WO2024114906A1 - Anticorps dirigés contre une protéine de type speck associée à l'apoptose contenant une card (asc) et leurs utilisations - Google Patents

Anticorps dirigés contre une protéine de type speck associée à l'apoptose contenant une card (asc) et leurs utilisations Download PDF

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WO2024114906A1
WO2024114906A1 PCT/EP2022/083921 EP2022083921W WO2024114906A1 WO 2024114906 A1 WO2024114906 A1 WO 2024114906A1 EP 2022083921 W EP2022083921 W EP 2022083921W WO 2024114906 A1 WO2024114906 A1 WO 2024114906A1
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seq
antibody
antigen
binding fragment
identity
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PCT/EP2022/083921
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Niccolo PENGO
Danaelle CAPRON
Dimitri BIELI
Micha BEETZ
Doug Phillips
Ilca TUDOR
Tiziana SONATI
Alcide Barberis
Natascha WUILLEMIN
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Mabylon Ag
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Priority to PCT/EP2022/083921 priority Critical patent/WO2024114906A1/fr
Priority to PCT/EP2023/083828 priority patent/WO2024115700A1/fr
Publication of WO2024114906A1 publication Critical patent/WO2024114906A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], 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
    • 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
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • 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
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • 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 relates to antibodies binding to apoptosis-associated speck-like protein 10 containing a CARD (ASC; also referred to as PYCARD), in particular to the PYRIN-PAAD- DAPIN domain (PYD) of ASC.
  • ASC also referred to as PYCARD
  • PYD PYRIN-PAAD- DAPIN domain
  • the present invention also relates to compositions and kits comprising such antibodies.
  • the present invention also relates to the use of such antibodies, compositions and kits, e.g. for preventing or treating a neurodegenerative or neuroinflammatory disease.
  • Neurodegenerative diseases are typically chronic, progressive disorders characterized by the gradual loss of neurons in discrete areas of the central nervous system (CNS), such as the brain. Such neuronal damage may involve progressive degeneration and death of neurons.
  • CNS central nervous system
  • neurodegenerative diseases it is estimated that 55 million people worldwide had 20 dementia in 2019, and that by 2050 this figure will increase to 139 million people. In the United States, it is estimated that 6.5 million older adults suffer from Alzheimer's disease (AD) and about a million Americans suffer from Parkinson's disease. As of today, neurodegenerative diseases are not curable.
  • AD Alzheimer's disease
  • Parkinson's disease As of today, neurodegenerative diseases are not curable.
  • neuroinflammation has attracted increasing attention as common feature in the development of neurodegenerative diseases (Guzman-Martinez L, Maccioni RB, Andrade V, Navarrete LP, Pastor MG, Ramos-Escobar N. Neuroinflammation as a Common 30 Feature of Neurodegenerative Disorders. Front Pharmacol.2019 Sep 12;10:1008. doi: 10.3389/fphar.2019.01008; Amor S, Puentes F, Baker D, van der Valk P. Inflammation in neurodegenerative diseases. Immunology.2010 Feb;129(2):154-69.
  • Inflammasomes are cytosolic multiprotein complexes, which assemble in a stimulus-specific manner and, upon assembly, activate an inflammatory cascade.
  • the assembly of an inflammasome is driven by self-association and oligomerization of three types of proteins: sensor, adaptor, and effector.
  • Inflammasome sensors self-associate upon activation by danger signals and prompt the oligomerization of the inflammasome adaptor ASC (apoptosis- associated speck-like protein containing a CARD). Accordingly, inflammasome assembly usually requires ASC as adaptor protein.
  • ASC is composed of two protein-protein interaction domains: a N-terminal PYRIN-PAAD-DAPIN domain (PYD) and a C-terminal caspase- recruitment domain (CARD).
  • PYD and CARD domains are members of the six-helix bundle death domain-fold superfamily that mediates assembly of large signaling complexes in the inflammatory and apoptotic signaling pathways via the activation of caspase.
  • upstream sensor proteins such as NLRP3 and AIM2
  • recruit ASC via interactions between their PYDs.
  • ASC then interacts with caspase 1 via CARD/CARD interactions.
  • ASC with its two domains, PYD and CARD therefore represents the core structure of the inflammasome.
  • NLRP3 inhibitory compounds which target the NLRP3 sensor and are, thus, specific for NLRP3 inflammasomes, but not other inflammasomes (Voet S, Srinivasan S, Lamkanfi M, van Loo G. Inflammasomes in neuroinflammatory and neurodegenerative diseases. EMBO Mol Med. 2019 Jun;11 (6):e10248. doi: 10.15252/emmm.201810248). Recently, antibodies against ASC were developed (Schmidt Fl, Lu A, Chen JW, Ruan J, Tang C, Wu ⁇ - ⁇ , Ploegh HL.
  • a single domain antibody fragment that recognizes the adaptor ASC defines the role of ASC domains in inflammasome assembly.
  • IC100 a novel anti-ASC monoclonal antibody improves functional outcomes in an animal model of multiple sclerosis. J Neuroinflammation.2020 May 4;17(1):143. doi: 10.1186/st 2974-020-01826-0), which may be useful to target ASC.
  • x means x ⁇ 10%, for example, x + 5%, or x + 7%, or x ⁇ 10%, or x ⁇ 12%, or x ⁇ 15%, or x ⁇ 20%.
  • disorder and “condition” (as in medical condition), in that all reflect an abnormal condition of the human or animal body or of one of its parts that impairs normal functioning, is typically manifested by distinguishing signs and symptoms, and causes the human or animal to have a reduced duration or quality of life.
  • a subject or patient is intended to include prevention, prophylaxis, attenuation, amelioration and therapy.
  • subject or patient are used interchangeably herein to mean all mammals including humans. Examples of subjects include humans, cows, dogs, cats, horses, goats, sheep, pigs, and rabbits. Preferably, the subject or patient is a human. Doses are often expressed in relation to the bodyweight. Thus, a dose which is expressed as [g, mg, or other unit]/kg (or g, mg etc.) usually refers to [g, mg, or other unit] “per kg (or g, mg etc.) bodyweight", even if the term “bodyweight” is not explicitly mentioned.
  • binding and similar reference usually means “specifically binding”, which does not encompass non-specific sticking.
  • specific binding of an antibody means that the antibody recognizes its target antigen and binds its target with greater affinity (or at lower antibody concentrations, e.g. EC50) than it does to a structurally different antigen and/or to an antigen with a modified or mutated sequence.
  • a "greater” affinity may be at least 2fold, 3fold, 4fold, Sfold, 10fold, 15fold, 20fold, 25fold, 50fold, 75fold, 100fold 150fold, 200fold, SOOfold, 750fold, 1,000fold, 1,500fold, 2,000fold, 5,000fold, 7,500fold, 10,000fold or even higher affinity as compared to the binding to a control antigen.
  • antibody-binding to the control antigen may be undetectable (below detection threshold), while antibody-binding to the specific antigen may be well detected/determined.
  • the term “antibody” encompasses various forms of antibodies including, without being limited to, whole antibodies, antibody fragments (such as antigen binding fragments), human antibodies, chimeric antibodies, humanized antibodies, recombinant antibodies and genetically engineered antibodies (e.g., variant or mutant antibodies) as long as the characteristic properties according to the invention are retained.
  • the antibody is a humanized antibody.
  • the antibody is a monoclonal antibody.
  • the antibody may be a humanized monoclonal antibody.
  • the term “antibody” generally also includes antibody fragments. Fragments of the antibodies may retain the antigen-binding activity of the antibodies. Such fragments are referred to as "antigen-binding fragments".
  • Antigen-binding fragments include, but are not limited to, single chain antibodies, Fab, Fab', F(ab')2, Fv or scFv. Fragments of the antibodies can be obtained from the antibodies by methods that include digestion with enzymes, such as pepsin or papain, and/or by cleavage of disulfide bonds by chemical reduction. Alternatively, fragments of the antibodies can be obtained by recombinant means, for example by cloning and expressing a part (fragment) of the sequences of the heavy and/or light chain. The invention also encompasses single-chain Fv fragments (scFv) derived from the heavy and light chains of an antibody of the invention.
  • scFv single-chain Fv fragments
  • the invention includes a scFv comprising the CDRs from an antibody of the invention.
  • heavy or light chain monomers and dimers include single domain heavy chain antibodies, single domain light chain antibodies, as well as single chain antibodies, e.g., single chain Fv in which the heavy and light chain variable domains are joined by a peptide linker.
  • Antibody fragments of the invention may be contained in a variety of structures known to the person skilled in the art.
  • the sequences of the invention may be a component of multispecific molecules in which the sequences of the invention target the epitopes of the invention and other regions of the molecule bind to other targets.
  • antibody includes all categories of antibodies, namely, antigen binding fragment(s), antibody fragment(s), variant(s) and derivative(s) of antibodies.
  • variable region denotes each of the pair of light and heavy chains which is involved directly in binding the antibody to the antigen.
  • Antibodies of the invention can be of any isotype (e.g., IgA, IgG, IgM i.e.
  • the antibody is of the IgG type or the IgA type.
  • antibodies may be Igd, lgG2, lgG3 or lgG4 subclass, preferably Igd or lgG4.
  • Antibodies of the invention may have a Kor a A light chain.
  • Antibodies according to the present invention may be provided in purified form.
  • the antibody will be present in a composition that is substantially free of other polypeptides e.g., where less than 90% (by weight), usually less than 60% and more usually less than 50% of the composition is made up of other polypeptides.
  • Antibodies according to the present invention may be immunogenic in human and/or in non-human (or heterologous) hosts e.g., in mice.
  • the antibodies may have an idiotope that is immunogenic in non-human hosts, but not in a human host.
  • Antibodies of the invention for human use include those that cannot be easily isolated from hosts such as mice, goats, rabbits, rats, non-primate mammals, etc. and cannot generally be obtained by humanization or from xeno-mice.
  • the term "antigen" refers to any structural substance which serves as a target for the receptors of an adaptive immune response, in particular as a target for antibodies, T cell receptors, and/or B cell receptors.
  • an “epitope”, also known as “antigenic determinant”, is the part (or fragment) of an antigen that is recognized by the immune system, in particular by antibodies, T cell receptors, and/or B cell receptors.
  • one antigen has at least one epitope, i.e. a single antigen has one or more epitopes.
  • An antigen may be (i) a peptide, a polypeptide, or a protein, (ii) a polysaccharide, (iii) a lipid, (iv) a lipoprotein or a lipopeptide, (v) a glycolipid, (vi) a nucleic acid, or (vii) a small molecule drug or a toxin.
  • an antigen may be a peptide, a protein, a polysaccharide, a lipid, a combination thereof including lipoproteins and glycolipids, a nucleic acid (e.g. DNA, siRNA, shRNA, antisense oligonucleotides, decoy DNA, plasmid), or a small molecule drug (e.g. cyclosporine A, paclitaxel, doxorubicin, methotrexate, 5-aminolevulinic acid), or any combination thereof.
  • a nucleic acid e.g. DNA, siRNA, shRNA, antisense oligonucleotides, decoy DNA, plasmid
  • a small molecule drug e.g. cyclosporine A, paclitaxel, doxorubicin, methotrexate, 5-aminolevulinic acid
  • the antigen is selected from (i) a peptide, a polypeptide, or a protein, (ii) a polysaccharide, (iii) a lipid, (iv) a lipoprotein or a lipopeptide and (v) a glycolipid; more preferably, the antigen is a peptide, a polypeptide, or a protein.
  • the term "mutation” relates to a change in the nucleic acid sequence and/or in the amino acid sequence in comparison to a reference sequence, e.g. a corresponding genomic sequence. A mutation, e.g.
  • mutation in comparison to a genomic sequence, may be, for example, a (naturally occurring) somatic mutation, a spontaneous mutation, an induced mutation, e.g. induced by enzymes, chemicals or radiation, or a mutation obtained by site- directed mutagenesis (molecular biology methods for making specific and intentional changes in the nucleic acid sequence and/or in the amino acid sequence).
  • mutation or “mutating” shall be understood to also include physically making a mutation, e.g. in a nucleic acid sequence or in an amino acid sequence.
  • a mutation includes substitution, deletion and insertion of one or more nucleotides or amino acids as well as inversion of several successive nucleotides or amino acids.
  • a mutation may be introduced into the nucleotide sequence encoding said amino acid sequence in order to express a (recombinant) mutated polypeptide.
  • a mutation may be achieved e.g., by altering, e.g., by site-directed mutagenesis, a codon of a nucleic acid molecule encoding one amino acid to result in a codon encoding a different amino acid, or by synthesizing a sequence variant, e.g., by knowing the nucleotide sequence of a nucleic acid molecule encoding a polypeptide and by designing the synthesis of a nucleic acid molecule comprising a nucleotide sequence encoding a variant of the polypeptide without the need for mutating one or more nucleotides of a nucleic acid molecule.
  • sequence variant refers to any alteration in comparison to a reference sequence.
  • sequence variant includes nucleotide sequence variants and amino acid sequence variants.
  • a reference sequence is any of the sequences listed in the "Table of Sequences and SEQ ID Numbers" (Sequence listing), i.e. SEQ ID NO: 1 to SEQ ID NO: 199.
  • a "sequence variant” shares (over the whole length of the sequence) at least 70% or at least 75%, preferably at least 80% or at least 85%, more preferably at least 90% or at least 93%, even more preferably at least 95% or at least 96%, still more preferably at least 97% or at least 98%, particularly preferably at least 99% sequence identity with its reference sequence.
  • the "sequence variant" shares at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.
  • sequence identity In general, i.e.
  • sequence variant maintains the (biological) function of the reference sequence.
  • sequence variants relating to antibodies of the invention preferably maintain the specific binding to ASC, in particular to the PYD domain ofASC.
  • other functions such as anti-ASC-fibrillation activity and/or reducing or blocking of ASC-Ap interaction may also be maintained.
  • Sequence identity may be calculated as described below. Usually a sequence variant may preserve the specific function of the reference sequence.
  • an amino acid sequence variant has an altered sequence in which one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of the amino acids in the reference sequence is deleted or substituted, or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acids are inserted into or added to the sequence of the reference amino acid sequence.
  • the amino acid sequence variant has an amino acid sequence which is at least 70% or at least 75%, preferably at least 80% or at least 85%, more preferably at least 90% or at least 93%, even more preferably at least 95% or at least 96%, still more preferably at least 97% or at least 98%, particularly preferably at least 99% identical to the reference sequence.
  • variant sequences which are at least 90% identical have no more than 10 alterations, i.e., any combination of deletions, insertions or substitutions, per 100 amino acids of the reference sequence.
  • the "% identity" of the sequence variant is usually determined with respect to the reference sequence. It is usually calculated with regard to the full length of the reference sequence (i.e. the sequence recited in the application).
  • the substitutions are preferably conservative amino acid substitutions, wherein the substituted amino acid has similar structural or chemical properties with the corresponding amino acid in the reference sequence.
  • conservative amino acid substitutions involve substitution of one aliphatic or hydrophobic amino acids, e.g.
  • alanine, valine, leucine and isoleucine with another; substitution of one hydoxyl-containing amino acid, e.g. serine and threonine, with another; substitution of one acidic residue, e.g. glutamic acid or aspartic acid, with another; replacement of one amide-containing residue, e.g. asparagine and glutamine, with another; replacement of one aromatic residue, e.g. phenylalanine and tyrosine, with another; replacement of one basic residue, e.g.
  • the present invention provides an (isolated) antibody, or an antigen-binding fragment thereof, which (specifically) binds to ASC, in particular to the PYD domain of ASC.
  • the ASC is human ASC (SEQ ID NO: 1) and the PYD domain is the PYD domain (SEQ ID NO: 143) of human ASC.
  • Apoptosis-associated speck-like protein containing a CARD is an adaptor protein that is composed of two protein- protein interaction domains: PYRIN-PAAD-DAPIN domain (PYD) and a caspase-recruitment domain (CARD).
  • PYD and CARD domains are members of the six-helix bundle death domain-fold superfamily that mediates assembly of large signaling complexes in the inflammatory and apoptotic signaling pathways via the activation of caspase.
  • the antibody, or the antigen-binding fragment thereof, of the present invention binds specifically to the PYD domain of ASC.
  • the antibody, or the antigen-binding fragment thereof, of the present invention binds in particular to a polypeptide or protein having an amino acid sequence according to SEQ ID NO: 1 (human ASC), in particular to SEQ ID NO: 143 (PYD of human ASC). Accordingly, the antibody, or an antigen-binding fragment thereof, of the present invention does preferably not bind (specifically) to the CARD domain (SEQ ID NO: 144) ofASC.
  • the antibody, or the antigen-binding fragment thereof, of the present invention binds (specifically) to monomeric ASC. It is also preferred that the antibody, or the antigen- binding fragment thereof, of the present invention binds (specifically) to ASC specks.
  • ASC can assemble into a large protein complex, which is termed "speck”.
  • ASC specks are structures formed by ASC, which can reach a size of around 1 pm and which are characteristic of inflammasome activation. More preferably, the antibody, or the antigen-binding fragment thereof, of the present invention binds (specifically) to monomeric ASC and to ASC specks.
  • ASC may be of any origin, for example from the species from which it is originally derived from (e.g. human ASC may be of human origin; murine ASC may be of murine origin and the like), it may be recombinantly expressed or a synthetic ASC peptide.
  • the antibody, or the antigen-binding fragment thereof, of the present invention binds (specifically) to physiological/endogenous ASC, in particular to human and/or murine physiological/endogenous ASC, e.g. physiological/endogenous ASC of human macrophages and/or of mouse bone-marrow derived macrophages (BMDMs). Binding to physiological/endogenous ASC may be tested by using cell lysates of human macrophages (such as TH P-1 cells) and/or of mouse bone-marrow derived macrophages (BMDMs), e.g. as described in the appended examples.
  • human macrophages such as TH P-1 cells
  • BMDMs mouse bone-marrow derived macrophages
  • ASC can occur in four different isoforms, namely (1) ASC (or "full-length ASC”; SEQ ID NO: 1); (2) ASC-b (SEQ ID NO: 145); (3) ASC-c (SEQ ID NO: 146) and (4) ASC-d (SEQ ID NO: 147).
  • ASC or "full-length ASC”; SEQ ID NO: 1
  • ASC-b SEQ ID NO: 145
  • ASC-c SEQ ID NO: 146)
  • ASC-d SEQ ID NO: 147.
  • the antibody, or the antigen-binding fragment thereof, of the present invention (specifically) binds to (human) ASC and (human) ASC-b.
  • the antibody, or the antigen-binding fragment thereof, of the present invention does preferably not (specifically) bind to (human) ASC-c and to (human) ASC-d.
  • the antibody, or the antigen-binding fragment thereof, of the present invention binds (specifically) to ASCK21 A K22AK26A (mutated ASC with K21 A, K22A and K26A mutations). Furthermore, it is preferred that the antibody, or the antigen-binding fragment thereof, of the present invention binds (specifically) to ASC Y146A (mutated ASC with Y146A mutation).
  • the antibody, or the antigen-binding fragment thereof, of the present invention binds (specifically) to a fragment or a region of human ASC, which includes amino acids 36 to 85 of human ASC (SEQ ID NO: 1), more preferably to a fragment or a region of human ASC, which includes amino acids 41 to 85 of human ASC (SEQ ID NO: 1). Accordingly, the antibody, or the antigen-binding fragment thereof, of the present invention does preferably not bind (specifically) to a region (or fragment) of ASC comprised in (or corresponding to) amino acids 1 to 35 of ASC (SEQ ID NO: 1).
  • the antibody, or the antigen-binding fragment thereof, of the present invention does not (specifically) bind to the ASC peptide according to SEQ ID NO: 148 (ASC aa 1-20 peptide). Additionally (or alternatively), the antibody, or the antigen-binding fragment thereof, of the present invention may (also) not (specifically) bind to the ASC peptide according to SEQ ID NO: 149 (ASC aa 11 -30 peptide). In some embodiments, the antibody, or the antigen-binding fragment thereof, of the present invention does not (specifically) bind to the ASC peptide according to SEQ ID NO: 150 (ASC aa 21-40 peptide).
  • the antibody, or the antigen-binding fragment thereof, of the present invention does preferably not bind (specifically) to a region (or fragment) of ASC comprised in (or corresponding to) amino acids 86 to 195 of ASC (SEQ ID NO: 1).
  • the epitope in the PYD of human ASC, to which the antibody, or the antigen-binding fragment thereof, of the present invention binds to is preferably located within amino acids 36 to 85 ofASC (SEQ ID NO: 1), more preferably within amino acids 41 to 85 ofASC (SEQ ID NO: 1).
  • the antibody, or the antigen-binding fragment thereof, of the present invention binds (specifically) to ASC of different species.
  • the antibody, or the antigen- binding fragment thereof, of the present invention binds (specifically) to human ASC (SEQ ID NO: 1)andmurineASC(SEQID NO: 151).
  • Standard methods to assess binding of the antibody according to the present invention, or the antigen-binding fragment thereof are known to those skilled in the art and include, for example, ELISA (enzyme-1 inked immunosorbent assay). Thereby, the relative affinities of antibody binding may be determined by measuring the concentration of the antibody (ECso) required to achieve 50% maximal binding at saturation.
  • ELISA enzyme-1 inked immunosorbent assay
  • the antibody, or an antigen-binding fragment thereof, according to the present invention may comprise three heavy chain complementarity determining regions (CDRs) and three light chain CDRs.
  • complementarity determining regions (CDRs) are the hypervariable regions present in the heavy chain variable domain and in the light chain variable domains.
  • the CDRs of a heavy chain and the connected light chain of an antibody together form the antigen receptor.
  • the three CDRs (CDR1, CDR2, and CDR3) are arranged non-consecutively in the variable domain. Since antigen receptors are typically composed of two variable domains (on two different polypeptide chains, i.e.
  • heavy and light chain heavy chain variable region (VH) and light chain variable region (VL)
  • CDRs for each antigen receptor
  • heavy chain CDRH1, CDRH2, and CDRH3
  • light chain CDRL1, CDRL2, and CDRL3
  • the CDRs in the VH and VL are usually separated by framework regions, whereby a framework region (FR) is a region in the variable domain which is less "variable” than the CDR.
  • FR framework region
  • a variable region or each variable region, respectively
  • the sequences of the heavy chains and light chains of exemplary antibodies of the invention, comprising three different CDRs on the heavy chain and three different CDRs on the light chain were determined.
  • ASC_Ab001, ASC_Ab002, ASC_Ab003, ASC_Ab004, ASC_Ab005, ASC_Ab006, ASC_Ab007, ASC_Ab008 and ASC_Ab009 are shown in Table 1 below.
  • Table 1 SEQ ID NOs for CDR and VHA/L sequences of exemplary antibodies ASC_Ab001, ASC_Ab002, ASC_Ab003, ASC_Ab004, ASC_Ab005, ASC_Ab006, ASC_Ab007, ASC_Ab008 and ASC_Ab009.
  • the antibody of the invention comprises a CDRH1, a CDRH2, a CDRH3, a CDRL1, a CDRL2 and a CDRL3 of the specific combinations of the specific VH and VL sequences of exemplary antibodies ASC_Ab001, ASC_Ab002, ASC_Ab003, ASC_Ab004, ASC_Ab005, ASC_Ab006, ASC_Ab007, ASC_Ab008 and ASC_Ab009 shown in Table 1.
  • the antibody of the invention comprises a CDRH1, a CDRH2 and a CDRH3 of a VH according to SEQ ID NO: 8 and a CDRL1, a CDRL2 and a CDRL3 of a VL according to SEQ ID NO: 9.
  • the CDRs in the variable regions of SEQ ID NOs 8 and 9 may be determined by different numbering schemes known in the art, such as Kabat, Chothia, EU, IMGT, Paratome and AHo numbering.
  • the antibody of the invention comprises heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively.
  • These CDR sequences were determined as described above.
  • the present inventions also encompasses antibodies comprising a CDRH1, a CDRH2, a CDRH3, a CDRL1, a CDRL2 and a CDRL3 of a VH according to SEQ ID NO: 8 and of a VL according to SEQ ID NO: 9, wherein the CDRs are determined by a distinct method.
  • an antibody e.g., ASC_Ab001
  • binds specifically to the PYD domain of human ASC exhibits anti-ASC-fibrillation activity, blocks ASC-AR interaction and blocks the pro-inflammatory function of extracellular ASC fibrils.
  • such an antibody of the invention may comprise (i) a heavy chain variable region (VH) comprising an amino acid sequence having 70% or more (e.g., at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identity to SEQ ID NO: 8 and a light chain variable region (VL) comprising an amino acid sequence having 70% or more (e.g., at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at
  • the CDR sequences as defined above are preferably maintained.
  • the antibody, or an antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 8 and a light chain variable region comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 9.
  • the antibody of the invention comprises a CDRH1, a CDRH2 and a CDRH3 of a VH according to SEQ ID NO:15 and a CDRL1, a CDRL2 and a CDRL3 of a VL according to SEQ ID NO: 16.
  • the CDRs in the variable regions of SEQ ID NOs 15 and 16 may be determined by different numbering schemes known in the art, such as Kabat, Chothia, EU, IMGT, Paratome and AHo numbering.
  • the antibody of the invention comprises heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 2, SEQ ID NO: 10, and SEQ ID NO: 11, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively.
  • These CDR sequences were determined as described above.
  • the present inventions also encompasses antibodies comprising a CDRH1, a CDRH2, a CDRhl3, a CDRL1, a CDRL2 and a CDRL3 of a VH according to SEQ ID NO: 15 and of a VL according to SEQ ID NO: 16, wherein the CDRs are determined by a distinct method.
  • an antibody e.g., ASC_Ab002
  • such an antibody of the invention may comprise (i) a heavy chain variable region (VH) comprising an amino acid sequence having 70% or more (e.g., at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identity to SEQ ID NO: 15 and a light chain variable region (VL) comprising an amino acid sequence having 70% or more (e.g., at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at
  • the CDR sequences as defined above are preferably maintained.
  • the antibody, or an antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 15 and a light chain variable region comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 16.
  • the antibody of the invention comprises a CDRH1, a CDRH2 and a CDRH3 of a VH according to SEQ ID NO: 22 and a CDRL1, a CDRL2 and a CDRL3 of a VL according to SEQ ID NO: 23.
  • the CDRs in the variable regions of SEQ ID NOs 22 and 23 may be determined by different numbering schemes known in the art, such as Kabat, Chothia, EU, IMGT, Paratome and Ahlo numbering.
  • the antibody of the invention comprises heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 2, SEQ ID NO: 17, and SEQ ID NO: 18, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively.
  • These CDR sequences were determined as described above.
  • the present inventions also encompasses antibodies comprising a CDRH1, a CDRH2, a CDRH3, a CDRL1, a CDRL2 and a CDRL3 of a VH according to SEQ ID NO: 22 and of a VL according to SEQ ID NO: 23, wherein the CDRs are determined by a distinct method.
  • an antibody e.g., ASC_Ab003
  • such an antibody of the invention, or the antigen-binding fragment thereof may comprise (i) a heavy chain variable region (VH) comprising an amino acid sequence having 70% or more (e.g., at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identity to SEQ ID NO: 22 and a light chain variable region (VL) comprising an amino acid sequence having 70% or more (e.g., at least 70%, at least 71%, at least 72%, at least 74%, at least 7
  • the CDR sequences as defined above are preferably maintained.
  • the antibody, or an antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 22 and a light chain variable region comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 23.
  • the antibody of the invention comprises a CDRH1, a CDRH2 and a CDRH3 of a VH according to SEQ ID NO: 30 and a CDRL1, a CDRL2 and a CDRL3 of a VL according to SEQ ID NO: 31.
  • the CDRs in the variable regions of SEQ ID NOs 30 and 31 may be determined by different numbering schemes known in the art, such as Kabat, Chothia, EU, IMGT, Paratome and Ahlo numbering.
  • the antibody of the invention comprises heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29, respectively.
  • These CDR sequences were determined as described above.
  • the present inventions also encompasses antibodies comprising a CDRH1, a CDRH2, a CDRH3, a CDRL1, a CDRL2 and a CDRL3 of a VH according to SEQ ID NO: 30 and of a VL according to SEQ ID NO: 31, wherein the CDRs are determined by a distinct method.
  • an antibody e.g., ASC_Ab004
  • binds specifically to the PYD domain of human ASC exhibits anti-ASC-fibrillation activity and blocks ASC-AR interaction.
  • such an antibody of the invention may comprise (i) a heavy chain variable region (VH) comprising an amino acid sequence having 70% or more (e.g., at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identity to SEQ ID NO: 30 and a light chain variable region (VL) comprising an amino acid sequence having 70% or more (e.g., at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at
  • the CDR sequences as defined above are preferably maintained.
  • the antibody, or an antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 30 and a light chain variable region comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 31.
  • the antibody of the invention comprises a CDRH1, a CDRH2 and a CDRH3 of a VH according to SEQ ID NO: 38 and a CDRL1, a CDRL2 and a CDRL3 of a VL according to SEQ ID NO: 39.
  • the CDRs in the variable regions of SEQ ID NOs 38 and 39 may be determined by different numbering schemes known in the art, such as Kabat, Chothia, EU, IMGT, Paratome and Ahlo numbering.
  • the antibody of the invention comprises heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37, respectively.
  • These CDR sequences were determined as described above.
  • the present inventions also encompasses antibodies comprising a CDRH1, a CDRH2, a CDRH3, a CDRL1, a CDRL2 and a CDRL3 of a VH according to SEQ ID NO: 38 and of a VL according to SEQ ID NO: 39, wherein the CDRs are determined by a distinct method.
  • an antibody e.g., ASC_Ab005
  • such an antibody of the invention, or the antigen-binding fragment thereof may comprise (i) a heavy chain variable region (VH) comprising an amino acid sequence having 70% or more (e.g., at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identity to SEQ ID NO: 38 and a light chain variable region (VL) comprising an amino acid sequence having 70% or more (e.g., at least 70%, at least 71 %, at least 72%, at least 7
  • the CDR sequences as defined above are preferably maintained.
  • the antibody, or an antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 38 and a light chain variable region comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 39.
  • the antibody of the invention comprises a CDRH1, a CDRH2 and a CDRH3 of a VH according to SEQ ID NO: 46 and a CDRL1, a CDRL2 and a CDRL3 of a VL according to SEQ ID NO: 47.
  • the CDRs in the variable regions of SEQ ID NOs 46 and 47 may be determined by different numbering schemes known in the art, such as Kabat, Chothia, EU, IMGT, Paratome and AHo numbering.
  • the antibody of the invention comprises heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 45, respectively.
  • These CDR sequences were determined as described above.
  • the present inventions also encompasses antibodies comprising a CDRH1, a CDRh-12, a CDRH3, a CDRL1, a CDRL2 and a CDRL3 of a VH according to SEQ ID NO: 46 and of a VL according to SEQ ID NO: 47, wherein the CDRs are determined by a distinct method.
  • an antibody e.g., ASC_Ab006
  • such an antibody of the invention may comprise (i) a heavy chain variable region (VH) comprising an amino acid sequence having 70% or more (e.g., at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identity to SEQ ID NO: 46 and a light chain variable region (VL) comprising an amino acid sequence having 70% or more (e.g., at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at
  • the CDR sequences as defined above are preferably maintained.
  • the antibody, or an antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 46 and a light chain variable region comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 47.
  • the antibody of the invention comprises a CDRH1, a CDRH2 and a CDRH3 of a VH according to SEQ ID NO: 54 and a CDRL1, a CDRL2 and a CDRL3 of a VL according to SEQ ID NO: 55.
  • the CDRs in the variable regions of SEQ ID NOs 54 and 55 may be determined by different numbering schemes known in the art, such as Kabat, Chothia, EU, IMGT, Paratome and Ahlo numbering.
  • the antibody of the invention comprises heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 48, SEQ ID NO: 49, and SEQ ID NO: 50, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53, respectively.
  • These CDR sequences were determined as described above.
  • the present inventions also encompasses antibodies comprising a CDRH1, a CDRH2, a CDRH3, a CDRL1, a CDRL2 and a CDRL3 of a VH according to SEQ ID NO: 54 and of a VL according to SEQ ID NO: 55, wherein the CDRs are determined by a distinct method.
  • an antibody e.g., ASC_Ab007
  • such an antibody of the invention, or the antigen-binding fragment thereof may comprise (i) a heavy chain variable region (VH) comprising an amino acid sequence having 70% or more (e.g., at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identity to SEQ ID NO: 54 and a light chain variable region (VL) comprising an amino acid sequence having 70% or more (e.g., at least 70%, at least 71 %, at least 72%, at least 93%, at
  • the CDR sequences as defined above are preferably maintained.
  • the antibody, or an antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 54 and a light chain variable region comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 55.
  • the antibody of the invention comprises a CDRH1, a CDRH2 and a CDRH3 of a VH according to SEQ ID NO: 61 and a CDRL1, a CDRL2 and a CDRL3 of a VL according to SEQ ID NO: 62.
  • the CDRs in the variable regions of SEQ ID NOs 61 and 62 may be determined by different numbering schemes known in the art, such as Kabat, Chothia, EU, IMGT, Paratome and AHo numbering.
  • the antibody of the invention comprises heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 56, SEQ ID NO: 57, and SEQ ID NO: 58, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 59, SEQ ID NO: 52, and SEQ ID NO: 60, respectively.
  • CDR sequences were determined as described above.
  • the present inventions also encompasses antibodies comprising a CDRH1, a CDRH2, a CDRH3, a CDRL1, a CDRL2 and a CDRL3 of a VH according to SEQ ID NO: 61 and of a VL according to SEQ ID NO: 62, wherein the CDRs are determined by a distinct method.
  • an antibody e.g., ASC_Ab008
  • such an antibody of the invention may comprise (i) a heavy chain variable region (VH) comprising an amino acid sequence having 70% or more (e.g., at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identity to SEQ ID NO: 61 and a light chain variable region (VL) comprising an amino acid sequence having 70% or more (e.g., at least 70%, at least 71 %, at least 72%,
  • the CDR sequences as defined above are preferably maintained.
  • the antibody, or an antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 61 and a light chain variable region comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 62.
  • the antibody of the invention comprises a CDRH1, a CDRH2 and a CDRH3 of a VH according to SEQ ID NO: 68 and a CDRL1, a CDRL2 and a CDRL3 of a VL according to SEQ ID NO: 69.
  • the CDRs in the variable regions of SEQ ID NOs 68 and 69 may be determined by different numbering schemes known in the art, such as Kabat, Chothia, EU, IMGT, Paratome and AHo numbering.
  • the antibody of the invention comprises heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 66, SEQ ID NO: 36, and SEQ ID NO: 67, respectively.
  • CDR sequences were determined as described above.
  • the present inventions also encompasses antibodies comprising a CDRH1, a CDRH2, a CDRH3, a CDRL1, a CDRL2 and a CDRL3 of a VH according to SEQ ID NO: 68 and of a VL according to SEQ ID NO: 69, wherein the CDRs are determined by a distinct method.
  • an antibody e.g., ASC_Ab009
  • such an antibody of the invention, or the antigen-binding fragment thereof may comprise (i) a heavy chain variable region (Vhl) comprising an amino acid sequence having 70% or more (e.g., at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identity to SEQ ID NO: 68 and a light chain variable region (VL) comprising an amino acid sequence having 70% or more (e.g., at least 70%, at least 71 %, at least 72%
  • the CDR sequences as defined above are preferably maintained.
  • the antibody, or an antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 68 and a light chain variable region comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 69.
  • the antibody, or the antigen-binding fragment thereof, of the invention may exhibit anti-ASC- fibrillation activity.
  • Antibodies reducing or inhibiting ASC fibrillation provide a way to block either de novo or seeded ASC assembly, and thus the downstream effect of ASC speck formation such as inflammation and/or amyloid seeding.
  • Anti-fibrillation activity may be tested in a fibrillation assay or in a sedimentation assay, e.g. as described in the appended examples. Accordingly, preferred fibrillation assay is described in the examples of the present specification. Briefly, ASC assembles in large cytoplasmic macromolecular assemblies called ASC specks.
  • ASC e.g. including a marker such as green fluorescent protein (GFP)
  • GFP green fluorescent protein
  • Protein aggregates of the antibody to be tested may be compared to those obtained with controls, such as ASC only and an unrelated antibody (which are both not expected to decrease ASC fibrillation). If significantly less fibrillation is observed for the antibody to be tested as compared to an unrelated antibody, the antibody to be tested exhibits anti-fibrillation activity.
  • the antibody, or the antigen-binding fragment thereof, of the invention may reduce or block ASC-Ap interaction.
  • ASC-Ap interaction is assumed to be at the basis of increased A(3 aggregation and amyloid deposition in Alzheimer disease (Venegas C, Kumar S, Franklin BS, Dierkes T, Brinkschulte R, Tejera D, Vieira-Saecker A, Schwartz S, Santarelli F, Kummer MP, Griep A, Gelpi E, Beilharz M, Riedel D, Golenbock DT, Geyer M, Walter J, Latz E, Heneka MT. Microglia-derived ASC specks cross-seed amyloid-(3 in Alzheimer's disease. Nature. 2017 Dec 20;552(7685):355-361.
  • ASC fibrils may be formed by incubating ASC monomers, e.g. at about 37°C for about 1 h. Fibrillary ASC may then be incubated with Ap, e.g. including a marker such as TAMRA ((5- Carboxy)tetramethylrhodamine), in the presence of the antibody to be tested (and appropriate controls as described below), e.g.
  • Ap e.g. including a marker such as TAMRA ((5- Carboxy)tetramethylrhodamine
  • protein aggregates may be determined in view of the marker used (e.g. by flow cytometry in case of TAMRA). Protein aggregates obtained after incubation of ASC+AR+anti-ASC antibodies may be compared to those obtained with controls, such as ASC only, ASC+Ap and an unrelated antibody.
  • ASC-AR inhibition may be determined as percentage in view the binding (aggregation) observed for the ASC+Ap (without antibody) control and the ASC only control. While no inhibition (0%) is assumed for the ASC+AR control, 100% inhibition is assumed for the ASC only group (because no interaction (aggregation) is expected for ASC without Ap).
  • the %-inhibition of ASC-Ap interaction for the antibody to be tested may thus be calculated in view thereof and compared to the effects of an unrelated antibody to determine whether or not significant inhibition ofASC-AR interaction is observed.
  • the antibody, or the antigen-binding fragment thereof, of the invention may reduce or block Ap aggregation.
  • the antibody, or the antigen- binding fragment thereof, of the invention may reduce or block protein aggregation, in particular in the context of inflammatory processes. Without being bound to any theory, it is assumed that ASC acts as a "catalyzer" on the aggregation of proteins, such as AR.
  • antibodies reducing or blocking the interaction of ASC with such (aggregating) proteins - in particular by binding to ASC - are assumed to reduce or block the aggregation of proteins (which is otherwise catalyzed by ASC).
  • the antibody, or the antigen-binding fragment thereof, of the invention may reduce or block the pro-inflammatory function of extracellular ASC fibrils.
  • ASC fibrils and ASC specks can propagate inflammation (Franklin BS, Bossaller L, De Nardo D, Ratter JM, Stutz A, Engels G, Brenker C, Nordhoff M, Mirandola SR, Al-Amoudi A, Mangan MS, Zimmer S, Monks BG, Fricke M, Schmidt RE, Espevik T, Jones B, Jarnicki AC, Hansbro PM, Busto P, Marshak-Rothstein A, Hornemann S, Aguzzi A, KastenmullerW, Latz E.
  • the adaptor ASC has extracellular and 'prionoid' activities that propagate inflammation. Nat Immunol. 2014 Aug;15(8):727-37.
  • THP-1 efficacy assay may be used.
  • a preferred THP-1 efficacy assay is described in the examples of the present specification. Briefly, wt and asc/ ⁇ T ⁇ - ⁇ P-' ⁇ monocytes may be seeded, e.g. at a density of about 10'OQO cells/well, in a multi-well plate. Monocytes may then be differentiated into macrophages, e.g. by incubation in 100 ng/ml phorbol 12-myristate-13-acetate (PMA)- containing medium, e.g. for about 4 days at about 37°C and about 5% C02.
  • PMA phorbol 12-myristate-13-acetate
  • ASC fibrils may be prepared by incubating ASC monomers, e.g. at about 37°C for about 1 h. Before addition of the ASC fibrils to the cells, they may be pre-incubated with the antibody to be tested, e.g. for about 15 min at about 22°C. After incubation (e.g. about 16 h at about 37°C and about 5% C02), secreted IL-1(3 may be measured, e.g. by Lumit Human IL-1 Immunoassay (Promega, W6010). The amount of IL-1 p secretion may be compared to that obtained when an unrelated control antibody was used in the same assay in wt THP-1 cells.
  • the antibody, or the antigen-binding fragment thereof is a humanized antibody.
  • Methods for humanization of antibodies are well-known in the art.
  • a humanized antibody is engineered to comprise one or more human framework regions in the variable region together with non-human (e.g., rabbit) CDRs of the heavy and/or light chain.
  • a humanized antibody comprises sequences that are entirely human except for the CDR regions.
  • a humanized antibody may comprise residues that are found neither in the human form of the antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance.
  • a humanized antibody comprises a VH and a VL, in which all or essentially all of the CDR regions correspond to those of a parental non-human (e.g. rabbit) antibody and all or essentially all of the FR regions are those of a human antibody or a human immunoglobulin consensus sequence.
  • the FR region can be modified in any manner known in the art and/or provided herein.
  • the humanized antibody may also comprise a human Fc moiety.
  • Humanized antibodies preferably exhibit reduced immunogenicity when introduced into the human body.
  • ,1-lumanized” antibodies may be prepared by creating a ,,chimeric" antibody (non-human Fab grafted onto human Fc) as an initial step and selective mutation of the (non- CDR) amino acids in the Fab portion of the molecule.
  • ,,humanized” antibodies can be obtain directly by grafting appropriate ,,donor" CDR coding segments derived from a non-human animal onto a human antibody ,,acceptor” scaffold, and optionally mutating (non-CDR) amino acids for optimized binding.
  • the antibody, or the antigen-binding fragment thereof is a monoclonal antibody.
  • the antibody, or the antigen-binding fragment thereof may be a humanized monoclonal antibody.
  • the variable regions or the CDRs of the antibody as defined herein are derived from a rabbit antibody and grafted in a scaffold of a human IgG or IgA antibody.
  • the variable regions, portions thereof or the CDRs may be rabbit and grafted in an antibody framework, which is preferably of human origin.
  • the rabbit-derived portions of the variable regions that are grafted into the antibody framework comprise the CDRs.
  • IgG IgGI and lgG4 are preferred.
  • the skilled person may use the (rabbit) VHA/L sequences of the exemplary antibodies, as described in Table 1, and determine "extended" rabbit CDRs in said VHA/L sequences of Table 1 by combining two or more (e.g., 3, 4, 5 or 6) different antibody numbering schemes (e.g., selected from Kabat, Chothia, EU, IMGT, Paratome and AHo), such that each "extended" CDR covers the respective CDRs as defined in each of the combined numbering schemes.
  • two or more e.g., 3, 4, 5 or 6
  • different antibody numbering schemes e.g., selected from Kabat, Chothia, EU, IMGT, Paratome and AHo
  • Such "extended" CDRs determined by combining different numbering schemes may, thus, include amino acid residues of the framework region adjacent to a CDR (in particular when a single numbering scheme is used).
  • an "extended" CDR may correspond to the CDR as determined by a single numbering scheme. This applies in particular, if the CDR as determined by the single numbering scheme contains (or corresponds to) the corresponding CDR determined by one or more other numbering schemes.
  • Kabat, IMGT and Paratome may be used to obtain extended (rabbit) CDR sequences (based on the VHA/L sequences of the exemplified antibodies of Table 1 above), as described in Zhang and Ho, 2017 (Zhang YF, Ho M.
  • Table 2 SEQ ID NOs of exemplary extended (rabbit) CDRs of the exemplary antibodies ASC_Ab001 (two versions of extended CDRs), ASC_Ab002, ASC_Ab003, ASC_Ab004, ASC_Ab005, ASC_Ab006, ASC_Ab007, ASC_Ab008 and ASC_Ab009 Specific examples of VHA/L sequences of humanized versions of the exemplified antibodies are shown in Table 3 below.
  • Table 3 Exemplary humanized VHA/L sequences of the exemplary antibodies ASC_Ab001, ASC_Ab002, ASC_Ab003, ASC_Ab004, ASC_Ab005, ASC_Ab006, ASC_Ab007, ASC_Ab008 and ASC_Ab009 and human germlined V and J gene versions suitable for each of these antibodies.
  • the antibody of the invention comprises an extended CDRH1, an extended CDRH2, an extended CDRH3, an extended CDRL1, an extended CDRL2 and an extended CDRL3 of exemplary antibodies ASC_Ab001 , ASC_Ab002, ASC_Ab003, ASC_Ab004, ASC_Ab005, ASC_Ab006, ASC_Ab007, ASC_Ab008 and ASC_Ab009 shown in Table 2.
  • an "extended" CDRs may include amino acid residues of the framework region adjacent to the CDRs shown in Table 1.
  • the antibody of the invention comprises an extended CDRH1, an extended CDRH2 and an extended CDRH3 as set forth in SEQ ID NO: 159, SEQ ID NO: 3, and SEQ ID NO: 160, respectively; and an extended CDRL1, an extended CDRL2 and an extended CDRL3 as set forth in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively.
  • the antibody of the invention comprises an extended CDRH1, an extended CDRH2 and an extended CDRH3 as set forth in SEQ ID NO: 194, SEQ ID NO: 195, and SEQ ID NO: 196, respectively; and an extended CDRL1, an extended CDRL2 and an extended CDRL3 as set forth in SEQ ID NO: 197, SEQ ID NO: 198, and SEQ ID NO: 199, respectively.
  • the antibody of the invention comprises heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively; and humanized framework regions, wherein the heavy chain V gene IGHV3-66, the heavy chain J gene IGh-IJ2, the light chain V gene IGKV1-5 and the light chain J gene IGKJ4 are used to obtain the humanized framework regions.
  • such an antibody or the antigen- binding fragment thereof, comprises an extended CDRH1, an extended CDRH2 and an extended CDRH3 as set forth in SEQ ID NO: 159, SEQ ID NO: 3, and SEQ ID NO: 160, respectively; and an extended CDRL1, an extended CDRL2 and an extended CDRL3 as set forth in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively.
  • such an antibody or the antigen-binding fragment thereof, comprises an extended CDRH1, an extended CDRH2 and an extended CDRH3 as set forth in SEQ ID NO: 194, SEQ ID NO: 195, and SEQ ID NO: 196, respectively; and an extended CDRL1, an extended CDRL2 and an extended CDRL3 as set forth in SEQ ID NO: 197, SEQ ID NO: 198, and SEQ ID NO:199, respectively.
  • such an antibody of the invention, or the antigen-binding fragment thereof may comprise (i) a heavy chain variable region (VH) comprising an amino acid sequence having 70% or more (e.g., at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identity to SEQ ID NO: 157 and a light chain variable region (VL) comprising an amino acid sequence having 70% or more (e.g., at least 70%, at least 71 %, at least 72%,
  • CDR sequences as defined above are preferably maintained.
  • the extended CDR sequences as defined above are maintained in the sequence variant of SEQ ID NO: 157 and in the sequence variant of SEQ ID NO: 158, respectively.
  • the antibody, or an antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 157 and a light chain variable region comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 158.
  • such an antibody of the invention, or the antigen-binding fragment thereof may comprise (i) a heavy chain variable region (VH) comprising an amino acid sequence having 70% or more (e.g., at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identity to SEQ ID NO: 176 and a light chain variable region (VL) comprising an amino acid sequence having 70% or more (e.g., at least 70%, at least 71 %, at least 72%,
  • CDR sequences as defined above are preferably maintained.
  • the extended CDR sequences as defined above are maintained in the sequence variant of SEQ ID NO: 176 and in the sequence variant of SEQ ID NO: 177, respectively.
  • the antibody, or an antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 176 and a light chain variable region comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 177.
  • the antibody of the invention, or the antigen-binding fragment thereof comprises an extended CDRH1, an extended CDRH2 and an extended CDRH3 as set forth in SEQ ID NO: 161, SEQ ID NO: 10, and SEQ ID NO: 162, respectively; and an extended CDRL1, an extended CDRL2 and an extended CDRL3 as set forth in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively.
  • the antibody of the invention comprises heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 2, SEQ ID NO: 10, and SEQ ID NO: 11, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively; and humanized framework regions, wherein the heavy chain V gene IGHV3-23, the heavy chain J gene IGHJ2, the light chain V gene IGKV1 -16 and the light chain J gene IGKJ4 are used to obtain the humanized framework regions.
  • such an antibody or the antigen-binding fragment thereof, comprises an extended CDRH1, an extended CDRH2 and an extended CDRH3 as set forth in SEQ ID NO: 161, SEQID NO: 10, and SEQ ID NO: 162, respectively; and an extended CDRL1, an extended CDRL2 and an extended CDRL3 as set forth in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively.
  • such an antibody of the invention, or the antigen-binding fragment thereof may comprise (i) a heavy chain variable region (VH) comprising an amino acid sequence having 70% or more (e.g., at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identity to SEQ ID NO: 178 and a light chain variable region (VL) comprising an amino acid sequence having 70% or more (e.g., at least 70%, at least 71 %, at least 72%, at least
  • CDR sequences as defined above are preferably maintained.
  • the extended CDR sequences as defined above are maintained in the sequence variant of SEQ ID NO: 178 and the sequence variant of SEQ ID NO: 179.
  • the antibody, or an antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 178 and a light chain variable region comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 179.
  • the antibody of the invention, or the antigen-binding fragment thereof comprises an extended CDRH1, an extended CDRH2 and an extended CDRH3 as set forth in SEQ ID NO: 161, SEQ ID NO: 17, and SEQ ID NO: 163, respectively; and an extended CDRL1, an extended CDRL2 and an extended CDRL3 as set forth in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively.
  • the antibody of the invention comprises heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 2, SEQ ID NO: 17, and SEQ ID NO: 18, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively; and humanized framework regions, wherein the heavy chain V gene IGHV3-66, the heavy chain J gene IGHJ2, the light chain V gene IGKV1 -5 and the light chain J gene IGKJ4 are used to obtain the humanized framework regions.
  • such an antibody or the antigen- binding fragment thereof, comprises an extended CDRH1, an extended CDRH2 and an extended CDRH3 as set forth in SEQ ID NO: 161, SEQ ID NO: 17, and SEQ ID NO: 163, respectively; and an extended CDRL1 , an extended CDRL2 and an extended CDRL3 as set forth in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively.
  • such an antibody of the invention, or the antigen-binding fragment thereof may comprise (i) a heavy chain variable region (VH) comprising an amino acid sequence having 70% or more (e.g., at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identity to SEQ ID NO: 180 and a light chain variable region (VL) comprising an amino acid sequence having 70% or more (e.g., at least 70%, at least 71 %, at least 72%, at least 93%, at
  • the CDR sequences as defined above are preferably maintained.
  • the extended CDR sequences as defined above are maintained in the sequence variant of SEQ ID NO: 180 and the sequence variant of SEQ ID NO: 181.
  • the antibody, or an antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 180 and a light chain variable region comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 181.
  • the antibody of the invention, or the antigen-binding fragment thereof comprises an extended CDRH1, an extended CDRhl2 and an extended CDRH3 as set forth in SEQ ID NO: 164, SEQ ID NO: 25, and SEQ ID NO: 165, respectively; and an extended CDRL1, an extended CDRL2 and an extended CDRL3 as set forth in SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29, respectively.
  • the antibody of the invention comprises heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29, respectively; and humanized framework regions, wherein the heavy chain V gene IGHV3-66, the heavy chain J gene IGh-IJ2, the light chain V gene IGKV1 -6 and the light chain J gene IGKJ4 are used to obtain the humanized framework regions.
  • such an antibody or the antigen- binding fragment thereof, comprises an extended CDRH1, an extended CDRH2 and an extended CDRH3 as set forth in SEQ ID NO: 164, SEQ ID NO: 25, and SEQ ID NO: 165, respectively; and an extended CDRL1, an extended CDRL2 and an extended CDRL3 as set forth in SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29, respectively.
  • such an antibody of the invention may comprise (i) a heavy chain variable region (VH) comprising an amino acid sequence having 70% or more (e.g., at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identity to SEQ ID NO: 182 and a light chain variable region (VL) comprising an amino acid sequence having 70% or more (e.g., at least 70%, at least 71 %, at least 72%,
  • CDR sequences as defined above are preferably maintained.
  • the extended CDR sequences as defined above are maintained in the sequence variant of SEQ ID NO: 182 and the sequence variant of SEQ ID NO: 183.
  • the antibody, or an antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 182 and a light chain variable region comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 183.
  • the antibody of the invention, or the antigen-binding fragment thereof comprises an extended CDRH1, an extended CDRH2 and an extended CDRH3 as set forth in SEQ ID NO: 166, SEQ ID NO: 33, and SEQ ID NO: 167, respectively; and an extended CDRL1, an extended CDRL2 and an extended CDRL3 as set forth in SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37, respectively.
  • the antibody of the invention comprises heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37, respectively; and humanized framework regions, wherein the heavy chain V gene IGh-IV3-33, the heavy chain J gene IGHJ2, the light chain V gene IGKV1 -5 and the light chain J gene IGKJ4 are used to obtain the humanized framework regions.
  • such an antibody or the antigen- binding fragment thereof, comprises an extended CDRH1, an extended CDRhl2 and an extended CDRH3 as set forth in SEQ ID NO: 166, SEQ ID NO: 33, and SEQ ID NO: 167, respectively; and an extended CDRL1, an extended CDRL2 and an extended CDRL3 as set forth in SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37, respectively.
  • such an antibody of the invention, or the antigen-binding fragment thereof may comprise (i) a heavy chain variable region (VH) comprising an amino acid sequence having 70% or more (e.g., at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identity to SEQ ID NO: 184 and a light chain variable region (VL) comprising an amino acid sequence having 70% or more (e.g., at least 70%, at least 71 %, at least 72%,
  • the CDR sequences as defined above are preferably maintained.
  • the extended CDR sequences as defined above are maintained in the sequence variant of SEQ ID NO: 184 and the sequence variant of SEQ ID NO: 185.
  • the antibody, or an antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 184 and a light chain variable region comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 185.
  • the antibody of the invention, or the antigen-binding fragment thereof comprises an extended CDRHI1, an extended CDRH2 and an extended CDRH3 as set forth in SEQ ID NO: 168, SEQ ID NO: 41, and SEQ ID NO: 169, respectively; and an extended CDRL1, an extended CDRL2 and an extended CDRL3 as set forth in SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 45, respectively.
  • the antibody of the invention comprises heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 40, SEQ ID NO: 41 , and SEQ ID NO: 42, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 45, respectively; and humanized framework regions, wherein the heavy chain V gene IGHV3-23, the heavy chain J gene IGhIJ2, the light chain V gene IGKV1-27 and the light chain J gene IGKJ4 are used to obtain the humanized framework regions.
  • such an antibody or the antigen-binding fragment thereof, comprises an extended CDRH1, an extended CDRH2 and an extended CDRH3 as set forth in SEQ ID NO: 168, SEQ ID NO: 41, and SEQ ID NO: 169, respectively; and an extended CDRL1, an extended CDRL2 and an extended CDRL3 as set forth in SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 45, respectively.
  • such an antibody of the invention, or the antigen-binding fragment thereof may comprise (i) a heavy chain variable region (VH) comprising an amino acid sequence having 70% or more (e.g., at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identity to SEQ ID NO: 186 and a light chain variable region (VL) comprising an amino acid sequence having 70% or more (e.g., at least 70%, at least 71 %, at least 72%, at least
  • CDR sequences as defined above are preferably maintained.
  • the extended CDR sequences as defined above are maintained in the sequence variant of SEQ ID NO: 186 and the sequence variant of SEQ ID NO: 187.
  • the antibody, or an antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 186 and a light chain variable region comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 187.
  • the antibody of the invention, or the antigen-binding fragment thereof comprises an extended CDRH1, an extended CDRH2 and an extended CDRH3 as set forth in SEQ ID NO: 170, SEQ ID NO: 49, and SEQ ID NO: 171, respectively; and an extended CDRL1, an extended CDRL2 and an extended CDRL3 as set forth in SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53, respectively.
  • the antibody of the invention comprises heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 48, SEQ ID NO: 49, and SEQ ID NO: 50, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53, respectively; and humanized framework regions, wherein the heavy chain V gene IGHV3-15, the heavy chain J gene IGHJ2, the light chain V gene IGKV1-8 and the light chain J gene IGKJ4are used to obtain the humanized framework regions.
  • such an antibody or the antigen- binding fragment thereof, comprises an extended CDRH1, an extended CDRH2 and an extended CDRH3 as set forth in SEQ ID NO: 170, SEQ ID NO: 49, and SEQ ID NO: 171, respectively; and an extended CDRL1, an extended CDRL2 and an extended CDRL3 as set forth in SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53, respectively.
  • such an antibody of the invention, or the antigen-binding fragment thereof may comprise (i) a heavy chain variable region (VH) comprising an amino acid sequence having 70% or more (e.g., at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identity to SEQ ID NO: 188 and a light chain variable region (VL) comprising an amino acid sequence having 70% or more (e.g., at least 70%, at least 71 %, at least 72%,
  • CDR sequences as defined above are preferably maintained.
  • the extended CDR sequences as defined above are maintained in the sequence variant of SEQ ID NO: 188 and the sequence variant of SEQ ID NO: 189.
  • the antibody, or an antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 188 and a light chain variable region comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 189.
  • the antibody of the invention, or the antigen-binding fragment thereof comprises an extended CDRH1, an extended CDRH2 and an extended CDRH3 as set forth in SEQ ID NO: 172, SEQ ID NO: 57, and SEQ ID NO: 173, respectively; and an extended CDRL1, an extended CDRL2 and an extended CDRL3 as set forth in SEQ ID NO: 59, SEQ ID NO: 52, and SEQ ID NO: 60, respectively.
  • the antibody of the invention comprises heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 56, SEQ ID NO: 57, and SEQ ID NO: 58, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 59, SEQ ID NO: 52, and SEQ ID NO: 60, respectively; and humanized framework regions, wherein the heavy chain V gene IGHV3-33, the heavy chain J gene IGHJ2, the light chain V gene IGKV1-12 and the light chain J gene IGKJ4 are used to obtain the humanized framework regions.
  • such an antibody or the antigen-binding fragment thereof, comprises an extended CDRH1, an extended CDRH2 and an extended CDRH3 as set forth in SEQ ID NO: 172, SEQ ID NO: 57, and SEQ ID NO: 173, respectively; and an extended CDRL1, an extended CDRL2 and an extended CDRL3 as set forth in SEQ ID NO: 59, SEQ ID NO: 52, and SEQ ID NO: 60, respectively.
  • such an antibody of the invention, or the antigen-binding fragment thereof may comprise (i) a heavy chain variable region (VH) comprising an amino acid sequence having 70% or more (e.g., at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identity to SEQ ID NO: 190 and a light chain variable region (VL) comprising an amino acid sequence having 70% or more (e.g., at least 70%, at least 71 %, at least 72%,
  • the CDR sequences as defined above are preferably maintained.
  • the extended CDR sequences as defined above are maintained in the sequence variant of SEQ ID NO: 190 and the sequence variant of SEQ ID NO: 191.
  • the antibody, or an antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 190 and a light chain variable region comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 191.
  • the antibody of the invention, or the antigen-binding fragment thereof comprises an extended CDRFI1, an extended CDRH2 and an extended CDRH3 as set forth in SEQ ID NO: 174, SEQ ID NO: 64, and SEQ ID NO: 175, respectively; and an extended CDRL1, an extended CDRL2 and an extended CDRL3 as set forth in SEQ ID NO: 66, SEQ ID NO: 36, and SEQ ID NO: 67, respectively.
  • the antibody of the invention comprises heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65, respectively; and light chain CDR1 , CDR2, and CDR3 sequences as set forth in SEQ ID NO: 66, SEQ ID NO: 36, and SEQ ID NO: 67, respectively; and humanized framework regions, wherein the heavy chain V gene IGHV3-23, the heavy chain J gene IGHJ6, the light chain V gene IGKV1-27 and the light chain J gene IGKJ3 are used to obtain the humanized framework regions.
  • such an antibody or the antigen-binding fragment thereof, comprises an extended CDRH1, an extended CDRH2 and an extended CDRH3 as set forth in SEQ ID NO: 174, SEQ ID NO: 64, and SEQ ID NO: 175, respectively; and an extended CDRL1, an extended CDRL2 and an extended CDRL3 as set forth in SEQ ID NO: 66, SEQ ID NO: 36, and SEQ ID NO: 67, respectively.
  • such an antibody of the invention, or the antigen-binding fragment thereof may comprise (i) a heavy chain variable region (VH) comprising an amino acid sequence having 70% or more (e.g., at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identity to SEQ ID NO: 192 and a light chain variable region (VL) comprising an amino acid sequence having 70% or more (e.g., at least 70%, at least 71 %, at least 72%,
  • the CDR sequences as defined above are preferably maintained.
  • the extended CDR sequences as defined above are maintained in the sequence variant of SEQ ID NO: 192 and the sequence variant of SEQ ID NO: 193.
  • the antibody, or an antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 192 and a light chain variable region comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 193.
  • humanization may include back mutations, i.e. wherein amino acids of the human framework sequences are mutated back to the corresponding amino acids of the rabbit framework sequences, in particular if the result of grafting of the (extended) CDRs onto human IgG framework regions is not satisfactory.
  • the goal of humanization is always to maintain as many human sequences/amino acids as possible.
  • the amino acid residues of the vernier zones (vernier zone residues) of the human VH and VL frame work regions may be back mutated to the corresponding rabbit amino acids (of the VHA/L regions as shown in Table 1).
  • the first two amino acid residues in the light chain may be back mutated (from human to rabbit), if required.
  • the first 1, 2 or 3 residues at the N terminus of the HV4/LV4 loops (light chain LV4 loop: Kabat positions 66-70; and heavy chain HV4 loop: Kabat positions 72-75 or 76) may be back mutated (from human to rabbit), if required.
  • an extra cysteine in position 80 (Kabat numbering) of the rabbit VL may be removed or replaced (substituted) in the humanized version.
  • T23 (Kabat numbering) of the rabbit VH is replaced, preferably by the corresponding amino acid of the human VH.
  • the antibody according to the present invention, or an antigen bind ing fragment thereof may comprise an Fc moiety.
  • the Fc moiety may be of any origin, preferably it is derived from human origin, e.g. from human IgAor IgG, such as Igd, lgG2, lgG3, and/or lgG4, e.g. human Igd.
  • an Fc moiety refers to a sequence derived from the portion of an immunoglobulin heavy chain beginning in the hinge region just upstream of the papain cleavage site (e.g., residue 216 in native IgG, taking the first residue of heavy chain constant region to be 114) and ending at the C-terminus of the immunoglobulin heavy chain. Accordingly, an Fc moiety may be a complete Fc moiety or a portion (e.g., a domain) thereof. A complete Fc moiety comprises at least a hinge domain, a CH2 domain, and a CH3 domain (e.g., EU amino acid positions 216-446).
  • an Fc moiety comprises at least one of: a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant, portion, or fragment thereof.
  • a hinge e.g., upper, middle, and/or lower hinge region
  • An Fc moiety may comprise at least a hinge domain, a CH2 domain or a CH3 domain.
  • the Fc moiety may be a complete Fc moiety.
  • the Fc moiety may also comprises one or more amino acid insertions, deletions, or substitutions relative to a naturally-occurring Fc moiety.
  • the antibody, or antigen binding fragment thereof, according to the present invention comprises an Fc region.
  • Fc region refers to the portion of an immunoglobulin formed by two or more Fc moieties of antibody heavy chains.
  • the Fc region may be monomeric or "single-chain" Fc region (i.e., a scFc region).
  • Single chain Fc regions are comprised of Fc moieties linked within a single polypeptide chain (e.g., encoded in a single contiguous nucleic acid sequence).
  • Exemplary scFc regions are disclosed in WO 2008/143954 A2.
  • the Fc region is dimeric.
  • a "dimeric Fc region” or “dcFc” refers to the dimer formed by the Fc moieties of two separate immunoglobulin heavy chains.
  • the dimeric Fc region may be a homodimer of two identical Fc moieties (e.g., an Fc region of a naturally occurring immunoglobulin) or a heterodimer of two non-identical Fc moieties.
  • the Fc moiety, or the Fc region comprises or consists of an amino acid sequence derived from a human immunoglobulin sequence (e.g., from an Fc region or Fc moiety from a human IgG molecule).
  • the Fc moiety, or the Fc region may comprise one or more amino acids from another mammalian species.
  • a primate Fc moiety or a primate binding site may be included in the antibody, or antigen-binding fragment.
  • one or more murine amino acids may be present in the Fc moiety or in the Fc region.
  • the Fc moieties of the Fc region may be of the same or different class and/or subclass.
  • the Fc moieties may be derived from an immunoglobulin (e.g., a human immunoglobulin) of an IgGt, lgG2, lgG3 or lgG4 subclass.
  • an immunoglobulin e.g., a human immunoglobulin
  • the Fc moiety, or Fc region may be modified such that it varies in amino acid sequence from the complete Fc moiety, or Fc region, of a naturally occurring immunoglobulin molecule.
  • the engineered Fc moiety, or Fc region retains at least one desirable function conferred by the natural ly-occurring Fc moiety.
  • Fc functions which may be modified by Fc engineering, or which may be retained, include Fc receptor (FcR) binding, antibody half-life modulation, ADCC function, protein A binding, protein G binding, and complement binding.
  • FcR Fc receptor
  • ADCC function protein A binding
  • protein G binding protein G binding
  • complement binding The portions of naturally occurring Fc moieties, or Fc regions, which are responsible and/or essential for such functions are well known by those skilled in the art.
  • Various Fc modifications are known in the art and described, for example, in Saunders KO. Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life. Front Immunol.2019 Jun 7;10:1296. doi: 10.3389/fimmu.2019.01296, which is incorporated herein by reference in its entirety.
  • the antibody according to the present invention comprises a (complete) Fc moiety/Fc region.
  • the (complete) Fc moiety/Fc region may be engineered by one or more specific amino acid substitutions. In other embodiments, the may not be engineered (i.e., as occurring in a natural immunoglobulin, e.g. Igd or lgG4).
  • the Fc moiety/Fc region is engineered (e.g., based on a human IgG, such as lgG1 or lgG4, Fc moiety/Fc region) to diminish Fc effector functions, in particular to reduce or inhibit FcyR and complement interactions.
  • the antibody of the present invention may comprise an Fc modification selected from Leu235Glu, Leu234Ala/Leu235Ala, Ser228Pro/Leu235Glu, Gly237Ala, Glu318Ala, Leu234Ala/Leu235Ala/Pro329Gly, Pro331Ser/Leu234Glu/Leu235Phe, Asp265Ala, Glu233Pro, Gly236Arg/Leu328Arg, lgG2- lgG4 cross-subclass, Ala330Leu, His268Gln/Val309Leu/Ala330Ser/Pro331Ser, Val234Ala/Gly237Ala/Pro238Ser/His268Ala/Val309Leu/Ala330Ser/Pro331Ser, Asp270Ala, Leu234Ala/L235Ala/Gly237Ala/P238Ser/His268Ala/Ala330Ser/Pro331S
  • LALA L1A
  • L234A L235A
  • CH2 L4A CH2 L5A mutation
  • the "LALA” mutation abolishes antibody binding to FcyRI, FcyRII and FcyRllla (Lund J, Winter G, Jones PT, Pound JD, Tanaka T, Walker MR et al.
  • Human Fc gamma Rl and Fc gamma Rll interact with distinct but overlapping sites on human IgG. J Immunol.1991 ;147:2657-2662).
  • An exemplary amino acid sequence of CH1-CH2-CH3 comprising the "LALA” mutation is set forth in SEQ ID NO: 141. Accordingly, the antibody preferably comprises an amino acid sequence according to SEQ ID NO: 141 or a sequence variant thereof, as described herein, wherein the "LALA" mutation is maintained. In some embodiments, the "LALA” mutation may be combined with further mutations, such as P329G or G236R (EU numbering).
  • the antibody includes the "LALA" mutation (L234A and L235A), while P329 and/or G236 are maintained (i.e., not mutated).
  • binding of the antibody to an Fc receptor may be assessed by various methods known to the skilled person, such as ELISA (h-Iessell AJ, Hangartner L, Hunter M, Havenith CEG, Beurskens FJ, BakkerJM, Lanigan CMS, Landucci G, Forthal DN, Parren PWHI, etal.: Fc receptor but not complement binding is important in antibody protection against HIV.
  • Antibodies of the invention can be of any isotype (e.g., IgA, IgG, IgM i.e. an a, y or ⁇ 1 heavy chain).
  • the antibody may be of the IgA or IgG type.
  • antibodies may be IgGt, lgG2, lgG3 or lgG4 subclass, preferably IgGt or lgG4.
  • Exemplified sequences for Igd and lgG4 constant regions which may be useful in the antibody as described herein, are provided in SEQ ID NO: 70 (Igd) and SEQ ID NO: 142 (lgG4).
  • the antibody of the invention may comprise an amino acid sequence according to SEQ ID NO: 70 or SEQ ID NO: 142, or a sequence variant thereof as described herein.
  • the human lgG4 constant region sequence of SEQ ID NO: 142 comprises the stable hinge mutation S228P (S. Angal, D.J.
  • Antibodies of the invention may have a Kor a X light chain.
  • the antibody of the invention may comprise an amino acid sequence according to SEQ ID NO: 71 or 72, or a sequence variant thereof as described herein.
  • the antibody of the invention has a K light chain, e.g. having the constant region comprising or consisting of SEQ ID NO: 71.
  • the present invention encompasses antigen-binding fragments.
  • An antigen-binding fragment may or may not comprise an Fc moiety, in particular a portion of a complete Fc region.
  • the antibody, or antigen-binding fragment thereof is selected from Fab, Fab', F(ab')2, Fv or scFv.
  • F(ab')2 which may be obtained by pepsin cleavage or recombinant expression
  • Fab' which can be obtained from F(ab')2 or by recombinant expression
  • the antibody, or antigen-binding fragment may be a single-chain antibody (or fragment).
  • the single-chain antibody (or fragment) may encode the complete set of six CDRs, i.e. include the three heavy chain CDRs as well as the three light chain CDRs.
  • the single-chain antibody may include a heavy chain variable region (VH) as well as a light chain variable region (VL), for example including the VH and VL sequences as described above.
  • VH heavy chain variable region
  • VL light chain variable region
  • variants of the sequences recited in the application are also included within the scope of the invention.
  • variants include natural variants generated by somatic mutation in vivo during the immune response or in vitrou ⁇ on culture of immortalized B cell clones. Alternatively, variants may arise due to the degeneracy of the genetic code or may be produced due to errors in transcription or translation.
  • Antibodies of the invention, or antigen-binding fragments thereof, may be provided in purified form.
  • the antibody, or antigen-binding fragment will be present in a composition that is substantially free of other polypeptides e.g., where less than 90% (by weight), usually less than 60% and more usually less than 50% of the composition is made up of other polypeptides.
  • Antibodies of the invention may be immunogenic in non-human (or heterologous) hosts e.g., in mice.
  • the antibodies may have an idiotope that is immunogenic in non-human hosts, but not in a human host.
  • antibodies of the invention for human use include those that cannot be easily isolated from hosts such as mice, goats, rabbits, rats, non-primate mammals, etc. and cannot generally be obtained by humanization or from xeno-mice.
  • Antibodies of the invention also include hybrid antibody molecules that comprise the six CDRs from an antibody of the invention as defined above and one or more CDRs from another antibody to an antigen.
  • the antibody may be multispecific.
  • the multispecific antibody may comprise a first antigen-binding site according to the present invention (binding to ASC, in particular PYD of ASC as described herein) and a second antigen-binding site, which is different from the first antigen-binding site, i.e. which may bind to a distinct target.
  • the antibody, or the antigen-binding fragment thereof may be monospecific.
  • the invention also provides a nucleic acid molecule comprising a polynucleotide encoding the antibody according to the present invention, or an antigen- binding fragment thereof, as described above.
  • the nucleic acid molecule comprises one or more polynucleotide(s) encoding the exemplified antibodies of the invention (e.g., as described above, in particular in Table 1), or a sequence variant thereof as described herein (e.g., having at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity as described above).
  • Table 4 Exemplified nucleic acid sequences encoding the CDR and Vhl/VL sequences of exemplified antibodies as described herein are shown in Table 4 below.
  • Table 4 SEQ ID NOs for polynucleotide sequences encoding the CDR and VHA/L sequences of exemplary antibodies ASC_Ab001, ASC_Ab002, ASC_Ab003, ASC_Ab004, ASC_Ab005, ASC_Ab006, ASC_Ab007, ASC_Ab008 and ASC_Ab009.
  • nucleic acid molecules and/or polynucleotides include, e.g., a recombinant polynucleotide, a vector, an oligonucleotide, an RNA molecule such as an rRNA, an mRNA, an miRNA, an siRNA, or a tRNA, or a DNA molecule such as a cDNA.
  • Nucleic acids may encode the light chain and/or the heavy chain of an antibody (or a single chain antibody).
  • the light chain and the heavy chain of the antibody may be encoded by the same nucleic acid molecule (e.g., for single chain antibodies or for antibodies with separate heavy and light chains in bicistronic manner or an expression cassette containing more than one ribosome entry site such as IRES).
  • the light chain and the heavy chain of the antibody may be encoded by distinct nucleic acid molecules. Due to the redundancy of the genetic code, the present invention also comprises sequence variants of nucleic acid sequences, which encode the same amino acid sequences.
  • the polynucleotide encoding the antibody (or the complete nucleic acid molecule) may be optimized for expression of the antibody.
  • nucleic acid molecule may comprise heterologous elements (i.e., elements, which in nature do not occur on the same nucleic acid molecule as the coding sequence for the (heavy or light chain of) an antibody.
  • a nucleic acid molecule may comprise a heterologous promotor, a heterologous enhancer, heterologous UTR (e.g., for optimal translation/expression), a heterologous poly-A-tail, heterologous DNA insulator elements and the like.
  • a nucleic acid molecule is a molecule comprising nucleic acid components.
  • nucleic acid molecule usually refers to DNA or RNA molecules. It may be used synonymous with the term "polynucleotide", i.e. the nucleic acid molecule may consist of a polynucleotide encoding the antibody. Alternatively, the nucleic acid molecule may also comprise further elements in addition to the polynucleotide encoding the antibody. Typically, a nucleic acid molecule is a polymer comprising or consisting of nucleotide monomers which are covalently linked to each other by phosphodiester-bonds of a sugar/phosphate-backbone.
  • nucleic acid molecule also encompasses modified nucleic acid molecules, such as base- modified, sugar-modified or backbone-modified etc. DNA or RNA molecules.
  • the nucleic acid molecule may be manipulated to insert, delete or alter certain nucleic acid sequences. Changes from such manipulation include, but are not limited to, changes to introduce restriction sites, to amend codon usage, to add or optimize transcription and/or translation regulatory sequences, etc. It is also possible to change the nucleic acid to alter the encoded amino acids. For example, it may be useful to introduce one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid substitutions, deletions and/or insertions into the antibody's amino acid sequence.
  • Such point mutations can modify effector functions, antigen-binding affinity, post-translational modifications, immunogenicity, etc., can introduce amino acids for the attachment of covalent groups (e.g., labels) or can introduce tags (e.g., for purification purposes).
  • a mutation in a nucleic acid sequence may be "silent", i.e. not reflected in the amino acid sequence due to the redundancy of the genetic code.
  • mutations can be introduced in specific sites or can be introduced at random, followed by selection (e.g., molecular evolution).
  • one or more nucleic acids encoding any of the light or heavy chains of an (exemplary) antibody can be randomly or directionally mutated to introduce different properties in the encoded amino acids.
  • the polynucleotide encoding the antibody, or an antigen-binding fragment thereof, (or the (complete) nucleic acid molecule) may be codon-optimized.
  • nucleic acid molecule of the invention may comprise a nucleic acid sequence as set forth in any one of SEQ ID NOs 73 - 140; or a sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.
  • the nucleic acid molecule may encode any one of the exemplified antibodies ASC_Ab001, ASC_Ab002, ASC_Ab003, ASC_Ab004, ASC_Ab005, ASC_Ab006, ASC_Ab007, ASC_Ab008 and ASC_Ab009 (by combining the sequences as shown in Table 2), or a sequence variant thereof as described herein.
  • the present invention also provides a plurality of nucleic acid molecules encoding the antibody, or an antigen-binding fragment thereof, as described herein, wherein each of the nucleic acid molecules (of the plurality of nucleic acid molecules) comprises a polynucleotide encoding an immunoglobulin chain of the antibody, or an antigen-binding fragment thereof.
  • each of the nucleic acid molecules comprises a polynucleotide encoding an immunoglobulin chain of the antibody, or an antigen-binding fragment thereof.
  • the plurality of nucleic acid molecules encoding the antibody, or an antigen-binding fragment thereof, as described herein may be a combination of a first and a second nucleic acid molecule, wherein the first nucleic acid molecule comprises a polynucleotide encoding the heavy chain of the antibody, or an antigen-binding fragment thereof, of the present invention; and the second nucleic acid molecule comprises a polynucleotide encoding the corresponding light chain of the same antibody, or the same antigen-binding fragment thereof.
  • the above description regarding the (general) features of the nucleic acid molecule of the invention applies accordingly to the nucleic acid molecules of the plurality of nucleic acid molecules.
  • one or more of the polynucleotides encoding the immunoglobulin chains of the antibody, or an antigen-binding fragment thereof may be codon-optimized.
  • the plurality may comprise a nucleic acid sequence as set forth in any one of SEQ ID NOs 73 - 140;or a sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.
  • the plurality of nucleic acid molecules may encode any one of the exemplified antibodies ASC_Ab001, ASC_Ab002, ASC_Ab003, ASC_Ab004, ASC_Ab005, ASC_Ab006, ASC_Ab007, ASC_Ab008 and ASC_Ab009 (by combining the sequences as shown in Table 2), or a sequence variant thereof as described herein.
  • Vectors Further included within the scope of the invention are vectors, for example, expression vectors, comprising a nucleic acid molecule according to the present invention or the plurality of nucleic acid molecules according to the present invention.
  • a vector comprises a nucleic acid molecule as described above.
  • the present invention also provides a plurality of vectors comprising the plurality of nucleic acid molecules according to invention as described above.
  • each vector of the plurality of vectors may contain one or more nucleic acid molecules of the plurality of nucleic acid molecules according to invention as described above.
  • the plurality of vectors may be a combination of a first and a second vector, wherein the first vector comprises a first nucleic acid molecule as described above (for the combination of nucleic acid molecules) and the second vector comprises a second nucleic acid molecule as described above (for the combination of nucleic acid molecules).
  • a vector is usually a recombinant nucleic acid molecule, i.e. a nucleic acid molecule which does not occur in nature.
  • the vector may comprise heterologous elements (i.e., sequence elements of different origin in nature).
  • the vector may comprise a multiple cloning site, a heterologous promotor, a heterologous enhancer, a heterologous selection marker (to identify cells comprising said vector in comparison to cells not comprising said vector), heterologous origin of replications, heterologous DNA insulator elements and the like.
  • a vector in the context of the present invention is suitable for incorporating or harboring a desired nucleic acid sequence.
  • Such vectors may be storage vectors, expression vectors, cloning vectors, transfer vectors etc.
  • a storage vector is a vector which allows the convenient storage of a nucleic acid molecule.
  • the vector may comprise a sequence corresponding, e.g., to a (heavy and/or light chain of a) desired antibody according to the present invention.
  • An expression vector may be used for production of expression products such as RNA, e.g. mRNA, or peptides, polypeptides or proteins.
  • an expression vector may comprise sequences needed for transcription of a sequence stretch of the vector, such as a (heterologous) promoter sequence.
  • a cloning vector is typically a vector that contains a cloning site, which may be used to incorporate nucleic acid sequences into the vector.
  • a cloning vector may be, e.g., a plasmid vector or a bacteriophage vector.
  • a transfer vector may be a vector which is suitable for transferring nucleic acid molecules into cells or organisms, for example, viral vectors.
  • a vector in the context of the present invention may be, e.g., an RNA vector or a DNA vector.
  • a vector in the sense of the present application comprises a cloning site, a selection marker, such as an antibiotic resistance factor, and a sequence suitable for multiplication of the vector, such as an origin of replication.
  • a vector in the context of the present application may be a plasmid vector.
  • the term "vector” may also refer to a delivery vector, e.g. for viral or non-viral delivery of a nucleic acid of the invention.
  • the present invention also provides a delivery vector/system comprising the nucleic acid molecule as described above (or comprising an expression vector as described above).
  • the delivery vector/system may be viral or non-viral.
  • Various examples of viral and non-viral delivery vectors/systems are known in the art and described, for example, in Nayerossadat N, Maedeh T, Ali PA. Viral and nonviral delivery systems for gene delivery. Adv Biomed Res.2012;1:27. doi:10.4103/2277-9175.98152, which is incorporated herein by reference.
  • Non-limiting examples of viral delivery vectors/systems include retroviral vectors; adenoviral vectors; adeno-associated viral (AAV) vectors, including helper-dependentadenoviral vectors and hybrid adenoviral vectors; herpes simplex virus vectors; lentivirus vectors; poxvirus vectors and Epstein-Barr virus vectors.
  • retroviral vectors adenoviral vectors
  • adeno-associated viral (AAV) vectors including helper-dependentadenoviral vectors and hybrid adenoviral vectors
  • herpes simplex virus vectors lentivirus vectors
  • poxvirus vectors and Epstein-Barr virus vectors Epstein-Barr virus vectors.
  • Non-limiting examples of non-viral delivery vectors/systems include chemical and non-chemical methods.
  • Non-chemical delivery includes physical methods, such as electroporation and other methods for transient penetration of the cell membrane by mechanical, electrical, ultrasonic, hydrodynamic, or laser-based energy; naked DNAor RNA delivery; gene gun; hydrodynamic delivery; ultrasound delivery and magnetofection.
  • Chemical non-viral delivery systems include cationic particles, in particular cationic lipids/liposomes, cationic polymers and lipid/polymer systems. Among non-viral vectors/systems, cationic liposomes are preferred.
  • Cells also provides a (host) cell expressing the antibody according to the present invention, or an antigen-binding fragment thereof; and/or comprising the vector (or the plurality of vectors) according the present invention.
  • the (host) cell may be an isolated cell, which is not part of a human or animal body, e.g. a cell line or an engineered cell.
  • the cell may express the nucleic acid(s) or vector(s) of the invention in a recombinant manner, e.g. in a heterologous manner (i.e., the cell/cell type does not express the antibody or the antigen-binding fragment in nature).
  • examples of such cells include, but are not limited to, eukaryoticcells, e.g., yeast cells, animal cells or plant cells.
  • Other examples of such cells include, but are not limited to, prokaryotic cells, e.g. E. coll.
  • the cells are mammalian cells, such as a mammalian cell line.
  • mammalian cells such as a mammalian cell line.
  • examples include human cells, CHO cells, HEK293 cells, PER.C6 cells, NSO cells, human liver cells, myeloma cells or hybridoma cells.
  • the cell may be transfected with a vector according to the present invention, for example with an expression vector.
  • the term "transfection” refers to the introduction of nucleic acid molecules, such as DNA or RNA (e.g. mRNA) molecules, into cells, e.g. into eukaryotic or prokaryotic cells.
  • the term "transfection” encompasses any method known to the skilled person for introducing nucleic acid molecules into cells, such as into mammalian cells. Such methods encompass, for example, electroporation, lipofection, e.g. based on cationic lipids and/or liposomes, calcium phosphate precipitation, nanoparticle based transfection, virus based transfection, or transfection based on cationic polymers, such as DEAE-dextran or polyethylenimine etc.
  • the introduction is non-viral.
  • the cells of the present invention may be transfected stably or transiently with the vector according to the present invention, e.g. for expressing the antibody according to the present invention.
  • the cells are stably transfected with the vector according to the present invention encoding the antibody according to the present invention.
  • the cells are transiently transfected with the vector according to the present invention encoding the antibody according to the present invention.
  • the present invention also provides a recombinant host cell, which heterologously expresses the antibody of the invention or the antigen-binding fragment thereof.
  • the cell may be of another species than the antibody (e.g., CHO cells expressing human antibodies).
  • the eel I type of the eel I does not express (such) antibodies in nature.
  • the host cell may impart a post-translational modification (PTM; e.g., glycosylation) on the antibody that is not present in their native state.
  • PTM post-translational modification
  • theantibodyofthe invention, or the antigen-binding fragment thereof may have a post-translational modification, which is distinct from the naturally produced antibody (e.g., an antibody of an immune response in a human).
  • Production of antibodies Antibodies according to the invention can be made by any method known in the art. For example, the general methodology for making monoclonal antibodies using hybridoma technology is well known (Kohler, G.
  • DNA sequences encoding the antibodies or antigen-binding fragments of the present invention may be synthesized completely or in part, e.g., using oligonucleotide synthesis techniques. Site-directed mutagenesis and polymerase chain reaction (PCR) techniques may be used as appropriate.
  • Any suitable host cell/vector system may be used for expression of the DNA sequences encoding the antibody molecules of the present invention. Eukaryotic, e.g., mammalian, host cell expression systems may be used for production of antibody molecules, such as complete antibody molecules.
  • Suitable mammalian host cells include, but are not limited to, CHO, HEK293, PER.C6, NSO, myeloma or hybridoma cells.
  • prokaryotic, e.g. bacterial host cell expression systems may be used for the production of antibody molecules, such as complete antibody molecules.
  • Suitable bacteria] host cells include, but are not limited to, E. co//eel Is. Accordingly, the present invention provides a method for preparing the antibody, or an antigen-binding fragment or an immunoglobulin chain(s) thereof, according to the present invention, said method comprising (i) culturing the host cell as described above; and (ii) isolating the antibody or immunoglobulin chain(s) thereof from the culture.
  • the present invention also provides a process for the production of an antibody molecule according to the present invention comprising culturing a (heterologous) host cell comprising a vector encoding a nucleic acid of the present invention, in particular under conditions suitable for expression of protein from DNA encoding the antibody molecule of the present invention, and isolating the antibody molecule.
  • a host cell such as a cell line, may be transfected with two vectors, a first vector encoding a light chain polypeptide and a second vector encoding a heavy chain polypeptide, e.g. as described above.
  • the invention also provides a method for preparing a recombinant cell, comprising the steps of: (i) providing one or more nucleic acids that encode(s) the antibody of the invention; (ii) inserting the nucleic acid into an expression vector and (iii) transfecting the vector into a (heterologous) host cell in order to permit expression of the antibody of interest in that host cell.
  • the nucleic acid of step (i) may, but need not, be manipulated to introduce restriction sites, to change codon usage, and/or to optimize transcription and/or translation regulatory sequences.
  • the invention also provides a method of preparing a transfected host cell, comprising the step of transfecting a host cell with one or more nucleic acids that encode an antibody of interest.
  • the procedures for first preparing the nucleic acid(s) and then using it to transfect a host cell can be peri:ormed at different times by different people in different places (e.g., in different countries).
  • These recombinant cells of the invention can then be used for expression and culture purposes. They are particularly useful for expression of antibodies for large-scale pharmaceutical production.
  • the transfected host cell may be a eukaryotic cell, including yeast and animal cells, particularly mammalian cells (e.g., CHO cells, NSO cells, human cells such as PER.C6, HEK293 or HKB-11 cells, myeloma cells, or a human liver cell), as well as plant cells.
  • the transfected host cell is a mammalian cell, such as a human cell.
  • expression hosts can glycosylate the antibody of the invention, particularly with carbohydrate structures that are not themselves immunogenic in humans.
  • the transfected host cell may be able to grow in serum-free media.
  • the transfected host cell may be able to grow in culture without the presence of animal-derived products.
  • the transfected host cell may also be cultured to give a cell line.
  • the invention also provides a method of preparing the antibody of interest comprising the steps of: culturing or sub-culturing a transfected host cell population, e.g. a stably transfected host cell population, under conditions where the antibody of interest is expressed and, optionally, purifying the antibody of interest.
  • the transfected host cell population may be prepared by (i) providing nucleic acid(s) encoding a selected antibody of interest, (ii) inserting the nucleic acid(s) into an expression vector, (iii) transfecting the vector in a host cell that can express the antibody of interest, and (iv) culturing or sub-culturing the transfected host cell comprising the inserted nucleic acids to produce the antibody of interest.
  • antibodies according to the invention may be produced by (i) expressing a nucleic acid sequence according to the invention in a host cell, e.g. by use of a vector (or host cell) according to the present invention, and (ii) isolating the expressed antibody product.
  • the method may include (iii) purifying the isolated antibody. Accordingly, after production, the antibodies may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography. Techniques for purification of antibodies, e.g., monoclonal antibodies, including techniques for producing pharmaceutical-grade antibodies, are well known in the art.
  • compositions and kits The present invention also provides a composition comprising one or more of: (i) the antibody of the present invention, or an antigen-binding fragment thereof; (ii) the nucleic acid or the plurality of nucleic acids of the present invention; (iii) the vector or the plurality of vectors of the present invention; or (iv) the cell expressing the antibody according to the present invention or comprising the vector according to the present invention.
  • the composition may be used for treatment or diagnostic purposes. Accordingly, the composition may be a pharmaceutical composition or a diagnostic composition.
  • the composition may comprise a (pharmaceutical ly acceptable) excipient, diluent or carrier.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody according to the present invention, or an antigen-binding fragment thereof, the nucleic acid or the plurality of nucleic acids of the present invention, the vector or the plurality of vectors of the present invention, and/or the cell according to the present invention.
  • the pharmaceutical composition comprises the antibody, or an antigen-binding fragment thereof, according to the present invention.
  • the pharmaceutical composition may optionally also contain a pharmaceutically acceptable carrier, diluent and/or excipient. Although the carrier or excipient may facilitate administration, it should not itself induce the production of antibodies harmful to the individual receiving the composition. Nor should it be toxic.
  • Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles.
  • the pharmaceutical ly acceptable carrier, diluent and/or excipient in the pharmaceutical composition is not an active component in respect to ASC-related diseases or disorders.
  • the pharmaceutically acceptable carrier, diluent and/or excipient in the pharmaceutical composition is not an active component in respect to a neurodegenerative or neuroinflammatory disorder.
  • Pharmaceutical ly acceptable salts can be used, for example mineral acid salts, such as hydrochlorides, hydrobromides, phosphates and sulphates, or salts of organic acids, such as acetates, propionates, malonates and benzoates.
  • Pharmaceutical ly acceptable carriers in a pharmaceutical composition may additionally contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents or pH buffering substances, may be present in such compositions. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries and suspensions, for ingestion by the subject. Pharmaceutical compositions may be prepared in various forms.
  • the compositions may be prepared as injectables, either as liquid solutions or suspensions.
  • Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared (e.g., a lyophilized composition, similar to SynagisTM and Herceptin®, for reconstitution with sterile water containing a preservative).
  • the composition may be prepared for topical administration e.g., as an ointment, cream or powder.
  • the composition may be prepared for oral administration e.g., as a tablet or capsule, as a spray, or as a syrup (optionally flavored).
  • the composition may be prepared for pulmonary administration e.g., as an inhaler, using a fine powder or a spray.
  • the composition may be prepared as a suppository or pessary.
  • the composition may be prepared for nasal, aural or ocular administration e.g., as drops.
  • the composition may be in kit form, designed such that a combined composition is reconstituted just prior to administration to a subject.
  • a lyophilized antibody may be provided in kit form with sterile water or a sterile buffer.
  • the (only) active ingredient in the composition is the antibody, or the antigen-binding fragment thereof, of the present invention. As such, it may be susceptible to degradation in the gastrointestinal tract.
  • the composition may contain agents which protect the antibody from degradation but which release the antibody once it has been absorbed from the gastrointestinal tract.
  • agents which protect the antibody from degradation but which release the antibody once it has been absorbed from the gastrointestinal tract are available in Gennaro (2000) Remington: The Science and Practice of Pharmacy, 20th edition, ISBN: 0683306472.
  • the present invention also provides a method of preparing a pharmaceutical composition comprising the steps of: (i) preparing an antibody of the invention; and (ii) admixing the purified antibody with one or more pharmaceutically acceptable excipients, diluents or carriers.
  • a method of preparing a pharmaceutical composition comprises the step of: admixing an antibody with one or more pharmaceutically-acceptable carriers, wherein the antibody is a monoclonal antibody.
  • Pharmaceutical compositions may generally have a pH between 5.5 and 8.5, in some embodiments this may be between 6 and 8, for example about 7. The pH may be maintained by the use of a buffer.
  • the composition may be sterile and/or pyrogen free.
  • the composition may be isotonic with respect to humans.
  • pharmaceutical compositions are supplied in hermetically-sealed containers.
  • compositions present in several forms of administration include, but are not limited to, those forms suitable for parenteral administration, e.g., by injection or infusion, for example by bolus injection or continuous infusion.
  • parenteral administration e.g., by injection or infusion
  • the product may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle and it may contain formulatory agents, such as suspending, preservative, stabilizing and/or dispersing agents.
  • the antibody may be in dry form, for reconstitution before use with an appropriate sterile liquid.
  • a vehicle is typically understood to be a material that is suitable for storing, transporting, and/or administering a compound, such as a pharmaceutical ly active compound, in particular the antibodies as described herein.
  • the vehicle may be a physiologically acceptable liquid, which is suitable for storing, transporting, and/or administering a pharmaceutically active compound, in particular the antibodies as described herein.
  • the compositions can be administered directly to the subject.
  • the compositions are adapted for administration to mammalian, e.g., human subjects.
  • Pharmaceutical compositions may include an antimicrobial, particularly if packaged in a multiple dose format. They may comprise detergent e.g., a Tween (polysorbate), such as Tween 80.
  • Detergents are generally present at low levels e.g., less than 0.01 %.
  • Compositions may also include sodium salts (e.g., sodium chloride) to give tonicity.
  • sodium salts e.g., sodium chloride
  • concentration of 10+2mg/ml NaCI is typical.
  • pharmaceutical compositions may comprise a sugar alcohol (e.g., mannitol) or a disaccharide (e.g., sucrose or trehalose) e.g., at around 15-30 mg/ml (e.g., 25 mg/ml), particularly if they are to be lyophilized or if they include material which has been reconstituted from lyophilized material.
  • the pH of a composition for lyophilization may be adjusted to between 5 and 8, or between 5.5 and 7, or around 6.1 prior to lyophilization.
  • the pharmaceutical compositions may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intraperitoneal, intrathecal, intraventricular, transdermal, transcutaneous, topical, subcutaneous, intranasal, enteral, sublingual, intravaginal or rectal routes.
  • the pharmaceutical composition may be prepared for oral administration, e.g. as tablets, capsules and the like, for topical administration, or as injectable, e.g. as liquid solutions or suspensions.
  • the pharmaceutical composition is an injectable.
  • Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection are also encompassed, for example the pharmaceutical composition may be in lyophilized form.
  • the active ingredient may be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection.
  • Preservatives, stabilizers, buffers, antioxidants and/or other additives may be included, as required.
  • administration is usually in an "effective amount", e.g. in a "prophylactically effective amount” or a “therapeutically effective amount” (as the case may be), this being sufficient to show benefit to the individual.
  • an "effective amount” e.g. in a "prophylactically effective amount” or a “therapeutically effective amount” (as the case may be)
  • the actual amount administered, and rate and time-course of administration will depend on the nature and severity of what is being treated.
  • the pharmaceutical composition may be provided for example in a pre-filled syringe.
  • the pharmaceutical composition may also be administered orally in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions.
  • carriers commonly used include lactose and corn starch.
  • Lubricating agents such as magnesium stearate, are also typically added.
  • useful diluents include lactose and dried cornstarch.
  • the active ingredient i.e. the antibody as defined above
  • emulsifying and suspending agents include lactose and dried cornstarch.
  • certain sweetening, flavoring or coloring agents may also be added.
  • the pharmaceutical composition may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, e.g. including accessible epithelial tissue. Suitable topical formulations are readily prepared for each of these areas or organs.
  • the pharmaceutical composition may be formulated in a suitable ointment, containing the pharmaceutical composition, particularly its components as defined above, suspended or dissolved in one or more carriers.
  • Carriers for topical administration include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water.
  • the pharmaceutical composition can be formulated in a suitable lotion or cream.
  • Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
  • Dosage treatment may be a single dose schedule or a multiple dose schedule.
  • a single dose e.g. a daily, weekly or monthly dose
  • the amount of the antibody in the pharmaceutical composition may not exceed 1 g or 500 mg.
  • the amount of the antibody in the pharmaceutical composition may not exceed 200 mg, or 100 mg.
  • the amount of the antibody in the pharmaceutical composition may not exceed 50 mg.
  • the composition may include antibodies of the invention, wherein the antibodies may make up at least 50% by weight (e.g., 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) of the total protein in the composition.
  • the antibodies may be in purified form.
  • nucleic acid typically DNA
  • Suitable gene therapy and nucleic acid delivery vectors are known in the art.
  • Pharmaceutical compositions typically include an "effective" amount of one or more antibodies as described herein, i.e. an amount that is sufficient to treat, ameliorate, attenuate, decrease or prevent a desired disease or condition, or to exhibit a detectable therapeutic effect.
  • Therapeutic effects also include reduction or attenuation in pathogenic potency or physical symptoms.
  • an effective dose may generally be from about 0.005 to about 100 mg/kg, for example from about 0.0075 toabout50 mg/kgorfrom about 0.01 to about 10mg/kg. In some embodiments, the effective dose will be from about 0.02 to about 5 mg/kg, of the antibody (e.g. amount of the antibody in the pharmaceutical composition) in relation to the bodyweight (e.g., in kg) of the individual to which it is administered.
  • the pharmaceutical composition may also comprise an additional active component, which may be a further antibody or a component, which is not an antibody.
  • the pharmaceutical composition may not comprise an additional active component (in addition to the antibody of the invention or respective nucleic acids, vectors or cells as described above).
  • the pharmaceutical composition may comprise one or more of the additional active components.
  • the antibody of the invention can be present either in the same pharmaceutical composition as the additional active component or, alternatively, the antibody may be comprised by a first pharmaceutical composition and the additional active component may be comprised by a second pharmaceutical composition different from the first pharmaceutical composition. Accordingly, if more than one additional active component is envisaged, each additional active component and the antibody may be comprised in a different pharmaceutical composition.
  • Such different pharmaceutical compositions may be administered either combined/simultaneously or at separate times or at separate locations (e.g. separate parts of the body), optionally by different routes of administration.
  • the antibody and the additional active component may provide an additive therapeutic effect, such as a synergistic therapeutic effect.
  • the term "synergy” is used to describe a combined effect of two or more active agents that is greater than the sum of the individual effects of each respective active agent. Thus, where the combined effect of two or more agents results in "synergistic inhibition" of an activity or process, it is intended that the inhibition of the activity or process is greater than the sum of the inhibitory effects of each respective active agent.
  • the term "synergistic therapeutic effect” refers to a therapeutic effect observed with a combination of two or more therapies wherein the therapeutic effect (as measured by any of a number of parameters) is greater than the sum of the individual therapeutic effects observed with the respective individual therapies.
  • the present invention also provides a diagnostic composition comprising an antibody according to the present invention, a nucleic acid(s) according to the present invention, a vector(s) according to the present invention, and/or a cell according to the present invention.
  • the diagnostic composition may optionally comprise suitable means for detection, such as reagents conventionally used in immuno- or nucleic acid based diagnostic methods.
  • the antibodies described herein are, for example, suited for diagnostic purposes.
  • immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier.
  • Such immunoassays may be competitive or non- competitive immunoassays; in either a direct or in an indirect format.
  • immunoassays include, but are not limited to, radioimmunoassay (RIA), enzyme-linked immunoassay (ELISA), sandwich (immunometric assay), immunohistochemistry, flow cytometry and Western blot assay.
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunoassay
  • sandwich immunometric assay
  • immunohistochemistry e.g. as described above.
  • the present invention also provides a kit comprising one or more of (i) the antibody according to the present invention, or an antigen-binding fragment thereof, as described above, (ii) the nucleic acid molecule (or the plurality of nucleic acid molecules) according to the present invention as described above, (iii) the vector (or the plurality of vectors) according to the present invention as described above, (iv) the cell according to the present invention as described above, and/or (v) the composition according to the present invention as described above.
  • the kit may comprise means for administration of the antibody, or an antigen binding fragment thereof, according to the present invention, the nucleic acid according to the present invention, the vector according to the present invention, the cell according to the present invention or the pharmaceutical composition according to the present invention, such as a syringe or a vessel, a leaflet, and/or a co-agent to be administered as described herein.
  • the kit may contain a leaflet, e.g. comprising instructions for use.
  • the kit may comprise one or more reagents, e.g. for use in appropriate diagnostic assays.
  • the kit may contain a reference agent or control.
  • the composition of the invention may be provided in kit form, e.g., designed such that a combined composition is reconstituted just prior to administration to a subject.
  • a lyophilized antibody may be provided in kit form with sterile water or a sterile buffer (e.g., in a separate container).
  • Medical treatments and other uses the present invention provides the use of the antibody according to the present invention, or an antigen-binding fragment thereof, the nucleic acid molecule (or the plurality of nucleic acid molecules) according to the present invention, the vector (or the plurality of vectors) according to the present invention, the cell according to the present invention or the (pharmaceutical) composition according to the present invention as a medicament.
  • the antibody according to the present invention, or an antigen- binding fragment thereof, the nucleic acid molecule (or the plurality of nucleic acid molecules) according to the present invention, the vector (or the plurality of vectors) according to the present invention, the cell according to the present invention or the (pharmaceutical) composition according to the present invention may be used in prophylaxis and/or treatment of a neurodegenerative or neuroinflammatory disease.
  • the present invention also provides a method of treating, ameliorating or reducing a neurodegenerative or neuroinflammatory disease or a symptom of a neurodegenerative or neuroinflammatory disease, or lowering the risk of (occurrence of) a neurodegenerative or neuroinflammatory disease or a symptom of a neurodegenerative or neuroinflammatory disease, comprising: administering to a subject (in need thereof), (a therapeutical ly effective amount of) the antibody, or an antigen-binding fragment thereof, according to the present invention, a nucleic acid molecule (or the plurality of nucleic acid molecules) according to the present invention, a vector (or the plurality of vectors) according to the present invention, a cell according to the present invention or a (pharmaceutical) composition according to the present invention.
  • the present invention also provides the use of an antibody according to the present invention, or an antigen-binding fragment thereof, a nucleic acid molecule (or the plurality of nucleic acid molecules) according to the present invention, a vector (or the plurality of vectors) according to the present invention, a cell according to the present invention, or a pharmaceutical composition according to the present invention in the manufacture of a medicament for prophylaxis, treatment or attenuation of a neurodegenerative or neuroinflammatory disease or of a symptom of a neurodegenerative or neuroinflammatory disease.
  • the terms "treat” or “treatment” include therapeutic treatment and prophylactic or preventative measures.
  • Prophylaxis of a neurodegenerative or neuroinflammatory disease refers in particular to prophylactic settings, wherein the subject was either not diagnosed with a neurodegenerative or neuroinflammatory disease (either no diagnosis was performed or diagnosis results were negative) and/or the subject does not show symptoms of a neurodegenerative or neuroinflammatory disease.
  • the subject is typically diagnosed with a neurodegenerative or neuroinflammatory disease and/or showing symptoms of a neurodegenerative or neuroinflammatory disease.
  • treatment and “therapy'V'therapeutic” include (complete) cure as well as attenuation/reduction of a neurodegenerative or neuroinflammatory disease and/or related symptoms.
  • the object of the "treatment” may be to decrease, ameliorate, inhibit, prevent or slow down (lessen or delay) an undesired physiological change or disorder, such as the neurodegenerative or neuroinflammatory disease or a symptom of a neurodegenerative or neuroinflammatory disease.
  • beneficial or desired clinical results of a treatment include, but are not limited to, alleviation of symptoms, diminishmentof the extent of a disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • Treatment can also mean prolonging survival, e.g. as compared to expected survival if not receiving treatment.
  • Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the manifestation of the condition or disorder or the risk thereof is to be decreased, delayed or prevented.
  • One way of checking efficacy of therapeutic treatment involves monitoring disease symptoms after administration of the antibody or of the composition.
  • Treatment can be a single dose schedule or a multiple dose schedule.
  • an antibody, antibody fragment, nucleic acid/ vector, cell, or composition as described herein may be administered to a subject in need of such treatment.
  • a subject includes, but is not limited to, one who is particularly at risk of, or susceptible to, a neurodegenerative or neuroinflammatory disease or a symptom of a neurodegenerative or neuroinflammatory disease.
  • the antibody according to the present invention, or an antigen-binding fragment thereof, the nucleic acid molecule (or the plurality of nucleic acid molecules) according to the present invention, the vector (or the plurality of vectors) according to the present invention, the cell according to the present invention or the (pharmaceutical) composition according to the present invention may be used for human and also for veterinary medical purposes, preferably for human medical purposes.
  • the term ,,subject", ,,patient” or ,,individual” as used herein thus generally includes humans and non-human animals and preferably mammals (e.g., non- human primates, including marmosets, tamarins, spider monkeys, owl monkeys, vervet monkeys, squirrel monkeys, and baboons, macaques, chimpanzees, orangutans, gorillas; cows; horses; sheep; pigs; chicken; cats; dogs; mice; rat; rabbits; guinea pigs; etc.), including chimeric and transgenic animals and disease models.
  • the term ,,subject” preferably refers a non-human primate or a human.
  • the subject is a human.
  • the human may be selected from the group of a human suffering from a neurodegenerative or neuroinflammatory disease, a human at risk of developing a neurodegenerative or neuroinflammatory disease and a human of unknown clinical history for a neurodegenerative or neuroinflammatory disease.
  • the "neurodegenerative or neuroinflammatory disease” is characterized by or associated with the formation of ASC aggregates ("specks") and/or amyloid-P (Ap)-related pathology, in particular the formation and spreading of amyloid-P aggregates.
  • Ap- related pathology refers to the abnormal production, deposition and aggregation ofamyloid- p in the brain.
  • the neurodegenerative or neuroinflammatory disease may involve interaction of ASC and amyloid-P.
  • the neurodegenerative or neuroinflammatory disease is associated with the fibrillation activity of ASC.
  • the neurodegenerative or neuroinflammatory disease involves ASC-induced inflammation.
  • Neurodegenerative diseases are typically chronic, progressive disorders characterized by the gradual loss of neurons in discrete areas of the central nervous system (CNS), such as the brain.
  • the neurodegenerative diseases envisaged to be treated may preferably be characterized and/or accompanied by dementia. "Dementia" is a general term for a decline in mental ability severe enough to interfere with daily life.
  • Dementia may include decline or loss of memory, communication and language, ability to focus and pay attention, reasoning and judgment, visual perception, or a combination thereof. It may be caused by neurodegeneration in a variety of neurodegenerative diseases. Neuroinflammation is the inflammation of nervous tissue. It may be initiated in response to a variety of cues, including infection, traumatic brain injury, toxic metabolitesorautoimmunity. Neuroinflammatory diseases are characterized by inflammatory processes in the nervous system, in particular in the CNS, which may result in the gradual damage and loss of neurons. Neuroinflammation and neurodegeneration often result from the aberrant deposition of aggregated host proteins that can activate inflammasomes. ASC is a central component of the inflammasome.
  • the neurodegenerative or neuroinflammatory disease is selected from Alzheimer's Disease, Parkinsons's Disease, Huntington's disease, Multiple System Atrophy, Amyotrophic Lateral Sclerosis, Multiple Sclerosis, traumatic brain injury, Sinocerebellar ataxia, Frontotemporal Dementia, Frontotemporal Lobar Degeneration, Mild Cognitive Impairment, Parkinson-plus syndromes, Pick disease, Progressive isolated aphasia, Grey-matter degeneration [Alpers], Subacute necrotizing encephalopathy, or Lewy body dementia.
  • Alzheimer's Disease (AD) and Mild Cognitive Impairment (MCI) are particularly preferred.
  • "Alzheimer's Disease” is a neurodegenerative brain disease that is a major cause of dementia among the elderly.
  • Symptoms of AD may include progressive loss of learning and memory functions, personality changes, neuromuscular changes, seizures and occasionally psychotic behaviour.
  • Alzheimer's disease is characterized by the deposition of amyloid-p plaques in areas of the brain that are critical for memory and other cognitive functions. It is believed that the deposition of amyloid-R plaques, in these critical areas of the brain, interferes with brain functions.
  • Mild Cognitive Impairment is a syndrome defined as a subjective and objective decline in cognition and function greater than expected for an individual's age and education level that may not meet the criteria for a diagnosis of dementia.
  • MCI Alzheimer's disease
  • AD Alzheimer's disease
  • FDD frontotemporal dementia
  • DLB dementia with Lewy bodies
  • MCI is often referred to as an objective cognitive complaint for age, in a person with essentially normal functional activities, who does not have dementia. It affects 19% of people aged 65 and over. Around 46% of people with MCI develop dementia within 3 years compared with 3% of the population of the same age.
  • neurodegenerative or neuroinflammatory diseases envisaged for prophylaxis or treatment according to the present invention include hereditary ataxia, congenital nonprogressive ataxia, early-onset cerebellar ataxia, late-onset cerebellar ataxia, cerebellar ataxia with defective DNA repair, hereditary spastic paraplegia, infantile spinal muscular atrophy, type I [Werdnig-Hoffman], inherited spinal muscular atrophy, systemic atrophies primarily affecting the central nervous system, paraneoplastic neuromyopathy and neuropathy, postpolio syndrome, Degenerative diseases of basal ganglia, Hallervorden-Spatz disease, progressive supranuclear ophthalmoplegia [Steele-Richardson- Olszewski], Neurogenic orthostatic hypotension [Shy-Drager], dystonia, tremor, chorea, Restless legs syndrome, Stiff-man syndrome, extrapyramidal and movement disorders, Multiple sclerosis, acute disseminated demye
  • any gene therapy approaches may be used, e.g. the antibody according to the present invention, or an antigen-binding fragment thereof, may be administered as nucleic acid or vector encoding said antibody, e.g. using viral or non-viral vectors as described above.
  • the antibody according to the present invention, or an antigen-binding fragment thereof, the nucleic acid molecule (or the plurality of nucleic acid molecules) according to the present invention, the vector (or the plurality of vectors) according to the present invention, the cell according to the present invention or the pharmaceutical composition according to the present invention may be administered systemically, for example by intravenous or subcutaneous administration.
  • Antibodies and fragments thereof as described herein may also be used for the (in-vitro) diagnosis of a neurodegenerative or neuroinflammatory disease.
  • Methods of diagnosis may include contacting an antibody with a sample. Such samples may be isolated from a subject, for example an isolated blood sample, such as whole blood, plasma or serum.
  • the methods of diagnosis may also include the detection of an antigen/antibody complex, in particular following the contacting of an antibody with a sample.
  • it may be tested, whether the sample contains antibodies competing with the antibodies as described herein, e.g. for allergen binding. This is typically performed in vitro, i.e. without any contact to the human or animal body.
  • diagnosis may be performed in vitro, for example by using an isolated sample as described above (and an in vitro analysis step as described above).
  • the present invention also provides the use of the antibody, or an antigen- binding fragment thereof, according to the present invention, the nucleic acid molecule or the plurality of nucleic acid molecules according to the present invention, the vector or the plurality of vectors according to the present invention, the cell according to the present invention, the composition according to the present invention, or the kit according to the present invention in (in-vitro) diagnosis of a neurodegenerativeorneuroinflammatory disease.
  • the present invention also provides the use of the antibody, or an antigen- binding fragment thereof, according to the present invention, the nucleic acid molecule or the plurality of nucleic acid molecules according to the present invention, the vector or the plurality of vectors according to the present invention, the cell according to the present invention, the composition according to the present invention, or the kit according to the present invention in (in-vitro) diagnosis of a neurodegenerativeorneuroinflammatory disease.
  • Figure 1 shows for Example 3 binding curves of exemplary antibody ASC_Ab001 to full length ASC, ASC specks, CARD domain, PYD domain, murine ASC and BSA as tested by ELISA.
  • Figure 2 shows for Example 4 an alignment of the different ASC isoforms: full-length ASC (SEQ ID NO: 1); ASC-b (SEQ ID NO: 145); ASC-c (SEQ ID NO: 146) and ASC-d (SEQ ID NO: 147).
  • Figure3 shows for Example 4 representative images of binding patterns of (A) ASC_Ab001, (B) ASC_Ab008, (C) ASC_Ab004, and (D) control antibody (AL- 177, adipogen).
  • lane 1 shows binding to ASC-b 20kDa
  • lane 2 shows binding to ASC-d 11.8kDa
  • lane 3 shows binding to ASC-c 15kDa
  • lane 4 shows binding to ASC Y146A 21.5kDa
  • lane 5 shows binding to ASC K21A K22A K26A21,5kDa
  • lane 6 shows binding to UT Ctrl
  • lane 7 shows binding to ASC-HIS purified 23kDa.
  • Figure 4 shows for Example 4 the binding of anti-ASC antibodies and control antibodies as indicated to ASC peptides as indicated.
  • A Binding of ASC_Ab001, ASC_Ab002 clonotype, ASC_Ab004, ASC_Ab005, ASC_Ab006, ASC_Ab007, ASC_Ab008 clonotype, AL-177 and VHI-IASC to peptides covering amino acids 1 to 20 and 11 to 30 of ASC.
  • B Binding of ASC_Ab001, ASC_Ab002, AL-177, IC100 and VHHASC to peptides covering amino acids 1 to 20, 11 to 30 and 21 to 40 of ASC.
  • Figure5 shows for Example 5 ELISA binding curves of antibodies ASC_Ab001, ASC_Ab002, AL-177, VHHASC and IC100 to human ASC.
  • Figure 6 shows for Example 5 ELISA binding curves of antibodies ASC_Ab001, ASC_Ab002, AL-177, VHHASC and IC100 to murine ASC.
  • Figure 7 shows for Example 5 ELISA binding curves of antibodies ASC_Ab001, ASC_Ab002, AL-177, VHHASC and IC100 to ASC specks.
  • Figures shows for Example 5 ELISA binding curves of antibodies ASC_Ab001, ASC_Ab002, AL-177, VHHASC and IC100 to the PYD domain of ASC.
  • Figure 9 shows for Example 6 (A) the quantification of ASC specks detected in Thp1 wt or asc-/' cells primed with LPS (1ug/mL), stimulated with Nigericin (5uM) and stained with 1ug/mL of anti-ASC antibodies or isotype control and (B) representative immunofluorescence pictures ofTHP-1 asc +/+ and THP-1 asc- /- cells stained with an ASC_Ab001 antibody.
  • Figure 10 shows for Example 7 Western blot images of antibodies ASC_Ab001, ASC_Ab004, ASC_Ab007 and ASC_Ab009 on cell lysates of wt and asc-/- human THP-1 monocytes.
  • Figure 11 shows for Example 7 Western blot images of antibodies ASC_Ab001, ASC_Ab004, ASC_Ab007 and ASC_Ab009 on cell lysates of mouse bone- marrow derived macrophages (BMDMs).
  • Figure 12 shows for Example 8 that treatment with ASC_Ab001, ASC_Ab004, ASC_Ab007, ASC_Ab008 and ASC_Ab009 significantly impaired ASC fibrillation.
  • the effect of anti-ASC antibodies on ASC fibrillation was assessed by IF of ASC fibrils formed after incubation of ASC-gfp with anti-ASC antibodies or isotype control 1h at 37°C.
  • FIG. 14 shows for Example 9 that the anti-ASC antibodies block ASC-A(3 interaction. Percentage of inhibition of ASC- A? binding after treatment with anti-ASC antibodies (3.5uM) or isotype control (3.5uM).
  • FIG. 15 shows for Example 10 that ASC_Ab001 blocks the pro-inflammatory function of extracellular ASC fibrils.
  • the in-vitro efficacy of ASC_Ab001 was tested in a cell-based assay using differentiated human Th-IP-1 macrophages that were treated with extracellular ASC fibrils. Before addition of the ASC fibrils to the cells, ASC fibrils were pre-incubated with ASC_Ab001 and isotype control antibody. IL-1R measurement was used as a readout for inflammasome activation.
  • ASC_Ab001 significantly reduced the ASC fibrils-induced production of IL-1 (3 compared to the isotype control.
  • Figure 16 shows for Example 11 that humanized ASC_Ab001 preserves binding to ASC. Binding of humanized ASC_Ab001 to ASC-His was assessed by ELISA. Similar binding of the humanized antibodies was observed compared to the rabbit IgG format.
  • EXAMPLES In the following, particular examples illustrating various embodiments and aspects of the invention are presented. However, the present invention shall not to be limited in scope by the specific embodiments described herein. The following preparations and examples are given to enable those skilled in the art to more clearly understand and to practice the present invention. The present invention, however, is not limited in scope by the exemplified embodiments, which are intended as illustrations of single aspects of the invention only, and methods which are functionally equivalent are within the scope of the invention.
  • suspension HEK293 cells were transiently transfected with expression plasmids encoding full length human ASC or ASC-GFP using linear PEI 40kDa. After 7 days of expression at 37°C and 5% €02, cells were harvested and resuspended in Buffer A (320 mM sucrose, 20 mM HEPES-KOH (pH 7.5), 10 mM KCI, 1.5 mM MgCl2, 1 mM, EDTA, 1 mM EGTA. Cells were lysed by syringing (1 Ox 20G, 20x 25G), freeze-thawing (3 x), followed by subsequent syringing (20x 25G).
  • Buffer A 320 mM sucrose, 20 mM HEPES-KOH (pH 7.5), 10 mM KCI, 1.5 mM MgCl2, 1 mM, EDTA, 1 mM EGTA.
  • the lysate was centrifuged at 400g for 8min, the pellet was re-suspended in 2x CHAPS buffer (40 mM HEPES-KOH (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.2 mM PMSF, 0.2 % CHAPS) and filtered using a 5 pm centrifugal filters at2000gfor 10 min.
  • the filtrate was then diluted and gently mixed with 1 volume of 2x CHAPS buffer and centrifuged at 2300g for 8 min.
  • the resulting pellet was resuspended in 1 ml of 1x ChlAPS buffer and centrifuged at SOOOg for 8 min. This washing step was repeated twice.
  • pellets were resuspended in 50 mM phosphate, 300 mM NaCl, 6M Gua-HCI, 2 mM DTT, pH 7.5 for 30min at RT. Afterwards the suspension was centrifuged at 14000g at 4°C for 30min to remove residual insoluble cell debris.
  • the supernatant was then incubated with Nickel beads (Themo, #88221) for 3h at RT.
  • the beads were washed once with 50 mM phosphate, 300 mM NaCI, 6M Gua-HCl, 2 mM DTT, pH 7.5 and followed by 50 mM phosphate, 300 mM NaCl, 6M Gua-HCI, 2 mM DTT, 20mM imidazole pH 7.5.
  • beads were incubated with 50 mM phosphate, 300 mM NaCI, 6M Gua-HCI, 2 mM DTT, SOOmM imidazole pH 7.5.
  • the pH of the eluate was adjusted to pH 3.8 with diluted HCI.
  • the eluate was dialysed against 50 mM Glycine, 150 mM NaCl, pH 3.8 in 3500 Da cassette 0/N at 4°C.
  • the dialysed samples were purified using a preparative SEC column (HiLoad 16/600 Superdex 75 pg).
  • the monomeric proteins were finally concentrated using a VivaSpin 3000 Da column.
  • ELISA High binding clear flat bottom 384-well plate (Greiner bio-one, 781061) were coated o/n (4°C) with 10 ⁇ jl/well ofASC CARD domain (1.25ug/mL; SEQ ID NO: 156), ASC PYD domain (1.25ug/mL; SEQ ID NO: 155), murine ASC (Cusabio, CSB-EP861664 MO, 1.25ug/mL; SEQ ID NO: 151), ASC-His (1ug/mL; SEQ ID NO: 153), ASC-gfp specks (50000 specks/well) or BSA(1
  • both human ASC and murine ASC were covalently immobilized onto AR2G biosensors (Sartorius AC) via EDC/NHS-mediated amine coupling.
  • both human ASC and murine ASC were immobilized at a concentration as low as 0.625 [jg/mL in 10 mM sodium phosphate buffer ph-l 5, for 300 s, to ensure the interaction with the analyte taking place predominantly at 1:1 stoichiometric ratio.
  • a biosensor for which buffer was injected as analyte throughout the entire run was used as a reference for subtraction.
  • the association (kon) and dissociation (koff) constants were calculated using a 1:1 binding model, with a global fit.
  • human ASC and murine ASC were immobilized at concentrations ranging from 1.25
  • Antibodies were binned in sets of up to 7 antibodies per experiment, in a two-dimensional matrix format, with the antibodies added sequentially, in pairs, at saturating concentrations. The biosensors were regenerated between consecutive antibody pairs with regeneration buffer.
  • Cell lysates were prepared by lysing the cells with ice-cold CHAPS buffer supplemented with proteases inhibitors. After clearing the lysates by centrifugation at 17'000 x G for 15 min at 4°C, supernatant was transferred to a new set of pre-chilled tubes. Protein concentration was determined by BCA assay (PierceTM BCA Protein Assay Kit, Thermofisher Scientific). NuPAGE LDS sample buffer (4X) (Invitrogen) and NuPAGE Sample reducing agent (1 OX) (Invitrogen) was added to the samples and they were boiled at 95°C for 5 min.
  • Protein transfer (Blotting): Gels were placed onto AmershamTM ProtranTM Premium 0.45um Nitrocellulose Blotting membrane (Cytiva) and protei ns transferred at 100 V for 1 h using Mini Trans-BlotTM Cell system (Bio-Rad) and 1X Transfer buffer (25mM Tris-Base, 192mM glycine, 10% methanol). After blotting, membranes were blocked in 5% non-fat dry milk in PBS-T (0.1 % Tween-20) for 1 h at RT. Staining: After blocking, primary antibodies were added at 1 ug/ml in 1% non-fat dry milk in PBS-T to the membranes for 2h at RT.
  • Membranes were washed 3x in PBS-T for 10 min each time and then incubated in secondary antibody in 1% non-fat dry milk in PBS-Tfor 1h at RT. Membranes were washed 3x in PBS-T and subsequently incubated in SuperSignalTM West Pico PLUS Chemiluminescent Substrate (Thermofisher Scientific).
  • Immunofluorescence of ASC specks TH P-1 wt and asc-/- cells were seeded at a density of 10000 cells/ well in 50uL of RPMI medium supplemented with Glutamax, 10%FBS, 1%P/S, 100ug/mL Normocin, 100ug/mL Zeocin in a 384 well plate (Greiner bio-one 781986).
  • the cells were primed with 1 ug/mL LPS (Sigma, cat: L4391 -1 MG) for 3h. After priming, the formation of ASC specks was induced by treatment with 5uM Nigericin (Invivogen cat: tlrl-nig) for 1 h.
  • the cells were fixed and permealized using BD cytofix/cytoperm (BD biosciences, cat: 554722) solution for 20min at RT. The cells were then washed twice with PBS and blocked with 2% BSA supplemented with human Fc block reagent (Miltenyi cat: 130-059-901).
  • the cells were incubated with 1 ug/mL of anti-ASC antibodies or isotype control 2h at RT, washed twice with PBS and further incubated with a detection secondary antibody coupled with Alexa fluor 647 (Jackson Immunoresearch, 111-607-003) for th at RT.4',6-Diamidino-2'-phenylindol- dihydrochloride (DAP! (Sigma, cat: MBD0015-1ML) was used as a counterstain at 1 ug/mL for 10 min in PBS. Images were taken using a 10X objective. All images were acquired using a fluorescence microscope. Image processing and ASC speck count was accomplished using Fiji ImageJ and Cellprofiler.
  • Cell lysates were prepared by lysing the cells (wt and asc/ ⁇ human TH P-1 monocytes and mouse BMDMs) with ice-cold lysis buffer containing 1% Triton X-100 for 15 min on ice. After clearing the lysates by centrifugation at 17'000 x G for 15 min at 4°C, supernatant was transferred to a new set of pre-chilled tubes. Protein concentration was determined by BCA assay (PierceTM BCA Protein Assay Kit, Thermofisher Scientific).
  • NuPAGE LDS sample buffer (4X) (Invitrogen) and NuPAGE Sample reducing agent (1 OX) (Invitrogen) was added to the samples and boiled at 95°C for 5 min.
  • -SDS PAGE 10 ug of protein were loaded onto NuPAGE 4-12% Bis-Tris Gels (Invitrogen). Gels were run in MES SDS Running buffer (Invitrogen) at 80 V for 10 min and then at 120 V for another 110 min. Gel electrophoresis was performed using the Invitrogen XCell SureLockTM Electrophoresis cell chamber system.
  • -Protein transfer (Blotting): Gels were placed onto AmershamTM ProtranTM Premium 0.45um Nitrocellulose Blotting membrane (Cytiva) and proteins transferred at 100 V for 1 h using Mini Trans-BlotTM Cell system (Bio-Rad) and 1 X Transfer buffer (25mM Tris-Base, 192 mM glycine, 10% methanol). After blotting, membranes were blocked in 5% non-fat dry milk in PBS-T (0.1 % Tween-20) for 1 h at RT. -Staining: After blocking, primary antibodies were added at 1 ug/ml in 1 % non-fat dry milk in PBS-T to the membranes for 2h at RT.
  • Membranes were washed 3x in PBS-T for 10 min each time and then incubated in secondary antibody in 1 % non-fat dry milk in PBS-T for 1 h at RT. Membranes were washed 3x in PBS-T and subsequently incubated in SuperSignalTM West Pico PLUS Chemiluminescent Substrate (Thermofisher Scientific). Fibrillation Assay: 1,75uM ASC-GFP-His was incubated with 3.5uM of anti-ASC antibody or isotype control 1 h at37°C in protein LoBindtube (Eppendorf, cat 0030109.116)).
  • Membranes were washed 3x in PBS-T for 10 min each time and then incubated in secondary antibody in 1 % non-fat dry milk in PBS-T for 1 h at RT. Membranes were washed 3x in PBS-T and subsequently incubated in SuperSignalTM West Pico PLUS Chemiluminescent Substrate (Thermofisher Scientific). Blocking ofASC-Ap interaction ASC-H is fibrils were formed by incubatingASC-hlis monomerat37°Cfor 1 h in protein LoBind tubes (Eppendorf, 0030109.116).
  • Fibrillary ASC d.75uM was then incubated with 1 uM 5'- TAMRA-Apl-42 (Bachem) with and without 3.5uM anti-ASC antibodies or isotype control for 4h at 37°C. After incubation, protein aggregates were analyzed by flow cytometry using a Novocyte Advanteon (Agilent) and Flowjo v10.7.1.
  • ASC_Ab001 THP-1 efficacy data - Wt and asc/ ⁇ THP-1 monocytes were seeded at a density of 10'OQO cells/well in RPMI medium supplemented with Glutamax, 10%FBS, 1%P/S, 100 ug/mL Normocin, 100 ug/mL Zeocin in a 384-well plate.
  • PMA phorbol 12-myristate-t 3-acetate
  • ASC fibrils were prepared by incubating ASC-h-Iis monomers in sterile PBS at37°Cfor 1 h. Before addition of the ASC fibrils (3.5uM) to the cells, they were pre-incubated with antibodies (3.5uM) for 15 min at RT. After 16 h incubation at 37°C and 5% C02, secreted IL-1p was measured by Lumit Human IL-1 Immunoassay (Promega, W6010) according to manufacturer's protocol.
  • Example 1 Identification and production of antibodies binding to ASC Immunization of Rabbits Immunization of rabbits with untagged, HEK derived ASC specks, was carried out by dosing rabbits on day 1, 7, 14, 21 and 35, with spleen and bone marrow cell harvesting 7 days after the final dose. Immunization of rabbits with the His tagged PYD or CARD domain of ASC was carried out by dosing rabbits on day 1, 7, 14, 21 and 28, with spleen and bone marrow cell harvesting 5 days after the final dose. Cells were cryopreserved.
  • Rabbit bone marrow or spleen cells were screened for plasmablasts secreting antibodies specific for ASC specks, human ASC (SEQ ID NO: 1), mouse ASC (SEQ ID NO: 151), or ASC PYD or CARD domains (SEQ ID NOs 143 and 144, respectively), which were then sorted in 10
  • RNA lysis and reverse transcription of RNA was carried out using maxima H minus reverse transcriptase (ThermoFisher, USA), and DNA encoding the variable domain heavy (VH) and light (VL) sequences was amplified by 2 step PCR using primers annealing to the ends of rabbit variable framework regions, sequenced (Microsynth, Switzerland), translated into amino acid sequences, annotated and aligned (Geneious Biologies) and cloned into expression vectors containing rabbit constant heavy or light regions.
  • the expression vector pDB contains an Epstein-Barr virus origin of replication (oriP), which allows episomal replication of the transfected in cells expressing Epstein-Barr virus Nuclear Antigen! (EBNA1).
  • oriP Epstein-Barr virus origin of replication
  • the plasmid pDB has a cytomegalovirus- chicken actin-fusion promoter (CAG Promoter), which results in high expression of recombinant proteins.
  • CAG Promoter cytomegalovirus- chicken actin-fusion promoter
  • Small scale antibody expression was carried out in suspension HEK293 cells. A few minutes prior to transfection, cells were seeded into a 24-well plate SOOpl cell suspension at 0.8-1 mio cells/ml in FreeStyle medium supplemented with 0.1% Pluronic F68, 1% Glutamax and 1x Geniticin (all ThermoFisher, USA).250 ng of heavy chain plasmid DNA and 250 ng of light chain plasmid DNA was mixed in a total volume of 15
  • High binding clear flat bottom 384-well plates (Greiner bio-one , 781061) were coated overnight at 4°C with 10 ⁇ jl/well of ASC CARD domain (1.25ug/mL), ASC PYD domain (1.25ug/mL), murine ASC (Cusabio, CSB-EP861664 MO, 1.25ug/mL), ASC-His (1ug/mL), ASC-GFP specks (50000 specks/well) or BSA (1 ⁇ ig/ml) diluted in PBS. After overnight incubation, wells were aspirated and washed once with 100 pl/well of wash buffer (0.05% Tween-20 in PBS).
  • Wells were blocked for an hour with 50 pl of 2% BSA diluted in PBS at RT. After blocking, wells were incubated 2 hours at RT with 10 [jl of test anti body and serially diluted. Wells were washed four times and consecutively incubated with 10 [jl/well of detection antibody for an hour at RT. Unbound detection antibody was removed by washing four times, and the wells were incubated with 20 [jl TMB for 5 min. The reaction was stopped by adding 10 pl of 1M H2S04 per well. Absorbance was immediately read at 450 nm using EnSpire 2300 microplate reader (Perkin Elmer).
  • VH and VL sequences as well as CDR sequences of the exemplary antibodies identified as described above are shown in Table 5 below.
  • Table 5 SEQ ID NOs for CDR and VH/VL sequences of exemplary antibodies ASC_Ab001, ASC_Ab002, ASC_Ab003, ASC_Ab004, ASC_Ab005, ASC_Ab006, ASC_Ab007, ASC_Ab008 and ASC_Ab009.
  • Antibody Expression and Purification Larger scale antibody expression was carried out in suspension HEK293 cells, using a method scalable from 20 to 1000 mL expression volumes. Transfection was achieved by adding a mixture of polyethylenimine (PEI Max, Polysciences, USA) and DNA encoding rabbit light and heavy chain IgG to cells.
  • PEI Max polyethylenimine
  • the transfected cells are supplemented with Tryptone N1 (OrganoTechni, France) after 24 hours. After expression for 5-7 days the supernatant, which contains the expressed antibodies, was harvested. Purification was carried out using a HiTrap Protein A HIP column (Cytiva, US) or Protein A Sepharose 4 Fast Flow beads (Cytiva, US). These beads contain Staphylococcus Protein A immobilized on an agarose matrix. Protein A has a strong affinity to the Fc region of IgG. Binding of protein A to IgG occurs at a neutral pH, and antibody is eluted using acidic phi.
  • Example 2 Cross-competition of antibodies binding to PYD of ASC To identify whether antibodies ASC_Ab001, ASC_Ab002, ASC_Ab003, ASC_Ab004, ASC_Ab005, ASC_Ab006, ASC_Ab007, ASC_Ab008 and ASC_Ab009 bind to the same or distinct epitope(s), a cross-competition assay was performed. To this end, cloned monoclonal antibodies were tested by biolayer interferometry (BLI) on full length ASC coupled to BLI sensors.
  • BLI biolayer interferometry
  • Results are summarized in Table 6 below, wherein “ ⁇ ” means (full) competition, “-” means no competition; and “pc” means partial competition: T , ASC_Ab004, ASC_Ab005, ASC_Ab006, ASC_Ab007, ASC_Ab008 and ASC_Ab009
  • T ASC_Ab004, ASC_Ab005, ASC_Ab006, ASC_Ab007, ASC_Ab008 and ASC_Ab009
  • antibodies ASC_Ab001, ASC_Ab002 and ASC_Ab003 compete for the same epitope on ASC, indicating that antibodies ASC_Ab001, ASC_Ab002 and ASC_Ab003 bind to the same epitope.
  • Antibodies were assigned to different binning groups named "PYD-1”, //PYD-2", “PYD-4", TYD-6", //PYD- 7a", “PYD-7b” and //PYD-7c", wherein each binning group represents a distinct, non- overlapping ASC epitope, except for //PYD-7a" and “PYD-7b” and “PYD-7b” and “P ⁇ D-7c", which epitopes are partially overlapping, as shown in Table 7 below: Table 7: Binning groups based on cross-competition results
  • Example 3 Characterization of antibody binding For further characterization of the binding of antibodies ASC_Ab001, ASC_Ab002, ASC_Ab003, ASC_Ab004, ASC_Ab005, ASC_Ab006, ASC_Ab007, ASC_Ab008 and ASC_Ab009, an ELISAwas performed as described above, wherein binding of the antibodies to monomeric human ASC (SEQ ID NO: 1), HEK-
  • Table 8 ECSO-values determined for antibodies ASC_Ab001, ASC_Ab002, ASC_Ab003, ASC_Ab004, ASC_Ab005, ASC_Ab006, ASC_Ab007, ASC_Ab008 and ASC_Ab009 These data demonstrate that antibodies ASC_Ab001, ASC_Ab002, ASC_Ab003, ASC_Ab004, ASC_Ab005, ASC_Ab006, ASC_Ab007, ASC_Ab008 and ASC_Ab009 bind specifically to monomeric ASC as well as to ASC specks.
  • Figure 1 shows the binding curves exemplarily for antibody ASC_Ab001, additionally including the results for binding to the PYD domain and the CARD domain.
  • the data shown in Figure 1 demonstrate that exemplary antibody ASC_Ab001 specifically binds to human monomeric ASC, ASC specks, murine ASC and to the PYD domain of ASC, whereas no binding was detected to the CARD domain of ASC and to the bovine serum albumin (BSA) control.
  • BSA bovine serum albumin
  • Bio-Layer Interferometry as described above, was used to test antibodies ASC_Ab001, ASC_Ab002, ASC_Ab003, ASC_Ab004, ASC_Ab005, ASC_Ab006, ASC_Ab007, ASC_Ab008 and ASC_Ab009 for their binding affinity to human full-length ASC captured on BLI sensors.
  • the KD, off-rates and on-rates were calculated using a 1:1 binding model with a global fit.
  • FIG. 1 shows an alignment of the different ASC isoforms. While full-length ASC and ASC-b contain a complete PYD domain, ASC-c lacks amino acids 26 to 85 of full-length ASC, while ASC-d contains only the N-terminal 35 amino acids of the PYD domain of ASC.
  • Figure 3 shows representative images of binding patterns of ASC_Ab001, ASC_Ab004, ASC_Ab008 and control antibody (AL-177, adipogen).
  • binding of antibodies ASC_Ab001, ASC_Ab002, ASC_Ab004, ASC_Ab005, ASC_Ab006, ASC_Ab007, ASC_Ab008 and control antibody AL-177 to ASC peptides covering amino acid 1 to 20 (SEQ ID NO: 148) or 11 to 30 (SEQ ID NO: 149) was assessed by ELISA.
  • nanobody VHI-IASC as described in Schmidt et al., 2016 Schomidt Fl, Lu A, Chen JW, Ruan J, Tang C, Wu H, Ploegh HL.
  • a single domain antibody fragment that recognizes the adaptor ASC defines the role of ASC domains in inflammasome assembly.
  • Figure 4(B) shows the results obtained for ASC peptides covering amino acid 1 to 20 (SEQ ID NO: 148), 11 to 30 (SEQ ID NO: 149) or 21 to 40 (SEQ ID NO: 150).
  • AL-177 was found to bind to the ASC peptides covering amino acid 1 to 20 (SEQ ID NO: 148) and 11 to 30 (SEQ ID NO: 149), while none of VHHASC, ASC_Ab001 and ASC_Ab002 bound to this peptide.
  • Antibody IC-100 did not bind to the most N-terminal peptide (amino acids 1-20; SEQ ID NO: 148), but was found to bind to the peptides covering amino acids 11 to 30 (SEQ ID NO: 149) or 21 to 40 (SEQ ID NO: 150). None of the other antibodies bound to the peptide covering 21 to 40 (SEQ ID NO: 150). In summary, this ASC peptide analysis shows that AL-177 binds to an epitope within amino acids 1 - 30 of ASC, while IC100 binds to an epitope within amino acids 11 - 40 of ASC. None of the antibodies of the present invention was found to bind to such an N-terminal peptide.
  • Example 5 Comparison with antibodies of the prior art As described above (see also Schmidt et al., 2016) and demonstrated in Example 4, antibodies AL-177, IC100 and anti-CARD antibody VHHASC bind to distinct epitopes of ASC as compared to the antibodies of the present invention.
  • binding of antibodies ASC_Ab001 and ASC_Ab002 as well as prior art antibodies AL-177, VHHASC and IC100 obtained as described in Example 4 above) to (i) human ASC, (ii) murine ASC, (iii) ASC specks and (iv) the PYD domain of ASC was assessed by ELISA as described above.
  • Results are shown in Figures 5 (human ASC), 6 (murine ASC), 7 (ASC specks) and 8 (PYD domain ofASC). All tested antibodies were found to bind to human ASC and ASC specks, except for IC100. Surprisingly, despite the data previously obtained by others for an antibody "IC100" or "IC- 100" (see US 10,961,306 B2 and Desu HL, Plastini M, llliano P, Bramlett HM, Dietrich WD, de Rivero Vaccari JP, Brambilla R, Keane RW. IC100: a novel anti-ASC monoclonal antibody improves functional outcomes in an animal model of multiple sclerosis. J Neuroinflammation.
  • Example 4 demonstrates binding of IC100 to peptides of SEQ ID NOs 149 and 150, no binding to full-length human ASC, murine ASC, ASC specks and the (entire) PYD domain of ASC could be detected for IC100 (human Igd antibody containing the VH of SEQ ID NO: 19 and the VL of SEQ ID NO: 30 of US 10,961,306 B2, as described in US 10,961,306 B2 column 40, lines 60-64).
  • THP-1 asc-/- cells were compared to THP-1 wt cells.
  • THP-1 is human macrophage cell line, which is commercially available.
  • ThIP-1 asc-/-cells lack expression ofASC, in contrast to TH P- 1 wt cells, such that physiological ASC speck formation is only expected in THP-1 wt cells, while THP-1 asc-/- cells represent negative controls.
  • formation of ASC specks was induced in THP-1 wt and THP-1 asc-/- cells.
  • the cells were then incubated with the anti-ASC antibodies ASC_Ab001, ASC_Ab002, ASC_Ab003, ASC_Ab004, ASC_Ab005, ASC_Ab006, ASC_Ab007, ASC_Ab008, ASC_Ab009 or isotype control, as described above in detail. Results were obtained by fluorescent imaging. Results are shown in Figure 9. These data show that each of the antibodies ASC_Ab001, ASC_Ab002, ASC_Ab003, ASC_Ab004, ASC_Ab005, ASC_Ab006, ASC_Ab007, ASC_Ab008 and ASC_Ab009 binds to physiological ASC speck.
  • Example 7 Binding specificity of antibodies To confirm binding of antibodies ASC_Ab001, ASC_Ab004, ASC_Ab007 and ASC_Ab009 to endogenous ASC and species cross-reactivity, antibodies were tested by Western blot on wt and a5 ⁇ r/-human TH P-1 monocytes and mouse bone-marrow derived macrophages (BMDMs), as described in detail above. Results are shown in Figures 10 (wt and a5c-/-human THP-1 cells) and 11 (BMDMs). All of the tested antibodies were binding to human and murine endogenous ASC in cell lysatesfrom wt cells, while there was no binding observed in the asc/ ⁇ ce ⁇ lysates.
  • BMDMs mouse bone-marrow derived macrophages
  • Example 8 Treatment with antibodies of the invention significantly impaired ASC fibrillation ASC assembles in large cytoplasmic macromolecular assemblies called ASC specks. This is driven by ASC-ASC interaction and can be reconstituted in vitroby incubating ASC monomers at 37°C. Under these conditions ASC will rapidly fibrillate and retain their physiological and pathological functions (Friker LL, Scheibling H, Hochheiser IV, Brinkschulte R, Riedel D, Latz E, Geyer M, Heneka MT. P-Amyloid Clustering around ASC Fibrils Boosts Its Toxicity in Microglia. Cell Rep.2020 Mar 17;30(11):3743-3754.e6.
  • ASC_Ab001, ASC_Ab004, ASC_Ab007, ASC_Ab008 and ASC_Ab009 were assessed in an ASC fibrillation assay as described above. Briefly, ASC-GFP-His was incubated with the antibody to be tested and protein aggregates were imaged. Results are shown in Figure 12.
  • ASC_Ab001 Treatment with ASC_Ab001 lead to more soluble ASC (supernatant fraction) when compared to ASC only or isotype control after 30 min of incubation at 37°C. Or in other words, ASC_Ab001 shifts the equilibrium towards the soluble ASC in the supernatant fraction. Similarly as in the ASC fibrillation assay described above, treatment with an anti-CARD antibody had no effect on the fibrillation.
  • Example 9 _Anti-ASC antibodies block ASC -AB interaction ASC -A?
  • ASC Ab001 blocks the pro-inflammatory function of extracellular ASC fibrils
  • ASC_Ab001 blocks the pro-inflammatory function of extracellular ASC fibrils
  • the in-vitro efficacy of ASC_Ab001 was tested in a cell-based assay using differentiated human THP-1 macrophages that were treated with extracellular ASC fibrils obtained as described above.
  • ASC fibrils and ASC specks can propagate inflammation (Franklin BS, Bossaller L, De Nardo D, Ratter JM, Stutz A, Engels G, Brenker C, Nordhoff M, Mirandola SR, Al-Amoudi A, Mangan MS, Zimmer S, Monks BG, Fricke M, Schmidt RE, Espevik T, Jones B, Jarnicki AC, Hansbro PM, Busto P, Marshak-Rothstein A, Hornemann S, Aguzzi A, Kastenmuller W, Latz E.
  • the adaptor ASC has extracellular and 'prionoid' activities that propagate inflammation. Natlmmunol.2014 Aug;15(8):727-37.
  • ASC_Ab001 significantly reduced the ASC fibrils-induced production of IL-1R compared to the isotype control, demonstrating the efficacy of ASC_Ab001. This reduction was not dependent on antibody Fc function, as no difference between the wt IgG and the PGLALA (Fc-inactive) format was observed. Treatment with antibody alone did not influence IL-1 R production.
  • Example 11 Humanization of antibodies preserves binding to ASC
  • ASC_Ab001 humanized contained the VH according to SEQ ID NO: 157 (which comprises heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 4, respectively) and the VL according to SEQ ID NO: 158 (which comprises light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively).

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Abstract

La présente invention concerne des anticorps se liant à une protéine de type speck associée à l'apoptose contenant une CARD (ASC), en particulier au domaine PYRINE-PAAD-DAPIN (PYD) de ASC. La présente invention concerne également des acides nucléiques codant pour de tels anticorps, des vecteurs comprenant de tels acides nucléiques et des cellules hôtes exprimant lesdits anticorps. L'invention concerne en outre des compositions et des kits comprenant de tels anticorps. De plus, la présente invention concerne également l'utilisation de tels anticorps, par exemple pour la prophylaxie ou le traitement d'une maladie neurodégénérative ou neuroinflammatoire.
PCT/EP2022/083921 2022-11-30 2022-11-30 Anticorps dirigés contre une protéine de type speck associée à l'apoptose contenant une card (asc) et leurs utilisations WO2024114906A1 (fr)

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