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Combining Local Immunotoxins Targeting Mesothelin with CTLA-4 Blockade Synergistically Eradicates Murine Cancer by Promoting Anticancer Immunity.

ABSTRACT: Immune checkpoint blockade using antibodies to cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) benefits a limited number of cancer patients. SS1P and LMB-100 are immunotoxins that target mesothelin. We observed delayed responses to SS1P in patients with mesothelioma suggesting that antitumor immunity was induced. Our goal was to stimulate antitumor immunity by combining SS1P or LMB-100 with anti-CTLA-4. We constructed a BALB/c breast cancer cell line expressing human mesothelin (66C14-M), which was implanted in one or two locations. SS1P or LMB-100 was injected directly into established tumors and anti-CTLA-4 administered i.p. In mice with two tumors, one tumor was injected with immunotoxin and the other was not. The complete regression rate was 86% for the injected tumors and 53% for the uninjetced tumors. No complete regressions occurred when drugs were given separately. In regressing tumors, dying and dead tumor cells were intermingled with PMNs and surrounded by a collar of admixed eosinophils and mononuclear cells. Tumor regression was associated with increased numbers of tumor-infiltrating CD8+ cells and blocked by administration of antibodies to CD8. Surviving mice were protected from tumor rechallenge by 66C14 cells not expressing mesothelin, indicating the development of antitumor immunity. The antitumor effect was abolished when a mutant noncytotoxic variant was used instead of LMB-100, showing that the antitumor response is not mediated by recognition of a foreign bacterial protein. Our findings support developing a therapy composed of immunotoxins and checkpoint inhibitors for patients. Cancer Immunol Res; 5(8); 685-94. ©2017 AACR.

SUBMITTER: Leshem Y

PROVIDER: S-EPMC5549555 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Combining Local Immunotoxins Targeting Mesothelin with CTLA-4 Blockade Synergistically Eradicates Murine Cancer by Promoting Anticancer Immunity.

Leshem Yasmin Y O'Brien James J Liu Xiufen X Bera Tapan K TK Terabe Masaki M Berzofsky Jay A JA Bossenmaier Birgit B Niederfellner Gerhard G Tai Chin-Hsien CH Reiter Yoram Y Pastan Ira I

Cancer immunology research 20170703 8

Immune checkpoint blockade using antibodies to cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) benefits a limited number of cancer patients. SS1P and LMB-100 are immunotoxins that target mesothelin. We observed delayed responses to SS1P in patients with mesothelioma suggesting that antitumor immunity was induced. Our goal was to stimulate antitumor immunity by combining SS1P or LMB-100 with anti-CTLA-4. We constructed a BALB/c breast cancer cell line expressing human mesothelin (66C14-M), w ...[more]

PMID: 28674083

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Project description:Anti-mesothelin Pseudomonas exotoxin A-based recombinant immunotoxins (RITs) present a potential treatment modality for pancreatic ductal adenocarcinoma (PDAC). To study mechanisms of resistance, the sensitive PDAC cell line KLM-1 was intermittently exposed to the anti-mesothelin SS1-LR-GGS RIT. Surviving cells were resistant to various anti-mesothelin RITs (IC50s >1 μg/ml), including the novel de-immunized RG7787. These resistant KLM-1-R cells were equally sensitive to the anti-CD71 HB21(Fv)-PE40 RIT as KLM-1, indicating resistance was specific to anti-mesothelin RITs. Mesothelin gene expression was partially down-regulated in KLM-1-R, resulting in 5-fold lower surface protein levels and decreased cellular uptake of RG7787 compared to KLM-1. Bisulfite sequencing analysis found that the mesothelin promoter region was significantly more methylated in KLM-1-R (59 ± 3.6%) compared to KLM-1 (41 ± 4.8%), indicating hypermethylation as a mechanism of mesothelin downregulation. The DNA methyltransferase inhibitor 5-azacytidine restored original mesothelin surface expression to more than half in KLM-1-R and increased sensitivity to RG7787 (IC50 = 722.4 ± 232.6 ng/ml), although cells remained significantly less sensitive compared to parental KLM-1 cells (IC50 = 4.41 ± 0.38 ng/ml). Mesothelin cDNA introduction in KLM-1-R led to 5-fold higher surface protein levels and significantly higher RG7887 uptake compared to KLM-1. As a result, the original sensitivity to RG7787 was fully restored (IC50 = 4.49 ± 1.11 ng/ml). A significantly higher RG7787 uptake was thus required to reach the original cytotoxicity in resistant cells, hinting that intracellular RIT trafficking is also a limiting factor. RNA deep sequencing analysis of KLM-1 and KLM-1-R cells supported our experimental findings; compared to KLM-1, resistant cells displayed differential expression of genes linked to intracellular transport and an expression pattern that matched a more general hypermethylation status. In conclusion, resistance to anti-mesothelin RITs in KLM-1 is linked to a methylation-associated down-regulation of mesothelin, while aberrations in RIT trafficking could also play a role.

Project description:Melanoma remains a critical public health problem worldwide. Patients with stage IV disease have very poor prognosis and their 1-year survival rate is only 25%. Until recently, systemic treatments with a positive impact on overall survival (OS) had remained elusive. In recent years, the United States Food and Drug Administration (FDA) - approved several novel agents targeting the RAS/RAF/MEK/ERK pathway (vemurafenib, dabrafenib, and trametinib) - critical in cell division and proliferation of melanoma, and an immune checkpoint inhibitor (ipilimumab) directed against the cytotoxic T lymphocyte Antigen - (CTLA-4). Moreover, recent reports of clinical trials studying other immune checkpoint modulating agents will most likely result in their FDA approval within the next months. This review focuses on ipilimumab, its safety and efficacy, and future considerations. Ipilimumab has demonstrated a positive OS impact after a several-year follow-up. It is also recognized that due to its mechanism of action, the response patterns to ipilimumab can differ from those observed in patients following treatment with conventional cytotoxic agents and even the most recently approved BRAF inhibitors. Most patients (84.8%) experience drug-related adverse events (AEs) of any grade; most of these are mild to moderate and immune mediated. However, a minority of patients may also experience severe and life-threatening AEs. In clinical studies, AEs were managed according to guidelines that emphasized close clinical monitoring and early use of corticosteroids when appropriate. Preliminary results have taught us the potential greater toxicity when in combination with vemurafenib, and the greater antitumor efficacy when combined with nivolumab, a monoclonal antibody directed against programmed death receptor-1 (PD-1), another immune checkpoint inhibitor. Future challenges include the optimization of dosing and toxicities when used as a single agent, and studying the safety and efficacy of combinations with targeted small molecules and other monoclonal antibodies to treat patients with melanoma and other malignancies.

Dataset Information

Combining Local Immunotoxins Targeting Mesothelin with CTLA-4 Blockade Synergistically Eradicates Murine Cancer by Promoting Anticancer Immunity.

Publications

Combining Local Immunotoxins Targeting Mesothelin with CTLA-4 Blockade Synergistically Eradicates Murine Cancer by Promoting Anticancer Immunity.

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