mTOR Kinase Inhibitor AZD8055 Enhances the Immunotherapeutic Activity of an Agonist CD40 Antibody in Cancer Treatment

mTOR plays a central role in regulation of cell growth and proliferation by monitoring nutrient availability, cellular energy levels, oxygen levels, and mitogenic signals (1). It exists in 2 distinct complexes—mTOR Complex 1 (mTORC1) and mTOR Complex 2 (mTORC2). mTORC1 is characterized by the classic features of mTOR as a nutrient/energy/redox sensor and regulator of protein synthesis (1); mTORC2 has been shown to function as an important regulator of the actin cytoskeleton and Akt activation (2, 3). The mTOR pathway is dysregulated in many kinds of human diseases, especially certain cancers, which suggests it could be a good target for cancer therapy (4, 5). Rapamycin and its pharmacologic analogs (e.g., sirolimus and RAD001) are preferentially effective at blocking mTORC1, which could also have the unwanted side effect of disengaging the mTORC1 negative feedback loop, thereby causing PI3K–AKT overactivation and impairment of overall antitumor efficacy (6). Therefore, ATP-competitive inhibitors of mTOR kinase, which target both mTORC1 and mTORC2 (7) and more profoundly inhibit of 4E-BP1 phosphorylation by mTORC1 (8, 9), are anticipated to have broader application and, perhaps, increased activity when used in combination with other antitumor agents.

Besides direct effects on tumor cells, studies in the past few years have reported a broad spectrum of regulatory effects on both innate and adaptive immune cells by mTOR inhibition (10). Rapamycin exerts its potent immunosuppressive effects through restricting proliferation of T cells and natural killer (NK) cells (11, 12) and inhibiting differentiation, maturation, and function of innate immune cells in vitro, including dendritic cells (DC) and macrophages (13–15). These types of effects could be presumed to impair antitumor immune responses. However, rapamycin-mediated inhibition of mTOR can also cause an increase in the production of proinflammatory cytokines, such as interleukin 12 (IL-12), IL-23, and IL-6 by innate immune cells, and can also decrease the production of the anti-inflammatory cytokine IL-10 following LPS stimulation. Moreover, even though rapamycin can act directly on T cells to suppress their proliferation and effector function, it also has been shown to increase the ability of LPS-stimulated monocytes to prime Th1 cells in vitro (16). Finally, rapamycin-induced autophagy has been reported to increase antigen presentation in DCs in vivo (17). These immune modulating effects of mTOR inhibition suggest mTOR-inhibiting drugs could synergize with more traditional immune modulators for induction of antitumor responses.

CD40 is a TNF receptor family member that plays a critical role in both humoral and cellular immune responses (18). It has a broad pattern of expression, including antigen-presenting cells (APC), B cells, endothelial cells, and some tumor cells (18). Agonistic αCD40 antibodies, a potent mimic of the natural ligand CD154, have been shown to promote T-cell–mediated immunity in treatment of cancers in experimental animal models through activation of APCs (19, 20). In some tumor models, the effector cells were shown to be CD8⁺ T cells and macrophages, and it has been reported that a stimulatory αCD40 antibody indirectly activated NK cells by IL-12 secreted by CD40⁺ DCs and monocytes, which resulted in significant antitumor activity (21). The successes achieved with agonistic αCD40 monoclonal antibodies (mAb) in preclinical models has led to clinical evaluation of anti-human CD40 mAbs as a potential treatment for cancer (22, 23).

In this study, we hypothesized that combining the ATP-competitive inhibitor AZD8055 with αCD40 antibody would induce more efficient antitumor effects by a combination of direct tumor killing and subsequent release of tumor-associated antigens to APCs and coincident modulation of immune cell functions in vivo. The results of our study show that in a syngeneic mouse metastatic renal cell carcinoma (RCC) model, AZD8055 and αCD40 synergize for tumor regression by activating macrophages and DCs and inducing strong Th1 immune responses in the tumor microenvironment.

AZD8055 was supplied by AstraZeneca. Rapamycin was purchased from LC Labs. For in vitro studies, AZD8055 was prepared as a 10 mmol/L stock solution in dimethyl sulfoxide. For studies in mice, AZD8055 and rapamycin were prepared in sterile water with 0.5% hydroxypropylmethylcellulose (HPMC), 0.1% polysorbate 80, and one-third of overall final volume of glass beads and then shaken overnight to generate a homogenous suspension. Agonist rat anti-mouse CD40 (clone FGK115B3) was purified from ascites, as previously described (24). Endotoxin was less than 1 EU/mg antibody, as determined by chromogenic Limulus Amebocyte Lysate kit (Cambrex). Purified rat IgG (immunoglobulin G) was purchased from Jackson ImmunoResearch Laboratories. Monoclonal antibodies obtained from BD PharMingen included anti-mouse CD3ϵ (clone 145-2C11, clone 500A2), anti-mouse CD8 (clone 53-6.7), anti-mouse CD86 (clone GL-1), anti-mouse MHC Class II (I-A/I-E; clone M5/114.15.2), anti-mouse CD69 (clone H1.2F3), and anti-mouse IL-12 (p40/p70; clone C15.6). Monoclonal antibodies obtained from eBiosciences included anti-mouse F4/80 (clone BM8), anti-mouse NKp46 (clone 29A1.4), anti-mouse CD11c (clone N418), NKG2D (clone CX5), anti-mouse IFN-γ (clone XMG1.2), and anti-mouse TNFα (clone MP6-XT22). Pacific orange–conjugated rat anti-mouse CD45 (clone 30-F11) was purchased from Invitrogen.

BALB/c wild-type mice were obtained from the Animal Production Area of the National Cancer Institute-Frederick (NCI-Frederick) Cancer Research and Development Center (Frederick, MD). BALB/c IFN-γ KO (GKO) mice were obtained from the Jackson Laboratories and then bred at NCI-Frederick. Mutant alleles were confirmed by PCR genotyping. All mice were maintained in a dedicated pathogen-free environment and used between 7 and 10 weeks of age in accordance with an approved NCI-Frederick Institutional Animal Care and Use protocol. For tumor cell lines used in this study, Renca (murine renal carcinoma) was obtained from Dr. Pontes (25); the streptozotocin-induced RCC cell line was developed by our lab (26); and the B16-F10 mouse melanoma was obtained from Dr. Josh Fidler (1982). All cell lines were tested by using the molecular testing of biological materials assay for murine cells in 2007. Tumor cell lines were maintained in RPMI 1640 medium with 10% FBS, 2 mmol/L l-glutamine, 1×nonessential amino acids, and 1 mmol/L sodium pyruvate. For studies in mice, Renca tumors were maintained by serial intraperitoneal (i.p.) passage in syngeneic mice. Enhanced green fluorescent protein (eGFP)-Renca was prepared by transducing Renca cells with lentiviral pLenti6/EF1a/eGFP expression vector in the presence of 5 μg/mL polybrene (Sigma), which was created by using Gateway Technology (Invitrogen) by site-specific recombination between pDEST/eGFP, pDEST-5′-EFIα promoter, and pLenti6/R4R2/V5—DEST vectors. RENCA/eGFP clones were selected by adding 5 μg/mL blasticidin to the RPMI media and then confirming more than 90% eGFP expression by flow cytometry.

Renca cells were injected intrasplenically at a dose of 0.4 × 10⁵ cells (Renca) or 2 × 10⁵ (GFP-Renca) on day 0, and splenectomies were done on all mice immediately after tumor injection. Mice were then treated with vehicle control for AZD8055 (0.5% HPMC, 0.1% polysorbate 80, oral gavage) and IgG control for CD40 antibody (i.p.), CD40 antibody (65 μg, i.p.), AZD8055 (20 mg/kg weight, oral gavage), or rapamycin (2 mg/kg weight, oral gavage), according to the schedules outlined for each experiment. All mice were then euthanized on day 17, livers were harvested into Bouin's solution, and the number of tumor nodules was counted by using a dissecting microscope.

Livers were dissected and fixed in 10% neutral buffered formalin and then embedded in paraffin. Sections were cut at 5 μm in thickness, deparaffinized in xylene, and serially dehydrated in decreasing concentrations of ethanol. Sections were stained with Hematoxylin and eosin (H&E) and examined under light microscopy to detect lymphocyte infiltration and evaluate tissue structure.

CD8⁺ T cells were depleted in vivo using i.p. injections of rat anti-mouse CD8 (clone 19–178), starting on day 2 and continuing 3 times weekly (≥90% depletion). Control mice received 10% normal rat serum. To deplete NK cells, mice were given rabbit anti-asialo-GM1 i.v. (Wako Chemicals) on days 2, 4, 6, 9, and 13 after tumor cell implantation in the liver (≥90% depletion). Control mice received 10% normal rabbit serum. For macrophage depletion, mice were injected i.v. with 100 μL of clodronate liposomes on days -1, 2, 6, 10, and 14 after tumor cell implantation into the liver (≥90% depletion). Clodronate liposomes were obtained from Dr. N. van Rooijen (Vrije Universiteit, Amsterdam, Netherlands). PBS-containing liposomes were used as a control.

On day 14 or 17, mice were euthanized and the livers flushed with 10 mL of HBSS through the portal vein, removed, and homogenized by using a GentleMACS Dissociator (Miltenyi Biotec) and then digested in RPMI containing 5% fetal calf serum, 250 U/mL type IV collagenase (Invitrogen), 100 μg/mL DNase I (Roche Molecular Biochemicals), and 1 mmol/L EDTA (pH 8.0), at 37°C for 20 minutes. The homogenate was then processed in a tissue stomacher-80 (Seward) for 30 seconds, washed with HBSS (BioWhittaker), and resuspended in 40% Percoll (Amersham Pharmacia) in Dulbecco's modified Eagle's medium (BioWhittaker). The suspension was underlaid with 80% Percoll and centrifuged for 25 minutes at 1,000 × g. Leukocytes were collected from the interphase, filtered with a 70-μm Nylon cell strainer (BD Biosciences), washed, and enumerated on a Sysmex KX-21 (Roche) automated cell counter.

Flow cytometric analysis was done on liver leukocytes according to a previously described procedure (27). Briefly, cells (1 × 10⁶) were incubated in cell staining buffer (0.1% bovine serum albumin, 0.1% sodium azide) containing 250 μg/mL 2.4G2 ascites for 25 minutes. Cells were stained with diluted fluorescently conjugated antibodies for 30 minutes followed by 2 washes in staining buffer. Labeled cells were analyzed on an LSR-II flow cytometer (Becton Dickinson). For Ki67 staining, fluorescein isothiocyanate (FITC)-mouse anti-human Ki67 set (BD PharMingen) was used according to the manufacturer's suggested protocol. For IFN-γ intracellular staining, hepatic mononuclear cells (MNC) were restimulated with phorbol 12-myristate 13-acetate (5 ng/mL)/Ionomycin (1 μg/mL) for 4 hours and 1 μg/mL Brefeldin A (GolgiPlug) for another 5 hours at 37°C in 5% CO₂. For the detection for TNFα and IL-12, the restimulation conditions were 3 hours for LPS (100 ng/mL) plus 5 hours for brefeldin A, and 2 hours for LPS (1 μg/mL) plus 10 hours for brefeldin A, respectively. After surface protein staining, cells were fixed by using the Cytofix/Cytoperm Kit (PharMingen) and then stained with optimally titered anti-mouse IFN-γ, TNFα, and IL-12.

Livers obtained on day 17 were placed in RNALater (Ambion). Total RNA was isolated from liver homogenates by using TRIzol (Invitrogen), precipitated, and then reverse transcribed (Applied Biosystems). RT2 real-time SYBR Green/Rox PCR master mix and RT2 Profiler PCR array were used for murine cytokines and chemokines detection (SuperArray). Genes of interest were subsequently examined with greater sensitivity by real-time reverse transcriptase PCR by using TaqMan Universal PCR Master Mix and the ABI Prism 7300 Detection System (TaqMan; Applied Biosystems) according to the manufacturer's instructions. Primer sets for mouse included IL-12(p35; Mm00434169), TNFα (Mm00443258), IFN-γ (Mm99999071), CCL5 (Mm01302428), CXCL10 (Mm00445235), and CXCL9 (Mm00434946; Applied Biosystems). Gene expression was normalized to the level of the β-actin housekeeping gene. Data were analyzed by using the ΔΔC_t method (28) and expressed as a fold change in mRNA expression relative to control values.

Values of P were determined by unpaired student's t test. The value of P < 0.05 was considered statistically significant.

On the basis of reports that AZD8055 induces a profound growth inhibition, autophagy, and cell death in many human tumor cell lines (29, 30) and our previous study showing that Renca tumor cells express functional CD40 (24), we first tested the direct effect of AZD8055 and αCD40 on the survival of Renca tumor cells in vitro. We found that only AZD8055 inhibited Renca cell growth, whereas αCD40 could neither inhibit Renca growth directly nor enhance the inhibitory ability of AZD8055 (Fig. 1A). To evaluate the antitumor effect in vivo, we designed and evaluated a variety of different schedules for the administration of AZD8055 in combination with αCD40 (Fig. 1B), in an experimental Renca liver metastasis model. The results showed that priming the immune system with αCD40 prior to the use of AZD8055 which directly targets the tumor cells (Fig. 1B schedule a) was the most active approach among the schedules utilized (Fig. 1C). These findings suggest administration of AZD8055 before or at the same time αCD40 delivery might suppress some immune functions and thereby impair the immune-priming function of subsequently delivered αCD40. Therefore, schedule a (αCD40 prior to AZD8055) which yielded the best overall antitumor effect was used in all subsequent studies, although significant tumor reduction was also observed with each of the other schedules tested.

In contrast to the antitumor effects of AZD8055 and αCD40 alone, we found that AZD8055/αCD40 combination treatment induced a substantial reduction in both number (Fig. 2A) and size (data not shown) of tumor nodules in liver, although both agents administered alone also had some antitumor effects. The enhanced antitumor efficacy of the combination was also shown in a Renca orthotopic model in which Renca cells were injected under the kidney capsule and allowed to metastasize spontaneously (Supplementary Fig. S1). We also observed that the antitumor responses achieved by AZD8055/αCD40 were largely abrogated in CD40^−/− mice bearing Renca (Supplementary Fig. S2), which indicates that host CD40 expression is necessary for the antitumor response in vivo. We also hypothesized that a major contribution of mTOR inhibitor–induced tumor apoptosis would be the liberation of tumor antigen from killed cells, which could further promote maturation and T-cell–priming function of APCs in response to αCD40. Therefore, we expected similar enhanced antitumor effects to be achieved when αCD40 was combined with different mTOR inhibitors. However, when αCD40 was combined with the classical mTOR inhibitor rapamycin, the antitumor responses achieved by the combination were indistinguishable from those achieved by rapamycin alone (Fig. 2A). Importantly, we observed a remarkable infiltration of CD8⁺ T cells, DCs, and macrophages into the livers of AZD8055/αCD40-treated mice, but not in the group treated with rapamycin/αCD40 (Fig. 2B), when compared with either vehicle control or αCD40 alone treated groups. These results showed that the AZD8055 ATP-competitive mTOR kinase inhibitor, but not rapamycin, induced enhanced antitumor activities when combined with αCD40, suggesting substantially different mechanisms of action on the immune system for AZD8055, compared with rapamycin.

We also investigated the antitumor effects of AZD8055/αCD40 in several other tumor models. By using the streptozotocin-induced, CD40 positive, RCC cell line that we characterized previously (26), we observed that AZD8055/αCD40 exhibited enhanced antitumor efficacy in vivo that was comparable to that observed for Renca (Supplementary Fig. S3A). We also used the CD40 negative B16 melanoma cell line to potentially address whether tumor-associated CD40 expression might contribute to the αCD40-mediated immune or antitumor effects. In contrast to the 2 RCC models previously described, neither AZD8055 nor αCD40, or the combination of AZD8055/αCD40, had any appreciable ability to reduce the number of B16 tumor nodules in the liver (Supplementary Fig. S3B). It is noteworthy, however, that regardless of the tumor model, the AZD8055/αCD40 combination still elicited significant immune cell infiltration in vivo. Taken together, it seems that RCC, may be particularly amenable to the AZD8055/αCD40 combination therapy.

To further delineate the immune effects induced by AZD8055 and αCD40, we examined the livers of treated mice and found that although αCD40 alone induced some recruitment of immune cells to the liver, αCD40 plus AZD8055 induced a more pronounced lymphocytic infiltration. In addition, there was a pronounced localization of leukocytes around tumor foci in response to αCD40/AZD8055 whereas few leukocytes were found in or around tumor foci in the vehicle control and AZD8055-treated groups (Fig. 3A). To determine the activation of CD8⁺ T cells and NK cells, hepatic MNCs of tumor-bearing mice were isolated on day 14 and subjected to flow cytometric analysis. AZD8055 plus αCD40 induced a significant elevation in the expression of CD69 and NKG2D on CD8⁺ T cells (Fig. 3B). Among NK cells, CD69⁺ and NKGD2⁺ cell frequencies were also moderately increased in the combination-treated group (Fig. 3B). Furthermore, there was a notable upregulation of Ki67 protein in both NK cells and CD8⁺ T cells from AZD8055/αCD40-treated mice (Fig. 3C), suggesting that the expansion of these cell types might be at least partially due to proliferation. Moreover, we also found significant induction of IFN-γ expression in CD8⁺ T cells and NK cells derived from AZD8055/αCD40-treated mice (Fig. 3D).

Because AZD8055 could significantly increase the frequency of macrophages and DCs in liver when combined with αCD40 (Fig. 2B), we next investigated the effect of AZD8055/αCD40 on the status of antigen-presenting DCs and macrophages in vivo. By using eGFP-expressing Renca cells, we detected increased efficiency of processing of tumor-derived antigen by macrophages and DCs in AZD8055/αCD40-treated mice (Fig. 4A). In addition, the maturation markers CD86 and MHC II were significantly upregulated on macrophages and DCs (Fig. 4B). Increased production of IL-12 was also observed in macrophages and DCs from the AZD8055-alone group and in macrophages from the combination group (Fig. 4C). In addition, TNFα production was only significantly induced in macrophages by AZD8055/αCD40 treatment (Fig. 4D). Overall, these results showed that macrophages and DCs were matured and activated in the AZD8055/αCD40-treated group.

We next used antibodies to deplete CD8⁺ T cells and NK cells before AZD8055/αCD40 treatment and found that depletion of either cell type could partially mitigate the antitumor response. The depletion of both cell types could substantially reduce the antitumor response to the level achieved with AZD8055 alone (Fig. 5A), suggesting that CD8⁺ T cells and NK cells might contribute to the enhanced antitumor effect induced by the combination treatment. Furthermore, by using macrophage depletion, we showed a dramatic loss of antitumor activity by AZD8055/αCD40, thereby confirming a unique and indispensable role of macrophages in the antitumor immune responses induced by AZD8055/αCD40 combination treatment (Fig. 5B).

Because we observed a significant degree of infiltration and activation of immune cells in the AZD8055/αCD40-treated tumor-bearing mice, we hypothesized that this process would be associated with increased levels of cytokines and chemokines induced by AZD8055/αCD40. Therefore, we analyzed liver tissue from treated mice for gene expression by quantitative PCR (qPCR). AZD8055/αCD40 treatment significantly increased Th1 cytokines and Th1-associated chemokine expression in the liver, including IL-12, IFN-γ, TNFα, CCL5/RANTES, CXCL10/IP-10, and CXCL9/MIG (Fig. 6A), whereas αCD40 alone only slightly induced IL-12, CCL5, and CXCL9 production, as compared with AZD8055 alone and vehicle control groups. Because of the high levels of IFN-γ production and previous studies showing the role of IFN-γ in this Renca model (31), we evaluated its role in the in vivo therapy. As shown in Figure 6B, AZD8055/αCD40-mediated antitumor responses were completely abolished in IFN-γ KO mice as was as the activation of CD8⁺ T cells and NK cells (Fig. 6C). When the number of CD8⁺ T cells was evaluated, no significant differences between WT mice and IFN-γ KO mice were observed (Fig. 6C), which implied that IFN-γ might have no effect on survival and proliferation of CD8⁺ T cells in this model. The previously observed increase in macrophages was also dependent on IFN-γ (Fig. 6D), but no impairment of DC priming was detected in IFN-γ KO mice (data not shown). In addition to its immunoregulatory roles, we also found that IFN-γ could directly enhance inhibition of tumor cell growth by AZD8055 in vitro (Supplementary Fig. S4).

Inhibitors of the mTOR pathway have been used individually and in combination with a variety of cancer therapeutic agents, including chemotherapy, IGF-IR inhibitors, and trastuzumab (32–34). Our article outlines an innovative immunotherapeutic approach combining a novel ATP-competitive mTOR inhibitor with an agonist αCD40 antibody in an experimental model of metastatic RCC. We showed that combining the mTORC1/mTORC2 inhibitor AZD8055 with αCD40 induced significant infiltration, proliferation, and activation of CD8⁺ T cells and NK cells in the liver. Th1 cytokines and related chemokines were also significantly elevated by the combination treatment over levels achieved by either agent alone. In addition, the frequency of activated macrophages and DCs in tumor foci was substantially increased following AZD8055/αCD40 treatment, which facilitated a transition of the tumor microenvironment from predominantly immunosuppressive to one that is strongly Th1 in composition.

Recently, the use of the rapamycin analog temsirolimus to inhibit mTORC1, in combination with cancer vaccine therapy also showed enhanced antitumor responses (35). However, temsirolimus was not able to regulate innate immune cells as we have observed by using AZD8055. Furthermore, we show that the mTORC1 inhibitor rapamycin was able to modestly inhibit tumor growth to a similar extent as did AZD8055 alone, but unlike AZD8055, it was unable to synergize with αCD40 for enhanced induction of immunomodulatory responses. Although the efficacy of AZD8055 as a single agent was similar to that of rapamycin alone, it remains unclear whether this is due to the suboptimal concentrations of the inhibitors used in our study or whether there are differences with respect to efficacious dose range with mTORC1/2 versus mTORC1 inhibitors. Nevertheless, our data makes clear that the distinctive benefits of AZD8055, when used in combination with αCD40, cannot be simply attributed to quantitatively increased antigen release by AZD8055-induced tumor destruction that could simply contribute to further maturation of αCD40-primed APC and promotion of subsequent Th1 antitumor responses. Instead, when compared with rapamycin, the ATP-competitive mTOR inhibitor more dramatically inhibited protein synthesis and induced G1 cell-cycle arrest in several cancer cell lines not only by targeting both mTORC1 and mTORC2 equally, but also by more completely inhibiting 4E-BP1 phosphorylation, which causes more profound inhibition of mTORC1 (8, 9, 36, 37). These different effects on tumor cells or endothelial cells of AZD8055 may result in qualitatively different tumor-derived products, including distinct cytokine and chemokines, which could therefore contribute indirectly to differences in immune cell recruitment and activation. Another possible explanation for the increase of DCs and macrophages induced by AZD8055 but not rapamycin in the liver is that 4E-BP1 dephosphorylation caused by AZD8055 may contribute to the myeloid differentiation program and thereby alter some biological functions of subsets of mononuclear phagocytes (38). Moreover, we found IFN-γ played important roles in the combination therapy through both directly mediating antitumor response and indirectly regulating immune cell survival and functions. The induction of key IFN-γ–inducible proteins ISG15 (interferon-stimulated gene 15) and CXCL10 that mediate IFN responses are enhanced in 4E-BP1(−/−) mouse epithelial fibroblasts, suggesting that AZD8055 inhibition on 4E-BP1 activity may exhibit important regulatory effects in the generation of IFN responses (39). Recently, divergent effects of rapamycin versus ATP-competitive mTOR inhibitors have been described for tumor cells (6). Our studies underscore the need for additional investigation as to the different roles these agents play in immune system modulation, particularly within certain tumor microenvironments, which may inform new approaches to the use of these agents for treatment of cancer or autoimmune diseases, or for transplantation.

Recent studies documented several promising features of mTOR inhibition that may enhance antitumor immunity (10). First, mTOR inhibition can promote IL-12 production by DCs in the presence of LPS (16). By using intracellular staining, we observed only a slight enhancement of LPS-induced IL-12 production by macrophages and DCs from AZD8055-treated mice. Thus, our data are more consistent with a mechanism whereby the increased level of IL-12 in the combination treatment group is mainly due to an overall increase in the number of IL-12–producing macrophages and DCs present in the liver. Second, mTOR inhibition enhances antigen presentation through induction of autophagy (17). Consistently, we also found upregulation of CD86 and MHC II expression and presentation of tumor-derived GFP protein in DCs and macrophages in AZD8055/αCD40-treated mice, which would be expected to contribute to more effective T-cell–mediated responses. The upregulation of NKG2D expression on CD8⁺ T cells and NK cells by AZD8055/αCD40 treatment may also contribute to their cytotoxic activity against tumors (40, 41). Rapamycin has also been reported to promote CD8⁺ memory T-cell differentiation (42). However, we found that most CD8⁺ T cells induced by AZD8055/αCD40 in our model were CD8⁺ effector T cells (CD44^high, CD62L^low, data not shown). On the other hand, although mTOR inhibitors are generally considered to have an overall immunosuppressive effect (13, 43, 44), we observed no significant reduction in the number of immune cells (Fig. 2B), the activation, or proliferation of CD8 and NK cells (Fig. 3) as well as the expression of maturation markers by DCs (Fig. 4), when results from AZD8055 or rapamycin alone were compared with the vehicle control group. We also observed an increased number of Tregs in the livers of AZD8055/αCD40-treated mice (Supplementary Fig. S5), that is consistent with the earlier observation that mTOR inhibition can promote Treg differentiation (45). Despite the presence of Treg cells, enhanced proliferation and activation of CD8⁺ T cells and NK cells was observed following combination treatment. Therefore, we conclude that the immunosuppressive effects of mTOR inhibitors are not dominant in the setting of their combination with powerful immunotherapeutic stimulants such as αCD40.

Although systemic administration of agonist αCD40 has been used in cancer patients to boost CD8⁺ T cell or NK cell responses to tumors and to break peripheral self-tolerance (21, 46), there is also evidence that it can induce immune suppression in some settings of tumor growth or virus infection (47, 48). We did detect transient infiltration of CD8⁺ T cells, NK cells, and macrophages into liver during the early stage of single agent αCD40 treatment, and long-term treatment accelerated the deletion of all these antitumor immune cells (data not shown). We propose that persisting functions of CD8⁺ T cells restored in the AZD8055/αCD40 treatment group result from the enhanced priming by an increased frequency of APCs acquiring antigens derived from AZD8055-triggered tumor destruction, and activated by proinflammatory cytokines elevated by the combination treatment (47). The mechanism by which AZD8055 administration prevents the loss of macrophages in vivo induced by long-term αCD40 treatment still needs further investigation. In addition, possible proangiogenic effects may result in some settings following CD40 engagement which could be perceived as a limitation of αCD40 for cancer therapy (49). However, we found AZD8055/αCD40 had an overall antiangiogenic effect (data not shown), which could also contribute to an overall antitumor and antimetastatic effect. One of our previous studies showed that direct ligation of CD40 on Renca tumor cells triggered cytokine production, leukocyte recruitment, and antitumor responses which could be independent of host CD40 expression (24). In this study, we used Renca-bearing CD40 KO mice to show that Renca-associated CD40 expression was not sufficient for the antitumor effects of this combination therapy (Supplementary Fig. S2). Furthermore, we also observed significant expansion of the immune system by using the CD40 negative B16 tumor cell line, which further suggests that tumor-derived CD40 expression is not sufficient for immune responses induced by αCD40-based therapy. However, because the antitumor effects of AZD8055/αCD40 were also lost in the B16 tumor model (Supplementary Fig. S3B), we cannot exclude the possibility that CD40 ligation by tumor cells contributes to enhanced immune sensitivity of some tumors. Future studies should be aimed at addressing the striking sensitivity that RCC seems to have for the combined effects of mTOR and CD40 targeted agents. Ongoing studies will also explore the sensitivities of other tumor models toward mTOR and/or CD40-based combination therapies.

In conclusion, our study shows that combining the ATP-competitive mTOR inhibitor AZD8055 with αCD40 induced strongly enhanced Th1 antitumor responses through the recruitment and activation of macrophages and DCs in the tumor microenvironment. These events were critical for the overall enhanced antitumor effects of this novel combination because depletion of macrophages substantially reduced the overall antitumor effects. Overall, these results show novel immune-stimulating effects that contribute to enhanced antitumor activity, suggesting this combination may also distinctively restructure the tumor microenvironment of RCC in a manner that favors tumor regression.

No potential conflicts of interest were disclosed.

The authors thank Anjan Thakurta (formerly of AstraZeneca) for helpful discussions.

This project was funded in whole or part by the Intramural Research Program of NIH/NCI.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.