Combination of loco-regional radiotherapy with a TIM-3 aptamer improves survival in diffuse midline glioma models

Pediatric diffuse midline gliomas (DMG) with H3-K27M-altered are aggressive brain tumors that arise during childhood. Despite advances in genomic knowledge and the significant number of clinical trials testing new targeted therapies, patient outcomes are still insufficient. Immune checkpoint blockades with small molecules, such as aptamers, are opening new therapeutic options that represent hope for this orphan disease. Here, we demonstrated that a TIM-3 aptamer as monotherapy increased the immune infiltration and elicited a strong specific immune response with a tendency to improve the overall survival of treated DMG-bearing mice. Importantly, combining TIM-3 Apt with radiotherapy increased the overall median survival and led to long-term survivor mice in two pediatric DMG orthotopic murine models. Interestingly, TIM-3 aptamer administration increased the number of myeloid populations and the pro-inflammatory ratios of CD8: Tregs in the tumor microenvironment as compared to non-treated groups after radiotherapy. Importantly, the depletion of T-cells led to a major loss of the therapeutic effect achieved by the combination. This work uncovers TIM-3 targeting as an immunotherapy approach to improve the radiotherapy outcome in DMGs and offers a strong foundation for propelling a phase I clinical trial using radiotherapy and TIM-3 blockade combination as a treatment for these tumors.


Introduction
Diffuse midline glioma (DMG) is an aggressive brain tumor and the leading cause of pediatric death caused by cancer (1).DMGs are defined as an infiltrative high-grade glioma, located in the brain midline (usually brainstem, spinal cord, cerebellum, or thalamus) with astrocytic differentiation and K27M mutation in either H3.3 (H3F3A) or H3.1 (HIST1H3B/C) (2).However, despite great strides in the understanding of this disease, survival outcomes after treatment are dismal.The standard of care has not changed for more than 50 years, with focal radiotherapy (RT) being the main treatment for DMG (3).Radiotherapy is not curative providing tumor stabilization and temporary reduction of symptoms extending the average survival to approximately 3 months (3,4).This overall lack of response to traditional treatments including chemotherapeutic agents, target therapies and radiotherapy underscores the need for new therapies targeting the unique biology of DMG tumor microenvironment (5).
In the last years, aptamers have emerged as a therapeutic alternative to antibody therapy (27).Aptamers are single-strand oligonucleotide ligands that bind to their target with affinity and specificity similar to those of antibodies.However, they pose some differences: aptamers can be easily produced via in vitro transcription or at a higher cost by direct chemical synthesis, while antibodies are cell-based products (28).
Oligonucleotides are immunologically less prone to induced anti-drug antibodies (ADA), which is a current problem with many types of monoclonal antibodies hindering the efficacy of the treatment after repetitive administration.Aptamers are smaller, and although they display a shorter half-life, they could likely get deeper into the target tissue than antibodies.All the above features uncover oligonucleotide-based therapy as a potential therapeutic tool for solid tumors, including brain tumors (29).
In this study, we showed that targeting TIM-3 with a 2F-pyrimidine-RNA oligonucleotide-aptamer (Apt1) (30) in combination with locoregional radiotherapy (standard of care of DMGs) resulted in a significant antitumor effect accompanied by immune memory acquisition in immunocompetent murine glioma models.

TIM-3 local targeting in DMGs with an inhibitory aptamer enhances tumor immunity with limited antitumor effect.
First, we evaluated the therapeutic value of TIM-3 antagonist monovalent aptamer (Apt1) as monotherapy in a DMG pre-clinical immunocompetent model.Apt1 is a short 62 nucleotide RNA 2F-Py modified oligonucleotide (Figure 1A) that binds to TIM-3 and counteracts the TIM-3 inhibitor signal (30).We have previously demonstrated that an intratumoral administration of an antiTIM-3 monoclonal antibody into the brainstem is more efficacious than a single systemic administration due to the integrity of the brainblood barrier (BBB) in this disease (31).In fact, in the context of DMGs, alternative routes of administration have been evaluated in the clinic, such as Convention Enhanced Delivery (CED) or direct injection of therapeutic agents such as oncolytic viruses.for these tumors (32); however, giving more than one administration in the clinic harbored its challenges due to the risk of accessing the brainstem (32,33).Thus, mice bearing orthotopically NP53 DMG-cells were treated with a single intratumoral injection of Apt1 (380 pmol/mouse; 5 days after implantation) or its corresponding controls (Saline and control Apt group) and with three subsequent intravenous doses (8,11, and 14 days after tumor implantation; 320 pmol/mouse) (Figure 1B).Under this experimental setting, Apt1 treatment as monotherapy did not result in a statistically significant increase in the median survival of NP53 tumor-bearing mice (Figure 1C).To elucidate if the TIM-3 Apt1 treatment affected the immune infiltrate in the tumor microenvironment, we characterized the adaptative immune populations at the endpoint by immunochemistry (Figure 1D).
Apt1-treated mice showed a significant increase in the number of CD3+ T-cells compared to control Apt and saline-treated groups (Figure 1E).This increase in T-cells in the Apt1treated mice was due to a significant increase in the number of CD8 lymphocytes (Figure 1F).No differences in the number of total CD4 T lymphocytes in the tumor of treated mice were observed.However, there was a drastic reduction in the number of Tregs (Foxp3+) in the Apt1-treated group compared to the control mice (Control Apt and saline groups; Figure 1G).Apt1 treatment significantly increased the proinflammatory CD8 + /Foxp3+ T cell ratio (Figure 1H).These results indicate that Apt1 treatment causes an increase in the number of CD8 T-cells and a reduction of Treg in the TME, but this is insufficient to promote a robust antitumor effect in the DMG model.

TIM-3 blockade with Apt1 promotes the release of effector cytokines
To determine whether Apt1 could enhance the endogenous specific effector immune response against NP53 antigens, we performed IFN-γ ELISPOT and 3 H thymidine proliferation assays to study the importance of the T-cell systemic immune response in a distant secondary lymphoid organ (spleen) or proximal ones (lymph nodes) to the tumor.
We detected a significant increase in IFN-γ spot number measured by ELISPOT as well as higher T proliferative rates measured by 3 H thymidine (CPM) in the splenocytes of animals treated with Apt1 as compared to the control Apt group (Figure 2A).We performed similar experiments with lymphocytes from tumor-draining lymph nodes, and we also obtained significant differences between the different treatment groups (Figure 2B).To better assess the type of immune response elicited by TIM-3 Atp1 treatment, we performed a cytokine MACSplex analysis.Splenocytes from Apt1-treated mice secrete significantly higher concentrations of cytokines IL-2 and IFN-γ, indicative of an effectoractivated Th1 response (Figure 2C).In addition, T-cells also produced higher levels of IL-17A associated with a Th17 response (Figure 2D).However, Apt1 treatment also led to a higher Th2 response, as cytokines IL-4, IL-5, and IL-10 were induced upon Apt1 treatment (Figure 2E).Interestingly, Apt1 increases the production of GM-CSF (Figure 2F), which functions in the recruitment and activation of myeloid cells, including dendritic cells and microglia.Taken together, these data suggest that TIM-3 targeting promotes a multi-pronged immune response activating helper effector arms of the immune system.This is probably due to the ubiquitous expression of TIM-3 in all the different immune cell types.

Radiotherapy increases TIM-3 expression in diffuse midline gliomas.
Because radiotherapy is the standard of care for DMG, next, we evaluated the impact of this approach on TIM-3 expression.It has been previously described that TIM-3 was expressed in different tumor types, including DIPGs (34) and gliomas, although at low levels (35), and its expression could be associated with hypoxia in brain damage responses (35).Thus, we wanted to determine whether radiotherapy would increase TIM-3 expression, creating a potential synergistic effect when combined with TIM-3 targeting agents.We irradiated NP53 DMG cells with different Gys (3, 6, and 12), and we evaluated TIM-3 expression at the RNA and protein levels at different time points, 24 and 48h, postradiotherapy.At the RNA level, we did not observe an increase in the expression of TIM-3 at 24h post-RT (Figure S1A).Importantly, at 48 hours post-RT, the cells displayed a significant increase in TIM-3 expression in both mRNA (Figure 3A) and protein (Figure 3B) levels at all the doses.Of importance, TIM-3 expression was significantly increased in a DMG orthotopic model after 6Gy radiotherapy treatment compared to the control measured at mRNA (Figure S1B) and protein levels (Figure 3C).Interestingly, after radiotherapy, TIM3 expression increased (percentage and mean fluorescence intensity) in several immune populations of the TME including microglia (Figure 3D), macrophages, dendritic cells (Figure 3E), NK cells (Figure 3F), conventional CD4, Tregs, and CD8 (Figure 3G).No differences were found in the expression of TIM-3 in monocytes or B cells after radiotherapy (Figure S1D).
These data demonstrate that TIM-3 expression in DMG is induced after radiotherapy and opens the possibility of enhancing its therapeutic window with the combination of an anti-TIM-3 agent.

Apt1 TIM-3 blockade enhances the antitumor radiotherapy efficacy in DMG models
To determine whether locoregional radiotherapy could synergize with TIM-3 aptamer, we perform locoregional radiotherapy in combination with TIM-3 Apt-1 in two different immunocompetent orthotopic aggressive DMG models (NP53 and XFM).Mice were treated with an intratumoral neoadjuvant dose of Apt1 (380pmol/mouse) two days before radiotherapy and then with 3 intravenous subsequent doses (320pmol/mouse; Figure 4A).
Treatment with radiotherapy resulted in a significant increase in the median survival in both radiotherapy groups (RT+Control apt and RT+TIM-3 Apt-1) compared to saline and led to long-term survival in mice bearing NP53 tumors.We observed a significant increase in median survival (30 days RT+Control Apt vs 58 days RT+TIM-3 Apt) with the appearance of a considerable number of long-term survivors (25% RT+control apt vs 50% RT+TIM-3 Apt) (Figure 4B) indicating a potential synergistic effect between radiotherapy and TIM-3 targeting by Apt1.Mice sacrificed 15 days after tumor implantation showed that the tumor size of groups treated with radiotherapy and the combination of radiotherapy and TIM-3 Apt was significantly smaller (Figure 4C).Then, we performed a rechallenge experiment to ascertain whether treated mice developed antiglioma immune memory.We observed that 100% of the mice cured from the treatment were protected from a rechallenge with a lethal dose of glioma cells indicating the existence of an immune memory in the treated mice (Figure 4D).In agreement with these data, anatomopathological analyses of the brains showed that long-term survivors were free of tumors (Figure 4E).Treatment of mice bearing XFM tumors with the combination treatment also led to an increase in median survival compared to the saline group (Saline = 17 vs.RT+TIM-3 Apt = 25 days; p = 0.002) and RT+control Apt (20 days; p=0.016), leading to 20% long-term survival (Figure 4F).In addition, mice sacrificed 10 days after tumor implantation demonstrated that the tumor size of groups treated with radiotherapy and the combination of radiotherapy and TIM-3 Apt was significantly smaller (Figure 4G).
Overall, our data support the value of targeting TIM-3 with an aptamer in combination with radiotherapy in diffuse midline gliomas.

A combination of radiotherapy and anti-TIM-3 aptamer results in an increase in immune infiltration in the tumor microenvironment
We analyzed the tumor immune infiltration to understand better the immune mechanism that underlines the anti-tumor efficacy of radiotherapy and TIM-3 Apt combo.NP53 tumor cells were implanted orthotopically, and the mice were randomized to either of the three groups of treatment (Saline, RT+Control apt, and RT+TIM-3 Apt-1).Mice were sacrificed 15 days after the implantation of the tumor following the same schedule as in the survival experiment (Figure 4A), and immune populations were analyzed by flow cytometry (Figure 5) and immunochemistry (Figure S2).Furthermore, radiotherapy and TIM-3 combo led to an increase in the number of total immune cells (CD45 high ) per mg of tumor (Figure 5A).In addition, we observed a significant increase in the number and proliferative state measured by Ki67 of microglia (CD45 med CD11b + ) compared to the other groups (Figure 5B).Radiotherapy and TIM-3 Apt combination also increased the number of NK and B cells (Figure 5C and S2B) innate immune populations compared with the other two treatment groups.
Regarding the myeloid cells, the combination increased the number of monocytes and dendritic cells (Figure 5D); however, no differences were found in the number of macrophages (Figure S2A) in the tumor microenvironment.Locoregional radiotherapytreated mice (both groups) displayed a significant increase in the accumulation of proliferative T-cells per mg of tumor compared with saline-treated brains (Figure 5E).
We observed a significant increase in the number of conventional CD4 and CD8 T cells in both radiotherapy groups (Figure 5F).However, although the number of T cells, including conventional CD4 and CD8 lymphocytes, remained constant between the radiotherapy groups, we only saw a significant increase in the proliferative status in the combination group (Figure 5G).Moreover, we observed a substantial decrease in the regulatory T-cell (Treg) population and its proliferative capacity in the RT-TIM-3 Apt-1 treated group (Figure 5H).Only combination treatment significantly increased the proinflammatory CD8+ T cell: Treg and CD4+ T cells: Treg ratios at day 15 after tumor implantation (Figure S2C).We confirmed these results by immunohistochemistry (Figures S2, D, and E).Besides, the serum cytokine analysis performed 15 days after implanting the cells in the NP53 model did not reveal any significant difference (Figures S3, and B).To further rule out the roles of the T cells in therapeutic efficacy, we used immunodeficient Rag2 -/-mice, which lack functional T cells but have macrophage and microglial populations.Interestingly, survival studies demonstrated a significant loss of the therapeutic effect in the combination groups (Figure 5I).We still observed a significant impact in mice treated with RT+TIM-3 Apt, probably due to the effect of radiotherapy on tumor cells and TIM-3 blockade on myeloid cells (Figure 5I).All these data suggest that myeloid cells, T cells, and Tregs could be one of the main mechanisms of action of the Apt-1 by modifying the tumor microenvironment and counteracting the immunosuppression mediated by both populations (36).

Discussion
Diffuse midline glioma meagre survival has not changed despite the combination of radiotherapy with targeted therapies (5), emphasizing the urgent need for effective treatments.Immune-checkpoint blockade therapy, including anti-CTLA-4 and PD(L)-1 antibodies, is the most successful immunotherapy approach in many cancer patients (37).
Nonetheless, brain tumor patients remain elusive to this type of treatment (38,39), probably due to the unique, highly immunosuppressive immune tumor microenvironment of these types of tumors (40).In this work, we show that targeting the TIM-3 axis is a vulnerability to DMG tumors, favoring the induction of a potent systemic antitumor immune response that can be efficacious in controlling tumor progression when combined with loco-regional radiotherapy.TIM-3 is an attractive target for cancer immunotherapy (41) due to its expression in cells of the adaptive (42,43) and innate (9, 44) immune systems, including the tumor cells (45)(46)(47).TIM-3 blockade by oligomeric aptamer has already been shown to control tumor growth in murine models of CT26, alone or combination with a PD-1 antibody, by significantly increasing the pro-inflammatory CD8/Tregs ratio in the tumor microenvironment (48).
In the current orthotopic glioma models, TIM-3 targeting with Apt1 also considerably increased the CD8/Treg ratio.The therapeutic effect of inhibiting TIM-3 in monotherapy seems to be modest.Still, TIM-3 Apt1 treatment promotes a pool of effector cytokine secretion activating all the effector arms of the immune system, including a Th2 immune response.The increase of IL-10 after treatment intrigues us because it is overexpressed in GBM patients (49), and it is associated with increased glioma cell proliferation and invasion in pre-clinical models (50).Additionally, IL-10 has been shown as one of the main cytokines with anti-inflammatory effects in human tumors due to its ability to suppress T-cells (51).This upregulation of IL-10 may dilute the pro-inflammatory effect of Apt1, decreasing the therapeutic effect of TIM-3 blockade and opening the possibility of combining IL10 blockade with anti-TIM-3 agents to improve the therapeutic outcome in future studies.Radiotherapy is the standard of care for DMG (3) and also a fundamental requirement in any first-line DMG clinical trial (33,52), so we decided to combine our TIM-3 Apt with locoregional radiotherapy.TIM-3 blockade using a monoclonal antibody in combination with radiotherapy and anti-PD-1 has already shown promising efficacy in preclinical models of glioblastoma multiforme (GBM) (53), although other molecules such as aptamers have never been used.Moreover, the combination in DMG models of radiotherapy and TIM-3 blockade had never been tested.
In our work, the antitumor effect of a radiotherapy and TIM-3 aptamer combination was remarkable, increasing the median overall survival of treated mice associated with higher infiltration of proinflammatory immune cell populations in the TME.Radiotherapy has been described as capable of increasing the infiltration of T cells into the microenvironment of brain tumors (54).However, radiotherapy not only causes the infiltration of proinflammatory T cells but also increases the infiltration of Tregs (55).In brain tumors, tumor-infiltrating Tregs percentage in patients is strongly correlated with the WHO grade.It demonstrates that the accumulation of Tregs in glioblastomas contributes to the dismal immune responses observed in these tumors (56).Interestingly, treatment with TIM-3 Apt1 after radiotherapy causes a significant decrease in the infiltration of Tregs, which is promoted by radiotherapy.Therefore, we speculate that TIM-3 targeting may directly affect Tregs due to the importance of this receptor in their phenotype and function (36).Furthermore, our combination treatment also improves the expansion of myeloid cells, according to previous works demonstrating this population's fundamental role after the TIM-3 blockade (31).
In summary, we provide evidence that the combination of radiotherapy and TIM-3 targeting is capable of inducing a significant increase in overall median survival in DMG models.This leads to the expansion of myeloid populations and T cells in the TME and the generation of immune memory.Additionally, we demonstrate the importance of regulatory T cells in TIM-3 aptamer targeting.
Our study examined male and female animals in the same proportion; similar findings are reported for both sexes.

Animal studies
The orthotopic DMG model was engrafted by injection into the pons using a guide-screw system (Taconic Farms, Inc) (58).The NP53 cells (10 4 cells) were implanted in transgenic mice kindly provided by Dr. Oren Becher, and the XFM (10 3 cells) were implanted in balb/c mice.The cells were administered in 3 μl of PBS.The animals were randomly assigned to the following three groups: control mice injected with saline or aptamer control apt and mice injected with TIM-3 aptamer (Apt1).Apt1 was administered intratumorally in 3 μl (380pmol/mouse) and three times intravenous (320pmol/mouse) 5, 8, 11, and 14 days after the cell implantation, respectively.In the radiotherapy experiments, animals were randomly assigned to the following three groups: saline, RT+control apt, and +TIM-3 Apt.6Gy of radiotherapy was given on day 7, and the aptamer was injected using the same schedule as the previous experiment.In the case of immunocompetent murine models, in which kinetics are very fast, we consider long-term survivors to be animals that live at least three times longer than the median survival of the control animals.For rechallenge experiments, mice that survived three times longer than the median survival time of the control group were orthotopically re-implanted with the same number of tumor cells in the brain.

TIM-3 aptamer production
The Atp-1 anti-TIM-3 use in the study was previously described (30) and it is 2F'pyrimidine RNA aptamer: 5'-GGGAGAGGACCAUGUAGCCACUAUGGUGUU GGAGCUAGCGGCAGAGCGUCGCGGUCCCUCCC-3'.As control Apt in the experiments it was used as a randomized 2′F-RNA 25N aptamer flanked containing the constant regions at 5′ and 3′ than the Apt-1 TIM-3 aptamer.Both aptamers were produced by transcription from a double-stranded DNA oligonucleotide template generated from hybridization of two partially complementary sequences and amplified by PCR with the primers Fwd: GGGGAATTCTAATACGACTCACTATAGGGAGAGGACCATGTA and Rev: GGGAGGGACCGCGACGCTCTG.The Fwd primed includes the T7 promoter to allow its transcription using the T7 Durascribe Kit (Epicentre, Madison, WI).The aptamers were purified by polyacrylamide gel electrophoresis (PAGE) and refolded by hitting.

IFN-gamma ELISPOT
NP53 cells were incubated with murine recombinant IFN-gamma (100IU/mL).24h later, splenocytes and lymph nodes were isolated from mice and co-cultured with NP53 cells (ratio of 10:1) for 24 hours in a 96-well plate.A mouse IFN-γ ELISPOT set (551083 BD) was used according to the manufacturer's instructions, and the results were measured using an Immunospot S6 Analyzer (Macro, Immunospot).The results of the IFN-gamma ELISPOT were normalized per 10 4 cells.
Flow cytometry NP53 single-cell suspensions were stained for flow cytometry.Dead cells were excluded by PromoFluor-840 staining (1:10,000, PK-PF840-3-01).Tumor cells were stained using TIM-3-PE (Biolegend Cat: 119703, Clone: RMT3-23; 1:200).For immune characterization, excised tumors in the experiment were weighted and mechanically dissociated using a scalpel, incubated with collagenase IV/DNase I (17018-029 Gibco/11284932001 Roche) with rotation for 15 minutes, and then incubated twice for 10 minutes at 37ºC.The solution was filtered through a 70-mm cell strainer (Thermo Fisher Scientific, Waltham, Massachusetts, USA).After the addition of a 30% Percoll solution (17-0891-01 GE Healthcare, Chicago, IL, USA), tumor cells were isolated by centrifugation at 500 g for 20 minutes.Single-cell suspensions were then stained for flow cytometry.Dead cells were excluded by PromoFluor-840 staining (1:10,000, PK-PF840-3-01).Our previous published work lists the fluorochrome-tagged monoclonal antibodies (mAbs) used in this assay (58).Cells were fixed and permeabilized for nuclear staining using BD Cytofix/Cytoperm Plus (555028 BD Biosciences) and then stained according to the manufacturer's instructions.The remaining samples were then analyzed using CytoFLEX (Beckman Coulter) and FlowJo software (BD Biosciences).The flow gating strategy used for tumor microenvironment immune population characterization experiments was explained in Ausejo-Mauleon et al. (58).Immune population data was normalized against the weight of the tumors after removal from the brain.

MACSPlex Cytokine assay
Splenocytes were isolated from mice and co-cultured with NP53 cells (ratio of 10:1) for 24 hours in a 96-well plate.Supernatants were analyzed by the MACSPlex 12 cytokine Kit (Miltenyi Biotec, Germany).Serum was collected from mice bearing NP53 tumors 15 days after tumor implantation.All working steps were carried out according to the manufacturer's instructions, and washing procedures were performed with a centrifuge.Flow analysis was performed using MACS Quant Analyzer (Miltenyi Biotec, Germany).Data analysis was performed using Flow Logic™ (V7.2.1) and BeadLogic™ (V7) (Miltenyi Biotec, Germany).

RNA extraction and real-time PCR
Total RNA was extracted from isolated cells using TRIzol according to the manufacturer's instructions (Life Technologies, Carlsbad, CA, USA).RNA samples were quantified using a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific) and stored at −80°C.One microgram of RNA was reverse transcribed using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Thermo Fisher, Bedford, MA, USA).Afterward, cDNA was amplified using SYBR-Green Master Mix (Applied Biosystems).The gene-specific assay was murine TIM-3.HPRT1 was used as the housekeeping control gene, and all samples were run in triplicate.The sequences of the primers for TIM-3 are Fwd 5´-TCAGGTCTTACCCTCAACTGTG-3´ and Rv 5´-GGGCAGATAGGCATTTTTACCA-3´.Real-time PCR was monitored using an ABI 7700 sequence detection system (Applied Biosystems).The fold changes in the expression of the genes of interest were calculated as the mean values calculated using the 2 -ΔΔCT method.

Statistical analysis
For the in vitro experiments, data are expressed as the mean ± SD, and comparisons were evaluated by the two-tailed Student's t-test or one-way ANOVA.The effect of TIM-3 Apt and radiotherapy, alone or in combination, in glioma orthotopic models was assessed by plotting survival curves using the Kaplan-Meier method.Survival in different treatment groups was compared using the log-rank test.GraphPad software (Prism version 9.3.1)was used for the statistical analysis.

Study approval
Ethical approval for the animal studies was granted by the Animal Ethical Committee of the University of Navarra (CEEA; Comité Etico de Experimentación Animal) under the protocol numbers CEEA/069-13.All animal studies were performed at the veterinary facilities of the Center for Applied Medical Research in accordance with institutional, regional, and national laws and ethical guidelines for experimental animal care.

Data availability
Data are available in the "Supporting data values" XLS file.

Figure 4 .
Figure 4. Evaluation of the antitumor effect of radiotherapy and Apt1 TIM-3

Figure 5 .
Figure 5. Characterization of innate and adaptative immune response modulation