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WO2009147384A2 - Peptide - Google Patents

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Publication number
WO2009147384A2
WO2009147384A2 PCT/GB2009/001374 GB2009001374W WO2009147384A2 WO 2009147384 A2 WO2009147384 A2 WO 2009147384A2 GB 2009001374 W GB2009001374 W GB 2009001374W WO 2009147384 A2 WO2009147384 A2 WO 2009147384A2
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peptide
seq
nucleic acid
acid molecule
expression
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PCT/GB2009/001374
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WO2009147384A3 (fr
Inventor
Rossen Mintchev Donev
Bryan Paul Morgan
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University College Cardiff Consultants Limited
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Publication of WO2009147384A3 publication Critical patent/WO2009147384A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to peptides which are of use in the treatment of cancer, to nucleic acids encoding the peptides, constructs containing the nucleic acids and cells transformed or transfected with the constructs.
  • the invention relates to peptides derived from the neural-restrictive silencer factor (REST) which are of use in modulating expression of the complement regulator CD59 on tumour cells and so sensitising the tumour cells to immunotherapy.
  • REST neural-restrictive silencer factor
  • mAb therapy, but often relapse into a multidrug resistant state. Outcome in neuroblastoma patients is particularly poor. Sensitising tumours to immune attack, for example by blocking complement regulators, might aid therapy, but current approaches, while effective in vitro (siRNA) or in xenograft animal models (blocking mAb) of human cancer, cannot easily be applied in vivo.
  • siRNA in vitro
  • blocking mAb mAb
  • Complement is a major component of innate immunity (Walport, 2001). Complement can be activated on tumour cells by antibodies (Magyarlaki et al., 1996), immune complexes (Lucas et al., 1996), as a consequence of apoptosis (Matsumoto et al., 1997) or through proteolytic processes (Bj ⁇ rge et al., 1997). Normal and malignant cells are protected by membrane-bound complement regulators (mCReg) that act as physiologic brakes to complement amplification either by limiting formation of the C3/C5 convertase enzymes (CD35, CD46, CD55), or of the assembly of the cytolytic membrane attack complex (CD59) (Walport, 2001).
  • mCReg membrane-bound complement regulators
  • mCReg expression is greater than in normal surrounding tissue (Bj ⁇ rge et al., 1997; Rushmere et al., 2004). Consequently, the increased complement resistance conferred by these mCReg has been proposed as a mechanism that facilitates survival of the tumour or the metastasising tumour cell when it enters the circulation (Gorter and Meri, 1999).
  • complement For the majority of therapeutic mAb, complement likely plays little or no role in tumour clearance because the tumour abundantly expresses mCReg (Imai et al., 2005). Indeed, repeated sub-optimal Rituximab treatment caused resistance to complement killing in the B-cell line RAMOS by inducing increased expression of CD55 and CD59 (Takei et al., 2006). Blocking of CD55 and CD59 increased the effectiveness of therapeutic mAb killing in B-cell lines by 5-6 fold, confirming the protective role of mCReg (Golay et al., 2001). Although blocking of the mCReg with mAbs enhances complement-mediated immunoclearance of tumours, their high molecular mass and the ubiquitous expression of their targets are serious limitations for their application in humans.
  • the present invention results from an extension of this work and in particular from the identification of additional and novel molecular mechanisms leading to overexpression of CD59 in neuroblastoma.
  • REST neural- restrictive silencer factor
  • REST has been found to be a target for several different types of mutations in neuroblastoma (Palm et al., 1999), small cell lung carcinoma (Coulson et al., 2000) and colorectal cancer (Westbrook et al., 2005).
  • the present invention has its basis in our finding that REST is involved in modulation of CD59 expression in neuroblastoma and in particular relates to novel REST peptides that target identified transcriptional regulator sites of CD59, reduce CD59 expression and sensitise tumour cells to complement- mediated killing triggered by a mAb used in neuroblastoma immunotherapy.
  • a peptide comprising SEQ ID NO: 2 or a homologue or derivative thereof; provided that the peptide is not full-length REST.
  • homologues or derivatives of the peptide of the invention will also find use in the context of the present invention, ie as suppressors of CD59 gene expression.
  • proteins or polypeptides which include one or more additions, deletions, substitutions or the like are encompassed by the present invention.
  • a program like BLASTx will align the longest stretch of similar sequences and assign a value to the fit. It is thus possible to obtain a comparison where several regions of similarity are found, each having a different score. Both types of analysis are contemplated in the present invention.
  • a homologue of a peptide such as those of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 will be at least 70% homologous to that peptide, but more suitably it will have at least 80% and, increasing order of suitability, 85%, 90%, 95% or 99% homology to the given peptide.
  • SEQ ID NO: 2 consists of zinc-finger domains 6, 7 and 8 of full-length REST plus their interdomain sequences.
  • the peptide may consist solely of the sequence of SEQ ID NO: 2 or a homologue or derivative thereof. Alternatively, however, the peptide may comprise the sequence of SEQ ID NO: 2 or a homologue or derivative thereof. Alternatively, however, the peptide may comprise the sequence of SEQ ID NO: 2 or a homologue or derivative thereof. Alternatively, however, the peptide may comprise the sequence of SEQ ID NO: 2 or a homologue or derivative thereof. Alternatively, however, the peptide may comprise the sequence of SEQ ID NO: 2 or a homologue or derivative thereof. Alternatively, however, the peptide may comprise the sequence of SEQ ID NO: 2 or a homologue or derivative thereof. Alternatively, however, the peptide may comprise the sequence of SEQ ID NO: 2 or a homologue or derivative thereof. Alternatively, however, the peptide may comprise the sequence of SEQ ID NO: 2 or a homologue or derivative thereof. Alternatively, however, the peptide may comprise the sequence of SEQ
  • SEQ ID NO: 2 or a homologue or derivative thereof linked at the 5' and/or 3' end to a nuclear localisation signal, which is optionally separated from SEQ ID NO: 2 by a spacer.
  • the nuclear localisation signal may be derived from the full-length REST protein, in which case it is suitably zinc-finger domain 5 of full-length REST.
  • An example of such a peptide is REST58 (SEQ ID NO: 4) or a homologue or derivative thereof, which comprises domain 5 of full length REST, which is responsible for nuclear localisation, and domains 6, 7 and 8 ( Figure 4A) that are essential for DNA binding.
  • REST58 is delivered to the nucleus via a REST- specific receptor (Shimojo and Hersh, 2003) recognised by the zinc-finger domain 5 of the protein.
  • the REST-specific receptor has been identified only recently and its efficacy in delivering REST to the nucleus has not been investigated.
  • the peptide may additionally comprise an N' terminal methionine residue and an example of such a peptide is shown as SEQ ID NO: 6.
  • Alternative nuclear localisation signals may also be used, for example in the peptide REST68 (SEQ ID NO: 8) or a homologue or derivative thereof.
  • This peptide is similar to REST58 except that domain 5 has been replaced by the classical nuclear localisation signal (NLS) which has the sequence PKKKRKV and which delivers proteins to the nucleus via importin ⁇ 1 (Nadler et al., 1997).
  • NLS classical nuclear localisation signal
  • nucleic acid molecule encoding a peptide of the first aspect of the invention or which hybridises under stringent conditions to nucleic acid encoding a peptide of the first aspect of the invention.
  • the nucleic acid may encode a peptide having SEQ ID NO: 2, SEQ ID NO:
  • SEQ ID NO: 4 SEQ ID NO: 6 or SEQ ID NO: 8.
  • SEQ ID NO: 1 which encodes SEQ ID NO: 2, SEQ ID NO: 3, which encodes SEQ ID NO: 4, SEQ ID NO: 5, which encodes SEQ ID NO: 6 or SEQ ID NO: 7, which encodes SEQ ID NO:8 or homologues thereof or nucleic acid which hybridises to SEQ ID NO: 1 , 3, 5 or 7 under stringent conditions.
  • BESTFIT both from the Wisconsin Genetics Computer Group (GCG) software package
  • BESTFIT compares two sequences and produces an optimal alignment of the most similar segments.
  • GAP enables sequences to be aligned along their whole length and finds the optimal alignment by inserting spaces in either sequence as appropriate.
  • the comparison is made by alignment of the sequences along their whole length.
  • a homologue of a nucleic acid sequence of the invention for example, SEQ ID NO: 1 , SEQ ID NO: 3 or SEQ ID NO: 5 will be at least 70% homologous to that nucleic acid sequence, but more suitably it will have at least 80% and, increasing order of suitability, 85%, 90%, 95% or 99% homology.
  • Reference herein to stringent conditions includes reference to either increasing the temperature of incubation to at least 45 0 C but more suitably to 65 0 C and/or washing the annealed molecules using a salt solution having an ionic strength of from 1.0M sodium chloride to 0.02M sodium chloride.
  • the invention also provides a construct comprising the nucleic acid molecule of the second aspect of the invention and, further, the invention also provides a cell transformed or transfected with the construct. Furthermore, there is provided a process for the preparation of a peptide according to the first aspect of the invention, the process comprising expressing the nucleic acid molecule of the second aspect of the invention.
  • REST68 is particularly effective and has a 4-fold effect at mRNA level and a 2.5-fold effect at protein level ( Figure 4). Taking into account the fact that cell-bound CD59 turns over relatively rapidly (Davies et al., 2005), and that transfected neuroblastoma cells were cultured for up to two weeks before studying the effect of REST68, the observed discrepancy between mRNA and protein change might be a result of compensatory mechanism(s) that affects either the stability or the expression of CD59 at protein level.
  • nucleic acid of the second aspect of the invention or a construct comprising said nucleic acid for use in medicine, particularly in the treatment of cancer.
  • cancers which may be treated include especially neuroblastoma but also other types of cancer such as small cell lung carcinoma and colorectal cancer.
  • the peptide, nucleic acid molecule or construct will generally be used in combination with an immunotherapeutic agent targeted to the type of tumour being treated.
  • the invention also provides the use of a peptide of the first aspect of the invention, nucleic aGid molecule of the second aspect of the invention or a construct comprising said nucleic acid molecule in the preparation of an agent for the treatment of cancer, in particular neuroblastoma but also other types of cancer such as small cell lung carcinoma and colorectal cancer.
  • nucleic acid molecule of the second aspect of the invention comprising a construct comprising said nucleic acid molecule and an immunotherapeutic agent as a combined preparation for simultaneous, separate or sequential use in the treatment of cancer.
  • a pharmaceutical composition comprising a peptide of the first aspect of the invention, nucleic acid molecule of the second aspect of the invention or a construct comprising said nucleic acid molecule together with a pharmaceutically acceptable excipient.
  • the composition may contain an immunotherapeutic suitable for the treatment of cancer, particularly a cancer of one of the types specified above.
  • an immunotherapeutic suitable for the treatment of cancer particularly a cancer of one of the types specified above.
  • a method for the treatment of cancer comprising administering to a patient in need of such treatment effective amounts of a first agent selected from the group consisting of a peptide of the first aspect of the invention, nucleic acid molecule of the second aspect of the invention and a construct comprising said nucleic acid molecule; and a second agent which is an immunotherapeutic agent.
  • the cancer to be treated is suitably neuroblastoma but the method may also be used for the treatment of other types of cancer such as small cell lung carcinoma and colorectal cancer.
  • Peptides and related agents will be administered in different ways, such as intravenously, intraperitoneal ⁇ , intramuscularly, subcutaneously, topically or orally as a powder formulation or more suitably as a liquid formulation, as appropriate for the tumour type.
  • directed delivery into the supplying artery or even direct intrat ⁇ moural administration may be possible.
  • the immunotherapeutic agent is suitably chosen such that it is appropriate for use in the treatment of the particular type of cancer being targeted.
  • it may be an antibody, especially a monoclonal antibody and will bind specifically to cells of the targeted tumour.
  • FIG. 1 A 35bp positive responsive element from the CD59 promoter is essential for overexpression of the gene in neuroblastoma.
  • A Schematic presentation of the CD59 promoter fragments used in the EGFP-reporter assays. Four different fragments containing parts of exon 1 (solid box) along with variable portions of the 5 1 flanking region (represented by lines) were prepared and ligated into a promoterless pEGFP-1 vector upstream of the EGFP gene. The arrow points in the 3' direction.
  • B Kelly cells were transfected with the constructs shown in (A) and the expression of EGFP was assessed by flow cytometry. Values are mean ⁇ SD for three independent experiments.
  • C The 35bp sequence that differs between the -35 and -70 constructs and upregulates expression of the reporter EGFP gene.
  • FIG. 1 Expression of truncated REST in neuroblastoma cells correlates with high expression of CD59.
  • A RT-PCR analysis of expression of REST isoforms in different neuroblastoma cell lines and in primary neurons. Specific primers either annealed on each side of the inserted sequence or a nested reverse primer, were designed within the insert amplifying only the cDNA encoding the truncated REST isoform.
  • B Expression of CD59 on the cell surface of neuroblastoma cells was assessed by flow cytometry. The data are mean ⁇ SD of triplicates and representative of two experiments.
  • C RT-PCR analysis of expression of REST isoforms in clinical neuroblastoma samples (NT1 - NT10). GAPDH was monitored as an indicator for the quality of RNA prepared from clinical tissue samples.
  • EMSA with oligonucleotides labelled with biotin incubated with 5 ⁇ g of nuclear protein extracts from normal brain and neuroblastoma cell lines Kelly and
  • FIG. 4 Peptides derived from the DNA-binding domain of REST suppress expression of CD59.
  • the REST repressor protein is shown schematically (left hand-side), illustrating the eight zinc finger DNA binding domains and two repressor domains. Domains included in REST58 (110 amino acid residues, AA) and REST68 (includes 82AA from the REST plus the 7AA- long NLS) are shown.
  • RT-PCR analysis of expression of CD59 mRNA in IMR32 and Kelly cells transfected either with REST58, REST68, or an empty expression vector.
  • C Flow cytometry analysis of expression of CD59 on the surface of IMR32 and Kelly cells transfected as above.
  • D Flow cytometry analysis of expression of CD59 on the surface of NMB7, La-N-1 , La1-5S, SH5Y, SK-N-ER, SK-N-SH, and La1-55N 96h after cells were transfected either with REST68 or an empty expression vector. Bars represent mean ( ⁇ SD) from two independent measurements ( * , p ⁇ 0.001).
  • REST68 peptide suppresses expression of CD59 gene by blocking binding of transcriptional activators to the 35bp responsive element.
  • ChIP was performed on Kelly cells transfected with either REST68 expression construct or empty vector with antibodies against Sp1, AP2, CPBP, and REST, lmmunoprecipitation with non-immune rabbit IgG was carried out as a control for the assay background.
  • the pulled-down DNA was characterised for presence of the 35bp element by QPCR. Highest levels of binding for each transcription factor, regardless of the plasmid with which cells were transfected, were set as 100%. Values are mean ⁇ SD for two independent ChIP experiments each analysed in duplicate (*, p ⁇ 0.001).
  • FIG. 6 REST68 peptide sensitises neuroblastoma cells to C-mediated lysis.
  • Kelly, La1-5S, SK-N-ER, and NMB7 cells transfected either with REST68 expression construct (- ⁇ -) or empty vector (-A-) were tested for their sensitivity to C-dependent cytotoxicity triggered by anti-GD2 mAb.
  • Lysis assay with preincubation of both cell lines (REST68, - ⁇ -; empty vector, - ⁇ -) with CD59- blocking Fab fragment was carried out as a control. Values are mean ⁇ SD for three independent experiments.
  • Example 1 Identification of a 35bp Positive Regulatory Sequence within the CD59 Promoter in Neuroblastoma
  • Similar investigations have been carried out previously for human Burkitt lymphoma and chronic myelogenous leukemia using luciferase-reporter constructs (Holguin et al., 1996).
  • Stimulating protein 1 SP1 -64 to -50
  • Pleomorphic adenoma gene (PLAG) 1 -60 to -40
  • Stimulating protein 1 SP1 -57 to -35
  • Activator protein 2 (AP2) -55 to -39
  • Stimulating protein 1 Cell lines, patient samples and preparation of nuclear lysates Human neuroblastoma cell lines IMR32, SH5Y, Kelly, La-N-1 , La1-55N, SK-N- SH, La1-5S (European Collection of Animal Cell Cultures, Salisbury, UK), NMB7, and SK-N-ER (kind gift from Dr. P. Gasque, University of Ia Reunion, Saint Denis, lie de Ia Reunion) were maintained in RPM11640 with 10% heat- inactivated FCS, supplemented with glutamine, penicillin, and streptomycin (Invitrogen, Paisley, UK).
  • SP1 Stimulating protein 1
  • Neuroblastoma clinical samples (NT1 - NT10) were obtained via the CCLG Biological Studies Tumor Bank, UK (Study number: 2007 BS 08). Nuclear protein extracts were prepared from all neuroblastoma cell lines as described previously (Donev et al., 2003).
  • Expression constructs were prepared by ligating the CD59 promoter fragments into the pEGFP-1 vector (Clontech, United Kingdom). This promoter- less vector contains a cloning site immediately upstream of the EGFP reporter gene.
  • the promoter fragments were amplified from human genomic DNA using a common reverse primer containing restriction site (underlined) for Age I enzyme (GCACCGGTAAGATCCTCTTCCAGCCTCGA; SEQ ID NO: 9) and a series of forward primers with Kpn I restriction site (underlined):
  • the amplified fragments and the plasmid were digested with Age. I and Kpn I. After ligation into pEGFP-1 , the nucleotide sequence of the inserts was determined by sequencing to ensure that PCR artefacts had not been introduced.
  • the reporter constructs were transfected ⁇ into Kelly neuroblastoma cells using the Effectene reagent (Qiagen, Crawley, Westshire, United Kingdom).
  • Transfected cells were selected by inclusion of 400 ⁇ g/ml G418 (Clontech) in the culture medium. Cells were then analysed for expression of EGFP by flow cytometry.
  • REST is expressed as a truncated form in neuroblastoma tumours due to an insertion within the gene that introduces a stop codon (Palm et al., 1999). Recently it was suggested that expression of this truncated isoform plays a role in tumour progression (Coulson, 2005; Majumder, 2006). Taking into account that full-length REST may bind within the 35bp responsive element in the CD59 promoter, we addressed here the role of the expression of truncated REST in CD59 overexpression in neuroblastoma.
  • RT-PCR analysis of expression of REST using a primer pair that anneals on both sides of the inserted sequence showed that eight of nine studied neuroblastoma cell lines express predominantly the truncated isoform. We did not detect the insertion in primary neurons and IMR32 cells indicating that they expressed exclusively or predominantly full-length REST. However, when we carried out a nested RT-PCR with reverse primer within the REST insertion, the insertion was detected at low level in the IMR32 cells but not in primary neurons. Importantly, we found that high expression level of CD59 coincided with expression of the truncated REST isoform in the various cell types (Fig. 2B).
  • RT-PCR analysis of expression of REST was tested in ten clinical neuroblastoma samples (Fig. 2C). Truncated REST was present in nine of ten specimens. The sample without truncated REST (N1) contained only trace amounts of full-length REST. These findings demonstrate the clinical relevance of the switch to expression of truncated REST in neuroblastoma. Two of the tumors (NT6 and NT7) showed presence of an additional alternative REST transcript that is not further defined.
  • ESA electrophoretic mobility shift assay
  • Electrophoretic Mobility Shift Assay Biotinylated sense and antisense strands of the 35bp regulatory sequence ( Figure 1C) were purchased from Biomers.net GmbH (UIm, Germany). Oligonucleotides (200pmol each) were mixed in equimolar amounts in 50 ⁇ l of annealing buffer (5OmM KCI, 1.5mM MgCI 2 , Tris-HCI, pH 8.3), placed in a boiling water bath for 2 minutes, and allowed to cool slowly to room temperature.
  • annealing buffer 5OmM KCI, 1.5mM MgCI 2 , Tris-HCI, pH 8.3
  • the annealed DNA probe (l Opmol per reaction) was incubated with 5 ⁇ g of nuclear protein extracts from IMR32, Kelly, or normal human brain (Active Motif, Rixensart, Belgium) as previously described (Enukashvily et al., 2005). Each reaction contained 0.1 mg/ml poly(deoxyinosinicdeoxycytidylic acid) to block non-specific electrostatic interactions and 1 mg/ml salmon sperm DNA as a competitor (both purchased from Sigma-Aldrich). In some of the reactions, a rabbit polyclonal anti-REST (H-290) antibody raised against amino acids 1-290 of the protein (Santa Cruz Biotechnology, Santa Cruz, California, USA) was added to identify complexes containing the protein.
  • H-290 rabbit polyclonal anti-REST
  • This antibody recognises both the full-length and the truncated REST isoforms. Each reaction was separated in 6% polyacrylamide gels and transferred onto nitrocellulose membranes. DNA was detected using ExtrAvidin-HRP (Sigma-Aldrich) followed by chemiluminescence development (Bio-Rad, Herts, United Kingdom).
  • a siRNA sequence efficiently knocking-down expression of REST was designed into a short hairpin RNA expressing plasmid pLKO.1- TRC (Addgene Inc, Cambridge, MA, USA).
  • Sense and antisense oligonucleotides containing either the active sequence (GATGCACAAACTGTTCTTC; SEQ ID NO: 14) or the scrambled one (CATTCGCGTTTACGACTAA; SEQ ID NO: 15) were purchased from Biomers.net GmbH. Single stranded oligonucleotides were annealed as described above for the EMSA.
  • Transfected cells were then selected by adding of 1 ⁇ g/ml or 10 ⁇ g/ml of puromycin (Invitrogen) to the culture medium of IMR32 and Kelly cells, respectively, and cells were analysed for expression of REST, CD59 and GAPDH.
  • puromycin Invitrogen
  • REST binds within the 35bp responsive element to suppress expression of
  • REST58 comprises domain 5, which is responsible for nuclear localisation, and domains 6, 7 and 8 ( Figure 4A) that are essential for DNA binding.
  • REST68 domain 5 has been replaced by the classical nuclear localisation signal (NLS) (Nadler et al., 1997).
  • NLS nuclear localisation signal
  • CD59 several pDR2 ⁇ EF1 ⁇ -based constructs for expression in mammalian cells were designed. Sequence encoding REST58 was amplified from IMR32 cDNA using the following pair of primers: CGATCTAGAGCCACCATGTATAAATGTGAACTT (SEQ ID NO: 16) forward, and:
  • Forward and reverse primers contained restriction sites (underlined) for Xbal and Bam HI, respectively, which were used for cloning into the pDR2 ⁇ EF1 ⁇ vector after digestion with the same enzymes.
  • the forward primer also contains a Kozak sequence to improve RNA translation.
  • For the REST68 expression construct domain 5 from REST58 was replaced with a NLS.
  • the same reverse primer was used; however, to accommodate the long extension at the 5'-end, a series of five forward primers was designed, F1 to F5, that partially overlap, and five sequential amplifications, starting with F1 and finishing with F5, were performed.
  • the sequences of these primers were: F1 - GGGTGGTGGTTTTAAATGTGATCAGT (SEQ ID NO: 18);
  • F4 AGCCACCATGCCAAAAAAAACGTA (SEQ ID NO: 21); F5 - CGATCTAGAGCCACCATGCCAAAA (SEQ ID NO: 22).
  • F5 primer contained Xbal restriction site (underlined) and Kozak sequence. All constructs and products were sequenced to ensure their fidelity.
  • Kelly cells transfected either with the REST68 expression construct or the vector alone as a control were fixed for 10 minutes at room temperature in tissue culture medium containing 1% formaldehyde. All further steps of this assay have been described previously (Orlando and Paro, 1993). Chromatin was sonicated to produce DNA fragments in the range of 200 to 800 bp (electrophoretically determined in 1.5% agarose).
  • the lmmunoprecipitation was carried out either with rabbit polyclonal anti-Sp1 (Merck, Nottingham, United Kingdom), rabbit polyclonal anti-AP2 (Merck), sheep polyclonal anti-CPBP (R&D Systems Europe Ltd, Abingdon, United Kingdom), or a mixture of rabbit polyclonal anti-REST (H- 290) and goat polyclonal anti-REST (P-18) (Santa Cruz Biotechnology) raised against peptides mapping within the N-terminus and the internal region of the protein, respectively. Mixing both anti-REST antibodies was necessary to ensure recognition of both the truncated isoform of REST and the REST68 peptide.
  • Non-immune rabbit IgG was used as a control for the background of these experiments.
  • the naked co-immunoprecipitated DNAs were then used as templates in QPCR assays as described below. Statistical significance of the data was assessed by the Student's t test.
  • RNA from frozen patient samples and neuroblastoma cell lines was purified using the GenElute Mammalian Total RNA Miniprep kit (Sigma-Aldrich). Total RNA from normal primary neurons was obtained from TCS Cellworks (Buckingham, United Kingdom).
  • GenElute Mammalian Total RNA Miniprep kit Sigma-Aldrich
  • Total RNA from normal primary neurons was obtained from TCS Cellworks (Buckingham, United Kingdom).
  • a conventional RT-PCR was performed using either a pair of primers on each side of the mutated sequence or with a nested reverse primer within the insert (Coulson et al., 2000).
  • CD59-specific primers (TGCAATTTCAACGACGTCACA; SEQ ID NO: 25 - forward, and: GAAATGGAGTCACCAGCAGAAGA; SEQ ID NO: 26 - reverse) and a GAPDH specific primer pair (Coulson et al, 1999) as a control, were used.
  • cDNA were synthesised using TaqMan Reverse Transcription reagents (Applied Biosystems, Warrington, United Kingdom) and the amplification was carried out with Platinum Blue PCR SuperMix (Invitrogen).
  • Neuroblastoma cells transfected with either empty pDR2 ⁇ EF1 ⁇ vector or constructs expressing REST68 were suspended in RPMI 1640 culture medium with no FCS and transferred into 96-well plates (10 4 cells/well) with anti-GD2 monoclonal antibody, clone 14.2Ga (Chemicon, Chandlers Ford, Hampshire, United Kingdom) at a concentration of 10 ⁇ g/ml, which was previously shown to yield a maximum lysis effect at this conditions (Mujoo et al., 1987).
  • % Lysis [(measured LDH release - spontaneous release) / (maximum release - spontaneous release)] x 100 The experiment was replicated twice and data were analysed by Student's test.
  • CD59a is the primary regulator of membrane attack complex assembly in the mouse. J. Immunol. 173, 3684- 3692.
  • CD59 expressed on a tumor cell surface modulates decay-accelerating factor expression and enhances tumor growth in a rat model of human neuroblastoma. Cancer Res. 60, 3013-3018.
  • a splice variant of the neuron-restrictive silencer factor repressor is expressed in small cell lung cancer: 2 potential role in derepression of neuroendocrine genes and a useful clinical marker. Cancer Res. 60, 1840-1844.
  • the mouse complement regulator CD59b is significantly expressed only in testis and plays roles in sperm acrosome activation and motility. MoI. Immunol. 45, 534-542.
  • Gazouli M., Kokotas, S., Zoumpourlis, V., Zacharatos, P., Mariatos, G., Kletsas, D., Perunovic, B., Athanasiou, A., Kittas, C, and Gorgoulis, V.
  • LFA-3, CD58 lymphocyte function-associated antigen-3
  • CD20 levels determine the in vitro susceptibility to rituximab and complement of B-cell chronic lymphocytic leukemia: further regulation by CD55 and CD59. Blood 98, 3383-
  • NRSF Neuron-restrictive silencer factor
  • Disialoganglioside GD2 on human neuroblastoma cells target antigen for monoclonal antibody-mediated cytolysis and suppression of tumor growth.
  • NRSF neuron-restrictive silencer factor
  • Decay-accelerating factor (CD55) is expressed by neurons in response to chronic but not acute autoimmune central nervous system inflammation associated with complement activation. J. Immunol. 774, 2353-2365. Walport, M.J. (2001). Complement. First of two parts. N. Engl. J. Med.
  • a genetic screen for candidate tumor suppressors identifies REST. Cell 121, 837-848.

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Abstract

L'invention porte sur des peptides issus d'un facteur silenceur de restriction neurale qui modulent l'expression de CD59 et affectent ainsi la cytotoxicité du complément médiée par des anticorps dans des tumeurs.
PCT/GB2009/001374 2008-06-07 2009-06-01 Peptide WO2009147384A2 (fr)

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