Mutant interleukin-2 polypeptides

• US 9,266,938 B2
• Filed: 02/07/2012
• Issued: 02/23/2016
• 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, Umana, Pablo, Nicolini, Valerla G.
• Primary Examiner(s): Xie, Xiaozhen
• Application Number:
  US13/367,843
• Publication Number:
  US 20120244112A1
• Time in Patent Office: 1,477 Days
• US Class Current: 1/1
• 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 isolated mutant interleukin-2 (IL-2) polypeptide, wherein said mutant IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO:

1 with up to five amino acid substitutions, wherein said up to five amino acid substitutions comprise three amino acid substitutions that abolish or reduce affinity of the mutant IL-2 polypeptide to a high-affinity IL-2 receptor and preserve affinity of the mutant IL-2 polypeptide to an intermediate-affinity IL-2 receptor, as compared to a wild-type IL-2 polypeptide, wherein said three amino acid substitutions comprise;

(a) a first amino acid substitution at position 72 of SEQ ID NO;

1, selected from the group of L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K;

(b) a second amino acid substitution at position 42 of SEQ ID NO;

1, selected from the group of F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, and F42K; and

(c) a third amino acid substitution at position 45 of SEQ ID NO;

1, selected from the group of Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R and Y45K.

2. The mutant interleukin-2 polypeptide of claim 1, wherein said first amino acid substitution is L72G.

3. The mutant interleukin-2 polypeptide of claim 1, wherein said second amino acid substitution is F42A.

4. The mutant interleukin-2 polypeptide of claim 1, wherein said third amino acid substitution is Y45A.

5. The mutant interleukin-2 polypeptide of claim 1, wherein said three amino acid substitutions are F42A, Y45A and L72G.

6. The mutant interleukin-2 polypeptide of claim 1, further comprising an amino acid substitution at position 3 of SEQ ID NO:

1, wherein said amino acid substitution eliminates the O-glycosylation site of IL-2 at position 3 of SEQ ID NO;

1.

7. The mutant interleukin-2 polypeptide of claim 6, wherein said amino acid substitution at position 3 of SEQ ID NO:

1 is selected from the group of T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, and T3P.

8. The mutant interleukin-2 polypeptide of claim 6, wherein said amino acid substitution at position 3 of SEQ ID NO:

1 is T3A.

9. The mutant interleukin-2 polypeptide of claim 1, further comprising an amino acid substitution at position 125 of SEQ ID NO:

1.

10. The mutant interleukin-2 polypeptide of claim 9, wherein said amino acid substitution at position 125 of SEQ ID NO:

1 is selected from the group of C125A, C125S, C125T and C125V.

11. The mutant interleukin-2 polypeptide of claim 9, wherein said amino acid substitution at position 125 of SEQ ID NO:

1 is C125A.

12. The mutant interleukin-2 polypeptide of claim 1, comprising the sequence of SEQ ID NO:

15 or SEQ ID NO;

19.

13. The mutant interleukin-2 polypeptide of claim 1, comprising the sequence of SEQ ID NO:

19.

14. The mutant interleukin-2 polypeptide of claim 1, wherein said mutant IL-2 polypeptide is linked to a non-IL-2 moiety.

15. The mutant interleukin-2 polypeptide of claim 14, wherein said non-IL-2 moiety is an antigen binding moiety.

16. The mutant interleukin-2 polypeptide of claim 15, wherein said mutant IL-2 polypeptide shares an amino- or carboxy-terminal peptide bond with said antigen binding moiety.

17. The mutant interleukin-2 polypeptide of claim 15, wherein said antigen binding moiety is an antibody or an antibody fragment.

18. The mutant interleukin-2 polypeptide of claim 15, wherein said antigen binding moiety is selected from a Fab molecule and a scFv molecule.

19. The mutant interleukin-2 polypeptide of claim 15, wherein said antigen binding moiety is an immunoglobulin molecule.

20. The mutant interleukin-2 polypeptide of claim 19, wherein said immunoglobulin molecule is an IgG molecule.

21. The mutant interleukin-2 polypeptide of claim 19, wherein said mutant IL-2 polypeptide shares an amino-terminal peptide bond with one of the immunoglobulin heavy chains.

22. The mutant interleukin-2 polypeptide of claim 15, wherein said antigen binding moiety is directed to an antigen presented on a tumor cell or in a tumor cell environment.

23. The mutant interleukin-2 polypeptide of claim 22, wherein said antigen is selected from the group of Fibroblast Activation Protein (FAP), the A1 domain of Tenascin-C (TNC A1), the A2 domain of Tenascin-C (TNC A2), the Extra Domain B of Fibronectin (EDB), Carcinoembryonic Antigen (CEA) and the Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP).

24. An isolated mutant IL-2 polypeptide produced by a method comprising:

(i) culturing a host cell comprising a polynucleotide encoding the mutant IL-2 polypeptide of claim 1 under conditions suitable for the expression of the mutant IL-2 polypeptide and (ii) optionally recovering the mutant IL-2 polypeptide.

25. A pharmaceutical composition comprising the mutant IL-2 polypeptide of claim 1 and a pharmaceutically acceptable carrier.