WO2017076864A1 - Polythérapie à agent neutralisant ntn1 comprenant des médicaments inhibant une régulation épigénétique - Google Patents

Polythérapie à agent neutralisant ntn1 comprenant des médicaments inhibant une régulation épigénétique Download PDF

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Publication number
WO2017076864A1
WO2017076864A1 PCT/EP2016/076350 EP2016076350W WO2017076864A1 WO 2017076864 A1 WO2017076864 A1 WO 2017076864A1 EP 2016076350 W EP2016076350 W EP 2016076350W WO 2017076864 A1 WO2017076864 A1 WO 2017076864A1
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ntn1
composition
neutralizing agent
dapk1
netrin
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PCT/EP2016/076350
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English (en)
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Mélodie GRANDIN
Robert DANTE
Patrick Mehlen
Stéphane DEPIL
Jordan GUYON
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Netris Pharma
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A61K31/167—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the nitrogen of a carboxamide group directly attached to the aromatic ring, e.g. lidocaine, paracetamol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/18—Sulfonamides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A61K31/4045—Indole-alkylamines; Amides thereof, e.g. serotonin, melatonin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/15—Depsipeptides; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/179—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention relates to novel pharmaceutical or anticancerous compositions.
  • compositions may be useful in treating a cancer having a DNA methylation dependent loss of expression of NTN1 , UNC5H or DAPK1 .
  • the invention also relates to a method of treatment of such cancers. Background to the invention
  • dependence receptors that may constrain cancer progression.
  • NTN1 secreted netrin-1
  • NTN1 secreted netrin-1
  • an autocrine expression of NTN1 provides a similar tumor growth selective advantage by abolishing their dependency on NTN1 availability in the micro-environment (Llambi et al., 2001 ).
  • This gain of NTN1 may represent an opportunity for therapeutic intervention: disrupting the NTN1 binding to its receptors should induce apoptotic cell death. Proof of this concept has been obtained in vitro and in vivo using several cancer cells and animal models.
  • DAPK1 a serine threonine kinase responsible for UNC5H- induced apoptosis (Llambi et al., 2005; Guenebeaud et al., 2010), is downregulated in various cancers (Raval et al., 2007; Pulling et al., 2009; Kilinc et al., 2012).
  • DNMT1 inhibitors Decitabine (5'aza-2-deoxycytidine) and Azacytidine (5'azacytidine); these molecules are nucleoside analogs.
  • HDAC histone deacetylase
  • SAHA Vorinostat
  • Belinostat Belinostat
  • cyclic peptides such as Romidepsin.
  • US 8,530,445 discloses combination of a DNA methyltransferase inhibitor, e.g. azacitidine or decitabine, and 1 -(2-C- cyano-2-dioxy-3-D-arabino-pentofuranosyl)-N4-palmitoyl cytosine.
  • WO 02/067681 discloses combination of a DNA methylation inhibitor and an anti-neoplastic agent whose activity as an anti-neoplastic agent in vivo is adversely affected by aberrant DNA methylation.
  • the effect of drug combination is inherently unpredictable. There is often a propensity for one drug to partially or completely inhibit the effects of the other.
  • the present invention is based on the surprising observation that inhibition of DNA methylation by decitabine or azacitidine restores the pro-apoptotic machinery linked to the NTN1 dependence receptor signaling death pathway and that combining decitabine or azacitidine with a NTN1 neutralizing antibody empowers tumor cell death in vitro and tumor growth inhibition in mice.
  • the unexpected absence of any such antagonist interference is critical for clinical application and is remarkable in the present context where one of the drugs is supposed, in accordance with the invention, to reverse a selective advantage of some cancers based on inhibition of a pro-apoptotic pathway.
  • the combination according to the invention allows reversion of inhibition of the pro-apoptotic pathway linked to NTN1 receptors.
  • the surprising nature of this observation is unexpected from the prior art. This opens the way to combination therapy in human and animal, especially to induce NTN1 dependence receptors mediated cancer cell apoptosis.
  • the invention provides a combination comprising at least one epigenetic drug (or drug inhibiting epigenetic control) and at least one NTN1 neutralizing agent.
  • Said epigenetic drug may be an inhibitor of DNA methylation, such as a DNA methyltransferase (DNMT) inhibitor, preferably a DNMT1 inhibitor or a mixture of DNMT1 inhibitors, or an inhibitor having histone inhibitory activity, such as an HDAC inhibitor, or a mixture of HDAC inhibitors, or a mixture of DNMT1 and HDAC inhibitors.
  • DNMT DNA methyltransferase
  • the NTN1 neutralizing agent may be especially an antibody binding to NTN1 .
  • a second aspect provides a pharmaceutical composition
  • a pharmaceutical composition comprising a combination according to the invention admixed with a pharmaceutically acceptable carrier, diluent or excipient.
  • a third aspect relates to a combination according to the invention for use in the treatment of cancer.
  • a fourth aspect relates to the use of a combination according to the invention in the preparation of a medicament for treatment of cancer.
  • a fifth aspect relates to a pharmaceutical composition
  • a pharmaceutical composition comprising at least one epigenetic drug, especially DNA methyltransferase and/or HDAC inhibitor, and at least one NTN1 neutralizing agent, as a combined preparation for simultaneous, sequential or separate use in therapy, especially for treatment of cancer.
  • a sixth aspect relates to a method for treating a cancer, said method comprising simultaneously, sequentially or separately administering a sufficient amount of at least one epigenetic drug, especially DNA methyltransferase and/or HDAC inhibitor, and of at least one NTN1 neutralizing agent, to a subject.
  • a seventh aspect relates to an epigenetic drug, especially a DNA methyltransferase or HDAC inhibitor, for use, or the use of an epigenetic drug, especially a DNA methyltransferase or HDAC inhibitor, in the preparation of a medicament for use, in the treatment of a cancer, wherein said treatment comprises simultaneously, sequentially or separately administering a NTN1 neutralizing agent, to a subject.
  • An eighth aspect relates to the use of an epigenetic drug, especially a DNA methyltransferase or HDAC inhibitor, for use, or the use of an epigenetic drug, especially a DNA methyltransferase or HDAC inhibitor, in the preparation of a medicament for use, in the treatment of a cancer, wherein the epigenetic drug is for use in combination therapy with a NTN1 neutralizing agent.
  • a ninth aspect relates to a NTN1 neutralizing antibody for use, or the use of a NTN1 neutralizing agent in the preparation of a medicament for use, in the treatment of a cancer, wherein said treatment comprises simultaneously, sequentially or separately administering an epigenetic drug, especially a DNA methyltransferase or HDAC inhibitor, to a subject.
  • an epigenetic drug especially a DNA methyltransferase or HDAC inhibitor
  • An tenth aspect relates to the use of a NTN1 neutralizing agent for use, or the use of NTN1 neutralizing agent in the preparation of a medicament for use, in the treatment of a cancer, wherein the NTN1 neutralizing agent is for use in combination therapy with an epigenetic drug, especially a DNA methyltransferase or HDAC inhibitor.
  • An eleven aspect of the invention is the use of a NTN1 neutralizing agent for use, or the use of NTN1 neutralizing agent, to prime tumors to netrin-1 addiction.
  • NTN1 is up-regulated, and through an autocrine secretion, blocks apoptosis through its so-called dependence receptors DCC and UNC5H and thus promotes tumor progression.
  • the selective inhibition of this dependence receptor death pathway relies on the silencing of either the receptors or the downstream pro-apoptotic effector proteins.
  • the inventors show here that a large fraction of human breast tumors exhibits simultaneous DNA methylation-dependent loss of expression of NTN1 and of DAPK1 , a serine threonine kinase known to transduce the NTN1 dependence receptor pro-apoptotic pathway.
  • NTN1 -low cancer cells restores the expression of both NTN1 and DAPK1 . Consequently, the combination of decitabine with NTN1 silencing strategies or with an anti-NTN1 neutralizing antibody potentiates tumor cell death and efficiently blocks tumor growth in different animal models. Thus, combining DNA methylation inhibitors with NTN1 neutralizing agents appears to be a valuable strategy for combating cancer.
  • the combination of an epigenetic drug and a NTN1 inhibitor is in particular for use on cancers which exhibits DNA methylation-dependent loss of expression of NTN1 , or of UNC5H (especially UNC5B) or of DAPK1 , or simultaneous DNA methylation- dependent loss of expression of any two or three of NTN1 , UNC5H (especially UNC5B) and DAPK1 .
  • the combination is for use on every cancer expressing one or several NTN1 receptors such as DCC and/or UNC5H, especially UNC5B, wherein there is a phenomena of escaping NTN1 receptor mediated apoptosis.
  • NTN1 receptors such as DCC and/or UNC5H, especially UNC5B
  • an epigenetic drug such as a DNA methyltransferase inhibitor or an HDAC inhibitor
  • a NTN1 neutralizing agent allows reversion of inhibition of the pro-apoptotic pathway linked to NTN1 receptors in cancers having DNA methylation-dependent loss of expression of NTN1 and of DAPK1 .
  • the combination allows efficient NTN1 receptor mediated cell death or apoptosis of cancer cells in cancers in which NTN1 is up-regulated and blocks apoptosis through its dependence receptors.
  • a first group of cancer types concerned by the invention is one whose signature is to exhibit simultaneous DNA methylation-dependent loss of expression of NTN1 , or of UNC5H (especially UNC5B) or of DAPK1 , or simultaneous DNA methylation-dependent loss of expression of any two or three of NTN1 , UNC5H (especially UNC5B) and DAPK1 .
  • a method of determining a cancer which exhibits simultaneous DNA methylation- dependent loss of expression of NTN1 , UNC5H (especially UNC5B), and/or of DAPK1 can include analysis of the methylome using routinely based methods, or a more specific method investigating direct methylation of the promoters of NTN1 , UNC5H (especially UNC5B) and DAPK as exemplified (see Figure 1 A and corresponding description).
  • Some embodiments of such cancers include breast cancer, non-small cell lung cancer, aggressive neuroblastoma, pancreatic adenocarcinoma, primary melanoma, melanoma metastasis, ovarian cancers, glioblastoma, acute myeloid leukaemia, chronic lymphocytic leukaemia, aggressive B-cell lymphoma, sarcoma, renal adenocarcinoma, head and neck cancers, testicular cancers (e.g. embryonal carcinoma, teratoma, yolk sac tumors), kidney cancers, stomach cancers, uterus cancers.
  • testicular cancers e.g. embryonal carcinoma, teratoma, yolk sac tumors
  • a second group of cancer types concerned by the invention is one whose signature is NTN1 blocks apoptosis through its dependence receptors, this being generally due to up- regulation of NTN1 by the cancer cells or the stroma.
  • Methods of determining whether cells express NTN1 dependence receptors DCC and/or UNC5 on the surface and shows significant presence of NTN1 or up-regulation of NTN1 gene expression are well known in the art and comprise, but are not limited to, IHC (Immunohistochemistry) of FACS (Fluorescence activated cell sorting), quantitative PCR (e.g.
  • NTN1 overexpression may thus be measured through RT-PCR using suitable primers as those disclosed and provided herein, with respect to normal tissue or to a similar cancer which does not overexpress NTN1 .
  • Some embodiments of such cancers include metastatic breast cancer, non-small cell lung cancer, aggressive neuroblastoma, pancreatic adenocarcinoma, primary melanoma, melanoma metastasis, ovarian cancers, glioblastoma, acute myeloid leukaemia, chronic lymphocytic leukaemia, aggressive B-cell lymphoma, sarcoma, renal adenocarcinoma, head and neck cancers, testicular cancers (e.g. embryonal carcinoma, teratoma, yolk sac tumors), kidney cancers, stomach cancers, uterus cancers.
  • testicular cancers e.g. embryonal carcinoma, teratoma, yolk sac tumors
  • the NTN1 neutralizing agents are NTN1 neutralizing agents.
  • the NTN1 neutralizing agent is a drug which interferes with the netrin-1 ability to interact with a netrin-1 receptor, preferably blocks the binding between netrin-1 and its receptor, or which interferes with the ability of netrin-1 to induce dimerisation or multimerization of netrin-1 receptor.
  • the person skilled in the art may refer to WO2007/099133, incorporated herein by reference, which discloses interference between netrin-1 and its receptors, either a decrease or an inhibition of interaction or binding between netrin-1 and receptors, or a decrease or an inhibition of the ability of netrin-1 to induce dimerization or multimerization of netrin-1 receptor, whereby netrin-1 receptors-induced apoptosis is promoted.
  • the NTN1 neutralizing agent is a small interfering RNA or siRNA which is a double stranded RNA (dsRNA) (that may have namely from 10 to 50 nucleotides in length) and which reduces expression of the gene coding for netrin-1 .
  • dsRNA double stranded RNA
  • Portions of the first strand are complementary to the target gene, i.e. it has sufficient complementarity to hybridize to the target gene, for example there is at least 80% identity to the target gene or to a portion thereof.
  • AP human Netrin-1 mRNA sequence accession number: NM 004822.
  • the NTN1 neutralizing agent is a molecule (e.g. antibody, polypeptide, small molecule, and the like) which binds to netrin-1 and netrin-1 is rendered unable to bind to its receptors or to induce dimerization/multimerization of the netrin-1 receptors, especially DCC and/or UNC5.
  • the NTN1 neutralizing agent is a molecule (e.g. antibody, polypeptide, small molecule, and the like) which binds to a netrin-1 receptor, this binding allowing NTN1 receptor- mediated apoptosis.
  • this NTN1 neutralizing agent inhibits NTN1 binding to receptor or dimerization/ multimerization of the receptor.
  • the netrin- 1 receptors may be in particular DCC, UNC5A, UNC5B, UNC5C or UNC5D.
  • the NTN1 neutralizing agent is an antibody binding to netrin-1 . It is preferably a polyclonal or monoclonal antibody specifically binding to netrin-1 .
  • a NTN1 polyclonal antibody may, inter alia, be obtained by immunizing an animal such as a rabbit, a mouse and the like with the aid of the selected amino acid sequence, collecting and then depleting the antiserum obtained on, for example, an immunoadsorbent containing the receptor according to methods known per se to a person skilled in the art.
  • netrin-1 amino acid sequence is as depicted on SEQ ID NO:1 and netrin-1 may be used in whole or in part to design antibodies.
  • monoclonal antibodies may be obtained according to the conventional method of lymphocyte fusion and hybridoma culture described by Kohler and Milstein, (Nature, 1975, 256(5517): 495-7). Other methods for preparing monoclonal antibodies are also known (Harlow et al., ed., 1988 "Antibodies: a laboratory manual”).
  • the monoclonal antibodies may be prepared by immunizing a mammal (for example a mouse, a rat, a rabbit or even a human being, and the like) and using the lymphocyte fusion technique leading to hybridoma (Kohler and Milstein, 1975). Alternative techniques to this customary technique exist.
  • the NTN1 neutralizing antibody is one disclosed in
  • WO2015/104360 which reference is incorporated herein by reference. It is an antibody that specifically binds to a NTN1 epitope or polypeptide having the amino acid sequence SEQ ID NO: 3 or 35 or a variant thereof. These antibodies have the property of binding to NTN1 and induce cell death or apoptosis of a tumor cell via an UNC5 or DCC receptor. These antibodies are preferably monoclonal antibodies. Various forms of antibodies (including fragments and combination thereof) will be described later herein.
  • the antibody comprises a CDR1 -H of sequence SEQ ID NO: 5, a CDR2-H of sequence SEQ ID NO: 6, a CDR3-H of sequence SEQ ID NO: 7, a CDR1 -L of sequence SEQ ID NO: 8, a CDR2-L of sequence YAS and a CDR3-L of sequence SEQ ID NO: 9.
  • the antibody comprises a CDR1 -H of sequence SEQ ID NO: 28, a CDR2-H of sequence SEQ ID NO: 29, a CDR3-H of sequence SEQ ID NO: 30, a CDR1 -L of sequence SEQ ID NO: 31 , a CDR2-L of sequence SEQ ID NO:32 and a CDR3-L of sequence SEQ ID NO: 9.
  • the polypeptide or antibody of the invention comprises an amino acid sequence SEQ ID NO: 10, 1 1 , 12 or 13. Typically, it comprises both sequences SEQ ID NO: 10 and 1 1 , or SEQ ID NO: 12 and 13.
  • the antibody is humanized. Preferably it comprises an amino acid sequence selected from the group of SEQ ID NO: 14 to 19 (VL) and/or from the group of SEQ ID NO: 20 to 27 (VH) . Typically, the antibody is humanized and comprises an amino acid sequence selected from the group of SEQ ID NO: 14 to 19 and an amino acid sequence selected from the group of SEQ ID NO: 20 to 27.
  • HUM monoclonal antibodies having variable human framework regions. In a typical embodiment, HUM03 is used.
  • the table gives also a reference for the CH and CL of a human lgG1 .
  • this drug is a compound comprising an extracellular domain of a netrin-1 receptor or a fragment of said extracellular domain.
  • the amino acid sequence of the extracellular domain of a netrin-1 receptor or a fragment of said extracellular domain are given in UniProt Sequence ID [extracellular domain position range]: UNC5A : Q6ZN44 [aas 26-306, or fragment 34-240]; UNC5B : Q8IZJ1 [aas 27-377 or fragment 29- 244]; UNC5C : 095185 [aas 41 -380 or fragment 61 -258]; UNC5D : Q6UXZ4 [aas 33-379]; DCC : P43146 [aas 26-1097].
  • the netrin-1 receptors may be DCC, UNC5A, UNC5B, UNC5C or UNC5D.
  • the extracellular domain or part thereof is bound to an antibody Fc part.
  • the Fc part is the Fc or part thereof of a human IgG.
  • the human IgG may be namely lgG1 , lgG2A, lgG2B, lgG3.
  • the IgG is lgG1 .
  • the fusion protein is single chain, which means that the protein is made of a DCC or a UNC5 fragment comprising or constituted of respectively the fourth or fifth fibronectin-like domain of DCC or the two Ig-like domains of UNC5 and of a peptidic or protein sequence improving the pharmaceutical parameters of the compound.
  • the fusion protein is double chain, which means that the fusion protein is made of two chains each comprising or constituted of respectively the fourth or fifth fibronectin-like domain of DCC or the two Ig-like domains of UNC5 and of an antibody Fc part, wherein both chains are linked together, preferably by one or more, e.g. two, disulfide bonds.
  • the drug comprises the fifth fibronectin domain (Fn5 or 5Fbn) of DCC.
  • the drug comprises a DCC-fusion protein comprising this Fn5 fused to an antibody Fc part.
  • the Fc part is the Fc or part thereof of a human IgG.
  • the human IgG may be namely lgG1 , lgG2A, lgG2B, lgG3.
  • the IgG is lgG1 .
  • the DCC gene is available for example from NCBI, under ID 1630 (as updated on July 14, 2012), it encodes the DCC receptor protein as Uniprot P43146, updated July 1 1 , 2012.
  • a DCC-fusion protein useful in the invention and comprising the Fn5 is described in WO2012025618, incorporated herein by reference.
  • the fusion protein has the amino acid sequence SEQ ID NO: 2, 3 or 4 in WO2012025618.
  • the fusion protein is encoded by the DNA sequence SEQ ID NO: 1 in WO2012025618.
  • Other examples of fusion proteins comprising the Fn5 are the DCC-5- fibronectin fusion protein with Glutathione-S-transferase (DCC-5Fbn-GST) described in WO2007099133.
  • the drug comprises the two Ig-like domains of a UNC5.
  • the drug comprises an UNC5-fusion protein comprising the two Ig-like domains of a UNC5 fused to an antibody Fc part.
  • the human IgG may be namely lgG1 , lgG2A, lgG2B, lgG3.
  • the IgG is lgG1 .
  • UNC5 is UNC5A.
  • UNC5 is UNC5B.
  • UNC5 is UNC5C.
  • UNC5D UNC5D.
  • the UNC5A protein in UNC5A-fusion comprises or consists of the amino acids 20 to 217 of SEQ ID NO: 1 in WO2014/041088, which document is incorporated herein by reference.
  • This fusion protein may further comprise the lgG1 Fc comprising or consisting of amino acids 220 to 446 of this SEQ ID NO: 1 .
  • This Fc is fused to the UNC5A protein, for example through a linker, such as GT.
  • the present invention relates to an UNC5A-fusion of UNC5A protein comprising or consisting of the amino acid sequence of this SEQ ID NO: 1 : Kappa2 signal peptide sequence: aas 1 to 19; Ig-like domains of UNC5A: aas 20 to 217; Linker: aas 218-219; Human lgG1 Fc: aas 220 to 446.
  • the mature fusion protein does not comprise the Kappa2 signal peptide sequence.
  • the fusion protein is double chain.
  • the present invention also encompasses variant sequences having a percentage of identity which is equal or more than 90%, preferably than 96, 95, 94, 93, 92 or 91 %, on the whole length of the 20-217 amino acid sequence, or of amino acids 20-446 of this SEQ ID NO: 1 .
  • Amino acid substitutions may for example occur at one or several of positions 9, 72, 74, 87, 144, 164, 170, 193 and/or 210 on the whole length of the 20-217 amino acid sequence, or of this SEQ ID NO: 1 .
  • the UNC5B protein in UNC5B-fusion comprises or consists of the amino acids 20 to 215 of SEQ ID NO: 2 in WO2014/041088.
  • This fusion protein may further comprise the lgG1 Fc comprising or consisting of amino acids 218 to 444 of this SEQ ID NO: 2.
  • This Fc is fused to the UNC5A protein, for example through a linker, such as GT.
  • the present invention relates to an UNC5B-fusion of UNC5B protein comprising or consisting of the amino acid sequence of this SEQ ID NO: 2: Kappa2 signal peptide sequence: aas 1 to 19; Ig-like domains of UNC5B: aas 20 to 215; Linker: aas 216- 217; Human lgG1 Fc: aas 218 to 444.
  • the mature fusion protein does not comprise the Kappa2 signal peptide sequence.
  • the fusion protein is double chain.
  • the present invention encompasses variant sequences having a percentage of identity which is equal or more than 90%, preferably than 96, 95, 94, 93, 92 or 91 %, on the whole length of the 20-215 amino acid sequence, or of amino acids 20-444 of this SEQ ID NO: 2.
  • Amino acid substitutions may for example occur at one or several of positions 29, 74, 100, 109, 1 13, 146, 149, 155, 172, 184, 189, 201 , 213 and/or 214 on the whole length of the 20-215 amino acid sequence, or of this SEQ ID NO: 2.
  • the UNC5C protein in UNC5C-fusion comprises or consists of the amino acids 20 to 217 of SEQ ID NO: 3 in WO2014/041088.
  • This fusion protein may further comprise the lgG1 Fc comprising or consisting of amino acids 220 to 446 of this SEQ ID NO: 3.
  • This Fc is fused to the UNC5A protein, for example through a linker, such as GT.
  • the present invention relates to an UNC5C-fusion of UNC5C protein comprising or consisting of the amino acid sequence of this SEQ ID NO: 3: Kappa2 signal peptide sequence: aas 1 to 19; Ig-like domains of UNC5C: aas 20 to 217; Linker: aas 218-219; Human lgG1 Fc: aas 220 to 446.
  • the mature fusion protein does not comprise the Kappa2 signal peptide sequence.
  • the fusion protein is double chain.
  • the present invention encompasses variant sequences having a percentage of identity which is equal or more than 90%, preferably than 96, 95, 94, 93, 92 or 91 %, on the whole length of the 20-217 amino acid sequence, or of amino acids 20-446 of this SEQ ID NO: 3.
  • Amino acid substitutions may for example occur at one or several of positions 33, 66, 109, 129, 136, 178, 189 and/or 21 1 on the whole length of the 20-217 amino acid sequence, or of this SEQ ID NO: 3.
  • the UNC5D protein in UNC5D-fusion comprises or consists of the amino acids 20 to 217 of SEQ ID NO: 4 in WO2014/041088.
  • This fusion protein may further comprise the lgG1 Fc comprising or consisting of amino acids 220 to 446 of this SEQ ID NO: 4.
  • This Fc is fused to the UNC5A protein, for example through a linker, such as GT.
  • the present invention relates to an UNC5D-fusion of UNC5D protein comprising or consisting of the amino acid sequence of this SEQ ID NO: 4: Kappa2 signal peptide sequence: aas 1 to 19; Ig-like domains of UNC5D: aas 20 to 217; Linker: aas 218-219; Human lgG1 Fc: aas 220 to 446.
  • the mature fusion protein does not comprise the Kappa2 signal peptide sequence.
  • the fusion protein is double chain.
  • the present invention encompasses variant sequences having a percentage of identity which is equal or more than 90%, preferably than 96, 95, 94, 93, 92 or 91 %, on the whole length of the 20-217 amino acid sequence, or of amino acids 20-446 of this SEQ ID NO: 4.
  • Amino acid substitutions may for example occur at one or several of positions 38, 79, 80, 1 15, 131 , 178, 186, 201 and/or 212 on the whole length of the 20-217 amino acid sequence, or of this SEQ ID NO: 4.
  • the invention may provide for administration of a nucleic acid encoding those polypeptides, rather than the polypeptide itself.
  • Vectors able to express the polypeptide in the patient may be used, as it is usual.
  • the nucleic acid molecule of the present invention may be cloned into a vector.
  • the person skilled in the art may refer to WO2007/099133 and WO2014/041088 which describe vectors and methods of preparing vectors and their use, which can be used in carrying out the present invention.
  • the term "vector” as used herein particularly refers to plasmids, cosmids, viruses, bacteriophages and other vectors commonly used in genetic engineering.
  • these vectors are suitable for the transformation of cells, eukaryotic cells like fungal cells, cells of microorganisms such as yeast or prokaryotic cells.
  • such vectors are suitable for stable transformation of bacterial cells, for example to transcribe the nucleic acid molecule of the present invention.
  • the vector may be pUC18.
  • WO2007/099133 discloses vectors expressing fusion proteins based on DCC, such as the vectors identified as 7800 and 7809 in Example 1 of WO2007/099133.
  • the present invention thus provides for a vector encoding the fusion protein of SEQ ID NO: 2, 3 or 4 in WO2012025618, or for a vector the DNA sequence SEQ ID NO: 1 in WO2012025618, for the DCC fusion protein.
  • WO2014/041088 also provides for a vector such as pUC18 containing a nucleic acid molecule of the present invention coding for a fusion protein as described and provided herein.
  • the present invention therefore relates to a vector such a pUC18 containing a nucleic acid molecule encoding the amino acids 20-217 and 220-446 of SEQ ID NO: 1 fused together, or the sequence SEQ ID NO:1 ; the amino acids 20-215 and 218-444 of SEQ ID NO: 2 fused together, or the sequence SEQ ID NO: 2; the amino acids 20-217 and 220-446 of SEQ ID NO: 3 fused together, or the sequence SEQ ID NO:3; or the amino acids 20-217 and 220-446 of SEQ ID NO: 4 fused together, or the sequence SEQ ID NO:4.
  • the present invention provides for a vector such as pUC18 containing a nucleic acid molecule comprising the nucleotide sequence 73-666 of SEQ ID NO : 5, or 73- 660 of SEQ ID NO : 6, or 73-666 of SEQ ID NO : 7 or 73-666 of SEQ ID NO : 8.
  • These vectors also comprise the nucleotide sequence of SEQ ID NO : 9, particularly nucleotides 7- 693.
  • the vector may be capable of expressing said nucleic acid molecule in a eukaryotic host cell.
  • SEQ ID NO: refer to those disclosed in WO2014/041088.
  • the polypeptides intended to be placed in a composition according to the invention are preferably produced by recombinant means.
  • the protein expression is in eukaryotic cell with subsequent isolation of the polypeptide and usually purification to a pharmaceutically acceptable purity.
  • nucleic acids encoding the protein thereof are inserted into expression vectors by standard methods. Expression is performed in appropriate stable eukaryotic host cells like CHO cells, NS0 cells, SP2/0 cells, HEK293 cells, COS cells, and the protein is recovered from the cells (supernatant or cells after lysis). HEK293 cells appeared to be very suitable for this aim and forms a particular embodiment.
  • the nucleic acid molecule of the present invention and/or the vector into which the polynucleotide described herein is cloned may be transduced, transformed or transfected or otherwise introduced into a host cell.
  • the host cell is a eukaryotic or a prokaryotic cell, preferably a eukaryotic cell.
  • the host cell is a mammalian cell.
  • the host cell described herein is intended to be particularly useful for generating the UNC5-fusion proteins described and provided in the present invention.
  • the host cell described hereinabove may be a prokaryotic or eukaryotic cell, preferably a eukaryotic cell, comprising a nucleic acid molecule provided in the present invention or the vector described herein or a cell derived from such a cell and containing the nucleic acid molecule or the vector described herein.
  • the host cell comprises, i.e. is genetically modified with the nucleic acid molecule of the present invention or the vector described herein in such a way that it contains the nucleic acid molecule of the present invention integrated into the genome.
  • such host cell described herein may be a human, yeast, or fungus cell.
  • the host cell is capable to transcribe the nucleic acid molecule of the present invention.
  • An overview of examples of different corresponding expression systems to be used for generating the host cell described herein is for instance contained in Methods in Enzymology 153 (1987), 385-516, in Bitter (Methods in Enzymology 153 (1987), 516-544), in Sawers (Applied Microbiology and Biotechnology 46 (1996), Billman-Jacobe (Current Opinion in Biotechnology 7 (1996), 500-4), Hockney (Trends in Biotechnology 12 (1994), 456-463), and in Griffiths (Methods in Molecular Biology 75 (1997), 427-440).
  • the transformation or genetically engineering of the host cell with a nucleic acid molecule of the present invention or vector described herein can be carried out by standard methods, as for instance described in Sambrook and Russell (2001 ), Molecular Cloning : A Laboratory Manual, CHS Press, Cold Spring Harbor, NY USA ; Methods in Yeast Genetics, A Laboratory Course Manual, Cold Spring Harbor Laboratory Press, 1990.
  • the host cell comprising the nucleic acid molecule provided herein or a vector described herein may be a HEK293 cell or a HEK293-Freestyle cell (Human embryonic kidney cell line 293, Invitrogen).
  • the present invention thus provides for a method for producing the DCC and UNC5-fusion proteins as provided and described herein. This method comprises the steps of expressing a nucleic acid molecule as provided and described herein in a suitable host cell, especially as described herein, and recovering the DCC or UNC5-fusion protein from said cell or the cell culture supernatant.
  • the DNA methyltransferase inhibitor (or DNA methylation inhibitor) is a nucleoside analogue, in particular a cytosine analogue such as azacitidine and decitabine.
  • a nucleoside analogue such as azacitidine and decitabine.
  • cytosine analogue such as azacitidine and decitabine.
  • the nucleoside analogue is incorporated into the DNA and then form a suicidal covalent complex with the DNA methyltransferases.
  • Various embodiments of existing nucleoside analogues (refer to US 8,530,445 and J. Fahy et al., Expert Opin. Ther.
  • Patents 2012, 22(12):1427-1442, both incorporated herein by reference) are 5-azacytidine (azacitidine; trade name Vidaza); 5-aza-2'-deoxycytidine (decitabine; trade name Dacogen); Zebularine; 5,6-dihydro-5-azacitidine; 5-fluoro-2'-deoxycitidine; pro-drugs of 5-azacytidine, such as NPEOC-DAC, a 5-azacytidine derivative in which the N4 amino function is protected by a 2-(p-nitrophenyl)ethoxycarbonyl group that is removed by the cellular carboxylesterases; CP-4200, an elaidic acid ester of azacitidine; pro-drugs of decitabine, such as decitabine-p-deoxyguanosine (SGI-1 10).
  • NPEOC-DAC a 5-azacytidine derivative in which the N4 amino function is protected by a 2-(p-nitrophenyl)eth
  • the DNA methyltransferase inhibitor is azacitidine-, including a pro-drug or derivative thereof having similar effect than azacitidine.
  • the DNA methyltransferase inhibitor is decitabine, including a pro-drug or derivative thereof having similar effect than decitabine.
  • the DNA methyltransferase inhibitor (or DNA methylation inhibitor) is a non-nucleoside analogue; refer also to US 8,530,445 and J. Fahy et al.).
  • This compound may be selected e.g. from procainamide, procaine, hydralazine, ((-)- epigallocatechin-3-gallate (EGCG), RG108 also known as N-phthalyl-1 -tryptophan, SGI- 1027.
  • the HDAC inhibitor is an hydroxamic acid having histone inhibitory activity, e.g. vorinostat (SAHA), trichostatine A, panobinostat or belinostat, or is a cyclic peptide having histone inhibitory activity, such as Romidepsin.
  • SAHA vorinostat
  • trichostatine A panobinostat
  • belinostat or is a cyclic peptide having histone inhibitory activity, such as Romidepsin.
  • a sequence "at least 85% identical to a reference sequence” is a sequence having, on its entire length, 85%, or more, in particular 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with the entire length of the reference sequence.
  • a percentage of "sequence identity" may be determined by comparing the two sequences, optimally aligned over a comparison window, wherein the portion of the polypeptide sequence in the comparison window may comprise additions or deletions (i.e. gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • Optimal alignment of sequences for comparison is conducted by global pairwise alignment, e.g. using the algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48: 443.
  • a “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain group with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein.
  • Examples of groups of amino acids that have side chains with similar chemical properties include 1 ) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine and methionine.
  • Conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine- tryptophane, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.
  • an “antibody” may be a natural or conventional antibody in which two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond.
  • Each chain contains distinct sequence domains.
  • the light chain includes two domains or regions, a variable domain (VL) and a constant domain (CL).
  • the heavy chain includes four domains, a variable domain (VH) and three constant domains (CH1 , CH2 and CH3, collectively referred to as CH).
  • variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen.
  • the constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR).
  • the Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain.
  • the specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant.
  • Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from non- hypervariable or framework regions (FR) influence the overall domain structure and hence the combining site.
  • CDRs refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site.
  • the light and heavy chains of an immunoglobulin each have three CDRs, designated CDR1 -L, CDR2-L, CDR3-L and CDR1 -H, CDR2-H, CDR3-H, respectively.
  • a conventional antibody antigen-binding site therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region.
  • FRs Framework Regions
  • the light and heavy chains of an immunoglobulin each have four FRs, designated FR1 -L, FR2-L, FR3-L, FR4-L, and FR1 -H, FR2-H, FR3-H, FR4-H, respectively.
  • a "human framework region” is a framework region that is substantially identical (about 85%, or more, in particular 90%, 95%, 97%, 99% or 100%) to the framework region of a naturally occurring human antibody.
  • CDR/FR definition in an immunoglobulin light or heavy chain is to be determined based on IMGT definition (Lefranc et al. (2003) Dev Comp Immunol. 27(1 ):55-77; www.imgt.org).
  • antibody denotes conventional antibodies and fragments thereof, as well as single domain antibodies and fragments thereof, in particular variable heavy chain of single domain antibodies, and chimeric, humanized, bispecific or multispecific antibodies.
  • antibody or immunoglobulin also includes "single domain antibodies" which have been more recently described and which are antibodies whose complementary determining regions are part of a single domain polypeptide.
  • single domain antibodies include heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional four-chain antibodies, engineered single domain antibodies.
  • Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, goat, rabbit and bovine.
  • Single domain antibodies may be naturally occurring single domain antibodies known as heavy chain antibody devoid of light chains.
  • Camelidae species for example camel, dromedary, llama, alpaca and guanaco, produce heavy chain antibodies naturally devoid of light chain.
  • Camelid heavy chain antibodies also lack the CH1 domain.
  • VHH variable heavy chain of these single domain antibodies devoid of light chains
  • VHHs Similar to conventional VH domains, VHHs contain four FRs and three CDRs.
  • Nanobodies have advantages over conventional antibodies: they are about ten times smaller than IgG molecules, and as a consequence properly folded functional nanobodies can be produced by in vitro expression while achieving high yield. Furthermore, nanobodies are very stable, and resistant to the action of proteases. The properties and production of nanobodies have been reviewed by Harmsen and De Haard (Harmsen and De Haard (2007) Appl. Microbiol. Biotechnol. 77:13-22).
  • monoclonal antibody refers to an antibody molecule of a single amino acid composition that is directed against a specific antigen, and is not to be construed as requiring production of the antibody by any particular method.
  • a monoclonal antibody may be produced by a single clone of B cells or hybridoma, but may also be recombinant, i.e. produced by protein engineering.
  • “Fragments” of (conventional) antibodies comprise a portion of an intact antibody, in particular the antigen binding region or variable region of the intact antibody.
  • antibody fragments include Fv, Fab, F(ab') 2 , Fab', dsFv, (dsFv) 2 , scFv, sc(Fv) 2 , diabodies, bispecific and multispecific antibodies formed from antibody fragments.
  • a fragment of a conventional antibody may also be a single domain antibody, such as a heavy chain antibody or VHH.
  • Fab denotes an antibody fragment having a molecular weight of about 50,000 Da and antigen binding activity, in which about a half of the N-terminal side of H chain and the entire L chain, among fragments obtained by treating IgG with a protease, papaine, are bound together through a disulfide bond.
  • F(abV' refers to an antibody fragment having a molecular weight of about 100,000 Da and antigen binding activity, which is slightly larger than the Fab bound via a disulfide bond of the hinge region, among fragments obtained by treating IgG with a protease, pepsin.
  • a single chain Fv (“scFv”) polypeptide is a covalently linked VH::VL heterodimer which is usually expressed from a gene fusion including VH and VL encoding genes linked by a peptide-encoding linker.
  • the human scFv fragment of the invention includes CDRs that are held in appropriate conformation, in particular by using gene recombination techniques.
  • Divalent and multivalent antibody fragments can form either spontaneously by association of monovalent scFvs, or can be generated by coupling monovalent scFvs by a peptide linker, such as divalent sc(Fv) 2 .
  • dsFv is a VH::VL heterodimer stabilized by a disulphide bond.
  • BsAb denotes an antibody which combines the antigen-binding sites of two antibodies within a single molecule. Thus, BsAbs are able to bind two different antigens simultaneously. Genetic engineering has been used with increasing frequency to design, modify, and produce antibodies or antibody derivatives with a desired set of binding properties and effector functions as described for instance in EP 2 050 764 A1 .
  • multispecific antibody denotes an antibody which combines the antigen- binding sites of two or more antibodies within a single molecule.
  • diabodies refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light- chain variable domain (VL) in the same polypeptide chain (VH-VL).
  • VH heavy-chain variable domain
  • VL light- chain variable domain
  • linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen- binding sites.
  • the epitope-binding fragment is selected from the group consisting of Fv, Fab, F(ab') 2 , Fab', dsFv, (dsFv) 2 , scFv, sc(Fv) 2 , diabodies and VHH.
  • a “chimeric antibody”, as used herein, is an antibody in which the constant region, or a portion thereof, is altered, replaced, or exchanged, so that the variable region is linked to a constant region of a different species, or belonging to another antibody class or subclass.
  • Chimeric antibody also refers to an antibody in which the variable region, or a portion thereof, is altered, replaced, or exchanged, so that the constant region is linked to a variable region of a different species, or belonging to another antibody class or subclass.
  • humanized antibody refers to an antibody which is initially wholly or partially of non-human origin and which has been modified to replace certain amino acids, in particular in the framework regions of the heavy and light chains, in order to avoid or minimize an immune response in humans.
  • the constant domains of a humanized antibody are most of the time human CH and CL domains.
  • a humanized antibody has constant domains of human origin.
  • the term “humanized antibody” refers to a chimeric antibody which contain minimal sequence derived from non-human immunoglobulin, e.g. the CDRs.
  • NTN1 binding antibody is used to encompass all these kinds of antibodies, fragments or combination thereof.
  • Humanized antibodies or antibodies adapted for non-rejection by other mammals, may be produced using several technologies such as resurfacing and CDR grafting.
  • the resurfacing technology uses a combination of molecular modeling, statistical analysis and mutagenesis to alter the non- CDR surfaces of antibody variable regions to resemble the surfaces of known antibodies of the target host.
  • Antibodies can be humanized using a variety of other techniques including CDR- grafting (EP0239400; WO91 /09967; U.S. Patent Nos.
  • treating means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition.
  • treating cancer is meant the inhibition of the growth of malignant cells of a tumour and/or the progression of metastases from said tumor.
  • Such treatment can also lead to the regression of tumor growth, i.e., the decrease in size of a measurable tumor.
  • such treatment leads to a partial regression of the tumor or metastasis.
  • such treatment leads to the complete regression of the tumor or metastasis.
  • treatment prevents metastasis.
  • the term "patient” or “patient in need thereof” is intended for a human or non-human mammal affected or likely to be affected with a malignant tumor.
  • the patient to be treated may have been previously treated with other anti-cancer treatments.
  • the patient to be treated may have been previously treated with an oxaliplatin-, cisplatin-, a carboplatin-, and/or a paclitaxel- docetaxel-based regimen.
  • a “therapeutically effective amount” is meant a sufficient amount of the active agents to treat said cancer disease, at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the active agents will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific polypeptide or antibody employed; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific active agents employed; the duration of the treatment; drugs used in combination or coincidental with the specific active agents employed; and like factors well known in the medical arts.
  • said therapeutically effective amount of the active agents administered to the patient is a dose ranging from 5 mg/m 2 to 500 mg/m 2 , more particularly ranging from 150 mg/m 2 to 450 mg/m 2 of body surface area.
  • the active agents of the invention is administered repeatedly according to a protocol that depends on the patient to be treated (age, weight, treatment history, etc.), which can be determined by a skilled physician.
  • “Pharmaceutically” or “pharmaceutically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate.
  • a pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • compositions including the polypeptide or antibody of the invention and the route of administration naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and gender of the patient, etc.
  • the active agents of the invention can be formulated for a topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration and the like.
  • the active agents of the invention are administered intravenously
  • the pharmaceutical compositions including the active agents of the invention may contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
  • an effective amount of the active agents of the invention may be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like) and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants, stabilizing agents, cryoprotectants or antioxidants.
  • the prevention of the action of microorganisms can be brought about by antibacterial and antifungal agents. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.
  • Sterile injectable solutions are prepared by incorporating the active agents in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.
  • aqueous solutions For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage could be dissolved in 1 ml_ of isotonic NaCI solution and either added to 1000 ml_ of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • “simultaneously” is used to mean that the two agents are administered concurrently, whereas the term “in combination” is used to mean they are administered, if not simultaneously, then “sequentially” within a timeframe that they both are available to act therapeutically within the same time-frame.
  • administration “sequentially” may permit one agent to be administered within 5 minutes, 10 minutes or a matter of hours after the other provided the circulatory half-life of the first administered agent is such that they are both concurrently present in therapeutically effective amounts.
  • the time delay between administration of the components will vary depending on the exact nature of the components, the interaction therebetween, and their respective half-lives.
  • the DNA methyltransferase inhibitor is administered sequentially or separately prior to the NTN1 neutralizing agent or NTN1 antagonist.
  • the DNA methyltransferase inhibitor is administered at least 4 hours before the antagonist, and more preferably at least 72 hours before the antagonist.
  • the antagonist is administered sequentially or separately prior to the DNA methyltransferase inhibitor.
  • the antagonist is administered at least one hour before the DNA methyltransferase inhibitor, and more preferably at least 24 hours before the DNA methyltransferase inhibitor.
  • the antagonist and the DNA methyltransferase inhibitor interact in a synergistic manner.
  • the term “synergistic” means that the antagonist and the DNA methyltransferase inhibitor produce a greater effect when used in combination than would be expected from adding the individual effects of the two components.
  • a synergistic interaction may allow for lower doses of each component to be administered to a patient, thereby decreasing the toxicity of chemotherapy, whilst producing and/or maintaining the same therapeutic effect.
  • each component can be administered in a sub-therapeutic amount.
  • NTN1 amino acid MMRA VWEALAALAA VACL VGA VRGG PG LSM FAGQAAQ sequence (seq.) with PDPCSDENGHPRRCIPDFVNAAFGKDVRVSSTCGRPPA signal peptide in bold RYCVVSERGEERLRSCHLCNASDPKKAHPPAFLTDLNNP and linear epitope HNLTCWQSENYLQFPHNVTLTLSLGKKFEVTYVSLQFCS mapping in bold and PRPESMAIYKSMDYGRTWVPFQFYSTQCRKMYNRPHRA underlined PITKQNEQEAVCTDSHTDMRPLSGGLIAFSTLDGRPSAH
  • NTN1 nucleic acid ATGATGCGCGCAGTGTGGGAGGCGCTGGCGGCGCTG seq. GCGGCGGTGGCGTGCCTGGTGGGCGCGGTGCGCGG
  • NTN1 aa epitopic seq.
  • NTN1 epitopic cDNA GTGGCCTGTAACTGCAACCTGCATGCCCGGCGCTGCC seq.
  • VL aa sequence of EIVMTQSPATLSVSPGERATLSCKASQSVSNDVAWYQQK humanized variant of PGKAPKLLIYYASNRYTGIPPRFSGSGYGTDFTLTINNIES 4C1 1 EDAAYYFCQQDYSSPWTFGQG
  • VL aa sequence of DIQMTQSPSSLSASVGDRVTITCKASQSVSNDVAWFQQR humanized variant of PGQSPRRLIYYASNRYTGVPSRFSGSGSGTDFTFTISSLE 4C1 1 AEDAATYYCQQDYSSPWTFGQG
  • VL aa sequence of DIQMTQSPSSLSASVGDRVTITCKASQSVSNDVAWYQQK humanized variant of PGQAPRLLIYYASNRYTGIPPRFSGSGYGTDFTLTINNIES 4C1 1 EDAAYYFCQQDYSSPWTFGQG
  • VL aa sequence of DIVMTQTPLSLPVTPGEPASISCKASQSVSNDVAWYQQK humanized variant of PGQAPRLLIYYASNRYTGIPPRFSGSGYGTDFTLTINNIES 4C1 1 EDAAYYFCQQDYSSPWTFGQG
  • Figure 1 NGS methylation profiles in cancer cell lines.
  • Tissue microarrays (70 sections) from human breast carcinoma were immuno- stained with antibodies against DAPK1 , UNC5B, and netrin-1 . Samples were classified in quartiles according to the expression level of netrin-1 . The expression levels of DAPK1 and UNC5B (index constructed from the percentage of sections exhibiting a positive staining) in the first and fourth quartiles of netrin-1 expression groups were compared using a Chi2 test.
  • HMEC Human mammary epithelial cells
  • RRBS data from Encode database
  • the methylation profiles result from MeDP- sequencing experiments : MDA-MB231 , HMLER and H460 cell lines. After sequencing, data were obtained in bigwig formats. The peaks represent the Bound vs. Input results visualized in Integrated Genome Browser (IgB). Bars indicate the predicted location of CpG island at the gene promoter (data obtained from UCSC). Black plain square indicate the coding exon whereas small ones indicate the 5' UTR regions. The grey squares indicate the position of the pyrosequenced CpG.
  • IgB Integrated Genome Browser
  • NTN1 CpG island DNA methylation profiles of the NTN1 promoter region. There are variations of DNA methylation profiles according to the cell line and nucleotide position. Globally, NTN1 CpG island is unmethylated in HMEC cells while it is highly methylated in the three other cell lines, especially in MDA-MB231 .
  • DAPK1 CpG island is unmethylated in HMEC, highly methylated in MDA-MB231 cells, but only the 3' region of the CpG island is methylated in both HMLER and H460 cells lines. It is postulated that there is a possible correlation between DNA methylation at the 3' region of the CpG island and gene expression in human cell lines.
  • D-F Gene expression in human cancer cell lines treated or not with decitabine (DAC) (72h, 10 ⁇ ).
  • Quantitative real-time RT-PCR was performed using total RNA with specific human
  • DAPK1 DAPK1 , UNC5B and NTN1 primers. Gene expression is shown in percentage of expression of the housekeeping gene PBGD.
  • DAPK1 Gene expression profiles in HMLER cells; DAPK1 is not expressed in untreated cells as well as NTN-1 , but the demethylating agent cell treatment reinduces these both gene expression. UNC5B is expressed in this cell before and after decitabine treatment.
  • DAPK1 and NTN1 are expressed at a very low level and the dependence receptor UNC5B is well expressed. After decitabine treatment, there is an important increase of expression of both DAPK1 and NTN-1 , and no statistical difference of expression for UNC5B.
  • NTN1 blocking antibody "net-1 mAb” triggered apoptosis in hypomethylated breast and lung cancer cell lines.
  • MDA-MB-231 , HMLER, and H460 cell lines were treated with decitabine (DAC, 10 ⁇ , 72h), and/or net-1 mAb (10 g/mL, 48h) and/or recombinant NTN1 (5 g/mL, 48h).
  • DAC decitabine
  • NTN1 5 g/mL, 48h
  • A Caspase-3 activities were determined in 3 independent experiments, Error bars, SEM; p-values, two-tailed unpaired Student's t test.
  • B TUNEL from 3 independent experiments; p-values, Mann Whitney test.
  • FIG 3. Decitabine (DAC) treatments up-regulated genes involved in the netrin-1 receptor signaling pathways and, when combined with netrin-1 blocking antibodies, triggered apoptosis in MDA-MB-231 and H460 xenografts, in immune-compromised mice.
  • Figure 3. Decitabine (DAC) treatments up-regulated genes involved in the netrin-1 receptor signaling pathways and, when combined with netrin-1 blocking antibodies, triggered apoptosis in MDA-MB-231 and H460 xenografts, in immune-compromised mice.
  • Tumor xenografts were fixed in formalin, paraffin embedded, sliced into 4 mm sections.
  • E-H netrin-1 neutralizing antibody
  • DAC low dose decitabine
  • mice were injected subcutaneously with decitabine (0.4mg/kg) or PBS and/or intraperitoneal ⁇ with netl - mAb (10 mg/kg).
  • Figure 4 Primer sequences used in the experimental part.
  • Figure 5 (A) Levels of DAPK1 , UNC5B, and netrin-1 were measured in paraffin embedded tumor xenografts by immunohistochemistry staining from at least 3 independent tumors per treated groups, and expressed as percentage of total tumor surface; p-values, Mann Whitney test.
  • C-D net-1 mAb, combined with decitabine (DAC) reduces human breast tumor, in immuno-compromised mice.
  • FIG. 6 Cells were treated with decitabine (DAC, 10 ⁇ , 72hr), and/or NET1 -H- mAb (10 g/mL, 48 hr) and/or Iso-mAb (10 g/mL, 48 hr).
  • PI staining (A) and apoptotic indexes (B) were determined in 3 independent experiments, Error bars, SEM; p values, two- tailed unpaired Student's t test.
  • mice Schematic representation of treatment schedules of mice with pre-established H460 subcutaneous tumors. When tumors reached 100 mm3, mice were injected subcutaneously with decitabine (0.5 mg/kg) or PBS and/or intraperitoneal ⁇ with NET1 -H- mAb (10 mg/kg) or Iso-mAb (10 mg/kg).
  • Quantitative real-time RT-PCR was performed using total RNA with specific human DAPK1 , UNC5B and NTN1 primers. Gene expression is shown in percentage of expression of the housekeeping gene PBGD.
  • Figure 9 DNA methylation, angiogenesis and proliferation in vivo.
  • H460 xenografted tumors arising from mice treated with PBS or decitabine.
  • A Levels of vessels, and (B) cell proliferation were measured by immunohistochemistry staining using CD31 and KI67 antibodies from 4 independent tumors per treated groups, and expressed as percentage of total tumor surface or ratio between proliferative and non-proliferative cells; p values, Mann Whitney test.
  • C Tumor weights; statistical significance of differences between groups was determined by two-tailed unpaired Student's t test. Error bars, SEM.
  • mice Five-week-old female athymic Swiss nu/nu mice were obtained from Charles River (Ecully, France). Mice were housed in a specific pathogen-free animal facility. 2.10 6 cells resuspended in 200 ⁇ PBS of MDA-MB-231 or MDA-MB-231 -Luc shRNA cell lines were implanted into the fat pad of the mammary gland. H460 cells (2.10 s cells in 200 ⁇ PBS) were subcutaneously injected into the right flank of mice. When tumors reached a volume of about 100 mm 3 , 10 mg/kg of NTN1 -targeting antibody net-1 mAb or an equal volume of PBS was injected intraperitoneal ⁇ , twice a week for 3 weeks.
  • HMLER and MDA-MB231 were maintained in Dulbecco's Minimum Essential Medium F12 Glutamax (DMEM-F12 Glutamax) (Life Technologies), supplemented with 10% FBS (Lonza, Basel, Switzerland) and 1 % penicillin/ streptomycin (InVitrogen, Carlsbad, CA, USA).
  • human EGF 10 ng/ml Promocell, Heidelberg, Germany
  • hydrocortisone 0.5 ⁇ g/ml
  • puromycin 0.5 ⁇ g/ml
  • insulin 10 ⁇ g/ml insulin 10 ⁇ g/ml
  • MDA-MB-231 -Luc cells (Cell Biolabs, San Diego, CA, USA) were maintained in Dulbecco's Minimum Essential Medium F12 Glutamax (DMEM-F12 Glutamax) (Life Technologies), supplemented with 10% FBS (Lonza) and 1 % penicillin/ streptomycin (InVitrogen).
  • Stable shRNA cell lines derived from MDA-MB-231 -Luc cells were maintained in the same medium supplemented by 2 ⁇ g/ml of puromycin (InVitrogen).
  • the H460 lung cancer cell line was maintained in RPMI Glutamax (Life Technologies, Carlsbad, CA), supplemented with 10% FBS (Lonza, Basel, Switzerland) and 1 % penicillin/ streptomycin (InVitrogen).
  • Stable shRNA cell lines derived from H460 cells were maintained in the same medium RPMI Glutamax (Life Technologies), 10% FBS (Lonza, Basel, Switzerland) and 1 % penicillin/ streptomycin (InVitrogen) supplemented by 2 ⁇ g/ml of puromycin (InVitrogen) for shRNA selection.
  • cell lines were grown 3 days in fresh medium containing 10 ⁇ decitabine (Sigma-Aldrich) or 5 ⁇ 5-azacytidine (Sigma-Aldrich) renewed every day.
  • Cells (6.10 4 per well in six-well plates) were treated with 10 ⁇ decitabine or 5 ⁇ 5- azacytidine, and then cultured in presence of net-1 mAb anti-NTN1 antibody ⁇ g/mL (Netris-Pharma) in presence or not of recombinant Flag-tagged NTN1 ⁇ g/mL (Adipogen) in serum deprived condition for 48h.
  • Net-1 mAb anti-NTN1 antibody ⁇ g/mL Netris-Pharma
  • Flag-tagged NTN1 ⁇ g/mL Adipogen
  • DAPK1 pcDNA3.1 vector coding for DAPK1 (Llambi et al., 2005) to enforce the expression of DAPK1 in HMLER cells.
  • Empty plasmid or plasmid containing a HA-tagged DAPK construct was transfected into HMLER cells using Lipofectamine 2000 (Invitrogen).
  • NTN1 siRNA has been previously described (Delloye-Bourgeois et al., 2009a), and was transfected using Lipofectamine 2000 in HMLER cells previously treated or not with decitabine for 72 hours.
  • Scramble siRNA (Sigma) was used as a control.
  • DAPK1, UNC5B and NTN1 shRNA plasmids were transfected in sub-confluent cells using Lipofectamine 2000 (Invitrogen), according to the manufacturer's protocol. After transfection, 2 ⁇ g/mL puromycin was added to the fresh medium as a selection factor. Transfected cells selection occurred during 5 days, cells were then trypsinized and diluted to obtain 0-2 clones per well in 96 wells plates. Puromycin selection (2 ⁇ g/mL) was kept during the shRNA transfection, selection and during all of the subsequent experiments in order to conserve the gene downregulation.
  • Immunohistochemistry staining was performed on an automated immunostainer (Ventana Discovery XT, Roche, Meylan, France) using DABmap Kit according to the manufacturer's instructions. Tissue samples were fixed in 10% buffered formalin and embedded in paraffin. After antigen unmasking (citrate buffer pH 7.3, 98 °C for 35 min), immunostainings were performed with a rat anti-mouse NTN1 targeting antibody (R&D), a rabbit polyclonal anti-DAPK1 (Acris) or an UNC5B antibody (Sigma), using the Novolink kit (Leica) for revelation.
  • R&D rat anti-mouse NTN1 targeting antibody
  • Acris rabbit polyclonal anti-DAPK1
  • UNC5B antibody Novolink kit
  • Apoptotic cells staining was performed using a rabbit cleaved caspase- 3 specific antibody (Cell Signaling) and the In Situ Cell Death Detection kit POD (Roche).
  • Angiogenesis was measured using a rabbit polyclonal CD31 (Platelet Endothelial Cell Adhesion Molecule-1 ) antibody (ANASPEC), and cell proliferation rate was determined using a mouse monoclonal Mib1 clone specific antibody (DAKO).
  • Image analysis was performed using a light microscope (Eclipse E400, Nikon France,
  • Quantitative values were determined by morphometric analysis (Histolab, Microvision Instruments, Evry, France) of at least 40 fields per staining at a X200 magnification for the xenograft tumor sections, and one field per staining of the total tissue section at X40 magnification for the tissue microarray. The total surface occupied by tumor tissue was measured and each surface of positive staining was expressed as a percentage of the total surface for each field.
  • RNA Isolation and Quantitative Real-Time RT-PCR (Q-RT-PCR): Total RNA was extracted from biopsies using the TRIzol-Reagent (Ambion, Life Technologies), and from cell lines using the Nucleospin RNAII kit (Macherey-Nagel, Hoerdt, Germany) according to the manufacturers' instructions. One ⁇ g of RNA was reverse- transcribed, using the iScript cDNA Synthesis Kit (BioRad, Ivry, France). Quantitative RT- PCR (Q-RT-PCR) was performed using a Mini opticon (Biorad) and the SYBR supermix qPCR kit (Biorad).
  • Q-RT-PCR Quantitative RT-PCR
  • Biopsies samples and xenografted cell lines were cryogrinded in liquid nitrogen and DNA was extracted and purified using the Nucleospin tissue DNA extraction kit (Macherey Nagel) according to the manufacturer's protocol. DNA from cell lines was directly extracted using the standard protocol (Auriol et al., 2005)
  • genomic DNA Two hundred nanograms of genomic DNA were added to 1 .8 ⁇ g of standard plasmid DNA (pGL3 Basic) and converted using the Epitect Bisulfite kit (Qiagen), according to the manufacturer's protocol.
  • Thermal profiles were as follows: 95°C for 10 minutes followed by 50 cycles of 95°C for 30 sec, 50°C (NTN1) or 52°C (DAPK1) or 58°C (LINE- 1 or 57°C (GAPDH) for 30 sec and 1 min 30 sec (NTN1 and DAPK1) or 30 sec (LINE-1 and GAPDH) of extension followed by a 10 min final elongation.
  • the primer sequences are shown in Figure 4. The quality and quantity of the PCR product was confirmed by agarose gel (2%) electrophoresis before the pyrosequencing analysis.
  • PCR products were pyrosequenced using Pyromark kit (Qiagen). Reverse single- stranded biotinylated templates were isolated using the PyroMark Vacuum Prep Workstation (Qiagen). Forty microliters of PCR product were added to 38 ⁇ _ of binding buffer (Qiagen) and 2 ⁇ _ streptavidin sepharose high-performance beads (GE Healthcare ® ). The mixtures were shaken for 10 min at 1400 rpm (revolution per minute). After agitation, beads covered by biotinylated DNA were collected and retained on filter probes by permanent vacuum.
  • Pyromark kit Qiagen
  • Reverse single- stranded biotinylated templates were isolated using the PyroMark Vacuum Prep Workstation (Qiagen). Forty microliters of PCR product were added to 38 ⁇ _ of binding buffer (Qiagen) and 2 ⁇ _ streptavidin sepharose high-performance beads (GE Healthcare ® ). The mixtures were shaken
  • the filter probes were successively immerged in different baths: in ethanol 70% for 5 s, in PyroMark denaturation solution for 5 s and in PyroMark wash buffer 1 x for 15 s (Qiagen). Then the vacuum was turned off and the beads fixing DNA strands were released into a 96 well plate containing 25 ⁇ of annealing buffer with 0.3 ⁇ of sequencing primer in each well. The sequencing plate was kept at 80°C for 2 min and at room temperature for 5 min. Pyrosequencing reactions were performed in a PyroMark Q96 system using PyroGold reagents (Qiagen). Results were analyzed using PyroMark Software.
  • the average LINE-1 methylation level was calculated as the mean of the proportions of C (%) at the 3 CpG sites analysed.
  • Caspase-3 activity was measured as previously described (Llambi et al., 2005) using the Ac-DEVD-AFC substrate assay (Gentaur Biovision, Brussel, Belgium). Alternatively, cell death percentage was measured by acridine orange and DAPI staining, using the NucleoCounter NC-3000 system (ChemoMetec A/S, Allerod, Denmark).
  • the apoptotic index was measured using Cellplayer 96 well kinetic caspase 3/7 apoptosis kit, according to the manufacturer's protocol (Essen bioscience, Hertfordshire, UK). Three thousands cells were plated in 96 wells plate, treated with decitabine (DAC 10 ⁇ final) or vehicle (PBS) for 48h, then cells were incubated in serum free medium enriched kinetic apoptosis reagent (1/5000e) with net-1 mAb (5 g/mL) and/or recombinant NTN1 (5 ⁇ 9/ ⁇ _), DAC or PBS Cells were placed in an IncuCyte FLR or ZOOM with a 10X objective in a standard cell culture incubator at 37°C and 5% C02 for 48 additional hours.
  • the total number of DNA containing objects was counted at the final time point using Vybrant Green. This number was used to calculate the "apoptotic index", defined as the number of caspase-3/7 positive objects divided by the total number of DNA containing objects.
  • TUNEL Terminal deoxynucleodityl- transferase mediated dUTP-biotin Nick End Labeling
  • Genomic DNAs (1 ⁇ g) were sheared to an average length of 300-600 bp. Methylated DNA fragments were isolated using MBD domain containing beads according to manufacturer's recommendations (MethylMiner, InVitrogen). After sequencing using the lllumina 2000 High throughput Sequencing technology by BGI service (Beijing, China), 30-40 millions of 50 bp reads were obtained from each Input and Bound fraction and analyzed using R and bioconductor packages.
  • Hvpermethylation of DAPK1 and NTN1 is associated with gene silencing in human breast cancers.
  • UNC5B/H2 triggers apoptosis through the activation of its pro-apoptotic partner DAPK1 (Llambi et al., 2005; Guenebeaud et al., 2010; Delloye-Bourgeois et al., 2009b; Delloye- Bourgeois et al., 2009a) in mammary and lung cancer cell lines.
  • DAPK1 pro-apoptotic partner
  • DMRs differentially methylated regions
  • DNA methylation patterns of three cancer cell lines were determined by parallel sequencing of methylated DNA fragments selectively pulled-down with a recombinant protein containing the Methyl-CpG-binding domain of MBD2 (Methyl-Cap-seq).
  • MDA-MB-231 cell line derived from human breast cancer
  • HMLER cell line constructed from in vitro transformation of human mammary cells (Elenbaas et al., 2001 ; Morel et al., 2008), as well as a non small-cell lung cancer cell line, H460 were analyzed.
  • DAC global methylation level upon decitabine
  • the pro-apoptotic activity induced by the combination of the net-1 mAb and decitabine was not limited to decitabine per se.
  • Treatments of the MDA-MB-231 , HMLER, and H460 cell lines with the 5-azacytidine, a DNA methylation inhibitor led to similar modifications of gene expression and also sensitized the cell lines to the anti-NTN1 antibody.
  • the inhibition of DNA methylation allows the induction of apoptosis upon NTN1 neutralization in these cell lines.
  • the combination of the NTN1 neutralizing antibody with decitabine inhibits tumor growth in mice.
  • TUNEL assays displayed a higher percentage of cells with DNA fragmentation in xenografts from mice receiving the combined treatment ( Figure 3A-D).
  • the H460 tumors were shown to be insensitive to NTN1 interference in immuno-compromised mice (Delloye-Bourgeois et al., 2009a). As shown in Figures 3H, while decitabine alone or net-1 mAb alone had no significant effect on tumor growth, combination of both decitabine and net-1 mAb nicely inhibited tumor growth.
  • mice model bearing a human tumor (PDX model).
  • PDX model mice model bearing a human tumor
  • combination of both decitabine and net- 1 mAb stimulated DAPK1 , UNC5B, and NTN1 expression ( Figure 5A) and inducted a pro- apoptotic activity ( Figure 5B).
  • Figure 5A the combination failed to be associated with changes in tumor cell proliferation or with reduced angiogenesis, the combination was associated with tumor cell death in the patient derived xenograft.
  • combined treatment strongly reduced the growth of patient-derived tumors ( Figure 5C-D).
  • NTN1 and DAPK1 were hypermethylated at their 5' end in respectively 33% and 41 % of tumors, when compared to the paired normal tissues.
  • H460 cells that were shown to be non-responsive to NET-1 interference (Delloye-Bourgeois et al., 2009a), were assessed for DAPK1, UNC5h2 and NTN1 expression by Q-RT-PCR and DNA methylation by pyrosequencing. While DAPK1 is not expressed in these cells, treatment with decitabine (DAC), a DNMT1 inhibitor, leads to a loss of general DNA methylation and an increased expression rate of both DAPK1 and NTN1 while the NTN1 dependence receptors expression was not affected by DAC treatment. Thus, chemically-induced hypomethylation directly impacts on NET1 and its dependence receptor pathway expression, even though it cannot exclude that the effect of DAC shown here occurs via indirect re-expression of master transcription regulators.
  • DAC decitabine
  • This assay related to gene expression in human cancer cell lines treated or not with different HDAC inhibitors. Quantitative real-time RT-PCR was performed using total RNA with specific human DAPK1 , UNC5B and NTN1 primers.
  • MDA-MB-231 cells were treated with panobinostat (20 nM, 48 hr) and/or net1 -mAb (10 g/mL, 48 hr) and the level of cell death was measured. (Figure 7D)
  • the current results provide the first animal proof of concept that inhibition of DNA methylation can sensitize solid tumors to antibodies mediating tumor cell apoptosis.
  • experimental downregulation of NTN1 using siRNA
  • NTN1 trapping using decoy molecules are able to induce apoptotic cell death in vitro and tumor growth reduction of human xenografts in nude mice, cancer cells which show low NTN1 level are resistant to NTN1 neutralization both in vitro and in vivo.
  • NTN1 dependence receptors in NTN1 low tumors may occur at different levels, either by downregulation of the receptors as largely exemplified in colorectal cancer, or by simultaneous downregulation of key signaling pathway partners.
  • low NTN1 expression is associated with an hypermethylation of the CpG island located at the 5' end of this gene.
  • DAPK1 an essential partner in NTN1 dependence receptor pathway.
  • the fully human NTN1 antibody (NET1 -H-mAb) is in late regulatory preclinical evaluation with in particular no sign of toxicity in rodent or cynomolgus monkeys.
  • NET1 -H-mAb The fully human NTN1 antibody (NET1 -H-mAb) is in late regulatory preclinical evaluation with in particular no sign of toxicity in rodent or cynomolgus monkeys.
  • Early clinical trial that is scheduled in 2016, and should rapidly take advantage of the data reported here supporting the view that patients with breast cancer with hypermethylation of both NTN1 and DAPK1 promoters should respond to a combination of inhibitors of DNA methylation with this anti-NTN1 antibody.

Abstract

La présente invention concerne une composition pharmaceutique ou anticancéreuse qui est utile dans le traitement d'un cancer présentant une perte d'expression de NTN1, UNC5H ou DAPK1, dépendant de la méthylation de l'ADN. L'invention concerne également une méthode de traitement de tels cancers. Ladite composition comprend un médicament épigénétique et un agent neutralisant NTN1 ou un vecteur capable d'exprimer un agent neutralisant NTN1 in vivo, dans un transporteur ou un véhicule pharmaceutiquement acceptable. L'agent neutralisant NTN1, tel qu'un anticorps se liant à la nétrine-1 ou à un récepteur de la nétrine-1, favorise l'apoptose induite par des récepteurs de la nétrine-1. Le médicament épigénétique peut être un inhibiteur de l'ADN méthyltransférase ou un inhibiteur de la HDAC.
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