WO2008006893A1 - MUTÉINES DE hNGF, UTILISATIONS THÉRAPEUTIQUES, ET COMPOSITIONS PHARMACEUTIQUES - Google Patents

MUTÉINES DE hNGF, UTILISATIONS THÉRAPEUTIQUES, ET COMPOSITIONS PHARMACEUTIQUES Download PDF

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WO2008006893A1
WO2008006893A1 PCT/EP2007/057215 EP2007057215W WO2008006893A1 WO 2008006893 A1 WO2008006893 A1 WO 2008006893A1 EP 2007057215 W EP2007057215 W EP 2007057215W WO 2008006893 A1 WO2008006893 A1 WO 2008006893A1
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hngf
mutein
ngf
human ngf
mice
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PCT/EP2007/057215
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English (en)
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Antonino Cattaneo
Simona Capsoni
Sonia Covaceuszach
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Lay Line Genomics S.P.A.
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Publication of WO2008006893A1 publication Critical patent/WO2008006893A1/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
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/48—Nerve growth factor [NGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to muteins of hNGF, their therapeutic uses and pharmaceutical compositions.
  • the muteins of the invention are distinguishable from endogenous hNGF and can be recognised by specific antibodies.
  • the invention relates to mutated coding sequences with point mutation(s) that do(es) not constitute an attack site for nucleases. The resulting protein does not provoke undesired immune reactions when administered for therapeutic or diagnostic purposes.
  • the mutation introduced in hNGF is the substitution of the pro line amino acid in position 61 with a serine and/or a mutation in residue position 100, that provides further advantages to the mutein, with respect to hNGF.
  • NGF nerve growth factor
  • SWISS PROT No POl 138 protein has numerous potential therapeutic applications with regard to the central and peripheral nervous system.
  • NGF could be used for treating Alzheimer's Disease (Tuszynski et al, 2005), multiple sclerosis (Villoslada et al., 2004; Caggiula et al., 2006, ), schizophrenia (Shoval et al., 2005), Rett's Syndrome (Riikonen et al., 1999), cerebral ischemia (Semkova et al., 1999), peripheral neuronal azotemia (Terenghi, 1999), diabetic neuropathies (Apfel, 1999) and paralyses of the facial nerve (Chao et al., 2006).
  • NGF may be used in the treatment of ocular pathologies.
  • NGF in topical application, is used in the treatment of corneal ulcers (Lambiase et al., 1998), neuritis of the optic nerve and neuropathies of the optic nerve from traumatic or ischemic origins (Lambiase et al., 2005).
  • NGF is therapeutically beneficial in cutaneous and pressure ulcers (Landi et al, 2003) and other dermatology indications.
  • NGF neuropeptide kinase kinase kinase kinase
  • cholinergic neurons of the basal forebrain depends on NGF to survive (Hefti, 1986).
  • This concept has, moreover, been reinforced by the production and characterisation of a murine model, in which the deprivation of NGF, by using transgenic antibodies directly against NGF, induces the onset of a phenotype of progressive neurodegeneration, very similar to that encountered in the brains of patients suffering from Alzheimer's Disease (Capsoni et al, (2000; WO01/10203)).
  • NGF neurotrophic factor
  • the use of NGF as a therapeutic agent in the treatment of Alzheimer's disease has already been analysed in patients suffering from this pathology.
  • the first two clinical trials realised by means of intra-cerebro ventricular infusion of NGF, have demonstrated an improvement in the patients' conditions indicated by an increase in the cerebral flow and nicotine receptors in the brain's frontal and temporal cortices (Olson et al., 1992, 1993).
  • the second trial although reporting an improvement in the cerebral functions, also provided evidence of side effects, such as weight loss and the occurrence of pain in the lumbar region that resulted in the discontinuation of the treatment (Eriksdottir- Johangen et al., 1998).
  • Rett's affected patients show a reduced NGF concentration either in the cerebrospinal liquid (Riikonen et al., 1999) or in the brain (Lipani et al., 2000) and the plasma (Calamandrei et al., 2001; Guideri et al, 2004).
  • Rett's syndrome patients therefore, may benefit from having NGF administrations.
  • CIPA insensitivity to pain with anhidrosis
  • CIPA is the first human genetic disorder implicated in the neurotrophin signal transduction system and NGF would be of therapeutic benefit for these patients as well.
  • NGF neurotrophic factor
  • NGF has a pro-nociceptive (or algogenic) action on the sensory neurons of dorsal root ganglia and spinal cord, on which it modulates the transmission of pain signals, and a pro -inflammatory action on inflammatory cells (including mastocytes), on which it activates the release of inflammation mediators (Bennett, 2001).
  • NGF In rodents, NGF, administered topically or systemically, causes hyperalgesia in response to heat stimuli which lasts over time (Lewin et al., 1994; Delia Seta et al., 1994)). In humans, the capacity of NGF to cause pain has been demonstrated in the clinical trials in AD patients, described above, as well as during clinical trials undertaken to explore the potential use of NGF in poly-neuropathies. The subcutaneous injection of NGF into the arms of healthy volunteers produces allodynia and hypersensitivity of the skin surrounding the point of injection. Furthermore, a general sensation of muscular pain is present in individuals treated with NGF and not with placebo (Dyck et al., 1997).
  • NGF neurotrophic factor
  • the maximum dose of NGF that does not cause hyperalgesia is 0.03 ⁇ g/kg (Petty et al., 1994). However, such dose is too low for NGF to access the CNS. In fact, only 2.4 ng out of 0.24 mg of NGF administered intravenously to a 25Og rat (therefore, equal to 0.001% of the injected dose) will pass the blood brain barrier and reach the brain. This quantity is twenty times lower than the 55 ng that are found in the brain after intranasal administration of the same dose of 0.24 mg (Thome and Frey, 2001).
  • NGF is a treatment that is potentially effective for patients with corneal ulcers (Lambiase et al., 1998). Furthermore, in animal models of ocular diseases, the intraocular administration of NGF resulted in an improvement of the degeneration of the ganglia cells in the retina observed after transection of the optic nerve, ocular ischemia or induced ocular hypertension (Carmignoto et al, 1989; Siliprandi et al. 1993), putting NGF forward as a potential treatment for glaucoma.
  • a pharmacokinetic study of ophthalmic applications (Lambiase et al 2005) describes the application of NGF by the topical conjunctival route and demonstrates that NGF is vehiculated at the level of the optic nerve.
  • the study shows that even in the case of conjunctival local administration of NGF drops, significant doses of NGF are found at the systemic level in the serum of the animals.
  • This study demonstrates that, following the conjunctival topical application of NGF into an eye, the levels of NGF accumulate in the serum and in the other eye, demonstrating that NGF crosses the haematic ocular barrier.
  • compositions and methods of administration that facilitate access to the desired target and to design a therapeutic product that can easily be measured, specifically and selectively, to optimise the therapeutic dose and to monitor it under the various conditions, and against the variable background of endogenous NGF.
  • the state of the art describes methods of administration, in rodents preclinical studies, by means of osmotic mini-pumps or intraventricular catheters.
  • Such methods require repeated infusions into the brain, and mini-pumps must be refilled every time that the reservoir needs to be substituted or the injection syringe reinserted into the catheters. These methods may also introduce infection.
  • the state of the art also describes direct injections of a host's cells transformed with viral vectors to induce the production of NGF by means of in vivo gene therapy.
  • This technique is limited since there is a risk of mutagenesis and tumorigenesis. In addition, this technique does not allow NGF secretion regulation and can cause undesired side effects.
  • the use of cells engineered to secrete NGF was applied in clinical trials, during which coetaneous fibroblasts secreting NGF were implanted into Alzheimer's patients (Tuszynski et al., 2005). Despite the encouraging therapeutic results obtained using this technique, significant limits were demonstrated in connection with the extremely invasive method of administration that in 2 patients has lead to cerebral haemorrhages with consequent hemiparesis.
  • Kordower et al. (1994) discloses the use of modified BHK cells expressing hNGF, but with the main limit of introducing animal proteins that represent potential immunogens for humans.
  • the international patent application WO2005/068498 describes a method for the implantation of encapsulated human cells in order to secrete a predetermined dose of NGF directly into the brain. Such a method requires general anaesthesia and an invasive neurosurgery and does not allow the NGF dose to be adjusted during the course of the treatment since the number of cells implanted is decided beforehand. Furthermore, all transplants of in vivo cells should allow treatment to be interrupted if needed, for instance in the case of side effects, the end of the treatment or a change of treatment strategy.
  • patent EP 1 137 401 relates to the use of a neurological agent or of one of its biologically active variants as a pharmaceutical product to be inoculated into the nasal cavity at a dose of 0.1 nmol up to 1000 nmol in order to protect and treat a CNS cell, in which the neurotrophic agent is selected from the group of neurotrophins (IGF, NGF, FGF and their biologically active variants).
  • the patent application CN 1616087 relates to NGF liposomes that have a greater intra- body stability than NGF, a quadruple half-life and a greater concentration in the cerebral tissue than NGF. The NGF liposomes are administered into the nasal cavity by means of gel spray.
  • the patent application EP 1 539 208 relates to compositions and methods that include a biologically active agent and a permeating agent that promotes the administration of the biologically active agent, i.e. NGF by the mucous route.
  • NGF neurotrophic factor
  • endogenous NGF concentrations in the blood and tissue vary greatly between individuals, either because NGF is bound to plasmatic proteins, thus limiting its availability to the action site, or due to the variability in its production in relation to the individual conditions that influence the synthesis of this protein and the expression of its receptors, for example stress (Aloe et al, 2002) or the patient's hormonal balance (Lanlua et al., 2001).
  • exogenous NGF protein administered needs to have a bioactivity that is similar in every respect to that of the endogenous NGF.
  • the protein should not be immunogenic and, therefore as similar as possible in terms of its amino acid sequence to endogenous NGF. However such protein would not be distinguishable from endogenous NGF. It is, therefore, necessary to identify an NGF for which an optimal therapeutic dose can be established and that can be detected.
  • NGF-based treatment for pathologies of the CNS for example Alzheimer's (AD), multiple sclerosis and Rett's syndrome, and for ocular pathologies must overcome the problems mentioned above and allow the therapeutic dose of NGF to be optimised, on an individual basis and over time, both to maximise the therapeutic dose, for the various types of administration that may be on offer and to minimise the undesirable side effects.
  • AD Alzheimer's
  • multiple sclerosis and Rett's syndrome ocular pathologies
  • ocular pathologies must overcome the problems mentioned above and allow the therapeutic dose of NGF to be optimised, on an individual basis and over time, both to maximise the therapeutic dose, for the various types of administration that may be on offer and to minimise the undesirable side effects.
  • a therapeutic NGF in addition to having the same biological activity of the wild type endogenous NGF should have at least one of the following properties and, ideally, all of them: i) to be distinguishable from endogenous NGF by a sensitive and selective method, whilst being bioactive to the same degree as endogenous NGF; ii) not to provoke undesired immune reactions if administered for therapeutic or diagnostic purposes; iii) to be used with co-adjuvants for blocking any hyperalgesic effects; in the course of optimising the dose or of the treatment; iv) to be further mutated to avoid causing pain.
  • peptide tags have the disadvantage of being immunogenic, because they alter the protein's immunological profile. Tags can also be easily proteolitically removed from the protein, thus losing their function as indicators of such protein. Peptide tags are relatively long sequences and, therefore, may alter the protein's structural and functional profile, particularly if inserted within the protein sequence. For this reason tags are usually located at the N terminus or the C terminus of the protein, but in such positions they are more easily proteolitically cleaved off.
  • the smallest known antibody tag (epitope) described is made up of three amino acids, such as, for example, the C-terminal GAE tag recognised by th monoclonal antibody 423 (Khuebachova et al, 2002). In addition, antibody 423 also recognises an epitope of the tau protein (Skrabana et al., 2004) therefore cannot be used because of its non-specificity.
  • NGF neuropeptide tag
  • a peptide tag would have to be inserted within the coding sequence for NGF since both the N- and the C- terminal parts are proteolitically processed during the course of the biosynthetic maturation of NGF (by N-terminal processing: Edwards et al., 1988; Seidah et al., 1996 and by C-terminal processing: Kruttgen et al., 1997 ).
  • the insertion of a tag in the N-terminal portion would have the further disadvantage of interfering with the binding between NGF and TrkA receptor (Woo et al., 1995; Wiesmann et al., 1999; Robertson et al., 2001).
  • the insertion of a peptide tag within the coding sequence of mature NGF would result in a drastic alteration to its structure and, therefore, its function.
  • Patent-related and scientific literature cite numerous muteins of NGF with various characteristics as listed below: i) Pan neurotrophins: muteins that combine on NGF key residues of other neurotrophins, to obtain a neurotrophin that acts on three receptors, TrkA, TrkB and TrkC. In particular muteins in position 16, 18, 20, 23, 79, 81, 84, 86, 88, in various combinations are disclosed. These muteins are described in the international patent application WO2005068498; ii) Mutant NGF with a reduced capacity to bind the p75 receptor. The involved residues are residues in positions 32,34, 35, and 95.
  • WO2005/040335 identified from genomic DNA of patients with a pathology of slight insensitivity to pain.
  • the corresponding peptide presents a mutation in position 100 of the deduced amino acid sequence of NGF
  • the authors of the present invention have a panel of antibodies (directed against NGF, or its TrkA receptor). These antibodies are able to block the biological effects of NGF mediated by the binding to TrkA, including peripheral nociceptive effects.
  • Two reagents alfaDl l (anti-NGF, Cattaneo et al 1988; WO 2005/061540) and MNAC 13 (anti-TrkA, Cattaneo et al 1999, EP 1 181 318, WO 2005/061540) are of particular interest because they are available in a "humanised" form which can be used in humans.
  • NGF mutein further mutated to avoid causing pain, but having the same potency and neurotrophic activity as hNGF on its neuronal targets would be of great advantage, avoiding the unwanted side effects that limit its therapeutic uses.
  • mutein of human NGF refers to muteins of the whole human pro-NGF or of the truncated mature human NGF, lacking first 121 amino acids.
  • therapeutically active refers to a biologically active molecule providing a therapeutic benefit in the following human diseases: Alzheimer's disease, Parkinson's disease, Chronic Insensitivity to pain with anhydrosis (CIPA), multiple sclerosis, peripheral nerve injury and neuropathies, Rett's syndrome, Schizofrenia, pathologies of peripheral nervous system, ulcers from diabetis or decubitus, Vasculitis associated reumatoid arthritis, congestive heart failure, corneal ulcer and/or age-related immunodeficiency.
  • CIPA Chronic Insensitivity to pain with anhydrosis
  • multiple sclerosis peripheral nerve injury and neuropathies
  • Rett's syndrome Rett's syndrome
  • Schizofrenia pathologies of peripheral nervous system
  • ulcers from diabetis or decubitus ulcers from diabetis or decubitus
  • the term "undesired immune reactions” refers to a molecule lacking induction of antibodies against endogenous human NGF and against mutein of human NGF and lacking induction of allergic reactions.
  • the mutein is obtained by at least one amino acid substitution residing in loop III of hNGF, more the substituted amino acid in loop III is pro line at position 61, most preferably the amino acid substitution of proline in position 61 is with a serine.
  • the mutein of human NGF as above described further comprising at least one amino acid substitution able to substantially reduce its nociceptive activity.
  • the mutein of human NGF further comprises at least one amino acid substitution leading to a reduced interaction of the mutein with the p75 receptor.
  • the amino acid substitution is at any of positions 95-101.
  • the amino acid substitution is that of the arginine in position 100, even more preferably the amino acid substitution of the arginine in position 100 is with an acidic amino acid or tryptophane.
  • the acidic amino acid may be glutamic or aspartic acid.
  • the amino acid substitution is that of the glutamine in position 96.
  • the amino acid substitution is that of the tryptophan in position 99.
  • the amino acid substitution is that of the phenylalanine in position 101. It is a further object of the invention the mutein of human NGF as above described for therapeutic use.
  • a medicament for the treatment of pathologies of the central and peripheral nervous system.
  • CIPA Chronic Insensitivity to pain with anhydrosis
  • multiple sclerosis preferably for Alzheimer's disease, Parkinson's disease, Chronic Insensitivity to pain with anhydrosis (CIPA), multiple sclerosis, peripheral nerve injury and neuropathies, Rett's syndrome, schizophrenia, ulcers from diabetes or decubitus, vasculitis associated rheumatoid arthritis, congestive heart failure, corneal ulcer and/or age-related immunodeficiency.
  • a pharmaceutical composition comprising a pharmaceutically acceptable and effective dose of the mutein of human NGF as above described and suitable diluent and/or carrier.
  • the dose is determined according to the concentration of endogenous human NGF.
  • the pharmaceutical composition as above described is for final vehiculation to the central nervous system, for intranasal administration, for intraocular or ophthalmic administration, for systemic administration or topical administration. Even more preferably the mutein of human NGF is associated with nanostructures or with liposomes.
  • the pharmaceutical composition further comprises a pain-killer agent. More preferably the pain-killer agent is a neutralising anti-NGF or anti-TrkA antibody.
  • the neutralising anti-NGF or anti-TrkA antibody is an antibody that blocks the biological activity of NGF or TrkA, and in particular the nociceptive action.
  • the pain-killer agent is formulated separately.
  • the specific reagent is a monoclonal antibody or a synthetic or a biotechno logical or a humanised antibody, or a fragment thereof. More preferably it is an antibody able to selectively recognise a mutein of hNGF having a serine at position 61.
  • FIGURE 1 (A) Multiple alignment of the primary structures of NGF from various analysed species: mouse NGF, mNGF, rat NGF, rNGF, human NGF, hNGF (B) and (C)
  • FIGURE 2 Structural alignment of the tertiary structure of mNGF (shown in grey) and the hNGF (in black): loop III is indicated by a dotted line; figures produced using Pymol
  • FIGURE 3 Absence of interaction between loop III (dotted area) and domain d5 of receptor TrkA (A) and P75 (B).
  • FIGURE 4 Comparison of binding activity of the monoclonal antibody 4GA to NGF in various species A) comparison between rat NGF (rNGF) and mouse (mNGF); B) comparison between mouse (mNGF) and human (hNGF) NGF and their precursors mouse
  • m proNGF human (h proNGF) proNGF .
  • HPR Horseradish Peroxidase
  • FIGURE 5 Comparison of binding activity of the monoclonal antibody 4GA to point muteins of rNGF (rat wt) in non-conserved amino acid positions 40 (G40A), 61 (P61S) and 117 (Al 17T), demonstrated in comparative analyses between mNGF and rNGF.
  • P61S mutation of the rNGF confers reactivity towards the 4GA antibody that is fully comparable with that of the natural antigen (mNGF).
  • the measured OD(450nm) is related to 4GA binding activity as explained in Figure 4.
  • FIGURE 6 Comparison of binding activity of the monoclonal antibody 4GA to the point mutein of hNGF (human wt) in non-conserved amino acid position 61 (P61S) demonstrated in comparative analyses with wildtype human NGF.
  • the point mutation Pro ⁇ lSer in hNGF is able to restore the reactivity of the monoclonal 4GA antibody, even with regards to the hNGF.
  • FIGURE 7 The affinity for the receptors TrkA and p75 of mutein form hNGF Pro ⁇ lSer results similar to that of wildtype hNGF.
  • FIGURE 8 The induction of TrkA phosphorylation of the mutein form hNGF Pro ⁇ lSer results similar to that of wild type hNGF.
  • TrkA phosphorylation levels of TrkA induced by serial dilutions (shown on x axis) of wild type hNGF and the mutein form hNGF Pro ⁇ lSer (indicated on y axis) are detected by a rabbit antibody that recognizes phospho-TrkA, which is in turn revealed by an anti-rabbit antibody that is conjugated to the enzyme Horseradish Peroxidase (HPR): The measured OD (450nm), plotted on z axis, is related to TrkA phosphorylation levels as explained in Figure 4.
  • HPR Horseradish Peroxidase
  • FIGURE 9 Proliferative response of the TFl cell line to NGF.
  • the TFl proliferation assay was performed in quadruplicate in microtiter plates by incubating 15,000 cells per well in the presence of the indicated concentrations of huNGF and hNGF-61. After a 40-hour culture period, 10 ⁇ l of MTT substrate solution was added for an additional 4 hours of incubation and the OD was determined at 570 nm in a microtiter plate reader, 16 h following the addition of detergent reagent (100 ⁇ l per well). huNGF and hNGF-61 dose/response curves were substantially super-imposable.
  • FIGURE 10 Scheme of TrkA signalling pathways.
  • FIGURE 11 Western blot analysis of TrkA and Akt phosphorylation on Y490 and S473, respectively, in extracts from BALB/C 3T3 cells incubated in presence of huNGF R&D (commercial source, produced in mammalian cells), hNGF (produced in E. coli following the same procedure as NGF-61, see examples) and hNGF-61.
  • the three NGF variants induce the same degree of activation.
  • FIGURE 12 Position of the residue ArglOO (RlOO, dotted area) in the crystallographic structures of the complexes between wild type hNGF (shown in dark grey) and its receptors: A) p75 (left part of the complex) and B) domain d5 of receptor TrkA (right- hand part of the complex); figures produced using Pymol (DeLano, 2002).
  • FIGURE 13 Analysis of the binding interface between hNGF and p75 receptor, that involves the ArglOO residue (underlined and in bold).
  • the table lists all the residues that mediates the binding interface, in particular first column: residue number, second column: residue type; third column: molecule (NGF monomer: NGF A; p75 respectively); forth and fifth columns: interface mediated by each residue (respectively in absolute and % value); sixth column: hydrogen bonds mediated by each residue (if present, 1).
  • FIGURE 14 (A) Elution profile from Hitrap SP sepharose XL of huproNGF WT after pulsed refolding: the area below the curve is proportional to the total amount of purified protein. (B) Comparison of elution profiles from Hitrap SP sepharose XL of huproNGF muteins in position 100, after pulsed refolding: the areas below each curve is proportional to the total amount of each purified mutein.
  • FIGURE 15 Affinity to TrkA of hNGF muteins in position 100: response curves of serial dilutions (from 50OnM to 4nM) of A) hNGF, B) hNGF ArglOOLys, C) hNGF ArglOOAla, D) hNGF ArglOOGln and E) hNGF ArglOOGlu All the graphs plot the resonance units (RU) on the y axis versus the time on the x axis.
  • RU resonance units
  • FIGURE 16 Affinity to p75 of hNGF muteins in position 100: response curves of serial dilutions (from 50OnM to 4nM) of A) hNGF, B) hNGF ArglOOLys, C) hNGF ArglOOAla, D) hNGF ArglOOGln and E) hNGF ArglOOGlu.
  • AU the graphs plot the resonance units (RU) on the y axis versus the time on the x axis.
  • FIGURE 17 Analysis of the affinity of muteins NGF-61 RlOO for TrkA (A, C, E, G, I) and p75 receptors (B, D, F, H, J).
  • FIGURE 19 Western blot analysis of TrkA, Akt and PLC- ⁇ l phosphorylation on Y490, S473 and Y783, respectively, in extracts from BALB/C 3T3 cells incubated in presence of different RlOO muteins.
  • FIGURE 20 Densitometric analysis of western blot analysis for (A) TrkA phosphorylated in position Y490, shc/Ras pathway, (B) Akt phosphorylated in position S473 and (C) PLC- ⁇ l phosphorylated in position Y783. Bars are representative of the ratio between values obtained for muteins and wild type huNGF.
  • FIGURE 21 The differentiation of PC 12 cells is induced by 100 ng/ml (A) hNGF and (B) NGF-61. (C) No differentiation was shown in PC 12 cultures grown in absence of NGF.
  • the muteins (D) huNGF ArglOOLys and (E) NGF-61 ArglOOLys induce a similar differentiation with respect to huNGF and NGF-61, while the muteins (F) huNGF ArglOOGlu and (G) NGF-61 ArglOOGlu are less effective in inducing differentiation in PC12 cells.
  • FIGURE 22 Treatment with NGF-61 administered intranasally improves the number of cholinergic neurons in the basal forebrain in ADI l anti-NGF mice, a transgenic mouse model for Alzheimer's disease.
  • FIGURE 23 Treatment with NGF-61 administered intranasally reduces the number of cells that express phosphorylated tau in ADl 1 anti-NGF mice, a transgenic mouse model for Alzheimer's disease.
  • FIGURE 24 Treatment with NGF-61 administered intranasally reduces the number of dystrophic neurites that contain ⁇ amyloid.
  • * P ⁇ 0.05 ADI l vs. non-transgenic mice; #, P ⁇ 0.05 ADl 1 treated with rhNGF or hNGF-61 vs. ADl 1 treated with placebo).
  • FIGURE 25 Discrimination index in the object recognition test in ADI l anti-NGF mice after treatment with hNGF-61 . Non-transgenic mice mainly explore the new object and, consequently, have a positive discrimination index. ADI l mice treated with PBS do not discriminate between the new and old object and, therefore, have a negative discrimination index.
  • FIGURE 26 The intranasal administration of huNGF and hNGF-61 to 15 months old ADl 1 mice determines: the increase in cholinergic neurons in (A) the medial septum (MS) and diagonal band of Broca (DBH); (B) the decrease of ⁇ -amyloid burden in the hippocampus and (C) the decrease of tangle-like cells over-expressing phosphorylated tau.
  • FIGURE 27 The monoclonal antibody 4Ga is able to distinguish different concentrations of exogenous hNGF-61 from endogenous wild type human NGF in human blood serum (A) The antibody 4GA allows to distinguish hNGF-61 from endogenous NGF in human serums, whereas (B) the antibody ⁇ Dl l does not distinguish between hNGF-61 and endogenous NGF. Bars represent the mean value ⁇ SEM for each exogenous hNGF-61 concentration.
  • FIGURE 28 Absence of production of anti-NGF IgG in mice treated with hNGF61.
  • the level of anti-NGF IgG were measured by ELISA in different dilutions of the serum.
  • the level of optical density (O. D.) represented on the Y axis directly correlates with the amount of IgG in the serum. Bars represent the mean value ⁇ SEM for each serum dilution.
  • FIGURE 29 Absence of increase of IgE (expressed on the Y axis as ⁇ g/ml) in the serum of mice treated with hNGF61 intranasally with respect to mice treated with PBS. Bars represent the mean value ⁇ SEM for each group of treatment.
  • FIGURE 30 Effects of intra-cerebroventricular injection (I. CV.) of NGF-61 on the response to pain assessed using the hot plate test. I. C. V. NGF61 administration decreases the latency to paw licking in rats. The reduction in pain threshold is prevented by administration of the antibody ⁇ Dl 1.
  • FIGURE 31 Intermitting dosing in ADI l mice.
  • the intranasal administration of NGF-61 to 6 months old ADl 1 mice three times per week (3 W) determines the rescue of the object recognition memory deficit, indicated by a higher discrimination index (on the Y axis).
  • a more delayed administration [once per week (IW) or twice per week (2W)] is not effective.
  • FIGURE 32 The intranasal administration of NGF-61 to 6 months old ADI l mice three times per week determines the rescue all neurodegenerative markers, while a more delayed administration is effective only in rescuing the increase in A ⁇ deposits.
  • C Percentage of hippocampal area occupied by ⁇ amyloid clusters and
  • D Number of phosphotau-positive dystrophic neurites in the entorhinal cortex.
  • FIGURE 33 (A) The intranasal administration of huNGF and NGF-61 to 15 months old ADI l mice three times per week determines the rescue object recognition deficit (a) and this amelioration persists after one month during which the mice were not treated (b). (B) After 1 month of was-out the number of cholinergic neurons in the basal forebrain was not improved both by huNGF and NGF-61. (C) After 1 month washout, a decrease in the percentage of hippocampal area occupied by ⁇ -amyloid was shown in ADI l mice treated with NGF-61, but not huNGF.
  • FIGURE 34 The intranasal (in) and conjunctival (oc) administration of NGF-61 to 6 months old ADI l mice three times per week determines the rescue of the object recognition memory deficit.
  • FIGURE 35 The intranasal (in) administration of NGF-61 to 6 months old ADI l mice three times per week determines the rescue all neurodegenerative markers.
  • FIGURE 36 Absence of loss of body weight in ADl 1 mice treated intranasally for 15 days with 480 ng/kg of hNGF-61.
  • FIGURE 37 Area in ⁇ m 2 (mean ⁇ S. E. M.) of calcitonin gene-related peptide- immunoreactive fibers in the spinal cord in ADI l mice treated intranasally with placebo, huNGF (480 ng/kg) or NGF-61 (480 ng/kg) for 1 month.
  • FIGURE 38 The intranasal administration of NGF-61 ArglOOGlu and NGF-61 to 6 months old ADI l mice three times per week determines the rescue of the object recognition memory deficit (*, P ⁇ 0.05 ADl 1 vs WT mice; # P ⁇ 0.05 ADl 1 treated with
  • FIGURE 39 The intranasal administration of NGF-61 ArglOOGlu to 6 months old ADI l mice three times per week determines the rescue all neurodegenerative markers with the same efficacy as NGF-61.
  • FIGURE 40 Hot plate jumping response of male mice injected IV with either NGF-61 (2.5 ⁇ g/g), NGF-61 ArglOOGlu (2.5 ⁇ g/g) or cytochrome c (CYT, 2.5 ⁇ g/g) in repeated tests (n
  • NGF NGF-like growth factor
  • a variant of NGF needs to be identified. It should have the following properties: i) same bioactivity than wild human NGF; ii) minimum possible number of differences in the composition and sequence of amino acids compared to wild human NGF, but selectively and quantitatively distinguishable from endogenous NGF.
  • NGF proteins of any known species are also bioactive on human receptors as measured in a large number of in vitro quantitative tests.
  • murine NGF is active on humans, under therapeutic conditions, for example in the treatment of corneal ulcers (Lambiase et al 1998). The reason for this similar bioactivity may be explained by the fact that NGF proteins of these species are highly conserved, both from the point of view of the primary sequence, especially in the protein regions involved in the interactions with TrkA e p75 receptors, and from the structural point of view.
  • murine NGF is considered to be the golden standard in terms of bioactivity, also in respect to the bioactivity of recombinant human NGF. Therefore, the authors proceeded to identify, by means of a primary sequence alignment, the difference in amino acid residues between NGF of three species, mice, rats and humans (mNGF, rNGF and hNGF). A multiple alignment of the primary structures of the mNGF, rNGF and hNGF was carried out, as reported in Figure IA. The comparative analysis indicated a total of 5 mutations that differentiate mNGF from both hNGF and rNGF (indicated in bold and underlined in Figure IA).
  • Example 1 Structural comparative analysis of mNGF and hNGF of the loop III region containing position 61
  • the region including position 61 is not defined in the structure of hNGF, in which the entire loop III is completely lacking (see black C-alpha trace underlined by a circle).
  • the absence of loop III in the experimental structures of hNGF complexes indicates the scarce definition of the electronic density at this level of the crystallographic structures and is an index of disorder and flexibility, which is not found in the structure of mNGF, where such a region is perfectly defined.
  • the only difference between mNGF and hNGF is represented by Pro 61Ser, it is reasonable to suppose that the presence of Pro in position 61 is responsible for the increased flexibility of this loop in the human form, compared to that of murine. It can also be considered that the introduction of the residue Ser into position 61 of hNGF, in place of residue Pro could stabilise the entire loop III, consequently facilitating its crystallisation.
  • loop III of NGF which contains position 61, is not directly involved in the structural interaction between NGF and its two receptors TrkA and p75 is demonstrated in Figure 3.
  • mNGF was substituted in the experimental complexes between hNGF and the receptors, to indicate loop III given that, in the case of hNGF it cannot be seen.
  • Figure 3 clearly shows that loop III does not significantly interact with either of the two receptors TrkA and p75.
  • position 61 of hNGF and, in particular, the Pro ⁇ lSer substitution may lead to the engineering of an NGF variant capable of solving the problem described.
  • the variant of NGF modified in position 61 has a bioactivity comparable to that of hNGF (and mNGF), since such a position is not involved in the interaction with its two receptors, and since the residue that is naturally present in position 61 (proline 61) in huNGF is substituted by a residue (serine 61) naturally present in mNGF, which represents the golden standard in terms of NGF bioactivity and potency.
  • position 61 distinguishes hNGF from mNGF.
  • the most convenient method involve the use of a specific binder, i.e. an antibody, directed against an epitope comprising position 61.
  • antibodies that were capable of distinguishing a variant of hNGF comprising the Serine antibody in position 61 from the corresponding wild hNGF protein were selected.
  • the strategy used to obtain such antibodies was to implement a combined approach: immunising rats with the murine NGF protein (according to the already described protocol to isolate monoclonal antibodies directed against NGF (Cattaneo et al, 1988), benefiting from the fact that mNGF protein naturally contains the residue Ser 61, whilst the rat and human proteins (rNGF, hNGF) do not, and testing the antibodies obtained for their ability to differentiate between i) mNGF from rNGF and hNGF and ii) an hNGF-61 variant, containing S er61 , from wild hNGF .
  • the immunisation protocol and the immunisation methods used are implemented as described in Cattaneo et al 1988 and allowed the isolation of an antibody denominated 4GA.
  • Example 2 The monoclonal 4GA antibody is species-specific for mNGF
  • the immunoglobulins to be tested were expressed in the supernatant using culture of hybridoma cells deriving from the fusion of the lymphocytes obtained from the spleens of rats immunised with myeloma cells, and concentrated using precipitation with ammonium sulphate 29% followed by dialysis in PBS. Next, they were purified by means of affinity chromatography using a column of Protein G Sepharose (Pharmacy).
  • a two-site sandwich ELISA test was developed to compare the reactivity to murine NGF (mNGF) and the ortolog rat form (rNGF), immobilising the monoclonal 4GA antibody on Maxi sorb 96 well plates, by means of incubation at 37°C for 2 hours and in a concentration of 10 ⁇ g/ml in sodium carbonate buffer 0.1M at pH 9.6.
  • mNGF murine NGF
  • rNGF ortolog rat form
  • the aforementioned 4 antigens (mNGF, Alomone, Israel, or hNGF, hproNGF, mproNGF produced according to Rattenholl et ah, (2001)) at a concentration of 10 ⁇ g/ml in sodium carbonate buffer 0.1M, pH 9.6 were directly immobilised on Maxi sorb 96 well plates by means of incubation overnight at 4°C.
  • the monoclonal 4GA antibody is able to interact only with the murine NGF (mNGF) and its precursor (m-proNGF) and not with the human NGF (hNGF) or its precursor (h- proNGF, Figure 4B).
  • Example 3 Mapping the epitope of the monoclonal 4GA antibody To identify the epitope recognised by the 4GA antibody, three point rat NGF muteins were generated by site-specific mutagenesis according to Stratagene's protocol. Such mutations restore the murine amino acid in the backbone of r NGF: Gly40Ala, Pro ⁇ lSer, Alal 17Thr. The resulting mutagenesis was controlled by DNA sequencing.
  • each mutein including rNGF
  • 250,000 COS cells were transfected with 1 ⁇ g of coding plasmid DNA for each mutein by FuGENE, as advised in the protocol (Roche).
  • each supernatants containing the transfected mutein expressed by the host cells were collected, then concentrated using Centriprep 50 (Amicon): the quantity of each mutein was standardised by dot blot. Briefly, 5 ⁇ l of each concentrated supernatant was applied to nitrocellulose membrane, together with serial dilutions of known concentration of mNGF (positive control and standard) and in parallel to the concentrated supernatant of non-transfected COS cells (negative control).
  • both cDNA were cloned in a vector for prokaryotic expression (pETMl l) in the form of a precursor (hproNGF), and the corresponding proteins expressed in inclusion bodies and re- naturated by means of the pulsed refolding protocol of Rattenholl et ah, (2001).
  • pETMl l prokaryotic expression
  • hproNGF precursor
  • corresponding proteins expressed in inclusion bodies and re- naturated by means of the pulsed refolding protocol of Rattenholl et ah, (2001).
  • Each precursor underwent proteolytic cleavage with trypsin (enzyme substrate ratio of 1 :250) to obtain mature NGF that, after addition of protease inhibitors, was purified by means of gel chromatography using a Superdex 75 column (Pharmacy) and quantified by a Lo wry test (BIORAD).
  • the biological activity of the mutein hNGF Pro ⁇ lSer was compared to that of the wild type hNGF by means of TrkA phosphorylation test in BALB/C 3T3 cells stably transfected with the human TrkA receptor. Briefly, 10 6 cells from this stable line were plated for each experiment and incubated for an hour in DMEM with the serum removed and supplemented with 0.05% BSA. Therefore, serial equimolar dilutions of wild type hNGF and mutein hNGF Pro ⁇ lSer (from 1 to 20 nM) are incubated for 10 minutes at 37°C.
  • the cells were collected in 0.25ml of cold RIPA buffer supplemented by protease inhibitors and phosphates (5OmM Tris pH 7.4, 15OmM NaCl, 1% Triton XlOO, 1% Na deoxycholate, 1OmM EDTA, cocktails of protease inhibitors (Roche), 1 mM sodium orthovanadate, 50 mM NaF, 1 nM okadaic acid).
  • protease inhibitors and phosphates 5OmM Tris pH 7.4, 15OmM NaCl, 1% Triton XlOO, 1% Na deoxycholate, 1OmM EDTA, cocktails of protease inhibitors (Roche), 1 mM sodium orthovanadate, 50 mM NaF, 1 nM okadaic acid.
  • the insoluble fraction was removed by centrifuging at 10,000 rpm for 5 minutes.
  • the extracts were quantified using the Lowry test (BIORAD) and standardised in
  • This test includes the immobilization of the monoclonal anti-TrkA MNAC 13 antibody (Cattaneo et al., 1999) on Maxi sorb 96 well plates, by incubation at 4°C for 16 hours and a concentration of 10 ⁇ g/ml in sodium carbonate buffer 0.1M pH 9.6. After an hour of blocking with PBS containing 3% milk (MPBS) at room temperature, the standardised quantities of each extract were incubated for two hours at room temperature.
  • MPBS PBS containing 3% milk
  • TrkA cell signalling
  • TrkA cell signalling
  • secondary antibody anti-rabbit peroxidase conjugated
  • Neurite outgrowth in PC 12 cells was also evaluated by treating for a week 25,000 cells plated on collagenated plates with 100 ng/ml of hNGF or of hNGF P61S, changing the culture medium every three days.
  • the capacity of hNGFPro ⁇ lSer mutein to induce differentiation is indistinguishable from that of the wild type Hngf (Fig. 21).
  • hNGF ⁇ l protein has been assessed using a survival and differentiation test in dorsal root ganglia (DRG) from chicken embryos 7-9 days old and paravertebral sympathetic ganglia from chicken embryos aged 10-12 days, after incubation with wild hNGF and hNGF-61, at concentration intervals between 1 and 100 ng/ml. After dissection, the ganglia were left on a Hepes buffer (HBSS) for then to be cultivated on plates treated with poly- lysine (1%), in DMEM with 10% FBS, 2mM L-glutamine and antibiotics. The treatment with hNGF and hNGF-61 was performed at the beginning, then repeated every 48 hours for 2-3 weeks.
  • DDG dorsal root ganglia
  • HBSS Hepes buffer
  • DMEM poly- lysine
  • hNGF and hNGF-61 The effect of hNGF and hNGF-61 on the neurite growth (time of appearance and length of neurites) after 24 hours was similar in the two treatments.
  • PC 12 cells, DRG and SCG bioassays represent the classical assays to measure the potency of NGF activity.
  • the biological activity of hNGF ⁇ l was analyzed in TFl erythro leukemic cells.
  • This human cell line express human TrkA receptors in the absence of detectable p75 (Chevalier et al, Blood 83: 1479-1485, 1994).
  • NGF induces autophosphorylation of TrkA and could substitute for granulocyte-monocyte colony-stimulating factor (GM-CSF) to trigger the proliferation of the TFI cell line.
  • GM-CSF granulocyte-monocyte colony-stimulating factor
  • hNGF-61 and wild type hNGF were synthesized according to the procedure described above.
  • hNGF-61 was equally potent as hNGF in inducing the proliferative response in TFl cells, the latter were purchased from ATCC (ATCC #CRL-2003, United Kingdom) and cultured according to the protocol established by Kitamura T , et al. (J. Cell. Physiol. 140: 323-334, 1989).
  • Cells were cultured for 1 week in the following medium (ATCC #30- 2001): RPMI 1640 medium with 2 mM L-glutamine adjusted to contain 1.5 g/1 sodium bicarbonate, 4.5 g/1 glucose, 10 mM HEPES, and 1 mM sodium pyruvate (90 %), supplemented with 2 ng/ml recombinant human GM-CSF (R&D Systems #215-GM), and 10% fetal bovine serum.
  • the cell culture were maintained at constant temperature of 37 ° C under an atmosphere of 95% air and 5% CO 2 .
  • the TFI proliferation assay was performed in 96 wells microtiter plates by incubating 15,000 cells per well in the presence of several doses of either huNGF or hNGF-61 ranging between 50,000 and 5 pg/ml. Cells were seeded Ih before adding treatments.
  • a MTT Cell proliferation assay kit (ATCC #30- 1010K) was employed to evaluate cell response: after a 40-hour culture period, 10 ⁇ l of MTT solution was added for an additional 4-hour incubation, according to the previously described colorimetric assay (Mosmann, J Immunol Methods 65:55, 1983).
  • the optical density (OD) was determined at 570 nm in a microtiter plate reader, 16 h (O/N), following addition of detergent reagent (lOO ⁇ l per well).
  • hNGF-61 was able to induce proliferation TFl cells in a dose/dependent manner and that its potency was identical to the one showed by wild type hNGF ( Figure 9) and to the one reported in literature (Chevalier et al., Blood 83: 1479- 1485, 1994).
  • concentration of NGF determining a half maximum effect on cell proliferation was equal to 0.80 ng/ml for hNGF-61, 0.78 for hNGF and to 1 ng/ml for mNGF (Chevalier et al., Blood 83: 1479-1485, 1994), which reprecent the golden standard for NGF potency and bioactivity.
  • the potency of hNGF-61 is identical to that of huNGF and of mNGF also on a quantitative potency assay involving human TrkA receptors.
  • TrkA pathway Activation of TrkA pathway It is known that NGF induces cell survival through the activation of the Akt pathway and that the activation of cytoplasmic domain of TrkA Y490 promotes cell growth through the activation of the she pathway (reviewed in Reichardt, Phil. Trans. R. Soc. B, 361 : 1545- 1564, 2006). The induction of cell growth and of cell differentiation and plasticity is also mediated by the activation of PKC pathway through PLC ⁇ (reviewed in Reichardt, Phil. Trans. R. Soc. B, 361 : 1545-1564, 2006).
  • hNGF-61 was able to activate different signalling pathways summarized in Figure 10
  • BALB/C 3T3 cells transfected with the human TrkA receptor were seeded and cultured in DMEM medium without serum and supplemented with 0.05% BSA.
  • commercial hNGF hNGF R&D
  • hNGF and hNGF-61 produced from E. coli were added at different concentrations ranging from 5 to 25 ng/ml for 10 min at 37°C.
  • TrkA anti Pi TrkA Y490; Cell Signaling
  • Akt anti-pi AkT Ser 473, Cell Signaling
  • Figure 11 shows that hNGF-61 induce a perfectly comparable activation of TrkAY490 domain and of Akt to that induced by other NGF species and sources tested.
  • Example 5 Structural analyses of the complexes hNGF with TrkA and p75 receptors at the level of residue RlOO
  • the position of residue Arg 100 in hNGF has been analysed at the level of NGF known tertiary structures in complex with its natural receptors.
  • the structure of the complex between hNGF and the extra-cellular domain of p75 receptor was analysed (code PDB ISGl, He and Garcia, (2004)); as demonstrated in Figure 12 A, the residue ArglOO (dotted area shaded in grey) is directly involved in salt bridge with residue 75 of p75 (left-hand part of the complex) and is located within a large interface described in Figure 13.
  • Figure 13 lists all the residues that mediate NGF-p75 interaction, pointing out the contribution of each residue to the binding interface.
  • variants of wild type hNGF or hNGF Pro ⁇ lSer were engineered in which the residue ArglOO was substituted with Trp, Ala, GIu, Lys and GIn respectively.
  • the residue ArglOO was substituted in the wild type hNGF or hNGF-61 proteins by site-specific mutagenesis according to the Stratagene protocol. The occurrence of the mutagenesis was verified by DNA sequencing.
  • the cDNA was cloned in the prokaryotic vector pETMl l and expressed and purified according to the pulsed-refolding protocol described by Rattenholl et al. (2001).
  • the refolded protein yields of the different mutants were compared to those obtained for wild type huproNGF (Fig. 14A).
  • Mutants ArglOOLys and ArglOOGln (Fig. 14B) gave the same yields as wild type huProNGF, while ArglOOTrp gave a low expression yield (Fig. 14 B).
  • the expression of ARg 100 Ala and ARGIOOGIu gave an intermediate amount of protein with respect to huproNGF and ArglOOTrp (Fig. 14B).
  • TrkA phosphorylation The ability of the hNGF muteins in position 100 to induce overall TrkA phosphorylation in 3T3 BALB/C cells as described above was evaluated. hNGF muteins in position 100 show similar activity compared to that of the wild type hNGF ( Figure 18). However, muteins ArglOOAla and ArglOOGlu display a slight reduction in TrkA phosphorylation.
  • PBK/ AKT and PCL- ⁇ l pathways are particularly interesting since the activation of TrkA receptor through these pathways results in hypersensitization of the TRP channel to thermal and mechanical stimuli and thus results in induction of pain (Chuang et al, Nature 411, 957-962, 2001; Prescott and Julius Science 300, 1284-1288, 2003; Bonnington and McNaughton J Physiol (London) 551, 433-446, 2003).
  • an ideal NGF mutant should be able to activate Akt and Shc/Ras pathways, while having a reduced efficacy in activating the PCL- ⁇ l pathway.
  • BALB/C 3T3 cells expressing the human TrkA receptor were incubated with NGF RlOO mutants under the conditions described before. All mutants were used in the range of 50- 100 ng/ml. Data shown are for 100 ng/ml.
  • FIG. 21 shows that both hNGF (Fig. 21A) and hNGF-61 (Fig 21B) were able to induce and differentiation in PC 12 cells, while in the absence of hNGF or hNGF 61 PC 12 cells did not differentiate (Fig. 21C).
  • a pro-differentiation effect was obtained by hNGF ArglOOLys (Fig. 21D) and by hNGF-61 ArglOOLys (Fig. 21E).
  • the mutation ArglOOGlu in line with the results obtained in the TrkA phosphorylation assay described above, showed a reduced differentiation activity with respect to hNGF and hNGF-61 (Fig. 21 F and 2 IG, respectively).
  • TFl proliferation The biological activity of hNGF mutants and hNGF-61 mutants in position RlOO was also tested in the proliferation assay on TFl cells, according to the protocol described above. As positive controls, hNGF R&D (commercial from R&D systems) , hNGF synthetized according to the procedure described in example (hNGF) and hNGF-61 were used .
  • Table 2 reports the concentrations of hNGF mutants at which the half-maximum (50%) proliferative response it was observed.
  • the mutation was performed on the NGF-61 background, the same result was obtained for hNGF-61 ArglOOGlu and ArglOOLys, while the mutation hNGF-61 ArglOOGln provoked a shift of the concentration to 3.23 ng/ml.
  • hNGF derivatives mutated in position 100 and possibly in other positions, showing an identical capacity than wild type hNGF in inducing of survival, differentiation and proliferation, but a differential ability in activating the signalling pathways of TrkA receptor linked to nociception.
  • the mutants ArglOOTrp and ArglOOGlu do not activate the PCL- ⁇ l and Shc/Ras pathways. This conclusion has been verified both on wild type huNGF and NGF-61 backbone.
  • Example 7 Efficacy of hNGF-61 in a murine model of Alzheimer's disease
  • hNGF-61 The efficacy of hNGF-61 was evaluated after intranasal administration in 6 month old ADI l anti-NGF mice (Ruberti et al, 2000; Capsoni et al. 2000). hNGF-61 was administered every 2 days for 2 months.
  • human recombinant NGF was administered (rhNGF; Alomone Laboratories, Jerusalem).
  • rhNGF human recombinant NGF
  • the mice were anaesthetised with an intraperitoneal injection of 2,2,2- tribromoethanol (400 mg/kg).
  • NGF-61 and rhNGF were administered in a 10 ⁇ M dose, mixed into 40 mM PBS, pH 7.4 for a total volume of 48 ⁇ l. This volume was subdivided into 3 ⁇ l drops, which were then allowed to fall into nostrils, alternating between nostrils, for a period of 30 min.
  • ELISA was used to check that sufficient quantities of hNGF-61 had reached the brain.
  • the effects on the Alzheimer's phenotype were assessed by incubating sections of alfaDl 1 mice brain with antibodies directed against the transfected acetylcholine enzyme
  • hNGF-61 and rhNGF induced an increase in the levels of free NGF equal to double that observed in alfaDl l mice not treated with NGF-61 or rhNGF (mice treated with NGF-61 : 23 ng/mg tissue; mice treated with rhNGF: 20 ng/mg tissue, mice treated with vehicle: 8 ng/mg tissue).
  • hNGF 61 As with hNGF, the administration of hNGF 61 induced an increase in the number of cholinergic neurons in the basal proencephalon of alfaDl l mice, which have, therefore, recovered the cholinergic deficit ( Figure 22). Furthermore, the administration of hNGF-61 induced a the reduction in the number of neurons that express the tau protein in its phosphorylated form ( Figure 23) and the number of groups of dystrophic neurites in the hippocampus ( Figure 24). Therefore, hNGF-61 effectively improves Alzheimer's phenotype.
  • mice were anaesthetised with an intraperitoneal injection of 2,2,2-tribromoetanolo (400 mg/kg).
  • hNGF-61 and rhNGF were administered at a dose of 10 ⁇ M, mixed in 40 mM PBS, pH 7.4 at a dose of 480 ng/kg.
  • the control mice received an equal volume of PBS.
  • mice explored for 5 minutes a Plexiglas square labyrinth with black walls and washable surfaces. The session was repeated after 30-minutes. On the next day, the mice explored 2 identical objects (Al and A2) for 10 minutes and were removed from the labyrinth. After twenty- four hours, the mice were placed again in the labyrinth in the presence of an object identical to those of the previous day (Al or A2) and of a second object (B), never explored before and different from the previous one in terms of shape and colour.
  • the results of the test demonstrate that ADI l mice treated with PBS have extensively explored the old object, resulting in a negative discrimination index, statistically different from that of non-transgenic mice. Mice treated with rhNGF and hNGF-61 have a discrimination index around zero. There is a significant difference between animals treated with hNGF-61 and those treated with PBS.
  • hNGF-61 and recombinant hNGF hNGF, Alomone labs
  • the administration of hNGF-61 and hNGF determined a similar rescue of cholinergic neurons in the medial septum and diagonal band of Broca (Fig. 26A).
  • the administration of hNGF- 61 was also effective in reducing the percentage of hippocampal area occupied by beta amyloid plaques (Fig. 26B).
  • the number of tau-positive neurons in the lateral entorhinal cortex was revealed using mAB AT8, recognizing phosphorylated form of Ser 202, and mAB AT270 (Pierce Endogen), a monoclonal antibody that recognizes the phosphorylated form of Thrl81, known to be one of the phosphorylated aminoacids in paired helical filaments-tau proteins.
  • Example 8 Efficacy of the 4GA antibody in discriminating between hNGF-61 and endogenous NGF.
  • hNGF-61 was added at a range of concentrations from 1 to 1000 pg/ml.
  • human serum was added in quadruplicates. A duplicate contained only the human serum, the second duplicate contained the serum with known quantities of hNGF-61 equal to 5, 10, 25, 50 pg/ml.
  • the serums were incubated (2 hours at ambient temperature) with the supernatant of the hybridoma secreting the detector antibody 4GA diluted 1 :10 in phosphate buffer and 2% milk.
  • the secondary antibody anti-rat peroxidase conjugated IgG, DAKO
  • the binding activity was measured by reading the optic density at 450 nm (OD450) by incubation with substrate TMB (Sigma).
  • the colorimetric reaction is blocked by adding a volume of 0.5M H 2 SO 4 and spectrophotometric reading is realised using an ELISA Reader.
  • the experiment was carried out in parallel, using as a detecting antibody, the monoclonal alfaDl l antibody.
  • the results demonstrate that 4GA is able to recognise only hNGF-61, and not endogenous NGF (Figure 27A).
  • the alfaDl l antibody is unable to distinguish hNGF-61 from endogenous NGF.
  • the values obtained in the serum correspond to the sum of the values for endogenous NGF plus the quantity of NGF-61 that was exogenously added to the sera (Figure 27B).
  • Example 9 Absence of immunogenic effects of hNGF-61 in mice. The absence of immunogenic effects has been evaluated after intranasal administration over 2 months. At the end of the treatment, blood was collected from the mice, centrifuged to obtain the serum and the level of anti-NGF IgG was measured using ELISA. The solid phase was obtained by incubating the columns overnight at 4°C with a solution containing 5 ⁇ g/ml of murine NGF mixed in 0.1 M of pH 9.5 sodium carbonate buffer. After 3 washes in phosphate buffers (0.1 M, pH 7.4) and 3 washes in buffer and 0.1 % Tween 20, the column was incubated in a solution constituted by phosphate buffer and 2% milk for 2 hours at 37°C.
  • the mouse blood was added in various dilutions.
  • the monoclonal alfaDl l antibody diluted 1 :1000, 1 :5000, 1 :10,000.
  • the columns were incubated (2 hours at ambient temperature) with an anti-rat IgG antibody (in the case of shafts with mAb alfaDl l) or with an anti-mouse IgG antibody. Both antibodies were connected with horseradish peroxides (HRP). After incubation with the secondary antibody, the reaction was revealed by incubation with substrate TMB (Sigma).
  • Example 10 Absence of allergogenic effects. The absence of allergogenic effects has been verified by analysing the blood of mice after administration of hNGF-61 for 2 months. The blood was left to coagulate at an ambient temperature and then centrifuged at 1200 g for 10 min. The quantity of total IgE was measured using ELISA, as described by Braun et al., (Eur J Immunol 28, pp. 3240-3251 (1998)). No increase in IgE production was observed when compared to the mice treated with PBS (Fig. 29).
  • a fourth group of rats received PBS through the osmotic pump and is the control group.
  • the behavioural test performed to determine the effects on pain was the hot plate test.
  • the rats underwent this test for 5 consecutive days prior to the osmotic pumps being implanted.
  • the test was carried out with a hot plate temperature of 54°C and the latency time taken for the rats to start licking their rear paws was measured.
  • Example 12 Efficacy of hNGF-61 in a murine model of Rett's syndrome The efficacy of hNGF-61 was evaluated after intranasal administration in 6-week old protein Mecp-2 knock-out mice (Chen et al, 2001). hNGF-61 was administered every day for 1 month at a dose of lmg/kg in association with the antibody ⁇ Dl l (2 mg/kg, i.v) or MNAC 13 (0.4 mg/kg, s.c).
  • NGF-61 was able to ameliorate the general behavioural alterations of the knockout mice, while the histological analysis of the brain showed no alterations of the basal levels cytoskeletal and synaptic proteins (such as MAP2, NF200, synaptophysin and PS95) in Mecp2 mice treated with placebo or NGF-61 with respect to wild type mice.
  • cytoskeletal and synaptic proteins such as MAP2, NF200, synaptophysin and PS95
  • Mecp-2 mice treated with placebo the expression of NRl and GIuRl was unchanged with respect tto wild type mice and mice treated with NGF-61.
  • the number of neurons expressing glutamate receptors subunits NR2A was decreased in Mecp2 mice with respect to wild type mice, whereas the expression of the NR2B receptor was increased.
  • the treatment with NGF-61 determined a recovery by the expression of this receptors, with lelvels of expression comparable to those of wild type mice. Acetyl cholinesterase levels were also mildly decreased in Mecp2 mice and the treatment with NGF-61 recovered the levels of this enzyme.
  • Example 13 Intermitting dosing The frequency of administration is a critical parameter to obtain the desired therapeutic effect.
  • the analysis of the brains was performed as described above.
  • the neurostereological counts of the number of cholinergic neurons in the basal forebrain nuclei confirmed that a statistically significant increase in the number of ChAT -positive neurons in MS/DBH and NBM was obtained only in ADl 1 mice treated three times per week (Fig. 32A and 32B), even if dosing twice a week gives the same trend even if not statistically significant because of higher variability.
  • the calculation of the percentage of hippocampal area occupied by ⁇ -amyloid clusters was significantly decreased in all groups of ADI l treated with NGF-61 (Fig 32C).
  • Example 14 The rescue of the neurodegenerative phenotype by hNGF-61 is maintained after 1 month of wash-out in a mouse model for Alzheimer's disease
  • a disease-modifying intervention is typically considered to be the one that can reduce disease progression rate, due to an effect of the drug on the pathophysiological mechanism of the disease and resulting in long-lasting changes in disability (Vellas et al, 2007). This contrasts with interventions that arc only able to relieve impairments as measured by the signs and symptoms of the disease (Vellas ⁇ t al, 2007).
  • a real disease modifying agent should not only improve cognitive deficit but also stop or slow down the progression of neurodegeneration, in a way that this improvement is maintained after the discontinuation of the administration of the therapeutic agent. Wash out studies in experimental models are essential to assess the disease modifying property of a molecule.
  • mice 15 months old ADI l mice were treated with hNGF or hNGF-61 for 1 month and analyzed for memory in the object recognition test (ORT). The results show that there is a rescue of the memory deficit both when huNGF LLG and hNGF-61 were administered (Fig.33Aa). At the end of the ORT, mice were maintained for 1 month without treatment (wash-out) and tested again for memory deficits. Fig33Ab showed that the amelioration of the memory deficit was persistent.
  • mice were sacrificed and the brain tissue processed for immunohistochemical analysis to quantify the number of cholinergic neurons in the basal forebrain, the percentage of hippocampal area occupied by ⁇ -amyloid plaques and the number of phosphor-tau positive neurons in the entorhinal cortex.
  • the results showed that there was no difference among the three groups of ADI l mice in the number of cholinergic neurons in the basal forebrain (Fig. 33B) while a marked decrease in the percentage of hippocampal area occupied by ⁇ -amyloid plaques was observed in mice treated with HNGF-61 but not with hNGF (Fig. 33C).
  • the immunohostochemistry against phospho-tau revealed that the number of neurons expressing phosphorylated tau was decreased in both ADl 1 mice treated with hNGF and with NGF-61 (Fig. 33D).
  • mice were not anaesthetized and were placed on their belly with the head elevated with respect to the rest of the body. Drops were placed on the border of inferior eyelid. As controls, WT mice and a group of ADl 1 mice received eye drops of placebo.
  • mice After 15 days of administration, with a frequency of 3 administrations per week, the mice were analysed in the object recognition test. The results showed that both intranasal and ocular treatment were able to rescue the behavioural deficit (Fig. 34). At the end of the behavioural test, mice were perfused with 4% paraformaldehyde and the brains processed for histological analysis as described before.
  • Example 16 Absence of loss of body weight after intranasal administration of hNGF- 61 It is known that one of the side effects observed when NGF was administered to Alzheimer's patients during a clinical trial is the loss of body weight (Eriksdotter et al, 1998). Such side effect lead to the end of the trial.
  • Example 17 Absence of induction of sensory hyper-innervation by hNGF-61 in mouse spinal cord
  • the spinal cord sections were viewed using a Nikon microscope.
  • the cumulative area occupied by calcitonin-gene relate peptide (CGRP)-positive fibers in regions of the gray matter of the spinal cord was obtained using the morphometry LUCIA program (Laboratory Imaging Ltd., Prague, Czechoslovakia).
  • the numbers of stained pixels in each section were summed and converted into square micrometers via pre-programmed calibration standards. Each pixel represented an area of 0.24 ⁇ m 2 . This degree of resolution allowed immunoreactivity to be detected in single fibers.
  • Example 18 Effects of treatment with hNGF-61 ArglOOGlu on the neurodegenerative phenotype in ADIl mice
  • ADI l mice were treated for 15 days with intranasal hNGF-61 or hNGF-61 ArglOOGlu at the dose of 480 ng/kg ( 0.45 pmol) and 540 ng/kg (0.51 pmol), respectively.
  • mice were analyzed for the cognitive deficit using the object recognition test, as described above.
  • mice were perfused with 4% paraformaldehyde and the brains processed for histological analysis as described before.
  • the histological analysis showed that the intranasal delivery of hNGF-61 ArglOOGlu was as effective as hNGF-61 in determining the rescue of the cholinergic neurons in the MS + DBH (Fig. 39A) and in NBM (Fig. 39B).
  • hNGF-61 ArglOOGlu showed the same efficacy as hNGF-61 in diminishing the number of ⁇ -amyloid clusters of dystrophic neurites (Fig. 39C) and in decreasing the number of phosphotau-positive dystrophic neurites (Fig. 39D).
  • the mutation in position ArglOOGlu is comparable to hNGF61 and is effective in rescuing the neurodegeneration in ADl 1 mice.
  • the compound was administered to CD-I mice at the dose of 2.5 ⁇ g/g i.v.
  • As positive control the same dose of hNGF-61 was used.
  • Cytochrome c was used as negative control since it is usually employed as a control treatment due to its similar physicochemical characteristics to NGF but without neurotrophic activity.
  • each mouse was placed in the center of a glass-covered cylinder (diameter 19 cm) hot plate apparatus (Ugo Basile Inc., Comerio, Italy ) maintained at 52 ⁇ 0.1 0 C. Mice were tested on the hot plate 15, 30, 60, 180, and 360 min after the injection. hNGF-61 -treated animals showed increased nociceptive response when compared to cytochrome c-treated mice (Fig. 40). In particular, the number of jumps in hNGF-61 - treated group was significantly higher than in the control group (Fig. 40A) and the latency to the first jump was shorter in hNGF-61 -treated mice (Fig. 40B).
  • the reduced nociceptive activity of mutein hNGF-61 ArglOOGlu allows to increase the amount of mutein deliverable by a systemic route without induction of pain.
  • a higher systemic dosage can be used, allowing to increase the percentage of mutein crossing the blood brain barrier up to a therapeutical effective dose (Thome and Frey, 2001).

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Abstract

Mutéines du facteur hNGF, leurs utilisations thérapeutiques, et compositions pharmaceutiques. En particulier, les mutéines décrites sont reconnues sélectivement au moyen d'un réactif spécifique par rapport au facteur hNGF endogène. Ces mutéines sont issues d'au moins une substitution d'acide aminé dans la boucle III du facteur hNGF.
PCT/EP2007/057215 2006-07-13 2007-07-12 MUTÉINES DE hNGF, UTILISATIONS THÉRAPEUTIQUES, ET COMPOSITIONS PHARMACEUTIQUES WO2008006893A1 (fr)

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WO2021052926A1 (fr) 2019-09-17 2021-03-25 Chiesi Farmaceutici S.P.A. Agent destiné à être utilisé dans le traitement ou la prévention de troubles ophtalmiques
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