WO2024256583A1 - Anticorps anti-fibrilles - Google Patents

Anticorps anti-fibrilles Download PDF

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Publication number
WO2024256583A1
WO2024256583A1 PCT/EP2024/066449 EP2024066449W WO2024256583A1 WO 2024256583 A1 WO2024256583 A1 WO 2024256583A1 EP 2024066449 W EP2024066449 W EP 2024066449W WO 2024256583 A1 WO2024256583 A1 WO 2024256583A1
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Prior art keywords
seq
monoclonal antibody
domain
sequence identity
humanised
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PCT/EP2024/066449
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English (en)
Inventor
Mark Brian Pepys
David Langley
Jennifer Jackson
Jack William WADE
Daniel Christ
Jake HENRY
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Immutrin Ltd
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Immutrin Ltd
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Priority claimed from GBGB2308884.2A external-priority patent/GB202308884D0/en
Priority claimed from GBGB2308898.2A external-priority patent/GB202308898D0/en
Application filed by Immutrin Ltd filed Critical Immutrin Ltd
Priority to KR1020257043905A priority Critical patent/KR20260026033A/ko
Priority to CN202480038031.8A priority patent/CN121487964A/zh
Priority to AU2024304161A priority patent/AU2024304161A1/en
Publication of WO2024256583A1 publication Critical patent/WO2024256583A1/fr
Priority to IL325202A priority patent/IL325202A/en
Priority to MX2025014924A priority patent/MX2025014924A/es
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention provides novel antibodies with broad specificity for the different types of amyloid fibrils that cause all the major forms of human systemic amyloidosis, humanised monoclonal antibodies which target multiple amyloid fibril types, as well as methods for using such antibodies and uses of the antibodies in therapeutic applications.
  • Amyloidosis is a serious disease caused by extracellular deposition of insoluble abnormal fibrils, derived from aggregation of misfolded autologous proteins [Pepys, M. B. (2006). Annu. Rev. Med., 57, 223-241 ; Pepys, M. B. and P. N. Hawkins (2020). Amyloidosis. Oxford Textbook of Medicine. J. Firth, C. Conlon and T. Cox, Oxford University Press], About 30 different proteins are known to form amyloid fibrils in vivo in humans, each associated with clinically distinct conditions (https://doi.org/10.1080/13506129.2020.1835263).
  • Amyloidosis can be acquired as a complication of pre-existing primary disease that produces either an inherently amyloidogenic abnormal protein or greatly increased exposure to a normal but potentially amyloidogenic protein, or it is caused in the elderly by the normal expression of wild type transthyretin, which is inherently amyloidogenic.
  • Hereditary amyloidosis is caused by mutant genes that encode variant proteins that happen to be amyloidogenic.
  • Amyloid deposits can be local, restricted to a particular organ or tissue, or systemic, with amyloid deposits throughout the body except within the brain.
  • Systemic amyloidosis is overwhelmingly either AL or ATTR type; the various hereditary types are rare.
  • Systemic AA amyloidosis which is a complication of chronic infections and other inflammatory conditions, is now rare in developed countries but remains more prevalent elsewhere.
  • SAP serum amyloid P component
  • amyloid fibrils of all types are an attractive target antigen but the actual purpose of antibody immunotherapy is removal of the amyloid fibrils themselves.
  • amyloid fibrils have long been known to be very poorly immunogenic. Patients with amyloidosis almost never produce specific anti-amyloid fibril antibodies, and experimental animals respond poorly if at all, even when vigorously immunised with xenogeneic fibrils.
  • ex vivo amyloid fibrils of all types share very similar morphology, ultrastructure and protein fold, especially the cross-p core structure that is common to all ex vivo amyloid fibril types, regardless of their completely unrelated protein sequences [Sunde, M., et al., J. Mol. Biol., 1997.
  • amyloid fibrils may share potentially epitopic structures but, although some putatively cross-reactive antibodies have been claimed, no genuinely broad spectrum anti-amyloid fibril antibodies have been reported in the prior art.
  • a monoclonal antibody or antigen binding portion thereof which specifically binds to amyloid fibrils with broad anti-fibril specificity.
  • the antibody of the invention is capable of binding to at least 9 different types of amyloid fibrils, for example including ALK, ALA, ATTR wild type, ATTR variant, AA, AApoAl, ALys, AP2m and AFib amyloid fibrils.
  • the antibody of the invention successfully binds to fibrils which cause or are involved in systemic amyloidosis.
  • the antibody does not bind to Ap fibrils present in the CNS and particularly in the brain in patients with Alzheimer’s disease.
  • the antibody according to the invention is preferably an antibody or antigen binding portion thereof wherein CDRs in the variable domain of the heavy chain have a sequence identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99% or 100% with SEQ ID No 6, SEQ ID No. 7 and SEQ ID NO. 8.
  • the antibody according to the invention is preferably an antibody or antigen binding portion thereof wherein CDRs in the variable domain of the light chain have a sequence identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99% or 100% with SEQ ID No 9, SEQ ID No. 10 and SEQ ID NO. 11.
  • the antibody according to the invention is preferably an antibody or antigen binding portion thereof wherein CDRs in the variable domain of an alternative light chain have a sequence identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99% or 100% with SEQ ID No 12, SEQ ID No. 13 and SEQ ID NO. 14.
  • the antibody according to the invention is preferably an antibody or antigen binding portion thereof wherein CDRs in the variable domain of the light chain have a sequence of SEQ ID No 9, SEQ ID No. 10 and SEQ ID NO. 11 optionally comprising one amino acid change.
  • the antibody according to this aspect of the invention can comprise heavy chain CDRs having SEQ ID Nos 6 to 8 and light chain CDRs having SEQ ID Nos 9 to 11.
  • the antibody according to this aspect of the invention can comprise heavy chain CDRs having SEQ ID Nos 6 to 8 and light chain CDRs having SEQ ID Nos 12 to 14.
  • the antibody of the invention comprises a heavy chain variable region having a sequence identity of at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, at least 90%, 91%, 92%, 93%, 94%, at least 95%, 96%, 97%, 98%, at least 99% or 100% with SEQ ID No. 2.
  • the antibody of the invention comprises a light chain variable region having a sequence identity of at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, at least 90%, 91%, 92%, 93%, 94%, at least 95%, 96%, 97%, 98%, at least 99% or 100% with SEQ ID No. 4.
  • the antibody of the invention comprises a light chain variable region having a sequence identity of at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, at least 90%, 91%, 92%, 93%, 94%, at least 95%, 96%, 97%, 98%, at least 99% or 100% with SEQ ID No. 6.
  • the antibody is of human isotope lgG1 or murine isotype lgG2.
  • the antibody can be selected from a human antibody, a chimeric antibody containing a human variable region, a humanized antibody, a bispecific antibody, or a single chain antibody, as well as antigen-binding fragments thereof.
  • antibody of the invention is specific for a conformational epitope commonly presented on amyloid fibrils.
  • the antibody is able to bind to an amyloid-specific epitope independently of the linear polypeptide structure of the fibril.
  • the conformational epitope is present on at least three different amyloid fibrils. Preferably, it is present on 4, 5, 6, 7, 8 or 9 different amyloid fibrils, selected from ALK, ALA, ATTR wild type, ATTR variant, AA, AApoAl, ALys, AP2m and AFib amyloid fibrils.
  • the epitope is formed at the C-terminus of the proteins which compose the fibrils.
  • the epitope comprises at least one charged amino acid, preferably at least 2 charged amino acids, comprising a charged side-chain.
  • Amino acids with charged side-chains include aspartic acid, histidine, glutamic acid, lysine and arginine.
  • the conformational epitope comprises a C-terminal carboxyl group, preferably a free C-terminal carboxyl group.
  • the conformational epitope can be mimicked by both malonate and citrate ions.
  • the invention provides a method for generating an antibody according to the first aspect of the invention, comprising immunising a mammal in which SAP gene has been deleted.
  • a SAP knockout mouse which does not express the murine SAP protein, is immunised with human synthetic ATTR fibril material.
  • a SAP knockout mouse which does not express the murine SAP protein, is immunised with human synthetic ATTR fibril material coated with human SAP.
  • the humanised antibody of the invention successfully binds to fibrils which cause or are involved in systemic amyloidosis.
  • the humanised antibody does not bind to Ap fibrils present in the CNS and particularly in the brain in patients with Alzheimer’s disease.
  • the humanised monoclonal antibody binds to at least ALK, ATTR wild type and ATTR variant fibrils.
  • the humanised monoclonal antibody binds to at least 4, 5, 6, 7, 8 or 9 types of amyloid fibrils, which are selected from ALK, ALA, ATTR wild type, ATTR variant, AA, AApoAl, ALys, Ap2m and AFib amyloid fibrils.
  • the humanised antibody of the invention does not compete with SAP, preferably human SAP, for binding to the amyloid fibrils.
  • SAP preferably human SAP
  • the epitope bound by the antibody of the invention is not identical to the epitope bound by SAP.
  • the humanised monoclonal antibody of the invention binds to fibrils which cause or are involved in systemic amyloidosis, but does not bind to soluble, native peptides from which the fibrils are derived.
  • the epitope bound by the antibody of the invention comprises the C- terminus of the proteins which compose the fibrils.
  • the epitope comprises at least one charged amino acid, preferably at least 2 charged amino acids, comprising a charged side-chain.
  • Amino acids with charged side-chains include aspartic acid, glutamic acid, lysine and arginine.
  • the epitope comprises a C-terminal carboxyl group, preferably a free C- terminal carboxyl group.
  • a citrate and/or a malonate ion can be located in the binding site of the antibody according to the invention, when examined by x-ray crystallography.
  • the humanised monoclonal antibody or antigen binding portion thereof may have CDRH1 , CDRH2 and CDRH3 in the variable domain of the heavy chain with a sequence identity of at least 90% with SEQ ID No 6, SEQ ID No. 7 and SEQ ID No. 8 respectively.
  • the humanised monoclonal antibody or antigen binding portion thereof may have CDRL1 , CDRL2 and CDRL3 in the variable domain of the light chain with a sequence identity of at least 90% with SEQ ID No 9, SEQ ID No. 10 and SEQ ID No. 11 respectively.
  • the humanised monoclonal antibody or antigen binding portion thereof may have CDRH1 , CDRH2 and CDRH3 in the variable domain of the heavy chain with a sequence identity of at least 90% with SEQ ID No 6, SEQ ID No. 7 and SEQ ID NO. 8 respectively. Additionally, the humanised monoclonal antibody or antigen binding portion thereof may have CDRL1 , CDRL2 and CDRL3 in the variable domain of the light chain with a sequence identity of at least 90% with SEQ ID No 9, SEQ ID No. 10 and SEQ ID No. 11 respectively.
  • the antibody according to the invention may have one optimal amino acid change in one or more CDRs, compared to the SEQ IDs of the CDRs as set forth herein.
  • the humanised monoclonal antibody may have human framework regions derived from antibody genes selected from SEQ ID Nos. 12 and 13.
  • the isolated humanised monoclonal antibody may have framework residues mutated to match murine residues. These mutated residues may include VL residues I2, L39, A40, Q44, A49, V101, N66, T85 and F87, and VH residues M39, A80, L55, I66, V25, D85, E69, A45, P46, G47 and K48.
  • the isolated humanised monoclonal antibody may have the VH region comprising the mutations L55K and I66K.
  • the isolated humanised monoclonal antibody may have a combination of light and heavy chain framework mutations.
  • the light chain framework mutations may consist of I2K, A40Y, N66K, T85N and F87Y.
  • the heavy chain framework mutations may consist of L55K, I66K, V25A, D85N and E69P.
  • the isolated humanised monoclonal antibody may have a combination of light and heavy chain framework mutations.
  • the light chain framework mutations may consist of I2K, L39M, A40Y, N66K, T85N and F87Y.
  • the heavy chain framework mutations may consist of M39I, A80T, L55K, I66K, E69P, A45R, P46T, G47E and K48Q.
  • the isolated humanised monoclonal antibody may have a combination of light and heavy chain framework mutations.
  • the light chain framework mutations may consist of I2K, A40Y, N66K, T85N and F87Y.
  • the heavy chain framework mutations may consist of M39I, A80T, L55K, I66K, V25A, D85N and E69P.
  • the isolated humanised monoclonal antibody may have a combination of light and heavy chain framework mutations.
  • the light chain framework mutations may consist of I2K, L39M, A40Y and N66K.
  • the heavy chain framework mutations may consist of L55K, I66K, and E69P.
  • the isolated humanised monoclonal antibody may have a combination of light and heavy chain framework mutations.
  • the light chain framework mutations may consist of A40Y.
  • the heavy chain framework mutations may consist of L55K, I66K, V25A, D85N and E69P.
  • the isolated humanised monoclonal antibody may have a combination of light and heavy chain framework mutations.
  • the light chain framework mutations may consist of A40Y.
  • the heavy chain framework mutations may consist of L55K and I66K.
  • the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 14 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 15.
  • the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 16 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 17.
  • the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 18 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 19.
  • the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 20 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 21.
  • the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 22 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 23.
  • the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 24 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 25.
  • the humanised monoclonal antibody may be further modified to alter the charge of the immunoglobulin.
  • positive charge in the immunoglobulin may be reduced, for example by reducing positive charge such that the next charge over the Fv region is +4 or less.
  • the VL domain may be modified by including mutations Q44E, A49S, V101T, or omitting mutations L39M or I2K and L39M.
  • the VH domain may comprise changes including the mutations and L55K or I66K, but not both; adding E69P; and mutants at D57E and/or D62E, in HCDR2.
  • the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 26 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 27.
  • the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 28 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 29.
  • the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 30 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 31.
  • the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 32 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 33.
  • the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 34 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 35.
  • the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 36 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 37.
  • the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 38 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 39.
  • the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 40 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 41.
  • the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 42 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 43.
  • the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 44 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 45.
  • the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 46 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 47.
  • the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 48 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 49.
  • the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 50 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 51.
  • the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 52 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 53.
  • the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 54 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 55.
  • the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 56 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 57.
  • the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 58 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 59.
  • the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 60 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 61.
  • the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 62 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 63.
  • the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 64 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 65.
  • the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 66 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 67.
  • the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 68 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 69.
  • the VL domain of the isolated humanised monoclonal antibody may have a sequence identity of at least 90% with SEQ ID No. 70 and the VH domain may have a sequence identity of at least 90% with SEQ ID No. 71.
  • “at least 90%” is to be understood as 90% or more, optionally 91% or more, 92% or more, optionally 93% or more, optionally 94% or more, optionally 95% or more, optionally 96% or more, optionally 97% or more, optionally 98% or more, or optionally 99% or more, up to 100% identity with the recited SEQ ID.
  • the monoclonal antibody or antigen binding portion thereof may be selected from an IgG, IgA, or an antigen binding antibody fragment selected from an antibody single variable domain polypeptide, dAb, FAb, F(ab’)2, an scFv, an Fv, a VHH domain (such as a Nanobody® or other camelid immunoglobulin domain) or a disulfide-bonded Fv, a human antibody, a chimeric antibody preferably containing a human variable region, a humanized antibody, a bispecific antibody or a single chain antibody.
  • the humanised monoclonal antibody or antigen binding portion thereof may have the Fc region of the antibody derived from the human lgG1 isotype.
  • the antibody is of hl gG 1 isotype.
  • the humanised monoclonal antibody or antigen binding portion thereof may also effectively promote regression of systemic murine AA amyloid deposits when administered parenterally to mice with experimentally induced systemic AA amyloidosis.
  • the humanised monoclonal antibody or antigen binding portion thereof may have in vivo efficacy that is complement activation dependent.
  • the humanised monoclonal antibody or antigen binding portion thereof may have in vivo efficacy that is Fey receptor binding dependent.
  • the antibody of the invention is indicated for use in the treatment of systemic amyloidosis, and there is accordingly provided a pharmaceutical composition comprising an antibody as defined herein for use in the treatment of systemic amyloidosis.
  • an antibody as defined herein in the manufacture of a composition for the treatment of systemic amyloidosis.
  • a method for treating a subject suffering from systemic amyloidosis comprising administering to a subject in need thereof a composition comprising an antibody specific for amyloid fibrils as described herein.
  • the pharmaceutical composition is co-administered together with a supporting treatment for amyloidosis.
  • Amyloidosis therapy in the prior art typically aims to remove or reduce the presence or production of amyloid precursors, and the present antibody is designed to remove of established amyloid deposits. Together with existing or novel therapies to remove amyloid, the antibodies of the invention thus provide a more complete treatment for amyloidosis.
  • the antibody of the invention may be administered independently of other amyloidosis treatments, for example where the occurrence of amyloid precursor has been minimised and it is desired to deplete established amyloid fibrils,
  • amyloidosis therapy is selected from any existing systemic AL amyloidosis therapies, including those listed by Bianchi et al., JACC CardioOncol. 2021 Oct; 3(4): 467-487.
  • Fig. 1 Generation of best-in-class anti-amyloid antibody 2E5 with broad specificity against amyloid deposits.
  • Anti-amyloid fibril antibody 2E5 binds to mouse AA amyloid and removes amyloid in vivo.
  • B) Antibody 2E5 removes amyloid in vivo. Liver amyloid load scores in systemic AA amyloidotic mice 16 days after single IP injection of 4.8 mg/mouse 2E5 compared with untreated controls. Mann-Whitney test: Control vs 2E5: p 0.01278
  • a BLI assay was set up as depicted and deletions of peptide 99-127 tested for binding to 2E5. The results are shown in graphical and check-box form.
  • a BLI assay was performed with variants of 99-127 in which C-terminal amino acids were replaced with alanine.
  • the three C-terminal amino acids (P, K, E) are increasingly essential for binding by 2E5.
  • Fig. 6 Competition ELISA between 2E5 mAb and hSAP for coated ATTR fibrils.
  • a fixed concentration of 2E5 mAb was incubated with increasing concentrations of hSAP within a physiologically relevant window. Binding of hSAP and 2E5 mAb is observed to ATTR fibrils, and the 2E5 binding signal is stable in the presence of bound hSAP, indicating that 2E5 has a non-overlapping epitope with hSAP on ATTR fibrils.
  • CDR grafts show 100-fold decrease in binding compared to 2E5.
  • Fibrils are synthetic amyloid derived from the truncated fragment of immunoglobulin light chain (AL55-133) and mutant forms of the transthyretin protein (S52P TTR) and beta2- microglobulin (D76N Abeta2-m) as well as AA (Amyloid A).
  • an “antibody” may be selected from, but not limited to, an IgG, IgA, or an antigen binding antibody fragment selected from an antibody single variable domain polypeptide, dAb, FAb, F(ab’)2, an scFv, an Fv, a VHH domain (such as a Nanobody® or other camelized immunoglobulin domain) or a disulfide-bonded Fv.
  • any of the above antibody types or fragments thereof may be prepared from one or more of a mammalian species selected from, but not limited to mouse, rat, rabbit, human. Such antibodies can be humanized for use in humans.
  • any of the above antibody types or fragments thereof may be provided as heteroconjugates, bispecific, single-chain, chimeric or humanized molecules having affinity for amyloid fibrils.
  • any of the aforementioned antibody/antibodies binds to amyloid with a dissociation coefficient of 100nM or less, 75nM or less, 50nM or less, 25nM or less, such as 10nM or less, 5nM or less, 1nM or less, or in embodiments 500pM or less, 100pM or less, 50pM or less or 25pM or less.
  • Antibodies may be monospecific, with narrow or broad specificity; or multispecific, such as bispecific, such that they possess two distinct epitope specificities in a single antibody molecule. Cocktails of antibodies may be targeted at two or more specific epitopes.
  • Antibody cocktails may be prepared by a mixture of one or more monoclonal antibodies.
  • an antibody cocktail contains two, three, four or more monoclonal antibodies each of which recognises a plurality of amyloid fibrils.
  • the antibody is monoclonal and binds to at least two, at least three, at least four, at least five, at least 6, at least 7, at least 8, at least 9 or at least 10 different amyloid fibril types.
  • it binds substantially to all systemic amyloid fibril types.
  • the antibody or antibodies of the invention are formulated for intravenous (iv) or intramuscular (im) administration.
  • Antibodies administered iv should extravasate from the circulation in order to enter the interstitial tissue space and bind to their cognate target.
  • the antibody in one embodiment, is an antibody fragment such as a scFv, dAb or VHH antibody. Small antibody fragments are extravasated much more readily into tissue, and for this reason can perform better than IgG or other larger antibodies. However, smaller fragments are also cleared faster from the circulation. A compromise must be struck between tissue accessibility and clearance. For example, see Wang et al., Clinical pharmacology & Therapeutics, 84:5, 2008, 548-558.
  • an antibody fragment or derivative is an antibody fragment or derivative which is appropriately modified to activate complement.
  • fragment is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide.
  • a fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.
  • isolated refers to material that is free to varying degrees from components which normally accompany it as found in its native state. “Isolate” denotes a degree of separation from original source or surroundings. “Purify” denotes a degree of separation that is higher than isolation.
  • a “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography.
  • the term “purified” can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel.
  • modifications for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.
  • isolated polynucleotide is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene.
  • the term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences.
  • the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.
  • an “isolated polypeptide” is meant a polypeptide of the invention that has been separated from components that naturally accompany it.
  • the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated.
  • the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention.
  • An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
  • Amyloidosis and amyloid fibrils are known in the art. About 30 different proteins are known to form amyloid fibrils in vivo in humans, each associated with clinically distinct conditions. For reviews and definitions, see Pepys, M. B. and P. N. Hawkins (2020). Amyloidosis. Oxford Textbook of Medicine. J. Firth, C. Conlon and T. Cox, Oxford University Press], and the Nomenclature Report of the International Society for Amyloidosis (https://doi.org/10.1080/13506129.2020.1835263).
  • Amyloid fibrils are aggregates of proteins, which typically assemble in a beta sheet.
  • the termini of proteins can be exposed in beta sheet structures, and it is postulated that exposed termini of the proteins which compose the amyloid fibrils are responsible for the binding of pan-fibril-specific antibodies to the amyloid.
  • the C-termini of the proteins are bound by the antibody; a free C-terminal carboxyl group in the TTR peptide is essential for binding, indicating that a charged ligand is required by 2E5.
  • the amyloid epitope formed by the C-terminal amino acids of TTR can be mimicked by both a citrate ion and a malonate ion in space and charge interactions in the antibody binding domain.
  • the structure of the C-terminus of TTR (PKE) is very similar to the structure of citrate and malonate. Citrate forms binding interactions with N30, F90, T91 , Y104, N105 and W106 in the 2E5 binding cleft; citrate and malonate bind in an almost identical fashion, but are not large enough to contact the heavy chain.
  • the term "monoclonal antibody” refers to an antibody obtained from a single clone of B lymphocyte derived plasma cells producing a homogeneous antibody of a single heavy and light chain class and epitope specificity.
  • Monoclonal antibodies are typically highly specific, and are directed against a single antigenic site (epitope), in contrast to conventional antibodies within an antiserum induced in a whole animal by immunisation with a particular antigen.
  • Such conventional antibodies are derived from many different clones of B lymphocytes which recognise either the same or different epitopes on the immunising antigen, and are known as polyclonal antibodies.
  • monoclonal antibodies are readily produced in pure form uncontaminated by other immunoglobulins, whereas isolation of specific antibodies from a polyclonal antiserum requires demanding immunopurification procedures.
  • Monoclonal antibodies may be prepared by the hybridoma method (see Kohler et al., Nature, 256:495-7, 1975), or by recombinant DNA methods. The monoclonal antibodies may even be isolated from phage antibody libraries using well known techniques.
  • the monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567;
  • a host animal typically a mouse
  • Lymphocytes harvested from the immunised animal are then fused in vitro with a continuous line of myeloma cells grown in vitro to form so-called hybridoma cells.
  • myeloma cells include, but are not limited to, human myeloma and mouse-human heteromyeloma cell lines which have been described for the production of human monoclonal antibodies.
  • the culture medium from the growing hybridoma cells may be assayed for monoclonal antibodies directed against the antigen.
  • the binding specificity of the antibodies produced by the cells may be determined by various methods - such as immunoprecipitation or an in vitro binding assay - such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA) or immunoradiometric assay (IRMA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • IRMA immunoradiometric assay
  • the clones may be subcloned by limiting dilution procedures and grown by standard methods.
  • the monoclonal antibodies secreted by the subclones are separated from the culture medium or serum by well-known immunoglobulin purification procedures - such as protein A-Sepharose, gel electrophoresis, dialysis, hydroxyapatite chromatography or affinity chromatography.
  • the antibodies of the invention also encompass variants of such antibodies and fragments thereof.
  • Variants include peptides and polypeptides comprising one or more amino acid sequence substitutions, deletions, and/or additions that have the same or substantially the same affinity and specificity of epitope binding as the anti-amyloid antibody or fragments thereof.
  • the deletions, insertions or substitutions of amino acid residues may produce a silent change and result in a functionally equivalent substance.
  • Deliberate ammo acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues.
  • negatively charged amino acids include aspartic acid and glutamic acid
  • positively charged amino acids include lysine and arginine
  • amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.
  • Homologous substitution substitution and replacement are both used herein to mean the interchange of an existing amino acid residue, with an alternative residue
  • substitution and replacement may occur i.e. like-for-like substitution such as basic for basic, acidic for acidic, polar for polar etc.
  • Non- homologous substitution may also occur i.e. from one class of residue to another or alternatively involving the inclusion of unnatural amino acids - such as ornithine (hereinafter referred to as Z), diami- nobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as O), pyriylalanine, thienylalanine, naphthylalanine and phenylglycine.
  • Z ornithine
  • B diami- nobutyric acid ornithine
  • O norleucine ornithine
  • pyriylalanine pyriylalanine
  • Replacements may also be made by unnatural amino acids include; alpha* and alphadisubstituted* amino acids, N-alkyl amino acids*, lactic acid*, halide derivatives of natural amino acids such as trifluorotyrosine*, p-CI-phe- nylalanine*, p-Br-phenylalanine*, p-l- phenylalanine*, L-allyl-glycine*, p-alanine*, L-a-amino butyric acid*, L-y-amino bu- tyric acid*, L-a-amino isobutyric acid*, L-e-amino caproic acid#, 7-amino heptanoic acid*, L- methionine sulfone#*, L- norleucine*, L-norvaline*, p-nitro-L-phenylalanine*, L- hydroxyproline#, L-thioproline*
  • variants may include peptides and polypeptides comprising one or more amino acid sequence substitutions, deletions, and/or additions to the antibodies and fragments of the invention wherein such substitutions, deletions and/or additions do not cause substantial changes in affinity and specificity of epitope binding.
  • a variant of an antibody or fragment thereof may result from one or more changes to the antibody or fragment thereof, where the changed antibody or fragment thereof has the same or substantially the same affinity and specificity of epitope binding as the starting sequence.
  • Variants may be naturally occurring, such as allelic or splice variants, or may be artificially constructed. Variants may be prepared from the corresponding nucleic acid molecules encoding said variants.
  • Variants of the antibodies or fragments thereof may have changes in light and/or heavy chain amino acid sequences that are naturally occurring or are introduced by in vitro engineering of native sequences using recombinant DNA techniques.
  • Naturally occurring variants include "somatic" variants which are generated in vivo in the corresponding germ line nucleotide sequences during the generation of an antibody response to a foreign antigen.
  • Variants of antibodies and binding fragments may also be prepared by mutagenesis techniques. For example, amino acid changes may be introduced at random throughout an antibody coding region and the resulting variants may be screened for binding affinity for amyloid or for another property. Alternatively, amino acid changes may be introduced into selected regions of the antibody, such as in the light and/or heavy chain CDRs, and/or in the framework regions, and the resulting antibodies may be screened for binding to amyloid or some other activity. Amino acid changes encompass one or more amino acid substitutions in a CDR, ranging from a single amino acid difference to the introduction of multiple permutations of amino acids within a given CDR. Also encompassed are variants generated by insertion of amino acids to increase the size of a CDR.
  • Amyloid specific antibodies or fragments thereof may be provided with a modified Fc region where a naturally-occurring Fc region is modified to increase the half-life of the antibody or fragment in a biological environment, for example, the serum half-life or a half-life measured by an in vitro assay.
  • Fc modification can also be employed to alter antibody biodistribution in vivo, which can direct the antibody to amyloid-bearing tissues.
  • Variants also include antibodies or fragments thereof comprising a modified Fc region, wherein the modified Fc region comprises at least one amino acid modification relative to a wild-type Fc region.
  • the variant Fc region may be designed, relative to a comparable molecule comprising the wild-type Fc region, so as to bind Fc receptors with a greater or lesser affinity.
  • the antibodies and fragments thereof may comprise a modified Fc region.
  • Fc region refers to naturally-occurring or synthetic polypeptides homologous to the IgG C-terminal domain that is produced upon papain digestion of IgG.
  • IgG Fc has a molecular weight of approximately 50 kD. In the antibodies and fragments, an entire Fc region can be used, or only a half-life enhancing portion.
  • the antibodies and fragments thereof also encompass derivatives of the antibodies, fragments and sequences disclosed herein.
  • Derivatives include polypeptides or peptides, or variants, fragments or derivatives thereof, which have been chemically modified. Examples include covalent attachment of one or more polymers - such as water soluble polymers, N- linked, or O-linked carbohydrates, sugars, phosphates, and/or other such molecules.
  • the derivatives are modified in a manner that is different from naturally occurring or starting peptide or polypeptides, either in the type or location of the molecules attached. Derivatives further include deletion of one or more chemical groups which are naturally present on the peptide or polypeptide.
  • the present invention also encompasses amyloid specific antibodies that include two full length heavy chains and two full length light chains.
  • the antibodies may be constructs such as single chain antibodies or "mini" antibodies that retain binding activity to amyloid.
  • Such constructs may be prepared by methods well known in the art.
  • fragments are modified to enable complement activation.
  • DNA is cloned into a plasmid vector system.
  • a plasmid vector system uses a bacteriophage lambda vector system having a leader sequence that causes the expressed Fab protein to migrate to the periplasmic space (between the bacterial cell membrane and the cell wall) or to be secreted.
  • Fab fragments with specificity for amyloid are specifically encompassed within the amyloid specific antibodies and fragments thereof.
  • amyloid binding antibodies and fragments thereof may be humanized or human engineered antibodies.
  • a humanized antibody or antigen binding fragment thereof, is a recombinant polypeptide that comprises a portion of an antigen binding site from a non-human antibody and a portion of the framework and/or constant regions of a human antibody.
  • a human engineered antibody or antibody fragment is a non-human (e.g., mouse) antibody that has been engineered by modifying (e.g., deleting, inserting, or substituting) amino acids at specific positions so as to reduce or eliminate any detectable immunogenicity of the modified antibody in a human.
  • Humanized antibodies include chimeric antibodies and CDR-grafted antibodies.
  • Chimeric antibodies are antibodies that include a non-human antibody variable region linked to a human constant region. Thus, in chimeric antibodies, the variable region is mostly non- human, and the constant region is human. Chimeric antibodies and methods for making them are described in, for example, Proc. Natl. Acad. Sci. USA, 81: 6841-6855 (1984). Although, they can be less immunogenic than a mouse monoclonal antibody, administrations of chimeric antibodies have been associated with human immune responses (HAMA) to the non-human portion of the antibodies.
  • HAMA human immune responses
  • Chimeric antibodies can also be produced by splicing the genes from a mouse antibody molecule of appropriate antigenbinding specificity together with genes from a human antibody molecule of appropriate biological activity, such as the ability to activate human complement and mediate antibodydependent cellular phagocytosis (ADCP).
  • ADCP antibodydependent cellular phagocytosis
  • One example is the replacement of a Fc region with that of a different isotype.
  • CDR-grafted antibodies are antibodies that include the CDRs from a non-human "donor” antibody linked to the framework region from a human “recipient” antibody.
  • CDR- grafted antibodies include more human antibody sequences than chimeric antibodies because they include both constant region sequences and variable region (framework) sequences from human antibodies.
  • a CDR-grafted humanized antibody of the invention can comprise a heavy chain that comprises a contiguous amino acid sequence (e.g., about 5 or more, 10 or more, or even 15 or more contiguous amino acid residues) from the framework region of a human antibody (e.g., FR-I, FR-2, or FR-3 of a human antibody) or, optionally, most or all of the entire framework region of a human antibody.
  • a human antibody e.g., FR-I, FR-2, or FR-3 of a human antibody
  • CDR-grafted antibodies and methods for making them are described in Nature, 321 : 522-525 (1986). Methods that can be used to produce humanized antibodies also are described in, for example, US 5,721 ,367 and 6,180,377.
  • Veneered antibodies are non-human or humanized (e.g., chimeric or CDR-grafted antibodies) antibodies that have been engineered to replace certain solvent-exposed amino acid residues so as to reduce their immunogenicity or enhance their function. Veneering of a chimeric antibody may comprise identifying solvent-exposed residues in the non-human framework region of a chimeric antibody and replacing at least one of them with the corresponding surface residues from a human framework region.
  • Veneering can be accomplished by any suitable engineering technique.
  • humanized or human engineered antibodies are IgG, IgM, IgE, IgA, and IgD antibodies.
  • the antibodies may be of any class (IgG, IgA, IgM, IgE, IgD, etc.) or isotype and can comprise a kappa or lambda light chain.
  • a human antibody may comprise an IgG heavy chain or defined fragment, such as at least one of isotypes, lgG1 , lgG2, lgG3 or lgG4.
  • the antibodies or fragments thereof can comprise an IgG 1 heavy chain and a kappa or lambda light chain.
  • Human antibodies to target amyloid can be produced using transgenic animals that have no endogenous immunoglobulin production and are engineered to contain human immunoglobulin loci, as described in WO 98/24893 and WO 91/00906.
  • an immune response can be produced to a selected antigenic molecule, and antibody producing cells can be removed from the animal and used to produce hybridomas that secrete human monoclonal antibodies.
  • Immunization protocols, adjuvants, and the like are known in the art, and are used in immunization of, for example, a transgenic mouse.
  • the antibodies produced by phage technology are produced as antigen binding fragments- usually Fv or Fab fragments-in bacteria and thus lack effector functions.
  • Effector functions can be introduced by one of two strategies: the fragments can be engineered either into complete antibodies for expression in mammalian cells, or into bispecific antibody fragments with a second binding site capable of triggering an effector function.
  • Human antibodies may be generated through the in vitro screening of antibody display libraries (J Mol. Biol. (1991) 227: 381). Various antibody-containing phage display libraries have been described and may be readily prepared. Libraries may contain a diversity of human antibody sequences, such as human Fab, Fv, and scFv fragments, that may be screened against an appropriate target. Phage display libraries may comprise peptides or proteins other than antibodies which may be screened to identify agents capable of selective binding to amyloid.
  • Phage-display processes mimic immune selection through the display of antibody repertoires on the surface of filamentous bacteriophage, and subsequent selection of phage by their binding to an antigen of choice.
  • One such method is described in WO 99/10494.
  • Anti-amyloid antibodies can be isolated by screening of a recombinant combinatorial antibody library, preferably a scFv phage display library, prepared using human VL and VH cDNAs prepared from mRNA derived from human lymphocytes. Methodologies for preparing and screening such libraries are known in the art. There are commercially available kits for generating phage display libraries.
  • amyloid binding antibodies and fragments thereof may comprise one or more portions that do not bind amyloid but instead are responsible for other functions, such as circulating half-life, direct cytotoxic effect, detectable labelling, or activation of the recipient’s endogenous complement cascade or endogenous cellular cytotoxicity.
  • the antibodies or fragments thereof may comprise all or a portion of the constant region and may be of any isotype, including IgA (e.g., IgAI or lgA2), IgD, IgE, IgG (e.g. lgG1, lgG2, lgG3 or lgG4), or IgM.
  • antigen-binding compounds of the invention may include an epitope tag, a salvage receptor epitope, a label moiety for diagnostic or purification purposes, or a cytotoxic moiety such as a radionuclide or toxin.
  • the anti- amyloid antibody or fragment thereof may be modified in order to increase its serum half-life, for example, by adding molecules - such as PEG or other water soluble polymers, including polysaccharide polymers to increase the half-life.
  • the amyloid binding antibodies and fragments thereof may be bispecific.
  • bispecific antibodies may resemble single antibodies (or antibody fragments) but have two different antigen binding sites (variable regions).
  • Bis- pecific antibodies can be produced by various methods - such as chemical techniques, "polydoma” techniques or re- combinant DNA techniques.
  • Bispecific antibodies may have binding specificities for at least two different epitopes, at least one of which is an epitope of amyloid.
  • Amyloid binding antibodies and fragments may be heteroantibodies.
  • Heteroantibodies are two or more anti- bodies, or antibody binding fragments (Fab) linked together, each antibody or fragment having a different specificity.
  • antibody fragments refers to portions of an intact full length antibody - such as an antigen binding or variable region of the intact antibody.
  • antibody fragments include Fab, Fab’, F(ab’)2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); multispecific antibody fragments such as bispecific, trispecific, and multispecific antibodies (e.g., diabodies, triabodies, tetrabodies); binding-domain immunoglobulin fusion proteins; camelized antibodies; minibodies; chelating recombinant antibodies; tribodies or bibodies; intrabodies; nanobodies; small modular immunopharmaceuticals (SMIP), VHH containing antibodies; and any other polypeptides formed from antibody fragments.
  • SMIP small modular immunopharmaceuticals
  • anti- amyloid antibody and amyloid binding antibody encompass amyloid binding antibody fragments comprising any part of the heavy or light chain sequences of the full length antibodies, and which bind amyloid.
  • fragments refers to fragments capable of binding amyloid, for example any of at least 3 contiguous amino acids (e.g., at least 4, 5, 6, 7, 8, 9 or 10 or more contiguous amino acids, for example from a CDR) of the antibody involved in antigen binding, and encompasses Fab, Fab’, F(ab’)2, and F(v) fragments, or the individual light or heavy chain variable regions or portion thereof.
  • Amyloid binding fragments include, for example, Fab, Fab’, F(ab’)2, Fv and scFv. These fragments lack the Fc fragment of an intact antibody, clear more rapidly from the circulation, and can have less non-specific tissue binding than an intact antibody. These fragments can be produced from intact antibodies using well known methods, for example by proteolytic cleavage with enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab’)2 fragments).
  • amyloid binding antibodies and fragments also encompass single-chain antibody fragments (scFv) that bind to amyloid.
  • An scFv comprises an antibody heavy chain variable region (VH) operably linked to an antibody light chain variable region (VL) wherein the heavy chain variable region and the light chain variable region, together or individually, form a binding site that binds amyloid.
  • An scFv may comprise a VH region at the amino-terminal end and a VL region at the carboxy-terminal end.
  • scFv may comprise a VL region at the amino-terminal end and a VH region at the carboxy-terminal end.
  • the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv).
  • An scFv may optionally further comprise a polypeptide linker between the heavy chain variable region and the light chain variable region.
  • amyloid binding antibodies and fragments also encompass domain antibody (dAb) fragments as described in Nature 341:544-546 (1989) which consist of a VH domain.
  • dAb domain antibody
  • amyloid binding antibodies and fragments also encompass heavy chain antibodies (HCAb). These antibodies can apparently form antigen-binding regions using only heavy chain variable region, in that these functional antibodies are dimers of heavy chains only (referred to as “heavy-chain antibodies” or “HCAbs”). Accordingly, amyloid binding antibodies and fragments may be heavy chain antibodies (HCAb) that specifically bind to amyloid.
  • HCAb heavy chain antibodies
  • amyloid binding antibodies and fragments also encompass antibodies that are SMIPs or binding domain immunoglobulin fusion proteins specific for amyloid protein. These constructs are single-chain polypeptides comprising antigen binding domains fused to immunoglobulin domains necessary to carry out antibody effector functions (see W003/041600).
  • amyloid binding antibodies and fragments also encompass diabodies. These are bivalent antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain. This forces the domains to pair with complementary domains of another chain and thereby creates two antigen binding sites (see, for example, WO 93/11161). Diabodies can be bispecific or monospecific.
  • the amyloid binding antibodies and fragments thereof also encompass immunoadhesins.
  • One or more CDRs may be incorporated into a molecule either covalently or noncovalently to make it an immunoadhesin.
  • An immunoadhesin may incorporate the CDR(s) as part of a larger polypeptide chain, may covalently link the CDR(s) to another polypeptide chain, or may incorporate the CDR(s) noncovalently.
  • the CDRs permit the immunoadhesin to specifically bind to amyloid.
  • amyloid binding antibodies and fragments thereof also encompass antibody mimics comprising one or more amyloid binding portions built on an organic or molecular scaffold (such as a protein or carbohydrate scaffold).
  • an organic or molecular scaffold such as a protein or carbohydrate scaffold.
  • Proteins having relatively defined three- dimensional structures commonly referred to as protein scaffolds, may be used as reagents for the design of antibody mimics.
  • These scaffolds typically contain one or more regions which are amenable to specific or random sequence variation, and such sequence randomization is often carried out to produce libraries of proteins from which desired products may be selected.
  • an antibody mimic can comprise a chimeric nonimmunoglobulin binding polypeptide having an immunoglobulin-like domain containing scaffold having two or more solvent exposed loops containing a different CDR from a parent antibody inserted into each of the loops and exhibiting selective binding activity toward a ligand bound by the parent antibody.
  • Non-immunoglobulin protein scaffolds have been proposed for obtaining proteins with novel binding properties.
  • Anti- amyloid antibodies or antibody fragments thereof typically bind to human amyloid with high affinity (e.g., as determined with BIACORE), such as for example with an equilibrium binding dissociation constant (KD) for amyloid of about 15nM or less, 10 nM or less, about 5 nM or less, about 1 nM or less, about 500 pM or less, about 250 pM or less, about 100 pM or less, about 50 pM or less, or about 25 pM or less, about 10 pM or less, about 5 pM or less, about 3 pM or less about 1 pM or less, about 0.75 pM or less, or about 0.5 pM or less.
  • KD equilibrium binding dissociation constant
  • the antibodies and antibody fragments described herein can be prepared by any suitable method. Suitable methods for preparing such antibodies and antibody fragments are known in the art.
  • the antibody or antibody fragment may be isolated or purified to any degree.
  • humanised forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
  • humanised antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
  • donor antibody such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
  • FR residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • CDRs of the antibodies described herein may be transferred; for example, it is possible to retain human acceptor CDRs as long as the donor CDR H3 is transferred.
  • the members of the immunoglobulin superfamily all share a similar fold for their polypeptide chain.
  • antibodies are highly diverse in terms of their primary sequence, comparison of sequences and crystallographic structures has revealed that, contrary to expectation, five of the six antigen binding loops of antibodies (H1 , H2, L1 , L2, L3) adopt a limited number of main-chain conformations, or canonical structures (Chothia and Lesk (1987) J. Mol. Biol., 196: 901 ; Chothia et al. (1989) Nature, 342: 877).
  • a pharmaceutical composition may comprise, in addition to the antibody, one or more pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, stabilizers, preservatives, lubricants, or other materials well known to those skilled in the art.
  • suitable materials will be sterile and pyrogen-free, with a suitable isotonicity and stability. Examples include sterile saline (e.g. 0.9% NaCI), water, dextrose, glycerol, ethanol or the like or combinations thereof. Such materials should be non-toxic and should not interfere with the efficacy of the active compound.
  • Suitable materials will be sterile and pyrogen free, with a suitable isotonicity and stability. Examples include sterile saline (e.g. 0.9% NaCI), water, dextrose, glycerol, ethanol or the like or combinations thereof.
  • the composition may further contain auxiliary substances such as wetting agents, emulsifying agents, pH buffering agents or the like.
  • Suitable carriers, excipients, etc. can be found in standard pharmaceutical texts, for example, Remington’s Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990.
  • pharmaceutically acceptable refers to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject (e.g. human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • a subject e.g. human
  • Each carrier, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation.
  • the antibodies may be provided in a lyophilized form for reconstitution prior to administration.
  • lyophilized reagents may be reconstituted in sterile water and mixed with saline prior to administration to a subject.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active compound with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active compound with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.
  • Formulations may be in the form of liquids, solutions, suspensions, emulsions, and the like.
  • other therapeutic or prophylactic agents may be included in a pharmaceutical composition or formulation.
  • Treatment may be any treatment and therapy, whether of a human or an animal (e.g. in veterinary applications), in which some desired therapeutic effect is achieved, for example, the inhibition or delay of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, amelioration of the condition, cure or remission (whether partial or total) of the condition, preventing, delaying, abating or arresting one or more symptoms and/or signs of the condition or prolonging survival of a subject or patient beyond that expected in the absence of treatment.
  • some desired therapeutic effect is achieved, for example, the inhibition or delay of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, amelioration of the condition, cure or remission (whether partial or total) of the condition, preventing, delaying, abating or arresting one or more symptoms and/or signs of the condition or prolonging survival of a subject or patient beyond that expected in the absence of treatment.
  • Treatment as a prophylactic measure is also included.
  • a subject susceptible to or at risk of the occurrence or re-occurrence of amyloidosis may be treated as described herein. Such treatment may prevent or delay the occurrence or reoccurrence of amyloidosis in the subject.
  • treatment may include inhibiting amyloid deposition, including complete amyloid deposition reversal.
  • Antibodies may be administered as described herein in therapeutically-effective amounts.
  • terapéuticaally-effective amount refers to that amount of an active compound, or a combination, material, composition or dosage form comprising an active compound, which is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit/risk ratio.
  • appropriate dosages of the active compounds can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects of the administration.
  • the selected dosage level will depend on a variety of factors including, but not limited to, the route of administration, the time of administration, the rate of excretion of the active compound, other drugs, compounds, and/or materials used in combination, and the age, sex, weight, condition, general health, and prior medical history of the patient.
  • the amount of active compounds and route of administration will ultimately be at the discretion of the physician, although generally the dosage will be to achieve concentrations of the active compound at a site of therapy without causing substantial harmful or deleterious sideeffects.
  • a suitable dose of the active compound is in the range of about 100 pg to about 250 mg per kilogram body weight of the subject per day.
  • the active compound is a salt, an ester, prodrug, or the like
  • the amount administered is calculated on the basis of the parent compound and so the actual weight to be used is increased proportionately.
  • Administration in vivo can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals). Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the physician.
  • Multiple doses of antibody may be administered, for example 2, 3, 4, 5 or more than 5 doses may be administered.
  • compositions comprising the active compounds may be formulated in suitable dosage unit formulations appropriate for the intended route of administration.
  • Formulations suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active compound; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion; as a bolus; as an electuary; or as a paste.
  • a tablet may be made by conventional means, e.g., compression or moulding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active compound in a free-flowing form such as a powder or granules, optionally mixed with one or more binders (e.g. povidone, gelatin, acacia, sorbitol, tragacanth, hydroxypropylmethyl cellulose); fillers or diluents (e.g. lactose, microcrystalline cellulose, calcium hydrogen phosphate); lubricants (e.g. magnesium stearate, talc, silica); disintegrants (e.g.
  • Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active compound therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile.
  • Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.
  • Formulations suitable for parenteral administration include aqueous and nonaqueous isotonic, pyrogen-free, sterile injection solutions which may contain anti-oxidants, buffers, preservatives, stabilizers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs.
  • Suitable isotonic vehicles for use in such formulations include Sodium Chloride Injection, Ringer’s Solution, or Lactated Ringer’s Injection.
  • concentration of the active compound in the solution is from about 1 ng/ml to about 10 pg/ml, for example from about 10 ng/ml to about 1 pg/ml.
  • the formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.
  • Formulations may be in the form of liposomes or other microparticulate systems which are designed to target the active compound to blood components or one or more organs.
  • compositions may be prepared in the form of a concentrate for subsequent dilution, or may be in the form of divided doses ready for administration.
  • the reagents may be provided separately within a kit, for mixing prior to administration to a human or animal subject.
  • Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity.
  • Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule.
  • hybridize is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency.
  • complementary polynucleotide sequences e.g., a gene described herein
  • stringent salt concentration will ordinarily be less than about 750 mM NaCI and 75 mM trisodium citrate, preferably less than about 500 mM NaCI and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCI and 25 mM trisodium citrate.
  • Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide.
  • Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, more preferably of at least about 37° C, and most preferably of at least about 42° C.
  • Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art.
  • concentration of detergent e.g., sodium dodecyl sulfate (SDS)
  • SDS sodium dodecyl sulfate
  • Various levels of stringency are accomplished by combining these various conditions as needed.
  • hybridization will occur at 30° C in 750 mM NaCI, 75 mM trisodium citrate, and 1% SDS.
  • hybridization will occur at 37° C in 500 mM NaCI, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 .mu.g/ml denatured salmon sperm DNA (ssDNA).
  • hybridization will occur at 42° C in 250 mM NaCI, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 pg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
  • wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature.
  • stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCI and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCI and 1.5 mM trisodium citrate.
  • Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25 °C, more preferably of at least about 42° C, and even more preferably of at least about 68 °C.
  • wash steps will occur at 25° C in 30 mM NaCI, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42 °C in 15 mM NaCI, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68° C in 15 mM NaCI, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad.
  • substantially identical is meant a polypeptide or nucleic acid molecule exhibiting at least 90% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein).
  • a reference amino acid sequence for example, any one of the amino acid sequences described herein
  • nucleic acid sequence for example, any one of the nucleic acid sequences described herein.
  • such a sequence is at least 91%, more preferably 92% or 93%, and more preferably 94%, 95%, 96%, 97%, 98% or even 99% and up to 100% identical at the amino acid level or nucleic acid to the sequence used for comparison.
  • Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e -3 and e -100 indicating a closely related sequence.
  • sequence analysis software for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Bio
  • Ranges provided herein are understood to be shorthand for all of the values within the range.
  • a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.
  • Antibody 2E5 was produced by immunisation of a SAP-/- mouse (Bickerstaff et al., Nat Med 1999 Jun;5(6):694-7) using a synthetic human ATTR amyloid fibril immunogen by a variation of the classic hybridoma approach, originally developed by Milstein and Koehler (Fig. 1A).
  • ATTR fibrils in PBS were mixed with the adjuvant RIBI and injected at two-weekly intervals with 50 .g ATTR in week zero, followed by 25 .g ATTR in week two and 25 .g ATTR complexed with SAP in week 4.
  • the original 2E5 mouse monoclonal isotype was lgG2c, which is homologous to human IgG 1 and potently complement activating. This is a necessary property for in vivo antibody-mediated amyloid removal which we have demonstrated to be complement dependent [Bodin, K., et al., Nature, 2010. 468(7320): p. 93-97, Richards, D.B., et al., N. Engl. J. Med., 2015. 373(12): p. 1106-1114, Milde, R., et al., Cell Rep., 2015. 13(9): p. 1937-1948], Indeed, administration of 2E5 to mice with established systemic AA amyloidosis, produced notable amyloid clearance (Fig. 2B).
  • Crystal structure data from 2E5 showed both malonate and citrate ions locating in the binding cleft of the antibody, adopting a structure which mimics the C-terminal structure of TTR polypeptides.
  • peptide truncation studies and alanine scans were preformed using an TTR peptide (99-127) previously shown to behave as an analogue for the full-length TTR protein.
  • Peptides were synthesised with a biotin-SGSG N-terminal tag, based on the human TTR sequence as follows:
  • a stock solution (4mg/ml) of each peptide was prepared, dissolving 2mg of peptide in 500ul of:
  • the peptides were diluted to 10ug/ml in PBS for mapping using a bio-layer interferometry (BLI) biosensor.
  • BBI bio-layer interferometry
  • 2E5-1B7 antibody (murine lgG2a) was prepared by recombinant expression, and diluted to 40ug/ml in PBS for BLI
  • the binding of the antibody to each of the peptides was analysed by pre-soaking the streptavidin BLI biosensors for 10min in PBS + 0.1% BSA, equilibrating the peptides and mAb to room temperature, and performing BLI measurement using a Blitz instrument (Blitz pro software), applying advanced kinetics as follows:
  • Antibodies 1-3 were tested by ELISA to determine binding to amyloid TTR fibrils in comparison to murine 2E5. All of the CDR-grafted antibodies showed about 100 fold decrease in binding. See Figure 7 A and B.
  • Antibodies P029_Ab0019 and P029_Ab0008 were chosen as the base clones for modification.
  • VH Heavy-chain variable region
  • VH Heavy-chain variable region
  • VL Light-chain variable region
  • VL Light-chain variable region
  • VL Light-chain variable region
  • APKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARAYYSNYNWFAYWGQGTLVTVSS SEQ ID No. 62

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Abstract

L'invention concerne un anticorps monoclonal isolé ou une partie de liaison à l'antigène de celui-ci qui se lie spécifiquement à des fibrilles amyloïdes et permet efficacement la clairance de dépôts amyloïdes extracellulaires de tissus in vivo, ainsi que des anticorps humanisés ayant les mêmes propriétés.
PCT/EP2024/066449 2023-06-14 2024-06-13 Anticorps anti-fibrilles Pending WO2024256583A1 (fr)

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AU2024304161A AU2024304161A1 (en) 2023-06-14 2024-06-13 Anti-fibril antibodies
IL325202A IL325202A (en) 2023-06-14 2025-12-07 Antibodies against fibers (fibrils)
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