Fibroblast antibodies

Abstract

The present invention relates to an isolated monoclonal antibody or an antigen-binding portion thereof, which specifically binds to amyloid fibrils and effectively promotes removal of extracellular amyloid deposits from in vivo tissues, as well as a humanized antibody having the same characteristics.

Description

[0001] The present invention provides novel antibodies with broad specificity against different types of amyloid fibrils causing all major forms of human systemic amyloidosis, humanized monoclonal antibodies targeting multiple amyloid fibril types, and methods for using such antibodies and uses of the antibodies in therapeutic applications. BACKGROUND

[0002] Amyloidosis is a serious disease caused by extracellular deposition of insoluble abnormal fibrils caused by aggregation of misfolded self-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 humans, each associated with a clinically distinct disease (https: / / doi.org / 10.1080 / 13506129.2020.1835263). Systemic amyloidosis, in which amyloid deposits in internal organs, blood vessel walls and connective tissue, is usually fatal and is the cause of about one in a thousand deaths in developed countries. Cardiac transthyretin amyloidosis, known as ATTR, is a fatal disease that occurs predominantly in older men and has recently been found to have a prevalence of 3-5% in those over 75 years of age. Diagnosis of all forms of amyloidosis is challenging in the clinic and is often delayed, with the result that outcomes are often poor, compounded by the limited efficacy and significant toxicity of many existing treatments. Amyloidosis is therefore a major unmet medical need.

[0003] Amyloidosis can be acquired as a complication of a pre-existing primary disease that produces an intrinsic amyloidogenic abnormal protein or greatly increases exposure to a normal but potentially amyloidogenic protein, or in the elderly is caused by normal expression of a wild-type transthyretin protein that is inherently amyloidogenic. Hereditary amyloidosis is caused by a mutant gene that encodes a variant protein that happens to be amyloidogenic. Amyloid deposits can be localized, confined to a particular organ or tissue, or can be systemic, with amyloid deposits throughout the body (except in the brain). Systemic amyloidosis is overwhelmingly AL or ATTR type; the various hereditary types are rare. Systemic AA amyloidosis is a complication of chronic infection and other inflammatory conditions, and is currently rare in developed countries but remains more prevalent elsewhere.

[0004] Tissue and organ damage manifesting as clinical disease in amyloidosis is directly caused by the persistent accumulation of extracellular fibrillar amyloid deposits. They disrupt the structure of affected tissues, and thus the function of said affected tissues. For reasons that are not understood, the normal mechanisms for clearing debris from the extracellular space remove amyloid deposits very slowly, if at all. Thus, existing management of amyloidosis involves (a) supportive therapy to maintain the function of damaged organs, up to and including multi-organ transplantation, and (b) measures to reduce the abundance of the corresponding amyloid fibril precursor protein and / or to stabilize its native fold to prevent fibril formation. The goal is to reduce or stop the persistent accumulation of amyloid, and it is hoped that then, in some patients, amyloid will slowly spontaneously but ultimately clinically beneficially resolve. However, the range and efficacy of interventions aimed at preventing amyloid formation is limited, and for many different types of amyloidosis and for many patients, no specific treatment exists.

[0005] A direct approach to remove amyloid deposits in the body is known in the art, in particular targeting serum amyloid P component (SAP), an invariant, normal, constitutive plasma protein, ubiquitously present in all human amyloid deposits due to its strong specific calcium-dependent binding to all types of amyloid fibrils [Pepys, M.B., Front. Immunol., 2018. 9: p. 2382.]. SAP bound to amyloid fibrils contributes to their persistence in the body [Tennent, G.A., et al., Proc. Natl. Acad. Sci. USA, 1995. 92(10): p. 4299-4303]. Therefore, the small molecule drug miridesap was 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 completely depletes circulating SAP, it fails to remove all SAP from amyloid deposits and cannot accelerate amyloid clearance [Gillmore, J.D., et al., Br. J. Haematol., 2010. 148(5): p. 760-767]. However, the depletion of circulating SAP by miridesap enabled the safe and effective administration of anti-SAP antibodies to target all types of amyloid deposits [Bodin, K., et al., Nature, 2010. 468(7320): p. 93-97]. The obligate therapeutic partnership between miridesap and anti-SAP antibodies 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 therapy has not progressed so far to a licensed drug.

[0006] SAPs that bind to amyloid fibrils of all types are an attractive target antigen, but the practical goal of antibody immunotherapy is to remove the amyloid fibrils themselves. Unfortunately, amyloid fibrils have been considered to be poorly immunogenic. Patients with amyloidosis almost never produce specific anti-amyloid fibril antibodies, and experimental animals, even when strongly immunized with heterologous fibrils, respond poorly, if at all. On the other hand, all types of amyloid fibrils share very similar morphologies, ultrastructures, and protein folding, especially the cross-beta core structure common to all types of amyloid fibrils, regardless of their completely unrelated protein sequences [Sunde, M. et al., J. Mol. Biol., 1997. 273: p. 729-739]. Thus, we and others have hypothesized that amyloid fibrils can share underlying epitope structures, but despite claims of some putative cross-reactive antibodies, there have been no reports of truly broad-spectrum anti-amyloid fibril antibodies in the prior art. SUMMARY

[0007] In a first aspect, a monoclonal antibody or antigen binding portion thereof is provided that specifically binds to amyloid fibrils with broad antigenic fibril specificity. In one embodiment, the antibody of the present invention is capable of binding to at least 9 different types of amyloid fibrils, including, for example, ALK, ALA, ATTR wild-type, ATTR variant, AA, AApoAI, ALys, Aβ2m, and AFib amyloid fibrils. The antibody of the present invention successfully binds to fibrils that cause or are involved in systemic amyloidosis. The antibody does not bind to Aβ fibrils that are present in the CNS, and particularly in the brain of patients with Alzheimer's disease.

[0008] The antibody according to the present invention is preferably an antibody or antigen binding portion thereof, wherein the CDRs in the variable domain of the heavy chain have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% sequence identity with SEQ ID No. 6, SEQ ID No. 7, and SEQ ID NO. 8.

[0009] In embodiments, the antibody according to the present invention is preferably an antibody or antigen binding portion thereof, wherein the CDRs in the variable domain of the light chain have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% sequence identity with SEQ ID No. 9, SEQ ID No. 10, and SEQ ID NO. 11.

[0010] In an embodiment, the antibody according to the application is preferably an antibody or an antigen binding portion thereof, wherein the CDRs in the variable domain of the alternative light chain have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% sequence identity with SEQ ID No. 12, SEQ ID No. 13, and SEQ ID NO. 14.

[0011] In one embodiment, the antibody according to the application is preferably an antibody or an antigen binding portion thereof, wherein the CDRs in the variable domain of the light chain have the sequence of SEQ ID No. 9, SEQ ID No. 10, and SEQ ID NO. 11, optionally comprising one amino acid change.

[0012] In one embodiment, the antibody according to this aspect of the application can comprise heavy chain CDRs having SEQ ID No. 6 to 8 and light chain CDRs having SEQ ID No. 9 to 11.

[0013] In one embodiment, the antibody according to this aspect of the application can comprise heavy chain CDRs having SEQ ID No. 6 to 8 and light chain CDRs having SEQ ID No. 12 to 14.

[0014] In one aspect, the antibody of the application comprises a heavy chain variable region having 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% sequence identity with SEQ ID No. 2.

[0015] In one aspect, the antibody of the application comprises a light chain variable region having 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% sequence identity with SEQ ID No. 4.

[0016] In one aspect, the antibody of the application comprises a heavy chain variable region having 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% sequence identity to SEQ ID No. 5.

[0017] In embodiments, the antibody is a human isotope IgGl or murine isotype IgG2.

[0018] In the above aspects of the application, the antibody can be selected from a human antibody, a chimeric antibody comprising a human variable region, a humanized antibody, a bispecific antibody or a single chain antibody, and antigen-binding fragments thereof.

[0019] In embodiments, the antibody of the application is specific for a conformational epitope commonly present on amyloid fibrils. Thus, the antibody is capable of binding to the amyloid specific epitope independent of the linear polypeptide structure of the fibril.

[0020] 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 the group consisting of ALK, AL, ATTR wild type, ATTR variant, AA, AApoAI, ALys, A 2m and AFib amyloid fibrils.

[0021] In embodiments, the epitope is formed at the C-terminus of the protein comprising the fibril. 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 a charged side chain include aspartic acid, histidine, glutamic acid, lysine and arginine.

[0022] In embodiments, the conformational epitope comprises a C-terminal carboxyl group, preferably a free C-terminal carboxyl group.

[0023] In embodiments, the conformational epitope can be mimicked by both malonate and citrate ions.

[0024] In a further aspect, the application provides a method for producing an antibody according to the first aspect of the application, the method comprising immunizing a mammal in which the SAP gene has been deleted.

[0025] In one embodiment, SAP knockout mice, which do not express the murine SAP protein, are immunized with human synthetic ATTR fibril material.

[0026] In another embodiment, SAP knockout mice, which do not express the murine SAP protein, are immunized with human synthetic ATTR fibril material coated with human SAP.

[0027] The humanized antibodies of the present application successfully bind to fibrils that cause or are involved in systemic amyloidosis. The humanized antibodies do not bind to Aβ fibrils that are present in the CNS, and in particular in the brain of patients suffering from Alzheimer's disease.

[0028] For example, the humanized monoclonal antibody binds to at least ALκ, ATTR wild type and ATTR variant fibrils.

[0029] In another embodiment, the humanized monoclonal antibody binds to at least 4, 5, 6, 7, 8 or 9 amyloid fibrils selected from the group consisting of ALκ, ALλ, ATTR wild type, ATTR variant, AA, AApoAI, ALys, Aβ2m and AFib amyloid fibrils.

[0030] In embodiments, the humanized antibodies of the present application do not compete with SAP, preferably human SAP, for binding to amyloid fibrils. Thus, the epitope bound by the antibodies of the present application is not identical to the epitope bound by SAP.

[0031] The humanized monoclonal antibodies of the present application bind to fibrils that cause or are involved in systemic amyloidosis, but do not bind to the soluble native peptides from which the fibrils are derived.

[0032] In embodiments, the epitope bound by the antibodies of the present application comprises the C-terminus of the protein that constitutes the fibril. In embodiments, the epitope comprises at least one charged amino acid, preferably at least 2 charged amino acids, which comprise a charged side chain. Amino acids with a charged side chain include aspartic acid, glutamic acid, lysine and arginine.

[0033] In embodiments, the epitope comprises a C-terminal carboxyl group, preferably a free C-terminal carboxyl group.

[0034] In embodiments, citrate ions and / or malonate ions can be located in the binding site of the antibodies according to the present application when examined by x-ray crystallography.

[0035] The humanized monoclonal antibody or antigen-binding portion thereof can have CDRH1, CDRH2 and CDRH3 in the variable domain of the heavy chain having at least 90% sequence identity to SEQ ID No. 6, SEQ ID No. 7 and SEQ ID No. 8, respectively.

[0036] Additionally or alternatively, the humanized monoclonal antibody or antigen-binding portion thereof can have CDRL1, CDRL2 and CDRL3 in the variable domain of the light chain having at least 90% sequence identity to SEQ ID No. 9, SEQ ID No. 10 and SEQ ID No. 11, respectively.

[0037] In another embodiment, the humanized monoclonal antibody or antigen-binding portion thereof can have CDRH1, CDRH2 and CDRH3 in the variable domain of the heavy chain having at least 90% sequence identity to SEQ ID No. 6, SEQ ID No. 7 and SEQ ID No. 8, respectively. Additionally, the humanized monoclonal antibody or antigen-binding portion thereof can have CDRL1, CDRL2 and CDRL3 in the variable domain of the light chain having at least 90% sequence identity to SEQ ID No. 9, SEQ ID No. 10 and SEQ ID No. 11, respectively.

[0038] In another embodiment, the antibody according to the application can have one optimal amino acid change in one or more CDRs compared to the SEQ ID of the CDRs as shown herein.

[0039] In another optional embodiment, the humanized monoclonal antibody can have human framework regions derived from the antibody genes selected from the group consisting of SEQ ID No. 12 and 13.

[0040] The isolated humanized monoclonal antibody can have mutations to match the framework residues of the murine residues. These mutated residues can 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.

[0041] The isolated humanized monoclonal antibody can have a VH region comprising the mutations L55K and I66K.

[0042] The isolated humanized monoclonal antibody can have a combination of light chain framework mutations and heavy chain framework mutations. The light chain framework mutations can consist of I2K, A40Y, N66K, T85N, and F87Y. The heavy chain framework mutations can consist of L55K, I66K, V25A, D85N, and E69P.

[0043] Optionally, the isolated humanized monoclonal antibody can have a combination of light chain framework mutations and heavy chain framework mutations. The light chain framework mutations can consist of I2K, L39M, A40Y, N66K, T85N, and F87Y. The heavy chain framework mutations can consist of M39I, A80T, L55K, I66K, E69P, A45R, P46T, G47E, and K48Q.

[0044] In another optional embodiment, the isolated humanized monoclonal antibody can have a combination of light chain framework mutations and heavy chain framework mutations. The light chain framework mutations can consist of I2K, A40Y, N66K, T85N, and F87Y. The heavy chain framework mutations can consist of M39I, A80T, L55K, I66K, V25A, D85N, and E69P.

[0045] In yet another optional embodiment, the isolated humanized monoclonal antibody can have a combination of light chain framework mutations and heavy chain framework mutations. The light chain framework mutations can consist of I2K, L39M, A40Y, and N66K. The heavy chain framework mutations can consist of L55K, I66K, and E69P.

[0046] The isolated humanized monoclonal antibody can have a combination of light chain framework mutations and heavy chain framework mutations. The light chain framework mutations can consist of A40Y. The heavy chain framework mutations can consist of L55K, I66K, V25A, D85N, and E69P.

[0047] Optionally, the isolated humanized monoclonal antibody can have a combination of light chain framework mutations and heavy chain framework mutations. The light chain framework mutations can consist of A40Y. The heavy chain framework mutations can consist of L55K and I66K.

[0048] In another optional embodiment, the VL domain of the isolated humanized monoclonal antibody is capable of having at least 90% sequence identity to SEQ ID No. 14, and the VH domain is capable of having at least 90% sequence identity to SEQ ID No. 15.

[0049] Alternatively, the VL domain of the isolated humanized monoclonal antibody is capable of having at least 90% sequence identity to SEQ ID No. 16, and the VH domain is capable of having at least 90% sequence identity to SEQ ID No. 17.

[0050] In another optional embodiment, the VL domain of the isolated humanized monoclonal antibody is capable of at least 90% sequence identity to SEQ ID No. 18 and the VH domain is capable of at least 90% sequence identity to SEQ ID No. 19.

[0051] Optionally, the VL domain of the isolated humanized monoclonal antibody is capable of at least 90% sequence identity to SEQ ID No. 20 and the VH domain is capable of at least 90% sequence identity to SEQ ID No. 21.

[0052] Alternatively, the VL domain of the isolated humanized monoclonal antibody is capable of at least 90% sequence identity to SEQ ID No. 22 and the VH domain is capable of at least 90% sequence identity to SEQ ID No. 23.

[0053] Alternatively, the VL domain of the isolated humanized monoclonal antibody is capable of at least 90% sequence identity to SEQ ID No. 24 and the VH domain is capable of at least 90% sequence identity to SEQ ID No. 25.

[0054] In embodiments, the humanized monoclonal antibody can be further modified to alter the charge of the immunoglobulin. For example, the positive charge in the immunoglobulin can be reduced, for example by reducing the positive charge such that the next charge above the Fv region is +4 or less.

[0055] For example, the VL domain can be modified by including the mutations Q44E, A49S, V101T, or omitting the mutations L39M or I2K and L39M.

[0056] The VH domain can include changes including the mutations and L55K or I66K, but not both; the addition E69P; and the mutations D57E and / or D62E in HCDR2.

[0057] In embodiments, the VL domain of the isolated humanized monoclonal antibody is capable of at least 90% sequence identity to SEQ ID No. 26 and the VH domain is capable of at least 90% sequence identity to SEQ ID No. 27.

[0058] Alternatively, the VL domain of the isolated humanized monoclonal antibody is capable of at least 90% sequence identity to SEQ ID No. 28 and the VH domain is capable of at least 90% sequence identity to SEQ ID No. 29.

[0059] Alternatively, the VL domain of the isolated humanized monoclonal antibody is capable of having at least 90% sequence identity to SEQ ID No. 30 and the VH domain is capable of having at least 90% sequence identity to SEQ ID No. 31.

[0060] Alternatively, the VL domain of the isolated humanized monoclonal antibody is capable of having at least 90% sequence identity to SEQ ID No. 32 and the VH domain is capable of having at least 90% sequence identity to SEQ ID No. 33.

[0061] Alternatively, the VL domain of the isolated humanized monoclonal antibody is capable of having at least 90% sequence identity to SEQ ID No. 34 and the VH domain is capable of having at least 90% sequence identity to SEQ ID No. 35.

[0062] Alternatively, the VL domain of the isolated humanized monoclonal antibody is capable of having at least 90% sequence identity to SEQ ID No. 36 and the VH domain is capable of having at least 90% sequence identity to SEQ ID No. 37.

[0063] Alternatively, the VL domain of the isolated humanized monoclonal antibody is capable of having at least 90% sequence identity to SEQ ID No. 38 and the VH domain is capable of having at least 90% sequence identity to SEQ ID No. 39.

[0064] Alternatively, the VL domain of the isolated humanized monoclonal antibody is capable of having at least 90% sequence identity to SEQ ID No. 40 and the VH domain is capable of having at least 90% sequence identity to SEQ ID No. 41.

[0065] Alternatively, the VL domain of the isolated humanized monoclonal antibody is capable of having at least 90% sequence identity to SEQ ID No. 42 and the VH domain is capable of having at least 90% sequence identity to SEQ ID No. 43.

[0066] Alternatively, the VL domain of the isolated humanized monoclonal antibody is capable of having at least 90% sequence identity to SEQ ID No. 44 and the VH domain is capable of having at least 90% sequence identity to SEQ ID No. 45.

[0067] Alternatively, the VL domain of the isolated humanized monoclonal antibody is capable of having at least 90% sequence identity with SEQ ID No. 48 and the VH domain is capable of having at least 90% sequence identity with SEQ ID No. 49.

[0068] Alternatively, the VL domain of the isolated humanized monoclonal antibody is capable of having at least 90% sequence identity with SEQ ID No. 48 and the VH domain is capable of having at least 90% sequence identity with SEQ ID No. 49.

[0069] Alternatively, the VL domain of the isolated humanized monoclonal antibody is capable of having at least 90% sequence identity with SEQ ID No. 50 and the VH domain is capable of having at least 90% sequence identity with SEQ ID No. 51.

[0070] Alternatively, the VL domain of the isolated humanized monoclonal antibody is capable of having at least 90% sequence identity with SEQ ID No. 52 and the VH domain is capable of having at least 90% sequence identity with SEQ ID No. 53.

[0071] Alternatively, the VL domain of the isolated humanized monoclonal antibody is capable of having at least 90% sequence identity with SEQ ID No. 54 and the VH domain is capable of having at least 90% sequence identity with SEQ ID No. 55.

[0072] Alternatively, the VL domain of the isolated humanized monoclonal antibody is capable of having at least 90% sequence identity with SEQ ID No. 56 and the VH domain is capable of having at least 90% sequence identity with SEQ ID No. 57.

[0073] Alternatively, the VL domain of the isolated humanized monoclonal antibody is capable of having at least 90% sequence identity with SEQ ID No. 58 and the VH domain is capable of having at least 90% sequence identity with SEQ ID No. 59.

[0074] Alternatively, the VL domain of the isolated humanized monoclonal antibody is capable of having at least 90% sequence identity with SEQ ID No. 60 and the VH domain is capable of having at least 90% sequence identity with SEQ ID No. 61.

[0075] Alternatively, the VL domain of the isolated humanized monoclonal antibody can have at least 90% sequence identity to SEQ ID No. 62 and the VH domain can have at least 90% sequence identity to SEQ ID No. 63.

[0076] Alternatively, the VL domain of the isolated humanized monoclonal antibody can have at least 90% sequence identity to SEQ ID No. 64 and the VH domain can have at least 90% sequence identity to SEQ ID No. 65.

[0077] Alternatively, the VL domain of the isolated humanized monoclonal antibody can have at least 90% sequence identity to SEQ ID No. 66 and the VH domain can have at least 90% sequence identity to SEQ ID No. 67.

[0078] Alternatively, the VL domain of the isolated humanized monoclonal antibody can have at least 90% sequence identity to SEQ ID No. 68 and the VH domain can have at least 90% sequence identity to SEQ ID No. 69.

[0079] Alternatively, the VL domain of the isolated humanized monoclonal antibody can have at least 90% sequence identity to SEQ ID No. 70 and the VH domain can have at least 90% sequence identity to SEQ ID No. 71.

[0080] In the preceding 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 to the SEQ ID.

[0081] The monoclonal antibody or antigen binding portion thereof can be selected from an IgG, IgA or an antigen binding antibody fragment selected from the group consisting of an antibody single variable domain polypeptide, a dAb, a FAb, a F(ab')2, a scFv, a Fv, a VHH domain (such as a Nanobody® or other Camelidae immunoglobulin domain) or disulfide bonded Fv, a human antibody, preferably a chimeric antibody containing human variable regions, a humanized antibody, a bispecific antibody or a single chain antibody.

[0082] The humanized monoclonal antibody or antigen binding portion thereof can have a Fc region derived from an antibody of human IgGl isotype. In a particular embodiment, the antibody is of hlgGl isotype.

[0083] The humanized monoclonal antibody, or antigen-binding portion thereof, can also be effective in promoting resolution of systemic murine AA amyloid deposits when administered parenterally to mice with experimentally induced systemic AA amyloidosis.

[0084] The humanized monoclonal antibody, or antigen-binding portion thereof, can have complement activation-dependent in vivo efficacy.

[0085] Alternatively or additionally, the humanized monoclonal antibody, or antigen-binding portion thereof, can have Fcy receptor binding-dependent in vivo efficacy.

[0086] In further embodiments, the antibody of the application is indicated for the treatment of systemic amyloidosis, and thus there is provided a pharmaceutical composition comprising an antibody as defined herein for use in the treatment of systemic amyloidosis.

[0087] 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 having systemic amyloidosis, the method comprising administering to a subject in need thereof a composition comprising an antibody specific for amyloid fibrils as described herein.

[0088] In a preferred embodiment, the pharmaceutical composition is co-administered with supportive therapy for amyloidosis. Amyloidosis therapies in the art are generally aimed at removing or reducing the presence or production of amyloid precursor, and the present antibody is aimed at removing established amyloid deposits. Together with existing or novel therapies for removing amyloid, the antibody of the application thus provides a more complete treatment for amyloidosis.

[0089] In some embodiments, the antibody of the application can be administered independently of other amyloidosis treatments, for example in cases where the appearance of amyloid precursor has already been minimised and there is a need to deplete established amyloid fibrils.

[0090] In another optional embodiment, the amyloidosis therapy is selected from any existing systemic AL amyloidosis therapy, including those listed in Bianchi et al., JACC CardioOncol. 2021 Oct; 3(4): 467-487.

[0091] It should be noted that in this disclosure, and particularly in the claims and / or paragraphs, terms such as “comprises,” “comprised,” and “comprising” may have the meanings conferred upon them under patent law; for example, they may mean “includes,” “included,” and “including.” Furthermore, terms such as “consisting essentially of” and “consists essentially of” have the meanings conferred upon them under patent law, for example, they allow elements not explicitly referenced but exclude elements present in the prior art or affecting the essential or novel characteristics of the invention.

[0092] These and other implementations are disclosed or apparent from the following detailed description and are covered by the following detailed description. Attached Figure Description

[0093] Figure 1 . Generates best-in-class anti-amyloid antibody 2E5 with broad specificity against amyloid deposits.

[0094] A) 2E5 antibody was generated using a novel mouse fibrillary immunization strategy. B) Antibody 2E5 at 10 µg / ml specifically binds to ATTR fibrils in ELISA, but not to immobilized soluble, globular, human or mouse transthyretin (TTR). C) Antibody 2E5 at 10 µg / ml specifically stains tissue deposits of major forms of human systemic amyloid, AL, ATTR, and AA, as well as rare genetic forms (image labeled AF488). Strong fluorescence counterstained with Congo red indicates the location of amyloid in the same field.

[0095] Figure 2 Anti-amyloid fibrillary antibody 2E5 binds to and removes amyloid protein (AA) in mice in vivo.

[0096] A) Antibody 2E5 at 10 µg / ml specifically stained mouse AA amyloid deposits (image labeled AF488) identified by Congo red staining in serial sections. B) Antibody 2E5 removed amyloid in vivo. Liver amyloid loading score in mice with systemic AA amyloidosis 16 days after a single IP injection of 4.8 mg / mouse 2E5 compared to the untreated control. Mann-Whitney test: Control vs. 2E5: p = 0.01278 Figure 3. Malonate and citrate binding to 2E5 A: Binding of citrate in the antibody binding cleft of 2E5 Fab, crystal structure.

[0097] B: Space between malonate, citrate and TTR C-terminus and comparison of changes.

[0098] Figure 4 . BLI assay of antibody 2E5 binding to deletion of TTR 99-127 BLI assay was set up as depicted and the deletion of peptide 99-127 was tested for binding to 2E5. Results are shown in graphical and checkbox format.

[0099] Deletion of the C-terminus of the peptide abrogates binding to 2E5. Similarly, amidation of the C-terminal amino acid causes loss of binding, indicating that the free C-terminal COOH is critical for binding.

[0100] Figure 5 . BLI assay of alanine scan of TTR 99-127 BLI assay was performed using variants of 99-127 in which the C-terminal amino acids were replaced by alanine. The three C-terminal amino acids (P, K, E) are increasingly critical for binding to 2E5.

[0101] Figure 6 . Competition ELISA between 2E5 mAb and hSAP for coated ATTR fibrils.

[0102] A fixed concentration of 2E5 mAb was incubated with increasing concentrations of hSAP within a physiologically relevant window. Binding of both hSAP and 2E5 mAb to ATTR fibrils was observed and the 2E5 binding signal was stable in the presence of bound hSAP, indicating that 2E5 has a non-overlapping epitope with hSAP on ATTR fibrils.

[0103] Figure 7 . Binding of humanized antibodies to ATTR fibrils ELISA of antibodies binding to ATTR fibrils after initial humanization experiments compared to murine 2E5 antibody.

[0104] A: CDR grafts show 100-fold reduced binding compared to 2E5.

[0105] B: Control in the absence of ATTR.

[0106] Figure 8 . Binding of CDR grafted variants to ATTR fibrils Elisa of optimized CDR grafted antibodies binding to 10 ug / ml ATTR fibrils.

[0107] Figure 9 Elisa of CDR grafted antibodies binding to different amyloid fibrils Elisa plot of humanized clones interaction with synthetic and native amyloid fibrils. Fibrils are synthetic amyloid derived from a truncated fragment of immunoglobulin light chain (AL55-133) and mutant forms of transthyretin (S52P TTR) and beta2 microglobulin (D76N Abeta2-m) and AA (amyloid A). DETAILED DESCRIPTION

[0108] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art in the field of peptide chemistry, cell culture and phage display, nucleic acid chemistry, and biochemistry. The following references provide one of ordinary skill with the general definitions of many terms used in this application: 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 Edition, 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 unless specified otherwise.

[0109] Standard techniques are used for molecular biology, genetic and biochemical methods ("Molecular Cloning: A Laboratory Manual", 2nd Ed., ed. 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 suitably adapted to produce the polynucleotides and polypeptides of the application, and thus, can be considered in making and practicing the application. In the next section, particularly useful techniques for particular embodiments will be discussed.

[0110] The "antibody" can be selected from, but not limited to, IgG, IgA, or an antigen binding antibody fragment selected from the group consisting of: antibody single variable domain polypeptides, dAbs, FAb, F(ab')2, scFv, Fv, V HH domains (such as Nanobody® or other camelized immunoglobulin domains) or disulfide bonded Fv. In certain embodiments, any of the above antibody types or fragments thereof can be prepared from one or more mammalian species selected from, but not limited to, mouse, rat, rabbit, human. Such antibodies can be humanized for use in humans.

[0111] In certain embodiments, any of the above antibody types or fragments thereof can be provided as heteroconjugates, bispecific, single chain, chimeric or humanized molecules with affinity for amyloid fibrils.

[0112] In certain embodiments, any of the foregoing antibody / antibodies binds to amyloid with a dissociation constant of 100 nM or less, 75 nM or less, 50 nM or less, 25 nM or less, such as 10 nM or less, 5 nM or less, 1 nM or less, or in embodiments, 500 pM or less, 100 pM or less, 50 pM or less, or 25 pM or less.

[0113] Antibodies can be monospecific, having a narrow specificity or a broad specificity; or multispecific, such as bispecific, such that they have two different epitope specificities in a single antibody molecule. Mixtures of antibodies can target two or more specific epitopes. Antibody cocktails can be prepared by a mixture of one or more monoclonal antibodies. In one embodiment, the antibody cocktail contains two, three, four or more monoclonal antibodies, wherein each monoclonal antibody recognizes a plurality of amyloid fibrils.

[0114] 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.

[0115] In one aspect, an antibody or antibodies of the present invention are formulated for intravenous (iv) or intramuscular (im) administration. Antibodies administered iv should extravasate from the circulation to enter the interstitial tissue space and bind to their cognate targets.

[0116] In one embodiment, the antibody is an antibody fragment, such as a scFv, dAb or V HH Antibodies. Smaller antibody fragments extravasate more easily into tissues and can therefore perform better than IgG or other larger antibodies. However, smaller fragments also clear more rapidly from the circulation. A compromise must be struck between tissue accessibility and clearance. See, e.g., Wang et al., Clinical pharmacology & Therapeutics, 84:5, 2008, 548-558. Several antibody conjugates have been described that use various strategies to extend half-life, for example by conjugation to albumin, such as human serum albumin. See Kontermann et al., BioDrugs April 2009, Volume 23, Issue 2, pages 93-109.

[0117] It is postulated that the mechanism by which antibodies according to the present application promote the removal of amyloid deposits necessarily involves complement activation of antibodies bound to amyloid fibrils, leading to the recruitment of macrophages, which 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 a unique phenotype that enables them to destroy extracellular amyloid deposits that are extremely large relative 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). Therefore, antibody fragments, whole antibodies, and any other constructs that, when they bind to amyloid fibrils, cannot effectively activate the classical complement pathway are advantageously modified or further modified to promote complement activation. In a preferred embodiment, the antibody fragment or derivative is an antibody fragment or derivative that is suitably modified to activate complement.

[0118] "Fragment" means a portion of a polypeptide or nucleic acid molecule. The portion preferably contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the full length of the reference nucleic acid molecule or polypeptide. A fragment can 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.

[0119] The terms "isolated," "purified," or "biologically pure" refer to material which is free from components which normally accompany the material as it is found in its native state. "Isolated" indicates a degree of separation from the original source or from the surrounding environment. "Purified" indicates a higher degree of separation than isolated. 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 the application is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques or substantially free of chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, e.g., polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can indicate that a nucleic acid or protein produces substantially one band on an electrophoretic gel. For proteins that can undergo modifications (e.g., phosphorylation or glycosylation), different modifications can produce different isolated proteins, which can be purified individually.

[0120] An "isolated polynucleotide" means a nucleic acid (e.g., DNA) of a gene that is not found in the genome of the organism from which the nucleic acid molecule of the application is derived, flanked by the nucleic acid of the gene on both sides. Thus, the term includes, for example, a recombinant DNA that is incorporated into a vector, an autonomously replicating plasmid, or a virus, or that is integrated into the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (e.g., as 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 transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequences.

[0121] An "isolated polypeptide" means a polypeptide of the application that has been separated from components which naturally accompany it. Generally, a polypeptide is isolated when it is at least 60% by weight (excluding protein and other naturally associated organic molecules of nature) free from the components which naturally accompany it. Preferably, the preparation is at least 75% by weight, more preferably at least 90% by weight, and most preferably at least 99% by weight of the polypeptide of the application. An isolated polypeptide of the application can be obtained, for example, from natural sources by extraction, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemical synthesis of the protein. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

[0122] Amyloid Amyloidosis and amyloid fibrils are known in the art. About 30 different proteins are known to form amyloid fibrils in the human body, each associated with a clinically distinct disease. For a review and definition, see Pepys, M. B. and P. N. Hawkins (2020). Amyloidosis. Oxford Textbook of Medicine . J. Firth, C. Conlon and T. Cox, Oxford University Press] International Society for Amyloidosis nomenclature report (https: / / doi.org / 10.1080 / 13506129.2020.1835263).

[0123] Amyloid fibrils are aggregates of proteins, usually assembled into beta sheets. The termini of the proteins can be exposed in the beta sheet structure, and it is hypothesized that the exposed termini of the proteins that make up amyloid fibrils are responsible for the binding of pan-fibril specific antibodies to amyloid. In embodiments, the C-terminus of the protein is bound by the antibody; the free C-terminal carboxyl in the TTR peptide is critical for binding, indicating that 2E5 requires a charged ligand.

[0124] The amyloid epitope formed by the C-terminal amino acid of TTR can be mimicked by both the citrate and malonate ions 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 nearly the same way, but not large enough to touch the heavy chain.

[0125] Antibodies The term “monoclonal antibody” refers to an antibody obtained from a single clone of B-lymphocyte-derived plasma cells that produce a homogeneous antibody of a single heavy and light chain class and epitope specificity.

[0126] Monoclonal antibodies are generally highly specific, targeting a single antigenic site (epitope), in contrast to conventional antibodies within an antiserum induced by immunization of an entire animal with a particular antigen. Such conventional antibodies are derived from many different clones of B lymphocytes that recognize the same or different epitopes on the immunizing antigen, and are referred to as polyclonal antibodies. In addition to their very limited specificity, monoclonal antibodies are readily produced in pure form, free from contamination by other immunoglobulins, whereas isolation of specific antibodies from polyclonal antisera requires rigorous immunopurification procedures.

[0127] Monoclonal antibodies can be made by the hybridoma method (see Kohler et al., Nature, 256:495-7, 1975) or by recombinant DNA methods. Monoclonal antibodies can even be isolated from phage antibody libraries using well-known techniques.

[0128] 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 a corresponding sequence in an antibody 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 a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as the antibodies exhibit the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984)).

[0129] In the hybridoma method, a host animal (usually a mouse) is immunized with the desired antigen to induce the production of clones of B lymphocytes that produce or are capable of producing antibodies that will specifically bind to that antigen. Lymphocytes taken from the immunized animal are then fused with an in vitro grown continuous bone marrow tumor cell line to form so-called hybridoma cells in vitro. These cells are then selected by growth in a suitable medium that only allows the survival of the fused cells and not the un-fused parental bone marrow tumor cells. Examples of myeloma cells include, but are not limited to, human myeloma and mouse-human heteromyeloma cell lines that have been described for the production of human monoclonal antibodies.

[0130] Monoclonal antibodies to the antigen can be assayed in the medium from growing hybridoma cells. The binding specificity of the antibodies produced by the cells can be determined by various methods, such as immunoprecipitation or in vitro binding assays, such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), or immunoradiometric assay (IRMA).

[0131] After the hybridoma cells that produce the desired antibody are identified, the clones can 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 standard methods, including immunoglobulin purification procedures, such as protein A-agarose, gel electrophoresis, dialysis, hydroxylapatite chromatography, or affinity chromatography.

[0132] Antibodies of the present application also encompass variants of such antibodies and fragments thereof. Variants include peptides and polypeptides that comprise one or more amino acid sequence substitutions, deletions, and / or additions that have the same or substantially the same affinity and specificity for epitope binding as the anti-amyloid antibody or fragment thereof.

[0133] Deletions, insertions, or substitutions of amino acid residues can produce silent changes, and produce functionally equivalent substances. Intentional amino acid substitutions can 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 that include uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

[0134] Homologous substitutions (both substitution and replacement as used herein mean the exchange of one amino acid residue for a replacement residue) can occur, i.e., conservative substitutions, such as basic for basic, acidic for acidic, polar for polar, etc. Non-homologous substitutions can also occur, i.e., from one class of residues to another class of residues or alternatively involve unnatural amino acids, such as ornithine (hereinafter Z), diaminobutyric acid ornithine (hereinafter B), norleucine ornithine (hereinafter O), pyridylalanine, thiophenylalanine, naphthylalanine, and phenylglycine.

[0135] Substitutions can also be made by unnatural amino acids including: alpha* and alpha-disubstituted* amino acids, N-alkyl amino acids, lactic acid, halide derivatives of natural amino acids such as trifluorotyrosine, p-Cl-phenylalanine, p-Br-phenylalanine, p-l-phenylalanine, L-allyl-glycine, beta-alanine, L-alpha-aminobutyric acid, L-gamma-aminobutyric acid, L-alpha- aminoisobutyric acid, L-epsilon-aminohexanoic acid, 7-aminoheptanoic 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, penta- methyl-Phe, L-phe (4-amino), L-Tyr (methyl), L-phenylalanine (4-isopropyl), L-Dic (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid), L-diaminopropionic acid, and L-Phe (4-benzyl). The symbol*is used for the purposes of the above discussion (in relation to homologous or non-homologous substitutions) to indicate the hydrophilic-hydrophobic nature of the derivative, while #is used to indicate the hydrophilic nature of the derivative, and #*to indicate amphipathic properties.

[0136] Accordingly, variants can include peptides and polypeptides comprising one or more amino acid sequence substitutions, deletions, and / or additions in the antibodies and fragments of the application, wherein such substitutions, deletions, and / or additions do not cause a substantial change in the affinity and specificity of epitope binding. For example, a variant of an antibody or fragment thereof can be one that results from one or more changes to the antibody or fragment thereof, wherein the altered antibody or fragment thereof has the same or essentially the same affinity and specificity of epitope binding as the starting sequence. Variants can be naturally occurring, such as allelic or splice variants, or can be artificially constructed. Variants can be prepared from corresponding nucleic acid molecules encoding the variant. Variants of an antibody or fragment thereof can have changes in the light and / or heavy chain amino acid sequences that are naturally occurring or introduced by in vitro engineering of the natural sequence using recombinant DNA technology. Naturally occurring variants include "somatic" variants that are generated in vivo during an antibody response to a foreign antigen in the corresponding germline nucleotide sequence.

[0137] Variants of antibodies and binding fragments can also be made by mutagenesis techniques. For example, amino acid changes can be introduced randomly throughout the antibody coding region, and the resulting variants can be screened for binding affinity to amyloid or another property. Alternatively, amino acid changes can be introduced into selected regions of the antibody, such as the light and / or heavy chain CDRs, and / or the framework regions, and the resulting antibodies can 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. Variants produced by increasing the size of a CDR through the insertion of amino acids are also encompassed.

[0138] Amyloid-specific antibodies or fragments thereof can be equipped with a modified Fc region, wherein the naturally occurring Fc region is modified to increase the half-life of the antibody or fragment in a biological environment, e.g., serum half-life or half-life as measured by an in vitro assay. Fc modifications can also be used to alter the biodistribution of the antibody in vivo, which can direct the antibody to tissues carrying amyloid.

[0139] 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. Relative to a comparable molecule comprising a wild-type Fc region, a variant Fc region can be designed to bind to an Fc receptor with greater or lesser affinity. For example, antibodies and fragments thereof can comprise a modified Fc region. The Fc region refers to a naturally occurring or synthetic polypeptide that is homologous to the IgG C-terminal domain resulting from papain digestion of IgG. IgG Fc has a molecular weight of approximately 50 kD. In antibodies and fragments, the entire Fc region can be used, or only the half-life enhancing portion.

[0140] Antibodies and fragments thereof also encompass derivatives of the antibodies, fragments, and sequences disclosed herein. Derivatives include polypeptides or peptides that have been chemically modified, or variants, fragments, or derivatives thereof. 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. Derivatives are modified in a manner different from the naturally occurring or starting peptide or polypeptide, whether in the type or location of the attached molecule. Derivatives further include the deletion of one or more chemical groups naturally occurring on the peptide or polypeptide.

[0141] The present application also encompasses amyloid-specific antibodies that include two full-length heavy chains and two full-length light chains. Alternatively, the antibodies can be constructs such as single-chain antibodies or "minibodies" that retain binding activity to amyloid. Such constructs can be made by methods well known in the art. Advantageously, such fragments are modified to effect complement activation.

[0142] For amyloid-specific antibodies and fragments thereof, methods for creating recombinant DNA versions of the antigen-binding regions of antibody molecules that bypass the production of monoclonal antibodies are contemplated. The DNA is cloned into a plasmid vector system. One example of such technology is the use of the bacteriophage lambda vector system with a leader sequence that causes the expressed Fab protein to migrate into the periplasmic space (between the bacterial cell membrane and cell wall) or be secreted. Large numbers of functional Fab fragments that bind to amyloid can be rapidly produced and screened. Such amyloid-binding agents (Fab fragments specific for amyloid) are specifically encompassed within amyloid-specific antibodies and fragments thereof.

[0143] Amyloid-binding antibodies and fragments thereof can be humanized or human engineered antibodies. As used herein, a "humanized antibody" or antigen-binding fragment thereof is a recombinant polypeptide that includes a portion of the antigen-binding site from a non-human antibody and a portion of the framework and / or constant region of a human antibody. A human engineered antibody or antibody fragment is a non-human (e.g., mouse) antibody engineered by modifying (e.g., deleting, inserting, or substituting) amino acids at particular positions to reduce or eliminate any detectable immunogenicity of the modified antibody in humans.

[0144] Humanized antibodies include chimeric antibodies and CDR-grafted antibodies. Chimeric antibodies are antibodies that include non-human variable regions linked to human constant regions. Thus, in a chimeric antibody, the variable regions are primarily non-human and the constant regions are human. Chimeric antibodies and methods for making them are described, for example, in Proc. Natl. Acad. Sci. USA, 81 :6841-6855 (1984). Although they can not be as immunogenic as mouse monoclonal antibodies, administration of chimeric antibodies is associated with a human immune response (HAMA) to the non-human portion of the antibody. Chimeric antibodies can also be produced by splicing the genes from a mouse antibody molecule of appropriate antigen-binding specificity together with genes from a human antibody molecule of appropriate biological activity (e.g., the ability to activate human complement and mediate antibody-dependent cellular cytotoxicity (ADCC)). One example is replacing the Fc region with a different isotype.

[0145] CDR-grafted antibodies are antibodies that include CDRs from a non-human "donor" antibody linked to framework regions from a human "recipient" antibody. Generally, CDR-grafted antibodies include more human antibody sequences than chimeric antibodies, as the CDR-grafted antibodies include both constant region sequences and variable region (framework) sequences from human antibodies. Thus, for example, a CDR-grafted, humanized antibody of the application can comprise a heavy chain that includes contiguous amino acid sequences from a framework region of a human antibody (e.g., FR-I, FR-2, or FR-3 of a human antibody) or optionally a substantial portion or all of a contiguous amino acid sequence from an entire framework region of a human antibody (e.g., about 5 or more, 10 or more, or even 15 or more contiguous amino acid residues). CDR-grafted antibodies, and methods for making the same, are described in Nature, 321 : 522-525 (1986). Methods that can be used to generate humanized antibodies are also described, for example, in US 5,721,367 and 6,180,377.

[0146] A "veneered antibody" is a non-human or humanized (e.g., chimeric or CDR-grafted antibody) antibody that has been engineered to replace certain solvent-exposed amino acid residues to reduce its immunogenicity or enhance its function. Veneering of a chimeric antibody can comprise identifying solvent-exposed residues in the non-human framework regions of the chimeric antibody, and replacing at least one of the residues with a corresponding surface residue from a human framework region.

[0147] Veneering can be accomplished by any suitable engineering technique.

[0148] Further details regarding antibodies, humanized antibodies, human engineered antibodies, and methods for their preparation can be found in Antibody Engineering, Springer, New York, NY, 2001.

[0149] Examples of humanized or human engineered antibodies are IgG, IgM, IgE, IgA, and IgD antibodies. The antibodies can be of any class (IgG, IgA, IgM, IgE, IgD, etc.) or isotype, and can include either kappa or lambda light chains. For example, a human antibody can include an IgG heavy chain or a defined fragment, such as at least one of the isotypes IgGl, IgG2, IgG3, or IgG4. As a further example, an antibody or fragment thereof can include an IgG 1 heavy chain and a kappa or lambda light chain.

[0150] Human antibodies targeting amyloid can be produced using transgenic animals that do not produce endogenous immunoglobulins, and engineered to contain human immunoglobulin loci, as described in WO 98 / 24893 and WO 91 / 00906.

[0151] Using the transgenic animals described above, an immune response can be generated to a selected antigenic molecule, and cells producing antibodies can be removed from the animal and used to generate hybridomas that secrete human monoclonal antibodies. Immunization protocols, adjuvants, etc. are known in the art and are used, for example, to immunize transgenic mice.

[0152] The development of technologies for making libraries of recombinant human antibody genes, and the display of encoded antibody fragments on the surface of filamentous phage, provides a means for directly making human antibodies.

[0153] Antibodies produced by phage technology are produced as antigen-binding fragments, typically Fv or Fab fragments in bacteria, and thus lack effector functions.

[0154] Effector functions can be introduced by one of two strategies: fragments can be engineered into whole antibodies for expression in mammalian cells, or engineered as bispecific antibody fragments with a second binding site capable of triggering effector functions.

[0155] Human antibodies can be produced by in vitro screening of antibody display libraries (J Mol. Biol. (1991) 227:381). Various antibody-containing phage display libraries have been described and can be readily made. Libraries can contain a variety of human antibody sequences (such as human Fab, Fv, and scFv fragments) that can be screened against appropriate targets. Phage display libraries can comprise peptides or proteins other than antibodies that can be screened to identify agents capable of selectively binding to amyloid.

[0156] The phage display process mimics immune selection by displaying a repertoire of antibodies on the surface of filamentous phage and then selecting phage by their binding to selected antigens. One such method is described in WO 99 / 10494. Anti-amyloid antibodies can be isolated by screening recombinant combinatorial antibody libraries, preferably using a scFv phage display library made from human VL and VH cDNAs prepared from mRNA derived from human lymphocytes. Methods for making and screening such libraries are known in the art. There are commercially available kits for producing phage display libraries.

[0157] Amyloid-binding antibodies and fragments thereof can comprise one or more moieties that do not bind to amyloid but are responsible for other functions, such as circulating half-life, direct cytotoxic effects, detectable labels, or activation of the recipient's endogenous complement cascade or endogenous cytotoxicity. Antibodies or fragments thereof can comprise all or a portion of a constant region and can be of any isotype, including IgA (e.g., IgAl or IgA2), IgD, IgE, IgG (e.g., IgGl, IgG2, IgG3, or IgG4), or IgM. In addition to, or as an alternative to, comprising a constant region, antigen-binding compounds of the application can include an epitope tag, a salvage receptor epitope, a labeling moiety for diagnostic or purification purposes, or a cytotoxic moiety (such as a radionuclide or a toxin).

[0158] Anti-amyloid antibodies or fragments thereof can be modified to increase their serum half-life, for example, by adding a molecule such as PEG or other water-soluble polymer, including polysaccharide polymers, to increase half-life.

[0159] Amyloid-binding antibodies and fragments thereof can be bispecific. For example, a bispecific antibody can resemble a single antibody (or antibody fragment) but have two different antigen-binding sites (variable regions). Bispecific antibodies can be produced by various methods, such as chemical techniques, "polydoma" techniques, or recombinant DNA techniques. Bispecific antibodies can have binding specificity for at least two different epitopes, at least one of which is an epitope of amyloid.

[0160] Amyloid-binding antibodies and fragments can be heteroantibodies. Heteroantibodies are two or more antibodies or antibody-binding fragments (Fabs) linked together, each with a different specificity. As used herein, the term "antibody fragment" refers to a portion of an intact full-length antibody, such as an antigen-binding or variable region of an 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 multi-specific 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 (SMIPs), VHH-containing antibodies; and any other polypeptide formed from antibody fragments.

[0161] In the context of the present application, the terms anti-amyloid antibody and amyloid binding antibody encompass amyloid binding antibody fragments comprising any portion of the heavy chain sequence or light chain sequence of a full-length antibody and bind to amyloid.

[0162] As used herein, the term "fragment" refers to a fragment capable of binding to amyloid, such as at least 3 contiguous amino acids (e.g., at least 4, 5, 6, 7, 8, 9, or 10 or more contiguous amino acids, e.g., from a CDR) of any of the antibody involved in antigen binding, and encompasses Fab, Fab', F(ab')2, and F(v) fragments, or their individual light or heavy chain variable regions or portions. Amyloid binding fragments include, for example, Fab, Fab', F(ab')2, Fv, and scFv. These fragments lack the Fc fragment of intact antibodies, clear more rapidly from the circulation, and can have less non-specific tissue binding than intact antibodies. These fragments can be produced from whole antibodies using well-known methods, such as proteolytic cleavage, e.g., using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).

[0163] Amyloid binding antibodies and fragments also encompass single-chain antibody fragments (scFv) that bind to amyloid. scFv comprise an antibody heavy chain variable region (VH) operably linked to an antibody light chain variable region (VL), wherein the heavy chain variable region and light chain variable region together or individually form a binding site that binds to amyloid. The scFv can comprise the VH region at the amino terminus and the VL region at the carboxy terminus. Alternatively, the scFv can comprise the VL region at the amino terminus and the VH region at the carboxy terminus. 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)). The scFv can optionally further comprise a polypeptide linker between the heavy chain variable region and the light chain variable region.

[0164] Amyloid binding antibodies and fragments also encompass domain antibody (dAb) fragments consisting of a VH domain as described in Nature 341 :544-546 (1989).

[0165] Amyloid binding antibodies and fragments also encompass heavy chain antibodies (HCAb). These antibodies can apparently form antigen binding regions using only heavy chain variable regions, as these functional antibodies are only dimers of heavy chains (referred to as "heavy chain antibodies" or "HCAb"). Thus, amyloid binding antibodies and fragments can be heavy chain antibodies (HCAb) that specifically bind to amyloid.

[0166] Amyloid binding antibodies and fragments also encompass antibodies that are SMIPs or binding domain immunoglobulin fusion proteins specific for amyloid. These constructs are single chain polypeptides comprising an antigen binding domain fused to an immunoglobulin domain required for antibody effector function (see WO 03 / 041600).

[0167] Amyloid binding antibodies and fragments also encompass diabodies. These are bivalent antibodies in which VHand VLdomains 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 the complementarity domains of another chain and thereby creates two antigen binding sites (see, e.g., WO 93 / 11161). Diabodies can be bispecific or monospecific.

[0168] Amyloid binding antibodies and fragments thereof also encompass immunoadhesins. One or more CDRs can be covalently or noncovalently incorporated into a molecule to make it an immunoadhesin. The immunoadhesin can incorporate the CDRs as part of a larger polypeptide chain, can covalently link the CDRs to another polypeptide chain, or can noncovalently incorporate the CDRs. The CDRs allow the immunoadhesin to specifically bind to amyloid.

[0169] Amyloid binding antibodies and fragments thereof also encompass antibody mimetics comprising one or more amyloid binding moieties constructed on an organic or molecular scaffold, such as a protein or carbohydrate scaffold. Proteins having a relatively defined three-dimensional structure, often referred to as protein scaffolds, can be used as reagents for the design of antibody mimetics. These scaffolds often contain one or more regions susceptible to variation by specific or random sequence variation, and often such sequence randomization is performed to generate libraries of proteins from which desired products can be selected. For example, an antibody mimetic can comprise a chimeric non-immunoglobulin binding polypeptide having a scaffold comprising an immunoglobulin-like domain having two or more solvent exposed loops containing CDRs different from those of a parent antibody inserted into each of the loops and exhibiting selective binding activity for a ligand bound by the parent antibody. Non-immunoglobulin protein scaffolds have been proposed to obtain proteins with novel binding properties.

[0170] The anti-amyloid antibodies or antibody fragments thereof typically bind to human amyloid with high affinity (e.g., as determined using BIACORE), such as, for example, an equilibrium binding dissociation constant (KD) for amyloid of about 15 nM 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, or about 1 pM or less, about 0.75 pM or less, or about 0.5 pM or less.

[0171] 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 antibodies or antibody fragments can be isolated or purified to any degree.

[0172] Humanization “Humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized 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 non-human 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 can comprise residues not found in the recipient antibody or donor antibody. These modifications are made to further refine antibody performance. In general, a 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 constant region.

[0173] Some or all of the CDRs of the antibodies described herein can be transferred; for example, human acceptor CDRs can be retained so long as the donor CDR H3 is transferred. Members of the immunoglobulin superfamily all share similar folding of their polypeptide chains. For example, although the primary sequences of antibodies are highly diverse, comparison of sequences with crystal structures revealed, contrary to expectation, that five of the six antigen-binding loops of antibodies (H1, H2, L1, L2, L3) adopt a limited number of backbone conformations or canonical structures (Chothia and Lesk (1987) J. Mol. Biol., 196: 901; Chothia et al. (1989) Nature, 342: 877). Thus, analysis of loop length and key residues enabled prediction of the backbone conformations of H1, H2, L1, L2, and L3 found in most 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).

[0174] Pharmaceutical compositions In addition to the antibody, the pharmaceutical composition can also include one or more pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, stabilizers, preservatives, lubricants, or other materials well known in the art. Suitable materials should be nontoxic and biocompatible to the extent that they will not diminish the efficacy of the active compound. Examples include sterile saline (e.g., 0.9% NaCl), water, dextrose, glycerol, ethanol, etc., or combinations thereof. Such materials should be nontoxic 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 can be by infusion, injection or any other suitable route, as discussed below. Suitable materials should be nontoxic and biocompatible to the extent that they will not diminish the efficacy of the active compound. Examples include sterile saline (e.g., 0.9% NaCl), water, dextrose, glycerol, ethanol, etc., or combinations thereof. The composition can further contain auxiliary substances such as wetting agents, emulsifiers, pH buffering agents, and the like.

[0175] Suitable carriers, excipients, etc. can be found in standard pharmaceutical texts, e.g., Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Company, Easton, Pa., 1990.

[0176] As used herein, the term "pharmaceutically acceptable" means, within the scope of sound medical judgment, a compound, material, composition, and / or dosage form that is suitable for use in contact with the tissues of a subject (e.g., a 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.

[0177] In some embodiments, the antibody can be provided in lyophilized form for reconstitution prior to administration. For example, the lyophilized reagent can be reconstituted in sterile water and mixed with saline prior to administration to a subject.

[0178] The formulations can conveniently be presented in unit dosage form and can 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 a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product.

[0179] The formulations can be in the form of a liquid, solution, suspension, emulsion, etc.

[0180] Optionally, other therapeutic or prophylactic agents can be included in the pharmaceutical composition or formulation.

[0181] The treatment can be any treatment and therapy, whether in humans or in animals (e.g., in veterinary applications), in which some desired therapeutic effect is achieved, for example, inhibition or delay of progression of a condition, and includes a decrease in the rate of progression, stasis in the rate of progression, improvement of the condition, cure or remission of the condition, whether partial or total, prevention, delay, mitigation, or prevention of one or more symptoms and / or signs of the condition, or lengthening of survival beyond that expected without treatment.

[0182] Treatment as a prophylactic measure (i.e., prophylaxis) is also included. For example, a subject susceptible to, or at risk of, developing or recurring amyloidosis can be treated as described herein. Such treatment can prevent or delay the development or recurrence of amyloidosis in the subject.

[0183] In particular, the treatment can include inhibition of amyloid deposition, including complete reversal of amyloid deposition.

[0184] The antibody can be administered in a therapeutically effective amount as described herein.

[0185] As used herein, the term "therapeutically effective amount" relates to the amount of an active compound or combination, material, composition, or dosage form comprising an active compound that is effective to produce some desired therapeutic effect, commensurate with a reasonable benefit / risk ratio.

[0186] It will be appreciated that a proper dosage of an active compound can vary between patients. Determining the optimal dosage will generally involve balancing the level of therapeutic benefit achieved by the administration with any risk or deleterious side effects. 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 active compounds used in combination, the rate of excretion of the other drugs, compounds and / or materials used in the combination, and the age, sex, weight, condition, general health and prior medical history of the patient. The amount of active compound and route of administration will ultimately be at the discretion of the physician, although generally the dosage will be such that a concentration of the active compound is achieved at the site of therapy that does not cause substantial harmful or deleterious side effects.

[0187] Generally, a suitable dose of an active compound is in the range of about 100 μg to about 250 mg per kilogram body weight of the subject per day. Where the active compound is a salt, an ester, a prodrug, etc., the amount administered is calculated on the basis of the parent compound and thus the actual weight used will be proportionally increased.

[0188] In vivo administration can be in a single dose, continuously or intermittently (e.g., in divided doses at appropriate intervals). Methods of determining the most effective dosage and regimen are well known to those skilled 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.

[0189] Multiple doses of the antibody can be administered, for example, 2, 3, 4, 5, or more than 5 doses can be administered.

[0190] Pharmaceutical compositions comprising an active compound can be formulated to suit the intended route of administration in suitable dosage unit formulations.

[0191] Formulations suitable for oral administration (e.g., by ingestion) can be presented in 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 a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion; as an ampoule; as a bolus; as a paste; or as a gel.

[0192] Tablets can be prepared optionally with one or more accessory ingredients by conventional means, e.g., compression or molding. Compressed tablets can 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 a binder (e.g., povidone, gelatin, acacia, sorbitol, tragacanth, hydroxypropylmethyl cellulose); a filler (e.g., lactose, microcrystalline cellulose, calcium hydrogen phosphate); a lubricant (e.g., magnesium stearate, talc, silica); a disintegrant (e.g., sodium starch glycolate, Primogel, sodium starch glycolate); a surface-active or dispersing or wetting agent (e.g., sodium lauryl sulfate); and a preservative (e.g., methyl paraben, propyl paraben, sorbic acid). Molded tablets can be prepared by molding, in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent. Tablets can optionally be coated or scored and can be formulated so as to provide a sustained or controlled release of the active compound, e.g., different proportions of hydroxypropylmethyl cellulose, to provide the desired release profile. Tablets can optionally be provided with an enteric coating, to provide release in parts of the intestine other than the stomach.

[0193] 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 in a concentration of about 1 ng / ml to about 10 μg / ml, e.g., about 10 ng / ml to about 1 μg / ml; and aqueous and nonaqueous sterile suspensions which can include suspending agents and thickening agents. Examples of suitable isotonic vehicles for use in such formulations include sodium chloride injection, Ringer's Solution, or lactated Ringer's injection. Generally, the concentration of active compound in the solution is sufficient to provide the dosage required by the patient. The formulations can be presented in unit-dose or multi-dose sealed containers, for example, ampules and vials, and can be stored in a freeze-dried (lyophilized) condition using, e.g., sterile powders, which can be reconstituted into a solution or suspension upon addition of a sterile liquid carrier, e.g., water for injection. Preparations for injection can contain added excipients, such as are described above. Injectables can be prepared from sterile powders, granules, or tablets. Formulations can be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and can 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 can be prepared from sterile powders, granules, and tablets. Formulations can be presented in lipid or other microparticulate systems which are designed to target the active compound to blood components or one or more organs.

[0194] The compositions can be prepared in a concentrate form for subsequent dilution, or can be prepared in a divided dosage form ready for administration. Alternatively, the agents can be provided separately within a kit for mixing prior to administration to a human or animal subject.

[0195] Identity Nucleic acid molecules useful in the methods of the application include any nucleic acid molecule that encodes a polypeptide of the application or a fragment thereof. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but will generally exhibit substantial identity. A polynucleotide that has "substantial identity" to an endogenous sequence is generally capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the application include any nucleic acid molecule that encodes a polypeptide of the application or a fragment thereof. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but will generally exhibit substantial identity. A polynucleotide that has "substantial identity" to an endogenous sequence is generally capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. By "hybridize" is meant a pair-wise sequence of complementary polynucleotides (e.g., genes described herein) or portions thereof that form a double-stranded molecule 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).

[0196] For example, stringent salt concentration will typically be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent (e.g., formamide), whereas 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 typically involve temperatures of at least about 30°C, more preferably at least about 37°C, and most preferably at least about 42°C. Different additional parameters, such as hybridization time, concentration of detergent (e.g., sodium dodecyl sulfate (SDS)), and inclusion or exclusion of carrier DNA are well known to those of skill in the art. Different levels of stringency are achieved by combining these different conditions as needed. In a preferred embodiment, hybridization will be performed at 30°C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will be performed at 37°C in 500 mM NaCl, 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 be performed at 42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200.mu.g / ml ssDNA. Useful variations on these conditions will be apparent to those of skill in the art.

[0197] For most applications, the washing step after hybridization will also vary in stringency. Washing stringency conditions can be defined by salt concentration and temperature. Washing stringency can be increased by decreasing salt concentration or by increasing temperature, as described above. For example, stringent salt concentrations for the washing step will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the washing step will typically include temperatures 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, the washing step will be performed at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the washing step will be performed at 42°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the washing step will be performed at 68°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations of these conditions will be 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.

[0198] By "substantially identical" is meant a polypeptide or nucleic acid molecule that exhibits at least 90% identity with a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or nucleic acid sequence (e.g., any of the nucleic acid sequences described herein). Preferably, such sequences can be 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.

[0199] Sequence identity is typically measured using sequence analysis software (e.g., the sequence analysis software package of the Genetics Computer Group (GCG) of the 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 method for determining degrees of identity, the BLAST program can be used, with a probability score of e -3 and e -100 between indicate closely related sequences.

[0200] The ranges provided herein are to be understood as shorthand for all values within the range. For example, a range of 1 to 50 is to be understood as including any number, combination of numbers, or sub-range from the group consisting of: 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.

[0201] Modifications to the above embodiments, additional embodiments, and modifications of them will be apparent to those skilled in the art from a reading of this disclosure, and it is intended to cover all such modifications and embodiments that come within the scope of the present application.

[0202] All documents and sequence database entries cited in this specification are hereby incorporated by reference in their entirety for all purposes.

[0203] The present application is further described below with reference to the following examples.

[0204] Example Example 1 Antibody 2E5 was generated by immunizing SAP- / - mice with a synthetic human ATTR amyloid fibrillocyte immunogen, a variant of the classic hybridoma method originally developed by Milstein and Koehler (Bickerstaff et al., NatMed 1999 June;5(6):694-7). Figure 1 A).

[0205] ATTR fibrils in PBS were mixed with adjuvant RIBI, and 50 μg ATTR was injected at week 0 every two weeks, followed by 25 μg ATTR at week 2, and 25 μg ATTR combined with SAP at week 4.

[0206] This method provides a range of monoclonal antibodies, including 2E5. ELISA screening confirmed specific binding to human ATTR fibrils, but not to globular human TTR, fibril precursor protein, or mouse TTR. Figure 1 B). 2E5 also showed similarities to all major types of human systemic amyloid (B). Figure 1 C) and experimentally induced [Bodin, K. et al., Nature, 2010. 468(7320): 93-97, Botto, M. et al., Nature Med., 1997. 3(8): 855-859] mouse AA amyloid deposits ( Figure 2 A) unprecedented specific binding. The original 2E5 mouse monoclonal isotype is IgG2c, which is homologous to human IgG1 and has a strong complement-activating effect. This is a necessary characteristic for antibody-mediated amyloid removal in vivo, which has been shown to be complement-dependent [Bodin, K. et al., Nature, 2010. 468(7320): 93-97, Richards, DB et al., N. Engl. J. Med., 2015. 373(12): 1106-1114, Milde, R. et al., Cell Rep., 2015.13(9): 1937-1948]. In fact, administration of 2E5 to mice with confirmed systemic AA amyloidosis produced significant amyloid clearance ( Figure 2 B).

[0207] Example 2 Crystal structure data from 2E5 showed that both malonate and citrate ions were located in the binding cleft of the antibody, adopting a structure that mimicked the C-terminal structure of the TTR polypeptide. To identify the epitope bound by 2E5, peptide truncation studies and alanine scanning were performed using TTR peptides (99-127) previously shown to behave as analogues of the full-length TTR protein.

[0208] Peptides were synthesized 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-terminus Stock solutions (4mg / ml) of each peptide were prepared, dissolving 2mg of peptide in 500ul of: - 50% acetonitrile - 10% glacial acetic acid - 40% H2O.

[0209] Peptides were diluted to 10ug / ml in PBS for mapping using a Biolayer Interferometry (BLI) biosensor.

[0210] The 2E5-1B7 antibody (murine IgG2a) was prepared by recombinant expression and diluted to 40ug / ml in PBS for BLI.

[0211] Peptides and mAb were equilibrated to room temperature by pre-soaking a streptavidin BLI biosensor in PBS + 0.1% BSA for 10 minutes and BLI measurements were performed using a Blitz instrument (Blitz pro software) applying the following advanced kinetics to analyse antibody binding to each of the peptides: 1. 30 second baseline (PBS) 2. 60 seconds load biotinylated peptide at 10 ug / ml onto streptavidin biosensor 3. 30 seconds baseline (PBS) 4. 120 seconds association, 2E5-1B7 at 40 ug / ml 5. 120 seconds dissociation in PBS Reference runs were performed using the huTTR 99-127 peptide at step 2 (above) and association with PBS only at step 4 (above).

[0212] Binding affinity data were calculated within the Blitz pro software and raw BLI trace data exported for plotting.

[0213] The results show that deletion of the C-terminus of the 99-127 peptide abolishes binding of the antibody to the peptide. Thus, while peptides 99-127, 113-127, 118-127 and 123-127 retain the same binding properties (p Figure 4 ), 105-115 truncation and 99-127 E127A fail to be bound by 2E5.

[0214] Furthermore, amidation of the C-terminus and removal of the carboxyl group causes loss of binding, indicating that the charged nature of the acidic C-terminus is essential for binding.

[0215] Performing an alanine scan (AlaX, where X is the amino acid position) on the C-terminus Figure 5 ) shows that the T123A and N124A variants retain normal binding, but this binding is progressively reduced in the P125A, K126A and E127A variants. Thus, binding of 2E5 to the TTR peptide depends on the presence of charged amino acids at the C-terminus of the protein fibril.

[0216] Example 3 2E5 does not compete with SAP for binding fibrils To determine whether 2E5 and SAP bind to the same epitope in amyloid fibrils, a competition assay was performed to determine the binding of 2E5 at 0.01 μg / ml to 0.5 μg coated ATTR fibrils in the presence of different concentrations of SAP at 0.08 μg / ml to 50 μg / ml and in the presence of an irrelevant control antibody. Antibody detection was performed by the Fc domain using anti-mouse Fc HRP. It was found that 2E5 binds to the fibrils in a manner independent of SAP, indicating that SAP and 2E5 do not compete for the same epitope on ATTR fibrils.

[0217] Figure 6 Results are shown.

[0218] Example 4: CDR grafting of 2E5 The initial CDR transplantation experiment yielded three humanized antibodies, as shown in Table 1: Table 1

[0219] Antibodies 1-3 were tested by ELISA to determine their binding to amyloid TTR fibrils compared to mouse 2E5. Binding to antibodies in all CDR transplants showed an approximately 100-fold reduction. See also Figure 7 A and B.

[0220] Sequence and structural analysis of 2E5 was performed to identify key residues responsible for providing structure to the CDR in the mouse framework. These key residues were identified and then mutated back into mouse residues. Twenty humanized antibodies were generated and tested by ELISA.

[0221] Figure 8 The results are shown.

[0222] The results are ranked as shown in Table 2.

[0223]

[0224] Six of the most promising antibodies showed strong binding to fibrils, comparable to mouse 2E5 (see [link to antibody list]). Figure 4 Select P029_Ab004, 006, 008, 016, 019, and 021 for further development.

[0225] Example 5 Reduced charge in humanized antibodies As shown in Table 3, humanized antibodies with high affinity are associated with high positive charge. To reduce this charge to a level considered more suitable for drug development, the humanized clones were further modified, as shown in Table 4. This included removing positively charged residues by replacing them with non-positively charged residues frequently observed in human antibodies. Adding negatively charged residues was also employed to reduce the overall positive charge. In addition to charge, mutations were made at isomerization sites to improve the developability of antibodies in CDR-H2 (details are described in Table 3).

[0226] Antibodies P029_Ab0019 and P029_Ab0008 were selected as base clones for modification.

[0227]

[0228] Table 3

[0229] Table 4 Table 5 shows the results of the modification procedure. In several cases, the net charge was reduced to or below +4.

[0230]

[0231] Table 5 To determine if 2E5 maintained broad reactivity against different amyloid types after humanization, the interaction of the humanized clones with synthetic fibrils and native amyloid was determined by ELISA. This included synthetic amyloid derived from a truncated fragment of the immunoglobulin light chain (AL55-133) and mutant forms of transthyretin (S52P TTR), beta2 microglobulin (D76N Abeta2-m), and AA (amyloid A) which is present in spleen extracts from mice. ELISA characterization confirmed that the humanized 2E5 clones were able to interact with these amyloid forms in a dose dependent manner (Figure 6). Figure 9 ).

[0232] Sequence Listing SEQ ID No. 1 Heavy chain variable region (VH) Nucleotide sequence GAGGTTCAGCTGCTGCAGTCTGGGGCAGAGCTTGTGAAGCCAGGGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCTTCAAGATTAAAGACTTCTATATACACTGGGTGAAACAGAGGACTGAACAGGGCCTGGACTGGATTGGAAAGATTGATCCTGAGGATGGTAAAACTAAATATGCCCCGAAATTCCAGGGCAAGGCCACTATAACAACAGACACATCCTCCAATACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTGCTAGAGCCTACTATAGTAACTACAATTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAG SEQ ID No. 2 Heavy chain variable region (VH) Amino acid sequence EVQLLQSGAELVKPGASVKLSCTASGFKIKDFYIHWVKQRTEQGLDWIGKIDPEDGKTKYAPKFQGKATITTDTSSNTAYLQLSSLTSEDTAVYYCARAYYSNYNWFAYWG QGTLVTVSA SEQ ID No. 3 Light chain variable region (VL) Nucleotide sequence GAAAAAGTGCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCTAGGGGAGAAGGTCACCATGAGCTGCAGGGCCAGCTCAAGTGTAAATTACATGTACTGGTACCAGGAGAAGTCAGATGCCTCCCCCAAACTATGGATTTATTACACATCCAAGTTGGCTCCTGGAGTCCCAGCTCGCTTCAGTGGCAGTGGGTCTGGGAACTCTTATTCTCTCACAATCAGCAGCATGGAGGGTGAAGATGCTGCCACTTATTACTGCCAGCAGTTTACTAGTTCCCCATACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAAC SEQ ID No. 4 Light chain variable region (VL) Amino acid sequence EKVLTQSPAIMSASLGEKVTMSCRASSSVNYMYWYQEKSDASPKLWIYYTSKLAPGVPARFSGSGSGNSYSLTISSMEGEDAATYYCQQFTSSPYTFGGGTKLEIK SEQ ID No. 5 Light chain variable region (VL) Alternative nucleotide sequence GAAATTGTGCTCACCCAGTCTCCAACCACCATGGCTGCATCTCCCGGGGAGAAGATCACTATCACCTGCAGTGCCAGCTCAAGTATAAGTTCCAATTACTTGCATTGGTATCAGCAGAAGCCAGGATTCTCCCCTAAACTCTTGATTTATAGGACATCCAATCTGGCTTCTGGAGTCCCAGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATTGGCACCATGGAGGCTGAAGATGTTGCCACTTACTACTGCCAGCAGGGTAGTAGTATACCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAAC 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 EVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCWGQGTLVTVSS SEQ ID No. 13 Light chain V gene, CDR-grafted antibody EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCFGQGTKLEIK SEQ ID No. 14 Clone P029_Ab04, VL EIVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQQKPGQAPRLLIYYTSKRATGIPARFSGSGSGNDYTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK SEQ ID No. 15 Clone P029_Ab04, VH EVQLVQSGAEVKKPGATVKISCKASGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTKYAPKFQGRVTITADTSTNTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS SEQ ID No. 16 Clone P029_Ab06, VL EIVLTQSPATLSLSPGERATLSCRASSSVNYMYWYQEKPGQSPRLLIYYTSKRATGIPARFSGSGSGNDYTLTISSLEPEDFATYYCQQFTSSPYTFGQGTKLEIK SEQ ID No. 17 Clone P029_Ab06, VH EVQLVQSGAEVKKPGATVKISCKASGFKIKDFYIHWVQQRTEQGLEWMGKIDPEDGKTKYAPKFQGRVTITTDTSTNTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS SEQ ID No. 18 Clone P029_Ab08, VL EIVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQQKPGQAPRLLIYYTSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK SEQ ID No. 19 Clone P029_Ab08, VH EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTKYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS SEQ ID No. 20 Clone P029_Ab16, VL EKVLTQSPATLSLSPGERATLSCRASSSVNYMYWYQEKPGQSPRLLIYYTSKRATGIPARFSGSGSGNDYTLTISSLEPEDFATYYCQQFTSSPYTFGQGTKLEIK SEQ ID No. 21 Clone P029_Ab16, VH EVQLVQSGAEVKKPGATVKISCKASGFKIKDFYIHWVQQAPGKGLEWMGKIDPEDGKTKYAPKFQGRVTITTDTSTNTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS SEQ ID No. 22 Clone P029_Ab19, VL EKVLTQSPATLSLSPGERATLSCRASSSVNYMYWYQQKPGQAPRLLIYYTSKRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK SEQ ID No. 23 Clone P029_Ab19, VH EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTKYAPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS SEQ ID No. 24 Clone P029_Ab21, VL EIVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQQKPGQAPRLLIYYTSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK SEQ ID No. 25 Clone P029_Ab21, VH EVQLVQSGAEVKKPGATVKISCKASGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTKYAPKFQGRVTITADTSTNTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS SEQ ID No. 26 Clone P029_Ab25, VL EIVLTQSPATLSLSPGERATLSCRASSSVNYMYWYQQKPGQAPRLLIYYTSKRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK SEQ ID No. 27 Clone P029_Ab25, VH EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTKYAPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS SEQ ID No. 28 Clone P029_Ab26, VL EKVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQQKPGQAPRLLIYYTSKRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK SEQ ID No. 29 Clone P029_Ab26, VH EVQLVQSGAEVKKPGASVKISCKASGFTFSSNYWMNWVRQAPGQGLEWMGKIDPENGDTEYAPKFQGRTITADTSTDTAYMELSSLRSEDTAVYYCAKQYVSSVWYFDVWGQGTLVTVSS SEQ ID No. 30 Clone P029_Ab27, VL EIVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQQKPGQAPRLLIYYTSKRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK SEQ ID No. 31 Clone P029_Ab27, VH EVQLVQSGAEVKKPGASVKISCKASGFTFSSNYWMNWVRQAPGQGLEWMGKIDPENGDTEYAPKFQGRTITADTSTDTAYMELSSLRSEDTAVYYCAKQYVSSVWYFDVWGQGTLVTVSS SEQ ID No. 32 Clone P029_Ab28, VL EIVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQQKPGQAPRLLIYYTSKRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK SEQ ID No. 33 Clone P029_Ab28, VH EVQLVQSGAEVKKPGASVKISCKASGFTFSSNYWMNWVRQAPGQGLEWMGKIDPENGDTEYAPKFQGRTITADTSTDTAYMELSSLRSEDTAVYYCAKQYVSSVWYFDVWGQGTLVTVSS SEQ ID No. 34 Clone P029_Ab29, VL EIVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQQKPGQAPRLLIYYTSKRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK SEQ ID No. 35 Clone P029_Ab29, VH EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS SEQ ID No. 36 Clone P029_Ab29, VL EIVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQQKPGQAPRLLIYYTSKRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK SEQ ID No. 37 Clone P029_Ab30, VH EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGLIDPEDGKTKYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS SEQ ID No. 38 Clone P029_Ab30, VL EIVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQQKPGQAPRLLIYYTSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK SEQ ID No. 39 Clone P029_Ab30, VH EVQLVQSGAEVKKPGASVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTKYAPKFQG RVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS SEQ ID No. 40 Clone P029_Ab32, VH EIVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQQKPGQAPRLLIYYTSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK SEQ ID No. 41 Clone P029_Ab32, VH EVQLVQSGAEVKKPGASVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTKYAPKFQG RVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS SEQ ID No. 42 Clone P029_Ab33, VL EIVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQQKPGQAPRLLIYYTSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK SEQ ID No. 43 Clone P029_Ab33, VH EVQLVQSGAEVKKPGASVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGLIDPEDGKTKYAPKFQG RVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS SEQ ID No. 44 Clone P029_Ab34, VL EIVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQQKPGQAPRLLIYYTSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK SEQ ID No. 45 Clone P029_Ab34, VH EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS SEQ ID No. 46 Clone P029_Ab35, VL EIVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQQKPGQAPRLLIYYTSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK SEQ ID No. 47 Clone P029_Ab35, VH EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGLIDPEDGKTKYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS SEQ ID No. 48 Clone P029_Ab36, VL EKVLTQSPATLSLSPGERATLSCRASSSVNYMYWYQQKPGQAPRLLIYYTSKRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK SEQ ID No. 49 Clone P029_Ab36, VH EVQLVQSGAEVKKPGASVKISCKASGFTFSSNYWISWVRQAPGQGLEWMGKIDPISDGTY YADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAADNTAYYFDYWGQGTLVTVSS SEQ ID No. 50 Clone P029_Ab37, VL EKVLTQSPATLSLSPGERATLSCRASSSVNYMYWYQQKPGQAPRLLIYYTSKRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK SEQ ID No. 51 Clone P029_Ab37, VH EVQLVQSGAEVKKPGASVKISCKASGFTFSSNYWISWVRQAPGQGLEWMGKIDPISDGTY YADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAADNTAYYFDYWGQGTLVTVSS SEQ ID No. 52 Clone P029_Ab38, VL EKVLTQSPATLSLSPGERATLSCRASSSVNYMYWYQQKPGQAPRLLIYYTSKRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK SEQ ID No. 53 Clone P029_Ab38, VH EVQLVQSGAEVKKPGASVKISCKASGFTFSSNYWISWVRQAPGQGLEWMGKIDPISDGTY YADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAADNTAYYFDYWGQGTLVTVSS SEQ ID No. 54 Clone P029_Ab39, VL EVQLVQSGAEVKKPGESLEKISCKGSFSLDTYMYWVQQRPGQGLEWMGKIDPQDGKTYYPDFINNAYYLDHRLVTVSEI SKYGPPCPSCPAFQGTYTMDVGGGDRFSSYIHWYQQKPGQPPKLLIYRNSNRPSLAVSFGTQVQVYVMDVWDGKTYTSSVQKVDN AKLQDGFFPWYFDSWGQGTTVTVSS SEQ ID No. 55 Clone P029_Ab39, VH EVQLVQSGAEVKKPGESLEKISCKGSFSLDTYMYWVQQRPGQGLEWMGKIDPQDGKTYYPDFINNAYYLDHRLVTVSEI SKYGPPCPSCPAFQGTYTMDVGGGDRFSSYIHWYQQKPGQPPKLLIYRNSNRPSLAVSFGTQVQVYVMDVWDGKTYTSSVQKVDN AKLQDGFFPWYFDSWGQGTTVTVSS SEQ ID No. 56 Clone P029_Ab40, VL EKVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQEKPGQAPRLLIYYTSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK SEQ ID No. 57 Clone P029_Ab41, VH EVQLVQSGAEVKKPGESLEKISCKGSFSLDTYMYWVQQRPGQGLEWMGKIDPQDGKTYYPDFINNAYYLDHRLVTVSEI SKYGPPCPSCPAFQGTYTMDVGGGDRFSSYIHWYQQKPGQPPKLLIYRNSNRPSLAVSFGTQVQVYVMDVWDGKTYTSSVQKVDN AKLQDGFFPWYFDSWGQGTTVTVSS SEQ ID No. 58 Clone P029_Ab42, VL EKVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQEKPGQAPRLLIYYTSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK SEQ ID No. 59 Clone P029_Ab42, VH EVQLVQSGAEVKKPGASVKISCKASGFTFSSNYWISWVRQAPGQGLEWMGKIDPISDGTY YADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAADNTAYYFYFDYWGQGTTVTV S S SEQ ID No. 60 Clone P029_Ab43, VL EKVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQEKPGQAPRLLIYYTSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK SEQ ID No. 61 Clone P029_Ab43, VH EVQLVQSGAEVKKPGASVKISCKASGFTFSSNYWISWVRQAPGQGLEWMGKIDPISDGTY YADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAADNTAYYFYFDYWGQGTTVTV S S SEQ ID No. 62 Clone P029_Ab44, VL EKVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQEKPGQAPRLLIYYTSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK SEQ ID No. 63 Clone P029_Ab44, VH EVQLVQSGAEVKKPGASVKISCKASGFTFSSNYWISWVRQAPGQGLEWMGKIDPISDGTY YADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAADNTAYYFYFDYWGQGTTVTV S S SEQ ID No. 64 Clone P029_Ab45, VL EVQLVQSGAEVKKPGESLEKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIEPEDGKTI YAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLV TVSS SEQ ID No. 65 Clone P029_Ab45, VL EVQLVQSGAEVKKPGESLEKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIEPEDGKTI YAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLV TVSS SEQ ID No. 66 Clone P029_Ab45, VH EVQLVQSGAEVKKPGESLEKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIEPEDGKTI YAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLV TVSS SEQ ID No. 67 Clone P029_Ab46, VL EVQLVQSGAEVKKPGESLEKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIEPEDGKTI YAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLV TVSS SEQ ID No. 68 Clone P029_Ab46, VH EVQLVQSGAEVKKPGESLEKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIEPEDGKTI YAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLV TVSS SEQ ID No. 69 Clone P029_Ab47, VL EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIDPEEGKTKYAPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS SEQ ID No. 70 Clone P029_Ab48, VL EKVLTQSPATLSLSPGERATLSCRASSSVNYMYWYQQKPGQAPRLLIYYTSKRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK SEQ ID No. 71 Clone P029_Ab48, VH EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTKYAPKFQGRVTITADTSTDTAYMELSSLTSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

Claims

1. An isolated monoclonal antibody or its antigen-binding portion thereof, said isolated monoclonal antibody or its antigen-binding portion thereof specifically binding to at least three different types of amyloid fibrils selected from the following: ALκ, ALλ, ATTR wild-type, ATTR variant, AA, AApoAI, ALys, Aβ2m and AFib amyloid fibrils.

2. The isolated monoclonal antibody or its antigen-binding portion according to claim 1, wherein the isolated monoclonal antibody or its antigen-binding portion is bound to at least 4, 5, 6, 7, 8 or 9 different types of amyloid fibrils selected from the following: ALκ, ALλ, ATTR wild-type, ATTR variant, AA, AApoAI, ALys, Aβ2m and AFib amyloid fibrils.

3. An isolated monoclonal antibody or its antigen-binding moiety thereof, said isolated monoclonal antibody or its antigen-binding moiety specifically binding to amyloid fibrils, wherein each of the variable domains of the heavy chain, CDRH1, CDRH2, and CDRH3, is present. (a) Has at least 90% sequence identity with SEQ ID No. 6, SEQ ID No. 7 and SEQ ID No. 8 respectively; or (b) optionally having a single amino acid change relative to SEQ ID No. 6, SEQ ID No. 7 and SEQ ID No. 8, respectively.

4. An isolated monoclonal antibody or its antigen-binding moiety thereof, said isolated monoclonal antibody or its antigen-binding moiety specifically binding to amyloid fibrils, wherein each of the variable domains of the light chain, CDRL1, CDRL2, and CDRL3, is present. (a) Has at least 90% sequence identity with SEQ ID No. 9, SEQ ID No. 10 and SEQ ID No. 11 respectively; or (b) optionally having a single amino acid change relative to SEQ ID No. 9, SEQ ID No. 10 and SEQ ID No. 11, respectively.

5. An isolated monoclonal antibody or its antigen-binding moiety thereof, wherein the isolated monoclonal antibody or its antigen-binding moiety specifically binds to amyloid fibrils, wherein... (a) Each of CDRH1, CDRH2, and CDRH3 in the variable domain of the heavy chain has at least 90% sequence identity 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 has at least 90% sequence identity 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 relative 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 relative to SEQ ID No. 9, SEQ ID No. 10 and SEQ ID No. 11, respectively.

6. An isolated monoclonal antibody or its antigen-binding moiety thereof, said isolated monoclonal antibody or its antigen-binding moiety specifically binding to amyloid fibrils, wherein the heavy chain variable region has at least 70% sequence identity with SEQ ID No.

2.

7. An isolated monoclonal antibody or its antigen-binding moiety thereof, said isolated monoclonal antibody or its antigen-binding moiety specifically binding to amyloid fibrils, wherein the light chain variable region has at least 70% sequence identity with SEQ ID No.

4.

8. An isolated monoclonal antibody or its antigen-binding moiety thereof, said isolated monoclonal antibody or its antigen-binding moiety specifically binding to amyloid fibrils, wherein the heavy chain variable region has at least 70% sequence identity with SEQ ID No. 2, and the light chain variable region has at least 70% sequence identity with SEQ ID No.

4.

9. The isolated monoclonal antibody or its antigen-binding fragment according to any one of the preceding claims, wherein the isolated monoclonal antibody or its antigen-binding fragment... (a) Binds to epitopes present in the amyloid fibrils, but not to native peptides that form the fibrils; and / or (b) Does not bind to Aβ fibrils present in the CNS and / or brain of patients with Alzheimer's disease; and / or (c) Does not compete with SAP for binding fibrils.

10. The isolated monoclonal antibody or its antigen-binding fragment according to claim 9, wherein the epitope is present in at least three amyloid fibrils selected from the following: ALκ, ALλ, ATTR wild-type, ATTR variant, AA, AApoAI, ALys, Aβ2m and AFib amyloid fibrils.

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

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

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

14. The isolated monoclonal antibody or its antigen-binding fragment according to any one of claims 11 to 13, wherein citrate ions and / or malonate ions are localized within the antibody binding site when examined by X-ray crystallography.

15. The isolated monoclonal antibody or its antigen-binding portion according to any one of the preceding claims, wherein the antibody is selected from IgG, IgA or an antigen-binding antibody fragment selected from: antibody monovariable domain polypeptide, dAb, FAb, F(ab')2, scFv, Fv, V HH Domains (such as Nanobody® or other camel-derived immunoglobulin domains) or disulfide-bound Fv, human antibodies, preferably chimeric antibodies containing human variable regions, humanized antibodies, bispecific antibodies or single-chain antibodies.

16. The isolated monoclonal antibody according to any one of the preceding claims, wherein the Fc region of the antibody is derived from mouse IgG2 or human IgG1 isotype.

17. The isolated monoclonal antibody according to claim 15 or claim 16, wherein the isolated monoclonal antibody is a humanized antibody.

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

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

20. The humanized monoclonal antibody or its antigen-binding portion according to claim 19 or 20, wherein each of CDRH1, CDRH2, and CDRH3 in the variable domain of the heavy chain has at least 90% sequence identity 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 at least 90% sequence identity with SEQ ID No. 9, SEQ ID No. 10, and SEQ ID No. 11, respectively.

21. The humanized monoclonal antibody according to any one of claims 19 to 21, wherein the human framework region is derived from the antibody gene selected from SEQ ID No. 12 and 13.

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

23. The humanized monoclonal antibody according to any one of claims 18 to 22, wherein the VH region comprises mutant L55K and I66K.

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

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

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

27. The humanized monoclonal antibody according to any one of claims 18 to 23, wherein the humanized monoclonal antibody has a combination of light chain framework mutations and heavy chain framework mutations, the combination comprising a light chain framework mutation consisting of I2K, L39M, A40Y and N66K and a heavy chain framework mutation consisting of L55K, I66K and E69P.

28. The humanized monoclonal antibody according to any one of claims 18 to 24, wherein the humanized monoclonal antibody has a combination of light chain framework mutations and heavy chain framework mutations, the combination comprising a light chain framework mutation consisting of A40Y and a heavy chain framework mutation consisting of L55K, I66K, V25A, D85N and E69P.

29. The humanized monoclonal antibody according to any one of claims 18 to 23, wherein the humanized monoclonal antibody has a combination of light chain framework mutations and heavy chain framework mutations, the combination comprising a light chain framework mutation consisting of A40Y and a heavy chain framework mutation consisting of L55K and I66K.

30. The humanized monoclonal antibody according to claim 17, wherein the VL domain has at least 90% sequence identity with SEQ ID No. 14, and the VH domain has at least 90% sequence identity with SEQ ID No.

15.

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

17.

32. The humanized monoclonal antibody according to claim 17, wherein the VL domain has at least 90% sequence identity with SEQ ID No. 18, and the VH domain has at least 90% sequence identity with SEQ ID No.

19.

33. The humanized monoclonal antibody according to claim 17, wherein the VL domain has at least 90% sequence identity with SEQ ID No. 20, and the VH domain has at least 90% sequence identity with SEQ ID No.

21.

34. The humanized monoclonal antibody according to claim 17, wherein the VL domain has at least 90% sequence identity with SEQ ID No. 22, and the VH domain has at least 90% sequence identity with SEQ ID No.

23.

35. The humanized monoclonal antibody according to claim 17, wherein the VL domain has at least 90% sequence identity with SEQ ID No. 24, and the VH domain has at least 90% sequence identity with SEQ ID No.

25.

36. The humanized monoclonal antibody according to claim 17, wherein the VL domain of the isolated humanized monoclonal antibody has at least 90% sequence identity with SEQ ID No. 26, and the VH domain has at least 90% sequence identity with SEQ ID No.

27.

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

29.

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

31.

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

33.

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

35.

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

37.

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

39.

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

41.

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

43.

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

45.

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

47.

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

49.

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

51.

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

53.

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

55.

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

57.

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

59.

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

61.

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

63.

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

65.

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

67.

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

69.

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

71.

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

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

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

62. The humanized monoclonal antibody or antigen-binding fragment thereof according to any one of claims 59 to 61, wherein citrate ions and / or malonate ions are localized at the antibody binding site when examined by X-ray crystallography.

63. The humanized monoclonal antibody or its antigen-binding portion according to any one of claims 17 to 62, wherein the antibody is selected from IgG, IgA or an antigen-binding antibody fragment selected from the following: antibody monovariable domain polypeptide, dAb, FAb, F(ab')2, scFv, Fv, V HH Domains (such as Nanobody® or other camel-derived immunoglobulin domains) or disulfide-bound Fv.

64. The humanized monoclonal antibody according to any one of claims 17 to 63, wherein the Fc region of the antibody is derived from the human IgG1 isotype.

65. The humanized monoclonal antibody or its antigen-binding portion according to claim 66, wherein the antibody belongs to the hIgG1 isotype.

66. When the isolated monoclonal antibody or humanized antibody or its antigen-binding portion according to any one of the preceding claims is administered parenterally to mice suffering from experimentally induced systemic AA amyloidosis, the isolated monoclonal antibody or humanized antibody or its antigen-binding portion also effectively promotes the regression of systemic murine AA amyloid deposits.

67. The isolated monoclonal antibody or humanized antibody or antigen-binding moiety according to any one of the preceding claims, wherein the in vivo efficacy of the isolated monoclonal antibody or humanized antibody or antigen-binding moiety is complement-dependent.

68. The isolated monoclonal antibody or humanized antibody or its antigen-binding portion according to any one of the preceding claims, wherein the in vivo efficacy of the isolated monoclonal antibody or humanized antibody or its antigen-binding portion is Fcγ receptor binding dependent.

69. The isolated monoclonal antibody or humanized antibody according to any one of the preceding claims, wherein the isolated monoclonal antibody or humanized antibody is used to treat a disease.

70. The isolated monoclonal antibody or humanized antibody for use according to claim 22, wherein the disease is amyloidosis.

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

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

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