WO2005060996A2 - Inhibiteurs - Google Patents

Inhibiteurs Download PDF

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Publication number
WO2005060996A2
WO2005060996A2 PCT/GB2004/005396 GB2004005396W WO2005060996A2 WO 2005060996 A2 WO2005060996 A2 WO 2005060996A2 GB 2004005396 W GB2004005396 W GB 2004005396W WO 2005060996 A2 WO2005060996 A2 WO 2005060996A2
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Prior art keywords
binding
inhibitor
sequence
seq
ezrin
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PCT/GB2004/005396
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English (en)
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WO2005060996A3 (fr
Inventor
Kjetil TASKÉN
Anja Ruppelt
Mikaela GRÖNHOLM
Einar Martin Aandahl
Derek Tobin
Cathrine Carlson
Olli CARPÉN
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Lauras As
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Priority claimed from GB0329869A external-priority patent/GB0329869D0/en
Application filed by Lauras As filed Critical Lauras As
Publication of WO2005060996A2 publication Critical patent/WO2005060996A2/fr
Publication of WO2005060996A3 publication Critical patent/WO2005060996A3/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
    • 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
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)

Definitions

  • the present invention relates to inhibitors which abolish the function of cAMP dependent protein kinase (PKA) , type I and their use to produce pharmaceutical preparations to treat or prevent immunosuppressive diseases. More specifically, the present invention provides inhibitors of binding between specific binding partners of the PKA type I complex involved in PKA type I signalling. In particular inhibitors of the binding between ezrin and PKA type I, ezrin and EBP50 and EBP50 and Cbp/PAG are provided.
  • the immune system of mammals has evolved different strategies to defend the organism against ,the variety of potentially * infectious agents. The ability to acquire specific and anamnestic responses against intruders relies on the adaptive immune system.
  • the main players in the adaptive immune system are B and T lymphocytes, and the specific recognition of antigen by these cells is mediated by receptors with some degree of structural similarity, yet which are functionally very different.
  • the different receptor specificities are made possible through somatic rearrangement of a limited number of genes and are clonally distributed.
  • the main strategy of this system is to generate a nearly unlimited number of specificities to cover the recognition of almost any foreign antigen.
  • Immunological memory is partly a result of clonal expansion of subsets of T and B cells reacting with a particular antigen, and enables the organism to respond more quickly at the second encounter with the same antigen.
  • Cell proliferation is used as a marker of immune activation.
  • Cyclic AMP-dependent protein kinase is an enzyme present in all cells. Hormones and neurotransmitters binding to specific receptors stimulate the generation of the second messenger 3 ' , 5 ' - cyclic adenosine monophosphate (cAMP) . Cyclic AMP is one of the most common and versatile second messengers. The best characterized and major downstream effector mechanism whereby cAMP exerts its effects involves binding to and activating PKA. PKA is a serine/threonine protein kinase which phosphorylates a number of different proteins within the cell, and thereby regulates their activity.
  • PKA PKA regulates a vast variety of cellular processes such as metabolism, proliferation, differentiation and regulation of gene transcription.
  • the great diversity of cellular processes mediated by cAMP and PKA strongly suggests that there exists mechanisms that provide the required sensitivity and specificity of the effector pathway to ensure that rapid and precise signalling processes take place.
  • Specificity can be achieved by tissue- and cell-type specific expression of PKA isoforms with different biochemical properties.
  • targeting of PKA isoforms by A- kinase anchoring proteins (AKAPs) provides a higher level of specificity to the signalling process by localizing PKA to defined subcellular sites in close proximity to the substrate.
  • Anchoring of PKA by AKAPs may also tune the sensitivity of the signal pathway by recruiting PKA into multiprotein complexes that include phosphodiesterases and protein phosphatases as well as other signal proteins in addition to PKA (Michel and Scott, 2002, Ann. Rev. Pharmacol. Toxicol . , 42, p235- 257) .
  • PKA is made up of four different subunits, a regulatory (R) subunit dimer and two catalytic (C) subunits.
  • R regulatory
  • C catalytic
  • two main classes of PKA isozymes, PKA type I and PKA type II (PKAI and PKAII, respectively) have been described.
  • PKAI and PKAII can be distinguished by their R subunits, designated RI and RII.
  • Isoforms of RI and RII are referred to as Rio * , Rl ⁇ , Rll ⁇ and Rll ⁇ .
  • the C subunits also exist as isoforms referred to as Co., C ⁇ and Cy.
  • the different subunits may form multiple forms of PKA (isozymes) with potentially more than 18 different forms .
  • Activation occurs upon binding of cAMP to the R subunits followed by the release of the active catalytic subunit.
  • PKA type II is mainly particulate and associated with AKAPs whereas PKA type I is both soluble and particulate although PKA type I anchoring has remained more elusive.
  • PKA type I is recruited to the lipid raft fraction of the cell membrane upon T- cell activation, and colocalizes with the TCR-CD3 complex (Skalhegg et al. , 1994, Science, 263, p84-87) .
  • Lipid rafts are specialised membrane domains enriched in certain lipids, cholesterol and proteins. Their primary function is believed to be in signalling transduction. In particular they are considered to be the site of recruitment for various signalling molecules crucial for T cell activation.
  • PKA is a key negative regulator of lymphocyte function. The present inventors and others have shown that cAMP inhibits T lymphocyte proliferation induced through the T cell antigen receptor/CD3 complex (TCR/CD3) .
  • T cells express both PKAI and PKAII. However, only the selective activation of PKAI is sufficient to mediate the inhibitory effect of cAMP. In addition, we have demonstrated that PKAI, but not PKAII, redistributes to, colocalizes with and inhibits signalling through antigen receptors on T and B cells and natural killer cells and regulates mitogenic responses in T and B cells and acute cytotoxic responses in NK cells.
  • PKA type I mediates an inhibitory effect on the T cell activation cascade that involves activation of C- terminal Src kinase (Csk) by phosphorylating residue S364 (Vang et al, 2001, J. Exp. Med., 193, p497-507) .
  • PKAI serves as a key negative regulator of lymphocyte functions, e.g. mitogenic and cytotoxic responses initiated through antigen receptors.
  • the inventors' work has led to the conclusion that modulation of normal immune responsiveness by activation of PKA type I is a negative feedback mechanism. Dysregulation of this system may lead to immunological overshoot or impaired immune functions .
  • HIV Human immunodeficiency virus
  • CVI common variable immunodeficiency
  • T cell dysfunction is the immunological hallmark of HIV infection.
  • Defective lymphocyte cytokine production and impaired proliferative responses on stimulation are early signs of immunodeficiency in these patients, manifested even before a decline in CD4+ lymphocytes counts is observed.
  • B cell dysfunction with impaired antibody synthesis is the major immunological characteristic of CVI patients.
  • the immunological abnormalities in CVI are not restricted to B cells, but often also involve T cell dysfunction, e.g. impaired proliferative responses on stimulation.
  • the B cells in CVI patients are not necessarily intrinsically defective, and impaired T cell "help" may be of importance for the B cell defects in these patients. T cell dysfunction may also be of importance for certain clinical manifestations in these patients not necessarily related to defective antibody production, e.g. increased incidence of granulomata and malignancies.
  • antiretroviral therapy is the main component in the treatment of HIV- infected patients.
  • potent antiretroviral combination therapy may markedly increase the CD4+ and CD8+ lymphocytes counts in HIV-infected patients, impaired T cell function seems to persist, as indicated in the observations made in Example 1, table I and 2B of WO98/48807.
  • Immunoglobulin substitution is the main component in the treatment of CVI patients.
  • this substitution therapy does not restore the defective T and B cell function.
  • some clinical complications e.g. noncaseating granulomata and persistent viral infections, there is a need for therapy which may more directly enhance T cell function.
  • impaired T cell function is a well recognized immunological feature of both HIV infection and CVI, the exact molecular mechanism for this T cell impairment was not known.
  • Therapeutic modalities directed against such intracellular defects are expected to be of major importance in the treatment of these patients and may have the potential to restore important immunological defects in HIV-infected patients and in patients with CVI.
  • protein kinase A type I redistributes to and colocalizes with the antigen receptor complex upon activation and capping of T and B cells (Skalhegg et al , 1994, supra) .
  • This anchoring of protein kinase A type I supports a role for this specific isozyme in modulation of immune responses mediated through receptors on lymphoid cells.
  • discoveries by the inventors and as described in WO98/48809 show that there is an increased activation of protein kinase A type I in T cells from patients with HIV infection or CVI.
  • the inventors further demonstrated that activation of this isozyme of PKA leads to inhibition of immune function that can be reversed by selectively inhibiting the type I and not the type II isozymes of PKA.
  • the inventors sought compounds aimed at reversing the inappropriate activation of protein kinase A type I in immunodeficiencies (such as HIV, CVI) and thereby restoring T cell function and immune responsiveness.
  • the present inventors therefore set about developing strategies to specifically increase T cell immune function and reverse T cell dysfunction in human immunodeficiency virus infected and common variable immunodeficiency patients by using suitable compounds interfering with the cAMP/PKA pathway in T cells.
  • PKA type I as an immune modulator is shared among all lymphoid cells (T cells, B cells, NK cells) disruption of the cAMP/PKA pathway is also expected to be relevant to B and NK cell function.
  • Appropriate mechanisms for improving T cell function as developed by the inventors are described in WO98/48809, which is incorporated herein by reference.
  • the strategies relied on disruption of the cAMP-induced inhibition of T cell immune responses by abolishing PKA type I/RIoc signalling. Specifically, a number of specific mechanisms for disrupting the effects mediated by cAMP dependent protein kinase and hence stimulation of immune function were described.
  • protein kinase A type I function was inhibited by removal of the activated enzyme from substrates in the antigen receptor complex that are relevant for inhibition of immune function in T cells.
  • the search for new and improved modulators of PKA type I signalling and hence of immune function continues.
  • the inventors have now identified the involvement of a number of molecules not previously known to be involved in signalling via PKA type I which thus constitute attractive targets to mediate cell signalling.
  • the inventors have now identified a new AKAP which recruits and binds PKA type I and is hence involved in PKA type I signalling. This molecule's involvement in the signalling pathway provides a number of molecules which may be targeted to intervene in the signalling process.
  • Ezrin is an AKAP responsible for anchoring PKA type I to the TCR-CD3 complex during T cell activation and capping.
  • Ezrin also binds to EBP50 (ERM-binding phosphoprotein of 50kDa) which in turn binds to Cbp/PAG (Csk-binding protein or phosphoprotein associated with GEMs) which binds Csk which is involved in PKA type I signalling as mentioned previously.
  • EBP50 EM-binding phosphoprotein of 50kDa
  • Cbp/PAG Csk-binding protein or phosphoprotein associated with GEMs
  • Ezrin is a 78kDa protein belonging to the ezrin- radixin-moesin (ERM) -family of proteins that plays structural and regulatory roles in the assembly and stabilization of the plasma membrane by linking microfilaments to the membrane (cortical actin layer) .
  • Ezrin is often located at actin-enriched foci; for instance during membrane polarization, lymphocyte migration and polarized secretion of cytokines .
  • ERM proteins have a high degree of homology in their N- terminal regions and bind directly to a number of transmembrane proteins including CD44, layilin, Na/H exchanger NHE1, CD43 and intercellular adhesion molecules (ICAMs) (Tsukita S, 1994, J.
  • IAMs intercellular adhesion molecules
  • the N-terminal ERM domain also binds to signalling molecules in the Rho pathway, including Rho guanine dinucleotide dissociation domain (Rho-GDI) (Hirao et al . , 1996, J. Cell. Biol., 135, p37-51) .
  • Rho-GDI Rho guanine dinucleotide dissociation domain
  • the carboxyl terminus of ERM proteins interacts with filamentous actin (F-actin) and thereby links the cytoskeleton to the plasma membrane.
  • ERM proteins Dependent on the phosphorylation status, ERM proteins can undergo intracellular interactions by association of the N- and carboxyl-termini which leads to masking of binding domains essential for intermolecular interactions .
  • the F-actin binding site and sites for interaction with EBP50 and RhoGDI have been shown to be masked in the dormant, inactive monomer.
  • Ezrin had previously been identified as an AKAP for PKA type II. However, there was no suggestion that this molecule could associate with PKA type I or act as an AKAP for that molecule. The presence of ezrin in lipid rafts had not been observed.
  • EBP50 is an abundant cytoplasmic protein containing 2 N-terminal PDZ domains and a C-terminal domain known to bind ERM-proteins .
  • Cbp/PAG is a Csk- associated membrane adapter protein exclusively localized in lipid rafts. Cbp/PAG-Csk complex formation increases Csk activity through a binding and conformational mechanism.
  • Cbp/PAG is phosphorylated in resting, cells and TCR stimulation induces dephosphorylation and dissociation of Csk which activates Src kinases .
  • Cbp/PAG associates via its C-terminal residues (TRL) to one or both of the PDZ domains in EBP50 (Itoh et al .
  • the present invention provides a variety of modes of interrupting PKA type I-mediated signal transduction by affecting the binding between PKA type I and ezrin, between ezrin and EBP50 and/or between EBP50 and Cbp/PAG.
  • Suitable modes of interruption include the use of inhibitors of those interactions, and modification of the wild-type forms to impair normal binding .
  • the present invention provides a method of altering the PKA type I signalling pathway in a cell by administration of an inhibitor (or a molecule encoding an inhibitor) preferably as defined herein, which reduces or inhibits the binding between one or more of the following binding partners : i) ezrin and PKA type I, ii) ezrin and EBP50, and iii) EBP50 and Cbp/PAG.
  • PKA type I signalling pathway refers to a series of signalling events in which PKA type I is activated resulting in increased kinase activity of this enzyme.
  • This signalling pathway is intended to include molecular events from activation of PKA to end effects such as reduced proliferation or IL-2 production, or intermediate effects such as inactivation of Src kinases.
  • altering the activity of the PKA type I signalling pathway is intended to mean the alteration of one or more signalling elements in the pathway (e.g. to affect its enzymatic or other functional properties) which affects downstream signalling events. Alteration of the signalling elements refers to the ability to form interactions with other molecules, e.g. protein-protein interactions.
  • the ultimate effect is to down-regulate downstream events which typify PKA signalling.
  • Alteration of said signalling pathway may be assessed by determining the extent of activation of a molecule involved in said pathway, e.g. phosphorylation of a kinase, e.g. Csk, or examination of levels of molecules whose levels are dependent on the activity of said pathway, e.g. IL-2.
  • a molecule involved in said pathway e.g. phosphorylation of a kinase, e.g. Csk
  • IL-2 e.g. IL-2
  • binding refers to the interaction or association of at least two moieties in a reversible or irreversible reaction, wherein said binding is preferably specific and selective.
  • Specific binding refers to binding which relies on specific features of the molecules involved to achieve binding, ie. does not occur when a non-specific molecule is used (ie. shows significant binding relative to background levels) and is selective insofar as binding occurs between those partners in preference to binding to any of the majority of other molecules which may be present.
  • a "binding partner” refers to a molecule which recognizes and binds specifically (ie. in preference to binding to other molecules) through a binding site to its binding partner. Such binding pairs when bound together form a complex.
  • a “reduced” binding refers to a decrease in binding, e.g. as manifest by reduced affinity for one another and/or an increased concentration of one of the binding pair required to achieve binding. Reduction includes a slight decrease as well as absolute abrogation of specific binding. A total reduction of specific binding is considered to equate to a prevention of binding.
  • “Inhibited” binding refers to adversely affecting (e.g. by competitive interference) the binding of the binding partners by use of an inhibitor molecule which serves to reduce the partners' binding. A reduction in binding or inhibition of binding may be assessed by any appropriate technique which directly or indirectly measures binding between the binding partners . Thus relative affinity may be assessed, or indirect effects reliant on that binding may be assessed.
  • the binding of the 2 binding partners in isolated form may be assessed in the presence of an inhibitor or by using a modified version of one or more of the binding partners which results from the use of an inhibitor, as described hereinafter.
  • tests may be conducted in which the signalling achieved by the PKA type I pathway is examined (see e.g. Figure 9 which examines IL-2 release) or by assessing disrupted localization as evident from displacement of one or more binding partners from biochemical subcellular fractionation such as lipid raft purification or delocalization evident by immunofluorescent staining and epifluorescence microscopy.
  • Inhibitors according to the present invention are intended for use in affecting the PKA type I signalling pathway.
  • inhibitors may affect PKA type II signalling if used in sufficiently high concentrations .
  • preferred inhibitors are those which affect only the PKA type I signalling pathway and not the PKA type II signalling pathway regardless of the concentration used.
  • Inhibitors which may be used are preferably selective for PKA type I relative to PKA type II signalling. If said inhibitors are able to reduce or inhibit both PKA type I and PKA type II signalling, they may be used at an effective concentration such that they are selective for the PKA type I pathway relative to the PKA type II pathway. Appropriate concentrations may be determined in the above described or other appropriate tests. Especially preferably the inhibitors are selective for (or used at concentrations which are selective for) PKA type Io. relative to PKA type I ⁇ signalling.
  • selectivity is present (for inhibition of e.g. type I relative to type II or type Io. relative to type I ⁇ ) when at least a 5-fold lower concentration of said inhibitor is required to reduce binding between the binding partners by 50%. Especially preferably at least a 10 or 100 fold lower concentration is required.
  • said binding may be assessed according to the K D between the binding partners .
  • Said binding may alternatively be assessed according to the K D between the inhibitor and the binding site of the relevant binding partner.
  • the K D should be l-200nM when assessed in vitro .
  • the amino acid sequence of the human form of ezrin appears in SEQ ID NO.
  • the amino acid sequence of the human form of EBP50 appears in SEQ ID NO . 3 : msadaaagap Iprlcclekg pngygfhlhg ekgklgqyir Ivepgspaek agllagdrlv evngenveke thqqwsrir aalnavrllv vdpetdeqlq klgvqvreel lraqeapgqa 1 eppaaaevqg agneneprea dkshpeqrel rprlctmkkg psgygfnlhs dkskpgqfir 1 svdpdspaea sglraqdriv evngvcmegk qhgdwsair aggdetkllv vdretdeffk 1 kcrvipsqeh Ingplpvpft
  • the binding partners ezrin, PKAI, EBP50 and Cbp/PAG as described herein refer to a polypeptide comprising SEQ ID NO . 1, 2, 3 and 4, respectively, and their functionally equivalent variants, derivatives or fragments.
  • the binding partners are those molecules which occur endogenously .
  • variants, derivatives and fragments are described hereinafter with particular reference to inhibitors of the invention.
  • variants, derivatives and fragments of the binding partners as mentioned herein are similarly defined.
  • such variants include naturally occurring variants such as comparable proteins found in other species or more particularly variants and alleles found within humans. Conveniently, said variants may be described as having more than 75%, e.g.
  • the present invention provides a method of altering the PKA type I signalling pathway in a cell by administration of an inhibitor (or a molecule encoding an inhibitor) as defined herein, which reduces or inhibits binding between one or more of the following binding partners : i) a polypeptide comprising the sequence as set forth in SEQ ID No. 1 or a sequence with 95% similarity thereto or a sequence encoded by a nucleotide sequence which hybridizes under conditions of high stringency to the nucleotide sequence encoding the amino acid sequence of SEQ ID No.
  • polypeptide comprising the sequence as set forth in SEQ ID No. 2 or a sequence with 95% similarity thereto or a sequence encoded by a nucleotide sequence which hybridizes under conditions of high stringency to the nucleotide sequence encoding the amino acid sequence of SEQ ID No . 2 , or a functionally equivalent fragment thereof; ii) a polypeptide comprising the sequence as set forth in SEQ ID No. 1 or a sequence with 95% similarity thereto or a sequence encoded by a nucleotide sequence which hybridizes under conditions of high stringency to the nucleotide sequence encoding the amino acid sequence of SEQ ID No .
  • polypeptide comprising the sequence as set forth in SEQ ID No. 3 or a sequence with 95% similarity thereto or a sequence encoded by a nucleotide sequence which hybridises under conditions of high stringency to the nucleotide sequence encoding the amino acid sequence of SEQ ID No . 3 , or a functionally equivalent fragment thereof; iii) a polypeptide comprising the sequence as set forth in SEQ ID No. 3 or a sequence with 95% similarity thereto or a sequence encoded by a nucleotide sequence which hybridizes under conditions of high stringency to the nucleotide sequence encoding the amino acid sequence of SEQ ID No.
  • sequence similarity refers to sequences which have the stated value when assessed using e.g.
  • sequences encoded by a sequence which hybridizes to a particular sequence refers to the polypeptide sequence encoded by the complementary sequence of a sequence which hybridizes to the particular sequence.
  • hydridizing sequences may be described as those which exhibit at least 70%, preferably at least 80 or 90%, e.g. at least 95% sequence identity (as determined by, e.g.
  • binding partners consist of, or comprise, specific fragments (functionally equivalent fragments) of said above described polypeptides which correspond to the relevant binding sites .
  • Ezrin binds to PKA type I through bases in the region of 356 to 470 (particularly in the region 363 to 470) .
  • residues 413 or 414 to 443 (containing the amphipathic helix binding domain at residues 413-430) are involved in binding to PKA type I.
  • a second region has also been identified that provides specificity and additional affinity in anchoring the enzyme. This region is found at residues 356 to 397 (particularly 367 to 390) and is described in more detail in Example 3 and Figure 10. This region has been found to comprise two separate binding elements, namely from residues 356 to 376 and 378 to 397 (see Example 4) . Binding to PKA involves at least residues 1-50 of that enzyme. Residues 1-286 of ezrin bind to residues 329-358 of EBP50.
  • said binding partner polypeptide consists of at least the following amino acid sequence or a sequence with 95% similarity thereto or a sequence encoded by a nucleotide sequence which hybridizes under conditions of high stringency thereto: (a) amino acids 356 to 470, (e.g. 363-470) (for binding to SEQ ID No. 2 or its variants) or 1 to 286 (for binding to SEQ ID No . 3 or its variants) of SEQ ID No. 1; (b) amino acids 1 to 50 of SEQ ID No . 2 (for binding to SEQ ID No.
  • binding may be reduced by modifying the endogenous molecules taking part in binding.
  • Both inhibitors and the molecules used to achieve an alteration in the form or expression of one or more of the endogenous binding partners are referred to herein as inhibitors even though in the latter case their mode of interaction is not necessarily to achieve direct inhibition of the binding site (ie. a steric inhibitor) .
  • molecules which act to ultimately achieve inhibition of binding between binding partners are referred to herein as inhibitors .
  • Such molecules include peptides and proteins as well as nucleic acid molecules, such as those which encode peptide/protein inhibitors which may be used to express the peptide/protein inhibitors within the cell or may be used to derive sense of antisense nucleotide sequences to cause co-suppression or suppression to reduce expression of the endogenous binding partner.
  • the present invention provides an inhibitor molecule which reduces or inhibits the binding between one or more of the binding partners as defined hereinbefore and which preferably is capable of altering the PKA type I signalling pathway in a cell.
  • direct inhibitors of binding are antagonists of binding between the specific binding pairs .
  • Such inhibitors may themselves bind to one of the binding pair at the binding site (e.g.
  • an "antagonist” is a molecule or complex of molecules which by virtue of structural similarity to one molecule of a binding pair competes with that molecule for binding to the other molecule of the binding pair.
  • the inhibitors may be molecules which specifically recognize the binding site, such as antibodies (or fragments thereof) , or proteins or peptides which associate with that region or associate sufficiently close to affect accessibility by the other binding partner.
  • molecules, particularly peptides or larger molecules, which mimic the binding site (or contain a region which mimics the binding site) may be used to act instead as the pseudo-binding partner to reduce the extent of binding between the true endogenous binding partners .
  • Preferred mimics are peptides that comprise the relevant binding site as described herein.
  • Molecules which are complementary to the binding site, e.g. such that they bind to that site may also be used.
  • Molecules which affect the binding site indirectly, by binding at a distant part of the molecule, but which affect the conformation of the binding site and thus the ability to form relevant interactions with its binding partner may also be used.
  • Appropriate molecules may for example be proteins or peptides or other molecules which can affect binding, or a nucleic acid molecule which encodes such a product may be used to generate the inhibitor. Conveniently small inhibitor molecules may be used. However, where desired, large molecules containing the binding site may be employed, e.g. larger molecules which mimic the entire binding partner (e.g. the soluble portion thereof) , but which have preferably been modified such that one or more properties required for involvement in the signalling pathway is missing or altered to lose or impair its ability to be involved in signalling. For example in the case of EBP50 a large molecule absent only the 30 C-terminal amino acids may be used, which would allow functional binding with Cbp/PAG, but not ezrin.
  • the present invention further extends to an inhibitor which is a polypeptide (or the nucleic acid molecules encoding it) containing one or more mutations in one or more binding sites described herein, in which said mutation results in a molecule which has impaired binding at that site to the relevant binding partner relative to the same molecule without the mutation.
  • one or more of the valine and/or isoleucine residues in the PKA binding site of the ezrin molecule may be mutated, preferably to a proline residue, and the resultant molecule used as an inhibitor to interfere with the ability of the endogenous enzyme to bind to its binding partner PKA.
  • the present invention thus also extends to novel modified, e.g. mutated binding partners or functionally equivalent variants, derivatives or fragments thereof and the nucleic acid molecules which encode them.
  • the present invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding a binding partner, or a functionally equivalent variant, derivative or fragment thereof, as defined above, wherein said sequence is modified as defined above to alter its ability to bind to its binding partner as defined above.
  • Appropriate inhibitors for use in methods of the invention may be identified or tested using appropriate screening tests.
  • the present invention provides a method of screening for, or testing the ability or efficacy of, a molecule to reduce or inhibit binding between any one of the aforementioned binding partners, wherein said test molecule is contacted with said binding partners and the extent of binding is assessed.
  • binding partners are present in isolated form, for example the first of said binding pair may be immobilized on a solid support and the ability of the second of said pair to bind to said first binding partner may be assessed in the presence or absence of said test molecule, ie. by competition.
  • said binding partners may be present in endogenous form, e.g. in a cell and the ability of said test molecule to affect PKA type I signalling by examination of an indicator of said signalling, e.g. T-cell proliferation or IL-2 release, may be examined.
  • Preferred inhibitors of binding between the ezrin and PKA type I binding partners as described hereinbefore mimic the PKA type I binding site on ezrin, ie.
  • inhibitors are derived from and/or mimic said binding site.
  • said inhibitor may comprise or may encode one or more of the residues mentioned above and/or e.g.
  • inhibitors as described herein consist of less than 100 amino acids, e.g. are or encode peptides which consist of less than 100 amino acids, but are preferably shorter, e.g. less than 50, e.g.
  • “Functionally equivalent” variants, derivatives or fragments thereof refers to molecules, preferably peptides, related to, or derived from the above described amino acid sequences, where the amino acid sequence has been modified by single or multiple amino acid (e.g. at 1 to 10, e.g. l to 5, preferably 1 or 2 residues) substitution, addition and/or deletion or chemical modification, including deglycosylation or glycosylation, but which nonetheless retain functional activity, insofar as they act as inhibitors, e.g.
  • substitution variants are included amino and/or carboxyl terminal fusion proteins or polypeptides, comprising an additional protein or polypeptide or other molecule fused to the inhibitor (or binding partner) sequence.
  • substitution variants preferably involve the replacement of one or more amino acids with the same number of amino acids and making conservative substitutions .
  • Such functionally-equivalent variants mentioned above include in particular naturally occurring biological variations (e.g. allelic variants or geographical variations within a species, most particularly variants and alleles found within humans) and derivatives prepared using known techniques.
  • inhibitors (or binding partners) described herein extend to inhibitors (or binding partners) which are functional in (or present in) , or derived from proteins isolatable from, different genera or species than the specific inhibitors and binding partner molecules mentioned herein.
  • Preferred "derivatives" or “variants” include those in which instead of the naturally occurring amino acid the amino acid which appears in the sequence is a structural analog thereof .
  • Amino acids used in the sequences may also be derivatized or modified, e.g. labelled, glycosylated or methylated, providing the function of the inhibitor (or binding partner) is not significantly adversely affected.
  • Derivatives particularly include peptidomimetics which may be prepared using techniques known in the art.
  • non-standard amino acids such as ot- aminobutyric acid, penicillamine, pyroglutamic acid or conformationally restricted analogs, e.g. such as Tic (to replace Phe) , Aib (to replace Ala) or pipecolic acid (to replace Pro) may be used.
  • Tic to replace Phe
  • Aib to replace Ala
  • pipecolic acid to replace Pro
  • Other alterations may be made when the inhibitor is to be used in the methods of the invention described hereinafter (or the binding partners are to be used for screening for inhibitors) . In such cases, the stability of the inhibitor (or binding partner), e.g. peptide, may be enhanced, e.g.
  • D-amino acids or amide isosteres (such as N- methyl amide, retro-inverse amid, thioamide, thioester, phosphonate, ketomethylene, hydroxymethylene, fluorovinyl, (E) -vinyl, methyleneamino, methylenethio or alkane) which protect the peptides against proteolytic degradation.
  • Di (oligo) peptidomimetics may also be prepared.
  • Precursors of the inhibitors (or binding partners) are also encompassed by the term functionally equivalent variants and include molecules which are larger than the inhibitors (or binding partners) and which may optionally be processed, e.g. by proteolysis to yield the inhibitor (or binding partner) .
  • Additional moieties may also be added to the inhibitors (or binding partners) to provide a required function, e.g. a moiety may be attached to assist or facilitate entry of the inhibitor into the cell .
  • Derivatives and variants as described above may be prepared during synthesis of the inhibitor (or binding partner if isolated binding partners are to be used) , e.g. peptide, or by post-production modification, or when the peptide is in recombinant form, using the known techniques of site-directed mutagenesis including deletion, random mutagenesis and/or ligation of nucleic acids .
  • Functionally-equivalent "fragments" according to the invention may be made by truncation, e.g.
  • fragments may be derived from the inhibitor peptides (or binding partners) described above, or may be derived from a functionally equivalent peptide as described above, but which retain the ability to act as an inhibitor (or binding partner) according to the method of the invention.
  • fragments are between 6 and 30 residues in length, e.g. 6 to 25 or 10 to 15 residues.
  • these fragments satisfy the homology (relative to a comparable region) or hybridizing conditions mentioned herein.
  • functional variants according to the invention have an amino acid sequence which has more than 75%, e.g. 75 or 80%, preferably more than 85%, e.g.
  • nucleotide sequences are the degenerate sequences which encode the recited polypeptides or their variants or fragments .
  • peptide inhibitors in this aspect comprise the following sequence or a functionally equivalent variant, derivative or fragment thereof, based on residues 413- 430:
  • each of Xi to X s is any amino acid, wherein Xi is preferably arginine, glutamic acid, glutamine, threonine, phenylalanine, lysine or tyrosine, especially preferably glutamine;
  • X 2 is preferably arginine, lysine, glycine, phenylalanine, alanine, serine or asparagine, especially preferably alanine;
  • X 3 is preferably arginine, glutamic acid, glutamine, threonine, lysine, serine, valine, alanine or leucine, especially preferably glutamic acid;
  • X 4 is preferably glutamic acid, glutamine, lysine, leucine, serine, aspartic acid, alanine, glycine or asparagine, especially preferably glutamic acid;
  • X 5 is preferably tyrosine;
  • X s is preferably thre
  • said sequence is:
  • said sequence may be based on ezrin residues 367-385 and may have the form: D/EX 1 X 2 X 3 X 4 X 5 LX 6 X 7 X 8 EEX 9 X ;L0 X 11 X. L2 X 13 EE
  • each of X_ and X 2 is any amino acid, wherein X ⁇ is preferably arginine, glutamine, alanine, glutamic acid or isoleucine, preferably glutamine; X 2 is preferably asparagine, isoleucine, glutamic acid or glutamine, preferably isoleucine; and wherein X 3 is glutamic acid, glutamine, valine, arginine or phenylalanine, preferably glutamine; X 4 is glutamic acid, arginine, leucine or methionine, preferably arginine; X s is glycine, alanine, arginine or glutamic acid, preferably alanine,- X 6 is aspartic acid, glutamine, lysine or arginine, preferably glutamine; X 7 is arginine, leucine, methionine or lysine, preferably leucine; X B is asparagine, glutamic acid,
  • said sequence is : D/ EX I X 2 X 3 X 4 ALQX 7 EEEX X ⁇ QRX I X I3 EE .
  • inhibitors or their encoding nucleic acid sequences form further preferred aspects of the invention for use in methods of the invention such as a method of altering the PKA type I signalling pathway in a cell by administration of said inhibitor.
  • Preferred inhibitors of binding between the ezrin and EBP50 binding partners as described hereinbefore mimic the ezrin binding site on EBP50 or the EBP50 binding site on ezrin, ie. residues 329 to 358 of SEQ ID No. 3 (EBP50) , or residues 1-286 of SEQ ID No. 1 (ezrin) , respectively, or variants as described hereinbefore .
  • the inhibitors are derived from and/or mimic said binding sites.
  • said inhibitor may comprise or encode one or more of residues 1-20, 20- 40, 40-60, 60-80, 80-100, 100-120, 120-140, 140-160, 160-180, 180-200, 200-220, 220-240, 240-260 or 260-286 of SEQ ID No. 1 or 329 to 349 or 339 to 358 of SEQ ID No. 3 or functionally equivalent variants, derivatives or fragments of such peptides as described hereinbefore.
  • Preferred inhibitors of binding between the EBP50 and Cbp/PAG binding partners as described hereinbefore mimic the EBP50 binding site on Cbp/PAG or the Cbp/PAG binding site on EBP50, ie . 429 to 432 residues of SEQ ID No.
  • inhibitors are derived from and/or mimic said binding sites.
  • said inhibitor may comprise or encode one or more of residues 429-432 of SEQ ID No. 4 or residues 11-30, 31-50, 51-70, 71-90, 80-97, 149-170, 171-190, 191-210, 211-230 or 215-236 of SEQ ID No. 3 or functionally equivalent variants, derivatives or fragments of such peptides as described hereinbefore .
  • inhibitors may where appropriate include full length molecules identical or closely related to those found endogenously to compete with the wild-type molecule.
  • appropriate inhibitors include any of the aforementioned binding partner proteins in complete form.
  • the present invention also extends to antibodies (monoclonal or polyclonal) and their antigen-binding fragments (e.g F(ab) 2 , Fab and Fv fragments ie. fragments of the "variable" region of the antibody, which comprises the antigen binding site) directed to the binding sites or inhibitor peptides as defined hereinbefore, ie .
  • inhibitors as described herein are conveniently proteinaceous, however the use of other types of inhibitors of the binding is contemplated, e.g. molecules which match the binding sites' spatial and conformational structure.
  • inhibitor molecules as described hereinbefore are conveniently added to a cell . This may be achieved by relying on spontaneous uptake of the inhibitor into the cells or appropriate carrier means may be provided.
  • Exogenous peptides or proteins may thus be introduced by any suitable technique known in the art such as in a liposome, niosome or nanoparticle or attached to a carrier or targetting molecule (see hereinafter) .
  • the protein may be tagged with a suitable sequence that allows the protein to cross the cell membrane.
  • An example of such a tag is the HIV tat sequence or a stretch of e.g. 11 arginines . It will be appreciated that the level of exogenous molecules introduced into a cell will need to be controlled to avoid adverse effects .
  • the inhibitor may be transported into the cell in the form of the inhibitor or in the form of a precursor, e.g.
  • the inhibitors may be administered to a cell by transfection of a cell with a nucleic acid molecule encoding the peptide or protein inhibitor.
  • the present invention thus extends to nucleic acid molecules comprising a sequence which encodes the peptide/polypeptide inhibitors described herein and their use in methods described herein.
  • said nucleic acid molecules are contained in a vector, e.g. an expression vector.
  • the nucleic acid molecules described above may be operatively linked to an expression control sequence, or a recombinant DNA cloning vehicle or vector containing such a recombinant DNA molecule.
  • This allows intracellular expression of the inhibitor as a gene product, the expression of which is directed by the gene(s) introduced into cells of interest.
  • Gene expression is directed from a promoter active in the cells of interest and may be inserted in any form of linear or circular DNA vector for incorporation in the genome or for independent replication or transient transfection/expression. Alternatively, the naked DNA molecule may be injected directly into the cell.
  • Appropriate expression vectors include appropriate control sequences such as for example translational (e.g.
  • vectors may include plasmids and viruses (including both bacteriophage and eukaryotic viruses) .
  • Suitable viral vectors include baculovirus and also adenovirus, adeno-associated virus, herpes and vaccinia/pox viruses . Many other viral vectors are described in the art.
  • Preferred vectors include bacterial and mammalian expression vectors pGEX-KG, pEF- neo and pEF-HA.
  • the nucleic acid molecule may conveniently be fused with DNA encoding an additional polypeptide, e.g. glutathione-S-transferase, to produce a fusion protein on expression.
  • an additional polypeptide e.g. glutathione-S-transferase
  • the present invention provides a vector comprising a nucleic acid molecule as defined above.
  • Other aspects of the invention include methods for preparing recombinant nucleic acid molecules according to the invention, comprising inserting nucleotide sequences encoding the modified substrate into vector nucleic acid.
  • Nucleic acid molecules of the invention preferably contained in a vector, may be introduced into a cell by any appropriate means . Suitable transformation or transfection techniques are well described in the literature.
  • a variety of techniques are known and may be used to introduce such vectors into prokaryotic or eukaryotic cells for expression.
  • Preferred host cells for this purpose include insect cell lines, eukaryotic cell lines or E. coli , such as strain BL21/DE3.
  • the invention also extends to transformed or transfected prokaryotic or eukaryotic host cells containing a nucleic acid molecule, particularly a vector as defined above.
  • a further aspect of the invention provides a method of preparing a peptide or protein inhibitor of the invention as hereinbefore defined, which comprises culturing a host cell containing a nucleic acid molecule as defined above, under conditions whereby said inhibitor is expressed and recovering said inhibitor thus produced.
  • the expressed protein product forms a further aspect of the invention.
  • the invention also extends to an inhibitor encoded by a nucleic acid molecule as hereinbefore described. This may be produced by expression of a host cell as described above. Cells containing inhibitors of the invention, introduced directly as inhibitors or by expression of encoding nucleic acid material form further aspects of the invention. Nucleic acid molecules which may be used according to the invention may be single or double stranded DNA, cDNA or RNA, preferably DNA and include degenerate, substantially homologous and hybridizing sequences as described before. Ideally however genomic DNA or cDNA is employed. Inhibitors as described above may be prepared by conventional modes of synthesis including genetic or chemical means .
  • Chemical syntheses may be performed by methods well known in the art involving in the case of peptides cyclic sets of reactions of selection deprotection of the functional groups of a terminal amino acid and coupling of selectively protected amino acid residues, followed finally by complete deprotection of all functional groups. Synthesis may be performed in solution or on a solid support using suitable solid phases known in the art, such as the well known Merrifield solid phase synthesis procedure.
  • the inhibitors of the invention are substantially purified, e.g. pyrogen-free, e.g. more than 70%, especially preferably more than 90% pure (as assessed for example, in the case of peptides or proteins, by an appropriate technique such as peptide mapping, sequencing or chromatography) .
  • Purification may be performed for example by chromatography (e.g. HPLC, size-exclusion, ion-exchange, affinity, hydrophobic interaction, reverse-phase) or capillary electrophoresis .
  • Alternative methods of reducing binding between the binding partners as defined hereinbefore includes modification of endogenous molecules taking part in said binding.
  • the invention extends to modifying the endogenous binding partner as described hereinbefore in a cell . This may be achieved for example by manipulation of the wild-type gene, by manipulating expression of the gene (e.g. by affecting transcription or translation) or by manipulating the expressed product. This could for example be achieved by using antisense oligonucleotides comprising nucleic acid sequences as described hereinbefore (ie.
  • binding partners or of relevant protein/peptide inhibitors or their complementary sequences, ribozymes, RNAi or antibodies and the invention extends to such molecules and their uses.
  • this could be performed for example by somatic cell gene therapy with homologous recombination to for example remove or mutate the binding site.
  • This could be performed on for example hematopoietic stem cells or on blood cells ex vivo or in vivo .
  • Mutation of one or more of the leucine or valine residues to a proline residue in the ezrin binding site for PKA for example could be performed to generated proteins that have reduced binding to PKA.
  • wild-type or mutated sequences may be used to cause co-suppression of the naturally occurring molecule.
  • exogenous molecules may be administered to cells as described hereinbefore.
  • the above described methods of the invention may be used in vi tro, for example in cell or organ culture, particularly for affecting PKA type I signalling pathways which have been activated or to reduce the extent of endogenous signalling.
  • the method may also be used ex vivo, on animal parts or products, for example organs or collected blood, cells or tissues, particularly when it is contemplated that these will be reintroduced into the body from which they are derived.
  • levels may be normalized, e.g.
  • sample refers to any material obtained from a human or non-human animal, including tissues and body fluid.
  • Body fluids in this case include in particular blood, spinal fluid and lymph and "tissues” include tissue obtained by surgery or other means .
  • tissues include tissue obtained by surgery or other means .
  • Such methods are particularly useful when the inhibitor is to be introduced into the body by expression of an appropriate nucleic acid molecule, or the inhibitor is itself a nucleic acid molecule. T cells for example, could be treated in this way.
  • the methods of treatment of the invention as described hereinafter comprise the initial step of obtaining a sample from an individual, contacting cells from said sample with an inhibitor (or a nucleic acid molecule encoding an inhibitor) of the invention and administering said cells of said sample to the individual .
  • the step of contacting refers to the use of any suitable technique which results in the presence of said inhibitor in cells of the sample.
  • the method may also be used in vivo for the treatment or prevention of diseases in which abnormal PKA type I signalling occurs and this will be discussed in more detail below. As described previously the methods of altering PKA type I signalling have utility in a variety of clinical indications in which abnormal PKA type I signalling is exhibited.
  • the inhibitors which abolish the function of PKA type I may be used to produce pharmaceutical preparations to treat immunosuppressive disease.
  • the inhibitors may be used to treat or prevent disorders typified by aberrant PKA type I signalling such as immunosuppressive disorders (such as HIV infection, AIDS or common variable immunodeficiency) or proliferative diseases in which PKA type I signalling has been implicated, e.g. cancers such as colorectal carcinoma, pancreatic carcinoma, hepatocellular carcinoma, cancer mamma, ovarian cancer and non-small cell carcinoma of the lung) .
  • the inhibitors as described herein may therefore be formulated as pharmaceutical compositions in which the inhibitor may be provided as a pharmaceutically acceptable salt.
  • Pharmaceutically acceptable salts may be readily prepared using counterions and techniques well known in the art.
  • the invention thus further extends to pharmaceutical compositions comprising one or more inhibitors (e.g. nucleic acid molecules, peptides or proteins, such as an antisense oligonucleotide, ribozyme or antibody, nucleic acid molecule or peptide/protein) as defined above and one or more pharmaceutically acceptable excipients and/or diluents .
  • pharmaceutically acceptable is meant that the ingredient must be compatible with other ingredients in the composition as well as physiologically acceptable to the recipient.
  • the active ingredient for administration may be appropriately modified for use in a pharmaceutical composition.
  • peptides such as peptidomimetics as described hereinbefore.
  • the active ingredient may also be stabilized for example by the use of appropriate additives such as salts or non-electrolytes, acetate, SDS, EDTA, citrate or acetate buffers, mannitol, glycine, HSA or polysorbate.
  • Conjugates may be formulated to provide improved lipophilicity, increase cellular transport, increase solubility or allow targeting. Conjugates may be made terminally or on side portions of the molecules, e.g. on side chains of amino acids.
  • conjugates may be cleavable such that the conjugate behaves as a pro-drug. Stability may also be conferred by use of appropriate metal complexes, e.g. with Zn, Ca or Fe .
  • the active ingredient may be formulated in an appropriate vehicle for delivery or for targeting particular cells, organs or tissues.
  • the pharmaceutical compositions may take the form of microemulsions, liposomes, niosomes or nanoparticles with which the active ingredient may be absorbed, adsorbed, incorporated or bound. This can effectively convert the product to an insoluble form.
  • These particulate forms have utility for transfer of nucleic acid molecules and/or protein/peptides and may overcome both stability (e.g. enzymatic degradation) and delivery problems .
  • These particles may carry appropriate surface molecules to improve circulation time (e.g. serum components, surfactants, polyoxamine908 , PEG etc.) or moieties for site-specific targeting, such as ligands to particular cell borne receptors .
  • Appropriate techniques for drug delivery and for targeting are well known in the art and are described in W099/62315.
  • site directed targeting see for example Schafer et al . , 1992, Pharm. Res., 9, p541-546 in which nanoparticles can be accumulated in HIV-infected macrophages.
  • Such derivatized or conjugated active ingredients are intended to fall within the definition of inhibitory molecules which form aspects of this invention.
  • compositions for use according to the invention may be formulated in conventional manner using readily available ingredients.
  • the active ingredient may be incorporated, optionally together with other active substances as a combined preparation, with one or more conventional carriers, diluents and/or excipients, to produce conventional galenic preparations such as tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions (as injection or infusion fluids) , emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium) , ointments, soft and hard gelatin capsules, suppositories, sterile injectable solutions, sterile packaged powders, and the like.
  • Biodegradable polymers may also be used for solid implants.
  • the compositions may be stabilized by use of freeze-drying, undercooling or Permazyme .
  • Suitable excipients, carriers or diluents are lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, calcium carbonate, calcium lactose, corn starch, aglinates, tragacanth, gelatin, calcium silicate, macrocrystalline cellulose, polyvinylpyrrolidone, cellulose, water syrup, water, water/ethanol, water/glycol, water/polyethylene, glycol, propylene glycol, methyl cellulose, methylhydroxybenzoates, propyl hydroxybenzoates, talc, magnesium stearate, mineral oil or fatty substances such as hard fat or suitable mixtures thereof.
  • compositions may additionally include lubricating agents, wetting agents, viscosity increasing agents, colouring agents, granulating agents, disintegrating agents, binding agents, osmotic active agents, emulsifying agents, suspending agents, preserving agents, sweetening agents, flavouring agents, adsorption enhancers, e.g. for nasal delivery (bile salts, lecithins, surfactants, fatty acids, chelators) and the like.
  • lubricating agents wetting agents, viscosity increasing agents, colouring agents, granulating agents, disintegrating agents, binding agents, osmotic active agents, emulsifying agents, suspending agents, preserving agents, sweetening agents, flavouring agents, adsorption enhancers, e.g. for nasal delivery (bile salts, lecithins, surfactants, fatty acids, chelators) and the like.
  • compositions of the invention may be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration of the patient by employing procedures well known in the art .
  • the active ingredient in such compositions may comprise from about 0.01% to about 99% by weight of the formulation, preferably from about 0.1 to about 50%, for example 10%.
  • the invention also extends to pharmaceutical compositions as described above for use as a medicament. In methods of the invention, inhibitors should be used at appropriate concentrations such that a significant number of the relevant binding partners' interactions, are prevented.
  • the pharmaceutical composition is formulated in a unit dosage form, e.g. with each dosage containing from about 0.1 to 500mg of the active ingredient.
  • an effective dose may lie in the range of from about O.Olmg/kg to 20mg/kg, depending on the animal to be treated, and the substance being administered (e.g 0.1 to 7mg/kg for antisense oligonucleotides) , taken as a single dose.
  • suitable dosages of said inhibitor are 25-100nM or lower, such as 10-50nM or 5-25nM.
  • the administration may be by any suitable method known in the medicinal arts, including for example oral, parenteral (e.g. intramuscular, subcutaneous, intraperitoneal or intravenous) percutaneous, buccal, rectal or topical administration or administration by inhalation.
  • parenteral e.g. intramuscular, subcutaneous, intraperitoneal or intravenous
  • the preferred administration forms will be administered orally, rectally or by injection or infusion.
  • oral administration has its limitations if the active ingredient is digestible. To overcome such problems, ingredients may be stabilized as mentioned previously and see also the review by Bernkop-Schn ⁇ rch, 1998, J. Controlled Release, 52, pl-16. It will be appreciated that since the active ingredient for performance of the invention takes a variety of forms, e.g.
  • compositions may be used for treating or preventing conditions in which the PKA type I signalling pathway is abnormal, in particular when the activity of this pathway is elevated.
  • the present invention provides a method of treating or preventing disorders exhibiting abnormal PKA type I signalling activity, preferably immunosuppressive disorders or proliferative diseases, in a human or non-human animal wherein a pharmaceutical composition as described hereinbefore is administered to said animal .
  • the present invention provides the use of a pharmaceutical composition as defined above for the preparation of a medicament for the treatment or prevention of immunosuppressive disorders or proliferative diseases.
  • a "disorder" or “disease” refers to an underlying pathological disturbance in a symptomatic or asymptomatic organism relative to a normal organism, which may result, for example, from infection or an acquired or congenital genetic imperfection.
  • a “condition” refers to a state of the mind or body of an organism which has not occurred through disease, e.g. the presence of a moiety in the body such as a toxin, drug or pollutant.
  • an “immunosuppressive disorder” refers to a disorder which is typified by impaired function of cells involved in normal immune responses, particularly B and T cells, and is also referred to herein as immunodeficiency or immune dysfunction.
  • immunodeficiency disorders are treated.
  • Preferred conditions for treatment according to the invention include infection by retroviruses, particularly HIV (and infection by related viruses in other animals, e.g.
  • the methods described herein may be used to reverse cAMP hyperactivation that produces T cell dysfunction in immunodeficiencies by interrupting cAMP and PKA-mediated signal transduction in T cells, particularly T cell lipid rafts.
  • proliferative diseases refers to those diseases in which aberrant proliferation of cells occurs.
  • said cells are cells involved in the generation or maintenance of immune responses, particularly B or T cells. Such diseases concern decreases in proliferation relative to normal levels .
  • Subjects which may be treated are preferably mammalian, preferably humans and companion or agricultural animals such as dogs, cats, monkeys, horses, sheep, goats, cows, rabbits, rats and mice.
  • treating refers to the reduction, alleviation or elimination, preferably to normal levels, of one or more of the symptoms of said disease, disorder or condition which is being treated, e.g. infectivity or a reduction or alleviation of immune dysfunction, relative to the symptoms prior to treatment.
  • Preventing refers to absolute prevention, ie . absence of detectable infectious agents, e.g. virus and/or maintenance of normal levels with reference to the extent or appearance of a particular symptom (e.g.
  • the method of treatment according to the invention may advantageously be combined with administration of one or more active ingredients which are effective in treating the disorder or disease to be treated.
  • additional active ingredients are cAMP antagonists e.g. a thiosubstituted cAMP analog (such as derivatives of adenosine-3 ', 5 ' -cyclic monophosphorothioate, Rp isomer, such as Rp-8-Br-cAMPS or Rp-8-Cl-cAMPS) , or COX-2 inhibitors as described in WO02/07721, which is incorporated herein by reference.
  • cAMP antagonists e.g. a thiosubstituted cAMP analog (such as derivatives of adenosine-3 ', 5 ' -cyclic monophosphorothioate, Rp isomer, such as Rp-8-Br-cAMPS or Rp-8-Cl-cAMPS)
  • COX-2 inhibitors as described in WO02/07
  • compositions of the invention may additionally contain one or more of such active ingredients .
  • the present invention provides methods and/or compositions which combine one or more inhibitors as described herein with compounds that improve the tolerability of the active ingredient, especially during long term treatment.
  • Typical compounds include antihistamine and proton pump inhibitors .
  • Typical compounds include antihistamine and proton pump inhibitors .
  • Figure 1 shows the identification of AKAPs in T cell lipid rafts in which lysed Jurkat T cells fractionated on sucrose gradients were resolved on SDS-PAGE, blotted to PVDF-membranes, subjected to Rll-overlay assay (A) and probed with the indicated antibodies (B) .
  • Immunodetection of LAT-antibody was used as a marker for lipid rafts. Lanes refer to fraction numbers following sucrose gradient centrifugation of the lysate and fractionation from the top. Mobility of molecular weight markers are indicated;
  • Figure 2 shows deletional mapping of the PKA anchoring domain in ezrin.
  • Schematic view of ezrin fragments expressed and tested for interaction (A, left) . Depicted are the N-terminal (dark shaded) ; oi-helical (light shaded) and C-terminal (black) domains of Ezrin. Fragments were tested for interaction with Rio. and RIIo; in the yeast-2 -hybrid system by growing on selective media and examining ⁇ -gal (A, middle panel) . GST-linked ezrin fragments as indicated were incubated separately with Rio; and RIIo. proteins and precipitated using glutathione-agarose beads (A, right) .
  • Figure 3 shows the determination of association and dissociation rate constants for the binding of Ezrin to the Rio;-subunit .
  • Figure 4 shows the effect of actin disruption by cytochalasin D on distribution of AKAPs in lipid rafts.
  • Lipid rafts were purified from T lymphocytes by sucrose gradient centrifugation following incubation in the absence or presence of cytochalasin D (80 mM) . The same experiment without cytochalasin treatment was performed in parallel. Fractions were resolved on SDS-PAGE, blotted to PVDF-membranes and probed with the indicated antibodies (A, B two different fractions) . Lanes as indicated in Figure 1. Immunoblotting with anti-LAT antibody indicated lipid raft containing fractions. Molecular weight markers are indicated;
  • Figure 5 shows that ezrin interacts with PKA type Io. in T-cell lipid rafts.
  • Lipid raft fractions of T lymphocytes were isolated, pooled and subjected to precipitation with mouse monoclonal antibodies against Rio, RIIo or control IgG (A) . Precipitates were analyzed by 10% SDS-PAGE followed by immunoblotting with mouse monoclonal antibodies against Ezrin (upper panel) , RIo;, RIIoc and PKA-C subunits (lower panels) . Similarly, lipid raft fractions were subjected to immunoprecipitation with anti-ezrin antibodies, precipitated and immunoblotted with the indicated antibodies (B) ;
  • Figure 6 shows that ezrin colocalizes with the RIo.- subunit of PKA in T cell lipid rafts .
  • Jurkat-T cells were immunostained using (A) a mouse monoclonal antibody against RIo; and (B) rabbit polyclonal antibody against the lipid raft specific protein LAT; rabbit polyclonal antibody against ezrin (D,G) and a mouse monoclonal antibody against RIo; (E) or RIIo (H) . Merged images show overlapping subcellular distribution (C,F,I);
  • Figure 7 shows that the RIo;-subunit of PKA is distributed with VSVG-tagged ezrin.
  • Jurkat T-lymphocytes transfected with cDNA constructs coding for VSVG-tagged (full-length) ezrin were stained with mouse monoclonal antibody VSVG (A) and mouse monoclonal IgG 2A antibody against RIo. (B) . Colocalization of RIo; with Ezrin-VSVG appears as signal in the merged image (C) . Control immunocytochemical analysis showed no antibody signal. Scale bar, 5 ⁇ m;
  • Figure 8 shows that ezrin is colocalized with PKA, EBP50 and Csk in T-cell lipid rafts.
  • Lipid raft fractions of T lymphocytes were isolated, pooled and subjected to precipitation with rabbit polyclonal against Csk or control IgG (A) . Precipitates were analyzed by 10% SDS-PAGE followed by immunoblotting with mouse monoclonal antibodies against ezrin (upper panel) or EBP50 (lower panel) . Similarly, lipid raft fractions were subjected to immunoprecipitation with mouse monoclonal anti-EBP50 antibodies, precipitated and immunoblotted with antibodies to Csk or PKA-C (B) .
  • Figure 8C is a schematic of the complex formed during PKA type I signalling;
  • Figure 9 shows the expression of soluble R-binding domains of ezrin and competition of Ht31 to remove anchored RIx from lipid rafts and disrupt cAMP inhibition of T cell function.
  • T lymphocytes were transfected with mammalian expression vectors encoding PKA-binding domains of ezrin, or empty vector and incubated for 16 h. Subsequently, cells were incubated with or without 50 ⁇ M (15 min) cAMP and next stimulated with anti-CD3£ mAb OKT3 and incubated for 20 h. Supernatants were harvested and IL-2 levels were determined by ELISA and the effect of anchoring disruption on cAMP-mediated inhibition of IL-2 production assessed (A) . The release from inhibition by cAMP was also calculated as fold increase in IL-2 production upon disruption of PKA anchoring (B) ;
  • Figure 10 shows the identification of a putative upstream type I regulatory subunit binding domain (RI- AKAP specifier region) in PKA type I binding AKAPs using bioinformatics .
  • the alignment was performed using the Clustal-W program.
  • the names of the AKAPs and residue numbers of the binding domains are indicated on the right. Boxed text shows conserved residues with similar functional properties.
  • the down-stream amphipathic helix binding domain that may confer binding to both PKA types I and II (albeit with different specificities) is shown.
  • the locations of additional residues with structural similarity are marked by stars,-
  • Figure 11 shows analysis of binding of PKA type I and II to various parts of ezrin by peptide array. Scanning arrays of the ezrin polypeptide were synthesized (Autospot, Intavis AG) where each peptide (spot) represents a 20-mer with a 2-residue frame shift from the previous peptide. The immobilized peptides were analyzed for R binding by either RII- 32 P-overlay with wild type RIIo. or by overlay with RI- 32 P RIo.(A98S) or 1-11, 1-15 or 1-24 N-terminal deletions of RIo;(A98S) as indicated above the columns. Binding of 32 P-labeled RI or RII was detected by autoradiography. Regions that appear to bind in the scan analysis are shown;
  • Figure 12 shows lowered sensitivity to cAMP in inhibition of immune responses in mature T cells with Ezrin knockdown.
  • EXAMPLE 1 Identification of Ezrin as an anchoring protein for PKA type I and EBP50
  • Saccharomyces cerevisiae yeast strains were grown at 30°C in standard liquid YPD medium or minimal SD synthetic medium with appropriate supplement amino acids (Clontech Laboratories, Inc.) .
  • Plasmids were retrans ormed into yeast reporter strain HF7c (genotype: MATa, ura3-52, his3-200, lys2-801, ade2-101, trpl-901, leu2-3, 112, gal4-542, gal80-538, LYS : :GAL-HIS3 , URA3 : : (GAL4 17mers) 3 - CYCl-lacZ from Clontech) with plasmid pGBT-PBR to test for histidine prototrophy and ⁇ -galactosidase activity (Clontech, manual) .
  • the cDNA inserts from the positive clones were sequenced.
  • Ezrin constructs were cloned into the EG202 bait vector and RIo; and RIIo; into the JG4-5 prey vector. Mating assays and detection of interactions were performed as described (Gronholm et al . , 1999, J. Cell Sci., 112, p895-904) . Cell Cul ture, Stimulation, and Transfection of Jurkat TAg
  • the human leukemic T cell line Jurkat TAg a derivative of the Jurkat cell line was kept in logarithmic growth in RPMI medium supplemented with 10% fetal calf serum and antibiotics.
  • T-cells (2 x IO 7 ) in 0.4 ml of Opti-MEM were mixed with 20 ⁇ g of Ht31-cDNA construct in electroporation cuvettes with a 0.4 cm electrode gap (Bio-Rad) and subjected to an electric field of 250 V/cm with 960-microfarad capacitance .
  • the cells were expanded in complete medium and harvested after 20 h.
  • Peripheral blood CD3+ T cells were purified by negative selection from buffycoats from normal healthy donors (Ullevaal University Hospital Blood Center, Oslo, Norway) . Briefly, peripheral blood mononuclear cells were isolated by density gradient (Lymphoprep, NycoMed, Oslo, Norway) centrifugation followed by negative selection using monodisperse magnetic beads directly coated with antibodies to CD14 and CD19 and rat anti- mouse IgG beads coated with antibodies to CD56 and a magnet. Magnetic beads were all from Dynal (Oslo, Norway, cat. no. 111.12, 111.04 and 110.11, respectively) whereas anti-CD56 antibody was from Pharmingen (San Diego, CA, cat. no. 31660. d) . All steps were performed at 4°C. Cell suspensions were analyzed by flow cytometry and routinely shown to consist of more than 90% CD3+ cells.
  • T-cells (5xl0 6 ) in 0.1 ml of Nucleofection solution (Amaxa) were mixed with 2 ⁇ g of each cDNA construct and subjected to electroporation in a Nucleofector (Amaxa) following the manufacturer's protocol. The cells were expanded in complete medium and incubated for 16h at 37°C. 4x 10 s T-cells were treated with 50 ⁇ M cAMP for 15 min (37°C) and activated by the addition of 5 g/ml anti-CD3 ⁇ mAb OKT-3. After 16 h incubation at 37°C the level of secreted IL-2 was determined by ELISA.
  • T-cells or Jurkat T-Cells (3x10 s cells obtained from confluent culture flasks) were washed in PBS, resuspended in 1ml of MES containing 80 ⁇ g/ml Cytochalasin D and pelleted after 30 min at 37°C.
  • T- cells were resuspended in 1 ml ice-cold lysis buffer (25 mM MES, pH 6.5, 100 mM NaCl, 5 mM EDTA, 0,7% Triton X-100 with 1 mM sodium orthovanadate, 1 mM PMSF, 10 mM sodium pyrophosphate, and 50 mM sodium fluoride) containing 0.7% Triton X100 and lysed 10 min on ice.
  • 1 ice-cold lysis buffer 25 mM MES, pH 6.5, 100 mM NaCl, 5 mM EDTA, 0,7% Triton X-100 with 1 mM sodium orthovanadate, 1 mM PMSF, 10 mM sodium pyrophosphate, and 50 mM sodium fluoride
  • the lysate was mixed 1:1 with 80% (w/v) sucrose and placed at the bottom of a 5.2 ml polyallomer centrifuge tube (Beckman Instruments), then carefully overlaid with 2.0 ml of 30% (w/v) sucrose in MNE-buffer and finally with 1 ml of 5% (w/v) sucrose in MNE-buffer.
  • Centrifugation was performed at 4°C in a Beckman SW55Ti rotor (20 h, 46000 rpm) . Twelve 0.4 ml fractions were collected gradually from the top of the gradient, proteins were separated by SDS-PAGE and analyzed by immunoblotting.
  • Ezrin fragments fused to GST were produced in Escherichia coli BL21 cells following stimulation with IPTG (3h) .
  • BL21 cells were pelleted from 10 ml of broth, lysed and after removal of insoluble pellet, glutathione beads were added for lh at 4 Q C. The beads were then washed and finally boiled in SDS-PAGE buffer. Proteins were separated on 10% polyacrylamide gel .
  • RI overlay membranes were blocked in "blotto" overnight at 4°C, 100 nM GST-cleaved RIo: was added to "blotto" and incubated with the membrane for 4-6 h at 4°C. Membranes were then washed with PBS and blocked for Western blot analysis against RIo;.
  • RIIo overlay was performed as previously described (Carr & Scott, 1992, Trends Biochem. Sci., 17, p246-249). Briefly, bovine RIIo; was radiolabelled with [ ⁇ - 3Z P] -ATP and unincorporated ATP was removed by gel filtration. Labelled RIIo was then incubated with the membrane overnight and washed extensively to remove non-specific binding. GST-precipitation assay
  • Fragments of ezrin fused to GST were expressed in Escherichia coli BL21 cells, induced using 0.4 mM IPTG and purified on glutathione-sepharose (Amersham Pharmacia Biotech) .
  • 2.5 ⁇ g of purified ezrin fragments and R subunits were incubated at a 1:1 ratio (50 ⁇ M each) in 100 ⁇ l pull-down buffer (300 mM NaCl, 0.1 % Triton X-100, lmM PMSF, lmM EDTA, 5mM benzamidine, 5 mM DTT, lO ⁇ g/ml of antipain, chymostain, leupeptin and pepstatin A) overnight at 4°C.
  • Biol., 327, p609-618) identified 12 clones that interacted with the full-length but not the deleted RIo; bait. Of the 12 clones, 2 were identified as ezrin, 2 were identified as PAP7 , a itochondrial AKAP for PKA type I as earlier reported (Li et al . , 2001, Mol. Endocrinol . , 15, p2211-2228) and the remaining 8 were checked out as false positives.
  • RII overlay screening of a Jurkat T cell Lambda Zap expression library was performed using radio- labelled RIIo; as a probe.
  • this screening identified AKAPs 95, 79, 149 and a fragment of AKAP450 in T cells, which is consistent with the recent report by Carr and co-workers on PKA type II AKAPs in T cells (Schillace et al . , 2002, supra).
  • the screening with RIo; as a bait identified a different set of AKAPs in T cells to those identified by the RII overlay screening.
  • AKAPs that specifically would serve to anchor the pool of PKA type I identified in T cell lipid rafts and that are involved in the PKA type I- Csk inhibitory pathway that regulates TCR signalling
  • sucrose gradient fractionation of Jurkat T cell lysates and performed far-Western experiments (RII overlay) using radio-labelled PKA RIIo; subunit.
  • Fig. 1A shows the result of this experiment, which identified R- binding proteins of approximately 80 and 150 kDa.
  • AKAP149 The approximately 150-kDa band detected in Fig. 1A was further identified as AKAP149 (D-AKAP1) .
  • Ezrin was identified both in a yeast two- hybrid screen and as an R-binding protein purified from lipid rafts;
  • AKAP149 was less abundant in lipid rafts versus non-raft fractions (compare fractions 4 to 5 and 9 to 12) ; and
  • iii) Disruption of PKA-AKAP149 signalling complexes did not appear to affect signalling through the PKA-Csk-Lck regulatory pathway in T cell lipid rafts (not shown) .
  • lipid rafts from T cells in the presence and absence of the actin-depolymerizing drug cytochalasin D.
  • actin cytoskeleton was disrupted, we observed increased amounts of ezrin in lipid rafts (Fig. 4A)..
  • PKA RIo. and some levels of PKA RIIcc were present in the same raft preparations (Fig. 4A) .
  • the ezrin-binding protein EBP50 also co-migrated with ezrin in the density gradient separation and localised to lipid rafts (Fig. 4B) .
  • ezrin and RIo co-localized well with the expressed ezrin (Figs. 7A-C) .
  • overexpression of ezrin resulted in numerous needle-shaped processes at the cell surface (philopodia) as reported elsewhere earlier.
  • RIo also localized to these needle-shaped ezrin containing processes at the cell surface.
  • EBP50 bridges ezrin and Cbp/PAG to yield a scaffold for the PKA-Csk signalling pathway in T cell lipid rafts
  • EXAMPLE 2 Inhibition of PKA Type I signalling by disruption of ezrin binding
  • Ht31 anchoring disruptor containing the R-binding site from AKAP-Lbc is well established and widely used to compete localization of PKA.
  • Ht31 has a higher affinity for PKA type II, it can also be used to disrupt PKA type I interaction with AKAPs (Fig. 3C, Herberg et al . , 2000, J. Biol. Chem., 298, p329-339) .
  • T cells were transfected with a construct directing the expression of the Ht31 fragment (Lester et al . , 1997, PNAS, 94, pl4942-14947) this displaced both PKA types I and II from lipid rafts (not shown) .
  • Peripheral blood T cells were transfected by nucleofection (Amaxa) with constructs directing the expression of a soluble ezrin fragment containing the cx-helical domain mock-transfected.
  • Transfected and control-transfected cells were either left untreated, activated by cross ligation of the TCR/CD3 complex or pretreated with a concentration of cAMP that produced an 80% maximal inhibition of IL-2 production, and subsequently activated and cultured for 16 hours after which IL-2 levels secreted to the supernatant were assessed.
  • Results from these experiments show that T cells expressing soluble fragments of ezrin were less sensitive to cAMP-mediated inhibition of IL-2 production (Fig 9A) .
  • IL-2 production in the presence of cAMP increased 3-fold by introducing soluble fragments of ezrin (Fig. 9B) .
  • EXAMPLE 3 Identification of a putative upstream type I regulatory subunit binding domain (RI-AKAP specifier region) in AKAPs
  • Peptide arrays were synthesized on cellulose paper by using an Autospot Robot ASP222 (ABiMED, Langenfeld, Germany) as described (Frank, R. , 1992, Tetrahedron 48, 123-132) .
  • R-overlays were conducted as described, using 3Z P-labeled recombinant murine RIIo; (Hausken, Z. E. , et al . , 1998 in Protein Targeting Protocols, ed. Clegg, R. A. (Humana, Totowa, NJ) , Vol. 88, pp. 47-64), recombinant bovine RI (A98S) or deletion mutants.
  • radiolabeled RI as a probe, binding to regions 356-375 and 378-397 was detected. This overlapped with the predicted upstream RI-AKAP specifier region in ezrin. Furthermore, this region appeared to have a tandem binding motif. In contrast, no binding to this region was detected with radiolabeled RII or with any of the RI deletion mutants indicating that the RI-AKAP specifier region interacts with the N-terminus of RIo.
  • RII- 32 P overlay demonstrated binding to the most N-terminal part of this region whereas RI- 32 P overlay gave weak binding to the more C-terminal part of the helix. The RI-binding was enhanced when the N-terminus had been removed.
  • EXAMPLE 5 siRNA-mediated knockdown of Ezrin disrupts cAMP inhibition of T cell proliferation
  • siRNA duplexes targeting different positions within human ezrin mRNA were designed and synthesized in-house. Following initial screening of siRNA activity in cell culture, mismatched control siRNAs for the best siRNA were also designed. SiRNAs are named according to the position of the 5 ' nucleotide of the sense strand relative to the reference sequences of the respective target mRNAs and the sequences are:

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Abstract

L'invention se rapporte à un procédé de modification de la voie de signalisation de la PKA de type I dans une cellule, qui comprend l'étape d'administration d'un inhibiteur, ou d'une molécule codant un inhibiteur, qui réduit ou inhibe la liaison entre un ou plusieurs des partenaires de liaison suivants: i) ezrin et PKA de type I, ii) ezrin et EBP50, et iii) EBP50 et Cbp/PAG. L'invention se rapporte également à des molécules inhibitrices conçues pour être utilisées dans le procédé décrit ci-dessus ainsi qu'à des compositions pharmaceutiques contenant ces inhibiteurs. L'invention se rapporte en outre à des méthodes de traitement de troubles ou de maladies dans lesquels se produit une signalisation anormale de la PKA de type I tels que l'infection par VIH, le SIDA, l'immunodéficience variable commune (IVC) ou le cancer au moyen de tels inhibiteurs.
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Cited By (7)

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WO2010059400A1 (fr) * 2008-10-30 2010-05-27 The Translational Genomics Research Institute Procédés et kits pour identifier un glioblastome invasif
US20130102758A1 (en) * 2007-07-19 2013-04-25 Biomerieux Ezrin assay method for the in vitro diagnosis of colorectal cancer
US9388404B2 (en) 2008-07-10 2016-07-12 Biomerieux Protein disulfide isomerase assay method for the in vitro diagnosis of colorectal cancer
WO2016172583A1 (fr) * 2015-04-23 2016-10-27 Novartis Ag Traitement du cancer à l'aide de protéine récepteur antigénique chimérique et un inhibiteur de protéine kinase
US9726670B2 (en) 2007-07-19 2017-08-08 Biomerieux Method for the assay of liver fatty acid binding protein, ACE and CA 19-9 for the in vitro diagnosis of colorectal cancer
US9891223B2 (en) 2007-07-19 2018-02-13 Biomerieux Method of assaying leukocyte elastase inhibitor for the in vitro diagnosis of colorectal cancer
US10591482B2 (en) 2007-07-19 2020-03-17 Biomerieux Method of assaying Apolipoprotein AI for the in vitro diagnosis of colorectal cancer

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130102758A1 (en) * 2007-07-19 2013-04-25 Biomerieux Ezrin assay method for the in vitro diagnosis of colorectal cancer
US9726670B2 (en) 2007-07-19 2017-08-08 Biomerieux Method for the assay of liver fatty acid binding protein, ACE and CA 19-9 for the in vitro diagnosis of colorectal cancer
US9891223B2 (en) 2007-07-19 2018-02-13 Biomerieux Method of assaying leukocyte elastase inhibitor for the in vitro diagnosis of colorectal cancer
US9890196B2 (en) * 2007-07-19 2018-02-13 Biomerieux Ezrin assay method for the in vitro diagnosis of colorectal cancer
US10591482B2 (en) 2007-07-19 2020-03-17 Biomerieux Method of assaying Apolipoprotein AI for the in vitro diagnosis of colorectal cancer
US9388404B2 (en) 2008-07-10 2016-07-12 Biomerieux Protein disulfide isomerase assay method for the in vitro diagnosis of colorectal cancer
WO2010059400A1 (fr) * 2008-10-30 2010-05-27 The Translational Genomics Research Institute Procédés et kits pour identifier un glioblastome invasif
US8962581B2 (en) 2008-10-30 2015-02-24 The Translational Genomics Research Institute Methods and kits to identify invasive glioblastoma
WO2016172583A1 (fr) * 2015-04-23 2016-10-27 Novartis Ag Traitement du cancer à l'aide de protéine récepteur antigénique chimérique et un inhibiteur de protéine kinase

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