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Reduced Shedding of Surface Mesothelin Improves Efficacy of Mesothelin-Targeting Recombinant Immunotoxins.

ABSTRACT: Mesothelin (MSLN) is a differentiation antigen that is highly expressed in many epithelial cancers. MSLN is an important therapeutic target due to its high expression in cancers and limited expression in normal human tissues. Although it has been assumed that shed antigen is a barrier to immunotoxin action, a modeling study predicted that shed MSLN may enhance the action of MSLN-targeting recombinant immunotoxins such as SS1P and similar therapeutics by facilitating their redistribution within tumors. We aimed to determine whether shed MSLN enhances or reduces the antitumor effect of MSLN-targeting immunotoxins SS1P and RG7787. We engineered a cell line, A431/G9 (TACE mutant) that expresses a mutant form of MSLN in which the TNF-converting enzyme protease site is replaced with GGGS. We compared the response of the TACE-mutant cells with immunotoxins SS1P and RG7787 with that of the parental A431/H9 cell line. We show that TACE-mutant cells shed 80% less MSLN than A431/H9 cells, that TACE-mutant cells show a 2- to 3-fold increase in MSLN-targeted immunotoxin uptake, and that they are about 5-fold more sensitive to SS1P killing in cell culture. Tumors with reduced shedding respond significantly better to treatment with SS1P and RG7787. Our data show that MSLN shedding is an impediment to the antitumor activity of SS1P and RG7787. Approaches that decrease MSLN shedding could enhance the efficacy of immunotoxins and immunoconjugates targeting MSLN-expressing tumors. Mol Cancer Ther; 15(7); 1648-55. ©2016 AACR.

SUBMITTER: Awuah P

PROVIDER: S-EPMC4936933 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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Reduced Shedding of Surface Mesothelin Improves Efficacy of Mesothelin-Targeting Recombinant Immunotoxins.

Awuah Prince P Bera Tapan K TK Folivi Messan M Chertov Oleg O Pastan Ira I

Molecular cancer therapeutics 20160518 7

Mesothelin (MSLN) is a differentiation antigen that is highly expressed in many epithelial cancers. MSLN is an important therapeutic target due to its high expression in cancers and limited expression in normal human tissues. Although it has been assumed that shed antigen is a barrier to immunotoxin action, a modeling study predicted that shed MSLN may enhance the action of MSLN-targeting recombinant immunotoxins such as SS1P and similar therapeutics by facilitating their redistribution within t ...[more]

PMID: 27196771

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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.

Dataset Information

Reduced Shedding of Surface Mesothelin Improves Efficacy of Mesothelin-Targeting Recombinant Immunotoxins.

Publications

Reduced Shedding of Surface Mesothelin Improves Efficacy of Mesothelin-Targeting Recombinant Immunotoxins.

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