WO2024251983A1

WO2024251983A1 - Il-18 mimetics

Info

Publication number: WO2024251983A1
Application number: PCT/EP2024/065796
Authority: WO
Inventors: Stefan ZIELONKA; Lukas PEKAR; Paul ARRAS; Britta LIPINSKI; Laura UNMUTH
Original assignee: Merck Patent GmbH
Current assignee: Merck Patent GmbH
Priority date: 2023-06-09
Filing date: 2024-06-07
Publication date: 2024-12-12
Anticipated expiration: 2025-12-09
Also published as: CN121666402A; AU2024286186A1

Abstract

The present disclosure relates to novel, VHH-based binders of IL-18Rα and/or IL-18Rβ with favorable characteristics. Moreover, the present disclosure relates to pharmaceutical compositions comprising such a compound.

Description

IL-18 MIMETICS

FIELD OF THE INVENTION

The present disclosure relates to novel, VHH-based binders of IL-18Ra and/or IL-18RP with favorable characteristics. Moreover, the present disclosure relates to pharmaceutical compositions comprising such a compound.

BACKGROUND OF THE INVENTION

Cytokines are potent immunomodulatory proteins comprising huge therapeutic potential. Consequently, several different molecules were granted marketing approval for the treatment of different diseases (Propper and Balkwill, 2022; Pires et al., 2021; Berraondo et al., 2019).

However, the pleiotropic mechanism of action of many cytokines, their short half-life combined with often dose-limiting toxicities when administered systemically hamper their therapeutic applicability (Pires et al., 2021; Amin et al., 2014).

To address these inherent limitations, "next-generation cytokines" were generated (Zheng et al., 2022). For instance, muteins of IL-2 were engineered, which do not target the a-subunit of the IL-2 receptor (CD25) in order to eliminate the intrinsic bias of this cytokine for activating regulatory T cells (Tregs) (Klein et al., 2017; Dolgin, 2022). To optimize the poor pharmacokinetics of cytokines, fusions with the (effector silenced or attenuated) Fc portion of IgGs were engineered (Jazayeri and Carroll, 2008) as well as conjugates with polyethylene glycol (Eliason, 2001). Besides, bifunctional antibody cytokine fusion proteins were constructed aiming at accumulating the immunomodulatory function of cytokines at the site of disease (Murer and Neri, 2019; Neri and Sondel, 2016).

Another interesting approach regarding "next-generation cytokines" relies on exploiting antibody-derivatives that mimic the function of cytokines, referred to as cytokine mimetics or surrogate cytokines (Saxton et al., 2023). In this respect, bispecific antibody derivatives (diabodies) have been developed that dimerize the Erythropoietin receptor (EpoR) (Moraga et al., 2015). Intriguingly, different diabodies induced differential EpoR phosphorylation ranging from weak and partial agonism to full agonism. In addition to this, single domain antibody (sdAb)-based bispecifics were generated that activate signalling through the heterodimeric IL- 2 receptor Py (IL-2RPy). These bispecific antibodies (bsAbs) mimic the function of IL-2 but without preferential activation of Tregs via IL-2 receptor a binding (Harris et al., 2021). Garcia and co-workers recently described the engineering of VHH-derived surrogate agonists targeting IL-2RPy as well as type I IFN-mimicking bsAbs (Yen et al., 2022). Importantly, the group constructed surrogate agonists displaying functional diversification in terms of receptor downstream signalling compared to the natural cytokine. Moreover, also bsAbs were constructed that trigger agonistic activity through binding to IL-2RP and IL-10RP, a receptor heterodimer that naturally does not exist. This is clearly demonstrating that the modular assembly of bsAbs enables the generation of cytokine mimetics with tailor-made functionalities which might be versatile building blocks for drug discovery.

IL-18 is a proinflammatory cytokine belonging to the IL-1 family of cytokines that mediates signalling through heterodimerization of the receptor subunits IL-18Ra and IL-18RP (Yasuda et al., 2019; Dinarello et al., 2013). IL-18 stimulates IFN-y production in innate lymphoid cells as well as antigen-experienced T cells in synergy with IL-12 (Nakamura et al., 2020). Recombinant (r)IL-18 has been assessed in clinical trials and demonstrated a favorable toxicity profile but limited efficacy as monotherapy (Atallah-Yunes and Robertson, 2022; Robertson et al., 2006; Tarhini et al., 2009). Physiologically, the activity of IL-18 is balanced via the high- affinity neutralizing and naturally occurring IL-18 binding protein (IL-18BP) (Dinarello et al., 2013; Nakamura et al., 2020). After treatment with (r)IL-18, substantially 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 show that IL18BP is expressed in the tumor microenvironment of several tumor types (Zhou et al., 2020). Moreover, the authors found increased IL-18BP concentrations in the serum of patients with non-small cell lung cancer which were further elevated post PD-1 or PD-L1 treatment. In the same study, the group engineered a decoyresistant IL-18 mutein that still triggered IL-18 receptor activation. This next-generation IL-18 derivative showed superior antitumor efficacy in preclinical models as monotherapy as well as in combination with immune checkpoint inhibition (Zhou et al., 2020).

IL-18 is a proinflammatory cytokine promoting NK cell activation as well as effector T cell maturation and function (Holder et al., 2022). Consequently, recombinant human (rh) IL-18 emerged as promising potential therapeutic inducing antitumor immunity, (rh) IL-18 was assessed in several clinical trials either as single agent or as 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). (rh) IL-18 therapy has been well tolerated, however, clinical efficacy has been limited. A possible explanation for the lack of therapeutic efficacy relies in the inhibition of IL-18 by the IL-18BP decoy receptor (Dinarello et al., 2013). IL-18BP binds to an overlapping site on IL-18 with IL-18Ra albeit with much higher affinities. Accordingly, it antagonizes signalling of IL- 18 by blocking the IL-18Ra interaction with IL-18. Recently, Ring and colleagues described the generation of a decoy resistant mutein of IL-18 showing great promise for anti-cancer therapy in preclinical models (Zhou et al., 2020). This entity is currently in clinical investigations (NCT04787042).

Despite the relevance of IL- 18 in the formation of tumors and the potential in targeting IL- 18 for cancer treatment, at present approaches that allow to mimic IL-18 and its functions in a taylor-made fashion are still lacking.

Accordingly, there is a need in the art for improved IL-18 receptor agonists. Moreover, there is a need in the art for improved ways to mimic the function of IL-18 in a taylor-made fashion, e.g. specifically with respect to different potencies and magnitudes of activation of IL-18R receptor activation, different potencies and magnitudes for the release of IFN-y or resistance to inhibition by IL-18BP.

The present disclosure overcomes the above-described problems and addresses the abovedescribed needs.

SUMMARY OF THE INVENTION

The present disclosure addresses the needs described above in the section "Background of the Invention" by the different aspects and embodiments described below.

The present invention is, in part, based on the surprising observation that novel bispecific surrogate agonists can be obtained that are based on sdAbs (sdAb: single-domain antibody, also referred to herein as VHH or VHH), target both IL-18Ra and IL-18RP and mimic the functionality of IL-18. Moreover, it was found that it is possible to obtain such IL-18 mimics that at the same time are resistant to the decoy receptor IL-18BP. Moreover, it was surprisingly observed that molecules in which the overall design architecture with respect to valencies as well as to the spatial orientation of individual VHH-based paratopes is engineered within the molecule allow to obtain IL-18 mimetics in which the agonistic activities are tailor-made, e.g. with respect to their potency, the magnitude of the induced activation of IL-18R and IFN-y release or the tolerance to inhibition by IL-18BP receptor decoy.

In an aspect, the present disclosure relates to a VHH antibody domain or a fragment thereof, wherein

(a) said VHH antibody domain or 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) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 as defined in (a) with modification, wherein the modification is that the sequence of at least one of CDR1, CDR2 and CDR3 is humanized; or

(c) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 as defined in (a) with modification, wherein the modification is

- the replacement, addition or deletion of up to three amino acids in CDR1,

- the replacement, addition or deletion of up to three amino acids in CDR2 and/or

- the replacement, addition or deletion of up to three amino acids in CDR3; Table of CDRs:

In another aspect, the present disclosure relates to a VHH antibody domain or a fragment thereof, wherein

(A) said 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, VHHIO and VHH11 as shown in the Table of VHH Sequences;

(B) said VHH antibody domain comprises a VHH sequence as defined in (A) with modification, wherein the modification is that said sequence is humanized;

(C) said VHH antibody domain comprises a VHH sequence as defined in (A) with modification, wherein the modification is the replacement, addition or deletion of up to 25 amino acids; or

(D) said VHH antibody domain comprises a VHH sequence that is at least 75% identical to a VHH sequence referred to in (A);

Table of VHH Sequences:

In another aspect, aspect, the present disclosure relates to a VHH antibody domain or a fragment thereof, wherein

(A) said VHH antibody domain consists of 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) said VHH antibody domain consists of a VHH sequence as defined in (A) with modification, wherein the modification is that said sequence is humanized;

(C) said VHH antibody domain consists of a VHH sequence as defined in (A) with modification, wherein the modification is the replacement, addition or deletion of up to 25 amino acids; or

(D) said VHH antibody domain consists of a VHH sequence that is at least 75% identical to a VHH sequence referred to in (A). In another aspect, aspect, the present disclosure relates to a VHH antibody domain or a fragment thereof, wherein

(d) said VHH antibody domain or fragment thereof comprises 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;

(e) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 as defined in (d) with modification, wherein the modification is that the sequence of at least one of CDR1, CDR2 and CDR3 is humanized; or

(f) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 as defined in (d) with modification, wherein the modification is

- the replacement, addition or deletion of up to three amino acids in CDR1,

- the replacement, addition or deletion of up to three amino acids in CDR3.

(E) said VHH antibody domain comprises the amino acid sequence of a 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) said VHH antibody domain comprises a VHH sequence as defined in (E) with modification, wherein the modification is that said sequence is humanized;

(G) said VHH antibody domain comprises a VHH sequence as defined in (E) with modification, wherein the modification is the replacement, addition or deletion of up to 25 amino acids; or

(H) said VHH antibody domain comprises a VHH sequence that is at least 75% identical to a VHH sequence referred to in (E).

In another aspect, aspect, the present disclosure relates to a VHH antibody domain or a fragment thereof, wherein (E) said VHH antibody domain consists of the amino acid sequence of a 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) said VHH antibody domain consists of a VHH sequence as defined in (E) with modification, wherein the modification is that said sequence is humanized;

(G) said VHH antibody domain consists of a VHH sequence as defined in (E) with modification, wherein the modification is the replacement, addition or deletion of up to 25 amino acids; or

(H) said VHH antibody domain consists of a VHH sequence that is at least 75% identical to a VHH sequence referred to in (E).

In another aspect, aspect, the present disclosure relates to a compound comprising

- a VHH antibody domain or fragment thereof according to any one of embodiments 1 to

188.

- a VHH antibody domain or fragment thereof according to any one of claims 189 to 374.

- a first binding module which is a VHH antibody domain or fragment thereof, wherein

(a) said VHH antibody domain or 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 CDR;

- the replacement, addition or deletion of up to three amino acids in CDR1, - the replacement, addition or deletion of up to three amino acids in CDR2 and/or

- the replacement, addition or deletion of up to three amino acids in CDR3;

- a second binding module which is a VHH antibody domain or a fragment thereof, wherein

- the replacement, addition or deletion of up to three amino acids in CDR1,

- the replacement, addition or deletion of up to three amino acids in CDR3.

(A) said 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) said VHH antibody domain comprises a VHH sequence as defined in (A) with modification, wherein the modification is that said sequence is humanized; 258 (C) said VHH antibody domain comprises a VHH sequence as defined in (A) with

259 modification, wherein the modification is the replacement, addition or

260 deletion of up to 25 amino acids; or

261 (D) said VHH antibody domain comprises a VHH sequence that is at least 75%

262 identical to a VHH sequence referred to in (A);

263 - a second binding module which is a VHH antibody domain or a fragment thereof, wherein

264 (E) said VHH antibody domain comprises the amino acid sequence of a VHH

265 selected from the group consisting of VHH12, VHH13, VHH14, VHH15,

266 VHH16, VHH17, VHH18, VHH20, VHH21 and VHH22 as shown in the

267 Table of VHH Sequences;

268 (F) said VHH antibody domain comprises a VHH sequence as defined in (E) with

269 modification, wherein the modification is that said sequence is humanized;

270 (G) said VHH antibody domain comprises a VHH sequence as defined in (E) with

271 modification, wherein the modification is the replacement, addition or

Til deletion of up to 25 amino acids; or

273 (H) said VHH antibody domain comprises a VHH sequence that is at least 75%

274 identical to a VHH sequence referred to in (E).

U

276 In another aspect, aspect, the present disclosure relates to a compound comprising

277 - a first binding module which is a VHH antibody domain or fragment thereof, wherein

278 (A) said VHH antibody domain consists of the amino acid sequence of a VHH

279 selected from the group consisting of VHH1, VHH2, VHH3, VHH4, VHH5,

280 VHH6, VHH8, VHH9, VHH10 and VHH11 as shown in the Table of VHH

281 Sequences;

282 (B) said VHH antibody domain consists of a VHH sequence as defined in (A)

283 with modification, wherein the modification is that said sequence is

284 humanized;

285 (C) said VHH antibody domain consists of a VHH sequence as defined in (A)

286 with modification, wherein the modification is the replacement, addition or

287 deletion of up to 25 amino acids; or

288 (D) said VHH antibody domain consists of a VHH sequence that is at least 75%

289 identical to a VHH sequence referred to in (A);

290 - a second binding module which is a VHH antibody domain or a fragment thereof, wherein (E) said VHH antibody domain consists of the amino acid sequence of a 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;

In another aspect, the present disclosure relates to a compound which is a bispecific antibody molecule comprising an IL-18Ra-binding VHH with an amino acid sequence as defined in the present disclosure below (First binding module) and an IL18RP-binding VHH with an amino acid sequence as defined in the present disclosure below (Second binding module).

In another aspect, the present disclosure relates to a bispecific antibody molecule prepared by the SEED (strand-exchange engineered domain) technology, said molecule comprising

- an AG chain linked to an IL-18Ra-binding VHH, wherein said AG chain linked to said IL-18Ra-binding VHH has an amino acid sequence as defined in the present disclosure below ("VHH-SEED-AG-1") and

- a GA chain linked to an IL18RP-binding VHH, wherein said GA chain linked to said

IL18RP-binding VHH has an amino acid sequence as defined in the present disclosure below ("VHH-SEED-GA-1").

In another aspect, the present disclosure relates to a pharmaceutical composition comprising the compound or bispecific antibody molecule according to the present disclosure.

In another aspect, the present disclosure relates to the use of a compound or bispecific antibody molecule according to the present disclosure for activating IL-18R receptor. In another aspect, the present disclosure relates to the use of a compound or bispecific antibody molecule according to the present disclosure for inducing IL-y release from cells carrying an IL-18R receptor.

BRIEF DESCRIPTION OF THE FIGURES

In the following, reference is made to the figures. All methods referred to in the figure descriptions below were carried out as described in detail in the examples.

Figure 1 summarizes the overall strategy applied in the present disclosure for the generation of tailor-made cytokine mimetics based on sdAb-derived bispecifics and YSD-enabled antibody discovery. (A) Bottom/Left panel: IL-18 (blue, §) triggers IL-18R downstream signalling by consecutive binding to IL-18Ra (yellow, *; the two "lobes" of IL-18Ra are each labelled with *) followed by IL-18RP (lime, #) recruitment. IL-18BP (dark grey, $) inhibits IL-18 by high- affinity binding and hence, by blocking the IL-18/IL-18Ra interaction. Top/Right panel: Upon reformatting camelid-derived sdAbs with IL-18Ra-targeting functionality (orange, &) and IL- 18RP-targeting functionality (green, J) into an IgG-like bispecific, resulting cytokine mimetics cross-link the IL18R subunits IL-18Ra (yellow, *; the two "lobes" of IL-18Ra are each labelled with *) and IL-18RP (lime, #) and hence elicit down-stream signalling. Essentially, generated IL-18 mimetics are resistant to inhibition by IL-18BP (dark grey, $). Generated with PyMOL software version 2.3.0 based on PDB entries YYY and ZZZ and modified using www.biorender.com (B) Immunization of cam elids followed by YSD facilitate the enrichment of sdAbs specific to (rh) IL-18Ra and (rh) IL-18RP. One sublibrary was generated for each specimen (huarizo and llama) and sorted separately. In the first round of selection, a mixture of both (rh) receptor subunits was exploited at a concentration of 250 nM. In the subsequent second sorting round, enriched libraries were sorted separately against each antigen at 250 nM. A two-dimensional sorting strategy was applied to select for full-length VHH display in addition to antigen binding. Percentage of cells in sorting gates are shown. Plots show 5xl0⁴ events. (C) Graphical alignments of 55 unique sdAb clones addressing IL-18Ra (top) as well as 101 independent clones targeting IL-18RP (bottom) retrieved from YSD library sorting. Complementarity-determining regions (CDRs) are highlighted. Red bars indicate high sequence diversity at the amino acid level and green bars represent high sequence conservation at a given position. Alignment conducted with MUSCLE alignment tool using Geneious Prime 2021.1.1.

Figure 2 shows data obtained with a reporter assay, confirming that combinatorial reformatting of monospecific (1+0) SEEDbodies into strictly monovalent (1+1) bsAbs enables the identification of IL- 18 mimetics with attenuated capacities to trigger NFKB reporter activity on IL-18 reporter cells. (A) HEK-Blue™ reporter cells were incubated with increasing conentrations of reformatted bsAbs, as exemplarily shown for IL18R_VHHa2pi5, IL18R_VHHa8pi5, IL18R_VHHa2pi7 and IL18R_VHHa8pi7. Secreted embryonic alkaline phosphatase activity was monitored by determining the OD640. Reporter activity was normalized to maximal IL-18 read-out. (B) Heatmap of NFKB reporter activitation elicited by combinatorial reformatted (1+1) bsAbs. Molecules failing initial quality control (target monomer peak in SEC < 96% post protein A purification given in dark grey, functionally inactive bsAbs shown in red (J), minimally active surrogate agonists (NFKB reporter activitation < 50% compared to (rh) IL-18at 1 nM or EC50 > 0.1 nM) in yellow (#) and moderately active IL-18 mimetics (NFKB reporter activitation > 50% compared to (rh) IL-18at 1 nM or EC50 < 0.1 nM) depicted in green (*).

Figure 3 depicts data from an IFN-y release assay, confirming that bispecific (1+1) surrogate agonists trigger IFN-y release on PBMCs isolated from healthy donors. (A) IFN-y production of PBMCs stimulated with bsAbs 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. Graph shows box and whisker plots as superimpositions with dot plots of IFN-y release of ten different donors. ****p < 0.0001,***p < 0.001, **p < 0.01, *p < 0.05. IL18R_VHHaipi6 was used as negative control (given in red). Four leading candidates used for further characterization shown in green, purple, blue and orange. (B) TOP4 candidates evoke a dose dependent IFN-y read-out on PBMCs in the presence of low dose (rh) IL-12 (10 ng/ml). IL18R_VHHaipi6 as negative control shown in red was used at a fixed concentration of 1 pM. Mean values ± SEM of ten independent experiments are shown.

Figure 4 shows schematic depictions of the architecture of antibodies prepared and summarizes data to characterize such antibodies. These data confirm that antibody engineering enables the generation of IL-18 mimetics with augmented agonism capacities. (A) Schematic depiction of main different bispecific antibody architectures that were constructed within this work. Fusion of an anti-IL18Ra VHHa2 to the hinge region of the AG chain of the SEEDbody as well as engraftment of an anti-IL18Rp VHHP15 onto the GA chain results in the initially generated (1+1) format IL18R_VHHa2pi5. Replacing the VH and VLz. of an effector silenced IgG by VHHa2 and VHHpi5, respectively facilitates the generation of the IL18R_sdIgGa2pi5 architecture (2+2). Within the IL18R_tanVHHa2pi5 design (2+2), VHHa2 and VHHP15 are arranged in tandem (from A-terminus to C-terminus) and separated by a five amino acid Gly4Ser linker. The tandem is fused to the hinge region of an effector silenced IgGl Fc fragment. Of note, also the opposite orientation was constructed (VHHpi5 followed by VHHa2, IL18R_tanVHHpi5a2). In addition, all four molecules were also produced harboring the E430G mutation for on-target hexamerization. Structure models generated with PyMOL software version 2.3.0. (B) Distinct surrogate agonist formats of the same paratopes (VHHa2 and VHHP15) display differential properties in eliciting a functional IFN-y response on human PBMCs isolated from healthy donors at fixed concentrations. Experiments were performed at two different concentrations (10 nM and 1 nM) in the presence of 10 ng/ml (rh) IL-12. Graph shows box and whisker plots as superimpositions with dot plots of IFN-y release of six different donors. ****p < 0.0001,***p < 0.001, **p < 0.01, *p < 0.05. (C) Surrogate agonists arranged in tandem (IL18R_tanVHHa2pi5 and IL18R_tanVHHpi5a2) elicit enhanced IFN-y production in terms of potencies and magnitude on PBMCs isolated from healthy donors, resulting in a variant with increased potencies compared with (rh) IL-18. All experiments were performed in the presence of low dose (rh) IL-12 (10 ng/ml). IL18R_VHHaipi6 as negative control shown in red was used at a fixed concentration of 1 pM. Mean values ± SEM of 13 independent experiments are shown. (D) Potency augmented tandem IL- 18 mimetics are resistant to inhibition by (rh) IL-18BP, whereas (rh) IL-18 is efficiently blocked from signalling. PBMCs of healthy human donors were stimulated either with (rh) IL- 18 or IL18R_tanVHHa2pi5 and IL18R_tanVHHpi5a2 at a fixed concentration of 0.5 nM in the presence of (rh) IL-12 (10 ng/ml) and different concentrations of (rh) IL-18BP. Five independent experiments were performed and mean values ± SEM are shown.

Figure 5 summarizes the sorting procedure applied to obtain sdABs. YSD enables the enrichment of sdAbs targeting (rh) IL-18Ra and (rh) IL-18RP. Sorting output after two sorting rounds of each library against (rh) IL-18Ra and (rh) IL-18RP. A two-dimensional sorting strategy was applied do detect full-length VHH display simultaneous to the binding functionality at a concentration of 250 nM. Plots show 5xl0³ events of the corresponding sorting output to visualize enrichment.

Figure 6 depicts the results of binding expreiments examining if monospecific (1+0) VHH SEEDbodies show specific binding to (rh) IL-18Ra (red) or IL-18RP (green). (A) Schematic depiction of monospecific VHHs targeting (rh) IL-18Ra (red) or (rh) IL-18RP (green) engrafted onto the hinge region of the SEED backbone. sdAbs targeting IL-18Ra were fused to the hinge region of the AG chain (msVHHaX), while VHHs addressing the IL-18RP subunit were grafted onto the GA chain (msVHHpX). (B) Binding of respective monospecific VHH SEEDbodies to (rh) IL-18Ra (red) or (rh) IL-18RP (green) as determined by BLI. Either (rh) IL-18Ra or (rh) IL-18RP were loaded to HIS IK biosensors at a concentraion of 3 pg/ml followed by a first association of monospecific VHH SEEDbodies at 100 nM for 300 s. Subsequently, dissociation was measured for 100 s in kinetics buffer. Schemes generated via www.biorender.com.

Figure 7 presents data from a reporter assay suggesting that combinatorial reformatting of monospecific (1+0) SEEDbodies into strictly monovalent (1+1) bsAbs enables the identification of IL- 18 mimetics with attenuated capacities to trigger NFKB reporter activity on IL-18 reporter cells. HEK-Blue™ reporter cells were incubated with increasing conentrations of reformatted bsAbs or (rh) IL-18 and secreted embryonic alkaline phosphatase activity was monitored by determining the OD640. Reporter activity was normalized to maximal IL-18 readout. (A) Exemplary dose-dependent IL-18R agonism triggered by surrogate agonists IL18R_VHHa2pi5, IL18R_VHHa8pi5, IL18R_VHHa2pi7 and IL18R_VHHa8pi7 in direct comparison to (rh) IL-18. (B) Dose-dependent receptor agonism mediated by all 44 cytokine mimetics.

Figure 8 depicts data from an IFN-y release assay, showing that bispecific (1+1) surrogate agonists do not elicit IFN-y release on PBMCs isolated from healthy donors without the presence of low dose (rh) IL-12. IFN-y production of PBMCs stimulated with bsAbs at a fixed concentration of 100 nM or with (rh) IL-18 at 1 nM in the absence of (rh)IL-12. Graph dot plots of IFN-y release of eight different donors. IL18R_VHHaipi6 was used as negative control (given in red). Four leading candidates used for further characterization shown in green, purple, blue and orange. Figure 9 shows binding kinetics data obtained with bispecific (1+1) TOP4 surrogate agonists against receptor subunits IL-18Ra and IL-18RP. IL-18 mimetics were loaded on AHC biosensor tips at a concentration of 5 pg/ml. Interactions against both receptor subunits were measured at a concentration of 100 nM, 50 nM, 25 nM and 12.5 nM for 180 s followed by a dissociation in kinetics buffer for 300 s.

Figure 10 summarizes analytical size exclusion chromatography profiles of the generated surrogate agonist formats, indicating adequate purities following downstream purification. SEC-HPLC profiles of different formats as well as the corresponding target monomer peaks are shown. Absorbance at 214 nm was used for purity determination. (A) SEC profiles of all eight formats post protein A purification, indicating purities below 90% target species for IL18R_VHHa2pi5_E430G and IL18R_sdIgGa2pi5_E430G. (B) Analysis of IL18R_VHHa2pi5_E430G and IL18R_sdIgGa2pi5_E430G following second step SEC purification.

Figure 11 depicts data obtained by differential scanning fluorimetry to characterize the thermal unfolding of IL-18R agonistic bsAbs in different formats. Overlays of the melting curves for different formats of IL-18R agonists were recorded utilizing a temperature gradient from 20°C to 95°C at a slope of l°C/min. First derivatives of 350 nm / 330 nm curves are shown.

Figure 12 shows data obtained upon stimulation of PBMCs with the full dose-range of compound concentrations, indicating a strong hooking effect for (rh) IL 18 while for the different surrogate agonist formats it is not as pronounced. IFN-y read-out on PBMCs in the presence of low dose (rh) IL-12 (10 ng/ml). IL18R_VHHaipi6 as negative control shown in red was used at a fixed concentration of 1 pM. Mean values ± SEM of 13 independent experiments are shown.

Figure 13 shows data examining the binding of engineered tandem IL-18 mimetics. Such engineered tandem IL-18 mimetics exhibit strong binding to both receptor subunits in an avidity driven setting, (rh) IL-18Ra or (rh) IL-18RP were loaded onto HIS1K sensor tips at 5 pg/ml. A first association step was performed with different surrogate agonist formats at 100 nM, 33.3 nM and 11.1 nM for 180 s followed by a dissociation step in kinetics buffer fpr 300 s. Figure 14 shows biolayer interferometry (BLI) binding data for the binding of bispecific surrogate agonists to (rh)IL-18BP. Bispecific surrogate agonists do not bind to (rh)IL-18BP as determined, whereas (rh) IL-18BP binds to (rh)IL-18 with high affinities. BsAbs were loaded on AHC biosensor tips at 5 pg/ml. Subsequently, association was monitored using 1 pM IL- 18BP for 180 s, followed by dissociation in kinetics buffer for 180 s.

Figure 15 summarizes binding data showing that bispecific cytokine mimetics compete with (rh)IL-18 for binding to (rh)IL-18Ra. Surrogate agonists were loaded on AHC biosensor tips, followed by association using IL-18Ra at 200 nM for 300 s. Subsequently, a second association step was performed for 180 s using either (rh)IL-18 at 100 nM (red) or kinetics buffer (black).

SUMMARY OF SEQUENCES (in case of any difference between the below table and the cofiled WIPO ST.26 compliant sequence listing, the information of the below table shall prevail)

SEQ ID NO: 1 to 11 provide the amino acid sequences of VHH1 to VHH11 (corresponding to al to al 1 according to the present disclosure). The CDRs of these VHHs are provided in SEQ ID NO: 23 to 55.

SEQ ID NO: 12 to 22 provide the amino acid sequences of VHH12 to VHH22 (corresponding to pi2 to P22 according to the present disclosure). The CDRs of these VHHs are provided in SEQ ID NO: 56 to 88.

SEQ ID NO: 89 to 99 provide the amino acid sequences of SEED AG chains based on VHH1 to VHH11 (al to al 1) according to the present disclosure. SEQ ID NO: 100 to 110 provide the amino acid sequences of SEED GA chains based on VHH12 to VHH22 (P 12 to P22) according to the present disclosure.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

Although the present disclosure is described in detail above and below, it is to be understood that this disclosure is not limited to the particular methodologies, protocols and reagents described by the present disclosure, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

In the following, certain elements of the present disclosure will be described in more detail, including the description of specific embodiments. However, the variously described examples and preferred embodiments should not be construed to limit the present disclosure to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and/or preferred elements and in any manner. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application except for where this leads to logical contradictions or the context indicates otherwise.

Unless defined otherwise herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclatures and techniques referred to in the present disclosure, e.g. nomenclatures and techniques of organic chemistry, chemical synthesis, biology, medicinal and pharmaceutical chemistry, medicine, pharmacology or toxicology, are those well-known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well-known in the art and as described in the references cited and discussed throughout the present disclosure unless otherwise indicated.

First aspect of the present disclosure (also referred to as "Embodiment 1"): According to a first aspect, the present disclosure relates to a VHH antibody domain or a fragment thereof, wherein

- the replacement, addition or deletion of up to three amino acids in CDR1,

- the replacement, addition or deletion of up to three amino acids in CDR3;

Table of CDRs:

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

In primates such as humans, a heavy and the light chain variable domain of an antibody combine to specifically bind the antigen. Generally, a naturally occurring primate (e.g., human) or murine immunoglobulin has heavy (H) chains and light (L) chains interconnected by disulfide bonds. There are two types of light chain, lambda ( ) and kappa (K). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Primate antibodies can be class switched.

Certain IgG antibodies from members of the camel and dromedary (Camelus bactrianus and Calelus dromaderius) family including new world members such as llama species (Lama paccos. Lama glama and Lama vicugna) of mammals as found in nature lack light chains, and are thus structurally distinct from the typical four chain quaternary structure having two heavy and two light chains, for antibodies from other animals. See PCT/EP93/02214 (WO 94/04678 published 3 March 1994). Such IgG subtypes of the llamas lack the light chains and the CHI domain and are called heavy chain antibodies. These naturally occurring camelid antibodies consisting of only a heavy chain are functional and stable in the absence of light chain. The antigen-binding site of these heavy chain antibodies is formed only by a single domain, referred to as "VHH" (Kbnning et al., 2017) or, used synonymously herein, "VHH antibody domain".

Each light and heavy chain of an antibody, contains constant domains and variable domains. References to "VH" or "VH" refer to the variable region of an immunoglobulin heavy chain, including that of an antibody fragment. References to "VL" or "VL" refer to the variable region of an immunoglobulin light chain, such as in a primate antibody. The variable domain of a heavy chain antibody is called VHH. The VHH is composed of only one polypeptide chain of 15 kDa and is considered the smallest known natural domain with full antigen-binding capacity. Light and heavy chain variable domains contain a "framework" region interrupted by three hypervariable regions, also called "complementarity determining regions" or "CDRs" (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991). The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space. The CDRs are primarily responsible for antigen binding.

The CDRs are typically referred to as CDR1, CDR2, and CDR3 (from the N-terminus to C- terminus), and are also typically identified by the chain in which the particular CDR is located. Thus, a VH CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, whereas a VL CDR1 is the 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 LI, CDR L2, and CDR L3. Heavy chain CDRs are sometimes referred to as CDR Hl, CDR H2, and CDR H3. VHH monoclonal antibodies have only a heavy chain, and thus include only one CDR1, CDR2 and CDR3. Generally, the CDR3 is primarily responsible for antigen specificity.

A VHH includes in an N- to C- direction, the following structural regions: N - FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 - C, wherein FR denotes a framework region amino acid sequence and CDR denotes a complementary determining region amino acid sequence (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991).

As used in the present disclosure, a "VHH antibody domain" refers to the domain formed by the complete VHH, including (in an N- to C- direction) N - FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 - C. A VHH antibody domain can be part of a larger molecule to which the VHH antibody domain is covalently linked. Alternative, a VHH antibody domain can be a molecule of its own that is not covalently linked to any other molecular structure.

Since resulting paratopes are devoid of light chains, sdAbs afford the benefit of multiple reformatting options in a ‘plug-and-play’ manner involving beads-on-string assemblies or facile combinations with existing Fab-based paratopes for the generation of bispecifics (Lipinski et al., 2023; Chanier and Chames, 2019). The extent of the framework region and CDRs have been defined (see, Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991). The CDRs of the heavy chain variable domain are located at residues 31-35 (CDR-H1), residues 50-65 (CDR-H2) and residues 95-102 (CDR-H3) according to the Kabat numbering system. In antibodies (such as primate antibodies) that include a light chain, such as a primate antibody, the CDRs of the light chain variable domain are located at residues 24-34 (CDR-L1), residues 50-56 (CDR-L2) and residues 89-97 (CDR-L3) according to the Kabat numbering system. The Kabat database is now maintained online. The location of camelid CDRs can also be determined (see, for example, Sircar et al., J. Immunol. 186: 6357-6367, 2011); a program to determine camelid antibody structure, the Rosetta Antibody program, is available on the internet.

A "monoclonal antibody" is an antibody produced by a single clone of B-lymphocytes or by a cell into which the heavy chain gene (and optionally a light chain gene, such as for a primate antibody) of a single antibody have been transfected. Monoclonal antibodies may be obtained using a variety of techniques known to those skilled in the art, including standard hybridoma technology (see e.g. Kohler and Milstein, Eur. J. Immunol. (1976), vol. 5, p. 511-519; Antibodies: A Laboratory Manual, 2nd edition (2014), editor Greenfield, Cold Spring Harbor Laboratory Press (USA); Immunobiology, 5th ed. (2001), editors Janeway et al., Garland Publishing (USA)) and e.g. expression from a eukaryotic host cell transfected with a DNA molecule coding for the homogeneous antibody or from a prokaryotic host cell transfected with a DNA molecule coding for the homogeneous antibody.

VHH antibody domains can be obtained by genetic engineering to yield a small protein having high affinity for a target, resulting in a low molecular weight antibody derived protein. See e.g. 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).

In some embodiments, the VHH molecules can be produced as recombinant monoclonal antibodies or antigen binding fragments in different expression platforms, avoiding the use of hybridomas and mice. VHH monoclonal antibodies can be humanized monoclonal antibodies. In some embodiments, monoclonal antibodies can be chimeric antibodies. Without being bound by theory, a VHH monoclonal antibody has a molecular weight approximately one-tenth that of a human IgG molecule, and the protein has a physical diameter of only a few nanometers.

One consequence of the small size is the ability of the VHH monoclonal antibody to bind to antigenic sites that are functionally invisible to larger antibody proteins, such that VHH monoclonal antibodies are useful as reagents to detect antigens that are otherwise cryptic using classical immunological techniques, and thus are of use as therapeutic agents. Thus, yet another consequence of small size is that a camelid VHH monoclonal antibody can inhibit as a result of binding to a specific site in a groove or narrow cleft of a target protein, and hence can serve in a capacity that more closely resembles the function of a classical low molecular weight drug than that of a classical antibody.

Without being bound by theory, low molecular weight and compact size further result in camelid VHH monoclonal antibodies being extremely thermostable, stable to extreme pH and to proteolytic digestion, and poorly antigenic. Further, these molecules can be fully expressed in prokaryotic cells such as E. coli and are expressed as fusion proteins with bacteriophage and are functional.

"Humanizing" an antib ody/antibody sequence, as used herein, refers to the process of "germlining" where a non-human (such as camelid, 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 the corresponding amino acids of a human antibody sequence. Typically, as human antibody sequence will be selected that is particularly close (i.e. has a high degree of sequence homology) to the non-human sequence. Such a human antibody sequence can be identified e.g. by a BLAST search. The corresponding amino acids can then be identified by a 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. A VHH antibody domain is easily humanized based on the human VH domain, which has a sequence that is highly homologous to the sequence of the VHH antibody domain. Furthermore, a VHH sequence can be adapted to reduce sequence liabilities. This may comprise, e.g., replacing amino acids susceptible for glycosylation, deamination, oxidization or isomerization by exchanging the respective amino acid(s) in the VHH sequence with the corresponding amino acid in the closest human germline sequence or with another amino acid (e.g. alanine). Similarly, the hydrophobic patches at the cell surface can be reduced by exchanging the respective amino acid(s) with less hydrophobic amino acid(s). Again, the amino acid replacements are chosen such that after the adaption the VHH sequence still binds to the same antigen and with similar characteristics as the VHH sequence before adaption.

VHHs can be used as modular building blocks for generating multivalent and/or multispecific antibody constructs, whereby "multivalent" means that the construct encompasses more than one single domain antibody and "multispecific" means that it encompasses single domain antibodies of more than one binding specificity.

At some occasions, the present disclosure states that a certain protein/amino acid sequence A is a "fragment" of another protein/amino acid sequence B. This means that, compared to protein/amino acid B, the protein/amino acid sequence A lacks one or more amino acids at the N-terminus and/or one or more amino acids at the C-terminus. Whether a protein/amino acid sequence lacks, compared to another protein/amino acid sequence, one or more amino acids at the N-terminus and/or one or more amino acids at the C-terminus can for example readily be determined upon forming a sequence alignment e.g. with the BLAST family of programs.

As the skilled person understands, when the present disclosure refers to an "VHH antibody domain or a fragment thereof', said fragment is an antigen-binding fragment. Thus, said fragment binds to the same antigen as the "full-length" VHH antibody domain according to the present disclosure from which said fragment is derived (i.e. to IL-18Ra resp. IL-18RP). Preferably, said fragment of said VHH antibody domain is a C-terminal fragment. This means that compared to the "complete" VHH antibody domain sequence said fragment lacks amino acids at the N-terminus.

When the present disclosure indicates that a VHH antibody domain or fragment thereof "of one VHH selected from the group consisting of VHH1, VHH2 and VHH3 as shown in the Table of CDRs" (or a corresponding wording), this means that said VHH antibody domain comprises the combination of CDRs or either VHH1 or VHH2 or VHH3, but not a mixture of CDRs selected from different of the listed VHHs. Thus, said VHH antibody domain or fragment thereof includes e.g. the combination of CDR1, CDR2 and CDR3 of VHH1 (SEQ ID NO: 23, 24 and 25), the combination of CDR1, CDR2 and CDR3 of VHH2 (SEQ ID NO: 26, 27, and 28), or the combination of CDR1, CDR2 and CDR3 of VHH3 (SEQ ID NO: 29, 30, and 31) etc., but not the combination of CDR1 and CDR2 of VHH1 and CDR3 of VHH2 (SEQ ID NO: 23, 24 and 28).

When the present disclosure defines that a VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 as defined in e.g. (a) with modification, wherein the modification is e.g. that the sequence of at least one of CDR1, CDR2 and CDR3 is humanized, the skilled person is aware that this humanization exists compared to the corresponding sequence in the Table of CDRs that provides the combinations of CDR sequences without modification.

If the present disclosure indicates the existence of a modification which is "replacement, addition or deletion" of a certain number of amino acids (e.g. up to three), the skilled person understands that this is an individual replacement, addition or deletion of the indicated number of amino acids. Thus, the replaced, added or deleted amino acids may be at neighboring positions or at independent, isolated positions within the amino acid sequence. Moreover, as above, the skilled person is aware that this definition indicates the replacement, addition or deletion compared to the unmodified sequence in the Table of CDRs.

Embodiment 2: The VHH antibody domain or fragment thereof according to embodiment 1, wherein the modification in (b) is that the sequence of CDR1 and/or CDR2, but not the sequence of CDR3 is humanized.

Embodiment 3: The VHH antibody domain or fragment thereof according to any one of embodiments 1 or 2, wherein the modification in (b) is that the sequence of CDR1 is humanized, but not the sequence of CDR2 and CDR3.

Embodiment 4: The VHH antibody domain or fragment thereof according to any one of embodiments 1 or 2, wherein the modification in (b) is that the sequence of CDR2 is humanized, but not the sequence of CDR1 and CDR3. Embodiment 5: The VHH antibody domain or fragment thereof according to any one of embodiments 1 to 4, wherein the modification in (b) is that the sequence of one, but not more than one of CDR1, CDR2 and CDR3 is humanized.

Embodiment 6: The VHH antibody domain or fragment thereof according to any one of embodiments 1 to 5, wherein said humanization of said CDR(s) is by replacing at least one amino acid in the sequence of said CDR by the corresponding amino acid of a human VH domain.

Embodiment 7: The VHH antibody domain or fragment thereof according to any one of embodiments 1 to 6, wherein said humanization of said CDR(s) is by replacing up to three amino acids in the sequence of said CDR by the corresponding amino acid of a human VH domain.

Embodiment 8: The VHH antibody domain or fragment thereof according to any one of embodiments 1 to 6, wherein said humanization of said CDR(s) is 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 by the corresponding amino acid of a human VH domain.

Embodiment 9: The VHH antibody domain or fragment thereof according to any one of embodiments 1 to 6, wherein said humanization of said CDR(s) is by replacing up to two amino acids in the sequence of said CDR by the corresponding amino acid of a human VH domain.

Embodiment 10: The VHH antibody domain or fragment thereof according to any one of embodiments 1 to 6, wherein said humanization of said CDR(s) is 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 by the corresponding amino acid of a human VH domain.

Embodiment 11 : The VHH antibody domain or fragment thereof according to any one of embodiments 1 to 10, wherein said humanization of said CDR(s) is by replacing one amino acid in the sequence of said CDR by the corresponding amino acid of a human VH domain.

Embodiment 12: The VHH antibody domain or fragment thereof according to any one of embodiments 1 to 11, wherein the modification in (c) is - the replacement, addition or deletion of up to three amino acids in CDR1,

- the replacement, addition or deletion of up to one amino acid in CDR3.

Embodiment 13: The VHH antibody domain or fragment thereof according to any one of embodiments 1 to 11, wherein the modification in (c) is

- the replacement, addition or deletion of up to two amino acids in CDR1,

- the replacement, addition or deletion of up to two amino acids in CDR2 and/or

- the replacement, addition or deletion of up to two amino acids in CDR3;

Embodiment 14: The VHH antibody domain or fragment thereof according to any one of embodiments 1 to 11, wherein the modification in (c) is

- the replacement, addition or deletion of up to two amino acids in CDR1;

- the replacement, addition or deletion of up to two amino acids in CDR2; and/or

- the replacement, addition or deletion of up to one amino acid in CDR3.

Embodiment 15: The VHH antibody domain or fragment thereof according to any one of embodiments 1 to 11, wherein the modification in (c) is

- the replacement, addition or deletion of up to two amino acids in CDR1 and/or

- the replacement, addition or deletion of up to two amino acids in CDR2; wherein the sequence of CDR3 is unmodified.

As a skilled person understands, the indication that the "sequence of CDR3 is unmodified" means that the sequence is unmodified compared to the sequence provided for CDR3 for the VHH at issue in the Table of CDRs.

Embodiment 16: The VHH antibody domain or fragment thereof according to any one of embodiments 1 to 11, wherein the modification in (c) is

- the replacement, addition or deletion of up to two amino acids in CDR1, wherein the sequence of CDR2 and CDR3 is unmodified.

Embodiment 17: The VHH antibody domain or fragment thereof according to any one of embodiments 1 to 11, wherein the modification in (c) is

- the replacement, addition or deletion of up to two amino acids in CDR2, wherein the sequence of CDR1 and CDR3 is unmodified.

Embodiment 18: The VHH antibody domain or fragment thereof according to any one of embodiments 1 to 11, wherein the modification in (c) is

- the replacement, addition or deletion of up to one amino acid in CDR1;

- the replacement, addition or deletion of up to one amino acid in CDR2; and/or

- the replacement, addition or deletion of up to one amino acid in CDR3.

Embodiment 19: The VHH antibody domain or fragment thereof according to any one of embodiments 1 to 11, wherein the modification in (c) is

- the replacement, addition or deletion of one amino acid in CDR1 and/or

- the replacement, addition or deletion of one amino acid in CDR2; wherein the sequence of CDR3 is unmodified.

Embodiment 20: The VHH antibody domain or fragment thereof according to any one of embodiments 1 to 11, wherein the modification in (c) is

- the replacement, addition or deletion of one amino acid in CDR1, wherein the sequence of CDR2 and CDR3 is unmodified.

Embodiment 21 : The VHH antibody domain or fragment thereof according to any one of embodiments 1 to 11, wherein the modification in (c) is

- the replacement, addition or deletion of one amino acid in CDR2, wherein the sequence of CDR1 and CDR3 is unmodified.

Embodiment 22: The VHH antibody domain or fragment thereof according to any one of embodiments 1 to 21, wherein the modification in (c) comprises only the replacement, but not the addition or deletion of amino acids.

Embodiment 23: The VHH antibody domain or fragment thereof according to any one of embodiments 1 to 22, wherein said replacement is a conservative amino acid replacement.

As used herein, a "conservative amino acid replacement" refers to the replacement of an amino acid by another, biologically similar amino acid. Conservative replacements are not likely to change the shape or characteristics of a protein/amino acid sequence. Examples of conservative replacements include the replacement of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine.

Second aspect of the present disclosure (also referred to as "Embodiment 24"): According to a second aspect, the present disclosure relates to a VHH antibody domain or a fragment thereof, wherein

Table of VHH Sequences:

Third aspect of the present disclosure (also referred to as "Embodiment 25"): According to a third aspect, the present disclosure relates to a VHH antibody domain or a fragment thereof, wherein

(D) said VHH antibody domain consists of a VHH sequence that is at least 75% identical to a VHH sequence referred to in (A). To the second and third aspects of the present disclosure and the embodiments referring thereto, the same explanations and definitions apply accordingly as for the first aspect of the present disclosure and the embodiments referring thereto.

As the skilled person is aware, if the present disclosure indicates that a VHH antibody domain comprises e.g. "the VHH 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", this means that said VHH antibody domain comprises one and (not multiple or all) of the sequences listed in the Table of VHH Sequences.

Thus, if the present disclosure indicates that a VHH antibody domain or fragment thereof comprises/consists e.g. of a VHH sequence as defined in (A) "with modification, wherein the modification is that said sequence is humanized", the skilled person is aware that this means that this humanization exists compared to the corresponding sequence in the Table of VHH Sequences that provides the sequences without modification.

If the present disclosure states that a certain sequence A "is at least x % identical" to another sequence B, this is synonymous to the statement that sequence A "has x % identity" to sequence B. The statement reflects a relationship between the two polypeptide sequences A and B determined by comparing the sequences. In general, identity refers to an exact amino acid to amino acid correspondence of the two polypeptide sequences, respectively, over the length of the sequences being compared. For sequences where there is not an exact correspondence, a percentage to which the two sequences are identical may be determined. In general, the two sequences to be compared are aligned to give a maximum correlation between the sequences. This may include inserting "gaps" in either one or both sequences, to enhance the degree of alignment. A % identity may be determined over the whole length of each of the sequences being compared (so-called global alignment), that is particularly suitable for sequences of the same or very similar length, or over shorter, defined lengths (so-called local alignment), that is more suitable for sequences of unequal length.

Methods for comparing the identity of two or more sequences are well known in the art. Thus, for instance, programs available in the Wisconsin Sequence Analysis Package, version 9.1 (Devereux J et al., 1984), for example the programs BESTFIT and GAP, may be used to determine the % identity between two polynucleotides and the % identity between two polypeptide sequences. BESTFIT uses the "local homology" algorithm of Smith and Waterman (1981) and finds the best single region of similarity between two sequences. Other programs for determining identity sequences are also known in the art, for instance the BLAST family of programs (Altschul S F et al, 1990, Altschul S F et al, 1997, accessible through the home page of the NCBI at www.ncbi.nlm.nih.gov) and FASTA (Pearson WR, 1990). Preferably, % identity according to the present disclosure is determined according to the BLAST family of programs (Altschul S F et al, 1990, Altschul S F et al, 1997, accessible through the home page of the NCBI at www.ncbi.nlm.nih.gov).

Embodiment 26: The VHH antibody domain or fragment thereof according to any one of embodiments 24 or 25, wherein said fragment of said VHH antibody domain comprises at least 75% of the amino acids of the sequence of said VHH antibody domain. As the skilled person understands, this means that that fragment lacks up to a quarter of the total number of amino acids of said VHH antibody domain, wherein, compared to the "complete" VHH antibody domain sequence said amino acids are lacking either at the N-terminus or at the C-terminus.

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

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

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

Embodiment 30: The VHH antibody domain or fragment thereof according to any one of embodiments 24 or 25, wherein said fragment of said VHH antibody domain comprises at least 95% of the amino acids of the sequence of said VHH antibody domain. 973 Embodiment 31 : The VHH antibody domain or fragment thereof according to any one of

974 embodiments 24 or 25, wherein said fragment of said VHH antibody domain comprises at least

975 98% of the amino acids of the sequence of said VHH antibody domain.

976

977 Embodiment 32: The VHH antibody domain or fragment thereof according to any one of

978 embodiments 24 or 25, wherein said fragment of said VHH antibody domain comprises at least

979 99% of the amino acids of the sequence of said VHH antibody domain.

980

981 Embodiment 33: The VHH antibody domain or fragment thereof according to any one of

982 embodiments 24 to 32, wherein said fragment of said VHH antibody domain comprises the

983 complementarity determining regions CDR1, CDR2 and CDR3.

984

985 Embodiment 34: The VHH antibody domain or fragment thereof according to any one of

986 embodiments 24 to 33, wherein said fragment of said VHH antibody domain comprises at least

987 the sequence from the N-terminus of CDR1 to the C-terminus of CDR3 of said VHH antibody

988 domain.

989

990 Embodiment 35: The VHH antibody domain or fragment thereof according to any one of

991 embodiments 24 to 34, wherein in (A) said fragment of said VHH antibody domain comprises

992 all the complementarity determining regions (CDRs) of said VHH antibody domain.

993

994 Embodiment 36: The VHH antibody domain or fragment thereof according to any one of

995 embodiments 24 to 35, wherein in (B) said humanization of said sequence is by replacing at

996 least one amino acid of said sequence by the corresponding amino acid of a human VH (variable

997 heavy) domain.

998

999 Embodiment 37: The VHH antibody domain or fragment thereof according to any one of

1000 embodiments 24 to 36, wherein in (B) said humanization of said sequence is by (individually)

1001 replacing up to 25 amino acids of said sequence by the corresponding amino acids of a human

1002 VH domain.

1003

1004 As the skilled person understands, if the present disclosure refers to the replacement of an amino

1005 acid of a certain sequence/domain A by the "corresponding" amino acid of a certain

1006 sequence/domain B, this designates that said amino acid of sequence/domain A is replaced by 1007 the amino acid in sequence/domain B that in an alignment of the two sequences aligns with said

1008 amino acid of sequence/domain A.

1009

1010 Embodiment 38: The VHH antibody domain or fragment thereof according to any one of

1011 embodiments 24 to 36, wherein in (B) said humanization of said sequence is by replacing up to

1012 20 amino acids of said sequence by the corresponding amino acids of a human VH domain.

1013

1014 Embodiment 39: The VHH antibody domain or fragment thereof according to any one of

1015 embodiments 24 to 36, wherein in (B) said humanization of said sequence is by replacing up to

1016 15 amino acids of said sequence by the corresponding amino acids of a human VH domain.

1017

1018 Embodiment 40: The VHH antibody domain or fragment thereof according to any one of

1019 embodiments 24 to 36, wherein in (B) said humanization of said sequence is by replacing up to

1020 10 amino acids of said sequence by the corresponding amino acids of a human VH domain.

1021

1022 Embodiment 41 : The VHH antibody domain or fragment thereof according to any one of

1023 embodiments 24 to 36, wherein in (B) said humanization of said sequence is by replacing up to

1024 5 amino acids of said sequence by the corresponding amino acids of a human VH domain.

1025

1026 Embodiment 42: The VHH antibody domain or fragment thereof according to any one of

1027 embodiments 24 to 36, wherein in (B) said humanization of said sequence is by replacing up to

1028 3 amino acids of said sequence by the corresponding amino acids of a human VH domain.

1029

1030 Embodiment 43: The VHH antibody domain or fragment thereof according to any one of

1031 embodiments 24 to 36, wherein in (B) said humanization of said sequence is by replacing up to

1032 2 amino acids of said sequence by the corresponding amino acids of a human VH domain.

1033

1034 Embodiment 44: The VHH antibody domain or fragment thereof according to any one of

1035 embodiments 24 to 36, wherein in (B) said humanization of said sequence is by replacing one

1036 amino acid of said sequence by the corresponding amino acid of a human VH domain.

1037

1038 Embodiment 45: The VHH antibody domain or fragment thereof according to any one of

1039 embodiments 24 to 44, wherein in (B) said humanization is within the framework regions of

1040 said VHH antibody domain and/or within the CDRs of said VHH antibody domain. 1041

1042 Embodiment 46: The VHH antibody domain or fragment thereof according to any one of

1043 embodiments 24 to 44, wherein in (B) said humanization is within the framework regions of

1044 said VHH antibody domain, but not within the CDRs of said VHH antibody domain.

1045

1046 Embodiment 47: The VHH antibody domain or fragment thereof according to any one of

1047 embodiments 24 to 36, wherein in (B) said humanization is within the CDRs of said VHH

1048 antibody domain, but not within the framework regions of said VHH antibody domain.

1049

1050 Embodiment 48: The VHH antibody domain or fragment thereof according to any one of

1051 embodiments 24 to 45 or 47, wherein in (B) said humanization within the CDRs of said VHH

1052 antibody domain is within CDR1, CDR2 and/or CDR3.

1053

1054 Embodiment 49: The VHH antibody domain or fragment thereof according to any one of

1055 embodiments 24 to 45 or 47 to 48, wherein in (B) said humanization within the CDRs of said

1056 VHH antibody domain is within CDR1 and/or CDR2.

1057

1058 Embodiment 50: The VHH antibody domain or fragment thereof according to any one of

1059 embodiments 24 to 45 or 47 to 49, wherein in (B) said humanization within the CDRs of said

1060 VHH antibody domain is within CDR1.

1061

1062 Embodiment 51: The VHH antibody domain or fragment thereof according to any one of

1063 embodiments 24 to 45 or 47 to 50, wherein in (B) said humanization within the CDRs of said

1064 VHH antibody domain is within CDR2.

1065

1066 Embodiment 52: The VHH antibody domain or fragment thereof according to any one of

1067 embodiments 24 to 51, wherein in (B) said humanization within the CDRs of said VHH

1068 antibody domain is not within CDR3.

1069

1070 Embodiment 53: The VHH antibody domain or fragment thereof according to any one of

1071 embodiments 24 to 52, wherein in (C) the modification is the replacement, addition or deletion

1072 of up to 20 amino acids.

1073 1074 Embodiment 54: The VHH antibody domain or fragment thereof according to any one of

1075 embodiments 24 to 52, wherein in (C) the modification is the replacement, addition or deletion

1076 of up to 15 amino acids.

1077

1078 Embodiment 55: The VHH antibody domain or fragment thereof according to any one of

1079 embodiments 24 to 52, wherein in (C) the modification is the replacement, addition or deletion

1080 of up to 10 amino acids.

1081

1082 Embodiment 56: The VHH antibody domain or fragment thereof according to any one of

1083 embodiments 24 to 52, wherein in (C) the modification is the replacement, addition or deletion

1084 of up to 5 amino acids.

1085

1086 Embodiment 57: The VHH antibody domain or fragment thereof according to any one of

1087 embodiments 24 to 52, wherein in (C) the modification is the replacement, addition or deletion

1088 of up to 3 amino acids.

1089

1090 Embodiment 58: The VHH antibody domain or fragment thereof according to any one of

1091 embodiments 24 to 52, wherein in (C) the modification is the replacement, addition or deletion

1092 of up to 2 amino acids.

1093

1094 Embodiment 59: The VHH antibody domain or fragment thereof according to any one of

1095 embodiments 24 to 52, wherein in (C) the modification is the replacement, addition or deletion

1096 of one amino acid.

1097

1098 Embodiment 60: The VHH antibody domain or fragment thereof according to any one of

1099 embodiments 24 to 59, wherein the modification in (C) comprises only the replacement, but not

1100 the addition or deletion of amino acids.

1101

1102 Embodiment 61 : The VHH antibody domain or fragment thereof according to any one of

1103 embodiments 24 to 60, wherein in (A) said VHH antibody domain or fragment thereof

1104 comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH

1105 selected from the group consisting of VHH1, VHH2, VHH3, VHH4, VHH5, VHH6, VHH8,

1106 VHH9, VHH10 and VHH11 as shown in the Table of CDRs.

1107 1108 Embodiment 62: The VHH antibody domain or fragment thereof according to any one of

1109 embodiments 24 to 61, wherein in (B) to (D)

1110 (a) said VHH antibody domain or fragment thereof comprises the complementarity

1111 determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group

1112 consisting of VHH1, VHH2, VHH3, VHH4, VHH5, VHH6, VHH8, VHH9, VHH10

1113 and VHH11 as shown in the Table of CDRs;

1114 (b) said VHH antibody domain or fragment thereof comprises the complementarity

1115 determining regions CDR1, CDR2 and CDR3 as defined in (a) with modification,

1116 wherein the modification is that the sequence of at least one of CDR1, CDR2 and

1117 CDR3 is humanized;

1118 (c) said VHH antibody domain or fragment thereof comprises the complementarity

1119 determining regions CDR1, CDR2 and CDR3 as defined in (a) with modification,

1120 wherein the modification is

1121 - the replacement, addition or deletion of up to three amino acids in CDR1,

1122 - the replacement, addition or deletion of up to three amino acids in CDR2

1123 and/or

1124 - the replacement, addition or deletion of up to three amino acids in CDR3.

1125

1126 Embodiment 63 : The VHH antibody domain or fragment thereof according to embodiment 62,

1127 wherein the modification in (b) is that the sequence of CDR1 and/or CDR2, but not the sequence

1128 of CDR3 is humanized.

1129

1130 Embodiment 64: The VHH antibody domain or fragment thereof according to any one of

1131 embodiments 62 or 63, wherein the modification in (b) is that the sequence of CDR1 is

1132 humanized, but not the sequence of CDR2 and CDR3.

1133

1134 Embodiment 65: The VHH antibody domain or fragment thereof according to any one of

1135 embodiments 62 or 63, wherein the modification in (b) is that the sequence of CDR2 is

1136 humanized, but not the sequence of CDR1 and CDR3.

1137

1138 Embodiment 66: The VHH antibody domain or fragment thereof according to any one of

1139 embodiments 62 to 65, wherein the modification in (b) is that the sequence of one, but not more

1140 than one of CDR1, CDR2 and CDR3 is humanized.

1141 1142 Embodiment 67: The VHH antibody domain or fragment thereof according to any one of

1143 embodiments 62 to 66, wherein said humanization of said CDR(s) is by replacing at least one

1144 amino acid in the sequence of said CDR by the corresponding amino acid of a human VH

1145 domain.

1146

1147 Embodiment 68: The VHH antibody domain or fragment thereof according to any one of

1148 embodiments 62 to 67, wherein said humanization of said CDR(s) is by replacing up to three

1149 amino acids in the sequence of said CDR by the corresponding amino acid of a human VH

1150 domain.

1151

1152 Embodiment 69: The VHH antibody domain or fragment thereof according to any one of

1153 embodiments 62 to 67, wherein said humanization of said CDR(s) is by replacing up to three

1154 amino acids in the sequence of CDR1 and/or CDR2 and up to one amino acid in the sequence

1155 of CDR3 by the corresponding amino acid of a human VH domain.

1156

1157 Embodiment 70: The VHH antibody domain or fragment thereof according to any one of

1158 embodiments 62 to 67, wherein said humanization of said CDR(s) is by replacing up to two

1159 amino acids in the sequence of said CDR by the corresponding amino acid of a human VH

1160 domain.

1161

1162 Embodiment 71 : The VHH antibody domain or fragment thereof according to any one of

1163 embodiments 62 to 67, wherein said humanization of said CDR(s) is by replacing up to two

1164 amino acids in the sequence of CDR1 and/or CDR2 and up to one amino acid in the sequence

1165 of CDR3 by the corresponding amino acid of a human VH domain.

1166

1167 Embodiment 72: The VHH antibody domain or fragment thereof according to any one of

1168 embodiments 62 to 71, wherein said humanization of said CDR(s) is by replacing one amino

1169 acid in the sequence of said CDR by the corresponding amino acid of a human VH domain.

1170

1171 Embodiment 73: The VHH antibody domain or fragment thereof according to any one of

1172 embodiments 62 to 72, wherein the modification in (c) is

1173 - the replacement, addition or deletion of up to three amino acids in CDR1,

1174 - the replacement, addition or deletion of up to three amino acids in CDR2 and/or

1175 - the replacement, addition or deletion of up to two amino acids in CDR3. 1176

1177 Embodiment 74: The VHH antibody domain or fragment thereof according to any one of

1178 embodiments 62 to 72, wherein the modification in (c) is

1179 - the replacement, addition or deletion of up to three amino acids in CDR1,

1180 - the replacement, addition or deletion of up to three amino acids in CDR2 and/or

1181 - the replacement, addition or deletion of up to one amino acid in CDR3.

1182

1183 Embodiment 75: The VHH antibody domain or fragment thereof according to any one of

1184 embodiments 62 to 72, wherein the modification in (c) is

1185 - the replacement, addition or deletion of up to three amino acids in CDR1, and/or

1186 - the replacement, addition or deletion of up to three amino acids in CDR2,

1187 wherein the sequence of CDR3 is unmodified.

1188

1189 Embodiment 76: The VHH antibody domain or fragment thereof according to any one of

1190 embodiments 62 to 72, wherein the modification in (c) is

1191 - the replacement, addition or deletion of up to two amino acids in CDR1,

1192 - the replacement, addition or deletion of up to two amino acids in CDR2 and/or

1193 - the replacement, addition or deletion of up to two amino acids in CDR3;

1194

1195 Embodiment 77: The VHH antibody domain or fragment thereof according to any one of

1196 embodiments 62 to 72, wherein the modification in (c) is

1197 - the replacement, addition or deletion of up to two amino acids in CDR1;

1198 - the replacement, addition or deletion of up to two amino acids in CDR2; and/or

1199 - the replacement, addition or deletion of up to one amino acid in CDR3.

1200

1201 Embodiment 78: The VHH antibody domain or fragment thereof according to any one of

1202 embodiments 62 to 72, wherein the modification in (c) is

1203 - the replacement, addition or deletion of up to two amino acids in CDR1 and/or

1204 - the replacement, addition or deletion of up to two amino acids in CDR2;

1205 wherein the sequence of CDR3 is unmodified.

1206

1207 As the skilled person is aware, "unmodified" means unmodified compared to the sequence in

1208 the Table of CDRs.

1209 1210 Embodiment 79: The VHH antibody domain or fragment thereof according to any one of

1211 embodiments 62 to 72, wherein the modification in (c) is

1212 - the replacement, addition or deletion of up to two amino acids in CDR1,

1213 wherein the sequence of CDR2 and CDR3 is unmodified.

1214

1215 Embodiment 80: The VHH antibody domain or fragment thereof according to any one of

1216 embodiments 62 to 72, wherein the modification in (c) is

1217 - the replacement, addition or deletion of up to two amino acids in CDR2,

1218 wherein the sequence of CDR1 and CDR3 is unmodified.

1219

1220 Embodiment 81 : The VHH antibody domain or fragment thereof according to any one of

1221 embodiments 62 to 72, wherein the modification in (c) is

1222 - the replacement, addition or deletion of up to one amino acid in CDR1;

1223 - the replacement, addition or deletion of up to one amino acid in CDR2; and/or

1224 - the replacement, addition or deletion of up to one amino acid in CDR3.

1225

1226 Embodiment 82: The VHH antibody domain or fragment thereof according to any one of

1227 embodiments 62 to 72, wherein the modification in (c) is

1228 - the replacement, addition or deletion of one amino acid in CDR1 and/or

1229 - the replacement, addition or deletion of one amino acid in CDR2;

1230 wherein the sequence of CDR3 is unmodified.

1231

1232 Embodiment 83: The VHH antibody domain or fragment thereof according to any one of

1233 embodiments 62 to 72, wherein the modification in (c) is

1234 - the replacement, addition or deletion of one amino acid in CDR1,

1235 wherein the sequence of CDR2 and CDR3 is unmodified.

1236

1237 Embodiment 84: The VHH antibody domain or fragment thereof according to any one of

1238 embodiments 62 to 72, wherein the modification in (c) is

1239 - the replacement, addition or deletion of one amino acid in CDR2,

1240 wherein the sequence of CDR1 and CDR3 is unmodified.

1241 1242 Embodiment 85: The VHH antibody domain or fragment thereof according to any one of

1243 embodiments 62 to 84, wherein the modification in (c) comprises only the replacement, but not

1244 the addition or deletion of amino acids.

1245

1246 Embodiment 86: The VHH antibody domain or fragment thereof according to any one of

1247 embodiments 62 to 85, wherein said replacement is a conservative amino acid replacement.

1248

1249 Embodiment 87: The VHH antibody domain or fragment thereof according to any one of

1250 embodiments 1 to 86, wherein said fragment consists of at least 100 amino acids.

1251

1252 Embodiment 88: The VHH antibody domain or fragment thereof according to any one of

1253 embodiments 1 to 86, wherein said fragment consists of at least 105 amino acids.

1254

1255 Embodiment 89: The VHH antibody domain or fragment thereof according to any one of

1256 embodiments 1 to 86, wherein said fragment consists of at least 110 amino acids.

1257

1258 Embodiment 90: The VHH antibody domain or fragment thereof according to any one of

1259 embodiments 1 to 86, wherein said fragment consists of at least 115 amino acids.

1260

1261 Embodiment 91 : The VHH antibody domain or fragment thereof according to any one of

1262 embodiments 1 to 90, wherein said VHH antibody domain is an anti-IL-18Ra VHH antibody

1263 domain.

1264

1265 Embodiment 92: The VHH antibody domain or fragment thereof according to any one of

1266 embodiments 1 to 91, wherein said VHH antibody domain is specific for IL-18Ra.

1267

1268 Embodiment 93: The VHH antibody domain or fragment thereof according to any one of

1269 embodiments 1 to 92, wherein said VHH antibody domain or fragment thereof binds to IL-

1270 18Ra.

1271

1272 Embodiment 94: The VHH antibody domain or fragment thereof according to any one of

1273 embodiments 1 to 93, wherein said VHH antibody domain or fragment thereof binds

1274 specifically to IL-18Ra.

1275 1276 Embodiment 95: The VHH antibody domain or fragment thereof according to any one of

1277 embodiments 1 to 94, wherein said VHH antibody domain or fragment thereof does not bind to

1278 IL-18RP.

1279

1280 Embodiment 96: The VHH antibody domain or fragment thereof according to any one of

1281 embodiments 1 to 95, wherein said VHH antibody domain or fragment thereof is capable of

1282 specifically binding to IL-18Ra ECD.

1283

1284 In some embodiments, if the present disclosure states that a first molecule/molecular group (e.g.

1285 an antibody/antibody component) "is capable of specifically binding"/" specifically binds" to a

1286 second molecule/molecular group (e.g. an antigen of interest), this means that the first

1287 molecule/molecular group (in this example the antibody) binds to said second

1288 molecule/molecular group (in this example the antigen of interest) with an affinity that is at

1289 least ten-fold greater than its affinity for other molecules/molecular groups, in particular other

1290 molecule/molecular group in the human body (in this example at least ten-fold greater than its

1291 affinity for binding to non-specific antigens (e.g., BSA, casein) other than said antigen of

1292 interest (or closely related antigens)). In a preferred embodiment, a first molecule/molecular

1293 group (e.g. an antibody/antibody component) that "specifically binds" to a second

1294 molecule/molecular group (e.g. an antigen of interest) binds to said antigen with an affinity that

1295 is at least 100-fold greater than its affinity for other molecules/molecular groups, in particular

1296 other molecule/molecular group in the human body (in this example at least 100-fold greater

1297 than its affinity for binding to non-specific antigens other than said antigen of interest (or

1298 closely related antigens)). Typically said binding will be determined under physiological

1299 conditions. A first molecule/molecular group that "specifically binds" to a second

1300 molecule/molecular group may bind to that second molecule/molecular group with a KD of

1301 1 * 10'⁷ M or stronger.

1302

1303 Embodiment 97: The VHH antibody domain or fragment thereof according to any one of

1304 embodiments 1 to 96, wherein said VHH antibody domain or fragment thereof binds to

1305 recombinant human IL-18Ra ECD with a KD of 1x1 O'⁶ M or stronger.

1306

1307 Embodiment 98: The VHH antibody domain or fragment thereof according to any one of

1308 embodiments 1 to 97, wherein said VHH antibody domain or fragment thereof binds to

1309 recombinant human IL-18Ra ECD with a KD of 1x1 O'⁷ M or stronger. 1310

1311 Embodiment 99: The VHH antibody domain or fragment thereof according to any one of

1312 embodiments 1 to 97, wherein said VHH antibody domain or fragment thereof binds to

1313 recombinant human IL-18Ra ECD with a KD of 5x1 O'⁸ M or stronger.

1314

1315 Embodiment 100: The VHH antibody domain or fragment thereof according to any one of

1316 embodiments 1 to 97, wherein said VHH antibody domain or fragment thereof binds to

1317 recombinant human IL-18Ra ECD with a KD of 1.5x1 O'⁸ M or stronger.

1318

1319 Embodiment 101 : The VHH antibody domain or fragment thereof according to any one of

1320 embodiments 1 to 96, wherein said VHH antibody domain or fragment thereof binds to

1321 recombinant human IL-18Ra ECD with a KD of 1x1 O'⁸ M or stronger.

1322

1323 Embodiment 102: The VHH antibody domain or fragment thereof according to any one of

1324 embodiments 1 to 96, wherein said VHH antibody domain or fragment thereof binds to

1325 recombinant human IL-18Ra ECD with a KD of 5x1 O'⁹ M or stronger.

1326

1327 Embodiment 103: The VHH antibody domain or fragment thereof according to any one of

1328 embodiments 1 to 96, wherein said VHH antibody domain or fragment thereof binds to

1329 recombinant human IL-18Ra ECD with a KD of 1x1 O'⁹ M or stronger.

1330

1331 Embodiment 104: The VHH antibody domain or fragment thereof according to any one of

1332 embodiments 1 to 103, wherein said fragment of said VHH antibody domain binds to

1333 recombinant human IL-18Ra ECD with at least 90% of the affinity (as determined by KD value)

1334 with which said VHH antibody domain binds to recombinant human IL-18Ra ECD.

1335

1336 Embodiment 105: The VHH antibody domain or fragment thereof according to any one of

1337 embodiments 1 to 104, wherein said fragment of said VHH antibody domain binds to IL-18Ra

1338 ECD with an affinity (as determined from the KD value) that is at least equal to the affinity

1339 with which VHH2 (SEQ ID NO: 2) binds to IL-18Ra ECD or stronger.

1340

1341 Such binding can be determined by in vitro binding experiments as described in Example 1

1342 below (by biolayer interferometry).

1343 1344 Embodiment 106: The VHH antibody domain or fragment thereof according to any one of

1345 embodiments 1 to 23 or 62 to 105, wherein in (b) and (c) the VHH antibody domain or fragment

1346 thereof binds to recombinant human IL-18Ra ECD with an affinity (KD value) that is by not

1347 more than a factor of 10 weaker than the binding of the corresponding VHH antibody domain

1348 without modification.

1349

1350 Embodiment 107: The VHH antibody domain or fragment thereof according to any one of

1351 embodiments 1 to 23 or 62 to 105, wherein in (b) and (c) the VHH antibody domain or fragment

1352 thereof binds to recombinant human IL-18Ra ECD with an affinity (KD value) that is by not

1353 more than a factor of 5 weaker than the binding of the corresponding VHH antibody domain

1354 without modification.

1355

1356 Embodiment 108: The VHH antibody domain or fragment thereof according to any one of

1357 embodiments 1 to 23 or 62 to 105, wherein in (b) and (c) the VHH antibody domain or fragment

1358 thereof binds to recombinant human IL-18Ra ECD with an affinity (KD value) that is by not

1359 more than a factor of 2 weaker than the binding of the corresponding VHH antibody domain

1360 without modification.

1361

1362 Embodiment 109: The VHH antibody domain or fragment thereof according to any one of

1363 embodiments 1 to 23 or 62 to 105, wherein in (b) and (c) the VHH antibody domain or fragment

1364 thereof binds to recombinant human IL-18Ra ECD with an affinity (KD value) that is by not

1365 more than a factor of 1.5 weaker than the binding of the corresponding VHH antibody domain

1366 without modification.

1367

1368 Embodiment 110: The VHH antibody domain or fragment thereof according to any one of

1369 embodiments 1 to 23 or 62 to 109, wherein in (b) and (c) the VHH antibody domain or fragment

1370 thereof binds to recombinant human IL-18Ra ECD with an affinity (KD value) that is by not

1371 more than a factor of 10 stronger than the binding of the corresponding VHH antibody domain

1372 without modification.

1373

1374 Embodiment 111 : The VHH antibody domain or fragment thereof according to any one of

1375 embodiments 1 to 23 or 62 to 109, wherein in (b) and (c) the VHH antibody domain or fragment

1376 thereof binds to recombinant human IL-18Ra ECD with an affinity (KD value) that is by not 1377 more than a factor of 5 stronger than the binding of the corresponding VHH antibody domain

1378 without modification.

1379

1380 Embodiment 112: The VHH antibody domain or fragment thereof according to any one of

1381 embodiments 1 to 23 or 62 to 109, wherein in (b) and (c) the VHH antibody domain or fragment

1382 thereof binds to recombinant human IL-18Ra ECD with an affinity (KD value) that is by not

1383 more than a factor of 2 stronger than the binding of the corresponding VHH antibody domain

1384 without modification.

1385

1386 Embodiment 113: The VHH antibody domain or fragment thereof according to any one of

1387 embodiments 1 to 23 or 62 to 109, wherein in (b) and (c) the VHH antibody domain or fragment

1388 thereof binds to recombinant human IL-18Ra ECD with an affinity (KD value) that is by not

1389 more than a factor of 1.5 stronger than the binding of the corresponding VHH antibody domain

1390 without modification.

1391

1392 Embodiment 114: The VHH antibody domain or fragment thereof according to any one of

1393 embodiments 24 to 113, wherein in (B) and (C) the VHH antibody domain binds to recombinant

1394 human IL-18Ra ECD with an affinity (KD value) that is by not more than a factor of 10 weaker

1395 than the binding of the corresponding VHH antibody domain without modification.

1396

1397 Embodiment 115: The VHH antibody domain or fragment thereof according to any one of

1398 embodiments 24 to 113, wherein in (B) and (C) the VHH antibody domain binds to recombinant

1399 human IL-18Ra ECD with an affinity (KD value) that is by not more than a factor of 5 weaker

1400 than the binding of the corresponding VHH antibody domain without modification.

1401

1402 Embodiment 116: The VHH antibody domain or fragment thereof according to any one of

1403 embodiments 24 to 113, wherein in (B) and (C) the VHH antibody domain binds to recombinant

1404 human IL-18Ra ECD with an affinity (KD value) that is by not more than a factor of 2 weaker

1405 than the binding of the corresponding VHH antibody domain without modification.

1406

1407 Embodiment 117: The VHH antibody domain or fragment thereof according to any one of

1408 embodiments 24 to 113, wherein in (B) and (C) the VHH antibody domain binds to recombinant

1409 human IL-18Ra ECD with an affinity (KD value) that is by not more than a factor of 1.5 weaker

1410 than the binding of the corresponding VHH antibody domain without modification. 1411

1412 Embodiment 118: The VHH antibody domain or fragment thereof according to any one of

1413 embodiments 24 to 117, wherein in (B) and (C) the VHH antibody domain binds to recombinant

1414 human IL- 18Ra ECD with an affinity (KD value) that i s by not more than a factor of 10 stronger

1415 than the binding of the corresponding VHH antibody domain without modification.

1416

1417 Embodiment 119: The VHH antibody domain or fragment thereof according to any one of

1418 embodiments 24 to 117, wherein in (B) and (C) the VHH antibody domain binds to recombinant

1419 human IL-18Ra ECD with an affinity (KD value) that is by not more than a factor of 5 stronger

1420 than the binding of the corresponding VHH antibody domain without modification.

1421

1422 Embodiment 120: The VHH antibody domain or fragment thereof according to any one of

1423 embodiments 24 to 117, wherein in (B) and (C) the VHH antibody domain binds to recombinant

1424 human IL-18Ra ECD with an affinity (KD value) that is by not more than a factor of 2 stronger

1425 than the binding of the corresponding VHH antibody domain without modification.

1426

1427 Embodiment 121 : The VHH antibody domain or fragment thereof according to any one of

1428 embodiments 24 to 117, wherein in (B) and (C) the VHH antibody domain binds to recombinant

1429 human IL-18Ra ECD with an affinity (KD value) that is by not more than a factor of 1.5

1430 stronger than the binding of the corresponding VHH antibody domain without modification.

1431

1432 Embodiment 122: The VHH antibody domain or fragment thereof according to any one of

1433 embodiments 24 to 121, wherein in (D) the affinity (KD value) of the binding of the VHH

1434 antibody domain to recombinant human IL-18RaECD is by not more than a factor of 10 weaker

1435 than the affinity (KD value) of the binding to recombinant human IL-18Ra ECD of a VHH

1436 antibody domain consisting of the sequence from the Table of VHH Sequences that has the

1437 highest degree of sequence identity with the sequence of said VHH antibody domain of (D).

1438 The degree of sequence identity can be determined by sequence alignment.

1439

1440 Embodiment 123: The VHH antibody domain or fragment thereof according to any one of

1441 embodiments 24 to 121, wherein in (D) the affinity (KD value) of the binding of the VHH

1442 antibody domain to recombinant human IL-18Ra ECD is by not more than a factor of 5 weaker

1443 than the affinity (KD value) of the binding to recombinant human IL-18Ra ECD of a VHH

1444 antibody domain consisting of the sequence from the Table of VHH Sequences that has the 1445 highest degree of sequence identity with the sequence of said VHH antibody domain of (D).

1446 The degree of sequence identity can be determined by sequence alignment.

1447

1448 Embodiment 124: The VHH antibody domain or fragment thereof according to any one of

1449 embodiments 24 to 121, wherein in (D) the affinity (KD value) of the binding of the VHH

1450 antibody domain to recombinant human IL-18Ra ECD is by not more than a factor of 2 weaker

1451 than the affinity (KD value) of the binding to recombinant human IL-18Ra ECD of a VHH

1452 antibody domain consisting of the sequence from the Table of VHH Sequences that has the

1453 highest degree of sequence identity with the sequence of said VHH antibody domain of (D).

1454

1455 Embodiment 125: The VHH antibody domain or fragment thereof according to any one of

1456 embodiments 24 to 121, wherein in (D) the affinity (KD value) of the binding of the VHH

1457 antibody domain to recombinant human IL-18Ra ECD is by not more than a factor of 1.5

1458 weaker than the affinity (KD value) of the binding to recombinant human IL-18Ra ECD of a

1459 VHH antibody domain consisting of the sequence from the Table of VHH Sequences that has

1460 the highest degree of sequence identity with the sequence of said VHH antibody domain of (D).

1461

1462 Embodiment 126: The VHH antibody domain or fragment thereof according to any one of

1463 embodiments 24 to 125, wherein in (D) the affinity (KD value) of the binding of the VHH

1464 antibody domain to recombinant human IL-18Ra ECD is by not more than a factor of 10

1465 stronger than the affinity (KD value) of the binding to recombinant human IL-18Ra ECD of a

1466 VHH antibody domain consisting of the sequence from the Table of VHH Sequences that has

1467 the highest degree of sequence identity with the sequence of said VHH antibody domain of (D).

1468

1469 Embodiment 127: The VHH antibody domain or fragment thereof according to any one of

1470 embodiments 24 to 125, wherein in (D) the affinity (KD value) of the binding of the VHH

1471 antibody domain to recombinant human IL-18Ra ECD is by not more than a factor of 5 stronger

1472 than the affinity (KD value) of the binding to recombinant human IL-18Ra ECD of a VHH

1473 antibody domain consisting of the sequence from the Table of VHH Sequences that has the

1474 highest degree of sequence identity with the sequence of said VHH antibody domain of (D).

1475

1476 Embodiment 128: The VHH antibody domain or fragment thereof according to any one of

1477 embodiments 24 to 125, wherein in (D) the affinity (KD value) of the binding of the VHH

1478 antibody domain to recombinant human IL-18Ra ECD is by not more than a factor of 2 stronger 1479 than the affinity (KD value) of the binding to recombinant human IL-18Ra ECD of a VHH

1480 antibody domain consisting of the sequence from the Table of VHH Sequences that has the

1481 highest degree of sequence identity with the sequence of said VHH antibody domain of (D).

1482

1483 Embodiment 129: The VHH antibody domain or fragment thereof according to any one of

1484 embodiments 24 to 125, wherein in (D) the affinity (KD value) of the binding of the VHH

1485 antibody domain to recombinant human IL-18Ra ECD is by not more than a factor of 1.5

1486 stronger than the affinity (KD value) of the binding to recombinant human IL-18Ra ECD of a

1487 VHH antibody domain consisting of the sequence from the Table of VHH Sequences that has

1488 the highest degree of sequence identity with the sequence of said VHH antibody domain of (D).

1489

1490 Embodiment 130: The VHH antibody domain or fragment thereof according to any one of

1491 embodiments 91 to 129, wherein said KD value is determined in in vitro binding experiments

1492 through biolayer interferometry.

1493

1494 Embodiment 131 : The VHH antibody domain or fragment thereof according to any one of

1495 embodiments 91 to 130, wherein said KD value is measured by kinetic measurements by

1496 biolayer interferometry at 25°C and 1000 rpm in KB Buffer (PBS + 0.1 % Tween-20 +

1497 1% BSA).

1498

1499 Embodiment 132: The VHH antibody domain or fragment thereof according to any one of

1500 embodiments 24 to 131, wherein said VHH antibody domain of (A) comprises one VHH

1501 sequence selected from the group consisting of VHH1, VHH2, VHH3, VHH4, VHH5, VHH6,

1502 VHH8, VHH9 and VHH10 shown in the Table of VHH Sequences.

1503

1504 Embodiment 133: The VHH antibody domain or fragment thereof according to any one of

1505 embodiments 24 to 131, wherein said VHH antibody domain of (A) comprises one VHH

1506 sequence selected from the group consisting of VHH1, VHH2, VHH5, VHH6, VHH8, VHH9

1507 and VHH10 shown in the Table of VHH Sequences.

1508

1509 Embodiment 134: The VHH antibody domain or fragment thereof according to any one of

1510 embodiments 24 to 131, wherein said VHH antibody domain of (A) comprises one VHH

1511 sequence selected from the group consisting of VHH2, VHH6, VHH8 and VHH10 shown in

1512 the Table of VHH Sequences. 1513

1514 Embodiment 135: The VHH antibody domain or fragment thereof according to any one of

1515 embodiments 24 to 131, wherein said VHH antibody domain of (A) comprises one VHH

1516 sequence selected from the group consisting of VHH6 and VHHl 1 shown in the Table of VHH

1517 Sequences.

1518

1519 Embodiment 136: The VHH antibody domain or fragment thereof according to any one of

1520 embodiments 24 to 131, wherein said VHH antibody domain of (A) comprises one VHH

1521 sequence selected from the group consisting of VHHl, VHH2, VHH5, VHH6, VHH8 and

1522 VHH10 shown in the Table of VHH Sequences.

1523

1524 Embodiment 137: The VHH antibody domain or fragment thereof according to any one of

1525 embodiments 24 to 131, wherein said VHH antibody domain of (A) comprises one VHH

1526 sequence selected from the group consisting of VHH2, VHH6, VHH8 and VHH10 shown in

1527 the Table of VHH Sequences.

1528

1529 Embodiment 138: The VHH antibody domain or fragment thereof according to any one of

1530 embodiments 24 to 131, wherein said VHH antibody domain of (A) comprises one VHH

1531 sequence selected from the group consisting of VHH2 and VHH8 shown in the Table of VHH

1532 Sequences.

1533

1534 Embodiment 139: The VHH antibody domain or fragment thereof according to any one of

1535 embodiments 24 to 131, wherein said VHH antibody domain of (A) comprises the VHH

1536 sequence VHHl shown in the Table of VHH Sequences.

1537

1538 Embodiment 140: The VHH antibody domain or fragment thereof according to any one of

1539 embodiments 24 to 131, wherein said VHH antibody domain of (A) comprises the VHH

1540 sequence VHH2 shown in the Table of VHH Sequences.

1541

1542 Embodiment 141 : The VHH antibody domain or fragment thereof according to any one of

1543 embodiments 24 to 131, wherein said VHH antibody domain of (A) comprises the VHH

1544 sequence VHH3 shown in the Table of VHH Sequences.

1545 1546 Embodiment 142: The VHH antibody domain or fragment thereof according to any one of

1547 embodiments 24 to 131, wherein said VHH antibody domain of (A) comprises the VHH

1548 sequence VHH4 shown in the Table of VHH Sequences.

1549

1550 Embodiment 143: The VHH antibody domain or fragment thereof according to any one of

1551 embodiments 24 to 131, wherein said VHH antibody domain of (A) comprises the VHH

1552 sequence VHH5 shown in the Table of VHH Sequences.

1553

1554 Embodiment 144: The VHH antibody domain or fragment thereof according to any one of

1555 embodiments 24 to 131, wherein said VHH antibody domain of (A) comprises the VHH

1556 sequence VHH6 shown in the Table of VHH Sequences.

1557

1558 Embodiment 145: The VHH antibody domain or fragment thereof according to any one of

1559 embodiments 24 to 131, wherein said VHH antibody domain of (A) comprises the VHH

1560 sequence VHH8 shown in the Table of VHH Sequences.

1561

1562 Embodiment 146: The VHH antibody domain or fragment thereof according to any one of

1563 embodiments 24 to 131, wherein said VHH antibody domain of (A) comprises the VHH

1564 sequence VHH9 shown in the Table of VHH Sequences.

1565

1566 Embodiment 147: The VHH antibody domain or fragment thereof according to any one of

1567 embodiments 24 to 131, wherein said VHH antibody domain of (A) comprises the VHH

1568 sequence VHH10 shown in the Table of VHH Sequences.

1569

1570 Embodiment 148: The VHH antibody domain or fragment thereof according to any one of

1571 embodiments 24 to 131, wherein said VHH antibody domain of (A) comprises the VHH

1572 sequence VHH11 shown in the Table of VHH Sequences.

1573

1574 Embodiment 149: The VHH antibody domain or fragment thereof according to any one of

1575 embodiments 1 to 23 or 62 to 148, wherein in (a) said VHH antibody domain or fragment

1576 thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one

1577 VHH selected from the group consisting of VHH1, VHH2, VHH3, VHH4, VHH5, VHH6,

1578 VHH8, VHH9 and VHH10 as shown in the Table of CDRs.

1579 1580 Embodiment 150: The VHH antibody domain or fragment thereof according to any one of

1581 embodiments 1 to 23 or 62 to 148, wherein in (a) said VHH antibody domain or fragment

1582 thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one

1583 VHH selected from the group consisting of VHH1, VHH2, VHH5, VHH6, VHH8, VHH9 and

1584 VHH10 as shown in the Table of CDRs.

1585

1586 Embodiment 151 : The VHH antibody domain or fragment thereof according to any one of

1587 embodiments 1 to 23 or 62 to 148, wherein in (a) said VHH antibody domain or fragment

1588 thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of

1589 VHH2, VHH6, VHH8 and VHH10 as shown in the Table of CDRs.

1590

1591 Embodiment 152: The VHH antibody domain or fragment thereof according to any one of

1592 embodiments 1 to 23 or 62 to 148, wherein in (a) said VHH antibody domain or fragment

1593 thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one

1594 VHH selected from the group consisting of VHH6 and VHH11.

1595

1596 Embodiment 153: The VHH antibody domain or fragment thereof according to any one of

1597 embodiments 1 to 23 or 62 to 148, wherein in (a) said VHH antibody domain or fragment

1598 thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one

1599 VHH selected from the group consisting of VHH1, VHH2, VHH5, VHH6, VHH8 and VHH10

1600 as shown in the Table of CDRs.

1601

1602 Embodiment 154: The VHH antibody domain or fragment thereof according to any one of

1603 embodiments 1 to 23 or 62 to 148, wherein in (a) said VHH antibody domain or fragment

1604 thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one

1605 VHH selected from the group consisting of VHH2, VHH6, VHH8, VHH10 as shown in the

1606 Table of CDRs.

1607

1608 Embodiment 155: The VHH antibody domain or fragment thereof according to any one of

1609 embodiments 1 to 23 or 62 to 148, wherein in (a) said VHH antibody domain or fragment

1610 thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one

1611 VHH selected from the group consisting of VHH2 or VHH8 as shown in the Table of CDRs.

1612 1613 Embodiment 156: The VHH antibody domain or fragment thereof according to any one of

1614 embodiments 1 to 23 or 62 to 148, wherein in (a) said VHH antibody domain or fragment

1615 thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH1

1616 as shown in the Table of CDRs.

1617

1618 Embodiment 157: The VHH antibody domain or fragment thereof according to any one of

1619 embodiments 1 to 23 or 62 to 148, wherein in (a) said VHH antibody domain or fragment

1620 thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH2

1621 as shown in the Table of CDRs.

1622

1623 Embodiment 158: The VHH antibody domain or fragment thereof according to any one of

1624 embodiments 1 to 23 or 62 to 148, wherein in (a) said VHH antibody domain or fragment

1625 thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH3

1626 as shown in the Table of CDRs.

1627

1628 Embodiment 159: The VHH antibody domain or fragment thereof according to any one of

1629 embodiments 1 to 23 or 62 to 148, wherein in (a) said VHH antibody domain or fragment

1630 thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH4

1631 as shown in the Table of CDRs.

1632

1633 Embodiment 160: The VHH antibody domain or fragment thereof according to any one of

1634 embodiments 1 to 23 or 62 to 148, wherein in (a) said VHH antibody domain or fragment

1635 thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH5

1636 as shown in the Table of CDRs.

1637

1638 Embodiment 161 : The VHH antibody domain or fragment thereof according to any one of

1639 embodiments 1 to 23 or 62 to 148, wherein in (a) said VHH antibody domain or fragment

1640 thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH6

1641 as shown in the Table of CDRs.

1642

1643 Embodiment 162: The VHH antibody domain or fragment thereof according to any one of

1644 embodiments 1 to 23 or 62 to 148, wherein in (a) said VHH antibody domain or fragment

1645 thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH8

1646 as shown in the Table of CDRs. 1647

1648 Embodiment 163: The VHH antibody domain or fragment thereof according to any one of

1649 embodiments 1 to 23 or 62 to 148, wherein in (a) said VHH antibody domain or fragment

1650 thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH9

1651 as shown in the Table of CDRs.

1652

1653 Embodiment 164: The VHH antibody domain or fragment thereof according to any one of

1654 embodiments 1 to 23 or 62 to 148, wherein in (a) said VHH antibody domain or fragment

1655 thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of

1656 VHH10 as shown in the Table of CDRs.

1657

1658 Embodiment 165: The VHH antibody domain or fragment thereof according to any one of

1659 embodiments 1 to 23 or 62 to 148, wherein in (a) said VHH antibody domain or fragment

1660 thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of

1661 VHH11 as shown in the Table of CDRs.

1662

1663 Embodiment 166: The VHH antibody domain or fragment thereof according to any one of

1664 embodiments 1 to 165, wherein said VHH antibody domain or fragment thereof competes with

1665 VHH2 for binding to recombinant human IL-18Ra ECD.

1666

1667 Embodiment 167: The VHH antibody domain or fragment thereof according to any one of

1668 embodiments 1 to 165, wherein said VHH antibody domain or fragment thereof partially

1669 competes with VHH2 for binding to recombinant human IL-18Ra ECD.

1670

1671 Embodiment 168: The VHH antibody domain or fragment thereof according to any one of

1672 embodiments 1 to 167, wherein said VHH antibody domain or fragment thereof does not

1673 compete with VHH15 for binding to recombinant human IL-18RP ECD.

1674

1675 Embodiment 169: The VHH antibody domain or fragment thereof according to any one of

1676 embodiments 1 to 168, wherein said humanization is by germlining.

1677

1678 Embodiment 170: The VHH antibody domain or fragment thereof according to embodiment

1679 169, wherein said germlining involves 1680 - identifying the human VH germline sequence that is closest to the sequence to be

1681 humanized with respect to its sequence similarity; and

1682 - replacing amino acid(s) in the sequence to be humanized by the amino acid(s) at the

1683 corresponding sequence position(s) in the closest human VH germline sequence.

1684

1685 Sequence similarity and corresponding sequence positions can be determined by sequence

1686 alignment as described above.

1687

1688 Embodiment 171 : The VHH antibody domain or fragment thereof according to embodiment

1689 169, wherein said germlining consists of

1690 - identifying the human VH germline sequence that is closest to the sequence to be

1691 humanized with respect to its sequence similarity; and

1692 - replacing amino acid(s) in the sequence to be humanized by the amino acid(s) at the

1693 corresponding sequence position(s) in the closest human VH germline sequence.

1694

1695 Embodiment 172: The VHH antibody domain or fragment thereof according to any one of

1696 embodiments 1 to 171, wherein said VHH antibody domain (resp. fragment thereof) with

1697 humanization binds to human IL-18Ra ECD with an affinity (KD value) that is not weaker by

1698 a factor of more than 10 compared to the binding of a corresponding VHH antibody domain

1699 (resp. fragment thereof) without humanization to human IL-18Ra ECD.

1700

1701 As a skilled person understands, the term "corresponding VHH antibody domain without

1702 humanization" refers to a VHH antibody domain that differs only by the amino acid changes

1703 introduced in order to carry out said humanization, but that is otherwise identical.

1704

1705 Embodiment 173: The VHH antibody domain or fragment thereof according to any one of

1706 embodiments 1 to 171, wherein said VHH antibody domain (resp. fragment thereof) with

1707 humanization binds to human IL-18Ra ECD with an affinity (KD value) that is not weaker by

1708 a factor of more than 5 compared to the binding of a corresponding VHH antibody domain

1709 (resp. fragment thereof) without humanization to human IL-18Ra ECD.

1710

1711 Embodiment 174: The VHH antibody domain or fragment thereof according to any one of

1712 embodiments 1 to 171, wherein said VHH antibody domain (resp. fragment thereof) with

1713 humanization binds to human IL-18Ra ECD with an affinity (KD value) that is not weaker by 1714 a factor of more than 2 compared to the binding of a corresponding VHH antibody domain

1715 (resp. fragment thereof) without humanization to human IL-18Ra ECD.

1716

1717 Embodiment 175: The VHH antibody domain or fragment thereof according to any one of

1718 embodiments 1 to 171, wherein said VHH antibody domain (resp. fragment thereof) with

1719 humanization binds to human IL-18Ra ECD with an affinity (KD value) that is not weaker than

1720 the binding of a corresponding VHH antibody domain (resp. fragment thereof) without

1721 humanization to human IL-18Ra ECD.

1722

1723 Embodiment 176: The VHH antibody domain or fragment thereof according to any one of

1724 embodiments 172 to 175, wherein said affinity is determined by KD measurement.

1725

1726 Embodiment 177: The VHH antibody domain or fragment thereof according to any one of

1727 embodiments 24 to 176, wherein said VHH antibody domain comprises a VHH antibody

1728 domain according to (A), (B) or (C)/said fragment of a VHH antibody domain comprises a

1729 fragment of a VHH antibody domain according to (A), (B) or (C).

1730

1731 Embodiment 178: The VHH antibody domain or fragment thereof according to any one of

1732 embodiments 24 to 176, wherein said VHH antibody domain comprises a VHH antibody

1733 domain according to (A), (B) or (D)/said fragment of a VHH antibody domain comprises a

1734 fragment of a VHH antibody domain according to (A), (B) or (D).

1735

1736 Embodiment 179: The VHH antibody domain or fragment thereof according to any one of

1737 embodiments 24 to 176, wherein said VHH antibody domain comprises a VHH antibody

1738 domain according to (A), (C) or (D)/said fragment of a VHH antibody domain comprises a

1739 fragment of a VHH antibody domain according to (A), (C) or (D).

1740

1741 Embodiment 180: The VHH antibody domain or fragment thereof according to any one of

1742 embodiments 24 to 176, wherein said VHH antibody domain comprises a VHH antibody

1743 domain according to (A) or (B)/said fragment of a VHH antibody domain comprises a fragment

1744 of a VHH antibody domain according to (A) or (B).

1745

1746 Embodiment 181 : The VHH antibody domain or fragment thereof according to any one of

1747 embodiments 24 to 176, wherein said VHH antibody domain comprises a VHH antibody 1748 domain according to (A) or (C)/said fragment of a VHH antibody domain comprises a fragment

1749 of a VHH antibody domain according to (A) or (C).

1750

1751 Embodiment 182: The VHH antibody domain or fragment thereof according to any one of

1752 embodiments 24 to 176, wherein said VHH antibody domain comprises a VHH antibody

1753 domain according to (A) or (D)/said fragment of a VHH antibody domain comprises a fragment

1754 of a VHH antibody domain according to (A) or (D).

1755

1756 Embodiment 183: The VHH antibody domain or fragment thereof according to any one of

1757 embodiments 24 to 176, wherein said VHH antibody domain comprises a VHH antibody

1758 domain according to (A)/said fragment of a VHH antibody domain comprises a fragment of a

1759 VHH antibody domain according to (A).

1760

1761 Embodiment 184: The VHH antibody domain or fragment thereof according to any one of

1762 embodiments 24 to 176, wherein said VHH antibody domain comprises a VHH antibody

1763 domain according to (B)/said fragment of a VHH antibody domain comprises a fragment of a

1764 VHH antibody domain according to (B).

1765

1766 Embodiment 185: The VHH antibody domain or fragment thereof according to any one of

1767 embodiments 1 to 23 or 62 to 184, wherein said VHH antibody domain or fragment thereof

1768 comprises complementarity determining regions according to (a) or (b).

1769

1770 Embodiment 186: The VHH antibody domain or fragment thereof according to any one of

1771 embodiments 1 to 23 or 62 to 184, wherein said VHH antibody domain or fragment thereof

1772 comprises complementarity determining regions according to (a) or (c).

1773

1774 Embodiment 187: The VHH antibody domain or fragment thereof according to any one of

1775 embodiments 1 to 23 or 62 to 184, wherein said VHH antibody domain or fragment thereof

1776 comprises complementarity determining regions according to (a).

1777

1778 Embodiment 188: The VHH antibody domain or fragment thereof according to any one of

1779 embodiments 1 to 23 or 62 to 184, wherein said VHH antibody domain or fragment thereof

1780 comprises complementarity determining regions according to (b).

1781 1782 Fourth aspect of the present disclosure (also referred to as "Embodiment 189"): According to a

1783 fourth aspect, the present disclosure relates to a VHH antibody domain or a fragment thereof,

1784 wherein

1785 (d) said VHH antibody domain or fragment thereof comprises the complementarity

1786 determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group

1787 consisting of VHH12, VHH13, VHH14, VHH15, VHH16, VHH17, VHH18,

1788 VHH20, VHH21 and VHH22 as shown in the Table of CDRs;

1789 (e) said VHH antibody domain or fragment thereof comprises the complementarity

1790 determining regions CDR1, CDR2 and CDR3 as defined in (d) with modification,

1791 wherein the modification is that the sequence of at least one of CDR1, CDR2 and

1792 CDR3 is humanized; or

1793 (f) said VHH antibody domain or fragment thereof comprises the complementarity

1794 determining regions CDR1, CDR2 and CDR3 as defined in (d) with modification,

1795 wherein the modification is

1796 - the replacement, addition or deletion of up to three amino acids in CDR1,

1797 - the replacement, addition or deletion of up to three amino acids in CDR2

1798 and/or

1799 - the replacement, addition or deletion of up to three amino acids in CDR3;

1800

1801 Table of CDRs:

1802

1803

1804 To the fourth aspect of the present disclosure and the embodiments referring thereto, the same

1805 explanations and definitions apply accordingly as for the first to third aspects of the present

1806 disclosure and the embodiments referring thereto.

1807

1808 Embodiment 190: The VHH antibody domain or fragment thereof according to embodiment

1809 189, wherein the modification in (e) is that the sequence of CDR1 and/or CDR2, but not the

1810 sequence of CDR3 is humanized.

1811 1812 Embodiment 191 : The VHH antibody domain or fragment thereof according to any one of

1813 embodiments 189 or 190, wherein the modification in (e) is that the sequence of CDR1 is

1814 humanized, but not the sequence of CDR2 and CDR3.

1815

1816 Embodiment 192: The VHH antibody domain or fragment thereof according to any one of

1817 embodiments 189 or 190, wherein the modification in (e) is that the sequence of CDR2 is

1818 humanized, but not the sequence of CDR1 and CDR3.

1819

1820 Embodiment 193: The VHH antibody domain or fragment thereof according to any one of

1821 embodiments 189 to 192, wherein the modification in (e) is that the sequence of one, but not

1822 more than one of CDR1, CDR2 and CDR3 is humanized.

1823

1824 Embodiment 194: The VHH antibody domain or fragment thereof according to any one of

1825 embodiments 189 to 193, wherein said humanization of said CDR(s) is by replacing at least one

1826 amino acid in the sequence of said CDR by the corresponding amino acid of a human VH

1827 domain.

1828

1829 Embodiment 195: The VHH antibody domain or fragment thereof according to any one of

1830 embodiments 189 to 194, wherein said humanization of said CDR(s) is by replacing up to three

1831 amino acids in the sequence of said CDR by the corresponding amino acid of a human VH

1832 domain.

1833

1834 Embodiment 196: The VHH antibody domain or fragment thereof according to any one of

1835 embodiments 189 to 194, wherein said humanization of said CDR(s) is by replacing up to three

1836 amino acids in the sequence of CDR1 and/or CDR2 and up to one amino acid in the sequence

1837 of CDR3 by the corresponding amino acid of a human VH domain.

1838

1839 Embodiment 197: The VHH antibody domain or fragment thereof according to any one of

1840 embodiments 189 to 194, wherein said humanization of said CDR(s) is by replacing up to two

1841 amino acids in the sequence of said CDR by the corresponding amino acid of a human VH

1842 domain.

1843

1844 Embodiment 198: The VHH antibody domain or fragment thereof according to any one of

1845 embodiments 189 to 194, wherein said humanization of said CDR(s) is by replacing up to two 1846 amino acids in the sequence of CDR1 and/or CDR2 and up to one amino acid in the sequence

1847 of CDR3 by the corresponding amino acid of a human VH domain.

1848

1849 Embodiment 199: The VHH antibody domain or fragment thereof according to any one of

1850 embodiments 189 to 198, wherein said humanization of said CDR(s) is by replacing one amino

1851 acid in the sequence of said CDR by the corresponding amino acid of a human VH domain.

1852

1853 Embodiment 200: The VHH antibody domain or fragment thereof according to any one of

1854 embodiments 189 to 199, wherein the modification in (f) is

1855 - the replacement, addition or deletion of up to three amino acids in CDR1,

1856 - the replacement, addition or deletion of up to three amino acids in CDR2 and/or

1857 - the replacement, addition or deletion of up to one amino acid in CDR3.

1858

1859 Embodiment 201 : The VHH antibody domain or fragment thereof according to any one of

1860 embodiments 189 to 199, wherein the modification in (f) is

1861 - the replacement, addition or deletion of up to two amino acids in CDR1,

1862 - the replacement, addition or deletion of up to two amino acids in CDR2 and/or

1863 - the replacement, addition or deletion of up to two amino acids in CDR3;

1864

1865 Embodiment 202: The VHH antibody domain or fragment thereof according to any one of

1866 embodiments 189 to 199, wherein the modification in (f) is

1867 - the replacement, addition or deletion of up to two amino acids in CDR1;

1868 - the replacement, addition or deletion of up to two amino acids in CDR2; and/or

1869 - the replacement, addition or deletion of up to one amino acid in CDR3.

1870

1871 Embodiment 203: The VHH antibody domain or fragment thereof according to any one of

1872 embodiments 189 to 199, wherein the modification in (f) is

1873 - the replacement, addition or deletion of up to two amino acids in CDR1 and/or

1874 - the replacement, addition or deletion of up to two amino acids in CDR2;

1875 wherein the sequence of CDR3 is unmodified.

1876

1877 As a skilled person understands, the indication that the "sequence of CDR3 is unmodified"

1878 means that the sequence is unmodified compared to the sequence provided for CDR3 for the

1879 VHH at issue in the Table of CDRs. 1880

1881 Embodiment 204: The VHH antibody domain or fragment thereof according to any one of

1882 embodiments 189 to 199, wherein the modification in (f) is

1883 - the replacement, addition or deletion of up to two amino acids in CDR1,

1884 wherein the sequence of CDR2 and CDR3 is unmodified.

1885

1886 Embodiment 205: The VHH antibody domain or fragment thereof according to any one of

1887 embodiments 189 to 199, wherein the modification in (f) is

1888 - the replacement, addition or deletion of up to two amino acids in CDR2,

1889 wherein the sequence of CDR1 and CDR3 is unmodified.

1890

1891 Embodiment 206: The VHH antibody domain or fragment thereof according to any one of

1892 embodiments 189 to 199, wherein the modification in (f) is

1893 - the replacement, addition or deletion of up to one amino acid in CDR1;

1894 - the replacement, addition or deletion of up to one amino acid in CDR2; and/or

1895 - the replacement, addition or deletion of up to one amino acid in CDR3.

1896

1897 Embodiment 207: The VHH antibody domain or fragment thereof according to any one of

1898 embodiments 189 to 199, wherein the modification in (f) is

1899 - the replacement, addition or deletion of one amino acid in CDR1 and/or

1900 - the replacement, addition or deletion of one amino acid in CDR2;

1901 wherein the sequence of CDR3 is unmodified.

1902

1903 Embodiment 208: The VHH antibody domain or fragment thereof according to any one of

1904 embodiments 189 to 199, wherein the modification in (f) is

1905 - the replacement, addition or deletion of one amino acid in CDR1,

1906 wherein the sequence of CDR2 and CDR3 is unmodified.

1907

1908 Embodiment 209: The VHH antibody domain or fragment thereof according to any one of

1909 embodiments 189 to 199, wherein the modification in (f) is

1910 - the replacement, addition or deletion of one amino acid in CDR2,

1911 wherein the sequence of CDR1 and CDR3 is unmodified.

1912 1913 Embodiment 210: The VHH antibody domain or fragment thereof according to any one of

1914 embodiments 189 to 209, wherein the modification in (f) comprises only the replacement, but

1915 not the addition or deletion of amino acids.

1916

1917 Embodiment 211 : The VHH antibody domain or fragment thereof according to any one of

1918 embodiments 189 to 210, wherein said replacement is a conservative amino acid replacement.

1919

1920 Fifth aspect of the present disclosure (also referred to as "Embodiment 212"): According to a

1921 fifth aspect, the present disclosure relates to a VHH antibody domain or a fragment thereof,

1922 wherein

1923 (E) said VHH antibody domain comprises the amino acid sequence of a VHH selected

1924 from the group consisting of VHH12, VHH13, VHH14, VHH15, VHH16, VHH17,

1925 VHH18, VHH20, VHH21 and VHH22 as shown in the Table of VHH Sequences;

1926 (F) said VHH antibody domain comprises a VHH sequence as defined in (E) with

1927 modification, wherein the modification is that said sequence is humanized;

1928 (G) said VHH antibody domain comprises a VHH sequence as defined in (E) with

1929 modification, wherein the modification is the replacement, addition or deletion of up

1930 to 25 amino acids; or

1931 (H) said VHH antibody domain comprises a VHH sequence that is at least 75% identical

1932 to a VHH sequence referred to in (E);

1933

1934 Table of VHH Sequences:

1935

1936

1937 Sixth aspect of the present disclosure (also referred to as "Embodiment 213"): According to a

1938 sixth aspect, the present disclosure relates to a VHH antibody domain or a fragment thereof,

1939 wherein

1940 (E) said VHH antibody domain consists of the amino acid sequence of a VHH selected

1941 from the group consisting of VHH12, VHH13, VHH14, VHH15, VHH16, VHH17,

1942 VHH18, VHH20, VHH21 and VHH22 as shown in the Table of VHH Sequences;

1943 (F) said VHH antibody domain consists of a VHH sequence as defined in (E) with

1944 modification, wherein the modification is that said sequence is humanized;

1945 (G) said VHH antibody domain consists of a VHH sequence as defined in (E) with

1946 modification, wherein the modification is the replacement, addition or deletion of up

1947 to 25 amino acids; or 1948 (H) said VHH antibody domain consists of a VHH sequence that is at least 75% identical

1949 to a VHH sequence referred to in (E).

1950

1951 To the fifth and sixth aspects of the present disclosure and the embodiments referring thereto,

1952 the same explanations and definitions apply accordingly as for the first to fourth aspects of the

1953 present disclosure and the embodiments referring thereto.

1954

1955 Embodiment 214: The VHH antibody domain or fragment thereof according to any one of

1956 embodiments 212 or 213, wherein said fragment of said VHH antibody domain comprises at

1957 least 75% of the amino acids of the sequence of said VHH antibody domain. As the skilled

1958 person understands, this means that that fragment lacks up to a quarter of the total number of

1959 amino acids of said VHH antibody domain, wherein, compared to the "complete" VHH

1960 antibody domain sequence said amino acids are lacking either at the N-terminus or at the C-

1961 terminus.

1962

1963 Embodiment 215: The VHH antibody domain or fragment thereof according to any one of

1964 embodiments 212 or 213, wherein said fragment of said VHH antibody domain comprises at

1965 least 80% of the amino acids of the sequence of said VHH antibody domain.

1966

1967 Embodiment 216: The VHH antibody domain or fragment thereof according to any one of

1968 embodiments 212 or 213, wherein said fragment of said VHH antibody domain comprises at

1969 least 85% of the amino acids of the sequence of said VHH antibody domain.

1970

1971 Embodiment 217: The VHH antibody domain or fragment thereof according to any one of

1972 embodiments 212 or 213, wherein said fragment of said VHH antibody domain comprises at

1973 least 90% of the amino acids of the sequence of said VHH antibody domain.

1974

1975 Embodiment 218: The VHH antibody domain or fragment thereof according to any one of

1976 embodiments 212 or 213, wherein said fragment of said VHH antibody domain comprises at

1977 least 95% of the amino acids of the sequence of said VHH antibody domain.

1978

1979 Embodiment 219: The VHH antibody domain or fragment thereof according to any one of

1980 embodiments 212 or 213, wherein said fragment of said VHH antibody domain comprises at

1981 least 98% of the amino acids of the sequence of said VHH antibody domain. 1982

1983 Embodiment 220: The VHH antibody domain or fragment thereof according to any one of

1984 embodiments 212 or 213, wherein said fragment of said VHH antibody domain comprises at

1985 least 99% of the amino acids of the sequence of said VHH antibody domain.

1986

1987 Embodiment 221 : The VHH antibody domain or fragment thereof according to any one of

1988 embodiments 212 to 220, wherein said fragment of said VHH antibody domain comprises the

1989 complementarity determining regions CDR1, CDR2 and CDR3.

1990

1991 Embodiment 222: The VHH antibody domain or fragment thereof according to any one of

1992 embodiments 212 to 221, wherein said fragment of said VHH antibody domain comprises at

1993 least the sequence from the N-terminus of CDR1 to the C-terminus of CDR3 of said VHH

1994 antibody domain.

1995

1996 Embodiment 223: The VHH antibody domain or fragment thereof according to any one of

1997 embodiments 212 to 222, wherein in (E) said fragment of said VHH antibody domain comprises

1998 all the complementarity determining regions (CDRs) of said VHH antibody domain.

1999

2000 Embodiment 224: The VHH antibody domain or fragment thereof according to any one of

2001 embodiments 212 to 223, wherein in (F) said humanization of said sequence is by replacing at

2002 least one amino acid of said sequence by the corresponding amino acid of a human VH (variable

2003 heavy) domain.

2004

2005 Embodiment 225: The VHH antibody domain or fragment thereof according to any one of

2006 embodiments 212 to 224, wherein in (F) said humanization of said sequence is by (individually)

2007 replacing up to 25 amino acids of said sequence by the corresponding amino acids of a human

2008 VH domain.

2009

2010 Embodiment 226: The VHH antibody domain or fragment thereof according to any one of

2011 embodiments 212 to 224, wherein in (F) said humanization of said sequence is by replacing up

2012 to 20 amino acids of said sequence by the corresponding amino acids of a human VH domain.

2013 2014 Embodiment 227: The VHH antibody domain or fragment thereof according to any one of

2015 embodiments 212 to 224, wherein in (F) said humanization of said sequence is by replacing up

2016 to 15 amino acids of said sequence by the corresponding amino acids of a human VH domain.

2017

2018 Embodiment 228: The VHH antibody domain or fragment thereof according to any one of

2019 embodiments 212 to 224, wherein in (F) said humanization of said sequence is by replacing up

2020 to 10 amino acids of said sequence by the corresponding amino acids of a human VH domain.

2021

2022 Embodiment 229: The VHH antibody domain or fragment thereof according to any one of

2023 embodiments 212 to 224, wherein in (F) said humanization of said sequence is by replacing up

2024 to 5 amino acids of said sequence by the corresponding amino acids of a human VH domain.

2025

2026 Embodiment 230: The VHH antibody domain or fragment thereof according to any one of

2027 embodiments 212 to 224, wherein in (F) said humanization of said sequence is by replacing up

2028 to 3 amino acids of said sequence by the corresponding amino acids of a human VH domain.

2029

2030 Embodiment 231 : The VHH antibody domain or fragment thereof according to any one of

2031 embodiments 212 to 224, wherein in (F) said humanization of said sequence is by replacing up

2032 to 2 amino acids of said sequence by the corresponding amino acids of a human VH domain.

2033

2034 Embodiment 232: The VHH antibody domain or fragment thereof according to any one of

2035 embodiments 212 to 224, wherein in (F) said humanization of said sequence is by replacing one

2036 amino acid of said sequence by the corresponding amino acid of a human VH domain.

2037

2038 Embodiment 233: The VHH antibody domain or fragment thereof according to any one of

2039 embodiments 212 to 232, wherein in (F) said humanization is within the framework regions of

2040 said VHH antibody domain and/or within the CDRs of said VHH antibody domain.

2041

2042 Embodiment 234: The VHH antibody domain or fragment thereof according to any one of

2043 embodiments 212 to 232, wherein in (F) said humanization is within the framework regions of

2044 said VHH antibody domain, but not within the CDRs of said VHH antibody domain.

2045 2046 Embodiment 235: The VHH antibody domain or fragment thereof according to any one of

2047 embodiments 212 to 224, wherein in (F) said humanization is within the CDRs of said VHH

2048 antibody domain, but not within the framework regions of said VHH antibody domain.

2049

2050 Embodiment 236: The VHH antibody domain or fragment thereof according to any one of

2051 embodiments 212 to 233 or 235, wherein in (F) said humanization within the CDRs of said

2052 VHH antibody domain is within CDR1, CDR2 and/or CDR3.

2053

2054 Embodiment 237: The VHH antibody domain or fragment thereof according to any one of

2055 embodiments 212 to 233 or 235 to 236, wherein in (F) said humanization within the CDRs of

2056 said VHH antibody domain is within CDR1 and/or CDR2.

2057

2058 Embodiment 238: The VHH antibody domain or fragment thereof according to any one of

2059 embodiments 212 to 233 or 235 to 237, wherein in (F) said humanization within the CDRs of

2060 said VHH antibody domain is within CDR1.

2061

2062 Embodiment 239: The VHH antibody domain or fragment thereof according to any one of

2063 embodiments 212 to 233 or 235 to 238, wherein in (F) said humanization within the CDRs of

2064 said VHH antibody domain is within CDR2.

2065

2066 Embodiment 240: The VHH antibody domain or fragment thereof according to any one of

2067 embodiments 212 to 239, wherein in (F) said humanization within the CDRs of said VHH

2068 antibody domain is not within CDR3.

2069

2070 Embodiment 241: The VHH antibody domain or fragment thereof according to any one of

2071 embodiments 212 to 240, wherein in (G) the modification is the replacement, addition or

2072 deletion of up to 20 amino acids.

2073

2074 Embodiment 242: The VHH antibody domain or fragment thereof according to any one of

2075 embodiments 212 to 240, wherein in (G) the modification is the replacement, addition or

2076 deletion of up to 15 amino acids.

2077 2078 Embodiment 243: The VHH antibody domain or fragment thereof according to any one of

2079 embodiments 212 to 240, wherein in (G) the modification is the replacement, addition or

2080 deletion of up to 10 amino acids.

2081

2082 Embodiment 244: The VHH antibody domain or fragment thereof according to any one of

2083 embodiments 212 to 240, wherein in (G) the modification is the replacement, addition or

2084 deletion of up to 5 amino acids.

2085

2086 Embodiment 245: The VHH antibody domain or fragment thereof according to any one of

2087 embodiments 212 to 240, wherein in (G) the modification is the replacement, addition or

2088 deletion of up to 3 amino acids.

2089

2090 Embodiment 246: The VHH antibody domain or fragment thereof according to any one of

2091 embodiments 212 to 240, wherein in (G) the modification is the replacement, addition or

2092 deletion of up to 2 amino acids.

2093

2094 Embodiment 247: The VHH antibody domain or fragment thereof according to any one of

2095 embodiments 212 to 240, wherein in (G) the modification is the replacement, addition or

2096 deletion of one amino acid.

2097

2098 Embodiment 248: The VHH antibody domain or fragment thereof according to any one of

2099 embodiments 212 to 247, wherein the modification in (G) comprises only the replacement, but

2100 not the addition or deletion of amino acids.

2101

2102 Embodiment 249: The VHH antibody domain or fragment thereof according to any one of

2103 embodiments 212 to 248, wherein in (E) said VHH antibody domain or fragment thereof

2104 comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH

2105 selected from the group consisting of VHH12, VHH13, VHH14, VHH15, VHH16, VHH17,

2106 VHH18, VHH20, VHH21 and VHH22 as shown in the Table of CDRs.

2107

2108 Embodiment 250: The VHH antibody domain or fragment thereof according to any one of

2109 embodiments 212 to 249, wherein in (F) to (H)

2110 (d) said VHH antibody domain or fragment thereof comprises the complementarity

2111 determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group 2112 consisting of VHH12, VHH13, VHH14, VHH15, VHH16, VHH17, VHH18,

2113 VHH20, VHH21 and VHH22 as shown in the Table of CDRs;

2114 (e) said VHH antibody domain or fragment thereof comprises the complementarity

2115 determining regions CDR1, CDR2 and CDR3 as defined in (d) with modification,

2116 wherein the modification is that the sequence of at least one of CDR1, CDR2 and

2117 CDR3 is humanized;

2118 (f) said VHH antibody domain or fragment thereof comprises the complementarity

2119 determining regions CDR1, CDR2 and CDR3 as defined in (d) with modification,

2120 wherein the modification is

2121 - the replacement, addition or deletion of up to three amino acids in CDR1,

2122 - the replacement, addition or deletion of up to three amino acids in CDR2

2123 and/or

2124 - the replacement, addition or deletion of up to three amino acids in CDR3.

2125

2126 Embodiment 251 : The VHH antibody domain or fragment thereof according to embodiment

2127 250, wherein the modification in (e) is that the sequence of CDR1 and/or CDR2, but not the

2128 sequence of CDR3 is humanized.

2129

2130 Embodiment 252: The VHH antibody domain or fragment thereof according to any one of

2131 embodiments 250 or 251, wherein the modification in (e) is that the sequence of CDR1 is

2132 humanized, but not the sequence of CDR2 and CDR3.

2133

2134 Embodiment 253: The VHH antibody domain or fragment thereof according to any one of

2135 embodiments 250 or 251, wherein the modification in (e) is that the sequence of CDR2 is

2136 humanized, but not the sequence of CDR1 and CDR3.

2137

2138 Embodiment 254: The VHH antibody domain or fragment thereof according to any one of

2139 embodiments 250 to 253, wherein the modification in (e) is that the sequence of one, but not

2140 more than one of CDR1, CDR2 and CDR3 is humanized.

2141

2142 Embodiment 255: The VHH antibody domain or fragment thereof according to any one of

2143 embodiments 250 to 254, wherein said humanization of said CDR(s) is by replacing at least one

2144 amino acid in the sequence of said CDR by the corresponding amino acid of a human VH

2145 domain. 2146

2147 Embodiment 256: The VHH antibody domain or fragment thereof according to any one of

2148 embodiments 250 to 255, wherein said humanization of said CDR(s) is by replacing up to three

2149 amino acids in the sequence of said CDR by the corresponding amino acid of a human VH

2150 domain.

2151

2152 Embodiment 257: The VHH antibody domain or fragment thereof according to any one of

2153 embodiments 250 to 255, wherein said humanization of said CDR(s) is by replacing up to three

2154 amino acids in the sequence of CDR1 and/or CDR2 and up to one amino acid in the sequence

2155 of CDR3 by the corresponding amino acid of a human VH domain.

2156

2157 Embodiment 258: The VHH antibody domain or fragment thereof according to any one of

2158 embodiments 250 to 255, wherein said humanization of said CDR(s) is by replacing up to two

2159 amino acids in the sequence of said CDR by the corresponding amino acid of a human VH

2160 domain.

2161

2162 Embodiment 259: The VHH antibody domain or fragment thereof according to any one of

2163 embodiments 250 to 255, wherein said humanization of said CDR(s) is by replacing up to two

2164 amino acids in the sequence of CDR1 and/or CDR2 and up to one amino acid in the sequence

2165 of CDR3 by the corresponding amino acid of a human VH domain.

2166

2167 Embodiment 260: The VHH antibody domain or fragment thereof according to any one of

2168 embodiments 250 to 259, wherein said humanization of said CDR(s) is by replacing one amino

2169 acid in the sequence of said CDR by the corresponding amino acid of a human VH domain.

2170

2171 Embodiment 261 : The VHH antibody domain or fragment thereof according to any one of

2172 embodiments 250 to 260, wherein the modification in (f) is

2173 - the replacement, addition or deletion of up to three amino acids in CDR1,

2174 - the replacement, addition or deletion of up to three amino acids in CDR2 and/or

2175 - the replacement, addition or deletion of up to two amino acids in CDR3.

2176

2177 Embodiment 262: The VHH antibody domain or fragment thereof according to any one of

2178 embodiments 250 to 260, wherein the modification in (f) is

2179 - the replacement, addition or deletion of up to three amino acids in CDR1, 2180 - the replacement, addition or deletion of up to three amino acids in CDR2 and/or

2181 - the replacement, addition or deletion of up to one amino acid in CDR3.

2182

2183 Embodiment 263: The VHH antibody domain or fragment thereof according to any one of

2184 embodiments 250 to 260, wherein the modification in (f) is

2185 - the replacement, addition or deletion of up to three amino acids in CDR1, and/or

2186 - the replacement, addition or deletion of up to three amino acids in CDR2,

2187 wherein the sequence of CDR3 is unmodified.

2188

2189 Embodiment 264: The VHH antibody domain or fragment thereof according to any one of

2190 embodiments 250 to 260, wherein the modification in (f) is

2191 - the replacement, addition or deletion of up to two amino acids in CDR1,

2192 - the replacement, addition or deletion of up to two amino acids in CDR2 and/or

2193 - the replacement, addition or deletion of up to two amino acids in CDR3;

2194

2195 Embodiment 265: The VHH antibody domain or fragment thereof according to any one of

2196 embodiments 250 to 260, wherein the modification in (f) is

2197 - the replacement, addition or deletion of up to two amino acids in CDR1;

2198 - the replacement, addition or deletion of up to two amino acids in CDR2; and/or

2199 - the replacement, addition or deletion of up to one amino acid in CDR3.

2200

2201 Embodiment 266: The VHH antibody domain or fragment thereof according to any one of

2202 embodiments 250 to 260, wherein the modification in (f) is

2203 - the replacement, addition or deletion of up to two amino acids in CDR1 and/or

2204 - the replacement, addition or deletion of up to two amino acids in CDR2;

2205 wherein the sequence of CDR3 is unmodified.

2206

2207 As the skilled person is aware, "unmodified" means unmodified compared to the sequence in

2208 the Table of CDRs.

2209

2210 Embodiment 267: The VHH antibody domain or fragment thereof according to any one of

2211 embodiments 250 to 260, wherein the modification in (f) is

2212 - the replacement, addition or deletion of up to two amino acids in CDR1,

2213 wherein the sequence of CDR2 and CDR3 is unmodified. 2214

2215 Embodiment 268: The VHH antibody domain or fragment thereof according to any one of

2216 embodiments 250 to 260, wherein the modification in (f) is

2217 - the replacement, addition or deletion of up to two amino acids in CDR2,

2218 wherein the sequence of CDR1 and CDR3 is unmodified.

2219

2220 Embodiment 269: The VHH antibody domain or fragment thereof according to any one of

2221 embodiments 250 to 260, wherein the modification in (f) is

2222 - the replacement, addition or deletion of up to one amino acid in CDR1;

2223 - the replacement, addition or deletion of up to one amino acid in CDR2; and/or

2224 - the replacement, addition or deletion of up to one amino acid in CDR3.

2225

2226 Embodiment 270: The VHH antibody domain or fragment thereof according to any one of

2227 embodiments 250 to 260, wherein the modification in (f) is

2228 - the replacement, addition or deletion of one amino acid in CDR1 and/or

2229 - the replacement, addition or deletion of one amino acid in CDR2;

2230 wherein the sequence of CDR3 is unmodified.

2231

2232 Embodiment 271 : The VHH antibody domain or fragment thereof according to any one of

2233 embodiments 250 to 260, wherein the modification in (f) is

2234 - the replacement, addition or deletion of one amino acid in CDR1,

2235 wherein the sequence of CDR2 and CDR3 is unmodified.

2236

2237 Embodiment 272: The VHH antibody domain or fragment thereof according to any one of

2238 embodiments 250 to 260, wherein the modification in (f) is

2239 - the replacement, addition or deletion of one amino acid in CDR2,

2240 wherein the sequence of CDR1 and CDR3 is unmodified.

2241

2242 Embodiment 273: The VHH antibody domain or fragment thereof according to any one of

2243 embodiments 250 to 272, wherein the modification in (f) comprises only the replacement, but

2244 not the addition or deletion of amino acids.

2245

2246 Embodiment 274: The VHH antibody domain or fragment thereof according to any one of

2247 embodiments 250 to 273, wherein said replacement is a conservative amino acid replacement. 2248

2249 Embodiment 275: The VHH antibody domain or fragment thereof according to any one of

2250 embodiments 189 to 274, wherein said fragment consists of at least 100 amino acids.

2251

2252 Embodiment 276: The VHH antibody domain or fragment thereof according to any one of

2253 embodiments 189 to 274, wherein said fragment consists of at least 105 amino acids.

2254

2255 Embodiment 277: The VHH antibody domain or fragment thereof according to any one of

2256 embodiments 189 to 274, wherein said fragment consists of at least 110 amino acids.

2257

2258 Embodiment 278: The VHH antibody domain or fragment thereof according to any one of

2259 embodiments 189 to 274, wherein said fragment consists of at least 115 amino acids.

2260

2261 Embodiment 279: The VHH antibody domain or fragment thereof according to any one of

2262 embodiments 189 to 278, wherein said VHH antibody domain is an anti-IL-18RP VHH

2263 antibody domain.

2264

2265 Embodiment 280: The VHH antibody domain or fragment thereof according to any one of

2266 embodiments 189 to 279, wherein said VHH antibody domain is specific for IL-18RP.

2267

2268 Embodiment 281 : The VHH antibody domain or fragment thereof according to any one of

2269 embodiments 189 to 280, wherein said VHH antibody domain or fragment thereof binds to IL-

2270 18RP.

2271

2272 Embodiment 282: The VHH antibody domain or fragment thereof according to any one of

2273 embodiments 189 to 281, wherein said VHH antibody domain or fragment thereof binds

2274 specifically to IL-18Rp.

2275

2276 Embodiment 283: The VHH antibody domain or fragment thereof according to any one of

2277 embodiments 189 to 282, wherein said VHH antibody domain or fragment thereof does not

2278 bind to IL-18Ra.

2279 2280 Embodiment 284: The VHH antibody domain or fragment thereof according to any one of

2281 embodiments 189 to 283, wherein said VHH antibody domain or fragment thereof is capable

2282 of specifically binding to IL-18RP ECD.

2283

2284 Embodiment 285: The VHH antibody domain or fragment thereof according to any one of

2285 embodiments 189 to 284, wherein said VHH antibody domain or fragment thereof binds to

2286 recombinant human IL-18RP ECD with a KD of 1x1 O'⁶ M or stronger.

2287

2288 Embodiment 286 The VHH antibody domain or fragment thereof according to any one of

2289 embodiments 189 to 284, wherein said VHH antibody domain or fragment thereof binds to

2290 recombinant human IL-18RP ECD with a KD of 1x1 O'⁷ M or stronger.

2291

2292 Embodiment 287: The VHH antibody domain or fragment thereof according to any one of

2293 embodiments 189 to 284, wherein said VHH antibody domain or fragment thereof binds to

2294 recombinant human IL-18RP ECD with a KD of 5x1 O'⁸ M or stronger.

2295

2296 Embodiment 288: The VHH antibody domain or fragment thereof according to any one of

2297 embodiments 189 to 284, wherein said VHH antibody domain or fragment thereof binds to

2298 recombinant human IL-18RP ECD with a KD of 1.5x1 O'⁸ M or stronger.

2299

2300 Embodiment 289: The VHH antibody domain or fragment thereof according to any one of

2301 embodiments 189 to 284, wherein said VHH antibody domain or fragment thereof binds to

2302 recombinant human IL-18RP ECD with a KD of 1x1 O'⁸ M or stronger.

2303

2304 Embodiment 290: The VHH antibody domain or fragment thereof according to any one of

2305 embodiments 189 to 284, wherein said VHH antibody domain or fragment thereof binds to

2306 recombinant human IL-18RP ECD with a KD of 5x1 O'⁹ M or stronger.

2307

2308 Embodiment 291 : The VHH antibody domain or fragment thereof according to any one of

2309 embodiments 189 to 284, wherein said VHH antibody domain or fragment thereof binds to

2310 recombinant human IL-18RP ECD with a KD of 1x1 O'⁹ M or stronger.

2311

2312 Embodiment 292: The VHH antibody domain or fragment thereof according to any one of

2313 embodiments 189 to 291, wherein said fragment of said VHH antibody domain binds to 2314 recombinant human IL-18RP ECD with at least 90% of the affinity (as determined by KD value)

2315 with which said VHH antibody domain binds to recombinant human IL-18RP ECD.

2316

2317 Embodiment 293: The VHH antibody domain or fragment thereof according to any one of

2318 embodiments 189 to 292, wherein said fragment of said VHH antibody domain binds to IL-

2319 18Ra ECD with an affinity (as determined from the KD value) that is at least equal to the

2320 affinity with which VHH2 (SEQ ID NO: 2) binds to IL-18Ra ECD or stronger. Such binding

2321 can be determined by in vitro binding experiments as described in Example 1 below (by

2322 biolayer interferometry).

2323

2324 Embodiment 294: The VHH antibody domain or fragment thereof according to any one of

2325 embodiments 189 to 211 or 250 to 293, wherein in (e) and (f) the VHH antibody domain or

2326 fragment thereof binds to recombinant human IL-18RP ECD with an affinity (KD value) that

2327 is by not more than a factor of 10 weaker than the binding of the corresponding VHH antibody

2328 domain without modification.

2329

2330 Embodiment 295: The VHH antibody domain or fragment thereof according to any one of

2331 embodiments 189 to 211 or 250 to 293, wherein in (e) and (f) the VHH antibody domain or

2332 fragment thereof binds to recombinant human IL-18RP ECD with an affinity (KD value) that

2333 is by not more than a factor of 5 weaker than the binding of the corresponding VHH antibody

2334 domain without modification.

2335

2336 Embodiment 296: The VHH antibody domain or fragment thereof according to any one of

2337 embodiments 189 to 211 or 250 to 293, wherein in (e) and (f) the VHH antibody domain or

2338 fragment thereof binds to recombinant human IL-18RP ECD with an affinity (KD value) that

2339 is by not more than a factor of 2 weaker than the binding of the corresponding VHH antibody

2340 domain without modification.

2341

2342 Embodiment 297: The VHH antibody domain or fragment thereof according to any one of

2343 embodiments 189 to 211 or 250 to 293, wherein in (e) and (f) the VHH antibody domain or

2344 fragment thereof binds to recombinant human IL-18RP ECD with an affinity (KD value) that

2345 is by not more than a factor of 1.5 weaker than the binding of the corresponding VHH antibody

2346 domain without modification.

2347 2348 Embodiment 298: The VHH antibody domain or fragment thereof according to any one of

2349 embodiments 189 to 211 or 250 to 297, wherein in (e) and (f) the VHH antibody domain or

2350 fragment thereof binds to recombinant human IL-18RP ECD with an affinity (KD value) that

2351 is by not more than a factor of 10 stronger than the binding of the corresponding VHH antibody

2352 domain without modification.

2353

2354 Embodiment 299: The VHH antibody domain or fragment thereof according to any one of

2355 embodiments 189 to 211 or 250 to 297, wherein in (e) and (f) the VHH antibody domain or

2356 fragment thereof binds to recombinant human IL-18RP ECD with an affinity (KD value) that

2357 is by not more than a factor of 5 stronger than the binding of the corresponding VHH antibody

2358 domain without modification.

2359

2360 Embodiment 300: The VHH antibody domain or fragment thereof according to any one of

2361 embodiments 189 to 211 or 250 to 297, wherein in (e) and (f) the VHH antibody domain or

2362 fragment thereof binds to recombinant human IL-18RP ECD with an affinity (KD value) that

2363 is by not more than a factor of 2 stronger than the binding of the corresponding VHH antibody

2364 domain without modification.

2365

2366 Embodiment 301 : The VHH antibody domain or fragment thereof according to any one of

2367 embodiments 189 to 211 or 250 to 297, wherein in (e) and (f) the VHH antibody domain or

2368 fragment thereof binds to recombinant human IL-18RP ECD with an affinity (KD value) that

2369 is by not more than a factor of 1.5 stronger than the binding of the corresponding VHH antibody

2370 domain without modification.

2371

2372 Embodiment 302: The VHH antibody domain or fragment thereof according to any one of

2373 embodiments 212 to 301, wherein in (F) and (G) the VHH antibody domain binds to

2374 recombinant human IL-18RP ECD with an affinity (KD value) that is by not more than a factor

2375 of 10 weaker than the binding of the corresponding VHH antibody domain without

2376 modification.

2377

2378 Embodiment 303: The VHH antibody domain or fragment thereof according to any one of

2379 embodiments 212 to 301, wherein in (F) and (G) the VHH antibody domain binds to

2380 recombinant human IL-18RP ECD with an affinity (KD value) that is by not more than a factor

2381 of 5 weaker than the binding of the corresponding VHH antibody domain without modification. 2382

2383 Embodiment 304: The VHH antibody domain or fragment thereof according to any one of

2384 embodiments 212 to 301, wherein in (F) and (G) the VHH antibody domain binds to

2385 recombinant human IL-18RP ECD with an affinity (KD value) that is by not more than a factor

2386 of 2 weaker than the binding of the corresponding VHH antibody domain without modification.

2387

2388 Embodiment 305: The VHH antibody domain or fragment thereof according to any one of

2389 embodiments 189 to 301, wherein in (F) and (G) the VHH antibody domain binds to

2390 recombinant human IL-18RP ECD with an affinity (KD value) that is by not more than a factor

2391 of 1.5 weaker than the binding of the corresponding VHH antibody domain without

2392 modification.

2393

2394 Embodiment 306: The VHH antibody domain or fragment thereof according to any one of

2395 embodiments 212 to 305, wherein in (F) and (G) the VHH antibody domain binds to

2396 recombinant human IL-18RP ECD with an affinity (KD value) that is by not more than a factor

2397 of 10 stronger than the binding of the corresponding VHH antibody domain without

2398 modification.

2399

2400 Embodiment 307: The VHH antibody domain or fragment thereof according to any one of

2401 embodiments 212 to 305, wherein in (F) and (G) the VHH antibody domain binds to

2402 recombinant human IL-18RP ECD with an affinity (KD value) that is by not more than a factor

2403 of 5 stronger than the binding of the corresponding VHH antibody domain without

2404 modification.

2405

2406 Embodiment 308: The VHH antibody domain or fragment thereof according to any one of

2407 embodiments 212 to 305, wherein in (F) and (G) the VHH antibody domain binds to

2408 recombinant human IL-18RP ECD with an affinity (KD value) that is by not more than a factor

2409 of 2 stronger than the binding of the corresponding VHH antibody domain without

2410 modification.

2411

2412 Embodiment 309: The VHH antibody domain or fragment thereof according to any one of

2413 embodiments 212 to 305, wherein in (F) and (G) the VHH antibody domain binds to

2414 recombinant human IL-18RP ECD with an affinity (KD value) that is by not more than a factor 2415 of 1.5 stronger than the binding of the corresponding VHH antibody domain without

2416 modification.

2417

2418 Embodiment 310: The VHH antibody domain or fragment thereof according to any one of

2419 embodiments 212 to 309, wherein in (H) the affinity (KD value) of the binding of the VHH

2420 antibody domain to recombinant human IL-18RP ECD is by not more than a factor of 10 weaker

2421 than the affinity (KD value) of the binding to recombinant human IL-18RP ECD of a VHH

2422 antibody domain consisting of the sequence from the Table of VHH Sequences that has the

2423 highest degree of sequence identity with the sequence of said VHH antibody domain of (H).

2424 The degree of sequence identity can be determined by sequence alignment.

2425

2426 Embodiment 311 : The VHH antibody domain or fragment thereof according to any one of

2427 embodiments 212 to 309, wherein in (H) the affinity (KD value) of the binding of the VHH

2428 antibody domain to recombinant human IL-18RP ECD is by not more than a factor of 5 weaker

2429 than the affinity (KD value) of the binding to recombinant human IL-18RP ECD of a VHH

2430 antibody domain consisting of the sequence from the Table of VHH Sequences that has the

2431 highest degree of sequence identity with the sequence of said VHH antibody domain of (H).

2432 The degree of sequence identity can be determined by sequence alignment.

2433

2434 Embodiment 312: The VHH antibody domain or fragment thereof according to any one of

2435 embodiments 212 to 309, wherein in (H) the affinity (KD value) of the binding of the VHH

2436 antibody domain to recombinant human IL-18RP ECD is by not more than a factor of 2 weaker

2437 than the affinity (KD value) of the binding to recombinant human IL-18RP ECD of a VHH

2438 antibody domain consisting of the sequence from the Table of VHH Sequences that has the

2439 highest degree of sequence identity with the sequence of said VHH antibody domain of (H).

2440

2441 Embodiment 313: The VHH antibody domain or fragment thereof according to any one of

2442 embodiments 212 to 309, wherein in (H) the affinity (KD value) of the binding of the VHH

2443 antibody domain to recombinant human IL-18RP ECD is by not more than a factor of 1.5

2444 weaker than the affinity (KD value) of the binding to recombinant human IL-18RP ECD of a

2445 VHH antibody domain consisting of the sequence from the Table of VHH Sequences that has

2446 the highest degree of sequence identity with the sequence of said VHH antibody domain of (H).

2447 2448 Embodiment 314 The VHH antibody domain or fragment thereof according to any one of

2449 embodiments 212 to 313, wherein in (H) the affinity (KD value) of the binding of the VHH

2450 antibody domain to recombinant human IL-18RP ECD is by not more than a factor of 10

2451 stronger than the affinity (KD value) of the binding to recombinant human IL-18RP ECD of a

2452 VHH antibody domain consisting of the sequence from the Table of VHH Sequences that has

2453 the highest degree of sequence identity with the sequence of said VHH antibody domain of (H).

2454

2455 Embodiment 315: The VHH antibody domain or fragment thereof according to any one of

2456 embodiments 212 to 313, wherein in (H) the affinity (KD value) of the binding of the VHH

2457 antibody domain to recombinant human IL-18RP ECD is by not more than a factor of 5 stronger

2458 than the affinity (KD value) of the binding to recombinant human IL-18RP ECD of a VHH

2459 antibody domain consisting of the sequence from the Table of VHH Sequences that has the

2460 highest degree of sequence identity with the sequence of said VHH antibody domain of (H).

2461

2462 Embodiment 316: The VHH antibody domain or fragment thereof according to any one of

2463 embodiments 212 to 313, wherein in (H) the affinity (KD value) of the binding of the VHH

2464 antibody domain to recombinant human IL-18RP ECD is by not more than a factor of 2 stronger

2465 than the affinity (KD value) of the binding to recombinant human IL-18RP ECD of a VHH

2466 antibody domain consisting of the sequence from the Table of VHH Sequences that has the

2467 highest degree of sequence identity with the sequence of said VHH antibody domain of (H).

2468

2469 Embodiment 317: The VHH antibody domain or fragment thereof according to any one of

2470 embodiments 212 to 313, wherein in (H) the affinity (KD value) of the binding of the VHH

2471 antibody domain to recombinant human IL-18RP ECD is by not more than a factor of 1.5

2472 stronger than the affinity (KD value) of the binding to recombinant human IL-18RP ECD of a

2473 VHH antibody domain consisting of the sequence from the Table of VHH Sequences that has

2474 the highest degree of sequence identity with the sequence of said VHH antibody domain of (H).

2475

2476 Embodiment 318: The VHH antibody domain or fragment thereof according to any one of

2477 embodiments 279 to 317, wherein said KD value is determined in in vitro binding experiments

2478 through biolayer interferometry.

2479

2480 Embodiment 319: The VHH antibody domain or fragment thereof according to any one of

2481 embodiments 279 to 318, wherein said KD value is measured by kinetic measurements by 2482 biolayer interferometry at 25°C and 1000 rpm in KB Buffer (PBS + 0.1 % Tween-20 +

2483 1% BSA).

2484

2485 Embodiment 320: The VHH antibody domain or fragment thereof according to any one of

2486 embodiments 212 to 319, wherein said VHH antibody domain of (E) comprises one VHH

2487 sequence selected from the group consisting of VHH12, VHH13, VHH14, VHH15, VHH17,

2488 VHH18, VHH20, VHH21 and VHH22 shown in the Table of VHH Sequences.

2489

2490 Embodiment 321 : The VHH antibody domain or fragment thereof according to any one of

2491 embodiments 212 to 319, wherein said VHH antibody domain of (E) comprises one VHH

2492 sequence selected from the group consisting of VHH12, VHH14, VHH15, VHH17 and VHH22

2493 shown in the Table of VHH Sequences.

2494

2495 Embodiment 322: The VHH antibody domain or fragment thereof according to any one of

2496 embodiments 212 to 319, wherein said VHH antibody domain of (E) comprises one VHH

2497 sequence selected from the group consisting of VHH6 and VHH11 shown in the Table of VHH

2498 Sequences.

2499

2500 Embodiment 323: The VHH antibody domain or fragment thereof according to any one of

2501 embodiments 212 to 319, wherein said VHH antibody domain of (E) comprises one VHH

2502 sequence selected from the group consisting of VHH13, VHH16 and VHH22 shown in the

2503 Table of VHH Sequences.

2504

2505 Embodiment 324: The VHH antibody domain or fragment thereof according to any one of

2506 embodiments 212 to 319, wherein said VHH antibody domain of (E) comprises one VHH

2507 sequence selected from the group consisting of VHH12, VHH13, VHH15, VHH17, VHH18

2508 and VHH21 shown in the Table of VHH Sequences.

2509

2510 Embodiment 325: The VHH antibody domain or fragment thereof according to any one of

2511 embodiments 212 to 319, wherein said VHH antibody domain of (E) comprises one VHH

2512 sequence selected from the group consisting of VHH15 and VHH17 shown in the Table of

2513 VHH Sequences.

2514 2515 Embodiment 326: The VHH antibody domain or fragment thereof according to any one of

2516 embodiments 212 to 319, wherein said VHH antibody domain of (E) comprises the VHH

2517 sequence VHH12 shown in the Table of VHH Sequences.

2518

2519 Embodiment 327: The VHH antibody domain or fragment thereof according to any one of

2520 embodiments 212 to 319, wherein said VHH antibody domain of (E) comprises the VHH

2521 sequence VHH13 shown in the Table of VHH Sequences.

2522

2523 Embodiment 328: The VHH antibody domain or fragment thereof according to any one of

2524 embodiments 212 to 319, wherein said VHH antibody domain of (E) comprises the VHH

2525 sequence VHH14 shown in the Table of VHH Sequences.

2526

2527 Embodiment 329: The VHH antibody domain or fragment thereof according to any one of

2528 embodiments 212 to 319, wherein said VHH antibody domain of (E) comprises the VHH

2529 sequence VHH15 shown in the Table of VHH Sequences.

2530

2531 Embodiment 330: The VHH antibody domain or fragment thereof according to any one of

2532 embodiments 212 to 319, wherein said VHH antibody domain of (E) comprises the VHH

2533 sequence VHH16 shown in the Table of VHH Sequences.

2534

2535 Embodiment 331 : The VHH antibody domain or fragment thereof according to any one of

2536 embodiments 212 to 319, wherein said VHH antibody domain of (E) comprises the VHH

2537 sequence VHH17 shown in the Table of VHH Sequences.

2538

2539 Embodiment 332: The VHH antibody domain or fragment thereof according to any one of

2540 embodiments 212 to 319, wherein said VHH antibody domain of (E) comprises the VHH

2541 sequence VHH18 shown in the Table of VHH Sequences.

2542

2543 Embodiment 333: The VHH antibody domain or fragment thereof according to any one of

2544 embodiments 212 to 319, wherein said VHH antibody domain of (E) comprises the VHH

2545 sequence VHH20 shown in the Table of VHH Sequences.

2546 2547 Embodiment 334: The VHH antibody domain or fragment thereof according to any one of

2548 embodiments 212 to 319, wherein said VHH antibody domain of (E) comprises the VHH

2549 sequence VHH21 shown in the Table of VHH Sequences.

2550

2551 Embodiment 335: The VHH antibody domain or fragment thereof according to any one of

2552 embodiments 212 to 319, wherein said VHH antibody domain of (E) comprises the VHH

2553 sequence VHH22 shown in the Table of VHH Sequences.

2554

2555 Embodiment 336: The VHH antibody domain or fragment thereof according to any one of

2556 embodiments 189 to 211 or 250 to 335, wherein in (d) said VHH antibody domain or fragment

2557 thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one

2558 VHH selected from the group consisting of VHH12, VHH13, VHH14, VHH15, VHH17,

2559 VHH18, VHH20, VHH21 or VHH22 as shown in the Table of CDRs.

2560

2561 Embodiment 337: The VHH antibody domain or fragment thereof according to any one of

2562 embodiments 189 to 211 or 250 to 335, wherein in (d) said VHH antibody domain or fragment

2563 thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one

2564 VHH selected from the group consisting of VHH12, VHH14, VHH15, VHH17 or VHH22 as

2565 shown in the Table of CDRs.

2566

2567 Embodiment 338: The VHH antibody domain or fragment thereof according to any one of

2568 embodiments 189 to 211 or 250 to 335, wherein in (d) said VHH antibody domain or fragment

2569 thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH6

2570 or VHH11 as shown in the Table of CDRs.

2571

2572 Embodiment 339: The VHH antibody domain or fragment thereof according to any one of

2573 embodiments 189 to 211 or 250 to 335, wherein in (d) said VHH antibody domain or fragment

2574 thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one

2575 VHH selected from the group consisting of VHH13, VHH16 or VHH22 as shown in the Table

2576 of CDRs.

2577

2578 Embodiment 340: The VHH antibody domain or fragment thereof according to any one of

2579 embodiments 189 to 211 or 250 to 335, wherein in (d) said VHH antibody domain or fragment

2580 thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one 2581 VHH selected from the group consisting of VHH 12, VHH13, VHH15, VHH17, VHH18 or

2582 VHH21 as shown in the Table of CDRs.

2583

2584 Embodiment 341 : The VHH antibody domain or fragment thereof according to any one of

2585 embodiments 189 to 211 or 250 to 335, wherein in (d) said VHH antibody domain or fragment

2586 thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one

2587 VHH selected from the group consisting of VHH15 or VHH17 as shown in the Table of CDRs.

2588

2589 Embodiment 342: The VHH antibody domain or fragment thereof according to any one of

2590 embodiments 189 to 211 or 250 to 335, wherein in (d) said VHH antibody domain or fragment

2591 thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of

2592 VHH12 as shown in the Table of CDRs.

2593

2594 Embodiment 343: The VHH antibody domain or fragment thereof according to any one of

2595 embodiments 189 to 211 or 250 to 335, wherein in (d) said VHH antibody domain or fragment

2596 thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of

2597 VHH13 as shown in the Table of CDRs.

2598

2599 Embodiment 344: The VHH antibody domain or fragment thereof according to any one of

2600 embodiments 189 to 211 or 250 to 335, wherein in (d) said VHH antibody domain or fragment

2601 thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of

2602 VHH14 as shown in the Table of CDRs.

2603

2604 Embodiment 345: The VHH antibody domain or fragment thereof according to any one of

2605 embodiments 189 to 211 or 250 to 335, wherein in (d) said VHH antibody domain or fragment

2606 thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of

2607 VHH15 as shown in the Table of CDRs.

2608

2609 Embodiment 346: The VHH antibody domain or fragment thereof according to any one of

2610 embodiments 189 to 211 or 250 to 335, wherein in (d) said VHH antibody domain or fragment

2611 thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of

2612 VHH16 as shown in the Table of CDRs.

2613 2614 Embodiment 347: The VHH antibody domain or fragment thereof according to any one of

2615 embodiments 189 to 211 or 250 to 335, wherein in (d) said VHH antibody domain or fragment

2616 thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of

2617 VHH17 as shown in the Table of CDRs.

2618

2619 Embodiment 348: The VHH antibody domain or fragment thereof according to any one of

2620 embodiments 189 to 211 or 250 to 335, wherein in (d) said VHH antibody domain or fragment

2621 thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of

2622 VHH18 as shown in the Table of CDRs.

2623

2624 Embodiment 349: The VHH antibody domain or fragment thereof according to any one of

2625 embodiments 189 to 211 or 250 to 335, wherein in (d) said VHH antibody domain or fragment

2626 thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of

2627 VHH20 as shown in the Table of CDRs.

2628

2629 Embodiment 350: The VHH antibody domain or fragment thereof according to any one of

2630 embodiments 189 to 211 or 250 to 335, wherein in (d) said VHH antibody domain or fragment

2631 thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of

2632 VHH21 as shown in the Table of CDRs.

2633

2634 Embodiment 351: The VHH antibody domain or fragment thereof according to any one of

2635 embodiments 189 to 211 or 250 to 335, wherein in (d) said VHH antibody domain or fragment

2636 thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of

2637 VHH22 as shown in the Table of CDRs.

2638

2639 Embodiment 352: The VHH antibody domain or fragment thereof according to any one of

2640 embodiments 189 to 351, wherein said VHH antibody domain or fragment thereof competes

2641 with VHH15 for binding to recombinant human IL-18RP ECD.

2642

2643 Embodiment 353: The VHH antibody domain or fragment thereof according to any one of

2644 embodiments 189 to 351, wherein said VHH antibody domain or fragment thereof partially

2645 competes with VHH15 for binding to recombinant human IL-18RP ECD.

2646 2647 Embodiment 354: The VHH antibody domain or fragment thereof according to any one of

2648 embodiments 189 to 353, wherein said VHH antibody domain or fragment thereof does not

2649 compete with VHH2 for binding to recombinant human IL-18Ra ECD.

2650

2651 Embodiment 355: The VHH antibody domain or fragment thereof according to any one of

2652 embodiments 189 to 354, wherein said humanization is by germlining.

2653

2654 Embodiment 356: The VHH antibody domain or fragment thereof according to embodiment

2655 355, wherein said germlining involves

2656 - identifying the human VH germline sequence that is closest to the sequence to be

2657 humanized with respect to its sequence similarity; and

2658 - replacing amino acid(s) in the sequence to be humanized by the amino acid(s) at the

2659 corresponding sequence position(s) in the closest human VH germline sequence.

2660

2661 Sequence similarity and corresponding sequence positions can be determined by sequence

2662 alignment as described above.

2663

2664 Embodiment 357: The VHH antibody domain or fragment thereof according to embodiment

2665 355, wherein said germlining consists of

2666 - identifying the human VH germline sequence that is closest to the sequence to be

2667 humanized with respect to its sequence similarity; and

2668 - replacing amino acid(s) in the sequence to be humanized by the amino acid(s) at the

2669 corresponding sequence position(s) in the closest human VH germline sequence.

2670

2671 Embodiment 358: The VHH antibody domain or fragment thereof according to any one of

2672 embodiments 189 to 357, wherein said VHH antibody domain (resp. fragment thereof) with

2673 humanization binds to human IL-18RP ECD with an affinity (KD value) that is not weaker by

2674 a factor of more than 10 compared to the binding of a corresponding VHH antibody domain

2675 (resp. fragment thereof) without humanization to human IL-18RP ECD.

2676

2677 As a skilled person understands, the term "corresponding VHH antibody domain without

2678 humanization" refers to a VHH antibody domain that differs only by the amino acid changes

2679 introduced in order to carry out said humanization, but that is otherwise identical.

2680 2681 Embodiment 359: The VHH antibody domain or fragment thereof according to any one of

2682 embodiments 189 to 357, wherein said VHH antibody domain (resp. fragment thereof) with

2683 humanization binds to human IL-18RP ECD with an affinity (KD value) that is not weaker by

2684 a factor of more than 5 compared to the binding of a corresponding VHH antibody domain

2685 (resp. fragment thereof) without humanization to human IL-18RP ECD.

2686

2687 Embodiment 360: The VHH antibody domain or fragment thereof according to any one of

2688 embodiments 189 to 357, wherein said VHH antibody domain (resp. fragment thereof) with

2689 humanization binds to human IL-18RP ECD with an affinity (KD value) that is not weaker by

2690 a factor of more than 2 compared to the binding of a corresponding VHH antibody domain

2691 (resp. fragment thereof) without humanization to human IL-18RP ECD.

2692

2693 Embodiment 361 : The VHH antibody domain or fragment thereof according to any one of

2694 embodiments 189 to 357, wherein said VHH antibody domain (resp. fragment thereof) with

2695 humanization binds to human IL-18RP ECD with an affinity (KD value) that is not weaker than

2696 the binding of a corresponding VHH antibody domain (resp. fragment thereof) without

2697 humanization to human IL-18RP ECD.

2698

2699 Embodiment 362: The VHH antibody domain or fragment thereof according to any one of

2700 embodiments 358 to 361, wherein said affinity is determined by KD measurement.

2701

2702 Embodiment 363: The VHH antibody domain or fragment thereof according to any one of

2703 embodiments 212 to 362, wherein said VHH antibody domain comprises a VHH antibody

2704 domain according to (E), (F) or (G)/said fragment of a VHH antibody domain comprises a

2705 fragment of a VHH antibody domain according to (E), (F) or (G).

2706

2707 Embodiment 364: The VHH antibody domain or fragment thereof according to any one of

2708 embodiments 212 to 362, wherein said VHH antibody domain comprises a VHH antibody

2709 domain according to (E), (F) or (H)/said fragment of a VHH antibody domain comprises a

2710 fragment of a VHH antibody domain according to (E), (F) or (H).

2711

2712 Embodiment 365: The VHH antibody domain or fragment thereof according to any one of

2713 embodiments 212 to 362, wherein said VHH antibody domain comprises a VHH antibody 2714 domain according to (E), (G) or (H)/said fragment of a VHH antibody domain comprises a

2715 fragment of a VHH antibody domain according to (E), (G) or (H).

2716

2717 Embodiment 366: The VHH antibody domain or fragment thereof according to any one of

2718 embodiments 212 to 362, wherein said VHH antibody domain comprises a VHH antibody

2719 domain according to (E) or (F)/said fragment of a VHH antibody domain comprises a fragment

2720 of a VHH antibody domain according to (E) or (F).

2721

2722 Embodiment 367: The VHH antibody domain or fragment thereof according to any one of

2723 embodiments 212 to 362, wherein said VHH antibody domain comprises a VHH antibody

2724 domain according to (E) or (G)/said fragment of a VHH antibody domain comprises a fragment

2725 of a VHH antibody domain according to (E) or (G).

2726 1 1 Embodiment 368: The VHH antibody domain or fragment thereof according to any one of

2728 embodiments 212 to 362, wherein said VHH antibody domain comprises a VHH antibody

2729 domain according to (E) or (H)/said fragment of a VHH antibody domain comprises a fragment

2730 of a VHH antibody domain according to (E) or (H).

2731

2732 Embodiment 369: The VHH antibody domain or fragment thereof according to any one of

2733 embodiments 212 to 362, wherein said VHH antibody domain comprises a VHH antibody

2734 domain according to (E)/said fragment of a VHH antibody domain comprises a fragment of a

2735 VHH antibody domain according to (E).

2736

2737 Embodiment 370: The VHH antibody domain or fragment thereof according to any one of

2738 embodiments 212 to 362, wherein said VHH antibody domain comprises a VHH antibody

2739 domain according to (F)/said fragment of a VHH antibody domain comprises a fragment of a

2740 VHH antibody domain according to (F).

2741

2742 Embodiment 371 : The VHH antibody domain or fragment thereof according to any one of

2743 embodiments 189 to 211 or 250 to 370, wherein said VHH antibody domain or fragment thereof

2744 comprises complementarity determining regions according to (d) or (e).

2745 2746 Embodiment 372: The VHH antibody domain or fragment thereof according to any one of

2747 embodiments 189 to 211 or 250 to 370, wherein said VHH antibody domain or fragment thereof

2748 comprises complementarity determining regions according to (d) or (f).

2749

2750 Embodiment 373: The VHH antibody domain or fragment thereof according to any one of

2751 embodiments 189 to 211 or 250 to 370, wherein said VHH antibody domain or fragment thereof

2752 comprises complementarity determining regions according to (d).

2753

2754 Embodiment 374: The VHH antibody domain or fragment thereof according to any one of

2755 embodiments 189 to 211 or 250 to 370, wherein said VHH antibody domain or fragment thereof

2756 comprises complementarity determining regions according to (e).

2757

2758 Seventh aspect of the present disclosure (also referred to as "Embodiment 375"): According to

2759 a seventh aspect, the present disclosure relates to a compound comprising

2760 - a VHH antibody domain or fragment thereof according to any one of embodiments 1 to

2761 188.

2762

2763 Eighth aspect of the present disclosure (also referred to as "Embodiment 376"): According to a

2764 eighth aspect, the present disclosure relates to a compound comprising

2765 - a VHH antibody domain or fragment thereof according to any one of claims 189 to 374.

2766

2767 Nineth aspect of the present disclosure (also referred to as "Embodiment 377"): According to a

2768 nineth aspect, the present disclosure relates to a compound comprising

2769 - a first binding module which is a VHH antibody domain or fragment thereof, wherein

2770 (a) said VHH antibody domain or fragment thereof comprises the

2771 complementarity determining regions CDR1, CDR2 and CDR3 of one VHH

2772 selected from the group consisting of VHH1, VHH2, VHH3, VHH4, VHH5,

2773 VHH6, VHH8, VHH9, VHH10 and VHH11 as shown in the Table of CDR;

2774 (b) said VHH antibody domain or fragment thereof comprises the

2775 complementarity determining regions CDR1, CDR2 and CDR3 as defined in

2776 (a) with modification, wherein the modification is that the sequence of at least

2777 one of CDR1, CDR2 and CDR3 is humanized; or 2778 (c) said VHH antibody domain or fragment thereof comprises the

2779 complementarity determining regions CDR1, CDR2 and CDR3 as defined in

2780 (a) with modification, wherein the modification is

2781 - the replacement, addition or deletion of up to three amino acids in

2782 CDR1,

2783 - the replacement, addition or deletion of up to three amino acids in

2784 CDR2 and/or

2785 - the replacement, addition or deletion of up to three amino acids in

2786 CDR3;

2787 - a second binding module which is a VHH antibody domain or a fragment thereof, wherein

2788 (d) said VHH antibody domain or fragment thereof comprises the

2789 complementarity determining regions CDR1, CDR2 and CDR3 of one VHH

2790 selected from the group consisting of VHH12, VHH13, VHH14, VHH15,

2791 VHH16, VHH17, VHH18, VHH20, VHH21 and VHH22 as shown in the

2792 Table of CDRs;

2793 (e) said VHH antibody domain or fragment thereof comprises the

2794 complementarity determining regions CDR1, CDR2 and CDR3 as defined in

2795 (d) with modification, wherein the modification is that the sequence of at least

2796 one of CDR1, CDR2 and CDR3 is humanized; or

2797 (f) said VHH antibody domain or fragment thereof comprises the

2798 complementarity determining regions CDR1, CDR2 and CDR3 as defined in

2799 (d) with modification, wherein the modification is

2800 - the replacement, addition or deletion of up to three amino acids in

2801 CDR1,

2802 - the replacement, addition or deletion of up to three amino acids in

2803 CDR2 and/or

2804 - the replacement, addition or deletion of up to three amino acids in

2805 CDR3.

2806

2807 To the seventh to nineth aspect of the present disclosure and the embodiments referring thereto,

2808 the same explanations and definitions apply accordingly as for the first to sixth aspects of the

2809 present disclosure and the embodiments referring thereto.

2810 2811 For embodiment 377 and embodiments referring thereto, with respect to said first binding

2812 module further embodiments of said VHH antibody domain or fragment thereof of said first

2813 binding module are as defined in embodiments 1 to 188.

2814

2815 For embodiment 377 and embodiments referring thereto, with respect to said second binding

2816 module further embodiments of said VHH antibody domain or fragment thereof of said second

2817 binding module are as defined in embodiments 189 to 374.

2818

2819 Tenth aspect of the present disclosure (also referred to as "Embodiment 378"): According to a

2820 tenth aspect, the present disclosure relates to a compound comprising

2821 - a first binding module which is a VHH antibody domain or fragment thereof, wherein

2822 (A) said VHH antibody domain comprises the amino acid sequence of a VHH

2823 selected from the group consisting of VHH1, VHH2, VHH3, VHH4, VHH5,

2824 VHH6, VHH8, VHH9, VHH10 and VHH11 as shown in the Table of VHH

2825 Sequences;

2826 (B) said VHH antibody domain comprises a VHH sequence as defined in (A) with

2827 modification, wherein the modification is that said sequence is humanized;

2828 (C) said VHH antibody domain comprises a VHH sequence as defined in (A) with

2829 modification, wherein the modification is the replacement, addition or

2830 deletion of up to 25 amino acids; or

2831 (D) said VHH antibody domain comprises a VHH sequence that is at least 75%

2832 identical to a VHH sequence referred to in (A);

2833 - a second binding module which is a VHH antibody domain or a fragment thereof, wherein

2834 (E) said VHH antibody domain comprises the amino acid sequence of a VHH

2835 selected from the group consisting of VHH12, VHH13, VHH14, VHH15,

2836 VHH16, VHH17, VHH18, VHH20, VHH21 and VHH22 as shown in the

2837 Table of VHH Sequences;

2838 (F) said VHH antibody domain comprises a VHH sequence as defined in (E) with

2839 modification, wherein the modification is that said sequence is humanized;

2840 (G) said VHH antibody domain comprises a VHH sequence as defined in (E) with

2841 modification, wherein the modification is the replacement, addition or

2842 deletion of up to 25 amino acids; or

2843 (H) said VHH antibody domain comprises a VHH sequence that is at least 75%

2844 identical to a VHH sequence referred to in (E). 2845

2846 To the tenth aspect of the present disclosure and the embodiments referring thereto, the same

2847 explanations and definitions apply accordingly as for the first to nineth aspects of the present

2848 disclosure and the embodiments referring thereto.

2849

2850 For embodiment 378 and embodiments referring thereto, with respect to said first binding

2851 module further embodiments of said VHH antibody domain or fragment thereof of said first

2852 binding module are as defined in embodiments 1 to 188.

2853

2854 For embodiment 378 and embodiments referring thereto, with respect to said second binding

2855 module further embodiments of said VHH antibody domain or fragment thereof of said second

2856 binding module are as defined in embodiments 189 to 374.

2857

2858 Eleventh aspect of the present disclosure (also referred to as "Embodiment 379"): According to

2859 an eleventh aspect, the present disclosure relates to a compound comprising

2860 - a first binding module which is a VHH antibody domain or fragment thereof, wherein

2861 (A) said VHH antibody domain consists of the amino acid sequence of a VHH

2862 selected from the group consisting of VHH1, VHH2, VHH3, VHH4, VHH5,

2863 VHH6, VHH8, VHH9, VHH10 and VHH11 as shown in the Table of VHH

2864 Sequences;

2865 (B) said VHH antibody domain consists of a VHH sequence as defined in (A)

2866 with modification, wherein the modification is that said sequence is

2867 humanized;

2868 (C) said VHH antibody domain consists of a VHH sequence as defined in (A)

2869 with modification, wherein the modification is the replacement, addition or

2870 deletion of up to 25 amino acids; or

2871 (D) said VHH antibody domain consists of a VHH sequence that is at least 75%

2872 identical to a VHH sequence referred to in (A);

2873 - a second binding module which is a VHH antibody domain or a fragment thereof, wherein

2874 (E) said VHH antibody domain consists of the amino acid sequence of a VHH

2875 selected from the group consisting of VHH12, VHH13, VHH14, VHH15,

2876 VHH16, VHH17, VHH18, VHH20, VHH21 and VHH22 as shown in the

2877 Table of VHH Sequences; 2878 (F) said VHH antibody domain consists of a VHH sequence as defined in (E)

2879 with modification, wherein the modification is that said sequence is

2880 humanized;

2881 (G) said VHH antibody domain consists of a VHH sequence as defined in (E)

2882 with modification, wherein the modification is the replacement, addition or

2883 deletion of up to 25 amino acids; or

2884 (H) said VHH antibody domain consists of a VHH sequence that is at least 75%

2885 identical to a VHH sequence referred to in (E).

2886

2887 To the eleventh aspect of the present disclosure and the embodiments referring thereto, the same

2888 explanations and definitions apply accordingly as for the first to tenth aspects of the present

2889 disclosure and the embodiments referring thereto.

2890 For embodiment 379 and embodiments referring thereto, with respect to said first binding

2891 module further embodiments of said VHH antibody domain or fragment thereof of said first

2892 binding module are as defined in embodiments 1 to 188.

2893

2894 For embodiment 379 and embodiments referring thereto, with respect to said second binding

2895 module further embodiments of said VHH antibody domain or fragment thereof of said second

2896 binding module are as defined in embodiments 189 to 374.

2897

2898 "Compound", as used in the present disclosure, is not particularly limited and refers to a

2899 chemical entity of any chemical class, provided that it includes the binding module(s) as defined

2900 above. Thus, the compound can e.g. be an organic compound or a compound composed of an

2901 organic and an inorganic part, it can be a protein composed of a single amino acid chain, a

2902 protein composed of multiple amino acid chains that are either non-covalently or covalently

2903 associated, or a non-covalent complex including an inorganic component. The compound can

2904 consist of the amino acid sequence of the binding module(s) referred to in the above

2905 embodiments alone or it can in addition include further amino acid(s) that may be covalently

2906 or non-covalently attached, or it can be associated with inorganic components. Preferably, the

2907 compound is a molecule.

2908

2909 For example, in embodiment 375, with optional modifications from embodiments 1 to 188 and

2910 further embodiments referring to embodiment 375, (resp. in embodiment 376, with optional

2911 modifications from embodiments 189 to 374 and further embodiments referring to embodiment 2912 376), the compound can be e.g. a monovalent, monospecific antibody molecule in which an

2913 anti-IL-18Ra VHH according to the present disclosure (resp. an anti-IL-18RP VHH according

2914 to the present disclosure) is linked covalently to an Fc region of an IgG. Alternatively, it could

2915 be a bispecific molecule in which an anti-IL-18Ra VHH according to the present disclosure

2916 (resp. an anti-IL-18RP VHH according to the present disclosure) is linked covalently to an IgGl

2917 antibody lacking one of its "arms", such that the molecule has two binding modules, namely

2918 the anti-IL-18Ra VHH according to the present disclosure (resp. anti-IL-18Rp VHH according

2919 to the present disclosure) and the remaining "original" binding module of the "one-armed" IgGl

2920 antibody. Or in another example, the compound can be a bispecific molecule comprising the

2921 anti-IL-18Ra VHH according to the present disclosure (resp. anti-IL-18RP VHH according to

2922 the present disclosure) and a targeting module.

2923

2924 For example, in embodiments 377 to 379 (with optional modifications for the first binding

2925 module defined in embodiments 1 to 188 and optional modifications for the second binding

2926 module defined in embodiments 189 to 374, with further optional modifications defined in the

2927 embodiments referring to embodiments 377 to 379), the compound may e.g. be an antibody

2928 with two binding modules (an anti-IL-18Ra VHH according to the present disclosure and an

2929 anti-IL-18RP VHH according to the present disclosure) prepared in the SEED format, resulting

2930 in a bispecific antibody with a structure e.g. as shown in Fig. 4A (left). The compound of

2931 embodiment 377 to 379 may in addition comprise further molecular components, e.g. further

2932 copies of the first and second bindings modules as exemplified in Fig. 4A (middle and right)

2933 where molecules with two copies of the first binding module and two copies of the second

2934 binding module in different molecular arrangements are shown. As the skilled person will

2935 understand, many other formats of the compound are possible, provided that the resulting

2936 compound format does not interfere with the function of the VHH antibody domain(s) or

2937 fragment(s) thereof according to the invention (i.e. in the case of the compound of embodiment

2938 377 to 379: the molecular format does not interfere with the binding of the first binding module

2939 to IL18Ra and the binding of the second binding module to IL18RP, as described in the present

2940 disclosure).

2941

2942 The compound of the present disclosure can be prepared by standard methods of genetic

2943 engineering and recombinant protein technology known to the skilled person (see e.g. Green

2944 and Sambrook, "Molecular Cloning: A Laboratory Manual", 2014; Coligan et al., "Current 2945 Protocols in Protein Science", 1997). Exemplary methods are also described in the Examples

2946 section of the present disclosure.

2947

2948 In cases where the compound cannot be expressed in a single piece, individual parts can be

2949 prepared individually and later either covalently coupled, for example by a chemical reaction

2950 with appropriate reactive groups (e.g. linkage by maleimide chemistry) or by enzymatic linkage

2951 (e.g. transglutaminase-catalyzed linkage). For example, the VHH antibody domain(s) or

2952 fragment(s) thereof can be prepared by recombinant protein expression and subsequently linked

2953 to an antibody or antibody fragment, resulting in a bispecific antibody compound as described

2954 in the Examples section.

2955

2956 If the compound comprises components that are not biomolecules (such as a peptide mimetics

2957 or a small molecule), these components may be obtained e.g. by standard methods of synthetic

2958 organic chemistry.

2959

2960 Embodiment 380: The compound according to embodiment 377,

2961 - wherein said VHH antibody domain or fragment thereof defined in (a) comprises the

2962 complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected

2963 from the group consisting of VHH1, VHH2, VHH5, VHH6, VHH8, VHH9 and VHH10

2964 as shown in the Table of CDRs and

2965 - wherein said VHH antibody domain or fragment thereof defined in (d) comprises the

2966 complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected

2967 from the group consisting of VHH12, VHH13, VHH14, VHH15, VHH17, VHH18,

2968 VHH20, VHH21 and VHH22 as shown in the Table of CDRs, wherein said VHH

2969 antibody domain or fragment thereof defined in (d) is selected as follows:

2970 if the VHH antibody domain of (a) or the fragment thereof comprises the

2971 complementarity determining regions CDR1, CDR2 and CDR3 of VHH1, then

2972 the VHH antibody domain of (d) or the fragment thereof comprises the

2973 complementarity determining regions CDR1, CDR2 and CDR3 of one VHH

2974 selected from the group consisting of VHH12, VHH13, VHH15, VHH17,

2975 VHH20, VHH21 and VHH22 as shown in the Table of CDRs;

2976 if the VHH antibody domain of (a) or the fragment thereof comprises the

2977 complementarity determining regions CDR1, CDR2 and CDR3 of VHH2, then

2978 the VHH antibody domain of (d) or the fragment thereof comprises the 2979 complementarity determining regions CDR1, CDR2 and CDR3 of one VHH

2980 selected from the group consisting of VHH12, VHH13, VHH15, VHH17,

2981 VHH18, VHH20, VHH21 and VHH22 as shown in the Table of CDRs;

2982 if the VHH antibody domain of (a) or the fragment thereof comprises the

2983 complementarity determining regions CDR1, CDR2 and CDR3 of VHH5, then

2984 the VHH antibody domain of (d) or the fragment thereof comprises the

2985 complementarity determining regions CDR1, CDR2 and CDR3 of one VHH

2986 selected from the group consisting of VHH13 and VHH14 as shown in the Table

2987 of CDRs;

2988 if the VHH antibody domain of (a) or the fragment thereof comprises the

2989 complementarity determining regions CDR1, CDR2 and CDR3 of VHH6, then

2990 the VHH antibody domain of (d) or the fragment thereof comprises the

2991 complementarity determining regions CDR1, CDR2 and CDR3 of one VHH

2992 selected from the group consisting of VHH17, VHH20, VHH21 and VHH22 as

2993 shown in the Table of CDRs;

2994 if the VHH antibody domain of (a) or the fragment thereof comprises the

2995 complementarity determining regions CDR1, CDR2 and CDR3 of VHH8, then

2996 the VHH antibody domain of (d) or the fragment thereof comprises the

2997 complementarity determining regions CDR1, CDR2 and CDR3 of one VHH

2998 selected from the group consisting of VHH12, VHH13, VHH17, VHH18, VHH20

2999 and VHH21 as shown in the Table of CDRs;

3000 if the VHH antibody domain of (a) or the fragment thereof comprises the

3001 complementarity determining regions CDR1, CDR2 and CDR3 of VHH9, then

3002 the VHH antibody domain of (d) or the fragment thereof comprises the

3003 complementarity determining regions CDR1, CDR2 and CDR3 of one VHH

3004 selected from the group consisting of VHH12, VHH13, VHH15, VHH17,

3005 VHH18, VHH20, VHH21 and VHH22 as shown in the Table of CDRs;

3006 if the VHH antibody domain of (a) or the fragment thereof comprises the

3007 complementarity determining regions CDR1, CDR2 and CDR3 of VHHIO, then

3008 the VHH antibody domain of (d) or the fragment thereof comprises the

3009 complementarity determining regions CDR1, CDR2 and CDR3 of one VHH

3010 selected from the group consisting of VHH13, VHH20 and VHH21 as shown in

3011 the Table of CDRs.

3012 3013 Thus, according this embodiment, the compound of embodiment 377 (and the embodiments

3014 referring thereto) can comprise any of the combinations VHHa and VHHP shown in Fig. 4B in

3015 green (surrogate agonists triggering either a normalized NFKB reporter activation of more than

3016 50% relative to IL18 or displaying potencies of less than 0.1 nM).

3017

3018 Embodiment 381 : The compound according to embodiment 377,

3019 - wherein said VHH antibody domain or fragment thereof defined in (a) comprises the

3020 complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected

3021 from the group consisting of VHH1, VHH2, VHH3, VHH4, VHH5, VHH6, VHH8,

3022 VHH9 and VHH10 as shown in the Table of CDRs and

3023 - wherein said VHH antibody domain or fragment thereof defined in (d) comprises the

3024 complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected

3025 from the group consisting of VHH12, VHH13, VHH14, VHH15, VHH16, VHH17,

3026 VHH18, VHH20, VHH21 and VHH22 as shown in the Table of CDRs, wherein said

3027 VHH antibody domain or fragment thereof defined in (d) is selected as follows:

3028 if the VHH antibody domain of (a) or the fragment thereof comprises the

3029 complementarity determining regions CDR1, CDR2 and CDR3 of VHH1, then

3030 the VHH antibody domain of (d) or the fragment thereof comprises the

3031 complementarity determining regions CDR1, CDR2 and CDR3 of one VHH

3032 selected from the group consisting of VHH14, VHH16 and VHH18 as shown in

3033 the Table of CDRs;

3034 if the VHH antibody domain of (a) or the fragment thereof comprises the

3035 complementarity determining regions CDR1, CDR2 and CDR3 of VHH2, then

3036 the VHH antibody domain of (d) or the fragment thereof comprises the

3037 complementarity determining regions CDR1, CDR2 and CDR3 of one VHH

3038 selected from the group consisting of VHH14 and VHH16 as shown in the Table

3039 of CDRs;

3040 if the VHH antibody domain of (a) or the fragment thereof comprises the

3041 complementarity determining regions CDR1, CDR2 and CDR3 of VHH3, then

3042 the VHH antibody domain of (d) or the fragment thereof comprises the

3043 complementarity determining regions CDR1, CDR2 and CDR3 of one VHH

3044 selected from the group consisting of VHH12, VHH13, VHH14, VHH15,

3045 VHH16, VHH17, VHH18, VHH20, VHH21 and VHH22 as shown in the Table

3046 of CDRs; 3047 if the VHH antibody domain of (a) or the fragment thereof comprises the

3048 complementarity determining regions CDR1, CDR2 and CDR3 of VHH4, then

3049 the VHH antibody domain of (d) or the fragment thereof comprises the

3050 complementarity determining regions CDR1, CDR2 and CDR3 of one VHH

3051 selected from the group consisting of VHH12, VHH13, VHH14, VHH15,

3052 VHH16, VHH17, VHH18, VHH20, VHH21 and VHH22 as shown in the Table

3053 of CDRs;

3054 if the VHH antibody domain of (a) or the fragment thereof comprises the

3055 complementarity determining regions CDR1, CDR2 and CDR3 of VHH5, then

3056 the VHH antibody domain of (d) or the fragment thereof comprises the

3057 complementarity determining regions CDR1, CDR2 and CDR3 of one VHH

3058 selected from the group consisting of of VHH12, VHH15, VHH16, VHH17,

3059 VHH18, VHH20, VHH21 and VHH22 as shown in the Table of CDRs;

3060 if the VHH antibody domain of (a) or the fragment thereof comprises the

3061 complementarity determining regions CDR1, CDR2 and CDR3 of VHH6, then

3062 the VHH antibody domain of (d) or the fragment thereof comprises the

3063 complementarity determining regions CDR1, CDR2 and CDR3 of one VHH

3064 selected from the group consisting of VHH14, VHH16 and VHH18 as shown in

3065 the Table of CDRs;

3066 if the VHH antibody domain of (a) or the fragment thereof comprises the

3067 complementarity determining regions CDR1, CDR2 and CDR3 of VHH8, then

3068 the VHH antibody domain of (d) or the fragment thereof comprises the

3069 complementarity determining regions CDR1, CDR2 and CDR3 of VHH16 as

3070 shown in the Table of CDRs;

3071 if the VHH antibody domain of (a) or the fragment thereof comprises the

3072 complementarity determining regions CDR1, CDR2 and CDR3 of VHH9, then

3073 the VHH antibody domain of (d) or the fragment thereof comprises the

3074 complementarity determining regions CDR1, CDR2 and CDR3 of one VHH

3075 selected from the group consisting of VHH14, VHH16 and VHH18 as shown in

3076 the Table of CDRs;

3077 if the VHH antibody domain of (a) or the fragment thereof comprises the

3078 complementarity determining regions CDR1, CDR2 and CDR3 of VHH10, then

3079 the VHH antibody domain of (d) or the fragment thereof comprises the

3080 complementarity determining regions CDR1, CDR2 and CDR3 of one VHH 3081 selected from the group consisting of VHH14, VHH15, VHH16 and VHH18 as

3082 shown in the Table of CDRs.

3083

3084 Thus, according to this embodiment, the compound of embodiment 377 (and the embodiments

3085 referring thereto) can comprise any of the combinations VHHa and VHHP shown in Fig. 4B in

3086 red.

3087

3088 Embodiment 382: The compound according to embodiment 377,

3089 - wherein said VHH antibody domain or fragment thereof defined in (a) comprises the

3090 complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected

3091 from the group consisting of VHH6 and VHH11 as shown in the Table of CDRs and

3092 - wherein said VHH antibody domain or fragment thereof defined in (d) comprises the

3093 complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected

3094 from the group consisting of VHH12, VHH13, VHH14, VHH15, VHH16, VHH17,

3095 VHH18, VHH20, VHH21 and VHH22 as shown in the Table of CDRs, wherein said

3096 VHH antibody domain or fragment thereof defined in (d) is selected as follows:

3097 if the VHH antibody domain of (a) or the fragment thereof comprises the

3098 complementarity determining regions CDR1, CDR2 and CDR3 of VHH6, then

3099 the VHH antibody domain of (d) or the fragment thereof comprises the

3100 complementarity determining regions CDR1, CDR2 and CDR3 of one VHH

3101 selected from the group consisting of VHH12 and VHH13 as shown in the Table

3102 of CDRs;

3103 if the VHH antibody domain of (a) or the fragment thereof comprises the

3104 complementarity determining regions CDR1, CDR2 and CDR3 of VHH11, then

3105 the VHH antibody domain of (d) or the fragment thereof comprises the

3106 complementarity determining regions CDR1, CDR2 and CDR3 of one VHH

3107 selected from the group consisting of VHH12, VHH13, VHH14, VHH15,

3108 VHH16, VHH17, VHH18, VHH20, VHH21 and VHH22 as shown in the Table

3109 of CDRs.

3110

3111 Thus, according to this embodiment, the compound of embodiment 377 (and the embodiments

3112 referring thereto) can comprise any of the combinations VHHa and VHHP shown in Fig. 4B in

3113 black. 3115 Embodiment 383: The compound according to embodiment 377,

3116 - wherein said VHH antibody domain or fragment thereof defined in (a) comprises the

3117 complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected

3118 from the group consisting of VHH2, VHH6, VHH8 and VHH10 as shown in the Table

3119 of CDRs and

3120 - wherein said VHH antibody domain or fragment thereof defined in (d) comprises the

3121 complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected

3122 from the group consisting of VHH 12, VHH 14, VHH 15, VHH 17 and VHH22 as shown

3123 in the Table of CDRs, wherein said VHH antibody domain or fragment thereof defined

3124 in (d) is selected as follows:

3125 if the VHH antibody domain of (a) or the fragment thereof comprises the

3126 complementarity determining regions CDR1, CDR2 and CDR3 of VHH2, then

3127 the VHH antibody domain of (d) or the fragment thereof comprises the

3128 complementarity determining regions CDR1, CDR2 and CDR3 of VHH22 as

3129 shown in the Table of CDRs;

3130 if the VHH antibody domain of (a) or the fragment thereof comprises the

3131 complementarity determining regions CDR1, CDR2 and CDR3 of VHH6, then

3132 the VHH antibody domain of (d) or the fragment thereof comprises the

3133 complementarity determining regions CDR1, CDR2 and CDR3 of VHH15 as

3134 shown in the Table of CDRs;

3135 if the VHH antibody domain of (a) or the fragment thereof comprises the

3136 complementarity determining regions CDR1, CDR2 and CDR3 of VHH8, then

3137 the VHH antibody domain of (d) or the fragment thereof comprises the

3138 complementarity determining regions CDR1, CDR2 and CDR3 of one VHH

3139 selected from the group consisting of VHH14, VHH15 and VHH22 as shown in

3140 the Table of CDRs;

3141 if the VHH antibody domain of (a) or the fragment thereof comprises the

3142 complementarity determining regions CDR1, CDR2 and CDR3 of VHH10, then

3143 the VHH antibody domain of (d) or the fragment thereof comprises the

3144 complementarity determining regions CDR1, CDR2 and CDR3 of one VHH

3145 selected from the group consisting of VHH12, VHH17 and VHH22 as shown in

3146 the Table of CDRs.

3147 3148 Thus, according to this embodiment, the compound of embodiment 377 (and the embodiments

3149 referring thereto) can comprise any of the combinations VHHa and VHHP shown in Fig. 4B in

3150 orange (active surrogate agonists eliciting less than 50% of NFKB reporter activation

3151 normalized to (rh) IL-18 at 1 nM or EC50 higher than 0.1 nM).

3152

3153 Embodiment 384: The compound according to embodiment 377, wherein said VHH antibody

3154 domain or fragment thereof defined in (a) comprises the complementarity determining regions

3155 CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHH2 and VHH8

3156 as shown in the Table of CDRs and wherein said VHH antibody domain or fragment thereof

3157 defined in (d) comprises the complementarity determining regions CDR1, CDR2 and CDR3 of

3158 one VHH selected from the group consisting of VHH15 and VHH17 as shown in the Table of

3159 CDRs.

3160

3161 Embodiment 385: The compound according to embodiment 377,

3162 - wherein said VHH antibody domain or fragment thereof defined in (a) comprises the

3163 complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected

3164 from the group consisting of VHH2 and VHH8 as shown in the Table of CDRs and

3165 - wherein said VHH antibody domain or fragment thereof defined in (d) comprises the

3166 complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected

3167 from the group consisting of VHH15 and VHH17 as shown in the Table of CDRs,

3168 wherein said VHH antibody domain or fragment thereof defined in (d) is selected as

3169 follows:

3170 if the VHH antibody domain of (a) or the fragment thereof comprises the

3171 complementarity determining regions CDR1, CDR2 and CDR3 of VHH2, then

3172 the VHH antibody domain of (d) or the fragment thereof comprises the

3173 complementarity determining regions CDR1, CDR2 and CDR3 of one VHH

3174 selected from the group consisting of VHH15 and VHH17 as shown in the Table

3175 of CDRs;

3176 if the VHH antibody domain of (a) or the fragment thereof comprises the

3177 complementarity determining regions CDR1, CDR2 and CDR3 of VHH8, then

3178 the VHH antibody domain of (d) or the fragment thereof comprises the

3179 complementarity determining regions CDR1, CDR2 and CDR3 of VHH17 as

3180 shown in the Table of CDRs.

3181 3182 Embodiment 386: The compound according to embodiment 377, wherein said VHH antibody

3183 domain or fragment thereof defined in (a) comprises the complementarity determining regions

3184 CDR1, CDR2 and CDR3 of VHH2 as shown in the Table of CDRs and wherein said VHH

3185 antibody domain or fragment thereof defined in (d) comprises the complementarity determining

3186 regions CDR1, CDR2 and CDR3 of VHH15 as shown in the Table of CDRs.

3187

3188 Embodiment 387: The compound according to embodiment 377,

3189 - wherein said VHH antibody domain or fragment thereof defined in (a) comprises the

3190 complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected

3191 from the group consisting of VHH1, VHH2, VHH5, VHH6, VHH8 and VHH 10 as

3192 shown in the Table of CDRs and

3193 - wherein said VHH antibody domain or fragment thereof defined in (d) comprises the

3194 complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected

3195 from the group consisting of VHH13, VHH16 and VHH22 as shown in the Table of

3196 CDRs, wherein said VHH antibody domain or fragment thereof defined in (d) is selected

3197 as follows:

3198 if the VHH antibody domain of (a) or the fragment thereof comprises the

3199 complementarity determining regions CDR1, CDR2 and CDR3 of VHH1, then

3200 the VHH antibody domain of (d) or the fragment thereof comprises the

3201 complementarity determining regions CDR1, CDR2 and CDR3 of VHH16 as

3202 shown in the Table of CDRs;

3203 if the VHH antibody domain of (a) or the fragment thereof comprises the

3204 complementarity determining regions CDR1, CDR2 and CDR3 of VHH2, then

3205 the VHH antibody domain of (d) or the fragment thereof comprises the

3206 complementarity determining regions CDR1, CDR2 and CDR3 of VHH22 as

3207 shown in the Table of CDRs;

3208 if the VHH antibody domain of (a) or the fragment thereof comprises the

3209 complementarity determining regions CDR1, CDR2 and CDR3 of VHH5, then

3210 the VHH antibody domain of (d) or the fragment thereof comprises the

3211 complementarity determining regions CDR1, CDR2 and CDR3 of VHH13 as

3212 shown in the Table of CDRs;

3213 if the VHH antibody domain of (a) or the fragment thereof comprises the

3214 complementarity determining regions CDR1, CDR2 and CDR3 of VHH6, then

3215 the VHH antibody domain of (d) or the fragment thereof comprises the 3216 complementarity determining regions CDR1, CDR2 and CDR3 of VHH22 as

3217 shown in the Table of CDRs;

3218 if the VHH antibody domain of (a) or the fragment thereof comprises the

3219 complementarity determining regions CDR1, CDR2 and CDR3 of VHH8, then

3220 the VHH antibody domain of (d) or the fragment thereof comprises the

3221 complementarity determining regions CDR1, CDR2 and CDR3 of VHH22 as

3222 shown in the Table of CDRs;

3223 if the VHH antibody domain of (a) or the fragment thereof comprises the

3224 complementarity determining regions CDR1, CDR2 and CDR3 of VHH10, then

3225 the VHH antibody domain of (d) or the fragment thereof comprises the

3226 complementarity determining regions CDR1, CDR2 and CDR3 of VHH22 as

3227 shown in the Table of CDRs.

3228

3229 Embodiment 388: The compound according to embodiment 377,

3230 - wherein said VHH antibody domain or fragment thereof defined in (a) comprises the

3231 complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected

3232 from the group consisting of VHH2, VHH6, VHH8 and VHH10 as shown in the Table

3233 of CDRs and

3234 - wherein said VHH antibody domain or fragment thereof defined in (d) comprises the

3235 complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected

3236 from the group consisting of VHH12 and VHH15 as shown in the Table of CDRs, wherein

3237 said VHH antibody domain or fragment thereof defined in (d) is selected as follows:

3238 if the VHH antibody domain of (a) or the fragment thereof comprises the

3239 complementarity determining regions CDR1, CDR2 and CDR3 of VHH2, then the

3240 VHH antibody domain of (d) or the fragment thereof comprises the

3241 complementarity determining regions CDR1, CDR2 and CDR3 of VHH12 as

3242 shown in the Table of CDRs;

3243 if the VHH antibody domain of (a) or the fragment thereof comprises the

3244 complementarity determining regions CDR1, CDR2 and CDR3 of VHH6, then the

3245 VHH antibody domain of (d) or the fragment thereof comprises the

3246 complementarity determining regions CDR1, CDR2 and CDR3 of one VHH

3247 selected from the group consisting of VHH12 and VHH15 as shown in the Table of

3248 CDRs; 3249 if the VHH antibody domain of (a) or the fragment thereof comprises the

3250 complementarity determining regions CDR1, CDR2 and CDR3 of VHH8, then the

3251 VHH antibody domain of (d) or the fragment thereof comprises the

3252 complementarity determining regions CDR1, CDR2 and CDR3 of one VHH

3253 selected from the group consisting of VHH13 and VHH18 as shown in the Table of

3254 CDRs;

3255 if the VHH antibody domain of (a) or the fragment thereof comprises the

3256 complementarity determining regions CDR1, CDR2 and CDR3 of VHH10, then

3257 the VHH antibody domain of (d) or the fragment thereof comprises the

3258 complementarity determining regions CDR1, CDR2 and CDR3 of VHH12 as

3259 shown in the Table of CDRs.

3260

3261 Embodiment 389: The compound according to any one of embodiments 377 or 380 to 388,

3262 wherein in the first binding module said VHH antibody domain or fragment thereof comprises

3263 complementarity determining regions according to (a) or (b).

3264

3265 If the present disclosure states that "in the first binding module said VHH antibody domain or

3266 fragment thereof comprises complementarity determining regions according to (a) or (b)", then

3267 this is meant to designate that in this embodiment only (a) and (b) are considered as options for

3268 the VHH antibody domain (or fragment thereof) of the first binding module, whereas option (c)

3269 is excluded. If the present disclosure states that "in the first binding module said VHH antibody

3270 domain or fragment thereof comprises complementarity determining regions according to (a)

3271 or (c)", then this is meant to designate that in this embodiment only (a) and (c) are considered

3272 as options for the VHH antibody domain (or fragment thereof) of the first binding module,

3273 whereas option (b) is excluded. The alternative wording below is to be understood accordingly.

3274 If the present disclosure states that "in the first binding module said VHH antibody domain or

3275 fragment thereof comprises complementarity determining regions according to (a)", then this

3276 is meant to designate that in this embodiment only (a) is considered as option for the VHH

3277 antibody domain (or fragment thereof) of the first binding module, whereas options (b) and (c)

3278 are excluded. Further variations of this wording below are to be understood accordingly.

3279

3280 Embodiment 390: The compound according to any one of embodiments 377 or 380 to 388,

3281 wherein in the first binding module said VHH antibody domain or fragment thereof comprises

3282 complementarity determining regions according to (a) or (c). 3283

3284 Embodiment 391 : The compound according to any one of embodiments 377 or 380 to 388,

3285 wherein in the first binding module said VHH antibody domain or fragment thereof comprises

3286 complementarity determining regions according to (a).

3287

3288 Embodiment 392: The compound according to any one of embodiments 377 or 380 to 388,

3289 wherein in the first binding module said VHH antibody domain or fragment thereof comprises

3290 complementarity determining regions according to (b).

3291

3292 Embodiment 393: The compound according to any one of embodiments 377 or 380 to 392,

3293 wherein in the second binding module said VHH antibody domain or fragment thereof

3294 comprises complementarity determining regions according to (d) or (e).

3295

3296 Embodiment 394: The compound according to any one of embodiments 377 or 380 to 392,

3297 wherein in the second binding module said VHH antibody domain or fragment thereof

3298 comprises complementarity determining regions according to (d) or (f).

3299

3300 Embodiment 395: The compound according to any one of embodiments 377 or 380 to 392,

3301 wherein in the second binding module said VHH antibody domain or fragment thereof

3302 comprises complementarity determining regions according to (d).

3303

3304 Embodiment 396: The compound according to any one of embodiments 377 or 380 to 392,

3305 wherein in the second binding module said VHH antibody domain or fragment thereof

3306 comprises complementarity determining regions according to (e).

3307

3308 Embodiment 397: The compound according to any one of embodiments 378 or 379,

3309 - wherein said VHH antibody domain of (A) comprises the amino acid sequence of one

3310 VHH selected from the group consisting of VHH1, VHH2, VHH5, VHH6, VHH8,

3311 VHH9 and VHH10 shown in the Table of VHH Sequences and

3312 - wherein said VHH antibody domain of (E) comprises the amino acid sequence of one

3313 VHH selected from the group consisting of VHH12, VHH13, VHH14, VHH15,

3314 VHH17, VHH18, VHH20, VHH21 and VHH22 shown in the Table of VHH Sequences,

3315 wherein said VHH antibody domain of (E) is selected as follows: 3316 if the VHH antibody domain of (A) comprises the amino acid sequence of VHH1,

3317 then the VHH antibody domain of (E) comprises the amino acid sequence of one

3318 VHH selected from the group consisting of VHH12, VHH13, VHH15, VHH17,

3319 VHH20, VHH21 and VHH22 shown in the Table of VHH Sequences;

3320 if the VHH antibody domain of (A) comprises the amino acid sequence of VHH2,

3321 then the VHH antibody domain of (E) comprises the amino acid sequence of one

3322 VHH selected from the group consisting of VHH12, VHH13, VHH15, VHH17,

3323 VHH18, VHH20, VHH21 and VHH22 shown in the Table of VHH Sequences;

3324 if the VHH antibody domain of (A) comprises the amino acid sequence of VHH5,

3325 then the VHH antibody domain of (E) comprises the amino acid sequence of one

3326 VHH selected from the group consisting of VHH13 and VHH14 shown in the

3327 Table of VHH Sequences;

3328 if the VHH antibody domain of (A) comprises the amino acid sequence of VHH6,

3329 then the VHH antibody domain of (E) comprises the amino acid sequence of one

3330 VHH selected from the group consisting of VHH17, VHH20, VHH21 and VHH22

3331 shown in the Table of VHH Sequences;

3332 if the VHH antibody domain of (A) comprises the amino acid sequence of VHH8,

3333 then the VHH antibody domain of (E) comprises the amino acid sequence of one

3334 VHH selected from the group consisting of VHH12, VHH13, VHH17, VHH18,

3335 VHH20 and VHH21 shown in the Table of VHH Sequences;

3336 if the VHH antibody domain of (A) comprises the amino acid sequence of VHH9,

3337 then the VHH antibody domain of (E) comprises the amino acid sequence of one

3338 VHH selected from the group consisting of VHH12, VHH13, VHH15, VHH17,

3339 VHH18, VHH20, VHH21 and VHH22 shown in the Table of VHH Sequences;

3340 if the VHH antibody domain of (A) comprises the amino acid sequence of VHH10,

3341 then the VHH antibody domain of (E) comprises the amino acid sequence of one

3342 VHH selected from the group consisting of VHH13, VHH20 and VHH21 shown

3343 in the Table of VHH Sequences.

3344

3345 Thus, according to this embodiment, the compound of embodiments 378 to 379 (and the

3346 embodiments referring thereto) can comprise any of the combinations VHHa and VHHP shown

3347 in Fig. 4B in green (surrogate agonists triggering either a normalized NFKB reporter activation

3348 of more than 50% relative to IL18 or displaying potencies of less than 0.1 nM).

3349 3350 Embodiment 398: The compound according to any one of embodiments 378 or 379,

3351 - wherein said VHH antibody domain of (A) comprises the amino acid sequence of one

3352 VHH selected from the group consisting of VHH1, VHH2, VHH3, VHH4, VHH5,

3353 VHH6, VHH8, VHH9 and VHH10 shown in the Table of VHH Sequences and

3354 - wherein said VHH antibody domain of (E) comprises the amino acid sequence of one

3355 VHH selected from the group consisting of VHH12, VHH13, VHH14, VHH15,

3356 VHH16, VHH17, VHH18, VHH20, VHH21 and VHH22 shown in the Table of VHH

3357 Sequences, wherein said VHH antibody domain of (E) is selected as follows:

3358 if the VHH antibody domain of (A) comprises the amino acid sequence of VHH1,

3359 then the VHH antibody domain of (E) comprises the amino acid sequence of one

3360 VHH selected from the group consisting of VHH14, VHH16 and VHH18 shown

3361 in the Table of VHH Sequences;

3362 if the VHH antibody domain of (A) comprises the amino acid sequence of VHH2,

3363 then the VHH antibody domain of (E) comprises the amino acid sequence of one

3364 VHH selected from the group consisting of VHH14 and VHH16 shown in the

3365 Table of VHH Sequences;

3366 if the VHH antibody domain of (A) comprises the amino acid sequence of VHH3,

3367 then the VHH antibody domain of (E) comprises the amino acid sequence of one

3368 VHH selected from the group consisting of VHH12, VHH13, VHH14, VHH15,

3369 VHH16, VHH17, VHH18, VHH20, VHH21 and VHH22 shown in the Table of

3370 VHH Sequences;

3371 if the VHH antibody domain of (A) comprises the amino acid sequence of VHH4,

3372 then the VHH antibody domain of (E) comprises the amino acid sequence of one

3373 VHH selected from the group consisting of VHH12, VHH13, VHH14, VHH15,

3374 VHH16, VHH17, VHH18, VHH20, VHH21 and VHH22 shown in the Table of

3375 VHH Sequences;

3376 if the VHH antibody domain of (A) comprises the amino acid sequence of VHH5,

3377 then the VHH antibody domain of (E) comprises the amino acid sequence of one

3378 VHH selected from the group consisting of VHH12, VHH15, VHH16, VHH17,

3379 VHH18, VHH20, VHH21 and VHH22 shown in the Table of VHH Sequences;

3380 if the VHH antibody domain of (A) comprises the amino acid sequence of VHH6,

3381 then the VHH antibody domain of (E) comprises the amino acid sequence of one

3382 VHH selected from the group consisting of VHH14, VHH16 and VHH18 shown

3383 in the Table of VHH Sequences; 3384 if the VHH antibody domain of (A) comprises the amino acid sequence of VHH8,

3385 then the VHH antibody domain of (E) comprises the amino acid sequence of

3386 VHH16 shown in the Table of VHH Sequences;

3387 if the VHH antibody domain of (A) comprises the amino acid sequence of VHH9,

3388 then the VHH antibody domain of (E) comprises the amino acid sequence of one

3389 VHH selected from the group consisting of VHH14, VHH16 and VHH18 shown

3390 in the Table of VHH Sequences;

3391 if the VHH antibody domain of (A) comprises the amino acid sequence of VHH10,

3392 then the VHH antibody domain of (E) comprises the amino acid sequence of one

3393 VHH selected from the group consisting of VHH14, VHH15, VHH16 and VHH18

3394 shown in the Table of VHH Sequences.

3395

3396 Thus, according to this embodiment, the compound of embodiments 378 to 379 (and the

3397 embodiments referring thereto) can comprise any of the combinations VHHa and VHHP shown

3398 in Fig. 4B in red.

3399

3400 Embodiment 399: The compound according to any one of embodiments 378 or 379,

3401 - wherein said VHH antibody domain of (A) comprises the amino acid sequence of one

3402 VHH selected from the group consisting of VHH6 and VHH11 shown in the Table of

3403 VHH Sequences and

3404 - wherein said VHH antibody domain of (E) comprises the amino acid sequence of one

3405 VHH selected from the group consisting of VHH12, VHH13, VHH14, VHH15,

3406 VHH16, VHH17, VHH18, VHH20, VHH21 and VHH22 shown in the Table of VHH

3407 Sequences, wherein said VHH antibody domain of (E) is selected as follows:

3408 if the VHH antibody domain of (A) comprises the amino acid sequence of VHH6,

3409 then the VHH antibody domain of (E) comprises the amino acid sequence of one

3410 VHH selected from the group consisting of VHH12 and VHH13 shown in the

3411 Table of VHH Sequences;

3412 if the VHH antibody domain of (A) comprises the amino acid sequence of VHH11,

3413 then the VHH antibody domain of (E) comprises the amino acid sequence of one

3414 VHH selected from the group consisting of VHH12, VHH13, VHH14, VHH15,

3415 VHH16, VHH17, VHH18, VHH20, VHH21 and VHH22 shown in the Table of

3416 VHH Sequences.

3417 3418 Thus, according to this embodiment, the compound of embodiments 378 to 379 (and the

3419 embodiments referring thereto) can comprise any of the combinations VHHa and VHHP shown

3420 in Fig. 4B in black.

3421

3422 Embodiment 400: The compound according to any one of embodiments 378 or 379,

3423 - wherein said VHH antibody domain of (A) comprises the amino acid sequence of one

3424 VHH selected from the group consisting of VHH2, VHH6, VHH8 and VHH10 shown

3425 in the Table of VHH Sequences and

3426 - wherein said VHH antibody domain of (E) comprises the amino acid sequence of one

3427 VHH selected from the group consisting of VHH 12, VHH14, VHH15, VHH17 and

3428 VHH22 shown in the Table of VHH Sequences, wherein said VHH antibody domain of

3429 (E) is selected as follows:

3430 if the VHH antibody domain of (A) comprises the amino acid sequence of VHH2,

3431 then the VHH antibody domain of (E) comprises the amino acid sequence of

3432 VHH22 shown in the Table of VHH Sequences;

3433 if the VHH antibody domain of (A) comprises the amino acid sequence of VHH6,

3434 then the VHH antibody domain of (E) comprises the amino acid sequence of

3435 VHH15 shown in the Table of VHH Sequences;

3436 if the VHH antibody domain of (A) comprises the amino acid sequence of VHH8,

3437 then the VHH antibody domain of (E) comprises the amino acid sequence of one

3438 VHH selected from the group consisting of VHH14, VHH15 and VHH22 shown

3439 in the Table of VHH Sequences;

3440 if the VHH antibody domain of (A) comprises the amino acid sequence of VHH10,

3441 then the VHH antibody domain of (E) comprises the amino acid sequence of one

3442 VHH selected from the group consisting of VHH12, VHH17 and VHH22 shown

3443 in the Table of VHH Sequences.

3444

3445 Thus, according to this embodiment, the compound of embodiments 378 to 379 (and the

3446 embodiments referring thereto) can comprise any of the combinations VHHa and VHHP shown

3447 in Fig. 4B in orange (active surrogate agonists eliciting less than 50% of NFKB reporter

3448 activation normalized to (rh) IL-18 at 1 nM or EC50 higher than 0.1 nM).

3449

3450 Embodiment 401 : The compound according to any one of embodiments 378 or 379, wherein

3451 said VHH antibody domain of (A) comprises the amino acid sequence of one VHH selected 3452 from the group consisting of VHH2 and VHH8 shown in the Table of VHH Sequences and

3453 wherein said VHH antibody domain of (E) comprises the amino acid sequence of one VHH

3454 selected from the group consisting of VHH15 and VHH17 shown in the Table of VHH

3455 Sequences.

3456

3457 Embodiment 402: The compound according to any one of embodiments 378 or 379,

3458 - wherein said VHH antibody domain of (A) comprises the amino acid sequence of one

3459 VHH selected from the group consisting of VHH2 and VHH8 shown in the Table of

3460 VHH Sequences and

3461 - wherein said VHH antibody domain of (E) comprises the amino acid sequence of one

3462 VHH selected from the group consisting of VHH15 and VHH17 shown in the Table of

3463 VHH Sequences, wherein said VHH antibody domain of (E) is selected as follows:

3464 if the VHH antibody domain of (A) comprises the amino acid sequence of VHH2,

3465 then the VHH antibody domain of (E) comprises the amino acid sequence of one

3466 VHH selected from the group consisting of VHH15 and VHH17 shown in the

3467 Table of VHH Sequences;

3468 if the VHH antibody domain of (A) comprises the amino acid sequence of VHH8,

3469 then the VHH antibody domain of (E) comprises the amino acid sequence of

3470 VHH17 shown in the Table of VHH Sequences.

3471

3472 Embodiment 403: The compound according to any one of embodiments 378 or 379, wherein

3473 said VHH antibody domain of (A) comprises the amino acid sequence of VHH2 shown in the

3474 Table of VHH Sequences and wherein said VHH antibody domain of (E) comprises the amino

3475 acid sequence of VHH15 shown in the Table of VHH Sequences.

3476

3477 Embodiment 404: The compound according to any one of embodiments 378 or 379,

3478 - wherein said VHH antibody domain of (A) comprises the amino acid sequence of one

3479 VHH selected from the group consisting of VHH1, VHH2, VHH5, VHH6, VHH8 and

3480 VHH10 shown in the Table of VHH Sequences and

3481 - wherein said VHH antibody domain of (E) comprises the amino acid sequence of one

3482 VHH selected from the group consisting of VHH13, VHH16 and VHH22 shown in the

3483 Table of VHH Sequences, wherein said VHH antibody domain of (E) is selected as

3484 follows: 3485 if the VHH antibody domain of (A) comprises the amino acid sequence of VHH1,

3486 then the VHH antibody domain of (E) comprises the amino acid sequence of

3487 VHH16 shown in the Table of VHH Sequences;

3488 if the VHH antibody domain of (A) comprises the amino acid sequence of VHH2,

3489 then the VHH antibody domain of (E) comprises the amino acid sequence of

3490 VHH22 shown in the Table of VHH Sequences;

3491 if the VHH antibody domain of (A) comprises the amino acid sequence of VHH5,

3492 then the VHH antibody domain of (E) comprises the amino acid sequence of

3493 VHH13 shown in the Table of VHH Sequences;

3494 if the VHH antibody domain of (A) comprises the amino acid sequence of VHH6,

3495 then the VHH antibody domain of (E) comprises the amino acid sequence of

3496 VHH22 shown in the Table of VHH Sequences;

3497 if the VHH antibody domain of (A) comprises the amino acid sequence of VHH8,

3498 then the VHH antibody domain of (E) comprises the amino acid sequence of

3499 VHH22 shown in the Table of VHH Sequences;

3500 if the VHH antibody domain of (A) comprises the amino acid sequence of VHH10,

3501 then the VHH antibody domain of (E) comprises the amino acid sequence of

3502 VHH22 shown in the Table of VHH Sequences.

3503

3504 Embodiment 405: The compound according to any one of embodiments 378 or 379,

3505 - wherein said VHH antibody domain of (A) comprises the amino acid sequence of one

3506 VHH selected from the group consisting of VHH2, VHH6, VHH8 and VHH10 shown

3507 in the Table of VHH Sequences and

3508 - wherein said VHH antibody domain of (E) comprises the amino acid sequence of one

3509 VHH selected from the group consisting of VHH12, VHH13, VHH15 and VHH18

3510 shown in the Table of VHH Sequences, wherein said VHH antibody domain of (E) is

3511 selected as follows:

3512 if the VHH antibody domain of (A) comprises the amino acid sequence of VHH2,

3513 then the VHH antibody domain of (E) comprises the amino acid sequence of

3514 VHH12 shown in the Table of VHH Sequences;

3515 if the VHH antibody domain of (A) comprises the amino acid sequence of VHH6,

3516 then the VHH antibody domain of (E) comprises the amino acid sequence of one

3517 VHH selected from the group consisting of VHH15 and VHH12 shown in the

3518 Table of VHH Sequences; 3519 if the VHH antibody domain of (A) comprises the amino acid sequence of VHH8,

3520 then the VHH antibody domain of (E) comprises the amino acid sequence of one

3521 VHH selected from the group consisting of VHH13 and VHH18 shown in the

3522 Table of VHH Sequences;

3523 if the VHH antibody domain of (A) comprises the amino acid sequence of VHH10,

3524 then the VHH antibody domain of (E) comprises the amino acid sequence of

3525 VHH12 shown in the Table of VHH Sequences.

3526

3527 Embodiment 406: The compound according to any one of embodiments 378 to 379 or 397 to

3528 405, wherein in the first binding module said VHH antibody domain or fragment thereof

3529 comprises a VHH antibody domain or fragment thereof according to (A), (B) or (C).

3530

3531 If the present disclosure states that " in the first binding module said VHH antibody domain or

3532 fragment thereof comprises a VHH antibody domain or fragment thereof according to (A), (B)

3533 or (C)", then this is meant to designate that in this embodiment only (A), (B) and (C) are

3534 considered as options for the VHH antibody domain (or fragment thereof) of the first binding

3535 module, whereas the option (D) is excluded. If the present disclosure states that " in the first

3536 binding module said VHH antibody domain or fragment thereof comprises a VHH antibody

3537 domain or fragment thereof according to (A), (C) or (D)", then this is meant to designate that

3538 in this embodiment only (A), (C) and (D) are considered as options for the VHH antibody

3539 domain (or fragment thereof) of the first binding module, whereas the option (B) is excluded.

3540 If the present disclosure states that " in the first binding module said VHH antibody domain or

3541 fragment thereof comprises a VHH antibody domain or fragment thereof according to (A)",

3542 then this is meant to designate that in this embodiment only (A) is considered as option for the

3543 VHH antibody domain (or fragment thereof) of the first binding module, whereas the options

3544 (B), (C) and (D) are excluded. Further variations of this wording below are to be understood

3545 accordingly.

3546

3547 Embodiment 407: The compound according to any one of embodiments 378 to 379 or 397 to

3548 405, wherein in the first binding module said VHH antibody domain or fragment thereof

3549 comprises a VHH antibody domain or fragment thereof according to (A), (B) or (D).

3550 3551 Embodiment 408: The compound according to any one of embodiments 378 to 379 or 397 to

3552 405, wherein in the first binding module said VHH antibody domain or fragment thereof

3553 comprises a VHH antibody domain or fragment thereof according to (A), (C) or (D).

3554

3555 Embodiment 409: The compound according to any one of embodiments 378 to 379 or 397 to

3556 405, wherein in the first binding module said VHH antibody domain or fragment thereof

3557 comprises a VHH antibody domain or fragment thereof according to (A) or (B).

3558

3559 Embodiment 410: The compound according to any one of embodiments 378 to 379 or 397 to

3560 405, wherein in the first binding module said VHH antibody domain or fragment thereof

3561 comprises a VHH antibody domain or fragment thereof according to (A) or (C).

3562

3563 Embodiment 411 : The compound according to any one of embodiments 378 to 379 or 397 to

3564 405, wherein in the first binding module said VHH antibody domain or fragment thereof

3565 comprises a VHH antibody domain or fragment thereof according to (A) or (D).

3566

3567 Embodiment 412: The compound according to any one of embodiments 378 to 379 or 397 to

3568 405, wherein in the first binding module said VHH antibody domain or fragment thereof

3569 comprises a VHH antibody domain or fragment thereof according to (A).

3570

3571 Embodiment 413: The compound according to any one of embodiments 378 to 379 or 397 to

3572 405, wherein in the first binding module said VHH antibody domain or fragment thereof

3573 comprises a VHH antibody domain or fragment thereof according to (B).

3574

3575 Embodiment 414: The compound according to any one of embodiments 378 to 379 or 397 to

3576 413, wherein in the second binding module said VHH antibody domain or fragment thereof

3577 comprises a VHH antibody domain or fragment thereof according to (E), (F) or (G).

3578

3579 Embodiment 415: The compound according to any one of embodiments 378 to 379 or 397 to

3580 413, wherein in the second binding module said VHH antibody domain or fragment thereof

3581 comprises a VHH antibody domain or fragment thereof according to (E), (F) or (H).

3582 3583 Embodiment 416: The compound according to any one of embodiments 378 to 379 or 397 to

3584 413, wherein in the second binding module said VHH antibody domain or fragment thereof

3585 comprises a VHH antibody domain or fragment thereof according to (E), (G) or (H).

3586

3587 Embodiment 417: The compound according to any one of embodiments 378 to 379 or 397 to

3588 413, wherein in the second binding module said VHH antibody domain or fragment thereof

3589 comprises a VHH antibody domain or fragment thereof according to (E) or (F).

3590

3591 Embodiment 418: The compound according to any one of embodiments 378 to 379 or 397 to

3592 413, wherein in the second binding module said VHH antibody domain or fragment thereof

3593 comprises a VHH antibody domain or fragment thereof according to (E) or (G).

3594

3595 Embodiment 419: The compound according to any one of embodiments 378 to 379 or 397 to

3596 413, wherein in the second binding module said VHH antibody domain or fragment thereof

3597 comprises a VHH antibody domain or fragment thereof according to (E) or (H).

3598

3599 Embodiment 420: The compound according to any one of embodiments 378 to 379 or 397 to

3600 413, wherein in the second binding module said VHH antibody domain or fragment thereof

3601 comprises a VHH antibody domain or fragment thereof according to (E).

3602

3603 Embodiment 421 : The compound according to any one of embodiments 378 to 379 or 397 to

3604 413, wherein in the second binding module said VHH antibody domain or fragment thereof

3605 comprises a VHH antibody domain or fragment thereof according to (F).

3606

3607 Embodiment 422: The compound according to any one of embodiments 375 to 421, wherein

3608 said compound is a molecule.

3609

3610 Embodiment 423: The compound according to any one of embodiments 375 to 422, wherein

3611 said compound comprises or is a protein.

3612

3613 By stating that the compound "comprises" a protein, the present disclosure designates that the

3614 compound includes a part within its chemical structure that is a protein. A compound that

3615 comprises a protein may or may not comprise a part that is not a protein.

3616 3617 Embodiment 424: The compound according to any one of embodiments 375 to 422, wherein

3618 said compound is a protein.

3619

3620 By stating that the compound "is" a protein, the present disclosure designates that the compound

3621 consists only of protein and does not comprise a part that is not a protein.

3622

3623 Embodiment 425: The compound according to any one of embodiments 375 to 424, wherein

3624 said compound further comprises a targeting moiety.

3625

3626 As used herein, the term "targeting moiety" refers to a moiety (i.e. a molecular group or

3627 chemical structure) that is (typically covalently) associated with said compound and that binds

3628 a target site, wherein said binding allows to recruit the compound to said target site (e.g. a

3629 moiety that specifically binds to EGFR and thus targets the compound to cells expressing EGFR

3630 at their cell surface). The target site will typically be a biological molecule or a certain part of

3631 a biological molecule. An example of a targeting moiety is an antigen-binding antibody

3632 fragment that is covalently linked to an IL-18Ra-binding and/or IL-18RP-binding VHH

3633 antibody domain to form a compound according to the present disclosure, wherein the antigen¬

3634 binding fragment binds to a certain receptor present at the surface of a certain cell type (its

3635 antigen), and wherein binding of the antigen-binding fragment to this receptor results in

3636 recruitment of the compound to this cell.

3637

3638 Non-targeted drugs typically reach their site of action by whole-body distribution and passive

3639 diffusion. In contrast, targeted compounds are not distributed evenly across the whole body.

3640 Due to the interaction of targeting moiety with its target molecule, a compound including a

3641 targeting moiety is concentrated preferentially at its site target site. Therefore, e.g. therapeutic

3642 compounds with a targeting moiety require lower dosages to be therapeutically effective, thus

3643 improving the therapeutic window.

3644

3645 Embodiment 426: The compound according to embodiment 425, wherein said targeting moiety

3646 is a protein, a peptide, a peptide mimetic, a nucleic acid, an oligonucleotide or a small molecule.

3647

3648 As used herein, the term "peptide mimetic" refers to a peptide-like chain which is designed to

3649 mimic a peptide. An example of a peptide mimetic is a D-peptide mimetic containing a D- 3650 amino acid, but is not limited thereto. As used herein, a "small molecule", is a molecule with a

3651 molecular weight < 1000 Da.

3652

3653 Embodiment 427: The compound according to any one of embodiments 375 to 426, wherein

3654 said compound is a monospecific molecule.

3655

3656 Embodiment 428: The compound according to any one of embodiments 375 to 426, wherein

3657 said compound is a bispecific molecule.

3658

3659 Embodiment 429: The compound according to any one of embodiments 375 to 426 or 428,

3660 wherein said compound is a bispecific antibody molecule.

3661

3662 The term "bispecific antibody molecule", as used in the present disclosure, refers to an antibody

3663 molecule that is capable of specifically binding to two different epitopes at the same time. This

3664 does not exclude the possibility that said bispecific antibody molecule is linked to further

3665 domains or moieties.

3666

3667 The terms "epitope" or "antigenic determinant" are used interchangeably herein and refer to the

3668 portion of an antigen that is recognized and specifically bound by a particular antibody. When

3669 the antigen is a polypeptide, epitopes can be formed both from contiguous amino acids and

3670 noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from

3671 contiguous amino acids are typically retained upon protein denaturing, whereas epitopes

3672 formed by tertiary folding are typically lost upon protein denaturing. An epitope typically

3673 includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial

3674 conformation.

3675

3676 Typically, in the compounds of the present disclosure, one of the two epitopes to which the

3677 compound of the present disclosure binds is an epitope on IL-18Ra whereas the other of the

3678 two epitopes to which the compound of the present disclosure binds is an epitope on IL-18Rp.

3679

3680 Methods for making bispecific antibodies are known in the art. For example, bispecific

3681 antibodies can be produced recombinantly using the co-expression of two immunoglobulin

3682 heavy chain/light chain pairs (see e.g. Milstein et al., Nature (1983), vol. 305, p. 537-539).

3683 Alternatively, bispecific antibodies can be prepared using chemical linkage (see e.g. Brennan 3684 et al., Science (1985), vol. 229, p. 81). A bispecific antibody can also for example be prepared

3685 by the SEED technology (see below).

3686

3687 Embodiment 430: The compound according to any one of embodiments 375 to 426 or 428 to

3688 429, wherein one binding site of said bispecific antibody molecule is formed by a VHH

3689 antibody domain or fragment thereof according to the present disclosure that is specific for IL-

3690 18Ra, and one binding site of said bispecific antibody is formed by a VHH antibody domain or

3691 fragment thereof according to the present disclosure that is specific for IL-18Rp.

3692

3693 Embodiment 431: The compound according to any one of embodiments 375 to 426 or 428 to

3694 429, wherein one binding site of said bispecific antibody molecule is a VHH antibody domain

3695 or fragment thereof according to the present disclosure and the other binding site of said

3696 bispecific antibody molecule is selected from the group consisting of a Fab, a Fab', a (Fab')2, a

3697 Fv, a scFv, a diabody and a VHH.

3698

3699 "Fab" fragments are obtained by papain digestion of an antibody, which produces two identical

3700 antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment, a designation

3701 reflecting the ability to crystallize readily. The Fab fragment consists of an entire L chain along

3702 with the variable region domain of the H chain (VH), and the first constant domain of one heavy

3703 chain (CHI). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a

3704 single antigen-binding site.

3705

3706 "F(ab')2" fragments are obtained by pepsin treatment of an antibody, which yields a single large

3707 F(ab')2 fragment which roughly corresponds to two disulfide linked Fab fragments having

3708 different antigen-binding activity and is still capable of cross-linking antigen.

3709

3710 "Fab¹ " fragments differ from Fab fragments by having a few additional residues at the carboxy

3711 terminus of the CHI domain including one or more cysteines from the antibody hinge region.

3712 Fab'-SH is the designation for Fab' in which the cysteine residue(s) of the constant domains

3713 bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab'

3714 fragments which have hinge cysteines between them. Other chemical couplings of antibody

3715 fragments are also known.

3716 3717 The Fc fragment comprises the carboxy-terminal portions of both H chains held together by

3718 disulfides. The effector functions of antibodies are determined by sequences in the Fc region,

3719 the region which is also recognized by Fc receptors (FcR) found on certain types of cells.

3720

3721 "Fv" is the minimum antibody fragment which contains a complete antigen-recognition and -

3722 binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region

3723 domain in tight, non-covalent association. From the folding of these two domains emanate six

3724 hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid

3725 residues for antigen binding and confer antigen binding specificity to the antibody. However,

3726 even a single variable domain (or half of an Fv comprising only three HVRs specific for an

3727 antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire

3728 binding site.

3729

3730 "Single-chain Fv", also abbreviated as "scFv", are antibody fragments that comprise the VH

3731 and VL antibody domains connected into a single polypeptide chain. Preferably, the scFv

3732 polypeptide further comprises a polypeptide linker between the VH and VL domains which

3733 enables the scFv to form the desired structure for antigen binding. For a review of the scFv, see

3734 Pluckthun, in: The Pharmacology of Monoclonal Antibodies, vol. 113 (1994), editors

3735 Rosenburg and Moore, Springer-Verlag (New York), p. 269-315.

3736

3737 The term "diabody" refers to a small antibody fragment prepared by constructing scFv

3738 fragments (see preceding paragraph) with short linkers (about 5-25 residues) between the VH

3739 and VL domains such that inter-chain but not intra-chain pairing of the V domains is achieved,

3740 thereby resulting in a bivalent fragment, i.e., a fragment having two antigen-binding sites.

3741 Bispecific diabodies are heterodimers of two "crossover" scFv fragments in which the VH and

3742 VL domains of the two antibodies are present on different polypeptide chains. Diabodies are

3743 described in greater detail in, for example, EP 0404097; WO 93/11161; Hollinger et al., Proc.

3744 Natl. Acad. Sci. USA (1993), vol. 90, p. 6444-6448.

3745

3746 Embodiment 432: The compound according to any one of embodiments 375 to 426 or 430 to

3747 431, wherein said compound is a multispecific molecule.

3748

3749 Embodiment 433: The compound according to any one of embodiments 375 to 426 or 428 to

3750 430, wherein said compound is a bispecific molecule that is specific for IL-18Ra and IL-18Rp. 3751

3752 Embodiment 434: The compound according to any one of embodiments 375 to 426 or 428 to

3753 430 or 433, wherein said compound is a bispecific molecule that binds to IL-18Ra and IL-

3754 18R .

3755

3756 Embodiment 435: The compound according to any one of embodiments 375 to 426 or 428 to

3757 430 or 433 to 434, wherein said compound is a bispecific molecule directed to IL-18Ra and IL-

3758 18R .

3759

3760 Embodiment 436: The compound according to any one of embodiments 375 to 426 or 428 to

3761 430 or 433 to 435, wherein said compound is a bispecific antibody molecule that is specific for

3762 IL-18Ra and IL-18Rp.

3763

3764 If the present disclosure refers to "IL-18Ra" without indicating a specific organism from which

3765 said IL-18Ra originates, then this refers to human IL-18Ra. If the present disclosure refers to

3766 "IL-18RP" without indicating a specific organism from which said IL-18RP originates, then

3767 this refers to human IL-18RP.

3768

3769 According to a twelfth aspect, the present disclosure relates to any of the following (To the

3770 twelfth aspect of the present disclosure and the embodiments referring thereto, the same

3771 explanations and definitions apply accordingly as for the first to eleventh aspects of the present

3772 disclosure and the embodiments referring thereto.):

3773

3774 Embodiment 437: A compound which is a bispecific antibody molecule comprising an IL-

3775 18Ra-binding VHH with the amino acid sequence of SEQ ID NO: 1 (First binding module) and

3776 an IL18RP-binding VHH with the amino acid sequence of SEQ ID NO: 12 (Second binding

3777 module).

3778

3779 Embodiment 438: A compound which is a bispecific antibody molecule comprising an IL-

3780 18Ra-binding VHH with the amino acid sequence of SEQ ID NO: 1 (First binding module) and

3781 an IL18RP-binding VHH with the amino acid sequence of SEQ ID NO: 13 (Second binding

3782 module).

3783 3784 Embodiment 439: A compound which is a bispecific antibody molecule comprising an IL-

3785 18Ra-binding VHH with the amino acid sequence of SEQ ID NO: 1 (First binding module) and

3786 an IL18RP-binding VHH with the amino acid sequence of SEQ ID NO: 15 (Second binding

3787 module).

3788

3789 Embodiment 440: A compound which is a bispecific antibody molecule comprising an IL-

3790 18Ra-binding VHH with the amino acid sequence of SEQ ID NO: 1 (First binding module) and

3791 an IL18RP-binding VHH with the amino acid sequence of SEQ ID NO: 17 (Second binding

3792 module).

3793

3794 Embodiment 441: A compound which is a bispecific antibody molecule comprising an IL-

3795 18Ra-binding VHH with the amino acid sequence of SEQ ID NO: 1 (First binding module) and

3796 an IL18RP-binding VHH with the amino acid sequence of SEQ ID NO: 20 (Second binding

3797 module).

3798

3799 Embodiment 442: A compound which is a bispecific antibody molecule comprising an IL-

3800 18Ra-binding VHH with the amino acid sequence of SEQ ID NO: 1 (First binding module) and

3801 an IL18RP-binding VHH with the amino acid sequence of SEQ ID NO: 21 (Second binding

3802 module).

3803

3804 Embodiment 443: A compound which is a bispecific antibody molecule comprising an IL-

3805 18Ra-binding VHH with the amino acid sequence of SEQ ID NO: 1 (First binding module) and

3806 an IL18RP-binding VHH with the amino acid sequence of SEQ ID NO: 22 (Second binding

3807 module).

3808

3809 Embodiment 444: A compound which is a bispecific antibody molecule comprising an IL-

3810 18Ra-binding VHH with the amino acid sequence of SEQ ID NO: 2 (First binding module) and

3811 an IL18RP-binding VHH with the amino acid sequence of SEQ ID NO: 12 (Second binding

3812 module).

3813

3814 Embodiment 445: A compound which is a bispecific antibody molecule comprising an IL-

3815 18Ra-binding VHH with the amino acid sequence of SEQ ID NO: 2 (First binding module) and

3816 an IL18RP-binding VHH with the amino acid sequence of SEQ ID NO: 13 (Second binding

3817 module). 3818

3819 Embodiment 446: A compound which is a bispecific antibody molecule comprising an IL-

3820 18Ra-binding VHH with the amino acid sequence of SEQ ID NO: 2 (First binding module) and

3821 an IL18RP-binding VHH with the amino acid sequence of SEQ ID NO: 15 (Second binding

3822 module).

3823

3824 Embodiment 447: A compound which is a bispecific antibody molecule comprising an IL-

3825 18Ra-binding VHH with the amino acid sequence of SEQ ID NO: 2 (First binding module) and

3826 an IL18RP-binding VHH with the amino acid sequence of SEQ ID NO: 17 (Second binding

3827 module).

3828

3829 Embodiment 448: A compound which is a bispecific antibody molecule comprising an IL-

3830 18Ra-binding VHH with the amino acid sequence of SEQ ID NO: 2 (First binding module) and

3831 an IL18RP-binding VHH with the amino acid sequence of SEQ ID NO: 18 (Second binding

3832 module).

3833

3834 Embodiment 449: A compound which is a bispecific antibody molecule comprising an IL-

3835 18Ra-binding VHH with the amino acid sequence of SEQ ID NO: 2 (First binding module) and

3836 an IL18RP-binding VHH with the amino acid sequence of SEQ ID NO: 20 (Second binding

3837 module).

3838

3839 Embodiment 450: A compound which is a bispecific antibody molecule comprising an IL-

3840 18Ra-binding VHH with the amino acid sequence of SEQ ID NO: 2 (First binding module) and

3841 an IL18RP-binding VHH with the amino acid sequence of SEQ ID NO: 21 (Second binding

3842 module).

3843

3844 Embodiment 451: A compound which is a bispecific antibody molecule comprising an IL-

3845 18Ra-binding VHH with the amino acid sequence of SEQ ID NO: 5 (First binding module) and

3846 an IL18RP-binding VHH with the amino acid sequence of SEQ ID NO: 13 (Second binding

3847 module).

3848

3849 Embodiment 452: A compound which is a bispecific antibody molecule comprising an IL-

3850 18Ra-binding VHH with the amino acid sequence of SEQ ID NO: 5 (First binding module) and 3851 an IL18RP-binding VHH with the amino acid sequence of SEQ ID NO: 14 (Second binding

3852 module).

3853

3854 Embodiment 453: A compound which is a bispecific antibody molecule comprising an IL-

3855 18Ra-binding VHH with the amino acid sequence of SEQ ID NO: 6 (First binding module) and

3856 an IL18RP-binding VHH with the amino acid sequence of SEQ ID NO: 17 (Second binding

3857 module).

3858

3859 Embodiment 454: A compound which is a bispecific antibody molecule comprising an IL-

3860 18Ra-binding VHH with the amino acid sequence of SEQ ID NO: 6 (First binding module) and

3861 an IL18RP-binding VHH with the amino acid sequence of SEQ ID NO: 20 (Second binding

3862 module).

3863

3864 Embodiment 455: A compound which is a bispecific antibody molecule comprising an IL-

3865 18Ra-binding VHH with the amino acid sequence of SEQ ID NO: 6 (First binding module) and

3866 an IL18RP-binding VHH with the amino acid sequence of SEQ ID NO: 21 (Second binding

3867 module).

3868

3869 Embodiment 456: A compound which is a bispecific antibody molecule comprising an IL-

3870 18Ra-binding VHH with the amino acid sequence of SEQ ID NO: 6 (First binding module) and

3871 an IL18RP-binding VHH with the amino acid sequence of SEQ ID NO: 22 (Second binding

3872 module).

3873

3874 Embodiment 457: A compound which is a bispecific antibody molecule comprising an IL-

3875 18Ra-binding VHH with the amino acid sequence of SEQ ID NO: 8 (First binding module) and

3876 an IL18RP-binding VHH with the amino acid sequence of SEQ ID NO: 12 (Second binding

3877 module).

3878

3879 Embodiment 458: A compound which is a bispecific antibody molecule comprising an IL-

3880 18Ra-binding VHH with the amino acid sequence of SEQ ID NO: 8 (First binding module) and

3881 an IL18RP-binding VHH with the amino acid sequence of SEQ ID NO: 13 (Second binding

3882 module).

3883 3884 Embodiment 459: A compound which is a bispecific antibody molecule comprising an IL-

3885 18Ra-binding VHH with the amino acid sequence of SEQ ID NO: 8 (First binding module) and

3886 an IL18RP-binding VHH with the amino acid sequence of SEQ ID NO: 17 (Second binding

3887 module).

3888

3889 Embodiment 460: A compound which is a bispecific antibody molecule comprising an IL-

3890 18Ra-binding VHH with the amino acid sequence of SEQ ID NO: 8 (First binding module) and

3891 an IL18RP-binding VHH with the amino acid sequence of SEQ ID NO: 18 (Second binding

3892 module).

3893

3894 Embodiment 461: A compound which is a bispecific antibody molecule comprising an IL-

3895 18Ra-binding VHH with the amino acid sequence of SEQ ID NO: 8 (First binding module) and

3896 an IL18RP-binding VHH with the amino acid sequence of SEQ ID NO: 20 (Second binding

3897 module).

3898

3899 Embodiment 462: A compound which is a bispecific antibody molecule comprising an IL-

3900 18Ra-binding VHH with the amino acid sequence of SEQ ID NO: 8 (First binding module) and

3901 an IL18RP-binding VHH with the amino acid sequence of SEQ ID NO: 21 (Second binding

3902 module).

3903

3904 Embodiment 463: A compound which is a bispecific antibody molecule comprising an IL-

3905 18Ra-binding VHH with the amino acid sequence of SEQ ID NO: 9 (First binding module) and

3906 an IL18RP-binding VHH with the amino acid sequence of SEQ ID NO: 12 (Second binding

3907 module).

3908

3909 Embodiment 464: A compound which is a bispecific antibody molecule comprising an IL-

3910 18Ra-binding VHH with the amino acid sequence of SEQ ID NO: 9 (First binding module) and

3911 an IL18RP-binding VHH with the amino acid sequence of SEQ ID NO: 13 (Second binding

3912 module).

3913

3914 Embodiment 465: A compound which is a bispecific antibody molecule comprising an IL-

3915 18Ra-binding VHH with the amino acid sequence of SEQ ID NO: 9 (First binding module) and

3916 an IL18RP-binding VHH with the amino acid sequence of SEQ ID NO: 15 (Second binding

3917 module). 3918

3919 Embodiment 466: A compound which is a bispecific antibody molecule comprising an IL-

3920 18Ra-binding VHH with the amino acid sequence of SEQ ID NO: 9 (First binding module) and

3921 an IL18RP-binding VHH with the amino acid sequence of SEQ ID NO: 17 (Second binding

3922 module).

3923

3924 Embodiment 467: A compound which is a bispecific antibody molecule comprising an IL-

3925 18Ra-binding VHH with the amino acid sequence of SEQ ID NO: 9 (First binding module) and

3926 an IL18RP-binding VHH with the amino acid sequence of SEQ ID NO: 20 (Second binding

3927 module).

3928

3929 Embodiment 468: A compound which is a bispecific antibody molecule comprising an IL-

3930 18Ra-binding VHH with the amino acid sequence of SEQ ID NO: 9 (First binding module) and

3931 an IL18RP-binding VHH with the amino acid sequence of SEQ ID NO: 21 (Second binding

3932 module).

3933

3934 Embodiment 469: A compound which is a bispecific antibody molecule comprising an IL-

3935 18Ra-binding VHH with the amino acid sequence of SEQ ID NO: 9 (First binding module) and

3936 an IL18RP-binding VHH with the amino acid sequence of SEQ ID NO: 22 (Second binding

3937 module).

3938

3939 Embodiment 470: A compound which is a bispecific antibody molecule comprising an IL-

3940 18Ra-binding VHH with the amino acid sequence of SEQ ID NO: 10 (First binding module)

3941 and an IL18RP-binding VHH with the amino acid sequence of SEQ ID NO: 13 (Second binding

3942 module).

3943

3944 Embodiment 471 : A compound which is a bispecific antibody molecule comprising an IL-

3945 18Ra-binding VHH with the amino acid sequence of SEQ ID NO: 10 (First binding module)

3946 and an IL18RP-binding VHH with the amino acid sequence of SEQ ID NO: 20 (Second binding

3947 module).

3948

3949 Embodiment 472: A compound which is a bispecific antibody molecule comprising an IL-

3950 18Ra-binding VHH with the amino acid sequence of SEQ ID NO: 10 (First binding module) 3951 and an IL18RP-binding VHH with the amino acid sequence of SEQ ID NO: 21 (Second binding

3952 module).

3953

3954 Embodiment 473: The compound according to any one of embodiments 375 or 377 to 472,

3955 wherein said compound is monovalent for binding to IL-18Ra.

3956

3957 Embodiment 474: The compound according to any one of embodiments 375 or 377 to 472,

3958 wherein said compound is bivalent for binding to IL-18Ra.

3959

3960 Embodiment 475: The compound according to any one of embodiments 375 or 377 to 472,

3961 wherein said compound is multivalent for binding to IL-18Ra.

3962

3963 Embodiment 476: The compound according to any one of embodiments 376 to 475, wherein

3964 said compound is monovalent for binding to IL-18RP.

3965

3966 Embodiment 477: The compound according to any one of embodiments 376 to 475, wherein

3967 said compound is bivalent for binding to IL-18RP.

3968

3969 Embodiment 478: The compound according to any one of embodiments 376 to 475, wherein

3970 said compound is multivalent for binding to IL-18RP.

3971

3972 Embodiment 479: The compound according to any one of embodiments 377 to 478, wherein

3973 said compound comprises one copy of said first binding module.

3974

3975 Embodiment 480: The compound according to any one of embodiments 377 to 478, wherein

3976 said compound comprises two copies of said first binding module.

3977

3978 Embodiment 481: The compound according to any one of embodiments 377 to 478, wherein

3979 said compound comprises two or more copies of said first binding module.

3980

3981 Embodiment 482: The compound according to any one of embodiments 377 to 479, wherein

3982 said compound comprises not more than one copy of said first binding module.

3983 3984 Embodiment 483: The compound according to any one of embodiments 377 to 478 or 480,

3985 wherein said compound comprises not more than two copies of said first binding module.

3986

3987 Embodiment 484: The compound according to any one of embodiments 377 to 483, wherein

3988 said compound comprises one copy of said second binding module.

3989

3990 Embodiment 485: The compound according to any one of embodiments 377 to 483, wherein

3991 said compound comprises two copies of said second binding module.

3992

3993 Embodiment 486: The compound according to any one of embodiments 377 to 483, wherein

3994 said compound comprises two or more copies of said second binding module.

3995

3996 Embodiment 487: The compound according to any one of embodiments 377 to 484, wherein

3997 said compound comprises not more than one copy said second binding module.

3998

3999 Embodiment 488: The compound according to any one of embodiments 377 to 483 or 485,

4000 wherein said compound comprises not more than two copies of said second binding module.

4001

4002 Embodiment 489: The compound according to any one of embodiments 375 to 488, wherein

4003 said compound comprises only one molecular structure that binds to IL-18Ra.

4004

4005 Embodiment 490: The compound according to any one of embodiments 375 to 489, wherein

4006 said compound comprises only one molecular structure that binds to IL-18RP.

4007

4008 Embodiment 491: The compound according to any one of embodiments 375 to 490, wherein

4009 all components of said compound are covalently linked.

4010

4011 Embodiment 492: The compound according to any one of embodiments 377 to 491, wherein

4012 said first binding module is a VHH antibody domain. As the skilled person understands, by

4013 stating that the first binding module "is a VHH antibody domain", the present disclosure

4014 indicates that the first binding module is a full VHH antibody domain, not just a fragment

4015 thereof.

4016 4017 Embodiment 493: The compound according to any one of embodiments 377 to 492, wherein

4018 said second binding module is a VHH antibody domain.

4019

4020 Embodiment 494: The compound according to any one of embodiments 377 to 491, wherein

4021 said first binding module consists of a fragment of a VHH antibody domain. As the skilled

4022 person understands, by stating that the first binding module "consists of a fragment of a VHH

4023 antibody domain", the present disclosure indicates that the first binding module is a fragment

4024 of a VHH antibody domain, not a full-length VHH antibody domain.

4025

4026 Embodiment 495: The compound according to any one of embodiments 377 to 491 or 494,

4027 wherein said second binding module consists of a fragment of a VHH antibody domain.

4028

4029 Embodiment 496: The compound according to any one of embodiments 375 to 495, wherein

4030 said compound comprises an antibody Fc region.

4031

4032 As used herein, the term "antibody Fc region" refers to the portion of a native immunoglobulin

4033 formed by the Fc domains of its two heavy chains (which includes a heavy chain constant region

4034 1 (CHI), a heavy chain constant region 2 (CH2) and a heavy chain constant region 3 (CH3) of

4035 an immunoglobulin, but does not include variable regions of the heavy and light chains and a

4036 light chain constant region 1 (CL1) of an immunoglobulin). A native Fc region is homodimeric.

4037 In some embodiments, the term includes variant Fc regions with one or more alterations relative

4038 to a native Fc region. An Fc region may be altered by amino acid substitutions, additions and/or

4039 deletions, linkage of additional moieties, and/or alteration of the native glycans. The term

4040 encompasses Fc regions wherein each of the constituent Fc domains is different. Examples of

4041 heterodimeric Fc regions include, without limitation, Fc regions made using the "knobs into

4042 holes" technology as described in, for example US Patent No. 8,216,805 or by the SEED

4043 technology as described in WO 2016/087650.

4044

4045 Embodiment 497: The compound according to any one of embodiments 375 to 495, wherein

4046 said compound comprises an antibody Fc region that is competent in Fc receptor binding.

4047

4048 An antibody Fc region is "competent in FC receptor binding" if said antibody Fc region is

4049 capable of binding to at least one of the Fc receptors (FcyRI, FcyRII, and FcyRIII subclasses,

4050 including allelic variants and alternatively spliced forms of these receptors). 4051

4052 Embodiment 498: The compound according to any one of embodiments 375 to 495, wherein

4053 said compound comprises an antibody Fc region that is not competent in Fc receptor binding.

4054

4055 Embodiment 499: The compound according to any one of embodiments 375 to 495, wherein

4056 said compound does not comprise an effector-competent antibody Fc region.

4057

4058 An "effector-competent" Fc region is an Fc region having the functional ability to bind proteins

4059 and/or cells of the immune system and mediate biological effects normally induced following

4060 the binding of an antibody to a corresponding antigen. Such biological effects include e.g. the

4061 ability to bind a complement protein (e.g. Clq), resulting in activation of the classical

4062 complement system leading to the opsonisation and lysis of cell pathogens (complement¬

4063 dependent cytotoxicity, CDCC). Other biological effects are endocytosis of immune

4064 complexes, engulfment and destruction of antibody-coated particles or microorganisms (also

4065 called antibody-dependent phagocytosis, or ADCP), clearance of immune complexes, lysis of

4066 antibody-coated target cells by killer cells (called antibody-dependent cell-mediated

4067 cytotoxicity, or ADCC), release of inflammatory mediators, regulation of immune system cell

4068 activation or control of immunoglobulin production.

4069

4070 Embodiment 500: The compound according to any one of embodiments 375 to 497, wherein

4071 said compound comprises an effector-competent antibody Fc region.

4072

4073 Embodiment 501 : The compound according to any one of embodiments 375 to 495 or 498 to

4074 499, wherein said compound does not comprise an antibody Fc region capable of inducing

4075 ADCC (antibody-dependent cellular cytotoxicity).

4076

4077 Embodiment 502: The compound according to any one of embodiments 375 to 497 or 500,

4078 wherein said compound comprises an antibody Fc region capable of inducing ADCC.

4079

4080 Embodiment 503: The compound according to any one of embodiments 375 to 499, wherein

4081 said compound comprises an antibody Fc region that is not capable of inducing ADCC.

4082

4083 Embodiment 504: The compound according to any one of embodiments 375 to 503, wherein

4084 that antibody Fc region carries a mutation E430G. 4085

4086 This mutation is described in de Jong et al., 2016 and has been reported to drive antibody

4087 hexamerization upon target cell binding.

4088

4089 Embodiment 505: The compound according to any one of embodiments 377 to 504, wherein

4090 said compound is a bispecific antibody molecule prepared by the SEED technology (i.e. a

4091 SEEDbody).

4092

4093 The SEED (strand-exchange engineered domain) technology is an approach for generation of

4094 bispecific antibodies in which structurally related sequences within the conserved CH3 domains

4095 of human IgA and IgG are exchanged to form two asymmetric but complementary domains

4096 (see the Examples section). For details about the SEED technology are described in WO

4097 2016/087650 and Davis et al., 2010. A bispecific antibody prepared by the SEED technology

4098 is herein also referred to as "SEEDbody".

4099

4100 Embodiment 506: The compound according to any one of embodiments 377 to 505, wherein

4101 the VHH antibody domain or fragment thereof of the first binding module is formed by a

4102 sequence selected from the group consisting of SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO:

4103 91, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ

4104 ID NO: 98 and SEQ ID NO: 99,

4105 and wherein the VHH antibody domain or fragment thereof of the second binding module is

4106 formed by a sequence selected from the group consisting of SEQ ID NO: 100, SEQ ID NO:

4107 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO:

4108 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109 and SEQ ID NO: 110.

4109

4110 SEQ ID NO: 89 to 99 define the amino acid sequences of SEED AG chains based on VHH1 to

4111 VHH11 (al to pi l). SEQ ID NO: 100 to 110 define the amino acid sequences of SEED GA

4112 chains based on VHH12 to VHH22 (pi2 to P22).

4113

4114 Embodiment 507: The compound according to any one of embodiments 377 to 506, wherein

4115 said compound is a bispecific antibody with a strictly monovalent (1+1) bispecific sdAb (single¬

4116 domain antibody) architecture.

4117 4118 Embodiment 508: The compound according to any one of embodiments 377 to 507, wherein

4119 said compound comprises two VHHs of different binding specificity, and each of these VHHs

4120 is present in said compound in only one copy. As the skilled person understands, if the

4121 compound is a bispecific antibody comprising a first and a second binding module, then

4122 typically one VHH forms the first binding module of said compound whereas the other VHH

4123 forms the second binding module of said compound.

4124

4125 Embodiment 509: The compound according to any one of embodiments 377 to 504 or 506 to

4126 508, wherein said compound is a single domain-based IgG (sdlgG).

4127

4128 Embodiment 510: The compound according to embodiment 509, wherein a sdlgG is a

4129 conventional IgG antibody in which the two VH regions are replaced by VHH domains of one

4130 binding specificity, and the two VL regions are replaced by VHH domains of a different binding

4131 specificity, resulting in a bispecific, tetravalent antibody.

4132

4133 The single domain-based IgG (sdlgG) format is described in detail in Pekar et al., 2020.

4134

4135 Embodiment 511: The compound according to any one of embodiments 509 to 510, wherein

4136 said sdlgG is a conventional IgG antibody in which each of the two VH regions is replaced by

4137 a copy of said first binding module, and each of the two VL regions is replaced by a copy of

4138 said second binding module, resulting in a bispecific, tetravalent antibody.

4139

4140 Embodiment 512: The compound according to embodiment 511, wherein the first binding

4141 module is a VHH that has been grafted onto the CHI domain of the heavy chain of an IgGl and

4142 the second binding module is a VHH that has been grafted onto the constant region of the

4143 lambda light chain of an IgGl .

4144

4145 Embodiment 513: The compound according to any one of embodiments 510 to 512, wherein

4146 said IgG/IgGl is a human IgGl.

4147

4148 Embodiment 514: The compound according to any one of embodiments 377 to 504 or 507 to

4149 to 513, wherein the IL-18Ra-specific VHH2 (SEQ ID NO: 2) has been grafted onto the CHI

4150 domain of the heavy chain of an IgGl and the IL-18RP-specific VHH15 (SEQ ID NO: 15) has

4151 been grafted onto the constant region of the lambda light chain of an IgGl. 4152

4153 Embodiment 515: The compound according to any one of embodiments 377 to 514, wherein

4154 said heavy chain of said IgG is effector-silenced.

4155

4156 Embodiment 516: The compound according to any one of embodiments 377 to 504 or 515,

4157 wherein said compound consists of a IgG Fc region to which two copies of a tandem-VHH

4158 arrangement are covalently linked at the hinge-region, wherein said tandem-VHH arrangement

4159 consists of a first VHH antibody domain that is covalently linked to a second VHH antibody

4160 domain, wherein said second VHH antibody domain is covalently linked to the hinge region of

4161 said Fc region, resulting in a bispecific, tetravalent compound.

4162

4163 Embodiment 517: The compound according to embodiment 516, wherein said IgG Fc region is

4164 an effector-silenced IgGl Fc region.

4165

4166 Embodiment 518: The compound according to any one of embodiments 516 to 517, wherein

4167 said first binding module forms said first VHH antibody domain and said second binding

4168 module forms said second VHH antibody domain.

4169

4170 Embodiment 519: The compound according to any one of embodiments 516 to 518, wherein

4171 said first VHH antibody domain and said second VHH antibody domain are separated by a

4172 linker.

4173

4174 Embodiment 520: The compound according to any one of embodiments 516 to 519, wherein

4175 said first VHH antibody domain and said second VHH antibody domain are separated by a five

4176 amino acid Gly4Ser-linker.

4177

4178 Embodiment 521: The compound according to any one of embodiments 516 to 520, wherein

4179 said first VHH antibody domain is specific for IL-18Ra and said second VHH antibody domain

4180 is specific for IL-18RP.

4181

4182 Embodiment 522: The compound according to any one of embodiments 516 to 521, wherein

4183 said first VHH antibody domain has the amino acid sequence of VHH2 (SEQ ID NO: 2) and

4184 said second VHH antibody domain has the amino acid sequence of VHH15 (SEQ ID NO: 15).

4185 4186 Embodiment 523: The compound according to any one of embodiments 516 to 520, wherein

4187 said first VHH antibody domain is specific for IL-18RP and said second VHH antibody domain

4188 is specific for IL-18Ra.

4189

4190 Embodiment 524: The compound according to any one of embodiments 516 to 520 or 523,

4191 wherein said first VHH antibody domain has the amino acid sequence of VHH15 (SEQ ID NO:

4192 15) and said second VHH antibody domain has the amino acid sequence of VHH2 (SEQ ID

4193 NO: 2).

4194

4195 Embodiment 525: The compound according to any one of embodiments 516 to 524, wherein

4196 the orientation of the first and second VHH antibody domains is the first VHH antibody

4197 domains followed by the second VHH antibody domain (from N-terminus to C-terminus).

4198

4199 Embodiment 526: The compound according to any one of embodiments 516 to 525, wherein

4200 the orientation of the first and second VHH antibody domains is the second VHH antibody

4201 domains followed by the first VHH antibody domain (from N-terminus to C-terminus).

4202

4203 Embodiment 527: The compound according to any one of embodiments 375 to 526, wherein

4204 said compound is an IL-18 mimetic.

4205

4206 As used herein, the term "mimetic" refers to a compound that imitates certain aspects of the

4207 function of another compound. Provided herein are, among other things, IL-18 mimetics. IL-

4208 18 is a proinflammatory cytokine belonging to the IL-1 family of cytokines that mediates

4209 signalling through heterodimerization of the receptor subunits IL-18Ra and IL-18RP (Yasuda

4210 et al., 2019; Dinarello et al., 2013). IL-18 stimulates IFN-y production in innate lymphoid cells

4211 as well as antigen-experienced T cells in synergy with IL-12. The aspects of the function of

4212 IL 18 that such IL- 18 mimetics imitate is the induction of heterodimerization of IL-18R (from

4213 IL-18Ra and IL-18RP) in the presence of IL-12 (or IL-15). Without being bound by theory, it

4214 is speculated that by binding to both IL-18Ra and IL-18RP, the IL-18 mimetics of the present

4215 disclosure crosslink IL-18Ra and IL-18RP, thus leading to dimerization and activation of IL-

4216 18R in the presence of IL-12 (or IL-15). Thus, the IL-18 mimetics of the present disclosure

4217 imitate the biological function of IL-18, for example the function of IL-18 to induce IFN-y

4218 release (in the presence of IL-12 or IL-15).

4219 4220 Embodiment 528: The compound according to embodiment 527, wherein said IL- 18 mimetic

4221 is not affected from inhibition by IL-18BP (IL-18 binding protein).

4222

4223 Embodiment 529: The compound according to any one of embodiments 375 to 528, wherein

4224 said compound is a IL-18 receptor (IL-18R) agonist.

4225

4226 Embodiment 530: The compound according to any one of embodiments 375 to 529, wherein

4227 said compound is a cytokine mimetic targeting IL-18Ra and IL-18RP.

4228

4229 Embodiment 531: The compound according to any one of embodiments 375 to 530, wherein

4230 said compound binds to IL-18Ra.

4231

4232 Embodiment 532: The compound according to any one of embodiments 375 to 531, wherein

4233 said compound specifically binds to IL-18Ra.

4234

4235 Embodiment 533: The compound according to any one of embodiments 375 to 532, wherein

4236 said compound is capable of binding to IL-18Ra with an affinity that is at least equal to the

4237 affinity with which VHH1 (SEQ ID NO: 1), VHH2 (SEQ ID NO: 2), VHH3 (SEQ ID NO: 3),

4238 VHH4 (SEQ ID NO: 4), VHH5 (SEQ ID NO: 5), VHH6 (SEQ ID NO: 6), VHH8 (SEQ ID NO:

4239 8), VHH9 (SEQ ID NO: 9) or VHH10 (SEQ ID NO: 10) binds to IL-18Ra (or stronger).

4240

4241 Embodiment 534: The compound according to any one of embodiments 375 to 533, wherein

4242 said compound binds to IL-18RP.

4243

4244 Embodiment 535: The compound according to any one of embodiments 375 to 534, wherein

4245 said compound specifically binds to IL-18RP.

4246

4247 Embodiment 536: The compound according to any one of embodiments 375 to 535, wherein

4248 said compound is capable of binding to IL-18RP with an affinity that is at least equal to the

4249 affinity with which VHH12 (SEQ ID NO: 12), VHH13 (SEQ ID NO: 13), VHH14 (SEQ ID

4250 NO: 14), VHH15 (SEQ ID NO: 15), VHH16 (SEQ ID NO: 16), VHH17 (SEQ ID NO: 17),

4251 VHH18 (SEQ ID NO: 18), VHH20 (SEQ ID NO: 20), VHH21 (SEQ ID NO: 21) or VHH22

4252 (SEQ ID NO: 22) binds to IL-18RP (or stronger).

4253 4254 Embodiment 537: The compound according to any one of embodiments 375 to 536, wherein

4255 said compound binds to IL-18Ra and IL-18RP.

4256

4257 Embodiment 538: The compound according to any one of embodiments 375 to 537, wherein

4258 said compound specifically binds to IL-18Ra and IL-18Rp.

4259

4260 Embodiment 539: The compound according to any one of embodiments 375 to 538, wherein

4261 said compound is capable of crosslinking IL-18Ra and IL-18RP.

4262

4263 Embodiment 540: The compound according to any one of embodiments 375 to 539, wherein

4264 said compound is capable of inducing heterodimerization of IL-18Ra and IL-18RP.

4265

4266 Such binding/crosslinking/heterodimerization can be determined by in vitro binding

4267 experiments as described in Example 1 below (by biolayer interferometry).

4268

4269 Embodiment 541: The compound according to any one of embodiments 375 to 540, wherein

4270 said compound binds to IL-18Ra ECD with a KD of 100 nM or stronger.

4271

4272 Embodiment 542: The compound according to any one of embodiments 375 to 540, wherein

4273 said compound binds to IL-18Ra ECD with a KD of 50 nM or stronger.

4274

4275 Embodiment 543: The compound according to any one of embodiments 375 to 540, wherein

4276 said compound binds to IL-18Ra ECD with a KD of 25 nM or stronger.

4277

4278 Embodiment 544: The compound according to any one of embodiments 375 to 540, wherein

4279 said compound binds to IL-18Ra ECD with a KD of 10 nM or stronger.

4280

4281 Embodiment 545: The compound according to any one of embodiments 375 to 540, wherein

4282 said compound binds to IL-18Ra ECD with a KD of 1 nM or stronger.

4283

4284 Embodiment 546: The compound according to any one of embodiments 375 to 540, wherein

4285 said compound binds to IL-18Ra ECD with a KD of 500 pM or stronger.

4286 4287 Embodiment 547: The compound according to any one of embodiments 375 to 540, wherein

4288 said compound binds to IL-18Ra ECD with a KD of 100 pM or stronger.

4289

4290 Embodiment 548: The compound according to any one of embodiments 375 to 540, wherein

4291 said compound binds to IL-18Ra ECD with a KD of 10 pM or stronger.

4292

4293 Embodiment 549: The compound according to any one of embodiments 375 to 548, wherein

4294 said compound binds to IL-18RP ECD with a KD of 100 nM or stronger.

4295

4296 Embodiment 550: The compound according to any one of embodiments 375 to 548, wherein

4297 said compound binds to IL-18RP ECD with a KD of 50 nM or stronger.

4298

4299 Embodiment 551: The compound according to any one of embodiments 375 to 548, wherein

4300 said compound binds to IL-18RP ECD with a KD of 25 nM or stronger.

4301

4302 Embodiment 552: The compound according to any one of embodiments 375 to 548, wherein

4303 said compound binds to IL-18RP ECD with a KD of 10 nM or stronger.

4304

4305 Embodiment 553: The compound according to any one of embodiments 375 to 548, wherein

4306 said compound binds to IL-18RP ECD with a KD of 1 nM or stronger.

4307

4308 Embodiment 554: The compound according to any one of embodiments 375 to 548, wherein

4309 said compound binds to IL-18RP ECD with a KD of 500 pM or stronger.

4310

4311 Embodiment 555: The compound according to any one of embodiments 375 to 548, wherein

4312 said compound binds to IL-18RP ECD with a KD of 100 pM or stronger.

4313

4314 Embodiment 556: The compound according to any one of embodiments 375 to 548, wherein

4315 said compound binds to IL-18RP ECD with a KD of 10 pM or stronger.

4316

4317 Embodiment 557: The compound according to any one of embodiments 375 to 556, wherein

4318 said compound shows competitive binding with recombinant human IL-18 for targeting IL-

4319 18Ra.

4320 4321 Embodiment 558: The compound according to any one of embodiments 375 to 557, wherein

4322 said compound shows competitive binding with recombinant human IL-18 for targeting IL-

4323 18R .

4324

4325 Embodiment 559: The compound according to any one of embodiments 375 to 558, wherein

4326 said compound is capable of activating the IL-18 receptor IL-18R.

4327

4328 Embodiment 560: The compound according to any one of embodiments 375 to 559, wherein

4329 said compound is capable of activating the IL-18 receptor IL-18R by binding to IL-18Ra and

4330 IL-18RP.

4331

4332 Embodiment 561: The compound according to any one of embodiments 375 to 560, wherein

4333 said compound is capable of activating the IL-18 receptor IL-18R by cross-linking the IL-18

4334 receptor subunits IL-18Ra and IL-18RP.

4335

4336 Embodiment 562: The compound according to any one of embodiments 375 to 558, wherein

4337 said compound is does not activate the IL-18 receptor IL-18R.

4338

4339 Embodiment 563: The compound according to any one of embodiments 375 to 562, wherein

4340 said compound triggers dose-dependent IL-18R downstream signaling on IL-18 reporter cells.

4341

4342 Activation of IL-18R and whether a compound triggers dose-dependent IL-18R downstream

4343 signaling on IL-18 reporter cells can for example be determined as described in Example 1,

4344 section "Human IL-18 HEK reporter assay".

4345

4346 Embodiment 564: The compound according to any one of embodiments 375 to 563, wherein

4347 said compound induces at a concentration of 50 nM at least 1.5-fold higher NFKB activation

4348 than TNF-a at a concentration of 50 nM.

4349

4350 Embodiment 565: The compound according to any one of embodiments 375 to 564, wherein

4351 said compound shows a lower potency of IL-18R receptor activation (as determined from the

4352 ECso of NFKB reporter activation) compared with IL-18.

4353 4354 Embodiment 566: The compound according to any one of embodiments 375 to 565, wherein

4355 said compound shows a lower magnitude of IL-18 activation (as determined by maximal NFKB

4356 activation) compared with IL-18.

4357

4358 Whether a compound shows a higher potency of IL-18R receptor activation (as determined

4359 from the ECso of NFKB reporter activation) compared with IL-18 and/or shows lower

4360 magnitude of IL- 18 activation (as determined by maximal NFKB activation) compared with IL-

4361 18 can be determined based on an assay as described in Example 3 and Example 1, section

4362 "Human IL- 18 HEK reporter assay".

4363

4364 Embodiment 567: The compound according to any one of embodiments 375 to 566, wherein

4365 said IL-18 is recombinant human IL-18.

4366

4367 Embodiment 568: The compound according to any one of embodiments 375 to 567, wherein

4368 said compound induces at a concentration of 1 nM a normalized NFKB reporter activation of

4369 more than 50% relative to the NFKB reporter activation induced by recombinant human IL18

4370 at a concentration of 1 nM or wherein said compound has an ECso for NFKB reporter activation

4371 of less than 0.1 nM.

4372

4373 Embodiment 569: The compound according to any one of embodiments 375 to 568, wherein

4374 said compound induces at a concentration of 1 nM a normalized NFKB reporter activation of

4375 more than 50% relative to the NFKB reporter activation induced by recombinant human IL18

4376 at a concentration of 1 nM.

4377

4378 Embodiment 570: The compound according to any one of embodiments 375 to 569, wherein

4379 said compound has an ECso for NFKB reporter activation of less than 0.1 nM.

4380

4381 Embodiment 571: The compound according to any one of embodiments 375 to 567, wherein

4382 said compound induces at a concentration of 1 nM a normalized NFKB reporter activation of

4383 less than 50% relative to the NFKB reporter activation induced by recombinant human IL 18 at

4384 a concentration of 1 nM or wherein said compound has an ECso for NFKB reporter activation

4385 of more than 0.1 nM.

4386 4387 Embodiment 572: The compound according to any one of embodiments 375 to 567 or 571,

4388 wherein said compound induces at a concentration of 1 nM a normalized NFKB reporter

4389 activation of less than 50% relative to the NFKB reporter activation induced by recombinant

4390 human IL 18 at a concentration of 1 nM.

4391

4392 Embodiment 573: The compound according to any one of embodiments 375 to 567 or 571 to

4393 572, wherein said compound has an ECso for NFKB reporter activation of more than 0.1 nM.

4394

4395 Whether a compound induces at a concentration of 1 nM a normalized NFKB reporter activation

4396 of more or less than 50% relative to the NFKB reporter activation induced by recombinant

4397 human IL 18 at a concentration of 1 nM and/or has an ECso for NFKB reporter activation of less

4398 or more than 0.1 nM can be determined based on an assay as described in Example 3 and

4399 Example 1, section "Human IL-18 HEK reporter assay".

4400

4401 Embodiment 574: The compound according to any one of embodiments 375 to 573, wherein

4402 said compound is capable of inducing IFN-y release by peripheral blood mononuclear cells

4403 (PBMCs) in the presence of low-dose IL-12.

4404

4405 Embodiment 575: The compound according to embodiment 574, wherein said low-dose IL-12

4406 is 10 ng/ml IL-12.

4407

4408 Embodiment 576: The compound according to any one of embodiments 375 to 575, wherein

4409 said IL-12 is recombinant human IL-12.

4410

4411 Details how to determine whether a compound is capable of inducing IFN-y release by

4412 peripheral blood mononuclear cells (PBMCs) in the presence of low-dose IL-12 are described

4413 in Example 1, section "IFN-y release assay".

4414

4415 Embodiment 577: The compound according to any one of embodiments 375 to 576, wherein

4416 said compound has an EC50 for the release of IFN-y from human PBMCs of less than 150 nM.

4417

4418 Embodiment 578: The compound according to any one of embodiments 375 to 576, wherein

4419 said compound has an EC50 for the release of IFN-y from human PBMCs of less than 100 nM.

4420 4421 Embodiment 579: The compound according to any one of embodiments 375 to 576, wherein

4422 said compound has an EC50 for the release of IFN-y from human PBMCs of less than 50 nM.

4423

4424 Embodiment 580: The compound according to any one of embodiments 375 to 576, wherein

4425 said compound has an EC50 for the release of IFN-y from human PBMCs of less than 20 nM.

4426

4427 Embodiment 581: The compound according to any one of embodiments 375 to 576, wherein

4428 said compound has an EC50 for the release of IFN-y from human PBMCs of less than 150 pM.

4429

4430 Embodiment 582: The compound according to any one of embodiments 375 to 576, wherein

4431 said compound has an EC50 for the release of IFN-y from human PBMCs of less than 100 pM.

4432

4433 Embodiment 583: The compound according to any one of embodiments 375 to 576, wherein

4434 said compound has an EC50 for the release of IFN-y from human PBMCs of less than 50 pM.

4435

4436 Embodiment 584: The compound according to any one of embodiments 375 to 576, wherein

4437 said compound has an EC50 for the release of IFN-y from human PBMCs of less than 20 pM.

4438

4439 Embodiment 585: The compound according to any one of embodiments 375 to 584, wherein

4440 the capacity of the compound to trigger IFN-y release on PBMCs (as measured by ECso) is

4441 increased compared to recombinant human IL-18. As the skilled person understands, this means

4442 that the ECso of said compound to trigger IFN-y release on PBMCs is smaller than that of

4443 recombinant human IL-18.

4444

4445 Embodiment 586: The compound according to any one of embodiments 375 to 585, wherein

4446 the EC50 of said compound for the release of IFN-y from human PBMCs is less than 50% of

4447 the EC50 of (rh) IL-18 for the release of IFN-y from human PBMCs.

4448

4449 As used herein, "rh" in the context of a molecule stands for "recombinant human".

4450

4451 Embodiment 587: The compound according to any one of embodiments 375 to 585, wherein

4452 the EC50 of said compound for the release of IFN-y from human PBMCs is less than 200% of

4453 the EC50 of (rh) IL-18 for the release of IFN-y from human PBMCs.

4454 4455 Embodiment 588: The compound according to any one of embodiments 565 to 587, wherein

4456 said EC50 is measured in the presence of low dose (rh) IL-12.

4457

4458 Embodiment 589: The compound according to any one of embodiments 375 to 588, wherein

4459 said compound causes a release of IFN-y that is at least 200% of the IFN-y release caused by

4460 (rh) IL-18.

4461

4462 Embodiment 590: The compound according to any one of embodiments 375 to 588, wherein

4463 said compound causes a release of IFN-y that is at least 150% of the IFN-y release caused by

4464 (rh) IL-18.

4465

4466 Embodiment 591: The compound according to any one of embodiments 375 to 588, wherein

4467 said compound causes a release of IFN-y that is at least 100% of the IFN-y release caused by

4468 (rh) IL-18.

4469

4470 Embodiment 592: The compound according to any one of embodiments 375 to 588, wherein

4471 said compound causes a release of IFN-y that is at least 80% of the IFN-y release caused by (rh)

4472 IL-18.

4473

4474 Embodiment 593: The compound according to any one of embodiments 375 to 588, wherein

4475 said compound causes a release of IFN-y that is at least 50% of the IFN-y release caused by (rh)

4476 IL-18.

4477

4478 Embodiment 594: The compound according to any one of embodiments 375 to 588, wherein

4479 said compound causes a release of IFN-y that is at least 10% of the IFN-y release caused by (rh)

4480 IL-18.

4481

4482 Embodiment 595: The compound according to any one of embodiments 574 to 594, wherein

4483 said IFN-y release is measured upon stimulation of human peripheral blood mononuclear cells

4484 (PBMCs) with said compound (resp. with IL-18) in the presence of low dose recombinant

4485 human IL-12 (10 ng/ml).

4486

4487 Embodiment 596: The compound according to embodiment 595, wherein said compound resp.

4488 said IL-18 is used at a concentration of 1 nM. 4489

4490 Embodiment 597: The compound according to any one of embodiments 574 to 596, wherein

4491 said release of IFN-y is the release of IFN-y at a concentration of 1 nM of said compound resp.

4492 said IL-18.

4493

4494 Embodiment 598: The compound according to any one of embodiments 574 to 595, wherein

4495 said release of IFN-y is the maximum release of IFN-y.

4496

4497 Further details about the assay are described in the Examples section, in particular Example 1,

4498 section "IFN-y release assay".

4499

4500 Embodiment 599: The compound according to any one of embodiments 375 to 598, wherein

4501 said compound is not affected by the presence of IL-18 binding protein (IL-18BP).

4502

4503 Embodiment 600: The compound according to any one of embodiments 375 to 599, wherein

4504 said compound does not bind to recombinant human IL-18 binding protein (IL-18BP).

4505

4506 Embodiment 601: The compound according to any one of embodiments 375 to 600, wherein

4507 binding of said compound to IL-18Ra and IL-18RP is not affected by the presence of IL-18

4508 binding protein (IL-18BP).

4509

4510 Embodiment 602: The compound according to any one of embodiments 375 to 601, wherein

4511 said activation of IL-18R by said compound is not affected by the presence of IL-18 binding

4512 protein (IL-18BP).

4513

4514 Embodiment 603: The compound according to any one of embodiments 375 to 602, wherein

4515 said capacity of said compound to induce IFN-y release is not affected by the presence of IL-

4516 18BP under conditions where the capacity of IL-18 to induce IFN-y release is inhibited by the

4517 presence of IL-18BP.

4518

4519 Embodiment 604: The compound according to any one of embodiments 375 to 603, wherein

4520 the EC50 of said compound for the release of IFN-y from human PBMCs is not increased by

4521 the presence of (rh) IL-18BP (compared to the absence of (rh) IL-18BP) under conditions where 4522 the EC50 of IL-18 for the release of IFN-y from human PBMCs is increased by the presence of

4523 (rh) IL-18BP (compared to the absence of (rh) IL-18BP).

4524

4525 Embodiment 605: The VHH antibody domain or fragment thereof according to any one of

4526 embodiments 1 to 374 or the compound according to any one of embodiments 375 to 604,

4527 wherein said binding is determined by biolayer interferometry at 25°C and 1000 rpm in KB

4528 Buffer (PBS + 0.1 % Tween-20 + 1% BSA).

4529

4530 Embodiment 606: The VHH antibody domain or fragment thereof according to any one of

4531 embodiments 1 to 374 or the compound according to any one of embodiments 375 to 604,

4532 wherein said competition is determined by biolayer interferometry at 25°C and 1000 rpm in KB

4533 Buffer (PBS + 0.1 % Tween-20 + 1% BSA).

4534

4535 Embodiment 607: The VHH antibody domain or fragment thereof according to any one of

4536 embodiments 1 to 374 or the compound according to any one of embodiments 375 to 604,

4537 wherein said crosslinking/heterodimerization is determined by biolayer interferometry at 25°C

4538 and 1000 rpm in KB Buffer (PBS + 0.1 % Tween-20 + 1% BSA).

4539

4540 Embodiment 608: The VHH antibody domain or fragment thereof according to any one of

4541 embodiments 1 to 374 or the compound according to any one of embodiments 375 to 604,

4542 wherein said KD value is measured by kinetic measurements by biolayer interferometry at 25°C

4543 and 1000 rpm in KB Buffer (PBS + 0.1 % Tween-20 + 1% BSA).

4544

4545 According to a thirteenth aspect, the present disclosure relates to any of the following (To the

4546 thirteenth aspect of the present disclosure and the embodiments referring thereto, the same

4547 explanations and definitions apply accordingly as for the first to twelfth aspects of the present

4548 disclosure and the embodiments referring thereto.):

4549

4550 Embodiment 609: A bispecific antibody molecule prepared by the SEED (strand-exchange

4551 engineered domain) technology, said molecule comprising

4552 - an AG chain linked to an IL-18Ra-binding VHH, wherein said AG chain linked to said

4553 IL-18Ra-binding VHH has the amino acid sequence of SEQ ID NO: 89 ("VHH-SEED-

4554 AG-l") and 4555 - a GA chain linked to an IL18RP-binding VHH, wherein said GA chain linked to said

4556 IL18RP-binding VHH has the amino acid sequence of SEQ ID NO: 100 ("VHH-SEED-

4557 GA-1").

4558

4559 Embodiment 610: A bispecific antibody molecule prepared by the SEED (strand-exchange

4560 engineered domain) technology, said molecule comprising

4561 - an AG chain linked to an IL-18Ra-binding VHH, wherein said AG chain linked to said

4562 IL-18Ra-binding VHH has the amino acid sequence of SEQ ID NO: 89 ("VHH-SEED-

4563 AG-l") and

4564 - a GA chain linked to an IL18RP-binding VHH, wherein said GA chain linked to said

4565 IL18RP-binding VHH has the amino acid sequence of SEQ ID NO: 101 ("VHH-SEED-

4566 GA-2").

4567

4568 Embodiment 611 : A bispecific antibody molecule prepared by the SEED (strand-exchange

4569 engineered domain) technology, said molecule comprising

4570 - an AG chain linked to an IL-18Ra-binding VHH, wherein said AG chain linked to said

4571 IL-18Ra-binding VHH has the amino acid sequence of SEQ ID NO: 89 ("VHH-SEED-

4572 AG-l") and

4573 - a GA chain linked to an IL18RP-binding VHH, wherein said GA chain linked to said

4574 IL18RP-binding VHH has the amino acid sequence of SEQ ID NO: 103 ("VHH-SEED-

4575 GA-4").

4576

4577 Embodiment 612: A bispecific antibody molecule prepared by the SEED (strand-exchange

4578 engineered domain) technology, said molecule comprising

4579 - an AG chain linked to an IL-18Ra-binding VHH, wherein said AG chain linked to said

4580 IL-18Ra-binding VHH has the amino acid sequence of SEQ ID NO: 89 ("VHH-SEED-

4581 AG-l") and

4582 - a GA chain linked to an IL18RP-binding VHH, wherein said GA chain linked to said

4583 IL18RP-binding VHH has the amino acid sequence of SEQ ID NO: 105 ("VHH-SEED-

4584 GA-6").

4585

4586 Embodiment 613: A bispecific antibody molecule prepared by the SEED (strand-exchange

4587 engineered domain) technology, said molecule comprising 4588 - an AG chain linked to an IL-18Ra-binding VHH, wherein said AG chain linked to said

4589 IL-18Ra-binding VHH has the amino acid sequence of SEQ ID NO: 89 ("VHH-SEED-

4590 AG-l") and

4591 - a GA chain linked to an IL18RP-binding VHH, wherein said GA chain linked to said

4592 IL18RP-binding VHH has the amino acid sequence of SEQ ID NO: 108 ("VHH-SEED-

4593 GA-9").

4594

4595 Embodiment 614: A bispecific antibody molecule prepared by the SEED (strand-exchange

4596 engineered domain) technology, said molecule comprising

4597 - an AG chain linked to an IL-18Ra-binding VHH, wherein said AG chain linked to said

4598 IL-18Ra-binding VHH has the amino acid sequence of SEQ ID NO: 89 ("VHH-SEED-

4599 AG-l") and

4600 - a GA chain linked to an IL18RP-binding VHH, wherein said GA chain linked to said

4601 IL18RP-binding VHH has the amino acid sequence of SEQ ID NO: 109 ("VHH-SEED-

4602 GA-10").

4603

4604 Embodiment 615: A bispecific antibody molecule prepared by the SEED (strand-exchange

4605 engineered domain) technology, said molecule comprising

4606 - an AG chain linked to an IL-18Ra-binding VHH, wherein said AG chain linked to said

4607 IL-18Ra-binding VHH has the amino acid sequence of SEQ ID NO: 89 ("VHH-SEED-

4608 AG-l") and

4609 - a GA chain linked to an IL18RP-binding VHH, wherein said GA chain linked to said

4610 IL18RP-binding VHH has the amino acid sequence of SEQ ID NO: 110 ("VHH-SEED-

4611 GA-11").

4612

4613 Embodiment 616: A bispecific antibody molecule prepared by the SEED (strand-exchange

4614 engineered domain) technology, said molecule comprising

4615 - an AG chain linked to an IL-18Ra-binding VHH, wherein said AG chain linked to said

4616 IL-18Ra-binding VHH has the amino acid sequence of SEQ ID NO: 90 ("VHH-SEED-

4617 AG-2") and

4618 - a GA chain linked to an IL18RP-binding VHH, wherein said GA chain linked to said

4619 IL18RP-binding VHH has the amino acid sequence of SEQ ID NO: 100 ("VHH-SEED-

4620 GA-1").

4621 4622 Embodiment 617: A bispecific antibody molecule prepared by the SEED (strand-exchange

4623 engineered domain) technology, said molecule comprising

4624 - an AG chain linked to an IL-18Ra-binding VHH, wherein said AG chain linked to said

4625 IL-18Ra-binding VHH has the amino acid sequence of SEQ ID NO: 90 ("VHH-SEED-

4626 AG-2") and

4627 - a GA chain linked to an IL18RP-binding VHH, wherein said GA chain linked to said

4628 IL18RP-binding VHH has the amino acid sequence of SEQ ID NO: 101 ("VHH-SEED-

4629 GA-2").

4630

4631 Embodiment 618: A bispecific antibody molecule prepared by the SEED (strand-exchange

4632 engineered domain) technology, said molecule comprising

4633 - an AG chain linked to an IL-18Ra-binding VHH, wherein said AG chain linked to said

4634 IL-18Ra-binding VHH has the amino acid sequence of SEQ ID NO: 90 ("VHH-SEED-

4635 AG-2") and

4636 - a GA chain linked to an IL18RP-binding VHH, wherein said GA chain linked to said

4637 IL18RP-binding VHH has the amino acid sequence of SEQ ID NO: 103 ("VHH-SEED-

4638 GA-4").

4639

4640 Embodiment 619: A bispecific antibody molecule prepared by the SEED (strand-exchange

4641 engineered domain) technology, said molecule comprising

4642 - an AG chain linked to an IL-18Ra-binding VHH, wherein said AG chain linked to said

4643 IL-18Ra-binding VHH has the amino acid sequence of SEQ ID NO: 90 ("VHH-SEED-

4644 AG-2") and

4645 - a GA chain linked to an IL18RP-binding VHH, wherein said GA chain linked to said

4646 IL18RP-binding VHH has the amino acid sequence of SEQ ID NO: 105 ("VHH-SEED-

4647 GA-6").

4648

4649 Embodiment 620: A bispecific antibody molecule prepared by the SEED (strand-exchange

4650 engineered domain) technology, said molecule comprising

4651 - an AG chain linked to an IL-18Ra-binding VHH, wherein said AG chain linked to said

4652 IL-18Ra-binding VHH has the amino acid sequence of SEQ ID NO: 90 ("VHH-SEED-

4653 AG-2") and 4654 - a GA chain linked to an IL18RP-binding VHH, wherein said GA chain linked to said

4655 IL18RP-binding VHH has the amino acid sequence of SEQ ID NO: 106 ("VHH-SEED-

4656 GA-7").

4657

4658 Embodiment 621 : A bispecific antibody molecule prepared by the SEED (strand-exchange

4659 engineered domain) technology, said molecule comprising

4660 - an AG chain linked to an IL-18Ra-binding VHH, wherein said AG chain linked to said

4661 IL-18Ra-binding VHH has the amino acid sequence of SEQ ID NO: 90 ("VHH-SEED-

4662 AG-2") and

4663 - a GA chain linked to an IL18RP-binding VHH, wherein said GA chain linked to said

4664 IL18RP-binding VHH has the amino acid sequence of SEQ ID NO: 108 ("VHH-SEED-

4665 GA-9").

4666

4667 Embodiment 622: A bispecific antibody molecule prepared by the SEED (strand-exchange

4668 engineered domain) technology, said molecule comprising

4669 - an AG chain linked to an IL-18Ra-binding VHH, wherein said AG chain linked to said

4670 IL-18Ra-binding VHH has the amino acid sequence of SEQ ID NO: 90 ("VHH-SEED-

4671 AG-2") and

4672 - a GA chain linked to an IL18RP-binding VHH, wherein said GA chain linked to said

4673 IL18RP-binding VHH has the amino acid sequence of SEQ ID NO: 109 ("VHH-SEED-

4674 GA-10").

4675

4676 Embodiment 623: A bispecific antibody molecule prepared by the SEED (strand-exchange

4677 engineered domain) technology, said molecule comprising

4678 - an AG chain linked to an IL-18Ra-binding VHH, wherein said AG chain linked to said

4679 IL-18Ra-binding VHH has the amino acid sequence of SEQ ID NO: 93 ("VHH-SEED-

4680 AG-5") and

4681 - a GA chain linked to an IL18RP-binding VHH, wherein said GA chain linked to said

4682 IL18RP-binding VHH has the amino acid sequence of SEQ ID NO: 101 ("VHH-SEED-

4683 GA-2").

4684

4685 Embodiment 624: A bispecific antibody molecule prepared by the SEED (strand-exchange

4686 engineered domain) technology, said molecule comprising 4687 - an AG chain linked to an IL-18Ra-binding VHH, wherein said AG chain linked to said

4688 IL-18Ra-binding VHH has the amino acid sequence of SEQ ID NO: 93 ("VHH-SEED-

4689 AG-5") and

4690 - a GA chain linked to an IL18RP-binding VHH, wherein said GA chain linked to said

4691 IL18RP-binding VHH has the amino acid sequence of SEQ ID NO: 102 ("VHH-SEED-

4692 GA-3").

4693

4694 Embodiment 625: A bispecific antibody molecule prepared by the SEED (strand-exchange

4695 engineered domain) technology, said molecule comprising

4696 - an AG chain linked to an IL-18Ra-binding VHH, wherein said AG chain linked to said

4697 IL-18Ra-binding VHH has the amino acid sequence of SEQ ID NO: 94 ("VHH-SEED-

4698 AG-6") and

4699 - a GA chain linked to an IL18RP-binding VHH, wherein said GA chain linked to said

4700 IL18RP-binding VHH has the amino acid sequence of SEQ ID NO: 105 ("VHH-SEED-

4701 GA-6").

4702

4703 Embodiment 626: A bispecific antibody molecule prepared by the SEED (strand-exchange

4704 engineered domain) technology, said molecule comprising

4705 - an AG chain linked to an IL-18Ra-binding VHH, wherein said AG chain linked to said

4706 IL-18Ra-binding VHH has the amino acid sequence of SEQ ID NO: 94 ("VHH-SEED-

4707 AG-6") and

4708 - a GA chain linked to an IL18RP-binding VHH, wherein said GA chain linked to said

4709 IL18RP-binding VHH has the amino acid sequence of SEQ ID NO: 108 ("VHH-SEED-

4710 GA-9").

4711

4712 Embodiment 627: A bispecific antibody molecule prepared by the SEED (strand-exchange

4713 engineered domain) technology, said molecule comprising

4714 - an AG chain linked to an IL-18Ra-binding VHH, wherein said AG chain linked to said

4715 IL-18Ra-binding VHH has the amino acid sequence of SEQ ID NO: 94 ("VHH-SEED-

4716 AG-6") and

4717 - a GA chain linked to an IL18RP-binding VHH, wherein said GA chain linked to said

4718 IL18RP-binding VHH has the amino acid sequence of SEQ ID NO: 109 ("VHH-SEED-

4719 GA-10").

4720 4721 Embodiment 628: A bispecific antibody molecule prepared by the SEED (strand-exchange

4722 engineered domain) technology, said molecule comprising

4723 - an AG chain linked to an IL-18Ra-binding VHH, wherein said AG chain linked to said

4724 IL-18Ra-binding VHH has the amino acid sequence of SEQ ID NO: 94 ("VHH-SEED-

4725 AG-6") and

4726 - a GA chain linked to an IL18RP-binding VHH, wherein said GA chain linked to said

4727 IL18RP-binding VHH has the amino acid sequence of SEQ ID NO: 110 ("VHH-SEED-

4728 GA-11").

4729

4730 Embodiment 629: A bispecific antibody molecule prepared by the SEED (strand-exchange

4731 engineered domain) technology, said molecule comprising

4732 - an AG chain linked to an IL-18Ra-binding VHH, wherein said AG chain linked to said

4733 IL-18Ra-binding VHH has the amino acid sequence of SEQ ID NO: 96 ("VHH-SEED-

4734 AG-8") and

4735 - a GA chain linked to an IL18RP-binding VHH, wherein said GA chain linked to said

4736 IL18RP-binding VHH has the amino acid sequence of SEQ ID NO: 100 ("VHH-SEED-

4737 GA-1").

4738

4739 Embodiment 630: A bispecific antibody molecule prepared by the SEED (strand-exchange

4740 engineered domain) technology, said molecule comprising

4741 - an AG chain linked to an IL-18Ra-binding VHH, wherein said AG chain linked to said

4742 IL-18Ra-binding VHH has the amino acid sequence of SEQ ID NO: 96 ("VHH-SEED-

4743 AG-8") and

4744 - a GA chain linked to an IL18RP-binding VHH, wherein said GA chain linked to said

4745 IL18RP-binding VHH has the amino acid sequence of SEQ ID NO: 101 ("VHH-SEED-

4746 GA-2").

4747

4748 Embodiment 631 : A bispecific antibody molecule prepared by the SEED (strand-exchange

4749 engineered domain) technology, said molecule comprising

4750 - an AG chain linked to an IL-18Ra-binding VHH, wherein said AG chain linked to said

4751 IL-18Ra-binding VHH has the amino acid sequence of SEQ ID NO: 96 ("VHH-SEED-

4752 AG-8") and 4753 - a GA chain linked to an IL18RP-binding VHH, wherein said GA chain linked to said

4754 IL18RP-binding VHH has the amino acid sequence of SEQ ID NO: 105 ("VHH-SEED-

4755 GA-6").

4756

4757 Embodiment 632: A bispecific antibody molecule prepared by the SEED (strand-exchange

4758 engineered domain) technology, said molecule comprising

4759 - an AG chain linked to an IL-18Ra-binding VHH, wherein said AG chain linked to said

4760 IL-18Ra-binding VHH has the amino acid sequence of SEQ ID NO: 96 ("VHH-SEED-

4761 AG-8") and

4762 - a GA chain linked to an IL18RP-binding VHH, wherein said GA chain linked to said

4763 IL18RP-binding VHH has the amino acid sequence of SEQ ID NO: 106 ("VHH-SEED-

4764 GA-7").

4765

4766 Embodiment 633: A bispecific antibody molecule prepared by the SEED (strand-exchange

4767 engineered domain) technology, said molecule comprising

4768 - an AG chain linked to an IL-18Ra-binding VHH, wherein said AG chain linked to said

4769 IL-18Ra-binding VHH has the amino acid sequence of SEQ ID NO: 96 ("VHH-SEED-

4770 AG-8") and

4771 - a GA chain linked to an IL18RP-binding VHH, wherein said GA chain linked to said

4772 IL18RP-binding VHH has the amino acid sequence of SEQ ID NO: 108 ("VHH-SEED-

4773 GA-9").

4774

4775 Embodiment 634: A bispecific antibody molecule prepared by the SEED (strand-exchange

4776 engineered domain) technology, said molecule comprising

4777 - an AG chain linked to an IL-18Ra-binding VHH, wherein said AG chain linked to said

4778 IL-18Ra-binding VHH has the amino acid sequence of SEQ ID NO: 96 ("VHH-SEED-

4779 AG-8") and

4780 - a GA chain linked to an IL18RP-binding VHH, wherein said GA chain linked to said

4781 IL18RP-binding VHH has the amino acid sequence of SEQ ID NO: 109 ("VHH-SEED-

4782 GA-10").

4783

4784 Embodiment 635: A bispecific antibody molecule prepared by the SEED (strand-exchange

4785 engineered domain) technology, said molecule comprising 4786 - an AG chain linked to an IL-18Ra-binding VHH, wherein said AG chain linked to said

4787 IL-18Ra-binding VHH has the amino acid sequence of SEQ ID NO: 97 ("VHH-SEED-

4788 AG-9") and

4789 - a GA chain linked to an IL18RP-binding VHH, wherein said GA chain linked to said

4790 IL18RP-binding VHH has the amino acid sequence of SEQ ID NO: 100 ("VHH-SEED-

4791 GA-1").

4792

4793 Embodiment 636: A bispecific antibody molecule prepared by the SEED (strand-exchange

4794 engineered domain) technology, said molecule comprising

4795 - an AG chain linked to an IL-18Ra-binding VHH, wherein said AG chain linked to said

4796 IL-18Ra-binding VHH has the amino acid sequence of SEQ ID NO: 97 ("VHH-SEED-

4797 AG-9") and

4798 - a GA chain linked to an IL18RP-binding VHH, wherein said GA chain linked to said

4799 IL18RP-binding VHH has the amino acid sequence of SEQ ID NO: 101 ("VHH-SEED-

4800 GA-2").

4801

4802 Embodiment 637: A bispecific antibody molecule prepared by the SEED (strand-exchange

4803 engineered domain) technology, said molecule comprising

4804 - an AG chain linked to an IL-18Ra-binding VHH, wherein said AG chain linked to said

4805 IL-18Ra-binding VHH has the amino acid sequence of SEQ ID NO: 97 ("VHH-SEED-

4806 AG-9") and

4807 - a GA chain linked to an IL18RP-binding VHH, wherein said GA chain linked to said

4808 IL18RP-binding VHH has the amino acid sequence of SEQ ID NO: 103 ("VHH-SEED-

4809 GA-4").

4810

4811 Embodiment 638: A bispecific antibody molecule prepared by the SEED (strand-exchange

4812 engineered domain) technology, said molecule comprising

4813 - an AG chain linked to an IL-18Ra-binding VHH, wherein said AG chain linked to said

4814 IL-18Ra-binding VHH has the amino acid sequence of SEQ ID NO: 97 ("VHH-SEED-

4815 AG-9") and

4816 - a GA chain linked to an IL18RP-binding VHH, wherein said GA chain linked to said

4817 IL18RP-binding VHH has the amino acid sequence of SEQ ID NO: 105 ("VHH-SEED-

4818 GA-6").

4819 4820 Embodiment 639: A bispecific antibody molecule prepared by the SEED (strand-exchange

4821 engineered domain) technology, said molecule comprising

4822 - an AG chain linked to an IL-18Ra-binding VHH, wherein said AG chain linked to said

4823 IL-18Ra-binding VHH has the amino acid sequence of SEQ ID NO: 97 ("VHH-SEED-

4824 AG-9") and

4825 - a GA chain linked to an IL18RP-binding VHH, wherein said GA chain linked to said

4826 IL18RP-binding VHH has the amino acid sequence of SEQ ID NO: 108 ("VHH-SEED-

4827 GA-9").

4828

4829 Embodiment 640: A bispecific antibody molecule prepared by the SEED (strand-exchange

4830 engineered domain) technology, said molecule comprising

4831 - an AG chain linked to an IL-18Ra-binding VHH, wherein said AG chain linked to said

4832 IL-18Ra-binding VHH has the amino acid sequence of SEQ ID NO: 97 ("VHH-SEED-

4833 AG-9") and

4834 - a GA chain linked to an IL18RP-binding VHH, wherein said GA chain linked to said

4835 IL18RP-binding VHH has the amino acid sequence of SEQ ID NO: 109 ("VHH-SEED-

4836 GA-10").

4837

4838 Embodiment 641 : A bispecific antibody molecule prepared by the SEED (strand-exchange

4839 engineered domain) technology, said molecule comprising

4840 - an AG chain linked to an IL-18Ra-binding VHH, wherein said AG chain linked to said

4841 IL-18Ra-binding VHH has the amino acid sequence of SEQ ID NO: 97 ("VHH-SEED-

4842 AG-9") and

4843 - a GA chain linked to an IL18RP-binding VHH, wherein said GA chain linked to said

4844 IL18RP-binding VHH has the amino acid sequence of SEQ ID NO: 110 ("VHH-SEED-

4845 GA-11").

4846

4847 Embodiment 642: A bispecific antibody molecule prepared by the SEED (strand-exchange

4848 engineered domain) technology, said molecule comprising

4849 - an AG chain linked to an IL-18Ra-binding VHH, wherein said AG chain linked to said

4850 IL-18Ra-binding VHH has the amino acid sequence of SEQ ID NO: 98 ("VHH-SEED-

4851 AG-10") and 4852 - a GA chain linked to an IL18RP-binding VHH, wherein said GA chain linked to said

4853 IL18RP-binding VHH has the amino acid sequence of SEQ ID NO: 101 ("VHH-SEED-

4854 GA-2").

4855

4856 Embodiment 643: A bispecific antibody molecule prepared by the SEED (strand-exchange

4857 engineered domain) technology, said molecule comprising

4858 - an AG chain linked to an IL-18Ra-binding VHH, wherein said AG chain linked to said

4859 IL-18Ra-binding VHH has the amino acid sequence of SEQ ID NO: 98 ("VHH-SEED-

4860 AG-10") and

4861 - a GA chain linked to an IL18RP-binding VHH, wherein said GA chain linked to said

4862 IL18RP-binding VHH has the amino acid sequence of SEQ ID NO: 108 ("VHH-SEED-

4863 GA-9").

4864

4865 Embodiment 644: A bispecific antibody molecule prepared by the SEED (strand-exchange

4866 engineered domain) technology, said molecule comprising

4867 - an AG chain linked to an IL-18Ra-binding VHH, wherein said AG chain linked to said

4868 IL-18Ra-binding VHH has the amino acid sequence of SEQ ID NO: 98 ("VHH-SEED-

4869 AG-10") and

4870 - a GA chain linked to an IL18RP-binding VHH, wherein said GA chain linked to said

4871 IL18RP-binding VHH has the amino acid sequence of SEQ ID NO: 109 ("VHH-SEED-

4872 GA-10").

4873

4874 According to a fourteenth aspect, the present disclosure relates to a pharmaceutical composition

4875 comprising the compound or bispecific antibody molecule according to any one of the aspects

4876 or embodiments described above.

4877

4878 Methods for preparing pharmaceutical compositions are known to a skilled person in the art

4879 (Remington: The Science and Practice of Pharmacy, 22nd ed. (2012), Pharmaceutical Press).

4880

4881 In some embodiments, said pharmaceutical composition comprises a pharmaceutically

4882 acceptable carrier, diluent and/or excipient.

4883

4884 The term "pharmaceutically acceptable" designates that said carrier, diluent or excipient is a

4885 non-toxic, inert material that is compatible with the other ingredients of the pharmaceutical 4886 composition and not harmful to the patient that the pharmaceutical composition is administered

4887 to, such that it can be used in a pharmaceutical product. Substances suitable as carriers, diluents

4888 or excipients in pharmaceutical compositions are known to a skilled person in the art

4889 (Remington: The Science and Practice of Pharmacy, 22nd ed. (2012), Pharmaceutical Press).

4890 The pharmaceutical composition may further include e.g. additional adjuvants, antioxidants,

4891 buffering agents, bulking agents, colorants, emulsifiers, fillers, flavoring agents, preservatives,

4892 stabilizers, suspending agents and/or other customary pharmaceutical auxiliaries.

4893

4894 According to a fifteenth aspect, the present disclosure relates to the use of a compound or

4895 bispecific antibody molecule according to any one of the aspects or embodiments described

4896 above for activating IL-18R receptor.

4897

4898 According to a sixteenth aspect, the present disclosure relates to the use of a compound or

4899 bispecific antibody molecule according to any one of the aspects or embodiments described

4900 above for inducing IL-y release from cells carrying an IL-18R receptor.

4901

4902 Preferably, said use occurs in the presence of IL-12.

4903

4904 Preferably, said cells carrying an IL-18R receptor are T cells.

4905

4906 Preferably, said cells carrying an IL-18R receptor are Thl T cells.

4907

4908

4909 EXAMPLES

4910

4911 The following examples describe the preparation and characterization of IL18 mimetics as

4912 disclosed in the present disclosure, as well as related compounds and methods, along with

4913 comparative disclosure. It is understood that various embodiments of the disclosure reflected

4914 in the examples may be practiced, given the general description provided above. Although the

4915 foregoing invention has been described in some detail by way of illustration and example for

4916 purposes of clarity of understanding, the description and examples should not be construed as

4917 limiting the scope of the invention.

4918

4919 Example 1 4920 Camelid immunization

4921 For the immunization procedure, one llama (lama glama) with an age of about 12.5 years and

4922 one male huarizo (lama glama x vicugna pacos) of about 11 years of age were immunized with

4923 a cocktail of recombinant human (rh) IL18Ra extracellular domain (ECD) as Fc-fusion (R&D

4924 Systems) as well as rh IL18RP ECD (Sino Biologies) with a poly histidine-tag. For

4925 administration, the antigens were diluted to a stock concentration of 1 mg/mL in PBS and

4926 emulsified for initial immunization with Complete Freund's Adjuvant or with Incomplete

4927 Freund's Adjuvant for subsequent immunizations. The antigens were injected subcutaneously

4928 at three sites with 200 pg material in total (1 : 1 ratio of IL18Ra and ILR18P). This procedure

4929 was conducted four times over a period of 35 days, i.e., administration at dO, dl4, d24 and d35.

4930 One week after final administration (d42), a volume of 150 ml blood was collected from each

4931 specimen for subsequent RNA extraction and cDNA synthesis. Of note, processes involving

4932 animals were performed at preclinics GmbH, Germany and in accordance with local regulations

4933 and animal welfare protection laws, hence immunized animals remained alive after final blood

4934 collection.

4935

4936 Yeast strains and media

4937 For antibody surface display, the yeast Saccharomyces cerevisiae strain EBY100 (MAT a

4938 URA3-52 trpl leu2Al his3A200 pep4::HIS3 prblA1.6R canl GAL (pIU211:URA3)) (Thermo

4939 Fisher Scientific) was employed. The cells were cultivated in yeast extract-peptone-dextrose

4940 (YPD) medium composed of 10 g/L yeast extract, 20 g/L peptone and 20 g/L dextrose,

4941 additionally supplemented with 10 mg/mL penicillin-streptomycin (Gibco). Cells harboring

4942 the library plasmids (pDisp) after gap repair cloning were cultivated in minimal synthetic

4943 defined (SD)-base (Takara Bio) medium supplemented with 5.4 g/L Na2HPO4 and 8.6 g/L

4944 NaH2PO4 H2O, also comprising the corresponding dropout mix (Takara Bio) composed of all

4945 essential amino acids except for tryptophan (-Trp) for selection. To induce antibody gene

4946 expression, dextrose was replaced by galactose as carbon source, thus cells were transferred

4947 into SG dropout medium (-Trp) consisting of SG-base medium (Takara Bio) and 10% (w/v)

4948 polyethylene glycol 8000 (PEG 8000).

4949

4950 Plasmids for yeast surface display and library generation 4951 Homologous recombination-based cloning, referred to as gap repair cloning, was utilized for

4952 the generation of the VHH libraries in yeast. For this, our group already described the specific

4953 PCR amplification of VHH fragments as well as library construction detailed elsewhere (Roth

4954 et al., 2020). In short, digestion of a stuffer sequence in the pDisp with specific restriction

4955 enzyme Bsal allow for the subsequent genetic fusion of VHH library candidates in frame to

4956 Aga2p by gap repair cloning, ultimately enabling the sdAb presentation on the surface of the

4957 yeast cell. Additionally, monitoring of proper full-length VHH presentation on yeast surface

4958 was realized by a HA epitope C-terminally linked to Aga2p on the pDisp backbone.

4959

4960 Library sorting

4961 EBY100 cells were grown overnight in SD medium with dropout mix lacking tryptophan (-

4962 Trp) at 30 °C and 120 rpm. Next day, cells were transferred into SG medium with dropout mix

4963 (-Trp) at 107 cells/mL to induce VHH surface expression and incubated for another 48 h at 20

4964 °C and 120 rpm. The fluorescence activated cell sorting (FACS) procedures were conducted on

4965 a BD FACSAria™ Fusion cell sorter (BD Biosciences) device. For library sorting purposes,

4966 full-length VHH surface expression was monitored by application of an Alexa Fluor 488

4967 labeled mouse monoclonal anti-HA antibody (R&D Systems, diluted 1 :20). A two-dimensional

4968 sorting strategy was enabled by simultaneous antigen binding detection using indirect

4969 immunofluorescence staining with 250 nM rh his-tagged IL18Ra (Sino Biological) or 250 nM

4970 rh his-tagged ILR18RP ECD (Sino Biological) in combination with murine anti -his detection

4971 antibody (Penta His Alexa Fluor 647 Conjugate, Qiagen, diluted 1 :20, for sorting round I or

4972 Allophycocyanin anti-His Tag Antibody, BioLegend, diluted 1 :20, for sorting round II),

4973 respectively (Fig. IB). Control samples of cells incubated with secondary labeling reagents and

4974 an unrelated antigen or cells incubated with secondary labeling only as well as untreated cells

4975 were employed in every FACS analysis, enabling an adequate gate adjustment for the desired

4976 cell population.

4977

4978 Protein Expression, Purification and Analytics

4979 After clone selection based on sequencing results obtained from FACS enriched populations,

4980 the VHH variants directed against IL18Ra ECD were N-terminally fused to the hinge region of

4981 Fc immune effector-silenced (eff-) SEED AG chains, while the VHHs targeting IL18RP ECD

4982 were fused accordingly to eff- SEED GA chains prior to cloning into pTT5 mammalian 4983 expression vector (Durocher, 2002), ultimately enabling the production of eff- monospecific

4984 (IL18R_msVHH) and bispecific SEEDbodies (1+1 IL18R_VHHaP). For small-scale

4985 production of the proteins (25 mL scale), Expi293 cells were transiently transfected with

4986 respective pTT5 vectors according to the manufacturer's instructions (Thermo Fisher

4987 Scientific). Six days post transfection the protein containing supernatants were harvested by

4988 centrifugation prior purification via Mab Select antibody purification chromatography resin (GE

4989 Healthcare). A buffer exchange step to PBS pH 6.8 overnight using Pur-A-Lyzer™ Maxi 3500

4990 Dialysis Kit (Sigma Aldrich) was employed, followed by sterile filtration with Ultrafree®-CL

4991 GV 0.22 pm centrifugal devices (Merck Millipore) and measurement of resulting molecule

4992 concentrations using Nanodrop ND-1000 (Peqlab). Protein purities were afterwards determined

4993 by analytical size exclusion chromatography (SEC) on a TSKgel UP-SW3000 column (2 pm,

4994 4.6 x 300 mm, Tosoh Bioscience) using an Agilent HPLC 1260 Infinity system. 7.5 pg protein

4995 per sample were injected and run at a flow rate of 0.35 ml/min using 50 mM sodium phosphate,

4996 0.4 M NaC104 pH 6.3 as mobile phase.

4997

4998 Bispecific proteins generated for the assessment of antibody format engineering on IL-18

4999 cytokine mimetics (Fig. 4A) were generated by the replacement of VH and VL+ of an effector

5000 silenced IgG by VHHa and VHHp, respectively, thereby facilitating the generation of the

5001 IL18R_sdIgGaP architecture (2+2). Tandem arrangement of the respective VHHs was achieved

5002 by N-terminal fusion to the hinge region of an effector silenced IgGl Fc fragment, whereby the

5003 VHHs were separated by a five amino acid Gly4Ser linker, consequently leading to the (2+2)

5004 IL18R_tanVHHaP design (2+2). Moreover, tandem arrangement was constructed for both

5005 VHH orientations, i.e., aP and Pa. Of note, all four molecules were also produced harboring the

5006 E430G mutation for on-target hexamerization.

5007

5008 These antibodies were transiently expressed from ExpiCHO cells (Thermo Fisher Scientific)

5009 following a 11-day protocol at 250 mL scale according to the manufacturer’s instructions (Max

5010 Titer Protocol). The cultivation protocol included a temperature shift from 36.5 °C to 32.0 °C

5011 after addition of ExpiFectamine CHO Enhancer and first ExpiCHO Feed at day 1 while

5012 incubating at 5 % CO2 and 80 rpm. Plasmids for transfections (pTT5 backbone) were used at

5013 0.8 mg/L and were mixed 1 : 1 HC:LC (IgGs) or 2: 1 AG:GA (SEEDs) for constructs composed

5014 of multiple antibody chains. Antibody containing supernatants were harvested by centrifugation

5015 at 4000 x g for 20 min at 4°C and afterwards sterile filtered. Protein purification was done by

5016 affinity capture on Protein A resin (HiTrap MabSelect SuRe, 5 ml) and acidic elution with 50 5017 mM acetic acid pH 3.0 at 5 ml/min using an GE Healthcare AKTAxpress system followed by

5018 a desalting step at 10 ml/min (HiPrep 26/10 columns) into PBS pH 6.8. Protein purities were

5019 afterwards determined by analytical SEC on a TSKgel UP-SW3000 column (2 pm, 4.6 x 300

5020 mm, Tosoh Bioscience) using an Agilent HPLC 1260 Infinity system. 7.5 pg protein per sample

5021 were injected and run at a flow rate of 0.35 ml/min using 50 mM sodium phosphate, 0.4 M

5022 NaClO4 pH 6.3 as mobile phase. For proteins with purities below 90%, a polishing step by

5023 preparative SEC (HiLoad 26/600 or 16/600 Superdex 200 pg) was applied. Final samples were

5024 sterile filtered over 0.2 pm, flash frozen in liquid nitrogen and stored at -80°C until further use.

5025 To assure no quality loss after freeze/thaw, a final purity determination by analytical SEC was

5026 done. Furthermore, thermal unfolding of the antibodies was assessed by differential scanning

5027 fluorimetry (DSF) on a Prometheus NT.PLEX nanoDSF instrument (NanoTemper). Samples

5028 were measured in duplicates using nanoDSF Standard Capillary Chips. A temperature gradient

5029 from 20°C to 95°C at a slope of l°C/min was used while recording fluorescence at 350 and 330

5030 nm. Unfolding transition midpoints (Tm) were determined from the first derivative of the

5031 fluorescence ratio 350 nm/330 nm.

5032

5033 Biolayer interferometry

5034 For binding assays, kinetic measurements, competition assays as well as simultaneous binding

5035 with recombinant proteins, the Octet RED96 system (ForteBio, Pall Life Science) was

5036 employed using 25 °C and 1000 rpm agitation settings. In order to determine binding kinetics

5037 of monospecific molecules, human IL18Ra-His (Sino Biological) and human IL18RP-His

5038 (Sino Biological) were loaded on anti-Penta His (EHS1K) biosensors at 3 pg/mL in PBS for 180

5039 s followed by 60 s sensor rinsing in kinetics buffer (KB; PBS + 0.1% Tween-20 and 1% bovine

5040 serum albumin, BSA). Afterwards, binding to monospecific molecules at 100 nM in KB was

5041 measured for 300 s followed by dissociation for 100 s in KB. In each experiment, one negative

5042 control using irrelevant antibody and a second reference by incubating the IL-18R subunits in

5043 KB instead of the monospecific molecules was measured. Kinetic constants (KD) of bispecific

5044 molecules to determine affinities were measured by loading bispecific molecules on anti-human

5045 Fc (AHC) biosensors for 180s at 5 pg/mL in PBS. After sensor rinsing in KB for 45 s,

5046 interaction was tested by association of human IL18Ra-His or human IL18RP-His in decreasing

5047 concentrations from 100 nM to 12.5 nM for top four molecules and 100 nM to 11 nM for

5048 engineered constructs in KB for 180 s, followed by dissociation for 300 s in KB. Avidity effects

5049 of engineered tandem constructs were shown loading human IL18Ra-His and human IL18RP- 5050 His on HIS IK biosensors at 5 pg/mL in PBS for 180 s, followed by 45 s sensor rinsing in KB.

5051 Association of engineered tandem constructs as well as parental molecule was measured in

5052 ranging concentrations from 100 nM to 11 nM for 180 s in KB followed by dissociation for

5053 300s in KB. Simultaneous binding capacities of IL18R subunits for engineered tandem

5054 constructs and parental molecule were measured by loading molecules on AHC biosensors for

5055 180 s at 5 pg/mL in PBS. After sensor rinsing a first binding step was performed using human

5056 IL18Ra-His or human IL18RP-His at 200 nM to saturate the binding capacity of the loaded

5057 molecule for 300 s. Afterward, the association was determined against other IL18R subunit for

5058 180 s using varying concentrations from 100 nM to 11 nM. To avoid a dissociation of first

5059 bound IL18R subunit in this step, 100 nM of first bound subunit was used for every

5060 concentration tested in association. For the following dissociation step (300 s), first bound

5061 subunit was also used at 100 nM to assess dissociation against second IL18R subunit only. To

5062 determine KD of human IL-18 (R&D Systems) on human IL-18BP His (Aero

5063 Biosytems)/human IL-18BP Fc Tag (R&D Systems) was loaded on HIS IK/ AHC biosensors at

5064 5 pg/mL in PBS for 180 s. After sensor rinsing in KB for 45 s, association of human IL- 18 was

5065 measured in decreasing concentrations from 10 nM to 0.3125 nM in KB for 180 s. Dissociation

5066 of human IL-18 was measured in KB for 300 s. Parallel control measurements for each

5067 association step of biosensors incubated in KB instead were utilized. Data were fitted and

5068 analyzed with ForteBio data analysis software 8.0 using a 1 : 1 binding model after Savitzky-

5069 Golay filtering. Epitope binning experiment of top 4 molecules on IL18R subunits were

5070 performed by loading human IL18Ra-His or human IL18RP-His on HIS IK biosensors at 5

5071 pg/mL in PBS for 180s, followed by 45 s sensor rinsing in KB. Binding to first bispecific

5072 molecule at 200 nM in KB was measured for 300 s to saturate binding capacities of loaded

5073 IL18R subunit, followed by a second association step of another bispecific molecule at 200nM

5074 in KB for another 180 s. To avoid a dissociation of first bound bispecific molecule in second

5075 association, 100 nM of first bound bispecific molecule was used for every association of second

5076 bispecific molecule. To analyze competitive binding of top 4 molecules and engineered tandem

5077 constructs vs. human IL-18 on human IL18Ra-His, molecules were loaded at 5 pg/mL in PBS

5078 for 180 s on AHC biosensors, followed by 45 s sensor rinsing in KB. Association of the

5079 molecules at 200 nM for 300 s in KB was followed by an additional association step with human

5080 IL- 18 at 100 nM for another 180 s in KB. Control values using an unrelated antibody as well

5081 as controls using KB buffer were included. To illustrate that our top 4 molecules and engineered

5082 tandem molecules are not affected by human IL-18BP His, molecules were loaded on AHC

5083 biosensors at 5 pg/mL for 180 s in PBS. After a rinsing step in KB for 45 s, human IL-18BP 5084 His was associated at 1000 nM for 180 s and dissociated in KB for 180 s. To show binding of

5085 human IL-18 to human IL-18BP, human IL-18BP His was loaded at 5 pg/mL in PBS for 180 s

5086 on HIS IK biosensors, followed by a rinsing step in KB for 45 s. Binding to human IL-18 at

5087 1000 nM in KB was measured for 180 s followed by dissociation for 180 s in KB.

5088

5089 Human IL-18 HEK reporter assay

5090 For detection of the activation of JAK-STAT pathway by bispecific IL18R_VHHaP

5091 SEEDbodies the IL-18 HEK-Blue assay (InvivoGen, hkb-hmill8) was performed according to

5092 the manufacturer’s instructions. In brief, 5^ 104 cells were seeded into each well of a 96-well

5093 plate and stimulated initially with 50 nM of selected IL18R_VHHaP for 24 hours at 37 °C and

5094 5 % CO2 including incubation with TNF-a and IL-18 as controls. For EC50 determination,

5095 samples were titrated with 1 :5 serial dilution with concentrations ranging from 100 nM to

5096 5.12x10-5 nM. Additionally, an IL-18 titration was performed with 1 :5 serial dilution with

5097 concentrations ranging from 100 nM to 5.24x10-12 nM. Twenty microliters of cell culture

5098 supernatant were then taken from each well and mixed with 180 pl QUANTLBlue medium in

5099 a 96-well plate, incubated for 3 h at 37 °C and 5 % CO2, and then read in a microplate reader

5100 at 640 nm.

5101

5102 IFN-y release assay

5103 For functional assays to measure IFN-y release, freshly isolated human peripheral mononuclear

5104 cells (PBMCs) from healthy donors were used. Isolation of PBMCs from whole blood samples

5105 was performed using SepMate-50 tubes (StemCell Technologies) and Lymphoprep medium

5106 (StemCell Technologies) according to StemCell Technologies’ SepMate PBMC Isolation

5107 protocol (2019). Human PBMCs were used directly for functional assays after isolation and

5108 cultivated in AIM V medium (Gibco) at 37°C and 5% CO2 in a humidity box for 24 h during

5109 functional assays. For initial Cytokine Release Assay of top 16 molecules 1x106 human

5110 PBMCs per well were stimulated with bispecific molecules (100 nM) or human IL-18 (1 nM,

5111 R&D Systems) in AIM V medium in 96-well white-opaque cultivation plates (Perkin&Elmer).

5112 All stimulations were performed once in the presence of human IL- 12 (10 ng/mL, R&D

5113 Systems) and once in the absence of human IL-12. To determine EC50 values of top 4

5114 molecules 1x106 human PBMCs per well were stimulated with a range of 1000 nM to 0.122

5115 nM of bi specific molecules and human IL-12 (10 ng/mL). For first functional test of engineered 5116 constructs based on parental molecule IL18R_VHHa2pi5 1x106 human PBMCs per well were

5117 stimulated with two concentrations of 10 nM and 1 nM for engineered constructs and 1 nM/0.1

5118 nM for human IL-18. All stimulations were performed in the presence of human IL- 12 (10

5119 ng/ml). To determine EC50 values of top engineered constructs 1x106 human PBMCs were

5120 stimulated with ranging concentrations of engineered constructs (1000 nM to 0.000209 nM)

5121 and IL-18 (0.457 nM to 0.000209 nM) as well as human IL-12 (10 ng/ml). For human IL-18BP

5122 Assay 1x106 human PBMCs were stimulated with a fixed concentration of IL-18 (0.5 nM) or

5123 engineered tandem constructs (0.5 nM) and range of IL-18BP concentrations (0.00001 nM to

5124 100 nM). All stimulations were performed in the presence of human IL-12 (10 ng/ml). As

5125 controls for every functional assay a non-functional binder that serves as negative control as

5126 well as human PBMCs only stimulated with human IL-12 as basal stimulation reference were

5127 used. Quantification of IFN-y released by human PBMCs was performed using human cytokine

5128 HTRF kits (Cisbio) as previously described by our group (Pekar et al., 2020). After 24 h

5129 incubation, PBMC cells were sedimented in 96-well plates by centrifugation, and cytokine¬

5130 containing supernatants were further processed according to the manufacturer’s instructions.

5131 Assay plates were measured with a PHERAstar FSX device (BMG Labtech). HTRF optical

5132 entity using excitation at 337 nm and emission at 620 nm as well as 665 nm was used. Analyses

5133 and fitting of resulting data were facilitated by MARS software (v3.32, BMG) enabling a four-

5134 parameter logistic (4PL l/y2) model fitting of the standard curve following the kit

5135 manufacturer’s instructions.

5136

5137 Data processing and statistical analysis

5138 Graphical and statistical analyses were conducted with GraphPad Prism 8 software. P-values

5139 were calculated utilizing repeated measures 2way ANOVA and the Bonferroni as

5140 recommended, p < 0.05 were regarded as statistically significant.

5141

5142 Example 2

5143 sdAbs targeting IL-18Ra and IL-18RP

5144 For the isolation of VHHs targeting IL-18Ra and IL-18RP one llama and one huarizo (llama x

5145 alpaca) were immunized with a cocktail of the extracellular domains (ECDs) of both receptor

5146 subunits. Afterwards, for each specimen a YSD library was generated as described previously

5147 (Roth et al., 2020; Klausz et al., 2022) and both libraries were sorted individually. A two- 5148 dimensional sorting strategy was applied to select for full-length VHH display via HA tag

5149 staining simultaneous to the binding functionality by exploiting an antigen concentration of

5150 250 nM for each respective receptor subunit. In the first round of selection, a mixture of both

5151 receptor subunits was utilized (Fig. IB). In the second selection round the enriched libraries

5152 were selected against both antigens separately and for each library and each receptor subunit

5153 enrichment for an antigen-binding population could be achieved (Fig. IB, Fig. 5). From each

5154 library, 96 clones were sent out for sequencing for each antigen. This resulted in 55 unique

5155 clones in total enriched for binding to IL-18Ra and 101 unique clones for IL- 18RP, respectively

5156 (Fig. 1C). By applying a clonotyping strategy based on CDR3 diversity, eleven VHHs targeting

5157 each receptor subunit, respectively, were selected for expression.

5158

5159 To this end, all 22 sdAbs were expressed as one-armed (1+0) antibodies by employing the

5160 SEED technology which relies on beta-strand exchanges of IgG and IgA CH3 constant

5161 domains, resulting in preferential heavy chain heterodimerization (Fig. 6A) (Davis et al., 2010).

5162 sdAbs enriched for IL-18Ra binding were grafted onto the hinge region of the AG chain of the

5163 SEEDbody and produced with a paratope-less GA chain, while sdAbs targeting IL-18RP were

5164 fused to the GA chain and combined with a non-targeted AG chain. An effector silenced

5165 derivative of the SEEDbody Fc region was exploited in order to abolish the potential of

5166 antibody-dependent cell-mediated cytotoxicity (ADCC) in primary cell assays (and to verify

5167 that abolishment of ADCC by such an approach also functions in this antibody set-up).

5168

5169 Following expression in ExpiCHO™ cells and protein A purification, we assessed binding to

5170 the ECDs of IL-18Ra and IL-18RP by biolayer interferometry (BLI) using an antigen

5171 concentration of 100 nM (Fig. 6B).

5172

5173 As expected, (given the low sequence identity of approximately 21% between IL-18Ra ECD

5174 and IL-18RP ECD), monospecific molecules obtained from the IL-18Ra sorts

5175 (ms_IL18R_VHHal-l 1) only showed binding to IL-18Ra ECD but not to IL-18RP ECD which

5176 was vice versa for monospecific clones selected from the IL-18RP enrichments

5177 (ms_IL18R_VHHpi2-22). In addition to this, ms_IL18R_VHHa7 only displayed negligible

5178 binding to IL-18Ra ECD, whereas IL-18RP-directed paratope ms_IL18R_VHHpi9 exhibited

5179 no binding at all. Consequently, both sdAbs were excluded from further consideration.

5180 5181 Thus, ten sdAbs specific for IL-18Ra (VHH1, VHH2, VHH3, VHH4, VHH5, VHH6, VHH8,

5182 VHH9, VHH10, VHH11) and ten sdAbs specific for IL-18RP (VHH12, VHH13, VHH14,

5183 VHH15, VHH16, VHH17, VHH18, VHH20, VHH21, VHH22) were obtained.

5184

5185 Example 3

5186 Identification of IL-18 receptor agonists after combinatorial reformatting of sdAbs targeting

5187 individual IL-18 receptor subunits as (1+1) bsAbs

5188 For functional characterization, all ten VHHs targeting IL-18Ra (engrafted to the AG chain of

5189 the SEED architecture) as well as all ten sdAbs specific for IL-18RP (fused to the GA chain)

5190 were recombined and expressed as bispecific SEEDbodies, resulting in 100 bispecific sdAb-

5191 derived antibody derivatives.

5192

5193 As initial cut-off in terms of biophysical properties, we only considered bispecifics for further

5194 investigation that comprised more than 86% target peak in analytical size exclusion

5195 chromatography post protein A purification, resulting in twelve molecules that were excluded.

5196 Interestingly, this encompassed all bsAbs that harbored IL18R_VHHal 1, indicating liabilities

5197 of the respective sdAb paratope. Notwithstanding, 88 VHH-derived bsAbs were assessed

5198 regarding their agonistic potential exploiting IL-18 reporter cells, stably expressing IL-18Ra

5199 and IL-18RP (HEK-Blue™ IL-18 cells).

5200

5201 In an initial experiment, bsAbs as well as all monospecific SEEDbodies (either series

5202 ms_IL18R_VHHal-l 1 or ms_IL18R_VHHp 12-22) were used at a concentration of 50 nM.

5203 Recombinant human (rh) TNF-a was employed as negative control as well as (rh) IL- 18 as

5204 positive control, showing a dose-dependent activation of IL-18 reporter cells (Fig. 7A). As

5205 expected, none of the monospecific SEEDbodies was able to agonize IL-18 reporter cells,

5206 whereas 44 out of the 88 sdAb-based bispecifics induced NFKB activation that was at least 1.5-

5207 fold higher than for TNF-a at this fixed concentration (data not shown). These molecules were

5208 further characterized exploiting the reporter cell assay in a dose-dependent manner. To this end,

5209 we also included IL18R_VHHaipi6 which did not show agonism potential but specific binding

5210 to the respective receptor subunits as negative control (Fig. 2A). Intriguingly, all 44 different

5211 bispecifics were able to agonize the IL-18 reporter cells to a certain extent, whereas

5212 IL18R_VHHaipi6 did not induce activation, as exemplarily shown for surrogate agonists

5213 IL18R_VHHa2pi5, IL18R_VHHa8pi5, IL18R_VHHa2pi7 and IL18R_VHHa8pi7 in 5214 Figure 2A. However, compared with (rh) IL-18, potencies (EC50 of NFKB reporter activation)

5215 as well as the magnitude (maximal NFKB activation) were attenuated for all bispecifics tested

5216 (Fig. 7A, B). According to their agonism potential, all bispecifics were ranked into four groups:

5217 molecules discarded due to low purity (grey), functionally inactive bispecifics (red), minimally

5218 active agonists eliciting less than 50% of NFKB reporter activation normalized to (rh) IL-18 at

5219 1 nM or EC50 higher than 0.1 nM (yellow) and moderately active surrogate agonists triggering

5220 either a normalized NFKB reporter activation of more than 50% relative to IL 18 or displaying

5221 potencies of less than 0.1 nM (green, Fig. 2B).

5222

5223 Example 4

5224 Dose-dependent release of IFN-y by bispecific sdAb-derived IL-18 mimetics in PBMC-based

5225 assays

5226 To evaluate the functionality of the engineered bispecific cytokine mimetics in primary cell

5227 assays, we stimulated human peripheral blood mononuclear cells (PBMCs) either with (rh) IL-

5228 18 or with a selection of 16 different surrogate agonists that showed at least some level of

5229 agonistic activity in the IL-18 reporter cell assay (ranging from minimally active to moderately

5230 active agonists) in combination with low dose (rh) IL-12. Again, IL18R_VHHaipi6 was

5231 included as negative control. To this end, we exploited a fixed concentration of 100 nM of each

5232 of bsAbs scrutinized, whereas for (rh) IL-18 due to an anticipated much stronger potency, we

5233 used a concentration of 1 nM. As functional read out we used PBMC-derived production of

5234 IFN-y (Fig. 3A). Stimulation of PBMCs with (rh) IL-18 triggered a robust release of IFN-y

5235 (mean release at 1 nM of 2135.8 pg/ml), whereas IFN-y production was negligible for the

5236 treatment with IL18R_VHHaipi6 (mean release at 100 nM of 39.5 pg/ml). For the 16

5237 bispecific surrogate agonists we observed a quite diverse release of IFN-y at 100 nM. While for

5238 five bsAbs (IL18R_VHHa5pi3, IL18R_VHHa2p22, IL18R_VHHa6p22, IL18R_VHHa8p22,

5239 IL18R_VHHal0p22), the magnitude of IFN-y production was not appreciably different from

5240 the negative control (IL18R_VHHaipi6), there was a trend for higher proinflammatory

5241 cytokine release for the vast majority of bispecific surrogate agonists tested. Most importantly,

5242 sdAb-based IL-18 mimetics IL18R_VHHa2pi5, IL18R_VHHa2pi7 and IL18R_VHHa8pi7

5243 evoked a statistically significant higher IFN-y production compared to IL18R_VHHaipi6,

5244 with IL18R_VHHa2pi5 triggering the most prominent release (mean release 1390.2 pg/ml).

5245 We also assessed IFN-y production of all the molecules without low-dose (rh) IL-12 (Fig. 8).

5246 Neither (rh) IL-18, nor the generated bispecifics triggered IFN-y production in the absence of 5247 (rh) IL-12, which is in accordance with the finding, that IL-18 induces IFN-y either with IL-12

5248 or IL- 15 (Dinarello et al., 2013).

5249

5250 To further narrow down surrogate agonists for in-depth characterization, we only focused on

5251 the three molecules that triggered significant release of IFN-y as well as IL18R_VHHa8pi5

5252 which also elicited a moderate production of IFN-y, albeit not being statistically significant.

5253 Remarkably, those four bispecifics were composed of different combinations of two sdAbs

5254 targeting IL-18Ra (VHHa2 and VHHa8) as well as two VHHs specific for the IL-18RP subunit

5255 (VHHP15 and VHHP17). For this, we stimulated human PBMCs isolated from healthy donors

5256 with the four bispecific candidates as well as (rh) IL-18 using a range of different concentrations

5257 in combination with low dose (rh) IL-12 (Fig. 3B). All four bispecific IL-18 mimetics triggered

5258 a dose-dependent release of IFN-y with potencies (EC50 of IFN-y production) of 9.1 nM -

5259 -107 nM (Table 1). Of note, IL18R_VHHa8pi5 did not reach a maximum IFN-y release at the

5260 highest concentration tested (1 pM). Hence, we haven’t been able to determine the EC50 of this

5261 bsAb accurately.

5262

5263 Besides functional characterization, we also determined affinities of the TOP4 IL-18 mimetics

5264 against each receptor subunit (Table 1, Fig. 9). Affinities for binding to IL-18Ra ranged from

5265 5.2 nM for IL18R_VHHa2pi5 to 35.6 nM for IL18R_VHHa8pi6. Also binding to the IL-18RP

5266 subunit ranged from the single digit picomolar range to binding in the lower double digit

5267 nanomolar range (for IL18R_VHHa2pi5). Interestingly, there was a trend towards higher

5268 potencies and efficacies in terms of IFN-y production for cytokine mimetics harboring a VHH

5269 with higher binding affinities to IL-18Ra which were mainly driven by an improved off-rate

5270 (VHHa2 vs. VHHa8).

5271

5272 Table 1: Binding kinetics and functional properties of the herein generated four leading bispecific IL-18 mimetics.

5273

5274 Example 5

5275 IL-18 cytokine mimetics with engineered antibody formats

5276 Next, we also set out to further augment the functional properties of the herein generated

5277 bispecific surrogate agonists. To this end, we focused on IL18R_VHHa2pi5 that triggered the

5278 most prominent production of IFN-y on human PBMCs. In particular, we aimed at investigating

5279 the influence of paratope valencies as well as the spatial orientation of individual paratopes

5280 within the overall antibody design architecture on triggering a functional IFN-y response. The

5281 different antibody designs are shown in Fig. 4A. Besides the strictly monovalent (1+1) VHH

5282 SEED format, that was exploited for initial IL- 18 mimetic generation and characterization

5283 (IL18R_VHHa2pi5), we employed two additional designs. In 2020, our group described a

5284 VHH-based IgG-like bi- and multispecific antibody platform that relies on the replacement of

5285 the VH and the VL regions of a conventional antibody by two independently functioning VHH

5286 domains, resulting in a bispecific, tetravalent antibody derivative, herein referred to as single

5287 domain-based IgG (sdlgG) (Pekar et al., 2020). We engrafted the IL-18Ra-specific VHHa2

5288 onto the CHI domain of the effector silenced heavy chain (IgGl) and the IL-18RP-targeting

5289 VHHP15 onto the constant region of the lambda light chain (IL18R_sdIgGa2pi5). In addition

5290 to this IgG-like design, we also constructed tandem- VHH arrangements, grafted onto the hinge¬

5291 region of an effector-silenced IgGl Fc region (2+2). The two independent VHH domains were

5292 separated by a five amino acid Gly4Ser-linker. For this, we assessed both orientations, VHHa2

5293 followed by VHHP15 (from N-terminus to C-terminus, IL18R_tanVHHa2pi5) and vice versa

5294 (IL18R_tanVHHpi5a2). In addition to these four molecules in total (including the initial 1+1

5295 SEED design), all formats were also produced harboring the E340G mutation, that was first

5296 described by Parren and colleagues (de Jong et al., 2016). This mutation enhances antibody

5297 hexamer formation on the target cell surface after antigen binding and was initially utilized to

5298 induce conditional complement-dependent cytotoxicity. In this regard, we speculated that on-

5299 target hexamerization of the different surrogate agonist formats would result into enhanced

5300 receptor clustering and consequently into an improved IFN-y response.

5301

5302 Expression yields after protein A purification for the different designs ranged from 12 mg/L for

5303 IL18R_sdIgGa2pi5_E430G to up to 375.2 mg/L for IL18R_tanVHHa2pi5 (Table 2).

5304 Generally, we observed a trend towards lower expression for molecules harboring the E430G

5305 mutation. Interestingly, the different formats comprising the VHH tandem arrangements

5306 without the E430G amino acid exchanges displayed expression yields of more than 300 mg/L, 5307 indicating high production profiles for transient antibody expression. Most of the different

5308 bispecific IL-18 mimetic designs also showed quite favorable aggregation properties as

5309 indicated by SEC profiles above 90% target peak post protein A purification. Only for two

5310 molecules with implemented E430G mutations (IL18R_VHHa2pi5_E430G and

5311 IL18R_sdIgGa2pi5_E430G), we observed SEC purities of 79.3% and 82.6%, respectively.

5312 Hence, both molecules were further polished by preparative SEC, yielding final purities of

5313 97.3% and 98.3% (Fig. 10). Additionally, also the thermal stabilities of the engineered designs

5314 were monitored by measuring the tonset that is the lowest temperature at which a protein starts

5315 to unfold. Generally, all molecules not harboring the E430G exchanges showed thermal

5316 stabilities (tonsets) above 50 °C, indicating favorable biophysical properties, whereas all designs

5317 with incorporated E430G mutations showed diminished stabilities by 4 °C - 8.6 °C (Table 2,

5318 Fig. 11)

5319

5320

21 Table 2: Biophysical, biochemical and functional attributes of engineered cytokine mimetic formats.

22 *only for IL18R_VHHa2pi5_E430G and IL18R_sdIgGa2pi5_E430G preparative SEC was applied for polishing.

5323 Subsequently, all different antibody designs based on VHHa2 and VHHpi5 were analyzed by

5324 means of triggering IFN-y production on human PBMCs isolated from six different donors in

5325 total. The different formats were used at two different concentrations (10 nM and 1 nM).

5326 Intriguingly, profound differences were unveiled for the different design architectures (Fig.

5327 4B) The engineered sdlgG-based cytokine mimetic (IL18R_sdIgGa2pi5) did not induce a

5328 substantially improved IFN-y response compared to the initial (1+1) SEED design termed

5329 IL18R_VHHa2pi5 (332.1 pg/ml and 376.1 pg/ml for IL18R_sdIgGa2pi5 at 10 nM and 1 nM,

5330 respectively vs 418.2 pg/ml and 115.1 pg/ml). Hence, we de-prioritized this format for further

5331 analysis. In contrast to this, stimulation of PBMCs with both tandem arrangements

5332 IL18R_tanVHHpi5a2 and IL18R_tanVHHa2pi5 provoked a significantly augmented IFN-y

5333 production with IL18R_tanVHHa2pi5 eliciting the strongest release (1116.5 pg/ml and 1238.2

5334 pg/ml at 10 nM and 1 nM, respectively) within the set of engineered IL- 18 mimetic

5335 architectures. In general, implementation of the E430G amino acid exchanges did not seem to

5336 improve IFN-y release. Consequently, we also discarded all formats harboring the E430G

5337 mutation for additional characterization.

5338

5339 Both formats comprising the VHH arranged in tandem (IL18R_tanVHHpi5a2 and

5340 IL18R_tanVHHa2pi5) as well as the initial 1+1 SEED design (IL18R_ VHHa2pi5) were

5341 directly compared to (rh) IL- 18 regarding their potential to elicit a functional IFN-y response

5342 (Fig. 4C, Table 2). In comparison with (rh) IL-18, the capacity of IL18R_VHHa2pi5 to trigger

5343 IFN-y release on PBMCS was clearly attenuated (ECso of 2.4 nM for IL18R_VHHa2pi5 v.s

5344 61 pM for (rh) IL-18).

5345

5346 In contrast to this, the potential to evoke IFN-y production of both mimetics harboring the

5347 tandem arrangements were substantially augmented (ECso of 16 pM for IL18R_tanVHHpi5a2

5348 and 6 pM for IL 18R_tanVHHa2p 15), resulting in molecules that were significantly more potent

5349 than (rh) IL-18. While potencies were fairly similar between both tandem formats, the

5350 magnitude IFN-y release was quite different. In this regard, IL18R_tanVHHa2pi5 caused a

5351 maximum release that was quite similar to (rh)IL-18 (2130 pg/ml vs 2097 pg/ml), whereas

5352 IL18R_tanVHHpi5a2 elicited a significantly reduced maximum IFN-y production of 1095

5353 pg/ml. Of note, we observed a strong hooking effect (bell shaped curve) for (rh) IL-18, i.e.,

5354 reduced IFN-y release with high and increasing compound concentrations (Fig. 12). This effect

5355 was clearly not as pronounced for the different surrogate agonist formats tested.

5356 5357 To investigate whether biofunctional differences also translate into differential biochemical

5358 attributes, we performed BLI experiments that were aimed at evaluating target affinities as well

5359 as binding avidities of IL18R_VHHa2pi5, IL18R_tanVHHa2pi5 and IL18R_tanVHHpi5a2.

5360

5361 For determining affinities, bispecific cytokine mimetics were captured on the sensor tips and

5362 the respective IL-18 receptor subunits were exploited as analyte (Table 3). Compared with

5363 IL18R_VHHa2pi5, binding affinities for the inner paratopes of tandem IL-18 mimetics were

5364 slightly reduced, whereas kinetics for the outer sdAb remained largely unaffected.

5365

5366 To evaluate avidities, the respective receptor subunits were captured on the sensor tips and

5367 binding was assessed for the monovalent (for each target) bsAb IL18R_VHHa2pi5 as well as

5368 for the bivalent (for binding to each receptor chain) surrogate agonists IL18R_tanVHHa2pi5

5369 and IL18R_tanVHHpi5a2 (Table 3, Figure 13). In this experimental setting,

5370 IL18R_VHHa2pi5 displayed binding to both respective receptor chains in the lower double

5371 digit nanomolar range. In contrast to this, apparent binding affinities for both tandem IL-18

5372 mimetics could not be determined, which was driven by the fact that no dissociation from the

5373 receptor subunits was observed. This is giving strong evidence for high avidity binding of both

5374 tandem surrogate agonists.

75 Table 3: Apparent binding affinities of selected surrogate agonists in an affinity driven BLI experiment and in an avidity driven setting.

76 77

Example 6

Bispecific IL-18 mimetics are resistant to inhibition by IL-18BP

Finally, we wanted to analyze whether the herein generated IL-18 mimetics are inhibited by IL- 18BP receptor decoy. To this end, we employed the initially generated TOP4 cytokine mimetics, IL18R_VHHa2pi5, IL18R_VHHa2pi7, IL18R_VHHa8pi7 and IL18R_VHHa8pi5 as well as both tandem-engineered surrogate agonists IL18R_tanVHHpi5a2 and IL18R_tanVHHpi5a2 (which are format engineered derivatives of IL18R_VHHa2pi5). As determined by BLI, no binding interaction of (rh) IL-18BP was measurable to any of the IL-18 mimetics exploited (Fig. 14A). Opposed to this, (rh) IL-18BP showed high-affinity binding to (rh)IL-18 in the picomolar range (Fig. 14B). Both, IL-18BP and IL-18Ra bind to IL-18 at an overlapping interface (Zhou et al., 2020). Of note, all six surrogate agonists showed competitive binding with (rh) IL-18 for targeting IL-18Ra, indicating a similar or at least a partially overlapping epitope on IL-18Ra (Fig. 15 and Table 1).

Ultimately, we evaluated the resistance of the engineered IL-18 mimetics to IL-18BP inhibition on PBMC stimulation. Therefore, we focused on both tandem IL-18 mimetics, showing potencies at a similar level as (rh)IL-18 in provoking an IFN-y response. Both cytokine mimetics as well as (rh)IL-18 were utilized at a fixed concentration of 0.5 nM combined with low dose (rh)IL-12 (10 ng/ml) and increasing concentrations of (rh)IL-18BP were titrated (Fig. 4D). Contrary to (rh)IL18 that was efficiently inhibited by (rh)IL-18BP (IC50 of 2.2 nM), both tandem IL-18 mimetics, IL18R_tanVHHpi5a2 and IL18R_tanVHHpi5a2 triggered a robust IFN-y release, regardless of the IL-18BP receptor decoy concentration tested.

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Claims

1. A compound comprising

(a) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHH1, VHH2, VHH5, VHH6, VHH8, VHH9 and VHH10 as shown in the Table of CDRs;

- the replacement, addition or deletion of up to three amino acids in CDR1,

- the replacement, addition or deletion of up to three amino acids in CDR3;

(d) said VHH antibody domain or fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 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 CDRs, wherein said VHH antibody domain or fragment thereof defined in (d) is selected as follows: if the VHH antibody domain of (a) or the fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH1, then the VHH antibody domain of (d) or the fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH 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 of (a) or the fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH2, then the VHH antibody domain of (d) or the fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH 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 of (a) or the fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH5, then the VHH antibody domain of (d) or the fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHH13 and VHH14 as shown in the Table of CDRs; if the VHH antibody domain of (a) or the fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH6, then the VHH antibody domain of (d) or the fragment thereof comprises 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 of (a) or the fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH8, then the VHH antibody domain of (d) or the fragment thereof comprises 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 of (a) or the fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH9, then the VHH antibody domain of (d) or the fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH 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 of (a) or the fragment thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH10, then the VHH antibody domain of (d) or the fragment thereof comprises 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;

- the replacement, addition or deletion of up to one amino acid in CDR1 ,

- the replacement, addition or deletion of up to one amino acid in CDR2 and/or

- the replacement, addition or deletion of up to one amino acid in CDR3;

Table of CDRs:

2. The compound according to claim 1, wherein said VHH antibody domain or fragment thereof defined in (a) comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHH2 and VHH8 as shown in the Table of CDRs and wherein said VHH antibody domain or fragment thereof defined in (d) comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH selected from the group consisting of VHH15 and VHH17 as shown in the Table of CDRs.

3. The compound according to claim 1, wherein said VHH antibody domain or fragment thereof defined in (a) comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH2 as shown in the Table of CDRs and wherein said VHH antibody domain or fragment thereof defined in (d) comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH15 as shown in the Table of CDRs.

4. A compound comprising

(A) said VHH antibody domain comprises the amino acid sequence of a VHH selected from the group consisting of VHH1, VHH2, VHH5, VHH6, VHH8, VHH9 and VHH10 as shown in the Table of VHH Sequences;

(E) said VHH antibody domain comprises the amino acid sequence of a 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 said VHH antibody domain of (E) is selected as follows: if the VHH antibody domain of (A) comprises the amino acid sequence of VHH1, then the VHH antibody domain of (E) comprises the amino acid sequence of one VHH selected from the group consisting of VHH12, VHH13, VHH15, VHH17, VHH20, VHH21 and VHH22 shown in the Table of VHH Sequences; if the VHH antibody domain of (A) comprises the amino acid sequence of VHH2, then the VHH antibody domain of (E) comprises the amino acid sequence of one VHH selected from the group consisting of VHH12, VHH13, VHH15, VHH17, VHH18, VHH20, VHH21 and VHH22 shown in the Table of VHH Sequences; if the VHH antibody domain of (A) comprises the amino acid sequence of VHH5, then the VHH antibody domain of (E) comprises the amino acid sequence of one VHH selected from the group consisting of VHH13 and VHH14 shown in the Table of VHH Sequences; if the VHH antibody domain of (A) comprises the amino acid sequence of VHH6, then the VHH antibody domain of (E) comprises the amino acid sequence of one VHH selected from the group consisting of VHH17, VHH20, VHH21 and VHH22 shown in the Table of VHH Sequences; if the VHH antibody domain of (A) comprises the amino acid sequence of VHH8, then the VHH antibody domain of (E) comprises the amino acid sequence of one VHH selected from the group consisting of VHH12, VHH13, VHH17, VHH18, VHH20 and VHH21 shown in the Table of VHH Sequences; if the VHH antibody domain of (A) comprises the amino acid sequence of VHH9, then the VHH antibody domain of (E) comprises the amino acid sequence of one VHH selected from the group consisting of VHH12, VHH13, VHH15, VHH17, VHH18, VHH20, VHH21 and VHH22 shown in the Table of VHH Sequences; if the VHH antibody domain of (A) comprises the amino acid sequence of VHH10, then the VHH antibody domain of (E) comprises the amino acid sequence of one VHH selected from the group consisting of VHH13, VHH20 and VHH21 shown in the Table of VHH Sequences;

(G) said VHH antibody domain comprises a VHH sequence as defined in (E) with modification, wherein the modification is the replacement, addition or deletion of up to 10 amino acids; or

(H) said VHH antibody domain comprises a VHH sequence that is at least 90% identical to a VHH sequence referred to in (E);

Table of VHH Sequences:

5. The compound according to claim 4, wherein said VHH antibody domain of (A) comprises the amino acid sequence of one VHH selected from the group consisting of VHH2 and VHH8 shown in the Table of VHH Sequences and wherein said VHH antibody domain of (E) comprises the amino acid sequence of one VHH selected from the group consisting of VHH15 and VHH17 shown in the Table of VHH Sequences.

6. The compound according to claim 4, wherein said VHH antibody domain of (A) comprises the amino acid sequence of VHH2 shown in the Table of VHH Sequences and wherein said VHH antibody domain of (E) comprises the amino acid sequence of VHH15 shown in the Table of VHH Sequences.

7. The compound according to any one of claims 1 to 6, wherein said compound is a bispecific antibody molecule prepared by the SEED technology.

8. The compound according to any one of claims 1 to 6, wherein said compound is a single domain-based IgG (sdlgG), wherein said sdlgG is a conventional IgG antibody in which each of the two VH regions is replaced by a copy of said first binding module, and each of the two VL regions is replaced by a copy of said second binding module, resulting in a bispecific, tetravalent antibody.

9. The compound according to any one of claims 1 to 6, wherein said compound consists of a IgG Fc region to which two copies of a tandem -VHH arrangement are covalently linked at the hinge-region, wherein said tandem-VHH arrangement consists of a first VHH antibody domain that is covalently linked to a second VHH antibody domain, wherein said second VHH antibody domain is covalently linked to the hinge region of said Fc region, resulting in a bispecific, tetravalent compound.

10. The compound according to any one of claims 1 to 9, wherein said compound binds to IL-18Ra ECD with a KD of 100 nM or stronger.

11. The compound according to any one of claims 1 to 10, wherein said compound binds to IL-18RP ECD with a KD of 100 nM or stronger.

12. The compound according to any one of claims 1 to 11, wherein said compound is capable of activating the IL-18 receptor IL-18R by binding to IL-18Ra and IL-18RP.

13. The compound according to any one of claims 1 to 12, wherein said compound is capable of triggering dose-dependent IL-18R downstream signaling on IL- 18 reporter cells.

14. The compound according to any one of claims 1 to 13, wherein said compound is capable of inducing IFN-y release by peripheral blood mononuclear cells (PBMCs) in the presence of low-dose IL-12 (10 ng/ml).

15. A pharmaceutical composition comprising the compound according to any one of claims 1 to 14.