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.

Keywords: Antigens; B7-H1 Antigen; Carcinoma; Hepatocellular (HCC); Immune Checkpoint Inhibitors; Neoplasm; Oncolytic virotherapy; Transposases; Vesicular stomatitis Indiana virus (VSV).

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.

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.

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.

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.

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.

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.

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 ASMEL and SB HCC 1,2&3 Libraries was validated using PCR from the equivalent of 10⁸ pfu of the VSV-ASMEL 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) 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.