Synergy between chemotherapeutic agents and CTLA-4 blockade in preclinical tumor models

Abstract

Ipilimumab, a cytotoxic T-lymphocyte antigen-4 (CTLA-4) binding agent, has proven to be an effective monotherapy for metastatic melanoma and has shown antitumor activity in trials when administered with other therapeutic agents. We hypothesized that the combination of ipilimumab with chemotherapeutic agents, such as ixabepilone, paclitaxel, etoposide, and gemcitabine, may produce therapeutic synergy based on distinct but complementary mechanisms of action for each drug and unique cellular targets. This concept was investigated using a mouse homolog of ipilimumab in preclinical murine tumor models, including SA1N fibrosarcoma, EMT-6 mammary carcinoma, M109 lung carcinoma, and CT-26 colon carcinoma. Results of CTLA-4 blockade in combination with one of various chemotherapeutic agents demonstrate that synergy occurs in settings where either agent alone was not effective in inducing tumor regression. Furthermore, when combined with CTLA-4 blockade, ixabepilone, etoposide, and gemcitabine elicited prolonged antitumor effects in some murine models with induction of a memory immune response. Future investigations are warranted to determine which specific chemo-immunotherapy combinations, if any, will produce synergistic antitumor effects in the clinical setting.

Fig. 1

Therapeutic synergy observed with CTLA-4 blockade in combination with ixabepilone and paclitaxel in tumor models. In the SA1N fibrosarcoma model (a), combination of anti-mCTLA-4 mAb with either ixabepilone or paclitaxel resulted in therapeutic synergy, with the majority of mice displaying substantially delayed tumor growth over time. In the EMT-6 mammary carcinoma model (b), combination of anti-mCTLA-4 mAb with ixabepilone yielded synergistic effects over time, resulting in complete regression of tumors on Day 18; anti-mCTLA-4 mAb paired with paclitaxel improved antitumor activity without achieving a synergistic effect. Anti-mCTLA-4 mAb in combination with ixabepilone expanded T lymphocytes with cytolytic function by Day 19 (CD8⁺CD107⁺), supporting synergistic efficacy in the EMT-6 mammary carcinoma model (c, d). In the M109 lung carcinoma model (e), tumor-free mice previously treated with ixabepilone monotherapy or in combination with anti-mCTLA-4 mAb were rechallenged on Day 95 with live tumor cells. The majority of mice (75 %) treated with the combination of anti-mCTLA-4 mAb and ixabepilone rejected the tumor rechallenge, suggestive of a memory immune response. In the CT-26 colon carcinoma model (f), treatment of mice with anti-mCTLA-4 mAb and either ixabepilone or paclitaxel resulted in synergy between CTLA-4 blockade and these chemotherapeutic agents. Expansion of activated T cells (CD8⁺/CD69⁺ and CD4⁺/CD69⁺) was observed with anti-mCTLA-4 mAb alone and in combination with either ixabepilone or paclitaxel (g, h)

Fig. 1

Therapeutic synergy observed with CTLA-4 blockade in combination with ixabepilone and paclitaxel in tumor models. In the SA1N fibrosarcoma model (a), combination of anti-mCTLA-4 mAb with either ixabepilone or paclitaxel resulted in therapeutic synergy, with the majority of mice displaying substantially delayed tumor growth over time. In the EMT-6 mammary carcinoma model (b), combination of anti-mCTLA-4 mAb with ixabepilone yielded synergistic effects over time, resulting in complete regression of tumors on Day 18; anti-mCTLA-4 mAb paired with paclitaxel improved antitumor activity without achieving a synergistic effect. Anti-mCTLA-4 mAb in combination with ixabepilone expanded T lymphocytes with cytolytic function by Day 19 (CD8⁺CD107⁺), supporting synergistic efficacy in the EMT-6 mammary carcinoma model (c, d). In the M109 lung carcinoma model (e), tumor-free mice previously treated with ixabepilone monotherapy or in combination with anti-mCTLA-4 mAb were rechallenged on Day 95 with live tumor cells. The majority of mice (75 %) treated with the combination of anti-mCTLA-4 mAb and ixabepilone rejected the tumor rechallenge, suggestive of a memory immune response. In the CT-26 colon carcinoma model (f), treatment of mice with anti-mCTLA-4 mAb and either ixabepilone or paclitaxel resulted in synergy between CTLA-4 blockade and these chemotherapeutic agents. Expansion of activated T cells (CD8⁺/CD69⁺ and CD4⁺/CD69⁺) was observed with anti-mCTLA-4 mAb alone and in combination with either ixabepilone or paclitaxel (g, h)

Fig. 2

Therapeutic synergy observed with CTLA-4 blockade in combination with etoposide in tumor models. In the SA1N fibrosarcoma model (a), M109 lung carcinoma model (b), and CT-26 colon carcinoma model (c), therapeutic synergy was observed with the administration of anti-mCTLA-4 mAb in combination with etoposide relative to the treatment with either monotherapy. Efficacy was evaluated either by tumor volume (a, c) or by survival measurement (b)

Fig. 3

In vivo cytotoxic activity toward a CT-26 tumor antigen. In the CT-26 colon carcinoma mouse model, anti-mCTLA-4 mAb was administered at a dose of 20 mg/kg on Days 8, 12, and 16. Etoposide was administered at a dose of 50 mg/kg on Days 7, 14, and 21, whereas gemcitabine was administered at a dose of 120 mg/kg on Days 7, 11, and 15. In vivo cell kill was determined on Days 18 and 23 post-implant. One day prior to analysis, a 50:50 mixture of peptide-pulsed ([H] SPSYVYHQF [OH], Sigma Genosys) and peptide non-pulsed carboxyfluorescein diacetate succinimidyl ester (CSFE)-labeled splenocytes from naive BALB/c donors were adoptively transferred via IV tail injection into treated animals; 24 h later, spleens were removed and analyzed via flow cytometry to determine the percent cell kill of peptide-pulsed cells. In vivo cytotoxicity against a CT-26 tumor antigen showed a slight increase in the CTLA-4 blockade and etoposide combination group without reaching statistical significance versus anti-mCTLA-4 mAb alone. At these time points, there were no significant enhancements of the cytotoxic activity against a CT-26 tumor antigen with the combination of anti-mCTLA-4 mAb and gemcitabine when compared with either treatment alone

Fig. 4

Therapeutic synergy observed with CTLA-4 blockade in combination with gemcitabine in tumor models. In the SA1N fibrosarcoma model (a), the combination of anti-mCTLA-4 mAb with gemcitabine did not produce therapeutic synergy. In the M109 lung carcinoma model (b) and CT-26 colon carcinoma model (c), therapeutic synergy was observed with administration of anti-mCTLA-4 mAb in combination with gemcitabine relative to the treatment of mice with either monotherapy