivo. Splenocytes from mice vaccinated with separate VSV-IFNß-TAA did not generate any detectable Th17 responses following in vitro re-stimulation with a 1:1:1 mix of three Sleeping Beauty HCC explants recovered from untreated tumor bearing mice (SB-HCC 1,2,3) (Fig. 5D). However, splenocytes from mice vaccinated with VSV-IFNß-Lcn2 generated a Th1-like, IFN-γ response to these tumor targets which was significantly greater than that generated by VSV-IFNß alone (Fig. 5E), suggesting that this TAA may be a potential HCC_TAA in this system.

Figure 5. Putative HCC_TAA selected for high level expression and MHC binding affinity.

A. SB-HCC tumor-bearing mice were sacrificed on day 18 with livers harvested for RNA extraction. RNA samples were then subjected to RNA-seq analysis, identifying 10 highest relative gene expression compared to non-tumor bearing livers. B-C. The 10 most-expressed genes identified and full-length sequences were filtered through NET MHC 2.0 binding affinity algorithm to identify octamer or nonamer peptides whose binding affinity for H2Kb (B) or H2Kd (C) was below a threshold of 500 nM, and whose corresponding wild-type peptides had a binding affinity to the same molecules above 500 nM. These corresponded to Lcn2, Lect2, and SMAGP. D-E C57Bl/6 mice (n=12, 3 per group) were injected intravenously with 10⁷ plaque-forming units (pfu) of VSV-mIFNß, VSV-Lcn2, VSV-Lect2, or VSV-SMAGP. 10 days later 10⁶ splenocytes were co-cultured with a 1:1:1 mixture of SB-HCC explants 1, 2 and 3 at an effector:target (E:T) ratio of 10:1. Supernatants were was assayed 48 hours later for IL-17 (D) or IFNγ (E). Significance was determined through ordinary one-way ANOVA with Tukey’s multiple comparison tests. Statistical significance was set with * indicating a p value less than 0.05, ** < 0.01, *** < 0.001, and **** < 0.0001.

VSV expressing a single putative HCC_TAA may boost a pre-existing HCC_TAA T cell response.

From these data we reasoned that a treatment regimen in which early ICI was administered prior to virus treatment would optimize the expansion of the endogenous anti-tumor T cell response prior to a boosting effect with VSV-TAA. In this setting, and as before, anti-PD-L1 ICI alone cured ~ 50% of mice bearing Sleeping Beauty HCC tumors, a therapeutic effect which was completely and rapidly eradicated by the addition of VSV-IFN-ß (Fig. 6A). Interestingly, treatment with VSV-IFNß-Lcn2 in combination with anti-PD-L1 ICI was significantly more therapeutic than VSV-IFN-ß + anti-PD-L1 (Fig. 6A). However, although VSV-IFNß-Lcn2 in combination with anti-PD-L1 did not ablate the therapy seen with anti-PD-L1 ICI alone, it did not increase therapy either (Fig. 6A). Similarly, treatment with (VSV-IFNß-Lect2 + VSV-IFNß-Lcn2 + VSV-IFNß-Smagp) in a ratio of 1:1:1 in combination with anti-PD-L1 ICI was also significantly more effective than VSV-IFN-ß + anti-PD-L1, but also did not improve upon, the therapy of anti-PD-L1 alone (Fig. 6A). Consistent with the results of Figs. 5D–E, splenocytes from all groups showed no detectable Th17 anti-SB-HCC 1,2,3 responses (Fig. 6B). In contrast, splenocytes from mice treated with anti-PD-L1 alone showed significant Th-1-like IFN-γ recall responses against live SB-HCC 1,2,3 explants (Fig. 6C). Splenocytes from mice treated with VSV-Lcn2 + anti-PD-L1, showed a trend towards a recall Th-1 response against live SB-HCC 1,2,3 explants but this did not reach significance compared to splenocytes from mice treated with VSV-IFNß + anti-PD-L1 (Fig. 6C). Finally, splenocytes from mice treated with the combination of all three putative HCC_TAA (VSV-IFNß-Lect2 + VSV-IFNß-Lcn2 + VSV-IFNß-Smagp) + anti-PD-L1 showed no detectable response to SB-HCC 1,2,3 explants (Fig. 6C), suggesting that addition of the Lect2 and/or Smagp antigens may even exert an inhibitory effect upon anti-tumor immunity as generated by Lcn2 alone.

Figure 6. VSV expressing the putative HCC_TAA Lcn2 improves upon therapy with VSV-IFNß in combination with ICI.

A. Following hydrodynamic injection of hMet + S45Y ß-Catenin (day 0), mice were treated with anti-PD-L1 (200μg/injection; days 5,7,9,12,14,16) followed by 10⁷pfu of VSV-IFNß, VSV-IFNß-Lcn2 or with 3×10⁶ pfu each of (VSV-IFNß-Lect2 + VSV-IFNß-Lcn2 + VSV-IFNß-Smagp) (day 21,22,23). N=32. B-C. In a repeat of the protocol of A, spleens were harvested 10 days following the last dose of virus and 10⁶ splenocytes were co-cultured with SB-HCC 1,2,3 tumor targets at an effector:target ratio of 10:1 with supernatant assayed 48 hours later for (B)IL-17 and (C) IFNγ.

VSV expressing multiple undefined HCC_TAA boosts anti-tumor CD8⁺ T cell responses expanded by ICI.

One possible interpretation of these data is that, when at least one potentially immunogenic HCC_TAA, such as Lcn2, was added to VSV-IFNß + anti-PD-L1, early treatment with anti-PD-L1 ICI allowed for expansion of potentially tumor reactive CD8⁺ T cells (Cluster 17 in Fig. 3). Thereafter, the anti-Lcn2 component of the anti-tumor T cell response could then be boosted by late vaccination with VSV-IFNß-Lcn2. If this model were true, we predicted that by adding multiple further HCC_TAA to the vaccinating VSV-IFNß virus, an increased number of anti-HCC_TAA T cells could be boosted by late VSV-IFNß-HCC_TAA vaccination. Therefore, cDNA from three separate Sleeping Beauty explants mixed at a 1:1:1 ratio was cloned into the VSV-IFNß virus to give a viral stock of VSV-IFNß-SB-HCC 1,2,3 cDNA (Fig. 7A). Presence of the three most highly expressed SB-HCC genes, Lcn2, Lect2, and Smagp (identified by RNAseq, Fig. 5) in the VSV-IFNß-SB-HCC 1,2,3 cDNA library, but not in the ASMEL VSV-cDNA library constructed previously from melanoma cells³⁶, was confirmed by PCR. Conversely, the melanoma associated genes gp100 or TYRP1 were abundant in the ASMEL library but were essentially undetectable in the VSV-IFNß-SB-HCC 1,2,3 cDNA library (Fig. 7B).

Figure 7. VSV-SB-HCC1,2,3 cures mice with SB-HCC mediated by CD8 T cells and associated with Th1 and Th17 responses.

A. cDNA from three murine Sleeping Beauty HCC tumor-bearing livers, (SB HCC 1,2&3 Library), was pooled, cloned and amplified by PCR. The SB HCC 1,2&3 Library was then cloned into the VSV backbone plasmid between the Xho1 and Nhe1 sites. B. Composition of the VSV-derived ASMEL³⁶ and SB HCC 1,2&3 Libraries was validated using PCR from the equivalent of 10⁸ pfu of the VSV-ASMEL³⁶ or VSV-SB HCC 1,2&3 with gene specific primers to the HCC-specific Lcn2, Lect2, and Smagp genes or to the melanoma specific TYRP1 and GP100 genes. C Following hydrodynamic injection of hMet + S45Y ß-Catenin (day 0), animals were treated starting on days 21,23,25,28,30,32 with anti-PD-L1 (100μg/injection) with, or without, 10⁷ pfu of VSV-SB-HCC1,2,3 or 3×10⁶ pfu each of (VSV-IFNß-Lect2 + VSV-IFNß-Lcn2 + VSV-IFNß-Smagp) on days 38,40,42. (N=32, 8 mice per group). D-E Spleens were harvested and 10⁶ splenocytes were co-cultured with a 1:1;1 micture of live SB-HCC 1,2,3 explant cells as targets at an effector:target ratio of 10:1 with supernatant assayed 48 hours later for (D) IL-17 and (E) IFNγ. (N=12, 3 mice per group) F Following hydrodynamic injection of hMet + S45Y ß-Catenin (day 0), animals were treated on days 21,23,25,28,30,32 with control isotype IgG or with anti-PD-L1 (200μg/injection) with, or without, PBS or 10⁷pfu ASMEL (VSV-melanoma cDNA library)³⁶ or 10⁷pfu VSV-SB-HCC1,2,3 on days 38,40,42. Depleting antibodies were given concurrently with either anti-PD-L1 or IgG isotype control for 6 doses. (N=65, 8 mice per group except (VSV-SB-HCC1,2,3 + Control IgG) where n= 9). For clarity, significance comparisons for all groups are included in the Supplement. Significance for PCR (B) was determined using 2-way ANOVA with multiple comparisons using Tukey’s multiple comparisons test. Significance for survival experiments (C&F) was determined through survival curve comparison testing using a log-rank Mantel-Cox test. Significance for ELISA experiments (D&E) was determined through ordinary one-way ANOVA with Tukey’s multiple comparison tests. Statistical significance was set with * indicating a p value less than 0.05, ** < 0.01, *** < 0.001, and **** < 0.0001.

Treatment of SB-HCC tumor bearing mice with a reduced dosage of anti-PD-L1 ICI (100μg/mouse/injection instead of 200μg/mouse/per injection) in order to accentuate any combinatorial therapeutic effects, generated some long-term survivors (Fig. 7C). As before, addition of VSV expressing at least one putative HCC_TAA(Lcn2) no longer ablated, but neither did it improve, the therapy with ICI alone (Fig. 7C). However, with Early anti-PD-L1 ICI + Late VSV-SB-HCC1,2,3, for the first time, a combination of VSV and anti-PD-L1 ICI generated significantly improved therapy over anti-PD-L1 ICI treatment alone with 100% of mice surviving to day 150 (Fig. 7C). Interestingly, treatment with VSV-SB-HCC1,2,3 alone (no anti-PD-L1) was also very effective at generating long term cures (Fig. 7C). These data suggest that VSV-mediated display of multiple antigens was sufficient of itself to boost slow developing anti-HCC_TAA CD8⁺T cell responses even in the absence of expansion by anti-PD-L1 treatment. Consistent with this, splenocytes from mice treated with anti-PD-L1 ICI alone re-stimulated in vitro with live SB-HCC Explants 1,2,3 cells secreted IFN-γ but not IL-17 (Figs. 7D&E). In contrast, splenocytes from mice treated with either VSV-SB-HCC1,2,3 alone (no anti-PD-L1 ICI), or with VSV-SB-HCC1,2,3 + anti-PD-L1, generated a Th17 recall response when re-stimulated in vitro with SB-HCC cells (Fig. 7D). Splenocytes from mice treated with VSV-SB-HCC1,2,3 alone generated a Th1/IFN-γ recall response that was equivalent to that from splenocytes from mice treated with anti-PD-L1 alone (Fig. 7E), whereas splenocytes from mice treated with VSV-IFNß-SB-HCC1,2,3 + anti-PD-L1 generated a significantly increased Th1/IFN-γ recall response to SB-HCC cell explants (Fig. 7E). To confirm the mechanism associated with the highly significant therapy induced by treatment with VSV-SB-HCC1,2,3, SB-HCC tumor-bearing mice were depleted of different immune subsets prior to treatment with VSV-SB-HCC1,2,3 +/− ICI (Fig. 7F). As before, anti-PD-L1 ICI alone generated cures in just under 50% of the mice (Fig. 7F, light blue line) and mice treated with a control isotype IgG all succumbed to tumor (Fig. 7F, dark blue line). Also, as before, treatment with Early anti-PD-L1 + Late VSV-SB-HCC1,2,3 cured 100% of mice (Fig. 7F, brown line). Treatment with VSV-SB-HCC1,2,3 alone was not significantly different from Early anti-PD-L1 + Late VSV-SB-HCC1,2,3, although one mouse in this experiment succumbed to tumor (Fig. 7F, black line). Depletion of neither NK, nor CD4⁺, cells from mice treated with the Early anti-PD-L1 + Late VSV-SB-HCC1,2,3 regimen significantly decreased overall therapy, although both groups fell from 100% survival by day 150 (Fig. 7F, red and green lines respectively). However, depletion of CD8⁺ T cells almost completely abolished therapy (Fig. 7F, purple line). Finally, treatment of tumor bearing mice with Early anti-PD-L1 and Late VSV-cDNA library in which the cDNA was sourced from a melanoma cell line (VSV-ASMEL) was also ineffective (Fig. 7F, orange line), showing that CD8⁺ T cell boosting against cancer type specific antigens is critical for these effects.

Taken together, these data show that optimal therapy requires ICI-induced expansion of anti-HCC_TAA- specific CD8⁺ T cells, which are subsequently further expanded by VSV-IFNß-HCC_TAA boosting, through induction of both Th17 and Th1 component mechanisms.

DISCUSSION

Here, we used a Sleeping Beauty Transposon-based model of HCC which re-capitulates many features of the human disease genetically, phenotypically, and in its partial sensitivity to anti-PD-L1 ICI therapy. We show that, in a tumor model where there is a pre-existing, weak/immuno-subdominant anti-HCC_TAA CD8⁺ T cell response, addition of a highly immuno-dominant oncolytic virus effectively occluded the anti-tumor response with a rapid effector anti-viral CD8⁺ T cell response. However, by encoding a range of HCC_TAA within the virus in the form of an HCC-derived cDNA library, the potent immune adjuvant properties of the virus allowed for boosting of the pre-existing anti-HCC_TAA CD8⁺ T cells leading to high rates of tumor cures.

With the rationale of disrupting the immune-suppressive TME of HCC and converting it into a virus-inflamed immune infiltrated TME, we tested VSV-IFNß gene as an oncolytic and immunotherapeutic agent^{15, 17, 18, 19, 20, 21, 24, 34} in a clinical trial [NCT01628640] in which VSV-IFNß was administered to patients with sorafenib-refractory HCC. In that study, we observed modest activity associated with VSV-IFNß virus alone. Therefore, we sought to improve clinical utility of the virus in the context of the current standard of care regimen atezolizumab/bevacizumab which includes ICI. We used a slow developing model of HCC, which resembles the etiology of human HCC, in which a Sleeping Beauty transposon integrates the ß-catenin and hMet oncogenes into the livers of neonatal mice resulting in multi-focal tumor formation over 100–150 days (Fig. 1). Tumors grew progressively in the liver, showed characteristics of immune suppression, expressed PD-L1, and were partially susceptible to anti-PD-L1 therapy (~ 50% long term cures), thereby mimicking several aspects of the human disease and its current therapy (Figs. 1&2A–C).

Contrary to our initial hypothesis, the addition of a highly immunogenic oncolytic virus to a weak, pre-existing anti-PD-L1-sensitive anti-tumor immune response inhibited the therapeutic anti-HCC_TAA immune response (Fig. 2D–E) irrespective of treatment sequencing and the combination of virus and ICI therapy was unable to improve on the efficacy of ICI alone. One possible interpretation of these data is that a highly immuno-dominant anti-viral CD8⁺ T cell response induced by VSV-IFNß out competes, or interferes with, the weak, immuno-subdominant, slow developing, anti-PD-L1-sensitive anti-HCC_TAA T cell response. Using CyTOF analysis, Fig. 3 showed that VSV-IFNß treatment did indeed generate a highly significant expansion of one, or a few, dominant anti-viral effector CD8⁺ T cell populations with the concomitant relative disappearance of those putative anti-tumor T cell populations which are the likely target of anti-PD-L1 treatment. The immune profile of the virally induced CD8⁺ T cell populations matches that of anti-viral effector CD8⁺ T cells, as granzyme B characterizes antiviral CD8⁺ T cell responses^{49, 50}. Other markers also are consistent with this population being an anti-viral effector CD8 population. IL-7R has been shown to be consistently overexpressed on antigen-specific effector CD8⁺ T cells during viral infections⁵¹, while Bat3 promotes immune cell proliferation and cytokine production in persistent viral infections by promoting a T helper type 1 response^{52, 53}. Taken together, these data strongly argue that the abolition of therapeutic benefit achieved with ICI alone by addition of virus to ICI therapy was due to a rapid induction/expansion of anti-viral effector CD8 cells at the expense of anti-tumor, PD-1 expressing terminal effector CD8⁺ T cells. Moreover, the repolarization of the effector response of CD8⁺ T cells away from anti-HCC_TAA CD8⁺ T cells towards a population of anti-viral effector CD8⁺ T cells suggest that inappropriately timed anti-PD-L1 ICI could actually lead to in vivo enhancement of the immunodominant anti-VSV T cell response over the immuno-subdominant anti-HCC_TAA T cell response – expanding anti-viral T cells and not anti-HCC_TAA T cells. An implication of these data is that early, prolonged ICI may allow for strengthening/expansion of the immuno-subdominant anti-HCC_TAA T cell response with time such that a later immuno-dominant anti-viral T cell response is less able to out-compete it. However, the data of Fig. 2E suggest that even late virus treatment has a dominant negative effect on the anti-tumor T cell response.

Our previous studies have shown that inclusion of a tumor antigen within VSV induces CD8⁺ T cell dependent anti-tumor therapy directed specifically against the virally encoded antigen as a result of the potent immune stimulating adjuvant properties of infection with VSV^{19, 34, 35, 36}. Therefore, we hypothesized that it would be possible to amalgamate the potent anti-viral response with an anti-tumor T cell response by expressing immunologically relevant tumor associated antigens from within the virus. As a model system, we incorporated the OVALBUMIN antigen into the Sleeping Beauty ß-catenin/hMet system such that tumors would express the OVA protein. ß-catenin/hMet/OVA tumors generated spontaneous anti-HCC_TAA(OVA) endogenous T cells in both spleen and tumor and addition of anti-PD-L1 ICI expanded these CD8⁺ T cell responses (Fig. 4). By encoding the putative HCC_TAA(OVA) within oncolytic VSV-ova highly significant numbers of anti HCC_TAA(OVA) T cells were generated in vivo even in the absence of ICI. In addition, those anti-HCC_TAA(OVA) CD8⁺ T cell responses were substrates for expansion by anti-PD-L1 ICI therapy. Moreover, in contrast to the ablation of the anti-HCC_TAA CD8⁺ T cell responses by the addition of VSV virotherapy observed in Figs. 2&3, combined treatment with virus_(TAA) and ICI was able to expand the anti-HCC_TAA CD8⁺ T cell response preferentially over at least one component of the immuno-dominant anti-viral response (Fig. 4). Nevertheless, in this system, OVA represents a highly immunogenic, non-self, non-tolerized HCC_TAA and does not reflect the situation in which HCC_TAA are likely to be highly immuno-subdominant with low numbers of precursor T cells available to recognize and reject them (Fig. 3). Therefore, using RNAseq analysis of Sleeping Beauty tumors recovered from untreated mice, we identified three separate antigens predicted to be strong MHC Class I binders and, therefore, potential T cell targets when over-expressed in tumors. Of these three candidates, only one, Lcn2, generated detectable Th1-like T cell responses following expression from VSV (Fig. 5). Treatment of SB-HCC tumors with VSV-IFNß-Lcn2 VSV was still not able to enhance therapy compared to anti-PD-L1 alone, although the presence of Lcn2 did prevent the inhibition of the effects of anti-PD-L1 alone (Fig. 6).

Cumulatively, these data are consistent with a model in which the immuno-dominant anti-VSV CD8⁺ T cell response ablates the weaker, immuno-subdominant anti-HCC_TAA CD8⁺ T cell response causing loss of the ICI therapy alone when ICI was combined with VSV-IFNß (Figs. 2&3). However, when at least one potentially immunogenic HCC_TAA, such as Lcn2, was added to VSV-IFNß + anti-PD-L1, early treatment with anti-PD-L1 ICI allowed for expansion of potentially tumor reactive CD8⁺ T cells (Cluster 17 in Fig. 3), the anti-Lcn2 component of which could then be boosted by late vaccination with VSV-IFNß-Lcn2. In contrast, late VSV-IFNß with no additional HCC_TAA simply activates a rapid new anti-viral CD8⁺ T cell population, which replaces/outcompetes the developing, anti-PD-L1-expanded HCC_TAA-specific CD8⁺ T cells.

In this model, an endogenous anti-HCC_TAA CD8⁺ T cell response against multiple (weak) HCC_TAA, expanded by early ICI treatment, would be further boosted by VSV-HCC_TAA vaccination. If this model were true, we predicted that by adding multiple further HCC_TAA to the vaccinating VSV-IFNß virus, an increased number of anti-HCC_TAA T cells could be boosted by late VSV-IFNß-HCC_TAA vaccination. In the absence of effective models to predict the nature of multiple possible HCC_TAA (Fig. 5), we hypothesized that cloning a cDNA library from Sleeping Beauty HCC tumor lines into VSV would allow for the highly immunogenic display of multiple HCC_TAA with the advantage that the HCC_TAA would not need to be molecularly identified beforehand to be effective^{35, 36}. Therefore, cDNA from three separate Sleeping Beauty explants mixed at a 1:1:1 ratio was cloned into the VSV-IFNß virus to give a viral stock of VSV-IFNß-SB-HCC1,2,3cDNA (Fig. 7A). By expressing a multitude of putative, uncharacterized, tumor antigens within VSV, for the first time, a combination of VSV and anti-PD-L1 ICI generated significantly improved therapy over anti-PD-L1 ICI treatment alone with 100% of mice surviving to day 150 (Fig. 7C). Anti-tumor therapy against HCC was not observed if the VSV-cDNA was derived from melanoma instead of HCC tumors, was dependent upon CD8⁺ but less so on CD4⁺ T cells and was associated with a Th17 phenotype of T cell activity (Fig. 7). Interestingly, treatment with VSV-IFNß-SB-HCC1,2,3cDNA alone (no anti-PD-L1) was also very effective at generating long term cures. These data suggest that VSV-mediated display of multiple antigens was sufficient of itself to boost slow developing anti HCC_TAA CD8⁺ T cell responses even in the absence of expansion by prior anti-PD-L1 treatment.

A major advantage of using a tumor-derived cDNA library as the source of multiple HCC_TAA is that there is no need to define the identity of multiple TAA from each patient. On the other hand, a concern about the use of a cDNA library is that tolerance may be broken to multiple normal liver associated antigens - although we did not observe any toxicity associated with autoimmune liver disease in the mice cured of their tumors when treated with VSV-IFNß-SB-HCC1,2,3cDNA either with, or without, anti-PD-L1. We believe that by constructing the cDNA library from tumor tissue the representation of normal self-antigens against which any non-tolerized T cells exist in vivo may be reduced relative to tumor associated markers which can form immunological targets. Further studies are underway to assess the full range of toxicities associated with VSV-IFNß-SB-HCC1,2,3cDNA-mediated liver damage.

Our results here provide a cautionary message for the use of highly immunogenic viruses as tumor-specific immune-therapeutics. We show here that oncolytic virotherapy can induce anti-viral T cell responses which can actively inhibit, or obscure, pre-existing weak anti-tumor T cell responses leading, potentially, to decreased anti-tumor therapy with ICI which targets that immuno-subdominant anti-tumor T cell population. However, by ensuring that at least a proportion of the anti-viral T cell response can also act to boost an anti-tumor response by encoding multiple relevant tumor antigens within the virus the CD8⁺ T cell response associated with oncolytic virotherapy can be purposed for very effective tumor clearance. In the model system used here, oncolytic virotherapy was used in the context of a poorly immunogenic developing tumor against which an endogenous T cell response existed and which could be enhanced by ICI therapy. We are currently investigating the alternative situation in which a developing tumor is completely non- immunogenic – that is it does not raise any anti-tumor T cell response. In such a case it may be that oncolysis by the virus helps to generate a previously non-existent anti-tumor T cell response upon which subsequent ICI treatment may be able to work. Our findings can, therefore, inform the rational sequencing of a combinatorial use of oncolytic virotherapy and ICI therapy such that viral oncolysis is used productively to generate anti-tumor T cell populations upon which immune checkpoint blockade can effectively work.

In summary, our data are consistent with a model in which a poorly immunogenic tumor is partially sensitive to ICI therapy through the selective re-invigoration/expansion of anti-tumor CD8⁺ T cell populations presumably recognizing weak, sub-dominant antigens. In these circumstances, treatment with an OV encoding a set of highly immunogenic, immuno-dominant viral antigens induces populations of anti-viral CD8⁺ T cells which overwhelm the pre-existing anti-tumor CD8⁺ T cell populations and which, therefore, significantly inhibit therapy associated with ICI alone. However, by expressing multiple TAA within the OV, the immune adjuvant properties of the virus become advantageous rather than detrimental by promoting boosting of the pre-existing anti-TAA CD8⁺ T cell populations in vivo through induction of both Th17 and Th1 component mechanisms. In this way, the anti-viral T cell response is chimerized to become, at least in part, an anti-tumor T cell response leading to a positive interaction between ICI therapy and oncolytic virotherapy.

METHODS

Pathology.

Livers were fixed in 10% formalin and stained with hematoxylin and eosin by the Mayo Clinic Histology Core Facility. Immunofluorescence staining was as described previously⁵⁴. PD-L1 antibody was purchased from BioX-cell, clone 10F.9G2.

Cell lines and viruses.

VSV expressing murine IFN-ß (VSV-IFN-ß), ovalbumin (VSV-OVA), Lcn2 (VSV-IFNß-Lcn2), Lect2 (VSV-IFNß-Lect2), SMAGP (VSV-IFNß-SMAGP), or green fluorescent protein (VSV-GFP) were rescued from the pXN2 cDNA plasmid using the established reverse genetics system in BHK cells as described previously^{21, 22, 34}. In brief, BHK cells were infected with MVA-T7 at an MOI of 1. Cells were incubated at 37°C and 5% CO₂. After 1 h, cells were transfected with pVSV-XN2 genomic VSV plasmid (10 μg), pBluescript (pBS)-encoding VSV-N (3 μg), pBS-encoding VSV-P (5 μg), and pBS-encoding VSV L proteins (1 μg) using Fugene6 according to the manufacturer’s recommendations. Cells were incubated at 37°C and 5% CO2 for 48 h. After 48 h, supernatant was collected and clarified by passing through a 0.2-μm filter. All transgenes were inserted between viral G and L genes using the XhoI and NheI restriction sites with the murine IFN-ß gene cloned between the viral M and G genes. Virus titers were determined by plaque assay on BHK cells. VSV-cDNA libraries were generated as described previously^{35, 36}. Briefly, cDNA from two human melanoma cell lines, Mel624 and Mel888, (ASMEL library)³⁶ or from three murine Sleeping Beauty HCC tumor-bearing livers, (SB HCC 1,2&3 Library), was pooled, cloned (Clone Miner cDNA Construction kit) (Invitrogen) and amplified by PCR. The PCR-amplified cDNA molecules were size fractionated to below 4 kbp for ligation into the parental VSV genomic plasmid pVSV-XN2⁵⁵ between the G and L genes since lower sized cDNA inserts were associated with both higher viral titers and lower proportions of Defective Interfering particles. The complexity of the ASMEL and SB HCC 1,2&3 cDNA libraries cloned into the VSV backbone plasmid between the Xho1 and Nhe1 sites was 7.0 × 10^{6 36} and 4.1 × 10⁶ colony forming units respectively (at dilutions of 10^{− 6} and 10^{− 5} there were 8 and 74 colonies with the SB HCC 1,2&3 Library). For the SB-HCC1,2,3 Library, of 30 colonies picked at random, 1 had no insert, 12 had an insert of less than 0.5 kbp and 17 had inserts between 0.5kbp and 4kbp. Virus was generated from BHK cells by co-transfection of pVSV-XN2-cDNA library DNA along with plasmids encoding viral genes as described⁵⁵. Virus was expanded by a single round of infection of BHK cells and purified by sucrose gradient centrifugation.

Mice.

All experiments utilized 6 to 8-week-old male and female C57Bl/6 mice (stock 000664) obtained from The Jackson Laboratory with the exception of Fig. 1H, which utilized FVB mice (stock 001800) obtained from The Jackson Laboratory. All animals were maintained in a specific pathogen-free BSL2 biohazard facility. Experimental mice were co-housed and exposed to a 12:12-h light-dark cycle with unrestricted access to water and food. The ambient temperature was restricted to 68°F to 79°F and the room humidity ranged from 30–70%. All animal studies were conducted in accordance with and approved by the Institutional Animal Care and Use Committee at Mayo Clinic.

In vivoexperiments.

All in vivo studies were approved by the Institutional Animal Care and Use Committee at Mayo Clinic. Our study uses a murine model of human hepatocellular carcinoma (HCC) developed by Dr. Satdarshan Monga in which human Met (hMet) and an activating ß-catenin mutant drive tumorigenesis³³. Specifically, hMet and ß-catenin point mutants, in this case S45Y, are co-expressed in hepatocytes utilizing the Sleeping Beauty (SB) transposable element system⁵⁶. C57Bl6 mice were injected with pT3-EF5a-hMet-V5 (20μg), pT3-EF5a- Human ß-catenin with S45Y mutation (20μg), and pCMV/SB (the hyperactive sleeping beauty expression vector) 1.6μg (25:1 ratio of oncogenes to SB) diluted in 2ml of normal saline (0.9% NaCl), filtered through 0.22 mm filter and injected into the lateral tail vein in 5 to 7 seconds. For HCC-OVA tumors, an OVA-expressing plasmid, pT3-EF5a-OVA-V5 (20μg), was added to the pT3-EF5a-hMet-V5 (20μg), pT3-EF5a- Human ß catenin with S45Y mutation (20μg), and pCMV/SB 1.6μg plasmids diluted in 2ml of normal saline prior to hydrodynamic injection.

Following hydrodynamic injection, animals were treated as described in the text. Specific antibodies used include anti-mouse PD-L1 (BioX-Cell, clone 10F.9G2), CD8α (BioX-Cell, clone 2.43), CD4 (BioX-Cell, clone GK1.5), NK1.1 (BioX-Cell, clone PK136), and IgG2A isotype (BioX-Cell, clone 2A3). All treatments were given i.v. by tail vein unless otherwise specified.

Immune Cell Isolation.

Livers or spleens from C57Bl/6 mice were immediately excised upon euthanasia. Single-cell suspensions were generated in vitro via mechanical dissociation. Red blood cells were lysed by resuspension in ammonium-chloride-potassium lysis buffer and incubating at room temperature for 2 min.

Flow Cytometry/CyTOF analysis.

Tumor-infiltrating lymphocytes were isolated as above in Flow Cytometry staining buffer. Immune cells where analyzed using FlowJo 10. Mouse cells were stained with fluorochrome-conjugated antibodies against combinations of the following antigens: CD3 (Biolegend clone 145–2C11), CD8α (Biolegend clone 53 − 6.7), CD4 (Biolegend clone GK1.5), PD1 (Biolegend clone RMP1–30), TIM3 (Biolegend clone RMT3–23), CD11c (BD Biosciences clone HL3), I-A/I-E (MHCII) (Biolegend M5/114.15.2), CD86 (Biolegend #105037, clone GL-1), and fixable live dead viability dye (Zombie NIR). Cells were stained with the H-2Kb VSV NP_52–59 RGYVYQGL (Brilliant Violet 421–labeled) tetramer at a dilution of 1:500 or the H-2Kb chicken ova_257–264 SIINFEKL (APC-labeled) tetramer at a dilution of 1:150, which were obtained from the National Institutes of Health Tetramer Core Facility.

CyTOF analysis was conducted through the Mayo Clinic CyTOF Core using a 44-label combined T cell panel. CyTOF analysis was conducted using RStudio and Cytofkit, with individual populations isolated using Rphenograph and FlowSOM tSNE analyses for 22 and 9 populations, respectively.

ELISA.

Following isolation, splenocytes were resuspended at a concentration of 1 × 10⁶ cells/mL in Iscove’s modified Dulbecco’s medium (Gibco) supplemented with 5% FBS, 1% penicillin-streptomycin, and 40 μmol/L 2-mercaptoethanol. Splenocytes were re-stimulated with 10⁵ live tumor cells which were a 1:1:1 mix of three different sleeping beauty tumor cell lines recovered from untreated mice at euthanasia (between 75 and 100 days post hydrodynamic injection). Supernatants were collected and assayed for IL-17 and IFNγ by enzyme-linked immunosorbent assay (ELISA) as per the manufacturer’s instructions (Mouse IL-17 or Mouse IFN-γ ELISA Kit, OptEIA, BD Biosciences).

RNA-Sequencing Analysis of Gene Expression.

hMet + S45Y ß-Catenin Sleeping Beauty transposon system liver tumor-induced mice sacrificed on day 18 with livers harvested for RNA extraction (Rneasy, Qiagen). RNA samples were then subjected to RNA-seq analysis at the Genome Analysis Core, Mayo Clinic. The 10 most-expressed genes identified and full-length sequences were filtered through NET MHC 2.0 binding affinity algorithm to identify octamer or nonamer peptides whose binding affinity for H2K^b or H2K^d was below a threshold of 500 nM, and whose corresponding wild-type peptides had a binding affinity to the same molecules above 500 nM. A list of 10 candidates was further refined using the EMBL-EBI Expression Atlas for expression in liver tissue to identify 4 high affinity peptides. This methodology is discussed in detail in our previous work⁵⁷.

PCR Validation of ASMEL and SB-HCC1,2&3 Libraries.

RNA was prepared from the equivalent of 10⁸ pfu of the VSV-based ASMEL³⁶ or SB-HCC1,2&3 Library with the QIAGEN RNA extraction kit. 1μg total RNA was reverse transcribed in a 20μl volume using oligo-(dT) as a primer. A cDNA equivalent of 1ng RNA was amplified by PCR with gene specific primers to the HCC-specific Lcn2, Lect2, and Smagp, or melanoma specific TYRP1 and GP100 genes.

Statistical analysis.

All analysis was performed within GraphPad Prism software (GraphPad). Multiple comparisons were analyzed using one- or two-way analysis of variances with a Tukey’s post hoc multi-comparisons test. Survival data were assessed using a log rank Mantel-Cox test. Data are expressed as group mean ± SD unless otherwise stated.

DATA AVAILABILITY STATEMENT

Data not published within this article will be made available by request from any qualified investigator.