Novel NK Cytotoxicity Assay Enables Drug Discovery For Hemophagocytic Lymphohistiocytosis

Abstract

RationaleDrugs correcting cytotoxic deficiency are currently unavailable but highly desirable for treating hemophagocytic lymphohistiocytosis (HLH), a condition characterized by various degree of fundamental defect in lymphocyte cytotoxicity. Discovery efforts have been thwarted by lack of a high throughput screening (HTS) assay. We hypothesized that a novel granzyme B specific biosensor would allow HTS of NK cell cytotoxicity and facilitate drug development.MethodsBiosensor was created by linking the termini of firefly luciferase with a granzyme B cleavage site, which rendered the luciferase inactive until cleaved by granzyme B. NK cytotoxic biosensor assay was performed in 96-well plate by mixing the effector cell NK92 with the biosensor expressing- target K562, whereby the biosensor detects the delivery of granzyme B into the live targets in real time.ResultsLuciferase activity rose within 10 minutes of NK92:K562 contact, plateaued at 75 minutes and gradually declined. The biosensor detected induction of luciferase at effector:target ratio as low as 0.1:1 and target number lower than 600/well. The biosensor was suppressed by granzyme B inhibitor C20, and shRNA knockdown of granzyme B. Activated NK cells from PBMC also triggered the biosensor and the luminescent signal correlated with 51Cr release assay. HTS of NK92 cells with the biosensor assay in 384-well plate identified signal enhancing hits from 1591 small molecule compounds.ConclusionsGranzyme B specific luciferase biosensor assay is a sensitive and specific real time cytotoxic assay that is suitable for HTS of NK cytotoxicity. The assay allows further discovery and development of drugs for treatment of HLH. RationaleDrugs correcting cytotoxic deficiency are currently unavailable but highly desirable for treating hemophagocytic lymphohistiocytosis (HLH), a condition characterized by various degree of fundamental defect in lymphocyte cytotoxicity. Discovery efforts have been thwarted by lack of a high throughput screening (HTS) assay. We hypothesized that a novel granzyme B specific biosensor would allow HTS of NK cell cytotoxicity and facilitate drug development. Drugs correcting cytotoxic deficiency are currently unavailable but highly desirable for treating hemophagocytic lymphohistiocytosis (HLH), a condition characterized by various degree of fundamental defect in lymphocyte cytotoxicity. Discovery efforts have been thwarted by lack of a high throughput screening (HTS) assay. We hypothesized that a novel granzyme B specific biosensor would allow HTS of NK cell cytotoxicity and facilitate drug development. MethodsBiosensor was created by linking the termini of firefly luciferase with a granzyme B cleavage site, which rendered the luciferase inactive until cleaved by granzyme B. NK cytotoxic biosensor assay was performed in 96-well plate by mixing the effector cell NK92 with the biosensor expressing- target K562, whereby the biosensor detects the delivery of granzyme B into the live targets in real time. Biosensor was created by linking the termini of firefly luciferase with a granzyme B cleavage site, which rendered the luciferase inactive until cleaved by granzyme B. NK cytotoxic biosensor assay was performed in 96-well plate by mixing the effector cell NK92 with the biosensor expressing- target K562, whereby the biosensor detects the delivery of granzyme B into the live targets in real time. ResultsLuciferase activity rose within 10 minutes of NK92:K562 contact, plateaued at 75 minutes and gradually declined. The biosensor detected induction of luciferase at effector:target ratio as low as 0.1:1 and target number lower than 600/well. The biosensor was suppressed by granzyme B inhibitor C20, and shRNA knockdown of granzyme B. Activated NK cells from PBMC also triggered the biosensor and the luminescent signal correlated with 51Cr release assay. HTS of NK92 cells with the biosensor assay in 384-well plate identified signal enhancing hits from 1591 small molecule compounds. Luciferase activity rose within 10 minutes of NK92:K562 contact, plateaued at 75 minutes and gradually declined. The biosensor detected induction of luciferase at effector:target ratio as low as 0.1:1 and target number lower than 600/well. The biosensor was suppressed by granzyme B inhibitor C20, and shRNA knockdown of granzyme B. Activated NK cells from PBMC also triggered the biosensor and the luminescent signal correlated with 51Cr release assay. HTS of NK92 cells with the biosensor assay in 384-well plate identified signal enhancing hits from 1591 small molecule compounds. ConclusionsGranzyme B specific luciferase biosensor assay is a sensitive and specific real time cytotoxic assay that is suitable for HTS of NK cytotoxicity. The assay allows further discovery and development of drugs for treatment of HLH. Granzyme B specific luciferase biosensor assay is a sensitive and specific real time cytotoxic assay that is suitable for HTS of NK cytotoxicity. The assay allows further discovery and development of drugs for treatment of HLH.