Antibodies against CARD-containing apoptosis-associated speck-like protein (ASC) and uses thereof

Abstract

The present invention provides antibodies that bind to CARD-containing apoptosis-associated speck-like protein (ASC), particularly to the PYRIN-PAAD-DAPIN domain (PYD) of ASC. The present invention also provides nucleic acids encoding such antibodies, vectors containing such nucleic acids, and host cells expressing the antibodies. Additionally, compositions and kits containing such antibodies are provided. The present invention also relates to the use of such antibodies, for example, for the prevention or treatment of neurodegenerative or neuroinflammatory diseases, or peripheral inflammatory disorders.

Description

Detailed Description of the Invention

[0001] The present invention relates to antibodies that bind to apoptosis-associated speck-like protein containing a CARD (ASC; also known as PYCARD), particularly the PYRIN-PAAD-DAPIN domain (PYD) of ASC. The present invention also relates to compositions and kits comprising such antibodies. Furthermore, the present invention relates to the use of such antibodies, compositions, and kits for, for example, preventing or treating neurodegenerative or neuroinflammatory diseases, or peripheral inflammatory disorders.

[0002] Neurodegenerative diseases are chronic, progressive disorders typically characterized by the gradual loss of neurons in discrete regions of the central nervous system (CNS), such as the brain. Such neuronal damage includes progressive degeneration and death of neurons. Dementia, a type of neurodegenerative disease, is estimated to have affected 55 million people worldwide in 2019, increasing to 139 million by 2050. In the United States, an estimated 6.5 million older adults suffer from Alzheimer's disease (AD), and approximately 1 million Americans suffer from Parkinson's disease. Neurodegenerative diseases are currently incurable. While certain treatments can help alleviate some of the physical or mental symptoms associated with neurodegenerative diseases, they currently cannot even slow their progression.

[0003] In the case of the snowflakes of the snowflakes, the snowflakes are smooth (Guzman-Martinez [ PMC free article ] [ PubMed ] [ Cross Ref ] Maccioni RB, Andrade V, Navarrete LP, Ramos-Escobar N. Neuroinflammation as a Common Feature of Neurodegenerative Disorders. Baker D, van der Valk P. Inflammation in neurodegenerative diseases 2010 Feb;129(2):154-69. 2017 Oct 2;127(10):3577-3587;Voet S, Srinivasan S, Lamkanfi M, Van Loo G. Inflammasomes in neuroinflammatory and neurodegenerative diseases. 10.15252 / emmm.201810248;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-β in Alzheimer's disease. Nature. 2017 Dec 20;552(7685):355-361; Hulse J, Bhaskar K. Crosstalk Between the NLRP3 Inflammasome / ASC Speck and Amyloid Protein Aggregates Drives Disease Progression in Alzheimer's and Parkinson's Disease. Front Mol Neurosci. 2022 Feb 3;15:805169. doi: 10.3389 / fnmol.2022.805169). Neurodegenerative diseases often result from the intracellular deposition of self-aggregating proteins, such as amyloid-β (Aβ). These self-aggregated products are typically toxic to neurons, eventually leading to widespread toxicity. Aggregated proteins are known to activate inflammasomes and induce the production of inflammatory mediators.

[0004] Inflammasomes are cytoplasmic multiprotein complexes that assemble in a stimulus-specific manner and activate the inflammatory cascade upon assembly. Inflammasome assembly is driven by the self-association and oligomerization of three types of proteins: sensor, adaptor, and effector. Upon activation by danger signals, the inflammasome sensor self-associates and promotes the oligomerization of the inflammasome adaptor ASC (CARD-containing apoptosis-associated speck-like protein). Therefore, ASC is normally required as an adaptor protein for inflammasome assembly. ASC consists of two protein-protein interaction domains: an N-terminal PYRIN-PAAD-DAPIN domain (PYD) and a C-terminal caspase recruitment domain (CARD). The PYD and CARD domains are members of the six-helix bundle death domain fold superfamily, which mediate the assembly of large signaling complexes in inflammatory and apoptotic signaling pathways via caspase activation. During inflammasome assembly, upstream sensor proteins such as NLRP3 and AIM2 recruit ASC through interactions between their PYDs, and ASC then interacts with caspase-1 via a CARD / CARD interaction. Thus, ASC, which contains two domains, PYD and CARD, is a central structure of the inflammasome.

[0005] Despite growing interest in targeting inflammasomes in neurodegenerative and neuroinflammatory diseases, most of the compounds developed in this context are NLRP3 inhibitors, which target the NLRP3 sensor and are therefore specific for the NLRP3 inflammasome but not for 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).

[0006] Recently, antibodies against ASC have been developed (Schmidt FI, Lu A, Chen JW, Ruan J, Tang C, Wu H, Ploegh HL. A single domain antibody fragment that recognizes the adapter ASC defines the role of ASC domains in inflammasome assembly. J Exp Med. 2016 May 2;213(5):771-90. doi: 10.1084 / jem.20151790;Desu HL, Plastini M, Illiano 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. 2020 May 4;17(1):143. doi: 10.1186 / s12974-020-01826-0), these antibodies may be useful for targeting ASCs. However, these antibodies are in the early stages of development and the epitopes targeted by ASCs have not yet been fully characterized. Further strategies for targeting ASCs are needed, especially in the context of neuroinflammatory or neurodegenerative diseases.

[0007] Therefore, an object of the present invention is to overcome the drawbacks of the prior art. In particular, an object of the present invention is to provide an antibody that specifically binds to the PYD domain of ASC. Another object of the present invention is to provide an antibody that can reduce or block the fibrillogenic activity of ASC. Another object of the present invention is to provide an antibody that can reduce or block the interaction between ASC and β-amyloid (Aβ).

[0008] This object is achieved by the subject matter described below and in the appended claims.

[0009] Although the present invention is described in detail below, it should be understood that the present invention is not limited to the particular methodology, protocols, and reagents described herein, as these may vary. It should also be understood that the terminology used herein is not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0010] The elements of the present invention are described below. While these elements are listed with specific embodiments, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The various described examples and embodiments should not be construed as limiting the invention to only the explicitly described embodiments. The specification should be understood to support and encompass embodiments combining any number of the disclosed elements with the explicitly described embodiments. Furthermore, any permutation and combination of all described elements in this application should be deemed to be disclosed by the description in this application, unless the context indicates otherwise.

[0011] Throughout this specification and the claims that follow, unless the context dictates otherwise, the term "comprise" and variations such as "comprises" and "comprising" will be understood to mean the inclusion of stated elements, integers, or steps, but not the exclusion of other unrecited elements, integers, or steps. The term "consisting of" is a specific embodiment of the term "comprises," excluding other unrecited elements, integers, or steps. In the context of the present invention, the term "comprising" encompasses the term "consisting of." Thus, the term "comprising" encompasses "including" as well as "consisting." For example, a composition "comprising" X may consist solely of X, or may include additional elements, integers, or steps, such as X+Y.

[0012] As used in the context of describing the present invention (particularly in the context of the claims), the terms "a," "an," and "the," and similar articles, are intended to cover both the singular and the plural, unless otherwise specified herein or clearly contradicted by context. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method for individually referencing each discrete value falling within the range. Unless otherwise specified herein, each discrete value is incorporated herein as if individually set forth herein. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0013] The term "substantially" does not exclude "completely." For example, a composition that is "substantially free" of Y may be completely free of Y. Where necessary, the term "substantially" may be omitted from the definition of the invention.

[0014] The term "about" in reference to a numerical value x means x±10%, for example x±5%, or x±7%, or x±10%, or x±12%, or x±15%, or x±20%.

[0015] As used herein, the term "disease" is intended to be generally synonymous with, and used interchangeably with, the terms "disorder" and "condition" (in medical conditions), in that all of these terms reflect an abnormal condition of the human or animal body or parts thereof that impairs normal function, is typically manifested by characteristic signs and symptoms, and reduces the duration or quality of the human or animal's life.

[0016] As used herein, reference to "treatment" of a subject or patient is intended to include prevention, prophylaxis, attenuation, amelioration, and therapy. As used herein, the terms "subject" or "patient" are used interchangeably 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 human.

[0017] Doses are often expressed relative to body weight. Thus, a dose expressed as [g, mg, or other unit] / kg (or g, mg, etc.) usually means [g, mg, or other unit] "per kg (or g, mg, etc.) of body weight," even if the term "body weight" is not explicitly stated.

[0018] The term "binding" and similar references generally mean "specifically bind" and do not encompass nonspecific attachment. In particular, specific binding of an antibody means that the antibody recognizes its target antigen and binds to its target with higher affinity (or lower antibody concentration, e.g., EC50) than a structurally different antigen and / or an antigen with an altered or mutated sequence. Hereby, "higher" affinity means at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold, 500-fold, 750-fold, 1,000-fold, 1,500-fold, 2,000-fold, 5,000-fold, 7,500-fold, 10,000-fold, or more affinity compared to binding to a control antigen. In some cases, antibody binding to a control antigen may not be detected (below the detection threshold), while antibody binding to a specific antigen may be sufficiently detected / determined.

[0019] As used herein, the term "antibody" encompasses various forms of antibodies, including, but not 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., engineered or mutated antibodies), so long as the characteristic properties of the present invention are maintained. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a monoclonal antibody. For example, the antibody may be a humanized monoclonal antibody.

[0020] As mentioned above, the term "antibody" generally also includes antibody fragments. Antibody fragments may retain the antigen-binding activity of the antibody. 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. Antibody fragments can be obtained from antibodies by methods including digestion with enzymes such as pepsin or papain, and / or by cleavage of disulfide bonds by chemical reduction. Alternatively, antibody fragments can be obtained recombinantly, for example, by cloning and expressing portions (fragments) of the heavy and / or light chain sequences. The present invention also encompasses single-chain Fv fragments (scFv) derived from the heavy and light chains of the antibodies of the present invention. For example, the present invention includes scFvs comprising CDRs derived from the antibodies of the present invention. The present invention also encompasses heavy or light chain monomers and dimers, single-domain heavy chain antibodies, single-domain light chain antibodies, and single-chain antibodies, such as single-chain Fvs in which the heavy and light chain variable domains are linked by a peptide linker. Antibody fragments of the present invention may be included in a variety of structures known to those skilled in the art. Furthermore, sequences of the present invention may be components of multispecific molecules in which sequences of the present invention target epitopes of the present invention and other regions of the molecule bind to other targets. While this specification, including the claims, may explicitly refer to antigen-binding fragments, antibody fragments, antibody variants, and / or antibody derivatives, the term "antibody" is understood to include all categories of antibodies, i.e., antigen-binding fragments, antibody fragments, antibody variants, and antibody derivatives.

[0021] As used herein, the term "variable region" (V L ), the variable region of the heavy chain (V H )) indicates each of the light and heavy chain pairs that are directly involved in binding between the antibody and the antigen.

[0022] The antibodies of the present invention may be of any isotype (e.g., IgA, IgG, IgM, i.e., α heavy chain, γ heavy chain, or μ heavy chain). Preferably, the antibodies are of the IgG or IgA type. Within the IgG isotype, the antibodies may be of the IgG1, IgG2, IgG3, or IgG4 subclass, preferably IgG1 or IgG4. The antibodies of the present invention may have a κ or λ light chain.

[0023] Antibodies of the present invention may be provided in purified form. Typically, the antibodies will be present in a composition that is substantially free of other polypeptides, e.g., less than 90% (by weight) of the composition being made up of other polypeptides, usually less than 60%, and more usually less than 50%.

[0024] Antibodies of the invention may be immunogenic in humans and / or non-human (or heterologous) hosts, such as mice. For example, an antibody may have an idiodide that is immunogenic in a non-human host but not in a human host. Antibodies of the invention for use in humans include those that cannot be readily isolated from hosts such as mice, goats, rabbits, rats, non-primate mammals, and generally cannot be obtained by humanization or from xenomurine.

[0025] As used herein, the term "antigen" refers to any structural entity that serves as a target for receptors of the adaptive immune response, particularly antibodies, T cell receptors, and / or B cell receptors. An "epitope," also known as an "antigenic determinant," is a portion (or fragment) of an antigen that is recognized by the immune system, particularly antibodies, T cell receptors, and / or B cell receptors. Thus, an antigen has at least one epitope, i.e., an antigen has one or more epitopes. An antigen can be (i) a peptide, polypeptide, or protein, (ii) a polysaccharide, (iii) a lipid, (iv) a lipoprotein or lipopeptide, (v) a glycolipid, (vi) a nucleic acid, or (vii) a small molecule drug or toxin. Thus, the antigen can be a peptide, a protein, a polysaccharide, a lipid, a combination thereof including a lipoprotein and a glycolipid, a nucleic acid (e.g., DNA, siRNA, shRNA, antisense oligonucleotide, decoy DNA, plasmid), or a small molecule drug (e.g., cyclosporin A, paclitaxel, doxorubicin, methotrexate, 5-aminolevulinic acid), or any combination thereof. Preferably, the antigen is selected from (i) a peptide, polypeptide, or protein, (ii) a polysaccharide, (iii) a lipid, (iv) a lipoprotein or lipopeptide, and (v) a glycolipid; more preferably, the antigen is a peptide, polypeptide, or protein.

[0026] As used herein, the term "mutation" refers to a change in a nucleic acid and / or amino acid sequence compared to a reference sequence, e.g., a corresponding genomic sequence. For example, a mutation compared to a genomic sequence can 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 (a molecular biology method for generating specific and deliberate changes in nucleic acid and / or amino acid sequences). Therefore, the term "mutation" or "mutating" is understood to include, for example, physically altering a nucleic acid or amino acid sequence. Mutations include substitutions, deletions, and insertions of one or more nucleotides or amino acids, as well as inversions of multiple consecutive nucleotides or amino acids. To achieve a mutation in an amino acid sequence, mutations can be introduced into a nucleotide sequence encoding said amino acid sequence, and a (recombinant) mutant polypeptide can be expressed. For example, mutations may be achieved by changing a codon in a nucleic acid molecule that encodes one amino acid, e.g., by site-directed mutagenesis, to result in a codon that encodes a different amino acid, or by synthesizing a sequence variant, e.g., by knowing the nucleotide sequence of a nucleic acid molecule that encodes a polypeptide and designing the synthesis of a nucleic acid molecule that includes a nucleotide sequence that encodes a variant of the polypeptide, without having to mutate one or more nucleotides of the nucleic acid molecule.

[0027] As used herein (i.e., throughout the specification), the term "sequence variant" refers to any alteration compared to a reference sequence. The term "sequence variant" includes nucleotide sequence variants and amino acid sequence variants. Preferably, the reference sequence is any of the sequences listed in the "Table of Sequences and SEQ ID NOs" (Sequence Listing), i.e., SEQ ID NO: 1 to SEQ ID NO: 199. In particular, a "sequence variant" shares 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%, and particularly preferably at least 99% sequence identity (over the entire length of the sequence) with its reference sequence. In some embodiments, a "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. Generally, and throughout this specification, the higher the percent identity of a sequence variant, the more preferred. For example, a sequence variant having at least 84% sequence identity with a reference sequence is preferred over a sequence variant having at least 75% sequence identity with the reference sequence.

[0028] In some embodiments, "sequence variants" maintain the (biological) function of the reference sequence. For example, sequence variants of the antibodies of the present invention preferably maintain specific binding to ASC, particularly to the PYD domain of ASC. In addition, other functions, such as anti-ASC fibrillogenic activity and / or reducing or blocking ASC-Aβ interaction, may also be maintained.

[0029] Sequence identity can be calculated as described below. Typically, sequence variants can retain certain functions of the reference sequence. In some embodiments, amino acid sequence variants have an altered sequence in which one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acids in the reference sequence have been deleted or substituted, or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acids have been inserted or added to the sequence of the reference amino acid sequence. As a result of the alterations, the amino acid sequence variant has an amino acid sequence that 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%, and particularly preferably at least 99% identical to the reference sequence. For example, a variant sequence that is at least 90% identical will have no more than 10 alterations, i.e., any combination of deletions, insertions, or substitutions, per 100 amino acids of the reference sequence. Of course, the same applies to nucleic acid sequences as well.

[0030] The "% identity" of a sequence variant is usually determined relative to a reference sequence. The % identity is usually calculated with respect to the entire length of the reference sequence (i.e., the sequence described in this application). The percent identity referred to herein can be determined by methods known in the art, such as, for example, BLAST using the default parameters specified by NCBI (National Center for Biotechnology Information; http: / / www.ncbi.nlm.nih.gov / ) [Blosum 62 matrix; gap opening penalty = 11, and gap extension penalty = 1].

[0031] Generally, although it is also possible to have non-conservative amino acid substitution, substitution is preferably conservative amino acid substitution, where substituted amino acid has similar structural or chemical properties with the corresponding amino acid in reference sequence.For example, conservative amino acid substitution includes: substituting a certain aliphatic amino acid or hydrophobic amino acid, such as alanine, valine, leucine and isoleucine, with another aliphatic amino acid or hydrophobic amino acid; substituting a certain hydroxyl-containing amino acid, such as serine and threonine, with another hydroxyl-containing amino acid; substituting a certain acidic residue, such as glutamic acid or aspartic acid, with another acidic residue; substituting a certain amide-containing residue, such as asparagine and glutamine, with another amide-containing residue; substituting a certain aromatic residue, such as phenylalanine and tyrosine, with another aromatic residue; substituting a certain basic residue, such as lysine, arginine and histidine, with another basic residue; and substituting a certain small amino acid, such as alanine, serine, threonine, cysteine ​​and glycine, with another small amino acid.

[0032] Throughout the text of this specification, several documents are cited. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention.

[0033] It is understood that the present invention is not limited to the particular methodology, protocols, and reagents described herein, as these may vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0034] [Antibodies and antigen-binding fragments thereof] In a first aspect, the present invention provides an (isolated) antibody or antigen-binding fragment thereof that (specifically) binds to ASC, particularly the PYD domain of ASC. Preferably, the ASC is human ASC (SEQ ID NO: 1), and the PYD domain is the PYD domain of human ASC (SEQ ID NO: 143). Apoptosis-associated speck-like protein containing a CARD (ASC; also referred to as PYCARD) is an adaptor protein composed of two protein-protein interaction domains: a PYRIN-PAAD-DAPIN domain (PYD) and a caspase recruitment domain (CARD). The PYD and CARD domains are members of the six-helix bundle death domain fold superfamily, which mediate the assembly of large signaling complexes in inflammatory and apoptotic signaling pathways via caspase activation. In particular, the antibody or antigen-binding fragment thereof of the present invention specifically binds to the PYD domain of ASC. Preferably, the antibodies or antigen-binding fragments thereof of the present invention specifically bind to a polypeptide or protein having the amino acid sequence set forth in SEQ ID NO: 1 (human ASC), in particular SEQ ID NO: 143 (PYD of human ASC). Accordingly, the antibodies or antigen-binding fragments thereof of the present invention preferably do not (specifically) bind to the CARD domain of ASC (SEQ ID NO: 144).

[0035] Preferably, the antibody or antigen-binding fragment thereof of the present invention (specifically) binds to monomeric ASC. It is also preferred that the antibody or antigen-binding fragment thereof (specifically) binds to ASC specks. ASC can assemble into large protein complexes called "specks." ASC specks are structures formed by ASC, which can reach approximately 1 μm in size and are characteristic of inflammasome activation. More preferably, the antibody or antigen-binding fragment thereof of the present invention (specifically) binds to monomeric ASC and ASC specks.

[0036] The ASC may be of any origin, e.g., derived from the species in which it originally originated (e.g., human ASC may be of human origin; mouse ASC may be of mouse origin, etc.), recombinantly expressed, or a synthetic ASC peptide. Preferably, the antibodies or antigen-binding fragments thereof of the present invention bind (specifically) to physiological / endogenous ASC, in particular to human and / or murine physiological / endogenous ASC, e.g., to the physiological / endogenous ASC of human macrophages and / or mouse bone marrow-derived macrophages (BMDMs). Binding to physiological / endogenous ASC can be tested using cell lysates of human macrophages (e.g., THP-1 cells) and / or mouse bone marrow-derived macrophages (BMDMs), e.g., as described in the accompanying Examples.

[0037] ASC can occur in four different isoforms: (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). Preferably, the antibody or antigen-binding fragment thereof of the present invention (specifically) binds to (human) ASC and (human) ASC-b. Furthermore, the antibody or antigen-binding fragment thereof preferably does not (specifically) bind to (human) ASC-c and (human) ASC-d. Also, it is preferred that the antibody or antigen-binding fragment thereof (specifically) binds to ASC K21A K22A K26A (mutant ASC with K21A, K22A, and K26A mutations). Furthermore, it is preferred that the antibody or antigen-binding fragment thereof (specifically) binds to ASC Y146A (mutant ASC with Y146A mutation).

[0038] More preferably, antibodies or antigen-binding fragments thereof of the present invention (specifically) bind to a fragment or region of human ASC comprising amino acids 36-85 of human ASC (SEQ ID NO: 1), and more preferably to a fragment or region of human ASC comprising amino acids 41-85 of human ASC (SEQ ID NO: 1). Thus, antibodies or antigen-binding fragments thereof preferably do not (specifically) bind to a region (or fragment) of ASC comprised in (or corresponding to) amino acids 1-35 of ASC (SEQ ID NO: 1). In some embodiments, antibodies or antigen-binding fragments thereof do not (specifically) bind to the ASC peptide set forth in SEQ ID NO: 148 (ASC aa 1-20 peptide). Additionally (or alternatively), antibodies or antigen-binding fragments thereof may not (specifically) bind to the ASC peptide set forth in SEQ ID NO: 149 (ASC aa 11-30 peptide). In some embodiments, antibodies or antigen-binding fragments thereof of the present invention do not (specifically) bind to the ASC peptide set forth in SEQ ID NO: 150 (ASC aa 21-40 peptide). Furthermore, antibodies or antigen-binding fragments thereof preferably do not (specifically) bind to a region (or fragment) of ASC contained in (or corresponding to) amino acids 86 to 195 of ASC (SEQ ID NO: 1). In other words, the epitope in PYD of human ASC to which antibodies or antigen-binding fragments thereof of the present invention bind is preferably located within amino acids 36 to 85 of ASC (SEQ ID NO: 1), more preferably within amino acids 41 to 85 of ASC (SEQ ID NO: 1).

[0039] Preferably, the antibodies or antigen-binding fragments thereof of the present invention (specifically) bind to ASC of different species, more preferably, the antibodies or antigen-binding fragments thereof (specifically) bind to human ASC (SEQ ID NO: 1) and mouse ASC (SEQ ID NO: 151).

[0040] Standard methods for assessing binding of the antibodies or antigen-binding fragments thereof of the present invention are known to those skilled in the art and include, for example, ELISA (enzyme-linked immunosorbent assay), whereby the relative affinity of antibody binding can be determined as the antibody concentration required to achieve 50% of maximal binding at saturation (EC 50) can be determined. An example of an ELISA that can be used to assess antibody binding is described in the accompanying Examples.

[0041] Generally, an antibody or antigen-binding fragment thereof according to the present invention may comprise three heavy chain complementarity-determining regions (CDRs) and three light chain CDRs. Generally, complementarity-determining regions (CDRs) are hypervariable regions present in the heavy chain variable domain and the light chain variable domain. Typically, the CDRs of an antibody's heavy chain and the CDRs of its associated light chain together form an antigen receptor. Typically, three CDRs (CDR1, CDR2, and CDR3) are non-contiguously arranged in the variable domain. Since an antigen receptor typically consists of two variable domains (on two different polypeptide chains, i.e., heavy chain variable region (VH) and light chain variable region (VL)), each antigen receptor typically contains six CDRs (heavy chain: CDRH1, CDRH2, and CDRH3; light chain: CDRL1, CDRL2, and CDRL3). The CDRs in VH and VL are usually separated by framework regions. Here, framework regions (FRs) are regions in a variable domain that are less "variable" than the CDRs. Typically, a variable region (or each variable region) consists of four framework regions separated by three CDRs.

[0042] The numbering of amino acids in variable regions and the identification of CDRs, various numbering schemes are known in the art, including Kabat, Chothia, EU, IMGT, Paratome, and AHo (Kabat, EA; National Institutes of Health (US) Office of the Director. Sequences of Proteins of Immunological Interest, 5th ed.; DIANE Publishing: Collingdale, PA, USA, 1991; Tai Te Wu, Elvin A. Kabat; An analysis of the sequences of the variable regions of Bence Jones proteins and myeloma light chains and their implications for antibody complementarity. J Exp Med 1 August 1970; 132 (2): 211-250; George Johnson, Tai Te Wu, Kabat Database and its applications: 30 years after the first variability plot, Nucleic Acids Research, Volume 28, Issue 1, 1 January 2000, Pages 214-218; Chothia C, Lesk AM. Canonical structures for the hypervariable regions of immunoglobulins. J Mol Biol. (1987) 196:901-17. doi: 10.1016 / 0022-2836(87)90412-8; Edelman, GM; Cunningham, BA; Gall, WE; Gottlieb, PD; Rutishauser, U.; Waxdal, MJ The covalent structure of an entire gammaG immunoglobulin molecule.Proc. Natl. Acad. Sci. USA 1969, 63, 78-85; Lefranc, MP; Giudicelli, V.; Ginestoux, C.; Bodmer, J.; Muller, W.; Bontrop, R.; Lemaitre, M.; Malik, A.; Barbie, V.; Chaume, D. Imgt, the international immunogenetics database. Nucleic Acids Res. 1999, 27, 209-212; Kunik V, Ashkenazi S, Ofran Y. Paratome: an online tool for systematic identification of antigen-binding regions in antibodies based on sequence or structure. Nucleic Acids Res. 2012 Jul;40(Web Server issue):W521-4. doi: 10.1093 / nar / gks480; Honegger A, Pluckthun A. Yet another numbering scheme for immunoglobulin variable domains: an automatic (Modeling and analysis tool. J Mol Biol. 2001 Jun 8;309(3):657-70. doi: 10.1006 / jmbi.2001.4662). Therefore, CDRs of the same variable region (VH or VL) may be numbered according to different numbering schemes.

[0043] The heavy and light chain sequences of exemplary antibodies of the invention have been determined, comprising three different CDRs on the heavy chain and three different CDRs on the light chain. The CDR amino acid sequences of heavy chain CDR1 (CDRH1), heavy chain CDR2 (CDRH2), heavy chain CDR3 (CDRH3), light chain CDR1 (CDRL1), light chain CDR2 (CDRL2), and light chain CDR3 (CDRL3) for exemplary antibodies 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. To define the CDR regions, the Kabat CDR definition was applied (Tai Te Wu, Elvin A. Kabat; An analysis of the sequences of the variable regions of Bence Jones proteins and myeloma light chains and their implications for antibody complementarity. J Exp Med 1 August 1970; 132 (2): 211-250; George Johnson, Tai Te Wu, Kabat Database and its applications: 30 years after the first variability plot, Nucleic Acids Research, Volume 28, Issue 1, 1 January 2000, Pages 214-218).

[0044] [Table 1]

[0045] Preferably, the antibodies or antigen-binding fragments thereof of the present invention comprise the specific combinations of CDRH1, CDRH2, CDRH3, CDRL1, CDRL1, CDRH2, and CDRH3 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.

[0046] Preferably, the antibody or antigen-binding fragment thereof of the present invention comprises CDRH1, CDRH2, and CDRH3 of the VH set forth in SEQ ID NO: 8, and CDRL1, CDRL2, and CDRL3 of the VL set forth in SEQ ID NO: 9. As described above, the CDRs in the variable regions of SEQ ID NOs: 8 and 9 can be determined according to various numbering schemes known in the art, such as Kabat, Chothia, EU, IMGT, Paratome, and AHo numbering. Preferably, the antibody or antigen-binding fragment thereof of the present invention comprises heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 2, 3, and 4, respectively; and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 5, 6, and 7, respectively. These CDR sequences were determined as described above. The present invention also encompasses antibodies comprising CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of a VH set forth in SEQ ID NO: 8 and a VL set forth in SEQ ID NO: 9, wherein it is understood that the CDRs are determined by separate methods.

[0047] As shown in the accompanying examples, such antibodies (e.g., ASC_Ab001) specifically bind to the PYD domain of human ASC, exhibit anti-ASC fibrillogenic activity, block ASC-Aβ interactions, and block the pro-inflammatory function of extracellular ASC fibrils.

[0048] In some embodiments, such antibodies or antigen-binding fragments thereof of the invention comprise: (i) an amino acid sequence having 70% or more identity (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%) to SEQ ID NO:8; and a light chain variable region (VL) comprising an amino acid sequence having 70% or more identity to SEQ ID NO:9 (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%). Thereby, the CDR sequences defined above (heavy chain CDR1 sequences, heavy chain CDR2 sequences, and heavy chain CDR3 sequences set forth in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively; and light chain CDR1 sequences, light chain CDR2 sequences, and light chain CDR3 sequences set forth in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively) are preferably maintained.

[0049] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO:8, and a light chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO:9.

[0050] In a specific embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises CDRH1, CDRH2, and CDRH3 of the VH set forth in SEQ ID NO: 15, and CDRL1, CDRL2, and CDRL3 of the VL set forth in SEQ ID NO: 16. As described above, the CDRs in the variable regions of SEQ ID NOs: 15 and 16 may be determined according to various numbering schemes known in the art, such as Kabat, Chothia, EU, IMGT, Paratome, and AHo numbering. Preferably, the antibody or antigen-binding fragment thereof of the present invention comprises heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 2, 10, and 11, respectively; and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 12, 13, and 14, respectively. These CDR sequences were determined as described above. The present invention also encompasses antibodies comprising CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of VH set forth in SEQ ID NO: 15 and VL set forth in SEQ ID NO: 16, where it is understood that the CDRs are determined by separate methods.

[0051] As shown in the accompanying examples, such antibodies (eg, ASC_Ab002) specifically bind to the PYD domain of human ASC and block ASC-Aβ interactions.

[0052] In some embodiments, such antibodies or antigen-binding fragments thereof of the invention comprise: (i) an amino acid sequence having 70% or more identity (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%) to SEQ ID NO: 15; and a light chain variable region (VL) comprising an amino acid sequence having 70% or more identity to SEQ ID NO: 16 (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%). Thereby, the CDR sequences defined above (heavy chain CDR1 sequences, heavy chain CDR2 sequences, and heavy chain CDR3 sequences set forth in SEQ ID NO: 2, SEQ ID NO: 10, and SEQ ID NO: 11, respectively; and light chain CDR1 sequences, light chain CDR2 sequences, and light chain CDR3 sequences set forth in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively) are preferably maintained.

[0053] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 15, and a light chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 16.

[0054] In a specific embodiment, an antibody or antigen-binding fragment thereof of the present invention comprises CDRH1, CDRH2, and CDRH3 of the VH set forth in SEQ ID NO: 22, and CDRL1, CDRL2, and CDRL3 of the VL set forth in SEQ ID NO: 23. As described above, the CDRs in the variable regions of SEQ ID NOs: 22 and 23 may be determined according to various numbering schemes known in the art, such as Kabat, Chothia, EU, IMGT, Paratome, and AHo numbering. Preferably, an antibody or antigen-binding fragment thereof of the present invention comprises heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 2, 17, and 18, respectively; and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 19, 20, and 21, respectively. These CDR sequences were determined as described above. The present invention also encompasses antibodies comprising CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of VH set forth in SEQ ID NO: 22 and VL set forth in SEQ ID NO: 23, wherein it is understood that the CDRs are determined by separate methods.

[0055] As shown in the accompanying examples, such antibodies (eg, ASC_Ab003) specifically bind to the PYD domain of human ASC and block ASC-Aβ interactions.

[0056] In some embodiments, such antibodies or antigen-binding fragments thereof of the invention comprise: (i) an amino acid sequence having 70% or more identity (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%) to SEQ ID NO:22; and a light chain variable region (VL) comprising an amino acid sequence having 70% or more identity to SEQ ID NO:23 (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%). Thereby, the CDR sequences defined above (heavy chain CDR1 sequences, heavy chain CDR2 sequences, and heavy chain CDR3 sequences set forth in SEQ ID NO: 2, SEQ ID NO: 17, and SEQ ID NO: 18, respectively; and light chain CDR1 sequences, light chain CDR2 sequences, and light chain CDR3 sequences set forth in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively) are preferably maintained.

[0057] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 22, and a light chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 23.

[0058] In a specific embodiment, an antibody or antigen-binding fragment thereof of the present invention comprises CDRH1, CDRH2, and CDRH3 of the VH set forth in SEQ ID NO: 30, and CDRL1, CDRL2, and CDRL3 of the VL set forth in SEQ ID NO: 31. As described above, the CDRs in the variable regions of SEQ ID NOs: 30 and 31 may be determined according to various numbering schemes known in the art, such as Kabat, Chothia, EU, IMGT, Paratome, and AHo numbering. Preferably, an antibody or antigen-binding fragment thereof of the present invention comprises heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 24, 25, and 26, respectively; and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 27, 28, and 29, respectively. These CDR sequences were determined as described above. The present invention also encompasses antibodies comprising CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of a VH set forth in SEQ ID NO: 30 and a VL set forth in SEQ ID NO: 31, wherein it is understood that the CDRs are determined by separate methods.

[0059] As shown in the accompanying Examples, such antibodies (eg, ASC_Ab004) specifically bind to the PYD domain of human ASC, exhibit anti-ASC fibrillogenic activity, and block ASC-Aβ interactions.

[0060] In some embodiments, such antibodies or antigen-binding fragments thereof of the invention comprise: (i) an amino acid sequence having 70% or more identity (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%) to SEQ ID NO: 30; and a light chain variable region (VL) comprising an amino acid sequence having 70% or more identity to SEQ ID NO: 31 (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%). Thereby, the CDR sequences defined above (heavy chain CDR1 sequences, heavy chain CDR2 sequences, and heavy chain CDR3 sequences set forth in SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, respectively; and light chain CDR1 sequences, light chain CDR2 sequences, and light chain CDR3 sequences set forth in SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29, respectively) are preferably maintained.

[0061] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 30, and a light chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 31.

[0062] In a specific embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises CDRH1, CDRH2, and CDRH3 of the VH set forth in SEQ ID NO: 38, and CDRL1, CDRL2, and CDRL3 of the VL set forth in SEQ ID NO: 39. As described above, the CDRs in the variable regions of SEQ ID NOs: 38 and 39 may be determined according to various numbering schemes known in the art, such as Kabat, Chothia, EU, IMGT, Paratome, and AHo numbering. Preferably, the antibody or antigen-binding fragment thereof of the present invention comprises heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 32, 33, and 34, respectively; and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 35, 36, and 37, respectively. These CDR sequences were determined as described above. The present invention also encompasses antibodies comprising CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of VH set forth in SEQ ID NO: 38 and VL set forth in SEQ ID NO: 39, where it is understood that the CDRs are determined by separate methods.

[0063] As shown in the accompanying examples, such antibodies (eg, ASC_Ab005) specifically bind to the PYD domain of human ASC and block ASC-Aβ interactions.

[0064] In some embodiments, such antibodies or antigen-binding fragments thereof of the invention comprise: (i) an amino acid sequence having 70% or more identity (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%) to SEQ ID NO: 38; and a light chain variable region (VL) comprising an amino acid sequence having 70% or more identity to SEQ ID NO: 39 (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%). Thereby, the CDR sequences defined above (heavy chain CDR1 sequences, heavy chain CDR2 sequences, and heavy chain CDR3 sequences set forth in SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34, respectively; and light chain CDR1 sequences, light chain CDR2 sequences, and light chain CDR3 sequences set forth in SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37, respectively) are preferably maintained.

[0065] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 38, and a light chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 39.

[0066] In a specific embodiment, an antibody or antigen-binding fragment thereof of the present invention comprises CDRH1, CDRH2, and CDRH3 of the VH set forth in SEQ ID NO: 46, and CDRL1, CDRL2, and CDRL3 of the VL set forth in SEQ ID NO: 47. As described above, the CDRs in the variable regions of SEQ ID NOs: 46 and 47 can be determined according to various numbering schemes known in the art, such as Kabat, Chothia, EU, IMGT, Paratome, and AHo numbering. Preferably, an antibody or antigen-binding fragment thereof of the present invention comprises heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 40, 41, and 42, respectively; and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 43, 44, and 45, respectively. These CDR sequences were determined as described above. The present invention also encompasses antibodies comprising CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of VH set forth in SEQ ID NO: 46 and VL set forth in SEQ ID NO: 47, where it is understood that the CDRs are determined by separate methods.

[0067] As shown in the accompanying examples, such antibodies (eg, ASC_Ab006) specifically bind to the PYD domain of human ASC and block ASC-Aβ interactions.

[0068] In some embodiments, such antibodies or antigen-binding fragments thereof of the invention comprise: (i) an amino acid sequence having 70% or more identity (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%) to SEQ ID NO: 46; and a light chain variable region (VL) comprising an amino acid sequence having 70% or more identity to SEQ ID NO:47 (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%). Thereby, the CDR sequences defined above (heavy chain CDR1 sequences, heavy chain CDR2 sequences, and heavy chain CDR3 sequences set forth in SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42, respectively; and light chain CDR1 sequences, light chain CDR2 sequences, and light chain CDR3 sequences set forth in SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 45, respectively) are preferably maintained.

[0069] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 46, and a light chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 47.

[0070] In a specific embodiment, an antibody or antigen-binding fragment thereof of the present invention comprises CDRH1, CDRH2, and CDRH3 of the VH set forth in SEQ ID NO: 54, and CDRL1, CDRL2, and CDRL3 of the VL set forth in SEQ ID NO: 55. As described above, the CDRs in the variable regions of SEQ ID NOs: 54 and 55 can be determined according to various numbering schemes known in the art, such as Kabat, Chothia, EU, IMGT, Paratome, and AHo numbering. Preferably, an antibody or antigen-binding fragment thereof of the present invention comprises heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 48, 49, and 50, respectively; and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 51, 52, and 53, respectively. These CDR sequences were determined as described above. The present invention also encompasses antibodies comprising CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of VH set forth in SEQ ID NO: 54 and VL set forth in SEQ ID NO: 55, wherein it is understood that the CDRs are determined by separate methods.

[0071] As shown in the accompanying Examples, such antibodies (eg, ASC_Ab007) specifically bind to the PYD domain of human ASC and exhibit anti-ASC fibrogenesis activity.

[0072] In some embodiments, such antibodies or antigen-binding fragments thereof of the invention comprise: (i) an amino acid sequence having 70% or more identity (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%) to SEQ ID NO: 54; and a light chain variable region (VL) comprising an amino acid sequence having 70% or more identity to SEQ ID NO:55 (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%). Thereby, the CDR sequences defined above (heavy chain CDR1 sequences, heavy chain CDR2 sequences, and heavy chain CDR3 sequences set forth in SEQ ID NO: 48, SEQ ID NO: 49, and SEQ ID NO: 50, respectively; and light chain CDR1 sequences, light chain CDR2 sequences, and light chain CDR3 sequences set forth in SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53, respectively) are preferably maintained.

[0073] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 54, and a light chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 56.

[0074] In a specific embodiment, an antibody or antigen-binding fragment thereof of the present invention comprises CDRH1, CDRH2, and CDRH3 of the VH set forth in SEQ ID NO: 61, and CDRL1, CDRL2, and CDRL3 of the VL set forth in SEQ ID NO: 62. As described above, the CDRs in the variable regions of SEQ ID NOs: 61 and 62 may be determined according to various numbering schemes known in the art, such as Kabat, Chothia, EU, IMGT, Paratome, and AHo numbering. Preferably, an antibody or antigen-binding fragment thereof of the present invention comprises heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 56, 57, and 58, respectively; and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 59, 52, and 60, respectively. These CDR sequences were determined as described above. The present invention also encompasses antibodies comprising CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of VH set forth in SEQ ID NO: 61 and VL set forth in SEQ ID NO: 62, where it is understood that the CDRs are determined by separate methods.

[0075] As shown in the accompanying Examples, such antibodies (eg, ASC_Ab008) specifically bind to the PYD domain of human ASC and exhibit anti-ASC fibrogenesis activity.

[0076] In some embodiments, such antibodies or antigen-binding fragments thereof of the invention comprise: (i) an amino acid sequence having 70% or more identity (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%) to SEQ ID NO: 61; and a light chain variable region (VL) comprising an amino acid sequence having 70% or more identity to SEQ ID NO:62 (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%). Thereby, the CDR sequences defined above (heavy chain CDR1 sequences, heavy chain CDR2 sequences, and heavy chain CDR3 sequences set forth in SEQ ID NO: 56, SEQ ID NO: 57, and SEQ ID NO: 58, respectively; and light chain CDR1 sequences, light chain CDR2 sequences, and light chain CDR3 sequences set forth in SEQ ID NO: 59, SEQ ID NO: 52, and SEQ ID NO: 60, respectively) are preferably maintained.

[0077] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 61, and a light chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 62.

[0078] In a specific embodiment, an antibody or antigen-binding fragment thereof of the present invention comprises CDRH1, CDRH2, and CDRH3 of the VH set forth in SEQ ID NO: 68, and CDRL1, CDRL2, and CDRL3 of the VL set forth in SEQ ID NO: 69. As described above, the CDRs in the variable regions of SEQ ID NOs: 68 and 69 may be determined according to various numbering schemes known in the art, such as Kabat, Chothia, EU, IMGT, Paratome, and AHo numbering. Preferably, an antibody or antigen-binding fragment thereof of the present invention comprises heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 63, 64, and 65, respectively; and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 66, 36, and 67, respectively. These CDR sequences were determined as described above. The present invention also encompasses antibodies comprising CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of VH set forth in SEQ ID NO: 68 and VL set forth in SEQ ID NO: 69, where it is understood that the CDRs are determined by separate methods.

[0079] As shown in the accompanying Examples, such antibodies (eg, ASC_Ab009) specifically bind to the PYD domain of human ASC and exhibit anti-ASC fibrogenesis activity.

[0080] In some embodiments, such antibodies or antigen-binding fragments thereof of the invention comprise: (i) an amino acid sequence having 70% or more identity (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%) to SEQ ID NO: 68; and a light chain variable region (VL) comprising an amino acid sequence having 70% or more identity to SEQ ID NO:69 (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%). Thereby, the CDR sequences defined above (heavy chain CDR1 sequences, heavy chain CDR2 sequences, and heavy chain CDR3 sequences set forth in SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65, respectively; and light chain CDR1 sequences, light chain CDR2 sequences, and light chain CDR3 sequences set forth in SEQ ID NO: 66, SEQ ID NO: 36, and SEQ ID NO: 67, respectively) are preferably maintained.

[0081] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 68, and a light chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 69.

[0082] The antibodies or antigen-binding fragments thereof of the present invention may exhibit anti-ASC fibrillogenesis activity. Antibodies that reduce or inhibit ASC fibrillogenesis provide a method for blocking either new or disseminated ASC assembly, thus blocking downstream effects of ASC speck formation, such as inflammation and / or amyloid dissemination. Anti-fibrogenic activity can be tested using a fibrillogenesis assay or sedimentation assay, for example, as described in the accompanying Examples. Accordingly, preferred fibrillogenesis assays are described in the Examples herein. Briefly, ASC assembles into large cytoplasmic macromolecular assemblies called ASC specks. This is driven by ASC-ASC interactions and can be reconstituted in vitro by incubating ASC monomers at 37°C. Under these conditions, ASC rapidly forms fibrils and retains their physiological and pathological functions. Thus, to examine the effect of an antibody or its antigen-binding fragment on ASC fibril formation, ASC containing a marker, such as green fluorescent protein (GFP), can be incubated in the presence of the antibody being tested (and an appropriate control, as described below) at about 37°C for, e.g., about 1 hour, and protein aggregates can be determined in terms of the marker used (e.g., by fluorescent imaging in the case of GFP). Protein aggregates from the antibody being tested can be compared to aggregates obtained with a control, such as ASC alone or an irrelevant antibody (neither of which would be expected to reduce ASC fibril formation). If a significant reduction in fibril formation is observed with the antibody being tested compared to the irrelevant antibody, the antibody being tested demonstrates anti-fibrogenic activity.

[0083] The antibody or antigen-binding fragment thereof of the present invention may reduce or block ASC-Aβ interaction. ASC-Aβ interactions are thought to underlie not only the increased Aβ aggregation and amyloid deposition in Alzheimer's 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-β in Alzheimer's disease. Nature. 2017 Dec 20;552(7685):355-361. doi: 10.1038 / nature25158), but also the increased neuroinflammation caused by these ASC-Aβ complexes (Friker LL, Scheiblich H, Hochheiser IV, Brinkschulte R, Riedel D, Latz E, Geyer M, Heneka MT. β-Amyloid Clustering around ASC Fibrils Boosts Its Toxicity in Microglia. Cell Rep. 2020 Mar 17;30(11):3743-3754.e6. doi: 10.1016 / j.celrep.2020.02.025). Therefore, antibodies that reduce or block ASC-Aβ interactions may reduce or block its downstream effects, particularly Aβ aggregation. Thereby, such antibodies may reduce or block increased amyloid deposition and neuroinflammation.

[0084] The effect on ASC-Aβ interaction can be tested using an ASC-Aβ interaction assay. A preferred ASC-Aβ interaction assay is described in the Examples section of this specification. Briefly, ASC fibrils can be formed by incubating ASC monomers at about 37°C for about 1 hour. The fibrillar ASC can then be incubated with Aβ containing a marker, such as TAMRA ((5-carboxy)tetramethylrhodamine), in the presence of the antibody to be tested (and appropriate controls, described below) for about 4 hours at about 37°C, and protein aggregation can be determined based on the marker used (e.g., by flow cytometry in the case of TAMRA). Protein aggregates obtained after incubation with ASC, Aβ, and anti-ASC antibody can be compared with protein aggregates obtained using controls such as ASC alone, ASC, Aβ, and an irrelevant antibody. ASC-Aβ inhibition can be determined as a percentage of the binding (aggregation) observed in the ASC and Aβ (no antibody) control and the ASC only control. The ASC+Aβ control assumes no inhibition (0%), whereas the ASC-only group assumes 100% inhibition (as no interaction (aggregation) is expected with ASC without Aβ). Therefore, the % inhibition of ASC-Aβ interaction for the antibody being tested can be calculated taking this into account to determine whether significant inhibition of ASC-Aβ interaction is observed compared to the effect of an irrelevant antibody.

[0085] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention may reduce or block Aβ aggregation. In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention may reduce or block protein aggregation, particularly in the context of an inflammatory process. Without being bound by any theory, it is believed that ASC acts as a "catalyst" for the aggregation of proteins such as Aβ. Thus, antibodies that reduce or block the interaction of ASC with such (aggregating) proteins are believed to reduce or block protein aggregation (which would otherwise be catalyzed by ASC), particularly by binding to ASC.

[0086] The antibodies or antigen-binding fragments thereof of the present invention may reduce or block the pro-inflammatory function of extracellular ASC fibers. ASC fibers and specks have been shown to 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 AG, 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. Nat Immunol. 2014 Aug;15(8):727-37. doi: 10.1038 / ni.2913; Baroja-Mazo A, Martin-Sanchez F, Gomez AI, Martinez CM, Amores-Iniesta J, Compan V, Barbera-Cremades M, Yague J, Ruiz-Ortiz E, Anton J, Bujan S, Couillin I, Brough D, Arostegui JI, Pelegrin P. The NLRP3 inflammasome is released as a particulate danger signal that amplifies the inflammatory response. Nat Immunol. 2014 Aug;15(8):738-48. doi: 10.1038 / ni.2919). To test the effect of antibodies on the pro-inflammatory function of extracellular ASC fibers, a THP-1 efficacy assay can be used. A preferred THP-1 efficacy assay is described in the Examples section herein. Briefly, wild-type and asc fibrils were tested.- / - THP-1 monocytes can be seeded into multiwell plates at a density of, for example, about 10,000 cells / well. The monocytes can then be differentiated into macrophages by, for example, incubating in a medium containing 100 ng / ml of phorbol 12-myristate-13-acetate (PMA) at about 37°C and about 5% CO for about 4 days. After differentiation, the cells can be washed, and ASC fibers can be prepared by incubating with ASC monomers at about 37°C for about 1 hour. Before adding the ASC fibers to the cells, they can be preincubated with the antibody to be tested for, for example, about 15 minutes at about 22°C. After incubation (for example, for about 16 hours at about 37°C and about 5% CO), secreted IL-1β can be measured, for example, by the Lumit Human IL-1 Immunoassay (Promega, W6010). The amount of IL-1β secreted can be compared to that obtained in the same assay in wt THP-1 cells using an irrelevant control antibody.

[0087] In some embodiments, the antibody or antigen-binding fragment thereof is a humanized antibody. Methods for humanizing antibodies are well known in the art. Generally, in a "humanized" antibody, a minimal portion of a non-human antibody (such as six CDRs, or (a portion of) a VH and VL comprising six CDRs) is introduced into an otherwise human antibody. Preferably, a humanized antibody is engineered to contain one or more human framework regions in the variable regions along with non-human (e.g., rabbit) CDRs in the heavy and / or light chains. More preferably, a humanized antibody contains entirely human sequences with the exception of the CDR regions. However, amino acids in the framework regions (FR) of a human immunoglobulin may be substituted with corresponding non-human residues. Furthermore, a humanized antibody may contain residues that are found neither in the human form of the antibody nor in the imported CDR or framework sequences, but that are included to further refine and optimize antibody performance. Preferably, a humanized antibody comprises a VH and a VL, in which all or substantially all of the CDR regions correspond to those of a parent non-human (e.g., rabbit) antibody, and all or substantially all of the FR regions are those of a human antibody or human immunoglobulin consensus sequence. The FR regions can be modified by any method known in the art and / or provided herein. A humanized antibody may also comprise a human Fc portion. Humanized antibodies preferably exhibit reduced immunogenicity when introduced into humans. "Humanized" antibodies can be prepared by first generating a "chimeric" antibody (a non-human Fab grafted onto a human Fc) and selectively mutating (non-CDR) amino acids in the Fab portion of the molecule. Alternatively, "humanized" antibodies can be obtained directly by grafting appropriate "donor" CDR-encoding segments from a non-human animal onto a human antibody "acceptor" scaffold and appropriately mutating (non-CDR) amino acids for optimized binding. In some embodiments, the antibody or antigen-binding fragment thereof is a monoclonal antibody. For example, the antibody or antigen-binding fragment thereof may be a humanized monoclonal antibody.

[0088] In some embodiments, the variable regions or CDRs of the antibodies defined herein are derived from rabbit antibodies and grafted onto a human IgG or IgA antibody scaffold. Thus, the variable regions, portions thereof, or CDRs may be rabbit and grafted onto an antibody framework, where the antibody framework is preferably of human origin. Typically, the rabbit-derived portions of the variable regions grafted onto the antibody framework comprise the CDRs. Among IgGs, IgG1 and IgG4 are preferred.

[0089] Specific methods for humanizing rabbit antibodies are known in the art, and are described, for example, in Borras L, Gunde T, Tietz J, Bauer U, Hulmann-Cottier V, Grimshaw JP, Urech DM. Generic approach for the generation of stable humanized single-chain Fv fragments from rabbit monoclonal antibodies. J Biol Chem. 2010 Mar 19;285(12):9054-66. doi: 10.1074 / jbc.M109.072876; WO 2009 / 155726 A2; and Zhang YF, Ho M. Humanization of rabbit monoclonal antibodies via grafting combined Kabat / IMGT / Paratome complementarity-determining regions: Rationale and examples. MAbs. 2017 Apr;9(3):419-429. doi: 10.1080 / 19420862.2017.1289302, which are incorporated herein by reference in their entirety.

[0090] In particular, for humanizing an exemplary antibody of the present invention, one skilled in the art can use the (rabbit) VH / VL sequence of an exemplary antibody as set forth in Table 1 and combine 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) so that each "extended" CDR covers each CDR defined in each of the combined numbering schemes. Thus, such "extended" CDRs determined by combining different numbering schemes may include amino acid residues in the framework regions adjacent to the CDR (particularly when a single numbering scheme is used). In some embodiments, an "extended" CDR may correspond to a CDR determined by a single numbering scheme. This applies particularly when a CDR determined by a single numbering scheme includes (or corresponds to) a corresponding CDR determined by one or more other numbering schemes. In some embodiments, extended (rabbit) CDR sequences (based on the VH / VL sequences of the exemplary antibodies in Table 1 above) can be obtained using Kabat, IMGT, and Paratome, as described in Zhang and Ho, 2017 (Zhang YF, Ho M. Humanization of rabbit monoclonal antibodies via grafting combined Kabat / IMGT / Paratome complementarity-determining regions: Rationale and examples. MAbs. 2017 Apr;9(3):419-429. doi: 10.1080 / 19420862.2017.1289302). The extended rabbit CDRs can then be grafted into human (germline) IgG framework regions.

[0091] Specific examples of extended CDR sets for exemplary antibodies are shown in Table 2 below.

[0092] [Table 2]

[0093] Specific examples of VH / VL sequences for humanized versions of exemplary antibodies are shown in Table 3 below.

[0094] [Table 3]

[0095] Preferably, antibodies or antigen-binding fragments thereof of the invention comprise the extended CDRH1, extended CDRH2, extended CDRH3, extended CDRL1, extended CDRL2, and 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. As noted above, the "extended" CDRs may include amino acid residues of the framework regions adjacent to the CDRs shown in Table 1.

[0096] Preferably, the antibody or antigen-binding fragment thereof of the invention comprises an extended CDRH1, CDRH2, and CDRH3 as set forth in SEQ ID NO: 159, SEQ ID NO: 3, and SEQ ID NO: 160, respectively; and an extended CDRL1, CDRL2, and CDRL3 as set forth in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively. More preferably, the antibody or antigen-binding fragment thereof of the invention comprises an extended CDRH1, CDRH2, and CDRH3 as set forth in SEQ ID NO: 194, SEQ ID NO: 195, and SEQ ID NO: 196, respectively; and an extended CDRL1, CDRL2, and CDRL3 as set forth in SEQ ID NO: 197, SEQ ID NO: 198, and SEQ ID NO: 199, respectively.

[0097] In some embodiments, an antibody or antigen-binding fragment thereof of the present invention comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 sequences set forth in SEQ ID NOs: 2, 3, and 4, respectively; and light chain CDR1, light chain CDR2, and light chain CDR3 sequences set forth in SEQ ID NOs: 5, 6, and 7, 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. Preferably, such an antibody or antigen-binding fragment thereof comprises extended CDRH1, CDRH2, and CDRH3 sequences set forth in SEQ ID NOs: 159, 3, and 160, respectively; and extended CDRL1, CDRL2, and CDRL3 sequences set forth in SEQ ID NOs: 5, 6, and 7, respectively. More preferably, such an antibody or antigen-binding fragment thereof comprises an extended CDRH1, an extended CDRH2, and an extended CDRH3 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 set forth in SEQ ID NO: 197, SEQ ID NO: 198, and SEQ ID NO: 199, respectively.

[0098] In some embodiments, such antibodies or antigen-binding fragments thereof of the invention comprise: (i) an amino acid sequence having 70% or more identity (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%) to SEQ ID NO: 157; and a light chain variable region (VL) comprising an amino acid sequence having 70% or more identity to SEQ ID NO: 158 (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%). Thereby, the CDR sequences defined above (heavy chain CDR1 sequences, heavy chain CDR2 sequences, and heavy chain CDR3 sequences set forth in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively; and light chain CDR1 sequences, light chain CDR2 sequences, and light chain CDR3 sequences set forth in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively) are preferably maintained.More preferably, the extended CDR sequences defined above (extended CDRH1, extended CDRH2 and extended CDRH3 set forth in SEQ ID NO: 159, SEQ ID NO: 3 and SEQ ID NO: 160, respectively, and extended CDRL1, extended CDRL2 and extended CDRL3 set forth in SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, respectively; even more preferably extended CDRH1, extended CDRH2 and extended CDRH3 set forth in SEQ ID NO: 194, SEQ ID NO: 195 and SEQ ID NO: 196, respectively, and extended CDRL1, extended CDRL2 and extended CDRL3 set forth in SEQ ID NO: 197, SEQ ID NO: 198 and SEQ ID NO: 199, respectively) are maintained in the sequence variant of SEQ ID NO: 157 and the sequence variant of SEQ ID NO: 158, respectively.

[0099] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 157, and a light chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 158.

[0100] In some embodiments, such antibodies or antigen-binding fragments thereof of the invention comprise: (i) an amino acid sequence having 70% or more identity (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%) to SEQ ID NO: 176; and a light chain variable region (VL) comprising an amino acid sequence having 70% or more identity to SEQ ID NO: 177 (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%). Thereby, the CDR sequences defined above (heavy chain CDR1 sequences, heavy chain CDR2 sequences, and heavy chain CDR3 sequences set forth in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively; and light chain CDR1 sequences, light chain CDR2 sequences, and light chain CDR3 sequences set forth in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively) are preferably maintained.More preferably, the extended CDR sequences defined above (extended CDRH1, extended CDRH2 and extended CDRH3 set forth in SEQ ID NO: 159, SEQ ID NO: 3 and SEQ ID NO: 160, respectively, and extended CDRL1, extended CDRL2 and extended CDRL3 set forth in SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, respectively; even more preferably extended CDRH1, extended CDRH2 and extended CDRH3 set forth in SEQ ID NO: 194, SEQ ID NO: 195 and SEQ ID NO: 196, respectively, and extended CDRL1, extended CDRL2 and extended CDRL3 set forth in SEQ ID NO: 197, SEQ ID NO: 198 and SEQ ID NO: 199, respectively) are maintained in the sequence variant of SEQ ID NO: 176 and the sequence variant of SEQ ID NO: 177, respectively.

[0101] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 176, and a light chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 177.

[0102] In certain aspects, the antibody or antigen-binding fragment thereof of the present invention comprises an extended CDRH1, an extended CDRH2, and an extended CDRH3 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 set forth in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively.

[0103] In some embodiments, an antibody or antigen-binding fragment thereof of the present invention comprises heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 2, 10, and 11, respectively; and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 12, 13, and 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. Preferably, such an antibody or antigen-binding fragment thereof comprises extended CDRH1, CDRH2, and CDRH3 sequences set forth in SEQ ID NOs: 161, 10, and 162, respectively; and extended CDRL1, CDRL2, and CDRL3 sequences set forth in SEQ ID NOs: 12, 13, and 14, respectively.

[0104] In some embodiments, such antibodies or antigen-binding fragments thereof of the invention comprise: (i) an amino acid sequence having 70% or more identity (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%) to SEQ ID NO: 178; and a light chain variable region (VL) comprising an amino acid sequence having 70% or more identity to SEQ ID NO: 179 (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%). Thereby, the CDR sequences defined above (heavy chain CDR1 sequences, heavy chain CDR2 sequences, and heavy chain CDR3 sequences set forth in SEQ ID NO: 2, SEQ ID NO: 10, and SEQ ID NO: 11, respectively; and light chain CDR1 sequences, light chain CDR2 sequences, and light chain CDR3 sequences set forth in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively) are preferably maintained.More preferably, the extended CDR sequences defined above (extended CDRH1, extended CDRH2 and extended CDRH3 set forth in SEQ ID NO: 161, SEQ ID NO: 10 and SEQ ID NO: 162, respectively; and extended CDRL1, extended CDRL2 and extended CDRL3 set forth in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively) are maintained in the sequence variant of SEQ ID NO: 178 and in the sequence variant of SEQ ID NO: 179.

[0105] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 178 and a light chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 179.

[0106] In certain aspects, the antibody or antigen-binding fragment thereof of the present invention comprises an extended CDRH1, an extended CDRH2, and an extended CDRH3 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 set forth in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively.

[0107] In some embodiments, an antibody or antigen-binding fragment thereof of the present invention comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 sequences set forth in SEQ ID NOs: 2, 17, and 18, respectively; and light chain CDR1, light chain CDR2, and light chain CDR3 sequences set forth in SEQ ID NOs: 19, 20, and 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. Preferably, such an antibody or antigen-binding fragment thereof comprises extended CDRH1, CDRH2, and CDRH3 sequences set forth in SEQ ID NOs: 161, 17, and 163, respectively; and extended CDRL1, CDRL2, and CDRL3 sequences set forth in SEQ ID NOs: 19, 20, and 21, respectively.

[0108] In some embodiments, such antibodies or antigen-binding fragments thereof of the invention comprise: (i) an amino acid sequence having 70% or more identity (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%) to SEQ ID NO: 180; and a light chain variable region (VL) comprising an amino acid sequence having 70% or more identity to SEQ ID NO: 181 (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%). Thereby, the CDR sequences defined above (heavy chain CDR1 sequences, heavy chain CDR2 sequences, and heavy chain CDR3 sequences set forth in SEQ ID NO: 2, SEQ ID NO: 17, and SEQ ID NO: 18, respectively; and light chain CDR1 sequences, light chain CDR2 sequences, and light chain CDR3 sequences set forth in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively) are preferably maintained.More preferably, the extended CDR sequences defined above (extended CDRH1, extended CDRH2 and extended CDRH3 set forth in SEQ ID NO: 161, SEQ ID NO: 17 and SEQ ID NO: 163, respectively; and extended CDRL1, extended CDRL2 and extended CDRL3 set forth in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively) are maintained in the sequence variant of SEQ ID NO: 180 and in the sequence variant of SEQ ID NO: 181.

[0109] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 180, and a light chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 181.

[0110] In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises an extended CDRH1, an extended CDRH2, and an extended CDRH3 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 set forth in SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29, respectively.

[0111] In some embodiments, an antibody or antigen-binding fragment thereof of the present invention comprises heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 24, 25, and 26, respectively; and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 27, 28, and 29, respectively; and humanized framework regions, wherein the heavy chain V gene IGHV3-66, the heavy chain J gene IGHJ2, the light chain V gene IGKV1-6, and the light chain J gene IGKJ4 are used to obtain the humanized framework regions. Preferably, such an antibody or antigen-binding fragment thereof comprises extended CDRH1, CDRH2, and CDRH3 sequences set forth in SEQ ID NOs: 164, 25, and 165, respectively; and extended CDRL1, CDRL2, and CDRL3 sequences set forth in SEQ ID NOs: 27, 28, and 29, respectively.

[0112] In some embodiments, such antibodies or antigen-binding fragments thereof of the invention comprise: (i) an amino acid sequence having 70% or more identity (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%) to SEQ ID NO: 182; and a light chain variable region (VL) comprising an amino acid sequence having 70% or more identity to SEQ ID NO: 183 (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%). Thereby, the CDR sequences defined above (heavy chain CDR1 sequences, heavy chain CDR2 sequences, and heavy chain CDR3 sequences set forth in SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, respectively; and light chain CDR1 sequences, light chain CDR2 sequences, and light chain CDR3 sequences set forth in SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29, respectively) are preferably maintained.More preferably, the extended CDR sequences defined above (extended CDRH1, extended CDRH2 and extended CDRH3 set forth in SEQ ID NO: 164, SEQ ID NO: 25 and SEQ ID NO: 165, respectively; and extended CDRL1, extended CDRL2 and extended CDRL3 set forth in SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, respectively) are maintained in the sequence variant of SEQ ID NO: 182 and in the sequence variant of SEQ ID NO: 183.

[0113] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 182, and a light chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 183.

[0114] In certain aspects, the antibody or antigen-binding fragment thereof of the present invention comprises an extended CDRH1, an extended CDRH2, and an extended CDRH3 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 set forth in SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37, respectively.

[0115] In some embodiments, an antibody or antigen-binding fragment thereof of the present invention comprises heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 32, 33, and 34, respectively; and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 35, 36, and 37, respectively; and humanized framework regions, wherein the heavy chain V gene IGHV3-33, the heavy chain J gene IGHJ2, the light chain V gene IGKV1-5, and the light chain J gene IGKJ4 are used to obtain the humanized framework regions. Preferably, such an antibody or antigen-binding fragment thereof comprises extended CDRH1, CDRH2, and CDRH3 sequences set forth in SEQ ID NOs: 166, 33, and 167, respectively; and extended CDRL1, CDRL2, and CDRL3 sequences set forth in SEQ ID NOs: 35, 36, and 37, respectively.

[0116] In some embodiments, such antibodies or antigen-binding fragments thereof of the invention comprise: (i) an amino acid sequence having 70% or more identity (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%) to SEQ ID NO: 184; and a light chain variable region (VL) comprising an amino acid sequence having 70% or more identity to SEQ ID NO: 185 (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%). Thereby, the CDR sequences defined above (heavy chain CDR1 sequences, heavy chain CDR2 sequences, and heavy chain CDR3 sequences set forth in SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34, respectively; and light chain CDR1 sequences, light chain CDR2 sequences, and light chain CDR3 sequences set forth in SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37, respectively) are preferably maintained.More preferably, the extended CDR sequences defined above (extended CDRH1, extended CDRH2 and extended CDRH3 set forth in SEQ ID NO: 166, SEQ ID NO: 33 and SEQ ID NO: 167, respectively; and extended CDRL1, extended CDRL2 and extended CDRL3 set forth in SEQ ID NO: 35, SEQ ID NO: 36 and SEQ ID NO: 37, respectively) are maintained in the sequence variant of SEQ ID NO: 184 and in the sequence variant of SEQ ID NO: 185.

[0117] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 184, and a light chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 185.

[0118] In certain aspects, the antibody or antigen-binding fragment thereof of the present invention comprises an extended CDRH1, an extended CDRH2, and an extended CDRH3 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 set forth in SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 45, respectively.

[0119] In some embodiments, an antibody or antigen-binding fragment thereof of the present invention comprises heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 40, 41, and 42, respectively; and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 43, 44, and 45, respectively; and humanized framework regions, wherein the heavy chain V gene IGHV3-23, the heavy chain J gene IGHJ2, the light chain V gene IGKV1-27, and the light chain J gene IGKJ4 are used to obtain the humanized framework regions. Preferably, such an antibody or antigen-binding fragment thereof comprises extended CDRH1, CDRH2, and CDRH3 sequences set forth in SEQ ID NOs: 168, 41, and 169, respectively; and extended CDRL1, CDRL2, and CDRL3 sequences set forth in SEQ ID NOs: 43, 44, and 45, respectively.

[0120] In some embodiments, such antibodies or antigen-binding fragments thereof of the invention comprise: (i) an amino acid sequence having 70% or more identity (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%) to SEQ ID NO: 186; and a light chain variable region (VL) comprising an amino acid sequence having 70% or more identity to SEQ ID NO: 187 (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%). Thereby, the CDR sequences defined above (heavy chain CDR1 sequences, heavy chain CDR2 sequences, and heavy chain CDR3 sequences set forth in SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42, respectively; and light chain CDR1 sequences, light chain CDR2 sequences, and light chain CDR3 sequences set forth in SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 45, respectively) are preferably maintained.More preferably, the extended CDR sequences defined above (extended CDRH1, extended CDRH2 and extended CDRH3 set forth in SEQ ID NO: 168, SEQ ID NO: 41 and SEQ ID NO: 169, respectively; and extended CDRL1, extended CDRL2 and extended CDRL3 set forth in SEQ ID NO: 43, SEQ ID NO: 44 and SEQ ID NO: 45, respectively) are maintained in the sequence variant of SEQ ID NO: 186 and in the sequence variant of SEQ ID NO: 187.

[0121] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 186, and a light chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 187.

[0122] In certain aspects, the antibody or antigen-binding fragment thereof of the present invention comprises an extended CDRH1, an extended CDRH2, and an extended CDRH3 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 set forth in SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53, respectively.

[0123] In some embodiments, an antibody or antigen-binding fragment thereof of the present invention comprises heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 48, 49, and 50, respectively; and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 51, 52, and 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 IGKJ4 are used to obtain the humanized framework regions. Preferably, such an antibody or antigen-binding fragment thereof comprises extended CDRH1, CDRH2, and CDRH3 sequences set forth in SEQ ID NOs: 170, 49, and 171, respectively; and extended CDRL1, CDRL2, and CDRL3 sequences set forth in SEQ ID NOs: 51, 52, and 53, respectively.

[0124] In some embodiments, such antibodies or antigen-binding fragments thereof of the invention comprise: (i) an amino acid sequence having 70% or more identity (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%) to SEQ ID NO: 188; and a light chain variable region (VL) comprising an amino acid sequence having 70% or more identity to SEQ ID NO: 189 (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%). Thereby, the CDR sequences defined above (heavy chain CDR1 sequences, heavy chain CDR2 sequences, and heavy chain CDR3 sequences set forth in SEQ ID NO: 48, SEQ ID NO: 49, and SEQ ID NO: 50, respectively; and light chain CDR1 sequences, light chain CDR2 sequences, and light chain CDR3 sequences set forth in SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53, respectively) are preferably maintained.More preferably, the extended CDR sequences defined above (extended CDRH1, extended CDRH2 and extended CDRH3 set forth in SEQ ID NO: 170, SEQ ID NO: 49 and SEQ ID NO: 171, respectively; and extended CDRL1, extended CDRL2 and extended CDRL3 set forth in SEQ ID NO: 51, SEQ ID NO: 52 and SEQ ID NO: 53, respectively) are maintained in the sequence variant of SEQ ID NO: 188 and in the sequence variant of SEQ ID NO: 189.

[0125] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 188 and a light chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 189.

[0126] In certain aspects, the antibody or antigen-binding fragment thereof of the present invention comprises an extended CDRH1, an extended CDRH2, and an extended CDRH3 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 set forth in SEQ ID NO: 59, SEQ ID NO: 52, and SEQ ID NO: 60, respectively.

[0127] In some embodiments, an antibody or antigen-binding fragment thereof of the present invention comprises heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 56, 57, and 58, respectively; and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 59, 52, and 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. Preferably, such an antibody or antigen-binding fragment thereof comprises extended CDRH1, CDRH2, and CDRH3 sequences set forth in SEQ ID NOs: 172, 57, and 173, respectively; and extended CDRL1, CDRL2, and CDRL3 sequences set forth in SEQ ID NOs: 59, 52, and 60, respectively.

[0128] In some embodiments, such antibodies or antigen-binding fragments thereof of the invention comprise: (i) an amino acid sequence having 70% or more identity (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%) to SEQ ID NO: 190; and a light chain variable region (VL) comprising an amino acid sequence having 70% or more identity to SEQ ID NO: 191 (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%). Thereby, the CDR sequences defined above (heavy chain CDR1 sequences, heavy chain CDR2 sequences, and heavy chain CDR3 sequences set forth in SEQ ID NO: 56, SEQ ID NO: 57, and SEQ ID NO: 58, respectively; and light chain CDR1 sequences, light chain CDR2 sequences, and light chain CDR3 sequences set forth in SEQ ID NO: 59, SEQ ID NO: 52, and SEQ ID NO: 60, respectively) are preferably maintained.More preferably, the extended CDR sequences defined above (extended CDRH1, extended CDRH2 and extended CDRH3 set forth in SEQ ID NO: 172, SEQ ID NO: 57 and SEQ ID NO: 173, respectively; and extended CDRL1, extended CDRL2 and extended CDRL3 set forth in SEQ ID NO: 59, SEQ ID NO: 52 and SEQ ID NO: 60, respectively) are maintained in the sequence variant of SEQ ID NO: 190 and in the sequence variant of SEQ ID NO: 191.

[0129] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 190, and a light chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 191.

[0130] In certain aspects, the antibody or antigen-binding fragment thereof of the present invention comprises an extended CDRH1, an extended CDRH2, and an extended CDRH3 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 set forth in SEQ ID NO: 66, SEQ ID NO: 36, and SEQ ID NO: 67, respectively.

[0131] In some embodiments, an antibody or antigen-binding fragment thereof of the present invention comprises heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 63, 64, and 65, respectively; and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 66, 36, and 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. Preferably, such an antibody or antigen-binding fragment thereof comprises extended CDRH1, CDRH2, and CDRH3 sequences set forth in SEQ ID NOs: 174, 64, and 175, respectively; and extended CDRL1, CDRL2, and CDRL3 sequences set forth in SEQ ID NOs: 66, 36, and 67, respectively.

[0132] In some embodiments, such antibodies or antigen-binding fragments thereof of the invention comprise: (i) an amino acid sequence having 70% or more identity (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%) to SEQ ID NO: 192; and a light chain variable region (VL) comprising an amino acid sequence having 70% or more identity to SEQ ID NO: 193 (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%). Thereby, the CDR sequences defined above (heavy chain CDR1 sequences, heavy chain CDR2 sequences, and heavy chain CDR3 sequences set forth in SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65, respectively; and light chain CDR1 sequences, light chain CDR2 sequences, and light chain CDR3 sequences set forth in SEQ ID NO: 66, SEQ ID NO: 36, and SEQ ID NO: 67, respectively) are preferably maintained.More preferably, the extended CDR sequences defined above (extended CDRH1, extended CDRH2 and extended CDRH3 set forth in SEQ ID NO: 174, SEQ ID NO: 64 and SEQ ID NO: 175, respectively; and extended CDRL1, extended CDRL2 and extended CDRL3 set forth in SEQ ID NO: 66, SEQ ID NO: 36 and SEQ ID NO: 67, respectively) are maintained in the sequence variant of SEQ ID NO: 192 and in the sequence variant of SEQ ID NO: 193.

[0133] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 192, and a light chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 193.

[0134] In some embodiments, humanization may involve backmutation. That is, amino acids in the human framework sequences are backmutated to the corresponding amino acids in the rabbit framework sequences, especially when the results of grafting (extended) CDRs into human IgG framework regions are unsatisfactory. In this context, it is understood that the goal of humanization is always to maintain as many human sequences / amino acids as possible. In some embodiments, amino acid residues in the Vernier zones of the human VH and VL framework regions (Vernier zone residues) may be backmutated to the corresponding rabbit amino acids (of the VH / VL regions shown in Table 1). In some embodiments, the first two amino acid residues of the light chain may be backmutated (from human to rabbit), if desired. Alternatively, or in addition, the first one, two, or three residues at the N-terminus of the HV4 / LV4 loop (light chain LV4 loop: positions 66-70 by Kabat; and heavy chain HV4 loop: positions 72-75 or 76 by Kabat) may be backmutated (from human to rabbit), if desired. In some embodiments, the extra cysteine ​​at position 80 (Kabat numbering) of the rabbit VL (compared to the human VL) may be removed or substituted in the humanized version. In some embodiments, T23 (Kabat numbering) of the rabbit VH is substituted, preferably with the corresponding amino acid in the human VH.

[0135] In some embodiments of the humanized (ASC_AB001) antibody or antigen-binding fragment thereof of the invention, amino acid residues in the Vernier zone of the human VH framework region (e.g., Vernier zone FR3) may be backmutated to the corresponding rabbit amino acid (see Figure 21, graft ID Ab001_VHhum3). In some embodiments, the backmutated amino acid residues (e.g., Vernier zone FR3) are refined, e.g., to (at least partially) human sequences (see Figure 21, graft ID Ab001_VHhum23). In some embodiments, IGHJ2 to IGHJ1 / 4 / 5 mutations are introduced in framework region FR4 (see Figure 21, graft ID Ab001_VHhum23). In certain embodiments, the above mutations are combined (see Figure 21, graft ID Ab001_VHhum23).

[0136] In some embodiments, light chain CDRL2 (DTSSLAS) may be humanized (DASSLAS) (see Figure 22, e.g., graft ID Ab001_VLhum4). In some embodiments, light chain CDRL1 (QSSESVYNNSRLS) may be humanized (RASESVYNNSRLS) (see Figure 22, e.g., graft ID Ab001_VLhum5). In some embodiments, an IGKJ4 to IGKJ1 (G→Q) mutation is introduced in framework region FR4 (see Figure 22, e.g., graft ID Ab001_VLhum6). In some embodiments, an N29A mutation is introduced in LCDR1, thereby removing a glycosylation motif (see Figure 22, e.g., graft ID Ab001_VLhum10). In some embodiments, an N29Q mutation is introduced in LCDR1 (see Figure 22, graft ID Ab001_VLhum11). In some embodiments, an S31A mutation is introduced into LCDR1 (see Figure 22, graft ID Ab001_VLhum12). In certain embodiments, one or more of the above mutations are combined (see Figure 22, graft ID Ab001_VLhum5, VLhum6, VLhum10, VLhum11, VLhum12).

[0137] Therefore, in certain embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises CDRH1 set forth in SEQ ID NO: 2, CDRH2 set forth in SEQ ID NO: 3, CDRH3 set forth in SEQ ID NO: 4, CDRL1 set forth in SEQ ID NO: 5 or any one of SEQ ID NOs: 230 to 233, CDRL2 set forth in SEQ ID NO: 234, and CDRL3 set forth in SEQ ID NO: 7.

[0138] In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises a CDRH1 set forth in SEQ ID NO: 2, a CDRH2 set forth in SEQ ID NO: 3, a CDRH3 set forth in SEQ ID NO: 4, a CDRL1 set forth in SEQ ID NO: 5, a CDRL2 set forth in SEQ ID NO: 234, and a CDRL3 set forth in SEQ ID NO: 7.

[0139] In a further specific embodiment, the antibody or antigen-binding fragment thereof of the invention comprises a CDRH1 set forth in SEQ ID NO: 2, a CDRH2 set forth in SEQ ID NO: 3, a CDRH3 set forth in SEQ ID NO: 4, a CDRL1 set forth in SEQ ID NO: 230, a CDRL2 set forth in SEQ ID NO: 234, and a CDRL3 set forth in SEQ ID NO: 7.

[0140] In a further specific embodiment, the antibody or antigen-binding fragment thereof of the invention comprises a CDRH1 set forth in SEQ ID NO: 2, a CDRH2 set forth in SEQ ID NO: 3, a CDRH3 set forth in SEQ ID NO: 4, a CDRL1 set forth in SEQ ID NO: 231, a CDRL2 set forth in SEQ ID NO: 234, and a CDRL3 set forth in SEQ ID NO: 7.

[0141] In a preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises a CDRH1 set forth in SEQ ID NO: 2, a CDRH2 set forth in SEQ ID NO: 3, a CDRH3 set forth in SEQ ID NO: 4, a CDRL1 set forth in SEQ ID NO: 232, a CDRL2 set forth in SEQ ID NO: 234, and a CDRL3 set forth in SEQ ID NO: 7.

[0142] In a further preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises a CDRH1 set forth in SEQ ID NO: 2, a CDRH2 set forth in SEQ ID NO: 3, a CDRH3 set forth in SEQ ID NO: 4, a CDRL1 set forth in SEQ ID NO: 233, a CDRL2 set forth in SEQ ID NO: 234, and a CDRL3 set forth in SEQ ID NO: 7.

[0143] Thus, in certain embodiments, an antibody or antigen-binding fragment thereof of the invention comprises a VH having at least 70% identity (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% identity), preferably at least 80% identity (e.g., 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% identity), more preferably at least 90% identity (e.g., 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 the sequence set forth in any one of SEQ ID NO:200, SEQ ID NO:201, and SEQ ID NO:202; and a VL having at least 70% identity (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% identity) to the sequence set forth in any one of SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, and SEQ ID NO:209 (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% identity), preferably at least 80% identity (e.g., 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% identity), more preferably at least 90% identity (e.g., 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). Here, the above-mentioned CDRH1 to 3 sequences and CDRL1 to 3 sequences are preferably maintained.

[0144] In a preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises a VH comprising the amino acid sequence set forth in any one of SEQ ID NO: 200, SEQ ID NO: 201, and SEQ ID NO: 202; and a VL comprising the amino acid sequence set forth in any one of SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, and SEQ ID NO: 209.

[0145] In a preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention has at least 70% identity (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% identity) to SEQ ID NO: 202, preferably at least 80% identity (e.g., 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% identity), more preferably at least 90% identity (e.g., 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). 9% identity) to SEQ ID NO: 208; and a VL having at least 70% identity (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% identity), preferably at least 80% identity (e.g., 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% identity), more preferably at least 90% identity (e.g., 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: 208. Here, the CDRH1 to 3 sequences (described in SEQ ID NOs: 2, 3 and 4) and the CDRL1 to 3 sequences (described in SEQ ID NOs: 232, 234 and 7) are preferably maintained.

[0146] In a more preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 202; and a VL comprising the amino acid sequence set forth in SEQ ID NO: 208.

[0147] In a further preferred embodiment, the antibody or antigen-binding fragment thereof of the invention has at least 70% identity (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% identity) to SEQ ID NO: 202, preferably at least 80% identity (e.g., 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% identity), more preferably at least 90% identity (e.g., 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: 209; and a VL having at least 70% identity (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% identity), preferably at least 80% identity (e.g., 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% identity), more preferably at least 90% identity (e.g., 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: 209. Here, the CDRH1 to 3 sequences (described in SEQ ID NOs: 2, 3 and 4) and the CDRL1 to 3 sequences (described in SEQ ID NOs: 233, 234 and 7) are preferably maintained.

[0148] In a more preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 202; and a VL comprising the amino acid sequence set forth in SEQ ID NO: 209.

[0149] In some embodiments of the humanized (ASC_AB002) antibody, the light chain CDRL2 (GASTLAS) may be humanized (GASSLAS) (see Figure 23, graft ID Ab002_VLhum6). In some embodiments, a mutation (F→Y) is introduced in the framework region FR2 (see Figure 23, graft IDs Ab002_VLhum11, VLhum12). In some embodiments, an additional mutation (G→A) is introduced in CDRL2 (see Figure 23, graft IDs Ab001_VLhum11, VLhum12). In some embodiments, the light chain CDRL1 (QSSQNVYSNNHLS) may be humanized (RASQNVYSNNHLS) (see Figure 23, graft ID Ab002_VLhum12). In certain embodiments, one or more of the above mutations are combined (see Figure 23, graft IDs Ab002_VLhum6, VLhum11, VLhum12). In a preferred embodiment, all of the above mutations are present in the VL of the antibody or antigen-binding fragment thereof (see Figure 23, graft ID Ab002_VLhum12).

[0150] Thus, in certain embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises a CDRH1 set forth in SEQ ID NO: 2, a CDRH2 set forth in SEQ ID NO: 10, a CDRH3 set forth in SEQ ID NO: 11, a CDRL1 set forth in SEQ ID NO: 12 or SEQ ID NO: 235, a CDRL2 set forth in SEQ ID NO: 13, SEQ ID NO: 236 or SEQ ID NO: 237, and a CDRL3 set forth in SEQ ID NO: 14.

[0151] In a further specific embodiment, the antibody or antigen-binding fragment thereof of the invention comprises a CDRH1 set forth in SEQ ID NO: 2, a CDRH2 set forth in SEQ ID NO: 10, a CDRH3 set forth in SEQ ID NO: 11, a CDRL1 set forth in SEQ ID NO: 12, a CDRL2 set forth in SEQ ID NO: 236, and a CDRL3 set forth in SEQ ID NO: 14.

[0152] In a further specific embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises a CDRH1 set forth in SEQ ID NO: 2, a CDRH2 set forth in SEQ ID NO: 10, a CDRH3 set forth in SEQ ID NO: 11, a CDRL1 set forth in SEQ ID NO: 12, a CDRL2 set forth in SEQ ID NO: 237, and a CDRL3 set forth in SEQ ID NO: 14.

[0153] In a preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises a CDRH1 set forth in SEQ ID NO: 2, a CDRH2 set forth in SEQ ID NO: 10, a CDRH3 set forth in SEQ ID NO: 11, a CDRL1 set forth in SEQ ID NO: 235, a CDRL2 set forth in SEQ ID NO: 237, and a CDRL3 set forth in SEQ ID NO: 14.

[0154] In certain embodiments, an antibody or antigen-binding fragment thereof of the invention comprises a VH having at least 70% identity (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% identity), preferably at least 80% identity (e.g., 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% identity), and more preferably at least 90% identity (e.g., 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 the sequence set forth in SEQ ID NO: 210; and The VLs include those having at least 70% identity (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% identity) to the sequences set forth in any one of SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, and SEQ ID NO:214, preferably at least 80% identity (e.g., 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% identity), and more preferably at least 90% identity (e.g., 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). Here, the CDRH1 to 3 sequences (described in SEQ ID NOs: 2, 10, and 11, respectively) and CDRL1 to 3 sequences (described in SEQ ID NOs: 12 / 235; SEQ ID NOs: 13 / 236 / 237; and SEQ ID NO: 14, respectively) are preferably maintained.

[0155] Thus, in certain embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 210; and a VL comprising the amino acid sequence set forth in any one of SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, and SEQ ID NO: 214.

[0156] In a preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention has at least 70% identity (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% identity) to SEQ ID NO: 210, preferably at least 80% identity (e.g., 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% identity), more preferably at least 90% identity (e.g., 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). 9% identity) to SEQ ID NO: 214; and a VL having at least 70% identity (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% identity), preferably at least 80% identity (e.g., 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% identity), more preferably at least 90% identity (e.g., 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: 214. Here, the CDRH1 to 3 sequences (set forth in SEQ ID NOs: 2, 10, and 11, respectively) and the CDRL1 to 3 sequences (set forth in SEQ ID NOs: 235, 237, and 14, respectively) are preferably maintained.

[0157] Therefore, in a more preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 210; and a VL comprising the amino acid sequence set forth in SEQ ID NO: 214.

[0158] The antibody or antigen-binding fragment thereof of the present invention may comprise an Fc portion, which may be of any origin, preferably the Fc portion is of human origin, such as human IgA or IgG, e.g., IgG1, IgG2, IgG3, and / or IgG4, e.g., human IgG1.

[0159] As used herein, the term "Fc portion" refers to a sequence derived from a portion of an immunoglobulin heavy chain beginning with the hinge region immediately upstream of the papain cleavage site (e.g., residue 216 of native IgG, with the first residue of the heavy chain constant region being 114) and ending at the C-terminus of the immunoglobulin heavy chain. Thus, the Fc portion may be the entire Fc portion or a portion thereof (e.g., a domain). A complete Fc portion includes at least the hinge domain, CH2 domain, and CH3 domain (e.g., amino acids 216-446 of EU). An additional lysine residue (K) may be present at the extreme C-terminus of the Fc portion, but is often cleaved from mature antibodies.

[0160] In some embodiments, in the context of the present invention, an Fc portion comprises at least one of a hinge (e.g., upper, middle, and / or lower hinge region), CH2 domain, CH3 domain, or a variant, portion, or fragment thereof. The Fc portion may comprise at least a hinge domain, a CH2 domain, or a CH3 domain. The Fc portion may be a complete Fc portion. The Fc portion may also comprise an insertion, deletion, or substitution of one or more amino acids relative to a naturally occurring Fc portion. For example, at least one of the hinge domain, the CH2 domain, or the CH3 domain (or a portion thereof) may be deleted.

[0161] In some embodiments, an antibody or antigen-binding fragment thereof of the present invention comprises an Fc region. As used herein, the term "Fc region" refers to the portion of an immunoglobulin formed by two or more Fc portions of antibody heavy chains. For example, the Fc region may be a monomeric or "single-chain" Fc region (i.e., an scFc region). A single-chain Fc region is composed of Fc portions linked in a single polypeptide chain (e.g., encoded by a single contiguous nucleic acid sequence). Exemplary scFc regions are disclosed in WO 2008 / 143954 A2. Preferably, the Fc region is dimeric. A "dimeric Fc region" or "dcFc" refers to a dimer formed by the Fc portions of two separate immunoglobulin heavy chains. A dimeric Fc region may be a homodimer of two identical Fc portions (e.g., Fc regions of naturally occurring immunoglobulins) or a heterodimer of two non-identical Fc portions.

[0162] In some embodiments, the Fc portion or Fc region comprises or consists of an amino acid sequence derived from a human immunoglobulin sequence (e.g., derived from an Fc region or Fc portion from a human IgG molecule). However, the Fc portion or Fc region may also comprise one or more amino acids derived from other mammalian species. For example, a primate Fc portion or primate binding site may be included in the antibody or antigen-binding fragment. Alternatively, one or more murine amino acids may be present in the Fc portion or Fc region.

[0163] The Fc portions of the Fc region may be of the same or different class and / or subclass, for example, the Fc portion may be derived from an immunoglobulin (e.g., a human immunoglobulin) of the IgG1, IgG2, IgG3, or IgG4 subclass.

[0164] Those skilled in the art will understand that an Fc portion or Fc region may be engineered such that it differs in amino acid sequence from the complete Fc portion or Fc region of a naturally occurring immunoglobulin molecule. In some embodiments, the engineered Fc portion or Fc region retains at least one desirable function imparted by a naturally occurring Fc portion. Fc functions that may be altered or retained by Fc engineering include Fc receptor (FcR) binding, antibody half-life modulation, ADCC function, protein A binding, protein G binding, and complement fixation. The portions of naturally occurring Fc portions or Fc regions responsible for such functions and / or essential portions are well known to those skilled in the art. Various Fc modifications are known in the art and are 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.

[0165] In some embodiments, an antibody according to the invention comprises an (intact) Fc moiety / Fc region. The (intact) Fc moiety / Fc region may be engineered by one or more specific amino acid substitutions. In other embodiments, it may not be engineered (i.e., it may be present in a naturally occurring immunoglobulin, e.g., IgG1 or IgG4).

[0166] Preferably, the Fc portion / Fc region is engineered (e.g., based on a human IgG, such as IgG1 or IgG4, Fc portion / Fc region) to reduce Fc effector function, in particular to reduce or inhibit FcγR and complement interactions. It has been demonstrated that autoantibodies against ASCs opsonize ASC specks (via their Fc region), thereby increasing 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 AG, 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. Nat Immunol. 2014 Aug;15(8):727-37. doi: 10.1038 / ni.2913). Various Fc modifications for reducing or inhibiting FcγR and complement interactions are known in the art and may be useful for avoiding opsonization of anti-ASC antibodies. Specifically, Table 3 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 (incorporated herein by reference) provides examples of Fc modifications for silencing antibody effector functions. Such Fc modifications may be included in the antibodies of the present invention.Therefore, the antibody of the present invention has the following properties: Saunders KO. Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life. Front Immunol. 2019 Jun 7; 10:1296. doi: Leu235Glu, Leu234Ala / Leu235Ala, Ser228Pro / Leu235Glu, Gly237Ala, Glu318Ala, Leu234Ala / Leu235Ala / Pro329Gly, Pro331Ser / Leu234Glu / Leu235Phe, Asp265Ala, Glu233Pro, Gly236Arg / Leu328Arg, IgG2-IgG4 cross-subclass, Ala330Leu, His268Gln / Val309Leu / Ala330Ser / Pro331Ser, Val23 The antibody may comprise an Fc modification selected from 4Ala / Gly237Ala / Pro238Ser / His268Ala / Val309Leu / Ala330Ser / Pro331Ser, Asp270Ala, Leu234Ala / L235Ala / Gly237Ala / P238Ser / His268Ala / Ala330Ser / Pro331Ser, Lys322Ala, Pro329Ala, Pro331Ala, IgG2-IgG3 cross-subclass, high mannose glycosylation, Val264Ala, Phe241Ala, Asn297Ala or Gly or Gln, and S228P / Phe234Ala / Leu235Ala.

[0167] Among such Fc modifications that reduce effector function, the "LALA" mutation is preferred. The "LALA" mutation refers to two substitutions, L234A and L235A (EU numbering; also known as CH2 L4A and CH2 L5A mutations). The "LALA" mutation abolishes antibody binding to FcγRI, FcγRII, and FcγRIIIa (Lund J, Winter G, Jones PT, Pound JD, Tanaka T, Walker MR et al. Human Fc gamma RI and Fc gamma RII interact with distinct but overlapping sites on human IgG. J Immunol. 1991; 147:2657-2662). An exemplary amino acid sequence of CH1-CH2-CH3 containing the "LALA" mutation is set forth in SEQ ID NO: 141. Therefore, the antibody preferably comprises the amino acid sequence set forth in SEQ ID NO: 141, or a sequence variant thereof described herein, wherein the "LALA" mutation is maintained.

[0168] In some embodiments, the "LALA" mutation may be combined with additional mutations, such as P329G or G236R (EU numbering). In other embodiments, the antibody (Fc portion / Fc region) comprises the "LALA" mutations (L234A and L235A), while P329 and / or G236 are maintained (i.e., not mutated).

[0169] In general, antibody binding to Fc receptors can be assessed by various methods known to those skilled in the art, such as ELISA (Hessell AJ, Hangartner L, Hunter M, Havenith CEG, Beurskens FJ, Bakker JM, Lanigan CMS, Landucci G, Forthal DN, Parren PWHI, et al.: Fc receptor but not complement binding is important in antibody protection against HIV. Nature 2007, 449:101-104; Grevys A, Bern M, Foss S, Bratlie DB, Moen A, Gunnarsen KS, Aase A, Michaelsen TE, Sandlie I, Andersen JT: Fc Engineering of Human IgG1 for Altered Binding to the Neonatal Fc Receptor Affects Fc Effector Functions. 2015, 194:5497-5508), or flow cytometry (Perez LG, Costa MR, Todd CA, Haynes BF, Montefiori DC: Utilization of immunoglobulin G Fc receptors by human immunodeficiency virus type 1: a specific role for antibodies against the membrane-proximal external region of gp41. J Virol 2009, 83:7397-7410; Piccoli L, Campo I, Fregni CS, Rodriguez BMF, Minola A, Sallusto F, Luisetti M, Corti D, Lanzavecchia A: Neutralization and clearance of GM-CSF by autoantibodies in pulmonary alveolar proteinosis.(Nat Commun 2015, 6:1-9).

[0170] Antibodies of the invention may be of any isotype (e.g., IgA, IgG, IgM, i.e., α, γ, or μ heavy chains). Preferably, antibodies may be of the IgA or IgG type. Within the IgG isotype, antibodies may be of the IgG1, IgG2, IgG3, or IgG4 subclass, preferably IgG1 or IgG4. Exemplary sequences of IgG1 and IgG4 constant regions that may be useful in the antibodies described herein are provided in SEQ ID NO: 70 (IgG1) and SEQ ID NO: 142 (IgG4). Thus, antibodies of the invention may comprise the amino acid sequence set forth in SEQ ID NO: 70 or SEQ ID NO: 142, or sequence variants thereof described herein. The human IgG4 constant region sequence of SEQ ID NO: 142 contains the stabilizing hinge mutation S228P (S. Angal, DJ King, MW Bodmer, A. Turner, ADG Lawson, G. Roberts, B. Pedley, JR Adair, A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody, Molecular Immunology, Volume 30, Issue 1, 1993, Pages 105-108, ISSN 0161-5890, https: / / doi.org / 10.1016 / 0161-5890(93)90432-B).

[0171] Antibodies of the invention may have a κ light chain or a λ light chain. Exemplary sequences of κ and λ light chain constant regions that may be useful in the antibodies described herein are provided in SEQ ID NO: 71 (Ckappa) and SEQ ID NO: 72 (Clambda). Thus, antibodies of the invention may comprise the amino acid sequence set forth in SEQ ID NO: 71 or 72, or sequence variants thereof described herein. Preferably, antibodies of the invention have a κ light chain, e.g., a constant region comprising or consisting of SEQ ID NO: 71.

[0172] As noted above, the present invention encompasses antigen-binding fragments. Antigen-binding fragments may or may not include a portion of the Fc portion, particularly the complete Fc region. In some embodiments, the antibody or antigen-binding fragment thereof is selected from Fab, Fab', F(ab')2, Fv, or scFv. For example, F(ab')2 (which may be obtained by pepsin cleavage or recombinant expression) and Fab' (which may be obtained from F(ab')2 or by recombinant expression) typically include the hinge region.

[0173] In some embodiments, the antibody or antigen-binding fragment may be a single-chain antibody (or fragment). A single-chain antibody (or fragment) may encode a complete set of six CDRs, i.e., three heavy chain CDRs and three light chain CDRs. More particularly, the single-chain antibody (or fragment) may comprise a heavy chain variable region (VH) and a light chain variable region (VL), for example, comprising the VH and VL sequences described above.

[0174] Mutant antibodies are also within the scope of the present invention. Accordingly, variants of the sequences described herein are also within the scope of the present invention. Such variants include naturally occurring variants that arise from in vivo somatic mutation during an immune response or from in vitro somatic mutation associated with the cultivation of immortalized B-cell clones. Alternatively, variants may arise due to the degeneracy of the genetic code or may be generated by transcription or translation errors.

[0175] The antibodies or antigen-binding fragments thereof of the present invention may be provided in purified form. Typically, the antibodies or antigen-binding fragments will be present in a composition that is substantially free of other polypeptides, e.g., less than 90% (by weight) of the composition being made up of other polypeptides, generally less than 60%, and more generally less than 50%.

[0176] The antibodies of the invention may be immunogenic in a non-human (or heterologous) host, such as a mouse. In particular, the antibody may have an idiodide that is immunogenic in a non-human host but not in a human host. In particular, antibodies of the invention for use in humans include those that cannot be readily isolated from hosts such as mice, goats, rabbits, rats, non-primate mammals, and generally cannot be obtained by humanization or from xenomurine.

[0177] The antibodies of the present invention also include hybrid antibody molecules comprising six CDRs from an antibody of the present invention as defined above and one or more CDRs from another antibody against the antigen. For example, the antibody may be multispecific. A multispecific antibody may comprise a first antigen-binding site according to the present invention (which binds to the PYD of the ASC, particularly the ASC described herein) and a second antigen-binding site that is different from the first antigen-binding site, i.e., may bind to a different target. In other embodiments, the antibody or antigen-binding fragment thereof may be monospecific.

[0178] [Nucleic acid] In another aspect, the present invention also provides a nucleic acid molecule comprising a polynucleotide encoding the above-described antibody or antigen-binding fragment thereof according to the present invention.

[0179] In some embodiments, nucleic acid molecules comprise one or more polynucleotides encoding exemplary antibodies of the invention (e.g., those described above, particularly in Table 1), or sequence variants thereof described herein (e.g., those 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).

[0180] Exemplary nucleic acid sequences encoding the CDR and VH / VL sequences of the exemplary antibodies described herein are shown in Table 4 below.

[0181] [Table 4]

[0182] Examples of nucleic acid molecules and / or polynucleotides include, for example, recombinant polynucleotides, vectors, oligonucleotides, RNA molecules such as rRNA, mRNA, miRNA, siRNA, or tRNA, or DNA molecules such as cDNA. The nucleic acid may encode the light chain and / or the heavy chain of an antibody (or a single-chain antibody). In other words, the light and heavy chains of an antibody may be encoded by the same nucleic acid molecule (e.g., in the case of a single-chain antibody, or an antibody having separate heavy and light chains in a bicistronic format, or in the case of an expression cassette containing multiple ribosome entry sites such as an IRES). Alternatively, the light and heavy chains of an antibody may be encoded by separate nucleic acid molecules.

[0183] Due to redundancy in the genetic code, the present invention also encompasses sequence variants of nucleic acid sequences that encode the same amino acid sequence. A polynucleotide (or complete nucleic acid molecule) encoding an antibody can be optimized for antibody expression. For example, codon optimization of the nucleotide sequence can be used to improve translation efficiency in an expression system for antibody production. Furthermore, a nucleic acid molecule can contain heterologous elements (i.e., elements that are not naturally present on the same nucleic acid molecule as the antibody (heavy or light chain) coding sequence). For example, a nucleic acid molecule can contain a heterologous promoter, a heterologous enhancer, a heterologous UTR (e.g., for optimal translation / expression), a heterologous poly(A) tail, a heterologous DNA insulator element, etc.

[0184] A nucleic acid molecule is a molecule that contains a nucleic acid component. The term nucleic acid molecule typically refers to a DNA molecule or an RNA molecule. The term "polynucleotide" is sometimes used synonymously with nucleic acid molecule; that is, a nucleic acid molecule may consist of a polynucleotide that encodes an antibody. Alternatively, a nucleic acid molecule may contain additional elements in addition to a polynucleotide that encodes an antibody. Typically, a nucleic acid molecule is a polymer that contains or consists of nucleotide monomers covalently linked to each other by sugar / phosphate backbone phosphodiester bonds. The term "nucleic acid molecule" encompasses modified nucleic acid molecules, such as DNA molecules or RNA molecules with base modifications, sugar modifications, or backbone modifications.

[0185] Generally, nucleic acid molecules can be engineered to insert, delete, or modify specific nucleic acid sequences. Such engineered modifications include, but are not limited to, modifications that introduce restriction sites, modifications that modify codon usage, and modifications that add or optimize transcriptional and / or translational regulatory sequences. Nucleic acids can also be altered to modify 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 amino acid sequence of an antibody. Such point mutations can modify effector function, antigen-binding affinity, post-translational modification, immunogenicity, etc., and can introduce amino acids for attaching covalent groups (e.g., labels) or introduce tags (e.g., for purification purposes). Alternatively, mutations in nucleic acid sequences can be "silent," i.e., not reflected in the amino acid sequence due to redundancy in the genetic code. Generally, mutations can be introduced at specific sites or randomly, followed by selection (e.g., molecular evolution). For example, one or more nucleic acids encoding either the light or heavy chain of an (exemplary) antibody may be randomly or directionally mutated to introduce different characteristics into the encoded amino acids. Such changes may be the result of an iterative process in which initial changes are retained and new changes are introduced at other nucleotide positions. Furthermore, changes achieved in independent steps may be combined.

[0186] In some embodiments, a polynucleotide (or (complete) nucleic acid molecule) encoding an antibody or antigen-binding fragment thereof may be codon-optimized. Various tools for codon optimization are known to those skilled in the art and are described, for example, in: Ju Xin Chin, Bevan Kai-Sheng Chung, Dong-Yup Lee, Codon Optimization OnLine (COOL): a web-based multi-objective optimization platform for synthetic gene design, Bioinformatics, Volume 30, Issue 15, 1 August 2014, Pages 2210-2212; or Grote A, Hiller K, Scheer M, Munch R, Nortemann B, Hempel DC, Jahn D, JCat: a novel tool to adapt codon usage of a target gene to its potential expression host. Nucleic Acids Res. 2005 Jul 1; 33(Web Server issue):W526-31; or, for example, OptimumGene from Genscript. TM Algorithm (described in U.S. Patent Application Publication No. 2011 / 0081708 A1).

[0187] For example, a nucleic acid molecule of the invention can comprise any one of the nucleic acid sequences set forth in SEQ ID NOs: 73-140, or sequence variants 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, such that the nucleic acid molecule can encode any one of exemplary antibodies ASC_Ab001, ASC_Ab002, ASC_Ab003, ASC_Ab004, ASC_Ab005, ASC_Ab006, ASC_Ab007, ASC_Ab008, and ASC_Ab009 (in combination with the sequences shown in Table 2), or sequence variants thereof described herein.

[0188] In certain embodiments, the nucleic acid molecule of the present invention may comprise any one of the nucleic acid sequences set forth in SEQ ID NOs: 215-229, or sequence variants 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, thereby encoding any one of the exemplary humanized antibodies ASC_Ab001_hum (RP02-F8) or ASC_Ab002_hum (RP03-C12).

[0189] The present invention also provides a plurality of nucleic acid molecules encoding an antibody or antigen-binding fragment thereof 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 antigen-binding fragment thereof. The plurality of nucleic acid molecules thereby collectively encodes (all of the immunoglobulin chains of) the antibody or antigen-binding fragment thereof described herein. In some embodiments, the plurality of nucleic acid molecules encoding the antibody or antigen-binding fragment thereof described herein may be a combination of a first nucleic acid molecule and a second nucleic acid molecule, wherein the first nucleic acid molecule comprises a polynucleotide encoding a heavy chain of an antibody or antigen-binding fragment thereof of the 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.

[0190] In general, the above descriptions of (general) features of nucleic acid molecules of the invention also apply, as appropriate, to nucleic acid molecules of a plurality of nucleic acid molecules. Thus, one or more of the polynucleotides encoding the immunoglobulin chains of an antibody or antigen-binding fragment thereof may be codon-optimized. For example, the plurality may comprise a nucleic acid sequence 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. Thus, the plurality of nucleic acid molecules may encode any one of exemplary antibodies ASC_Ab001, ASC_Ab002, ASC_Ab003, ASC_Ab004, ASC_Ab005, ASC_Ab006, ASC_Ab007, ASC_Ab008, and ASC_Ab009 (by combining the sequences shown in Table 2), or sequence variants thereof as described herein.

[0191] In certain embodiments, the plurality of the present invention may comprise a nucleic acid sequence set forth in any one of SEQ ID NOs: 215-229, 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, such that the nucleic acid molecule may encode any one of exemplary humanized antibodies ASC_Ab001_hum (RP02-F8) or ASC_Ab002_hum (RP03-C12).

[0192] 〔vector〕 Also included within the scope of the present invention is a vector, such as an expression vector, comprising a nucleic acid molecule according to the invention or a plurality of nucleic acid molecules according to the invention. Generally, the vector comprises a nucleic acid molecule as described above.

[0193] The present invention also provides a plurality of vectors comprising the plurality of nucleic acid molecules of the present invention. Thus, each vector of the plurality of vectors may comprise one or more of the plurality of nucleic acid molecules of the present invention. In some embodiments, the plurality of vectors may be a combination of a first vector and a second vector, wherein the first vector (for a combination of nucleic acid molecules) comprises the first nucleic acid molecule described above, and the second vector (for a combination of nucleic acid molecules) comprises the second nucleic acid molecule described above.

[0194] A vector is typically a recombinant nucleic acid molecule, i.e., a nucleic acid molecule that does not occur in nature. Thus, a vector may contain heterologous elements (i.e., sequence elements of a different origin than in nature). For example, a vector may contain a multiple cloning site, a heterologous promoter, a heterologous enhancer, a heterologous selection marker (for identifying cells containing the vector compared to cells not containing the vector), a heterologous origin of replication, a heterologous DNA insulator element, etc. A vector in the context of the present invention is suitable for incorporating or carrying a desired nucleic acid sequence. Such vectors may be storage vectors, expression vectors, cloning vectors, transfer vectors, etc. Storage vectors are vectors that allow for convenient storage of nucleic acid molecules. Thus, a vector may contain, for example, sequences corresponding to a desired antibody (heavy and / or light chain) of the present invention. Expression vectors can be used to produce expression products such as RNA, e.g., mRNA, or peptides, polypeptides, or proteins. For example, an expression vector may contain sequences necessary for transcription of sequence extensions of the vector, such as (heterologous) promoter sequences. A cloning vector is typically a vector that contains a cloning site, which can be used to incorporate a nucleic acid sequence into the vector. A cloning vector may be, for example, a plasmid vector or a bacteriophage vector. A transfer vector may be a vector suitable for transferring a nucleic acid molecule into a cell or organism, such as a viral vector. A vector in the context of the present invention may be, for example, an RNA vector or a DNA vector. For example, a vector in the sense of the present application comprises a cloning site, a selection marker such as an antibiotic resistance factor, and sequences suitable for amplifying the vector, such as an origin of replication. A vector in the sense of the present application may be a plasmid vector.

[0195] As used herein, the term "vector" may refer to, for example, a delivery vector for viral or nonviral delivery of a nucleic acid of the present invention. Alternatively, the term "vector" may refer to a viral or nonviral delivery system. Accordingly, the present invention also provides a delivery vector / system comprising the above-described nucleic acid molecule (or comprising the above-described expression vector). The delivery vector / system may be viral or nonviral. Various examples of viral and nonviral delivery vectors / systems are known in the art and are 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-dependent adenoviral vectors and hybrid adenoviral vectors; herpes simplex viral vectors; lentiviral vectors; poxvirus vectors; and Epstein-Barr viral vectors. Among viral vectors, adenoviral vectors and adeno-associated viral (AAV) vectors are preferred. 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 cell membranes by mechanical, electrical, ultrasonic, hydrodynamic, or laser-based energy; naked DNA or RNA delivery; gene guns; hydrodynamic delivery; ultrasonic delivery; and magnetofection. Chemical non-viral delivery systems include cationic particles, particularly cationic lipids / liposomes, cationic polymers, and lipid / polymer systems. Among non-viral vectors / systems, cationic liposomes are preferred.

[0196] 〔cell〕 In a further aspect, the present invention also provides a (host) cell expressing an antibody or antigen-binding fragment thereof according to the invention; and / or comprising a vector(s) according to the invention. The (host) cell may be an isolated cell that is not part of the human or animal body, e.g., a cell line or an artificial cell. The cell may recombinantly, e.g., heterologously, express the nucleic acid(s) or vector(s) of the invention (i.e., the cell / cell type does not naturally express the antibody or antigen-binding fragment).

[0197] Examples of such cells include, but are not limited to, eukaryotic cells, such as yeast cells, animal cells, or plant cells. Other examples of such cells include, but are not limited to, prokaryotic cells, such as E. coli. In some embodiments, the cells are mammalian cells, such as mammalian cell lines. Examples include human cells, CHO cells, HEK293 cells, PER.C6 cells, NS0 cells, human hepatocytes, myeloma cells, or hybridoma cells.

[0198] The cell may be transfected with a vector, e.g., an expression vector, according to the invention. The term "transfection" refers to the introduction of a nucleic acid molecule, such as a DNA or RNA (e.g., mRNA) molecule, into a cell, e.g., a eukaryotic or prokaryotic cell. In the context of the present invention, the term "transfection" encompasses any method known to those skilled in the art for introducing a nucleic acid molecule into a cell, e.g., a mammalian cell. Such methods include, for example, electroporation, lipofection, e.g., based on cationic lipids and / or liposomes, calcium phosphate precipitation, nanoparticle-based transfection, viral-based transfection, or transfection based on cationic polymers, e.g., DEAE-dextran or polyethyleneimine. In some embodiments, the introduction is non-viral.

[0199] Furthermore, cells of the invention may be stably or transiently transfected with vectors of the invention, e.g., to express antibodies of the invention. In some embodiments, cells are stably transfected with vectors of the invention encoding antibodies of the invention. In other embodiments, cells are transiently transfected with vectors of the invention encoding antibodies of the invention.

[0200] Accordingly, the present invention also provides recombinant host cells heterologously expressing an antibody or antigen-binding fragment thereof of the present invention. For example, the cell may be of a species other than the antibody (e.g., a CHO cell expressing a human antibody). In some embodiments, the cell type does not naturally express the antibody. Furthermore, the host cell may impart a post-translational modification (PTM; e.g., glycosylation) to the antibody that is not naturally present. Such PTMs may result in functional differences (e.g., reduced immunogenicity). Thus, the antibody or antigen-binding fragment thereof of the present invention may have post-translational modifications that differ from those of naturally produced antibodies (e.g., antibodies of the human immune response).

[0201] [Antibody production] The antibodies of the present invention can be produced by any method known in the art, for example, the general methodology for producing monoclonal antibodies using hybridoma technology is well known (Kohler, G. and Milstein, C., 1975; Kozbar et al. 1983).

[0202] Standard techniques of molecular biology can be used to prepare DNA sequences encoding the antibodies or antigen-binding fragments of the invention. For example, the desired DNA sequence can be synthesized in whole or in part using oligonucleotide synthesis techniques. Site-directed mutagenesis and polymerase chain reaction (PCR) techniques may also be used, as appropriate.

[0203] Any suitable host cell / vector system can be used to express DNA sequences encoding the antibody molecules of the present invention. Eukaryotic, e.g., mammalian, host cell expression systems can be used to produce antibody molecules, such as complete antibody molecules. Suitable mammalian host cells include, but are not limited to, CHO cells, HEK293 cells, PER.C6 cells, NS0 cells, myeloma cells, or hybridoma cells. Prokaryotic, e.g., bacterial, host cell expression systems can also be used to produce antibody molecules, such as complete antibody molecules. Suitable bacterial host cells include, but are not limited to, Escherichia coli (E. coli) cells.

[0204] Accordingly, the present invention provides a method for preparing an antibody or antigen-binding fragment thereof, or an immunoglobulin chain thereof according to the present invention, said method comprising: (i) culturing the host cell; and (ii) isolating the antibodies or immunoglobulin chains thereof from the culture; Includes.

[0205] In other words, the present invention also provides a method for producing an antibody molecule according to the invention, which method comprises culturing a (heterologous) host cell containing a vector encoding a nucleic acid of the invention under conditions suitable for the expression of a protein, in particular from DNA encoding an antibody molecule of the invention, and isolating the antibody molecule.

[0206] To produce antibodies containing both heavy and light chains, host cells, such as cell lines, can be transfected with two vectors, one encoding a light chain polypeptide and a second vector encoding a heavy chain polypeptide, e.g., as described above. Alternatively, a single vector can be used that contains sequences encoding both light and heavy chain polypeptides (e.g., for single-chain antibodies or in a bicistronic format).

[0207] Thus, the present invention also provides a method for preparing a recombinant cell, comprising the steps of: (i) providing one or more nucleic acids encoding an antibody of the present invention; (ii) inserting the nucleic acids into an expression vector; and (iii) transfecting the vector into a (heterologous) host cell to allow expression of the antibody of interest in the host cell. The nucleic acids of step (i) can be, but are not required to be, manipulated to introduce restriction sites, change codon usage, and / or optimize transcriptional and / or translational regulatory sequences.

[0208] Furthermore, the present invention provides methods for preparing transfected host cells, comprising the step of transfecting a host cell with one or more nucleic acids encoding an antibody of interest. In this manner, the steps of first preparing the nucleic acid(s) and then transfecting the host cell therewith can be carried out at different times, by different people, and in different places (e.g., different countries).

[0209] These recombinant cells of the present invention can be used for expression and culture purposes. The recombinant cells of the present invention are particularly useful for expressing antibodies for large-scale pharmaceutical production. The recombinant cells of the present invention can also be used as an active ingredient in pharmaceutical compositions. Any suitable culture technique can be used, including, but not limited to, static culture, roller bottle culture, ascites, hollow fiber bioreactor cartridges, modular minifermentors, stirred tanks, microcarrier culture, ceramic core perfusion, etc.

[0210] The transfected host cell can be a eukaryotic cell, including yeast cells and animal cells, particularly mammalian cells (e.g., CHO cells, NS0 cells, human cells such as PER.C6 cells, HEK293 cells, or HKB-11 cells, myeloma cells, or human hepatocytes), as well as plant cells. In some embodiments, the transfected host cell is a mammalian cell, such as a human cell. In some embodiments, the expression host is capable of glycosylation of the antibody of the invention with a carbohydrate structure that is not itself immunogenic, particularly in humans. In some embodiments, the transfected host cell is capable of growth in serum-free medium. In further embodiments, the transfected host cell is capable of growth in culture without the presence of animal-derived products. The transfected host cell can also be cultured to obtain a cell line.

[0211] The present invention also provides a method of preparing an antibody of interest, comprising the steps of culturing or subculturing a transfected host cell population, e.g., a stably transfected host cell population, under conditions in which the antibody of interest is expressed; and, optionally, purifying the antibody of interest. The transfected host cell population can be prepared by (i) providing nucleic acid encoding a selected antibody of interest, (ii) inserting the nucleic acid(s) into an expression vector, (iii) transfecting the vector into a host cell capable of expressing the antibody of interest, and (iv) culturing or subculturing the transfected host cells containing the inserted nucleic acid to produce the antibody of interest.

[0212] In some embodiments, antibodies of the invention can be produced by (i) expressing a nucleic acid sequence of the invention in a host cell, for example, by using a vector (or host cell) of the invention, and (ii) isolating the expressed antibody product. The method may further include (iii) purifying the isolated antibody.

[0213] Thus, after production, antibodies may be further purified, if desired, using filtration, centrifugation, and various chromatographic methods, such as HPLC or affinity chromatography. Techniques for purifying antibodies, e.g., monoclonal antibodies, are well known in the art, including techniques for producing pharmaceutical-grade antibodies.

[0214] Compositions and Kits The present invention also provides compositions comprising one or more of the following: (i) an antibody or antigen-binding fragment thereof of the present invention; (ii) a nucleic acid or nucleic acids of the invention; (iii) a vector or vectors of the invention; or (iv) A cell expressing an antibody according to the invention or comprising a vector according to the invention.

[0215] The composition can be used for therapeutic or diagnostic purposes. Thus, the composition may be a pharmaceutical composition or a diagnostic composition. The composition may comprise a (pharmaceutically acceptable) excipient, diluent or carrier.

[0216] Accordingly, the present invention also provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to the invention, a nucleic acid or nucleic acids according to the invention, a vector or vectors according to the invention, and / or a cell according to the invention. Preferably, the pharmaceutical composition comprises an antibody or antigen-binding fragment thereof according to the invention.

[0217] The pharmaceutical composition may also optionally include a pharmaceutically acceptable carrier, diluent, and / or excipient. The carrier or excipient may facilitate administration, but should not itself induce the production of antibodies harmful to the individual receiving the composition. Furthermore, none of them should 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. In some embodiments, the pharmaceutically acceptable carrier, diluent, and / or excipient in the pharmaceutical composition is not an active ingredient with respect to an ASC-associated disease or disorder. In some embodiments, the pharmaceutically acceptable carrier, diluent, and / or excipient in the pharmaceutical composition is not an active ingredient with respect to a neurodegenerative or neuroinflammatory disease, or a peripheral inflammatory disorder.

[0218] Pharmaceutically acceptable salts can be, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates and sulfates, or organic acid salts such as acetates, propionates, malonates and benzoates.

[0219] Pharmaceutically acceptable carriers in pharmaceutical compositions may further 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 a subject.

[0220] Pharmaceutical compositions can be prepared in a variety of forms. For example, the compositions can 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., Synagis TMand lyophilized compositions for reconstitution with sterile water containing a preservative, similar to Herceptin®). The compositions can be prepared for topical administration, for example, as an ointment, cream, or powder. The compositions can be prepared for oral administration, for example, as a tablet or capsule, as a spray, or as an (optionally flavored) syrup. The compositions can be prepared for pulmonary administration, for example, as an inhaler using a fine powder or spray. The compositions can be prepared as a suppository or pessary. The compositions can be prepared for nasal, otic, or ocular administration, for example, as drops. The compositions may be in the form of a kit designed so that the combined composition is reconstituted immediately prior to administration to a subject. For example, a lyophilized antibody may be provided in the form of a kit together with sterile water or a sterile buffer.

[0221] In some embodiments, the (only) active ingredient in the composition is an antibody or antigen-binding fragment thereof of the present invention. As such, the active ingredient may be susceptible to degradation in the digestive tract. Therefore, when the composition is administered via a route that uses the digestive tract, the composition may contain a drug that protects the antibody from degradation but releases the antibody once it is absorbed from the digestive tract.

[0222] A thorough discussion of pharmaceutically acceptable carriers can be found in Gennaro (2000) Remington: The Science and Practice of Pharmacy, 20th edition, ISBN: 0683306472.

[0223] The present invention also provides a method for preparing a pharmaceutical composition, comprising the steps of: (i) preparing an antibody of the present invention; and (ii) admixing the purified antibody with one or more pharmaceutically acceptable excipients, diluents, or carriers.

[0224] In other embodiments, methods for preparing a pharmaceutical composition comprise admixing an antibody with one or more pharmaceutically acceptable carriers, wherein the antibody is a monoclonal antibody.

[0225] Pharmaceutical compositions generally have a pH of 5.5 to 8.5, and in some embodiments, the pH may be 6 to 8, for example, about 7. The pH may be maintained using a buffer. The composition may be sterile and / or pyrogen-free. The composition may be isotonic for humans. In some embodiments, the pharmaceutical composition is supplied in a sealed container.

[0226] The scope of the present invention includes compositions present in several dosage forms, including, but not limited to, forms suitable for parenteral administration, e.g., by injection or infusion, e.g., by bolus injection or continuous infusion. When the product is for injection or infusion, it can take the form of a suspension, solution, or emulsion in an oily or aqueous vehicle, and the product can contain formulatory agents such as suspending agents, preservatives, stabilizers, and / or dispersing agents. Alternatively, the antibody can be in a dry form for reconstitution with an appropriate sterile liquid before use.

[0227] A vehicle is typically understood to be a material suitable for storing, transporting, and / or administering a pharmaceutically active compound, particularly a compound such as an antibody described herein. For example, the vehicle may be a physiologically acceptable liquid suitable for storing, transporting, and / or administering a pharmaceutically active compound, particularly an antibody described herein. Once formulated, the composition can be administered directly to a subject. In some embodiments, the composition is adapted for administration to a mammal, e.g., a human subject.

[0228] Pharmaceutical compositions may also contain antibacterial agents, especially when packaged in multi-dose formats. Pharmaceutical compositions may also contain surfactants, such as Tween (polysorbate), e.g., Tween 80. Surfactants are generally present at low levels, e.g., less than 0.01%. Compositions may also contain sodium salts (e.g., sodium chloride) to impart tonicity. For example, an NaCl concentration of 10±2 mg / ml is typical.

[0229] Additionally, pharmaceutical compositions, particularly when lyophilized or containing reconstituted lyophilized material, may contain a sugar alcohol (e.g., mannitol) or a disaccharide (e.g., sucrose or trehalose), e.g., about 15-30 mg / ml (e.g., 25 mg / ml). The pH of the lyophilized composition may be adjusted to 5-8, or 5.5-7, or about 6.1 prior to lyophilization.

[0230] Pharmaceutical compositions can be administered by any number of routes, including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intraperitoneal, intrathecal, intraventricular, transdermal, transcutaneous, topical, subcutaneous, nasal, enteral, sublingual, vaginal, or rectal. Optionally, pharmaceutical compositions can be formulated for oral administration, e.g., as tablets, capsules, etc., for topical administration, or as an injectable preparation, e.g., as a liquid solution or suspension. In some embodiments, the pharmaceutical composition is an injectable preparation. Solid forms suitable for solution or suspension in a liquid vehicle prior to injection are also encompassed; for example, the pharmaceutical composition may be in a lyophilized form.

[0231] For injection, e.g., intravenous, cutaneous, or subcutaneous injection, or injection into an affected area, the active ingredient may be in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has appropriate pH, isotonicity, and stability. Those skilled in the art can prepare appropriate solutions using isotonic vehicles, such as sodium chloride injection, Ringer's injection, lactated Ringer's injection, etc. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as needed. Whether an antibody, peptide, nucleic acid molecule, or another pharmaceutically useful compound is administered to an individual, the administration is typically in an "effective amount," e.g., a "prophylactically effective amount" or a "therapeutically effective amount" (as the case may be), sufficient to demonstrate benefit to the individual. The actual amount administered, as well as the rate and time-course of administration, will depend on the nature and severity of what is being treated. For injection, the pharmaceutical composition may be provided, for example, in a prefilled syringe.

[0232] The pharmaceutical composition can also be orally administered in any orally acceptable dosage form, including, but not limited to, capsules, tablets, aqueous suspensions, or solutions. For tablets for oral use, commonly used carriers include lactose and corn starch. Lubricants such as magnesium stearate are also typically added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. When an aqueous suspension is required for oral use, the active ingredient, i.e., the antibody defined above, is combined with an emulsifying and suspending agent. If desired, certain sweeteners, flavorings, or coloring agents can also be added.

[0233] Pharmaceutical compositions can also be administered topically, particularly when the area or organ to be treated includes an area or organ easily accessible by topical application, such as accessible epithelial tissue. Suitable topical formulations are readily prepared for each of these areas or organs. For topical application, the pharmaceutical composition can be formulated into 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 paraffin, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying wax, and water. Alternatively, the pharmaceutical composition can be formulated into 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.

[0234] The prescription therapy may be a single-dose schedule or a multiple-dose schedule. For a single administration, such as daily, weekly, or monthly administration, the amount of antibody in the pharmaceutical composition may not exceed 1 g or 500 mg. In some embodiments, for a single administration, the amount of antibody in the pharmaceutical composition may not exceed 200 mg or 100 mg. For example, for a single administration, the amount of antibody in the pharmaceutical composition may not exceed 50 mg.

[0235] In some embodiments, a composition can comprise an antibody of the invention, wherein the antibody comprises 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 antibody in the composition can be in purified form.

[0236] As an alternative to delivering an antibody for therapeutic purposes, a nucleic acid (typically DNA) encoding the monoclonal antibody of interest can be delivered to the subject, where it is expressed in situ in the subject to produce the desired therapeutic effect. Suitable gene therapy and nucleic acid transfer vectors are known in the art.

[0237] Pharmaceutical compositions typically contain an "effective" amount of one or more antibodies described herein, i.e., an amount sufficient to treat, ameliorate, attenuate, reduce, or prevent the desired disease or condition, or to exhibit a detectable therapeutic effect. A therapeutic effect also includes a reduction or attenuation of pathogenic potency or physical symptoms. The precise effective amount for any particular subject will depend on the subject's size, weight, and health, the nature and extent of the condition, and the therapeutic agent or combination of therapeutic agents selected for administration. The effective amount for a given situation will be determined by routine experimentation and is within the judgment of the clinician. An effective amount generally may be about 0.005 to about 100 mg / kg, e.g., about 0.0075 to about 50 mg / kg, or about 0.01 to about 10 mg / kg. In some embodiments, an effective amount may be about 0.02 to about 5 mg / kg of antibody (e.g., the amount of antibody in the pharmaceutical composition) relative to the body weight (e.g., in kilograms) of the individual to whom the antibody is administered.

[0238] In some embodiments, the pharmaceutical composition may also comprise an additional active ingredient, which may be a further antibody or a non-antibody component, hi other embodiments, the pharmaceutical composition may not comprise an additional active ingredient (in addition to the antibody, or respective nucleic acid, vector, or cell of the invention described above).

[0239] Thus, a pharmaceutical composition may contain one or more additional active ingredients. An antibody of the present invention may be present in the same pharmaceutical composition as the additional active ingredient, or the antibody may be contained in a first pharmaceutical composition and the additional active ingredient may be contained in a second pharmaceutical composition different from the first pharmaceutical composition. Thus, when multiple additional active ingredients are contemplated, each of the additional active ingredients and the antibody may be contained in a different pharmaceutical composition. Such different pharmaceutical compositions may be administered in combination / simultaneously or at different times or in different locations (e.g., different parts of the body), optionally by different administration routes.

[0240] The antibody and the additional active ingredient may provide an additive therapeutic effect, such as a synergistic therapeutic effect. The term "synergistic" is used to describe a manner in which the combined effect of two or more active agents is greater than the sum of the individual effects of each active agent. Thus, when the combined effect of two or more drugs results in "synergistic inhibition" of an activity or process, it is intended that the inhibition of that activity or process is greater than the sum of the inhibitory effects of each active agent. The term "synergistic therapeutic effect" refers to a therapeutic effect observed with the combination of two or more therapies, where the therapeutic effect (as measured by any of a number of parameters) is greater than the sum of the individual therapeutic effects observed with each of the therapies individually.

[0241] The present invention also provides a diagnostic composition comprising an antibody according to the invention, a nucleic acid(s) according to the invention, a vector(s) according to the invention, and / or a cell according to the invention. The diagnostic composition may optionally comprise suitable means for detection, such as reagents conventionally used in immunological or nucleic acid-based diagnostic methods.

[0242] The antibodies described herein are suitable, for example, for diagnostic purposes. Accordingly, the antibodies described herein can be used in immunoassays, where they can be utilized in liquid phase or bound to a solid-phase carrier. Such immunoassays can be competitive or non-competitive immunoassays; they can be in either direct or indirect formats. Examples of such immunoassays include, but are not limited to, radioimmunoassays (RIA), enzyme-linked immunoassays (ELISA), sandwich (immunometric), immunohistochemistry, flow cytometry, and Western blot assays. For this purpose, the antibodies can be labeled, for example, as described above.

[0243] In further aspects, the present invention also provides kits comprising one or more of the following: (i) the antibody or antigen-binding fragment thereof according to the present invention; (ii) a nucleic acid molecule (or a plurality of nucleic acid molecules) according to the present invention as defined above, (iii) the vector(s) according to the invention as described above, (iv) the cells according to the present invention, and / or (v) The composition according to the present invention.

[0244] Furthermore, the kit may include a means for administering the antibody or antigen-binding fragment thereof of the present invention, the nucleic acid of the present invention, the vector of the present invention, the cell of the present invention, or the pharmaceutical composition of the present invention, such as a syringe or container, a leaflet, and / or a co-agent to be administered as described herein. For example, the kit may include, for example, a leaflet containing instructions for use. Additionally or alternatively, the kit may include one or more reagents, such as those for use in an appropriate diagnostic assay. In some cases, the kit may also include a reference drug or control. In some embodiments, the compositions of the present invention may be provided in the form of a kit, e.g., designed to be reconstituted immediately prior to administration to a subject. For example, a lyophilized antibody may be provided in the form of a kit together with sterile water or a sterile buffer (e.g., in a separate container).

[0245] [Medical treatment and other uses] In a further aspect, the present invention provides the use of an antibody or antigen-binding fragment thereof according to the invention, a nucleic acid molecule(s) according to the invention, a vector(s) according to the invention, a cell according to the invention, or a (pharmaceutical) composition according to the invention as a medicament. In particular, an antibody or antigen-binding fragment thereof according to the invention, a nucleic acid molecule(s) according to the invention, a vector(s) according to the invention, a cell according to the invention, or a (pharmaceutical) composition according to the invention can be used in the prevention and / or treatment of neurodegenerative or neuroinflammatory diseases, or peripheral inflammatory disorders.

[0246] Accordingly, the present invention also provides a method for treating, ameliorating or alleviating a neurodegenerative or neuroinflammatory disease, or a peripheral inflammatory disorder, or treating, ameliorating or alleviating the symptoms of a neurodegenerative or neuroinflammatory disease, or a peripheral inflammatory disorder, or reducing the risk of (developing) a neurodegenerative or neuroinflammatory disease, or a peripheral inflammatory disorder, or reducing the risk of (developing) a symptom of a neurodegenerative or neuroinflammatory disease, or a peripheral inflammatory disorder, the method comprising administering to a subject (in need thereof) a (therapeutically effective amount) of an antibody or antigen-binding fragment thereof according to the present invention, a nucleic acid molecule (or nucleic acid molecules) according to the present invention, a vector (or vectors) according to the present invention, a cell according to the present invention, or a (pharmaceutical) composition according to the present invention. Furthermore, the present invention also provides the use of an antibody or antigen-binding fragment thereof according to the invention, a nucleic acid molecule (or nucleic acid molecules) according to the invention, a vector (or vectors) according to the invention, a cell according to the invention, or a pharmaceutical composition according to the invention in the manufacture of a medicament for the prevention, treatment or attenuation of a neurodegenerative or neuroinflammatory disease, or a peripheral inflammatory disorder, or for the prevention, treatment or attenuation of symptoms of a neurodegenerative or neuroinflammatory disease, or a peripheral inflammatory disorder.

[0247] As used herein, the terms "treat" or "treatment" include therapeutic treatment as well as prophylactic or preventative treatment. Prevention of a neurodegenerative or neuroinflammatory disease particularly refers to a prophylactic setting in which the subject has not been diagnosed with a neurodegenerative or neuroinflammatory disease, or a peripheral inflammatory disorder (no diagnosis has been made or the diagnosis has been negative) and / or the subject does not exhibit symptoms of a neurodegenerative or neuroinflammatory disease. In contrast, in a therapeutic setting, the subject typically has been diagnosed with a neurodegenerative or neuroinflammatory disease and / or exhibits symptoms of a neurodegenerative or neuroinflammatory disease or a peripheral inflammatory disorder. It should be noted that the terms "treatment" and "therapy" / "therapeutic" include (complete) cure and attenuation / alleviation of a neurodegenerative or neuroinflammatory disease, or a peripheral inflammatory disorder, and / or associated symptoms.

[0248] Generally, the purpose of "treatment" can be to reduce, ameliorate, inhibit, prevent, or slow (alleviate or delay) an undesired physiological change or disorder, such as a neurodegenerative or neuroinflammatory disease, or a symptom of a neurodegenerative or neuroinflammatory disease or a peripheral inflammatory disorder. Beneficial or desired clinical results of treatment include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, reduction in the extent of the disease, stabilization of the disease state (i.e., not worsening), delay or slowing of disease progression, improvement or palliation of the disease state, and remission (whether partial or total). "Treatment" can also mean prolonging survival, for example, as compared to expected survival if not receiving treatment. Those in need of treatment include those already with a condition or disorder, as well as those prone to having the condition or disorder, or those in whom the onset or risk of the condition or disorder is to be reduced, delayed, or prevented.

[0249] One method for determining the effectiveness of therapeutic treatment is to monitor disease symptoms after administration of the antibody or composition. Treatment can be a single-dose schedule or a multiple-dose schedule. In some embodiments, the antibodies, antibody fragments, nucleic acids, vectors, cells, or compositions described herein can be administered to a subject in need of such treatment. Such subjects include, but are not limited to, those at particular risk for or susceptible to neurodegenerative or neuroinflammatory diseases or symptoms of neurodegenerative or neuroinflammatory diseases or symptoms of peripheral inflammatory disorders.

[0250] The antibody or antigen-binding fragment thereof of the present invention, the nucleic acid molecule(s) of the present invention, the vector(s) of the present invention, the cell(s) of the present invention, or the (pharmaceutical) composition of the present invention can be used for human medical purposes as well as for veterinary medical purposes, preferably for human medical purposes. Thus, as used herein, the term "subject," "patient," or "individual" generally includes humans and non-human animals, preferably mammals (e.g., marmosets, tamarins, spider monkeys, opossums, vervet monkeys, squirrel monkeys, and non-human primates, including baboons, macaques, chimpanzees, orangutans, gorillas; cows; horses; sheep; pigs; chickens; cats; dogs; mice; rats; rabbits; guinea pigs; etc.), chimeric and transgenic animals, and disease models. In the context of the present invention, the term "subject" preferably refers to a non-human primate or a human. Most preferably, the subject is a human. The human may be selected from the group of humans suffering from a neurodegenerative or neuroinflammatory disease, or a peripheral inflammatory disorder, humans at risk of developing a neurodegenerative or neuroinflammatory disease, or a peripheral inflammatory disorder, and humans with an unknown clinical history of a neurodegenerative or neuroinflammatory disease, or a peripheral inflammatory disorder.

[0251] Preferably, the "neurodegenerative or neuroinflammatory disease" is characterized by or associated with the formation of ASC aggregates ("specks") and / or amyloid-β (Aβ)-related pathology, particularly the formation and spread of amyloid-β aggregates. The term "Aβ-related pathology" refers to the abnormal production, deposition, and aggregation of amyloid-β in the brain. In particular, the neurodegenerative or neuroinflammatory disease may involve the interaction of ASC with amyloid-β. In some embodiments, the neurodegenerative or neuroinflammatory disease is associated with the fibrillogenic activity of ASC. In some embodiments, the neurodegenerative or neuroinflammatory disease involves ASC-induced inflammation.

[0252] Neurodegenerative diseases are typically chronic, progressive disorders characterized by the gradual loss of nerve cells in discrete regions of the central nervous system (CNS), such as the brain. The neurodegenerative diseases contemplated for treatment are preferably characterized by and / or may be accompanied by dementia. "Dementia" is a general term referring to a decline in intellectual ability severe enough to interfere with daily life. Dementia includes a decline or loss of memory, communication skills, language skills, concentration, attention, reasoning, judgment, visual perception, or a combination thereof. Dementia can be caused by neurodegeneration in various neurodegenerative diseases.

[0253] Neuroinflammation is the inflammation of nervous tissue. Neuroinflammation can be triggered by a variety of triggers, including infection, traumatic brain injury, toxic metabolites, or autoimmunity. Neuroinflammatory diseases are characterized by inflammatory processes in the nervous system, particularly the CNS, resulting in gradual neuronal damage and loss. Neuroinflammation and neurodegeneration are often caused by the abnormal deposition of aggregated host proteins, which activate inflammasomes. ASC is a central component of inflammasomes.

[0254] Preferably, the neurodegenerative or neuroinflammatory disease is selected from Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple system atrophy, amyotrophic lateral sclerosis, multiple sclerosis, traumatic brain injury, spinocerebellar ataxia, frontotemporal dementia, frontotemporal lobar degeneration, mild cognitive impairment, Parkinson's plus syndrome, Pick's disease, primary progressive aphasia, gray matter degeneration [Alpers], subacute necrotizing encephalopathy, or dementia with Lewy bodies, with Alzheimer's disease (AD) and mild cognitive impairment (MCI) being particularly preferred.

[0255] Alzheimer's disease (AD) is a neurodegenerative brain disorder and the leading cause of dementia in the elderly. Symptoms of AD include progressive decline in learning and memory function, personality changes, neuromuscular changes, seizures, and sometimes psychotic behavior. Alzheimer's disease is characterized by the deposition of amyloid-β plaques in areas of the brain important for memory and other cognitive functions. The deposition of amyloid-β plaques in these critical brain regions is thought to impair brain function.

[0256] "Mild cognitive impairment" (MCI) is a syndrome defined by subjective and objective declines in cognitive function beyond those expected for an individual's age and educational level, but does not meet the diagnostic criteria for dementia. However, older adults diagnosed with MCI are at high risk for dementia, particularly Alzheimer's disease (AD), as well as other types of dementia, such as vascular dementia, frontotemporal dementia (FTD), and dementia with Lewy bodies (DLB). MCI is often referred to as an objective cognitive complaint for age in individuals who are not dementia-affected and have essentially normal functional activity. 19% of people aged 65 and older suffer from MCI. Approximately 46% of MCI patients develop dementia within three years, compared with 3% of the population of the same age.

[0257] Further non-limiting examples of neurodegenerative or neuroinflammatory diseases that may be prevented or treated according to the present invention include hereditary ataxias, congenital non-progressive ataxias, early-onset cerebellar ataxia, late-onset cerebellar ataxia, cerebellar ataxia with DNA repair deficiency, hereditary spastic paraplegia, infantile spinal muscular atrophy type I [Werdnig-Hoffman], hereditary spinal muscular atrophy, systemic atrophies primarily affecting the central nervous system, paraneoplastic neuromuscular and neurological disorders, post-polio syndrome, degenerative diseases of the 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 demyelination, neuromyelitis optica [Devic], acute and subacute hemorrhagic leukoencephalitis [Hurst], periaxonal encephalitis, Schilder's disease, central demyelination of the corpus callosum, central pontine myelolysis, acute transverse myelitis in central nervous system demyelinating diseases, subacute necrotizing myelitis, concentric sclerosis, epilepsy, focal-related (focal) (partial) idiopathic epilepsies and epilepsy syndromes with focal seizures, focal-related (focal) (partial) symptomatic epilepsies and epilepsy syndromes with simple partial seizures, focal-related (focal) (partial) symptomatic epilepsies and epilepsy syndromes with complex partial seizures, myoclonic epilepsy of infancy, neonatal convulsions (familial), childhood absence epilepsy, absence epilepsy, myoclonic epilepsy petit mal], myoclonic absence epilepsy, myoclonic-quiescent seizures, infantile convulsions, Lennox-Gastaut syndrome, Salam seizures, symptomatic early myoclonic encephalopathy, West syndrome, partial epilepsy of continuum [Kozhevnikof], grand mal seizures, petit mal seizures, status epilepticus, grand mal status epilepticus, petit mal status epilepticus, complex partial status epilepticus, migraine, cluster headache syndrome, vascular headache, tension-type headache, chronic post-traumatic headache, narcolepsy and cataplexy, Kleine-Levin syndrome.

[0258] Examples of peripheral inflammatory disorders include, but are not limited to, inflammatory bowel disease, non-alcoholic steatohepatitis, Behcet's disease, sarcoidosis, lupus erythematosus, vasculitis, and rheumatoid arthritis.

[0259] The antibody or antigen-binding fragment thereof of the present invention, the nucleic acid molecule(s) of the present invention, the vector(s) of the present invention, the cell(s) of the present invention, or the pharmaceutical composition of the present invention can be administered by any route of administration, including, but not limited to, oral, intravenous, intramuscular, intraarterial, intraperitoneal, transdermal, transcutaneous, topical, subcutaneous, nasal, enteral, sublingual, or rectal. In addition, any gene therapy approach can be used; for example, the antibody or antigen-binding fragment thereof of the present invention can be administered using a nucleic acid or vector encoding the antibody, such as a viral or non-viral vector as described above. In some embodiments, the antibody or antigen-binding fragment thereof of the present invention, the nucleic acid molecule(s) of the present invention, the vector(s) of the present invention, the cell(s) of the present invention, or the pharmaceutical composition of the present invention can be administered systemically, for example, by intravenous or subcutaneous administration.

[0260] [Further uses] The antibodies and fragments thereof described herein can also be used for the (in vitro) diagnosis of neurodegenerative or neuroinflammatory diseases, or peripheral inflammatory disorders. Diagnostic methods include contacting an antibody with a sample. Such a sample can be isolated from a subject, e.g., an isolated blood sample such as whole blood, plasma, or serum. Diagnostic methods can also include detection of antigen / antibody complexes, particularly after contacting the antibody with the sample. Furthermore, the sample can be tested for antibodies that compete with the antibodies described herein, e.g., for allergen binding. This is typically performed in vitro, i.e., without contact with the human or animal body. Examples of analytical methods are well known to those skilled in the art and include immunoassays such as flow cytometry, dot or slot blots, Western blots, ELISA (enzyme-linked immunosorbent assay), immunohistochemistry, and immunoprecipitation followed by SDS-PAGE immunocytochemistry. Thus, diagnosis can be performed in vitro, e.g., by using the isolated sample (and the in vitro analysis steps described above).

[0261] Accordingly, the present invention also provides the use of an antibody or antigen-binding fragment thereof according to the invention, a nucleic acid molecule or nucleic acid molecules according to the invention, a vector or vectors according to the invention, a cell according to the invention, a composition according to the invention, or a kit according to the invention in the (in vitro) diagnosis of a neurodegenerative or neuroinflammatory disease, or a peripheral inflammatory disorder.

[0262] BRIEF DESCRIPTION OF THE DRAWINGS A brief description of the accompanying drawings will now be given, which are intended to explain the invention in more detail, but which are not intended to limit the subject matter of the invention in any way.

[0263] FIG. 1 shows binding curves of an exemplary antibody, ASC_Ab001, to full-length ASC, ASC speck, CARD domain, PYD domain, mouse ASC, and BSA, as tested by ELISA.

[0264] Figure 2 shows an alignment of various ASC isoforms for Example 4: 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).

[0265] Figure 3 shows representative images of binding patterns for Example 4: (A) ASC_Ab001, (B) ASC_Ab008, (C) ASC_Ab004, and (D) control antibody (AL-177, Adipogen). In all blots, lane 1 shows binding to ASC-b 20 kDa, lane 2 shows binding to ASC-d 11.8 kDa, lane 3 shows binding to ASC-c 15 kDa, lane 4 shows binding to ASC Y146A 21.5 kDa, lane 5 shows binding to ASC K21A K22A K26A 21.5 kDa, lane 6 shows binding to UT control, and lane 7 shows binding to purified ASC-HIS 23 kDa.

[0266] Figure 4 shows binding of the indicated anti-ASC antibodies and control antibodies to the indicated ASC peptides for Example 4. (A) Binding of ASC_Ab001, ASC_Ab002 clonotype, ASC_Ab004, ASC_Ab005, ASC_Ab006, ASC_Ab007, ASC_Ab008 clonotype, AL-177 and VHH to peptides covering amino acids 1-20 and amino acids 11-30 of ASC. ASC (B) Binding of ASC_Ab001, ASC_Ab002, AL-177, IC100 and VHH to peptides covering amino acids 1-20, 11-30 and 21-40 of ASC. ASC Combination of.

[0267] FIG. 5 shows the results of Example 5, which show the results of antibodies ASC_Ab001, ASC_Ab002, AL-177, and VHH against human ASC. ASC and IC100 ELISA binding curves are shown.

[0268] FIG. 6 shows the results of Example 5, which shows the results of antibodies ASC_Ab001, ASC_Ab002, AL-177, and VHH against mouse ASC.ASC and IC100 ELISA binding curves are shown.

[0269] FIG. 7 shows the results of Example 5, which show the results of antibodies ASC_Ab001, ASC_Ab002, AL-177, and VHH against ASC speck. ASC and IC100 ELISA binding curves are shown.

[0270] FIG. 8 shows the results of Example 5, which show the results of antibodies ASC_Ab001, ASC_Ab002, AL-177, and VHH against the PYD domain of ASC. ASC and IC100 ELISA binding curves are shown.

[0271] FIG. 9 shows, for Example 6, (A) Thp1 wt or asc primed with LPS (1 μg / mL), stimulated with nigericin (5 μM), and stained with 1 μg / mL anti-ASC antibody or isotype control. - / - (B) Quantification of ASC specks detected in cells is shown, and (B) representative immunofluorescence photographs of THP-1 asc + / + and THP-1 asc − / − cells stained with ASC_Ab001 antibody are shown.

[0272] FIG. 10 shows the results of Example 7 for wt and asc - / - 1 shows a Western blot image of antibodies ASC_Ab001, ASC_Ab004, ASC_Ab007 and ASC_Ab009 on cell lysates of human THP-1 monocytes.

[0273] FIG. 11 shows Western blot images of antibodies ASC_Ab001, ASC_Ab004, ASC_Ab007, and ASC_Ab009 on cell lysates of mouse bone marrow-derived macrophages (BMDM) for Example 7.

[0274] Figure 12 shows that treatment with ASC_Ab001, ASC_Ab004, ASC_Ab007, ASC_Ab008, and ASC_Ab009 significantly inhibited ASC fibril formation for Example 8. The effect of anti-ASC antibodies on ASC fibril formation was assessed by IF of ASC fibers formed after incubating ASC-gfp with anti-ASC antibodies or an isotype control for 1 hour at 37°C. (A) Representative photographs, and (B) quantification of the number of ASC fibers per well treated with anti-ASC antibodies. Pooled data from n = 1 or 2 independent experiments. One-way ANOVA followed by Dunnett's multiple comparison test. ns: not significant, ****: p-value < 0.0001.

[0275] Figure 13 shows the results of a precipitation assay to evaluate the anti-fibrogenic properties of anti-ASC antibodies for Example 8. Soluble and insoluble fractions of ASC were separated by high-speed centrifugation after treatment with anti-ASC antibodies or an isotype control. After 30 minutes of incubation at 37°C, treatment with ASC_Ab001 yielded more soluble ASC (supernatant fraction) compared with treatment with ASC alone or an isotype control. In other words, ASC_Ab001 shifts the equilibrium toward soluble ASC in the supernatant fraction. Treatment with anti-CARD antibodies did not affect fibrogenesis.

[0276] Figure 14 shows that anti-ASC antibody blocks ASC-Aβ interaction for Example 9. Inhibition rate of ASC-Aβ binding after treatment with anti-ASC antibody (3.5uM) or isotype control (3.5uM). One-way ANOVA followed by Dunnett's multiple comparison test. ***: p-value < 0.001.

[0277] Figure 15 (Example 10) shows that ASC_Ab001 blocks the pro-inflammatory function of extracellular ASC fibers. The in vitro efficacy of ASC_Ab001 was tested in a cell-based assay using differentiated human THP-1 macrophages treated with extracellular ASC fibers. Before adding ASC fibers to the cells, the ASC fibers were preincubated with ASC_Ab001 and an isotype control antibody. Measurement of IL-1β was used as a readout of inflammasome activation. ASC_Ab001 significantly reduced ASC fiber-induced IL-1β production compared to the isotype control.

[0278] FIG. 16 shows quantification of IL-1 beta release by ASC fibers and differentiated human macrophages treated with 0.0035 nM or 35 nM of anti-ASC antibodies (ASC_Ab001 to ASC_Ab009) and isotype control, respectively, in an ASC-seeded inflammation assay for Example 11.

[0279] FIG. 17 for Example 12 shows that anti-ASC antibodies (ASC_Ab001 to ASC_Ab009) do not bind to inflammasome proteins AIM2 and NRLP3, which have sequences homologous to ASC, as determined by binding ELISA.

[0280] Figure 18 (Example 13) shows that anti-ASC antibodies ASC_Ab001 and ASC_Ab002 ameliorate the severity of dextran sulfate sodium (DSS)-induced colitis in an acute inflammatory bowel disease model. (A) Body weight measurement; (B) Disease Activity Index measurement; and (C) Colon length measurement. Data represent mean ± SEM. Multiple comparisons with isotype were performed using two-way ANOVA. *p<0.05, ***p<0.01, ***p<0.001, ***p<0.0001.

[0281] Figure 19 shows representative photographs of hematoxylin-eosin stained colons from N=8 mice per group (A: healthy; B: isotype control; C: ASC_Ab001) for Example 13. Figure 19D shows inflammatory cell infiltration into the mucosa of healthy, isotype-, and Asc_Ab001-treated mice.

[0282] Figure 20 shows that humanized ASC_Ab001 retains binding to ASC for Example 14. Binding of humanized ASC_Ab001 to ASC-His was assessed by ELISA. Similar binding of the humanized antibody was observed compared to the rabbit IgG format.

[0283] Figure 21 shows the VH amino acid sequences of the humanized Ab001 antibody. All heavy chain sequences were based on the IGHV3-66 germline, which has the highest homology with the original rabbit Ab001 heavy chain framework and has also been reported to be a good acceptor framework for rabbit CDRs. In Example 15, humanized Ab001 antibodies, including Ab001_VHhum23, were used.

[0284] Figure 22 shows the VL amino acid sequences of the humanized antibodies. All light chain sequences were based on the IGKV1-5 germline, which has the highest homology to the original rabbit Ab001 light chain framework and is very similar to the IGKV1-27*01 germline, which was previously used as a template for the universal acceptor frame of the rabbit light chain CDRs. In Example 15, humanized Ab001 antibodies were used, including Ab001_VLhum11 and Ab001_VLhum12, respectively.

[0285] Figure 23 shows the amino acid sequences of the VH and VL of the humanized Ab002 antibody. The sequence shown for the heavy chain is based on the IGHV3-66 germline, which has the highest homology to the original rabbit Ab002 heavy chain framework and has also been reported to be a good acceptor framework for rabbit CDRs. All heavy chain sequences are based on the IGKV1-27 germline, which has the highest homology to the original rabbit Ab002 light chain framework and was previously used as a template for the universal acceptor frame for rabbit light chain CDRs. Humanized Ab002 antibodies, including Ab002_VHhum17 and Ab002_VLhum12, were used in Example 15.

[0286] Figure 24 shows kinetic measurements of humanized Ab001 Fab: Ab001 VH23 / VL11 (A), and Ab001 VH23 / VL12 (B) versus non-humanized Ab001 VHrab / VLrab (C) for Example 15. By biolayer interferometry (BLI) using a RED96e OCTET system (Sartorius AG).

[0287] Figure 25 shows kinetic measurements of humanized Ab002 Fab: Ab002 VH16 / VL12 (A) vs. non-humanized Ab002 VHrab / VLrab (B) for Example 15. By biolayer interferometry (BLI) using a RED96e OCTET system (Sartorius AG).

[0288] Figure 26 shows binding of fully IgG1 humanized Ab001 antibodies Ab001 VH23 / VL11 and Ab001 VH23 / VL12, and Ab001 rb-huIgG (non-humanized), to human ASC (A) and mouse ASC (B) for Example 16. Measured by binding ELISA.

[0289] Figure 27 shows binding of fully IgG1 humanized Ab002 antibody Ab002 VH17 / VL12 and Ab002 rb-huIgG (non-humanized) to human ASC (A) and mouse ASC (B) for Example 16. Measured by binding ELISA.

[0290] [Example] Below are given specific examples illustrating various embodiments and aspects of the present invention. However, the present invention is not limited in scope by the specific embodiments described herein. The following preparations and examples are provided to enable those skilled in the art to more clearly understand and to practice the present invention. However, the present invention is not limited in scope by the exemplified embodiments, which are intended only to illustrate single aspects of the invention, and functionally equivalent methods are within the scope of the invention. Indeed, various modifications of the present invention in addition to those described herein will be readily apparent to those skilled in the art from the foregoing description, the accompanying drawings, and the following examples. All such modifications are within the scope of the appended claims.

[0291] 〔method〕 [Production of recombinant ASC specks in HEK293] Full-length, tag-free human ASC (SEQ ID NO: 1) and ASC-GFP (SEQ ID NO: 152) were produced as intracellular specks in HEK293 cells and purified according to the procedure described in Martin-Sanchez F, Gomez AI, Pelegrin P. Isolation of Particles of Recombinant ASC and NLRP3. Bio Protoc. 2015 May 20;5(10):e1480. doi: 10.21769 / BioProtoc.1480. Briefly, suspension HEK293 cells were transiently transfected with expression plasmids encoding full-length human ASC or ASC-GFP using linear PEI 40 kDa. After 7 days of expression at 37°C and 5% CO2, cells were harvested and resuspended in buffer A (320 mM sucrose, 20 mM HEPES-KOH (pH 7.5), 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA). Cells were lysed by syringe (10 x 20G, 20 x 25G), freeze-thawing (3x), and then syringe (20 x 25G). The lysate was then centrifuged at 400g for 8 minutes, and the pellet was resuspended 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 at 2000g for 10 minutes using a 5 μm centrifugal filter. The filtrate was then diluted, gently mixed with 1 volume of 2x CHAPS buffer, and centrifuged at 2300g for 8 minutes. The resulting pellet was resuspended in 1 ml of 1x CHAPS buffer and centrifuged at 5000g for 8 minutes. This washing step was repeated twice. The pellet was then resuspended in 1x CHAPS buffer, carefully placed on top of 40% Percoll, and centrifuged at 16000g for 10 minutes. The interface layer containing ASC-speck particles was carefully collected and washed once by centrifugation at 5000g for 3 minutes and resuspension in 1x CHAPS buffer. Finally, fluorescent particles were quantified under a fluorescence microscope using a Burkel chamber.

[0292] [Production of monomeric ASC in E. coli] Monomeric human ASC-His (SEQ ID NO: 153), ASC-GFP-His (SEQ ID NO: 154), and the ASC PYD domain (ASCPYD-His-SII; SEQ ID NO: 155) and ASC CARD domain (ASCCARD-His-SII; SEQ ID NO: 156) were expressed in E. coli and purified from inclusion bodies via nickel beads. Therefore, E. coli strain BL21(DE3) was transformed with pET-based vectors encoding his-tagged ASC variants. Expression was performed in autoinduction medium (MagicMedia). TM The incubation was performed in a 5000 x g (Invitrogen) buffer at 37°C for 1 hour and at 20°C for 67 hours. Cells were then harvested by centrifugation at 4000 rpm for 30 minutes. For cell lysis, the pellet was resuspended in 50 mM phosphate, 300 mM NaCl, pH 7.5, and sonicated on ice for 10 minutes at 40% power (2-second pulse / pause). The suspension was centrifuged at 14000 x g for 30 minutes at 4°C, and the pellet containing inclusion bodies was collected. To solubilize the inclusion bodies, the pellet was resuspended in 50 mM phosphate, 300 mM NaCl, 6 M Gua-HCl, 2 mM DTT, pH 7.5, for 30 minutes at room temperature. The suspension was then centrifuged at 14000 x g for 30 minutes at 4°C to remove any remaining insoluble cell debris. The supernatant was then incubated with nickel beads (Themo, #88221) for 3 hours at room temperature. The beads were washed once with 50 mM phosphate, 300 mM NaCl, 6 M Gua-HCl, 2 mM DTT, pH 7.5, followed by another wash with 50 mM phosphate, 300 mM NaCl, 6 M Gua-HCl, 2 mM DTT, 20 mM imidazole, pH 7.5. To elute the His-tagged protein, the beads were incubated with 50 mM phosphate, 300 mM NaCl, 6 M Gua-HCl, 2 mM DTT, 500 mM imidazole, pH 7.5. The pH of the eluate was adjusted to pH 3.8 with dilute hydrochloric acid. The eluate was then dialyzed overnight at 4°C in a 3500 Da cassette against 50 mM glycine, 150 mM NaCl, pH 3.8. To remove higher-order aggregates, the dialyzed sample was purified using a preparative SEC column (HiLoad 16 / 600 Superdex 75 pg). The monomeric protein was finally concentrated using a VivaSpin 3000Da column.

[0293] [ELISA] High-binding, clear, flat-bottom 384-well plates (Greiner Bio-One, 781061) were coated overnight (4°C) with 10 μl / well of ASC CARD domain (1.25 μg / mL; SEQ ID NO: 156), ASC PYD domain (1.25 μg / mL; SEQ ID NO: 155), mouse ASC (Cusabio, CSB-EP861664 MO, 1.25 μg / mL; SEQ ID NO: 151), ASC-His (1 μg / mL; SEQ ID NO: 153), ASC-gfp specks (50,000 specs / well), or BSA (1 μg / mL) diluted in PBS. To test binding to ASC peptides (SEQ ID NOs: 148–150), the same plates were precoated with 1 μg / mL streptavidin (Sigma, cat. S4762) and then incubated with 1 μg / mL of peptide overnight (4°C). After overnight incubation, wells were aspirated and washed once with 100 μl / well of wash buffer (0.05% Tween-20 in PBS). Wells were blocked with 50 μl of 2% BSA diluted in PBS for 1 hour at room temperature. After blocking, wells were incubated with 10 μl of serially diluted test antibody for 2 hours at room temperature. Wells were washed four times and subsequently incubated with 10 μl / well of detection antibody for 1 hour at room temperature. Unbound detection antibody was removed by washing four times, and wells were incubated with 20 μl of TMB for 5 minutes. The reaction was stopped by adding 10 μl of 1M H2SO4 per well. Absorbance at 450 nm was immediately measured using an EnSpire 2300 microplate reader (Perkin Elmer).

[0294] [BLI Assay] Kinetic measurements and epitope binning experiments were performed by biolayer interferometry (BLI) using the RED96e OCTET system (Sartorius AG). In both setups, in-house expressed recombinant human or mouse ASC (Cusabio, CSB-EP861664 MO) was covalently immobilized on an AR2G biosensor (Sartorius AG) via EDC / NHS-mediated amine coupling.

[0295] For kinetic measurements, both human and mouse ASCs were immobilized at a low concentration of 0.625 μg / mL in 10 mM sodium phosphate buffer, pH 5, for 300 s, ensuring that the interaction with the analyte occurred predominantly at a 1:1 stoichiometry. Binding and dissociation kinetics were measured for 150 s and 500 s, respectively, at antibody concentrations of 50 nM, 16.67 nM, 5.55 nM, 1.85 nM, 0.617 nM, 0.206 nM, and 0 nM in 1x PBS buffer (pH 7.4) supplemented with 0.1% BSA and 0.02% Tween. The antibody was released from the ASCs by immersing the biosensor in a regeneration buffer (10 mM glycine, pH 3.0). The regeneration buffer was selected according to a regeneration scouting procedure as the mildest drug that resulted in a complete recovery of baseline and complete maintenance of ligand binding capacity. This allowed the use of a single immobilized biosensor for the entire concentration series of each antibody. The analytical section used a biosensor injected with buffer as the analyte throughout the entire run as a reference for subtraction. The binding constant (k on ), and the dissociation constant (k off ) was calculated by global fit using a 1:1 binding model.

[0296] In the setup used for epitope binning experiments, human and mouse ASCs were immobilized at concentrations ranging from 1.25 μg / mL to 20 μg / mL. Antibodies were binned in a two-dimensional matrix format, with sets of up to seven antibodies per experiment. Antibodies were added sequentially in duplicate at saturating concentrations. The biosensor was regenerated with regeneration buffer between successive antibody sets. For data analysis, a condition in which the same antibody was injected as both the first and second antibody was selected as a reference and subtracted. Only subsequent antibodies that produced additive binding responses greater than the Bmax of the first antibody alone were considered non-competing. Distinct and overlapping bins were manually defined based on the relative degree of competition between the antibodies involved.

[0297] [Binding to ASC isoforms] Protein expression and cell lysates: ASC-b, ASC-c, ASC-d (described in Bryan et al., 2010), ASC-Y146A (described in Prather et al., 2022), and ASC K21A K22A K26A (described in Venegas-Maldonado et al., 2017) were expressed in HEK293 cells by transfection with PEI. Cells were lysed in ice-cold CHAPS buffer supplemented with protease inhibitors to prepare cell lysates. After centrifugation at 17,000 × G for 15 min at 4°C, the lysates were removed and the supernatants were transferred to fresh pre-chilled tubes. Protein concentrations were determined by BCA assay (Pierce TM NuPAGE LDS sample buffer (4x) (Invitrogen) and NuPAGE sample reducing agent (10x) (Invitrogen) were added to the samples and boiled at 95°C for 5 minutes.

[0298] SDS PAGE: 15 μg of lysate or 150 ng of ASC-His monomer was loaded onto a NuPAGE 4-12% Bis-Tris gel (Invitrogen). Gels were run at 120 V for 40 minutes using MES SDS running buffer (Invitrogen). Gel electrophoresis was performed using an Invitrogen XCell SureLock TM This was performed using an electrophoresis cell chamber system.

[0299] Protein transfer (blotting): Gels were transferred to Amersham TM Protran TM Place on a Premium 0.45 μm nitrocellulose blotting membrane (Cytiva) and perform Mini Trans-Blot TM Proteins were transferred using a Cell System (Bio-Rad) and 1x transfer buffer (25 mM Tris-Base, 192 mM glycine, 10% methanol) at 100 V for 1 hour. After blotting, the membrane was blocked with 5% nonfat dry milk in PBS-T (0.1% Tween-20) for 1 hour at room temperature.

[0300] Staining: After blocking, primary antibody was added to the membrane at 1µg / ml in 1% non-fat dry milk in PBS-T for 2 hours at room temperature. The membrane was washed three times with PBS-T for 10 minutes each time, and then incubated with secondary antibody in 1% non-fat dry milk in PBS-T for 1 hour at room temperature. The membrane was washed three times with PBS-T and then incubated with SuperSignal TM and incubated with West Pico PLUS chemiluminescent substrate (Thermofisher Scientific).

[0301] [Immunofluorescence analysis of ASC speck] THP-1 wt and asc- / - cells were seeded at a density of 10,000 cells / well in 50 μL of RPMI medium supplemented with Glutamax, 10% FBS, 1% P / S, 100 μg / mL Normocin, and 100 μg / mL Zeocin in a 384-well plate (Greiner Bio-One 781986). Cells were primed with 1 μg / mL LPS (Sigma, cat: L4391-1MG) for 3 hours. After priming, cells were treated with 5 μM nigericin (Invivogen, cat: tlrl-nig) for 1 hour to induce ASC speck formation. Cells were then fixed and permeabilized with BD cytofix / cytoperm (BD Biosciences, cat: 554722) solution for 20 minutes at room temperature. The cells were then washed twice with PBS and blocked with 2% BSA supplemented with human Fc blocking reagent (Miltenyi cat: 130-059-901). After blocking, the cells were incubated with 1 μg / mL anti-ASC antibody or isotype control for 2 hours at room temperature, washed twice with PBS, and further incubated with a detection secondary antibody coupled to Alexa fluor 647 (Jackson Immunoresearch, 111-607-003) for 1 hour at room temperature. 4',6-diamidino-2'-phenylindole-dihydrochloride (DAPI) (Sigma, cat: MBD0015-1ML) was used as a counterstain at 1 μg / mL in PBS for 10 minutes. Images were taken using a 10x objective. All images were captured under a fluorescence microscope. Image processing and ASC speck counting were performed using Fiji ImageJ and Cellprofiler.

[0302] Western blot of selected antibodies against THP-1 cells and BMDM. - Cell lysis: Cell lysates were prepared by lysing cells (wt and asc - / -Human THP-1 monocytes and mouse BMDMs were lysed on ice for 15 min in ice-cold lysis buffer containing 1% Triton X-100. After centrifugation at 17,000 x g for 15 min at 4°C to remove the lysate, the supernatant was transferred to a fresh pre-chilled tube. Protein concentration was determined by BCA assay (Pierce TM NuPAGE LDS sample buffer (4x) (Invitrogen) and NuPAGE sample reducing agent (10x) (Invitrogen) were added to the samples and boiled at 95°C for 5 minutes.

[0303] -SDS PAGE: 10 μg of protein was loaded onto a NuPAGE 4-12% Bis-Tris gel (Invitrogen). The gel was run at 80 V for 10 minutes and then at 120 V for 110 minutes in MES SDS running buffer (Invitrogen). Gel electrophoresis was performed using an Invitrogen XCell SureLock TM This was performed using an electrophoresis cell chamber system.

[0304] - Protein transfer (blotting): The gel was transferred to Amersham TM Protran TM Place on a Premium 0.45 μm nitrocellulose blotting membrane (Cytiva) and perform Mini Trans-Blot TM Proteins were transferred using a Cell System (Bio-Rad) and 1x transfer buffer (25 mM Tris-Base, 192 mM glycine, 10% methanol) at 100 V for 1 hour. After blotting, the membrane was blocked with 5% nonfat dry milk in PBS-T (0.1% Tween-20) for 1 hour at room temperature.

[0305] - Staining: After blocking, primary antibody was added to the membrane at 1µg / ml in 1% non-fat dry milk in PBS-T for 2 hours at room temperature. The membrane was washed three times with PBS-T for 10 minutes each time, and then incubated with secondary antibody in 1% non-fat dry milk in PBS-T for 1 hour at room temperature. The membrane was washed three times with PBS-T and then incubated with SuperSignal TM and incubated with West Pico PLUS chemiluminescent substrate (Thermofisher Scientific).

[0306] Fibrogenesis assay 1.75 μM ASC-GFP-His was incubated with 3.5 μM anti-ASC antibody or isotype control in a Protein LoBind tube (Eppendorf, cat. 0030109.116) for 1 h at 37°C. The mixture was then transferred to a non-binding clear-bottom 384-well plate (Greiner bio-one, cat. 781906), and protein aggregates were photographed. Images were taken using a 10x objective on a fluorescence microscope, and image processing was performed using Fiji ImageJ.

[0307] Sedimentation Assay 1.75 μM ASC-GFP-His monomer was incubated with 3.5 μM MY_anti-ASC antibody or isotype control in a Protein LoBind tube (Eppendorf, cat. 0030109.116) at 37°C for 30 min. After incubation, the tube was centrifuged at 17,000 × g for 15 min at 4°C. The supernatant was transferred to a new tube. The pellet was washed once with PBS and centrifuged at 17,000 × g for 15 min at 4°C, and the supernatant was discarded. Samples were prepared for Western blotting by adding NuPAGE LDS sample buffer (4×) (Invitrogen) and NuPAGE sample reducing agent (10×) (Invitrogen) and boiling at 95°C for 5 min before loading onto a NuPAGE 4-12% Bis-Tris gel (Invitrogen). The gel was run at 80 V for 10 minutes and then at 120 V for 110 minutes using MES SDS running buffer (Invitrogen). Gel electrophoresis was performed using an Invitrogen XCell SureLock TM Electrophoresis was performed using a cell chamber system. Gels were prepared using Amersham TM Protran TM Place on a Premium 0.45 μm nitrocellulose blotting membrane (Cytiva) and perform Mini Trans-Blot TM Proteins were transferred using a Cell System (Bio-Rad) in 1x transfer buffer (25mM Tris-Base, 192mM glycine, 10% methanol) at 100V for 1 hour. After blotting, the membrane was blocked with 5% non-fat dry milk in PBS-T (0.1% Tween-20) for 1 hour at room temperature. Primary antibody was added to the membrane at 1µg / ml in 1% non-fat dry milk in PBS-T for 2 hours at room temperature. The membrane was washed three times with PBS-T for 10 minutes each time, and then incubated with secondary antibody in 1% non-fat dry milk in PBS-T for 1 hour at room temperature. The membrane was washed three times with PBS-T and then incubated with SuperSignal TM and incubated with West Pico PLUS chemiluminescent substrate (Thermofisher Scientific).

[0308] [Blocking ASC-Aβ interaction] ASC-His fibrils were formed by incubating ASC-His monomers in Protein LoBind tubes (Eppendorf, 0030109.116) at 37°C for 1 h. Filamentous ASC (1.75 μM) was incubated with 1 μM 5'-TAMRA-Aβ1-42 (Bachem) and 3.5 μM anti-ASC antibody or isotype control for 4 h at 37°C. After incubation, protein aggregation was analyzed by flow cytometry using Novocyte Advanteon (Agilent) and FlowJo v10.7.1.

[0309] [THP-1 efficacy data for ASC_Ab001] - Wt and asc - / - THP-1 monocytes were seeded at a density of 10,000 cells / well in 384-well plates in RPMI medium supplemented with Glutamax, 10% FBS, 1% P / S, 100 μg / mL Normocin, and 100 μg / mL Zeocin. To differentiate monocytes into macrophages, cells were incubated in medium containing 100 ng / mL phorbol 12-myristate-13-acetate (PMA) at 37°C and 5% CO2 for 4 days. After differentiation, cells were washed twice with fresh cell culture medium (10 min each time). ASC fibers were prepared by incubating ASC-His monomers in sterile PBS at 37°C for 1 hour. Before adding ASC fibers (3.5 μM) to the cells, they were preincubated with antibody (3.5 μM) for 15 minutes at room temperature. After 16 h of incubation at 37°C and 5% CO , secreted IL-1β was measured using the Lumit Human IL-1 Immunoassay (Promega, W6010) according to the manufacturer's protocol.

[0310] Example 1: Identification and production of antibodies that bind to ASC [Rabbit immunization] Rabbits were immunized with untagged HEK-derived ASC specks on days 1, 7, 14, 21, and 35, with splenocytes and bone marrow cells harvested 7 days after the final injection. Rabbits were immunized with His-tagged PYD or CARD domains of ASC on days 1, 7, 14, 21, and 28, with splenocytes and bone marrow cells harvested 5 days after the final injection. Cells were cryopreserved.

[0311] [Identification and cloning of rabbit ASC-specific antibodies] Rabbit bone marrow or spleen cells were screened for plasmablasts secreting antibodies specific for ASC speck, human ASC (SEQ ID NO: 1), mouse ASC (SEQ ID NO: 151), or the ASC PYD or CARD domain (SEQ ID NOs: 143 and 144, respectively) and sorted in 10 μl of first-strand buffer (ThermoFisher, USA). Cell lysis and reverse transcription of RNA were performed using maxima H minus reverse transcriptase (ThermoFisher, USA). DNA encoding the variable domain heavy (VH) and variable domain light (VL) sequences was amplified by two-step PCR using primers annealing to the ends of the rabbit variable framework regions, sequenced (Microsynth, Switzerland), translated into amino acid sequences, annotated, and aligned (Geneious Biologics), and cloned into expression vectors containing the rabbit constant heavy or light regions. The expression vector pDB contains the Epstein-Barr virus origin of replication (oriP), allowing episomal replication upon transfection in cells expressing Epstein-Barr virus nuclear antigen 1 (EBNA1). Additionally, the plasmid pDB contains the cytomegalovirus-chicken actin fusion promoter (CAG promoter), resulting in high expression of recombinant proteins.

[0312] Small-scale antibody expression was performed in suspension HEK293 cells. A few minutes before transfection, 500 μl of a cell suspension at 0.8–1 million cells / ml was seeded into a 24-well plate in FreeStyle medium supplemented with 0.1% Pluronic F68, 1% Glutamax, and 1× Geniticin (all ThermoFisher, USA). 250 ng of heavy chain plasmid DNA and 250 ng of light chain plasmid DNA were mixed in a total volume of 15 μl of supplement-free FreeStyle medium. 2 μl of TransIT-LT1 (Mirus, USA) was added to 50 μl of pure FreeStyle medium, mixed, and incubated for 5 minutes. The mixture was then added dropwise to the plated cells and incubated at room temperature for an additional 15 minutes. The mixture was then added dropwise to the plated cells and cultured for approximately 3–4 days in a humidified incubator at 37°C with 5% CO2. After centrifugation at 8000 rpm for 5 to 10 minutes, the supernatant was collected.

[0313] Initially, unpurified cell supernatants were tested by ELISA to confirm binding specificity. High-binding clear flat-bottom 384-well plates (Greiner Bio-One, 781061) were coated overnight at 4°C with 10 μl / well of ASC CARD domain (1.25 μg / mL), ASC PYD domain (1.25 μg / mL), mouse ASC (Cusabio, CSB-EP861664 MO, 1.25 μg / mL), ASC-His (1 μg / mL), ASC-GFP specks (50,000 specs / well), or BSA (1 μg / mL) diluted in PBS. After overnight incubation, wells were aspirated and washed once with 100 μl / well of wash buffer (0.05% Tween-20 in PBS). The wells were blocked with 50 μl of 2% BSA diluted in PBS for 1 hour at room temperature. After blocking, wells were incubated with 10 μl of serially diluted test antibody for 2 hours at room temperature. Wells were washed four times and subsequently incubated with 10 μl / well of detection antibody for 1 hour at room temperature. Unbound detection antibody was removed by washing four times, and wells were incubated with 20 μl of TMB for 5 minutes. The reaction was stopped by adding 10 μl of 1M H2SO4 per well. Absorbance was immediately measured at 450 nm using an EnSpire 2300 microplate reader (Perkin Elmer).

[0314] Antibodies ASC_Ab001, ASC_Ab002, ASC_Ab003, ASC_Ab005, ASC_Ab006, ASC_Ab007, ASC_Ab008, and ASC_Ab009 were identified and selected for further characterization, which were found to bind to the PYD domain of ASC. H and V L The sequences as well as the CDR sequences are shown in Table 5 below.

[0315] [Table 5]

[0316] Antibody Expression and Purification Larger-scale antibody expression was performed in suspension HEK293 cells using a method scalable to expression volumes of 20–1000 mL. Transfection was achieved by adding a mixture of polyethylenimine (PEI Max, Polysciences, USA) and DNA encoding rabbit IgG light and heavy chains to the cells. To achieve high levels of IgG, transfected cells were supplemented with Tryptone N1 (OrganoTechni, France) after 24 hours. After 5–7 days of expression, the supernatant containing the expressed antibody was harvested. Purification was performed using a HiTrap Protein A HP column (Cytiva, USA) or Protein A Sepharose 4 Fast Flow beads (Cytiva, USA). These beads contain Staphylococcal protein A immobilized on an agarose matrix. Protein A has a strong affinity for the Fc region of IgG. Protein A binds to IgG at neutral pH, and the antibody is eluted at acidic pH. The purified antibody was dialyzed against PBS, concentrated, and stored at -80°C.

[0317] Example 2: Cross-competition of antibodies binding to PYD of ASC To determine whether antibodies ASC_Ab001, ASC_Ab002, ASC_Ab003, ASC_Ab005, ASC_Ab004, ASC_Ab006, ASC_Ab007, ASC_Ab008, and ASC_Ab009 bind to the same or different epitopes, a cross-competition assay was performed. To this end, cloned monoclonal antibodies were tested by biolayer interferometry (BLI) on full-length ASC bound to a BLI sensor. Briefly, 1.25 μg / ml of monomeric human ASC was immobilized to an ARG2G biosensor (Sartorius AG) via amine coupling. First, a blocking antibody was applied at a concentration of 266.7 nM to saturate the ASC binding sites. To monitor competition, subsequent antibody binding was measured at a concentration of 66.7 nM. Only subsequent antibodies that produced an additive binding response greater than the maximal response of the blocking antibody alone were considered non-competing. Partial competition was observed in combinations where the subsequent antibody increased the response, but was somewhat reduced from that predicted by the expected increase in response level.

[0318] The results are summarized in Table 6 below, where "X" means (complete) competition, "-" means no competition, and "pc" means partial competition.

[0319] [Table 6]

[0320] As can be seen from Table 4, antibodies ASC_Ab001, ASC_Ab002, and ASC_Ab003 competed for the same epitope on ASC, indicating that antibodies ASC_Ab001, ASC_Ab002, and ASC_Ab003 bind to the same epitope. Partial competition was observed between antibodies ASC_Ab008 and ASC_Ab009, and between ASC_Ab008 and ASC_Ab001, ASC_Ab002, and ASC_Ab003, indicating that these antibodies bind to partially overlapping epitopes. No competition was observed for the remaining antibodies, indicating that these antibodies bind to distinct, non-overlapping epitopes on ASC.

[0321] In this regard, antibodies were divided into groups based on the ASC epitopes they target. The antibodies were assigned to different binning groups designated "PYD-1," "PYD-2," "PYD-4," "PYD-6," "PYD-7a," "PYD-7b," and "PYD-7c." Each binning group represents a distinct, non-overlapping ASC epitope, with the exception of "PYD-7a," "PYD-7b," "PYD-7b," and "PYD-7c," which have partially overlapping epitopes, as shown in Table 7 below.

[0322] [Table 7]

[0323] Example 3: Characterization of antibody binding To further characterize the binding of antibodies ASC_Ab001, ASC_Ab002, ASC_Ab003, ASC_Ab004, ASC_Ab005, ASC_Ab006, ASC_Ab007, ASC_Ab008, and ASC_Ab009, ELISAs were performed as described above to determine antibody binding to monomeric human ASC (SEQ ID NO: 1), HEK-derived ASC-GFP-Speck, and mouse ASC (SEQ ID NO: 151). EC50 values ​​were calculated using Graphpad Prism. The results are shown in Table 8 below.

[0324] [Table 8]

[0325] These data demonstrate that antibodies ASC_Ab001, ASC_Ab002, ASC_Ab003, ASC_Ab004, ASC_Ab005, ASC_Ab006, ASC_Ab007, ASC_Ab008, and ASC_Ab009 specifically bind to monomeric ASC and ASC speck.

[0326] Figure 1 shows an exemplary binding curve for antibody ASC_Ab001, and additionally shows binding results for the PYD and CARD domains. The data shown in Figure 1 demonstrate that exemplary antibody ASC_Ab001 specifically binds to human monomeric ASC, ASC speck, mouse ASC, and the PYD domain of ASC, while no binding was detected to the CARD domain of ASC or the control bovine serum albumin (BSA).

[0327] Next, antibodies ASC_Ab001, ASC_Ab002, ASC_Ab003, ASC_Ab004, ASC_Ab005, ASC_Ab006, ASC_Ab007, ASC_Ab008, and ASC_Ab009 were tested for binding affinity to human full-length ASC captured on a BLI sensor using biolayer interferometry (BLI) as described above. KD, dissociation rates, and association rates were calculated using a 1:1 binding model with a global fit. The results are shown in Table 9.

[0328] [Table 9]

[0329] Example 4: ASC isoforms and peptide mapping To further characterize the binding of antibodies ASC_Ab001, ASC_Ab004, and ASC_Ab008 to ASC, binding to 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) and to (i) human ASC with the mutations K21A, K22A, and K26A (“ASC K21A K22A K26A”) or (ii) human ASC with the mutation Y146A (“ASC Y146A”) was investigated by Western blot using lysates of HEK293 cells transfected with expression plasmids for the expression of the different ASC isoforms or (i) ASC with the mutations K21A, K22A, and K26A; or (ii) ASC with the mutation Y146A. The polyclonal antibody AL-177 (AdipoGen, product number AG-25B-0006-C100, Liestal, Switzerland), known to target an epitope at the N-terminus of the PYD domain present in all four isoforms, was used as a control.

[0330] Figure 2 shows an alignment of the different ASC isoforms. Full-length ASC and ASC-b contain the entire PYD domain, whereas ASC-c lacks amino acids 26-85 of full-length ASC, and ASC-d contains only the N-terminal 35 amino acids of the PYD domain of ASC.

[0331] FIG. 3 shows representative images of the binding patterns of ASC_Ab001, ASC_Ab004, ASC_Ab008, and a control antibody (AL-177, adipogen).

[0332] The control antibody AL-177 bound to each of the four ASC isoforms and to both mutant ASC, while antibodies ASC_Ab001, ASC_Ab004, and ASC_Ab008 bound to full-length ASC, ASC-b, and the two mutant ASCs, but not to isoforms ASC-c and ASC-d.

[0333] This demonstrates that the epitopes bound by antibodies ASC_Ab001, ASC_Ab004, and ASC_Ab008 are present in the region spanning amino acids 36 to 85 of ASC (not present in ASC-c and ASC-d).

[0334] To confirm and extend these findings to further antibodies of the invention, antibodies ASC_Ab001, ASC_Ab002, ASC_Ab004, ASC_Ab005, ASC_Ab006, ASC_Ab007, ASC_Ab008, and control antibody AL-177 were assessed by ELISA for binding to ASC peptides covering amino acids 1-20 (SEQ ID NO: 148) or amino acids 11-30 (SEQ ID NO: 149). As an additional control, we used the nanobody VHH described in Schmidt et al., 2016 (Schmidt FI, 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. J Exp Med. 2016 May 2;213(5):771-90. doi: 10.1084 / jem.20151790). ASC As described in Schmidt et al., 2016, VHH ASC binds to the CARD domain of ASC, and therefore is not expected to bind to the N-terminal peptide.

[0335] In further experiments, antibodies ASC_Ab001 and ASC_Ab002 against the ASC peptide covering amino acids 1 to 20 (SEQ ID NO: 148), amino acids 11 to 30 (SEQ ID NO: 149) or amino acids 21 to 40 (SEQ ID NO: 150) were tested against the prior art antibodies AL-177 and VHH ASCThe binding of the antibody was assessed by ELISA. Additionally, as described in U.S. Patent No. 10,961,306 B2, column 40, lines 60-64, a human IgG1 antibody was generated containing a VH of SEQ ID NO: 19 and a VL of SEQ ID NO: 30 in U.S. Patent No. 10,961,306 B2, which can be designated "IC-100." According to U.S. Patent No. 10,961,306 B2, this antibody is designated "IC100" and was included in this experiment.

[0336] The results of the peptide binding experiments are shown in Figure 4. Figure 4(A) shows the results of the first experiment. Only the control antibody AL-177 bound to the N-terminal peptide, which was expected since it binds to all four ASC isoforms. As expected, VHHs that bind to CARD ASC No binding to the N-terminal peptide was observed for antibodies ASC_Ab001, ASC_Ab002, ASC_Ab004, ASC_Ab005, ASC_Ab006, ASC_Ab007, and ASC_Ab008. This confirmed the results obtained with the distinct ASC isoforms ASC_Ab001, ASC_Ab004, and ASC_Ab008, and demonstrated that ASC_Ab002, ASC_Ab005, ASC_Ab006, and ASC_Ab007 also do not bind to the N-terminal portion of ASC. Figure 4(B) shows the results obtained with ASC peptides covering amino acids 1-20 (SEQ ID NO: 148), amino acids 11-30 (SEQ ID NO: 149), or amino acids 21-40 (SEQ ID NO: 150). As in the previous experiment, AL-177 was found to bind to the ASC peptide covering amino acids 1-20 (SEQ ID NO: 148) and amino acids 11-30 (SEQ ID NO: 149), but not to the VHH ASC Neither ASC_Ab001 nor 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 peptides covering amino acids 11-30 (SEQ ID NO: 149) or amino acids 21-40 (SEQ ID NO: 150). None of the other antibodies bound to the peptide covering amino acids 21-40 (SEQ ID NO: 150).

[0337] In summary, this ASC peptide analysis showed that AL-177 binds to an epitope within amino acids 1 to 30 of ASC, and IC100 binds to an epitope within amino acids 11 to 40 of ASC. None of the antibodies of the present invention were found to bind to such N-terminal peptides.

[0338] Example 5: Comparison with prior art antibodies As described above (see also Schmidt et al., 2016) and demonstrated in Example 4, antibodies AL-177, IC100 and anti-CARD antibody VHH ASC binds to a distinct epitope of ASC compared to the antibodies of the present invention. For further comparison of the antibodies of the present invention with prior art antibodies, antibodies ASC_Ab001 and ASC_Ab002, as well as prior art antibody AL-177, VHH ASC and IC100 (obtained as described in Example 4 above) to (i) human ASC, (ii) mouse ASC, (iii) ASC speck, and (iv) the PYD domain of ASC were assessed by ELISA as described above.

[0339] The results are shown in Figure 5 (human ASC), Figure 6 (mouse ASC), Figure 7 (ASC speck), and Figure 8 (PYD domain of ASC).

[0340] All tested antibodies were found to bind to human ASC and ASC speck, except for IC100. Surprisingly, no antibody "IC100" or "IC-100" was found, despite previous data obtained by other researchers (U.S. Patent No. 10,961,306 B2, and Desu HL, Plastini M, Illiano 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. 2020 May 4;17(1):143. doi: 10.1186 / s12974-020-01826-0) and despite the data obtained in Example 4 showing IC100 binding to peptides of SEQ ID NO: 149 and SEQ ID NO: 150, no IC100 was detectable for binding to the (entire) PYD domain of full-length human ASC, mouse ASC, ASC speck and ASC (human IgG1 antibody containing VH of SEQ ID NO: 19 and VL of SEQ ID NO: 30 in U.S. Pat. No. 10,961,306 B2, as described in column 40, lines 60-64 of U.S. Pat. No. 10,961,306 B2). ASC No binding was detected for ASC, which was expected since this antibody has previously been reported to bind to the CARD domain of ASC.

[0341] For mouse ASC, only antibodies ASC_Ab001 and ASC_Ab002 showed binding, whereas AL-177, IC100, and VHH ASC Therefore, only antibodies ASC_Ab001 and ASC_Ab002 showed binding to mouse ASC (in addition to human ASC), and therefore AL-177, IC100, and VHH ASCshowed favorable cross-reactivity not seen in

[0342] Example 6: Antibody binding to physiological ASC specks To examine the binding of the antibodies of the present invention to physiological ASC speck, THP-1 asc - / - The cells were compared with THP-1 wt cells. THP-1 is a human macrophage cell line and is commercially available. THP-1 asc - / - In contrast to THP-1 wt cells, THP-1 cells lack ASC expression, so physiological ASC speck formation is expected only in THP-1 wt cells, and not in THP-1 asc - / - Cells represent a negative control.

[0343] As detailed above, THP-1 wt cells and THP-1 asc - / - Cells were induced to form ASC specks. Then, cells were incubated with anti-ASC antibodies ASC_Ab001, ASC_Ab002, ASC_Ab003, ASC_Ab004, ASC_Ab005, ASC_Ab006, ASC_Ab007, ASC_Ab008, ASC_Ab009, or an isotype control, as detailed above. Results were obtained by fluorescence imaging.

[0344] The results are shown in Figure 9. These data demonstrate 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 specks.

[0345] Example 7: Antibody binding specificity To confirm the binding and species cross-reactivity of antibodies ASC_Ab001, ASC_Ab004, ASC_Ab007, and ASC_Ab009 to endogenous ASC, the antibodies were tested against wild-type and ascAb as detailed above. - / - The antibodies were tested by Western blot using human THP-1 monocytes as well as mouse bone marrow-derived macrophages (BMDM).

[0346] The results are shown in Figure 10 (wt and asc - / - The results are shown in Figure 11 (human THP-1 cells) and Figure 11 (BMDM). All tested antibodies bound to endogenous human and mouse ASC in cell lysates of wt cells, but not to asc - / - No binding was observed in cell lysates.

[0347] Example 8: Treatment with antibodies of the present invention significantly inhibited ASC fibrogenesis ASCs assemble into large cytoplasmic macromolecular assemblies called ASC specks, which are driven by ASC-ASC interactions and can be reconstituted in vitro by incubating ASC monomers at 37°C. Under these conditions, ASCs rapidly undergo fibrillogenesis and retain their physiological and pathological functions (Friker LL, Scheiblich H, Hochheiser IV, Brinkschulte R, Riedel D, Latz E, Geyer M, Heneka MT. β-Amyloid Clustering around ASC Fibrils Boosts Its Toxicity in Microglia. Cell Rep. 2020 Mar 17;30(11):3743-3754.e6. doi: 10.1016 / j.celrep.2020.02.025). The effect of the antibody on fibril formation provides a way to block either new or disseminated ASC assembly, and therefore the downstream effects of ASC speck formation, such as inflammation and / or amyloid dissemination.

[0348] To examine the effects of the anti-ASC antibodies ASC_Ab001, ASC_Ab004, ASC_Ab007, ASC_Ab008, and ASC_Ab009 on ASC fibrillogenesis, we performed an ASC fibrillogenesis assay as described above. Briefly, ASC-GFP-His was incubated with the antibody to be tested, and protein aggregates were photographed.

[0349] The results are shown in Figure 12. These data show that each of the antibodies ASC_Ab001, ASC_Ab004, ASC_Ab007, ASC_Ab008, and ASC_Ab009 significantly inhibited ASC fibril formation compared to the isotype control. The anti-ASC-CARD antibody used as a control had no effect, but the anti-CARD antibody VHH ASC increased ASC fibrogenesis.

[0350] Furthermore, a precipitation assay to evaluate the anti-fibrogenic properties of the antibodies was performed as described above. Briefly, the soluble and insoluble fractions of ASCs treated with anti-ASC antibodies or isotype controls were separated by high-speed centrifugation.

[0351] The results are shown in Figure 13. Treatment with ASC_Ab001 resulted in more soluble ASC (supernatant fraction) after 30 minutes of incubation at 37°C compared to ASC alone or the isotype control. In other words, ASC_Ab001 shifts the equilibrium toward soluble ASC in the supernatant fraction. Similar to the ASC fibrillogenesis assay described above, treatment with anti-CARD antibodies did not affect fibrillogenesis.

[0352] Example 9: Anti-ASC antibodies block ASC-Aβ interaction ASC-Aβ interactions are thought to underlie not only the increased Aβ aggregation and amyloid deposition in Alzheimer's 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-β in Alzheimer's disease. Nature. 2017 Dec 20;552(7685):355-361. doi: 10.1038 / nature25158), but also the increased neuroinflammation caused by these ASC-Aβ complexes (Friker LL, Scheiblich H, Hochheiser IV, Brinkschulte R, Riedel D, Latz E, Geyer M, Heneka MT. β-Amyloid Clustering around ASC Fibrils Boosts Its Toxicity in Microglia. Cell Rep. 2020 Mar 17;30(11):3743-3754.e6. doi: 10.1016 / j.celrep.2020.02.025). Therefore, measuring the effect of antibodies on ASC-Aβ interactions can test the potential of antibodies to block the downstream effects of amyloid deposition and increased neuroinflammation.

[0353] Therefore, we investigated whether the antibodies of the present invention can reduce or inhibit ASC-Aβ binding. For this purpose, an ASC-Aβ interaction assay was performed as described above. Protein aggregates of the tested antibodies were then separated into ASC only, ASC + Aβ, VHH ASCThe binding (aggregation) was compared with that obtained with controls such as an isotype control antibody and an ASC-Aβ antibody. ASC-Aβ inhibition was determined as a percentage of the binding (aggregation) observed with the ASC + Aβ (no antibody) control and the ASC only control.

[0354] The results are shown in Figure 14. These data show that antibodies ASC_Ab001, ASC_Ab002, ASC_Ab003, ASC_Ab004, ASC_Ab005, and ASC_Ab006 significantly reduced ASC-Aβ interactions, while the anti-CARD antibody VHH ASC further increases ASC-Aβ interaction.

[0355] Example 10: ASC_Ab001 inhibits the pro-inflammatory function of extracellular ASC fibers. The in vitro efficacy of ASC_Ab001 was tested in a cell-based assay using differentiated human THP-1 macrophages treated with extracellular ASC fibers obtained as described above. ASC fibers and specks have been shown to be able to 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 AG, 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. Nat Immunol. 2014 Aug;15(8):727-37. doi: 10.1038 / ni.2913; Baroja-Mazo A, Martin-Sanchez F, Gomez AI, Martinez CM, Amores-Iniesta J, Compan V, Barbera-Cremades M, Yague J, Ruiz-Ortiz E, Anton J, Bujan S, Couillin I, Brough D, Arostegui JI, Pelegrin P. The NLRP3 inflammasome is released as a particulate danger signal that amplifies the inflammatory response. Nat Immunol. 2014 Aug;15(8):738-48. doi: 10.1038 / ni.2919).As detailed above, ASC fibers were preincubated with ASC_Ab001 and an isotype control antibody, each with or without the Leu234Ala / Leu235Ala / Pro329Gly ("PGLALA") Fc modification, before being added to cells. Measurement of IL-1β was used as a readout for inflammasome activation.

[0356] The results are shown in Figure 15. ASC_Ab001 significantly reduced ASC fiber-induced IL-1β production compared to the isotype control, demonstrating the efficacy of ASC_Ab001. This reduction was independent of the Fc function of the antibody, with no difference observed between wt IgG and PGLALA (Fc inactive) formats. Treatment with antibody alone did not affect IL-1β production.

[0357] Example 11: Anti-ASC antibodies dose-dependently reduced IL-1β release in an ASC-seeded inflammation assay. Wild-type THP-1 monocytes were seeded at a density of 20,000 cells / well in 384-well plates in RPMI medium supplemented with Glutamax, 10% FBS, 1% P / S, 100 μg / mL Normocin, and 100 μg / mL Zeocin. To differentiate monocytes into macrophages, cells were incubated in medium containing 100 ng / mL phorbol 12-myristate-13-acetate (PMA) at 37°C and 5% CO2 for 4 days. After differentiation, cells were washed twice with fresh cell culture medium (10 min each time) and primed with LPS (100 ng / mL) for 3 hours. ASC fibers were prepared by incubating ASC-his monomers in sterile PBS for 1 hour at 37°C. Before adding ASC fibers to the cells, they were preincubated with antibodies for 15 minutes at room temperature. After 48 h of incubation at 37°C and 5% CO , secreted IL-1β was measured using the Lumit Human IL-1 Immunoassay (Promega, W6010) according to the manufacturer's protocol.

[0358] As shown in FIG. 16, all of the tested anti-ASC antibodies (ASC_AB001 to ASC_Ab009) reduced IL-1β release in a dose-dependent manner.

[0359] Example 12: Anti-ASC antibodies do not bind to proteins with sequences homologous to ASC The binding of anti-ASC antibodies (ASC_AB001-ASC_Ab009) to the inflammasome proteins AIM2 and NLRP3 was tested by binding ELISA. High-binding clear flat-bottom 384-well plates (Greiner Bio-One, 781061) were coated overnight (4°C) with 10 μl / well of AIM2 (1 μg / mL, Sino Biological, cat: 11654-H09B) or NLRP3 (1 μg / mL, cusabio, CSB-EP822275HU1) diluted in PBS. After overnight incubation, wells were aspirated and washed once with 100 μl / well of wash buffer (0.05% Tween-20 in PBS). The wells were blocked with 50 μl of 2% BSA diluted in PBS for 1 hour at room temperature. After blocking, the wells were incubated with 10 μl of serially diluted test antibodies for 2 hours at room temperature. The wells were washed four times and incubated consecutively with 10 μl / well of detection antibody for 1 hour at room temperature. Unbound detection antibody was removed by washing four times, and the wells were incubated with 20 μl of TMB for 5 minutes. The reaction was stopped by adding 10 μl of 1 M H2SO4 per well. The absorbance at 450 nm was immediately measured using an EnSpire 2300 microplate reader (Perkin Elmer).

[0360] As shown in Figure 17, the anti-ASC antibodies did not bind to the inflammasome proteins AIM2 and NLRP3, which contain sequences homologous to ASC, confirming the specificity of the anti-ASC antibodies (ASC_AB001 to ASC_Ab009) to ASC.

[0361] Example 13: Anti-ASC antibody ameliorates the severity of dextran sulfate sodium (DSS)-induced colitis. Acute colitis was induced in 8-week-old female C57BL / 6 mice by administering 2.5% dextran sodium sulfate (DSS) in their drinking water for 8 days. Healthy animals (n = 8) received normal drinking water. Two hours before colitis induction, anti-ASC antibody (n = 8 per group) or normal IgG (n = 8) as a control was injected intraperitoneally. Mice were sacrificed on day 8. The disease activity index (DAI) was measured based on the following parameters: weight change, stool consistency, and the presence or absence of fecal occult blood during DSS administration. For H&E staining, sections were cut from paraffin blocks of colon tissue pretreated with 10% neutral buffered formalin (Fujifilm Wako Pure Chemical Corporation, Japan) and stained with Lilly-Meyer's hematoxylin (Muto Chemical Co., Japan) and eosin solution (Fujifilm Wako Pure Chemical Corporation). Representative photographs of the observed changes were taken from each group.

[0362] As shown in Figure 18 (A: body weight measurement; B: Disease Activity Index (DAI) score measurement; C: colon length measurement), the anti-ASC antibodies ASC_Ab001 and ASC_Ab002 ameliorate the severity of DSS-induced colitis compared to the isotype control.

[0363] This finding is also supported by Figure 19, where Figures 19A-C (hematoxylin and eosin staining of colon) show the beneficial effect of ASC_Ab001 on mucosal morphology of isolated intestinal segments in DSS-induced colitis, and Figure 19D shows reduced infiltration of inflammatory cells into the intestinal mucosa in animals treated with anti-ASC antibody versus isotype (normal IgG).

[0364] Example 14: Humanization of antibodies maintains binding to ASC To investigate the effect of humanization, a humanized version of ASC_Ab001 was generated. The humanized antibody (ASC_Ab001 humanized) contained a VH set forth in SEQ ID NO: 157 (comprising the heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively) and a VL set forth in SEQ ID NO: 158 (comprising the light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively).

[0365] Binding of humanized ASC_Ab001 to human ASC was compared to (rabbit) ASC_Ab001 having the VH and VL sequences set forth in SEQ ID NO: 8 and SEQ ID NO: 9, respectively, in an ELISA as described above.

[0366] The results are shown in Figure 20. These data show that similar binding of the humanized antibodies was observed compared to the rabbit IgG format.

[0367] Example 15: Construction and evaluation of further humanized anti-ASC antibodies and antigen-binding fragments thereof Further humanized antibodies were designed based on the "parent" antibodies ASC_Ab001 and ASC_Ab002, respectively.

[0368] The amino acid sequences of the humanized variable heavy chains (VH) based on ASC_Ab001 (Ab001_VHhum1, Ab001_VHhum3, and Ab001_VHhum23) are shown in Figure 21. All heavy chain sequences were based on the IGHV3-66 germline, which has the highest homology to the original rabbit Ab001 heavy chain framework and has also been reported to be a good acceptor framework for rabbit CDRs. Kinetic and binding studies used humanized constructs containing Ab001_VHhum23, respectively.

[0369] The amino acid sequences of the humanized variable light chains (VL) based on ASC_Ab001 (Ab001_VLhum1, Ab001_VLhum4, Ab001_VLhum5, Ab001_VLhum6, Ab001_VLhum10, Ab001_VLhum11, and Ab001_VLhum12) are shown in Figure 22. All light chain sequences were based on the IGKV1-5 germline, which shares the highest homology with the original rabbit Ab001 light chain framework and is very similar to the IGKV1-27*01 germline, which was previously used as a template for the universal acceptor frame of the rabbit light chain CDRs. Humanized constructs containing Ab001_VLhum11 and Ab001_VLhum12, respectively, were used in kinetic and binding studies.

[0370] The amino acid sequences of the humanized variable heavy chain (VH) (Ab002_VHhum17) based on ASC_Ab002 and the humanized variable light chain (VL) (Ab002_VLhum1, Ab002_VLhum6, Ab002_VLhum11, Ab002_VLhum12) based on ASC_Ab002 are shown in Figure 23. The sequences shown for the heavy chains are based on the IGHV3-66 germline, which has the highest homology to the original rabbit Ab002 heavy chain framework and has also been reported to be a good acceptor framework for rabbit CDRs. All heavy chain sequences are based on the IGKV1-27 germline, which has the highest homology to the original rabbit Ab002 light chain framework and has previously been used as a template for the universal acceptor frame for rabbit light chain CDRs. Kinetic measurements and binding studies used humanized constructs containing Ab002_VHhum17 and Ab002_VLhum12, respectively.

[0371] [Fab kinetics measurement] Fab kinetic measurements were performed by biolayer interferometry (BLI) using a RED96e OCTET system (Sartorius AG). In-house expressed recombinant human autosomal dominant cell (hASC) was covalently immobilized to an AR2G biosensor (Sartorius AG) at a concentration of 1.25 μg / mL in 10 mM sodium phosphate buffer, pH 5, for 300 s via EDC / NHS-mediated amine coupling. Binding and dissociation kinetics were measured for 200 s and 500 s, respectively, at Fab concentrations of 50 nM, 16.67 nM, 5.55 nM, 1.85 nM, 0.617 nM, and 0 nM in 1x PBS buffer (pH 7.4) supplemented with 0.1% BSA and 0.02% Tween. The antibody was released from the ASC by immersing the biosensor in regeneration buffer (10 mM glycine, pH 3.0). The binding rate constant (k on ), and the dissociation rate constant (k off ) was calculated using a 1:1 binding model with a global fit. on rate constant, and k off The rate constants are shown in Table 10 (for ASC_Ab001 Fab) and Table 11 (for ASC_Ab002 Fab), respectively.

[0372] [Table 10]

[0373] [Table 11]

[0374] The binding and dissociation curves are shown in Figure 24 (for Fabs containing Ab001 VH23 / VL11, Ab001 VH23 / VL12, and Ab001 VHrab / VLrab, respectively) and Figure 25 (for Fabs containing Ab002 VH17 / VL12 and Ab002 VHrab / VLrab, respectively).

[0375] As shown in Table 10 and Figure 24, the tested humanized Ab001 (RP02-F8) Fab has a superior binding rate but a poorer dissociation rate than the parental rabbit Fab, thus the overall affinity of the humanized Ab001 Fab for hASCs is only slightly reduced compared to the parental rabbit Fab.

[0376] As shown in Table 11 and Figure 25, the humanized Ab002 (RP03-12) Fab tested exhibited binding and dissociation rates similar to those of the parental Fab.

[0377] These data indicate that Fabs containing humanized VH and VL domains (Ab001 VH23 / VL11, Ab001 VH23 / VL12, Ab002 VH17 / VL12) have overall affinities for hASCs similar to those of their respective parental (non-humanized) rabbit antibodies (Ab001 VHrab / VLrab, Ab002 VHrab / VLrab).

[0378] Example 16: Humanized IgG1 antibodies have affinity for human and mouse ASC The binding of the whole IgG1 humanized ASC_Ab001 and ASC_Ab002 antibodies to human and mouse ASC was compared to Ab001 rb-huIgG (rabbit) and Ab002 rb-huIgG (non-humanized), respectively, in the ELISA described above.

[0379] The results are shown in Figure 26 (for Ab001 VH23 / VL11, Ab001 VH23 / VL12 compared to Ab001 rb-huIgG) and Figure 27 (for Ab002 VH17 / VL12 compared to Ab002 rb-huIgG).

[0380] These data demonstrate that the affinity of the humanized (bivalent) Ab001 IgG1 antibody to human and mouse ASC is superior compared to the rabbit Ab001 IgG format (Figure 26), and that the apparent affinity of the humanized (bivalent) Ab002 IgG1 antibody is similar compared to the rabbit Ab002 IgG format (Figure 27).

[0381] [Table 12] [Table 13] [Table 14] [Table 15] [Table 16] [Table 17] [Table 18] [Table 19] [Table 20] [Table 21] [Table 22] [Table 23] [Table 24] [Table 25] [Table 26] [Table 27] [Table 28] [Table 29] [Table 30] [Table 31] [Table 32] [Table 33] [Table 34] [Brief explanation of the drawings]

[0382] [Figure 1] FIG. 1 shows binding curves of an exemplary antibody, ASC_Ab001, to full-length ASC, ASC speck, CARD domain, PYD domain, mouse ASC, and BSA, as tested by ELISA. [Figure 2]Figure 2 shows an alignment of various ASC isoforms for Example 4: 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). [Figure 3] Figure 3 shows representative images of binding patterns for Example 4: (A) ASC_Ab001, (B) ASC_Ab008, (C) ASC_Ab004, and (D) control antibody (AL-177, Adipogen). In all blots, lane 1 shows binding to ASC-b 20 kDa, lane 2 shows binding to ASC-d 11.8 kDa, lane 3 shows binding to ASC-c 15 kDa, lane 4 shows binding to ASC Y146A 21.5 kDa, lane 5 shows binding to ASC K21A K22A K26A 21.5 kDa, lane 6 shows binding to UT control, and lane 7 shows binding to purified ASC-HIS 23 kDa. [Figure 4] Figure 4 shows binding of the indicated anti-ASC antibodies and control antibodies to the indicated ASC peptides for Example 4. (A) Binding of ASC_Ab001, ASC_Ab002 clonotype, ASC_Ab004, ASC_Ab005, ASC_Ab006, ASC_Ab007, ASC_Ab008 clonotype, AL-177, and VHHASC to peptides covering amino acids 1-20 and amino acids 11-30 of ASC. (B) Binding of ASC_Ab001, ASC_Ab002, AL-177, IC100, and VHHASC to peptides covering amino acids 1-20, amino acids 11-30, and amino acids 21-40 of ASC. [Figure 5] FIG. 5 shows ELISA binding curves of antibodies ASC_Ab001, ASC_Ab002, AL-177, VHHASC and IC100 against human ASC for Example 5. [Figure 6] FIG. 6 shows ELISA binding curves of antibodies ASC_Ab001, ASC_Ab002, AL-177, VHHASC, and IC100 against mouse ASC for Example 5. [Figure 7]FIG. 7 shows the ELISA binding curves of antibodies ASC_Ab001, ASC_Ab002, AL-177, VHHASC, and IC100 against ASC speck for Example 5. [Figure 8] FIG. 8 shows ELISA binding curves of antibodies ASC_Ab001, ASC_Ab002, AL-177, VHHASC, and IC100 against the PYD domain of ASC for Example 5. [Figure 9] Figure 9 shows (A) quantification of ASC specks detected in Thp1 wt or asc- / - cells primed with LPS (1 μg / mL), stimulated with nigericin (5 μM), and stained with 1 μg / mL of anti-ASC antibody or isotype control, and (B) representative immunofluorescence photographs of THP-1 asc + / + and THP-1 asc- / - cells stained with ASC_Ab001 antibody, for Example 6. [Figure 10] FIG. 10 shows 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 for Example 7. [Figure 11] FIG. 11 shows Western blot images of antibodies ASC_Ab001, ASC_Ab004, ASC_Ab007, and ASC_Ab009 on cell lysates of mouse bone marrow-derived macrophages (BMDM) for Example 7. [Figure 12] Figure 12 shows that treatment with ASC_Ab001, ASC_Ab004, ASC_Ab007, ASC_Ab008, and ASC_Ab009 significantly inhibited ASC fibril formation for Example 8. The effect of anti-ASC antibodies on ASC fibril formation was assessed by IF of ASC fibers formed after incubating ASC-gfp with anti-ASC antibodies or an isotype control for 1 hour at 37°C. (A) Representative photographs, and (B) quantification of the number of ASC fibers per well treated with anti-ASC antibodies. Pooled data from n = 1 or 2 independent experiments. One-way ANOVA followed by Dunnett's multiple comparison test. ns: not significant, ****: p-value < 0.0001. [Figure 13]Figure 13 shows the results of a precipitation assay to evaluate the anti-fibrogenic properties of anti-ASC antibodies for Example 8. Soluble and insoluble fractions of ASC were separated by high-speed centrifugation after treatment with anti-ASC antibodies or an isotype control. After 30 minutes of incubation at 37°C, treatment with ASC_Ab001 yielded more soluble ASC (supernatant fraction) compared with treatment with ASC alone or an isotype control. In other words, ASC_Ab001 shifts the equilibrium toward soluble ASC in the supernatant fraction. Treatment with anti-CARD antibodies did not affect fibrogenesis. [Figure 14] Figure 14 shows that anti-ASC antibody blocks ASC-Aβ interaction for Example 9. Inhibition rate of ASC-Aβ binding after treatment with anti-ASC antibody (3.5uM) or isotype control (3.5uM). One-way ANOVA followed by Dunnett's multiple comparison test. ***: p-value < 0.001. [Figure 15] Figure 15 (Example 10) shows that ASC_Ab001 blocks the pro-inflammatory function of extracellular ASC fibers. The in vitro efficacy of ASC_Ab001 was tested in a cell-based assay using differentiated human THP-1 macrophages treated with extracellular ASC fibers. Before adding ASC fibers to the cells, the ASC fibers were preincubated with ASC_Ab001 and an isotype control antibody. Measurement of IL-1β was used as a readout of inflammasome activation. ASC_Ab001 significantly reduced ASC fiber-induced IL-1β production compared to the isotype control. [Figure 16] FIG. 16 shows quantification of IL-1 beta release by ASC fibers and differentiated human macrophages treated with 0.0035 nM or 35 nM of anti-ASC antibodies (ASC_Ab001 to ASC_Ab009) and isotype control, respectively, in an ASC-seeded inflammation assay for Example 11. [Figure 17] FIG. 17 for Example 12 shows that anti-ASC antibodies (ASC_Ab001 to ASC_Ab009) do not bind to inflammasome proteins AIM2 and NRLP3, which have sequences homologous to ASC, as determined by binding ELISA. [Figure 18] Figure 18 (Example 13) shows that anti-ASC antibodies ASC_Ab001 and ASC_Ab002 ameliorate the severity of dextran sulfate sodium (DSS)-induced colitis in an acute inflammatory bowel disease model. (A) Body weight measurement; (B) Disease Activity Index measurement; and (C) Colon length measurement. Data represent mean ± SEM. Multiple comparisons with isotype were performed using two-way ANOVA. *p<0.05, ***p<0.01, ***p<0.001, ***p<0.0001. [Figure 19] Figure 19 shows representative photographs of hematoxylin-eosin stained colons from N=8 mice per group (A: healthy; B: isotype control; C: ASC_Ab001) for Example 13. Figure 19D shows inflammatory cell infiltration into the mucosa of healthy, isotype-, and Asc_Ab001-treated mice. [Figure 20] Figure 20 shows that humanized ASC_Ab001 retains binding to ASC for Example 14. Binding of humanized ASC_Ab001 to ASC-His was assessed by ELISA. Similar binding of the humanized antibody was observed compared to the rabbit IgG format. [Figure 21] Figure 21 shows the VH amino acid sequences of the humanized Ab001 antibody. All heavy chain sequences were based on the IGHV3-66 germline, which has the highest homology with the original rabbit Ab001 heavy chain framework and has also been reported to be a good acceptor framework for rabbit CDRs. In Example 15, humanized Ab001 antibodies, including Ab001_VHhum23, were used. [Figure 22] Figure 22 shows the VL amino acid sequences of the humanized antibodies. All light chain sequences were based on the IGKV1-5 germline, which has the highest homology to the original rabbit Ab001 light chain framework and is very similar to the IGKV1-27*01 germline, which was previously used as a template for the universal acceptor frame of the rabbit light chain CDRs. In Example 15, humanized Ab001 antibodies were used, including Ab001_VLhum11 and Ab001_VLhum12, respectively. [Figure 23] Figure 23 shows the amino acid sequences of the VH and VL of the humanized Ab002 antibody. The sequence shown for the heavy chain is based on the IGHV3-66 germline, which has the highest homology to the original rabbit Ab002 heavy chain framework and has also been reported to be a good acceptor framework for rabbit CDRs. All heavy chain sequences are based on the IGKV1-27 germline, which has the highest homology to the original rabbit Ab002 light chain framework and was previously used as a template for the universal acceptor frame for rabbit light chain CDRs. Humanized Ab002 antibodies, including Ab002_VHhum17 and Ab002_VLhum12, were used in Example 15. [Figure 24] Figure 24 shows kinetic measurements of humanized Ab001 Fab: Ab001 VH23 / VL11 (A), and Ab001 VH23 / VL12 (B) versus non-humanized Ab001 VHrab / VLrab (C) for Example 15. By biolayer interferometry (BLI) using a RED96e OCTET system (Sartorius AG). [Figure 25] Figure 25 shows kinetic measurements of humanized Ab002 Fab: Ab002 VH16 / VL12 (A) vs. non-humanized Ab002 VHrab / VLrab (B) for Example 15. By biolayer interferometry (BLI) using a RED96e OCTET system (Sartorius AG). [Figure 26] Figure 26 shows binding of fully IgG1 humanized Ab001 antibodies Ab001 VH23 / VL11 and Ab001 VH23 / VL12, and Ab001 rb-huIgG (non-humanized), to human ASC (A) and mouse ASC (B) for Example 16. Measured by binding ELISA. [Figure 27] Figure 27 shows binding of fully IgG1 humanized Ab002 antibody Ab002 VH17 / VL12 and Ab002 rb-huIgG (non-humanized) to human ASC (A) and mouse ASC (B) for Example 16. Measured by binding ELISA.

Claims

1. An antibody or antigen-binding fragment thereof that binds to the PYRIN-PAAD-DAPIN domain (PYD) of CARD-containing apoptosis-associated speck-like protein (ASC).

2. the antibody or antigen-binding fragment thereof binds to the PYD domain of human ASC (SEQ ID NO: 1); The antibody or antigen-binding fragment thereof described in claim 1.

3. The antibody or antigen-binding fragment thereof specifically binds to ASC-b (SEQ ID NO: 145). The antibody or antigen-binding fragment thereof according to claim 1 or 2.

4. The antibody or antigen-binding fragment thereof does not specifically bind to ASC-c (SEQ ID NO: 146).

10. The antibody or antigen-binding fragment thereof of any one of the preceding claims.

5. The antibody or antigen-binding fragment thereof does not specifically bind to ASC-d (SEQ ID NO: 147).

10. The antibody or antigen-binding fragment thereof of any one of the preceding claims.

6. the antibody or the antigen-binding fragment thereof binds to human ASC K21A K22A K26A and / or human ASC Y146A; 10. The antibody or antigen-binding fragment thereof of any one of the preceding claims.

7. The antibody or the antigen-binding fragment thereof does not bind to the ASC peptide set forth in SEQ ID NO: 148 (ASC aa 1-20 peptide) or the ASC peptide set forth in SEQ ID NO: 149 (ASC aa 11-30 peptide).

10. The antibody or antigen-binding fragment thereof of any one of the preceding claims.

8. The antibody or the antigen-binding fragment thereof does not bind to the ASC peptide set forth in SEQ ID NO: 150 (ASC aa 21-40 peptide).

10. The antibody or antigen-binding fragment thereof of any one of the preceding claims.

9. the antibody or antigen-binding fragment thereof binds to an epitope located in a region of the PYD domain spanning amino acids 36-85 of human ASC set forth in SEQ ID NO: 1, preferably an epitope located in a region of the PYD domain spanning amino acids 41-85 of human ASC set forth in SEQ ID NO: 1; 10. The antibody or antigen-binding fragment thereof of any one of the preceding claims.

10. the antibody or the antigen-binding fragment thereof binds to mouse ASC; 10. The antibody or antigen-binding fragment thereof of any one of the preceding claims.

11. The antibody or the antigen-binding fragment thereof exhibits anti-ASC fibrogenesis activity.

10. The antibody or antigen-binding fragment thereof of any one of the preceding claims.

12. the antibody or the antigen-binding fragment thereof reduces or blocks ASC-Aβ interaction; 10. The antibody or antigen-binding fragment thereof of any one of the preceding claims.

13. the antibody or the antigen-binding fragment thereof reduces or blocks the pro-inflammatory function of extracellular ASC fibers; 10. The antibody or antigen-binding fragment thereof of any one of the preceding claims.

14. the antibody or the antigen-binding fragment thereof comprises a CDRH1 set forth in SEQ ID NO:2, a CDRH2 set forth in SEQ ID NO:3, a CDRH3 set forth in SEQ ID NO:4, a CDRL1 set forth in SEQ ID NO:5, a CDRL2 set forth in SEQ ID NO:6, and a CDRL3 set forth in SEQ ID NO:7; 10. The antibody or antigen-binding fragment thereof of any one of the preceding claims.

15. the antibody or antigen-binding fragment thereof comprises an extended CDRH1 set forth in SEQ ID NO: 159, an extended CDRH2 set forth in SEQ ID NO: 3, an extended CDRH3 set forth in SEQ ID NO: 160, an extended CDRL1 set forth in SEQ ID NO: 5, an extended CDRL2 set forth in SEQ ID NO: 6, and an extended CDRL3 set forth in SEQ ID NO: 7; The antibody or antigen-binding fragment thereof described in claim 14.

16. The antibody or antigen-binding fragment thereof may comprise an extended CDRH1 (comprising CDRH1 and adjacent framework residues) set forth in SEQ ID NO: 194, an extended CDRH2 (comprising CDRH2 and adjacent framework residues) set forth in SEQ ID NO: 195, an extended CDRH3 (comprising CDRH3 and adjacent framework residues) set forth in SEQ ID NO: 196, an extended CDRL1 (comprising CDRL1 and adjacent framework residues) set forth in SEQ ID NO: 197, an extended CDRL2 (comprising CDRL2 and adjacent framework residues) set forth in SEQ ID NO: 198, and an extended CDRL3 (comprising CDRL3 and adjacent framework residues) set forth in SEQ ID NO:

199. An antibody or antigen-binding fragment thereof according to claim 14 or 15.

17. The antibody or antigen-binding fragment thereof comprises a VH having at least 70% identity to SEQ ID NO: 8; and a VL having at least 70% identity to SEQ ID NO:

9. An antibody or antigen-binding fragment thereof according to any one of claims 14 to 16.

18. The antibody or antigen-binding fragment thereof comprises a VH having at least 80% identity to SEQ ID NO: 8; and a VL having at least 80% identity to SEQ ID NO:

9. An antibody or antigen-binding fragment thereof according to any one of claims 14 to 16.

19. The antibody or antigen-binding fragment thereof comprises a VH having at least 90% identity to SEQ ID NO:8; and a VL having at least 90% identity to SEQ ID NO:

9. An antibody or antigen-binding fragment thereof according to any one of claims 14 to 16.

20. the antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 8, and a VL comprising the amino acid sequence set forth in SEQ ID NO: 9; An antibody or antigen-binding fragment thereof according to any one of claims 14 to 16.

21. The antibody or antigen-binding fragment thereof comprises a VH having at least 70% identity to SEQ ID NO: 157; and a VL having at least 70% identity to SEQ ID NO:

158. An antibody or antigen-binding fragment thereof according to any one of claims 14 to 16.

22. The antibody or antigen-binding fragment thereof comprises a VH having at least 80% identity to SEQ ID NO: 157; and a VL having at least 80% identity to SEQ ID NO:

158. An antibody or antigen-binding fragment thereof according to any one of claims 14 to 16.

23. The antibody or antigen-binding fragment thereof comprises a VH having at least 90% identity to SEQ ID NO: 157; and a VL having at least 90% identity to SEQ ID NO:

158. An antibody or antigen-binding fragment thereof according to any one of claims 14 to 16.

24. the antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 157, and a VL comprising the amino acid sequence set forth in SEQ ID NO: 158; An antibody or antigen-binding fragment thereof according to any one of claims 14 to 16.

25. The antibody or antigen-binding fragment thereof comprises a VH having at least 70% identity to SEQ ID NO: 176; and a VL having at least 70% identity to SEQ ID NO:

177. An antibody or antigen-binding fragment thereof according to any one of claims 14 to 16.

26. The antibody or antigen-binding fragment thereof comprises a VH having at least 80% identity to SEQ ID NO: 176; and a VL having at least 80% identity to SEQ ID NO:

177. An antibody or antigen-binding fragment thereof according to any one of claims 14 to 16.

27. The antibody or antigen-binding fragment thereof comprises a VH having at least 90% identity to SEQ ID NO: 176; and a VL having at least 90% identity to SEQ ID NO:

177. An antibody or antigen-binding fragment thereof according to any one of claims 14 to 16.

28. the antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 176, and a VL comprising the amino acid sequence set forth in SEQ ID NO: 177; An antibody or antigen-binding fragment thereof according to any one of claims 14 to 16.

29. the antibody or the antigen-binding fragment thereof comprises a CDRH1 set forth in SEQ ID NO: 2, a CDRH2 set forth in SEQ ID NO: 10, a CDRH3 set forth in SEQ ID NO: 11, a CDRL1 set forth in SEQ ID NO: 12, a CDRL2 set forth in SEQ ID NO: 13, and a CDRL3 set forth in SEQ ID NO: 14; An antibody or antigen-binding fragment thereof according to any one of claims 1 to 13.

30. the antibody or antigen-binding fragment thereof comprises an extended CDRH1 set forth in SEQ ID NO: 161, an extended CDRH2 set forth in SEQ ID NO: 10, an extended CDRH3 set forth in SEQ ID NO: 162, an extended CDRL1 set forth in SEQ ID NO: 12, an extended CDRL2 set forth in SEQ ID NO: 13, and an extended CDRL3 set forth in SEQ ID NO: 14; 30. The antibody or antigen-binding fragment thereof of claim 29.

31. The antibody or antigen-binding fragment thereof comprises a VH having at least 70% identity to SEQ ID NO: 15; and a VL having at least 70% identity to SEQ ID NO:

16.

31. The antibody or antigen-binding fragment thereof of claim 29 or 30.

32. The antibody or antigen-binding fragment thereof comprises a VH having at least 80% identity to SEQ ID NO: 15; and a VL having at least 80% identity to SEQ ID NO:

16.

31. The antibody or antigen-binding fragment thereof of claim 29 or 30.

33. The antibody or antigen-binding fragment thereof comprises a VH having at least 90% identity to SEQ ID NO: 15; and a VL having at least 90% identity to SEQ ID NO:

16.

31. The antibody or antigen-binding fragment thereof of claim 29 or 30.

34. the antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 15, and a VL comprising the amino acid sequence set forth in SEQ ID NO: 16; 31. The antibody or antigen-binding fragment thereof of claim 29 or 30.

35. The antibody or antigen-binding fragment thereof comprises a VH having at least 70% identity to SEQ ID NO: 178; and a VL having at least 70% identity to SEQ ID NO:

179.

31. The antibody or antigen-binding fragment thereof of claim 29 or 30.

36. The antibody or antigen-binding fragment thereof comprises a VH having at least 80% identity to SEQ ID NO: 178; and a VL having at least 80% identity to SEQ ID NO:

179.

31. The antibody or antigen-binding fragment thereof of claim 29 or 30.

37. The antibody or antigen-binding fragment thereof comprises a VH having at least 90% identity to SEQ ID NO: 178; and a VL having at least 90% identity to SEQ ID NO:

179.

31. The antibody or antigen-binding fragment thereof of claim 29 or 30.

38. The antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 178, and a VL comprising the amino acid sequence set forth in SEQ ID NO:

179.

31. The antibody or antigen-binding fragment thereof of claim 29 or 30.

39. the antibody or the antigen-binding fragment thereof comprises a CDRH1 set forth in SEQ ID NO: 2, a CDRH2 set forth in SEQ ID NO: 17, a CDRH3 set forth in SEQ ID NO: 18, a CDRL1 set forth in SEQ ID NO: 19, a CDRL2 set forth in SEQ ID NO: 20, and a CDRL3 set forth in SEQ ID NO: 21; An antibody or antigen-binding fragment thereof according to any one of claims 1 to 13.

40. the antibody or antigen-binding fragment thereof comprises an extended CDRH1 set forth in SEQ ID NO: 161, an extended CDRH2 set forth in SEQ ID NO: 17, an extended CDRH3 set forth in SEQ ID NO: 163, an extended CDRL1 set forth in SEQ ID NO: 19, an extended CDRL2 set forth in SEQ ID NO: 20, and an extended CDRL3 set forth in SEQ ID NO: 21; 40. The antibody or antigen-binding fragment thereof of claim 39.

41. The antibody or antigen-binding fragment thereof comprises a VH having at least 70% identity to SEQ ID NO: 22; and a VL having at least 70% identity to SEQ ID NO:

23.

41. An antibody or antigen-binding fragment thereof according to claim 39 or 40.

42. The antibody or antigen-binding fragment thereof comprises a VH having at least 80% identity to SEQ ID NO: 22; and a VL having at least 80% identity to SEQ ID NO:

23.

41. An antibody or antigen-binding fragment thereof according to claim 39 or 40.

43. The antibody or antigen-binding fragment thereof comprises a VH having at least 90% identity to SEQ ID NO: 22; and a VL having at least 90% identity to SEQ ID NO:

23.

41. An antibody or antigen-binding fragment thereof according to claim 39 or 40.

44. the antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 22, and a VL comprising the amino acid sequence set forth in SEQ ID NO: 23; 41. An antibody or antigen-binding fragment thereof according to claim 39 or 40.

45. The antibody or antigen-binding fragment thereof comprises a VH having at least 70% identity to SEQ ID NO: 180; and a VL having at least 70% identity to SEQ ID NO:

181.

41. An antibody or antigen-binding fragment thereof according to claim 39 or 40.

46. The antibody or antigen-binding fragment thereof comprises a VH having at least 80% identity to SEQ ID NO: 180; and a VL having at least 80% identity to SEQ ID NO:

181.

41. An antibody or antigen-binding fragment thereof according to claim 39 or 40.

47. The antibody or antigen-binding fragment thereof comprises a VH having at least 90% identity to SEQ ID NO: 180; and a VL having at least 90% identity to SEQ ID NO:

181.

41. An antibody or antigen-binding fragment thereof according to claim 39 or 40.

48. The antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 180, and a VL comprising the amino acid sequence set forth in SEQ ID NO:

181.

41. An antibody or antigen-binding fragment thereof according to claim 39 or 40.

49. the antibody or antigen-binding fragment thereof comprises a CDRH1 set forth in SEQ ID NO: 24, a CDRH2 set forth in SEQ ID NO: 25, a CDRH3 set forth in SEQ ID NO: 26, a CDRL1 set forth in SEQ ID NO: 27, a CDRL2 set forth in SEQ ID NO: 28, and a CDRL3 set forth in SEQ ID NO: 29; An antibody or antigen-binding fragment thereof according to any one of claims 1 to 13.

50. the antibody or antigen-binding fragment thereof comprises an extended CDRH1 set forth in SEQ ID NO: 164, an extended CDRH2 set forth in SEQ ID NO: 25, an extended CDRH3 set forth in SEQ ID NO: 165, an extended CDRL1 set forth in SEQ ID NO: 27, an extended CDRL2 set forth in SEQ ID NO: 28, and an extended CDRL3 set forth in SEQ ID NO: 29; 50. The antibody or antigen-binding fragment thereof of claim 49.

51. The antibody or antigen-binding fragment thereof comprises a VH having at least 70% identity to SEQ ID NO: 30; and a VL having at least 70% identity to SEQ ID NO:

31.

51. An antibody or antigen-binding fragment thereof according to claim 49 or 50.

52. The antibody or antigen-binding fragment thereof comprises a VH having at least 80% identity to SEQ ID NO: 30; and a VL having at least 80% identity to SEQ ID NO:

31.

51. An antibody or antigen-binding fragment thereof according to claim 49 or 50.

53. The antibody or antigen-binding fragment thereof comprises a VH having at least 90% identity to SEQ ID NO: 30; and a VL having at least 90% identity to SEQ ID NO:

31.

51. An antibody or antigen-binding fragment thereof according to claim 49 or 50.

54. the antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 30, and a VL comprising the amino acid sequence set forth in SEQ ID NO: 31; 51. An antibody or antigen-binding fragment thereof according to claim 49 or 50.

55. The antibody or antigen-binding fragment thereof comprises a VH having at least 70% identity to SEQ ID NO: 182; and a VL having at least 70% identity to SEQ ID NO:

183.

51. An antibody or antigen-binding fragment thereof according to claim 49 or 50.

56. The antibody or antigen-binding fragment thereof comprises a VH having at least 80% identity to SEQ ID NO: 182; and a VL having at least 80% identity to SEQ ID NO:

183.

51. An antibody or antigen-binding fragment thereof according to claim 49 or 50.

57. The antibody or antigen-binding fragment thereof comprises a VH having at least 90% identity to SEQ ID NO: 182; and a VL having at least 90% identity to SEQ ID NO:

183.

51. An antibody or antigen-binding fragment thereof according to claim 49 or 50.

58. The antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 182, and a VL comprising the amino acid sequence set forth in SEQ ID NO:

183.

51. An antibody or antigen-binding fragment thereof according to claim 49 or 50.

59. the antibody or antigen-binding fragment thereof comprises a CDRH1 set forth in SEQ ID NO: 32, a CDRH2 set forth in SEQ ID NO: 33, a CDRH3 set forth in SEQ ID NO: 34, a CDRL1 set forth in SEQ ID NO: 35, a CDRL2 set forth in SEQ ID NO: 36, and a CDRL3 set forth in SEQ ID NO: 37; An antibody or antigen-binding fragment thereof according to any one of claims 1 to 13.

60. the antibody or antigen-binding fragment thereof comprises an extended CDRH1 set forth in SEQ ID NO: 166, an extended CDRH2 set forth in SEQ ID NO: 33, an extended CDRH3 set forth in SEQ ID NO: 167, an extended CDRL1 set forth in SEQ ID NO: 35, an extended CDRL2 set forth in SEQ ID NO: 36, and an extended CDRL3 set forth in SEQ ID NO: 37; 60. The antibody or antigen-binding fragment thereof of claim 59.

61. The antibody or antigen-binding fragment thereof comprises a VH having at least 70% identity to SEQ ID NO: 38; and a VL having at least 70% identity to SEQ ID NO:

39.

61. An antibody or antigen-binding fragment thereof according to claim 59 or 60.

62. The antibody or antigen-binding fragment thereof comprises a VH having at least 80% identity to SEQ ID NO: 38; and a VL having at least 80% identity to SEQ ID NO:

39.

61. An antibody or antigen-binding fragment thereof according to claim 59 or 60.

63. The antibody or antigen-binding fragment thereof comprises a VH having at least 90% identity to SEQ ID NO: 38; and a VL having at least 90% identity to SEQ ID NO:

39.

61. An antibody or antigen-binding fragment thereof according to claim 59 or 60.

64. the antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 38, and a VL comprising the amino acid sequence set forth in SEQ ID NO: 39; 61. An antibody or antigen-binding fragment thereof according to claim 59 or 60.

65. The antibody or antigen-binding fragment thereof comprises a VH having at least 70% identity to SEQ ID NO: 184; and a VL having at least 70% identity to SEQ ID NO:

185.

61. An antibody or antigen-binding fragment thereof according to claim 59 or 60.

66. The antibody or antigen-binding fragment thereof comprises a VH having at least 80% identity to SEQ ID NO: 184; and a VL having at least 80% identity to SEQ ID NO:

185.

61. An antibody or antigen-binding fragment thereof according to claim 59 or 60.

67. The antibody or antigen-binding fragment thereof comprises a VH having at least 90% identity to SEQ ID NO: 184; and a VL having at least 90% identity to SEQ ID NO:

185.

61. An antibody or antigen-binding fragment thereof according to claim 59 or 60.

68. The antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 184, and a VL comprising the amino acid sequence set forth in SEQ ID NO:

185.

61. An antibody or antigen-binding fragment thereof according to claim 59 or 60.

69. the antibody or antigen-binding fragment thereof comprises a CDRH1 set forth in SEQ ID NO: 40, a CDRH2 set forth in SEQ ID NO: 41, a CDRH3 set forth in SEQ ID NO: 42, a CDRL1 set forth in SEQ ID NO: 43, a CDRL2 set forth in SEQ ID NO: 44, and a CDRL3 set forth in SEQ ID NO: 45; An antibody or antigen-binding fragment thereof according to any one of claims 1 to 13.

70. the antibody or antigen-binding fragment thereof comprises an extended CDRH1 set forth in SEQ ID NO: 168, an extended CDRH2 set forth in SEQ ID NO: 41, an extended CDRH3 set forth in SEQ ID NO: 169, an extended CDRL1 set forth in SEQ ID NO: 43, an extended CDRL2 set forth in SEQ ID NO: 44, and an extended CDRL3 set forth in SEQ ID NO: 45; 70. The antibody or antigen-binding fragment thereof of claim 69.

71. The antibody or antigen-binding fragment thereof comprises a VH having at least 70% identity to SEQ ID NO: 46; and a VL having at least 70% identity to SEQ ID NO:

47.

71. An antibody or antigen-binding fragment thereof according to claim 69 or 70.

72. The antibody or antigen-binding fragment thereof comprises a VH having at least 80% identity to SEQ ID NO: 46; and a VL having at least 80% identity to SEQ ID NO:

47.

71. An antibody or antigen-binding fragment thereof according to claim 69 or 70.

73. The antibody or antigen-binding fragment thereof comprises a VH having at least 90% identity to SEQ ID NO: 46; and a VL having at least 90% identity to SEQ ID NO:

47.

71. An antibody or antigen-binding fragment thereof according to claim 69 or 70.

74. the antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 46, and a VL comprising the amino acid sequence set forth in SEQ ID NO: 47; 71. An antibody or antigen-binding fragment thereof according to claim 69 or 70.

75. The antibody or antigen-binding fragment thereof comprises a VH having at least 70% identity to SEQ ID NO: 186; and a VL having at least 70% identity to SEQ ID NO:

187.

71. An antibody or antigen-binding fragment thereof according to claim 69 or 70.

76. The antibody or antigen-binding fragment thereof comprises a VH having at least 80% identity to SEQ ID NO: 186; and a VL having at least 80% identity to SEQ ID NO:

187.

71. An antibody or antigen-binding fragment thereof according to claim 69 or 70.

77. The antibody or antigen-binding fragment thereof comprises a VH having at least 90% identity to SEQ ID NO: 186; and a VL having at least 90% identity to SEQ ID NO:

187.

71. An antibody or antigen-binding fragment thereof according to claim 69 or 70.

78. The antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 186, and a VL comprising the amino acid sequence set forth in SEQ ID NO:

187.

71. An antibody or antigen-binding fragment thereof according to claim 69 or 70.

79. the antibody or antigen-binding fragment thereof comprises a CDRH1 set forth in SEQ ID NO: 48, a CDRH2 set forth in SEQ ID NO: 49, a CDRH3 set forth in SEQ ID NO: 50, a CDRL1 set forth in SEQ ID NO: 51, a CDRL2 set forth in SEQ ID NO: 52, and a CDRL3 set forth in SEQ ID NO: 53; An antibody or antigen-binding fragment thereof according to any one of claims 1 to 13.

80. the antibody or antigen-binding fragment thereof comprises an extended CDRH1 set forth in SEQ ID NO: 170, an extended CDRH2 set forth in SEQ ID NO: 49, an extended CDRH3 set forth in SEQ ID NO: 171, an extended CDRL1 set forth in SEQ ID NO: 51, an extended CDRL2 set forth in SEQ ID NO: 52, and an extended CDRL3 set forth in SEQ ID NO: 53; 80. The antibody or antigen-binding fragment thereof of claim 79.

81. The antibody or antigen-binding fragment thereof comprises a VH having at least 70% identity to SEQ ID NO: 54; and a VL having at least 70% identity to SEQ ID NO:

55.

81. An antibody or antigen-binding fragment thereof according to claim 79 or 80.

82. The antibody or antigen-binding fragment thereof comprises a VH having at least 80% identity to SEQ ID NO: 54; and a VL having at least 80% identity to SEQ ID NO:

55.

81. An antibody or antigen-binding fragment thereof according to claim 79 or 80.

83. The antibody or antigen-binding fragment thereof comprises a VH having at least 90% identity to SEQ ID NO: 54; and a VL having at least 90% identity to SEQ ID NO:

55.

81. An antibody or antigen-binding fragment thereof according to claim 79 or 80.

84. the antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 54, and a VL comprising the amino acid sequence set forth in SEQ ID NO: 55; 81. An antibody or antigen-binding fragment thereof according to claim 79 or 80.

85. The antibody or antigen-binding fragment thereof comprises a VH having at least 70% identity to SEQ ID NO: 188; and a VL having at least 70% identity to SEQ ID NO:

189.

81. An antibody or antigen-binding fragment thereof according to claim 79 or 80.

86. The antibody or antigen-binding fragment thereof comprises a VH having at least 80% identity to SEQ ID NO: 188; and a VL having at least 80% identity to SEQ ID NO:

189.

81. An antibody or antigen-binding fragment thereof according to claim 79 or 80.

87. The antibody or antigen-binding fragment thereof comprises a VH having at least 90% identity to SEQ ID NO: 188; and a VL having at least 90% identity to SEQ ID NO:

189.

81. An antibody or antigen-binding fragment thereof according to claim 79 or 80.

88. The antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 188, and a VL comprising the amino acid sequence set forth in SEQ ID NO:

189.

81. An antibody or antigen-binding fragment thereof according to claim 79 or 80.

89. the antibody or antigen-binding fragment thereof comprises a CDRH1 set forth in SEQ ID NO: 56, a CDRH2 set forth in SEQ ID NO: 57, a CDRH3 set forth in SEQ ID NO: 58, a CDRL1 set forth in SEQ ID NO: 59, a CDRL2 set forth in SEQ ID NO: 52, and a CDRL3 set forth in SEQ ID NO: 60; An antibody or antigen-binding fragment thereof according to any one of claims 1 to 13.

90. the antibody or antigen-binding fragment thereof comprises an extended CDRH1 set forth in SEQ ID NO: 172, an extended CDRH2 set forth in SEQ ID NO: 57, an extended CDRH3 set forth in SEQ ID NO: 173, an extended CDRL1 set forth in SEQ ID NO: 59, an extended CDRL2 set forth in SEQ ID NO: 52, and an extended CDRL3 set forth in SEQ ID NO: 60; 90. The antibody or antigen-binding fragment thereof of claim 89.

91. The antibody or antigen-binding fragment thereof comprises a VH having at least 70% identity to SEQ ID NO: 61; and a VL having at least 70% identity to SEQ ID NO:

62.

91. An antibody or antigen-binding fragment thereof according to claim 89 or 90.

92. The antibody or antigen-binding fragment thereof comprises a VH having at least 80% identity to SEQ ID NO: 61; and a VL having at least 80% identity to SEQ ID NO:

62.

91. An antibody or antigen-binding fragment thereof according to claim 89 or 90.

93. The antibody or antigen-binding fragment thereof comprises a VH having at least 90% identity to SEQ ID NO: 61; and a VL having at least 90% identity to SEQ ID NO:

62.

91. An antibody or antigen-binding fragment thereof according to claim 89 or 90.

94. the antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 61, and a VL comprising the amino acid sequence set forth in SEQ ID NO: 62; 91. An antibody or antigen-binding fragment thereof according to claim 89 or 90.

95. The antibody or antigen-binding fragment thereof comprises a VH having at least 70% identity to SEQ ID NO: 190; and a VL having at least 70% identity to SEQ ID NO:

191.

91. An antibody or antigen-binding fragment thereof according to claim 89 or 90.

96. The antibody or antigen-binding fragment thereof comprises a VH having at least 80% identity to SEQ ID NO: 190; and a VL having at least 80% identity to SEQ ID NO:

191.

91. An antibody or antigen-binding fragment thereof according to claim 89 or 90.

97. The antibody or antigen-binding fragment thereof comprises a VH having at least 90% identity to SEQ ID NO: 190; and a VL having at least 90% identity to SEQ ID NO:

191.

91. An antibody or antigen-binding fragment thereof according to claim 89 or 90.

98. The antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 190, and a VL comprising the amino acid sequence set forth in SEQ ID NO:

191.

91. An antibody or antigen-binding fragment thereof according to claim 89 or 90.

99. the antibody or antigen-binding fragment thereof comprises a CDRH1 set forth in SEQ ID NO: 63, a CDRH2 set forth in SEQ ID NO: 64, a CDRH3 set forth in SEQ ID NO: 65, a CDRL1 set forth in SEQ ID NO: 66, a CDRL2 set forth in SEQ ID NO: 36, and a CDRL3 set forth in SEQ ID NO: 67; An antibody or antigen-binding fragment thereof according to any one of claims 1 to 13.

100. the antibody or antigen-binding fragment thereof comprises an extended CDRH1 set forth in SEQ ID NO: 174, an extended CDRH2 set forth in SEQ ID NO: 64, an extended CDRH3 set forth in SEQ ID NO: 175, an extended CDRL1 set forth in SEQ ID NO: 66, an extended CDRL2 set forth in SEQ ID NO: 36, and an extended CDRL3 set forth in SEQ ID NO: 67; 100. The antibody or antigen-binding fragment thereof of claim 99.

101. The antibody or antigen-binding fragment thereof comprises a VH having at least 70% identity to SEQ ID NO: 68; and a VL having at least 70% identity to SEQ ID NO:

69.

101. An antibody or antigen-binding fragment thereof according to claim 99 or 100.

102. The antibody or antigen-binding fragment thereof comprises a VH having at least 80% identity to SEQ ID NO: 68; and a VL having at least 80% identity to SEQ ID NO:

69.

101. An antibody or antigen-binding fragment thereof according to claim 99 or 100.

103. The antibody or antigen-binding fragment thereof comprises a VH having at least 90% identity to SEQ ID NO: 68; and a VL having at least 90% identity to SEQ ID NO:

69.

101. An antibody or antigen-binding fragment thereof according to claim 99 or 100.

104. the antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 68, and a VL comprising the amino acid sequence set forth in SEQ ID NO: 69; 101. An antibody or antigen-binding fragment thereof according to claim 99 or 100.

105. The antibody or antigen-binding fragment thereof comprises a VH having at least 70% identity to SEQ ID NO: 192; and a VL having at least 70% identity to SEQ ID NO:

193.

101. An antibody or antigen-binding fragment thereof according to claim 99 or 100.

106. The antibody or antigen-binding fragment thereof comprises a VH having at least 80% identity to SEQ ID NO: 192; and a VL having at least 80% identity to SEQ ID NO:

193.

101. An antibody or antigen-binding fragment thereof according to claim 99 or 100.

107. The antibody or antigen-binding fragment thereof comprises a VH having at least 90% identity to SEQ ID NO: 192; and a VL having at least 90% identity to SEQ ID NO:

193.

101. An antibody or antigen-binding fragment thereof according to claim 99 or 100.

108. The antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 192, and a VL comprising the amino acid sequence set forth in SEQ ID NO:

193.

101. An antibody or antigen-binding fragment thereof according to claim 99 or 100.

109. the antibody or the antigen-binding fragment thereof comprises a CDRH1 set forth in SEQ ID NO: 2, a CDRH2 set forth in SEQ ID NO: 3, a CDRH3 set forth in SEQ ID NO: 4, a CDRL1 set forth in SEQ ID NO: 5 or any one of SEQ ID NOs: 230-233, a CDRL2 set forth in SEQ ID NO: 234, and a CDRL3 set forth in SEQ ID NO: 7; An antibody or antigen-binding fragment thereof according to any one of claims 1 to 13.

110. The antibody or antigen-binding fragment thereof comprises a VH having at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity to the sequence set forth in any one of SEQ ID NO:200, SEQ ID NO:201, and SEQ ID NO:202; and a VL having at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity to the sequence set forth in any one of SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, and SEQ ID NO:

209.

110. The antibody or antigen-binding fragment thereof of claim 109.

111. The antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence set forth in any one of SEQ ID NOs: 200, 201, and 202; and a VL comprising the amino acid sequence set forth in any one of SEQ ID NOs: 203, 204, 205, 206, 207, 208, and 209.

111. An antibody or antigen-binding fragment thereof according to claim 109 or 110.

112. The antibody or antigen-binding fragment thereof comprises a VH having at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity to SEQ ID NO: 202; and a VL having at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity to SEQ ID NO:

208. An antibody or antigen-binding fragment thereof according to any one of claims 109 to 111.

113. The antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 202; and a VL comprising the amino acid sequence set forth in SEQ ID NO:

208.

113. The antibody or antigen-binding fragment thereof of claim 112.

114. The antibody or antigen-binding fragment thereof comprises a VH having at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity to SEQ ID NO: 202; and a VL having at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity to SEQ ID NO:

209. An antibody or antigen-binding fragment thereof according to any one of claims 109 to 111.

115. The antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 202; and a VL comprising the amino acid sequence set forth in SEQ ID NO:

209. An antibody or antigen-binding fragment thereof described in claim 114.

116. the antibody or antigen-binding fragment thereof comprises a CDRH1 set forth in SEQ ID NO: 2, a CDRH2 set forth in SEQ ID NO: 10, a CDRH3 set forth in SEQ ID NO: 11, a CDRL1 set forth in SEQ ID NO: 12 or SEQ ID NO: 235, a CDRL2 set forth in SEQ ID NO: 13, SEQ ID NO: 236 or SEQ ID NO: 237, and a CDRL3 set forth in SEQ ID NO: 14; An antibody or antigen-binding fragment thereof according to any one of claims 1 to 13.

117. The antibody or antigen-binding fragment thereof comprises a VH having at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity to the sequence set forth in SEQ ID NO:210; and a VL having at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity to the sequence set forth in any one of SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, and SEQ ID NO:

214.

117. The antibody or antigen-binding fragment thereof of claim 116.

118. The antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence set forth in SEQ ID NO:210; and a VL comprising the amino acid sequence set forth in any one of SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, and SEQ ID NO:

214.

118. An antibody or antigen-binding fragment thereof according to claim 116 or 117.

119. The antibody or antigen-binding fragment thereof comprises a VH having at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity to SEQ ID NO: 210; and a VL having at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity to SEQ ID NO:

214. An antibody or antigen-binding fragment thereof according to any one of claims 116 to 118.

120. The antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 210; and a VL comprising the amino acid sequence set forth in SEQ ID NO:

214.

120. The antibody or antigen-binding fragment thereof of claim 119.

121. the antibody or the antigen-binding fragment thereof is a humanized antibody; 10. The antibody or antigen-binding fragment thereof of any one of the preceding claims.

122. the antibody or the antigen-binding fragment thereof is a monoclonal antibody; 10. The antibody or antigen-binding fragment thereof of any one of the preceding claims.

123. The antibody of any one of the preceding claims, wherein the antibody comprises an Fc portion.

124. 10. The antibody of any one of the preceding claims, wherein the antibody is of the IgG type.

125. The antibody of claim 124, wherein the antibody is of the IgG1 or IgG4 type.

126. the antibody comprises the mutations L234A and L235A in its Fc portion; An antibody according to any one of the preceding claims.

127. the antibody or the antigen-binding fragment thereof is purified; 10. The antibody or antigen-binding fragment thereof of any one of the preceding claims.

128. the antibody or the antigen-binding fragment thereof is a single chain antibody; 10. The antibody or antigen-binding fragment thereof of any one of the preceding claims.

129. the antibody or the antigen-binding fragment thereof is an scFv; 129. The antibody or antigen-binding fragment thereof of claim 128.

130. the antibody or the antigen-binding fragment thereof is selected from Fab, Fab', F(ab')2, and Fv; An antibody or antigen-binding fragment thereof according to any one of claims 1 to 127.

131. For use as a medicine, 10. The antibody or antigen-binding fragment thereof of any one of the preceding claims.

132. For use in the prevention or treatment of a neurodegenerative disease, a neuroinflammatory disease or a peripheral inflammatory disorder, 132. The antibody or antigen-binding fragment thereof of claim 131.

133. A polynucleotide encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 130. Nucleic acid molecule.

134. A nucleic acid sequence set forth in any one of SEQ ID NOs: 73-140 and 215-229; or sequence variants thereof, including said sequence variants having at least 50%, at least 55%, at least 60%, at least 65%, 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 of claim 133.

135. A plurality of nucleic acid molecules encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 130, each of the nucleic acid molecules comprises a polynucleotide encoding an immunoglobulin chain of the antibody or antigen-binding fragment thereof; A plurality of nucleic acid molecules.

136. A nucleic acid sequence set forth in any one of SEQ ID NOs: 73-140 and 215-229; or sequence variants thereof, including said sequence variants having at least 50%, at least 55%, at least 60%, at least 65%, 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. A plurality of nucleic acid molecules according to claim 133.

137. A vector comprising the nucleic acid molecule of claim 133 or 134.

138. A plurality of vectors comprising a plurality of nucleic acid molecules according to claim 135 or 136.

139. 137. A vector comprising a vector combination according to claim 138, which expresses an antibody or antigen-binding fragment thereof according to any one of claims 1 to 130, or a vector according to claim 137 or a vector combination according to claim 138. host cell.

140. 131. A method for preparing an antibody or antigen-binding fragment thereof, or an immunoglobulin chain thereof according to any one of claims 1 to 130, said method comprising: (i) culturing the host cell of claim 139; and (ii) isolating the antibody or immunoglobulin chain thereof from the culture; A method comprising:

141. The antibody or antigen-binding fragment thereof of any one of claims 1 to 130, the nucleic acid molecule or molecules of any one of claims 133 to 136, the vector or vectors of claim 137 or 138, or the cell of claim 139. composition.

142. further comprising a pharmaceutically acceptable excipient, diluent or carrier, 142. The composition of claim 141.

143. (i) an antibody or antigen-binding fragment thereof according to any one of claims 1 to 130; (ii) a nucleic acid molecule according to any one of claims 133 to 136; (iii) the vector of claim 137 or 138; (iv) a cell according to claim 139; and / or (v) the composition of claim 141 or 142; A kit comprising one or more of:

144. 142. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 130, a nucleic acid molecule or nucleic acid molecules according to any one of claims 133 to 136, a vector or vectors according to claim 137 or 138, a cell according to claim 139, a composition according to claim 141 or 142, or a kit according to claim 143 for use as a medicament.

145. 142. The antibody or antigen-binding fragment thereof of any one of claims 1 to 130, the nucleic acid molecule or nucleic acid molecules of any one of claims 133 to 136, the vector or vectors of claim 137 or 138, the cell of claim 139, the composition of claim 141 or 142, or the kit of claim 143 for use in the prevention or treatment of a neurodegenerative or neuroinflammatory disease, or a peripheral inflammatory disorder.

146. the neurodegenerative disease or the neuroinflammatory disease is associated with the formation of ASC aggregates and / or amyloid-β aggregates; 146. An antibody or antigen-binding fragment thereof, nucleic acid or nucleic acids, vector or vectors, cell, or composition for use as described in claim 145.

147. The neurodegenerative disease or the neuroinflammatory disease is characterized by and / or is associated with dementia; 147. An antibody or antigen-binding fragment thereof, nucleic acid or nucleic acids, vector or vectors, cell, or composition for use as described in claim 145 or 146.

148. The neurodegenerative disease or the neuroinflammatory disease is selected from Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple system atrophy, multiple sclerosis, traumatic brain injury, amyotrophic lateral sclerosis, spinocerebellar ataxia, frontotemporal dementia, frontotemporal lobar degeneration, mild cognitive impairment, Parkinson's plus syndrome, Pick's disease, primary progressive aphasia, gray matter degeneration [Alpers], subacute necrotizing encephalopathy, and dementia with Lewy bodies; 148. An antibody or antigen-binding fragment thereof, nucleic acid or nucleic acids, vector or vectors, cell, or composition for use according to any one of claims 145 to 147.

149. The neurodegenerative disease is Alzheimer's disease.

149. An antibody or antigen-binding fragment thereof, nucleic acid or nucleic acids, vector or vectors, cell, or composition for use according to any one of claims 145 to 148.

150. The neurodegenerative disease is mild cognitive impairment.

149. An antibody or antigen-binding fragment thereof, nucleic acid or nucleic acids, vector or vectors, cell, or composition for use according to any one of claims 145 to 148.

151. The peripheral inflammatory disorder is inflammatory bowel disease.

146. An antibody or antigen-binding fragment thereof, nucleic acid or nucleic acids, vector or vectors, cell, or composition for use as described in claim 145.

152. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 130, or the composition according to claim 141 or 142, in the (in vitro) diagnosis of a neurodegenerative or neuroinflammatory disease, or a peripheral inflammatory disorder, use.

153. an antibody or antigen-binding fragment thereof according to any one of claims 1 to 130, a nucleic acid molecule or nucleic acid molecules according to any one of claims 133 to 136, a vector or vectors according to claim 137 or 138, a cell according to claim 139, a composition according to claim 141 or 142, or a kit according to claim 143 in the manufacture of a medicament for the prevention, treatment or attenuation of a neurodegenerative or neuroinflammatory disease, or a peripheral inflammatory disorder; use.

154. 1. A method of treating, ameliorating, or alleviating a neurodegenerative or neuroinflammatory disease, or a peripheral inflammatory disorder, or reducing the risk of a neurodegenerative or neuroinflammatory disease, or a peripheral inflammatory disorder, comprising: Administering to a subject in need thereof an antibody or antigen-binding fragment thereof according to any one of claims 1 to 130, a nucleic acid molecule or molecules according to any one of claims 133 to 136, a vector or vectors according to claim 137 or 138, a cell according to claim 139, a composition according to claim 141 or 142, or a kit according to claim 143. method.

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