WO2024256583A1 - Anti-fibril antibodies - Google Patents

Abstract

The invention relates to an isolated monoclonal antibody or antigen binding portion thereof which specifically binds to amyloid fibrils and effectively promotes clearance of extracellular amyloid deposits from the tissues in vivo, as well as humanised antibodies having the same properties.

Description

Anti-Fibril Antibodies

The present invention provides novel antibodies with broad specificity for the different types of amyloid fibrils that cause all the major forms of human systemic amyloidosis, humanised monoclonal antibodies which target multiple amyloid fibril types, as well as methods for using such antibodies and uses of the antibodies in therapeutic applications.

Introduction

Amyloidosis is a serious disease caused by extracellular deposition of insoluble abnormal fibrils, derived from aggregation of misfolded autologous proteins [Pepys, M. B. (2006). Annu. Rev. Med., 57, 223-241 ; Pepys, M. B. and P. N. Hawkins (2020). Amyloidosis. Oxford Textbook of Medicine. J. Firth, C. Conlon and T. Cox, Oxford University Press], About 30 different proteins are known to form amyloid fibrils in vivo in humans, each associated with clinically distinct conditions (https://doi.org/10.1080/13506129.2020.1835263). Systemic amyloidosis, with amyloid deposits in the viscera, blood vessel walls and connective tissue, is usually fatal and is the cause of about one per thousand deaths in developed countries. Cardiac transthyretin amyloidosis, known as ATTR, is a fatal disease, predominantly of elderly men, recently recognised to have a prevalence of 3-5% over the age of 75. The diagnosis of all forms of amyloidosis is clinically challenging and usually delayed, contributing, together with the limited efficacy and notable toxicity of many existing treatments, to frequently poor outcomes. Amyloidosis is thus a major unmet medical need.

Amyloidosis can be acquired as a complication of pre-existing primary disease that produces either an inherently amyloidogenic abnormal protein or greatly increased exposure to a normal but potentially amyloidogenic protein, or it is caused in the elderly by the normal expression of wild type transthyretin, which is inherently amyloidogenic. Hereditary amyloidosis is caused by mutant genes that encode variant proteins that happen to be amyloidogenic. Amyloid deposits can be local, restricted to a particular organ or tissue, or systemic, with amyloid deposits throughout the body except within the brain. Systemic amyloidosis is overwhelmingly either AL or ATTR type; the various hereditary types are rare. Systemic AA amyloidosis, which is a complication of chronic infections and other inflammatory conditions, is now rare in developed countries but remains more prevalent elsewhere.

The tissue and organ damage that manifests as clinical disease in amyloidosis is directly caused by remorseless accumulation of the extracellular fibrillar amyloid deposits. They disrupt the structure and thus the function of the affected tissues. For reasons that are not known, the normal mechanisms for clearance of debris from the extracellular space remove amyloid deposits very slowly if at all. Existing management of amyloidosis therefore comprises (a) supportive therapy to sustain function of damaged organs, up to and including multiple organ transplantation, and (b) measures to reduce the abundance of the respective amyloid fibril precursor protein and/or stabilise its native fold to prevent fibrillogenesis. The aim is to reduce or arrest the ongoing accumulation of amyloid and hope for the slow spontaneous but ultimately clinically beneficial regression of amyloid that then occurs in some patients. However, the range and efficacy of interventions intended to prevent amyloid formation, are limited and no specific treatments exist for many different types of amyloidosis and for many patients.

Direct approaches to removal of amyloid deposits in vivo are known in the art, specifically targeting serum amyloid P component (SAP), an invariant, normal, constitutive plasma protein that is universally present in all human amyloid deposits due to its avid specific calcium dependent binding to all types of amyloid fibrils [Pepys, M.B., Front. Immunol., 2018. 9: p. 2382.]. SAP binding to amyloid fibrils promotes their persistence in vivo [Tennent, G.A., et al., Proc. Natl. Acad. Sci. USA, 1995. 92(10): p. 4299-4303], A small molecule drug, miridesap, was therefore created, which is specifically bound by SAP [Pepys, M.B., et al., Nature, 2002. 417(6886): p. 254-259], It was intended to remove SAP from amyloid deposits. However, although miridesap almost totally depletes circulating SAP, it cannot remove all SAP from amyloid deposits and it did not accelerate amyloid clearance [Gillmore, J.D., et al., Br. J. Haematol., 2010. 148(5): p. 760-767], Nevertheless, the depletion of circulating SAP by miridesap enabled the safe and effective administration of anti-SAP antibodies to target amyloid deposits of all types [Bodin, K., et al., Nature, 2010. 468(7320): p. 93-97], The obligate therapeutic partnership between miridesap and anti-SAP antibody established proof of concept for safe and clinically beneficial amyloid removal by complement activating IgG antibodies in patients with different forms of systemic amyloidosis [Richards, D.B., et al., N. Engl. J. Med., 2015. 373(12): p. 1106-1114, Richards, D.B., et al., Sci. Transl. Med., 2018. 10(422): p. eaan3128.]. Despite this unprecedented and very encouraging result, the treatment has so far not progressed to a licensed medicine.

The SAP bound to amyloid fibrils of all types is an attractive target antigen but the actual purpose of antibody immunotherapy is removal of the amyloid fibrils themselves. Unfortunately, amyloid fibrils have long been known to be very poorly immunogenic. Patients with amyloidosis almost never produce specific anti-amyloid fibril antibodies, and experimental animals respond poorly if at all, even when vigorously immunised with xenogeneic fibrils. On the other hand, ex vivo amyloid fibrils of all types share very similar morphology, ultrastructure and protein fold, especially the cross-p core structure that is common to all ex vivo amyloid fibril types, regardless of their completely unrelated protein sequences [Sunde, M., et al., J. Mol. Biol., 1997. 273: p. 729-739], We and others have therefore hypothesised that amyloid fibrils may share potentially epitopic structures but, although some putatively cross-reactive antibodies have been claimed, no genuinely broad spectrum anti-amyloid fibril antibodies have been reported in the prior art.

Summary of the Invention

In a first aspect, there is provided a monoclonal antibody or antigen binding portion thereof which specifically binds to amyloid fibrils with broad anti-fibril specificity. In one embodiment, the antibody of the invention is capable of binding to at least 9 different types of amyloid fibrils, for example including ALK, ALA, ATTR wild type, ATTR variant, AA, AApoAl, ALys, AP2m and AFib amyloid fibrils. The antibody of the invention successfully binds to fibrils which cause or are involved in systemic amyloidosis. The antibody does not bind to Ap fibrils present in the CNS and particularly in the brain in patients with Alzheimer’s disease.

The antibody according to the invention is preferably an antibody or antigen binding portion thereof wherein CDRs in the variable domain of the heavy chain have a sequence identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99% or 100% with SEQ ID No 6, SEQ ID No. 7 and SEQ ID NO. 8.

In embodiments, the antibody according to the invention is preferably an antibody or antigen binding portion thereof wherein CDRs in the variable domain of the light chain have a sequence identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99% or 100% with SEQ ID No 9, SEQ ID No. 10 and SEQ ID NO. 11.

In embodiments, the antibody according to the invention is preferably an antibody or antigen binding portion thereof wherein CDRs in the variable domain of an alternative light chain have a sequence identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99% or 100% with SEQ ID No 12, SEQ ID No. 13 and SEQ ID NO. 14.

In one embodiment, the antibody according to the invention is preferably an antibody or antigen binding portion thereof wherein CDRs in the variable domain of the light chain have a sequence of SEQ ID No 9, SEQ ID No. 10 and SEQ ID NO. 11 optionally comprising one amino acid change. In one embodiment, the antibody according to this aspect of the invention can comprise heavy chain CDRs having SEQ ID Nos 6 to 8 and light chain CDRs having SEQ ID Nos 9 to 11.

In one embodiment, the antibody according to this aspect of the invention can comprise heavy chain CDRs having SEQ ID Nos 6 to 8 and light chain CDRs having SEQ ID Nos 12 to 14.

In one aspect, the antibody of the invention comprises a heavy chain variable region having a sequence identity of at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, at least 90%, 91%, 92%, 93%, 94%, at least 95%, 96%, 97%, 98%, at least 99% or 100% with SEQ ID No. 2.

In one aspect, the antibody of the invention comprises a light chain variable region having a sequence identity of at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, at least 90%, 91%, 92%, 93%, 94%, at least 95%, 96%, 97%, 98%, at least 99% or 100% with SEQ ID No. 4.

In one aspect, the antibody of the invention comprises a light chain variable region having a sequence identity of at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, at least 90%, 91%, 92%, 93%, 94%, at least 95%, 96%, 97%, 98%, at least 99% or 100% with SEQ ID No. 6.

In embodiments, the antibody is of human isotope lgG1 or murine isotype lgG2.

In the above aspects of the invention, the antibody can be selected from a human antibody, a chimeric antibody containing a human variable region, a humanized antibody, a bispecific antibody, or a single chain antibody, as well as antigen-binding fragments thereof.

In embodiments, antibody of the invention is specific for a conformational epitope commonly presented on amyloid fibrils. Thus, the antibody is able to bind to an amyloid-specific epitope independently of the linear polypeptide structure of the fibril.

In embodiments, the conformational epitope is present on at least three different amyloid fibrils. Preferably, it is present on 4, 5, 6, 7, 8 or 9 different amyloid fibrils, selected from ALK, ALA, ATTR wild type, ATTR variant, AA, AApoAl, ALys, AP2m and AFib amyloid fibrils.

In embodiments, the epitope is formed at the C-terminus of the proteins which compose the fibrils. In embodiments, the epitope comprises at least one charged amino acid, preferably at least 2 charged amino acids, comprising a charged side-chain. Amino acids with charged side-chains include aspartic acid, histidine, glutamic acid, lysine and arginine. In embodiments, the conformational epitope comprises a C-terminal carboxyl group, preferably a free C-terminal carboxyl group.

In embodiments, the conformational epitope can be mimicked by both malonate and citrate ions.

In a further aspect, the invention provides a method for generating an antibody according to the first aspect of the invention, comprising immunising a mammal in which SAP gene has been deleted.

In one embodiment, a SAP knockout mouse, which does not express the murine SAP protein, is immunised with human synthetic ATTR fibril material.

In another embodiment, a SAP knockout mouse, which does not express the murine SAP protein, is immunised with human synthetic ATTR fibril material coated with human SAP.

The humanised antibody of the invention successfully binds to fibrils which cause or are involved in systemic amyloidosis. The humanised antibody does not bind to Ap fibrils present in the CNS and particularly in the brain in patients with Alzheimer’s disease.

For example, the humanised monoclonal antibody binds to at least ALK, ATTR wild type and ATTR variant fibrils.

In another embodiment, the humanised monoclonal antibody binds to at least 4, 5, 6, 7, 8 or 9 types of amyloid fibrils, which are selected from ALK, ALA, ATTR wild type, ATTR variant, AA, AApoAl, ALys, Ap2m and AFib amyloid fibrils.

In embodiments, the humanised antibody of the invention does not compete with SAP, preferably human SAP, for binding to the amyloid fibrils. Thus, the epitope bound by the antibody of the invention is not identical to the epitope bound by SAP.

The humanised monoclonal antibody of the invention binds to fibrils which cause or are involved in systemic amyloidosis, but does not bind to soluble, native peptides from which the fibrils are derived.

In embodiments, the epitope bound by the antibody of the invention comprises the C- terminus of the proteins which compose the fibrils. In embodiments, the epitope comprises at least one charged amino acid, preferably at least 2 charged amino acids, comprising a charged side-chain. Amino acids with charged side-chains include aspartic acid, glutamic acid, lysine and arginine. In embodiments, the epitope comprises a C-terminal carboxyl group, preferably a free C- terminal carboxyl group.

In embodiments, a citrate and/or a malonate ion can be located in the binding site of the antibody according to the invention, when examined by x-ray crystallography.

The humanised monoclonal antibody or antigen binding portion thereof may have CDRH1 , CDRH2 and CDRH3 in the variable domain of the heavy chain with a sequence identity of at least 90% with SEQ ID No 6, SEQ ID No. 7 and SEQ ID No. 8 respectively.

Additionally, or alternatively, the humanised monoclonal antibody or antigen binding portion thereof may have CDRL1 , CDRL2 and CDRL3 in the variable domain of the light chain with a sequence identity of at least 90% with SEQ ID No 9, SEQ ID No. 10 and SEQ ID No. 11 respectively.

In another embodiment, the humanised monoclonal antibody or antigen binding portion thereof may have CDRH1 , CDRH2 and CDRH3 in the variable domain of the heavy chain with a sequence identity of at least 90% with SEQ ID No 6, SEQ ID No. 7 and SEQ ID NO. 8 respectively. Additionally, the humanised monoclonal antibody or antigen binding portion thereof may have CDRL1 , CDRL2 and CDRL3 in the variable domain of the light chain with a sequence identity of at least 90% with SEQ ID No 9, SEQ ID No. 10 and SEQ ID No. 11 respectively.

In another embodiment, the antibody according to the invention may have one optimal amino acid change in one or more CDRs, compared to the SEQ IDs of the CDRs as set forth herein.

In another optional embodiment, the humanised monoclonal antibody may have human framework regions derived from antibody genes selected from SEQ ID Nos. 12 and 13.

The isolated humanised monoclonal antibody may have framework residues mutated to match murine residues. These mutated residues may include VL residues I2, L39, A40, Q44, A49, V101, N66, T85 and F87, and VH residues M39, A80, L55, I66, V25, D85, E69, A45, P46, G47 and K48.

The isolated humanised monoclonal antibody may have the VH region comprising the mutations L55K and I66K.

The isolated humanised monoclonal antibody may have a combination of light and heavy chain framework mutations. The light chain framework mutations may consist of I2K, A40Y, N66K, T85N and F87Y. The heavy chain framework mutations may consist of L55K, I66K, V25A, D85N and E69P. Optionally, the isolated humanised monoclonal antibody may have a combination of light and heavy chain framework mutations. The light chain framework mutations may consist of I2K, L39M, A40Y, N66K, T85N and F87Y. The heavy chain framework mutations may consist of M39I, A80T, L55K, I66K, E69P, A45R, P46T, G47E and K48Q.

In another optional embodiment, the isolated humanised monoclonal antibody may have a combination of light and heavy chain framework mutations. The light chain framework mutations may consist of I2K, A40Y, N66K, T85N and F87Y. The heavy chain framework mutations may consist of M39I, A80T, L55K, I66K, V25A, D85N and E69P.

In yet another optional embodiment, the isolated humanised monoclonal antibody may have a combination of light and heavy chain framework mutations. The light chain framework mutations may consist of I2K, L39M, A40Y and N66K. The heavy chain framework mutations may consist of L55K, I66K, and E69P.

The isolated humanised monoclonal antibody may have a combination of light and heavy chain framework mutations. The light chain framework mutations may consist of A40Y. The heavy chain framework mutations may consist of L55K, I66K, V25A, D85N and E69P.

Optionally, the isolated humanised monoclonal antibody may have a combination of light and heavy chain framework mutations. The light chain framework mutations may consist of A40Y. The heavy chain framework mutations may consist of L55K and I66K.

In another optional embodiment, the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 14 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 15.

Alternatively, the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 16 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 17.

In another optional embodiment, the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 18 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 19.

Optionally, the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 20 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 21.

Alternatively, the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 22 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 23. Alternatively, the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 24 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 25.

In embodiments, the humanised monoclonal antibody may be further modified to alter the charge of the immunoglobulin. For example, positive charge in the immunoglobulin may be reduced, for example by reducing positive charge such that the next charge over the Fv region is +4 or less.

For example, the VL domain may be modified by including mutations Q44E, A49S, V101T, or omitting mutations L39M or I2K and L39M.

The VH domain may comprise changes including the mutations and L55K or I66K, but not both; adding E69P; and mutants at D57E and/or D62E, in HCDR2.

In embodiments, the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 26 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 27.

Alternatively, the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 28 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 29.

Alternatively, the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 30 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 31.

Alternatively, the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 32 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 33.

Alternatively, the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 34 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 35.

Alternatively, the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 36 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 37.

Alternatively, the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 38 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 39. Alternatively, the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 40 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 41.

Alternatively, the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 42 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 43.

Alternatively, the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 44 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 45.

Alternatively, the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 46 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 47.

Alternatively, the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 48 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 49.

Alternatively, the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 50 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 51.

Alternatively, the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 52 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 53.

Alternatively, the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 54 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 55.

Alternatively, the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 56 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 57.

Alternatively, the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 58 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 59.

Alternatively, the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 60 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 61.

Alternatively, the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 62 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 63.

Alternatively, the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 64 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 65.

Alternatively, the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 66 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 67.

Alternatively, the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 68 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 69.

Alternatively, the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 70 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 71.

In the foregoing embodiments, “at least 90%” is to be understood as 90% or more, optionally 91% or more, 92% or more, optionally 93% or more, optionally 94% or more, optionally 95% or more, optionally 96% or more, optionally 97% or more, optionally 98% or more, or optionally 99% or more, up to 100% identity with the recited SEQ ID.

The monoclonal antibody or antigen binding portion thereof may be selected from an IgG, IgA, or an antigen binding antibody fragment selected from an antibody single variable domain polypeptide, dAb, FAb, F(ab’)2, an scFv, an Fv, a VHH domain (such as a Nanobody® or other camelid immunoglobulin domain) or a disulfide-bonded Fv, a human antibody, a chimeric antibody preferably containing a human variable region, a humanized antibody, a bispecific antibody or a single chain antibody.

The humanised monoclonal antibody or antigen binding portion thereof may have the Fc region of the antibody derived from the human lgG1 isotype. In a specific embodiment, the antibody is of hl gG 1 isotype.

The humanised monoclonal antibody or antigen binding portion thereof may also effectively promote regression of systemic murine AA amyloid deposits when administered parenterally to mice with experimentally induced systemic AA amyloidosis. The humanised monoclonal antibody or antigen binding portion thereof may have in vivo efficacy that is complement activation dependent.

Alternatively, or in addition, the humanised monoclonal antibody or antigen binding portion thereof may have in vivo efficacy that is Fey receptor binding dependent.

In a further embodiment, the antibody of the invention is indicated for use in the treatment of systemic amyloidosis, and there is accordingly provided a pharmaceutical composition comprising an antibody as defined herein for use in the treatment of systemic amyloidosis.

In a further aspect, there is provided the use of an antibody as defined herein in the manufacture of a composition for the treatment of systemic amyloidosis. In a further aspect, there is provided a method for treating a subject suffering from systemic amyloidosis, comprising administering to a subject in need thereof a composition comprising an antibody specific for amyloid fibrils as described herein.

In a preferred embodiment, the pharmaceutical composition is co-administered together with a supporting treatment for amyloidosis. Amyloidosis therapy in the prior art typically aims to remove or reduce the presence or production of amyloid precursors, and the present antibody is designed to remove of established amyloid deposits. Together with existing or novel therapies to remove amyloid, the antibodies of the invention thus provide a more complete treatment for amyloidosis.

In some embodiments, the antibody of the invention may be administered independently of other amyloidosis treatments, for example where the occurrence of amyloid precursor has been minimised and it is desired to deplete established amyloid fibrils,

In another optional embodiment, the amyloidosis therapy is selected from any existing systemic AL amyloidosis therapies, including those listed by Bianchi et al., JACC CardioOncol. 2021 Oct; 3(4): 467-487.

It is noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as "comprises", "comprised", "comprising" and the like can have the meaning attributed to it in Patent law; e.g., they can mean "includes", "included", "including", and the like; and that terms such as "consisting essentially of and "consists essentially of have the meaning ascribed to them in Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.

These and other embodiments are disclosed or are obvious from and encompassed by, the following Detailed Description. Brief description of the Figures

Fig. 1. Generation of best-in-class anti-amyloid antibody 2E5 with broad specificity against amyloid deposits.

A) Generation of 2E5 antibody through novel mouse fibril immunization strategies. B) Antibody 2E5 at 10 pg/ml specifically binds to ATTR fibrils in ELISA, but not to soluble, globular, human or mouse transthyretin (TTR) that have been immobilised. C) Antibody 2E5 at 10 pg/ml specifically stains tissue deposits of the major forms of human systemic amyloid, AL, ATTR and AA, as well as rare hereditary forms (images labelled AF488). Amyloid location in the same fields is shown by intense fluorescence of Congo red counterstain.

Fig. 2. Anti-amyloid fibril antibody 2E5 binds to mouse AA amyloid and removes amyloid in vivo.

A) Antibody 2E5 at 10 pg/ml specifically stains mouse AA amyloid deposits (images labelled AF488) identified by Congo red staining in serial sections. B) Antibody 2E5 removes amyloid in vivo. Liver amyloid load scores in systemic AA amyloidotic mice 16 days after single IP injection of 4.8 mg/mouse 2E5 compared with untreated controls. Mann-Whitney test: Control vs 2E5: p=0.01278

Fig. 3. Binding of Malonate and Citrate to 2E5

A: binding of citrate in antibody binding cleft of 2E5 Fab, crystal structure.

B: space and change comparisons between malonate, citrate and TTR C-terminus

Fig. 4. BLI assay of binding of antibody 2E5 to deletions of TTR 99-127

A BLI assay was set up as depicted and deletions of peptide 99-127 tested for binding to 2E5. The results are shown in graphical and check-box form.

Deletion of the C-terminus of the peptide abolishes binding to 2E5. Similarly, amidation of the C-terminal amino acid result in loss of binding, indicating that a free C-terminal COOH is essential for binding.

Fig. 5. BLI assay of alanine scan of TTR 99-127

A BLI assay was performed with variants of 99-127 in which C-terminal amino acids were replaced with alanine. The three C-terminal amino acids (P, K, E) are increasingly essential for binding by 2E5.

Fig. 6. Competition ELISA between 2E5 mAb and hSAP for coated ATTR fibrils. A fixed concentration of 2E5 mAb was incubated with increasing concentrations of hSAP within a physiologically relevant window. Binding of hSAP and 2E5 mAb is observed to ATTR fibrils, and the 2E5 binding signal is stable in the presence of bound hSAP, indicating that 2E5 has a non-overlapping epitope with hSAP on ATTR fibrils.

Fig. 7. Binding to ATTR Fibrils of Humanised Antibodies

ELISA of antibodies binding to ATTR fibrils after initial humanisation experiments, compared to murine 2E5 antibody.

A: CDR grafts show 100-fold decrease in binding compared to 2E5.

B: Control in absence of ATTR

Fig. 8. CDR grafted variants binding to ATTR fibrils

Elisa of optimised CDR-grafted antibodies binding to 10ug/ml ATTR fibrils.

Fig. 9. Elisa of CDR-grafted antibodies binding to different amyloid fibrils

Elisa plot of interaction of humanized clones to synthetic fibrils and native amyloid. Fibrils are synthetic amyloid derived from the truncated fragment of immunoglobulin light chain (AL55-133) and mutant forms of the transthyretin protein (S52P TTR) and beta2- microglobulin (D76N Abeta2-m) as well as AA (Amyloid A).

Detailed description of the invention

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art, such as in the arts of peptide chemistry, cell culture and phage display, nucleic acid chemistry and biochemistry. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

Standard techniques are used for molecular biology, genetic and biochemical methods (“Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989);

“Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction” (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991)), which are incorporated herein by reference. These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

An “antibody” may be selected from, but not limited to, an IgG, IgA, or an antigen binding antibody fragment selected from an antibody single variable domain polypeptide, dAb, FAb, F(ab’)2, an scFv, an Fv, a VHH domain (such as a Nanobody® or other camelized immunoglobulin domain) or a disulfide-bonded Fv. In certain embodiments, any of the above antibody types or fragments thereof may be prepared from one or more of a mammalian species selected from, but not limited to mouse, rat, rabbit, human. Such antibodies can be humanized for use in humans.

In certain embodiments, any of the above antibody types or fragments thereof may be provided as heteroconjugates, bispecific, single-chain, chimeric or humanized molecules having affinity for amyloid fibrils.

In certain embodiments, any of the aforementioned antibody/antibodies binds to amyloid with a dissociation coefficient of 100nM or less, 75nM or less, 50nM or less, 25nM or less, such as 10nM or less, 5nM or less, 1nM or less, or in embodiments 500pM or less, 100pM or less, 50pM or less or 25pM or less.

Antibodies may be monospecific, with narrow or broad specificity; or multispecific, such as bispecific, such that they possess two distinct epitope specificities in a single antibody molecule. Cocktails of antibodies may be targeted at two or more specific epitopes.

Antibody cocktails may be prepared by a mixture of one or more monoclonal antibodies. In one embodiment, an antibody cocktail contains two, three, four or more monoclonal antibodies each of which recognises a plurality of amyloid fibrils.

In one embodiment, the antibody is monoclonal and binds to at least two, at least three, at least four, at least five, at least 6, at least 7, at least 8, at least 9 or at least 10 different amyloid fibril types. Advantageously, it binds substantially to all systemic amyloid fibril types.

In one aspect, the antibody or antibodies of the invention are formulated for intravenous (iv) or intramuscular (im) administration. Antibodies administered iv should extravasate from the circulation in order to enter the interstitial tissue space and bind to their cognate target. The antibody, in one embodiment, is an antibody fragment such as a scFv, dAb or VHH antibody. Small antibody fragments are extravasated much more readily into tissue, and for this reason can perform better than IgG or other larger antibodies. However, smaller fragments are also cleared faster from the circulation. A compromise must be struck between tissue accessibility and clearance. For example, see Wang et al., Clinical pharmacology & Therapeutics, 84:5, 2008, 548-558. Several antibody conjugates have been described which have extended half-life using a variety of strategies, for example through conjugation to albumin (such as human serum albumin). See Kontermann et al., BioDrugs April 2009, Volume 23, Issue 2, pp 93-109.

The mechanism by which antibodies according to the present invention promote removal of amyloid deposits is postulated to obligatorily involve complement activation by the antibody bound to the amyloid fibrils, leading to recruitment of macrophages that then fuse into multinucleated giant cells (Bodin, K., et al. (2010). "Antibodies to human serum amyloid P component eliminate visceral amyloid deposits." Nature 468(7320): 93-97). These have the unique phenotype that enables them to destroy the extracellular amyloid deposits that are overwhelmingly massive in relation to single cells (Milde, R., et al. (2015). "Multinucleated giant cells are specialized for complement-mediated phagocytosis and large target destruction." Cell Rep. 13(9): 1937-1948). Accordingly, antibody fragments, intact antibodies and any other constructs that do not efficiently activate the classical complement pathway when they have bound to amyloid fibrils, are advantageously modified, or further modified to promote complement activation. In a preferred embodiment, an antibody fragment or derivative is an antibody fragment or derivative which is appropriately modified to activate complement.

By “fragment” is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

The terms “isolated,” “purified,” or “biologically pure” refer to material that is free to varying degrees from components which normally accompany it as found in its native state. “Isolate” denotes a degree of separation from original source or surroundings. “Purify” denotes a degree of separation that is higher than isolation. A “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term “purified” can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.

By “isolated polynucleotide” is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.

By an “isolated polypeptide” is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

Amyloid

Amyloidosis and amyloid fibrils are known in the art. About 30 different proteins are known to form amyloid fibrils in vivo in humans, each associated with clinically distinct conditions. For reviews and definitions, see Pepys, M. B. and P. N. Hawkins (2020). Amyloidosis. Oxford Textbook of Medicine. J. Firth, C. Conlon and T. Cox, Oxford University Press], and the Nomenclature Report of the International Society for Amyloidosis (https://doi.org/10.1080/13506129.2020.1835263).

Amyloid fibrils are aggregates of proteins, which typically assemble in a beta sheet. The termini of proteins can be exposed in beta sheet structures, and it is postulated that exposed termini of the proteins which compose the amyloid fibrils are responsible for the binding of pan-fibril-specific antibodies to the amyloid. In embodiments, the C-termini of the proteins are bound by the antibody; a free C-terminal carboxyl group in the TTR peptide is essential for binding, indicating that a charged ligand is required by 2E5.

The amyloid epitope formed by the C-terminal amino acids of TTR can be mimicked by both a citrate ion and a malonate ion in space and charge interactions in the antibody binding domain. The structure of the C-terminus of TTR (PKE) is very similar to the structure of citrate and malonate. Citrate forms binding interactions with N30, F90, T91 , Y104, N105 and W106 in the 2E5 binding cleft; citrate and malonate bind in an almost identical fashion, but are not large enough to contact the heavy chain.

Antibodies

The term "monoclonal antibody" refers to an antibody obtained from a single clone of B lymphocyte derived plasma cells producing a homogeneous antibody of a single heavy and light chain class and epitope specificity.

Monoclonal antibodies are typically highly specific, and are directed against a single antigenic site (epitope), in contrast to conventional antibodies within an antiserum induced in a whole animal by immunisation with a particular antigen. Such conventional antibodies are derived from many different clones of B lymphocytes which recognise either the same or different epitopes on the immunising antigen, and are known as polyclonal antibodies. In addition to their very restricted specificity, monoclonal antibodies are readily produced in pure form uncontaminated by other immunoglobulins, whereas isolation of specific antibodies from a polyclonal antiserum requires demanding immunopurification procedures.

Monoclonal antibodies may be prepared by the hybridoma method (see Kohler et al., Nature, 256:495-7, 1975), or by recombinant DNA methods. The monoclonal antibodies may even be isolated from phage antibody libraries using well known techniques.

The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567;

Morrison et al., Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984)).

In the hybridoma method, a host animal, typically a mouse, is immunized with the desired antigen to induce generation of clones of B lymphocytes that produce or are capable of producing antibodies that will specifically bind to that antigen. Lymphocytes harvested from the immunised animal are then fused in vitro with a continuous line of myeloma cells grown in vitro to form so-called hybridoma cells. These are then selected by growth in a suitable culture medium that permits survival only of fused cells and not the unfused, parental myeloma cells. Examples of myeloma cells include, but are not limited to, human myeloma and mouse-human heteromyeloma cell lines which have been described for the production of human monoclonal antibodies.

The culture medium from the growing hybridoma cells may be assayed for monoclonal antibodies directed against the antigen. The binding specificity of the antibodies produced by the cells may be determined by various methods - such as immunoprecipitation or an in vitro binding assay - such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA) or immunoradiometric assay (IRMA).

After hybridoma cells are identified that produce the desired antibodies, the clones may be subcloned by limiting dilution procedures and grown by standard methods. The monoclonal antibodies secreted by the subclones are separated from the culture medium or serum by well-known immunoglobulin purification procedures - such as protein A-Sepharose, gel electrophoresis, dialysis, hydroxyapatite chromatography or affinity chromatography.

The antibodies of the invention also encompass variants of such antibodies and fragments thereof. Variants include peptides and polypeptides comprising one or more amino acid sequence substitutions, deletions, and/or additions that have the same or substantially the same affinity and specificity of epitope binding as the anti-amyloid antibody or fragments thereof.

The deletions, insertions or substitutions of amino acid residues may produce a silent change and result in a functionally equivalent substance. Deliberate ammo acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

Homologous substitution (substitution and replacement are both used herein to mean the interchange of an existing amino acid residue, with an alternative residue) may occur i.e. like-for-like substitution such as basic for basic, acidic for acidic, polar for polar etc. Non- homologous substitution may also occur i.e. from one class of residue to another or alternatively involving the inclusion of unnatural amino acids - such as ornithine (hereinafter referred to as Z), diami- nobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as O), pyriylalanine, thienylalanine, naphthylalanine and phenylglycine.

Replacements may also be made by unnatural amino acids include; alpha* and alphadisubstituted* amino acids, N-alkyl amino acids*, lactic acid*, halide derivatives of natural amino acids such as trifluorotyrosine*, p-CI-phe- nylalanine*, p-Br-phenylalanine*, p-l- phenylalanine*, L-allyl-glycine*, p-alanine*, L-a-amino butyric acid*, L-y-amino bu- tyric acid*, L-a-amino isobutyric acid*, L-e-amino caproic acid#, 7-amino heptanoic acid*, L- methionine sulfone#*, L- norleucine*, L-norvaline*, p-nitro-L-phenylalanine*, L- hydroxyproline#, L-thioproline*, methyl derivatives of phenylalanine (Phe) such as 4-methyl- Phe*, pentamethyl-Phe*, L-Phe (4-amino)#, L-Tyr (methyl)*, L-Phe (4-isopropyl)*, L-Tic (1 ,2,3,4- tetrahydroisoquinoline-3-carboxyl acid)*, L-diaminopropionic acid# and L-Phe (4- benzyl)*. The notation * has been utilised for the purpose of the discussion above (relating to homologous or non-homologous substitution), to indicate the hydro- phobic nature of the derivative whereas # has been utilised to indicate the hydrophilic nature of the derivative, #* indicates amphipathic characteristics.

Thus, variants may include peptides and polypeptides comprising one or more amino acid sequence substitutions, deletions, and/or additions to the antibodies and fragments of the invention wherein such substitutions, deletions and/or additions do not cause substantial changes in affinity and specificity of epitope binding. For example, a variant of an antibody or fragment thereof may result from one or more changes to the antibody or fragment thereof, where the changed antibody or fragment thereof has the same or substantially the same affinity and specificity of epitope binding as the starting sequence. Variants may be naturally occurring, such as allelic or splice variants, or may be artificially constructed. Variants may be prepared from the corresponding nucleic acid molecules encoding said variants. Variants of the antibodies or fragments thereof may have changes in light and/or heavy chain amino acid sequences that are naturally occurring or are introduced by in vitro engineering of native sequences using recombinant DNA techniques. Naturally occurring variants include "somatic" variants which are generated in vivo in the corresponding germ line nucleotide sequences during the generation of an antibody response to a foreign antigen.

Variants of antibodies and binding fragments may also be prepared by mutagenesis techniques. For example, amino acid changes may be introduced at random throughout an antibody coding region and the resulting variants may be screened for binding affinity for amyloid or for another property. Alternatively, amino acid changes may be introduced into selected regions of the antibody, such as in the light and/or heavy chain CDRs, and/or in the framework regions, and the resulting antibodies may be screened for binding to amyloid or some other activity. Amino acid changes encompass one or more amino acid substitutions in a CDR, ranging from a single amino acid difference to the introduction of multiple permutations of amino acids within a given CDR. Also encompassed are variants generated by insertion of amino acids to increase the size of a CDR.

Amyloid specific antibodies or fragments thereof may be provided with a modified Fc region where a naturally-occurring Fc region is modified to increase the half-life of the antibody or fragment in a biological environment, for example, the serum half-life or a half-life measured by an in vitro assay. Fc modification can also be employed to alter antibody biodistribution in vivo, which can direct the antibody to amyloid-bearing tissues.

Variants also include antibodies or fragments thereof comprising a modified Fc region, wherein the modified Fc region comprises at least one amino acid modification relative to a wild-type Fc region. The variant Fc region may be designed, relative to a comparable molecule comprising the wild-type Fc region, so as to bind Fc receptors with a greater or lesser affinity. For example, the antibodies and fragments thereof may comprise a modified Fc region. Fc region refers to naturally-occurring or synthetic polypeptides homologous to the IgG C-terminal domain that is produced upon papain digestion of IgG. IgG Fc has a molecular weight of approximately 50 kD. In the antibodies and fragments, an entire Fc region can be used, or only a half-life enhancing portion.

The antibodies and fragments thereof also encompass derivatives of the antibodies, fragments and sequences disclosed herein. Derivatives include polypeptides or peptides, or variants, fragments or derivatives thereof, which have been chemically modified. Examples include covalent attachment of one or more polymers - such as water soluble polymers, N- linked, or O-linked carbohydrates, sugars, phosphates, and/or other such molecules. The derivatives are modified in a manner that is different from naturally occurring or starting peptide or polypeptides, either in the type or location of the molecules attached. Derivatives further include deletion of one or more chemical groups which are naturally present on the peptide or polypeptide.

The present invention also encompasses amyloid specific antibodies that include two full length heavy chains and two full length light chains. Alternatively, the antibodies may be constructs such as single chain antibodies or "mini" antibodies that retain binding activity to amyloid. Such constructs may be prepared by methods well known in the art. Advantageously, such fragments are modified to enable complement activation.

Methods for creating recombinant DNA versions of the antigen-binding regions of antibody molecules which bypass the generation of monoclonal antibodies are contemplated for the amyloid specific antibodies and fragments thereof. DNA is cloned into a plasmid vector system. One example of such a technique uses a bacteriophage lambda vector system having a leader sequence that causes the expressed Fab protein to migrate to the periplasmic space (between the bacterial cell membrane and the cell wall) or to be secreted. One can rapidly generate and screen great numbers of functional Fab fragments for those which bind amyloid. Such amyloid binding agents (Fab fragments with specificity for amyloid) are specifically encompassed within the amyloid specific antibodies and fragments thereof.

The amyloid binding antibodies and fragments thereof may be humanized or human engineered antibodies. As used herein, "a humanized antibody", or antigen binding fragment thereof, is a recombinant polypeptide that comprises a portion of an antigen binding site from a non-human antibody and a portion of the framework and/or constant regions of a human antibody. A human engineered antibody or antibody fragment is a non-human (e.g., mouse) antibody that has been engineered by modifying (e.g., deleting, inserting, or substituting) amino acids at specific positions so as to reduce or eliminate any detectable immunogenicity of the modified antibody in a human.

Humanized antibodies include chimeric antibodies and CDR-grafted antibodies. Chimeric antibodies are antibodies that include a non-human antibody variable region linked to a human constant region. Thus, in chimeric antibodies, the variable region is mostly non- human, and the constant region is human. Chimeric antibodies and methods for making them are described in, for example, Proc. Natl. Acad. Sci. USA, 81: 6841-6855 (1984). Although, they can be less immunogenic than a mouse monoclonal antibody, administrations of chimeric antibodies have been associated with human immune responses (HAMA) to the non-human portion of the antibodies. Chimeric antibodies can also be produced by splicing the genes from a mouse antibody molecule of appropriate antigenbinding specificity together with genes from a human antibody molecule of appropriate biological activity, such as the ability to activate human complement and mediate antibodydependent cellular phagocytosis (ADCP). One example is the replacement of a Fc region with that of a different isotype.

CDR-grafted antibodies are antibodies that include the CDRs from a non-human "donor" antibody linked to the framework region from a human "recipient" antibody. Generally, CDR- grafted antibodies include more human antibody sequences than chimeric antibodies because they include both constant region sequences and variable region (framework) sequences from human antibodies. Thus, for example, a CDR-grafted humanized antibody of the invention can comprise a heavy chain that comprises a contiguous amino acid sequence (e.g., about 5 or more, 10 or more, or even 15 or more contiguous amino acid residues) from the framework region of a human antibody (e.g., FR-I, FR-2, or FR-3 of a human antibody) or, optionally, most or all of the entire framework region of a human antibody. CDR-grafted antibodies and methods for making them are described in Nature, 321 : 522-525 (1986). Methods that can be used to produce humanized antibodies also are described in, for example, US 5,721 ,367 and 6,180,377.

"Veneered antibodies" are non-human or humanized (e.g., chimeric or CDR-grafted antibodies) antibodies that have been engineered to replace certain solvent-exposed amino acid residues so as to reduce their immunogenicity or enhance their function. Veneering of a chimeric antibody may comprise identifying solvent-exposed residues in the non-human framework region of a chimeric antibody and replacing at least one of them with the corresponding surface residues from a human framework region.

Veneering can be accomplished by any suitable engineering technique.

Further details on antibodies, humanized antibodies, human engineered antibodies, and methods for their preparation can be found in Antibody Engineering, Springer, New York, NY, 2001.

Examples of humanized or human engineered antibodies are IgG, IgM, IgE, IgA, and IgD antibodies. The antibodies may be of any class (IgG, IgA, IgM, IgE, IgD, etc.) or isotype and can comprise a kappa or lambda light chain. For example, a human antibody may comprise an IgG heavy chain or defined fragment, such as at least one of isotypes, lgG1 , lgG2, lgG3 or lgG4. As a further example, the antibodies or fragments thereof can comprise an IgG 1 heavy chain and a kappa or lambda light chain.

Human antibodies to target amyloid can be produced using transgenic animals that have no endogenous immunoglobulin production and are engineered to contain human immunoglobulin loci, as described in WO 98/24893 and WO 91/00906.

Using a transgenic animal described above, an immune response can be produced to a selected antigenic molecule, and antibody producing cells can be removed from the animal and used to produce hybridomas that secrete human monoclonal antibodies. Immunization protocols, adjuvants, and the like are known in the art, and are used in immunization of, for example, a transgenic mouse.

The development of technologies for making repertoires of recombinant human antibody genes, and the display of the encoded antibody fragments on the surface of filamentous bacteriophage, has provided a means for making human antibodies directly.

The antibodies produced by phage technology are produced as antigen binding fragments- usually Fv or Fab fragments-in bacteria and thus lack effector functions.

Effector functions can be introduced by one of two strategies: the fragments can be engineered either into complete antibodies for expression in mammalian cells, or into bispecific antibody fragments with a second binding site capable of triggering an effector function.

Human antibodies may be generated through the in vitro screening of antibody display libraries (J Mol. Biol. (1991) 227: 381). Various antibody-containing phage display libraries have been described and may be readily prepared. Libraries may contain a diversity of human antibody sequences, such as human Fab, Fv, and scFv fragments, that may be screened against an appropriate target. Phage display libraries may comprise peptides or proteins other than antibodies which may be screened to identify agents capable of selective binding to amyloid.

Phage-display processes mimic immune selection through the display of antibody repertoires on the surface of filamentous bacteriophage, and subsequent selection of phage by their binding to an antigen of choice. One such method is described in WO 99/10494. Anti-amyloid antibodies can be isolated by screening of a recombinant combinatorial antibody library, preferably a scFv phage display library, prepared using human VL and VH cDNAs prepared from mRNA derived from human lymphocytes. Methodologies for preparing and screening such libraries are known in the art. There are commercially available kits for generating phage display libraries.

The amyloid binding antibodies and fragments thereof may comprise one or more portions that do not bind amyloid but instead are responsible for other functions, such as circulating half-life, direct cytotoxic effect, detectable labelling, or activation of the recipient’s endogenous complement cascade or endogenous cellular cytotoxicity. The antibodies or fragments thereof may comprise all or a portion of the constant region and may be of any isotype, including IgA (e.g., IgAI or lgA2), IgD, IgE, IgG (e.g. lgG1, lgG2, lgG3 or lgG4), or IgM. In addition to, or instead of, comprising a constant region, antigen-binding compounds of the invention may include an epitope tag, a salvage receptor epitope, a label moiety for diagnostic or purification purposes, or a cytotoxic moiety such as a radionuclide or toxin.

The anti- amyloid antibody or fragment thereof may be modified in order to increase its serum half-life, for example, by adding molecules - such as PEG or other water soluble polymers, including polysaccharide polymers to increase the half-life.

The amyloid binding antibodies and fragments thereof may be bispecific. For example, bispecific antibodies may resemble single antibodies (or antibody fragments) but have two different antigen binding sites (variable regions). Bis- pecific antibodies can be produced by various methods - such as chemical techniques, "polydoma" techniques or re- combinant DNA techniques. Bispecific antibodies may have binding specificities for at least two different epitopes, at least one of which is an epitope of amyloid.

Amyloid binding antibodies and fragments may be heteroantibodies. Heteroantibodies are two or more anti- bodies, or antibody binding fragments (Fab) linked together, each antibody or fragment having a different specificity. As used herein, the term "antibody fragments" refers to portions of an intact full length antibody - such as an antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab’, F(ab’)2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); multispecific antibody fragments such as bispecific, trispecific, and multispecific antibodies (e.g., diabodies, triabodies, tetrabodies); binding-domain immunoglobulin fusion proteins; camelized antibodies; minibodies; chelating recombinant antibodies; tribodies or bibodies; intrabodies; nanobodies; small modular immunopharmaceuticals (SMIP), VHH containing antibodies; and any other polypeptides formed from antibody fragments.

In the context of the present invention, the terms anti- amyloid antibody and amyloid binding antibody encompass amyloid binding antibody fragments comprising any part of the heavy or light chain sequences of the full length antibodies, and which bind amyloid. The term "fragments" as used herein refers to fragments capable of binding amyloid, for example any of at least 3 contiguous amino acids (e.g., at least 4, 5, 6, 7, 8, 9 or 10 or more contiguous amino acids, for example from a CDR) of the antibody involved in antigen binding, and encompasses Fab, Fab’, F(ab’)2, and F(v) fragments, or the individual light or heavy chain variable regions or portion thereof. Amyloid binding fragments include, for example, Fab, Fab’, F(ab’)2, Fv and scFv. These fragments lack the Fc fragment of an intact antibody, clear more rapidly from the circulation, and can have less non-specific tissue binding than an intact antibody. These fragments can be produced from intact antibodies using well known methods, for example by proteolytic cleavage with enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab’)2 fragments).

The amyloid binding antibodies and fragments also encompass single-chain antibody fragments (scFv) that bind to amyloid. An scFv comprises an antibody heavy chain variable region (VH) operably linked to an antibody light chain variable region (VL) wherein the heavy chain variable region and the light chain variable region, together or individually, form a binding site that binds amyloid. An scFv may comprise a VH region at the amino-terminal end and a VL region at the carboxy-terminal end. Alternatively, scFv may comprise a VL region at the amino-terminal end and a VH region at the carboxy-terminal end. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv). An scFv may optionally further comprise a polypeptide linker between the heavy chain variable region and the light chain variable region.

The amyloid binding antibodies and fragments also encompass domain antibody (dAb) fragments as described in Nature 341:544-546 (1989) which consist of a VH domain.

The amyloid binding antibodies and fragments also encompass heavy chain antibodies (HCAb). These antibodies can apparently form antigen-binding regions using only heavy chain variable region, in that these functional antibodies are dimers of heavy chains only (referred to as "heavy-chain antibodies" or "HCAbs"). Accordingly, amyloid binding antibodies and fragments may be heavy chain antibodies (HCAb) that specifically bind to amyloid.

The amyloid binding antibodies and fragments also encompass antibodies that are SMIPs or binding domain immunoglobulin fusion proteins specific for amyloid protein. These constructs are single-chain polypeptides comprising antigen binding domains fused to immunoglobulin domains necessary to carry out antibody effector functions (see W003/041600).

The amyloid binding antibodies and fragments also encompass diabodies. These are bivalent antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain. This forces the domains to pair with complementary domains of another chain and thereby creates two antigen binding sites (see, for example, WO 93/11161). Diabodies can be bispecific or monospecific.

The amyloid binding antibodies and fragments thereof also encompass immunoadhesins. One or more CDRs may be incorporated into a molecule either covalently or noncovalently to make it an immunoadhesin. An immunoadhesin may incorporate the CDR(s) as part of a larger polypeptide chain, may covalently link the CDR(s) to another polypeptide chain, or may incorporate the CDR(s) noncovalently. The CDRs permit the immunoadhesin to specifically bind to amyloid.

The amyloid binding antibodies and fragments thereof also encompass antibody mimics comprising one or more amyloid binding portions built on an organic or molecular scaffold (such as a protein or carbohydrate scaffold). Proteins having relatively defined three- dimensional structures, commonly referred to as protein scaffolds, may be used as reagents for the design of antibody mimics. These scaffolds typically contain one or more regions which are amenable to specific or random sequence variation, and such sequence randomization is often carried out to produce libraries of proteins from which desired products may be selected. For example, an antibody mimic can comprise a chimeric nonimmunoglobulin binding polypeptide having an immunoglobulin-like domain containing scaffold having two or more solvent exposed loops containing a different CDR from a parent antibody inserted into each of the loops and exhibiting selective binding activity toward a ligand bound by the parent antibody. Non-immunoglobulin protein scaffolds have been proposed for obtaining proteins with novel binding properties.

Anti- amyloid antibodies or antibody fragments thereof typically bind to human amyloid with high affinity (e.g., as determined with BIACORE), such as for example with an equilibrium binding dissociation constant (KD) for amyloid of about 15nM or less, 10 nM or less, about 5 nM or less, about 1 nM or less, about 500 pM or less, about 250 pM or less, about 100 pM or less, about 50 pM or less, or about 25 pM or less, about 10 pM or less, about 5 pM or less, about 3 pM or less about 1 pM or less, about 0.75 pM or less, or about 0.5 pM or less.

The antibodies and antibody fragments described herein can be prepared by any suitable method. Suitable methods for preparing such antibodies and antibody fragments are known in the art. The antibody or antibody fragment may be isolated or purified to any degree.

Humanisation

Humanised forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanised antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

Some or all of the CDRs of the antibodies described herein may be transferred; for example, it is possible to retain human acceptor CDRs as long as the donor CDR H3 is transferred. The members of the immunoglobulin superfamily all share a similar fold for their polypeptide chain. For example, although antibodies are highly diverse in terms of their primary sequence, comparison of sequences and crystallographic structures has revealed that, contrary to expectation, five of the six antigen binding loops of antibodies (H1 , H2, L1 , L2, L3) adopt a limited number of main-chain conformations, or canonical structures (Chothia and Lesk (1987) J. Mol. Biol., 196: 901 ; Chothia et al. (1989) Nature, 342: 877). Analysis of loop lengths and key residues has therefore enabled prediction of the mainchain conformations of H1 , H2, L1 , L2 and L3 found in the majority of human antibodies (Chothia et al. (1992) J. Mol. Biol., 227: 799; Tomlinson et al. (1995) EMBO J., 14: 4628; Williams et al. (1996) J. Mol. Biol., 264: 220). Pharmaceutical Composition

A pharmaceutical composition may comprise, in addition to the antibody, one or more pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, stabilizers, preservatives, lubricants, or other materials well known to those skilled in the art. Suitable materials will be sterile and pyrogen-free, with a suitable isotonicity and stability. Examples include sterile saline (e.g. 0.9% NaCI), water, dextrose, glycerol, ethanol or the like or combinations thereof. Such materials should be non-toxic and should not interfere with the efficacy of the active compound. The precise nature of the carrier or other material will depend on the route of administration, which may be by infusion, injection or any other suitable route, as discussed below. Suitable materials will be sterile and pyrogen free, with a suitable isotonicity and stability. Examples include sterile saline (e.g. 0.9% NaCI), water, dextrose, glycerol, ethanol or the like or combinations thereof. The composition may further contain auxiliary substances such as wetting agents, emulsifying agents, pH buffering agents or the like.

Suitable carriers, excipients, etc. can be found in standard pharmaceutical texts, for example, Remington’s Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990.

The term “pharmaceutically acceptable” as used herein pertains to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject (e.g. human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation.

In some embodiments, the antibodies may be provided in a lyophilized form for reconstitution prior to administration. For example, lyophilized reagents may be reconstituted in sterile water and mixed with saline prior to administration to a subject.

The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active compound with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active compound with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.

Formulations may be in the form of liquids, solutions, suspensions, emulsions, and the like. Optionally, other therapeutic or prophylactic agents may be included in a pharmaceutical composition or formulation.

Treatment may be any treatment and therapy, whether of a human or an animal (e.g. in veterinary applications), in which some desired therapeutic effect is achieved, for example, the inhibition or delay of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, amelioration of the condition, cure or remission (whether partial or total) of the condition, preventing, delaying, abating or arresting one or more symptoms and/or signs of the condition or prolonging survival of a subject or patient beyond that expected in the absence of treatment.

Treatment as a prophylactic measure (i.e. prophylaxis) is also included. For example, a subject susceptible to or at risk of the occurrence or re-occurrence of amyloidosis may be treated as described herein. Such treatment may prevent or delay the occurrence or reoccurrence of amyloidosis in the subject.

In particular, treatment may include inhibiting amyloid deposition, including complete amyloid deposition reversal.

Antibodies may be administered as described herein in therapeutically-effective amounts.

The term “therapeutically-effective amount" as used herein, pertains to that amount of an active compound, or a combination, material, composition or dosage form comprising an active compound, which is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit/risk ratio.

It will be appreciated that appropriate dosages of the active compounds can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects of the administration. The selected dosage level will depend on a variety of factors including, but not limited to, the route of administration, the time of administration, the rate of excretion of the active compound, other drugs, compounds, and/or materials used in combination, and the age, sex, weight, condition, general health, and prior medical history of the patient. The amount of active compounds and route of administration will ultimately be at the discretion of the physician, although generally the dosage will be to achieve concentrations of the active compound at a site of therapy without causing substantial harmful or deleterious sideeffects.

In general, a suitable dose of the active compound is in the range of about 100 pg to about 250 mg per kilogram body weight of the subject per day. Where the active compound is a salt, an ester, prodrug, or the like, the amount administered is calculated on the basis of the parent compound and so the actual weight to be used is increased proportionately.

Administration in vivo can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals). Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the physician.

Multiple doses of antibody may be administered, for example 2, 3, 4, 5 or more than 5 doses may be administered.

The pharmaceutical compositions comprising the active compounds may be formulated in suitable dosage unit formulations appropriate for the intended route of administration.

Formulations suitable for oral administration (e.g. by ingestion) may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active compound; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion; as a bolus; as an electuary; or as a paste.

A tablet may be made by conventional means, e.g., compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active compound in a free-flowing form such as a powder or granules, optionally mixed with one or more binders (e.g. povidone, gelatin, acacia, sorbitol, tragacanth, hydroxypropylmethyl cellulose); fillers or diluents (e.g. lactose, microcrystalline cellulose, calcium hydrogen phosphate); lubricants (e.g. magnesium stearate, talc, silica); disintegrants (e.g. sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose); surface-active or dispersing or wetting agents (e.g. sodium lauryl sulfate); and preservatives (e.g. methyl p-hydroxybenzoate, propyl p- hydroxybenzoate, sorbic acid). Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active compound therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach. Formulations suitable for parenteral administration (e.g. by injection, including cutaneous, subcutaneous, intramuscular, intravenous and intradermal), include aqueous and nonaqueous isotonic, pyrogen-free, sterile injection solutions which may contain anti-oxidants, buffers, preservatives, stabilizers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs. Examples of suitable isotonic vehicles for use in such formulations include Sodium Chloride Injection, Ringer’s Solution, or Lactated Ringer’s Injection. Typically, the concentration of the active compound in the solution is from about 1 ng/ml to about 10 pg/ml, for example from about 10 ng/ml to about 1 pg/ml. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. Formulations may be in the form of liposomes or other microparticulate systems which are designed to target the active compound to blood components or one or more organs.

Compositions may be prepared in the form of a concentrate for subsequent dilution, or may be in the form of divided doses ready for administration. Alternatively, the reagents may be provided separately within a kit, for mixing prior to administration to a human or animal subject.

Identity

Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By “hybridize” is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).

For example, stringent salt concentration will ordinarily be less than about 750 mM NaCI and 75 mM trisodium citrate, preferably less than about 500 mM NaCI and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCI and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, more preferably of at least about 37° C, and most preferably of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred: embodiment, hybridization will occur at 30° C in 750 mM NaCI, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37° C in 500 mM NaCI, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 .mu.g/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42° C in 250 mM NaCI, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 pg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.

For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCI and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCI and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25 °C, more preferably of at least about 42° C, and even more preferably of at least about 68 °C. In a preferred embodiment, wash steps will occur at 25° C in 30 mM NaCI, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42 °C in 15 mM NaCI, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68° C in 15 mM NaCI, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961 , 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

By “substantially identical” is meant a polypeptide or nucleic acid molecule exhibiting at least 90% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 91%, more preferably 92% or 93%, and more preferably 94%, 95%, 96%, 97%, 98% or even 99% and up to 100% identical at the amino acid level or nucleic acid to the sequence used for comparison.

Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e^-3 and e^-100 indicating a closely related sequence.

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

Modifications of the above embodiments, further embodiments and modifications thereof will be apparent to the skilled person on reading this disclosure, and as such these are within the scope of the present invention.

All documents and sequence database entries mentioned in this specification are incorporated herein by reference in their entirety for all purposes.

The invention is further described below, with reference to the following examples.

EXAMPLES Example 1

Antibody 2E5 was produced by immunisation of a SAP-/- mouse (Bickerstaff et al., Nat Med 1999 Jun;5(6):694-7) using a synthetic human ATTR amyloid fibril immunogen by a variation of the classic hybridoma approach, originally developed by Milstein and Koehler (Fig. 1A).

ATTR fibrils in PBS were mixed with the adjuvant RIBI and injected at two-weekly intervals with 50 .g ATTR in week zero, followed by 25 .g ATTR in week two and 25 .g ATTR complexed with SAP in week 4.

This approach provided a range of monoclonal antibodies, including 2E5. ELISA screening confirmed specific binding to human ATTR fibrils but not to globular human TTR, the fibril precursor protein, or to mouse TTR (Fig. 1 B). 2E5 also shows unprecedented specific binding to all the major types of human systemic amyloid (Fig. 1 C) and to experimentally induced [Bodin, K., et al., Nature, 2010. 468(7320): p. 93-97, Botto, M., et al., Nature Med., 1997. 3(8): p. 855-859] mouse AA amyloid deposits (Fig. 2A). The original 2E5 mouse monoclonal isotype was lgG2c, which is homologous to human IgG 1 and potently complement activating. This is a necessary property for in vivo antibody-mediated amyloid removal which we have demonstrated to be complement dependent [Bodin, K., et al., Nature, 2010. 468(7320): p. 93-97, Richards, D.B., et al., N. Engl. J. Med., 2015. 373(12): p. 1106-1114, Milde, R., et al., Cell Rep., 2015. 13(9): p. 1937-1948], Indeed, administration of 2E5 to mice with established systemic AA amyloidosis, produced notable amyloid clearance (Fig. 2B).

Example 2

Crystal structure data from 2E5 showed both malonate and citrate ions locating in the binding cleft of the antibody, adopting a structure which mimics the C-terminal structure of TTR polypeptides. In order to identify the epitope to which 2E5 binds, peptide truncation studies and alanine scans were preformed using an TTR peptide (99-127) previously shown to behave as an analogue for the full-length TTR protein.

Peptides were synthesised with a biotin-SGSG N-terminal tag, based on the human TTR sequence as follows:

- huTTR 99-127

- huTTR 105-115

- huTTR 113-127

- huTTR 118-127 - huTTR 123-127

- huTTR 99-127 T123A

- huTTR 99-127 N124A

- huTTR 99-127 P125A

- huTTR 99-127 K126A

- huTTR 99-127 E127A

- huTTR 99-127 amidated C-Term

A stock solution (4mg/ml) of each peptide was prepared, dissolving 2mg of peptide in 500ul of:

- 50% acetonitrile

- 10% glacial acetic acid

- 40% H2O

The peptides were diluted to 10ug/ml in PBS for mapping using a bio-layer interferometry (BLI) biosensor.

2E5-1B7 antibody (murine lgG2a) was prepared by recombinant expression, and diluted to 40ug/ml in PBS for BLI

The binding of the antibody to each of the peptides was analysed by pre-soaking the streptavidin BLI biosensors for 10min in PBS + 0.1% BSA, equilibrating the peptides and mAb to room temperature, and performing BLI measurement using a Blitz instrument (Blitz pro software), applying advanced kinetics as follows:

1. 30sec baseline (PBS)

2. 60sec Loading biotinylated peptide at 10ug/ml onto streptavidin biosensor

3. 30sec baseline (PBS)

4. 120sec Association, 2E5-1B7 at 40ug/ml

5. 120sec Dissociation in PBS

A reference run was performed using huTTR 99-127 peptide at Step 2 (above), and association with PBS only at Step 4 (above). Binding affinity data was calculated within the Blitz pro software, and the raw BLI trace data exported for plotting.

The results show that deletion of the C-terminus of the 99-127 peptide abolishes binding of the antibody to the peptide. Thus, whilst peptides 99-127, 113-127, 118-127 and 123-127 retain identical binding properties (Figure 4), the 105-115 truncation and 99-127 E127A failed to be bound by 2E5.

Moreover, amidation of the C-terminus and removal of the carboxyl group results in loss of binding, indicating that the charged nature of the acidic C-terminus is essential for binding.

Performing an alanine scan of the C-terminus (Figure 5) showed that T123A and N124A variants retained normal binding, but that binding was progressively reduced in P125A, K126A and E127A variants. Accordingly, binding of 2E5 to the TTR peptide is dependent on the presence of charged amino acids at the C-terminus of the protein fibril.

Example 3

2E5 does not compete with SAP for binding to fibrils

In order to determine whether 2E5 and SAP bind to the same epitope in amyloid fibrils, a competition assay was conducted to assay binding of 2E5 at 0.01 .g/ml to 0.5 ig coated ATTR fibrils in the presence of SAP at a varying concentration of 0.08 to 50 pg/ml, and in the presence of an irrelevant control antibody. Antibody detection was conducted through the Fc domain using anti-mouse Fc HRP. 2E5 was found to bind to fibrils in a SAP-independent manner, indicating that SAP and 2E5 do not compete for the same epitope on ATTR fibrils.

The results are shown in Figure 6.

Example 4: CDR Grafting of 2E5

Initial CDR grafting experiments produced three humanised antibodies, as illustrated in table 1 :

TABLE 1

Antibodies 1-3 were tested by ELISA to determine binding to amyloid TTR fibrils in comparison to murine 2E5. All of the CDR-grafted antibodies showed about 100 fold decrease in binding. See Figure 7 A and B.

A sequence and structural analysis of 2E5 was performed, to identify the key residues responsible for providing structure to the CDRs in the murine framework. Key residues were identified and mutated back to murine residues. 20 humanised antibodies were generated, and tested by ELISA. The results are shown in Figure 8.

Results were ranked as shown in Table 2.

The 6 most promising antibodies show strong binding to fibrils, comparable to murine 2E5 (See Figure 4). P029_Ab004, 006, 008, 016, 019 and 021 were selected for further development.

Example 5

Charge reduction in humanised antibodies

As shown in Table 3, the higher affinity humanised antibodies were associated with a high positive charge. In order to reduce this charge to a charge which is considered more suitable for pharmaceutical development, further modifications were carried out to the humanised clones, as shown in Table 4. This included removal of positively charged residues by substituting them with non-positively charged residues observed at high frequency in human antibodies. The addition of negatively charged residues was also employed to reduce overall positive charge. In addition to charge, mutations were made in isomerization sites to improve developability of the antibody in CDR-H2 (Details described in Table 3).

Antibodies P029_Ab0019 and P029_Ab0008 were chosen as the base clones for modification.

Antibody Net charge across Fv

P029_Ab004 8

P029_Ab005 0

P029_Ab006 6

P029_Ab007 5

P029_Ab008 4 P029_Ab009 5

P029_Ab010 4

P029_Ab011 3

PO29_AbO12 5

P029_Ab013 5

P029_Ab014 2

P029_Ab015 3

P029_Ab016 7

P029_Ab017 6

P029_Ab018 8

P029_Ab019 7

P029_Ab020 5

P029_Ab021 6

PO29_AbO22 2

P029_Ab023 2

Table 3

Clone VL changes VH changes

Table 4 The results of the modification procedure are shown in table 5. Net charge was reduced to or below +4 in several instances.

Net charge VL Changes from

Table 5 To determine whether the broad reactivity of 2E5 was maintained to different amyloid types following humanization, the interaction of the humanized clones to synthetic fibrils and native amyloid was determined by ELISA. This includes synthetic amyloid derived from the truncated fragment of immunoglobulin light chain (AL55-133) and mutant forms of the transthyretin protein (S52P TTR) and beta2-microglobulin (D76N Abeta2-m) as well as AA (Amyloid A) present in the spleen extract from mice. ELISA characterization confirmed that humanized 2E5 clones were able to interact with these amyloid forms in a dose-dependent manner (Fig 9).

Sequence Listing

SEQ ID No. 1

Heavy-chain variable region (VH)

Nucleotide sequence

GAGGTTCAGCTGCTGCAGTCTGGGGCAGAGCTTGTGAAGCCAGGGGCCTCAG TCAAGTTGTCCTGCACAGCTTCTGGCTTCAAGATTAAAGACTTCTATATACACTG GGTGAAACAGAGGACTGAACAGGGCCTGGACTGGATTGGAAAGATTGATCCTG AGGATGGTAAAACTAAATATGCCCCGAAATTCCAGGGCAAGGCCACTATAACAA CAGACACATCCTCCAATACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAG GACACTGCCGTCTATTACTGTGCTAGAGCCTACTATAGTAACTACAATTGGTTT GCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAG

SEQ ID No. 2

Heavy-chain variable region (VH)

Amino acid sequence

EVQLLQSGAELVKPGASVKLSCTASGFKIKDFYIHWVKQRTEQGLDWIGKIDPEDG KTKYAPKFQGKATITTDTSSNTAYLQLSSLTSEDTAVYYCARAYYSNYNWFAYWG QGTLVTVSA

SEQ ID No. 3

Light-chain variable region (VL)

Nucleotide sequence

GAAAAAGTGCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCTAGGGGAGAA GGTCACCATGAGCTGCAGGGCCAGCTCAAGTGTAAATTACATGTACTGGTACC AGGAGAAGTCAGATGCCTCCCCCAAACTATGGATTTATTACACATCCAAGTTGG

CTCCTGGAGTCCCAGCTCGCTTCAGTGGCAGTGGGTCTGGGAACTCTTATTCT

CTCACAATCAGCAGCATGGAGGGTGAAGATGCTGCCACTTATTACTGCCAGCA

GTTTACTAGTTCCCCATACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAAC

SEQ ID No. 4

Light-chain variable region (VL)

Amino acid sequence

EKVLTQSPAIMSASLGEKVTMSCRASSSVNYMYWYQEKSDASPKLWIYYTSKLAPGVPAR

FSGSGSGNSYSLTISSMEGEDAATYYCQQFTSSPYTFGGGTKLEIK

SEQ ID No. 5

Light-chain variable region (VL) alternative

Nucleotide sequence

GAAATTGTGCTCACCCAGTCTCCAACCACCATGGCTGCATCTCCCGGGGAGAA

GATCACTATCACCTGCAGTGCCAGCTCAAGTATAAGTTCCAATTACTTGCATTG

GTATCAGCAGAAGCCAGGATTCTCCCCTAAACTCTTGATTTATAGGACATCCAA

TCTGGCTTCTGGAGTCCCAGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTT

ACTCTCTCACAATTGGCACCATGGAGGCTGAAGATGTTGCCACTTACTACTGCC

AGCAGGGTAGTAGTATACCGTACACGTTCGGAGGGGGGACCAAGCTGGAAAT AAAAC

SEQ ID No. 6

Heavy Chain Variable Region CDRH1

GFKIKDFY

SEQ ID No. 7

Heavy Chain Variable Region CDRH2

IDPEDGKT SEQ ID No. 8

Heavy Chain Variable Region CDRH3

ARAYYSNYNWFAY

SEQ ID No. 9

Light Chain Variable Region CDRL1

SSVNY

SEQ ID No. 10

Light Chain Variable Region CDRL2

YTS

SEQ ID No. 11

Light Chain Variable Region CDRL3

QQFTSSPYT

SEQ ID No. 12

Heavy chain V gene, CDR-grafted antibody

EVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIY

AEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCWGQGTLVTVSS

SEQ ID No. 13

Light chain V gene, CDR Grafted Antibody

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARF

SGSGSGTDFTLTISSLEPEDFAVYYCFGQGTKLEIK

SEQ ID No. 14

Clone P029_Ab04, VL

EKVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQQKPGQAPRLLIYYTSKRATGIPARFS

GSGSGNDYTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK SEQ ID No. 15

Clone P029_Ab04, VH

EVQLVQSGAEVKKPGATVKISCKASGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTKY

APKFQGRVTITADTSTNTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

SEQ ID No. 16

Clone P029_Ab06, VL

EKVLTQSPATLSLSPGERATLSCRASSSVNYMYWYQEKPGQSPRLLIYYTSKRATGIPARF

SGSGSGNDYTLTISSLEPEDFATYYCQQFTSSPYTFGQGTKLEIK

SEQ ID No. 17

Clone P029_Ab06, VH

EVQLVQSGAEVKKPGATVKISCKASGFKIKDFYIHWVQQRTEQGLEWMGKIDPEDGKTKYA

PKFQGRVTITTDTSTNTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

SEQ ID No. 18

Clone P029_Ab08, VL

EIVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQQKPGQAPRLLIYYTSNRATGIPARFS

GSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

SEQ ID No. 19

Clone P029_Ab08, VH

EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTKY

AEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

SEQ ID No. 20

Clone P029_Ab16, VL

EKVLTQSPATLSLSPGERATLSCRASSSVNYMYWYQEKPGQSPRLLIYYTSKRATGIPARF

SGSGSGNDYTLTISSLEPEDFATYYCQQFTSSPYTFGQGTKLEIK

SEQ ID No. 21

Clone P029_Ab16, VH

EVQLVQSGAEVKKPGATVKISCKASGFKIKDFYIHWVQQAPGKGLEWMGKIDPEDGKTKYA

PKFQGRVTITTDTSTNTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS SEQ ID No. 22

Clone P029_Ab19, VL

EKVLTQSPATLSLSPGERATLSCRASSSVNYMYWYQQKPGQAPRLLIYYTSKRATGIPARF SGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

SEQ ID No. 23

Clone P029_Ab19, VH

EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTKY

APKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

SEQ ID No. 24

Clone P029_Ab21, VL

EIVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQQKPGQAPRLLIYYTSNRATGIPARFS

GSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

SEQ ID No. 25

Clone P029_Ab21, VH

EVQLVQSGAEVKKPGATVKISCKASGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTKY

APKFQGRVTITADTSTNTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

SEQ ID No. 26

Clone P029_Ab25, VL

EIVLTQSPATLSLSPGERATLSCRASSSVNYMYWYQQKPGQAPRLLIYYTSKRATGIPARFS

GSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

SEQ ID No. 27

Clone P029_Ab25, VH

EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTKY

APKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS SEQ ID No. 28

Clone P029_Ab26, VL

EKVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQQKPGQAPRLLIYYTSKRATGIPARFS

GSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

SEQ ID No. 29

Clone P029_Ab26, VH

EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTKY

APKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

SEQ ID No. 30

Clone P029_Ab27, VL

EKVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQQKPGQAPRLLIYYTSNRATGIPARFS

GSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

SEQ ID No. 31

Clone P029_Ab27, VH

EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTKY

APKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

SEQ ID No. 32

Clone P029_Ab28, VL

EIVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQQKPGQAPRLLIYYTSKRATGIPARFS

GSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

SEQ ID No. 33

Clone P029_Ab28, VH

EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTKY

APKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS SEQ ID No. 34

Clone P029_Ab29, VL

EIVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQQKPGQAPRLLIYYTSKRATGIPARFS

GSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

SEQ ID No. 35

Clone P029_Ab29, VH

EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTIY

AEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

SEQ ID No. 36

Clone P029_Ab30, VL

EIVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQQKPGQAPRLLIYYTSKRATGIPARFS

GSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

SEQ ID No. 37

Clone P029_Ab30, VH

EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGLIDPEDGKTKY

AEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

SEQ ID No. 38

Clone P029_Ab31, VL

EIVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQQKPGQAPRLLIYYTSNRATGIPARFS

GSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

SEQ ID No. 39

Clone P029_Ab31, VH EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTKY

APKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

SEQ ID No. 40

Clone P029_Ab32, VH

EIVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQQKPGQAPRLLIYYTSNRATGIPARFS

GSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

SEQ ID No. 41

Clone P029_Ab32, VH

EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTIY

APKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

SEQ ID No. 42

Clone P029_Ab33, VL

EIVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQQKPGQAPRLLIYYTSNRATGIPARFS

GSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

SEQ ID No. 43

Clone P029_Ab33, VH

EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGLIDPEDGKTKY

APKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

SEQ ID No. 44

Clone P029_Ab34, VL

EIVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQQKPGQAPRLLIYYTSNRATGIPARFS

GSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

SEQ ID No. 45 Clone P029_Ab34, VH

EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTIY

AEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

SEQ ID No. 46

Clone P029_Ab35, VL

EIVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQQKPGQAPRLLIYYTSNRATGIPARFS

GSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

SEQ ID No. 47

Clone P029_Ab35, VH

EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGLIDPEDGKTKY

AEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

SEQ ID No. 48

Clone P029_Ab36, VL

EKVLTQSPATLSLSPGERATLSCRASSSVNYMYWYQQKPGQAPRLLIYYTSKRATGIPARF

SGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

SEQ ID No. 49

Clone P029_Ab36, VH

EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTIY

APKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

SEQ ID No. 50

Clone P029_Ab37, VL

EKVLTQSPATLSLSPGERATLSCRASSSVNYMYWYQQKPGQAPRLLIYYTSKRATGIPARF

SGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK SEQ ID No. 51

Clone P029_Ab37, VH

EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGLIDPEDGKTKY

APKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

SEQ ID No. 52

Clone P029_Ab38, VL

EKVLTQSPATLSLSPGERATLSCRASSSVNYMYWYQQKPGQAPRLLIYYTSKRATGIPARF

SGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

SEQ ID No. 53

Clone P029_Ab38, VH

EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTKY

AEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

SEQ ID No. 54

Clone P029_Ab39, VL

EKVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQEKPGQSPRLLIYYTSNRATGIPARFS

GSGSGTDFTLTISSLEPEDFATYYCQQFTSSPYTFGQGTKLEIK

SEQ ID No. 55

Clone P029_Ab39, VH

EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTKY

APKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

SEQ ID No. 56

Clone P029_Ab40, VL EKVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQEKPGQAPRLLIYYTSNRATGIPARFS

GSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

SEQ ID No. 57

Clone P029_Ab41, VH

EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTKY

APKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

SEQ ID No. 58

Clone P029_Ab42, VL

EKVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQEKPGQAPRLLIYYTSNRATGIPARFS

GSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

SEQ ID No. 59

Clone P029_Ab42, VH

EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTIY

APKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

SEQ ID No. 60

Clone P029_Ab43, VL

EKVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQEKPGQAPRLLIYYTSNRATGIPARFS

GSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

SEQ ID No. 61

Clone P029_Ab43, VH

EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGLIDPEDGKTKY

APKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS SEQ ID No. 62

Clone P029_Ab44, VL

EKVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQEKPGQAPRLLIYYTSNRATGIPARFS

GSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

SEQ ID No. 63

Clone P029_Ab44, VH

EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTKY

AEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

SEQ ID No. 64

Clone P029_Ab45, VL

EKVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQEKPGQAPRLLIYYTSNRATGIPARFS

GSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

SEQ ID No. 65

Clone P029_Ab45, VH

EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTIY

AEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

SEQ ID No. 66

Clone P029_Ab46, VL

EKVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQEKPGQAPRLLIYYTSNRATGIPARFS

GSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

SEQ ID No. 67

Clone P029_Ab46, VH EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIEPEDGKTKY

APKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

SEQ ID No. 68

Clone P029_Ab47, VL

EKVLTQSPATLSLSPGERATLSCRASSSVNYMYWYQQKPGQAPRLLIYYTSKRATGIPARF

SGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

SEQ ID No. 69

Clone P029_Ab47, VH

EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIDPEEGKTKY

APKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

SEQ ID No. 70

Clone P029_Ab48, VL

EKVLTQSPATLSLSPGERATLSCRASSSVNYMYWYQQKPGQAPRLLIYYTSKRATGIPARF

SGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

SEQ ID No. 71

Clone P029_Ab48, VH

EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTKY

APKFQGRVTITADTSTDTAYMELSSLTSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

Claims

Claims:

1. An isolated monoclonal antibody or antigen binding portion thereof which specifically binds to at least 3 different types of amyloid fibrils, selected from ALK, ALA, ATTR wild type, ATTR variant, AA, AApoAl, ALys, AP2m and AFib amyloid fibrils.

2. An isolated monoclonal antibody or antigen binding portion thereof according to claim 1 , which binds to at least 4, 5, 6, 7, 8 or 9 different types of amyloid fibrils, selected from ALK, ALA, ATTR wild type, ATTR variant, AA, AApoAl, ALys, AP2m and AFib amyloid fibrils.

3. An isolated monoclonal antibody or antigen binding portion thereof which specifically binds to amyloid fibrils and wherein each of CDRH1 , CDRH2 and CDRH3 in the variable domain of the heavy chain has

(a) a sequence identity of at least 90% with SEQ ID No 6, SEQ ID No. 7 and SEQ ID NO. 8 respectively; or

(b) optionally a single amino acid change with respect to SEQ ID No 6, SEQ ID No. 7 and SEQ ID NO. 8 respectively.

4. An isolated monoclonal antibody or antigen binding portion thereof which specifically binds to amyloid fibrils and wherein each of CDRL1, CDRL2 and CDRL3 in the variable domain of the light chain has

(a) a sequence identity of at least 90% with SEQ ID No 9, SEQ ID No. 10 and SEQ ID NO. 11 respectively; or

(b) optionally a single amino acid change with respect to SEQ ID No 9, SEQ ID No. 10 and SEQ ID NO. 11 respectively.

5. An isolated monoclonal antibody or antigen binding portion thereof which specifically binds to amyloid fibrils wherein

(a) each of CDRH1 , CDRH2 and CDRH3 in the variable domain of the heavy chain has a sequence identity of at least 90% with SEQ ID No 6, SEQ ID No. 7 and SEQ ID NO. 8 respectively and CDRL1, CDRL2 and CDRL3 in the variable domain of the light chain have a sequence identity of at least 90% with SEQ ID No 9, SEQ ID No. 10 and SEQ ID NO. 11 respectively; or

(b) each of CDRH1 , CDRH2 and CDRH3 in the variable domain of the heavy chain optionally has a single amino acid change with respect to SEQ ID No 6, SEQ ID No. 7 and SEQ ID NO. 8 respectively, and CDRL1, CDRL2 and CDRL3 in the variable domain of the light chain optionally have a single amino acid change with respect to SEQ ID No 9, SEQ ID No. 10 and SEQ ID NO. 11 respectively.

6. An isolated monoclonal antibody or antigen binding portion thereof which specifically binds to amyloid fibrils wherein the heavy chain variable region has a sequence identity of at least 70% with SEQ ID No. 2.

7. An isolated monoclonal antibody or antigen binding portion thereof which specifically binds to amyloid fibrils wherein the light chain variable region has a sequence identity of at least 70% with SEQ ID No. 4.

8. An isolated monoclonal antibody or antigen binding portion thereof which specifically binds to amyloid fibrils wherein the heavy chain variable region has a sequence identity of at least 70% with SEQ ID No. 2 and the light chain variable region has a sequence identity of at least 70% with SEQ ID No. 4.

9. An isolated monoclonal antibody or antigen-binding fragment thereof according to any preceding claim, which

(a) binds to an epitope present in the amyloid fibril but does not bind to the native peptide from which the fibril is formed; and/or

(b) does not bind to Ap fibrils present in the CNS and/or the brain in patients with Alzheimer’s disease; and/or

(c) does not compete with SAP for binding to fibrils.

10. An isolated monoclonal antibody or antigen-binding fragment thereof according to claim 9, wherein the epitope is present in at least 3 amyloid fibrils selected from ALK, ALA, ATTR wild type, ATTR variant, AA, AApoAl, ALys, AP2m and AFib amyloid fibrils.

11. An isolated monoclonal antibody or antigen-binding fragment thereof according to claim 9 or claim 10, wherein the epitope comprises the C-terminus of the fibril.

12. An isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 9 to 11 , wherein the epitope comprises at least one charged amino acid.

13. An isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 9 to 12, wherein the epitope comprises a C-terminal carboxyl group.

14. An isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 11 to 13, wherein a citrate ion and/or a malonate ion can be located within the antibody binding site when examined by x-ray crystallography.

15. The isolated monoclonal antibody or antigen binding portion thereof according to any preceding claim wherein the antibody is selected from an IgG, IgA, or an antigen binding antibody fragment selected from an antibody single variable domain polypeptide, dAb, FAb, F(ab’)2, an scFv, an Fv, a VHH domain (such as a Nanobody® or other camelized immunoglobulin domain) or a disulfide-bonded Fv, a human antibody, a chimeric antibody preferably containing a human variable region, a humanized antibody, a bispecific antibody or a single chain antibody.

16. The isolated monoclonal antibody according to any preceding claim wherein the Fc region of the antibody is derived from either the mouse lgG2 or human lgG1 isotype.

17. The isolated monoclonal antibody according to claim 15 or claim 16, which is a humanised antibody.

18. A humanised monoclonal antibody or antigen binding portion thereof according to claim 17, wherein CDRH1 , CDRH2 and CDRH3 in the variable domain of the heavy chain have a sequence identity of at least 90% with SEQ ID No 6, SEQ ID No. 7 and SEQ ID No. 8 respectively.

19. A humanised monoclonal antibody or antigen binding portion thereof according to claim 17 or claim 18, wherein CDRL1, CDRL2 and CDRL3 in the variable domain of the light chain have a sequence identity of at least 90% with SEQ ID No 9, SEQ ID No. 10 and SEQ ID No. 11 respectively.

20. A humanised monoclonal antibody or antigen binding portion thereof according to claim 19 or claim 20, wherein CDRH1 , CDRH2 and CDRH3 in the variable domain of the heavy chain have a sequence identity of at least 90% with SEQ ID No 6, SEQ ID No. 7 and SEQ ID NO. 8 respectively and CDRL1, CDRL2 and CDRL3 in the variable domain of the light chain have a sequence identity of at least 90% with SEQ ID No 9, SEQ ID No. 10 and SEQ ID No. 11 respectively.

21. A humanised monoclonal antibody according to any one of claims 19 to 21 , wherein the human framework regions are derived from antibody genes selected from SEQ ID Nos. 12 and 13.

22. A humanised monoclonal antibody according to any one of claims 18 to 21 , wherein the framework residues mutated to match murine residues include resides selected from VL residues I2, L39, A40, Q44, A49, V101 , N66, T85 and F87, and residues selected from VH residues M39, A80, L55, I66, V25, D85, E69, A45, P46, G47 and K48.

23. A humanised monoclonal antibody according to any one of claims 18 to 22, wherein the VH region comprises the mutations L55K and I66K.

24. A humanised monoclonal antibody according to any one of claims 18 to 23, which has a combination of light and heavy chain framework mutations comprising light chain framework mutations consisting of I2K, A40Y, N66K, T85N and F87Y and heavy chain framework mutations consisting of L55K, I66K, V25A, D85N and E69P.

25. A humanised monoclonal antibody according to any one of claims 1 to 8, which has a combination of light and heavy chain framework mutations comprising light chain framework mutations consisting of I2K, L39M, A40Y, N66K, T85N and F87Y and heavy chain framework mutations consisting of M39I, A80T, L55K, I66K, E69P, A45R, P46T, G47E and K48Q.

26. A humanised monoclonal antibody according to any one of claims 18 to 23, which has a combination of light and heavy chain framework mutations comprising light chain framework mutations consisting of I2K, A40Y, N66K, T85N and F87Y and heavy chain framework mutations consisting of M39I, A80T, L55K, I66K, V25A, D85N and E69P.

27. A humanised monoclonal antibody according to any one of claims 18 to 23 which has a combination of light and heavy chain framework mutations comprising light chain framework mutations consisting of I2K, L39M, A40Y and N66K and heavy chain framework mutations consisting of L55K, I66K, and E69P.

28. A humanised monoclonal antibody according to any one of claims 18 to 24, which has a combination of light and heavy chain framework mutations comprising light chain framework mutations consisting of A40Y and heavy chain framework mutations consisting of L55K, I66K, V25A, D85N and E69P.

29. A humanised monoclonal antibody according to any one of claims 18 to 23, which has a combination of light and heavy chain framework mutations comprising light chain framework mutations consisting of A40Y and heavy chain framework mutations consisting of L55K and I66K.

30. A humanised monoclonal antibody according to claim 17, wherein the VL domain has a sequence identity of at least 90% with SEQ ID No. 14 and the VH domain has a sequence identity of at least 90% with SEQ ID No. 15.

31. A humanised monoclonal antibody according to claim 17, wherein the VL domain has a sequence identity of at least 90% with SEQ ID No. 16 and the VH domain has a sequence identity of at least 90% with SEQ ID No. 17.

32. A humanised monoclonal antibody according to claim 17, wherein the VL domain has a sequence identity of at least 90% with SEQ ID No. 18 and the VH domain has a sequence identity of at least 90% with SEQ ID No. 19.

33. A humanised monoclonal antibody according to claim 17, wherein the VL domain has a sequence identity of at least 90% with SEQ ID No. 20 and the VH domain has a sequence identity of at least 90% with SEQ ID No. 21.

34. A humanised monoclonal antibody according to claim 17, wherein the VL domain has a sequence identity of at least 90% with SEQ ID No. 22 and the VH domain has a sequence identity of at least 90% with SEQ ID No. 23.

35. A humanised monoclonal antibody according to claim 17, wherein the VL domain has a sequence identity of at least 90% with SEQ ID No. 24 and the VH domain has a sequence identity of at least 90% with SEQ ID No. 25.

36. A humanised monoclonal antibody according to claim 17, wherein the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 26 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 27.

37. A humanised monoclonal antibody according to claim 17, wherein the VL domain of the isolated humanised monoclonal antibody has a sequence identity of at least 90% with SEQ ID No. 28 and the VH domain has a sequence identity of at least 90% with SEQ ID No. 29.

38. A humanised monoclonal antibody according to claim 17, wherein the VL domain of the isolated humanised monoclonal antibody has a sequence identity of at least 90% with SEQ ID No. 30 and the VH domain has a sequence identity of at least 90% with SEQ ID No. 31.

39. A humanised monoclonal antibody according to claim 17, wherein the VL domain of the isolated humanised monoclonal antibody has a sequence identity of at least 90% with SEQ ID No. 32 and the VH domain has a sequence identity of at least 90% with SEQ ID No. 33.

40. A humanised monoclonal antibody according to claim 17, wherein the VL domain of the isolated humanised monoclonal antibody has a sequence identity of at least 90% with SEQ ID No. 34 and the VH domain has a sequence identity of at least 90% with SEQ ID No.

41 . A humanised monoclonal antibody according to claim 17, wherein the VL domain of the isolated humanised monoclonal antibody has a sequence identity of at least 90% with SEQ ID No. 36 and the VH domain has a sequence identity of at least 90% with SEQ ID No.

37.

42. A humanised monoclonal antibody according to claim 17, wherein the VL domain of the isolated humanised monoclonal antibody has a sequence identity of at least 90% with SEQ ID No. 38 and the VH domain has a sequence identity of at least 90% with SEQ ID No.

39.

43. A humanised monoclonal antibody according to claim 17, wherein the VL domain of the isolated humanised monoclonal antibody has a sequence identity of at least 90% with SEQ ID No. 40 and the VH domain has a sequence identity of at least 90% with SEQ ID No.

41.

44. A humanised monoclonal antibody according to claim 17, wherein the VL domain of the isolated humanised monoclonal antibody has a sequence identity of at least 90% with SEQ ID No. 42 and the VH domain has a sequence identity of at least 90% with SEQ ID No.

43.

45. A humanised monoclonal antibody according to claim 17, wherein the VL domain of the isolated humanised monoclonal antibody has a sequence identity of at least 90% with SEQ ID No. 44 and the VH domain has a sequence identity of at least 90% with SEQ ID No.

45.

46. A humanised monoclonal antibody according to claim 17, wherein the VL domain of the isolated humanised monoclonal antibody has a sequence identity of at least 90% with SEQ ID No. 46 and the VH domain has a sequence identity of at least 90% with SEQ ID No.

47.

47. A humanised monoclonal antibody according to claim 17, wherein the VL domain of the isolated humanised monoclonal antibody has a sequence identity of at least 90% with SEQ ID No. 48 and the VH domain has a sequence identity of at least 90% with SEQ ID No.

49.

48. A humanised monoclonal antibody according to claim 17, wherein the VL domain of the isolated humanised monoclonal antibody has a sequence identity of at least 90% with SEQ ID No. 50 and the VH domain has a sequence identity of at least 90% with SEQ ID No.

51.

49. A humanised monoclonal antibody according to claim 17, wherein the VL domain of the isolated humanised monoclonal antibody has a sequence identity of at least 90% with SEQ ID No. 52 and the VH domain has a sequence identity of at least 90% with SEQ ID No.

53.

50. A humanised monoclonal antibody according to claim 17, wherein the VL domain of the isolated humanised monoclonal antibody has a sequence identity of at least 90% with SEQ ID No. 54 and the VH domain has a sequence identity of at least 90% with SEQ ID No.

55.

51 . A humanised monoclonal antibody according to claim 17, wherein the VL domain of the isolated humanised monoclonal antibody has a sequence identity of at least 90% with SEQ ID No. 56 and the VH domain has a sequence identity of at least 90% with SEQ ID No.

57.

52. A humanised monoclonal antibody according to claim 17, wherein the VL domain of the isolated humanised monoclonal antibody has a sequence identity of at least 90% with SEQ ID No. 58 and the VH domain has a sequence identity of at least 90% with SEQ ID No.

59.

53. A humanised monoclonal antibody according to claim 17, wherein the VL domain of the isolated humanised monoclonal antibody has a sequence identity of at least 90% with SEQ ID No. 60 and the VH domain has a sequence identity of at least 90% with SEQ ID No.

61.

54. A humanised monoclonal antibody according to claim 17, wherein the VL domain of the isolated humanised monoclonal antibody has a sequence identity of at least 90% with SEQ ID No. 62 and the VH domain has a sequence identity of at least 90% with SEQ ID No.

63.

55. A humanised monoclonal antibody according to claim 17, wherein the VL domain of the isolated humanised monoclonal antibody has a sequence identity of at least 90% with SEQ ID No. 64 and the VH domain has a sequence identity of at least 90% with SEQ ID No.

65.

56. A humanised monoclonal antibody according to claim 17, wherein the VL domain of the isolated humanised monoclonal antibody has a sequence identity of at least 90% with SEQ ID No. 66 and the VH domain has a sequence identity of at least 90% with SEQ ID No.

67.

57. A humanised monoclonal antibody according to claim 17, wherein the VL domain of the isolated humanised monoclonal antibody has a sequence identity of at least 90% with SEQ ID No. 68 and the VH domain has a sequence identity of at least 90% with SEQ ID No. 69.

58. A humanised monoclonal antibody according to claim 17, wherein the VL domain of the isolated humanised monoclonal antibody has a sequence identity of at least 90% with SEQ ID No. 70 and the VH domain has a sequence identity of at least 90% with SEQ ID No. 71.

59. A humanised monoclonal antibody or antigen-binding fragment thereof according to any one of claims 18 to 58, which binds to an epitope on amyloid fibrils, wherein the epitope comprises the C-terminus of the fibril.

60. A humanised monoclonal antibody or antigen-binding fragment thereof according to claim 59, wherein the epitope comprises at least one charged amino acid.

61 . A humanised monoclonal antibody or antigen-binding fragment thereof according to claim 59 or claim 60, wherein the epitope comprises a C-terminal carboxyl group.

62. A humanised monoclonal antibody or antigen-binding fragment thereof according to any one of claims 59 to 61 , wherein a citrate ion and/or a malonate ion can be located in the antibody binding site when examined by x-ray crystallography.

63. The humanised monoclonal antibody or antigen binding portion thereof according to any one of claims 17 to 62, wherein the antibody is selected from an IgG, IgA, or an antigen binding antibody fragment selected from an antibody single variable domain polypeptide, dAb, FAb, F(ab’)2, an scFv, an Fv, a HH domain (such as a Nanobody® or other camelized immunoglobulin domain) or a disulfide-bonded Fv.

64. The humanised monoclonal antibody according to any one of claims 17 to 63, wherein the Fc region of the antibody is derived from a human lgG1 isotype.

65. The humanised monoclonal antibody or antigen binding portion thereof according to claim 66, wherein the antibody is of hlgG 1 isotype.

66. An isolated monoclonal antibody or a humanised antibody or antigen binding portion thereof according to any preceding claim, which also effectively promotes regression of systemic murine AA amyloid deposits when administered parenterally to mice with experimentally induced systemic AA amyloidosis.

67. An isolated monoclonal antibody or a humanised antibody or antigen binding portion according to any preceding claim, the in vivo efficacy of which is complement dependent.

68. An isolated monoclonal antibody or a humanised antibody or antigen binding portion thereof according to any preceding claim, the in vivo efficacy of which is Fey receptor binding dependent.

69. An isolated monoclonal antibody or a humanised antibody according to any preceding claim, for use in the treatment of disease.

70. An isolated monoclonal antibody or a humanised antibody for use according to claim 22, wherein the disease is amyloidosis.

71. A method for removing amyloid deposits from the tissues in a subject suffering from systemic amyloidosis, comprising administering at least one isolated monoclonal antibody or a humanised antibody according to any one of claims 1 to 70.

72. A pharmaceutical composition comprising an isolated monoclonal antibody or a humanised antibody according to any one of claims 1 to 70 for use in the treatment of systemic amyloidosis.