Mutant interleukin-2 polypeptides

• US 10,323,098 B2
• Filed: 01/15/2016
• Issued: 06/18/2019
• Est. Priority Date: 02/10/2011
• Status: Active Grant

• Current Assignee: Roche Glycart AG (Roche Holding AG)
• Original Assignee: Roche Glycart AG (Roche Holding AG)
• Inventors: Ast, Oliver, Bruenker, Peter, Freimoser-Grundschober, Anne, Herter, Sylvia, Hofer, Thomas U., Hosse, Ralf, Klein, Christian, Moessner, Ekkehard, Nicolini, Valeria G., Umana, Pablo
• Primary Examiner(s): Xie, Xiaozhen
• Application Number:
  US14/996,789
• Publication Number:
  US 20160208017A1
• Time in Patent Office: 1,250 Days
• Field of Search: None
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 38/2013 IL-2
• CPC Class Codes: A61K 39/39558 against tumor tissues, cells, antigens
• CPC Class Codes: A61K 47/6813 the drug being a peptidic cytokine, e.g. an interleukin or interferon
• CPC Class Codes: A61K 47/6849 the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
• CPC Class Codes: A61K 47/6871 the antibody targeting an enzyme
• CPC Class Codes: A61K 47/6891 Pre-targeting systems involving an antibody for targeting specific cells
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/04 Immunostimulants
• CPC Class Codes: C07K 14/55 IL-2
• CPC Class Codes: C07K 16/28 against receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: C07K 16/30 from tumour cells
• CPC Class Codes: C07K 16/40 against enzymes
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 15/09 Recombinant DNA-technology
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins

Abstract

The present invention generally relates to mutant interleukin-2 polypeptides that exhibit reduced affinity to the α-subunit of the IL-2 receptor, for use as immunotherapeutic agents. In addition, the invention relates to immunoconjugates comprising said mutant IL-2 polypeptides, polynucleotide molecules encoding the mutant IL-2 polypeptides or immunoconjugates, and vectors and host cells comprising such polynucleotide molecules. The invention further relates to methods for producing the mutant IL-2 polypeptides or immunoconjugates, pharmaceutical compositions comprising the same, and uses thereof.

Claims

1. An immunoconjugate comprising a mutant interleukin-2 (IL-2) polypeptide and an immunoglobulin molecule that specifically binds Fibroblast Activation Protein (FAP), wherein said mutant IL-2 polypeptide comprises a mutant form of SEQ ID NO:

1, wherein said mutant form of SEQ ID NO;

1 comprises up to five amino acid substitutions of SEQ ID NO;

1, wherein said up to five amino acid substitutions comprise;

alanine at amino acid residue 42 of SEQ ID NO;

1, alanine at amino acid residue 45 of SEQ ID NO;

1, and glycine at amino acid residue 72 of SEQ ID NO;

1; and

wherein said immunoglobulin molecule comprises a heavy chain variable region sequence of SEQ ID NO;

143 and a light chain variable region sequence of SEQ ID NO;

141.

2. The immunoconjugate of claim 1, wherein said up to five amino acid substitutions further comprise:

(a) zero additional amino acid substitution;

(b) one additional amino acid substitution at amino acid residue 3 or 125 of SEQ ID NO;

1;

or

(c) two additional amino acid substitutions at amino acid residues 3 and 125 of SEQ ID NO;

1.

3. The immunoconjugate of claim 1, wherein said up to five amino acid substitutions comprise:

alanine at amino acid residue 42 of SEQ ID NO;

1, alanine at amino acid residue 45 of SEQ ID NO;

1, glycine at amino acid residue 72 of SEQ ID NO;

1, and alanine at amino acid residue 3 of SEQ ID NO;

1.

4. The immunoconjugate of claim 1, wherein said up to five amino acid substitutions comprise:

(a) alanine at amino acid residue 42 of SEQ ID NO;

1, alanine at amino acid residue 45 of SEQ ID NO;

1, glycine at amino acid residue 72 of SEQ ID NO;

1, and alanine, glycine, glutamine, glutamic acid, asparagine, aspartic acid, arginine, lysine, or proline at amino acid residue 3 of SEQ ID NO;

1, or

(b) alanine at amino acid residue 42 of SEQ ID NO;

1, alanine at amino acid residue 45 of SEQ ID NO;

1, glycine at amino acid residue 72 of SEQ ID NO;

1, and serine, alanine, threonine, or valine at amino acid residue 125 of SEQ ID NO;

1.

5. The immunoconjugate of claim 1, wherein said up to five amino acid substitutions comprise:

alanine at amino acid residue 42 of SEQ ID NO;

1, alanine at amino acid residue 45 of SEQ ID NO;

1, glycine at amino acid residue 72 of SEQ ID NO;

1; and

two additional amino acid substitutions, wherein

one of said two additional amino acid substitutions is alanine, glycine, glutamine, glutamic acid, asparagine, aspartic acid, arginine, lysine, or proline at amino acid residue 3 of SEQ ID NO;

1;

and the other one of said two additional amino acid substitutions is serine, alanine, threonine, or valine at amino acid residue 125 of SEQ ID NO;

1.

6. The immunoconjugate of claim 1, wherein said immunoglobulin molecule is an IgG class immunoglobulin molecule.

7. The immunoconjugate of claim 1, wherein said immunoglobulin molecule is an IgG₁ subclass immunoglobulin molecule.

8. The immunoconjugate of claim 1, wherein said mutant IL-2 polypeptide is joined at its amino-terminal amino acid to the carboxy-terminal amino acid of one of the heavy chains of said immunoglobulin molecule.

9. The immunoconjugate of claim 8, wherein the immunoconjugate comprises not more than one mutant IL-2 polypeptide.

10. The immunoconjugate of claim 1, wherein the immunoconjugate comprises not more than one mutant IL-2 polypeptide.

11. The immunoconjugate of claim 10, consisting of the one mutant IL-2 polypeptide and the immunoglobulin molecule, joined by a linker sequence.

12. The immunoconjugate of claim 1, wherein said immunoglobulin molecule comprises a modification in the Fc domain promoting heterodimerization of the two immunoglobulin heavy chains.

13. The immunoconjugate of claim 12, wherein the modification is a knob-into-hole modification, comprising a knob modification in one of the immunoglobulin heavy chains and a hole modification in the other one of the immunoglobulin heavy chains.

14. The immunoconjugate of claim 13, wherein said immunoglobulin molecule is an IgG₁ subclass immunoglobulin molecule and wherein the knob modification comprises the amino acid substitution T366W in one of the two immunoglobulin heavy chains, and the hole modification comprises the amino acid substitutions T366S, L368A and Y407V in the other one of the two immunoglobulin heavy chains, according to the EU numbering of Kabat.

15. The immunoconjugate of claim 14, wherein the immunoglobulin heavy chain comprising the knob modification further comprises the amino acid substitution S354C, and the immunoglobulin heavy chain comprising the hole modification further comprises the amino acid substitution Y349C, according to the EU numbering of Kabat.

16. The immunoconjugate of claim 1, wherein the immunoglobulin molecule comprises one or more amino acid mutations in its Fc domain that reduces the binding affinity of the immunoconjugate to an Fcγ

RIIIa, Fcγ

RI or Fcγ

RIIa receptor.

17. The immunoconjugate of claim 16, wherein said immunoglobulin molecule is an IgG₁ subclass immunoglobulin molecule and wherein the immunoglobulin molecule comprises the amino acid mutations L234A, L235A and P329G according to the EU numbering of Kabat.

18. A pharmaceutical composition comprising the immunoconjugate of claim 1 and a pharmaceutically acceptable carrier.

19. An immunoconjugate comprising a mutant interleukin-2 (IL-2) polypeptide and an immunoglobulin molecule that specifically binds Fibroblast Activation Protein (FAP),

wherein said mutant IL-2 polypeptide comprises the sequence of SEQ ID NO:

19; and

wherein said immunoglobulin molecule comprises a heavy chain variable region sequence of SEQ ID NO;

143 and a light chain variable region sequence of SEQ ID NO;

141.

20. The immunoconjugate of claim 19, wherein said mutant IL-2 polypeptide is joined at its amino-terminal amino acid to the carboxy-terminal amino acid of one of the heavy chains of said immunoglobulin molecule.

21. The immunoconjugate of claim 19, wherein the immunoconjugate comprises not more than one mutant IL-2 polypeptide.

22. The immunoconjugate of claim 21, wherein the immunoglobulin molecule comprises a modification in the Fc domain promoting heterodimerization of the two immunoglobulin heavy chains.

23. The immunoconjugate of claim 22, wherein the modification is a knob-into-hole modification, comprising a knob modification in one of the immunoglobulin heavy chains and a hole modification in the other one of the immunoglobulin heavy chains.

24. The immunoconjugate of claim 23, wherein said immunoglobulin molecule is an IgG₁ subclass immunoglobulin molecule and wherein the knob modification comprises the amino acid substitution T366W in one of the two immunoglobulin heavy chains, and the hole modification comprises the amino acid substitutions T366S, L368A and Y407V in the other one of the two immunoglobulin heavy chains, according to the EU numbering of Kabat.

25. The immunoconjugate of claim 24, wherein the immunoglobulin heavy chain comprising the knob modification further comprises the amino acid substitution S354C, and the immunoglobulin heavy chain comprising the hole modification further comprises the amino acid substitution Y349C, according to the EU numbering of Kabat.

26. The immunoconjugate of claim 19, wherein the immunoglobulin molecule comprises one or more amino acid mutations in its Fc domain that reduces the binding affinity of the immunoconjugate to an Fcγ

RIIIa, Fcγ

RI or Fcγ

RIIa receptor.

27. The immunoconjugate of claim 26, wherein said immunoglobulin molecule is an IgG₁ subclass immunoglobulin molecule and wherein the immunoglobulin molecule comprises the amino acid mutations L234A, L235A and P329G according to the EU numbering of Kabat.

28. A pharmaceutical composition comprising the immunoconjugate of claim 19 and a pharmaceutically acceptable carrier.

29. An immunoconjugate comprising a mutant interleukin-2 (IL-2) polypeptide and an immunoglobulin molecule that specifically binds Fibroblast Activation Protein (FAP), wherein the immunoconjugate comprises a polypeptide sequence of SEQ ID NO:

299, a polypeptide sequence of SEQ ID NO;

297, and a polypeptide sequence of SEQ ID NO;

233.

30. A pharmaceutical composition comprising the immunoconjugate of claim 29 and a pharmaceutically acceptable carrier.