IL-18 Mimetic

Abstract

This disclosure relates to novel, advantageously characterized VHH-based binders for IL-18Rα and / or IL-18Rβ. Furthermore, this disclosure relates to pharmaceutical compositions comprising such compounds.

Description

[Technical Field]

[0001] Field of Invention This disclosure relates to novel, advantageously characterized VHH-based binders for IL-18Rα and / or IL-18Rβ. Furthermore, this disclosure relates to pharmaceutical compositions comprising such compounds. [Background technology]

[0002] Background of the Invention Cytokines are potent immunomodulatory proteins with enormous therapeutic potential. Therefore, several different molecules have been granted marketing authorization for the treatment of various diseases (Propper and Balkwill, 2022; Pires et al., 2021; Berraondo et al., 2019).

[0003] However, the multifaceted mechanisms of action of many cytokines, combined with their short half-lives and dose-limiting toxicity, often hinder their therapeutic application when administered systemically (Pires et al., 2021; Amin et al., 2014).

[0004] To address these inherent limitations, "next-generation cytokines" have been developed (Zheng et al., 2022). For example, an IL-2 mutain was designed that does not target the α-subunit (CD25) of the IL-2 receptor to eliminate the intrinsic bias of this cytokine in activating regulatory T cells (Tregs) (Klein et al., 2017; Dolgin, 2022). To optimize the pharmacokinetics of cytokines, fusions with the Fc moiety of IgG (with its effector function silenced or attenuated) (Jazayeri and Carroll, 2008) and conjugates with polyethylene glycol (Eliason, 2001) have been designed. Meanwhile, bifunctional antibody-cytokine fusion proteins have been constructed with the aim of accumulating the immunomodulatory function of cytokines at the disease site (Murer and Neri, 2019; Neri and Sondel, 2016).

[0005] Another interesting approach to “next-generation cytokines” relies on developing antibody derivatives that mimic the function of cytokines, which are referred to as cytokine mimetic or surrogate cytokines (Saxton et al., 2023). In this regard, bispecific antibody derivatives (diabodies) that dimerize the erythropoietin receptor (EpoR) have been developed (Moraga et al., 2015). Interestingly, different diabodies induced differential EpoR phosphorylation ranging from weak and partial agonism to complete agonism. In addition, single-domain antibody (sdAb)-based bispecifics have been created that activate signaling through heterodimer IL-2 receptor βγ (IL-2Rβγ). These bispecific antibodies (bsAbs) mimic the function of IL-2 but do not involve preferential activation of Treg via IL-2 receptor α-binding (Harris et al., 2021). Garcia and collaborators recently described the design of a VHH-derived surrogate agonist targeting IL-2Rβγ, as well as a bsAb mimicking type I IFN (Yen et al., 2022). Importantly, the group constructed a surrogate agonist that exhibits diversification of function with respect to downstream signaling of the receptor compared to the native cytokine. Furthermore, they constructed bsAbs that induce agonist activity through binding to receptor heterodimers, IL-2Rβ and IL-10Rβ, which do not exist in nature. This clearly demonstrates that modular assemblies of bsAbs enable the creation of cytokine mimetic compounds with tailor-made functionality that can serve as versatile components for drug delivery.

[0006] IL-18 is a pro-inflammatory cytokine belonging to the IL-1 family of cytokines, mediating signal transduction through heterodimerization of receptor subunits IL-18Rα and IL-18Rβ (Yasuda et al., 2019; Dinarello et al., 2013). IL-18 stimulates IFN-γ production in innate lymphoid cells and in antigen-experienced T cells in synergistic effects with IL-12 (Nakamura et al., 2020). Recombinant(r)IL-18 has been evaluated in clinical trials and shows a favorable toxicity profile, but its efficacy as monotherapy is limited (Atallah-Yunes and Robertson, 2022; Robertson et al., 2006; Tarhinini et al., 2009). Physiologically, IL-18 activity is balanced via high-affinity neutralization and the naturally occurring IL-18 binding protein (IL-18BP) (Dinarello et al., 2013; Nakamura et al., 2020). (r) Following treatment with IL-18, subsequent elevated levels of IL-18BP were found in the serum of patients (Robertson et al., 2006; Robertson et al. 2008). Ring and colleagues were able to demonstrate that IL-18BP is expressed in the tumor microenvironment of several tumor types (Zhou et al., 2020). Furthermore, the authors found increased IL-18BP concentrations in the serum of patients with non-small cell lung cancer, which were further elevated after PD-1 or PD-L1 treatment. In the same study, the group designed a decoy-resistant IL-18 mutain that still induces IL-18 reporter activation. This next-generation IL-18 derivative demonstrated excellent antitumor efficacy in preclinical models, both as monotherapy and in combination with immune checkpoint inhibitors (Zhou et al., 2020).

[0007] IL-18 is a pro-inflammatory cytokine that promotes NK cell activation and the maturation and function of effector T cells (Holder et al., 2022). Therefore, recombinant human (rh)IL-18 has emerged as a promising potential therapeutic agent for inducing anti-tumor immunity. (rh)IL-18 has been evaluated in several clinical trials, either as a monotherapy or in combination therapy (see, e.g., Tarhini et al., 2009; Robertson et al., 2008; Robertson et al., 2013; Simpkins et al., 2013; Robertson et al., 2018). While (rh)IL-18 therapy has been well tolerated, its clinical efficacy has been limited. A possible explanation for the lack of therapeutic efficacy depends on the inhibition of IL-18 by the IL-18BP decoy receptor (Dinarello et al., 2013). Despite its much higher affinity, IL-18BP binds to IL-18 at a position overlapping with IL-18Rα. Therefore, it antagonizes IL-18 signaling by blocking the interaction of IL-18Rα with IL-18. Recently, Ring and colleagues described the creation of an IL-18 decoy-resistant mutaine that shows great potential for anti-cancer therapy in preclinical models (Zhou et al., 2020). This entity is currently in clinical research (NCT04787042).

[0008] Despite the relevance of IL-18 to major tumor formation and its potential for targeting for cancer treatment, approaches that would allow for the tailor-made mimicry of IL-18 and its functions are still lacking.

[0009] Therefore, in the art, there is a need for improved IL-18 receptor agonists. Further, in the art, there is a need for improved methods of mimicking the function of IL-18 in a tailor-made fashion with respect to, for example, the various potencies and magnitudes of activation of IL-18R reporter activation, the various potencies and magnitudes of IFN-γ release, or resistance to inhibition by IL-18BP.

[0010] The present disclosure overcomes the above problems and addresses the above needs. SUMMARY OF THE INVENTION

[0011] SUMMARY OF THE INVENTION The present disclosure addresses the needs described in the "Background of the Invention" section above in the following various aspects and embodiments.

[0012] The present invention is based, in part, on the surprising observation that novel bispecific surrogate agonists can be obtained that target both IL-18Rα and IL-18Rβ and mimic the functionality of IL-18, based on sdAbs (sdAbs: single domain antibodies, also referred to herein as V H H or VHH). Furthermore, it has been found that it is possible to obtain such IL-18 mimics that are simultaneously resistant to the decoy receptor IL-18BP. Furthermore, surprisingly, molecules in which the overall design architecture regarding the valency and spatial orientation of individual VHH-based paratopes has been engineered within the molecule enable the obtaining of IL-18 mimetics that are tailor-made with respect to (e.g., their potency, the magnitude of IL-18R-induced activation and IFN-γ release, or resistance to inhibition by the IL-18BP receptor decoy).

[0013] In one aspect, the present disclosure relates to a VHH antibody domain or a fragment thereof, where (a) The VHH antibody domain or a fragment thereof comprises the complementarity determining regions CDR1, CDR2, and CDR3 of one VHH selected from the group consisting of VHH1, VHH2, VHH3, VHH4, VHH5, VHH6, VHH8, VHH9, VHH10, and VHH11 as shown in the table of CDRs; (b) The VHH antibody domain or a fragment thereof comprises complementarity determining regions CDR1, CDR2, and CDR3 as defined in (a) and having modifications, where the modification is such that the sequence of at least one of CDR1, CDR2, and CDR3 is humanized; or (c) The VHH antibody domain or a fragment thereof comprises complementarity determining regions CDR1, CDR2, and CDR3 as defined in (a) and having modifications, where the modification is - Substitutions, additions, or deletions of up to 3 amino acids in CDR1, - Substitutions, additions, or deletions of up to 3 amino acids in CDR2, and / or - Substitutions, additions, or deletions of up to 3 amino acids in CDR3 is.

[0014] Table of CDRs: [Table A-1] [Table A-2] [Table A-3]

[0015] In another aspect, the present disclosure relates to a VHH antibody domain or a fragment thereof, where (A) The VHH antibody domain comprises the amino acid sequence of a VHH selected from the group consisting of VHH1, VHH2, VHH3, VHH4, VHH5, VHH6, VHH8, VHH9, VHH10, and VHH11 as shown in the table of VHH sequences; (B) The VHH antibody domain comprises a VHH sequence as defined in (A) and having modifications, wherein the modification is humanization of the sequence; (C) The VHH antibody domain comprises a VHH sequence as defined in (A) and having modifications, wherein the modifications are substitutions, additions, or deletions of up to 25 amino acids; or, (D) The VHH antibody domain comprises a VHH sequence that is at least 75% identical to the VHH sequence mentioned in (A);

[0016] Table of VHH sequences: [Table B-1] [Table B-2] [Table B-3]

[0017] In another respect, this disclosure relates to the VHH antibody domain or its fragments, hereby (A) The VHH antibody domain consists of an amino acid sequence of VHH selected from the group consisting of VHH1, VHH2, VHH3, VHH4, VHH5, VHH6, VHH8, VHH9, VHH10, and VHH11, as shown in the VHH sequence table; (B) The VHH antibody domain consists of a VHH sequence as defined in (A) and is modified, wherein the modification is humanization of the sequence; (C) The VHH antibody domain consists of a VHH sequence as defined in (A) and is modified, wherein the modification is the substitution, addition, or deletion of up to 25 amino acids; or, (D) The VHH antibody domain consists of a VHH sequence that is at least 75% identical to the VHH sequence mentioned in (A).

[0018] In another respect, this disclosure relates to the VHH antibody domain or its fragments, hereby (d) The VHH antibody domain or fragment thereof includes one VHH complementarity-determining region CDR1, CDR2, and CDR3 selected from the group consisting of VHH12, VHH13, VHH14, VHH15, VHH16, VHH17, VHH18, VHH20, VHH21, and VHH22 as shown in the table of CDRs; (e) The VHH antibody domain or fragment thereof includes the complementarity-determining regions CDR1, CDR2 and CDR3 as defined in (d), wherein the modification is that at least one of the sequences of CDR1, CDR2 and CDR3 is humanized; or (f) The VHH antibody domain or fragment thereof includes the complementarity-determining regions CDR1, CDR2 and CDR3 as defined in (d) and which are modified, where the modification is - Substitution, addition, or deletion of up to three amino acids in CDR1, - Substitution, addition or deletion of up to three amino acids in CDR2, and / or - Substitution, addition, or deletion of up to three amino acids in CDR3 That is the case.

[0019] In another respect, this disclosure relates to the VHH antibody domain or its fragments, hereby (E) The VHH antibody domain contains an amino acid sequence of VHH selected from the group consisting of VHH12, VHH13, VHH14, VHH15, VHH16, VHH17, VHH18, VHH20, VHH21, and VHH22 as shown in the VHH sequence table; (F) The VHH antibody domain comprises a VHH sequence as defined in (E) and having modifications, wherein the modification is humanization of the sequence; (G) The VHH antibody domain comprises a VHH sequence as defined in (E) and having modifications, wherein the modifications are substitutions, additions, or deletions of up to 25 amino acids; or, (H) The VHH antibody domain comprises a VHH sequence that is at least 75% identical to the VHH sequence referenced in (E).

[0020] In another respect, this disclosure relates to the VHH antibody domain or its fragments, hereby (E) The VHH antibody domain consists of an amino acid sequence of VHH selected from the group consisting of VHH12, VHH13, VHH14, VHH15, VHH16, VHH17, VHH18, VHH20, VHH21, and VHH22 as shown in the table of VHH sequences; (F) The VHH antibody domain consists of a VHH sequence as defined in (E) and is modified, wherein the modification is humanization of the sequence; (G) The VHH antibody domain consists of a VHH sequence as defined in (E) and is modified, wherein the modification is the substitution, addition, or deletion of up to 25 amino acids; or, (H) The VHH antibody domain consists of a VHH sequence that is at least 75% identical to the VHH sequence referenced in (E).

[0021] In another respect, this disclosure is: - VHH antibody domain or fragment thereof according to any one of embodiments 1 to 188 This relates to compounds containing [the specified compound]. In another respect, this disclosure is: - VHH antibody domain or fragment thereof according to any one of claims 189 to 374 This relates to compounds containing [the specified compound].

[0022] In another aspect, this disclosure relates to compounds including: -A first binding module which is a VHH antibody domain or a fragment thereof, where, (a) The VHH antibody domain or fragment thereof includes one VHH complementarity-determining region CDR1, CDR2, and CDR3 selected from the group consisting of VHH1, VHH2, VHH3, VHH4, VHH5, VHH6, VHH8, VHH9, VHH10, and VHH11 as shown in the table of CDRs; (b) The VHH antibody domain or fragment thereof includes the complementarity-determining regions CDR1, CDR2 and CDR3 as defined in (a), wherein the modification is that at least one of the sequences of CDR1, CDR2 and CDR3 is humanized; or (c) The VHH antibody domain or fragment thereof includes the complementarity-determining regions CDR1, CDR2 and CDR3 as defined in (a) and which are modified, wherein the modifications are - Substitution, addition, or deletion of up to three amino acids in CDR1, - Substitution, addition or deletion of up to three amino acids in CDR2, and / or - Substitution, addition, or deletion of up to three amino acids in CDR3 is; - A second binding module which is the VHH antibody domain or a fragment thereof, where, (d) The VHH antibody domain or fragment thereof includes one VHH complementarity-determining region CDR1, CDR2, and CDR3 selected from the group consisting of VHH12, VHH13, VHH14, VHH15, VHH16, VHH17, VHH18, VHH20, VHH21, and VHH22 as shown in the table of CDRs; (e) The VHH antibody domain or fragment thereof includes the complementarity-determining regions CDR1, CDR2 and CDR3 as defined in (d), wherein the modification is that at least one of the sequences of CDR1, CDR2 and CDR3 is humanized; or (f) The VHH antibody domain or fragment thereof includes the complementarity-determining regions CDR1, CDR2 and CDR3 as defined in (d) and which are modified, where the modification is - Substitution, addition, or deletion of up to three amino acids in CDR1, - Substitution, addition or deletion of up to three amino acids in CDR2, and / or - Substitution, addition, or deletion of up to three amino acids in CDR3 That is the case.

[0023] In another aspect, this disclosure relates to compounds including: -A first binding module which is a VHH antibody domain or a fragment thereof, where, (A) The VHH antibody domain contains an amino acid sequence of VHH selected from the group consisting of VHH1, VHH2, VHH3, VHH4, VHH5, VHH6, VHH8, VHH9, VHH10 and VHH11 as shown in the VHH sequence table; (B) The VHH antibody domain comprises a VHH sequence as defined in (A) and having modifications, wherein the modification is humanization of the sequence; (C) The VHH antibody domain comprises a VHH sequence as defined in (A) and having modifications, wherein the modifications are substitutions, additions, or deletions of up to 25 amino acids; or, (D) The VHH antibody domain comprises a VHH sequence that is at least 75% identical to the VHH sequence mentioned in (A); - A second binding module which is the VHH antibody domain or a fragment thereof, where, (E) The VHH antibody domain contains an amino acid sequence of VHH selected from the group consisting of VHH12, VHH13, VHH14, VHH15, VHH16, VHH17, VHH18, VHH20, VHH21, and VHH22 as shown in the VHH sequence table; (F) The VHH antibody domain comprises a VHH sequence as defined in (E) and having modifications, wherein the modification is humanization of the sequence; (G) The VHH antibody domain comprises a VHH sequence as defined in (E) and having modifications, wherein the modifications are substitutions, additions, or deletions of up to 25 amino acids; or, (H) The VHH antibody domain comprises a VHH sequence that is at least 75% identical to the VHH sequence referenced in (E).

[0024] In another aspect, this disclosure relates to compounds including: -A first binding module which is a VHH antibody domain or a fragment thereof, where, (A) The VHH antibody domain consists of an amino acid sequence of VHH selected from the group consisting of VHH1, VHH2, VHH3, VHH4, VHH5, VHH6, VHH8, VHH9, VHH10, and VHH11, as shown in the VHH sequence table; (B) The VHH antibody domain consists of a VHH sequence as defined in (A) and is modified, wherein the modification is humanization of the sequence; (C) The VHH antibody domain consists of a VHH sequence as defined in (A) and is modified, wherein the modification is the substitution, addition, or deletion of up to 25 amino acids; or, (D) The VHH antibody domain consists of a VHH sequence that is at least 75% identical to the VHH sequence mentioned in (A); - A second binding module which is the VHH antibody domain or a fragment thereof, where, (E) The VHH antibody domain consists of an amino acid sequence of VHH selected from the group consisting of VHH12, VHH13, VHH14, VHH15, VHH16, VHH17, VHH18, VHH20, VHH21, and VHH22 as shown in the table of VHH sequences; (F) The VHH antibody domain consists of a VHH sequence as defined in (E) and is modified, wherein the modification is humanization of the sequence; (G) The VHH antibody domain consists of a VHH sequence as defined in (E) and is modified, wherein the modification is the substitution, addition, or deletion of up to 25 amino acids; or, (H) The VHH antibody domain consists of a VHH sequence that is at least 75% identical to the VHH sequence referenced in (E).

[0025] In another aspect, the present disclosure relates to a compound that is a bispecific antibody molecule comprising an IL-18Rα-binding VHH (first binding module) having the amino acid sequence defined below in this disclosure, and an IL18Rβ-binding VHH (second binding module) having the amino acid sequence defined below in this disclosure.

[0026] In another aspect, this disclosure relates to a bispecific antibody molecule characterized by SEED (strand-exchange engineered domain) technology, the molecule comprising: - An AG chain linked to VHH that binds to IL-18Rα, wherein the AG chain linked to VHH that binds to IL-18Rα has the amino acid sequence as defined below in this disclosure ("VHH-SEED-AG-1"), and - A GA chain linked to VHH bound to IL18Rβ, wherein the GA chain linked to VHH bound to IL18Rβ has the amino acid sequence as defined below in this disclosure ("VHH-SEED-GA-1").

[0027] In another aspect, this disclosure relates to pharmaceutical compositions comprising the compounds or bispecific antibody molecules relating to this disclosure. In another aspect, this disclosure relates to the use of the compounds or bispecific antibody molecules according to this disclosure for activating the IL-18R receptor. In another aspect, the disclosure relates to the use of the compounds or bispecific antibody molecules according to the disclosure for inducing the release of IL-γ from cells carrying the IL-18R receptor.

[0028] References to drawings are made below. All methods mentioned in the description of the drawings below were carried out as described in detail in the examples. [Brief explanation of the drawing]

[0029] [Figure 1A] Figure 1 summarizes the overall strategy applied in this disclosure for the creation of tailor-made cytokine mimetic based on antibody discovery enabled by sdAb-derived bispecificity and YSD. (A) Bottom / left panel: IL-18 (blue, §) triggers downstream signaling of IL-18R by sequential binding to IL-18Rα (yellow, *; the two “lobes” of IL-18Ra are each labeled with *) and subsequent recruitment of IL-18Rβ (yellow-green, #). IL-18BP (dark gray, $) inhibits IL-18 by high-affinity binding and thereby by blocking the IL-18 / IL-18Rα interaction. Top / Right Panel: When camel-derived sdAbs are reformatted to IgG-like bispecificity via IL-18Rα targeting (orange, &) and IL-18Rβ targeting (green, ‡), the resulting cytokine mimetic bridges the IL18R subunits IL-18Rα (yellow, *; the two "lobes" of IL-18Ra are each labeled with *) and IL-18Rβ (yellow-green, #), thereby inducing downstream signaling. Essentially, the generated IL-18 mimetic is resistant to inhibition by IL-18BP (dark gray, $). Created using PyMOL software version 2.3.0, modified using www.biorender.com based on PDB entries YYY and ZZZ. [Figure 1B]Figure 1 summarizes the overall strategy applied in this disclosure for the creation of tailor-made cytokine mimetic based on antibody discovery enabled by bispecificity and YSD derived from sdAb. (B) Camel immunization and subsequent YSD facilitate the enrichment of sdAb specific to (rh)IL-18Rα and (rh)IL-18Rβ. One sublibrary was prepared for each specimen (huarizo and llama) and sorted separately. In the first round of selection, a mixture of both (rh) receptor subunits was utilized at a concentration of 250 nM. In the subsequent second round of sorting, the enriched libraries were sorted separately for each antigen at 250 nM. A two-dimensional sorting strategy was applied to select full-length VHH display in addition to antigen binding. Percentage of cells at the sorting gate is shown. The plot shows 5 × 10⁴ events. [Figure 1C] Figure 1 summarizes the overall strategy applied in this disclosure for the creation of tailor-made cytokine mimetic based on antibody discovery enabled by sdAb-derived bispecificity and YSD. (C) Graph alignment of 55 unique sdAb clones directed at IL-18Rα (top) and 101 independent clones targeting IL-18Rβ (bottom) recovered from YSD library sorting. Complementarity-determining regions (CDRs) are highlighted. Red bars indicate high sequence diversity at the amino acid level, and green bars indicate high sequence conservation at a given position. Alignment was performed using the MUSCLE alignment tool with Geneious Prime 2021.1.1.

[0030] [Figure 2]Figure 2 shows data obtained by reporter assays, confirming that combinatorial reformatting of single-specific (1+0) SEED bodies to strictly monovalent (1+1) bsAb allows for the identification of IL-18 mimetic cells with reduced ability to induce NFκB reporter activity in IL-18 reporter cells. (A) HEK-Blue® reporter cells were incubated with increasing concentrations of reformatted bsAb, as illustrated for IL18R_VHHα2β15, IL18R_VHHα8β15, IL18R_VHHα2β17, and IL18R_VHHα8β17. Secreted embryonic alkaline phosphatase activity was monitored by determining OD640. Reporter activity was normalized to the maximum IL-18 reading. (B) Heatmap of NFκB reporter activation induced by combinatorially reformatted (1+1)bsAb. Molecules that failed initial quality control after protein A purification (target monomer peak <96% at SEC) are shown in dark gray, functionally inactive bsAb are shown in red (‡), minimally active surrogate agonists (50% compared to (rh)IL-18 at NFκB reporter activation <1 nM, or EC50 ≥ 0.1 nM) are shown in yellow (#), and moderately active IL-18 mimetic (50% compared to (rh)IL-18 at NFκB reporter activation ≥ 1 nM, or EC50 < 0.1 nM) are shown in green (*).

[0031] [Figure 3]Figure 3 shows data from the IFN-γ release assay, confirming that a bispecific (1+1) surrogate agonist induces IFN-γ release in PBMCs isolated from healthy donors. (A) IFN-γ production in PBMCs stimulated with bsAb at a fixed concentration of 100 nM or with (rh)IL-18 at 1 nM. Experiments were performed in the presence of 10 ng / ml (rh)IL-12. The graph shows box plots overlaid with dot plots of IFN-γ release from 10 different donors. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. IL18R_VHHα1β16 was used as a negative control (shown in red). Four primary candidates used for further characterization are shown in green, purple, blue, and orange. (B) The top four candidates induce dose-dependent IFN-γ readings in PBMCs in the presence of low doses of (rh)IL-12 (10 ng / ml). IL18R_VHHα1β16, shown in red, was used as a negative control at a fixed concentration of 1 μM. Mean ± SEM results from 10 independent experiments are shown.

[0032] [Figure 4A-B]Figure 4 shows a schematic depiction of the antibody architecture and summarizes the data for characterizing such antibodies. These data confirm that antibody manipulation enables the creation of IL-18 mimetic with enhanced agonism ability. (A) Schematic depiction of the main different bispecific antibody architectures constructed in this study. Fusion of anti-IL18RαVHHα2 to the hinge region of the SEED body AG chain and engraving of anti-IL18RβVHHβ15 onto the GA chain results in the initially constructed (1+1) format IL18R_VHHα2β15. Replacing the VH and VLλ of the IgG with VHHα2 and VHHβ15, respectively, facilitates the creation of the IL18R_sdIgGα2β15 architecture (2+2). In the design (2+2) of IL18R_tanVHHα2β15, VHHα2 and VHHβ15 are arranged in tandem (N-terminus to C-terminus) and separated by a five-amino acid Gly4Ser linker. The tandem is fused to the hinge region of an IgG1 Fc fragment whose effector function is silenced. It should be noted that the reverse orientation was also constructed (VHHβ15 followed by VHHα2, IL18R_tanVHHβ15α2). In addition, all four molecules were also generated carrying the E430G mutation for on-target hexamerization. Structural models were created using PyMOL software version 2.3.0. (B) Different surrogate agonist formats of the same paratope (VHHα2 and VHHβ15) exhibit differential characteristics in inducing a functional IFN-γ response at fixed concentrations in human PBMCs isolated from healthy donors. Experiments were performed in the presence of 10 ng / ml (rh)IL-12 at two different concentrations (10 nM and 1 nM). The graph shows box plots overlaid with dot plots of IFN-γ release from six different donors. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. [Figure 4C-D]Figure 4 shows a schematic depiction of the antibody architecture and summarizes the data for characterizing such antibodies. These data confirm that antibody manipulation enables the creation of IL-18 mimetic with enhanced agonistic ability. (C) Surrogate agonists (IL18R_tanVHHα2β15 and IL18R_tanVHHβ15α2), arranged in tandem, induced enhanced IFN-γ production in terms of potency and magnitude against PBMCs isolated from healthy donors, resulting in variants with increased potency compared to (rh)IL-18. All experiments were performed in the presence of low doses of (rh)IL-12 (10 ng / ml). IL18R_VHHα1β16, shown in red as a negative control, was used at a fixed concentration of 1 μM. Mean ± SEM of 13 independent experiments are shown. (D) The enhanced tandem IL-18 mimetic is resistant to inhibition by (rh)IL-18BP, while (rh)IL-18 is efficiently blocked from signaling. PBMCs from healthy human donors were stimulated with either (rh)IL-18 at a fixed concentration of 0.5 nM or with either IL18R_tanVHHα2β15 or IL18R_tanVHHβ15α2 in the presence of (rh)IL-12 (10 ng / ml) and various concentrations of (rh)IL-18BP. Five independent experiments were conducted, and mean ± SEM results are shown.

[0033] [Figure 5] Figure 5 summarizes the sorting procedure applied to obtain sdAB. YSD enables enrichment of sdAbs targeting (rh)IL-18Rα and (rh)IL-18Rβ. Sorting output after two rounds of sorting of each library against (rh)IL-18Rα and (rh)IL-18Rβ. A two-dimensional sorting strategy was applied to detect binding functionality simultaneously with full-length VHH display at a concentration of 250 nM. The plot shows 5 × 10³ events of the corresponding sorting output to visualize enrichment.

[0034] [Figure 6] Figure 6 shows the results of binding experiments to test whether single-specific (1+0)VHH SEED bodies exhibit specific binding to (rh)IL-18Rα (red) or (rh)IL-18Rβ (green). (A) Schematic depiction of single-specific VHH targeting (rh)IL-18Rα (red) or (rh)IL-18Rβ (green) transplanted into the hinge region of the SEED skeleton. An sdAb targeting IL-18Rα was fused to the hinge region of the AG chain (msVHHαX), while a VHH directed towards the IL-18Rβ subunit was transplanted onto the GA chain (msVHHβX). (B) Binding of each single-specific VHH SEED body to (rh)IL-18Rα (red) or (rh)IL-18Rβ (green) was determined by BLI. Either (rh)IL-18Rα or (rh)IL-18Rβ was loaded into a HIS1K biosensor at a concentration of 3 μg / ml, followed by the first association of single-specific VHH seed bodies at 100 nM for 300 seconds. Dissociation was then measured in kinetic buffer for 100 seconds. The scheme was developed via www.biorender.com.

[0035] [Figure 7A]Figure 7 shows data from the reporter assay, suggesting that combinatorial reformatting of single-specific (1+0) SEED bodies to strictly monovalent (1+1) bsAb allows for the identification of IL-18 mimetic cells with reduced ability to induce NFκB reporter activity in IL-18 reporter cells. HEK-Blue® reporter cells were incubated with increasing concentrations of reformatted bsAb or (rh)IL-18, and secreted embryonic alkaline phosphatase activity was monitored by determining the OD640. Reporter activity was normalized against the maximum IL-18 reading. Exemplary dose-dependent IL-18R agonisms induced by surrogate agonists IL18R_VHHα2β15, IL18R_VHHα8β15, IL18R_VHHα2β17, and IL18R_VHHα8β17, compared directly with (A)(rh)IL-18. [Figure 7B] Figure 7 shows data from a reporter assay, suggesting that combinatorial reformatting of a single-specific (1+0) SEED body to strictly monovalent (1+1) bsAb allows for the identification of IL-18 mimetic cells with reduced ability to induce NFκB reporter activity in IL-18 reporter cells. HEK-Blue® reporter cells were incubated with increasing concentrations of reformatted bsAb or (rh)IL-18, and secreted embryonic alkaline phosphatase activity was monitored by determining OD640. Reporter activity was normalized against the maximum IL-18 reading. (B) Dose-dependent receptor agonism mediated by all 44 cytokine mimetic cells.

[0036] [Figure 8]Figure 8 shows data from the IFN-γ release assay, demonstrating that the bispecific (1+1) surrogate agonist does not induce IFN-γ release in PBMCs isolated from healthy donors in the absence of a low dose of (rh)IL-12. IFN-γ production in PBMCs stimulated with bsAb at a fixed concentration of 100 nM or (rh)IL-18 at 1 nM in the absence of (rh)IL-12. Dot plot graph of IFN-γ release from eight different donors. IL18R_VHHα1β16 was used as a negative control (shown in red). Four main candidates used for further characterization are shown in green, purple, blue, and orange.

[0037] [Figure 9] Figure 9 shows the binding kinetics data obtained using the top four surrogate agonists with bispecificity (1+1) for the receptor subunits IL-18Rα and IL-18Rβ. IL-18 mimetic was loaded into an AHC biosensor chip at a concentration of 5 μg / ml. Interactions with both receptor subunits were measured at concentrations of 100 nM, 50 nM, 25 nM, and 12.5 nM over 180 seconds, followed by dissociation in kinetics buffer over 300 seconds.

[0038] [Figure 10] Figure 10 summarizes the analytical size exclusion chromatography profiles of the prepared surrogate agonist formats, showing appropriate purity after downstream purification. SEC-HPLC profiles of various formats, as well as the corresponding target monomer peaks, are shown. Absorbance at 214 nm was used to determine purity. (A) SEC profiles after protein A purification for all eight formats, showing target species with purity below 90% for IL18R_VHHα2β15_E430G and IL18R_sdIgGα2β15_E430G. (B) Analysis of IL18R_VHHα2β15_E430G and IL18R_sdIgGα2β15_E430G after the second step of SEC purification.

[0039] [Figure 11] Figure 11 shows data obtained by differential scanning fluorescence quantification to characterize the thermal unfolding of the IL-18R agonist bsAb in various formats. The superposition of melting curves for the IL-18R agonist in various formats was recorded using a temperature gradient of 20°C to 95°C with a slope of 1°C / min. The curve for the first derivative at 350 nm / 330 nm is shown.

[0040] [Figure 12] Figure 12 shows data obtained from PBMC stimulation with compound concentrations across the full dose range, demonstrating a strong hooking effect for (rh)IL-18, while this is less pronounced for different surrogate agonist formats. IFN-γ readings for PBMCs in the presence of low doses of (rh)IL-12 (10 ng / ml). IL18R_VHHα1β16 was used as a negative control at a fixed concentration of 1 μM, shown in red. Mean ± SEM values ​​from 13 independent experiments are shown.

[0041] [Figure 13] Figure 13 shows data for testing the binding of an engineered tandem IL-18 mimetic. Such an engineered tandem IL-18 mimetic exhibits strong binding to both receptor subunits in an avidity-driven setting. (rh)IL-18Rα or (rh)IL-18Rβ was loaded onto a HIS1K sensor chip at 5 μg / ml. A first association step was performed for 180 seconds at 100 nM, 33.3 nM, and 11.1 nM using various surrogate agonist formats, followed by a dissociation step for 300 seconds in kinetic buffer.

[0042] [Figure 14]Figure 14 shows bilayer interference (BLI) binding data for the binding of a bispecific surrogate agonist to (rh)IL-18BP. It was determined that the bispecific surrogate agonist did not bind to (rh)IL-18BP, while (rh)IL-18BP bound to (rh)IL-18 with high affinity. BsAb was loaded at 5 μg / ml onto an AHC biosensor chip. Subsequently, association was monitored for 180 seconds using 1 μM IL-18BP, followed by dissociation in kinetic buffer for 180 seconds.

[0043] [Figure 15] Figure 15 summarizes binding data showing that the bispecific cytokine mimetic competes with (rh)IL-18 for binding to (rh)IL-18Rα. The surrogate agonist was loaded onto an AHC biosensor chip, and then associated with IL-18Rα at 200 nM for 300 seconds. Subsequently, a second association step was performed for 180 seconds using either (rh)IL-18 at 100 nM (red) or kinetic buffer (black). [Modes for carrying out the invention]

[0044] Summary of Sequences (If there are any discrepancies between the table below and the so-called WIPO ST.26 compliant sequence list, the information in the table below should take precedence.) [Table C-1] [Table C-2] [Table C-3] [Table C-4] [Table C-5] [Table C-6] [Table C-7] [Table C-8] [Table C-9]

[0045] Sequence IDs 1-11 provide the amino acid sequences of VHH1-VHH11 (corresponding to α1-α11 in this disclosure). The CDRs of these VHH sequences are provided in Sequence IDs 23-55.

[0046] Sequence IDs 12-22 provide amino acid sequences for VHH12-VHH22 (corresponding to β12-β22 in this disclosure). The CDRs for these VHH sequences are provided in Sequence IDs 56-88.

[0047] Sequence IDs 89-99 provide the amino acid sequences of the SEED AG chain based on VHH1-VHH11(α1-α11) according to this disclosure. Sequence IDs 100-110 provide the amino acid sequences of the SEED GA chain based on VHH12-VHH22(β12-β22) according to this disclosure.

[0048] Detailed description of a certain aspect While this disclosure is described in detail above and below, it should be understood that this disclosure is not limited to the specific methodologies, protocols, and reagents described herein, because they are subject to change. It should also be understood that the terms used herein are intended to describe only specific aspects and are not intended to limit the scope of this disclosure, which will be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0049] In the following, certain elements of this disclosure will be described in more detail, which will include descriptions of specific embodiments. However, the various examples and preferred embodiments described should not be construed as limiting this disclosure to only the embodiments expressly described. This description should be understood as supporting and encompassing embodiments that combine the expressly described embodiments with any number of disclosed and / or preferred elements in any manner. Furthermore, any permutation and combination of all elements described in this application should be considered disclosed by the description of this application unless this would result in a logical contradiction or the context indicates otherwise.

[0050] Unless otherwise defined herein, scientific and technical terms used in conjunction with this disclosure should have meanings generally understood by those skilled in the art. Generally, the nomenclature and techniques referred to herein, such as those in organic chemistry, chemical synthesis, biology, medicinal chemistry and pharmaceutical chemistry, medicine, pharmacology, or toxicology, are well known and commonly used in the art. Unless otherwise indicated, the methods and techniques described herein are generally carried out in accordance with prior art, as described in the references cited and discussed throughout this disclosure.

[0051] A first aspect of this disclosure (also referred to as “Aspect 1”): According to the first aspect, this disclosure relates to a VHH antibody domain or a fragment thereof, hereby (a) The VHH antibody domain or fragment thereof includes one VHH complementarity-determining region CDR1, CDR2, and CDR3 selected from the group consisting of VHH1, VHH2, VHH3, VHH4, VHH5, VHH6, VHH8, VHH9, VHH10, and VHH11 as shown in the table of CDRs; (b) The VHH antibody domain or fragment thereof includes the complementarity-determining regions CDR1, CDR2 and CDR3 as defined in (a), wherein the modification is that at least one of the sequences of CDR1, CDR2 and CDR3 is humanized; or (c) The VHH antibody domain or fragment thereof includes the complementarity-determining regions CDR1, CDR2 and CDR3 as defined in (a) and which are modified, wherein the modifications are - Substitution, addition, or deletion of up to three amino acids in CDR1, - Substitution, addition or deletion of up to three amino acids in CDR2, and / or - Substitution, addition, or deletion of up to three amino acids in CDR3 That is the case.

[0052] CDR table: [Table D-1] [Table D-2] [Table D-3]

[0053] An "antibody" is an immunoglobulin gene, a polypeptide substantially encoded by an immunoglobulin gene, or an antigen-binding fragment thereof, which specifically binds to and recognizes an analyte (antigen). Immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable region genes.

[0054] In primates such as humans, the heavy and light chain variable domains of antibodies combine to specifically bind to antigens. Generally, naturally occurring immunoglobulins in primates (e.g., humans) or murids have heavy (H) and light (L) chains linked to each other by disulfide bonds. Two types of light chains exist: lambda (λ) and kappa (κ). There are five main heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Antibodies in primates can be transclassified.

[0055] Certain IgG antibodies from members of the Camelidae and Dromedariidae families (Camelus bactrianus and Calelus dromaderius), including New World members such as naturally occurring mammalian species of llama (Lama paccos, Lama glama, and Lama vicugna), lack a light chain and are therefore structurally distinguishable from the typical four-chain quaternary structure with two heavy chains and two light chains found in antibodies from other animals. See PCT / EP93 / 02214 (WO 94 / 04678, published March 3, 1994). Such llama IgG subtypes lack a light chain and CH1 domain and are referred to as heavy-chain antibodies. These naturally occurring camel antibodies, consisting only of heavy chains, are functional and stable in the absence of a light chain. The antigen-binding sites of these heavy-chain antibodies are formed by a single domain and are referred to as "VHH" (Koenning et al., 2017), or are used herein synonymously with "VHH antibody domain."

[0056] Each light and heavy chain of an antibody contains a constant domain and a variable domain. The reference "VH" or "VH" refers to the variable region of the immunoglobulin heavy chain, which includes that of antibody fragments. The reference "VL" or "VL" refers to the variable region of the immunoglobulin light chain, such as that found in antibodies of primates. The variable domain of a heavy chain antibody is called VHH. VHH consists of only one polypeptide chain of 15 kDa and is considered the smallest known native domain capable of binding to a full-length antigen.

[0057] The light and heavy chain variable domains contain a “framework” region interrupted by three hypervariable regions, also known as the “complementarity-determining region” or “CDR” (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991). The sequences of different light or heavy chain framework regions are relatively conserved within a species. The antibody framework region, which is a combined framework region of its constituent light and heavy chains, helps to position and align the CDR in three-dimensional space. The CDR is primarily responsible for antigen binding.

[0058] CDRs are typically referred to as CDR1, CDR2, and CDR3 (from N-terminus to C-terminus) and are also typically identified by the chain in which a particular CDR is located. Thus, VH CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, while VL CDR1 is CDR1 from the variable domain of the light chain of the antibody in which it is found. Light chain CDRs are sometimes referred to as CDR L1, CDR L2, and CDR L3. Heavy chain CDRs are sometimes referred to as CDR H1, CDR H2, and CDR H3. VHH monoclonal antibodies have only a heavy chain and therefore contain only one CDR1, CDR2, and CDR3. Generally, CDR3 is primarily responsible for antigen specificity.

[0059] VHH encompasses the following structural region in the N or C direction: N-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-C, where FR represents the amino acid sequence of the framework region and CDR represents the amino acid sequence of the complementarity-determining region (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991).

[0060] As used in this disclosure, “VHH antibody domain” refers to a domain formed by a complete VHH, which includes N-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-C (in the N or C direction). A VHH antibody domain may be part of a larger molecule to which it is covalently linked. Alternatively, a VHH antibody domain may be a molecule of its own, not covalently linked to any other molecular structure.

[0061] Because the resulting paratopes lack light chains, sdAb offers the advantage of multiple reformatting options in a "plug-and-play" manner involving bead-on-string assembly, or facilitates combination with existing Fab-based paratopes for the generation of bisingularities (Lipinski et al., 2023; Chanier and Chames, 2019).

[0062] The framework region and CDR limits are defined (see Kabat et al., Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991). The CDRs of the heavy chain variable domain are located at residues 31-35 (CDR-H1), 50-65 (CDR-H2), and 95-102 (CDR-H3) according to the Kabat numbering system. In antibodies containing light chains, such as antibodies from primates, the CDRs of the light chain variable domain are located at residues 24-34 (CDR-L1), 50-56 (CDR-L2), and 89-97 (CDR-L3) according to the Kabat numbering system. The Kabat database is currently maintained online. The location of the camel CDR can also be determined (see, for example, Sircar et al., J. Immunol. 186:6357-6367, 2011); the Rosetta Antibody program, a program for determining the structure of camel antibodies, is available online.

[0063] A "monoclonal antibody" is an antibody produced by a single clone of B lymphocytes or by cells transfected with the heavy chain gene (and optionally the light chain gene (for primate antibodies)) of a single antibody. Monoclonal antibodies may be obtained using a variety of techniques known to those skilled in the art, including standard hybridoma techniques (see, for example: Kohler and Milstein, Eur. J. Immunol. (1976), vol. 5, p. 511-519; Antibodies: A Laboratory Manual, 2nd edition (2014), edited by Greenfield, Cold Spring Harbor Laboratory Press (USA); Immunobiology, 5th edition (2001), edited by Janeway et al., Garland Publishing (USA)), and expression from eukaryotic host cells transfected with a DNA molecule encoding an alloantibody, or from prokaryotic host cells transfected with a DNA molecule encoding an alloantibody.

[0064] VHH antibody domains can be obtained through genetic engineering to acquire the smallest protein with high affinity to a target, resulting in proteins derived from low molecular weight antibodies. See, for example, Sellmann et al., 2020; U.S. Patent No. 5,759,808 issued June 2, 1998; see also Dumoulin et al., (2003); Pleschberger et al., (2003); Cortez-Retamozo et al., (2002); and Lauwereys et al., (1998).

[0065] In some embodiments, VHH molecules can be generated as recombinant monoclonal antibodies or antigen-binding fragments on various expression platforms, avoiding the use of hybridomas and mice. VHH monoclonal antibodies may be humanized monoclonal antibodies. In some embodiments, monoclonal antibodies may be chimeric antibodies.

[0066] Without being constrained by theory, VHH monoclonal antibodies have a molecular weight of approximately one-tenth that of human IgG molecules, and the proteins have a physical diameter of only a few nanometers.

[0067] One consequence of small size is the ability of VHH monoclonal antibodies to bind to antigenic sites that are functionally invisible to larger antibody proteins. Therefore, VHH monoclonal antibodies are useful as reagents for detecting antigens that would be negative by other methods using classical immunological techniques, and thus useful as therapeutic agents. Another consequence of small size is that camel VHH monoclonal antibodies can inhibit this by binding to specific sites on the target protein in the groove or narrow cleft of the target protein, and thus can be useful in their ability to more closely resemble the function of classical small molecular weight drugs than the function of classical antibodies.

[0068] Without being constrained by theory, the low molecular weight and compact size further result in camel VHH monoclonal antibodies that are extremely thermally stable, stable to extreme pH and proteolytic digestion, and have low antigenicity. Furthermore, these molecules can be fully expressed in prokaryotic cells such as E. coli, and are functional when expressed as fusion proteins with bacteriophages.

[0069] "Humanizing" an antibody / antibody sequence, as used herein, refers to the germlining process, in which a non-human (such as camel, llama, or synthetic) antibody sequence is adapted to be more similar to a human antibody sequence by replacing one or more individual amino acids with corresponding amino acids from a human antibody sequence. Typically, a human antibody sequence that is particularly close to (i.e., has a high degree of sequence homology to) the non-human sequence will be selected. Such a human antibody sequence can be identified, for example, by BLAST search. The corresponding amino acids can then be identified by pairwise sequence alignment between the selected human antibody sequence and the non-human antibody sequence to be humanized. After humanization, the humanized antibody still binds to the same antigen as the original non-human antibody before humanization. Humanized immunoglobulins can be constructed by means of genetic engineering. VHH antibody domains are readily humanized based on human VH domains having sequences that are highly homologous to the sequence of the VHH antibody domain.

[0070] Furthermore, VHH sequences can be adapted by reducing their sequence liability. This may include, for example, replacing each amino acid (one or more) in the VHH sequence that is prone to glycosylation, deamination, oxidation, or isomerization with the corresponding amino acid in the nearest human germline sequence, or with another amino acid (e.g., alanine). Similarly, the hydrophobic patch on the cell surface can be reduced by replacing each amino acid (one or more) with a less hydrophobic amino acid (one or more). Again, the amino acid replacements are selected so that, after adaptation, the VHH sequence still binds to the same antigen and with characteristics similar to the pre-adaptation VHH sequence.

[0071] VHH can be used as a modular component to create multivalent and / or multispecific antibody constructs, where "multivalent" means the construct contains one or more single-domain antibodies, and "multispecific" means it contains one or more single-domain antibodies with different binding specificities.

[0072] In some cases, this disclosure describes a particular protein / amino acid sequence A as a “fragment” of another protein / amino acid sequence B. This means that, compared to protein / amino acid B, protein / amino acid sequence A is missing one or more amino acids at its N-terminus and / or one or more amino acids at its C-terminus. Whether a protein / amino acid sequence is missing one or more amino acids at its N-terminus and / or one or more amino acids at its C-terminus compared to another protein / amino acid sequence can be easily determined, for example, by forming a sequence alignment using a program of the BLAST family.

[0073] As those skilled in the art will understand, where this disclosure refers to a “VHH antibody domain or fragment thereof,” the fragment is a fragment that binds to an antigen. Thus, the fragment binds to the same antigen as the original “full-length” VHH antibody domain from which the fragment according to this disclosure is derived (i.e., IL-18Rβ to IL-18Rα). Preferably, the fragment of the VHH antibody domain is a C-terminal fragment. This means that the fragment has an amino acid deletion at the N-terminus compared to the “complete” VHH antibody domain sequence.

[0074] Where this disclosure refers to a VHH antibody domain or fragment (or corresponding phrase) of "one VHH selected from the group consisting of VHH1, VHH2, and VHH3 as shown in the table of CDRs," this means that the VHH antibody domain does not include a combination of CDRs or a mixture of CDRs selected from VHH1, VHH2, or VHH3, but not from the VHHs listed. Therefore, the VHH antibody domain or its fragments include, for example, combinations of CDR1, CDR2, and CDR3 of VHH1 (SEQ ID NOs. 23, 24, and 25), combinations of CDR1, CDR2, and CDR3 of VHH2 (SEQ ID NOs. 26, 27, and 28), or combinations of CDR1, CDR2, and CDR3 of VHH3 (SEQ ID NOs. 29, 30, and 31), but do not include combinations of CDR1 and CDR2 of VHH1 and CDR3 of VHH2 (SEQ ID NOs. 23, 24, 29, and 28).

[0075] Where this disclosure defines that a VHH antibody domain or fragment thereof includes a modified complementarity-determining region CDR1, CDR2, and CDR3 as defined in (a), for example, where modification means, for example, that at least one of the sequences of CDR1, CDR2, and CDR3 is humanized, and a person skilled in the art will understand that this humanization exists compared to the corresponding sequence in a table of CDRs that provides combinations of unmodified CDR sequences.

[0076] This disclosure indicates the presence of a modification which is a “substitution, addition, or deletion” of a specific number of amino acids (e.g., up to three), and those skilled in the art will understand that this refers to individual substitutions, additions, or deletions of the indicated number of amino acids. Thus, the substituted, added, or deleted amino acids may be adjacent or independently separated within the amino acid sequence. Furthermore, as stated above, those skilled in the art will understand that this definition refers to substitutions, additions, or deletions compared to the unmodified sequence in the CDR table.

[0077] Aspect 2: The modification in (b) is such that the sequences of CDR1 and / or CDR2 are humanized, but the sequence of CDR3 is not humanized, the VHH antibody domain or fragment according to Aspect 1.

[0078] A VHH antibody domain or fragment according to either Aspect 1 or 2, wherein the modification in Aspect 3 (b) is such that the sequence of CDR1 is humanized, but the sequences of CDR2 and CDR3 are not humanized.

[0079] A VHH antibody domain or fragment according to either Aspect 4:(b), wherein the modification in Aspect 4 is such that the sequence of CDR2 is humanized, but the sequences of CDR1 and CDR3 are not humanized.

[0080] A VHH antibody domain or fragment according to any one of Aspects 1 to 4, wherein the modification in Aspect 5(b) is such that one of the sequences CDR1, CDR2, and CDR3 is humanized, but more than one is not.

[0081] Embodiment 6: A VHH antibody domain or fragment according to any one of Embodiments 1 to 5, wherein the humanization of the CDR(s) is performed by replacing at least one amino acid in the sequence of the CDR with the corresponding amino acid of a human VH domain.

[0082] Embodiment 7: A VHH antibody domain or fragment according to any one of Embodiments 1 to 6, wherein the humanization of the CDR(s) is performed by replacing at least three amino acids in the sequence of the CDR with corresponding amino acids of a human VH domain.

[0083] Embodiment 8: A VHH antibody domain or fragment according to any one of Embodiments 1 to 6, wherein the humanization of the CDR(s) is performed by replacing up to three amino acids in the sequence of CDR1 and / or CDR2 and up to one amino acid in the sequence of CDR3 with the corresponding amino acids of a human VH domain.

[0084] Embodiment 9: A VHH antibody domain or fragment according to any one of Embodiments 1 to 6, wherein the humanization of the CDR(s) is performed by replacing up to two amino acids in the sequence of the CDR with corresponding amino acids of a human VH domain.

[0085] Embodiment 10: A VHH antibody domain or fragment according to any one of Embodiments 1 to 6, wherein the humanization of the CDR(s) is performed by replacing up to two amino acids in the sequence of CDR1 and / or CDR2 and up to one amino acid in the sequence of CDR3 with the corresponding amino acids of a human VH domain.

[0086] Embodiment 11: A VHH antibody domain or fragment according to any one of Embodiments 1 to 10, wherein the humanization of the CDR(s) is performed by replacing one amino acid in the sequence of the CDR with the corresponding amino acid of a human VH domain.

[0087] The modification in aspect 12:(c) is - Substitution, addition, or deletion of up to three amino acids in CDR1, - Substitution, addition or deletion of up to three amino acids in CDR2, and / or - Substitution, addition, or deletion of up to one amino acid in CDR3 A VHH antibody domain or fragment thereof according to any one of embodiments 1 to 11.

[0088] The modification in aspect 13:(c) is - Substitution, addition, or deletion of up to two amino acids in CDR1, - Substitution, addition or deletion of up to two amino acids in CDR2, and / or - Substitution, addition, or deletion of up to two amino acids in CDR3; A VHH antibody domain or fragment thereof according to any one of embodiments 1 to 11.

[0089] The modification in aspect 14:(c) is - Substitution, addition, or deletion of up to two amino acids in CDR1; - Substitution, addition, or deletion of up to two amino acids in CDR2; and / or - Substitution, addition, or deletion of up to one amino acid in CDR3 A VHH antibody domain or fragment thereof according to any one of embodiments 1 to 11.

[0090] The modification in aspect 15:(c) is - Substitution, addition or deletion of up to two amino acids in CDR1, and / or - Substitution, addition, or deletion of up to two amino acids in CDR2; And here, the sequence of CDR3 is unmodified. A VHH antibody domain or a fragment thereof according to any one of embodiments 1 to 11.

[0091] As a person skilled in the art would understand, the instruction "the sequence of CDR3 is unmodified" means that the sequence is not modified compared to the sequence provided in the table of CDRs for the VHH in question.

[0092] The modification in aspect 16:(c) is - Substitution, addition, or deletion of up to two amino acids in CDR1 Here, the sequences of CDR2 and CDR3 are unmodified. A VHH antibody domain or a fragment thereof according to any one of embodiments 1 to 11.

[0093] The modification in aspect 17:(c) is - Substitution, addition, or deletion of up to two amino acids in CDR2 Here, the sequences of CDR1 and CDR3 are unmodified. A VHH antibody domain or a fragment thereof according to any one of embodiments 1 to 11.

[0094] The modification in aspect 18:(c) is - Substitution, addition, or deletion of up to one amino acid in CDR1; - Substitution, addition, or deletion of up to one amino acid in CDR2; and / or - Substitution, addition, or deletion of up to one amino acid in CDR3 A VHH antibody domain or fragment thereof according to any one of embodiments 1 to 11.

[0095] The modification in aspect 19:(c) is - Substitution, addition or deletion of one amino acid in CDR1, and / or - Substitution, addition, or deletion of a single amino acid in CDR2; And here, the sequence of CDR3 is unmodified. A VHH antibody domain or a fragment thereof according to any one of embodiments 1 to 11.

[0096] The modification in aspect 20:(c) is - Substitution, addition, or deletion of one amino acid in CDR1, Here, the sequences of CDR2 and CDR3 are unmodified. A VHH antibody domain or a fragment thereof according to any one of embodiments 1 to 11.

[0097] The modification in aspect 21:(c) is - Substitution, addition, or deletion of one amino acid in CDR2, Here, the sequences of CDR1 and CDR3 are unmodified. A VHH antibody domain or a fragment thereof according to any one of embodiments 1 to 11.

[0098] Embodiment 22: A VHH antibody domain or fragment thereof according to any one of Embodiments 1 to 21, wherein the modification in (c) comprises only amino acid substitutions and does not involve the addition or deletion of amino acids.

[0099] Embodiment 23: A VHH antibody domain or fragment thereof according to any one of Embodiments 1 to 22, wherein the substitution is a conservative amino acid substitution.

[0100] As used herein, “conservative amino acid substitution” refers to the substitution of one amino acid with another biologically similar amino acid. Conservative substitutions are unlikely to alter the shape or characteristics of a protein / amino acid sequence. Examples of conservative substitutions include the substitution of one hydrophobic residue, such as isoleucine, valine, leucine, or methionine, with another, or the substitution of one polar residue with another, such as arginine to lysine, glutamic acid to aspartic acid, or glutamine to asparagine.

[0101] A second aspect of this disclosure (also referred to as "Aspect 24"): According to the second aspect, this disclosure relates to a VHH antibody domain or fragment thereof, hereby (A) The VHH antibody domain contains an amino acid sequence of VHH selected from the group consisting of VHH1, VHH2, VHH3, VHH4, VHH5, VHH6, VHH8, VHH9, VHH10 and VHH11 as shown in the VHH sequence table; (B) The VHH antibody domain comprises a VHH sequence as defined in (A) and having modifications, wherein the modification is humanization of the sequence; (C) The VHH antibody domain comprises a VHH sequence as defined in (A) and having modifications, wherein the modifications are substitutions, additions, or deletions of up to 25 amino acids; or, (D) The VHH antibody domain comprises a VHH sequence that is at least 75% identical to the VHH sequence mentioned in (A);

[0102] Table of VHH sequences: [Table E-1] [Table E-2] [Table E-3]

[0103] A third aspect of this disclosure (also referred to as "Aspect 25"): According to the third aspect, this disclosure relates to VHH antibody domains or fragments thereof, hereby (A) The VHH antibody domain consists of an amino acid sequence of VHH selected from the group consisting of VHH1, VHH2, VHH3, VHH4, VHH5, VHH6, VHH8, VHH9, VHH10, and VHH11 as shown in the VHH sequence table; (B) The VHH antibody domain consists of a VHH sequence as defined in (A) and having modifications, wherein the modification is such that the sequence is humanized; (C) The VHH antibody domain consists of a VHH sequence as defined in (A) and is modified, wherein the modification is the substitution, addition or deletion of up to 25 amino acids; or (D) The VHH antibody domain consists of a VHH sequence that is at least 75% identical to the VHH sequence mentioned in (A).

[0104] The same descriptions and definitions apply to the first aspect of this disclosure and the aspects referring thereto, as well as to the second and third aspects of this disclosure and the aspects referring thereto.

[0105] As those skilled in the art will understand, where this disclosure indicates that a VHH antibody domain includes, for example, "a VHH sequence of VHH selected from the group consisting of VHH1, VHH2, VHH3, VHH4, VHH5, VHH6, VHH8, VHH9, VHH10, and VHH11 as shown in the Table of VHH Sequences," this means that the VHH antibody domain includes one of the sequences listed in the Table of VHH Sequences, but not more than one (and not all of them).

[0106] Therefore, if the VHH antibody domain or fragment thereof in this disclosure includes / consists of a VHH sequence having modifications as defined in (A), where the modifications are that the sequence is humanized, a person skilled in the art will understand that this humanization exists in comparison to the corresponding sequence in a table of VHH sequences that provide unmodified sequences.

[0107] Where this disclosure states that a particular sequence A is “at least x% identical” to another sequence B, this is equivalent to the statement that sequence A has “x% identity” with sequence B. Such statements reflect the relationship between two polypeptide sequences A and B as determined by comparing the sequences. Generally, identity refers to the exact amino acid-to-amino acid match of each of the two polypeptide sequences over the length of the sequence being compared. For sequences where an exact match does not exist, the percentage of identity between the two sequences may be determined. Generally, two sequences to be compared are aligned to obtain the greatest possible correlation between them. This may involve inserting “gaps” in one or both sequences to enhance the degree of alignment. % identity may be determined over the entire length of each of the sequences being compared (so-called global alignment), which is particularly preferred for sequences of the same or very similar lengths, or it may be determined over a shorter, predetermined length (so-called local alignment), which is more preferred for sequences of unequal lengths.

[0108] Methods for comparing the identity of two or more sequences are well known in the art. Therefore, programs available in the Wisconsin Sequence Analysis Package, version 9.1 (Devereux J et al., 1984), such as BESTFIT and GAP, may be used to determine the % identity between two polynucleotides and between two polypeptide sequences. BESTFIT uses the "local homology" algorithm of Smith and Waterman (1981) to find the single region of similarity between two sequences. Other programs for determining identical sequences are also well known in the art, such as the BLAST family of programs (Altschul SF et al., 1990, Altschul SF et al., 1997: accessible through the NCBI homepage at www.ncbi.nlm.nih.gov) and FASTA (Pearson WR, 1990). Preferably, the % identity provided in this disclosure is determined in accordance with the BLAST family program (Altschul SF et al, 1990, Altschul SF et al, 1997: accessible through the NCBI homepage at www.ncbi.nlm.nih.gov).

[0109] Embodiment 26: A VHH antibody domain or fragment thereof according to either Embodiment 24 or 25, wherein the fragment of the VHH antibody domain comprises at least 75% of the amino acids of the sequence of the VHH antibody domain. As those skilled in the art will understand, this means that the fragment is missing up to one-quarter of the total number of amino acids of the VHH antibody domain, where the amino acids are missing at the N-terminus or C-terminus compared to the “complete” VHH antibody domain sequence.

[0110] Embodiment 27: A VHH antibody domain or fragment thereof according to any one of Embodiment 24 or 25, wherein the fragment of the VHH antibody domain comprises at least 80% of the amino acids of the sequence of the VHH antibody domain.

[0111] Embodiment 28: A VHH antibody domain or fragment thereof according to any one of Embodiment 24 or 25, wherein the fragment of the VHH antibody domain comprises at least 85% of the amino acids of the sequence of the VHH antibody domain.

[0112] Embodiment 29: A VHH antibody domain or fragment thereof according to any one of Embodiment 24 or 25, wherein the fragment of the VHH antibody domain comprises at least 90% of the amino acids of the sequence of the VHH antibody domain.

[0113] Embodiment 30: A VHH antibody domain or fragment thereof according to any one of Embodiments 24 or 25, wherein the fragment of the VHH antibody domain comprises at least 95% of the amino acids of the sequence of the VHH antibody domain.

[0114] Embodiment 31: A VHH antibody domain or fragment thereof according to any one of Embodiments 24 or 25, wherein the fragment of the VHH antibody domain comprises at least 98% of the amino acids of the sequence of the VHH antibody domain.

[0115] Embodiment 32: A VHH antibody domain or fragment thereof according to any one of Embodiment 24 or 25, wherein the fragment of the VHH antibody domain comprises at least 99% of the amino acids of the sequence of the VHH antibody domain.

[0116] Embodiment 33: A VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 32, wherein the fragment of the VHH antibody domain includes complementarity-determining regions CDR1, CDR2 and CDR3.

[0117] Embodiment 34: A VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 33, wherein the fragment of the VHH antibody domain includes at least the sequence from the N-terminus of CDR1 to the C-terminus of CDR3 of the VHH antibody domain.

[0118] Embodiment 35: A VHH antibody domain or fragment according to any one of Embodiments 24 to 34, wherein the fragment of the VHH antibody domain includes all of the complementarity-determining regions (CDRs) of the VHH antibody domain.

[0119] Aspect 36:(B) wherein the humanization of the sequence is performed by replacing at least one amino acid of the sequence with the corresponding amino acid of the human VH (variable heavy chain) domain, a VHH antibody domain or fragment according to any one of aspects 24 to 35.

[0120] Aspect 37:(B) wherein the humanization of the sequence is performed by replacing up to 25 amino acids of the sequence with the corresponding amino acids of a human VH domain (individually), a VHH antibody domain or fragment according to any one of aspects 24 to 36.

[0121] As those skilled in the art will understand, where this disclosure refers to the substitution of an amino acid in a particular sequence / domain A with a “corresponding” amino acid in a particular sequence / domain B, this means that the amino acid in sequence / domain A is substituted with an amino acid in sequence / domain B that aligns with the amino acid in sequence / domain A in the alignment of the two sequences.

[0122] A VHH antibody domain or fragment according to any one of Aspects 24 to 36, wherein the humanization of the sequence is performed by replacing up to 20 amino acids of the sequence with the corresponding amino acids of a human VH domain, in Aspect 38:(B).

[0123] A VHH antibody domain or fragment according to any one of Aspects 24 to 36, wherein the humanization of the sequence is performed by replacing up to 15 amino acids of the sequence with the corresponding amino acids of a human VH domain in Aspect 39 (B).

[0124] A VHH antibody domain or fragment according to any one of Aspects 24 to 36, wherein the humanization of the sequence is performed by replacing up to 10 amino acids of the sequence with the corresponding amino acids of a human VH domain, in Aspect 40:(B).

[0125] A VHH antibody domain or fragment according to any one of Aspects 24 to 36, wherein the humanization of the sequence is performed by replacing up to five amino acids of the sequence with the corresponding amino acids of a human VH domain, in Aspect 41:(B).

[0126] Embodiment 42:(B), the humanization of the sequence is performed by replacing up to three amino acids of the sequence with corresponding amino acids of a human VH domain, a VHH antibody domain or fragment according to any one of Embodiments 24 to 36.

[0127] A VHH antibody domain or fragment according to any one of Aspects 24 to 36, wherein the humanization of the sequence is performed by replacing up to two amino acids of the sequence with corresponding amino acids of a human VH domain, in Aspect 43:(B).

[0128] A VHH antibody domain or fragment according to any one of Aspects 24 to 36, wherein the humanization of the sequence is performed by replacing one amino acid of the sequence with the corresponding amino acid of a human VH domain, in Aspect 44:(B).

[0129] Embodiment 45:(B), wherein the humanization is located within the framework region of the VHH antibody domain and / or within the CDR of the VHH antibody domain, according to any one of Embodiments 24 to 44.

[0130] Embodiment 46:(B), wherein the humanization is within the framework region of the VHH antibody domain but not within the CDR of the VHH antibody domain, and is a VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 44.

[0131] Embodiment 47:(B), wherein the humanization is within the CDR of the VHH antibody domain but not within the framework region of the VHH antibody domain, and is a VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 36.

[0132] Embodiment 48:(B), wherein the humanization of the VHH antibody domain within the CDR is within CDR1, CDR2 and / or CDR3, a VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 45 or 47.

[0133] Embodiment 49:(B), wherein the humanization of the VHH antibody domain within the CDR is within CDR1 and / or CDR2, a VHH antibody domain or fragment thereof according to any one of Embodiments 24-45 or 47-48.

[0134] Embodiment 50:(B), wherein the humanization of the VHH antibody domain within the CDR is within CDR1, a VHH antibody domain or fragment thereof according to any one of Embodiments 24-45 or 47-49.

[0135] Embodiment 51:(B), wherein the humanization of the VHH antibody domain within the CDR is within the CDR2, a VHH antibody domain or fragment thereof according to any one of Embodiments 24-45 or 47-50.

[0136] Embodiment 52:(B), wherein the humanization of the VHH antibody domain within the CDR is not within CDR3, but is a VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 51.

[0137] Embodiment 53:(C), the modification is a VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 52, wherein the modification is a substitution, addition, or deletion of up to 20 amino acids.

[0138] Embodiment 54:(C), the modification is a VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 52, wherein the modification is a substitution, addition, or deletion of up to 15 amino acids.

[0139] Embodiment 55:(C), the modification is a VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 52, wherein the modification is a substitution, addition, or deletion of up to 10 amino acids.

[0140] Embodiment 56:(C), the modification is a VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 52, wherein the modification is a substitution, addition, or deletion of up to five amino acids.

[0141] Embodiment 57:(C), the modification is a VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 52, wherein the modification is a substitution, addition, or deletion of up to three amino acids.

[0142] Embodiment 58:(C), the modification is a VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 52, wherein the modification is a substitution, addition, or deletion of up to two amino acids.

[0143] Embodiment 59:(C), the modification is a VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 52, wherein the modification is a substitution, addition, or deletion of one amino acid.

[0144] Embodiment 60: A VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 59, wherein the modification in (C) comprises only amino acid substitutions and does not involve the addition or deletion of amino acids.

[0145] Aspect 61:(A), wherein the VHH antibody domain or fragment thereof comprises one VHH complementarity-determining region CDR1, CDR2, and CDR3 selected from the group consisting of VHH1, VHH2, VHH3, VHH4, VHH5, VHH6, VHH8, VHH9, VHH10, and VHH11 as shown in the table of CDRs, a VHH antibody domain or fragment thereof according to any one of aspects 24 to 60.

[0146] Embodiment 62: A VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 61, wherein in (B) to (D), (a) The VHH antibody domain or fragment thereof includes one VHH complementarity-determining region CDR1, CDR2, and CDR3 selected from the group consisting of VHH1, VHH2, VHH3, VHH4, VHH5, VHH6, VHH8, VHH9, VHH10, and VHH11 as shown in the table of CDRs; (b) The VHH antibody domain or fragment thereof includes the complementarity-determining regions CDR1, CDR2 and CDR3 as defined in (a), wherein the modification is that at least one of the sequences of CDR1, CDR2 and CDR3 is humanized; (c) The VHH antibody domain or fragment thereof includes the complementarity-determining regions CDR1, CDR2 and CDR3 as defined in (a) and which are modified, wherein the modifications are - Substitution, addition, or deletion of up to three amino acids in CDR1, - Substitution, addition or deletion of up to three amino acids in CDR2, and / or - Substitution, addition, or deletion of up to three amino acids in CDR3 That is the case.

[0147] A VHH antibody domain or fragment according to Aspect 62, wherein the modification in Aspect 63(b) is such that the sequences of CDR1 and / or CDR2 are humanized, but the sequence of CDR3 is not humanized.

[0148] A VHH antibody domain or fragment thereof according to either one of Aspect 62 or 63, wherein the modification in Aspect 64:(b) is such that the sequence of CDR1 is humanized, but the sequences of CDR2 and CDR3 are not humanized.

[0149] A VHH antibody domain or fragment thereof according to either one of Aspect 62 or 63, wherein the modification in Aspect 65:(b) is such that the sequence of CDR2 is humanized, but the sequences of CDR1 and CDR3 are not humanized.

[0150] A VHH antibody domain or fragment according to any one of embodiments 62 to 65, wherein the modification in embodiment 66:(b) is such that one of the sequences CDR1, CDR2, and CDR3 is humanized, but more than one is not.

[0151] Embodiment 67: A VHH antibody domain or fragment thereof according to any one of Embodiments 62 to 66, wherein the humanization of the CDR(s) is performed by replacing at least one amino acid in the sequence of the CDR with the corresponding amino acid of a human VH domain.

[0152] Embodiment 68: A VHH antibody domain or fragment thereof according to any one of Embodiments 62 to 67, wherein the humanization of the CDR(s) is performed by replacing at least three amino acids in the sequence of the CDR with corresponding amino acids of a human VH domain.

[0153] Embodiment 69: A VHH antibody domain or fragment according to any one of Embodiments 62 to 67, wherein the humanization of the CDR(s) is performed by replacing up to three amino acids in the sequence of CDR1 and / or CDR2 and up to one amino acid in the sequence of CDR3 with the corresponding amino acids of a human VH domain.

[0154] Embodiment 70: A VHH antibody domain or fragment thereof according to any one of Embodiments 62 to 67, wherein the humanization of the CDR(s) is performed by replacing up to two amino acids in the sequence of the CDR with corresponding amino acids of a human VH domain.

[0155] Embodiment 71: A VHH antibody domain or fragment thereof according to any one of Embodiments 62 to 67, wherein the humanization of the CDR(s) is performed by replacing up to two amino acids in the sequence of CDR1 and / or CDR2 and up to one amino acid in the sequence of CDR3 with the corresponding amino acids of a human VH domain.

[0156] Embodiment 72: A VHH antibody domain or fragment thereof according to any one of Embodiments 62 to 71, wherein the humanization of the CDR(s) is performed by replacing one amino acid in the sequence of the CDR with the corresponding amino acid of a human VH domain.

[0157] The modification in aspect 73:(c) is - Substitution, addition, or deletion of up to three amino acids in CDR1, - Substitution, addition or deletion of up to three amino acids in CDR2, and / or - Substitution, addition, or deletion of up to two amino acids in CDR3 A VHH antibody domain or fragment thereof according to any one of embodiments 62 to 72.

[0158] The modification in aspect 74:(c) is - Substitution, addition, or deletion of up to three amino acids in CDR1, - Substitution, addition or deletion of up to three amino acids in CDR2, and / or - Substitution, addition, or deletion of up to one amino acid in CDR3 A VHH antibody domain or fragment thereof according to any one of embodiments 62 to 72.

[0159] The modification in aspect 75:(c) is - Substitution, addition or deletion of up to 3 amino acids in CDR1, and / or - Substitution, addition, or deletion of up to three amino acids in CDR2 And here, the sequence of CDR3 is unmodified. A VHH antibody domain or fragment thereof according to any one of embodiments 62 to 72.

[0160] The modification in aspect 76:(c) is - Substitution, addition, or deletion of up to two amino acids in CDR1, - Substitution, addition or deletion of up to two amino acids in CDR2, and / or - Substitution, addition, or deletion of up to two amino acids in CDR3 A VHH antibody domain or fragment thereof according to any one of embodiments 62 to 72.

[0161] The modification in aspect 77:(c) is - Substitution, addition, or deletion of up to two amino acids in CDR1; - Substitution, addition, or deletion of up to two amino acids in CDR2; and / or - Substitution, addition, or deletion of up to one amino acid in CDR3 A VHH antibody domain or fragment thereof according to any one of embodiments 62 to 72.

[0162] The modification in aspect 78:(c) is - Substitution, addition or deletion of up to two amino acids in CDR1, and / or - Substitution, addition, or deletion of up to two amino acids in CDR2; And here, the sequence of CDR3 is unmodified. A VHH antibody domain or fragment thereof according to any one of embodiments 62 to 72.

[0163] As those skilled in the art will recognize, "unmodified" means that it has not been modified compared to its arrangement in the CDR table.

[0164] The modification in aspect 79:(c) is - Substitution, addition, or deletion of up to two amino acids in CDR1 Here, the sequences of CDR2 and CDR3 are unmodified. A VHH antibody domain or fragment thereof according to any one of embodiments 62 to 72.

[0165] The modification in aspect 80:(c) is - Substitution, addition, or deletion of up to two amino acids in CDR2 Here, the sequences of CDR1 and CDR3 are unmodified. A VHH antibody domain or fragment thereof according to any one of embodiments 62 to 72.

[0166] The modification in aspect 81:(c) is - Substitution, addition, or deletion of up to one amino acid in CDR1; - Substitution, addition, or deletion of up to one amino acid in CDR2; and / or - Substitution, addition, or deletion of up to one amino acid in CDR3 A VHH antibody domain or fragment thereof according to any one of embodiments 62 to 72.

[0167] The modification in aspect 82:(c) is - Substitution, addition or deletion of one amino acid in CDR1, and / or - Substitution, addition, or deletion of a single amino acid in CDR2; And here, the sequence of CDR3 is unmodified. A VHH antibody domain or fragment thereof according to any one of embodiments 62 to 72.

[0168] The modification in aspect 83:(c) is - Substitution, addition, or deletion of one amino acid in CDR1 Here, the sequences of CDR2 and CDR3 are unmodified. A VHH antibody domain or fragment thereof according to any one of embodiments 62 to 72.

[0169] The modification in aspect 84:(c) is - Substitution, addition, or deletion of one amino acid in CDR2 Here, the sequences of CDR1 and CDR3 are unmodified. A VHH antibody domain or fragment thereof according to any one of embodiments 62 to 72.

[0170] A VHH antibody domain or fragment thereof according to any one of Aspects 62 to 84, wherein the modification in Aspect 85:(c) comprises only amino acid substitutions and does not involve the addition or deletion of amino acids.

[0171] Embodiment 86: A VHH antibody domain or fragment thereof according to any one of Embodiments 62 to 85, wherein the substitution is a conservative amino acid substitution.

[0172] Embodiment 87: A VHH antibody domain or fragment thereof according to any one of Embodiments 1 to 86, wherein the fragment consists of at least 100 amino acids.

[0173] Embodiment 88: A VHH antibody domain or fragment thereof according to any one of Embodiments 1 to 86, wherein the fragment consists of at least 105 amino acids.

[0174] Aspect 89: The VHH antibody domain or a fragment thereof according to any one of Aspects 1 to 86, wherein the fragment consists of at least 110 amino acids.

[0175] Aspect 90: The VHH antibody domain or a fragment thereof according to any one of Aspects 1 to 86, wherein the fragment consists of at least 115 amino acids.

[0176] Aspect 91: The VHH antibody domain or a fragment thereof according to any one of Aspects 1 to 90, wherein the VHH antibody domain is an anti-IL-18Rα VHH antibody domain.

[0177] Aspect 92: The VHH antibody domain or a fragment thereof according to any one of Aspects 1 to 91, wherein the VHH antibody domain is specific for IL-18Rα.

[0178] Aspect 93: The VHH antibody domain or a fragment thereof according to any one of Aspects 1 to 92, wherein the VHH antibody domain or the fragment binds to IL-18Rα.

[0179] Aspect 94: The VHH antibody domain or a fragment thereof according to any one of Aspects 1 to 93, wherein the VHH antibody domain or the fragment specifically binds to IL-18Rα.

[0180] Aspect 95: The VHH antibody domain or a fragment thereof according to any one of Aspects 1 to 94, wherein the VHH antibody domain or the fragment does not bind to IL-18Rβ.

[0181] Aspect 96: The VHH antibody domain or a fragment thereof according to any one of Aspects 1 to 95, wherein the VHH antibody domain or the fragment can specifically bind to IL-18Rα ECD.

[0182] In some embodiments, where the Disclosure describes a first molecule / group of molecules (e.g., an antibody / antibody component) as "capable of specifically binding" to a second molecule / group of molecules (e.g., the antigen of interest), this means that the first molecule / group of molecules (in this example, the antibody) binds to the second molecule / group of molecules (in this example, the antigen of interest) with an affinity at least 10 times higher than its affinity for other molecules / groups of molecules, particularly other molecules / groups of molecules in the human body (in this example, at least 10 times higher than its affinity for binding to nonspecific antigens other than the antigen of interest (or closely related antigens) (e.g., BSA, casein)). In a preferred embodiment, a first molecule / molecule group (e.g., an antibody / antibody component) that "specifically binds" to a second molecule / molecule group (e.g., the antigen of interest) binds to the antigen with an affinity at least 100 times higher than its affinity for other molecules / molecule groups, particularly other molecules / molecule groups in the human body (in this example, at least 100 times higher than its affinity for binding to nonspecific antigens other than the antigen of interest (or closely related antigen)). Typically, such binding will be determined under physiological conditions. The first molecule / molecule group that "specifically binds" to the second molecule / molecule group binds to that second molecule / molecule group with an affinity of 1 × 10⁻¹⁶. -7 It may be bound with M or a stronger KD.

[0183] Embodiment 97: The VHH antibody domain or its fragment is 1 × 10 -6 A VHH antibody domain or fragment thereof, according to any one of embodiments 1 to 96, that binds to recombinant human IL-18RαECD with M or a stronger KD.

[0184] Embodiment 98: The VHH antibody domain or its fragment is 1 × 10 -7 A VHH antibody domain or fragment thereof, according to any one of embodiments 1 to 97, that binds to recombinant human IL-18RαECD with M or a stronger KD.

[0185] Aspect 99: The VHH antibody domain or a fragment thereof binds to recombinant human IL-18RαECD with a KD of 5×10 -8 M or stronger, and is a VHH antibody domain or a fragment thereof according to any one of Aspects 1 to 97.

[0186] Aspect 100: The VHH antibody domain or a fragment thereof binds to recombinant human IL-18RαECD with a KD of 1.5×10 -8 M or stronger, and is a VHH antibody domain or a fragment thereof according to any one of Aspects 1 to 97.

[0187] Aspect 101: The VHH antibody domain or a fragment thereof binds to recombinant human IL-18RαECD with a KD of 1×10 -8 M or stronger, and is a VHH antibody domain or a fragment thereof according to any one of Aspects 1 to 96.

[0188] Aspect 102: The VHH antibody domain or a fragment thereof binds to recombinant human IL-18RαECD with a KD of 5×10 -9 M or stronger, and is a VHH antibody domain or a fragment thereof according to any one of Aspects 1 to 96.

[0189] Aspect 103: The VHH antibody domain or a fragment thereof binds to recombinant human IL-18RαECD with a KD of 1×10 -9 M or stronger, and is a VHH antibody domain or a fragment thereof according to any one of Aspects 1 to 96.

[0190] Aspect 104: The fragment of the VHH antibody domain binds to recombinant human IL-18RαECD with at least 90% of the affinity (determined by the KD value) of the VHH antibody domain for binding to recombinant human IL-18RαECD, and is a VHH antibody domain or a fragment thereof according to any one of Aspects 1 to 103.

[0191] Embodiment 105: The VHH antibody domain or fragment thereof according to any one of Embodiments 1 to 104, wherein the fragment of the VHH antibody domain binds to IL-18RαECD with an affinity (determined from the KD value) that is at least equivalent to or stronger than the affinity that VHH2 (SEQ ID NO: 2) has for binding to IL-18RαECD.

[0192] Such binding can be determined by in vitro binding experiments (by bilayer interference) as described in Example 1 below.

[0193] Aspect 106: A VHH antibody domain or fragment according to any one of aspects 1-23 or 62-105, wherein the VHH antibody domain or fragment binds to recombinant human IL-18RαECD with an affinity (KD value) not more than 10 times weaker than the binding of the corresponding VHH antibody domain without modification.

[0194] Aspect 107: A VHH antibody domain or fragment according to any one of aspects 1-23 or 62-105, wherein the VHH antibody domain or fragment binds to recombinant human IL-18RαECD with an affinity (KD value) not more than 5 times weaker than the binding of the corresponding VHH antibody domain without modification.

[0195] Aspect 108: A VHH antibody domain or fragment according to any one of aspects 1-23 or 62-105, wherein the VHH antibody domain or fragment binds to recombinant human IL-18RαECD with an affinity (KD value) not more than twice weaker than the binding of the corresponding VHH antibody domain without modification.

[0196] Aspect 109: A VHH antibody domain or fragment according to any one of aspects 1 to 23 or 62 to 105, wherein the VHH antibody domain or fragment binds to recombinant human IL-18RαECD with an affinity (KD value) not more than 1.5 times weaker than the binding of the corresponding VHH antibody domain without modification.

[0197] Aspect 110: A VHH antibody domain or fragment according to any one of aspects 1 to 23 or 62 to 109, wherein the VHH antibody domain or fragment binds to recombinant human IL-18RαECD with an affinity (KD value) not more than 10 times stronger than the binding of the corresponding VHH antibody domain without modification.

[0198] Aspect 111: A VHH antibody domain or fragment according to any one of aspects 1-23 or 62-109, wherein the VHH antibody domain or fragment binds to recombinant human IL-18RαECD with an affinity (KD value) not more than 5 times stronger than the binding of the corresponding VHH antibody domain without modification.

[0199] Aspect 112: A VHH antibody domain or fragment according to any one of aspects 1-23 or 62-109, wherein the VHH antibody domain or fragment binds to recombinant human IL-18RαECD with an affinity (KD value) not more than twice as strong as the binding of the corresponding VHH antibody domain without modification.

[0200] Aspect 113: A VHH antibody domain or fragment according to any one of aspects 1-23 or 62-109, wherein the VHH antibody domain or fragment binds to recombinant human IL-18RαECD with an affinity (KD value) not greater than 1.5 times stronger than the binding of the corresponding VHH antibody domain without modification.

[0201] Aspect 114: In (B) and (C), a VHH antibody domain or a fragment thereof according to any one of Aspects 24 to 113, which binds to recombinant human IL-18RαECD with an affinity (KD value) that is not more than 10-fold weaker than the binding of the corresponding VHH antibody domain without modification.

[0202] Aspect 115: In (B) and (C), a VHH antibody domain or a fragment thereof according to any one of Aspects 24 to 113, which binds to recombinant human IL-18RαECD with an affinity (KD value) that is not more than 5-fold weaker than the binding of the corresponding VHH antibody domain without modification.

[0203] Aspect 116: In (B) and (C), a VHH antibody domain or a fragment thereof according to any one of Aspects 24 to 113, which binds to recombinant human IL-18RαECD with an affinity (KD value) that is not more than 2-fold weaker than the binding of the corresponding VHH antibody domain without modification.

[0204] Aspect 117: In (B) and (C), a VHH antibody domain or a fragment thereof according to any one of Aspects 24 to 113, which binds to recombinant human IL-18RαECD with an affinity (KD value) that is not more than 1.5-fold weaker than the binding of the corresponding VHH antibody domain without modification.

[0205] Aspect 118: In (B) and (C), a VHH antibody domain or a fragment thereof according to any one of Aspects 24 to 117, which binds to recombinant human IL-18RαECD with an affinity (KD value) that is not more than 10-fold stronger than the binding of the corresponding VHH antibody domain without modification.

[0206] Aspect 119: A VHH antibody domain or fragment thereof according to any one of aspects 24 to 117, wherein the VHH antibody domain binds to recombinant human IL-18RαECD with an affinity (KD value) not more than 5 times stronger than the binding of the corresponding VHH antibody domain without modification.

[0207] Aspect 120: A VHH antibody domain or fragment thereof according to any one of aspects 24 to 117, wherein the VHH antibody domain binds to recombinant human IL-18RαECD with an affinity (KD value) not more than twice as strong as the binding of the corresponding VHH antibody domain without modification.

[0208] Aspect 121: A VHH antibody domain or fragment thereof according to any one of aspects 24 to 117, wherein the VHH antibody domain binds to recombinant human IL-18RαECD with an affinity (KD value) not more than 1.5 times stronger than the binding of the corresponding VHH antibody domain without modification.

[0209] Embodiment 122: A VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 121, wherein in (D), the affinity (KD value) of the VHH antibody domain to recombinant human IL-18RαECD is not more than 10 times weaker than the affinity (KD value) of the VHH antibody domain to recombinant human IL-18RαECD consisting of a sequence from a table of VHH sequences that has the highest degree of sequence identity with the sequence of the VHH antibody domain in (D). The degree of sequence identity can be determined by sequence alignment.

[0210] Embodiment 123: A VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 121, wherein in (D), the affinity (KD value) of the VHH antibody domain to recombinant human IL-18RαECD is not more than 5 times weaker than the affinity (KD value) of the VHH antibody domain to recombinant human IL-18RαECD consisting of a sequence from a table of VHH sequences that has the highest degree of sequence identity with the sequence of the VHH antibody domain in (D). The degree of sequence identity can be determined by sequence alignment.

[0211] Embodiment 124: A VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 121, wherein in (D), the affinity (KD value) of the VHH antibody domain to recombinant human IL-18RαECD is not more than twice weaker than the affinity (KD value) of the VHH antibody domain to recombinant human IL-18RαECD consisting of a sequence from a table of VHH sequences having the highest degree of sequence identity with the sequence of the VHH antibody domain in (D).

[0212] Embodiment 125: A VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 121, wherein in (D), the affinity (KD value) of the VHH antibody domain to recombinant human IL-18RαECD is not more than 1.5 times weaker than the affinity (KD value) of the VHH antibody domain to recombinant human IL-18RαECD consisting of a sequence from a table of VHH sequences that has the highest degree of sequence identity with the sequence of the VHH antibody domain in (D).

[0213] Embodiment 126: A VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 125, wherein in (D), the affinity (KD value) of the VHH antibody domain to recombinant human IL-18RαECD is not more than 10 times stronger than the affinity (KD value) of the VHH antibody domain to recombinant human IL-18RαECD consisting of a sequence from a table of VHH sequences that has the highest degree of sequence identity with the sequence of the VHH antibody domain in (D).

[0214] Embodiment 127: A VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 125, wherein in (D), the affinity (KD value) of the VHH antibody domain to recombinant human IL-18RαECD is not more than 5 times stronger than the affinity (KD value) of the VHH antibody domain to recombinant human IL-18RαECD consisting of a sequence from a table of VHH sequences that has the highest degree of sequence identity with the sequence of the VHH antibody domain in (D).

[0215] Embodiment 128: A VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 125, wherein in (D), the affinity (KD value) of the VHH antibody domain to recombinant human IL-18RαECD is not more than twice as strong as the affinity (KD value) of the VHH antibody domain to recombinant human IL-18RαECD consisting of a sequence from a table of VHH sequences that has the highest degree of sequence identity with the sequence of the VHH antibody domain in (D).

[0216] Embodiment 129: A VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 125, wherein in (D), the affinity (KD value) of the VHH antibody domain to recombinant human IL-18RαECD is not more than 1.5 times stronger than the affinity (KD value) of the VHH antibody domain to recombinant human IL-18RαECD consisting of a sequence from a table of VHH sequences that has the highest degree of sequence identity with the sequence of the VHH antibody domain in (D).

[0217] Embodiment 130: A VHH antibody domain or fragment thereof according to any one of Embodiments 91 to 129, wherein the KD value is determined in an in vitro binding experiment via bilayer interference.

[0218] Embodiment 131: A VHH antibody domain or fragment thereof according to any one of Embodiments 91 to 130, wherein the KD value is measured by kinetic measurement by bilayer interference in KB buffer (PBS + 0.1% Tween 20 + 1% BSA) at 25°C and 1000 rpm.

[0219] Embodiment 132: A VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 131, wherein the VHH antibody domain of (A) includes one VHH sequence selected from the group consisting of VHH1, VHH2, VHH3, VHH4, VHH5, VHH6, VHH8, VHH9, and VHH10 as shown in the table of VHH sequences.

[0220] Embodiment 133: A VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 131, wherein the VHH antibody domain of (A) comprises one VHH sequence selected from the group consisting of VHH1, VHH2, VHH5, VHH6, VHH8, VHH9, and VHH10 as shown in the table of VHH sequences.

[0221] Embodiment 134: A VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 131, wherein the VHH antibody domain of (A) comprises one VHH sequence selected from the group consisting of VHH2, VHH6, VHH8, and VHH10 as shown in the VHH sequence table.

[0222] Embodiment 135: A VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 131, wherein the VHH antibody domain of (A) comprises one VHH sequence selected from the group consisting of VHH6 and VHH11 as shown in the VHH sequence table.

[0223] Embodiment 136: A VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 131, wherein the VHH antibody domain of (A) comprises one VHH sequence selected from the group consisting of VHH1, VHH2, VHH5, VHH6, VHH8 and VHH10 as shown in the table of VHH sequences.

[0224] Embodiment 137: A VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 131, wherein the VHH antibody domain of (A) comprises one VHH sequence selected from the group consisting of VHH2, VHH6, VHH8, and VHH10 as shown in the VHH sequence table.

[0225] Embodiment 138: A VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 131, wherein the VHH antibody domain of (A) comprises one VHH sequence selected from the group consisting of VHH2 and VHH8 as shown in the VHH sequence table.

[0226] Embodiment 139: A VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 131, wherein the VHH antibody domain of (A) includes the VHH sequence VHH1 shown in the VHH sequence table.

[0227] Embodiment 140: A VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 131, wherein the VHH antibody domain of (A) includes the VHH sequence VHH2 shown in the VHH sequence table.

[0228] Embodiment 141: A VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 131, wherein the VHH antibody domain of (A) includes the VHH sequence VHH3 shown in the VHH sequence table.

[0229] Embodiment 142: A VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 131, wherein the VHH antibody domain of (A) includes the VHH sequence VHH4 shown in the VHH sequence table.

[0230] Embodiment 143: A VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 131, wherein the VHH antibody domain of (A) includes the VHH sequence VHH5 shown in the VHH sequence table.

[0231] Embodiment 144: A VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 131, wherein the VHH antibody domain of (A) includes the VHH sequence VHH6 shown in the VHH sequence table.

[0232] Embodiment 145: A VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 131, wherein the VHH antibody domain of (A) includes the VHH sequence VHH8 shown in the VHH sequence table.

[0233] Embodiment 146: A VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 131, wherein the VHH antibody domain of (A) includes the VHH sequence VHH9 shown in the VHH sequence table.

[0234] Embodiment 147: A VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 131, wherein the VHH antibody domain of (A) includes the VHH sequence VHH10 shown in the VHH sequence table.

[0235] Embodiment 148: A VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 131, wherein the VHH antibody domain of (A) includes the VHH sequence VHH11 shown in the VHH sequence table.

[0236] A VHH antibody domain or fragment according to any one of Aspects 1 to 23 or 62 to 148, wherein the VHH antibody domain or fragment comprises one VHH complementarity-determining region CDR1, CDR2 and CDR3 selected from the group consisting of VHH1, VHH2, VHH3, VHH4, VHH5, VHH6, VHH8, VHH9 and VHH10 as shown in the table of CDRs.

[0237] A VHH antibody domain or fragment according to any one of Aspects 1 to 23 or 62 to 148, wherein the VHH antibody domain or fragment comprises one VHH complementarity-determining region CDR1, CDR2 and CDR3 selected from the group consisting of VHH1, VHH2, VHH5, VHH6, VHH8, VHH9 and VHH10 as shown in the table of CDRs.

[0238] Aspect 151:(a) wherein the VHH antibody domain or fragment thereof comprises complementarity-determining regions CDR1, CDR2, and CDR3 of VHH2, VHH6, VHH8, and VHH10 as shown in the table of CDRs, according to any one of aspects 1 to 23 or 62 to 148.

[0239] A VHH antibody domain or fragment according to any one of Aspects 1 to 23 or 62 to 148, wherein the VHH antibody domain or fragment comprises one VHH complementarity-determining region CDR1, CDR2, and CDR3 selected from the group consisting of VHH6 and VHH11.

[0240] A VHH antibody domain or fragment according to any one of Aspects 1 to 23 or 62 to 148, wherein the VHH antibody domain or fragment comprises one VHH complementarity-determining region CDR1, CDR2 and CDR3 selected from the group consisting of VHH1, VHH2, VHH5, VHH6, VHH8 and VHH10 as shown in the table of CDRs.

[0241] Aspect 154:(a) A VHH antibody domain or fragment according to any one of aspects 1 to 23 or 62 to 148, wherein the VHH antibody domain or fragment comprises one VHH complementarity-determining region CDR1, CDR2, and CDR3 selected from the group consisting of VHH2, VHH6, VHH8, and VHH10 as shown in the table of CDRs.

[0242] A VHH antibody domain or fragment according to any one of Aspects 1 to 23 or 62 to 148, wherein the VHH antibody domain or fragment comprises one VHH complementarity-determining region CDR1, CDR2, and CDR3 selected from the group consisting of VHH2 or VHH8 as shown in the table of CDRs.

[0243] Aspect 156:(a) wherein the VHH antibody domain or fragment thereof comprises the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH1 as shown in the table of CDRs, according to any one of aspects 1 to 23 or 62 to 148.

[0244] A VHH antibody domain or fragment according to any one of Aspects 1 to 23 or 62 to 148, wherein the VHH antibody domain or fragment comprises the complementarity-determining regions CDR1, CDR2 and CDR3 of VHH2 as shown in the table of CDRs.

[0245] Aspect 158:(a) wherein the VHH antibody domain or fragment thereof comprises the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH3 as shown in the table of CDRs, according to any one of aspects 1 to 23 or 62 to 148.

[0246] Aspect 159:(a) wherein the VHH antibody domain or fragment thereof comprises the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH4 as shown in the table of CDRs, according to any one of aspects 1 to 23 or 62 to 148.

[0247] Aspect 160:(a) wherein the VHH antibody domain or fragment thereof comprises the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH5 as shown in the table of CDRs, according to any one of aspects 1 to 23 or 62 to 148.

[0248] A VHH antibody domain or fragment according to any one of Aspects 1 to 23 or 62 to 148, wherein the VHH antibody domain or fragment comprises the complementarity-determining regions CDR1, CDR2 and CDR3 of VHH6 as shown in the table of CDRs.

[0249] Aspect 162:(a) A VHH antibody domain or fragment according to any one of aspects 1 to 23 or 62 to 148, wherein the VHH antibody domain or fragment comprises the complementarity-determining regions CDR1, CDR2 and CDR3 of VHH8 as shown in the table of CDRs.

[0250] A VHH antibody domain or fragment according to any one of Aspects 1 to 23 or 62 to 148, wherein the VHH antibody domain or fragment comprises the complementarity-determining regions CDR1, CDR2 and CDR3 of VHH9 as shown in the table of CDRs.

[0251] Aspect 164:(a) A VHH antibody domain or fragment according to any one of aspects 1 to 23 or 62 to 148, wherein the VHH antibody domain or fragment comprises complementarity-determining regions CDR1, CDR2 and CDR3 of VHH10 as shown in the table of CDRs.

[0252] Aspect 165:(a) A VHH antibody domain or fragment according to any one of aspects 1 to 23 or 62 to 148, wherein the VHH antibody domain or fragment comprises complementarity-determining regions CDR1, CDR2 and CDR3 of VHH11 as shown in the table of CDRs.

[0253] Embodiment 166: A VHH antibody domain or fragment according to any one of Embodiments 1 to 165, wherein the VHH antibody domain or fragment competes with VHH2 for binding to recombinant human IL-18RαECD.

[0254] Embodiment 167: A VHH antibody domain or fragment according to any one of Embodiments 1 to 165, wherein the VHH antibody domain or fragment partially competes with VHH2 for binding to recombinant human IL-18RαECD.

[0255] Embodiment 168: The VHH antibody domain or its fragment is recombinant human IL- 18Rβ A VHH antibody domain or fragment thereof, according to any one of embodiments 1 to 167, that does not compete with VHH15 for binding to ECD.

[0256] Embodiment 169: A VHH antibody domain or fragment thereof according to any one of Embodiments 1 to 168, wherein the humanization is performed by germline lining.

[0257] Aspect 170: A VHH antibody domain or fragment thereof according to Aspect 169, wherein the germlineization is as follows: - To identify the sequence to be humanized and the closest human VH germline sequence in terms of sequence similarity; and - Replacing one or more amino acids in a sequence to be humanized with one or more amino acids at the corresponding sequence position in the nearest human VH germline sequence. Includes.

[0258] Sequence similarity and the positions of corresponding sequences can be determined by sequence alignment as described above.

[0259] Aspect 171: A VHH antibody domain or fragment thereof according to Aspect 169, wherein the germlineization is as follows: - To identify the sequence to be humanized and the closest human VH germline sequence in terms of sequence similarity; and - Replacing one or more amino acids in a sequence to be humanized with one or more amino acids at the corresponding sequence position in the nearest human VH germline sequence. It consists of.

[0260] Embodiment 172: A VHH antibody domain or fragment according to any one of Embodiments 1 to 171, wherein the humanized VHH antibody domain (each fragment thereof) binds to human IL-18RαECD with an affinity (KD value) that is not more than 10 times weaker than the binding of the corresponding unhumanized VHH antibody domain (each fragment thereof) to human IL-18RαECD.

[0261] As those skilled in the art will understand, the term "corresponding non-humanized VHH antibody domain" refers to a VHH antibody domain that is identical in all other respects, differing only in the alteration of amino acids introduced to carry out the humanization.

[0262] Embodiment 173: A VHH antibody domain or fragment according to any one of Embodiments 1 to 171, wherein the humanized VHH antibody domain (each fragment thereof) binds to human IL-18RαECD with an affinity (KD value) that is not more than 5 times weaker than the binding of the unhumanized corresponding VHH antibody domain (each fragment thereof) to human IL-18RαECD.

[0263] Embodiment 174: A VHH antibody domain or fragment according to any one of Embodiments 1 to 171, wherein the humanized VHH antibody domain (each fragment thereof) binds to human IL-18RαECD with an affinity (KD value) that is not weaker than slightly less than twice the binding of the unhumanized corresponding VHH antibody domain (each fragment thereof) to human IL-18RαECD.

[0264] Embodiment 175: A VHH antibody domain or fragment according to any one of Embodiments 1 to 171, wherein the humanized VHH antibody domain (each fragment thereof) binds to human IL-18RαECD with an affinity (KD value) that is no weaker than the binding of the unhumanized corresponding VHH antibody domain (each fragment thereof) to human IL-18RαECD.

[0265] Embodiment 176: A VHH antibody domain or fragment thereof according to any one of Embodiments 172 to 175, wherein the affinity is determined by KD measurement.

[0266] Embodiment 177: A VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 176, wherein the VHH antibody domain comprises a VHH antibody domain according to (A), (B), or (C) / the fragment of the VHH antibody domain comprises a fragment of the VHH antibody domain according to (A), (B), or (C).

[0267] Embodiment 178: A VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 176, wherein the VHH antibody domain comprises a VHH antibody domain according to (A), (B), or (D) / the fragment of the VHH antibody domain comprises a fragment of the VHH antibody domain according to (A), (B), or (D).

[0268] Embodiment 179: A VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 176, wherein the VHH antibody domain comprises a VHH antibody domain according to (A), (C), or (D) / the fragment of the VHH antibody domain comprises a fragment of the VHH antibody domain according to (A), (C), or (D).

[0269] Embodiment 180: A VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 176, wherein the VHH antibody domain comprises a VHH antibody domain according to (A) or (B) / the fragment of the VHH antibody domain comprises a fragment of the VHH antibody domain according to (A) or (B).

[0270] Embodiment 181: A VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 176, wherein the VHH antibody domain comprises a VHH antibody domain according to (A) or (C) and the fragment of the VHH antibody domain comprises a fragment of the VHH antibody domain according to (A) or (C).

[0271] Embodiment 182: A VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 176, wherein the VHH antibody domain comprises a VHH antibody domain according to (A) or (D) and the fragment of the VHH antibody domain comprises a fragment of the VHH antibody domain according to (A) or (D).

[0272] Embodiment 183: A VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 176, wherein the VHH antibody domain comprises a VHH antibody domain according to (A) and the fragment of the VHH antibody domain comprises a fragment of the VHH antibody domain according to (A).

[0273] Embodiment 184: A VHH antibody domain or fragment thereof according to any one of Embodiments 24 to 176, wherein the VHH antibody domain comprises a VHH antibody domain according to (B) and the fragment of the VHH antibody domain comprises a fragment of the VHH antibody domain according to (B).

[0274] Embodiment 185: A VHH antibody domain or fragment thereof according to any one of Embodiments 1 to 23 or 62 to 184, wherein the VHH antibody domain or fragment thereof includes a complementarity determination region according to (a) or (b).

[0275] Embodiment 186: A VHH antibody domain or fragment thereof according to any one of Embodiments 1 to 23 or 62 to 184, wherein the VHH antibody domain or fragment thereof includes a complementarity determination region according to (a) or (c).

[0276] Embodiment 187: A VHH antibody domain or fragment thereof according to any one of Embodiments 1 to 23 or 62 to 184, wherein the VHH antibody domain or fragment thereof includes a complementarity determination region according to (a).

[0277] Embodiment 188: A VHH antibody domain or fragment according to any one of Embodiments 1 to 23 or 62 to 184, wherein the VHH antibody domain or fragment includes a complementarity determination region according to (b).

[0278] A fourth aspect of this disclosure (also referred to as “Aspect 189”): According to the fourth aspect, this disclosure relates to VHH antibody domains or fragments thereof, hereby (d) The VHH antibody domain or fragment thereof includes one VHH complementarity-determining region CDR1, CDR2, and CDR3 selected from the group consisting of VHH12, VHH13, VHH14, VHH15, VHH16, VHH17, VHH18, VHH20, VHH21, and VHH22 as shown in the table of CDRs; (e) The VHH antibody domain or fragment thereof includes the complementarity-determining regions CDR1, CDR2 and CDR3 as defined in (d), wherein the modification is that at least one of the sequences of CDR1, CDR2 and CDR3 is humanized; or (f) The VHH antibody domain or fragment thereof includes the complementarity-determining regions CDR1, CDR2 and CDR3 as defined in (d) and which are modified, where the modification is - Substitution, addition, or deletion of up to three amino acids in CDR1, - Substitution, addition or deletion of up to three amino acids in CDR2, and / or - Substitution, addition, or deletion of up to three amino acids in CDR3 is;

[0279] CDR table: [Table F-1] [Table F-2] [Table F-3]

[0280] The same descriptions and definitions as those for the first to third aspects of this disclosure and the aspects referencing thereto apply accordingly.

[0281] A VHH antibody domain or fragment according to Aspect 189, wherein the modification in Aspect 190:(e) is such that the sequences of CDR1 and / or CDR2 are humanized, but the sequence of CDR3 is not humanized.

[0282] A VHH antibody domain or fragment thereof according to either one of Aspect 189 or 190, wherein the modification in Aspect 191:(e) is such that the sequence of CDR1 is humanized, but the sequences of CDR2 and CDR3 are not humanized.

[0283] A VHH antibody domain or fragment thereof according to either one of Aspect 189 or 190, wherein the modification in Aspect 192:(e) is such that the sequence of CDR2 is humanized, but the sequences of CDR1 and CDR3 are not humanized.

[0284] A VHH antibody domain or fragment according to any one of the embodiments 189 to 192, wherein the modification in embodiment 193:(e) is such that one of the sequences CDR1, CDR2, and CDR3 is humanized, but more than one is not.

[0285] Embodiment 194: A VHH antibody domain or fragment thereof according to any one of Embodiments 189 to 193, wherein the humanization of the CDR(s) is performed by replacing at least one amino acid in the sequence of the CDR with the corresponding amino acid of a human VH domain.

[0286] Embodiment 195: A VHH antibody domain or fragment according to any one of Embodiments 189 to 194, wherein the humanization of the CDR(s) is performed by replacing at least three amino acids in the sequence of the CDR with corresponding amino acids of a human VH domain.

[0287] Embodiment 196: A VHH antibody domain or fragment according to any one of Embodiments 189 to 194, wherein the humanization of the CDR(s) is performed by replacing up to three amino acids in the sequence of CDR1 and / or CDR2 and up to one amino acid in the sequence of CDR3 with the corresponding amino acids of a human VH domain.

[0288] Embodiment 197: A VHH antibody domain or fragment according to any one of Embodiments 189 to 194, wherein the humanization of the CDR(s) is performed by replacing up to two amino acids in the sequence of the CDR with corresponding amino acids of a human VH domain.

[0289] Embodiment 198: A VHH antibody domain or fragment according to any one of Embodiments 189 to 194, wherein the humanization of the CDR(s) is performed by replacing up to two amino acids in the sequence of CDR1 and / or CDR2 and up to one amino acid in the sequence of CDR3 with the corresponding amino acids of a human VH domain.

[0290] Embodiment 199: A VHH antibody domain or fragment according to any one of Embodiments 189 to 198, wherein the humanization of the CDR(s) is performed by replacing one amino acid in the sequence of the CDR with the corresponding amino acid of a human VH domain.

[0291] The modification in aspect 200:(f) is - Substitution, addition, or deletion of up to three amino acids in CDR1, - Substitution, addition or deletion of up to three amino acids in CDR2, and / or - Substitution, addition, or deletion of up to one amino acid in CDR3 A VHH antibody domain or fragment thereof according to any one of embodiments 189 to 199.

[0292] The modification in aspect 201:(f) is - Substitution, addition, or deletion of up to two amino acids in CDR1, - Substitution, addition or deletion of up to two amino acids in CDR2, and / or - Substitution, addition, or deletion of up to two amino acids in CDR3 That is, VHH antibody domain or fragment thereof according to any one of embodiments 189 to 199

[0293] The modification in aspect 202:(f) is - Substitution, addition, or deletion of up to two amino acids in CDR1; - Substitution, addition, or deletion of up to two amino acids in CDR2; and / or - Substitution, addition, or deletion of up to one amino acid in CDR3 That is, A VHH antibody domain or fragment thereof according to any one of embodiments 189 to 199.

[0294] The modification in aspect 203:(f) is - Substitution, addition or deletion of up to two amino acids in CDR1, and / or - Substitution, addition, or deletion of up to two amino acids in CDR2; And here, the sequence of CDR3 is unmodified. A VHH antibody domain or fragment thereof according to any one of embodiments 189 to 199.

[0295] As a person skilled in the art would understand, the instruction “the sequence of CDR3 is unmodified” means that the sequence is not modified compared to the sequence provided in the table of CDRs for CDR3 for the VHH in question.

[0296] The modification in aspect 204:(f) is - Substitution, addition, or deletion of up to two amino acids in CDR1, Here, the sequences of CDR2 and CDR3 are unmodified. A VHH antibody domain or fragment thereof according to any one of embodiments 189 to 199.

[0297] The modification in aspect 205:(f) is - Substitution, addition, or deletion of up to two amino acids in CDR2, Here, the sequences of CDR1 and CDR3 are unmodified. A VHH antibody domain or fragment thereof according to any one of embodiments 189 to 199.

[0298] The modification in aspect 206:(f) is - Substitution, addition, or deletion of up to one amino acid in CDR1; - Substitution, addition, or deletion of up to one amino acid in CDR2; and / or - Substitution, addition, or deletion of up to one amino acid in CDR3 That is, A VHH antibody domain or fragment thereof according to any one of embodiments 189 to 199.

[0299] The modification in aspect 207:(f) is - Substitution, addition or deletion of one amino acid in CDR1, and / or - Substitution, addition, or deletion of a single amino acid in CDR2; And here, the sequence of CDR3 is unmodified. A VHH antibody domain or fragment thereof according to any one of embodiments 189 to 199.

[0300] The modification in aspect 208:(f) is - Substitution, addition, or deletion of one amino acid in CDR1, Here, the sequences of CDR2 and CDR3 are unmodified. A VHH antibody domain or fragment thereof according to any one of embodiments 189 to 199.

[0301] The modification in aspect 209:(f) is - Substitution, addition, or deletion of one amino acid in CDR2, Here, the sequences of CDR1 and CDR3 are unmodified. A VHH antibody domain or fragment thereof according to any one of embodiments 189 to 199.

[0302] Aspect 210: A VHH antibody domain or fragment thereof according to any one of aspects 189 to 209, wherein the modification in (f) comprises only amino acid substitutions and does not involve the addition or deletion of amino acids.

[0303] Embodiment 211: A VHH antibody domain or fragment thereof according to any one of Embodiments 189 to 210, wherein the substitution is a conservative amino acid substitution.

[0304] A fifth aspect of this disclosure (also referred to as “Aspect 212”): According to the fifth aspect, this disclosure relates to VHH antibody domains or fragments thereof, hereby (E) The VHH antibody domain contains an amino acid sequence of VHH selected from the group consisting of VHH12, VHH13, VHH14, VHH15, VHH16, VHH17, VHH18, VHH20, VHH21, and VHH22 as shown in the VHH sequence table; (F) The VHH antibody domain comprises a VHH sequence as defined in (E) and having modifications, wherein the modification is humanization of the sequence; (G) The VHH antibody domain comprises a VHH sequence as defined in (E) and having modifications, wherein the modifications are substitutions, additions, or deletions of up to 25 amino acids; or, (H) The VHH antibody domain comprises a VHH sequence that is at least 75% identical to the VHH sequence referenced in (E);

[0305] Table of VHH sequences: [Table G-1] [Table G-2] [Table G-3]

[0306] A sixth aspect of this disclosure (also referred to as “Aspect 213”): According to the sixth aspect, this disclosure relates to VHH antibody domains or fragments thereof, hereby (E) The VHH antibody domain consists of an amino acid sequence of VHH selected from the group consisting of VHH12, VHH13, VHH14, VHH15, VHH16, VHH17, VHH18, VHH20, VHH21, and VHH22 as shown in the table of VHH sequences; (F) The VHH antibody domain consists of a VHH sequence as defined in (E) and is modified, wherein the modification is humanization of the sequence; (G) The VHH antibody domain consists of a VHH sequence as defined in (E) and is modified, wherein the modification is the substitution, addition, or deletion of up to 25 amino acids; or, (H) The VHH antibody domain consists of a VHH sequence that is at least 75% identical to the VHH sequence referenced in (E).

[0307] The same descriptions and definitions as those for the first to fourth aspects of this disclosure and the aspects referencing them apply accordingly to the fifth and sixth aspects of this disclosure and the embodiments referencing them.

[0308] Embodiment 214: A VHH antibody domain or fragment thereof according to either Embodiment 212 or 213, wherein the fragment of the VHH antibody domain comprises at least 75% of the amino acids of the sequence of the VHH antibody domain. As those skilled in the art will understand, this means that the fragment is missing up to one-quarter of the total number of amino acids in the VHH antibody domain, where the amino acids are missing at the N-terminus or C-terminus compared to the “complete” VHH antibody domain sequence.

[0309] Embodiment 215: A VHH antibody domain or fragment thereof according to either Embodiment 212 or 213, wherein the fragment of the VHH antibody domain comprises at least 80% of the amino acids of the sequence of the VHH antibody domain.

[0310] Embodiment 216: A VHH antibody domain or fragment thereof according to either Embodiment 212 or 213, wherein the fragment of the VHH antibody domain comprises at least 85% of the amino acids of the sequence of the VHH antibody domain.

[0311] Embodiment 217: A VHH antibody domain or fragment thereof according to either Embodiment 212 or 213, wherein the fragment of the VHH antibody domain comprises at least 90% of the amino acids of the sequence of the VHH antibody domain.

[0312] Embodiment 218: A VHH antibody domain or fragment thereof according to either Embodiment 212 or 213, wherein the fragment of the VHH antibody domain comprises at least 95% of the amino acids of the sequence of the VHH antibody domain.

[0313] Embodiment 219: A VHH antibody domain or fragment thereof according to either Embodiment 212 or 213, wherein the fragment of the VHH antibody domain comprises at least 98% of the amino acids of the sequence of the VHH antibody domain.

[0314] Embodiment 220: A VHH antibody domain or fragment thereof according to either Embodiment 212 or 213, wherein the fragment of the VHH antibody domain comprises at least 99% of the amino acids of the sequence of the VHH antibody domain.

[0315] Embodiment 221: A VHH antibody domain or fragment thereof according to any one of Embodiments 212 to 220, wherein the fragment of the VHH antibody domain includes complementarity-determining regions CDR1, CDR2 and CDR3.

[0316] Embodiment 222: A VHH antibody domain or fragment thereof according to any one of Embodiments 212 to 221, wherein the fragment of the VHH antibody domain includes at least the sequence from the N-terminus of CDR1 to the C-terminus of CDR3 of the VHH antibody domain.

[0317] Embodiment 223:(E), a VHH antibody domain or fragment thereof according to any one of Embodiments 212 to 222, wherein the fragment of the VHH antibody domain includes all of the complementarity-determining regions (CDRs) of the VHH antibody domain.

[0318] Embodiment 224:(F), wherein the humanization of the sequence is performed by replacing at least one amino acid of the sequence with the corresponding amino acid of the human VH (variable heavy chain) domain, a VHH antibody domain or fragment according to any one of Embodiments 212 to 223.

[0319] Embodiment 225:(F), wherein the humanization of the sequence is performed by replacing up to 25 amino acids of the sequence with the corresponding amino acids of a human VH domain (individually), a VHH antibody domain or fragment according to any one of Embodiments 212 to 224.

[0320] Embodiment 226:(F), wherein the humanization of the sequence is performed by replacing up to 20 amino acids of the sequence with the corresponding amino acids of a human VH domain, a VHH antibody domain or fragment according to any one of Embodiments 212 to 224.

[0321] Embodiment 227:(F), wherein the humanization of the sequence is performed by replacing up to 15 amino acids of the sequence with the corresponding amino acids of a human VH domain, a VHH antibody domain or fragment according to any one of Embodiments 212 to 224.

[0322] Embodiment 228:(F), wherein the humanization of the sequence is performed by replacing up to 10 amino acids of the sequence with the corresponding amino acids of a human VH domain, a VHH antibody domain or fragment according to any one of Embodiments 212 to 224.

[0323] Embodiment 229:(F), the humanization of the sequence is performed by replacing up to five amino acids of the sequence with the corresponding amino acids of a human VH domain, a VHH antibody domain or fragment according to any one of Embodiments 212 to 224.

[0324] Embodiment 230:(F), wherein the humanization of the sequence is performed by replacing up to three amino acids of the sequence with corresponding amino acids of a human VH domain, a VHH antibody domain or fragment according to any one of Embodiments 212 to 224.

[0325] Embodiment 231:(F), wherein the humanization of the sequence is performed by replacing up to two amino acids of the sequence with corresponding amino acids of a human VH domain, a VHH antibody domain or fragment according to any one of Embodiments 212 to 224.

[0326] Embodiment 232:(F), wherein the humanization of the sequence is performed by replacing one amino acid of the sequence with the corresponding amino acid of a human VH domain, a VHH antibody domain or fragment according to any one of Embodiments 212 to 224.

[0327] Embodiment 233: A VHH antibody domain or fragment thereof according to any one of Embodiments 212 to 232, wherein the humanization is located within the framework region of the VHH antibody domain and / or within the CDR of the VHH antibody domain.

[0328] Embodiment 234:(F), wherein the humanization is within the framework region of the VHH antibody domain but not within the CDR of the VHH antibody domain, wherein the VHH antibody domain or its fragment is according to any one of Embodiments 212 to 232.

[0329] Embodiment 235:(F), wherein the humanization is within the CDR of the VHH antibody domain but not within the framework region of the VHH antibody domain, according to any one of Embodiments 212 to 224, the VHH antibody domain or its fragment.

[0330] Embodiment 236:(F), wherein the humanization of the VHH antibody domain within the CDR is within CDR1, CDR2 and / or CDR3, a VHH antibody domain or fragment thereof according to any one of Embodiments 212-233 or 235.

[0331] Embodiment 237:(F), wherein the humanization of the VHH antibody domain within the CDR is within CDR1 and / or CDR2, according to any one of Embodiments 212-233 or 235-236.

[0332] Embodiment 238:(F), wherein the humanization of the VHH antibody domain within the CDR is within CDR1, a VHH antibody domain or fragment thereof according to any one of Embodiments 212-233 or 235-237.

[0333] Embodiment 239:(F), wherein the humanization of the VHH antibody domain within the CDR is within CDR2, a VHH antibody domain or fragment thereof according to any one of Embodiments 212-233 or 235-238.

[0334] Embodiment 240:(F), wherein the humanization of the VHH antibody domain within the CDR is not within CDR3, and is a VHH antibody domain or fragment thereof according to any one of Embodiments 212 to 239.

[0335] Embodiment 241:(G), a VHH antibody domain or fragment thereof according to any one of Embodiments 212 to 240, wherein the modification is the substitution, addition, or deletion of up to 20 amino acids.

[0336] Embodiment 242:(G), a VHH antibody domain or fragment thereof according to any one of Embodiments 212 to 240, wherein the modification is the substitution, addition, or deletion of up to 15 amino acids.

[0337] Embodiment 243:(G), a VHH antibody domain or fragment thereof according to any one of Embodiments 212 to 240, wherein the modification is the substitution, addition, or deletion of up to 10 amino acids.

[0338] Embodiment 244:(G), a VHH antibody domain or fragment thereof according to any one of Embodiments 212 to 240, wherein the modification is the substitution, addition, or deletion of up to five amino acids.

[0339] Embodiment 245:(G), a VHH antibody domain or fragment thereof according to any one of Embodiments 212 to 240, wherein the modification is the substitution, addition, or deletion of up to three amino acids.

[0340] Embodiment 246:(G), a VHH antibody domain or fragment thereof according to any one of Embodiments 212 to 240, wherein the modification is the substitution, addition, or deletion of up to two amino acids.

[0341] Embodiment 247:(G), a VHH antibody domain or fragment thereof according to any one of Embodiments 212 to 240, wherein the modification is the substitution, addition, or deletion of one amino acid.

[0342] Embodiment 248: A VHH antibody domain or fragment thereof according to any one of Embodiments 212 to 247, wherein the modification in (G) consists only of amino acid substitutions and does not involve the addition or deletion of amino acids.

[0343] Aspect 249:(E) A VHH antibody domain or fragment according to any one of aspects 212 to 248, wherein the VHH antibody domain or fragment comprises one VHH complementarity-determining region CDR1, CDR2, and CDR3 selected from the group consisting of VHH12, VHH13, VHH14, VHH15, VHH16, VHH17, VHH18, VHH20, VHH21, and VHH22 as shown in the table of CDRs.

[0344] Embodiment 250: A VHH antibody domain or fragment thereof according to any one of Embodiments 212 to 249, wherein in (F) to (H), (d) The VHH antibody domain or fragment thereof includes one VHH complementarity-determining region CDR1, CDR2, and CDR3 selected from the group consisting of VHH12, VHH13, VHH14, VHH15, VHH16, VHH17, VHH18, VHH20, VHH21, and VHH22 as shown in the table of CDRs; (e) The VHH antibody domain or fragment thereof includes the complementarity-determining regions CDR1, CDR2 and CDR3 as defined in (d), wherein the modification is that at least one of the sequences of CDR1, CDR2 and CDR3 is humanized; (f) The VHH antibody domain or fragment thereof includes the complementarity-determining regions CDR1, CDR2 and CDR3 as defined in (d) and which are modified, where the modification is - Substitution, addition, or deletion of up to three amino acids in CDR1, - Substitution, addition or deletion of up to three amino acids in CDR2, and / or - Substitution, addition, or deletion of up to three amino acids in CDR3 That is the case.

[0345] A VHH antibody domain or fragment according to Aspect 250, wherein the modification in Aspect 251:(e) is such that the sequences of CDR1 and / or CDR2 are humanized, but the sequence of CDR3 is not humanized.

[0346] A VHH antibody domain or fragment thereof according to either one of Aspect 250 or 251, wherein the modification in Aspect 252:(e) is such that the sequence of CDR1 is humanized, but the sequences of CDR2 and CDR3 are not humanized.

[0347] A VHH antibody domain or fragment thereof according to either one of Aspect 250 or 251, wherein the modification in Aspect 253:(e) is such that the sequence of CDR2 is humanized, but the sequences of CDR1 and CDR3 are not humanized.

[0348] A VHH antibody domain or fragment according to any one of Aspects 250 to 253, wherein the modification in Aspect 254:(e) is such that one of the sequences CDR1, CDR2, and CDR3 is humanized, but more than one is not.

[0349] Embodiment 255: A VHH antibody domain or fragment thereof according to any one of Embodiments 250 to 254, wherein the humanization of the CDR(s) is performed by replacing at least one amino acid in the sequence of the CDR with the corresponding amino acid of a human VH domain.

[0350] Embodiment 256: A VHH antibody domain or fragment thereof according to any one of Embodiments 250 to 255, wherein the humanization of the CDR(s) is performed by replacing at least three amino acids in the sequence of the CDR with corresponding amino acids of a human VH domain.

[0351] Embodiment 257: A VHH antibody domain or fragment according to any one of Embodiments 250 to 255, wherein the humanization of the CDR(s) is performed by replacing up to three amino acids in the sequence of CDR1 and / or CDR2 and up to one amino acid in the sequence of CDR3 with the corresponding amino acids of a human VH domain.

[0352] Embodiment 258: A VHH antibody domain or fragment thereof according to any one of Embodiments 250 to 255, wherein the humanization of the CDR(s) is performed by replacing up to two amino acids in the sequence of the CDR with corresponding amino acids of a human VH domain.

[0353] Embodiment 259: A VHH antibody domain or fragment according to any one of Embodiments 250 to 255, wherein the humanization of the CDR(s) is performed by replacing up to two amino acids in the sequence of CDR1 and / or CDR2 and up to one amino acid in the sequence of CDR3 with the corresponding amino acids of a human VH domain.

[0354] Embodiment 260: A VHH antibody domain or fragment thereof according to any one of Embodiments 250 to 259, wherein the humanization of the CDR(s) is performed by replacing one amino acid in the sequence of the CDR with the corresponding amino acid of a human VH domain.

[0355] The modification in aspect 261:(f) is - Substitution, addition, or deletion of up to three amino acids in CDR1, - Substitution, addition or deletion of up to three amino acids in CDR2, and / or - Substitution, addition, or deletion of up to two amino acids in CDR3 That is, A VHH antibody domain or a fragment thereof according to any one of embodiments 250 to 260.

[0356] The modification in aspect 262:(f) is - Substitution, addition, or deletion of up to three amino acids in CDR1, - Substitution, addition or deletion of up to three amino acids in CDR2, and / or - Substitution, addition, or deletion of up to one amino acid in CDR3 That is, A VHH antibody domain or a fragment thereof according to any one of embodiments 250 to 260.

[0357] The modification in aspect 263:(f) is - Substitution, addition or deletion of up to 3 amino acids in CDR1, and / or - Substitution, addition, or deletion of up to three amino acids in CDR2 And here, the sequence of CDR3 is unmodified. A VHH antibody domain or a fragment thereof according to any one of embodiments 250 to 260.

[0358] The modification in aspect 264:(f) is - Substitution, addition, or deletion of up to two amino acids in CDR1, - Substitution, addition or deletion of up to two amino acids in CDR2, and / or - Substitution, addition, or deletion of up to two amino acids in CDR3 That is, A VHH antibody domain or a fragment thereof according to any one of embodiments 250 to 260.

[0359] The modification in aspect 265:(f) is - Substitution, addition, or deletion of up to two amino acids in CDR1; - Substitution, addition, or deletion of up to two amino acids in CDR2; and / or - Substitution, addition, or deletion of up to one amino acid in CDR3 That is, A VHH antibody domain or a fragment thereof according to any one of embodiments 250 to 260.

[0360] The modification in aspect 266:(f) is - Substitution, addition or deletion of up to two amino acids in CDR1, and / or - Substitution, addition, or deletion of up to two amino acids in CDR2; And here, the sequence of CDR3 is unmodified. A VHH antibody domain or a fragment thereof according to any one of embodiments 250 to 260.

[0361] As those skilled in the art will recognize, "unmodified" means that it has not been modified compared to its arrangement in the CDR table.

[0362] The modification in aspect 267:(f) is - Substitution, addition, or deletion of up to two amino acids in CDR1, Here, the sequences of CDR2 and CDR3 are unmodified. A VHH antibody domain or a fragment thereof according to any one of embodiments 250 to 260.

[0363] The modification in aspect 268:(f) is - Substitution, addition, or deletion of up to two amino acids in CDR2, Here, the sequences of CDR1 and CDR3 are unmodified. A VHH antibody domain or a fragment thereof according to any one of embodiments 250 to 260.

[0364] The modification in aspect 269:(f) is - Substitution, addition, or deletion of up to one amino acid in CDR1; - Substitution, addition, or deletion of up to one amino acid in CDR2; and / or - Substitution, addition, or deletion of up to one amino acid in CDR3 That is, A VHH antibody domain or a fragment thereof according to any one of embodiments 250 to 260.

[0365] The modification in aspect 270:(f) is - Substitution, addition or deletion of one amino acid in CDR1, and / or - Substitution, addition, or deletion of a single amino acid in CDR2; And here, the sequence of CDR3 is unmodified. A VHH antibody domain or a fragment thereof according to any one of embodiments 250 to 260.

[0366] The modification in aspect 271:(f) is - Substitution, addition, or deletion of one amino acid in CDR1, Here, the sequences of CDR2 and CDR3 are unmodified. A VHH antibody domain or a fragment thereof according to any one of embodiments 250 to 260.

[0367] The modification in aspect 272:(f) is - Substitution, addition, or deletion of one amino acid in CDR2, Here, the sequences of CDR1 and CDR3 are unmodified. A VHH antibody domain or a fragment thereof according to any one of embodiments 250 to 260.

[0368] Aspect 273: A VHH antibody domain or fragment thereof according to any one of aspects 250 to 272, wherein the modification in (f) comprises only amino acid substitutions and does not involve the addition or deletion of amino acids.

[0369] Embodiment 274: A VHH antibody domain or fragment thereof according to any one of Embodiments 250 to 273, wherein the substitution is a conservative amino acid substitution.

[0370] Embodiment 275: A VHH antibody domain or fragment thereof according to any one of Embodiments 189 to 274, wherein the fragment consists of at least 100 amino acids.

[0371] Embodiment 276: A VHH antibody domain or fragment thereof according to any one of Embodiments 189 to 274, wherein the fragment consists of at least 105 amino acids.

[0372] Embodiment 277: A VHH antibody domain or fragment thereof according to any one of Embodiments 189 to 274, wherein the fragment consists of at least 110 amino acids.

[0373] Embodiment 278: A VHH antibody domain or fragment thereof according to any one of Embodiments 189 to 274, wherein the fragment consists of at least 115 amino acids.

[0374] Embodiment 279: A VHH antibody domain or fragment thereof according to any one of Embodiments 189 to 278, wherein the VHH antibody domain is an anti-IL-18RβVHH antibody domain.

[0375] Embodiment 280: A VHH antibody domain or fragment thereof according to any one of Embodiments 189 to 279, wherein the VHH antibody domain is specific to IL-18Rβ.

[0376] Embodiment 281: A VHH antibody domain or fragment thereof according to any one of Embodiments 189 to 280, wherein the VHH antibody domain or fragment thereof binds to IL-18Rβ.

[0377] Embodiment 282: A VHH antibody domain or fragment according to any one of Embodiments 189 to 281, wherein the VHH antibody domain or fragment specifically binds to IL-18Rβ.

[0378] Embodiment 283: A VHH antibody domain or fragment according to any one of Embodiments 189 to 282, wherein the VHH antibody domain or fragment does not bind to IL-18Rα.

[0379] Embodiment 284: A VHH antibody domain or fragment according to any one of Embodiments 189 to 283, wherein the VHH antibody domain or fragment can specifically bind to IL-18RβECD.

[0380] Embodiment 285: The VHH antibody domain or its fragment is 1 × 10 -6 A VHH antibody domain or fragment thereof, according to any one of embodiments 189 to 284, which binds to recombinant human IL-18RβECD with M or a stronger KD.

[0381] Embodiment 286 The VHH antibody domain or its fragment is 1 × 10 -7 A VHH antibody domain or fragment thereof, according to any one of embodiments 189 to 284, which binds to recombinant human IL-18RβECD with M or a stronger KD.

[0382] Embodiment 287: The VHH antibody domain or its fragment is 5 × 10 -8 Recombinant human IL-18R with M or a stronger KD β A VHH antibody domain or fragment thereof, according to any one of embodiments 189 to 284, that binds to ECD.

[0383] Embodiment 288: The VHH antibody domain or its fragment is 1.5 × 10 -8 Recombinant human IL-18R with M or a stronger KD β A VHH antibody domain or fragment thereof, according to any one of embodiments 189 to 284, that binds to ECD.

[0384] Embodiment 289: The VHH antibody domain or its fragment is 1 × 10 -8 Recombinant human IL-18R with M or a stronger KD β A VHH antibody domain or fragment thereof, according to any one of embodiments 189 to 284, that binds to ECD.

[0385] Embodiment 290: The VHH antibody domain or its fragment is 5 × 10 -9 Recombinant human IL-18R with M or a stronger KD β A VHH antibody domain or fragment thereof, according to any one of embodiments 189 to 284, that binds to ECD.

[0386] Embodiment 291: The VHH antibody domain or its fragment is 1 × 10 -9 Recombinant human IL-18R with M or a stronger KD β A VHH antibody domain or fragment thereof, according to any one of embodiments 189 to 284, that binds to ECD.

[0387] Embodiment 292: A VHH antibody domain or fragment thereof according to any one of Embodiments 189 to 291, wherein the fragment of the VHH antibody domain binds to recombinant human IL-18RβECD with an affinity (determined by the KD value) of at least 90% of the affinity (determined by the KD value) of the VHH antibody domain to recombinant human IL-18RβECD.

[0388] Embodiment 293: A VHH antibody domain or fragment according to any one of Embodiments 189 to 292, wherein the fragment of the VHH antibody domain binds to IL-18RαECD with at least the same affinity (determined from the KD value) as VHH2 (SEQ ID NO: 2) binds to IL-18RαECD, or a stronger affinity. Such binding can be determined by an in vitro binding experiment (by bilayer interference) as described in Example 1 below.

[0389] Aspect 294: A VHH antibody domain or fragment according to any one of aspects 189-211 or 250-293, wherein the VHH antibody domain or fragment binds to recombinant human IL-18RβECD with an affinity (KD value) not more than 10 times weaker than the binding of the corresponding VHH antibody domain without modification.

[0390] Aspect 295: A VHH antibody domain or fragment according to any one of aspects 189-211 or 250-293, wherein the VHH antibody domain or fragment binds to recombinant human IL-18RβECD with an affinity (KD value) not more than 5 times weaker than the binding of the corresponding VHH antibody domain without modification.

[0391] Aspect 296: A VHH antibody domain or fragment according to any one of aspects 189-211 or 250-293, wherein the VHH antibody domain or fragment binds to recombinant human IL-18RβECD with an affinity (KD value) not more than twice weaker than the binding of the corresponding VHH antibody domain without modification.

[0392] Aspect 297: A VHH antibody domain or fragment according to any one of aspects 189-211 or 250-293, wherein the VHH antibody domain or fragment binds to recombinant human IL-18RβECD with an affinity (KD value) not more than 1.5 times weaker than the binding of the corresponding VHH antibody domain without modification.

[0393] Aspect 298: A VHH antibody domain or fragment according to any one of aspects 189-211 or 250-297, wherein the VHH antibody domain or fragment binds to recombinant human IL-18RβECD with an affinity (KD value) not more than 10 times stronger than the binding of the corresponding VHH antibody domain without modification.

[0394] Aspect 299: A VHH antibody domain or fragment according to any one of aspects 189-211 or 250-297, wherein the VHH antibody domain or fragment binds to recombinant human IL-18RβECD with an affinity (KD value) not more than 5 times stronger than the binding of the corresponding VHH antibody domain without modification.

[0395] Aspect 300: A VHH antibody domain or fragment according to any one of aspects 189-211 or 250-297, wherein the VHH antibody domain or fragment binds to recombinant human IL-18RβECD with an affinity (KD value) not more than twice as strong as the binding of the corresponding VHH antibody domain without modification.

[0396] Aspect 301: A VHH antibody domain or fragment according to any one of aspects 189-211 or 250-297, wherein the VHH antibody domain or fragment binds to recombinant human IL-18RβECD with an affinity (KD value) not greater than 1.5 times stronger than the binding of the corresponding VHH antibody domain without modification.

[0397] Aspect 302: A VHH antibody domain or fragment thereof according to any one of aspects 212 to 301, wherein in (F) and (G), the VHH antibody domain binds to recombinant human IL-18RβECD with an affinity (KD value) not more than 10 times weaker than the binding of the corresponding VHH antibody domain without modification.

[0398] Aspect 303: A VHH antibody domain or fragment thereof according to any one of aspects 212 to 301, wherein the VHH antibody domain binds to recombinant human IL-18RβECD with an affinity (KD value) not more than 5 times weaker than the binding of the corresponding VHH antibody domain without modification.

[0399] Aspect 304: A VHH antibody domain or fragment thereof according to any one of aspects 212 to 301, wherein in (F) and (G), the VHH antibody domain binds to recombinant human IL-18RβECD with an affinity (KD value) not more than twice weaker than the binding of the corresponding VHH antibody domain without modification.

[0400] Aspect 305: A VHH antibody domain or fragment thereof according to any one of aspects 189 to 301, wherein in (F) and (G), the VHH antibody domain binds to recombinant human IL-18RβECD with an affinity (KD value) not more than 1.5 times weaker than the binding of the corresponding VHH antibody domain without modification.

[0401] Aspect 306: A VHH antibody domain or fragment thereof according to any one of aspects 212 to 305, wherein in (F) and (G), the VHH antibody domain binds to recombinant human IL-18RβECD with an affinity (KD value) not more than 10 times stronger than the binding of the corresponding VHH antibody domain without modification.

[0402] Aspect 307: A VHH antibody domain or fragment thereof according to any one of aspects 212 to 305, wherein the VHH antibody domain binds to recombinant human IL-18RβECD with an affinity (KD value) not more than 5 times stronger than the binding of the corresponding VHH antibody domain without modification.

[0403] Aspect 308: A VHH antibody domain or fragment thereof according to any one of aspects 212 to 305, wherein the VHH antibody domain binds to recombinant human IL-18RβECD with an affinity (KD value) not more than twice as strong as the binding of the corresponding VHH antibody domain without modification.

[0404] Aspect 309: A VHH antibody domain or fragment thereof according to any one of aspects 212 to 305, wherein in (F) and (G), the VHH antibody domain binds to recombinant human IL-18RβECD with an affinity (KD value) not greater than 1.5 times stronger than the binding of the corresponding VHH antibody domain without modification.

[0405] Embodiment 310: A VHH antibody domain or fragment thereof according to any one of Embodiments 212 to 309, wherein in (H), the affinity (KD value) of the binding of the VHH antibody domain to recombinant human IL-18RβECD is not more than 10 times weaker than the affinity (KD value) of the binding of a VHH antibody domain consisting of a sequence from a table of VHH sequences that has the highest degree of sequence identity with the sequence of the VHH antibody domain in (H) to recombinant human IL-18RβECD. The degree of sequence identity can be determined by sequence alignment.

[0406] Embodiment 311: A VHH antibody domain or fragment thereof according to any one of Embodiments 212 to 309, wherein in (H), the affinity (KD value) of the binding of the VHH antibody domain to recombinant human IL-18RβECD is not more than 5 times weaker than the affinity (KD value) of the binding of a VHH antibody domain to recombinant human IL-18RβECD consisting of a sequence from a table of VHH sequences that has the highest degree of sequence identity with the sequence of the VHH antibody domain in (H). The degree of sequence identity can be determined by sequence alignment.

[0407] Embodiment 312: A VHH antibody domain or fragment thereof according to any one of Embodiments 212 to 309, wherein (H) is recombinant human IL-18R β The affinity (KD value) of binding the VHH antibody domain to ECD is not more than twice as weak as the affinity (KD value) of binding the VHH antibody domain to recombinant human IL-18RβECD, which consists of a sequence from the VHH sequence table that has the highest degree of sequence identity with the VHH antibody domain sequence of (H) above.

[0408] Embodiment 313: A VHH antibody domain or fragment thereof according to any one of Embodiments 212 to 309, wherein in (H), the affinity (KD value) of the binding of the VHH antibody domain to recombinant human IL-18RβECD is not more than 1.5 times weaker than the affinity (KD value) of the binding of a VHH antibody domain to recombinant human IL-18RβECD consisting of a sequence from a table of VHH sequences that has the highest degree of sequence identity with the sequence of the VHH antibody domain in (H).

[0409] Embodiment 314: A VHH antibody domain or fragment thereof according to any one of Embodiments 212 to 313, wherein in (H), the affinity (KD value) of the binding of the VHH antibody domain to recombinant human IL-18RβECD is not more than 10 times stronger than the affinity (KD value) of the binding of a VHH antibody domain to recombinant human IL-18RβECD consisting of a sequence from a table of VHH sequences that has the highest degree of sequence identity with the sequence of the VHH antibody domain in (H).

[0410] Embodiment 315: A VHH antibody domain or fragment thereof according to any one of Embodiments 212 to 313, wherein in (H), the affinity (KD value) of the binding of the VHH antibody domain to recombinant human IL-18RβECD is not more than 5 times stronger than the affinity (KD value) of the binding of a VHH antibody domain to recombinant human IL-18RβECD consisting of a sequence from a table of VHH sequences that has the highest degree of sequence identity with the sequence of the VHH antibody domain in (H).

[0411] Embodiment 316: A VHH antibody domain or fragment thereof according to any one of Embodiments 212 to 313, wherein in (H), the affinity (KD value) of the binding of the VHH antibody domain to recombinant human IL-18RβECD is not more than twice as strong as the affinity (KD value) of the binding of a VHH antibody domain to recombinant human IL-18RβECD consisting of a sequence from a table of VHH sequences that has the highest degree of sequence identity with the sequence of the VHH antibody domain in (H).

[0412] Embodiment 317: A VHH antibody domain or fragment thereof according to any one of Embodiments 212 to 313, wherein in (H), the affinity (KD value) of the binding of the VHH antibody domain to recombinant human IL-18RβECD is not stronger than 1.5 times the affinity (KD value) of the binding of a VHH antibody domain to recombinant human IL-18RβECD consisting of a sequence from a table of VHH sequences that has the highest degree of sequence identity with the sequence of the VHH antibody domain in (H).

[0413] Embodiment 318: A VHH antibody domain or fragment thereof according to any one of Embodiments 279 to 317, wherein the KD value is determined in an in vitro binding experiment via bilayer interference.

[0414] Embodiment 319: A VHH antibody domain or fragment thereof according to any one of Embodiments 279 to 318, wherein the KD value is measured by kinetic measurement by bilayer interference in KB buffer (PBS + 0.1% Tween - 20% + 1% BSA) at 25°C and 1000 rpm.

[0415] Embodiment 320: A VHH antibody domain or fragment thereof according to any one of Embodiments 212 to 319, wherein the VHH antibody domain of (E) comprises one VHH sequence selected from the group consisting of VHH12, VHH13, VHH14, VHH15, VHH17, VHH18, VHH20, VHH21, and VHH22 as shown in the table of VHH sequences.

[0416] Embodiment 321: A VHH antibody domain or fragment thereof according to any one of Embodiments 212 to 319, wherein the VHH antibody domain of (E) comprises one VHH sequence selected from the group consisting of VHH12, VHH14, VHH15, VHH17, and VHH22 as shown in the VHH sequence table.

[0417] Embodiment 322: A VHH antibody domain or fragment thereof according to any one of Embodiments 212 to 319, wherein the VHH antibody domain of (E) comprises one VHH sequence selected from the group consisting of VHH6 and VHH11 as shown in the table of VHH sequences.

[0418] Embodiment 323: A VHH antibody domain or fragment thereof according to any one of Embodiments 212 to 319, wherein the VHH antibody domain of (E) comprises one VHH sequence selected from the group consisting of VHH13, VHH16, and VHH22 as shown in the VHH sequence table.

[0419] Embodiment 324: A VHH antibody domain or fragment thereof according to any one of Embodiments 212 to 319, wherein the VHH antibody domain of (E) comprises one VHH sequence selected from the group consisting of VHH12, VHH13, VHH15, VHH17, VHH18, and VHH21 as shown in the table of VHH sequences.

[0420] Embodiment 325: A VHH antibody domain or fragment thereof according to any one of Embodiments 212 to 319, wherein the VHH antibody domain of (E) comprises one VHH sequence selected from the group consisting of VHH15 and VHH17 as shown in the VHH sequence table.

[0421] Embodiment 326: A VHH antibody domain or fragment thereof according to any one of Embodiments 212 to 319, wherein the VHH antibody domain of (E) includes the VHH sequence VHH12 shown in the VHH sequence table.

[0422] Embodiment 327: A VHH antibody domain or fragment thereof according to any one of Embodiments 212 to 319, wherein the VHH antibody domain of (E) includes the VHH sequence VHH13 shown in the VHH sequence table.

[0423] Embodiment 328: A VHH antibody domain or fragment thereof according to any one of Embodiments 212 to 319, wherein the VHH antibody domain of (E) includes the VHH sequence VHH14 shown in the VHH sequence table.

[0424] Embodiment 329: A VHH antibody domain or fragment thereof according to any one of Embodiments 212 to 319, wherein the VHH antibody domain of (E) includes the VHH sequence VHH15 shown in the VHH sequence table.

[0425] Embodiment 330: A VHH antibody domain or fragment thereof according to any one of Embodiments 212 to 319, wherein the VHH antibody domain of (E) includes the VHH sequence VHH16 shown in the VHH sequence table.

[0426] Embodiment 331: A VHH antibody domain or fragment thereof according to any one of Embodiments 212 to 319, wherein the VHH antibody domain of (E) includes the VHH sequence VHH17 shown in the VHH sequence table.

[0427] Embodiment 332: A VHH antibody domain or fragment thereof according to any one of Embodiments 212 to 319, wherein the VHH antibody domain of (E) includes the VHH sequence VHH18 shown in the VHH sequence table.

[0428] Embodiment 333: A VHH antibody domain or fragment thereof according to any one of Embodiments 212 to 319, wherein the VHH antibody domain of (E) includes the VHH sequence VHH20 shown in the VHH sequence table.

[0429] Embodiment 334: A VHH antibody domain or fragment thereof according to any one of Embodiments 212 to 319, wherein the VHH antibody domain of (E) includes the VHH sequence VHH21 shown in the VHH sequence table.

[0430] Embodiment 335: A VHH antibody domain or fragment thereof according to any one of Embodiments 212 to 319, wherein the VHH antibody domain of (E) includes the VHH sequence VHH22 shown in the VHH sequence table.

[0431] Aspect 336:(d) wherein the VHH antibody domain or fragment thereof comprises one VHH complementarity-determining region CDR1, CDR2, and CDR3 selected from the group consisting of VHH12, VHH13, VHH14, VHH15, VHH17, VHH18, VHH20, VHH21, or VHH22 as shown in the table of CDRs, according to any one of aspects 189 to 211 or 250 to 335.

[0432] Aspect 337:(d) A VHH antibody domain or fragment according to any one of aspects 189-211 or 250-335, wherein the VHH antibody domain or fragment comprises one VHH complementarity-determining region CDR1, CDR2, and CDR3 selected from the group consisting of VHH12, VHH14, VHH15, VHH17, or VHH22 as shown in the table of CDRs.

[0433] Aspect 338:(d) wherein the VHH antibody domain or fragment thereof comprises complementarity-determining regions CDR1, CDR2, and CDR3 of VHH6 or VHH11 as shown in the table of CDRs, according to any one of aspects 189-211 or 250-335.

[0434] Aspect 339:(d) A VHH antibody domain or fragment according to any one of aspects 189-211 or 250-335, wherein the VHH antibody domain or fragment comprises one VHH complementarity-determining region CDR1, CDR2, and CDR3 selected from the group consisting of VHH13, VHH16, or VHH22 as shown in the table of CDRs.

[0435] Aspect 340:(d) A VHH antibody domain or fragment according to any one of aspects 189-211 or 250-335, wherein the VHH antibody domain or fragment comprises one VHH complementarity-determining region CDR1, CDR2, and CDR3 selected from the group consisting of VHH12, VHH13, VHH15, VHH17, VHH18, or VHH21 as shown in the table of CDRs.

[0436] A VHH antibody domain or fragment according to any one of Aspects 189-211 or 250-335, wherein the VHH antibody domain or fragment comprises one VHH complementarity-determining region CDR1, CDR2, and CDR3 selected from the group consisting of VHH15 or VHH17 as shown in the table of CDRs.

[0437] Aspect 342:(d) A VHH antibody domain or fragment according to any one of aspects 189-211 or 250-335, wherein the VHH antibody domain or fragment comprises complementarity-determining regions CDR1, CDR2, and CDR3 of VHH12 as shown in the table of CDRs.

[0438] Aspect 343:(d) wherein the VHH antibody domain or fragment thereof comprises the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH13 as shown in the table of CDRs, according to any one of aspects 189-211 or 250-335.

[0439] Aspect 344:(d) A VHH antibody domain or fragment according to any one of aspects 189-211 or 250-335, wherein the VHH antibody domain or fragment comprises complementarity-determining regions CDR1, CDR2, and CDR3 of VHH14 as shown in the table of CDRs.

[0440] Aspect 345:(d) A VHH antibody domain or fragment according to any one of aspects 189-211 or 250-335, wherein the VHH antibody domain or fragment comprises complementarity-determining regions CDR1, CDR2 and CDR3 of VHH15 as shown in the table of CDRs.

[0441] Aspect 346:(d) A VHH antibody domain or fragment according to any one of aspects 189-211 or 250-335, wherein the VHH antibody domain or fragment comprises complementarity-determining regions CDR1, CDR2 and CDR3 of VHH16 as shown in the table of CDRs.

[0442] Aspect 347:(d) A VHH antibody domain or fragment according to any one of aspects 189-211 or 250-335, wherein the VHH antibody domain or fragment comprises complementarity-determining regions CDR1, CDR2 and CDR3 of VHH17 as shown in the table of CDRs.

[0443] Aspect 348:(d) wherein the VHH antibody domain or fragment thereof comprises complementarity-determining regions CDR1, CDR2, and CDR3 of VHH18 as shown in the table of CDRs, according to any one of aspects 189-211 or 250-335.

[0444] Aspect 349:(d) wherein the VHH antibody domain or fragment thereof comprises complementarity-determining regions CDR1, CDR2, and CDR3 of VHH20 as shown in the table of CDRs, according to any one of aspects 189-211 or 250-335.

[0445] Aspect 350:(d) A VHH antibody domain or fragment thereof according to any one of aspects 189-211 or 250-335, wherein the VHH antibody domain or fragment thereof comprises complementarity-determining regions CDR1, CDR2 and CDR3 of VHH21 as shown in the table of CDRs.

[0446] Aspect 351:(d) wherein the VHH antibody domain or fragment thereof comprises complementarity-determining regions CDR1, CDR2, and CDR3 of VHH22 as shown in the table of CDRs, according to any one of aspects 189-211 or 250-335.

[0447] Embodiment 352: The VHH antibody domain or its fragment is recombinant human IL-18R β A VHH antibody domain or fragment thereof, according to any one of embodiments 189 to 351, that competes with VHH15 for binding to ECD.

[0448] Embodiment 353: The VHH antibody domain or its fragment is recombinant human IL-18R β A VHH antibody domain or fragment thereof according to any one of embodiments 189 to 351, which partially competes with VHH15 for binding to ECD.

[0449] Embodiment 354: A VHH antibody domain or fragment according to any one of Embodiments 189 to 353, wherein the VHH antibody domain or fragment does not compete with VHH2 for binding to recombinant human IL-18RαECD.

[0450] Embodiment 355: A VHH antibody domain or fragment thereof according to any one of Embodiments 189 to 354, wherein the humanization is performed by germline fusion.

[0451] Embodiment 356: A VHH antibody domain or fragment thereof according to Embodiment 355, wherein the germlineization includes: - To identify the sequence to be humanized and the closest human VH germline sequence in terms of sequence similarity; and - Replacing one or more amino acids in a sequence to be humanized with one or more amino acids at the corresponding sequence position in the nearest human VH germline sequence.

[0452] Sequence similarity and the positions of corresponding sequences can be determined by sequence alignment as described above.

[0453] Embodiment 357: A VHH antibody domain or fragment thereof according to Embodiment 355, wherein the germlineization comprises: - To identify the sequence to be humanized and the closest human VH germline sequence in terms of sequence similarity; and - Replacing one or more amino acids in a sequence to be humanized with one or more amino acids at the corresponding sequence position in the nearest human VH germline sequence.

[0454] Aspect 358: A VHH antibody domain or fragment thereof according to any one of Aspects 189 to 357, wherein the humanized VHH antibody domain (each fragment thereof) is equivalent to the human IL-18R of the corresponding unhumanized VHH antibody domain (each fragment thereof). β Compared to binding to ECD, human IL-18R has an affinity (KD value) that is not weaker than 10 times. β Connect to ECD.

[0455] As those skilled in the art will understand, the term "corresponding non-humanized VHH antibody domain" refers to a VHH antibody domain that is identical in all other respects, but differs only in the alteration of amino acids introduced to carry out the humanization.

[0456] Aspect 359: A VHH antibody domain or fragment thereof according to any one of Aspects 189 to 357, wherein the humanized VHH antibody domain (each fragment thereof) is equivalent to the human IL-18R of the corresponding unhumanized VHH antibody domain (each fragment thereof). β Compared to binding to ECD, human IL-18R has an affinity (KD value) that is not weak, exceeding 5 times. β Connect to ECD. with an

[0457] Aspect 360: A VHH antibody domain or fragment thereof according to any one of aspects 189 to 357, wherein the humanized VHH antibody domain (each fragment thereof) binds to human IL-18RβECD with an affinity (KD value) that is not more than twice as weak as the binding of the corresponding non-humanized VHH antibody domain (each fragment thereof) to human IL-18RβECD. β Connect to ECD.

[0458] Aspect 361: A VHH antibody domain or fragment thereof according to any one of Aspects 189 to 357, wherein the humanized VHH antibody domain (each fragment thereof) binds to human IL-18RβECD with an affinity (KD value) no weaker than that of the corresponding unhumanized VHH antibody domain (each fragment thereof) to human IL-18RβECD. β Connect to ECD.

[0459] Embodiment 362: A VHH antibody domain or fragment thereof according to any one of Embodiments 358 to 361, wherein the affinity is determined by KD measurement.

[0460] Embodiment 363: The VHH antibody domain comprises a VHH antibody domain according to (E), (F), or (G) / the fragment of the VHH antibody domain comprises a fragment of the VHH antibody domain according to any one of Embodiments 212 to 362.

[0461] Embodiment 364: The VHH antibody domain comprises a VHH antibody domain according to (E), (F), or (H) / the fragment of the VHH antibody domain comprises a fragment of the VHH antibody domain according to any one of Embodiments 212 to 362.

[0462] Embodiment 365: The VHH antibody domain comprises a VHH antibody domain according to (E), (G), or (H) / the fragment of the VHH antibody domain comprises a fragment of the VHH antibody domain according to any one of Embodiments 212 to 362.

[0463] Embodiment 366: The VHH antibody domain comprises a VHH antibody domain according to (E) or (F) / the fragment of the VHH antibody domain comprises a fragment of the VHH antibody domain according to any one of Embodiments 212 to 362.

[0464] Embodiment 367: The VHH antibody domain comprises a VHH antibody domain according to (E) or (G) / the fragment of the VHH antibody domain comprises a fragment of the VHH antibody domain according to any one of Embodiments 212 to 362.

[0465] Embodiment 368: The VHH antibody domain comprises a VHH antibody domain according to (E) or (H) / the fragment of the VHH antibody domain comprises a fragment of the VHH antibody domain according to any one of Embodiments 212 to 362.

[0466] Embodiment 369: The VHH antibody domain comprises a VHH antibody domain according to (E) / the fragment of the VHH antibody domain comprises a fragment of the VHH antibody domain according to any one of Embodiments 212 to 362.

[0467] Embodiment 370: The VHH antibody domain comprises a VHH antibody domain according to (F) / the fragment of the VHH antibody domain comprises a fragment of the VHH antibody domain according to any one of Embodiments 212 to 362.

[0468] Embodiment 371: A VHH antibody domain or fragment thereof according to any one of Embodiments 189-211 or 250-370, wherein the VHH antibody domain or fragment thereof includes a complementarity determination region according to (d) or (e).

[0469] Embodiment 372: A VHH antibody domain or fragment thereof according to any one of Embodiments 189-211 or 250-370, wherein the VHH antibody domain or fragment thereof includes a complementarity determination region according to (d) or (f).

[0470] Embodiment 373: A VHH antibody domain or fragment thereof according to any one of Embodiments 189-211 or 250-370, wherein the VHH antibody domain or fragment thereof includes a complementarity determination region according to (d).

[0471] Embodiment 374: A VHH antibody domain or fragment thereof according to any one of Embodiments 189-211 or 250-370, wherein the VHH antibody domain or fragment thereof includes a complementarity determination region according to (e).

[0472] Aspect seven of this disclosure (also referred to as “Aspect 375”): According to aspect seven, this disclosure is as follows: - A VHH antibody domain or fragment thereof according to any one of embodiments 1 to 188 This relates to compounds, including those mentioned above.

[0473] The eighth aspect of this disclosure (also referred to as “Aspect 376”): According to the eighth aspect, this disclosure is as follows: - A VHH antibody domain or fragment thereof according to any one of claims 189 to 374 This relates to compounds, including those mentioned above.

[0474] Aspect 9 of the present disclosure (also referred to as “Aspect 377”): According to aspect 9, the present disclosure relates to compounds including: -A first binding module which is a VHH antibody domain or a fragment thereof, where, (a) The VHH antibody domain or fragment thereof includes one VHH complementarity-determining region CDR1, CDR2, and CDR3 selected from the group consisting of VHH1, VHH2, VHH3, VHH4, VHH5, VHH6, VHH8, VHH9, VHH10, and VHH11 as shown in the table of CDRs; (b) The VHH antibody domain or fragment thereof includes the complementarity-determining regions CDR1, CDR2 and CDR3 as defined in (a), wherein the modification is that at least one of the sequences of CDR1, CDR2 and CDR3 is humanized; or (c) The VHH antibody domain or fragment thereof includes the complementarity-determining regions CDR1, CDR2 and CDR3 as defined in (a) and which are modified, wherein the modifications are - Substitution, addition, or deletion of up to three amino acids in CDR1, - Substitution, addition or deletion of up to three amino acids in CDR2, and / or - Substitution, addition, or deletion of up to three amino acids in CDR3 is; - A second binding module which is the VHH antibody domain or a fragment thereof, where, (d) The VHH antibody domain or fragment thereof includes one VHH complementarity-determining region CDR1, CDR2, and CDR3 selected from the group consisting of VHH12, VHH13, VHH14, VHH15, VHH16, VHH17, VHH18, VHH20, VHH21, and VHH22 as shown in the table of CDRs; (e) The VHH antibody domain or fragment thereof includes the complementarity-determining regions CDR1, CDR2 and CDR3 as defined in (d), wherein the modification is that at least one of the sequences of CDR1, CDR2 and CDR3 is humanized; or (f) The VHH antibody domain or fragment thereof includes the complementarity-determining regions CDR1, CDR2 and CDR3 as defined in (d) and which are modified, where the modification is - Substitution, addition, or deletion of up to three amino acids in CDR1, - Substitution, addition or deletion of up to three amino acids in CDR2, and / or - Substitution, addition, or deletion of up to three amino acids in CDR3 That is the case.

[0475] The same descriptions and definitions as those for aspects 1 through 6 of this disclosure and aspects relating thereto apply accordingly to aspects 7 through 9 of this disclosure and aspects relating thereto.

[0476] With respect to the first binding module, with respect to Embodiment 377 and embodiments referencing it, further embodiments of the VHH antibody domain or fragment of the first binding module are as defined in Embodiments 1 to 188.

[0477] Appearance 377 and Appearances Referring Thereto With respect to the second binding module, further embodiments of the VHH antibody domain or fragment of the second binding module are as defined in Appearances 189 to 374.

[0478] The tenth aspect of this disclosure (also referred to as “Aspect 378”): According to the tenth aspect, this disclosure relates to compounds including: -A first binding module which is a VHH antibody domain or a fragment thereof, where, (A) The VHH antibody domain contains an amino acid sequence of VHH selected from the group consisting of VHH1, VHH2, VHH3, VHH4, VHH5, VHH6, VHH8, VHH9, VHH10 and VHH11 as shown in the VHH sequence table; (B) The VHH antibody domain comprises a VHH sequence as defined in (A) and having modifications, wherein the modification is humanization of the sequence; (C) The VHH antibody domain comprises a VHH sequence as defined in (A) and having modifications, wherein the modifications are substitutions, additions, or deletions of up to 25 amino acids; or, (D) The VHH antibody domain comprises a VHH sequence that is at least 75% identical to the VHH sequence mentioned in (A); - A second binding module which is the VHH antibody domain or a fragment thereof, where, (E) The VHH antibody domain contains an amino acid sequence of VHH selected from the group consisting of VHH12, VHH13, VHH14, VHH15, VHH16, VHH17, VHH18, VHH20, VHH21, and VHH22 as shown in the VHH sequence table; (F) The VHH antibody domain comprises a VHH sequence as defined in (E) and having modifications, wherein the modification is humanization of the sequence; (G) The VHH antibody domain comprises a VHH sequence as defined in (E) and having modifications, wherein the modifications are substitutions, additions, or deletions of up to 25 amino acids; or, (H) The VHH antibody domain comprises a VHH sequence that is at least 75% identical to the VHH sequence referenced in (E).

[0479] The same descriptions and definitions as those for aspects 1 through 9 of this disclosure and aspects relating thereto apply accordingly to the tenth aspect of this disclosure and aspects relating thereto.

[0480] For Embodiment 378 and embodiments referencing it, with respect to the first binding module, further embodiments of the VHH antibody domain or fragment of the first binding module are as defined in Embodiments 1 to 188.

[0481] For embodiment 378 and embodiments referencing it, with respect to the second binding module, further embodiments of the VHH antibody domain or fragment of the second binding module are as defined in embodiments 189 to 374.

[0482] Aspect 11 of the present disclosure (also referred to as “Aspect 379”): According to Aspect 11, the present disclosure relates to compounds including: -A first binding module which is a VHH antibody domain or a fragment thereof, where, (A) The VHH antibody domain consists of an amino acid sequence of VHH selected from the group consisting of VHH1, VHH2, VHH3, VHH4, VHH5, VHH6, VHH8, VHH9, VHH10, and VHH11, as shown in the VHH sequence table; (B) The VHH antibody domain consists of a VHH sequence as defined in (A) and is modified, wherein the modification is humanization of the sequence; (C) The VHH antibody domain consists of a VHH sequence as defined in (A) and is modified, wherein the modification is the substitution, addition, or deletion of up to 25 amino acids; or, (D) The VHH antibody domain consists of a VHH sequence that is at least 75% identical to the VHH sequence mentioned in (A); - A second binding module which is the VHH antibody domain or a fragment thereof, where, (E) The VHH antibody domain consists of an amino acid sequence of VHH selected from the group consisting of VHH12, VHH13, VHH14, VHH15, VHH16, VHH17, VHH18, VHH20, VHH21, and VHH22 as shown in the table of VHH sequences; (F) The VHH antibody domain consists of a VHH sequence as defined in (E) and is modified, wherein the modification is humanization of the sequence; (G) The VHH antibody domain consists of a VHH sequence as defined in (E) and is modified, wherein the modification is the substitution, addition, or deletion of up to 25 amino acids; or, (H) The VHH antibody domain consists of a VHH sequence that is at least 75% identical to the VHH sequence referenced in (E).

[0483] The same descriptions and definitions as those for the first to tenth aspects of this disclosure and the aspects referencing them apply accordingly to the eleventh aspect of this disclosure and the embodiments referencing it.

[0484] With respect to the first binding module, with respect to the 379th embodiment and embodiments referencing it, further embodiments of the VHH antibody domain or fragment of the first binding module are as defined in embodiments 1 to 188.

[0485] With respect to the second binding module, with respect to the second binding module, further embodiments of the VHH antibody domain or fragment of the second binding module are as defined in embodiments 189 to 374.

[0486] As used in this disclosure, “compound” means any chemical entity of any chemical class, which includes the binding module(s) defined above. Thus, a compound may be, for example, an organic compound, or a compound comprising organic and inorganic parts, which may be a protein comprising a single amino acid chain, a protein comprising multiple amino acid chains associated by either non-covalent or covalent bonds, or a non-covalent complex comprising inorganic components. A compound may consist of the amino acid sequence of the binding module(s) referred to alone in the above embodiments, or it may also include further amino acids(s), which may be attached by covalent or non-covalent bonds, or which may be associated by inorganic components. Preferably, a compound is a molecule.

[0487] For example, in Embodiment 375 having any modification from Embodiments 1 to 188, and in further embodiments referring to Embodiment 375 (corresponding in Embodiment 376 to any modification having any modification from Embodiments 189 to 374, and in further embodiments referring to Embodiment 376), the compound may be, for example, a monovalent, monospecific antibody molecule in which anti-IL-18RαVHH (anti-IL-18RβVHH according to the Disclosure, respectively) is covalently linked to the Fc region of IgG. Alternatively, it may be a bispecific molecule in which anti-IL-18RαVHH according to the Disclosure (each anti-IL-18RβVHH according to the Disclosure) is covalently linked to an IgG1 antibody lacking one of its "arms," ​​so that the molecule has two binding modules, namely, anti-IL-18RαVHH according to the Disclosure (corresponding to anti-IL-18RβVHH according to the Disclosure) and the remaining "original" binding module of a "one-armed" IgG1 antibody. Or, in another example, the compound may be a bispecific molecule comprising anti-IL-18RαVHH according to the Disclosure and a targeting module (corresponding to anti-IL-18RβVHH according to the Disclosure).

[0488] For example, in embodiments 377-379, (having any modifications to the first binding module as defined in embodiments 1-188 and any modifications to the second binding module as defined in embodiments 189-374, and having any further modifications as defined in embodiments referring to embodiments 377-379), the compound may be, for example, an antibody having two binding modules (anti-IL-18RαVHH and anti-IL-18RβVHH as defined herein) prepared in SEED format, which results in a bispecific antibody having the structure shown, for example, in Figure 4A (left). The compounds of embodiments 377-379 may also include further molecular components, for example, further copies of the first and second binding modules, as exemplified in Figure 4A (center and right), which show molecules having two copies of the first binding module and two copies of the second binding module in different configurations. As those skilled in the art will understand, many other compound formats are possible, however, the resulting compound format does not interfere with the function of the VHH antibody domain(s) or fragment(s) according to the present invention (i.e., in the case of the compounds of aspects 377-379: as described herein, the molecular format does not interfere with the binding of the first binding module to IL18Rα and the binding of the second binding module to IL18Rβ).

[0489] The compounds of this disclosure can be prepared by standard methods of genetic engineering and recombinant protein techniques known to those skilled in the art (see, for example, Green and Sambrook, "Molecular Cloning: A Laboratory Manual," 2014; Coligan et al., "Current Protocols in Protein Science" (1997)). Exemplary methods are also described in the Examples section of this disclosure.

[0490] If a compound cannot be expressed as a single piece, the individual parts may be prepared separately and then coupled later by covalent bonding, for example, by a chemical reaction using an appropriate reactive group (e.g., linking using maleimide chemistry), or by enzymatic linking (e.g., linking catalyzed by transglutaminase). For example, as described in the Examples section, one or more VHH antibody domains or fragments thereof may be prepared by recombinant protein expression and then linked to an antibody or antibody fragment to produce a bispecific antibody compound.

[0491] If a compound contains non-biomolecular components (such as peptidomimetic molecules or small molecules), these components may be obtained, for example, by standard methods of synthetic organic chemistry.

[0492] Aspect 380: A compound according to Aspect 377, - Herein, the VHH antibody domain or fragment defined in (a) includes one VHH complementarity-determining region CDR1, CDR2 and CDR3 selected from the group consisting of VHH1, VHH2, VHH5, VHH6, VHH8, VHH9 and VHH10 as shown in the table of CDRs, and, - Herein, the VHH antibody domain or fragment defined in (d) includes one VHH complementarity-determining region CDR1, CDR2 and CDR3 selected from the group consisting of VHH12, VHH13, VHH14, VHH15, VHH17, VHH18, VHH20, VHH21 and VHH22 as shown in the table of CDRs, wherein the VHH antibody domain or fragment defined in (d) is selected as follows: If the VHH antibody domain or fragment of (a) contains the VHH1 complementarity-determining regions CDR1, CDR2, and CDR3, then the VHH antibody domain or fragment of (d) contains one VHH complementarity-determining region CDR1, CDR2, and CDR3 selected from the group consisting of VHH12, VHH13, VHH15, VHH17, VHH20, VHH21, and VHH22 as shown in the table of CDRs; If the VHH antibody domain or fragment of (a) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH2, then the VHH antibody domain or fragment of (d) contains one VHH complementarity-determining region CDR1, CDR2, and CDR3 selected from the group consisting of VHH12, VHH13, VHH15, VHH17, VHH18, VHH20, VHH21, and VHH22 as shown in the table of CDRs; If the VHH antibody domain or fragment of (a) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH5, then the VHH antibody domain or fragment of (d) contains one VHH complementarity-determining region CDR1, CDR2, and CDR3 selected from the group consisting of VHH13 and VHH14 as shown in the table of CDRs; If the VHH antibody domain or fragment of (a) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH6, then the VHH antibody domain or fragment of (d) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of one VHH selected from the group consisting of VHH17, VHH20, VHH21, and VHH22 as shown in the table of CDRs; If the VHH antibody domain or fragment of (a) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH8, then the VHH antibody domain or fragment of (d) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of one VHH selected from the group consisting of VHH12, VHH13, VHH17, VHH18, VHH20, and VHH21 as shown in the table of CDRs; If the VHH antibody domain or fragment of (a) contains the VHH9 complementarity-determining regions CDR1, CDR2, and CDR3, then the VHH antibody domain or fragment of (d) contains one VHH complementarity-determining region CDR1, CDR2, and CDR3 selected from the group consisting of VHH12, VHH13, VHH15, VHH17, VHH18, VHH20, VHH21, and VHH22 as shown in the table of CDRs; If the VHH antibody domain or fragment of (a) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH10, then the VHH antibody domain or fragment of (d) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of one VHH selected from the group consisting of VHH13, VHH20, and VHH21 as shown in the table of CDRs.

[0493] Therefore, according to this embodiment, the compounds of Embodiment 377 (and embodiments referencing them) may include any combination of VHHα and VHHβ shown in green in Figure 4B (surrogate agonists that induce normalized NFκB reporter activation at a level higher than 50% compared to IL18, or that exhibit an action strength of less than 0.1 nM).

[0494] Aspect 381: A compound according to Aspect 377, - Herein, the VHH antibody domain or fragment as defined in (a) includes one VHH complementarity-determining region CDR1, CDR2 and CDR3 selected from the group consisting of VHH1, VHH2, VHH3, VHH4, VHH5, VHH6, VHH8, VHH9 and VHH10 as shown in the table of CDRs, and, - Herein, the VHH antibody domain or fragment defined in (d) includes one VHH complementarity-determining region CDR1, CDR2 and CDR3 selected from the group consisting of VHH12, VHH13, VHH14, VHH15, VHH16, VHH17, VHH18, VHH20, VHH21 and VHH22 as shown in the table of CDRs, wherein the VHH antibody domain or fragment defined in (d) is selected as follows: If the VHH antibody domain or fragment of (a) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH1, then the VHH antibody domain or fragment of (d) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of one VHH selected from the group consisting of VHH14, VHH16, and VHH18 as shown in the table of CDRs; If the VHH antibody domain or fragment of (a) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH2, then the VHH antibody domain or fragment of (d) contains one VHH complementarity-determining region CDR1, CDR2, and CDR3 selected from the group consisting of VHH14 and VHH16 as shown in the table of CDRs; If the VHH antibody domain or fragment of (a) contains the VHH3 complementarity-determining regions CDR1, CDR2, and CDR3, then the VHH antibody domain or fragment of (d) contains one VHH complementarity-determining region CDR1, CDR2, and CDR3 selected from the group consisting of VHH12, VHH13, VHH14, VHH15, VHH16, VHH17, VHH18, VHH20, VHH21, and VHH22 as shown in the table of CDRs; If the VHH antibody domain or fragment of (a) contains the VHH4 complementarity-determining regions CDR1, CDR2, and CDR3, then the VHH antibody domain or fragment of (d) contains one VHH complementarity-determining region CDR1, CDR2, and CDR3 selected from the group consisting of VHH12, VHH13, VHH14, VHH15, VHH16, VHH17, VHH18, VHH20, VHH21, and VHH22 as shown in the table of CDRs; If the VHH antibody domain or fragment of (a) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH5, then the VHH antibody domain or fragment of (d) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of one VHH selected from the group consisting of VHH12, VHH15, VHH16, VHH17, VHH18, VHH20, VHH21, and VHH22 as shown in the table of CDRs; If the VHH antibody domain or fragment of (a) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH6, then the VHH antibody domain or fragment of (d) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of one VHH selected from the group consisting of VHH14, VHH16, and VHH18 as shown in the table of CDRs; If the VHH antibody domain or fragment of (a) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH8, then the VHH antibody domain or fragment of (d) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH16 as shown in the table of CDRs; If the VHH antibody domain or fragment of (a) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH9, then the VHH antibody domain or fragment of (d) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of one VHH selected from the group consisting of VHH14, VHH16, and VHH18 as shown in the table of CDRs; If the VHH antibody domain or fragment of (a) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH10, then the VHH antibody domain or fragment of (d) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of one VHH selected from the group consisting of VHH14, VHH15, VHH16, and VHH18 as shown in the table of CDRs.

[0495] Therefore, according to this embodiment, the compounds of embodiment 377 (and embodiments referencing them) may include any combination of VHHα and VHHβ shown in red in Figure 4B.

[0496] Aspect 382: A compound according to Aspect 377, - Herein, the VHH antibody domain or fragment defined in (a) includes one VHH complementarity-determining region CDR1, CDR2 and CDR3 selected from the group consisting of VHH6 and VHH11 as shown in the table of CDRs, and, - Herein, the VHH antibody domain or fragment defined in (d) includes one VHH complementarity-determining region CDR1, CDR2 and CDR3 selected from the group consisting of VHH12, VHH13, VHH14, VHH15, VHH16, VHH17, VHH18, VHH20, VHH21 and VHH22 as shown in the table of CDRs, wherein the VHH antibody domain or fragment defined in (d) is selected as follows: If the VHH antibody domain or fragment of (a) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH6, then the VHH antibody domain or fragment of (d) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of one VHH selected from the group consisting of VHH12 and VHH13 as shown in the table of CDRs; If the VHH antibody domain or fragment of (a) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH11, then the VHH antibody domain or fragment of (d) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of one VHH selected from the group consisting of VHH12, VHH13, VHH14, VHH15, VHH16, VHH17, VHH18, VHH20, VHH21, and VHH22 as shown in the table of CDRs.

[0497] Therefore, according to this embodiment, the compounds of embodiment 377 (and embodiments referencing them) may include any combination of VHHα and VHHβ shown in black in Figure 4B.

[0498] Embodiment 383: A compound according to Embodiment 377, - Herein, the VHH antibody domain or fragment defined in (a) includes one VHH complementarity-determining region CDR1, CDR2 and CDR3 selected from the group consisting of VHH2, VHH6, VHH8 and VHH10 as shown in the table of CDRs, and, - Herein, the VHH antibody domain or fragment defined in (d) includes one VHH complementarity-determining region CDR1, CDR2 and CDR3 selected from the group consisting of VHH12, VHH14, VHH15, VHH17 and VHH22 as shown in the table of CDRs, wherein the VHH antibody domain or fragment defined in (d) is selected as follows: If the VHH antibody domain or fragment of (a) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH2, then the VHH antibody domain or fragment of (d) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH22 as shown in the table of CDRs; If the VHH antibody domain or fragment of (a) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH6, then the VHH antibody domain or fragment of (d) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH15 as shown in the table of CDRs; If the VHH antibody domain or fragment of (a) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH8, then the VHH antibody domain or fragment of (d) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of one VHH selected from the group consisting of VHH14, VHH15, and VHH22 as shown in the table of CDRs; If the VHH antibody domain or fragment of (a) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH10, then the VHH antibody domain or fragment of (d) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of one VHH selected from the group consisting of VHH12, VHH17, and VHH22 as shown in the table of CDRs.

[0499] Therefore, according to this embodiment, the compounds of Embodiment 377 (and embodiments referencing them) may include any combination of VHHα and VHHβ shown in orange in Figure 4B (active surrogate agonists that induce less than 50% of NFκB reporter activation normalized to (rh)IL-18 at 1 nM, or have an EC50 greater than 0.1 nM).

[0500] Embodiment 384: A compound according to Embodiment 377, wherein the VHH antibody domain or fragment as defined in (a) comprises one VHH complementarity-determining region CDR1, CDR2 and CDR3 selected from the group consisting of VHH2 and VHH8 as shown in the table of CDR, and wherein the VHH antibody domain or fragment as defined in (d) comprises one VHH complementarity-determining region CDR1, CDR2 and CDR3 selected from the group consisting of VHH15 and VHH17 as shown in the table of CDR.

[0501] Aspect 385: A compound according to Aspect 377, - Herein, the VHH antibody domain or fragment defined in (a) comprises one VHH complementarity-determining region CDR1, CDR2 and CDR3 selected from the group consisting of VHH2 and VHH8 as shown in the table of CDRs, and - Herein, the VHH antibody domain or fragment defined in (d) includes one VHH complementarity-determining region CDR1, CDR2 and CDR3 selected from the group consisting of VHH15 and VHH17 as shown in the table of CDRs, wherein the VHH antibody domain or fragment defined in (d) is selected as follows: If the VHH antibody domain or fragment of (a) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH2, then the VHH antibody domain or fragment of (d) contains one VHH complementarity-determining region CDR1, CDR2, and CDR3 selected from the group consisting of VHH15 and VHH17 as shown in the table of CDRs; If the VHH antibody domain or fragment of (a) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH8, then the VHH antibody domain or fragment of (d) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH17 as shown in the table of CDRs.

[0502] Embodiment 386: A compound according to Embodiment 377, wherein the VHH antibody domain or fragment as defined in (a) comprises the complementarity-determining regions CDR1, CDR2 and CDR3 of VHH2 as shown in the table of CDRs, and wherein the VHH antibody domain or fragment as defined in (d) comprises the complementarity-determining regions CDR1, CDR2 and CDR3 of VHH15 as shown in the table of CDRs.

[0503] Aspect 387: A compound according to Aspect 377, - Herein, the VHH antibody domain or fragment as defined in (a) includes one VHH complementarity-determining region CDR1, CDR2 and CDR3 selected from the group consisting of VHH1, VHH2, VHH5, VHH6, VHH8 and VHH10 as shown in the table of CDRs, and - Herein, the VHH antibody domain or fragment defined in (d) comprises one VHH complementarity-determining region CDR1, CDR2, and CDR3 selected from the group consisting of VHH13, VHH16, and VHH22 as shown in the table of CDRs, wherein the VHH antibody domain or fragment defined in (d) is selected as follows: If the VHH antibody domain or fragment of (a) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH1, then the VHH antibody domain or fragment of (d) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH16 as shown in the table of CDRs; If the VHH antibody domain or fragment of (a) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH2, then the VHH antibody domain or fragment of (d) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH22 as shown in the table of CDRs; If the VHH antibody domain or fragment of (a) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH5, then the VHH antibody domain or fragment of (d) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH13 as shown in the table of CDRs; If the VHH antibody domain or fragment of (a) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH6, then the VHH antibody domain or fragment of (d) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH22 as shown in the table of CDRs; If the VHH antibody domain or fragment of (a) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH8, then the VHH antibody domain or fragment of (d) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH22 as shown in the table of CDRs; If the VHH antibody domain or fragment of (a) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH10, then the VHH antibody domain or fragment of (d) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH22 as shown in the table of CDRs.

[0504] Aspect 388: A compound according to Aspect 377, - Herein, the VHH antibody domain or fragment defined in (a) comprises one VHH complementarity-determining region CDR1, CDR2 and CDR3 selected from the group consisting of VHH2, VHH6, VHH8 and VHH10 as shown in the table of CDRs, and - Herein, the VHH antibody domain or fragment defined in (d) comprises one VHH complementarity-determining region CDR1, CDR2 and CDR3 selected from the group consisting of VHH12 and VHH15 as shown in the table of CDRs, wherein the VHH antibody domain or fragment defined in (d) is selected as follows: If the VHH antibody domain or fragment of (a) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH2, then the VHH antibody domain or fragment of (d) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH12 as shown in the table of CDRs; If the VHH antibody domain or fragment of (a) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH6, then the VHH antibody domain or fragment of (d) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of one VHH selected from the group consisting of VHH12 and VHH15 as shown in the table of CDRs; If the VHH antibody domain or fragment of (a) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH8, then the VHH antibody domain or fragment of (d) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of one VHH selected from the group consisting of VHH13 and VHH18 as shown in the table of CDRs; If the VHH antibody domain or fragment of (a) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH10, then the VHH antibody domain or fragment of (d) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH12 as shown in the table of CDRs.

[0505] Embodiment 389: A compound according to any one of Embodiments 377 or 380-388, wherein in the first binding module, the VHH antibody domain or its fragment includes a complementarity determination region according to (a) or (b).

[0506] Where this disclosure states that "in the first binding module, the VHH antibody domain or fragment thereof includes a complementarity-determining region according to (a) or (b)," this is intended to indicate that, in this embodiment, only (a) and (b) are considered options for the VHH antibody domain (or fragment thereof) of the first binding module, while option (c) is excluded. Where this disclosure states that "in the first binding module, the VHH antibody domain or fragment thereof includes a complementarity-determining region according to (a) or (c)," this is intended to indicate that, in this embodiment, only (a) and (c) are considered options for the VHH antibody domain (or fragment thereof) of the first binding module, while option (b) is excluded. The alternative words below should be understood to fit. Where this disclosure states that "in the first binding module, the VHH antibody domain or fragment thereof includes a complementarity-determining region by (a)", it is intended to indicate that, in this embodiment, only (a) is considered an option for the VHH antibody domain (or fragment thereof) of the first binding module, while options (b) and (c) are excluded. Further variations of this wording below should be understood to fit this.

[0507] Embodiment 390: A compound according to any one of Embodiments 377 or 380-388, wherein in the first binding module, the VHH antibody domain or its fragment includes a complementarity determination region according to (a) or (c).

[0508] Embodiment 391: A compound according to any one of Embodiments 377 or 380-388, wherein in the first binding module, the VHH antibody domain or its fragment includes a complementarity determination region according to (a).

[0509] Embodiment 392: A compound according to any one of Embodiments 377 or 380-388, wherein in the first binding module, the VHH antibody domain or its fragment includes a complementarity determination region according to (b).

[0510] Embodiment 393: A compound according to any one of Embodiments 377 or 380-392, wherein the VHH antibody domain or fragment thereof comprises a complementarity determination region according to (d) or (e).

[0511] Embodiment 394: A compound according to any one of Embodiments 377 or 380-392, wherein the second binding module, the VHH antibody domain or its fragment, comprises a complementarity determination region according to (d) or (f).

[0512] Embodiment 395: A compound according to any one of Embodiments 377 or 380-392, wherein the second binding module, the VHH antibody domain or its fragment, includes a complementarity determination region according to (d).

[0513] Embodiment 396: A compound according to any one of Embodiments 377 or 380-392, wherein the second binding module, the VHH antibody domain or its fragment, comprises a complementarity determination region according to (e).

[0514] Embodiment 397: A compound according to either Embodiment 378 or 379, - Here, the VHH antibody domain of (A) includes the amino acid sequence of one VHH selected from the group consisting of VHH1, VHH2, VHH5, VHH6, VHH8, VHH9 and VHH10 shown in the table of VHH sequences, and - Here, the VHH antibody domain of (E) comprises an amino acid sequence of one VHH selected from the group consisting of VHH12, VHH13, VHH14, VHH15, VHH17, VHH18, VHH20, VHH21, and VHH22 as shown in the table of VHH sequences, wherein the VHH antibody domain of (E) is selected as follows: If the VHH antibody domain of (A) contains the amino acid sequence of VHH1, then the VHH antibody domain of (E) contains the amino acid sequence of one VHH selected from the group consisting of VHH12, VHH13, VHH15, VHH17, VHH20, VHH21, and VHH22 as shown in the table of VHH sequences; If the VHH antibody domain of (A) contains the amino acid sequence of VHH2, then the VHH antibody domain of (E) contains the amino acid sequence of one VHH selected from the group consisting of VHH12, VHH13, VHH15, VHH17, VHH18, VHH20, VHH21, and VHH22 as shown in the table of VHH sequences; If the VHH antibody domain of (A) contains the amino acid sequence of VHH5, then the VHH antibody domain of (E) contains the amino acid sequence of one VHH selected from the group consisting of VHH13 and VHH14 as shown in the table of VHH sequences; If the VHH antibody domain of (A) contains the amino acid sequence of VHH6, then the VHH antibody domain of (E) contains the amino acid sequence of one VHH selected from the group consisting of VHH17, VHH20, VHH21, and VHH22 as shown in the table of VHH sequences; If the VHH antibody domain of (A) contains the amino acid sequence of VHH8, then the VHH antibody domain of (E) contains the amino acid sequence of one VHH selected from the group consisting of VHH12, VHH13, VHH17, VHH18, VHH20, and VHH21 as shown in the table of VHH sequences; If the VHH antibody domain of (A) contains the amino acid sequence of VHH9, then the VHH antibody domain of (E) contains the amino acid sequence of one VHH selected from the group consisting of VHH12, VHH13, VHH15, VHH17, VHH18, VHH20, VHH21, and VHH22 as shown in the table of VHH sequences; If the VHH antibody domain of (A) contains the amino acid sequence of VHH10, then the VHH antibody domain of (E) contains the amino acid sequence of one VHH selected from the group consisting of VHH13, VHH20, and VHH21 as shown in the table of VHH sequences.

[0515] Therefore, according to this embodiment, the compounds of embodiments 378-379 (and embodiments referencing them) may include any combination of VHHα and VHHβ shown in green in Figure 4B (surrogate agonists that induce normalized NFκB reporter activation at a level higher than 50% compared to IL18, or that exhibit an action strength of less than 0.1 nM).

[0516] Embodiment 398: A compound according to either Embodiment 378 or 379, - Here, the VHH antibody domain of (A) includes the amino acid sequence of one VHH selected from the group consisting of VHH1, VHH2, VHH3, VHH4, VHH5, VHH6, VHH8, VHH9 and VHH10 as shown in the table of VHH sequences, and, - Here, the VHH antibody domain of (E) comprises an amino acid sequence of one VHH selected from the group consisting of VHH12, VHH13, VHH14, VHH15, VHH16, VHH17, VHH18, VHH20, VHH21, and VHH22 as shown in the table of VHH sequences, wherein the VHH antibody domain of (E) is selected as follows: If the VHH antibody domain of (A) contains the amino acid sequence of VHH1, then the VHH antibody domain of (E) contains the amino acid sequence of one VHH selected from the group consisting of VHH14, VHH16, and VHH18 as shown in the table of VHH sequences; If the VHH antibody domain of (A) contains the amino acid sequence of VHH2, then the VHH antibody domain of (E) contains the amino acid sequence of one VHH selected from the group consisting of VHH14 and VHH16 as shown in the table of VHH sequences; If the VHH antibody domain of (A) contains the amino acid sequence of VHH3, then the VHH antibody domain of (E) contains the amino acid sequence of one VHH selected from the group consisting of VHH12, VHH13, VHH14, VHH15, VHH16, VHH17, VHH18, VHH20, VHH21, and VHH22 as shown in the table of VHH sequences; If the VHH antibody domain of (A) contains the amino acid sequence of VHH4, then the VHH antibody domain of (E) contains the amino acid sequence of one VHH selected from the group consisting of VHH12, VHH13, VHH14, VHH15, VHH16, VHH17, VHH18, VHH20, VHH21, and VHH22 as shown in the table of VHH sequences; If the VHH antibody domain of (A) contains the amino acid sequence of VHH5, then the VHH antibody domain of (E) contains the amino acid sequence of one VHH selected from the group consisting of VHH12, VHH15, VHH16, VHH17, VHH18, VHH20, VHH21, and VHH22 as shown in the table of VHH sequences; If the VHH antibody domain of (A) contains the amino acid sequence of VHH6, then the VHH antibody domain of (E) contains the amino acid sequence of one VHH selected from the group consisting of VHH14, VHH16, and VHH18 as shown in the table of VHH sequences; If the VHH antibody domain of (A) contains the amino acid sequence of VHH8, then the VHH antibody domain of (E) contains the amino acid sequence of VHH16 as shown in the table of VHH sequences; If the VHH antibody domain of (A) contains the amino acid sequence of VHH9, then the VHH antibody domain of (E) contains the amino acid sequence of one VHH selected from the group consisting of VHH14, VHH16, and VHH18 as shown in the table of VHH sequences; If the VHH antibody domain of (A) contains the amino acid sequence of VHH10, then the VHH antibody domain of (E) contains the amino acid sequence of one VHH selected from the group consisting of VHH14, VHH15, VHH16, and VHH18 as shown in the table of VHH sequences.

[0517] Therefore, according to this embodiment, the compounds of embodiments 378 to 379 (and embodiments referencing them) may include any combination of VHHα and VHHβ shown in red in Figure 4B.

[0518] Embodiment 399: A compound according to either Embodiment 378 or 379, - Here, the VHH antibody domain of (A) comprises the amino acid sequence of one VHH selected from the group consisting of VHH6 and VHH11 shown in the table of VHH sequences, and, - Here, the VHH antibody domain of (E) comprises an amino acid sequence of one VHH selected from the group consisting of VHH12, VHH13, VHH14, VHH15, VHH16, VHH17, VHH18, VHH20, VHH21, and VHH22 as shown in the table of VHH sequences, wherein the VHH antibody domain of (E) is selected as follows: If the VHH antibody domain of (A) contains the amino acid sequence of VHH6, then the VHH antibody domain of (E) contains the amino acid sequence of one VHH selected from the group consisting of VHH12 and VHH13 as shown in the table of VHH sequences; If the VHH antibody domain of (A) contains the amino acid sequence of VHH11, then the VHH antibody domain of (E) contains one amino acid sequence of VHH selected from the group consisting of VHH12, VHH13, VHH14, VHH15, VHH16, VHH17, VHH18, VHH20, VHH21, and VHH22 as shown in the table of VHH sequences.

[0519] Therefore, according to this embodiment, the compounds of embodiments 378 to 379 (and embodiments referencing them) may include any combination of VHHα and VHHβ shown in black in Figure 4B.

[0520] Aspect 400: A compound according to either aspect 378 or 379, - Here, the VHH antibody domain of (A) comprises the amino acid sequence of one VHH selected from the group consisting of VHH2, VHH6, VHH8 and VHH10 shown in the table of VHH sequences, and - Here, the VHH antibody domain of (E) comprises an amino acid sequence of one VHH selected from the group consisting of VHH12, VHH14, VHH15, VHH17, and VHH22 as shown in the table of VHH sequences, wherein the VHH antibody domain of (E) is selected as follows: If the VHH antibody domain of (A) contains the amino acid sequence of VHH2, then the VHH antibody domain of (E) contains the amino acid sequence of VHH22 as shown in the table of VHH sequences; If the VHH antibody domain of (A) contains the amino acid sequence of VHH6, then the VHH antibody domain of (E) contains the amino acid sequence of VHH15 as shown in the VHH sequence table; If the VHH antibody domain of (A) contains the amino acid sequence of VHH8, then the VHH antibody domain of (E) contains the amino acid sequence of one VHH selected from the group consisting of VHH14, VHH15, and VHH22 as shown in the table of VHH sequences; If the VHH antibody domain of (A) contains the amino acid sequence of VHH10, then the VHH antibody domain of (E) contains the amino acid sequence of one VHH selected from the group consisting of VHH12, VHH17, and VHH22 as shown in the table of VHH sequences.

[0521] Therefore, according to this embodiment, the compounds of embodiments 378-379 (and embodiments referencing them) may include any combination of VHHα and VHHβ shown in orange in Figure 4B (active surrogate agonists that induce less than 50% of NFκB reporter activation normalized to (rh)IL-18 at 1 nM, or have an EC50 greater than 0.1 nM).

[0522] Embodiment 401: A compound according to either Embodiment 378 or 379, wherein the VHH antibody domain of (A) comprises an amino acid sequence of one VHH selected from the group consisting of VHH2 and VHH8 as shown in the VHH sequence table, and wherein the VHH antibody domain of (E) comprises an amino acid sequence of one VHH selected from the group consisting of VHH15 and VHH17 as shown in the VHH sequence table.

[0523] Embodiment 402: A compound according to either Embodiment 378 or 379, - Here, the VHH antibody domain of (A) includes the amino acid sequence of one VHH selected from the group consisting of VHH2 and VHH8 shown in the table of VHH sequences, and - Here, the VHH antibody domain of (E) comprises an amino acid sequence of one VHH selected from the group consisting of VHH15 and VHH17 as shown in the table of VHH sequences, wherein the VHH antibody domain of (E) is selected as follows: If the VHH antibody domain of (A) contains the amino acid sequence of VHH2, then the VHH antibody domain of (E) contains the amino acid sequence of one VHH selected from the group consisting of VHH15 and VHH17 as shown in the table of VHH sequences; If the VHH antibody domain of (A) contains the amino acid sequence of VHH8, then the VHH antibody domain of (E) contains the amino acid sequence of VHH17 as shown in the VHH sequence table.

[0524] Aspect 403: A compound according to either aspect 378 or 379, wherein the VHH antibody domain of (A) comprises the amino acid sequence of VHH2 as shown in the VHH sequence table, and wherein the VHH antibody domain of (E) comprises the amino acid sequence of VHH15 as shown in the VHH sequence table.

[0525] Embodiment 404: A compound according to either Embodiment 378 or 379, - Here, the VHH antibody domain of (A) includes the amino acid sequence of one VHH selected from the group consisting of VHH1, VHH2, VHH5, VHH6, VHH8 and VHH10 as shown in the table of VHH sequences, and - Here, the VHH antibody domain of (E) comprises an amino acid sequence of one VHH selected from the group consisting of VHH13, VHH16, and VHH22 as shown in the table of VHH sequences, wherein the VHH antibody domain of (E) is selected as follows: If the VHH antibody domain of (A) contains the amino acid sequence of VHH1, then the VHH antibody domain of (E) contains the amino acid sequence of VHH16 as shown in the table of VHH sequences; If the VHH antibody domain of (A) contains the amino acid sequence of VHH2, then the VHH antibody domain of (E) contains the amino acid sequence of VHH22 as shown in the table of VHH sequences; If the VHH antibody domain of (A) contains the amino acid sequence of VHH5, then the VHH antibody domain of (E) contains the amino acid sequence of VHH13 as shown in the table of VHH sequences; If the VHH antibody domain of (A) contains the amino acid sequence of VHH6, then the VHH antibody domain of (E) contains the amino acid sequence of VHH22 as shown in the VHH sequence table; If the VHH antibody domain of (A) contains the amino acid sequence of VHH8, then the VHH antibody domain of (E) contains the amino acid sequence of VHH22 as shown in the VHH sequence table; If the VHH antibody domain of (A) contains the amino acid sequence of VHH10, then the VHH antibody domain of (E) contains the amino acid sequence of VHH22 as shown in the VHH sequence table.

[0526] Embodiment 405: A compound according to either Embodiment 378 or 379, - Here, the VHH antibody domain of (A) comprises the amino acid sequence of one VHH selected from the group consisting of VHH2, VHH6, VHH8 and VHH10 shown in the table of VHH sequences, and - Here, the VHH antibody domain of (E) comprises an amino acid sequence of one VHH selected from the group consisting of VHH12, VHH13, VHH15, and VHH18 as shown in the table of VHH sequences, wherein the VHH antibody domain of (E) is selected as follows: If the VHH antibody domain of (A) contains the amino acid sequence of VHH2, then the VHH antibody domain of (E) contains the amino acid sequence of VHH12 as shown in the VHH sequence table; If the VHH antibody domain of (A) contains the amino acid sequence of VHH6, then the VHH antibody domain of (E) contains the amino acid sequence of one VHH selected from the group consisting of VHH15 and VHH12 as shown in the table of VHH sequences; If the VHH antibody domain of (A) contains the amino acid sequence of VHH8, then the VHH antibody domain of (E) contains the amino acid sequence of one VHH selected from the group consisting of VHH13 and VHH18 as shown in the table of VHH sequences; If the VHH antibody domain of (A) contains the amino acid sequence of VHH10, then the VHH antibody domain of (E) contains the amino acid sequence of VHH12 as shown in the VHH sequence table.

[0527] Embodiment 406: A compound according to any one of Embodiments 378-379 or 397-405, wherein in the first binding module, the VHH antibody domain or fragment thereof comprises a VHH antibody domain or fragment thereof according to (A), (B), or (C).

[0528] Where this disclosure states that "in the first binding module, the VHH antibody domain or fragment thereof comprises a VHH antibody domain or fragment thereof by (A), (B), or (C)," it is intended to indicate that, in this embodiment, only (A), (B), and (C) are considered options for the VHH antibody domain (or fragment thereof) of the first binding module, while option (D) is excluded. Where this disclosure states that "in the first binding module, the VHH antibody domain or fragment thereof comprises a VHH antibody domain or fragment thereof by (A), (C), or (D)," it is intended to indicate that, in this embodiment, only (A), (C), and (D) are considered options for the VHH antibody domain (or fragment thereof) of the first binding module, while option (B) is excluded. Where this disclosure states that "in the first binding module, the VHH antibody domain or fragment thereof comprises the VHH antibody domain or fragment thereof by (A)", it is intended to indicate that, in this embodiment, only (A) is considered an option for the VHH antibody domain (or fragment thereof) of the first binding module, while options (B), (C), and (D) are excluded. Further variations of this wording below should be understood to conform to this.

[0529] Embodiment 407: A compound according to any one of Embodiments 378-379 or 397-405, wherein in the first binding module, the VHH antibody domain or fragment thereof comprises a VHH antibody domain or fragment thereof according to (A), (B), or (D).

[0530] Embodiment 408: A compound according to any one of Embodiments 378-379 or 397-405, wherein in the first binding module, the VHH antibody domain or fragment thereof comprises a VHH antibody domain or fragment thereof according to (A), (C), or (D).

[0531] Embodiment 409: A compound according to any one of Embodiments 378-379 or 397-405, wherein in the first binding module, the VHH antibody domain or fragment thereof comprises a VHH antibody domain or fragment thereof according to (A) or (B).

[0532] Embodiment 410: A compound according to any one of Embodiments 378-379 or 397-405, wherein in the first binding module, the VHH antibody domain or fragment thereof comprises a VHH antibody domain or fragment thereof according to (A) or (C).

[0533] Embodiment 411: A compound according to any one of Embodiments 378-379 or 397-405, wherein in the first binding module, the VHH antibody domain or fragment thereof comprises a VHH antibody domain or fragment thereof according to (A) or (D).

[0534] Embodiment 412: A compound according to any one of Embodiments 378-379 or 397-405, wherein in the first binding module, the VHH antibody domain or fragment thereof comprises a VHH antibody domain or fragment thereof according to (A).

[0535] Embodiment 413: A compound according to any one of Embodiments 378-379 or 397-405, wherein in the first binding module, the VHH antibody domain or fragment thereof comprises a VHH antibody domain or fragment thereof according to (B).

[0536] Embodiment 414: A compound according to any one of Embodiments 378-379 or 397-413, wherein in the second binding module, the VHH antibody domain or fragment thereof comprises a VHH antibody domain or fragment thereof according to (E), (F), or (G).

[0537] Embodiment 415: A compound according to any one of Embodiments 378-379 or 397-413, wherein in the second binding module, the VHH antibody domain or fragment thereof comprises a VHH antibody domain or fragment thereof according to (E), (F), or (H).

[0538] Embodiment 416: A compound according to any one of Embodiments 378-379 or 397-413, wherein in the second binding module, the VHH antibody domain or fragment thereof comprises a VHH antibody domain or fragment thereof according to (E), (G), or (H).

[0539] Embodiment 417: A compound according to any one of Embodiments 378-379 or 397-413, wherein in the second binding module, the VHH antibody domain or fragment thereof comprises a VHH antibody domain or fragment thereof according to (E) or (F).

[0540] Embodiment 418: A compound according to any one of Embodiments 378-379 or 397-413, wherein in the second binding module, the VHH antibody domain or fragment thereof comprises a VHH antibody domain or fragment thereof according to (E) or (G).

[0541] Embodiment 419: A compound according to any one of Embodiments 378-379 or 397-413, wherein in the second binding module, the VHH antibody domain or fragment thereof comprises a VHH antibody domain or fragment thereof according to (E) or (H).

[0542] Embodiment 420: A compound according to any one of Embodiments 378-379 or 397-413, wherein in the second binding module, the VHH antibody domain or fragment thereof comprises a VHH antibody domain or fragment thereof according to (E).

[0543] Embodiment 421: A compound according to any one of Embodiments 378-379 or 397-413, wherein in the second binding module, the VHH antibody domain or fragment thereof comprises a VHH antibody domain or fragment thereof according to (F).

[0544] Embodiment 422: A compound according to any one of Embodiments 375 to 421, wherein the compound is a molecule.

[0545] Embodiment 423: A compound according to any one of Embodiments 375 to 422, wherein the compound contains a protein or is a protein.

[0546] By describing a compound as "containing" a protein, this disclosure indicates that the compound includes a protein-containing portion in its chemical structure. A protein-containing compound may or may not contain a non-protein portion.

[0547] Embodiment 424: A compound according to any one of Embodiments 375 to 422, wherein the compound is a protein.

[0548] By describing the compound as "being" a protein, this disclosure indicates that the compound consists solely of protein and contains no non-protein portions.

[0549] Embodiment 425: A compound according to any one of Embodiments 375 to 424, wherein the compound further comprises a targeting moiety.

[0550] As used herein, the term “targeting moiety” means a moiety (i.e., a group of molecules or chemical structure) that associates with the compound and binds to a target site (typically by covalent bond), wherein the binding enables the recruitment of the compound to the target site (e.g., a moiety that specifically binds to EGFR, thereby targeting the compound to cells expressing EGFR on their cell surface). The target site is typically a biomolecule or a specific part of a biomolecule. An example of a targeting moiety is an antibody fragment that binds to an antigen, covalently linked to a VHH antibody domain that binds to IL-18Rα and / or IL-18Rβ, thereby forming the compound according to this disclosure, wherein the antigen-binding fragment binds to a specific receptor present on the surface of a particular cell type (its antigen), and wherein the binding of the antigen-binding fragment to this receptor results in the recruitment of the compound to this cell.

[0551] Non-targeted drugs typically reach their sites of action through systemic distribution and passive diffusion. In contrast, targeted compounds are not uniformly distributed throughout the body. Due to the interaction between the targeted moiety and its target molecule, the molecule containing the targeted moiety is preferentially enriched at its target site. Therefore, for example, a therapeutic compound with a targeted moiety requires a lower dose to be therapeutically effective, thus improving the therapeutic window.

[0552] Embodiment 426: A compound according to Embodiment 425, wherein the targeted portion is a protein, peptide, peptidomimetic, nucleic acid, oligonucleotide, or small molecule.

[0553] As used herein, the term “peptidomimetic” refers to a peptide-like chain designed to mimic a peptide. Examples of peptidomimetics include, but are not limited to, D-peptidomimetics containing D-amino acids. As used herein, “small molecule” refers to a molecule with a molecular weight < 1000 Da.

[0554] Embodiment 427: A compound according to any one of Embodiments 375 to 426, wherein the compound is a single specific molecule.

[0555] Embodiment 428: A compound according to any one of Embodiments 375 to 426, wherein the compound is a bispecific molecule.

[0556] Embodiment 429: A compound according to any one of Embodiments 375 to 426 or 428, wherein the compound is a bispecific antibody molecule.

[0557] The term “bispecific antibody molecule,” as used in this disclosure, refers to an antibody molecule that can simultaneously and specifically bind to two different epitopes. This does not preclude the possibility that the bispecific antibody molecule may be ligated to further domains or portions.

[0558] The terms “epitope” and “antigenicity determinant” are used interchangeably herein and refer to a portion of an antigen that is recognized by a particular antibody and thereby specifically bound to it. When the antigen is a polypeptide, epitopes can be formed from both consecutive amino acids and discontinuous amino acids juxtaposed by the three-dimensional folding of the protein. Epitopes formed from consecutive amino acids are typically retained during protein denaturation, while epitopes formed by three-dimensional folding are typically lost during protein denaturation. Epitopes typically contain at least 3, and more typically at least 5 or 8-10, amino acids in their unique spatial structure.

[0559] Typically, in the compounds of the disclosed herein, one of the two epitopes to which the compound of the disclosed herein binds is an epitope on IL-18Rα, while the other of the two epitopes to which the compound of the disclosed herein binds is an epitope on IL-18Rβ.

[0560] Methods for producing bispecific antibodies are known in the field. For example, bispecific antibodies can be produced by recombination using the co-expression of two immunoglobulin heavy / light chain pairs (see, e.g., Milstein et al., Nature (1983), vol. 305, p. 537-539). Alternatively, bispecific antibodies can be prepared using chemical crosslinking (see, e.g., Brennan et al., Science (1985), vol. 229, p. 81). Bispecific antibodies can also be prepared, for example, by SEED technology (see below).

[0561] Aspect 430: A compound according to any one of Aspects 375-426 or 428-429, wherein one binding site of the bispecific antibody molecule is formed by a VHH antibody domain or fragment thereof according to the Disclosure that is specific to IL-18Rα, and the other binding site of the bispecific antibody is formed by a VHH antibody domain or fragment thereof according to the Disclosure that is specific to IL-18Rβ.

[0562] Embodiment 431: A compound according to any one of Embodiments 375-426 or 428-429, wherein one binding site of the bispecific antibody molecule is a VHH antibody domain or a fragment thereof according to the present disclosure, and the other binding site of the bispecific antibody molecule is selected from the group consisting of Fab, Fab', (Fab')2, Fv, scFv, diabody, and VHH.

[0563] The "Fab" fragment is obtained by papain digestion of the antibody, which produces two identical antigen-binding fragments, referred to as the "Fab" fragment, and the remaining "Fc" fragment (its naming reflects its readily catalyzed ability). The Fab fragment consists of the entire light chain with a variable region domain (VH) of the heavy chain and a first constant domain (CH1) of the heavy chain. Each Fab fragment is monovalent with respect to antigen binding, meaning it has a single antigen-binding site.

[0564] The "F(ab')2" fragment is obtained by pepsin treatment of the antibody, which yields a single large F(ab')2 fragment, which is roughly equivalent to two disulfide-linked Fab fragments with different antigen-binding activities and can still crosslink antigens.

[0565] The "Fab'" fragment differs from the Fab fragment by having several additional residues at the carboxyl terminus of the CH1 domain, containing one or more cysteines from the antibody hinge region. Fab'-SH is a designation for Fab' fragments where the cysteine ​​residue(s) of the constant domain have a free thiol group. The F(ab')2 antibody fragment was originally generated as a pair of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0566] The Fc fragment contains the carboxyl terminals of both H chains held together by a disulfide. The effector function of the antibody is determined by the sequence in the Fc region, which is also recognized by an Fc receptor (FcR) found on specific types of cells.

[0567] "Fv" is the smallest antibody fragment containing both a complete antigen recognition site and an antigen binding site. This fragment consists of a dimer in a robust non-covalent association of one heavy chain variable domain and one light chain variable domain. The folding of these two domains gives rise to six hypervariable loops (three from the H chain and three from the L chain), which contribute amino acid residues to antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of Fv containing only three HVRs specific to the antigen) has the ability to recognize and bind to the antigen, although its affinity is lower than that of the entire binding site.

[0568] "Single-chain Fv" is also abbreviated as "scFv" and is an antibody fragment containing VH and VL antibody domains linked to a single polypeptide chain. Preferably, the scFv polypeptide further contains a polypeptide linker between the VH and VL domains. This allows the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun (Rosenburg and Moore, eds., The Pharmacology of Monoclonal Antibodies, Vol. 113 (1994), Springer-Verlag (New York), pp. 269-315).

[0569] The term "diabody" refers to a small antibody fragment prepared by constructing an scFv fragment (see preceding paragraph) such that a short linker (approximately 5-25 residues) between the VH and VL domains enables interchain pairing of the V domains but not intrachain pairing, thereby resulting in a bivalent fragment, i.e., a fragment with two antigen-binding sites. A bispecific diabody is a heterodimer of two "crossover" scFv fragments, where the VH and VL domains of two antibodies are located on different polypeptide chains. Diabodies are described in more detail, for example, EP 0404097;WO 93 / 11161;Hollinger et al., Proc. Natl. Acad. Sci. USA (1993), vol. 90, p. 6444-6448.

[0570] Embodiment 432: A compound according to any one of Embodiments 375-426 or 430-431, wherein the compound is a multispecific molecule.

[0571] Embodiment 433: A compound according to any one of Embodiments 375-426 or 428-430, wherein the compound is a bispecific molecule that is specific to IL-18Rα and IL-18Rβ.

[0572] Embodiment 434: A compound according to any one of Embodiments 375-426, 428-430, or 433, wherein the compound is a bispecific molecule that binds to IL-18Rα and IL-18Rβ.

[0573] Embodiment 435: A compound according to any one of Embodiments 375-426, 428-430, or 433-434, wherein the compound is a bispecific molecule directed toward IL-18Rα and IL-18Rβ.

[0574] Embodiment 436: A compound according to any one of Embodiments 375-426, 428-430, or 433-435, wherein the compound is a bispecific antibody molecule that is specific to IL-18Rα and IL-18Rβ.

[0575] Where this disclosure refers to “IL-18Rα” and does not specify a particular organism from which IL-18Rα originates, this refers to human IL-18Rα. Where this disclosure refers to “IL-18Rβ” and does not specify a particular organism from which IL-18Rβ originates, this refers to human IL-18Rβ.

[0576] According to Aspect 12, this disclosure relates to any of the following (the same descriptions and definitions as those for Aspects 1-11 of this disclosure and the embodiments relating thereto apply accordingly):

[0577] Embodiment 437: A compound that is a bispecific antibody molecule comprising an IL-18Rα-binding VHH (first binding module) having the amino acid sequence of SEQ ID NO: 1 and an IL18Rβ-binding VHH (second binding module) having the amino acid sequence of SEQ ID NO: 12.

[0578] Embodiment 438: A compound that is a bispecific antibody molecule comprising an IL-18Rα-binding VHH (first binding module) having the amino acid sequence of SEQ ID NO: 1 and an IL18Rβ-binding VHH (second binding module) having the amino acid sequence of SEQ ID NO: 13.

[0579] Embodiment 439: A compound that is a bispecific antibody molecule comprising an IL-18Rα-binding VHH (first binding module) having the amino acid sequence of SEQ ID NO: 1 and an IL18Rβ-binding VHH (second binding module) having the amino acid sequence of SEQ ID NO: 15.

[0580] Embodiment 440: A compound that is a bispecific antibody molecule comprising an IL-18Rα-binding VHH (first binding module) having the amino acid sequence of SEQ ID NO: 1 and an IL18Rβ-binding VHH (second binding module) having the amino acid sequence of SEQ ID NO: 17.

[0581] Embodiment 441: A compound that is a bispecific antibody molecule comprising an IL-18Rα-binding VHH (first binding module) having the amino acid sequence of SEQ ID NO: 1 and an IL18Rβ-binding VHH (second binding module) having the amino acid sequence of SEQ ID NO: 20.

[0582] Embodiment 442: A compound that is a bispecific antibody molecule comprising an IL-18Rα-binding VHH (first binding module) having the amino acid sequence of SEQ ID NO: 1 and an IL18Rβ-binding VHH (second binding module) having the amino acid sequence of SEQ ID NO: 21.

[0583] Embodiment 443: A compound that is a bispecific antibody molecule comprising an IL-18Rα-binding VHH (first binding module) having the amino acid sequence of SEQ ID NO: 1 and an IL18Rβ-binding VHH (second binding module) having the amino acid sequence of SEQ ID NO: 22.

[0584] Embodiment 444: A compound that is a bispecific antibody molecule comprising an IL-18Rα-binding VHH (first binding module) having the amino acid sequence of SEQ ID NO: 2 and an IL18Rβ-binding VHH (second binding module) having the amino acid sequence of SEQ ID NO: 12.

[0585] Embodiment 445: A compound that is a bispecific antibody molecule comprising an IL-18Rα-binding VHH (first binding module) having the amino acid sequence of SEQ ID NO: 2 and an IL18Rβ-binding VHH (second binding module) having the amino acid sequence of SEQ ID NO: 13.

[0586] Embodiment 446: A compound that is a bispecific antibody molecule comprising an IL-18Rα-binding VHH (first binding module) having the amino acid sequence of SEQ ID NO: 2 and an IL18Rβ-binding VHH (second binding module) having the amino acid sequence of SEQ ID NO: 15.

[0587] Embodiment 447: A compound that is a bispecific antibody molecule comprising an IL-18Rα-binding VHH (first binding module) having the amino acid sequence of SEQ ID NO: 2 and an IL18Rβ-binding VHH (second binding module) having the amino acid sequence of SEQ ID NO: 17.

[0588] Embodiment 448: A compound that is a bispecific antibody molecule comprising an IL-18Rα-binding VHH (first binding module) having the amino acid sequence of SEQ ID NO: 2 and an IL18Rβ-binding VHH (second binding module) having the amino acid sequence of SEQ ID NO: 18.

[0589] Embodiment 449: A compound that is a bispecific antibody molecule comprising an IL-18Rα-binding VHH (first binding module) having the amino acid sequence of SEQ ID NO: 2 and an IL18Rβ-binding VHH (second binding module) having the amino acid sequence of SEQ ID NO: 20.

[0590] Embodiment 450: A compound that is a bispecific antibody molecule comprising an IL-18Rα-binding VHH (first binding module) having the amino acid sequence of SEQ ID NO: 2 and an IL18Rβ-binding VHH (second binding module) having the amino acid sequence of SEQ ID NO: 21.

[0591] Embodiment 451: A compound that is a bispecific antibody molecule comprising an IL-18Rα-binding VHH (first binding module) having the amino acid sequence of SEQ ID NO: 5 and an IL18Rβ-binding VHH (second binding module) having the amino acid sequence of SEQ ID NO: 13.

[0592] Embodiment 452: A compound that is a bispecific antibody molecule comprising an IL-18Rα-binding VHH (first binding module) having the amino acid sequence of SEQ ID NO: 5 and an IL18Rβ-binding VHH (second binding module) having the amino acid sequence of SEQ ID NO: 14.

[0593] Embodiment 453: A compound that is a bispecific antibody molecule comprising an IL-18Rα-binding VHH (first binding module) having the amino acid sequence of SEQ ID NO: 6 and an IL18Rβ-binding VHH (second binding module) having the amino acid sequence of SEQ ID NO: 17.

[0594] Embodiment 454: A compound that is a bispecific antibody molecule comprising an IL-18Rα-binding VHH (first binding module) having the amino acid sequence of SEQ ID NO: 6 and an IL18Rβ-binding VHH (second binding module) having the amino acid sequence of SEQ ID NO: 20.

[0595] Embodiment 455: A compound that is a bispecific antibody molecule comprising an IL-18Rα-binding VHH (first binding module) having the amino acid sequence of SEQ ID NO: 6 and an IL18Rβ-binding VHH (second binding module) having the amino acid sequence of SEQ ID NO: 21.

[0596] Embodiment 456: A compound that is a bispecific antibody molecule comprising an IL-18Rα-binding VHH (first binding module) having the amino acid sequence of SEQ ID NO: 6 and an IL18Rβ-binding VHH (second binding module) having the amino acid sequence of SEQ ID NO: 22.

[0597] Embodiment 457: A compound that is a bispecific antibody molecule comprising an IL-18Rα-binding VHH (first binding module) having the amino acid sequence of SEQ ID NO: 8 and an IL18Rβ-binding VHH (second binding module) having the amino acid sequence of SEQ ID NO: 12.

[0598] Embodiment 458: A compound that is a bispecific antibody molecule comprising an IL-18Rα-binding VHH (first binding module) having the amino acid sequence of SEQ ID NO: 8 and an IL18Rβ-binding VHH (second binding module) having the amino acid sequence of SEQ ID NO: 13.

[0599] Embodiment 459: A compound that is a bispecific antibody molecule comprising an IL-18Rα-binding VHH (first binding module) having the amino acid sequence of SEQ ID NO: 8 and an IL18Rβ-binding VHH (second binding module) having the amino acid sequence of SEQ ID NO: 17.

[0600] Embodiment 460: A compound that is a bispecific antibody molecule comprising an IL-18Rα-binding VHH (first binding module) having the amino acid sequence of SEQ ID NO: 8 and an IL18Rβ-binding VHH (second binding module) having the amino acid sequence of SEQ ID NO: 18.

[0601] Embodiment 461: A compound that is a bispecific antibody molecule comprising an IL-18Rα-binding VHH (first binding module) having the amino acid sequence of SEQ ID NO: 8 and an IL18Rβ-binding VHH (second binding module) having the amino acid sequence of SEQ ID NO: 20.

[0602] Embodiment 462: A compound that is a bispecific antibody molecule comprising an IL-18Rα-binding VHH (first binding module) having the amino acid sequence of SEQ ID NO: 8 and an IL18Rβ-binding VHH (second binding module) having the amino acid sequence of SEQ ID NO: 21.

[0603] Embodiment 463: A compound that is a bispecific antibody molecule comprising an IL-18Rα-binding VHH (first binding module) having the amino acid sequence of SEQ ID NO: 9 and an IL18Rβ-binding VHH (second binding module) having the amino acid sequence of SEQ ID NO: 12.

[0604] Embodiment 464: A compound that is a bispecific antibody molecule comprising an IL-18Rα-binding VHH (first binding module) having the amino acid sequence of SEQ ID NO: 9 and an IL18Rβ-binding VHH (second binding module) having the amino acid sequence of SEQ ID NO: 13.

[0605] Embodiment 465: A compound that is a bispecific antibody molecule comprising an IL-18Rα-binding VHH (first binding module) having the amino acid sequence of SEQ ID NO: 9 and an IL18Rβ-binding VHH (second binding module) having the amino acid sequence of SEQ ID NO: 15.

[0606] Embodiment 466: A compound that is a bispecific antibody molecule comprising an IL-18Rα-binding VHH (first binding module) having the amino acid sequence of SEQ ID NO: 9 and an IL18Rβ-binding VHH (second binding module) having the amino acid sequence of SEQ ID NO: 17.

[0607] Embodiment 467: A compound that is a bispecific antibody molecule comprising an IL-18Rα-binding VHH (first binding module) having the amino acid sequence of SEQ ID NO: 9 and an IL18Rβ-binding VHH (second binding module) having the amino acid sequence of SEQ ID NO: 20.

[0608] Embodiment 468: A compound that is a bispecific antibody molecule comprising an IL-18Rα-binding VHH (first binding module) having the amino acid sequence of SEQ ID NO: 9 and an IL18Rβ-binding VHH (second binding module) having the amino acid sequence of SEQ ID NO: 21.

[0609] Embodiment 469: A compound that is a bispecific antibody molecule comprising an IL-18Rα-binding VHH (first binding module) having the amino acid sequence of SEQ ID NO: 9 and an IL18Rβ-binding VHH (second binding module) having the amino acid sequence of SEQ ID NO: 22.

[0610] Embodiment 470: A compound that is a bispecific antibody molecule comprising an IL-18Rα-binding VHH (first binding module) having the amino acid sequence of SEQ ID NO: 10 and an IL18Rβ-binding VHH (second binding module) having the amino acid sequence of SEQ ID NO: 13.

[0611] Embodiment 471: A compound that is a bispecific antibody molecule comprising an IL-18Rα-binding VHH (first binding module) having the amino acid sequence of SEQ ID NO: 10 and an IL-18Rβ-binding VHH (second binding module) having the amino acid sequence of SEQ ID NO: 20.

[0612] Embodiment 472: A compound that is a bispecific antibody molecule comprising an IL-18Rα-binding VHH (first binding module) having the amino acid sequence of SEQ ID NO: 10 and an IL18Rβ-binding VHH (second binding module) having the amino acid sequence of SEQ ID NO: 21.

[0613] Embodiment 473: A compound according to any one of Embodiments 375 or 377-472, wherein the bond to IL-18Rα is monovalent.

[0614] Embodiment 474: A compound according to any one of Embodiments 375 or 377-472, wherein the bond to IL-18Rα is divalent.

[0615] Embodiment 475: A compound according to any one of Embodiments 375 or 377-472, which is polyvalent in its binding to IL-18Rα.

[0616] Aspect 476: A compound according to any one of Aspects 376 to 475, wherein the binding to IL-18Rβ is monovalent.

[0617] Aspect 477: A compound according to any one of Aspects 376 to 475, wherein the binding to IL-18Rβ is divalent.

[0618] Embodiment 478: A compound according to any one of Embodiments 376 to 475, which is polyvalent in its binding to IL-18Rβ.

[0619] Embodiment 479: A compound according to any one of Embodiments 377 to 478, wherein the compound comprises one copy of the first bonding module.

[0620] Embodiment 480: A compound according to any one of Embodiments 377 to 478, wherein the compound comprises two copies of the first bonding module.

[0621] Embodiment 481: A compound according to any one of Embodiments 377 to 478, wherein the compound comprises two or more copies of the first bonding module.

[0622] Embodiment 482: A compound according to any one of Embodiments 377 to 479, which does not contain more than one copy of the first bonding module.

[0623] Embodiment 483: A compound according to any one of Embodiments 377-478 or 480, which does not contain more than two copies of the first bonding module.

[0624] Embodiment 484: A compound according to any one of Embodiments 377 to 483, comprising one copy of the second bonding module.

[0625] Embodiment 485: A compound according to any one of Embodiments 377 to 483, comprising two copies of the second bonding module.

[0626] Embodiment 486: A compound according to any one of Embodiments 377 to 483, comprising two or more copies of the second bonding module.

[0627] Embodiment 487: A compound according to any one of Embodiments 377 to 484, which does not contain one or more copies of the second bonding module.

[0628] Embodiment 488: A compound according to any one of Embodiments 377 to 483 or 485, which does not contain two or more copies of the second bonding module.

[0629] Embodiment 489: A compound according to any one of Embodiments 375 to 488, comprising only one molecular structure that binds to IL-18Rα.

[0630] Aspect 490: A compound according to any one of Aspects 375 to 489, comprising only one molecular structure that binds to IL-18Rβ.

[0631] Embodiment 491: A compound according to any one of Embodiments 375 to 490, wherein all components of the compound are linked by covalent bonds.

[0632] Embodiment 492: A compound according to any one of Embodiments 377 to 491, wherein the first binding module is a VHH antibody domain. As those skilled in the art will understand, by stating that the first binding module "is a VHH antibody domain", the disclosure indicates that the first binding module is a full-length VHH antibody domain and not merely a fragment thereof.

[0633] Embodiment 493: A compound according to any one of Embodiments 377 to 492, wherein the second binding module is a VHH antibody domain.

[0634] Embodiment 494: A compound according to any one of Embodiments 377 to 491, wherein the first binding module consists of a fragment of a VHH antibody domain. As those skilled in the art will understand, by stating that the first binding module "consists of a fragment of a VHH antibody domain", the disclosure indicates that the first binding module is a fragment of a VHH antibody domain and not a full-length VHH antibody domain.

[0635] Embodiment 495: A compound according to any one of Embodiments 377 to 491 or 494, wherein the second binding module consists of a fragment of a VHH antibody domain.

[0636] Embodiment 496: A compound according to any one of Embodiments 375 to 495, wherein the compound includes an antibody Fc region.

[0637] As used herein, the term “antibody Fc region” refers to a portion of a native immunoglobulin formed by the Fc domains of its two heavy chains (which include the constant heavy chain regions 1 (CH1), 2 (CH2), and 3 (CH3) of the immunoglobulin, but not the variable regions of the heavy and light chains of the immunoglobulin, or the constant light chain region 1 (CL1)). The native Fc region is a homodimer. In some embodiments, the term encompasses variant Fc regions that have been modified in one or more ways from the native Fc region. The Fc region may be modified by amino acid substitution, addition and / or deletion, linking of additions, and / or modification of the native glycan. The term encompasses Fc regions in which each of the constituent Fc domains is distinct. Examples of heterodimer Fc regions include, without limitation, Fc regions fabricated using the "KIH (knobs into holes)" technique as described in U.S. Patent No. 8,216,805, or by the SEED technique as described in WO 2016 / 087650.

[0638] Embodiment 497: A compound according to any one of Embodiments 375 to 495, comprising an antibody Fc region that retains function in Fc receptor binding.

[0639] The antibody Fc region "retains function in FC receptor binding" if it can bind to at least one of the Fc receptors (FcγRI, FcγRII, and FcγRIII subclasses; including allele variants and alternatively spliced ​​forms of these receptors).

[0640] Aspect 498: A compound according to any one of Aspects 375 to 495, comprising an antibody Fc region that does not retain function in Fc receptor binding.

[0641] Embodiment 499: A compound according to any one of Embodiments 375 to 495, which does not contain the effector function-retaining antibody Fc region.

[0642] An "effector-competent" Fc region is an Fc region that possesses the functional ability to bind to proteins and / or cells of the immune system and mediate biological effects that are typically induced after the binding of an antibody to a corresponding antigen. Such biological effects include, for example, the ability to bind to complement proteins (e.g., C1q) to result in the activation of the classical complement system, leading to opsonization and lysis of cellular pathogens (complement-dependent cytotoxicity, CDCC). Other biological effects include endocytosis of immune complexes, phagocytosis and destruction of antibody-coated particles or microorganisms (also referred to as antibody-dependent phagocytosis, or ADCP), clearance of immune complexes, lysis of antibody-coated target cells by killer cells (referred to as antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, regulation of immune system cell activation, or control of immunoglobulin production.

[0643] Embodiment 500: A compound according to any one of Embodiments 375 to 497, comprising an effector function-retaining antibody Fc region.

[0644] Embodiment 501: A compound according to any one of Embodiments 375-495 or 498-499, which does not contain an antibody Fc region capable of inducing ADCC (antibody-dependent cytotoxicity).

[0645] Embodiment 502: A compound according to any one of Embodiments 375-497 or 500, comprising an antibody Fc region capable of inducing ADCC.

[0646] Embodiment 503: A compound according to any one of Embodiments 375 to 499, comprising an antibody Fc region that cannot induce ADCC.

[0647] Embodiment 504: A compound according to any one of Embodiments 375 to 503, wherein the antibody Fc region carries the mutation E430G.

[0648] This mutation was described in de Jong et al., 2016, and it has been reported that it drives antibody hexamerization upon binding to target cells.

[0649] Embodiment 505: A compound according to any one of Embodiments 377 to 504, which is a bispecific antibody molecule (i.e., a SEED body) prepared by SEED technology.

[0650] SEED (Chain Exchange Domain) technology is an approach for producing bispecific antibodies in which structurally related sequences within the conserved CH3 domains of human IgA and IgG are exchanged to form two asymmetric but complementary domains (see the Examples section). Further details on SEED technology are described in WO 2016 / 087650 and Davis et al., 2010. Bispecific antibodies prepared by SEED technology are referred to herein as “SEED bodies.”

[0651] Applicable aspect 506: A compound according to any one of Applicable aspects 377 to 505, wherein, The VHH antibody domain or fragment of the first binding module is formed by a sequence selected from the group consisting of SEQ ID NOs: 89, 90, 91, 92, 94, 95, 96, 97, 98, and 99. And here, the VHH antibody domain or fragment of the second binding module is formed by a sequence selected from the group consisting of SEQ ID NOs: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, and 110. The aforementioned compound.

[0652] Sequence IDs 89-99 define the amino acid sequence of the SEED AG chain based on VHH1-VHH11 (α1-β11). Sequence IDs 100-110 define the amino acid sequence of the SEED GA chain based on VHH12-VHH22 (β12-β22).

[0653] Embodiment 507: A compound according to any one of Embodiments 377 to 506, which is a bispecific antibody having a strictly monovalent (1+1) bispecific sdAb (single-domain antibody) architecture.

[0654] Embodiment 508: A compound according to any one of Embodiments 377 to 507, wherein the compound comprises two VHHs of different binding specificities, each of which is present in only one copy in the compound. As those skilled in the art will understand, if the compound is a bispecific antibody comprising a first and a second binding module, typically one VHH forms the first binding module of the compound and the other VHH forms the second binding module of the compound.

[0655] Embodiment 509: A compound according to any one of Embodiments 377-504 or 506-508, which is a single-domain-based IgG (sdIgG).

[0656] Aspect 510: A compound according to Aspect 509, wherein sdIgG is a conventional IgG antibody in which two VH regions are replaced by one binding-specific VHH domain and two VL regions are replaced by VHH domains of different binding-specificities, resulting in a bispecific tetravalent antibody. The single-domain-based IgG (sdIgG) format is described in detail in Pekar et al., 2020.

[0657] Embodiment 511: A compound according to any one of Embodiments 509 to 510, wherein the sdIgG is a conventional IgG antibody, wherein each of the two VH regions is replaced by one copy of the first binding module, and each of the two VL regions is replaced by one copy of the second binding module, resulting in a bispecific tetravalent antibody.

[0658] Embodiment 512: A compound according to Embodiment 511, wherein the first binding module is a VHH transplanted onto the CH1 domain of the heavy chain of IgG1, and the second binding module is a VHH transplanted onto the constant region of the lambda light chain of IgG1.

[0659] Embodiment 513: A compound according to any one of Embodiments 510 to 512, wherein the IgG / IgG1 is human IgG1.

[0660] Embodiment 514: A compound according to any one of Embodiments 377-504 or 507-513, wherein IL-18Rα-specific VHH2 (SEQ ID NO: 2) is transplanted onto the CH1 domain of the heavy chain of IgG1, and IL-18Rβ-specific VHH15 (SEQ ID NO: 15) is transplanted onto the constant region of the lambda light chain of IgG1.

[0661] Embodiment 515: A compound according to any one of Embodiments 377 to 514, wherein the heavy chain of the IgG has its effector function silenced.

[0662] Embodiment 516: A compound according to any one of Embodiments 377 to 504 or 515, wherein the compound comprises an IgG Fc region in which two copies of a tandem VHH configuration are covalently linked to a hinge region, wherein the tandem VHH configuration comprises a first VHH antibody domain covalently linked to a second VHH antibody domain, wherein the second VHH antibody domain is covalently linked to the hinge region of the Fc region, resulting in a bispecific tetravalent compound.

[0663] Embodiment 517: A compound according to Embodiment 516, wherein the IgG Fc region is an IgG1 Fc region in which the effector function is silenced.

[0664] Embodiment 518: A compound according to any one of Embodiments 516 to 517, wherein the first binding module forms the first VHH antibody domain and the second binding module forms the second VHH antibody domain.

[0665] Embodiment 519: A compound according to any one of Embodiments 516 to 518, wherein the first VHH antibody domain and the second VHH antibody domain are separated by a linker.

[0666] Embodiment 520: A compound according to any one of Embodiments 516 to 519, wherein the first VHH antibody domain and the second VHH antibody domain are separated by a 5-amino acid Gly4Ser linker.

[0667] Embodiment 521: A compound according to any one of Embodiments 516 to 520, wherein the first VHH antibody domain is specific to IL-18Rα and the second VHH antibody domain is specific to IL-18Rβ.

[0668] Embodiment 522: A compound according to any one of Embodiments 516 to 521, wherein the first VHH antibody domain has the amino acid sequence of VHH2 (SEQ ID NO: 2), and the second VHH antibody domain has the amino acid sequence of VHH15 (SEQ ID NO: 15).

[0669] Embodiment 523: A compound according to any one of Embodiments 516 to 520, wherein the first VHH antibody domain is specific to IL-18Rβ and the second VHH antibody domain is specific to IL-18Rα.

[0670] Embodiment 524: A compound according to any one of Embodiments 516 to 520 or 523, wherein the first VHH antibody domain has the amino acid sequence of VHH15 (SEQ ID NO: 15) and the second VHH antibody domain has the amino acid sequence of VHH2 (SEQ ID NO: 2).

[0671] Embodiment 525: A compound according to any one of Embodiments 516 to 524, wherein the orientation of the first and second VHH antibody domains is such that the second VHH antibody domain follows the first VHH antibody domain (from N-terminus to C-terminus).

[0672] Embodiment 526: A compound according to any one of Embodiments 516 to 525, wherein the orientation of the first and second VHH antibody domains is such that the first VHH antibody domain follows the second VHH antibody domain (from N-terminus to C-terminus).

[0673] Embodiment 527: A compound according to any one of Embodiments 375 to 526, wherein the compound is IL-18 mimetic.

[0674] As used herein, the term “mimetic” refers to a compound that mimics a particular aspect of the function of another compound. Provided herein is the IL-18 mimetic. IL-18 is a pro-inflammatory cytokine belonging to the IL-1 family of cytokines, which mediates signal transduction through heterodimerization of receptor subunits IL-18Rα and IL-18Rβ (Yasuda et al., 2019; Dinarello et al., 2013). IL-18 stimulates IFN-γ production in synergy with IL-12 in innate lymphoid cells and antigen-experienced T cells. The aspect of IL-18 function that such an IL-18 mimetic mimics is the induction of IL-18R heterodimerization (from IL-18Rα and IL-18Rβ) in the presence of IL-12 (or IL-15). Without being constrained by theory, it is hypothesized that by binding to both IL-18Rα and IL-18Rβ, the IL-18 mimetic of this disclosure bridges IL-18Rα and IL-18Rβ, thereby resulting in the dimerization and activation of IL-18R in the presence of IL-12 (or IL-15). Thus, the IL-18 mimetic of this disclosure mimics the biological function of IL-18, such as the function by which IL-18 induces IFN-γ release (in the presence of IL-12 or IL-15).

[0675] Embodiment 528: A compound according to Embodiment 527, wherein the IL-18 mimetic is unaffected by inhibition by IL-18BP (IL-18 binding protein).

[0676] Embodiment 529: A compound according to any one of Embodiments 375 to 528, wherein the compound is an IL-18 receptor (IL-18R) agonist.

[0677] Embodiment 530: A compound according to any one of Embodiments 375 to 529, wherein the compound is a cytokine mimetic targeting IL-18Rα and IL-18Rβ.

[0678] Embodiment 531: A compound according to any one of Embodiments 375 to 530, wherein the compound is bound to IL-18Rα.

[0679] Embodiment 532: A compound according to any one of Embodiments 375 to 531, wherein the compound specifically binds to IL-18Rα.

[0680] Embodiment 533: A compound according to any one of Embodiments 375 to 532, wherein the compound can bind to IL-18Rα with an affinity at least equivalent to that of VHH1 (SEQ ID NO: 1), VHH2 (SEQ ID NO: 2), VHH3 (SEQ ID NO: 3), VHH4 (SEQ ID NO: 4), VHH5 (SEQ ID NO: 5), VHH6 (SEQ ID NO: 6), VHH8 (SEQ ID NO: 8), VHH9 (SEQ ID NO: 9), or VHH10 (SEQ ID NO: 10) that binds to IL-18Rα (or has a stronger affinity).

[0681] Embodiment 534: A compound according to any one of Embodiments 375 to 533, wherein the compound is bound to IL-18Rβ.

[0682] Embodiment 535: A compound according to any one of Embodiments 375 to 534, wherein the compound specifically binds to IL-18Rβ.

[0683] Embodiment 536: A compound according to any one of Embodiments 375 to 535, wherein the compound can bind to IL-18Rβ with an affinity at least equivalent to the affinity of VHH12 (SEQ ID NO: 12), VHH13 (SEQ ID NO: 13), VHH14 (SEQ ID NO: 14), VHH15 (SEQ ID NO: 15), VHH16 (SEQ ID NO: 16), VHH17 (SEQ ID NO: 17), VHH18 (SEQ ID NO: 18), VHH20 (SEQ ID NO: 20), VHH21 (SEQ ID NO: 21), or VHH22 (SEQ ID NO: 22) that binds to IL-18Rβ (or has a stronger affinity).

[0684] Embodiment 537: A compound according to any one of Embodiments 375 to 536, which binds to IL-18Rα and IL-18Rβ.

[0685] Embodiment 538: A compound according to any one of Embodiments 375 to 537 that specifically binds to IL-18Rα and IL-18Rβ.

[0686] Embodiment 539: A compound according to any one of Embodiments 375 to 538 that can crosslink IL-18Rα and IL-18Rβ.

[0687] Embodiment 540: A compound according to any one of Embodiments 375 to 539 that can induce heterodimer formation between IL-18Rα and IL-18Rβ. Such binding / crosslinking / heterodimer formation can be determined by in vitro binding experiments (by bilayer interference) as described in Example 1 below.

[0688] Aspect 541: A compound according to any one of Aspects 375 to 540, which binds to IL-18RαECD with a KD of 100 nM or stronger.

[0689] Embodiment 542: A compound according to any one of Embodiments 375 to 540, which is bound to IL-18RαECD with a KD of 50 nM or stronger.

[0690] Embodiment 543: A compound according to any one of Embodiments 375 to 540, which is bound to IL-18RαECD with a KD of 25 nM or stronger.

[0691] Aspect 544: A compound according to any one of Aspects 375 to 540, which is bound to IL-18RαECD with a KD of 10 nM or stronger.

[0692] Aspect 545: A compound according to any one of Aspects 375 to 540, which is bound to IL-18RαECD with 1 nM or stronger KD.

[0693] Aspect 546: A compound according to any one of Aspects 375 to 540, which binds to IL-18RαECD with a KD of 500 pM or stronger.

[0694] Aspect 547: A compound according to any one of Aspects 375 to 540, which binds to IL-18RαECD with a KD of 100 pM or stronger.

[0695] Aspect 548: A compound according to any one of Aspects 375 to 540, which binds to IL-18RαECD with a KD of 10 pM or stronger.

[0696] Aspect 549: A compound according to any one of Aspects 375 to 548, which binds to IL-18RβECD with a KD of 100 nM or stronger.

[0697] Embodiment 550: A compound according to any one of Embodiments 375 to 548, which binds to IL-18RβECD with a KD of 50 nM or stronger.

[0698] Embodiment 551: A compound according to any one of Embodiments 375 to 548, which binds to IL-18RβECD with a KD of 25 nM or stronger.

[0699] Embodiment 552: A compound according to any one of Embodiments 375 to 548, which binds to IL-18RβECD with a KD of 10 nM or stronger.

[0700] Aspect 553: A compound according to any one of Aspects 375 to 548, which binds to IL-18RβECD with 1 nM or stronger KD.

[0701] Aspect 554: A compound according to any one of Aspects 375 to 548, which binds to IL-18RβECD with a KD of 500 pM or stronger.

[0702] Aspect 555: A compound according to any one of Aspects 375 to 548, which binds to IL-18RβECD with a KD of 100 pM or stronger.

[0703] Aspect 556: A compound according to any one of Aspects 375 to 548, which binds to IL-18RβECD with a KD of 10 pM or stronger.

[0704] Embodiment 557: A compound according to any one of Embodiments 375 to 556 that exhibits competitive binding with recombinant human IL-18 with respect to targeting IL-18Rα.

[0705] Embodiment 558: A compound according to any one of Embodiments 375 to 557 that exhibits competitive binding to recombinant human IL-18 with respect to targeting IL-18Rβ.

[0706] Embodiment 559: A compound according to any one of Embodiments 375 to 558 that can activate the IL-18 receptor IL-18R.

[0707] Embodiment 560: A compound according to any one of Embodiments 375 to 559, which can activate the IL-18 receptor IL-18R by binding to IL-18Rα and IL-18Rβ.

[0708] Embodiment 561: A compound according to any one of Embodiments 375 to 560, which can activate the IL-18 receptor IL-18R by crosslinking the IL-18 receptor subunits IL-18Rα and IL-18Rβ.

[0709] Embodiment 562: A compound according to any one of Embodiments 375 to 558 that does not activate the IL-18 receptor IL-18R.

[0710] Embodiment 563: A compound according to any one of Embodiments 375 to 562 that induces dose-dependent downstream signaling of IL-18R on IL-18 reporter cells. Whether IL-18R is activated and whether the compound can induce dose-dependent downstream signaling of IL-18R on IL-18 reporter cells can be determined, for example, as described in the section "Human IL-18 HEK Reporter Assay" in Example 1.

[0711] Embodiment 564: A compound according to any one of Embodiments 375 to 563, wherein the compound induces NFκB activation at a concentration of 50 nM that is at least 1.5 times higher than that of TNF-α at a concentration of 50 nM.

[0712] Embodiment 565: IL-18R reporter activation with lower efficacy compared to IL-18 (EC of NFκB reporter activation) 50 A compound according to any one of embodiments 375 to 564, which is determined from the above.

[0713] Aspect 566: A compound according to any one of Aspects 375 to 565 that exhibits a lower level of IL-18 activation (determined by maximum NFκB activation) compared to IL-18.

[0714] Whether the compound exhibits higher-intensity IL-18R reporter activation compared to IL-18 (EC of NFκB reporter activation) 50 Whether or not it exhibits a lower level of IL-18 activation compared to IL-18 (as determined by maximum NFκB activation) can be determined based on the assay as described in the sections of Example 3 and Example 1, “Human IL-18 HEK Reporter Assay.”

[0715] Embodiment 567: A compound according to any one of Embodiments 375 to 566, wherein IL-18 is recombinant human IL-18.

[0716] Embodiment 568: A compound according to any one of Embodiments 375 to 567, wherein the compound induces normalized NFκB reporter activation at a concentration of 1 nM, which is greater than 50% relative to the NFκB reporter activation induced by recombinant human IL18 at a concentration of 1 nM, or the compound has an EC of less than 0.1 nM relative to the NFκB reporter activation. 50 The compound having the above-mentioned properties.

[0717] Aspect 569: A compound according to any one of aspects 375 to 568 that induces normalized NFκB reporter activation at a concentration of 1 nM, which is higher than 50% relative to NFκB reporter activation induced by recombinant human IL18 at a concentration of 1 nM.

[0718] Apparatus 570: NFκB reporter activation with EC less than 0.1 nM 50 A compound having any one of embodiments 375 to 569.

[0719] Embodiment 571: A compound according to any one of Embodiments 375 to 567, wherein the compound induces normalized NFκB reporter activation of less than 50% relative to NFκB reporter activation induced by recombinant human IL18 at a concentration of 1 nM, or wherein the compound has an EC higher than 0.1 nM with respect to NFκB reporter activation. 50 The compound having the above-mentioned properties.

[0720] Aspect 572: A compound according to any one of aspects 375-567 or 571, which induces normalized NFκB reporter activation of less than 50% relative to NFκB reporter activation induced by recombinant human IL18 at a concentration of 1 nM, at a concentration of 1 nM.

[0721] Apparatus 573: Activation of NFκB reporter with EC higher than 0.1 nM 50 A compound having any one of embodiments 375-567 or 571-572.

[0722] Whether the compound induces normalized NFκB reporter activation at a concentration of 1 nM, which is greater than or less than 50% of the NFκB reporter activation induced by recombinant human IL18 at a concentration of 1 nM, and / or whether the EC is greater than or less than 0.1 nM for NFκB reporter activation. 50 Whether or not it has can be determined based on the assay as described in the sections of Example 3 and Example 1, “Human IL-18 HEK Reporter Assay”.

[0723] Embodiment 574: A compound according to any one of Embodiments 375 to 573 that can induce IFN-γ release by peripheral blood mononuclear cells (PBMCs) in the presence of a low dose of IL-12.

[0724] Embodiment 575: The compound according to Embodiment 574, wherein the low dose of IL-12 is 10 ng / ml of IL-12.

[0725] Embodiment 576: A compound according to any one of Embodiments 375 to 575, wherein the IL-12 is recombinant human IL-12. Details of a method for determining whether a compound can induce IFN-γ release by peripheral blood mononuclear cells (PBMCs) in the presence of a low dose of IL-12 are described in Example 1, Section “IFN-γ Release Assay”.

[0726] Embodiment 577: A compound according to any one of Embodiments 375 to 576, having an EC50 of less than 150 nM for IFN-γ release from human PBMCs.

[0727] Embodiment 578: A compound according to any one of Embodiments 375 to 576, having an EC50 of less than 100 nM for the release of IFN-γ from human PBMCs.

[0728] Aspect 579: A compound according to any one of Aspects 375 to 576, having an EC50 of less than 50 nM for IFN-γ release from human PBMCs.

[0729] Embodiment 580: A compound according to any one of Embodiments 375 to 576, having an EC50 of less than 20 nM for the release of IFN-γ from human PBMCs.

[0730] Aspect 581: A compound according to any one of Aspects 375 to 576, having an EC50 of less than 150 pM for IFN-γ release from human PBMCs.

[0731] Embodiment 582: A compound according to any one of Embodiments 375 to 576, having an EC50 of less than 100 pM for IFN-γ release from human PBMCs.

[0732] Embodiment 583: A compound according to any one of Embodiments 375 to 576, having an EC50 of less than 50 pM for IFN-γ release from human PBMCs.

[0733] Embodiment 584: A compound according to any one of Embodiments 375 to 576, having an EC50 of less than 20 pM for IFN-γ release from human PBMCs.

[0734] Embodiment 585: The ability of a compound to induce IFN-γ release on PBMCs (EC 50 A compound according to any one of embodiments 375 to 584, wherein the EC of the compound (as measured by) is increased compared to recombinant human IL-18. As those skilled in the art will understand, this is the EC of the compound for inducing IFN-γ release on PBMCs. 50 However, this means it is smaller than that of recombinant human IL-18.

[0735] Embodiment 586: The EC50 of the compound for the release of IFN-γ from human PBMCs is equal to the EC50 of (rh)IL-18 for the release of IFN-γ from human PBMCs. 50 A compound according to any one of embodiments 375 to 585, which is less than 50% of the above. As used herein, "rh" in relation to molecules is an abbreviation for "recombinant human."

[0736] Embodiment 587: The EC50 of the compound for the release of IFN-γ from human PBMCs is equal to the EC50 of (rh)IL-18 for the release of IFN-γ from human PBMCs. 50 A compound according to any one of embodiments 375 to 585, which is less than 200% of the above.

[0737] Aspect 588: The EC 50A compound according to any one of embodiments 565 to 587, measured in the presence of a low dose of (rh)IL-12.

[0738] Embodiment 589: A compound according to any one of Embodiments 375 to 588 that causes the release of IFN-γ, which is at least 200% of the IFN-γ release induced by (rh)IL-18.

[0739] Embodiment 590: A compound according to any one of Embodiments 375 to 588 that causes the release of IFN-γ, which is at least 150% of the IFN-γ release induced by (rh)IL-18.

[0740] Embodiment 591: A compound according to any one of Embodiments 375 to 588 that causes the release of IFN-γ which is at least 100% of the IFN-γ release induced by (rh)IL-18.

[0741] Embodiment 592: A compound according to any one of Embodiments 375 to 588 that causes the release of IFN-γ, which is at least 80% of the IFN-γ release induced by (rh)IL-18.

[0742] Embodiment 593: A compound according to any one of Embodiments 375 to 588 that causes the release of IFN-γ, which is at least 50% of the IFN-γ release induced by (rh)IL-18.

[0743] Embodiment 594: A compound according to any one of Embodiments 375 to 588 that causes the release of IFN-γ, which is at least 10% of the IFN-γ release induced by (rh)IL-18.

[0744] Embodiment 595: A compound according to any one of Embodiments 574 to 594, wherein the IFN-γ release is measured in the presence of a low dose of recombinant human IL-12 (10 ng / ml) upon stimulation of human peripheral blood mononuclear cells (PBMCs) with the compound (corresponding to IL-18 (resp. with IL-18)).

[0745] Embodiment 596: The compound according to Embodiment 595, wherein the compound (corresponding to IL-18) is used at a concentration of 1 nM.

[0746] Embodiment 597: A compound according to any one of Embodiments 574 to 596, wherein the release of IFN-γ is equivalent to the release of IFN-γ at a concentration of 1 nM of IL-18.

[0747] Embodiment 598: A compound according to any one of Embodiments 574 to 595, wherein the release of IFN-γ is the maximum release of IFN-γ. Further details about the assay are provided in the examples section, in particular in Example 1, section “IFN-γ release assay”.

[0748] Aspect 599: A compound according to any one of aspects 375 to 598, which is not affected by the presence of IL-18 binding protein (IL-18BP).

[0749] Embodiment 600: A compound according to any one of Embodiments 375 to 599 that does not bind to recombinant human IL-18 binding protein (IL-18BP).

[0750] Embodiment 601: A compound according to any one of Embodiments 375 to 600, wherein the binding of the compound to IL-18Rα and IL-18Rβ is not affected by the presence of IL-18 binding protein (IL-18BP).

[0751] Embodiment 602: A compound according to any one of Embodiments 375 to 601, wherein the activation of IL-18R by the compound is not affected by the presence of IL-18 binding protein (IL-18BP).

[0752] Embodiment 603: A compound according to any one of Embodiments 375 to 602, wherein, under conditions in which the ability of IL-18 to induce IFN-γ release is inhibited by the ability of IL-18BP, the compound's ability to induce IFN-γ release is not affected by the ability of IL-18BP.

[0753] Aspect 604: A compound according to any one of Aspects 375 to 603, wherein, under conditions in which the EC50 of IL-18 for the release of IFN-γ from human PBMCs increases in the presence of (rh)IL-18BP (compared to the absence of (rh)IL-18BP), the EC50 of the compound for the release of IFN-γ from human PBMCs does not increase in the presence of (rh)IL-18BP (compared to the absence of (rh)IL-18BP).

[0754] Embodiment 605: A VHH antibody domain or fragment thereof according to any one of Embodiments 1 to 374 or a compound according to any one of Embodiments 375 to 604, wherein the binding is determined by bilayer interference at 25°C and 1000 rpm in KB buffer (PBS + 0.1% Tween-20 + 1% BSA).

[0755] Embodiment 606: The competition is determined by bilayer interference at 25°C and 1000 rpm in KB buffer (PBS + 0.1% Tween-20 + 1% BSA) by any one of Embodiments 1 to 374, wherein the VHH antibody domain or a fragment thereof or a compound according to any one of Embodiments 375 to 604.

[0756] Embodiment 607: A VHH antibody domain or fragment thereof according to any one of Embodiments 1 to 374 or a compound according to any one of Embodiments 375 to 604, wherein the crosslinking / heterodimerization is determined by bilayer interference at 25°C and 1000 rpm in KB buffer (PBS + 0.1% Tween-20 + 1% BSA).

[0757] Embodiment 608: A VHH antibody domain or fragment thereof according to any one of Embodiments 1 to 374, or a compound according to any one of Embodiments 375 to 604, wherein the KD value is measured by kinetic measurement by bilayer interference in KB buffer (PBS + 0.1% Tween-20 + 1% BSA) at 25°C and 1000 rpm.

[0758] According to Aspect 13, the Disclosure relates to any of the following (the same descriptions and definitions as those for Aspects 1-12 of the Disclosure and those for which it refers apply accordingly):

[0759] Apparatus 609: A bispecific antibody molecule prepared by SEED (chain exchange operation domain) technology, wherein: -AG chain linked to VHH bound to IL-18Rα, where the AG chain linked to the VHH bound to IL-18Rα has the amino acid sequence of SEQ ID NO: 89 ("VHH-SEED-AG-1"), and - A GA chain linked to VHH bound to IL18Rβ, wherein the GA chain linked to VHH bound to IL18Rβ has the amino acid sequence of SEQ ID NO: 100 ("VHH-SEED-GA-1"). The molecule containing the above.

[0760] Apparatus 610: A bispecific antibody molecule prepared by SEED (chain exchange operation domain) technology, wherein: -AG chain linked to VHH bound to IL-18Rα, where the AG chain linked to the VHH bound to IL-18Rα has the amino acid sequence of SEQ ID NO: 89 ("VHH-SEED-AG-1"), and - A GA chain linked to VHH bound to IL18Rβ, wherein the GA chain linked to VHH bound to IL18Rβ has the amino acid sequence of SEQ ID NO: 101 ("VHH-SEED-GA-2"). The molecule containing the above.

[0761] Apparatus 611: A bispecific antibody molecule prepared by SEED (chain exchange operation domain) technology, wherein: -AG chain linked to VHH bound to IL-18Rα, where the AG chain linked to the VHH bound to IL-18Rα has the amino acid sequence of SEQ ID NO: 89 ("VHH-SEED-AG-1"), and - A GA chain linked to VHH bound to IL18Rβ, wherein the GA chain linked to VHH bound to IL18Rβ has the amino acid sequence of SEQ ID NO: 103 ("VHH-SEED-GA-4"). The molecule containing the above.

[0762] Apparatus 612: A bispecific antibody molecule prepared by SEED (chain exchange operation domain) technology, wherein: -AG chain linked to VHH bound to IL-18Rα, where the AG chain linked to the VHH bound to IL-18Rα has the amino acid sequence of SEQ ID NO: 89 ("VHH-SEED-AG-1"), and - A GA chain linked to VHH bound to IL18Rβ, wherein the GA chain linked to VHH bound to IL18Rβ has the amino acid sequence of SEQ ID NO: 105 ("VHH-SEED-GA-6"). The molecule containing the above.

[0763] Apparatus 613: A bispecific antibody molecule prepared by SEED (chain exchange operation domain) technology, wherein: -AG chain linked to VHH bound to IL-18Rα, where the AG chain linked to the VHH bound to IL-18Rα has the amino acid sequence of SEQ ID NO: 89 ("VHH-SEED-AG-1"), and - A GA chain linked to VHH bound to IL18Rβ, wherein the GA chain linked to VHH bound to IL18Rβ has the amino acid sequence of SEQ ID NO: 108 ("VHH-SEED-GA-9"). The molecule containing the above.

[0764] Apparatus 614: A bispecific antibody molecule prepared by SEED (chain exchange operation domain) technology, wherein: -AG chain linked to VHH bound to IL-18Rα, where the AG chain linked to the VHH bound to IL-18Rα has the amino acid sequence of SEQ ID NO: 89 ("VHH-SEED-AG-1"), and - A GA chain linked to VHH bound to IL18Rβ, wherein the GA chain linked to VHH bound to IL18Rβ has the amino acid sequence of SEQ ID NO: 109 ("VHH-SEED-GA-10"). The molecule containing the above.

[0765] Apparatus 615: A bispecific antibody molecule prepared by SEED (chain exchange operation domain) technology, wherein: -AG chain linked to VHH bound to IL-18Rα, where the AG chain linked to the VHH bound to IL-18Rα has the amino acid sequence of SEQ ID NO: 89 ("VHH-SEED-AG-1"), and - A GA chain linked to VHH bound to IL18Rβ, wherein the GA chain linked to VHH bound to IL18Rβ has the amino acid sequence of SEQ ID NO: 100 ("VHH-SEED-GA-11"). The molecule containing the above.

[0766] Apparatus 616: A bispecific antibody molecule prepared by SEED (chain exchange operation domain) technology, wherein: -AG chain linked to VHH bound to IL-18Rα, where the AG chain linked to the VHH bound to IL-18Rα has the amino acid sequence of SEQ ID NO: 90 ("VHH-SEED-AG-2"), and - A GA chain linked to VHH bound to IL18Rβ, wherein the GA chain linked to VHH bound to IL18Rβ has the amino acid sequence of SEQ ID NO: 100 ("VHH-SEED-GA-1"). The molecule containing the above.

[0767] Apparatus 617: A bispecific antibody molecule prepared by SEED (chain exchange operation domain) technology, wherein: -AG chain linked to VHH bound to IL-18Rα, where the AG chain linked to the VHH bound to IL-18Rα has the amino acid sequence of SEQ ID NO: 90 ("VHH-SEED-AG-2"), and - A GA chain linked to VHH bound to IL18Rβ, wherein the GA chain linked to VHH bound to IL18Rβ has the amino acid sequence of SEQ ID NO: 101 ("VHH-SEED-GA-2"). The molecule containing the above.

[0768] Apparatus 618: A bispecific antibody molecule prepared by SEED (chain exchange operation domain) technology, wherein: -AG chain linked to VHH bound to IL-18Rα, where the AG chain linked to the VHH bound to IL-18Rα has the amino acid sequence of SEQ ID NO: 90 ("VHH-SEED-AG-2"), and - A GA chain linked to VHH bound to IL18Rβ, wherein the GA chain linked to VHH bound to IL18Rβ has the amino acid sequence of SEQ ID NO: 103 ("VHH-SEED-GA-4"). The molecule containing the above.

[0769] Apparatus 619: A bispecific antibody molecule prepared by SEED (chain exchange operation domain) technology, wherein: -AG chain linked to VHH bound to IL-18Rα, where the AG chain linked to the VHH bound to IL-18Rα has the amino acid sequence of SEQ ID NO: 90 ("VHH-SEED-AG-2"), and - A GA chain linked to VHH bound to IL18Rβ, wherein the GA chain linked to VHH bound to IL18Rβ has the amino acid sequence of SEQ ID NO: 105 ("VHH-SEED-GA-6"). The molecule containing the above.

[0770] Apparatus 620: A bispecific antibody molecule prepared by SEED (chain exchange operation domain) technology, wherein: -AG chain linked to VHH bound to IL-18Rα, where the AG chain linked to the VHH bound to IL-18Rα has the amino acid sequence of SEQ ID NO: 90 ("VHH-SEED-AG-2"), and - A GA chain linked to VHH bound to IL18Rβ, wherein the GA chain linked to VHH bound to IL18Rβ has the amino acid sequence of SEQ ID NO: 106 ("VHH-SEED-GA-7"). The molecule containing the above.

[0771] Apparatus 621: A bispecific antibody molecule prepared by SEED (chain exchange operation domain) technology, wherein: -AG chain linked to VHH bound to IL-18Rα, where the AG chain linked to the VHH bound to IL-18Rα has the amino acid sequence of SEQ ID NO: 90 ("VHH-SEED-AG-2"), and - A GA chain linked to VHH bound to IL18Rβ, wherein the GA chain linked to VHH bound to IL18Rβ has the amino acid sequence of SEQ ID NO: 108 ("VHH-SEED-GA-9"). The molecule containing the above.

[0772] Apparatus 622: A bispecific antibody molecule prepared by SEED (chain exchange operation domain) technology, wherein: -AG chain linked to VHH bound to IL-18Rα, where the AG chain linked to the VHH bound to IL-18Rα has the amino acid sequence of SEQ ID NO: 90 ("VHH-SEED-AG-2"), and - A GA chain linked to VHH bound to IL18Rβ, wherein the GA chain linked to VHH bound to IL18Rβ has the amino acid sequence of SEQ ID NO: 109 ("VHH-SEED-GA-10"). The molecule containing the above.

[0773] Apparatus 623: A bispecific antibody molecule prepared by SEED (chain exchange operation domain) technology, wherein: -AG chain linked to VHH bound to IL-18Rα, where the AG chain linked to the VHH bound to IL-18Rα has the amino acid sequence of SEQ ID NO: 93 ("VHH-SEED-AG-5"), and - A GA chain linked to VHH bound to IL18Rβ, wherein the GA chain linked to VHH bound to IL18Rβ has the amino acid sequence of SEQ ID NO: 101 ("VHH-SEED-GA-2"). The molecule containing the above.

[0774] Apparatus 624: A bispecific antibody molecule prepared by SEED (chain exchange operation domain) technology, wherein: -AG chain linked to VHH bound to IL-18Rα, where the AG chain linked to the VHH bound to IL-18Rα has the amino acid sequence of SEQ ID NO: 93 ("VHH-SEED-AG-5"), and - A GA chain linked to VHH bound to IL18Rβ, wherein the GA chain linked to VHH bound to IL18Rβ has the amino acid sequence of SEQ ID NO: 102 ("VHH-SEED-GA-3"). The molecule containing the above.

[0775] Apparatus 625: A bispecific antibody molecule prepared by SEED (chain exchange operation domain) technology, wherein: -AG chain linked to VHH bound to IL-18Rα, where the AG chain linked to VHH bound to IL-18Rα has the amino acid sequence of SEQ ID NO: 94 ("VHH-SEED-AG-6"), and - A GA chain linked to VHH bound to IL18Rβ, wherein the GA chain linked to VHH bound to IL18Rβ has the amino acid sequence of SEQ ID NO: 105 ("VHH-SEED-GA-6"). The molecule containing the above.

[0776] Apparatus 626: A bispecific antibody molecule prepared by SEED (chain exchange operation domain) technology, wherein: -AG chain linked to VHH bound to IL-18Rα, where the AG chain linked to VHH bound to IL-18Rα has the amino acid sequence of SEQ ID NO: 94 ("VHH-SEED-AG-6"), and - A GA chain linked to VHH bound to IL18Rβ, wherein the GA chain linked to VHH bound to IL18Rβ has the amino acid sequence of SEQ ID NO: 108 ("VHH-SEED-GA-9"). The molecule containing the above.

[0777] Apparatus 627: A bispecific antibody molecule prepared by SEED (chain exchange operation domain) technology, wherein: -AG chain linked to VHH bound to IL-18Rα, where the AG chain linked to VHH bound to IL-18Rα has the amino acid sequence of SEQ ID NO: 94 ("VHH-SEED-AG-6"), and - A GA chain linked to VHH bound to IL18Rβ, wherein the GA chain linked to VHH bound to IL18Rβ has the amino acid sequence of SEQ ID NO: 109 ("VHH-SEED-GA-10"). The molecule containing the above.

[0778] Apparatus 628: A bispecific antibody molecule prepared by SEED (chain exchange operation domain) technology, wherein: -AG chain linked to VHH bound to IL-18Rα, where the AG chain linked to VHH bound to IL-18Rα has the amino acid sequence of SEQ ID NO: 94 ("VHH-SEED-AG-6"), and - A GA chain linked to VHH bound to IL18Rβ, wherein the GA chain linked to VHH bound to IL18Rβ has the amino acid sequence of SEQ ID NO: 100 ("VHH-SEED-GA-11"). The molecule containing the above.

[0779] Apparatus 629: A bispecific antibody molecule prepared by SEED (chain exchange operation domain) technology, wherein: -AG chain linked to VHH bound to IL-18Rα, where the AG chain linked to the VHH bound to IL-18Rα has the amino acid sequence of SEQ ID NO: 96 ("VHH-SEED-AG-8"), and - A GA chain linked to VHH bound to IL18Rβ, wherein the GA chain linked to VHH bound to IL18Rβ has the amino acid sequence of SEQ ID NO: 100 ("VHH-SEED-GA-1"). The molecule containing the above.

[0780] Apparatus 630: A bispecific antibody molecule prepared by SEED (chain exchange operation domain) technology, wherein: -AG chain linked to VHH bound to IL-18Rα, where the AG chain linked to the VHH bound to IL-18Rα has the amino acid sequence of SEQ ID NO: 96 ("VHH-SEED-AG-8"), and - A GA chain linked to VHH bound to IL18Rβ, wherein the GA chain linked to VHH bound to IL18Rβ has the amino acid sequence of SEQ ID NO: 101 ("VHH-SEED-GA-2"). The molecule containing the above.

[0781] Apparatus 631: A bispecific antibody molecule prepared by SEED (chain exchange operation domain) technology, wherein: -AG chain linked to VHH bound to IL-18Rα, where the AG chain linked to the VHH bound to IL-18Rα has the amino acid sequence of SEQ ID NO: 96 ("VHH-SEED-AG-8"), and - A GA chain linked to VHH bound to IL18Rβ, wherein the GA chain linked to VHH bound to IL18Rβ has the amino acid sequence of SEQ ID NO: 105 ("VHH-SEED-GA-6"). The molecule containing the above.

[0782] Apparatus 632: A bispecific antibody molecule prepared by SEED (chain exchange operation domain) technology, wherein: -AG chain linked to VHH bound to IL-18Rα, where the AG chain linked to the VHH bound to IL-18Rα has the amino acid sequence of SEQ ID NO: 96 ("VHH-SEED-AG-8"), and - A GA chain linked to VHH bound to IL18Rβ, wherein the GA chain linked to VHH bound to IL18Rβ has the amino acid sequence of SEQ ID NO: 106 ("VHH-SEED-GA-7"). The molecule containing the above.

[0783] Apparatus 633: A bispecific antibody molecule prepared by SEED (chain exchange operation domain) technology, wherein: -AG chain linked to VHH bound to IL-18Rα, where the AG chain linked to the VHH bound to IL-18Rα has the amino acid sequence of SEQ ID NO: 96 ("VHH-SEED-AG-8"), and - A GA chain linked to VHH bound to IL18Rβ, wherein the GA chain linked to VHH bound to IL18Rβ has the amino acid sequence of SEQ ID NO: 108 ("VHH-SEED-GA-9"). The molecule containing the above.

[0784] Apparatus 634: A bispecific antibody molecule prepared by SEED (chain exchange operation domain) technology, wherein: -AG chain linked to VHH bound to IL-18Rα, where the AG chain linked to the VHH bound to IL-18Rα has the amino acid sequence of SEQ ID NO: 96 ("VHH-SEED-AG-8"), and - A GA chain linked to VHH bound to IL18Rβ, wherein the GA chain linked to VHH bound to IL18Rβ has the amino acid sequence of SEQ ID NO: 109 ("VHH-SEED-GA-10"). The molecule containing the above.

[0785] Apparatus 635: A bispecific antibody molecule prepared by SEED (chain exchange operation domain) technology, wherein: -AG chain linked to VHH bound to IL-18Rα, where the AG chain linked to the VHH bound to IL-18Rα has the amino acid sequence of SEQ ID NO: 97 ("VHH-SEED-AG-9"), and - A GA chain linked to VHH bound to IL18Rβ, wherein the GA chain linked to VHH bound to IL18Rβ has the amino acid sequence of SEQ ID NO: 100 ("VHH-SEED-GA-1"). The molecule containing the above.

[0786] Apparatus 636: A bispecific antibody molecule prepared by SEED (chain exchange operation domain) technology, wherein: -AG chain linked to VHH bound to IL-18Rα, where the AG chain linked to the VHH bound to IL-18Rα has the amino acid sequence of SEQ ID NO: 97 ("VHH-SEED-AG-9"), and - A GA chain linked to VHH bound to IL18Rβ, wherein the GA chain linked to VHH bound to IL18Rβ has the amino acid sequence of SEQ ID NO: 101 ("VHH-SEED-GA-2"). The molecule containing the above.

[0787] Apparatus 637: A bispecific antibody molecule prepared by SEED (chain exchange operation domain) technology, wherein: -AG chain linked to VHH bound to IL-18Rα, where the AG chain linked to the VHH bound to IL-18Rα has the amino acid sequence of SEQ ID NO: 97 ("VHH-SEED-AG-9"), and - A GA chain linked to VHH bound to IL18Rβ, wherein the GA chain linked to VHH bound to IL18Rβ has the amino acid sequence of SEQ ID NO: 103 ("VHH-SEED-GA-4"). The molecule containing the above.

[0788] Apparatus 638: A bispecific antibody molecule prepared by SEED (chain exchange operation domain) technology, wherein: -AG chain linked to VHH bound to IL-18Rα, where the AG chain linked to the VHH bound to IL-18Rα has the amino acid sequence of SEQ ID NO: 97 ("VHH-SEED-AG-9"), and - A GA chain linked to VHH bound to IL18Rβ, wherein the GA chain linked to VHH bound to IL18Rβ has the amino acid sequence of SEQ ID NO: 105 ("VHH-SEED-GA-6"). The molecule containing the above.

[0789] Apparatus 639: A bispecific antibody molecule prepared by SEED (chain exchange operation domain) technology, wherein: -AG chain linked to VHH bound to IL-18Rα, where the AG chain linked to the VHH bound to IL-18Rα has the amino acid sequence of SEQ ID NO: 97 ("VHH-SEED-AG-9"), and - A GA chain linked to VHH bound to IL18Rβ, wherein the GA chain linked to VHH bound to IL18Rβ has the amino acid sequence of SEQ ID NO: 108 ("VHH-SEED-GA-9"). The molecule containing the above.

[0790] Apparatus 640: A bispecific antibody molecule prepared by SEED (chain exchange operation domain) technology, wherein: -AG chain linked to VHH bound to IL-18Rα, where the AG chain linked to the VHH bound to IL-18Rα has the amino acid sequence of SEQ ID NO: 97 ("VHH-SEED-AG-9"), and - A GA chain linked to VHH bound to IL18Rβ, wherein the GA chain linked to VHH bound to IL18Rβ has the amino acid sequence of SEQ ID NO: 109 ("VHH-SEED-GA-10"). The molecule containing the above.

[0791] Apparatus 641: A bispecific antibody molecule prepared by SEED (chain exchange operation domain) technology, wherein: -AG chain linked to VHH bound to IL-18Rα, where the AG chain linked to the VHH bound to IL-18Rα has the amino acid sequence of SEQ ID NO: 97 ("VHH-SEED-AG-9"), and - A GA chain linked to VHH bound to IL18Rβ, wherein the GA chain linked to VHH bound to IL18Rβ has the amino acid sequence of SEQ ID NO: 100 ("VHH-SEED-GA-11"). The molecule containing the above.

[0792] Apparatus 642: A bispecific antibody molecule prepared by SEED (chain exchange operation domain) technology, wherein: -AG chain linked to VHH bound to IL-18Rα, where the AG chain linked to the VHH bound to IL-18Rα has the amino acid sequence of SEQ ID NO: 98 ("VHH-SEED-AG-10"), and - A GA chain linked to VHH bound to IL18Rβ, wherein the GA chain linked to VHH bound to IL18Rβ has the amino acid sequence of SEQ ID NO: 101 ("VHH-SEED-GA-2"). The molecule containing the above.

[0793] Apparatus 643: A bispecific antibody molecule prepared by SEED (chain exchange operation domain) technology, wherein: -AG chain linked to VHH bound to IL-18Rα, where the AG chain linked to the VHH bound to IL-18Rα has the amino acid sequence of SEQ ID NO: 98 ("VHH-SEED-AG-10"), and - A GA chain linked to VHH bound to IL18Rβ, wherein the GA chain linked to VHH bound to IL18Rβ has the amino acid sequence of SEQ ID NO: 108 ("VHH-SEED-GA-9"). The molecule containing the above.

[0794] Apparatus 644: A bispecific antibody molecule prepared by SEED (chain exchange operation domain) technology, wherein: -AG chain linked to VHH bound to IL-18Rα, where the AG chain linked to the VHH bound to IL-18Rα has the amino acid sequence of SEQ ID NO: 98 ("VHH-SEED-AG-10"), and - A GA chain linked to VHH bound to IL18Rβ, wherein the GA chain linked to VHH bound to IL18Rβ has the amino acid sequence of SEQ ID NO: 109 ("VHH-SEED-GA-10"). The molecule containing the above.

[0795] According to the fourteenth aspect, the present disclosure relates to a pharmaceutical composition comprising a compound or a bispecific antibody molecule according to any one of the aspects or embodiments described above.

[0796] Methods for preparing pharmaceutical compositions are known to those skilled in the art (Remington: The Science and Practice of Pharmacy, 22nd ed. (2012), Pharmaceutical Press).

[0797] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, diluent, and / or excipient.

[0798] The term "pharmaceutically acceptable" means that the carrier, diluent, or excipient is a non-toxic, inert material that is compatible with other components of the pharmaceutical composition, is not harmful to the patient to whom the pharmaceutical composition is administered, and is therefore usable in the pharmaceutical product. Publicly known substances as carriers, diluents, or excipients ...

Claims

1. below: - A first binding module which is a VHH antibody domain or a fragment thereof, where, (a) The VHH antibody domain or fragment thereof includes one VHH complementarity-determining region CDR1, CDR2, and CDR3 selected from the group consisting of VHH1, VHH2, VHH5, VHH6, VHH8, VHH9, and VHH10 as shown in the table of CDRs; (b) The VHH antibody domain or fragment thereof includes the complementarity-determining regions CDR1, CDR2 and CDR3 as defined in (a), wherein the modification is that at least one of the sequences of CDR1, CDR2 and CDR3 is humanized; or (c) The VHH antibody domain or fragment thereof includes the complementarity-determining regions CDR1, CDR2 and CDR3 as defined in (a) and having modifications, wherein the modifications are - Substitution, addition, or deletion of up to three amino acids in CDR1, - Substitution, addition or deletion of up to three amino acids in CDR2, and / or - Substitution, addition, or deletion of up to three amino acids in CDR3 It is; - A second binding module which is the VHH antibody domain or a fragment thereof, where, (d) The VHH antibody domain or fragment thereof comprises one VHH complementarity-determining region CDR1, CDR2 and CDR3 selected from the group consisting of VHH12, VHH13, VHH14, VHH15, VHH17, VHH18, VHH20, VHH21 and VHH22 as shown in the table of CDR, wherein the VHH antibody domain or fragment thereof as defined in (d) is selected as follows: If the VHH antibody domain or fragment of (a) contains the VHH1 complementarity-determining regions CDR1, CDR2, and CDR3, then the VHH antibody domain or fragment of (d) contains one VHH complementarity-determining region CDR1, CDR2, and CDR3 selected from the group consisting of VHH12, VHH13, VHH15, VHH17, VHH20, VHH21, and VHH22 as shown in the table of CDRs; If the VHH antibody domain or fragment of (a) contains the VHH2 complementarity-determining regions CDR1, CDR2, and CDR3, then the VHH antibody domain or fragment of (d) contains one VHH complementarity-determining region CDR1, CDR2, and CDR3 selected from the group consisting of VHH12, VHH13, VHH15, VHH17, VHH18, VHH20, VHH21, and VHH22 as shown in the table of CDRs; If the VHH antibody domain or fragment of (a) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH5, then the VHH antibody domain or fragment of (d) contains one VHH complementarity-determining region CDR1, CDR2, and CDR3 selected from the group consisting of VHH13 and VHH14 as shown in the table of CDRs; If the VHH antibody domain or fragment of (a) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH6, then the VHH antibody domain or fragment of (d) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of one VHH selected from the group consisting of VHH17, VHH20, VHH21, and VHH22 as shown in the table of CDRs; If the VHH antibody domain or fragment of (a) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH8, then the VHH antibody domain or fragment of (d) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of one VHH selected from the group consisting of VHH12, VHH13, VHH17, VHH18, VHH20, and VHH21 as shown in the table of CDRs; If the VHH antibody domain or fragment of (a) contains the VHH9 complementarity-determining regions CDR1, CDR2, and CDR3, then the VHH antibody domain or fragment of (d) contains one VHH complementarity-determining region CDR1, CDR2, and CDR3 selected from the group consisting of VHH12, VHH13, VHH15, VHH17, VHH18, VHH20, VHH21, and VHH22 as shown in the table of CDRs; If the VHH antibody domain or fragment of (a) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of VHH10, then the VHH antibody domain or fragment of (d) contains the complementarity-determining regions CDR1, CDR2, and CDR3 of one VHH selected from the group consisting of VHH13, VHH20, and VHH21 as shown in the table of CDRs; (e) The VHH antibody domain or fragment thereof includes the complementarity-determining regions CDR1, CDR2 and CDR3 as defined in (d), wherein the modification is that at least one of the sequences of CDR1, CDR2 and CDR3 is humanized; or (f) The VHH antibody domain or fragment thereof includes the complementarity-determining regions CDR1, CDR2 and CDR3 as defined in (d) and having modifications, wherein the modifications are - Substitution, addition, or deletion of up to one amino acid in CDR1, - Substitution, addition or deletion of up to one amino acid in CDR2, and / or - Substitution, addition, or deletion of up to one amino acid in CDR3 It is; CDR table: Table 1-1 Table 1-2 Table 1-3 A compound containing [this compound].

2. The compound according to claim 1, wherein the VHH antibody domain or fragment defined in (a) comprises one VHH complementarity-determining region CDR1, CDR2 and CDR3 selected from the group consisting of VHH2 and VHH8 as shown in the table of CDR, wherein the VHH antibody domain or fragment defined in (d) comprises one VHH complementarity-determining region CDR1, CDR2 and CDR3 selected from the group consisting of VHH15 and VHH17 as shown in the table of CDR.

3. The compound according to claim 1, wherein the VHH antibody domain or fragment as defined in (a) comprises the complementarity-determining regions CDR1, CDR2 and CDR3 of VHH2 as shown in the table of CDRs, and wherein the VHH antibody domain or fragment as defined in (d) comprises the complementarity-determining regions CDR1, CDR2 and CDR3 of VHH15 as shown in the table of CDRs.

4. below: - A first binding module which is a VHH antibody domain or a fragment thereof, where, (A) The VHH antibody domain contains an amino acid sequence of VHH selected from the group consisting of VHH1, VHH2, VHH5, VHH6, VHH8, VHH9 and VHH10 as shown in the table of VHH sequences; (B) The VHH antibody domain comprises a VHH sequence as defined in (A) and having modifications, wherein the modification is humanization of the sequence; (C) The VHH antibody domain comprises a VHH sequence as defined in (A) and having modifications, wherein the modifications are substitutions, additions, or deletions of up to 25 amino acids; or, (D) The VHH antibody domain comprises a VHH sequence that is at least 75% identical to the VHH sequence mentioned in (A); - A second binding module which is the VHH antibody domain or a fragment thereof, where, (E) The VHH antibody domain comprises an amino acid sequence of VHH selected from the group consisting of VHH12, VHH13, VHH14, VHH15, VHH17, VHH18, VHH20, VHH21 and VHH22 as shown in the table of VHH sequences, wherein the VHH antibody domain of (E) is selected as follows: If the VHH antibody domain of (A) contains the amino acid sequence of VHH1, then the VHH antibody domain of (E) contains the amino acid sequence of one VHH selected from the group consisting of VHH12, VHH13, VHH15, VHH17, VHH20, VHH21, and VHH22 as shown in the table of VHH sequences; If the VHH antibody domain of (A) contains the amino acid sequence of VHH2, then the VHH antibody domain of (E) contains the amino acid sequence of one VHH selected from the group consisting of VHH12, VHH13, VHH15, VHH17, VHH18, VHH20, VHH21, and VHH22 as shown in the table of VHH sequences; If the VHH antibody domain of (A) contains the amino acid sequence of VHH5, then the VHH antibody domain of (E) contains the amino acid sequence of one VHH selected from the group consisting of VHH13 and VHH14 as shown in the table of VHH sequences; If the VHH antibody domain of (A) contains the amino acid sequence of VHH6, then the VHH antibody domain of (E) contains the amino acid sequence of one VHH selected from the group consisting of VHH17, VHH20, VHH21, and VHH22 as shown in the VHH sequence table; If the VHH antibody domain of (A) contains the amino acid sequence of VHH8, then the VHH antibody domain of (E) contains the amino acid sequence of one VHH selected from the group consisting of VHH12, VHH13, VHH17, VHH18, VHH20, and VHH21 as shown in the table of VHH sequences; If the VHH antibody domain of (A) contains the amino acid sequence of VHH9, then the VHH antibody domain of (E) contains the amino acid sequence of one VHH selected from the group consisting of VHH12, VHH13, VHH15, VHH17, VHH18, VHH20, VHH21, and VHH22 as shown in the table of VHH sequences; If the VHH antibody domain of (A) contains the amino acid sequence of VHH10, then the VHH antibody domain of (E) contains the amino acid sequence of one VHH selected from the group consisting of VHH13, VHH20, and VHH21 as shown in the VHH sequence table; (F) The VHH antibody domain comprises a VHH sequence as defined in (E) and having modifications, wherein the modification is humanization of the sequence; (G) The VHH antibody domain comprises a VHH sequence as defined in (E) and having modifications, wherein the modifications are substitutions, additions, or deletions of up to 10 amino acids; or (H) The VHH antibody domain comprises a VHH sequence that is at least 90% identical to the VHH sequence referenced in (E); Table of VHH sequences: Table 2-1 Table 2-2 A compound containing [this compound].

5. The compound according to claim 4, wherein the VHH antibody domain of (A) comprises an amino acid sequence of one VHH selected from the group consisting of VHH2 and VHH8 shown in the VHH sequence table, and wherein the VHH antibody domain of (E) comprises an amino acid sequence of one VHH selected from the group consisting of VHH15 and VHH17 shown in the VHH sequence table.

6. The compound according to claim 4, wherein the VHH antibody domain of (A) comprises the amino acid sequence of VHH2 as shown in the VHH sequence table, and wherein the VHH antibody domain of (E) comprises the amino acid sequence of VHH15 as shown in the VHH sequence table.

7. The compound according to claim 1 or 6, which is a bispecific antibody molecule prepared by SEED technology.

8. The compound according to claim 1 or 6, wherein the compound is a single-domain-based IgG (sdIgG), in which, in a conventional IgG antibody, each of the two VH regions is replaced by a copy of the first binding module, and each of the two VL regions is replaced by a copy of the second binding module, resulting in a bispecific tetravalent antibody.

9. The compound according to claim 1 or 6, wherein the compound comprises an IgG Fc region in which two copies of a tandem VHH configuration are covalently linked to a hinge region, the tandem VHH configuration comprising a first VHH antibody domain covalently linked to a second VHH antibody domain, the second VHH antibody domain being covalently linked to the hinge region of the Fc region, resulting in a bispecific tetravalent compound.

10. The compound according to any one of claims 1 to 9, wherein the compound is bound to IL-18RαECD at a KD of 100 nM or stronger.

11. The compound according to any one of claims 1 to 10, wherein the compound is bound to IL-18RβECD at a KD of 100 nM or stronger.

12. The compound according to any one of claims 1 to 11, wherein the compound can activate the IL-18 receptor IL-18R by binding to IL-18Rα and IL-18Rβ.

13. The compound according to any one of claims 1 to 12, wherein the compound can induce dose-dependent downstream signaling of IL-18R in IL-18 reporter cells.

14. The compound according to any one of claims 1 to 13, which can induce IFN-γ release by peripheral blood mononuclear cells (PBMCs) in the presence of a low dose of IL-12 (10 ng / ml).

15. A pharmaceutical composition comprising the compound described in any one of claims 1 to 14.

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