Fl ELD OF THE I NVENTI ON

The present invention relates to the administration of anti-beta-amyloid (Αβ) antibodies with Serum Amyloid P component (SAP) depleting compounds for the treatment or prophylaxis of diseases associated with amyloid deposition where the amyloid deposits comprise or consist of oligomers or fibrils derived from Αβ.

BACKGROUND TO TH E I NVENTI ON

Alzheimer's disease (AD) is the most common cause of age-related cognitive decline, affecting greater than 12 million individuals worldwide (Citron M (2002) Nat. Neurosci 5, Suppl 1055-1057). The earliest stages of the disease are characterized by a progressive loss of memory with associated cognitive decline and language and behavioural deficits. In the later stages of the disease, patients develop global amnesia and have greatly reduced motor function. Death typically occurs 9 years following diagnosis and is often associated with other conditions, typically pneumonia (Davis K.L. and Samules S.C. (1998) in Pharmacological Management of Neurological and Psychiatric Disorders eds Enna SJ. and Coyle J.T. (McGraw-Hill, New York pp267-316)). Current therapies represent symptomatic approaches, focussing on alleviating the cognitive impairment and ameliorating the behavioural symptoms associated with the progressing disease aetiology. In practice these treatments provide only a short lived cognitive benefit with the level of cognitive impairment reported only to last up to 2 years. The potential for a disease-modifying therapy that slows and possibly halts the progression of the disease is enormous.

Genetic, histological and functional evidence suggests that the β-amyloid peptide (Αβ) is key to the progression of Alzheimer's disease (Selkoe, D. J. (2001) Physiological Reviews 81 : 741-766).

Αβ is known to be produced through the cleavage of the beta amyloid precursor protein

(also known as APP) by an aspartyl protease enzyme known as BACE1 (also known as β-secretase, Asp2 or Memapsin-2) (De Strooper, B. and Konig, G. (1999) Nature 402: 471-472). In addition to the parenchymal and vascular deposition, soluble oligomeric forms of Αβ have been postulated to contribute to the onset of Alzheimer's Disease (AD) and they may affect neuronal function initially by impairing synaptic function (Lambert et. al. (1998) Proceedings of the National Academy of Science, U.S.A. 95 : 6448-6453). Although insoluble amyloid plaques are found early in AD and in Mild Cognitive Impairment (MCI), the levels of soluble Αβ aggregates (referred to as oligomers or Αβ- derived diffusible ligands (ADDLs)) are also increased in these individuals, and soluble Αβ levels correlate better with neurofibrillary degeneration, and the loss of synaptic markers than do amyloid plaques (Naslund et. al. (2000) J Am Med Assoc 283: 1571-1577, Younkin, S. (2001) Nat. Med. 1 : 8- It has become increasingly apparent that the transport of exogenous Αβ between the central nervous system (CNS) and plasma plays a role in the regulation of brain amyloid levels (Shibata, et al (2000) J Clin Invest 106 : 1489-1499), with CSF Αβ being rapidly transported from CSF to plasma.

Amyloid deposits are composed predominantly of amyloid fibrils, but also contain heparin, dermatan proteoglycans, and amyloid P component, which is identical to and derived from the normal circulating plasma protein of the pentraxin family, serum amyloid P component (SAP) (Pepys 1994).

Human SAP is a constitutive protein in the plasma, at a concentration of around 20-40 mg/l (Nelson et al. (1991) Clin. Chim. Acta, 200: 191-200). Animal experiments and in vitro studies suggest a role for circulating SAP in host defence (Noursadeghi et al. (2000) PNAS, 97: 14584- 14589)). SAP is also a normal tissue matrix constituent associated with elastic fibres and the glomerular basement membrane although its function there is not known.

SAP bound to amyloid fibrils is in equilibrium with SAP in the circulation and extracellular fluid. SAP undergoes avid (Kd ~1 μιηοΙ/Ι), calcium-dependent, reversible binding to amyloid fibrils of all types, which makes both the fibrils and the SAP more resistant to proteolysis [Tennent 1995]. In this way SAP may contribute to the persistence of amyloid deposits in vivo.

In addition to binding to amyloid fibrils, human SAP binds to and enters cerebral neurones and causes neuronal apoptosis in vitro and in vivo (Urbanyi, Z., et al., (1994). Eur. J. Pharmacol., 270: 375-387, Duong, T., et al., (1998) Brain Res., 813: 303-312, Urbanyi, Z., et al., (2003) Brain Res., 988: 69-77, Urbanyi, Z., et al., (2007) Brain Res., 1145: 221-226, Pisalyaput, K. Et al.,(2008). J. Neurochem., 104: 696-707). It has been shown that unique, pharmaceutical grade pure human SAP (Pepys, M.B., et al., (2012) J. Immunol. Methods, 384: 92-102) disrupts synaptic transmission, causing abnormal paired pulse ratio and long term potentiation in organotypic rodent brain slices in vitro. The cerebral neurotoxicity of human SAP is therefore likely to contribute to neurodegeneration in humans Urbanyi, Z., et al., (1994). Eur. J. Pharmacol., 270: 375-387, Duong, T., et al., (1998) Brain Res., 813: 303-312, Urbanyi, Z., et al., (2003) Brain Res., 988: 69-77, Urbanyi, Z., et al., (2007) Brain Res., 1145: 221-226, Crawford, J.R., et al., (2012) Neurochem. Res., 37: 795-801).

Depletion of circulating SAP by carboxy pyrrolidine hexanoyl pyrrolidine carboxylate (CPHPC) also leads to depletion of SAP from amyloid deposits owing to the reversible nature of SAP binding to amyloid deposits. There is clinical evidence that this may limit further amyloid accumulation (Gilmore 2010). It is also possible that depletion of SAP from amyloid deposits may expose epitopes on Αβ, which may have previously been masked by the bound SAP, leading to greater targeting of amyloid deposits by anti-Αβ antibodies.

Reducing the levels of Αβ and SAP might reduce the load and formation of amyloid deposits and offer neuroprotection against the effects of SAP on synaptic transmission. It will be appreciated that therapy using two therapeutics with differing mechanisms of action may offer advantages over the use of a single therapeutic or multiple therapeutics all with the same mechanism of action.

Administration of an antibody specific to Αβ with a compound that depletes SAP could potentially lead to treatment of conditions where the amyloid deposits comprise or consist of oligomers or fibrils derived from Αβ: Age-related macular degeneration (AMD), Alzheimer's disease (AD), cerebral amyloid angiopathy (CAA), Fragile X syndrome, Down's syndrome, Autism, Huntington's disease, Parkinson's disease, mild cognitive impairment, hereditary cerebral haemorrhage with β-amyloidosis of the Dutch type, cerebral β-amyloid angiopathy and various types of degenerative dementias, such as those associated with progressive supranuclear palsy and cortical basal degeneration.

In AMD Αβ fibrils have been detected in drusen, protein lipid deposits that sit below the retinal pigment epithelial layer [Isas, 2010].

International Patent Applications WO2007/113172 and provisional patent application US 62/167437 disclose anti-Αβ antibodies, methods for their manufacture and improved formulations. In particular, anti-Αβ antibodies are disclosed with variable heavy chain and variable light chain regions comprising the following CDRs: CDRH1 (SEQ ID NO: l), CDRH2 (SEQ ID NO:2), CDRH3 (SEQ ID NO:3) within the heavy chain variable region and; CDRL1 (SEQ ID NO:4), CDRL2 (SEQ ID NO:5), CDRL3 (SEQ ID NO:6) within the light chain variable region.

Also described in WO2007/113172 is an antibody with a humanised heavy chain variable region variant H2 (SEQ ID No:28 therein, SEQ ID NO:7 herein) and a humanised light chain variable region variant LI (SEQ ID No:32 therein, SEQ ID NO:8 herein).

Also described in WO2007/113172 is an antibody with a mature heavy chain amino acid sequence (Fc double mutation, bold) (SEQ ID No:36 therein; SEQ ID NO:9 herein) and a mature light chain amino acid sequence (SEQ ID No:40 therein; SEQ ID NO: 10 herein).

US provisional patent application US 62/167437 describes such antibodies in an improved formulation comprising a solution containing 50mM sodium acetate, 0.02% polysorbate 80, 1% arginine, 51mM sodium chloride, at pH 5.5.

Other examples of anti-Αβ antibodies include solanezumab, crenezumab, bapineuzumab, gantenerumab and aducanumab.

European patent application EP0915088 discloses D-proline derivative compounds that are competitive inhibitors of binding of SAP to amyloid fibrils, as well as methods for their manufacture. A preferred compound disclosed in EP0915088 is (R)-l-[6-[(R)-2-Carboxy-pyrrolidin-l-yl]-6-oxo oxohexanoyl] pyrrolidine-2-carboxylic acid (CPHPC) according to Formula (I):

International patent application PCT/EP2015/058998 and priority application GB1518950.9 disclose two forms of orally bioavailable CPHPC.

PCT/EP2015/058998 discloses a compound (2R,2'R)-bis(((((tetrahydro-2H-pyran-4- yl)oxy)carbonyl)oxy)methyl) l,l'-adipoylbis(pyrrolidine-2-carboxylate) according to Formula (II):

Priority application GB1518950.9 discloses a compound (2R,2'R)-bis(((tetrahydro-2H-pyran 4-carbonyl)oxy)methyl) l,l'-adi to Formula (III):

CPHPC and its orally bioavailable forms (as disclosed in the patent applications listed above) bind with high affinity to human SAP. This triggers rapid clearance of the complex by the liver, depleting almost all circulating SAP for as long as the drug is administered. SUMMARY OF TH E I NVENTI ON

The present invention provides the use of an anti-Αβ antibody and a SAP depleting compound for the treatment or prophylaxis of diseases associated with amyloid deposition where the amyloid deposits comprise or consist of oligomers or fibrils derived from Αβ.

The present invention also provides methods of preventing and/or treating a subject susceptible to or afflicted with a disease associated with amyloid deposition, which method comprises the step of administering a prophylactically or therapeutically effective amount of an anti- Αβ antibody and a SAP depleting compound to said subject. DETAI LED DESCRI PTI ON OF THE I NVENTI ON

The present invention provides the use of an anti-Αβ antibody and a SAP depleting compound for the treatment or prophylaxis of diseases associated with amyloid deposition where the amyloid deposits comprise or consist of oligomers or fibrils derived from Αβ.

In one aspect the present invention provides the use of an anti-Αβ antibody and a SAP depleting compound for the treatment or prophylaxis of diseases associated with amyloid deposition where the amyloid deposits comprise oligomers or fibrils derived from Αβ, wherein the anti-Αβ antibody has variable heavy and variable light chain regions comprising the following CDRs: CDRH1 (SEQ ID NO: l), CDRH2 (SEQ ID NO:2), CDRH3 (SEQ ID NO:3) within the heavy chain variable region and; CDRLl (SEQ ID NO:4), CDRL2 (SEQ ID NO:5), CDRL3 (SEQ ID NO:6) within the light chain variable region.

In one aspect the present invention provides the use of an anti-Αβ antibody and a SAP depleting compound for the treatment or prophylaxis of diseases associated with amyloid deposition where the amyloid deposits consist of oligomers or fibrils derived from Αβ, wherein the anti-Αβ antibody has variable heavy and variable light chain regions comprising the following CDRs: CDRH1 (SEQ ID NO: l), CDRH2 (SEQ ID NO:2), CDRH3 (SEQ ID NO:3) within the heavy chain variable region and; CDRLl (SEQ ID NO:4), CDRL2 (SEQ ID NO:5), CDRL3 (SEQ ID NO:6) within the light chain variable region.

In another aspect the present invention provides the use of an anti-Αβ antibody and a SAP depleting compound for the treatment or prophylaxis of diseases associated with amyloid deposition where the amyloid deposits comprise oligomers or fibrils derived from Αβ, wherein the anti-Αβ antibody comprises a variable heavy region comprising a polypeptide having an amino acid sequence of SEQ ID NO:7 and a light chain variable region comprising a polypeptide having an amino acid sequence of SEQ ID NO:8.

In another aspect the present invention provides the use of an anti-Αβ antibody and a SAP depleting compound for the treatment or prophylaxis of diseases associated with amyloid deposition where the amyloid deposits consist of oligomers or fibrils derived from Αβ, wherein the anti-Αβ antibody comprises a variable heavy region comprising a polypeptide having an amino acid sequence of SEQ ID NO:7 and a light chain variable region comprising a polypeptide having an amino acid sequence of SEQ ID NO:8.

In another aspect the present invention provides the use of an anti-Αβ antibody and a SAP depleting compound for the treatment or prophylaxis of diseases associated with amyloid deposition where the amyloid deposits comprise oligomers or fibrils derived from Αβ, wherein the anti-Αβ antibody has mature heavy and light chains having amino acid sequences of SEQ ID NO 9: and 10, respectively..

In another aspect the present invention provides the use of an anti-Αβ antibody and a SAP depleting compound for the treatment or prophylaxis of diseases associated with amyloid deposition where the amyloid deposits consist of oligomers or fibrils derived from Αβ, wherein the anti-Αβ antibody has mature heavy and light chains having amino acid sequences of SEQ ID NO 9: and 10, respectively.

In another aspect the present invention provides the use of an anti-Αβ antibody and a SAP depleting compound for the treatment or prophylaxis of diseases associated with amyloid deposition where the amyloid deposits comprise oligomers or fibrils derived from Αβ, wherein the anti-Αβ antibody is selected from solanezumab, crenezumab, bapineuzumab, gantenerumab and aducanumab.

In another aspect the present invention provides the use of an anti-Αβ antibody and a SAP depleting compound for the treatment or prophylaxis of diseases associated with amyloid deposition where the amyloid deposits consist of oligomers or fibrils derived from Αβ, wherein the anti-Αβ antibody is selected from solanezumab, crenezumab, bapineuzumab, gantenerumab and aducanumab.

In another aspect the present invention provides the use of an anti-Αβ antibody and a SAP depleting compound for the treatment or prophylaxis of diseases associated with amyloid deposition where the amyloid deposits comprise oligomers or fibrils derived from Αβ, wherein the anti-Αβ antibody comprises the CDRs or the variable regions from any one of solanezumab, crenezumab, bapineuzumab, gantenerumab and aducanumab. In another aspect the present invention provides the use of an anti-Αβ antibody and a SAP depleting compound for the treatment or prophylaxis of diseases associated with amyloid deposition where the amyloid deposits consist of oligomers or fibrils derived from Αβ, wherein the anti-Αβ antibody comprises the CDRs or the variable regions from any one of solanezumab, crenezumab, bapineuzumab, gantenerumab and aducanumab. In an embodiment, the anti-Αβ antibody comprises all six CDRs from any one of solanezumab, crenezumab, bapineuzumab, gantenerumab and aducanumab. In an embodiment, the CDRs are defined by any one of the standard numbering conventions for CDR sequences, including "Kabat" (Sequences of Proteins of Immunological Interest, 5th Ed., U.S. Department of Health and Human Services, National Institutes of Health (1991)), "Chothia" ((1989) Nature 342: 877-883), "AbM" (University of Bath) and "contact" (University College London) methods. In an embodiment, the CDRs are the Kabat CDRs. In an embodiment, the anti-Αβ antibody comprises the heavy chain variable region and the light chain variable region from any one of solanezumab, crenezumab, bapineuzumab, gantenerumab and aducanumab.

In another aspect the present invention provides the use of an anti-Αβ antibody and a SAP depleting compound for the treatment or prophylaxis of diseases associated with amyloid deposition where the amyloid deposits comprise oligomers or fibrils derived from Αβ, wherein the anti-Αβ antibody is a single domain antibody fused to an Fc region (a "dAb-Fc").

In another aspect the present invention provides the use of an anti-Αβ antibody and a SAP depleting compound for the treatment or prophylaxis of diseases associated with amyloid deposition where the amyloid deposits consist of oligomers or fibrils derived from Αβ, wherein the anti-Αβ antibody is a single domain antibody fused to an Fc region (a "dAb-Fc").

In another aspect the present invention provides the use of an anti-Αβ antibody and a SAP depleting compound for the treatment or prophylaxis of diseases associated with amyloid deposition where the amyloid deposits comprise oligomers or fibrils derived from Αβ, wherein the anti-Αβ antibody comprises a wild-type Fc region.

In another aspect the present invention provides the use of an anti-Αβ antibody and a SAP depleting compound for the treatment or prophylaxis of diseases associated with amyloid deposition where the amyloid deposits consist of oligomers or fibrils derived from Αβ, wherein the anti-Αβ antibody comprises a wild-type Fc region.

In another aspect the present invention provides the use of an anti-Αβ antibody and a SAP depleting compound for the treatment or prophylaxis of diseases associated with amyloid deposition where the amyloid deposits comprise oligomers or fibrils derived from Αβ, wherein the anti-Αβ antibody comprises an enhanced Fc region. In another aspect the present invention provides the use of an anti-Αβ antibody and a SAP depleting compound for the treatment or prophylaxis of diseases associated with amyloid deposition where the amyloid deposits consist of oligomers or fibrils derived from Αβ, wherein the anti-Αβ antibody comprises an enhanced Fc region. In an embodiment, the enhanced Fc region enhances complement-dependant cytotoxic activity. In an embodiment, the enhanced Fc region enhances Fc-mediated phagocytosis.

In another aspect the present invention provides the use of an anti-Αβ antibody and a SAP depleting compound for the treatment or prophylaxis of diseases associated with amyloid deposition where the amyloid deposits comprise oligomers or fibrils derived from Αβ, wherein the SAP-depleting compound is selected from (R)-l-[6-[(R)-2-Carboxy-pyrrolidin-l-yl]-6-oxo oxohexanoyl] pyrrolidine- 2-carboxylic acid (CPHPC, Formula (I)) or a diester thereof, (2R,2'R)-bis(((((tetrahydro-2H-pyran-4- yl)oxy)carbonyl)oxy)methyl) l,l'-adipoylbis(pyrrolidine-2-carboxylate) (Formula(II)) and (2R,2'R)- bis(((tetrahydro-2H-pyran-4-carbonyl)oxy)methyl) 1, l'-adipoylbis(pyrrolidine-2-carboxylate)

(Formula (III)). In another aspect the present invention provides the use of an anti-Αβ antibody and a SAP depleting compound for the treatment or prophylaxis of diseases associated with amyloid deposition where the amyloid deposits consist of oligomers or fibrils derived from Αβ, wherein the SAP- depleting compound is selected from (R)-l-[6-[(R)-2-Carboxy-pyrrolidin-l-yl]-6-oxo oxohexanoyi] pyrrolidine-2-carboxylic acid (CPHPC, Formula (I)) or a diester thereof, (2R,2'R)-bis(((((tetrahydro- 2H-pyran-4-yl)oxy)carbonyl)oxy)methyl) l,l'-adipoylbis(pyrrolidine-2-carboxylate) (Formula(II)) and (2R,2'R)-bis(((tetrahydro-2H-pyran-4-carbonyl)oxy)methyl) l,l'-adipoylbis(pyrrolidine-2-carboxylate) (Formula (III)).

In another aspect the present invention provides the use of an anti-Αβ antibody and a SAP depleting compound for the treatment or prophylaxis of diseases associated with amyloid deposition where the amyloid deposits comprise oligomers or fibrils derived from Αβ, wherein the SAP-depleting compound is (R)-l-[6-[(R)-2-Carboxy-pyrrolidin-l-yl]-6-oxo oxohexanoyi] pyrrolidine-2-carboxylic acid (CPHPC, Formula (I)) or a diester thereof.

In another aspect the present invention provides the use of an anti-Αβ antibody and a SAP depleting compound for the treatment or prophylaxis of diseases associated with amyloid deposition where the amyloid deposits consist of oligomers or fibrils derived from Αβ, wherein the SAP- depleting compound is (R)-l-[6-[(R)-2-Carboxy-pyrrolidin-l-yl]-6-oxo oxohexanoyi] pyrrolidine-2- carboxylic acid (CPHPC, Formula (I)) or a diester thereof.

In another aspect the present invention provides the use of an anti-Αβ antibody and a SAP depleting compound for the treatment or prophylaxis of diseases associated with amyloid deposition where the amyloid deposits comprise oligomers or fibrils derived from Αβ, wherein the SAP-depleting compound is (2R,2'R)-bis(((((tetrahydro-2H-pyran-4-yl)oxy)carbonyl)oxy)m ethyl) 1,1'- adipoylbis(pyrrolidine-2-carboxylate) (Formula (II)).

In another aspect the present invention provides the use of an anti-Αβ antibody and a SAP depleting compound for the treatment or prophylaxis of diseases associated with amyloid deposition where the amyloid deposits consist of oligomers or fibrils derived from Αβ, wherein the SAP- depleting compound is (2R,2'R)-bis(((((tetrahydro-2H-pyran-4-yl)oxy)carbonyl)oxy)m ethyl) 1,1'- adipoylbis(pyrrolidine-2-carboxylate) (Formula (II)).

In another aspect the present invention provides the use of an anti-Αβ antibody and a SAP depleting compound for the treatment or prophylaxis of diseases associated with amyloid deposition where the amyloid deposits comprise oligomers or fibrils derived from Αβ, wherein the SAP-depleting compound is (2R,2'R)-bis(((tetrahydro-2H-pyran-4-carbonyl)oxy)methyl) l,l'-adipoylbis(pyrrolidine- 2-carboxylate) (Formula (III)).

In another aspect the present invention provides the use of an anti-Αβ antibody and a SAP depleting compound for the treatment or prophylaxis of diseases associated with amyloid deposition where the amyloid deposits consist of oligomers or fibrils derived from Αβ, wherein the SAP- depleting compound is (2R,2'R)-bis(((tetrahydro-2H-pyran-4-carbonyl)oxy)methyl) 1,1'- adipoylbis(pyrrolidine-2-carboxylate) (Formula (III)).

In another aspect the present invention provides the use of an anti-Αβ antibody and a SAP depleting compound for the treatment or prophylaxis of diseases associated with amyloid deposition where the amyloid deposits comprise oligomers or fibrils derived from Αβ, wherein the disease associated with amyloid deposition is selected from Age-related macular degeneration (AMD),

Alzheimer's disease (AD), cerebral amyloid angiopathy (CAA), Fragile X syndrome, Downs syndrome,

Autism, Huntington's, and Parkinson's diseases.

In another aspect the present invention provides the use of an anti-Αβ antibody and a SAP depleting compound for the treatment or prophylaxis of diseases associated with amyloid deposition where the amyloid deposits consist of oligomers or fibrils derived from Αβ, wherein the disease associated with amyloid deposition is selected from Age-related macular degeneration (AMD),

Alzheimer's disease (AD), cerebral amyloid angiopathy (CAA), Fragile X syndrome, Downs syndrome,

Autism, Huntington's, and Parkinson's diseases.

In another aspect the present invention provides the use of an anti-Αβ antibody and a SAP depleting compound for the treatment or prophylaxis of diseases associated with amyloid deposition where the amyloid deposits comprise oligomers or fibrils derived from Αβ, wherein the disease associated with amyloid deposition is Alzheimer's Disease.

In another aspect the present invention provides the use of an anti-Αβ antibody and a SAP depleting compound for the treatment or prophylaxis of diseases associated with amyloid deposition where the amyloid deposits consist of oligomers or fibrils derived from Αβ, wherein the disease associated with amyloid deposition is Alzheimer's Disease.

In another aspect the present invention provides the use of an anti-Αβ antibody and a SAP depleting compound for the treatment or prophylaxis of diseases associated with amyloid deposition where the amyloid deposits comprise oligomers or fibrils derived from Αβ, wherein the disease associated with amyloid deposition is Age-related macular degeneration.

In another aspect the present invention provides the use of an anti-Αβ antibody and a SAP depleting compound for the treatment or prophylaxis of diseases associated with amyloid deposition where the amyloid deposits consist of oligomers or fibrils derived from Αβ, wherein the disease associated with amyloid deposition is Age-related macular degeneration.

In another aspect the present invention provides the use of an anti-Αβ antibody and a SAP depleting compound for the treatment or prophylaxis of diseases associated with amyloid deposition where the amyloid deposits comprise oligomers or fibrils derived from Αβ, wherein the disease associated with amyloid deposition is cerebral amyloid angiopathy.

In another aspect the present invention provides the use of an anti-Αβ antibody and a SAP depleting compound for the treatment or prophylaxis of diseases associated with amyloid deposition where the amyloid deposits consist of oligomers or fibrils derived from Αβ, wherein the disease associated with amyloid deposition is cerebral amyloid angiopathy.

The present invention also provides methods of preventing and/or treating a subject susceptible to or afflicted with a disease associated with amyloid deposition, which method comprises the step of administering a prophylactically or therapeutically effective amount of an anti- Αβ antibody and a SAP depleting compound to said subject.

In another aspect the present invention provides a method of preventing and/or treating a subject susceptible to or afflicted with a disease associated with amyloid deposition, which method comprises the step of administering a prophylactically or therapeutically effective amount of an anti- Αβ antibody and a SAP depleting compound to said subject, wherein the anti-Αβ antibody has variable heavy and variable light chain regions comprising the following CDRs: CDRHl (SEQ ID No: l), CDRH2 (SEQ ID NO:2), CDRH3 (SEQ ID NO:3) within the heavy chain variable region and; CDRL1 (SEQ ID NO:4), CDRL2 (SEQ ID No:5),CDRL3 (SEQ ID No:6) within the light chain variable region.

In another aspect the present invention provides a method of preventing and/or treating a subject susceptible to or afflicted with a disease associated with amyloid deposition, which method comprises the step of administering a prophylactically or therapeutically effective amount of an anti- Αβ antibody and a SAP depleting compound to said subject, wherein the anti-Αβ antibody is selected from selected from solanezumab, crenezumab, bapineuzumab, gantenerumab and aducanumab.

In another aspect the present invention provides a method of preventing and/or treating a subject susceptible to or afflicted with a disease associated with amyloid deposition, which method comprises the step of administering a prophylactically or therapeutically effective amount of an anti- Αβ antibody and a SAP depleting compound to said subject, wherein the SAP-depleting compound is selected from a compound of Formula (I), (R)-l-[6-[(R)-2-Carboxy-pyrrolidin-l-yl]-6-oxo oxohexanoyl] pyrrolidine-2-carboxylic acid (CPHPC) or a diester thereof, a compound of Formula (II), (2R,2'R)-bis(((((tetrahydro-2H-pyran-4-yl)oxy)carbonyl)oxy)m ethyl) l,l'-adipoylbis(pyrrolidine- 2-carboxylate) and a compound of Formula (III), compound (2R,2'R)-bis(((tetrahydro-2H-pyran-4- carbonyl)oxy)methyl) l,l'-adipoylbis(pyrrolidine-2-carboxylate).

In another aspect the present invention provides a method of preventing and/or treating a subject susceptible to or afflicted with a disease associated with amyloid deposition, which method comprises the step of administering a prophylactically or therapeutically effective amount of an anti- Αβ antibody and a SAP depleting compound to said subject, wherein the SAP-depleting compound is a compound of Formula (I), (R)-l-[6-[(R)-2-Carboxy-pyrrolidin-l-yl]-6-oxo oxohexanoyl] pyrrolidine- 2-carboxylic acid (CPHPC) or a diester thereof.

In another aspect the present invention provides a method of preventing and/or treating a subject susceptible to or afflicted with a disease associated with amyloid deposition, which method comprises the step of administering a prophylactically or therapeutically effective amount of an anti- Αβ antibody and a SAP depleting compound to said subject, wherein the SAP-depleting compound is a compound of Formula (II), (2R,2'R)-bis(((((tetrahydro-2H-pyran-4-yl)oxy)carbonyl)oxy)m ethyl) l,l'-adipoylbis(pyrrolidine-2-carboxylate).

In another aspect the present invention provides a method of preventing and/or treating a subject susceptible to or afflicted with a disease associated with amyloid deposition, which method comprises the step of administering a prophylactically or therapeutically effective amount of an anti- Αβ antibody and a SAP depleting compound to said subject, wherein the SAP-depleting compound is a compound of Formula (III), compound (2R,2'R)-bis(((tetrahydro-2H-pyran-4-carbonyl)oxy)methyl) l,l'-adipoylbis(pyrrolidine-2-carboxylate).

The term "antibody" is used herein in the broadest sense to refer to molecules with an immunoglobulin-like domain and includes monoclonal, recombinant, polyclonal, chimeric, humanised, bispecific and heteroconjugate antibodies; a single variable domain, a domain antibody, a single variable domain fused to an Fc region (dAb-Fc), antigen binding fragments, immunologically effective fragments, single chain Fv, diabodies, Tandabs™, etc. (for a summary of alternative "antibody" formats see Holliger and Hudson, Nature Biotechnology, 2005, Vol 23, No. 9, 1126- 1136).

The term "domain" refers to a folded protein structure which retains its tertiary structure independent of the rest of the protein. Generally domains are responsible for discrete functional properties of proteins and in many cases may be added, removed or transferred to other proteins without loss of function of the remainder of the protein and/or of the domain.

The term "single variable domain" refers to a folded polypeptide domain comprising sequences characteristic of antibody variable domains. It therefore includes complete antibody variable domains such as VH, VHH and VL and modified antibody variable domains, for example, in which one or more loops have been replaced by sequences which are not characteristic of antibody variable domains, or antibody variable domains which have been truncated or comprise N- or C- terminal extensions, as well as folded fragments of variable domains which retain at least the binding activity and specificity of the full-length domain. A single variable domain is capable of binding an antigen or epitope independently of a different variable region or domain. A "domain antibody" or "dAb ^(TM) " may be considered the same as a "single variable domain". A single variable domain may be a human single variable domain, but also includes single variable domains from other species such as rodent nurse shark and Camelid VHH dAbs™. Camelid VHH are immunoglobulin single variable domain polypeptides that are derived from species including camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain antibodies naturally devoid of light chains. Such VHH domains may be humanised according to standard techniques available in the art, and such domains are considered to be "single variable domains". As used herein VH includes camelid VHH domains. As herein defined, the term 'dAb-Fc' describes an engineered immunoglobulin molecule comprising a single variable domain attached to one or more constant region domains and/or hinge. In an embodiment, a dAb-Fc comprises a single variable domain fused to an Fc region of an antibody. Such constant region(s) or Fc region permits Fc receptor binding (e.g. to one or both of Fc receptors CD64 and CD32) and complement activation via the interaction with Clq, whilst at the same time providing the molecule with a longer half-life than a single variable heavy chain domain in isolation. The CH3 domain facilitates the interaction of a dAb-Fc with Fc receptors whilst the CH2 domain permits the interaction of a dAb-Fc with Clq, thus facilitating the activation of the complement system. In an embodiment a dAb-Fc comprises the constant region domains CH2 and/or CH3. In an embodiment, the Fc region is the Fc region of an IgG molecule, such as an IgGl, IgG2, IgG3, iGG4 or IgG4PE; or an IgA antibody. For the avoidance of doubt, dAb-Fc molecules according to the invention are single chain molecules.

The binding affinity of the anti-Αβ antibody may be measured by BIAcore™, for example by antigen capture with Αβ coupled onto a carboxymethydextran chip by primary amine coupling and antibody capture onto this surface. Alternatively, the binding affinity can be measured by BIAcore™ by binding of anti-Αβ antibodies to human Αβ immobilised on a CM5 chip.

"CDRs" are defined as the complementarity determining region amino acid sequences of an antibody. These are the hypervariable regions of immunoglobulin heavy and light chains. There are three heavy chain and three light chain CDRs (or CDR regions) in the variable portion of an immunoglobulin. Thus, "CDRs" as used herein refers to all three heavy chain CDRs, all three light chain CDRs, all heavy and light chain CDRs, or at least two CDRs.

The interaction between the Fc region of an antibody and various Fc receptors is believed to mediate phagocytosis and half-life/clearance of an antibody or antibody fragment.

The term "Effector Function" as used herein is meant to refer to one or more of Antibody dependant cell mediated cytotoxic activity (ADCC), Complement-dependant cytotoxic activity (CDC) mediated responses, Fc-mediated phagocytosis or antibody dependant cellular phagocytosis (ADCP) and antibody recycling via the FcRn receptor.

The interaction between the constant region of an antibody and various Fc receptors (FcR) including FcyRI (CD64), FcyRII (CD32) and FcyRIII (CD16) is believed to mediate the effector functions of the antibody. Significant biological effects can be a consequence of effector functionality. Usually, the ability to mediate effector function requires binding of the antibody to an antigen and not all antibodies will mediate every effector function.

Effector function can be measured in a number of ways including for example via binding of the FcyRIII to Natural Killer cells or via FcyRI to monocytes/macrophages to measure for ADCC effector function. For example an antigen binding protein of the present invention can be assessed for ADCC effector function in a Natural Killer cell assay. Practical approaches to evaluate ADCC and/or CDC function can be found in Kellner et al (Methods, 2014, 65(1): 105-13). Human IgGl constant regions containing specific mutations or altered glycosylation on residue Asn297 have also been described to enhance binding to Fc receptors. In some cases these mutations have also been shown to enhance ADCC and CDC, see for example, Kellner (2014).

In one embodiment of the present invention, such mutations are in one or more of positions selected from 239, 332 and 330 (IgGl), or the equivalent positions in other IgG isotypes. Examples of suitable mutations are S239D and I332E and A330L. In one embodiment the antibody of the invention herein described is mutated at positions 239 and 332, for example S239D and I332E or in a further embodiment it is mutated at three or more positions selected from 239 and 332 and 330, for example S239D and I332E and A330L (EU index numbering).

In one embodiment of the present invention there is provided an antibody comprising a chimaeric heavy chain constant region, for example an antibody comprising a chimaeric heavy chain constant region with at least one CH2 domain from IgG3 such that the antibody has enhanced effector function, for example wherein it has enhanced ADCC or enhanced CDC, or enhanced ADCC and CDC functions. In one such embodiment, the antibody may comprise one CH2 domain from IgG3 or both CH2 domains may be from IgG3.

PHARMACEUTICAL COMPOSITIONS

Purified preparations of an antibody as described herein may be incorporated into pharmaceutical compositions for use in the treatment of the human diseases, disorders and conditions described herein. The terms diseases, disorders and conditions are used interchangeably. The pharmaceutical composition can be used in the treatment of any diseases where amyloid deposits are present in the tissues and contribute to structural and functional damage leading to clinical illness.

The pharmaceutical preparation may comprise an antibody in combination with a pharmaceutically acceptable carrier. The antibody may be administered alone, or as part of a pharmaceutical composition.

Typically such compositions comprise a pharmaceutically acceptable carrier as known and called for by acceptable pharmaceutical practice, see e.g. Remingtons Pharmaceutical Sciences, 16th edition (1980) Mack Publishing Co. Examples of such carriers include sterilised carriers such as saline, Ringers solution or dextrose solution, optionally buffered with suitable buffers to a pH within a range of 5 to 8.

Pharmaceutical compositions may be administered by injection or continuous infusion (e.g. intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular or intraportal). Such compositions are suitably free of visible particulate matter. Pharmaceutical compositions may also be administered orally, specifically those containing orally available CPHPC. Pharmaceutical compositions may comprise between lmg to lOg of antibody, for example between 5 mg and 1 g of antibody. Alternatively, the composition may comprise between 5 mg and 500 mg, for example between 5 mg and 50 mg.

Methods for the preparation of such pharmaceutical compositions are well known to those skilled in the art. Pharmaceutical compositions may comprise between 1 mg to 10 g of antibody in unit dosage form, optionally together with instructions for use. Pharmaceutical compositions may be lyophilised (freeze dried) for reconstitution prior to administration according to methods well known or apparent to those skilled in the art. Where antibodies have an IgGl isotype, a chelator of copper, such as citrate (e.g. sodium citrate) or EDTA or histidine, may be added to the pharmaceutical composition to reduce the degree of copper-mediated degradation of antibodies of this isotype, see EP0612251. Pharmaceutical compositions may also comprise a solubiliser such as arginine base, a detergent/anti-aggregation agent such as polysorbate 80, and an inert gas such as nitrogen to replace vial headspace oxygen.

Effective doses and treatment regimes for administering the antibody are generally determined empirically and may be dependent on factors such as the age, weight and health status of the patient and disease or disorder to be treated. Such factors are within the purview of the attending physician. Guidance in selecting appropriate doses may be found in e.g. Smith et al (1977) Antibodies in human diagnosis and therapy, Raven Press, New York.

The dosage of antibody administered to a subject is generally between 1 μg/kg to 150 mg/kg, between 0.1 mg/kg and 100 mg/kg, between 0.5 mg/kg and 50 mg/kg, between 1 and 25 mg/kg or between 1 and 10 mg/kg of the subject's body weight. For example, the dose may be 10 mg/kg, 30 mg/kg, or 60 mg/kg. The antibody may be administered parenterally, for example subcutaneously, intravenously or intramuscularly.

If desired, the effective daily dose of a therapeutic composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.

The antibody may be administered in a single large dose or in smaller repeated doses.

The administration of a dose may be by slow continuous infusion over a period of from 2 to 24 hours, such as from 2 to 12 hours, or from 2 to 6 hours. This may result in reduced toxic side effects.

The administration of a dose may be repeated one or more times as necessary, for example, three times daily, once every day, once every 2 days, once a week, once a fortnight, once a month, once every 3 months, once every 6 months, or once every 12 months. The antibodies may be administered by maintenance therapy, for example once a week for a period of 6 months or more. The antibodies may be administered by intermittent therapy, for example for a period of 3 to 6 months and then no dose for 3 to 6 months, followed by administration of antibodies again for 3 to 6 months, and so on in a cycle.

For example, the dose may be administered subcutaneously, once every 14 or 28 days in the form of multiple sub-doses on each day of administration.

The antibody may be administered to the subject in such a way as to target therapy to a particular site. For example, the antibody may be injected locally into a circumscribed local amyloid mass in the tissues, or infused into the blood supply to an amyloidotic organ.

The anti-Αβ antibody and SAP depleting compound may be co-administered or sequentially administered.

Sequential administration may involve two or more sequential treatments with SAP depleting compound followed by two or more sequential treatments with the anti-Αβ antibody, or vice versa.

The sequential/subsequent dose may be an amount that is more than the initial/previous dose or less than the initial/previous dose.

The SAP-depleting compound of Formula (I) (CPHPC) may be parenterally administered at a dose of between 0.1 mg/kg and 2 mg/kg, depending on its activity. The SAP-depleting compound may be administered as a fixed dose, independent of a dose per subject weight ratio. The SAP- depleting compound may be administered in one or more separate, simultaneous or sequential parenteral doses of 100 mg or less, of 50 mg or less, 25 mg or less, or 10 mg or less.

Tablets and capsules for oral administration may be in unit dose form, and may contain conventional excipients, such as binding agents (e.g. pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g. lactose, microcrystalline cellulose or calcium hydrogen phosphate); tabletting lubricants (e.g. magnesium stearate, talc or silica); disintegrants (e.g. potato starch or sodium starch glycollate); and acceptable wetting agents (e.g. sodium lauryl sulphate). The tablets may be coated according to methods well known in normal pharmaceutical practice.

Oral liquid preparations may be in the form of, for example, oily suspension, non-aqueous solutions, emulsions, syrups or elixirs, or may be in the form of a dry product for reconstitution with suitable aqueous or non-aqueous vehicle immediately prior to administration. Such liquid preparations may contain conventional additives such as suspending agents (e.g. sorbitol syrup, cellulose derivatives or hydrogenated edible fats), emulsifying agents (e.g. lecithin or acacia), nonaqueous vehicles (which may include edible oils e.g. almond oil, oily esters, ethyl alcohol or fractionated vegetable oils), preservatives (e.g. methyl or propyl-p-hydroxybenzoates or sorbic acid), and, if desired, conventional flavourings or colorants, buffer salts and sweetening agents as appropriate. Preparations for oral administration may be suitably formulated to give controlled release of the active compound. In another embodiment, the dosage form for compounds of Formula (II) and Formula (III) is a tablet or a capsule.

The composition may contain from 0.1% to 99% by weight, preferably from 10 to 60% by weight, of a compound of Formula (II) or Formula (III), depending on the method of administration. The dose of the compound used in the treatment of the aforementioned disorders will vary in the usual way with the seriousness of the disorders, the weight of the sufferer, and other similar factors. However, as a general guide suitable unit doses may be 0.05 to 5000 mg, 1.0 to 1000 mg, or 100 to 600 mg, for example 100, 200 or 300 mg, and such unit doses may be administered more than once a day, for example two or three times a day. Such therapy may extend for a number of days, weeks, months or years.

Accordingly, the administration may use a pre-determined or routine schedule for administration, thereby resulting in a predetermined designated period of time between dose administrations. The schedule may encompass periods of time which are identical or which differ in length, as long as the schedule is predetermined. Any particular combination would be covered by the schedule as long as it is determined ahead of time that the appropriate schedule involves administration on a certain day.

The pharmaceutical composition may comprise a kit of parts of the antibody together with other medicaments, optionally with instructions for use. For convenience, the kit may comprise the reagents in predetermined amounts with instructions for use.

The terms "individual", "subject" and "patient" are used herein interchangeably. The subject may be a primate (e.g. a marmoset or monkey). The subject is typically a human.

Treatment can be therapeutic, prophylactic or preventative. The subject will be one who is in need thereof. Those in need of treatment may include individuals already suffering from a particular medical disease in addition to those who may develop the disease in the future.

Thus, the use of a SAP depleting compound with the anti-Αβ antibody described herein can be used for prophylactic or preventative treatment. In this case, the sequential treatments described herein are administered to an individual in order to prevent or delay the onset of one or more aspects or symptoms of the disease. The subject can be asymptomatic or may have a genetic predisposition to the disease, as amyloid deposits are known to be present in the tissues and to accumulate for periods of time before they cause sufficient damage to produce clinical symptoms. Such sub-clinical amyloid deposition can be detected by histological examination of tissue biopsies or by non-invasive imaging procedures, including radiolabeled Αβ scintigraphy, echocardiography and magnetic resonance imaging. A prophylactically effective amount of the anti-Αβ antibody and SAP depleting compound is administered to such an individual. A prophylactically effective amount is an amount which prevents or delays the onset of one or more aspects or symptoms of a disease described herein.

The antibodies and SAP depleting compounds described herein are to be used in methods of therapy. The term "therapy" encompasses alleviation, reduction, or prevention of at least one aspect or symptom of a disease. For example, to ameliorate or reduce one or more aspects or symptoms of a disease described herein.

The use of an anti-Αβ antibody and a SAP depleting compound need not affect a complete cure, or eradicate every symptom or manifestation of the disease to constitute a viable therapeutic treatment. As is recognised in the pertinent field, drugs employed as therapeutic agents may reduce the severity of a given disease state, but need not abolish every manifestation of the disease to be regarded as useful therapeutic agents. Similarly, a prophylactically administered treatment need not be completely effective in preventing the onset of a disease in order to constitute a viable prophylactic agent. Simply reducing the impact of a disease (for example, by reducing the number or severity of its symptoms, or by increasing the effectiveness of another treatment, or by producing another beneficial effect), or reducing the likelihood that the disease will occur (for example by delaying the onset of the disease) or worsen in a subject, is sufficient.

The term "amyloid" refers to extracellular deposits in the tissues of insoluble protein fibres composed of fibrils with characteristic ultrastructural morphology, a cross-β sheet core structure and the pathognomonic histochemical tinctorial property of binding Congo red dye from alkaline alcoholic solution and then giving red-green dichroism when viewed microscopically in strong cross polarised light. About 25 different unrelated proteins are known to form amyloid fibrils which deposit in human tissues and share all these typical properties. Amyloid deposits in the brain substance, cerebral amyloid, differ somewhat from amyloid deposits elsewhere in the body in that they are always focal and microscopic in size, and are commonly referred to as amyloid plaques.

EXAMPLES

It will be readily apparent to those skilled in the art that the SAP depleting compounds and anti-Αβ antibodies may be prepared using methods analogous to those outlined below. Example 1 -taken from EP091 5088 ( Example 8)

(R)-l-r6-r(RV2-Carboxy-pyrrolidin-l-yl1-6-oxo-hexanoyl1-pyrr olidine-2-carboxylic acid (a) (R)-l-r6-r(R)-2-Benzyloxycarbonyl-pyrrolidin-l-yl1-6-oxo-hex anoyl1-pyrrolidine-2- carboxylic acid benzyl ester

0.97g (10 mmol) D-proline-benzylester hydrochloride in 70 ml dichloromethane were stirred with 0.92g (5 mmol) adipoyl chloride and 2.8 ml (20 mmol) triethylamine over the weekend under argon at room temperature. Extraction with 2N hydrochloric acid and water, drying with sodiumsulfate, evaporation and chromatography over silicagel with acetoacetate to yielded 0.42g (16%) (R)-l-[6-[(R)-2-benzyloxycarbonyl-pyrrolidin-l-yl]-6-oxo-hex anoyl)-pyrrolidine-2-carboxylic acid benzyl ester as colourless oil.

MS-ISP: 521 (M+H) ⁺ .

b) (RVl-r6-r(RV2-Carboxy-pyrrolidin-l-yl1-6-oxo-hexanoyl1-pyrro lidine-2-carboxylic acid

410mg (0.79 mmol) (R)-l-[6-[(R)-2-benlyloxycarbonyl-pyrrolidin-l-yl]-6-oxo-hex anoyl]- pyrrolidine-2-carboxylic acid benzyl ester in 100 ml methanol were hydrogenated in the presence of 50mg 5% Pd on carbon. Filtration and evaporation of the solvent yielded 160mg (59%) (R)-l-[6- [(R)-2-carboxy-pyrrolidin-l-yl]-6-oxo-hexanoyl]-pyrrolidine- 2- carboxylic acid as colourless oil.

MS: 339 (M-H).

Example 2 - taken from PCT/ EP2015/ 058998

(2R,2'R)-Bis(((((tetrahydro-2H-pyran-4-yl)oxy)carbonyl)oxy)m ethyl)l,r- ad i poyl bisfpyrrolidi ne-2-ca rboxylate)

H and ¹³ C NMR spectra were recorded on a Bruker 300 or 400 MHz. Chemical shifts are reported in parts per million (ppm, units). High-resolution mass spectra were recorded on a Micromass LCT (TOF) spectrometer coupled to analytical high performance liquid chromatography (HPLC). HPLC was conducted on a Waters X-Terra MS C18 column (3.5μιη 30 x 4.6 mm id) eluting with 0.01M ammonium acetate in water (solvent A) and 100% acetonitrile (solvent B), using the following elution gradient 0-0.5 minutes 5% B, 0.5-3.75 minutes 5% to 100% B, 3.75-4.5 100% B, 4.5-5 100% to 5% B, 5-5.5 5% B at a flow rate of 1.3 ml/minute at 40°C. The mass spectra (MS) were recorded on a Waters LCT mass spectrometer using electrospray positive ionisation [ES ⁺ ve to give MH ⁺ molecular ions] or electrospray negative ionisation [ES-ve to give (M-H) ^" molecular ions] modes.

Analytical HPLC was conducted on a XSelect XP C18 column (2.5μιη 30 x 4.6 mm id) eluting with 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B), using the following elution gradient 0-3.2 minutes: 5% to 100% B, 3.2-4.0 minutes 100% B, at a flow rate of 1.8 ml/minute at 40°C. The mass spectra (MS) were recorded on a Waters ZQ mass spectrometer using electrospray positive ionisation [ES ⁺ to give MH ⁺ molecular ions] or electrospray negative ionisation [ES ^" to give (M-H) ^" molecular ions] modes. Intermediate 1: Chloromethyl (tetrahydro-2H-pyran-4-yl) carbonate.

To a solution of tetrahydro-2H-pyran-4-ol (40 g, 392 mmol) in diethyl ether (500 mL) was added pyridine (38.0 mL, 470 mmol) and the solution was cooled to 0°C. Chloromethyl carbonochloridate (41.8 mL, 470 mmol) was added dropwise leading to the formation of a white solid. The reaction mixture was stirred at RT for 18 h. The reaction mixture was washed with water (200 mL), with HCI 0.5N (200 mL), and then with a saturated solution of NaHC0 ₃ (200 mL). The organic phase was dried over Na ₂ S0 ₄ and concentrated under reduced pressure.

Toluene was added and the solution was concentrated under reduced pressure (to remove chloromethyl carbonochloridate in excess). Chloromethyl (tetrahydro-2H-pyran-4-yl) carbonate (Intermediate 1) was obtained as a colourless oil (70 g, 360 mmol, 92 % yield). Η NMR (400 MHz, CDCI ₃ ) δ ppm 5.75 (s, 2 H), 4.92 (m, 1 H), 3.95 (m, 2 H), 3.56 (m, 2 H), 2.02 (m, 2 H), 1.80 (m, 2 H).

Intermediate 1 (Alternative preparation): Chloromethyl (tetrahydro-2H-pyran-4-yl) carbonate. To a solution of chloromethyl carbonochloridate (5 g, 38.8 mmol) in DCM (50 mL) was added tetrahydro-2H-pyran-4-ol (3.96 g, 38.8 mmol) and the solution was cooled to 0 °C. DMAP (4.97 g, 40.7 mmol) was added then the reaction mixture was stirred at RT for 18 h. The reaction mixture was diluted with water and extracted with DCM (3 x 100 mL). The organic phases were combined, dried over Na ₂ S0 ₄ and concentrated under reduced pressure. The pale yellow oil was purified by chromatography eluting with 10% EtOAc in cyclohexane. The appropriate fractions were combined and concentrated in vacuo to give the required product as a colorless oil (2.2 g, 11.3 mmol, 29.2 % yield).

Η NMR (300 MHz, CDCI ₃ ) δ ppm 5.76 (s, 2 H), 4.92 (m, 1 H), 3.94 (m, 2 H), 3.57 (m, 2 H), 2.02 (m, 2 H), 1.80 (m, 2 H).

Example 2: (2R,2'R)-Bis(((((tetrahydro-2H-pyran-4-yl)oxy)carbonyl)oxy)m ethyl) 1,1'- ad i poyl bisfpyrrolidi ne-2-ca rboxylate)

Solution A: Potassium carbonate (29.8 g, 216 mmol) was added to a stirred suspension of (2R,2'R)-l, -adipoylbis(pyrrolidine-2-carboxylic acid) (35 g, 103 mmol) in 1,4-dioxane (1 L) and the reaction mixture was stirred at 80°C for 30 min.

Solution B: TBAI (7.60 g, 20.57 mmol) was added to a solution of chloromethyl (tetrahydro-

2H-pyran-4-yl) carbonate (42.0 g, 216 mmol) in dioxane (50 mL) and the mixture was stirred at RT for 15 min.

The solution B was added to the solution A. The reaction mixture was stirred at 80°C for 18 h.

The reaction mixture was filtered and concentrated under reduced pressure. The residue was taken up in EtOAc (400 mL) and washed with an aq. solution of NaHCC>3 (2 x 100 mL), an aq. solution of sodium thiosulfate (50 mL) and with 0.5N HCI (100 mL). The organic layer was dried over anhydrous Na ₂ S0 ₄ , filtered and concentrated in vacuo. The yellow oil was solubilized in 2- MeTHF(100 mL) and sonicated until crystallization occurred. The mixture was left to stand for 1 h at RT. The precipitate was filtered and washed with a mixture 2-MeTHF/iPr ₂ 0 70/30 to afford (2R,2'R)- bis(((((tetrahydro-2H-pyran-4-yl)oxy)carbonyl)oxy)methyl) l,l'-adipoylbis(pyrrolidine-2-carboxylate) (Example 2) as an off white powder (42 g, 64.0 mmol, 62.2 % yield). The product was dried under reduced pressure (5 mbar) and 35°C for 12 h.

*H NMR (400 MHz, CDCI ₃ ) δ ppm 5.88 (d, J = 5.5 Hz, 2 H), 5.73 (d, J = 5.5 Hz, 2 H), 4.87 (m, 2 H), 4.50 (m, 2 H), 3.93 (m, 4 H), 3.65 (m, 2 H), 3.55 (m, 6 H), 2.42 - 1.90 (m, 16 H), 1.84 - 1.60 (m, 8H).

Some minor peaks were observed due to the presence of rotamers.

Recrystallisation of (2R,2'R)-bis(((((tetrahydro-2H-pyran-4-yl)oxy)carbonyl)oxy)m ethyl) 1,1'- adipoylbis(pyrrolidine-2-carboxylate)

(2R,2'R)-bis(((((tetrahydro-2H-pyran-4-yl)oxy)carbonyl)oxy)m ethyl)l,r- adipoylbis(pyrrolidine-2-carboxylate) (170 g, 259 mmol) was suspended in 2-MeTHF and heated to 90°C until complete dissolution. The solution was filtered when still hot and allowed to cool to RT. The precipitate was filtered and washed with a mixture of 2-MeTHF/iPr ₂ 0 70/30 to afford (2R,2'R)- bis(((((tetrahydro-2H-pyran-4-yl)oxy)carbonyl)oxy)methyl) l,l'-adipoylbis(pyrrolidine-2-carboxylate) (130 g, 198 mmol, 76 % yield) as an off white crystalline solid. The product was dried under reduced pressure (5 mbar) and 35°C for 24 h.

LC/MS : m/z 657 [M+H] ⁺ , Rt 1.98 min.

Η NMR (400 MHz, CDCI ₃ ) δ ppm 5.87 (d, J = 5.5 Hz, 2 H), 5.71 (d, J = 5.5 Hz, 2 H), 4.86 (m, 2 H), 4.49 (m, 2 H), 3.91 (m, 4 H), 3.63 (m, 2 H), 3.54 (m, 6 H), 2.43 - 1.85 (m, 16 H), 1.84 - 1.59 (m, 8H).

Some minor peaks were observed due to the presence of rotamers.

¹³ C NMR (100 MHz, CDCI3) 171.69, 170.85, 153.12, 82.26, 77.36, 77.05, 76.72, 73.94, 65.02, 58.41, 46.95, 34.11, 31.47, 29.02, 24.80, 24.17.

HRMS : m/z calculated for C30H45N2O14 [M+H] ⁺ 657.2870, found 657.2883.

XRPD data were acquired on a PANalytical X'Pert Pro powder diffractometer, model PW3040/60 using an X'Celerator detector. The acquisition conditions were: radiation: Cu Ka, generator tension: 40 kV, generator current: 45 mA, start angle: 2.0° 2Θ, end angle: 40.0° 2Θ, step size: 0.0167° 2Θ, time per step: 31.75 seconds. The sample was prepared by mounting a few milligrams of sample (compound of Formula (II)) on a silicon wafer (zero background plate), resulting in a thin layer of powder.

Characteristic XRPD angles and d-spacings for the compound of Formula (II) are recorded in Table 1. The margin of error is approximately ± 0.1° 2Θ for each of the peak assignments. Peak intensities may vary from sample to sample due to preferred orientation.

Peak positions were measured using Highscore software.

27.1 3.3

Table 1: XRPD diffraction angles and d-spacings for compound of Formula (II)

Example 3 taken from GB1 51 8950.9

(2R,2'R)-bis(((tetrahydro-2H-pyran-4-carbonyl)oxy)methyl) l,l'-adipoylbis(pyrrolidine-2- carboxyl

H and ¹³ C NMR spectra were recorded on a Bruker 300 or 400 MHz NMR spectrometer. Chemical shifts are reported in parts per million (ppm, units). High-resolution mass spectra were recorded on a Micromass LCT (TOF) spectrometer coupled to analytical high performance liquid chromatography (HPLC). HPLC was conducted on a Waters X-Terra MS C18 column (3.5 μιη 30 x 4.6 mm id) eluting with 0.01M ammonium acetate in water (solvent A) and 100% acetonitrile (solvent B), using the following elution gradient 0-0.5 minutes 5% B, 0.5-3.75 minutes 5% to 100% B, 3.75-4.5 100% B, 4.5-5 100% to 5% B, 5-5.5 5% B at a flow rate of 1.3 mL/minute at 40 °C. The mass spectra (MS) were recorded on a Waters LCT mass spectrometer using electrospray positive ionisation [ES ⁺ ve to give MH ⁺ molecular ions] or electrospray negative ionisation [ES-ve to give (M-H) ^" molecular ions] modes.

Analytical HPLC was conducted on a XSelect XP C18 column (2.5 μιη 30 x 4.6 mm id) eluting with 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B), using the following elution gradient 0-3.2 minutes: 5% to 100% B, 3.2-4.0 minutes 100% B, at a flow rate of 1.8 mL/minute at 40 °C. The mass spectra (MS) were recorded on a Waters ZQ mass spectrometer using electrospray positive ionisation [ES ⁺ to give MH ⁺ molecular ions] or electrospray negative ionisation [ES ^" to give (M-H) ^" molecular ions] modes.

Intermediate 1

Ch lo ro methyl tetra hyd ro-2 H - pyra n -4-ca rboxy late

A 3L flask equipped with a mechanical stirrer was charged with a suspension of tetrahydro- 2H-pyran-4-carboxylic acid (50 g, 384 mmol) in water (50 mL) then a solution of Na ₂ CC>3 (163 g, 1537 mmol) in water (600 mL) was slowly added. The colourless aqueous solution was cooled down to 0 °C and tetrabutylammonium bromide (12.39 g, 38.4 mmol) was added. A solution of chloromethyl sulfochloridate (127 g, 768 mmol) in DCM (250 mL) was added drop wise over 1 h at 0 °C with vigourous stirring. The reaction mixture was stirred at 0 °C for 1 h then allowed to rise to RT and stirred overnight at that temperature. The precipitate formed during the reaction was filtered off and the filtrate was extracted with DCM (2 x 300 mL). The organic phases were gathered, dried over anhydrous Na ₂ S0 ₄ and concentrated under reduced pressure.

The yellow oily residue was dissolved in DCM (100 mL), loaded onto a silica pad (500 g) and eluted with 10% EtOAc in cyclohexane (1 L) then DCM (250 mL) to afford the title compound as a pale yellow oil (24 g, 35%).

H NMR (400 MHz, CDCI ₃ ) δ ppm 5.74 (s, 2 H), 3.97 (m, 2 H), 3.45 (m, 2 H), 2.64 (m, 1 H), 1.86 (m, 4 H).

Minor peaks were observed due to the presence of side product.

Example 3 : (2R,2'R)-bis(((tetrahydro-2H-pyran-4-carbonyl)oxy)methyl) 1,1'- adipoylb

A 3 L flask equipped with a mechanical stirrer was charged with a suspension of (2R,2'R)- l, -adipoylbis(pyrrolidine-2-carboxylic acid) (30 g, 88 mmol) in 1,4-dioxane (200 mL). K ₂ C0 ₃ (30.5 g, 220 mmol) was added to stirred suspension at RT and the reaction mixture was stirred at RT for 10 min then heated to 80 °C. Tetrabutylammonium iodide (6.51 g, 17.63 mmol) was added to a solution of chloromethyl tetrahydro-2H-pyran-4-carboxylate (36.2 g, 203 mmol) dissolved in 1,4- dioxane (100 mL). After 10 min stirring at RT, the precipitate was filtered and the orange filtrate was added drop wise over 30 min in the reaction mixture prepared above. After 8 h at 80 °C, the reaction mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was taken up in EtOAc (300 mL) and washed with NaHCC>3 aq. solution (1 x 100 mL), sodium sulfite aq. solution (1 x 100 mL), 0.5 N HQ (1 x 50 mL), water (1 x 100 mL) and brine. The organic layer was dried over anhydrous Na ₂ S0 ₄ , filtered then stirred 15 min with vegetal charcoal, filtered over celite bed and concentrated in vacuo to afford the title compound as a pale yellow gum which crystallized. The amorphous solid was taken up in iPr ₂ 0 and filtered to afford the title compound as off-white powder (28 g, 50.9 %).

Crystallisation

Final purification

Several batches of (2R,2'R)-Bis(((tetrahydro-2H-pyran-4-carbonyl)oxy)methyl) 1,1'- adipoylbis(pyrrolidine-2-carboxylate) (106 g, 170 mmol) were gathered and diluted in ethyl acetate (200 mL) and heated to reflux in a 2 L flask with magnetic stirrer. After 20 min the solid was dissolved and the flask was taken off the heating system, filtered and allowed to cool down naturally to RT. When the temperature fell to 50 °C some crystals began to appear. The product was left to stand overnight at RT without stirring to complete the crystallisation process. The mixture was filtered, washed successively with iPr ₂ 0 (1 x 150 mL) and pentane (2 x 100 mL). The product was dried at 35 °C and 5 mbars for 5 h to afford the product as a white powder (82.5 g, 80 %).

LC/MS : m/z 625 [M+H] ⁺ , Rt 2.68 min.

*H NMR (400 MHz, CDQ ₃ ) δ ppm 5.84 (d, J = 5.5 Hz, 2 H), 5.74 (d, J = 5.5 Hz, 2 H), 4.46 (m, 2 H), 4.01-3.91 (m, 4 H), 3.70-3.59 (m, 2 H), 3.58-3.38 (m, 6 H), 2.67-2.55 (m, 2H), 2.43 - 1.54 (m, 24 H).

Some minor peaks were observed due to the presence of rotamers.

¹³ C NMR (100 MHz, CDQ ₃ ) 173.20, 171.41, 171.21, 79.55, 66.33, 58.53, 46.95, 33.68, 29.11, 28.53, 24.88 , 24.20 , 22.52.

HRMS : m/z calculated for C ₃₀ H ₄₅ N ₂ Oi ₂ [M+H] ⁺ 625.2972, found 625.3010.

XRPD data were acquired on a PANalytical X'Pert Pro powder diffractometer, model PW3040/60 using an X'Celerator detector. The acquisition conditions were: radiation: Cu Ka, generator tension: 40 kV, generator current: 45 mA, start angle: 2.0° 2Θ, end angle: 40.0° 2Θ, step size: 0.0167° 2Θ, time per step: 31.75 seconds. The sample was prepared by mounting a few milligrams of sample (compound of Formula (III)) on a silicon wafer (zero background plate), resulting in a thin layer of powder.

Characteristic XRPD angles and d-spacings for Form I of the compound of Formula (III) are recorded in Table 2. The margin of error is approximately ± 0.1° 2Θ for each of the peak assignments. Peak intensities may vary from sample to sample due to preferred orientation.

Peak positions were measured using Highscore software. Compound of formula (III)

2Θ / ° d-spacings/ A

3.6 24.4

7.2 12.3

10.8 8.2

14.4 6.2

16.3 5.4

17.0 5.2

18.0 4.9

18.6 4.8

21.0 4.2

22.8 3.9

Table 2: XRPD diffraction angles and d-spacings for Form I of compound of Formula (III)

Example 4

Construction of Humanised Heavy and Light Chain DNA as described in WO WO2007/113172

Humanised V regions were synthesised de novo by build up of overlapping oligos and PCR amplification. Restriction sites for cloning into mammalian expression vectors RLD-bshe and RLN- bshe and human immunoglobulin signal sequences derived from the chosen human acceptor frameworks were included. The DNAs encoding the humanised V regions (HI (SEQ ID NO:27 therein), H2 (SEQ ID NO:29 therein), H3 (SEQ ID NO:31 therein), LI (SEQ ID NO:33 therein)) together with signal sequences and restriction sites were then cloned in frame into mammalian expression vectors: HI, H2 and H3 into RLD-bshe to generate DNA encoding three full length human IgGl Fc mutated heavy chains each containing mutations L235A and G237A, full length HI (SEQ ID NO:35 therein), full length H2 (SEQ ID NO:37 therein) and full length H3 (SEQ ID NO:39 therein); LI was cloned in frame into RLN-bshe containing the DNA encoding human kappa constant region to generate DNA encoding a full length human kappa light chain (SEQ ID NO:41 therein).

Representative Examples of Expression of Humanised Heavy and Light Chain Antibody Combinations as described in WO WO2007/113172

CHOK1 cells were transiently transfected at small scale with all combinations of humanised light and heavy chain DNA constructs: Ll+Hl, L1+H2, L1+H3 (SEQ ID NOs: 35 + 41, 37 + 41, 39 + 41 therein) in 6-well plates. CHOK1 cells passaged in DMEM F12, with 5% ultra low IgG foetal bovine serum and 2mM glutamine were grown to confluency in 6-well plates. The confluent cells were transfected with a total of 7.5 μg DNA: 30 μg Transfast lipid (Promega) in Optimem Glutamax medium (Invitrogen). Transfected cells were incubated at 37°C with 5% C02. At 72 hours supernatants were harvested and assayed for antibody concentration and then tested for binding to human Αβ by ELISA. Humanized LI combined with the three humanized heavy chains all expressed complete antibody that bound to human Αβ.

Humanized antibodies were also expressed in large scale transient CHOK1 cell transfections using liposomal delivery of DNA (eg TransFast (Promega)) and expression in culture bottles. For optimization of expression levels in transient transfections a heavy to light chain expression vector DNA ratio of 1:6 was used. Material from transient transfection was purified using ProSepA columns or FPLC with ProSepA HiTrap columns.

SEQUENCE LI STI NG

SEP ID NO: l

DNGMA SEP ID NO:2

FISNLAYSIDYADTVTG

SEP ID NO:3

GTWFAY

SEP ID NP:4

RVSQSLLHSNGYTYLH

SEP ID NP:5

KVSNRFS

SEP ID NP:6

SQTRHVPYT SEP ID NP:7

EVPLVESGGGLVPPGGSLRLSCAVSGFTFSDNGMAWVRPAPGKGLEWVSFISNLAYSIDY ADTVTGRFTISRDN AKN SLYLQM N SLRAEDTAVYYCVSGT FAYWGQGTLVTVSS SEP ID NP:8

DIVMTPSPLSLPVTPGEPASISCRVSPSLLHSNGYTYLHWYLPKPGPSPPLLIYKVSNRF SGVPDRFSGSGSGTD FTLKISRVEAEDVGVYYCSPTRHVPYTFGGGTKVEIK SEP ID NP:9

EVPLVESGGGLVPPGGSLRLSCAVSGFTFSDNGMAWVRPAPGKGLEWVSFISNLAYSIDY ADTVTGRFTISRDN AKNSLYLQMNSLRAEDTAVYYCVSGT F WGQGTLVTVSSASTKGPSVFPU\PSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDKTHT CPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNST YRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTK NQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK SEP ID NO: 10

DIVMTOSPLSLPVTPGEPASISCRVSOSLLHSNGYTYLHWYLOKPGOSPOLLIYKVSNRF SGVPDRFSGSGSGTD FTLKISRVEAEDVGVYYCSOTRHVPYTFGGGTKVEIKRTVAAPSVFIFPPSDEOLKSGTA SWCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC