WO2014056934A1 - Modulateur d'acide quinolinique destiné à être utilisé dans le traitement d'une affection neuropathologique - Google Patents

Modulateur d'acide quinolinique destiné à être utilisé dans le traitement d'une affection neuropathologique Download PDF

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WO2014056934A1
WO2014056934A1 PCT/EP2013/070970 EP2013070970W WO2014056934A1 WO 2014056934 A1 WO2014056934 A1 WO 2014056934A1 EP 2013070970 W EP2013070970 W EP 2013070970W WO 2014056934 A1 WO2014056934 A1 WO 2014056934A1
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modulator
antagonist
quin
disease
quinolinic acid
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PCT/EP2013/070970
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Alban BESSÈDE
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Immusmol Sas
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    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0007—Nervous system antigens; Prions
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G01N33/9406—Neurotransmitters
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6081—Albumin; Keyhole limpet haemocyanin [KLH]
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2835—Movement disorders, e.g. Parkinson, Huntington, Tourette
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/285—Demyelinating diseases; Multipel sclerosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2857—Seizure disorders; Epilepsy

Definitions

  • the present invention is related to a modulator of Quinolinic Acid for use in the treatment of a neuropathological disease.
  • europathological diseases encompasses, among others, neurodegenerative diseases, neuro inflammatory diseases or seizure disorders.
  • Neurodegenerative diseases are characterized by progressive loss of structure and/or function of neurons, including death of neurons.
  • Many neurodegenerative diseases including Parkinson's, Alzheimer's, Huntington's, Amyotrophic lateral sclerosis and Multiple Sclerosis occur as a result of neurodegenerative processes.
  • Many similarities appear which relate these diseases to one another on a sub-cellular level. Discovering these similarities offers hope for therapeutic advances that could ameliorate many diseases simultaneously (Rubinstein DC 2006; Bredesen et al 2006).
  • There are many parallels between different neurodegenerative disorders including atypical protein assemblies as well as induced cell death. Neuro degeneration can further be found in many different levels of neuronal circuitry ranging from molecular to systemic.
  • Neurodegenerative diseases and “Neuroinflammatory diseases” have a partially overlapping scope. Inflammatory responses are a hallmark of neurodegenerative disease and participate, or contribute, through different mechanisms in the neuronal cell death.
  • the tryptophan catabolism along the kynurenine pathway (KP) represents one of these mechanisms.
  • Seizure disorders are brain disorders, which are characterized by abnormal signaling between brain cells. Seizure disorders can affect part of the brain (Partial seizures) or the entire brain (Generalized seizures). The most prominent Seizure disorder is epilepsy. To date, the therapeutic options to treat neurodegenerative diseases, neuroinflammatory diseases or seizure disorders are limited, and some diseases cannot be treated at all.
  • a modulator of QUIN for use in the treatment of a neuropathological disease, or a disease which is characterized by an over-production of Quinolinic Acid, is provided.
  • Quinolinic Acid is a dicarboxylic acid and, as such, a downstream kynurenine pathway metabolite of tryptophan, which has excitatory properties.
  • the excitatory properties of QUIN are due to its direct, selective stimulation of NMDAR, which is a specific type of ionotropic glutamate receptor.
  • NMDA N-methyl-D-aspartate
  • NMDA receptors Activation of NMDA receptors results in the opening of an ion channel that is nonselective to cations with an equilibrium potential near 0 mV.
  • a property of the NMDA receptor is its voltage-dependent activation, a result of ion channel block by extracellular Mg2+ ions. This allows the flow of Na+ and small amounts of Ca2+ ions into the cell and K+ out of the cell to be voltage-dependent
  • QUIN also induces the production of reactive oxygen species such as hydrogen peroxide, superoxide and hydroxyl radicals and stimulates lipid peroxidation, an effect strictly dependent on the presence of Fe 2+ ions.
  • the inventors have, for the first time, suggested to make, and use, a modulator of QUIN (Quinolinic Acid) for the treatment of a neuropathological disease, or a disease which is characterized by an over-production of Quinolinic Acid.
  • QUIN Quinolinic Acid
  • module of QUIN refers to a substance that affects at least one selected from the group consisting of the formation, concentration, availability metabolization and/or effect of QUIN (Quinolinic Acid).
  • QUIN can either be in a soluble form, or attached to another moiety (e.g., bound to a membrane or the like, attached to a cofactor, or the like).
  • the term "over-production of Quinolinic Acid” means a condition in which an organism produces more Quinolinic Acid than normal.
  • Quinolinic acid is a byproduct of the kynurenine pathway, which is related to the tryptophan metabolism. Tryptophan metabolism exaggeration is defined as a decrease in tryptophan level and/or increase of the kynurenine pathway metabolites when compared to healthy subjects while Quinolinic over-production is characterized by increase in Quinolinic level when compared to healthy subjects.
  • over-production of Quinolinic Acid is used herein synonymously with the term “activation of the kynurenine pathway”.
  • Over-production of Quinolinic Acid leads to levels of > 20 nM Quinolinic Acid in cerebrospinal fluid, preferably > 30 nM, > 40 nM, > 50 nM, > 60 nM, > 70 nM, > 80 nM, > 90 nM, > 100 nM, > 110 nM, > 120 nM, > 130 nM, > 140 nM, > 150 nM.
  • the highest levels encountered in cerebrospinal fluid are in the range of 500 nM.
  • the term "neuropathological disease” shall encompass neurodegenerative diseases, neuro inflammatory diseases and seizure disorders.
  • the modulator according to the invention is an antagonist to QUIN (Quinolinic Acid), or a conjugate thereof.
  • the term "antagonist” shall encompass all moieties that have an affinity to QUIN (Quinolinic Acid), or a conjugate thereof (e.g, Quinolinic Acid conjugated to BSA) but no efficacy. This means, for example, that (i) upon binding of said antagonist to the QUIN (Quinolinic Acid) no physiological function is elicited, or a dampened or altered physiological function is elicited, (ii) binding of said antagonist to QUIN (Quinolinic Acid) inhibits the binding thereof to its physiological counterpart, or (iii) binding of said antagonist to QUIN (Quinolinic Acid) inhibits metabolization thereof. Binding will thus disrupt the interaction between QUIN (Quinolinic Acid), or a conjugate thereof to its physiological target, and thus inhibit its role in pathological processes.
  • administration of said modulator and/or antagonist to a human or animal body affects at least one selected from the group consisting of the formation, concentration, availability metabolization and/or effect of QUIN (Quinolinic Acid) in said human or animal body.
  • QUIN Quinolinic Acid
  • Quinolinic Acid is a dicarboxylic acid and, as such, a downstream kynurenine pathway metabolite of tryptophan, which has excitatory properties.
  • the excitatory properties of QUIN are due to its direct, selective stimulation of NMDAR, which is a specific type of ionotropic glutamate receptor.
  • NMDA N-methyl-D-aspartate
  • NMDA receptors Activation of NMDA receptors results in the opening of an ion channel that is nonselective to cations with an equilibrium potential near 0 mV.
  • a property of the NMDA receptor is its voltage-dependent activation, a result of ion channel block by extracellular Mg2+ ions. This allows the flow of Na+ and small amounts of Ca2+ ions into the cell and K+ out of the cell to be voltage-dependent
  • QUIN also induces the production of reactive oxygen species such as hydrogen peroxide, superoxide and hydroxyl radicals and stimulates lipid peroxidation, an effect strictly dependent on the presence of Fe 2+ ions.
  • said modulator and/or antagonist is at least one selected from the group consisting of
  • mAb monoclonal antibody
  • mAb monoclonal antibody
  • mAb shall refer to an antibody composition having a homogenous antibody population, i.e., a homogeneous population consisting of a whole immunoglobulin, or a fragment or derivative thereof.
  • mAb is selected from the group consisting of IgG, IgD, IgE, IgA and/or IgM, or a fragment or derivative thereof.
  • fragment shall refer to fragments of such antibody retaining, in some cases, target binding capacities, e.g.
  • IgG heavy chain consisting of VH, CHI, hinge, CH2 and CH3 regions
  • IgG light chain consisting of VL and CL regions
  • derivative shall refer to protein constructs being structurally different from, but still having some structural relationship to, the common antibody concept, e.g., scFv, Fab and/or F(ab) 2 , as well as bi-, tri- or higher specific antibody constructs. All these items are explained below.
  • Fab relates to an IgG fragment comprising the antigen binding region, said fragment being composed of one constant and one variable domain from each heavy and light chain of the antibody
  • F(ab) 2 relates to an IgG fragment consisting of two Fab fragments connected to one another by disulfide bonds.
  • scFv relates to a single-chain variable fragment being a fusion of the variable regions of the heavy and light chains of immunoglobulins, linked together with a short linker, usually serine (S) or glycine (G). This chimeric molecule retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of a linker peptide.
  • new antibody formats encompasses, for example bi- or trispecific antibody constructs, Diabodies, Camelid Antibodies, Domain Antibodies, bivalent homodimers with two chains consisting of scFvs, IgAs (two IgG structures joined by a J chain and a secretory component), shark antibodies, antibodies consisting of new world primate framework plus non-new world primate CDR, dimerised constructs comprising CH3+VL+VH, and antibody conjugates (e.g., antibody or fragments or derivatives linked to a toxin, a cytokine, a radioisotope or a label).
  • antibody mimetic relates to target binding proteins, which are not related to immunoglobulins. Many of the above mentioned techniques, like phage display, are applicable for these molecules as well.
  • antibody mimetics are for example derived from Ankyrin Repeat Proteins, C-Type Lectins, A-domain proteins of Staphylococcus aureus, Transferrins, Lipocalins, Fibronectins, Kunitz domain protease inhibitors, Ubiquitin, Cysteine knots or knottins, thioredoxin A, and so forth, and are known to the skilled person in the art from the respective literature.
  • aptamer relates to nucleic Acid species, which are capable of binding to molecular targets such as small molecules, proteins, nucleic Acids, and even cells, tissues and organisms. Aptamers are useful in biotechno logical and therapeutic applications as they offer molecular recognition properties that rival that of the commonly used biomolecule, antibodies. In addition to their discriminate recognition, aptamers offer advantages over antibodies or other target binders as they can be engineered completely in a test tube, are readily produced by chemical synthesis, possess desirable storage properties, and elicit little or no immunogenicity in therapeutic applications.
  • Aptamers can for example be produced through repeated rounds of in vitro selection or equivalent ly, SELEX (systematic evolution of ligands by exponential enrichment) to bind
  • small molecule antagonist relates to a low molecular weight organic compound, which is by definition not a polymer.
  • small molecule especially within the field of pharmacology, is usually restricted to a molecule that also binds with high affinity to a biopolymer such as protein, nucleic Acid, or polysaccharide and in addition alters the activity or function of the biopolymer.
  • the upper molecular weight limit for a small molecule is often set at 800 Daltons, which allows for the possibility to rapidly diffuse across cell membranes so that they can reach intracellular sites of action.
  • this molecular weight cutoff is a necessary but insufficient condition for oral bioavailability.
  • Small molecules acting as antagonists against a given target e.g., QUIN (Quinolinic Acid ) can be found by high throughput screening of respective libraries comprising a large variety of different small molecular candidates.
  • the modulator and/or antagonist is a monoclonal antibody selected from the group consisting of: ⁇ 3C10-E5
  • the modulator and/or antagonist is a monoclonal antibody having an half maximal effective concentration EC50 towards Quinolinic Acid, or a conjugate thereof, as measured with an Elisa competition assay, of ⁇ 5* 10 "10 M.
  • said EC 50 is ⁇ 1 * 10 -11 M, ⁇ 5* 10 "n M, ⁇ 1 * 10 ⁇ 12 M, ⁇ 5* 10 ⁇ 12 M, ⁇ 1 * 10 "13 M, ⁇ 5* 10 ⁇ 13 M, ⁇ 1 * 10 ⁇ 14 M, ⁇ 5* 10 ⁇ 14 M, ⁇ 1 * 10 ⁇ 15 M, ⁇ 5* 10 ⁇ 15 M, ⁇ 1 * 10 ⁇ 16 M, or ⁇ 5* 10 ⁇ 16 M.
  • said EC 50 is ⁇ 2,22* 10 ⁇ 10 M, ⁇ 4,99* 10 -11 M, ⁇ 3,5* 10 -11 M, ⁇ 3, 17* 10 " 11 M, or ⁇ 1,77* 10 "15 M.
  • the modulator and/or antagonist is a monoclonal antibody being capable of inhibiting a physiological response towards Quinolinic Acid.
  • Said physiological response towards Quinolinic Acid is, for example, the Ca 2+ influx mediated by Quinolinic Acid in cortical neurons, which, under pathological conditions, may lead to neuron death.
  • the modulator and/or antagonist is a monoclonal antibody being capable of inhibiting such physiological response towards Quinolinic Acid by > 10 %.
  • it is capable of inhibiting a physiological response towards Quinolinic Acid, by > 1 1; 12; 13; 14; 15; 16; 17; 18; 19; 20; 21; 22; 23; 24; 25; 26; 27; 28; 29; 30; 31; 32; 33; 34; 35; 36; 37; 38; 39; 40; 41 ; 42; 43; 44; 45; 46; 47; 48; 49; 50; 51; 52; 53; 54; 55; 56; 57; 58; 59; 60; 61 ; 62; 63; 64; 65; 66; 67; 68; 69; 70; 71 ; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81 ; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100 %.
  • said modulator and/or antagonist is conjugated to a given carrier.
  • Said conjugation to a given carrier may serve to increase the bioavailability, the efficiency and/or the serum half- life of the modulator and/or antagonist according to the invention.
  • PEGylation involves the modification of a protein, peptide, or non-peptide molecule by linking of one or more polyethylene glycol chains to it, and thus results in a prolonged serum half-life particularly of smaller protein drugs, like antibody fragments, as for example put into practice in the pegylated Fab fragment Certolizumab pegol.
  • a similar effect is obtained by N-glycosylation of respective domains in a given protein therapeutic (see above definition).
  • additional N-glycosylation sites are introduced into said protein therapeutic.
  • N-glycosylation motifs i.e., tripeptide sequences Asn-X-Ser or Asn-X-Thr, where X can be any amino Acid (although Pro and Asp are rarely found).
  • additional N-glycosylation motifs i.e., tripeptide sequences Asn-X-Ser or Asn-X-Thr, where X can be any amino Acid (although Pro and Asp are rarely found).
  • the antibody, or fragment or derivative thereof has, somewhere in its chain, the motif "Gly-X-Ser”, one could substitute "Gly” by "Asn”, on order to create an additional N-glycosylation site. It is of course necessary to make sure that the said substitution does not affect important properties of the protein, like target affinity, binding by Fc gamma receptors (FcyRs) or the like.
  • FcyRs Fc gamma receptors
  • Increasing half-life can further be obtained by conjugating the said Quin (Quinolinic Acid) antagonist to a polypeptidic carrier such as Poly-L-Lysine or modified Poly-L-Lysine.
  • a polypeptidic carrier such as Poly-L-Lysine or modified Poly-L-Lysine.
  • This method encompasses the covalent binding of the antagonist to Poly-Lysine and/or modified Poly-L-Lysine.
  • Modified Poly-L-Lysine can be obtained by adding at least one other moiety.
  • Conjugating the antagonist to Poly-Lysine can be performed by using crosslinkers that react with the free amine group of the Poly-L-Lysine and reactive functions on the antagonist. Grafting the antagonist on modified poly-l-lysine can be performed by crosslinking of the reactive groupement of antagnist to new reactive functions of the Pol-L-Lysine.
  • Crosslinkers can be for example glutaraldehyde (NH 2 to NH 2 ), EDC (NH 2 to COOH), SMCC (NH 2 to SH). This conjugate would be able to limit liver and kidney filtration of the therapeutic protein as well as limiting its degradation by specific enzymes and therefore increasing its efficiency.
  • the use of the modulator and/or antagonist according to the invention for the treatment of a disease which is characterized by an overproduction of Quinolinic Acid is provided.
  • the use of the modulator and/or antagonist according to the invention for the treatment of a neuropathologic disease is provided.
  • the use of the modulator and/or antagonist according to any of the aforementioned claims in the diagnosis, prognosis, risk assessment and/or prediction of a neuropathologic disease, a disease which is characterized by an overproduction of Quinolinic Acid, is provided.
  • QUIN Quinolinic Acid
  • said prognosis, risk assessment and/or prediction method is carried out in a tissue sample and/or in a liquid sample.
  • tissue sample can be a slice, a biopsy, a needle aspirate, a smear sample, a scratch sample or the like.
  • liquid sample can be taken from a biological fluid like blood, serum, urine, cell supernatants, cerebrospinal fluid, saliva, interstitial fluid, or the like.
  • the presence and/or concentration of QUIN (Quinolinic Acid) in a tissue sample and/or in a liquid sample is determined by at least one method selected from the group of
  • Solid phase immonoassays like Immunohistochemistry, ELISA and/or Immunofluorescence , and/or
  • said modulator and/or antagonist is labeled.
  • Such label is, preferably, selected from the group consisting of a Radiolabel, Fluorescent label, a Luminescent label and/or an enzyme label.
  • Said labeling can be direct, i.e., the antagonist itself is labeled.
  • the labeling can be indirect (e.g., by means of a labeled secondary antibody which detects the antagonist antibody).
  • the use of QUIN (Quinolinic Acid ), or a conjugate thereof with at least one carrier is provided to induce an immune response in a human or animal body.
  • said immune response leads to the production of a monoclonal antibody for therapeutic or diagnostic purposes.
  • Said embodiment in the following called option (i) can be used to produce a monoclonal antibody against Quin (Quinolinic Acid ), e.g., according to the Kohler Milstein technique (Kohler and Milstein 1975), or by immunization of a transgenic mammal having the human immunoglobulin gene repertoire (Lonberg 2005). Because an antibody response can probably be directed against epitopes on both the carrier protein and hapten, it is important to plan carefully how hapten-specific antibodies will be identified and purified from the final immunized serum.
  • Quin Quinolinic Acid
  • said immune response serves to reduce the risk, of a subject, to develop a neuropathologic disease, or a disease which is characterized by an over-production of Quinolinic Acid, or forms at least part of a therapy for the treatment of a neuropathologic disease, or a disease which is characterized by an over-production of Quinolinic Acid.
  • the use thereof as a vaccine in a human or animal patient can be provided.
  • This embodiment describes a classical vaccine. The concept is that a patient is immunized in such way that he develops an immune response against Quin (Quinolinic Acid ). If, in such patient, a pathologic process leads to overproduction of Quin (Quinolinic Acid ), the immune system should be capable of either eliminating the overabundant enzymes and/or metabolites, or eliminating the cells responsible for such overabundancy.
  • Quin Quinolinic Acid
  • a self-antigen as they form an intrinsic part of the human proteome and the human physiology. Further it is a small molecule, which does not evoke an immune response at all due to their small size.
  • Quin (Quinolinic Acid) to a carrier, such as a protein.
  • Said carrier may be one that does not elicit an immune response by itself either.
  • the conjugate thus produced is immunogenic despite the self-antigen character and/or the low molecular weight of Quin (Quinolinic Acid ).
  • the small-molecule hapten, or the self-antigen may also be detected by the antibodies produced.
  • the overabundant enzymes and/or metabolites, or the cells responsible for such overabundancy marked by such antibody can be eliminated by the immune system.
  • Such carrier protein can be, principally, any peptide or protein, preferably of a size above 1 kD, that can be coupled with Quin (Quinolinic Acid ).
  • the carrier protein because it is large and complex, confers immunogenicity to the conjugated hapten, resulting in antibodies being produced against epitopes on the hapten and carrier.
  • proteins can be used as carriers and are chosen based on immunogenicity, solubility, and availability of useful functional groups through which conjugation with the hapten can be achieved.
  • some carriers are more useful with respect to above mentioned option (i), i.e., for use to produce a monoclonal antibody, e.g., according to the Kohler Milstein (Kohler & Milstein 1975) technique, or by immunization of a transgenic mammal having the human immunoglobulin gene repertoire, while others are more useful with respect to above mentioned option (ii), i.e., for use as a vaccine.
  • option (i) i.e., for use to produce a monoclonal antibody
  • option (ii) e.g., for use to produce a monoclonal antibody
  • option (ii) e.g., for use to produce a monoclonal antibody
  • option (ii) e.g., for use to produce a monoclonal antibody
  • option (ii) e.g., for use to produce a monoclonal antibody
  • option (ii) e.g., for use to produce a monoclonal antibody
  • a carrier which forms a strong antigen is useful, because it results in the immediate production of antibodies.
  • a carrier which forms a somehow weaker antigen may be acceptable, because the purpose of this apporach is to merely teach the immune system, not to actually make antibodies.
  • the weak immunogenicity is thus a small disadvantage only, particularly if the respective carrier results in smaller side effects and has better pharmacological acceptance.
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • KLH is the most widely used carrier protein.
  • the copper- containing polypeptide belongs to a group of non-heme proteins called hemocyanins, which are found in arthropods and mollusks.
  • KLH is isolated from keyhole limpets (Megathura crenulata). Because KLH is from a class of proteins and a group of organisms that are evolutionarily distant from mammals, it is very "foreign" to the mammalian immune system.
  • the protein is also highly immunogenic because of its very large size and complex structure.
  • the molecule is composed of 350kDa and 390kDa subunits that associate to form aggregates ranging from 0.5 to 8 million daltons.
  • KLH protein molecule contains several hundred surface lysine groups that provide primary amines as targets for covalent attachment of haptens or self-antigens using a variety of crosslinking techniques. These features make KLH an extremely immunogenic and effective carrier protein for immunogen preparation. Although the large protein is sometimes difficult to work with because it has limited solubility, the commercial availability of stabilized and pre-activated formulations makes it convenient to use.
  • Blue Carrier* Protein is a purified preparation of Concholepas concholepas hemocyanin (CCH).
  • CCH Concholepas concholepas hemocyanin
  • KLH keyhole limpet hemocyanin
  • the CCH protein is composed of two very large polypeptide subunits (404 and 351kDa) that form an extremely stable heterodidecameric structure even in the absence of divalent cations. (By contrast, KLH has a less stable and soluble homodidecameric structure).
  • the complex molecular arrangement of CCH subunits contains diverse repeated antigenic structures that elicit a strong immune reaction mediated by T and B lymphocytes. Because of their large size and molecular complexity, KLH and CCH hemocyanins are carrier proteins of choice for use as immunogens to produce antibodies against haptens or self antigens. Moreover, studies suggest that the strong DTH immune response elicited by hemocyanins in animals and in humans may have beneficial therapeutic effects in certain types of immunotherapy New developments in the immunotherapy of cancer, autoimmune diseases and neuropathological diseases have taken advantage of the unique immunogenic properties of hemocyanins in the development of novel conjugate vaccines for treatment of emerging diseases.
  • Bovine serum albumin belongs to the class of serum proteins called albumins. Albumins constitute about half the protein content of plasma and are quite stable and soluble. BSA is much smaller than KLH but is nonetheless fully immunogenic. It is a popular carrier protein for weakly antigenic compounds. BSA exists as a single polypeptide with 59 lysine residues, 30 to 35 of which have primary amines that are capable of reacting with a conjugation reagent. Numerous carboxylate groups give BSA its net negative charge (pi 5.1).
  • BSA is commonly used in development of immunoassays because it is readily available, is fully soluble and has numerous functional groups useful for crosslinking to small molecules that otherwise would not coat efficiently in polystyrene microplates. These same characteristics that make BSA easy to use in immunoassay development also make it simple to use for preparing and testing conjugation efficiency of carrier- hapten/self antigen conjugates.
  • Cationized bovine serum albumin (cBSA) is prepared by modifying native BSA with excess ethylenediamine, essentially capping all negatively-charged carboxyl groups with positively- charged primary amines. The result is a highly positively-charged protein (pi > 11) that has significantly increased immunogenicity compared to native BSA. In addition, the increased number of primary amines provides for a greater number of antigen molecules to be conjugated with typical crosslinking methods.
  • Another suitable carrier is Ovalbumin (OVA; 45kDa). Also known as egg albumin, ovalbumin constitutes 75% of protein in hen egg whites. OVA contains 20 lysine groups and is most often used as a secondary (screening) carrier rather than for immunization, although it is somewhat immunogenic.
  • the protein also contains 14 aspartic Acid and 33 glutamic Acid residues that afford carboxyl groups. These groups can be used as targets for conjugation with haptens or self-antigens.
  • Ovalbumin exists as a single polypeptide chain having many hydrophobic residues and a pi of 4.63. The protein denatures at temperatures above 56°C or when subject to electric current or vigorous shaking. OVA is unusual among proteins in being soluble in high concentrations of the organic solvent DMSO, enabling conjugation to haptens or self antigens that are not easily soluble in aqueous buffers.
  • Muramyl dipeptide Acetylmuramyl-Alanyl-Isoglutamine (NAc-Mur-L-ala- D-isoGln), or derivatives therof, like Murabutide (NAcMur-L-Ala-D-Gln-alpha-n-butyl- ester).
  • Muramyl dipeptide is a peptidoglycan constituent of both Gram positive and Gram negative bacteria. It is composed of N-acetylmuramic Acid linked by its lactic Acid moiety to the N-terminus of an L-alanine D-isoglutamine dipeptide.
  • Suitable carriers encompass multi-poly (DL-alanine)--poly(L-lysine).
  • proteins and peptides have primary amines (the N-terminus and the side chain of lysine residues), carboxylic groups (C-terminus or the side chain of aspartic Acid and glutamic Acid), and sulfhydryls (side chain of cysteine residues) that can be targeted for conjugation.
  • primary amines the N-terminus and the side chain of lysine residues
  • carboxylic groups C-terminus or the side chain of aspartic Acid and glutamic Acid
  • sulfhydryls side chain of cysteine residues
  • crosslinking encompass, but are not restricted to, bifunctional or derivatizing agents, such as EDC Conjugation (carboxyl and amine crosslinking with the carbodiimide crosslinker EDC), Maleimide Conjugation (sulfhydryl crosslinking with, for example, the heterobifunctional crosslinker Sulfo-SMCC which contains a maleimide group that will react with free sulfhydryls, plus a succinimidyl (NHS) ester that will react with primary amines, glutaraldehyde conjugation (amine-to-amine crosslinking via amines on the respective polypeptides), or crosslinking with disuccinimidyl suberate (DSS) and its water- soluble analog, BS3 or pegylated versions of BS3 (Amine-to-amine crosslinking).
  • EDC Conjugation carboxyl and amine crosslinking with the carbodiimide crosslinker EDC
  • Maleimide Conjugation
  • Non-protein carriers e.g., colloidal gold.
  • the administration of Quin (Quinolinic Acid ), or a conjugate thereof with at least one carrier is complemented, in a coordinated fashion, by administration of a suitable adjuvant.
  • a suitable adjuvant to enhance the immune response to an immunogen, both to (i) to produce a monoclonal antibody against Quin (Quinolinic Acid ), e.g., according to the Kohler Milstein technique, or by immunization of a transgenic mammal having the human immunoglobiulin gene repertoire, or (ii) for the use as a vaccine in a human or animal patient, adjuvants are commonly used.
  • an adjuvant will enhance the immune response.
  • adjuvant is not a substitute for a carrier protein because it enhances the immune response to immunogens but cannot itself render haptens immunogenic.
  • Adjuvants are nonspecific stimulators of the immune response, helping to deposit or sequester the injected material and causing a dramatic increase in the antibody response.
  • CFA complete Freund's adjuvant
  • FCA complete Freund's adjuvant
  • This reagent consists of a water-in-oil emulsion and killed Mycobacterium. The oil-and-water emulsion localizes the antigen for an extended period of time, and the Mycobacterium attracts macrophages and other appropriate cells to the injection site. Complete Freund's adjuvant is often used for the first injection (immunization).
  • peptide-carrier protein conjugates are immunogenic, and alum provides significant stimulation for them.
  • Alum is safer to use than Freund's adjuvants, as it is much less likely to cause tissue necrosis at the injection site.
  • Yet another adjuvant is Muramyl dipeptide.
  • adjuvants encompass squalene-based oil-in-water nano-emulsions, Muramyl dipeptide (Acetylmuramyl-Alanyl-Isoglutamine (NAc-Mur-L-ala-D-isoGln), the latter preferably N- Glycolated, or PRR (Patterns recognition Receptors) ligands like TLR (Toll like receptor) agonists.
  • TLR9 agonists such as CpG-ODN Typs A and/or B
  • TLR4 agonists modified Lipid A, e.g.
  • TLR5 agonists Flagellin, preferably recombinant
  • TLR7 and/or TLR8 agonists e.g., Imidazoquinoline compounds
  • TLR1/2 agonists e.g., Synthetic triacylated lipoprotein
  • TLR3 agonists e.g., Polyinosine-polycytidylic acid
  • the administration of QUIN (Quinolinic Acid) or a conjugate thereof with at least one carrier is complemented, in a coordinated fashion, by administration of a suitable adjuvant.
  • an immunotherapeutic comprising QUIN (Quinolinic Acid), or a conjugate thereof with at least one carrier.
  • immunotherapeutic relates to a vaccine of type b) or c) as discussed above.
  • immunotherapeutic can be administered, in a coordinated fashion, with an adjuvant (see above).
  • Such immunotherapeutic can either work by activating B cells (humoral immune response), or by activating T cells (cell-mediated immune response).
  • the use of QUIN (Quinolinic Acid), or a conjugate thereof, for the development of a modulator and/or antagonist against QUIN is provided, said modulator and/or antagonist being useful as a therapeutic, an immunotherapeutic and/or a diagnostic agent with respect to a neuropathological disease.
  • said modulator and/or antagonist is at least one modulator and/or antagonist according to the invention.
  • said development of a modulator and/or antagonist comprises at least one step of screening at least one library against QUIN (Quinolinic Acid), or a conjugate thereof.
  • QUIN Quinolinic Acid
  • Such library can be an antibody library, e.g., as it is used for phage display or retrocyte display (see e.g. Hogenboom 2005).
  • Such library can however also be a small molecule library, as e.g. described by Inglese et al (2007).
  • a method of treatment of a neuropathological disease in a human or animal patient comprises the modulation of at least one parameter selected from the group consisting of the formation, concentration, availability and/or effect of QUIN (Quinolinic Acid ).
  • said method comprises the administration of a modulator and/or antagonist according to the invention.
  • a method of diagnosis, prognosis, risk assessment and/or prediction of a neuropathological disease in which method the presence and/or concentration of QUIN (Quinolinic Acid ), or a conjugate thereof, in a given sample is determined by means of an the modulator and/or antagonist according to the invention.
  • the presence of QUIN (Quinolinic Acid ), which is causative for, or at least related to, the neuropathologic diseases discussed herein, has also diagnostic, prognostic or predictive value.
  • the presence and/or concentration of QUIN is determined in a tissue sample and/or in a liquid sample.
  • tissue sample can be a slice, a biopsy, a needle aspirate, a smear sample, a scratch sample or the like.
  • liquid sample can be taken from a biological fluid like blood, serum, urine, cell supernatants, cerebrospinal fluid, saliva, interstitial fluid, or the like.
  • the presence and/or concentration of QUIN (Quinolinic Acid) in a tissue sample and/or in a liquid sample is determined by at least one method selected from the group of
  • Solid phase immonoassays like Immunohistochemistry, ELISA and/or Immunofluorescence , and/or
  • a modulator, antagonist, use, immunotherapeutic or method according to the invention wherein said neuropathological disease is a neurodegenerative disease, a neuromflammatory disease or a seizure disorder.
  • said neurodegenerative and/or neuromflammatory disease is at least one selected from the group consisting of
  • Amyotrophic lateral sclerosis Other neurodegenerative and/or neuromflammatory diseases falling under the above defmtion encompass traumatic brain injury, ataxia telangiectasia, Coackayne syndrome, corticobasal degeneration, Creutzfeldt- Jakob disease, spinocerebellare ataxia type 3, neuroborreliosis, primary lateral sclerosis, progressive supranuclear palsy, Schilder's disease, subacute combined degeneration of spinal cord secondary to pernicious anaemia, drug-induced demyelination, radiation induced demyelination, spinal muscular atrophy, tabes dorsales, spinal cord injury, chronic inflammatory demyelinating neuropathy, a congenital metabolic disorder, polymyositis, temporal arteritis, vasculitis, autism, and interstitial cystitis, Hurler's Syndrome, Scheie's Syndrome, Hunter's Syndrome, San Fillipo's Syndrome, Maroteaux-Lany Syndrome, Sly Syndrome, Fu
  • said seizure disorder is at least one selected from the group consisting of:
  • Anti-QUIN antagonist To test the activity of an anti-QUIN antagonist we decided to use a specific antibody directed against QUIN. Several conjugated QUIN antibodies are commercially available.
  • Anti-QUIN antibody We decided to use the Anti-Quinolinic Acid Antibody MAB6512 obtained from Abnova (called herein "anti-QUIN mAb”). This is a murine monoclonal antibody detecting conjugated Quinolinic Acid. Although QUIN is present, in the body, mainly in adsorbed form (i.e., bound to a macromelucule or a cellular structure), said antibody will even bind to adsorbed QUIN, and is thus a suitable tool to investigate the therapeutic potential of an anti Quin antagonist in the teratement of neuropathologic diseases.
  • QUIN is conjugated to bovine serum albumine (BSA) by means of a carbodiimide crosslinker, which reacts with the carboxylic functions of QUIN and free amine functions of the BSA, to form a stable amide bond.
  • BSA bovine serum albumine
  • the "Imject BSA and EDC Conjugation Kit” provided by Thermo Scientific is used for this purpose.
  • Monoclonal antibodies are generated following the established method of Kohler and Milstein (1975). Briefly, lymphocytes are isolated from mice immunized three times with the QUIN- BSA conjugates. These lymphocytes were then fused with murine myeloma cells (SP20-Ag) with poly ethyl eneglycol (PEG 1500) to obtain hybridoma cells. The selection of hybridomas cells was realized by enzyme-linked immunosorbent anssay (ELISA).
  • ELISA enzyme-linked immunosorbent anssay
  • Clones are selected based on their affinity and specificity towards QUIN.
  • Monoclonal antibodies to QUIN can be obtained which have an affinity of up to 10 "10 M (calculated based on the conjugates amount used to make the competition assay in ELISA, wherein the amount of conjugates is related to amount of BSA).
  • the amount of conjugates is related to amount of BSA.
  • a competitive assay with other metabolites of the Kyrunenine pathway it becomes evident that the selected antibody has no cross reactions (Fig- 1).
  • Quinolinic-PC conjugated Quinolinic Acid
  • the carrier protein is BSA (Bovine Serum Albumine).
  • the specifity was measured as half maximal effective concentration (EC 50 ) with an ELISA competition assay.
  • EC50s values of these monoclonal antibodies Values were obtained after linear regression on GraphPad prism (v6.0).
  • Another antibody, 5D1-G1 was created which detects the isolated protein carrier, i.e., BSA, and has no affinity for Quinolinic Acid.
  • Fig. 4 shows the immunochemical characterization of said antibodies targeting protein carrier conjugated Quinolinic Acid (hereinafter called "Quinolinic-PC").
  • the carrier protein is BSA.
  • Fig. 4A shows an affinity comparison of the different monoclonal antibodies as measured in an ELISA competition assay. According to this experiment, the clone 3B2-C7 demonstrated the highest affinity.
  • Fig. 4B shows a specificity comparison the clone 3C10-E5 as measured in an ELISA competition assay. No cross reactions with the Picolinic acid conjugate ("Picolini- PC") was observed, while the Quinolinic conjugate was recognized at an affinity ⁇ 10 ⁇ n M. 3. Production of a humanized anti QUIN antibody
  • murine monoclonal antibodies in clinical settings is limited by the human anti- murine antibodies (HAMA) response against both variable and constant regions of the the murine Abs (Reynolds et al. 1989).
  • murine antibodies can be humanized.
  • the first technology to addresss this need was the grafting of the complementary determining residues (CDRs) onto the variable light (VL) and variable heavy (VH) frameworks of human immunoglobulin molecules (Winter et al 1993).
  • VL variable light
  • VH variable heavy
  • the remaining mice CDRs of the humanized antibodies can still generate an anti-idiotypic (anti- Id) response in patients.
  • SDRs specificity determining residues
  • the most important CDR residues in the antibody-ligand binding are thus grafted.
  • the last method is used to generate a humanized anti QUIN antibody.
  • the humanized anti QUIN antibody is able to recognize the conjugated 3HAA with the same affinity as the murine anti QUIN antibody, and is still specific enough when tested against other metabolites of the kynurenine pathway. 4. Production of a fully human anti QUIN antibody in transgenic mice
  • a transgenic mouse (HuMab TM mouse developed by Medarex) is used.
  • the endologoues immunoglobulin gene repertoire has been replaced by its human counterpart, so that, after immunization, said mouse produces fully-human
  • irradiated BALB/c Rag2-/-IL-2Ryc-/- mice reconstituted with human hematopoietic progenitor cells (hHPC) (Shultz et al 2007) are used.
  • mice are immunized with between 10 and 100 ⁇ g of QUIN/BSA conjugates solubilised in ⁇ in NaCl 9g/l and emulsified with ⁇ of Freund complete adjuvant (1st immunization) and in Freund incomplete adjuvant, for the 2nd and 3rd immunizations. Immunizations are repeated every 2 weeks. The mice are sacrificed 2 weeks after the 3 rd immunization and the spleens recovered and splenocytes are isolated. The latter are then fused with a human myeloma cell (Karpas 707, see Karpas et al. 2001) line to generate hybridoma cell lines.
  • a human myeloma cell Kerpas 707, see Karpas et al. 2001
  • Methods to produce a human antibody against a hapten or self antigen by means of phage display are, e.g., discussed in Brichta et al (2005), Kerrm et al (2003), Keith et al. (2001) or Sheedy et al (2007).
  • QUIN can be conjugated to either keyhole limpet haemocyanin (KLH) or bovine serum albumin (BSA) via linkage to 2-mercaptoethylamine, and hapten load/carrier protein is determined to be between eight and 10 haptens per BSA molecule using matrix assisted laser desorption spectrometry.
  • KLH keyhole limpet haemocyanin
  • BSA bovine serum albumin
  • hapten load/carrier protein is determined to be between eight and 10 haptens per BSA molecule using matrix assisted laser desorption spectrometry.
  • the resulting conjugates are assayed for protein according to standard protocols.
  • the Griffin library (MRC Laboratories, Cambridge, UK) consists of the majority of human VH and VL chain gene segments used in vivo, with CDR3 diversity generated using synthetic oligonucleotides (semi-synthetic).
  • the Tomlinson library (MRC Laboratories, Cambridge, UK) is based on a single human framework with side chain diversity (DVT encoded) incorporated at 18 amino Acid positions in the antigen binding site (synthetic).
  • the antibodies are displayed as scFv fragments on the coat protein of filamentous bacteria in the phagemid vector pHEN II.
  • the phagemid clones are maintained and propagated in T-phage resistant E. coli TGlTr (Stratagene).
  • Antibody fragments are expressed using the dicistronic, expression vector pIMS147.
  • the vector is inducible with isopropyl L-D-thiogalactosidase (IPTG) and downstream from the scFv genes contains a human CU domain (forming a single chain antibody or scAb) for immunodetection and a hexahistidine tail for purification by nickel chelate affinity chromatography.
  • the antibody expression vector pIMS147 is maintained in E. coli strain XL-1 Blue (Stratagene).
  • glycerol stock One hundred microlitres of either glycerol stock are inoculated into 100 ml 2xTY broth containing 1 % glucose and 100 ⁇ g ml "1 ampicillin (2xTY-glu-amp), and incubated with shaking at 37°C to an OD 600 of 0.4.
  • KM13 helper phage (2xlO n pfu) are added to 50 ml of each library culture and the mixture incubated at 37°C without shaking for 30 min.
  • Infected cells are pelleted, resuspended in 100 ml 2xTY broth-0.1 % glu-amp-50 ⁇ g ml "1 kanamycin, and incubated overnight with shaking at 30°C. Phage particles are concentrated from each culture supernatant by precipitation with 20 ml polyethylene glycol in 2.5 M NaCl (20% w/v) as described previously.
  • Two immunotubes are coated overnight with 100 ⁇ g ml "1 QUIN -BSA in phosphate buffered saline (PBS), washed with PBS and blocked with 2% skimmed milk-PBS at room temperature for 2 h.
  • the concentrated phage particles (approximately lxlO 13 ) from each library (Griffin or Tomlinson) are added to the immunotubes.
  • Specific scFv phage bound to the antigen, and the unbound phages are removed by washing.
  • the bound scFv phage are eluted from the immunotube with triethylamine (TEA) and infected into exponential phase TGI cell culture suspension in 2xTY broth for 30 min before being pelleted and plated onto agar plates of TYE-glu-amp and incubated at 30°C overnight.
  • the colonies are scraped into 5 ml of 2xTY- glu-amp-15% glycerol and stored at -80°C. Fifty microlitres of this stock are used to inoculate 50 ml fresh 2xTY-glu-amp and phage grown, infected and rescued as described above.
  • pan 2 QUIN-KLH (100 ⁇ g ml "1 )
  • pan 3 QUIN-BSA (1 ⁇ ⁇ ⁇ 1 ) 5.4. Screening and selection of phage antibodies to QUIN
  • Phage antibody clones that only recognise QUIN conjugates and not BSA or KLH alone are further characterised using a monoclonal binding ELISA where the phage antibodies are added to the plate in the presence or absence of free QUIN.
  • phAbs showing reduction of binding compared with phAbs added to the plate alone are sequenced in both directions on an ABI377 automated DNA sequencer (P.E. Applied Biosystems, Foster City, CA, USA). DNA from clones found to have different H or L chain sequences are digested with Ncol and Notl, and the scFv genes cloned into the similarly digested soluble expression vector pIMS147 before transformation into electrocompetent E. coli XL-1 Blue. Antibodies are then identified which show binding to QUIN.
  • the anti-Quinolinic Acid antibody 3B2-C7 does not detect free floating, unconjugated Quinolinic Acid in a biological fluid.
  • a biological sample with 3B2-C7 e.g., for analytic or diagnostic purposes, it is thus required to use a supplementary step of derivatization.
  • the biological sample has to be pretreated, accordingly, after it has been obtained from a patient. This process can be achieved, e.g., by means of carbodiimide. Other derivatizing agents may however also be used.
  • Results are demonstrated in Fig. 8, which shows Area Under Curves (AUC) for 300 seconds after cortical neurons treatment.
  • Data are plotted as Mean ⁇ SEM (standard error of the mean). All antibodies were added at 0, ⁇ g/ml. Among the antibodies tested, 3C10-E5 and 3B2-C7 have the highest blocking efficacy.
  • EAE Experimental autoimmune encephalomyelitis
  • CNS central nervous system
  • ADAM acute disseminated encephalomyelitis
  • EAE can be induced in a number of species, including mice, rats, guinea pigs, rabbits and primates.
  • the most commonly used antigens for inducing EAE in rodents are spinal cord homogenate (SCH), purified myelin, myelin protein such as MBP, PLP and MOG (Myelin oligodendrocyte glycoprotein), or peptides of these proteins.
  • rodents can display a monophasic bout of EAE, a relapsing-remitting form, or chronic EAE.
  • the typical susceptible rodent will debut with clinical symptoms around two weeks after immunization and present with a relapsing- remitting disease.
  • the archetypical first clinical symptom is weakness of tail tonus that progresses to paralysis of the tail, followed by a progression up the body to affect the hind limbs and finally the forelimbs.
  • the disease symptoms reflect the anatomical location of the inflammatory lesions, and may also include emotional liability, sensory loss, optic neuritis, difficulties with coordination and balance (ataxia), and muscle weakness and spasms.
  • Recovery from symptoms can be complete or partial and the time varies with symptoms and disease severity.
  • rats can have up to 3 bouts of disease within an experimental period.
  • the administration of the said antigens can take place in emulsified form in an adjuvant such as complete Freund's adjuvant.
  • an adjuvant such as complete Freund's adjuvant.
  • the presence of the adjuvant allows the generation of inflammatory responses to the protein/peptides.
  • mice are coinjected with pertussis toxin to break down the Blood-brain barrier and allow immune cells access to the CNS tissue.
  • EAE was induced as previously described.
  • mice Female C57BL/6 mice (8-10 weeks) were challenged with MOG (myelin oligodendrocyte glycoprotein) 35-55 peptide (Sigma Aldrich) emulsified in incomplete Freund adjuvant (Sigma- Aldrich) supplemented with heat inactivated mycobacterium tuberculosis (Difco) at 1 mg/ml. 100 ⁇ g of peptide were injected subcutaneously close to the lumbar spinal cord followed by intraperitoneal injection of 200 ⁇ g pertussis toxin (Sigma Aldrich). At day 2, mice were rechallenged with the same amount of pertussis toxin.
  • MOG myelin oligodendrocyte glycoprotein 35-55 peptide
  • Difco heat inactivated mycobacterium tuberculosis
  • mice were treated with either saline as a control, or with anti conjugated QUIN IgG. 100 ⁇ g were subcutaneously injected at day 9, 14 and 16.
  • mice were observed daily from day 8 to day 20 and body weight and clinical score were monitored according to Veldhoen et al (2006) as follows: 0: unaffected;
  • Fig. 2 we observed a decrease of the disease severity after 3B2-C7 IgG injection when compared to saline treated mice.
  • treatment with an anti-QUIN antagonist can provide a meaningful therapeutic option for the treatment of neurodegenerative diseases in general, and multiple sclerosis in particular.
  • Parkinson's disease is a progressive neurodegenerative disorder that affects several regions of the central and peripheral nervous system.
  • the symptoms of Parkinson's disease encompass the classic parkinsonian triad (tremor, bradykinesia, and rigidity) associated with dopaminergic denervation, other motor signs associated with non- dopaminergic transmission (postural instability and impairment of gait, speech, and posture), and non-motor symptoms (NMS).
  • the mean age at onset is 55, and the incidence increases markedly with age, from 20/100,000 overall to 120/100,000 at age 70.
  • sporadic genetic linkage
  • the disease is inherited. Over time, symp-toms worsen, and prior to the introduction of levodopa, the mortality rate among PD patients was three times that of the normal age- matched subjects.
  • 6-hydroxydopamine the first animal model of PD associated with substantia nigra pars compacta (SNpc) dopaminergic neuronal death, was introduced more than 30 years ago (Ungerstedt, 1968). 6-OHDA-induced toxicity is relatively selective for monoaminergic neurons, resulting from preferential uptake by DA and noradrenergic transporters (Luthman et al, 1989). Reminiscent of PD, there is a range of sensitivity to 6- OHDA between the ventral midbrain dopaminergic neuronal groups; greatest loss is observed in the SNpc, while tuberoinfundibular neurons are almost completely resistant.
  • 6-OHDA Inside neurons, 6-OHDA accumulates in the cytosol, generating ROS and inactivating biological macro molecules by generating quinones that attack nucleophilic groups. Because 6-OHDA cannot cross the blood-brain barrier, it must be administered by local stereotaxic injection into the substantia nigra. After 6-OHDA injections into substantia nigra , dopaminergic neurons start degenerating within 24 hr and die without apoptotic morphology (Jeon et al, 1995). These injections produce an asymmetric circling behavior in the animals, the magnitude of which depends on the degree of the nigrostriatal lesion.
  • mice are treated subcutaneoulsy with Anti-QUIN mAb at 10( ⁇ g/mouse; treatment is mainained once weekly until the end of the experiment (56 days after 6-ODA injection).
  • body weight rotarod performance
  • cognitive function Y-maze test
  • animals are sacrified and brains are subjected to IHC (Immunohistochemistry) to evaluate the number of tyrosine hydroxylase positive neurons in the SNc.
  • 6-OHDA treated mice have a significant decrease in Tyrosine hydroxylase positive neurons that is largely abrogated by preventive administration of QUIN mAb.
  • QUIN mAb is able to reestablish partially dopamine level in the striatum of 6-OHDA treated mice. Motor and memory functions are ameliorated as shown by rotarod experiment and Y-maze test.
  • Anti-QUIN mAb can represent therefore a suitable solution to limit motor and cognitive impairement induced by dopaminergic neuronal cell death.
  • treatment with an anti-QUIN antagonist can provide a meaningful therapeutic option for the treatment of neuropathological diseases in general, and Parkinson's disease in particular.
  • Alzheimer's disease Disease description Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Extracellular senile plaques, intracellular neurofibrillary tangles, and neuronal loss represent the main histological hallmarks of AD. Amyloid- ⁇ ( ⁇ ) peptides, the main components of senile plaques, result from the sequential endoproteo lytic cleavage of amyloid precursor protein (APP) by ⁇ -secretase (BACE-1) and the presenilin (PS)-dependent- secretase complex (Checler, 1995). An increased level of ⁇ is considered as a key event contributing to AD etiology. As a support of the amyloid cascade hypothesis, most of the mutations in APP and PS1/PS2 responsible for early-onset familial AD (FAD) modulate ⁇ production (Bekris et al, 2010).
  • FAD early-onset familial AD
  • Tg2576 is a transgenic mouse model, where mice overexpress a mutant form of APP, APPK670/671L (APP-swe). These mice are used as an experimental model of AD, a model linked to the FAD. They develop amyloid plaques and progressive cognitive deficits. These mice are available from Charles River laboratories. They show impaired cognitive functions in the contextual fear conditioning (CFC) test, coinciding with the increased cortical and hippocampal soluble beta amyloid levels starting from 5 months of age.
  • CFC contextual fear conditioning
  • mice At the age of 5 months, mice (WT, Tg2576 + vehicle and Tg2576 + QUIN mAb) are subjected to the CFC test and soluble ⁇ 1-42 are measured from cortex, hippocampus, plasma and Cerebrospinal fluid (CSF).It s expected that Memory and motor functions of Tg2576 mice are largely diminished when compared to wild type mice using CFC test (memory for both context and cue) and evaluation of the total distance travelled. When mice are treated with QUIN mAb, there is a partial restoration of these defects. This is associated with a decrease of soluble ⁇ in both serum and hippocampe.
  • CSF Cerebrospinal fluid
  • Anti-QUIN mAb may thus represent a suitable solution to limit cognitive functions impairment and molecular alteration of the APP.
  • treatment with an anti-QUIN antagonist can provide a meaningful therapeutic option for the treatment of neuropathological diseases in general, and Alzheimer's disease in particular. Huntington's disease
  • HD Huntington's disease
  • CAG trinucleotide poly
  • HTT huntingtin
  • Expanded poly(CAG) HTT leads to production of huntingtin protein with an equally expanded polyglutamine (polyQ) stretch near the N terminus. It is well established that mutant polyQ huntingtin (mHTT) exerts a gain of toxic function through aberrant protein-protein interactions.
  • polyQ polyglutamine
  • the transgenic R6/2 mouse [B6CBA-Tg(HDexonl)62Gpb/lJ] contains N- terminally truncated mutant HTT (mHTT) with CAG repeat expansion (-125 repeats) within the huntingtin gene exon 1.
  • mHTT N- terminally truncated mutant HTT
  • CAG repeat expansion -125 repeats
  • the R6/2 mouse is the most commonly used HD model to assess compound efficacy for alleviating the deficits of the phenotype in short and longitudinal studies.
  • the R6/2 mouse rapidly develops disease signs of HD-like symptoms, including decreased body weight as well as motor and cognitive deficits, starting as early as 6-8 week of age. Experimental protocol and expected result. Evaluation of anti-QUIN mAb benefits is performed by Charles River.
  • mice are treated weekly at the age of 6 weeks by 100 ⁇ g anti-QUIN mAb subcutaneous injection until death. Mice are monitored by means of survival, body weight and evaluation of motor deficits (rotarod performance and open field test) as well as evaluation of cognitive deficits (CFC tests).
  • Another experimental scheme is repeated to evaluate, at the age of 20 weeks, the level of mutated huntingtin and dopaminergic neurotransmission by means of immunohistochemistry (IHC). It is expected that survival analysis shows that administration of QUIN mAb delay the death of mice with a protection of a part of mice (when followed for 6 months); this is associated with a decrease of body weight lost. Motor and cognitive performances were as well ameliorated as shown by rotarod and CFC tests. We monitored level of mutated huntingtin, and no effect of the QUIN mAb is reported. This suggests that QUIN mAb doesn't affect the production of mutated HTT but its deleterious effects.
  • IHC immunohistochemistry
  • Anti-QUIN mAb may thus represent a suitable solution to limit motor and cognitive functions impairment of R6/2 mice induced by mutated huntingtin.
  • treatment with an anti-QUIN antagonist can provide a meaningful therapeutic option for the treatment of neuropathological diseases in general, and Huntington's disease in particular
  • ALS Amyotrophic lateral sclerosis
  • Lou Gehrig's disease is the most common adult-onset motor neuron disease.
  • ALS is characterized by a selective and progressive degeneration of the motor neurons in the corticospinal tracts. Failure of the respiratory muscles is generally the fatal event, occurring within 1-5 years of onset.
  • ALS is a disease of mature adults, with a median age of onset of 55 years. Due to its uniform lethality, ALS appears as a rare disease (prevalence of 4-6 per 100 000 each year), although its incidence is of one to two per 100 000 each year. Most cases (90%) are classified as sporadic ALS (SALS), as they are not associated with a documented family history.
  • FALS familial ALS
  • SODl superoxidedismutase
  • Model description The model which is used to investigate the effect of a modulator of QUIN in the treatment of Amyotrophic lateral sclerosis disease is a transgenic mouse based on a mutation on a superoxide dismutase (SOD1-G93A) that was discovered in human ALS patients by Rosen et al.
  • mice are viable and fertile, with transgenic expression of a G93A mutant form of human SODl .
  • This founder line (often referred to as G1H) is reported to have high transgene copy number.
  • Hemizygotes exhibit a phenotype similar to amyotrophic lateral sclerosis (ALS) in humans; becoming paralyzed in one or more limbs with paralysis due to loss of motor neurons from the spinal cord.
  • ALS amyotrophic lateral sclerosis
  • mice are characterized by a loss of motor functions associated with a loss of motor neurons and a reduced survival.
  • Experimental protocol and expected result Evaluation of the anti-QUIN mAb benefits is performed by Charles River.
  • anti-QUIN mAb is administered weekly at the disease onset by subcutanous adminstration of 100 ⁇ g purified antibody.
  • Body weight, motor performace (rotarod test) and survival are monitored.
  • Severeal mice are sacrificed, and the number of motorneurons in spinal cord sections is evaluated by means of immunohistochemistry (IHC) using SMI-32 marker, which is a Neurofilament H Non- Phosphorylated (SMI 32) Monoclonal Antibody for research purposes provided by Covance. It is expected that survival of SOD1-G93A mice is ameliorated when they are treated with QUIN mAb at the disease onset.
  • motor functions are ameliorated as follow by rotarod assay (time spent on the running-wheel). Analysis of number of motorneurons in spinal cord sections reveals a higher number in QUIN mAb treated mice than in isotype treated mice.
  • Anti-QUIN mAb may therefore represent a suitable solution to limit motor dysfunction and increase survival of mice harbouring the SOD1-G93A mutation.
  • treatment with an anti-QUIN antagonist can provide a meaningful therapeutic option for the treatment of neuropathological diseases in general, and Amyotrophic lateral sclerosis in particular.
  • Figures Fig. 1 Cross reactivity of the monoclonal antibody AB6512.
  • X axis shows the concentration of conjugates added for the competitive assay
  • Y axis shows the part of the response obtained by QUIN mAb reaction on its specific antigen, QUIN-BSA.
  • QUIN-BSA conjugate is immobilized on a solid support (coating step) and the QUIN mAb is added at a given dilution.
  • the affinity of the QUIN mAb is evaluated by addition of soluble QUIN-BSA with QUIN mAb to evaluate how the free form displaces the reaction between the free QUIN-BSA and the immobilized one.
  • the specificity was evaluated by addition of each conjugates (one by one), as free form, with the QUIN mAb.
  • the different conjugates was not able to displace the reaction between QUIN mAb and the immobilized QUIN-BSA ; the QUIN mA bis therfore specific.
  • Fig. 2 Preventive administration of QUIN mAB prevents EAE crisis. EAE was induced at day 0 by MOG injection and mice (8/group) were treated s.c either with 10( ⁇ g non immune IgG or QUIN IgG at days 5, 7 and 9 (arrows on the graph indicate time of injections). Clinical scores are shown on the Y axis. See text for further explanations.
  • Fig. 3 Overview of the kynurenine pathway with its enzymes and metabolites.
  • the enzymes are as follows: i) Kynurenine formamidase, a) Kynurenine amino-transferase, b) Kynurenine 3-hydroxylase (also called Kynurenine mono-oxygenase), c) Kynureninase (also called L- Kynurenine hydrolase), d) Kynurenine amino-transferase, e) Kynureninase (also called L- Kynurenine hydrolase), and f) 3-Hydroxyanthranilic Acid oxygenase (also called 3- Hydroxanthranilate dioxygenase).
  • the metabolites are as follows: L-Formylkynurenine, Kynuramine, L-Kynurenine, Kynurenic Acid, 3-hydroxyL-kynurenine, Anthranilic Acid, 3-hydroxyanthranilic Acid, Xanthurenic Acid, Quinaldic Acid, Picolinioc Acid and/or Quinolinic Acid Please note that some metabolites and enzymes of the kynurenine pathway are not shown.
  • Fig. 4 Immunochemical characterization of monoclonal antibodies targeting protein carrier conjugated Quinolinic Acid (Quinolinic-PC).
  • Affinity comparison of the different monoclonal antibodies was performed with an ELISA competition assay and shows a higher affinity for the 3B2-C7.
  • B) Specificity of the 3C10-E5 antibody was evaluated with a competition assay and shown no cross reaction with the Picolinic acid conjugate while the Quinolinic conjugate was recognized at an affinity ⁇ 10 ⁇ n M.
  • Fig. 5 Detection of derivatized Quinolinic Acid using ELISA competition assay while free Quinolinic was not recognized.
  • A An increasing concentration of Quinolinic was derivatized by mean of carbodiimide in MES buffer for lhour and then incubated over night at 4°C with a fixed amount of 3B2-C7. The mixed solution was incubated for lh30 at 37°C on a previously coated maxisorp plate with Quinolinic Acid conjugate. HRP conjugated anti mouse IgG were added for 45 minutes at 37°C and the reaction was revealed using TetraMethylBenzidine (TMB).
  • TMB TetraMethylBenzidine
  • Fig. 6 Area Under Curves (AUC) for 300 seconds after either cortical or striatal neurons treatment were calculated from mean traces of calcium signal variations in the non treated (CTRL), QA ⁇ , QA 10 ⁇ , QA ⁇ and QA ImM experimental groups and plotted as Mean ⁇ Sem
  • Fig. 7 Area Under Curves (AUC) for 300 seconds after cortical neurons treatment were calculated from mean traces of calcium signal variations in the non treated (CTRL), QA ⁇ and QA ⁇ + MK801 10 ⁇ experimental groups and plotted as Mean ⁇ standard error of the mean (sem)
  • Fig. 8 Area Under Curves (AUC) for 300 seconds after cortical neurons treatment were calculated from mean traces of calcium signal variations in the Glutamate (10 ⁇ ), QA ⁇ , QA ⁇ + 5D1-G1, QA ⁇ + 3C10-E5, QA ⁇ + 3B2-C7, QA ⁇ + 1H1-E3, QA ⁇ + 1A6-F6, and QA ⁇ + 4E11-G3 experimental groups and plotted as Mean ⁇ Sem. All antibodies were added at 0, ⁇ g/ml. References mentioned in the text

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Abstract

La présente invention se rapporte à un modulateur de QUIN (acide quinolinique) destiné à être utilisé dans le traitement d'une affection neuropathologique.
PCT/EP2013/070970 2012-10-08 2013-10-08 Modulateur d'acide quinolinique destiné à être utilisé dans le traitement d'une affection neuropathologique WO2014056934A1 (fr)

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WO2020160251A1 (fr) * 2019-01-30 2020-08-06 Arizona Board Of Regents On Behalf Of The University Of Arizona Compositions et procédés pour le traitement de maladies ou de pathologies liées à l'âge

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CA2035088A1 (fr) * 1990-01-29 1991-07-30 Michael D. Dan Anticorps humain monoclonaux pour le diagnostic et le traitement des tumeurs cerebrales
WO1995003815A1 (fr) * 1993-07-30 1995-02-09 Georgetown University Anticorps specifiques de l'acide quinolinique

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CA2035088A1 (fr) * 1990-01-29 1991-07-30 Michael D. Dan Anticorps humain monoclonaux pour le diagnostic et le traitement des tumeurs cerebrales
WO1995003815A1 (fr) * 1993-07-30 1995-02-09 Georgetown University Anticorps specifiques de l'acide quinolinique

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HEYES MELVYN P ET AL: "Quinolinic acid in tumors, hemorrhage and bacterial infections of the central nervous system in children", JOURNAL OF NEUROLOGICAL SCIENCES, ELSEVIER SCIENTIFIC PUBLISHING CO, AMSTERDAM, NL, vol. 133, no. 1-2, 1 January 1995 (1995-01-01), pages 112 - 118, XP009172448, ISSN: 0022-510X, DOI: 10.1016/0022-510X(95)00164-W *
STONE T W: "Development and therapeutic potential of kynurenic acid and kynurenine derivatives for neuroprotection", TRENDS IN PHARMACOLOGICAL SCIENCES, ELSEVIER, HAYWARTH, GB, vol. 21, no. 4, 1 April 2000 (2000-04-01), pages 149 - 154, XP004196017, ISSN: 0165-6147, DOI: 10.1016/S0165-6147(00)01451-6 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020160251A1 (fr) * 2019-01-30 2020-08-06 Arizona Board Of Regents On Behalf Of The University Of Arizona Compositions et procédés pour le traitement de maladies ou de pathologies liées à l'âge

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