Effective antibody therapy induces host-protective antitumor immunity that is augmented by TLR4 agonist treatment

Abstract

Toll-like receptors are potent activators of the innate immune system and generate signals leading to the initiation of the adaptive immune response that can be utilized for therapeutic purposes. We tested the hypothesis that combined treatment with a Toll-like receptor agonist and an antitumor monoclonal antibody is effective and induces host-protective antitumor immunity. C57BL/6 human mutated HER2 (hmHER2) transgenic mice that constitutively express kinase-deficient human HER2 under control of the CMV promoter were established. These mice demonstrate immunological tolerance to D5-HER2, a syngeneic human HER2-expressing melanoma cell line. This human HER2-tolerant model offers the potential to serve as a preclinical model to test both antibody therapy and the immunization potential of human HER2-targeted therapeutics. Here, we show that E6020, a Toll-like receptor-4 (TLR4) agonist effectively boosted the antitumor efficacy of the monoclonal antibody trastuzumab in immunodeficient C57BL/6 SCID mice as well as in C57BL/6 hmHER2 transgenic mice. E6020 and trastuzumab co-treatment resulted in significantly greater inhibition of tumor growth than was observed with either agent individually. Furthermore, mice treated with the combination of trastuzumab and the TLR4 agonist were protected against rechallenge with human HER2-transfected tumor cells in hmHER2 transgenic mouse strains. These findings suggest that combined treatment with trastuzumab and a TLR4 agonist not only promotes direct antitumor effects but also induces a host-protective human HER2-directed adaptive immune response, indicative of a memory response. These data provide an immunological rationale for testing TLR4 agonists in combination with antibody therapy in patients with cancer.

Fig. 1

Construction of a human mutated HER2 transgene and identification of C57BL/6 human mutated HER2 (hmHER2) transgenic mice. a Map of the 7734-bp CMV-regulated human mutated HER2 transgene. b Detection of human HER2 RNA in different tissues of hmHER2 transgenic mice. Total RNA was extracted and analyzed for relative levels of HER2 RNA by RT-PCR. (1. kidney, 2. liver, 3. lung, 4. heart, 5. DNA ladder) c Immunohistochemical analysis of human HER2 expression by kidney from hmHER2 transgenic (left panel) and wild-type (right panel) mice. Original magnification: ×200

Fig. 2

Establishment of a syngeneic mouse model system. a Kinetics of D5 and D5-HER2 syngeneic melanoma cell growth in mice. C57BL/6 SCID, C57BL/6, and C57BL/6 hmHER2 Tg mice; 10³ or 10⁴ D5 (filled diamond) and D5-HER2 cells (filled square) were inoculated, and tumor growth was monitored. b Anti-human HER2 antibody induction associated with D5-HER2 tumor growth was measured. Sera from untreated C57BL/6 SCID, C57BL/6, and C57BL/6 hmHER2 Tg tumor-bearing mice were collected 21 days after tumor inoculation and used to stain D5-HER2 cells. Bound antibody was detected by flow cytometry, as described in “Materials and methods.” The results are expressed as the mean fluorescence of individual samples. c Immunohistochemical detection of human HER2 expression by D5-HER2 tumors and D5 tumors from tumor-bearing C57BL/6 SCID, C57BL/6 wild-type, and C57BL/6 hmHER2 Tg mice. Original magnification: ×400

Fig. 3

E6020 enhances the efficacy of therapy with trastuzumab in C57BL/6 SCID mice and hmHER2 transgenic mice. Cohorts of 10-12 C57BL/6 SCID mice a, and hmHER2 mice b were challenged with 10³ D5-HER2 tumor cells s.c. on day 0, and treated starting on day 1 twice weekly by intraperitoneal injections of PBS, 10 μg E6020 alone, 200 μg trastuzumab alone, or combined E6020 and trastuzumab. The percentage of tumor-free mice was recorded. The data are presented as Kaplan–Meier survival curves indicating the percentage of tumor-free mice. Similar results were found in three independent experiments. **P < 0.001, *P < 0.05 when combined therapy was compared with trastuzumab, E6020, or PBS

Fig. 4

TLR4 agonist exposure activates FcγR-dependent functions on macrophages and dendritic cells but not NK cells. In vivo therapy with E6020 induces upregulation of FcγRs and CD80 costimulatory molecules. Cohorts of HmHER2 Tg mice received 10 μg E6020, CD11b⁺ peritoneal macrophages, splenic NK1.1⁺ cells or CD11c⁺ dendritic cells were analyzed for murine FcγR expression. Flow cytometry profiles of CD11b⁺, NK1.1⁺ or CD11c⁺ cells gated for FcγR I, FcγR II/III and CD80 expression analysis demonstrate upregulation of FcγRs and the activation of macrophage from E6020-injected hmHER2 Tg mouse. FcγR I but not FcγR II/III is upregulated on CD11c⁺ dendritic cells by E6020 treatment. E6020 shows no effect on FcγR expression in NK cells

Fig. 5

Macrophages mediate the antitumor effect of combination therapy with trastuzumab plus E6020. a Depletion of macrophages impairs the enhanced efficacy of combination therapy with trastuzumab plus E6020. Cohorts of 10-12 C57BL/6 hmHER2 mice were challenged with 10³ D5-HER2 tumor cells s.c. on day 0 and treated starting on day 1 twice weekly by intraperitoneal injections of either PBS or combination therapy with trastuzumab plus E6020. Macrophages were depleted by i.p. injection of 200 μl of clodronate-containing liposomes the day prior to tumor cell inoculation and thereafter every 5 days. The percentage of tumor-free mice was recorded. *P < 0.01 when combination therapy with macrophages depletion was compared to that without macrophage depletion. b E6020 enhances macrophage phagocytosis in vitro. Peritoneal macrophages were isolated from hmHER2 Tg mice and were pre-incubated with 0, 5, and 10 μg/ml E6020 for 20 h then tested in an ADCP assay with 4D5-coated D5-HER2 cells. D5-HER2 cells were labeled with CMFDA-green for tracking purposes and treated with non-binding control IgG or 4D5 MAb at 0, 10 μg/ml and mixed with macrophages. Macrophages were stained with APC-conjugated anti-CD11b. Flow cytometry was used to calculate the percentage of tumor cell engulfment by macrophages. The effector/ target ratio used was 4:1. Percent phagocytosis was determined as the number of double-positive cells divided by the total number of CMFDA-positive cells

Fig. 6

NK cells are not responsible for the antitumor effect of combination therapy with trastuzumab plus E6020. a Cohorts of 10-12 C57BL/6 hmHER2 mice were challenged with 10³ D5-HER2 tumor cells s.c. on day 0 and treated starting on day 1 twice weekly by intraperitoneal injections of either PBS or combined E6020 and trastuzumab. NK cells were depleted by i.p. injection of anti-Asialo GM1 antibody the day prior to tumor cell inoculation and thereafter every 5 days. The percentage of tumor-free mice was recorded. b Splenocytes were isolated from hmHER2 Tg mice that received 10 μg E6020. The ADCC assay was measured using a standard ⁵¹Cr release assay as described in “Materials and Methods.” A representative assay and the mean ± SD of triplicate samples are shown; each assay was repeated at least twice

Fig. 7

Combination therapy with trastuzumab plus E6020 enhances host-protective HER2-directed adaptive immune responses. a C57BL/6 hmHER2 mice treated with either the combination of trastuzumab and E6020 or trastuzumab alone were protected from D5-HER2 but not D5 rechallenge. C57BL/6 hmHER2 mice were s.c. inoculated with 1 × 10³ D5-HER2 tumor cells on day 0. Treatment with trastuzumab plus E6020 or trastuzumab was initiated on day 1 and continued for 30 days, as described in “Materials and Methods.” One hundred and twenty days following the initial challenge, tumor-free mice that previously received combined treatment with 200 μg trastuzumab and 10 μg E6020 or treatment with 200 μg trastuzumab alone were inoculated with 5 × 10³ D5-HER2 cells or D5 cells (i.e., a fivefold higher challenge than was initially administered) in a site opposite that of the first s.c. injection. Tumor recurrence was monitored by palpation for a minimum of 2 months. The data are presented as Kaplan–Meier survival curves indicating the percentage of tumor-free mice. *P = 0.005 when initial combined therapy was compared with trastuzumab. The data are combined from at least two replicate experiments. b, c Both CD4⁺ and CD8⁺ T cells are required for D5-HER2 tumor rejection upon tumor rechallenge. C57BL/6 hmHER Tg mice that rejected D5-HER2 tumors after initial treatment with trastuzumab plus E6020 (Fig. 7b) or trastuzumab alone (Fig. 7c) were depleted of CD4⁺, CD8⁺ T cells or both as described in “Materials and Methods.” Animals were then rechallenged in the opposite flank with D5-HER2 cells (fivefold higher dose than the initial challenge). The mice were monitored for the development of palpable tumors, and the percentage of tumor-free mice was recorded. The data from two separate experiments with similar outcomes were combined. d Antigen specificity was detected by intracellular IFNγ cytokine analysis. Tumor-free Tg mice from both the trastuzumab-treated group and the combination-treated group were rechallenged as described above. Two weeks after tumor rechallenge, mice were killed and splenocytes were isolated. Splenocytes were restimulated with HER2 peptide mix (ECD or ICD peptide mix) and assayed for IFN-γ production using an intracellular cytokine staining. The percentage of CD4⁺/IFN-γ⁺ T cells is shown