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EP-4739701-A2 - MUTEINS OF HUMAN INTERLEUKIN 12

EP4739701A2EP 4739701 A2EP4739701 A2EP 4739701A2EP-4739701-A2

Abstract

The present invention refers, inter alia, to i) a mutein of the α-subunit of human Interleukin 12 (hIL-12), wherein at least one of the amino acid residue(s) of said α-subunit of hIL-12 selected from the group consisting of sequence position(s) 70, 71, 72, 185, 187 and 189 is/are mutated, ii) a mutein of the β-subunit of hIL-12, wherein at least one of the amino acid residue(s) of said β-subunit of hIL-12 selected from the group consisting of sequence position(s) 37, 38, 39, 67, 81, 82, 103, 106, 115, 122, 123, 216, 221, 232 and 312 is/are mutated, iii) a mutein of hIL-12, comprising an α-subunit (p35) and a β-subunit (p40), wherein the α-subunit is a mutein of the α-subunit of hIL-12 according to the present invention, and/or wherein the β-subunit is a mutein of the β-subunit of hIL-12 according to the present invention, and iv) uses thereof.

Inventors

  • FEIGE, MATTHIAS
  • Liebl, Korbinian

Assignees

  • Technische Universität München, in Vertretung des Freistaats Bayern

Dates

Publication Date
20260513
Application Date
20240705

Claims (15)

  1. 1. 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 the a-subunit of human Interleukin 12 selected from the group consisting of sequence positions 70, 71 , 72, 185, 187 and 189 is/are mutated.
  2. 2. The mutein of claim 1 , wherein said mutein comprises at least 90% sequence identity to the a-subunit of human Interleukin 12 (SEQ ID NO: 1 ).
  3. 3. The mutein of claim 1 or 2, 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, preferably 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 and 187 is/are mutated.
  4. 4. The mutein of any one of the preceding claims, 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, 187 and 189, preferably selected 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).
  5. 5. 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 of human Interleukin 12 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.
  6. 6. The mutein of claim 5, wherein said mutein comprises at least 90% sequence identity to the [3-subunit of human Interleukin 12 (SEQ ID NO: 2).
  7. 7. The mutein of claim 5 or claim 6, 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, preferably selected from the group consisting of sequence positions 67, 115, 122, 123, 221 , 232 and 312, is/are mutated.
  8. 8. The mutein of any one of claims 5 to 7, 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 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(s) in SEQ ID NO: 2, preferably is/are replaced by glycine (G).
  9. 9. The mutein of any one of claims 5 to 8, wherein 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(s) in SEQ ID NO: 2, preferably is/are replaced by alanine (A).
  10. 10. The mutein of any one of claims 5 to 9, 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 position(s) 37, 38, 39, 67, 81 , 82, 106, 115, 216 and 221 , preferably selected from the group consisting of sequence position(s) 38, 39, 67, 106, 115 and 221 , more preferably 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(s) in SEQ ID NO: 2, preferably is/are replaced by alanine (A).
  11. 11. 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 claims 1 to 4, 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 claims 5 to 10.
  12. 12. A nucleic acid molecule comprising i) a nucleotide sequence encoding the mutein of human Interleukin 12 of claim 11 or ii) a nucleotide sequence encoding a) the mutein of the a-subunit of human Interleukin 12 of any one of claims 1 to 4, and/or b) the mutein of the [3-subunit of human Interleukin 12 of any one of claims 5 to 10, 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.
  13. 13. A pharmaceutical composition comprising a mutein of any one of claims 1 to 11 , preferably further comprising a pharmaceutically acceptable carrier.
  14. 14. The mutein of any one of claims 1 to 11 , for use as a medicament.
  15. 15. The mutein of any one of claims 1 to 11 , 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.

Description

MUTEINS OF HUMAN INTERLEUKIN 12 FIELD OF THE INVENTION [001] 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. In a further aspect, 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. In yet another aspect, 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. In yet another embodiment, 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. In yet another aspect, 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. In yet another embodiment, 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. In yet another embodiment, the present invention relates to a mutein according to the present invention for use in the treatment of a disease. In yet another embodiment, 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. In yet another aspect, 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 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 [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 (b) introducing the obtained nucleic acid molecule for expression into a host cell or into a cell extract or into a cell lysate. BACKGROUND OF THE INVENTION [002] 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. A remarkable feature of the IL-12 family is that nature uses extensive sharing of only five subunits to build the four heterodimers. Likewise, this combinatorial complexity applies to the IL-12 family receptors, which are also heterodimers formed by five different chains (IL-12R[31 , IL- 12R|32, IL-23R, IL-27Ra, gp130). Binding of the suitable interleukin induces receptor chain dimerization thereby activ