Modulation of CD59 Expression by Restrictive Silencer Factor–Derived Peptides in Cancer Immunotherapy for Neuroblastoma

Abstract

Tumor cells escape clearance by complement by abundantly expressing CD59 and other membrane complement regulators. Existing strategies for blocking/knocking down these regulators can contribute to tumor immunoclearance in vitro; however, there are numerous difficulties restricting their use in vivo. Here, we report a new strategy for suppression of CD59 expression in neuroblastoma using peptides that target regulators of CD59 expression. We identified the neuralrestrictive silencer factor (REST) as a target for modulation of CD59 expression in neuroblastoma. We next designed plasmids that encoded peptides comprising different DNA-binding domains of REST and transfected them into neuroblastoma cell lines. These peptides suppressed CD59 expression, sensitizing neuroblastoma to complement-mediated killing triggered by anti-GD2 therapeutic monoclonal antibody. These CD59-modulating peptides might be effective therapeutic adjuvants to therapeutic monoclonal antibodies used for treatment of neuroblastoma and other cancer types sharing the same mechanism for regulation of CD59 expression.

Application of a novel inhibitor of human CD59 for the enhancement of complement-dependent cytolysis on cancer cells

“…39 Meanwhile, in an effort to enhance the efficacy of mAb-induced immunotherapies in other tumor cell lines, a variety of methods, such as Ab, siRNA and peptide treatments, have been used to neutralize or decrease the expression of CD59. [40][41][42] However, these treatments often produce multiple side effects because they target both tumor cells and normal cells. We selected the novel hCD59 inhibitor rILYd4, which has been shown to have a high affinity to CD59 5 and has not shown signs of cytoxicity in normal cells (unpublished data), to improve or sensitize the resistant lymphoma cell lines in vitro.…”

The dual role of complement in cancer and its implication in anti-tumor therapy

“…Other groups (88,89) have successfully blocked CD59 using mAbs in a model of NSCLC that led to a similar synergistic effect. Donev et al (90) used a different approach and identified neural-restrictive silencer factor (REST), as an important regulatory component of the transcriptional machinery of the CD59 gene. They inhibited REST using peptides against the promoter region of CD59 where REST binds.…”

Upregulating CD59: a new strategy for protection of neurons from complement-mediated degeneration

“…A detailed description of this procedure was given previously. 23 All measurements were made at least in duplicate and each experiment was replicated twice. Results were combined and statistically analysed by Student's t-test.…”

“…Immunoprecipitated DNAs were used as templates (10 ng per reaction) in a quantitative assay with a primer pair for detection of the responsive element within the cd59 promoter. 23 The C t value calculated for the input DNA used in ChIPs was used to normalize the cell number. Two independent analyses of immunoprecipitated DNAs were carried out for each antibody.…”

“…23 Different dilutions of normal human serum (NHS) or conditioned medium from primary human oligodendroglia were used as the source of complement. As negative controls for the complement-mediated lysis, we either inactivated complement by heat treatment (15 min at 56 1C) of NHS and the conditioned medium or depleted the NHS/conditioned medium of C8 using a monoclonal affinity column.…”