WO2024251983A1 - Mimétiques d'il-18 - Google Patents

Mimétiques d'il-18 Download PDF

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Publication number
WO2024251983A1
WO2024251983A1 PCT/EP2024/065796 EP2024065796W WO2024251983A1 WO 2024251983 A1 WO2024251983 A1 WO 2024251983A1 EP 2024065796 W EP2024065796 W EP 2024065796W WO 2024251983 A1 WO2024251983 A1 WO 2024251983A1
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vhh
antibody domain
fragment
vhh antibody
sequence
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Stefan ZIELONKA
Lukas PEKAR
Paul ARRAS
Britta LIPINSKI
Laura UNMUTH
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Merck Patent GmbH
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Merck Patent GmbH
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Priority to CN202480051902.XA priority Critical patent/CN121666402A/zh
Priority to AU2024286186A priority patent/AU2024286186A1/en
Publication of WO2024251983A1 publication Critical patent/WO2024251983A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • 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.
  • 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).
  • cytokine fusion proteins 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).
  • Tegs regulatory T cells
  • 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).
  • 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).
  • cytokine mimetics or surrogate cytokines
  • bispecific antibody derivatives diabodies
  • EpoR Erythropoietin receptor
  • single domain antibody (sdAb)-based bispecifics were generated that activate signalling through the heterodimeric IL- 2 receptor Py (IL-2RPy).
  • sdAbs single domain antibody
  • bsAbs bispecific antibodies
  • bsAbs mimic the function of IL-2 but without preferential activation of Tregs via IL-2 receptor a binding
  • 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).
  • the group constructed surrogate agonists displaying functional diversification in terms of receptor downstream signalling compared to the natural cytokine.
  • 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).
  • IL-18BP high- affinity neutralizing and naturally occurring IL-18 binding protein
  • 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.
  • IL-18BP IL-18BP decoy receptor
  • 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.
  • 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).
  • IL-18 receptor agonists 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.
  • 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.
  • sdAb single-domain antibody
  • the present disclosure relates to a VHH antibody domain or a fragment thereof, wherein
  • 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;
  • 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
  • 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 present disclosure relates to a VHH antibody domain or a fragment thereof, wherein
  • 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;
  • VHH antibody domain comprises a VHH sequence as defined in (A) with modification, wherein the modification is that said sequence is humanized;
  • 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;
  • said VHH antibody domain comprises a VHH sequence that is at least 75% identical to a VHH sequence referred to in (A);
  • the present disclosure relates to a VHH antibody domain or a fragment thereof, wherein
  • 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;
  • VHH antibody domain consists of a VHH sequence as defined in (A) with modification, wherein the modification is that said sequence is humanized;
  • 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;
  • VHH antibody domain consists of a VHH sequence that is at least 75% identical to a VHH sequence referred to in (A).
  • present disclosure relates to a VHH antibody domain or a fragment thereof, wherein
  • 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;
  • 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
  • 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 present disclosure relates to a VHH antibody domain or a fragment thereof, wherein
  • 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;
  • said VHH antibody domain comprises a VHH sequence as defined in (E) with modification, wherein the modification is that said sequence is humanized;
  • 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;
  • said VHH antibody domain comprises a VHH sequence that is at least 75% identical to a VHH sequence referred to in (E).
  • 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;
  • VHH antibody domain consists of a VHH sequence as defined in (E) with modification, wherein the modification is that said sequence is humanized;
  • 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;
  • said VHH antibody domain consists of a VHH sequence that is at least 75% identical to a VHH sequence referred to in (E).
  • the present disclosure relates to a compound comprising
  • VHH antibody domain or fragment thereof according to any one of embodiments 1 to
  • the present disclosure relates to a compound comprising
  • VHH antibody domain or fragment thereof according to any one of claims 189 to 374.
  • the present disclosure relates to a compound comprising
  • 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;
  • 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
  • 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
  • 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;
  • 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
  • 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 present disclosure relates to a compound comprising
  • 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;
  • 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
  • said VHH antibody domain comprises a VHH sequence that is at least 75%
  • VHH antibody domain comprises the amino acid sequence of a VHH
  • VHH12 selected from the group consisting of VHH12, VHH13, VHH14, VHH15,
  • VHH16, VHH17, VHH18, VHH20, VHH21 and VHH22 as shown in the
  • VHH antibody domain comprises a VHH sequence as defined in (E) with
  • said VHH antibody domain comprises a VHH sequence as defined in (E) with
  • said VHH antibody domain comprises a VHH sequence that is at least 75%
  • the present disclosure relates to a compound comprising
  • VHH antibody domain consists of the amino acid sequence of a VHH
  • VHH6, VHH8, VHH9, VHH10 and VHH11 as shown in the Table of VHH
  • VHH antibody domain consists of a VHH sequence as defined in (A)
  • VHH antibody domain consists of a VHH sequence as defined in (A)
  • VHH antibody domain consists of a VHH sequence that is at least 75%
  • 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;
  • VHH antibody domain consists of a VHH sequence as defined in (E) with modification, wherein the modification is that said sequence is humanized;
  • 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;
  • said VHH antibody domain consists of a VHH sequence that is at least 75% identical to a VHH sequence referred to in (E).
  • 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).
  • the present disclosure relates to a bispecific antibody molecule prepared by the SEED (strand-exchange engineered domain) technology, said molecule comprising
  • VHH-SEED-AG-1 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
  • VHH-SEED-GA-1 amino acid sequence as defined in the present disclosure below
  • the present disclosure relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the compound or bispecific antibody molecule according to the present disclosure.
  • 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.
  • 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.
  • IL-18 blue, ⁇
  • IL-18Ra yellow, *; the two "lobes" of IL-18Ra are each labelled with *
  • IL-18RP lime, #
  • IL-18BP dark grey, $
  • 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, $).
  • 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-BlueTM 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.
  • 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.
  • VHHa2 and VHHpi5 respectively facilitates the generation of the IL18R_sdIgGa2pi5 architecture (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.
  • the opposite orientation was constructed (VHHpi5 followed by VHHa2, IL18R_tanVHHpi5a2).
  • (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.
  • 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 5xl0 3 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).
  • 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-BlueTM 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.
  • IL18R_VHHa2pi5 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.
  • 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.
  • 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.
  • FIG 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.
  • BBI biolayer interferometry
  • 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).
  • 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.
  • the present disclosure relates to a VHH antibody domain or a fragment thereof, wherein
  • 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;
  • 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
  • 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
  • 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.
  • a heavy and the light chain variable domain of an antibody combine to specifically bind the antigen.
  • a naturally occurring primate e.g., human
  • murine immunoglobulin has heavy (H) chains and light (L) chains interconnected by disulfide bonds.
  • Primate antibodies can be class switched.
  • IgG subtypes of the llamas lack the light chains and the CHI domain and are called heavy chain antibodies.
  • 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”.
  • VHH variable domain of a heavy chain antibody
  • VHH 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).
  • CDRs complementarity determining regions
  • 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.
  • a VH CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found
  • 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.
  • 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).
  • 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.
  • a VHH antibody domain can be a molecule of its own that is not covalently linked to any other molecular structure.
  • 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.
  • antibodies such as primate antibodies
  • 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.
  • 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).
  • 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.
  • monoclonal antibodies can be chimeric antibodies.
  • 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.
  • VHH monoclonal antibody 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.
  • 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.
  • Humanizing an antib ody/antibody sequence 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • the hydrophobic patches at the cell surface can be reduced by exchanging the respective amino acid(s) with less hydrophobic amino acid(s).
  • 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.
  • a certain protein/amino acid sequence A is a "fragment" of another protein/amino acid sequence 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.
  • 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.
  • VHH antibody domain when the present disclosure refers to an "VHH antibody domain or a fragment thereof', said fragment is an antigen-binding fragment.
  • 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).
  • 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.
  • 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)
  • 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.
  • said VHH antibody domain or fragment thereof includes e.g.
  • 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.
  • 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.
  • the replaced, added or deleted amino acids may be at neighboring positions or at independent, isolated positions within the amino acid sequence.
  • 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,
  • Embodiment 13 The VHH antibody domain or fragment thereof according to any one of embodiments 1 to 11, wherein the modification in (c) is
  • Embodiment 14 The VHH antibody domain or fragment thereof according to any one of embodiments 1 to 11, wherein the modification in (c) is
  • 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 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
  • Embodiment 17 The VHH antibody domain or fragment thereof according to any one of embodiments 1 to 11, wherein the modification in (c) is
  • Embodiment 18 The VHH antibody domain or fragment thereof according to any one of embodiments 1 to 11, wherein the modification in (c) is
  • Embodiment 19 The VHH antibody domain or fragment thereof according to any one of embodiments 1 to 11, wherein the modification in (c) is
  • Embodiment 20 The VHH antibody domain or fragment thereof according to any one of embodiments 1 to 11, wherein the modification in (c) is
  • Embodiment 21 The VHH antibody domain or fragment thereof according to any one of embodiments 1 to 11, wherein the modification in (c) is
  • 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.
  • 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
  • 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;
  • VHH antibody domain comprises a VHH sequence as defined in (A) with modification, wherein the modification is that said sequence is humanized;
  • 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;
  • said VHH antibody domain comprises a VHH sequence that is at least 75% identical to a VHH sequence referred to in (A);
  • the present disclosure relates to a VHH antibody domain or a fragment thereof, wherein
  • 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;
  • VHH antibody domain consists of a VHH sequence as defined in (A) with modification, wherein the modification is that said sequence is humanized;
  • 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;
  • said VHH antibody domain consists of a VHH sequence that is at least 75% identical to a VHH sequence referred to in (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.
  • 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"
  • modification is that said sequence is humanized
  • sequence A is at least x % identical to another sequence B
  • sequence A has x % identity
  • the statement reflects a relationship between the two polypeptide sequences A and B determined by comparing the sequences.
  • 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.
  • a percentage to which the two sequences are identical may be determined.
  • 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.
  • 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
  • FASTA Pearson WR, 1990
  • % 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.
  • 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.
  • Embodiment 31 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 32 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 33 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 34 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 35 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 36 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 37 The VHH antibody domain or fragment thereof according to any one of
  • 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
  • VHH antibody domain or fragment thereof according to any one of
  • Embodiment 39 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 40 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 41 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 42 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 43 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 44 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 45 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 46 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 47 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 48 The VHH antibody domain or fragment thereof according to any one of
  • 1052 antibody domain is within CDR1, CDR2 and/or CDR3.
  • Embodiment 49 The VHH antibody domain or fragment thereof according to any one of
  • VHH antibody domain is within CDR1 and/or CDR2.
  • Embodiment 50 The VHH antibody domain or fragment thereof according to any one of
  • VHH antibody domain is within CDR1.
  • VHH antibody domain or fragment thereof according to any one of
  • VHH antibody domain or fragment thereof according to any one of
  • 1068 antibody domain is not within CDR3.
  • Embodiment 53 The VHH antibody domain or fragment thereof according to any one of
  • VHH antibody domain or fragment thereof according to any one of
  • VHH antibody domain or fragment thereof according to any one of
  • Embodiment 56 The VHH antibody domain or fragment thereof according to any one of
  • VHH antibody domain or fragment thereof according to any one of
  • VHH antibody domain or fragment thereof according to any one of
  • Embodiment 59 The VHH antibody domain or fragment thereof according to any one of
  • VHH antibody domain or fragment thereof according to any one of
  • Embodiment 61 The VHH antibody domain or fragment thereof according to any one of
  • 1104 comprises the complementarity determining regions CDR1, CDR2 and CDR3 of one VHH
  • VHH9, VHH10 and VHH11 as shown in the Table of CDRs.
  • Embodiment 62 The VHH antibody domain or fragment thereof according to any one of
  • VHH antibody domain or fragment thereof comprises the complementarity
  • VHH1 consisting of VHH1, VHH2, VHH3, VHH4, VHH5, VHH6, VHH8, VHH9, VHH10
  • VHH antibody domain or fragment thereof comprises the complementarity
  • VHH antibody domain or fragment thereof comprises the complementarity
  • Embodiment 63 The VHH antibody domain or fragment thereof according to embodiment 62,
  • Embodiment 64 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 65 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 66 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 67 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 68 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 69 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 70 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 71 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 72 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 73 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 74 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 75 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 76 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 77 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 78 The VHH antibody domain or fragment thereof according to any one of
  • unmodified means unmodified compared to the sequence in
  • Embodiment 79 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 80 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 81 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 82 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 83 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 84 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 85 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 86 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 87 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 88 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 89 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 90 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 91 The VHH antibody domain or fragment thereof according to any one of
  • VHH antibody domain is an anti-IL-18Ra VHH antibody
  • Embodiment 92 The VHH antibody domain or fragment thereof according to any one of
  • VHH antibody domain is specific for IL-18Ra.
  • Embodiment 93 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 94 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 95 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 96 The VHH antibody domain or fragment thereof according to any one of
  • a first molecule/molecular group e.g.
  • an antibody/antibody component "is capable of specifically binding"/" specifically binds" to a
  • molecule/molecular group e.g. an antigen of interest
  • a first molecule/molecular group that "specifically binds" to a second molecule/molecular group that "specifically binds" to a second molecule/molecular group
  • 1300 molecule/molecular group may bind to that second molecule/molecular group with a KD of
  • Embodiment 97 The VHH antibody domain or fragment thereof according to any one of
  • VHH antibody domain or fragment thereof binds to
  • Embodiment 98 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 99 The VHH antibody domain or fragment thereof according to any one of
  • VHH antibody domain or fragment thereof binds to
  • Embodiment 100 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 101 The VHH antibody domain or fragment thereof according to any one of
  • VHH antibody domain or fragment thereof binds to
  • Embodiment 102 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 103 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 104 The VHH antibody domain or fragment thereof according to any one of
  • VHH antibody domain binds to recombinant human IL-18Ra ECD.
  • Embodiment 105 The VHH antibody domain or fragment thereof according to any one of
  • VHH2 SEQ ID NO: 2
  • IL-18Ra ECD 1339 with which VHH2 (SEQ ID NO: 2) binds to IL-18Ra ECD or stronger.
  • Embodiment 106 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 107 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 108 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 109 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 110 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 111 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 112 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 113 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 114 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 115 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 116 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 117 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 118 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 119 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 120 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 121 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 122 The VHH antibody domain or fragment thereof according to any one of
  • the degree of sequence identity can be determined by sequence alignment.
  • Embodiment 123 The VHH antibody domain or fragment thereof according to any one of
  • the degree of sequence identity can be determined by sequence alignment.
  • Embodiment 124 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 125 The VHH antibody domain or fragment thereof according to any one of
  • VHH antibody domain consisting of the sequence from the Table of VHH Sequences that has
  • Embodiment 126 The VHH antibody domain or fragment thereof according to any one of
  • VHH antibody domain consisting of the sequence from the Table of VHH Sequences that has
  • Embodiment 127 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 128 The VHH antibody domain or fragment thereof according to any one of
  • 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
  • Embodiment 129 The VHH antibody domain or fragment thereof according to any one of
  • VHH antibody domain consisting of the sequence from the Table of VHH Sequences that has
  • Embodiment 130 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 131 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 132 The VHH antibody domain or fragment thereof according to any one of
  • VHH antibody domain of (A) comprises one VHH
  • VHH8, VHH9 and VHH10 shown in the Table of VHH Sequences.
  • Embodiment 133 The VHH antibody domain or fragment thereof according to any one of
  • VHH antibody domain of (A) comprises one VHH
  • Embodiment 134 The VHH antibody domain or fragment thereof according to any one of
  • VHH antibody domain of (A) comprises one VHH
  • VHH2 1511 sequence selected from the group consisting of VHH2, VHH6, VHH8 and VHH10 shown in
  • Embodiment 135 The VHH antibody domain or fragment thereof according to any one of
  • VHH antibody domain of (A) comprises one VHH
  • VHH6 1516 sequence selected from the group consisting of VHH6 and VHHl 1 shown in the Table of VHH
  • Embodiment 136 The VHH antibody domain or fragment thereof according to any one of
  • VHH antibody domain of (A) comprises one VHH
  • VHHl 1521 sequence selected from the group consisting of VHHl, VHH2, VHH5, VHH6, VHH8 and
  • Embodiment 137 The VHH antibody domain or fragment thereof according to any one of
  • VHH antibody domain of (A) comprises one VHH
  • Embodiment 138 The VHH antibody domain or fragment thereof according to any one of
  • VHH antibody domain of (A) comprises one VHH
  • VHH2 1531 sequence selected from the group consisting of VHH2 and VHH8 shown in the Table of VHH
  • Embodiment 139 The VHH antibody domain or fragment thereof according to any one of
  • VHH antibody domain of (A) comprises the VHH
  • Embodiment 140 The VHH antibody domain or fragment thereof according to any one of
  • VHH antibody domain of (A) comprises the VHH
  • Embodiment 141 The VHH antibody domain or fragment thereof according to any one of
  • VHH antibody domain of (A) comprises the VHH
  • Embodiment 142 The VHH antibody domain or fragment thereof according to any one of
  • VHH antibody domain of (A) comprises the VHH
  • Embodiment 143 The VHH antibody domain or fragment thereof according to any one of
  • VHH antibody domain of (A) comprises the VHH
  • Embodiment 144 The VHH antibody domain or fragment thereof according to any one of
  • VHH antibody domain of (A) comprises the VHH
  • Embodiment 145 The VHH antibody domain or fragment thereof according to any one of
  • VHH antibody domain of (A) comprises the VHH
  • Embodiment 146 The VHH antibody domain or fragment thereof according to any one of
  • VHH antibody domain of (A) comprises the VHH
  • Embodiment 147 The VHH antibody domain or fragment thereof according to any one of
  • VHH antibody domain of (A) comprises the VHH
  • Embodiment 148 The VHH antibody domain or fragment thereof according to any one of
  • VHH antibody domain of (A) comprises the VHH
  • Embodiment 149 The VHH antibody domain or fragment thereof according to any one of
  • VHH selected from the group consisting of VHH1, VHH2, VHH3, VHH4, VHH5, VHH6,
  • Embodiment 150 The VHH antibody domain or fragment thereof according to any one of
  • VHH selected from the group consisting of VHH1, VHH2, VHH5, VHH6, VHH8, VHH9 and
  • Embodiment 151 The VHH antibody domain or fragment thereof according to any one of
  • VHH2, VHH6, VHH8 and VHH10 1589 VHH2, VHH6, VHH8 and VHH10 as shown in the Table of CDRs.
  • Embodiment 152 The VHH antibody domain or fragment thereof according to any one of
  • VHH selected from the group consisting of VHH6 and VHH11.
  • Embodiment 153 The VHH antibody domain or fragment thereof according to any one of
  • VHH selected from the group consisting of VHH1, VHH2, VHH5, VHH6, VHH8 and VHH10
  • Embodiment 154 The VHH antibody domain or fragment thereof according to any one of
  • VHH selected from the group consisting of VHH2, VHH6, VHH8, VHH10 as shown in the
  • Embodiment 155 The VHH antibody domain or fragment thereof according to any one of
  • VHH selected from the group consisting of VHH2 or VHH8 as shown in the Table of CDRs.
  • Embodiment 156 The VHH antibody domain or fragment thereof according to any one of
  • VHH1 comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH1
  • Embodiment 157 The VHH antibody domain or fragment thereof according to any one of
  • VHH2 comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH2
  • Embodiment 158 The VHH antibody domain or fragment thereof according to any one of
  • 1625 thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH3
  • Embodiment 159 The VHH antibody domain or fragment thereof according to any one of
  • 1630 thereof comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH4
  • Embodiment 160 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 161 The VHH antibody domain or fragment thereof according to any one of
  • VHH6 comprises the complementarity determining regions CDR1, CDR2 and CDR3 of VHH6
  • Embodiment 162 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 163 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 164 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 165 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 166 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 167 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 168 The VHH antibody domain or fragment thereof according to any one of
  • VHH antibody domain or fragment thereof does not
  • Embodiment 169 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 170 The VHH antibody domain or fragment thereof according to embodiment
  • Embodiment 171 The VHH antibody domain or fragment thereof according to embodiment
  • Embodiment 172 The VHH antibody domain or fragment thereof according to any one of
  • 1702 humanization refers to a VHH antibody domain that differs only by the amino acid changes
  • Embodiment 173 The VHH antibody domain or fragment thereof according to any one of
  • 1707 humanization binds to human IL-18Ra ECD with an affinity (KD value) that is not weaker by
  • Embodiment 174 The VHH antibody domain or fragment thereof according to any one of
  • 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
  • Embodiment 175 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 176 The VHH antibody domain or fragment thereof according to any one of
  • Embodiment 177 The VHH antibody domain or fragment thereof according to any one of
  • VHH antibody domain comprises a VHH antibody
  • 1728 domain according to (A), (B) or (C)/said fragment of a VHH antibody domain comprises a
  • Embodiment 178 The VHH antibody domain or fragment thereof according to any one of
  • VHH antibody domain comprises a VHH antibody
  • 1733 domain according to (A), (B) or (D)/said fragment of a VHH antibody domain comprises a
  • Embodiment 179 The VHH antibody domain or fragment thereof according to any one of
  • VHH antibody domain comprises a VHH antibody
  • 1738 domain according to (A), (C) or (D)/said fragment of a VHH antibody domain comprises a
  • Embodiment 180 The VHH antibody domain or fragment thereof according to any one of
  • VHH antibody domain comprises a VHH antibody
  • 1743 domain according to (A) or (B)/said fragment of a VHH antibody domain comprises a fragment
  • Embodiment 181 The VHH antibody domain or fragment thereof according to any one of
  • VHH antibody domain comprises a VHH antibody 1748 domain according to (A) or (C)/said fragment of a VHH antibody domain comprises a fragment
  • Embodiment 182 The VHH antibody domain or fragment thereof according to any one of
  • VHH antibody domain comprises a VHH antibody
  • 1753 domain according to (A) or (D)/said fragment of a VHH antibody domain comprises a fragment
  • Embodiment 183 The VHH antibody domain or fragment thereof according to any one of
  • VHH antibody domain comprises a VHH antibody
  • 1758 domain according to (A)/said fragment of a VHH antibody domain comprises a fragment of a
  • Embodiment 184 The VHH antibody domain or fragment thereof according to any one of
  • VHH antibody domain comprises a VHH antibody
  • 1763 domain according to (B)/said fragment of a VHH antibody domain comprises a fragment of a
  • Embodiment 185 The VHH antibody domain or fragment thereof according to any one of
  • 1768 comprises complementarity determining regions according to (a) or (b).

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  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Peptides Or Proteins (AREA)
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Abstract

La présente divulgation concerne de nouveaux composés de liaison à l'IL-18Rα et/ou l'IL-18Rβ à base de VHH présentant des caractéristiques favorables. De plus, la présente divulgation concerne des compositions pharmaceutiques comprenant un tel composé.
PCT/EP2024/065796 2023-06-09 2024-06-07 Mimétiques d'il-18 Pending WO2024251983A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN202480051902.XA CN121666402A (zh) 2023-06-09 2024-06-07 Il-18模拟物
AU2024286186A AU2024286186A1 (en) 2023-06-09 2024-06-07 Il-18 mimetics

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EP23178542.9 2023-06-09
EP23178542 2023-06-09

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WO2024251983A1 true WO2024251983A1 (fr) 2024-12-12

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AU (1) AU2024286186A1 (fr)
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