IP Library › Granted Patent US 12,398,188
Granted Patent B2
US 12,398,188 · App. 18/346,250 · Granted Aug 26, 2025

Cytokine fusion proteins, and their pharmaceutical compositions and therapeutic applications

Inventors: Ziyang Zhong (Belmont, CA); Fan Ye (Mountain View, CA); Matthew Siegel (Menlo Park, CA); Jianing Huang (San Mateo, CA)
Assignee: Anwita Biosciences, Inc.
C07K14/55C07K16/00A61K38/00C07K2317/565C07K2317/569C07K2319/31C07K2319/33
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Quick Facts
Patent No.
US 12,398,188
App. No.
18/346,250
Granted
Aug 26, 2025
Kind
B2
Abstract

Provided herein are a fusion protein comprising first and second cytokine domains, and a half-life extension domain; and a pharmaceutical composition thereof. Also provided herein are methods of their use for treating, preventing, or ameliorating one or more symptoms of a proliferative disease.

Claims (37)

1. A method of activating STAT3 and STAT5 signaling pathways in a subject with cancer, comprising administering to the subject a therapeutically effective amount of a fusion protein comprising an interleukin-2 domain, an interleukin-21 domain, and an albumin binding domain:

wherein the interleukin-2 domain comprises the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, or 5; or wherein the interleukin-2 domain is an N-glycosylated interleukin-2 comprising one, two, three, or four substitutions at position P34, K35, L36, T37, R38, M39, L40, T41, F42, K43, F44, Y45, M46, P47, E61, E62, L63, K64, P65, L66, E67, E68, V69, L70, N71, L72, A73, Q74, Y107, D109, and/or T111 as set forth in the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, or 5;

wherein the interleukin-21 domain comprises the amino acid sequence of SEQ ID NO: 156, 157, or 158; and

wherein the C-terminus of the interleukin-2 domain is connected to the N-terminus of the interleukin-21 domain directly or via a peptide linker; and the C-terminus of the interleukin-21 domain is connected to the N-terminus of the albumin binding domain directly or via a peptide linker; or

wherein the C-terminus of the interleukin-21 domain is connected to the N-terminus of the interleukin-2 domain directly or via a peptide linker; and the C-terminus of the interleukin-2 domain is connected to the N-terminus of the albumin binding domain directly or via a peptide linker; or

wherein the C-terminus of the interleukin-2 domain is connected to the N-terminus of the albumin binding domain directly or via a peptide linker; and the C-terminus of the albumin binding domain is connected to the N-terminus of the interleukin-21 domain directly or via a peptide linker; or

wherein the C-terminus of the interleukin-21 domain is connected to the N-terminus of the albumin binding domain directly or via a peptide linker; and the C-terminus of the albumin binding domain is connected to the N-terminus of the interleukin-2 domain directly or via a peptide linker; or

wherein the C-terminus of the albumin binding domain is connected to the N-terminus of the interleukin-2 domain directly or via a peptide linker; and the C-terminus of the interleukin-2 domain is connected to the N-terminus of the interleukin-21 domain directly or via a peptide linker; or

wherein the C-terminus of the albumin binding domain is connected to the N-terminus of the interleukin-21 domain directly or via a peptide linker; and the C-terminus of the interleukin-21 domain is connected to the N-terminus of the interleukin-2 domain directly or via a peptide linker.

2. The method of claim 1 , wherein the fusion protein comprises an interleukin-2 domain, an interleukin-21 domain, an albumin binding domain, and optionally first and second peptide linkers; wherein the C-terminus of the interleukin-2 domain is connected to the N-terminus of the albumin binding domain directly or via the first peptide linker; and the C-terminus of the albumin binding domain is connected to the N-terminus of the interleukin-21 domain directly or via the second peptide linker.

3. The method of claim 1 , wherein the fusion protein comprises an interleukin-2 domain, an interleukin-21 domain, an albumin binding domain, and optionally first and second peptide linkers; wherein the C-terminus of the interleukin-21 domain is connected to the N-terminus of the albumin binding domain directly or via the first peptide linker; and the C-terminus of the albumin binding domain is connected to the N-terminus of the interleukin-2 domain directly or via the second peptide linker.

4. The method of claim 1 , wherein the albumin binding domain of the fusion protein is a single domain antibody.

5. The method of claim 4 , wherein the single domain antibody of the fusion protein comprises: (i) CDR1 of SEQ ID NO: 101, CDR2 of SEQ ID NO: 102, and CDR3 of SEQ ID NO: 103; or (ii) CDR1 of SEQ ID NO: 109, CDR2 of SEQ ID NO: 110, and CDR3 of SEQ ID NO: 111.

6. The method of claim 1 , wherein the interleukin-2 domain of the fusion protein comprises one, two, three, or four, substitutions at position P34, K35, L36, T37, R38, M39, L40, T41, F42, K43, F44, Y45, M46, P47, E61, E62, L63, K64, P65, L66, E67, E68, V69, L70, N71, L72, A73, Q74, Y107, D109, and/or T111 as set forth in the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, or 5.

7. The method of claim 6 , wherein the interleukin-2 domain of the fusion protein comprises four substitutions at positions R38, L40, F42, and Y45 as set forth in the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, or 5.

8. The method of claim 1 , wherein the interleukin-2 domain of the fusion protein comprises an N-glycosylation site having the amino acid sequence of NXT or NXS, wherein each X is independently A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y.

9. The method of claim 1 , wherein the interleukin-2 domain of the fusion protein comprises an N-glycosylation site having the amino acid sequence of NMT or NMS, each independently starting at position 38 or 45 as set forth in the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, or 5.

10. The method of claim 1 , wherein the interleukin-2 domain of the fusion protein comprises an N-glycosylation site at an interface residue between an interleukin-2 and an interleukin-2 receptor-α chain.

11. The method of claim 10 , wherein the interface residue is R38 as set forth in the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, or 5.

12. The method of claim 1 , wherein the interleukin-2 domain of the fusion protein comprises two amino acid substitutions: (i) R38N and (ii) L40T or L40S as set forth in the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, or 5.

13. The method of claim 1 , wherein the interleukin-2 domain of the fusion protein comprises three amino acid substitutions: (i) R38N, (ii) L40T or L40S, and (iii) F42A, Y45A, E61A, or E62A, as set forth in the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, or 5.

14. The method of claim 1 , wherein the interleukin-2 domain of the fusion protein comprises four amino acid substitutions: (i) R38N, (ii) L40T or L40S, (iii) K43N, or (iv) Y45T or Y45S, as set forth in the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, or 5.

15. The method of claim 1 , wherein the interleukin-2 domain of the fusion protein comprises the amino acid sequence selected from SEQ ID NOs: 1 to 5 and 18.

16. The method of claim 1 , wherein the interleukin-2 domain of the fusion protein comprises the amino acid sequence selected from SEQ ID NOs: 1 to 5.

17. The method of claim 1 , wherein the interleukin-2 domain of the fusion protein comprises the amino acid sequence of SEQ ID NO: 18.

18. The method of claim 1 , wherein the interleukin-21 domain of the fusion protein comprises the amino acid sequence of SEQ ID NO: 156 or 157.

19. The method of claim 1 , wherein each peptide linker is independently a peptide linker having the amino acid sequence of GSG or one of SEQ ID NOs: 124 to 155.

20. The method of claim 1 , wherein the fusion protein comprises one interleukin-2 domain having the amino acid sequence of any one of SEQ ID NOs: 1 to 5 and 18; one interleukin-21 domain having the amino acid sequence of any one of SEQ ID NOs: 156 to 158; one V H H single domain antibody having the amino acid sequence of SEQ ID NO: 108 or 115; and optionally one or two peptide linkers, each independently having the amino acid sequence of GSG or any one of SEQ ID NOs: 124 to 155.

21. The method of claim 1 , wherein the fusion protein comprises one interleukin-2 domain having the amino acid sequence of SEQ ID NO: 2 or 18; one interleukin-21 domain having the amino acid sequence of SEQ ID NO: 156 or 157; one V H H single domain antibody having the amino acid sequence of SEQ ID NO: 108 or 115; and optionally one or two peptide linkers, each independently having the amino acid sequence of SEQ ID NO: 126 or 133.

22. The method of claim 1 , wherein the fusion protein comprises one interleukin-2 domain having the amino acid sequence of any one of SEQ ID NOs: 1 to 5 and 18; one interleukin-21 domain having the amino acid sequence of any one of SEQ ID NOs: 156 to 158; one V H H single domain antibody having the amino acid sequence of SEQ ID NO: 108 or 115; and one peptide linker having the amino acid sequence of GSG or any one of SEQ ID NOs: 124 to 155; wherein the C-terminus of the interleukin-2 domain is connected to the N-terminus of the peptide linker, the C-terminus of the peptide linker is connected to the N-terminus of the V H H single domain antibody, and the C-terminus of the V H H single domain antibody is connected to the N-terminus of the interleukin-21 domain; or wherein the C-terminus of the interleukin-21 domain is connected to the N-terminus of the V H H single domain antibody, the C-terminus of the V H H single domain antibody is connected to the N-terminus of the peptide linker, and the C-terminus of the peptide linker is connected to the N-terminus of the interleukin-2 domain.

23. The method of claim 1 , wherein the fusion protein comprises one interleukin-2 domain having the amino acid sequence of SEQ ID NO: 2 or 18; one interleukin-21 domain having the amino acid sequence of SEQ ID NO: 156 or 157; one V H H single domain antibody having the amino acid sequence of SEQ ID NO: 108 or 115; and one peptide linker having the amino acid sequence of SEQ ID NO: 126 or 133; wherein the C-terminus of the interleukin-2 domain is connected to the N-terminus of the peptide linker, the C-terminus of the peptide linker is connected to the N-terminus of the V H H single domain antibody, and the C-terminus of the V H H single domain antibody is connected to the N-terminus of the interleukin-21 domain; or wherein the C-terminus of the interleukin-21 domain is connected to the N-terminus of the V H H single domain antibody, the C-terminus of the V H H single domain antibody is connected to the N-terminus of the peptide linker, and the C-terminus of the peptide linker is connected to the N-terminus of the interleukin-2 domain.

24. The method of claim 1 , wherein the fusion protein has the amino acid sequence selected from SEQ ID NOs: 159 to 163 and 166 to 171.

25. The method of claim 1 , wherein the cancer is metastatic cancer.

26. The method of claim 1 , wherein the cancer is renal cell carcinoma (RCC).

27. The method of claim 26 , wherein the cancer is metastatic renal cell carcinoma (RCC).

28. The method of claim 4 , wherein the single domain antibody of the fusion protein has the amino acid sequence of SEQ ID NO: 108.

29. The method of claim 4 , wherein the single domain antibody of the fusion protein has the amino acid sequence of SEQ ID NO: 115.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Oct 10, 2023
From: ZHONG, ZIYANG; YE, FAN; SIEGEL, MATTHEW; HUANG, JIANING
To: ANWITA BIOSCIENCES, INC.
Reel/Frame 065167/0842 →
Continuity (3)
Division 16952079 · Nov 19, 2020
Provisional Application 62938275 · Nov 20, 2019
Related Publication 20230348549A1 · Nov 2, 2023
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