WO2023118497A1 - Nouveaux variants d'il-18 - Google Patents

Nouveaux variants d'il-18 Download PDF

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WO2023118497A1
WO2023118497A1 PCT/EP2022/087596 EP2022087596W WO2023118497A1 WO 2023118497 A1 WO2023118497 A1 WO 2023118497A1 EP 2022087596 W EP2022087596 W EP 2022087596W WO 2023118497 A1 WO2023118497 A1 WO 2023118497A1
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variant
amino acid
wild
mature human
human
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Martin SIEGEMUND
Stefan HAAK
Ting Su
Tim KÜKENSHÖNER
Lisa HEPTING
Stefan Grüner
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Pieris Pharmaceuticals Gmbh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/35—Fusion polypeptide containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin

Definitions

  • Interleukin-18 (IL-18; UniProt Q14116) is a proinflammatory cytokine that is primarily involved in type-1 T cell and natural killer (NK) cell immune responses. Upon binding to IL-18 receptor 1 (IL-18R1) and IL-18 receptor accessory protein (IL-18RAP), IL-18 forms a ternary signaling complex which activates NF-KB, triggering synthesis of inflammatory mediators. Furthermore, IL-18 synergizes with interleukin-12 (IL-12) to induce interferon-gamma (IFN-y) secretion from type-1 T cells and NK cells.
  • IL-18 is produced as an inactive precursor (pro-IL-18) that is enzymatically processed into a mature form by caspase-1 (Caspl).
  • Different cells such as macrophages, dendritic cells (DCs), microglial cells, synovial fibroblasts, and epithelial cells, express pro-IL-18, and the production of bioactive IL-18 is mainly regulated at the processing level.
  • the natural inhibitor of IL-18 is IL-18 binding protein (IL-18BP), whose production is enhanced by IFN-y and IL-27.
  • IL-18BP further regulates IL-18 activity in the extracellular environment.
  • IL-18 for cytokine-based therapeutic approaches, e.g., in the treatment of cancer, is hampered by toxicity risks and low therapeutic efficacy and precision, especially in the case of non-targeted systemic approaches. Accordingly, there is a need for variants of IL-18 with particular activity profiles that are, for example, suitable for targeted cytokine therapy.
  • interleukin- 18 or “IL-18” means human IL-18 (also referred to as hulL-18 herein).
  • Human IL-18 means a full-length protein defined by UniProt Q14116-1 (version 193 of September 29, 2021), a fragment thereof, or a naturally occurring variant thereof (e.g., isoform 2 lacking amino acid residues 27-30 with UniProt Q14116-2 and other naturally-occurring variant forms, such as alternatively spliced forms and naturally-occurring allelic variants).
  • Human IL-18 is encoded by the IL18 gene.
  • IL-18 of non-human species e.g., mouse IL-18, is used.
  • mature human IL-18 refers to the enzymatically processed, active form of hulL-18 lacking amino acid residues 1-36 of hulL-18 (referred to as “propeptide”).
  • propeptide The amino acid sequence of mature hulL-18 is shown in SEQ ID NO: 1.
  • IL-18 and mature IL- 18 may be used interchangeably herein.
  • interleukin-18 receptor 1 or “IL- 18R1” means human IL-18R1 (hulL-18R1).
  • Human IL-18R1 means a full-length protein defined by UniProt Q13478 (version 181 of September 29, 2021), a fragment thereof, or a naturally occurring variant thereof (e.g., alternatively spliced forms and naturally-occurring allelic variants).
  • Human IL-18R1 is encoded by the IL18R1 gene.
  • IL- 18R1 of non-human species e.g., mouse IL-18R1, is used.
  • the amino acid sequence of hulL- 18R1 is shown in SEQ ID NO: 2.
  • IL-18R1 refers to mature IL-18R1 lacking the signal peptide (amino acid residues 1-18).
  • IL-18 binding protein or “IL- 18BP” means human IL-18BP (hulL-18BP).
  • Human IL-18BP means a full-length protein defined by UniProt 095998 (version 172 of September 29, 2021), a fragment thereof, or a naturally occurring variant thereof (e.g., alternatively spliced forms and naturally-occurring allelic variants).
  • Human IL-18BP is encoded by the IL18BP gene.
  • IL- 18BP of non-human species e.g., mouse IL-18BP, is used.
  • the amino acid sequence of hulL- 18BP is shown in SEQ ID NO: 3.
  • IL-18BP refers to mature IL-18BP lacking the signal peptide (amino acid residues 1-30).
  • downstream signaling pathways of IL-18 refers to cellular signaling pathways activated by the binding of IL-18 to IL-18R1 , as described, e.g., in Rex et al., J Cell Commun Signal, 2020, 14(2): 257-266, which is incorporated herein by reference in its entirety.
  • the downstream signaling pathways of IL-18 comprise the activation of NF-KB and/or the secretion of IFN-y from T cells (e.g., CD4 + T cells) and NK cells.
  • binding affinity describes the ability of a biomolecule (e.g., a polypeptide or a protein) of the disclosure (e.g., an IL-18 variant, a fusion protein, or any other peptide or protein) to bind a selected target (and form a complex).
  • a biomolecule e.g., a polypeptide or a protein
  • the disclosure e.g., an IL-18 variant, a fusion protein, or any other peptide or protein
  • Binding affinity is measured by a number of methods known to those skilled in the art including, but not limited to, fluorescence titration, enzyme-linked immunosorbent assay (ELISA)-based assays, including direct and competitive ELISA, calorimetric methods, such as isothermal titration calorimetry (ITC), quartz crystal microbalance (QCM), bio-layer interferometry (BLI), and surface plasmon resonance (SPR). These methods are well-established in the art and some examples of such methods are further described herein. Binding affinity is thereby reported as a value of dissociation constant (KD), half maximal effective concentration (ECso), or half maximal inhibitory concentration (IC50) measured using such methods. A lower KD, EC50, or IC50 value reflects better (higher) binding ability (affinity).
  • KD dissociation constant
  • ECso half maximal effective concentration
  • IC50 half maximal inhibitory concentration
  • the term “detect,” “detection,” “detectable,” or “detecting” is understood both on a quantitative and a qualitative level, as well as a combination thereof. It thus includes quantitative, semi-quantitative, and qualitative measurements performed on a biomolecule of the disclosure.
  • “detectable affinity” generally means the binding ability between a biomolecule and its target, reported by a KD, EC50, or IC50 value, is at most about 10' 5 M or lower.
  • a binding affinity, reported by a KD, EC50, or IC50 value, higher than 10' 5 M is generally no longer measurable with common methods such as ELISA and SPR and is therefore of secondary importance.
  • “detectable affinity” may refer to a KD value of about 10 -5 M or lower as determined by ELISA or SPR, preferably SPR.
  • binding affinity reported by a KD, EC50, or IC50 value may vary within a certain experimental range, depending on the method and experimental setup. This means that there may be a slight deviation in the measured KD, EC50, or IC50 values or a tolerance range depending, for example, on whether such values were determined by ELISA (including direct or competition ELISA), by SPR, or by another method.
  • binding specificity relates to the ability of a biomolecule to discriminate between the desired target and one or more reference targets. It is understood that such specificity is not an absolute but a relative property and can be determined, for example, by means of SPR, western blots, ELISA, fluorescence activated cell sorting (FACS), radioimmunoassay (RIA), electrochemiluminescence (ECL), immunoradiometric assay (IRMA), ImmunoHistoChemistry (IHC), and peptide scans.
  • variant relates to derivatives of a protein or polypeptide that include one or more mutations or mutated amino acid residues, for example by amino acid substitution(s), deletion(s), insertion(s), and/or one or more chemical modifications of an amino acid sequence or nucleotide sequence. Such substitutions may be conservative, i.e., an amino acid residue is replaced with a chemically similar amino acid residue.
  • conservative substitutions are the replacements among the members of the following groups: 1) alanine, serine, and threonine; 2) aspartic acid and glutamic acid; 3) asparagine and glutamine; 4) arginine and lysine; 5) isoleucine, leucine, methionine, and valine; and 6) phenylalanine, tyrosine, and tryptophan.
  • Such variants also include proteins or polypeptides, wherein one or more amino acids have been substituted by their respective D-stereoisomers or by amino acids other than the naturally occurring 20 amino acids, such as, for example, ornithine, hydroxyproline, citrulline, homoserine, hydroxylysine, norvaline.
  • variants also include, for instance, proteins or polypeptides in which one or more amino acid residues are added or deleted at the N- and/or C-terminus.
  • a variant has at least about 50%, 60%, 70%, 75%, 80%, 82.5%, 85%, 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least about 99% amino acid sequence identity with a reference protein or polypeptide, e.g., the native (wild-type) sequence protein or polypeptide.
  • a “variant” may also be referred to as a “mutein” or “mutated” entity herein.
  • variant as used herein with respect to IL-18 variants of the disclosure, generally relates to IL-18 or a fragment thereof, respectively, that has one or more, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or more, amino acid substitutions, deletions and/or insertions in comparison to the native sequence of IL-18 (wild-type IL-18), such as human IL-18 as defined by UniProt Q14116-1 (version 193 of September 29, 2021).
  • an IL-18 variant has an amino acid sequence identity of at least about 80.0%, 82.5%, 85%, 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% to the amino acid sequence of native (wild-type) IL-18 or a fragment thereof, e.g., wild-type mature human IL-18 of SEQ ID NO: 1.
  • mutant and wild-type e.g., in connection with an amino acid or nucleotide sequence, generally refer to something that is naturally occurring (i.e. , it can be derived/isolated from nature).
  • a “fragment” with respect to IL-18 and/or the IL-18 variants of the disclosure refers to N-terminally and/or C-terminally truncated IL-18 and/or IL-18 variants. Fragments of IL- 18 and/or IL-18 variants as described herein retain the functionality of the full-length IL-18 or IL- 18 variants, i.e., they are functional fragments.
  • mutagenesis refers to the introduction of mutations into a polynucleotide or amino acid sequence. Mutations are preferably introduced under experimental conditions such that the amino acid naturally occurring at a given position of the protein or polypeptide sequence can be altered, for example substituted by at least one amino acid.
  • the term “mutagenesis” also includes the (additional) modification of the length of sequence segments by deletion or insertion of one or more amino acids. Thus, it is within the scope of the disclosure that, for example, one amino acid at a chosen sequence position is replaced by a stretch of three amino acids, leading to an addition of two amino acid residues compared to the length of the respective segment of the native protein or polypeptide amino acid sequence. Such an insertion or deletion may be introduced independently from each other in any of the sequence segments that can be subjected to mutagenesis in the disclosure.
  • random mutagenesis means that no predetermined mutation (alteration of an amino acid) is present at a certain sequence position but that at least two amino acids can be incorporated with a certain probability at a predefined sequence position during mutagenesis.
  • sequence identity denotes a property of sequences that measures their similarity or relationship.
  • sequence identity or “identity” as used in the present disclosure means the percentage of pair-wise identical residues - following (homologous) alignment of a sequence of a polypeptide of the disclosure with a sequence in question - with respect to the number of residues in the longer of these two sequences. Sequence identity is measured by dividing the number of identical amino acid residues by the total number of residues and multiplying the product by 100.
  • sequence homology or “homology” has its usual meaning, and a homologous amino acid includes identical amino acids as well as amino acids which are regarded to be conservative substitutions at equivalent positions in the linear amino acid sequence of a protein or a polypeptide of the disclosure (e.g., any IL-18 variant of the disclosure).
  • BLAST Altschul et al., Nucleic Acids Res, 1997, 25, 3389-402
  • BLAST2 Altschul et al., J Mol Biol, 1990, 215, 403-10
  • Smith- Waterman Smith and Waterman, J Mol Biol, 1981 , 147, 195-7
  • the percentage of sequence homology or sequence identity can, for example, be determined herein using the program BLASTP, version 2.2.5, November 16, 2002 (Altschul et al., Nucleic Acids Res, 1997, 25, 3389-402).
  • the percentage of homology is based on the alignment of the entire protein or polypeptide sequences (matrix: BLOSUM 62; gap costs: 11.1 ; cutoff value set to 10 -3 ) including the propeptide sequences, preferably using the wild-type protein scaffold as reference in a pairwise comparison. It is calculated as the percentage of numbers of “positives” (homologous amino acids) indicated as result in the BLASTP program output divided by the total number of amino acids selected by the program for the alignment.
  • a wild-type sequence of IL-18 can serve as “subject sequence” or “reference sequence,” while the amino acid sequence of an IL- 18 variant different from wild-type IL- 18 described herein serves as “query sequence.”
  • the terms “wild-type sequence,” “reference sequence,” and “subject sequence” may be used interchangeably herein.
  • a preferred wild-type sequence of IL-18 is the sequence of mature hulL- 18 as shown in SEQ ID NO: 1.
  • Gaps are spaces in an alignment that are the result of additions or deletions of amino acids. Thus, two copies of exactly the same sequence have 100% identity, but sequences that are less highly conserved, and have deletions, additions, or replacements (substitutions), may have a lower degree of sequence identity.
  • position means the position of either an amino acid within an amino acid sequence disclosed herein or the position of a nucleotide within a nucleic acid sequence disclosed herein. It is to be understood that when the term “correspond” or “corresponding” is used herein in the context of the amino acid sequence positions of one or more IL-18 variants, a corresponding position is not only determined by the number of the preceding nucleotides or amino acids. Accordingly, the absolute position of a given amino acid in accordance with the disclosure may vary from the corresponding position due to deletion or addition of amino acids elsewhere in a (variant or wild-type) protein.
  • the absolute position of a given nucleotide in accordance with the present disclosure may vary from the corresponding position due to deletions or additional nucleotides elsewhere in a variant or wildtype IL18 5’-untranslated region (UTR) including the promoter and/or any other regulatory sequences or gene regions (including exons and introns).
  • UTR untranslated region
  • nucleotides or amino acids may differ from adjacent nucleotides or amino acids but said adjacent nucleotides or amino acids which may have been exchanged, deleted, or added may be comprised by the same one or more “corresponding positions”.
  • positions of nucleotides or amino acids of an IL-18 variant can structurally correspond to the positions elsewhere in a reference protein (wild-type IL-18) or another IL-18 variant, even if they may differ in the absolute position numbers, as appreciated by the skilled person.
  • conjugate refers to the joining together of two or more subunits, through all forms of covalent or non-covalent linkage, by means including, but not limited to, genetic fusion, chemical conjugation, coupling through a linker or a cross-linking agent, and non-covalent association.
  • fusion polypeptide or “fusion protein” as used interchangeably herein refers to a polypeptide or protein comprising two or more subunits.
  • a fusion polypeptide as described herein comprises two or more subunits, wherein at least one of these subunits comprises or is an IL-18 variant as described herein.
  • a fusion polypeptide as described herein comprises at least two subunits, wherein one subunit comprises or is an IL-18 variant as described herein, and wherein another subunit comprises or is a moiety targeting immune cells, in particular a moiety having binding affinity towards (being specific for) an antigen associated with and/or being specific for the immune cells.
  • these subunits may be linked by covalent or non-covalent linkage.
  • the fusion polypeptide is a translational fusion between the two or more subunits.
  • the translational fusion may be generated by genetically engineering the coding sequence for one subunit in a reading frame with the coding sequence of a further subunit. Both subunits may be interspersed by a nucleotide sequence encoding a linker.
  • the subunits of a fusion polypeptide of the present disclosure may also be linked through chemical conjugation.
  • the subunits forming the fusion polypeptide are typically linked to each other as follows: C-terminus of one subunit to N-terminus of another subunit, or C-terminus of one subunit to C-terminus of another subunit, or N-terminus of one subunit to N-terminus of another subunit, or N-terminus of one subunit to C-terminus of another subunit.
  • the subunits forming the fusion polypeptide may also be linked to each other via one or more amino acid side chains of one or more of the subunits, e.g., through chemical conjugation.
  • the subunits of the fusion polypeptide can be linked in any order and may include more than one of any of the constituent subunits.
  • fusion polypeptide may also refer to the polypeptide comprising the fused sequences and all other polypeptide chain(s) of the protein (complex).
  • a fusion protein/polypeptide disclosed herein refers to a single protein or a separate polypeptide chain, which can form a stable folded structure by itself and defines a unique function of providing a binding motif towards a target.
  • a preferred subunit of the disclosure is an IL-18 variant as described herein.
  • the linkage can be covalent or non-covalent.
  • a preferred covalent linkage is via a peptide bond, such as a peptide bond between amino acids.
  • a preferred linker is a peptide linker. Accordingly, in a preferred embodiment, said linker comprises one or more amino acids, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids.
  • Preferred peptide linkers are described herein, including glycine-serine (Gly/Ser; GS) linkers, glycosylated GS linkers, proline-alanine-serine polymer (PAS) linkers, alpha helical linkers comprising the sequence motif A(EAAAK) X A or A(EAAAR) X A, wherein x is an integer between (and including) 2 and 6, and hybrid linkers composed of glycine-serine linker sequences and alpha helical linker sequences, such as (G4S) x A(EAAAK) y A(G4S) z , (GSG) x A(EAAAK) y A(GSG) z , (G4S) x A(EAAAR) y A(G4S) z , or (GSG) x A(EAAAR) y A(GSG) z , wherein x, y and z are independently selected from integers
  • a GS linker is used to join together the subunits of a fusion polypeptide, wherein, preferably, the GS linker has the general formula (G4S) X or (G2SG2) X , wherein x is an integer between (and including) 2 and 8.
  • an “immune cell”, as used herein, refers to a cell that is part of the immune system and helps the body fight infections and other diseases.
  • Immune cells include neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, DCs, natural killer (NK) cells, and lymphocytes, such as B cells and/or T cells.
  • Preferred immune cells are T cells and NK cells.
  • the T cell may be a CD8 + T cell.
  • the T cell may be a CD4 + T cell.
  • immune cells are preferably enriched in a target tissue and/or in proximity to a target tissue (e.g., a diseased tissue and/or an injured tissue).
  • the immune cells are enriched in a tumor and/or in a tumor microenvironment.
  • the immune cells are tumor- associated immune cells enriched within the tumor microenvironment.
  • moiety targeting immune cells refers to a molecule having binding specificity for a target (e.g., a target protein) associated with and/or being specific for immune cells.
  • a target e.g., a target protein
  • Such moiety/molecule may be selected from the group consisting of antibodies and antigen-binding fragments thereof, antibody mimetics, small molecules and other antigen-binding molecules, such as aptamers.
  • the antibody mimetics are selected from the group consisting of Affibody molecules, Affilins, Affimers, Affitins, Alphabodies, lipocalin muteins (also referred to as Anticalin® proteins), Avimers, DARPins, Fynomers, Kunitz domain peptides, Monobodies and nanoCI_AMPs.
  • the term refers to a molecule having binding affinity towards an antigen associated with and/or being specific for immune cells.
  • the antigen is an antigen expressed on the surface of the immune cells.
  • antigens include, but are not limited to of PD-1 , LAG3, PD-L1 , CTLA4, TIM3, TIGIT, VISTA, IGOS, GITR, 4-1 BB, 0X40, CD56, CD40L, and CD40.
  • the antigen is selected from the group consisting of PD-1, LAG3, CTLA4, TIM3, TIGIT, IGOS, GITR, 4-1BB, 0X40, CD56, and CD40L.
  • tumor microenvironment or “TME”, as used herein, collectively refers to a variety of resident and infiltrating host cells (including immune cells and stromal cells), secreted factors and extracellular matrix proteins within and around a tumor (in contrast to the heterogeneous population of cancer cells). Tumor progression is profoundly influenced by interactions of cancer cells with this environment that ultimately determine whether the primary tumor is eradicated, metastasizes or establishes dormant micrometastases, as reviewed, for example, in Anderson and Simon, Current Biology, 2020, 30: R905-R931, which is incorporated herein by reference in its entirety.
  • antibody includes whole antibodies or any antigen-binding fragment (or portion) or single chain thereof.
  • a whole antibody refers to a glycoprotein comprising at least two heavy chains (HCs) and two light chains (LCs) inter-connected by disulfide bonds.
  • Each heavy chain is comprised of a heavy chain variable domain (VH or HCVR) and a heavy chain constant region (CH).
  • the heavy chain constant region is comprised of three domains, CHI , CH2 and CH3.
  • Each light chain is comprised of a light chain variable domain (VL or LCVR) and a light chain constant region (CL).
  • the light chain constant region is comprised of one domain, CL.
  • VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged in the following order from the amino-terminus to the carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies may optionally mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.
  • antigen-binding fragment or “antigen-binding portion” of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
  • binding fragments encompassed within the term “antigen-binding fragment” of an antibody include (i) a Fab fragment consisting of the VH, VL, CL and CHI domains; (ii) a F(ab')2 fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fab' fragment consisting of the VH, VL, CL and CHI domains and the region between CHI and CH2 domains; (iv) an Fd fragment consisting of the VH and CHI domains; (v) a single-chain Fv fragment consisting of the VH and VL domains of a single arm of an antibody, (vi) a dAb fragment (Ward et al., Nature, 1989, 341, 544-546) consisting of a VH domain; and (vii) an isolated complementarity determining region (CDR) or a combination of two or more isolated CDRs which may optionally be joined by a synthetic linker; (viii)
  • Antibodies may be polyclonal or monoclonal; xenogeneic, allogeneic, or syngeneic; or modified forms thereof (e.g., humanized, chimeric, or multispecific). Antibodies may also be fully human.
  • framework or “FR” refers to the variable domain residues other than the hypervariable region (CDR) residues.
  • Fc region refers to the C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy-chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof (numbering according to EU index of Kabat (Johnson and Wu, Nucleic Acids Res, 2000, 28, 214-8).
  • the C-terminal lysine (residue 447 according to EU index of Kabat) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Accordingly, a composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue.
  • Suitable native-sequence Fc regions for use in the antibodies of the disclosure include human lgG1 , lgG2 (lgG2A, lgG2B), lgG3, and lgG4.
  • Fc receptor or “FcR” refers to a receptor that binds to the Fc region of an antibody.
  • isolated antibody refers to an antibody that is substantially free of its natural environment. For instance, an isolated antibody is substantially free of cellular material and other proteins from the cell or tissue source from which it is derived. An “isolated antibody” further refers to an antibody that is substantially free of other antibodies having different antigenic specificities. In an illustrative example, an isolated antibody that binds specifically target X is substantially free of antibodies that specifically bind antigens other than target X. However, an isolated antibody that specifically binds target X may have cross-reactivity with other antigens, such as target X molecules from other species. [0042] As used herein, “monoclonal antibody” refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
  • humanized antibody refers to an antibody that consists of the CDRs of antibodies derived from mammals other than human, and the FR region and the constant region of a human antibody.
  • a humanized antibody is useful as an effective component in a therapeutic agent due to the reduced antigenicity.
  • human antibody includes antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences.
  • the human antibodies of the disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • the term “human antibody”, as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • lipocalin refers to a monomeric protein of approximately 18-20 kDa in weight, having a cylindrical p-pleated sheet supersecondary structural region comprising a plurality of p-strands (preferably eight p-strands designated A to H) connected pair-wise by a plurality of (preferably four) loops at one end to thereby comprise a ligand-binding pocket and define the entrance to the ligand-binding pocket.
  • the loops comprising the ligand-binding pocket used in the present disclosure are loops connecting the open ends of p-strands A and B, C and D, E and F, and G and H, and are designated loops AB, CD, EF, and GH.
  • lipocalin As used herein include, but are not limited to, tear lipocalin, Lipocalin-2 or neutrophil gelatinase- associated lipocalin, apolipoprotein D, and Von Ebner's gland protein.
  • lipocalin mutein refers to a “mutein,” a “mutated” entity (whether protein or nucleic acid), or “mutant” of a wild-type lipocalin, wherein the lipocalin mutein has binding specificity for a target other than the natural target(s) of the respective lipocalin.
  • the present disclosure explicitly encompasses lipocalin muteins having a cylindrical p- pleated sheet supersecondary structural region comprising eight p-strands connected pair-wise by four loops at one end to thereby comprise a ligand-binding pocket and define the entrance of the ligand-binding pocket, wherein at least one amino acid of each of at least three of said four loops has been mutated as compared to the native lipocalin sequence.
  • Lipocalin muteins of the present disclosure preferably have the function of binding a target associated with an immune cell as described herein.
  • Proteins falling in the definition of “lipocalin” as used herein include, but are not limited to, human tear lipocalin (Tic, Lcn1), Lipocalin-2 (Lcn2) or neutrophil gelatinase-associated lipocalin (NGAL), apolipoprotein D (ApoD), apolipoprotein M, ai-acid glycoprotein, ai-microglobulin, complement component 8y, retinol-binding protein, the epididymal retinoic acid-binding protein, glycodelin, odorant-binding protein, prostaglandin D synthase, with human tear lipocalin (Tic, Lcn1) and human neutrophil gelatinase-associated lipocalin (NGAL) being preferred.
  • Tic, Lcn1 human tear lipocalin
  • Lcn2 Lipocalin-2
  • NGAL neutrophil gelatinase-associated lipocalin
  • NGAL neutrophil gelatinase-associated
  • Exemplary lipocalin muteins specific for LAG3 are described in WO 2017/009456 and WO 2018/134274, exemplary lipocalin muteins specific for CTLA-4 are described in WO 2006/056464 and WO 2012/072806, exemplary lipocalin muteins specific for 4-1BB are described in WO 2016/177762, all which are incorporated herein by reference in their entirety
  • tissue lipocalin refers to human tear lipocalin (hTIc) and further refers to mature human tear lipocalin.
  • a “mature hTIc” of the instant disclosure refers to the mature form of human tear lipocalin, which is free from the signal peptide. Mature hTIc is described by residues 19-176 of the sequence deposited with the SWISS-PROT Data Bank under Accession Number P31025, and its amino acid sequence is shown in SEQ ID NO: 12.
  • a mutein of hTIc typically has at least about 60%, preferably at least about 70%, in some cases at least about 80% sequence identity to SEQ ID NO: 12.
  • Lipocalin-2 or “neutrophil gelatinase-associated lipocalin” refers to human Lipocalin-2 (hLcn2) or human neutrophil gelatinase-associated lipocalin (hNGAL) and further refers to mature human Lipocalin-2 or mature human neutrophil gelatinase-associated lipocalin.
  • a “mature hNGAL” of the instant disclosure refers to the mature form of human neutrophil gelatinase-associated lipocalin, which is free from the signal peptide.
  • Mature hNGAL is described by residues 21-198 of the sequence deposited with the SWISS-PROT Data Bank under Accession Number P80188, and its amino acid sequence is shown in SEQ ID NO: 13.
  • a mutein of hNGAL typically has at least about 60%, preferably at least about 70%, in some cases at least about 80% sequence identity to SEQ ID NO: 13.
  • the term “small molecule”, as used herein, generally refers to a low molecular weight (e.g., ⁇ 900 Daltons) organic compound.
  • pharmaceutically acceptable refers to the non-toxicity of a material which, in certain exemplary embodiments, does also not interact with the action of the active agent(s) of the pharmaceutical composition.
  • carrier refers to an organic or inorganic component of natural origin or synthetic nature, in which the active agent(s) of a pharmaceutical composition is/are provided in order to facilitate, enhance or enable its/their application.
  • carrier may include one or more solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to a subject.
  • excipient is intended to include all substances which may be present in a pharmaceutical composition and which are not pharmaceutically active ingredients, such as salts, binders, fillers, lubricants, thickeners, surfactants, preservatives, emulsifiers, or buffer substances.
  • treat or “treatment” or “therapy” refers to clinical intervention designed to alter the natural course of the subject being treated during the course of a physiological condition or disorder or clinical pathology.
  • a treatment may be a therapeutic treatment and/or a prophylactic or preventative measure, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the growth, development or spread of a hyperprol iterative condition, such as cancer.
  • Desired effects of treatment include, but are not limited to, decreasing the rate of disease progression, ameliorating or palliating the disease state, alleviating symptoms, stabilizing or not worsening the disease state, and remission of improved prognosis, whether detectable or undetectable. Desired effects of treatment also include prolonging survival as compared to expected survival if not receiving treatment.
  • a subject in need of treatment includes a subject already with the condition or disorder or prone to have the condition or disorder or a subject in which the condition or disorder is to be prevented.
  • a “subject” or “organism”, as referred to herein, is a vertebrate, preferably a mammal, more preferably a human.
  • the term “mammal” is used herein to refer to any animal classified as a mammal, including, without limitation, humans, domestic and farm animals, and zoo, sports, or pet animals, such as sheep, dogs, horses, cats, cows, rats, pigs, apes such as cynomolgus monkeys, to name only a few illustrative examples.
  • the “mammal” used herein is human.
  • an “effective amount” is an amount sufficient to yield beneficial or desired results of treatment.
  • An effective amount can be administered in one or more individual administrations or doses.
  • An effective amount can be administered alone with one agent or in combination with one or more additional agents.
  • cancer and “cancerous” refers to the physiological condition in mammals that is typically characterized by unregulated cell growth.
  • a “tumor” may comprise one or more cancerous cells.
  • a “lesion” is a localized change in a tissue or an organ. The terms “cancer”, “tumor”, and “lesion” are used interchangeably herein.
  • infectious disease refers to a disorder resulting from an infection.
  • An infection is the invasion of an organism's body tissue(s) by disease-causing agents (e.g., bacteria, viruses, fungi or parasites), their multiplication, and the reaction of host tissues to the infectious agents and the toxins they produce.
  • disease-causing agents e.g., bacteria, viruses, fungi or parasites
  • metabolic disease refers to a disorder that negatively alters an organism’s body's processing and distribution of nutrients, such as proteins, fats, and carbohydrates.
  • autoimmune disease refers to a condition arising from an abnormal immune response to a functioning part of an organism’s body, e.g., a particular type of cells, specific tissue(s) and/or one or more organs.
  • the term “about”, “approximately” or “similar to” means within 20%, preferably within 15%, preferably within 10%, and more preferably within 5% of a given value or range. It also includes the concrete number, i.e., “about 20” includes the number of 20. The term “at least about” as used herein includes the concrete number, i.e., “at least about 20” includes 20.
  • Figure 1 shows production of wild-type mature human IL-18 and of an exemplary IL-18 variant of the disclosure (IL-18v2; SEQ ID NO: 5; K53L).
  • IL-18v2 an exemplary IL-18 variant of the disclosure
  • SEQ ID NO: 5 an exemplary IL-18 variant of the disclosure
  • Lane 1 6xHis-SUMO-hulL-18, non-reducing conditions
  • lane 2 GST- ULP1 digested 6xHis-SUMO-hulL-18, non-reducing conditions
  • lane 3 hulL-18, non-reducing conditions
  • lane 4 hulL-18, reducing conditions.
  • Lane 1 SUMO-hulL-18v2-6xHis, non-reducing conditions
  • lane 2 GST-ULP1 digested SUMO-hulL-18v2-6xHis, non-reducing conditions
  • lane 3 hulL-18v2-6xHis, non-reducing conditions
  • lane 4 hulL-18v2-6xHis, reducing conditions.
  • B right panel
  • Figure 2 shows stimulation of IL-18 downstream signaling pathways by IL-18 variants of the disclosure in HEK-Blue IL-18 reporter cells.
  • HEK-Blue IL-18 reporter cells were stimulated as described in Example 2 by titrations of wild-type mature human IL-18 or indicated hulL-18 variants with (dashed lines) or without addition of 200 nM hulL-18BP-6xHis for competition (solid lines).
  • the plotted conversion of QUANTI-Blue substrate by secreted embryonic alkaline phosphatase (SEAP) measured at a wavelength of 655 nm correlates with the bioactivity and concentration of wild-type mature hulL-18 (A-D), hulL-18v1-6xHis (K53H) (A), hulL-18v2-6xHis (K53L) (B), hulL-18v3-6xHis (K53A) (C) and hulL-18v4-6xHis (C38S/K53A/P57T/M60A/C68S/C127S) (D).
  • SEAP secreted embryonic alkaline phosphatase
  • the present disclosure relates to the identification and generation of IL- 18 variants with the purpose of treating diseases in a specifically targeted manner, e.g., by using fusion molecules comprising an IL- 18 variant as disclosed herein and a moiety targeting an antigen associated with and/or being specific for immune cells, particularly immune cells enriched in a target tissue and/or in proximity to a target tissue, e.g., a tumor and/or the tumor microenvironment.
  • the IL-18 variants disclosed herein preferably have (a) strongly reduced binding to the IL-18 binding protein (IL-18BP) ensuring therapeutic activity and (b) reduced binding to IL-18 receptor 1 (IL-18R1) focusing the activity of the targeted variants on- target.
  • IL-18BP IL-18 binding protein
  • IL-18R1 IL-18 receptor 1
  • the present disclosure provides a variant of human interleukin-18 (IL-18), wherein the variant comprises an amino acid sequence having at least about 80% sequence identity to the amino acid sequence of wild-type mature human IL-18 (SEQ ID NO: 1), and wherein the variant comprises, at a position corresponding to position 53 of the linear amino acid sequence of wild-type mature human IL-18 (SEQ ID NO: 1), a mutated amino acid residue (K53X).
  • X is an amino acid residue other than R, G, S, or T.
  • X is an amino acid residue other than R, G, S, T, or A.
  • the variant comprises an amino acid sequence having at least about 82.5%, 85%, 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of wild-type mature human IL-18 (SEQ ID NO: 1).
  • the variant comprises, at a position corresponding to position 53 of the linear amino acid sequence of wild-type mature human IL-18 (SEQ ID NO: 1), a mutated amino acid residue selected from the group consisting of K53H, K53L, and K53A.
  • the mutated amino acid residue is K53H or K53L.
  • the mutated amino acid residue is K53A.
  • the variant further comprises up to 31 , 30, 29, 28, 27, 26,
  • the variant further comprises, at positions corresponding to the respective positions of the linear amino acid sequence of wild-type mature human IL-18 (SEQ ID NO: 1), one or more of the following mutated amino acid residues: Y1A, S10R, M51A, M51Y, S55L, S55Y, P57T, M60A, M60E, M60H, Q103S, M113A, M113E, or N155Y.
  • the variant further comprises, at positions corresponding to the respective positions of the linear amino acid sequence of wild-type mature human IL-18 (SEQ ID NO: 1), the mutated amino acid residues P57T and M60A.
  • the variant further comprises, at positions corresponding to the respective positions of the linear amino acid sequence of wildtype mature human IL-18 (SEQ ID NO: 1), the mutated amino acid residues G3Y, S10K, and M51Q.
  • the mutated amino acid residue is K53A
  • the variant comprises one of the following sets of mutated amino acid residues: (a) K53A, P57T, and M60A; or (b) G3Y, S10K, M51Q, and K53A.
  • the variant comprises at least one amino acid substitution of a native cysteine residue by another amino acid.
  • the variant comprises, at positions corresponding to the respective positions of the linear amino acid sequence of wild-type mature human IL-18 (SEQ ID NO: 1), the mutated amino acid residues C38S, C68S, and C127S. [0076] In some embodiments, the variant comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-8 or a fragment or variant thereof.
  • the variant comprises or consists of an amino acid sequence having at least about 85%, 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-8.
  • the variant has binding affinity towards IL-18 receptor 1 (IL-18R1), wherein the binding affinity towards IL-18R1 is similar to that of wild-type mature human IL-18 or reduced as compared to that of wild-type mature human IL-18, and/or the variant has a binding affinity towards IL-18 binding protein (IL-18BP) which is reduced as compared to that of wild-type mature human IL-18.
  • IL-18BP IL-18 binding protein
  • the variant has detectable affinity towards IL-18R1 (e.g., the respective KD is ⁇ 10 -5 M).
  • the variant does not have higher binding affinity towards IL-18R1 as compared to that of wildtype mature human IL-18 towards IL-18R1.
  • the binding affinity towards IL-18R1 is reduced as compared to that of wild-type mature human IL-18. In some embodiments, the binding affinity towards IL-18R1 is reduced as compared to that of wild-type mature human IL-18, and the binding affinity towards IL-18BP is reduced as compared to that of wild-type mature human IL- 18.
  • the present disclosure provides an IL-18 variant which (i) binds and activates IL-18R1 and activates downstream signaling pathways of IL-18 to a lesser degree (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% less) than wild-type mature human IL-18 and (ii) is resistant to inhibition by IL-18BP (e.g., as indicated by a significantly reduced binding affinity towards IL-18BP).
  • the binding affinity towards IL-18R1 is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% as compared to that of wild-type mature human IL-18.
  • the binding affinity towards IL-18R1 is reduced by at least about 90% as compared to that of wild-type mature human IL-18.
  • the binding affinity towards IL-18R1 is reduced at least about 2-fold, at least about 5-fold, at least about 7-fold, at least about 10-fold, at least about 12- fold, at least about 15-fold, or at least about 20-fold as compared to that of wild-type mature human IL-18.
  • the variant has a capability to activate IL-18R1 and downstream signaling pathways of IL-18, wherein the capability is reduced as compared to that of wild-type mature human IL-18, e.g., as shown in a HEK-Blue IL-18 reporter cell assay as essentially described in Example 2.
  • the capability to activate IL-18R1 and downstream signaling pathways of IL-18 is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% as compared to that of wild-type mature human IL-18.
  • the capability to activate IL-18R1 and downstream signaling pathways of IL-18 is reduced at least about 2-fold, at least about 5-fold, at least about 7-fold, at least about 10- fold, at least about 12-fold, at least about 15-fold, or at least about 20-fold as compared to that of wild-type mature human IL- 18.
  • the binding affinity towards IL-18BP is reduced by at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.6%, at least about 99,7%, at least about 99.8%, at least about 99.9%, at least about 99.95%, at least about 99.96%, at least about 99.97%, at least about 99.98%, at least about 99.99%, at least about 99.995%, at least about 99.996%, at least about 99.997%, at least about 99.998%, at least about 99.999%, at least about 99.9999%, at least about 99.99999%, at least about 99.999999%, at least about 99.999999%, at least about 99.999999%, at least about 99.99999999%, at least about 99.999999%, at least about 99.999999%, at least about 99.99999999%, at least about 99.999999%
  • the binding affinity towards IL-18BP is reduced at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, or at least about 30-fold as compared to that of wild-type mature human IL-18.
  • the binding affinity towards IL-18BP is less than about 1 %, less than about 0.1%, less than about 0.01 %, less than about 0.001%, less than about 0.0001%, less than about 0.00001%, less than about 0.000001%, less than about 0.0000001%, less than about 0.00000001%, less than about 0.000000001%, less than about 0.0000000001%, less than about 0.00000000001%, or less than about 0.000000000001% of the binding affinity of wild-type mature human IL-18.
  • the variant binds to IL-18BP with a KD of about 10' 8 M or higher, about 10' 7 M or higher, or about 10' 6 M or higher.
  • the variant does not bind to IL-18BP. In some embodiments, the variant has no detectable affinity towards IL-18BP (e.g., the respective KD is >10’ 5 M).
  • the variant is resistant to inhibition by IL-18BP.
  • the variant is conjugated to a compound selected from the group consisting of an organic molecule, an enzyme label, a radioactive label, a colored label, a fluorescent label, a chromogenic label, a luminescent label, a hapten, digoxigenin, biotin, a cytostatic agent, a toxin, a metal complex, a metal, and colloidal gold.
  • the variant is fused at its N-terminus and/or its C-terminus to a fusion partner that is a protein, a protein domain, or a peptide.
  • the variant is fused at its N-terminus and/or its C-terminus to a tag (e.g., a protein, protein domain or peptide tag) allowing or facilitating the solubilization, isolation and/or immobilization of the variant.
  • a tag e.g., a protein, protein domain or peptide tag
  • the tag is selected from the group consisting of poly(His) (e.g., 6* His), Strep-tag®, Strep-tag II®, Twin-Strep-tag®, SUMO, thioredoxin (TRX), FLAG, myc, V5, HA, chitin binding protein (CBP), maltose binding protein (MBP), glutathione-S-transferase (GST) and combinations thereof.
  • Such tag(s) may be fused to the variant either directly or via a linker as described herein.
  • the variant is fused at its N-terminus and/or its C-terminus to a fusion partner that is an antibody or an antigen-binding fragment thereof.
  • the variant is fused at its N-terminus and/or its C-terminus to a fusion partner that is a lipocalin mutein.
  • the fusion partner has binding affinity towards an antigen associated with and/or being specific for a particular cell, cell type, tissue and/or organ.
  • the variant is conjugated to a compound that extends the serum half-life of the variant.
  • the compound that extends the serum half-life is selected from the group consisting of a polyethylene glycol (PEG) molecule, hydroxyethyl starch, an Fc part of an immunoglobulin, a CH3 domain of an immunoglobulin, a CH4 domain of an immunoglobulin, an albumin binding peptide, an albumin binding protein, conformationally disordered peptide sequences composed of the amino acids Pro, Ala, and/or Ser (“PASylation”), and neonatal Fc receptor (FcRn) binding peptides or proteins.
  • PEG polyethylene glycol
  • the albumin is a mammalian albumin, such as human serum albumin or bovine serum albumin or rat serum albumin.
  • the present disclosure provides a fusion protein comprising at least two subunits, wherein one subunit comprises an IL-18 variant as defined above, and wherein another subunit comprises a moiety targeting (being specific for) immune cells, in particular a moiety having binding affinity towards (being specific for) an antigen associated with and/or being specific for the immune cells.
  • the immune cells are enriched in a target tissue and/or in proximity to a target tissue.
  • the target tissue is a diseased tissue and/or an injured tissue.
  • the immune cells are enriched in a tumor and/or in a tumor microenvironment.
  • the immune cells are selected from the group consisting of T cells (e.g., CD4 + or CD8 + T cells) and NK cells.
  • the antigen is an antigen expressed on the surface of the immune cells.
  • the antigen is selected from the group consisting of PD-1 , LAG3, CTLA4, TIM3, TIGIT, ICOS, GITR, 4-1 BB, 0X40, CD56, and CD40L.
  • the moiety targeting (being specific for) the immune cells in particular the moiety having binding affinity towards (being specific for) an antigen associated with and/or being specific for the immune cells, comprises an antibody or an antigen-binding fragment thereof.
  • the moiety targeting (being specific for) the immune cells comprises a lipocalin mutein.
  • the present disclosure provides a nucleic acid molecule comprising a nucleotide sequence encoding an IL-18 variant as defined above or a fusion protein as defined above.
  • the disclosure is not limited to a specific nucleic acid molecule encoding an IL-18 variant or fusion protein as described herein, but rather encompasses all nucleic acid molecules that include nucleotide sequences encoding a functional IL-18 variant or fusion protein.
  • the nucleic acid molecule may be DNA or RNA (e.g., mRNA).
  • a nucleic acid molecule may be in the form of a molecule which is single-stranded or double-stranded.
  • a nucleic acid molecule according to the invention may be linear or covalently closed to form a circle.
  • a nucleic acid molecule such as DNA
  • An operable linkage is a linkage in which the regulatory sequence elements and the sequence to be expressed are connected in a way that enables gene expression.
  • promoter regions necessary for gene expression may vary among species, but in general these regions include a promoter, which, in prokaryotes, contains both the promoter per se, i.e., DNA elements directing the initiation of transcription, as well as DNA elements which, when transcribed into RNA, will signal the initiation of translation.
  • promoter regions normally include 5’ non-coding sequences involved in initiation of transcription and translation, such as the -35/-10 boxes and the Shine-Dalgarno element in prokaryotes or the TATA box, CAAT sequences, and 5’-capping elements in eukaryotes.
  • These regions can also include enhancer or repressor elements as well as translated signal and leader sequences for targeting the native protein to a specific compartment of a host cell.
  • non-coding sequences may contain regulatory elements involved in transcriptional termination, polyadenylation or the like. If, however, these termination sequences are not satisfactorily functional in a particular host cell, then they may be substituted with signals functional in that cell.
  • a nucleic acid molecule of the disclosure may be “operably linked” to one or more regulatory sequences, such as a promoter sequence, to allow expression of this nucleic acid molecule.
  • a nucleic acid molecule of the disclosure includes a promoter sequence and a transcriptional termination sequence.
  • Suitable prokaryotic promoters are, for example, the tet promoter, the lacllV5 promoter or the T7 promoter. Examples of promoters useful for expression in eukaryotic cells are the SV40 promoter or the CMV promoter.
  • a nucleic acid molecule encoding an IL-18 variant as disclosed herein may be “operably linked” to another nucleic acid molecule encoding a moiety or domain of the disclosure to allow expression of a fusion protein as disclosed herein.
  • provided nucleic acid molecules can also be part of a vector or any other kind of cloning vehicle, such as a plasmid, a phagemid, a phage, a baculovirus, a cosmid or an artificial chromosome.
  • a provided nucleic acid molecule can also be comprised in the genomic DNA of a host cell.
  • a provided nucleic acid molecule can be comprised in an expression vector.
  • Such expression vector may be a viral vector.
  • Viral vectors for expression in animal cells, such as mammalian cells are known in the art.
  • the nucleic acid molecule can be comprised in a nanoparticle.
  • the nucleic acid molecule can be comprised in a liposome or lipoplex.
  • DNA or RNA e.g., mRNA
  • encoding an IL-18 variant or a fusion protein of the disclosure can be comprised in a nanoparticle or in a liposome or lipoplex.
  • a provided nucleic acid molecule may be included in a phagemid.
  • a phagemid vector denotes a vector encoding the intergenic region of a temperate phage, such as M13 or f1 , or a functional part thereof fused to the cDNA of interest.
  • phagemid vector e.g., M13K07, VCS-M13 or R408
  • phagemid vector e.g., M13K07, VCS-M13 or R408
  • intact phage particles are produced, thereby enabling physical coupling of the encoded heterologous cDNA to its corresponding polypeptide displayed on the phage surface (Lowman, Annu Rev Biophys Biomol Struct, 1997, 26, 401-24, Rodi and Makowski, Curr Opin Biotechnol, 1999, 10, 87-93).
  • cloning vehicles can include, aside from the regulatory sequences described above and a nucleotide sequence encoding an IL-18 variant or fusion protein as described herein, replication and control sequences derived from a species compatible with the host cell that is used for expression as well as selection markers conferring a selectable phenotype on transformed or transfected cells. Large numbers of suitable cloning vectors are known in the art and are commercially available.
  • the present disclosure provides a host cell containing a nucleic acid molecule as defined above or an expression vector as defined above.
  • Suitable host cells include prokaryotic cells, such as bacterial cells, and eukaryotic cells, such as yeast cells, fungal cells, or mammalian cells (e.g., cells from humans, mice, hamsters, pigs, goats, or primates).
  • Suitable bacterial cells include, but are not limited to, cells from gram-negative bacterial strains, such as strains of Escherichia coli (E. coli), Proteus, and Pseudomonas, and gram-positive bacterial strains, such as strains of Bacillus, Streptomyces, Staphylococcus, and Lactococcus.
  • Suitable fungal cells include, but are not limited to, cells from the species of Trichoderma, Neurospora, and Aspergillus.
  • Suitable yeast cells include, but are not limited to, cells from the species of Saccharomyces (for example, Saccharomyces cerevisiae), Schizosaccharomyces (for example, Schizosaccharomyces pombe), Pichia (for example, Pichia pastoris and Pichia methanolica), and Hansenula.
  • Suitable mammalian cells include, but are not limited to, for example, CHO cells, BHK cells, HeLa cells, COS cells and H EK-293 cells. However, amphibian cells, insect cells, plant cells, and any other cells used in the art for the expression of heterologous proteins can be used as host cells as well.
  • the host cell is an immune cell (in particular a human immune cell), e.g., a T cell or an NK cell, which may be recombinant.
  • the immune cell in particular the T cell, may comprise a recombinant antigen receptor.
  • a recombinant antigen receptor may be a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the host cell may be a CAR T cell, wherein, preferably, the CAR T cell is equipped with the capacity to secrete an IL-18 variant or fusion protein as disclosed herein.
  • the host cell may be a CAR NK cell.
  • the present disclosure provides a method of producing an IL- 18 variant as defined above or a fusion protein as defined above, wherein the variant or the fusion protein is produced starting from the nucleic acid molecule encoding the variant or the fusion protein, respectively.
  • a provided method can be carried out in vivo, wherein a provided IL-18 variant or fusion protein can, for example, be produced in a bacterial or eukaryotic host organism.
  • the IL-18 variant or fusion protein may further be isolated from the host organism or its culture. It is also possible to produce an IL-18 variant or fusion protein of the disclosure in vitro, for example, using an in vitro translation system.
  • a nucleic acid molecule encoding an IL-18 variant or fusion protein may be introduced into a suitable bacterial or eukaryotic host organism using recombinant DNA technology well known in the art.
  • a DNA molecule encoding an IL-18 variant or fusion protein as described herein, and in particular a cloning vector containing the coding sequence of such an IL-18 variant or fusion protein can be transformed into a host cell capable of expressing the gene. Transformation can be performed using standard techniques. The host cell is then cultured under conditions, which allow expression of the heterologous DNA and thus the synthesis of the corresponding polypeptide. Subsequently, the polypeptide is recovered either from the cell or the cultivation medium.
  • host cells can be prokaryotic, such as E. coli or Bacillus subtilis, or eukaryotic, such as Saccharomyces cerevisiae, Pichia pastoris, SF9 or High5 insect cells, immortalized mammalian cell lines (e.g., HeLa cells or CHO cells) or primary mammalian cells.
  • prokaryotic such as E. coli or Bacillus subtilis
  • eukaryotic such as Saccharomyces cerevisiae, Pichia pastoris, SF9 or High5 insect cells
  • immortalized mammalian cell lines e.g., HeLa cells or CHO cells
  • primary mammalian cells e.g., primary mammalian cells.
  • an IL-18 variant or fusion protein of the disclosure includes intramolecular disulfide bonds
  • an oxidizing environment may be provided by the periplasm of Gram-negative bacteria such as E. coli, in the extracellular milieu of Gram-positive bacteria or the lumen of the endoplasmic reticulum of eukaryotic cells and usually favors the formation of structural disulfide bonds.
  • an IL-18 variant or fusion protein of the disclosure in the cytosol of a host cell, preferably E. coli.
  • a provided IL- 18 variant or fusion protein can either be directly obtained in a soluble and folded state or recovered in the form of inclusion bodies, followed by renaturation in vitro.
  • a further option is the use of specific host strains having an oxidizing intracellular milieu, which may thus allow the formation of disulfide bonds in the cytosol (Venturi et al., J Mol Biol, 2002, 315, 1-8).
  • an IL-18 variant or fusion protein as described herein may not necessarily be generated or produced only by use of genetic engineering. Rather, such polypeptide can also be obtained by chemical synthesis such as Merrifield solid phase polypeptide synthesis or by in vitro transcription and translation. Methods for the solid phase and solution phase synthesis of polypeptides/proteins are well known in the art (see, e.g., Bruckdorfer et al., Curr Pharm Biotechnol, 2004, 5, 29-43). In another embodiment, the IL-18 variant or fusion protein of the disclosure may be produced by in vitro transcription/translation employing well- established methods known to those skilled in the art.
  • IL-18 variants and fusion proteins contemplated by the present disclosure but whose protein or nucleic acid sequences are not explicitly disclosed herein.
  • Potential additional modifications of the amino acid sequence include, e.g., directed mutagenesis of single amino acid positions to simplify subcloning of a protein gene or its parts by incorporating cleavage sites for certain restriction enzymes.
  • mutations can be introduced to modulate one or more characteristics of the protein such as to improve folding stability, serum stability, protein resistance or water solubility or to reduce aggregation tendency, if necessary.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising an IL-18 variant as defined above, a fusion protein as defined above, a nucleic acid molecule as defined above, an expression vector as defined above, or a host cell as defined above.
  • the pharmaceutical composition further comprises one or more carriers and/or excipients, all of which are pharmaceutically acceptable.
  • the present disclosure provides a method of binding and activating IL-18R1 and activating downstream signaling pathways of IL-18, comprising applying an IL-18 variant as defined above, a fusion protein as defined above, a nucleic acid molecule as defined above, an expression vector as defined above, a host cell as defined above, or a pharmaceutical composition as defined above.
  • the present disclosure provides a method of stimulating an immune response, such as a T cell (e.g., CD4 + or CD8 + T cell) immune response and/or an NK cell immune response, in a subject, comprising applying an IL-18 variant as defined above, a fusion protein as defined above, a nucleic acid molecule as defined above, an expression vector as defined above, a host cell as defined above, or a pharmaceutical composition as defined above.
  • an immune response such as a T cell (e.g., CD4 + or CD8 + T cell) immune response and/or an NK cell immune response
  • an immune response such as a T cell (e.g., CD4 + or CD8 + T cell) immune response and/or an NK cell immune response
  • an immune response such as a T cell (e.g., CD4 + or CD8 + T cell) immune response and/or an NK cell immune response
  • the present disclosure provides an IL-18 variant as defined above, a fusion protein as defined above, a nucleic acid molecule as defined above, an expression vector as defined above, a host cell as defined above, or a pharmaceutical composition as defined above for use in therapy.
  • the use is in the treatment of cancer, an infectious disease, a metabolic disease, or an autoimmune disease.
  • the present disclosure provides the use of an IL- 18 variant as defined above, a fusion protein as defined above, a nucleic acid molecule as defined above, an expression vector as defined above, a host cell as defined above, or a pharmaceutical composition as defined above for the manufacture of a medicament.
  • the medicament is for the treatment of cancer, an infectious disease, a metabolic disease, or an autoimmune disease.
  • the present disclosure provides a method of treating a disease comprising administering to a subject in need thereof an effective amount of an IL-18 variant as defined above, a fusion protein as defined above, a nucleic acid molecule as defined above, an expression vector as defined above, a host cell as defined above, or a pharmaceutical composition as defined above.
  • the disease is cancer, an infectious disease, a metabolic disease, or an autoimmune disease.
  • DNA constructs for prokaryotic expression of the IL-18 variants were generated by cloning of coding sequences generated by gene synthesis (Thermo Fisher) into the pET-24a expression vector.
  • the IL- 18 variants (IL-18v1-v4 of SEQ ID NOs: 4-8) were expressed as fusion proteins with a SUMO tag at the N-terminus and a 6* His tag at the C- terminus of the IL-18v amino acid sequence, with a 5-amino acid Gly/Ser linker being introduced between the C-terminus of IL-18v and the 6*His tag (SEQ ID NO: 11).
  • Wild-type mature human IL-18 was additionally expressed as a 6xHis-SUMO-IL-18 fusion protein (i.e., with the N-terminal tag of SEQ ID NO: 9) to yield non-tagged reference IL-18 (SEQ ID NO: 1) after protease cleavage.
  • the expression constructs were transformed into E. coli BL21(DE3), and protein expression was induced by addition of 0.5 mM IPTG, followed by a cultivation at 16 °C for 20 h. After harvest, cells were lysed by sonication, and cell lysates were subjected to Ni- NTA IMAC to isolate the 6xHis-tagged SUMO-IL-18v fusion proteins.
  • the SUMO tag was then cleaved from the SUMO-IL-18 fusion proteins by proteolytic digest with recombinantly produced GST-tagged S. cerevisiae ULP1 protease.
  • the preparation was further purified by a second IMAC step which resulted in binding and elution of IL-18v-6xHis or capture of 6xHis-SUMO in case of the 6xHis-SUMO-IL-18 fusion.
  • the polypeptides were further subjected to preparative size exclusion chromatography for removal of aggregates using a Hi Load 16/600 Superdex 75 pg or a Superdex 75 Increase 10/300 GL FPLC column.
  • the purity and monomer content of the polypeptides were analyzed by reducing/non-reducing SDS-PAGE and size exclusion chromatography conducted on a HPLC instrument equipped with a Superdex 75 Increase 3.2/300 column.
  • Figure 1 shows the SDS-PAGE and size exclusion chromatography analysis for wild-type mature human IL-18 (A) and for the exemplary IL-18 variant of SEQ ID NO: 5 (IL- 18v2; K53L) (B), demonstrating successful expression and purification of the polypeptides.
  • Example 2 Bioactivity of IL-18 variants in a HEK-Blue IL-18 reporter cell assay
  • HEK-BlueTM IL-18 reporter cells (Invivogen) were utilized. This cell line allows a specific readout of IL-18 cytokine activity via a fully functional signaling pathway which is not responding to human TNF-a nor to IL-1p. The quantification of the signaling activity is achieved by colorimetric measurement of the enzyme activity of secreted embryonic alkaline phosphatase (SEAP) expressed under the control of the native transcription factor NF-KB.
  • SEAP secreted embryonic alkaline phosphatase
  • the HEK-Blue IL- 18 cells were seeded into 384-well cell culture plates at a density of 8.4x10 3 cells per well.
  • wild-type mature human IL-18 SEQ ID NO: 1
  • IL-18 variants IL-18v1-v4 of SEQ ID NOs: 4-7, each with the C-terminal 6xHis tag of SEQ ID NO: 11
  • SEQ ID NO: 1 wild-type mature human IL-18
  • IL-18 variants IL-18v1-v4 of SEQ ID NOs: 4-7, each with the C-terminal 6xHis tag of SEQ ID NO: 11
  • IL-18 binding protein IL-18 binding protein
  • SEQ ID NO: 3 For competition with IL-18 binding protein (IL-18BP; SEQ ID NO: 3), recombinant hulL-18BP-6xHis was added to the cells at a constant concentration of 200 nM 20 min before adding the IL-18v samples. After stimulation of HEK Blue IL-18 cells, the SEAP-containing cell culture supernatant was removed. The SEAP enzyme activity was quantified by mixing of supernatants with QUANTI-Blue substrate solution and incubation for 2 h at 37 °C, followed by measurement of absorption at a wavelength of 620-655 nm.
  • HulL-18v3- 6xHis showed activation of the reporter system comparable/similar to wild-type IL-18 in combination with a reduced susceptibility against inactivation by IL-18BP.
  • Example 3 Binding affinity of IL-18 variants towards human IL-18R1 and IL- 18BP determined by SPR
  • the anti-human IgG Fc antibody (Cytiva) was immobilized on a CM5 sensor chip using standard amine chemistry: the carboxyl groups on the chip were activated using 1 -ethyl-3- (3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Subsequently, anti-human IgG Fc antibody solution (Cytiva) at a concentration of 25 pg/mL in 10 mM sodium acetate (pH 5) was applied at a flow rate of 5 pL/min until an immobilization level of 5.500- 10.000 resonance units (RU) was achieved.
  • EDC 1 -ethyl-3- (3-dimethylaminopropyl)-carbodiimide
  • NHS N-hydroxysuccinimide
  • IL-18 variants For affinity determination of the IL-18 variants, dilutions of each variant at various concentrations, typically ranging from 1.17 to 1000 nM, were prepared in HBS-EP+ buffer and applied to the prepared chip surface for affinity measurement to human IL-18R1 and human IL- 18BP. hulL-18 was used as a control. The binding assay was carried out with a contact time of 180 s, a dissociation time of 900-1200 s and a flow rate of 30 pL/min. All measurements were performed at 25°C. Regeneration of the chip surface was achieved with injections of 3 M MgCh for 120 s at a flow rate of 10 pL/min followed by an extra wash with running buffer. Prior to the protein measurements, three startup cycles were performed for conditioning purposes. Data were evaluated with Biacore Insight Evaluation software. Double referencing was used, and the 1:1 binding model was used to fit the raw data.
  • Table 2 List of Sequences.
  • Embodiments illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein.
  • the terms “comprising,” “including,” “containing,” etc. shall be read expansively and without limitation.
  • the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed.

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Abstract

La présente divulgation concerne des variants de l'interleukine-18 humaine (IL-18) avec des profils d'activité particuliers ainsi que des protéines de fusion comprenant de tels variants d'IL-18. La divulgation concerne en outre des molécules d'acide nucléique, des cellules hôtes et des compositions pharmaceutiques correspondantes ainsi que des procédés et des utilisations.
PCT/EP2022/087596 2021-12-22 2022-12-22 Nouveaux variants d'il-18 WO2023118497A1 (fr)

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Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0404097A2 (fr) 1989-06-22 1990-12-27 BEHRINGWERKE Aktiengesellschaft Récepteurs mono- et oligovalents, bispécifiques et oligospécifiques, ainsi que leur production et application
WO1993011161A1 (fr) 1991-11-25 1993-06-10 Enzon, Inc. Proteines multivalentes de fixation aux antigenes
WO2002101049A2 (fr) * 2001-03-08 2002-12-19 Ares Trading S.A. Production et utilisation de mutants d'interleukine-18
WO2004091517A2 (fr) * 2003-04-15 2004-10-28 Smithkline Beecham Corporation Conjugues comprenant l'interleukine 18 humaine il 18 et mutants de ces conjugues par substitution
WO2006056464A2 (fr) 2004-11-26 2006-06-01 Pieris Ag Compose a affinite pour l'antigene associe au lymphocyte t cytotoxique (ctla-4)
US7250297B1 (en) 1997-09-26 2007-07-31 Pieris Ag Anticalins
WO2012072806A1 (fr) 2010-12-02 2012-06-07 Pieris Ag Mutéines dérivées de la lipocaline 2 humaine à affinité pour ctla-4
WO2016113203A1 (fr) 2015-01-12 2016-07-21 Pieris Ag Lymphocyte t produits par génie génétique et leurs utilisations
WO2016177762A1 (fr) 2015-05-04 2016-11-10 Pieris Pharmaceuticals Gmbh Protéines spécifiques de cd137
WO2017009456A1 (fr) 2015-07-15 2017-01-19 Pieris Pharmaceuticals Gmbh Nouvelles protéines spécifiques à lag-3
WO2018134274A1 (fr) 2017-01-18 2018-07-26 Pieris Pharmaceuticals Gmbh Mutéines de lipocaline ayant une affinité de liaison pour lag-3
US20190070262A1 (en) * 2017-09-06 2019-03-07 Yale University Interleukin-18 variants and methods of use

Patent Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0404097A2 (fr) 1989-06-22 1990-12-27 BEHRINGWERKE Aktiengesellschaft Récepteurs mono- et oligovalents, bispécifiques et oligospécifiques, ainsi que leur production et application
WO1993011161A1 (fr) 1991-11-25 1993-06-10 Enzon, Inc. Proteines multivalentes de fixation aux antigenes
US7250297B1 (en) 1997-09-26 2007-07-31 Pieris Ag Anticalins
WO2002101049A2 (fr) * 2001-03-08 2002-12-19 Ares Trading S.A. Production et utilisation de mutants d'interleukine-18
WO2004091517A2 (fr) * 2003-04-15 2004-10-28 Smithkline Beecham Corporation Conjugues comprenant l'interleukine 18 humaine il 18 et mutants de ces conjugues par substitution
WO2006056464A2 (fr) 2004-11-26 2006-06-01 Pieris Ag Compose a affinite pour l'antigene associe au lymphocyte t cytotoxique (ctla-4)
WO2012072806A1 (fr) 2010-12-02 2012-06-07 Pieris Ag Mutéines dérivées de la lipocaline 2 humaine à affinité pour ctla-4
WO2016113203A1 (fr) 2015-01-12 2016-07-21 Pieris Ag Lymphocyte t produits par génie génétique et leurs utilisations
WO2016177762A1 (fr) 2015-05-04 2016-11-10 Pieris Pharmaceuticals Gmbh Protéines spécifiques de cd137
WO2017009456A1 (fr) 2015-07-15 2017-01-19 Pieris Pharmaceuticals Gmbh Nouvelles protéines spécifiques à lag-3
WO2018134274A1 (fr) 2017-01-18 2018-07-26 Pieris Pharmaceuticals Gmbh Mutéines de lipocaline ayant une affinité de liaison pour lag-3
US20190070262A1 (en) * 2017-09-06 2019-03-07 Yale University Interleukin-18 variants and methods of use

Non-Patent Citations (20)

* Cited by examiner, † Cited by third party
Title
"SWISS-PROT", Database accession no. P31025
"UniProt", Database accession no. Q14116-2
ALTSCHUL ET AL., J MOL BIOL, vol. 215, 1990, pages 403 - 10
ALTSCHUL ET AL., NUCLEIC ACIDS RES, vol. 25, 1997, pages 3389 - 402
BRUCKDORFER ET AL., CURR PHARM BIOTECHNOL, vol. 5, 2004, pages 29 - 43
CHMIELEWSKI ET AL., CANCER RES, vol. 71, no. 17, 2011, pages 5697 - 706
FLOWER ET AL., BIOCHIM BIOPHYS ACTA, vol. 1482, 2000, pages 337 - 24
FLOWER, BIOCHEM J, vol. 318, 1996, pages 1 - 14
HOLLIGER ET AL., PROC NATL ACAD SCI U S A, vol. 90, no. 14, 1993, pages 6444 - 6448
JOHNSONWU, NUCLEIC ACIDS RES, vol. 28, 2000, pages 214 - 8
LOWMAN, ANNU REV BIOPHYS BIOMOL STRUCT, vol. 26, 1997, pages 401 - 24
PEGRAM ET AL., BLOOD, vol. 119, no. 18, 2012, pages 4133 - 41
PEGRAM ET AL., LEUKEMIA, vol. 29, no. 2, 2014, pages 415 - 22
REX ET AL., J CELL COMMUN SIGNAL, vol. 14, no. 2, 2020, pages 257 - 266
RODIMAKOWSKI, CURR OPIN BIOTECHNOL, vol. 10, 1999, pages 87 - 93
SMITHWATERMAN, J MOL BIOL, vol. 147, 1981, pages 195 - 7
VENTURI ET AL., J MOL BIOL, vol. 315, 2002, pages 1 - 8
WARD ET AL., NATURE, vol. 341, 1989, pages 544 - 546
XIANGZHI MENG ET AL: "Variola virus IL-18 binding protein interacts with three human IL-18 residues that are part of a binding site for human IL-18 receptor alpha subunit", VIROLOGY, ELSEVIER, AMSTERDAM, NL, vol. 358, no. 1, 1 January 2007 (2007-01-01), pages 211 - 220, XP008157403, ISSN: 0042-6822, [retrieved on 20060918], DOI: 10.1016/J.VIROL.2006.08.019 *
ZHANG ET AL., MOL THER, vol. 19, no. 4, 2011, pages 751 - 9

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