Cytokine fusion proteins, and their pharmaceutical compositions and therapeutic applications
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.
1. 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 fusion protein of claim 1 , comprising 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 fusion protein of claim 1 , comprising 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 fusion protein of claim 1 , wherein the albumin binding domain is a single domain antibody.
5. The fusion protein of claim 4 , wherein the single domain antibody 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 fusion protein of claim 1 , wherein the interleukin-2 domain 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 fusion protein of claim 6 , wherein the interleukin-2 domain 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 fusion protein of claim 1 , wherein the interleukin-2 domain 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 fusion protein of claim 1 , wherein the interleukin-2 domain 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 fusion protein of claim 1 , wherein the interleukin-2 domain comprises an N-glycosylation site at an interface residue between an interleukin-2 and an interleukin-2 receptor-α chain.
11. The fusion protein of claim 10 , wherein the interface residue is R38 as set forth in in the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, or 5.
12. The fusion protein of claim 1 , wherein the interleukin-2 domain 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 fusion protein of claim 1 , wherein the interleukin-2 domain 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 fusion protein of claim 1 , wherein the interleukin-2 domain 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 fusion protein of claim 1 , wherein the interleukin-2 domain comprises the amino acid sequence selected from SEQ ID NOs: 1 to 5 and 18.
16. The fusion protein of claim 1 , wherein the interleukin-2 domain comprises the amino acid sequence selected from SEQ ID NOs: 1 to 5.
17. The fusion protein of claim 1 , wherein the interleukin-2 domain comprises the amino acid sequence of SEQ ID NO: 18.
18. The fusion protein of claim 1 , wherein the interleukin-21 domain comprises the amino acid sequence of SEQ ID NO: 156 or 157.
19. The fusion protein of claim 1 , comprising 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.
20. The fusion protein of claim 1 , comprising 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.
21. The fusion protein of claim 1 , comprising 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.
22. The fusion protein of claim 1 , comprising 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.
23. The fusion protein of claim 1 , having the amino acid sequence selected from SEQ ID NOs: 159 to 163 and 166 to 171.
24. A pharmaceutical composition comprising the fusion protein of claim 1 , and a pharmaceutically acceptable excipient.
25. The fusion protein of claim 4 , wherein the single domain antibody has the amino acid sequence of SEQ ID NO: 108 or 115.
26. The fusion protein of claim 4 , wherein the single domain antibody has the amino acid sequence of SEQ ID NO: 108.
27. The fusion protein of claim 4 , wherein the single domain antibody has the amino acid sequence of SEQ ID NO: 115.
28. The fusion protein of claim 6 , wherein the interleukin-2 domain comprises an amino acid substitution 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, or T111 as set forth in the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, or 5.
29. The fusion protein of claim 6 , wherein the interleukin-2 domain comprises two amino acid substitutions at positions 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.
30. The fusion protein of claim 6 , wherein the interleukin-2 domain comprises two amino acid substitutions at positions P34, K35, L36, T37, R38, L40, T41, F42, K43, F44, Y45, M46, P47, E61, E62, L63, P65, L66, E67, E68, V69, L70, N71, L72, Q74, Y107, D109, and/or T111 as set forth in the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, or 5.
31. The fusion protein of claim 1 , wherein the interleukin-2 domain comprises one, two, three, or four substitutions selected from P34N, K35N, T37N, R38N, M39N, T41N, F42N, K43N, F44N, Y45N, E61N, E62N, K64N, P65N, L66N, E68N, V69N, L72N, Y107N, and D109N as set forth in the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, or 5.
32. The fusion protein of claim 31 , wherein the interleukin-2 domain comprises a substitution selected from P34N, K35N, T37N, R38N, M39N, T41N, F42N, K43N, F44N, Y45N, E61N, E62N, K64N, P65N, L66N, E68N, V69N, L72N, Y107N, and D109N as set forth in the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, or 5.
33. The fusion protein of claim 31 , wherein the interleukin-2 domain comprises two substitutions selected from P34N, K35N, T37N, R38N, M39N, T41N, F42N, K43N, F44N, Y45N, E61N, E62N, K64N, P65N, L66N, E68N, V69N, L72N, Y107N, and D109N as set forth in the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, or 5.
34. The fusion protein 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.