Fusion protein including BDNF

Patent metadata

Field	Value
Publication number	US-12214007-B2
Application number	US-201716473816-A
Country	US
Kind code	B2
Filing date	Dec 26, 2017
Priority date	Dec 26, 2016
Publication date	Feb 4, 2025
Grant date	Feb 4, 2025

Abstract

The present invention provides a fusion protein of BDNF and an anti-human transferrin receptor antibody, in which in a heavy chain variable region of the antibody, (a) CDR1 includes an amino acid sequence of SEQ ID NO: 66 or SEQ ID NO: 67, (b) CDR2 includes an amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14, and (c) CDR3 includes an amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16.

First claim

The invention claimed is:

1. A fusion protein of human brain-derived neurotrophic factor (human BDNF) and an anti-human transferrin receptor antibody, selected from (1) or (2) below:
(1) the fusion protein wherein in a heavy chain variable region of the antibody,
(a) CDR1 comprises the amino acid sequence of SEQ ID NO: 66,
(b) CDR2 comprises the amino acid sequence of SEQ ID NO: 13,
(c) CDR3 comprises the amino acid sequence of SEQ ID NO: 15, and
(d) framework region 3 comprises the amino acid sequence of SEQ ID NO: 68,

wherein in a light chain variable region of the antibody,
(a) CDR1 comprises the amino acid sequence of SEQ ID NO: 6,
(b) CDR2 comprises the amino acid sequence of SEQ ID NO: 8, and
(c) CDR3 comprises the amino acid sequence of SEQ ID NO: 10, and

wherein the human BDNF binds to the N-terminus of the heavy chain of the antibody via a linker;
(2) the fusion protein wherein in a heavy chain variable region of the antibody,
(a) CDR1 comprises the amino acid sequence of SEQ ID NO: 67,
(b) CDR2 comprises the amino acid sequence of SEQ ID NO: 14,
(c) CDR3 comprises the amino acid sequence of SEQ ID NO: 16, and
(d) framework region 3 comprises the amino acid sequence of SEQ ID NO: 68, and

wherein in a light chain variable region of the antibody,
(a) CDR1 comprises the amino acid sequence of SEQ ID NO: 7,
(b) CDR2 comprises the amino acid sequence of SEQ ID NO: 9, and
(c) CDR3 comprises the amino acid sequence of SEQ ID NO: 10, and

wherein the human BDNF binds to the N-terminus of the heavy chain of the antibody via a linker.

2. The fusion protein according to claim 1 ,
wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 69.

3. The fusion protein according to claim 2 ,
wherein the heavy chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 70 or SEQ ID NO: 72.

4. The fusion protein according to claim 1 ,
wherein the light chain variable region of the antibody comprises the amino acid sequence of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22.

5. The fusion protein according to claim 1 ,
wherein the light chain of the antibody comprises the amino acid sequence of SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, or SEQ ID NO: 29.

6. The fusion protein according to claim 1 ,
wherein the antibody is a Fab antibody, a F(ab′) 2 antibody, or a F(ab′) antibody.

7. The fusion protein according to claim 1 ,
wherein the antibody is a single-chain antibody selected from the group consisting of scFab, scF(ab′), scF(ab′) 2 , and scFv.

8. The fusion protein according to claim 7 ,
wherein the light chain binds to a C-terminus of the heavy chain of the antibody via a linker sequence.

9. The fusion protein according to claim 8 ,
wherein the linker sequence is formed of 8 to 50 amino acid residues.

10. The fusion protein according to claim 8 ,
wherein the linker sequence is selected from the group consisting of the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence Gly-Gly-Gly, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, and the amino acid sequence of SEQ ID NO: 5, the amino acid sequence corresponding to three consecutive amino acid sequences of SEQ ID NO: 3, and the amino acid sequence consisting of 2 to 10 of the aforementioned amino acid sequences consecutively linked.

11. The fusion protein according to claim 1 ,
wherein the linker is a peptide including 1 to 50 amino acid residues.

12. The fusion protein according to claim 1 ,
wherein the linker is a peptide including the amino acid sequence selected from the group consisting of the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, and the amino acid sequence consisting of 2 to 10 of the aforementioned amino acid sequences consecutively linked.

13. The fusion protein according to claim 1 ,
wherein the human BDNF comprises
the amino acid sequence of SEQ ID NO: 51.

14. The fusion protein according to claim 1 ,
wherein the fusion protein having affinity to both of an extracellular region of a human transferrin receptor and an extracellular region of a monkey transferrin receptor.

15. The fusion protein according to claim 14 ,
wherein a dissociation constant of the anti-human transferrin receptor antibody with the extracellular region of the human transferrin receptor is equal to or less than 1×10 −10 M, and a dissociation constant of the anti-human transferrin receptor antibody with the extracellular region of the monkey transferrin receptor is equal to or less than 1×10 −9 M.

16. The fusion protein according to claim 1 ,
wherein the antibody is an antigen-binding fragment.

17. The fusion protein according to claim 16 ,
wherein the human BDNF binds to the N-terminus of the antigen-binding fragment via the linker.

18. The fusion protein according to claim 16 ,
wherein the antigen-binding fragment is a single-chain antibody.

19. The fusion protein according to claim 18 ,
wherein the light chain variable region binds to the C-terminus of the heavy chain variable region via a linker sequence.

20. The fusion protein according to claim 19 ,
wherein the linker sequence includes 8 to 50 amino acid residues.

21. The fusion protein according to claim 19 ,
wherein the linker sequence is selected from the group consisting of the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence Gly-Gly-Gly, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, the amino acid sequence corresponding to three consecutive amino acid sequences of SEQ ID NO: 3, and the amino acid sequence consisting of 2 to 10 of the aforementioned amino acid sequences consecutively linked.

22. The fusion protein according to claim 18 ,
wherein the antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 69 and a light chain variable region having the amino acid sequence of SEQ ID NO: 18.

23. The fusion protein according to claim 22 ,
wherein the antibody is formed of the amino acid sequence of SEQ ID NO: 57, and the human BDNF binds to the N-terminus of the heavy chain of the antibody via the linker.

24. The fusion protein according to claim 23 ,
wherein the antibody is formed of the amino acid sequence of SEQ ID NO: 57, the human BDNF is human pro-BDNF, and binds to the N-terminus of the heavy chain of the antibody via the linker, and the fusion protein comprises the amino acid sequence of SEQ ID NO: 59.

25. The fusion protein according to claim 23 ,
wherein the antibody is formed of the amino acid sequence of SEQ ID NO: 57, the human BDNF binds to the N-terminus of the heavy chain of the antibody via the linker, and the fusion protein comprises the amino acid sequence of SEQ ID NO: 60.

26. The fusion protein according to claim 16 ,
wherein the antigen-binding fragment is any one of Fab, F(ab′) 2 , or F(ab′).

27. The fusion protein according to claim 26 ,
wherein the human BDNF binds to the N-terminus of a heavy chain of any one of Fab, F(ab′) 2 , or F(ab′) via the linker.

28. The fusion protein according to cl

Assignees

• Japan Chem Res

Inventors

• Sonoda Hiroyuki
• Takahashi Kenichi

Classifications

• C12N15/66

  General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease · CPC title

• C07K2319/00

  Fusion polypeptide · CPC title

• C07K2317/62

  comprising only variable region components · CPC title

• C07K2317/567

  Framework region [FR] · CPC title

• C07K2317/565

  Complementarity determining region [CDR] · CPC title