Anti-linear antibodies

Abstract

The present invention relates to isolated monoclonal antibodies or their antigen-binding moieties that specifically bind to amyloid fibrils and effectively promote the clearance of extracellular amyloid deposits from tissues in vivo, as well as to humanized antibodies having the same properties.

Description

Technical Field

[0001] The present invention provides novel antibodies having broad specificity for different types of amyloid fibrils that cause all major forms of human systemic amyloidosis, humanized monoclonal antibodies targeting multiple types of amyloid fibrils, and methods for using such antibodies and their use in the therapeutic applications of antibodies.

[0002] Introduction Amyloidosis is a severe disease caused by extracellular deposition of insoluble abnormal fibrils resulting from 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]. Approximately 30 different proteins are known to form amyloid fibrils in vivo in humans, each associated with clinically distinct conditions (https: / / doi.org / 10.1080 / 13506129.2020.1835263). Systemic amyloidosis with amyloid deposition in internal organs, blood vessel walls, and connective tissues is usually fatal and causes approximately 1 death per 1000 people in developed countries. Cardiac transthyretin amyloidosis, known as ATTR, is a predominantly fatal disease in elderly men, and recently the prevalence in those over 75 years old has been recognized to be 3 - 5%. The diagnosis of all forms of amyloidosis is clinically difficult, usually leading to a delayed diagnosis, with limited effectiveness of many existing treatments, significant toxicity, and often resulting in poor outcomes. Therefore, there is a large unmet medical need for amyloidosis.

[0003] Amyloidosis can be acquired as a complication of a pre-existing primary disease that either produces amyloid-forming abnormal proteins or is normal but potentially significantly increases exposure to amyloid-forming proteins, or it can be caused in older adults by the normal expression of wild-type transthyretin, which is inherently amyloid-forming. Hereditary amyloidosis is caused by a mutated gene that codes for a variant protein that leads to amyloid formation. Amyloid deposition may be localized, limited to specific organs or tissues, or it may be systemic, occurring throughout the body except in the brain. Systemic amyloidosis is overwhelmingly either AL or ATTR type, with various hereditary types being rare. Systemic AA amyloidosis, a complication of chronic infections and other inflammatory diseases, is now rare in developed countries but remains more common in other regions.

[0004] The tissue and organ damage that manifests as a clinical disease in amyloidosis is directly caused by the relentless accumulation of extracellular fibril amyloid deposits. These destroy the structure of the affected tissue and, therefore, its function. For unknown reasons, the normal mechanisms for the clearance of debris from the extracellular space remove amyloid deposits very slowly, if at all. Thus, existing management of amyloidosis includes (a) supportive therapies to maintain the function of the damaged organs, including multiple organ transplants, and (b) measures to reduce the abundance of each amyloid fibril precursor protein and / or stabilize its original folding to prevent fibril formation. The goal is to reduce or halt the ongoing accumulation of amyloid and to expect a slow, spontaneous, but ultimately clinically beneficial regression of amyloid that occurs in some patients. However, the scope and effectiveness of interventions intended to prevent amyloid formation are limited, and there are no specific treatments for the many different types of amyloidosis and for many patients.

[0005] A direct approach to the removal of amyloid deposits in vivo is known in the art, specifically targeting serum amyloid-P component (SAP), which is an invariant, normal, constitutive plasma protein universally present in all human amyloid deposits due to its intense, specific, calcium-dependent binding to all types of amyloid fibrils [Pepys, MB, Front.Immunol., 2018.9:p.2382.]. SAP binding to amyloid fibrils promotes their persistence in vivo [Tennent, GA, et al., Proc.Natl.Acad.Sci.USA, 1995.92(10):p.4299-4303]. Therefore, a small molecule drug called miridesap was created, which specifically binds to SAP [Pepys, MB, et al., Nature, 2002. 417(6886): p. 254-259]. It was intended to remove SAP from amyloid deposits. However, while miridesap almost completely depletes circulating SAP, it does not remove all SAP from amyloid deposits and does not promote amyloid clearance [Gillmore, JD, et al., Br. J. Haematol., 2010. 148(5): p. 760-767]. Nevertheless, the depletion of circulating SAP by myridesap has enabled the safe and effective administration of anti-SAP antibodies targeting all types of amyloid deposits [Bodin, K., et al., Nature, 2010. 468(7320): p. 93-97]. The essential therapeutic partnership between myridesap and anti-SAP antibodies has established proof of concept for safe and clinically beneficial amyloid removal with complement-activated IgG antibodies in patients with different forms of systemic amyloidosis [Richards, DB, et al., N.Engl.J.Med., 2015. 373(12): p. 1106-1114, Richards, DB, et al., Sci.Transl.Med., 2018. 10(422): p. eaan3128.]. Despite these unprecedented and highly promising results, the treatment has not yet reached the stage of approved drugs.

[0006] While SAPs bound to all types of amyloid fibrils are attractive target antigens, the actual goal of antibody immunotherapy is the removal of amyloid fibrils themselves. Unfortunately, it has long been known that amyloid fibrils are very unimmunogenic. Patients with amyloidosis produce very few specific anti-amyloid fibril antibodies, and experimental animals show little response even when strongly immunized with heterofibrils. On the other hand, all types of ex vivo amyloid fibrils share very similar morphology, ultrastructure, and protein folding, and in particular, the cross-β-core structure is common to all types of ex vivo amyloid fibrils, regardless of their completely unrelated protein sequences [Sunde, M., et al., J. Mol. Biol., 1997. 273: p. 729-739]. Therefore, we and others hypothesize that amyloid fibrils may potentially share epitope structures, but despite claims of several putative cross-reactive antibodies, no truly broad-spectrum anti-amyloid fibril antibodies have been reported in the prior art. [Overview of the project]

[0007] In a first embodiment, a monoclonal antibody or its antigen-binding moiety is provided that has broad antigen fibril specificity and specifically binds to amyloid fibrils. In one embodiment, the antibody of the present invention can bind to at least nine different types of amyloid fibrils, including, for example, ALκ, ALλ, 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 Ab fibrils present in the CNS, particularly in the brains of patients with Alzheimer's disease.

[0008] The antibody according to the present invention preferably has an antibody or its antigen-binding moiety in which the CDR within the variable domain of the heavy chain has 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 its antigen-binding moiety in which the CDR within the variable domain of the light chain has 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 embodiments, the antibody according to the present invention is preferably an antibody or its antigen-binding moiety in which the CDR within the variable domain of the surrogate light chain has 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 present invention is preferably an antibody or its antigen-binding moiety in which the CDR within the variable domain of the light chain has the sequence of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, which optionally includes one amino acid change.

[0012] In one embodiment, the antibody according to this aspect of the present invention may include heavy chain CDRs having SEQ ID NOs. 6-8 and light chain CDRs having SEQ ID NOs. 9-11.

[0013] In one embodiment, the antibody according to this aspect of the present invention may include heavy chain CDRs having SEQ ID NOs. 6-8 and light chain CDRs having SEQ ID NOs. 12-14.

[0014] In one embodiment, the antibody of the present invention comprises a heavy chain variable region having sequence identity of at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, at least 90%, 91%, 92%, 93%, 94%, at least 95%, 96%, 97%, 98%, at least 99%, or 100% with SEQ ID NO: 2.

[0015] In one embodiment, the antibody of the present invention comprises a light chain variable region having sequence identity of at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, at least 90%, 91%, 92%, 93%, 94%, at least 95%, 96%, 97%, 98%, at least 99%, or 100% with SEQ ID NO: 4.

[0016] In one embodiment, the antibody of the present invention comprises a light chain variable region having sequence identity of at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, at least 90%, 91%, 92%, 93%, 94%, at least 95%, 96%, 97%, 98%, at least 99%, or 100% with SEQ ID NO: 6.

[0017] In this embodiment, the antibody is either human isotope IgG1 or mouse isotype IgG2.

[0018] In the above embodiments of the present invention, the antibody can be selected from human antibodies, chimeric antibodies containing a human variable region, humanized antibodies, bispecific antibodies, or single-chain antibodies, as well as their antigen-binding fragments.

[0019] In the embodiments, the antibody of the present invention is specific to the three-dimensional epitopes commonly presented in amyloid fibrils. Therefore, the antibody can bind to amyloid-specific epitopes regardless of the linear polypeptide structure of the fibrils.

[0020] In the embodiment, the three-dimensional 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 ALκ, ALλ, ATTR wild-type, ATTR mutant, AA, AApoAI, Alys, Aβ2m, and AFib amyloid fibrils.

[0021] In the embodiment, the epitope is formed at the C-terminus of the protein constituting the fibril. In the embodiment, the epitope comprises at least one charged amino acid, preferably at least two charged amino acids, including a charged side chain. Examples of amino acids having a charged side chain include aspartic acid, histidine, glutamic acid, lysine, and arginine.

[0022] In the embodiment, the stereostructure epitope includes a C-terminal carboxyl group, preferably a free C-terminal carboxyl group.

[0023] In the embodiment, the stereochemical epitope can be mimicked by both the malonate ion and the citrate ion.

[0024] In a further embodiment, the present invention provides a method for producing antibodies according to a first embodiment of the present invention, which includes immunizing a mammal lacking the SAP gene.

[0025] In one embodiment, SAP knockout mice that do not express the mouse SAP protein are immunized with human synthetic ATTR fibrillary material.

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

[0027] The humanized antibody of the present invention binds well to the fibrils that cause or are involved in systemic amyloidosis. The humanized antibody does not bind to the Ab fibrils present in the CNS, particularly in the brains of Alzheimer's disease patients.

[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 ALκ, ALλ, ATTR wild-type, ATTR variant, AA, AApoAI, ALys, Aβ2m, and AFib amyloid fibrils.

[0030] In an embodiment, the humanized antibody of the present invention does not compete with SAP, preferably human SAP, for binding to amyloid fibrils. Therefore, the epitope bound by the antibody of the present invention is not the same as the epitope bound by SAP.

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

[0032] In an embodiment, the epitope bound by the antibody of the present invention includes the C-terminus of the protein constituting the fibril. In an embodiment, the epitope includes at least one charged amino acid, preferably at least two charged amino acids, including a charged side chain. Examples of amino acids having a charged side chain include aspartic acid, glutamic acid, lysine, and arginine.

[0033] In an embodiment, the epitope includes a C-terminal carboxyl group, preferably a free C-terminal carboxyl group.

[0034] In the embodiment, citrate ions and / or malonate ions can be located at the binding site of the antibody according to the present invention when examined by X-ray crystallography.

[0035] Humanized monoclonal antibodies or their antigen-binding moieties may have CDRH1, CDRH2, and CDRH3 within the variable domain of the heavy chain, each having at least 90% sequence identity with SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively.

[0036] Furthermore, or alternatively, a humanized monoclonal antibody or its antigen-binding moiety may have CDRL1, CDRL2, and CDRL3 within the variable domain of the light chain, each having at least 90% sequence identity with SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively.

[0037] In another embodiment, the humanized monoclonal antibody or its antigen-binding moiety may have CDRH1, CDRH2, and CDRH3 within the variable domain of the heavy chain, each having at least 90% sequence identity with SEQ ID NOs. 6, 7, and 8, respectively. Furthermore, the humanized monoclonal antibody or its antigen-binding moiety may have CDRL1, CDRL2, and CDRL3 within the variable domain of the light chain, each having at least 90% sequence identity with SEQ ID NOs. 9, 10, and 11, respectively.

[0038] In another embodiment, the antibody according to the present invention may have one optimal amino acid change in one or more CDRs compared with the sequence IDs of the CDRs described herein.

[0039] In another optional embodiment, the humanized monoclonal antibody may have a human framework region derived from an antibody gene selected from SEQ ID NOs: 12 and 13.

[0040] Isolated humanized monoclonal antibodies may have framework residues that have been mutated to match mouse residues. These mutated residues may include VL residues I2, L39, A40, Q44, A49, V101, N66, T85, and F87, and VH residues M39, A80, L55, I66, V25, D85, E69, A45, P46, G47, and K48.

[0041] Isolated humanized monoclonal antibodies may have VH regions containing mutants L55K and I66K.

[0042] Isolated humanized monoclonal antibodies may have combinations of light-chain and heavy-chain framework mutations. Light-chain framework mutations may consist of I2K, A40Y, N66K, T85N, and F87Y. Heavy-chain framework mutations may consist of L55K, I66K, V25A, D85N, and E69P.

[0043] Selectively isolated humanized monoclonal antibodies may contain combinations of light-chain and heavy-chain framework mutations. Light-chain framework mutations may consist of I2K, L39M, A40Y, N66K, T85N, and F87Y. Heavy-chain framework mutations may consist of M39I, A80T, L55K, I66K, E69P, A45R, P46T, G47E, and K48Q.

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

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

[0046] Isolated humanized monoclonal antibodies may have combinations of light-chain and heavy-chain framework mutations. Light-chain framework mutations may consist of A40Y. Heavy-chain framework mutations may consist of L55K, I66K, V25A, D85N, and E69P.

[0047] Selectively isolated humanized monoclonal antibodies may contain combinations of light-chain and heavy-chain framework mutations. Light-chain framework mutations may consist of A40Y. Heavy-chain framework mutations may consist of L55K and I66K.

[0048] In another optional embodiment, the VL domain of the isolated humanized monoclonal antibody may have at least 90% sequence identity with SEQ ID NO: 14, and the VH domain may have at least 90% sequence identity with SEQ ID NO: 15.

[0049] Alternatively, the VL domain of the isolated humanized monoclonal antibody may have at least 90% sequence identity with SEQ ID NO: 16, and the VH domain may have at least 90% sequence identity with SEQ ID NO: 17.

[0050] In another optional embodiment, the VL domain of the isolated humanized monoclonal antibody may have at least 90% sequence identity with SEQ ID NO: 18, and the VH domain may have at least 90% sequence identity with SEQ ID NO: 19.

[0051] Optionally, the VL domain of the isolated humanized monoclonal antibody may have at least 90% sequence identity with SEQ ID NO: 20, and the VH domain may have at least 90% sequence identity with SEQ ID NO: 21.

[0052] Alternatively, the VL domain of the isolated humanized monoclonal antibody may have at least 90% sequence identity with SEQ ID NO: 22, and the VH domain may have at least 90% sequence identity with SEQ ID NO: 23.

[0053] Alternatively, the VL domain of the isolated humanized monoclonal antibody may have at least 90% sequence identity with SEQ ID NO: 24, and the VH domain may have at least 90% sequence identity with SEQ ID NO: 25.

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

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

[0056] The VH domain may include mutations containing L55K or I66K, but not both, the addition of E69P, and variants of D57E and / or D62E in HCDR2.

[0057] In this embodiment, the VL domain of the isolated humanized monoclonal antibody may have at least 90% sequence identity with SEQ ID NO: 26, and the VH domain may have at least 90% sequence identity with SEQ ID NO: 27.

[0058] Alternatively, the VL domain of the isolated humanized monoclonal antibody may have at least 90% sequence identity with SEQ ID NO: 28, and the VH domain may have at least 90% sequence identity with SEQ ID NO: 29.

[0059] Alternatively, the VL domain of the isolated humanized monoclonal antibody may have at least 90% sequence identity with SEQ ID NO: 30, and the VH domain may have at least 90% sequence identity with SEQ ID NO: 31.

[0060] Alternatively, the VL domain of the isolated humanized monoclonal antibody may have at least 90% sequence identity with SEQ ID NO: 32, and the VH domain may have at least 90% sequence identity with SEQ ID NO: 33.

[0061] Alternatively, the VL domain of the isolated humanized monoclonal antibody may have at least 90% sequence identity with SEQ ID NO: 34, and the VH domain may have at least 90% sequence identity with SEQ ID NO: 35.

[0062] Alternatively, the VL domain of the isolated humanized monoclonal antibody may have at least 90% sequence identity with SEQ ID NO: 36, and the VH domain may have at least 90% sequence identity with SEQ ID NO: 37.

[0063] Alternatively, the VL domain of the isolated humanized monoclonal antibody may have at least 90% sequence identity with SEQ ID NO: 38, and the VH domain may have at least 90% sequence identity with SEQ ID NO: 39.

[0064] Alternatively, the VL domain of the isolated humanized monoclonal antibody may have at least 90% sequence identity with SEQ ID NO: 40, and the VH domain may have at least 90% sequence identity with SEQ ID NO: 41.

[0065] Alternatively, the VL domain of the isolated humanized monoclonal antibody may have at least 90% sequence identity with SEQ ID NO: 42, and the VH domain may have at least 90% sequence identity with SEQ ID NO: 43.

[0066] Alternatively, the VL domain of the isolated humanized monoclonal antibody may have at least 90% sequence identity with SEQ ID NO: 44, and the VH domain may have at least 90% sequence identity with SEQ ID NO: 45.

[0067] Alternatively, the VL domain of the isolated humanized monoclonal antibody may have at least 90% sequence identity with SEQ ID NO: 46, and the VH domain may have at least 90% sequence identity with SEQ ID NO: 47.

[0068] Alternatively, the VL domain of the isolated humanized monoclonal antibody may have at least 90% sequence identity with SEQ ID NO: 48, and the VH domain may have at least 90% sequence identity with SEQ ID NO: 49.

[0069] Alternatively, the VL domain of the isolated humanized monoclonal antibody may have at least 90% sequence identity with SEQ ID NO: 50, and the VH domain may have at least 90% sequence identity with SEQ ID NO: 51.

[0070] Alternatively, the VL domain of the isolated humanized monoclonal antibody may have at least 90% sequence identity with SEQ ID NO: 52, and the VH domain may have at least 90% sequence identity with SEQ ID NO: 53.

[0071] Alternatively, the VL domain of the isolated humanized monoclonal antibody may have at least 90% sequence identity with SEQ ID NO: 54, and the VH domain may have at least 90% sequence identity with SEQ ID NO: 55.

[0072] Alternatively, the VL domain of the isolated humanized monoclonal antibody may have at least 90% sequence identity with SEQ ID NO: 56, and the VH domain may have at least 90% sequence identity with SEQ ID NO: 57.

[0073] Alternatively, the VL domain of the isolated humanized monoclonal antibody may have at least 90% sequence identity with SEQ ID NO: 58, and the VH domain may have at least 90% sequence identity with SEQ ID NO: 59.

[0074] Alternatively, the VL domain of the isolated humanized monoclonal antibody may have at least 90% sequence identity with SEQ ID NO: 60, and the VH domain may have at least 90% sequence identity with SEQ ID NO: 61.

[0075] Alternatively, the VL domain of the isolated humanized monoclonal antibody may have at least 90% sequence identity with SEQ ID NO: 62, and the VH domain may have at least 90% sequence identity with SEQ ID NO: 63.

[0076] Alternatively, the VL domain of the isolated humanized monoclonal antibody may have at least 90% sequence identity with SEQ ID NO: 64, and the VH domain may have at least 90% sequence identity with SEQ ID NO: 65.

[0077] Alternatively, the VL domain of the isolated humanized monoclonal antibody may have at least 90% sequence identity with SEQ ID NO: 66, and the VH domain may have at least 90% sequence identity with SEQ ID NO: 67.

[0078] Alternatively, the VL domain of the isolated humanized monoclonal antibody may have at least 90% sequence identity with SEQ ID NO: 68, and the VH domain may have at least 90% sequence identity with SEQ ID NO: 69.

[0079] Alternatively, the VL domain of the isolated humanized monoclonal antibody may have at least 90% sequence identity with SEQ ID NO: 70, and the VH domain may have at least 90% sequence identity with SEQ ID NO: 71.

[0080] In the embodiments described above, "at least 90%" should be understood as identity with the enumerated array IDs of 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 a maximum of 100%.

[0081] The monoclonal antibody or its antigen-binding portion may be selected from an antigen-binding antibody fragment selected from IgG, IgA, or an antibody single variable domain polypeptide, dAb, FAb, F(ab')2, scFv, Fv, VHH domain (such as Nanobody® or other camelid immunoglobulin domains), or a disulfide-linked Fv, a human antibody, preferably a chimeric antibody containing a human variable region, a humanized antibody, a bispecific antibody, or a single-chain antibody.

[0082] A humanized monoclonal antibody or its antigen-binding moiety may have the Fc region of an antibody derived from a human IgG1 isotype. In certain embodiments, the antibody is an hIgG1 isotype.

[0083] Humanized monoclonal antibodies or their antigen-binding molars, when administered parenterally to mice with experimentally induced systemic AA amyloidosis, can also effectively promote the regression of systemic mouse AA amyloidosis.

[0084] Humanized monoclonal antibodies or their antigen-binding moieties may possess complement activation-dependent in vivo efficacy.

[0085] Alternatively, or in addition, humanized monoclonal antibodies or their antigen-binding moieties may have in vivo efficacy that is Fcγ receptor-dependent.

[0086] In further embodiments, the antibodies of the present invention have been shown to be used for the treatment of systemic amyloidosis, and thus a pharmaceutical composition comprising the antibodies defined herein for use in the treatment of systemic amyloidosis is provided.

[0087] In a further embodiment, the use of antibodies as defined herein in the preparation of a composition for the treatment of systemic amyloidosis is provided. In a further embodiment, a method for treating a subject suffering from systemic amyloidosis is provided, comprising administering a composition comprising antibodies specific to amyloid fibrils as described herein to the subject in need of treatment.

[0088] In preferred embodiments, the pharmaceutical composition is co-administered with supportive therapy for amyloidosis. Prior art amyloidosis therapies typically aim to eliminate or reduce the presence or production of amyloid precursors, and the antibody is designed to remove established amyloid deposits. Therefore, together with existing or novel therapies for removing amyloid, the antibody of the present invention provides a more complete treatment for amyloidosis.

[0089] In some embodiments, the antibodies of the present invention can be administered independently of other amyloidosis treatments, for example, when it is desirable to minimize the generation of amyloid precursors and deplete established amyloid fibrils.

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

[0091] In this disclosure, particularly in the claims and / or paragraphs, terms such as “comprises,” “comprised,” and “comprising” may have the meanings assigned to them in patent law, for example, they may mean “includes,” “included,” and “including,” and terms such as “consisting essentially of” and “consists essentially of” may have the meanings assigned to them in patent law. For example, they may allow elements that are not explicitly enumerated but exclude elements found in the prior art or elements that affect the basic or novel features of the present invention.

[0092] These and other embodiments are disclosed or evident from the following detailed description and are included therein. [Brief explanation of the drawing]

[0093] [Figure 1] Generation of best-in-class anti-amyloid antibody 2E5 with broad specificity for amyloid deposits. A) Generation of 2E5 antibody by a novel mouse fibrillary immunotherapy strategy. B) 10 μg / mL antibody 2E5 specifically binds to ATTR fibrillaries in ELISA but does not bind to immobilized soluble, spherical, human or mouse transthyretin (TTR). C) 10 μg / mL antibody 2E5 specifically stains tissue deposits of major forms of human systemic amyloid, AL, ATTR, and AA, as well as rare hereditary forms (image labeled AF488). Amyloid locations within the same field of view are indicated by strong fluorescence with Congo red counterstaining. [Figure 2]Anti-amyloid fibril antibody 2E5 binds to mouse AA amyloid and removes amyloid in vivo. A) 10 μg / mL of antibody 2E5 specifically stains mouse AA amyloid deposits (image labeled AF488) identified by Congo red staining in serial sections. B) Antibody 2E5 removes amyloid in vivo. Liver amyloid load score in systemic AA amyloidosis mice 16 days after a single IP injection of 4.8 mg / mouse 2E5 compared to untreated controls. Mann-Whitney test: control vs. 2E5: p=0.01278 [Figure 3] Binding of malonic acid and citrate to 2E5. A: Binding of citrate in the antibody binding groove of 2E5 Fab, crystal structure. B: Comparison of the spatial arrangement and changes of malonic acid, citrate, and the TTR C-terminus. [Figure 4] The BLI assay for antibody 2E5 binding to deletion of TTR99-127 was set up as shown in the figure, and the deletion of peptide 99-127 was tested for binding to 2E5. The results are shown in graph and checkbox format. Deletion of the C-terminus of the peptide eliminates binding to 2E5. Similarly, amidation of the C-terminal amino acid results in loss of binding, indicating that the free C-terminal COOH is essential for binding. [Figure 5] BLI assay of TTR99-127 alanine scan: The BLI assay was performed on a variant of 99-127 in which the C-terminal amino acid was substituted with alanine. The three C-terminal amino acids (P, K, E) are increasingly essential for 2E5 binding. [Figure 6] Competitive ELISA between 2E5 mAb and hSAP on coated ATTR fibrils. Within a physiologically relevant window, a constant concentration of 2E5 mAb was incubated with increasing concentrations of hSAP. Binding of 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. [Figure 7]ELISA of antibodies binding to ATTR fibrils after the initial humanization experiment, compared to mouse 2E5 antibody, showing the binding of humanized antibodies to ATTR fibrils. A: CDR grafts show binding to 1 / 100th compared to 2E5. B: Control in the absence of ATTR. [Figure 8] ELISA for an optimized CDR transplant antibody that binds to ATTR fibrils, 10 ug / ml. [Figure 9] ELISA plots of interactions between humanized clones and synthetic amyloid fibrils of CDR-transplanted antibodies binding to various amyloid fibrils. The fibrils are truncated fragments of immunoglobulin light chains (AL55-133), as well as variants of transthyretin protein (S52PTTR) and beta-2-microglobulin (D76N Abeta2-m), and synthetic amyloid derived from AA (amyloid A). [Modes for carrying out the invention]

[0094] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art, such as in the fields of peptide chemistry, cell culture and phage display, nucleic acid chemistry and biochemistry. The following references provide many common definitions of terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994), The Cambridge Dictionary of Science and Technology (Walker ed., 1988), The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991), and Hale & Marham, The Harper Collins Dictionary of Biology (1991). Where used herein, unless otherwise specified, the following terms have the meanings given to them below.

[0095] Standard techniques used in molecular biology, genetic, and biochemical methods ("Molecular Cloning: A Laboratory Manual", second edition (Sambrook, 1989), "Oligonucleotide Synthesis" (Gait, 1984), "Animal Cell Culture" (Freshney, 1987), "Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1996), "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987), "Current Protocols in Molecular Biology" (Ausubel, 1987), "PCR: The Polymerase Chain Reaction" (Mullis, 1994), "Current Protocols in Immunology" (Coligan, 1991)) are incorporated herein by reference. These techniques are applicable to the production of polynucleotides and polypeptides of the present invention and can therefore be considered when constructing and carrying out the present invention. Techniques particularly useful for specific embodiments are described in the following sections.

[0096] "Antibody" refers to IgG, IgA, or antibody single variable domain polypeptide, dAb, FAb, F(ab')2, scFv, Fv, V HH The antibody type or fragment may be selected from a domain (such as Nanobody® or other camelized immunoglobulin domains) or an antigen-binding antibody fragment selected from a disulfide-linked Fv. In certain embodiments, either the antibody type or fragment thereof may be prepared from one or more mammalian species selected from, but not limited to, mouse, rat, rabbit, and human. Such antibodies can be humanized for use in humans.

[0097] In certain embodiments, either the antibody type or fragment thereof may be provided as a heteroconjugate, bispecific, single-stranded, chimeric, or humanized molecule having affinity for amyloid fibrils.

[0098] In a particular embodiment, any of the aforementioned antibodies bind to amyloid with a dissociation coefficient of 100 nM or less, 75 nM or less, 50 nM or less, 25 nM or less, for example, 10 nM or less, 5 nM or less, 1 nM or less, or in an embodiment, 500 pM or less, 100 pM or less, 50 pM or less, or 25 pM or less.

[0099] Antibodies may be monospecific, having narrow or broad specificity, or they may be multispecific, such as bispecific, having two different epitope specificities in a single antibody molecule. An antibody cocktail may target two or more specific epitopes. An antibody cocktail may 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, each recognizing multiple amyloid fibrils.

[0100] In one embodiment, the antibody is monoclonal and binds to at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten different amyloid fibrillary types. Advantageously, it substantially binds to all systemic amyloid fibrillary types.

[0101] In one embodiment, the antibody of the present invention is formulated for intravenous (IV) or intramuscular (IM) administration. The IV-administered antibody should be released from circulation to enter the interstitial tissue space and bind to its homologous targets.

[0102] In one embodiment, the antibody is scFv, dAb, or V HHThese are antibody fragments, such as antibodies. Small antibody fragments are much easier to exfiltrate into tissues, and for this reason, they can perform better than IgG or other larger antibodies. However, smaller fragments are also eliminated from circulation more quickly. A compromise must be found between tissue accessibility and clearance. See, for example, Wang et al., Clinical pharmacology & Therapeutics, 84:5, 2008, 548-558. Several antibody conjugates have been described that have extended half-lives using various strategies, such as conjugation to albumin (e.g., human serum albumin). See, for example, Kontermann et al., BioDrugs April 2009, Volume 23, Issue 2, pp93-109.

[0103] The mechanism by which the antibodies according to the present invention promote the removal of amyloid deposits is presumed to involve complement activation by antibodies bound to amyloid fibrils, leading to the recruitment of macrophages, which then fuse with 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 possess a unique phenotype that enables the destruction of overwhelmingly large extracellular amyloid deposits in relation to single cells (Milde, R., et al. (2015). "Multinucleated giant cells are specialized for complement-mediated phagocytosis and large target destruction." Cell Rep. 13(9):1937-1948). Therefore, antibody fragments, intact antibodies, and any other constructs that do not efficiently activate the classical complement pathway when bound to amyloid fibrils are favorably modified or further modified to promote complement activation. In preferred embodiments, the antibody fragment or derivative is an antibody fragment or derivative that is appropriately modified to activate complement.

[0104] A "fragment" means a portion of a polypeptide or nucleic acid molecule. This portion preferably contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the total length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

[0105] The terms “isolated,” “purified,” or “biologically pure” refer to substances from which components normally associated with them, as found in their natural state, have been removed to varying degrees. “Isolation” indicates a degree of separation from the original source or surroundings. “Purification” indicates a higher degree of separation than isolation. A “purified” or “biologically pure” protein contains so little other material that any impurities will not significantly affect the protein’s biological properties or cause other harmful consequences. That is, the nucleic acids or peptides of this invention are purified if, when produced by recombinant DNA techniques, they are substantially free of cellular material, viral material, or culture medium, or if, when chemically synthesized, they are substantially free of chemical precursors or other chemicals. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term “purified” can mean that the nucleic acid or protein produces essentially one band on an electrophoretic gel. In the case of proteins that can undergo modifications, such as phosphorylation or glycosylation, different modifications may result in different isolated proteins, which can be purified separately.

[0106] "Isolated polynucleotide" means nucleic acid (e.g., DNA) that does not contain genes adjacent to the gene in the naturally occurring genome of the organism from which the nucleic acid molecule of the present invention originates. Therefore, this term includes recombinant DNA that is, for example, incorporated into a vector, incorporated into an autonomously replicating plasmid or virus, incorporated into the genomic DNA of a prokaryote or eukaryote, or existing as a separate molecule independently of other sequences (e.g., cDNA or a genome or cDNA fragment produced by PCR or restriction nuclease digestion). Furthermore, this term includes RNA molecules transcribed from DNA molecules, and recombinant DNA that is part of a hybrid gene encoding an additional polypeptide sequence.

[0107] "Isolated polypeptide" means the polypeptide of the present invention isolated from its naturally associated components. Typically, a polypeptide is isolated if it does not contain at least 60% by weight of naturally associated proteins and naturally occurring organic molecules. Preferably, the preparation contains at least 75% by weight, more preferably at least 90% by weight, and most preferably at least 99% by weight, the polypeptide of the present invention. The isolated polypeptide of the present invention can be obtained, for example, by extraction from a natural source, by expression of recombinant nucleic acids encoding such polypeptides, or by chemical synthesis of proteins. Purity can be measured by any suitable method, such as column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0108] Amyloid Amyloidosis and amyloid fibrils are well known in the art. Approximately 30 different proteins are known to form amyloid fibrils in vivo in humans, each associated with a clinically distinct condition. For an overview and definition, see Pepys, MB and PNHawkins (2020). Amyloidosis. Oxford Textbook of Medicine. J. Firth, C. Conlon and T. Cox, Oxford University Press, and the Nomenclature Report of the International Society for Amyloidosis (https: / / doi.org / 10.1080 / 13506129.2020.1835263).

[0109] Amyloid fibrils are typically aggregates of proteins assembled into beta sheets. The ends of the proteins can be exposed in the beta-sheet structure, and it is hypothesized that these exposed ends of the proteins constituting the amyloid fibrils are involved in the binding of panfibril-specific antibodies to amyloid. In embodiments, the C-terminus of the protein is bound by the antibody, and the free C-terminal carboxyl group in the TTR peptide is essential for binding, requiring a charged ligand 2E5.

[0110] The amyloid epitope formed by the C-terminal amino acids of TTR can be mimicked by both citrate and malonate ions in spatial and charge interactions within the antibody-binding domain. The C-terminal structure of TTR(PKE) is very similar to that of citrate and malonate. Citrate forms binding interactions with N30, F90, T91, Y104, N105, and W106 in the 2E5 binding groove, and citrate and malonate bind in almost identical ways, but are not large enough to contact the heavy chain.

[0111] antibody The term "monoclonal antibody" refers to an antibody obtained from a single clone of a B lymphocyte-derived plasma cell that produces an antibody with uniform heavy and light chain classes and epitope specificity.

[0112] Monoclonal antibodies are typically highly specific and directed against a single antigenic site (epitope), in contrast to conventional antibodies in antiserum that are induced in the whole animal by immunization with a specific antigen. Such conventional antibodies, derived from many different clones of B lymphocytes that recognize either the same or different epitopes on the immunized antigen, are known as polyclonal antibodies. In addition to their very limited specificity, monoclonal antibodies are readily produced in a pure form uncontaminated with other immunoglobulins, whereas the isolation of specific antibodies from polyclonal antiserum requires demanding immunopurification procedures.

[0113] Monoclonal antibodies can be prepared by hybridoma (see Kohler et al., Nature, 256:495-7, 1975) or by recombinant DNA. Monoclonal antibodies can also be isolated from phage antibody libraries using well-known techniques.

[0114] The monoclonal antibodies used herein specifically include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain is identical 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 insofar as they exhibit the desired biological activity (U.S. Patent No. 4,816,567, Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

[0115] In the hybridoma method, a host animal, typically a mouse, is immunized with a desired antigen to induce the production of clones of B lymphocytes that produce antibodies specifically binding to that antigen, or that are capable of producing such antibodies. Lymphocytes collected from the immunized animal are then fused in vitro with a serial line of myeloma cells grown in vitro to form so-called hybridoma cells. These are then selected by growth in a suitable medium that allows only the fused cells to survive, while preventing the survival of unfused parental myeloma cells. Examples of myeloma cells include, but are not limited to, human myeloma cell lines and mouse-human heterozygous myeloma cell lines described for the production of human monoclonal antibodies.

[0116] Culture media derived from proliferating hybridoma cells may be assayed for monoclonal antibodies that target antigens. The binding specificity of antibodies produced by 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).

[0117] After hybridoma cells producing the desired antibody are identified, the clones may be subcloned by limiting dilution procedures and proliferated by standard methods. The monoclonal antibodies secreted by the subclones are isolated from the culture medium or serum by well-known immunoglobulin purification procedures such as protein A-Sepharose, gel electrophoresis, dialysis, hydroxyapatite chromatography, or affinity chromatography.

[0118] The antibodies of the present invention also include variants of such antibodies and fragments thereof. Examples of variants include peptides and polypeptides comprising substitutions, deletions, and / or additions of one or more amino acid sequences having the same or substantially the same epitope binding affinity and specificity as the anti-amyloid antibody or fragment thereof.

[0119] Deletion, insertion, or substitution of amino acid residues can result in silent changes and functionally equivalent substances. Intentional ammo acid substitutions can be made based on the similarity of residues in terms of polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity. For example, negatively charged amino acids include aspartic acid and glutamic acid, positively charged amino acids include lysine and arginine, and amino acids with similar hydrophilic values ​​and uncharged head groups include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

[0120] Homologous substitutions (wherein used both terms substitution and replacement to mean the exchange of an existing amino acid residue with a substitute residue), i.e., substitutions of the same kind, such as basic to basic, acid to acid, or polar to polar, may occur. Non-homologous substitutions, i.e., substitutions from one class of residue to another, or alternatively, the inclusion of non-natural amino acids such as ornithine (Z in this specification), ornithine diaminobutyrate (B in this specification), norleucine ornithine (O in this specification), pyrylalanine, thienylalanine, naphthylalanine, and phenylglycine, may also occur.

[0121] Substitution with non-natural amino acids is also possible, including alpha* and alpha-disubstituted* amino acids, N-alkyl amino acids*, halogenated derivatives of natural amino acids such as lactic acid* and trifluorotyrosine*, p-Cl-phenylalanine*, p-Br-phenylalanine*, pI-phenylalanine*, L-allyl-glycine*, β-alanine*, L-α-aminobutyric acid*, L-γ-aminobutyric acid*, L-α-aminoisobutyric acid*, L-ε-aminocaproic acid#, 7-aminoheptanoic acid*, L-methionine sulfone#*, L-norleucine*, L-norvaline*, p-nitro-L-phenylalanine*, L-Hydro Examples include roxyproline#, L-thioproline*, methyl derivatives of phenylalanine (Phe) (4-methyl-Phe*, pentamethyl-Phe*, L-Phe(4-amino)#, L-Tyr(methyl)*, L-Phe(4-isopropyl)*, L-Tic(1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid)*, L-diaminopropionic acid#, and L-Phe(4-benzyl)*. The notation * is used to indicate the hydrophobicity of the derivative for the purposes of the above explanation (regarding homologous or non-homologous substitution), # is used to indicate the hydrophilicity of the derivative, and #* indicates amphiphilic properties.

[0122] Accordingly, variants may include peptides and polypeptides comprising substitutions, deletions, and / or additions of one or more amino acid sequences to the antibodies and fragments of the present invention, such substitutions, deletions, and / or additions not causing substantial changes in epitope binding affinity and specificity. For example, variants of an antibody or fragment thereof may arise from one or more alterations to the antibody or fragment thereof, and the altered antibody or fragment thereof has the same or substantially the same epitope binding affinity and specificity as the starting sequence. Variants may be naturally occurring, such as alleles or splice variants, or may be artificially constructed. Variants may be prepared from the corresponding nucleic acid molecule encoding the variant. Variants of an antibody or fragment thereof may have alterations in the light chain and / or heavy chain amino acid sequences, which may be naturally occurring or introduced by in vitro manipulation of the natural sequence using recombinant DNA technology. Examples of naturally occurring variants include “somatic cell” variants that are generated in vivo in the corresponding germline nucleotide sequence during the production of an antibody response to a foreign antigen.

[0123] Antibody and binding fragment variants may also be prepared by mutagenesis techniques. For example, amino acid changes may be introduced randomly throughout the antibody coding region, and the resulting variants may be screened for binding affinity to amyloid or other properties. Alternatively, amino acid changes may be introduced into selected regions of the antibody, e.g., light chain and / or heavy chain CDRs, and / or framework regions, and the resulting antibodies may be screened for binding to amyloid or some other activity. Amino acid changes encompass one or more amino acid substitutions within a CDR, ranging from single amino acid differences to the introduction of multiple permutations of amino acids within a given CDR. Variants generated by inserting amino acids to increase the size of the CDR are also included.

[0124] Amyloid-specific antibodies or fragments thereof may have a modified Fc region in which a naturally occurring Fc region is modified to increase the half-life of the antibody or fragment in the biological environment, for example, the serum half-life or half-life measured by an in vitro assay. The modification of Fc can also be used to alter the in vivo distribution of the antibody, thereby directing the antibody to tissues containing amyloid.

[0125] The variant also comprises an antibody or fragment thereof containing a modified Fc region, the modified Fc region containing at least one amino acid modification relative to the wild-type Fc region. The variant Fc region may be designed to bind to the Fc receptor with higher or lower affinity compared to an equivalent molecule containing the wild-type Fc region. For example, the antibody and fragment thereof may contain a modified Fc region. The Fc region refers to a natural or synthetic polypeptide homologous to the IgG C-terminal domain produced during papain digestion of IgG. IgG Fc has a molecular weight of approximately 50 kD. In the antibody and fragment, either the entire Fc region or only the half-life-enhancing portion may be used.

[0126] Antibodies and their fragments also encompass derivatives of antibodies, fragments, and sequences disclosed herein. Derivatives include chemically modified polypeptides or peptides, or their variants, fragments, or derivatives. Examples include covalent bonds 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 those naturally occurring or from the starting peptide or polypeptide, either in terms of the type or position of the attached molecule. Derivatives also include deletions of one or more chemical groups naturally present on the peptide or polypeptide.

[0127] The present invention also encompasses amyloid-specific antibodies comprising two full-length heavy chains and two full-length light chains. Alternatively, the antibody may be a construct such as a single-chain antibody or a “mini” antibody that retains amyloid-binding activity. Such constructs can be prepared by methods known in the art. Advantageously, such fragments are modified to enable complement activation.

[0128] A method for producing recombinant DNA versions of the antigen-binding region of antibody molecules, bypassing the production of monoclonal antibodies, is envisioned for amyloid-specific antibodies and their fragments. The DNA is cloned into a plasmid vector system. One example of such a technique uses a bacteriophage-lambda vector system with a leader sequence that moves or secretes the expressed Fab protein into the periplasmic space (the space between the bacterial cell membrane and the cell wall). A large number of functional Fab fragments that bind to amyloid can be rapidly generated and screened. Such amyloid-binding agents (Fab fragments with amyloid specificity) are specifically incorporated within amyloid-specific antibodies and their fragments.

[0129] Amyloid-binding antibodies and their fragments may be humanized antibodies or human-manipulated antibodies. As used herein, "humanized antibody" or its antigen-binding fragment is a recombinant polypeptide comprising a portion of an antigen-binding site derived from a non-human antibody and a portion of the framework and / or constant region of a human antibody. Human-manipulated antibodies or antibody fragments are non-human (e.g., mouse) antibodies that have been manipulated by modifying (e.g., deleting, inserting, or substituting) amino acids at specific positions to reduce or eliminate any detectable immunogenicity of the modified antibody in humans.

[0130] Humanized antibodies include chimeric antibodies and CDR-transplanted antibodies. Chimeric antibodies are antibodies that contain a non-human antibody variable region linked to a human constant region. Therefore, in chimeric antibodies, the variable region is mostly non-human, and the constant region is human. Chimeric antibodies and methods for producing them are described, for example, in Proc. Natl. Acad. Sci. USA, 81:6841-6855 (1984). Although they may have lower immunogenicity than 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 a gene from a mouse antibody molecule with appropriate antigen-binding specificity together with a gene from a human antibody molecule with appropriate biological activity, such as the ability to activate human complement and mediate antibody-dependent cell phagocytosis (ADCP). One example is replacing the Fc region with an Fc region of a different isotype.

[0131] CDR-transplanted antibodies are antibodies containing a CDR from a non-human "donor" antibody linked to a framework region from a human "recipient" antibody. Generally, CDR-transplanted antibodies contain more human antibody sequences than chimeric antibodies because they include both constant region sequences and variable region (framework) sequences derived from human antibodies. Therefore, for example, the CDR-transplanted humanized antibody of the present invention may include a continuous amino acid sequence (e.g., about 5 or more, 10 or more, or 15 or more continuous amino acid residues) from the framework region of a human antibody (e.g., FR-I, FR-2, or FR-3 of a human antibody), or optionally, a heavy chain containing most or all of the entire framework region of the human antibody. CDR-transplanted antibodies and methods for producing them are described in Nature, 321:522-525 (1986). Methods that can be used to produce humanized antibodies are also described, for example, in US5,721,367 and 6,180,377.

[0132] A "veneered antibody" is a non-humanized or humanized (e.g., chimeric or CDR-transplanted antibody) antibody that has been manipulated to replace specific solvent-exposed amino acid residues in order to reduce their immunogenicity or enhance their function. Veneering of a chimeric antibody may involve identifying solvent-exposed residues within the non-human framework region of the chimeric antibody and replacing at least one of them with a corresponding surface residue from the human framework region.

[0133] Veneer construction can be achieved by any suitable engineering technique.

[0134] Further details on antibodies, humanized antibodies, humanized antibodies, and methods for preparing them can be found in Antibody Engineering, Springer, New York, NY, 2001.

[0135] Examples of humanized or human-manipulated antibodies include IgG, IgM, IgE, IgA, and IgD antibodies. Antibodies may be of any class (IgG, IgA, IgM, IgE, IgD, etc.) or isotype and may contain kappa or lambda light chains. For example, a human antibody may contain an IgG heavy chain or a defined fragment, such as at least one of the isotypes IgG1, IgG2, IgG3, or IgG4. As a further example, an antibody or fragment thereof may contain an IgG1 heavy chain and a kappa or lambda light chain.

[0136] Human antibodies targeting amyloid can be produced using transgenic animals that lack endogenous immunoglobulin production and are engineered to contain human immunoglobulin loci, as described in WO98 / 24893 and WO91 / 00906.

[0137] The transgenic animals described above can be used to induce an immune response to selected antigen molecules, remove antibody-producing cells from the animals, and produce hybridomas that secrete human monoclonal antibodies. Immunization protocols, adjuvants, etc., are known in the art and are used, for example, for immunization of transgenic mice.

[0138] The development of techniques for creating a repertoire of recombinant human antibody genes, and the presentation of encoded antibody fragments on the surface of filamentous bacteriophages, provided a means for directly producing human antibodies.

[0139] Antibodies produced by phage technology are generated as antigen-binding fragments (usually Fv fragments or Fab fragments) within bacteria and therefore lack effector function.

[0140] Effector function can be introduced by one of two strategies: the fragment can be modified into either a complete antibody for expression in mammalian cells, or a bispecific antibody fragment having a second binding site capable of inducing effector function.

[0141] Human antibodies can be generated 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 easily prepared. The libraries may contain diverse human antibody sequences, such as human Fab, Fv, and scFv fragments, which can be screened for appropriate targets. Phage display libraries may contain peptides or proteins other than antibodies and can be screened to identify drugs that can selectively bind to amyloid.

[0142] The phage display process mimics immunoselection through the presentation of an antibody repertoire on the surface of filamentous bacteriophages and subsequent selection of phages by their binding to a select antigen. One such method is described in WO99 / 10494. Anti-amyloid antibodies can be isolated by screening recombinant combined antibody libraries, preferably scFv phage display libraries, prepared using human VL and VHcDNA prepared from human lymphocyte-derived mRNA. Methodologies for preparing and screening such libraries are known in the art. There are commercially available kits for preparing phage display libraries.

[0143] Amyloid-binding antibodies and their fragments may contain one or more moieties that do not bind to amyloid but instead perform other functions such as circulating half-life, direct cytotoxic effect, detectable labeling, or activation of the recipient's endogenous complement cascade or endogenous cytotoxicity. The antibody or its fragment may contain all or part of a constant region and may be any isotype including IgA (e.g., IgAl or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3 or IgG4), or IgM. In addition to, or instead of containing, a constant region, the antigen-binding compound of the present invention may contain an epitope tag, a salvage receptor epitope, a labeling moiety for diagnostic or purification purposes, or a cytotoxic moiety such as a radionuclide or toxin.

[0144] Anti-amyloid antibodies or fragments thereof may be modified to increase their serum half-life, for example, by adding molecules such as PEG or other water-soluble polymers containing polysaccharide polymers.

[0145] Amyloid-binding antibodies and their fragments may be bispecific. For example, a bispecific antibody may 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. A bispecific antibody may have binding specificity to at least two different epitopes, at least one of which is an amyloid epitope.

[0146] Amyloid-binding antibodies and fragments may be heteroantibodies. A heteroantibody is two or more antibodies or antibody-binding fragments (Fabs) linked together, where each antibody or fragment has a different specificity. As used herein, the term “antibody fragment” refers to a portion of an intact full-length antibody, e.g., the 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, tripspecific, and multispecific antibodies (e.g., diabodies, triabodies, tetrabodies); binding domain immunoglobulin fusion proteins; camelized antibodies; minibodies; chelated recombinant antibodies; tribodies or bibodies; intrabody antibodies; nanobodies; small modular immunopharmaceuticals (SMIPs); VHH-containing antibodies; and any other polypeptides formed from antibody fragments.

[0147] In connection with the present invention, the terms anti-amyloid antibody and amyloid-binding antibody encompass amyloid-binding antibody fragments that include any portion of the heavy chain or light chain sequence of a full-length antibody and bind to amyloid.

[0148] As used herein, the term “fragment” refers to any fragment capable of binding to amyloid, such as at least three consecutive amino acids of an antibody involved in antigen binding (e.g., at least four, five, six, seven, eight, nine, or ten, or more, consecutive amino acids from a CDR), and includes Fab, Fab', F(ab')2, and F(v) fragments, or individual light chain or heavy chain variable regions or parts thereof. Examples of amyloid-binding fragments include Fab, Fab', F(ab')2, Fv, and scFv. These fragments lack the Fc fragment of the intact antibody, are eliminated from circulation more rapidly, and exhibit less nonspecific tissue binding than the intact antibody. These fragments can be produced from an intact antibody using well-known methods, for example, by enzymatic proteolytic cleavage using papain (to produce the Fab fragment) or pepsin (to produce the F(ab')2 fragment).

[0149] Amyloid-binding antibodies and fragments also include single-chain antibody fragments (scFv) that bind to amyloid. The scFv comprises an antibody heavy-chain variable region (VH) operably linked to an antibody light-chain variable region (VL), where the heavy-chain and light-chain variable regions, together or individually, form a binding site for amyloid. The scFv may contain the VH region at the amino terminus or the VL region at the carboxyl terminus. Alternatively, the scFv may contain the VL region at the amino terminus and the VH region at the carboxyl terminus. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked by a synthetic linker that allows them to be constructed as a single protein chain (known as single-chain Fv (scFv)) where the VL and VH regions pair to form a monovalent molecule, using a recombination method. The scFv may optionally further contain a polypeptide linker between the heavy-chain and light-chain variable regions.

[0150] Amyloid-binding antibodies and fragments also include domain antibody (dAb) fragments consisting of the VH domain, as described in Nature 341:544-546 (1989).

[0151] Amyloid-binding antibodies and fragments also include heavy chain antibodies (HCAb). Since these antibodies are dimers of only the heavy chain (referred to as "heavy chain antibodies" or "HCAb"), it appears clear that they can form an antigen-binding region using only the variable region of the heavy chain. Therefore, amyloid-binding antibodies and fragments may also be heavy chain antibodies (HCAb) that specifically bind to amyloid.

[0152] Amyloid-binding antibodies and fragments also include antibodies that are SMIP or binding domain immunoglobulin fusion proteins specific to amyloid proteins. These constructs are single-chain polypeptides containing an antigen-binding domain fused to an immunoglobulin domain necessary for performing antibody effector function (see WO03 / 041600).

[0153] Amyloid-binding antibodies and fragments also contain diabodies. These are bivalent antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but which use a linker that is too short to allow pairing between the two domains on the same chain. This causes one domain to pair with a complementary domain on another chain, thereby creating two antigen-binding sites (see, for example, WO93 / 11161). Diabodies can be bispecific or monospecific.

[0154] Amyloid-binding antibodies and their fragments also contain immunoadhesins. One or more CDRs may be incorporated into a molecule either covalently or noncovalently to form an immunoadhesin. The immunoadhesin may incorporate the CDR as part of a larger polypeptide chain, the CDR may be covalently bonded to another polypeptide chain, or the CDR may be incorporated noncovalently. The CDR enables the immunoadhesin to specifically bind to amyloid.

[0155] Amyloid-binding antibodies and their fragments also encompass antibody mimetics containing one or more amyloid-binding moieties constructed on organic or molecular scaffolds (such as protein or carbohydrate scaffolds). Proteins with relatively clear three-dimensional structures, commonly referred to as protein scaffolds, can be used as reagents for the design of antibody mimetics. These scaffolds typically contain one or more regions to accommodate specific or random sequence mutations, and such sequence randomization is often performed to generate a library of proteins from which the desired product can be selected. For example, an antibody mimetic may include a chimeric non-immunoglobulin-binding polypeptide containing immunoglobulin-like domains having two or more solvent-exposed loops that contain different CDRs from the parent antibody inserted into each loop and exhibit selective binding activity to the ligand bound by the parent antibody. Non-immunoglobulin protein scaffolds have been proposed to obtain proteins with novel binding properties.

[0156] Anti-amyloid antibodies or their antibody fragments typically bind to human amyloid with high affinity (e.g., measured by BIACORE), and the equilibrium binding dissociation constant (KD) for amyloid is approximately ≤15 nM, ≤10 nM, ≤5 nM, ≤1 nM, ≤500 pM, ≤250 pM, ≤100 pM, ≤50 pM, or ≤25 pM, ≤10 pM, ≤50 pM, or ≤25 pM, ≤10 pM, ≤5 pM, ≤3 pM, ≤1 pM, ≤0.75 pM, or ≤0.5 pM.

[0157] 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 extent.

[0158] Humanization Humanized versions of non-human (e.g., mouse) antibodies are chimeric antibodies containing the smallest sequence derived from non-human immunoglobulins. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient's hypervariable region are replaced with residues from the hypervariable region of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, possessing the desired specificity, affinity, and capability. In some cases, FR residues of human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues not found in the recipient or donor antibody. These modifications are made to further improve antibody performance. Generally, a humanized antibody contains substantially all of the variable domains, at least one, typically two, with all or substantially all of the hypervariable region corresponding to the hypervariable region of a non-human immunoglobulin, and all or substantially all of the FR region being the FR region of a human immunoglobulin sequence. Humanized antibodies will also, optionally, contain at least a portion of the constant region (Fc) of an immunoglobulin, typically a portion of the constant region (Fc) of a human immunoglobulin.

[0159] Some or all of the CDRs of the antibodies described herein may be migrated, and it is possible to retain human acceptor CDRs, for example, as long as donor CDRH3 is migrated. All members of the immunoglobulin superfamily share similar folding of their polypeptide chains. For example, although antibodies are highly diverse with respect to their primary sequences, comparisons of sequences and crystallographic structures have unexpectedly revealed that five of the six antigen-binding loops of antibodies (H1, H2, L1, L2, L3) employ a limited number of main chain conformations, i.e., canonical structures (Chothia and Lesk (1987) J.Mol.Biol., 196:901, Chothia et al. (1989) Nature, 342:877). Therefore, analysis of loop length and major residues has made it possible to predict the H1, H2, L1, L2, and L3 main chain conformations 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).

[0160] Pharmaceutical composition The pharmaceutical composition may include, in addition to the antibody, one or more pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, stabilizers, preservatives, lubricants, or other materials well known to those skilled in the art. Preferred substances are sterile, pyrogen-free, and possess suitable isotonicity and stability. Examples include sterile saline (e.g., 0.9% NaCl), water, dextrose, glycerol, ethanol, etc., or combinations thereof. Such materials must be non-toxic and must not interfere with the efficacy of the active compound. The exact properties of the carrier or other materials will depend on the route of administration, which may be by infusion, injection, or any other suitable route, as described below. Preferred substances are sterile, pyrogen-free, and possess suitable isotonicity and stability. Examples include sterile saline (e.g., 0.9% NaCl), water, dextrose, glycerol, ethanol, etc., or combinations thereof. The composition may further contain auxiliary substances such as wetting agents, emulsifiers, and pH buffers.

[0161] Suitable carriers, excipients, etc., can be found in standard pharmaceutical textbooks, such as Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990.

[0162] As used herein, the term “pharmaceutically acceptable” refers to compounds, materials, compositions, and / or dosage forms suitable for use in contact with the tissues of a subject (e.g., human) within the bounds of sound medical judgment, without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio. Each carrier, excipient, etc., must also be “acceptable” in the sense that it is compatible with the other components of the formulation.

[0163] In some embodiments, the antibody may be provided in a lyophilized form for reconstitution before administration. For example, the lyophilized reagent may be reconstituted in sterile water and mixed with physiological saline before administration to the subject.

[0164] Formulations may be conveniently presented in unit dosage forms and may be prepared by any method well known in the field of pharmacy. Such methods include the step of associating an active compound with a carrier constituting one or more auxiliary components. Generally, formulations are prepared by homogeneously and closely associating an active compound with a liquid carrier or a finely divided solid carrier or both, and then shaping the product as needed.

[0165] The formulation may be in the form of a liquid, solution, suspension, emulsion, or the like.

[0166] Other therapeutic or prophylactic agents may be included in the pharmaceutical composition or formulation at the discretion of the party.

[0167] Treatment may be a treatment or therapy in either a human or an animal (e.g., in a veterinary use) that includes several desired therapeutic effects, such as inhibiting or delaying the progression of a disease, reducing the rate of progression, stopping the rate of progression, improving the disease, curing or relieving the disease (whether partially or completely), preventing, delaying, reducing or stopping one or more symptoms and / or signs of the disease, or extending the survival of a subject or patient beyond the expected survival in the absence of treatment.

[0168] This also includes treatment as a preventative measure (i.e., prevention). For example, subjects who are susceptible to or at risk of developing or relapsing amyloidosis may be treated as described herein. Such treatment may prevent or delay the development or relapsing of amyloidosis in the subject.

[0169] In particular, treatment may include inhibiting amyloid deposition, including the complete reversal of amyloid deposition.

[0170] The antibody may be administered in a therapeutically effective dose as described herein.

[0171] As used herein, the term “therapeutic dose” refers to the amount of an active compound, or combination, material, composition, or dosage form containing an active compound, that is effective in producing several desired therapeutic effects in proportion to a reasonable benefit-to-risk ratio.

[0172] It should be understood that the appropriate dose of the active compound may vary from patient to patient. Determining the optimal dose generally requires balancing the level of therapeutic benefit against any risks or adverse side effects of the administration. The selected dose level depends on a variety of factors, including but not limited to the route of administration, time of administration, excretion rate of the active compound, other drugs, compounds, and / or substances used concomitantly, and the patient's age, sex, weight, condition, overall health, and prior medical history. The amount and route of administration of the active compound are ultimately determined at the physician's discretion, but generally, the dose should achieve a concentration of the active compound at the treatment site without causing serious harm or adverse side effects.

[0173] Generally, the preferred dose of an active compound is in the range of approximately 100 μg to 250 mg per kg of body weight per day. If the active compound is a salt, ester, prodrug, etc., the dosage is calculated based on the parent compound, and therefore the actual weight used increases proportionally.

[0174] In vivo administration can be performed in a single dose, either continuously or intermittently (e.g., in divided doses at appropriate intervals). Methods for determining the most effective means and dose of administration are well known to those skilled in the art and will vary depending on the formulation used for treatment, the purpose of treatment, the target cells being treated, and the subject being treated. Single or multiple doses can be administered using dose levels and patterns selected by the physician.

[0175] Multiple doses of the antibody may be administered; for example, two, three, four, five, or more than five doses may be administered.

[0176] The pharmaceutical composition containing the active compound may be formulated into a suitable dosage unit formulation appropriate to the intended route of administration.

[0177] Formulations suitable for oral administration (e.g., ingestion) may be presented as individual units such as capsules, cachetes, or tablets, each containing a predetermined amount of the active compound; as powders or granules; as solutions or suspensions in aqueous or non-aqueous liquids; as oil-in-water or water-in-oil liquid emulsions; as boluses; as licks; or as pastes.

[0178] Tablets can be prepared by conventional means, such as compression or molding, using one or more optional auxiliary components. Compressed tablets can be prepared by compressing an active compound in a free-flowing form, such as powder or granules, mixed with one or more optionally selected binders (e.g., povidone, gelatin, acacia, sorbitol, tragacanth, hydroxypropyl methylcellulose); fillers or diluents (e.g., lactose, microcrystalline cellulose, calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, silica); disintegrants (e.g., sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethylcellulose); surfactants, dispersants, or wetting agents (e.g., sodium lauryl sulfate); and preservatives (e.g., p-methyl hydroxybenzoate, p-propyl hydroxybenzoate, sorbic acid), using a suitable machine. Molded tablets can be prepared by molding a mixture of powder compounds moistened with an inert liquid diluent, using a suitable machine. The tablets may optionally be coated or scored, and may be formulated to provide a slow or controlled release of the active compound therein, for example, using varying proportions of hydroxypropyl methylcellulose, in order to provide a desired release profile. The tablets may optionally have an enteric coating to provide release to the intestinal portion other than the stomach.

[0179] Formulations suitable for parenteral administration (e.g., by injection, including cutaneous, subcutaneous, intramuscular, intravenous, and intradermal) include aqueous and non-aqueous isotonic, pyrogen-free, sterile injection solutions that may contain antioxidants, buffers, preservatives, stabilizers, bacteriostatic agents, and solutes that make the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents and thickeners, and liposomes or other particulate systems designed to target the compound to blood components or one or more organs. Examples of suitable isotonic vehicles for use in such formulations include sodium chloride injection, Ringer's solution, or lactated Ringer's injection. Typically, the concentration of the active compound in the solution is about 1 ng / ml to about 10 μg / ml, for example, about 10 ng / ml to about 1 μg / ml. The formulations may be presented in sealed containers of one or more doses, such as ampoules and vials, and may be stored in a freeze-dried state requiring only the addition of a sterile liquid carrier, such as water for injection, immediately before use. Instantaneous injectable solutions and suspensions may be prepared from sterile powders, granules, and tablets. The formulations may also be in the form of liposomes or other particulate systems designed to target blood components or one or more organs with the active compound.

[0180] The composition may be prepared in the form of a concentrate for subsequent dilution, or in the form of divided doses ready for administration. Alternatively, the reagents may be provided separately in a kit for mixing before administration to human or animal subjects.

[0181] identity Nucleic acid molecules useful in the methods of the present invention include any nucleic acid molecules encoding the polypeptide or a fragment thereof of the present invention. Such nucleic acid molecules do not need to be 100% identical to the endogenous nucleic acid sequence, but typically exhibit substantial identity. Polynucleotides having “substantial identity” with the endogenous sequence can typically hybridize with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the present invention include any nucleic acid molecules encoding the polypeptide or a fragment thereof of the present invention. Such nucleic acid molecules do not need to be 100% identical to the endogenous nucleic acid sequence, but typically exhibit substantial identity. Polynucleotides having “substantial identity” with the endogenous sequence can typically hybridize with at least one strand of a double-stranded nucleic acid molecule. “Hybridize” means a pair for forming a double-stranded molecule between complementary polynucleotide sequences (e.g., the genes described herein) or a portion thereof under various stringency conditions. (See, for example, Wahl, G.M. and S. B. L. Berger (1987) Methods Enzymol. 152:399 and Kimmel, AR (1987) Methods Enzymol. 152:507).

[0182] For example, stringent salt concentrations are typically less than about 750 mM NaCl and less than 75 mM trisodium citrate, preferably less than about 500 mM NaCl and less than 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and less than 25 mM trisodium citrate. Low-stringency hybridization can be obtained in the absence of an organic solvent, such as formamide, while high-stringency hybridization can be obtained in the presence of at least about 35% formamide, more preferably at least about 50% formamide. Stringent temperature conditions typically include temperatures of at least about 30°C, more preferably at least about 37°C, and most preferably at least about 42°C. Various additional parameters such as hybridization time, detergent concentration (e.g., sodium dodecyl sulfate (SDS)), and inclusion or exclusion of carrier DNA are well known to those skilled in the art. Various levels of stringency can be achieved by combining these various conditions as needed. In a preferred embodiment, hybridization is carried out at 30°C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization is carried out at 37°C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA). In the most preferred embodiment, hybridization is carried out at 42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA. Useful variations of these conditions will be readily apparent to those skilled in the art.

[0183] In most applications, the stringency of the washing step following hybridization also varies. Washing stringency conditions can be defined by salt concentration and temperature. As described above, washing stringency can be increased by decreasing the salt concentration or increasing the temperature. For example, the stringent salt concentration for the washing step is preferably less than about 30 mM NaCl and less than 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and less than 1.5 mM trisodium citrate. The stringent temperature conditions for the washing step typically include temperatures of at least about 25°C, more preferably at least about 42°C, and even more preferably at least about 68°C. In a preferred embodiment, the washing step is carried out at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the washing step is carried out at 42°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the washing step is carried out at 68°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations of these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977), Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975), Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001), Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York), and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0184] "Substantially identical" means a polypeptide or nucleic acid molecule that exhibits at least 90% identity with respect to a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or a nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). Preferably, such a sequence is identical to the sequence used for comparison by at least 91%, more preferably 92% or 93%, more preferably 94%, 95%, 96%, 97%, 98%, or even 99%, and up to 100%, at the amino acid level or nucleic acid level.

[0185] Sequence identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. An exemplary approach to determining the degree of identity may use the BLAST program, e -3 from e -100 The probability scores between these sequences indicate closely related sequences.

[0186] The ranges provided herein are understood to be abbreviated representations of all values ​​within that range. For example, the range 1 to 50 is understood to include any number, combination of numbers, or subrange 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.

[0187] Modifications to the above embodiments, further embodiments and their modifications will be apparent to those skilled in the art when reading this disclosure, and therefore they are within the scope of the present invention.

[0188] All documents and array database entries mentioned herein are incorporated herein in their entirety by reference for all purposes.

[0189] The present invention is further described below with reference to the following embodiments. [Examples]

[0190] Example 1 Antibody 2E5 was produced by immunization of SAP- / - mice using synthetic human ATTR amyloid fibrillary immunogen, via a variation of the classic hybridoma approach originally developed by Milstein and Koehler (Bickerstaff et al., Nat Med 1999 Jun;5(6):694-7) (Figure 1A).

[0191] ATTR fibrils in PBS were mixed with the adjuvant RIBI, and 50 mg of ATTR was injected at 2-week intervals: 25 mg of ATTR in week 0, followed by 25 mg of ATTR in week 2, and 25 mg of ATTR conjugated with SAP in week 4.

[0192] This approach provided a variety of monoclonal antibodies, including 2E5. ELISA screening confirmed specific binding to human ATTR fibrils, but not to the fibril precursor proteins, globular human TTR or mouse TTR (Figure 1B). 2E5 also showed unprecedented specific binding to all major types of human whole-body amyloid (Figure 1C) and experimentally induced [Bodin, K., et al., Nature, 2010.468(7320):p.93-97, Botto, M., et al., Nature Med., 1997.3(8):p.855-859] mouse AA amyloid deposits (Figure 2A). The original 2E5 mouse monoclonal isotype was IgG2c, homologous to human IgG1 and potently complement-activating. This is a necessary property for antibody-mediated amyloid removal in vivo, and we have demonstrated that it is complement-dependent [Bodin, K., et al., Nature, 2010.468(7320):p.93-97, Richards, DB, et al., N.Engl.J.Med., 2015.373(12):p.1106-1114, Milde, R., et al., Cell Rep., 2015.13(9):p.1937-1948]. In fact, administration of 2E5 to mice with established systemic AA amyloidosis resulted in significant amyloid clearance (Figure 2B).

[0193] Example 2 Crystallographic data from 2E5 showed that both the malonate and citrate ions are located in the antibody binding groove and adopt a structure that mimics the C-terminal structure of the TTR polypeptide. To identify the epitope to which 2E5 binds, peptide truncation studies and alanine scans were performed using TTR peptides (99-127), which have been previously shown to act as analogs of the full-length TTR protein.

[0194] Based on the human TTR sequence, the peptide was synthesized using a biotin-SGSG N-terminal tag as follows: -huTTR99-127 -huTTR105-115 -huTTR113-127 -huTTR118-127 -huTTR123-127 -huTTR99-127T123A -huTTR99-127N124A -huTTR99-127P125A -huTTR99-127K126A -huTTR99-127 E127A -huTTR99-127 Amidated C-terminus

[0195] For each peptide, use a storage solution (4 mg / ml) and add 2 mg of peptide to 500 µl or less: -50% acetonitrile -10% glacial acetic acid Prepared by dissolving in -40% H2O.

[0196] The peptide was diluted to 10 ug / ml in PBS for mapping using a biolayer interferometry (BLI) biosensor.

[0197] Recombinant expression was used to prepare 2E5-1B7 antibody (mouse IgG2a), which was then diluted to 40 ug / ml in PBS for BLI.

[0198] The binding of antibodies to each peptide was analyzed by pre-immersing a streptavidin BLI biosensor in PBS + 0.1% BSA for 10 minutes, equilibrating the peptides and mAbs to room temperature, and performing BLI measurements using a Blitz instrument (Blitz pro software), applying advanced kinetics as follows. 1.30 second baseline (PBS) 2. Fill the streptavidin biosensor with 10 ug / ml of biotinylated peptide over 2.60 seconds. 3.30 second baseline (PBS) 4.120-second meeting, 2E5-1B7, 40 ug / ml 5. Dissociation in PBS for 120 seconds.

[0199] In the reference run, the huTTR99-127 peptide was used in step 2 (above), and association with PBS only was performed in step 4 (above).

[0200] The binding affinity data was calculated within the Blitz Pro software, and the raw BLI trace data was exported for plotting.

[0201] The results indicate that the C-terminal deletion of peptide 99-127 invalidates antibody binding to the peptide. Therefore, peptides 99-127, 113-127, 118-127, and 123-127 retain identical binding properties (Figure 4), while truncation of 105-115 and 99-127 E127A could not be bound by 2E5.

[0202] Furthermore, C-terminal amidation and removal of the carboxyl group result in bond loss, indicating that the charged nature of the acidic C-terminus is essential for bonding.

[0203] A C-terminal alanine scan (Figure 5) showed that the T123A and N124A variants maintained normal binding, while binding progressively decreased in the P125A, K126A, and E127A variants. Therefore, the binding of 2E5 to the TTR peptide depends on the presence of charged amino acids at the C-terminus of the proteinifibrillar.

[0204] Example 3 2E5 does not compete with SAP in terms of binding to the raw fiber. To determine whether 2E5 and SAP bind to the same epitopes within amyloid fibrils, a competitive assay was performed to test the binding of 0.01 mg / ml of 2E5 to 0.5 mg of coated ATTR fibrils in the presence of various concentrations of SAP ranging from 0.08 to 50 mg / ml, and in the presence of an unrelated control antibody. Antibody detection was performed via the Fc domain using anti-mouse FcHRP. 2E5 was found to bind to fibrils in an SAP-independent manner, indicating that SAP and 2E5 do not compete for the same epitopes on ATTR fibrils.

[0205] The results are shown in Figure 6.

[0206] Example 4: CDR transfer of 2E5 Early CDR transplantation experiments produced three humanized antibodies, as illustrated in Table 1. [Table 1]

[0207] Antibodies 1-3 were tested by ELISA, and their binding to amyloid TTR fibrils was determined by comparing them to mouse 2E5. All CDR-implanted antibodies showed a binding reduction of approximately 1 / 100th. See Figures 7A and 7B.

[0208] Sequence and structural analysis of 2E5 was performed to identify key residues involved in providing structure to the CDR within the mouse framework. Key residues were identified and mutated back to mouse residues. Twenty humanized antibodies were generated and tested by ELISA.

[0209] The results are shown in Figure 8.

[0210] The results were ranked as shown in Table 2. [Table 2]

[0211] The six most promising antibodies showed strong binding to fibrils, comparable to mouse 2E5 (see Figure 4). P029_Ab004, 006, 008, 016, 019, and 021 were selected for further development.

[0212] Example 5 Charge reduction in humanized antibodies As shown in Table 3, higher affinity humanized antibodies were associated with higher positive charge. To reduce this charge to a level more favorable for pharmaceutical development, further modifications were made to the humanized clones, as shown in Table 4. This included the removal of positively charged residues by substitution with non-positively charged residues frequently observed in human antibodies. The addition of negatively charged residues was also used to reduce the overall positive charge. In addition to charge, mutations were introduced at the isomerization site to improve the developmental potential of the antibody in CDR-H2 (details are shown in Table 3).

[0213] Antibodies P029_Ab0019 and P029_Ab0008 were selected as base clones for modification. [Table 3] [Table 4]

[0214] The results of the modification procedure are shown in Table 5. The net charge was reduced to +4 or less in several examples. [Table 5]

[0215] To determine whether the broad reactivity of 2E5 to various amyloid types was maintained after humanization, the interactions of the humanized clone with synthetic fibrils and native amyloid were determined by ELISA. This included synthetic amyloid derived from the truncated fragment of immunoglobulin light chain (AL55-133), transthyretin protein (S52PTTR), a variant of beta-2-microglobulin (D76N Abeta2-m), and synthetic amyloid derived from AA (amyloid A) present in spleen extracts from mice. ELISA characterization confirmed that the humanized 2E5 clone could interact with these amyloid morphologies in a dose-dependent manner (Figure 9).

[0216] Sequence List

[0217] Sequence ID 1 Heavy chain variable region (VH) Nucleotide sequence GAGGTTCAGCTGCTGCAGTCTGGGGCAGAGCTTGTGAAGCCAGGGGCCTCAG TCAAGTTGTCCTGCACAGCTTCTGGCTTCAAGATTAAAGACTTCTATATACACTG GGTGAAACAGAGGACTGAACAGGGCCTGGACTGGATTGGAAAGATTGATCCTG AGGATGGTAAAACTAAATATGCCCCGAAATTCCAGGGCAAGGCCACTATAACAA CAGACACATCCTCCAATACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAG GACACTGCCGTCTATTACTGTGCTAGAGCCTACTATAGTAACTACAATTGGTTT GCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAG

[0218] Sequence ID 2 Heavy chain variable region (VH) amino acid sequence EVQLLQSGAELVKPGASVKLSCTASGFKIKDFYIHWVKQRTEQGLDWIGKIDPEDG KTKYAPKFQGKATITTDTSSNTAYLQLSSLTSEDTAVYYCARAYYSNYNWFAYWG QGTLVTVSA

[0219] Sequence ID 3 Light chain variable region (VL) Nucleotide sequence GAAAAAGTGCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCTAGGGGAGAA GGTCACCATGAGCTGCAGGGCCAGCTCAAGTGTAAATTACATGTACTGGTACC AGGAGAAGTCAGATGCCTCCCCCAAACTATGGATTTATTACACATCCAAGTTGG CTCCTGGAGTCCCAGCTCGCTTCAGTGGCAGTGGGTCTGGGAACTCTTATTCT CTCACAATCAGCAGCATGGAGGGTGAAGATGCTGCCACTTATTACTGCCAGCA GTTTACTAGTTCCCCATACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAAC

[0220] Sequence ID 4 Light chain variable region (VL) amino acid sequence EKVLTQSPAIMSASLGEKVTMSCRASSSVNYMYWYQEKSDASPKLWIYYTSKLAPGVPAR FSGSGNSYSLTISSMEGEDAATYYCQQFTSSPYTFGGGTKLEIK

[0221] Sequence ID 5 Light chain variable region (VL) substitute Nucleotide sequence GAAATTGTGCTCACCCAGTCTCCAACCACCATGGCTGCATCTCCCGGGGAGAA GATCACTATCACCTGCAGTGCCAGCTCAAGTATAAGTTCCAATTACTTGCATTG GTATCAGCAGAAGCCAGGATTCTCCCCTAAACTCTTGATTTATAGGACATCCAA TCTGGCTTCTGGAGTCCCAGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTT ACTCTCTCACAATTGGCACCATGGAGGCTGAAGATGTTGCCACTTACTACTGCC AGCAGGGTAGTAGTATACCGTACACGTTCGGAGGGGGGACCAAGCTGGAAAT AAAAC

[0222] Sequence ID 6 Heavy chain variable region CDRH1 GFKIKDFY

[0223] Sequence ID 7 Heavy chain variable region CDRH2 IDPEDGKT

[0224] Sequence ID 8 Heavy chain variable region CDRH3 ARAYYSNYNWFAY

[0225] Sequence ID 9 Light chain variable region CDRL1 SSVNY

[0226] Sequence ID 10 Light chain variable region CDRL2 YTS

[0227] Sequence ID 11 Light chain variable region CDRL3 QQFTSSPYT

[0228] Sequence ID 12 Heavy chain V gene, CDR transplant antibody EVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCWGQGTLVTVSS

[0229] Sequence ID 13 Light chain V gene, CDR transplant antibody EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCFGQGTKLEIK

[0230] Sequence ID 14 Clone P029_Ab04, VL EKVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQQKPGQAPRLLIYYTSKRATGIPARFSGSGSGNDYTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

[0231] Sequence ID 15 Clone P029_Ab04, VH EVQLVQSGAEVKKPGATVKISCKASGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTKYAPKFQGRVTITADTSTNTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

[0232] Sequence ID 16 Clone P029_Ab06, VL EKVLTQSPATLSLSPGERATLSCRASSSVNYMYWYQEKPGQSPRLLIYYTSKRATGIPARFSGSGSGNDYTLTISSLEPEDFATYYCQQFTSSPYTFGQGTKLEIK

[0233] Sequence ID 17 Clone P029_Ab06, VH EVQLVQSGAEVKKPGATVKISCKASGFKIKDFYIHWVQQRTEQGLEWMGKIDPEDGKTKYAPKFQGRVTITTDTSNTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

[0234] Sequence ID 18 Clone P029_Ab08, VL EIVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQQKPGQAPRLLIYYTSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

[0235] Sequence ID 19 Clone P029_Ab08, VH EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTKYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

[0236] Sequence ID 20 Clone P029_Ab16, VL EKVLTQSPATLSLSPGERATLSCRASSSVNYMYWYQEKPGQSPRLLIYYTSKRATGIPARFSGSGSGNDYTLTISSLEPEDFATYYCQQFTSSPYTFGQGTKLEIK

[0237] Sequence ID 21 Clone P029_Ab16, VH EVQLVQSGAEVKKPGATVKISCKASGFKIKDFYIHWVQQAPGKGLEWMGKIDPEDGKTKYAPKFQGRVTITTDTSNTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

[0238] Sequence ID 22 Clone P029_Ab19, VL EKVLTQSPATLLSPGERATLSCRASSSVNYMYWYQQKPGQAPRLLIYYTSKRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

[0239] Sequence ID 23 Clone P029_Ab19, VH EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTKYAPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

[0240] Sequence ID 24 Clone P029_Ab21, VL EIVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQQKPGQAPRLLIYYTSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

[0241] Sequence ID 25 Clone P029_Ab21, VH EVQLVQSGAEVKKPGATVKISCKASGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTKYAPKFQGRVTITADTSTNTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

[0242] Sequence ID 26 Clone P029_Ab25, VL EIVLTQSPATLLSPGERATLSCRASSSVNYMYWYQQKPGQAPRLLIYYTSKRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

[0243] Sequence ID 27 Clone P029_Ab25, VH EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTKYAPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

[0244] Sequence number 28 Clone P029_Ab26, VL EKVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQQKPGQAPRLLIYYTSKRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

[0245] Sequence number 29 Clone P029_Ab26, VH EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTKYAPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

[0246] Sequence number 30 Clone P029_Ab27, VL EKVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQQKPGQAPRLLIYYTSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

[0247] Sequence number 31 Clone P029_Ab27, VH EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTKYAPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

[0248] Sequence number 32 Clone P029_Ab28, VL EIVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQQKPGQAPRLLIYYTSKRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

[0249] Sequence number 33 Clone P029_Ab28, VH EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTKYAPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

[0250] Sequence number 34 Clone P029_Ab29, VL EIVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQQKPGQAPRLLIYYTSKRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

[0251] Sequence number 35 Clone P029_Ab29, VH EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

[0252] Sequence number 36 Clone P029_Ab30, VL EIVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQQKPGQAPRLLIYYTSKRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

[0253] Sequence number 37 Clone P029_Ab30, VH EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGLIDPEDGKTKYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

[0254] Sequence ID 38 Clone P029_Ab31, VL EIVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQQKPGQAPRLLIYYTSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

[0255] Sequence ID 39 Clone P029_Ab31, VH EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTKYAPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

[0256] Sequence ID 40 Clone P029_Ab32, VH EIVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQQKPGQAPRLLIYYTSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

[0257] Sequence ID 41 Clone P029_Ab32, VH EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTIYAPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

[0258] Sequence ID 42 Clone P029_Ab33, VL EIVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQQKPGQAPRLLIYYTSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

[0259] Sequence number 43 Clone P029_Ab33, VH EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGLIDPEDGKTKYAPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

[0260] Sequence number 44 Clone P029_Ab34, VL EIVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQQKPGQAPRLLIYYTSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

[0261] Sequence number 45 Clone P029_Ab34, VH EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

[0262] Sequence number 46 Clone P029_Ab35, VL EIVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQQKPGQAPRLLIYYTSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

[0263] Sequence number 47 Clone P029_Ab35, VH EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGLIDPEDGKTKYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

[0264] Sequence ID 48 Clone P029_Ab36, VL EKVLTQSPATLLSPGERATLSCRASSSVNYMYWYQQKPGQAPRLLIYYTSKRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

[0265] Sequence ID 49 Clone P029_Ab36, VH EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTIYAPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

[0266] Sequence ID 50 Clone P029_Ab37, VL EKVLTQSPATLLSPGERATLSCRASSSVNYMYWYQQKPGQAPRLLIYYTSKRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

[0267] Sequence ID 51 Clone P029_Ab37, VH EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGLIDPEDGKTKYAPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

[0268] Sequence ID 52 Clone P029_Ab38, VL EKVLTQSPATLLSPGERATLSCRASSSVNYMYWYQQKPGQAPRLLIYYTSKRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

[0269] Sequence ID 53 Clone P029_Ab38, VH EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTKYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

[0270] Sequence ID 54 Clone P029_Ab39, VL EKVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQEKPGQSPRLLIYYTSNRATGIPARFSGSGSGTDFTLTISSLEPEDFATYYCQQFTSSPYTFGQGTKLEIK

[0271] Sequence ID 55 Clone P029_Ab39, VH EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTKYAPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

[0272] Sequence ID 56 Clone P029_Ab40, VL EKVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQEKPGQAPRLLIYYTSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

[0273] Sequence ID 57 Clone P029_Ab41, VH EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTKYAPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

[0274] Sequence ID 58 Clone P029_Ab42, VL EKVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQEKPGQAPRLLIYYTSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

[0275] Sequence ID 59 Clone P029_Ab42, VH EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTIYAPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

[0276] Sequence ID 60 Clone P029_Ab43, VL EKVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQEKPGQAPRLLIYYTSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

[0277] Sequence ID 61 Clone P029_Ab43, VH EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGLIDPEDGKTKYAPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

[0278] Sequence ID 62 Clone P029_Ab44, VL EKVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQEKPGQAPRLLIYYTSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

[0279] Sequence ID 63 Clone P029_Ab44, VH EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTKYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

[0280] Sequence ID 64 Clone P029_Ab45, VL EKVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQEKPGQAPRLLIYYTSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

[0281] Sequence ID 65 Clone P029_Ab45, VH EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

[0282] Sequence ID 66 Clone P029_Ab46, VL EKVLTQSPATLSLSPGERATLSCRASSSVNYLYWYQEKPGQAPRLLIYYTSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

[0283] Sequence ID 67 Clone P029_Ab46, VH EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIEPEDGKTKYAPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

[0284] Sequence ID 68 Clone P029_Ab47, VL EKVLTQSPATLLSPGERATLSCRASSSVNYMYWYQQKPGQAPRLLIYYTSKRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

[0285] Sequence ID 69 Clone P029_Ab47, VH EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIDPEEGKTKYAPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

[0286] Sequence ID 70 Clone P029_Ab48, VL EKVLTQSPATLLSPGERATLSCRASSSVNYMYWYQQKPGQAPRLLIYYTSKRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFTSSPYTFGQGTKLEIK

[0287] Sequence ID 71 Clone P029_Ab48, VH EVQLVQSGAEVKKPGATVKISCKVSGFKIKDFYMHWVQQAPGKGLEWMGKIDPEDGKTKYAPKFQGRVTITADTSTDTAYMELSSLTSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS

Claims

1. ALκ, ALλ, ATTR wild type, ATTR variant, AA, AApoAI, ALys, Aβ ２ An isolated monoclonal antibody or its antigen-binding moiety that specifically binds to at least three different amyloid fibrils selected from m and AFib amyloid fibrils.

2. ALκ, ALλ, ATTR wild type, ATTR variant, AA, AApoAI, ALys, Aβ ２ An isolated monoclonal antibody or antigen-binding moiety thereof according to claim 1, which binds to at least 4, 5, 6, 7, 8, or 9 different amyloid fibrils selected from m and AFib amyloid fibrils.

3. An isolated monoclonal antibody or its antigen-binding moiety that specifically binds to amyloid fibrils, wherein each of CDRH1, CDRH2, and CDRH3 in the variable domain of the heavy chain is (a) At least 90% sequence identity with sequence number 6, sequence number 7 and sequence number 8, respectively, or (b) An isolated monoclonal antibody or its antigen-binding moiety, optionally having a single amino acid change with respect to SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively.

4. An isolated monoclonal antibody or its antigen-binding moiety that specifically binds to amyloid fibrils, wherein each of CDRL1, CDRL2, and CDRL3 within the variable domain of the light chain is (a) At least 90% sequence identity with sequence number 9, sequence number 10 and sequence number 11, respectively, or (b) An isolated monoclonal antibody or its antigen-binding moiety having, optionally, a single amino acid change with respect to SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively.

5. An isolated monoclonal antibody or its antigen-binding moiety that specifically binds to amyloid fibrils, (a) Each of CDRH1, CDRH2, and CDRH3 within 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 within 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) An isolated monoclonal antibody or its antigen-binding moiety, wherein each of CDRH1, CDRH2, and CDRH3 in the variable domain of the heavy chain optionally has a single amino acid change with respect to SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, and each of CDRL1, CDRL2, and CDRL3 in the variable domain of the light chain optionally has a single amino acid change with respect to SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively.

6. An isolated monoclonal antibody or its antigen-binding moiety that specifically binds 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 that specifically binds 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 that specifically binds 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. (a) The above It binds to epitopes present in amyloid fibrils, but does not bind to the natural peptides that form the fibrils, and / or (b) Ab fibrils that do not bind to the CNS and / or brain in patients with Alzheimer's disease, and / or (c) An isolated monoclonal antibody or antigen-binding fragment thereof according to any one of the prior claims, which does not compete with SAP for binding to fibrils.

10. The aforementioned epitopes are ALκ, ALλ, ATTR wild-type, ATTR variant, AA, AApoAI, ALys, and Aβ. ２ An isolated monoclonal antibody or antigen-binding fragment thereof according to claim 9, present in at least three amyloid fibrils selected from m and AFib amyloid fibrils.

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

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

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

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

15. The antibody is IgG, IgA, or an antibody single variable domain polypeptide, dAb, FAb, F(ab')2, scFv, Fv, V ＨＨ An isolated monoclonal antibody or its antigen-binding moiety according to any one of the prior claims, selected from an antigen-binding antibody fragment selected from a domain (e.g., Nanobody® or other camelid immunoglobulin domain) or a disulfide bond Fv, a human antibody, a chimeric antibody containing a human variable region, a humanized antibody, a bispecific antibody, or a single-chain antibody.

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

17. An isolated monoclonal antibody according to claim 15 or 16, which is a humanized antibody.

18. The humanized monoclonal antibody or its antigen-binding moiety according to claim 17, wherein CDRH1, CDRH2, and CDRH3 within 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 moiety according to claim 17 or 18, wherein CDRL1, CDRL2, and CDRL3 within 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. A humanized monoclonal antibody or its antigen-binding moiety according to claim 19 or 20, 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, 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. A humanized monoclonal antibody according to any one of claims 19 to 21, wherein the human framework region is derived from an antibody gene selected from SEQ ID NOs: 12 and 13.

22. A 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 VL residues I2, L39, A40, Q44, A49, V101, N66, T85 and F87, and residues selected from VH residues M39, A80, L55, I66, V25, D85, E69, A45, P46, G47 and K48.

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

24. A humanized monoclonal antibody according to any one of claims 18 to 23, having a combination of light chain and heavy chain framework mutations, 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. A humanized monoclonal antibody according to any one of claims 1 to 8, having a combination of light chain and heavy chain framework mutations, including 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. A humanized monoclonal antibody according to any one of claims 18 to 23, having a combination of light chain and heavy chain framework mutations, 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. A humanized monoclonal antibody according to any one of claims 18 to 23, having a combination of light chain and heavy chain framework mutations, including 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. A humanized monoclonal antibody according to any one of claims 18 to 24, having a combination of light chain and heavy chain framework mutations, comprising a light chain framework mutation consisting of A40Y and a heavy chain framework mutation consisting of L55K, I66K, V25A, D85N, and E69P.

29. A humanized monoclonal antibody according to any one of claims 18 to 23, having a combination of light chain and heavy chain framework mutations, including 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 may have at least 90% sequence identity with SEQ ID NO: 26, and the VH domain may have 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. A humanized monoclonal antibody or antigen-binding fragment thereof according to any one of claims 18 to 58, wherein the antibody binds to an epitope on an amyloid fibril, and the epitope includes 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 60, wherein the epitope comprises a C-terminal carboxyl group.

62. A humanized monoclonal antibody or antigen-binding fragment thereof according to any one of claims 59 to 61, wherein the citrate ion and / or malonate ion can be located at the antibody binding site during X-ray crystallography.

63. The antibody is IgG, IgA, or an antibody single variable domain polypeptide, dAb, FAb, F(ab')2, scFv, Fv, V ＨＨ A humanized monoclonal antibody or its antigen-binding moiety according to any one of claims 17 to 62, selected from an antigen-binding antibody fragment selected from a domain (such as Nanobody® or other camelid immunoglobulin domains) or a disulfide bond Fv.

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

65. The humanized monoclonal antibody or antigen-binding moiety thereof according to claim 66, wherein the antibody is the hIgG1 isotype.

66. An isolated monoclonal antibody or humanized antibody or its antigen-binding moiety according to any one of the prior claims, which, when administered parenterally to mice having experimentally induced systemic AA amyloidosis, also effectively promotes regression of systemic mouse AA amyloid deposits.

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

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

69. An isolated monoclonal antibody or humanized antibody according to any one of the prior claims, for use in the treatment of 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 tissue affected by systemic amyloidosis, comprising administering at least one isolated monoclonal antibody or humanized antibody as described in any one of claims 1 to 70.

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

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