CA2876284A1

CA2876284A1 - Optimum dose regime of an anti-nogo-a antibody in the treatment of amyotrophic lateral sclerosis

Info

Publication number: CA2876284A1
Application number: CA2876284A
Authority: CA
Inventors: Jonathan BULLMAN; David KRULL
Original assignee: Glaxo Group Ltd
Current assignee: Glaxo Group Ltd
Priority date: 2012-07-05
Filing date: 2013-07-03
Publication date: 2014-01-09
Also published as: EP2870177A1; IN2014KN02952A; CN104395343A; BR112014032987A2; JP2015521992A; AU2013285493A1; KR20150036398A; RU2014150265A; WO2014006105A1

Abstract

The invention relates to a method of treatment or prophylaxis of a neurological disorder, in particular but not exclusively amyotrophic lateral sclerosis (ALS), comprising administration of an anti-Nogo-A antibody.

Description

OPTIMUM DOSE REGIME OF AN ANTI-NOGO-A ANTIBODY IN THE TREATMENT OF AMYOTROPIC
LATERALSCLEROSIS
FIELD OF THE INVENTION
The invention relates to a method of treatment or prophylaxis of a neurological disorder, in particular but not exclusively amyotrophic lateral sclerosis (ALS), comprising administration of an anti-Nogo-A antibody.
BACKGROUND OF THE INVENTION
Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's Disease or Maladie de Charcot, is the most common adult-onset motor neuron disease. The primary disease hallmark is the progressive degeneration of the upper and lower motor neurons in the corticospinal tracts. Dysfunction of lower motor neurons (in the brainstem and spinal cord) triggers generalized weakness, muscle atrophy and paralysis. Failure of the respiratory muscles is generally the fatal event, occurring within 1-5 years of onset.
ALS is the most common motor neuron disease in adults affecting approximately 30,000 people in the United States and 5,000 in the United Kingdom each year (Leigh & Swash, 1991). The typical age of onset is between 50 and 70 years, although sometimes occurring at a younger age. Most cases (90-95%) are classified as sporadic ALS (sALS) and the remainder are inherited and referred to as familial ALS (fALS). Sporadic and familial forms are clinically and pathologically similar, suggesting a common pathogenesis (Bruijn et al, 2004).

However, the precise cause for most cases is still unknown, and there is no effective remedy to stop the course of the disease.
Nogo, also known as Reticulon-4 or Neurite outgrowth inhibitor, is a protein that in humans is encoded by the RTN4 gene that has been identified as an inhibitor of neurite outgrowth specific to the central nervous system.
Three forms of human Nogo have been identified: Nogo-A having 1192 amino acid residues (GenBank accession no. A3251383); Nogo-B, a splice variant which lacks residues 186 to 1004 in the putative extracellular domain (GenBank accession no. A3251384) and a shorter splice variant, Nogo-C, which also lacks residues 186 to 1004 and also has smaller, alternative amino terminal domain (GenBank accession no. A3251385) (Prinjha R et al (2000) Nature 403, 383-384). Inhibition of the CNS inhibitory proteins such as Nogo may provide a therapeutic means to ameliorate neuronal damage and promote neuronal repair and growth thereby potentially assisting recovery from neuronal injury such as that sustained in stroke. Examples of such Nogo inhibitors may include small molecules, peptides and antibodies.
It has been reported that a murine monoclonal antibody, IN-1 , that was raised against NI-220/250, a myelin protein which is a potent inhibitor of neurite growth (and subsequently shown to be fragment of Nogo-A), promotes axonal regeneration (Caroni, P and Schwab, ME (1988) Neuron 1 85-96; Schnell, L and Schwab, ME (1990) Nature 343 269-272; Bregman, BS et al (1995) Nature 378 498-501 and Thallmair, M et al (1998) Nature Neuroscience 1 124-131 ). It has also been reported that Nogo-A is the antigen for IN-1 (Chen et al (2000) Nature 403 434-439). Administration of IN-1 Fab fragment or humanised IN-1 enhanced recovery in rats that have undergone spinal cord transection (Fiedler, M et al (2002) Protein Eng 15 931-941; Brosamle, C et al (2000) J.
Neuroscience 20 8061-8068).
Monoclonal antibodies which bind to Nogo are described in WO 2004/052932, WO 2005/028508, WO 2005/061544 and WO 2007/068750. WO 2004/052932 discloses a murine antibody 11C7 which binds to certain forms of human Nogo with high affinity. WO 2005/061544 also discloses high affinity monoclonal antibodies, including a murine monoclonal antibody 2A10, and generally discloses humanised variants thereof, for example H1 L11 (the sequences for the H1 and L11 are provided in SEQ ID NOs. 33 and 34 respectively (VH or VL
sequences only)). The antibodies disclosed bind to human Nogo-A with high affinity. WO 2007/068750 discloses a number of high affinity humanised monoclonal anti-Nogo antibodies, including H28L16 (the sequences for the H28 and L16 are provided in SEQ ID NOs. 49 and 14, respectively (VH or VL
sequences only)). The antibodies disclosed were indicated to be useful in the treatment of neurological diseases.

Despite the art providing high affinity anti-Nogo antibodies for the treatment of neurological disorders, it remains a highly desirable goal to optimize the therapeutic regime for such disorders.
SUMMARY OF THE INVENTION
According to a first aspect of the invention, there is provided a method of treatment or prophylaxis of a neurological disorder in a patient which comprises administering to said patient an anti-Nogo-A antibody, or a functional fragment io thereof, at a dosage to achieve and maintain a level of co-localisation of said antibody with human Nogo-A on the cell membrane in said patient of greater than 90%.
According to a second aspect of the invention, there is provided an anti-Nogo-A
antibody for use in the treatment or prophylaxis of a neurological disorder in a patient which comprises administering said anti-Nogo-A antibody, or a functional fragment thereof, at a dosage to achieve and maintain a level of co-localisation of said antibody with human Nogo-A on the cell membrane in said patient of greater than 90%.
According to a third aspect of the invention, there is provided a pharmaceutical composition comprising an anti-Nogo-A antibody, or a functional fragment thereof, for use in the treatment or prophylaxis of a neurological disorder in a patient which comprises administering said anti-Nogo-A antibody, or a functional fragment thereof, at a dosage to achieve and maintain a level of co-localisation of said antibody with human Nogo-A on the cell membrane in said patient of greater than 90%.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1: Laser scanning cytometry (LSC) well scan to identify the tissue section (grey image) and four regions (cyan boxes) were placed in each section. High-resolution field scans produced images of the location of the three dyes. Individual proteins are colored blue (gamma sarcoglycan), red (Nogo A) and green (H28L16).

Figure 2: Gating of Event Populations (pre dose biopsy). Scatter plots were created to separate the pixels of dye for features based on fluorescence intensity.
Figure 3: Gating of Event Populations (pre dose biopsy). This figure is a field image showing the process of "back gating" which is used to show the gated events on the tissue section.
Figure 4: Gating of Event Populations (post dose biopsy). Scatter plots were created as in Figure 2 however following dosing with 15 mg/kg H28L16.
Figure 5: Gating of Event Populations (post dose biopsy). Back gating was performed as with Figure 3 however following dosing with 15 mg/kg H28L16.
Figure 6: NogoA/H28L16 Expression (gamma sarcoglycan channel not displayed). The individual fluorescent channels are shown. The top row shows target only (Nogo A), second row shows H28L16 only and third row shows Nogo A and H28L16.
Figure 7: Percent of Nogo A on the membrane co-localised with H28L16. Shows the percentage of Nogo A on the muscle fibre membrane that is co-localised with H28L16. The box highlighted "15mg/kg" shows the greatest amount of co-localisation was achieved post-dosing with 15 mg/kg H28L16.
Each bar represents one section.
Figure 8: Median Percent Membrane Nogo-A Co-localised with H28L16.
Data from 2.5 mg/kg repeat-dose (left panel), 15 mg/kg repeat-dose (central 3 panels) and 15 mg/kg single dose (right panel). Median of triplicate sections from biopsies from repeat-dose cohorts, with a single section from biopsies from single dose cohorts.

Figure 9: Model Estimated Relationship (Emax) between Plasma H28L16 Concentrations and percentage of Membrane Nogo-A Co-Localised with H28L16.
5 Figure 10: Predicted Median (5th and 95th Percentile) H28L16 Plasma Concentration Time Profiles for 15mg/kg H28L16 administered every 4 weeks.
Figure 11: Predicted Median (5th and 95th Percentile) H28L16 Plasma Concentration Time Profiles for 15mg/kg H28L16 administered every 2 weeks.
Figure 12: Variable heavy and variable light chain regions of the H28L16 antibody.
DETAILED DESCRIPTION OF THE INVENTION
According to a first aspect of the invention, there is provided a method of treatment or prophylaxis of a neurological disorder in a patient which comprises administering to said patient an anti-Nogo-A antibody, or a functional fragment thereof, at a dosage to achieve and maintain a level of co-localisation of said antibody with human Nogo-A on the cell membrane in said patient of greater than 90%.
It will be appreciated that references herein to "co-localisation" refer to the proportion of membrane bound Nogo-A being localized, associated with or bound to the anti-Nogo-A antibody. For example, a value of 100% co-localisation equates to all membrane Nogo-A being co-localised with anti-Nogo-A antibody.
The skilled person will understand that co-localisation does not equate to target occupancy, however, it does provide an extremely useful measure of likely pharmacodynamic effect in order to ascertain an optimal dosage regime.
Data is presented herein which demonstrates that co-localisation of Nogo-A on the cell membrane with anti-Nogo-A antibody at a level of greater than 90%
achieved the maximum pharmacodynamic effect.

References herein to "anti-Nogo-A antibody" refer to any antibody or variant form thereof, including but not limited to, antibody fragment, domain antibody or single chain antibody capable of binding to Nogo-A. An anti-Nogo-A antibody may be a neutralising anti-Nogo-A antibody. An anti-Nogo-A may be murine, chimeric, humanized, or fully human antibody or fragment thereof.
"Neutralising" and grammatical variations thereof refers to inhibition, either total or partial, of any Nogo function, and more particularly, any Nogo-A function.
References to "Treatment" as used herein refer to the reduction or elimination of disease symptoms associated with and/or causes of amyotrophic lateral sclerosis, including the reduction in or elimination of the progressive degeneration of the neurons in the corticospinal tracts, the denervation of muscle fibres, and/or muscle weakness and/or spasticity.
References to "Prophylaxis" as used herein refer to the retardation, prevention or minimization of disease symptoms associated with amyotrophic lateral sclerosis, including the retardation, prevention or minimization of the progressive degeneration of the neurons in the corticospinal tracts, the denervation of muscle fibres, and/or muscle weakness and/or spasticity.
It will be appreciated that the amount and frequency of anti-Nogo-A antibody to be administered will be chosen to ensure that co-localisation with Nogo-A on the cell membrane of greater than 90% is achieved and maintained.
WO 2010/004031 discloses that a suitable dosage amount of an anti-Nogo-A
antibody for treating ALS or other neurological diseases in a human will be in the range of 0.1 mg/kg to 300 mg/kg, usually from about 2 mg/kg to about 40 mg/kg. Thus, in one embodiment, the dosage of anti-Nogo-A antibody is between 0.1 mg/kg and 300 mg/kg. In a further embodiment, the dosage of anti-Nogo-A antibody is between 2 mg/kg and 40 mg/kg.
Furthermore, WO 2007/068750 discloses that a suitable dosage amount of an anti-Nogo-A antibody for treating stroke and other neurological diseases in a human will be in the range of 700 mg to 3500 mg per 70 kg body weight. Thus, in one embodiment, the dosage of anti-Nogo-A antibody is between 10 mg/kg and 50 mg/kg.
In one particular embodiment of the invention, the dosage of anti-Nogo-A
antibody is 15 mg/kg. Data is presented herein which surprisingly shows beneficial results for a dosage amount of 15 mg/kg. In particular, this dosage amount is particularly amenable to convenient frequency of dosage to achieve the greater than the threshold of the invention of 90% co-localisation with Nogo-A on the cell membrane.
Data is presented herein for the particularly advantageous dosage amount of 15 mg/kg of the invention, which shows that co-localisation with Nogo-A on the cell membrane increased to greater than 90% for a duration of 8 days following administration and dropped to approximately 70% between 22 and 27 days following administration. Therefore, in one embodiment, when the dosage is 15 mg/kg, a suitable dosage interval to maintain greater than 90% co-localisation is between 8 and 22 days. In a further embodiment, when the dosage is 15 mg/kg, a suitable dosage interval to maintain greater than 90% co-localisation is between 10 and 18 days.
In one particular embodiment of the invention when the dosage is 15 mg/kg, a suitable dosage interval to maintain greater than 90% co-localisation is 14 days.
It will be appreciated that slight variation of the dosage interval of 14 days will be acceptable and dosing could occur at 14 days +/- up to 3 days either before or after the optimum 14 day dosage interval. Thus, in one embodiment, when the dosage is 15 mg/kg, a suitable dosage interval to maintain greater than 90%
co-localisation is between 11 and 17 days (i.e. 3 days either before or after the optimum 14 day dosage interval). In an alternative embodiment, when the dosage is 15 mg/kg, a suitable dosage interval to maintain greater than 90% co-localisation is between 12 and 16 days (i.e. 2 days either before or after the optimum 14 day dosage interval). In an alternative embodiment, when the dosage is 15 mg/kg, a suitable dosage interval to maintain greater than 90% co-localisation is between 13 and 15 days (i.e. 1 day either before or after the optimum 14 day dosage interval).

According to a further aspect of the invention, there is provided a method of treatment or prophylaxis of a neurological disorder in a patient which comprises administering to said patient an anti-Nogo-A antibody at a dosage of 15 mg/kg every 14 days (+/- 3 days).
Examples of anti-Nogo-A antibodies which may be used in accordance with the invention are described in WO 2004/052932, WO 2005/028508, WO
2005/061544 and WO 2007/068750, the anti-Nogo-A antibodies of which are herein incorporated by reference.
In one embodiment, the anti-Nogo-A antibody is a monoclonal antibody.
In one embodiment, the anti-Nogo-A antibody is a humanised antibody.
References herein to the term "humanized antibody" refer to a type of engineered antibody having its CDRs derived from a non-human donor immunoglobulin, the remaining immunoglobulin-derived parts of the molecule being derived from one (or more) human immunoglobulin(s). In addition, framework support residues may be altered to preserve binding affinity (see, e.g., Queen etal., Proc. Natl Acad Sci USA, 86:10029-10032 (1989), Hodgson et al., Bio/Technology, 9:421 (1991 )). A suitable human acceptor antibody may be one selected from a conventional database, e.g., the KABATC) database, Los Alamos database, and Swiss Protein database, by homology to the nucleotide and amino acid sequences of the donor antibody (in this case the murine donor antibody 2A10). A human antibody characterized by a homology to the framework regions of the donor antibody (on an amino acid basis) may be suitable to provide a heavy chain constant region and/or a heavy chain variable framework region for insertion of the donor CDRs. A suitable acceptor antibody capable of donating light chain constant or variable framework regions may be selected in a similar manner. It should be noted that the acceptor antibody heavy and light chains are not required to originate from the same acceptor antibody. EP-A-0239400 and EP-A-054951, inter alia, describe several ways of producing such humanised antibodies.

The term "donor antibody" refers to a non-human antibody which contributes the amino acid sequences of its variable regions, CDRs, or other functional fragments or analogs thereof to the humanised antibody, and thereby provide the humanised antibody with the antigenic specificity and neutralizing activity characteristic of the donor antibody.
The term "acceptor antibody" refers to an antibody heterologous to the donor antibody, which provides the amino acid sequences of its heavy and/or light chain framework regions and/or its heavy and/or light chain constant regions to the humanised antibody. The acceptor antibody may be derived from any mammal provided that it is non-immunogenic in humans. In one embodiment, the acceptor antibody is a human antibody.
Alternatively, humanisation may be achieved by a process of "veneering". A
statistical analysis of unique human and murine immunoglobulin heavy and light chain variable regions revealed that the precise patterns of exposed residues are different in human and murine antibodies, and most individual surface positions have a strong preference for a small number of different residues (see PadIan E.A. et al; (1991 ) Mol.Immunol.28, 489-498 and Pedersen J.T. et al (1994) J.
MoI. Biol. 235; 959-973). Therefore it is possible to reduce the immunogenicity of a non-human Fv by replacing exposed residues in its framework regions that differ from those usually found in human antibodies. Because protein antigenicity can be correlated with surface accessibility, replacement of the surface residues may be sufficient to render the mouse variable region "invisible" to the human immune system (see also Mark G. E. et al (1994) in Handbook of Experimental Pharmacology vol.113: The pharmacology of monoclonal Antibodies, Springer-Verlag, pp105-134). This procedure of humanisation is referred to as "veneering" because only the surface of the antibody is altered, the supporting residues remain undisturbed. A further alternative approach is set out in WO
2004/006955.
"CDRs" are defined as the complementarity determining region amino acid sequences of an antibody which are the hypervariable regions of immunoglobulin heavy and light chains. See, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., U.S. Department of Health and Human Services, National Institutes of Health (1987). There are three heavy chain and three light chain CDRs (or CDR regions) in the variable portion of an 5 immunoglobulin. Thus, "CDRs" as used herein refers to all three heavy chain CDRs, or all three light chain CDRs (or both all heavy and all light chain CDRs, if appropriate). The structure and protein folding of the antibody may mean that other residues are considered part of the antigen binding region and would be understood to be so by a skilled person. See for example Chothia et al., (1989) 10 Conformations of immunoglobulin hypervariable regions; Nature 342, p877-883.
In one embodiment, the anti-Nogo-A antibody is a neutralizing anti-Nogo-A
antibody or a fragment thereof such as murine antibodies 2A10, 15C3 and 2C4 (as described in WO 2005/016544, the content of which are incorporated herein by reference it its entirety).
In one embodiment, the anti-Nogo-A antibody is any of the antibodies described in WO 2004/052932 (the content of which are incorporated herein by reference it its entirety). Examples of antibodies disclosed in WO 2004/052932 are 11C7, including humanised variants thereof.
In one embodiment, the anti-Nogo-A antibody is any of the human anti-Nogo-A
antibodies as described in WO 2005/028508 and WO 2009/056509 (the content of which are incorporated herein by reference it its entirety). In a further embodiment, the anti-Nogo-A antibody is human anti-Nogo-A antibody 6A3 as described in WO 2009/056509.
In one embodiment, the anti-Nogo-A antibody comprises an anti-Nogo-A
antibody described in WO 2007/068750. In a further embodiment, the anti-Nogo-A antibody comprises a humanised antibody such as a humanised variant of 2A10, for example, H20L16, H28L16, H28L13 and H27L16 (as described in WO 2007/068750, the content of which are incorporated herein by reference it its entirety), a human antibody, or a fragment thereof.

In a further embodiment, the anti-Nogo-A antibody comprises H27L16 (SEQ ID
NO: 48 and SEQ ID NO: 14 of WO 2007/068750), H28L13 (SEQ ID NO: 49 and SEQ ID NO: 13 of WO 2007/068750) or H28L16 (SEQ ID NO: 49 and SEQ ID
NO: 14 of WO 2007/068750). In a yet further embodiment, the anti-Nogo-A
antibody comprises H28L16 (SEQ ID NO: 49 and SEQ ID NO: 14 of WO
2007/068750, respectively, SEQ ID NO:1 and SEQ ID NO:2 herein).
In a further embodiment, the anti-Nogo-A antibody comprises H27FL L16FL
(SEQ ID NO: 54 and SEQ ID NO: 18 of WO 2007/068750), H28FL L13FL (SEQ ID
NO: 55 and SEQ ID NO: 17 of WO 2007/068750) or H28FL L16FL (SEQ ID NO:
55 and SEQ ID NO: 18 of WO 2007/068750). In a yet further embodiment, the anti-Nogo-A antibody comprises H28FL L16FL (SEQ ID NO: 55 and SEQ ID NO:
18 of WO 2007/068750).
In a further embodiment, the anti-Nogo-A antibody comprises H28L16 (SEQ ID
NO: 49 and SEQ ID NO: 14 of WO 2007/068750) or H28FL L16FL (SEQ ID NO:
55 and SEQ ID NO: 18 of WO 2007/068750). In a yet further embodiment, the anti-Nogo-A antibody comprises H28L16 (SEQ ID NO: 49 and SEQ ID NO: 14 of WO 2007/068750).
In a further embodiment, the anti-Nogo-A antibody or fragment thereof comprises one or more, optionally six, of the CDRs of 2A10, H28L16 or 6A3. In a further embodiment, the anti-Nogo-A antibody or fragment thereof is an antibody that binds to the same human Nogo-A epitope as H28L16 (human Nogo-A 610-621aa, which includes SEQ ID NO:6 from WO 2010/004031) or competes with the binding of H28L16 to human Nogo-A.
Examples of neurological disorders which may be treated in accordance with the dosage regime of the invention are selected from: stroke (ischemic or haemorrhagic), traumatic brain injury, spinal cord injury, Alzheimer's disease, fronto-temporal dementias (tauopathies), peripheral neuropathy, Parkinson's disease, Creutzfeldt-Jakob disease (C3D), Schizophrenia, amyotrophic lateral sclerosis (ALS), multiple sclerosis, Huntington's disease, multiple sclerosis, inclusion body myositis, polymyositis, dermatomyositis, morphologically nonspecific myopathies, congestive heart failure and neuropathic pain.
In one embodiment, the neurological disorder is amyotrophic lateral sclerosis (ALS).
The mode of administration of the anti-Nogo-A antibody or fragment thereof of the invention may be any suitable route which delivers the agent to the host.
The antibodies, and pharmaceutical compositions of the invention are particularly useful for parenteral administration, i.e., subcutaneously (s.c), intrathecally, intraperitoneally (i.p.), intramuscularly (i.m.), intravenously (i.v.), or intranasally (i.n.). In one embodiment, the anti-Nogo-A antibody or fragment thereof is administered intravenously. In an alternative embodiment, the anti-Nogo-A antibody or fragment thereof is administered subcutaneously.
According to a second aspect of the invention, there is provided an anti-Nogo-A
antibody for use in the treatment or prophylaxis of a neurological disorder in a patient which comprises administering said anti-Nogo-A antibody, or a functional fragment thereof, at a dosage to achieve and maintain a level of co-localisation of said antibody with human Nogo-A on the cell membrane in said patient of greater than 90%.
According to a third aspect of the invention, there is provided a pharmaceutical composition comprising an anti-Nogo-A antibody, or a functional fragment thereof, for use in the treatment or prophylaxis of a neurological disorder in a patient which comprises administering said anti-Nogo-A antibody, or a functional fragment thereof, at a dosage to achieve and maintain a level of co-localisation of said antibody with human Nogo-A on the cell membrane in said patient of greater than 90%.
The pharmaceutical composition of the invention typically contains an effective amount of the antibody of the invention as an active ingredient in a pharmaceutically acceptable carrier. In the prophylactic agent of the invention, an aqueous suspension or solution containing the engineered antibody, typically buffered at physiological pH, in a form ready for injection is preferred. The compositions for parenteral administration will commonly comprise a solution of the antibody of the invention or a cocktail thereof dissolved in a pharmaceutically acceptable carrier, typically an aqueous carrier. A variety of aqueous carriers may be employed, e.g., 0.9% saline, 0.3% glycine, and the like. These solutions are sterile and generally free of particulate matter.
These solutions may be sterilized by conventional, well known sterilization techniques (e.g., filtration). The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, etc. The concentration of the antibody of the invention in such pharmaceutical formulation can vary widely, i.e., from less than about 0.5%, usually at or at least about 1 % to as much as 15 or 20% by weight and will be selected primarily based on fluid volumes, viscosities, etc., according to the particular mode of administration selected.
Thus, a pharmaceutical composition of the invention for intramuscular injection could be prepared to contain 1 mL sterile buffered water, and between about 1 ng to about 100 mg, e.g. about 50 ng to about 30 mg or more, such as, about 5 mg to about 25 mg, of an antibody of the invention. Similarly, a pharmaceutical composition of the invention for intravenous infusion could be made up to contain about 250 ml of 0.9% normal saline (sodium chloride) solution, and about 1 to about 30 and typically 5 mg to about 25 mg of an engineered antibody of the invention per ml of 0.9% normal saline (sodium chloride) solution. Actual methods for preparing parenterally administrable compositions are well known or will be apparent to those skilled in the art and are described in more detail in, for example, Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pennsylvania. For the preparation of intravenously administrable antibody formulations of the invention see Lasmar U and Parkins D
"The formulation of Biopharmaceutical products", Pharma. Sci.Tech.today, page 129-137, Vol.3 (3rd April 2000), Wang, W "Instability, stabilisation and formulation of liquid protein pharmaceuticals", Int. J. Pharm 185 (1999) 129-188, Stability of Protein Pharmaceuticals Part A and B ed Ahem T.3., Manning M.
C, New York, NY: Plenum Press (1992), Akers.M.3. "Excipient-Drug interactions in Parenteral Formulations", 3.Pharm Sci 91 (2002) 2283-2300, Imamura, K eta!

"Effects of types of sugar on stabilization of Protein in the dried state", J
Pharm Sci 92 (2003) 266-274, lzutsu, Kkojima, S. "Excipient crystalinity and its protein- structure-stabilizing effect during freeze-drying", J Pharm.
Pharmacol, 54 (2002) 1033-1039, Johnson, R, "Mannitol-sucrose mixtures-versatile formulations for protein lyophilization", J. Pharm. Sci, 91 (2002) 914-922.
Ha, E
Wang W, Wang YJ. "Peroxide formation in polysorbate 80 and protein stability", J. Pharm Sci, 91 , 2252-2264,(2002) the entire contents of which are incorporated herein by reference and to which the reader is specifically referred.
io The antibodies described herein can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional immunoglobulins and art-known lyophilization and reconstitution techniques can be employed.
In one embodiment, the anti-Nogo-A antibody is administered in combination with a further therapeutic agent. In a further embodiment, the further therapeutic agent is an agent having efficacy against amyotrophic lateral sclerosis (ALS). In a yet further embodiment, the agent having efficacy against amyotrophic lateral sclerosis (ALS) is dexpramipexole. Dexpramipexole (KNS-760704; (R)-N6-propy1-4,5,6,7-tetrahydrobenzo[d]thiazole-2,6-diamine) is a drug in development by Knopp Biosciences and Biogen. A 2010 Phase II clinical trial involving 102 patients showed a slowing of ALS disease progression and phase III trials are currently in progress.
In one embodiment, the anti-Nogo-A antibody is administered with a compound having anti-glutamate activity. In a specific embodiment, the compound having anti-glutamate activity is riluzole. In another embodiment, the compound having anti-glutamate activity is an antagonist of an AMPA receptor, such as a 2,3-benzodiazepine compound, in particular, talampanel. In another embodiment, the compound having anti-glutamate activity is TRO 19622 or ceftriaxone. The anti-Nogo-A antibody and the compound having anti-glutamate activity may be administered to the patient simultaneously, sequentially or separately. Where the compound having anti-glutamate activity is riluzole, about 50mg to about 150 or 200mg riluzole may be administered to the patient daily, typically 100mg riluzole is administered to the patient daily. Riluzole is typically orally administered. Where the compound having anti-glutamate activity is Talampanel, Talampanel is administered, typically orally, at about 10mg to about 250mg, from once to five times per day. In one embodiment, Talampanel is 5 administered at a dosage of 25mg or 50mg, from once to five times per day, optionally three times per day.
The invention is illustrated with reference to the following studies:
10 Example 1: Co-Localisation Analysis of Nogo A and H28L16 Laser Scanning Cytometry (LSC) uses lasers to excite fluorescent dyes to produce an image of the dye's location in cells and tissue sections which enables the measurement of expression at the location. The dyes are attached to antibodies that bind to targeted proteins. A commercial anti-Nogo A antibody 15 attached to Alexa 647 dye was used to identify the target of H28L16. The use of the commercial Nogo A antibody allowed the measurement of the target in the tissue sections. It was also desired to know how much of the target (Nogo A) was located on the muscle fibre membrane. This was performed by staining sections with antibodies to the muscle fibre membrane protein anti-gamma sarcoglycan attached to an Alexa 488 dye.
This allows the identification and measurement of the amounts of target (Nogo A) co-localised with the muscle fibre membrane and not colocalised with the membrane. Next, the amount of H28L16 in the dosed muscle sections is required to be quantified and co-localisation with its target (Nogo A). In order to provide this quantification of H28L16, a non-neutralizing anti-idiotype antibody against H28L16, labelled with Alexa 555 dye was used to label muscle section.
Once the three components are labeled with three different dyes, the LSC
software can be used to separate and measure each feature.
A well scan was performed to identify the tissue section (gray image) and four regions (cyan boxes) were placed on the each section. High-resolution field scans produced images of the location of the three dyes.

Figure 1 shows an LSC region image in which colours were assigned to each channel. For example, the individual proteins are colored blue (gamma sarcoglycan), red (Nogo A) and green (H28L16). The rectangular boxes outlined in white are the individual field images that make up the region image. Each region image contained 10 field images.
Scatter plots were created to separate the pixels of dye for features based on fluorescence intensity. The pixels were threshold (background noise was separated from specific signal) using a process known as "gating". The gating is displayed as colored lines on the scatter plots and the gate is labeled R1, R2, and R3...etc, according to the order that the gate was created (Figure 2). The scatter plots of Figure 2 were created by plotting two channels against each other. Membrane and non-membrane areas of the tissue were separated by plotting the "green maxpixel" against the "green intensity". The brightest pixels in R1 are on the membrane that was labelled with the gamma sarcoglycan antibody. The dimmer pixels in R5 are the unlabelled fibres. The long red intensity values were plotted against the membrane values (green maxpixel) to obtain the amount of the Nogo A that was co-localised with membrane R2. To obtain the amount of drug on the membrane, green maxpixel was plotted against the yellow intensity. Since this is pre-dose biopsy, there are no events in R3 that would contain drug on membrane if present. The final plot shows drug that would be co-localised with Nogo A on the membrane.
The groups of pixels within a gated population can be displayed on the image as a colored box. This process is known as "back gating". It is a quality control task to verify the most accurate placement of the gates (Figure 3). The gated populations in the scatter plots in Figure 2 are displayed in the image in Figure 3. It should be noted from Figure 3 that these data are from a patient dosed with vehicle only, so there is minimal background staining with the anti-idiotype antibody against H28L16 and little evidence of co-localisation with Nogo-A
(red boxes).
When compared with Figures 4 and 5 which show data from a patient dosed with 15 mg/kg H28L16, it will be noted that back gating shows green boxes showing the location of H28L16 that is not co-localised with Nogo A and red boxes that show co-localisation of H28L16 with Nogo A. Thus, Figures 4 and 5 show positive drug events in R3 and co-localisation of drug and Nogo A.
Figure 6 shows the individual fluorescent channels. The pre-dose biopsy is red and there is no orange/yellow fluorescence compared to the post-dose biopsy that shows co-localisation of H28L16 with Nogo A and excess H28L16 that is not co-localised with Nogo A. High co-localisation occurred along the fibre membrane.
Figure 7 is a graph that shows the percentage of Nogo A on the muscle fibre membrane that is co-localised with H28L16. Three sections were cut and evaluated for each patient biopsy (pre and post dosing with either drug or placebo). Some low level of co-localisation was seen in biopsies from undosed subjects (either pre-dose or placebo dosed) representing non-specific background staining seen with the anti-idiotype antibody co-localising with Nogo-A. Higher levels of anti-idiotype staining and co-localisation were consistently seen in post-drug doses samples, with co-localisation showing a dose-dependent increase. The section highlighted shows the highest levels of co-localisation of H28L16 with Nogo A on the muscle membrane, which was seen in biopsies collected from subjects 8-days post dose with 15mg/kg H28L16.
Example 2: Use of Co-Localisation Experiments to Investigate Optimum Dosage Regime of H 28116 for Amyotrophic Lateral Sclerosis (ALS) In the absence of a direct measure of pharmacodynamic response to H28L16 in muscle, co-localisation of H28L16 was used with Nogo-A at the target site on the membrane of skeletal muscle cells to inform dose-selection, as described in Example 1. For example, in order to estimate muscle levels of Nogo-A and the biodistribution of H28L16 to skeletal muscle, muscle biopsies were collected from specified cohorts in a "First Time in Human" (FTiH) study. Frozen biopsies of sufficient quality to support immunohistochemical analysis (primarily from 0.5 mg/kg, 2.5 mg/kg and 15 mg/kg cohorts) were stained with antibodies against Nogo-A, anti-idiotype antibodies against H28L16 and anti-gamma sarcoglycan (to define muscle membrane). Expression was visualised using fluorescently labelled secondary antibodies, and quantified using Laser Scanning Cytometry (LSC). Using this approach, the expression of Nogo-A on muscle membrane could be assessed and was distinguished from intra-cellular Nogo-A expression by co-location with gamma sarcoglycan. In addition, an estimate of the level of H28L16 in muscle was measured and an estimate of the degree of co-localisation of Nogo-A with H28L16 on the muscle membrane was derived.
Assessing H28L16 levels in muscle using anti-idiotype staining, a dose-dependent increase in drug staining was observed, with no measurable staining at 0.5 mg/kg and the highest staining seen in biopsies from the 15 mg/kg dosed subjects at post-dose Days 22-27 (consistent with immunoassay measurement of drug levels in muscle). Varying the biopsy collection timepoints in the 15 mg/kg repeat-dose cohort (cohort 8) allowed assessment of the timecourse of drug biodistribution to muscle post dose. These data showed initially lower levels of H28L16 in muscle at Day 1, increasing through Day 8 before falling slightly again by Day 23-24.
The additional measurement of membrane Nogo-A levels in these biopsies allowed an assessment of the degree of co-localisation of Nogo-A with H28L16 at the target site post dose. Figure 8 displays the median percentage of Nogo-A
co-localised with H28L16 at the muscle cell membrane in post-dose biopsies taken from the 2.5 mg/kg and 15 mg/kg cohorts (where 100% would equate to all membrane Nogo-A co-localised with H28L16).
The degree of co-localisation closely followed the measured levels of H28L16 in the muscle, with higher levels seen in biopsies from the 15 mg/kg treated subjects than in biopsies from the 2.5 mg/kg cohort. In the 15 mg/kg repeat dose cohort, co-localisation again correlated with H28L16 levels, with ,,,50%
median co-localisation at Day 1, increasing to >90% of the Nogo-A on the muscle membrane co-localised with study drug at Day 8 post-dose, before reducing back to ¨70% at Day 22-27.
Although co-localisation does not equate to target occupancy, it is believed that >90% co-localisation of muscle membrane Nogo-A with H28L16 is required over the whole dose interval in order to achieve the maximum pharmacodynamic effect. Based on the LSC data, a dose level of 15 mg/kg H28L16 is required to achieve >90% co-localisation of membrane Nogo-A with H28L16 at Day 8 post dose, although this degree of co-localisation was not maintained out to Day 23-24. Exploratory PK-PD modelling to investigate potential relationships between estimated plasma H28L16 concentrations (using a population PK model) at the time of biopsy with the corresponding level of co-localisation is shown in Figure 9. Application of an Emax model to these data estimated an EC50 in the region of ¨18 pg/mL and EC90 of ¨160pg/mL.
Based on population PK modelling and subsequent simulations to investigate dosing regimens to maintain plasma H28L16 concentrations above the EC90, it was evident that the dosing regimen utilised within a FTiH study (15 mg/kg once every 4 weeks) was predicted not to fully maintain, on average, plasma concentrations above 160 pg/mL over the dosing interval. However, increasing the dosing frequency to 15 mg/kg once every 2 weeks was predicted, on average, to maintain concentrations above 160 pg/mL over the whole dosing interval. Predicted plasma H28L16 concentration time profiles for 15 mg/kg administered every 2 and 4 weeks are displayed below in Figure 10 and Figure 11, respectively. Predicted plasma exposure of H28L16 (Cmax and AUC(o_tõ)) with regard to the proposed dosing regimen of 15 mg/kg every 2 weeks is displayed in Table 1.
Table 1 Predicted Median (5th and 95th Percentile) H28L16 plasma Cmax and AUC(0-tau) for 15mg/kg H28L16 administered every 2 weeks.
Predicted Predicted Cmax (pg/mL) AUC(0-tau) (pg.h/mL) Dose 1 376 47698 (216, 588) (35867, 60454) Dose 10 565 98027 (Steady-State) (387, 770) (60482, 145035) The predicted human systemic exposure at steady state following repeated dosing with 15 mg/kg at a frequency of once every 2 weeks is approximately double that after a single 15 mg /kg dose.
5 Experience from a FTiH study in ALS patients showed H28L16 was well tolerated at doses of 15 mg/kg once every 4 weeks. In the long-term 12-month toxicology study in cynomologous monkeys dosed up to 500 mg/kg intravenously every 14 days, no significant toxicities were observed. The monkey-to-human margins of exposure to H28L16 over the predicted human plasma exposure at steady state io in the proposed Phase 2 study, based on a dose of 15 mg/kg administered every 2 weeks are (i) 33-fold based on dose ratio, (ii) 10-fold based on steady state AUC ratio, (iii) 27-fold based on steady state Cmax ratio.
Based on the safety and systemic exposure margins from non-clinical studies;
15 clinical safety, PK-co-location relationship data from a FTiH study; and population PK modeling, the optimal dose for H28L16 appears to be 15 mg/kg once every 2 weeks. This dose, although expected to exceed exposure levels so far achieved in clinical studies, is well supported by non-clinical safety data and maximises the opportunity of delivering efficacy in ALS patients.

Claims

1. A method of treatment or prophylaxis of a neurological disorder in a patient which comprises administering to said patient an anti-Nogo-A antibody, or a functional fragment thereof, at a dosage to achieve and maintain a level of co-localisation of said antibody with human Nogo-A on the cell membrane in said patient of greater than 90%.

2. The method as defined in claim 1, wherein the dosage of anti-Nogo-A
antibody is between 0.1 mg/kg and 300 mg/kg.

3. The method as defined in claim 2, wherein the dosage of anti-Nogo-A
antibody is 15 mg/kg.

4. The method as defined in claim 3, wherein said greater than 90% co-localisation is maintained for between 8 and 22 days.

5. The method as defined in claim 4, wherein said greater than 90% co-localisation is maintained for between 10 and 18 days.

6. The method as defined in claim 5, wherein said greater than 90% co-localisation is maintained for between 11 and 17 days.

7. The method as defined in claim 6, wherein said greater than 90% co-localisation is maintained for between 12 and 16 days.

8. The method as defined in claim 6, wherein said greater than 90% co-localisation is maintained for between 13 and 15 days.

9. The method as defined in claim 8, wherein said greater than 90% co-localisation is maintained for 14 days.

10. A method of treatment of prophylaxis of a neurological disorder in a patient which comprises administering to said patient an anti-Nogo-A antibody, or a functional fragment thereof, at a dosage of 15 mg/kg every 14 days +/- 3 days.

11. The method as defined in any one of claims 1 to 10, wherein the anti-Nogo-A antibody is a monoclonal antibody.

12. The method as defined in any one of claims 1 to 11, wherein the anti-Nogo-A antibody is a humanised antibody.

13. The method as defined in any one of claims 1 to 12, wherein the anti-Nogo-A antibody comprises H28L16.

14. The method as defined in any one of claims 1 to 13, wherein the neurological disorder is selected from stroke (ischemic or haemorrhagic), traumatic brain injury, spinal cord injury, Alzheimer's disease, fronto-temporal dementias (tauopathies), peripheral neuropathy, Parkinson's disease, Creutzfeldt-Jakob disease (CJD), Schizophrenia, amyotrophic lateral sclerosis (ALS), multiple sclerosis, Huntington's disease, multiple sclerosis, inclusion body myositis, polymyositis, dermatomyositis, morphologically nonspecific myopathies, congestive heart failure and neuropathic pain.

15. The method as defined in claim 14, wherein the neurological disorder is amyotrophic lateral sclerosis (ALS).

16. The method as defined in any one of claims 1 to 15, which further comprises administration in combination with a further therapeutic agent.

17. The method as defined in claim 16, wherein the further therapeutic agent is an agent having efficacy against amyotrophic lateral sclerosis (ALS).

18. The method as defined in claim 17, wherein the agent having efficacy against amyotrophic lateral sclerosis (ALS) is dexpramipexole.

19. The method as defined in claim 16, wherein the further therapeutic agent is a compound having anti-glutamate activity.

20. The method as defined in claim 19, wherein the compound having anti-glutamate activity is riluzole.