Single-domain antibodies directed against aim2 and their use

Abstract

The present invention is concerned with single-domain antibodies directed against "absent in melanoma 2" (AIM2). The single-domain antibodies of the present invention can also be combined into a homobivalent or heterobivalent polypeptide comprising two of the provided single-domain antibodies of the present invention. The single-domain antibodies or the bivalent polypeptides can be used in medical applications, preferably for preventing and / or treating an inflammatory disease or condition in a subject.

Description

[0001] Rheinische Friedrich-Wilhelms-llniversitat Bonn December 4, 2025

[0002] Universitatsklinikum Bonn Simmons Ref.: P12539PC00

[0003] Boston Children’s Hospital NEW INTERNATIONAL APPLICATION

[0004] Single-domain antibodies directed against AIM2 and their use

[0005] TECHNICAL FIELD

[0006] The present invention is related to the field of biomedical science and provides singledomain antibodies directed against “absent in melanoma 2” (AIM2). The single-domain antibodies of the present invention can also be combined into a homobivalent or heterobivalent polypeptide comprising two of the provides single-domain antibodies of the present invention. The single-domain antibodies or the bivalent polypeptides can be used in medical applications, preferably for preventing and / or treating an inflammatory disease or condition in a subject.

[0007] BACKGROUND OF THE INVENTON

[0008] AIM2 (Absent in Melanoma 2) is a component of the innate immune system that plays a crucial role in the formation of inflammasomes. Inflammasomes are multiprotein oligomers that are responsible for the activation of inflammatory responses. AIM2 is a sensor protein that detects double-stranded DNA (dsDNA) in the cytosol, which can be a sign of either infection by pathogens such as bacteria and viruses, or cellular damage. Upon recognizing dsDNA, AIM2 can bind to it through its HIN-200 domain. This binding leads to a conformational change that allows AIM2 to interact with the adaptor protein ASC (Apoptosis-associated Speck-like protein containing a CARD). ASC acts as a bridge between AIM2 and the effector protein pro-caspase-1 . It has two domains: the PYD (Pyrin domain) and the CARD (Caspase activation and recruitment domain). The PYD of AIM2 oligomerizes and interacts with the PYD of ASC, whose polymerization is initiated by the seed. The CARD of ASC both cross-links ASCPYDfilaments and recruits pro-caspase-1 . These interactions lead to the formation of a large oligomeric complex known as the inflammasome. Within the inflammasome complex, in turn, caspase-1 is activated, which then cleaves 1) the pro-forms of the inflammatory cytokines interleukin-1 p (I L-1 ) and interleukin-18 (IL-18), into their active forms, and 2) the effector protein GSDMD, whose N-terminus oligomerizes to form pores in the plasma membrane that lead to a type of programmed cell death called pyroptosis. The combined inflammatory response is crucial for the clearance of pathogens and the response to cellular damage.

[0009] Pyroptosis is an inflammatory cell death usually caused by microbial infection, downstream of the activation of canonical inflammasomes described above, or as a consequence of LPS-induced non-canonical inflammasomes, e.g. by direct activation of caspase-4 by LPS. Pyroptosis exerts tumor suppression function and evokes anti-tumor immune responses. Therapeutic regimens, including chemotherapy, radiotherapy, targeted therapy and immune therapy, induce pyroptosis in cancer, which potentiate local and systemic anti-tumor immunity. On the other hand, pyroptosis of normal cells attributes to side effects of anti-cancer therapies. Furthermore, dysregulation of the inflammatory response is a key driver in many debilitating diseases linked to acute and chronic inflammation such as sepsis, atherosclerosis, inflammatory bowel disease, non-alcoholic steatohepatitis, lung cancer, psoriasis, and autoinflammatory diseases such as cryopyrin- associated periodic syndromes and familial Mediterranean fever. Inhibitors that specifically target pyroptotic cell death may therefore be therapeutically useful in the clinic for the treatment of these diseases.

[0010] The AIM2-ASC interaction is therefore a key event in the innate immune response, and understanding this interaction is important for developing treatments for diseases where the inflammasome plays a role in pathology, such as some autoinflammatory and autoimmune diseases, as well as certain cancers.

[0011] Therapeutically, targeting the AIM2 inflammasome is being explored for the treatment of various diseases. Since the AIM2 inflammasome can drive pathological inflammation, inhibitors of this pathway may be beneficial in conditions such as autoimmune diseases, atherosclerosis, and certain types of cancer where excessive or inappropriate inflammation is a factor. Additionally, since AIM2 is involved in the immune response to infections, modulating its activity could also be relevant in treating infectious diseases.

[0012] As described above, the pyrin domain (PYD) in AIM2 is crucial for its role in the innate immune response.

[0013] It was therefore a goal of the present invention to provide compositions which can inhibit or prevent the function of the PYD in AIM2, thereby disrupting the formation of the AIM2 inflammasome. Without the PYD-PYD interaction between two AIM2 molecules or the PYD-PYD interaction between AIM2 and ASC, the assembly of the inflammasome complex will be impaired, preventing the activation of caspase-1 . As a result, the maturation and secretion of IL-1 and IL-18 will be reduced, leading to a diminished inflammatory response. This could have potential therapeutic implications; for example, inhibiting the PYD of AIM2 might be beneficial in diseases where excessive inflammasome activation contributes to pathology, such as certain autoinflammatory condition. SUMMARY OF THE INVENTION

[0014] In the following, the present invention is described in detail. The features of the present invention are described in individual paragraphs. This, however, does not mean that a feature described in a paragraph stands isolated from a feature or features described in other paragraphs. Rather, a feature described in a paragraph can be combined with a feature or features described in other paragraphs.

[0015] The present invention is concerned with single-domain antibodies directed against AIM2. The single-domain antibodies of the present invention are characterized by their ability to bind to AIM2, preferably the pyrin domain (PYD) in AIM2, in the cytosol of a cell.

[0016] The single-domain antibodies of the present invention are further characterized by comprising an amino acid sequence comprising framework region 1 (FR1 ), complementarity-determining region 1 (CDR1 ), FR2, CDR2, FR3, CDR3, and FR4. FR1 is located at the N-terminal side of the amino acid chain, and FR4 is located at the C-terminal side of the amino acid chain.

[0017] AIM2-1 and variants

[0018] In one embodiment, a single-domain antibody of the present invention is characterized by comprising CDR1 : GFTFNDYH (SEQ ID NO:2), CDR2: ITRTGGFT (SEQ ID NO:4), and CDR3: NAVSYELGRDF (SEQ ID NO: 6). Additionally, the single-domain antibody can comprise FR1 : QVQLVETGGGLVQPGGSLRLSCAAS (SEQ ID NO:1), FR2: MRWYRQAPGKERELVAG (SEQ ID NO:3), FR3:

[0019] NYGDSVKGRVTISRDDVKNTVYLQMSSLKAEDTAVYHC (SEQ ID NO:5), and FR4: WGQGTQVTVSS (SEQ ID NO: 7).

[0020] The single-domain antibody of the present invention can also be characterized by comprising the amino acid sequence as defined in SEQ ID NO: 8, which consists of SEQ NOS 1 to 7. The single-domain antibody of the present invention consisting of the amino acid sequence as defined by SEQ ID NO:8 is called VHH AIM2-1 .

[0021] The single-domain antibody of the present invention can also be characterized by being a variant of a single-domain antibody of the present invention characterized by comprising the amino acid sequence as defined in SEQ ID NO: 8. The variant is characterized by comprising an amino acid sequence, wherein the amino acid sequence is at least 80 %, 90 %, 95%, or 99% identical to the amino acid sequence as defined in SEQ ID NO: 8. In a preferred embodiment, the variant comprises the CDR regions as defined in SEQ ID NOS 2, 4, and 6, and the variation occurs in the frame work regions.

[0022] AIM2-2 and variants

[0023] In one embodiment, a single-domain antibody of the present invention is characterized by comprising CDR1 : GFTFSNYA (SEQ ID NQ:10), CDR2: IASSGRFT (SEQ ID NO:12), and CDR3: AKGGQSLRYLTPPI (SEQ ID NO: 14). Additionally, the single-domain antibody can comprise FR1 : QVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:9), FR2: MSWVRQAPGKGLEWVSA (SEQ ID NO:11), FR3:

[0024] NYADSVKGRFTISRDDAKNTLYLQMNSLKPEDTAVYYC (SEQ ID NO:13), and FR4: EGQGTQVTVSS (SEQ ID NO: 15).

[0025] The single-domain antibody of the present invention can also be characterized by comprising the amino acid sequence as defined in SEQ ID NO: 16, which consists of SEQ NOS 9 to 15. The single-domain antibody of the present invention consisting of the amino acid sequence as defined by SEQ ID NO:16 is called VHH AIM2-2.

[0026] The single-domain antibody of the present invention can also be characterized by being a variant of a single-domain antibody of the present invention characterized by comprising the amino acid sequence as defined in SEQ ID NO: 16. The variant is characterized by comprising an amino acid sequence, wherein the amino acid sequence is at least 80 %, 90 %, 95%, or 99% identical to the amino acid sequence as defined in SEQ ID NO: 16. In a preferred embodiment, the variant comprises the CDR regions as defined in SEQ ID NOS 10, 12, and 14, and the variation occurs in the frame work regions.

[0027] AIM2-3 and variants

[0028] In one embodiment, a single-domain antibody of the present invention is characterized by comprising CDR1 : GFAFSSYH as defined by SEQ ID NO:18, CDR2: ITNTGGFT as defined by SEQ ID NO:20, and CDR3: NAVSYVVGRDY as defined by SEQ ID NO: 22. Additionally, the single-domain antibody can comprise FR1 : QVQLVETGGGLVQPGGSLRLSCAAS as defined by SEQ ID NO:17, FR2: MRWYRQAPGKERELVAG, as defined by SEQ ID NO:19, FR3: NYPDSVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYHC as defined by SEQ ID NO:21 , and FR4: WGQGTQVTVSS as defined by SEQ ID NO: 23.

[0029] The single-domain antibody of the present invention can also be characterized by comprising the amino acid sequence as defined in SEQ ID NO: 24, which consists of SEQ NOS: 17 to 23. The single-domain antibody of the present invention consisting of the amino acid sequence as defined by SEQ ID NO:24 is called VHH AIM2-3.

[0030] The single-domain antibody of the present invention can also be characterized by being a variant of a single-domain antibody of the present invention characterized by comprising the amino acid sequence as defined in SEQ ID NO: 24. The variant is characterized by comprising an amino acid sequence, wherein the amino acid sequence is at least 80 %, 90 %, 95%, or 99% identical to the amino acid sequence as defined in SEQ ID NO: 24. In a preferred embodiment, the variant comprises the CDR regions as defined in SEQ ID NOS 18, 20, and 22, and the variation occurs in the frame work regions. Epitope of the single-domain antibodies of the present invention

[0031] Preferably, the single-domain antibody of the present invention is directed at an epitope located within the pyrin domain of AIM2 (AIM2PYD). In the context of inflammasome assembly, the PYD of AIM2 specifically interacts with the PYD of the adaptor protein ASC (Apoptosis-associated Speck-like protein containing a CARD). This interaction is necessary for the recruitment of ASC to the AIM2-DNA complex, which subsequently leads to the recruitment and activation of pro-caspase- 1 , resulting in the processing and secretion of pro-inflammatory cytokines. Therefore, a single-domain antibody of the present invention binding an epitope located within pyrin domain of AIM2 (AIM2PYD) can disrupt the formation of the AIM2 inflammasome, thereby preventing the activation of caspase-1 . As a result, the maturation and secretion of IL-1 and IL-18 would be reduced, leading to a diminished inflammatory response.

[0032] The single-domain antibody directed against AIM2 of the present invention can be capable of specifically binding an epitope within the pyrin domain of AIM2 (AIM2PYD), wherein the epitope is a discontinuous epitope comprising amino acids selected from a) D23, R24, K26, F27, F28, S30, D31 , T37, H41 , K71 , L72, and N73 of sequence of AIM2PYD(SEQ ID NO:25); or b) E7, I8, L11 , T12, D15, N16, T18, D19, E21 , N44, R45, I46, L76, R80, E83, E84, and K87 of AIM2PYD(SEQ ID NO:25):

[0033] MESKYKEILL LTGLDNITDE ELDRFKFFLS DEFNIATGKL HTANRIQVAT

[0034] LMIQNAGAVS AVMKTIRIFQ KLNYMLLAKR LQEEKEKVDK QYKSVTKPKP

[0035] LSQAEMSPAA SAAIRNDVAK QRAAPKVSPH VKPEQKQMVA QQESIREGFQ

[0036] KRCLPVMVLK AKKPFTFETQ EGKQEMFHAT VATEKEFFFV KVFNTLLKDK

[0037] FIPKRI I I IA RYYRHSGFLE VNSASRVLDA ESDQKVNVPL NI IRKAGETP

[0038] KINTLQTQPL GTIVNGLFVV QKVTEKKKNI LFDLSDNTGK MEVLGVRNED

[0039] TMKCKEGDKV RLTFFTLSKN GEKLQLTSGV HSTIKVIKAK KKT .

[0040] Bivalent polypeptides comprising the single-domain antibodies of the present invention

[0041] The present invention is also concerned with a polypeptide comprising a combination of two single-domain antibodies of the present invention. The bivalent polypeptides of the present invention are characterized by their improved inhibition of the AIMPYDmediated inflammasome formation.

[0042] In the present invention, the combination can lead to a homobivalent polypeptide, or a heterobivalent polypeptide. A homobivalent polypeptide of the present invention comprises the same single-domain antibody twice. A heterobivalent polypeptide of the present invention comprises a combination of one single-domain antibody of the present invention, with a further single-domain antibody of the present invention. In the present invention, polypeptide can comprise one of the following combinations:

[0043] (a) a combination of the single-domain antibody VHH AIM2-1 or a variant as defined herein with a further single-domain antibody VHH AIM2-1 or a variant as defined herein.

[0044] (b) a combination of the single-domain antibody VHH AIM2-1 or a variant as defined herein with the single-domain antibody of VHH AIM2-2 or a variant as defined herein.

[0045] (c) a combination of the single-domain antibody of VHH AIM2-1 or a variant as defined herein with the single-domain antibody VHH AIM2-3 or a variant as defined herein.

[0046] (d) a combination of the single-domain antibody VHH AIM2-2 or a variant as defined herein with a single-domain antibody VHH AIM2-3 or a variant as defined herein.

[0047] (e) a combination of the single-domain antibody VHH AIM2-2 or a variant as defined herein with a further single-domain antibody of VHH AIM2-2 or a variant as defined herein.

[0048] (f) a combination of the single-domain antibody VHH AIM2-3 or a variant as defined herein with a further single-domain antibody VHH AIM2-3 or a variant as defined herein.

[0049] In the present invention, the two single-domain antibodies can be linked with the peptide linker (GGGGS)s (SEQ ID NO:26).

[0050] The present invention is also concerned with a polynucleotide encoding the singledomain antibody of the present invention or the bivalent polypeptide of the present invention.

[0051] The present invention is also concerned with a polynucleotide comprising one or more nucleic acid sequence(s) encoding the single-domain antibody of the present invention or the bivalent polypeptide of the present invention. The polynucleotide of the present invention can be selected from RNA, such as mRNA, DNA, such as genomic DNA, cDNA, or synthetic DNA, analogs thereof, or a combination thereof. Preferably the polynucleotide is mRNA.

[0052] The polynucleotide of the present invention can be used to transfect a cell by a method as known in the art. For example, AAV-Mediated Gene Therapy can be used. Adeno-associated virus (AAV) is a small, nonenveloped virus that was adapted 30 years ago for use as a gene transfer vehicle. It is capable of transducing a wide range of species and tissues in vivo with no evidence of toxicity, and it generates relatively mild innate and adaptive immune responses. Upon transfection, the cell will produce the single-domain antibody of the present invention or the bivalent polypeptide of the present invention. The single-domain antibody or the bivalent polypeptide thus produced is capable of binding to cytosolic AIM2 in a cell, and thereby inhibiting inflammasome formation, and thus pyroptosis of the cell. Therefore, the present invention is also concerned with a single-domain antibody or a bivalent polypeptide, which is capable of binding to cytosolic AIM2 in a cell, and thereby inhibiting inflammasome formation, and thus inhibiting pyroptosis of the cell, wherein the single-domain antibody or the bivalent polypeptide directed against AIM2 is produced by the cell upon transfecting the cell with the nucleic acid encoding the single-domain antibody or the bivalent polypeptide of the present invention.

[0053] The present invention is also concerned with a host cell comprising the polynucleotide encoding the single-domain antibody or the bivalent polypeptide of the present invention, wherein optionally the host cell is selected from eukaryotic cells, which include, but are not limited to those e.g. hamster cell lines (CHO and their derivatives), mouse cell lines (such as C127, NSO, SP2 / 0, YB2 / 0, XB2 / 09 and derivatives of all of them), or human cell lines (such as HEK and their derivatives, e.g. EXPI293, HT-1080, PER.C6, or HuH-7). Also included are cell lines from monkeys, such as e.g., Vero cells and their derivatives and insect cells, such as SF-9 cells and their derivatives. The polynucleotide encoding the single-domain antibody or the bivalent polypeptide of the present invention can also be produced recombinantly in bacterial cell, or in yeast cells, such as in Pichia pastoris.

[0054] The present invention is also concerned with a pharmaceutical composition comprising the single-domain antibody or the bivalent polypeptide of the present invention or the polynucleotide encoding the single-domain antibody or the bivalent polypeptide of the present invention, and a pharmaceutically acceptable carrier.

[0055] The single-domain antibodies or the bivalent polypeptide of the present invention are capable of inhibiting inflammasome formation, and thus preventing pyroptosis. Inhibiting inflammasome formation can be a therapeutic approach to stop or prevent pyroptosis and the conditions and symptoms associated with pyroptosis. Therefore, the present invention is also concerned with the single-domain antibodies directed against AIM2 of the present invention, or the bivalent polypeptide of the present invention, or the polynucleotide encoding the single-domain antibodies of the present invention or the bivalent polypeptide of the present invention, or the pharmaceutical composition of the present invention for use in therapy.

[0056] In particular, the present invention is concerned with the single-domain antibodies directed against AIM2 of the present invention, or the bivalent polypeptide of the present invention, or the polynucleotide encoding the single-domain antibody of the present invention or the bivalent polypeptide of the present invention, or the pharmaceutical composition of the present invention for use in a method of treating or preventing an inflammatory disease or condition in a subject, wherein the inflammatory disease or condition is selected from the group comprising an acute inflammation, a chronic inflammation, sepsis, loss of the blood-brain barrier, in particular caused by sepsis, septic shock, non-alcoholic steatohepatitis, lung cancer, Familial Mediterranean Fever (FMF), autoinflammatory diseases, Cryoprin associated periodic syndrome (CAPS), non-alcoholic fatty liver disease, Alzheimer's disease, Parkinson's disease, age related macular degeneration, atherosclerosis, asthma and allergy airway inflammation, gout, Crohn's disease, ulcerative colitis, inflammatory bowel disease, psoriasis, hypertension, nephropathy, myocardial infarction, multiple sclerosis, experimental autoimmune encephalitis, hyperinflammation following influenza infection, graft versus host disease, stroke, silicosis, asbestosis, mesothelioma, type 1 diabetes, type 2 diabetes, obesity- induced inflammation, insulin resistance, rheumatoid arthritis, myelodysplastic syndrome, contact hypersensitivity, joint inflammation triggered by chikungunya virus and traumatic brain injury.

[0057] In a preferred embodiment, an mRNA encoding the single-domain antibody or the bivalent polypeptide of the present invention is used in the method of treatment. The mRNA can be taken up by the cells, and the cells will produce the single-domain antibody or the bivalent polypeptide of the present invention. The single-domain antibody or the bivalent polypeptide can then prevent inflammasome formation in the cytosol of the cell. Thereby, the process leading to pyroptosis can be prevented.

[0058] In the present invention, the sample can be selected from the group comprising serum, plasma, and whole blood.

[0059] DEFINITIONS

[0060] The term “comprise / s / ing”, as used herein, is meant to include or encompass the disclosed features and further features which are not specifically mentioned. The term “comprise / es / ing” is also meant in the sense of “consist / s / ing of” the indicated features, thus not including further features except the indicated features. Thus, the subject-matter of the present invention may be characterized by additional features in addition to the features as indicated.

[0061] The term “single-domain antibody" as used herein is interchangeable with the term “nanobody”, and defines a recombinant, antigen-specific antibody consisting of only one single monomeric variable antibody domain (normally, these correspond to the variable region (VHH) of a heavy-chain antibody). Single-domain antibodies can be derived from naturally occurring heavy chain antibodies. Due to their small size they offer several advantages over conventional antibodies. Sequence identity can be determined by the skilled person. For example, the sequence identity can be calculated using BLASTP as disclosed in the prior art (see e.g. Altschul et al. (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402; Altschul et al. (2005) “Protein database searches using compositionally adjusted substitution matrices.” FEBS J. 272:5101 -5109), preferably using version BLASTP 2.2.29+ (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi), preferably using the following settings:

[0062] • Field “Enter Query Sequence”: Query subrange: none

[0063] • Field “Choose Search Set”: Database: non-redundant protein sequences (nr); optional parameters: none

[0064] • Field “Program Selection”: Algorithm: blastp (protein-protein BLAST)

[0065] • Algorithm parameters: Field “General parameters”: Max target sequences: 100; Short queries: Automatically adjust parameters for short input sequences; Expect threshold: 10; Word size: 3; Max matches in a query range: 0

[0066] • Algorithm parameters: Field “Scoring parameters”: Matrix: BLOSUM62; Gap Costs: Existence: 1 1 Extension: 1 ; Compositional adjustments: Conditional compositional score matrix adjustment

[0067] • Algorithm parameters: Field “Filters and Masking”: Filter: none; Mask: none.

[0068] Results are filtered for sequences with more than 35 % query coverage.

[0069] Preferably, the variants can comprise one or more conservative substitutions for amino acids comprised in the exemplary sequences SEQ ID NO: 8, 16, or 24.

[0070] A "conservative substitution" refers to the substitution of one amino acid by another, wherein the replacement results in a silent alteration. This means that one or more amino acid residues within the amino acid sequence of the present invention can be substituted by another amino acid of a similar polarity which acts as a functional equivalent. Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs (i.e. a conservative substitution). For example, one polar amino acid can be substituted by another polar amino acid, one positively or negatively charged amino acid, respectively, can be substituted by another positively or negatively charged amino acid, respectively, et cetera. Classes of amino acids are for example, nonpolar (hydrophobic) amino acids including alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine; polar neutral amino acids including glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids including arginine, lysine and histidine; negatively charged (acidic) amino acids including aspartic acid and glutamic acid.

[0071] As it is used herein “derivative” and “derivatives” is to be understood as all descendant cell lines that have been derived from them or have emerged from them with modification or further development. Polypeptide expression using cellular systems can be performed by using diverse transfection systems. Non-limiting examples are for example lipid-based transfection or viral transduction techniques, which are very well known to a skilled person in the art.

[0072] The patient or subject of the present invention can be a mammal, preferably a human.

[0073] These therapeutic and prophylactic aspects of the present invention are preferably achieved by administering an effective amount of the single-domain antibody of the present invention, or the bivalent polypeptide of the invention, or the polynucleotide encoding the single-domain antibody of the present invention, or the pharmaceutical composition of the present invention, for a time and under conditions sufficient to appropriately achieve the therapeutic or prophylactic effect.

[0074] A “therapeutically effective amount” means an amount that is effective in prevention and / or therapy, or an amount sufficient to provide a preventive and / or therapeutic effect. An amount that is effective in therapy is an amount which produces a biological activity and will depend, among other things, on the individual. The amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of the individual to be treated, the degree of protection desired, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.

[0075] Reference herein to "treatment" and "prophylaxis" is to be considered in its broadest context. The term "treatment" does not necessarily imply that a subject is treated until total recovery. Similarly, "prophylaxis" does not necessarily mean that the subject will not eventually contract a disease condition. Accordingly, treatment and prophylaxis include amelioration of the symptoms of a particular condition or preventing or otherwise reducing the risk of developing a particular condition. The term "prophylaxis" may be considered as reducing the severity or onset of a particular condition. "Treatment" may also reduce or retard the severity or progression of an existing condition.

[0076] Administration of the single-domain antibody of the present invention, or the bivalent polypeptide of the invention, or the polynucleotide encoding the single-domain antibody or the bivalent polypeptide of the present invention, or the pharmaceutical composition of the present invention may be effected by different ways of administration. Non-limiting examples include, but are not limited to, for example, intravenous, intra-arterial, intraperitoneal, intramuscular, pulmonal, inhalative administration. The dosage regimen will be determined by the attending physician and other clinical factors. As well known the skilled person in art, the dosages for any one patient can vary and depend on many factors, including for example size, age, sex, time and router of administration and stage of the disease. The invention is further explained by the attached figures and examples, which are intended to illustrate, but not to limit the present invention.

[0077] FIGURES

[0078] Figure 1 shows identification of AIM2-specific single-domain antibodies. (A) Sequence alignment of the 18 AIM2-specific nanobody candidates identified. Complementaritydetermining regions (CDRs) are indicated. (B, C) ELISA to assess binding of recombinant single-domain antibodies: 1 pg / mL MBP-AIM2PYDor control protein MBP were immobilized on ELISA plates and binding 1 pM of HA-His-tagged single-domain antibodies was quantified by ELISA with anti-HA HRP antibody. Single-domain antibodies identified by phage display with full-length eukaryotically expressed AIM2-SH (B) or bacterially expressed MBP-AIM2PYD(C) were analyzed. (D, E) LUMIER assay to assess binding of single-domain antibodies in the cytosol: HEK 293T cells were co-transfected with expression vectors for the specified HA-tagged single-domain antibodies and the indicated proteins fused to Renilla luciferase. 24 h post-transfection, cells were lysed and VHH-HA was immuno-precipitated with immobilized anti-HA antibody. Coelenterazine h was added and luminescence of co-purified Renilla luciferase was measured. (F) Competition ELISA: MBP-AIM2PYDwas immobilized on ELISA plates and the binding of 100 nM of the indicated HA-His tagged single-domain antibodies in the presence of an increasing concentration of the indicated LPETG tagged nanobody was quantified by ELISA with anti-HA HRP antibodies. Data represent average values (with individual data points) from three independent experiments ± SEM.

[0079] Figure 2 shows a structural analysis of AIM2-specific single-domain antibodies. (A) Crystal structures of AIM2PYD-VHHAIM2-I complex solved at a resolution of 1 .85 A (left) , and AIM2PYD:VHHAIM2-3 complex solved at 2.15 A resolution (right). (B) The crystal structure of the AIM2PYD:VHHAIM2-2 complex solved at 2.08 A resolution. (C) Binding sites of VHHs relative to AIM2PYDpolymerization interfaces. A structure of the AIM2 PYD filament (pdb 7K3R) is shown at the left with each color corresponding to one strand of the three-stranded helical assembly. VHHAIM2-I / 3 and VHHAIM2-2 cover the lb interaction interface, and the Illa and la interfaces, respectively (right), obstructing PYD polymerization. (D) MBP-AIM2PYDwas incubated with TEV to remove MBP in the absence or presence of the indicated VHHs. The resulting structures were analyzed by negative staining and electron microscopy (EM). PYD filaments polymerized in the absence of VHHs, while AIM2 VHHs prevented polymerization. (E) AIM2PYDfilaments were assembled as in D, followed by incubation with the indicated VHHs. Samples were subjected to negative staining and EM. Incubation with VHHs disassembled pre-formed filaments. (F) Sequence alignment of AIM2 single-domain antibodies, highlighting VHH residues that form electrostatic interaction with AIM2PYD. (G) AIM2PYD:VHHs interfaces highlighting electrostatic interaction between residues with a buried surface area (BSA) > 40 A2.

[0080] Figure 3 shows a functional characterization of AIM2 inhibitory single-domain antibodies. (A) Scheme of reconstituted AIM2 inflammasome assay in HEK293T cells constitutively expressing ASC-EGFP (HEK293TASC EGFP), transiently transfected with expression vectors for AIM2 and single-domain antibodies. (B, C) HEK293TASC EGFPcells were co-transfected with empty vector (EV) or AIM2 expression vector in combination with expression vectors for the indicated monovalent VHH-HA (B) or bivalent VHH-(G4S)s-VHH- HA (C). ASC speck assembly was measured 24 h post-transfection by flow cytometry. (D) HEK293TASC EGFPcells were co-transfected with empty vector (EV) or NLRP3 expression vector in combination with expression vectors for the indicated monovalent VHH-HA or bivalent VHH-(G4S)s-VHH-HA. ASC speck assembly was quantified as in B and C. (E-F) Hela cells were co-transfected with expression vectors for AIM2PYD-EGFP and the indicated monovalent (E) and bivalent (F) VHH-HA. 24 h post transfection, cells were fixed and stained with Hoechst 33342 and anti-HA AF647. Representative images were recorded by confocal microscopy. Data representative of three independent experiments is shown. Scale bar = 5 pm. (G) AIM2PYD-EGFP filaments in HeLa cells positive for EGFP and AF647 were quantified and data from 3 independent experiments ± SEM is displayed (50 cells / experiment). (H) Genome structure of recombinant VACV strains expressing C1 C- EGFP from a J2R early promoter and bivalent single-domain antibodies from a synthetic early / late promoter. Transgenes were inserted into the TK locus. (I) Scheme of AIM2 inflammasome indicating all recruited components as well as VACV-encoded singledomain antibodies and tools to perturb or visualize inflammasomes. (J-L) PMA differentiated THP-1 cells were either left untreated or treated with IFN-y overnight and infected with VACV (MOI 5) expressing C1 C-EGFP and the indicated single-domain antibodies for 6 h in the presence (J) or absence (K, L) of VX765. Cells were harvested, fixed and ASC speck assembly was analyzed by flow cytometry (J). IL-ip in the supernatant was measured by Homogeneous Time-Resolved Fluorescence (HTRF) (K) and cell death was measured by LDH release, normalized to cells lysed in 1% Triton X-100 (L). (M) PMA-differentiated THP-1 cells were either left untreated or treated with IFN-y overnight and infected with VACV (MOI 5) expressing C1 C-EGFP and the indicated singledomain antibodies in the presence of 100 nM DRAQ7. DRAQ7 uptake was monitored over 6 h in an Incucyte Live-Cell Imaging system. Representative images after 6h (M) and graphs of normalized DRAQ7 uptake in untreated (N) and IFN-y treated (O) cells over 6 h are displayed. Scale bar: 100 pm. Graphs represent data from three independent experiments ± SEM. (P-R) Human monocytes-derived GM-CSF differentiated macrophages (P), Normal Human Epidermal Keratinocytes (Q), or CD14+monocytes (R) were left untreated or pre-treated with IFN-y overnight. Cells were infected with VACV (MOI5) expressing C1 C-EGFP and the indicated single-domain antibodies in the presence of VX765. 6 h post-infection, cells were harvested, fixed, and speck assembly was analyzed by flow cytometry. Data represent average values (with individual data points) from three independent donors ± SEM

[0081] Figure 4 shows identification of AIM2-specific single-domain antibodies. (A) The average distance tree was calculated based on the percentage identity between the indicated single-domain antibody sequences displayed in Fig 1 A. (B) ELISA to assess binding of recombinant single-domain antibodies: Data for individual single-domain antibodies displayed in Figure 1 , B and C, is shown in separate plots.

[0082] Figure 5 shows a structural analysis of AIM2-specific single-domain antibodies. (A) Crystal structures of AIM2PYD-VHHAIM2-I complex superimposed to AIM2PYD-VHHAIM2-2 complex. (B) Modeled poly-A loop (26A) from C-terminus of VHHAIM2-2 to N-terminus of VHHAIM2-I . The linear distance is » 58 A (length of 26A peptide » 98.4A). (C) Modeled poly- A loop (25A) from C-terminus of VHHA|M2-I to N-terminus of VHHAIM2-2- The linear distance is » 66A (length of 25A peptide » 95A).

[0083] Figure 6 shows in (A) a schematic representation of the AIM2PYDpolymerization assay. Soluble MBP-AIM2PYDis cleaved by TEV protease. The released AIM2PYDpolymerizes into insoluble filaments that can be sedimented by high speed centrifugation. (B) shows the results of an SDS-PAGE with colloidal Coomassie staining. MBP-AIM2PYDand a 1.5-fold molar excess of control or AIM2-inhibitory bivalent single-domain antibodies were incubated with TEV protease at RT for 16 h. AIM2PYDfilaments were sedimented at 20,000 x g, and supernatants and pellets were analyzed by SDS-PAGE and colloidal Coomassie staining. Input as well as supernatants and pellets from samples with bivalent single-domain antibodies are shown in this panel. The uncropped gels with samples of the reaction at t=0 h and 16 h as well as samples with monovalent single-domain antibodies are shown in Figure 7.

[0084] Figure 7 shows that AIM2 single-domain antibodies inhibit AIM2PYDoligomerization and filament formation. (A) 100 pg of MBP-AIM2PYD(34.4 pM) was incubated with TEV protease at RT for 16 h in the presence of 75 pg of monovalent (103.2 pM) or bivalent (51 .6 pM) control or AIM2-inhibitory single-domain antibodies as indicated. 2 pg of each input protein and samples before (t= Oh, To) and after incubation (t=16h, TI6) were analyzed by SDS-PAGE and displayed on the left. AIM2PYDfilaments were sedimented at 20,000 x g, and samples of supernatants and pellets are shown on the right. Excerpts from the entire gels shown here are displayed in Figure 6B. Control samples without TEV display that MBP-AIM2PYDis almost completely found in the supernatant, with very little fusion protein in the pellet (and no processing in the absence of TEV is observed). Note that AIM2PYDis exclusively found in the pellet fraction after release from MBP in the presence of bivalent control single-domain antibodies. In the presence of bivalent AIM2 single-domain antibodies, AIM2PYDis mostly found in the supernatant, indicating that single-domain antibody binding to AIM2PYDprevents filament formation. Monovalent single-domain antibodies cannot be distinguished from AIM2PYDby SDS-PAGE (compare input singledomain antibodies and pure AIM2PYDreleased at T16 in the presence of bivalent singledomain antibodies). In the presence of monovalent control single-domain antibodies, the VHH / AIM2PYDband in the pellet corresponds to the intensity of the AIM2PYDband in the pellet in the presence of bivalent control single-domain antibodies, indicating that the pellet mostly contains AIM2PYD. In the presence of inhibitory AIM2 single-domain antibodies, the band intensity of VHH / AIM2PYDin the supernatant is drastically increased, while the VHH / AIM2PYDband in the pellet is much weaker. Representative gels from at least 3 independent experiments are displayed.

[0085] EXAMPLES

[0086] Example 1 - Material and Methods

[0087] Cell lines

[0088] Human THP-1 cells (ATCC TIB-202, RRID: CVCL 0006) were cultured in RPMI 1640 GlutaMax medium (Thermo Fisher Scientific) containing 10% FBS, 50 pM 2- mercaptoethanol, and 100 LI / mL penicillin-streptomycin. Human HEK293T (ATCC CRL- 3216, RRID: CVCL_0063) and Vervet monkey BSC-40 (CRL-2761 , RRID:CVCL_3656) cells were cultivated in DMEM GlutaMax containing 10% FBS, and 100 LI / mL penicillinstreptomycin; BSC-40 media was supplemented with non-essential amino acids and 1 mM sodium pyruvate. Flp-ln 293 T-REx cells (Thermo Fisher Scientific, R78007, RRID:CVCL_U427), dox-inducibly expressing AIM2-SH or NLRP3-SH were generated according to manufacturer’s recommendation, and cultured in DMEM containing 10% FBS, GlutaMax, 4 pg / ml blasticidin S, and 50 pg / ml hygromycin B. Lentivirus produced with packaging vectors psPax2 and pMD2.G (kind gifts provided by Didier Trono, Ecole polytechnique federate de Lausanne, Switzerland), were used to generate genetically modified cell lines. THP-1 cell lines expressing caspase-1CARD-EGFP (C1 C-EGFP) or C1 C- TagBFP inflammasome reporter under a dox-inducible promoter were generated using lentiviral vectors derived from plnducer20 (a kind gift provided by Stephen Elledge, Harvard Medical School), and selected with 500 pg / mL geneticin (Thermo Fisher Scientific). Cell lines constitutively expressing transgenes under the control of the human elongation factor- 1a promoter (pEF1a) or human ubiquitin C promoter (pUbC) were generated using lentiviral vectors constructed by Gateway cloning (Thermo Fisher Scientific) using vectors modified from pRLL (a kind gift of Susan Lindquist, Whitehead Institute of Biomedical Research), followed by antibiotic selection.

[0089] Primary cells

[0090] For primary human cells isolation, whole blood buffy coats were obtained from the blood bank of the University Hospital Bonn, with consent of healthy donors and according to protocols accepted by the institutional review board of the University of Bonn. PBMCs were isolated using Ficoll-Paque™ PLUS (VWR) according to the manufacturer’s instruction and CD14+were isolated by positive selection using paramagnetic CD14 (human) MicroBeads (Miltenyi Biotec). 1 x 107CD14+monocytes were then differentiated into macrophages using either 100 ng / mL recombinant human M-CSF (Immunotools) or 500 U / mL recombinant human GM-CSF (Immunotools) for 4 days. Primary cells were cultured in RPMI 1640 GlutaMax medium supplemented with 10% FBS, 100 U / mL penicillin-streptomycin, and 1 mM sodium pyruvate.

[0091] Primary Normal Human Epidermal Keratinocytes (NHEK) were obtained from abdominal or breast skin removed during plastic and reconstructive surgery at the University Hospital of Bonn, with consent of patients and according to protocols accepted by the institutional review board of the University of Bonn. Keratinocytes were isolated from the skin utilizing a previously published protocol (Johansen 2017) and cultured in DermaCult™ Keratinocyte Expansion Medium (STEMCELL Technologies), supplemented with hydrocortisone (96 ng / ml, STEMCELL Technologies), penicillin-streptomycin (10 U / ml), gentamicin (5 pg / ml), and amphotericin B (2.5 pg / ml, all Thermo Fisher Scientific). NHEK cells expressing dox-inducible or constitutive caspase- 1CARD- EG FP (C1 C-EGFP) inflammasome reporter were generated by lentiviral transduction and antibiotic selection as described above.

[0092] Generation and production of recombinant VACV:

[0093] Recombinant VACV strains were generated based on VACV Western Reserve (WR) strain by transfecting infected cells with derivatives of plasmid pJS4 containing the flanking regions of the tk locus as well as sequences of genes of interest with viral promoters as described in (Chomczynski and Mackey 1995; Mercer and Helenius 2008). Recombinant VACV strains expressing C1 C-EGFP (WR E C1 C-EGFP) or EGFP (WR E EGFP) were generated with pJS4 derivatives encoding C1 C-EGFP / EGFP under the J2R early promoter and neomycin phosphotransferase under a synthetic early / late promoter (Chakrabarti, Sisler, and Moss 1997; Florian Ingo Schmidt et al. 2013). Recombinant VACV expressing inflammasome reporter and bivalent nanobodies were generated similarly using pJS4 derivatives encoding C1 C-EGFP or EGFP under the control of the J2R early promoter and the bivalent nanobodies under the control of a synthetic early / late promoter. Recombinant VACV strains were isolated by at least three rounds of fluorescent plaques purification. VACV mature virions (MVs) were produced using BSC-40 cells and purified from cytoplasmic lysates through a sucrose cushion as described in (Mercer and Helenius 2008). Virus titers were determined by plaque assay on BSC-40 cells. All work involving VACV and MPXV was performed according to BSL2 and BSL3 safety standards, respectively.

[0094] Plasmids

[0095] Expression vectors, lentiviral vectors, and plasmids for homologous recombination of VACV described in the individual experiments were generated by Gateway and Gibson cloning. Plasmid maps and oligonucleotide sequences are shared on request.

[0096] Protein expression and purification

[0097] Expression of His-MBP-AIM2PYDL 10A L11 A for immunization

[0098] To produce a soluble AIM2PYDmutant L10A L11A (Lu et al. 2014), we generated bacterial expression vector pEXPR His-MBP-AIM2 PYD L10A L1 1 A (encoding AA 1 -107 of AIM2) with a gateway-compatible derivative of modified pDB-His-MBP. Escherichia (E.) coll LOBSTR (PMID: 23852738) was transformed with pEXPR His-MBP-AIM2 PYD L10A L11 A and bacteria were grown in Terrific Broth, induced with 0.2 mM IPTG at an OD600 of 0.6, and cultivated for another 24 h at 18° C. Cells were lysed with a Bandelin Sonopuls HD2070 sonicator with a TT13 tip, followed by Ni-NTA affinity purification with Ni-NTA agarose (Qiagen) and size exclusion chromatography with a HiLoad 16 / 600 Superdex 75 pg column (GE Healthcare Life Sciences) in high salt HEPES gel filtration buffer (20 mM HEPES pH 7.4, 150 mM NaCI, and 10% glycerol).

[0099] Expression of His-MBP-AIM2PYDfor crystallization

[0100] Expression vector pET30a His-MBP-AIM2 PYD was a gift from Professor Tsan Sam Xiao, Case Western Reserve University (cite this https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC3650362 / ). E.coli BL21 (DE3) was transformed and was grown at 37° C until OD6oo reached 0.6. Cells were then transferred to 4° C for 30 minutes before IPTG was added to a final concentration of 1 mM and cells grown over night at 18° C. After 18h, cells were sedimented, lysed by sonication, and purified using Ni-NTA affinity purification and gel filtration on a Superdex 200 Increase 10 / 300 GL (GE Healthcare Life Sciences) in TRIS gel filtration buffer (20 mM TRIS pH 8.0, 100 mM NaCI, 5 mM maltose). Expression and purification of nanobodies

[0101] AIM2 nanobody sequences were cloned into pHEN6-based vectors for periplasmic expression with C-terminal LPETG-His or TEV.His for large scale, or C-terminal HA-His for small scale protein expression. Nanobodies were expressed and purified as described before (Koenig et al. 2021 ). E. coli \NKQ were transformed with nanobody expression vectors and grown in Terrific Broth, induced with 1 mM IPTG at an ODeoo of 0.6, and cultured at 30 °C for 16 h. Bacteria were sedimented and resuspended in TES buffer (200 mM Tris- HCI pH 8.0, 0.65 mM EDTA, 0.5 M sucrose), and incubated for 1 h at 4° C with shaking. Periplasmic extracts were obtained by osmotic shock in 0.25x TES overnight at 4°C. Nanobodies were purified with Ni-NTA agarose beads, followed by desalting with PD MiniT rap G-25 columns (GE Healthcare Life Sciences) for small scale expression, or by gel filtration with a HiLoad 16 / 600 Superdex 75 pg column in HEPES gel filtration buffer (20 mM HEPES pH 7.4, 150 mM NaCI, and 10% glycerol) or a Superdex 200 Increase 10 / 300 GL column in TRIS gel filtration buffer for large scale expressions. For crystallization, His- tags were removed by incubation with TEV protease at 4° C. Free His-tag and undigested nanobodies were removed with Ni-NTA resin. Flow through containing nanobodies were concentrated and purified by gel filtration with a Superdex 200 Increase 10 / 300 GL column.

[0102] Antibodies

[0103] The following antibodies were used: rabbit monoclonal anti-HA-Tag (Cell Signaling Technology Cat# 3724, RRID: AB_1549585), rabbit polyclonal anti-E-tag-HRP (Bethyl Cat# 5 A190-133P, RRID:AB_345222), mouse anti-HA.1 1 Epitope tag clone 16B12 (BioLegend Cat# 901503, RRID:AB_2565005), mouse anti-HA-HRP clone 6E2 (Cell Signaling Technology Cat# 2999S, RRID:AB_1264166).

[0104] Small compound inhibitors and reagents

[0105] The following small compound inhibitors and reagents were used: NLRP3 inhibitor CRID3 (MCC-950) (Tocris), doxycycline (Biomol), LPS-EK Ultrapure (Invivogen), human IFN-y (Immunotools), PMA (phorbol 12-myristate 13-acetate) (Sigma-Aldrich), Roche complete™ Mini protease Inhibitor Cocktail (Sigma-Aldrich), and Vx-765 / belnacasan (Selleckchem).

[0106] Nanobody library generation

[0107] To raise AIM2 specific VHHs, an alpaca was immunized six times with 200 pg MBP- AIM2PYDL10A L1 1 A using GERBU Adjuvant Fama (GERBU Biotechnik GmbH), according to locally authorized protocols (Landesuntersuchungsamt Rheinland-Pfalz, 23 177-07 / A 17-20-005 HP). The M13 phagemid vector pD (pJSC) was used to generate the VHH plasmid library as described before in (Koenig et al. 2021 ). In short, RNA from peripheral blood lymphocytes was isolated and used as a template to generate cDNA using three sets of primers (random hexamers, oligo(dT), and alpaca heavy chain gene specific primers). VHH coding sequences were amplified by PCR using VHH-specific primers, cut with Asci and Notl, and ligated into a linearized M13 phagemid vector (pJSC). E.coli TG1 cells (Agilent) were electroporated with ligation reactions and the obtained ampicillin-resistant colonies were harvested, pooled, and stored as glycerol stocks.

[0108] Nanobody screening by phage display

[0109] AIM2PYD-specific VHHs were obtained by phage display and panning as described in (Koenig et al. 2021 ). E.coli TG1 cells containing VHH library were infected with helper phage VCSM13 to produce phages displaying encoded VHHs as pill fusion proteins. Phages collected from the supernatant were purified and concentrated by precipitation. Phages displaying AIM2PYD-specific VHHs were enriched using two approaches; 1 ) phages were incubated with chemically biotinylated MBP-AIM2PYDimmobilized on amylose magnetic beads (New England Biolabs), and 2) phages were incubated with full length AIM2-SH expressed in HEK 293 Flp-ln T-REx cells and immobilized on MagStrep type 3 Strep-Tactin beads (IBA Lifesciences). Bound phages were eluted by low pH elution or biotin elution buffers, respectively, and used to infect E.coli ER2738, followed by a second round of panning. E.coli ER2837 colonies from the second round of panning were picked and grown in 96-well plates. VHH expression was induced with IPTG, and expressed VHHs that leaked into the supernatant were tested for specificity using ELISA plates coated with control protein MBP or MBP-AIM2PYDL10A L11A. VHH binding was detected with HRP- coupled rabbit anti-E-Tag antibodies (1 :10,000), and the chromogenic substrate tetramethylbenzidine (TMB) (Life Technologies). 1 M HCI was used to stop the reaction prior to recording absorption at 450 nm using a SpectraMax i3 instrument, and the SoftMax Pro 6.3 Software (Molecular Devices). Positive candidates were sequenced and representative VHHs were cloned into bacterial and mammalian expression vectors for further analysis.

[0110] ELISA

[0111] To test AIM2-specific VHHs, MBP or MBP-AIM2PYDL10A L11 A in PBS were immobilized on ELISA plates at a concentration of 1 pg / mL overnight. On the following day, the immobilized antigens were incubated with the HA-tagged VHHs in 10% FBS / PBS in a 10-fold dilution series ranging from 1 pM to 100 pM. Bound VHHs were detected using the mouse anti-HA HRP antibody (1 :5000) and developed with the chromogenic substrate TMB. 1 M HCI was used to stop the reaction prior to measuring absorption at 450 nm using a SpectraMax i3 instrument and the SoftMax Pro 6.3 Software (Molecular Devices).

[0112] LUMIER assay

[0113] To test the intracellular target binding of VHHs in the reducing environment of the cytosol, LUMIER assays was performed as previously described (Florian I. Schmidt et al. 2016). In short, Renilla luciferase fusions of target proteins were co-expressed with HA- tagged VHHs in HEK293T cells. VHHs from cell lysates were immunoprecipitated with anti- HA antibodies, and the co-purified luciferase activity was determined as a read out for cytosolic interaction of VHHs and target proteins. 2.5 105HEK293T cells per well were seeded into 24-well plates, and the following day co-transfected with 0.25 pg pCAGGS VHH-HA expression vectors and 0.25 pg of pcDNA3.1 -based expression vectors for the Renilla-fused target proteins human full length AIM2, mouse full length AIM2, human AIM2HIN200(aa 144-343), human AIM2PYD(aa 1 -107), or the control human NLRP1CARDusing transfection reagent PEI Max (Polysciences). High-binding Lumitrac 600 white 96- well plates (Greiner) were coated with 20 pg / mL of the mouse anti-HA.11 Epitope tag clone 16B12 antibody in PBS overnight. The next day, HEK293T cells were lysed in LUMIER lysis buffer (50 mM Hepes-KOH pH 7.9, 150 mM NaCI, 2 mM EDTA, 0.5% Triton X-100, 5% glycerol and Roche complete™ Mini protease Inhibitor Cocktail), and lysates were incubated in the anti-HA-coated Lumitrac 600 plates for one hour to immunoprecipitate HA- tagged VHHs. After repeated washing, Renilla luciferase substrate coelenterazine-h was added and luminescence was measured using a SpectraMax i3 instrument, and the SoftMax Pro 6.3 Software (Molecular Devices).

[0114] Crystallization and structural determination

[0115] MBP-AIM2PYDand nanobodies were mixed with a molar ration 1 :2 and incubated overnight at 4 °C in gentle shaking, followed by purification of MBP-AIM2PYDnanobodies complex by gel filtration. The peaks corresponding to AIM2-nanobodies complex were collected and concentrated with a concentration column with a 10kDa cut-off to a final concentration of 15 mg / ml. Purified AIM2-nanobodies complex were used for setting sittingdrop vapor-diffusion crystals in MRC two-well crystallization plates. Crystal trays were purchased from Molecular Dimension. Each well was fill with 75 pl of reservoirs and two drops were set with 1 :1 and 1 :3 protein :reservoir (v:v) ratio in a final volume of 250 nl and 500 nl respectively. Crystallization plates were then incubated at 20 °C. Rod-shaped crystals began to appear after 3-5 days.

[0116] AIM2PYD-VHHAIM2-I crystal were obtained in 0.1 M Ammonium sulfate, 0.3 M Sodium formate, 0.1 M Sodium cacodylate 6.5, 3 % w / v y-PGA (Na+ form, LM), 5 % w / v PEG 4000, with volume ration 1 :3. AIM2PYD-VHHAIM2-2 in 0.3 M Sodium acetate trihydrate, 0.1 M Tris 8.5, 10 % w / v PEG 8000, 10 % w / v PEG 1000 with volume ration 1 :1 . AIM2PYD-VHHAIM2-3 0.075 M Sodium bromide, 0.05 M Sodium fluoride, 0.1 M HEPES 7.8, 22.5 % v / v PEG Smear Broad, 0.075 M Sodium iodide, with volume ration 1 :3. 12% glycerol (v / v) was added to reservoir solution as cryoprotectant before freezing the crystals in liquid nitrogen.

[0117] X-ray crystallography data collection, structure solution and refinement

[0118] Data collection was performed at the Advanced Photon Source using Northeastern Collaborative Access Team (NE-CAT) beamlines 24-ID-C and 24-ID-E. Data were processed by XDS (Kabsch 2010), and molecular replacement solution was obtained from Phaser (Adams et al. 2010) using the PDB accession number 3VD8 (https: / / pubmed.ncbi.nlm.nih.gov / 23530044 / ) for AIM2 and 5H8D (https: / / pubmed.ncbi.nlm.nih.gov / 270691 17 / ) for nanobodies as search models.

[0119] Subsequent model building and refinement were carried out in Coot (Emsley et al. 2010) and PHENIX (Adams et al. 2010). Model was built by iterative refinement using coot (https: / / pubmed.ncbi.nlm.nih.gov / 15572765 / ) and Phenix. refine

[0120] (https: / / pubmed.ncbi.nlm.nih.gov / 20124702 / ). Structure was validated by Molprobity (Chen et al. 2010). Figures were generated using PyMOL (Delano 2002).

[0121] Virus infections

[0122] For virus infections in THP-1 cells, 3 105cells were differentiated into macrophage-like cells using 50 ng / mL PMA (Sigma-Aldrich) over night (16 h) in a 24-well plate. The following morning, medium was changed to PMA-free medium, where indicated containing 1 pg / mL dox to induce gene expression. For infection experiments in primary cells, we seeded 1.5-105primary macrophages, 5 104keratinocytes (both in 24-well plate), or 3 105CD14+monocytes (in 96-well plates). 24 h post seeding, adherent cells (differentiated THP-1 , primary macrophages and keratinocytes) were treated with 500 U / rnL IFN-y (Immunotools) overnight where indicated. CD14+monocytes were treated with IFN-y at the time of seeding. On the following day, cells were infected with 300 pL (for 24-well plate) and 100 pL (for 96-well plate) of virus inoculum containing the indicated MOI of freshly sonicated virus in serum-free medium. Cells were incubated for one hour with gentle rocking every 15 minutes. 1 h post infection, the virus inoculum was replaced with 500 pL full medium, and cells were cultivated for 6 h. CD14+monocytes were cultivated for 6 h without media change to enhance infection efficiency. Cells were analyzed by flow cytometry or microscopy, while supernatants were analyzed by HTRF (see below). Quantification of inflammasome assembly by flow cytometry

[0123] To quantify the assembly of inflammasomes, the assembly of ASC-EGFP specks or recruitment of caspase-1CARD-EGFP (C1 C-EGFP) or C1 C-TagBFP to endogenous ASC specks was quantified by flow cytometry. Reporter cell lines were either infected with VACV WT or transfected with the indicated expression vectors, or WT cells were infected with recombinant VACV expressing C1 C-EGFP. All infections were done in the presence of 40 pM VX to prevent cell death by pyroptosis. Infected cells were trypsinized, fixed in 4% formaldehyde for 20 minutes, resuspended in FACS buffer (PBS, 2% FBS, 5 mM EDTA, 0.02% NaN3), and analyzed using BD FACS Canto flow cytometer. To determine the frequency of cells with ASC or C1 C-EGFP / TagBFP specks, we first gated on single cells, followed by gating for EGFP positive cells. Only EGFP+or T agBFP+cells (except for mock- infected samples) were included for the analysis of inflammasome speck formation. We plotted the width against the height of EGFP / TagBFP and gated cells with C1 C specks as described in (Jenster et al. 2023). Flow cytometry data was analyzed using FlowJo 10.8.1 software.

[0124] Cell death quantification by LDH release (plasma membrane rupture)

[0125] To quantify release of cytosolic LDH as a consequence of cell death upon VACV infection, 3 105THP-1 cells were differentiated and infected in 24-well plates as described before. Cells were infected for 6 h in Opti-MEM™. Supernatants were collected, and LDH release was quantified using LDH Cytotoxicity Detection Kit (TakaRa) according to the manufacturer’s instructions. Absorption at 492 nm was measured using a SpectraMax i3 instrument and the SoftMax Pro 6.3 Software (Molecular Devices). Cell death was normalized to control well in which cells were lysed in 1 % Triton X-100, and background signal from medium was subtracted from all samples.

[0126] Cell death quantification by DRAQ7 uptake (membrane integrity)

[0127] To quantify VACV infection-induced loss of plasma membrane integrity overtime, 3 105THP-1 cells were differentiated and infected in 24-well plates as described before. Cells were infected with virus inoculum for 1 h, inoculum was removed and cells were covered with full medium containing 100 nM DRAQ7 (Biolegend). DRAQ7 uptake and infection (EGFP+cells) was recorded by taking four images per well every 15 minutes, for a total of 6 h using the Incucyte Live-Cell Imaging system (Sartorius). The number of DRAQ7-uptake, infection and cell confluency were analyzed using the Incucyte 2021 C software. For every single image, the cell death count was determined. The cell death was first corrected by subtracting cell death count at T=0 and was further normalized to the cell confluency, and average values from all 4 images were calculated and plotted over time. Cytokine quantification by homogenous time resolved fluorescence (HTRF)

[0128] To quantify IL-1 p secretion post VACV infection, 3 105cells were seeded in 24-well plates as described before. For IL-ip secretion quantification experiments, cells were infected for 6 h in Opti-MEM™ in the absence of VX. Supernatants were then collected, and IL-i levels were quantified using the Human IL1 HTRF kit (Ciscbio) according to the manufacturer’s instructions. Emissions at 620 and 665 nm were measured using a SpectraMax i3 instrument and I L- 1 p levels were calculated by the SoftMax Pro 6.3 Software (Molecular Devices) based on the standard curve.

[0129] Confocal microscopy

[0130] To quantify ASCPYD-EGFP filament formation by confocal microscopy, HEK293T cells were seeded on 12 mm cover slips in 24-well plates and co-transfected the next day with 0.25 pg of monovalent or bivalent VHH-HA, and AIM2PYD-EGFP expression vectors using Lipofectamine™ LTX transfection reagent (Thermo Fisher Scientific). 24 h later, transfected cells were fixed in 4% formaldehyde in PBS for 20 minutes, followed by permeabilization with permeabilization buffer (PBS with 0.5% Triton X-100, 10% goat serum) for 20 minutes. Cells were consecutively stained with primary rabbit anti-HA antibody (1 :1000), and secondary goat anti-rabbit IgG AF647 (1 :1000) in permeabilization buffer for 1 h each. DNA was stained with Hoechst 33342 (Thermo Fisher Scientific) (1 :5000), and images were recorded with the HC PL APO CS2 63x / 1 .20NA oil objective on a Leica SP8 Lightning confocal microscope. Images were processed and quantified using Imaged 2.3.0 software.

[0131] Example 2 - Identification of AIM2-specific single-domain antibodies

[0132] To visualize and functionally perturb human AIM2 inflammasomes, single-domain antibodies targeting the human AIM2 Pyrin domain, AIM2PYD, were generated. To enhance solubility of the protein and prevent oligomerization, bacterial expression vectors for fusion protein of His-tagged maltose binding protein (MBP) to the monomeric AIM2PYDmutant L10A L11 A (His-MBP-AIM2PYDL10A L11 A) were constructed. The protein was expressed in the cytosol of E. coli, and the protein was purified from bacterial lysates using Ni-NTA affinity purification and size exclusion chromatography with a Superdex S75 column. An alpaca was immunized six times with the purified protein and the coding sequences for the variable domains of heavy chain-only antibodies (VHHs) were cloned into a phagemid vector as described before.

[0133] Two strategies to identify AIM2PYD-specific single-domain antibodies by conventional phage display were employed: 1 ) First, full length human AIM2-Strep2-HA (AIM2-SH) was expressed in HEK 293 Flp-ln T-REx cells, AIM2-SH was immobilized from fresh lysates on StrepTactin beads and the coated beads were used to enrich for phages displaying AIM2- specific single-domain antibodies in two consecutive selection rounds. 2) In a second approach, phages were prepared from the library, phages reactive to MBP were depleted using recombinant MBP coated to a tissue culture flask, and subsequently recombinant His- MBP-AIM2PYDL10A L1 1A immobilized on magnetic amylose beads was used to enrich for phages displaying AIM2PYD-specific single-domain antibodies. 95 clones of single-domain antibodies of each phage display campaign were tested by ‘bug sup ELISA’ with immobilized His-MBP-AIM2PYDL10A L11 A and the corresponding single-domain antibodies to positive wells were sequenced. These efforts yielded 6 distinct single-domain antibody candidates from the phage display campaign with AIM2-Strep2-HA (VHHAIM2-I-VHHAIM2-6) as well as 12 single-domain antibody candidates from the phage display campaign with His- MBP-AIM2PYDL10A L1 1A (Fig. 1A, Fig. 4A).

[0134] All single-domain antibody candidates were expressed in the periplasm of bacteria and purified by a combination of Ni-NTA affinity chromatography and desalting. Next His-MBP alone or His-MBP-AIM2PYDL10A L1 1 A was immobilized to ELISA plates and ELISAs were performed to assess binding of recombinant single-domain antibodies (Fig. 1 , B and C, Fig. 4B). All single-domain antibody candidates identified using full length AIM2 expressed in human cells robustly bound to recombinant His-MBP-AIM2PYDL10A L1 1A, but did not bind MBP (Fig. 1 C). Most single-domain antibody candidates identified with the bacterially expressed AIM2PYDbound His-MBP-AIM2PYDL10A L11 A, although several clones exhibited some background binding to the control protein MBP, indicating some unspecific interactions. As not all single-domain antibodies function in the reducing environment of the cytosol, LUMIER assays were employed next to assess binding of single-domain antibodies to AIM2 in the cytosol of living cells: HEK293T cells were transfected with plasmids to coexpress HA-tagged single-domain antibodies as well as a fusion of full length AIM2 to Renilla luciferase. Single-domain antibodies from cell lysates were immunoprecipitated with immobilized anti-HA antibodies and co-immunoprecipitation of AIM2 was assessed by measuring the activity of Renilla luciferase using its specific substrate Coelenterazine h (Fig. 1 D). Three of the single-domain antibodies identified with eukaryotically expressed AIM2 (VHHAIM2-I , VHHAIM2-2, and VHHAIM2-3) bound full length AIM2 in LUMIER assays, while all other candidates did not function in the cytosol. Of note, although VHHA|M2-I and VHHAIM2-3exhibited differences in all CDRs, they were 90% identical and 94% similar, suggesting that they may be derived from the same ancestral clone and bind to AIM2 similarly. Further LUMIER assays revealed that all three single-domain antibodies bound to human AIMPYD, but not the HIN-200 domain of AIM2, as expected based on the antigen used for immunization. None of the single-domain antibodies bound to murine Aim2. Competition ELISAs revealed that an excess of untagged VHHAIM2-2interfered with binding of VHHAIM2-2- HA to AIM2PYDas expected (Fig. 1 E). Surprisingly, incubation with untagged VHHAIM2-2 enhanced binding of VHHAIM2-I and VHHAIM2-3, indicating that the identified single-domain antibodies bind to at least two non-overlapping epitopes defined by VHHAIM2-2and VHHA|M2- I / VHHAIM2-3- It is possible that binding of VHHAIM2-2reduced oligomerization of the AIM2PYDand therefore increased the availability of binding sites for the other two single-domain antibodies.

[0135] Example 3 - Structural analysis of AIM2-specific single-domain antibodies

[0136] To define the epitopes of the identified AIM2 single-domain antibodies in atomic detail, the X-ray crystallography structures of MBP-AIM2PYDin complex with VHHAIM2-I, VHHAIM2-2, and VHHAIM2-3were solved at resolutions of 1.85 A, 2.08 A, and 2.20 A, respectively (Fig. 2A, Table 1 ).

[0137] Table 1 : Data collection and refinement statistics.

[0138] VHHAIM2-I and VHHAIM2-3 take up a nearly identical 3D structure and bind to the same epitope of AIM2PYDthrough all the CDRs, masking the lb interaction interface of AIM2PYDfilaments (Fig. 2C). In the CDR1 , H33 and R35 of VHHAiM2-i / 3form salt bridges with D23 and D31 of the PYD, respectively. R35 and Y37 in the same CDR form hydrogen bonds with S30 and D31 in the PYD (Fig. 2G). The exact binding modes of CDR2 of VHHAIM2-I and VHHAIM2-3differed slightly: In CDR2 of VHHA|M2-I, R53 forms a salt bridge with D23 in the PYD (VHHAIM2-I contains Asparagine in position 53), while T54 and N59 form hydrogen bonds with R24 and K71 in the PYD. In CDR2 of VHHAIM2-3, N59 and Y60 both form hydrogen bonds with K71 . In the CD3 of VHHAIM2-I / 3, Y101 forms hydrogen bonds with S30 and H41 in the PYD, and D106 with T37 in the PYD. Overall, VHHAIM2-I / 3 thus bind an epitope of AIM2PYDcontaining D23, R24, K26, F27, F28, S30, D31 , T37, H41 , K71 , L72, and N73 (all >10 A2buried surface area, Fig. 2F).

[0139] VHHAIM2-2binds a distinct, non-overlapping epitope of AIM2PYDwith all its CDRs, masking the Illa and la interfaces that AIM2PYDemploys to assemble filaments (Fig. 2C). In the CDR1 , and extended network of hydrogen bonds between T28:K87, S30:R80, N31 :T23, N31 :R80, N31 :E83, N31 :E84, Y32:E84, and A33:L1 1 defines the interface of VHHAIM2-2:AIM2PYD. In the CDR2, R56 forms a salt bridge with E21 in the PYD, while S53:N16, S54:R80, and R56:N16 form hydrogen bonds in the VHHAIM2-2:AIM2PYDinterface. G100 and S102 in the CDR3 of VHHAIM2-2 bind D15 of AIM2PYDvia hydrogen bonds. Overall, VHHAIM2-2 masks an epitope comprised of E7, I8, L11 , T12, D15, N16, T18, D19, E21 , N44, R45, I46, L76, R80, E83, E84, and K87 of AIM2PYD(all >10 A2buried surface area, Fig. 2F).

[0140] While MBP-AIM2PYDexhibits moderate solubility, the PYD begins to polymerize into filaments once the TEV recognition site between MBP and PYD is cleaved with recombinant TEV protease. These filaments can be visualized by negative staining and electron microscopy (Fig. 2D). If MBP is removed in the presence of VHHA|M2-I, VHHAIM2-2, or VHHAIM2-3, no polymerization of AIM2PYDis observed, which is in line with their binding sites in the polymerization interfaces. When already polymerized AIM2PYDwas incubated with the individual single-domain antibodies, negative staining and electron microscopy revealed that all the single-domain antibodies were able to induce the de-polymerization of PYD filaments (Fig. 2E).

[0141] Taken together, the structural analysis suggested that the identified AIM2 singledomain antibodies bind to critical interfaces required for the oligomerization of the AIM2 PYD and consequently impair filament formation at least in vitro. Example 4 - Functional characterization of AIM2-specific single-domain antibodies in cells

[0142] Next, it was evaluated whether cytosolic expression of AIM2 single-domain antibodies interfered with AIM2 inflammasome assembly in living cells. First, AIM2 inflammasomes were reconstituted in HEK293T cells expressing ASC-EGFP (HEK293TASC EGFP) by overexpressing AIM2-FLAG (Fig. 3A). Transient transfection with AIM2-FLAG expression vectors provided double-stranded DNA (plasmid) as an AIM2 ligand and allowed transcription and translation of AIM2. Ligand-bound AIM2 oligomerizes and nucleates the assembly of ASC-EGFP specks. The latter can be detected by flow cytometry, exploiting the characteristic redistribution of EGFP fluorescence from broad cytoplasmic localization throughout the cell to a single spot per cell. While co-expression of a control single-domain antibody against influenza A virus NP (VHHNP-I) did not alter ASC speck assembly, coexpression of the three single-domain antibodies that are functional in the cytosol reduced inflammasome assembly to varying degrees, but not completely (Fig. 3B). Next, expression vectors were constructed for bivalent single-domain antibodies containing all possible combinations of VHHAIM2-I, VHHAIM2-2, and VHHAIM2-3as well as a bivalent control singledomain antibodies composed of two concatenated copies of VHHNp-i. In all cases, the 15 aa linker (GGGGS)3(SEQ ID NO:26) was used. All bivalent combinations substantially improved inhibition and most combinations completely inhibited AIM2 inflammasome assembly (Fig. 3C). In control experiments, NLRP3 inflammasomes were activated using a similar overexpression system in HEK293TASC EGFPcells (Fig. 3D). Of note, NLRP3 also recruits and nucleates ASC filaments and specks through its Pyrin domain (PYD). None of the AIM2 single-domain antibodies interfered with NLRP3 inflammasome assembly, indicating that the single-domain antibodies were specific for the PYD of AIM2. Most subsequent experiments were conducted with the homobivalent single-domain antibody VHHAIM2-I-VHHAIM2-I as well as the heterobivalent single-domain antibody VHHA|M2-I- VHHAIM2-2. Both single-domain antibody components of the latter should be able to bind the AIM2PYDat the same time. The structures predict that the distance between the ends of both domains is 58 A (VHHAIM2-I-VHHAIM2-2) or 66 A (VHHA|M2-2-VHHA|M2-I), corresponding to the length of idealized linker composed of 15 or 17 alanines, respectively (Fig. 5A, B, C).

[0143] AIM2PYD-EGFP overexpressed in HeLa cells also polymerizes into microscopically detectable filaments (Fig. 3E). While control single-domain antibodies did not affect PYD polymerization, overexpression of VHHA|M2-I, VHHAIM2-2, VHHAIM2-I-VHHA|M2-I, or VHHA|M2-I- VHHAIM2-2completely abrogated polymerization (Fig. 3E, F). To independently confirm inhibition of AIM2PYDoligomerization by AIM2 single-domain antibodies, we employed a biochemical assay based on the sedimentation of AIM2PYDfilaments. While MBP-AIM2PYDis a soluble protein, removal of the MBP fusion protein by cleavage with Tobacco Etch Virus (TEV) protease initiates the polymerization of AIM2PYDfilaments, which are no longer soluble (Fig. 6A). Insoluble filaments can be separated from soluble monomers and small oligomers by high-speed centrifugation, followed by analysis of pellet and supernatant fractions by SDS-PAGE and Coomassie staining. In the presence of bivalent control singledomain antibody, the entire pool of AIM2PYDwas sedimented upon MBP removal, indicating that all monomers polymerize into insoluble filaments (Fig. 6B, Fig. 7). In the presence of bivalent inhibitory AIM2 single-domain antibodies, AIM2PYDremained in the supernatant, indicating that single-domain antibody binding blocked oligomerization and filament formation (Fig. 6B, Fig. 7). Similar trends were observed with monovalent single-domain antibodies, although the interpretation was impaired by the similar molecular weight of monovalent single-domain antibodies and AIM2PYD, which prevented clear distinction by SDS-PAGE (Fig. EV4A). Together with the structural and biochemical data, this implies that the inhibitory AIM2 single-domain antibodies act by masking monomeric AIM2 and preventing its oligo- and polymerization.

[0144] While the contribution of individual proteins to a biological process in cell lines (or transgenic mice) can be evaluated with CRISPR / Cas9 knockout lines, this is not possible in most human primary cell types. It was therefore intended to employ antagonistic AIM2 single-domain antibodies to answer the outstanding question which inflammasome was activated by poxviruses in human myeloid cell types. To monitor inflammasome assembly in any cell type or tissue susceptible to infection, recombinant strains of vaccinia virus strain Western Reserve were genetically engineered to encode 1 ) the inflammasome reporter C1 C-EGFP or EGFP under the control of an J2R early promoter, as well as 2) different bivalent single-domain antibodies under the control of a strong synthetic early / late promoter (Fig. 3H). The bivalent VHHNP-I -VHHNP-I was used as a negative control, as it is not expected to interfere with vaccinia virus infection-induced inflammasome assembly. Bivalent VHHASC-VHHASC was used as a positive control, as we had shown earlier that VHHASC blocks ASC speck formation and the downstream consequences of inflammasome assembly (Fig. 3I). The recombinant virus strains were first tested in the human myeloid cell line THP-1 cells, which were differentiated into macrophage-like cells with PMA. It had been previously determined that incoming vaccinia virus genomes released from the viral core trigger AIM2 inflammasome assembly in THP-1 cells if cells are pre-treated with IFN-y. Infection was quantified by enumerating of C1 C-EGFP-positive cells with flow cytometry. Almost all cells were infected in all conditions (Fig. 3J). As expected, infection with vaccinia virus strains expressing control single-domain antibodies triggered inflammasome assembly in IFN-y-pretreated THP-1 cells. Inflammasome assembly was quantified based on the unique redistribution of C1 C-EGFP towards ASC specks (similar to the detection of ASC-EGFP specks earlier). Inflammasome assembly was completely blunted by viruses expressing ASC-specific single-domain antibodies, as well as by virus strains expressing VHHAIM2-I -VHHAIM2-I or VHHAIM2-I -VHHAIM2-2. Similarly, virus-triggered IL-ip secretion (as measured by HTRF) was substantially reduced after infection with viruses expressing VHHASC-VHHASC, VHHAIM2-I -VHHAIM2-I or VHHAIM2-I -VHHAIM2-2(Fig. 3K). Cell death (as measured by LDH release and uptake of DNA dye DRAQ7) was likewise reduced to background levels after infection with the same viruses (Fig 3L, M, O). This demonstrated that the designed poxvirus reporter strains were functional, as they allowed both detection and quantification of inflammasome assembly, and as they confirmed that inflammasome assembly in THP-1 cells required functional AIM2 and ASC.

[0145] Inflammasome assembly in human primary cells was investigated next using vaccinia strains expressing bivalent VHHNP-I -VHHNP-I , VHHASC-VHHASC, or VHHAIM2-I-VHHAIM2-I . Human macrophages differentiated from blood-derived monocytes with GM-CSF were robustly infected with the reporter viruses, although IFN-y pretreatment reduced infection by about 50% (Fig. 3P). Inflammasome assembly was specifically quantified in infected cells and was only observed in IFN-y-treated macrophages infected with viruses expressing control single-domain antibodies. Expression of bivalent ASC and AIM2 single-domain antibodies completely blocked inflammasome assembly. This indicates that inflammasome assembly in vaccinia virus-infected primary macrophages relied on AIM2, although it had previously been postulated that dsDNA activates NLRP3 in human primary cells. Similarly, normal human epidermal keratinocytes (NHEK) derived from human skin were robustly infected and only assembled inflammasomes after IFN-y pretreatment (Fig. 3Q). ASC and AIM2 single-domain antibodies completely abrogated inflammasome assembly, proving that this primary cell type also employs AIM2 to detect double-stranded DNA of vaccinia virus. Lastly, human CD14+monocytes purified from human blood were infected. Infection was robust, and was surprisingly even boosted by IFN-y pretreatment (Fig. 3R). Inflammasome assembly was detected in both untreated and IFN-y-treated monocytes. Viruses expressing ASC single-domain antibodies inhibited inflammasome assembly as expected for a readout that scores assembly of ASC specks. Of note, this result confirms that functional concentrations of single-domain antibodies are produced in infected monocytes. Yet, inflammasome activation was not altered when viruses expressed AIM2 single-domain antibodies, indicating that inflammasome assembly did not rely on the sensor AIM2. Instead, inflammasome assembly was sensitive to the NLRP3-specific inhibitor CRID3, proving that vaccinia virus infection is sensed by NLRP3 in human monocytes.

[0146] Taken together, our set of inhibitory AIM2 single-domain antibodies (in combination with a small molecule inhibitor of NLRP3) proves for the first time that poxvirus infections (and by extension dsDNA) is sensed by different mechanisms in inflammasome-competent cell types. While keratinocytes and macrophages employ IFN-y-induced AIM2 to nucleate inflammasomes, infection of monocytes trigger NLRP3 inflammasome assembly. Why and how cells that can express both AIM2 and NLRP3 rely on different sensors for inflammasome assembly, and what functional consequences this has, is the subject of ongoing investigations. The AIM2 single-domain antibodies described here are able to specifically inhibit the assembly of AIM2 inflammasomes triggered by dsDNA. Mechanistically, they impair the oligomerization of AIM2PYDfilaments by masking critical interfaces. They may therefore not only serve as a tool to precisely impair AIM2 in human primary cells and tissues that are not susceptible to CRISPR-mediated knockout. They can potentially also be used to therapeutically tackle pathologies relying on AIM2. In all cases, the coding sequences for the respective single-domain antibody construct (or the recombinant protein itself) must be delivered by transfection (DNA, mRNA, or protein), transduction with viral vectors (lentivirus, AAV), or other methods that e.g. rely on naturally occurring or artificially induced transient pores in the plasma membrane.

[0147] REFERENCES

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Claims

CLAIMS1 . Single-domain antibody directed against “absent in melanoma 2” (AIM2), wherein the single-domain antibody comprises an amino acid sequence comprising framework region 1 (FR1 ), complementarity-determining region 1 (CDR1 ), FR2, CDR2, FR3, CDR3, and FR4, and wherein the single-domain antibody comprises(a) CDR1 as defined by SEQ ID NO:2, CDR2 as defined by SEQ ID NO:4, and CDR3 as defined by SEQ ID NO:6; or(b) CDR1 as defined by SEQ ID NQ:10, CDR2 as defined by SEQ ID NO:12, and CDR3 as defined by SEQ ID NO:14; or(c) CDR1 as defined by SEQ ID NO:18, CDR2 as defined by SEQ ID NQ:20, and CDR3 as defined by SEQ ID NO:22.

2. The single-domain antibody directed against AIM2 of claim 1 , wherein the singledomain antibody comprises an amino acid sequence selected from the group comprising amino acid sequences as defined in SEQ ID NO: 8, 16, or 24, or variants thereof, wherein the variant comprises an amino acid sequence, which is at least 80%, 90%, 95%, or 99% identical to an amino acid sequence selected from the group comprising amino acid sequences as defined in SEQ ID NO: 8, 16, or 24.

3. The single-domain antibody directed against AIM2 of claim 1 or 2, wherein the singledomain antibody is capable of specifically binding an epitope within the pyrin domain of AIM2 (AIM2PYD), wherein the epitope is a discontinuous epitope comprising amino acids selected from: a) D23, R24, K26, F27, F28, S30, D31 , T37, H41 , K71 , L72, and N73 of sequence of AIM2PYD(SEQ ID NO:25); or b) E7, I8, L11 , T12, D15, N16, T18, D19, E21 , N44, R45, I46, L76, R80, E83, E84, and K87 of AIM2PYD(SEQ ID NO:25):MESKYKEILL LTGLDNITDE ELDRFKFFLS DEFNIATGKL HTANRIQVAT LMIQNAGAVS AVMKTIRIFQ KLNYMLLAKR LQEEKEKVDK QYKSVTKPKP LSQAEMSPAA SAAIRNDVAK QRAAPKVSPH VKPEQKQMVA QQESIREGFQ KRCLPVMVLK AKKPFTFETQ EGKQEMFHAT VATEKEFFFV KVFNTLLKDK FIPKRI I I IA RYYRHSGFLE VNSASRVLDA ESDQKVNVPL NI IRKAGETP KINTLQTQPL GTIVNGLFVV QKVTEKKKNI LFDLSDNTGK MEVLGVRNED TMKCKEGDKV RLTFFTLSKN GEKLQLTSGV HSTIKVIKAK KKT .

4. Polypeptide comprising a combination of two single-domain antibodies of any one of claims 1 to 3, wherein the combination can be one of the following:(a) a combination of the single-domain antibody of claim 1 a) with a further singledomain antibody of claim 1 a)(b) a combination of the single-domain antibody of claim 1 a) with a single-domain antibody of claim 1 b)(c) a combination of the single-domain antibody of claim 1 a) with a single-domain antibody of claim 1 c)(d) a combination of the single-domain antibody of claim 1 b) with a single-domain antibody of claim 1 c)(e) a combination of the single-domain antibody of claim 1 b) with a further singledomain antibody of claim 1 b)(f) a combination of the single-domain antibody of claim 1 c) with a further singledomain antibody of claim 1 c), wherein optionally the two single-domain antibodies are linked with the peptide linker (GGGGS)3(SEQ ID NO:26).

5. Polynucleotide encoding the single-domain antibody of any one of claims 1 to 3 or the polypeptide of claim 4.

6. The polynucleotide acid of claim 4, wherein the polynucleotide is selected from RNA, such as mRNA, DNA, such as genomic DNA, cDNA, or synthetic DNA, analogs thereof, or a combination thereof, wherein preferably the polynucleotide is mRNA.

7. The single-domain antibody directed against AIM2 of claim 1 to 3 or the polypeptide of claim 4, wherein the single-domain antibody or the polypeptide is capable of binding to cytosolic AIM2 in a cell, and thereby inhibiting pyroptosis of the cell, wherein the single-domain antibody or the polypeptide directed against AIM2 is produced by the cell upon transfecting the cell with the polynucleotide of claim 4 or 5.

8. Host cell comprising the polynucleotide of claim 5 or 6.

9. Pharmaceutical composition comprising the single-domain antibody directed against AIM2 of any one of claims 1 to 3, or the polypeptide of claim 4, or the polynucleotide of claim 5 or 6, and a pharmaceutically acceptable carrier.

10. The single-domain antibody directed against AIM2 of any one of claims 1 to 3, or the polypeptide of claim 4, or the polynucleotide of claim 5 or 6, or the pharmaceutical composition of claim 9 for use in therapy.1 1 . The single-domain antibody directed against AIM2 of any one of claims 1 to 3, or the polypeptide of claim 4, or the polynucleotide of claim 5 or 6, or the pharmaceuticalcomposition of claim 9, in a method of treating or preventing an inflammatory disease or condition in a subject, wherein the inflammatory disease or condition is selected from the group comprising an acute inflammation, a chronic inflammation, sepsis, loss of the blood-brain barrier, in particular caused by sepsis, septic shock, non-alcoholic steatohepatitis, lung cancer, Familial Mediterranean Fever (FMF), autoinflammatory diseases, Cryoprin associated periodic syndrome (CAPS), non-alcoholic fatty liver disease, Alzheimer's disease, Parkinson's disease, age related macular degeneration, atherosclerosis, asthma and allergy airway inflammation, gout, Crohn's disease, ulcerative colitis, inflammatory bowel disease, psoriasis hypertension, nephropathy, myocardial infarction, multiple sclerosis, experimental autoimmune encephalitis, hyperinflammation following influenza infection, graft versus host disease, stroke, silicosis, asbestosis, mesothelioma, type 1 diabetes, type 2 diabetes, obesity-induced inflammation, insulin resistance, rheumatoid arthritis, myelodysplastic syndrome, contact hypersensitivity, joint inflammation triggered by chikungunya virus and traumatic brain injury.

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