IJC International Journal of Cancer
Agonistic anti-CD137 antibody treatment leads to antitumor response in mice with liver cancer Vanessa Gauttier1, Jean-Paul Judor1, Valentin Le Guen1, Jeannette Cany2, Nicolas Ferry2 and Sophie Conchon1 1
^tel-Dieu, INSERM UMR1064, Centre for Research in Transplantation and Immunology—ITUN, Universite de Nantes, Centre Hospitalier Universitaire Ho Nantes, France 2 ^tel Dieu, Nantes, France INSERM UMR948, Hepatic Biotherapies, Universite de Nantes, Centre Hospitalier Universitaire Ho
Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and, due to limited treatment options, the third greatest cause of cancer death.1 Despite numerous attempts at amelioration, curative treatments continue to exhibit a very high recurrence rate.2 The liver possesses a specific immune system, which plays an intricate role in its functions and pathobiology, including the progressive evolution of chronic liver disease to fibrosis, Key words: combination immunotherapy, hepatocellular carcinoma, 4-1BB, liver cancer, therapy, MDSC Abbreviations: DEN: diethyl nitrosamine; HCC: hepatocellular carcinoma; IFNg: gamma interferon; mAb: monoclonal antibody; MDSC: myeloid derived suppressor cell; NPC: non-parenchymal cells; TNF: tumor necrosis factor; Treg: regulatory T lymphocytes Additional Supporting Information may be found in the online version of this article. Grant sponsor: IHU-Cesti; Grant number: ANR-10-IBHU-005; Grant sponsors: la Ligue Contre le Cancer; Inter-region GrandOuest; FUI-BPI France (Hepavac); INSERM; Region Pays de la Loire. DOI: 10.1002/ijc.28943 History: Received 13 Nov 2013; Accepted 16 Apr 2014; Online 2 May 2014 Correspondence to: Sophie Conchon, INSERM UMR1064 ITUN, CHU Hotel Dieu, 30 Bd Jean Monnet, 44093 Nantes Cedex 1, France. Tel.: 133-02-40-08-75-07, Fax: 133-02-40-08-74-11, E-mail: [email protected]
C 2014 UICC Int. J. Cancer: 00, 00–00 (2014) V
cirrhosis and finally to HCC.3 Work in recent years has demonstrated that local and systemic immunity can be harnessed to fight hepatic neoplastic development, and a variety of evidence points toward immunotherapy as a promising strategy against HCC. The presence of lymphocytic infiltrates has been shown to correlate with improved overall survival of HCC patients after surgical resection.4 T lymphocytes specific for some tumor antigens have been detected in HCC patients.5 In some patients, conventional treatment has been shown to enhance immune responses, suggesting that immunotherapy, as an adjuvant to, or following primary treatments,5,6 could be beneficial for the patient, especially as a solution to the high recurrence risk. Antigen specific immunotherapy could be a valid option for the treatment of HCC and a number of strategies are in development at a clinical level.7,8 Complementary to these approaches requiring a certain level of personalization (not all HCC express all tumor antigens), there is a place for nonspecific immunotherapy strategies: to block the suppressive mechanisms that arise during tumor development and impede the anti-tumor immune response, or to directly enhance this natural anti-tumor immune response. It is noteworthy that, to date, some of the most promising clinical results for HCC immunotherapy have been obtained by adoptive transfer of autologous T lymphocytes nonspecifically stimulated after surgical tumor resection.9 CD137 is a member of the tumor necrosis factor (TNF) receptor superfamily. It is expressed by many cell types
Tumor Immunology
Immunotherapy is a promising strategy against hepatocellular carcinoma (HCC). We assessed the therapeutic effects of stimulating CD137, a member of the TNF receptor family, with agonistic monoclonal antibodies (mAb). Agonistic anti-CD137 mAb treatment was tested on two in situ models of HCC in immunocompetent mice. We also studied the mediators involved at different time points. In an orthotopic HCC the treatment consistently leads to complete tumor regression in 40–60% of animals. The protection is long lasting in the animals responding to the treatment, which can reject a second tumor challenge more than 3 months after treatment and eradication of the first malignancy. The main mediators of the effect are T lymphocytes and NK cells, demonstrated through depletion experiments. In addition, adoptive transfer of splenocytes prepared from antiCD137 mAb-treated and -cured mice to naive mice allowed them to, in turn, reject the tumor. The efficacy of anti-CD137 mAb treatment is associated with early, sustained recruitment of iNOS-positive macrophages within tumor nodules. Moreover, in the absence of treatment, tumor development is accompanied by infiltration by myeloid derived suppressor cells (MDSC) and regulatory T lymphocytes. In mice responding to the anti-CD137 mAb treatment, this infiltration is very limited, and a combination treatment with a depletion of MDSC leads to the recovery of 80% of the mice. These results demonstrate that agonistic anti-CD137 mAb is a promising therapeutic strategy for anti-tumor immunity stimulation against HCC.
2
Agonistic anti-CD137 antibody for HCC immunotherapy
Tumor Immunology
What’s new? CD137 is a member of the tumor necrosis factor (TNF) receptor superfamily. In this study of hepatocellular carcinoma (HCC), the authors found that injections of an antibody that activates CD137 caused a reduction of tumor growth by 40 to 65% in mice, and that several types of immune cells, including cytotoxic T cells, NK cells, and macrophages play a major role in this effect. When myeloid derived suppressor cells (MDSC) were depleted, the anti-CD137 antibody led to 80% recovery. This combined approach may therefore have significant therapeutic potential.
including activated T cells, NK and NKT cells, dendritic cells, neutrophils or monocytes.10 The binding of its ligand CD137L to CD137 leads to the prolonged survival and sustained activation of CD8 T cells.11 Agonistic anti-CD137 monoclonal antibody fixation to CD137 enhances T-cell proliferation and cytokine production by T-helper lymphocytes, and protects CD8 T cells from activation-induced cell death.12,13 Agonistic anti-CD137 antibody induces regression of established tumors in various animal models,14–16 and prevents tumor recurrence in a model of melanoma.17 In low tumor burden conditions this nonspecific immunotherapy strategy could be well-suited to the treatment of HCC in order to stimulate in situ the natural anti-tumor immune response. To test this hypothesis, we investigated the antitumor effect of an agonistic anti-CD137 antibody treatment on murine models of HCC for which the tumor develops within the liver of immunocompetent animals. We show the therapeutic efficacy of this approach. The mechanistic analyses demonstrate that the treatment mobilizes T cells, NK cells and allows the early and sustained recruitment of macrophages in the tumor nodules and that the immune response elicited by the treatment keeps potentially harmful regulatory cells at bay.
Material and Methods Mice and reagents
C57Bl/6 mice were purchased from Janvier Laboratory (France), and were kept in the Nantes IFR26 animal facility. Animal housing and surgical procedures were conducted according to the guidelines of the French Agriculture Ministry and were approved by the regional ethical committee. Hepa1.6 cells were cultured in DMEM (Life Technologies), 10% FBS, glutamine and antibiotics at 37 C and 5% CO2. Cells were harvested and resuspended in FBS-free DMEM for animal inoculation. Anti-CD137 mAb was produced from the 3H3 hybridoma (kindly provided by Pr R.S. Mittler). Orthotopic model: anti-CD137 treatment, tumor challenge and adoptive transfer
Eight-weeks-old C57Bl/6J male mice received 2.5 3 106 Hepa1.6 cells in 100 mL through the portal vein, as described.18 Four and eight days after tumor inoculation, mice were injected with 100 mg of rat anti-CD137 mAb, or rat IgG (Sigma–Aldrich). For tumor challenge, mice received a second dose of 2.5 3 106 Hepa1.6 cells by intrasplenic
injection. For adoptive transfer, anti-CD137 cured mice and naive mice were sacrificed 10 days after rechallenge with Hepa1.6 cells. Total splenocytes were collected, single-cell suspensions were prepared and injected intravenously into naive mice, inoculated with Hepa1.6 cells on the same day. DEN model
Fifteen-day-old male F1[B6xC3H] mice received one intra peritoneal (i.p.) injection of 50 lg g21 DEN (Sigma). Immunizations with the agonistic anti-CD137 antibody (100 mg/injection) were performed at 4, 5 and 6 months post DEN-treatment. Depletion experiments
CD8, CD4 T cells and NK cells were depleted in vivo by i.p. injection of 500 mg of 2.43, GK1.5, or PK136 mAb (BioXCell), 2 days before and 5 days after tumor inoculation. For MDSC depletion, mice were injected i.p. with 250 mg of RB68C5 mAb (BioXCell) twice a week starting on day 4 after tumor inoculation, for 3 weeks. Flow cytometry
For liver non-parenchymal cell (NPC) preparation, the liver was digested with collagenase IV followed by Percoll densitygradient centrifugation and RBC lysis. Fluorochromeconjugated anti-mouse antibodies: CD3e (145-2C11), CD4 (RM4-5), CD8 (53-6.7), CD11b (M1/70), CD11c (HL3), CD45 (RA3-6B2), CD25 (PC61), NK1.1 (PK136), Gr1 (RB66C5) and IFNg (XMG1.2) (BD Pharmingen), FoxP3 (FJK16s, eBiosciences), F4/80 (CI:A3-1, AbDSerotec). FACS analysis was conducted using a BD Pharmingen LSR-II flow cytometer and FlowJo software (Tree Star). Proliferation test
Splenocytes or NPC were incubated with 1 mM Cell TraceTM Far Red DDAO-SE (Molecular Probes, Eugene, OR) followed by incubation with Hepa1.6 cell lysate for 72 hr in RPMI, 10% FBS and IL2 (50 U mL21). Cells were stimulated with 40 ng mL21 PMA, 200 ng mL21 ionomycin for 5 hr, collected and stained for FACS analysis. Quantitative real-time PCR
qRT-PCR was performed on RNA prepared from splenocytes or NPC with the Qiagen RNeasy Mini Kit (Qiagen). PCR reactions were done using SYBR Green Supermix (Primer sequences in Supporting Information Table 1).
C 2014 UICC Int. J. Cancer: 00, 00–00 (2014) V
3
Tumor Immunology
Gauttier et al.
Figure 1. Anti-tumor activity of agonistic anti-CD137 mAb on in situ HCC models. In the orthotopic model, C57Bl/6 male mice were injected with 2.5 3 106 Hepa 1.6 through the portal vein and received 100 mg of either the anti-CD137 mAb (n 5 7) or control rat IgG (n 5 6) at days 4 and 8 after tumor inoculation. At sacrifice (day 18), liver morphology was checked and the tumor burden was determined by the ratio of liver weight over body weight (a). In the DEN model, 15-day-old F1[B63C3H] males were injected with diethylnitosamine and received anti-CD137 mAb treatment (n 5 17) or control rat IgG (n 5 16) (100 mg) at month 4, 5 and 6 after DEN injection. At sacrifice (month 8), mouse and liver were weighed and nodules were counted and measured to determine a total tumor surface (b). Liver histology (H&E staining) was performed at the time of sacrifice in both HCC models. Some lymphocytic infiltrates were visible only in anti-CD137 treated mice (black arrows) (magnification 3100). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Immunohistochemistry/immunofluorescence
Liver samples were formalin-fixed, and prepared as previously described,19 or frozen in Tissue-Tek (Fisher Scientific) and cut into 10-mm sections. Immunohistochemistry was performed with anti-CD4 (RM4-5) and anti-CD8 (53-6.7) biotynilated antibodies (1:250) followed by streptavidin-
C 2014 UICC Int. J. Cancer: 00, 00–00 (2014) V
peroxidase and diaminobenzidine revelation. Presence of macrophages was assessed with primary anti-mouse Ab: F4/ 80 (CI:A3-1; AbDSerotec) and iNOS (Abcam). Slides were analyzed using standard fluorescence microscopy and AxioVision imaging software (Carl Zeiss, Oberkochen, Germany).
4
Statistical analysis
The significance of differences in ratio and FACS analyses between experimental groups was determined using the nonparametric Mann–Whitney test. A log-rank test was used to analyze overall survival. Two-sided p values
Agonistic anti-CD137 antibody treatment leads to antitumor response in mice with liver cancer Vanessa Gauttier1, Jean-Paul Judor1, Valentin Le Guen1, Jeannette Cany2, Nicolas Ferry2 and Sophie Conchon1 1
^tel-Dieu, INSERM UMR1064, Centre for Research in Transplantation and Immunology—ITUN, Universite de Nantes, Centre Hospitalier Universitaire Ho Nantes, France 2 ^tel Dieu, Nantes, France INSERM UMR948, Hepatic Biotherapies, Universite de Nantes, Centre Hospitalier Universitaire Ho
Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and, due to limited treatment options, the third greatest cause of cancer death.1 Despite numerous attempts at amelioration, curative treatments continue to exhibit a very high recurrence rate.2 The liver possesses a specific immune system, which plays an intricate role in its functions and pathobiology, including the progressive evolution of chronic liver disease to fibrosis, Key words: combination immunotherapy, hepatocellular carcinoma, 4-1BB, liver cancer, therapy, MDSC Abbreviations: DEN: diethyl nitrosamine; HCC: hepatocellular carcinoma; IFNg: gamma interferon; mAb: monoclonal antibody; MDSC: myeloid derived suppressor cell; NPC: non-parenchymal cells; TNF: tumor necrosis factor; Treg: regulatory T lymphocytes Additional Supporting Information may be found in the online version of this article. Grant sponsor: IHU-Cesti; Grant number: ANR-10-IBHU-005; Grant sponsors: la Ligue Contre le Cancer; Inter-region GrandOuest; FUI-BPI France (Hepavac); INSERM; Region Pays de la Loire. DOI: 10.1002/ijc.28943 History: Received 13 Nov 2013; Accepted 16 Apr 2014; Online 2 May 2014 Correspondence to: Sophie Conchon, INSERM UMR1064 ITUN, CHU Hotel Dieu, 30 Bd Jean Monnet, 44093 Nantes Cedex 1, France. Tel.: 133-02-40-08-75-07, Fax: 133-02-40-08-74-11, E-mail: [email protected]
C 2014 UICC Int. J. Cancer: 00, 00–00 (2014) V
cirrhosis and finally to HCC.3 Work in recent years has demonstrated that local and systemic immunity can be harnessed to fight hepatic neoplastic development, and a variety of evidence points toward immunotherapy as a promising strategy against HCC. The presence of lymphocytic infiltrates has been shown to correlate with improved overall survival of HCC patients after surgical resection.4 T lymphocytes specific for some tumor antigens have been detected in HCC patients.5 In some patients, conventional treatment has been shown to enhance immune responses, suggesting that immunotherapy, as an adjuvant to, or following primary treatments,5,6 could be beneficial for the patient, especially as a solution to the high recurrence risk. Antigen specific immunotherapy could be a valid option for the treatment of HCC and a number of strategies are in development at a clinical level.7,8 Complementary to these approaches requiring a certain level of personalization (not all HCC express all tumor antigens), there is a place for nonspecific immunotherapy strategies: to block the suppressive mechanisms that arise during tumor development and impede the anti-tumor immune response, or to directly enhance this natural anti-tumor immune response. It is noteworthy that, to date, some of the most promising clinical results for HCC immunotherapy have been obtained by adoptive transfer of autologous T lymphocytes nonspecifically stimulated after surgical tumor resection.9 CD137 is a member of the tumor necrosis factor (TNF) receptor superfamily. It is expressed by many cell types
Tumor Immunology
Immunotherapy is a promising strategy against hepatocellular carcinoma (HCC). We assessed the therapeutic effects of stimulating CD137, a member of the TNF receptor family, with agonistic monoclonal antibodies (mAb). Agonistic anti-CD137 mAb treatment was tested on two in situ models of HCC in immunocompetent mice. We also studied the mediators involved at different time points. In an orthotopic HCC the treatment consistently leads to complete tumor regression in 40–60% of animals. The protection is long lasting in the animals responding to the treatment, which can reject a second tumor challenge more than 3 months after treatment and eradication of the first malignancy. The main mediators of the effect are T lymphocytes and NK cells, demonstrated through depletion experiments. In addition, adoptive transfer of splenocytes prepared from antiCD137 mAb-treated and -cured mice to naive mice allowed them to, in turn, reject the tumor. The efficacy of anti-CD137 mAb treatment is associated with early, sustained recruitment of iNOS-positive macrophages within tumor nodules. Moreover, in the absence of treatment, tumor development is accompanied by infiltration by myeloid derived suppressor cells (MDSC) and regulatory T lymphocytes. In mice responding to the anti-CD137 mAb treatment, this infiltration is very limited, and a combination treatment with a depletion of MDSC leads to the recovery of 80% of the mice. These results demonstrate that agonistic anti-CD137 mAb is a promising therapeutic strategy for anti-tumor immunity stimulation against HCC.
2
Agonistic anti-CD137 antibody for HCC immunotherapy
Tumor Immunology
What’s new? CD137 is a member of the tumor necrosis factor (TNF) receptor superfamily. In this study of hepatocellular carcinoma (HCC), the authors found that injections of an antibody that activates CD137 caused a reduction of tumor growth by 40 to 65% in mice, and that several types of immune cells, including cytotoxic T cells, NK cells, and macrophages play a major role in this effect. When myeloid derived suppressor cells (MDSC) were depleted, the anti-CD137 antibody led to 80% recovery. This combined approach may therefore have significant therapeutic potential.
including activated T cells, NK and NKT cells, dendritic cells, neutrophils or monocytes.10 The binding of its ligand CD137L to CD137 leads to the prolonged survival and sustained activation of CD8 T cells.11 Agonistic anti-CD137 monoclonal antibody fixation to CD137 enhances T-cell proliferation and cytokine production by T-helper lymphocytes, and protects CD8 T cells from activation-induced cell death.12,13 Agonistic anti-CD137 antibody induces regression of established tumors in various animal models,14–16 and prevents tumor recurrence in a model of melanoma.17 In low tumor burden conditions this nonspecific immunotherapy strategy could be well-suited to the treatment of HCC in order to stimulate in situ the natural anti-tumor immune response. To test this hypothesis, we investigated the antitumor effect of an agonistic anti-CD137 antibody treatment on murine models of HCC for which the tumor develops within the liver of immunocompetent animals. We show the therapeutic efficacy of this approach. The mechanistic analyses demonstrate that the treatment mobilizes T cells, NK cells and allows the early and sustained recruitment of macrophages in the tumor nodules and that the immune response elicited by the treatment keeps potentially harmful regulatory cells at bay.
Material and Methods Mice and reagents
C57Bl/6 mice were purchased from Janvier Laboratory (France), and were kept in the Nantes IFR26 animal facility. Animal housing and surgical procedures were conducted according to the guidelines of the French Agriculture Ministry and were approved by the regional ethical committee. Hepa1.6 cells were cultured in DMEM (Life Technologies), 10% FBS, glutamine and antibiotics at 37 C and 5% CO2. Cells were harvested and resuspended in FBS-free DMEM for animal inoculation. Anti-CD137 mAb was produced from the 3H3 hybridoma (kindly provided by Pr R.S. Mittler). Orthotopic model: anti-CD137 treatment, tumor challenge and adoptive transfer
Eight-weeks-old C57Bl/6J male mice received 2.5 3 106 Hepa1.6 cells in 100 mL through the portal vein, as described.18 Four and eight days after tumor inoculation, mice were injected with 100 mg of rat anti-CD137 mAb, or rat IgG (Sigma–Aldrich). For tumor challenge, mice received a second dose of 2.5 3 106 Hepa1.6 cells by intrasplenic
injection. For adoptive transfer, anti-CD137 cured mice and naive mice were sacrificed 10 days after rechallenge with Hepa1.6 cells. Total splenocytes were collected, single-cell suspensions were prepared and injected intravenously into naive mice, inoculated with Hepa1.6 cells on the same day. DEN model
Fifteen-day-old male F1[B6xC3H] mice received one intra peritoneal (i.p.) injection of 50 lg g21 DEN (Sigma). Immunizations with the agonistic anti-CD137 antibody (100 mg/injection) were performed at 4, 5 and 6 months post DEN-treatment. Depletion experiments
CD8, CD4 T cells and NK cells were depleted in vivo by i.p. injection of 500 mg of 2.43, GK1.5, or PK136 mAb (BioXCell), 2 days before and 5 days after tumor inoculation. For MDSC depletion, mice were injected i.p. with 250 mg of RB68C5 mAb (BioXCell) twice a week starting on day 4 after tumor inoculation, for 3 weeks. Flow cytometry
For liver non-parenchymal cell (NPC) preparation, the liver was digested with collagenase IV followed by Percoll densitygradient centrifugation and RBC lysis. Fluorochromeconjugated anti-mouse antibodies: CD3e (145-2C11), CD4 (RM4-5), CD8 (53-6.7), CD11b (M1/70), CD11c (HL3), CD45 (RA3-6B2), CD25 (PC61), NK1.1 (PK136), Gr1 (RB66C5) and IFNg (XMG1.2) (BD Pharmingen), FoxP3 (FJK16s, eBiosciences), F4/80 (CI:A3-1, AbDSerotec). FACS analysis was conducted using a BD Pharmingen LSR-II flow cytometer and FlowJo software (Tree Star). Proliferation test
Splenocytes or NPC were incubated with 1 mM Cell TraceTM Far Red DDAO-SE (Molecular Probes, Eugene, OR) followed by incubation with Hepa1.6 cell lysate for 72 hr in RPMI, 10% FBS and IL2 (50 U mL21). Cells were stimulated with 40 ng mL21 PMA, 200 ng mL21 ionomycin for 5 hr, collected and stained for FACS analysis. Quantitative real-time PCR
qRT-PCR was performed on RNA prepared from splenocytes or NPC with the Qiagen RNeasy Mini Kit (Qiagen). PCR reactions were done using SYBR Green Supermix (Primer sequences in Supporting Information Table 1).
C 2014 UICC Int. J. Cancer: 00, 00–00 (2014) V
3
Tumor Immunology
Gauttier et al.
Figure 1. Anti-tumor activity of agonistic anti-CD137 mAb on in situ HCC models. In the orthotopic model, C57Bl/6 male mice were injected with 2.5 3 106 Hepa 1.6 through the portal vein and received 100 mg of either the anti-CD137 mAb (n 5 7) or control rat IgG (n 5 6) at days 4 and 8 after tumor inoculation. At sacrifice (day 18), liver morphology was checked and the tumor burden was determined by the ratio of liver weight over body weight (a). In the DEN model, 15-day-old F1[B63C3H] males were injected with diethylnitosamine and received anti-CD137 mAb treatment (n 5 17) or control rat IgG (n 5 16) (100 mg) at month 4, 5 and 6 after DEN injection. At sacrifice (month 8), mouse and liver were weighed and nodules were counted and measured to determine a total tumor surface (b). Liver histology (H&E staining) was performed at the time of sacrifice in both HCC models. Some lymphocytic infiltrates were visible only in anti-CD137 treated mice (black arrows) (magnification 3100). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Immunohistochemistry/immunofluorescence
Liver samples were formalin-fixed, and prepared as previously described,19 or frozen in Tissue-Tek (Fisher Scientific) and cut into 10-mm sections. Immunohistochemistry was performed with anti-CD4 (RM4-5) and anti-CD8 (53-6.7) biotynilated antibodies (1:250) followed by streptavidin-
C 2014 UICC Int. J. Cancer: 00, 00–00 (2014) V
peroxidase and diaminobenzidine revelation. Presence of macrophages was assessed with primary anti-mouse Ab: F4/ 80 (CI:A3-1; AbDSerotec) and iNOS (Abcam). Slides were analyzed using standard fluorescence microscopy and AxioVision imaging software (Carl Zeiss, Oberkochen, Germany).
4
Statistical analysis
The significance of differences in ratio and FACS analyses between experimental groups was determined using the nonparametric Mann–Whitney test. A log-rank test was used to analyze overall survival. Two-sided p values
