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

Abstract

In this study, an agonistic anti-CD40 monoclonal antibody was combined with monophosphoryl lipid A (MPL), a nontoxic derivative of lipopolysaccharide and agonist of toll-like receptor-4, to assess the immunomodulatory and antitumor synergy between the 2 agents in mice. Anti-CD40 was capable of priming macrophages to subsequent ex vivo activation by MPL in immunocompetent and T-cell-depleted mice. Intraperitoneal injections of anti-CD40+MPL induced additive to synergistic suppression of poorly immunogenic B16-F10 melanoma growing subcutaneously in syngeneic mice. When anti-CD40+MPL were injected directly into the subcutaneous tumor, the combination treatment was more effective, even with a 25-fold reduction in dose. Low-dose intratumoral treatment also slowed the growth of a secondary tumor growing simultaneously at a distant, untreated site. Antitumor effects were also induced in severe combined immunodeficiency mice and in T-cell-depleted C57BL/6 mice. Taken together, our results show that the antitumor effects of anti-CD40 are enhanced by subsequent treatment with MPL, even in T-cell-deficient hosts. These preclinical data suggest that an anti-CD40+MPL combined regimen is appropriate for clinical testing in human patients, including cancer patients who may be immunosuppressed from prior chemotherapy.

FIGURE 1

Synergistic activation of MΦ with anti-CD40 and MPL. A, Expression of TLR4 on MΦ from mice treated with anti-CD40. C57BL/6 mice were injected i.p. with anti-CD40 (0.5 mg/mouse in 0.5 ml PBS), and their PECs were collected 1, 3, or 5 days later and tested by flow cytometry for TLR4 expression. Results are presented as histograms of viable MΦ (PI⁻ F4/80⁺) from 1 representative mouse out of the 2 mice injected on each day. On histogram: black peak, baseline TLR4 expression from naïve mouse, MFI 9.6; open peaks, overlapping TLR4 expression 1 day or 5 days after anti-CD40, MFI 25 (1 d) and 24.4 (5 d); grey peak, TLR4 on MΦ collected 3 days after anti-CD40, MFI: 33.2. B, Tumoristatic synergy between anti-CD40 and MPL in vitro. C57BL/6 mice were injected with anti-CD40 (500 μg i.p.) or rat IgG (500 μg i.p.) on day 0. On day 3, PECs were collected and enriched for MΦ by allowing cells (2×10⁵ per well) to adhere to plastic wells for 2 hours, followed by gentle pipetting to remove non-adherent cells. Adherent cells were then co-cultured in triplicate wells for 48h with 10⁴ B16 cells and stimulated with either MPL (5μg/ml), CpG (5 μg/ml), or LPS (10ng/ml). Wells were pulsed with [³H] thymidine (1 μCi/well) for the last 6 h of incubation in order to measure proliferation of B16 cells. Results are presented as the mean total number of β counts over 5 min ± SE from 10 mice combined from 3 identical experiments. Cells from each mouse were tested in all 4 in vitro conditions. C, Anti-CD40+MPL-MΦ secrete NO in a synergistic manner. After 42 h of stimulation with MPL (5 μg/ml), CpG (5 μg/ml), or LPS (10 ng/ml), supernatants were tested for nitrite concentration using the Griess test. Results from 2 identical experiments are presented. Each bar represents the mean nitrite concentration ± SE from 6 IgG-treated mice and 6 anti-CD40-treated mice. Cells from each mouse were tested in all 4 in vitro conditions. D and E, Adherent PEC from anti-CD40-treated mice were pre-incubated with L-NAME, anti-TNFα, anti-TRAIL, or anti-FasL neutralizing antibodies, followed by 48 h of co-culture with B16 cells in the presence of MPL. [³H]-thymidine incorporation (D) and NO production (E) were tested as described for (B) and (C). The results of a representative experiment (of three experiments) is shown. A two-tailed Student’s t test was used to compare the populations indicated by the horizontal lines (*p<0.05; **p<0.01; ***p<0.001).

FIGURE 2

CD11b⁺ MΦ and monocytes are activated following anti-CD40 and MPL treatment. C57BL/6 mice were injected i.p. with 500 μg anti-CD40 on day 0. A, On day 3, mice were euthanized and their PECs were collected, stained, gated on CD11b^high cells and sorted into 4 sub-populations by a FACSAria cell sorter. The cells were divided as follows: (1) CD11b⁺ F4/80⁺⁺ Gr-1^low “monocytes”, (2) CD11b⁺ F4/80⁺ Gr-1^dim “activated MΦ”. A separate group of cells showed a CD11b⁺ F4/80⁻ Gr-1^high phenotype; these were further divided by forward and side scatter into populations (3) “granulocytes”, and (4) “monocytes”. B, Each cell population was stained with a Wright-Giemsa stain to identify cell types based on morphology. The relative sizes of each sorted cell population have been maintained, and these 4 micrographs all show 40x magnification. Each sorted population was tested in medium or MPL (5 μg/ml) for the cells’ ability to inhibit B16 proliferation ([³H] thymidine incorporation) (C) and produce NO (D). This experiment was performed 3 times; data from 1 representative experiment is shown. FACS sorting was also performed on PEC from tumor-bearing mice (TBM) (E – H). C57BL/6 mice were inoculated with 10⁵ B16 cells (i.p.) on day 0, treated i.p. with 500 μg anti-CD40 on day 4, and euthanized on day 7. B16 tumor cells (CD45⁻) were gated out, and CD45⁺ host cells were subsequently sorted (E) into the following 4 sub-populations: (1) CD11b⁺ Gr-1^low “macrophages”, (2) CD11b⁺ Gr-1^high “monocytes”, (3) CD19⁺ CD11b^int. Gr-1⁻ “activated B cells,” and (4) CD19⁺ CD11b^low Gr-1⁻ “naïve B cells”. Wright-Giemsa stains of these 4 populations are shown (F). Assays for [³H]-thymidine incorporation (G) and NO production (H) were performed for these 4 populations. The assays for tumor cytostasis and NO production shows activation with MPL as well as with CpG. A two-tailed Student’s T test was used to compare [³H]-TdR counts or nitrite concentration. (*p<0.05; **p<0.01; ***p<0.001).

FIGURE 3

Anti-CD40 and MPL synergistically induce antitumor effects in vivo. A, C57BL/6 mice (n=8–10, combined from two identical experiments) were injected s.c. with 10⁵ B16 melanoma cells on day 0 and treated i.p. with 0.5 mg anti-CD40 (or 0.5 mg rat IgG as control) on days 4 and 11. MPL (or PBS as control) was given on days 7 and 14. Graph shows the mean tumor volume ± SE. The tumor growth curve of each mouse was fit to an exponential curve, and the mean specific growth constant from the combination-treated mice was compared to each of the 3 other groups individually using a two-tailed Student’s T test. Asterisks indicate significant differences in growth rates (***p<0.001, Anti-CD40+MPL vs. IgG+PBS; *p<0.019, Anti-CD40+MPL vs. anti-CD40+PBS; ***p<0.001, Anti-CD40+MPL vs. MPL+IgG). B, Overall survival of the mice shown in (A) (**p<0.0075, Anti-CD40+MPL vs. IgG+PBS; p=0.27, Anti-CD40+MPL vs. anti-CD40+PBS; **p=0.0048, Anti-CD40+MPL vs. IgG+MPL). C, C57BL/6 mice (n=8–10, combined from two identical experiments) were implanted with a B16 tumor and treated i.t. with 20 μg anti-CD40 on days 5 and 12, and with 2 μg MPL on days 8 and 15. Control mice received 20 μg rat IgG or PBS, respectively, injected i.t. (***p<0.001, Anti-CD40+MPL vs. IgG+PBS; ***p<0.001, Anti-CD40+MPL vs. anti-CD40+PBS; **p=0.0099, Anti-CD40+MPL vs. MPL+IgG). D, Overall survival of the mice shown in (C) (***p<0.001, Anti-CD40+MPL vs. IgG+PBS; ***p<0.001, Anti-CD40+MPL vs. anti-CD40+PBS; **p=0.0032, Anti-CD40+MPL vs. IgG+MPL).

FIGURE 4

PECs from T cell deficient mice treated with anti-CD40 in vivo cause tumoristatic effects and produce NO after in vitro culture with MPL. A, C57BL/6 mice were injected i.p. with anti-CD4 (250 μg) and anti-CD8 (250 μg) mAbs on day -1. On day 0, mice were divided into two groups (n=4) and injected i.p. with either anti-CD40 or rat IgG (0.5 mg/mouse in 0.5 ml). Mice were euthanized on day 3. PECs were collected and enriched for adherent cells (MΦ) by plating 2×10⁵ cells per mouse into each well of a 96-well plate. Non-adherent cells were removed after 1.5 – 2 h and adherent cells were co-cultured with 10⁴ B16 cells per well and stimulated with MPL at a concentration of (5 μg/ml) for 48 hrs. Wells were pulsed with [³H]-thymidine (1 μCi/well) for the last 6h of incubation in order to measure proliferation of B16 cells. Results are presented as the mean total number of β counts over 5 min ± SE from 4 mice per group. B, T cells are not required for anti-CD40-induced NO production. After 42 h of stimulation, supernatants were taken from the wells described in (A) and tested for nitrite concentration using the Griess test. The experiments described in (A) and (B) were also repeated in SCID mice (C, D). C, [³H]-Thymidine incorporation into B16 cells in the presence of rat IgG- or anti-CD40-treated adherent SCID PEC. D, Nitrite production of the same PEC as in (C). Mean ± SE of 9 mice from 3 separate experiments are included in (C) and (D). In all panels, groups were compared using a two-tailed Student’s T test (*p<0.05; **p<0.01).

FIGURE 5

MPL with and without anti-CD40 slows tumor growth in SCID mice when injected i.t. A, CB-17 SCID mice (n=5) were implanted with a B16 tumor and treated i.t. with 20 μg anti-CD40 on days 5 and 12, and with 2 μg MPL on days 8 and 13. Control mice received (20 μg rat IgG or PBS, respectively, injected i.t.) The graph shows the mean tumor volume ± SE. The tumor growth curve of each mouse was fit to an exponential curve, and the mean specific growth constants from the combination-treated group and the rat IgG+MPL group were compared to the rat IgG+PBS control group using a two-tailed Student’s T test. Asterisks indicate significant differences in growth rates (*p<0.05). B, Survival of the mice shown in (A). Although the combination-treated mice and the rat IgG+MPL had higher median survival times than the other two groups, the survival benefit was not statistically significant by the log-rank test.

FIGURE 6

I.t. anti-CD40 and MPL treatment is effective against local and distant tumors in immunocompentent C57BL/6 mice, T cell-depleted C57BL/6 mice, and CB-17 SCID mice. Mice were implanted with 10⁵ B16 melanoma cells on day 0 and with a second inoculum of 10⁵ B16 into the opposite flank on day 3. Groups of mice received i.t. treatment with either the anti-CD40+MPL combination (triangles) or control rat IgG+PBS (circles) into the primary tumor for two rounds corresponding to days 5 and 12 for anti-CD40 and days 8 and 15 for MPL. A, Primary tumor growth of immunologically intact C57BL/6 mice (injected i.p. with 500 μg rat IgG on days 3, 9, and 14 as control for T cell depletion) (**p=0.008). B, Secondary tumor growth of the same mice as in (A) (*p=0.03). C, Survival of the same mice as in (A) and (B) (**p=0.0054). D, Primary tumor growth of C57BL/6 mice (injected i.p. with 250 μg of anti-CD4 and 250 μg anti-CD8 depleting mAbs on days 4, 9, and 14) (***p<0.001). E, Secondary tumor growth of the same mice as in (D) (**p=0.004). F, Survival of the same mice as in (D) and (E) (**p=0.0022). The experiments with C57BL/6 mice that are shown in (A) through (F) were performed in parallel using 5 – 7 mice per treatment group. G, In a separate experiment, CB-17 SCID mice (n=4 for rat IgG+PBS; n=5 for anti-CD40+MPL) were injected s.c. with 10⁵ B16 cells on day 0 and in the opposite flank on day 3, and treated i.t. with two rounds of treatment: anti-CD40 (20 μg) on day 5 and 12 and MPL (2 μg) on day 8 and 15 (*p<0.031). H, Secondary tumor growth of the same mice as in (G) (*p=0.012). I. Survival of the same mice as in (G) and (H) (**p=0.0054).