CN115073598B

CN115073598B - anti-BAFFR antibody and application thereof

Info

Publication number: CN115073598B
Application number: CN202110275249.3A
Authority: CN
Inventors: 秦春铃; 黄真真; 周青青; 杨珍; 姜晓玲
Original assignee: Sunho China Biopharmaceutical Co Ltd
Current assignee: Sunho China Biopharmaceutical Co Ltd
Priority date: 2021-03-15
Filing date: 2021-03-15
Publication date: 2024-02-20
Anticipated expiration: 2041-03-15
Also published as: WO2022194067A1; CN115073598A

Abstract

The invention discloses an anti-BAFFR antibody or an antigen binding fragment thereof, application thereof and a pharmaceutical composition. The anti-BAFFR antibody provided by the invention can be specifically combined with BAFFR, block a BAFFR-BAFF signal path, clear hyperproliferative B lymphocytes and inhibit the growth of the B cells.

Description

anti-BAFFR antibody and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an anti-BAFFR antibody and application thereof.

Background

BAFFR is encoded by TNFRSF13C gene, other abbreviations are: TNFRSF13C, BLyS-3, CD268, etc. The BAFFR belongs to the transmembrane protein of TNFR family, consists of 184 amino acids, and is divided into an extracellular part, a transmembrane region and an intracellular part, and the extracellular part is suitable for development of antibody medicines.

BAFFR is a high expression marker specific for B lymphocytes of different stages, and has a similar expression profile in B cells to CD20, expressed at a stage of development of other B cells than plasma cells (plasma), pro B cells (proB) and pre B (preB) cells, but a higher abundance of expression than CD 20. BAFFR is not expressed in bone marrow stem cells of B cell origin, and after the hyperproliferative B cells are cleared, the B cells can be replenished again after stopping the medicine, and the normal requirement of a human body is not influenced, so that the safety is good.

The B cell proliferation at different stages corresponds to different lymphoma indications, and the indications of the BAFFR target blocker include other lymphoma types besides ALL (acute lymphoblastic leukemia) and MM (multiple myeloma), such as MCL (mantle cell lymphoma), DLBCL (diffuse large B lymphoma, chronic stranguria, follicular lymphoma) and the like.

The only ligand of the BAFFR is BAFF, and after the BAFFR is combined with the BAFF, the BAFFR and the BAFF act together to activate NF-kB signal channels in B cells, so that proliferation and activation of the B cells are promoted. The BAFF-BAFFR signaling pathway and BCR pathway are key pathways for imatureb cell maturation and activation, and blocking of the dual pathway can completely clear peripheral B lymphocytes and inhibit development and proliferation of B lymphocytes.

Most of the current drugs for treating B cell lymphomas target CD19 and CD20, and aim at the BCR pathway of B cell growth, but the drugs are easy to generate drug resistance. Taking CD19 as an example, according to the prior report, about 40% of patients relapse due to loss of CD19 antigen on the surface of cancer cells. The BAFFR-BAFF signaling pathway is located downstream of the BCR pathway and is an essential survival pathway for cells, which can further inhibit B cell growth downstream.

Disclosure of Invention

In view of the shortcomings of the existing problems, in the present application, the inventors developed anti-BAFFR antibodies with good performance, which are capable of specifically recognizing/binding BAFFR, blocking BAFFR-BAFF signaling pathway, clearing hyperproliferative B lymphocytes, and inhibiting the growth of B cells.

It is a first object of the present invention to provide an anti-BAFFR antibody or antigen binding fragment thereof.

It is a second object of the present invention to provide a gene encoding the above anti-BAFFR antibody or antigen binding fragment thereof.

It is a third object of the present invention to provide the use of an anti-BAFFR antibody or antigen binding fragment thereof as described above.

A fourth object of the present invention is to provide a pharmaceutical composition.

The technical scheme adopted for solving the technical problems is as follows:

an anti-BAFFR antibody or antigen binding fragment thereof, wherein the antibody or antigen binding fragment thereof comprises a binding region in whole or in part of SEQ ID NO:145, and a polypeptide sequence described in seq id no.

Preferably, the antibody is a murine antibody, a single domain antibody, a chimeric antibody, a fully human antibody or a humanized antibody.

Preferably, the antibody is a monospecific, bispecific, multispecific antibody, antibody conjugate, or car molecule for cell therapy.

Preferably, the antibody conjugate is a cytotoxic moiety, radioisotope, drug or cytokine.

Preferably, the bispecific antibody comprises any of the antibodies or antigen binding fragments thereof described above, as well as antibodies or antigen binding fragments directed against other antigens and/or other antigenic epitopes.

Preferably, the antibody is a monoclonal antibody.

An anti-BAFFR monoclonal antibody or antigen binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises heavy chain complementarity determining regions CDR1, CDR2 and CDR3 and the light chain variable region comprises light chain complementarity determining regions CDR1, CDR2 and CDR3, wherein,

(a) CDR1 of the heavy chain variable region selected from SEQ ID NO:33-40, or an amino acid sequence that hybridizes to any one of SEQ ID NOs: 33-40, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to any one of SEQ ID NO:33-40, an amino acid sequence having one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence of any of seq id no;

(b) CDR2 of the heavy chain variable region selected from SEQ ID NO:41-48, or an amino acid sequence that hybridizes to any one of SEQ ID NOs: 41-48, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to any one of SEQ ID NO:41-48, an amino acid sequence having one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence;

(c) CDR3 of the heavy chain variable region selected from SEQ ID NO:49-56, or an amino acid sequence that hybridizes to any one of SEQ ID NOs: 49-56, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to any one of SEQ ID NO:49-56, an amino acid sequence having one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence;

(d) CDR1 of the light chain variable region selected from SEQ ID NO:57-64, or an amino acid sequence identical to any one of SEQ ID NOs: 57-64, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to any one of SEQ ID NO:57-64, an amino acid sequence having one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence;

(e) CDR2 of the light chain variable region selected from SEQ ID NO:65-72, or an amino acid sequence that hybridizes to any one of SEQ ID NOs: 65-72, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to any one of SEQ ID NO:65-72, an amino acid sequence having one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence;

(f) CDR3 of the light chain variable region selected from SEQ ID NO: 73-80; the antibody binds to siglec15, or to SEQ ID NO:73-80, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to any one of SEQ ID NO:73-80, an amino acid sequence having one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence;

preferably, the heavy and light chains comprise a heavy chain variable region comprising a framework region FR and CDR1, CDR2 and CDR3 of the heavy chain variable region; the frame region FR includes:

(a) Selected from SEQ ID NOs: 81-88 of any one of the amino acid sequences,

or with SEQ ID NO:81-88 has a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity,

or with SEQ ID NO:81-88, compared to an FR1 shown in an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) conservative amino acid mutations (preferably substitutions, insertions or deletions),

(b) Selected from SEQ ID NOs: 89-96 of any one of the amino acid sequences,

or with SEQ ID NO:89-96, a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to any one of the amino acid sequences given,

or with SEQ ID NO:89-96, compared to an FR2 represented by an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) conservative amino acid mutations (preferably substitutions, insertions or deletions),

(c) Selected from SEQ ID NOs: 97-104 of any one of the amino acid sequences,

or with SEQ ID NO:97-104 has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity,

or with SEQ ID NO:97-104, compared to an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) conservative amino acid mutations (preferably substitutions, insertions or deletions),

and

(d) Selected from SEQ ID NOs: 105-112,

or with SEQ ID NO:105-112 having a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity,

Or with a sequence selected from SEQ ID NO:105-112, compared to an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) conservative amino acid mutations (preferably substitutions, insertions or deletions);

preferably, the heavy and light chains comprise a heavy chain variable region and a light chain variable region comprising a framework region FR and CDR1, CDR2 and CDR3 of the light chain variable region described above; the frame region FR includes:

(e) Selected from SEQ ID NOs: 113-120,

or with SEQ ID NO:113-120 has a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity,

or with SEQ ID NO:113-120, compared to an FR1 shown in an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) conservative amino acid mutations (preferably substitutions, insertions or deletions),

(f) Selected from SEQ ID NOs: 121-128, or a fragment thereof,

or with SEQ ID NO:121-128, a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity,

Or with SEQ ID NO:121-128, compared to an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) conservative amino acid mutations (preferably substitutions, insertions or deletions),

(g) Selected from SEQ ID NOs: 129-136,

or with SEQ ID NO:129-136 having a sequence of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity,

or with SEQ ID NO:129-136, compared to an FR3 shown in an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) conservative amino acid mutations (preferably substitutions, insertions or deletions),

and

(h) Selected from SEQ ID NOs: 137-144 of the amino acid sequence,

or with SEQ ID NO:137-144, a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity,

or with a sequence selected from SEQ ID NO:137-144, compared to the amino acid sequence shown in amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) conservative amino acid mutations (preferably substitutions, insertions or deletions).

Preferably, CDR1, CDR2 and CDR3 of the heavy chain variable region consist of the amino acid sequences SEQ ID NO: 33. SEQ ID NO:41 and SEQ ID NO:49, and CDR1, CDR2, and CDR3 of the light chain variable region consist of SEQ ID NO: 57. SEQ ID NO:65 and SEQ ID NO: 73;

more preferably, the heavy chain variable region comprises the framework region FR and CDR1, CDR2 and CDR3 of the heavy chain variable region described above; more preferably, said frame region FR comprises: SEQ ID NO:91, FR1, SEQ ID NO:101, FR2, SEQ ID NO:111 and FR3 and SEQ ID NO:121, FR4.

More preferably, the light chain variable region comprises the framework region FR and CDR1, CDR2 and CDR3 of the light chain variable region described above; more preferably, said frame region FR comprises: SEQ ID NO:131, FR1, SEQ ID NO:139 FR2, SEQ ID NO:147 and FR3 and SEQ ID NO:155, FR4 as shown.

Preferably, CDR1, CDR2 and CDR3 of the heavy chain variable region consist of the amino acid sequences SEQ ID NO: 38. SEQ ID NO:48 and SEQ ID NO:58, and CDR1, CDR2 and CDR3 of the light chain variable region consist of SEQ ID NO: 68. SEQ ID NO:76 and SEQ ID NO: 84;

More preferably, the heavy chain variable region comprises the framework region FR and CDR1, CDR2 and CDR3 of the heavy chain variable region described above; more preferably, said frame region FR comprises: SEQ ID NO:92, FR1, SEQ ID NO:102, FR2, SEQ ID NO:112 and FR3 and SEQ ID NO:122, FR4.

More preferably, the light chain variable region comprises the framework region FR and CDR1, CDR2 and CDR3 of the light chain variable region described above; more preferably, said frame region FR comprises: SEQ ID NO:132, FR1, SEQ ID NO:140, FR2, SEQ ID NO:148 and FR3 shown in SEQ ID NO: FR4 indicated at 156.

Preferably, CDR1, CDR2 and CDR3 of the heavy chain variable region consist of the amino acid sequences SEQ ID NO: 39. SEQ ID NO:49 and SEQ ID NO:59, and CDR1, CDR2 and CDR3 of the light chain variable region consist of SEQ ID NO: 69. SEQ ID NO:77 and SEQ ID NO: 85;

more preferably, the heavy chain variable region comprises the framework region FR and CDR1, CDR2 and CDR3 of the heavy chain variable region described above; more preferably, said frame region FR comprises: SEQ ID NO:93, FR1, SEQ ID NO:103, FR2, SEQ ID NO:113 and FR3 and SEQ ID NO: FR4 indicated by 123.

More preferably, the light chain variable region comprises the framework region FR and CDR1, CDR2 and CDR3 of the light chain variable region described above; more preferably, said frame region FR comprises: SEQ ID NO:133, FR1, SEQ ID NO:141, FR2, SEQ ID NO:149 and FR3 shown in SEQ ID NO:157, FR4.

Preferably, CDR1, CDR2 and CDR3 of the heavy chain variable region consist of the amino acid sequences SEQ ID NO: 40. SEQ ID NO:50 and SEQ ID NO:60, and CDR1, CDR2 and CDR3 of the light chain variable region consist of SEQ ID NO: 70. SEQ ID NO:78 and SEQ ID NO: 86;

more preferably, the heavy chain variable region comprises the framework region FR and CDR1, CDR2 and CDR3 of the heavy chain variable region described above; more preferably, said frame region FR comprises: SEQ ID NO:84, FR1, SEQ ID NO:94, FR2, SEQ ID NO:100 and FR3 and SEQ ID NO: FR4 indicated at 108.

More preferably, the light chain variable region comprises the framework region FR and CDR1, CDR2 and CDR3 of the light chain variable region described above; more preferably, said frame region FR comprises: SEQ ID NO:116, FR1, SEQ ID NO:124, FR2, SEQ ID NO:132 and FR3 and SEQ ID NO:140, FR4.

Preferably, CDR1, CDR2 and CDR3 of the heavy chain variable region consist of the amino acid sequences SEQ ID NO: 37. SEQ ID NO:45 and SEQ ID NO:53, and CDR1, CDR2 and CDR3 of the light chain variable region consist of SEQ ID NO: 61. SEQ ID NO:69 and SEQ ID NO: 77;

more preferably, the heavy chain variable region comprises the framework region FR and CDR1, CDR2 and CDR3 of the heavy chain variable region described above; more preferably, said frame region FR comprises: SEQ ID NO:85, FR1, SEQ ID NO:93, FR2, SEQ ID NO:101 and FR3 and SEQ ID NO: FR4 indicated at 109.

More preferably, the light chain variable region comprises the framework region FR and CDR1, CDR2 and CDR3 of the light chain variable region described above; more preferably, said frame region FR comprises: SEQ ID NO:117, FR1, SEQ ID NO:125, FR2, SEQ ID NO:133 and FR3 and SEQ ID NO:141, FR4.

Preferably, CDR1, CDR2 and CDR3 of the heavy chain variable region consist of the amino acid sequences SEQ ID NO: 38. SEQ ID NO:46 and SEQ ID NO:54, and CDR1, CDR2, and CDR3 of the light chain variable region consist of SEQ ID NO: 62. SEQ ID NO:70 and SEQ ID NO: 78;

More preferably, the heavy chain variable region comprises the framework region FR and CDR1, CDR2 and CDR3 of the heavy chain variable region described above; more preferably, said frame region FR comprises: SEQ ID NO:86, FR1, SEQ ID NO:94, FR2, SEQ ID NO:102 and FR3 and SEQ ID NO:110, FR4.

More preferably, the light chain variable region comprises the framework region FR and CDR1, CDR2 and CDR3 of the light chain variable region described above; more preferably, said frame region FR comprises: SEQ ID NO:118, FR1, SEQ ID NO:126, FR2, SEQ ID NO:134 and FR3 and SEQ ID NO: FR4 indicated at 142.

Preferably, CDR1, CDR2 and CDR3 of the heavy chain variable region consist of the amino acid sequences SEQ ID NO: 39. SEQ ID NO:47 and SEQ ID NO:55, and CDR1, CDR2 and CDR3 of the light chain variable region consist of SEQ ID NO: 63. SEQ ID NO:71 and SEQ ID NO: 79;

more preferably, the heavy chain variable region comprises the framework region FR and CDR1, CDR2 and CDR3 of the heavy chain variable region described above; more preferably, said frame region FR comprises: SEQ ID NO:87, FR1, SEQ ID NO:95, FR2, SEQ ID NO:103 and FR3 and SEQ ID NO: FR4 indicated by 111.

More preferably, the light chain variable region comprises the framework region FR and CDR1, CDR2 and CDR3 of the light chain variable region described above; more preferably, said frame region FR comprises: SEQ ID NO:119, FR1, SEQ ID NO:127, FR2, SEQ ID NO:135 and FR3 and SEQ ID NO:143 FR4.

Preferably, CDR1, CDR2 and CDR3 of the heavy chain variable region consist of the amino acid sequences SEQ ID NO: 40. SEQ ID NO:48 and SEQ ID NO:56, and CDR1, CDR2 and CDR3 of the light chain variable region consist of SEQ ID NO: 64. SEQ ID NO:72 and SEQ ID NO: 80.

More preferably, the heavy chain variable region comprises the framework region FR and CDR1, CDR2 and CDR3 of the heavy chain variable region described above; more preferably, said frame region FR comprises: SEQ ID NO:88, FR1, SEQ ID NO:96, FR2, SEQ ID NO:104 and FR3 shown in SEQ ID NO: FR4 indicated at 112.

More preferably, the light chain variable region comprises the framework region FR and CDR1, CDR2 and CDR3 of the light chain variable region described above; more preferably, said frame region FR comprises: SEQ ID NO:120, FR1, SEQ ID NO:128, FR2, SEQ ID NO:136 and FR3 and SEQ ID NO: FR4 indicated at 144.

An anti-BAFFR monoclonal antibody, or antigen binding fragment thereof, comprising a heavy chain variable region and a light chain variable region; the method is characterized in that:

(a) The heavy chain variable region has the amino acid sequence of SEQ ID NO:1-8,

or with SEQ ID NO:1-8 has a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity,

or with SEQ ID NO:1-8, an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence;

(b) The light chain variable region has the amino acid sequence of SEQ ID NO:17-24, and any one of the amino acid sequences given in seq id no;

or with SEQ ID NO:17-24, a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity,

or with SEQ ID NO:17-24, having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence.

Preferably, the heavy chain variable region and the light chain variable region are selected from any one of the amino acid sequences of (1) to (8) below:

(1) SEQ ID NO:1 and SEQ ID NO:17;

(2) SEQ ID NO:2 and SEQ ID NO:18;

(3) SEQ ID NO:3 and SEQ ID NO:19;

(4) SEQ ID NO:4 and SEQ ID NO:20, a step of;

(5) SEQ ID NO:5 and SEQ ID NO:21, a step of;

(6) SEQ ID NO:6 and SEQ ID NO:22;

(7) SEQ ID NO:7 and SEQ ID NO:23;

(8) SEQ ID NO:8 and SEQ ID NO:24.

the invention discloses that an antibody or antigen binding fragment thereof of any of the above binds to a BAFFR protein.

The technical scheme of the invention is as follows:

1. an anti-BAFFR antibody or antigen binding fragment thereof, wherein the antibody or antigen binding fragment thereof comprises a binding region in whole or in part of SEQ ID NO:145, and a polypeptide sequence described in seq id no.

2. The antibody or antigen-binding fragment thereof according to scheme 1, wherein the antibody is a murine antibody, a single domain antibody, a chimeric antibody, a fully human antibody or a humanized antibody.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody is a monospecific, bispecific, multispecific antibody, antibody conjugate, or car molecule for cell therapy; preferably, the antibody conjugate is a cytotoxic moiety, radioisotope, drug or cytokine.

4. The antibody or antigen-binding fragment thereof of claim 3, wherein the bispecific antibody comprises an antibody or antigen-binding fragment thereof of any one of claims 1-3, and an antibody or antigen-binding fragment directed against other antigens and/or other epitopes.

5. The antibody or antigen-binding fragment thereof according to any one of schemes 1-2, wherein the antibody is a monoclonal antibody.

6. An anti-BAFFR monoclonal antibody or antigen binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises heavy chain complementarity determining regions CDR1, CDR2 and CDR3 and the light chain variable region comprises light chain complementarity determining regions CDR1, CDR2 and CDR3, wherein,

Preferably, CDR1, CDR2 and CDR3 of the heavy chain variable region consist of the amino acid sequences SEQ ID NO: 34. SEQ ID NO:42 and SEQ ID NO:50, and CDR1, CDR2 and CDR3 of the light chain variable region consist of SEQ ID NO: 58. SEQ ID NO:66 and SEQ ID NO: 74;

preferably, CDR1, CDR2 and CDR3 of the heavy chain variable region consist of the amino acid sequences SEQ ID NO: 35. SEQ ID NO:43 and SEQ ID NO:51, and CDR1, CDR2 and CDR3 of the light chain variable region consist of SEQ ID NO: 59. SEQ ID NO:67 and SEQ ID NO: 75;

preferably, CDR1, CDR2 and CDR3 of the heavy chain variable region consist of the amino acid sequences SEQ ID NO: 36. SEQ ID NO:44 and SEQ ID NO:52, and CDR1, CDR2 and CDR3 of the light chain variable region consist of SEQ ID NO: 60. SEQ ID NO:68 and SEQ ID NO: 76;

7. An anti-BAFFR monoclonal antibody or antigen binding fragment thereof according to claim 6 wherein the heavy and light chains comprise a heavy chain variable region comprising framework region FR and CDR1, CDR2 and CDR3 of the heavy chain variable region; the frame region FR includes:

(a) Selected from SEQ ID NOs: 81-88 of any one of the amino acid sequences,

(b) Selected from SEQ ID NOs: 89-96 of any one of the amino acid sequences,

(c) Selected from SEQ ID NOs: 97-104 of any one of the amino acid sequences,

and

(d) Selected from SEQ ID NOs: 105-112,

(e) Selected from SEQ ID NOs: 113-120,

(f) Selected from SEQ ID NOs: 121-128, or a fragment thereof,

(g) Selected from SEQ ID NOs: 129-136,

and

(h) Selected from SEQ ID NOs: 137-144 of the amino acid sequence,

8. An anti-BAFFR monoclonal antibody, or antigen binding fragment thereof, comprising a heavy chain variable region and a light chain variable region; the method is characterized in that:

9. An anti-BAFFR monoclonal antibody or antigen binding fragment thereof according to claim 8 wherein the heavy and light chain variable regions are selected from any one of the amino acid sequences of (1) - (8):

(1) SEQ ID NO:1 and SEQ ID NO:17;

(2) SEQ ID NO:2 and SEQ ID NO:18;

(3) SEQ ID NO:3 and SEQ ID NO:19;

(4) SEQ ID NO:4 and SEQ ID NO:20, a step of;

(5) SEQ ID NO:5 and SEQ ID NO:21, a step of;

(6) SEQ ID NO:6 and SEQ ID NO:22;

(7) SEQ ID NO:7 and SEQ ID NO:23;

(8) SEQ ID NO:8 and SEQ ID NO:24.

10. the antibody or antigen-binding fragment thereof according to any one of schemes 1-9, wherein the antibody or antigen-binding fragment thereof binds to a BAFFR protein.

11. The antibody or antigen-binding fragment thereof of claim 10, wherein the BAFFR protein is a human monkey BAFFR protein.

12. The antibody or antigen binding fragment thereof of claim 10 or 11, wherein the BAFFR protein forms part of a cell.

13. The antibody or antigen-binding fragment thereof of claim 12, wherein the BAFFR protein is expressed on the surface of the cell.

14. The antibody or antigen-binding fragment thereof of claim 12 or 13, wherein the cell is a lymphocyte.

15. The antibody or antigen binding fragment thereof according to any one of claims 12-14, wherein the cell is a B cell.

16. The antibody or antigen-binding fragment thereof of any one of claims 12-15, wherein the cell is a cancer cell.

17. The antibody or antigen-binding fragment thereof of claim 16, wherein the cancer cell is a lymphoma cell.

18. A gene encoding an anti-BAFFR antibody or antigen binding fragment thereof of any one of schemes 1-17.

19. The gene according to claim 18, wherein the gene is selected from any one of the following (1) to (8):

(1) Comprising the amino acid sequence as shown in SEQ ID NO:9 and a nucleotide sequence encoding a heavy chain variable region of an antibody as set forth in SEQ ID NO:25, a nucleotide sequence encoding a light chain variable region of said antibody;

(2) Comprising the amino acid sequence as shown in SEQ ID NO:10 and the nucleotide sequence encoding the heavy chain variable region of the antibody as set forth in SEQ ID NO:26, a nucleotide sequence encoding a light chain variable region of said antibody;

(3) Comprising the amino acid sequence as shown in SEQ ID NO:11 and the nucleotide sequence encoding the heavy chain variable region of the antibody as set forth in SEQ ID NO:27, a nucleotide sequence encoding a light chain variable region of said antibody;

(4) Comprising the amino acid sequence as shown in SEQ ID NO:12 and a nucleotide sequence encoding a heavy chain variable region of an antibody as set forth in SEQ ID NO:28, a nucleotide sequence encoding the antibody light chain variable region;

(5) Comprising the amino acid sequence as shown in SEQ ID NO:13 and a nucleotide sequence encoding a heavy chain variable region of an antibody as set forth in SEQ ID NO:29, a nucleotide sequence encoding a light chain variable region of said antibody;

(6) Comprising the amino acid sequence as shown in SEQ ID NO:14 and the nucleotide sequence encoding the heavy chain variable region of the antibody as set forth in SEQ ID NO:30, a nucleotide sequence encoding the antibody light chain variable region;

(7) Comprising the amino acid sequence as shown in SEQ ID NO:15 and a nucleotide sequence encoding a heavy chain variable region of an antibody as set forth in SEQ ID NO:31 encoding the light chain variable region of said antibody;

(8) Comprising the amino acid sequence as shown in SEQ ID NO:16 and the nucleotide sequence encoding the heavy chain variable region of the antibody as set forth in SEQ ID NO:32, and a nucleotide sequence encoding the light chain variable region of said antibody.

20. Recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing the genes described in scheme 19.

21. Application of the recombinant vector, the expression cassette, the transgenic cell line or the recombinant bacterium described in scheme 20 in preparation of anti-BAFFR antibody:

(a) Use of a drug that inhibits cancer cell migration;

(b) Use of a medicament for the treatment of cancer;

(c) The application of the medicine for treating autoimmune diseases.

22. A multifunctional fusion protein comprising the antibody or antigen-binding fragment thereof or antibody-conjugated drug of any one of schemes 1-17.

23. The multifunctional fusion protein according to claim 22, further comprising one or more secondary antibodies or antigen-binding portions thereof that specifically bind to other antigens.

24. The multifunctional fusion protein according to claim 23, wherein the antigen that binds to the second antibody or antigen binding portion thereof is selected from a tumor-associated antigen (TAA) or an immune checkpoint.

25. The multifunctional fusion protein of claim 24, wherein the immune checkpoint is CD3, 4-1BB, CD40, OX40, ICOS.

26. The multifunctional fusion protein according to any one of claims 23-25, further comprising a cytokine.

27. The multifunctional fusion protein according to claim 26, wherein the cytokine is selected from the group consisting of IL8, IL10, IL15, IL18, TGF, VEGF, IFN gamma, IFN alpha and GM-CSF.

28. The use of the antibody or antigen-binding fragment thereof of any one of schemes 1-17, the multifunctional fusion protein of any one of schemes 22-26 in the manufacture of a medicament for the treatment of cancer or an autoimmune disease.

29. The use of claim 28, wherein the cancer is lymphoma, leukemia, or myeloma.

30. The use according to claim 29, wherein the lymphoma is mantle cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, marginal zone lymphoma, or burkitt's lymphoma.

31. The use according to claim 29, wherein the leukemia is lymphoblastic leukemia, chronic lymphocytic leukemia or hairy cell leukemia.

32. The use of claim 29, wherein the myeloma is multiple myeloma.

33. The use according to claim 28, wherein the autoimmune disease is systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, idiopathic thrombocytopenic purpura, ulcerative colitis, sjogren's syndrome.

34. The use according to any one of claims 28-33, wherein the medicament is for use in combination with other medicaments or therapies.

35. The use according to claim 34, wherein the other therapy is selected from the group consisting of: chemotherapy, radiation therapy, targeted therapy, gene therapy, cell therapy, and stem cell therapy.

36. A method of modulating a function of a cell comprising delivering into the cell a polynucleotide sequence encoding the antibody or antigen binding fragment thereof of any one of schemes 1-17, or the multifunctional fusion protein of any one of schemes 22-27.

37. The method according to scheme 36, wherein the method is for gene therapy, CAR-T therapy or CRISPR therapy.

38. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of schemes 1-17 and an acceptable carrier, diluent or excipient.

39. A pharmaceutical composition comprising the multifunctional fusion protein of any one of claims 22-27 and an acceptable carrier, diluent or excipient.

40. A method of inhibiting cell proliferation, the method comprising:

(1) Contacting the cell with the BAFFR antibody of any one of claims 1-17 or the multifunctional fusion protein of any one of claims 22-27, thereby forming a contacted cell; and

(2) Allowing the BAFFR antibody or multifunctional fusion protein to bind to the BAFFR protein on the contacted cell, thereby inhibiting proliferation of the cell.

41. The method of claim 40, wherein the cell is a lymphocyte.

42. The method of claim 40 or 41, wherein the cell is a B cell.

43. The method of any one of claims 40-42, wherein the cell is a cancer cell.

44. The method of any one of claims 40-43, wherein the cell is a lymphoma cell.

Abbreviations and term definitions

The following abbreviations are used herein:

mAb: monoclonal antibodies

VH: antibody heavy chain variable region

VL: antibody light chain variable regions

CDR: complementarity determining regions in immunoglobulin variable regions

FR: antibody framework regions, i.e. amino acid residues other than CDR residues in the variable regions of antibodies

IgG: immunoglobulin G

ELISA: ELISA (enzyme-linked immunosorbent assay)

FACS: fluorescence activated cell sorting

And (2) PCR: polymerase chain reaction

Herein, the term "antibody" refers to a natural immunoglobulin or an immunoglobulin prepared by partial or complete synthesis. Antibodies can be isolated by reconstitution from natural sources such as plasma or serum in which the antibodies are naturally present, or culture supernatants of antibody-producing hybridoma cells, animal immune serum, or phage library screening. Alternatively, it may be partially or completely synthesized by using a technique of gene recombination or the like. Preferred antibodies include, for example, antibodies to the isotype of immunoglobulins or subclasses of these isotypes. Human immunoglobulins are known to include the 9 classes (isotypes) IgGl, igG2, igG3, igG4, igAl, igA2, igD, igE, igM. Among these isotypes, the antibodies of the invention may include IgGl, igG2, igG3, igG4.

The term "antigen-binding fragment" refers to a polypeptide fragment of an antibody that retains the ability to specifically bind to the same antigen to which a full-length antibody binds, and/or competes with a full-length antibody for specific binding to an antigen, also referred to as an "antigen-binding portion. Antigen binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen binding fragments include Fab, fab ', F (ab') 2, fd, fv, dAb, and Complementarity Determining Region (CDR) fragments, single chain antibodies (e.g., scFv), chimeric antibodies, diabodies, linear antibodies (linear antibodies), single domain antibodies, and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen binding capacity to the polypeptide.

The term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures and that are incorporated into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of a nucleic acid to which they are operably linked, referred to herein as "expression vectors".

The term "pharmaceutically acceptable carrier" includes any standard pharmaceutical carrier, such as phosphate buffered saline solutions, water and emulsions, as well as various types of wetting agents.

"percent (%) amino acid sequence identity" is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to obtain the maximum percent sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be performed in a variety of ways within the skill of the art, for example using publicly available computer software, such as the BLAST software or FASTA packages.

By "specific" is meant that one of the molecules involved in specific binding does not exhibit any significant binding to a molecule other than one or several of the binding partner molecules. In addition, the term is also used when the domain comprising the antibody variable region is specific for a particular epitope of a plurality of epitopes in an antigen. When an epitope to which a domain comprising an antibody variable region binds is contained in several different antigens, an antigen binding molecule comprising a domain comprising an antibody variable region can bind to various antigens having the epitope.

"cytotoxicity" is a simple cell killing event caused by a cell or chemical substance, independent of the cell death mechanism of apoptosis or necrosis. Sometimes it is desirable to conduct assays for cytotoxicity of specific substances, such as drug screening. Cytotoxicity assays are based on changes in cell membrane permeability, and are commonly used in several ways: MTT, XTT method; a method of LDH; other enzymatic methods: such as detecting alkaline phosphatase and acid phosphatase activity in the supernatant.

"epitope" means an antigenic determinant in an antigen, and refers to an antigenic site to which a domain of an antigen binding molecule comprising an antibody variable region disclosed in the present specification binds. Thus, epitopes can be defined according to their structure. Alternatively, the epitope may be defined in terms of antigen binding activity in an antigen binding molecule that recognizes the epitope. When the antigen is a peptide or polypeptide, the epitope may be specified by the amino acid residues that form the epitope; when the epitope is a sugar chain, the epitope can be determined by its specific sugar chain structure.

"BMK" is an abbreviation for Benchmark and refers to a positive control antibody that is capable of binding to a target protein or a natural or engineered cell expressing a target protein.

The recognition of the three-dimensional structure of an epitope by a test antigen binding molecule comprising a domain comprising an antibody variable region having BAFFR binding activity can be confirmed as follows. Cells expressing BAFFR were prepared for the above purpose. For example, when a test antigen binding molecule comprising a domain comprising an antibody variable region having BAFFR binding activity contacts a cell expressing BAFFR, it tightly binds to the cell, but on the other hand, there is a case in which the antigen binding molecule does not substantially bind to an immobilized linear peptide comprising an amino acid sequence of an extracellular domain constituting BAFFR. In these cases, "substantially not bound" means that the binding activity is 80% or less, usually 50% or less, preferably 30% or less, and particularly preferably 15% or less relative to the binding activity of cells expressing human BAFFR.

Examples of the method for measuring the binding activity of a test antigen binding molecule containing a BAFFR antigen binding domain to a cell expressing BAFFR include: antibodies: ALaboratory Manual (Ed Harlow, david Lane, cold Spring Harbor Laboratory (1988) 359-420). BR can be evaluated based on the principle of ELISA or Fluorescence Activated Cell Sorting (FACS) using BAFFR expressing cells as antigen.

The antibody variable region-containing domains in the antigen binding molecules of the invention may bind to the same epitope. Herein, the same epitope may be present in a polypeptide comprising SEQ ID N0:145 amino acid sequence. Alternatively, the antibody variable region-containing domains in the antigen binding molecules of the invention may each bind a different epitope. Herein, different epitopes may be present in the polypeptide comprising SEQ ID N0:145, and a protein having an amino acid sequence of 145.

More preferably, the present application provides an anti-BAFFR antibody or antigen binding fragment thereof, which antibody binds an epitope comprised in whole or in part in SEQ ID NO:145, and a polypeptide sequence described in seq id no.

The anti-BAFFR antibodies may be obtained as polyclonal or monoclonal antibodies using well known methods. As the anti-BAFFR antibody, a monoclonal antibody derived from a mammal can be preferably prepared. These monoclonal antibodies derived from mammals comprise antibodies produced by hybridomas, antibodies produced by screening by phage surface display technology, or produced by host cells transformed by genetic engineering techniques with expression vectors carrying antibody genes.

The monoclonal antibody is produced, for example, as follows. First, BAFFR protein was purchased from a conventional biochemical reagent store. BAFFR proteins are used as sensitizing antigens for immunization in mammals. Partial peptides of BAFFR can also be used as sensitising antigens. In this case, the partial peptide may also be obtained by chemical synthesis from the human BAFFR amino acid sequence. Furthermore, it can also be obtained by integrating a part of the BAFFR gene into an expression vector and expressing it. Furthermore, it can also be obtained by degrading BAFFR protein using a protease, but the region and size of BAFFR peptide used as a partial peptide are not particularly limited to specific embodiments. The number of amino acids constituting the peptide used as the sensitizing antigen is at least five or more, or preferably, for example, six or more, or seven or more. More specifically, a peptide consisting of 8 to 50 residues or preferably 10 to 30 residues may be used as the sensitizing antigen.

Alternatively, a fusion protein in which a desired partial polypeptide or peptide of the BAFFR protein is fused to a different polypeptide may be used as the sensitizing antigen. For preparing a fusion protein to be used as a sensitizing antigen, for example, an Fc fragment of an antibody, a peptide tag, or the like can be preferably used. Vectors for expressing the fusion proteins can be prepared by fusing genes encoding two or more desired polypeptide fragments thereof in-frame, and inserting the fused genes into expression vectors as described above.

The mammal immunized with the sensitization antigen is not limited to a specific animal. But is preferably selected in consideration of suitability with the parent cell for cell fusion. Generally, rodent animals such as mice, rats and hamsters, alpacas, rabbits and monkeys are preferably used.

To obtain the monoclonal antibodies of the invention by DNA immunization, first, DNA expressing BAFFR protein is administered to an immunized animal. The DNA encoding BAFFR can be synthesized by a known method such as PCR. The resulting BAFFR is inserted into an appropriate expression vector and administered to an immunized animal. As the expression vector, for example, a commercially available expression vector such as PCDNA3.1 can be suitably used. As a method of applying the carrier into a living body, a commonly used method can be utilized. For example, DNA immunization is performed by introducing gold particles to which expression vectors have been adsorbed into cells of an immunized animal by a gene gun.

An increase in BAFFR binding antibody titers was demonstrated in serum after immunization of the mammal as described above. Immune cells are then collected from the mammal and then subjected to cell fusion. As preferred immune cells, in particular spleen cells can be used.

As the cells fused with the above immune cells, myeloma cells of mammals can be used. Myeloma cells are preferably provided with appropriate selection markers for screening. Selectable markers refer to the property of being able (or unable) to survive under specific culture conditions. Well known selectable markers are: lack of hypoxanthine-guanine-phosphoribosyl transferase (hereinafter abbreviated as HGPRT), or lack of thymidine kinase (hereinafter abbreviated as TK), etc. Cells with the lack of HGPRT or TK have hypoxanthine-aminopterin-thymidine sensitivity (hereinafter omitted as HAT sensitivity). However, if fused with normal cells, DNA synthesis can be continued by the salvage pathway of normal cells, and thus can be proliferated in HAT selection culture.

Cells lacking HGPRT or lacking TK may be selected from a medium containing 6-thioguanine, 8-azaguanine (hereinafter abbreviated as 8 AG), or 5' -bromodeoxyuridine, respectively. Normal cells that ingest these pyrimidine analogs into DNA will die. At the same time, cells lacking these enzymes that do not ingest these pyrimidine analogs can survive in selective culture. Furthermore, a selectable marker called G418 resistance confers resistance to 2-deoxystreptamine antibiotics (gentamicin analogues) via a neomycin resistance gene. Various myeloma cells suitable for cell fusion are well known.

For example, myeloma cells including the following cells can be preferably used: p3 (P3x63Ag8.653) (J.Immunol. (1979) 123 (4), 1548-1550);

NS-1(C.Eur.J.Immunol.(1976)6(7)，511-519)；

SP2/0(Nature(1978)276(5685),269-270)；

r210 (Nature (1979) 277 (5692), 131-133), and the like.

Cell fusion of the immune cells with myeloma cells is carried out essentially according to known Methods, such as the method of Kohler and Milstein et al (Methods enzymol. (1981) 73,3-46), and the like.

More specifically, the above cell fusion can be carried out in a conventional nutrient medium, for example, in the presence of a cell fusion promoter. Fusion promoters include, for example, polyethylene glycol (PEG) and sendai virus (HVJ). Adjuvants such as dimethyl sulfoxide and the like may be added as needed to further improve the fusion efficiency.

The ratio of immune cells to myeloma cells can be arbitrarily set. For example, it is preferable to use 1 to 10 times as many immune cells as myeloma cells. As the culture medium used for the cell fusion, for example, RPMI1640 culture medium suitable for proliferation of the myeloma cell line, MEM culture medium, and a conventional culture medium used for cell culture of this kind can be used. And can be suitably added with serum replacement fluid such as Fetal Calf Serum (FCS).

For cell fusion, the predetermined amounts of the immune cells and myeloma cells may be mixed in the culture medium. Then, a PEG solution preheated to about 37℃was added at a concentration of usually 30 to 60% (w/v). The desired fused cells (hybridomas) can be formed by slowly mixing the mixed solution. Next, the above exemplified appropriate culture medium is gradually added to the cells, and it is repeatedly centrifuged to remove the supernatant. Cell fusion agents and the like which are detrimental to hybridoma growth can be removed.

Screening and single cloning of the desired antibodies can be performed by well known screening methods based on antigen-antibody reactions. For example, a monoclonal antibody that binds to BAFFR may bind to BAFFR expressed on the cell surface. Such monoclonal antibodies may be screened, for example, by Fluorescence Activated Cell Sorting (FACS). FACS refers to a system that allows measurement of antibodies bound to the surface of cells by measuring fluorescence emitted from each cell by analyzing cells contacted with fluorescent antibodies with a laser.

In order to screen hybridomas producing the monoclonal antibodies of the present invention by FACS, cells expressing BAFFR are first prepared. Preferred cells for screening are mammalian cells that are forced to express BAFFR. As a control, the binding activity of antibodies to BAFFR on the cell surface can be selectively detected using untransformed mammalian cells as host cells. That is, by selecting a hybridoma that produces an antibody that does not bind to the host cell and binds to a cell that forcibly expresses BAFFR, a hybridoma that produces a BAFFR monoclonal antibody can be obtained.

Alternatively, the binding activity of the antibody to immobilized BAFFR expressing cells can be evaluated based on the principles of ELISA. For example, cells expressing BAFFR are immobilized within wells of an ELISA plate. Antibodies bound to the immobilized cells are detected by contacting the culture supernatant of the hybridoma with the immobilized cells in the well. In the case where the monoclonal antibody is mouse-derived, the antibody bound to the cell can be detected by an anti-mouse immunoglobulin antibody. The hybridomas which are selected by the above screening and have antigen binding ability and produce the desired antibody can be cloned by limiting dilution method or the like.

The hybridoma is cultured according to a conventional method, and a desired monoclonal antibody can be obtained from the culture supernatant. Alternatively, the hybridoma can be administered to a mammal having adaptability thereto and proliferated, whereby a monoclonal antibody can be obtained from ascites thereof. The former method is suitable for obtaining high purity antibodies.

An antibody encoded by an antibody gene cloned from an antibody-producing cell such as a hybridoma can also be suitably used. By inserting the cloned antibody gene into an appropriate vector and introducing it into a host, the antibody encoded by the gene is expressed. Methods for isolation of antibody genes, insertion of genes into vectors, and transformation of host cells, methods for the preparation of recombinant antibodies are also well known.

For example, a cDNA encoding the variable region (V region) of an anti-BAFFR antibody can be prepared from hybridoma cells expressing the anti-BAFFR antibody. For this purpose, total RNA is usually first extracted from the hybridomas. As a method for extracting mRNA from cells, guanidine ultracentrifugation and AGPC can be used.

The extracted mRNA can be purified using an mRNA purification kit (GE Healthcare Bioscience) or the like. Alternatively, kits for directly extracting total mRNA from cells, such as QuickPrep mRNA purification kit (GE Healthcare Bioscience), are also commercially available. Such a kit can be used to obtain mRNA from a hybridoma. cDNA encoding the antibody V region can be synthesized from the prepared mRNA using reverse transcriptase. cDNA can be synthesized using AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Biochemical Co., ltd.) or the like. In addition, for the synthesis and amplification of cDNA, SMARTRACE cDNA amplification kit and PCR-based 5' -RACE method can be suitably used. In the synthesis of such cDNA, appropriate restriction enzyme sites to be described later may be introduced into both ends of the cDNA.

The cDNA fragment as a target was purified from the resulting PCR product, and then ligated with vector DNA. Thus, a recombinant vector was prepared and introduced into E.coli or the like. After selection of colonies, the desired recombinant vector can be prepared from E.coli forming the colonies. Further, whether or not the recombinant vector has a cDNA nucleotide sequence as a target can be confirmed by a known method, for example, a dideoxynucleotide chain termination method.

In order to isolate the gene encoding the variable region, it is convenient to use a 5' -RACE method using a primer for amplifying the variable region gene. First, cDNA was synthesized using RNA extracted from hybridoma cells as a template to obtain a 5' -RACE cDNA library. A commercially available kit such as SMARTRACE cDNA amplification kit can be suitably used for the synthesis of the 5' -RACE cDNA library.

The obtained 5' -RACE cDNA library was used as a template to amplify the antibody gene by PCR. Primers for mouse antibody gene amplification can be designed based on known antibody gene sequences. The nucleotide sequences of these primers vary depending on the subclass of immunoglobulin. Therefore, it is preferable to determine the subclass in advance using a commercially available kit such as the Iso Strip mouse monoclonal antibody isotype kit (Roche Diagnostics).

Specifically, primers capable of amplifying genes encoding Y1, Y2a, Y2b, Y3 as heavy chains, and k chain and & chain as light chains can be used to isolate genes encoding mouse IgG. In order to amplify the variable region gene of IgG, primers that anneal to the constant region near the variable region can be generally used as 3' -side primers. On the other hand, primers attached to the 5'RACE cDNA library construction kit were used as 5' side primers.

With the PCR product thus amplified, an immunoglobulin composed of a combination of heavy and light chains can be reconstituted. The desired antibodies can be screened using the BAFFR binding activity of the reconstituted immunoglobulin as an indicator. For example, when the purpose is to isolate an antibody against BAFFR, it is further preferred that the binding of the antibody to BAFFR is specific. Antibodies that bind to BAFFR can be screened, for example, by:

(1) Contacting an antibody comprising a V region encoded by cDNA isolated from the hybridoma with cells of BAFFR;

(2) Detecting binding of cells expressing BAFFR to the antibody;

(3) Antibodies that bind to cells expressing BAFFR are selected.

Methods for detecting binding of antibodies to cells expressing BAFFR are well known. Specifically, binding of the antibody to cells expressing BAFFR can be detected by the techniques such as FACS described above. For evaluating the binding activity of the antibody, a fixed sample of BAFFR expressing cells may be suitably used.

The screening method for an antibody having binding activity as an index also includes a panning method using a phage vector. In the case where the antibody genes are isolated from a library of heavy and light chain subclasses of a population of cells expressing polyclonal antibodies, a screening method using phage vectors is advantageous. Genes encoding the variable regions of the heavy and light chains may be joined by appropriate linker sequences to form a single chain Fv (scFv). Phage with scFv presented on the surface can be obtained by inserting the gene encoding scFv into phage vector. The phage is contacted with the antigen of interest. By collecting phage that bind to antigen, DNA encoding scFv with target binding activity can be isolated. The process is repeated as necessary to concentrate scFv having the desired binding activity.

After isolating the cDNA encoding the V region of the anti-BAFFR antibody of interest, the cDNA is digested by restriction enzymes recognizing restriction enzyme sites inserted into both ends of the cDNA. Preferred restriction enzymes recognize and cleave nucleotide sequences that occur less frequently in the nucleotide sequences of antibody genes. And preferably a restriction enzyme site imparting a cohesive end is introduced into the vector to insert a single copy of the digested fragment in the correct orientation. The cDNA encoding the V region of the anti-BAFFR antibody was digested as described above and inserted into a suitable expression vector to construct an antibody expression vector. In this case, if a gene encoding the antibody constant region (C region) and a gene encoding the above V region are fused in-frame, a chimeric antibody is obtained.

Herein, "chimeric antibody" means that the origin of the constant region is different from the origin of the variable region. Therefore, in addition to the mouse-human or other heterologous chimeric antibodies, human-human homologous chimeric antibodies are also included in the chimeric antibodies of the present invention. By inserting the above-described V region gene into an expression vector already having a constant region, a chimeric antibody expression vector can be constructed. Specifically, for example, the recognition sequence of a restriction enzyme that excises the above-described V region gene may be appropriately placed on the 5' side of an expression vector carrying DNA encoding a desired antibody constant region (C region). Chimeric antibody expression vectors were constructed by in-frame fusion of two genes digested with the same combination of restriction enzymes.

To prepare anti-BAFFR monoclonal antibodies, the antibody genes are inserted into an expression vector such that the genes are expressed under the control of an expression control region. Expression control regions for expressing antibodies include, for example, enhancers and promoters. In addition, an appropriate signal sequence may be added to the amino terminus to allow secretion of the expressed antibody outside the cell. Other suitable signal sequences are also attached. The expressed polypeptide is cleaved at the carboxy-terminal portion of the above sequence and the resulting polypeptide can be secreted extracellularly as a mature polypeptide. Next, by transforming an appropriate host cell with the expression vector, a recombinant cell expressing DNA encoding an anti-BAFFR antibody can be obtained.

For expression of antibody genes, the DNA encoding the heavy (H) and light (L) chains of the antibody are inserted into different expression vectors, respectively. Antibody molecules having H and L chains can be expressed by co-transfecting the same host cell with a vector into which the H and L chains have been inserted. Alternatively, a host cell can be transformed with a single expression vector into which DNA encoding H chain and L chain is inserted (see International publication WO 94/11523).

The respective combinations of host cells/expression vectors for the production of antibodies by introducing the isolated antibody genes into an appropriate host are well known. These expression systems can be used for isolation of domains comprising the antibody variable regions of the invention.

In addition, antibody gene expression systems using prokaryotic cells are also known. For example, when bacterial cells are used, bacterial cells such as E.coli (E.coli) and Bacillus subtilis can be suitably used. An expression vector comprising the target antibody gene is introduced into these cells by transformation. The desired antibody can be obtained from a culture of the transformed cells by culturing the transformed cells in vitro.

In addition to the host cells described above, transgenic animals can also be used to produce recombinant antibodies. That is, the antibody can be obtained from an animal into which a gene encoding a desired antibody has been introduced. For example, the antibody gene may be inserted in-frame into the interior of a gene encoding a protein that is inherently produced in milk to construct a fusion gene. As the protein secreted into the milk, for example, goat B casein or the like can be used. The DNA fragment containing the fusion gene into which the antibody gene is inserted is injected into the embryo of the goat, and then the embryo is introduced into the female goat. Transgenic goats are produced from goats that receive embryos, and the desired antibodies are obtained from the milk produced by the transgenic goats (or their offspring) as fusion proteins with milk proteins.

When the antigen binding molecules described herein are administered to humans, domains derived from genetically recombinant antibodies that have been artificially modified to reduce heterologous antigenicity against humans and the like can be suitably used as the domains of antigen binding molecules comprising antibody variable regions. Such genetically recombinant antibodies include, for example, humanized antibodies. These modified antibodies are suitably prepared by known methods.

Humanized antibodies, specifically, humanized antibodies obtained by grafting CDRs of a non-human animal antibody, for example, a mouse antibody, onto a human antibody, and the like are known. Conventional genetic recombination methods for obtaining humanized antibodies are also known. Specifically, as a method for grafting CDRs of a mouse antibody to human FR, for example, overlap sequence extension PCR (overlap extension PCR) is known. In overlap sequence extension PCR, a nucleotide sequence encoding the CDR of a mouse antibody to be grafted is added to a primer for synthesizing the FR of a human antibody. Primers were prepared for each of the 4 FRs. It is generally considered that when a mouse CDR is grafted into a human FR, it is advantageous to select a human FR having high identity with the mouse FR for maintaining the function of the CDR. That is, it is generally preferable to use human FRs comprising an amino acid sequence having high amino acid sequence identity to the FRs adjacent to the mouse CDR to be grafted.

In addition, techniques for preparing human antibodies by panning using human antibody libraries are also known. For example, the V region of a human antibody is expressed as a single chain antibody (scFv) on the phage surface by phage display methods. Phages expressing scFv that bind to antigen can be selected. The DNA sequence encoding the V region of the human antibody that binds to the antigen can be determined by analyzing the genes of the selected phage. The DNA sequence of scFv that binds to antigen was determined. Expression vectors can be made by fusing the V region sequence in frame with the sequence of the desired human antibody C region and inserting into an appropriate expression vector. The expression vector is introduced into cells suitable for expression, such as those described above. Human antibodies can be produced by expressing a gene encoding the human antibody in a cell.

The anti-BAFFR antibody provided by the invention can be specifically combined with BAFFR to block a BAFFR-BAFF signal path, clear hyperproliferative B lymphocytes and inhibit the growth of the B cells; effectively solves the drug resistance problem of the existing drugs for treating B cell lymphoma.

Drawings

The invention will be described in further detail with reference to the accompanying drawings and embodiments:

fig. 1: ELISA method for detecting the binding capacity of the anti-BAFFR murine antibody and the human BAFFR protein.

Fig. 2A: FACS detects the binding capacity of anti-BAFFR murine antibody to human BAFFR-CHO-K1 cells.

Fig. 2B: FACS detects the binding ability of the anti-BAFFR chimeric antibody to human BAFFR-CHO-K1 cells.

Fig. 3: anti-BAFFR chimeric antibodies block the ability of BAFF-BAFFR to bind.

Detailed Description

The present invention will be further described with reference to the accompanying drawings and specific examples, to which the invention is not limited. It is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention. Variations and advantages that will occur to those skilled in the art are included within the following claims and any equivalents thereof without departing from the spirit and scope of the inventive concept. In the description and claims of the present invention, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. The procedures, conditions, reagents, experimental methods, etc. for carrying out the present invention are common knowledge and common knowledge to those skilled in the art, except where specifically mentioned below, and the present invention is not particularly limited.

Example 1: immunization of animals

Immunization of mice: taking 3-5 female Balb/c mice with 6 weeks of age, and taking recombinant human BAFFR-Fc protein as immunogen for intraperitoneal or subcutaneous immunization. Negative serum is adopted 3 days in advance, the first immunization is carried out, 50 mug of recombinant human BAFFR-Fc protein fully emulsified by Freund's complete adjuvant is injected by intraperitoneal immunization, 25ug of recombinant human BAFFR-Fc protein fully emulsified by Freund's incomplete adjuvant is injected by intraperitoneal injection on 14 th and 35 th days, and the second immunization and the third immunization are carried out. After 6 days, the tail was collected and serum was diluted in titer and assayed for serum titer by ELISA. ELISA plates were coated overnight with recombinant human BAFFR-his protein, the coating solution was discarded, washed with PBST, added with serum dilution, washed with PBST, added with horseradish peroxidase-labeled goat anti-mouse secondary antibody, and washed with PBST. TMB color development was added and the optical density was measured at 450 nm. When the titer results meet the requirements, the spleen and lymph nodes of the mice can be harvested for cell fusion and phage library construction.

Alpaca immunization: the recombinant human BAFFR-Fc protein is taken as an immunogen, the antigen and the adjuvant are emulsified in a ratio of 1:1 to form a uniform mixture, the total antigen amount of each immunization is kept between 0.5 and 1mg, and the volume is less than 1-2 mL. The mixed antigen is injected in a small amount at multiple points at the left side and the right side near the alpaca neck lymph node, and the mixture is immunized once every 2 weeks for 7 times. Blood was collected from alpaca neck veins 5-7 days after 6 th and 7 th immunizations. The supernatant serum was removed by centrifugation. Titer of serum was diluted, serum titers were detected by ELISA, lymphocytes were isolated with cell separation fluid when the titer results met the requirements, cell numbers were counted using a hemocytometer, and lymphocytes isolated from the cell numbers were lysed using RNAiso Plus, ready for phage library construction.

Example 2: cell fusion

Mice were sacrificed to remove spleen and lymph nodes, and cells were pressure-ground through the mesh to obtain B lymphocytes and lymph node cells. B lymphocytes/and lymph node cells were mixed with myeloma cells SP2/0 at 2:1, and the suspension was centrifuged at 1000 rpm for 8 minutes to obtain a pellet, which was washed twice with electrofusion solution. The pellet was then taken and the electrofusion solution was brought to 9mL, and 9mL of cell suspension was injected into each electrode cuvette. It was fused to SP2/0 cells by conventional electrotransformation. The fusion was placed in complete DMEM medium containing HAT at 8% CO ₂ Culturing at 37deg.C.

Feeder cells used in the fusion selection culture were taken from macrophages in the abdominal cavity of the non-immunized animal to assist in new hybrid B lymphocyte hybridoma growth.

Example 3: construction of phage libraries

First, total lymphocyte RNA was extracted with RNAiso Plus reagent. Then, cDNA was reverse transcribed, a specific antibody fragment was amplified from the reverse transcribed cDNA, PCR amplification was performed using Taq DNAPolymerase Hot Start enzyme, the obtained PCR amplification product was subjected to 1% agarose gel electrophoresis, and a 0.7 kb-sized band cut gel was recovered by using a DNA purification recovery kit according to instructions. And amplifying specific antibody fragments again from the DNA fragments after the previous PCR amplification and recovery, and cutting and recovering. And (3) cutting the antibody gene sequence obtained by the amplification in the previous step and the phage vector by using SfiI enzyme, and cloning the antibody gene to phage plasmid in a connecting way.

Then the plasmid was electrotransformed into SS320 competent cells, immediately after electric shock 1mL of 2YT medium was added to the cuvette for resuscitation, the electric shock product was aspirated and the cuvette was washed with 2YT medium to obtain a resuscitated product, which was incubated at 37℃and 220rpm until OD600 reached 0.5. After addition of helper phage, incubation was continued for 30 min at 37℃and kanamycin and 0.2mM IPTG were added and incubated overnight at 30 ℃. Cells cultured overnight were centrifuged, and the supernatant was transferred to a new centrifuge tube, 1/4 volume of pre-chilled 5XPEG8000/NaCl was added and incubated on ice for 30 minutes. After centrifugation to remove the supernatant, 1mL of PBS buffer was added to dissolve the pellet. After adding 250. Mu.L of 5XPEG8000/NaCl again and incubating on ice for 10 minutes, the supernatant was removed by centrifugation and the pellet was dissolved in 1mL PBS to give phage library.

Example 4: screening for hybridoma-positive clones

Clones that bind to human BAFFR protein were screened for secreted antibodies using ELISA. The binding activity of the secreted antibody to huBAFFR-CHO-K1 was then examined by FACS and finally the species specificity of the hybridoma antibody for binding to BAFFR was examined.

(1) ELISA detection of binding of hybridoma anti-BAFFR antibodies to human BAFFR

Adding hybridoma cell culture supernatant into ELISA plate, 50 μl/well, taking SP2/0 cell supernatant as negative control, immune polyclonal antiserum as positive control, and water-bathing at 37deg.C for 2 hr; PBST wash 3 times; HRP-labeled goat anti-mouse IgG and IgM antibodies were added at working concentrations, 50 μl/well, in a 37 ℃ water bath for 1.5h; after washing, TMB was developed for 10min, showing the OD450 reading measured by the microplate reader after termination. The measured wells OD450 readings were more than twice as positive as the negative control.

(2) FACS detection of binding Activity of hybridoma anti-BAFFR antibodies to human BAFFR-CHO-K1 cells

The lentiviral vector carrying the human BAFFR gene is used for transduction into CHO-K1 cells to obtain cells which over-express the human BAFFR protein and are named as human BAFFR-CHO-K1 cells. Taking human BAFFR-CHO-K1 cells in logarithmic growth phase in a 1.5ml EP tube, washing with PBS, centrifuging twice, diluting positive clone antibody screened by ELISA detection to 40 mug/ml with PBS, diluting with 3 times 8 gradients, 100 mug of each tube, taking blank control with PBS, and incubating at 4 ℃ for 1 hour; washing with PBS, centrifuging twice, adding PBS diluted fluorescent antibody APC anti-human IgG FcAntibody, incubating for 45min at 4 ℃ with 100 μl each tube, washing with PBS, centrifuging twice; finally, 200. Mu.l PBS was used to re-suspend the cells for flow-through detection analysis.

(3) FACS detection of species specificity of hybridoma anti-BAFFR antibodies binding to BAFFR

The lentiviral vector carrying the BAFFR genes of mice and cynomolgus monkeys is used for transduction into CHO-K1 cells to obtain cells which over-express the BAFFR proteins of the mice or the cynomolgus monkeys and are named as the BAFFR-CHO-K1 cells of the mice/monkeys. The binding activity of the hybridoma antibodies to the murine, cynomolgus BAFFR protein was examined.

Example 5: phage library screening for anti-BAFFR positive antibodies

Phage 500. Mu.L was added to 1mL of 3% BSA and incubated for 2h at room temperature with rotation. Meanwhile, 2-3mL of 3% BSA was added to the human BAFFR-his coated immune tube, and the mixture was incubated at room temperature for 2 hours. The blocked immune tubes were washed 3 times with PBS containing 0.01% tween. The blocked phage library was added to the blocked immune tube, PBS was added until 2-3mL, and the incubation was performed at room temperature for 1h. The immunization after incubation of antigen and phage was washed 20 times with PBS containing 0.01% tween. 1mL of 100mM trimethyl imide was added to the immune tube, incubated at room temperature for 10 minutes, and the trimethyl imide was neutralized by adding 1M Tris-HCl, and the final 1.5mL of eluted phage was transferred to a new centrifuge tube.

Amplifying the eluted phage, repeating the screening process for 2 times, and gradually halving the antibody amount of the coated immune tube to obtain the eluted phage after 3 times of screening. After the phage obtained in the previous step is diluted by 106 times, 100 mu L of phage is added into SS320 bacterial liquid with OD600 of 0.5, the phage is cultured for 30min at 37 ℃ and then coated on a 2XYT culture plate containing tetracycline and ampicillin, and the phage is cultured overnight at 37 ℃ for the next day to obtain monoclonal colonies.

96 monoclonal colonies were selected and placed on 96-well cell culture plates containing 2XYT medium of tetracycline and ampicillin, cultured at 37℃for 3-4 hours, and after that, kanamycin and 20:1 helper phage were added to the culture wells, and cultured overnight at 30 ℃. The cell fluid after overnight culture was centrifuged to obtain a supernatant. The phage supernatant obtained in the previous step was added to a 96-well ELISA plate after overnight coating with antigen and blocking with 3% BSA, and incubated at room temperature for 1h. After 3 washes with PBS containing 0.05% Tween, the absorbance of each well was read at wavelength 450 after development with human BAFFR-his as primary antibody, and the corresponding secondary antibody anti-VHH-HRP or anti-mouse Fab-HRP, TMB. And (5) selecting the SS320 colony with the highest absorbance reading to send to sequencing to obtain the gene sequence of the antibody.

Example 6: hybridoma cell anti-BAFFR monoclonal antibody production

After obtaining a stable hybridoma cell line, monoclonal antibodies are obtained mainly by an in vitro culture method. Expanding the cell strain in T75 culture flask, culturing until cell coverage is 80-90%, discarding cell supernatant, adding 30ml hybrid-SFM, 37deg.C, 5% CO ₂ Culturing. After 2-3 days of culture, 30mL of hybrid-SFM was added, and fresh living cells were added if the cell viability was less than 30%. Culturing for 6-7 days, collecting culture supernatant after low-speed centrifugation until cell survival rate is lower than 20%, and storing at 4deg.C for use, and performing subsequent functional test to obtain candidate antibody.

Example 7: acquisition of hybridoma candidate antibody variable region sequences

The DNA sequence of the variable region of the mouse antibody expressed by the candidate hybridoma was determined by a method based on degenerate primer PCR. Hybridoma cell lines were individually grown in expansion, cells were collected by centrifugation at 1000rpm, and total RNA was extracted with Trizol. After synthesizing the first-strand cDNA by using the same as a template, the corresponding variable region DNA sequence is amplified by PCR using the first-strand cDNA as a subsequent template, and the PCR primers are based on Ig-primer sets. And (3) recovering and purifying PCR products, and sequencing amplified products to obtain the sequences of the heavy chain variable region and the light chain variable region of the candidate hybridoma.

Example 8: expression and purification of candidate sequence IgG chimeric antibodies

After antibody gene sequencing is carried out on candidate sequences obtained by screening hybridomas and phage libraries, gene synthesis is carried out on antibody fragments obtained by sequencing, the antibody fragments are constructed into a human IgG frame, then molecular cloning technology is utilized, the antibody fragments are inserted into vectors, mammalian cell expression plasmids are constructed, a liposome transfection mode is utilized, the antibody fragments are introduced into a host cell strain CHO cell, fermentation supernatant is obtained by utilizing a cell fed-batch, and fermentation supernatant is taken to carry out purification of a series of steps such as protein A affinity chromatography, ion exchange chromatography and the like, so that the purified IgG chimeric monoclonal antibody is finally obtained. And finally, determining the purity of the antibody by adopting an SDS-PAGE method and determining the concentration of the antibody by adopting an ultraviolet micro-spectrophotometry method.

Example 9: FACS detection of anti-BAFFR antibodies blocking BAFF binding to BAFFR

Antibodies against BAFFR were mixed with biotin-labeled human BAFF (300 ng/mL) protein and incubated for 1h at 4 ℃. Cells were added to the plate, centrifuged to remove supernatant, 100. Mu.L of a mixture of antibody and human BAFF was added to the cells, incubated at 4℃for 1h, and FACS buffer was used to wash the cells 3 times. 5. Mu.g/ml NA-PE antibody was added and incubated for 30 minutes at 4 ℃. After washing the cells 3 times with FACS buffer, the antibody was verified to be able to block the binding of human BAFF to BAFFR on the surface of human BAFFR-CHO-K1 cells by flow cytometry detection.

Example 10: anti-BAFFR antibodies and BAFFR affinity validation

(1) Curing and capturing:

the AHC sensor was equilibrated with 0.02% pbst (0.02% tween 20, ph7.4,1 x pbs) as buffer for 60s, BAFFR antibody in the cured sample plate for 300s, secondary equilibration buffer for 180s. The 100nm human BAFFR-hi protein binds to the BAFFR antibody for 300s and then dissociates for 600s. After dissociation, 10mM glycine (pH 2.0) was used as a regeneration buffer for 30s.

(2) Regeneration:

the sensor was regenerated with 10mM glycine (pH 2.0).

(3) Data analysis:

the result map of reference channel H1 is subtracted from the test result map. Experimental data were in accordance with the 1:1 binding model. Antibody affinity was calculated.

TABLE 1 anti-BAFFR chimeric antibodies and BAFFR affinity assay results

The affinity of the sequences 1-8 is about 10Nm, and the overall affinity is better.

Example 11: validation of anti-BAFFR antibody binding to B cells in PBMCs

(1) Antibody dilution

The Benchmark, anti-BAFFR antibodies and isotype control (purchased from roche) were taken and diluted to 10 μg/ml with PBS.

(2) Cell grouping

PBMC cells were taken, centrifuged (400G, 5 min) and the supernatant discarded, washed by adding PBS, and resuspended in PBS after centrifugation again. Blank (PBMC cells), pbmc+secondary antibodies (APC anti-human IgG Fc, purchased from Biolegend), pbmc+cd20, pbmc+cd20+secondary antibodies, pbmc+cd20+antibodies+secondary antibodies. Cell density was 1E6 cells/group, 100. Mu.l/group

(3) CD 20-labeled PBMC

Anti-human CD20 (purchased from BD) was added to the PBMC+CD20 group, PBMC+CD20+secondary antibody group, PBMC+CD20+antibody+secondary antibody group at 20 μl/well and incubated for 30min at room temperature in the dark.

(4) Antibody incubation

PBMC+CD20+ antibody+secondary antibody 120. Mu.l of antibody diluted to 10. Mu.g/ml (final concentration 5. Mu.g/ml) were added, respectively, and incubated for 1h at room temperature in the absence of light. Blank (PBMC), pbmc+secondary, pbmc+cd20, pbmc+cd20+secondary were incubated with equal volumes of PBS.

(5) Second antibody incubation

After 2 washes with PBS, 100. Mu.l PBS was added for resuspension, 5. Mu.l PBS was added to the blank (PBMC), PBMC+CD20 groups, 5. Mu.l secondary antibody was added to the remaining groups, and incubation was performed at room temperature for 30min in the absence of light.

(6) On-machine detection

After washing 2 times with PBS, 100. Mu.l PBS was added for resuspension, and the cells of each sample group were subjected to flow detection in sequence.

(7) Data analysis

The results of the anti-BAFFR antibody binding assays to B cells in PBMCs are shown in table 2. The results show that the sequences 1-8 have better binding activity with B cells.

TABLE 2 detection of B cell binding of anti-BAFFR antibodies to PBMC

Antibodies to	Mean-APC
		Benchmark	12642
Isotype control	783
		Sequence-1	13145
Sequence-2	12462
		Sequence-3	9244
Sequence-4	9616
		Sequence-5	12126
Sequence-6	10050
		Sequence-7	12882
Sequence-8	14436

The protection of the present invention is not limited to the above embodiments. Variations and advantages that would occur to one skilled in the art are included in the invention without departing from the spirit and scope of the inventive concept, and the scope of the invention is defined by the appended claims.

Sequence listing

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Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

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Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Glu Ile Ser Gly Tyr

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Leu Asn Trp Leu Gln Gln Lys Pro Asp Gly Thr Ile Lys Arg Leu Ile

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Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Asp Ile Gly Ser Ser

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Leu Asn Trp Leu Gln Gln Glu Pro Asp Gly Thr Ile Arg Arg Leu Ile

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Ser Arg Ser Gly Ser Glu Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser

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Glu Asp Phe Val Asp Tyr Tyr Cys Leu Gln Tyr Ala Asn Ser Pro Tyr

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Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly

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Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

Asn Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

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Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

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Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

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Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln

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Tyr Tyr Ile Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

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Lys

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Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

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Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Asp Ile Gly Ser Asn

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Leu Asn Trp Leu Gln Gln Glu Pro Asp Gly Thr Ile Lys Arg Leu Ile

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Tyr Ala Thr Ser Arg Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly

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Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser

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Glu Asp Phe Val Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Ser Pro Tyr

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Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

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<212> PRT

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Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

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Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Asp Ile Gly Asn Asn

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Leu Asn Trp Leu Gln Gln Glu Pro Asp Gly Thr Ile Lys Arg Leu Ile

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Phe Ala Thr Ser Arg Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser

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Glu Asp Phe Val Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Ser Pro Tyr

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Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

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<212> PRT

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Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Thr Val Ser Val Gly

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Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser

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Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

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Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

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Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

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Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln

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Tyr Tyr Ser Tyr Pro Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu

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Leu Lys

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Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Ile Gly

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Glu Lys Ile Thr Met Ser Cys Lys Ser Asn Gln Ser Leu Leu Tyr Ser

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Thr Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

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Ser Pro Lys Leu Leu Ile Tyr Trp Ala Phe Thr Arg Gly Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Tyr Tyr Ile Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

100 105 110

Lys

<210> 24

<211> 113

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 24

Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Tyr Tyr Thr Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

100 105 110

Lys

<210> 25

<211> 321

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 25

gacatccaga tgacccagtc tccatcctcc ttatctgcct ctctgggaga aagagtcagt 60

ctcacttgtc gggcaagtca ggaaattagt ggttacttaa actggcttca gcagaaacca 120

gatggaacta ttaaacgcct gatctacgcc gcatccactt tagattctgg tgtcccaaaa 180

aggttcagtg gcagtaggtc tgggtcagat tattctctca ccatcagcag ccttcagtct 240

gaagattttg cagactatta ctgtctacaa tatgctagtt atcctcggac gttcggtgga 300

ggcaccaagc tggaaatcaa a 321

<210> 26

<211> 321

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 26

gacatccaga tgacccagtc tccatcctcc ttatctgcct ctctgggaga aagagtcagt 60

ctcacttgtc gggcaagtca ggacattggt agtagcttaa actggcttca gcaggaacca 120

gatggaacta ttagacgtct gatctacgcc acatcccgtt tagattctgg tgtccccaag 180

aggttcagtg gcagtaggtc tggatcagaa tattctctca ccatcagcag ccttgagtct 240

gaagattttg tagactatta ctgtctacag tatgctaatt ctccgtacac gttcggaggg 300

gggaccaagc tggaaataga a 321

<210> 27

<211> 339

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 27

gacattgtga tgtcacagtc tccatcctcc ctagctgtgt cagttggaga gaaggttact 60

atgagctgca agtccagtca gagcctttta tacagtaaca atcaaaaaaa ctacttggcc 120

tggtaccagc agaaaccagg gcagtctcct aaactgctga tttactgggc atccactagg 180

gaatctgggg tccctgatcg cttcacaggc agtggatctg ggacagattt cactctcacc 240

atcagcagtg tgaaggctga agacctggca gtttattact gtcagcaata ttatatctat 300

ccgtacacgt tcggaggggg gaccaagctg gaaataaaa 339

<210> 28

<211> 321

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 28

gacatccaga tgacccagtc tccatcctcc ttatctgcct ctctgggaga aagagtcagt 60

ctcacttgtc gggcaagtca ggacattggt agtaacttaa actggcttca gcaggaacca 120

gatggaacta ttaaacgcct gatctacgcc acatccaggt tagattctgg tgtccccaaa 180

aggttcagtg gcagtaggtc tgggtcagat tattctctca ccatcagcag ccttgagtct 240

gaagattttg tagactatta ctgtctacaa tatgctagtt ctccgtacac gttcggaggg 300

gggaccaagc tggaaataaa a 321

<210> 29

<211> 321

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 29

gacatccaga tgacccagtc tccatcctcc ttatctgcct ctctgggaga aagagtcagt 60

ctcacttgtc gggcaagtca ggacattggt aataacttaa actggcttca gcaggaacca 120

gatggaacta ttaaacgcct gatcttcgcc acatcccgtt tagattctgg tgtccccaaa 180

aggttcagtg gcagtaggtc tgggtcagat tattctctca ccatcagcag ccttgagtct 240

gaagattttg tagactatta ctgtctacaa tatgctagtt ctccgtacac gttcggaggg 300

gggaccaagc tggaaataaa a 321

<210> 30

<211> 342

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 30

gacattgtga tgtcacagtc tccatcctcc ctaactgtgt cagttggaga gaaggttact 60

atgagctgca agtccagtca gagcctttta tatagtagca atcaaaagaa ctacttggcc 120

tggtaccagc agaaaccagg gcagtctcct aaactgctga tttactgggc atccactagg 180

gaatctgggg tccctgatcg cttcacaggc agtggatctg ggacagattt cactctcacc 240

atcagcagtg tgaaggctga agacctggca gtttattact gtcagcaata ttatagctat 300

cccccgctca cgttcggtgc tgggaccaag ctggagctga aa 342

<210> 31

<211> 339

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 31

gacattgtga tgtcacagtc tccatcctcc ctagctgtgt caattggaga gaagattact 60

atgagctgca agtccaatca gagcctttta tatagtacca atcaaaagaa ctacttggcc 120

tggtaccagc agaaaccagg gcagtctcct aaactgctga tttactgggc atttactagg 180

ggatctgggg tccctgatcg cttcacaggc agtggatctg ggacagattt cactctcacc 240

atcagcagtg tgaaggctga agacctggca gtttattact gtcagcaata ttatatctat 300

ccgctcacgt tcggtgctgg gaccaagctg gagctgaaa 339

<210> 32

<211> 339

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 32

gacattgtga tgtcacagtc tccatcctcc ctagctgtgt cagttggaga gaaggttact 60

atgagctgca agtccagtca gagcctttta tatagtggca atcaaaagaa ctacttggcc 120

tggtaccagc agaaaccagg gcagtctcct aaactgctga tttactgggc atccactagg 180

gaatctgggg tccctgatcg cttcacaggc agtggatctg ggacagattt cactctcacc 240

atcagcagtg tcaaggctga agacctggca gtttattact gtcagcaata ttatacctat 300

ccgctcacgt tcggtgctgg gaccaagctg gagctgaaa 339

<210> 33

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 33

Thr Phe Ala Met Gly Val Gly

1 5

<210> 34

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 34

Thr Phe Ala Met Gly Val Gly

1 5

<210> 35

<211> 5

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 35

Asp Tyr Tyr Met Ser

1 5

<210> 36

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 36

Thr Phe Gly Met Gly Gly Gly

1 5

<210> 37

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 37

Thr Phe Ala Met Gly Val Gly

1 5

<210> 38

<211> 5

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 38

Asp Tyr Tyr Met Ser

1 5

<210> 39

<211> 5

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 39

Asp Tyr Tyr Met Ser

1 5

<210> 40

<211> 5

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 40

Asp Tyr Tyr Met Asn

1 5

<210> 41

<211> 16

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 41

His Ile Trp Trp Asp Asp Val Lys Tyr Tyr Asn Pro Ala Leu Lys Ser

1 5 10 15

<210> 42

<211> 16

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 42

His Ile Trp Trp Asp Asp Val Lys Tyr Tyr Asn Pro Ala Leu Lys Ser

1 5 10 15

<210> 43

<211> 19

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 43

Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala Ser

1 5 10 15

Val Lys Gly

<210> 44

<211> 16

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 44

His Ile Trp Trp Asp Glu Asp Lys Tyr Tyr Asn Pro Ala Leu Lys Ser

1 5 10 15

<210> 45

<211> 16

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 45

His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Ser Val Leu Lys Ser

1 5 10 15

<210> 46

<211> 19

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 46

Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala Ser

1 5 10 15

Val Lys Gly

<210> 47

<211> 19

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 47

Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala Ser

1 5 10 15

Val Lys Gly

<210> 48

<211> 19

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 48

Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala Ser

1 5 10 15

Val Lys Gly

<210> 49

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 49

Thr Tyr Gly Tyr Asp Val Asp Tyr

1 5

<210> 50

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 50

Ile Gly Gly Tyr Gly Phe Asp Tyr

1 5

<210> 51

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 51

Leu Arg Ser Ala Leu Asp Tyr

1 5

<210> 52

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 52

Ile Ala Gly Tyr Ala Met Asp Tyr

1 5

<210> 53

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 53

Leu Gly Gly Tyr Ala Met Asp Tyr

1 5

<210> 54

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 54

Tyr Arg Leu Ala Phe Asp Tyr

1 5

<210> 55

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 55

Tyr Arg Asn Ala Met Asp Tyr

1 5

<210> 56

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 56

Tyr Lys Asn Ala Met Asp Tyr

1 5

<210> 57

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 57

Arg Ala Ser Gln Glu Ile Ser Gly Tyr Leu Asn

1 5 10

<210> 58

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 58

Arg Ala Ser Gln Asp Ile Gly Ser Ser Leu Asn

1 5 10

<210> 59

<211> 17

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 59

Lys Ser Ser Gln Ser Leu Leu Tyr Ser Asn Asn Gln Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 60

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 60

Arg Ala Ser Gln Asp Ile Gly Ser Asn Leu Asn

1 5 10

<210> 61

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 61

Arg Ala Ser Gln Asp Ile Gly Asn Asn Leu Asn

1 5 10

<210> 62

<211> 17

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 62

Lys Ser Ser Gln Ser Leu Leu Tyr Ser Ser Asn Gln Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 63

<211> 17

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 63

Lys Ser Asn Gln Ser Leu Leu Tyr Ser Thr Asn Gln Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 64

<211> 17

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 64

Lys Ser Ser Gln Ser Leu Leu Tyr Ser Gly Asn Gln Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 65

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 65

Ala Ala Ser Thr Leu Asp Ser

1 5

<210> 66

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 66

Ala Thr Ser Arg Leu Asp Ser

1 5

<210> 67

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 67

Trp Ala Ser Thr Arg Glu Ser

1 5

<210> 68

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 68

Ala Thr Ser Arg Leu Asp Ser

1 5

<210> 69

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 69

Ala Thr Ser Arg Leu Asp Ser

1 5

<210> 70

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 70

Trp Ala Ser Thr Arg Glu Ser

1 5

<210> 71

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 71

Trp Ala Phe Thr Arg Gly Ser

1 5

<210> 72

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 72

Trp Ala Ser Thr Arg Glu Ser

1 5

<210> 73

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 73

Leu Gln Tyr Ala Ser Tyr Pro Arg Thr

1 5

<210> 74

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 74

Leu Gln Tyr Ala Asn Ser Pro Tyr Thr

1 5

<210> 75

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 75

Gln Gln Tyr Tyr Ile Tyr Pro Tyr Thr

1 5

<210> 76

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 76

Leu Gln Tyr Ala Ser Ser Pro Tyr Thr

1 5

<210> 77

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 77

Leu Gln Tyr Ala Ser Ser Pro Tyr Thr

1 5

<210> 78

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 78

Gln Gln Tyr Tyr Ser Tyr Pro Pro Leu Thr

1 5 10

<210> 79

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 79

Gln Gln Tyr Tyr Ile Tyr Pro Leu Thr

1 5

<210> 80

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 80

Gln Gln Tyr Tyr Thr Tyr Pro Leu Thr

1 5

<210> 81

<211> 30

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 81

Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser

20 25 30

<210> 82

<211> 30

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 82

Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser

20 25 30

<210> 83

<211> 30

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 83

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Ser Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr

20 25 30

<210> 84

<211> 30

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 84

Gln Val Ser Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser

20 25 30

<210> 85

<211> 30

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 85

Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser

20 25 30

<210> 86

<211> 30

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 86

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asp

1 5 10 15

Ser Leu Ser Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr

20 25 30

<210> 87

<211> 30

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 87

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Ser Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr

20 25 30

<210> 88

<211> 30

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 88

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asp

1 5 10 15

Ser Leu Ser Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Thr

20 25 30

<210> 89

<211> 14

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 89

Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu Trp Leu Ala

1 5 10

<210> 90

<211> 14

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 90

Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu Trp Leu Ala

1 5 10

<210> 91

<211> 14

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 91

Trp Val Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Gly

1 5 10

<210> 92

<211> 14

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 92

Trp Val Arg Gln Pro Ser Gly Lys Gly Leu Glu Trp Leu Ala

1 5 10

<210> 93

<211> 14

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 93

Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu Trp Leu Ala

1 5 10

<210> 94

<211> 14

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 94

Trp Val Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Gly

1 5 10

<210> 95

<211> 14

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 95

Trp Val Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Gly

1 5 10

<210> 96

<211> 14

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 96

Trp Val Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Gly

1 5 10

<210> 97

<211> 32

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 97

Arg Leu Thr Ile Ser Lys Asp Thr Ser Asn His Gln Val Phe Leu Asn

1 5 10 15

Ile Ala Asn Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr Cys Gly Arg

20 25 30

<210> 98

<211> 32

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 98

Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Phe Leu Lys

1 5 10 15

Ile Ala Asn Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr Cys Gly Arg

20 25 30

<210> 99

<211> 32

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 99

Arg Phe Thr Ile Ser Arg Asp Lys Ser Gln Ser Ile Phe Tyr Leu Gln

1 5 10 15

Met Asn Ala Leu Arg Pro Glu Asp Ser Ala Thr Tyr Tyr Cys Ala Ser

20 25 30

<210> 100

<211> 32

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 100

Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Phe Leu Gln

1 5 10 15

Ile Ala Asn Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr Cys Ala Arg

20 25 30

<210> 101

<211> 32

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 101

Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Phe Leu Lys

1 5 10 15

Ile Ala Asn Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr Cys Ala Arg

20 25 30

<210> 102

<211> 32

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 102

Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Ile Val Tyr Leu Gln

1 5 10 15

Met Asn Ala Leu Arg Thr Glu Asp Ser Ala Thr Tyr Tyr Cys Ala Thr

20 25 30

<210> 103

<211> 32

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 103

Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Ile Leu Tyr Leu Gln

1 5 10 15

Met Asn Ala Leu Arg Ala Glu Asp Ser Ala Thr Tyr Tyr Cys Ala Gly

20 25 30

<210> 104

<211> 32

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 104

Arg Phe Thr Ile Ser Arg Asp Ile Ser Gln Thr Ile Val Tyr Leu Gln

1 5 10 15

Met Asn Ala Leu Arg Ala Glu Asp Ser Ala Thr Tyr Phe Cys Thr Ser

20 25 30

<210> 105

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 105

Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

1 5 10

<210> 106

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 106

Trp Gly Gln Gly Thr Ala Leu Thr Val Ser Ser

1 5 10

<210> 107

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 107

Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

1 5 10

<210> 108

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 108

Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

1 5 10

<210> 109

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 109

Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

1 5 10

<210> 110

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 110

Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

1 5 10

<210> 111

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 111

Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

1 5 10

<210> 112

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 112

Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

1 5 10

<210> 113

<211> 23

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 113

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Ser Leu Thr Cys

20

<210> 114

<211> 23

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 114

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Ser Leu Thr Cys

20

<210> 115

<211> 23

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 115

Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys

20

<210> 116

<211> 23

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 116

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Ser Leu Thr Cys

20

<210> 117

<211> 23

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 117

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Ser Leu Thr Cys

20

<210> 118

<211> 23

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 118

Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Thr Val Ser Val Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys

20

<210> 119

<211> 23

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 119

Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Ile Gly

1 5 10 15

Glu Lys Ile Thr Met Ser Cys

20

<210> 120

<211> 23

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 120

Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys

20

<210> 121

<211> 15

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 121

Trp Leu Gln Gln Lys Pro Asp Gly Thr Ile Lys Arg Leu Ile Tyr

1 5 10 15

<210> 122

<211> 15

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 122

Trp Leu Gln Gln Glu Pro Asp Gly Thr Ile Arg Arg Leu Ile Tyr

1 5 10 15

<210> 123

<211> 15

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 123

Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr

1 5 10 15

<210> 124

<211> 15

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 124

Trp Leu Gln Gln Glu Pro Asp Gly Thr Ile Lys Arg Leu Ile Tyr

1 5 10 15

<210> 125

<211> 15

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 125

Trp Leu Gln Gln Glu Pro Asp Gly Thr Ile Lys Arg Leu Ile Phe

1 5 10 15

<210> 126

<211> 15

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 126

Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr

1 5 10 15

<210> 127

<211> 15

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 127

Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr

1 5 10 15

<210> 128

<211> 15

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 128

Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr

1 5 10 15

<210> 129

<211> 32

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 129

Gly Val Pro Lys Arg Phe Ser Gly Ser Arg Ser Gly Ser Asp Tyr Ser

1 5 10 15

Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Asp Tyr Tyr Cys

20 25 30

<210> 130

<211> 32

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 130

Gly Val Pro Lys Arg Phe Ser Gly Ser Arg Ser Gly Ser Glu Tyr Ser

1 5 10 15

Leu Thr Ile Ser Ser Leu Glu Ser Glu Asp Phe Val Asp Tyr Tyr Cys

20 25 30

<210> 131

<211> 32

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 131

Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys

20 25 30

<210> 132

<211> 32

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 132

Gly Val Pro Lys Arg Phe Ser Gly Ser Arg Ser Gly Ser Asp Tyr Ser

1 5 10 15

Leu Thr Ile Ser Ser Leu Glu Ser Glu Asp Phe Val Asp Tyr Tyr Cys

20 25 30

<210> 133

<211> 32

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 133

Gly Val Pro Lys Arg Phe Ser Gly Ser Arg Ser Gly Ser Asp Tyr Ser

1 5 10 15

Leu Thr Ile Ser Ser Leu Glu Ser Glu Asp Phe Val Asp Tyr Tyr Cys

20 25 30

<210> 134

<211> 32

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 134

Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys

20 25 30

<210> 135

<211> 32

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 135

Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys

20 25 30

<210> 136

<211> 32

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 136

Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys

20 25 30

<210> 137

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 137

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

1 5 10

<210> 138

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 138

Phe Gly Gly Gly Thr Lys Leu Glu Ile Glu

1 5 10

<210> 139

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 139

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

1 5 10

<210> 140

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 140

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

1 5 10

<210> 141

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 141

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

1 5 10

<210> 142

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 142

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

1 5 10

<210> 143

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 143

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

1 5 10

<210> 144

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 144

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

1 5 10

<210> 145

<211> 78

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 145

Met Arg Arg Gly Pro Arg Ser Leu Arg Gly Arg Asp Ala Pro Ala Pro

1 5 10 15

Thr Pro Cys Val Pro Ala Glu Cys Phe Asp Leu Leu Val Arg His Cys

20 25 30

Val Ala Cys Gly Leu Leu Arg Thr Pro Arg Pro Lys Pro Ala Gly Ala

35 40 45

Ser Ser Pro Ala Pro Arg Thr Ala Leu Gln Pro Gln Glu Ser Val Gly

50 55 60

Ala Gly Ala Gly Glu Ala Ala Leu Pro Leu Pro Gly Leu Leu

65 70 75

Claims

1. An anti-BAFFR monoclonal antibody or antigen binding fragment thereof comprising a heavy chain comprising heavy chain complementarity determining regions CDR1, CDR2 and CDR3 and a light chain comprising light chain complementarity determining regions CDR1, CDR2 and CDR3, wherein CDR1, CDR2 and CDR3 of the heavy chain consist of the amino acid sequences SEQ ID NO: 37. SEQ ID NO:45 and SEQ ID NO:53, and CDR1, CDR2 and CDR3 of the light chain consist of SEQ ID NO: 61. seq id NO:69 and SEQ ID NO: 77.

2. An anti-BAFFR monoclonal antibody or antigen binding fragment thereof according to claim 1 wherein the heavy and light chains comprise heavy and light chain variable regions comprising framework region FR and CDR1, CDR2 and CDR3 of the heavy chain variable region; the frame region FR includes:

(a) As set forth in SEQ ID NO:85, the amino acid sequence FR1 shown in the specification,

(b) As set forth in SEQ ID NO:93, the amino acid sequence FR2 shown in the specification,

(c) As set forth in SEQ ID NO:101, and an amino acid sequence FR3 shown in the specification,

and

(d) As set forth in SEQ ID NO:109, and an amino acid sequence FR4;

the light chain variable region comprises a framework region FR and CDR1, CDR2 and CDR3 of the light chain variable region described above; the frame region FR includes:

(e) As set forth in SEQ ID NO:117, the amino acid sequence FR1 shown in,

(f) As set forth in SEQ ID NO:125, the amino acid sequence FR2 shown in the specification,

(g) As set forth in SEQ ID NO:133, the amino acid sequence FR3 indicated,

and

(h) As set forth in SEQ ID NO:141, and the amino acid sequence FR4 as shown.

3. An anti-BAFFR monoclonal antibody, or antigen binding fragment thereof, comprising a heavy chain variable region and a light chain variable region; wherein the antigen binding fragment is a Fab, fab ', F (ab') 2, fv, single chain antibody, chimeric antibody, or diabody, wherein:

(a) The heavy chain variable region is shown in SEQ ID NO:5, and a polypeptide sequence shown in the figure;

(b) The light chain variable region is shown in SEQ ID NO:21, and a polypeptide comprising the amino acid sequence shown in seq id no.

4. An antibody or antigen-binding fragment thereof according to any one of claims 1-3, wherein the antibody or antigen-binding fragment thereof binds to a BAFFR protein.