artARENA immunization induces a robust innate immune response and strong antigen-specific effector T-cell responses

Both artLCMV-E7E6 alone and artPICV-E7E6/artLCMV-E7E6 alternating prime-boost vaccination regimens have previously shown immunogenicity and therapeutic efficacy in preclinical mouse models.16 17 Interestingly, the order of administration influenced the outcome, with artPICV prime followed by artLCMV boost outperforming the inverse sequence.17 Therefore, we sought to analyze the immune response elicited by artPICV in comparison to artLCMV and uncover specific characteristics of each vector, that could ultimately inform about optimal regimens and promising combinational therapies. For this, naïve C57BL/6 mice were intravenously administered a single dose of either artLCMV-E7E6 or artPICV-E7E6, and the immune response was analyzed 24 hours and 7 days later (figure 1A).

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Figure 1

artARENA immunization induces a robust innate immune response, as well as strong antigen-specific effector T cell responses. (A) Experimental design (created with BioRender.com). C57BL/6 mice were immunized intravenously with 1×10⁵ (B, C) or with 1×10⁶ (D–H) RCV FFU of artLCMV-E7E6 or artPICV-E7E6 vectors. Control animals received formulation buffer. Blood and/or spleen were collected 24 hours and/or 7 days postimmunization. (B) Serum cytokines and chemokines measured by MSD multiplex assay. Log2 fold change calculated relative to control animals (n=5–7/group, t-test). (C) Immune cell activation measured by flow cytometry of splenocytes (mean±SD, n=5–7/group, one-way ANOVA and Tukey’s multiple comparisons test). (D–G) Characterization of E7-specific CD8⁺ T cell response, measured by flow cytometry of splenocytes, 7 days postimmunization (n=5/group). (D) Percentage of E7-specific CD8⁺ T cells. Boxplots show median±IQR, min/max, and individual mice (unpaired t-test). (E) Flow cytometry histograms showing PD-1 expression. (F) Expression of CD127 and KLRG1 on E7-specific CD8⁺ T cells (mean±SD; two-way ANOVA and Tukey’s multiple comparisons test). For control samples, CD8⁺ T cells were used. (G) Geometric mean fluorescence intensity (gMFI) of CD127 on E7-specific CD8⁺ T cells (mean±SD, unpaired t-test). (H) Polyfunctional CD8⁺ T cell response in immunized mice. Splenocytes harvested 7 days postimmunization were ex vivo restimulated with E6 or E7 peptides. Total percentage of IFN-y-expressing CD8⁺ T cells (left). Percentage of CD8⁺ T cells expressing different combinations of IFN-γ, TNF-α, and CD107a (right). Boxplots show median±IQR, min/max, and individual mice (n=5/group, one-way (left) or two-way (right) ANOVA and Dunnett’s multiple comparisons test). For each test: ns=non-significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANOVA, analysis of variance; artLCMV, arenavirus vectors based on lymphocytic choriomeningitis virus; artPICV, arenavirus vectors based on Pichinde virus.

Single immunization with either vector led to an increase in key systemic proinflammatory cytokines and chemokines 24 hours after treatment (figure 1B). Substantial levels of type I IFN (IFN-α, IFN-β) and robust induction of IFN-γ were detected in the serum (figure 1B). Increased levels of myeloid and lymphoid cell chemoattractant chemokines CCL2 and CXCL10 and other proinflammatory mediators further marked the increased inflammation shortly after treatment (figure 1B). Flow cytometry analysis of splenocytes revealed a concomitant increase in the activation of different subsets of myeloid cells as well as plasmacytoid dendritic cells (pDCs), as measured by CD86 expression (figure 1C, left). Of note, conventional DC1 (cDC1), key players in antitumor immunity,28 29 showed the highest increase in numbers and activation, with around 90% of these cells expressing CD86 following treatment with both vectors (figure 1C, online supplemental figure 1A). Lymphocyte populations also showed increased activation at this time point (figure 1C, right). Expression of early activation marker CD6930 was seen in all analyzed populations, including CD4⁺, CD8⁺ T, NK, NKT, and B cells (figure 1C, right). On the other hand, expression of granzyme B (GzmB), a potent cytotoxicity marker,31 was robustly upregulated in NK cells, and to a lower extent also in NKT and CD8⁺ T cells (figure 1C, right). Although a strong systemic immune activation was observed following immunization with either vector, significantly lower percentages of activated cells were detected following artPICV administration (figure 1B,C), possibly indicating a slower or overall lower induction of inflammation by this vector.

To further dissect the immunogenicity of each vector, de novo priming of antigen-specific T cells was analyzed 7 days post-treatment. Flow cytometry analysis of splenocytes revealed a significant increase in E7-specific T cells following administration of either vector, reaching in average 2%–3% of CD8⁺ T cells present (figure 1D, online supplemental figure 1B). These cells had increased expression of PD-1 (figure 1E), denoting their activated phenotype.32 While in control animals CD8⁺ T cells have a naïve KLRG1⁻ CD127⁺ phenotype, a large proportion (up to 90%) of treatment-induced E7-specific CD8⁺ T cells displayed KLGR1⁺ CD127⁻ and KLGR1⁻ CD127⁻ effector phenotypes (figure 1F, online supplemental figure 1C). A small but distinct population (3%–16%) expressed CD127 (figure 1F), a marker linked to the development of memory T cells.33 Interestingly, the CD127⁺ KLGR1⁻ T cell population was significantly larger following artPICV administration (figure 1F), and their CD127 expression levels per cell were also significantly higher when compared with artLCMV treatment-induced cells (figure 1G).

Finally, effector function was analyzed by measuring CD8⁺ T cell cytokines and CD107a expression after ex vivo restimulation with E6 and E7 peptides of splenocytes from treated animals (figure 1H). At this time point, E6-specific and E7-specific CD8⁺ T cells from animals treated with both vectors mainly coexpressed IFN-γ and CD107a, with or without TNF-α, and only a minor population expressed IFN-γ alone (figure 1H). E7-specific effector T cells were more abundant overall (figure 1H).

These data indicate that artARENA immunization quickly and efficiently induces systemic activation of innate and adaptive immune responses, which translate into robust antigen-specific T-cell responses. Seven days post-treatment, the majority of E6-specific and E7-specific CD8⁺ T cells were differentiated into an effector phenotype with polyfunctional cytokine expression, while artPICV immunization may have increased potential to develop long-lived antigen-specific memory T cells.

Human APCs are the preferential target for artARENA vectors, leading to T-cell activation through direct presentation of the encoded antigens on MHC class I molecules

While direct infection of APCs is known to mediate the strong immunogenicity triggered by artLCMV vectors,14 the infection pattern of arenavirus-based vectors, especially artPICV, toward different human myeloid cell subsets has not been fully investigated. Therefore, we sought to analyze the infection profile of both artLCMV and artPICV by infecting human PBMCs with vectors encoding GFP as a reporter gene. PBMCs were harvested 20 hours postinfection, and different myeloid populations were identified and analyzed by flow cytometry (figure 2A). All populations showed GFP expression after infection with either vector, indicating a similar infection profile (figure 2B). Infection rates were comparable and ranged from 50% to 80% for each cell type, with cDC1 infected at the highest rate (figure 2B). APC activation was analyzed by flow cytometry analysis of CD86, with all populations showing increased CD86 expression postinfection (figure 2C), providing strong costimulatory signals for T cells. Of note, artARENA vectors’ infection is specific to myeloid cells and pDCs, as little to no infection was observed in lymphocytes (data not shown).

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Figure 2

Human APCs are the preferential target for artARENA vectors and present encoded antigens on MHC class I, leading to T cell activation. (A–C) PBMCs from healthy donors were infected with artLCMV-GFP or artPICV-GFP vectors (MOI5) and analyzed 20 hours postinfection. (A) Flow cytometry gating strategy for obtaining the different human APC subsets. (B) GFP and (C) CD86 expression (mean fluorescence intensity) in human APC subsets analyzed by flow cytometry. Values indicate the percentage of GFP⁺ cells. Plots are representative of two replicates. Error bars, mean±SD of duplicate wells, one-way ANOVA. (D) Flow cytometry analysis of HLA-A2 expression on the human cell line ARPE-19 (top) stained with HLA-A2 antibody (gray) or isotype control (open histogram). Flow cytometry analysis of GFP expression in ARPE-19 cells (bottom) 24 hours postinfection with artLCMV-GFP or artPICV-GFP (MOI1). (E) artLCMV-E7E6 or artLCMV-GFP and artPICV-E6E7 or artPICV-GFP infected ARPE-19 cells (MOI2) were cocultured for 6 hours with Jurkat IL-2/NanoLuc reporter cells transgenic for HLA-A2-restricted TCRs against HPV E6 (TIHDIILECV)- and HPV E7 (YMLDLQPET)-derived epitopes. ELAGIGILTV (MART-1) was used as an irrelevant peptide control and TIH and YML peptide-loaded ARPE-19 cells were used as positive controls. Error bars, mean±SD of duplicate wells, one-way ANOVA and Dunnett’s multiple comparisons test. For each test: ns=non-significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANOVA, analysis of variance; APC, antigen-presenting cells; artLCMV, arenavirus vectors based on lymphocytic choriomeningitis virus; artPICV, arenavirus vectors based on Pichinde virus; PBMCs, peripheral blood mononuclear cells.

The capacity of infected cells to prime and activate T cells is crucial for the efficacy of the artARENA platform and for the development of antitumor immunity. Therefore, to investigate antigen expression and presentation in human cells following infection, we used ARPE-19 cells, a human HLA-A*02:01⁺ cell line amenable to artARENA infection (figure 2D). artLCMV-E7E6 or artPICV-E7E6 infected ARPE-19 cells were cocultured with E6-specific or E7-specific Jurkat reporter cells. These express an HLA-A*02:01-restricted E6-specific or E7-specific TCR and were engineered to express Nanoluc luciferase under the control of the interleukin-2 (IL-2) promoter. Six hours after coculture, we measured high levels of luciferase, indicating robust activation of the reporter cells (figure 2E).

Our results show that both artLCMV and artPICV vectors infect and activate human APC subsets. Moreover, encoded antigens are expressed, processed, and presented by human cells, inducing T-cell activation.

Therapeutic immunization with artARENA vectors mediates potent antitumor activity and control of bilateral tumors

To analyze the antitumor efficacy of artARENA vectors, C57BL/6 mice subcutaneously grafted with 1×10⁵ TC-1 cells (HPV16 E7/E6-expressing syngeneic tumor model) were intravenously or intratumorally administered 1×10⁵ RCV FFU of artLCMV-E7E6 or artPICV-E7E6 when tumors reached ~100 mm³ (figure 3A). Monitoring of tumor size showed that artARENA immunization suppressed tumor growth in all four treatment groups (figure 3B, online supplemental figure 2A). Further, survival was significantly prolonged, increasing 1.8-fold on average in this model (figure 3C). The observed antitumor effect was dependent on HPV-specific immunity induced by the artLCMV-E7E6 or artPICV-E7E6 vectors, as only a minor and transient delay in tumor growth and no survival benefit was observed in animals treated with vectors encoding an irrelevant protein (data not shown).16 34

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Figure 3

Therapeutic immunization with artARENA vectors mediates potent antitumor activity and control of bilateral tumors. (A) Experimental design (created with BioRender.com). TC-1 tumor-bearing C57BL/6 mice were immunized intravenously or intratumorally with 1×10⁵ RCV FFU of artLCMV-E7E6 or artPICV-E7E6 vectors when tumors reached a volume of ~100 mm³. Control animals received formulation buffer. Blood was collected 6 and 15 days postimmunization and cells were analyzed by flow cytometry. (B) Tumor volume monitored over time (mean±SEM, n=5/group). (C) Kaplan-Meier survival curves, with indicated median survival and long-term survivors (**p<0.01, Log-rank test). (D) Percentage of E7-specific CD8⁺ T cells. Boxplots show median±IQR, min/max, and individual mice. (E) Expression of CD127 and KLRG1 on E7-specific CD8⁺ T cells (mean±SD). For control animals, total CD8⁺ T cells were used. (F) Experimental design. TC-1 cells were grafted subcutaneously in both flanks of C57BL/6 mice. When tumors reached a volume of ~100 mm³, mice were immunized with 1×10⁵ RCV FFU of artLCMV-E7E6 or artPICV-E7E6 vectors. Immunization was done intravenously or intratumorally (on the right flank tumor only). Control animals received formulation buffer. (G) Tumor volume monitored over time (mean±SEM, n=10/group, data are shown until the time point at which more than 50% of the animals in the group were euthanized). Tumor growth data were analyzed by fitting a linear mixed model (fixed effect terms: treatment, day, and treatment:day interaction; subject was specified as a random factor nested within treatment). Data were log-transformed prior to analysis and a separate model was fitted for the pretreatment and post-treatment periods). From the model, the difference in mean effects between treatments was calculated and assessed by Tukey’s test. (H) Kaplan-Meier survival curves, with median survival indicated (ns=non-significant, log-rank test). artLCMV, arenavirus vectors based on lymphocytic choriomeningitis virus; artPICV, arenavirus vectors based on Pichinde virus.

Expansion of E7-specific CD8⁺ T cells was observed in the blood of immunized mice, 6 days post-treatment (figure 3D). While all treatment groups elicited comparable frequencies of E7-specific CD8⁺ T cells (ranging from 1% to 13%), two animals treated with artLCMV-E7E6 intravenously and intratumorally showed frequencies of 25% and 43%, respectively, which were maintained at d15 (figure 3D). Most treatment-induced E7-specific CD8⁺ T cells exhibited a CD127⁻ effector phenotype (figure 3E), likely contributing to the tumor control observed until d25 (figure 3B). At day 15, we observed a contraction of the early effector cell population, and a relative increase of both CD127⁺ KLRG1⁺ and CD127⁺ KLRG1⁻ cells (figure 3E), potentially indicating the development of long-term antitumor immunity.33 35 The increased frequency of memory CD8⁺ T cells was more pronounced following artPICV treatment via either administration route, whereas for artLCMV this increase was more pronounced for the intratumoral route (figure 3E).

Since combination therapies will likely be required for effective long-term tumor control, and improvement in response rates for current SoC treatments would have a greater and possibly more immediate impact on HPV-driven HNSCC patients, we sought to understand if cisplatin combination could be leveraged to increase the efficacy of artARENA vectors. As high doses of cisplatin were shown to have toxic side effects in the clinic,36 we investigated a lower dose for beneficial therapeutic effects. Although cisplatin treatment alone at this dose had a modest effect on tumor growth and median survival, we observed an improvement in the therapeutic responses when cisplatin was administered in combination with each vector (online supplemental figure 2B,C).

To explore the potential antitumor efficacy of each vector towards metastatic tumors, we compared local (intratumoral) and systemic (intravenous) vector administration in mice bearing TC-1 tumors in both the left and right flanks (figure 3F). Monitoring of tumor volume showed that both intratumoral administration into the right tumor and intravenous administration controlled the tumor growth in both flanks (figure 3G, online supplemental figure 2D). The rate of tumor control between flanks was comparable even in the intratumorally treated animals (figure 3G), and there were no significant differences in survival (figure 3H), suggesting that both regimens promote a systemic immune response. To analyze the biodistribution and transgene expression following either administration route, artARENA vectors encoding the Nanoluc luciferase were intravenously or intratumorally injected into tumor-bearing mice, and the bioluminescence was measured in vivo over time (online supplemental figure 2E,F), and ex vivo in different organs at 24 hours postimmunization (online supplemental figure 2G,H). We observed a similar infection pattern in multiple organs (online supplemental figure 2E–H). The spread of the vectors was similar for both administration routes and possibly explained the induction of systemic immunity in the intratumoral setting. Nanoluc expression was stable or even increased until the end of the observation period (online supplemental figure 2E,F). The in vivo bioluminescence was higher following artLCMV treatment.

Overall, the strong immunogenicity elicited by artARENA vectors translates into potent antitumor activity, with the potential for controlling tumor growth and prolonging survival in the presence of metastatic lesions, as monotherapy or in combination with chemotherapy.

artARENA immunization induces T-cell infiltration in tumors

CD8⁺ T-cell infiltration is prognostic for good clinical outcome and increased survival in several human cancers.37 38 Therefore, we analyzed the magnitude and phenotype of T-cell infiltration following treatment. For this, TC-1 tumor-bearing C57BL/6 mice were intravenously or intratumorally treated with either vector, tumors were harvested on days 7, 14, and 21 post-treatment, and tumor-infiltrating lymphocytes were analyzed by flow cytometry and IHC (figure 4A).

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Figure 4

artARENA immunization induces T cell infiltration in tumors. (A) Experimental design (created with BioRender.com). TC-1 tumor-bearing C57BL/6 mice were immunized intravenously or intratumorally with 1×10⁵ RCV FFU of artLCMV-E7E6 or artPICV-E7E6 vectors when tumors reached a volume of ~100 mm³. Control animals received formulation buffer. Tumors were harvested on days 7, 14, and 21 postimmunization. (B–E) Tumors were dissociated and cells were analyzed by flow cytometry (n=3–5/group, mean±SD). (B) Absolute numbers of CD8⁺ T cells, calculated per gram of tissue. (C) CD8⁺/Treg ratio, calculated by dividing the total number of CD8⁺ by the total number of CD4⁺CD25⁺FoxP3⁺ T cells. (D) Percentage of ICOS⁺ T cells (within CD8⁺T cell population) in TILs, spleen, and lymph node. (E) Percentage of PD-1⁺ subsets (low, intermediate, and high expression) in ICOS⁺CD8⁺ T cells. (F) Tumor tissue was subjected to immunohistochemical analysis. Representative whole staining scans (top, scale bar=1000 µm), and inserts (bottom, scale bar=50 µm) of respective conditions are shown (scans representative of 1–5 animals/group). Statistical analysis: two-way ANOVA and Dunnett’s multiple comparisons test, ns=non-significant, *p<0.05, **p<0.01, ***p<0.001,. ANOVA, analysis of variance.

Flow cytometry analysis revealed robust tumor infiltration of CD8⁺ T cells (figure 4B). Although some variability was observed, all artARENA-treated groups showed a comparable increase in infiltrating CD8⁺ T cells (around 300×10⁴ cells/g of tumor) 7 days postimmunization and promoted proinflammatory ratios of CD8⁺/Tregs within the TME (figure 4B,C). Around 60% of tumor-infiltrating CD8⁺ T cells expressed inducible T cell costimulator (ICOS), highlighting their activated effector phenotype (figure 4D).39 40 These cells were primarily accumulating at the tumor site, as low to no ICOS⁺ CD8⁺ T cells were detected in the spleen or lymph nodes of treated animals (figure 4D). However, T cell numbers tended to decrease over time, as fewer infiltrating CD8⁺ T cells were measured at days 14 and 21, along with a concomitant decrease in CD8⁺/Treg ratio, and increase in PD-1 expression (figure 4B,C and E). Cells expressing higher levels of PD-1 constituted most of the infiltrating CD8⁺ T cells by day 21 (figure 4E). Finally, IHC staining of tumor sections also showed robust infiltration of CD8⁺ T cells, penetrating the tumor mass (figure 4F). FoxP3⁺ T-cells were visible within the tumor mass at later time points (figure 4F). Total CD3⁺ and CD4⁺ T cell-infiltration can be seen in online supplemental figure 3A. Quantification of cells within the stained histological sections corroborated the flow cytometry results, with higher infiltration of CD8⁺ T cells at earlier time points, and gradually higher accumulation of Tregs at later time points (online supplemental figure 3B). In addition, quantification of Ki-67 expressing cells showed that these were reduced in vector-treated groups, 7 days post-treatment (online supplemental figure 3C).

These data show that both vectors triggered strong intratumoral infiltration of cytotoxic T cells; however, immunosuppression built up over time suggested that combination treatments might be required for adequate long-term tumor control.

Antitumor efficacy of artARENA is mediated by CD8⁺ T cells

To understand the relative contribution of the T cell and NK cell response in mediating tumor control, animals grafted with TC-1 cells were depleted of either CD4⁺, CD8⁺ T or NK cells, and tumor size was monitored following intravenous or intratumoral administration of either vector (figure 5A, online supplemental figure 4A). Depletion of CD8⁺ T cells abrogated the therapeutic effect from both vectors, suggesting a critical role for cytotoxic T cells in mediating tumor growth control (figure 5B,C). Depletion of CD4⁺ T cells had the opposite effect, leading to increased tumor growth control and to a moderate increase in survival for most treatment groups (figure 5B,C).

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Figure 5

Antitumor efficacy of artARENA is mediated by CD8⁺ T cells. (A) Experimental design (created with BioRender.com). TC-1 tumor-bearing C57BL/6 mice were immunized intravenously (B) or intratumorally (C) with 1×10⁵ RCV FFU of artLCMV-E7E6 or artPICV-E7E6 vectors when tumors reached a volume of ~100 mm³. Control animals received formulation buffer. Mice were depleted of CD8⁺, CD4⁺ T cells or NK cells by the addition of neutralizing antibodies three times over the course of the study. (B, C) Tumor volume monitored over time (left, mean±SEM, n=7/group, data are shown until the time point at which more than 50% of the animals in the group were euthanized). Kaplan-Meier survival curves, with indicated median survival and long-term survivors (right, *p<0.05, **p<0.01, ***p<0.001 Log-rank test). artLCMV, arenavirus vectors based on lymphocytic choriomeningitis virus; artPICV, arenavirus vectors based on Pichinde virus.

Unexpectedly, upon depletion of NK cells, the antitumor immune response was either not affected (intravenous groups) or further improved (intratumoral groups). This suggests that the NK cell response may be unfavorable in this model.

Overall, the antitumor effect of both artLCMV and artPICV vectors is mediated by CD8⁺ T cells, and CD4⁺ T cell-mediated immunosuppression may be partially impairing antitumor immunity following artARENA treatment.

artARENA treatment induces proinflammatory changes in the TME and reveals NKG2A blockade as a potential new target for combination

Having shown that the artARENA platform triggers T-cell infiltration, we sought to obtain a more comprehensive view of cancer and immune-related changes within the TME resulting from the two vectors and administration routes. For this, we performed NanoString nCounter PanCancer IO 360 analysis on tumor samples harvested 7 days post-treatment. This time point was chosen to analyze gene expression changes within the TME mediated by both, innate immune responses induced by the viral vectors as well as tumor antigen-specific T-cell responses.

Differential gene expression analysis identified a high number of genes upregulated in all treatment groups. Each sample’s gene expression profile was condensed into a set of pathway scores, whereas increasing scores correspond to mostly increasing expression (specifically, each pathway score has positive weights for at least half its genes). Pathway analysis revealed a deep rearrangement of the TME on administration of the vectors, but no distinct clustering of the different treatment groups (figure 6A,B). Pathways potentially promoting tumor growth including cell proliferation, autophagy, and DNA damage repair41 42 as well as TGF-ß signaling, which is known to potentiate an immunosuppressive TME,43 had a lower score in vector-treated groups. Conversely, pathways associated with immune activation scored higher (figure 6A). Enhanced activity of the myeloid and lymphoid compartments further underscored the increased immune infiltration observed previously (figure 4). Higher scores of costimulatory signaling, cytotoxicity, and antigen presentation indicated increased activation of the infiltrating immune cells. Additional pathways (figure 6A—other) with multiple described roles in the function of both tumor and immune cells were enhanced in treated samples; however, further studies are needed to better dissect their contribution to the artARENA-mediated antitumor response.

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Figure 6

artARENA treatment induces proinflammatory changes in the TME and identifies NKG2A blockade as a potential new target for combination. (A, B) Similar to previous experiments, TC-1 tumor-bearing C57BL/6 mice were immunized intravenously or intratumorally with 1×10⁵ RCV FFU of artLCMV-E7E6 or artPICV-E7E6 vectors when tumors reached a volume of ~100 mm³. Control animals received formulation buffer. 7 days postimmunization tumors were harvested, and gene expression was analyzed using the nCounter PanCancer IO 360 Panel (Nanostring Technologies). (A) Heatmap of pathway scores of the differentially expressed genes in each sample is shown. Each column represents one sample (one mouse). Scores were normalized via z-transformation. (B) Volcano plots displaying each gene’s -log10 adjusted p value and log2 fold change between buffer and indicated treatment groups. The top 16 genes upregulated genes across all groups are displayed in red and indicated in the table. (C) TC-1 tumor-bearing C57BL/6 mice were immunized intravenously or intratumorally with 1×10⁵ RCV FFU of artLCMV-E7E6 or artPICV-E7E6 vectors when tumors reached a volume of ~100 mm³. αNKG2A antibody was administered intraperitoneally 3 times over the course of the study: on day 0 (same day of vector administration), on days 3–5, and on days 7–8 (data from three independent experiments). Control animals received formulation buffer. Tumor volume was monitored over time (left, mean±SEM, n=10/group, shown is one experiment representative of three independent experiments, data are shown until the time point at which more than 50% of the animals in the group were euthanized or maximum d23). Kaplan-Meier survival curves, with indicated median and long-term survivors (right, *p<0.05, Log-rank test, data pooled from three independent experiments). (D) Animals showing complete response were rechallenged with TC-1 cells in the opposite flank. Control animals correspond to age-matched naïve mice grafted with TC-1. Kaplan-Meier survival curves are shown. artLCMV, arenavirus vectors based on lymphocytic choriomeningitis virus; artPICV, arenavirus vectors based on Pichinde virus.

A closer look at the top 20 upregulated genes identified 16 to be consistently upregulated across all treated groups (figure 6B—red dots). In line with the pathways identified above, several of these were indicative of increased accumulation of T cells within the TME (Cd3g, Cxcl9), while others denoted their potentially increased cytotoxic capacity (Gzmb, Nkg7, Irf1). The upregulation of IFN-induced genes indicated a strong IFN response triggered post-treatment. Gbp2 and Gbp3 encode guanylate binding proteins which contribute to protective immunity during infection, inflammation, and cancer.44 IL18 binding protein (IL18bp), on the other hand, is a high-affinity decoy receptor that limits the proinflammatory antitumor activity of IL18.45 Its upregulation may, therefore, represent a potential therapeutic target for enhancing antitumor efficacy.46 In addition, multiple genes involved in antigen processing and presentation were identified (figure 6B). Upregulation of Psmb8 and Psmb9, immunoproteasome subunits involved in MHC class I antigen processing, and upregulation of Tap1, Tap2, and Tapbpl, involved in peptide loading onto MHC class I molecules, suggested a strong presence of functional APCs within the infiltrating myeloid cells.47 48

Among the top differentially expressed genes was the non-classical MHC class I molecule H2-T23, alternatively named Qa-1b (figure 6B—bold). This is a homolog of the HLA-E, the main ligand of inhibitory receptor NKG2A49 50 expressed in NK, NKT, and a subset of CD8⁺ T cells.51 The increased expression of H2-T23 following treatment prompted us to investigate whether a combination with an anti-NKG2A blocking antibody could enhance the therapeutic efficacy of artARENA vectors. Using the same TC-1 tumor model described in previous experiments, we observed that NKG2A blockade indeed improved the antitumor response in combination with intravenous administration of artLCMV-E7E6 (figure 6C, online supplemental figure 5A). We observed prolonged tumor growth control in this combination group and a significant increase in median survival, with 19% of animals achieving a complete response. Combination with artPICV-E7E6 had a less pronounced effect on tumor control, but we could still observe an increase in median survival (figure 6C, online supplemental figure 5A). Animals that remained tumor-free until the end of the study were rechallenged with TC-1 tumor cells in the opposite flank. All previously treated groups were protected from rechallenge (figure 6D), indicating the generation of long-term antitumor immunity.

Overall, transcriptomic characterization of tumors showed a profound proinflammatory shift within the TME, with active antigen presentation, influx of activated lymphocytes, and strong IFN response. Furthermore, it revealed that upregulation of an NKG2A ligand could be negatively impacting the immune response following treatment and identified the blockade of NKG2A as a potential new combination approach to further enhance the therapeutic efficacy of artARENA.