Intratumoral IL15 Improves Efficacy of Near-Infrared Photoimmunotherapy

This article is featured in Selected Articles from This Issue, p. 1113

IL15, a pleiotropic cytokine, is a potential therapeutic agent/adjuvant in the treatment of cancer as it stimulates host immune responses, including the differentiation and proliferation of CD8⁺ T and natural killer (NK) cells (1, 2). Although IL15 shares this function with IL2, it does not cause activation-induced cell death of memory T cells or activation of regulatory T cells (Tregs), making it a more promising immunotherapy for the induction of potent anticancer immunity (3). However, human clinical trials have demonstrated that intravenous infusion of human IL15 has modest efficacy but extensively expands peripheral NK cells, resulting in higher rates of severe dose-limiting toxicity (4). To overcome these limitations, studies have investigated the efficacy of various treatment strategies, including alternative routes of administration and the use of IL15 in combination therapy (5–7). Local administration of IL15 in the tumor can improve its delivery to the tumor microenvironment while reducing its systemic toxicity (8, 9). Also, given that IL15 monotherapy expands antigen-nonspecific immunity (10), it would more effectively work when combined with other cancer-killing therapies that substantially release tumor antigens and activate anticancer acquired immunity.

Near-infrared photoimmunotherapy (NIR-PIT) is a novel anticancer treatment that utilizes an antibody-photoabsorber conjugate (APC) to bind the tumor whereupon subsequent NIR light exposure triggers selective photochemical reactions leading to immunogenic cell death (ICD; refs. 11–13). The APC is constructed by conjugating the photoabsorber IRDye700DX (IR700; refs. 11–13), a silica-phthalocyanine dye, to mAbs directed against tumor antigens on the cancer cell membrane (11). NIR light irradiation provokes the dissociation of axial ligands within the IR700 molecule, converting it from highly hydrophilic to highly hydrophobic, causing aggregation of cell surface antigens bound to the APCs (14). These photochemical reactions immediately damage the membrane of cancer cells, leading to swelling, blebbing, and rupture (14–16). NIR-PIT induces ICD by inducing the rapid release of immunogenic signals, such as calreticulin, ATP, HSP 70, and HSP 90, which activate immature dendritic cells (DCs; ref. 17). Therefore, NIR-PIT is a potent anticancer therapy that not only directly kills cancer cells but also induces a potent anticancer immune response.

We hypothesized that local IL15 administration could increase the effectiveness of cancer cell–targeted NIR-PIT by promoting the immune response. The CD44 protein, which is overexpressed on various cancer cells, serves as an attractive target for cancer therapy but is also useful in syngeneic mouse models because it is commonly expressed (18–21). This study aimed to investigate in vivo anticancer efficacy of local IL15 administration combined with CD44-targeted NIR-PIT in syngeneic murine cancer models, specifically using models of IL15-auto-secretion and intratumoral IL15 injection.

APC was synthesized as described previously (19). In brief, 1 mg of anti-mouse/human CD44 monoclonal antibody (mAb) (6.7 nmol/L; clone IM7; BioXCell) was incubated with five-fold molar excess of IR700 NHS ester (10 mmol/L in DMSO; LI-COR Biosciences) in 100 mmol/L Na₂HPO₄ solution (pH 8.5) for 1 hour at room temperature. The mixture was purified with PD-10 columns containing Sephadex G25 resin (Cytiva). The resulting APC was abbreviated as CD44-IR700.

Murine cancer cell lines, MC38 (colon cancer) and Pan02-luc (pancreatic cancer), were used in this study. MC38 was kindly provided by Dr. Thomas Waldmann (NIH, Bethesda, MD). Pan02-luc was purchased from GenTarget Inc. Human IL15-secreting MC38 (hIL15-MC38) was constructed by introducing human IL15-coding plasmids into MC38 cells as described previously (22, 23). MC38 and Pan02-luc cells were cultured in RPMI1640 medium (Thermo Fisher Scientific) supplemented with 10% FBS (Thermo Fisher Scientific) and 100 IU/mL penicillin/streptomycin (Thermo Fisher Scientific). hIL15-MC38 cells were cultured in DMEM (ATCC) supplemented with 10% FBS and 100 IU/mL penicillin/streptomycin. All cells were cultured in a humidified incubator at 37°C in an atmosphere of 95% air and 5% carbon dioxide. MC38 was authenticated by STR profiling (IDEXX BioAnalytics) in 2021. Pan02-luc was used in our laboratory within 6 months after receipt from the vendor. All cell lines were tested for Mycoplasma using PCR; MC38 was examined by IDEXX BioAnalytics in 2021 and hIL15-MC38 and Pan02-luc were examined by Frederick National Laboratory for Cancer Research (Frederick, MD) in 2022. Cells were frozen down soon after the authentication and Mycoplasma testing, and thawed to be used in this study. After thawing, cells were maintained in culture for no more than 30 passages.

MC38 or hIL15-MC38 cells were seeded at 1 × 10⁵ cells in 1 mL of medium onto 24-well plates and incubated for 24 hours at 37°C. Cells were incubated in culture medium containing CD44-IR700 (10 μg/mL) for 1 hour at 37°C. After changing the medium into phenol-red-free medium, NIR laser-light (690 nm, 150 mW/cm²) was applied using an ML7710 laser system (Modulight). Cell-surface expression of calreticulin and HSP 70 was evaluated immediately after NIR light exposure. Cells were incubated for 30 minutes at 4°C with anti-mouse calreticulin antibody (rabbit poly; Bioss Antibodies) or its rabbit IgG1 isotype control (Bioss Antibodies) for calreticulin staining, and with anti-mouse/human HSP 70 antibody (clone REA349; Miltenyi Biotec) or its human IgG1 isotype control (clone REA293; Miltenyi Biotec) for HSP 70 staining. The fluorescence of cells was measured using BD FACSLyric (BD Biosciences) and FlowJo software (FlowJo LLC).

All procedures were performed in compliance with the Guide for the Care and Use of Laboratory Animals and approved by the local Animal Care and Use Committee (MIP-003–4-L). Six- to 8-week-old female C57BL/6 (strain #000664) and homozygote athymic nude mice were purchased from The Jackson Laboratory and Charles River, respectively. The lower part of the body of the mice was shaved before image analysis. Two million MC38, hIL15-MC38, or Pan02-luc cells were inoculated into the right dorsum. Mice with tumors reaching approximately 50 to 150 mm³ in volume were randomized for the experiments. For tumor rechallenge, 2 × 10⁶ MC38 cells were inoculated into the left dorsum. For bilateral models, 1 × 10⁶ MC38 and 2 × 10⁶ hIL15-MC38 cells were inoculated into the left and right sides of dorsal flank, respectively. Mice with a right tumor reaching approximately 30 to 50 mm³ and a left tumor reaching approximately 50 to 150 mm³ in volume were randomized for the experiment. Tumor volumes were defined as volume (mm³) = length × width² × 0.5 and measured three times a week by a caliper. The mice were euthanized with CO₂ when the tumor reached 2 cm in diameter or 2,000 mm³ in volume. For bilateral models, mice were euthanized when either tumor reached its endpoint. Mice survived 60 days after NIR-PIT were censored from the survival analysis. Tumor disappearance for four weeks or longer after treatment was defined as complete response (CR).

One million MC38 or hIL15-MC38 cells were incubated in 5 mL of medium at 37°C for 72 hours; then supernatants were collected. MC38 or hIL15-MC38 tumor-bearing mice were euthanized 14 days after inoculation. Tumors were harvested, and tumor homogenates were prepared. Peripheral blood samples were collected from mice, and plasma was prepared. Human IL15 concentration was measured using Human IL15 DuoSet ELISA kit (R&D Systems) according to the manufacturer's protocol.

Human IL15 (PeproTech) was administered to mice intraperitoneally (5 μg in 100 μL PBS) or intratumorally (5 μg in 20 μL PBS) every other day four times in total.

To compare the efficacy of NIR-PIT among MC38 and hIL15-MC38 tumors, mice were categorized into four groups as follows: (i) MC38 with no treatment (MC38/Control), (ii) MC38 treated with intravenous injection of CD44-IR700 (50 μg) followed by NIR light irradiation (MC38/NIR-PIT), (iii) hIL15-MC38 with no treatment (hIL15-MC38/Control), and (iv) hIL15-MC38 treated with intravenous injection of CD44-IR700 (50 μg) followed by NIR light irradiation (hIL15-MC38/NIR-PIT). To evaluate the efficacy of intratumoral IL15 injection combined with NIR-PIT, mice were randomized into four groups as follows: (i) no treatment (Control), (ii) intravenous injection of CD44-IR700 (50 μg) followed by NIR light irradiation (NIR-PIT), (iii) intratumoral IL15 injection (IL15-IT), and (iv) intravenous injection of CD44-IR700 (50 μg) followed by NIR light irradiation and intratumoral IL15 injection (Combination). For bilateral tumor model experiments, mice were categorized into two groups as follows: (i) no treatment (Control) and (ii) intravenous injection of CD44-IR700 (50 μg) followed by NIR light irradiation (NIR-PIT). 690-nm NIR light (150 mW/cm², 50 J/cm², day 0) was administered to the tumors 24 hours after injecting CD44-IR700. When NIR light was applied, mice were covered by aluminum foil except for a hole to expose only the target tumor. In mice with CR after intratumoral IL15 injection combined with NIR-PIT against MC38 tumors, 2 × 10⁶ MC38 cells were reinoculated into the contralateral dorsum 90 days after NIR-PIT. 700-nm fluorescence and white light images were obtained before and after NIR-PIT using a Pearl Imager (LI-COR Bioscience).

To compare tumor-infiltrating lymphocytes (TIL) between MC38 and hIL15-MC38 tumors, we euthanized mice when established tumors reached approximately 150 mm³ in volume. To assess the effect of intraperitoneal or intratumoral IL15 injection on host tumor immunity, we harvested tumors one day after IL15 injection. To assess host tumor immunity following CD44-targeted NIR-PIT, tumors and tumor draining lymph nodes (TDLN) were harvested two and four days after NIR-PIT. Single-cell suspension was prepared as previously described (24, 25). Cells were stained with the following antibodies: anti-CD3e (clone 145–2C11), anti-CD4 (clone RM4–5), anti-CD11b (clone M1/70), anti-CD11c (clone N418), anti-CD25 (clone PC61), anti-CD45 (clone 30-F11), anti-CD69 (clone H1.2F3), anti-CD83 (clone Michel-19), anti-CD86 (clone GL-1), anti-F4/80 (clone BM8), anti-Ki-67 (clone 10A8), anti-Ly6C (clone HK1.4), anti-I-A/I-E (clone M5/114.15.2), and anti-NK1.1 (clone PK136) were from BioLegend; anti-CD8α (clone 53–6.7), anti-CD45 (clone 30-F11), anti-Granzyme B (GZMB; clone NGZB), anti-FOXP3 (clone FJK-16s), anti-IL15Rα (clone DNT15Ra), anti-Ly6G (clone 1A8), and rat IgG1 kappa isotype control (clone eBRG1) were from Thermo Fisher Scientific; anti-IL15 (clone IC2471P) and mouse IgG1 isotype control (clone IC002P) were from R&D Systems. Dead cells were gated out from the analysis by Fixable Viability Dye staining (Thermo Fisher Scientific). For membrane-bound IL15 staining, single-cell suspension was incubated with human IL15 (500 pmol/L; PeproTech) for 15 minutes before antibody incubation. For intracellular staining of GZMB, cells were stimulated with eBioscience Cell Stimulation Cocktail plus protein transport inhibitors (Thermo Fisher Scientific) at 37°C for 4 hours before staining. For intracellular and intranuclear staining, cells were fixed and permeabilized with Intracellular Fixation & Permeabilization Buffer Set (Thermo Fisher Scientific). The stained cells were analyzed using FACSLyric (BD Biosciences) and FlowJo software (FlowJo LLC). Cell types were determined as follows; CD8⁺ T cells: CD45⁺/CD3⁺/CD8⁺/CD4⁻, CD4⁺ T cells: CD45⁺/CD3⁺/CD4⁺/CD8⁻, Tregs: CD45⁺/CD3⁺/CD4⁺/CD8⁻/FOXP3⁺, NK cells: CD45⁺/CD3⁻/NK1.1⁺, natural killer T (NKT) cells: CD45⁺/CD3⁺/NK1.1⁺, Myeloid cells: CD45⁺/CD11b⁺, DCs: CD45⁺/(F4/80)⁻/CD11c⁺/(I-A/I-E)⁺, Macrophages: CD11b⁺/(F4/80)⁺, and Monocytes: CD11b⁺/(F4/80)⁻/Ly6G⁻/Ly6C⁺. For IL15 and IL15Rα expression analyses, DCs were defined as CD11b⁻/(F4/80)⁻/CD11c⁺/(I-A/I-E)⁺.

To analyze TIL distribution after CD44-targeted NIR-PIT, we harvested tumors four days after NIR-PIT, and formalin-fixed, paraffin-embedded (FFPE) sections were prepared. Multiplex IHC was performed as described previously, using Opal Automation IHC Kit (Akoya Bioscience) and Bond RXm autostainer (Leica Biosystems; refs. 26, 27). The sections were stained with 4,6-diamino-2-phenyl indole (DAPI) and the following antibodies: anti-CD3 (clone SP7; 1:500 dilution; Novus Biologicals), anti-CD8 (clone EPR20305; 1:500 dilution; Abcam), anti-CD4 (clone EPR19514; 1:1,000 dilution; Abcam), anti-FOXP3 (clone 1054C; 1:1,000 dilution; Novus Biologicals), anti–pan-cytokeratin (pan-CK; rabbit poly; 1:250 dilution; Bioss Antibodies), anti-CD45 (clone D3F8Q; 1:500 dilution; Cell Signaling Technology), anti-GZMB (rabbit poly; 1:500 dilution; Abcam), and anti-Ki-67 (clone D3B5; 1:500 dilution; Cell Signaling Technology). Coverslips were mounted using ProLong Diamond Antifade Mountant (Thermo Fisher Scientific). Stained slides were analyzed with Mantra Quantitative Pathology Workstation (Akoya Biosystems) and inForm Tissue Finder software (Akoya Biosystems). inForm software was trained to detect tissues and cell phenotypes according to the following criteria: areas with pan-CK expression = tumor, other areas = stroma, pan-CK⁺/CD8⁻/CD4⁻ = cancer cells, pan-CK⁻/CD8⁺/CD4⁻ = CD8⁺ T cells, and pan-CK⁻/CD8⁻/CD4⁺ = CD4⁺ T cells, respectively. Four images were taken from each tumor sample, and cell density was calculated by combining areas for each tissue phenotype and cell counts of each cell phenotype. For GZMB expression analysis, cell phenotyping and GZMB expression analysis were performed separately for the same set of images, then cell segmentation data were consolidated and analyzed using phenoptrReports and phenoptr (Akoya Biosciences) to calculate the density of GZMB⁺CD8⁺ T cells. For Ki-67 expression analysis, cell phenotyping was performed as follows: pan-CK⁺CD45⁻ = cancer cells, pan-CK⁻CD45⁺ = hematopoietic cells, and pan-CK⁻CD45⁻ = other cells, respectively. The percentage of Ki-67 positive cells was computed among cancer cells. (See also Supplementary Table S1. List of key resource identifiers in this study)

Data are shown as mean ± SEM. GraphPad Prism 8.4.3 (GraphPad Software) was used for statistical analysis. A one-way ANOVA followed by Tukey test was performed to compare data among multiple groups. Tumor volumes were compared using a repeated measures two-way ANOVA followed by Tukey test. Survival percent was determined by a Kaplan–Meier method, and the results were compared using the log-rank test with Bonferroni correction. P < 0.05 was defined as statistically significant.

The data generated in this study are available within the article and its Supplementary Data or from the corresponding author upon reasonable request.

The therapeutic efficacy of local and systemic IL15 administration was evaluated using in vivo tumor models. Mice with MC38 tumors reaching approximately 50 to 150 mm³ in volume were divided into three groups: Control (untreated), IP (intraperitoneal IL15 injection), and IT (intratumoral IL15 injection). The IT group exhibited significantly slower tumor growth compared with the Control and IP groups (Fig. 1A). In addition, the IT group exhibited the best survival among the three groups (Fig. 1B). Subsequently, intratumoral immune responses were assessed one day after a single-dose treatment among the three groups (Fig. 1C; Supplementary Fig. S1). The IT group demonstrated a significant increase in the number of NK, myeloid, T, CD8⁺ T, and NKT cells and DCs compared with the Control and IP groups (Fig. 1C; Supplementary Fig. S1A and S1B). Furthermore, GZMB expressions in CD8⁺ T and NKT cells were significantly higher in the IT group compared with the Control and IP groups (Supplementary Fig. S1C). The number of Tregs was not significantly different among the three groups (Supplementary Fig. S1D).

Next, we evaluated the effects of local auto-secretion of IL15 on in vivo tumor growth using hIL15-MC38, a human IL15-secreting cell line. IL15 auto-secretion was confirmed in vitro (Fig. 2A) and in vivo (Fig. 2B). However, there was no significant difference in plasma IL15 levels between mice with hIL15-MC38 and MC38 tumors (Fig. 2C). On the basis of chronologic measurement of secreted IL15, the amount of IL15 secreted in a hIL15-MC38 tumor was estimated to be approximately 1/50 of that in the intratumoral IL15 injection (Supplementary Fig. S2). The growth of hIL15-MC38 tumors was significantly slower than that of the parental MC38 tumors (Fig. 2D). Mice with hIL15-MC38 tumors showed prolonged survival compared with those with MC38 tumors (Fig. 2E). However, hIL15-MC38 tumors showed significantly higher Ki-67 positivity compared with MC38 tumors (Supplementary Fig. S3). Similar body weight trends were observed between mice with hIL15-MC38 and MC38 tumors, suggesting an absence of acute toxicity in hIL15-MC38 tumor-bearing mice (Fig. 2F).

We subsequently compared intratumoral immune cell profiles between hIL15-MC38 and parental MC38 tumors (Fig. 2G; Supplementary Fig. S4). The number of NK, myeloid, T, CD8⁺ T, and NKT cells was significantly higher in hIL15-MC38 tumors than in MC38 tumors (Fig. 2G; Supplementary Fig. S4A). Although the number of Tregs was significantly higher in hIL15-MC38 tumors compared with MC38 tumors, the proportion of Tregs was not significantly different between them (Supplementary Fig. S4B). IL15Rα, an IL15-specific receptor with a high affinity, mediates trans-presentation of IL15, resulting in proliferation and activation of effector cells including NK and T cells. In both hIL15-MC38 and MC38 tumors, IL15Rα expression was slightly but significantly higher in monocytes compared to other immune cells (Fig. 2H). Similarly, a significantly higher level of membrane-bound IL15 was detected in monocytes in comparison with other immune cells (Fig. 2I). These results suggest that monocytes could be involved in IL15 trans-presentation in the IL15-auto-secretion model.

CD44 was highly expressed on the surface of hIL15-MC38 and MC38 cells in vitro (Supplementary Fig. S5A). Flow cytometric analysis confirmed the binding of CD44-IR700 to hIL15-MC38 and MC38 cells in vitro (Supplementary Fig. S5B). Next, we examined the cytotoxic efficacy of in vitro CD44-targeted NIR-PIT against hIL15-MC38 or MC38 cells. Microscopic examination showed that cellular swelling occurred in both cell lines immediately after NIR-PIT (Supplementary Fig. S5C). MTT assay showed a light-dose-dependent decrease in metabolic activity of hIL15-MC38 and MC38 cells following NIR-PIT (Supplementary Fig. S5D and S5E). Furthermore, Propidium iodide (PI) positivity, an indicator of cell membrane damage, increased after NIR-PIT in a light dose–dependent manner (Supplementary Fig. S5F and S5G). These results showed that CD44-targeted NIR-PIT induced cell death via cell membrane disruption in both hIL15-MC38 and MC38 cells.

The therapeutic efficacy of in vivo CD44-targeted NIR-PIT was compared with that of the control using hIL15-MC38 or parental MC38 tumor-bearing mice. The treatment and imaging schedule are shown in Fig. 3A. Mice with tumors reaching approximately 50 to 150 mm³ in volume were treated with or without NIR-PIT. NIR light was applied to tumors located on the right dorsal flank (Fig. 3B). Before NIR light irradiation, a 700-nm fluorescent signal was detected at the tumor site of mice in the NIR-PIT group due to the IR700 dye. After NIR light irradiation, this signal immediately decreased due to photoinduced bleaching of the conjugated IR700 (Fig. 3C). Among the four groups, the hIL15-MC38/NIR-PIT group showed the slowest tumor growth (Fig. 3D). Furthermore, survival was significantly prolonged in the hIL15-MC38/NIR-PIT group compared with the other groups (Fig. 3E). CR was achieved only in hIL15-MC38/NIR-PIT group (2/11 mice, 18%).

We tested the expression of ICD-related molecules after in vitro CD44-targeted NIR-PIT against hIL15-MC38 or parental MC38 cells. Calreticulin and HSP 70 expression significantly increased immediately after NIR-PIT in both cell lines, suggesting ICD (Fig. 4A and B; Supplementary Fig. S6).

Next, we evaluated host immune responses to CD44-targeted NIR-PIT in hIL15-MC38 or parental MC38 tumors. In both tumors, the number of antigen-presenting cells including DCs, monocytes, and macrophages in tumors did not differ with or without NIR-PIT two days after NIR-PIT (Supplementary Fig. S7A). To evaluate DC maturation after NIR-PIT, the expression of DC maturation markers in TDLN was analyzed by flow cytometry two days after NIR-PIT. CD83 and CD86 mean fluorescence intensity (MFI) in DCs significantly increased in the hIL15-MC38/NIR-PIT group compared with the other three groups (Fig. 4C). In addition, CD8⁺ T cell activation in TDLNs and tumors was also assessed two days after NIR-PIT. In TDLNs, CD69 and CD25 were significantly upregulated, and Ki-67 positivity was higher in the hIL15-MC38/NIR-PIT group (Fig. 4D). In tumors, upregulation of CD69, but not CD25, was observed in the hIL15-MC38/NIR-PIT group (Supplementary Fig. S7B). These results confirmed that CD8⁺ T cells in TDLNs and tumors were highly activated and proliferating in the hIL15-MC38/NIR-PIT group.

We further evaluated CD8⁺ T cell infiltration and its cytotoxic potential in tumors by flow cytometry four days after NIR-PIT. The number of CD8⁺ T cells was higher but equivalent in both the hIL15-MC38/Control and hIL15-MC38/NIR-PIT groups (Fig. 4E, left), regardless of NIR-PIT, suggesting that IL15 primarily promoted the proliferation of CD8⁺ T cells. GZMB expression in CD8⁺ T cells was also significantly higher in the MC38/NIR-PIT and hIL15-MC38/NIR-PIT groups (Fig. 4F, left), regardless of IL15 secretion, indicating that NIR-PIT contributed to the enhanced cytotoxicity of CD8⁺ T cells. The cytotoxic potential of NK and NKT cells was also analyzed by flow cytometry four days after NIR-PIT. The number of NK and NKT cells significantly increased in the hIL15-MC38/Control and hIL15-MC38/NIR-PIT groups (Fig. 4E, middle and right). GZMB expression in NK and NKT cells was significantly higher in the hIL15-MC38/NIR-PIT group (Fig. 4F, middle and right).

In flow cytometric analysis, the number of CD8⁺ T cells was comparable between the hIL15-MC38/Control and hIL15-MC38/NIR-PIT groups. Given the disparity in in vivo efficacy between them, we hypothesized that NIR-PIT may primarily alter the distribution of CD8⁺ T cells in tumors rather than their absolute number. Thus, we assessed TIL distribution after CD44-targeted NIR-PIT in hIL15-MC38 or parental MC38 tumors. Tumors were harvested four days after each treatment and analyzed by multiplex IHC (Fig. 4G). CD8⁺ T cell density in the tumor area was significantly higher in the hIL15-MC38/NIR-PIT group than in the other three groups (Fig. 4H, left). GZMB expression in CD8⁺ T cells was the highest in the hIL15-MC38/NIR-PIT group (Fig. 4H, right). These findings were not present in the peritumoral stromal area (Fig. 4I). In addition, we assessed Treg density and the ratio of CD8⁺ T cells to Tregs, a well-known index of strong anticancer immunity (28). As shown in Supplementary Fig. S8A and S8B, Treg density was comparable among the four groups in both the tumor and stroma areas. The hIL15-MC38/NIR-PIT group showed higher CD8⁺ T cells/Tregs ratio in the tumor area but not in the stromal area. Interestingly, a considerable number of GZMB⁺ CD8⁻ cells were observed in the hIL15-MC38/NIR-PIT group (Supplementary Fig. S8C). These cells were a mixture of CD3⁻ and CD3⁺, suggestive of cytotoxic NK and NKT cells, respectively.

In vivo therapeutic efficacy of CD44-targeted NIR-PIT, intratumoral IL15 injection, and the combination of the two was evaluated using mice with MC38 tumors reaching approximately 50 to 150 mm³ in volume. Figure 5A illustrates the treatment regimen and schedule. Tumors in the right dorsal flank were irradiated with NIR light and/or injected with IL15 (Fig. 5B). A 700-nm fluorescent signal at the tumor site of mice was bleached immediately after NIR light irradiation in mice treated with NIR-PIT (Fig. 5C). Tumor growth was significantly slower in the Combination group than in the other three groups (Fig. 5D). Moreover, the Combination group showed the longest survival, and 55% of the mice achieved CR (Fig. 5E). In the IL15-IT group, tumors were well controlled during IL15 administration (Fig. 5D), but grew rapidly after IL15 administration was completed. To evaluate the development of anticancer immunologic memory, mice achieving CR were reinoculated with MC38 cells approximately 13 weeks after the initial NIR-PIT on the contralateral dorsum (Fig. 5F). Although allograft rejection was not observed in control mice, all the CR mice completely rejected the reimplanted MC38 cells (Fig. 5G and H). These results indicated that anticancer immunologic memory was established after intratumoral IL15 injection combined with CD44-targeted NIR-PIT.

To further evaluate the role of CD8⁺ T cell immunity in the combination therapy, we tested the efficacy of intratumoral IL15 injection combined with CD44-targeted NIR-PIT using athymic nude mice which lack T cells but have functional NK cells (Supplementary Fig. S9A–S9C). Tumor growth was significantly slower in the combination group compared with the control group (Supplementary Fig. S9D), but the combination therapy did not improve survival, and no mice achieved CR (Supplementary Fig. S9E). These results suggest that CD8⁺ T cell immunity plays a pivotal role in anticancer immune responses in the setting of the combination therapy, albeit with a partial contribution from NK cells.

As intratumorally administered IL15 binds to intratumoral immune cells, it is postulated that intratumoral IL15 injection may be more efficacious in highly immunogenic tumors than in nonimmunogenic tumors. However, our previous studies have demonstrated that CD44-targeted NIR-PIT can convert poorly immunogenic tumor microenvironments into highly immunogenic ones (21). Thus, we hypothesized that intratumoral IL15 injection combined with CD44-targeted NIR-PIT might be effective against nonimmunogenic pancreatic cancer models that are refractory to standard anticancer therapeutics (29). Accordingly, we used Pan02-luc tumors as a pancreatic cancer model. Multiplex IHC showed a lower density of TILs in Pan02-luc tumors compared with MC38 tumors (Supplementary Fig. S10). Pan02-luc cells highly expressed CD44, and CD44-targeted NIR-PIT was effective against them in vitro (Supplementary Fig. S11). Next, we compared the therapeutic efficacy of CD44-targeted NIR-PIT, intratumoral IL15 injection, and their combination in vivo using mice with Pan02-luc tumors reaching approximately 50 to 150 mm³ in volume. The treatment schedule is shown in Fig. 6A. Confirmation of photobleaching at the tumor site via 700-nm fluorescent imaging was obtained upon exposure to NIR light (Fig. 6B). Tumor growth was significantly suppressed in the Combination group compared with the other three groups (Fig. 6C). Moreover, the Combination group showed significantly longer survival compared with the other three groups (Fig. 6D). However, none of the mice in the Combination group attained CR; while some tumors temporarily disappeared after treatment they recurred in 5 of the 10 mice (50%).

Finally, we assessed the development of systemic anticancer immunity using bilateral tumor models. To examine whether CD44-targeted NIR-PIT can induce abscopal effects, we treated only the right MC38 tumor, leaving the left MC38 tumor untreated (Supplementary Fig. S12). The growth of the untreated tumors was similar between the Control and NIR-PIT groups, indicating that CD44-targeted NIR-PIT did not induce abscopal effects in distant untreated tumors.

Subsequently, we tested whether local IL15 administration combined with CD44-targeted NIR-PIT showed anticancer effects in untreated tumors. Intratumoral IL15 injection may leak into contralateral untreated tumors. Thus, we developed mice with hIL15-MC38 tumors on the right dorsum and MC38 tumors on the left dorsum and administered NIR-PIT only to the right hIL15-MC38 tumor (Fig. 7A and B). Immediately after NIR-PIT, a marked reduction in the 700-nm fluorescent signal was observed in the right tumor site, while the left tumor site remained unchanged (Fig. 7C). The NIR-PIT group exhibited a significant decrease in the growth of the treated tumors compared with the Control group (Fig. 7D). Notably, the untreated tumors in the NIR-PIT group also displayed a slower rate of growth when compared with the Control group (Fig. 7D). In addition, the NIR-PIT group displayed a significantly prolonged survival rate in comparison with the Control group (Fig. 7E). We further analyzed TILs in the left untreated tumors four days after CD44-targeted NIR-PIT. The NIR-PIT group exhibited a significantly higher number of CD8⁺ T, NK, and NKT cells compared with the Control group (Fig. 7F; Supplementary Fig. S13). Moreover, the NIR-PIT group showed significantly higher GZMB expression in CD8⁺ T and NK cells compared with the Control group (Fig. 7G, left and middle). Although there was an increase in GZMB expression in NKT cells in the NIR-PIT group compared with the Control group, this difference was not statistically significant (Fig. 7G, right). These results indicated that local IL15 administration combined with CD44-targeted NIR-PIT elicited systemic anticancer immunity and abscopal effects in distant untreated tumors.

In this study, we demonstrate that locally administrated IL15, either injected intratumorally or produced by the cancer cells, exhibited synergistic therapeutic efficacy with CD44-targeted NIR-PIT in two mouse models. In both models, the combination therapy resulted in significantly slower tumor growth and prolonged survival compared with NIR-PIT alone. We confirmed that the combination therapy in the IL15 auto-secretion model resulted in enhanced DC maturation and tumor infiltration of cytotoxic CD8⁺, NK, and NKT cells. In mice that achieved CR after intratumoral IL15 and NIR-PIT, tumor rejection upon rechallenge was successful, indicating that immunologic memory had been generated. Furthermore, the combination therapy induced abscopal effects in distant untreated tumors in bilateral tumor models in which only one tumor was treated. Therefore, local IL15 administration is an effective adjuvant to NIR-PIT, and its combination with cancer cell-targeted NIR-PIT can be utilized as an effective anticancer treatment strategy.

Previously we reported the efficacy of intraperitoneal IL15 administration combined with CD44-targeted NIR-PIT using syngeneic murine cancer models (30). This combination therapy was effective, but it did not induce CR (30). In this study, the dose of CD44-IR700 was reduced to half of that in the previous study (30). Nevertheless, intratumoral IL15 injection combined with NIR-PIT could achieve a higher CR rate in the MC38 tumor model. This suggests that intratumoral IL15 administration is more efficacious as an anticancer immune booster when combined with NIR-PIT. By locally administering IL15, a high concentration of IL15 in the tumor microenvironment can directly stimulate intratumoral immune cells. Thus, NIR-PIT–activated cytotoxic CD8⁺ T cells could be efficiently expanded by intratumorally administered IL15.

Prior to combining it with NIR-PIT, we demonstrated that intratumoral injection of IL15 was more effective than intraperitoneal injection in suppressing tumor growth and inducing intratumoral immune responses. In the two models we used, IL15-auto-secretion and intratumoral IL15 injection, it is difficult to directly compare efficacy. For instance, establishing accurate intratumoral IL15 concentrations in the IL15 auto-secretion model is challenging. We speculated that intratumoral IL15 concentrations are likely lower in the IL15-auto-secretion model than in the intratumoral IL15 injection model (Supplementary Fig. S2). This may partially explain the variation in CR rates between the two models. Higher intratumoral IL15 concentrations are crucial for therapeutic effectiveness. In IL15-rich tumor microenvironments, IL15Rα-expressing monocytes can more efficiently trans-present IL15 to CD8⁺ T and NK cells expressing IL15Rβ/γC subunits (23, 31). In addition, a high concentration of IL15 may modestly stimulate CD8⁺ T and NK cells without IL15 trans-presentation (1). Thus, augmenting intratumoral concentrations of IL15 through molecular engineering may be a potential strategy for improving treatment success (9). Optimization of local administration methods, such as cancer-targeted vaccines and viruses that induce IL15 secretion (9), may further improve this combination therapy.

We revealed significant efficacy of local IL15 administration combined with CD44-targeted NIR-PIT in a Pan02-luc tumor model. Pancreatic cancer is well known to respond poorly to immunotherapy and is often refractory to many standard anticancer agents (32). Pan02-luc tumors share these features with clinical pancreatic cancer (29, 33). Although no mice achieved CR after the combination therapy, tumors temporarily disappeared, and tumor growth was well controlled in 50% of the mice. This result suggests that local IL15 administration combined with CD44-targeted NIR-PIT can treat nonimmunogenic tumors by inducing an immune response.

This study has several limitations. First, human IL15, but not murine IL15, was used in this study. However, the utilization of human IL15 in murine models is substantiated by multiple studies, wherein murine IL15Rα trans-presented human IL15 to murine CD8⁺ T cells, NK cells, and NKT cells, resulting in potent immunostimulatory effects that were comparable with those elicited by trans-presentation of murine IL15 (34–36). Second, CD44-targeted NIR-PIT may deplete a subset of antigen-presenting cells because they express CD44 (24). Because the number of intratumoral DCs, monocytes, and macrophages was not different between the hIL15-MC38/Control and hIL15-MC38/NIR-PIT groups two days after NIR-PIT, they would have been supplied from neighboring tissues and exposed to IL15 immediately after NIR-PIT. NIR-PIT using targets that can selectively kill only cancer cells might provide even higher therapeutic efficacy. Third, the phenotype of hIL15-MC38 cells is slightly different from that of MC38 cells. hIL15-MC38 tumors had significantly higher Ki-67 positivity than MC38 tumors, implying that hIL15-MC38 tumors were more biologically aggressive and exhibited more proliferation. We speculate that this might be the result of cancer cells overcoming a more immunogenic tumor microenvironment. This phenotypic difference may explain the low CR rate after CD44-targeted NIR-PIT against hIL15-MC38 tumors. Fourth, MC38 cells transfected with a control plasmid might have served as better control in this study. Fifth, orthotopic models, although technically difficult in our experiments, are more suitable for evaluating anticancer immunity induced by NIR-PIT (37). Finally, we did not determine the optimal therapeutic sequence of local IL15 administration and NIR-PIT.

In conclusion, we demonstrate that local IL15 administration combined with CD44-targeted NIR-PIT yielded synergistic anticancer immunity, resulting in eradication of the treated tumor, abscopal effects in distant untreated tumors, and establishment of immunologic memory. While technological advancements in local IL15 administration are necessary to efficiently administer IL15 and achieve higher intratumoral concentrations, our findings provide a rationale for exploring the therapeutic potential of local IL15 administration combined with cancer cell-targeted NIR-PIT in a clinical trial setting.

P.L. Choyke reports a patent for U.S. Government patents issued, licensed, and with royalties paid from Rakuten. No disclosures were reported by the other authors.

H. Fukushima: Investigation, methodology, and writing–original draft. A. Furusawa: Investigation, methodology, writing–review and editing. T. Kato: Investigation. H. Wakiyama: Investigation. S. Takao: Investigation. S. Okuyama: Investigation. P.L. Choyke: Supervision, writing–review and editing. H. Kobayashi: Resources, formal analysis, supervision, funding acquisition, validation, investigation, writing–original draft, project administration, writing–review and editing.

This work was supported by the Intramural Research Program of the NIH, NCI, Center for Cancer Research (award recipient: Hisataka Kobayashi, grant number: ZIA BC 011513).

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