IL-15 has recently emerged as a candidate immune-activator for the treatment of cancer [18]. IL-15 induces adaptive immune responses that include T-cell proliferation, the generation of cytotoxic T lymphocytes (CTL), stimulation of immunoglobulin synthesis by B cells, and the generation and persistence of natural killer (NK) cells. Some of its functions are shared with IL-2 [19]. However, IL-15 is not required for the maintenance of regulatory T cells (Tregs) that suppress antitumor immune responses, unlike IL-2 [20]. Therefore, IL-15 can induce protective effects against cancer development with minimal effects on Tregs. In preclinical studies, the administration of IL-15 has antitumor effects in several mouse tumor models [21–23]. We hypothesized that IL-15 administration following cancer-antigen targeted NIR-PIT could enhance tumor growth inhibition. The purpose of this study was to investigate the in vivo therapeutic efficacy of CD44-targted NIR-PIT and IL-15 treatment in syngeneic mouse models of cancer compared to either therapy alone.

#### **2. Results**

#### *2.1. E*ffi*cacy of CD44-Targeted NIR-PIT Combined with IL-15 Administration for MC38-luc Tumor*

In vitro and in vivo CD44 expression in all three tumor models (Figure S1) and cytotoxic effects of CD44 targeted NIR-PIT have been previously reported [24]. The NIR-PIT regimen and imaging protocol are depicted in Figure 1A. In the NIR-PIT treated group, 1 day after injection of anti-CD44-mAb-IR700, the tumors were exposed to 50 J/cm2 of NIR light on day 0. IR700 fluorescence signal in tumors decreased due to dispersion of fluorophore from dying cells and partial photo-bleaching in all cases (Figure 1B). To investigate tumor-killing efficacy after NIR-PIT, bioluminescence imaging was performed before and after treatment up to day 7 (Figure 1C). In most mice treated with NIR-PIT, luciferase activity decreased initially after NIR-PIT due to cell killing and then gradually increased as the tumor regrew (Figure 1C). In all treated groups luciferase activity was significantly lower at 2, 3, 4, 5, 6, and 7 days in the treatment groups than in the control group (*p* < 0.05, Tukey-Kramer test) (Figure 1D). CD44-targeted

NIR-PIT combined with IL-15 treatment showed significantly lower luciferase activity compared with CD44-targeted NIR-PIT alone at 5, 6, and 7 days after NIR-PIT, with IL-15 (*p* < 0.05, Tukey–Kramer test) (Figure 1D). Tumor volume in all treated groups was significantly less at 5, 7, and 10 days compared with controls (*p* < 0.05, Tukey–Kramer test) (Figure 1E). The combination group showed significantly greater tumor reduction compared with IL-15 treatment or CD44-targeted NIR-PIT groups at 5, 7, and 10 days (*p* < 0.05, Tukey–Kramer test) (Figure 1E). These data demonstrate that CD44-targeted NIR-PIT combined with IL-15 resulted in the slowest rate of tumor regrowth compared with other groups. The combined therapy also was associated with significantly prolonged survival compared with IL-15 treatment alone or CD44-targeted NIR-PIT alone (*p* < 0.05, log-rank test) (Figure 1F). Thus, our results demonstrate that CD44-targeted NIR-PIT combined with IL-15 administration produced superior in vivo therapeutic results compared with the other two monotherapies for MC38-luc tumors.

**Figure 1.** In vivo effect of CD44-targeted near infrared photoimmunotherapy (NIR-PIT) and interleukin-15 (IL-15) administration for MC38-luc tumor model. (**A**) NIR-PIT regimen. Bioluminescence and fluorescence images were obtained at each time point as indicated. i.p., intraperitoneal injection; i.v., intravenous injection. (**B**) Real-time in vivo IR700 fluorescence imaging of tumor-bearing mice before and approximately 10 min after NIR-PIT. The yellow arrows indicate the tumor locations. (**C**) In vivo bioluminescence imaging of tumor-bearing mice before and after treatment at the indicated timepoints. (**D**) Quantitative analysis of luciferase activity before and after treatment in tumor-bearing mice. *n* = 10/group, mean ± SEM; \*, *p* < 0.05, vs. the other groups; †, *p* < 0.05, vs. combination group; Tukey–Kramer test. (**E**) Tumor growth in control and treated groups. *n* = 10/group, mean ± SEM; \*, *p* < 0.05, vs. the other groups; †, *p* < 0.05, vs. combination group; Tukey–Kramer test. (**F**) Survival curves for control and treated groups. *n* = 10/group; \*, *p* < 0.05; NS, not significant; log-rank test with Bonferroni correction.

#### *2.2. Augmented CD8*+ *T Cells Infiltration in MC38-luc Tumors after CD44-Targeted NIR-PIT Combined with IL-15 Administration*

In order to assess the density of tumor infiltrating T cells, an important indicator of antitumor immune activity, multiplex immunohistochemistry (IHC) was performed in MC38-luc tumors (Figure 2, also see Figure S2). With IL-15 administration, the cell density of CD8 T cells within tumors significantly increased at day 4, however, this augmentation of CD8 T cells was less prominent at day 7, suggesting that the CD8 T-cell population seen at day 4 was short-lived. On the other hand, in the tumors treated with the combination therapy, the augmentation of CD8 T-cell infiltration was not observed at day 4. This result could be attributed to the cytotoxic effect of CD44-targeted PIT against activated immune cells which express CD44. However, the cell density of CD8 T cells inside the tumor tissue dramatically increased 7 days after the combination therapy. The infiltration of CD4 T cells also increased 7 days after the combination therapy. CD44 targeted NIR-PIT alone did not induce the increase of T cell population. These results indicated that only CD44-targeted NIR-PIT combined with IL-15 administration resulted in newly activated T cells within the tumor microenvironment.

The cell density of Tregs significantly increased in stroma 7 days after IL-15 administration and combination therapy. This was thought to be a secondary effect in response to the rapid increase of CD8 T cells.

We also tested the distribution of Granzyme B (Gzmb) expressing cytotoxic cells. With the combination of IL-15 and CD44-targeted NIR-PIT, the number of Gzmb-positive CD8 T cells in the tumor tissue significantly increased at day 7 after the therapy (Figure S3A,C). With IL-15 administration alone, we observed the increase of Gzmb-positive CD8 T cells was less significant. This result indicates that the combination therapy successfully increased the tumor-infiltration of fully differentiated effector T cells. In addition to CD8 T cells, after IL-15 administration or combination of IL-15 and CD44-targeted NIR-PIT, we also observed increased number of another Gzmb positive cytotoxic cells, which were not CD8- or CD4-positive, namely non-T cytotoxic cells (Figure S3A,B,D). Such non-T cytotoxic cells including NK cells were more prominent in day 4 than day 7, suggesting that IL-15 stimulates the cytotoxic activity of T and non-T cell population at acute and subacute phase of tumor rejection.

**Figure 2.** Immunohistochemical analysis after CD44-targeted NIR-PIT and/or IL-15 treatment in MC38-luc tumor model. (**A**) Representative multiplex immunohistochemistry (IHC) images in MC38-luc tumors 4 or 7 days after the treatments indicated. Antibody staining of CD8, CD4, FOXP3, and pan-Cytokeratin (pCK) are shown in magenta, green, yellow, and cyan respectively. Nucleus are stained with DAPI and shown in blue. Tumor areas are indicated in white dotted line. The inset in window d shows examples of CD8 T cell (gray filled arrowhead), CD4 T cell (open arrowhead), and Tregs (white filled arrowhead). Scale bar = 100 μm. (**B**) Cell density of CD8 T cells, CD4 T cells and Tregs in tumor and stroma calculated from IHC images. No Tx, no treatment; IL-15, IL-15 administration only; 44 PIT, CD44-targeted PIT only; combi, combination therapy of CD44-targeted PIT and IL-15 administration. Bars represent mean, dots represent individual samples, and error bars represent SEM. *n* = 4/group except *n* = 2 for CD44 day 4. \*\*, *p* < 0.05, \*\*, *p* < 0.01 \*\*\*, *p* < 0.0001; one-way ANOVA (followed by Dunnett's multiple comparison test, vs. no Tx).

#### *2.3. E*ffi*cacy of CD44-Targeted NIR-PIT Combined with IL-15 Administration for LL*/*2 Tumor*

The NIR-PIT regimen and imaging protocol are depicted in Figure 3A. In the NIR-PIT treated group, 1 day after injection of anti-CD44-mAb-IR700, the tumors were exposed to 50 J/cm2 of NIR light on day 0. IR700 tumor fluorescence signal decreased due to dispersion of fluorophore from dying cells and partial photo-bleaching (Figure 3B). Tumor volumes in the treated groups were significantly reduced at all time points after treatment compared to the control group (*p* < 0.05, Tukey–Kramer test) (Figure 3C). The combination group showed significantly greater tumor reduction compared with the IL-15 treatment group at 2, 5, 7, 10, and 12 days after NIR-PIT (*p* < 0.05, Tukey–Kramer test) (Figure 3C), and showed significantly greater tumor reduction compared with CD44-targeted NIR-PIT group at 5, 7, 10, and 12 days after NIR-PIT (*p* < 0.05, Tukey–Kramer test) (Figure 3C). In the long-term follow-up, the combination group had significantly prolonged survival after NIR-PIT compared with IL-15 treatment or CD44-targeted NIR-PIT group (*p* < 0.05, log-rank test) (Figure 4D). This combined therapy was superior therapeutically to the other two types of monotherapies in LL/2 tumors.

**Figure 3.** In vivo effect of CD44-targeted NIR-PIT and/or IL-15 administration in LL/2 tumor model. (**A**) NIR-PIT regimen. IR700 fluorescence images were obtained at each time point as indicated. i.p., intraperitoneal injection; i.v., intravenous injection. (**B**) Real-time in vivo IR700 fluorescence imaging of tumor-bearing mice before and approximately 10 min after NIR-PIT. The yellow arrows indicate the tumor locations. (**C**) Tumor growth in control and treated groups. *n* = 9/group, mean ± SEM; \*, *p* < 0.05, vs. the other groups; †, *p* < 0.05, vs. combination group; Tukey–Kramer test. (**D**) Survival curves for control and treated groups. *n* = 9/group; \*, *p* < 0.05; NS, not significant; log-rank test with Bonferroni correction.

#### *2.4. E*ffi*cacy of CD44-Targeted NIR-PIT Combined with IL-15 Administration for MOC1 Tumor*

Our previous study showed MOC1 tumor has low and more heterogeneous CD44 expression and CD44-targeted NIR-PIT was less effective against this type of tumor [13]. The NIR-PIT regimen and imaging protocol are depicted in Figure 4A. In the NIR-PIT treated group, 1 day after injection of anti-CD44-mAb-IR700, the tumors were exposed to 50 J/cm2 of NIR light on day 0. IR700 tumor fluorescence signal decreased due to dispersion of fluorophore from dying cells and partial photo-bleaching. (Figure 4B). The tumor volume in all treated groups was significantly reduced 4, 7, 11, 14, 21, 24, 28, and 31 days after NIR-PIT compared to the control group (*p* < 0.05, Tukey–Kramer test) (Figure 4C). The combination group showed significantly greater tumor reduction 17, 28, and 31 days after NIR-PIT compared to CD44-targeted NIR-PIT group (*p* < 0.05, Tukey–Kramer test). The combination therapy showed significantly prolonged survival compared to CD44-targeted NIR-PIT group, although IL-15 treatment group had no significant difference in survival compared to CD44-targeted NIR-PIT group (*p* < 0.05, log-rank test) (Figure 4D). This combined therapy was superior therapeutically to the other two types of single therapies in MOC1 tumors.

**Figure 4.** In vivo effect of CD44-targeted NIR-PIT and/or IL-15 administration in the MOC1 tumor model. (**A**) NIR-PIT regimen. IR700 fluorescence images were obtained at each time point as indicated. i.p., intraperitoneal injection; i.v., intravenous injection. (**B**) Real-time in vivo IR700 fluorescence imaging of tumor-bearing mice before and approximately 10 min after NIR-PIT. The yellow arrows indicate the tumor locations. (**C**) Tumor growth in control and treated groups. *n* = 10/group, mean ± SEM; \*, *p* < 0.05, vs. the other groups; †, *p* < 0.05, vs. combination group; Tukey–Kramer test. (**D**) Survival curves for control and treated groups. *n* = 10/group; \*, *p* < 0.05; NS, not significant; log-rank test with Bonferroni correction.

#### **3. Discussion**

The combination of IL-15 administration and CD44-targeted NIR-PIT showed superiority in survival to IL-15 treatment alone in all three tumor types tested here. IL-15 binds and sends signals through a heterotrimeric receptor composed of the IL-15-specific IL-15Rα, shared IL-2R/IL-15Rβ (CD122) with the IL-2 receptor, and the common type I cytokine receptor γ-chain (γc, CD132; [25,26]). IL-15 is highly bound through trans-presentation by IL-15Rα expressed on activated DCs or monocytes to the IL-2R/IL-15Rβ and γc on effector T, B, and NK cells [27]. When IL-15 binds its receptor, DCs can cause potent activation of the immune response [18,27]. Immunogenic cell death of cancer cells shortly after NIR-PIT releases damage-associated signals such as ATP, calreticulin, and high-mobility group box 1, which promotes DC maturation and subsequent effector T-cell activation mediated by IL-15 [3,14]. Thus, NIR-PIT and IL-15 administration trigger an immune response that is greater than either therapy alone.

In all tumor types used in this study, MC38-luc, LL/2, and MOC1, infiltration of Tregs was observed within the TME [24]. Tregs tend to attenuate antitumor activity and are, therefore, not necessarily a desired outcome. However, IL-15 may be a safer choice of cytokine than IL-2 because the former does not stimulate Tregs while the latter is required for Treg maintenance [20]. In our study, significant increases in stromal Tregs were observed after IL-15 administration and the combination therapy, which was likely to be a secondary effect in response to the rapid increase of CD8 T cells in the TME. Our results showed that CD44-targeted NIR-PIT combined with short-term IL-15 treatment increased the infiltration of fully differentiated CD8+ effector T-cells which contributed to significantly prolonged survival compared to

CD44-targeted NIR-PIT alone in all three types of tumors. Considering that administration of IL-15 alone only induced short-lived CD8 T cells, and CD44-targeted NIR-PIT alone did not increase the infiltration of T cells, the superior therapeutic effect of the combination therapy was thought to be the result of IL-15 mediated reinforcement of NIR-PIT-induced antitumor immune activation.

In addition to CD8 T cells, we observed increasing number of Gzmb-high non-T cytotoxic cells within a few days after IL-15 administration or the combination therapy. Since IL-15 is known to stimulate the cytotoxic function on both CD8 T and NK cells, these cells were most likely to be NK cells. Therefore, the combination therapy of CD44-targeted NIR-PIT and IL-15 administration demonstrate its therapeutic efficacy probably by stimulating both the NK-mediated innate immunity and T-cell mediated adoptive immunity.

This report represents an extension of previous studies which attempt to augment the immune response initiated by NIR-PIT. For instance, a previous study demonstrated that the combination of CD44-targeted NIR-PIT and either immune checkpoint blockade or Treg-targeted NIR-PIT showed inconsistent therapeutic efficacy among these three tumor models [13,14,24]. CD44-targeted NIR-PIT in combination with other therapies showed promising results in MC38-luc and LL/2 but was less effective against MOC1 tumor which has low CD44 expression and fewer CD44 positive tumor cells compared to MC38-luc and LL/2. On the other hand, in this study, the combination with IL-15 therapy was effective in all three tumor models including MOC1. Even though the release of damage-associated signals after NIR-PIT is likely to have been lower in MOC1 cells, IL-15 administration following NIR-PIT nevertheless contributed to enhancement of antitumor host immunity by activating tumor-attacking T and NK cells making this combination therapy similarly effective for all tumors. Further combination therapy of cancer-targeting NIR-PIT with both IL-15 and an immune-checkpoint blockade or Treg-targeting NIR-PIT could theoretically enhance tumor immunity. However, such therapies need to be designed cautiously since excessively enhanced immunity might induce cytokine storm as an adverse effect. Photodynamic therapy (PDT) is another therapeutic approach that utilizes light to treat cancer [28]. However, NIR-PIT is different from PDT in several aspects. Porphyrin-based photosensitizers used in conventional PDT do not selectively target cancer cells, resulting in damage to surrounding normal cells in organs or vasculatures. In contrast, because NIR-PIT induces selective immunogenic cell death only on targeted cancer cells in NIR light exposing tumor beds, it spares the normal cells including all immune cells in the TME allowing them to play a role in immune activation [3]. Therefore, rapid and effective activation of antitumor host immunity is induced by NIR-PIT [14], whereas such effects are not generally induced by nonselective cell killing with PDT.

This study had several limitations. First, we used subcutaneously implanted tumor models, whereas an orthotopic or transgenic mouse model would be considered as a superior clinically relevant animal model [29]. However, in the present study, it was technically important to obtain a consistent size, shape, and location of each tumor in order to fairly compare tumor growth between tested groups. The orthotopic model could produce variable results depending on surgical procedure for implanting tumor within the organ. Second, although CD44 is expressed on these syngeneic cancer cells, CD44 is also expressed on activated immune cells. CD44-targeted NIR-PIT could thus damage effector immune cells. Indeed, counts of all kinds of T cells decreased 4 days after CD44-targeted NIR-PIT alone. Thus, CD44 is not ideal as a tumor target. Since NIR-PIT is applicable to any antibody that targets tumor cell surface antigens [30], NIR-PIT using an anti-mouse EGFR antibody, the target molecule in NIR-PIT clinical trials, would have been preferable. However, there is no thoroughly established EGFR+ mouse tumor model, and commercially available anti-mouse EGFR Abs are too expensive for experimental cancer therapy studies. Further investigation is required to clarify the degree of enhancement of antitumor host immunity through NIR-PIT. Third, we may not have optimized the dosing of IL-15. IL-15 has a circulation half-life of around1h[31] that is longer than IL-2 (15 min), and only 5 μg of IL-15 was administered i.p. up to 1 week after CD44-targeted NIR-PIT. A more prolonged administration of IL-15 might have induced greater therapeutic effects albeit balanced by an increased risk of lymphoproliferative disorder or lymphoma, either of which was seen in the

IL-15 transgenic mouse [32]. In a previous article, Yu et al. reported that administration of IL-15 alone simultaneously activated both immune-system and negative regulatory checkpoints that might weaken the antitumor immune response [22]. To elucidate the reason why the treatment regimen of this combined NIR-PIT therapy with IL-15 was difficult to cure tumors, further investigation of the T cell status such as exhaustion, activation, and memory differentiation would be required.

#### **4. Materials and Methods**

#### *4.1. Cell Culture*

MC38 cells (murine colon cancer), which were generously provided by Dr. Thomas Waldmann, NIH [33] stably expressing luciferase (MC38-luc, generated via stable transduction with RediFect Red-Fluc lentivirus from PerkinElmer per manufacturer recommendations), which was purchased from ATCC (Rockville, MD, USA), LL/2 cells (murine Lewis lung carcinoma), which was purchased from ATCC (Rockville, MD, USA), and MOC1 cells (murine oral carcinoma), which were produced and generously provided by Dr. Clint Allen, NIH [34] were used in this study. Luciferase expression on the MC38-luc cells was confirmed through 10 passages. MC38-luc and LL/2 cells were cultured in RPMI1640 supplemented with 10% FBS and 1% penicillin–streptomycin (all Gibco brand, ThermoFisher Scientific, Waltman, MA, USA) in tissue culture flasks (182 cm2; CELLTREAT Scientific Products, Pepperell, MA, USA) in a humidified incubator at 37 ◦C in an atmosphere of 95% air and 5% carbon dioxide. MOC1 cells were cultured in HyClone Iscove's modified Dulbecco's medium (Crytiva, Marlborough, MA, USA)/HyClone Ham's Nutrient Mixture F12 (Crytiva, Marlborough, MA, USA) at a 2:1 mixture with 5% FBS, 1% penicillin/streptomycin, 3.5 ng/mL EGF (MilliporeSigma, Burlington, MA, USA), 40 ng/mL hydrocortisone (MilliporeSigma, Burlington, MA, USA), and 5 ng/mL insulin (MilliporeSigma, Burlington, MA, USA) in the tissue culture flasks in a humidified incubator at 37 ◦C in an atmosphere of 95% air and 5% carbon dioxide. Cells were authenticated via in vitro growth characteristics.

#### *4.2. Reagents*

Water soluble, silica-phthalocyanine derivative, IRDye700DX NHS ester was obtained from LI-COR Bioscience (Lincoln, NE, USA). An anti-mouse/human CD44 mAb (IM7) was purchased from Bio X Cell (Lebanon, NH, USA). All other chemicals were of reagent grade.

#### *4.3. Synthesis of IR700-Conjugated Anti-CD44 mAb*

Briefly, anti-CD44-IgG (1 mg, 6.7 nmol/L) was incubated with IR700 NHS ester (65.1 μg, 33.3 nmol, 10 mmol/L in DMSO) and 0.1 mol/L Na2HPO4 (pH 8.5) at room temperature for 1 h. The mixture was purified with a gel filtration column (Sephadex G 25 column, PD-10, Crytiva, Marlborough, MA, USA). The protein concentration was measured with Coomassie Plus protein assay kit (Thermo Fisher Scientific Inc, Waltman, MA, USA) by determining absorption at 595 nm with a UV-Vis spectroscopy system (8453 Value System; Agilent Technologies, Santa Clara, CA, USA). We abbreviate IR700-conjugated anti-CD44 mAb as anti-CD44-mAb-IR700.

#### *4.4. Animal Model*

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 on 12-02-2019 (MIP-003). Six- to eight-week-old female C57BL/6 mice (strain #000664) were purchased from the Jackson laboratory (Bar Harbor, ME, USA). The lower part of the body of the mice was shaved as fur can interfere with light activation of the conjugate and bioluminescence imaging. Mice with tumors reaching approximately 150 mm3 in volume were used for the experiments. Tumor volumes were calculated from the greatest longitudinal diameter (length) and the greatest transverse diameter (width) using the following formula; tumor volume = length × width2 × 0.5, based on caliper measurements. Mice were monitored each day and tumor volumes were measured three times a week for MC38-luc

and LL/2 tumors and twice a week for MOC1 tumors until the tumor volume reached 2000 mm3, whereupon the mice were euthanized by inhalation of carbon dioxide gas.

#### *4.5. In Vivo NIR-PIT*

MC38-luc cells (8 million), LL/2 cells (8 million), and MOC1 cells (4 million) were subcutaneously injected in the dorsum of mice. Once tumors reached volumes of approximately 150 mm3 mice were divided randomly into one of the following four experimental groups: (1) no treatment (control); (2) intraperitoneal injection of 5 μg murine IL-15 (PeproTech, Rocky Hill, NJ, USA) on days 0, 2, 4, and 6 following intravenous injection of 100 μg anti-CD44-mAb-IR700 (IL-15 treatment); (3) intravenous injection of 100 μg anti-CD44-mAb-IR700 followed by external NIR light irradiation at 50 J/cm2 on day 0 (CD44-targeted NIR-PIT); (4) intravenous injection of 100 μg anti-CD44-mAb-IR700 followed by external NIR light irradiation at 50 J/cm2 on day 0 and 100 J/cm2 on day 1 with intraperitoneal injection of 5 μg murine IL-15 on days 0, 2, 4, and 6 (combination). For the mice with MC38-luc tumor, LL/2 tumor, and MOC1 tumor in the NIR-PIT treated groups, intravenous injection of the anti-CD44-mAb-IR700 was performed 5, 5, and 28 days after tumor inoculation, respectively. NIR light was administered to tumor-bearing mice using a red-light emitting diode (LED), which emits light in the range of 670–710 nm wavelength (L690-66-60; Marubeni America Co., New York, NY, USA) at a power density of 50 mW/cm2 as measured with an optical power meter (PM 100, Thorlabs, Newton, NJ, USA). IR700 absorbs light at approximately 690 nm. IR700 fluorescence images were obtained before and after therapy.

#### *4.6. In Vivo Bioluminescence Imaging and IR700 Fluorescence Imaging*

To obtain bioluminescence images in MC38-luc tumor-bearing mice, D-luciferin (15 mg/mL, 150 mL; Goldbio, St Louis, MO, USA) was intraperitoneally injected. Luciferase activity was analyzed with a bioluminescence imaging system (Photon Imager; Biospace Lab, Nesles la Vallée, FRANCE) using units of relative light units (RLU). Regions of interests (ROIs) were placed over the entire tumor. The counts per minute of RLUs were calculated using M3 Vision Software (Biospace Lab) and converted to a percentage ratio based on the following formula: [(RLU after treatment)/(RLU before treatment) × 100 (%); [35]]. Bioluminescence imaging was performed before and after NIR-PIT (protocol below) on day 0 to day 7. In vivo IR700 fluorescence images were obtained with a Pearl Imager (LI-COR Biosciences, Lincoln, NE, USA) using the 700-nm fluorescence channel.

#### *4.7. Multicolor Immunofluorescence*

Multicolor immunofluorescence was performed using Opal 7-Color Automation immunohistochemistry staining Kit (Akoya Bioscience, Menlo Park, CA, USA) and BOND RXm auto stainer (Leica Biosystems, Wetzlar, Germany). The following antibodies were used: anti-CD44 (clone IM7; 1:5000 dilution; Bio X Cell, Lebanon, NH, USA), anti-CD8 (clone EPR20305; 1:500 dilution; Abcam, Cambridge, MA, USA), anti-CD4 (clone EPR19514; 1:1000 dilution; Abcam), anti-FOXP3 (clone 1054C; 1:1000 dilution; Novus Biologicals, Littleton, CO, USA), anti-pan cytokeratin (rabbit poly; 1:500 dilution, Bioss, Woburn, MA, USA), anti-CD45 (clone D3F8Q, 1:500 dilution, Cell Signaling Technology, Danvers, MA, USA), and anti-Granzyme B (rabbit poly; 1:500 dilution, Abcam). The staining was performed according to the Opal 7 color protocol provided by manufacturer with following modification: (i) antigen retrieval was performed using BOND ER2 solution (Leica Biosystems, Wetzlar, Germany) for 20 min and (ii) the ImmPRESS HRP anti-Rabbit IgG (Peroxidase) Polymer Detection Kit (Vector Laboratories, Burlingame, CA, USA) was used instead of anti-mouse/human secondary antibody provided in the kit. Stained slides were mounted with VECTASHIELD Hardset Antifade Mounting Medium (Vector Laboratories) and then imaged using Mantra Quantitative Pathology Workstation (Akoya Biosciences). Images were analyzed with inForm software (Akoya Biosciences). inForm software was trained to detect tissues and cell phenotypes according to following criteria: areas with pan-cytokeratin expression = tumor, other areas = stroma, CD4+FOXP3+ cells = Tregs, CD4+FOXP3− = CD4 T cells,

CD8+ = CD8+ T cells, respectively. At least five images were taken from each tumor sample, areas for each tissue phenotype and cell count of each phenotype were combined to calculate the cell density. As for Gzmb expression analysis, cell phenotyping (CD8+/CD45+ = CD8+ T cells, CD4+/CD45+ = CD4+ T cells, Gzmb+/CD8−/CD4− = non-T-cell cytotoxic cells) and Gzmb expression analysis (Gzmb + and other) were performed separately using inForm 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.

#### *4.8. Statistical Analysis*

Quantitative data were expressed as means ± SEM. The Mann–Whitney U test was used to compare differences between two groups. For multiple comparisons (≥3 groups), a one-way analysis of variance (ANOVA) followed by the Tukey–Kramer test or Dunnett's multiple comparison test was used. The cumulative survival was analyzed by the Kaplan–Meier survival curve analysis, and the differences between tested groups were compared using the log-rank test followed by Bonferroni correction. Statistical analysis was performed with JMP 13 software (SAS Institute, Cary, NC, USA). A *p* value of less than 0.05 was granted significant.

#### **5. Conclusions**

CD44-targeted NIR-PIT combined with IL-15 treatment showed superior in vivo therapeutic efficacy to either CD44-targeted NIR-PIT or IL-15 treatment alone in colon, lung, and oral cancer models. IL-15 might have potential to become a versatile adjuvant after cancer NIR-PIT.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2072-6694/12/9/2575/s1, Figure S1: CD44 expression within MC38-luc (A), LL/2 (B), and MOC1 (C) tumors, Figure S2: Tissue and cell phenotyping based on the multiplex immunohistochemistry (IHC), Figure S3: Distribution of Granzyme B (Gzmb) expressing cytotoxic cells within the treated tumors.

**Author Contributions:** Y.M. mainly designed and conducted experiments, performed analysis, and wrote the manuscript; A.F., R.O., F.I., H.W., T.K., and T.N. performed experiments and analysis; P.L.C. wrote the manuscript and supervised the project; and H.K. planned and initiated the project, designed and conducted experiments, wrote the manuscript, and supervised the entire project. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research (ZIA BC011513).

**Acknowledgments:** This research was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research (ZIA BC011513). FI was also supported with a grant from National Center for Global Health and Medicine Research Institute, Tokyo, Japan.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Review* **Nonreplicating Adenoviral Vectors: Improving Tropism and Delivery of Cancer Gene Therapy**

**Nayara Gusmão Tessarollo, Ana Carolina M. Domingues, Fernanda Antunes , Jean Carlos dos Santos da Luz, Otavio Augusto Rodrigues, Otto Luiz Dutra Cerqueira and Bryan E. Strauss \***

> Viral Vector Laboratory, Center for Translational Investigation in Oncology, Cancer Institute of São Paulo/LIM24, University of São Paulo School of Medicine, São Paulo 01246-000, Brazil<EMAIL_ADDRESS>(N.G.T<EMAIL_ADDRESS>(A.C.M.D<EMAIL_ADDRESS>(F.A<EMAIL_ADDRESS>(J.C.d.S.d.L<EMAIL_ADDRESS>(O.A.R<EMAIL_ADDRESS>(O.L.D.C.)

**\*** Correspondence<EMAIL_ADDRESS>or<EMAIL_ADDRESS>
**Simple Summary:** The treatment of cancer has progressed greatly with the advent of immunotherapy and gene therapy, including the use of nonreplicating adenoviral vectors to deliver genes with antitumor activity for cancer gene therapy. Even so, the successful application of these vectors may benefit from modifications in their design, including their molecular structure, so that specificity for the target cell is increased and off-target effects are minimized. With such improvements, we may find new opportunities for systemic administration of adenoviral vectors as well as the delivery of strategic antigen targets of an antitumor immune response. We propose that the improvement of nonreplicating adenoviral vectors will allow them to continue to hold a key position in cancer gene therapy and immunotherapy.

**Abstract:** Recent preclinical and clinical studies have used viral vectors in gene therapy research, especially nonreplicating adenovirus encoding strategic therapeutic genes for cancer treatment. Adenoviruses were the first DNA viruses to go into therapeutic development, mainly due to well-known biological features: stability in vivo, ease of manufacture, and efficient gene delivery to dividing and nondividing cells. However, there are some limitations for gene therapy using adenoviral vectors, such as nonspecific transduction of normal cells and liver sequestration and neutralization by antibodies, especially when administered systemically. On the other hand, adenoviral vectors are amenable to strategies for the modification of their biological structures, including genetic manipulation of viral proteins, pseudotyping, and conjugation with polymers or biological membranes. Such modifications provide greater specificity to the target cell and better safety in systemic administration; thus, a reduction of antiviral host responses would favor the use of adenoviral vectors in cancer immunotherapy. In this review, we describe the structural and molecular features of nonreplicating adenoviral vectors, the current limitations to their use, and strategies to modify adenoviral tropism, highlighting the approaches that may allow for the systemic administration of gene therapy.

**Keywords:** nonreplicating adenovirus vector; cancer; gene therapy; routes of delivery; virus coated with cancer cell membrane

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **1. Overview: Structural and Molecular Features of Nonreplicating Adenoviral Vectors**

Adenoviruses (Ads) are one of the most well-studied and widely used viral vectors, representing 17.5% (*n* = 573) of vectors used in gene therapy clinical trials [1]. The first gene therapy was approved in 2003 by the China Food and Drug Administration. Gendicine is a recombinant nonreplicating adenovirus encoding human p53 and, despite more than 17 years of commercial use, it has only been tested in clinical trials in China for the treatment of hepatocellular, nasopharyngeal, gastric, liver, lung, breast, prostate, ovarian, and head and neck cancer, either alone or in combination with radio- or chemotherapy [2]. This

#### **Citation:** Tessarollo, N.G.;

Domingues, A.C.M.; Antunes, F.; Luz, J.C.d.S.d.; Rodrigues, O.A.; Cerqueira, O.L.D.; Strauss, B.E. Nonreplicating Adenoviral Vectors: Improving Tropism and Delivery of Cancer Gene Therapy. *Cancers* **2021**, *13*, 1863. https://doi.org/10.3390/ cancers13081863

Academic Editors: Michael Kershaw and Clare Slaney

Received: 26 February 2021 Accepted: 6 April 2021 Published: 14 April 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

therapy serves to illustrate the potential for using nonreplicating adenoviral vectors as part of effective cancer treatment.

Recently, nonreplicating adenoviral vectors have gained attention due to their use in the development of vaccines, especially to combat SARS-CoV-2, the novel coronavirus. These vaccines include those based on recombinant adenovirus serotypes, such as human adenovirus vector 5 [3–7], chimpanzee adenovirus vector ChAdOx1 [8,9], and combined human serotypes vectors 5 and 26 [10,11]. The Ad vectors can elicit robust and durable cellular and humoral immune responses [12]. The induction of a balanced innate immune response makes the Ad vectors good candidates for vaccine platforms, and they can also play a role in cancer gene therapy.

Replicating adenoviral vectors are used for the induction of oncolysis (also referred to as oncolytic adenovirus or virotherapy), and these vectors have played a major role in showing the potential of adenoviruses in cancer immunotherapy. The use of nonreplicating adenoviral vectors also deserves particular attention. We argue that nonreplicating vectors perform quite well and may even offer advantages when compared to the use of their replicating counterparts, especially concerning the delivery of proimmune but antiviral transgenes. While we do not discount oncolytic viruses, we do support the continued development of nonreplicating adenoviral vectors for cancer immunotherapy. As shown in Table 1, many clinical trials that are underway involve the use of nonreplicating adenoviral vectors for cancer gene therapy. In this review, we focus on nonreplicating adenoviral vectors, discussing vector biology and current barriers to cancer gene therapy. We also propose some strategies that enhance vector performance, especially in terms of virus delivery and targeting, thus supporting the use of nonreplicating adenoviruses for cancer immunotherapy.

**Table 1.** Clinical trials using nonreplicating adenoviral vectors for cancer gene therapy.

**Table 1.** *Cont.*

Human adenoviruses (HAds) are subdivided into seven species (A–G) and >50 serotypes based on serological properties, DNA homology, genome organization, and oncogenicity [24]. More than 100 types of human adenovirus and >200 nonhuman Ad serotypes have been identified to date [25].

Ads carry a linear double-stranded DNA genome (26 to 46 kb in length) and core proteins inside an icosahedral capsid [26]. The Ad DNA genome contains two inverted terminal repeats (100–140 bp) and can be divided into five early genes and five late genes. The capsid facets are formed by structural proteins, mainly composed of hexons, and each vertex contains a penton base that anchors the trimeric protein fiber, divided into the fiber knob and shaft. The viral particles have around one million amino acid residues (weight around 150 MDa) and an average size of 90–100 nm [27]. In the infection process, the knob interacts with cell surface receptors such as the coxsackievirus and adenovirus receptor (CAR), CD46, CD80/86, and desmoglein 2 (DSG2) [28,29]. This interaction leads to viral particle immobilization, which facilitates the interaction between the penton base and integrins [30] and, thus, virion internalization. The DNA and some core proteins are transported through the microtubular complexes to the nuclear pore and are introduced into the cell's nucleus [31]. Inside the nucleus, the viral DNA remains episomal, and the expression of the early genes (E1A, E1B, E2, E3, and E4) suppresses transcription from the host genome, thus favoring adenovirus protein synthesis and replication. Then, the late genes (L1–L5) are expressed, leading to virus encapsulation and viral particle maturation in the nucleus during the completion of the lytic cycle. Nuclear and cytoplasmatic membranes are disrupted, and new virions are released from permissive cells 48–72 h after infection [32,33].

Many characteristics of adenoviruses, such as their safety, broad cell tropism, and ability to stimulate a robust immune response, favor their use as a viral vector platform employed as a gene delivery tool in gene therapy, as an oncolytic cancer treatment, and in the development of vaccines [12,32]. Moreover, the genome of Ads is well characterized, genetically stable, and does not integrate into the host's genome but remains as episomal DNA in the cell nucleus. In addition, adenoviral vectors are modified to control viral replication, have a large cloning capacity (up to 37 kb), can transduce both dividing and quiescent cells, and have high in vivo transduction efficiency [34–36]. Human adenovirus type 5 (HAd5) is the most frequently used adenovirus for the development of gene therapy vectors, which promote the expression of transgenes in the target cells yet have impaired replication and, hence, prevent unwanted virus spread. There are three generations of nonreplicating adenoviral vectors used in gene therapy. In the first generation, E1 and E3 early genes were deleted, rendering vector replication defective but maintaining the ability to transduce host cells without killing them and liberating ~8 kb of space in the genome for the genetic payload (transgene(s) plus regulatory sequences) [37,38]. Since the E1 region is essential for virus replication, E1A proteins induce the transcription of Ad genes, and E1B proteins inhibit cellular apoptosis. Vector production requires that the E1 gene be supplied by transcomplementation, using cell lines (such as HEK293 or PERC.6) that were modified to incorporate the viral E1 region [39,40]. For the second-generation adenoviral vectors, beyond E1/E3 deletions, E2 or E4 regions have also been removed, providing additional space for cargo sequences (~10.5 kb). Third-generation adenoviral vectors were generated after deletion of almost all viral sequences except for the ITRs, the packaging signal, and minimal sequences required for genome replication and encapsulation during vector production [41].

Therefore, nonreplicating adenoviral vectors, different from their replicating counterparts, do not provoke the same cellular responses due to their lack of viral protein expression, absence of viral genome replication, and deficiency in the ability to induce cytopathic effects.

#### **2. Current Applications of Nonreplicating Adenoviral Vectors in Cancer Immunotherapy**

There are two main routes to delivering gene therapy vectors: ex vivo and in vivo. In vivo gene transfer raises concerns related to the specificity of vector transduction and transgene transcription in the intended target cells in order to achieve the desired therapeutic outcome, a goal that may be compromised by off-target effects. Ex vivo gene transfer occurs outside the body, where the patient's cells are modified and reinfused. Here, we focus on the in vivo route, particularly the challenges associated with the antiviral immune response.

In general, gene therapy approaches that overcome the immunosuppressive tumor microenvironment (TME) and activate an antitumor immune response are expected to function as cancer immunotherapies. To this end, adenoviral vectors have been modified with a variety of immune-stimulating genes, such as cytokines, costimulatory molecules, tumor-associated antigens, and tumor-suppressor genes [42]. The purpose is not only the direct killing of tumor cells but also the activation of immune cells to attack the tumor. Thus, gene therapy may induce immunogenic cell death and/or the liberation of factors that will then go on to promote the immune response.

The adenoviral vector itself is expected to participate in the activation of an antiviral response that may be both an asset and a complication for gene therapy since attracting the immune response to the tumor site is desirable but the inhibition of viral activity may thwart treatment. After viral entry, pathogen-associated molecular patterns (PAMPs), including viral nucleic acids and viral capsids, are recognized by pattern recognition receptors (PRRs) and activate antiviral immune responses that result in the production of type-I interferons (IFNs), proinflammatory cytokines, and chemokines.

Another important signaling cascade stimulated by the interaction of the virus with CAR and αv integrins is nuclear factor-kB (NF-kB), which mediates the expression of chemokines and interleukin (IL)-1 [43]. Inside the cell, viral DNA is sensed by several cytosolic PRRs such as Toll-like receptor (TLR)-9 [44], DNA-dependent activator of IFNregulatory factors (DAIs) [45], cytosolic inflammasomes (NALP3) [46], and nucleotidebinding oligomerization domain-like receptors (NOD-like receptors (NLRs)) [47]. As a result, a signaling cascade is initiated, either dependent or independent of myeloid differentiation primary response gene 88 (MyD88), which culminates in the transcription factor (NF-kB, IRF3, IRF7)-mediated expression of IFN-α, IFN-β, and IL-6, among other proinflammatory cytokines and chemokines. In turn, the immunosuppressive TME is modulated to facilitate the recruitment of antigen-presenting cells (APCs) and helper and cytotoxic T-cells. Adenoviral vectors can be especially useful in the treatment of cold tumors [48], which lack immune infiltrate, although the increase in immune cell infiltration may not be enough for the eradication of the tumor [49]. Thus, the approach may be improved if the vector is armed with additional immune-stimulating factors.

Replication-deficient adenoviral vectors have been employed as vaccines and in cancer gene therapy due to strong humoral and T-cell responses to transgenes expressed by the vector [50]. Tatsis et al. (2007) showed that the application of replication-defective adenoviral vectors resulted in sustained levels of CD8+ T-cells specific for the transgene product and persistent levels of transcriptionally active adenoviral vector genomes at the site of inoculation in the liver and lymphatic tissues [51]. In comparisson, replicating adenoviral vectors mimic natural virus infection, resulting in the induction of cytokines and costimulatory molecules that provide a potent adjuvant effect [52]. Both nonreplicating and replicating Ad vectors have been shown to activate effector CD8+ T-cells and central memory T-cells in treated mice. For this, Osada et al. [53] compared Ad5[E1+]CEA, a replicating adenoviral vector carrying the carcinoembryonic antigen (CEA) with two nonreplicating vectors, Ad5[E1−] and Ad5[E1−, E2b−]. When used for the ex vivo transduction of human dendritic cells (DCs), they found that all three vectors yielded similar infectivity and temporal dynamics of transgene expression. In addition, replicating Ad5[E1+]CEA showed toxicity to DCs, eliciting less maturation of DCs and greater clearance by NK cells. Moreover, Ad5[E1−] and Ad5[E1−, E2b−] were superior to Ad5[E1+] in their capacity to induce and expand antigen-specific T-cell responses. The results suggest that increased replication of an Ad vector may result in diminished efficacy in this scenario, and the deletion of E1, E2, and E3 genes promoted a superior generation of CEA-specific T-cell responses in mice with pre-existing Ad5 immunity.

In the following discussion, we highlight some of the strategies for using nonreplicating adenoviral vectors as cancer immunotherapies in preclinical and clinical assays.

For example, preclinical outcomes in a prostate cancer model have revealed the benefit of immunotherapy based on a heterologous prime-boost, where the virus is injected as a vaccine with concomitant administration of a PD-1-blocking antibody. Similarly, a ChAdOx1–MVA vaccination strategy (a simian adenovirus, ChAdOx1, with the modified vaccinia Ankara virus, MVA) induced CD8+ T-cell responses to the tumor-specific selfantigen of prostate 1 (STEAP1) in murine models. The combination with the anti-PD-1 antibody improved the survival of the animals since tumors were abolished in 80% of the mice [54].

Our laboratory has developed an adenoviral vector, AdRGD-PG, with improved tropism and transgene expression. By including the RGD motif in the fiber knob, transduction no longer relies on CAR but instead uses integrins, which are more widely distributed. The use of a p53-responsive promoter (called PG) to control transgene expression resulted in high-level expression in the presence of wild-type p53 [55,56]. When used to deliver p19Arf (a functional partner of p53) and IFN-β, we observed cooperation between these genes for the induction of cell death in vitro and in vivo using the mouse model of melanoma, B16–F10 [56,57]. Moreover, only combined gene transfers conferred the emission of immunogenic cell death markers ATP, calreticulin, and HMGB1 [56]. The combination of p19Arf and IFN-β proved to be an effective immunotherapy since we confirmed the participation of natural killer (NK) cells and CD4+ and CD8+ T-lymphocytes in immune protection against B16-F10 tumor progression [58]. Other assays showed that the gene transfer of p19Arf and IFN-β using our nonreplicating Ad vector in the LLC1 mouse model of lung carcinoma was able to induce markers of immunogenic cell death. In situ gene therapy with IFN-β, either alone or in combination with p19Arf, could retard tumor progression, but only the combination approach limited the progression of challenge tumors, thus acting as an in situ vaccine [59]. Thus, the p19Arf + IFN-β gene transfer approach induces oncolysis and immune activation even in the absence of viral replication, functioning as a cancer vaccine and immunotherapy, at least in mice [60,61].

We have taken great care to use different models to demonstrate the functionality of our approach since it involves IFN-β, which is known to have species-specific activities [62]. To examine our approach in human melanoma cell lines, we used the AdRGD-PG backbone to construct vectors encoding the human cDNAs p14ARF and hIFN-β and showed immunogenic cell death characterized by the emission of critical markers in vitro as well as successful ex vivo priming of human T-cells [63].

Most of the clinical trials using nonreplicating Ads are in Phase I/II (Table 1). Kumon et al. [13] have demonstrated in preclinical and clinical data the benefit of in situ Ad-REIC (adenoviral vector carrying the human *REIC*/*Dkk*-*3* gene) treatment. In preclinical data, they showed that Ad-REIC induces selective toxicity in response to endoplasmic reticulum stress and IL-7 overproduction by infected normal cells, including cells of the TME. These cells can activate innate immunity, especially NK cells, as well as cytotoxic T-lymphocytes (CTLs). In addition, DCs induced by secreted REIC proteins can present cancer antigens from apoptotic cancer cells and induce tumor-associated antigen-specific CD8+ CTLs. In clinical settings, preliminary outcomes have shown cytopathic effects and tumor-infiltrating lymphocytes in patients with high-risk localized prostate cancer, undergoing radical prostatectomy, who received two ultrasound-guided intratumoral injections at 2-week intervals, followed by surgery six weeks after the second injection.

Another interesting approach is gene-mediated cytotoxic immunotherapy (GMCI), which uses an adenoviral vector expressing the herpes simplex virus (HSV) thymidine kinase (TK) gene (ADV/HSV-TK), followed by an antiherpetic prodrug. The HSV-TK protein has two principal functions: (1) nucleotide analog products of prodrug phosphorylation lead to the death of dividing cancer cells, and (2) TK is a superantigen that stimulates a potent immune reaction [64]. GMCI activates the stimulator of interferon genes (STING) pathway, enhancing the production of proinflammatory cytokines such as IFNs and promoting T-cell activation. The first study using ADV/HSV-TK plus ganciclovir for the treatment of human prostate cancer was conducted by Herman et al. [65]. The patients received a single injection of the vector (108 to 1011 vector particles) into the prostate gland in the region with the greatest concentration of tumor cells. Not only did the regimen prove safe, with minimal toxicity, but three patients that received 109–1011 viral particles had a decrease of more than 50% in serum prostate-specific antigen (PSA) levels for periods ranging from 45 to 330 days. The safety and efficacy of GMCI to convert the TME from cold to hot have been noted for studies in different tumor types, including glioma [66], retinoblastoma [67], and mesothelioma [68].

 This disease is an early form of bladder cancer, and the recommended treatment for these patients is the use of intravesical Bacillus Calmette-Guérin (BCG). However, data has shown that around 30% to 50% of cases will recur. The outcomes of BCG-unresponsive patients are poor, and total cystectomy (complete removal of the bladder) is the standard of care for patients who are operative candidates [69].

Alternatively, patients with bladder cancer are treated with nadofaragene firadenovec (rAd–IFN-α2b/Syn3), a replication-deficient recombinant Ad carrying the interferon-α gene, which can have both TRAIL- and non-TRAIL-mediated cytotoxic effects. The patients receive the treatment directly into the bladder using a catheter every three months, and there is elevated interferon production and, consequently, increased exposure to urotheliumenhanced cytotoxic activity. Among 157 patients with carcinoma in situ, 53% of patients achieved a complete response in as early as three months, and about 24% of patients remained free of high-grade recurrence at one year. The outcomes are encouraging and currently awaiting Food and Drug Administration (FDA) approval [70].

As mentioned before, replication-defective Ads are also used as cancer vaccine strategies. GVAX, a GM-CSF gene-modified tumor vaccine, was developed by transducing autologous tumor cells with E1/E3-deleted Ad vectors encoding GM-CSF in autologous tumor cells extracted from each patient. In a phase I/II trial, 33 patients with NSCLCs that were refractory to standard treatment received the GVAX vaccine consisting of 5–100 × 106 irradiated tumor cells per dose, every 2 weeks. This strategy was shown to be safe, and three patients had radiologically complete responses that lasted for more than six months [71].

Nonreplicating Ads have also been exploited as delivery vehicles in dendritic cells (DCs). Briefly, viral particles processed via proteasome result in the presentation of self and foreign antigens by MHCI and MHCII molecules to both CD8+ and CD4+ T-cells, inducing protective humoral and cellular immunity [72]. A phase I/II clinical trial tested the immune response against a vaccine consisting of autologous DCs obtained from patients, transduced ex vivo with Ads encoding the full-length melanoma antigen MART-1/Melan-A. This study pointed to an increase in CD8+ and CD4+ T-cells in 6/11 and 2/4 metastatic melanoma patients, respectively [73].

Another phase I clinical trial performed on patients with advanced NSCLC showed the induction of systemic tumor antigen-specific immune responses with enhanced CD8+ T-cell infiltration of tumors in 7/13 of patients. The treatment consisted of two intradermal injections of autologous DCs, transduced ex vivo with an Ad vector expressing the CCL21 gene [74]. Similar outcomes have been observed in small cell lung cancer, where 41.8% of patients presented specific anti-p53 immune responses when treated with a vaccine consisting of DCs transduced with an Ad encoding p53 [75]. These positive outcomes are not limited to solid tumors. In a phase II study, acute myeloid leukemia (AML) patients with early molecular relapse received a modified DC vaccine. The DCs were modified

with two tumor-associated antigens (TAAs), survivin and MUC1, plus secretory bacterial flagellin for DC maturation and RNA interference to suppress SOCS1. The complete remission rate was 83% among all relapsed AML patients [76].

TAAs are usually expressed in normal tissues at low levels but overexpressed in tumor cells. Many TAAs have been identified as targets for tumor-reactive T-cells and can be isolated from tumor-infiltrating lymphocytes (TILs) [77]. In contrast, tumor-specific antigens (TSAs) are only encoded in cancer cells as a consequence of somatic mutations that alter the amino acid sequence, resulting in foreign proteins that can be presented to the immune system. Therefore, the neoantigens are less susceptible to the mechanisms of immunological tolerance, comprising an interesting target for vaccination [78]. Thus, gene-based vaccination using Ad vectors as a delivery agent is emerging as one of the most promising approaches for loading antigens (TAA or TSA) onto DCs. Important advantages of this modified DC approach include persistent expression of the antigen that results in activation of CD4+ and CD8+ T-cells and the induction of antibody responses and the natural adjuvant stimulating effect that Ads mediate, which contributes to DC maturation [79].

As mentioned above, ex vivo modification of DCs followed by the reintroduction of these cells in the patient is a standard strategy for these vaccines. Even so, in situ targeting of DCs has been explored using either human or murine cells, though it can often be limited by the patient's pre-existing immunity against the adenovirus [80].

#### **3. Challenges of Using Adenoviral Vectors**

Even though the use of nonreplicating adenoviral vectors has shown great promise for cancer immunotherapy, several aspects of the virus and its delivery present barriers to its effectiveness. Ideally, the vector should transfer the gene to the intended cell type without causing undue antiviral host responses. In the following discussion, we present the molecular basis for Ad tropism, the anti-Ad immune responses, and the issues surrounding the systemic administration of Ad vectors. With a thorough understanding of these mechanisms, we can then explore solutions for the challenges that they pose.

#### *3.1. Tissue Tropism*

As mentioned previously, most human adenovirus serotypes use CAR as their primary receptor, which is expressed on several cell types, including hepatocytes, myocardiocytes, myoblasts, and epithelial and endothelial cells [51] (Table 2). Additionally, some Ads can bind to CD46, a complement regulatory protein that is present on most nucleated human cells, including hematopoietic stem cells and dendritic cells, as well as the costimulatory molecules CD80 and CD86, present on antigen-presenting cells [81–83]. Different primary receptors such as integrin αvβ5, heparin sulfate proteoglycans, sialic acid, and DSG2 and GD1a glycans have also been reported to support adenovirus internalization [81,84,85], with different primary receptors influencing the route of intracellular viral traffic [86].

**Table 2.** Classification and tropism of human adenoviruses.

CAR: coxsackie adenovirus receptor. DSG2: desmoglein-2. GD1a: GD1a ganglioside. HSPG, heparin sulfate proteoglycans. MHC1-a2: major histocompatibility complex-a2. SA: sialic acid. SR: scavenger receptor. VCAM-1, vascular cell adhesion molecule-1. Adapted from [85,87].

> Engineered HAd can transduce target cells and are internalized in a similar way to wild-type adenovirus infection. The internalization can be augmented by interactions between an arginine-glycine-aspartate (RGD) motif found in the penton base and integrins. Upon attaching to CAR, the fiber knob disassociates from the capsid, and the exposed penton base interacts with a secondary receptor, usually membrane integrins αvβ3 or αvβ5, responsible for virus internalization [86,88], followed by virion endocytosis via integrin-mediated signaling [86,89].

> Different subgroups of adenovirus can use different types of integrins as receptors, reinforcing their characteristic cell tropism [89,90]. The virus enters the cell by a clathrincoated vesicle and is transported in endosomes, where capsid disassembly occurs due to endosome acidity. The virion escapes the endosome and traffics to the nucleus by microtubular complexes, where replication occurs [31,85]. After 2 h, about 40% of the internalized wild-type virions arrive in the nucleus, ready to be transcribed due to their double-stranded genome. Additionally, 48–72 h after infection, nuclear and cytoplasmatic membranes are disrupted, and around 10,000 new virions are released [32].

> Different serotypes may favor particular receptors; for example, HAd5 from subgroup C has been shown to utilize CAR for facilitating entry into cells [91], while HAd11 and HAd35 from subgroup B utilize CD46 as their primary receptor [83]. As mentioned, the adenoviral vectors may be chosen due to their inherent tissue-specificity and compatibility with the intended route of administration. For example, when the virus is injected into the brain, tropism for specific cell populations depends on the interaction with CAR, and lower transduction is observed with vectors that bind neither to CAR nor integrins [92]. Even so, HAd5 has been shown to enter cells by CAR-independent mechanisms, including via a hexon–lactoferrin bridge [93,94]

> The native tropism of Ads for CAR on the cell surface and the interaction of viral vectors with nontarget tissues can result in toxicity and poor therapeutic efficacy. Thus, viral proteins can be genetically tailored to expand or restrict viral replication, and vector replication machinery can even be modified to augment or restrict viral replication in target cells [49]. Beyond that, nonhuman adenoviruses, such as canine (CAd2), bovine (BAd3), chimpanzee (ChAd1-7, ChAd68), and ovine (OAd7), can also be used to overcome the pre-existing immunity in human patients [95].

#### *3.2. Pre-Existing Immunity in the Host*

Due to the growing application of adenoviral vectors in gene therapy and vaccines, studies of seroprevalence in global populations are important. However, these studies may be limited by the lack of data from South America, Australasia, and most African countries [96]. Moreover, predominant HAdV types can change over time within a region [97], and transmission of new strains across continents appears to be frequent. A recent study conducted by Mennechet et al. [96] showed that HAdV-D26 seroprevalence appears to be relatively high in Africa and Asia and low in North America and Europe, while HAdV-B35 seroprevalence is low worldwide. HAdV-C5 is the most common serotype that infects humans, particularly in developing countries [98,99], and it is one the most commonly used adenoviral vectors; thus, the limitations on the applicability of the HadV-C5 vector, due to pre-existing immunity, have led to the construction of novel vectors derived from rare Ad serotypes [100].

HAd serotypes are often associated with specific diseases. For instance, serotypes 2–5, 7, and 21 commonly infect the respiratory tract of individuals [101–104], while serotypes 8, 19, and 37 are responsible for keratoconjunctivitis outbreaks [105–107]. Pharyngoconjunctivitis is often associated with serotypes 3, 4, and 7 [108,109] and acute gastroenteritis with serotypes 40 and 41 [110,111]. Likewise, neurological disorders and obesity seem to have some correlation with adenoviral infections [112–114]. Due to this frequent occurrence of these infections worldwide, humans have extensive preexisting immunity to adenoviruses [115–119].

HAd capsid proteins are very immunogenic, especially the hexon protein [120]. The host's adaptive immunity arm detects the hypervariable regions (HVRs) in the hexon protein and releases serotype-specific neutralizing antibodies (NAs) that appear to block a postentry step [121,122]. Thus, at second contact with the same adenovirus serotype, the host NAs may rapidly neutralize it. Interestingly, at the same time, coagulation factor X (FX) in the blood binds to the hexon protein and activates complements (C4 and C4BP in the classical and alternative complement pathways) against the adenoviruses. In a fair number of tested individuals, it also protected the virions from neutralization by serum components [123]. The HAd5 vector interacts with FX, which, in turn, binds cell surface heparan sulfate proteoglycans on hepatocytes; thus, FX is essential for intravenously injected Ad5 vectors to transduce the liver [124].

Even with FX binding the hexon proteins, anti-knob fiber and anti-penton base antibodies can also prevent the adenovirus from transducing cells [125]. Nevertheless, the HAd species C knob and penton base proteins have also been shown to induce serotype-specific NAs [126].

Beyond antibodies, strong and sustained CD8+ T-cell responses follow adenoviral infections [127]. Up to one-third of circulating T-cells against HAd have been reported to be CD4+ T-cells specific for a hexon epitope conserved between HAd serotypes. Hence, the host's preexisting CD4+ T-lymphocytes might promptly respond to various subsequent adenovirus serotypes in either blood or gut [128].

#### *3.3. Different Administration Routes and Their Particularities*

Although the administration of lower doses of Ads is well-tolerated, higher doses are known to overstimulate innate and adaptive immune responses, which might result in acute toxicity. For example, with HAd5 at the concentration 1 × 1011 PFU/kg, 70% of hepatocytes and 15% of Kupffer cells expressed transgene three days later [129]. Systemic delivery, which theoretically could solve the issue of reaching metastatic foci, is confounded by sequestration of the virus by the liver and the subsequent antiviral immune response as well as possible liver damage [130]. For example, at doses up to 4 × 1012 vp/kg of HAd5, approximately 98% of the injected virus was found in the liver 30 min after injection [131]. Thus, systemic delivery of adenoviral vectors is associated with dosedependent toxicity and a high risk of hepatotoxicity. Several studies have shown that the delivery of adenoviral vectors to immunocompetent mice by different routes, such as intravenous [132], intraperitoneal and intratracheal [133] or via direct injection into the pancreas, resulted in the production of neutralizing antibodies, decreasing the effectiveness of a second administration [134].

The relationship between the route of administration and viral load on CD8+ T-cell populations has already been studied. Holst et al. [135] administered adenoviral vectors

encoding β-galactosidase by intravenous or subcutaneous routes and then examined transgene-specific CD8+ T-cells. Independently of the route of administration, doses above 109 particles were disseminated systemically. In moderate doses, both routes induced a transient peak of IFN-γ produced by CD8+ T-cells 2 to 3 weeks postinfection. However, with intravenous administration, these cells were only detected in the liver. Additionally, after 2 to 4 months, the systemic immunization created dysfunctional transgene-specific CD8+ T-cells impaired in both cytokine production and in vivo effector functions as well as the accumulation of specific CD8+ T-cells in the spleen. Thus, the most important influence of adenovirus administration on CD8+ T cell response is the route of injection and not the total antigen load [135].

In another study, the intralymphnodal administration of a nonreplicating recombinant adenoviral vector encoding the LacZ reporter gene in canine lymphosarcoma was found to be safe, with no relevant adverse effects. This finding presents the potential for its administration to lymph node metastases in both animal and human models [136]. The examples above demonstrate that efficient gene delivery using adenoviral vectors can be performed without hepatic injury or systemic immunogenicity if off-target effects are avoided. To this end, several strategies have been developed to minimize interactions of the adenoviral vector with the liver and to protect the virus from neutralizing antibodies. Some of these approaches, such as the engineering of adenovirus capsid, hexon, or fiber proteins, use of nonhuman serotypes, and nanoformulation-coated adenoviral vectors will be discussed in more detail below.

#### **4. Strategies to Modify Adenovirus Tropism**

Although Ads infect many different types of cells, low (or no) expression of CAR, especially in tumor cells, confounds the attachment step and represents one of the hurdles to gene therapy using adenoviral vectors. Several strategies have been employed to overcome this barrier and redirect the Ads to the intended recipient and, consequently, decrease off-target effects (Figure 1).

**Figure 1.** Improvements in vector delivery to and targeting of cancer cells. (**a**) Several approaches can be used to modify viral attachment and entry, such as inhibiting the binding to natural receptors (detargeting) and creating tropism for neoplasms and their metastatic foci (retargeting). (**b**) An alternative to CAR-mediated viral attachment is modifying the fiber (for example, incorporation of the RGD sequence into the knob AB-loop motif). (**c**) Ad structure permits retargeting through the incorporation of synthetic molecules and antibody fragments within the virus capsid. (**d**) Both biological (e.g., cell membrane, liposome) and chemical (e.g., gold, silver, PEG) approaches may be used to coat the virus and improved delivery, especially for the systemic route. PEG: polyethylene glycol. Created with BioRender.com.

#### *4.1. Modifications in Viral Entry: Attachment Receptors and Virus Internalization*

To explore the targeting of adenovirus particles to tumor cells, initial events related to infection/transduction must be modulated: (i) viral attachment and (ii) viral entry. This strategy can target tissues by inhibiting binding to natural receptors (detargeting) in normal liver cells, for example, and, simultaneously, creating tropism for neoplasms and their metastatic foci (retargeting) [137]. Another strategy is pseudotyping, the creation of variants by recombining their capsid proteins. Here, we detail works that have used a variety of strategies to modify tropism.

The tropism of HAd5 is predominantly mediated by the interaction of fiber/knob with CAR. As an alternative to CAR-mediated viral attachment, one of the classical fiber modifications is the incorporation of the RGD sequence into the knob AB-loop motif, which greatly expands the spectrum of cell types that may be transduced [138]. Even so, the genetic incorporation of an RGD-4C peptide into the HI loop or the C-terminal end of the HAd5 fiber knob modifies the Ad knob domain without ablating native CAR-binding [139]. Another possibility is the insertion of positively charged polylysine motifs [140]. This modification permits the virus to target the tumor cell's heparan sulfate proteoglycans, common constituents of the cell surface, and the extracellular matrix, overexpressed in several different cancer types, including cervical cancer [141].

On the other hand, directing transduction and expression of the transgenes to occur only in tumor cells (but not in normal cells) should minimize the adverse effects of the therapy. Wickham and coworkers [142] successfully used bispecific antibodies to promote the targeting of an adenoviral vector to endothelial and smooth muscle cells. However, the attempt to noncovalently associate antibodies or molecules with the surface of the viral particle may be hampered by the instability of this binding, especially if used in vivo. For this reason, the adenovirus fiber gene sequence can be edited and, thus, the peptide ligands can be incorporated directly into the protein sequence [137,143–145].

Taking cues from the phage display technique, adenovirus libraries can be generated with random peptide combinations and screened for their ability to transduce a particular cell type, thus refining specificity to tumor populations in a strict manner [146–148]. Joung et al. [148] devised a technique for producing adenovirus with modified fibers that involved cotransfecting a packaging cell with a plasmid encoding a genetically fiber-less adenovirus with a plasmid containing the open reading frames (ORFs) of the fiber of interest. Moreover, Yoshida et al. [144] developed a Cre-lox-mediated recombination system using a plasmid library encoding modified fiber and the adenoviral genome. Using this approach, these authors inserted unique peptides, each with seven random amino acids, into the AB-loop of the fiber, and, after screening, they were successful in targeting these viral vectors to glioma cells [143,149]. Although the idea seems highly promising, there are technical complications that hinder this approach. The compaction and self-assembly of the protein monomers to form the adenovirus particle is a very delicate process. The insertion of random peptides can compromise the final structure of the viral particle as well as virus production [150].

Even though two receptors are required for the adenovirus particle to penetrate the target cells, each interaction is a distinct step. While attachment receptors apparently only recognize the target cell, the HAd5 penton base–αv integrin interaction activates signaling pathways such as p38MAPK [151,152] and Rho GTPases [153], which then trigger changes in the cell cytoskeleton for endocytosis mediated by clathrin [153,154]. Interestingly, mutation of the penton base RGD sequence slows but does not impair virus internalization and infection, nor does it prevent liver tropism [86].

#### *4.2. Pseudotyping the Capsid Using Components from Different Adenoviruses*

Chimeric adenoviruses are usually based on HAd5 with the fiber or its knob domain replaced by that of another serotype [155]. This creates perspectives for the recombination of these subtypes and, thus, the modulation of targeting: a concept known as pseudotyping. However, the resulting range of tropisms will be restricted to the respective serotypes used in the construction and may not necessarily contemplate the range of existing receptors

found in neoplasms. Table 3 summarizes important findings in studies that have employed adenovirus pseudotyping strategies.

**Table 3.** Different studies using adenovirus pseudotyping strategies.

PTD: Tat-PTD hexon modification.

#### *4.3. Encapsulation of Adenovirus Using Synthetic Polymers*

Shielding the virus with nanoparticles allows the Ad to escape immune recognition and avoid the undesirable accumulation of the vector in the liver upon systemic delivery. Furthermore, this approach can enhance the specific targeting of tumor cells. Several studies have been conducted to evaluate the efficacy of encapsulation of negatively charged Ads with cationic liposomes or particles that aim to prevent virus clearance from circulation [162].

Some Ad features, such as regular geometries, well-characterized surface properties, and nanoscale dimensions, make it a biocompatible scaffold for a wide variety of inorganic and biological structures. The Ad capsid has free lysines, the majority of them located on hexon, penton, and fiber proteins, which can be covalently linked to other molecules such as polymers, sugars, biotin, and fluorophores [163]. Polymers offer a wide range of conjugation and encapsulation that make them a safe option for immunotherapy. The main biopolymers studied are polyethylene glycol (PEG) and hydroxylpropyl methacrylamide (pHPMA), the latter being covalently bound to capsid proteins; thus, it can efficiently transduce solid tumors after intravenous injection into mice [164]. The first study using this polymer demonstrated passive tumor targeting of polymer-coated adenoviruses administered by intravenous injection; the authors observed that the coated virus accumulated inside solid subcutaneous AB22 mesothelioma tumors 40 times more than the unmodified virus [164].

As mentioned, Ad structure permits retargeting through the incorporation of synthetic molecules and antibody fragments within the virus capsid. For instance, PEG is an uncharged, hydrophilic, nonimmunogenic, synthetic linear polymer (CH2CH2O repetitions) [165] that is frequently utilized in the biopharmaceutical industry and can be useful to protect therapeutic molecules from proteolysis as well as humoral and cellular immune responses [166]. According to Fisher et al. [164], one advantage of vector PE-Gylation is the retention of viability after storage at various temperatures compared to conventional Ads. Covalent attachment of PEG to the adenovirus capsid may be achieved by using PEG activation mechanisms. PEG presents hydroxyl groups (OH) that make PEG-protein bonds impossible; thus, it is necessary to use chemical activation before protein attachment. In the specific case of adenoviruses, activation can be achieved through the use of tresyl-monomethoxypolyethylene glycol (TMPEG), succinimidyl succinatemonomethoxypolyethylene glycol (SSPEG), or cyanuric chloride-monomethoxypolyethylene glycol (CCPEG), which react preferentially with lysine residues in the capsid, thus supporting the formation of covalent bonds with PEG [167].

In addition to the PEGylation of the virus particle, ligands can also bind to the opposite extremity of PEG, thus providing a specific ligand to retarget the virus to the corresponding cellular receptor.

Such approaches also aid the vector in reaching distant tumor sites, as found by Eto et al. [167]. They used a cationic liposome that was composed of (1, 2-dioleoyloxypropyl)-N, N, N-trimethy-lammonium chloride:cholesterol to encapsulate the Ad vectors carrying the antiangiogenic gene (pigment epithelium-derived factor (PEDF)). The results showed that systemic administration of Ad-PEDF/liposome was well tolerated and caused the suppression of tumor growth. The coated Ad-PEDF increased apoptosis compared to uncoated Ad in the B16-F10 melanoma cell line and inhibited murine pulmonary metastasis in vivo. Moreover, Ad-luciferase encapsulated with liposome exhibited decreased liver tropism and increased transduction in the lung. Additionally, the anti-Ad IgG level after administration of the Ad-PEDF/liposome was significantly lower compared to Ad-PEDF alone. Eto et al. [167] showed that positively charged 14-nm gold nanoparticles increased the efficiency of Ad infection in mesenchymal stem cells, usually refractory to Ad transduction, mainly because CAR expression is absent or downregulated. The strategies described here support future exploration of additional formulations for liposome-encapsulated adenoviruses and their ability to target cancer cells.

#### *4.4. Cancer Cell Membrane-Coated Adenoviral Vectors*

Nanoparticle-based delivery systems have been extensively explored for improving cancer treatment. Cell membranes, which can be obtained from a variety of source cells, including leukocytes, platelets, red blood cells, and cancer cells, are being employed to encapsulate particles such as liposomes, polymers, silica, and Ad vectors in order to improve tumor-targeted drug delivery in addition to prolonged circulation time, reduced interaction with macrophages, and decreased nanoparticle uptake in the liver [168]. The membrane-based functions of cancer-related cells include extravasation, chemotaxis, and cancer cell adhesion [169]. As a source of cell membranes, cancer cells offer certain advantages. They can be obtained from cell lines or patient samples and possess a wide range of membrane surface proteins, such as MHC, TAAs, and neoantigens, that can program the immune system to attack local and distant tumor sites [170], as represented in Figure 2.

Tumors frequently develop a variety of mechanisms to subvert immune attack, resulting in an immune-suppressive TME. Although tumor cells can stimulate a variety of cell types, including fibroblasts, immune-inflammatory cells, and endothelial cells, through the production and secretion of stimulatory growth factors and cytokines [171], the TME can be modulated by the tumor cells themselves and tumor-infiltrating leukocytes (including regulatory T-cells (Tregs)), myeloid-derived suppressor cells (MDSCs), and alternatively activated (type 2) macrophages (M2), cytokines (IL-10, TGF-β), expression of inhibitory receptors (such as cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed deathligand 1 (PD-L1)) or impediment of T-cell function, resulting in the reduced effectiveness of immunotherapy [172]. Although many TAAs have been identified, their immunogenicity is generally insufficient to elicit potent antitumor responses. Typically, when the tumor reaches the malignant stage, the most immunogenic tumor-specific antigens have been eliminated via negative selection. Frequently, the nanoparticles are associated with adjuvants, secretory cytokines, antibodies, and/or viral vectors to improve the immune response [173].

**Figure 2.** Strategy used to improve the specificity of adenoviral vectors. An adenovirus coated with a cancer cell membrane has some advantages, such as the presence of TSA and TAA, which aids the anti-tumor immune response. Additionally, the membrane can be engineered to present specific molecules/receptors, improving the power of interaction with the tumor. Moreover, the viral coating can offer several benefits, including suppression of liver toxicity, increase of specific infectivity to cancer cells, preferential antitumor (not antiviral) immune response, and escape from pre-existing neutralizing antibodies in both routes of delivery (intratumoral and systemic). Systemic administration using virus coated with membranes could offer a highly desirable outcome: targeting metastatic foci. TAA: tumor-associated antigens; TSA: tumor-specific antigens. Created with BioRender.com.

Coating polymeric nanoparticles with cancer cell membranes can be used for different types of cancer therapy. For anticancer drug delivery, Zhuang et al. [173] showed that polymeric nanoparticle cores made of poly(lactic-co-glycolic acid) (PLGA), a polymer coated in an MDA-MB-435 membrane, significantly increased cellular adhesion to the source cells compared to naked nanoparticles due to a homotypic binding mechanism. For cancer immunotherapy, the authors demonstrated that a polymer coated with a B16−F10 membrane, which creates a stabilized particle, facilitated the uptake of membrane-bound tumor antigens and, consequently, the presentation and maturation of DCs. Another approach using a biohybrid (tumor-membrane-coated) nanoparticle was also able to elicit an antitumor immune response in melanoma models, changing the microenvironment profile. The administration of the vaccine enhanced the activation of APCs and increased the priming of CD8+ T-cells. When combining the nanovaccine with a checkpoint inhibitor, 87.5% of the animals responded, including two complete remissions, when compared to the immune checkpoint inhibitor alone. These results point to opportunities for the association of nanoparticles and immunomodulators to enhance T-cell responses [174].

The effects from the association of polymers, cancer cell membranes, and adjuvants were also observed by Fontana et al. [175]. PLGA nanoparticles were loaded with the TLR7 agonist and then coated with membranes from B16-OVA cancer cells since the presentation of foreign peptide OVA permits the tracking of responses. The nanovaccine was able to enhance uptake by antigen-presenting cells and showed efficacy in delaying tumor growth as a preventative vaccine besides displaying activity against established tumors when coadministered with the anti-programmed death 1 (PD-1) monoclonal antibody.

In another study, CpG oligodeoxynucleotide (CpG) was used as an immunological adjuvant and encapsulated into PLGA nanoparticle cores coated with membranes derived from B16–F10 mouse melanoma cells. The effect of nanoformulation on DC maturation was observed by the upregulated expression of costimulatory markers CD40, CD80, CD86, and MHC-II. Both prophylactic and therapeutic vaccines presented positive results. In the prophylactic study using the poorly immunogenic wild-type B16–F10 model, tumor occurrence was prevented in 86% of mice 150 days after challenge with tumor cells. Interestingly, mice vaccinated with the CpG-nanoformulation alone had tumor growth comparable to the control group and a median survival of 22 days. This reinforces the role of the cancer cell membrane in targeting the elimination of malignant cells by the immune system. In the therapeutic model, mice challenged with B16–F10 cells and subsequently treated with the nanoformulation presented a modest ability to control tumor growth. However, the combination of nanoformulation and a checkpoint blockade cocktail (anti-CTLA4 and anti-PD1) significantly enhanced tumor growth control. As such, the results encourage further research into nanoparticle vaccine formulation and possible associations with other immunotherapies that modulate different aspects of immunity [176].

In a different strategy, Fusciello et al. [177] combined an oncolytic virus (due to its natural adjuvant properties) and cancer cell membranes carrying tumor antigens. They found that viral transduction was significantly increased with the coated virus, implying an uptake mechanism different than that utilized by the naked virus, which requires CAR, representing a significant advantage for transducing CAR-negative cell lines. Additionally, the coated virus was better able to control tumor growth compared to other treatments. The vaccination using coated viruses created a highly specific anticancer immune response, redirecting the immune response against the tumor [177]. Thus, personalized cancer vaccines can represent an alternative approach to target cancer even without determining specific antigens for each patient. We hypothesize that this approach will also be applicable to nonreplicating adenoviral vectors, though this has not yet been shown.

#### *4.5. Association of Antibodies and Viral Structures*

The incorporation of antibodies into the viral structure is another interesting option for creating specificity. Despite the obstacles to the use of conventional antibodies (human, murine, and goat), smaller molecules from other species, such as alpacas, can be added to the structure of the Ad capsid without disturbing its synthesis and assembly. For example, van Erp et al. [178] generated a single domain camelid antibody against the human carcinoembryonic antigen present in human colorectal adenocarcinoma cells. They incorporated this molecule into the adenovirus capsid, achieving a more specific tropism for tumor cells and reducing off-target toxicity. Although the strategy was developed to retarget oncolytic viruses, it can also be used to improve nonreplicative adenoviral vectors.

Despite the cited possibilities, the need to re-engineer vectors de novo for each novel target may be an unnecessary and costly effort. Since it is possible to combat different kinds of cancers through similar molecular mechanisms, such as the induction of immunogenic cell death, the development of adaptable platforms may allow the establishment of virusbased therapies in a more scalable and affordable way. Such approaches may, in the future, permit low-effort adaptation of pre-existing therapies to target different cellular markers and treat other tumors.

A viable alternative may be the use of adapter molecules. Bhatia et al. [179] developed an anti-CXCR4 bispecific adapter (sCAR-CXCL12). Chemokine receptor type 4 (CXCR4) is known to be overexpressed in a wide variety of cancers, such as melanoma [180] and breast cancer [181], and it is associated with metastasis and poor overall survival. Bhatia et al. [179] designed a recombinant adapter molecule composed of an ectodomain portion of the human CAR, followed by a 5-peptide linker (GGPGS) and a 6-His tag sequence, fused to the mature human chemokine CXCL12/SDF-1a sequence (CXCR4 ligand). According to the researchers, this bispecific adapter attenuated liver infection in vivo and a promoted a considerable increase in cancer cell infection, as observed in xenograft tumors in mice.

In another interesting work, Schmid et al. [182] achieved, simultaneously, the retargeting of type 5 adenovirus tropism to a specific cancer marker and the reduction of its liver sequestration. Unlike other adapter strategies, they utilized designed ankyrin repeat proteins (DARPins). Similar to antibodies, these proteins can bind to a target with rather

good specificity. Moreover, these molecules can be engineered to target different antigens on the cell surface [183]. Schmid et al. [182] designed an adenovirus-antigen adapter composed of three monomers. Each monomer was made of a retargeting DARPin, a flexible linker, a knob-binding DARPin, and a trimerization motif [182,184]. The last component is responsible for the stability of the complex, allowing the coating of the adenovirus fiber knob and, consequently, impeding virus natural tropism. In addition, according to the researchers and some early works, the removal of CAR and integrin interactions may reduce liver tropism [185,186], an effect also observed when those capsid sites are blocked by DARPin adapters. Furthermore, this protein was able to hide the region responsible for adenovirus–liver interaction without disturbing the adenovirus–integrin interaction. Nonetheless, the researchers developed an adenovirus-binding molecule, named "shield", derived from humanized antihexon scFv, which was designed to bind to hexon proteins, effectively protecting them from neutralizing antibodies [182].

#### *4.6. Genetic and Chemical Capsid Modifications and Association with Polymers*

Other strategies have emerged that support the retargeting and detargeting of adenoviral vectors. For example, the CGKRK peptide mediates the targeting of tumor cells and tumor neovasculature and has been tested for its ability to retarget PEGylated adenoviral vectors: PEG molecules are conjugated to the surface of the viral vector; then, the peptide is attached via a chemical reaction, resulting in its conjugation to the functional group of PEG [187]. Moreover, Bonsted and colleagues demonstrated that a linker between the poly(2-(dimethylamino)ethyl methacrylate) (pDMAEMA) and the epidermal growth factor (EGF), commonly overexpressed in tumors, efficiently transduced CAR-deficient cells [188,189]. Additionally, an EGF mimetic peptide linked to the cationic PAMAM (polyamidoamine) dendrimer polymer through a PEG linker has been used to retarget dendrimer-coated Ad vectors; it has been shown to increase transgene expression in target cells compared with the untargeted vector [190].

Kreppel et al. [191] introduced a genetic–chemical concept for vector re- and detargeting. For that, the authors genetically modified the virus in order to present cysteine residues in the capsid, including the fiber HI-loop [191], protein IX [192], and hexon [193]. The cysteine residues were then covalently modified with thiol-reactive coupling moieties, including ligands, shielding polymers, carbohydrates, small molecules, and fluorescent dyes [194]. Kreppel et al. demonstrated that amine-based PEGylation and thiol-based coupling of transferrin to the fiber knob HI-loop successfully retargeted the modified Ad vectors to CAR-deficient cells [191].

These studies highlight the possibility of creating adenoviral vector platforms that need no further genetic modification; thus, a wide variety of target tissues may be explored with the aim of improving specificity and decreasing the neutralizing effects of preexisting antibodies.

#### **5. Conclusions and Future Perspectives**

Many features make Ads interesting vehicles for the delivery of foreign antigenic proteins or gene therapy: large cloning capacity, genetic stability, and high in vivo transduction capacity in both dividing and nondividing cells. The natural antiviral immune response can be useful to reprogram the tumor microenvironment from "cold" to "hot" by inducing T-cell-specific immune responses and proinflammatory cytokine expression [195]. The success of therapy depends on several other factors, such as the quality, intensity, specificity, and half-life of immune responses against the tumor. In this scenario, neoantigens have emerged as an attractive target for cancer therapy. Major advances in using the non-self-peptides are the absence of pre-existing central tolerance, potential strong immunogenicity, and lower risk of autoimmunity diseases [196]. We expect that continued refinement of Ad vector design and a deeper understanding of neoantigens will converge to provide an exceptional platform for cancer immunotherapy.

Even so, we point out some limitations for the use of neoantigens in personalized medicine: (1) neoantigens are limited by the diversity of somatic mutations in different tumor types and their individual specificity; (2) the probability that the neoantigens are shared between patients is very low; (3) identification and verification of neoantigens is still time-consuming and expensive [197]. In addition, the construction of adenoviral vectors encoding each neoantigen would be costly and time-consuming; thus, approaches that do not require vector construction may be preferable, including the use of peptides and membrane coatings.

Otherwise, the effectiveness in the use of neoantigens has already been observed in preclinical [198,199] and clinical data [200–202]. In addition, patients with high mutation burden tumors, like melanoma [203,204], non small-cell lung cancer [205], and bladder cancer [206], have had more clinical benefit from checkpoint-blockade therapy than those with lower mutation loads [196]. Moreover, the prediction of peptides binding to MHC molecules and, consequently, the identification of neoepitopes able to stimulate the immune response are emerging as novel approaches that could be associated with adenoviral vectors, reversing part of the tumor-induced immunosuppression.

Recently, D'Alise and collaborators [207] demonstrated the satisfactory benefits of genetic vaccines based on Ads derived from nonhuman great apes (GAd) encoding multiple neoantigens applied in the CT26 murine colon carcinoma model. Both prophylactic and early therapeutic vaccinations elicited strong and effective T-cell responses and controlled tumor growth in mice. The tumor-infiltrating T-cells were diversified in animals treated with GAd and anti-PD1 compared to anti-PD1 alone [207]. The big challenge of neoantigens is the complexity in identifying immunogenic antigens unique to each patient. However, more optimized sequencing platforms and bioinformatics tools are helping to make personalized therapy truly viable. All in all, the data presented here highlight new perspectives of cancer vaccines and gene therapy using modified nonreplicating adenoviruses and different strategies to turn the immune response against the tumor more specific and robust, contributing to local and distant control of tumor progression.

Although viral delivery systems are quite promising strategies in gene therapy, there are some limitations to their clinical application. The major barriers are host immune responses that result in the clearance of vectors, interaction with plasma proteins, liver sequestration, Ad CAR-dependence, and off-target effects [208]. Regarding these issues, a number of genetic manipulations have been exploited to redirect adenovirus binding to different cell surface receptors and, consequently, increase affinity for the target, with lower adverse effects [50].

In this scenario, different strategies using coated viruses have emerged in recent years, and both biological and chemical approaches can be used to coat the virus and improve delivery, especially for the systemic route. Since these strategies involve using cancer cell membranes that can be obtained directly from tumor cell lines, they provide greater biocompatibility with the tumor site and, consequently, specifically target these cells [209]. A growing body of evidence suggests that cancer cell membrane-coated viruses can be delivered by the systemic route, improving the targeting of metastases, with higher retention time, lower immune recognition, and decreased liver sequestration, toxicity, and accumulation in healthy tissues. The induction of immunogenic cell death by nonreplicating Ad vectors is associated with innate immune responses, antigen processing and presentation, and, finally, the activation of the cellular immune response. While few examples currently exist of using membrane-coated adenoviral vectors, we hypothesize that this approach will continue to be studied, including in nonreplicating vectors.

In summary, improvements in vector delivery and targeting will provide an even greater potential for the use of nonreplicating adenoviral vectors in cancer immunotherapy. In particular, we envision vectors adapted to support systemic delivery, achieve tumor specificity, induce tumor cell death and supply specific antigens to guide antitumor immune responses.

**Author Contributions:** N.G.T. conceived the review topic, wrote and edited the text, and prepared the figures; A.C.M.D. wrote and edited the text and prepared the figures, F.A., J.C.d.S.d.L., O.A.R., and O.L.D.C. wrote and edited the text; B.E.S. edited the text and provided funding. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Sao Paulo Research Foundation (FAPESP; grant 15/26580- 9 (B.E.S.) and fellowships 17/25290-2 (N.G.T.), 18/25555-9 (A.C.M.D.), 17/25284-2 (O.A.R.), and 17/23068-0 (O.L.D.C.)). Funding was also provided by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; fellowships 134305/2018-3 (J.C.d.S.d.L.) and 302888/2017/9 (B.E.S.)).

**Data Availability Statement:** No new data were created or analyzed in this study. Data sharing is not applicable to this article.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

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## *Review* **The Flt3L/Flt3 Axis in Dendritic Cell Biology and Cancer Immunotherapy**

**Francisco J. Cueto \* and David Sancho \***

Centro Nacional de Investigaciones Cardiovasculares (CNIC), 28029 Madrid, Spain **\*** Correspondence<EMAIL_ADDRESS>(F.J.C<EMAIL_ADDRESS>(D.S.)

**Simple Summary:** Cancer immunotherapy is currently focused mainly on the enhancement of the effector function of T cells. However, dendritic cells (DCs) are needed to prime T cells, suggesting that DCs can be an attractive target for immunotherapy. Flt3L/Flt3 is an essential pathway for DC development and function, although its potential in cancer immunotherapy is not yet clearly established. Herein, we will review the current evidence which suggests that the stimulation of DCs through the Flt3/Flt3L axis may contribute to improved cancer immunotherapy.

**Abstract:** Dendritic cells (DCs) prime anti-tumor T cell responses in tumor-draining lymph nodes and can restimulate T effector responses in the tumor site. Thus, in addition to unleashing T cell effector activity, current immunotherapies should be directed to boost DC function. Herein, we review the potential function of Flt3L as a tool for cancer immunotherapy. Flt3L is a growth factor that acts in Flt3-expressing multipotent progenitors and common lymphoid progenitors. Despite the broad expression of Flt3 in the hematopoietic progenitors, the main effect of the Flt3/Flt3L axis, revealed by the characterization of mice deficient in these genes, is the generation of conventional DCs (cDCs) and plasmacytoid DCs (pDCs). However, Flt3 signaling through PI3K and mTOR may also affect the function of mature DCs. We recapitulate the use of Flt3L in preclinical studies either as a single agent or in combination with other cancer therapies. We also analyze the use of Flt3L in clinical trials. The strong correlation between type 1 cDC (cDC1) infiltration of human cancers with overall survival in many cancer types suggests the potential use of Flt3L to boost expansion of this DC subset. However, this may need the combination of Flt3L with other immunomodulatory agents to boost cancer immunotherapy.

**Keywords:** Flt3; Flt3L; dendritic cells; cancer immunotherapy

#### **1. Introduction**

The development and refinement of immunotherapy constitutes a revolution in the treatment of cancer. Most of the current immunotherapies target and unleash the effector function of lymphocytes. However, lymphocyte functions depend on their previous activation by antigen-presenting cells, among which dendritic cells (DCs) stand out. DCs were initially characterized according to their morphology and the expression of major histocompatibility complex class II and CD11c [1,2]. DCs continuously sample their microenvironment, where they can take up antigens and present them to T cells [3,4]. The outcome of the DC–T cell interaction can lead to immunity or tolerance depending on costimulatory signals present during the priming phase. Thus, DCs act as decision nodes in the initiation of T cell responses [3,4].

DCs derive from hematopoietic stem cells in the bone marrow (Figure 1) [5]. These can give rise to Lin−IL7Ra−Sca1−cKit+FcgRloCD34+ common myeloid progenitors (CMPs), which can further differentiate into Lin−CX3CR1+CD11b−CSF1R+cKit+Flt3+ macrophage/DC progenitors (MDPs). MDPs can differentiate into Lin−CSF1R+cKitloFlt3+ common DC progenitors (CDPs) which are completely committed to the DC lineage. CDPs can give rise to type 1 and type 2 conventional DCs (cDC1s and cDC2s) and plasmacytoid DCs (pDCs) [6].

**Citation:** Cueto, F.J.; Sancho, D. The Flt3L/Flt3 Axis in Dendritic Cell Biology and Cancer Immunotherapy. *Cancers* **2021**, *13*, 1525. https:// doi.org/10.3390/cancers13071525

Academic Editors: Michael Kershaw and Clare Slaney

Received: 28 February 2021 Accepted: 23 March 2021 Published: 26 March 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**Figure 1.** Expression pattern of Flt3 across mouse hematopoietic lineages. We have simplified the lineage tree generated by Jojic et al. [5]. This tree includes all cell populations identified by the first project of ImmGen to express high levels of *Flt3* (relative expression > 1000). In the case of mature cDC1s, cDC2s and pDCs, an average of Flt3 expression has been used to color them, because different populations from different organs were characterized in this consortium.

cDC1 depends on basic leucine zipper ATF-like transcription factor 3 (BATF3) and interferon regulatory factor 8 (IRF8) and expresses X-C motif chemokine receptor 1 (XCR1) and dendritic cell natural killer lectin group receptor 1 (DNGR1; CLEC9A) in humans and mice, and BDCA3 (thrombomodulin; CD141) within the MHCII+CD11c+ population in humans. In mice, two cDC1 populations can be identified: a lymphoid tissue-resident population characterized by the expression of CD8α, and a peripheral population that expresses CD103. cDC1s stand out among other DC populations in their capacity to crosspresent antigens and their production of IL12, which drives CD8 T cell responses, but are also key in CD4 T cell priming [7]. Batf3−/− mice cannot generate peripheral cDC1s and the cell-associated cross-presentation by their resident cDC1s is impaired. These Batf3−/− mice fail at controlling the development of highly immunogenic tumors, similar to Rag1−/− mice that lack mature lymphocytes [8].

On the other hand, cDC2s constitute a group of cells that rely on V-rel reticuloendotheliosis viral oncogene homolog B (RELB), interferon regulatory factor 4 (IRF4), and zinc finger E-box-binding homeobox 2 (ZEB2) [9]. cDC2s express CD11b and signal regulatory protein alpha (SIRPα; CD172a) in mice, and BDCA1 (CD1c) in humans. Singlecell RNA sequencing technologies have enabled discrimination od a number of subsets within cDC2s [10,11]. These findings raise new questions about their differential developmental origin and their relevance in disease. Of note, a subset that expresses macrophage galactose N-acetyl-galactosamine specific lectin 2 (MGL2) displays outstanding antitumor capacities [11].

CDPs can also give rise to at least part of the pDCs, which can also derive from other lymphoid progenitors. pDCs are characterized by the expression of IL3 receptor alpha (IL3RA; CD123) and B220 in humans and mice, respectively. E2-2 and IRF8 are the main transcription factors involved in the development of pDCs. While pDCs are important producers of IFN-I during viral infections and IFN-I may have antitumor effects [12,13], cancers can disrupt the production of IFN-I by pDCs [14–16]. Furthermore, infiltration of pDCs within human tumors associates with poor prognosis [15,17].

The roles of the different DC subsets in orchestrating adaptive immune responses make them a very attractive target to boost antitumor immunity. Different strategies have been developed and evaluated in an effort to mobilize these populations, such as the administration of GM-CSF or the adoptive transfer of cells generated or stimulated in vitro. Among those efforts, we will focus this review on the use of Flt3L to enhance antitumor immunity.

#### **2. Expression Pattern of Flt3 and Flt3L**

The Fms-like tyrosine kinase receptor 3 (Flt3) was discovered as a surface protein intensely expressed on hematopoietic stem cells (HSCs) [18] (Figure 1). However, the improvements in the characterization of different hematopoietic progenitors identified CMPs and MDPs as the main expressors of Flt3 [19]. It should be noted that Flt3 expression is preserved in terminally differentiated cDCs and pDCs [20], which suggests that Flt3 signaling might have a functional impact on mature DCs. On the other hand, Flt3 is also expressed in some lymphoid progenitors. Although its function in this ontogeny branch has not been explored as profoundly, it plays a role in the early differentiation of B cells [21] and is re-expressed by B cells in the germinal center [22]. Moreover, new data indicate that the Flt3L/Flt3 axis is necessary for the development of NK and type 2 and 3 innate lymphoid cells [23] and their progenitors [24].

Flt3 belongs to the class III of tyrosine kinase receptors, characterized by a five immunoglobulin-domain extracellular region and a split tyrosine kinase domain. Class III tyrosine kinase receptors also include CSF1R, PDGFR, KIT and FMS [25,26]. Flt3 is encoded in chromosomes 13 in human and 5 in mice, encoding for 1000 and 993 aminoacid-long proteins, respectively [27].

Flt3−/− mice show decreased numbers of pre-DCs (CD11cintCD45RAloCD43int SIRP αintCD4−CD8−MHCII−), cDCs, and pDCs in their spleens [19], as well as a clear drop in peripheral CD103+ cDC1s, but not CD11b+ cDC2s [20]. However, Flt3−/− mice bear normal numbers of MDPs, indicating that Flt3 signaling is not required until the developmental stage of MDPs [19].

Two years after the discovery of Flt3, Lyman et al. identified a murine protein that could bind a soluble form of Flt3 [28]. This Flt3 ligand (Flt3L) promotes the expansion of Flt3+ primitive hematopoietic stem cells [28]. Soon afterwards, the human homolog of Flt3L (FLT3LG) was cloned, and a soluble version comprising its extracellular domain (amino acids 27 through 179) was found to induce proliferation in human CD34+ HSCs [29]. In the organism, Flt3L is expressed by multiple cell types, including stromal bone marrow and thymic cells [30,31], activated T lymphocytes [32], and NK cells [33], among others. Of note, blood levels of Flt3L are highly elevated in patients with aplastic anemia or receiving bone marrow-damaging chemotherapy or radiotherapy [34,35], and return to normal levels within three months from successful bone marrow transplantation [34].

In mice, three main isoforms have been reported [36]. Two of them contain a plasma membrane-spanning region and are tethered to the membrane [36]. The first one contains a cytoplasmic tail and its ectodomain can be cleaved to act as a soluble form [36]. The second membrane-bound isoform results from the retention of an intron during splicing, which limits the proteolytic release of the ectodomain [36]. TNFα-converting enzyme (TACE) mediates the shedding of the ectodomain of membrane-bound forms, which releases Flt3L ectodomain [37]. Accordingly, Tace−/− mice display reduced levels of Flt3L in serum [37]. A third isoform lacks the membrane-spanning region and can be directly released from the cell [36]. In humans, the complete and the soluble isoforms have been identified, although not the one lacking the cytoplasmic domain [36].

Mice deficient in Flt3L show a dramatic absence of cDC1s, cDC2s and pDCs in both lymphoid and nonlymphoid tissues [20,38]. Flt3L−/− mice also bear fewer CDPs and common lymphoid progenitors (CLPs, c-KitintFlt3+CSF1RloIL7Rα+) in their bone marrows [38]. Thus, the deficiency in the DC lineage in Flt3L−/− mice is more profound than in Flt3−/− mice, which has been attributed to a compensatory mechanism in Flt3−/− mice, where DC progenitors become more sensitive to CSF1R signaling, which can compensate for Flt3 deficiency and promote DC development [39]. Under physiological conditions, NK cells, as well as other lymphocytes, have been identified as an important source of Flt3L within the tumor microenvironment (TME) [33]. The use of tumors that overexpress Flt3L has been extensively used to expand systemic cDC1s [40], but also tumor-infiltrating cDC1s [41]. Of note, several reports could not reproduce the expansion of cDC1s within the TME with systemic administration of Flt3L [42,43], which raises the possibility of some tumors becoming impervious to circulating Flt3L.

#### **3. Flt3 Signaling in Response to Its Ligand**

One of the main driving mutations in acute myeloid and acute lymphoid leukemias occurs in Flt3. This genetic alteration consists of internal tandem duplication sequences and is associated with poor prognosis in leukemia patients. Therefore, signaling pathways downstream of Flt3 have been extensively studied in the context of leukemia. However, physiological signaling through Flt3 in the context of DC generation remains largely unexplored. In the context of DCpoiesis, we will focus on homeostatic Flt3L signaling through Flt3.

As in most tyrosine kinase receptors, unstimulated Flt3 is thought to appear as a monomer on the plasma membrane, which renders its tyrosine kinase domain inactive [44]. Human Flt3L promotes the dimerization of Flt3 after binding through a compact binding domain fitting with the lock-and-key model [45,46]. Upon binding, Flt3L promotes the internalization of Flt3 receptors, which can be detected as soon as five minutes after the addition of Flt3L [46]. The internalized receptors are processed, and the products of their degradation can be observed around 20 min after the engagement of Flt3 and its ligand. The interaction of Flt3 with its ligand promotes the autophosphorylation of Flt3 tyrosine residues [47].

The first studies on the signaling cascade downstream of FLT3 used chimeric receptors composed of the extracellular domain of CSF1R and the transmembrane and cytoplasmic modules of FLT3 [48,49]. Upon the engagement of CSF1, the cytoplasmic domain of these chimeric receptors was found to bind phospholipase C gamma 1 (PLCγ1), the p85 subunit of phosphatidyl inositol 3 -kinase (PI3K), growth factor receptor-bound protein 2 (Grb2), and SHC1 [48,49]. This led to the phosphorylation of Ras GTPase-activating protein (GAP), Vav, Nck, Signal transducer and activator of transcription 5a (STAT5a), and SH2 domaincontaining inositol phosphatase 1 (SHIP1), but no direct interaction with these proteins has been found [48–51]. Additionally, tyrosine residues in Gab1 and Gab2 become phosphorylated upon the FLT3/FLT3L engagement, which may act as adaptor proteins for Src homology region 2 domain-containing phosphatase 2 (SHP2), Grb2 and phosphatidylinositol 3 kinase (PI3K) [47]. Besides, Src homology 2 domain-containing transforming protein 1 (SHC) can interact and phosphorylate SHIP through its amino-terminal phosphotyrosine binding domain [50]. While most of the proteins identified downstream of Flt3 could potentially mediate its signaling, the phosphatases SHP2 and SHIP might act as negative regulators of the process.

Despite the great efforts directed to elucidating the signaling cascade triggered by Flt3, it has to be noted that most were performed in different cell lines that do not lead to the generation of mature DCs. The work in this context is more limited. Of note, Flt3L induces the phosphorylation of STAT3 during the generation of pDCs from bone marrow HSCs [52–54]. Accordingly, in mice, STAT3 is required for the development of pDCs, and its deficiency cannot be overcome by Flt3L administration [52].

Whether PI3K directly interacts or not with FLT3, the PI3K/mammalian target of the rapamycin (mTOR) cascade is clearly required for the generation of cDCs and pDCs by Flt3L [55]. In fact, CD11c-specific deletion of phosphatase and tensin homolog (PTEN), an Akt inhibitor that blocks the PI3K/mTOR pathway, expands the cDCs and pDCs [55]. In CD11cΔPten mice, both lymphoid-resident CD8α+ and peripheral CD103+ cDC1s display a stronger expansion than CD11b+ cDC2s, which can be reverted by rapamycin [55].

#### **4. Expansion of DCs with Flt3L**

Flt3L fosters the expansion of granulocyte-macrophage colony-forming units (CFU-GM) and granulocyte, erythrocyte, monocyte, megakaryocyte colony-forming units (CFU-GEMM) [56], which ultimately reinforces the generation of DCs [57]. Especially, Flt3L was found to strongly expand a splenic DC population characterized by the co-expression of MHCII, CD11c, DEC205 and CD8α [57]. Flt3L can drive the expansion of various bone marrow progenitor populations, which results in the differentiation of B cells, NK cells, monocytes, red pulp macrophages, granulocytes, and innate lymphoid cells [23,38,56,58,59]. However, Flt3 expression is only conserved in mature cDCs and pDCs; not on B cells, monocytes, neutrophils, NK cells, or other innate lymphoid cells. In fact, the expansive effect of Flt3L has been reported to rely on the trans-presentation of IL15 by expanded DCs [60]. Focusing on the DC lineage, the administration of Flt3L promotes the expansion of MDPs in mice, which contributes to the expansion of both cDCs and pDCs [19]. Similarly, Flt3L can expand the amount of blood pre-DCs (CD11c−IL3Rα+ or SSCloCD117+CD116+CD135+CD45RA+ CD115−) and CD141+ cDC1s, CD1c+ cDC2s and CD303+ pDCs in human volunteers, which can increase their frequencies even more than one order of magnitude [61,62].

The lymphoproliferative effects of Flt3L, together with the described mutations of the Flt3 receptor in different leukemias, suggest that its administration might promote lymphoproliferative malignancies. In mice, one report indicated this might be the case in mice inoculated with Flt3L-expressing retroviral vectors [63]. However, such effects have not been observed in the multiple studies where Flt3L was used for immunotherapeutic purposes. This might be caused by chronicity of the exposure to supraphysiologic levels of Flt3L and/or the use of irradiated mice and retroviral vectors, which can be tumorigenic on their own, or because of the local expression of Flt3L in the bone marrow. To the best of our knowledge, no clinical trial where Flt3L has been administered to patients or volunteers has reported the promotion of lymphoproliferative malignancies.

Addition of Flt3L to culture media drives the differentiation of bone marrow cell suspensions into the DC lineage [64]. Bone marrow cell cultures in the presence of Flt3L lead to mixtures of both cDC1s and cDC2s [64], with an important population of CD11c+B220+ pDCs that can produce IFN-I [65]. Flt3L culture-derived cDC1s resemble naturally occurring lymphoid tissue-resident cDC1s, but they lack the bona fide cDC1 markers CD8α and DEC205 [64,66]. Addition of Notch ligand Delta-like 1 to the standard Flt3L-supplemented media facilitates the generation of CD8α+DEC205+CD103+ cells, with capacity to migrate through CCR7 [67]. In addition, culturing CD34+ human cells with Flt3L required Notch signaling for full cDC1 generation, with granulocyte-macrophage colony-stimulating factor (GM-CSF) providing a synergistic effect [68]. Indeed, Flt3L can be used in combination with GM-CSF to generate CD103+ cDC1s from bone marrow cultures [69]. These CD103+ cDC1s are responsive to TLR stimulation, which drives the expression of activation markers such as CCR7, CD80 or CD86 [69].

#### **5. Preclinical Studies Involving Flt3L**

DCs are central to inducing T cell responses that might prevent cancer growth; therefore, Marakovsky et al. suggested that Flt3L might induce powerful antitumor immune responses [57]. Among the immune populations expanded by Flt3L, cDC1s stand out, whose infiltration within the TME has been extensively associated with patient survival. Flt3L did not affect the activation state of tumor-infiltrating cDC1s, because it did not affect the levels of CD40, CD86 or MHCII in tumor-infiltrating cDC1s [70]. However, it enhanced the proliferation of tumor-specific CD8 T cells at the tumor-draining lymph node [70], possibly by increasing the number of cross-presenting dendritic cells, especially cDC1s [41,71], although an effect of Flt3L on other immunomodulatory genes cannot be ruled out. Despite the frequent identification of cDC1s as the main cross-presenting DC population, other DC populations can promote antitumor CD8 T cell responses [41,71]. In fact, cDC1s express a cluster of BATF3-dependent genes, independent of IRF8-stabilization and cross-presentation, which are required for efficient antitumor immunity [72].

Indeed, administration of Flt3L as a single agent was demonstrated to delay or revert the growth of methylcholanthrene-induced fibrosarcomas [73], C3L5 breast tumors [74], B16 melanomas, and EL4 thymomas [75]. In these cases, the protection provided by Flt3L was associated with an expansion of DCs in both lymphoid and peripheral tissues, together with tumor antigen-specific T cell responses [73–75]. This observation has been recapitulated with tumor cell lines that stably express Flt3L, which regress after initial establishment [76]. However, some studies could not reproduce the protective effect of Flt3L as a single agent. An Flt3L-encoding adenovirus injected intravenously did not show any therapeutic effect on the cl-66 mammary tumor model [42]. Despite expanding DCs and other immune populations in spleen, this Flt3L-encoding adenovirus failed at promoting the infiltration of immune cells within tumors [42]. In this line, intraperitoneal administration of human recombinant Flt3L to mice failed at controlling the growth of CT26 and B16 tumors [43]. Once again, the administration of Flt3L did not increase the amount of MHCII+CD11c+ cells, which might include macrophages, within the TME, despite an intense increase in the number of circulating MHCII+CD11c+ DCs (up to 50-fold) [43]. In another study from the same laboratory, intraperitoneal administration of Flt3L enriched tumor-infiltrating cDC1s, but not cDC2s, within an ovalbumin-expressing B16 cell line [70]. The disparity of results obtained from preclinical models treated with Flt3L can be ascribed to differences in cancer models, Flt3L administration strategies, and dosage.

As with combination chemotherapy [77], a rational combination of immunotherapeutic strategies can synergize and provide increased protection against cancer [78]. In this case, it had been suggested that, by increasing the number of DCs, Flt3L might successfully synergize with other therapies that augment the availability of tumor antigens. Accordingly, Flt3L successfully increased survival after local radiation therapy in a metastasis

model of Lewis lung carcinoma, which relied on antitumor T cell responses [79]. cDC1s are required for the abscopal (out-of-field) effect of radiation therapy, supporting that this might explain these benefits [80]. Additionally, recombinant human Flt3L has been administered to mice in combination with immunostimulatory DNA and tumor antigens to raise efficient antitumor immune responses that rely on CD8+ T cells and NK cells [81].

In the context of immune checkpoint blockades, tumor-infiltrating cDC1s have also been identified to play a pivotal role. Mice deficient in cDC1s do not respond to immunotherapy with anti-CTLA4 blocking antibodies [82]. Accordingly, in B16 melanomas and TRAMP prostate adenocarcinomas, anti-CTLA4 therapy can be improved by the inoculation of an Flt3L-expressing Vaccinia virus administered either intratumorally or subcutaneously in the opposite flank [83]. cDC1s are also required for other immune checkpoint therapies targeting PD1, PDL1 or 41BB [33,70,71]. cDC1-deficient mice displayed a reduced expression of PD1 in their tumor-infiltrating CD8 T cells [71], suggesting that cDC1s are required for a basal activation of cytotoxic immune responses. Besides cross-presentation, cDC1s excel at producing IL12 in the TME [41,84]. IL12 is necessary for immune checkpoint therapy [85], but not sufficient to restore responsiveness to immune checkpoint therapy in cDC1-deficient mice [71]. In several studies, a synergistic effect of Flt3L with polyI:C has been shown, whereby the cross-presentation of tumor antigens and tumor control is improved [70,71,86]. Here, a combination of Flt3L with TLR3 agonists improved the efficacy of anti-PDL1, anti-PD1 and anti-41BB [70,71,86]. However, in these reports, the precise contribution of Flt3L to the combination with immune checkpoint therapy is difficult to discern, because the antitumor effects of polyI:C do not rely on cDC1s [87]. Finally, Batf3-dependent cDC1s are also required for efficacious adoptive T cell transfer therapy [88,89]. Thus, adoptive transfer of Flt3L-secreting CD8 T cells expands tumor-infiltrating cDC1s and potentiates immunotherapy with polyI:C and 41BB-activating antibodies [76]. Overall, Flt3L constitutes a potential therapeutic agent for the treatment of cancer; multiple studies indicate that it improves antitumor immunity and restricts tumor growth. However, a better understanding of its mechanism of action might help prevent undesired interactions with other cancer therapies.

#### **6. Flt3L in the Clinic**

The potential therapeutic effect of Flt3L has given it access to several clinical trials (Table 1). In patients with metastatic colon cancer, pre-resection administration of Flt3L expanded both blood and perilesional DCs, but not tumor-infiltrating DCs [90]. In that study, Flt3L administration was associated with enhanced T cell immunity, shown by the increased sensitivity to recall antigens, but no objective response to Flt3L was observed [90]. Moreover, Fong et al. [91] reported that subcutaneous administration of recombinant Flt3L expands circulating DCs (HLADR+CD3−CD14−CD19−CD56−) in carcinoembryonic antigen (CEA)+ cancer patients. The increase in blood DCs for the generation of a leukapheresis product was loaded with CEA and reinfused into the patient [91]. Fong et al. allowed a two-day gap where they kept the DCs in culture to promote the upregulation of the costimulatory molecules CD80, CD86, CD40, CD83 and CMRF-44 and the chemokine receptor CCR7 [91]. Out of 12 patients, two showed tumor regression, one showed a mixed response, and in two the disease stabilized for at least three months, which associated with an enhanced cytotoxic potential from their peripheral blood mononuclear cells [91]. In another phase I clinical trial, Flt3L was administered for 14 consecutive days monthly to patients with human epidermal growth factor receptor 2 (HER-2/neu)+ breast and ovarian cancers [92]. Flt3L boosted the efficacy of a vaccine based on HER-2/neu peptides administered with GM-CSF [92].

Among its concerning adverse effects, Flt3L was suggested to drive lymphoproliferative malignancies [63]. Flt3L might promote the proliferation of Flt3L-dependent/-addict cancers, which is a frequent feature of acute myeloid leukemia. However, even some acute myeloid leukemia models can benefit from the immunostimulatory effects of Flt3L [93]. Thus, caution should be taken when administrating Flt3L to immune cell malignancies. Among the reported unwanted consequences, inflammatory effects stand out. Pre-resection administration of Flt3L to patients with metastatic colon cancers, only local symptoms such as erythematous nodules with no arthralgia, myalgia or fever were reported [90]. Fong et al. indicated that Flt3L administration only caused minor adverse effects, while DC reinfusion caused self-limited low-grade rigors and diarrhea [91]. In their study with gynecological cancer patients, Disis et al. [92] indicated that only one out of five patients receiving Flt3L alone developed a low-grade rash. Here, two out of five patients receiving Flt3L and GM-CSF developed serological alterations, with one of them suffering Sicca syndrome [92]. This study also reported the development of transient nonspecific autoimmune adverse effects in some patients [92]. As mentioned before, Bhardwaj and colleagues [94] tested the effect of Flt3L on patients receiving polyICLC, and described several low-grade local and systemic side effects, and even some high-grade effects such as anemia, hypophosphatemia, syncope, skin ulceration and sepsis. However, these are compatible with the administration of polyICLC [94]. Therefore, despite the concerns on the tumorigenic potential of Flt3L, all its adverse effects are associated with inflammatory disorders. However, all these reports agree that Flt3L was well tolerated, and no dose-limiting toxicity was observed.

**Table 1.** Clinical Trials targeting the Flt3 receptor. Clinical trials registered in ClinicalTrials.gov where FLT3L has been used with immunostimulatory ends. This table includes clinical trials reported by 24 November 2020. Note that FLT3L is often used in a cytokine cocktail to expand hematopoietic precursors to improve the efficacy of bone marrow transplantation [95], but those have been excluded. s.c.: subcutaneous \* Estimated accrual.

**Table 1.** *Cont*.

**Table 1.** *Cont*.

In a recent study, in situ vaccination based on local administration of Flt3L, radiation therapy and the TLR3 ligand polyICLC showed increased CD8+ T cell responses in patients suffering indolent non-Hodgkin's lymphoma [86]. This study reported systemic tumor regression in some patients, but not in those that had not responded to previous treatments [86]. These studies show strong evidence for a role for Flt3L in boosting antitumor immune responses, but it is important to note that trials which prove its clinical efficacy are still lacking.

#### **7. Conclusions and Future Perspective**

Due to the importance of DCs in antigen-(cross-)presenting populations, and the association of their infiltration within cancer with a positive prognosis, different compounds that drive the expansion of these populations has been actively pursued in the last years. To that aim, Flt3L is currently being evaluated in multiple clinical trials (e.g., NCT03789097, NCT02839265, NCT01976585) [86], but no clear therapeutic benefit has been reported.