WO2025012105A2 - Mutéines d'interleukine 12 humaine - Google Patents

Mutéines d'interleukine 12 humaine Download PDF

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WO2025012105A2
WO2025012105A2 PCT/EP2024/068967 EP2024068967W WO2025012105A2 WO 2025012105 A2 WO2025012105 A2 WO 2025012105A2 EP 2024068967 W EP2024068967 W EP 2024068967W WO 2025012105 A2 WO2025012105 A2 WO 2025012105A2
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amino acid
subunit
mutein
sequence
seq
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WO2025012105A3 (fr
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Matthias Feige
Korbinian Liebl
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Technische Universitaet Muenchen
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Technische Universitaet Muenchen
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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]
    • C07K14/5434—IL-12
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to muteins of the human Interleukin 12 (hlL-12). More specifically, in a first aspect, the present invention relates to a mutein of the a- subunit of hlL-12 (SEQ ID NO: 1 ), wherein at least one of the amino acid residue(s) of said a-subunit selected from the group consisting of sequence positions 70, 71 , 72, 185, 187, and 189 is/are mutated.
  • the present invention relates to a mutein of the [3-subunit of hlL-12 (SEQ ID NO: 2), wherein at least one of the amino acid residue(s) of said [3-subunit selected from the group consisting of sequence positions 37, 38, 39, 67, 81 , 82, 103, 106, 115, 122, 123, 216, 221 , 232, and 312 is/are mutated.
  • the present invention relates to a mutein of hlL-12, comprising an a-subunit (p35) and a [3-subunit (p40), wherein the a-subunit is a mutein of the a-subunit of hlL-12 (SEQ ID NO: 1 ) according to the present invention and/or wherein the [3-subunit is a mutein of the [3-subunit of hlL-12 (SEQ ID NO: 2) according to the present invention.
  • the present invention relates to a nucleic acid molecule comprising i) a nucleotide sequence encoding the mutein of hlL-12 according to the present invention or ii) a nucleotide sequence encoding a) the mutein of the a-subunit of hlL-12 according to the present invention and/or b) the mutein of the [3-subunit of hlL-12 according to the present invention, a vector comprising said nucleic acid molecule, and a host cell comprising said nucleic acid molecule and/or said vector, respectively.
  • the present invention relates to an immune modulator comprising a mutein according to the present invention, or a pharmaceutical composition comprising a mutein according to the present invention and/or an immune modulator according to the present invention, preferably further comprising a pharmaceutically acceptable carrier.
  • the present invention relates to the use of a mutein according to the present invention for the manufacture of a medicament for treating a disease in a mammal.
  • the present invention relates to a mutein according to the present invention for use in the treatment of a disease.
  • the present invention relates to a method of treating an Interleukin 12-mediated disease in a mammal, comprising the step of administering a composition comprising a mutein according to the present invention, a pharmaceutical composition according to the present invention and/or an immune modulator according to the present invention to said mammal in need thereof.
  • the present invention relates to a method for producing a mutein according to the present invention comprising the steps of: (a) introducing into a nucleic acid molecule encoding a polypeptide, said polypeptide being (i) the hll_-12 a-subunit polypeptide (SEQ ID NO: 1 ), or (ii) a polypeptide comprising at least 90% sequence identity to the hlL-12 a-subunit polypeptide (SEQ ID NO: 1), or (iii) the hlL- 12 [3-subunit polypeptide (SEQ ID NO: 2), or (iv) a polypeptide comprising at least 90% sequence identity to the hlL-12 [3-subunit polypeptide (SEQ ID NO: 2), or (v) the hlL-12 polypeptide comprising an a-subunit (p35), and a [3-subunit (p40), a nucleotide sequence mutating at least one amino acid residue of said polypeptide in
  • Interleukins are key signaling molecules of the immune system that are classified into families based on structural similarities.
  • the interleukin 12 (IL-12) family consists of at least four members (IL-12, IL-23, IL-27, and IL-35) that are assigned to one family due to their unique heterodimeric character that separate these members from other ILs.
  • Each member is composed of an a-subunit that shows a cytokine-characteristic four-helix bundle fold (IL-12a, IL-23a, IL-27a) and a [3-subunit composed of two fibronectin (Fn) III domains (EBI3) with an additional immunoglobulin (Ig) domain in case of IL-12[3.
  • IL-12 family receptors which are also heterodimers formed by five different chains (IL-12R[31 , IL- 12R
  • Binding of interleukins to their respective receptors has been studied for example by Esch et al., 2020, and Georgy et al., 2021 , and, for example, Glassman et al., 2021 investigated the structural mechanism of receptor sharing used by IL-12 and IL-23 cytokines.
  • IL-12 subunit sharing on the level of cytokines and receptors may suggest closely related functions of the IL-12 family members, but the opposite holds true.
  • the effects of the four family members are surprisingly diverse and even opposing.
  • the mostly pro-inflammatory IL-12 and IL-23 are drivers of inflammation via Th1 differentiation and Th17 development, respectively.
  • IL-27 is an immunomodulatory cytokine, which on the one hand is able to promote Th1 differentiation and on the other hand suppresses pro-inflammatory Th17 cells and induces anti-inflammatory IL-10 producing T regulatory 1 cells.
  • IL-35 is the only strictly inhibitory family member and acts by suppression of conventional T cells.
  • Interleukins are secreted proteins that regulate immune cell functions. As such, they are of great interest to apply or inhibit in the clinics to modulate immune responses. Whereas antibody-based inhibition is well-established for certain interleukins, including IL-12 and IL-23 (Gaffen et al., 2014; Moschen et al., 2019), use of ILs as medicaments is still in its infancy. A major reason for this relates to the fact that ILs often have pleiotropic functions. Thus, potentially occurring organismwide side effects limit their usability as medicaments. A prime example for this is IL- 12, a heterodimeric cytokine composed of an a- and a [3-subunit.
  • IL-12 induces interferon gamma (IFNy) production and promotes the development of T cells into T cells with T helper 1 (Th1 ) phenotype, and as such IL-12 is considered an attractive molecule to re-activate or support immune responses in a generally immunosuppressive microenvironment as in case of solid tumors (Briukhovetska et al., 2021 ).
  • IFNy interferon gamma
  • Th1 T helper 1
  • IL-12 plays major roles in immune defense against intracellular pathogens by activating T cells and increasing antigen presentation and is moreover considered a potent anti-tumor molecule
  • IL-12 plays major roles in immune defense against intracellular pathogens by activating T cells and increasing antigen presentation and is moreover considered a potent anti-tumor molecule
  • the present invention provides a mutein of the a-subunit of human Interleukin 12 (SEQ ID NO: 1), wherein at least one of the amino acid residue(s) of said a-subunit selected from the group consisting of sequence positions 70, 71 , 72, 185, 187, and 189 is/are mutated.
  • said mutein comprises at least 90% sequence identity to the a-subunit of human Interleukin 12 (SEQ ID NO: 1).
  • At least one of the amino acid residue(s) of the a-subunit selected from the group consisting of sequence positions 70, 71 , 72, 185, and 187 is/are mutated, preferably from the group consisting of sequence positions 70, 71 , 72, and 187.
  • amino acid residue(s) of the a-subunit selected from the group consisting of sequence positions 70, 71 , 72, 185, 187, and 189, preferably from the group consisting of sequence positions 70, 71 , 72, and 187, is/are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A).
  • the present invention further provides a mutein of the [3-subunit of human Interleukin 12 (SEQ ID NO: 2), wherein at least one of the amino acid residue(s) of said [3-subunit selected from the group consisting of sequence positions 37, 38, 39, 67, 81 , 82, 103, 106, 115, 122, 123, 216, 221 , 232, and 312 is/are mutated.
  • said mutein comprises at least 90% sequence identity to the [3-subunit of human Interleukin 12 (SEQ ID NO: 2).
  • At least one of the amino acid residue(s) of the [3-subunit selected from the group consisting of sequence positions 38, 39, 67, 103, 106, 115, 122, 123, 221 , 232, and 312 is/are mutated, preferably from the group consisting of sequence positions 67, 115, 122, 123, 221 , 232, and 312.
  • amino acid residue(s) of the [3-subunit selected from the group consisting of sequence positions 103, 232, and 312, preferably at sequence position(s) 232 and/or 312, is/are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by glycine (G).
  • amino acid residue(s) of the [3-subunit at sequence position(s) 122 and/or 123 is/are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A).
  • amino acid residue(s) of the [3-subunit selected from the group consisting of sequence positions 37, 38, 39, 67, 81 , 82, 106, 115, 216, and 221 preferably from the group consisting of sequence positions 38, 39, 67, 106, 115, and 221 , more preferably from the group consisting of sequence positions 67, 115, and 221 , is/are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A).
  • the present invention further provides a mutein of human Interleukin 12, comprising an a-subunit (p35) and a [3-subunit (p40), wherein the a-subunit is a mutein of the a-subunit of human Interleukin 12 (SEQ ID NO: 1 ) according to the present invention and/or wherein the [3-subunit is a mutein of the [3-subunit of human Interleukin 12 (SEQ ID NO: 2) according to the present invention.
  • the present invention further provides a nucleic acid molecule comprising i) a nucleotide sequence encoding the mutein of human Interleukin 12 according to the present invention or ii) a nucleotide sequence encoding a) the mutein of the a- subunit of human Interleukin 12 according to the present invention, and/or b) the mutein of the [3-subunit of human Interleukin 12 according to the present invention, preferably wherein the nucleic acid molecule is operably linked to a regulatory sequence to allow expression of the nucleic acid molecule, wherein the regulatory sequence preferably comprises a promoter sequence.
  • the present invention further provides a pharmaceutical composition comprising a mutein according to the present invention, preferably further comprising a pharmaceutically acceptable carrier.
  • the present invention further provides a mutein according to the present invention for use as a medicament.
  • the present invention further provides a mutein according to the present invention for use in the treatment of a disease, wherein the disease is preferably a disease selected from the group consisting of an infectious disease, an autoimmune disease, cancer, a transplantation-related disease such as Graft-versus-Host- disease, a chronic inflammatory disease such as chronic inflammatory bowel disease, an acute inflammatory disease, sepsis, septic shock, diabetes or asthma.
  • a disease selected from the group consisting of an infectious disease, an autoimmune disease, cancer, a transplantation-related disease such as Graft-versus-Host- disease, a chronic inflammatory disease such as chronic inflammatory bowel disease, an acute inflammatory disease, sepsis, septic shock, diabetes or asthma.
  • Figure 1 shows modelling of the IL-12 : receptor complex.
  • Figure 1A shows in (i) IL-12 superposed on the IL-23 : receptor complex. The IL-23 receptor structure is depicted in light grey, the IL-12 components (IL-12a and IL-12
  • Figure 1 B shows the root mean squared deviation (RMSD) curves for three subparts of the system, in reference to the starting frame.
  • RMSD root mean squared deviation
  • Figure 2 shows the analysis of important residues within IL-12
  • Figure 2A shows the energetic contribution of every amino acid to the binding of IL-12
  • Figure 2B shows ten mutation candidates in IL-123 that are marked as light dots in Figure 2A. Values indicate energetic contributions to complex formation and are given in units of kcal/mol.
  • Figure 2C shows RMSF calculations for IL-123 in receptor-bound, and unbound form. Black dots indicate selected candidates, which are also shown in Figure 2A.
  • Figure 3 shows in Figure 3A the energetic contribution of every amino acid to the binding of IL-12a to IL-12R32 computed with MM/GBSA, and in Figure 3B the top six mutation candidates in IL-12a (most stabilizing contribution to binding). The values in Figure 3B indicate the energetic contribution to complex formation and are given in units of kcal/mol.
  • Figure 4 shows in silico engineered IL-12 mutants forming disulfide-bonded IL-12 heterodimers with similar structural properties compared to the wild-type protein.
  • Figure 4A shows the SDS-PAGE gels that confirmed purity and covalent linkage of IL-12 subunits for the different IL-12 mutants having one amino acid exchange in the IL-12
  • Figure 5 shows that IL-12-muteins activated natural killer (NK) cells in a concentration-dependent manner, showing reduced activity for IL-12
  • Figure 5A shows the stimulation of NK-92 cells with purified IL-12-muteins (10 ng/pl) and measurement of STAT4 phosphorylation via immunoblotting. Stimulation with PBS served as negative control, STAT4 signals served as loading control. All mutants were run on the same blot. Lines between blots indicate a change of the sample order for better visualization and mutant comparison.
  • Figure 5C shows the same as in Figure 5A, but NK-92 cells were stimulated with 1 ng/pl IL-12.
  • Figure 5D shows the same as in Figure 5B, with signal quantification of immunoblots shown in Figure 5C.
  • IL-12p E221A SEQ ID NO: 7
  • IL-12p L103G SEQ ID NO: 4
  • Figure 6 shows cell type specific IFNy secretion of primary human CD8 + T and NK cells induced by attenuated IL-12-muteins.
  • Figure 6A shows IL-12-mutein caused IFNy production by CD8 + T cells.
  • CD8 + T cells were isolated by MACS and stimulated with the indicated IL-12-muteins for 48 h.
  • IFNy secretion levels in the absence of IL-12 is shown as a horizontal dotted line.
  • Supernatant was analyzed for IFNy by ELISA.
  • Figure 6B shows that NK cells showed reduced IFNy secretion upon activation with IL-12-muteins. NK-cells were isolated by MACS and stimulated with the indicated IL-12-muteins for 48 h.
  • FIG. 6C shows exemplary EC50 graphs of cell responses after IL-12 stimulation of concentrations ranging from 0.01 pM to 10 nM, normalized to no IL-12 control. Arrows indicate differences of EC50 values of NK cell responses (squares) compared to CD8+ T cell responses (triangles) for the respective IL-12 variants.
  • the present invention relates to attenuated cytokine variants. More specifically, by combining computational modeling with insights obtained from experimental interleukin : receptor structures and dynamics, detailed molecular insights into the binding of human interleukins and human interleukin receptors were obtained. Based thereon, the inventors designed attenuated muteins of human interleukin 12 (hlL-12). It has been surprisingly found that the muteins according to the present invention show less activation of NK cells while maintaining CD8+ T cell activation compared to “native” hlL-12. Thus, the muteins according to the present invention have the advantage of providing a solution for making hlL-12 amenable to (medical) uses.
  • the inventors generated a high-resolution computational model of the hll_-12 : receptor complex that provided for the first time the opportunity of obtaining detailed insights into structure and dynamics of the human IL-12 : receptor complex.
  • These insights were successfully leveraged for engineering hlL-12 muteins for reduced receptor binding affinities compared to wild-type hlL-12 based on molecular dynamics simulations.
  • said hlL-12 muteins were experimentally validated showing that said muteins activate natural killer (NK) cells less compared to the hlL-12 wildtype while maintaining CD8 + T cell activation ability. This immunological signature is considered important for hlL-12-muteins suitable for cancer treatment, where natural killer cells contribute to severe side effects.
  • NK cells which are considered critical for systemic toxicity of hlL-12 by secretion of IFNy, have constant hlL-12 receptor levels, whereas the desired targets of hlL-12, T cells, increase surface expression of the hlL-12 receptor upon activation.
  • the engineered attenuated hlL-12 muteins thus are expected to provide a therapeutic hlL-12 window by preferentially activating T cells.
  • the hlL-12 muteins according to the present invention are expected to elicit fewer side-effects while maintaining relevant biological activity compared to wild-type hlL-12, said innovative hlL-12 muteins are advantageous, e.g., for medical purposes.
  • the term “mutein” refers to a variant of a native molecule, wherein said native molecule can be nucleic acid and/or amino acid molecule.
  • said mutein in case the mutein is a mutein of a native molecule being a nucleic acid molecule, said mutein has at least one nucleic acid residue mutated compared to the native molecule.
  • the mutein has at least one nucleic acid residue replaced by a nucleic acid residue other than the respective nucleic acid residue of the native molecule’s nucleic acid sequence at the respective position.
  • the mutein is a mutein of a native molecule being an amino acid molecule
  • said mutein has at least one amino acid residue mutated compared to the native molecule.
  • the mutein has at least one amino acid residue replaced by an amino acid residue other than the respective amino acid residue of the native molecule’s amino acid sequence at the respective position.
  • a mutation refers to a deletion of a residue, an addition of one or more residues, and/or a replacement of a residue, wherein said replacement may be a replacement of one residue by another residue or a replacement of one residue by more than one residue(s).
  • a mutation refers to a replacement of one residue by a single residue any other than the replaced residue, preferably by alanine (A) or glycine (G).
  • a mutated amino acid residue is an alanine (A) or glycine (G) that replaces the respective native amino acid residue.
  • A alanine
  • G glycine
  • a mutation can affect intracellular localization, functionality and/or activity of a molecule.
  • the term “mutein” refers to a mutein of a native molecule, wherein said mutein exhibits a different cellular, biochemical and/or immunological effect compared to the native molecule.
  • the native molecule is a molecule that can initiate a signal cascade in a cell upon binding to a receptor
  • a mutein of said native molecule preferably exhibits a lower or higher potential of initiating the signal cascade upon binding to the receptor compared to the native molecule.
  • Different mechanisms can be envisioned that can result in a change of the effect of a mutein compared to its native molecule.
  • muteins according to the present invention are secretion-competent muteins and thus, the muteins are able to perform a complete passage through the secretory pathway of a cell and through the cytoplasmic membrane.
  • hlL-12a refers to the native a-subunit of human Interleukin 12 with the sequence set forth in SEQ ID NO: 1
  • 3 refers to the native [3-subunit of human Interleukin 12 with the sequence set forth in SEQ ID NO: 2.
  • sequence set forth in SEQ ID NO: 1 refers to the “native” a- subunit of human Interleukin 12
  • sequence set forth in SEQ ID NO: 2 refers to the “native” [3-subunit of human Interleukin 12.
  • 3 as set forth in SEQ ID NO: 1 and 2, respectively, may also be understood as wild-type hlL-12a and wild-type hlL-12
  • Sequences set forth in SEQ ID NO: 1 and 2 are also deposited in UniProtKB, with the sequence of hlL-12a, SEQ ID NO: 1 , being also deposited under UniProtKB accession number P29459, and the sequence of hlL-12
  • a mutein according to the present invention has an attenuated activity compared to the activity of the native molecule. This is advantageous as hlL- 12 mute ins with attenuated and thus, reduced activity compared to native hlL-12 can pave the way for making hlL-12 amenable for (medical) uses.
  • the present invention relates to a mutein of the a-subunit of human Interleukin 12 (SEQ ID NO: 1 ), wherein at least one of the amino acid residue(s) of said a-subunit selected from the group consisting of sequence positions 70, 71 , 72, 185, 187, and 189 is/are mutated.
  • the mutein of hlL-12a has the amino acid sequence of hlL-12a as set forth in SEQ ID NO: 1 , except that at least one of the amino acid residues selected from the group consisting of sequence positions 70, 71 , 72, 185, 187, and 189 is mutated.
  • the numbering of sequence positions of said mutein of the a-subunit of human Interleukin 12 corresponds to the numbering of the sequence positions of the a-subunit of human Interleukin 12 according to SEQ ID NO: 1.
  • the mutein of hlL-12a can have the amino acid sequence of the native hlL-12a as set forth in SEQ ID NO: 1 , except that the amino acid residue(s) at position(s) 70, 71 , 72, 185, 187 and/or 189 is/are mutated.
  • the mutein of hlL-12a can have the amino acid sequence of the native hlL-12a as set forth in SEQ ID NO: 1 , except that the amino acid residue(s) at position(s) 70, 71 , 72, 185 and/or 187 is/are mutated.
  • the mutein of hlL-12a can have the amino acid sequence of the native hlL-12a as set forth in SEQ ID NO: 1 , except that the amino acid residue(s) at position(s) 70, 71 , 72 and/or 187 is/are mutated.
  • the mutein of hlL-12a can have the amino acid sequence of the native hlL-12a as set forth in SEQ ID NO: 1 , except that the amino acid residue(s) at position(s) 71 , 72 and/or 187 is/are mutated.
  • the mutein of hlL- 12a can have the amino acid sequence of the native hlL-12a as set forth in SEQ ID NO: 1 , except that the amino acid residue(s) at position(s) 72 and/or 187 is/are mutated.
  • the mutein of hlL-12a can have the amino acid sequence of the native hlL-12a as set forth in SEQ ID NO: 1 , except that the amino acid residues at positions 70 and 71 , 70 and 72, 71 and 72, 70 and 187, 71 and 187, or 72 and 187 are mutated.
  • the mutein of hlL-12a can have the amino acid sequence of the native hlL-12a as set forth in SEQ ID NO: 1 , except that the amino acid residues at positions 70, 71 and 72; 70, 71 and 187; 70, 72 and 187; or 71 , 72 and 187 are mutated.
  • the mutein of hlL-12a can have the amino acid sequence of the native hlL-12a as set forth in SEQ ID NO: 1 , except that the amino acid residues at positions 70, 71 , 72 and 187 are mutated.
  • the mutein of hlL-12a comprises at least 90% sequence identity to the a-subunit of human Interleukin 12 (SEQ ID NO: 1).
  • the mutein of hlL-12a has at least one of the amino acid residues of said a-subunit selected from the group consisting of sequence positions 70, 71 , 72, 185, 187, and 189 mutated and preferably comprises at least 90% sequence identity to hlL-12a (SEQ ID NO: 1 ).
  • the mutein of hlL-12a may have at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity to the a- subunit of human Interleukin 12 (SEQ ID NO: 1 ).
  • sequence identity refers to a property of sequences that measures their similarity or relationship.
  • sequence identity means the percentage of pair-wise identical residues - following (homology) alignment of a nucleic acid and/or amino acid sequence with a given reference sequence - with respect to the number of residues in the longer of the two compared sequences. Sequence identity is measured by dividing the number of identical residues by the total number of residues and multiplying the product by 100.
  • the percentage of sequence homology or sequence identity can, for example, be determined herein using the program BLASTP, version blastp 2.2.5 (November 16, 2002; cf. Altschul, S. F. et al. (1997) Nucl. Acids Res.25, 3389-3402).
  • the percentage of homology may be based on the alignment of the entire polypeptide sequences (matrix: BLOSUM 62; gap costs: 11.1 ; cutoff value set to 10' 3 ) including the respective sequences. It may be calculated as the percentage of numbers of "positives" (homologous amino acid residues) indicated as result in the BLASTP program output divided by the total number of amino acid residues selected by the program for the alignment.
  • the mutein of hlL-12a may be a mutein, wherein at least one of the amino acid residue(s) of the a-subunit selected from the group consisting of sequence positions 70, 71 , 72, 185, and 187 is/are mutated.
  • the mutein of hlL-12a may be a mutein, wherein at least one of the amino acid residue(s) of the a-subunit selected from the group consisting of sequence positions 70, 71 , 72, and 187 is/are mutated.
  • the mutein of hlL-12a may be a mutein, wherein i) at least one of the amino acid residue(s) of the a-subunit selected from the group consisting of sequence positions 70 to 72 is/are mutated, and/or wherein ii) the amino acid residue of the a-subunit at sequence position 187 is mutated.
  • the mutein of h I L- 12a may be a mutein, wherein i) at least one of the amino acid residue(s) of the a- subunit selected from the group consisting of sequence positions 70 to 72 is/are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , and/or wherein ii) the amino acid residue of the a-subunit at sequence position 187 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 .
  • the mutein of hlL-12a may be a mutein, wherein the amino acid residue of the a-subunit at sequence position 70 is mutated.
  • the mutein of hlL-12a may be a mutein, wherein the amino acid residue of the a-subunit at sequence position 70 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A).
  • the mutein of hlL-12a is a mutein, wherein the amino acid residue of the a-subunit at sequence position 70 is replaced by alanine.
  • the amino acid sequence of the mutein hlL-12a D70A is set forth herein as SEQ ID NO: 18.
  • the mutein of hlL-12a may be a mutein, wherein the amino acid residue of the a-subunit at sequence position 71 is mutated.
  • the mutein of hlL-12a may be a mutein, wherein the amino acid residue of the a-subunit at sequence position 71 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A).
  • the mutein of hlL-12a is a mutein, wherein the amino acid residue of the a-subunit at sequence position 71 is replaced by alanine.
  • the amino acid sequence of the mutein hlL-12a H71A is set forth herein as SEQ ID NO: 19.
  • the mutein of hlL-12a may be a mutein, wherein the amino acid residue of the a-subunit at sequence position 72 is mutated.
  • the mutein of hlL-12a may be a mutein, wherein the amino acid residue of the a-subunit at sequence position 72 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A).
  • the mutein of hlL-12a is a mutein, wherein the amino acid residue of the a-subunit at sequence position 72 is replaced by alanine.
  • the amino acid sequence of the mutein hlL-12a E72A is set forth herein as SEQ ID NO: 20.
  • the mutein of hlL-12a may be a mutein, wherein the amino acid residue of the a-subunit at sequence position 185 is mutated.
  • the mutein of hlL-12a may be a mutein, wherein the amino acid residue of the a-subunit at sequence position 185 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A).
  • the mutein of hlL-12a is a mutein, wherein the amino acid residue of the a-subunit at sequence position 185 is replaced by alanine.
  • the amino acid sequence of the mutein hlL-12a E185A is set forth herein as SEQ ID NO: 21.
  • the mutein of hlL-12a may be a mutein, wherein the amino acid residue of the a-subunit at sequence position 187 is mutated.
  • the mutein of hlL-12a may be a mutein, wherein the amino acid residue of the a-subunit at sequence position 187 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A).
  • the mutein of hlL-12a is a mutein, wherein the amino acid residue of the a-subunit at sequence position 187 is replaced by alanine.
  • the amino acid sequence of the mutein hlL-12a D187A is set forth herein as SEQ ID NO: 22.
  • the mutein of hlL-12a may be a mutein, wherein the amino acid residue of the a-subunit at sequence position 189 is mutated.
  • the mutein of hlL-12a may be a mutein, wherein the amino acid residue of the a-subunit at sequence position 189 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A).
  • the mutein of hlL-12a is a mutein, wherein the amino acid residue of the a-subunit at sequence position 189 is replaced by alanine.
  • the amino acid sequence of the mutein hlL-12a Y189A is set forth herein as SEQ ID NO: 23.
  • the present invention relates to a mutein of the [3-subunit of human Interleukin 12 (SEQ ID NO: 2), wherein at least one of the amino acid residue(s) of said [3-subunit selected from the group consisting of sequence positions 37, 38, 39, 67, 81 , 82, 103, 106, 115, 122, 123, 216, 221 , 232, and 312 is/are mutated.
  • the mutein of hlL-12[3 has the amino acid sequence of hlL-12[3 as set forth in SEQ ID NO: 2, except that at least one of the amino acid residue(s) selected from the group consisting of sequence positions 37, 38, 39, 67, 81 , 82, 103, 106, 115, 122, 123, 216, 221 , 232, and 312 is/are mutated.
  • the numbering of sequence positions of said mutein of the p-subunit of human Interleukin 12 corresponds to the numbering of the sequence positions of the p-subunit of human Interleukin 12 according to SEQ ID NO: 2.
  • the mutein of hlL-12[3 can have the amino acid sequence of the native hlL-12[3 as set forth in SEQ ID NO: 2, except that the amino acid residue(s) at positions 37, 38, 39, 67, 81 , 82, 103, 106, 115, 122, 123, 216, 221 , 232, and/or 312 is/are mutated.
  • the mutein of hlL-12[3 can have the amino acid sequence of the native hlL-12[3 as set forth in SEQ ID NO: 2, except that the amino acid residue(s) at positions 38, 39, 67, 81 , 82, 103, 106, 115, 122, 123, 216, 221 , 232, and/or 312 is/are mutated.
  • 3 can have the amino acid sequence of the native hlL-12(3 as set forth in SEQ ID NO: 2, except that the amino acid residue(s) at positions 39, 67, 81 , 82, 103, 106, 115, 122, 123, 216, 221 , 232, and/or 312 is/are mutated.
  • 3 can have the amino acid sequence of the native hll_-12(3 as set forth in SEQ ID NO: 2, except that the amino acid residue(s) at positions 67, 81 , 82, 103, 106, 115, 122, 123, 216, 221 , 232, and/or 312 is/are mutated.
  • 3 can have the amino acid sequence of the native hll_-12(3 as set forth in SEQ ID NO: 2, except that the amino acid residue(s) at positions 67, 82, 103, 106, 115, 122, 123, 216, 221 , 232, and/or 312 is/are mutated.
  • the mutein of hll_-12(3 can have the amino acid sequence of the native hll_-12
  • mutein of hll_-12(3 can have the amino acid sequence of the native hll_-12
  • 3 can have the amino acid sequence of the native hll_-12(3 as set forth in SEQ ID NO: 2, except that the amino acid residue(s) at positions 67, 103, 122, 123, 216, 221 , 232, and/or 312 is/are mutated.
  • the mutein of hll_-12(3 can have the amino acid sequence of the native hll_-12(3 as set forth in SEQ ID NO: 2, except that the amino acid residue(s) at positions 103, 122, 123, 216, 221 , 232, and/or 312 is/are mutated.
  • 3 can have the amino acid sequence of the native hll_-12(3 as set forth in SEQ ID NO: 2, except that the amino acid residue(s) at positions 122, 123, 216, 221 , 232, and/or 312 is/are mutated.
  • 3 can have the amino acid sequence of the native hll_-12(3 as set forth in SEQ ID NO: 2, except that the amino acid residue(s) at positions 123, 216, 221 , 232, and/or 312 is/are mutated.
  • 3 can have the amino acid sequence of the native hll_-12(3 as set forth in SEQ ID NO: 2, except that the amino acid residue(s) at positions 216, 221 , 232, and/or 312 is/are mutated.
  • the mutein of hll_-12(3 can have the amino acid sequence of the native hll_-12
  • the mutein of hll_-12(3 can have the amino acid sequence of the native hll_-12(3 as set forth in SEQ ID NO: 2, except that the amino acid residue(s) at positions 221 , 232, and/or 312 is/are mutated.
  • 3 can have the amino acid sequence of the native hll_-12
  • 3 can have the amino acid sequence of the native hll_-12
  • 3 comprises at least 90% sequence identity to the [3-subunit of human Interleukin 12 (SEQ ID NO: 2).
  • 3 has at least one of the amino acid residue(s) of said [3-subunit selected from the group consisting of sequence positions 37, 38, 39, 67, 81 , 82, 103, 106, 115, 122, 123, 216, 221 , 232, and 312 mutated and preferably comprises at least 90% sequence identity to hll_-12[3 (SEQ ID NO: 2).
  • the mutein of hll_-12[3 may have at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity to the [3- subunit of human Interleukin 12 (SEQ ID NO: 2).
  • the mutein of hll_-12[3 may be a mutein, wherein at least one of the amino acid residue(s) of the [3-subunit selected from the group consisting of sequence positions 38, 39, 67, 103, 106, 1 15, 122, 123, 221 , 232, and 312 is/are mutated.
  • the mutein of hlL-12[3 may be a mutein, wherein at least one of the amino acid residue(s) of the [3-subunit selected from the group consisting of sequence positions 67, 115, 122, 123, 221 , 232, and 312 is/are mutated.
  • the mutein of hlL-12[3 may be a mutein, wherein at least one of the amino acid residue(s) of the [3-subunit selected from the group consisting of sequence positions 103, 232 and 312 is/are mutated, preferably at sequence positions 232 and/or 312.
  • This is advantageous as it has been shown that a mutation of any of these sequence positions can entropically contribute to receptor binding and thus, IL-12
  • these three amino acid residues represent rigid positions in an unbound hlL-12
  • Replacing any of the amino acid residues at positions 103, 232 and 312, preferably at sequence positions 232 and/or 312, by any other amino acid residue than the one at the respective position in SEQ ID NO: 2, preferably by glycine (G), can alter the dynamics of hlL-12(3 and thus, can impede receptor binding.
  • said muteins are also advantageous as they allow engineering attenuated IL-12-muteins.
  • the mutein of hlL-12(3 may be a mutein, wherein the amino acid residues of the [3-subunit selected at sequence positions 122 and/or 123 is/are mutated.
  • This is advantageous, as it has been shown that a mutation of any of these sequence positions can entropically contribute to receptor binding and thus, IL-12
  • an amino acid residue replacement at any of these two sequence positions by any other amino acid residue than the amino acid residue at the respective position in SEQ ID NO: 2, preferably by alanine (A) can entropically destabilize receptor binding.
  • said muteins are also advantageous as they allow engineering attenuated IL-12-muteins.
  • the mutein of hlL-12(3 may be a mutein, wherein at least one of the amino acid residue(s) of the [3-subunit selected from the group consisting of sequence positions 37, 38, 39, 67, 81 , 82, 106, 115, 216 and 221 is/are mutated.
  • This is advantageous, as it has been shown that a mutation of any of these sequence positions can enthalpically contribute to receptor binding and thus, IL-120 : IL-12R01 complex formation.
  • 3 may be a mutein, wherein i) at least one of the amino acid residue(s) of the [3-subunit selected from the group consisting of sequence positions 103, 232, and 312 is/are mutated, and/or wherein ii) the amino acid residue(s) of the [3-subunit at sequence position(s) 122 and/or 123 is/are mutated, and/or wherein iii) at least one of the amino acid residue(s) of the [3-subunit selected from the group consisting of sequence positions 37, 38, 39, 67, 81 , 82, 106, 115, 216, and 221 is/are mutated.
  • the mutein of h I L-12[3 may be a mutein, wherein i) at least one of the amino acid residue(s) of the [3-subunit selected from the group consisting of sequence positions 103, 232, and 312 is/are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by glycine (G), and/or wherein ii) the amino acid residue(s) of the [3-subunit at sequence position(s) 122 and/or 123 is/are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A), and/or wherein iii) at least one of the amino acid residue(s) of the [3-subunit selected from the group consisting of sequence positions 37, 38, 39, 67, 81 , 82, 106, 115, 216, and 221 is replaced by an amino acid other than the amino acid
  • 3 may be a mutein, wherein i) at least one of the amino acid residue(s) of the [3-subunit selected from the group consisting of sequence positions 103, 232, and 312 is/are mutated, and/or wherein ii) the amino acid residue(s) of the [3-subunit at sequence position(s) 122 and/or 123 is/are mutated, and/or wherein iii) at least one of the amino acid residue(s) of the [3-subunit selected from the group consisting of sequence positions 38, 39, 67, 106, 115, and 221 is/are mutated.
  • the mutein of hlL-12[3 may be a mutein, wherein i) at least one of the amino acid residue(s) of the [3-subunit selected from the group consisting of sequence positions 103, 232, and 312 is/are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (G), and/or wherein ii) the amino acid residue(s) of the [3-subunit at sequence position(s) 122 and/or 123 is/are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A), and/or wherein iii) at least one of the amino acid residue(s) of the [3-subunit selected from the group consisting of sequence positions 38, 39, 67, 106, 115, and 221 is/are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO:
  • the mutein of hlL-12[3 may be a mutein, wherein i) the amino acid residue(s) of the [3-subunit at sequence position(s) 232 and/or 312 is/are mutated, and/or wherein ii) the amino acid residue(s) of the [3-subunit at sequence position(s) 122 and/or 123 is/are mutated, and/or wherein iii) at least one of the amino acid residue(s) of the [3-subunit selected from the group consisting of sequence positions 67, 115, and 221 is/are mutated.
  • the mutein of h I L-12[3 may be a mutein, wherein i) the amino acid residue(s) of the [3-subunit at sequence position(s) 232 and/or 312 is/are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by glycine (G), and/or wherein ii) the amino acid residue(s) of the [3-subunit at sequence position(s) 122 and/or 123 is/are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A), and/or wherein iii) at least one of the amino acid residue(s) of the [3-subunit selected from the group consisting of sequence positions 67, 115, and 221 is/are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A).
  • the mutein of hlL-12[3 may be a mutein, wherein the amino acid residue of the [3-subunit at sequence position 103 is mutated.
  • the mutein of hlL-12[3 may be a mutein, wherein the amino acid residue of the [3-subunit at sequence position 103 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by glycine (G).
  • the mutein of hlL-12[3 is a mutein, wherein the amino acid residue of the [3-subunit at sequence position 103 is replaced by glycine.
  • the amino acid sequence of the mutein hlL-12[3 L103G is set forth herein as SEQ ID NO: 4.
  • 3 may be a mutein, wherein the amino acid residue of the [3-subunit at sequence position 232 is mutated.
  • 3 may be a mutein, wherein the amino acid residue of the [3-subunit at sequence position 232 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by glycine (G).
  • the mutein of hlL-12[3 is a mutein, wherein the amino acid residue of the [3-subunit at sequence position 232 is replaced by glycine.
  • the amino acid sequence of the mutein hlL-12[3 I232G is depicted herein as SEQ ID NO: 8.
  • the mutein of hlL-12[3 may be a mutein, wherein the amino acid residue of the [3-subunit at sequence position 312 is mutated.
  • the mutein of hlL-12[3 may be a mutein, wherein the amino acid residue of the [3-subunit at sequence position 312 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by glycine (G).
  • the mutein of hlL-12[3 is a mutein, wherein the amino acid residue of the [3-subunit at sequence position 312 is replaced by glycine.
  • the amino acid sequence of the mutein hlL-12[3 D312G is depicted herein as SEQ ID NO: 9.
  • the mutein of hlL-12[3 may be a mutein, wherein the amino acid residue of the [3-subunit at sequence position 122 is mutated.
  • the mutein of hlL-12[3 may be a mutein, wherein the amino acid residue of the [3-subunit at sequence position 122 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A).
  • the mutein of hlL-12[3 is a mutein, wherein the amino acid residue of the [3-subunit at sequence position 122 is replaced by alanine.
  • the amino acid sequence of the mutein hlL-12[3 EI22A is depicted herein as SEQ ID NO: 10.
  • 3 may be a mutein, wherein the amino acid residue of the [3-subunit at sequence position 123 is mutated.
  • 3 may be a mutein, wherein the amino acid residue of the [3-subunit at sequence position 123 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A).
  • the mutein of hlL-12[3 is a mutein, wherein the amino acid residue of the [3-subunit at sequence position 123 is replaced by alanine.
  • the amino acid sequence of the mutein hlL-12[3 P123A is depicted herein as SEQ ID NO: 11.
  • the mutein of hlL-12[3 may be a mutein, wherein the amino acid residue of the [3-subunit at sequence position 82 is mutated.
  • the mutein of hlL-12[3 may be a mutein, wherein the amino acid residue of the [3-subunit at sequence position 82 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A).
  • the mutein of hlL-12[3 is a mutein, wherein the amino acid residue of the [3-subunit at sequence position 82 is replaced by alanine.
  • the amino acid sequence of the mutein hlL-12(3 F82A is depicted herein as SEQ ID NO: 12.
  • the mutein of hlL-12[3 may be a mutein, wherein the amino acid residue of the [3-subunit at sequence position 37 is mutated.
  • the mutein of hlL-12[3 may be a mutein, wherein the amino acid residue of the [3-subunit at sequence position 37 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A).
  • the mutein of hlL-12[3 is a mutein, wherein the amino acid residue of the [3-subunit at sequence position 37 is replaced by alanine.
  • the amino acid sequence of the mutein hlL-12(3 W37A is depicted herein as SEQ ID NO: 13.
  • 3 may be a mutein, wherein the amino acid residue of the [3-subunit at sequence position 39 is mutated.
  • 3 may be a mutein, wherein the amino acid residue of the [3-subunit at sequence position 39 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A).
  • the mutein of hlL-12[3 is a mutein, wherein the amino acid residue of the [3-subunit at sequence position 39 is replaced by alanine.
  • the amino acid sequence of the mutein hlL-12(3 P39A is depicted herein as SEQ ID NO: 14.
  • the mutein of hlL- 12[3 may be a mutein, wherein the amino acid residue of the [3-subunit at sequence position 106 is mutated.
  • the mutein of hlL- 12[3 may be a mutein, wherein the amino acid residue of the [3-subunit at sequence position 106 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A).
  • the mutein of hlL-12[3 is a mutein, wherein the amino acid residue of the [3-subunit at sequence position 106 is replaced by alanine.
  • the amino acid sequence of the mutein hlL-12[3 K106A is depicted herein as SEQ ID NO: 15.
  • the mutein of hlL- 12[3 may be a mutein, wherein the amino acid residue of the [3-subunit at sequence position 81 is mutated.
  • the mutein of hlL- 12[3 may be a mutein, wherein the amino acid residue of the [3-subunit at sequence position 81 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A).
  • the mutein of hlL-12[3 is a mutein, wherein the amino acid residue of the [3-subunit at sequence position 81 is replaced by alanine.
  • the amino acid sequence of the mutein hlL-12(3 E81A is depicted herein as SEQ ID NO: 16.
  • 3 may be a mutein, wherein the amino acid residue of the [3-subunit at sequence position 115 is mutated.
  • 3 may be a mutein, wherein the amino acid residue of the [3-subunit at sequence position 115 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A).
  • the mutein of hlL-12[3 is a mutein, wherein the amino acid residue of the [3-subunit at sequence position 115 is replaced by alanine.
  • the amino acid sequence of the mutein hlL-12[3 D115A is depicted herein as SEQ ID NO: 5.
  • the mutein of hlL- 12[3 may be a mutein, wherein the amino acid residue of the [3-subunit at sequence position 67 is mutated.
  • the mutein of hlL- 12[3 may be a mutein, wherein the amino acid residue of the [3-subunit at sequence position 67 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A).
  • the mutein of hlL-12[3 is a mutein, wherein the amino acid residue of the [3-subunit at sequence position 67 is replaced by alanine.
  • the amino acid sequence of the mutein hlL-12(3 E67A is depicted herein as SEQ ID NO: 3.
  • the mutein of hlL- 12[3 may be a mutein, wherein the amino acid residue of the [3-subunit at sequence position 216 is mutated.
  • the mutein of hlL- 12[3 may be a mutein, wherein the amino acid residue of the [3-subunit at sequence position 216 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A).
  • the mutein of hlL-12[3 is a mutein, wherein the amino acid residue of the [3-subunit at sequence position 216 is replaced by alanine.
  • the amino acid sequence of the mutein hlL-12[3 H216A is depicted herein as SEQ ID NO: 6.
  • the mutein of hlL- 12[3 may be a mutein, wherein the amino acid residue of the [3-subunit at sequence position 38 is mutated.
  • 3 may be a mutein, wherein the amino acid residue of the [3-subunit at sequence position 38 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A).
  • 3 is a mutein, wherein the amino acid residue of the [3-subunit at sequence position 38 is replaced by alanine.
  • 3 Y38A is depicted herein as SEQ ID NO: 17.
  • the mutein of hlL- 12[3 may be a mutein, wherein the amino acid residue of the [3-subunit at sequence position 221 is mutated.
  • the mutein of hlL- 12[3 may be a mutein, wherein the amino acid residue of the [3-subunit at sequence position 221 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A).
  • the mutein of hlL-12[3 is a mutein, wherein the amino acid residue of the [3-subunit at sequence position 221 is replaced by alanine.
  • the amino acid sequence of the mutein hlL-12[3 E221A is depicted herein as SEQ ID NO: 7.
  • the present invention relates to a mutein of human Interleukin 12, comprising an a-subunit (p35) and a [3-subunit (p40), wherein the a- subunit is a mutein of the a-subunit of human Interleukin 12 (SEQ ID NO: 1 ) according to the present invention and/or wherein the [3-subunit is a mutein of the [3- subunit of human Interleukin 12 (SEQ ID NO: 2) according to the present invention.
  • hlL-12a mutein and/or hlL-12[3 mutein according to the present invention the same applies as it has been described herein in connection with the muteins of the a-subunit of human Interleukin 12 (SEQ ID NO: 1 ) according to the present invention and/or the muteins of the [3-subunit of human Interleukin 12 (SEQ ID NO: 2) according to the present invention, respectively.
  • SEQ ID NO: 1 human Interleukin 12
  • SEQ ID NO: 2 [3-subunit of human Interleukin 12
  • the mutein of hll_-12 according to the present invention may comprise an a-subunit (p35), wherein said a-subunit may be a mutein of the a- subunit of human Interleukin 12 (SEQ ID NO: 1 ) according to the present invention.
  • the mutein of hlL-12 according to the present invention may comprise an a-subunit (p35), wherein said a-subunit may be a mutein of the a-subunit of human Interleukin 12 (SEQ ID NO: 1 ), wherein at least one of the amino acid residue(s) of said a-subunit selected from the group consisting of sequence positions 70, 71 , 72, 185, 187, and 189 is mutated, preferably selected from the group consisting of sequence positions 70, 71 , 72, and 187.
  • SEQ ID NO: 1 human Interleukin 12
  • the mutein of h IL-12 according to the present invention comprises an a-subunit (p35), wherein said a- subunit may be a mutein of the a-subunit of human Interleukin 12 (SEQ ID NO: 1 ) comprising at least 90% sequence identity to the a-subunit of human Interleukin 12 (SEQ ID NO: 1 ).
  • the hlL-12a mutein comprised in the hlL-12 mutein may have at least one of the amino acid residue(s) selected from the group consisting of sequence positions 70, 71 , 72, 185, 187, and 189, preferably selected from the group consisting of sequence positions 70, 71 , 72, and 187, which is/are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A).
  • A alanine
  • the hlL-12a mutein comprised in the hlL-12 mutein may have an amino acid residue at sequence positions 70, 71 , 72, 185, 187, and/or 189, which is/are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A).
  • the hlL-12a mutein comprised in the hlL-12 mutein may have an amino acid residue at sequence positions 70, 71 , 72, 185, and/or 187, which is/are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A).
  • the hlL-12a mutein comprised in the hll_-12 mutein may have an amino acid residue at sequence positions 70, 71 , 72, and/or 187, which is/are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A).
  • the hlL-12a mutein comprised in the hll_-12 mutein may have an amino acid residue at sequence positions 71 , 72, and/or 187, which is/are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A).
  • the hlL-12a mutein comprised in the hlL-12 mutein may have an amino acid residue at sequence positions 72 and/or 187, which is/are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A).
  • the mutein of hll_-12 according to the present invention comprises a [3-subunit (p40), wherein said [3-subunit may be a mutein of the [3- subunit of human Interleukin 12 (SEQ ID NO: 2) according to the present invention.
  • the mutein of h IL-12 may comprise a [3-subunit (p40), wherein said [3-subunit may be a mutein of the [3-subunit of human Interleukin 12 (SEQ ID NO: 2), wherein at least one of the amino acid residue(s) of the [3-subunit selected from the group consisting of sequence positions 37, 38, 39, 67, 81 , 82, 103, 106, 115, 122, 123, 216, 221 , 232, and 312 is/are mutated, preferably wherein the at least one of the amino acid residue(s) of the [3-subunit is selected from the group consisting of sequence positions 39, 67, 103, 106, 115, 122, 123, 221 , 232, and 312, more preferably wherein the at least one amino acid residue(s) of the [3-subunit is selected from the group consisting of sequence positions 39, 67, 103, 115,
  • the mutein of hlL-12 according to the present invention comprises a [3-subunit (p40), wherein said [3- subunit may be a mutein of the [3-subunit of human Interleukin 12 (SEQ ID NO: 2) comprising at least 90% sequence identity to the [3-subunit of human Interleukin 12 (SEQ ID NO: 2).
  • 3 mutein comprised in the hlL-12 mutein may have at least one of the amino acid residue(s) selected from the group consisting of sequence positions 37, 38, 39, 67, 81 , 82, 103, 106, 115, 122, 123, 216, 221 , 232, and 312, preferably the at least one of the amino acid residue(s) is/are selected from the group consisting of sequence positions 39, 67, 103, 106, 115, 122, 123, 221 , 232, and 312, more preferably the at least one of the amino acid residue(s) is/are selected from the group consisting of sequence positions 39, 67, 103, 115, 122, 123, 221 , 232, and 312 and/or is/are selected from the group consisting of sequence positions 67, 115, 122, 123, 221 , 232, and 312, which is/are replaced by
  • 3 mutein comprised in the hll_-12 mutein may have (an) amino acid residue(s) at sequence position(s) 37, 38, 39, 67, 81 , 82, 103, 106, 115, 122, 123, 216, 221 , 232, and/or 312, which is/are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A) or glycine (G).
  • A alanine
  • G glycine
  • 3 mutein comprised in the hll_-12 mutein may have (an) amino acid residue(s) at sequence position(s) 38, 39, 67, 81 , 82, 103, 106, 115, 122, 123, 216, 221 , 232, and/or 312, which is/are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A) or glycine (G).
  • A alanine
  • G glycine
  • 3 mutein comprised in the hll_-12 mutein may have (an) amino acid residue(s) at sequence position(s) 39, 67, 81 , 82, 103, 106, 115, 122, 123, 216, 221 , 232, and/or 312, which is/are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A) or glycine (G).
  • A alanine
  • G glycine
  • 3 mutein comprised in the hll_-12 mutein may have (an) amino acid residue(s) at sequence position(s) 67, 81 , 82, 103, 106, 115, 122, 123, 216, 221 , 232, and/or 312, which is/are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A) or glycine (G).
  • A alanine
  • G glycine
  • 3 mutein comprised in the hll_-12 mutein may have (an) amino acid residue(s) at sequence position(s) 81 , 82, 103, 106, 115, 122, 123, 216, 221 , 232, and/or 312, which is/are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A) or glycine (G).
  • A alanine
  • G glycine
  • 3 mutein comprised in the hll_-12 mutein may have (an) amino acid residue(s) at sequence position(s) 81 , 82, 103, 106, 115, 122, 123, 216, 221 , 232, and/or 312, which is/are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A) or glycine (G).
  • A alanine
  • G glycine
  • 3 mutein comprised in the hll_-12 mutein may have (an) amino acid residue(s) at sequence position(s) 82, 103, 106, 115, 122, 123, 216, 221 , 232, and/or 312, which is/are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A) or glycine (G).
  • A alanine
  • G glycine
  • the hll_-12(3 mutein comprised in the hll_-12 mutein may have (an) amino acid residue(s) at sequence position(s) 103, 106, 115, 122, 123, 216, 221 , 232, and/or 312, which is/are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A) or glycine (G).
  • A alanine
  • G glycine
  • 3 mutein comprised in the hll_-12 mutein may have (an) amino acid residue(s) at sequence position(s) 103, 115, 122, 123, 216, 221 , 232, and/or 312, which is/are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A) or glycine (G).
  • A alanine
  • G glycine
  • 3 mutein comprised in the hll_-12 mutein may have (an) amino acid residue(s) at sequence position(s) 103, 122, 123, 216, 221 , 232, and/or 312, which is/are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A) or glycine (G).
  • the hll_-12(3 mutein comprised in the hll_-12 mutein may have (an) amino acid residue(s) at sequence position(s) 122, 123, 216, 221 , 232, and/or 312, which is/are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A) or glycine (G).
  • 3 mutein comprised in the hll_-12 mutein may have (an) amino acid residue(s) at sequence position(s) 122, 123, 216, 221 , 232, and/or 312, which is/are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A) or glycine (G).
  • the hlL-12(3 mutein comprised in the hlL-12 mutein may have (an) amino acid residue(s) at sequence position(s) 123, 216, 221 , 232, and/or 312, which is/are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A) or glycine (G).
  • 3 mutein comprised in the hll_-12 mutein may have (an) amino acid residue(s) at sequence position(s) 216, 221 , 232, and/or 312, which is/are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A) or glycine (G).
  • the hll_-12(3 mutein comprised in the hll_-12 mutein may have (an) amino acid residue(s) at sequence position(s) 221 , 232, and/or 312, which is/are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A) or glycine (G).
  • the hll_-12(3 mutein comprised in the hll_-12 mutein may have (an) amino acid residue(s) at sequence position(s) 232 and/or 312, which is/are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A) or glycine (G).
  • the hll_-12(3 mutein comprised in the hll_-12 mutein may have an amino acid residue at sequence position 103, 232 or 312, which is/are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by glycine (G) and/or an amino acid residue at sequence position(s) 37, 38, 39, 67, 81 , 82, 106, 115, 122, 123, 216, and/or 221 , which is/are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A).
  • G glycine
  • A alanine
  • nucleic acid molecule refers to a, natural or artificial, single and/or double stranded nucleic acid molecule.
  • Said molecule is preferably a deoxyribonucleic acid (DNA) molecule and thus, preferably is built up of adenosine, guanosine, cytidine, and/or thymidine residues, herein also referred to as nucleotides.
  • DNA deoxyribonucleic acid
  • a nucleic acid molecule is advantageous to store genetic information that can be transcribed into ribonucleic acid (RNA) polyribonucleotides that carry the genetic information for protein synthesis, which can be translated into proteins.
  • RNA ribonucleic acid
  • nucleic acid molecules are also advantageous to enable expression of proteins like muteins according to the present invention, for example, when being introduced into a suitable host cell.
  • the present invention also provides a nucleic acid molecule comprising a nucleotide sequence encoding a mutein of the a-subunit of human Interleukin 12 according to the present invention and/or a mutein of the [3-subunit of human Interleukin 12 according to the present invention.
  • the nucleic acid molecule comprises a nucleotide sequence encoding a mutein of human Interleukin 12 according to the present invention.
  • the nucleic acid molecule of the present invention is operably linked to a regulatory sequence to allow expression of the nucleic acid molecule.
  • the regulatory sequence may comprise a promoter sequence.
  • promoter sequence refers to a DNA sequence, which initiates and directs the transcription of a gene into an RNA transcript in cells.
  • the nucleic acid molecule(s) according to the present invention may be comprised in a vector.
  • the term "vector” refers to a nucleic acid molecule capable of transporting another nucleic acid molecule, preferably a nucleic acid molecule according to the present invention, to which it has been linked.
  • One type of vector may be a plasmid, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • Another type of vector may be a viral vector, wherein additional DNA segments can be ligated into the viral genome.
  • the present invention also provides a vector comprising the nucleic acid molecule according to the present invention.
  • the nature of said nucleic acid molecule the same applies as it has been described herein in connection with the nucleic acid molecule according to the present invention.
  • the other features of such a nucleic acid molecule can be as described herein.
  • the present invention also provides a host cell comprising the nucleic acid molecule according to the present invention and/or the vector according to the present invention.
  • a host cell can be any prokaryotic (e.g., E. coli) or eukaryotic cell (e.g., insect cells, yeast or mammalian cells).
  • said host is a suitable host cell, more preferably a host cell suitable for expression of a nucleic acid molecule and/or of a vector according to the present invention.
  • the nature of the nucleic acid molecule and/or the vector the same applies as it has been described herein in connection with the nucleic acid molecule according to the present invention and the vector according to the present invention, respectively.
  • the other features of such a nucleic acid molecule and/or of such a vector can be as described herein.
  • the present invention also provides an immune modulator comprising a mutein according to the present invention.
  • the term “immune modulator” is intended to be understood as referring to any protein, substance or composition that is able to carry out immunomodulation, which is the adjustment of the immune- response to a desired level, as e.g. in immunopotentiation, immunosuppression, or induction of immunologic tolerance.
  • the immune modulator according to the present invention comprises a hll_-12a, hll_-12
  • the present invention also provides a pharmaceutical composition comprising a mutein according to the present invention and/or an immune modulator according to the present invention.
  • the pharmaceutical composition according to the present invention comprises a hlL-12a, hll_-12
  • an immune modulator according to the present invention.
  • said immune modulator and/or the mutein of the a-subunit of human Interleukin 12, of the [3-subunit of human Interleukin 12, and/or of the human Interleukin 12 the same applies as it has been described herein in connection with the immune modulator and the hll_-12a, hll_-12
  • the other features of such muteins and/or immune modulators can be as described herein.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to chemical compounds, materials, ingredients, and/or compositions, which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of a mammal, preferably of a human, without (excessive) toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • a pharmaceutically acceptable carrier relates to an inactive substance formulated alongside the pharmaceutically active substance, herein a mutein and/or an immune modulator according to the present invention, for facilitating its handling in view of dosage, adsorption, solubility or pharmacokinetic considerations.
  • suitable pharmaceutical acceptable carriers are well known in the art and include phosphate buffered saline solutions, buffer, water, emulsions, such as oil/water emulsions, various wetting agents, and sterile solutions.
  • the pharmaceutical composition comprises a mutein according to the present invention and/or an immune modulator according to the present invention in an effective amount, i.e. an amount sufficient to induce a detectable beneficial and/or therapeutic response in a mammal, preferably a human, to which the pharmaceutical composition is to be administered.
  • the present invention also provides the use of a mutein according to the present invention for the manufacture of a medicament for treating a disease in a mammal, preferably a human, wherein the disease is preferably a disease selected from the group consisting of an infectious disease, an autoimmune disease, cancer, a transplantation-related disease such as Graft-versus-Host-disease, a chronic inflammatory disease such as chronic inflammatory bowel disease, an acute inflammatory disease, sepsis, septic shock, diabetes or asthma.
  • a hll_-12a, hll_-12(3 and/or hlL-12 mutein according to the present invention is to be used.
  • the present invention also relates to a mutein according to the present invention for use as a medicament.
  • 3 and/or hll_-12 mutein according to the present invention are disclosed herein for use as a medicament.
  • said mutein of the a-subunit of human Interleukin 12, of the [3- subunit of human Interleukin 12, and/or of the human Interleukin 12 the same applies as it has been described herein in connection with the hll_-12a, hll_-12(3 and hlL-12 muteins according to the present invention, respectively.
  • the other features of such muteins can be as described herein.
  • the medicament is a medicament capable of preventing and/or treating a disease, preferably a Interleukin 12-mediated disease and/or a disease selected from the group consisting of an infectious disease, an autoimmune disease, cancer, a transplantation-related disease such as Graft-versus-Host-disease, a chronic inflammatory disease such as chronic inflammatory bowel disease, an acute inflammatory disease, sepsis, septic shock, diabetes or asthma.
  • a disease preferably a Interleukin 12-mediated disease and/or a disease selected from the group consisting of an infectious disease, an autoimmune disease, cancer, a transplantation-related disease such as Graft-versus-Host-disease, a chronic inflammatory disease such as chronic inflammatory bowel disease, an acute inflammatory disease, sepsis, septic shock, diabetes or asthma.
  • the present invention also relates to a mutein according to the present invention for use in the treatment of a disease, wherein the disease is preferably a disease selected from the group consisting of an infectious disease, an autoimmune disease, cancer, a transplantation-related disease such as Graft-versus-Host- disease, a chronic inflammatory disease such as chronic inflammatory bowel disease, an acute inflammatory disease, sepsis, septic shock, diabetes or asthma.
  • 3 and/or hlL-12 mutein according to the present invention are disclosed herein for use in the treatment of a disease.
  • the present invention also relates to a method of treating an Interleukin 12- mediated disease in a mammal, preferably a human, wherein the disease is preferably a disease selected from the group consisting of an infectious disease, an autoimmune disease, cancer, a transplantation-related disease such as Graft- versus-Host-disease, a chronic inflammatory disease, such as chronic inflammatory bowel disease, an acute inflammatory disease, sepsis, septic shock, diabetes or asthma, comprising the step of administering a composition comprising a mutein according to the present invention and/or a pharmaceutical composition according to the present invention and/or an immune modulator according to the present invention to said mammal in need thereof.
  • a disease selected from the group consisting of an infectious disease, an autoimmune disease, cancer, a transplantation-related disease such as Graft- versus-Host-disease, a chronic inflammatory disease, such as chronic inflammatory bowel disease, an acute inflammatory disease, sepsis, septic shock, diabetes or
  • 3 and/or hlL-12 mutein according to the present invention are disclosed herein for use in the treatment of a disease.
  • a composition comprising a mutein, a pharmaceutical composition and/or an immune modulator according to the present invention may be administered via a wide range of classes of forms of administration known to the skilled person, such as (needle) injection, by means of an inhalator, or in form of a cream, foam, gel, lotion and/or ointment. Dose and duration of action have to be deliberately adjusted in each case.
  • the present invention also relates to a method for producing a mutein according to the present invention as described herein, comprising the steps of:
  • the human Interleukin 12 polypeptide comprising an a-subunit (p35), preferably of (i) or (ii), and a [3-subunit (p40), preferably of (iii) or (iv), a nucleotide sequence mutating at least one amino acid residue(s) of said polypeptide in case of said a-subunit selected from the group consisting of sequence positions 70, 71 , 72, 185, 187, and 189 and/or in case of said 12 [3-subunit selected from the group consisting of sequence positions 37, 38, 39, 67, 81 , 82, 103, 106, 115, 122, 123, 216, 221 , 232, and 312, and
  • step (a) into the nucleic acid molecule encoding the polypeptide of (iii) or (iv) or (v) a nucleotide sequence is introduced mutating at least one of the amino acid residue(s) of the [3-subunit of hlL-12 selected from the group consisting of sequence positions
  • step (a) into the nucleic acid molecule encoding the polypeptide of (iii) or (iv) or (v) a nucleotide sequence is introduced mutating at least one of the amino acid residue(s) of the [3-subunit of hlL-12 selected from the group consisting of sequence positions
  • step (a) into the nucleic acid molecule encoding the polypeptide of (iii) or (iv) or (v) a nucleotide sequence is introduced mutating at least one of the amino acid residue(s) of the [3-subunit of hlL-12 selected from the group consisting of sequence positions 67, 115, 122, 123, 221 , 232, and 312.
  • step (a) into the nucleic acid molecule encoding the polypeptide of (iii) or (iv) or (v) a nucleotide sequence is introduced mutating i) at least one of the amino acid residue(s) of the [3-subunit of hll_-12 selected from the group consisting of sequence positions 103, 232, and 312, and/or ii) the amino acid residue(s) of the [3-subunit of hlL-12 at sequence position(s) 122 and/or 123, and/or iii) at least one of the amino acid residue(s) of the [3-subunit of hlL-12 selected from the group consisting of sequence positions 37, 38, 39, 67, 81 , 82, 106, 115, 216, and 221.
  • step (a) into the nucleic acid molecule encoding the polypeptide of (iii) or (iv) or (v) a nucleotide sequence is introduced replacing i) at least one of the amino acid residue(s) of the [3-subunit of hlL-12 selected from the group consisting of sequence positions 103, 232, and 312 by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by glycine (G), ii) the amino acid residue(s) of the [3-subunit of hlL-12 at sequence position(s) 122 and/or 123 by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A), and/or iii) at least one of the amino acid residue(s) of the [3-subunit of hlL-12 selected from the group consisting of sequence positions 37, 38, 39,
  • a nucleotide sequence is introduced mutating i) at least one of the amino acid residue(s) of the [3-subunit of hll_-12 selected from the group consisting of sequence positions 103, 232, and 312, ii) the amino acid residue(s) of the [3-subunit of hll_-12 at sequence position(s) 122 and/or 123, and/or iii) at least one of the amino acid residue(s) of the [3-subunit of hlL-12 selected from the group consisting of sequence positions 38, 39, 67, 106, 115, and 221.
  • step (a) into the nucleic acid molecule encoding the polypeptide of (iii) or (iv) or (v) a nucleotide sequence is introduced replacing i) at least one of the amino acid residue(s) of the [3-subunit of hlL-12 selected from the group consisting of sequence positions 103, 232, and 312 by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by glycine (G), ii) the amino acid residue(s) of the [3-subunit of hlL-12 at sequence position(s) 122 and/or 123 by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A), and/or iii) at least one of the amino acid residue(s) of the [3-subunit of hlL-12 selected from the group consisting of sequence positions 38, 39,
  • step (a) into the nucleic acid molecule encoding the polypeptide of (iii) or (iv) or (v) a nucleotide sequence is introduced mutating i) the amino acid residue(s) of the [3-subunit of hlL-12 at sequence position(s) 232 and/or 312, ii) the amino acid residue(s) of the [3-subunit of hll_-12 at sequence position(s) 122 and/or 123, and/or iii) at least one of the amino acid residue(s) of the [3-subunit of hll_-12 selected from the group consisting of sequence positions 67, 115, and 221.
  • step (a) into the nucleic acid molecule encoding the polypeptide of (iii) or (iv) or (v) a nucleotide sequence is introduced replacing i) the amino acid residue(s) of the [3-subunit of hlL-12 at sequence position(s) 232 and/or 312 by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by glycine (G), ii) the amino acid residue(s) of the [3-subunit of hlL-12 at sequence position(s) 122 and/or 123 by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A), and/or iii) at least one of the amino acid residue(s) of the [3-subunit of hlL-12 selected from the group consisting of sequence positions 67, 115, and 221 by an amino acid other than the
  • step (a) into the nucleic acid molecule encoding the polypeptide of (iii) or (iv) or (v) a nucleotide sequence is introduced mutating the amino acid residue of the [3-subunit of hlL-12 at sequence position 103.
  • step (a) into the nucleic acid molecule encoding the polypeptide of (iii) or (iv) or (v) a nucleotide sequence is introduced replacing the amino acid residue of the [3-subunit of hlL-12 at sequence position 103 by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by glycine (G).
  • step (a) into the nucleic acid molecule encoding the polypeptide of (iii) or (iv) or (v) a nucleotide sequence is introduced mutating the amino acid residue of the [3-subunit of hlL-12 at sequence position 232.
  • step (a) into the nucleic acid molecule encoding the polypeptide of (iii) or (iv) or (v) a nucleotide sequence is introduced replacing the amino acid residue of the [3-subunit of hlL-12 at sequence position 232 by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by glycine (G).
  • step (a) into the nucleic acid molecule encoding the polypeptide of (iii) or (iv) or (v) a nucleotide sequence is introduced mutating the amino acid residue of the [3-subunit of hlL-12 at sequence position 312.
  • step (a) into the nucleic acid molecule encoding the polypeptide of (iii) or (iv) or (v) a nucleotide sequence is introduced replacing the amino acid residue of the [3-subunit of hlL-12 at sequence position 312 by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by glycine (G).
  • step (a) into the nucleic acid molecule encoding the polypeptide of (iii) or (iv) or (v) a nucleotide sequence is introduced mutating the amino acid residue of the [3-subunit of hlL-12 at sequence position 122.
  • step (a) into the nucleic acid molecule encoding the polypeptide of (iii) or (iv) or (v) a nucleotide sequence is introduced replacing the amino acid residue of the [3-subunit of hlL-12 at sequence position 122 by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A).
  • step (a) into the nucleic acid molecule encoding the polypeptide of (iii) or (iv) or (v) a nucleotide sequence is introduced mutating the amino acid residue of the [3-subunit of hlL-12 at sequence position 123.
  • step (a) into the nucleic acid molecule encoding the polypeptide of (iii) or (iv) or (v) a nucleotide sequence is introduced replacing the amino acid residue of the [3-subunit of hlL-12 at sequence position 123 by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A).
  • step (a) into the nucleic acid molecule encoding the polypeptide of (iii) or (iv) or (v) a nucleotide sequence is introduced mutating the amino acid residue of the [3-subunit of h IL-12 at sequence position 82.
  • step (a) into the nucleic acid molecule encoding the polypeptide of (iii) or (iv) or (v) a nucleotide sequence is introduced replacing the amino acid residue of the [3-subunit of hlL-12 at sequence position 82 by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A).
  • step (a) into the nucleic acid molecule encoding the polypeptide of (iii) or (iv) or (v) a nucleotide sequence is introduced mutating the amino acid residue of the [3-subunit of hlL-12 at sequence position 37.
  • step (a) into the nucleic acid molecule encoding the polypeptide of (iii) or (iv) or (v) a nucleotide sequence is introduced replacing the amino acid residue of the [3-subunit of hlL-12 at sequence position 37 by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A).
  • step (a) into the nucleic acid molecule encoding the polypeptide of (iii) or (iv) or (v) a nucleotide sequence is introduced mutating the amino acid residue of the [3-subunit of hlL-12 at sequence position 39.
  • step (a) into the nucleic acid molecule encoding the polypeptide of (iii) or (iv) or (v) a nucleotide sequence is introduced replacing the amino acid residue of the [3-subunit of hlL-12 at sequence position 39 by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A).
  • step (a) into the nucleic acid molecule encoding the polypeptide of (iii) or (iv) or (v) a nucleotide sequence is introduced mutating the amino acid residue of the [3-subunit of hll_-12 at sequence position 106.
  • step (a) into the nucleic acid molecule encoding the polypeptide of (iii) or (iv) or (v) a nucleotide sequence is introduced replacing the amino acid residue of the [3-subunit of hlL-12 at sequence position 106 by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A).
  • step (a) into the nucleic acid molecule encoding the polypeptide of (iii) or (iv) or (v) a nucleotide sequence is introduced mutating the amino acid residue of the [3-subunit of hlL-12 at sequence position 81 .
  • step (a) into the nucleic acid molecule encoding the polypeptide of (iii) or (iv) or (v) a nucleotide sequence is introduced replacing the amino acid residue of the [3-subunit of hlL-12 at sequence position 81 by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A).
  • step (a) into the nucleic acid molecule encoding the polypeptide of (iii) or (iv) or (v) a nucleotide sequence is introduced mutating the amino acid residue of the [3-subunit of hlL-12 at sequence position 115.
  • step (a) into the nucleic acid molecule encoding the polypeptide of (iii) or (iv) or (v) a nucleotide sequence is introduced replacing the amino acid residue of the [3-subunit of hlL-12 at sequence position 115 by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A).
  • step (a) into the nucleic acid molecule encoding the polypeptide of (iii) or (iv) or (v) a nucleotide sequence is introduced mutating the amino acid residue of the [3-subunit of hlL-12 at sequence position 67.
  • step (a) into the nucleic acid molecule encoding the polypeptide of (iii) or (iv) or (v) a nucleotide sequence is introduced replacing the amino acid residue of the [3-subunit of hlL-12 at sequence position 67 by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A).
  • step (a) into the nucleic acid molecule encoding the polypeptide of (iii) or (iv) or (v) a nucleotide sequence is introduced mutating the amino acid residue of the [3-subunit of hlL-12 at sequence position 216.
  • step (a) into the nucleic acid molecule encoding the polypeptide of (iii) or (iv) or (v) a nucleotide sequence is introduced replacing the amino acid residue of the [3-subunit of hlL-12 at sequence position 216 by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A).
  • step (a) into the nucleic acid molecule encoding the polypeptide of (iii) or (iv) or (v) a nucleotide sequence is introduced mutating the amino acid residue of the [3-subunit of hlL-12 at sequence position 38.
  • step (a) into the nucleic acid molecule encoding the polypeptide of (iii) or (iv) or (v) a nucleotide sequence is introduced replacing the amino acid residue of the [3-subunit of hlL-12 at sequence position 38 by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A).
  • step (a) into the nucleic acid molecule encoding the polypeptide of (iii) or (iv) or (v) a nucleotide sequence is introduced mutating the amino acid residue of the [3-subunit of hlL-12 at sequence position 221 .
  • step (a) into the nucleic acid molecule encoding the polypeptide of (iii) or (iv) or (v) a nucleotide sequence is introduced replacing the amino acid residue of the [3-subunit of hlL-12 at sequence position 221 by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A).
  • the structure of the human IL-12 (hlL-12) receptor was modelled based on the crystal structures for human IL-12 (PDB: 1f45, Yoon et al. 2000) and the human IL-23 receptor (PDB: 6wdq, Glassman et al. 2021 ). This procedure was motivated by the similar molecular setup of IL-12 and IL-23 receptor complexes (Y. Bloch et al. 2017; Glassman et al. 2021 ). IL-12 and IL-23 both bind to the IL-12R[31 receptor chain, but IL-12 further assembles with IL-12R[32, while IL-23 binds to IL-23R to each form a fully functional receptor complex. Missing residues in the hlL-12 structure were incorporated using the MODELLER software (Webb and Sali 2016).
  • 31 was superposed onto the first domain of IL-12R
  • 32 model-structure was superposed onto IL-23R.
  • the superposed structures yield the final model for the hlL-12 : receptor complex for further analyses.
  • the resulting hlL-12 : receptor complex pdb-structure was processed with the pdb2pqr server (Dolinsky et al. 2004) to capture protonation states, and afterwards the simulation system was generated with the xleap-module of the Amber18 package (Case et al. 2014).
  • Sodium counterions were added to electrostatically minimize the system and a final salt concentration of 250 mM NaCI was adjusted.
  • the protein was described with the ff14SB force field (Maier et al. 2015). Hydrogen-mass repartitioning was activated with the parmed module.
  • the generated simulation system was energy-minimized in two steps. First, the system was minimized in 3,000 cycles (1 ,500 cycles steepest descent and 1 ,500 conjugate gradient method) employing harmonic position restraints with a force constant of 10.0 kcal/mol/A A 2. Second, the system was minimized in 20,000 cycles (10,000 cycles steepest descent and 10,000 conjugate gradient method) in absence of restraining forces.
  • the system was equilibrated in 8 consecutive molecular dynamics (MD) simulations.
  • the system was equilibrated from 100 K to 300 K (Berendsen-thermostat) in steps of 100 K, employing positional restraints with a force constant of 25.0 kcal/mol/A A 2 on the protein atoms.
  • the restraints were gradually removed in the subsequent five simulations and a Berendsen-barostat with a reference pressure of 1 bar was switched on.
  • the whole equilibration phase covers a simulation-time of 2.1 ns.
  • the output structure of the equilibration phase was used as starting structure for the production run, which covers a simulation time of 100 ns. In the production run, the atoms were free of external restraints and data was written out every 5,000 steps.
  • IL-12-muteins were produced using the ExpiCHO Expression System (Gibco) according to the manufacturer’s protocol. Subunit sequences for IL-12a and IL-12
  • the medium was supplemented with SigmaFAST protease inhibitor (Sigma-Aldrich), centrifuged (5,000 g, 30 min, 4 °C) and applied to a HisTrap HP column (Cytiva) in phosphate-buffered saline (PBS) buffer, pH 7.4. Elution was performed with an imidazole concentration gradient from 0 mM to 500 mM in PBS. His-tag cleavage was performed by TEV protease (containing a His-tag itself) overnight at 4 °C and followed by a subsequent HisTrap HP column in PBS buffer. A final purification step was performed by size exclusion chromatography using a HiLoad 26/600 Superdex 200 pg column (GE Healthcare) in PBS buffer, pH 7.4.
  • SigmaFAST protease inhibitor Sigma-Aldrich
  • CD Far-UV circular dichroism
  • Species-specific HRP-conjugated secondary antibodies (Santa Cruz Biotechnology, 1 :10,000 in 5% BSA) were used to detect the proteins. Blots were detected using Amersham ECL prime (Cytiva) and a Fusion- FX7.Edge V0.70 imager (Vilber).
  • the inventors used the NanoBRETTM assay for analysis of receptor chain dimerization upon cytokine binding.
  • COS-7 cells were seeded in uncoated tissue culture 6-well plates (VWR) and transfected using Metafectene PRO (Biontex) according to manufacturer’s protocol.
  • 2 pg receptor chain DNA pHTC HaloTag® (HT) CMV-neo IL-12R
  • transfected cells were detached by Accutase® (Sigma-Aldrich) and resuspended in assay medium (DMEM w/o Phenolred, 4 % (v/v) FBS) to 2.2 x 10 5 cells/ml. After division into two pools, 1 pl HaloTag® NanoLuc® 618 Ligand (Promega) or 1 pl DMSO per ml cells was added. 2 x 10 4 cells/well were seeded into a white bottom 96-well plate and incubated for 20 h. The IL-12 heterodimer variants or PBS were used to stimulate the cells for 30 min at final concentrations of 10-0.01 nM.
  • Nano-Gio® substrate After addition of the Nano-Gio® substrate, a CLARIOstar® platereader (BMG Labtech) was used for measurement of the bioluminescence resonance energy transfer (BRET) signal.
  • BRET bioluminescence resonance energy transfer
  • the normalized NanoBRETTM ratios were calculated by dividing blank-corrected acceptor emission (610 nm) by blank-corrected donor luminescence (450-480 nm) and multiplication by 1000. Normalized NanoBRETTM signals were determined averaging the ratios and subtracting the DMSO mean ratio from the experimental mean ratio. Samples are measured in technical triplicates.
  • PBMCs Human peripheral blood mononuclear cells
  • RPMI 1640 Roswell Park Memorial Institute Medium 1640
  • FCS fetal calve serum
  • Frozen cells were thawed and rested overnight in RPMI 1640 (Gibco) containing 10% FCS (Gibco), 1 % sodium pyruvate (100 mM, Gibco), 1% L-glutamin (200 mM, Gibco), 1x MEM non- essential amino acids (100x, Gibco), 1% penicillin/streptomycin (10000 lU/ml, Gibco), 0.01 M HEPES pH 7.4 (1 M, Gibco), 0.015 mg/ml Gentamicin (40 mg/ml, ratiopharm) and in the presence of 20 lll/ml IL-2 (Clingen).
  • CD8 + T cells and NK cells were then isolated from PBMCs by magnetic-activated cell sorting (MACS) separation using CD8 + T cell isolation kit (Miltenyi) and NK cell isolation kit with LS magnetic columns (Miltenyi Biotec).
  • MCS magnetic-activated cell sorting
  • CD8 + T cells were seeded at 80,000 cells/well in a 96-well flat bottom plate coated with 2 pg/ml anti-human CD3 antibody (eBioscience) and 0.5 pg/ml anti-human CD28 antibody (eBioscience). Cells were stimulated with 100 ILI/mL IL-2 with or without addition of the respective IL-12- mutein.
  • NK cells were seeded at 40,000 cells/well in a 96-well flat bottom plate, stimulated with 50 ng/mL IL-18 (R&D System) with or without addition of the respective IL-12-mutein. In each case, the supernatant was analyzed after 48 h using the IFNy human uncoated ELISA kit (ThermoFisher) and measured with an INFINITE F PLEX plate reader (TECAN).
  • Table 2 Tabular overview of mutated sequence positions.
  • RMSD root mean squared deviations
  • MM/GBSA Molecular Mechanics Generalized Bom Surface Area
  • the model of the IL-12 receptor complex provided several candidates for mutation to reduce receptor binding of IL-12.
  • wild-type IL-12 and nine of its variants were expressed in CHO cells and purified by meta I -affinity and size exclusion chromatography. Each mutein could be purified and formed disulfide-bridged covalent heterodimers as expected for IL-12 (Yoon et al. 2000) (see Fig.
  • amino acid sequence of “IL-12 wt ” corresponds to the SEQ ID NO: 2
  • _-12 E67A ” corresponds to the SEQ ID NO: 3
  • amino acid sequence of “IL-12 D115A ” corresponds to the SEQ ID NO: 5
  • amino acid sequence of “IL-12 H216A ” corresponds to the SEQ ID NO: 6
  • the amino acid sequence of “IL-12 E221 A ” corresponds to the SEQ ID NO: 7, wherein the amino acid sequence of “
  • muteins gave rise to far-UV circular dichroism (CD) spectra highly similar to the wild-type protein (see Fig. 4B) indicating that none of the muteins compromised structure formation.
  • CD far-UV circular dichroism
  • Using the selected panel of IL-12-muteins their capability was tested to induce STAT4 phosphorylation in human NK-92 cells. At cytokine concentrations of 10 ng/pl, most muteins showed wild-type-like signaling capabilities in NK cells (see Fig. 5A and 5B).
  • One exception in the investigated muteins was the L103G-mutein (SEQ ID NO: 4), that, even by this single point mutation, reduced signaling in NK cells by approximately 75% (see Fig. 5A, B).
  • L103G SEQ ID NO: 4
  • E221A SEQ ID NO: 7
  • a decreased production of IFNy by the NK cells indicating preferred T cell activation with attenuated NK cell activation that indicates a reduced systemic toxicity in vivo compared to wild-type IL-12.
  • IL-12-mutein concentrations ranging from 0.001 nM to 100 nM were used to activate CD8 + T cells and NK cells for 48 h.
  • different additives were used to induce IFNy release.
  • CD8 + T cells human anti-CD3, anti-CD28 antibodies, and IL-2 were added to enable a stimulatory signal mediated by the T cell receptor, while NK cells were incubated with additional IL-18 (Malek 2008; Oka et al. 2020; Romee et al. 2012). The concentration of produced IFNy in the supernatant was determined by ELISA.
  • Both L103G (SEQ ID NO: 4) and E221A (SEQ ID NO: 7) induced IFNy secretion from CD8 + T cells in a concentration dependent manner with significantly reduced secretion activity compared to the wild-type (see Fig. 6A).
  • NK cells were significantly less activated compared to the wild-type especially for lower concentrations of the muteins, with L103G (SEQ ID NO: 4) showing a stronger differentiation than E221A (SEQ ID NO: 7) (see Fig. 6B).
  • the IL-12 muteins showed substantial differences between the half-maximum effective concentration (EC50) and therefore in the potency on the different cell types (Fig. 6C).
  • L103G showed a stronger discrimination between NK cells and CD8+ T cells than E221A, although the magnitude of this effect was donordependent (Fig. 6D). Together, these data indicate that the tested IL-12 muteins are able to induce cell type-specific IFNy production as intended.
  • IL-12 is a key cytokine for immunity against intracellular pathogens and a highly promising candidate to reactive immune reactions in the immunosuppressive tumor microenvironment. As such, insights into the structure and dynamics of its receptor binding are of great interest. Using recently published high-resolution structures of IL-23 bound to its receptor (Y. Bloch et al. 2017; Glassman et al. 2021), a high-resolution structural model of the human IL-12 : receptor complex is provided by the inventors. Using this model combined with computer simulations, residues were identified that may reduce IL-12 : receptor binding. The chosen approach included flexibility analyses and thus entropic contributions to receptor binding, which have not been used thus far to engineer attenuated IL-12-muteins. These muteins may be of interest to provide IL-12 molecules with less systemic toxicity.
  • a mutein of the a-subunit of human Interleukin 12 (SEQ ID NO: 1 ), wherein at least one of the amino acid residue(s) of said a-subunit selected from the group consisting of sequence positions 70, 71 , 72, 185, 187, and 189 is/are mutated.
  • the mutein of any one of the preceding items wherein the amino acid residue of the a-subunit of human Interleukin 12 at sequence position 71 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 1 , preferably by alanine (A) (SEQ ID NO: 19).
  • A alanine
  • A alanine
  • A alanine
  • the mutein of item 19, wherein said mutein comprises at least 90% sequence identity to the [3-subunit of human Interleukin 12 (SEQ ID NO: 2).
  • the mutein of any one of items 19 to 21 wherein at least one of the amino acid residue(s) of the [3-subunit of human Interleukin 12 selected from the group consisting of sequence positions 67, 115, 122, 123, 221 , 232, and 312 is/are mutated.
  • G glycine
  • the mutein of any one of items 19 to 31 wherein the amino acid residue of the [3-subunit of human Interleukin 12 at sequence position 232 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by glycine (G) (SEQ ID NO: 8).
  • the mutein of any one of items 19 to 41 wherein the amino acid residue of the [3-subunit of human Interleukin 12 at sequence position 37 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A) (SEQ ID NO: 13).
  • the mutein of any one of items 19 to 51 wherein the amino acid residue of the [3-subunit of human Interleukin 12 at sequence position 67 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A) (SEQ ID NO: 3).
  • a mutein of human Interleukin 12 comprising an a-subunit (p35) and a [3- subunit (p40), wherein the a-subunit is a mutein of the a-subunit of human Interleukin 12 (SEQ ID NO: 1 ) of any one of items 1 to 18 and/or wherein the [3- subunit is a mutein of the [3-subunit of human Interleukin 12 (SEQ ID NO: 2) of any one of items 19 to 58.
  • the mutein of item 59 or item 60 wherein at least one of the amino acid residue(s) of said a-subunit selected from the group consisting of sequence positions 70, 71 , 72, 185, 187, and 189 is/are mutated.
  • the mutein of any one of items 59 to 61 wherein said a-subunit comprises at least 90% sequence identity to the a-subunit of human Interleukin 12 (SEQ ID NO: 1).
  • the mutein of any one of items 59 to 62, wherein at least one of the amino acid residue(s) of the a-subunit of human Interleukin 12 selected from the group consisting of sequence positions 70, 71 , 72, 185, and 187 is/are mutated.
  • any one of items 59 to 65 wherein i) at least one of the amino acid residue(s) of the a-subunit of human Interleukin 12 selected from the group consisting of sequence positions 70 to 72 is/are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A), and/or wherein ii) the amino acid residue of the a-subunit of human Interleukin 12 at sequence position 187 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A).
  • the mutein of any one of items 59 to 66, wherein the amino acid residue of the a-subunit of human Interleukin 12 at sequence position 70 is mutated.
  • the mutein of any one of items 59 to 67, wherein the amino acid residue of the a-subunit of human Interleukin 12 at sequence position 70 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A) (SEQ ID NO: 18).
  • A alanine
  • the mutein of any one of items 59 to 68, wherein the amino acid residue of the a-subunit of human Interleukin 12 at sequence position 71 is mutated.
  • the mutein of any one of items 59 to 71 wherein the amino acid residue of the a-subunit of human Interleukin 12 at sequence position 72 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A) (SEQ ID NO: 20).
  • A alanine
  • the mutein of any one of items 59 to 72, wherein the amino acid residue of the a-subunit of human Interleukin 12 at sequence position 185 is mutated.
  • the mutein of any one of items 59 to 81 wherein at least one of the amino acid residue(s) of the [3-subunit of human Interleukin 12 selected from the group consisting of sequence positions 38, 39, 67, 103, 106, 115, 122, 123, 221 , 232, and 312 is/are mutated.
  • any one of items 59 to 83 wherein i) at least one of the amino acid residue(s) of the [3-subunit of human Interleukin 12 selected from the group consisting of sequence positions 103, 232, and 312 is/are mutated, and/or wherein ii) the amino acid residue(s) of the [3-subunit of human Interleukin 12 at sequence position(s) 122 and/or 123 is/are mutated, and/or wherein iii) at least one of the amino acid residue(s) of the [3-subunit of human Interleukin 12 selected from the group consisting of sequence positions 37, 38, 39, 67, 81 , 82, 106, 115, 216, and 221 is/are mutated.
  • any one of items 59 to 84 wherein i) at least one of the amino acid residue(s) of the [3-subunit of human Interleukin 12 selected from the group consisting of sequence positions 103, 232, and 312 is/are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by glycine (G), and/or wherein ii) the amino acid residue(s) of the [3-subunit of human Interleukin 12 at sequence position(s) 122 and/or 123 is/are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A), and/or wherein iii) at least one of the amino acid residue(s) of the [3-subunit of human Interleukin 12 selected from the group consisting of sequence positions
  • any one of items 59 to 85 wherein i) at least one of the amino acid residue(s) of the [3-subunit of human Interleukin 12 selected from the group consisting of sequence positions 103, 232, and 312 is/are mutated, and/or wherein ii) the amino acid residue(s) of the [3-subunit of human Interleukin 12 at sequence position(s) 122 and/or 123 is/are mutated, and/or wherein iii) at least one of the amino acid residue(s) of the [3-subunit of human Interleukin 12 selected from the group consisting of sequence positions 38, 39, 67, 106, 115, and 221 is/are mutated.
  • any one of items 59 to 86 wherein i) at least one of the amino acid residue(s) of the [3-subunit of human Interleukin 12 selected from the group consisting of sequence positions 103, 232, and 312 is/are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by glycine (G), and/or wherein ii) the amino acid residue(s) of the [3-subunit of human Interleukin 12 at sequence position(s) 122 and/or 123 is/are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A), and/or wherein iii) at least one of the amino acid residue(s) of the [3-subunit of human Interleukin 12 selected from the group consisting of sequence positions
  • any one of items 59 to 88 wherein i) the amino acid residue(s) of the [3-subunit of human Interleukin 12 at sequence position(s) 232 and/or 312 is/are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by glycine (G), and/or wherein ii) the amino acid residue(s) of the [3-subunit of human Interleukin 12 at sequence position(s) 122 and/or 123 is/are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A), and/or wherein iii) at least one of the amino acid residue(s) of the [3-subunit of human Interleukin 12 selected from the group consisting of sequence positions 67, 115, and 221 is/are replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by alanine (A).
  • G g
  • the mutein of any one of items 59 to 89, wherein the amino acid residue of the [3-subunit of human Interleukin 12 at sequence position 103 is mutated.
  • the mutein of any one of items 59 to 90, wherein the amino acid residue of the [3-subunit of human Interleukin 12 at sequence position 103 is replaced by an amino acid other than the amino acid at the respective sequence position in SEQ ID NO: 2, preferably by glycine (G) (SEQ ID NO: 4).
  • G glycine
  • a nucleic acid molecule comprising i) a nucleotide sequence encoding the mutein of human Interleukin 12 of any one of items 59 to 1 19 or ii) a nucleotide sequence encoding a) the mutein of the a-subunit of human Interleukin 12 of any one of items 1 to 18 and/or b) the mutein of the [3-subunit of human Interleukin 12 of any one of items 19 to 58.
  • the nucleic acid molecule of item 120 wherein the nucleotide sequence encodes a mutein of the a-subunit of human Interleukin 12 comprising at least 90% sequence identity to the a-subunit of human Interleukin 12 (SEQ ID NO: 1 ).
  • the nucleic acid molecule of item 120 or 121 comprising a nucleotide sequence encoding a mutein of the a-subunit of human Interleukin 12 of any one of SEQ ID NOs: 18 to 24.
  • the nucleic acid molecule of any one of items 120 to 122 wherein the nucleotide sequence encodes a mutein of the [3-subunit of human Interleukin 12 comprising at least 90% sequence identity to the [3-subunit of human Interleukin 12 (SEQ ID NO: 2).
  • the nucleic acid molecule of any one of items 120 to 123 comprising a nucleotide sequence encoding a mutein of the [3-subunit of human Interleukin 12 of any one of SEQ ID NOs: 3 to 17.
  • the nucleic acid molecule of item 125 wherein the regulatory sequence comprises a promoter sequence.
  • a vector comprising the nucleic acid molecule of any one of items 120 to 126.
  • a host cell comprising the nucleic acid molecule of any one of items 120 to 126 and/or the vector of item 127.
  • An immune modulator comprising a mutein of any one of items 1 to 119.
  • a pharmaceutical composition comprising a mutein of any one of items 1 to 119 and/or an immune modulator of item 129, preferably further comprising a pharmaceutically acceptable carrier.
  • a mutein of any one of items 1 to 119 for the manufacture of a medicament for treating a disease in a mammal, preferably a human, wherein the disease is preferably a disease selected from the group consisting of an infectious disease, an autoimmune disease, cancer, a transplantation-related disease such as Graft-versus-Host-disease, a chronic inflammatory disease such as chronic inflammatory bowel disease, an acute inflammatory disease, sepsis, septic shock, diabetes or asthma.
  • the mutein of any one of items 1 to 119 for use as a medicament.
  • any one of items 1 to 119 for use in the treatment of a disease wherein the disease is preferably a disease selected from the group consisting of an infectious disease, an autoimmune disease, cancer, a transplantation- related disease such as Graft-versus-Host-disease, a chronic inflammatory disease such as chronic inflammatory bowel disease, an acute inflammatory disease, sepsis, septic shock, diabetes or asthma.
  • a disease selected from the group consisting of an infectious disease, an autoimmune disease, cancer, a transplantation- related disease such as Graft-versus-Host-disease, a chronic inflammatory disease such as chronic inflammatory bowel disease, an acute inflammatory disease, sepsis, septic shock, diabetes or asthma.
  • Method for producing a mutein according to any one of items 1 to 119 comprising the steps of:
  • the human Interleukin 12 polypeptide comprising an a-subunit (p35), preferably of (i) or (ii), and a [3-subunit (p40), preferably of (iii) or (iv), a nucleotide sequence mutating at least one amino acid residue(s) of said polypeptide in case of said a-subunit selected from the group consisting of sequence positions selected from the group consisting of sequence positions 70, 71 , 72, 185, 187, and 189 and/or in case of said 12 [3- subunit selected from the group consisting of sequence positions 37, 38, 39, 67, 81 , 82, 103, 106, 115, 122, 123, 216, 221 , 232, and 312, and
  • step (b) introducing the obtained nucleic acid molecule for expression into a host cell or into a cell extract or into a cell lysate.
  • step (a) into the nucleic acid molecule encoding the polypeptide of (i) or (ii) or (v), a nucleotide sequence is introduced mutating the amino acid residue of the a-subunit of human Interleukin 12 at sequence position 189.
  • sequence positions 103, 232, and 312 selected from the group consisting of sequence positions 103, 232, and 312, and/or ii) the amino acid residue(s) of the [3-subunit of human Interleukin 12 at sequence position(s) 122 and/or 123, and/or iii) at least one of the amino acid residue(s) of the [3-subunit of human Interleukin 12 selected from the group consisting of sequence positions 37, 38, 39, 67, 81 , 82, 106, 115, 216, and 221 .
  • sequence positions 103, 232, and 312 selected from the group consisting of sequence positions 103, 232, and 312, ii) the amino acid residue(s) of the [3-subunit of human Interleukin 12 at sequence position(s) 122 and/or 123, and/or iii) at least one of the amino acid residue(s) of the [3-subunit of human Interleukin 12 selected from the group consisting of sequence positions 38, 39, 67, 106, 115, and 221.
  • PDB2PQR an automated pipeline for the setup of Poisson-Boltzmann electrostatics calculations. Nucleic acids research 32, W665-667.
  • NKSF natural killer cell stimulatory factor

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Abstract

La présente invention concerne, entre autres, i) une mutéine de la sous-unité α de l'interleukine 12 humaine (hIL-12), au moins l'un du ou des résidus d'acide aminé de ladite sous-unité α de hIL-12 choisi dans le groupe constitué par la ou les positions de séquence 70, 71, 72, 185, 187 et 189 étant muté, ii) une mutéine de la sous-unité β de hIL-12, au moins l'un du ou des résidus d'acide aminé de ladite sous-unité β de hIL-12 choisi dans le groupe constitué par la ou les positions de séquence 37, 38, 39, 67, 81, 82, 103, 106, 115, 122, 123, 216, 221, 232 et 312 est mutée, iii) une mutéine de hIL-12, comprenant une sous-unité α (p35) et une sous-unité β (p40), la sous-unité α étant une mutéine de la sous-unité α de hIL-12 selon la présente invention, et/ou la sous-unité β étant une mutéine de la sous-unité β de hIL-12 selon la présente invention, et iv) leurs utilisations.
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