Most cancer immunotherapies include activation of either innate or adaptive immune responses. We hypothesized that the combined activation of both innate and adaptive immunity will result in better antitumor efficacy. We have previously shown the synergy of an agonistic anti-CD40 mAb (anti-CD40) and CpG-oligodeoxynucleotides in activating macrophages to induce tumor cell killing in mice. Separately, we have shown that a direct intratumoral injection of immunocytokine (IC), an anti-GD2 Ab linked to IL-2, can activate T and NK cells resulting in antitumor effects. We hypothesized that activation of macrophages with anti-CD40/CpG, and NK cells with IC, would cause innate tumor destruction, leading to increased presentation of tumor Ags and adaptive T cell activation; the latter could be further augmented by anti–CTLA-4 Ab to achieve tumor eradication and immunological memory. Using the mouse GD2+ B78 melanoma model, we show that anti-CD40/CpG treatment led to upregulation of T cell activation markers in draining lymph nodes. Anti-CD40/CpG + IC/anti–CTLA-4 synergistically induced regression of advanced s.c. tumors, resulting in cure of some mice and development of immunological memory against B78 and wild type B16 tumors. Although the antitumor effect of anti-CD40/CpG did not require T cells, the antitumor effect of IC/anti–CTLA-4 was dependent on T cells. The combined treatment with anti-CD40/CpG + IC/anti-CTLA-4 reduced T regulatory cells in the tumors and was effective against distant solid tumors and lung metastases. We suggest that a combination of anti-CD40/CpG and IC/anti-CTLA-4 should be developed for clinical testing as a potentially effective novel immunotherapy strategy.

Recent advances in cancer immunotherapy have shown it to be an effective strategy for treatment of certain cancers (1, 2). However, single-agent immunotherapeutic approaches can have limited efficacy, whereas combining two or more immunotherapeutic strategies can be synergistic in inducing antitumor effects (35).

One of the activators of innate and adaptive immune responses is agonistic anti-CD40 mAb (anti-CD40), which can induce antitumor effects in mice and in cancer patients (6). The clinical potential of anti-CD40 has been demonstrated by regression of primary and metastatic adenocarcinomas in 4 of 21 patients with pancreatic cancer (2). This clinical and preclinical activity of anti-CD40 against pancreatic cancer confirms our earlier findings showing the antitumor effect of anti-CD40 via macrophage activation in several mouse models (79). We have also demonstrated that the antitumor effect of anti-CD40 can be greatly potentiated by CpG-oligodeoxynucleotides, a TLR9 agonist, via synergistic activation of macrophages in mouse models of melanoma and neuroblastoma (10); however, complete responses were rarely achieved, suggesting that combining this approach with other immunotherapeutic modalities could be beneficial. Radiotherapy can convert tumor-associated suppressive M2 macrophages into effector M1 macrophages in the tumor microenvironment, facilitating T cell immunotherapy (11). We showed that immunotherapy with anti-CD40/CpG similarly converts M2 protumor macrophages into M1 antitumor effector macrophages (12), suggesting that this approach could also be effectively combined with T cell immunotherapy.

We have also shown that an intratumoral (IT) injection of immunocytokine (IC), which consists of an antitumor Ab linked to IL-2, can serve as an in situ vaccine; it enhances local antitumor effects and can generate an adaptive T cell response directed against distant tumors (13, 14). These in situ vaccine effects involve T cells as well as NK cells, and can result in T cell memory (13, 14).

T cell activation and function in the tumor microenvironment of cancer patients are suppressed (15, 16). Two inhibitory receptors on antitumor T cells, CTLA-4 and programmed death 1 (PD-1), play an important role in T suppression by the tumor (17, 18). Blockade of these inhibitory interactions, known as immune checkpoint blockade, with anti–CTLA-4, anti–PD-1, or both, counteracts the immunosuppression, results in augmenting endogenous tumor-specific T cell responses, and provides clinical benefit, particularly in melanoma patients (1921). CTLA-4 can synergize with NO produced by activated macrophages in inhibiting T cells via T regulatory cell (Treg) activation (22); anti–CTLA-4 Ab can deplete Tregs in the tumor (23). Therefore, our overall hypothesis was that a synergistic activation of innate and adaptive immunity could be achieved by combining anti-CD40/CpG (to activate macrophages), IT-IC (to activate NK cells and T cells), and anti–CTLA-4 checkpoint inhibitor (to counteract T cell suppression), resulting in strong antitumor effects.

Six- to ten-week-old female C57BL/6 and nude mice were obtained from Taconic Farms (Germantown, NY) or from The Jackson Laboratory (Bar Harbor, ME). Mice were housed in the University of Wisconsin-Madison animal facilities at the Wisconsin Institutes for Medical Research. Mice were used in accordance with the Guide for Care and Use of Laboratory Animals (National Institutes of Health publication no. 86-23, 1985; National Institutes of Health, Bethesda, MD).

Mouse B16-F10 melanoma cells (also referred to as B16) were transduced to express GD2 (B16-GD2) using a retroviral vector that encodes the GD2 mini-operon (MP9956:SFG.GD3synthase-2A-GD2synthase plasmid; a kind gift from Prof. Martin Pule from University College London). B16, B16-GD2, and B78 melanoma, a slow-growing derivative of B16 that expresses GD2 (24), cell lines were grown in RPMI 1640 cell culture medium supplemented with 10% FBS (Sigma-Aldrich, St. Louis, MO), 2 mM of l-glutamine, 100 U/ml penicillin/streptomycin, and 0.5 μM of 2-ME (Invitrogen Life Technologies, Carlsbad, CA) at 37°C in a humidified 5% CO2 atmosphere.

s.c. tumors were established by injecting 2 × 106 (B78) or 1–5 × 105 (B16 or B16-GD2) cells in 0.1 ml of PBS. Tumor size is reported as tumor volume (mm3) by measuring perpendicular diameters of the tumor and calculating as follows: (1/2) × tumor length × tumor width2; it is expressed as mean volume ± SEM of tumor volumes for all mice of each experimental group.

Agonistic anti-CD40 was obtained from ascites of nude mice injected with the FGK 45.5 hybridoma cells (a gift from Dr. F. Melchers, Basel Institute for Immunology, Basel, Switzerland) and enriched for IgG by ammonium sulfate precipitation. CpG1826 was purchased from TriLink Biotechnologies (San Diego, CA). The humanized hu14.18-IL-2 IC (APN301; Apeiron Biologics, Vienna, Austria) was supplied by the NCI Biologics Resources Branch (Frederick, MD) via a collaborative relationship with Apeiron Biologics. Hu14.18-IL-2 is an IC consisting of human IL-2 genetically linked to the carboxy termini of each human IgG1 H chain of the GD2-specific hu14.18 mAb (25). Anti–CTLA-4 clone UC10-4F10-11, IgG2b, was a gift of Dr. Jeffrey Bluestone, University of California, San Francisco (San Francisco, CA) (26). This UC10-4F10-11 IgG2b was used in all experiments requiring anti–CTLA-4 Ab, unless stated otherwise. Anti–CTLA-4 clone 9D9, provided as both IgG2a and IgG2b isotypes, was obtained from Bristol-Myers Squibb (Redwood City, CA) (23), and use of these two mAbs in Fig. 7 is clarified in the legend and labels. The anti-murine PD-1 mAb (clone 4H2) was obtained from Bristol-Myers Squibb (Redwood City, CA) (27).

FIGURE 7.

Systemic antitumor and antimetastatic effects of the combined therapy. (A) Comparison of different checkpoint Abs in combination with 14.18-IL-2 IC against B78 melanoma. C57BL/6 mice were injected s.c. with 2 × 106 B78 cells. 14.18-IL-2 IC (5 μg per mouse) was injected IT on days 12–16. Various checkpoint Abs (200 μg per mouse) were injected i.p. on days 12, 14, 16, 19, 21, and 23. (B and C) B78 melanoma cells (2 × 106) were injected s.c. into the right and left sides of the abdomen on day 0. Each tumor on the left was injected with CpG (25 μg) on days 12, 14, and 16 and with 14.18-IL-2 IC (25 μg) on days 12–16. These treatments were given in combination with i.p. injections of anti-CD40 (500 μg) on day 9 and anti–CTLA-4, IgG2a (200 μg) on days 12, 14, 16, 19, 21, and 23. Results are shown as means ± SEM of volumes of the left side (treated) (B) and right side (untreated) (C) tumors. (DF) C57BL/6 mice were injected s.c. with 2 × 106 B78 melanoma cells (day 0) and i.v. with 1 × 105 B16-F10 melanoma cells (day 1). Mice were treated with anti-CD40 (0.5 mg) i.p. on day 8, with CpG (25 μg) IT on days 11, 13, and 15, with 14.18-IL-2 IC (25 μg) IT on days 11–15, and with anti–CTLA-4 IgG2a (200 μg) i.p. on days 11, 13, 15, 18, 20, and 21. Results are shown as means ± SEM of s.c. tumor volumes (D) and survival (E) of five mice per group. Lung photographs taken on day 32 from a separate identical experiment are shown, contrasting visible metastases in control versus treated mice (F). Differences between the control versus treatment groups are shown. Statistical differences are indicated for the last day on the graph. *p < 0.05, **p < 0.01.

FIGURE 7.

Systemic antitumor and antimetastatic effects of the combined therapy. (A) Comparison of different checkpoint Abs in combination with 14.18-IL-2 IC against B78 melanoma. C57BL/6 mice were injected s.c. with 2 × 106 B78 cells. 14.18-IL-2 IC (5 μg per mouse) was injected IT on days 12–16. Various checkpoint Abs (200 μg per mouse) were injected i.p. on days 12, 14, 16, 19, 21, and 23. (B and C) B78 melanoma cells (2 × 106) were injected s.c. into the right and left sides of the abdomen on day 0. Each tumor on the left was injected with CpG (25 μg) on days 12, 14, and 16 and with 14.18-IL-2 IC (25 μg) on days 12–16. These treatments were given in combination with i.p. injections of anti-CD40 (500 μg) on day 9 and anti–CTLA-4, IgG2a (200 μg) on days 12, 14, 16, 19, 21, and 23. Results are shown as means ± SEM of volumes of the left side (treated) (B) and right side (untreated) (C) tumors. (DF) C57BL/6 mice were injected s.c. with 2 × 106 B78 melanoma cells (day 0) and i.v. with 1 × 105 B16-F10 melanoma cells (day 1). Mice were treated with anti-CD40 (0.5 mg) i.p. on day 8, with CpG (25 μg) IT on days 11, 13, and 15, with 14.18-IL-2 IC (25 μg) IT on days 11–15, and with anti–CTLA-4 IgG2a (200 μg) i.p. on days 11, 13, 15, 18, 20, and 21. Results are shown as means ± SEM of s.c. tumor volumes (D) and survival (E) of five mice per group. Lung photographs taken on day 32 from a separate identical experiment are shown, contrasting visible metastases in control versus treated mice (F). Differences between the control versus treatment groups are shown. Statistical differences are indicated for the last day on the graph. *p < 0.05, **p < 0.01.

Close modal

Tumor-bearing mice were treated with anti-CD40 mAb (0.25 mg/0.5 ml, unless indicated otherwise) i.p. and CpG (0.025 mg/0.1 ml, unless indicated otherwise) IT on different days as stated in the figure legends. CpG was given 3 d after anti-CD40 as in our previous studies (10, 12) because the maximal upregulation of TLR9 on macrophages occurs 3 d after anti-CD40 injection (10). Hu14.18-IL-2 IC was injected IT at doses of 5 or 25 μg in 0.1 ml of PBS (13). Anti–CTLA-4 (0.2 mg/0.2 ml PBS) was injected i.p. every other day three times a week for 2 wk. This combination is designated anti-CD40/CpG + IC/anti–CTLA-4.

C57BL/6 mice were injected s.c. on the left side of the abdomen with 2 × 106 B78 cells (day 0). Mice were injected with anti-CD40 on day 6 and CpG on day 9. Control tumor-bearing mice received rat IgG and PBS. Another group of control mice did not receive the tumor and treatments (“naive”). On day 10, left inguinal (draining) lymph nodes and right inguinal (contralateral) lymph nodes were removed and pooled from three mice per group. Lymph node cells were stained with anti–CD8-PE (clone 53-67), anti–CD69-allophycocyanin (clone H1.2F3), anti–CD44-allophycocyanin (clone IM7), anti–CD25-allophycocyanin (clone PC61; all from BioLegend, San Diego, CA), and anti-CD4-FITC (clone GK1.5; eBioscience, San Diego, CA). Data acquisition was performed on FACSCalibur flow cytometer with CellQuest software (BD, Franklin Lakes, NJ). Flow cytometry analysis was performed on FlowJo software (Tree Star, Ashland, OR) by gating on CD8+ or CD4+ cells. Results are presented as percent of positive cells or mean fluorescence intensity ratios.

B78 melanoma cells were injected s.c. into C57BL/6 mice on day 0. Anti-CD40 was injected i.p. on day 23; CpG was injected IT on days 26, 28, and 30; 14.18-IL-2 IC was given IT on days 26–30; and anti–CTLA-4 was injected i.p. on days 26, 28, 30, and 33. Control tumor-bearing mice received no treatment. On day 34, tumors were harvested, cut into small pieces, and incubated for 30 min at 37°C in dissociation solution containing HBSS supplemented with 5% FBS, 1 mg/ml collagenase type D, and 100 μg/ml DNase I (Sigma-Aldrich).

For cell surface staining, the cells were preincubated with mouse BD Fc block purified anti-mouse CD16/CD32 (clone 2.4G2; BD Biosciences) for 5 min at 4°C. After blocking, cells were incubated with CD4-FITC (clone GK1.5; eBioscience), CD8a-PE (clone 53-6.7; BioLegend), F4/80-FITC (clone BM8; eBioscience), or CD49b-PE (clone DX5; BD Biosciences) at 4°C for 30 min. The stained cells were washed and resuspended in PBS/1% FBS, propidium iodide was added, and data were acquired on a BD FACSCalibur.

For Treg staining, the cells were first incubated with CD4-FITC (clone GK1.5; eBioscience), CD45 eFluor450 (clone 30-F11; eBioscience), CD25-allophycocyanin (clone PC61; BioLegend), and fixable viability dye 506 (FVD506; eBioscience) at 4°C for 30 min. The stained cells were fixed in the eBioscience Foxp3/Transcription factor staining buffer set according to the manufacturer’s manual. After fixation overnight the cells were stained with Foxp3-PECy7 (clone FJK16s; eBioscience). Flow cytometry data were acquired using the MACSQuant Analyzer (Miltenyi Biotec) and analyzed using the software FlowJo version 10.1. Statistical analysis was performed using GraphPad Prism version 8.

An unpaired Student t test and ANOVA test with either Dunnett’s posttest or Tukey posttest were used to determine significance of differences between experimental and relevant control values within each experiment.

In our previous studies we showed a strong antitumor synergy between anti-CD40 and CpG when both agents were injected systemically (i.p.). It was reported that CpG given IT can result in T cell activation enhanced by additional T cell activation modalities including anti–CTLA-4 (28). Because our goal was to facilitate T cell activation by the means of innate immunity, we thought to combine IT CpG with systemic anti-CD40 treatment. The results show that anti-CD40/CpG caused suppression of B78 melanoma growth in syngeneic C57BL/6 mice (Fig. 1A) and in T cell–compromised nude mice (Fig. 1B), indicating that the antitumor effect of systemic anti-CD40 combined with local CpG does not require T cells. To determine which cell population is responsible for the antitumor effect after anti-CD40/CpG therapy, we injected C57BL/6 mice s.c. with B16 cells (day 0) and with anti-CD40 i.p. on day 4. Three days later (day 7), peritoneal cells were obtained, stained, gated on CD11bhigh cells, sorted into four subpopulations based on their expression of CD11b and Gr-1 markers, and further characterized by histological staining and antitumor activity in vitro. Similar to what we reported previously (29), antitumor effector cells were found to be CD11bhigh Gr-1 macrophages, as was confirmed by morphology and secretion of NO (data not shown).

FIGURE 1.

Antitumor effect of anti-CD40 + CpG is T cell independent. B78 melanoma cells (2 × 106) were injected s.c. into C57BL/6 (A) and nude mice (B) on day 0. Anti-CD40 (500 μg) was given i.p. on day 23. CpG (25 μg) was given IT on days 26, 28, and 30. Data shown are means ± SEM of five mice per group. Statistical differences are indicated for the last day on the graph. Differences between the control versus treatment groups are shown. *p < 0.05, **p < 0.01.

FIGURE 1.

Antitumor effect of anti-CD40 + CpG is T cell independent. B78 melanoma cells (2 × 106) were injected s.c. into C57BL/6 (A) and nude mice (B) on day 0. Anti-CD40 (500 μg) was given i.p. on day 23. CpG (25 μg) was given IT on days 26, 28, and 30. Data shown are means ± SEM of five mice per group. Statistical differences are indicated for the last day on the graph. Differences between the control versus treatment groups are shown. *p < 0.05, **p < 0.01.

Close modal

We hypothesized that T cell–independent anti-CD40/CpG therapy (Fig. 1) resulting in tumor cell killing via macrophages can also activate T cells, presumably by enhancing Ag presentation. To test this hypothesis, we injected C57BL/6 mice s.c. into the left side of the abdomen with B78 tumor cells (day 0), i.p with anti-CD40 (day 7), and IT with CpG (day 10). One day later, draining (left) and contralateral (right) inguinal lymph nodes were removed, pooled from three mice per group, and processed to single-cell suspensions. The cells were stained with anti-CD4 and anti-CD8 mAbs and mAbs to T cell activation markers (CD69, CD44, and CD25). The results in Fig. 2 show that anti-CD40 + CpG treatment upregulated the early T cell activation marker CD69 in both CD4 and CD8 T cells in draining lymph nodes. This upregulation was observed when anti-CD40 and CpG were given separately but was more pronounced when they were combined (data not shown). The upregulation of CD69 was more pronounced in draining lymph nodes (Fig. 2), but was also observed in contralateral lymph nodes (Supplemental Fig. 1), suggesting that this treatment induces both local and systemic activation of T cells. Similarly, anti-CD40 and CpG induced upregulation of other T cell activation markers, CD44 and CD25. These results suggest that anti-CD40/CpG therapy induced activation of T cells, although this activation was not essential for the early delay in B78 tumor growth as shown in Fig. 1.

FIGURE 2.

Anti-CD40 and CpG induce activation of T cells in draining lymph nodes. C57BL/6 mice (three mice per group) were injected s.c. into the left side of the abdomen with 2 × 106 B78 cells (day 0). On day 7, the mice were injected i.p. with anti-CD40, and on day 10, they received CpG IT. Control mice received PBS. On day 11, left inguinal lymph nodes were collected, pooled, and stained with anti-CD4, anti-CD8, and Abs against T cell activation markers. Results are shown as histograms of viable lymph node cells gated on either CD4+ or CD8+ cells. Gray areas show staining with specific Abs, and white areas are isotype controls. Numbers above indicate percentage of positive cells, and numbers below indicate mean fluorescence intensity.

FIGURE 2.

Anti-CD40 and CpG induce activation of T cells in draining lymph nodes. C57BL/6 mice (three mice per group) were injected s.c. into the left side of the abdomen with 2 × 106 B78 cells (day 0). On day 7, the mice were injected i.p. with anti-CD40, and on day 10, they received CpG IT. Control mice received PBS. On day 11, left inguinal lymph nodes were collected, pooled, and stained with anti-CD4, anti-CD8, and Abs against T cell activation markers. Results are shown as histograms of viable lymph node cells gated on either CD4+ or CD8+ cells. Gray areas show staining with specific Abs, and white areas are isotype controls. Numbers above indicate percentage of positive cells, and numbers below indicate mean fluorescence intensity.

Close modal

Having established that anti-CD40 and CpG activate T cells, we thought to combine this therapy with another T cell–activating approach using IT treatment with 14.18-IL-2 IC (14). Because IT-IC has been shown to activate T cells (13, 14), we first determined whether checkpoint blockade in tumor-bearing mice would augment the antitumor effect of 14.18-IL-2 IC. The results in Fig. 3A show that, indeed, IT-IC and anti–CTLA-4 synergistically induced regression of a 7-d B78 melanoma resulting in a survival rate of 40% of mice (Fig. 3B). This antitumor effect was T cell mediated because it was not observed in nude mice (Fig. 3C versus Fig. 3D). When the treatment with IT-IC and anti–CTLA-4 was used against more advanced tumors, that is, starting on day 12 (Fig. 3E) after tumor cell implantation (versus 7 d after tumor implantation; Fig. 3A–D), the antitumor effect was marginal, with no mice rejecting the tumor. This lack of potency against slightly larger tumors indicates that the strength of the combination of IT-IC + anti–CTLA-4 is limited and not effective against more established tumors. We thus sought to test whether adding additional immunotherapy could be beneficial in this setting.

FIGURE 3.

Antitumor effect of the combination of 14.18-IL-2 IC and anti–CTLA-4. (A and B) B78 melanoma cells were injected s.c. into C57BL/6 mice (day 0). 14.18-IL-2 IC (5 μg per mouse) was injected IT daily on days 7–11. Anti–CTLA-4 (200 μg per mouse) was injected i.p. on days 7, 9, 11, 14, 16, and 18. Data are shown as means ± SEM of tumor volumes (A) and survival (B) of five mice per group. (A) Differences between a combined treatment versus separate treatment groups are shown. (C and D) Role of T cells. B78 melanoma cells were injected s.c. into C57BL/6 (C) and Nude (D) mice on day 0. 14.18-IL-2 IC (5 μg) was given IT on days 6–10. Anti–CTLA-4 (200 μg) was given i.p. on days 6, 8, and 10. Data shown are means ± SEM of four to five mice per group. (E) Effect against advanced tumors. B78 cells were injected s.c. into C57BL/6 mice (day 0). Mice received 14.18-IL-2 IC IT (5 μg) on days 12–16 and anti–CTLA-4 i.p. on days 12, 14, 16, 19, 26, and 33. Data shown are means ± SEM of four to five mice per group. Differences between the control versus treatment groups are shown. Statistical differences are indicated for the last day on the graph. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 3.

Antitumor effect of the combination of 14.18-IL-2 IC and anti–CTLA-4. (A and B) B78 melanoma cells were injected s.c. into C57BL/6 mice (day 0). 14.18-IL-2 IC (5 μg per mouse) was injected IT daily on days 7–11. Anti–CTLA-4 (200 μg per mouse) was injected i.p. on days 7, 9, 11, 14, 16, and 18. Data are shown as means ± SEM of tumor volumes (A) and survival (B) of five mice per group. (A) Differences between a combined treatment versus separate treatment groups are shown. (C and D) Role of T cells. B78 melanoma cells were injected s.c. into C57BL/6 (C) and Nude (D) mice on day 0. 14.18-IL-2 IC (5 μg) was given IT on days 6–10. Anti–CTLA-4 (200 μg) was given i.p. on days 6, 8, and 10. Data shown are means ± SEM of four to five mice per group. (E) Effect against advanced tumors. B78 cells were injected s.c. into C57BL/6 mice (day 0). Mice received 14.18-IL-2 IC IT (5 μg) on days 12–16 and anti–CTLA-4 i.p. on days 12, 14, 16, 19, 26, and 33. Data shown are means ± SEM of four to five mice per group. Differences between the control versus treatment groups are shown. Statistical differences are indicated for the last day on the graph. *p < 0.05, **p < 0.01, ***p < 0.001.

Close modal

We hypothesized that combining two different strategies, anti-CD40/CpG to activate innate immunity and IT-IC/anti–CTLA-4 to activate adaptive immunity, would result in an additive or synergistic antitumor effect against advanced B78 tumors. The results in Fig. 4A show that when the treatment is started on day 23 post-tumor cell implantation (rather than on day 7 or 12, as in Fig. 3), anti-CD40/CpG or IC/anti–CTLA-4 given separately had only a slight ability to slow growth of these advanced B78 tumors. In contrast, the combination of all these treatment strategies (anti-CD40/CpG + IC/anti–CTLA-4) not only slowed tumor growth, but caused their regression (Fig. 4A), with 40% of animals becoming tumor-free and showing long-term survival (Fig. 4B). Examination of the cells in the tumor microenvironment revealed that the combined treatment resulted in the increase of CD4+ and CD8+ T cells, and dramatic reduction of Tregs, whereas the percentage of NK cells and macrophages remained unchanged (Fig. 4C).

FIGURE 4.

Synergistic antitumor effect of anti-CD40/CpG and 14.18-IL-2 IC/anti-CTLA-4. B78 melanoma cells (2 × 106) were injected s.c. into C57BL/6 mice on day 0. Anti-CD40 (500 μg) was injected i.p. on day 23; CpG (25 μg) was injected IT on days 26, 28, and 30. 14.18-IL-2 IC (25 μg) was given IT on days 26–30; anti–CTLA-4 (200 mg) was injected i.p. on days 26, 28, 30, 33, 35, and 40. Results are shown as means ± SEM of tumor volumes (A) and survival (B) of five mice per group. (A) Differences between a combined treatment versus separate treatment groups are shown. (C) C57BL/6 mice bearing B78 tumors received combined treatment with anti-CD40/CpG and IC/anti–CTLA-4 (clone 9D9, IgG2a) as described in (A) up to day 33. On day 34, tumors were removed, and single-cell suspensions were evaluated for indicated immune cells by flow cytometry. Results are shown as means ± SEM of five mice per group. (D) C57BL/6 mice were injected s.c. with 5 × 105 B16 or B16-GD2 cells. Anti-CD40 was injected i.p. on day 7; CpG was injected IT on days 10, 12, and 14. 14.18-IL-2 IC was given IT on days 10–14; anti–CTLA-4 (clone 9D9, IgG2a) was injected i.p. on days 10, 12, 14, 17, 19, and 21. Control mice received no treatment. Results are shown as survival of five mice per group. Differences between the control versus treatment groups are shown. Unless stated otherwise, statistical differences are indicated for the last day on the graph. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 4.

Synergistic antitumor effect of anti-CD40/CpG and 14.18-IL-2 IC/anti-CTLA-4. B78 melanoma cells (2 × 106) were injected s.c. into C57BL/6 mice on day 0. Anti-CD40 (500 μg) was injected i.p. on day 23; CpG (25 μg) was injected IT on days 26, 28, and 30. 14.18-IL-2 IC (25 μg) was given IT on days 26–30; anti–CTLA-4 (200 mg) was injected i.p. on days 26, 28, 30, 33, 35, and 40. Results are shown as means ± SEM of tumor volumes (A) and survival (B) of five mice per group. (A) Differences between a combined treatment versus separate treatment groups are shown. (C) C57BL/6 mice bearing B78 tumors received combined treatment with anti-CD40/CpG and IC/anti–CTLA-4 (clone 9D9, IgG2a) as described in (A) up to day 33. On day 34, tumors were removed, and single-cell suspensions were evaluated for indicated immune cells by flow cytometry. Results are shown as means ± SEM of five mice per group. (D) C57BL/6 mice were injected s.c. with 5 × 105 B16 or B16-GD2 cells. Anti-CD40 was injected i.p. on day 7; CpG was injected IT on days 10, 12, and 14. 14.18-IL-2 IC was given IT on days 10–14; anti–CTLA-4 (clone 9D9, IgG2a) was injected i.p. on days 10, 12, 14, 17, 19, and 21. Control mice received no treatment. Results are shown as survival of five mice per group. Differences between the control versus treatment groups are shown. Unless stated otherwise, statistical differences are indicated for the last day on the graph. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Close modal

Next, we asked whether GD2 expression on tumor cells is important for the antitumor activity of this combined treatment. Because B16-derived GD2+ B78 melanoma grows much slower than B16, we have virally transduced B16 cells to express GD2. These B16-GD2 tumors grew in C57BL/6 mice at a rate similar to parental B16 tumors. We compared the effect of the combined treatment in B16 versus B16-GD2–bearing mice. The results in Fig. 4D show that whereas anti-CD40/CpG + IC/anti–CTLA-4 therapy was effective in extending survival of mice with B16 tumors (p = 0.002), all treated mice died before day 60. In contrast, this same combined therapy was much more effective in mice bearing B16-GD2 tumors leading to 80% cure, suggesting that the anti-GD2 component of the IC plays a role in the antitumor effect of the combined treatment.

Next, we determined the role of T cells in the antitumor effect observed with the combined anti-CD40/CpG + IC/anti–CTLA-4 regimen. C57BL/6 and nude mice were injected with B78 cells and given various treatments. B78 tumors initially shrank in all C57BL/6 mice, as a result of treatment with anti-CD40/CpG and IC/anti–CTLA-4 (Fig. 5A, note the very small tumor volumes from days 28 to 35); however, in this experiment, the tumors subsequently regrew. In contrast, no tumors shrank in nude mice treated with the anti-CD40/CpG and IC/anti–CTLA-4 combination (Fig. 5B, note the larger mean tumor volumes on days 28–30 in these mice, compared with those in Fig. 5A), although their growth was statistically slower than in untreated nude mice (Fig. 5B). The antitumor effect of anti-CD40/CpG and IC/anti–CTLA-4 was more significant than all other treatments in C57BL/6 mice (Fig. 5A), but was not different from the potency of anti-CD40/CpG (without IC/anti–CTLA-4) in nude mice (Fig. 5B). These results were confirmed in Ab-depletion experiments. C57BL/6 mice were treated with anti-CD40/CpG and IC/anti–CTLA-4 between days 11 and 25 post-tumor cell implantation; one group was depleted of T cells by anti-CD4 and anti-CD8 mAbs, whereas the other group received rat IgG. Both these groups showed similar antitumor efficacy (compared with the control mice not receiving anti-CD40/CpG and IC/anti–CTLA-4) up through day 31 (Fig. 5C). However, after day 40, the tumors in treated mice depleted of T cells were not significantly different from control, whereas the tumors in treated nondepleted mice were still significantly smaller (Fig. 5C), indicating that T cells were not required for the antitumor effect during the initial 4 wk, but were involved in sustaining the antitumor effect later on.

FIGURE 5.

Role of T cells, but not NK cells, in the antitumor effect of the combined treatment with anti-CD40/CpG + 14.18-IL-2 IC/anti–CTLA-4. C57BL/6 (A) and Nude mice (B) were injected s.c. with 2 × 106 B78 cells. Mice were treated with anti-CD40 (500 mg i.p.) on day 16 and CpG (25 μg IT) on days 19, 21, and 23; anti–CTLA-4 (200 mg i.p.) on days 19, 21, 23, 26, 28, and 30 and 14.18-IL-2 IC (25 μg IT) on days 19–23; combination of all four agents or PBS (control). Results are shown as means ± SEM of tumor volumes of five mice per group. (C) C57BL/6 mice were injected s.c. with B78 cells. Treatment groups were injected with anti-CD40 on day 11; CpG on days 14, 16, and 18; 14.18-IL-2 IC on days 14–18; and anti–CTLA-4 on days 14, 16, 18, 21, 23, and 25. To deplete T cells, one group of treated mice received i.p. injections of both anti-CD4 and anti-CD8 (300 μg each) on days 10, 14, 18, 22, and 26, and another group of treated mice received rat IgG (600 μg) as a control for T cell depletion. (D) C57BL/6 mice were injected s.c. with B78 cells. All treatment groups were injected with anti-CD40 on day 10; CpG on days 13, 15, and 17; anti–CTLA-4 on days 13, 15, 17, 20, 22, and 24; and hu14.18-IL-2 IC on days 13–17 (designated as Combo). The mice received rat IgG, anti-CD4 + anti-CD8, anti-NK1.1, or a combination of anti-CD4/8 and anti-NK1.1 (all on days 9, 13, 17, 21, 25, 29, 33, 37, and 41). Results are shown as means ± SEM of tumor volumes. Differences between the control versus treatment groups are shown. Unless stated otherwise, statistical differences are indicated for the last day on the graph. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 5.

Role of T cells, but not NK cells, in the antitumor effect of the combined treatment with anti-CD40/CpG + 14.18-IL-2 IC/anti–CTLA-4. C57BL/6 (A) and Nude mice (B) were injected s.c. with 2 × 106 B78 cells. Mice were treated with anti-CD40 (500 mg i.p.) on day 16 and CpG (25 μg IT) on days 19, 21, and 23; anti–CTLA-4 (200 mg i.p.) on days 19, 21, 23, 26, 28, and 30 and 14.18-IL-2 IC (25 μg IT) on days 19–23; combination of all four agents or PBS (control). Results are shown as means ± SEM of tumor volumes of five mice per group. (C) C57BL/6 mice were injected s.c. with B78 cells. Treatment groups were injected with anti-CD40 on day 11; CpG on days 14, 16, and 18; 14.18-IL-2 IC on days 14–18; and anti–CTLA-4 on days 14, 16, 18, 21, 23, and 25. To deplete T cells, one group of treated mice received i.p. injections of both anti-CD4 and anti-CD8 (300 μg each) on days 10, 14, 18, 22, and 26, and another group of treated mice received rat IgG (600 μg) as a control for T cell depletion. (D) C57BL/6 mice were injected s.c. with B78 cells. All treatment groups were injected with anti-CD40 on day 10; CpG on days 13, 15, and 17; anti–CTLA-4 on days 13, 15, 17, 20, 22, and 24; and hu14.18-IL-2 IC on days 13–17 (designated as Combo). The mice received rat IgG, anti-CD4 + anti-CD8, anti-NK1.1, or a combination of anti-CD4/8 and anti-NK1.1 (all on days 9, 13, 17, 21, 25, 29, 33, 37, and 41). Results are shown as means ± SEM of tumor volumes. Differences between the control versus treatment groups are shown. Unless stated otherwise, statistical differences are indicated for the last day on the graph. *p < 0.05, **p < 0.01, ***p < 0.001.

Close modal

Given that anti-CD40 (7), CpG (30), and IC (14) can each activate NK cells, we next determined the role of NK cells in the antitumor effect of our combined treatment. Depletion of NK cells with anti-NK1.1 mAb did not reduce the antitumor effect of anti-CD40/CpG and IC/anti–CTLA-4. In fact, one of the five mice that received the combined treatment and anti-NK1.1 mAb rejected tumor and remained tumor-free. Furthermore, simultaneous depletion of both NK cells and T cells did not cause significant further reduction of the antitumor effect from the reduction of efficacy caused by depletion of T cells alone (Fig. 5D). Together, these results suggest that some cell populations other than NK cells, likely macrophages, activated by anti-CD40/CpG (7, 8, 10) are responsible for the initial tumor killing, whereas T cells further activated by IT-IC and anti–CTLA-4 are responsible for the subsequent tumor cell eradication after the combined treatment in the non-NK/T-depleted B78-bearing C57BL/6 mice (Fig. 5D).

We tested whether the mice that were made tumor-free either by IC/anti–CTLA-4 (Fig. 3B) or the combined treatment with anti-CD40/CpG + IC/anti–CTLA-4 (Fig. 4B) had generated immunological memory and would be able to reject secondary tumor challenge given 2–7 mo later. The results in Fig. 6 show that mice that rejected their B78 tumors after the combined treatment with anti-CD40/CpG + IC/anti–CTLA-4 (Fig. 6B) were resistant to rechallenge with B78 tumor cells, and mice that became tumor-free mice after IC/anti–CTLA-4 therapy were transiently resistant; they developed tumors after rechallenge (Fig. 6C), but much later than the control mice (Fig. 6C versus Fig. 6A). The more aggressive, rapidly growing, parental B16-F10, which in contrast with B78 does not express GD2, grew more slowly upon rechallenge in mice that previously rejected their B78 tumors after the combined treatment with anti-CD40/CpG + IC/anti–CTLA-4 (Fig. 6E) compared with IC/anti–CTLA-4–treated (Fig. 6F) and control mice (Fig. 6D). The experiment using mice that rejected their B78 tumors after the combined treatment with anti-CD40/CpG + IC/anti–CTLA-4 was repeated, demonstrating similar results. These results indicate that this combination of anti-CD40/CpG + IC/anti–CTLA-4 induces immunological memory in mice.

FIGURE 6.

Immunological memory in mice that rejected tumors after combined treatments. Naive C57BL/6 mice (A and D) and C57BL/6 mice that rejected their B78 tumors after treatment with either 14.18-IL-2 IC and anti–CTLA-4 (C and F) or anti-CD40/CpG + 14.18-IL-2 IC/anti–CTLA-4 (B and E) were injected s.c. with 2 × 106 B78 tumor cells (A–C) or 2 × 105 B16-F10 tumor cells (D–F). Tumor growth curves of individual mice (various lines and symbols) are shown. The mice in (E) (injected with B16-F10 cells) are the same mice as in (B) that were rechallenged with B78 cells and did not develop tumors. The number of mice in each group shown are four (A), two (B), two (C), four (D), two (E), and two (F). The time scales for (D)–(F) are smaller than for (A)–(C) because the B16-F10 grows far more quickly than does B78.

FIGURE 6.

Immunological memory in mice that rejected tumors after combined treatments. Naive C57BL/6 mice (A and D) and C57BL/6 mice that rejected their B78 tumors after treatment with either 14.18-IL-2 IC and anti–CTLA-4 (C and F) or anti-CD40/CpG + 14.18-IL-2 IC/anti–CTLA-4 (B and E) were injected s.c. with 2 × 106 B78 tumor cells (A–C) or 2 × 105 B16-F10 tumor cells (D–F). Tumor growth curves of individual mice (various lines and symbols) are shown. The mice in (E) (injected with B16-F10 cells) are the same mice as in (B) that were rechallenged with B78 cells and did not develop tumors. The number of mice in each group shown are four (A), two (B), two (C), four (D), two (E), and two (F). The time scales for (D)–(F) are smaller than for (A)–(C) because the B16-F10 grows far more quickly than does B78.

Close modal

The experiments described earlier have demonstrated the antitumor effect against a locally treated tumor. Given the evidence that this combination of anti-CD40/CpG + IC/anti–CTLA-4 induces immunological memory (Fig. 6), we hypothesized that this treatment may also be active against distant tumors. To potentially increase the antitumor effect, we first tested anti–CTLA-4 IgG2a (Fig. 7), which was reported to be more effective than anti–CTLA-4 IgG2b (the isotype we used in Figs. 36), likely by mediating a better reduction of Tregs within a tumor (23). We found that, when combined with IC, anti–CTLA-4 IgG2a was more effective than anti–CTLA-4 IgG2b against B78 melanoma (Fig. 7A). To test whether anti-CD40/CpG + IC/anti–CTLA-4 immunotherapy is effective against a distant solid tumor, we injected naive C57BL/6 mice on day 0 with B78 melanoma cells into both the left and the right sides of the abdomen. Treatment began on day 9 with IT injection of the tumor on the left side only with CpG and 14.18-IL-2 IC, and with i.p. administration of anti-CD40 and anti–CTLA-4, either IgG2a or IgG2b. The results in Fig. 7B show that anti-CD40/CpG + IC/anti–CTLA-4 IgG2a substantially suppressed growth of the tumor on the left side that had received the IT treatment. This same anti-CD40/CpG + IC/anti–CTLA-4 IgG2a treatment with IT CpG and IC to the left tumor also resulted in statistically significant reduction of the distant noninjected tumor on the right (Fig. 7C). When the anti–CTLA-4 IgG2b was substituted for the IgG2a in this same regimen, there was no significant tumor reduction for the noninjected tumor on the right (Fig. 7C). These results indicate that this anti-CD40/CpG + IC/anti–CTLA-4 IgG2a therapy had a systemic antitumor effect.

To confirm this systemic effect of the combined treatment, we tested it in a metastatic model, using i.v. injection of B16-F10 tumor cells, which has been shown previously to induce numerous lung metastases (31). We used GD2 parental B16-F10 cells rather than GD2+ B78 cells for metastasis induction to exclude a direct role of the anti-GD2 14.18 mAb, a part of 14.18-IL-2 IC, in the antimetastatic effect. In mice that received B78 cells s.c. on day 0 and B16-F10 cells i.v. on day 1, the mice in the control (untreated group) showed progressive growth of their s.c. tumor, yet died of metastatic disease before the s.c. tumors grew large enough to require euthanasia. The combined treatment of these mice with CpG and 14.18-IL-2 IC injected IT into the s.c. B78 tumor, and with anti-CD40 and anti–CTLA-4 (IgG2a) injected i.p., induced reduction of the primary tumors (Fig. 7D) and also had an antimetastatic effect, demonstrated by survival rate of 40% of treated mice, whereas all control mice died (Fig. 7E). Autopsies of dead mice confirmed the presence of metastases in the lungs or axillary lymph nodes. An additional identical experiment showed that the lungs removed on day 32 when some mice started dying exhibited melanoma metastases, which were more prevalent in the control group than the treatment group (Fig. 7F).

Most cancer immunotherapy strategies are focusing on activating adaptive immunity involving T cells. Some studies are targeting cells of the innate immune system; of these, most focus on activating NK cells (32). Some T cell approaches have recently shown substantial clinical benefit, such as CD19-directed chimeric Ag receptor–modified T cells for B cell malignancies (33) and the use of checkpoint blockade (CTLA-4 and PD-1) for melanoma and certain other malignancies (1921). Even so, most patients with cancer are not currently receiving immunotherapy or benefitting from it. Preclinical data suggest that combining two or more immunotherapeutic approaches may enable greater antitumor efficacy than treatment with a single immunotherapeutic agent (35, 34, 35). The majority of these combinatorial approaches, such as those adding STING (36) or FLT3 ligand (37) to other treatments, target T cell immunity, whereas some other approaches target innate immunity (38). In this study we tested the hypothesis that combining both immunological approaches, one targeting innate immunity and the other targeting T cells, will result in enhanced antitumor efficacy.

In previous studies we reported synergy between agonistic anti-CD40 and CpG via activation of innate immunity, mainly macrophages (10). This combination induced clear retardation of tumor growth but rarely resulted in complete tumor regression. We hypothesized that adding a strategy that enabled adaptive immune responses with T cell involvement would enhance antitumor efficacy and potentially induce immunological memory. We have previously developed an approach that involved tumor killing, partially by T cells, via IT administration of IC (13). However, before combining anti-CD40/CpG and IC, we considered ways to enhance the T cell–mediated antitumor effect of IC by using checkpoint blockade with anti–CTLA-4. Our results show a synergy between hu14.18-IL-2 IC and anti–CTLA-4 that resulted in a T cell–dependent rejection of B78 tumors. These results are in agreement with a report by Schwager et al. (39) showing that combining anti–CTLA-4 with a different IC, L19-IL-2, induced a better antitumor effect than these two agents given separately.

Despite the efficacy of IC combined with anti–CTLA-4 against small tumors (Fig. 3A–C), this treatment was less effective against larger tumors (Fig. 3E). Therefore, we combined 14.18-IL-2 IC and anti–CTLA-4 with anti-CD40 and CpG. Instead of injecting CpG i.p., as in our previous studies (10), we gave it in this study IT because it was shown that local treatment with CpG can induce both innate and adaptive immunity (28, 40). Similar to our previous studies (10), the antitumor effect of anti-CD40 given i.p. and CpG given IT was largely T cell independent (Fig. 1), suggesting that this combination activates macrophages, as shown in our previous studies (10, 12). The results of this study show that a combination of anti-CD40 and CpG (activating mostly innate immunity) with 14.18-IL-2 IC and anti–CTLA-4 (activating mostly adaptive immunity) induced a substantial antitumor effect resulting in regression of advanced tumors and survival rate of 40% of mice (Fig. 4A, 4B). This antitumor effect was systemic because a distant, untreated s.c. tumor (Fig. 7C) or lung metastases (Fig. 7E, 7F) were also inhibited. In addition, mice that became tumor-free, long-term survivors exhibited tumor-reactive immunological memory (Fig. 6B).

Checkpoint blockade treatment has shown clear clinical benefit, with FDA approval in several cancers (1, 21, 4143). There is a growing enthusiasm for testing checkpoint blockade in combination with other approaches to augment immune-mediated antitumor effects (5, 39). Anti–CTLA-4 has been combined in preclinical studies with each of the separate types of agents used in our study. Enhanced antitumor effects of checkpoint blockade and local treatment with CpG have been reported (44, 45). A combinatorial therapy using anti–CTLA-4 and agonistic anti-CD40 induced a stronger T cell–mediated antitumor effect than either treatment given individually (46, 47). Anti–CTLA-4 was synergistic with the IC L19-IL-2 (34). In this article, we show for the first time, to our knowledge, that a rational combination of all four of these immunomodulatory agents (anti-CD40, CpG, Ab-IL-2 IC, and anti-CTLA-4), each of which is either in clinical testing or already approved for clinical use, results in activation of innate and adaptive immunity and a synergistic antitumor effect resulting in more potent antitumor efficacy against well-established tumors. Addressing the mechanisms of this synergistic effect, we found that anti-CD40/CpG treatment of tumor-bearing mice induced local and systemic activation of T cells. This activation might be a result of local tumor destruction by activated macrophages and subsequent tumor Ag presentation. The mechanisms of anti–CTLA-4 augmenting T cell responses have been shown by others to be related to the blockade of the inhibitory activity of CTLA-4 on effector T cells (17, 18) and also to the depletion of CD4+ Tregs (46, 48), particularly for the anti–CTLA-4 IgG2a isotype. When tested in combination with 14.18-IL-2 IC against B78 melanoma, anti–CTLA-4 IgG2a was more effective than IgG2b, suggesting that better Treg depletion by anti–CTLA-4 IgG2a (23) is playing a beneficial role in our combined immunotherapy. We found that the combined treatment with anti-CD40/CpG + IC/anti–CTLA-4 substantially reduced Tregs and increased the number of CD4+ and CD8+ T cells (Fig. 4C); the relative contribution of individual treatments, and distinct T cell subpopulations, to this effect remains to be determined.

The combined treatment with anti-CD40/CpG + IC/anti–CTLA-4 induced much better antitumor effects in mice bearing GD2-expressing B16 tumor compared with parental B16 tumor, suggesting among other possibilities that 14.18 mAb in the IC plays a role in this antitumor effect. When 14.18-IL-2 IC was given i.v. it had a much greater antitumor effect than a combination of IL-2 and 14.18 mAb (49). In addition, when given IT as single-agent treatment, an IC able to bind to the tumor via its mAb component is more effective than either IT administration of IL-2 or IT administration of an IC consisting of a control mAb, unable to bind to the tumor (13). Even so, when these reagents are delivered in the tumor by IT injection, as in this study, the comparative efficacy of IC versus 14.18 mAb + IL-2 versus IL-2 alone, when given in combination with anti-CD40/CpG + anti-CTLA-4, is yet to be determined.

Overall, our results indicate that this combination of anti-CD40/CpG + IC/anti–CTLA-4 is more effective than its component parts and activates responses via both innate and adaptive immune effects. These findings also provide the preclinical justification for further development of this form of combined cancer immunotherapy strategy to pursue early-phase clinical testing.

We thank Lakeesha Carmichael for help with the statistical analysis of the data and Dr. Amy Erbe-Gurel for transfecting B16 cells with GD2.

This work was supported by grants from the National Institutes of Health (Grants CA032685, CA87025, CA166105, CA14520, CA197078, and GM067386), the Midwest Athletes for Childhood Cancer Fund, The Crawdaddy Foundation, The Evan Dunbar Foundation, the Hyundai Hope on Wheels Foundation, the University of Wisconsin-Madison Institute for Clinical and Translational Research (Grant 1TL1RR025013-01), Alex’s Lemonade Stand Foundation, The St. Baldrick’s Foundation, and Stand Up to Cancer–St. Baldrick’s Pediatric Dream Team Translational Research (Grant SU2C-AACR-DT1113). Stand Up to Cancer is a program of the Entertainment Industry Foundation administered by the American Association for Cancer Research.

The online version of this article contains supplemental material.

Abbreviations used in this article:

anti-CD40

anti-CD40 mAb

IC

immunocytokine

IT

intratumoral(ly)

PD-1

programmed death 1

Treg

T regulatory cell.

1
Couzin-Frankel
J.
2013
.
Breakthrough of the year 2013. Cancer immunotherapy.
Science
342
:
1432
1433
.
2
Beatty
G. L.
,
Chiorean
E. G.
,
Fishman
M. P.
,
Saboury
B.
,
Teitelbaum
U. R.
,
Sun
W.
,
Huhn
R. D.
,
Song
W.
,
Li
D.
,
Sharp
L. L.
, et al
.
2011
.
CD40 agonists alter tumor stroma and show efficacy against pancreatic carcinoma in mice and humans.
Science
331
:
1612
1616
.
3
Rini
B.
2014
.
Future approaches in immunotherapy.
Semin. Oncol.
41
(
Suppl. 5
):
S30
S40
.
4
Mahmood
S.
,
Upreti
D.
,
Sow
I.
,
Amari
A.
,
Nandagopal
S.
,
Kung
S. K.
.
2015
.
Bidirectional interactions of NK cells and dendritic cells in immunotherapy: current and future perspective.
Immunotherapy
7
:
301
308
.
5
Zamarin
D.
,
Postow
M. A.
.
2015
.
Immune checkpoint modulation: rational design of combination strategies.
Pharmacol. Ther.
150
:
23
32
.
6
Rakhmilevich
A. L.
,
Alderson
K. L.
,
Sondel
P. M.
.
2012
.
T-cell-independent antitumor effects of CD40 ligation.
Int. Rev. Immunol.
31
:
267
278
.
7
Buhtoiarov
I. N.
,
Lum
H.
,
Berke
G.
,
Paulnock
D.
,
Sondel
P. M.
,
Rakhmilevich
A. L.
.
2005
.
CD40 ligation induces antitumor reactivity of murine macrophages via an IFN gamma-dependent mechanism.
J. Immunol.
174
:
6013
6022
.
8
Lum
H. D.
,
Buhtoiarov
I. N.
,
Schmidt
B. E.
,
Berke
G.
,
Paulnock
D. M.
,
Sondel
P. M.
,
Rakhmilevich
A. L.
.
2006
.
In vivo CD40 ligation can induce T-cell-independent antitumor effects that involve macrophages.
J. Leukoc. Biol.
79
:
1181
1192
.
9
Rakhmilevich
A. L.
,
Buhtoiarov
I. N.
,
Malkovsky
M.
,
Sondel
P. M.
.
2008
.
CD40 ligation in vivo can induce T cell independent antitumor effects even against immunogenic tumors.
Cancer Immunol. Immunother.
57
:
1151
1160
.
10
Buhtoiarov
I. N.
,
Lum
H. D.
,
Berke
G.
,
Sondel
P. M.
,
Rakhmilevich
A. L.
.
2006
.
Synergistic activation of macrophages via CD40 and TLR9 results in T cell independent antitumor effects.
J. Immunol.
176
:
309
318
.
11
Klug
F.
,
Prakash
H.
,
Huber
P. E.
,
Seibel
T.
,
Bender
N.
,
Halama
N.
,
Pfirschke
C.
,
Voss
R. H.
,
Timke
C.
,
Umansky
L.
, et al
.
2013
.
Low-dose irradiation programs macrophage differentiation to an iNOS+/M1 phenotype that orchestrates effective T cell immunotherapy.
Cancer Cell
24
:
589
602
.
12
Buhtoiarov
I. N.
,
Sondel
P. M.
,
Wigginton
J. M.
,
Buhtoiarova
T. N.
,
Yanke
E. M.
,
Mahvi
D. A.
,
Rakhmilevich
A. L.
.
2011
.
Anti-tumour synergy of cytotoxic chemotherapy and anti-CD40 plus CpG-ODN immunotherapy through repolarization of tumour-associated macrophages.
Immunology
132
:
226
239
.
13
Johnson
E. E.
,
Lum
H. D.
,
Rakhmilevich
A. L.
,
Schmidt
B. E.
,
Furlong
M.
,
Buhtoiarov
I. N.
,
Hank
J. A.
,
Raubitschek
A.
,
Colcher
D.
,
Reisfeld
R. A.
, et al
.
2008
.
Intratumoral immunocytokine treatment results in enhanced antitumor effects.
Cancer Immunol. Immunother.
57
:
1891
1902
.
14
Yang
R. K.
,
Kalogriopoulos
N. A.
,
Rakhmilevich
A. L.
,
Ranheim
E. A.
,
Seo
S.
,
Kim
K.
,
Alderson
K. L.
,
Gan
J.
,
Reisfeld
R. A.
,
Gillies
S. D.
, et al
.
2012
.
Intratumoral hu14.18-IL-2 (IC) induces local and systemic antitumor effects that involve both activated T and NK cells as well as enhanced IC retention.
J. Immunol.
189
:
2656
2664
.
15
Becker
J. C.
,
Andersen
M. H.
,
Schrama
D.
,
Thor Straten
P.
.
2013
.
Immune-suppressive properties of the tumor microenvironment.
Cancer Immunol. Immunother.
62
:
1137
1148
.
16
Bazhin
A. V.
,
Bayry
J.
,
Umansky
V.
,
Werner
J.
,
Karakhanova
S.
.
2013
.
Overcoming immunosuppression as a new immunotherapeutic approach against pancreatic cancer.
OncoImmunology
2
:
e25736
.
17
Wolchok
J. D.
,
Saenger
Y.
.
2008
.
The mechanism of anti-CTLA-4 activity and the negative regulation of T-cell activation.
Oncologist
13
(
Suppl. 4
):
2
9
.
18
Weber
J.
2010
.
Immune checkpoint proteins: a new therapeutic paradigm for cancer--preclinical background: CTLA-4 and PD-1 blockade.
Semin. Oncol.
37
:
430
439
.
19
Ramsay
A. G.
2013
.
Immune checkpoint blockade immunotherapy to activate anti-tumour T-cell immunity.
Br. J. Haematol.
162
:
313
325
.
20
Kyi
C.
,
Postow
M. A.
.
2014
.
Checkpoint blocking antibodies in cancer immunotherapy.
FEBS Lett.
588
:
368
376
.
21
Ott
P. A.
,
Hodi
F. S.
,
Robert
C.
.
2013
.
CTLA-4 and PD-1/PD-L1 blockade: new immunotherapeutic modalities with durable clinical benefit in melanoma patients.
Clin. Cancer Res.
19
:
5300
5309
.
22
Deppong
C. M.
,
Bricker
T. L.
,
Rannals
B. D.
,
Van Rooijen
N.
,
Hsieh
C. S.
,
Green
J. M.
.
2013
.
CTLA4Ig inhibits effector T cells through regulatory T cells and TGF-β.
J. Immunol.
191
:
3082
3089
.
23
Selby
M. J.
,
Engelhardt
J. J.
,
Quigley
M.
,
Henning
K. A.
,
Chen
T.
,
Srinivasan
M.
,
Korman
A. J.
.
2013
.
Anti-CTLA-4 antibodies of IgG2a isotype enhance antitumor activity through reduction of intratumoral regulatory T cells.
Cancer Immunol. Res.
1
:
32
42
.
24
Straten
P. T.
,
Guldberg
P.
,
Seremet
T.
,
Reisfeld
R. A.
,
Zeuthen
J.
,
Becker
J. C.
.
1998
.
Activation of preexisting T cell clones by targeted interleukin 2 therapy.
Proc. Natl. Acad. Sci. USA
95
:
8785
8790
.
25
Gillies
S. D.
,
Reilly
E. B.
,
Lo
K. M.
,
Reisfeld
R. A.
.
1992
.
Antibody-targeted interleukin 2 stimulates T-cell killing of autologous tumor cells.
Proc. Natl. Acad. Sci. USA
89
:
1428
1432
.
26
Mokyr
M. B.
,
Kalinichenko
T.
,
Gorelik
L.
,
Bluestone
J. A.
.
1998
.
Realization of the therapeutic potential of CTLA-4 blockade in low-dose chemotherapy-treated tumor-bearing mice.
Cancer Res.
58
:
5301
5304
.
27
Woo
S. R.
,
Turnis
M. E.
,
Goldberg
M. V.
,
Bankoti
J.
,
Selby
M.
,
Nirschl
C. J.
,
Bettini
M. L.
,
Gravano
D. M.
,
Vogel
P.
,
Liu
C. L.
, et al
.
2012
.
Immune inhibitory molecules LAG-3 and PD-1 synergistically regulate T-cell function to promote tumoral immune escape.
Cancer Res.
72
:
917
927
.
28
Houot
R.
,
Levy
R.
.
2009
.
T-cell modulation combined with intratumoral CpG cures lymphoma in a mouse model without the need for chemotherapy.
Blood
113
:
3546
3552
.
29
Van De Voort
T. J.
,
Felder
M. A. R.
,
Yang
R. K.
,
Sondel
P. M.
,
Rakhmilevich
A. L.
.
2013
.
Intratumoral delivery of low doses of anti-CD40 mAb combined with monophosphoryl lipid A induces local and systemic antitumor effects in immunocompetent and T cell-deficient mice.
J. Immunother.
36
:
29
40
.
30
Buhtoiarov
I. N.
,
Sondel
P. M.
,
Eickhoff
J. C.
,
Rakhmilevich
A. L.
.
2007
.
Macrophages are essential for antitumour effects against weakly immunogenic murine tumours induced by class B CpG-oligodeoxynucleotides.
Immunology
120
:
412
423
.
31
Fidler
I. J.
,
Nicolson
G. L.
.
1976
.
Organ selectivity for implantation survival and growth of B16 melanoma variant tumor lines.
J. Natl. Cancer Inst.
57
:
1199
1202
.
32
Berrien-Elliott
M. M.
,
Romee
R.
,
Fehniger
T. A.
.
2015
.
Improving natural killer cell cancer immunotherapy.
Curr. Opin. Organ Transplant.
20
:
671
680
.
33
Heiblig
M.
,
Elhamri
M.
,
Michallet
M.
,
Thomas
X.
.
2015
.
Adoptive immunotherapy for acute leukemia: new insights in chimeric antigen receptors.
World J. Stem Cells
7
:
1022
1038
.
34
Spranger
S.
,
Gajewski
T.
.
2013
.
Rational combinations of immunotherapeutics that target discrete pathways.
J. Immunother. Cancer
1
:
16
.
35
Stone
G. W.
,
Barzee
S.
,
Snarsky
V.
,
Santucci
C.
,
Tran
B.
,
Langer
R.
,
Zugates
G. T.
,
Anderson
D. G.
,
Kornbluth
R. S.
.
2009
.
Nanoparticle-delivered multimeric soluble CD40L DNA combined with Toll-like receptor agonists as a treatment for melanoma.
PLoS One
4
:
e7334
.
36
Demaria
O.
,
De Gassart
A.
,
Coso
S.
,
Gestermann
N.
,
Di Domizio
J.
,
Flatz
L.
,
Gaide
O.
,
Michielin
O.
,
Hwu
P.
,
Petrova
T. V.
, et al
.
2015
.
STING activation of tumor endothelial cells initiates spontaneous and therapeutic antitumor immunity.
Proc. Natl. Acad. Sci. USA
112
:
15408
15413
.
37
Duraiswamy
J.
,
Freeman
G. J.
,
Coukos
G.
.
2013
.
Therapeutic PD-1 pathway blockade augments with other modalities of immunotherapy T-cell function to prevent immune decline in ovarian cancer.
Cancer Res.
73
:
6900
6912
.
38
Manrique
S. Z.
,
Dominguez
A. L.
,
Mirza
N.
,
Spencer
C. D.
,
Bradley
J. M.
,
Finke
J. H.
,
Lee
J. J.
,
Pease
L. R.
,
Gendler
S. J.
,
Cohen
P. A.
.
2016
.
Definitive activation of endogenous antitumor immunity by repetitive cycles of cyclophosphamide with interspersed Toll-like receptor agonists.
Oncotarget
7
:
42919
42942
.
39
Schwager
K.
,
Hemmerle
T.
,
Aebischer
D.
,
Neri
D.
.
2013
.
The immunocytokine L19-IL2 eradicates cancer when used in combination with CTLA-4 blockade or with L19-TNF.
J. Invest. Dermatol.
133
:
751
758
.
40
Veenstra
J. J.
,
Gibson
H. M.
,
Littrup
P. J.
,
Reyes
J. D.
,
Cher
M. L.
,
Takashima
A.
,
Wei
W. Z.
.
2014
.
Cryotherapy with concurrent CpG oligonucleotide treatment controls local tumor recurrence and modulates HER2/neu immunity.
Cancer Res.
74
:
5409
5420
.
41
Janakiram
M.
,
Pareek
V.
,
Cheng
H.
,
Narasimhulu
D. M.
,
Zang
X.
.
2016
.
Immune checkpoint blockade in human cancer therapy: lung cancer and hematologic malignancies.
Immunotherapy
8
:
809
819
.
42
Zibelman
M.
,
Ghatalia
P.
,
Geynisman
D. M.
,
Plimack
E. R.
.
2016
.
Checkpoint inhibitors for renal cell carcinoma: current landscape and future directions.
Immunotherapy
8
:
785
798
.
43
Xia
Y.
,
Medeiros
L. J.
,
Young
K. H.
.
2016
.
Immune checkpoint blockade: releasing the brake towards hematological malignancies.
Blood Rev.
30
:
189
200
.
44
Mangsbo
S. M.
,
Sandin
L. C.
,
Anger
K.
,
Korman
A. J.
,
Loskog
A.
,
Tötterman
T. H.
.
2010
.
Enhanced tumor eradication by combining CTLA-4 or PD-1 blockade with CpG therapy.
J. Immunother.
33
:
225
235
.
45
Marabelle
A.
,
Kohrt
H.
,
Sagiv-Barfi
I.
,
Ajami
B.
,
Axtell
R. C.
,
Zhou
G.
,
Rajapaksa
R.
,
Green
M. R.
,
Torchia
J.
,
Brody
J.
, et al
.
2013
.
Depleting tumor-specific Tregs at a single site eradicates disseminated tumors.
J. Clin. Invest.
123
:
2447
2463
.
46
Takeda
K.
,
Kojima
Y.
,
Uno
T.
,
Hayakawa
Y.
,
Teng
M. W.
,
Yoshizawa
H.
,
Yagita
H.
,
Gejyo
F.
,
Okumura
K.
,
Smyth
M. J.
.
2010
.
Combination therapy of established tumors by antibodies targeting immune activating and suppressing molecules.
J. Immunol.
184
:
5493
5501
.
47
Sckisel
G. D.
,
Mirsoian
A.
,
Bouchlaka
M. N.
,
Tietze
J. K.
,
Chen
M.
,
Blazar
B. R.
,
Murphy
W. J.
.
2015
.
Late administration of murine CTLA-4 blockade prolongs CD8-mediated anti-tumor effects following stimulatory cancer immunotherapy.
Cancer Immunol. Immunother.
64
:
1541
1552
.
48
Simpson
T. R.
,
Li
F.
,
Montalvo-Ortiz
W.
,
Sepulveda
M. A.
,
Bergerhoff
K.
,
Arce
F.
,
Roddie
C.
,
Henry
J. Y.
,
Yagita
H.
,
Wolchok
J. D.
, et al
.
2013
.
Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma.
J. Exp. Med.
210
:
1695
1710
.
49
Lode
H. N.
,
Xiang
R.
,
Varki
N. M.
,
Dolman
C. S.
,
Gillies
S. D.
,
Reisfeld
R. A.
.
1997
.
Targeted interleukin-2 therapy for spontaneous neuroblastoma metastases to bone marrow.
J. Natl. Cancer Inst.
89
:
1586
1594
.

H.L. declares employment and ownership interests in Apeiron Biologics Inc. S.D.G. declares employment and ownership interests in Provenance Biopharmaceuticals. A.J.K. declares employment and equity interests in Bristol-Myers Squibb. The other authors have no financial conflicts of interest.

Supplementary data