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WO2024175787A1 - Anti-inflammatory pannexin 1 channel inhibitors - Google Patents

Anti-inflammatory pannexin 1 channel inhibitors Download PDF

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Publication number
WO2024175787A1
WO2024175787A1 PCT/EP2024/054681 EP2024054681W WO2024175787A1 WO 2024175787 A1 WO2024175787 A1 WO 2024175787A1 EP 2024054681 W EP2024054681 W EP 2024054681W WO 2024175787 A1 WO2024175787 A1 WO 2024175787A1
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sequence
seq
set forth
amino acid
domain antibody
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Inventor
Mathieu Vinken
Nick Devoogdt
Raf VAN CAMPENHOUT
Timo DE GROOF
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Vrije Universiteit Brussel VUB
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Vrije Universiteit Brussel VUB
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • Pannexin1 Panx1 channels, composed of Panx1 proteins, form water-filled channels at the cell plasma membrane surface. They establish a pathway for paracrine communication by mediating the trafficking of a number of ions, such as potassium, and small molecules, including adenosine triphosphate (ATP), between the intracellular and extracellular environment. By doing so, Panx1 channels drive various physiological processes, yet they have been mainly studied in a pathological context.
  • Panx1 channels act as key players in inflammation and cell death.
  • Various triggers occurring during induction of inflammation including changes in extracellular potassium concentration and phosphorylation, induce opening of Panx1 channels leading to the extracellular release of ATP.
  • Panx1 channels also participate in the clearance of compromised cells. Apoptosis is associated with the activation of caspase-3 and 7, which results in cleavage of the cytosolic carboxyterminal tail of Panx1. This evokes a constitutively open Panx1 channel configuration liberating ATP, which acts as a “find me” signal for the recruitment of macrophages.
  • Panx1 channels of apoptotic cells stimulate the release of metabolites that serve as “good bye” signals.
  • Panx1 channels mediate inflammatory responses in healthy cells in the vicinity. Furthermore, Panx1 channels modulate other types of cell death, including pyroptosis. During pyroptosis, caspase 11 activates Panx1 channels by proteolytic cleavage, triggering ATP release, which in turn activates P2X7 receptors to cause cytotoxicity. Closing of Panx1 channels has been shown on many occasions to suppress inflammation. A variety of agents has been used for this purpose, such as carbenoxolone, lanthanum and, in particular, 10 Panx1. The latter is a synthetic decapeptide that mimics an amino acid sequence in the second extracellular loop of the Panx1 protein. However, 10 Panx1 suffers from low stability (i.e.
  • the present invention relates to single domain antibodies, in particular nanobodies, capable of specifically binding to Panx1.
  • the anti-Panx1 single domain antibodies have a high specificity, are capable of blocking Panx1 channels and serve as Panx1 channel inhibitors.
  • the single domain antibodies as described herein have been found to possess anti-inflammatory activity and can be used in diagnosis.
  • the present invention further relates to binding agents comprising the single domain antibodies described herein.
  • the present invention further relates to polynucleic acids encoding the single domain antibodies described herein or vectors comprising such polynucleic acids.
  • the present invention further relates to host cells comprising the single domain antibodies, polynucleic acids, vectors, or binding domains as described herein.
  • the present invention further relates to pharmaceutical compositions comprising the single domain antibodies, polynucleic acids, vectors, binding domains, or host cells as described herein.
  • the present invention further relates to kits comprising the single domain antibodies, polynucleic acids, vectors, binding domains, host cells, or pharmaceutical compositions as described herein.
  • the present invention further relates to the single domain antibodies, polynucleic acids, vectors, binding domains, host cells, or pharmaceutical compositions as described herein for use in therapy or diagnosis, such as in particular anti-inflammatory therapy or diagnosis of inflammation.
  • the present invention is in particular captured by any one or any combination of one or more of the below numbered statements 1 to 48, with any other statement and/or embodiments.
  • the single-domain antibody according to statement 1 wherein said single-domain antibody comprises a heavy chain variable domain derived from a heavy chain antibody (VHH). 3.
  • the single-domain antibody according to statement 2 wherein said VHH is a camelid VHH. 4.
  • K D dissociation constant
  • FR framework regions
  • CDR complementarity determining regions
  • the single-domain antibody according to any of statements 1 to 12, wherein said single-domain antibody comprises CDR1 having a sequence as set forth in SEQ ID NO: 1, CDR2 having a sequence as set forth in SEQ ID NO: 2, and CDR3 having a sequence as set forth in SEQ ID NO: 3; CDR1 having a sequence as set forth in SEQ ID NO: 4, CDR2 having a sequence as set forth in SEQ ID NO: 5, and CDR3 having a sequence as set forth in SEQ ID NO: 6; CDR1 having a sequence as set forth in SEQ ID NO: 7, CDR2 having a sequence as set forth in SEQ ID NO: 8, and CDR3 having a sequence as set forth in SEQ ID NO: 9; CDR1 having a sequence as set forth in SEQ ID NO: 10, CDR2 having a sequence as set forth in SEQ ID NO: 11, and CDR3 having a sequence as set forth in SEQ ID NO: 12; CDR1 having a sequence as set forth in SEQ ID NO: 13, CDR2 having
  • the single-domain antibody according to any of statements 1 to 13, wherein said single-domain antibody has a sequence as set forth in any of SEQ ID NOs: 25 to 32, or a sequence which is at least 80%, preferably at least 90%, identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to a sequence as set forth in any of SEQ ID NOs: 25 to 32, or a functional/antigen-binding fragment thereof. 15.
  • any one or more of FR1 to FR4 has a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence of FR1 to FR4 as set forth in any of SEQ ID NOs: 25 to 32, wherein FR1 to FR4 are annotated according to IMGT. 16.
  • a binding agent comprising a single-domain antibody according to any of statements 1 to 19. 21. The binding agent according to statement 20, wherein said single-domain antibody is fused to albumin. 22. The binding agent according to any of statements 20 to 21, wherein said single-domain antibody is fused to an Fc domain. 23.
  • binding agent according to any of statements 20 to 22, wherein said binding domain is PEGylated.
  • 25. A polynucleic acid encoding the single-domain antibody according to any of statements 1 to 19. 26.
  • 27. A recombinant vector comprising the polynucleic acid according to statement 25 or 26. 28.
  • a host cell comprising the polynucleic acid or vector according to any of statements 25 to 28.
  • a pharmaceutical composition comprising the single-domain antibody, binding agent, polynucleic acid, vector, or host cell according to any of statements 1 to 29 and one or more pharmaceutically acceptable excipients.
  • a kit comprising the single-domain antibody, binding agent, polynucleic acid, vector, host cell or pharmaceutical composition according to any of statements 1 to 31.
  • 33. The single-domain antibody, binding agent, polynucleic acid, vector, host cell or pharmaceutical composition according to any of statements 1 to 31 for use in therapy. 34.
  • 35. The single-domain antibody, binding agent, polynucleic acid, vector, host cell or pharmaceutical composition according to any of statements 1 to 31 for use in therapeutic treatment of an inflammatory disease or disorder.
  • 36. The single-domain antibody, binding agent, polynucleic acid, vector, host cell or pharmaceutical composition according to any of statements 1 to 31 for use in therapeutic treatment of a liver disease or disorder.
  • NASH non-alcoholic steatohepatitis
  • NAFLD non-alcoholic fatty liver disease
  • cholestatic disorder cholangitis
  • fibrotic/cirrhotic disorder or (liver) cancer 41.
  • the single-domain antibody, binding agent, polynucleic acid, vector, host cell or pharmaceutical composition for use according to any of statements 34 to 40, wherein said treatment comprises at least partial reduction of inflammation.
  • SV40 immortalised DUBCA wild-type (WT) cells were transduced with lentiviral vectors to express mouse Panx1 (mPanx1) or human Panx1 (hPanx1) using pASIET vectors containing Panx1 constructs (CMV-Panx-IRES- eGFP). Following transduction, cells were assessed by flow cytometry analysis for eGFP expression to confirm successful transduction. The fluorescence-activated cell sorting profile shown originates from cells grown for 2 days.
  • Figure 2 Analysis of Panx1 expression following transduction of DUBCA cells. DUBCA cells were transduced with lentiviral vectors to express mouse Panx1 (mPanx1) or human Panx1 (hPanx1).
  • Figure 3 Analysis of Panx1 Gly2 protein expression following transduction of DUBCA cells. DUBCA cells were transduced with lentiviral vectors to express mouse Panx1 (mPanx1) or human Panx1 (hPanx1).
  • Figure 4 Analysis of Panx1 protein localisation following transduction of DUBCA cells. DUBCA cells were transduced with lentiviral vectors to express mouse Panx1 (mPanx1) or human Panx1 (hPanx1).
  • DUBCA WT, DUBCA mPanx1 and DUBCA hPanx1 cells were prepared and subjected to immunocytochemistry analysis of Panx1 (white arrows) with nuclear counterstaining. Scale bars represent 500 ⁇ m.
  • Figure 5 Flow cytometry analysis with Panx1 nanobodies. DUBCA cells were transduced with lentiviral vectors to express mouse Panx1 (mPanx1) or human Panx1 (hPanx1). DUBCA WT, DUBCA mPanx1 and DUBCA hPanx1 cells were prepared and incubated with Panx1 nanobodies.
  • FIG. 1 Cells were incubated with allophycocyanin- conjugated hemagglutinin antibody and subjected to flow cytometry analysis. Gating of hemagglutinin positive cells was performed to identify Panx1 nanobodies.
  • Figure 6 Immunocytochemistry analysis with Panx1 nanobodies. DUBCA cells were transduced with lentiviral vectors to express human Panx1 (hPanx1). DUBCA WT and DUBCA hPanx1 cells were prepared and subjected to immunocytochemistry analysis with Panx1 nanobodies or R3b23 nanobody. Specificity of Panx1 nanobodies was assessed using Alexa Fluor® 594-conjugated hemagglutinin antibody with nuclear counterstaining. Scale bars represent 500 ⁇ m.
  • FIG. 7 Cell-based enzyme-linked immunosorbent assay with Panx1 nanobodies.
  • DUBCA cells were transduced with lentiviral vectors to express human Panx1 (hPanx1).
  • DUBCA WT and DUBCA hPanx1 cells were prepared and subjected to cell-based ELISA with Panx1 nanobodies or R3b23 nanobody in concentrations ranging from 0 nM to 600 nM.
  • Specificity of Panx1 nanobodies was assessed by measuring absorbance values at 450 nm (A).
  • the equilibrium dissociation constant (Kd) of the Panx1 nanobodies (B) was determined by plotting saturation curves with specific binding values (C).
  • C6 cells were transduced with lentiviral vectors to express rat Panx1 (Panx1).
  • C6 WT and C6 Panx1 cells were prepared and subjected to immunocytochemistry analysis of Panx1 (white arrows) with nuclear counterstaining. Scale bars represent 500 ⁇ m.
  • Figure 10 Cell-based enzyme-linked immunosorbent assay with C6 cells.
  • C6 cells were transduced with lentiviral vectors to express rat Panx1 (Panx1).
  • C6 WT and C6 Panx1 cells were prepared and subjected to cell-based ELISA with Panx1 nanobodies or R3b23 nanobody in concentrations ranging from 0 nM to 12000 nM.
  • Figure 11 Analysis of extracellular release of adenosine triphosphate with Panx1 nanobodies. DUBCA hPanx1 cells were cultured and exposed during 30 minutes to buffer.
  • CBX carbenoxolone disodium salt
  • La3+ lanthanum trichloride
  • 10Panx1 300-400 ⁇ M
  • Panx1 nanobodies and R3b23 nanobody (0-10,000 nM) were prepared ex tempore in buffer with or without increased potassium concentrations.
  • Cells were preconditioned with appropriate buffer for 15 minutes.
  • Cells were exposed to high potassium ion levels by replacing the buffer with preheated potassium-enriched buffer for another 30 minutes.
  • Extracellular ATP levels were measured and expressed as the percentage of ATP relative to the release level triggered by the potassium-enriched buffer.
  • THP-1 cells were differentiated with phorbol 12-myristate 13-acetate and consecutively exposed to 1 ⁇ g/mL LPS (4 hours), 25 ⁇ M Caspase-1 Inhibitor II; 10 ⁇ M MCC950; 1,000 nM Panx1 nanobodies or R3b23 nanobody (1 hour) and 5 mM ATP (30 minutes). Extracellular IL-1 ⁇ levels were measured by ELISA, expressed as percentage of IL-1 ⁇ relative to the release level triggered by LPS + ATP. Results were analysed by parametric 1-way analysis of variance (ANOVA) followed by post hoc tests with Dunnett’s correction. Data were expressed as means ⁇ standard error of the mean (SEM).
  • FIG. 14 Cell-based enzyme-linked immunosorbent assay with Panx1 nanobodies.
  • DUBCA cells were transduced with lentiviral vectors to express mouse Panx1 (mPanx1).
  • FIG. 15 Flow cytometry analysis with Panx1 nanobodies.
  • DUBCA cells were transduced with lentiviral vectors to express mouse Panx1 (mPanx1).
  • DUBCA WT and DUBCA mPanx1 cells were prepared and incubated with Panx1 nanobodies or R3b23 nanobody in concentrations ranging from 0 to 600 nM.
  • Cells were incubated with Alexa Fluor® 488-conjugated hemagglutinin antibody and Hoechst solution and subjected to flow cytometry analysis. Gating of Hoechst positive cells was performed to identify Panx1 nanobodies (A).
  • the equilibrium dissociation constant (Kd) of the Panx1 nanobodies was determined by plotting saturation curves with specific binding values (B).
  • Figure 16 Analysis of liver protein expression in acetaminophen-overdosed mice following Panx1 nanobody treatment. Wild type mice were injected with acetaminophen at 300 mg/kg body weight (APAP) or kept untreated (UTC). After 2 hours, some mice were additionally administrated either nanobody (Nb1, Nb3, Nb9, Nb30 or R3b23) at 10 mg/kg body weight or N-acetylcysteine at 200 mg/kg body weight (NAC). Sampling was performed 24 hours after acetaminophen overdosing.
  • APAP body weight
  • UTC untreated mice
  • Figure 17 Analysis of serum cytokines in acetaminophen-overdosed mice following Panx1 nanobody treatment. Wild type mice were injected with acetaminophen at 300 mg/kg body weight (APAP) or kept untreated (UTC). After 2 hours, some mice were additionally administrated either nanobody (Nb1, Nb3, Nb9, Nb30 or R3b23) at 10 mg/kg body weight or N-acetylcysteine at 200 mg/kg body weight (NAC). Sampling was performed 24 hours after acetaminophen overdosing.
  • APAP body weight
  • UTC untreated mice
  • GM-CSF granulocyte- macrophage colony-stimulating factor
  • IL interleukin
  • IFN- ⁇ interferon- ⁇
  • SDF-1 stromal cell derived factor-1
  • I-TAC interferon-inducible T-cell ⁇ chemoattractant
  • TCA-3 T-cell activation protein 3
  • KC growth-regulated ⁇ protein
  • TECK thymus-expressed chemokine
  • TIMP tissue inhibitor of metalloproteinase
  • BLC B lymphocyte chemoattractant
  • LIX lipopolysaccharide-induced CXC chemokine
  • CD30L CD30 ligand
  • TNF- ⁇ tumor necrosis factor ⁇
  • MCP-1 monocyte chemoattractant protein 1
  • sTNF RI/II soluble TNF receptor 1/2
  • MIG monokine induced by interferon- ⁇
  • MIP macrophage inflammatory protein
  • GCSF granulocyte-ma
  • Figure 18 Analysis of serum aminotransaminases in acetaminophen-overdosed mice following Panx1 nanobody treatment. Wild type and Panx1-/- mice were injected with acetaminophen at 300 mg/kg body weight (APAP) or kept untreated (UTC).
  • APAP body weight
  • UTC untreated
  • mice After 2 hours, some wild type mice were additionally administrated either nanobody (Nb1, Nb3, Nb9, Nb30 or R3b23) at 10 mg/kg body weight or N-acetylcysteine at 200 mg/kg body weight (NAC), while some Panx1-/- mice received an additional Panx1 nanobody treatment (Nb1, Nb3, Nb9 or Nb30) at 10 mg/kg body weight or N-acetylcysteine at 200 mg/kg body weight (NAC). Sampling was performed 24 hours after acetaminophen overdosing.
  • nanobody Nb1, Nb3, Nb9, Nb30 or R3b23
  • Panx1-/- mice received an additional Panx1 nanobody treatment (Nb1, Nb3, Nb9 or Nb30) at 10 mg/kg body weight or N-acetylcysteine at 200 mg/kg body weight (NAC).
  • Sampling was performed 24 hours after acetaminophen overdos
  • Figure 19 Examination of liver tissue in acetaminophen-overdosed mice following Panx1 nanobody treatment. Wild type mice were injected with acetaminophen at 300 mg/kg body weight (APAP) or kept untreated (UTC).
  • APAP body weight
  • UTC untreated
  • mice were additionally administrated either nanobody (Nb1, Nb3, Nb9, Nb30 or R3b23) at 10 mg/kg body weight or N-acetylcysteine at 200 mg/kg body weight (NAC).
  • NAC N-acetylcysteine
  • Sampling was performed 24 hours after acetaminophen overdosing.
  • Uptake levels of nanobodies were determined in blood, inguinal lymphnodes, cervical lymphnodes, cervical lymphnodes, salivary glands, thymus, heart, lungs, gallbladder, liver, pancreas, spleen, kidneys, stomach, small intestine, appendix, large intestine, skin, muscle, bone, tail and brain.
  • Panx1 protein expression was evaluated on 8 ⁇ m salivary glands and stomach sections. Sections were prepared and subjected to immunohistochemistry analysis of Panx1 with nuclear counterstaining. Scale bars represent 100 or 250 ⁇ m. (B). Results were analysed by parametric 1-way analysis of variance (ANOVA) followed by post hoc tests with Dunnett’s correction.
  • Figure 21 Full length sequences of single domain antibodies Nb16 (SEQ ID NO: 25), Nb31 (SEQ ID NO: 26), Nb6 (SEQ ID NO: 27), Nb9 (SEQ ID NO: 28), Nb20 (SEQ ID NO: 29), Nb30 (SEQ ID NO: 30), Nb3 (SEQ ID NO: 31), and Nb1 (SEQ ID NO: 32) aligned according to the IMGT numbering scheme (LeFranc, 2014; Frontiers in Immunology.5 (22): 1-22).
  • Panx1 is a protein belonging to the pannexin family of glycoproteins alongside Panx2 and Panx3.
  • Pannexins predominantly exist as multimeric, typically hexameric, transmembrane channels.
  • Panx1 comprises 4 transmembrane domains or regions, 2 extracellular loops, 1 intracellular loop, and an intracellular N-terminal and C-terminal domain/region.
  • Human Pannexin 1 (hPanx1) may have a sequence as set forth in NCBI reference sequence NP_056183.2.
  • Mouse Panx1 (mPanx1) may have a sequence as set forth in NCBI reference sequence NP_062355.2.
  • a first extracellular loop of hPanx1 may comprise or consist of a sequence as set forth in SEQ ID NO: 33.
  • a second extracellular loop of hPanx1 may comprise or consist of a sequence as set forth in SEQ ID NO: 34.
  • Corresponding sequences of extracellular loops of Panx1 from other organisms can be derived by suitable sequence alignments, as is known in the art.
  • Panx1 as used herein is human Panx1 (hPanx1).
  • Panx1 channels among others are believed to be responsible for channel-mediated ATP release. Accordingly, Panx1 channel activation, i.e. channel opening, may result in extracellular release of ATP.
  • Panx1 channel inhibition, i.e. channel closing or blocking may result in (partial or complete) reduction of extracellular release of ATP.
  • antibody refers to an immunoglobulin (Ig) molecule or a molecule comprising an immunoglobulin (Ig) domain, which specifically binds with an antigen, as well as multimers thereof.
  • Antibodies can be intact immunoglobulins or immunoreactive portions of intact immunoglobulins. The term encompasses naturally, recombinantly, semi-synthetically or synthetically produced antibodies.
  • an antibody can be present in or isolated from nature, e.g., produced or expressed natively or endogenously by a cell or tissue and optionally isolated therefrom; or an antibody can be recombinant, i.e., produced by recombinant DNA technology, and/or can be, partly or entirely, chemically or biochemically synthesised.
  • antibody fragment e.g., antigen-binding fragment
  • functional antibody fragment e.g., functional antibody fragment
  • active antibody fragment refer to a portion of any antibody that by itself has high affinity for an antigenic determinant, or epitope, and contains one or more complementarity determining regions (CDRs) accounting for such specificity.
  • antibody fragment and “antigen-binding fragment” and “active antibody fragment” and “functional antibody fragment” as used herein refer to a protein or peptide comprising an immunoglobulin domain or an antigen-binding domain capable of specifically binding to an antigen.
  • Non-limiting examples include immunoglobulin domains, Fab, F(ab)'2, scFv, heavy-light chain dimers, immunoglobulin single variable domains, Nanobodies (or VHH antibodies), domain antibodies, and single chain structures, such as a complete light chain or complete heavy chain.
  • the functional/antigen-binding fragment comprises at least CDR1, CDR2, and CDR3.
  • at least part of one or more framework regions or regions linking the CDRs is also present.
  • immunoglobulin (Ig) domain or more specifically “immunoglobulin variable domain” (abbreviated as “IVD”, also referred to herein as “variable domain”), means an immunoglobulin domain essentially consisting of four “framework regions” which are referred to in the art and herein below as “framework region 1” or “FR1”; as “framework region 2” or “FR2”; as “framework region 3” or “FR3”; and as “framework region 4” or “FR4”, respectively; which framework regions are interrupted by three “complementarity determining regions” or “CDRs”, which are referred to in the art and herein below as “complementarity determining region 1” or “CDR1”; as “complementarity determining region 2” or “CDR2”; and as “complementarity determining region 3” or “CDR3”, respectively.
  • an immunoglobulin variable domain can be indicated as follows: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4. It is the immunoglobulin variable domain(s) (IVDs), and in particular the CDRs therein, even more particularly CDR3 therein, that confer specificity to an antibody for the antigen by carrying the antigen- or epitope-binding site.
  • IVDs immunoglobulin variable domain
  • a heavy chain variable domain (VH) and a light chain variable domain (VL) interact to form an antigen-binding site.
  • the complementarity determining regions (CDRs) of both VH and VL contribute (although not necessarily evenly) to the antigen-binding site, i.e. a total of 6 CDRs will be involved in antigen- binding site formation.
  • the antigen-binding domain of a conventional 4-chain antibody such as an IgG, IgM, IgA, IgD or IgE molecule; known in the art
  • a Fab fragment, a F(ab')2 fragment, an Fv fragment such as a disulphide linked Fv or a scFv fragment, or a diabody (all known in the art) derived from such conventional 4-chain antibody will bind to the respective epitope of an antigen by a pair of (associated) immunoglobulin domains such as light and heavy chain variable domains, i.e., by a VH-VL pair of immunoglobulin domains, which jointly bind to an epitope of the respective antigen.
  • single-domain antibody is an antibody (fragment) comprising a single immunoglobulin domain, in particular a single variable domain.
  • the term “single- domain antibody may be used interchangeably with “single variable domain” or “immunoglobulin single variable domain”.
  • a “single-domain antibody” defines molecules wherein the antigen-binding site is present on, and formed by, a single immunoglobulin domain. This sets immunoglobulin single variable domains apart from “conventional” immunoglobulins or their fragments, wherein two immunoglobulin domains, in particular two variable domains, interact to form an antigen-binding site.
  • a single-domain antibody as used herein refers to a protein or peptide with an amino acid sequence comprising 4 framework regions (FR) and 3 complementary determining regions (CDR) according to the format of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the antigen-binding site of an immunoglobulin single variable domain is formed by a single VH/VHH or VL domain.
  • the antigen-binding site of an immunoglobulin single variable domain is formed by no more than three CDRs.
  • the single variable domain may be a light chain variable domain sequence (e.g., a VL-sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH-sequence or VHH sequence) or a suitable fragment thereof; as long as it is capable of forming a single antigen-binding unit (i.e., a functional antigen-binding unit that essentially consists of the single variable domain, such that the single antigen-binding domain does not need to interact with another variable domain to form a functional antigen-binding unit).
  • a light chain variable domain sequence e.g., a VL-sequence
  • a heavy chain variable domain sequence e.g., a VH-sequence or VHH sequence
  • the immunoglobulin single variable domains are heavy chain variable domain sequences (e.g., a VH-sequence or a VHH-sequence); more specifically, the immunoglobulin single variable domains can be heavy chain variable domain sequences that are derived from a conventional four-chain antibody or heavy chain variable domain sequences that are derived from a heavy chain antibody.
  • the immunoglobulin single variable domain may be a (single) domain antibody (or an amino acid sequence that is suitable for use as a (single) domain antibody), a variable domain of a heavy (VH) or light (VL) chain of a conventional antibody (also referred to as a “dAb”) (or an amino acid sequence that is suitable for use as a dAb) or a Nanobody (as defined herein, and including but not limited to a VHH); or any suitable fragment of any one thereof.
  • the single-domain antibody may be a Nanobody (as defined herein) or a suitable fragment thereof. Note: Nanobody®, Nanobodies® and Nanoclone® are registered trademarks of Ablynx N.V.
  • VHH domains also known as VHHs, VHH domains, VHH antibody fragments, and VHH antibodies, have originally been described as the antigen-binding immunoglobulin (Ig) (variable) domain of “heavy chain antibodies” (i.e., of “antibodies devoid of light chains”; Hamers-Casterman et al. 1993, Nature 363: 446-448).
  • Ig antigen-binding immunoglobulin
  • VHH domain has been chosen to distinguish these variable domains from the heavy chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as “VH domains”) and from the light chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as “VL domains”).
  • VH domains heavy chain variable domains that are present in conventional 4-chain antibodies
  • VL domains light chain variable domains that are present in conventional 4-chain antibodies
  • Nanobody in particular VHH sequences and partially humanized Nanobody
  • VHH sequences and partially humanized Nanobody can in particular be characterized by the presence of one or more “hallmark residues” in one or more of the framework sequences.
  • different numbering schemes can be applied. For example, numbering can be performed according to the AHo numbering scheme for all heavy (VH) and light chain variable domains (VL) given by Honegger & Plückthun (2001. J Mol Biol 309:657-70), as applied to VHH domains from camelids.
  • Alternative methods for numbering the amino acid residues of VH domains which can also be applied in an analogous manner to VHH domains, are known in the art.
  • the delineation of the FR and CDR sequences can be done by using the Kabat numbering system as applied to VHH domains from camelids by Riechmann & Muyldermans (1999. J Immunol Methods 231:25-38). It should be noted that - as is well known in the art for VH domains and for VHH domains - the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (that is, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering).
  • the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence.
  • the total number of amino acid residues in a VH domain and a VHH domain will usually be in the range of from 110 sequences may also be suitable for the purposes described herein.
  • the determination of the CDR regions in an antibody/immunoglobulin sequence generally depends on the algorithm/methodology applied. For example, determination of CDR regions may be done according to the designation based on contact analysis and binding site topography as described in MacCallum et al. (J. Mol. Biol.
  • Said annotations further include delineation of CDRs and framework regions (FRs) in immunoglobulin-domain-containing proteins, and are known methods and systems to a skilled artisan who thus can apply these annotations onto any antibody/immunoglobulin protein sequences without undue burden. Applying different methods to the same antibody/immunoglobulin sequence may give rise to different CDR amino acid sequences wherein the differences may reside in CDR sequence length and/or delineation within the antibody sequence.
  • FRs framework regions
  • CDRs can be described as the CDR sequences present in the antibodies or (functional) fragments thereof as described herein, as determined or delineated according to a well-known methodology such as according to any one of the Kabat-, Martin-, Chothia-, aHo, MacCallum et al.1996, AbM-, or IMGT-numbering scheme or method, such as preferably the IMGT numbering scheme or method.
  • VHHs or Nbs are often classified in different families according to amino acid sequences, or even in superfamilies, as to cluster the clonally related sequences derived from the same progenitor during B cell maturation (Deschaght et al.2017, Front Immunol 8:420).
  • each VHH or Nb family is defined as a cluster of (clonally) related sequences with a sequence identity threshold of the CDR3 region.
  • the CDR3 sequence is thus identical or very similar in amino acid composition, preferably with at least 80 % identity, or at least 85% identity, or at least 90 % identity in the CDR3 sequence, resulting in VHHs or Nbs of the same family binding to the same binding site, and having the same effect such as functional effect.
  • CDRs as described herein are preferably annotated or described according to the IMGT numbering scheme.
  • the single-domain antibodies as described herein can be subjected to humanization, i.e. to increase the degree of sequence identity with the closest human germline sequence.
  • humanized single-domain antibodies may be immunoglobulin single variable domains in which at least one amino acid residue is present (and in particular, at least one framework residue) that is and/or that corresponds to a humanizing substitution (as defined further herein).
  • Potentially useful humanizing substitutions can be ascertained by comparing the sequence of the framework regions of a naturally occurring (i.e. wild type) sequence with the corresponding framework sequence of one or more closely related human VH sequences, after which one or more of the potentially useful humanizing substitutions (or combinations thereof) thus determined can be introduced into said single-domain antibody sequence (in any manner known per se) and the resulting humanized single-domain antibody sequences can be tested for affinity for the target, for stability, for ease and level of expression, and/or for other desired properties.
  • a single-domain antibodies may be partially humanized or fully humanized.
  • Humanized single-domain antibodies may have several advantages, such as a reduced immunogenicity, compared to the corresponding naturally occurring single-domain antibodies.
  • humanized is meant mutated so that immunogenicity upon administration in human patients is minor or non-existent.
  • the humanizing substitutions should be chosen such that the resulting humanized amino acid sequence still retains the favourable properties of the parental (non-humanized) single-domain antibody, such as the antigen-binding capacity.
  • the skilled person will be able to select humanizing substitutions or suitable combinations of humanizing substitutions which optimize or achieve a desired or suitable balance between the favourable properties provided by the humanizing substitutions on the one hand and the favourable properties of naturally occurring single-domain antibodies on the other hand.
  • Such methods are known by the skilled person.
  • a human consensus sequence can be used as target sequence for humanization, but also other means are known in the art.
  • One alternative includes a method wherein the skilled person aligns a number of human germline alleles, such as for instance but not limited to the alignment of IGHV3 alleles, and to use said alignment for identification of residues suitable for humanization in the target sequence.
  • a subset of human germline alleles most homologous to the target sequence may be aligned as starting point to identify suitable humanisation residues.
  • the single-domain antibody is analyzed to identify its closest homologue in the human alleles and used for humanisation construct design.
  • a humanisation technique applied to single-domain antibodies, such as VHHs may also be performed by a method comprising the replacement of specific amino acids, either alone or in combination. Said replacements may be selected based on what is known from literature, from known humanization efforts, as well as from human consensus sequences compared to the natural single-domain antibody sequences, or from the human alleles most similar to the single-domain antibody sequence of interest.
  • a human-like class of Camelidae single domain antibodies contain the hydrophobic FR2 residues typically found in conventional antibodies of human origin or from other species, but compensating this loss in hydrophilicity by other substitutions at position 103 that substitutes the conserved tryptophan residue present in VH from double-chain antibodies.
  • peptides belonging to these two classes show a high amino acid sequence homology to human VH framework regions and said peptides might be administered to a human directly without expectation of an unwanted immune response therefrom, and without the burden of further humanisation.
  • Camelidae VHH sequences display a high sequence homology to human VH framework regions and therefore said VHH might be administered to patients directly without expectation of an immune response therefrom, and without the additional burden or need of humanization.
  • Suitable mutations, in particular substitutions can be introduced during humanization to generate a polypeptide with reduced binding to pre-existing antibodies (reference is made for example to WO 2012/175741 and WO2015/173325), for example at least one of the positions: 11, 13, 14, 15, 40, 41, 42, 82, 82a, 82b, 83, 84, 85, 87, 88, 89, 103, or 108.
  • the amino acid sequences and/or VHH of the invention may be suitably humanized at any framework residue(s), such as at one or more Hallmark residues or at one or more other framework residues (i.e. non-Hallmark residues) or any suitable combination thereof.
  • any framework residue(s) such as at one or more Hallmark residues or at one or more other framework residues (i.e. non-Hallmark residues) or any suitable combination thereof.
  • deletions and/or substitutions may also be designed in such a way that one or more sites for posttranslational modification (such as one or more glycosylation sites) are removed, as will be within the ability of the person skilled in the art.
  • substitutions or insertions may be designed so as to introduce one or more sites for attachment of functional groups (as described herein), for example to allow site-specific pegylation.
  • At least one of the typical Camelidae hallmark residues with hydrophilic characteristics at position 37, 44, 45 and/or 47 is replaced (see Table A-03 of WO2008/020079).
  • Another example of humanization includes substitution of residues in FR1, such as position 1, 5, 11, 14, 16, and/or 28; in FR3, such as positions 73, 74, 75, 76, 78, 79, 82b, 83, 84, 93 and/or 94; and in FR4, such as position 10103, 104, 108 and/or 111 (see Tables A-05 -A08 of WO2008/020079; all numbering according to the Kabat-methodology).
  • Binding means any interaction, be it direct or indirect.
  • a direct interaction implies a contact (e.g. physical or chemical) between two binding partners.
  • An indirect interaction means any interaction whereby the interaction partners interact in a complex of more than two molecules.
  • An interaction can be completely indirect (e.g. two molecules are part of the same complex with the help of one or more bridging molecules but don’t bind in the absence of the bridging molecule(s)).
  • An interaction may be partly direct or partly indirect: there is still a direct contact between two interaction partners, but such contact is e.g.
  • Specifically binding refers to the situation in which a molecule A is, at a certain concentration (e.g. sufficient to inhibit or neutralize a protein or process of interest) binding to a target of interest (e.g. protein) with higher affinity (e.g. at least 2-fold, 5-fold, or at least 10-fold higher affinity, e.g.
  • affinity generally refers to the degree to which one molecule (e.g. ligand, chemical, protein or peptide, antibody or antibody fragment) binds to another molecule (e.g.
  • binding agent generally relates to a molecule that is capable of binding to at least one other molecule, wherein said binding is preferably a specific binding, such as on a defined binding site, pocket or epitope.
  • the binding agent may be of any nature or type and is not dependent on its origin.
  • the binding agent may be chemically synthesized, naturally occurring, recombinantly produced (and optionally purified), as well as designed and synthetically produced (and optionally purified).
  • Said binding agent may hence be, e.g., a small molecule, a chemical, a peptide, a polypeptide, an antibody, or any derivative of any thereof, such as a peptidomimetic, an antibody mimetic, an active fragment, a chemical derivative, among others.
  • a functional fragment of a binding agent or a functional part of a binding agent refers to a fragment or part of that binding agent that is functionally equivalent to that binding agent.
  • such functional fragment or part of a binding agent as described herein ideally retains one or more of the functional features of that binding agent as outlined extensively elsewhere herein.
  • a functional fragment according to the resent invention typically comprises at least an antigen-binding fragment of a single-domain antibody as described herein.
  • fused to refers in one aspect to “genetic fusion”, e.g., by recombinant DNA technology, as well as to “chemical and/or enzymatic conjugation” resulting in a stable covalent link between two nucleic acid molecules.
  • inserted in wherein a fragment of one nucleic acid may be inserted in a second nucleic acid molecule by fusing or ligating the two sequences genetically, enzymatically or chemically.
  • Peptides or polypeptides can likewise be fused or connected to one another, such as via peptide bonds or via linking one peptide to a side chain of an amino acid in a second peptide. It will be understood that also non-genetic fusions (e.g. through chemical linkage) are encompassed.
  • Linkers may be used to fuse a herein identified single domain antibody (or (antigen- binding) fragment, variant, humanized form thereof, etc. as described herein) to an Fc domain such as the IgG1 and IgG2 Fc domain.
  • a “chimeric gene” or “chimeric construct” or “chimeric gene construct” is interchangeably meant a recombinant nucleic acid sequence in which a (gene) promoter or regulatory nucleic acid sequence is operably or operatively linked to, or associated with, a nucleic acid sequence of interest that codes for an RNA (e.g. a single-domain antibody as described herein), such that the regulatory nucleic acid sequence is able to regulate transcription or expression of the nucleic acid of interest.
  • RNA e.g. a single-domain antibody as described herein
  • an “Fc domain” as used herein refers to the fragment crystallizable region (Fc region) of a conventional antibody, which is the tail region known to interact with cell surface receptors called Fc receptors and some proteins of the complement system. Said Fc domain is composed of two identical protein fragments, derived from the second and third constant domains of the antibody's two heavy chains. All conventional antibodies comprise an Fc domain, hence, the Fc domain may be an Fc domain derived from or as a variant of the IgG, IgA and IgD antibody Fc regions, even more specifically derived from an IgG1, IgG2 or IgG4 antibody Fc region.
  • the hinge region of IgG2 may be replaced by the hinge of human IgG1 to generate single domain antibody fusion constructs, and vice versa.
  • Fc variants with known half-life extension may be used such as the M257Y/S259T/T261E (also known as YTE) or the LS variant (M428L combined with N434S). These mutations increase the binding of the Fc domain of a conventional antibody to the neonatal receptor (FcRn).
  • Humanized forms include but are not limited to the IgG humanization variants known in the art, such as C-terminal deletion of Lysine, alteration or truncation in the hinge region, LALA (L234A and L235A) or LALAPG (L234A, L235A, and P329G) mutations, among other substitutions in the IgG sequence.
  • the Fc domain as referred to herein is an IgG Fc domain, in particular a human IgG Fc domain, such as a (human) IgG1 Fc domain or a (human) IgG2 Fc domain.
  • protein protein
  • polypeptide and “peptide” are interchangeably used herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same; the sequential linear arrangement of the amino acids together resulting in/forming the “amino acid sequence” or “protein sequence”.
  • a “peptide” may also be referred to as a partial amino acid sequence derived from its original protein, for instance after enzymatic (e.g. tryptic) digestion. These terms apply to naturally-occurring amino acid polymers as well as to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally occurring amino acid, such as a chemical analogue of a corresponding naturally occurring amino acid.
  • proteins comprising one or more posttranslational modifications such as covalent addition of functional groups or proteins (such as glycosylation, phosphorylation, acetylation, ubiquitination, methylation, lipidation and nitrosylation) or such as proteolytic processing.
  • functional groups or proteins such as glycosylation, phosphorylation, acetylation, ubiquitination, methylation, lipidation and nitrosylation
  • proteolytic processing Based on the amino acid sequence and the modifications, the atomic or molecular mass or weight of a polypeptide is expressed in (kilo)dalton (kDa).
  • a further modification of proteins includes addition of a tag, such as a His-tag or sortag.
  • amino acid substitutions encompass amino acid alterations in which an amino acid is replaced with a different naturally-occurring amino acid residue. Such substitutions may be classified as "conservative ⁇ 1>, in which an amino acid residue contained in the wild- type protein is replaced with another naturally-occurring amino acid of similar character, for example Gly ⁇ Ala, Val ⁇ lle ⁇ Leu, Asp ⁇ Glu, Lys ⁇ Arg, Asn ⁇ Gln or Phe ⁇ Trp ⁇ Tyr.
  • substitutions encompassed by the present invention may also be "non- conservative", in which an amino acid residue which is present in the wild-type protein is substituted with an amino acid with different properties, such as a naturally-occurring amino acid from a different group (e.g., substituting a charged or hydrophobic amino acid with alanine.
  • Similar amino acids refers to amino acids that have similar amino acid side chains, i.e., amino acids that have polar, non-polar or practically neutral side chains.
  • Non-similar amino acids refers to amino acids that have different amino acid side chains, for example an amino acid with a polar side chain is non-similar to an amino acid with a non-polar side chain.
  • Polar side chains usually tend to be present on the surface of a protein where they can interact with the aqueous environment found in cells ("hydrophilic” amino acids).
  • non-polar amino acids tend to reside within the center of the protein where they can interact with similar non-polar neighbours (“hydrophobic” amino acids”).
  • amino acids that have polar side chains are arginine, asparagine, aspartate, cysteine, glutamine, glutamate, histidine, lysine, serine, and threonine (all hydrophilic, except for cysteine which is hydrophobic).
  • a “protein domain” is a distinct functional and/or structural unit in or part of a protein. Usually, a protein domain is responsible for a particular function or interaction, contributing to the overall (biological) role of a protein. Domains may exist in a variety of biological contexts, where similar domains can be found in different proteins with similar or different functions. Protein domains can have a rigid 3D- structure if confined by e.g. a number of intramolecular cysteines (e.g.
  • cysteine-knot proteins or can, depending on e.g. presence or absence of a bound ligand or e.g. presence or absence of a posttranslational modification, assume different 3D-conformations, or can have a less defined, more fluid 3D-structure.
  • polynucleic acid “nucleotide sequence(s)”, “polynucleotide(s)”, “nucleic acid sequence(s)”, “nucleic acid(s)”, “nucleic acid molecule” are used interchangeably herein and refer to nucleotides, either ribonucleotides or deoxyribonucleotides or a combination of both, in a polymeric unbranched form of any length.
  • Nucleic acid sequences include DNA, cDNA, genomic DNA, RNA, synthetic forms and mixed polymers, both sense and antisense strands, or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art.
  • This term refers only to the primary structure of the molecule. Thus, this term includes double- and single-stranded DNA, and RNA. It also includes known types of modifications, for example, methylation, “caps”, and substitution of one or more of the naturally occurring nucleotides with an analog. Modifications to nucleic acids can be introduced at one or more levels: phosphate linkage modification (e.g.
  • the polynucleic acid as referred to herein is an isolated polynucleic acid.
  • a “coding sequence” is a nucleotide sequence that can be transcribed into mRNA and/or translated into a polypeptide when placed under the control of appropriate (gene) regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5'-terminus and a translation stop codon at the 3'-terminus.
  • a coding sequence can include, but is not limited to mRNA, cDNA, recombinant nucleotide sequences or genomic DNA, while introns may be present as well under certain circumstances.
  • sequence when used herein relates to nucleotide sequence(s), polynucleotide(s), nucleic acid sequence(s), nucleic acid(s), nucleic acid molecule, peptides, polypeptides and proteins, depending on the context in which the term “sequence” is used.
  • Amino acids are presented herein by their 3- or 1-lettercode nomenclature and nucleotides/nucleosides are presented by their 1-lettercode nomenclature, both as defined and provided also in the IUPAC-IUB Joint Commission on Biochemical Nomenclature (Nomenclature and Symbolism for Amino Acids and Peptides. Eur. J.
  • Nucleotide designations are according to the table below.
  • a “percentage (of) sequence identity” is calculated by comparing two optimally aligned (amino acid or nucleic acid) sequences over the window of comparison, determining the number of positions at which the identical amino acid or nucleotide residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of (amino acid or nucleic acid) sequence identity.
  • a matched position refers to a position in which identical nucleotides or amino acids occur at the same position in aligned nucleic acid sequences.
  • a target nucleic acid or amino acid sequence is compared to the identified nucleic acid or amino acid sequence using the BLAST 2 Sequences (Bl2seq) program from the stand-alone version of BLASTZ containing BLASTN and BLASTP.
  • BLASTZ BLAST 2 Sequences
  • This stand-alone version of BLASTZ can be obtained from the U.S. government's National Center for Biotechnology Information web site (World Wide Web at ncbi.nlm.nih.gov). Instructions explaining how to use the Bl2seq program can be found in the readme file accompanying BLASTZ.
  • BI2seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm.
  • BLAST sequence alignments are performed according to the standard (i.e., default) settings (i.e., at the filing date of the present application).
  • BLASTN is used to compare nucleic acid sequences
  • BLASTP is used to compare amino acid sequences.
  • the options are set as follows: -i is set to a file containing the first nucleic acid sequence to be compared (e.g., C: ⁇ seq l .txt); -j is set to a file containing the second nucleic acid sequence to be compared (e.g., C: ⁇ seq2.txt); -p is set to blastn; -o is set to any desired file name (e.g., C : ⁇ output.txt); -q is set to - 1 ; -r is set to 2; and all other options are left at their default setting.
  • -i is set to a file containing the first nucleic acid sequence to be compared (e.g., C: ⁇ seq l .txt)
  • -j is set to a file containing the second nucleic acid sequence to be compared (e.g., C: ⁇ seq2.txt)
  • -p is set to blastn
  • -o
  • the following command will generate an output file containing a comparison between two sequences: C: ⁇ B12seq -i c: ⁇ seql .txt -j c: ⁇ seq2.txt -p blastn -o c: ⁇ output.txt - q - 1 -r 2. If the target sequence shares homology with any portion of the identified sequence, then the designated output file will present those regions of homology as aligned sequences. If the target sequence does not share homology with any portion of the identified sequence, then the designated output file will not present aligned sequences.
  • a length is determined by counting the number of consecutive nucleotides from the target sequence presented in alignment with the sequence from the identified sequence starting with any matched position and ending with any other matched position.
  • a matched position is any position where an identical nucleotide is presented in both the target and identified sequences. Gaps presented in the target sequence are not counted since gaps are not nucleotides. Likewise, gaps presented in the identified sequence are not counted since target sequence nucleotides are counted, not nucleotides from the identified sequence.
  • the percent identity over a particular length is determined by counting the number of matched positions over that length and dividing that number by the length followed by multiplying the resulting value by 100.
  • a (weighed) average of sequence identity may be applied to calculate total sequence identity (e.g. the total percentage of identity based on all partial alignment stretches combined).
  • the percent identity is determined over the entire length of the query sequence (or optionally the longer of the query or target sequence) by counting the number of matched positions over its entire length and dividing that number by the length followed by multiplying the resulting value by 100. It is noted that the percent identity value is rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2.
  • nucleic acid sequence e.g., DNA or genomic DNA
  • nucleic acid sequence identity to a reference sequence or having a sequence identity of at least 80%, e.g., at least 85%, 90%, 95%, 98%> or 99%> nucleic acid sequence identity to a reference sequence
  • said nucleotide sequence is considered substantially identical to the given nucleotide sequence and can be identified using hybridisation conditions.
  • nucleic acid sequence comprises one or more mutations compared to the given nucleotide sequence but still can be identified using stringent hybridisation conditions.
  • Stringent hybridisation conditions can be used to identify nucleotide sequences, which are substantially identical to a given nucleotide sequence. Stringent conditions are sequence dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequences at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridises to a perfectly matched probe. Typically, stringent conditions will be chosen in which the salt concentration is about 0.02 molar at pH 7 and the temperature is at least 60°C. Lowering the salt concentration and/or increasing the temperature increases stringency.
  • Stringent conditions for RNA-DNA hybridisations are for example those which include at least one wash in 0.2X SSC at 63°C for 20min, or equivalent conditions.
  • Stringent conditions for DNA-DNA hybridisation are for example those which include at least one wash (usually 2) in 0.2X SSC at a temperature of at least 50°C, usually about 55°C, for 20 min, or equivalent conditions. See also Sambrook et al. (1989) and Sambrook and Russell (2001).
  • high stringent hybridization conditions are conditions under which primarily only those nucleic acid molecules that have at least 90% or at least 95% sequence identity undergo hybridization. Such high stringent hybridization conditions are, for example: 4 x SSC at 65°C and subsequent multiple washes in 0.1 x SSC at 65°C for approximately 1 hour.
  • the term “high stringent hybridization conditions” as used herein may also mean: hybridization at 68°C in 0.25 M sodium phosphate, pH 7.2, 7 % SDS, 1 mM EDTA and 1 % BSA for 16 hours and subsequently washing twice with 2 x SSC and 0.1 % SDS at 68°C.
  • hybridization takes place under stringent conditions.
  • wild-type refers to a gene or gene product isolated from a naturally occurring source.
  • a wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designed the “normal” or “wild-type” form of the gene or gene product.
  • mutant refers to a gene or gene product that displays modifications (such as a substitution, mutation or variation, deletion or addition) in sequence, post-translational modifications and/or modification of biological or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product.
  • modifications such as a substitution, mutation or variation, deletion or addition
  • post-translational modifications i.e., modification of biological or functional properties
  • altered characteristics can solely reside at the sequence level, or can additionally confer altered biological and/or functional properties to the mutants or variants compared to the wild- type gene or gene product.
  • conservative amino acid substitutions can be introduced in a protein or polypeptide whereby such substitutions have no essential or substantial effect on the protein's activity.
  • Preferred conservative substitutions are those fulfilling the criteria defined for an accepted point mutation in Dayhoff et al., Atlas of Protein Sequence and Structure, 5, pp.345-352 (1978 & Supp.), which is incorporated herein by reference.
  • conservative substitutions are substitutions including but not limited to the following groups: (a) valine, glycine; (b) glycine, alanine; (c) valine, isoleucine, leucine; (d) aspartic acid, glutamic acid; (e) asparagine, glutamine; (f) serine, threonine; (g) lysine, arginine, methionine; and (h) phenylalanine, tyrosine.
  • a conservative substitution as referred to herein may be as defined according to the BLAST algorithm.
  • conservative substitutions may be described in the form of a Venn diagram (Livingstone C.D. and Barton G.J.
  • a “homologue”, or “homologues” of a protein of interest encompass(es) proteins having amino acid substitutions, deletions and/or insertions relative to an unmodified (e.g. native, wild-type) protein of interest and having essentially or substantially similar biological and functional activity as the unmodified protein from which it is/they are derived.
  • a “Fragment” is intended to mean a portion of a polynucleotide or polypeptide.
  • a “functional fragment” refers to a fragment of a polynucleotide or polypeptide capable of performing the same function as the polynucleotide or polypeptide it is derived from. It will be understood that, as used herein, a functional fragment of a polynucleotide refers to a polynucleotide which when translated results in a functional fragment of the polypeptide it is derived from.
  • polypeptide fragments in particular are polypeptide fragments of the single-domain antibodies as described herein (or polynucleotides encoding such polypeptide fragments) capable of performing the same function as the full length single-domain antibodies, i.e. Panx1 binding.
  • isolated or “purified” is meant material that is substantially or essentially free from components that normally accompany it in its native state.
  • an “isolated polypeptide” or “purified polypeptide” refers to a polypeptide which has been isolated or purified by any suitable means from a mixture of molecules comprising the to be isolated or to be purified polypeptide of interest.
  • An isolated or purified polypeptide or polynucleotide of interest can for instance be an immunoglobulin, antibody or nanobody, or a polynucleotide encoding an immunoglobulin, antibody or nanobody, and the mixture can be a mixture or molecules as present in a cell producing the immunoglobulin, antibody or nanobody, and/or the culture medium into which the immunoglobulin, antibody or nanobody is secreted into (likely together with other molecules secreted by the cell).
  • the term “vector”, “vector construct”, “expression vector”, “recombinant vector” or “gene transfer vector”, as used herein, is intended to refer to a nucleic acid molecule capable of carrying another nucleic acid molecule to which it has been linked.
  • Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g., circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art.
  • Said vectors may include a cloning or expression vector, as well as a delivery vehicle such as a viral, lentiviral or adenoviral vector.
  • Expression vectors may comprise plasmids as well as viral vectors and generally contain a desired coding sequence and appropriate DNA sequences necessary for the expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammal) or in in vitro expression systems.
  • an expression vector as described herein may comprise a nucleic acid molecule as described herein comprising a nucleic acid sequence encoding an antibody or an antigen-binding fragment as described herein operably linked to at least one regulatory sequence.
  • “Operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • Regulatory sequences are selected to direct the expression of the protein of interest, in particular the antibody or antigen-binding fragment, in a suitable host cell, and include promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences) as known to the skilled person.
  • the vector includes a promoter for driving expression of the nucleic acid of interest, optionally a nucleic acid sequence encoding a signal peptide that secretes the antibody or antigen-binding fragment, and optionally a nucleic acid sequence encoding a terminator.
  • the expression vector When the expression vector is manipulated in a production strain or cell line, the vector may or may not be integrated into the genome of the host cell when introduced into the host cell.
  • Cloning vectors are generally used to engineer and amplify a certain desired DNA fragment.
  • a cloning vectors may contain origin of replication that matches the cell type specified by the cloning vector, and may lack functional sequences needed for expression of the desired DNA fragments.
  • the vector contains one or more selection markers.
  • the choice of the selection markers may depend on the host cells of choice, although this is not critical to the present invention as is well known to persons skilled in the art.
  • the construction of expression vectors for use in transfecting cells is also well known in the art, and thus can be accomplished via standard techniques (see, for example, Sambrook, Fritsch, and Maniatis, in: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989; Gene Transfer and Expression Protocols, pp.109-128, ed. E. J. Murray, The Humana Press Inc., Clif ton, N.J.), and the Ambion 1998 Catalog (Ambion, Austin, Tex.).
  • vector refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques.
  • viral vector wherein virally-derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g., retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses).
  • Viral vectors also include polynucleotides carried by a virus for transfection into a host cell.
  • vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • Other vectors e.g., non-episomal mammalian vectors
  • said vector may include any vector known to the skilled person, including any suitable type, but not limited to, for instance, plasmid vectors, cosmid vectors, phage vectors, such as lambda phage, viral vectors, even more particular a lentiviral, adenoviral, AAV or baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or P1 artificial chromosomes (PAC).
  • BAC bacterial artificial chromosomes
  • YAC yeast artificial chromosomes
  • PAC P1 artificial chromosomes
  • expression vectors contain one or more expression cassette.
  • An "expression cassette” comprises any nucleic acid construct capable of directing the expression of a gene/coding sequence of interest, which is operably linked to a (gene) promoter.
  • Expression cassettes are generally DNA constructs preferably including (5’ to 3’ in the direction of transcription): a (gene) promoter region, a polynucleotide sequence of interest with a transcription initiation region, and a termination sequence including a stop signal for RNA polymerase and a polyadenylation signal; all these elements being operably or operatively linked meaning that all of these regions should be capable of operating (being expressed) in a cell, such as prokaryotic (e.g. bacterial) or eukaryotic (e.g.
  • the promoter region comprising the transcription initiation region, which preferably includes the RNA polymerase binding site, and the polyadenylation signal may be native to the cell to be transformed, may be derived from an alternative source, or may be synthetic, as long as it is functional in the cell.
  • Such expression cassettes can be constructed in e.g. a “vector” or “expression vector” (linear or circular nucleic acids, plasmids, cosmids, viral vectors, phagemids, etc.).
  • a "host cell” or a “recombinant host cell” or “transformed cell” are terms referring to a new individual cell (or organism) arising as a result of at least one nucleic acid molecule, having been introduced into said cell.
  • the host cell may be a prokaryotic or eukaryotic host cell.
  • the host cell may contain the nucleic acid as an extra-chromosomally (episomal) replicating molecule, or comprises the nucleic acid integrated in the nuclear or plastid genome of the host cell, or as introduced chromosome, e.g., minichromosome.
  • the host cell may comprise the single-domain antibody or binding agent as described herein or may comprise a polynucleotide encoding the single-domain antibody or binding agent as described herein and capable of expressing said the single-domain antibody or binding agent as described herein.
  • the polynucleotide may be comprised in a vector.
  • the host cell may comprise a (functional) fragment of the single-domain antibody or binding agent as described herein or may comprise a polynucleotide encoding such functional fragment and capable of expressing said functional fragment.
  • the host cell may also be a recombinant host cell, which involves a cell which has been genetically modified to contain an isolated nucleic acid molecule encoding the antibody or antigen-binding fragment of the invention.
  • Introduction of a vector in a host cell can be effected by, e.g., calcium phosphate transfection, virus infection, DEAE-dextran-mediated transfection, lipofectamin transfection or electroporation, and any person skilled in the art can select and use an introduction method suitable for the expression vector and host cell used.
  • Representative host cells that may be used to produce said antibodies or antigen-binding fragments include, but are not limited to, bacterial cells, yeast cells, plant cells and animal cells.
  • Bacterial host cells suitable for production of the antibodies or antigen-binding fragment of the invention include Escherichia spp. cells, Bacillus spp. cells, Streptomyces spp. cells, Erwinia spp.
  • yeast host cells suitable for use with the invention include species within Saccharomyces, Schizosaccharomyces, Kluyveromyces, Pichia (e.g. Pichia pastoris), Hansenula (e.g. Hansenula polymorpha), Yarowia, Schwaniomyces, Schizosaccharomyces, Zygosaccharomyces and the like. Saccharomyces cerevisiae, S. carlsbergensis and K. lactis are the most commonly used yeast hosts, and are convenient fungal hosts.
  • Animal host cells suitable for use with the invention include insect cells and mammalian cells (e.g. derived from Chinese hamster (e.g. CHO), and human cell lines, such as HeLa).
  • Exemplary insect cell lines include, but are not limited to, Sf9 cells, baculovirus-insect cell systems (e.g. review Jarvis, Virology Volume 310, Issue 1, 25 May 2003, Pages 1-7).
  • the host cells may also be (comprised in) transgenic animals or plants.
  • a “pharmaceutical composition” refers to a composition comprising at least one therapeutically active ingredient (an active pharmaceutical ingredient) in the context of treating a disease or otherwise pathological condition, i.e.
  • a pharmaceutical composition comprises one or more pharmaceutically acceptable carriers and/or excipients.
  • An “active pharmaceutical ingredient” refers to a biologically active component of a pharmaceutical composition, such as drug product (tablet, capsule, cream, injectable) that produces the intended effects.
  • An active pharmaceutical ingredient is any component that provides pharmacological activity or other direct effect in the diagnosis, cure, mitigation, or treatment of disease, or to affect the structure or any function of the body of man or animals.
  • pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the compound, in particular the single-domain antibodies or binding agents as described herein as well as the polynucleic acids, vectors or host cells etc. as described herein, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • a pharmaceutically acceptable carrier or excipient is preferably a carrier/excipient that is (relatively) non-toxic and innocuous to a patient at concentrations consistent with effective activity of the active ingredient so that any side effects ascribable to the carrier do not vitiate the beneficial effects of the active ingredient.
  • Suitable carriers or adjuvantia typically comprise one or more of the compounds included in the following non- exhaustive list: large slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles.
  • excipient as used herein, is intended to include all substances which may be present in a pharmaceutical composition and which are not active ingredients but may contribute to e.g.
  • Excipients may include, for example, salts, binders (e.g., lactose, dextrose, sucrose, trehalose, sorbitol, mannitol), lubricants, thickeners, surface active agents, preservatives, emulsifiers, buffer substances, stabilizing agents, flavouring agents or colorants.
  • a pharmaceutically effective amount of active ingredients such as the single-domain antibodies (or fragments) or binding agents as described herein, as well as the polynucleic acids, vectors, or host cells as described herein, is preferably that amount which produces a result or exerts an influence on the particular condition being treated.
  • the pharmaceutical composition of this invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use. When prepared as lyophilization or liquid, physiologically acceptable carrier, excipient, stabilizer need to be added into the pharmaceutical composition of the invention (Remington's Pharmaceutical Sciences 22nd edition, Ed. Allen, Loyd V, Jr. (2012).
  • the preparation containing pharmaceutical composition of this invention should be sterilized before injection. This procedure can be done using sterile filtration membranes before or after lyophilization and reconstitution.
  • the pharmaceutical composition can be packaged in a container or vial with sterile access port, such as an i.v. solution bottle with a rubber stopper – the pharmaceutical composition can be present as liquid, or the container or vial is filled with a liquid pharmaceutical composition that is subsequently lyophilized or dried; or can be packaged in a pre-filled syringe.
  • the pharmaceutical composition of the invention can be administered to any patient in accordance with standard techniques.
  • the administration can be by any appropriate mode, including oral, parenteral, topical, nasal, ophthalmic, intrathecal, intra- cerebroventricular, sublingual, rectal, vaginal, and the like. Still other techniques of formulation such as nanotechnology and aerosol and inhalant are also within the scope of this invention.
  • the dosage and frequency of administration will depend on the age, sex and condition of the patient, concurrent administration of other drugs, counter- indications and other parameters to be taken into account by the clinician.
  • the terms “therapy” or “treatment” refer to the alleviation or measurable lessening of one or more symptoms or measurable markers of a pathological condition such as a disease or disorder. Measurable lessening includes any statistically significant decline in a measurable symptom or marker.
  • the terms encompass both curative treatments and treatments directed to reduce symptoms and/or slow progression of the disease.
  • Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms associated with a disease, diminishment of extent of a disease, stabilisation of the disease, delay or slowing of the progression of a disease, amelioration or palliation of a disease, or combinations thereof.
  • the terms may relate to therapeutic treatments.
  • a “therapeutically effective amount” or “therapeutically effective dose” indicates an amount of active agent, in particular a single-domain antibody (or fragment), binding agent, nucleic acid, vector, host cell, or pharmaceutical composition that when administered to the subject brings about a clinical positive response with respect to a desired therapeutic treatment of the subject.
  • the active agent in particular a single-domain antibody (or fragment), binding agent, nucleic acid, vector, host cell, or pharmaceutical composition as described herein may need to be administered to a subject multiple times, such as with an interval of 1 week or 2 weeks; the interval being dictated by the pharmacokinetic behaviour or characteristics (e.g. half-time or half-life in the subject’s circulation) of the active agent, in particular a single-domain antibody (or fragment), binding agent, nucleic acid, vector, host cell, or pharmaceutical composition.
  • a single dose of a active agent in particular a single-domain antibody (or fragment), binding agent, nucleic acid, vector, host cell, or pharmaceutical composition as described herein is administered to the subject is envisaged.
  • the single dose may be in the range of 0.5 mg/kg to 25 mg/kg.
  • the term “subject”, “individual” or “patient”, used interchangeably herein, relates to any organism such as a vertebrate, particularly any mammal, including both a human and other mammals, for whom diagnosis or therapy is desired, e.g., an animal such as a rodent, a rabbit, a cow, a sheep, a horse, a dog, a cat, a lama, a pig, or a human or non- human primate (e.g., a monkey).
  • the rodent may be a mouse, rat, hamster, guinea pig, or chinchilla.
  • the subject is a human, a rat or a non-human primate.
  • the subject, individual, or patient may have any gender and any age.
  • the subject is a human.
  • a subject is a subject, such as a human subject, with or suspected of having inflammation, or an inflammatory disorder as described herein elsewhere, also designated ”patient” or “subject” herein.
  • the invention relates to a single-domain antibody capable of specifically binding to Panx1, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment.
  • the invention relates to a binding agent comprising a single-domain antibody capable of specifically binding to Panx1, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment.
  • the single domain antibody comprises a heavy chain variable domain derived from a heavy chain antibody (VHH), or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment.
  • the single domain antibody comprises a heavy chain variable domain derived from a camelid heavy chain antibody (VHH), or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment.
  • the camelid is a llama.
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment is capable of (specifically) binding to an extracellular part or domain of Panx1.
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment is capable of (specifically) binding to an extracellular part or domain of Panx1, wherein the single domain antibody comprises a heavy chain variable domain derived from a heavy chain antibody (VHH), preferably a camelid heavy chain antibody, such as a llama heavy chain antibody.
  • VHH heavy chain antibody
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment is capable of (specifically) binding to an extracellular loop of Panx1.
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment is capable of (specifically) binding to an extracellular loop of Panx1, wherein the single domain antibody comprises a heavy chain variable domain derived from a heavy chain antibody (VHH), preferably a camelid heavy chain antibody, such as a llama heavy chain antibody.
  • VHH heavy chain antibody
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment is capable of (specifically) binding to a sequence or epitope comprised in a sequence which is at least 90% identical, preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to a sequence as set forth in SEQ ID NO: 33 or 34.
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment is capable of (specifically) binding to a sequence or epitope comprised in a sequence which is at least 90% identical, preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to a sequence as set forth in SEQ ID NO: 33 or 34, wherein the single domain antibody comprises a heavy chain variable domain derived from a heavy chain antibody (VHH), preferably a camelid heavy chain antibody, such as a llama heavy chain antibody.
  • VHH heavy chain antibody
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment is capable of (specifically) binding to a sequence or epitope comprised in a sequence as set forth in SEQ ID NO: 33 or 34.
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment is capable of (specifically) binding to a sequence or epitope comprised in a sequence as set forth in SEQ ID NO: 33 or 34, wherein the single domain antibody comprises a heavy chain variable domain derived from a heavy chain antibody (VHH), preferably a camelid heavy chain antibody, such as a llama heavy chain antibody.
  • VHH heavy chain antibody
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment (specifically) binds to Panx1 with a Kd (dissociation constant) of at most 300 nM, preferably at most 200 nM, more preferably at most 100 nM, such as at most 50 nM, at most 40 nM, at most 30 nM, at most 20 nM, or at most 10 nM.
  • Kd dissociation constant
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment (specifically) binds to Panx1 with a Kd (dissociation constant) of at most 300 nM, preferably at most 200 nM, more preferably at most 100 nM, such as at most 50 nM, at most 40 nM, at most 30 nM, at most 20 nM, or at most 10 nM
  • the single domain antibody comprises a heavy chain variable domain derived from a heavy chain antibody (VHH), preferably a camelid heavy chain antibody, such as a llama heavy chain antibody.
  • VHH heavy chain antibody
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment (specifically) binds to an extracellular domain or region of Panx1 with a Kd (dissociation constant) of at most 300 nM, preferably at most 200 nM, more preferably at most 100 nM, such as at most 50 nM, at most 40 nM, at most 30 nM, at most 20 nM, or at most 10 nM.
  • Kd dissociation constant
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment (specifically) binds to an extracellular domain or region of Panx1 with a Kd (dissociation constant) of at most 300 nM, preferably at most 200 nM, more preferably at most 100 nM, such as at most 50 nM, at most 40 nM, at most 30 nM, at most 20 nM, or at most 10 nM
  • the single domain antibody comprises a heavy chain variable domain derived from a heavy chain antibody (VHH), preferably a camelid heavy chain antibody, such as a llama heavy chain antibody.
  • VHH heavy chain antibody
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment (specifically) binds to an extracellular loop of Panx1 with a Kd (dissociation constant) of at most 300 nM, preferably at most 200 nM, more preferably at most 100 nM, such as at most 50 nM, at most 40 nM, at most 30 nM, at most 20 nM, or at most 10 nM.
  • Kd dissociation constant
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment (specifically) binds to an extracellular loop of Panx1 with a Kd (dissociation constant) of at most 300 nM, preferably at most 200 nM, more preferably at most 100 nM, such as at most 50 nM, at most 40 nM, at most 30 nM, at most 20 nM, or at most 10 nM
  • the single domain antibody comprises a heavy chain variable domain derived from a heavy chain antibody (VHH), preferably a camelid heavy chain antibody, such as a llama heavy chain antibody.
  • VHH heavy chain antibody
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment (specifically) binds to a sequence or epitope comprised in a sequence which is at least 90% identical, preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to a sequence as set forth in SEQ ID NO: 33 or 34, with a Kd (dissociation constant) of at most 300 nM, preferably at most 200 nM, more preferably at most 100 nM, such as at most 50 nM, at most 40 nM, at most 30 nM, at most 20 nM, or at most 10 nM.
  • Kd dissociation constant
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment (specifically) binds to a sequence or epitope comprised in a sequence which is at least 90% identical, preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to a sequence as set forth in SEQ ID NO: 33 or 34, with a Kd (dissociation constant) of at most 300 nM, preferably at most 200 nM, more preferably at most 100 nM, such as at most 50 nM, at most 40 nM, at most 30 nM, at most 20 nM, or at most 10 nM, wherein the single domain antibody comprises a heavy chain variable domain derived from a heavy chain antibody (VHH), preferably a camelid heavy chain antibody, such as a llama
  • VHH heavy
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment (specifically) binds to a sequence or epitope comprised in a sequence as set forth in SEQ ID NO: 33 or 34, with a Kd (dissociation constant) of at most 300 nM, preferably at most 200 nM, more preferably at most 100 nM, such as at most 50 nM, at most 40 nM, at most 30 nM, at most 20 nM, or at most 10 nM.
  • Kd dissociation constant
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment (specifically) binds to a sequence or epitope comprised in a sequence as set forth in SEQ ID NO: 33 or 34, with a Kd (dissociation constant) of at most 300 nM, preferably at most 200 nM, more preferably at most 100 nM, such as at most 50 nM, at most 40 nM, at most 30 nM, at most 20 nM, or at most 10 nM
  • the single domain antibody comprises a heavy chain variable domain derived from a heavy chain antibody (VHH), preferably a camelid heavy chain antibody, such as a llama heavy chain antibody.
  • VHH heavy chain antibody
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, or the binding agent is capable of at least partially inhibiting Panx1 channel activity.
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, or the binding agent is capable of at least partially inhibiting Panx1 channel activity, wherein the single domain antibody comprises a heavy chain variable domain derived from a heavy chain antibody (VHH), preferably a camelid heavy chain antibody, such as a llama heavy chain antibody.
  • VHH heavy chain antibody
  • the inhibition of Panx1 channel activity is at least 20%, preferably at least 30%, more preferably at least 40%, most preferably at least 50%, such as at least 60%, at least 70%, at least 80%, at least 90%, or (substantially) 100%.
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, or the binding agent has a Panx1 channel activity inhibitory IC50 (half maximum inhibitory concentration) of at most 100 ⁇ M.
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, or the binding agent has a Panx1 channel activity inhibitory IC50 (half maximum inhibitory concentration) ranging from 100 ⁇ M to 100 nM.
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment is capable of at least partially inhibiting extracellular release of ATP from a cell.
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment is capable of at least partially inhibiting extracellular release of ATP from a cell
  • the single domain antibody comprises a heavy chain variable domain derived from a heavy chain antibody (VHH), preferably a camelid heavy chain antibody, such as a llama heavy chain antibody.
  • VHH heavy chain antibody
  • the inhibition of extracellular release of ATP from a cell is at least 20%, preferably at least 30%, more preferably at least 40%, most preferably at least 50%, such as at least 60%, at least 70%, at least 80%, at least 90%, or (substantially) 100%.
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, or the binding agent has an extracellular ATP release inhibitory IC50 (half maximum inhibitory concentration) of at most 100 ⁇ M.
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, or the binding agent has an extracellular ATP release inhibitory IC50 (half maximum inhibitory concentration) ranging from 100 ⁇ M to 100 nM.
  • the single-domain antibody comprises 4 framework regions (FR) and 3 complementarity determining regions (CDR) according to formula (I): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (I).
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment comprises CDR1 having a sequence as set forth in any of SEQ ID NOs: 1, 4, 7, 10, 13, 16, 19, or 21; CDR2 having a sequence as set forth in any of SEQ ID NOs: 2, 5, 8, 11, 14, 17, 20, or 23; and/or CDR3 having a sequence as set forth in any of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, or 24.
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment comprises CDR1 having a sequence as set forth in any of SEQ ID NOs: 1, 4, 7, 10, 13, 16, 19, or 21.
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment comprises CDR2 having a sequence as set forth in any of: 2, 5, 8, 11, 14, 17, 20, or 23.
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment comprises CDR3 having a sequence as set forth in any of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, or 24.
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment comprises CDR1 having a sequence as set forth in any of SEQ ID NOs: 1, 4, 7, 10, 13, 16, 19, or 21; CDR2 having a sequence as set forth in any of SEQ ID NOs: 2, 5, 8, 11, 14, 17, 20, or 23; and CDR3 having a sequence as set forth in any of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, or 24.
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment comprises CDR1 having a sequence as set forth in SEQ ID NO: 1, CDR2 having a sequence as set forth in SEQ ID NO: 2, and CDR3 having a sequence as set forth in SEQ ID NO: 3; CDR1 having a sequence as set forth in SEQ ID NO: 4, CDR2 having a sequence as set forth in SEQ ID NO: 5, and CDR3 having a sequence as set forth in SEQ ID NO: 6; CDR1 having a sequence as set forth in SEQ ID NO: 7, CDR2 having a sequence as set forth in SEQ ID NO: 8, and CDR3 having a sequence as set forth in SEQ ID NO: 9; CDR1 having a sequence as set forth in SEQ ID NO: 10, CDR2 having a sequence as set forth in SEQ ID NO: 11, and CDR3 having a sequence as set forth in SEQ ID NO: 12; CDR1 having a sequence as set forth in SEQ ID
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment comprises CDR1 having a sequence as set forth in SEQ ID NO: 1; CDR2 having a sequence as set forth in SEQ ID NO: 2; and CDR3 having a sequence as set forth in SEQ ID NO: 3.
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment comprises CDR1 having a sequence as set forth in SEQ ID NO: 4; CDR2 having a sequence as set forth in SEQ ID NO: 5; and CDR3 having a sequence as set forth in SEQ ID NO: 6.
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment comprises CDR1 having a sequence as set forth in SEQ ID NO: 7; CDR2 having a sequence as set forth in SEQ ID NO: 8; and CDR3 having a sequence as set forth in SEQ ID NO: 9.
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment comprises CDR1 having a sequence as set forth in SEQ ID NO: 10; CDR2 having a sequence as set forth in SEQ ID NO: 11; and CDR3 having a sequence as set forth in SEQ ID NO: 12.
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment comprises CDR1 having a sequence as set forth in SEQ ID NO: 13; CDR2 having a sequence as set forth in SEQ ID NO: 14; and CDR3 having a sequence as set forth in SEQ ID NO: 15.
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment comprises CDR1 having a sequence as set forth in SEQ ID NO: 16; CDR2 having a sequence as set forth in SEQ ID NO: 17; and CDR3 having a sequence as set forth in SEQ ID NO: 18.
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment comprises CDR1 having a sequence as set forth in SEQ ID NO: 19; CDR2 having a sequence as set forth in SEQ ID NO: 20; and CDR3 having a sequence as set forth in SEQ ID NO: 21.
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment comprises CDR1 having a sequence as set forth in SEQ ID NO: 22; CDR2 having a sequence as set forth in SEQ ID NO: 23; and CDR3 having a sequence as set forth in SEQ ID NO: 24.
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment comprises CDR1 having a sequence as set forth in SEQ ID NO: 19; CDR2 having a sequence as set forth in SEQ ID NO: 20; and CDR3 having a sequence as set forth in SEQ ID NO: 21.
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment comprises CDR1 having a sequence as set forth in SEQ ID NO: 10; CDR2 having a sequence as set forth in SEQ ID NO: 11; and CDR3 having a sequence as set forth in SEQ ID NO: 12.
  • the single-domain antibody comprises one or more of FR1 to FR4 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence of FR1 to FR4 as set forth in any of SEQ ID NOs: 25 to 32, wherein FR1 to FR4 are annotated according to IMGT.
  • the single-domain antibody comprises one or more of FR1 to FR4 having a sequence of FR1 to FR4 as set forth in any of SEQ ID NOs: 25 to 32, wherein FR1 to FR4 are annotated according to IMGT.
  • the single-domain antibody comprises one or more of FR1 to FR4 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence of FR1 to FR4 as set forth in Figure 21.
  • the single-domain antibody comprises one or more of FR1 to FR4 having a sequence of FR1 to FR4 as set forth in Figure 21.
  • the single-domain antibody comprises one or more of FR1 to FR4 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence of FR1 to FR4 as set forth in any of SEQ ID NOs: 35 to 66.
  • the single-domain antibody comprises one or more of FR1 to FR4 having a sequence of FR1 to FR4 as set forth in any of SEQ ID NOs: 35 to 66.
  • the single-domain antibody comprises FR1 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in any of SEQ ID NOs: 35, 39, 43, 47, 51, 55, 59, or 63.
  • the single-domain antibody comprises FR1 having a sequence as set forth in any of SEQ ID NOs: 35, 39, 43, 47, 51, 55, 59, or 63.
  • the single-domain antibody comprises FR2 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in any of SEQ ID NOs: 36, 40, 44, 48, 52, 56, 60, or 64.
  • the single-domain antibody comprises FR1 having a sequence as set forth in any of SEQ ID NOs: 36, 40, 44, 48, 52, 56, 60, or 64.
  • the single-domain antibody comprises FR3 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in any of SEQ ID NOs: 37, 41, 45, 49, 53, 57, 61, or 65.
  • the single-domain antibody comprises FR1 having a sequence as set forth in any of SEQ ID NOs: 37, 41, 45, 49, 53, 57, 61, or 65.
  • the single-domain antibody comprises FR4 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in any of SEQ ID NOs: 38, 42, 46, 50, 54, 58, 62, or 66.
  • the single-domain antibody comprises FR1 having a sequence as set forth in any of SEQ ID NOs: 38, 42, 46, 50, 54, 58, 62, or 66.
  • the single-domain antibody comprises FR1 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in any of SEQ ID NOs: 35, 39, 43, 47, 51, 55, 59, or 63, preferably a sequence as set forth in any of SEQ ID NOs: 35, 39, 43, 47, 51, 55, 59, or 63; FR2 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined)
  • the single-domain antibody comprises FR1 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in any of SEQ ID NOs: 35, 39, 43, 47, 51, 55, 59, or 63, preferably a sequence as set forth in any of SEQ ID NOs: 35, 39, 43, 47, 51, 55, 59, or 63; FR2 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined)
  • the single-domain antibody comprises FR1 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 35, preferably a sequence as set forth in SEQ ID NO: 35; FR2 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to the sequence as set forth in SEQ ID NO: 36, preferably a sequence as set forth in SEQ ID NO: 36; FR3 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most
  • the single-domain antibody comprises FR1 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 39, preferably a sequence as set forth in SEQ ID NO: 39; FR2 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 40, preferably a sequence as set forth in SEQ ID NO: 40; FR3 having a sequence which
  • the single-domain antibody comprises FR1 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 43, preferably a sequence as set forth in SEQ ID NO: 43; FR2 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to the sequence as set forth in SEQ ID NO: 44, preferably a sequence as set forth in SEQ ID NO: 44; FR3 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most
  • the single-domain antibody comprises FR1 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 47, preferably a sequence as set forth in SEQ ID NO: 47; FR2 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 48, preferably a sequence as set forth in SEQ ID NO: 48; FR3 having a sequence which
  • the single-domain antibody comprises FR1 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 51, preferably a sequence as set forth in SEQ ID NO: 51; FR2 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 52, preferably a sequence as set forth in SEQ ID NO: 52; FR3 having a sequence which
  • the single-domain antibody comprises FR1 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 55, preferably a sequence as set forth in SEQ ID NO: 55; FR2 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 56, preferably a sequence as set forth in SEQ ID NO: 56; FR3 having a sequence which
  • the single-domain antibody comprises FR1 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 59, preferably a sequence as set forth in SEQ ID NO: 59; FR2 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 60, preferably a sequence as set forth in SEQ ID NO: 60; FR3 having a
  • the single-domain antibody comprises FR1 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 63, preferably a sequence as set forth in SEQ ID NO: 63; FR2 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 64, preferably a sequence as set forth in SEQ ID NO: 64; FR3 having a
  • the single-domain antibody comprises FR1 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 59, preferably a sequence as set forth in SEQ ID NO: 59; FR2 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 60, preferably a sequence as set forth in SEQ ID NO: 60; FR3 having a sequence which is at
  • the single-domain antibody comprises FR1 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 47, preferably a sequence as set forth in SEQ ID NO: 47; FR2 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 48, preferably a sequence as set forth in SEQ ID NO: 48; FR3 having a sequence which
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment comprises CDR1 having a sequence as set forth in SEQ ID NO: 1; CDR2 having a sequence as set forth in SEQ ID NO: 2; CDR3 having a sequence as set forth in SEQ ID NO: 3; FR1 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 35, preferably a sequence as set forth in SEQ ID NO: 35; FR2 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 5, preferably at most
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment comprises CDR1 having a sequence as set forth in SEQ ID NO: 4; CDR2 having a sequence as set forth in SEQ ID NO: 5; CDR3 having a sequence as set forth in SEQ ID NO: 6; FR1 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 39, preferably a sequence as set forth in SEQ ID NO: 39; FR2 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 5, preferably at most
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment comprises CDR1 having a sequence as set forth in SEQ ID NO: 7; CDR2 having a sequence as set forth in SEQ ID NO: 8; CDR3 having a sequence as set forth in SEQ ID NO: 9; FR1 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 43, preferably a sequence as set forth in SEQ ID NO: 43; FR2 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 5, preferably at most
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment comprises CDR1 having a sequence as set forth in SEQ ID NO: 10; CDR2 having a sequence as set forth in SEQ ID NO: 11; CDR3 having a sequence as set forth in SEQ ID NO: 12; FR1 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 47, preferably a sequence as set forth in SEQ ID NO: 47; FR2 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 5, preferably at most
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment comprises CDR1 having a sequence as set forth in SEQ ID NO: 13; CDR2 having a sequence as set forth in SEQ ID NO: 14; CDR3 having a sequence as set forth in SEQ ID NO: 15; FR1 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 51, preferably a sequence as set forth in SEQ ID NO: 51; FR2 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 5, preferably at most
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment comprises CDR1 having a sequence as set forth in SEQ ID NO: 16; CDR2 having a sequence as set forth in SEQ ID NO: 17; CDR3 having a sequence as set forth in SEQ ID NO: 18; FR1 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 55, preferably a sequence as set forth in SEQ ID NO: 55; FR2 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 5, preferably at most
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment comprises CDR1 having a sequence as set forth in SEQ ID NO: 19; CDR2 having a sequence as set forth in SEQ ID NO: 20; CDR3 having a sequence as set forth in SEQ ID NO: 21; FR1 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 59, preferably a sequence as set forth in SEQ ID NO: 59; FR2 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment comprises CDR1 having a sequence as set forth in SEQ ID NO: 22; CDR2 having a sequence as set forth in SEQ ID NO: 23; CDR3 having a sequence as set forth in SEQ ID NO: 24; FR1 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 63, preferably a sequence as set forth in SEQ ID NO: 63; FR2 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment comprises CDR1 having a sequence as set forth in SEQ ID NO: 19; CDR2 having a sequence as set forth in SEQ ID NO: 20; CDR3 having a sequence as set forth in SEQ ID NO: 21; FR1 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 59, preferably a sequence as set forth in SEQ ID NO: 59; FR2 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5,
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment comprises CDR1 having a sequence as set forth in SEQ ID NO: 10; CDR2 having a sequence as set forth in SEQ ID NO: 11; CDR3 having a sequence as set forth in SEQ ID NO: 12.
  • FR1 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 47, preferably a sequence as set forth in SEQ ID NO: 47; FR2 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 48, preferably a sequence as set forth in SEQ ID NO: 48; FR3 having a sequence which is at least 70% identical, preferably at least
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment has a sequence as set forth in any of SEQ ID NOs: 25 to 32, or a sequence which is at least 80%, preferably at least 90%, identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to a sequence as set forth in any of SEQ ID NOs: 25 to 32, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment.
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment has a sequence as set forth in any of SEQ ID NOs: 25 to 32, or a sequence which is at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to a sequence as set forth in any of SEQ ID NOs: 25 to 32, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment.
  • amino acids deviating from the corresponding amino acids in SEQ ID NOs: 25 to 32 are confined to the framework regions.
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment has a sequence as set forth in any of SEQ ID NOs: 25 to 32.
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment has a sequence as set forth SEQ ID NO: 28 or 31, or a sequence which is at least 80%, preferably at least 90%, identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to a sequence as set forth in SEQ ID NO: 28 or 31, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment.
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment has a sequence as set forth in SEQ ID NO: 28 or 31, or a sequence which is at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to a sequence as set forth in SEQ ID NO: 28 or 31, or a fragment thereof, in particular a functional fragment such as an antigen- binding fragment.
  • amino acids deviating from the corresponding amino acids in SEQ ID NO: 28 or 31 are confined to the framework regions.
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment has a sequence as set forth in SEQ ID NOs: 28 or 31.
  • Embodiments of single-domain antibodies or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, of the invention are provided in Table A.
  • Framework regions are at least 80% identical, preferably at least 90% identical, more preferably at least 95% identical, most preferably identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the indicated SEQ ID NO.
  • the single-domain antibody comprises humanized framework regions.
  • the single-domain antibody comprises one or more mutation. In certain embodiments, the single-domain antibody comprises one or more mutation in one or more FR. In certain embodiments, the single-domain antibody comprises one or more mutation of amino acid residues prone to in vivo modification. In certain embodiments, the mutation results in reduced or abolished in vivo modification of said amino acid residue. In certain embodiments, the single-domain antibody comprises one or more mutation in one or more FR of amino acid residues prone to in vivo modification. In certain embodiments, the mutation results in reduced or abolished in vivo modification of said amino acid residue. In certain embodiments, the single-domain antibody comprises one or more amino acid residue mutation resulting in increased thermostability.
  • the single-domain antibody comprises one or more amino acid residue mutation in one or more FR resulting in increased thermostability.
  • the single-binding domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment is fused or linked to an Fc domain, preferably an IgG Fc domain, such as an IgG1 Fc domain, preferably human. As described herein elsewhere, such fusion or linkage may be genetic or non- genetic.
  • the binding agent comprises a single-binding domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, which is fused or linked to an Fc domain, preferably an IgG Fc domain, such as an IgG1 Fc domain, preferably human.
  • a functional fragment such as an antigen-binding fragment
  • the single-binding domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment is fused or linked to albumin, preferably human albumin.
  • such fusion or linkage may be genetic or non-genetic.
  • the binding agent comprises a single-binding domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, which is fused or linked to albumin, preferably human albumin. As described herein elsewhere, such fusion or linkage may be genetic or non-genetic.
  • the single-binding domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment is fused or linked to PEG (polyethylene glycol). As described herein elsewhere, such fusion or linkage may be genetic or non-genetic. Accordingly, in certain embodiments, the single-binding domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment is PEGylated.
  • the binding agent comprises a single-binding domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, which is fused or linked to PEG (polyethylene glycol). As described herein elsewhere, such fusion or linkage may be genetic or non-genetic. Accordingly, in certain embodiments, the binding agent is PEGylated. PEG chain length can be adjusted as known in the art for suitability in half-life extension.
  • the single-binding domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment is fused or linked to PAS (biosynthetic polymers made of the small L-amino acids Pro, Ala and/or Ser). As described herein elsewhere, such fusion or linkage may be genetic or non-genetic. Accordingly, in certain embodiments, the single-binding domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment is PASylated. PAS chain length can be adjusted as known in the art for suitability in half- life extension.
  • the binding agent comprises a single-binding domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, which is fused or linked to PAS (biosynthetic polymers made of the small L- amino acids Pro, Ala and/or Ser). As described herein elsewhere, such fusion or linkage may be genetic or non-genetic. Accordingly, in certain embodiments, the binding agent is PASylated. PAS chain length can be adjusted as known in the art for suitability in half- life extension.
  • the binding agent comprises at least two single-domain antibodies of the invention as described herein elsewhere, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment.
  • the binding agent comprises two single-domain antibodies of the invention as described herein elsewhere, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment. In certain embodiments, the binding agent comprises at least three single-domain antibodies of the invention as described herein elsewhere, or a fragment thereof, in particular a functional fragment such as an antigen- binding fragment. In certain embodiments, the binding agent comprises tree single- domain antibodies of the invention as described herein elsewhere, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment. In certain embodiments, the binding agent comprises a single-domain antibody or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, as described in Table A.
  • the invention relates to a polynucleic acid encoding a single-domain antibody as described herein, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment.
  • the invention relates to a polynucleic acid encoding a binding agent as described herein (i.e. comprising a single-domain antibody as described herein, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment).
  • the polynucleic acid comprises a sequence encoding a protein sequence as set forth in any of SEQ ID NO: 1 to 32, or a sequence which is at least 80%, preferably at least 90%, more preferably at least 95% identical to a sequence as set forth in any of SEQ ID NOs: 1 to 32, preferably SEQ ID NO: 28 or 31.
  • the polynucleic acid comprises a sequence encoding a single- domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, having a sequence as set forth in any of SEQ ID NO: 1 to 32, or a sequence which is at least 80%, preferably at least 90%, more preferably at least 95% identical to a sequence as set forth in any of SEQ ID NOs: 1 to 32, preferably SEQ ID NO: 28 or 31.
  • the polynucleic acid comprises a sequence encoding binding agent comprising a single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, having a sequence as set forth in any of SEQ ID NO: 1 to 32, or a sequence which is at least 80%, preferably at least 90%, more preferably at least 95% identical to a sequence as set forth in any of SEQ ID NOs: 1 to 32, preferably SEQ ID NO: 28 or 31.
  • the polynucleic acid comprises a sequence encoding a protein sequence as set forth in any of SEQ ID NO: 25 to 32, or a sequence which is at least 80%, preferably at least 90%, more preferably at least 95% identical to a sequence as set forth in any of SEQ ID NOs: 25 to 32, preferably SEQ ID NO: 28 or 31.
  • the polynucleic acid comprises a sequence encoding a single- domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, having a sequence as set forth in any of SEQ ID NO: 25 to 32, or a sequence which is at least 80%, preferably at least 90%, more preferably at least 95% identical to a sequence as set forth in any of SEQ ID NOs: 25 to 32, preferably SEQ ID NO: 28 or 31.
  • the polynucleic acid comprises a sequence encoding binding agent comprising a single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, having a sequence as set forth in any of SEQ ID NO: 25 to 32, or a sequence which is at least 80%, preferably at least 90%, more preferably at least 95% identical to a sequence as set forth in any of SEQ ID NOs: 25 to 32, preferably SEQ ID NO: 28 or 31.
  • the polynucleic acid comprises a sequence encoding a single- domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, having a CDR1 sequence as set forth in SEQ ID NO: 1, a CDR2 sequence as set forth in SEQ ID NO: 2, and a CDR3 sequence as set forth in SEQ ID NO: 3; a CDR1 sequence as set forth in SEQ ID NO: 4, a CDR2 sequence as set forth in SEQ ID NO: 5, and a CDR3 sequence as set forth in SEQ ID NO: 6; a CDR1 sequence as set forth in SEQ ID NO: 7, a CDR2 sequence as set forth in SEQ ID NO: 8, and a CDR3 sequence as set forth in SEQ ID NO: 9; a CDR1 sequence as set forth in SEQ ID NO: 10, a CDR2 sequence as set forth in SEQ ID NO: 11, and a CDR3 sequence as set forth in SEQ ID NO: 12; a CDR1 sequence as set forth in
  • the polynucleic acid comprises a sequence encoding binding agent comprising a single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, having a CDR1 sequence as set forth in SEQ ID NO: 1, a CDR2 sequence as set forth in SEQ ID NO: 2, and a CDR3 sequence as set forth in SEQ ID NO: 3; a CDR1 sequence as set forth in SEQ ID NO: 4, a CDR2 sequence as set forth in SEQ ID NO: 5, and a CDR3 sequence as set forth in SEQ ID NO: 6; a CDR1 sequence as set forth in SEQ ID NO: 7, a CDR2 sequence as set forth in SEQ ID NO: 8, and a CDR3 sequence as set forth in SEQ ID NO: 9; a CDR1 sequence as set forth in SEQ ID NO: 10, a CDR2 sequence as set forth in SEQ ID NO: 11, and a CDR3 sequence as set forth in SEQ ID NO: 12; a sequence encoding
  • the invention relates to a vector comprising one or more polynucleic acid according to the invention as described herein.
  • Suitable vectors include cloning vectors as well as expression vectors, as described herein elsewhere. It will be understood that expression vectors are configured for expression of the polynucleic acid by operative linkage of the polynucleic acid to a suitable regulatory sequence, such as a promoter.
  • the invention relates to a host cell comprising one or more polynucleic acid according to the invention as described herein.
  • the invention relates to a host cell comprising one or more vector according to the invention as described herein. Suitable host cell are described herein elsewhere.
  • the invention relates to a pharmaceutical composition comprising a single- domain antibody according to the invention as described herein, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment.
  • the invention relates to a pharmaceutical composition comprising a binding agent according to the invention as described herein.
  • the invention relates to a pharmaceutical composition comprising a polynucleic acid according to the invention as described herein.
  • the invention relates to a pharmaceutical composition comprising a vector according to the invention as described herein.
  • the invention relates to a pharmaceutical composition comprising a host cell according to the invention as described herein. Suitable pharmaceutical compositions are described herein elsewhere.
  • compositions typically additionally comprise a pharmaceutically acceptable excipients and/or carriers, as also described herein elsewhere.
  • the pharmaceutical composition further comprises one or more additional active pharmaceutical ingredient.
  • the second (or further) active pharmaceutical ingredient may have the same, a similar, or different activity than the single-domain antibody or binding agent of the invention.
  • the second (or further) active pharmaceutical ingredient may have a activity complementary to the activity of the single- domain antibody or binding agent of the invention.
  • the second (or further) active pharmaceutical ingredient may have a activity supplementary to the activity of the single- domain antibody or binding agent of the invention.
  • the second (or further) active pharmaceutical ingredient may have a activity which is synergistic with the activity of the single-domain antibody or binding agent of the invention.
  • the second (or further) active pharmaceutical ingredient may enhance the activity of the single-domain antibody or binding agent of the invention.
  • the second (or further) active pharmaceutical ingredient has anti-inflammatory activity.
  • the second (or further) active pharmaceutical ingredient mediates lipid metabolism.
  • the second (or further) active pharmaceutical ingredient activates or suppresses PPAR, FGF19/21, AMPK, DGAT2, ACC, FASN, MPC, and/or SCD1.
  • the second (or further) active pharmaceutical ingredient mediates sugar metabolism.
  • the second (or further) active pharmaceutical ingredient activates or suppresses GLP-1R, SGLT2, KHK, and/or MPC.
  • the second (or further) active pharmaceutical ingredient mediates cholesterol metabolism. In certain embodiments, the second (or further) active pharmaceutical ingredient mediates fibrosis. In certain embodiments, the second (or further) active pharmaceutical ingredient activates or suppresses ASK1, LOXL2, and/or GAL-3.
  • the invention relates to a kit comprising a single-domain antibody according to the invention as described herein, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment.
  • the invention relates to a kit comprising a binding agent according to the invention as described herein.
  • the invention relates to a kit comprising a polynucleic acid according to the invention as described herein.
  • kits comprising a vector according to the invention as described herein.
  • the invention relates to a kit comprising a host cell according to the invention as described herein.
  • the invention relates to a kit comprising a pharmaceutical composition according to the invention as described herein.
  • kits may further comprise instructions for use.
  • kits may be diagnostic kits, pharmaceutical kits or medicament kits which are comprising a container or vial (any suitable container or vial, such as a pharmaceutically acceptable container or vial) comprising an amount of single-domain antibody (fragment), binding agent, polynucleic acid, vector, host cell, or pharmaceutical composition, and further comprising e.g.
  • kits or medicament kits may further comprise e.g. a syringe for administering the single-domain antibody (fragment), binding agent, polynucleic acid, vector, host cell, or pharmaceutical composition as described herein to a subject.
  • the invention relates to a single-domain antibody according to the invention as described herein, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, for use in (curative) therapy.
  • the invention relates to a binding agent according to the invention as described herein for use in (curative) therapy.
  • the invention relates to a polynucleic acid according to the invention as described herein for use in (curative) therapy.
  • the invention relates to a vector according to the invention as described herein for use in (curative) therapy.
  • the invention relates to a host cell according to the invention as described herein for use in (curative) therapy.
  • the invention relates to a pharmaceutical composition according to the invention as described herein for use in (curative) therapy.
  • the invention relates to the use of a single-domain antibody according to the invention as described herein, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, for the manufacture of a medicament.
  • the invention relates to the use of a binding agent according to the invention as described herein for the manufacture of a medicament.
  • the invention relates to the use of a polynucleic acid according to the invention as described herein for the manufacture of a medicament.
  • the invention relates to the use of a vector according to the invention as described herein for the manufacture of a medicament.
  • the invention relates to the use of a host cell according to the invention as described herein for the manufacture of a medicament.
  • the invention relates to the use of a pharmaceutical composition according to the invention as described herein for the manufacture of a medicament.
  • the invention relates to the use of a single-domain antibody according to the invention as described herein, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, in diagnosis.
  • the invention relates to the use of a binding agent according to the invention as described herein in diagnosis.
  • the invention relates to the use of a polynucleic acid according to the invention as described herein in diagnosis.
  • the invention relates to the use of a vector according to the invention as described herein in diagnosis.
  • the invention relates to the use of a host cell according to the invention as described herein in diagnosis.
  • the invention relates to a (curative) method of treatment comprising administering to an individual in need thereof a single-domain antibody according to the invention as described herein, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment.
  • the invention relates to a (curative) method of treatment comprising administering to an individual in need thereof a binding agent according to the invention as described herein.
  • the invention relates to a (curative) method of treatment comprising administering to an individual in need thereof a polynucleic acid according to the invention as described herein.
  • the invention relates to a (curative) method of treatment comprising administering to an individual in need thereof a vector according to the invention as described herein.
  • the invention relates to a (curative) method of treatment comprising administering to an individual in need thereof a host cell according to the invention as described herein.
  • the invention relates to a (curative) method of treatment comprising administering to an individual in need thereof a pharmaceutical composition according to the invention as described herein.
  • Administration may be in accordance with standard techniques.
  • the administration can be by any appropriate mode, including intravenous injection, subcutaneous injection, oral, parenteral, topical, nasal (e.g. via inhalation or pulmonary delivery), ophthalmic, intrathecal, intra-cerebroventricular, sublingual, rectal, vaginal, and the like. Still other techniques of formulation such as nanotechnology and aerosol and inhalant are also within the scope of this invention.
  • the invention relates to a single-domain antibody according to the invention as described herein, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, for use in (curative) treatment or diagnosis of (liver) inflammation.
  • the invention relates to a binding agent according to the invention as described herein for use in (curative) treatment or diagnosis of (liver) inflammation.
  • the invention relates to a polynucleic acid according to the invention as described herein for use in (curative) treatment or diagnosis of (liver) inflammation.
  • the invention relates to a vector according to the invention as described herein for use in (curative) treatment or diagnosis of (liver) inflammation.
  • the invention relates to a host cell according to the invention as described herein for use in (curative) treatment or diagnosis of (liver) inflammation.
  • the invention relates to a pharmaceutical composition according to the invention as described herein for use in (curative) treatment of (liver) inflammation.
  • the invention relates to the use of a single-domain antibody according to the invention as described herein, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, for the manufacture of a medicament for (curative) treatment of (liver) inflammation.
  • the invention relates to the use of a binding agent according to the invention as described herein for the manufacture of a medicament for (curative) treatment of (liver) inflammation.
  • the invention relates to the use of a polynucleic acid according to the invention as described herein for the manufacture of a medicament for (curative) treatment of (liver) inflammation.
  • the invention relates to the use of a vector according to the invention as described herein for the manufacture of a medicament for (curative) treatment of (liver) inflammation.
  • the invention relates to the use of a host cell according to the invention as described herein for the manufacture of a medicament for (curative) treatment of (liver) inflammation.
  • the invention relates to the use of a pharmaceutical composition according to the invention as described herein for the manufacture of a medicament for (curative) treatment of (liver) inflammation.
  • a further aspect of the invention relates to a single-domain antibody (or fragment thereof) or binding agent as described herein for use in diagnosing, for use as a diagnostic agent, or for use in the manufacture of a diagnostic agent or diagnostic kit, such as an in vitro diagnostic agent or kit.
  • use of a single-domain antibody (or fragment thereof) or binding agent as described herein in the manufacture of a diagnostic agent/in vitro diagnostic agent is envisaged.
  • the binding agent as described herein is for use in identifying a medical condition as described herein, such as in a sample obtained from a subject, such as from a subject suspected to be afflicted with such medical condition.
  • a nucleic acid encoding a single-domain antibody (or fragment thereof) or binding agent as described herein, or a recombinant vector comprising such nucleic acid can likewise be used in or be for use in the manufacture of a diagnostic agent or diagnostic kit, such as an in vitro diagnostic agent or kit.
  • specific binding to activated (i.e. open) Panx1 channels may underly the diagnostic potential of the single-domain antibodies of the invention as described herein.
  • the invention relates to the use of a single-domain antibody according to the invention as described herein, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, for diagnosing (liver) inflammation.
  • the invention relates to the use of a binding agent according to the invention as described herein for diagnosing (liver) inflammation.
  • the invention relates to the use of a polynucleic acid according to the invention as described herein for diagnosing of (liver) inflammation.
  • the invention relates to the use of a vector according to the invention as described herein for diagnosing (liver) inflammation.
  • the invention relates to the use of a host cell according to the invention as described herein for diagnosing (liver) inflammation.
  • a further aspect of the invention relates to methods for detecting, identifying, or diagnosing a medical condition as described herein elsewhere, such as in a sample obtained from a subject, such as from a subject suspected to be afflicted with such medical condition.
  • Such methods usually may comprise detecting, determining, assessing, assaying, identifying or measuring binding of the single-domain antibody (or fragment thereof) or binding agent with Panx1 channels.
  • Such method may further comprise the steps of obtaining a sample and contacting the sample with a single-domain antibody (or fragment thereof) or binding agent as described herein.
  • the single-domain antibody (or fragment thereof) or binding agent as described herein is comprising a detectable moiety fused to it, bound to it, coupled to it, linked to it, complexed to it, or chelated to it.
  • a “detectable moiety” in general refers to a moiety that emits a signal or is capable of emitting a signal upon adequate stimulation, or to a moiety that is capable of being detected through binding or interaction with a further molecule (e.g. a tag, such as an affinity tag, that is specifically recognized by a labelled antibody), or is detectable by any means (preferably by a non-invasive means, if detection is in vivo/ inside the human body).
  • the detectable moiety may allow for computerized composition of an image, as such the detectable moiety may be called an imaging agent.
  • Detectable moieties include fluorescence emitters, phosphorescence emitters, positron emitters, radioemitters, etc., but are not limited to emitters as such moieties also include enzymes (capable of measurably converting a substrate) and molecular tags.
  • radioemitters/radiolabels examples include 68 Ga, 110m In, 18 F, 45 Ti, 44 Sc, 47 Sc, 61 Cu, 60 Cu, 62 Cu, 66 Ga, 64 Cu, 55 Ca, 72 As, 86 Y, 90 Y, 89 Zr, 125 I, 74 Br, 75 Br, 76 Br, 77 Br, 78 Br, 111 In, 114m In, 114 In, 99m Tc, 11 C, 32 Cl, 33 Cl, 34 Cl, 123 I, 124 I, 131 I, 186 Re, 188 Re, 177 Lu, 99 Tc, 212 Bi, 213 Bi, 212 Pb, 225 Ac, 153 Sm, and 67 Ga.
  • Fluorescence emitters include cyanine dyes (e.g. Cy5, Cy5.5, Cy7, Cy7.5), FITC, TRITC, coumarin, indolenine-based dyes, benzoindolenine-based dyes, phenoxazines, BODIPY dyes, rhodamines, Si-rhodamines, Alexa dyes, and derivatives of any thereof.
  • Affinity tags such as chitin binding protein (CBP), maltose binding protein (MBP), glutathione-S-transferase (GST), poly(His) (e.g., 6x His or His6), biotin or streptavidin, such as Strep-tag®, Strep-tag II® and Twin-Strep-tag®; solubilizing tags, such as thioredoxin (TRX), poly(NANP) and SUMO; chromatography tags, such as a FLAG-tag; epitope tags, such as V5-tag, myc-tag and HA-tag; fluorescent labels or tags (i.e., fluorochromes/-phores), such as fluorescent proteins (e.g., GFP, YFP, RFP etc.); luminescent labels or tags, such as luciferase, bioluminescent or chemiluminescent compounds (such as luminal, isoluminol, theromatic acridinium ester,
  • Binding agents as describe herein and comprising a detectable moiety may for example be used for in vitro, in vivo or in situ assays (including immunoassays known per se such as ELISA, RIA, EIA and other "sandwich assays", etc.) as well as in vivo imaging purposes (such as a SPECT-scan), depending on the choice of the specific label.
  • the invention relates to a (curative) method of treatment of inflammation comprising administering to an individual in need thereof a single-domain antibody according to the invention as described herein, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment.
  • the invention relates to a (curative) method of treatment of inflammation comprising administering to an individual in need thereof a binding agent according to the invention as described herein.
  • the invention relates to a (curative) method of treatment of inflammation comprising administering to an individual in need thereof a polynucleic acid according to the invention as described herein.
  • the invention relates to a (curative) method of treatment of inflammation comprising administering to an individual in need thereof a vector according to the invention as described herein.
  • the invention relates to a (curative) method of treatment of inflammation comprising administering to an individual in need thereof a host cell according to the invention as described herein.
  • the invention relates to a (curative) method of treatment of inflammation comprising administering to an individual in need thereof a pharmaceutical composition according to the invention as described herein.
  • Administration may be in accordance with standard techniques.
  • the administration can be by any appropriate mode, including intravenous injection, subcutaneous injection, oral, parenteral, topical, nasal (e.g. via inhalation or pulmonary delivery), ophthalmic, intrathecal, intra-cerebroventricular, sublingual, rectal, vaginal, and the like. Still other techniques of formulation such as nanotechnology and aerosol and inhalant are also within the scope of this invention.
  • inflammation is hepatitis, such as described herein elsewhere.
  • inflammation is caused by is steatohepatitis.
  • inflammation is caused by non-alcoholic steatohepatitis (NASH).
  • NASH non-alcoholic steatohepatitis
  • inflammation is caused by acute liver failure.
  • inflammation is acute.
  • inflammation is chronic.
  • the invention relates to a single-domain antibody according to the invention as described herein, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, for use in (curative) treatment or diagnosis of an inflammatory (liver) disease or disorder.
  • the invention relates to a binding agent according to the invention as described herein for use in (curative) treatment or diagnosis of an inflammatory (liver) disease or disorder.
  • the invention relates to a polynucleic acid according to the invention as described herein for use in (curative) treatment or diagnosis of an inflammatory (liver) disease or disorder.
  • the invention relates to a vector according to the invention as described herein for use in (pr curative) treatment or diagnosis of an inflammatory (liver) disease or disorder.
  • the invention relates to a host cell according to the invention as described herein for use in (curative) treatment or diagnosis of an inflammatory (liver) disease or disorder.
  • the invention relates to a pharmaceutical composition according to the invention as described herein for use in (curative) treatment of an inflammatory (liver) disease or disorder.
  • the invention relates to the use of a single-domain antibody according to the invention as described herein, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, for diagnosing or for the manufacture of a medicament for (curative) treatment of an inflammatory (liver) disease or disorder.
  • the invention relates to the use of a binding agent according to the invention as described herein for diagnosing or for the manufacture of a medicament for (curative) treatment of an inflammatory (liver) disease or disorder.
  • the invention relates to the use of a polynucleic acid according to the invention as described herein for diagnosing or for the manufacture of a medicament for (curative) treatment of an inflammatory (liver) disease or disorder.
  • the invention relates to the use of a vector according to the invention as described herein for diagnosing or for the manufacture of a medicament for (curative) treatment of an inflammatory (liver) disease or disorder.
  • the invention relates to the use of a host cell according to the invention as described herein for diagnosing or for the manufacture of a medicament for (curative) treatment of an inflammatory (liver) disease or disorder.
  • the invention relates to the use of a pharmaceutical composition according to the invention as described herein for the manufacture of a medicament for (curative) treatment of an inflammatory (liver) disease or disorder.
  • the invention relates to a (curative) method of treatment of an inflammatory disease or disorder comprising administering to an individual in need thereof a single- domain antibody according to the invention as described herein, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment.
  • the invention relates to a (curative) method of treatment of an inflammatory disease or disorder comprising administering to an individual in need thereof a binding agent according to the invention as described herein.
  • the invention relates to a (curative) method of treatment of an inflammatory disease or disorder comprising administering to an individual in need thereof a polynucleic acid according to the invention as described herein.
  • the invention relates to a (curative) method of treatment of an inflammatory disease or disorder comprising administering to an individual in need thereof a vector according to the invention as described herein.
  • the invention relates to a (curative) method of treatment of an inflammatory disease or disorder comprising administering to an individual in need thereof a host cell according to the invention as described herein.
  • the invention relates to a (curative) method of treatment of an inflammatory disease or disorder comprising administering to an individual in need thereof a pharmaceutical composition according to the invention as described herein.
  • Administration may be in accordance with standard techniques. The administration can be by any appropriate mode, including intravenous injection, subcutaneous injection, oral, parenteral, topical, nasal (e.g.
  • an inflammatory disease or disorder is a disease or disorder having an inflammatory component. Such disease or disorder is characterized by (among others) inflammation, either as a cause or as a consequence (i.e. as an initiating process or as an accompanying or resulting process).
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen- binding fragment, binding domain, polynucleic acid, vector, host cell, or pharmaceutical composition according to the invention as described herein may be for use or may be used (in a method) for treating the inflammatory component of an inflammatory disease or disorder.
  • the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, binding domain, polynucleic acid, vector, host cell, or pharmaceutical composition according to the invention as described herein may be for use or may be used (in a method) for treating inflammation in an inflammatory disease or disorder.
  • the inflammatory disease or disorder is an acute inflammatory disease or disorder.
  • the inflammatory disease or disorder is a chronic inflammatory disease or disorder.
  • the inflammatory disease or disorder is selected from autoimmune disorder, atherosclerosis, allergy, myopathy, cancer, pharmacologically induced inflammation (e.g. drug overdose), toxin- induced inflammation, radiation-induced inflammation, heavy metal-induced inflammation, sepsis, pathogen-induced (i.e.
  • diabetes type 1 or type 2
  • asthma rheumatoid artritis
  • obesity rheumatoid artritis
  • encephalitis lupus
  • myositis scleroderma
  • uveitis vasculitis
  • heart disease psoriasis
  • sarcoidosis meningitis, cystic fibrosis, gout, dermatitis, nephritis, pancreatitis, fibrosis, Crohn’s disease, ankylosing spondylitis, COPD.
  • the invention relates to a single-domain antibody according to the invention as described herein, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, for use in (curative) treatment of a liver disease or disorder, preferably an inflammatory liver disease or disorder.
  • the invention relates to a binding agent according to the invention as described herein for use in (curative) treatment of a liver disease or disorder, preferably an inflammatory liver disease or disorder.
  • the invention relates to a polynucleic acid according to the invention as described herein for use in (curative) treatment of a liver disease or disorder, preferably an inflammatory liver disease or disorder.
  • the invention relates to a vector according to the invention as described herein for use in (curative) treatment of a liver disease or disorder, preferably an inflammatory liver disease or disorder.
  • the invention relates to a host cell according to the invention as described herein for use in (curative) treatment of a liver disease or disorder, preferably an inflammatory liver disease or disorder.
  • the invention relates to a pharmaceutical composition according to the invention as described herein for use in (curative) treatment of a liver disease or disorder, preferably an inflammatory liver disease or disorder.
  • the invention relates to the use of a single-domain antibody according to the invention as described herein, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, for the manufacture of a medicament for (curative) treatment of a liver disease or disorder, preferably an inflammatory liver disease or disorder.
  • a binding agent according to the invention as described herein for the manufacture of a medicament for (curative) treatment of a liver disease or disorder, preferably an inflammatory liver disease or disorder.
  • the invention relates to the use of a polynucleic acid according to the invention as described herein for the manufacture of a medicament for (curative) treatment of a liver disease or disorder, preferably an inflammatory liver disease or disorder.
  • the invention relates to the use of a vector according to the invention as described herein for the manufacture of a medicament for (curative) treatment of a liver disease or disorder, preferably an inflammatory liver disease or disorder.
  • the invention relates to the use of a host cell according to the invention as described herein for the manufacture of a medicament for (curative) treatment of a liver disease or disorder, preferably an inflammatory liver disease or disorder.
  • the invention relates to the use of a pharmaceutical composition according to the invention as described herein for the manufacture of a medicament for (curative) treatment of a liver disease or disorder, preferably an inflammatory liver disease or disorder.
  • a (curative) method of treatment of a liver disease or disorder comprising administering to an individual in need thereof a single-domain antibody according to the invention as described herein, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment.
  • the invention relates to a (curative) method of treatment of a liver disease or disorder, preferably an inflammatory liver disease or disorder, comprising administering to an individual in need thereof a binding agent according to the invention as described herein.
  • the invention relates to a (curative) method of treatment of a liver disease or disorder, preferably an inflammatory liver disease or disorder, comprising administering to an individual in need thereof a polynucleic acid according to the invention as described herein.
  • the invention relates to a (curative) method of treatment of a liver disease or disorder, preferably an inflammatory liver disease or disorder, comprising administering to an individual in need thereof a vector according to the invention as described herein.
  • the invention relates to a (curative) method of treatment of a liver disease or disorder, preferably an inflammatory liver disease or disorder, comprising administering to an individual in need thereof a host cell according to the invention as described herein.
  • the invention relates to a (curative) method of treatment of a liver disease or disorder, preferably an inflammatory liver disease or disorder, comprising administering to an individual in need thereof a pharmaceutical composition according to the invention as described herein.
  • Administration may be in accordance with standard techniques.
  • the administration can be by any appropriate mode, including intravenous injection, subcutaneous injection, oral, parenteral, topical, nasal (e.g.
  • the (inflammatory) liver disease or disorder is steatohepatitis.
  • the (inflammatory) liver disease or disorder is non-alcoholic steatohepatitis (NASH).
  • the (inflammatory) liver disease or disorder is acute liver failure.
  • the (inflammatory) liver disease or disorder is cholestatic disorder. In certain embodiments, the (inflammatory) liver disease or disorder is cholangitis. In certain embodiments, the (inflammatory) liver disease or disorder is liver fibrosis. In certain embodiments, the (inflammatory) liver disease or disorder is liver cirrhosis. In certain embodiments, the (inflammatory) liver disease or disorder is liver cancer. In certain embodiments, the (inflammatory) liver disease or disorder is non-alcoholic fatty liver disease (NAFLD). In certain embodiments, the (inflammatory) liver disease or disorder is non-alcoholic fatty liver disease (NAFLD). In certain embodiments, the (inflammatory) liver disease or disorder is fatty liver disease.
  • NAFLD non-alcoholic fatty liver disease
  • the (inflammatory) liver disease or disorder is hepatitis or hepatic inflammation. In certain embodiments, the (inflammatory) liver disease or disorder is autoimmune hepatitis. In certain embodiments, the (inflammatory) liver disease or disorder is viral hepatitis. In certain embodiments, the (inflammatory) liver disease or disorder is alcoholic hepatitis. In certain embodiments, the (inflammatory) liver disease or disorder is fatty hepatitis. In certain embodiments, the (inflammatory) liver disease or disorder is toxin-induced hepatitis. In certain embodiments, the (inflammatory) liver disease or disorder is drug-induced hepatitis. In certain embodiments, the (inflammatory) liver disease or disorder is cholestatic liver disease.
  • embodiments of the medical indications to be treated or diagnosed according to the invention are listed in Table B.
  • Table B Embodiment Medical indication 1 inflammation 2 liver inflammation 3 inflammatory liver disease 4 (inflammation caused by) chronic liver disease 5 steatohepatitis 6 non-alcoholic steatohepatitis 7 (inflammation caused by) fatty liver disease 8 (inflammation caused by) non-alcoholic fatty liver disease 9 (inflammation caused by) acute liver failure 10 (inflammation caused by) cholestatic disorder 11 (inflammation caused by) fibrosis 12 (inflammation caused by) cirrhosis 13 (inflammation caused by) liver fibrosis 14 (inflammation caused by) liver cirrhosis 15 (inflammation caused by) liver cancer 16 cholangitis 17 hepatitis 18 autoimmune hepatitis 19 alcoholic hepatitis 20 viral hepatitis 21 fatty hepatitis 22 toxin-induced hepatitis 23 drug-induced hepatitis 24 acute
  • toxin-induced inflammation radiation-induced inflammation heavy metal-induced inflammation sepsis pathogen-induced (i.e. infection) inflammation arthritis (inflammation caused by) migraine (inflammation caused by) coeliac disease inflammatory bowel disease endometriosis (inflammation caused by) diabetes (type 1 or type 2) (inflammation caused by) asthma rheumatoid artritis (inflammation caused by) obesity (inflammation caused by) Alzheimer’ disease (inflammation caused by) Parkinson’s disease encephalitis (inflammation caused by) lupus myositis (inflammation caused by) scleroderma uveitis vasculitis (inflammation caused by) heart disease (inflammation caused by) psoriasis (inflammation caused by) multiple sclerosis (inflammation caused by) sarcoidosis meningitis (inflammation caused by) cystic fibrosis 62 (inflammation caused by) gout 63 dermatitis 64 n
  • the single domain antibody (or binding agent)/medical indication combination (with reference to respectively embodiments A to X of Table A and embodiments 1 to 68 of Table B) is selected from A-1, B-1, C-1, D-1, E-1, F-1, G-1, H-1, I-1, J-1, K-1, L-1, M-1, N-1, O-1, P-1, Q-1, R-1, S- 1, T-1, U-1, V-1, W-1, X-1, A-2, B-2, C-2, D-2, E-2, F-2, G-2, H-2, I-2, J-2, K-2, L-2, M-2, N-2, O-2, P-2, Q-2, R-2, S-2, T-2, U-2, V-2, W-2, X-2, A-3, B-3, C-3, D-3, E-3, F-3, G-3, H-3, I-3, J-3, K-3, L-3, M-3, N-3, O-3, P-3, Q-3, R-3, S-3, T-3, U-3, U-3,
  • EXAMPLE 1 Novel nanobody-based inhibitors of Panx1 channel activity reduce inflammation in vitro and in a mouse model of acute liver injury 1.
  • Generation of DUBCA Panx1 cells SV40 immortalised Caribbean camel (DUBCA) (Camelus dromedarius) fibroblasts were cultured in cell culture medium, Dulbecco’s modified Eagle’s medium (low glucose, GlutaMAX, pyruvate) (Thermo Fisher Scientific, Belgium) supplemented with 10% v/v fetal bovine serum (Thermo Fisher Scientific, Belgium), 50 ⁇ g/mL streptomycin sulphate (Merck, Germany) and 7.33 I.E./mL sodium benzyl penicillin (Continental Pharma, Belgium), at 37 °C with a constant supply of 5% CO2 and split every 3 days.
  • DUBCA Panx1 cells SV40 immortalised Caribbean camel (DUB
  • pRP expression vectors encoding mPanx1 or hPanx1 were used to design pASIET vectors (Addgene plasmid 17448, Trono Lab) containing Panx1 constructs. These transfer vectors were used for lentiviral production.
  • HEK human embryonal kidney
  • Viral vectors were harvested in the cell culture medium and concentrated with ultracentrifugation at 20,000 x g for 5 minutes. After transient transfection of HEK 293T cells, lentiviral particles harbouring the mPanx1 or hPanx1 coding sequence, containing a 6x His detection tag at the carboxyterminal tail, were used to transduce DUBCA cells.
  • DUBCA cells were cultured at a density of 100,000 cells/well in 1 mL/well of cell culture medium for 24 hours and transduced with lentiviral particles at a multiplicity of infection of 20 for 72 hours. Transduction of DUBCA cells was evaluated via flow cytometry of enhanced green fluorescent protein (eGFP) expression.
  • eGFP enhanced green fluorescent protein
  • Transduced cells are referred to as DUBCA mPanx1 and DUBCA hPanx1, non-transduced cells are named as DUBCA wild-type (WT) cells in this paper.
  • DUBCA mPanx1 and DUBCA hPanx1 non-transduced cells are named as DUBCA wild-type (WT) cells in this paper.
  • WT DUBCA wild-type cells.
  • 1.1.2.1. Immunoblot analysis For immunoblotting, cells were harvested from culture flasks by dissociation with TrypLE (Thermo Fisher Scientific, Belgium).
  • Proteins were isolated by homogenising cell pellets in radioimmunoprecipitation (RIPA) buffer (Thermo Fisher Scientific, Belgium) supplemented with 1% v/v ethylenediaminetetraacetic acid (EDTA) solution (Thermo Fisher Scientific, Belgium) and 1% v/v protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Belgium). Samples were mixed and placed on ice for 20 minutes. Thereafter, cell lysates were centrifugated at 14,000 x g for 20 minutes and proteins in supernatants were collected.
  • RIPA radioimmunoprecipitation
  • EDTA ethylenediaminetetraacetic acid
  • protease and phosphatase inhibitor cocktail Thermo Fisher Scientific, Belgium
  • Protein concentrations of each sample were determined according to the bicinchoninic acid (BCA) method using a BCA Protein Assay Kit (Thermo Fisher Scientific, Belgium) with bovine serum albumin (BSA) as a standard. Proteins were fractionated on sodium dodecyl sulphate (SDS) polyacrylamide gels (Bio- Rad Laboratories, USA) and blotted afterwards onto a nitrocellulose membrane (Bio-Rad Laboratories, USA). Membranes were incubated with a primary antibody directed against Panx1 (D9M1C) (Cell Signaling Technology, USA) (Table 1) followed by incubation with an appropriate horseradish peroxidase-conjugated secondary antibody (P0448) (Dako, USA) (Table 1).
  • BCA bicinchoninic acid
  • BSA bovine serum albumin
  • Panx1 signals in DUBCA mPanx1 and DUBCA hPanx1 cells were normalised against total protein loading and expressed as relative alterations compared to DUBCA WT cells.
  • 1.1.2.2. Immunocytochemistry analysis Immunocytochemistry analysis was performed by culturing DUBCA WT, DUBCA mPanx1 and DUBCA hPanx1 cells at a density of 25,000 cells/well in 750 ⁇ L/well of cell culture medium for 24 hours.
  • Panx1 nanobodies were produced by transforming bacteria with the pRP expression vector encoding mPanx1 to obtain endotoxin free plasmid.
  • a llama (Lama glama) (Lamasté, Belgium) was immunised 4 times at biweekly intervals with 2 mg of vectors expressing the mPanx1 gene and subsequently boosted 2 times with 2.107 DUBCA cells overexpressing mPanx1.
  • mRNA from peripheral blood lymphocytes of the llama was reverse transcribed to cDNA using the SuperScript II First- Strand Synthesis System for reverse transcription polymerase chain reaction (Thermo Fisher Scientific, Belgium).
  • Nanobody coding sequences were amplified by polymerase chain reaction analysis with CALL001 (5’-GTCCTGGCTGCTCTTCTACAAGG-3’ – SEQ ID NO: 67), CALL002 (5’-GGTACGTGCTGTTGAACTGTTCC-3’ – SEQ ID NO: 68), VHH-BACK (5’-GATGTGCAGCTGCAGGAGTCTGGRGGAGG-3’ – SEQ ID NO: 69) and PMCF (5’-CTAGTGCGGCCGCTGAGGAGACGGTGACCTGGGT-3’ – SEQ ID NO: 70) primers, and ligated into a variant of a pMECS phagemid vector using PstI, NotI and XbaI restriction enzymes (Thermo Fisher Scientific, Belgium) and Golden Gate cloning strategy.
  • CALL001 5’-GTCCTGGCTGCTCTTCTACAAGG-3’ – SEQ ID NO: 67
  • CALL002 (5’-GGTACGTGCTGTT
  • Electrocompetent Escherichia coli TG1 cells were transformed with the ligated material and grown for 24 hours to construct a library of nanobodies of 107 individual transformants.
  • M13K07 helper phages were added at multiplicity of infection of 20 for 30 minutes to display the nanobodies at the tip of the phages.
  • Escherichia coli TG1 cells were grown for 24 hours and centrifuged at 2,200 x g for 30 minutes to collect virus particles.
  • Panx1 nanobodies were retrieved from the resulting library of phage displayed nanobodies by biopanning on DUBCA cells. Selection rounds on DUBCA WT and DUBCA mPanx1 cells allowed to identify single individual colonies.
  • Nanobody periplasmic expression is started from a single colony of Escherichia coli WK6 cells transformed with pMECS- derived vectors containing nanobody gene. The expression of Panx1 nanobodies and R3b23 nanobody in periplasm was induced by isopropyl- ⁇ -D-thiogalactoside (Duchefa Biochemie, Netherlands).
  • Periplasmic extracts were collected via an osmotic shock, using a TES solution containing 25% v/v diluted TES buffer (500 mM sucrose (Duchefa Biochemie, Netherlands), 200 mM Tris-HCl (Merck, Germany) and 0.50 mM EDTA (Duchefa Biochemie, Netherlands)).
  • TES solution containing 25% v/v diluted TES buffer (500 mM sucrose (Duchefa Biochemie, Netherlands), 200 mM Tris-HCl (Merck, Germany) and 0.50 mM EDTA (Duchefa Biochemie, Netherlands)).
  • IMAC immobilized metal affinity chromatography
  • His-Select Nickel Affinity Gel Merck, Germany
  • 500 mM imidazole(Merck, Germany)-mediated elution was used to capture produced nanobodies from periplasmic extracts.
  • Non- permeabilized cells were incubated with Panx1 nanobodies or R3b23 nanobody (1,500 nM) in combination with Alexa Fluor® 594-conjugated hemagglutinin antibody (901511) (BioLegend, USA) (Table 1). Next, nuclear counterstaining was performed with Hoechst solution and Vectashield was used as mounting medium. Nanobody binding was detected using a fluorescence microscope Nikon Eclipse Ti. 1.1.6. Cell-based enzyme-linked immunosorbent assay The affinity of the Panx1 nanobodies was determined in DUBCA WT and DUBCA hPanx1 cells using cell-based ELISA.
  • DUBCA WT and DUBCA hPanx1 cells were cultured at a density of 12,000 cells/well in 200 ⁇ L/well cell culture medium. Cells were grown for 24 hours and fixed with 4% w/v paraformaldehyde in PBS and incubated with different concentrations of Panx1 nanobodies and R3b23 nanobody in the range of 0- 600 nM. Horseradish peroxidase-conjugated hemagglutinin antibody (901519) (BioLegend, USA) (Table 1) was added and cells were incubated with 1-Step Ultra TMB- ELISA (Thermo Fisher Scientific, Belgium).
  • Panx1 expression in C6 WT and C6 Panx1 cells was studied using immunoblot and immunocytochemistry analyses as described in section 1.1.2.1. and 1.1.2.2, respectively.
  • Cell-based ELISA was performed using C6 WT and C6 Panx1 cells.
  • Concentrations of the Panx1 nanobodies and R3b23 nanobody up to 12,000 nM were used.
  • Specificity of the Panx1 nanobodies was assessed by measuring absorbance values at 450 nm with a FLUOstar OPTIMA microplate reader. 1.1.7.
  • Panx1 channel activity assay The inhibitory effects of the Panx1 nanobodies on Panx1 channel activity were assessed following potassium-induced channel opening in DUBCA hPanx1 cells and measurement of extracellular release ATP.
  • DUBCA hPanx1 cells were cultured at a density of 12,000 cells/well in 200 ⁇ L/well cell culture medium (Thermo Fisher Scientific, Belgium) and grown for 24 hours.
  • Cell culture medium was changed with preheated buffer (137 mM NaCl, 2.68 mM KCl, 11.90 mM NaHCO3, 0.42 mM NaH2PO4.H2O, 1 mM MgCl2, 2 mM CaCl2.2H2O, 5 mM HEPES and 0.1% w/v glucose) ((Merck, Germany) for 30 minutes at 37 °C with a constant supply of 5% CO2.
  • Panx1 channels were opened by switching to a buffer with increased potassium concentration (22.93 mM NaCl, 5 mM KCl, 5.95 mM NaHCO3, 0.21 mM NaH2PO4.H2O, 1 mM MgCl2, 2 mM CaCl2.2H2O, 5 mM HEPES and 0.1% w/v glucose).
  • Extracellular ATP levels were assessed using an ATP Bioluminescent Assay Kit (213-579-1) (Merck, Germany). The amount of emitted light by the samples was immediately measured with a VICTOR3 Multilabel Plate Reader (PerkinElmer, USA). Extracellular ATP release was expressed as the percentage of ATP relative to the release level triggered by the buffer with increased potassium concentration. 1.1.8. In vitro inflammation assay The anti-inflammatory effects of the Panx1 nanobodies were tested in RAW264.7 and differentiated THP-1 cells using flow cytometry and ELISA analyses, respectively. RAW264.7 cells were cultured at a density of 106 cells/well in 1 mL/well culture medium for 24 hours.
  • THP-1 cells were differentiated with 0.2 ⁇ M phorbol 12-myristate 13-acetate (Merck, Germany) and subsequently exposed to 1 ⁇ g/mL LPS (4 hours), Panx1 nanobodies and R3b23 nanobody (1,000 nM) (1 hour) and 5 mM ATP (30 minutes). Detection of IL-1 ⁇ quantities in supernatants was performed with a with a FLUOstar OPTIMA microplate reader. For positive controls, RAW264.7 and differentiated THP-1 cells were treated with Caspase-1 Inhibitor II (5-25 ⁇ M) (Merck, Germany) and MCC950 (10-50 ⁇ M) (Merck, Germany) for 1 hour. 1.1.9.
  • Panx1 nanobodies Specificity and affinity of Panx1 nanobodies towards mouse Panx1
  • the specificity of 4 Panx1 nanobodies was determined in DUBCA WT and DUBCA mPanx1 cells using cell-based ELISA as described in section 1.1.6.
  • binding of Panx1 nanobodies was measured via flow cytometry analysis.
  • DUBCA WT and DUBCA mPanx1 cells were harvested from culture flasks by dissociation with TrypLE. Following centrifugation at 1,500 x g for 5 minutes, 105 cells were preincubated with different concentrations of Panx1 nanobodies and R3b23 nanobody in the range of 0-600 nM.
  • Panx1 nanobody in vivo testing 1.2.1. Set-up of the mouse model of acute liver injury Male C57BL/6 mice of approximately 2 months of age were used and housed in the animal facility of the Faculty of Medicine and Pharmacy of the VUB-Belgium. Wild type (Charles River Laboratories, France) and Panx1-/- animals were kept under controlled environmental conditions with free access to food and water.
  • mice were starved 16 hours prior to acetaminophen administration.
  • Acetaminophen (APAP) (Merck, Germany) was dissolved in PBS, slightly heated and injected (37 °C) intraperitoneally at 300 mg/kg body weight.1 group of animals was not injected with APAP. After 2 hours, 1 group of APAP- overdosed mice was additionally administered either Panx1 nanobody or R3b23 nanobody diluted in PBS at 10 mg/kg body weight through intraperitoneal injection.
  • another group of acetaminophen-overdosed mice was injected with N- acetylcysteine (Merck, Germany) in PBS at 200 mg/kg body weight.
  • mice All mice were euthanised 24 hours following APAP overdosing under ketamine and xylazine-induced anesthesia. Blood, collected by cardiac puncture, was centrifugated at 2,000 x g for 10 minutes, and serum was stored at -80 °C. Livers were excised and fragments were fixed in 4% phosphate-buffered formalin (ProSan, Belgium) or snap-frozen in liquid nitrogen with storage at -80°C. This protocol has been approved by the local Ethical Committee of the VUB-Belgium (project number 20-210-8) and all animals received human care according to the criteria by the guidelines provided by the Ethical Committee of the VUB- Belgium. 1.2.2.
  • Membranes were subsequently incubated with horseradish peroxidase- conjugated secondary antibodies (P0447 and P0448) (Dako, USA) (Table 1). Detection of liver protein expression was carried out by means of enhanced chemiluminescence. For semi-quantification purposes, liver protein expression levels were normalised against total protein loading. 1.2.3. Cytokine analysis Serum cytokine levels were measured using a mouse inflammation antibody array (ab133999) (Abcam, UK). After blocking the antibody array membranes with the provided blocking buffer for 30 minutes, membranes were incubated with collected serum samples for 24 hours. Thereafter, membranes were washed with provided washing buffers for 25 minutes and treated with the supplied cocktail of biotin-conjugated antibodies.
  • cytokine levels of 40 inflammatory factors were calculated and expressed as relative alterations compared to APAP-overdosed mice. 1.2.4. Analysis of serum aminotransaminases Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were measured using an AST Assay Kit (ARG81297) and an ALT Assay Kit (ARG82198) (Bio- Connect, Netherlands), respectively.
  • AST Aspartate aminotransferase
  • ALT alanine aminotransferase
  • Serum aminotransferases levels were assayed by adding distilled water in standard/blank wells and collected serum samples into corresponding sample wells of a flat bottomed 96-well microplate (Thermo Fisher Scientific, Belgium). After adding the provided blank working reagent and sample/standard reagent, absorbance values were measured at 340 nm with a FLUOstar OPTIMA microplate reader. Absorbance values for each set of blanks, standards and samples were used to calculate AST and ALT activity. Values were expressed in U/L. 1.2.5. Evaluation of liver tissue For microscopic evaluation, formalin-fixed liver tissue was embedded in paraffin (ProSan, Belgium).
  • Tissue sections of 10 ⁇ m were cut with a SM2010 R sliding microtome (Leica, Belgium) and placed on microscope adhesive glass slides (VWR International, Belgium). Thereafter, liver tissue sections were deparaffinized in xylene (Chem-Lab, Belgium) for 30 minutes, rehydrated in ethanol (Merck, Germany) by washing the slides in a series of 100%, 90% and 70% v/v ethanol solutions for 1 minute and washed in PBS for 5 minutes. Tissue samples were stained with hematoxylin and periodic acid Schiff base (H-PAS) by treating the samples subsequently with Schiff’s reagent (Merck, Germany) and hematoxylin (ProSan, Belgium) for 5 minutes.
  • H-PAS hematoxylin and periodic acid Schiff base
  • Panx1 nanobody coding sequences were recloned into a variant of a pHEN phagemid vector and transformed into Escherichia coli WK6 cells.
  • Panx1 nanobodies and R3b23 nanobody were produced as described in in section 1.1.3. Following production, purification and quality control, nanobody solutions were concentrated to 1 mg/mL using Vivaspin 5000 MW PES centrifugal concentrators (Satorius, Belgium). Next, Panx1 nanobodies and R3b23 nanobody solutions were labeled with Technetium-99m (99mTc).
  • 99mTc-tricarbonyl precursor was prepared from a 99mMo/99mTc generator (Drytec, UK) with a lyophilisation kit (IsoLink, Netherlands). An amount of 50 ⁇ g nanobody was mixed with 500 ⁇ L 99mTc-tricarbonyl precursor and incubated for 90 minutes at 50 °C to complete conjugation. Thereafter, radiolabeled nanobodies were purified from unbound 99mTc-tricarbonyl precursor and aggregates by filtration over a NAP-5 column (Cytiva, Belgium) and a 0.22 ⁇ m membrane filter (Merck, Germany), respectively.
  • the in vivo biodistribution of the 99mTc-labelled Panx1 nanobodies were measured using single- photon emission computerized tomography/computed tomography (SPECT/CT) imaging 1 hour post-injection.
  • SPECT/CT single- photon emission computerized tomography/computed tomography
  • mice Following whole-body imaging with a MiLabs VECTor/CT-device (MILabs, Netherlands), mice were sacrificed, organs were collected, weighed and radioactivity was measured using a Canberra ⁇ -counter Cobra Inspector 5003 (Global Medical Instrumentiation, USA) to assess the percentage of injected activity per gram in each organ. This protocol has been approved by the local Ethical Committee of the VUB- Belgium (project number 21-210-1) and all animals received human care according to the criteria by the guidelines provided by the Ethical Committee of the VUB-Belgium. 1.3. Statistical analysis All data were analysed using GraphPad Prism 7 software (GraphPad Software Inc., USA) and are presented as means ⁇ standard error of the mean (SEM).
  • DUBCA cells were transduced with lentiviral vectors to express mPanx1 and hPanx1 proteins and evaluated by means of flow cytometry (Figure 1).
  • Enhanced eGFP signals in DUBCA mPanx1 and DUBCA hPanx1 cells compared to DUBCA WT were observed.
  • Immunoblot analysis of DUBCA cell lysates revealed 3 Panx1 signals between 45 kDa and 55 kDa corresponding with the 3 glycosylated Panx1 variants (Gly0/1/2) ( Figure 2). Lysates of both DUBCA mPanx1 and DUBCA hPanx1 cells showed high Panx1 protein abundance in particular of the Gly2 variant ( Figure 3).
  • Panx1 nanobody generation and in vitro characterisation A llama was immunised with vectors expressing mPanx1 gene via DNA immunisation and subsequently boosted with DUBCA cells overexpressing mPanx1. Next, cDNA from peripheral blood lymphocytes of the immunised llama was generated and used for the construction of an immune nanobody library.
  • Panx1 nanobodies by phage display and panning on DUBCA mPanx1 cells was performed to screen for Panx1 nanobodies. By doing so, 7 different Panx1 nanobody families were identified. Individual clones of each family were selected for production and further characterisation. Since mouse and human Panx1 only differ by 6 amino acids in the extracellular loop regions, it was not surprising to identify cross-reactive nanobodies by flow cytometry (Figure 5). These results were also validated by immunocytochemistry experiments ( Figure 6).
  • Panx1 nanobodies While no binding was seen for the irrelevant R3b23 nanobody, 4 Panx1 nanobodies (Nb1, Nb3, Nb16 and Nb30) showed specific binding to DUBCA hPanx1 cells in the immunocytochemistry experiments (which is less sensitive than flow cytometry and is not necessarily capable to detect conformational epitopes). Affinity of the Panx1 nanobodies was evaluated in vitro by performing cell-based ELISA. DUBCA WT and DUBCA hPanx1 cells were incubated with individual Panx1 nanobody clones and R3b23 nanobody in different concentrations. Absorbance values were measured to determine Kd values ( Figure 7).Kd values were verified by flow cytometry.
  • Panx1 nanobodies and R3b23 nanobody were evaluated in this experimental set-up in concentrations ranging from 0 nM to 10,000 nM. Unlike for the non-targeting control R3b23 nanobody, inhibitory effects were noticed for all Panx1 nanobodies. For some of these Panx1 nanobodies, in particular Nb3, a clear concentration-dependent reduction of extracellular ATP levels was observed ( Figure 11).
  • Panx1 nanobodies The anti-inflammatory potential of Panx1 nanobodies was tested in RAW264.7 and differentiated THP-1 cells. LPS and ATP were used to trigger inflammation in vitro, whereafter detection of IL-1 ⁇ was carried out to measure anti-inflammatory effects. Inflammation blockers Caspase-1 Inhibitor II and MCC950 were used as positive controls. These compounds decreased IL-1 ⁇ intensity in both cell types.
  • Panx1 nanobodies, in particular Nb9, Nb20 and Nb30 possess anti-inflammatory potential as they lowered IL- 1 ⁇ signals in RAW264.7 ( Figure 12) and differentiated THP-1 cells ( Figure 13). Table 3 presents an overview of the in vitro results obtained with Panx1 nanobodies.
  • This human-relevant model is based on overdosing mice with the analgesic drug APAP (N-acetyl-para-aminophenol, i.e. paracetamol or acetaminophen).
  • APAP N-acetyl-para-aminophenol, i.e. paracetamol or acetaminophen.
  • APAP N-acetyl-para-aminophenol, i.e. paracetamol or acetaminophen.
  • APAP N-acetyl-para-aminophenol, i.e. paracetamol or acetaminophen
  • APAP lowered liver amounts of cytochrome P450 2E1 and pro-caspase 1, but did not affect Bax and Bcl-2.
  • Nb1 and Nb3 as well as N- acetylcysteine lowered NLRP3 quantities. None of the Panx1 nanobodies was found to affect protein levels of pro-caspase 1, IL-1 ⁇ or IFN- ⁇ ( Figure 16).
  • antibody array analysis of 40 cytokines was performed on collected serum samples.
  • N- acetylcysteine was found to reduce cytokine abundance in serum of wild type APAP- overdosed mice, which could support its use as a clinical antidote for the treatment of APAP poising. Inducing effects on the serum cytokines were observed for Nb30 and control nanobody R3b23. By contrast, both Nb3 and Nb9 showed pronounced overall anti-inflammatory effects, while being somewhat more subtle for Nb1 ( Figure 17). Collected serum samples were also used to determine serum aminotransaminases levels in wild type and Panx1-/- mice. AST and ALT serum levels were higher in APAP- overdosed wild type mice.

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Abstract

The present invention relates to single-domain antibodies directed to pannexin 1 (Panxl) and capable of inhibiting Panxl channel activity. The single-domain antibodies can be used to treat or diagnose inflammation.

Description

ANTI-INFLAMMATORY PANNEXIN 1 CHANNEL INHIBITORS FIELD OF THE INVENTION The invention relates to the field of medicine. The invention in particular relates to compounds for use in therapeutic treatment and diagnosis of inflammatory diseases, such as liver diseases. BACKGROUND OF THE INVENTION Pannexin1 (Panx1) channels, composed of Panx1 proteins, form water-filled channels at the cell plasma membrane surface. They establish a pathway for paracrine communication by mediating the trafficking of a number of ions, such as potassium, and small molecules, including adenosine triphosphate (ATP), between the intracellular and extracellular environment. By doing so, Panx1 channels drive various physiological processes, yet they have been mainly studied in a pathological context. Indeed, Panx1 channels act as key players in inflammation and cell death. Various triggers occurring during induction of inflammation, including changes in extracellular potassium concentration and phosphorylation, induce opening of Panx1 channels leading to the extracellular release of ATP. Panx1 channels also participate in the clearance of compromised cells. Apoptosis is associated with the activation of caspase-3 and 7, which results in cleavage of the cytosolic carboxyterminal tail of Panx1. This evokes a constitutively open Panx1 channel configuration liberating ATP, which acts as a “find me” signal for the recruitment of macrophages. Moreover, Panx1 channels of apoptotic cells stimulate the release of metabolites that serve as “good bye” signals. In this way, Panx1 channels mediate inflammatory responses in healthy cells in the vicinity. Furthermore, Panx1 channels modulate other types of cell death, including pyroptosis. During pyroptosis, caspase 11 activates Panx1 channels by proteolytic cleavage, triggering ATP release, which in turn activates P2X7 receptors to cause cytotoxicity. Closing of Panx1 channels has been shown on many occasions to suppress inflammation. A variety of agents has been used for this purpose, such as carbenoxolone, lanthanum and, in particular, 10Panx1. The latter is a synthetic decapeptide that mimics an amino acid sequence in the second extracellular loop of the Panx1 protein. However, 10Panx1 suffers from low stability (i.e. half-life of 1.5 minute) and may affect channels other than those composed of Panx1, including connexin46 hemichannels. Translational exploration in this direction is therefore impeded by the lack of specific and in vivo- applicable Panx1 channel inhibitors. It is an objective of the present invention to address the above-mentioned shortcomings. SUMMARY OF THE INVENTION The present invention relates to single domain antibodies, in particular nanobodies, capable of specifically binding to Panx1. Advantageously, the anti-Panx1 single domain antibodies have a high specificity, are capable of blocking Panx1 channels and serve as Panx1 channel inhibitors. Moreover, the single domain antibodies as described herein have been found to possess anti-inflammatory activity and can be used in diagnosis. This is a technological breakthrough, since currently used Panx1 channel inhibitors are small molecules and peptides, lacking specificity, stability, and/or in vivo applicability. The present invention further relates to binding agents comprising the single domain antibodies described herein. The present invention further relates to polynucleic acids encoding the single domain antibodies described herein or vectors comprising such polynucleic acids. The present invention further relates to host cells comprising the single domain antibodies, polynucleic acids, vectors, or binding domains as described herein. The present invention further relates to pharmaceutical compositions comprising the single domain antibodies, polynucleic acids, vectors, binding domains, or host cells as described herein. The present invention further relates to kits comprising the single domain antibodies, polynucleic acids, vectors, binding domains, host cells, or pharmaceutical compositions as described herein. The present invention further relates to the single domain antibodies, polynucleic acids, vectors, binding domains, host cells, or pharmaceutical compositions as described herein for use in therapy or diagnosis, such as in particular anti-inflammatory therapy or diagnosis of inflammation. The present invention is in particular captured by any one or any combination of one or more of the below numbered statements 1 to 48, with any other statement and/or embodiments. 1. A single-domain antibody capable of specifically binding to Panx1. 2. The single-domain antibody according to statement 1, wherein said single-domain antibody comprises a heavy chain variable domain derived from a heavy chain antibody (VHH). 3. The single-domain antibody according to statement 2, wherein said VHH is a camelid VHH. 4. The single-domain antibody according to any of statements 1 to 3, wherein said single-domain antibody is capable of binding to an extracellular part or domain of Panx1. 5. The single-domain antibody according to statement 4, wherein said extracellular domain is an extracellular loop. 6. The single-domain antibody according to any of statements 1 to 5, wherein said single-domain antibody is capable of binding to an epitope having a sequence or comprised in a sequence as set forth in any of SEQ ID NOs: 33 to 34, or a sequence which is at least 90% identical, preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to a sequence as set forth in any of SEQ ID NOs: 33 to 34. 7. The single-domain antibody according to any of statements 1 to 6, wherein said single-domain antibody is capable of at least partially inhibiting Panx1 channel activity. 8. The single-domain antibody according to any of statements 1 to 7, wherein said single-domain antibody is capable of at least partially inhibiting extracellular release of ATP from a cell. 9. The single-domain antibody according to any of statements 1 to 8, wherein said single-domain antibody is monoclonal. 10. The single-domain antibody according to any of statements 1 to 9, wherein said single domain antibody specifically binds to Panx1 with a dissociation constant (KD) of at most 300 nM, preferably at most 100 nM. 11. The single-domain antibody according to any of statements 1 to 10, wherein said single-domain antibody comprises 4 framework regions (FR) and 3 complementarity determining regions (CDR) according to formula (I): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (I). 12. The single-domain antibody according to any of statements 1 to 11, wherein the single-domain antibody comprises CDR1 having a sequence as set forth in any of SEQ ID NOs: 1, 4, 7, 10, 13, 16, 19, or 21; CDR2 having a sequence as set forth in any of SEQ ID NOs: 2, 5, 8, 11, 14, 17, 20, or 23; and CDR3 having a sequence as set forth in any of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, or 24. 13. The single-domain antibody according to any of statements 1 to 12, wherein said single-domain antibody comprises CDR1 having a sequence as set forth in SEQ ID NO: 1, CDR2 having a sequence as set forth in SEQ ID NO: 2, and CDR3 having a sequence as set forth in SEQ ID NO: 3; CDR1 having a sequence as set forth in SEQ ID NO: 4, CDR2 having a sequence as set forth in SEQ ID NO: 5, and CDR3 having a sequence as set forth in SEQ ID NO: 6; CDR1 having a sequence as set forth in SEQ ID NO: 7, CDR2 having a sequence as set forth in SEQ ID NO: 8, and CDR3 having a sequence as set forth in SEQ ID NO: 9; CDR1 having a sequence as set forth in SEQ ID NO: 10, CDR2 having a sequence as set forth in SEQ ID NO: 11, and CDR3 having a sequence as set forth in SEQ ID NO: 12; CDR1 having a sequence as set forth in SEQ ID NO: 13, CDR2 having a sequence as set forth in SEQ ID NO: 14, and CDR3 having a sequence as set forth in SEQ ID NO: 15; CDR1 having a sequence as set forth in SEQ ID NO: 16, CDR2 having a sequence as set forth in SEQ ID NO: 17, and CDR3 having a sequence as set forth in SEQ ID NO: 18; CDR1 having a sequence as set forth in SEQ ID NO: 19, CDR2 having a sequence as set forth in SEQ ID NO: 20, and CDR3 having a sequence as set forth in SEQ ID NO: 21; or CDR1 having a sequence as set forth in SEQ ID NO: 22, CDR2 having a sequence as set forth in SEQ ID NO: 23, and CDR3 having a sequence as set forth in SEQ ID NO: 24. 14. The single-domain antibody according to any of statements 1 to 13, wherein said single-domain antibody has a sequence as set forth in any of SEQ ID NOs: 25 to 32, or a sequence which is at least 80%, preferably at least 90%, identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to a sequence as set forth in any of SEQ ID NOs: 25 to 32, or a functional/antigen-binding fragment thereof. 15. The single-domain antibody according to any of statements 1 to 14, wherein any one or more of FR1 to FR4 has a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence of FR1 to FR4 as set forth in any of SEQ ID NOs: 25 to 32, wherein FR1 to FR4 are annotated according to IMGT. 16. The single-domain antibody according to any of statements 1 to 15, wherein said single-domain antibody comprises humanized framework regions. 17. The single-domain antibody according to any of statements 1 to 16, wherein said single-domain antibody comprises one or more mutation of amino acid residues prone to in vivo modification. 18. The single-domain antibody according to statement 17, wherein said mutation results in reduced or abolished in vivo modification of said amino acid residue. 19. The single-domain antibody according to any of statements 1 to 18, wherein said single-domain antibody comprises one or more amino acid residue mutation resulting in increased thermostability. 20. A binding agent comprising a single-domain antibody according to any of statements 1 to 19. 21. The binding agent according to statement 20, wherein said single-domain antibody is fused to albumin. 22. The binding agent according to any of statements 20 to 21, wherein said single-domain antibody is fused to an Fc domain. 23. The binding agent according to any of statements 20 to 22, wherein said binding domain is PEGylated. 24. The binding agent according to any of statements 20 to 23, wherein said binding domain is PASylated. 25. A polynucleic acid encoding the single-domain antibody according to any of statements 1 to 19. 26. A polynucleic acid encoding a polypeptide comprising a sequence as set forth in any of SEQ ID NOs: 1 to 32, or a sequence which is at least 80%, preferably at last 90% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to a sequence as set forth in any of SEQ ID NOs: 1 to 32. 27. A recombinant vector comprising the polynucleic acid according to statement 25 or 26. 28. The vector according to statement 27, which is an expression vector capable of expressing the polynucleotide. 29. A host cell comprising the polynucleic acid or vector according to any of statements 25 to 28. 30. A pharmaceutical composition comprising the single-domain antibody, binding agent, polynucleic acid, vector, or host cell according to any of statements 1 to 29 and one or more pharmaceutically acceptable excipients. 31. The pharmaceutical composition according to statement 30, further comprising a second active pharmaceutical ingredient. 32. A kit comprising the single-domain antibody, binding agent, polynucleic acid, vector, host cell or pharmaceutical composition according to any of statements 1 to 31. 33. The single-domain antibody, binding agent, polynucleic acid, vector, host cell or pharmaceutical composition according to any of statements 1 to 31 for use in therapy. 34. The single-domain antibody, binding agent, polynucleic acid, vector, host cell or pharmaceutical composition according to any of statements 1 to 31 for use in therapeutic or curative treatment of inflammation. 35. The single-domain antibody, binding agent, polynucleic acid, vector, host cell or pharmaceutical composition according to any of statements 1 to 31 for use in therapeutic treatment of an inflammatory disease or disorder. 36. The single-domain antibody, binding agent, polynucleic acid, vector, host cell or pharmaceutical composition according to any of statements 1 to 31 for use in therapeutic treatment of a liver disease or disorder. 37. The single-domain antibody, binding agent, polynucleic acid, vector, host cell or pharmaceutical composition according to any of statements 1 to 31 for use in therapeutic treatment of an inflammatory liver disease or disorder. 38. The single-domain antibody, binding agent, polynucleic acid, vector, host cell or pharmaceutical composition according to any of statements 1 to 31 for use in therapeutic treatment of hepatic inflammation. 39. The single-domain antibody, binding agent, polynucleic acid, vector, host cell or pharmaceutical composition for use according to any of statements 34 to 38, wherein said inflammation or disease or disorder is acute or chronic, such as an acute or a chronic liver disease or disorder. 40. The single-domain antibody, binding agent, polynucleic acid, vector, host cell or pharmaceutical composition according to any of statements 1 to 31 for use in therapeutic treatment of hepatitis, acute liver failure, (non-alcoholic) steatohepatitis (NASH) or (non-alcoholic) fatty liver disease (NAFLD), cholestatic disorder, cholangitis, fibrotic/cirrhotic disorder or (liver) cancer. 41. The single-domain antibody, binding agent, polynucleic acid, vector, host cell or pharmaceutical composition for use according to any of statements 34 to 40, wherein said treatment comprises at least partial reduction of inflammation. 42. Use of the single-domain antibody or binding agent according to any of statements 1 to 27 for diagnosing inflammation. 43. Use of the single-domain antibody or binding agent according to any of statements 1 to 27 for diagnosing an inflammatory disease or disorder. 44. Use of the single-domain antibody or binding agent according to any of statements 1 to 27 for diagnosing a liver disease or disorder. 45. Use of the single-domain antibody or binding agent according to any of statements 1 to 27 for diagnosing an inflammatory liver disease or disorder. 46. Use of the single-domain antibody or binding agent according to any of statements 1 to 27 for diagnosing hepatic inflammation. 47. Use according to any of statements 45 to 49, wherein said inflammation or disease or disorder is acute or chronic, such as an acute or a chronic liver disease or disorder. 48. Use of the single-domain antibody or binding agent according to any of statements 1 to 27 for diagnosing hepatitis, acute liver failure, (non-alcoholic) steatohepatitis (NASH) or (non-alcoholic) fatty liver disease (NAFLD), cholestatic disorder, cholangitis, fibrotic/cirrhotic disorder or (liver) cancer. DESCRIPTION OF THE DRAWINGS Figure 1: Transduction of DUBCA cells. SV40 immortalised DUBCA wild-type (WT) cells were transduced with lentiviral vectors to express mouse Panx1 (mPanx1) or human Panx1 (hPanx1) using pASIET vectors containing Panx1 constructs (CMV-Panx-IRES- eGFP). Following transduction, cells were assessed by flow cytometry analysis for eGFP expression to confirm successful transduction. The fluorescence-activated cell sorting profile shown originates from cells grown for 2 days. Figure 2: Analysis of Panx1 expression following transduction of DUBCA cells. DUBCA cells were transduced with lentiviral vectors to express mouse Panx1 (mPanx1) or human Panx1 (hPanx1). Protein levels of Panx1 were assessed by immunoblot analysis, normalised against the total protein content and expressed as relative alteration compared to non-transduced DUBCA cells. Results were analysed by unpaired t-tests with Welch’s correction. Data were expressed as means ± standard error of the mean (SEM). (p≤0.01; p≤0.0001; n = 3; N = 3). Figure 3: Analysis of Panx1 Gly2 protein expression following transduction of DUBCA cells. DUBCA cells were transduced with lentiviral vectors to express mouse Panx1 (mPanx1) or human Panx1 (hPanx1). Protein levels of Panx1 Gly2 were assessed by immunoblot analysis, normalised against the total protein content and expressed as relative alteration compared to non-transduced DUBCA cells. Results were analysed by unpaired t-tests with Welch’s correction. Data were expressed as means ±standard error of the mean (SEM). (p≤0.01; p≤0.0001; n = 3; N = 3). Figure 4: Analysis of Panx1 protein localisation following transduction of DUBCA cells. DUBCA cells were transduced with lentiviral vectors to express mouse Panx1 (mPanx1) or human Panx1 (hPanx1). DUBCA WT, DUBCA mPanx1 and DUBCA hPanx1 cells were prepared and subjected to immunocytochemistry analysis of Panx1 (white arrows) with nuclear counterstaining. Scale bars represent 500 µm. Figure 5: Flow cytometry analysis with Panx1 nanobodies. DUBCA cells were transduced with lentiviral vectors to express mouse Panx1 (mPanx1) or human Panx1 (hPanx1). DUBCA WT, DUBCA mPanx1 and DUBCA hPanx1 cells were prepared and incubated with Panx1 nanobodies. Cells were incubated with allophycocyanin- conjugated hemagglutinin antibody and subjected to flow cytometry analysis. Gating of hemagglutinin positive cells was performed to identify Panx1 nanobodies. Figure 6: Immunocytochemistry analysis with Panx1 nanobodies. DUBCA cells were transduced with lentiviral vectors to express human Panx1 (hPanx1). DUBCA WT and DUBCA hPanx1 cells were prepared and subjected to immunocytochemistry analysis with Panx1 nanobodies or R3b23 nanobody. Specificity of Panx1 nanobodies was assessed using Alexa Fluor® 594-conjugated hemagglutinin antibody with nuclear counterstaining. Scale bars represent 500 µm. Figure 7: Cell-based enzyme-linked immunosorbent assay with Panx1 nanobodies. DUBCA cells were transduced with lentiviral vectors to express human Panx1 (hPanx1). DUBCA WT and DUBCA hPanx1 cells were prepared and subjected to cell-based ELISA with Panx1 nanobodies or R3b23 nanobody in concentrations ranging from 0 nM to 600 nM. Specificity of Panx1 nanobodies was assessed by measuring absorbance values at 450 nm (A). The equilibrium dissociation constant (Kd) of the Panx1 nanobodies (B) was determined by plotting saturation curves with specific binding values (C). Data were expressed as means ± standard error of the mean (SEM) (n = 1; N = 3). Figure 8: Analysis of Panx1 expression following transduction of C6 cells. C6 cells were transduced with lentiviral vectors to express rat Panx1 (Panx1). Protein levels of Panx1 were assessed by immunoblot analysis, normalised against the total protein content and expressed as relative alteration compared to C6 WT cells. Results were analysed by unpaired t-tests with Welch’s correction. Data were expressed as means ±standard error of the mean (SEM). (p≤0.0001; n = 3; N = 3). Figure 9: Analysis of Panx1 localisation following transduction of C6 cells. C6 cells were transduced with lentiviral vectors to express rat Panx1 (Panx1). C6 WT and C6 Panx1 cells were prepared and subjected to immunocytochemistry analysis of Panx1 (white arrows) with nuclear counterstaining. Scale bars represent 500 µm. Figure 10: Cell-based enzyme-linked immunosorbent assay with C6 cells. C6 cells were transduced with lentiviral vectors to express rat Panx1 (Panx1). C6 WT and C6 Panx1 cells were prepared and subjected to cell-based ELISA with Panx1 nanobodies or R3b23 nanobody in concentrations ranging from 0 nM to 12000 nM. Specificity of the Panx1 nanobodies was assessed by measuring absorbance values at 450 nm. Data were expressed as means ± standard error of the mean (SEM) (n = 1; N = 4). Figure 11: Analysis of extracellular release of adenosine triphosphate with Panx1 nanobodies. DUBCA hPanx1 cells were cultured and exposed during 30 minutes to buffer. Stock solutions of carbenoxolone disodium salt (CBX) (100-200 µM), lanthanum trichloride (La3+) (100-200 µM), 10Panx1 (300-400 µM), Panx1 nanobodies and R3b23 nanobody (0-10,000 nM) were prepared ex tempore in buffer with or without increased potassium concentrations. Cells were preconditioned with appropriate buffer for 15 minutes. Cells were exposed to high potassium ion levels by replacing the buffer with preheated potassium-enriched buffer for another 30 minutes. Extracellular ATP levels were measured and expressed as the percentage of ATP relative to the release level triggered by the potassium-enriched buffer. Results were analysed by parametric 1-way analysis of variance (ANOVA) followed by post hoc tests with Dunnett’s correction. Data were expressed as means ± standard error of the mean (SEM). (non-significant (ns); p≤0.05; p≤0.01; p≤0.001; p≤0.0001; n = 3; N = 4). Figure 12: Inflammation assay with RAW264.7 cells. RAW264.7 cells were cultured and consecutively exposed to 1 µg/mL LPS (4 hours), 300 ng/mL brefeldin A (3 hours), 1,000 nM Panx1 nanobodies or R3b23 nanobody (1 hour) and 5 mM ATP (30 minutes). For positive controls, cells were preconditioned with Caspase-1 Inhibitor II (5 µM) or MCC950 (50 µM) for 1 hour. IL-1β signals were measured by flow cytometry analysis, expressed as percentage of IL-1β relative to the release level triggered by LPS + ATP. Results were analysed by parametric 1-way analysis of variance (ANOVA) followed by post hoc tests with Dunnett’s correction. Data were expressed as means ± standard error of the mean (SEM). (p≤0.05; p≤0.0001; n = 2-3; N = 3). Figure 13: Inflammation assay with THP-1 cells. THP-1 cells were differentiated with phorbol 12-myristate 13-acetate and consecutively exposed to 1 µg/mL LPS (4 hours), 25 µM Caspase-1 Inhibitor II; 10 µM MCC950; 1,000 nM Panx1 nanobodies or R3b23 nanobody (1 hour) and 5 mM ATP (30 minutes). Extracellular IL-1β levels were measured by ELISA, expressed as percentage of IL-1β relative to the release level triggered by LPS + ATP. Results were analysed by parametric 1-way analysis of variance (ANOVA) followed by post hoc tests with Dunnett’s correction. Data were expressed as means ± standard error of the mean (SEM). (p≤0.05; p≤0.01; p≤0.001 p≤0.0001; n = 1; N = 3). Figure 14: Cell-based enzyme-linked immunosorbent assay with Panx1 nanobodies. DUBCA cells were transduced with lentiviral vectors to express mouse Panx1 (mPanx1). DUBCA WT and DUBCA mPanx1 cells were prepared and subjected to cell-based ELISA with Panx1 nanobodies or R3b23 nanobody in concentrations ranging from 0 nM to 600 nM. Specificity of Panx1 nanobodies was assessed by measuring absorbance values at 450 nm. Data were expressed as means ± standard error of the mean (SEM) (n = 1; N = 3). Figure 15: Flow cytometry analysis with Panx1 nanobodies. DUBCA cells were transduced with lentiviral vectors to express mouse Panx1 (mPanx1). DUBCA WT and DUBCA mPanx1 cells were prepared and incubated with Panx1 nanobodies or R3b23 nanobody in concentrations ranging from 0 to 600 nM. Cells were incubated with Alexa Fluor® 488-conjugated hemagglutinin antibody and Hoechst solution and subjected to flow cytometry analysis. Gating of Hoechst positive cells was performed to identify Panx1 nanobodies (A). The equilibrium dissociation constant (Kd) of the Panx1 nanobodies was determined by plotting saturation curves with specific binding values (B). Figure 16: Analysis of liver protein expression in acetaminophen-overdosed mice following Panx1 nanobody treatment. Wild type mice were injected with acetaminophen at 300 mg/kg body weight (APAP) or kept untreated (UTC). After 2 hours, some mice were additionally administrated either nanobody (Nb1, Nb3, Nb9, Nb30 or R3b23) at 10 mg/kg body weight or N-acetylcysteine at 200 mg/kg body weight (NAC). Sampling was performed 24 hours after acetaminophen overdosing. Liver protein levels of Bax, Bcl-2, pro-caspase-1, CYP2E1, IL-1β, IFN-γ, NLRP3 and Panx1 were assessed by immunoblot analysis, normalised against the total protein content and expressed as relative alteration compared to compared to PAR mice (n = 4 (UTC and PAR) or 12 (R3b23, Nb1, Nb3, Nb9, Nb30 and NAC)). Results were analysed by Mann-Whitney tests or parametric 1- way analysis of variance (ANOVA) followed by post hoc tests with Dunnett’s correction. Data were expressed as means ± standard error of the mean (SEM). (non-significant (ns); p≤0,05; p≤0.01; p≤0.001; p≤0.0001). Figure 17: Analysis of serum cytokines in acetaminophen-overdosed mice following Panx1 nanobody treatment. Wild type mice were injected with acetaminophen at 300 mg/kg body weight (APAP) or kept untreated (UTC). After 2 hours, some mice were additionally administrated either nanobody (Nb1, Nb3, Nb9, Nb30 or R3b23) at 10 mg/kg body weight or N-acetylcysteine at 200 mg/kg body weight (NAC). Sampling was performed 24 hours after acetaminophen overdosing. Serum cytokine levels were measured using chemiluminescence detection and densitometric analysis. Cytokine levels were normalised and expressed as relative alterations compared to PAR mice (n = 4 (UTC and PAR) or 12 (R3b23, Nb1, Nb3, Nb9, Nb30 and NAC)). Data are presented in a heat map highlighting low values (< 1) and high values (> 1). (GM-CSF: granulocyte- macrophage colony-stimulating factor; IL: interleukin; IFN-γ: interferon-γ; SDF-1: stromal cell derived factor-1; I-TAC: interferon-inducible T-cell α chemoattractant; TCA-3: T-cell activation protein 3; KC: growth-regulated α protein; TECK: thymus-expressed chemokine; TIMP: tissue inhibitor of metalloproteinase; BLC: B lymphocyte chemoattractant; LIX; lipopolysaccharide-induced CXC chemokine; CD30L; CD30 ligand; TNF-α: tumor necrosis factor α; MCP-1: monocyte chemoattractant protein 1; sTNF RI/II: soluble TNF receptor 1/2; MIG: monokine induced by interferon-γ; MIP: macrophage inflammatory protein; GCSF: granulocyte-macrophage colony-stimulating factor). Figure 18: Analysis of serum aminotransaminases in acetaminophen-overdosed mice following Panx1 nanobody treatment. Wild type and Panx1-/- mice were injected with acetaminophen at 300 mg/kg body weight (APAP) or kept untreated (UTC). After 2 hours, some wild type mice were additionally administrated either nanobody (Nb1, Nb3, Nb9, Nb30 or R3b23) at 10 mg/kg body weight or N-acetylcysteine at 200 mg/kg body weight (NAC), while some Panx1-/- mice received an additional Panx1 nanobody treatment (Nb1, Nb3, Nb9 or Nb30) at 10 mg/kg body weight or N-acetylcysteine at 200 mg/kg body weight (NAC). Sampling was performed 24 hours after acetaminophen overdosing. Serum AST and ALT activity (U/L) was calculated by measuring absorbance values for each set of blanks, standards and samples (n = 4 (Wild type) and n = 2-3 (Panx1-/-). Data were expressed as means ± standard error of the mean (SEM). Figure 19: Examination of liver tissue in acetaminophen-overdosed mice following Panx1 nanobody treatment. Wild type mice were injected with acetaminophen at 300 mg/kg body weight (APAP) or kept untreated (UTC). After 2 hours, some mice were additionally administrated either nanobody (Nb1, Nb3, Nb9, Nb30 or R3b23) at 10 mg/kg body weight or N-acetylcysteine at 200 mg/kg body weight (NAC). Sampling was performed 24 hours after acetaminophen overdosing. The percentage of necrosis was estimated by measuring the areas of necrosis on 10 µm liver sections stained with hematoxylin and periodic acid Schiff base. Liver histopathology was evaluated by using Suzuki’s score method quantifying for congestion, vacuolization and necrosis on stained sections (Table 2) (n = 4 (UTC and PAR) or 12 (R3b23, Nb1, Nb3, Nb9, Nb30 and NAC)). Results were analysed by Mann-Whitney tests or parametric 1-way analysis of variance (ANOVA) followed by post hoc tests with Dunnett’s correction. Data were expressed as means ± standard error of the mean (SEM). (p≤0,05; p≤0.01; p≤0.0001). Figure 20: In vivo biodistribution of Panx1 nanobodies. Wild type mice were intravenously injected with Technetium-99m-labeled nanobodies (Nb1, Nb3, Nb9, Nb30 or R3b23). Single-photon emission computerized tomography/computed tomography imaging was performed 1 hour after injection (A). Following imaging, mice were sacrificed and organs were collected, weighed and radioactivity was measured using a γ-counter. Uptake levels of nanobodies were determined in blood, inguinal lymphnodes, cervical lymphnodes, cervical lymphnodes, salivary glands, thymus, heart, lungs, gallbladder, liver, pancreas, spleen, kidneys, stomach, small intestine, appendix, large intestine, skin, muscle, bone, tail and brain. Panx1 protein expression was evaluated on 8 µm salivary glands and stomach sections. Sections were prepared and subjected to immunohistochemistry analysis of Panx1 with nuclear counterstaining. Scale bars represent 100 or 250 µm. (B). Results were analysed by parametric 1-way analysis of variance (ANOVA) followed by post hoc tests with Dunnett’s correction. Data were expressed as means ± standard error of the mean (SEM). (p≤0,05; p≤0.01; p≤0.001; n = 3). Figure 21: Full length sequences of single domain antibodies Nb16 (SEQ ID NO: 25), Nb31 (SEQ ID NO: 26), Nb6 (SEQ ID NO: 27), Nb9 (SEQ ID NO: 28), Nb20 (SEQ ID NO: 29), Nb30 (SEQ ID NO: 30), Nb3 (SEQ ID NO: 31), and Nb1 (SEQ ID NO: 32) aligned according to the IMGT numbering scheme (LeFranc, 2014; Frontiers in Immunology.5 (22): 1-22). DETAILED DESCRIPTION OF THE INVENTION Before the aspects and embodiments of the present invention are described, it is to be understood that this invention is not limited to particular systems and methods or combinations described, since such systems and methods and combinations may, of course, vary. It is also to be understood that the terminology used herein is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise. The terms “comprising”, “comprises” and “comprised of” as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. It will be appreciated that the terms “comprising”, “comprises” and “comprised of” as used herein comprise the terms “consisting of”, “consists” and “consists of”, as well as the terms “consisting essentially of”, “consists essentially” and “consists essentially of”. The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints. The term “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of +/-20% or less, preferably +/-10% or less, more preferably +/- 5% or less, and still more preferably +/-1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed. Whereas the terms “one or more” or “at least one”, such as one or more or at least one member(s) of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any ≥3, ≥4, ≥5, ≥6 or ≥7 etc. of said members, and up to all said members. All references cited in the present specification are hereby incorporated by reference in their entirety. In particular, the teachings of all references herein specifically referred to are incorporated by reference. Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the present invention. Standard reference works setting forth the general principles of recombinant DNA technology include Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley- Interscience, New York, 1992 (with periodic updates) (“Ausubel et al., 1992”); the series Methods in Enzymology (Academic Press, Inc.); Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press: San Diego, 1990; PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995); Harlow and Lane, eds. (1988) Antibodies, a Laboratory Manual; and Animal Cell Culture (R.I. Freshney, ed. (1987). General principles of microbiology are set forth, for example, in Davis, B. D. et al., Microbiology, 3rd edition, Harper & Row, publishers, Philadelphia, Pa. (1980). In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous. Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some, but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the appended claims, any of the claimed embodiments can be used in any combination. In the following detailed description of the invention, reference is made to the accompanying drawings that form a part hereof, and in which are shown by way of illustration only of specific embodiments in which the invention may be practiced. It is to be understood that other embodiments may be utilised, and structural or logical changes may be made without departing from the scope of the present invention. The following detailed description, therefore, is not to be taken in a limiting sense, and the scope of the present invention is defined by the appended claims. Preferred statements (features) and embodiments of this invention are set herein below. Each of the statements and embodiments of the invention so defined may be combined with any other statement and/or embodiment unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features or statements indicated as being preferred or advantageous. Panx1 is a protein belonging to the pannexin family of glycoproteins alongside Panx2 and Panx3. Pannexins predominantly exist as multimeric, typically hexameric, transmembrane channels. Panx1 comprises 4 transmembrane domains or regions, 2 extracellular loops, 1 intracellular loop, and an intracellular N-terminal and C-terminal domain/region. Human Pannexin 1 (hPanx1) may have a sequence as set forth in NCBI reference sequence NP_056183.2. Mouse Panx1 (mPanx1) may have a sequence as set forth in NCBI reference sequence NP_062355.2. A first extracellular loop of hPanx1 may comprise or consist of a sequence as set forth in SEQ ID NO: 33. A second extracellular loop of hPanx1 may comprise or consist of a sequence as set forth in SEQ ID NO: 34. Corresponding sequences of extracellular loops of Panx1 from other organisms can be derived by suitable sequence alignments, as is known in the art. In preferred embodiments, Panx1 as used herein is human Panx1 (hPanx1). Panx1 channels among others are believed to be responsible for channel-mediated ATP release. Accordingly, Panx1 channel activation, i.e. channel opening, may result in extracellular release of ATP. Vice versa, Panx1 channel inhibition, i.e. channel closing or blocking, may result in (partial or complete) reduction of extracellular release of ATP. The term “antibody” refers to an immunoglobulin (Ig) molecule or a molecule comprising an immunoglobulin (Ig) domain, which specifically binds with an antigen, as well as multimers thereof. “Antibodies” can be intact immunoglobulins or immunoreactive portions of intact immunoglobulins. The term encompasses naturally, recombinantly, semi-synthetically or synthetically produced antibodies. Hence, for example, an antibody can be present in or isolated from nature, e.g., produced or expressed natively or endogenously by a cell or tissue and optionally isolated therefrom; or an antibody can be recombinant, i.e., produced by recombinant DNA technology, and/or can be, partly or entirely, chemically or biochemically synthesised. The terms “antibody fragment”, “antigen-binding fragment”, “functional antibody fragment” and "active antibody fragment" refer to a portion of any antibody that by itself has high affinity for an antigenic determinant, or epitope, and contains one or more complementarity determining regions (CDRs) accounting for such specificity. The terms “antibody fragment” and “antigen-binding fragment” and “active antibody fragment” and “functional antibody fragment” as used herein refer to a protein or peptide comprising an immunoglobulin domain or an antigen-binding domain capable of specifically binding to an antigen. Non-limiting examples include immunoglobulin domains, Fab, F(ab)'2, scFv, heavy-light chain dimers, immunoglobulin single variable domains, Nanobodies (or VHH antibodies), domain antibodies, and single chain structures, such as a complete light chain or complete heavy chain. Typically, the functional/antigen-binding fragment comprises at least CDR1, CDR2, and CDR3. Typically, at least part of one or more framework regions or regions linking the CDRs is also present. The term “immunoglobulin (Ig) domain”, or more specifically “immunoglobulin variable domain” (abbreviated as “IVD”, also referred to herein as “variable domain”), means an immunoglobulin domain essentially consisting of four “framework regions” which are referred to in the art and herein below as “framework region 1” or “FR1”; as “framework region 2” or “FR2”; as “framework region 3” or “FR3”; and as “framework region 4” or “FR4”, respectively; which framework regions are interrupted by three “complementarity determining regions” or “CDRs”, which are referred to in the art and herein below as “complementarity determining region 1” or “CDR1”; as “complementarity determining region 2” or “CDR2”; and as “complementarity determining region 3” or “CDR3”, respectively. Thus, the general structure or sequence of an immunoglobulin variable domain can be indicated as follows: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4. It is the immunoglobulin variable domain(s) (IVDs), and in particular the CDRs therein, even more particularly CDR3 therein, that confer specificity to an antibody for the antigen by carrying the antigen- or epitope-binding site. Typically, in conventional immunoglobulins, a heavy chain variable domain (VH) and a light chain variable domain (VL) interact to form an antigen-binding site. In this case, the complementarity determining regions (CDRs) of both VH and VL contribute (although not necessarily evenly) to the antigen-binding site, i.e. a total of 6 CDRs will be involved in antigen- binding site formation. In view of the above definition, the antigen-binding domain of a conventional 4-chain antibody (such as an IgG, IgM, IgA, IgD or IgE molecule; known in the art) or of a Fab fragment, a F(ab')2 fragment, an Fv fragment such as a disulphide linked Fv or a scFv fragment, or a diabody (all known in the art) derived from such conventional 4-chain antibody, will bind to the respective epitope of an antigen by a pair of (associated) immunoglobulin domains such as light and heavy chain variable domains, i.e., by a VH-VL pair of immunoglobulin domains, which jointly bind to an epitope of the respective antigen. As used herein, the term “single-domain antibody” is an antibody (fragment) comprising a single immunoglobulin domain, in particular a single variable domain. The term “single- domain antibody may be used interchangeably with “single variable domain” or “immunoglobulin single variable domain”. A “single-domain antibody” defines molecules wherein the antigen-binding site is present on, and formed by, a single immunoglobulin domain. This sets immunoglobulin single variable domains apart from “conventional” immunoglobulins or their fragments, wherein two immunoglobulin domains, in particular two variable domains, interact to form an antigen-binding site. A single-domain antibody as used herein, refers to a protein or peptide with an amino acid sequence comprising 4 framework regions (FR) and 3 complementary determining regions (CDR) according to the format of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The antigen-binding site of an immunoglobulin single variable domain is formed by a single VH/VHH or VL domain. Hence, the antigen-binding site of an immunoglobulin single variable domain is formed by no more than three CDRs. As such, the single variable domain may be a light chain variable domain sequence (e.g., a VL-sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH-sequence or VHH sequence) or a suitable fragment thereof; as long as it is capable of forming a single antigen-binding unit (i.e., a functional antigen-binding unit that essentially consists of the single variable domain, such that the single antigen-binding domain does not need to interact with another variable domain to form a functional antigen-binding unit). In certain embodiments, the immunoglobulin single variable domains are heavy chain variable domain sequences (e.g., a VH-sequence or a VHH-sequence); more specifically, the immunoglobulin single variable domains can be heavy chain variable domain sequences that are derived from a conventional four-chain antibody or heavy chain variable domain sequences that are derived from a heavy chain antibody. For example, the immunoglobulin single variable domain may be a (single) domain antibody (or an amino acid sequence that is suitable for use as a (single) domain antibody), a variable domain of a heavy (VH) or light (VL) chain of a conventional antibody (also referred to as a “dAb”) (or an amino acid sequence that is suitable for use as a dAb) or a Nanobody (as defined herein, and including but not limited to a VHH); or any suitable fragment of any one thereof. In embodiments, the single-domain antibody may be a Nanobody (as defined herein) or a suitable fragment thereof. Note: Nanobody®, Nanobodies® and Nanoclone® are registered trademarks of Ablynx N.V. (a Sanofi Company). For a general description of Nanobodies, reference is made to the further description below, as well as to the prior art cited herein, such as e.g. described in WO2008/020079. “VHH domains”, also known as VHHs, VHH domains, VHH antibody fragments, and VHH antibodies, have originally been described as the antigen-binding immunoglobulin (Ig) (variable) domain of “heavy chain antibodies” (i.e., of “antibodies devoid of light chains”; Hamers-Casterman et al. 1993, Nature 363: 446-448). The term “VHH domain” has been chosen to distinguish these variable domains from the heavy chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as “VH domains”) and from the light chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as “VL domains”). For a further description of VHHs and Nanobody, reference is made to the review article by Muyldermans (2001. Rev Mol Biotechnol 74: 277-302), as well as to the following patent applications, which are mentioned as general background art: WO 94/04678, WO 95/04079, WO 96/34103, WO 94/25591, WO 99/37681, WO 00/40968, WO 00/43507, WO 00/65057, WO 01/40310, WO 01/44301, EP 1134231, WO 02/48193, WO 97/49805, WO 01/21817, WO 03/035694, WO 03/054016, WO 03/055527, WO 03/050531, WO 01/90190, WO 03/025020 (= EP 1433793), WO 04/041867, WO 04/041862, WO 04/041865, WO 04/041863, WO 04/062551, WO 05/044858, WO 06/40153, WO 06/079372, WO 06/122786, WO 06/122787 and WO 06/122825. As described in these references, Nanobody (in particular VHH sequences and partially humanized Nanobody) can in particular be characterized by the presence of one or more “hallmark residues” in one or more of the framework sequences. For numbering of the amino acid residues of antibodies, such as the single-domain antibodies as described herein, different numbering schemes can be applied. For example, numbering can be performed according to the AHo numbering scheme for all heavy (VH) and light chain variable domains (VL) given by Honegger & Plückthun (2001. J Mol Biol 309:657-70), as applied to VHH domains from camelids. Alternative methods for numbering the amino acid residues of VH domains, which can also be applied in an analogous manner to VHH domains, are known in the art. For example, the delineation of the FR and CDR sequences can be done by using the Kabat numbering system as applied to VHH domains from camelids by Riechmann & Muyldermans (1999. J Immunol Methods 231:25-38). It should be noted that - as is well known in the art for VH domains and for VHH domains - the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (that is, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering). This means that, generally, the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence. The total number of amino acid residues in a VH domain and a VHH domain will usually be in the range of from 110 sequences may also be suitable for the purposes described herein. The determination of the CDR regions in an antibody/immunoglobulin sequence generally depends on the algorithm/methodology applied. For example, determination of CDR regions may be done according to the designation based on contact analysis and binding site topography as described in MacCallum et al. (J. Mol. Biol. (1996) 262, 732– 745), AbM (AbM is Oxford Molecular Ltd.'s antibody modelling package as described on http://www.bioinf.org.uk/abs/index.html), Chothia (Chothia and Lesk, 1987; Mol Biol. 196:901-17), Martin (Abhinandan, and Martin. Molecular Immunology 45 (2008) 3832– 3839; as shown in http://bioinf.org.uk/abs/info.html), Kabat (Kabat et al., 1991; 5th edition, NIH publication 91-3242), or IMGT (LeFranc, 2014; Frontiers in Immunology.5 (22): 1- 22). Said annotations further include delineation of CDRs and framework regions (FRs) in immunoglobulin-domain-containing proteins, and are known methods and systems to a skilled artisan who thus can apply these annotations onto any antibody/immunoglobulin protein sequences without undue burden. Applying different methods to the same antibody/immunoglobulin sequence may give rise to different CDR amino acid sequences wherein the differences may reside in CDR sequence length and/or delineation within the antibody sequence. These CDRs can be described as the CDR sequences present in the antibodies or (functional) fragments thereof as described herein, as determined or delineated according to a well-known methodology such as according to any one of the Kabat-, Martin-, Chothia-, aHo, MacCallum et al.1996, AbM-, or IMGT-numbering scheme or method, such as preferably the IMGT numbering scheme or method. VHHs or Nbs are often classified in different families according to amino acid sequences, or even in superfamilies, as to cluster the clonally related sequences derived from the same progenitor during B cell maturation (Deschaght et al.2017, Front Immunol 8:420). This classification is often based on the CDR sequence of the VHHs or Nbs, and wherein for instance each VHH or Nb family is defined as a cluster of (clonally) related sequences with a sequence identity threshold of the CDR3 region. Within a single VHH family defined herein, the CDR3 sequence is thus identical or very similar in amino acid composition, preferably with at least 80 % identity, or at least 85% identity, or at least 90 % identity in the CDR3 sequence, resulting in VHHs or Nbs of the same family binding to the same binding site, and having the same effect such as functional effect. As outlined above, many systems or methods (Kabat, MacCallum, IMGT, AbM, Chothia, Martin) exist for numbering amino acids in immunoglobulin protein sequences, including for delineation of CDRs and framework regions (FRs) in these protein sequences. These systems or methods are known to a skilled artisan who thus can apply these systems or methods on any immunoglobulin protein sequences without undue burden. CDRs as described herein are preferably annotated or described according to the IMGT numbering scheme. The single-domain antibodies as described herein can be subjected to humanization, i.e. to increase the degree of sequence identity with the closest human germline sequence. In particular, humanized single-domain antibodies may be immunoglobulin single variable domains in which at least one amino acid residue is present (and in particular, at least one framework residue) that is and/or that corresponds to a humanizing substitution (as defined further herein). Potentially useful humanizing substitutions can be ascertained by comparing the sequence of the framework regions of a naturally occurring (i.e. wild type) sequence with the corresponding framework sequence of one or more closely related human VH sequences, after which one or more of the potentially useful humanizing substitutions (or combinations thereof) thus determined can be introduced into said single-domain antibody sequence (in any manner known per se) and the resulting humanized single-domain antibody sequences can be tested for affinity for the target, for stability, for ease and level of expression, and/or for other desired properties. In this way, by means of a limited degree of trial and error, other suitable humanizing substitutions (or suitable combinations thereof) can be determined by the skilled person. Also, based on what is described before, (the framework regions of) a single-domain antibodies may be partially humanized or fully humanized. Humanized single-domain antibodies may have several advantages, such as a reduced immunogenicity, compared to the corresponding naturally occurring single-domain antibodies. By humanized is meant mutated so that immunogenicity upon administration in human patients is minor or non-existent. The humanizing substitutions should be chosen such that the resulting humanized amino acid sequence still retains the favourable properties of the parental (non-humanized) single-domain antibody, such as the antigen-binding capacity. Based on the description provided herein, the skilled person will be able to select humanizing substitutions or suitable combinations of humanizing substitutions which optimize or achieve a desired or suitable balance between the favourable properties provided by the humanizing substitutions on the one hand and the favourable properties of naturally occurring single-domain antibodies on the other hand. Such methods are known by the skilled person. A human consensus sequence can be used as target sequence for humanization, but also other means are known in the art. One alternative includes a method wherein the skilled person aligns a number of human germline alleles, such as for instance but not limited to the alignment of IGHV3 alleles, and to use said alignment for identification of residues suitable for humanization in the target sequence. Also a subset of human germline alleles most homologous to the target sequence may be aligned as starting point to identify suitable humanisation residues. Alternatively, the single-domain antibody is analyzed to identify its closest homologue in the human alleles and used for humanisation construct design. A humanisation technique applied to single-domain antibodies, such as VHHs, may also be performed by a method comprising the replacement of specific amino acids, either alone or in combination. Said replacements may be selected based on what is known from literature, from known humanization efforts, as well as from human consensus sequences compared to the natural single-domain antibody sequences, or from the human alleles most similar to the single-domain antibody sequence of interest. As can be seen from the data on the VHH entropy and VHH variability given in Tables A-5-A-8 of WO 08/020079, some amino acid residues in the framework regions are more conserved between human and Camelidae than others. Generally, although the invention in its broadest sense is not limited thereto, any substitutions, deletions or insertions (or additions) are preferably made at positions that are less conserved. Also, generally, amino acid substitutions are preferred over amino acid deletions or insertions. For instance, a human-like class of Camelidae single domain antibodies contain the hydrophobic FR2 residues typically found in conventional antibodies of human origin or from other species, but compensating this loss in hydrophilicity by other substitutions at position 103 that substitutes the conserved tryptophan residue present in VH from double-chain antibodies. As such, peptides belonging to these two classes show a high amino acid sequence homology to human VH framework regions and said peptides might be administered to a human directly without expectation of an unwanted immune response therefrom, and without the burden of further humanisation. Indeed, some Camelidae VHH sequences display a high sequence homology to human VH framework regions and therefore said VHH might be administered to patients directly without expectation of an immune response therefrom, and without the additional burden or need of humanization. Suitable mutations, in particular substitutions, can be introduced during humanization to generate a polypeptide with reduced binding to pre-existing antibodies (reference is made for example to WO 2012/175741 and WO2015/173325), for example at least one of the positions: 11, 13, 14, 15, 40, 41, 42, 82, 82a, 82b, 83, 84, 85, 87, 88, 89, 103, or 108. The amino acid sequences and/or VHH of the invention may be suitably humanized at any framework residue(s), such as at one or more Hallmark residues or at one or more other framework residues (i.e. non-Hallmark residues) or any suitable combination thereof. Depending on the host organism used to express the single-domain antibody amino acid sequence as described herein, such deletions and/or substitutions may also be designed in such a way that one or more sites for posttranslational modification (such as one or more glycosylation sites) are removed, as will be within the ability of the person skilled in the art. Alternatively, substitutions or insertions may be designed so as to introduce one or more sites for attachment of functional groups (as described herein), for example to allow site-specific pegylation. In some cases, at least one of the typical Camelidae hallmark residues with hydrophilic characteristics at position 37, 44, 45 and/or 47 is replaced (see Table A-03 of WO2008/020079). Another example of humanization includes substitution of residues in FR1, such as position 1, 5, 11, 14, 16, and/or 28; in FR3, such as positions 73, 74, 75, 76, 78, 79, 82b, 83, 84, 93 and/or 94; and in FR4, such as position 10103, 104, 108 and/or 111 (see Tables A-05 -A08 of WO2008/020079; all numbering according to the Kabat-methodology). Humanization typically only concerns substitutions, deletions or additions, in the FR and not in the CDRs, as this could/would impact binding affinity to the target and/or potency. “Binding” means any interaction, be it direct or indirect. A direct interaction implies a contact (e.g. physical or chemical) between two binding partners. An indirect interaction means any interaction whereby the interaction partners interact in a complex of more than two molecules. An interaction can be completely indirect (e.g. two molecules are part of the same complex with the help of one or more bridging molecules but don’t bind in the absence of the bridging molecule(s)). An interaction may be partly direct or partly indirect: there is still a direct contact between two interaction partners, but such contact is e.g. not stable, and is stabilized by the interaction with one or more additional molecules. The skilled person will understand that when reference is made herein to an antibody (or (functional) fragment thereof) which specifically binds to Panx1, such antibody (or (functional) fragment thereof) is capable of specifically binding to Panx1 under physiologically relevant conditions. “Specifically binding” refers to the situation in which a molecule A is, at a certain concentration (e.g. sufficient to inhibit or neutralize a protein or process of interest) binding to a target of interest (e.g. protein) with higher affinity (e.g. at least 2-fold, 5-fold, or at least 10-fold higher affinity, e.g. at least 20-, 50- or 100-fold or more higher affinity) than the affinity with which it is possibly (if at all) binding to other targets (targets not of interest). Specific binding does not mean exclusive binding. However, specific binding does mean that a binder has a certain increased affinity or preference for one or a few of its targets. Exclusivity of binding refers to the situation in which a binder is binding only to the target of interest. The term "affinity", as used herein, generally refers to the degree to which one molecule (e.g. ligand, chemical, protein or peptide, antibody or antibody fragment) binds to another molecule (e.g. (target) protein or peptide) so as to shift the equilibrium of single molecule monomers towards a complex formed by (specific)(non- covalent) binding of the two molecules. Non-covalent interaction or binding between 2 or more binding partners may involve interactions such as van der Waals interaction, hydrogen bonding, and salt bridges. A “binding agent” generally relates to a molecule that is capable of binding to at least one other molecule, wherein said binding is preferably a specific binding, such as on a defined binding site, pocket or epitope. The binding agent may be of any nature or type and is not dependent on its origin. The binding agent may be chemically synthesized, naturally occurring, recombinantly produced (and optionally purified), as well as designed and synthetically produced (and optionally purified). Said binding agent may hence be, e.g., a small molecule, a chemical, a peptide, a polypeptide, an antibody, or any derivative of any thereof, such as a peptidomimetic, an antibody mimetic, an active fragment, a chemical derivative, among others. A functional fragment of a binding agent or a functional part of a binding agent refers to a fragment or part of that binding agent that is functionally equivalent to that binding agent. In particular, such functional fragment or part of a binding agent as described herein ideally retains one or more of the functional features of that binding agent as outlined extensively elsewhere herein. A functional fragment according to the resent invention typically comprises at least an antigen-binding fragment of a single-domain antibody as described herein. The term “fused to” or “linked to” as used herein interchangeably with “connected to”, “conjugated to”, “ligated to” refers in one aspect to “genetic fusion”, e.g., by recombinant DNA technology, as well as to “chemical and/or enzymatic conjugation” resulting in a stable covalent link between two nucleic acid molecules. The same applies for the term “inserted in”, wherein a fragment of one nucleic acid may be inserted in a second nucleic acid molecule by fusing or ligating the two sequences genetically, enzymatically or chemically. Peptides or polypeptides can likewise be fused or connected to one another, such as via peptide bonds or via linking one peptide to a side chain of an amino acid in a second peptide. It will be understood that also non-genetic fusions (e.g. through chemical linkage) are encompassed. Linkers may be used to fuse a herein identified single domain antibody (or (antigen- binding) fragment, variant, humanized form thereof, etc. as described herein) to an Fc domain such as the IgG1 and IgG2 Fc domain. A non-limiting example of a linker comprises a Gly-Ser linker such as (G4S)n, with n=1-6, preferably 2-3. With a “chimeric gene” or “chimeric construct” or “chimeric gene construct” is interchangeably meant a recombinant nucleic acid sequence in which a (gene) promoter or regulatory nucleic acid sequence is operably or operatively linked to, or associated with, a nucleic acid sequence of interest that codes for an RNA (e.g. a single-domain antibody as described herein), such that the regulatory nucleic acid sequence is able to regulate transcription or expression of the nucleic acid of interest. The operable or operative linkage in a chimeric gene between the regulatory nucleic acid sequence and the nucleic acid sequence of interest is not found in nature. An “Fc domain” as used herein refers to the fragment crystallizable region (Fc region) of a conventional antibody, which is the tail region known to interact with cell surface receptors called Fc receptors and some proteins of the complement system. Said Fc domain is composed of two identical protein fragments, derived from the second and third constant domains of the antibody's two heavy chains. All conventional antibodies comprise an Fc domain, hence, the Fc domain may be an Fc domain derived from or as a variant of the IgG, IgA and IgD antibody Fc regions, even more specifically derived from an IgG1, IgG2 or IgG4 antibody Fc region. For example, the hinge region of IgG2, may be replaced by the hinge of human IgG1 to generate single domain antibody fusion constructs, and vice versa. In addition, Fc variants with known half-life extension may be used such as the M257Y/S259T/T261E (also known as YTE) or the LS variant (M428L combined with N434S). These mutations increase the binding of the Fc domain of a conventional antibody to the neonatal receptor (FcRn). Humanized forms, include but are not limited to the IgG humanization variants known in the art, such as C-terminal deletion of Lysine, alteration or truncation in the hinge region, LALA (L234A and L235A) or LALAPG (L234A, L235A, and P329G) mutations, among other substitutions in the IgG sequence. Preferably, the Fc domain as referred to herein is an IgG Fc domain, in particular a human IgG Fc domain, such as a (human) IgG1 Fc domain or a (human) IgG2 Fc domain. The terms “protein”, “polypeptide”, and “peptide” are interchangeably used herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same; the sequential linear arrangement of the amino acids together resulting in/forming the “amino acid sequence” or “protein sequence”. A “peptide” may also be referred to as a partial amino acid sequence derived from its original protein, for instance after enzymatic (e.g. tryptic) digestion. These terms apply to naturally-occurring amino acid polymers as well as to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally occurring amino acid, such as a chemical analogue of a corresponding naturally occurring amino acid. Also included are proteins comprising one or more posttranslational modifications such as covalent addition of functional groups or proteins (such as glycosylation, phosphorylation, acetylation, ubiquitination, methylation, lipidation and nitrosylation) or such as proteolytic processing. Based on the amino acid sequence and the modifications, the atomic or molecular mass or weight of a polypeptide is expressed in (kilo)dalton (kDa). A further modification of proteins includes addition of a tag, such as a His-tag or sortag. However, peptidomimetics of such proteins/polypeptides wherein amino acid(s) and/or peptide bond(s) have been replaced by functional analogs are also encompassed by the invention as well as other than the 20 gene-encoded amino acids, such as selenocysteine. Amino acid substitutions encompass amino acid alterations in which an amino acid is replaced with a different naturally-occurring amino acid residue. Such substitutions may be classified as "conservative<1>, in which an amino acid residue contained in the wild- type protein is replaced with another naturally-occurring amino acid of similar character, for example Gly<→Ala, Val<→lle<→Leu, Asp<→Glu, Lys<→Arg, Asn<→Gln or Phe<→ Trp<→Tyr. Substitutions encompassed by the present invention may also be "non- conservative", in which an amino acid residue which is present in the wild-type protein is substituted with an amino acid with different properties, such as a naturally-occurring amino acid from a different group (e.g., substituting a charged or hydrophobic amino acid with alanine. "Similar amino acids", as used herein, refers to amino acids that have similar amino acid side chains, i.e., amino acids that have polar, non-polar or practically neutral side chains. "Non-similar amino acids", as used herein, refers to amino acids that have different amino acid side chains, for example an amino acid with a polar side chain is non-similar to an amino acid with a non-polar side chain. Polar side chains usually tend to be present on the surface of a protein where they can interact with the aqueous environment found in cells ("hydrophilic" amino acids). On the other hand, "non-polar" amino acids tend to reside within the center of the protein where they can interact with similar non-polar neighbours ("hydrophobic" amino acids"). Examples of amino acids that have polar side chains are arginine, asparagine, aspartate, cysteine, glutamine, glutamate, histidine, lysine, serine, and threonine (all hydrophilic, except for cysteine which is hydrophobic). Examples of amino acids that have non-polar side chains are alanine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, and tryptophan (all hydrophobic, except for glycine which is neutral). A “protein domain” is a distinct functional and/or structural unit in or part of a protein. Usually, a protein domain is responsible for a particular function or interaction, contributing to the overall (biological) role of a protein. Domains may exist in a variety of biological contexts, where similar domains can be found in different proteins with similar or different functions. Protein domains can have a rigid 3D- structure if confined by e.g. a number of intramolecular cysteines (e.g. cysteine-knot proteins) or can, depending on e.g. presence or absence of a bound ligand or e.g. presence or absence of a posttranslational modification, assume different 3D-conformations, or can have a less defined, more fluid 3D-structure. The terms “polynucleic acid”, "nucleotide sequence(s)", "polynucleotide(s)", "nucleic acid sequence(s)", "nucleic acid(s)", "nucleic acid molecule" are used interchangeably herein and refer to nucleotides, either ribonucleotides or deoxyribonucleotides or a combination of both, in a polymeric unbranched form of any length. Nucleic acid sequences include DNA, cDNA, genomic DNA, RNA, synthetic forms and mixed polymers, both sense and antisense strands, or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art. This term refers only to the primary structure of the molecule. Thus, this term includes double- and single-stranded DNA, and RNA. It also includes known types of modifications, for example, methylation, “caps”, and substitution of one or more of the naturally occurring nucleotides with an analog. Modifications to nucleic acids can be introduced at one or more levels: phosphate linkage modification (e.g. introduction of one or more of phosphodiester, phosphoramidate or phosphorothioate bonds), sugar modification (e.g. introduction of one or more of LNA (locked nucleic acids), 2ʹ-O-methyl, 2ʹ-O-methoxy-ethyl, 2’-fluoro, S-constrained ethyl or tricyclo-DNA) and/or non-ribose modifications (e.g. introduction of one or more of phosphorodiamidate morpholinos or peptide nucleic acids). In certain embodiments, the polynucleic acid as referred to herein is an isolated polynucleic acid. A “coding sequence” is a nucleotide sequence that can be transcribed into mRNA and/or translated into a polypeptide when placed under the control of appropriate (gene) regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5'-terminus and a translation stop codon at the 3'-terminus. A coding sequence can include, but is not limited to mRNA, cDNA, recombinant nucleotide sequences or genomic DNA, while introns may be present as well under certain circumstances. The term "sequence" when used herein relates to nucleotide sequence(s), polynucleotide(s), nucleic acid sequence(s), nucleic acid(s), nucleic acid molecule, peptides, polypeptides and proteins, depending on the context in which the term "sequence" is used. Amino acids are presented herein by their 3- or 1-lettercode nomenclature and nucleotides/nucleosides are presented by their 1-lettercode nomenclature, both as defined and provided also in the IUPAC-IUB Joint Commission on Biochemical Nomenclature (Nomenclature and Symbolism for Amino Acids and Peptides. Eur. J. Biochem.138: 9-37 (1984)); as follows: Alanine (A or Ala), Cysteine (C or Cys), Aspartic acid (D or Asp), Glutamic acid (E or Glu), Phenylalanine (F or Phe), Glycine (G or Gly), Histidine (H or His), Isoleucine (I or Ile), Lysine (K or Lys), Leucine (L or Leu), Methionine (M or Met), Asparagine (N or Asn), Proline (P or Pro), Glutamine (Q or Gln), Arginine (R or Arg), Serine (S or Ser), Threonine (T or Thr), Valine (V or Val), Tryptophan (W or Trp), and Tyrosine (Y or Tyr). Nucleotide designations (with reference to the base contained by the nucleotide) as used herein are according to the table below. A Adenine C Cytosine G Guanine T (or U) Thymine (or Uracil) R A or G Y C or T S G or C W A or T K G or T M A or C B C or G or T D A or G or T H A or C or T V A or C or G N any base A “percentage (of) sequence identity” is calculated by comparing two optimally aligned (amino acid or nucleic acid) sequences over the window of comparison, determining the number of positions at which the identical amino acid or nucleotide residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of (amino acid or nucleic acid) sequence identity. A matched position refers to a position in which identical nucleotides or amino acids occur at the same position in aligned nucleic acid sequences. To determine percent sequence identity, a target nucleic acid or amino acid sequence is compared to the identified nucleic acid or amino acid sequence using the BLAST 2 Sequences (Bl2seq) program from the stand-alone version of BLASTZ containing BLASTN and BLASTP. This stand-alone version of BLASTZ can be obtained from the U.S. government's National Center for Biotechnology Information web site (World Wide Web at ncbi.nlm.nih.gov). Instructions explaining how to use the Bl2seq program can be found in the readme file accompanying BLASTZ. BI2seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm. Preferably, BLAST sequence alignments are performed according to the standard (i.e., default) settings (i.e., at the filing date of the present application). BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. To compare two nucleic acid sequences, the options are set as follows: -i is set to a file containing the first nucleic acid sequence to be compared (e.g., C:\seq l .txt); -j is set to a file containing the second nucleic acid sequence to be compared (e.g., C:\seq2.txt); -p is set to blastn; -o is set to any desired file name (e.g., C :\output.txt); -q is set to - 1 ; -r is set to 2; and all other options are left at their default setting. The following command will generate an output file containing a comparison between two sequences: C:\B12seq -i c:\seql .txt -j c:\seq2.txt -p blastn -o c:\output.txt - q - 1 -r 2. If the target sequence shares homology with any portion of the identified sequence, then the designated output file will present those regions of homology as aligned sequences. If the target sequence does not share homology with any portion of the identified sequence, then the designated output file will not present aligned sequences. Once aligned, a length is determined by counting the number of consecutive nucleotides from the target sequence presented in alignment with the sequence from the identified sequence starting with any matched position and ending with any other matched position. A matched position is any position where an identical nucleotide is presented in both the target and identified sequences. Gaps presented in the target sequence are not counted since gaps are not nucleotides. Likewise, gaps presented in the identified sequence are not counted since target sequence nucleotides are counted, not nucleotides from the identified sequence. The percent identity over a particular length is determined by counting the number of matched positions over that length and dividing that number by the length followed by multiplying the resulting value by 100. For example, if (i) a 500-base nucleic acid target sequence is compared to a subject nucleic acid sequence, (ii) the Bl2seq program presents 200 bases from the target sequence aligned with a region of the subject sequence where the first and last bases of that 200-base region are matches, and (iii) the number of matches over those 200 aligned bases is 180, then the 500-base nucleic acid target sequence contains a length of 200 and a sequence identity over that length of 90% (i.e. , 180 / 200 x 100 = 90). It will be appreciated that different regions within a single nucleic acid target sequence that aligns with an identified sequence can each have their own percent identity. A (weighed) average of sequence identity may be applied to calculate total sequence identity (e.g. the total percentage of identity based on all partial alignment stretches combined). Alternatively, and preferably, the percent identity is determined over the entire length of the query sequence (or optionally the longer of the query or target sequence) by counting the number of matched positions over its entire length and dividing that number by the length followed by multiplying the resulting value by 100. It is noted that the percent identity value is rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2. It also is noted that the length value will always be an integer. When reference is made to a nucleic acid sequence (e.g., DNA or genomic DNA) having "substantial sequence identity to" a reference sequence or having a sequence identity of at least 80%, e.g., at least 85%, 90%, 95%, 98%> or 99%> nucleic acid sequence identity to a reference sequence, in one embodiment said nucleotide sequence is considered substantially identical to the given nucleotide sequence and can be identified using hybridisation conditions. In another embodiment, the nucleic acid sequence comprises one or more mutations compared to the given nucleotide sequence but still can be identified using stringent hybridisation conditions. “Stringent hybridisation conditions" can be used to identify nucleotide sequences, which are substantially identical to a given nucleotide sequence. Stringent conditions are sequence dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequences at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridises to a perfectly matched probe. Typically, stringent conditions will be chosen in which the salt concentration is about 0.02 molar at pH 7 and the temperature is at least 60°C. Lowering the salt concentration and/or increasing the temperature increases stringency. Stringent conditions for RNA-DNA hybridisations (Northern blots using a probe of e.g., 100 nt) are for example those which include at least one wash in 0.2X SSC at 63°C for 20min, or equivalent conditions. Stringent conditions for DNA-DNA hybridisation (Southern blots using a probe of e.g., 100 nt) are for example those which include at least one wash (usually 2) in 0.2X SSC at a temperature of at least 50°C, usually about 55°C, for 20 min, or equivalent conditions. See also Sambrook et al. (1989) and Sambrook and Russell (2001). Examples of high stringent hybridization conditions are conditions under which primarily only those nucleic acid molecules that have at least 90% or at least 95% sequence identity undergo hybridization. Such high stringent hybridization conditions are, for example: 4 x SSC at 65°C and subsequent multiple washes in 0.1 x SSC at 65°C for approximately 1 hour. The term “high stringent hybridization conditions” as used herein may also mean: hybridization at 68°C in 0.25 M sodium phosphate, pH 7.2, 7 % SDS, 1 mM EDTA and 1 % BSA for 16 hours and subsequently washing twice with 2 x SSC and 0.1 % SDS at 68°C. Preferably, hybridization takes place under stringent conditions. Less stringent hybridization conditions are, for example: hybridizing in 4 x SSC at 37 °C and subsequent multiple washing in 1 x SSC at room temperature. As used herein, when reference is made to a sequence of a domain/protein/peptide/polynucleic acid, such as a single domain antibody, CDR, or FR of the invention as described herein, “having” (a certain (minimal) percentage sequence identity with) a sequence as set forth in a particular SEQ ID NO, such terminology may be used interchangeably with the sequence of such domain/protein/peptide/polynucleic acid comprising, consisting essentially of, or consisting of (a sequence which is at least a certain (minimal) percentage identical to) a sequence as set forth in a particular SEQ ID NO. Preferably, reference to a certain percentage sequence identity or a certain amount of substitutions/insertions/deletions as used herein entails mismatches only in FRs, and not in CDRs. The term “wild-type” or “native” refers to a gene or gene product isolated from a naturally occurring source. A wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designed the “normal” or “wild-type” form of the gene or gene product. In contrast, the term “modified”, “mutant”, “engineered” or “variant” refers to a gene or gene product that displays modifications (such as a substitution, mutation or variation, deletion or addition) in sequence, post-translational modifications and/or modification of biological or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. It is noted that naturally occurring mutants or variants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product. The altered characteristics can solely reside at the sequence level, or can additionally confer altered biological and/or functional properties to the mutants or variants compared to the wild- type gene or gene product. It is understood that conservative amino acid substitutions can be introduced in a protein or polypeptide whereby such substitutions have no essential or substantial effect on the protein's activity. Preferred conservative substitutions are those fulfilling the criteria defined for an accepted point mutation in Dayhoff et al., Atlas of Protein Sequence and Structure, 5, pp.345-352 (1978 & Supp.), which is incorporated herein by reference. Examples of conservative substitutions are substitutions including but not limited to the following groups: (a) valine, glycine; (b) glycine, alanine; (c) valine, isoleucine, leucine; (d) aspartic acid, glutamic acid; (e) asparagine, glutamine; (f) serine, threonine; (g) lysine, arginine, methionine; and (h) phenylalanine, tyrosine. A conservative substitution as referred to herein may be as defined according to the BLAST algorithm. Alternatively, conservative substitutions may be described in the form of a Venn diagram (Livingstone C.D. and Barton G.J. (1993) “Protein sequence alignments: a strategy for the hierarchical analysis of residue conservation” Comput. Appl. Biosci.9: 745-756) (Taylor W.R. (1986) “The classification of amino acid conservation” J. Theor. Biol. 119; 205-218). Conservative substitutions may be made, for example according to the table below which describes a generally accepted Venn diagram grouping of amino acids. Set Sub-set Hydrophobic F W Y H K M I L V A G C Aromatic F W Y H Aliphatic I L V Polar W Y H K R E D C S T N Q Charged H K R E D Positively charged H K R Negatively charged E D Small V C A G S P T N D Tiny A G S A “homologue”, or “homologues” of a protein of interest encompass(es) proteins having amino acid substitutions, deletions and/or insertions relative to an unmodified (e.g. native, wild-type) protein of interest and having essentially or substantially similar biological and functional activity as the unmodified protein from which it is/they are derived. "Fragment" is intended to mean a portion of a polynucleotide or polypeptide. A “functional fragment” refers to a fragment of a polynucleotide or polypeptide capable of performing the same function as the polynucleotide or polypeptide it is derived from. It will be understood that, as used herein, a functional fragment of a polynucleotide refers to a polynucleotide which when translated results in a functional fragment of the polypeptide it is derived from. In the context of the present invention, functional fragments in particular are polypeptide fragments of the single-domain antibodies as described herein (or polynucleotides encoding such polypeptide fragments) capable of performing the same function as the full length single-domain antibodies, i.e. Panx1 binding. By “isolated” or “purified” is meant material that is substantially or essentially free from components that normally accompany it in its native state. For example, an “isolated polypeptide” or “purified polypeptide” refers to a polypeptide which has been isolated or purified by any suitable means from a mixture of molecules comprising the to be isolated or to be purified polypeptide of interest. An isolated or purified polypeptide or polynucleotide of interest can for instance be an immunoglobulin, antibody or nanobody, or a polynucleotide encoding an immunoglobulin, antibody or nanobody, and the mixture can be a mixture or molecules as present in a cell producing the immunoglobulin, antibody or nanobody, and/or the culture medium into which the immunoglobulin, antibody or nanobody is secreted into (likely together with other molecules secreted by the cell). The term “vector”, “vector construct”, “expression vector”, “recombinant vector” or “gene transfer vector”, as used herein, is intended to refer to a nucleic acid molecule capable of carrying another nucleic acid molecule to which it has been linked. Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g., circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art. Said vectors may include a cloning or expression vector, as well as a delivery vehicle such as a viral, lentiviral or adenoviral vector. Expression vectors may comprise plasmids as well as viral vectors and generally contain a desired coding sequence and appropriate DNA sequences necessary for the expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammal) or in in vitro expression systems. In particular, an expression vector as described herein may comprise a nucleic acid molecule as described herein comprising a nucleic acid sequence encoding an antibody or an antigen-binding fragment as described herein operably linked to at least one regulatory sequence. “Operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). Regulatory sequences are selected to direct the expression of the protein of interest, in particular the antibody or antigen-binding fragment, in a suitable host cell, and include promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences) as known to the skilled person. Hence, in embodiments, the vector includes a promoter for driving expression of the nucleic acid of interest, optionally a nucleic acid sequence encoding a signal peptide that secretes the antibody or antigen-binding fragment, and optionally a nucleic acid sequence encoding a terminator. When the expression vector is manipulated in a production strain or cell line, the vector may or may not be integrated into the genome of the host cell when introduced into the host cell. Cloning vectors are generally used to engineer and amplify a certain desired DNA fragment. Thus, a cloning vectors may contain origin of replication that matches the cell type specified by the cloning vector, and may lack functional sequences needed for expression of the desired DNA fragments. Preferably, the vector contains one or more selection markers. The choice of the selection markers may depend on the host cells of choice, although this is not critical to the present invention as is well known to persons skilled in the art. The construction of expression vectors for use in transfecting cells is also well known in the art, and thus can be accomplished via standard techniques (see, for example, Sambrook, Fritsch, and Maniatis, in: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989; Gene Transfer and Expression Protocols, pp.109-128, ed. E. J. Murray, The Humana Press Inc., Clif ton, N.J.), and the Ambion 1998 Catalog (Ambion, Austin, Tex.). One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, wherein virally-derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g., retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses). Viral vectors also include polynucleotides carried by a virus for transfection into a host cell. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Accordingly, said vector may include any vector known to the skilled person, including any suitable type, but not limited to, for instance, plasmid vectors, cosmid vectors, phage vectors, such as lambda phage, viral vectors, even more particular a lentiviral, adenoviral, AAV or baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or P1 artificial chromosomes (PAC). The choice of the vector may be dependent amongst others on the nature of the host cell of choice. Typically, expression vectors contain one or more expression cassette. An "expression cassette" comprises any nucleic acid construct capable of directing the expression of a gene/coding sequence of interest, which is operably linked to a (gene) promoter. Expression cassettes are generally DNA constructs preferably including (5’ to 3’ in the direction of transcription): a (gene) promoter region, a polynucleotide sequence of interest with a transcription initiation region, and a termination sequence including a stop signal for RNA polymerase and a polyadenylation signal; all these elements being operably or operatively linked meaning that all of these regions should be capable of operating (being expressed) in a cell, such as prokaryotic (e.g. bacterial) or eukaryotic (e.g. mammalian, yeast, insect, fungal, plant, algal) cells, when transformed into that cell. The promoter region comprising the transcription initiation region, which preferably includes the RNA polymerase binding site, and the polyadenylation signal may be native to the cell to be transformed, may be derived from an alternative source, or may be synthetic, as long as it is functional in the cell. Such expression cassettes can be constructed in e.g. a “vector” or “expression vector” (linear or circular nucleic acids, plasmids, cosmids, viral vectors, phagemids, etc.). A "host cell" or a "recombinant host cell" or "transformed cell" are terms referring to a new individual cell (or organism) arising as a result of at least one nucleic acid molecule, having been introduced into said cell. The host cell may be a prokaryotic or eukaryotic host cell. The host cell may contain the nucleic acid as an extra-chromosomally (episomal) replicating molecule, or comprises the nucleic acid integrated in the nuclear or plastid genome of the host cell, or as introduced chromosome, e.g., minichromosome. The host cell may comprise the single-domain antibody or binding agent as described herein or may comprise a polynucleotide encoding the single-domain antibody or binding agent as described herein and capable of expressing said the single-domain antibody or binding agent as described herein. As described herein elsewhere, the polynucleotide may be comprised in a vector. The host cell may comprise a (functional) fragment of the single-domain antibody or binding agent as described herein or may comprise a polynucleotide encoding such functional fragment and capable of expressing said functional fragment. The host cell may also be a recombinant host cell, which involves a cell which has been genetically modified to contain an isolated nucleic acid molecule encoding the antibody or antigen-binding fragment of the invention. Introduction of a vector in a host cell can be effected by, e.g., calcium phosphate transfection, virus infection, DEAE-dextran-mediated transfection, lipofectamin transfection or electroporation, and any person skilled in the art can select and use an introduction method suitable for the expression vector and host cell used. Representative host cells that may be used to produce said antibodies or antigen-binding fragments, include, but are not limited to, bacterial cells, yeast cells, plant cells and animal cells. Bacterial host cells suitable for production of the antibodies or antigen-binding fragment of the invention include Escherichia spp. cells, Bacillus spp. cells, Streptomyces spp. cells, Erwinia spp. cells, Klebsiella spp. cells, Serratia spp. cells, Pseudomonas spp. cells, and Salmonella spp. cells. Yeast host cells suitable for use with the invention include species within Saccharomyces, Schizosaccharomyces, Kluyveromyces, Pichia (e.g. Pichia pastoris), Hansenula (e.g. Hansenula polymorpha), Yarowia, Schwaniomyces, Schizosaccharomyces, Zygosaccharomyces and the like. Saccharomyces cerevisiae, S. carlsbergensis and K. lactis are the most commonly used yeast hosts, and are convenient fungal hosts. Animal host cells suitable for use with the invention include insect cells and mammalian cells (e.g. derived from Chinese hamster (e.g. CHO), and human cell lines, such as HeLa). Exemplary insect cell lines include, but are not limited to, Sf9 cells, baculovirus-insect cell systems (e.g. review Jarvis, Virology Volume 310, Issue 1, 25 May 2003, Pages 1-7). Alternatively, the host cells may also be (comprised in) transgenic animals or plants. A “pharmaceutical composition” refers to a composition comprising at least one therapeutically active ingredient (an active pharmaceutical ingredient) in the context of treating a disease or otherwise pathological condition, i.e. an ingredient capable of exerting a curative effect, such as the single-domain antibodies, binding agents, polynucleic acids, vectors, or host cells as described herein. It will be understood that the active ingredient preferably is present in a concentration sufficient to exert at least partially such therapeutic effect. Multiple dosages may be required to be administered to exert maximal or optimal therapeutic effect. Typically, a pharmaceutical composition comprises one or more pharmaceutically acceptable carriers and/or excipients. An “active pharmaceutical ingredient” refers to a biologically active component of a pharmaceutical composition, such as drug product (tablet, capsule, cream, injectable) that produces the intended effects. An active pharmaceutical ingredient is any component that provides pharmacological activity or other direct effect in the diagnosis, cure, mitigation, or treatment of disease, or to affect the structure or any function of the body of man or animals. By “pharmaceutically acceptable” is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the compound, in particular the single-domain antibodies or binding agents as described herein as well as the polynucleic acids, vectors or host cells etc. as described herein, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. A pharmaceutically acceptable carrier or excipient is preferably a carrier/excipient that is (relatively) non-toxic and innocuous to a patient at concentrations consistent with effective activity of the active ingredient so that any side effects ascribable to the carrier do not vitiate the beneficial effects of the active ingredient. Suitable carriers or adjuvantia typically comprise one or more of the compounds included in the following non- exhaustive list: large slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles. The term “excipient”, as used herein, is intended to include all substances which may be present in a pharmaceutical composition and which are not active ingredients but may contribute to e.g. long-term stability, or therapeutic enhancement on the active ingredient (such as by facilitating drug absorption, reducing viscosity, or enhancing solubility). Excipients may include, for example, salts, binders (e.g., lactose, dextrose, sucrose, trehalose, sorbitol, mannitol), lubricants, thickeners, surface active agents, preservatives, emulsifiers, buffer substances, stabilizing agents, flavouring agents or colorants. A “diluent”, such as in particular a “pharmaceutically acceptable diluent”, includes vehicles such as water, saline, physiological salt solutions, glycerol, ethanol, etc. Auxiliary substances such as wetting or emulsifying agents, pH buffering substances, preservatives may be included in such vehicles. A pharmaceutically effective amount of active ingredients, such as the single-domain antibodies (or fragments) or binding agents as described herein, as well as the polynucleic acids, vectors, or host cells as described herein, is preferably that amount which produces a result or exerts an influence on the particular condition being treated. The pharmaceutical composition of this invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use. When prepared as lyophilization or liquid, physiologically acceptable carrier, excipient, stabilizer need to be added into the pharmaceutical composition of the invention (Remington's Pharmaceutical Sciences 22nd edition, Ed. Allen, Loyd V, Jr. (2012). The preparation containing pharmaceutical composition of this invention should be sterilized before injection. This procedure can be done using sterile filtration membranes before or after lyophilization and reconstitution. The pharmaceutical composition can be packaged in a container or vial with sterile access port, such as an i.v. solution bottle with a rubber stopper – the pharmaceutical composition can be present as liquid, or the container or vial is filled with a liquid pharmaceutical composition that is subsequently lyophilized or dried; or can be packaged in a pre-filled syringe. The pharmaceutical composition of the invention can be administered to any patient in accordance with standard techniques. The administration can be by any appropriate mode, including oral, parenteral, topical, nasal, ophthalmic, intrathecal, intra- cerebroventricular, sublingual, rectal, vaginal, and the like. Still other techniques of formulation such as nanotechnology and aerosol and inhalant are also within the scope of this invention. The dosage and frequency of administration will depend on the age, sex and condition of the patient, concurrent administration of other drugs, counter- indications and other parameters to be taken into account by the clinician. As used herein, the terms “therapy” or “treatment” refer to the alleviation or measurable lessening of one or more symptoms or measurable markers of a pathological condition such as a disease or disorder. Measurable lessening includes any statistically significant decline in a measurable symptom or marker. Generally, the terms encompass both curative treatments and treatments directed to reduce symptoms and/or slow progression of the disease. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms associated with a disease, diminishment of extent of a disease, stabilisation of the disease, delay or slowing of the progression of a disease, amelioration or palliation of a disease, or combinations thereof. In certain embodiments, the terms may relate to therapeutic treatments. A “therapeutically effective amount” or “therapeutically effective dose” indicates an amount of active agent, in particular a single-domain antibody (or fragment), binding agent, nucleic acid, vector, host cell, or pharmaceutical composition that when administered to the subject brings about a clinical positive response with respect to a desired therapeutic treatment of the subject. In order to achieve the therapeutic effect, the active agent, in particular a single-domain antibody (or fragment), binding agent, nucleic acid, vector, host cell, or pharmaceutical composition as described herein may need to be administered to a subject multiple times, such as with an interval of 1 week or 2 weeks; the interval being dictated by the pharmacokinetic behaviour or characteristics (e.g. half-time or half-life in the subject’s circulation) of the active agent, in particular a single-domain antibody (or fragment), binding agent, nucleic acid, vector, host cell, or pharmaceutical composition. Alternatively, therapeutic treatments in which a single dose of a active agent, in particular a single-domain antibody (or fragment), binding agent, nucleic acid, vector, host cell, or pharmaceutical composition as described herein is administered to the subject is envisaged. The single dose may be in the range of 0.5 mg/kg to 25 mg/kg. The term “subject”, “individual” or “patient”, used interchangeably herein, relates to any organism such as a vertebrate, particularly any mammal, including both a human and other mammals, for whom diagnosis or therapy is desired, e.g., an animal such as a rodent, a rabbit, a cow, a sheep, a horse, a dog, a cat, a lama, a pig, or a human or non- human primate (e.g., a monkey). The rodent may be a mouse, rat, hamster, guinea pig, or chinchilla. In one embodiment, the subject is a human, a rat or a non-human primate. The subject, individual, or patient may have any gender and any age. The skilled person will understand that for instance dosing of pharmaceutical compositions may depend on age or body weight, as is known in the art. Preferably, the subject is a human. In particular embodiments, a subject is a subject, such as a human subject, with or suspected of having inflammation, or an inflammatory disorder as described herein elsewhere, also designated ”patient” or “subject” herein. In an aspect, the invention relates to a single-domain antibody capable of specifically binding to Panx1, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment. In an aspect, the invention relates to a binding agent comprising a single-domain antibody capable of specifically binding to Panx1, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment. In certain embodiments, the single domain antibody comprises a heavy chain variable domain derived from a heavy chain antibody (VHH), or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment. In certain embodiments, the single domain antibody comprises a heavy chain variable domain derived from a camelid heavy chain antibody (VHH), or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment. In certain embodiments, the camelid is a llama. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, is capable of (specifically) binding to an extracellular part or domain of Panx1. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, is capable of (specifically) binding to an extracellular part or domain of Panx1, wherein the single domain antibody comprises a heavy chain variable domain derived from a heavy chain antibody (VHH), preferably a camelid heavy chain antibody, such as a llama heavy chain antibody. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, is capable of (specifically) binding to an extracellular loop of Panx1. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, is capable of (specifically) binding to an extracellular loop of Panx1, wherein the single domain antibody comprises a heavy chain variable domain derived from a heavy chain antibody (VHH), preferably a camelid heavy chain antibody, such as a llama heavy chain antibody. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, is capable of (specifically) binding to a sequence or epitope comprised in a sequence which is at least 90% identical, preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to a sequence as set forth in SEQ ID NO: 33 or 34. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, is capable of (specifically) binding to a sequence or epitope comprised in a sequence which is at least 90% identical, preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to a sequence as set forth in SEQ ID NO: 33 or 34, wherein the single domain antibody comprises a heavy chain variable domain derived from a heavy chain antibody (VHH), preferably a camelid heavy chain antibody, such as a llama heavy chain antibody. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, is capable of (specifically) binding to a sequence or epitope comprised in a sequence as set forth in SEQ ID NO: 33 or 34. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, is capable of (specifically) binding to a sequence or epitope comprised in a sequence as set forth in SEQ ID NO: 33 or 34, wherein the single domain antibody comprises a heavy chain variable domain derived from a heavy chain antibody (VHH), preferably a camelid heavy chain antibody, such as a llama heavy chain antibody. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, (specifically) binds to Panx1 with a Kd (dissociation constant) of at most 300 nM, preferably at most 200 nM, more preferably at most 100 nM, such as at most 50 nM, at most 40 nM, at most 30 nM, at most 20 nM, or at most 10 nM. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, (specifically) binds to Panx1 with a Kd (dissociation constant) of at most 300 nM, preferably at most 200 nM, more preferably at most 100 nM, such as at most 50 nM, at most 40 nM, at most 30 nM, at most 20 nM, or at most 10 nM, wherein the single domain antibody comprises a heavy chain variable domain derived from a heavy chain antibody (VHH), preferably a camelid heavy chain antibody, such as a llama heavy chain antibody. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, (specifically) binds to an extracellular domain or region of Panx1 with a Kd (dissociation constant) of at most 300 nM, preferably at most 200 nM, more preferably at most 100 nM, such as at most 50 nM, at most 40 nM, at most 30 nM, at most 20 nM, or at most 10 nM. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, (specifically) binds to an extracellular domain or region of Panx1 with a Kd (dissociation constant) of at most 300 nM, preferably at most 200 nM, more preferably at most 100 nM, such as at most 50 nM, at most 40 nM, at most 30 nM, at most 20 nM, or at most 10 nM, wherein the single domain antibody comprises a heavy chain variable domain derived from a heavy chain antibody (VHH), preferably a camelid heavy chain antibody, such as a llama heavy chain antibody. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, (specifically) binds to an extracellular loop of Panx1 with a Kd (dissociation constant) of at most 300 nM, preferably at most 200 nM, more preferably at most 100 nM, such as at most 50 nM, at most 40 nM, at most 30 nM, at most 20 nM, or at most 10 nM. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, (specifically) binds to an extracellular loop of Panx1 with a Kd (dissociation constant) of at most 300 nM, preferably at most 200 nM, more preferably at most 100 nM, such as at most 50 nM, at most 40 nM, at most 30 nM, at most 20 nM, or at most 10 nM, wherein the single domain antibody comprises a heavy chain variable domain derived from a heavy chain antibody (VHH), preferably a camelid heavy chain antibody, such as a llama heavy chain antibody. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, (specifically) binds to a sequence or epitope comprised in a sequence which is at least 90% identical, preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to a sequence as set forth in SEQ ID NO: 33 or 34, with a Kd (dissociation constant) of at most 300 nM, preferably at most 200 nM, more preferably at most 100 nM, such as at most 50 nM, at most 40 nM, at most 30 nM, at most 20 nM, or at most 10 nM. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, (specifically) binds to a sequence or epitope comprised in a sequence which is at least 90% identical, preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to a sequence as set forth in SEQ ID NO: 33 or 34, with a Kd (dissociation constant) of at most 300 nM, preferably at most 200 nM, more preferably at most 100 nM, such as at most 50 nM, at most 40 nM, at most 30 nM, at most 20 nM, or at most 10 nM, wherein the single domain antibody comprises a heavy chain variable domain derived from a heavy chain antibody (VHH), preferably a camelid heavy chain antibody, such as a llama heavy chain antibody. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, (specifically) binds to a sequence or epitope comprised in a sequence as set forth in SEQ ID NO: 33 or 34, with a Kd (dissociation constant) of at most 300 nM, preferably at most 200 nM, more preferably at most 100 nM, such as at most 50 nM, at most 40 nM, at most 30 nM, at most 20 nM, or at most 10 nM. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, (specifically) binds to a sequence or epitope comprised in a sequence as set forth in SEQ ID NO: 33 or 34, with a Kd (dissociation constant) of at most 300 nM, preferably at most 200 nM, more preferably at most 100 nM, such as at most 50 nM, at most 40 nM, at most 30 nM, at most 20 nM, or at most 10 nM, wherein the single domain antibody comprises a heavy chain variable domain derived from a heavy chain antibody (VHH), preferably a camelid heavy chain antibody, such as a llama heavy chain antibody. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, or the binding agent is capable of at least partially inhibiting Panx1 channel activity. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, or the binding agent is capable of at least partially inhibiting Panx1 channel activity, wherein the single domain antibody comprises a heavy chain variable domain derived from a heavy chain antibody (VHH), preferably a camelid heavy chain antibody, such as a llama heavy chain antibody. In certain embodiments, the inhibition of Panx1 channel activity is at least 20%, preferably at least 30%, more preferably at least 40%, most preferably at least 50%, such as at least 60%, at least 70%, at least 80%, at least 90%, or (substantially) 100%. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, or the binding agent has a Panx1 channel activity inhibitory IC50 (half maximum inhibitory concentration) of at most 100 µM. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, or the binding agent has a Panx1 channel activity inhibitory IC50 (half maximum inhibitory concentration) ranging from 100 µM to 100 nM. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, is capable of at least partially inhibiting extracellular release of ATP from a cell. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, is capable of at least partially inhibiting extracellular release of ATP from a cell, wherein the single domain antibody comprises a heavy chain variable domain derived from a heavy chain antibody (VHH), preferably a camelid heavy chain antibody, such as a llama heavy chain antibody. In certain embodiments, the inhibition of extracellular release of ATP from a cell is at least 20%, preferably at least 30%, more preferably at least 40%, most preferably at least 50%, such as at least 60%, at least 70%, at least 80%, at least 90%, or (substantially) 100%. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, or the binding agent has an extracellular ATP release inhibitory IC50 (half maximum inhibitory concentration) of at most 100 µM. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, or the binding agent has an extracellular ATP release inhibitory IC50 (half maximum inhibitory concentration) ranging from 100 µM to 100 nM. In certain embodiments, the single-domain antibody comprises 4 framework regions (FR) and 3 complementarity determining regions (CDR) according to formula (I): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (I). In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, comprises CDR1 having a sequence as set forth in any of SEQ ID NOs: 1, 4, 7, 10, 13, 16, 19, or 21; CDR2 having a sequence as set forth in any of SEQ ID NOs: 2, 5, 8, 11, 14, 17, 20, or 23; and/or CDR3 having a sequence as set forth in any of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, or 24. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, comprises CDR1 having a sequence as set forth in any of SEQ ID NOs: 1, 4, 7, 10, 13, 16, 19, or 21. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, comprises CDR2 having a sequence as set forth in any of: 2, 5, 8, 11, 14, 17, 20, or 23. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, comprises CDR3 having a sequence as set forth in any of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, or 24. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, comprises CDR1 having a sequence as set forth in any of SEQ ID NOs: 1, 4, 7, 10, 13, 16, 19, or 21; CDR2 having a sequence as set forth in any of SEQ ID NOs: 2, 5, 8, 11, 14, 17, 20, or 23; and CDR3 having a sequence as set forth in any of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, or 24. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, comprises CDR1 having a sequence as set forth in SEQ ID NO: 1, CDR2 having a sequence as set forth in SEQ ID NO: 2, and CDR3 having a sequence as set forth in SEQ ID NO: 3; CDR1 having a sequence as set forth in SEQ ID NO: 4, CDR2 having a sequence as set forth in SEQ ID NO: 5, and CDR3 having a sequence as set forth in SEQ ID NO: 6; CDR1 having a sequence as set forth in SEQ ID NO: 7, CDR2 having a sequence as set forth in SEQ ID NO: 8, and CDR3 having a sequence as set forth in SEQ ID NO: 9; CDR1 having a sequence as set forth in SEQ ID NO: 10, CDR2 having a sequence as set forth in SEQ ID NO: 11, and CDR3 having a sequence as set forth in SEQ ID NO: 12; CDR1 having a sequence as set forth in SEQ ID NO: 13, CDR2 having a sequence as set forth in SEQ ID NO: 14, and CDR3 having a sequence as set forth in SEQ ID NO: 15; CDR1 having a sequence as set forth in SEQ ID NO: 16, CDR2 having a sequence as set forth in SEQ ID NO: 17, and CDR3 having a sequence as set forth in SEQ ID NO: 18; CDR1 having a sequence as set forth in SEQ ID NO: 19, CDR2 having a sequence as set forth in SEQ ID NO: 20, and CDR3 having a sequence as set forth in SEQ ID NO: 21; or CDR1 having a sequence as set forth in SEQ ID NO: 22, CDR2 having a sequence as set forth in SEQ ID NO: 23, and CDR3 having a sequence as set forth in SEQ ID NO: 24. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, comprises CDR1 having a sequence as set forth in SEQ ID NO: 1; CDR2 having a sequence as set forth in SEQ ID NO: 2; and CDR3 having a sequence as set forth in SEQ ID NO: 3. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, comprises CDR1 having a sequence as set forth in SEQ ID NO: 4; CDR2 having a sequence as set forth in SEQ ID NO: 5; and CDR3 having a sequence as set forth in SEQ ID NO: 6. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, comprises CDR1 having a sequence as set forth in SEQ ID NO: 7; CDR2 having a sequence as set forth in SEQ ID NO: 8; and CDR3 having a sequence as set forth in SEQ ID NO: 9. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, comprises CDR1 having a sequence as set forth in SEQ ID NO: 10; CDR2 having a sequence as set forth in SEQ ID NO: 11; and CDR3 having a sequence as set forth in SEQ ID NO: 12. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, comprises CDR1 having a sequence as set forth in SEQ ID NO: 13; CDR2 having a sequence as set forth in SEQ ID NO: 14; and CDR3 having a sequence as set forth in SEQ ID NO: 15. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, comprises CDR1 having a sequence as set forth in SEQ ID NO: 16; CDR2 having a sequence as set forth in SEQ ID NO: 17; and CDR3 having a sequence as set forth in SEQ ID NO: 18. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, comprises CDR1 having a sequence as set forth in SEQ ID NO: 19; CDR2 having a sequence as set forth in SEQ ID NO: 20; and CDR3 having a sequence as set forth in SEQ ID NO: 21. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, comprises CDR1 having a sequence as set forth in SEQ ID NO: 22; CDR2 having a sequence as set forth in SEQ ID NO: 23; and CDR3 having a sequence as set forth in SEQ ID NO: 24. In a preferred embodiment, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, comprises CDR1 having a sequence as set forth in SEQ ID NO: 19; CDR2 having a sequence as set forth in SEQ ID NO: 20; and CDR3 having a sequence as set forth in SEQ ID NO: 21. In another preferred embodiment, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, comprises CDR1 having a sequence as set forth in SEQ ID NO: 10; CDR2 having a sequence as set forth in SEQ ID NO: 11; and CDR3 having a sequence as set forth in SEQ ID NO: 12. In certain embodiments, the single-domain antibody comprises one or more of FR1 to FR4 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence of FR1 to FR4 as set forth in any of SEQ ID NOs: 25 to 32, wherein FR1 to FR4 are annotated according to IMGT. In certain embodiments, the single-domain antibody comprises one or more of FR1 to FR4 having a sequence of FR1 to FR4 as set forth in any of SEQ ID NOs: 25 to 32, wherein FR1 to FR4 are annotated according to IMGT. In certain embodiments, the single-domain antibody comprises one or more of FR1 to FR4 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence of FR1 to FR4 as set forth in Figure 21. In certain embodiments, the single-domain antibody comprises one or more of FR1 to FR4 having a sequence of FR1 to FR4 as set forth in Figure 21. In certain embodiments, the single-domain antibody comprises one or more of FR1 to FR4 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence of FR1 to FR4 as set forth in any of SEQ ID NOs: 35 to 66. In certain embodiments, the single-domain antibody comprises one or more of FR1 to FR4 having a sequence of FR1 to FR4 as set forth in any of SEQ ID NOs: 35 to 66. In certain embodiments, the single-domain antibody comprises FR1 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in any of SEQ ID NOs: 35, 39, 43, 47, 51, 55, 59, or 63. In certain embodiments, the single-domain antibody comprises FR1 having a sequence as set forth in any of SEQ ID NOs: 35, 39, 43, 47, 51, 55, 59, or 63. In certain embodiments, the single-domain antibody comprises FR2 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in any of SEQ ID NOs: 36, 40, 44, 48, 52, 56, 60, or 64. In certain embodiments, the single-domain antibody comprises FR1 having a sequence as set forth in any of SEQ ID NOs: 36, 40, 44, 48, 52, 56, 60, or 64. In certain embodiments, the single-domain antibody comprises FR3 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in any of SEQ ID NOs: 37, 41, 45, 49, 53, 57, 61, or 65. In certain embodiments, the single-domain antibody comprises FR1 having a sequence as set forth in any of SEQ ID NOs: 37, 41, 45, 49, 53, 57, 61, or 65. In certain embodiments, the single-domain antibody comprises FR4 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in any of SEQ ID NOs: 38, 42, 46, 50, 54, 58, 62, or 66. In certain embodiments, the single-domain antibody comprises FR1 having a sequence as set forth in any of SEQ ID NOs: 38, 42, 46, 50, 54, 58, 62, or 66. In certain embodiments, the single-domain antibody comprises FR1 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in any of SEQ ID NOs: 35, 39, 43, 47, 51, 55, 59, or 63, preferably a sequence as set forth in any of SEQ ID NOs: 35, 39, 43, 47, 51, 55, 59, or 63; FR2 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in any of SEQ ID NOs: 36, 40, 44, 48, 52, 56, 60, or 64, preferably a sequence as set forth in any of SEQ ID NOs: 36, 40, 44, 48, 52, 56, 60, or 64; FR3 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in any of SEQ ID NOs: 37, 41, 45, 49, 53, 57, 61, or 65, preferably a sequence as set forth in any of SEQ ID NOs: 37, 41, 45, 49, 53, 57, 61, or 65; and/or FR4 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in any of SEQ ID NOs: 38, 42, 46, 50, 54, 58, 62, or 66, preferably a sequence as set forth in any of SEQ ID NOs: 38, 42, 46, 50, 54, 58, 62, or 66. In certain embodiments, the single-domain antibody comprises FR1 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in any of SEQ ID NOs: 35, 39, 43, 47, 51, 55, 59, or 63, preferably a sequence as set forth in any of SEQ ID NOs: 35, 39, 43, 47, 51, 55, 59, or 63; FR2 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in any of SEQ ID NOs: 36, 40, 44, 48, 52, 56, 60, or 64, preferably a sequence as set forth in any of SEQ ID NOs: 36, 40, 44, 48, 52, 56, 60, or 64; FR3 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in any of SEQ ID NOs: 37, 41, 45, 49, 53, 57, 61, or 65, preferably a sequence as set forth in any of SEQ ID NOs: 37, 41, 45, 49, 53, 57, 61, or 65; and FR4 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to the sequence as set forth in any of SEQ ID NOs: 38, 42, 46, 50, 54, 58, 62, or 66, preferably a sequence as set forth in any of SEQ ID NOs: 38, 42, 46, 50, 54, 58, 62, or 66. In certain embodiments, the single-domain antibody comprises FR1 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 35, preferably a sequence as set forth in SEQ ID NO: 35; FR2 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to the sequence as set forth in SEQ ID NO: 36, preferably a sequence as set forth in SEQ ID NO: 36; FR3 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 37, preferably a sequence as set forth in SEQ ID NO: 37; FR4 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 38, preferably a sequence as set forth in SEQ ID NO: 38. In certain embodiments, the single-domain antibody comprises FR1 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 39, preferably a sequence as set forth in SEQ ID NO: 39; FR2 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 40, preferably a sequence as set forth in SEQ ID NO: 40; FR3 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 41, preferably a sequence as set forth in SEQ ID NO: 41; FR4 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 42, preferably a sequence as set forth in SEQ ID NO: 42. In certain embodiments, the single-domain antibody comprises FR1 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 43, preferably a sequence as set forth in SEQ ID NO: 43; FR2 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to the sequence as set forth in SEQ ID NO: 44, preferably a sequence as set forth in SEQ ID NO: 44; FR3 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 45, preferably a sequence as set forth in SEQ ID NO: 45; FR4 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 46, preferably a sequence as set forth in SEQ ID NO: 46. In certain embodiments, the single-domain antibody comprises FR1 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 47, preferably a sequence as set forth in SEQ ID NO: 47; FR2 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 48, preferably a sequence as set forth in SEQ ID NO: 48; FR3 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 49, preferably a sequence as set forth in SEQ ID NO: 49; FR4 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 50, preferably a sequence as set forth in SEQ ID NO: 50. In certain embodiments, the single-domain antibody comprises FR1 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 51, preferably a sequence as set forth in SEQ ID NO: 51; FR2 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 52, preferably a sequence as set forth in SEQ ID NO: 52; FR3 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 53, preferably a sequence as set forth in SEQ ID NO: 53; FR4 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 54, preferably a sequence as set forth in SEQ ID NO: 54. In certain embodiments, the single-domain antibody comprises FR1 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 55, preferably a sequence as set forth in SEQ ID NO: 55; FR2 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 56, preferably a sequence as set forth in SEQ ID NO: 56; FR3 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 57, preferably a sequence as set forth in SEQ ID NO: 57; FR4 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 58, preferably a sequence as set forth in SEQ ID NO: 58. In certain embodiments, the single-domain antibody comprises FR1 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 59, preferably a sequence as set forth in SEQ ID NO: 59; FR2 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 60, preferably a sequence as set forth in SEQ ID NO: 60; FR3 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 61, preferably a sequence as set forth in SEQ ID NO: 61; FR4 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 62, preferably a sequence as set forth in SEQ ID NO: 62. In certain embodiments, the single-domain antibody comprises FR1 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 63, preferably a sequence as set forth in SEQ ID NO: 63; FR2 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 64, preferably a sequence as set forth in SEQ ID NO: 64; FR3 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 65, preferably a sequence as set forth in SEQ ID NO: 65; FR4 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 66, preferably a sequence as set forth in SEQ ID NO: 66. In a preferred embodiment, the single-domain antibody comprises FR1 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 59, preferably a sequence as set forth in SEQ ID NO: 59; FR2 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 60, preferably a sequence as set forth in SEQ ID NO: 60; FR3 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 61, preferably a sequence as set forth in SEQ ID NO: 61; FR4 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 62, preferably a sequence as set forth in SEQ ID NO: 62. In another preferred embodiment, the single-domain antibody comprises FR1 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 47, preferably a sequence as set forth in SEQ ID NO: 47; FR2 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 48, preferably a sequence as set forth in SEQ ID NO: 48; FR3 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 49, preferably a sequence as set forth in SEQ ID NO: 49; FR4 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 50, preferably a sequence as set forth in SEQ ID NO: 50. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, comprises CDR1 having a sequence as set forth in SEQ ID NO: 1; CDR2 having a sequence as set forth in SEQ ID NO: 2; CDR3 having a sequence as set forth in SEQ ID NO: 3; FR1 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 35, preferably a sequence as set forth in SEQ ID NO: 35; FR2 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 36, preferably a sequence as set forth in SEQ ID NO: 36; FR3 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 37, preferably a sequence as set forth in SEQ ID NO: 37; FR4 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 38, preferably a sequence as set forth in SEQ ID NO: 38. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, comprises CDR1 having a sequence as set forth in SEQ ID NO: 4; CDR2 having a sequence as set forth in SEQ ID NO: 5; CDR3 having a sequence as set forth in SEQ ID NO: 6; FR1 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 39, preferably a sequence as set forth in SEQ ID NO: 39; FR2 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 40, preferably a sequence as set forth in SEQ ID NO: 40; FR3 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 41, preferably a sequence as set forth in SEQ ID NO: 41; FR4 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 42, preferably a sequence as set forth in SEQ ID NO: 42. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, comprises CDR1 having a sequence as set forth in SEQ ID NO: 7; CDR2 having a sequence as set forth in SEQ ID NO: 8; CDR3 having a sequence as set forth in SEQ ID NO: 9; FR1 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 43, preferably a sequence as set forth in SEQ ID NO: 43; FR2 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 44, preferably a sequence as set forth in SEQ ID NO: 44; FR3 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 45, preferably a sequence as set forth in SEQ ID NO: 45; FR4 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 46, preferably a sequence as set forth in SEQ ID NO: 46. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, comprises CDR1 having a sequence as set forth in SEQ ID NO: 10; CDR2 having a sequence as set forth in SEQ ID NO: 11; CDR3 having a sequence as set forth in SEQ ID NO: 12; FR1 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 47, preferably a sequence as set forth in SEQ ID NO: 47; FR2 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 48, preferably a sequence as set forth in SEQ ID NO: 48; FR3 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 49, preferably a sequence as set forth in SEQ ID NO: 49; FR4 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 50, preferably a sequence as set forth in SEQ ID NO: 50. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, comprises CDR1 having a sequence as set forth in SEQ ID NO: 13; CDR2 having a sequence as set forth in SEQ ID NO: 14; CDR3 having a sequence as set forth in SEQ ID NO: 15; FR1 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 51, preferably a sequence as set forth in SEQ ID NO: 51; FR2 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 52, preferably a sequence as set forth in SEQ ID NO: 52; FR3 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 53, preferably a sequence as set forth in SEQ ID NO: 53; FR4 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 54, preferably a sequence as set forth in SEQ ID NO: 54. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, comprises CDR1 having a sequence as set forth in SEQ ID NO: 16; CDR2 having a sequence as set forth in SEQ ID NO: 17; CDR3 having a sequence as set forth in SEQ ID NO: 18; FR1 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 55, preferably a sequence as set forth in SEQ ID NO: 55; FR2 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 56, preferably a sequence as set forth in SEQ ID NO: 56; FR3 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 57, preferably a sequence as set forth in SEQ ID NO: 57; FR4 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 58, preferably a sequence as set forth in SEQ ID NO: 58. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, comprises CDR1 having a sequence as set forth in SEQ ID NO: 19; CDR2 having a sequence as set forth in SEQ ID NO: 20; CDR3 having a sequence as set forth in SEQ ID NO: 21; FR1 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 59, preferably a sequence as set forth in SEQ ID NO: 59; FR2 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 60, preferably a sequence as set forth in SEQ ID NO: 60; FR3 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 61, preferably a sequence as set forth in SEQ ID NO: 61; FR4 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 62, preferably a sequence as set forth in SEQ ID NO: 62. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, comprises CDR1 having a sequence as set forth in SEQ ID NO: 22; CDR2 having a sequence as set forth in SEQ ID NO: 23; CDR3 having a sequence as set forth in SEQ ID NO: 24; FR1 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 63, preferably a sequence as set forth in SEQ ID NO: 63; FR2 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 64, preferably a sequence as set forth in SEQ ID NO: 64; FR3 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 65, preferably a sequence as set forth in SEQ ID NO: 65; FR4 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 66, preferably a sequence as set forth in SEQ ID NO: 66. In a preferred embodiment, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, comprises CDR1 having a sequence as set forth in SEQ ID NO: 19; CDR2 having a sequence as set forth in SEQ ID NO: 20; CDR3 having a sequence as set forth in SEQ ID NO: 21; FR1 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 59, preferably a sequence as set forth in SEQ ID NO: 59; FR2 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 60, preferably a sequence as set forth in SEQ ID NO: 60; FR3 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 61, preferably a sequence as set forth in SEQ ID NO: 61; FR4 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 62, preferably a sequence as set forth in SEQ ID NO: 62. In another preferred embodiment, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, comprises CDR1 having a sequence as set forth in SEQ ID NO: 10; CDR2 having a sequence as set forth in SEQ ID NO: 11; CDR3 having a sequence as set forth in SEQ ID NO: 12. FR1 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 47, preferably a sequence as set forth in SEQ ID NO: 47; FR2 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 48, preferably a sequence as set forth in SEQ ID NO: 48; FR3 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 49, preferably a sequence as set forth in SEQ ID NO: 49; FR4 having a sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the sequence as set forth in SEQ ID NO: 50, preferably a sequence as set forth in SEQ ID NO: 50. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, has a sequence as set forth in any of SEQ ID NOs: 25 to 32, or a sequence which is at least 80%, preferably at least 90%, identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to a sequence as set forth in any of SEQ ID NOs: 25 to 32, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, has a sequence as set forth in any of SEQ ID NOs: 25 to 32, or a sequence which is at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to a sequence as set forth in any of SEQ ID NOs: 25 to 32, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment. Preferably, amino acids deviating from the corresponding amino acids in SEQ ID NOs: 25 to 32 are confined to the framework regions. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, has a sequence as set forth in any of SEQ ID NOs: 25 to 32. In a preferred embodiment, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, has a sequence as set forth SEQ ID NO: 28 or 31, or a sequence which is at least 80%, preferably at least 90%, identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to a sequence as set forth in SEQ ID NO: 28 or 31, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment. In a preferred embodiment, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, has a sequence as set forth in SEQ ID NO: 28 or 31, or a sequence which is at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to a sequence as set forth in SEQ ID NO: 28 or 31, or a fragment thereof, in particular a functional fragment such as an antigen- binding fragment. Preferably, amino acids deviating from the corresponding amino acids in SEQ ID NO: 28 or 31 are confined to the framework regions. In a preferred embodiment, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, has a sequence as set forth in SEQ ID NOs: 28 or 31. Embodiments of single-domain antibodies or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, of the invention are provided in Table A. Framework regions are at least 80% identical, preferably at least 90% identical, more preferably at least 95% identical, most preferably identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to the indicated SEQ ID NO. Table A Embodiment CDR1 CDR2 CDR3 FR1 FR2 FR3 FR4 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO NO NO NO NO NO NO A 1 2 3 B 4 5 6 C 7 8 9 D 10 11 12 E 13 14 14 F 16 17 18 G 19 20 21 H 22 23 24 I 1 2 3 35 36 37 38 J 4 5 6 39 40 41 42 K 7 8 9 43 44 45 46 L 10 11 12 47 48 49 50 M 13 14 14 51 52 53 54 N 16 17 18 55 56 57 58 O 19 20 21 59 60 61 62 P 22 23 24 63 64 65 66 Q 25 R 26 S 27 T 28 U 29 V 30 W 31 X 32 In certain embodiments, the single-domain antibody comprises humanized framework regions. In certain embodiments, the single-domain antibody comprises one or more mutation. In certain embodiments, the single-domain antibody comprises one or more mutation in one or more FR. In certain embodiments, the single-domain antibody comprises one or more mutation of amino acid residues prone to in vivo modification. In certain embodiments, the mutation results in reduced or abolished in vivo modification of said amino acid residue. In certain embodiments, the single-domain antibody comprises one or more mutation in one or more FR of amino acid residues prone to in vivo modification. In certain embodiments, the mutation results in reduced or abolished in vivo modification of said amino acid residue. In certain embodiments, the single-domain antibody comprises one or more amino acid residue mutation resulting in increased thermostability. In certain embodiments, the single-domain antibody comprises one or more amino acid residue mutation in one or more FR resulting in increased thermostability. In certain embodiments, the single-binding domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment is fused or linked to an Fc domain, preferably an IgG Fc domain, such as an IgG1 Fc domain, preferably human. As described herein elsewhere, such fusion or linkage may be genetic or non- genetic. In certain embodiments, the binding agent comprises a single-binding domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, which is fused or linked to an Fc domain, preferably an IgG Fc domain, such as an IgG1 Fc domain, preferably human. As described herein elsewhere, such fusion or linkage may be genetic or non-genetic. In certain embodiments, the single-binding domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment is fused or linked to albumin, preferably human albumin. As described herein elsewhere, such fusion or linkage may be genetic or non-genetic. In certain embodiments, the binding agent comprises a single-binding domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, which is fused or linked to albumin, preferably human albumin. As described herein elsewhere, such fusion or linkage may be genetic or non-genetic. In certain embodiments, the single-binding domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment is fused or linked to PEG (polyethylene glycol). As described herein elsewhere, such fusion or linkage may be genetic or non-genetic. Accordingly, in certain embodiments, the single-binding domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment is PEGylated. PEG chain length can be adjusted as known in the art for suitability in half-life extension. In certain embodiments, the binding agent comprises a single-binding domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, which is fused or linked to PEG (polyethylene glycol). As described herein elsewhere, such fusion or linkage may be genetic or non-genetic. Accordingly, in certain embodiments, the binding agent is PEGylated. PEG chain length can be adjusted as known in the art for suitability in half-life extension. In certain embodiments, the single-binding domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment is fused or linked to PAS (biosynthetic polymers made of the small L-amino acids Pro, Ala and/or Ser). As described herein elsewhere, such fusion or linkage may be genetic or non-genetic. Accordingly, in certain embodiments, the single-binding domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment is PASylated. PAS chain length can be adjusted as known in the art for suitability in half- life extension. In certain embodiments, the binding agent comprises a single-binding domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, which is fused or linked to PAS (biosynthetic polymers made of the small L- amino acids Pro, Ala and/or Ser). As described herein elsewhere, such fusion or linkage may be genetic or non-genetic. Accordingly, in certain embodiments, the binding agent is PASylated. PAS chain length can be adjusted as known in the art for suitability in half- life extension. In certain embodiments, the binding agent comprises at least two single-domain antibodies of the invention as described herein elsewhere, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment. In certain embodiments, the binding agent comprises two single-domain antibodies of the invention as described herein elsewhere, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment. In certain embodiments, the binding agent comprises at least three single-domain antibodies of the invention as described herein elsewhere, or a fragment thereof, in particular a functional fragment such as an antigen- binding fragment. In certain embodiments, the binding agent comprises tree single- domain antibodies of the invention as described herein elsewhere, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment. In certain embodiments, the binding agent comprises a single-domain antibody or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, as described in Table A. In an aspect, the invention relates to a polynucleic acid encoding a single-domain antibody as described herein, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment. In an aspect, the invention relates to a polynucleic acid encoding a binding agent as described herein (i.e. comprising a single-domain antibody as described herein, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment). In certain embodiments, the polynucleic acid comprises a sequence encoding a protein sequence as set forth in any of SEQ ID NO: 1 to 32, or a sequence which is at least 80%, preferably at least 90%, more preferably at least 95% identical to a sequence as set forth in any of SEQ ID NOs: 1 to 32, preferably SEQ ID NO: 28 or 31. In certain embodiments, the polynucleic acid comprises a sequence encoding a single- domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, having a sequence as set forth in any of SEQ ID NO: 1 to 32, or a sequence which is at least 80%, preferably at least 90%, more preferably at least 95% identical to a sequence as set forth in any of SEQ ID NOs: 1 to 32, preferably SEQ ID NO: 28 or 31. In certain embodiments, the polynucleic acid comprises a sequence encoding binding agent comprising a single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, having a sequence as set forth in any of SEQ ID NO: 1 to 32, or a sequence which is at least 80%, preferably at least 90%, more preferably at least 95% identical to a sequence as set forth in any of SEQ ID NOs: 1 to 32, preferably SEQ ID NO: 28 or 31. In certain embodiments, the polynucleic acid comprises a sequence encoding a protein sequence as set forth in any of SEQ ID NO: 25 to 32, or a sequence which is at least 80%, preferably at least 90%, more preferably at least 95% identical to a sequence as set forth in any of SEQ ID NOs: 25 to 32, preferably SEQ ID NO: 28 or 31. In certain embodiments, the polynucleic acid comprises a sequence encoding a single- domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, having a sequence as set forth in any of SEQ ID NO: 25 to 32, or a sequence which is at least 80%, preferably at least 90%, more preferably at least 95% identical to a sequence as set forth in any of SEQ ID NOs: 25 to 32, preferably SEQ ID NO: 28 or 31. In certain embodiments, the polynucleic acid comprises a sequence encoding binding agent comprising a single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, having a sequence as set forth in any of SEQ ID NO: 25 to 32, or a sequence which is at least 80%, preferably at least 90%, more preferably at least 95% identical to a sequence as set forth in any of SEQ ID NOs: 25 to 32, preferably SEQ ID NO: 28 or 31. In certain embodiments, the polynucleic acid comprises a sequence encoding a single- domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, having a CDR1 sequence as set forth in SEQ ID NO: 1, a CDR2 sequence as set forth in SEQ ID NO: 2, and a CDR3 sequence as set forth in SEQ ID NO: 3; a CDR1 sequence as set forth in SEQ ID NO: 4, a CDR2 sequence as set forth in SEQ ID NO: 5, and a CDR3 sequence as set forth in SEQ ID NO: 6; a CDR1 sequence as set forth in SEQ ID NO: 7, a CDR2 sequence as set forth in SEQ ID NO: 8, and a CDR3 sequence as set forth in SEQ ID NO: 9; a CDR1 sequence as set forth in SEQ ID NO: 10, a CDR2 sequence as set forth in SEQ ID NO: 11, and a CDR3 sequence as set forth in SEQ ID NO: 12; a CDR1 sequence as set forth in SEQ ID NO: 13, a CDR2 sequence as set forth in SEQ ID NO: 14, and a CDR3 sequence as set forth in SEQ ID NO: 15; a CDR1 sequence as set forth in SEQ ID NO: 16, a CDR2 sequence as set forth in SEQ ID NO: 17, and a CDR3 sequence as set forth in SEQ ID NO: 18; a CDR1 sequence as set forth in SEQ ID NO: 19, a CDR2 sequence as set forth in SEQ ID NO: 20, and a CDR3 sequence as set forth in SEQ ID NO: 21; or a CDR1 sequence as set forth in SEQ ID NO: 22, a CDR2 sequence as set forth in SEQ ID NO: 23, and a CDR3 sequence as set forth in SEQ ID NO: 14. In certain embodiments, the polynucleic acid comprises a sequence encoding binding agent comprising a single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, having a CDR1 sequence as set forth in SEQ ID NO: 1, a CDR2 sequence as set forth in SEQ ID NO: 2, and a CDR3 sequence as set forth in SEQ ID NO: 3; a CDR1 sequence as set forth in SEQ ID NO: 4, a CDR2 sequence as set forth in SEQ ID NO: 5, and a CDR3 sequence as set forth in SEQ ID NO: 6; a CDR1 sequence as set forth in SEQ ID NO: 7, a CDR2 sequence as set forth in SEQ ID NO: 8, and a CDR3 sequence as set forth in SEQ ID NO: 9; a CDR1 sequence as set forth in SEQ ID NO: 10, a CDR2 sequence as set forth in SEQ ID NO: 11, and a CDR3 sequence as set forth in SEQ ID NO: 12; a CDR1 sequence as set forth in SEQ ID NO: 13, a CDR2 sequence as set forth in SEQ ID NO: 14, and a CDR3 sequence as set forth in SEQ ID NO: 15; a CDR1 sequence as set forth in SEQ ID NO: 16, a CDR2 sequence as set forth in SEQ ID NO: 17, and a CDR3 sequence as set forth in SEQ ID NO: 18; a CDR1 sequence as set forth in SEQ ID NO: 19, a CDR2 sequence as set forth in SEQ ID NO: 20, and a CDR3 sequence as set forth in SEQ ID NO: 21; or a CDR1 sequence as set forth in SEQ ID NO: 22, a CDR2 sequence as set forth in SEQ ID NO: 23, and a CDR3 sequence as set forth in SEQ ID NO: 14. In an aspect, the invention relates to a vector comprising one or more polynucleic acid according to the invention as described herein. Suitable vectors include cloning vectors as well as expression vectors, as described herein elsewhere. It will be understood that expression vectors are configured for expression of the polynucleic acid by operative linkage of the polynucleic acid to a suitable regulatory sequence, such as a promoter. In an aspect, the invention relates to a host cell comprising one or more polynucleic acid according to the invention as described herein. In an aspect, the invention relates to a host cell comprising one or more vector according to the invention as described herein. Suitable host cell are described herein elsewhere. In an aspect, the invention relates to a pharmaceutical composition comprising a single- domain antibody according to the invention as described herein, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment. In an aspect, the invention relates to a pharmaceutical composition comprising a binding agent according to the invention as described herein. In an aspect, the invention relates to a pharmaceutical composition comprising a polynucleic acid according to the invention as described herein. In an aspect, the invention relates to a pharmaceutical composition comprising a vector according to the invention as described herein. In an aspect, the invention relates to a pharmaceutical composition comprising a host cell according to the invention as described herein. Suitable pharmaceutical compositions are described herein elsewhere. Pharmaceutical compositions typically additionally comprise a pharmaceutically acceptable excipients and/or carriers, as also described herein elsewhere. In certain embodiments, the pharmaceutical composition further comprises one or more additional active pharmaceutical ingredient. The second (or further) active pharmaceutical ingredient may have the same, a similar, or different activity than the single-domain antibody or binding agent of the invention. The second (or further) active pharmaceutical ingredient may have a activity complementary to the activity of the single- domain antibody or binding agent of the invention. The second (or further) active pharmaceutical ingredient may have a activity supplementary to the activity of the single- domain antibody or binding agent of the invention. The second (or further) active pharmaceutical ingredient may have a activity which is synergistic with the activity of the single-domain antibody or binding agent of the invention. The second (or further) active pharmaceutical ingredient may enhance the activity of the single-domain antibody or binding agent of the invention. In certain embodiments, the second (or further) active pharmaceutical ingredient has anti-inflammatory activity. In certain embodiments, the second (or further) active pharmaceutical ingredient mediates lipid metabolism. In certain embodiments, the second (or further) active pharmaceutical ingredient activates or suppresses PPAR, FGF19/21, AMPK, DGAT2, ACC, FASN, MPC, and/or SCD1. In certain embodiments, the second (or further) active pharmaceutical ingredient mediates sugar metabolism. In certain embodiments, the second (or further) active pharmaceutical ingredient activates or suppresses GLP-1R, SGLT2, KHK, and/or MPC. In certain embodiments, the second (or further) active pharmaceutical ingredient mediates cholesterol metabolism. In certain embodiments, the second (or further) active pharmaceutical ingredient mediates fibrosis. In certain embodiments, the second (or further) active pharmaceutical ingredient activates or suppresses ASK1, LOXL2, and/or GAL-3. In an aspect, the invention relates to a kit comprising a single-domain antibody according to the invention as described herein, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment. In an aspect, the invention relates to a kit comprising a binding agent according to the invention as described herein. In an aspect, the invention relates to a kit comprising a polynucleic acid according to the invention as described herein. In an aspect, the invention relates to a kit comprising a vector according to the invention as described herein. In an aspect, the invention relates to a kit comprising a host cell according to the invention as described herein. In an aspect, the invention relates to a kit comprising a pharmaceutical composition according to the invention as described herein. Such kits may further comprise instructions for use. Such kits may be diagnostic kits, pharmaceutical kits or medicament kits which are comprising a container or vial (any suitable container or vial, such as a pharmaceutically acceptable container or vial) comprising an amount of single-domain antibody (fragment), binding agent, polynucleic acid, vector, host cell, or pharmaceutical composition, and further comprising e.g. a kit insert such as a medical or diagnostic leaflet or package leaflet comprising information on e.g. intended indications (therapeutic treatment) and potential side-effects or diagnostic applications. Pharmaceutical kits or medicament kits may further comprise e.g. a syringe for administering the single-domain antibody (fragment), binding agent, polynucleic acid, vector, host cell, or pharmaceutical composition as described herein to a subject. In an aspect, the invention relates to a single-domain antibody according to the invention as described herein, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, for use in (curative) therapy. In an aspect, the invention relates to a binding agent according to the invention as described herein for use in (curative) therapy. In an aspect, the invention relates to a polynucleic acid according to the invention as described herein for use in (curative) therapy. In an aspect, the invention relates to a vector according to the invention as described herein for use in (curative) therapy. In an aspect, the invention relates to a host cell according to the invention as described herein for use in (curative) therapy. In an aspect, the invention relates to a pharmaceutical composition according to the invention as described herein for use in (curative) therapy. In an aspect, the invention relates to the use of a single-domain antibody according to the invention as described herein, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, for the manufacture of a medicament. In an aspect, the invention relates to the use of a binding agent according to the invention as described herein for the manufacture of a medicament. In an aspect, the invention relates to the use of a polynucleic acid according to the invention as described herein for the manufacture of a medicament. In an aspect, the invention relates to the use of a vector according to the invention as described herein for the manufacture of a medicament. In an aspect, the invention relates to the use of a host cell according to the invention as described herein for the manufacture of a medicament. In an aspect, the invention relates to the use of a pharmaceutical composition according to the invention as described herein for the manufacture of a medicament. In an aspect, the invention relates to the use of a single-domain antibody according to the invention as described herein, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, in diagnosis. In an aspect, the invention relates to the use of a binding agent according to the invention as described herein in diagnosis. In an aspect, the invention relates to the use of a polynucleic acid according to the invention as described herein in diagnosis. In an aspect, the invention relates to the use of a vector according to the invention as described herein in diagnosis. In an aspect, the invention relates to the use of a host cell according to the invention as described herein in diagnosis. In an aspect, the invention relates to a (curative) method of treatment comprising administering to an individual in need thereof a single-domain antibody according to the invention as described herein, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment. In an aspect, the invention relates to a (curative) method of treatment comprising administering to an individual in need thereof a binding agent according to the invention as described herein. In an aspect, the invention relates to a (curative) method of treatment comprising administering to an individual in need thereof a polynucleic acid according to the invention as described herein. In an aspect, the invention relates to a (curative) method of treatment comprising administering to an individual in need thereof a vector according to the invention as described herein. In an aspect, the invention relates to a (curative) method of treatment comprising administering to an individual in need thereof a host cell according to the invention as described herein. In an aspect, the invention relates to a (curative) method of treatment comprising administering to an individual in need thereof a pharmaceutical composition according to the invention as described herein. Administration may be in accordance with standard techniques. The administration can be by any appropriate mode, including intravenous injection, subcutaneous injection, oral, parenteral, topical, nasal (e.g. via inhalation or pulmonary delivery), ophthalmic, intrathecal, intra-cerebroventricular, sublingual, rectal, vaginal, and the like. Still other techniques of formulation such as nanotechnology and aerosol and inhalant are also within the scope of this invention. The dosage and frequency of administration will depend on the age, sex and condition of the patient, concurrent administration of other drugs, counter-indications and other parameters to be taken into account by the clinician. In an aspect, the invention relates to a single-domain antibody according to the invention as described herein, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, for use in (curative) treatment or diagnosis of (liver) inflammation. In an aspect, the invention relates to a binding agent according to the invention as described herein for use in (curative) treatment or diagnosis of (liver) inflammation. In an aspect, the invention relates to a polynucleic acid according to the invention as described herein for use in (curative) treatment or diagnosis of (liver) inflammation. In an aspect, the invention relates to a vector according to the invention as described herein for use in (curative) treatment or diagnosis of (liver) inflammation. In an aspect, the invention relates to a host cell according to the invention as described herein for use in (curative) treatment or diagnosis of (liver) inflammation. In an aspect, the invention relates to a pharmaceutical composition according to the invention as described herein for use in (curative) treatment of (liver) inflammation. In an aspect, the invention relates to the use of a single-domain antibody according to the invention as described herein, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, for the manufacture of a medicament for (curative) treatment of (liver) inflammation. In an aspect, the invention relates to the use of a binding agent according to the invention as described herein for the manufacture of a medicament for (curative) treatment of (liver) inflammation. In an aspect, the invention relates to the use of a polynucleic acid according to the invention as described herein for the manufacture of a medicament for (curative) treatment of (liver) inflammation. In an aspect, the invention relates to the use of a vector according to the invention as described herein for the manufacture of a medicament for (curative) treatment of (liver) inflammation. In an aspect, the invention relates to the use of a host cell according to the invention as described herein for the manufacture of a medicament for (curative) treatment of (liver) inflammation. In an aspect, the invention relates to the use of a pharmaceutical composition according to the invention as described herein for the manufacture of a medicament for (curative) treatment of (liver) inflammation. A further aspect of the invention relates to a single-domain antibody (or fragment thereof) or binding agent as described herein for use in diagnosing, for use as a diagnostic agent, or for use in the manufacture of a diagnostic agent or diagnostic kit, such as an in vitro diagnostic agent or kit. Alternatively, use of a single-domain antibody (or fragment thereof) or binding agent as described herein in the manufacture of a diagnostic agent/in vitro diagnostic agent is envisaged. In particular, the binding agent as described herein is for use in identifying a medical condition as described herein, such as in a sample obtained from a subject, such as from a subject suspected to be afflicted with such medical condition. A nucleic acid encoding a single-domain antibody (or fragment thereof) or binding agent as described herein, or a recombinant vector comprising such nucleic acid can likewise be used in or be for use in the manufacture of a diagnostic agent or diagnostic kit, such as an in vitro diagnostic agent or kit. Without wishing to be bound by theory, specific binding to activated (i.e. open) Panx1 channels may underly the diagnostic potential of the single-domain antibodies of the invention as described herein. In an aspect, the invention relates to the use of a single-domain antibody according to the invention as described herein, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, for diagnosing (liver) inflammation. In an aspect, the invention relates to the use of a binding agent according to the invention as described herein for diagnosing (liver) inflammation. In an aspect, the invention relates to the use of a polynucleic acid according to the invention as described herein for diagnosing of (liver) inflammation. In an aspect, the invention relates to the use of a vector according to the invention as described herein for diagnosing (liver) inflammation. In an aspect, the invention relates to the use of a host cell according to the invention as described herein for diagnosing (liver) inflammation. A further aspect of the invention relates to methods for detecting, identifying, or diagnosing a medical condition as described herein elsewhere, such as in a sample obtained from a subject, such as from a subject suspected to be afflicted with such medical condition. Such methods usually may comprise detecting, determining, assessing, assaying, identifying or measuring binding of the single-domain antibody (or fragment thereof) or binding agent with Panx1 channels. Such method may further comprise the steps of obtaining a sample and contacting the sample with a single-domain antibody (or fragment thereof) or binding agent as described herein. In certain embodiments, in the above diagnostic aspects, the single-domain antibody (or fragment thereof) or binding agent as described herein is comprising a detectable moiety fused to it, bound to it, coupled to it, linked to it, complexed to it, or chelated to it. A “detectable moiety” in general refers to a moiety that emits a signal or is capable of emitting a signal upon adequate stimulation, or to a moiety that is capable of being detected through binding or interaction with a further molecule (e.g. a tag, such as an affinity tag, that is specifically recognized by a labelled antibody), or is detectable by any means (preferably by a non-invasive means, if detection is in vivo/ inside the human body). Furthermore, the detectable moiety may allow for computerized composition of an image, as such the detectable moiety may be called an imaging agent. Detectable moieties include fluorescence emitters, phosphorescence emitters, positron emitters, radioemitters, etc., but are not limited to emitters as such moieties also include enzymes (capable of measurably converting a substrate) and molecular tags. Examples of radioemitters/radiolabels include 68Ga, 110mIn, 18F, 45Ti, 44Sc, 47Sc, 61Cu, 60Cu, 62Cu, 66Ga, 64Cu, 55Ca, 72As, 86Y, 90Y, 89Zr, 125I, 74Br, 75Br, 76Br, 77Br, 78Br, 111In, 114mIn, 114In, 99mTc, 11C, 32Cl, 33Cl, 34Cl, 123I, 124I, 131I, 186Re, 188Re, 177Lu, 99Tc, 212Bi, 213Bi, 212Pb, 225Ac, 153Sm, and 67Ga. Fluorescence emitters include cyanine dyes (e.g. Cy5, Cy5.5, Cy7, Cy7.5), FITC, TRITC, coumarin, indolenine-based dyes, benzoindolenine-based dyes, phenoxazines, BODIPY dyes, rhodamines, Si-rhodamines, Alexa dyes, and derivatives of any thereof. Affinity tags, such as chitin binding protein (CBP), maltose binding protein (MBP), glutathione-S-transferase (GST), poly(His) (e.g., 6x His or His6), biotin or streptavidin, such as Strep-tag®, Strep-tag II® and Twin-Strep-tag®; solubilizing tags, such as thioredoxin (TRX), poly(NANP) and SUMO; chromatography tags, such as a FLAG-tag; epitope tags, such as V5-tag, myc-tag and HA-tag; fluorescent labels or tags (i.e., fluorochromes/-phores), such as fluorescent proteins (e.g., GFP, YFP, RFP etc.); luminescent labels or tags, such as luciferase, bioluminescent or chemiluminescent compounds (such as luminal, isoluminol, theromatic acridinium ester, imidazole, acridinium salts, oxalate ester, dioxetane or GFP and its analogs); phosphorescent labels; a metal chelator; and (other) enzymatic labels (e.g., peroxidase, alkaline phosphatase, beta-galactosidase, urease or glucose oxidase). Binding agents as describe herein and comprising a detectable moiety may for example be used for in vitro, in vivo or in situ assays (including immunoassays known per se such as ELISA, RIA, EIA and other "sandwich assays", etc.) as well as in vivo imaging purposes (such as a SPECT-scan), depending on the choice of the specific label. In an aspect, the invention relates to a (curative) method of treatment of inflammation comprising administering to an individual in need thereof a single-domain antibody according to the invention as described herein, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment. In an aspect, the invention relates to a (curative) method of treatment of inflammation comprising administering to an individual in need thereof a binding agent according to the invention as described herein. In an aspect, the invention relates to a (curative) method of treatment of inflammation comprising administering to an individual in need thereof a polynucleic acid according to the invention as described herein. In an aspect, the invention relates to a (curative) method of treatment of inflammation comprising administering to an individual in need thereof a vector according to the invention as described herein. In an aspect, the invention relates to a (curative) method of treatment of inflammation comprising administering to an individual in need thereof a host cell according to the invention as described herein. In an aspect, the invention relates to a (curative) method of treatment of inflammation comprising administering to an individual in need thereof a pharmaceutical composition according to the invention as described herein. Administration may be in accordance with standard techniques. The administration can be by any appropriate mode, including intravenous injection, subcutaneous injection, oral, parenteral, topical, nasal (e.g. via inhalation or pulmonary delivery), ophthalmic, intrathecal, intra-cerebroventricular, sublingual, rectal, vaginal, and the like. Still other techniques of formulation such as nanotechnology and aerosol and inhalant are also within the scope of this invention. The dosage and frequency of administration will depend on the age, sex and condition of the patient, concurrent administration of other drugs, counter-indications and other parameters to be taken into account by the clinician. In a preferred embodiment, said inflammation is hepatitis, such as described herein elsewhere. In certain embodiments, inflammation is caused by is steatohepatitis. In certain embodiments, inflammation is caused by non-alcoholic steatohepatitis (NASH). In certain embodiments, inflammation is caused by acute liver failure. In certain embodiments, inflammation is acute. In certain embodiments, inflammation is chronic. In an aspect, the invention relates to a single-domain antibody according to the invention as described herein, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, for use in (curative) treatment or diagnosis of an inflammatory (liver) disease or disorder. In an aspect, the invention relates to a binding agent according to the invention as described herein for use in (curative) treatment or diagnosis of an inflammatory (liver) disease or disorder. In an aspect, the invention relates to a polynucleic acid according to the invention as described herein for use in (curative) treatment or diagnosis of an inflammatory (liver) disease or disorder. In an aspect, the invention relates to a vector according to the invention as described herein for use in (pr curative) treatment or diagnosis of an inflammatory (liver) disease or disorder. In an aspect, the invention relates to a host cell according to the invention as described herein for use in (curative) treatment or diagnosis of an inflammatory (liver) disease or disorder. In an aspect, the invention relates to a pharmaceutical composition according to the invention as described herein for use in (curative) treatment of an inflammatory (liver) disease or disorder. In an aspect, the invention relates to the use of a single-domain antibody according to the invention as described herein, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, for diagnosing or for the manufacture of a medicament for (curative) treatment of an inflammatory (liver) disease or disorder. In an aspect, the invention relates to the use of a binding agent according to the invention as described herein for diagnosing or for the manufacture of a medicament for (curative) treatment of an inflammatory (liver) disease or disorder. In an aspect, the invention relates to the use of a polynucleic acid according to the invention as described herein for diagnosing or for the manufacture of a medicament for (curative) treatment of an inflammatory (liver) disease or disorder. In an aspect, the invention relates to the use of a vector according to the invention as described herein for diagnosing or for the manufacture of a medicament for (curative) treatment of an inflammatory (liver) disease or disorder. In an aspect, the invention relates to the use of a host cell according to the invention as described herein for diagnosing or for the manufacture of a medicament for (curative) treatment of an inflammatory (liver) disease or disorder. In an aspect, the invention relates to the use of a pharmaceutical composition according to the invention as described herein for the manufacture of a medicament for (curative) treatment of an inflammatory (liver) disease or disorder. In an aspect, the invention relates to a (curative) method of treatment of an inflammatory disease or disorder comprising administering to an individual in need thereof a single- domain antibody according to the invention as described herein, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment. In an aspect, the invention relates to a (curative) method of treatment of an inflammatory disease or disorder comprising administering to an individual in need thereof a binding agent according to the invention as described herein. In an aspect, the invention relates to a (curative) method of treatment of an inflammatory disease or disorder comprising administering to an individual in need thereof a polynucleic acid according to the invention as described herein. In an aspect, the invention relates to a (curative) method of treatment of an inflammatory disease or disorder comprising administering to an individual in need thereof a vector according to the invention as described herein. In an aspect, the invention relates to a (curative) method of treatment of an inflammatory disease or disorder comprising administering to an individual in need thereof a host cell according to the invention as described herein. In an aspect, the invention relates to a (curative) method of treatment of an inflammatory disease or disorder comprising administering to an individual in need thereof a pharmaceutical composition according to the invention as described herein. Administration may be in accordance with standard techniques. The administration can be by any appropriate mode, including intravenous injection, subcutaneous injection, oral, parenteral, topical, nasal (e.g. via inhalation or pulmonary delivery), ophthalmic, intrathecal, intra-cerebroventricular, sublingual, rectal, vaginal, and the like. Still other techniques of formulation such as nanotechnology and aerosol and inhalant are also within the scope of this invention. The dosage and frequency of administration will depend on the age, sex and condition of the patient, concurrent administration of other drugs, counter-indications and other parameters to be taken into account by the clinician. As used herein, an inflammatory disease or disorder is a disease or disorder having an inflammatory component. Such disease or disorder is characterized by (among others) inflammation, either as a cause or as a consequence (i.e. as an initiating process or as an accompanying or resulting process). In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen- binding fragment, binding domain, polynucleic acid, vector, host cell, or pharmaceutical composition according to the invention as described herein may be for use or may be used (in a method) for treating the inflammatory component of an inflammatory disease or disorder. In certain embodiments, the single-domain antibody, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, binding domain, polynucleic acid, vector, host cell, or pharmaceutical composition according to the invention as described herein may be for use or may be used (in a method) for treating inflammation in an inflammatory disease or disorder. In certain embodiments, the inflammatory disease or disorder is an acute inflammatory disease or disorder. In certain embodiments, the inflammatory disease or disorder is a chronic inflammatory disease or disorder. In certain embodiments, the inflammatory disease or disorder is selected from autoimmune disorder, atherosclerosis, allergy, myopathy, cancer, pharmacologically induced inflammation (e.g. drug overdose), toxin- induced inflammation, radiation-induced inflammation, heavy metal-induced inflammation, sepsis, pathogen-induced (i.e. infection), arthritis, migraine coeliac disease, inflammatory bowel disease, endometriosis, diabetes (type 1 or type 2), asthma, rheumatoid artritis, obesity, Alzheimer’ disease, Parkinson’s disease, encephalitis, lupus, myositis, scleroderma, uveitis, vasculitis, heart disease, psoriasis, multiple sclerosis, sarcoidosis, meningitis, cystic fibrosis, gout, dermatitis, nephritis, pancreatitis, fibrosis, Crohn’s disease, ankylosing spondylitis, COPD. In an aspect, the invention relates to a single-domain antibody according to the invention as described herein, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, for use in (curative) treatment of a liver disease or disorder, preferably an inflammatory liver disease or disorder. In an aspect, the invention relates to a binding agent according to the invention as described herein for use in (curative) treatment of a liver disease or disorder, preferably an inflammatory liver disease or disorder. In an aspect, the invention relates to a polynucleic acid according to the invention as described herein for use in (curative) treatment of a liver disease or disorder, preferably an inflammatory liver disease or disorder. In an aspect, the invention relates to a vector according to the invention as described herein for use in (curative) treatment of a liver disease or disorder, preferably an inflammatory liver disease or disorder. In an aspect, the invention relates to a host cell according to the invention as described herein for use in (curative) treatment of a liver disease or disorder, preferably an inflammatory liver disease or disorder. In an aspect, the invention relates to a pharmaceutical composition according to the invention as described herein for use in (curative) treatment of a liver disease or disorder, preferably an inflammatory liver disease or disorder. In an aspect, the invention relates to the use of a single-domain antibody according to the invention as described herein, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment, for the manufacture of a medicament for (curative) treatment of a liver disease or disorder, preferably an inflammatory liver disease or disorder. In an aspect, the invention relates to the use of a binding agent according to the invention as described herein for the manufacture of a medicament for (curative) treatment of a liver disease or disorder, preferably an inflammatory liver disease or disorder. In an aspect, the invention relates to the use of a polynucleic acid according to the invention as described herein for the manufacture of a medicament for (curative) treatment of a liver disease or disorder, preferably an inflammatory liver disease or disorder. In an aspect, the invention relates to the use of a vector according to the invention as described herein for the manufacture of a medicament for (curative) treatment of a liver disease or disorder, preferably an inflammatory liver disease or disorder. In an aspect, the invention relates to the use of a host cell according to the invention as described herein for the manufacture of a medicament for (curative) treatment of a liver disease or disorder, preferably an inflammatory liver disease or disorder. In an aspect, the invention relates to the use of a pharmaceutical composition according to the invention as described herein for the manufacture of a medicament for (curative) treatment of a liver disease or disorder, preferably an inflammatory liver disease or disorder. In an aspect, the invention relates to a (curative) method of treatment of a liver disease or disorder, preferably an inflammatory liver disease or disorder, comprising administering to an individual in need thereof a single-domain antibody according to the invention as described herein, or a fragment thereof, in particular a functional fragment such as an antigen-binding fragment. In an aspect, the invention relates to a (curative) method of treatment of a liver disease or disorder, preferably an inflammatory liver disease or disorder, comprising administering to an individual in need thereof a binding agent according to the invention as described herein. In an aspect, the invention relates to a (curative) method of treatment of a liver disease or disorder, preferably an inflammatory liver disease or disorder, comprising administering to an individual in need thereof a polynucleic acid according to the invention as described herein. In an aspect, the invention relates to a (curative) method of treatment of a liver disease or disorder, preferably an inflammatory liver disease or disorder, comprising administering to an individual in need thereof a vector according to the invention as described herein. In an aspect, the invention relates to a (curative) method of treatment of a liver disease or disorder, preferably an inflammatory liver disease or disorder, comprising administering to an individual in need thereof a host cell according to the invention as described herein. In an aspect, the invention relates to a (curative) method of treatment of a liver disease or disorder, preferably an inflammatory liver disease or disorder, comprising administering to an individual in need thereof a pharmaceutical composition according to the invention as described herein. Administration may be in accordance with standard techniques. The administration can be by any appropriate mode, including intravenous injection, subcutaneous injection, oral, parenteral, topical, nasal (e.g. via inhalation or pulmonary delivery), ophthalmic, intrathecal, intra-cerebroventricular, sublingual, rectal, vaginal, and the like. Still other techniques of formulation such as nanotechnology and aerosol and inhalant are also within the scope of this invention. The dosage and frequency of administration will depend on the age, sex and condition of the patient, concurrent administration of other drugs, counter-indications and other parameters to be taken into account by the clinician. In certain embodiments, the (inflammatory) liver disease or disorder is steatohepatitis. In certain embodiments, the (inflammatory) liver disease or disorder is non-alcoholic steatohepatitis (NASH). In certain embodiments, the (inflammatory) liver disease or disorder is acute liver failure. In certain embodiments, the (inflammatory) liver disease or disorder is cholestatic disorder. In certain embodiments, the (inflammatory) liver disease or disorder is cholangitis. In certain embodiments, the (inflammatory) liver disease or disorder is liver fibrosis. In certain embodiments, the (inflammatory) liver disease or disorder is liver cirrhosis. In certain embodiments, the (inflammatory) liver disease or disorder is liver cancer. In certain embodiments, the (inflammatory) liver disease or disorder is non-alcoholic fatty liver disease (NAFLD). In certain embodiments, the (inflammatory) liver disease or disorder is non-alcoholic fatty liver disease (NAFLD). In certain embodiments, the (inflammatory) liver disease or disorder is fatty liver disease. In certain embodiments, the (inflammatory) liver disease or disorder is hepatitis or hepatic inflammation. In certain embodiments, the (inflammatory) liver disease or disorder is autoimmune hepatitis. In certain embodiments, the (inflammatory) liver disease or disorder is viral hepatitis. In certain embodiments, the (inflammatory) liver disease or disorder is alcoholic hepatitis. In certain embodiments, the (inflammatory) liver disease or disorder is fatty hepatitis. In certain embodiments, the (inflammatory) liver disease or disorder is toxin-induced hepatitis. In certain embodiments, the (inflammatory) liver disease or disorder is drug-induced hepatitis. In certain embodiments, the (inflammatory) liver disease or disorder is cholestatic liver disease. In certain embodiments, embodiments of the medical indications to be treated or diagnosed according to the invention are listed in Table B. Table B Embodiment Medical indication 1 inflammation 2 liver inflammation 3 inflammatory liver disease 4 (inflammation caused by) chronic liver disease 5 steatohepatitis 6 non-alcoholic steatohepatitis 7 (inflammation caused by) fatty liver disease 8 (inflammation caused by) non-alcoholic fatty liver disease 9 (inflammation caused by) acute liver failure 10 (inflammation caused by) cholestatic disorder 11 (inflammation caused by) fibrosis 12 (inflammation caused by) cirrhosis 13 (inflammation caused by) liver fibrosis 14 (inflammation caused by) liver cirrhosis 15 (inflammation caused by) liver cancer 16 cholangitis 17 hepatitis 18 autoimmune hepatitis 19 alcoholic hepatitis 20 viral hepatitis 21 fatty hepatitis 22 toxin-induced hepatitis 23 drug-induced hepatitis 24 acute inflammation 25 chronic inflammation 26 acute hepatitis 27 chronic hepatitis 28 (inflammation caused by) autoimmune disorder (inflammation caused by) atherosclerosis (inflammation caused by) allergy (inflammation caused by) myopathy (inflammation caused by) cancer pharmacologically induced inflammation (e.g. drug overdose) toxin-induced inflammation radiation-induced inflammation heavy metal-induced inflammation sepsis pathogen-induced (i.e. infection) inflammation arthritis (inflammation caused by) migraine (inflammation caused by) coeliac disease inflammatory bowel disease endometriosis (inflammation caused by) diabetes (type 1 or type 2) (inflammation caused by) asthma rheumatoid artritis (inflammation caused by) obesity (inflammation caused by) Alzheimer’ disease (inflammation caused by) Parkinson’s disease encephalitis (inflammation caused by) lupus myositis (inflammation caused by) scleroderma uveitis vasculitis (inflammation caused by) heart disease (inflammation caused by) psoriasis (inflammation caused by) multiple sclerosis (inflammation caused by) sarcoidosis meningitis (inflammation caused by) cystic fibrosis 62 (inflammation caused by) gout 63 dermatitis 64 nephritis 65 pancreatitis 66 (inflammation caused by) Crohn’s disease 67 ankylosing spondylitis 68 (inflammation caused by) COPD In certain embodiments, any of the single domain antibodies of the invention as described herein (or binding agents comprising the single-domain antibody), such as in particular according to embodiments A to X in Table A can be for use in the treatment of, can be used in a method for the treatment of, can be used in the manufacture for a medicament for the treatment of, or can be used in the diagnosis of any medical condition or indication as described in Table B. In certain embodiments, the single domain antibody (or binding agent)/medical indication combination (with reference to respectively embodiments A to X of Table A and embodiments 1 to 68 of Table B) is selected from A-1, B-1, C-1, D-1, E-1, F-1, G-1, H-1, I-1, J-1, K-1, L-1, M-1, N-1, O-1, P-1, Q-1, R-1, S- 1, T-1, U-1, V-1, W-1, X-1, A-2, B-2, C-2, D-2, E-2, F-2, G-2, H-2, I-2, J-2, K-2, L-2, M-2, N-2, O-2, P-2, Q-2, R-2, S-2, T-2, U-2, V-2, W-2, X-2, A-3, B-3, C-3, D-3, E-3, F-3, G-3, H-3, I-3, J-3, K-3, L-3, M-3, N-3, O-3, P-3, Q-3, R-3, S-3, T-3, U-3, V-3, W-3, X-3, A-4, B-4, C-4, D-4, E-4, F-4, G-4, H-4, I-4, J-4, K-4, L-4, M-4, N-4, O-4, P-4, Q-4, R-4, S-4, T- 4, U-4, V-4, W-4, X-4, A-5, B-5, C-5, D-5, E-5, F-5, G-5, H-5, I-5, J-5, K-5, L-5, M-5, N-5, O-5, P-5, Q-1, R-5, S-5, T-5, U-5, V-5, W-5, X-5, A-6, B-6, C-6, D-6, E-6, F-6, G-6, H-6, I-6, J-6, K-6, L-6, M-6, N-6, O-6, P-6, Q-2, R-6, S-6, T-6, U-6, V-6, W-6, X-6, A-7, B-7, C-7, D-7, E-7, F-7, G-7, H-7, I-7, J-7, K-7, L-7, M-7, N-7, O-7, P-7, Q-3, R-7, S-7, T-7, U- 7, V-7, W-7, X-7, A-8, B-8, C-8, D-8, E-8, F-8, G-8, H-8, I-8, J-8, K-8, L-8, M-8, N-8, O- 8, P-8, Q-4, R-8, S-8, T-8, U-8, V-8, W-8, X-8, A-9, B-9, C-9, D-9, E-9, F-9, G-9, H-9, I- 9, J-9, K-9, L-9, M-9, N-9, O-9, P-9, Q-1, R-9, S-9, T-9, U-9, V-9, W-9, X-9, A-10, B-10, C-10, D-10, E-10, F-10, G-10, H-10, I-10, J-10, K-10, L-10, M-10, N-10, O-10, P-10, Q- 2, R-10, S-10, T-10, U-10, V-10, W-10, X-10, A-11, B-11, C-11, D-11, E-11, F-11, G-11, H-11, I-11, J-11, K-11, L-11, M-11, N-11, O-11, P-11, Q-3, R-11, S-11, T-11, U-11, V-11, W-11, X-11, A-12, B-12, C-12, D-12, E-12, F-12, G-12, H-12, I-12, J-12, K-12, L-12, M- 12, N-12, O-12, P-12, Q-4, R-12, S-12, T-12, U-12, V-12, W-12, X-12, A-13, B-13, C-13, D-13, E-13, F-13, G-13, H-13, I-13, J-13, K-13, L-13, M-13, N-13, O-13, P-13, Q-1, R-13, S-13, T-13, U-13, V-13, W-13, X-13, A-14, B-14, C-14, D-14, E-14, F-14, G-14, H-14, I- 14, J-14, K-14, L-14, M-14, N-14, O-14, P-14, Q-2, R-14, S-14, T-14, U-14, V-14, W-14, X-14, A-15, B-15, C-15, D-15, E-15, F-15, G-15, H-15, I-15, J-15, K-15, L-15, M-15, N- 15, O-15, P-15, Q-3, R-15, S-15, T-15, U-15, V-15, W-15, X-15, A-16, B-16, C-16, D-16, E-16, F-16, G-16, H-16, I-16, J-16, K-16, L-16, M-16, N-16, O-16, P-16, Q-4, R-16, S-16, T-16, U-16, V-16, W-16, X-16, A-17, B-17, C-17, D-17, E-17, F-17, G-17, H-17, I-17, J- 17, K-17, L-17, M-17, N-17, O-17, P-17, Q-1, R-17, S-17, T-17, U-17, V-17, W-17, X-17, A-18, B-18, C-18, D-18, E-18, F-18, G-18, H-18, I-18, J-18, K-18, L-18, M-18, N-18, O- 18, P-18, Q-2, R-18, S-18, T-18, U-18, V-18, W-18, X-18, A-19, B-19, C-19, D-19, E-19, F-19, G-19, H-19, I-19, J-19, K-19, L-19, M-19, N-19, O-19, P-19, Q-3, R-19, S-19, T-19, U-19, V-19, W-19, X-19, A-20, B-20, C-20, D-20, E-20, F-20, G-20, H-20, I-20, J-20, K- 20, L-20, M-20, N-20, O-20, P-20, Q-4, R-20, S-20, T-20, U-20, V-20, W-20, X-20, A-21, B-21, C-21, D-21, E-21, F-21, G-21, H-21, I-21, J-21, K-21, L-21, M-21, N-21, O-21, P- 21, Q-1, R-21, S-21, T-21, U-21, V-21, W-21, X-21, A-22, B-22, C-22, D-22, E-22, F-22, G-22, H-22, I-22, J-22, K-22, L-22, M-22, N-22, O-22, P-22, Q-2, R-22, S-22, T-22, U-22, V-22, W-22, X-22, A-23, B-23, C-23, D-23, E-23, F-23, G-23, H-23, I-23, J-23, K-23, L- 23, M-23, N-23, O-23, P-23, Q-3, R-23, S-23, T-23, U-23, V-23, W-23, X-23, A-24, B-24, C-24, D-24, E-24, F-24, G-24, H-24, I-24, J-24, K-24, L-24, M-24, N-24, O-24, P-24, Q- 4, R-24, S-24, T-24, U-24, V-24, W-24, X-24, A-25, B-25, C-25, D-25, E-25, F-25, G-25, H-25, I-25, J-25, K-25, L-25, M-25, N-25, O-25, P-25, Q-1, R-25, S-25, T-25, U-25, V-25, W-25, X-25, A-26, B-26, C-26, D-26, E-26, F-26, G-26, H-26, I-26, J-26, K-26, L-26, M- 26, N-26, O-26, P-26, Q-2, R-26, S-26, T-26, U-26, V-26, W-26, X-26, A-27, B-27, C-27, D-27, E-27, F-27, G-27, H-27, I-27, J-27, K-27, L-27, M-27, N-27, O-27, P-27, Q-3, R-27, S-27, T-27, U-27, V-27, W-27, X-27, A-28, B-28, C-28, D-28, E-28, F-28, G-28, H-28, I- 28, J-28, K-28, L-28, M-28, N-28, O-28, P-28, Q-4, R-28, S-28, T-28, U-28, V-28, W-28, X-28, A-29, B-29, C-29, D-29, E-29, F-29, G-29, H-29, I-29, J-29, K-29, L-29, M-29, N- 29, O-29, P-29, Q-1, R-29, S-29, T-29, U-29, V-29, W-29, X-29, A-30, B-30, C-30, D-30, E-30, F-30, G-30, H-30, I-30, J-30, K-30, L-30, M-30, N-30, O-30, P-30, Q-2, R-30, S-30, T-30, U-30, V-30, W-30, X-30, A-31, B-31, C-31, D-31, E-31, F-31, G-31, H-31, I-31, J- 31, K-31, L-31, M-31, N-31, O-31, P-31, Q-3, R-31, S-31, T-31, U-31, V-31, W-31, X-31, A-32, B-32, C-32, D-32, E-32, F-32, G-32, H-32, I-32, J-32, K-32, L-32, M-32, N-32, O- 32, P-32, Q-4, R-32, S-32, T-32, U-32, V-32, W-32, X-32, A-33, B-33, C-33, D-33, E-33, F-33, G-33, H-33, I-33, J-33, K-33, L-33, M-33, N-33, O-33, P-33, Q-1, R-33, S-33, T-33, U-33, V-33, W-33, X-33, A-34, B-34, C-34, D-34, E-34, F-34, G-34, H-34, I-34, J-34, K- 34, L-34, M-34, N-34, O-34, P-34, Q-2, R-34, S-34, T-34, U-34, V-34, W-34, X-34, A-35, B-35, C-35, D-35, E-35, F-35, G-35, H-35, I-35, J-35, K-35, L-35, M-35, N-35, O-35, P- 35, Q-3, R-35, S-35, T-35, U-35, V-35, W-35, X-35, A-36, B-36, C-36, D-36, E-36, F-36, G-36, H-36, I-36, J-36, K-36, L-36, M-36, N-36, O-36, P-36, Q-4, R-36, S-36, T-36, U-36, V-36, W-36, X-36, A-37, B-37, C-37, D-37, E-37, F-37, G-37, H-37, I-37, J-37, K-37, L- 37, M-37, N-37, O-37, P-37, Q-1, R-37, S-37, T-37, U-37, V-37, W-37, X-37, A-38, B-38, C-38, D-38, E-38, F-38, G-38, H-38, I-38, J-38, K-38, L-38, M-38, N-38, O-38, P-38, Q- 2, R-38, S-38, T-38, U-38, V-38, W-38, X-38, A-39, B-39, C-39, D-39, E-39, F-39, G-39, H-39, I-39, J-39, K-39, L-39, M-39, N-39, O-39, P-39, Q-3, R-39, S-39, T-39, U-39, V-39, W-39, X-39, A-40, B-40, C-40, D-40, E-40, F-40, G-40, H-40, I-40, J-40, K-40, L-40, M- 40, N-40, O-40, P-40, Q-4, R-40, S-40, T-40, U-40, V-40, W-40, X-40, A-41, B-41, C-41, D-41, E-41, F-41, G-41, H-41, I-41, J-41, K-41, L-41, M-41, N-41, O-41, P-41, Q-1, R-41, S-41, T-41, U-41, V-41, W-41, X-41, A-42, B-42, C-42, D-42, E-42, F-42, G-42, H-42, I- 42, J-42, K-42, L-42, M-42, N-42, O-42, P-42, Q-2, R-42, S-42, T-42, U-42, V-42, W-42, X-42, A-43, B-43, C-43, D-43, E-43, F-43, G-43, H-43, I-43, J-43, K-43, L-43, M-43, N- 43, O-43, P-43, Q-3, R-43, S-43, T-43, U-43, V-43, W-43, X-43, A-44, B-44, C-44, D-44, E-44, F-44, G-44, H-44, I-44, J-44, K-44, L-44, M-44, N-44, O-44, P-44, Q-4, R-44, S-44, T-44, U-44, V-44, W-44, X-44, A-45, B-45, C-45, D-45, E-45, F-45, G-45, H-45, I-45, J- 45, K-45, L-45, M-45, N-45, O-45, P-45, Q-1, R-45, S-45, T-45, U-45, V-45, W-45, X-45, A-46, B-46, C-46, D-46, E-46, F-46, G-46, H-46, I-46, J-46, K-46, L-46, M-46, N-46, O- 46, P-46, Q-2, R-46, S-46, T-46, U-46, V-46, W-46, X-46, A-47, B-47, C-47, D-47, E-47, F-47, G-47, H-47, I-47, J-47, K-47, L-47, M-47, N-47, O-47, P-47, Q-3, R-47, S-47, T-47, U-47, V-47, W-47, X-47, A-48, B-48, C-48, D-48, E-48, F-48, G-48, H-48, I-48, J-48, K- 48, L-48, M-48, N-48, O-48, P-48, Q-4, R-48, S-48, T-48, U-48, V-48, W-48, X-48, A-49, B-49, C-49, D-49, E-49, F-49, G-49, H-49, I-49, J-49, K-49, L-49, M-49, N-49, O-49, P- 49, Q-1, R-49, S-49, T-49, U-49, V-49, W-49, X-49, A-50, B-50, C-50, D-50, E-50, F-50, G-50, H-50, I-50, J-50, K-50, L-50, M-50, N-50, O-50, P-50, Q-2, R-50, S-50, T-50, U-50, V-50, W-50, X-50, A-51, B-51, C-51, D-51, E-51, F-51, G-51, H-51, I-51, J-51, K-51, L- 51, M-51, N-51, O-51, P-51, Q-3, R-51, S-51, T-51, U-51, V-51, W-51, X-51, A-52, B-52, C-52, D-52, E-52, F-52, G-52, H-52, I-52, J-52, K-52, L-52, M-52, N-52, O-52, P-52, Q- 4, R-52, S-52, T-52, U-52, V-52, W-52, X-52, A-53, B-53, C-53, D-53, E-53, F-53, G-53, H-53, I-53, J-53, K-53, L-53, M-53, N-53, O-53, P-53, Q-1, R-53, S-53, T-53, U-53, V-53, W-53, X-53, A-54, B-54, C-54, D-54, E-54, F-54, G-54, H-54, I-54, J-54, K-54, L-54, M- 54, N-54, O-54, P-54, Q-2, R-54, S-54, T-54, U-54, V-54, W-54, X-54, A-55, B-55, C-55, D-55, E-55, F-55, G-55, H-55, I-55, J-55, K-55, L-55, M-55, N-55, O-55, P-55, Q-3, R-55, S-55, T-55, U-55, V-55, W-55, X-55, A-56, B-56, C-56, D-56, E-56, F-56, G-56, H-56, I- 56, J-56, K-56, L-56, M-56, N-56, O-56, P-56, Q-4, R-56, S-56, T-56, U-56, V-56, W-56, X-56, A-57, B-57, C-57, D-57, E-57, F-57, G-57, H-57, I-57, J-57, K-57, L-57, M-57, N- 57, O-57, P-57, Q-1, R-57, S-57, T-57, U-57, V-57, W-57, X-57, A-58, B-58, C-58, D-58, E-58, F-58, G-58, H-58, I-58, J-58, K-58, L-58, M-58, N-58, O-58, P-58, Q-2, R-58, S-58, T-58, U-58, V-58, W-58, X-58, A-59, B-59, C-59, D-59, E-59, F-59, G-59, H-59, I-59, J- 59, K-59, L-59, M-59, N-59, O-59, P-59, Q-3, R-59, S-59, T-59, U-59, V-59, W-59, X-59, A-60, B-60, C-60, D-60, E-60, F-60, G-60, H-60, I-60, J-60, K-60, L-60, M-60, N-60, O- 60, P-60, Q-4, R-60, S-60, T-60, U-60, V-60, W-60, X-60, A-61, B-61, C-61, D-61, E-61, F-61, G-61, H-61, I-61, J-61, K-61, L-61, M-61, N-61, O-61, P-61, Q-1, R-61, S-61, T-61, U-61, V-61, W-61, X-61, A-62, B-62, C-62, D-62, E-62, F-62, G-62, H-62, I-62, J-62, K- 62, L-62, M-62, N-62, O-62, P-62, Q-2, R-62, S-62, T-62, U-62, V-62, W-62, X-62, A-63, B-63, C-63, D-63, E-63, F-63, G-63, H-63, I-63, J-63, K-63, L-63, M-63, N-63, O-63, P- 63, Q-3, R-63, S-63, T-63, U-63, V-63, W-63, X-63, A-64, B-64, C-64, D-64, E-64, F-64, G-64, H-64, I-64, J-64, K-64, L-64, M-64, N-64, O-64, P-64, Q-4, R-64, S-64, T-64, U-64, V-64, W-64, X-64, A-65, B-65, C-65, D-65, E-65, F-65, G-65, H-65, I-65, J-65, K-65, L- 65, M-65, N-65, O-65, P-65, Q-1, R-65, S-65, T-65, U-65, V-65, W-65, X-65, A-66, B-66, C-66, D-66, E-66, F-66, G-66, H-66, I-66, J-66, K-66, L-66, M-66, N-66, O-66, P-66, Q- 2, R-66, S-66, T-66, U-66, V-66, W-66, X-66, A-67, B-67, C-67, D-67, E-67, F-67, G-67, H-67, I-67, J-67, K-67, L-67, M-67, N-67, O-67, P-67, Q-3, R-67, S-67, T-67, U-67, V-67, W-67, X-67, A-68, B-68, C-68, D-68, E-68, F-68, G-68, H-68, I-68, J-68, K-68, L-68, M- 68, N-68, O-68, P-68, Q-4, R-68, S-68, T-68, U-68, V-68, W-68, X-68. The aspects and embodiments of the invention are further supported by the following non-limiting examples. The following examples, including the experiments conducted and the results achieved, are provided for illustrative purposes only and are not constructed as limiting the present invention. The use of these and other examples anywhere in the specification is illustrative only and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to any particular preferred embodiments described here. Indeed, many modifications and variations of the invention may be apparent to those skilled in the art upon reading this specification, and such variations can be made without departing from the invention in spirit or in scope. The invention is therefore to be limited only by the terms of the appended claims along with the full scope of equivalents to which those claims are entitled. EXAMPLES EXAMPLE 1: Novel nanobody-based inhibitors of Panx1 channel activity reduce inflammation in vitro and in a mouse model of acute liver injury 1. Materials and methods 1.1. Panx1 nanobody generation and in vitro characterisation 1.1.1. Generation of DUBCA Panx1 cells SV40 immortalised Dubai camel (DUBCA) (Camelus dromedarius) fibroblasts were cultured in cell culture medium, Dulbecco’s modified Eagle’s medium (low glucose, GlutaMAX, pyruvate) (Thermo Fisher Scientific, Belgium) supplemented with 10% v/v fetal bovine serum (Thermo Fisher Scientific, Belgium), 50 µg/mL streptomycin sulphate (Merck, Germany) and 7.33 I.E./mL sodium benzyl penicillin (Continental Pharma, Belgium), at 37 °C with a constant supply of 5% CO2 and split every 3 days. To obtain cells expressing mouse Panx1 (mPanx1) or human Panx1 (hPanx1), DUBCA cells were transduced. Briefly, pRP expression vectors encoding mPanx1 or hPanx1 (VectorBuilder, USA) and gBlocks (Integrated DNA Technologies, USA) were used to design pASIET vectors (Addgene plasmid 17448, Trono Lab) containing Panx1 constructs. These transfer vectors were used for lentiviral production. A transient transfection mixture containing 4.5 µg/mL transfer plasmids, 3 µg/mL packaging plasmid pCMVΔR8.9 (Addgene plasmid 12263, Trono Lab), 1.5 µg/mL VSV.G encoding plasmid pMD.G (Addgene plasmid 12259, Trono Lab) and 18 µg/mL polyethylenimine (Polysciences, Germany) was dropwisely added to human embryonal kidney (HEK) 293T cells and incubated for 4 hours at 37 °C with CO2 supply. After 4 hours, the transient transfection mixture was removed and replaced with cell culture medium for 48 hours. Viral vectors were harvested in the cell culture medium and concentrated with ultracentrifugation at 20,000 x g for 5 minutes. After transient transfection of HEK 293T cells, lentiviral particles harbouring the mPanx1 or hPanx1 coding sequence, containing a 6x His detection tag at the carboxyterminal tail, were used to transduce DUBCA cells. DUBCA cells were cultured at a density of 100,000 cells/well in 1 mL/well of cell culture medium for 24 hours and transduced with lentiviral particles at a multiplicity of infection of 20 for 72 hours. Transduction of DUBCA cells was evaluated via flow cytometry of enhanced green fluorescent protein (eGFP) expression. Transduced cells are referred to as DUBCA mPanx1 and DUBCA hPanx1, non-transduced cells are named as DUBCA wild-type (WT) cells in this paper. 1.1.2. In vitro characterisation of DUBCA Panx1 cells Panx1 expression in DUBCA WT, DUBCA mPanx1 and DUBCA hPanx1 cells was studied using immunoblot (2.1.2.1.) and immunocytochemistry (2.1.2.2.) analyses. 1.1.2.1. Immunoblot analysis For immunoblotting, cells were harvested from culture flasks by dissociation with TrypLE (Thermo Fisher Scientific, Belgium). Proteins were isolated by homogenising cell pellets in radioimmunoprecipitation (RIPA) buffer (Thermo Fisher Scientific, Belgium) supplemented with 1% v/v ethylenediaminetetraacetic acid (EDTA) solution (Thermo Fisher Scientific, Belgium) and 1% v/v protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Belgium). Samples were mixed and placed on ice for 20 minutes. Thereafter, cell lysates were centrifugated at 14,000 x g for 20 minutes and proteins in supernatants were collected. Protein concentrations of each sample were determined according to the bicinchoninic acid (BCA) method using a BCA Protein Assay Kit (Thermo Fisher Scientific, Belgium) with bovine serum albumin (BSA) as a standard. Proteins were fractionated on sodium dodecyl sulphate (SDS) polyacrylamide gels (Bio- Rad Laboratories, USA) and blotted afterwards onto a nitrocellulose membrane (Bio-Rad Laboratories, USA). Membranes were incubated with a primary antibody directed against Panx1 (D9M1C) (Cell Signaling Technology, USA) (Table 1) followed by incubation with an appropriate horseradish peroxidase-conjugated secondary antibody (P0448) (Dako, USA) (Table 1). Detection of Panx1 was carried out by means of enhanced chemiluminescence. For semi-quantification purposes, a normalisation method based on total protein loading was used to overcome the drawbacks associated with the use of housekeeping proteins. Panx1 signals in DUBCA mPanx1 and DUBCA hPanx1 cells were normalised against total protein loading and expressed as relative alterations compared to DUBCA WT cells. 1.1.2.2. Immunocytochemistry analysis Immunocytochemistry analysis was performed by culturing DUBCA WT, DUBCA mPanx1 and DUBCA hPanx1 cells at a density of 25,000 cells/well in 750 µL/well of cell culture medium for 24 hours. After aspirating the supernatant from each well, cells were fixed with 4% w/v paraformaldehyde (Polysciences, USA) in phosphate-buffered saline (PBS) and permeabilised with PBS enriched with 0.1% v/v Triton X-100 (Thermo Fisher Scientific, Belgium). Cells were incubated with a primary antibody directed against Panx1 (ABN242) (Merck, Germany) (Table 1) followed by incubation with an Alexa Fluor® 594- conjugated secondary antibody donkey anti-rabbit (857218) (Thermo Fisher Scientific, Belgium) (Table 1). Nuclear counterstaining was performed with Hoechst solution (H3570) (Thermo Fisher Scientific, Belgium) and Vectashield (Vector laboratories, USA) was used as mounting medium. For negative controls, an identical procedure was followed, but the primary antibody was omitted. Detection was performed using a fluorescence microscope Nikon Eclipse Ti (Nikon, Japan). 1.1.3. Generation of Panx1 nanobodies Panx1 nanobodies were produced by transforming bacteria with the pRP expression vector encoding mPanx1 to obtain endotoxin free plasmid. A llama (Lama glama) (Lamasté, Belgium) was immunised 4 times at biweekly intervals with 2 mg of vectors expressing the mPanx1 gene and subsequently boosted 2 times with 2.107 DUBCA cells overexpressing mPanx1. Following immunisation, mRNA from peripheral blood lymphocytes of the llama was reverse transcribed to cDNA using the SuperScript II First- Strand Synthesis System for reverse transcription polymerase chain reaction (Thermo Fisher Scientific, Belgium). Nanobody coding sequences were amplified by polymerase chain reaction analysis with CALL001 (5’-GTCCTGGCTGCTCTTCTACAAGG-3’ – SEQ ID NO: 67), CALL002 (5’-GGTACGTGCTGTTGAACTGTTCC-3’ – SEQ ID NO: 68), VHH-BACK (5’-GATGTGCAGCTGCAGGAGTCTGGRGGAGG-3’ – SEQ ID NO: 69) and PMCF (5’-CTAGTGCGGCCGCTGAGGAGACGGTGACCTGGGT-3’ – SEQ ID NO: 70) primers, and ligated into a variant of a pMECS phagemid vector using PstI, NotI and XbaI restriction enzymes (Thermo Fisher Scientific, Belgium) and Golden Gate cloning strategy. Electrocompetent Escherichia coli TG1 cells were transformed with the ligated material and grown for 24 hours to construct a library of nanobodies of 107 individual transformants. M13K07 helper phages were added at multiplicity of infection of 20 for 30 minutes to display the nanobodies at the tip of the phages. After infecting, Escherichia coli TG1 cells were grown for 24 hours and centrifuged at 2,200 x g for 30 minutes to collect virus particles. Panx1 nanobodies were retrieved from the resulting library of phage displayed nanobodies by biopanning on DUBCA cells. Selection rounds on DUBCA WT and DUBCA mPanx1 cells allowed to identify single individual colonies. These colonies were screened through an enzyme-linked immunosorbent assay (ELISA) on DUBCA mPanx1 cells for antigen recognition. Positive clones were selected and sequenced. Next, 7 Panx1 nanobody clones and the non-targeting control nanobody clone R3b23 were selected for large-scale production. Nanobody periplasmic expression is started from a single colony of Escherichia coli WK6 cells transformed with pMECS- derived vectors containing nanobody gene. The expression of Panx1 nanobodies and R3b23 nanobody in periplasm was induced by isopropyl-β-D-thiogalactoside (Duchefa Biochemie, Netherlands). Periplasmic extracts were collected via an osmotic shock, using a TES solution containing 25% v/v diluted TES buffer (500 mM sucrose (Duchefa Biochemie, Netherlands), 200 mM Tris-HCl (Merck, Germany) and 0.50 mM EDTA (Duchefa Biochemie, Netherlands)). Next, immobilized metal affinity chromatography (IMAC) on His-Select Nickel Affinity Gel (Merck, Germany) combined with 500 mM imidazole(Merck, Germany)-mediated elution was used to capture produced nanobodies from periplasmic extracts. For further purification and quality control of nanobodies, size exclusion chromatography was performed on IMAC-eluted samples using an AKTAxpress chromatography system equipped with a HiLoad S75 (16/60) column (Cytiva, Belgium). 1.1.4. Flow cytometry To identify Panx1-specific and cross-reactive nanobodies, nanobodies were tested using a FACSCelesta flow cytometer (BD Biosciences, USA). DUBCA WT, DUBCA mPanx1 and DUBCA hPanx1 cells were harvested from culture flasks by dissociation with TrypLE. Following centrifugation at 1,500 x g for 5 minutes, 105 cells were preincubated with 1 µg of each Panx1 nanobody. Thereafter, cells were incubated with allophycocyanin- conjugated hemagglutinin antibody (901524) (BioLegend, USA) (Table 1) and binding of Panx1 nanobodies was detected via flow cytometry analysis. Besides R3b23, other negative controls were included by omitting nanobodies and incubating the cells with or without allophycocyanin-conjugated hemagglutinin antibody. 1.1.5. Immunocytochemistry In vitro targeting of Panx1 nanobodies was evaluated in DUBCA WT and DUBCA hPanx1 cells using immunocytochemistry analysis. DUBCA cells were cultured at a density of 25,000 cells/well in 750 µL/well culture medium for 24 hours. Non- permeabilized cells were incubated with Panx1 nanobodies or R3b23 nanobody (1,500 nM) in combination with Alexa Fluor® 594-conjugated hemagglutinin antibody (901511) (BioLegend, USA) (Table 1). Next, nuclear counterstaining was performed with Hoechst solution and Vectashield was used as mounting medium. Nanobody binding was detected using a fluorescence microscope Nikon Eclipse Ti. 1.1.6. Cell-based enzyme-linked immunosorbent assay The affinity of the Panx1 nanobodies was determined in DUBCA WT and DUBCA hPanx1 cells using cell-based ELISA. DUBCA WT and DUBCA hPanx1 cells were cultured at a density of 12,000 cells/well in 200 µL/well cell culture medium. Cells were grown for 24 hours and fixed with 4% w/v paraformaldehyde in PBS and incubated with different concentrations of Panx1 nanobodies and R3b23 nanobody in the range of 0- 600 nM. Horseradish peroxidase-conjugated hemagglutinin antibody (901519) (BioLegend, USA) (Table 1) was added and cells were incubated with 1-Step Ultra TMB- ELISA (Thermo Fisher Scientific, Belgium). Then, 2 M sulphuric acid (Merck, Germany) was added to stop the reaction and absorbance was measured at 450 nm with a FLUOstar OPTIMA microplate reader (BMG LABTECH, Germany) to assess the equilibrium dissociation constants (Kd) of the Panx1 nanobodies. The specificity of the Panx1 nanobodies was further tested by performing cell-based ELISA with rat glioma cells. Rat C6 wild-type cells and C6 cells stably expressing rat Panx1, referred to as C6 WT and C6 Panx1 cells, respectively, were kindly provided by Prof. Christian Naus (University of British Columbia, Canada). Panx1 expression in C6 WT and C6 Panx1 cells was studied using immunoblot and immunocytochemistry analyses as described in section 1.1.2.1. and 1.1.2.2, respectively. Cell-based ELISA was performed using C6 WT and C6 Panx1 cells. Concentrations of the Panx1 nanobodies and R3b23 nanobody up to 12,000 nM were used. Specificity of the Panx1 nanobodies was assessed by measuring absorbance values at 450 nm with a FLUOstar OPTIMA microplate reader. 1.1.7. Panx1 channel activity assay The inhibitory effects of the Panx1 nanobodies on Panx1 channel activity were assessed following potassium-induced channel opening in DUBCA hPanx1 cells and measurement of extracellular release ATP. DUBCA hPanx1 cells were cultured at a density of 12,000 cells/well in 200 µL/well cell culture medium (Thermo Fisher Scientific, Belgium) and grown for 24 hours. Cell culture medium was changed with preheated buffer (137 mM NaCl, 2.68 mM KCl, 11.90 mM NaHCO3, 0.42 mM NaH2PO4.H2O, 1 mM MgCl2, 2 mM CaCl2.2H2O, 5 mM HEPES and 0.1% w/v glucose) ((Merck, Germany) for 30 minutes at 37 °C with a constant supply of 5% CO2. Panx1 channels were opened by switching to a buffer with increased potassium concentration (22.93 mM NaCl, 5 mM KCl, 5.95 mM NaHCO3, 0.21 mM NaH2PO4.H2O, 1 mM MgCl2, 2 mM CaCl2.2H2O, 5 mM HEPES and 0.1% w/v glucose). Stock solutions of carbenoxolone disodium salt (100-200 µM) (Merck, Germany), lanthanum trichloride (100-200 µM) (Merck, Germany), 10Panx1 (300-400 µM) (Thermo Fisher Scientific, Belgium) and Panx1 nanobodies and R3b23 nanobody (0-1,000 nM) were prepared ex tempore in buffer with and without increased potassium concentration. Supernatants from each well were aspirated and preconditioned with appropriate preheated buffer samples in a humidified 5% CO2 incubator at 37 °C for 15 minutes. Cells were subsequently exposed to preheated buffer with higher potassium concentration for another 30 minutes at 37 °C with a constant supply of 5% CO2. Extracellular ATP levels were assessed using an ATP Bioluminescent Assay Kit (213-579-1) (Merck, Germany). The amount of emitted light by the samples was immediately measured with a VICTOR3 Multilabel Plate Reader (PerkinElmer, USA). Extracellular ATP release was expressed as the percentage of ATP relative to the release level triggered by the buffer with increased potassium concentration. 1.1.8. In vitro inflammation assay The anti-inflammatory effects of the Panx1 nanobodies were tested in RAW264.7 and differentiated THP-1 cells using flow cytometry and ELISA analyses, respectively. RAW264.7 cells were cultured at a density of 106 cells/well in 1 mL/well culture medium for 24 hours. Cell culture media was removed and RAW264.7 cells were consecutively exposed to 1 µg/mL LPS (4 hours) (L4391) (Merck, Germany), 300 ng/mL brefeldin A (3 hours) (Abcam, UK), Panx1 nanobodies and R3b23 nanobody (1,000 nM) (1 hour) and 5 mM ATP (30 minutes) (Thermo Fisher Scientific, Belgium) at 37 °C with a constant supply of 5% CO2. For assessing the anti-inflammatory effects of Panx1 nanobodies, RAW264.7 cells were incubated with a primary antibody directed against IL-1β (ab254360) (Abcam, UK) (Table 1). Following incubation with an Alexa Fluor® 488- conjugated secondary antibody goat anti-rabbit (ab150077) (Abcam, UK) (Table 1) and nuclear staining with Hoechst solution, detection of IL-1β was carried out on an Attune NxT flow cytometer (Thermo Fisher Scientific, Belgium). Anti-inflammatory effects of Panx1 nanobodies in THP-1 cells was determined by measuring IL-1β signals in supernatants using an IL-1β human ELISA Kit (88-7261-88) (Thermo Fisher Scientific, Belgium). THP-1 cells were differentiated with 0.2 µM phorbol 12-myristate 13-acetate (Merck, Germany) and subsequently exposed to 1 µg/mL LPS (4 hours), Panx1 nanobodies and R3b23 nanobody (1,000 nM) (1 hour) and 5 mM ATP (30 minutes). Detection of IL-1β quantities in supernatants was performed with a with a FLUOstar OPTIMA microplate reader. For positive controls, RAW264.7 and differentiated THP-1 cells were treated with Caspase-1 Inhibitor II (5-25 µM) (Merck, Germany) and MCC950 (10-50 µM) (Merck, Germany) for 1 hour. 1.1.9. Specificity and affinity of Panx1 nanobodies towards mouse Panx1 The specificity of 4 Panx1 nanobodies (Nb1, Nb3, Nb9 and Nb30) was determined in DUBCA WT and DUBCA mPanx1 cells using cell-based ELISA as described in section 1.1.6. For measuring the affinity of the Panx1 nanobodies, binding of Panx1 nanobodies was measured via flow cytometry analysis. DUBCA WT and DUBCA mPanx1 cells were harvested from culture flasks by dissociation with TrypLE. Following centrifugation at 1,500 x g for 5 minutes, 105 cells were preincubated with different concentrations of Panx1 nanobodies and R3b23 nanobody in the range of 0-600 nM. Thereafter, cells were incubated with Alexa Fluor® 488-conjugated hemagglutinin antibody (901509) (BioLegend, USA) (Table 1) and Hoechst solution. Binding of Panx1 nanobodies was detected on Hoechst positive cells using an Attune NxT flow cytometer. 1.2. Panx1 nanobody in vivo testing 1.2.1. Set-up of the mouse model of acute liver injury Male C57BL/6 mice of approximately 2 months of age were used and housed in the animal facility of the Faculty of Medicine and Pharmacy of the VUB-Belgium. Wild type (Charles River Laboratories, France) and Panx1-/- animals were kept under controlled environmental conditions with free access to food and water. Mice were starved 16 hours prior to acetaminophen administration. Acetaminophen (APAP) (Merck, Germany) was dissolved in PBS, slightly heated and injected (37 °C) intraperitoneally at 300 mg/kg body weight.1 group of animals was not injected with APAP. After 2 hours, 1 group of APAP- overdosed mice was additionally administered either Panx1 nanobody or R3b23 nanobody diluted in PBS at 10 mg/kg body weight through intraperitoneal injection. At the same time, another group of acetaminophen-overdosed mice was injected with N- acetylcysteine (Merck, Germany) in PBS at 200 mg/kg body weight. All mice were euthanised 24 hours following APAP overdosing under ketamine and xylazine-induced anesthesia. Blood, collected by cardiac puncture, was centrifugated at 2,000 x g for 10 minutes, and serum was stored at -80 °C. Livers were excised and fragments were fixed in 4% phosphate-buffered formalin (ProSan, Belgium) or snap-frozen in liquid nitrogen with storage at -80°C. This protocol has been approved by the local Ethical Committee of the VUB-Belgium (project number 20-210-8) and all animals received human care according to the criteria by the guidelines provided by the Ethical Committee of the VUB- Belgium. 1.2.2. Immunoblot analysis Flash frozen liver tissue was homogenised in RIPA buffer supplemented with 1% v/v EDTA and 1% v/v protease and phosphatase inhibitor cocktail. Following homogenization, samples were centrifugated at 14,000 x g for 20 minutes and protein concentrations were determined in the supernatants by means of a BCA assay. Following electrophoresis, blotting and blocking, membranes were incubated with primary antibody directed against Bax (sc-7480) (Santa Cruz, USA), Bcl-2 (554087) (BD Biosciences, USA), caspase-1 (sc-514) (Santa Cruz, USA), CYP2E1 (HPA009128) (Atlas Antibodies, Sweden), IL-1β (ab254360) (Abcam, UK), IFN-γ (ab216642) (Abcam, UK), NLRP3 (ab263899) (Abcam, UK) and Panx1 (D9M1C) (Cell Signaling Technology, USA) (Table 1). Membranes were subsequently incubated with horseradish peroxidase- conjugated secondary antibodies (P0447 and P0448) (Dako, USA) (Table 1). Detection of liver protein expression was carried out by means of enhanced chemiluminescence. For semi-quantification purposes, liver protein expression levels were normalised against total protein loading. 1.2.3. Cytokine analysis Serum cytokine levels were measured using a mouse inflammation antibody array (ab133999) (Abcam, UK). After blocking the antibody array membranes with the provided blocking buffer for 30 minutes, membranes were incubated with collected serum samples for 24 hours. Thereafter, membranes were washed with provided washing buffers for 25 minutes and treated with the supplied cocktail of biotin-conjugated antibodies. After 2 hours, antibody array membranes were washed for another 25 minutes. Serum cytokine levels were assayed by incubating membranes with the provided horseradish peroxidase-conjugated streptavidin solution for 2 hours and chemiluminescence detection solution for 2 minutes. Chemiluminescence signals were measured with a ChemiDoc MP Imaging System (Bio-Rad Laboratories, USA) and densitometric analysis was performed with Image Lab Software (Bio-Rad Laboratories, USA) according to the manufacturer’s instructions. After background subtraction and normalisation to the amount of biotin-conjugated IgG protein printed on each membrane, cytokine levels of 40 inflammatory factors were calculated and expressed as relative alterations compared to APAP-overdosed mice. 1.2.4. Analysis of serum aminotransaminases Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were measured using an AST Assay Kit (ARG81297) and an ALT Assay Kit (ARG82198) (Bio- Connect, Netherlands), respectively. Serum aminotransferases levels were assayed by adding distilled water in standard/blank wells and collected serum samples into corresponding sample wells of a flat bottomed 96-well microplate (Thermo Fisher Scientific, Belgium). After adding the provided blank working reagent and sample/standard reagent, absorbance values were measured at 340 nm with a FLUOstar OPTIMA microplate reader. Absorbance values for each set of blanks, standards and samples were used to calculate AST and ALT activity. Values were expressed in U/L. 1.2.5. Evaluation of liver tissue For microscopic evaluation, formalin-fixed liver tissue was embedded in paraffin (ProSan, Belgium). Tissue sections of 10 µm were cut with a SM2010 R sliding microtome (Leica, Belgium) and placed on microscope adhesive glass slides (VWR International, Belgium). Thereafter, liver tissue sections were deparaffinized in xylene (Chem-Lab, Belgium) for 30 minutes, rehydrated in ethanol (Merck, Germany) by washing the slides in a series of 100%, 90% and 70% v/v ethanol solutions for 1 minute and washed in PBS for 5 minutes. Tissue samples were stained with hematoxylin and periodic acid Schiff base (H-PAS) by treating the samples subsequently with Schiff’s reagent (Merck, Germany) and hematoxylin (ProSan, Belgium) for 5 minutes. Histological evaluation was carried out with an Olympus IX 81 bright field microscope (Olympus, Belgium) and liver samples were blindly analysed using ToupView software (ToupTek Photonics, China). The percentage of necrosis was estimated by measuring the necrotic area of microscopic fields compared to the cross-sectional areas over the entire section. Liver histopathology was evaluated by using Suzuki’s score-method quantifying for congestion, vacuolization and necrosis on stained sections (Table 2). 1.2.6. In vivo biodistribution of Panx1 nanobodies To examine the biodistribution of Panx1 nanobodies, Panx1 nanobody coding sequences were recloned into a variant of a pHEN phagemid vector and transformed into Escherichia coli WK6 cells. Panx1 nanobodies and R3b23 nanobody were produced as described in in section 1.1.3. Following production, purification and quality control, nanobody solutions were concentrated to 1 mg/mL using Vivaspin 5000 MW PES centrifugal concentrators (Satorius, Belgium). Next, Panx1 nanobodies and R3b23 nanobody solutions were labeled with Technetium-99m (99mTc). For the labeling, 99mTc-tricarbonyl precursor was prepared from a 99mMo/99mTc generator (Drytec, UK) with a lyophilisation kit (IsoLink, Netherlands). An amount of 50 µg nanobody was mixed with 500 µL 99mTc-tricarbonyl precursor and incubated for 90 minutes at 50 °C to complete conjugation. Thereafter, radiolabeled nanobodies were purified from unbound 99mTc-tricarbonyl precursor and aggregates by filtration over a NAP-5 column (Cytiva, Belgium) and a 0.22 µm membrane filter (Merck, Germany), respectively. Before injecting the nanobodies, radiochemical purity of labeled nanobodies was measured with instant thin-layer chromatography using silica gel impregnated glass fiber sheets (Pall Life Sciences, Belgium). Nanobodies showing high purity, i.e. at least 98%, were intravenously injected in healthy adult male C57BL/6 mice to determine their in vivo biodistribution. Mice of approximately 2 months of age were used and housed in the animal facility of the Faculty of Medicine and Pharmacy of the VUB-Belgium. The in vivo biodistribution of the 99mTc-labelled Panx1 nanobodies were measured using single- photon emission computerized tomography/computed tomography (SPECT/CT) imaging 1 hour post-injection. Following whole-body imaging with a MiLabs VECTor/CT-device (MILabs, Netherlands), mice were sacrificed, organs were collected, weighed and radioactivity was measured using a Canberra γ-counter Cobra Inspector 5003 (Global Medical Instrumentiation, USA) to assess the percentage of injected activity per gram in each organ. This protocol has been approved by the local Ethical Committee of the VUB- Belgium (project number 21-210-1) and all animals received human care according to the criteria by the guidelines provided by the Ethical Committee of the VUB-Belgium. 1.3. Statistical analysis All data were analysed using GraphPad Prism 7 software (GraphPad Software Inc., USA) and are presented as means ± standard error of the mean (SEM). The number of biological replicates (n) and technical repeats (N) are specified for each analysis. Results were statistically processed by unpaired t-tests with Welch’s correction, Mann-Whitney tests or parametric 1-way analysis of variance (ANOVA) followed by post hoc tests with Dunnett’s correction. Probability (p) values ≤ 0.05 were considered statistically significant. 2. Results 2.1. Characterisation of DUBCA WT, DUBCA mPanx1 and DUBCA hPanx1 cells The generation of Panx1 nanobodies involves the utilization of cells that overexpress Panx1 proteins. For this purpose, DUBCA cells were transduced with lentiviral vectors to express mPanx1 and hPanx1 proteins and evaluated by means of flow cytometry (Figure 1). Enhanced eGFP signals in DUBCA mPanx1 and DUBCA hPanx1 cells compared to DUBCA WT were observed. Immunoblot analysis of DUBCA cell lysates revealed 3 Panx1 signals between 45 kDa and 55 kDa corresponding with the 3 glycosylated Panx1 variants (Gly0/1/2) (Figure 2). Lysates of both DUBCA mPanx1 and DUBCA hPanx1 cells showed high Panx1 protein abundance in particular of the Gly2 variant (Figure 3). The latter is known to be located at the plasma membrane surface and is associated with Panx1 channel formation. This was confirmed in immunocytochemistry experiments following permeabilisation using a polyclonal Panx1 antibody recognising an epitope in the intracellular carboxyterminal tail of Panx1 (Figure 4). 2.2. Panx1 nanobody generation and in vitro characterisation A llama was immunised with vectors expressing mPanx1 gene via DNA immunisation and subsequently boosted with DUBCA cells overexpressing mPanx1. Next, cDNA from peripheral blood lymphocytes of the immunised llama was generated and used for the construction of an immune nanobody library. Selection of Panx1 nanobodies by phage display and panning on DUBCA mPanx1 cells was performed to screen for Panx1 nanobodies. By doing so, 7 different Panx1 nanobody families were identified. Individual clones of each family were selected for production and further characterisation. Since mouse and human Panx1 only differ by 6 amino acids in the extracellular loop regions, it was not surprising to identify cross-reactive nanobodies by flow cytometry (Figure 5). These results were also validated by immunocytochemistry experiments (Figure 6). While no binding was seen for the irrelevant R3b23 nanobody, 4 Panx1 nanobodies (Nb1, Nb3, Nb16 and Nb30) showed specific binding to DUBCA hPanx1 cells in the immunocytochemistry experiments (which is less sensitive than flow cytometry and is not necessarily capable to detect conformational epitopes). Affinity of the Panx1 nanobodies was evaluated in vitro by performing cell-based ELISA. DUBCA WT and DUBCA hPanx1 cells were incubated with individual Panx1 nanobody clones and R3b23 nanobody in different concentrations. Absorbance values were measured to determine Kd values (Figure 7).Kd values were verified by flow cytometry. These were for Nb1, Nb3 and Nb9, respectively 6.6 nM, 2.4 nM and 206.6 nM for mPanx1, and 6.9 nM, 0.7 nM and 277.4 nM for hPanx1. Further characterisation of the Panx1 nanobodies relied on the use of C6 WT cells and C6 cells overexpressing rat Panx1, as confirmed by immunoblot (Figure 8) and immunocytochemistry (Figure 9) analyses. ELISA experiments were carried out to evaluate the specificity of the Panx1 nanobodies. A total of 4 Panx1 nanobodies (Nb1, Nb3, Nb20 and Nb30) showed enhanced binding efficacy in C6 Panx1 cells with increased concentration (Figure 10). Inhibitory effects of the Panx1 nanobodies on Panx1 channel activity were assessed following potassium-induced channel opening in DUBCA hPanx1 cells and measurement of extracellular release ATP. Carbenoxolone, lanthanum and 10Panx1 reduced extracellular ATP, reflecting inhibition of Panx1 channel activity. Panx1 nanobodies and R3b23 nanobody were evaluated in this experimental set-up in concentrations ranging from 0 nM to 10,000 nM. Unlike for the non-targeting control R3b23 nanobody, inhibitory effects were noticed for all Panx1 nanobodies. For some of these Panx1 nanobodies, in particular Nb3, a clear concentration-dependent reduction of extracellular ATP levels was observed (Figure 11). The anti-inflammatory potential of Panx1 nanobodies was tested in RAW264.7 and differentiated THP-1 cells. LPS and ATP were used to trigger inflammation in vitro, whereafter detection of IL-1β was carried out to measure anti-inflammatory effects. Inflammation blockers Caspase-1 Inhibitor II and MCC950 were used as positive controls. These compounds decreased IL-1β intensity in both cell types. Panx1 nanobodies, in particular Nb9, Nb20 and Nb30, possess anti-inflammatory potential as they lowered IL- 1β signals in RAW264.7 (Figure 12) and differentiated THP-1 cells (Figure 13). Table 3 presents an overview of the in vitro results obtained with Panx1 nanobodies. These results were used to select 4 Panx1 nanobodies for further in vivo testing. For this purpose, specificity and affinity of Nb1, Nb3, Nb9 and Nb30 towards mPanx1 was measured. Cell-based ELISA experiments with DUBCA WT and DUBCA mPanx1 cells showed specific binding of Nb1, Nb3 and Nb30. No binding was observed for R3b23 and Nb9 for concentrations up to 600 nM (Figure 14). Next, flow cytometry analysis was used to evaluate the affinity of Panx1 nanobodies for mPanx1. The 4 selected Panx1 nanobodies possess affinity towards Panx1. Binding of Nb1, Nb3, Nb9 and Nb30 was noticed in DUBCA mPanx1 cells, while no binding was observed for R3b23. Affinity for mPanx1 was further evidenced by increasing the nanobody concentration. Obtained binding values were used to plot saturation curves and to define Kd values 2.3. In vivo testing of Panx1 nanobodies Based on the collective outcome of the in vitro testing, Panx1 nanobodies Nb1, Nb3, Nb9 and Nb30 were selected for in vivo experimentation. A mouse model of acute liver injury was used for proof-of-concept purposes in this study. This human-relevant model is based on overdosing mice with the analgesic drug APAP (N-acetyl-para-aminophenol, i.e. paracetamol or acetaminophen). This results in cytochrome P4502E1-mediated accumulation of its highly reactive N-acetyl-p-benzoquinone imine metabolite, which binds to cellular macromolecules, thereby triggering cell death and inflammation. Indeed, increased quantities of Panx1, NLRP3, IL-1β and IFN-γ were measured in mouse liver following acetaminophen overdosing. APAP lowered liver amounts of cytochrome P450 2E1 and pro-caspase 1, but did not affect Bax and Bcl-2. Treatment of APAP-overdosed mice with Nb3, Nb9 and Nb30 reduced hepatic Panx1 protein levels, while both the R3b23 nanobody and N-acetylcysteine had no effect. Nb1 and Nb3 as well as N- acetylcysteine lowered NLRP3 quantities. None of the Panx1 nanobodies was found to affect protein levels of pro-caspase 1, IL-1β or IFN-γ (Figure 16). In order to further investigate the effects of the Panx1 nanobodies on liver inflammation, antibody array analysis of 40 cytokines was performed on collected serum samples. N- acetylcysteine was found to reduce cytokine abundance in serum of wild type APAP- overdosed mice, which could support its use as a clinical antidote for the treatment of APAP poising. Inducing effects on the serum cytokines were observed for Nb30 and control nanobody R3b23. By contrast, both Nb3 and Nb9 showed pronounced overall anti-inflammatory effects, while being somewhat more subtle for Nb1 (Figure 17). Collected serum samples were also used to determine serum aminotransaminases levels in wild type and Panx1-/- mice. AST and ALT serum levels were higher in APAP- overdosed wild type mice. In case of Panx1-/- mice, only subtle changes in serum levels of AST and ALT levels were recorded after APAP overdosing. AST and ALT levels were lowered in APAP-overdosed wild type animals treated with N-acetylcysteine and Nb30. However, the relevance of this Nb30-mediated finding is not solely Panx1-dependent as reduced AST and ALT levels were noticed in APAP-overdosed Panx1-/- mice (Figure 18). The liver tissue was also evaluated to check if Panx1 nanobodies alleviate APAP- induced toxicity. The H-PAS-stained liver sections were examined microscopically. No necrosis was seen in UTC animals, whereas APAP group showed significant necrosis (20.83 % ± 0.73 %). However, there was no change in necrotic areas compared to any of the Panx1 nanobody-treated groups. These conditions of hepatocyte cell death initiate inflammation. The APAP-overdosed mice treated with Nb1, Nb3 and Nb9 showed significantly reduced score with the Suzuki method (Figure 19). In vivo biodistribution of Panx1 nanobodies was determined with 99mTc-labeled nanobodies in healthy adult mice. SPECT/CT imaging and γ-counting revealed low uptake levels for 99mTc-labeled nanobodies in nearly all organs, which is line with the ultrafast renal clearance of unbound nanobodies. However, a significantly increased uptake is seen for Panx1 nanobodies compared to R3b23 in salivary glands and stomach (Figure 20). It was not surprising to identify uptake of Panx1 nanobodies in these organs, as pronounced Panx1 protein expression in salivary glands and stomach was confirmed by immunohistochemistry analysis (Figure 20). Table 4 presents an overview of the in vitro results obtained with Panx1 nanobodies. Table 1: Overview antibodies Antibody Dilution Incubation Incubation period temperature 1.1.2.1. Immunoblot analysis Panx1 (D9M1C) (Cell Signaling 1/500 24 hours 4 °C Technology, USA) Goat anti-rabbit horseradish peroxidase- 1/500 1 hour Room conjugated antibody (P0448) (Dako, USA) temperature 1.1.2.2. Immunocytochemistry analysis Panx1 (ABN242) (Merck, Germany) 1/250 24 hours 4 °C Donkey anti-rabbit Alexa Fluor® 594- 1/250 1 hour Room conjugated antibody (857218) (Thermo temperature Fisher Scientific, Belgium) 1.1.4. Flow cytometry Hemagglutinin allophycocyanin- 1/25 20 minutes 4 °C conjugated antibody (901524) (BioLegend, USA) 1.1.5. Immunocytochemistry Hemagglutinin Alexa Fluor® 594- 1/250 24 hours 4 °C conjugated antibody (901511) (BioLegend, USA) 1.1.6. Cell-based enzyme-linked immunosorbent assay Hemagglutinin horseradish peroxidase- 1/5,000 1 hour Room conjugated antibody (901519) (BioLegend, temperature USA) 1.1.8. In vitro inflammation assay IL-1β (ab254360) (Abcam, UK) 1/500 24 hours 4 °C Goat anti-rabbit Alexa Fluor® 488- 1/500 1.5 hours 4 °C conjugated antibody (ab150077) (Abcam, UK) 1.1.9. Specificity and affinity of Panx1 nanobodies towards mouse Panx1 Hemagglutinin Alexa Fluor® 488- 1/1,000 20 minutes 4 °C conjugated antibody (901509) (BioLegend, USA) 1.2.3. Immunoblot analysis Bax (sc-7480) (Santa Cruz, USA) 1/500 24 hours 4 °C Bcl-2 (554087) (BD Biosciences, USA) 1/500 24 hours 4 °C Caspase-1 (sc-514) (Santa Cruz, USA) 1/500 24 hours 4 °C CYP2E1 (HPA009128) (Atlas Antibodies, 1/500 24 hours 4 °C Sweden) IL-1β (ab254360) (Abcam, UK) 1/500 24 hours 4 °C IFN-γ (ab216642) (Abcam, UK) 1/500 24 hours 4 °C NLRP3 (ab263899) (Abcam, UK) 1/500 24 hours 4 °C Panx1 (D9M1C) (Cell Signaling 1/500 24 hours 4 °C Technology, USA) Goat anti-mouse horseradish peroxidase- 1/1,000 1 hour Room conjugated antibody (P0447) (Dako, USA) temperature Goat anti-rabbit horseradish peroxidase- 1/1,000 1 hour Room conjugated antibody (P0448) (Dako, USA) temperature Table 2: Suzuki score method. Score Congestion Vacuolization Necrosis 0 None None None 1 Minimal Minimal Single cell necrosis 2 Mild Mild ± 30 % 3 Moderate Moderate ± 60 % 4 Severe Severe > 60 %
Table 3: Overview Panx1 nanobody in vitro testing (++ = very promising result, + = promising result, - = negative result). yr n o o t s i - a f e i t a P H mo d s 1 o m T b m; -i t mt c x o al y a n al f e f n n f s A n i f e a P a n nI ( s a ) 1 - - - + - + + + + y r o t s n e o i t ) 7 a f i a . mo d 4 - s i mt c 1 o m x b o m; 6 a y a 2 t n alf e f A n i f n e a n l f P a n nI s W ( s a A R - + + + + + + + + + + + ; s y a yr f e i s o o d l s t i s o 1 e a y A) 1 bi t c 1 h e x b x n ti n o n n vi C x B n n f I f e a n P a a n P a ( h t c c a Ua DP h + + + + + + + + + + f o y s ti e d e 6 c i i d s fi c 1 o b a C b ; A6 e x - n o l l S C) , 1 x p Sa n e I P a n C ( L T n E Wa P + + + + - - + + + + + s , d T f o e i y d e s W ti n 1 o b a b ; ) AAA 1 x if x f n o - n l l e S I C B Cn Aa P a n C ( L E UB Ua D DP h + + + + - - + + - + + - f o s o e t i y , d c ; T g o y r W ) o r t n i i t 1 o n t s AA 1 v e g x b u i CC x n r n o mm I a t a n P a n mI e B ( h c UB n Ua D DP h + + + - - + + - + + y - f o s , e c i ; T t i s if s y d y r W , ) i o c r ti v t i 1 o b e AA1 x A 1 x e c t c x p d S n a n o wo mCC o B B n a C B n a a e r a n P a l t n F ( y c UUPU D D m DP h + + + + + + + + + + + + + 1 3 6 9 6 0 0 b b b 1 2 3 N N N b N b N b N b N Table 4: Overview Panx1 nanobody in vivo testing (++ = very promising result, + = promising result, - = negative result). In vivo Mouse model of acute liver injury biodistribution of Panx1 nanobodies Evaluation Serum Analysis of serum Hepatic (SPECT/CT of liver cytokine aminotransferases protein levels imaging) tissue levels (ALT/AST) of IL-1β (Suzuki (Antibody (Immunoblot) score) array) Nb1 + + + - + Nb3 + ++ ++ - - Nb9 + ++ ++ - + Nb30 + - - - + * * * The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims. It is further to be understood that all values are approximate and are provided for description. Patents, patent applications, publications, product descriptions, and protocols are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties for all purposes.

Claims

CLAIMS 1. A single-domain antibody capable of specifically binding to pannexin 1 (Panx1), or an antigen-binding fragment thereof.
2. The single-domain antibody according to claim 1, wherein said single-domain antibody comprises a heavy chain variable domain derived from a heavy chain antibody (VHH).
3. The single-domain antibody according to any of claims 1 to 2, wherein said single- domain antibody is capable of at least partially inhibiting Panx1 channel activity.
4. The single-domain antibody according to any of claims 1 to 3, wherein said single domain antibody specifically binds to Panx1 with a KD of at most 300 nM, preferably at most 100 nM.
5. The single-domain antibody according to any of claims 1 to 4, wherein said single- domain antibody comprises CDR1 having a sequence as set forth in SEQ ID NO: 19, CDR2 having a sequence as set forth in SEQ ID NO: 20, and CDR3 having a sequence as set forth in SEQ ID NO: 21; CDR1 having a sequence as set forth in SEQ ID NO: 10, CDR2 having a sequence as set forth in SEQ ID NO: 11, and CDR3 having a sequence as set forth in SEQ ID NO: 12; CDR1 having a sequence as set forth in SEQ ID NO: 1, CDR2 having a sequence as set forth in SEQ ID NO: 2, and CDR3 having a sequence as set forth in SEQ ID NO: 3; CDR1 having a sequence as set forth in SEQ ID NO: 4, CDR2 having a sequence as set forth in SEQ ID NO: 5, and CDR3 having a sequence as set forth in SEQ ID NO: 6; CDR1 having a sequence as set forth in SEQ ID NO: 7, CDR2 having a sequence as set forth in SEQ ID NO: 8, and CDR3 having a sequence as set forth in SEQ ID NO: 9; CDR1 having a sequence as set forth in SEQ ID NO: 13, CDR2 having a sequence as set forth in SEQ ID NO: 14, and CDR3 having a sequence as set forth in SEQ ID NO: 15; CDR1 having a sequence as set forth in SEQ ID NO: 16, CDR2 having a sequence as set forth in SEQ ID NO: 17, and CDR3 having a sequence as set forth in SEQ ID NO: 18; or CDR1 having a sequence as set forth in SEQ ID NO: 22, CDR2 having a sequence as set forth in SEQ ID NO: 23, and CDR3 having a sequence as set forth in SEQ ID NO: 24.
6. The single-domain antibody according to any of claims 1 to 5, wherein said single- domain antibody has a sequence as set forth in any of SEQ ID NOs: 31, 28, 25, 26, 27, 29, 30, or 32, or a sequence which is at least 70% identical, preferably at least 80% identical, mor preferably at least 90% identical, most preferably at least 95% identical to, or having at most 5, preferably at most 3, more preferably at most 2, most preferably at most 1 (conservative) amino acid substitution, amino acid insertion and/or amino acid deletion (combined) compared to a sequence as set forth in any of SEQ ID NOs: 31, 28, 25, 26, 27, 29, 30, or 32.
7. A binding agent comprising a single-domain antibody or an antigen-binding fragment thereof according to any of claims 1 to 6.
8. The binding agent according to any of claims 6 to 7, wherein said single-domain antibody is fused to an Fc domain, albumin, PEG or PAS.
9. A polynucleic acid encoding the single-domain antibody according to any of claims 1 to 6, optionally wherein the polynucleic acid is comprised in a recombinant vector.
10. A host cell comprising the polynucleic acid or vector according claim 9.
11. The single-domain antibody, binding agent, polynucleic acid, vector, or host cell according to any of claims 1 to 10 for use in therapy.
12. The single-domain antibody, binding agent, polynucleic acid, vector, or host cell according to any of claims 1 to 10 for use in therapeutic treatment of inflammation.
13. The single-domain antibody, binding agent, polynucleic acid, vector, or host cell according to any of claims 1 to 10 for use in therapeutic treatment of a liver disease or disorder, preferably an inflammatory liver disease or disorder.
14. The single-domain antibody, binding agent, polynucleic acid, vector, host cell or pharmaceutical composition according to any of claims 1 to 10 for use in therapeutic treatment of hepatitis, acute liver failure, (non-alcoholic) steatohepatitis, (non-alcoholic) fatty liver disease, cholestatic disorder, cholangitis, fibrotic/cirrhotic disorder, or (liver) cancer.
15. Use of the single-domain antibody, binding agent, polynucleic acid, vector, host cell or pharmaceutical composition according to any of claims 1 to 10 for diagnosing inflammation or an inflammatory (liver) disease or disorder, preferably hepatitis, acute liver failure, (non-alcoholic) steatohepatitis, (non-alcoholic) fatty liver disease, cholestatic disorder, cholangitis, fibrotic/cirrhotic disorder, or (liver) cancer.
PCT/EP2024/054681 2023-02-24 2024-02-23 Anti-inflammatory pannexin 1 channel inhibitors Ceased WO2024175787A1 (en)

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