CN114057883B

CN114057883B - Bispecific antigen binding molecules and medical uses thereof

Info

Publication number: CN114057883B
Application number: CN202110868651.2A
Authority: CN
Inventors: 顾晓玲; 金薪盛; 叶鑫; 陶维康
Original assignee: Jiangsu Hengrui Medicine Co Ltd; Shanghai Hengrui Pharmaceutical Co Ltd
Current assignee: Jiangsu Hengrui Medicine Co Ltd; Shanghai Hengrui Pharmaceutical Co Ltd
Priority date: 2020-07-31
Filing date: 2021-07-30
Publication date: 2024-04-16
Anticipated expiration: 2041-07-30
Also published as: CN114057883A

Abstract

The present disclosure relates to bispecific antigen binding molecules and pharmaceutical uses. In particular, the disclosure relates to an isolated bispecific antigen binding molecule comprising at least one first antigen binding domain capable of specifically binding to PD-1, and at least one second antigen binding domain capable of specifically binding to VEGF, and its use as a medicament. In particular, the disclosure relates to the use of a bispecific antigen binding molecule in the manufacture of a medicament for the treatment of PD-1 and/or VEGF-related diseases or disorders.

Description

Bispecific antigen binding molecules and medical uses thereof

Technical Field

The present disclosure is in the field of biotechnology, more specifically, the disclosure relates to bispecific antigen binding molecules and uses thereof.

Background

The statements herein merely provide background information related to the present disclosure and may not necessarily constitute prior art.

Tumor immunotherapy is a treatment method for fully utilizing and mobilizing killer T cells in a tumor patient to kill tumors. At the same time, tumor cell escape is a great obstacle facing tumor immunotherapy, and tumor cells promote rapid growth of tumors by utilizing their own inhibitory effect on immune system. There is a very complex relationship between the immune escape mechanism of a tumor and the immune response of the body to the tumor. Tumor-specific killer T cells are biologically active in the early stages of tumor immunotherapy, but lose their killing function in the later stages as the tumor grows.

In vivo activation of T cells takes place in two signal pathway systems, requiring a series of co-stimulatory molecules to provide a second signal in addition to the first signal provided to T cells by antigen presenting cell presenting MHC-antigen peptides, to allow the T cells to mount a normal immune response. This dual signal pathway system plays a vital role in the balance of the immune system in vivo, and it tightly regulates the body's different immune responses to self and non-self antigens. Absent the second signal provided by the co-stimulatory molecule, a non-responsive or sustained specific immune response of the T cell will result, thereby producing tolerance. Thus, the second signaling pathway plays a very critical regulatory role throughout the immune response of the body.

Programmed death molecule 1 (PD-l) is a protein receptor expressed on the surface of T cells found in 1992 and is involved in the apoptotic process of cells. PD-l belongs to the CD28 family, has 23% amino acid homology with cytotoxic T lymphocyte antigen 4 (cytotoxic T Iymphocyte antigen, CTLA-4), but its expression is different from CTLA, mainly on activated T cells, B cells and myeloid cells. PD-1 has two ligands, PD-L1 and PD-L2, respectively. PD-L1 is expressed primarily on T cells, B cells, macrophages and Dendritic Cells (DCs), and expression on activated cells can be up-regulated. Whereas PD-L2 expression is relatively limited, primarily on antigen presenting cells, such as activated macrophages and dendritic cells.

The VEGF family is a kind of growth factors capable of promoting endothelial cell division and proliferation, promoting formation of new blood vessel and raising vascular permeability. In tumor tissue, tumor cells, tumor-invading macrophages and mast cells secrete high levels of VEGF, stimulating tumor vascular endothelial cells in paracrine fashion. VEGF family can promote vascular endothelial cell mitosis, stimulate endothelial cell proliferation and migration, induce angiogenesis, and promote tumor continuous growth. And the permeability of blood vessels, especially micro blood vessels, is improved, plasma proteins (including fibrinogen) are released, surrounding tissue fibrin deposition is caused, and good matrixes are provided for tumor growth, development and metastasis. The VEGF family can also inhibit the immune response of the organism and promote infiltration and metastasis of malignant tumors. Inhibition of tumor angiogenesis is thus considered one of the current methods of tumor treatment.

Disclosure of Invention

The present disclosure provides a bispecific antigen binding molecule and its medical uses.

In some alternative embodiments, the present disclosure provides a bispecific antigen binding molecule comprising at least one first antigen binding domain capable of specifically binding to PD-1, and at least one second antigen binding domain capable of specifically binding to VEGF;

Wherein the first antigen-binding domain capable of specifically binding to PD-1 comprises a heavy chain variable region PD-1-VH and a light chain variable region PD-1-VL, wherein:

the heavy chain variable region PD-1-VH has: comprising SEQ ID NO:65, comprising the amino acid sequence of SEQ ID NO:66, and a PD-1-HCDR2 comprising the amino acid sequence of SEQ ID NO:67, PD-1-HCDR3 of the amino acid sequence; and is also provided with

The light chain variable region PD-1-VL has: comprising SEQ ID NO:68, comprising the amino acid sequence of SEQ ID NO:12, and a PD-1-LCDR2 comprising the amino acid sequence of SEQ ID NO:69, and a PD-1-LCDR3 of the amino acid sequence of seq id no. The sequences are shown in Table 1 below:

TABLE 1

In some embodiments, the foregoing bispecific antigen binding molecules are at or below 10 ^-7 M dissociation equilibrium constant binds to human PD-1 at oneIn some embodiments, at or below 10 ^-8 M、10 ^-9 M、10 ^-10 M or 10 ^-11 The M dissociation equilibrium constant binds to human PD-1.

In some embodiments, the foregoing bispecific antigen binding molecules are at or below 10 ^-7 The M dissociation equilibrium constant binds to human VEGF, in some embodiments, at or below 10 ^-8 M、10 ^-9 M、10 ^-10 M or 10 ^-11 The M dissociation equilibrium constant binds to human VEGF. In some embodiments, the foregoing bispecific antigen binding molecules are at or below 10 ^-8 M、10 ^-9 M、10 ^-10 M or 10 ^-11 The M dissociation equilibrium constant binds to murine VEGF.

In some embodiments, the aforementioned bispecific antigen binding molecule, the heavy chain variable region PD-1-VH has: comprising SEQ ID NO:8, comprising the amino acid sequence of SEQ ID NO:9, and a PD-1-HCDR2 comprising the amino acid sequence of SEQ ID NO:10, PD-1-HCDR3 of the amino acid sequence of seq id no; and the light chain variable region PD-1-VL has: comprising SEQ ID NO:68, comprising the amino acid sequence of SEQ ID NO:12, comprising the amino acid sequence of SEQ ID NO:13, and a PD-1-LCDR3 of the amino acid sequence of 13.

In some alternative embodiments, the foregoing bispecific antigen binding molecule is a bispecific antigen binding molecule selected from any one of (a 1) to (e 1) as follows:

(a1) A bispecific antigen-binding molecule, the heavy chain variable region PD-1-VH having: comprising SEQ ID NO:8, comprising the amino acid sequence of SEQ ID NO:9, and a PD-1-HCDR2 comprising the amino acid sequence of SEQ ID NO:10, PD-1-HCDR3 of the amino acid sequence of seq id no; and the light chain variable region PD-1-VL has: comprising SEQ ID NO: 11. 47, 48, 49, 50, 51 or 52, comprising the amino acid sequence of SEQ ID NO:12, comprising the amino acid sequence of SEQ ID NO:13, PD-1-LCDR3 of the amino acid sequence of 13;

(b1) A bispecific antigen-binding molecule, the heavy chain variable region PD-1-VH having: comprising SEQ ID NO:14, comprising the amino acid sequence of SEQ ID NO:15, and a PD-1-HCDR2 comprising the amino acid sequence of SEQ ID NO:16, and a PD-1-HCDR3 of the amino acid sequence of 16; and the light chain variable region PD-1-VL has: comprising SEQ ID NO:17, comprising the amino acid sequence of SEQ ID NO:12, comprising the amino acid sequence of SEQ ID NO:18, PD-1-LCDR3 of the amino acid sequence of 18;

(c1) A bispecific antigen-binding molecule, the heavy chain variable region PD-1-VH having: comprising SEQ ID NO:21, comprising the amino acid sequence of SEQ ID NO:22, and a PD-1-HCDR2 comprising the amino acid sequence of SEQ ID NO:23, and a PD-1-HCDR3 of the amino acid sequence of seq id no; and the light chain variable region PD-1-VL has: comprising SEQ ID NO:24, comprising the amino acid sequence of SEQ ID NO:25, comprising the amino acid sequence of SEQ ID NO:26, and PD-1-LCDR3 of the amino acid sequence of 26;

(d1) A bispecific antigen-binding molecule, the heavy chain variable region PD-1-VH having: comprising SEQ ID NO:14, comprising the amino acid sequence of SEQ ID NO:15, and a PD-1-HCDR2 comprising the amino acid sequence of SEQ ID NO:16, and a PD-1-HCDR3 of the amino acid sequence of 16; and the light chain variable region PD-1-VL has: comprising SEQ ID NO: 11. 47, 48, 49, 50, 51 or 52, comprising the amino acid sequence of SEQ ID NO:12, comprising the amino acid sequence of SEQ ID NO:13, PD-1-LCDR3 of the amino acid sequence of 13; and

(e1) A bispecific antigen-binding molecule, the heavy chain variable region PD-1-VH having: comprising SEQ ID NO:8, comprising the amino acid sequence of SEQ ID NO:9, and a PD-1-HCDR2 comprising the amino acid sequence of SEQ ID NO:10, PD-1-HCDR3 of the amino acid sequence of seq id no; and the light chain variable region PD-1-VL has: comprising SEQ ID NO:17, comprising the amino acid sequence of SEQ ID NO:12, comprising the amino acid sequence of SEQ ID NO:13, and a PD-1-LCDR3 of the amino acid sequence of 13.

In some embodiments, the aforementioned bispecific antigen binding molecule, the heavy chain variable region PD-1-VH has: comprising SEQ ID NO:8, comprising the amino acid sequence of SEQ ID NO:9, and a PD-1-HCDR2 comprising the amino acid sequence of SEQ ID NO:10, PD-1-HCDR3 of the amino acid sequence of seq id no; and the light chain variable region PD-1-VL has: comprising SEQ ID NO:49, comprising the amino acid sequence of SEQ ID NO:12, comprising the amino acid sequence of SEQ ID NO:13, and a PD-1-LCDR3 of the amino acid sequence of 13.

In some embodiments, the aforementioned bispecific antigen binding molecule, the heavy chain variable region PD-1-VH has: comprising SEQ ID NO:21, comprising the amino acid sequence of SEQ ID NO:22, and a PD-1-HCDR2 comprising the amino acid sequence of SEQ ID NO:23, and a PD-1-HCDR3 of the amino acid sequence of seq id no; and the light chain variable region PD-1-VL has: comprising SEQ ID NO:24, comprising the amino acid sequence of SEQ ID NO:25, comprising the amino acid sequence of SEQ ID NO:26, and a PD-1-LCDR3 amino acid sequence of said polypeptide.

In some alternative embodiments, the present disclosure provides a bispecific antigen binding molecule selected from any one of f 1) to h 1) below:

f1 A bispecific antigen binding molecule whose heavy chain variable region PD-1-VH hybridizes with SEQ ID NO:4, and the heavy chain variable region of the amino acid sequence of SEQ ID NO:5, and the light chain variable region of the amino acid sequence of PD-1-LCDR1, PD-1-LCDR2 and PD-1-LCDR3 of the same sequence;

g1 A bispecific antigen binding molecule whose heavy chain variable region PD-1-VH hybridizes with SEQ ID NO:6, and the heavy chain variable region of the amino acid sequence of SEQ ID NO:7, the light chain variable region of the amino acid sequence has the same sequence of PD-1-LCDR1, PD-1-LCDR2 and PD-1-LCDR3; and

h1 A bispecific antigen binding molecule whose heavy chain variable region PD-1-VH hybridizes with SEQ ID NO:19, and the heavy chain variable region of the amino acid sequence of SEQ ID NO:20, and the light chain variable region of the amino acid sequence of 20 has the same sequence of PD-1-LCDR1, PD-1-LCDR2 and PD-1-LCDR3.

In some embodiments, the heavy chain variable region PD-1-VH or light chain variable region PD-1-VL of the bispecific antigen binding molecule comprises a framework region derived from a human antibody or a framework region variant thereof.

In some embodiments, the framework region variant is a back mutation having up to 11 amino acids based on the light chain framework region and/or heavy chain framework region, respectively, of a human antibody.

In some embodiments, the framework region variant comprises a mutation selected from any one of the following (j 1) to (l 1):

(j1) The light chain variable region PD-1-VL comprises a 2G amino acid back mutation and/or the heavy chain variable region PD-1-VH comprises one or more amino acid back mutations selected from the group consisting of 27Y, 48I, 67T, 69L, 82F and 93T;

(k1) The light chain variable region PD-1-VL comprises a 2V amino acid back mutation, and/or the heavy chain variable region PD-1-VH comprises one or more amino acid back mutations selected from the group consisting of 26D, 27F, 30T, 38K, 43H, 48I, 66K, 67A, 69L, 82F, and 93T; and

(l 1) one or more amino acid back mutations selected from 42G, 44V and 71Y are included in the light chain variable region PD-1-VL, and/or 1K and/or 94S amino acid back mutations are included in the heavy chain variable region PD-1-VH.

In some embodiments, the foregoing bispecific antigen binding molecules comprise an antibody variable region selected from the group consisting of:

(a2) The heavy chain variable region PD-1-VH comprises the sequence of SEQ ID NO: 8. SEQ ID NO:9 and SEQ ID NO:10, and the heavy chain framework regions comprise one or more amino acid back mutations of 27Y, 48I, 67T, 69L, 82F, and 93T, and

the light chain variable regions PD-1-VL comprise SEQ ID NOs: 12 and SEQ ID NO:13, and PD-1-LCDR2 and PD-1-LCDR3 of the amino acid sequence of SEQ ID NO: 11. 47, 48, 49, 50, 51 or 52, and the light chain framework region comprises a 2G amino acid back mutation;

(b2) The heavy chain variable region PD-1-VH comprises the sequence of SEQ ID NO: 14. SEQ ID NO:15 and SEQ ID NO:16, and the heavy chain framework regions comprise one or more amino acid back mutations selected from the group consisting of 26D, 27F, 30T, 38K, 43H, 48I, 66K, 67A, 69L, 82F, and 93T; and

the light chain variable regions PD-1-VL comprise SEQ ID NOs: 17. SEQ ID NO:12 and SEQ ID NO:18, PD-1-LCDR1, PD-1-LCDR2, and PD-1-LCDR3 of the amino acid sequence, and the light chain framework region comprises a 2V amino acid back mutation;

(c2) The heavy chain variable region PD-1-VH comprises the sequence of SEQ ID NO: 21. SEQ ID NO:22 and SEQ ID NO:23, and the heavy chain framework regions comprise 1K and/or 94S amino acid back mutations, and

the light chain variable regions PD-1-VL comprise SEQ ID NOs: 24. SEQ ID NO:25 and SEQ ID NO:26, PD-1-LCDR1, PD-1-LCDR2, and PD-1-LCDR3 of an amino acid sequence, and the light chain framework region comprises one or more amino acid back mutations selected from the group consisting of 42G, 44V, and 71Y.

In some embodiments, the bispecific antigen binding molecules described above comprise an antibody variable region selected from any one of (m 1) to (o 1) below;

(m 1) a heavy chain variable region PD-1-VH having SEQ ID NO:4 or an amino acid sequence identical to SEQ ID NO:4, and/or has at least 90% sequence identity, and/or

A light chain variable region PD-1-VL having the amino acid sequence of SEQ ID NO:5 or an amino acid sequence identical to SEQ ID NO:5 has at least 90% sequence identity;

(n 1) a heavy chain variable region PD-1-VH having SEQ ID NO:6 or amino acid sequence corresponding to SEQ ID NO:6 has at least 90% sequence identity, and/or

A light chain variable region PD-1-VL having the amino acid sequence of SEQ ID NO:7 or an amino acid sequence identical to SEQ ID NO:7 has at least 90% sequence identity;

(o 1) a heavy chain variable region PD-1-VH having SEQ ID NO:19 or amino acid sequence corresponding to SEQ ID NO:19 has at least 90% sequence identity, and/or

A light chain variable region PD-1-VL having the amino acid sequence of SEQ ID NO:20 or an amino acid sequence identical to SEQ ID NO:20 has at least 90% sequence identity;

(p 1) heavy chain variable region PD-1-VH having SEQ ID NO: 27. 30, 31 or 32 or an amino acid sequence corresponding to SEQ ID NO: 27. 30, 31 or 32 has at least 90% sequence identity, and/or

A light chain variable region PD-1-VL having the amino acid sequence of SEQ ID NO: 28. 29, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 or 64 or an amino acid sequence which hybridizes with SEQ ID NO: 28. 29, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, or 64 has at least 90% sequence identity;

(q 1) a heavy chain variable region PD-1-VH having SEQ ID NO: 33. 36, 37, 38, 39 or 40 or an amino acid sequence corresponding to SEQ ID NO: 33. 36, 37, 38, 39 or 40 has at least 90% sequence identity, and/or

A light chain variable region PD-1-VL having the amino acid sequence of SEQ ID NO: 34. 35, 28, 29, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 or 64 or an amino acid sequence which hybridizes with SEQ ID NO: 34. 35, 28, 29, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, or 64 has at least 90% sequence identity;

(r 1) a heavy chain variable region PD-1-VH having the amino acid sequence of SEQ ID NO: 41. 45 or 46 or an amino acid sequence corresponding to SEQ ID NO: 41. 45 or 46 has at least 90% sequence identity, and/or

A light chain variable region PD-1-VL having the amino acid sequence of SEQ ID NO: 42. 43 or 44 or an amino acid sequence corresponding to SEQ ID NO: 42. 43 or 44 has at least 90% sequence identity;

(s 1) a heavy chain variable region PD-1-VH having SEQ ID NO:70 or an amino acid sequence identical to SEQ ID NO:70, and/or has at least 90% sequence identity, and/or

A light chain variable region PD-1-VL having the amino acid sequence of SEQ ID NO:71 or amino acid sequence corresponding to SEQ ID NO:71 has at least 90% sequence identity;

(t 1) a heavy chain variable region PD-1-VH having the amino acid sequence of SEQ ID NO: 27. 30, 31 or 32 or an amino acid sequence corresponding to SEQ ID NO: 27. 30, 31 or 32 has at least 90% sequence identity, and/or

A light chain variable region PD-1-VL having the amino acid sequence of SEQ ID NO:34 or 35 or an amino acid sequence corresponding to SEQ ID NO:34 or 35 has at least 90% sequence identity;

wherein the sequence SEQ ID NO:70 and SEQ ID NO:71 is the general formula sequence shown in Table 2:

TABLE 2

In some embodiments, the bispecific antigen binding molecule of the foregoing, wherein the heavy chain variable region PD-1-VH comprises SEQ ID NO:27 or comprises an amino acid sequence identical to SEQ ID NO:27, and the light chain variable region PD-1-VL comprises an amino acid sequence having at least 95%,96%,97%,98% or 99% sequence identity to SEQ ID NO:55 or comprises an amino acid sequence identical to SEQ ID NO:55 has an amino acid sequence having at least 95%,96%,97%,98% or 99% sequence identity;

In some embodiments, the bispecific antigen binding molecule of the foregoing, wherein the heavy chain variable region PD-1-VH comprises SEQ ID NO:27, and the light chain variable region PD-1-VL comprises the amino acid sequence of SEQ ID NO: 55.

In some embodiments, the bispecific antigen binding molecule of the foregoing, wherein the heavy chain variable region PD-1-VH comprises SEQ ID NO:46 or comprises an amino acid sequence identical to SEQ ID NO:46, and the light chain variable region PD-1-VL comprises an amino acid sequence having at least 95%,96%,97%,98% or 99% sequence identity to SEQ ID NO:43 or comprises an amino acid sequence identical to SEQ ID NO:43 has an amino acid sequence having at least 95%,96%,97%,98% or 99% sequence identity;

in some embodiments, the bispecific antigen binding molecule of the foregoing, wherein the heavy chain variable region PD-1-VH has the amino acid sequence of SEQ ID NO:46, and the light chain variable region PD-1-VL has the amino acid sequence of SEQ ID NO: 43.

In some embodiments, the bispecific antigen binding molecules of the foregoing, wherein the bispecific antigen binding molecules further comprise a heavy chain constant region and a light chain constant region. In still other embodiments, the heavy chain constant region is selected from the group consisting of human IgG1, igG2, igG3, and IgG4 constant regions and conventional variants thereof, and the light chain constant region is selected from the group consisting of human antibody kappa and lambda chain constant regions and conventional variants thereof; in other embodiments, the heavy chain constant region comprises an IgG4 heavy chain constant region that introduces one or more mutations in S228P, F234A and L235A, e.g., three amino acid mutations in S228P, F234A and L235A; in other embodiments, the heavy chain constant region comprises SEQ ID NO:72, and the light chain constant region comprises the amino acid sequence of SEQ ID NO:73, and an amino acid sequence of 73.

In some embodiments, the bispecific antigen-binding molecule of the foregoing, wherein the second antigen-binding domain capable of specifically binding VEGF comprises a heavy chain variable region VEGF-VH and a light chain variable region VEGF-VL, wherein:

the heavy chain variable region VEGF-VH has: comprising SEQ ID NO:87, comprising the amino acid sequence of SEQ ID NO:88, and VEGF-HCDR2 of the amino acid sequence of SEQ ID NO:89, VEGF-HCDR3 of the amino acid sequence; and is also provided with

The light chain variable region VEGF-VL has: comprising SEQ ID NO:90, comprising the amino acid sequence of SEQ ID NO:91, and a VEGF-LCDR2 comprising the amino acid sequence of SEQ ID NO:92, and a VEGF-LCDR3 amino acid sequence.

HYGMN(SEQ ID NO：87)，

WINTYTGEPTYAADFKR(SEQ ID NO：88)，

YPYYYGTSHWYFDV(SEQ ID NO：89)，

SASQDISNYLN(SEQ ID NO：90)，

FTSSLHS(SEQ ID NO：91)，

QQYSTVPWT(SEQ ID NO：92)

In some embodiments, the bispecific antigen binding molecule of the foregoing, wherein the heavy chain variable region VEGF-VH comprises SEQ ID NO:85 or an amino acid sequence comprising a sequence identical to SEQ ID NO:85, and said light chain variable region VEGF-VL comprises an amino acid sequence having at least 95%,96%,97%,98% or 99% sequence identity to SEQ ID NO:86 or comprises an amino acid sequence as set forth in SEQ ID NO:86 has an amino acid sequence having at least 95%,96%,97%,98% or 99% sequence identity.

In some embodiments, the bispecific antigen binding molecule of the foregoing, wherein the heavy chain variable region VEGF-VH has the amino acid sequence of SEQ ID NO:85, and said light chain variable region VEGF-VL has the amino acid sequence set forth in SEQ ID NO:86, and a polypeptide having the amino acid sequence shown in seq id no.

EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSS(SEQ ID NO：85)

DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIK(SEQ ID NO：86)

In some embodiments, the bispecific antigen binding molecule of the foregoing, wherein the heavy chain variable region PD-1-VH is linked to the heavy chain variable region VEGF-VH directly or through a linker, and the light chain variable region PD-1-VL is linked to the light chain variable region VEGF-VL directly or through a linker. In some embodiments, the C-terminus of the heavy chain variable region PD-1-VH is linked directly or through a linker to the N-terminus of the heavy chain variable region VEGF-VH, and the C-terminus of the light chain variable region PD-1-VL is linked directly or through a linker to the N-terminus of the light chain variable region VEGF-VL.

In some embodiments, the bispecific antigen binding molecule of the foregoing, wherein the heavy chain variable region VEGF-VH and the light chain variable region VEGF-VL are linked directly or through a linker to form a VEGF-scFv antigen binding domain; and the heavy chain variable region PD-1-VH and the VEGF-scFv antigen-binding domain are linked to the heavy chain constant region either directly or through a linker, respectively. In some embodiments, the C-terminus of the heavy chain variable region VEGF-VH is linked to the N-terminus of the light chain variable region VEGF-VL directly or through a linker to form a VEGF-scFv antigen-binding domain; and the C-terminus of the heavy chain variable region PD-1-VH is linked directly or through a linker to the N-terminus of the heavy chain constant region, and the N-terminus of the VEGF-scFv antigen binding domain is linked directly or through a linker to the C-terminus of the heavy chain constant region.

In some embodiments, the bispecific antigen binding molecule described above, wherein the bispecific antigen binding molecule has the structure shown in the following formula or a combination thereof:

a [ heavy chain variable region PD-1-VH ] - [ linker ] - [ heavy chain variable region VEGF-VH ] - [ heavy chain constant region ]; or (b)

[ light chain variable region PD-1-VL ] - [ linker ] - [ light chain variable region VEGF-VL ] - [ light chain constant region ]; or (b)

A [ heavy chain variable region VEGF-VH ] - [ linker ] - [ heavy chain variable region PD-1-VH ] - [ heavy chain constant region ]; or (b)

[ light chain variable region VEGF-VL ] - [ linker ] - [ light chain variable region PD-1-VL ] - [ light chain constant region ].

[ heavy chain variable region PD-1-VH ] - [ heavy chain constant region ] - [ linker ] - [ heavy chain variable region VEGF-VH ] - [ linker ] - [ light chain variable region VEGF-VL ]; or (b)

[ heavy chain variable region VEGF-VH ] - [ linker ] - [ light chain variable region VEGF-VL ] - [ linker ] - [ heavy chain variable region PD-1-VH ] - [ heavy chain constant region ]; or (b)

[ light chain variable region PD-1-VL ] - [ light chain constant region ].

In some embodiments, the bispecific antigen binding molecules of the foregoing, the foregoing structures are arranged from N-terminus to C-terminus.

In some embodiments, the foregoing bispecific antigen binding molecules, wherein the bispecific antigen binding molecule has a first chain of the structure of formula (i) and a second chain of the structure of formula (ii),

(i) A [ heavy chain variable region PD-1-VH ] - [ linker ] - [ heavy chain variable region VEGF-VH ] - [ heavy chain constant region ];

(ii) [ light chain variable region PD-1-VL ] - [ linker ] - [ light chain variable region VEGF-VL ] - [ light chain constant region ];

wherein the formulas (i) and (ii) are arranged from the N-terminus to the C-terminus.

In some embodiments, the bispecific antigen binding molecules described above, wherein the bispecific antigen binding molecules have a first chain of the structure of formula (iii) and a second chain of the structure of formula (iv),

(iii) [ heavy chain variable region PD-1-VH ] - [ heavy chain constant region ] - [ linker ] - [ heavy chain variable region VEGF-VH ] - [ linker ] - [ light chain variable region VEGF-VL ];

(iv) [ light chain variable region PD-1-VL ] - [ light chain constant region ];

wherein the formulas (iii) and (iv) are arranged from the N-terminus to the C-terminus.

In some embodiments, the foregoing bispecific antigen binding molecule, wherein the second antigen binding domain capable of specifically binding VEGF comprises a polypeptide that binds to SEQ ID NO:94 has an amino acid sequence having at least 95%,96%,97%,98%,99% or 100% sequence identity.

In some embodiments, the foregoing bispecific antigen binding molecule, wherein the second antigen binding domain capable of specifically binding VEGF comprises SEQ ID NO:94 or SEQ ID NO:94, said variant comprising one or more amino acid substitutions selected from the group consisting of T100C, I101T, V116A, E C, V132A, L155C, G160Q, G160T, G160P, G162T, G162D, G162S, G164N, G164A, G164E, G166Y, G166A, K165C, S188C and T193C, wherein said variant has only one amino acid substitution at the same position.

In some embodiments, the foregoing bispecific antigen binding molecule, wherein the second antigen binding domain capable of specifically binding VEGF comprises the foregoing SEQ ID NO:94 or a variant thereof. In some embodiments, the truncated sequence comprises 100 to 204 amino acid residues.

In some embodiments, the foregoing bispecific antigen binding molecule, wherein the second antigen binding domain capable of specifically binding VEGF comprises a polypeptide that binds to SEQ ID NO:141 has an amino acid sequence having at least 95%,96%,97%,98%,99% or 100% sequence identity.

In some embodiments, the foregoing bispecific antigen binding molecule, wherein the second antigen binding domain capable of specifically binding VEGF comprises SEQ ID NO:141 or SEQ ID NO:141 comprising one or more amino acid substitutions selected from the group consisting of Y11C and T90C.

In some embodiments, the foregoing bispecific antigen binding molecule, wherein the second antigen binding domain capable of specifically binding VEGF has an amino acid sequence selected from the group consisting of SEQ ID NOs: 94 to SEQ ID NO: 100. SEQ ID NO:141 and SEQ ID NO:142, and a sequence of amino acids of the group consisting of seq id no.

SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK(SEQ ID NO：94)

SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNCIIDVVLSPSHGIELSVGEKLVLNCTARTCLNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK(SEQ ID NO：95)

SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAACSGLMCKKNSTFVRVHEK(SEQ ID NO：96)

SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDCKTQSGSEMKCFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK(SEQ ID NO：97)

SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTTIDVVLSPSHGIELSAGEKLVLNCTARTELNAGIDFNWEYPSSKHQHKKLVNRDLKTQSQSTMNKYLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK(SEQ ID NO：98)

SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSTSDMAKRLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK(SEQ ID NO：99)

SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSPSSMEKALSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK(SEQ ID NO：100)

SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNT(SEQ ID NO：141)

SDTGRPFVEMCSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKCNYLTHRQTNT(SEQ ID NO：142)

In some embodiments, the bispecific antigen binding molecule of the foregoing, wherein the second antigen binding domain capable of specifically binding VEGF is linked to the heavy chain constant region directly or through a linker. In some embodiments, the N-terminus of the second antigen binding domain capable of specifically binding VEGF is linked to the C-terminus of the heavy chain constant region directly or through a linker.

In some embodiments, the bispecific antigen-binding molecule of the foregoing, wherein the second antigen-binding domain capable of specifically binding VEGF is linked to the heavy chain variable region PD-1-VH directly or through a linker. In some embodiments, the C-terminus of the second antigen-binding domain capable of specifically binding VEGF is linked directly or through a linker to the N-terminus of the heavy chain variable region PD-1-VH.

In some embodiments, the bispecific antigen binding molecule described above, wherein the bispecific antigen binding molecule has the structure of the formula:

[ heavy chain variable region PD-1-VH ] - [ heavy chain constant region ] - [ linker ] - [ second antigen binding domain capable of specifically binding VEGF ]; or (b)

[ second antigen-binding domain capable of specifically binding VEGF ] - [ linker ] - [ heavy chain variable region PD-1-VH ] - [ heavy chain constant region ]; or (b)

[ light chain variable region PD-1-VL ] - [ light chain constant region ];

in some embodiments, the aforementioned bispecific antigen binding molecules, the structure is arranged from N-terminus to C-terminus.

In some embodiments, the bispecific antigen binding molecules described above, wherein the bispecific antigen binding molecules have a first chain of the structure of formula (v) and a second chain of the structure of formula (iv),

(v) [ heavy chain variable region PD-1-VH ] - [ heavy chain constant region ] - [ linker ] - [ second antigen binding domain capable of specifically binding VEGF ];

(iv) [ light chain variable region PD-1-VL ] - [ light chain constant region ];

wherein the formulas (v) and (iv) are arranged from the N-terminus to the C-terminus.

In some embodiments, the bispecific antigen binding molecules described above, wherein the bispecific antigen binding molecules have a first chain of the structure of formula (vi) and a second chain of the structure of formula (iv),

(vi) [ second antigen-binding domain capable of specifically binding VEGF ] - [ linker ] - [ heavy chain variable region PD-1-VH ] - [ heavy chain constant region ];

(iv) [ light chain variable region PD-1-VL ] - [ light chain constant region ];

wherein the formulas (vi) and (iv) are arranged from the N-terminus to the C-terminus.

In some embodiments, the bispecific antigen binding molecule of the foregoing, wherein the heavy chain variable region PD-1-VH comprises SEQ ID NO:27, and the light chain variable region PD-1-VL comprises the amino acid sequence of SEQ ID NO:55, an amino acid sequence of seq id no; and the second antigen-binding domain capable of specifically binding VEGF comprises a heavy chain variable region VEGF-VH and a light chain variable region VEGF-VL; wherein the heavy chain variable region VEGF-VH comprises the sequence set forth in SEQ ID NO:85, and said light chain variable region VEGF-VL comprises the amino acid sequence of SEQ ID NO:86, and a sequence of amino acids. In some embodiments, the bispecific antigen binding molecule further comprises a heavy chain constant region and a light chain constant region; wherein the heavy chain constant region comprises SEQ ID NO:72, and the light chain constant region comprises the amino acid sequence of SEQ ID NO:73, and an amino acid sequence of 73. In some embodiments, the bispecific antigen binding molecule has a first chain of the structure of formula (i) and a second chain of the structure of formula (ii),

wherein the formulas (i) and (ii) are arranged from the N-terminus to the C-terminus. In some embodiments, the bispecific antigen binding molecule of the foregoing, wherein the heavy chain variable region PD-1-VH comprises SEQ ID NO:27, and the light chain variable region PD-1-VL comprises the amino acid sequence of SEQ ID NO:55, an amino acid sequence of seq id no; and the second antigen binding domain capable of specifically binding VEGF comprises SEQ ID NO: 96. SEQ ID NO:141 or SEQ ID NO:142, and a fragment thereof. In some embodiments, the bispecific antigen binding molecule further comprises a heavy chain constant region and a light chain constant region; wherein the heavy chain constant region comprises SEQ ID NO:72, and the light chain constant region comprises the amino acid sequence of SEQ ID NO:73, and an amino acid sequence of 73. In some embodiments, the bispecific antigen binding molecule has a first chain of the structure of formula (v) and a second chain of the structure of formula (iv),

(v) [ PD-1-VH ] - [ heavy chain constant region ] - [ linker ] - [ second antigen-binding domain capable of specifically binding VEGF ];

(iv) [ PD-1-VL ] - [ light chain constant region ];

In some embodiments, the bispecific antigen binding molecule has a first chain of the structure of formula (vi) and a second chain of the structure of formula (iv),

(iv) [ light chain variable region PD-1-VL ] - [ light chain constant region ];

wherein the second antigen binding domain capable of specifically binding VEGF has an amino acid sequence selected from the group consisting of SEQ ID NOs: 95 to SEQ ID NO: 100. SEQ ID NO:141 and SEQ ID NO:142, and a sequence of amino acids of the group consisting of seq id no.

In some embodiments, the bispecific antigen-binding molecule of the foregoing, wherein the first antigen-binding domain capable of specifically binding to PD-1 comprises a heavy chain variable region PD-1-VH and a light chain variable region PD-1-VL, wherein:

The heavy chain variable region PD-1-VH has: comprising SEQ ID NO:8, comprising the amino acid sequence of SEQ ID NO:9, and a PD-1-HCDR2 comprising the amino acid sequence of SEQ ID NO:10, PD-1-HCDR3 of the amino acid sequence of seq id no; and is also provided with

The light chain variable region PD-1-VL has: comprising SEQ ID NO:49, comprising the amino acid sequence of SEQ ID NO:12, comprising the amino acid sequence of SEQ ID NO:13, PD-1-LCDR3 of the amino acid sequence of 13;

in some embodiments, the foregoing bispecific antigen binding molecules, the heavy chain variable region PD-1-VH has the amino acid sequence of SEQ ID NO:27, and the light chain variable region PD-1-VL has the amino acid sequence of SEQ ID NO: 55.

In some embodiments, the foregoing bispecific antigen binding molecules, the heavy chain variable region PD-1-VH has the amino acid sequence of SEQ ID NO:46, and the light chain variable region PD-1-VL has the amino acid sequence of SEQ ID NO: 43.

In some embodiments, the bispecific antigen binding molecules of the foregoing, wherein the bispecific antigen binding molecules further comprise a heavy chain constant region and a light chain constant region; in some embodiments, the heavy chain constant region comprises SEQ ID NO:72, and the light chain constant region comprises the amino acid sequence of SEQ ID NO:73, and an amino acid sequence of 73.

(iv) [ PD-1-VL ] - [ light chain constant region ];

(iv) [ light chain variable region PD-1-VL ] - [ light chain constant region ];

In some embodiments, the bispecific antigen binding molecules of the foregoing, wherein the linker is a peptide linker. In some embodiments, the linker is a flexible peptide linker. In some embodiments, the linkers each independently have L ₁ -(GGGGS) _n -L ₂ Wherein L is ₁ Is a bond, A, GS, GGS or GGGS, n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, L ₂ Is a bond, G, GG, GGG or GGGGGG. In some embodiments, the linker has an amino acid residue of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. In some embodiments, the linker has 8 to 24 or 10 to 19 amino acid residues.

In some embodiments, the bispecific antigen binding molecules of the foregoing, wherein each of the linkers independently has a sequence selected from the group consisting of SEQ ID NOs: 127 to SEQ ID NO:134, and a fragment thereof. The sequence is as follows:

GSGGGGSG(SEQ ID NO：127)，

GGGGSGGGGS(SEQ ID NO：128)，

GGGGSGGGGSGGGGSGGGG(SEQ ID NO：129)，

GGGGSGGGGSGGGG(SEQ ID NO：130)，

AGGGGSGGGGSGGGGSGGGGSG(SEQ ID NO：131)，

GGGGSGGGGSGGGGSGGGGSGGGG(SEQ ID NO：132)，

GGSGGGGSGGGGS (SEQ ID NO: 133), or

GGGGSGGGGSGGGGSGGGGS(SEQ ID NO：134)。

In some embodiments, the foregoing bispecific antigen binding molecules, wherein the linkers may be the same or different.

In some embodiments, the bispecific antigen binding molecule of the foregoing, wherein the bispecific antigen binding molecule has:

(a) Comprising a sequence selected from the group consisting of SEQ ID NOs: 105. SEQ ID NO: 107. SEQ ID NO: 109. SEQ ID NO: 111. SEQ ID NO:112 and SEQ ID NO:114 and a first strand comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 106. SEQ ID NO: 108. SEQ ID NO:110 and SEQ ID NO:113, and a second strand of an amino acid sequence of the group consisting of seq id no; or (b)

(b) Comprising a sequence selected from the group consisting of SEQ ID NOs: 115 to SEQ ID NO: 126. SEQ ID NO:138 and SEQ ID NO:140 and a first strand comprising the amino acid sequence of the group consisting of SEQ ID NO:75, and a second strand of an amino acid sequence of seq id no.

comprising SEQ ID NO:105, and a first strand comprising the amino acid sequence of SEQ ID NO:106, and a second strand of an amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:107, and a first strand comprising the amino acid sequence of SEQ ID NO:108, and a second strand of an amino acid sequence of 108; or (b)

Comprising SEQ ID NO:109, and a first strand comprising the amino acid sequence of SEQ ID NO:110, and a second strand of an amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:111, and a first strand comprising the amino acid sequence of SEQ ID NO:110, and a second strand of an amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:112, and a first strand comprising the amino acid sequence of SEQ ID NO:110, and a second strand of an amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:109, and a first strand comprising the amino acid sequence of SEQ ID NO:113, and a second strand of an amino acid sequence of 113; or (b)

Comprising SEQ ID NO:114, and a first strand comprising the amino acid sequence of SEQ ID NO:113, and a second strand of an amino acid sequence of 113; or (b)

Comprising SEQ ID NO:115, and a first strand comprising the amino acid sequence of SEQ ID NO:75, and a second strand of an amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:116, and a first strand comprising the amino acid sequence of SEQ ID NO:75, and a second strand of an amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:117, and a first strand comprising the amino acid sequence of SEQ ID NO:75, and a second strand of an amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:118, and a first strand comprising the amino acid sequence of SEQ ID NO:75, and a second strand of an amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:119, and a first strand comprising the amino acid sequence of SEQ ID NO:75, and a second strand of an amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:120, and a first strand comprising the amino acid sequence of SEQ ID NO:75, and a second strand of an amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:121, and a first strand comprising the amino acid sequence of SEQ ID NO:75, and a second strand of an amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:122, and a first strand comprising the amino acid sequence of SEQ ID NO:75, and a second strand of an amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:123, and a first strand comprising the amino acid sequence of SEQ ID NO:75, and a second strand of an amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:124, and a first strand comprising the amino acid sequence of SEQ ID NO:75, and a second strand of an amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:125, and a first strand comprising the amino acid sequence of SEQ ID NO:75, and a second strand of an amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:126, and a first strand comprising the amino acid sequence of SEQ ID NO:75, and a second strand of an amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:138, and a first strand comprising the amino acid sequence of SEQ ID NO:75, and a second strand of an amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:140, and a first strand comprising the amino acid sequence of SEQ ID NO:75, and a second strand of an amino acid sequence of seq id no.

comprising SEQ ID NO:137, and a first strand comprising the amino acid sequence of SEQ ID NO:78, and a second strand of an amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:139, and a first strand comprising the amino acid sequence of SEQ ID NO:78, and a second strand of the amino acid sequence of seq id no.

In some embodiments, the bispecific antigen binding molecules described above comprise two first chains and two second chains. In some embodiments, the bispecific antigen binding molecules described above comprise two identical first chains and two identical second chains.

In some embodiments, there is also provided a bispecific antigen binding molecule that competes with the bispecific antigen binding molecule segment of any one of the preceding claims for binding to human PD-1 and human VEGF.

In some embodiments, the present disclosure also provides a pharmaceutical composition comprising a therapeutically effective amount of a bispecific antigen binding molecule of any of the preceding claims, and one or more pharmaceutically acceptable carriers, diluents, buffers, or excipients. In some embodiments, the therapeutically effective amount comprises from 0.1 to 3000mg of a bispecific antigen binding molecule as described above in a unit dose of the composition.

In some embodiments, the disclosure also provides a nucleic acid molecule encoding the bispecific antigen binding molecule of any one of the preceding claims.

In some embodiments, the disclosure also provides a host cell comprising the aforementioned nucleic acid molecule.

In some embodiments, the disclosure also provides a method for immunodetection or assay of PD-1, the method comprising the step of contacting a subject or a sample from a subject with a bispecific antigen binding molecule of any one of the preceding claims.

In some embodiments, the disclosure also provides a kit comprising the aforementioned bispecific antigen binding molecule.

In some embodiments, there is also provided the use of the aforementioned bispecific antigen binding molecules in the preparation of a diagnostic agent for a PD-1 related disease.

In some embodiments, the disclosure also provides a method of treating a disease, the method comprising administering to a subject a therapeutically effective amount of a bispecific antigen binding molecule of any of the preceding, or a pharmaceutical composition of the preceding, or a nucleic acid molecule of the preceding.

In some embodiments, the disease is a tumor.

In other embodiments, the disease is selected from: head and neck squamous cell carcinoma, head and neck carcinoma, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system carcinoma, neuroendocrine tumor, laryngeal carcinoma, nasopharyngeal carcinoma, esophageal carcinoma, thyroid carcinoma, malignant pleural mesothelioma, lung carcinoma, breast carcinoma, liver carcinoma, hepatoma, hepatobiliary carcinoma, pancreatic carcinoma, stomach carcinoma, gastrointestinal carcinoma, intestinal carcinoma, colon carcinoma, colorectal carcinoma, renal carcinoma, clear cell renal cell carcinoma, ovarian carcinoma, endometrial carcinoma, cervical carcinoma, bladder carcinoma, prostate carcinoma, testicular carcinoma, skin carcinoma, melanoma, leukemia, lymphoma, bone carcinoma, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, myeloproliferative neoplasm, squamous cell carcinoma, ewing's sarcoma, systemic light chain amyloidosis, and merkel cell carcinoma; in some of these embodiments, the lymphoma is selected from: hodgkin's lymphoma, non-hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinum large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, T-cell/tissue cell enriched large B-cell lymphoma, and lymphoplasmacytic lymphoma, said lung cancer being selected from the group consisting of: non-small cell lung cancer and small cell lung cancer, said leukemia being selected from the group consisting of: chronic myeloid leukemia, acute myeloid leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, and myeloid leukemia; in other embodiments, the disease is selected from: PD-L1 positive melanoma, lung cancer, non-small cell lung cancer, breast cancer, gastric cancer, renal cancer, bladder cancer, intestinal cancer and colon cancer.

In some embodiments, the disclosure also provides the use of the aforementioned bispecific antigen binding molecules, or the aforementioned pharmaceutical compositions, or the aforementioned nucleic acid molecules in the preparation of a medicament for the treatment or prevention of a disease.

In some embodiments, the disease is a tumor.

In some embodiments, the disclosure also provides a bispecific antigen binding molecule of any one of the preceding claims, or a nucleic acid molecule of the preceding, or a pharmaceutical composition of the preceding, for use as a medicament.

In some embodiments, the medicament is for treating or preventing a disease associated with PD-1.

In some embodiments, the disease is a tumor.

The bispecific antigen binding molecules provided by the present disclosure have the characteristics of therapeutic activity, safety, pharmacokinetic properties, and good pharmaceutical properties (e.g., stability).

Drawings

Fig. 1: anti-PD-1 antibodies block the binding assay results of PD-1 to its ligand;

fig. 2: effect of anti-PD-1 antibodies on ifnγ secretion by PBMC cells;

fig. 3: therapeutic effect of anti-PD-1 antibody on mouse colon cancer MC38 engraftment;

fig. 4: effect of anti-PD-1 antibodies on mouse colon cancer MC38 tumor volume;

fig. 5: bispecific antibodies and antibody fusion proteins block PD-1/PD-L1 ligands;

fig. 6: fig. 6A: the effect of bispecific antibodies and antibody fusion proteins on ifnγ secretion by PBMC cells (donor 1); fig. 6B: the effect of bispecific antibodies and antibody fusion proteins on ifnγ secretion by PBMC cells (donor 2); fig. 6C: the effect of bispecific antibodies and antibody fusion proteins on ifnγ secretion by PBMC cells (donor 3); fig. 6D: the effect of bispecific antibodies and antibody fusion proteins on ifnγ secretion by PBMC cells (donor 4);

fig. 7: fig. 7A to 7F: bispecific antibodies and antibody fusion proteins inhibit VEGFA-induced HUVEC cell phosphorylation;

fig. 8: fig. 8A: the anti-tumor curative effect of the fusion protein in a mouse MDA-MB-231 model reconstructed by human PBMC; fig. 8B: the anti-tumor curative effect of the fusion protein in a mouse MDA-MB-231 model reconstructed by human PBMC;

Fig. 9: fig. 9A: a schematic structure of DVD-GS8, DVD-GS10, DVD-H19L14, DVD-H21L14, DVD-GS19, DVD-H24L19, 4-4-DVD; fig. 9B: the structural schematic diagrams of the IgG-GS10-scFv, the IgG-GS13-scFv and the IgG-GS 19-scFv; fig. 9C: structural schematic diagrams of Hu23-2TE, hu23-3ST, hu23-5LK, 3-6, 3-12, 6-37, hu33-V6, hu23-V6, hu33-WT and Hu 23-WT; fig. 9D: schematic structural diagrams of #2, #3 and # 5.

Detailed Description

Terminology

For easier understanding of the present disclosure, certain technical and scientific terms are specifically defined below. Unless defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

The terms "programmed death 1", "programmed cell death 1", "protein PD-1", "PDCD1" and "hPD-1" are used interchangeably and include variants, isoforms, species homologs of human PD-1, and analogs having at least one epitope in common with PD-1. The complete PD-1 sequence is found in GenBank accession U64863.

The term "programmed death ligand-1 (PD-L1)" is one of the two cell surface glycoprotein ligands of PD-1 (the other is PD-L2), which down-regulates T cell activation and cytokine secretion upon binding to PD-1. The term "PD-L1" as used herein includes human PD-L1 (hPD-L1), variants, isoforms, and interspecies homologs of hPD-L1, as well as 5 analogs having at least one epitope in common with hPD-L1. The complete hPD-L1 sequence is shown in GenBank accession number Q9 NZQ.

The term "vascular endothelial growth factor" or "VEGF" is used interchangeably and also includes known isoforms, species homologs, truncated forms, mutants and the like. Herein, VEGF may refer to VEGFA.

The amino acid three-letter codes and one-letter codes used in the present disclosure are as described in j.biol. Chem,243, p3558 (1968).

An "antigen binding molecule" as described in the present disclosure refers herein in the broadest sense to a molecule that specifically binds an antigen, examples of which are antibodies, antibody fragments, antibody fusion proteins or fusion proteins. Illustratively, bispecific antigen binding molecules herein include bispecific antibodies and antibody fusion proteins. Illustratively, bispecific antibodies and antibody fusion proteins herein may comprise a first chain and a second chain. The first chain may be a first polypeptide chain and the second chain may be a second polypeptide chain. The first polypeptide chain may be a heavy chain of an antibody or a polypeptide comprising a heavy chain of an antibody and the second polypeptide chain may be a light chain of an antibody or a polypeptide comprising a light chain of an antibody. Illustratively, the bispecific antibodies or antibody fusion proteins herein have the basic structure of a full length antibody. Illustratively, a bispecific antibody or antibody fusion protein herein is a tetrapeptide chain structure formed by two first polypeptide chains and two second polypeptide chains connected by interchain disulfide bonds.

The term "first antigen binding domain capable of specifically binding to PD-1" or "second antigen binding domain capable of specifically binding to VEGF" as used in the present disclosure refers to an antigen binding molecule comprising a region that specifically binds to all or part of PD-1 or VEGF. For example, the antigen binding domain may comprise one or more antibody variable regions or proteins. In particular, an antigen binding domain capable of specifically binding an antigen comprises an antibody light chain variable region and an antibody heavy chain variable region, which may form scFv, fab, or like configurations; alternatively, the antigen binding domain capable of specifically binding to an antigen comprises a protein capable of specifically binding to an antigen, such as VEGFtrap or a variant thereof.

The term "antibody" as used herein is used in its broadest sense and includes different antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, murine antibodies, chimeric antibodies, humanized antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity and specificity.

The term "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of the intact antibody that specifically binds to an antigen to which the intact antibody specifically binds. Examples of antibody fragments include, but are not limited to Fv, fab, fab ', fab ' -SH, F (ab ') ₂ Diabodies, linear antibodies, single chain antibody molecules (e.g., scFv or scFab), single domain antibodies (dAbs), and multispecific antibodies formed from antibody fragments.

"bispecific antibody" refers to an antibody (including an antibody or antigen-binding fragment thereof, such as a single chain antibody) that is capable of specifically binding to two different antigens or two different epitopes of the same antigen. Bispecific antibodies of various structures have been disclosed in the prior art, which can be classified into IgG-like bispecific antibodies and antibody fragment type bispecific antibodies according to the integrity of IgG molecules, bivalent, trivalent, tetravalent or more multivalent bispecific antibodies according to the number of antigen binding regions, and symmetrical structure bispecific antibodies and asymmetrical structure bispecific antibodies according to whether the structures are symmetrical or not. Among them, bispecific antibodies based on antibody fragments, such as Fab fragments lacking Fc fragments, are formed by binding 2 or more Fab fragments in one molecule, have low immunogenicity, have small molecular weight, have high tumor tissue permeability, and typical antibody structures of this type such as F (ab) 2, scFv-Fab, (scFv) 2-Fab, and the like; igG-like bispecific antibodies (e.g., having Fc fragments) of relatively large molecular weight, which facilitate later purification of the antibody and increase its solubility, stability, and which may also bind to the receptor FcRn, increasing antibody serum half-life, typical bispecific antibody structural models such as KiH, crossMAb, triomab quadrama, fcΔAdp, ART-Ig, biMAb, biclonics, BEAT, duoBody, azymetric, xmAb, 2:1TCBs, 1Fab-IgG TDB, fynomAb, two-in-one/DAF, scFv-Fab-IgG, DART-Fc, LP-DART, CODV-Fab-stability, E-BiTE, F (Ab) 2-CrossMAb, igG- (scFv) 2, bs4, DVD-Ig, tetravalent-DART-Fc, (scFv) 4-Fc, CODV-Ig, mAb2, F (Ab) 4-CrossMAb and the like bispecific antibodies (see Aran F. Nature Reviews Drug Discovery volume, lab.18-608; immunol 2019; thereby, J20141; 2019).

The terms "monovalent", "bivalent", "trivalent" or "multivalent" refer to the presence of a specified number of antigen binding sites in an antibody or polypeptide complex. For example, "monovalent antibody" means that an antigen binding site is present in the antibody, and "monovalent polypeptide complex" means that an antigen binding site is present in the polypeptide complex; "bivalent antibody" means that there are two antigen binding sites in the antibody, and "bivalent polypeptide complex" means that there are two antigen binding sites in the polypeptide complex; "trivalent antibody" means that there are three antigen binding sites in the antibody, and "trivalent polypeptide complex" means that there are three antigen binding sites in the polypeptide complex; "multivalent antibody" means that there are multiple (three or more) antigen binding sites in the antibody, and "multivalent polypeptide complex" means that there are multiple (two or more) antigen binding sites in the polypeptide complex.

The term "antibody fusion protein" refers to a fusion protein having biological activity formed by linking a protein (polypeptide) of interest to an antibody, said fusion protein having the biological activity of the linked protein as well as the immunoglobulin activity.

The "chimeric antibody (chimeric antibody)" described in the present disclosure is an antibody in which a variable region of a murine antibody and a constant region of a human antibody are fused, and can reduce an immune response induced by the murine antibody.

The term "humanized antibody (humanized antibody)" referred to herein, also known as CDR-grafted antibody (CDR-grafted antibody), refers to an antibody produced by grafting murine CDR sequences into the variable region framework of a human antibody, i.e., the framework sequences of a different type of human germline antibody. The heterologous reaction induced by chimeric antibodies due to the large amount of murine protein components can be overcome. To avoid a decrease in immunogenicity while at the same time causing a decrease in activity, the human antibody variable region framework sequences may be subjected to minimal reverse or back-mutations to maintain or enhance activity. Humanized antibodies of the present disclosure also include humanized antibodies that are further subjected to affinity maturation mutations of the CDRs by yeast display.

"human antibody" (HuMAb), "human antibody," "fully human antibody," are used interchangeably in this disclosure, and the amino acid sequence corresponds to that of an antibody produced by a human or human cell, or is derived from a non-human source using a repertoire of human antibodies or other human antibody coding sequences. The definition of human antibodies expressly excludes humanized antibodies that comprise non-human antigen binding residues.

An "antibody fragment" as described in the present disclosure refers to a molecule other than an intact antibody that comprises a portion of the intact antibody that specifically binds to an antigen to which the intact antibody specifically binds. Examples of antibody fragments include, but are not limited to Fv, fab, fab ', fab ' -SH, F (ab ') ₂ Diabodies, linear antibodies, single chain antibody molecules (e.g., scFv or scFab), single domain antibodies (dAbs), and multispecific antibodies formed from antibody fragments. The "antibodies" of the present disclosure include "whole antibodies" and "antibody fragments" thereof. Accordingly, the heavy chain of an antibody or the light chain of an antibody described in the present disclosure comprises a complete heavy chain or a complete light chain, as well as heavy chain fragments or light chain fragments of an antibody fragment.

The term "variable region" refers to the domains of the antibody heavy and light chains that are involved in antigen binding. In natural antibodies, the heavy chain variable region and the light chain variable region together form an antigen binding domain. Herein, the heavy chain variable region PD-1-VH refers to the heavy chain variable region of an anti-PD-1 antibody, the light chain variable region PD-1-VL refers to the light chain variable region of an anti-PD-1 antibody, the heavy chain variable region VEGF-VH refers to the heavy chain variable region of an anti-VEGF antibody, and the light chain variable region VEGF-VL refers to the light chain variable region of an anti-VEGF antibody.

The term "complementarity determining region", "CDR" or "hypervariable region" refers to one of the 6 hypervariable regions within the variable region of an antibody that contribute primarily to antigen binding. Typically, there are three CDRs (HCDR 1, HCDR2, HCDR 3) in each heavy chain variable region, and three CDRs (LCDR 1, LCDR2, LCDR 3) in each light chain variable region. The amino acid sequence boundaries of the CDRs can be determined using any of a variety of well-known protocols, including "Kabat" numbering convention (see Kabat et Al (1991), "Sequences of Proteins of Immunological Interest", 5 th edition, public Health Service, national Institutes of Health, bethesda, MD), "Chothia" numbering convention (see Al-Lazikani et Al, (1997) JMB 273:927-948), and ImMunoGenTics (IMGT) numbering convention (Lefranc m.p., immunology, 7, 132-136 (1999); lefranc, M.P., et Al, dev.Comp.Immunol.,27, 55-77 (2003) et Al, for example, for classical format, the CDR amino acid residues in the heavy chain variable region (VH) are numbered 31-35 (HCDR 1), 50-65 (HCDR 2) and 95-102 (HCDR 3), the CDR amino acid residues in the light chain variable region (VL) are numbered 24-34 (LCDR 1), 50-56 (LCDR 2) and 89-97 (LCDR 3) following the Chothia rule, the CDR amino acid residues in the VH are numbered 26-32 (HCDR 1), 52-56 (HCDR 2) and 95-102 (HCDR 3), and the amino acid residues in the VL are numbered 26-32 (LCDR 1), 50-52 (LCDR 2) and 91-96 (LCDR 3) are defined by combining CDRs of both bat and Chothia, the CDR amino acid residues in the CDR are numbered 26-32 (HCDR 1), 52-56 (HCDR 2) and 95-102 (HCDR 3) by amino acid residues in human being numbered 26-32 (HCDR 1), 50-52 (HCDR 2) and 95-35 (HCDR 3) 50-56 (LCDR 2) and 89-97 (LCDR 3). Following the IMGT rules, the CDR amino acid residues in VH are approximately 26-35 (CDR 1), 51-57 (CDR 2) and 93-102 (CDR 3), and the CDR amino acid residues in VL are approximately 27-32 (CDR 1), 50-52 (CDR 2) and 89-97 (CDR 3). Following IMGT rules, CDR regions of antibodies can be determined using the procedure IMGT/DomainGap alignment. Unless otherwise indicated, the antibody variable region and CDR sequences referred to in the examples of the present disclosure are all subject to the "Kabat" numbering convention. Herein, PD-1-HCDR1, PD-1-HCDR2, PD-1-HCDR3, PD-1-LCDR1, PD-1-LCDR2PD-1-LCDR3 refer to CDR combinations of anti-PD-1 antibodies; VEGF-HCDR1, VEGF-HCDR2, VEGF-HCDR3, VEGF-LCDR1, VEGF-LCDR2 VEGF-LCDR3 refer to the CDR combinations of anti-VEGF antibodies.

The term "framework" or "FR" refers to variable region residues other than Complementarity Determining Region (CDR) residues. The FR of the variable region generally consists of four FR domains: FR1, FR2, FR3 and FR4. Thus, CDR and FR sequences generally occur in VH (or VL) in the following order:

FR1-HCDR1(LCDR1)-FR2-HCDR2(LCDR2)-FR3-HCDR3(LCDR3)-FR4

the "constant region" of the present disclosure includes a light chain constant region of a kappa, lambda or variant thereof of human or murine origin, or a heavy chain constant region of a human or murine IgG1, igG2, igG3, igG4 or variant thereof. The constant regions of the present disclosure also include "conventional variants" of the human antibody heavy chain constant region and human antibody light chain constant region, which refer to variants of human-derived heavy chain constant region or light chain constant region that have been disclosed in the prior art that do not alter the structure and function of the antibody variable region, exemplary variants include IgG1, igG2, igG3, or IgG4 heavy chain constant region variants that are site-directed engineered and amino acid substituted for the heavy chain constant region, specific substitutions such as YTE mutations, L234A and/or L235A mutations, S228P mutations, and/or mutations that result in knob-into-hole structures (such that the antibody heavy chain has a knob-Fc and hole-Fc combination), which have been demonstrated to confer novel properties to the antibody, but do not alter the function of the antibody variable region. In one embodiment, the constant region comprises a human IgG1, igG2, or IgG4 heavy chain constant region, or an IgG1, igG2, or IgG4 variant using amino acid mutations (e.g., L234A and/or L235A mutations, and/or S228P mutations).

The terms "specific binding," "selective binding," "selectively binding," and "specifically binding" refer to binding of an antibody to an epitope on a predetermined antigen. Typically, the antibody is present at about less than 10 ^-8 M, e.g. less than about 10 ^-9 M、10 ^- ¹⁰ M、10 ^-11 M、10 ^-12 Affinity (KD) binding of M or less.

The term "KD" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. Typically, antibodies of the disclosure are administered at a ratio of less than about 10 ^-7 M, e.g. less than about 10 ^-8 M、10 ^-9 M、10 ^-10 M、10 ^-11 M、10 ^-12 The dissociation equilibrium constant (KD) of M binds to an antigen, e.g., as determined in a BIACORE instrument using Surface Plasmon Resonance (SPR) techniques.

The term "amino acid difference" or "amino acid mutation" refers to an alteration or mutation of an amino acid in a variant protein or polypeptide as compared to the original protein or polypeptide, including an insertion, deletion or substitution of 1, 2, 3 or more amino acids on the basis of the original protein or polypeptide.

The term "linker" refers to a linking unit that links two polypeptide fragments. In this context, the linkers present in the same structure may be the same or different. The linker may be a peptide linker comprising one or more amino acids, typically about 1-30, 2-24 or 8-24 amino acids. The linkers used herein may be the same or different.

When the term "bond" appears in a structural element, e.g. L ₁ Or L ₂ In (c), it means that the unit has no amino acid and that the units on both sides of the unit are directly linked.

When the term "compete" is used in the context of antigen binding proteins (e.g., neutralizing antigen binding proteins or neutralizing antibodies) that compete for the same epitope, it is meant that competition is between antigen binding proteins, as determined by the following assay: in such assays, the antigen binding protein (e.g., antibody or immunologically functional fragment thereof) to be detected prevents or inhibits (e.g., reduces) specific binding of the reference antigen binding protein (e.g., ligand or reference antibody) to a cognate antigen (e.g., PD-1 antigen or fragment thereof or VEGF antigen or fragment thereof). Numerous types of competitive binding assays can be used to determine whether one antigen binding protein competes with another, such as: solid phase direct or indirect Radioimmunoassay (RIA), solid phase direct or indirect Enzyme Immunoassay (EIA), sandwich competition assay (see, e.g., stahli et al, 1983,Methodsin Enzymology 9:242-253); solid phase direct biotin-avidin EIA (see, e.g., kirkland et al, 1986, J.Immunol.137:3614-3619), solid phase direct labeling assay, solid phase direct labeling sandwich assay (see, e.g., harlow and Lane,1988,Antibodies,A Laboratory Manual (antibodies, laboratory Manual), cold Spring Harbor Press); direct labelling of RIA with the solid phase of the I-125 label (see, e.g., morel et al, 1988, molecular. Immunol. 25:7-15); solid phase direct biotin-avidin EIA (see, e.g., cheung, et al, 1990, virology 176:546-552); and directly labeled RIA (Moldenhauer et al, 1990, scand. J. Immunol. 32:77-82). Typically the assay involves the use of purified antigen that binds to a solid surface or cell bearing either the unlabeled detection antigen binding protein or the labeled reference antigen binding protein. Competitive inhibition is measured by measuring the amount of label bound to a solid surface or cell in the presence of the antigen binding protein being measured. Typically the antigen binding protein to be tested is present in excess. Antigen binding proteins identified by competition assays (competing antigen binding proteins) include: an antigen binding protein that binds to the same epitope as the reference antigen binding protein; and an antigen binding protein that binds to an epitope in close proximity to the binding epitope of the reference antigen binding protein, the two epitopes spatially preventing binding from occurring. Additional details regarding methods for assaying competitive binding are provided in the examples herein. Typically, when the competing antigen binding protein is present in excess, it will inhibit (e.g., reduce) at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or more of the specific binding of the reference antigen binding protein to the cognate antigen. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

The term "nucleic acid molecule" as used herein refers to DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, preferably double-stranded DNA or single-stranded mRNA or modified mRNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one embodiment, the vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. In another embodiment, the vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. The vectors disclosed herein are capable of autonomous replication in a host cell into which they have been introduced (e.g., bacterial vectors and episomal mammalian vectors having a bacterial origin of replication) or may integrate into the genome of a host cell upon introduction into the host cell so as to replicate with the host genome (e.g., non-episomal mammalian vectors).

Methods for producing and purifying antibodies and antigen binding fragments are well known in the art, such as the guidelines for antibody experimentation in Cold spring harbor, chapters 5-8 and 15. For example, mice may be immunized with human PD-1 or a fragment thereof, the resulting antibodies may be renatured, purified, and amino acid sequenced using conventional procedures. Antigen binding fragments can likewise be prepared by conventional methods. The antibodies or antigen binding fragments of the invention are engineered to incorporate one or more human FR regions in the CDR regions of non-human origin. Human FR germline sequences can be obtained from ImMunoGeneTics (IMGT) website http:// IMGT. Cines. FR, or from the journal of immunoglobulins, 2001ISBN012441351 by aligning IMGT human antibody variable region germline gene databases with MOE (Molecular Operation Environment) software.

The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells may include bacterial, microbial, plant or animal cells. Bacteria that are susceptible to transformation include members of the Enterobacteriaceae family, such as strains of Escherichia coli (Escherichia coli) or Salmonella (Salmonella); the family of bacillus (bacillus) such as bacillus subtilis (Bacillus subtilis); pneumococci (pneumococci); streptococcus (Streptococcus) and haemophilus influenzae (Haemophilus influenzae). Suitable microorganisms include Saccharomyces cerevisiae (Saccharomyces cerevisiae) and Pichia pastoris (Pichia pastoris). Suitable animal host cell lines include CHO (chinese hamster ovary cell line) and NS0 cells.

The engineered antibodies or antigen binding fragments or bispecific antibodies or antibody fusion proteins of the disclosure can be prepared and purified using conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into GS expression vectors. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As a more recommended prior art, mammalian expression systems can lead to glycosylation of the antibody, particularly at the highly conserved N-terminal site of the Fc region. The means may include: stable clones were obtained and positive clones were grown up in serum-free medium in a bioreactor to produce antibodies. The antibody-secreting culture may be purified using conventional techniques. For example, purification is performed using an A or G Sepharose FF column containing conditioned buffer. Non-specifically bound components are washed away. The bound antibody was eluted by a pH gradient method, and the antibody fragment was detected by SDS-PAGE and collected. The antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange. The resulting product is either immediately frozen, e.g., -70 ℃, or lyophilized.

"administering," "administering," and "treating," when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to the contact of an exogenous drug, therapeutic, diagnostic, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "administration," "administration," and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of a cell includes contacting a reagent with the cell, and contacting the reagent with a fluid, wherein the fluid is in contact with the cell. "administration," "administration," and "treatment" also mean in vitro and ex vivo treatment of, for example, a cell by an agent, diagnosis, binding composition, or by another cell. "treatment" when applied to a human, veterinary or research subject refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

By "treating" is meant administering to a patient an internal or external therapeutic agent, such as a composition comprising any of the binding compounds of the present disclosure, that has one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the patient or population being treated to induce regression of such symptoms or to inhibit the development of such symptoms to any clinically measurable extent. The amount of therapeutic agent (also referred to as a "therapeutically effective amount") effective to alleviate any particular disease symptom can vary depending on a variety of factors, such as the disease state, age, and weight of the patient, and the ability of the drug to produce a desired therapeutic effect in the patient. Whether a disease symptom has been reduced can be assessed by any clinical test method that a physician or other healthcare professional typically uses to assess the severity or progression of the symptom. While embodiments of the present disclosure (e.g., therapeutic methods or articles of manufacture) may be ineffective in alleviating each target disease symptom, it should be determined according to any statistical test method known in the art, such as Student t-test, chi-square test, U-test according to Mann and Whitney, kruskal-Wallis test (H test), jonckheere-Terpstra test, and Wilcoxon test, that the target disease symptom should be alleviated in a statistically significant number of patients.

"conservative modifications" or "conservative substitutions or substitutions" refer to amino acids in other amino acid substituted proteins that have similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation, and rigidity, etc.) such that changes can be made frequently without altering the biological activity of the protein. Those skilled in The art know that in general, single amino acid substitutions in The non-essential region of a polypeptide do not substantially alter biological activity (see, e.g., watson et al (1987) Molecular Biology of The Gene, the Benjamin/Cummings pub. Co., page 224, (4 th edition)). In addition, substitution of structurally or functionally similar amino acids is unlikely to disrupt biological activity. Exemplary conservative substitutions are set forth in the following table "exemplary amino acid conservative substitutions".

TABLE 3 exemplary amino acid conservative substitutions

Original residue	Conservative substitutions
		Ala(A)	Gly；Ser
Arg(R)	Lys；His
		Asn(N)	Gln；His；Asp
Asp(D)	Glu；Asn
		Cys(C)	Ser；Ala；Val
Gln(Q)	Asn；Glu
		Glu(E)	Asp；Gln
Gly(G)	Ala
		His(H)	Asn；Gln
Ile(I)	Leu；Val
		Leu(L)	Ile；Val
Lys(K)	Arg；His
		Met(M)	Leu；Ile；Tyr
Phe(F)	Tyr；Met；Leu
		Pro(P)	Ala
Ser(S)	Thr
		Thr(T)	Ser
Trp(W)	Tyr；Phe
		Tyr(Y)	Trp；Phe
Val(V)	Ile；Leu

An "effective amount" or "effective dose" refers to the amount of a drug, compound, or pharmaceutical composition necessary to achieve any one or more beneficial or desired therapeutic results. For prophylactic use, beneficial or desired results include elimination or reduction of risk, lessening the severity, or delaying the onset of a disorder, including biochemical, histological and/or behavioral symptoms of the disorder, its complications, and intermediate pathological phenotypes that are exhibited during the development of the disorder. For therapeutic applications, beneficial or desired results include clinical results, such as reducing the incidence of or ameliorating one or more symptoms of various target antigen-related disorders of the present disclosure, reducing the dosage of other agents required to treat a disorder, enhancing the efficacy of another agent, and/or slowing the progression of a target antigen-related disorder of the present disclosure in a patient.

"exogenous" refers to substances produced outside of an organism, cell or human body as the case may be. "endogenous" refers to substances produced in cells, organisms or humans, as the case may be.

"homology" refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in both comparison sequences is occupied by the same base or amino acid monomer subunit, for example if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matched or homologous positions shared by the two sequences divided by the number of compared positions by 100. For example, when sequences are optimally aligned, if there are 6 matches or homologies at 10 positions in the two sequences, then the two sequences are 60% homologous; if there are 95 matches or homologies at 100 positions in the two sequences, then the two sequences are 95% homologous. Typically, the comparison is made when two sequences are aligned to give the greatest percent homology. For example, the comparison may be performed by the BLAST algorithm, wherein the parameters of the algorithm are selected to give a maximum match between the respective sequences over the entire length of the respective reference sequences. The following references relate to BLAST algorithms that are often used for sequence analysis: BLAST algorithm (BLAST ALGORITHMS) Altschul, S.F. et al, (1990) J.mol.biol.215:403-410; gish, W.et al, (1993) Nature Genet.3:266-272; madden, T.L. et al, (1996) meth.enzymol.266:131-141; altschul, S.F. et al, (1997) Nucleic Acids Res.25:3389-3402; zhang, J. Et al, (1997) Genome Res.7:649-656. Other conventional BLAST algorithms, such as those provided by NCBI BLAST, are also known to those skilled in the art.

The expressions "cell", "cell line" and "cell culture" are used interchangeably herein and all such designations include progeny. Thus, the words "transformant" and "transformed cell" include primary test cells and cultures derived therefrom, regardless of the number of transfers. It should also be understood that all offspring may not be exactly identical in terms of DNA content due to deliberate or unintentional mutations. Including mutant progeny having the same function or biological activity as screened in the original transformed cell. Where different names are meant, they are clearly visible from the context.

As used herein, "polymerase chain reaction" or "PCR" refers to a procedure or technique in which minute amounts of nucleic acids, RNA and/or DNA of a particular moiety are amplified as described, for example, in U.S. Pat. No. 4,683,195. In general, it is necessary to obtain sequence information from the end of the target region or beyond so that oligonucleotide primers can be designed; these primers are identical or similar in sequence to the corresponding strands of the template to be amplified. The 5' -terminal nucleotides of the 2 primers may correspond to the ends of the material to be amplified. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA, phage or plasmid sequences transcribed from total cellular RNA, etc. See generally Mullis et al (1987) Cold Spring Harbor Symp. Uant. Biol.51:263; erlich editions, (1989) PCR TECHNOLOGY (stock Press, n.y.). PCR as used herein is considered an example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, which includes using known nucleic acids and nucleic acid polymerases as primers to amplify or generate specific portions of the nucleic acid.

"isolated" refers to a purified state and in this case means that the specified molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates or other materials, such as cell debris and growth media. In general, the term "isolated" is not intended to refer to the complete absence of such materials or the absence of water, buffers, or salts, unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the compounds as described herein.

"optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable regions of a particular sequence may be, but need not be, present.

"pharmaceutical composition" means a mixture comprising one or more of the compounds described herein, which may be antigen binding molecules, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, such as physiological/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to organisms, facilitate the absorption of active ingredients and thus exert biological activity.

The term "pharmaceutically acceptable carrier" refers to any inactive substance suitable for use in a formulation for delivery of an antibody or antigen-binding fragment. The carrier may be an anti-adherent, binder, coating, disintegrant, filler or diluent, preservative (e.g., antioxidant, antimicrobial or antifungal), sweetener, absorption delaying agent, wetting agent, emulsifier, buffer, etc. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) dextrose, vegetable oils (e.g., olive oil), saline, buffers, buffered saline, and isotonic agents, such as sugars, polyols, sorbitol, and sodium chloride.

In addition, the present disclosure includes agents for treating diseases associated with an antigen of interest (e.g., PD-1) positive cell, the agents comprising an anti-PD-1 antibody or antigen-binding fragment thereof of the present disclosure as an active ingredient.

The disease associated with PD-1 or VEGF in the present disclosure is not limited so long as it is a disease associated with PD-1 or VEGF, e.g., a therapeutic response induced by a molecule of the present disclosure may be produced by binding to human PD-1 and then blocking the binding of PD-1 to its ligand PD-L1, PD-L2, or killing tumor cells that overexpress PD-1. Thus, the molecules of the present disclosure are very useful for those suffering from tumors or cancers, preferably melanoma, colon, breast, lung, stomach, intestinal, kidney, non-small cell lung, bladder, and the like, when in preparations and formulations suitable for therapeutic applications.

Furthermore, the present disclosure relates to methods for immunodetection or assay of an antigen of interest (e.g., PD-1 or VEGF), reagents for immunodetection or assay of an antigen of interest (e.g., PD-1 or VEGF), methods for immunodetection or assay of cells expressing an antigen of interest (e.g., PD-1 or VEGF), and diagnostic agents for diagnosis of diseases associated with antigen of interest (e.g., PD-1 or VEGF) positive cells, comprising as an active ingredient an antibody or antibody fragment of the present disclosure that specifically recognizes an antigen of interest (e.g., human PD-1 or VEGF) and binds to the amino acid sequence of the extracellular region or three-dimensional structure thereof.

In the present disclosure, the method for detecting or determining the amount of an antigen of interest (e.g., PD-1 or VEGF) may be any known method. For example, it includes immunological detection or assay methods.

The immunodetection or assay method is a method of detecting or assaying the amount of an antibody or an antigen using a labeled antigen or antibody. Examples of immunodetection or assay methods include radio-labeled immune antibody methods (RIA), enzyme immunoassays (EIA or ELISA), fluorescent Immunoassays (FIA), luminescent immunoassays, western immunoblotting, physicochemical methods, and the like.

For detecting the cells expressing the polypeptide, a known immunodetection method may be used, and immunoprecipitation, fluorescent cell staining, immunohistological staining, and the like are preferably used. Further, a fluorescent antibody staining method using FMAT8100HTS system (Applied Biosystem) or the like can be used.

In the present disclosure, a living sample for detecting or assaying an antigen of interest (e.g., PD-1 or VEGF) is not particularly limited as long as it has a possibility of containing cells expressing the antigen of interest (e.g., PD-1 or VEGF), such as tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, interstitial fluid, or culture fluid.

Diagnostic agents comprising monoclonal antibodies or antibody fragments thereof of the present disclosure may also contain reagents for performing antigen-antibody reactions or reagents for detecting reactions, depending on the desired diagnostic method. Reagents for performing the antigen-antibody reaction include buffers, salts, and the like. Reagents for detection include reagents commonly used in immunological detection or assay methods, such as a labeled secondary antibody that recognizes the monoclonal antibody, an antibody fragment thereof, or a conjugate thereof, a substrate corresponding to the label, and the like.

Preparation of PD-1 antigen

1. Antigen construction:

the fusion protein of human PD-1-IgG1Fc is designed and synthesized, wherein the N end is 150 amino acids of the extracellular region of human PD-1, and the C end is the Fc segment of human IgG1 (hIgG 1 Fc). The recombinant PD-1-Fc Protein with high purity can be obtained by purifying the Protein A by an affinity column and is used for detecting the combination of an anti-PD-1 antibody and an antigen.

Human PD-1-IgG1Fc (SEQ ID NO: 1):

annotation: the underlined part is the signal peptide, the orthotopic part is the human PD-1 extracellular domain, and the italic part is hIgG1Fc (signal peptide+extracellular domain+hIgG 1 Fc).

Human PD-1-his (SEQ ID NO: 2):

MEFGLSWLFLVAILKGVQCPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVGSSDYKDDDDKHHHHHH。

PD-1 antigen encoded by PD-1 nucleic acid of transfected cells (SEQ ID NO: 3):

MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL。

preparation of PD-1 antibodies

Anti-human PD-1 antibodies can be produced by immunizing mice, or can be obtained from an anti-human PD-1 phage mouse immune library.

The method for preparing anti-human PD-1 antibodies by immunizing mice is as follows:

1. immunization: the experimental SJL white mice, females, 6-8 weeks old and Balb/c white mice, females, 6-8 weeks old. Feeding environment: SPF stage. After the mice are purchased, the mice are fed in a laboratory environment for 1 week, the light/dark period is regulated for 12/12 hours, and the temperature is 20-25 ℃; humidity is 40-60%. The mice which are adapted to the environment are immunized according to different schemes, and 6-10 mice are used in each group. The immune antigen can be purified recombinant protein PD-1-IgG1Fc (see SEQ ID NO: 1), PD-1-his (see SEQ ID NO: 2), or PD-1 as antigen (see SEQ ID NO: 3) transfected Jurkat/CHO-PD-1 cells, can use a single antigen to match with different immune adjuvants or different types of immunogens for cross immunization. The immune site may be The abdominal cavity or the back is subcutaneously or alternatively immunized in both positions. ImmunoadjuvantGold Adjuvant (hereinafter Titermax, available from Sigma cat# T2684) was cross-immunized with Imject Alum Adjuvant (hereinafter Alum, available from Pierce cat# 77161). Antigen to adjuvant (Titermax) ratio of 1:1, antigen to adjuvant (Alum) ratio of 3:1, 25-50 μg/(priming), 50 μg/(boosting), or 1×10 ⁷ Jurkat/CHO-PD-1 cells/cell. Day 0 intraperitoneal injection of 25-50 μg/dose of emulsified antigen, once weekly or once every two weeks after the first immunization, alternating with Titermax and Alum for 5-8 times.

2. Cell fusion: mice with high antibody titer in serum are selected for spleen cell fusion, and the eyeballs of the mice after 72 hours of sprint immunization are exsanguinated, and the mice are killed by neck pulling and put into 75% ethanol for disinfection. The spleen lymphocytes and myeloma cells Sp2/0 cells (China academy of sciences) are fused by adopting an optimized PEG-mediated fusion step to obtain hybridoma cells. The fused hybridoma cells were resuspended in HAT complete medium (RPMI-1640 medium containing 20% FBS, 1 XHAT and 1 XOPI) and plated in 96-well cell culture plates (1X 10) ⁵ 150 μl/well), 37℃and 5% CO ₂ Incubating, and obtaining about 10-30 plates. HAT complete medium, 50. Mu.l/well, 37℃and 5% CO were added on day 5 after fusion ₂ And (5) incubating. From day 7 to day 8 after fusion, according to cell growth density, the whole solution was changed to 200. Mu.l/well, 37℃at 5% CO ₂ And (5) incubating.

3. Hybridoma cell selection: and (3) on the 7 th to 9 th days after fusion, ELISA detection of the combination of the antibody and the PD-1 is carried out according to the cell growth density, the detected positive hole cells are subjected to blocking ELISA detection of the combination of the PD-1/PDL1, the positive hole is replaced with liquid, and the positive hole cells are timely expanded into 24 pore plates according to the cell density. The cell lines engrafted into 24-well plates were retested for seed protection and first subcloning. Seed conservation is carried out when the first subclone screening is positive, and the second or third subclone is carried out until single cell clone is obtained. And obtaining hybridoma cells with the effect of blocking the combination of PD-1 and PDL1 through multiple times of fusion.

The method for obtaining anti-human PD-1 antibodies from an anti-human PD-1 phage mouse immune library is as follows:

1. construction of an anti-human PD-1 phage mouse immune library: spleens of mice with high antibody titers in serum were selected and total tissue RNA was extracted with Trizol (Invitrogen Cat No. 15596-018). Using PrimeScript ^TM The II 1st Strand cDNA Synthesis Kit kit (Takara Cat No. 6210A) was subjected to reverse transcription to obtain cDNA. Primers for constructing the library were designed and synthesized according to IMGT database. Single-chain antibody fragments were obtained by three rounds of PCR reactions. The single-chain antibody fragment and the engineered library carrier pCantab5E (Amersham Biosciences/GE Cat No. 27-9400-01) were digested with Sfi1 (NEB Cat No. #R0123L) and subjected to electrophoresis Gel Extraction Kit (Omega Cat No. D2500-02) was recovered by purification. Then, the mixture was ligated with T4 DNA ligase (NEB Cat No. # M0202L) at 16℃for 16-18 hours, and then purified and recovered by the above-mentioned kit, and finally eluted with deionized water. 1. Mu.g of the ligation product was mixed with 1 branch of electrotransformation competent TG1 (Lucigen Cat No. 60502-2), and the parameters of an electrotransformation instrument (Bio Rad Micropulser) were set to 2.5kV,200Ω, 25. Mu.F, and electrotransformation was performed. The transformation was repeated 10 times, plated, and incubated at 37℃for 16-18 hours in an inverted state. All colonies were scraped off and mixed together, glycerol was added at a final concentration of 15% and stored at-80℃until use.

2. Screening of anti-human PD-1 phage mouse immune library: the packaged anti-human PD-1 phage immune library (1X 10) ¹² -1×10 ¹³ ) With 100. Mu.l of streptavidin microbeads (Mi 1envi Biotec, auburn, calif.) 1m1 of 2% skim milk-phosphate buffer (abbreviated MPBS) were added and incubated for 1 hour at room temperature, and the supernatant was placed on a magnetic rack. The supernatant was incubated with 10. Mu.g/ml biotinylated human PD-1-ECD-his protein (from Sino Biological) for 1 hour at room temperature, and 100. Mu.l streptavidin-coated magnetic beads (1 ml MPBS pre-incubation) were added and incubated for 1 hour at room temperature. And loaded on a magnetic rack system for sorting, and the supernatant is sucked. 1ml of PBST (phosphate buffer solution containing 0.1% Tween-20) is added, and the mixture is turned over for several times and then added after the mixture is completely absorbed Fresh wash was added and repeated 11 times to remove unbound antibody fragments, 0.5ml of eluent (50. Mu.l 10mg/ml trypsin stock solution (stock) was added to 450. Mu.l PBS). Shake for 15min at room temperature. Placed on a magnetic rack and the supernatant aspirated into a new EP tube. TG1 was expanded to a bacterial density od600=0.4 in 2YT medium. 1.75ml of TG1 (OD 600 = 0.4) was added to each tube, and 250. Mu.l of post-elution phase (phage) was added, incubated for 30min in a 37℃water bath, and the plates were serially diluted for testing titers. The remaining TG1 solution was centrifuged, plated, and incubated overnight at 37 ℃.

Phage mouse immune library utilizes biotinylated human PD-1-ECD-his antigen, and through 2-3 rounds of MACS screening (streptomycin magnetic beads, invitrogen), monoclonal antibodies with the functions of binding PD-1 and blocking the binding of PD-1 and PD-L1 are finally obtained, and sequencing verification is carried out, so that the variable region sequence of the antibodies is obtained.

Purification of recombinant antigen proteins/antibodies

1. Hybridoma supernatant separation and purification/protein G affinity chromatography:

affinity chromatography is performed on the preferred ProteinG for purifying the mouse hybridoma supernatant, the cultured hybridoma is centrifuged to obtain the supernatant, and 1M Tris-HCl (pH 8.0-8.5) with the volume of 10-15% is added according to the volume of the supernatant to adjust the pH of the supernatant. Washing the ProteinG column with 6M guanidine hydrochloride for 3-5 times of column volume, and then washing with pure water for 3-5 times of column volume; the column is equilibrated for 3-5 column volumes using, for example, a 1 XPBS (pH 7.4) buffer system as an equilibration buffer; cell supernatants were combined using low flow loading, and the flow was controlled to allow retention time of about 1min or longer; washing the column with 1 XPBS (pH 7.4) for 3-5 times the column volume until the UV absorption falls back to baseline; sample elution was performed using 0.1M acetic acid/sodium acetate (pH 3.0) buffer, elution peaks were collected according to UV detection, and the eluted product was rapidly adjusted to pH 5-6 using 1M Tris-HCl (pH 8.0) for buffer storage. For the eluted product, solution displacement may be performed by methods well known to those skilled in the art, such as ultrafiltration concentration using an ultrafiltration tube and solution displacement to a desired buffer system, or desalting using a molecular exclusion column such as G-25 to a desired buffer system, or removing the polymer component in the eluted product using a high resolution molecular exclusion column such as Superdex 200 to increase the sample purity.

Protein a affinity chromatography purification of proteins or antibodies:

the supernatant is first harvested by high-speed centrifugation of cell culture supernatants expressing the antigen proteins or antibodies. The Protein A affinity column was washed 3-5 column volumes with 6M guanidine hydrochloride and then 3-5 column volumes with pure water. The column is equilibrated for 3-5 column volumes using, for example, a 1 XPBS (pH 7.4) buffer system as an equilibration buffer. Cell supernatants were loaded and bound using a low flow rate, the flow rate was controlled to allow a retention time of about 1min or longer, and after binding was completed the column was washed 3-5 column volumes with 1 XPBS (pH 7.4) until UV absorbance fell back to baseline. Sample elution is carried out by using 0.1M acetic acid/sodium acetate (pH 3.0-3.5) buffer solution, elution peaks are collected according to ultraviolet detection, and the eluting product is rapidly adjusted to pH 5-6 by using 1M Tris-HCl (pH 8.0) for temporary storage. For the eluted product, solution displacement may be performed by methods well known to those skilled in the art, such as ultrafiltration concentration using an ultrafiltration tube and solution displacement to a desired buffer system, or desalting using a molecular exclusion column such as G-25 to a desired buffer system, or removing the polymer component in the eluted product using a high resolution molecular exclusion column such as Superdex 200 to increase the sample purity.

Sequences of control antibodies or fusion proteins employed in the present disclosure

Lucentis：

Heavy chain amino acid sequence of Lucentis (SEQ ID NO: 83):

light chain amino acid sequence of Lucentis (SEQ ID NO: 84):

in the above-mentioned Lucentis antibody variable region sequence, the italic indicates a constant region, the underlined italic indicates a CDR sequence, and the sequence order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in order.

VEGFtrap-Fc(SEQ ID NO：93)：

SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

VP101

Heavy chain amino acid sequence of VP101 (SEQ ID NO: 101):

EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELK

the light chain amino acid sequence of VP101 (SEQ ID NO: 102):

DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECBy24.3

heavy chain amino acid sequence of By24.3 (SEQ ID NO: 103):

QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGSGGGGSGGGGSASDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK

light chain amino acid sequence of By24.3 (SEQ ID NO: 104):

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC embodiment

The present disclosure is further described below in connection with the examples, which are not intended to limit the scope of the present disclosure. Experimental methods for which specific conditions are not noted in the examples of the present disclosure are generally according to conventional conditions, such as an antibody technical laboratory manual for cold spring harbor, a molecular cloning manual; or according to the conditions recommended by the manufacturer of the raw materials or goods. The reagents of specific origin are not noted and are commercially available conventional reagents.

Example 1

Example 1-1 obtaining anti-human PD-1 murine antibody

The anti-human PD-1 murine antibody obtained by the method is subjected to an antigen binding experiment, and a plurality of antibodies with good activity are obtained by screening: including M23, M32 and M33, single cell clones were amplified, RNA was extracted, and reverse transcription amplification (RT-PCR) was performed using mouse-Ig degenerate primers to obtain the variable region sequences of the antibodies. The variable region sequence of the mouse antibody is connected with the constant region sequence of the human antibody, the chimeric antibody of the mouse monoclonal antibody is cloned and expressed in a recombination way, and an in vitro activity experiment is carried out to confirm that the variable region sequence of the obtained monoclonal antibody is correct.

The variable region sequences of murine antibodies M23, M32 and M33 were determined as follows:

heavy chain variable region of murine antibody M23 (SEQ ID NO: 4):

QVQLQQSGAELVRPGASVTLSCKASGYTFTDYEMHWVKQTPIHGLEWIGLIDPETGGTVYNQKFKDKTILTADKSSSTAYMEFRSLTSEDSAVYHCTRERFSYYGSTSDWYFDVWGTGTTVTVSS。

the light chain variable region of murine antibody M23 (SEQ ID NO: 5):

DGLMTQTPLSLPVSLGDHASISCRSSQSLVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGIYYCFQGSHVPYTFGGGTKLEIK。

heavy chain variable region of murine antibody M32 (SEQ ID NO: 6):

QVQLQQSGAELVRPGASVTLSCKASDFTFTDYEIHWVKQTPVHGLEWIGLFDPETGGIVYNQKFKGKAILTADKSSNTAYMEFRSLTSEDSAVYYCTREGYNRDWYFDVWGTGTTVTVSS。

the light chain variable region of murine antibody M32 (SEQ ID NO: 7):

DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGIYYCFQGSHVPYAFGGGTKLEIK。

heavy chain variable region of murine antibody M33: (SEQ ID NO: 19)

KVMLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPEKRLEWVATISGGGVDTYYQDNVQGRFTISRDNAKNTLYLQMSSLRSEDTALYYCASPYGHGYFDVWGTGTTVTVSS。

Light chain variable region of murine antibody M33: (SEQ ID NO: 20)

DIQMTQTTSSLSASLGDRVTISCRASQDINNFLNWYQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFGGGTKLEIK。

Remarks: in the heavy chain variable region and the light chain variable region sequences of the above antibody, CDR sequences determined by the Kabat numbering system are underlined, and FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 are in sequence.

TABLE 4 murine antibodies M23, M32 and M33 heavy and light chain CDR region sequences

Remarks: the antibody CDR sequences in the table are determined according to the Kabat numbering system.

Examples 1-2 humanization of anti-human PD-1 monoclonal antibodies

By comparing the germline gene database of the heavy and light chain variable regions of the IMGT human antibody and MOE software analysis, the germline genes of the heavy and light chain variable regions of the human germline with high sequence identity with the light and heavy chains of M23, M32 and M33 are respectively selected as templates, and the CDRs of the 3 murine antibodies are respectively transplanted into corresponding humanized antibody templates to respectively construct the corresponding humanized antibodies.

1. Humanized murine antibody M23

1.1 murine antibody M23 humanized framework selection

Humanized light chain templates of the murine antibody M23 are IGKV2-40 x 01 and IGKJ4 x 01, humanized heavy chain templates are IGHV1-69 x 02 and IGHJ6 x 01, and the sequence of a humanized modified variable region is as follows (underlined as a CDR sequence):

hu23VH-CDR grafting: (SEQ ID NO: 27)

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSDYEMHWVRQAPGQGLEWMGLIDPETGGTVYNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARERFSYYGSTSDWYFDVWGQGTTVTVSS

Hu23VL-CDR grafting: (SEQ ID NO: 28)

DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIK

1.2 humanized template selection and reverse mutation design of murine antibody M23

TABLE 5 reverse mutation of murine antibody M23 humanized antibody

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Note that: grafting (Grafted) represents implantation of murine antibody CDRs into human germline FR region sequences, amino acid residues are determined and annotated by the Kabat numbering system, as I2G represents mutating the Kabat numbered position 2I back to G according to the Kabat numbering system.

The humanized antibody light/heavy chain variable region sequence of M23 is as follows:

hu23VL1 (grafted with Hu23 VL-CDR): (SEQ ID NO: 28)

>Hu23VL2(SEQ ID NO：29)

DGVMTQTPLSLPVTPGEPASISCRSSQSLVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIK

Hu23VH1 (grafting onto Hu23 VH-CDR): (SEQ ID NO: 27)

>Hu23VH2(SEQ ID NO：30)

EVQLVQSGAEVKKPGSSVKVSCKASGYTFSDYEMHWVRQAPGQGLEWMGLIDPETGGTVYNQKFKDRVTLTADKSTSTAYMELSSLRSEDTAVYYCARERFSYYGSTSDWYFDVWGQGTTVTVSS

>Hu23VH3(SEQ ID NO：31)

EVQLVQSGAEVKKPGSSVKVSCKASGYTFSDYEMHWVRQAPGQGLEWIGLIDPETGGTVYNQKFKDRTTLTADKSTSTAYMEFSSLRSEDTAVYYCARERFSYYGSTSDWYFDVWGQGTTVTVSS

>Hu23VH4(SEQ ID NO：32)

EVQLVQSGAEVKKPGSSVKVSCKASGYTFSDYEMHWVRQAPGQGLEWIGLIDPETGGTVYNQKFKDRTTLTADKSTSTAYMEFSSLRSEDTAVYYCTRERFSYYGSTSDWYFDVWGQGTTVTVSS

1.3 humanized sequence combinations of murine antibody M23

The antibodies obtained after humanization of murine antibody M23 and the variable regions thereof are shown in the following table.

TABLE 6 humanized Hu23 antibody variable region combinations

Remarks: "Hu23-1" refers to an antibody in which the antibody light chain variable region is Hu23VL1 and the heavy chain variable region is Hu23VH1, and so on.

The antibody light/heavy chain variable region regions referred to in the above tables (e.g., hu 23-1) may be linked to antibody light/heavy chain constant regions, respectively, to form full length antibodies; in the present disclosure, the light chain variable region when formed into a full length antibody hybridizes to SEQ ID NO:73, and a heavy chain variable region linked to a Kappa chain constant region shown in SEQ ID NO:72 or the IgG4-AA heavy chain constant region shown in SEQ ID NO:79 and the designation of antibody light/heavy chain variable region combinations (e.g., hu 23-1) as indicated in the table with the suffix ". IgG4AA" indicates a full length antibody formed by the attachment of an IgG4-AA heavy chain constant region, and with the suffix ". IgG4P" indicates a full length antibody formed by the attachment of an IgG4-P heavy chain constant region, e.g., "Hu23-1.Igg4aa" indicates a variable region of a Hu23VH1 heavy chain and a variable region of a heavy chain as set forth in SEQ ID NO:72, and a heavy chain comprising a Hu23VL1 light chain variable region and an amino acid sequence as set forth in SEQ ID NO:73, a Kappa chain constant region, and a light chain formed by ligating the Kappa chain constant regions. "Hu23-1.IgG4P" means a heavy chain consisting of the Hu23VH1 variable region and a heavy chain consisting of the amino acid sequence as set forth in SEQ ID NO:79 and a heavy chain comprising an IgG4-P heavy chain constant region linked to a light chain variable region comprising Hu23VL1 and a light chain variable region as set forth in SEQ ID NO:73, a Kappa chain constant region, and a light chain formed by ligating the Kappa chain constant regions.

2. Humanized murine antibody M32

2.1 murine antibody M32 humanized framework selection

Humanized light chain templates of the murine antibody M32 are IGKV2-40 x 01 and IGKJ4 x 01, humanized heavy chain templates are IGHV1-69 x 02 and IGHJ6 x 01, and humanized variable region sequences are as follows (underlined as CDR sequences):

hu32VH-CDR grafting: (SEQ ID NO: 33) IGHV 1-69.times.02 and IGHJ 6.times.01

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSDYEIHWVRQAPGQGLEWMGLFDPETGGIVYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREGYNRDWYFDVWGQGTTVTVSS

Hu32VL-CDR grafting: (SEQ ID NO: 34)

DIVMTQTPLSLPVTPGEPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYAFGGGTKVEIK

2.2 humanized template selection and reverse mutation design of murine antibody M32

TABLE 7 reverse mutation of humanized antibody of murine antibody M32

Note that: grafting (Grafted) represents implantation of murine antibody CDRs into human germline FR region sequences. Amino acid residues are determined and annotated by the Kabat numbering system, as I2V denotes mutating the I at position 2 of the Kabat numbering back to V according to the Kabat numbering system.

The humanized antibody light and heavy chain variable region sequence of the murine antibody M32 is as follows:

hu32VL1 (grafting onto Hu32 VL-CDR): (SEQ ID NO: 34)

>Hu32VL2(SEQ ID NO：35)

DVVMTQTPLSLPVTPGEPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYAFGGGTKVEIK

Hu32VH1 (grafting onto Hu32 VH-CDR): (SEQ ID NO: 33)

>Hu32VH2(SEQ ID NO：36)

EVQLVQSGAEVKKPGSSVKVSCKASGFTFSDYEIHWVRQAPGQGLEWMGLFDPETGGIVYNQKFKGRVTLTADKSTSTAYMELSSLRSEDTAVYYCTREGYNRDWYFDVWGQGTTVTVSS

>Hu32VH3(SEQ ID NO：37)

EVQLVQSGAEVKKPGSSVKVSCKASDFTFSDYEIHWVRQAPGQGLEWMGLFDPETGGIVYNQKFKGRVTLTADKSTSTAYMELSSLRSEDTAVYYCTREGYNRDWYFDVWGQGTTVTVSS

>Hu32VH4(SEQ ID NO：38)

EVQLVQSGAEVKKPGSSVKVSCKASGFTFSDYEIHWVRQAPGQGLEWIGLFDPETGGIVYNQKFKGRATLTADKSTSTAYMEFSSLRSEDTAVYYCTREGYNRDWYFDVWGQGTTVTVSS

>Hu32VH5(SEQ ID NO：39)

EVQLVQSGAEVKKPGSSVKVSCKASDFTFTDYEIHWVRQAPGQGLEWIGLFDPETGGIVYNQKFKGRATLTADKSTSTAYMEFSSLRSEDTAVYYCTREGYNRDWYFDVWGQGTTVTVSS

>Hu32VH6(SEQ ID NO：40)

EVQLVQSGAEVKKPGSSVKVSCKASDFTFTDYEIHWVKQAPGHGLEWIGLFDPETGGIVYNQKFKGKATLTADKSTSTAYMEFSSLRSEDTAVYYCTREGYNRDWYFDVWGQGTTVTVSS

2.3 humanized sequence combinations of murine antibody M32

An antibody obtained after humanization of the murine antibody M32 and a variable region thereof.

TABLE 8 humanized antibody Hu32 light/heavy chain variable region combinations

Remarks: in the table, "Hu32-1" for example refers to the antibody light chain variable region of Hu32VL1 and the heavy chain variable region of Hu32VH1, and so on.

The antibody light/heavy chain variable region regions referred to in the above tables (e.g., hu 32-1) may be linked to antibody light/heavy chain constant regions, respectively, to form full length antibodies; in the present disclosure, the light chain variable region when formed into a full length antibody hybridizes to SEQ ID NO:73, and a heavy chain variable region linked to a Kappa chain constant region shown in SEQ ID NO:72 or the IgG4-AA heavy chain constant region shown in SEQ ID NO:79 and the designation of antibody light/heavy chain variable region combinations (e.g., hu 32-1) in the table with the suffix ". IgG4AA" indicates a full length antibody formed by linkage to an IgG4-AA heavy chain constant region, and with the suffix ". IgG4P" indicates a full length antibody formed by linkage to an IgG4-P heavy chain constant region, e.g., "Hu32-1.IgG4AA" indicates a polypeptide comprising a polypeptide of the inventionHu32VH1Heavy chain variable region and a sequence as set forth in SEQ ID NO:72, and a heavy chain comprising an IgG4-AA heavy chain constant region linked togetherHu32VL1Light chain variable region and a sequence as set forth in SEQ ID NO:73, a Kappa chain constant region, and a light chain formed by ligating the Kappa chain constant regions. "Hu32-1.IgG4P" means a polypeptide represented byHu32VH1Heavy chain variable region and a sequence as set forth in SEQ ID NO:79 to IgG4-P heavy chain constant regionsHeavy chain ofHu32VL1Light chain variable region and a sequence as set forth in SEQ ID NO:73, a Kappa chain constant region, and a light chain formed by ligating the Kappa chain constant regions.

3. Humanized murine antibody M33

3.1 murine antibody M33 humanized framework selection

The humanized light chain templates of the murine antibody M33 are IGKV1-39 x 01 and IGKJ4 x 01, the humanized heavy chain templates are IGHV3-7 x 01 and IGHJ6 x 01, and the humanized variable region sequences are as follows:

hu33VH-CDR grafting (SEQ ID NO: 41):

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVATISGGGVDTYYQDNVQGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPYGHGYFDVWGQGTTVTVSS

hu33VL-CDR grafting (SEQ ID NO: 42):

DIQMTQSPSSLSASVGDRVTITCRASQDINNFLNWYQQKPGKAPKLLIYYTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTFGGGTKVEIK

3.2 humanized template selection and reverse mutation design of murine antibody M33

TABLE 9 reverse mutation of murine antibody M33 humanized antibody

Note that: grafting (Grafted) represents implantation of murine antibody CDRs into human germline FR region sequences. Amino acid residues are determined and annotated by the Kabat numbering system, as F71Y denotes mutating F at position 71 of Kabat numbering back to Y according to the Kabat numbering system.

The humanized antibody light chain variable region and heavy chain variable region sequences of murine antibody M33 are as follows:

hu33VL1 (grafted with Hu33 VL-CDR): (SEQ ID NO: 42)

>Hu33VL2(SEQ ID NO：43)

DIQMTQSPSSLSASVGDRVTITCRASQDINNFLNWYQQKPGKAPKLLIYYTSSLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGGGTKVEIK

>Hu33VL3(SEQ ID NO：44)

DIQMTQSPSSLSASVGDRVTITCRASQDINNFLNWYQQKPGGAVKLLIYYTSSLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGGGTKVEIK

Hu33VH1 (grafted with Hu33 VH-CDR): (SEQ ID NO: 41)

>Hu33VH2(SEQ ID NO：45)

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVATISGGGVDTYYQDNVQGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASPYGHGYFDVWGQGTTVTVSS

>Hu33VH3(SEQ ID NO：46)

KVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVATISGGGVDTYYQDNVQGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASPYGHGYFDVWGQGTTVTVSS

3.3 humanized sequence combinations of murine antibody M33

TABLE 10 humanized antibody Hu33 light heavy chain variable region combinations

Remarks: in the table, "Hu33-6" refers to, for example, the antibody light chain variable region being Hu33VL2 and the heavy chain variable region being Hu33VH3, and so on.

The antibody light/heavy chain variable region regions referred to in the above tables (e.g., hu 33-6) may be linked to antibody light/heavy chain constant regions, respectively, to form full length antibodies; in the present disclosure, the light chain variable region when formed into a full length antibody hybridizes to SEQ ID NO:73, and a heavy chain variable region linked to a Kappa chain constant region shown in SEQ ID NO:72 or the IgG4-AA heavy chain constant region shown in SEQ ID NO:79 and by the name of the combination of light/heavy chain variable regions of the antibodies (e.g., hu 33-6) followed by the suffix ". IgG4AA" means heavy with IgG4AAA full length antibody formed by linking the chain constant regions is indicated by the suffix ". IgG4P" means a full length antibody formed by linking the IgG4-P heavy chain constant region, e.g., "Hu33-6.IgG4AA" means a polypeptide consisting ofHu33VH3Heavy chain variable region and a sequence as set forth in SEQ ID NO:72, and a heavy chain comprising an IgG4-AA heavy chain constant region linked togetherHu33VL2Light chain variable region and a sequence as set forth in SEQ ID NO:73, a Kappa chain constant region, and a light chain formed by ligating the Kappa chain constant regions. "Hu33-6.IgG4P" means a polypeptide represented byHu33VH3Heavy chain variable region and a sequence as set forth in SEQ ID NO:79 and a heavy chain comprising an IgG4-P heavy chain constant region linked theretoHu33VL2Light chain variable region and a sequence as set forth in SEQ ID NO:73, a Kappa chain constant region, and a light chain formed by ligating the Kappa chain constant regions.

4. Mutants of humanized antibodies

4.1 mutant antibodies of humanized Hu23 antibodies

Site-directed mutagenesis of amino acids at specific sites of the light chain LCDR1 (SEQ ID NO: 11) of Hu23 humanized antibodies was performed by computer simulation and the specific mutations are shown in Table 11:

TABLE 11 mutant sequences of Hu23 light chain LCDR1

Note that: hu23LCDR1 (N28Q) represents an LCDR1 mutant sequence in which the N at position 28 of Kabat numbering convention on Hu23VL1 or Hu23VL2 of the light chain variable region of Hu23 is mutated to Q, hu23LCDR1 (G29A) represents an LCDR1 mutant sequence in which the G at position 29 of Kabat numbering convention on Hu23VL1 or Hu23VL2 of the light chain variable region of Hu23 is mutated to A (the CDR is determined by the Kabat numbering convention).

The sequence of the light chain variable region of the Hu23 humanized antibody after LCDR1 mutation is as follows:

>Hu23VL1(N28Q)

DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSQGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIK(SEQ ID NO：53)

>Hu23VL1(N28L)

DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSLGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIK(SEQ ID NO：54)

>Hu23VL1(N28T)

DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSTGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIK(SEQ ID NO：55)

>Hu23VL1(N28D)

DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSDGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIK(SEQ ID NO：56)

>Hu23VL1(G29A)

DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSNANTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIK(SEQ ID NO：57)

>Hu23VL1(G29V)

DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSNVNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIK(SEQ ID NO：58)

>Hu23VL2(N28Q)

DGVMTQTPLSLPVTPGEPASISCRSSQSLVHSQGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIK(SEQ ID NO：59)

>Hu23VL2(N28L)

DGVMTQTPLSLPVTPGEPASISCRSSQSLVHSLGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIK(SEQ ID NO：60)

>Hu23VL2(N28T)

DGVMTQTPLSLPVTPGEPASISCRSSQSLVHSTGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIK(SEQ ID NO：61)

>Hu23VL2(N28D)

DGVMTQTPLSLPVTPGEPASISCRSSQSLVHSDGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIK(SEQ ID NO：62)

>Hu23VL2(G29A)

DGVMTQTPLSLPVTPGEPASISCRSSQSLVHSNANTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIK(SEQ ID NO：63)

>Hu23VL2(G29V)

DGVMTQTPLSLPVTPGEPASISCRSSQSLVHSNVNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIK(SEQ ID NO：64)

TABLE 12 Hu23 humanized antibody light/heavy chain variable region regions

Remarks: in the table, "Hu23-11" refers to, for example, the region of the antibody light chain variable region being Hu23VL1 (N28T) and the heavy chain variable region being Hu23VH1, and so on.

The antibody light/heavy chain variable region regions referred to in the above tables (e.g., hu 23-11) may be linked to antibody light/heavy chain constant regions, respectively, to form full length antibodies; in the present disclosure, the light chain variable region when formed into a full length antibody hybridizes to SEQ ID NO:73, and a heavy chain variable region linked to a Kappa chain constant region shown in SEQ ID NO:72 or the IgG4-AA heavy chain constant region shown in SEQ ID NO:79 and the designation of antibody light/heavy chain variable region combinations (e.g., hu 23-11) as indicated in the table with the suffix ". IgG4AA" indicates a full length antibody formed by the attachment of an IgG4-AA heavy chain constant region, and with the suffix ". IgG4P" indicates a full length antibody formed by the attachment of an IgG4-P heavy chain constant region, e.g., "Hu23-11.Igg4aa", indicates a variable region of Hu23VH1 and a variable region of SEQ ID NO:72 to a heavy chain comprising an IgG4-AA heavy chain constant region linked to a light chain variable region consisting of Hu23VL1 (N28T) and an amino acid sequence as set forth in SEQ ID NO:73, a Kappa chain constant region, and a light chain formed by ligating the Kappa chain constant regions. "Hu23-11.IgG4P" means a heavy chain consisting of the Hu23VH1 variable region and a heavy chain consisting of the amino acid sequence as set forth in SEQ ID NO:79, and a heavy chain comprising a Hu23VL1 (N28T) light chain variable region and an amino acid sequence as set forth in SEQ ID NO:73, a Kappa chain constant region, and a light chain formed by ligating the Kappa chain constant regions.

Experimental results show that humanized antibodies after site mutation of Hu23LCDR1 (N28Q), hu23LCDR1 (N28L), hu23LCDR1 (N28T), hu23LCDR1 (N28D), hu23LCDR1 (G29A), hu23LCDR1 (G29V) all maintain the binding capacity to PD-1 (Table 16).

4.2 mutant antibodies of Hu32 humanized antibodies

Through sequence analysis, the sequence identity of the series humanized antibodies Hu23 and Hu32 from M23 is higher, and the Hu23 light chain variable region and the Hu32 heavy chain variable region are combined into a new light and heavy chain variable region. The experimental results show that humanized antibodies comprising the newly combined light and heavy chain variable regions all retain binding capacity to PD-1 antigen (table 16).

TABLE 13 CDR general formulas for Hu32 and Hu23 antibodies

TABLE 14 combination of Hu32 heavy chain variable region and Hu23 light chain variable region

Remarks: in the table, "Hu32a-85" for example refers to the region of the antibody light/heavy chain variable region where the antibody light chain variable region is Hu23VL1 (N28T) and the heavy chain variable region is Hu32VH6, and so on.

The antibody light/heavy chain variable region regions referred to in the above tables (e.g., hu32 a-85) may be linked to antibody light/heavy chain constant regions, respectively, to form full length antibodies; in the present disclosure, the light chain variable region when formed into a full length antibody hybridizes to SEQ ID NO:73, and a heavy chain variable region linked to a Kappa chain constant region shown in SEQ ID NO:72 or the IgG4-AA heavy chain constant region shown in SEQ ID NO:79 and the designation of antibody light/heavy chain variable region combinations (e.g., hu32 a-85) followed by the suffix ". Lg4aa" means a full length antibody linked to an lg4-AA heavy chain constant region, followed by the suffix ". Lg4p" means a full length antibody linked to an lg4-P heavy chain constant region, e.g., "Hu32a-85.Igg4aa" means a variable region consisting of Hu32VH6 heavy chain and a variable region as set forth in SEQ ID NO:72 to a heavy chain comprising an IgG4-AA heavy chain constant region linked to a light chain variable region consisting of Hu23VL1 (N28T) and an amino acid sequence as set forth in SEQ ID NO:73, a Kappa chain constant region, and a light chain formed by ligating the Kappa chain constant regions. "Hu32a-85.IgG4P" means a heavy chain variable region consisting of Hu32VH6 and a polypeptide as set forth in SEQ ID NO:79, and a heavy chain comprising a Hu23VL1 (N28T) light chain variable region and an amino acid sequence as set forth in SEQ ID NO:73, a Kappa chain constant region, and a light chain formed by ligating the Kappa chain constant regions.

TABLE 15 combination of Hu23 heavy chain variable region and Hu32 light chain variable region

Remarks: in the table, "Hu23a-57" refers to, for example, the antibody light chain variable region being Hu32VL1 and the heavy chain variable region being Hu23VH1, and so on.

The antibody light/heavy chain variable region regions referred to in the above tables (e.g., hu23 a-57) may be linked to antibody light/heavy chain constant regions, respectively, to form full length antibodies; in the present disclosure, the light chain variable region when formed into a full length antibody hybridizes to SEQ ID NO:73, and a heavy chain variable region linked to a Kappa chain constant region shown in SEQ ID NO:72 or the IgG4-AA heavy chain constant region shown in SEQ ID NO:79 and the designation of antibody light/heavy chain variable region combinations (e.g., hu32 a-85) followed by the suffix ". Lg4aa" indicates a full length antibody linked to an lg4-AA heavy chain constant region, followed by the suffix ". Lg4p" indicates a full length antibody linked to an lg4-P heavy chain constant region, e.g., "Hu23a-57.Igg4aa" indicates a variable region of Hu23VH1 and a variable region of SEQ ID NO:72, and a heavy chain comprising a Hu32VL1 light chain variable region and an amino acid sequence as set forth in SEQ ID NO:73, a Kappa chain constant region, and a light chain formed by ligating the Kappa chain constant regions. "Hu23a-57.IgG4P" means a heavy chain variable region consisting of Hu23VH1 and a heavy chain sequence as set forth in SEQ ID NO:79 and a heavy chain comprising an IgG4-P heavy chain constant region linked to a light chain variable region comprising Hu32VL1 and a heavy chain variable region as set forth in SEQ ID NO:73, a Kappa chain constant region, and a light chain formed by ligating the Kappa chain constant regions.

5. Screening of humanized antibodies

The affinity assays of the different humanized antibodies were performed by Biacore (see test example 3 for methods), and the results are shown in table 16, which show that the different humanized antibodies retain binding capacity for PD-1, and the affinity of the partially humanized antibodies is even substantially similar to that of their murine antibodies.

TABLE 16 affinity of Hu23 humanized antibodies for human PD-1

Examples 1-3 construction and expression of PD-1 humanized antibodies

Designing primer PCR to build each humanized antibody VH/VK gene fragment, and then carrying out homologous recombination with an expression vector pHr (with signal peptide and constant region gene (CH 1-Fc/CL) fragment) to construct an antibody full-length expression vector VH-CH1-Fc-pHr/VK-CL-pHr. IgG4-P represents the S228P (corresponding to position 108 of the sequence SEQ ID NO:72 or SEQ ID NO: 79) mutation, igG4-AA represents the F234A (corresponding to position 114 of the sequence SEQ ID NO:72 or SEQ ID NO: 79), L235A (corresponding to position 115 of the sequence SEQ ID NO:72 or SEQ ID NO: 79) and S228P (corresponding to position 108 of the sequence SEQ ID NO:72 or SEQ ID NO: 79) mutation, and IgG4-AA and IgG4-P antibody forms can be obtained by simple point mutation of IgG4 antibody forms.

The IgG4-AA heavy chain constant region sequence is as follows (SEQ ID NO: 72):

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

the light chain (Kappa chain) constant region sequence of the antibody is as follows (SEQ ID NO: 73):

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

The sequence of the constructed full-length antibody of IgG4AA format is exemplified as follows:

hu23-11.IgG4AA antibody heavy chain (SEQ ID NO: 74):

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSDYEMHWVRQAPGQGLEWMGLIDPETGGTVYNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARERFSYYGSTSDWYFDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

hu23-11.Igg4aa light chain (SEQ ID NO: 75):

DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSTGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

hu32a-85.IgG4AA heavy chain (SEQ ID NO: 76):

EVQLVQSGAEVKKPGSSVKVSCKASDFTFTDYEIHWVKQAPGHGLEWIGLFDPETGGIVYNQKFKGKATLTADKSTSTAYMEFSSLRSEDTAVYYCTREGYNRDWYFDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK。

light chain of Hu32a-85.IgG4AA (light chain identical to Hu23-11.IgG4AA, SEQ ID NO: 75):

hu-6. IgG4AA heavy chain (SEQ ID NO: 77):

KVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVATISGGGVDTYYQDNVQGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASPYGHGYFDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

hu-6. IgG4AA light chain (SEQ ID NO: 78):

DIQMTQSPSSLSASVGDRVTITCRASQDINNFLNWYQQKPGKAPKLLIYYTSSLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

the heavy chain constant region sequence of IgG4-P is as follows (SEQ ID NO: 79):

the sequence of the constructed IgG4-P version of the full-length antibody is exemplified as follows:

hu23-11.IgG4P antibody heavy chain (SEQ ID NO: 80):

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSDYEMHWVRQAPGQGLEWMGLIDPETGGTVYNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARERFSYYGSTSDWYFDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

hu23-11.IgG4P light chain (light chain identical to Hu23-11.IgG4AA, SEQ ID NO: 75):

hu32a-85.Igg4p heavy chain (SEQ ID NO: 81):

EVQLVQSGAEVKKPGSSVKVSCKASDFTFTDYEIHWVKQAPGHGLEWIGLFDPETGGIVYNQKFKGKATLTADKSTSTAYMEFSSLRSEDTAVYYCTREGYNRDWYFDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

light chain of Hu32a-85.IgG4P (light chain identical to Hu23-11.IgG4AA, SEQ ID NO: 75):

hu-6. IgG4P heavy chain (SEQ ID NO: 82):

KVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVATISGGGVDTYYQDNVQGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASPYGHGYFDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

hu33-6.IgG4P light chain (same as Hu33-6.IgG4AA light chain, SEQ ID NO: 78):

test example 1

Test example 1-1 in vitro PD-1 ligand binding and binding blocking ELISA experiments with anti-PD-1 antibodies

PD-L1 on the surface of tumor cells can inhibit proliferation of T cells by combining with PD-1 on the surface of T cells. Antibodies to PD-1 block the signaling pathway of PD-L1/PD-1 by binding to PD-1, thereby stimulating proliferation of T cells. The binding blocking assay of PD-1/PD-L1 was used to detect the blocking activity of anti-PD-1 antibodies on signal pathways.

In the experiment, after PD-1-His protein (Cat. #10377H08H,Sino Biological) is coated on a 96-well plate, anti-PD-1 antibodies (including antibodies of Hu23-11.IgG4AA, hu32a-85.IgG4AA and Hu33-6.IgG4 AA) to be detected are respectively added, an incubation reaction is carried out on a positive control antibody of H005-1 (see H005-1 antibody in WO 2015085847), an HRP-labeled goat anti-human IgG (H+L) antibody (Cat. #109-035-003,Jackson ImmunoResearch) is added later, and after the incubation reaction, the binding amount of the HRP-labeled goat anti-human IgG (H+L) is detected, and the EC of the anti-PD-1 antibody on ligand PD-1 binding is calculated after the plate is washed ₅₀ Values.

In this experiment, after coating the extracellular region with Fc fused PD-1 protein (PD-1-Fc, sequence shown in SEQ ID NO: 1) in 96-well plate, anti-PD-1 antibodies (including antibodies: hu23-11.IgG4AA, hu32a-85.IgG4AA and Hu-6. IgG4 AA) to be tested, positive control antibody: H005-1 (see H005-1 antibody in WO 2015085847) were added, incubation reaction was performed, and then biotin-labeled PD-L1/PD-L2 was added, incubation reaction was performed, after washing the plate, the binding amount of biotin-labeled PD-L1/PD-L2 was detected, and IC for blocking the ligand PD-L1/PD-L2 binding by the anti-PD-1 antibody was calculated ₅₀ Values.

With pH 9.6CB buffer (1.59 g Na ₂ CO ₃ And 2.93g NaHCO ₃ In 1L of distilled water) PD-1-Fc was diluted to 1. Mu.g/ml, added to a 96-well plate at a volume of 100. Mu.l/well, and left at 4℃for 16h to 20h. After washing the plates 1 time with PBST (pH 7.4 PBS containing 0.05% tween 20) buffer, 120 μl/well PBST/1% mill was added and incubated for 1h at room temperature for blocking. After removing the blocking solution and washing the plate 1 time with PBST buffer, 90. Mu.l of anti-PD-1 antibody to be tested diluted to a suitable concentration with sample dilution (pH 7.4 PBS containing 5% BSA,0.05% Tween 20) was added and pre-incubated at 4℃for 1h. 10 Xconcentration of biotin-labeled PD-L1/PD-L2 (Beijing Yiqiao ShenzhouBiotechnology Co., ltd.) (10. Mu.g/ml), shaking on a shaker, mixing, and incubating at 37℃for 1 hour. After the reaction system was removed and the plate was washed 6 times with PBST, 100. Mu.l/well of strepitavidin-Peroxidase Polymer (Streptavidin-peroxidase polymer) diluted 1:400 in PBST buffer was added and incubated for 50 minutes at room temperature with shaking. After washing the plates 6 times with PBST, 100. Mu.l/well TMB was added and incubated for 5-10min at room temperature. Add 100. Mu.l/well 1M H ₂ SO ₄ The reaction was terminated. Reading the absorption value at 450nm by using an enzyme-labeled instrument, and calculating the IC of the anti-PD-1 antibody for ligand PD-L1/PD-L2 binding blocking ₅₀ Values. The data are detailed in table 17 below.

TABLE 17 anti-PD-1 antibodies and PD-1 binding of the present disclosure and ligand PD-L1/PD-L2 binding blocking ELISA

Exemplary anti-PD-1 antibodies of the disclosure, hu23-11.IgG4AA, hu32a-85.IgG4AA, and Hu33-6.IgG4AA, are all capable of effectively blocking the binding of PD-1 to PD-L1/PD-L2, with blocking activity similar to that of the positive control antibody.

Test examples 1-2 exemplary ligand blocking assay for antibodies

The blocking effect of antibodies on the binding of PD-1 to PD-L1 was investigated. The experimental procedure is briefly described as follows:

digestion of CHOK1/PD-L1 cells (Promega) 100. Mu.L/well in 96-well plates, placed at 37℃in 5% CO ₂ The incubator was incubated for 24 hours. The control and samples were diluted to the desired concentration using PBS. Counting Jurkat/PD-1 cells (Jurkat cells stably transferring PD-1), adding 10 μL/well of diluted antibody (antibody: hu23-11.IgG4AA, hu32a-85.IgG4AA and Hu33-6.IgG4AA, positive control antibody: H005-1) simultaneously to a cell culture plate (90 μL/well) of a certain proportion of CHOK1/PD-L1 cells, and subjecting to 5% CO at 37℃with a negative control IgG4 protein, antibody gradient dilution concentration of 0.3mg/ml, 3mg/ml, 30mg/ml ₂ The incubator was incubated for 5 hours. The cell culture plate was removed, left to stand at room temperature for 5 minutes, then 50. Mu.l of Bio-GloTMreagent was added to each well, incubated at room temperature for 5 minutes, and the plate read. Experimental results show that FIG. 1.

The results indicate that the exemplary anti-PD-1 antibodies of the present disclosure, hu23-11.IgG4AA, hu32a-85.IgG4AA, and Hu33-6.IgG4AA, are effective in blocking the binding of PD-1 to PD-L1.

Test examples 1-3 BIAcore antibody affinity experiments for exemplary antibodies

IgG was affinity captured using a Protein A biosensing chip (Cat. # 2927556, GE), human PD-1 antigen (Cat. #10377H08H,Sino Biological), cyno PD-1 antigen (purchased from Sino Biological) flowed across the chip surface, and the Biacore T200 instrument examined the PD-1 antibody and antigen PD-1 reaction signals in real time to obtain binding and dissociation curves. After completion of dissociation in each experimental cycle, the biochip was washed and regenerated with 10mM Glycine-HCl buffer pH 1.5. The experimental buffer system was 1 XHBS-EP buffer (Cat#BR-1001-88, GE). The data were fitted with the (1:1) Langmuir model using GE Biacore T200 Evaluation version 3.0 software after the end of the experiment to give affinity values, the results are shown in Table 18.

TABLE 18 affinity of anti-PD-1 antibodies to human PD-1 and monkey PD-1

The results show that the exemplary anti-PD-1 antibodies of the present disclosure, hu23-11.IgG4AA, hu32a-85.IgG4AA, and Hu33-6.IgG4AA, are each capable of binding to human PD-1 and monkey PD-1.

Test examples 1-4 secretion of cell IFNgamma by antibodies in PBMC-T lymphocyte activation experiments

To investigate the effect of anti-PD-1 antibodies on human primary T lymphocyte function, human Peripheral Blood Mononuclear Cells (PBMCs) were collected and purified, and cytokine ifnγ secretion levels were detected after 5 days of in vitro stimulation with Tuberculin (TB). The experimental procedure is briefly described as follows:

fresh blood was centrifuged using Ficoll-Hypaque (17-5442-02, GE), density gradient (StemCell Technologies) PBMC were obtained and cultured in RPMI 1640 (SH 30809.01, GE) medium supplemented with 10% (v/v) FBS (10099-141, gibco), 37℃and 5% CO ₂ Culturing under the condition.

Freshly isolated and purified PBMC were conditioned to a density of 2X 10 in RPMI 1640 medium ⁶ Mu.l tuberculin (97-8800, synbiotics) was added to 20mL of each/mL cell suspension at 37℃with 5% CO ₂ The incubator was cultured for 5 days. On day 5, the cultured cells were collected and centrifuged, resuspended in fresh RPMI 1640 medium, and the density was adjusted to 1.1X10% ⁶ Each mL was inoculated into 96-well cell culture plates, 90. Mu.l per well. Simultaneously, gradient diluted antibody samples (including antibodies of the present disclosure: hu23-11.IgG4AA, hu32a-85.IgG4AA, and Hu33-6.IgG4AA, positive control antibody H005-1, and negative control IgG4 protein) were added, and the antibody gradient diluted at 0.3mg/ml, 3mg/ml, 30mg/ml, and diluted with PBS (B320, shanghai source biosciences Co., ltd.) at 10. Mu.L per well. The cell culture plate was placed at 37℃with 5% CO ₂ Incubators were incubated for 3 days. The cell culture plates were removed, centrifuged (4000 rpm,10 min) to collect the cell culture supernatant, and the levels of IFN-gamma were detected by ELISA (human IFN-gamma detection kit (EHC 102g.96, xinbo) for specific procedures, see reagent instructions.

The results of the assay are shown in FIG. 2, which demonstrates that the anti-PD-1 antibodies of the present disclosure, hu23-11.IgG4AA, hu32a-85.IgG4AA, and Hu33-6.IgG4AA, are effective in activating IFN-gamma secretion.

Test examples 1-5. Effect of anti-PD-1 antibodies in transgenic PD-1 mouse colon cancer model MC38

MC38 cells 5×10 ⁵ Cells/mouse/100 μl were inoculated subcutaneously in the right rib of 90 hPD-1TG mice (baioser), and after 10 days, animals with excessively large and small tumor volumes were removed, and the mice were randomly divided into: blank (PBS), positive control H005-1 3mpk, hu32a-85.IgG4AA 1mpk, hu32a-85.IgG4AA 3mpk, hu23-11.IgG4AA 1mpk, hu23-11.IgG4AA 3mpk, hu33-6.IgG4AA 3mpk were 7 groups of 8. Day0 (Day 0) was followed by three intraperitoneal injections of each group of antibodies every week, with significant tumor inhibition observed after the end of the first week of administration, and the second and third weeks of administration were adjustedThe frequency was once per week and the administration was 5 times. Tumor volume, animal weight were monitored 2 times per week and data recorded. When the tumor volume exceeds 2000mm ³ Or when most tumors are broken or weight is reduced by 20%, euthanasia of the tumor-bearing animals is taken as the experimental end point.

Tumor Volume (TV) =1/2×l _{Long length} ×L _{Short length} ²

Tumor proliferation rate (T/C%) = (T-T0)/(C-C0) ×100%

Tumor inhibition (TGI%) =1-T/C%

Wherein T, T represents the tumor volume at the end of the test and at the beginning of the test of the antibody administration group, and C, C0 represents the tumor volume at the end of the test and at the beginning of the test of the blank group, respectively.

The test results are shown in Table 19 and FIG. 3, and the test results show that compared with a blank control, the antibody disclosed by the invention can obviously inhibit the growth of the mouse colon cancer MC38 transplanted tumor, wherein the tumor inhibition rate is the highest in the Hu32a-85.IgG4AA-3mpk group, and the tumor inhibition rate in the last measurement is 77.64%. When the dosing frequency was 3 times per week and tested on day seven, the results showed that the tumor inhibition rates of the antibodies of the present disclosure were all significantly better than the positive control antibody H005-1; the frequency of dosing was then reduced once a week and 2 times after dosing (Day 21), the efficacy of the antibodies of the present disclosure gradually pulled apart and showed a dose dependency, with Hu32a-85.Igg4aa being significantly better than the equivalent dose of H005-1 (p < 0.05). Moreover, the tumor-bearing mice can well tolerate the anti-PD-1 antibody, the weight of the tumor-bearing mice steadily rises in the whole administration process, and no obvious symptoms such as weight loss caused by medicines occur.

TABLE 19 influence of anti-PD-1 antibodies on tumor suppression Rate of mouse colon carcinoma MC38 (mm) ³ )

Test examples 1-6. Effect of anti-PD-1 antibodies in transgenic PD-1 mouse colon cancer model MC38

Transgenic PD-1 mice were derived from purchased transgenic PD-1 mice (ISIS INNOVATION LIMITED, university Offices, we)The fifth generation mice bred at Cephrim Biosciences, inc. MC38 cells were grown at 5X 10 ⁵ Mu.l/100. Mu.l/inoculated into the rear part of right rib of hPD-1 transgenic mice (male and female half) subcutaneously until the average tumor volume of the mice reaches 80-100mm ³ Animals with body weight, oversized tumor and undersized tumor were removed, and tumor-bearing mice were randomly divided into 5 groups (8 per group) according to tumor volume: negative controls hIgG control 30mpk, H005-1 10mpk, H005-1 30mpk, hu33-6.IgG4AA 10mpk, hu33-6.IgG4AA 30mpk. The group administration date is set to Day 0. Each drug was given intraperitoneally after grouping for 22 days with a total of 11 doses every two days. Tumor volumes were measured 2 times per week, weighed, and data recorded. Body weight and tumor volume of each group were expressed as Mean ± standard deviation (Mean ± SEM) and plotted using Graphpad Prism 5 and Excel software, using student t test statistical analysis.

Tumor Volume (TV) = 0.5236 ×l _{Long length} ×L _{Short length} ²

Tumor proliferation rate T/C% = (T-T0)/(C-C0). Times.100%

Tumor inhibition% tgi=1-T/C%

The test results are shown in table 20 and fig. 4, and the test results show that compared with the control group, the antibody disclosed by the disclosure can significantly inhibit the growth of the mouse colon cancer MC38 transplanted tumor, wherein the tumor inhibition rate is the highest in the Hu33-6.IgG4AA 30mpk group, and the tumor inhibition rate is 80.4% when measured on the 20 th day. In the low dose group (10 mpk), hu33-6.IgG4AA-10mpk was better than the positive control H005-1-10mpk.

TABLE 20 influence of anti-PD-1 antibodies on mouse colon carcinoma MC38 tumor volume

Remarks: the average tumor volume units for each group in the table are: mm (mm) ³ 。

Example 2

EXAMPLE 2-1 construction and expression of PD-1-VEGF bispecific antibodies

Construction of bispecific antibodies different forms of bispecific antibodies were constructed by different linker lengths based on the sequences of Hu23-11 and Lucentis. Different heavy chain and light chain gene fragments were synthesized based on the following sequences, and the heavy chain gene and the light chain gene were cloned into eukaryotic expression vectors to form heavy chain plasmids and light chain plasmids, respectively. And then introducing the expression vector into eukaryotes to express the bispecific antibody with two identical heavy chains and two identical light chains, and purifying the cultured supernatant by using a MabSelect Sure affinity column to obtain an expression product. The amino acid sequences of the heavy and light chains of the bispecific antibodies in the present disclosure are as follows:

DVD-GS8

Heavy chain amino acid sequence of DVD-GS8 (SEQ ID NO: 105):

light chain amino acid sequence of DVD-GS8 (SEQ ID NO: 106):

DVD-GS10

heavy chain amino acid sequence of DVD-GS10 (SEQ ID NO: 107):

light chain amino acid sequence of DVD-GS10 (SEQ ID NO: 108):

DVD-H19L14

heavy chain amino acid sequence of DVD-H19L14 (SEQ ID NO: 109):

light chain amino acid sequence of DVD-H19L14 (SEQ ID NO: 110):

DVD-H21L14

heavy chain amino acid sequence of DVD-H21L14 (SEQ ID NO: 111):

light chain amino acid sequence of DVD-H21L14 (SEQ ID NO: 110):

/>

DVD-GS14

heavy chain amino acid sequence of DVD-GS14 (SEQ ID NO: 112):

light chain amino acid sequence of DVD-GS14 (SEQ ID NO: 110):

DVD-GS19

heavy chain amino acid sequence of DVD-GS19 (SEQ ID NO: 109):

light chain amino acid sequence of DVD-GS19 (SEQ ID NO: 113):

DVD-H24L19

heavy chain amino acid sequence of DVD-H24L19 (SEQ ID NO: 114):

light chain amino acid sequence of DVD-H24L19 (SEQ ID NO: 113):

IgG-GS10-scFv

heavy chain amino acid sequence of IgG-GS10-scFv (SEQ ID NO: 115):

light chain amino acid sequence of IgG-GS10-scFv (SEQ ID NO: 75): igG-GS13-scFv

Heavy chain amino acid sequence of IgG-GS13-scFv (SEQ ID NO: 116):

light chain amino acid sequence of IgG-GS13-scFv (SEQ ID NO: 75): igG-GS19-scFv

Heavy chain amino acid sequence of IgG-GS19-scFv (SEQ ID NO: 117):

/>

light chain amino acid sequence of IgG-GS19-scFv (SEQ ID NO: 75):

4-4-DVD

heavy chain amino acid sequence of 4-4-DVD (SEQ ID NO: 135):

4-4-DVD light chain amino acid sequence (SEQ ID NO: 136):

( Remarks: the variable region is shown in single underline, the italics indicate the linker (linker) )

The structures of DVD-GS8, DVD-GS10, DVD-H19L14, DVD-H21L14, DVD-GS19, DVD-H24L19, 4-4-DVD are shown in FIG. 9A; the structures of the IgG-GS10-scFv, the IgG-GS13-scFv, and the IgG-GS19-scFv are shown in FIG. 9B.

EXAMPLE 2-2 construction and expression of PD-1-VEGFtrap antibody fusion proteins

Construction of antibody fusion proteins based on the sequences of Hu23-11 and VEGFtrap mutants, different forms of antibody fusion proteins were constructed by different linker lengths. Different heavy chain and light chain gene fragments were synthesized based on the following sequences, and the heavy chain gene and the light chain gene were cloned into eukaryotic expression vectors to form heavy chain plasmids and light chain plasmids, respectively. And then introducing the expression vector into eukaryotes to express the antibody fusion protein with two identical heavy chains and two identical light chains, and purifying the cultured supernatant by using a MabSelect Sure affinity column to obtain an expression product. The amino acid sequences of the heavy and light chains of the antibody fusion proteins of the present disclosure are as follows:

Hu23-2TE

Heavy chain amino acid sequence of Hu23-2TE (SEQ ID NO: 118):

light chain amino acid sequence of Hu23-2TE (SEQ ID NO: 75)

Hu23-3ST

Heavy chain amino acid sequence of Hu23-3ST (SEQ ID NO: 119):

light chain amino acid sequence of Hu23-3ST (SEQ ID NO: 75)

Hu23-5LK

Heavy chain amino acid sequence of Hu23-5LK (SEQ ID NO: 120):

/>

light chain amino acid sequence of Hu23-5LK (SEQ ID NO: 75) 3-6

3-6 heavy chain amino acid sequence (SEQ ID NO: 121):

3-6 light chain amino acid sequence (SEQ ID NO: 75)

3-12

Heavy chain amino acid sequence of >3-12 (SEQ ID NO: 122):

3-12 light chain amino acid sequence (SEQ ID NO: 75)

6-37

Heavy chain amino acid sequence of >6-37 (SEQ ID NO: 123):

/>

6-37 light chain amino acid sequence (SEQ ID NO: 75) #2

Heavy chain amino acid sequence of #2 (SEQ ID NO: 124):

light chain amino acid sequence of #2 (SEQ ID NO: 75)

#3

Heavy chain amino acid sequence of #3 (SEQ ID NO: 125):

light chain amino acid sequence of #3 (SEQ ID NO: 75)

#5

Heavy chain amino acid sequence of #5 (SEQ ID NO: 126):

light chain amino acid sequence of #5 (SEQ ID NO: 75)

Hu33-V6

Heavy chain amino acid sequence of Hu33-V6 (SEQ ID NO: 137):

light chain amino acid sequence of Hu33-V6 (SEQ ID NO: 78) Hu23-V6

Heavy chain amino acid sequence of Hu23-V6 (SEQ ID NO: 138):

/>

Light chain amino acid sequence of Hu23-V6 (SEQ ID NO: 75)

Hu33-WT

Heavy chain amino acid sequence of Hu33-WT (SEQ ID NO: 139):

light chain amino acid sequence of Hu33-WT (SEQ ID NO: 78)

Hu23-WT

Heavy chain amino acid sequence of Hu23-WT (SEQ ID NO: 140):

light chain amino acid sequence of Hu23-WT (SEQ ID NO: 75)

( Remarks: the variable region is shown in single underline, the linker (linker) is shown in italics, and VEGFtrap is shown in double underline )

The structures of Hu23-2TE, hu23-3ST, hu23-5LK, 3-6, 3-12, 6-37, hu33-V6, hu23-V6, hu33-WT and Hu23-WT are shown in FIG. 9C; the structures of #2, #3, #5 are shown in FIG. 9D.

Test example 2

Test example 2-1 ligand blocking assay for bispecific antibodies and antibody fusion proteins

Table 21 Experimental reagents and materials

Reagent or material name	Goods number	Manufacturer (S)
			PD-1/PD-L1 Blockade Bioassay	J1250	Promega
RPMI Medium1640	22400-089	Gibco
			F-12Nutrient Mixture(Ham)	11765-054	Gibco
Hygromycin B	10687-010	invitrogen
			Antibiotic G-418Sulfate Solution	V8091	Promega
Bio-GloTM Luciferase Assay System	G7940 or G7941	Promega
			FBS 10099-141	10099-141	Gibco
GloResponseTM NFAT-luc2/PD1 Jurkat	CS187102	Promega
			PD-L1+CHO-K1	CS187108	Promega

The CHO-K1/PD-L1 cells were digested, and finally the cells were resuspended using F-12Nutrient Mixture (Ham) complete medium, and the cell density was adjusted to 4X 10 using complete medium according to the cell count results ⁵ The cell suspension was transferred to a loading well at a volume of 100. Mu.L/well into a 96-well plate (Corning # 3903) using a multi-channel pipette, and placed at 37℃with 5% CO ₂ Culturing in an incubator for 20-24 hours. Meanwhile, according to the concentrations of the control and the sample, PBS is used for dilution to 300 mug/mL, 10-time gradient dilution is carried out, 3 concentration points are added, 2 wells are arranged for each concentration, and blank wells only added with an analysis culture medium are arranged. Centrifuging at 1000rpm for 5mins, and collecting Jurkat/PD1 fine Cell suspension, resuspending cells using assay medium based on cell count results, and adjusting cell density to 5.5X10 ⁵ Individual/mL or 1.25X10 ⁶ And each mL. The cell culture plate containing CHO-K1/PD-L1 cells was removed from the incubator, 95. Mu.L of the culture medium was removed per well using a multi-channel pipette, and Jurkat/PD1 cell suspension (5.5X10) ⁵ individual/mL) was transferred to a loading well, added to a cell culture plate at 90 μl/well, and then diluted control and sample were added at 10 μl/well, and placed at 37deg.C with 5% CO ₂ Culturing in an incubator for 5-6 h. The Bio-GloTMreagent was removed to return the temperature to room temperature, 50. Mu.L of the Bio-GloTMreagent was added to each well, and after 5 to 10 minutes, the plate was read with a Victor 3. Data were processed with GraphPad software.

The results are shown in FIG. 5. Ligand blocking experiments demonstrated that bispecific antibodies DVD-H19L14, DVD-H24L19, DVD-H21L14, DVD-GS19 and antibody fusion proteins 3-6, 6-37, #2, #3, #5 all blocked the binding of PD-1 to PD-L1 with blocking activity similar to that of control antibody Hu23-11. IgG4AA.

Test example 2-2 BIAcore antibody affinity experiments of bispecific antibodies and antibody fusion proteins

IgG was captured affinity using a Protein A biosensing chip (Cat. # 2927556, GE) and then passed over the antigen on the chip surface. The assay was performed in HBS-EP+10Xbuffer (Cat#BR-1001-88, GE) and binding and dissociation curves were obtained by real-time detection of bispecific antibodies and antibody fusion proteins and antigen response signals using a Biacore T200 instrument. After completion of dissociation in each experimental cycle, the biochip was washed and regenerated with 10mM Glycine-HCl (pH 1.5). The data fitting model uses a 1:1 model.

The antigen molecule samples were derived as follows:

Human VEGFA165(19.2KDa,Cat#11066-HNAH,Lot.#LC11DE0604,S.B)

Mouse VEGFA164(19.4kDa,Cat#5109-MNAB,Lot.#LC12OC2306,S.B)

Human VEGFB(20.3KDa,Cat.#VE6-H5225,Lot.#675-78AF1-JX,Acro)

Human PD1(17.4KDa,Cat.#10377-H08H,Lot.#LC13AU2111,S.B)

Cyno PD1(17.4KDa,Cat.#PD1-C5223,Lot.#643-7CTF1-US,S.B)

Human PIGF(14.9KDa,Cat#10274-HNAE1,Lot.#LC13SE0205,S.B)

Mouse PIGF(16.6kDa,Cat#50125-NCCH,Lot.#LC08MA2905,S.B)

table 22: affinity of bispecific antigen binding molecules to PD-1 proteins

Table 23-1: reactive affinity of bispecific antigen binding molecules to human VEGFA165 and murine VEGF164 proteins

Table 23-2: reactive affinity of bispecific antigen binding molecules to human VEGFA165, human VEGFB and murine VEGF164 protein binding

Molecules	hVEGFA165 KD(M)	hVEGFB KD(M)	mVEGF164 KD(M)
				Hu23-V6	2.48E-11	4.00E-11	1.25E-11
Hu23-WT	8.43E-12	3.40E-11	7.10E-12
				Hu33-V6	1.11E-11	2.01E-11	3.05E-12
Hu33-WT	7.28E-12	2.96E-11	1.46E-12

All bispecific antibodies and antibody fusion proteins were able to bind human PD-1 and monkey PD-1 with affinities comparable to PD-1 control antibody Hu23-11.Igg4aa, higher than control bispecific antibody VP101 and control antibody fusion protein by24.3. All bispecific antibodies and fusion proteins were able to bind human VEGFA165 with affinity comparable to control antibody Lucentis, control bispecific antibody VP 101. The antibody fusion proteins were both able to bind human VEGFAB and murine VEGFA164, with Hu23-3ST and Hu23-5LK and human VEGFA165 having slightly higher affinity than the control antibody fusion protein By24.3. Wherein the affinity of the antibody fusion protein (3-12, # 3) and murine VEGFA164 was comparable to the control antibody fusion protein By24.3.

Test examples 2-3 secretion of cell IFNgamma by bispecific antibodies and antibody fusion proteins in PBMC-T lymphocyte activation experiments

To investigate the effect of bispecific antibodies and antibody fusion proteins on human primary T lymphocyte function, human peripheral blood mononuclear cells (PBMC, 4 donors) were collected and purified, and cytokine ifnγ secretion levels were detected after 5 days of in vitro stimulation with Tuberculin (TB). The experimental procedure is described below:

fresh blood was centrifuged (Stem Cell Technologies) to obtain PBMC at RPMI 16 using Ficoll-Hypaque (17-5442-02, GE)40 (SH 30809.01, GE) in a medium supplemented with 10% (v/v) FBS (10099-141, gibco), 37℃and 5% CO ₂ Culturing under the condition.

Freshly isolated and purified PBMC were conditioned to a density of 2X 10 in RPMI 1640 medium ⁶ mu.L tuberculin (97-8800, synbiotics), 37℃and 5% CO were added to 20mL of cell suspension at a concentration of 40. Mu.L/mL ₂ The incubator was cultured for 5 days. On day 5, the cultured cells were collected and centrifuged, resuspended in fresh RPMI 1640 medium, and the density was adjusted to 1.1X10% ⁶ Each mL was inoculated into 96-well cell culture plates at 90. Mu.L per well. Simultaneously, a gradient of diluted antibody sample was added, and diluted with PBS (B320, shanghai Biotechnology Co., ltd.) to 10. Mu.L per well. The cell culture plate was placed at 37℃with 5% CO ₂ Incubators were incubated for 3 days. The cell culture plates were removed, centrifuged (4000 rpm,10 min) to collect cell culture supernatants, and the IFN-gamma levels were detected by ELISA (human IFN-gamma detection kit (EHC 102g.96, xinbo)). For specific operation reference is made to the reagent instructions.

The results (see FIG. 6) show that all bispecific antibodies and antibody fusion proteins can activate PBMC to secrete IFN-gamma in 4 donor experiments. In donor 2 (as in FIG. 6B), DVD-GS14 activated the secretion of IFN-gamma by PBMC with higher activity than the control antibody Hu23-11.IgG4AA.

Test examples 2-4 bispecific antibodies and antibody fusion proteins inhibit VEGFA-induced phosphorylation experiments on HUVEC cells

Table 24 laboratory apparatus and materials

HUVEC cells were cultured in HUVEC complete medium (HUVEC basal medium+additive+10% FBS) and passaged 2-3 times a week at a passaging ratio of 1:3 or 1:6. At passage, the medium was aspirated, the cell layer was rinsed with 5ml0.25% pancreatin, then pancreatin was aspirated, the cells were placed in an incubator to digest for 3-5 minutes, the digestion was stopped, centrifuged at 1500rpm for 5min, the supernatant was discarded, and fresh medium was added to resuspend the cells.

By cellsAfter counting by a counter, the cell suspension was adjusted to a density of 1.2X10 ⁵ cells/mL, 1 mL/well were plated in 12-well plates, with 120000 cells per well. The plates were incubated in an incubator for 24 hours (37 ℃,5% co ₂ ). Cell culture plates were removed, medium was discarded, washed once with PBS, replaced with 900. Mu.L/well of HUVEC basal medium containing 0.5% BSA, and incubated at room temperature for 25min. The samples to be tested were diluted with PBS to 2000nM (20 Xconcentration), 10-fold diluted 3 concentrations of 2000nM, 200nM and 20nM, respectively. 60 μl of the sample was added to 60 μl of VEGF165 (20 nM) 1:1 and mixed to give a 10 Xsample concentration. mu.L per well was added to the cell culture plate. The cell culture plates were incubated at room temperature for 15min. The culture was discarded and washed once with 4℃pre-chilled PBS. 1×lysis buffer was prepared: phospho-total protein lysis buffer was diluted 4-fold with double distilled water pre-chilled to 4 ℃, then 1/100 volume of Blocking Reagent was added and mixed well. 1 Xlysis buffer, 120. Mu.L/well, was added and incubated for 30 minutes at room temperature. mu.L of cell lysate per well was added to ELISA wells and incubated overnight at 4℃according to The experiments were performed with the specifications of Phospho-VEGFR-2 (Tyr 1175) Sandwich ELISA Kit.

The results (see FIG. 7, where IgG4 represents control molecule C25-IgG 4) show that all bispecific antibodies and antibody fusion proteins can inhibit VEGFA-induced phosphorylation of HUVEC cells and are dose dependent. Wherein the inhibitory activity of the antibody fusion protein (Hu 23-5 LK) was better than that of the control fusion protein VEGFtrap-Fc at low concentrations (FIG. 7E). The inhibitory activity of DVD-GS14 was better than that of DVD-GS8, DVD-GS10 and DVD-GS19 at high concentrations (FIG. 7F).

Test examples 2-5. Efficacy experiments of mouse MDA-MB-231 model reconstructed with human PBMC

This test example utilizes the NOD-SCID mouse (beijing villi-wa laboratory animal ltd) MDA-MB-231 model reconstituted with human PBMC to evaluate the antitumor efficacy of the disclosed test PD1-VEGFtrap fusion proteins in mice.

(a) MDA-MB-231 cells (3X 10) ⁶ Individual cells/mu.L/Only/200. Mu.L, containing 100. Mu.L Matrigel) were inoculated into the right flank skin of female NOD-SCID miceWhen the tumor volume of the tumor-bearing mice reaches 240mm ³ The mice were randomly grouped on either side, 7 mice per group, the Day of the grouping was defined as Day0 of the experiment, and the Day was followed by 5X 10 PBMCs stimulated with hCD3 antibody for 4 days ⁵ Cells/100. Mu.L/mouse intratumoral injection alone, remaining PBMCs were stopped at stimulation and cultured for one additional week at 5X 10 ⁶ Cells/100 μl/mouse i.p. injection was performed only, this is the first round of injection. After one more week a second round of injection was thus performed. The intraperitoneal injections of each antibody were started on the same day, 3 times per week for 3 weeks, 2 times per week for tumor volume, animal weight and data were recorded.

The results (see FIG. 8A) show that the tumor inhibition rates of the PD-1 antibodies Hu23-11.IgG4AA-10mpk and VEGFtrap-Fc-6.4mpk at the same molar doses of Day22 were 19.58% and 27.80% (p < 0.05), respectively.

The tumor inhibition rates of fusion proteins 6-37-13mpk and #3-12.9mpk at Day22 were 38.87% (p < 0.01) and 37.21% (p < 0.01), respectively, which are superior to equal molar doses of the PD-1 control antibody and VEGFtrap-Fc control fusion protein.

(b) The tumor volume of the tumor-bearing mice in the experiment (b) reaches 150mm ³ Random grouping is performed when left and right, and the rest steps are the same as those of the experiment (a).

The results (see FIG. 8B) show that the PD-1 antibodies Hu23-11.IgG4AA-10mpk and VEGFtrap-Fc-7mpk had tumor suppression rates of 12.59% and 14.00%, respectively, at the same molar dose of Day28, with no significant difference compared to the solvent control.

The in vivo tumor inhibition effect of the antibody fusion protein Hu23-5LK shows a dose-dependent effect, and the tumor inhibition rates at 13.2mpk and 4mpk doses at Day28 are 44.43% (p < 0.001) and 35.65% (p < 0.05), respectively, which are superior to the PD-1 control antibody and the VEGFtrap-Fc control fusion protein at the same molar dose.

The tumor inhibition rates of the antibody fusion proteins Hu23-5LK-13.2mpk, hu23-3ST-13.2mpk and 3-12-13.2mpk at Day28 were 44.43% (p < 0.01), 38.93% (p < 0.01) and 28.81% (p < 0.05), respectively, which are superior to the equivalent molar doses of the PD-1 control antibody and VEGFtrap-Fc control fusion protein.

Test examples 2-6. Efficacy experiments of mouse MDA-MB-231 model reconstructed with human PBMC

This test example utilizes the NOD-SCID mouse (beijing villi-hua experimental animal limited) MDA-MB-231 model reconstituted with human PBMC to evaluate the anti-tumor efficacy of the inventive test PD1-VEGF bispecific antibodies in mice.

MDA-231 cells in logarithmic growth phase were 3X 10 ⁶ Inoculating individual cells/cell/200. Mu.L (containing 100 μm Matrigel) subcutaneously in the right rib of 80 female NOD-SCID mice, when the tumor volume of the tumor-bearing mice reaches 190mm ³ On the left and right, tumor volumes that were too large, too small or less than 18g in weight were removed, and random groupings of PBS, DVD-GS 14/13 mpk, DVD-GS19 13mpk, DVD-GS24 13mpk, VP-101 13mpk, hu23-11.IgG4AA 10mpk were used. The Day of grouping was defined as the experimental Day0, and two volunteers stimulated with CD3 antibody for 3 days mixed with PBMCs at a 1:1 ratio at Day0, 5X 10 ⁵ Individual cells/100. Mu.L/injection into mouse tumor tissue alone, and the remaining PBMCs were stopped from stimulation and continued to culture at 5X 10 after 1 week ⁶ The individual cells/100. Mu.L/i.p. were injected into tumor-bearing mice, treated as round 1 injection, and co-injected with two rounds of PBMCs to the end of the experiment. Day0 was started twice weekly by intraperitoneal injection of each antibody, and was administered 9 times (table 1). Tumor volume, animal weight were monitored 2 times per week and data recorded. When the tumor volume exceeds 1000mm ³ Or when most tumors are broken or weight is reduced by 20%, euthanasia of the tumor-bearing animals is taken as the experimental end point.

The experimental results are shown in Table 25, the PD1-VEGF bispecific antibodies DVD-GS14, DVD-GS19 and DVD-GS24 can obviously inhibit the growth of human breast cancer MDA-MB-231 mice subcutaneous transplantable tumors, the efficacy of the 3 antibodies is basically equivalent, the tumor inhibition rates at the experimental end point are 70.60% (DVD-GS 24-13 mpk), 67.90% (DVD-GS 19-13 mpk), 67.08% (DVD-GS 14-13 mpk) and 57.98% (DVD-GS 14-4 mpk) respectively, and the two antibodies have extremely obvious differences with the PD-1 monoclonal antibody control Hu23-11.IgG4AA (10 mpk, TGI=13.18%) compared with the positive antibody VP-101 (13 mpk, TGI=63.12%) with the same dose.

TABLE 25 therapeutic efficacy of bispecific antigen binding molecules on human breast cancer cell MDA-MB-231 transplantations

***p＜0.001

Test examples 2-7 in vivo efficacy test of PD1-VEGF antibody against U87MG+PBMC-transplanted tumor nude mice

Subcutaneous inoculation of U87MG cells (1.5X10) into NOD-SCID mice right rib ⁶ PBMC, intraperitoneal injection, 4×10) on the day ⁶ And/or just. Day 19 of 6 months, second intraperitoneal injection of PBMC,4×10 ⁶ And/or just. After 8 days, the groups were 7 groups/group, 9 total groups. Average tumor volume per group on day of grouping was 79.26mm ³ . Day0 was started twice weekly by intraperitoneal injection of each antibody, for a total of 9 doses. Tumor volume, animal weight were monitored 2 times per week and data recorded.

The experimental results are shown in Table 26, and Day29 was observed for 8 times of intraperitoneal administration in this experiment. All three bifunctional molecules of the present disclosure are superior to the control molecule VP-101 (3 mpk) in tumor growth inhibition compared to the molar dose. In the administration process, the weight of each group of animals is not obviously reduced, which indicates that the tested medicine has no obvious toxic or side effect.

TABLE 26 therapeutic efficacy of bispecific antigen binding molecules on U87MG+ PBMC-transplants

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the description and examples should not be construed as limiting the scope of the disclosure. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

Sequence listing

<110> Jiangsu Hengrui medicine Co., ltd

SHANGHAI HENGRUI PHARMACEUTICAL Co.,Ltd.

<120> bispecific antigen binding molecules and medical uses thereof

<130> CN1016SH314EZS

<160> 142

<170> SIPOSequenceListing 1.0

<210> 1

<211> 401

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> human PD-1-IgG1Fc sequence

<400> 1

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp Asn

20 25 30

Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn

35 40 45

Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu

50 55 60

Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala

65 70 75 80

Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg Val

85 90 95

Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala

100 105 110

Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala

115 120 125

Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr

130 135 140

Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg

145 150 155 160

Pro Ala Gly Gln Phe Gln Thr Leu Val Glu Pro Lys Ser Ser Asp Lys

165 170 175

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

180 185 190

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

195 200 205

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

210 215 220

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

225 230 235 240

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

245 250 255

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

260 265 270

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

275 280 285

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

290 295 300

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

305 310 315 320

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

325 330 335

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

340 345 350

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

355 360 365

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

370 375 380

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

385 390 395 400

Lys

<210> 2

<211> 186

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> human PD-1-his sequence

<400> 2

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp Asn

20 25 30

Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn

35 40 45

Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu

50 55 60

Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala

65 70 75 80

Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg Val

85 90 95

Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala

100 105 110

Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala

115 120 125

Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr

130 135 140

Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg

145 150 155 160

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

165 170 175

Asp Asp Asp Lys His His His His His His

180 185

<210> 3

<211> 288

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> transfected cell PD-1 antigen sequence

<400> 3

Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln

1 5 10 15

Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp

20 25 30

Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp

35 40 45

Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val

50 55 60

Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala

65 70 75 80

Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg

85 90 95

Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg

100 105 110

Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu

115 120 125

Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val

130 135 140

Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro

145 150 155 160

Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly

165 170 175

Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys

180 185 190

Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro

195 200 205

Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly

210 215 220

Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro

225 230 235 240

Cys Val Pro Glu Gln Thr Glu Tyr Ala Thr Ile Val Phe Pro Ser Gly

245 250 255

Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg

260 265 270

Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu

275 280 285

<210> 4

<211> 125

<212> PRT

<213> murine sequence (Mus musculus)

<220>

<221> DOMAIN

<223> heavy chain variable region sequence of M23

<400> 4

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

1 5 10 15

Ser Val Thr Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Glu Met His Trp Val Lys Gln Thr Pro Ile His Gly Leu Glu Trp Ile

35 40 45

Gly Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Thr Ile Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Phe Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr His Cys

85 90 95

Thr Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe

100 105 110

Asp Val Trp Gly Thr Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 5

<211> 112

<212> PRT

<213> murine sequence (Mus musculus)

<220>

<221> DOMAIN

<223> light chain variable region sequence of M23

<400> 5

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

1 5 10 15

Asp His Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 6

<211> 120

<212> PRT

<213> murine sequence (Mus musculus)

<220>

<221> DOMAIN

<223> heavy chain variable region sequence of M32

<400> 6

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

1 5 10 15

Ser Val Thr Leu Ser Cys Lys Ala Ser Asp Phe Thr Phe Thr Asp Tyr

20 25 30

Glu Ile His Trp Val Lys Gln Thr Pro Val His Gly Leu Glu Trp Ile

35 40 45

Gly Leu Phe Asp Pro Glu Thr Gly Gly Ile Val Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Ile Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr

65 70 75 80

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

85 90 95

Thr Arg Glu Gly Tyr Asn Arg Asp Trp Tyr Phe Asp Val Trp Gly Thr

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 7

<211> 112

<212> PRT

<213> murine sequence (Mus musculus)

<220>

<221> DOMAIN

<223> light chain variable region sequence of M32

<400> 7

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

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Ala Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 8

<211> 5

<212> PRT

<213> murine sequence (Mus musculus)

<220>

<221> DOMAIN

<223> HCDR1 sequence of M23

<400> 8

Asp Tyr Glu Met His

1 5

<210> 9

<211> 17

<212> PRT

<213> murine sequence (Mus musculus)

<220>

<221> DOMAIN

<223> HCDR2 sequence of M23

<400> 9

Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe Lys

1 5 10 15

Asp

<210> 10

<211> 16

<212> PRT

<213> murine sequence (Mus musculus)

<220>

<221> DOMAIN

<223> HCDR3 sequence of M23

<400> 10

Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe Asp Val

1 5 10 15

<210> 11

<211> 16

<212> PRT

<213> murine sequence (Mus musculus)

<220>

<221> DOMAIN

<223> LCDR1 sequence of M23

<400> 11

Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Lys Thr Tyr Leu Glu

1 5 10 15

<210> 12

<211> 7

<212> PRT

<213> murine sequence (Mus musculus)

<220>

<221> DOMAIN

<223> LCDR2 sequence of M23/M32

<400> 12

Lys Val Ser Asn Arg Phe Ser

1 5

<210> 13

<211> 9

<212> PRT

<213> murine sequence (Mus musculus)

<220>

<221> DOMAIN

<223> LCDR3 sequence of M23

<400> 13

Phe Gln Gly Ser His Val Pro Tyr Thr

1 5

<210> 14

<211> 5

<212> PRT

<213> murine sequence (Mus musculus)

<220>

<221> DOMAIN

<223> HCDR1 sequence of M32

<400> 14

Asp Tyr Glu Ile His

1 5

<210> 15

<211> 17

<212> PRT

<213> murine sequence (Mus musculus)

<220>

<221> DOMAIN

<223> HCDR2 sequence of M32

<400> 15

Leu Phe Asp Pro Glu Thr Gly Gly Ile Val Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 16

<211> 11

<212> PRT

<213> murine sequence (Mus musculus)

<220>

<221> DOMAIN

<223> HCDR3 sequence of M32

<400> 16

Glu Gly Tyr Asn Arg Asp Trp Tyr Phe Asp Val

1 5 10

<210> 17

<211> 16

<212> PRT

<213> murine sequence (Mus musculus)

<220>

<221> DOMAIN

<223> LCDR1 sequence of M32

<400> 17

Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu

1 5 10 15

<210> 18

<211> 9

<212> PRT

<213> murine sequence (Mus musculus)

<220>

<221> DOMAIN

<223> LCDR3 sequence of M32

<400> 18

Phe Gln Gly Ser His Val Pro Tyr Ala

1 5

<210> 19

<211> 118

<212> PRT

<213> murine sequence (Mus musculus)

<220>

<221> DOMAIN

<223> heavy chain variable region sequence of M33

<400> 19

Lys Val Met Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Val Asp Thr Tyr Tyr Gln Asp Asn Val

50 55 60

Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

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

85 90 95

Ala Ser Pro Tyr Gly His Gly Tyr Phe Asp Val Trp Gly Thr Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 20

<211> 107

<212> PRT

<213> murine sequence (Mus musculus)

<220>

<221> DOMAIN

<223> light chain variable region sequence of M33

<400> 20

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

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Asn Asn Phe

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 21

<211> 5

<212> PRT

<213> murine sequence (Mus musculus)

<220>

<221> DOMAIN

<223> HCDR1 sequence of M33

<400> 21

Ser Tyr Ala Met Ser

1 5

<210> 22

<211> 17

<212> PRT

<213> murine sequence (Mus musculus)

<220>

<221> DOMAIN

<223> HCDR2 sequence of M33

<400> 22

Thr Ile Ser Gly Gly Gly Val Asp Thr Tyr Tyr Gln Asp Asn Val Gln

1 5 10 15

Gly

<210> 23

<211> 9

<212> PRT

<213> murine sequence (Mus musculus)

<220>

<221> DOMAIN

<223> HCDR3 sequence of M33

<400> 23

Pro Tyr Gly His Gly Tyr Phe Asp Val

1 5

<210> 24

<211> 11

<212> PRT

<213> murine sequence (Mus musculus)

<220>

<221> DOMAIN

LCDR1 sequence of <223> M33

<400> 24

Arg Ala Ser Gln Asp Ile Asn Asn Phe Leu Asn

1 5 10

<210> 25

<211> 7

<212> PRT

<213> murine sequence (Mus musculus)

<220>

<221> DOMAIN

LCDR2 sequence of <223> M33

<400> 25

Tyr Thr Ser Ser Leu His Ser

1 5

<210> 26

<211> 9

<212> PRT

<213> murine sequence (Mus musculus)

<220>

<221> DOMAIN

LCDR3 sequence of <223> M33

<400> 26

Gln Gln Gly Asn Thr Leu Pro Trp Thr

1 5

<210> 27

<211> 125

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> Hu23VH 1/Hu 23VH-CDR grafted sequence

<400> 27

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asp Tyr

20 25 30

Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 28

<211> 112

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> Hu23VL 1/Hu 23VL-CDR grafted sequences

<400> 28

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 29

<211> 112

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> Hu23VL2 sequence

<400> 29

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 30

<211> 125

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> Hu23VH2 sequence

<400> 30

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Tyr

20 25 30

Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 31

<211> 125

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> Hu23VH3 sequence

<400> 31

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Tyr

20 25 30

Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Thr Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 32

<211> 125

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> Hu23VH4 sequence

<400> 32

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Tyr

20 25 30

Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Thr Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Thr Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 33

<211> 120

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> Hu32 VH1/Hu 32VH-CDR grafted sequence

<400> 33

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asp Tyr

20 25 30

Glu Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Leu Phe Asp Pro Glu Thr Gly Gly Ile Val Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Glu Gly Tyr Asn Arg Asp Trp Tyr Phe Asp Val Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 34

<211> 112

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> Hu32 VL1/Hu 32VL-CDR grafted sequences

<400> 34

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 35

<211> 112

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> Hu32VL2 sequence

<400> 35

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 36

<211> 120

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> Hu32VH2 sequence

<400> 36

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Glu Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Leu Phe Asp Pro Glu Thr Gly Gly Ile Val Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Thr Arg Glu Gly Tyr Asn Arg Asp Trp Tyr Phe Asp Val Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 37

<211> 120

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> Hu32VH3 sequence

<400> 37

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Asp Phe Thr Phe Ser Asp Tyr

20 25 30

Glu Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Leu Phe Asp Pro Glu Thr Gly Gly Ile Val Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Thr Arg Glu Gly Tyr Asn Arg Asp Trp Tyr Phe Asp Val Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 38

<211> 120

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> Hu32VH4 sequence

<400> 38

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Glu Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Leu Phe Asp Pro Glu Thr Gly Gly Ile Val Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Thr Arg Glu Gly Tyr Asn Arg Asp Trp Tyr Phe Asp Val Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 39

<211> 120

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> Hu32VH5 sequence

<400> 39

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Asp Phe Thr Phe Thr Asp Tyr

20 25 30

Glu Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Leu Phe Asp Pro Glu Thr Gly Gly Ile Val Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Thr Arg Glu Gly Tyr Asn Arg Asp Trp Tyr Phe Asp Val Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 40

<211> 120

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> Hu32VH6 sequence

<400> 40

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Asp Phe Thr Phe Thr Asp Tyr

20 25 30

Glu Ile His Trp Val Lys Gln Ala Pro Gly His Gly Leu Glu Trp Ile

35 40 45

Gly Leu Phe Asp Pro Glu Thr Gly Gly Ile Val Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Thr Arg Glu Gly Tyr Asn Arg Asp Trp Tyr Phe Asp Val Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 41

<211> 118

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> Hu33VH1/Hu33VH-CDR grafted sequence

<400> 41

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

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

35 40 45

Ala Thr Ile Ser Gly Gly Gly Val Asp Thr Tyr Tyr Gln Asp Asn Val

50 55 60

Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

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

85 90 95

Ala Arg Pro Tyr Gly His Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 42

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> Hu33VL1/Hu33VL-CDR grafted sequence

<400> 42

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Asn Asn Phe

20 25 30

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

35 40 45

Tyr Tyr Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 43

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> Hu33VL2 sequence

<400> 43

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Asn Asn Phe

20 25 30

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

35 40 45

Tyr Tyr Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 44

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> Hu33VL3 sequence

<400> 44

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Asn Asn Phe

20 25 30

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

35 40 45

Tyr Tyr Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 45

<211> 118

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> Hu33VH2 sequence

<400> 45

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

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

35 40 45

Ala Thr Ile Ser Gly Gly Gly Val Asp Thr Tyr Tyr Gln Asp Asn Val

50 55 60

Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

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

85 90 95

Ala Ser Pro Tyr Gly His Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 46

<211> 118

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> Hu33VH3 sequence

<400> 46

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

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

35 40 45

Ala Thr Ile Ser Gly Gly Gly Val Asp Thr Tyr Tyr Gln Asp Asn Val

50 55 60

Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

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

85 90 95

Ala Ser Pro Tyr Gly His Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 47

<211> 16

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> Hu23LCDR1 (N33Q) sequence

<400> 47

Arg Ser Ser Gln Ser Leu Val His Ser Gln Gly Asn Thr Tyr Leu Glu

1 5 10 15

<210> 48

<211> 16

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> Hu23LCDR1 (N33L) sequence

<400> 48

Arg Ser Ser Gln Ser Leu Val His Ser Leu Gly Asn Thr Tyr Leu Glu

1 5 10 15

<210> 49

<211> 16

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> Hu23LCDR1 (N33T) sequence

<400> 49

Arg Ser Ser Gln Ser Leu Val His Ser Thr Gly Asn Thr Tyr Leu Glu

1 5 10 15

<210> 50

<211> 16

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> Hu23LCDR1 (N33D) sequence

<400> 50

Arg Ser Ser Gln Ser Leu Val His Ser Asp Gly Asn Thr Tyr Leu Glu

1 5 10 15

<210> 51

<211> 16

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> Hu23LCDR1 (G34A) sequence

<400> 51

Arg Ser Ser Gln Ser Leu Val His Ser Asn Ala Asn Thr Tyr Leu Glu

1 5 10 15

<210> 52

<211> 16

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> Hu23LCDR1 (G34V) sequence

<400> 52

Arg Ser Ser Gln Ser Leu Val His Ser Asn Val Asn Thr Tyr Leu Glu

1 5 10 15

<210> 53

<211> 112

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> Hu23VL1 (N33Q) sequence

<400> 53

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Gln Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 54

<211> 112

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> Hu23VL1 (N33L) sequence

<400> 54

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Leu Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 55

<211> 112

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> Hu23VL1 (N33T) sequence

<400> 55

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Thr Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 56

<211> 112

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> Hu23VL1 (N33D) sequence

<400> 56

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asp Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 57

<211> 112

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> Hu23VL1 (G34A) sequence

<400> 57

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

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

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 58

<211> 112

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> Hu23VL1 (G34V) sequence

<400> 58

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Val Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 59

<211> 112

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> Hu23VL2 (N33Q) sequence

<400> 59

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Gln Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 60

<211> 112

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> Hu23VL2 (N33L) sequence

<400> 60

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Leu Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 61

<211> 112

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> Hu23VL2 (N33T) sequence

<400> 61

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Thr Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 62

<211> 112

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> Hu23VL2 (N33D) sequence

<400> 62

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asp Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 63

<211> 112

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> Hu23VL2 (G34A) sequence

<400> 63

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

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

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 64

<211> 112

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> Hu23VL2 (G34V) sequence

<400> 64

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Val Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 65

<211> 5

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> HCDR1 general formula sequences of Hu32 and Hu23 antibodies

<220>

<221> DOMAIN

<222> (4)..(4)

<223> Xaa is selected from Ile or Met

<400> 65

Asp Tyr Glu Xaa His

1 5

<210> 66

<211> 17

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> HCDR2 general formula sequences of Hu32 and Hu23 antibodies

<220>

<221> DOMAIN

<222> (2)..(2)

<223> Xaa is selected from Phe or Ile

<220>

<221> DOMAIN

<222> (9)..(9)

<223> Xaa is selected from Ile or Thr

<220>

<221> DOMAIN

<222> (17)..(17)

<223> Xaa is selected from Gly or Asp

<400> 66

Leu Xaa Asp Pro Glu Thr Gly Gly Xaa Val Tyr Asn Gln Lys Phe Lys

1 5 10 15

Xaa

<210> 67

<211> 16

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> HCDR3 general sequences of Hu32 and Hu23 antibodies

<220>

<221> DOMAIN

<222> (2)..(2)

<223> Xaa is selected from Gly or Arg

<220>

<221> DOMAIN

<222> (3)..(3)

<223> Xaa is selected from Phe or a void

<220>

<221> DOMAIN

<222> (4)..(4)

<223> Xaa is selected from Ser or a void

<220>

<221> DOMAIN

<222> (5)..(5)

<223> Xaa is selected from Tyr or a void

<220>

<221> DOMAIN

<222> (7)..(7)

<223> Xaa is selected from Gly or a void

<220>

<221> DOMAIN

<222> (8)..(8)

<223> Xaa is selected from Ser or a void

<220>

<221> DOMAIN

<222> (9)..(9)

<223> Xaa is selected from Asn or Thr

<220>

<221> DOMAIN

<222> (10)..(10)

<223> Xaa is selected from Arg or Ser

<400> 67

Glu Xaa Xaa Xaa Xaa Tyr Xaa Xaa Xaa Xaa Asp Trp Tyr Phe Asp Val

1 5 10 15

<210> 68

<211> 16

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> LCDR1 general sequences of Hu32 and Hu23 antibodies

<220>

<221> DOMAIN

<222> (6)..(6)

<223> Xaa is selected from Ile or Leu

<220>

<221> DOMAIN

<222> (10)..(10)

<223> Xaa is selected from Asn, gln, leu, thr or Asp

<220>

<221> DOMAIN

<222> (11)..(11)

<223> Xaa is selected from Gly, ala or Val

<220>

<221> DOMAIN

<222> (12)..(12)

<223> Xaa is selected from Asn or Lys

<400> 68

Arg Ser Ser Gln Ser Xaa Val His Ser Xaa Xaa Xaa Thr Tyr Leu Glu

1 5 10 15

<210> 69

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> LCDR3 general sequences of Hu32 and Hu23 antibodies

<220>

<221> DOMAIN

<222> (9)..(9)

<223> Xaa is selected from Ala or Thr

<400> 69

Phe Gln Gly Ser His Val Pro Tyr Xaa

1 5

<210> 70

<211> 125

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> heavy chain variable region general sequences of Hu32 and Hu23 antibodies

<220>

<221> DOMAIN

<222> (26)..(26)

<223> Xaa is selected from Gly or Asp

<220>

<221> DOMAIN

<222> (27)..(27)

<223> Xaa is selected from Gly, phe or Tyr

<220>

<221> DOMAIN

<222> (30)..(30)

<223> Xaa is selected from Ser or Thr

<220>

<221> DOMAIN

<222> (34)..(34)

<223> Xaa is selected from Ile or Met

<220>

<221> DOMAIN

<222> (38)..(38)

<223> Xaa is selected from Arg or Lys

<220>

<221> DOMAIN

<222> (43)..(43)

<223> Xaa is selected from Gln or His

<220>

<221> DOMAIN

<222> (48)..(48)

<223> Xaa is selected from Ile or Met

<220>

<221> DOMAIN

<222> (51)..(51)

<223> Xaa is selected from Phe or Ile

<220>

<221> DOMAIN

<222> (58)..(58)

<223> Xaa is selected from Ile or Thr

<220>

<221> DOMAIN

<222> (66)..(66)

<223> Xaa is selected from Gly or Asp

<220>

<221> DOMAIN

<222> (67)..(67)

<223> Xaa is selected from Arg or Lys

<220>

<221> DOMAIN

<222> (68)..(68)

<223> Xaa is selected from Val, ala or Thr

<220>

<221> DOMAIN

<222> (70)..(70)

<223> Xaa is selected from Arg or Lys

<220>

<221> DOMAIN

<222> (83)..(83)

<223> Xaa is selected from Leu or Phe

<220>

<221> DOMAIN

<222> (97)..(97)

<223> Xaa is selected from Ala or Thr

<220>

<221> DOMAIN

<222> (100)..(100)

<223> Xaa is selected from Gly or Arg

<220>

<221> DOMAIN

<222> (101)..(101)

<223> Xaa is selected from Phe or a void

<220>

<221> DOMAIN

<222> (102)..(102)

<223> Xaa is selected from Ser or a void

<220>

<221> DOMAIN

<222> (103)..(103)

<223> Xaa is selected from Tyr or a void

<220>

<221> DOMAIN

<222> (105)..(105)

<223> Xaa is selected from Gly or a void

<220>

<221> DOMAIN

<222> (106)..(106)

<223> Xaa is selected from Ser or a void

<220>

<221> DOMAIN

<222> (107)..(107)

<223> Xaa is selected from Asn or Thr

<220>

<221> DOMAIN

<222> (108)..(108)

<223> Xaa is selected from Arg or Ser

<400> 70

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Xaa Xaa Thr Phe Xaa Asp Tyr

20 25 30

Glu Xaa His Trp Val Xaa Gln Ala Pro Gly Xaa Gly Leu Glu Trp Xaa

35 40 45

Gly Leu Xaa Asp Pro Glu Thr Gly Gly Xaa Val Tyr Asn Gln Lys Phe

50 55 60

Lys Xaa Xaa Xaa Thr Xaa Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Xaa Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Xaa Arg Glu Xaa Xaa Xaa Xaa Tyr Xaa Xaa Xaa Xaa Asp Trp Tyr Phe

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 71

<211> 112

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> light chain variable region general sequences of Hu32 and Hu23 antibodies

<220>

<221> DOMAIN

<222> (2)..(2)

<223> Xaa is selected from Ile, val or Gly

<220>

<221> DOMAIN

<222> (29)..(29)

<223> Xaa is selected from Ile or Leu

<220>

<221> DOMAIN

<222> (33)..(33)

<223> Xaa is selected from Asn, gln, leu, thr or Asp

<220>

<221> DOMAIN

<222> (34)..(34)

<223> Xaa is selected from Gly, ala or Val

<220>

<221> DOMAIN

<222> (35)..(35)

<223> Xaa is selected from Asn or Lys

<220>

<221> DOMAIN

<222> (102)..(102)

<223> Xaa is selected from Ala or Thr

<400> 71

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Xaa Val His Ser

20 25 30

Xaa Xaa Xaa Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Xaa Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 72

<211> 327

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> IgG4-AA heavy chain constant region sequence

<400> 72

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

1 5 10 15

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

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

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

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210> 73

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> antibody Kappa light chain constant region sequence

<400> 73

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 74

<211> 452

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> Hu23-11.IgG4AA antibody heavy chain sequences

<400> 74

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asp Tyr

20 25 30

Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

115 120 125

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

130 135 140

Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys

195 200 205

Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu

210 215 220

Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

225 230 235 240

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

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

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

405 410 415

Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Leu Gly Lys

450

<210> 75

<211> 219

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> Hu23-11.IgG4AA/Hu32a-85.IgG4AA/Hu23-11.IgG4P/Hu32a-85.IgG4P light chain sequence

<400> 75

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Thr Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 76

<211> 447

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> Hu32a-85.IgG4AA heavy chain sequence

<400> 76

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Asp Phe Thr Phe Thr Asp Tyr

20 25 30

Glu Ile His Trp Val Lys Gln Ala Pro Gly His Gly Leu Glu Trp Ile

35 40 45

Gly Leu Phe Asp Pro Glu Thr Gly Gly Ile Val Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Thr Arg Glu Gly Tyr Asn Arg Asp Trp Tyr Phe Asp Val Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

210 215 220

Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

325 330 335

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

340 345 350

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

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

405 410 415

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 77

<211> 445

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> Hu33-6.IgG4AA heavy chain sequence

<400> 77

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

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

35 40 45

Ala Thr Ile Ser Gly Gly Gly Val Asp Thr Tyr Tyr Gln Asp Asn Val

50 55 60

Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

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

85 90 95

Ala Ser Pro Tyr Gly His Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 78

<211> 214

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> Hu33-6.IgG4AA/Hu33-6.IgG4P light chain sequence

<400> 78

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Asn Asn Phe

20 25 30

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

35 40 45

Tyr Tyr Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

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

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 79

<211> 327

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> heavy chain constant region sequence of IgG4-P

<400> 79

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

1 5 10 15

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

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

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

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210> 80

<211> 452

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> Hu23-11.IgG4P antibody heavy chain sequence

<400> 80

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asp Tyr

20 25 30

Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

115 120 125

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

130 135 140

Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys

195 200 205

Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu

210 215 220

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

225 230 235 240

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

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

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

405 410 415

Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Leu Gly Lys

450

<210> 81

<211> 447

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> Hu32a-85.IgG4P heavy chain sequence

<400> 81

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Asp Phe Thr Phe Thr Asp Tyr

20 25 30

Glu Ile His Trp Val Lys Gln Ala Pro Gly His Gly Leu Glu Trp Ile

35 40 45

Gly Leu Phe Asp Pro Glu Thr Gly Gly Ile Val Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Thr Arg Glu Gly Tyr Asn Arg Asp Trp Tyr Phe Asp Val Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

210 215 220

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

325 330 335

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

340 345 350

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

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

405 410 415

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 82

<211> 445

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> Hu33-6.IgG4P heavy chain sequence

<400> 82

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

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

35 40 45

Ala Thr Ile Ser Gly Gly Gly Val Asp Thr Tyr Tyr Gln Asp Asn Val

50 55 60

Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

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

85 90 95

Ala Ser Pro Tyr Gly His Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 83

<211> 454

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> CHAIN

<223> heavy chain of Lucentis

<400> 83

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Asp Phe Thr His Tyr

20 25 30

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

35 40 45

Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe

50 55 60

Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Lys Tyr Pro Tyr Tyr Tyr Gly Thr Ser His Trp Tyr Phe Asp Val

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

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

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

195 200 205

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

210 215 220

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

225 230 235 240

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

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

275 280 285

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

305 310 315 320

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

325 330 335

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

340 345 350

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

355 360 365

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

370 375 380

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

385 390 395 400

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

405 410 415

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

420 425 430

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

435 440 445

Leu Ser Leu Pro Gly Lys

450

<210> 84

<211> 214

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> CHAIN

<223> light chain of Lucentis

<400> 84

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

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

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 85

<211> 123

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> heavy chain variable region of Lucentis

<400> 85

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Asp Phe Thr His Tyr

20 25 30

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

35 40 45

Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe

50 55 60

Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Lys Tyr Pro Tyr Tyr Tyr Gly Thr Ser His Trp Tyr Phe Asp Val

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 86

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> light chain variable region of Lucentis

<400> 86

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 87

<211> 5

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> HCDR1 of Lucentis

<400> 87

His Tyr Gly Met Asn

1 5

<210> 88

<211> 17

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> HCDR2 of Lucentis

<400> 88

Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe Lys

1 5 10 15

Arg

<210> 89

<211> 14

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> HCDR3 of Lucentis

<400> 89

Tyr Pro Tyr Tyr Tyr Gly Thr Ser His Trp Tyr Phe Asp Val

1 5 10

<210> 90

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> LCDR1 of Lucentis

<400> 90

Ser Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn

1 5 10

<210> 91

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> LCDR2 of Lucentis

<400> 91

Phe Thr Ser Ser Leu His Ser

1 5

<210> 92

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> LCDR3 of Lucentis

<400> 92

Gln Gln Tyr Ser Thr Val Pro Trp Thr

1 5

<210> 93

<211> 431

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<223> VEGFtrap-Fc

<400> 93

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg

85 90 95

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr

115 120 125

Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys

130 135 140

His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly

145 150 155 160

Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr

165 170 175

Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met

180 185 190

Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Asp Lys Thr

195 200 205

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

210 215 220

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

225 230 235 240

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

245 250 255

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

260 265 270

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

275 280 285

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

290 295 300

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

305 310 315 320

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

325 330 335

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

340 345 350

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

355 360 365

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

370 375 380

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

385 390 395 400

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

405 410 415

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

420 425 430

<210> 94

<211> 205

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<223> VEGFtrap

<400> 94

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg

85 90 95

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr

115 120 125

Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys

130 135 140

His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly

145 150 155 160

Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr

165 170 175

Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met

180 185 190

Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys

195 200 205

<210> 95

<211> 205

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<223> VEGFtrap mutants

<400> 95

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg

85 90 95

Gln Thr Asn Cys Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr

115 120 125

Cys Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys

130 135 140

His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly

145 150 155 160

Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr

165 170 175

Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met

180 185 190

Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys

195 200 205

<210> 96

<211> 205

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<223> VEGFtrap mutants

<400> 96

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg

85 90 95

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr

115 120 125

Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys

130 135 140

His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly

145 150 155 160

Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr

165 170 175

Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Cys Ser Gly Leu Met

180 185 190

Cys Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys

195 200 205

<210> 97

<211> 205

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<223> VEGFtrap mutants

<400> 97

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg

85 90 95

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr

115 120 125

Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys

130 135 140

His Gln His Lys Lys Leu Val Asn Arg Asp Cys Lys Thr Gln Ser Gly

145 150 155 160

Ser Glu Met Lys Cys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr

165 170 175

Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met

180 185 190

Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys

195 200 205

<210> 98

<211> 205

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<223> VEGFtrap mutants

<400> 98

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg

85 90 95

Gln Thr Asn Thr Thr Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Ala Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr

115 120 125

Glu Leu Asn Ala Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys

130 135 140

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

145 150 155 160

Ser Thr Met Asn Lys Tyr Leu Ser Thr Leu Thr Ile Asp Gly Val Thr

165 170 175

Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met

180 185 190

Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys

195 200 205

<210> 99

<211> 205

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<223> VEGFtrap mutants

<400> 99

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg

85 90 95

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr

115 120 125

Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys

130 135 140

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

145 150 155 160

Ser Asp Met Ala Lys Arg Leu Ser Thr Leu Thr Ile Asp Gly Val Thr

165 170 175

Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met

180 185 190

Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys

195 200 205

<210> 100

<211> 205

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<223> VEGFtrap mutants

<400> 100

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg

85 90 95

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr

115 120 125

Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys

130 135 140

His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Pro

145 150 155 160

Ser Ser Met Glu Lys Ala Leu Ser Thr Leu Thr Ile Asp Gly Val Thr

165 170 175

Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met

180 185 190

Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys

195 200 205

<210> 101

<211> 714

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> CHAIN

<223> heavy chain of VP101

<400> 101

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

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

35 40 45

Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe

50 55 60

Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Lys Tyr Pro His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp Val

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val

195 200 205

Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys

210 215 220

Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu

260 265 270

Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu

435 440 445

Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

450 455 460

Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu

465 470 475 480

Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe

485 490 495

Ala Phe Ser Ser Tyr Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys

500 505 510

Gly Leu Asp Trp Val Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr

515 520 525

Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

530 535 540

Lys Asn Asn Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

545 550 555 560

Ala Leu Tyr Tyr Cys Ala Asn Arg Tyr Gly Glu Ala Trp Phe Ala Tyr

565 570 575

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

580 585 590

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp

595 600 605

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

610 615 620

Arg Val Thr Phe Thr Cys Arg Ala Ser Gln Asp Ile Asn Thr Tyr Leu

625 630 635 640

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

645 650 655

Arg Ala Asn Arg Leu Val Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

660 665 670

Gly Ser Gly Gln Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

675 680 685

Asp Met Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Leu Thr

690 695 700

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

705 710

<210> 102

<211> 214

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> CHAIN

<223> light chain of VP101

<400> 102

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

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

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 103

<211> 659

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> CHAIN

<223> heavy chain of By24.3

<400> 103

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

1 5 10 15

Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser

20 25 30

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

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe

65 70 75 80

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

85 90 95

Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

100 105 110

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

115 120 125

Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp

130 135 140

Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr

145 150 155 160

Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr

165 170 175

Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys

180 185 190

Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp

195 200 205

Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala

210 215 220

Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

225 230 235 240

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

245 250 255

Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val

260 265 270

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

275 280 285

Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

290 295 300

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly

305 310 315 320

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

325 330 335

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr

340 345 350

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

355 360 365

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

370 375 380

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

385 390 395 400

Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe

405 410 415

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

420 425 430

Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

435 440 445

Gly Gly Gly Gly Ser Ala Ser Asp Thr Gly Arg Pro Phe Val Glu Met

450 455 460

Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg Glu Leu

465 470 475 480

Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr Leu Lys

485 490 495

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

500 505 510

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

515 520 525

Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr

530 535 540

Asn Tyr Leu Thr His Arg Gln Thr Asn Thr Ile Ile Asp Val Val Leu

545 550 555 560

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

565 570 575

Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe Asn Trp

580 585 590

Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn Arg Asp

595 600 605

Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser Thr Leu

610 615 620

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

625 630 635 640

Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val Arg Val

645 650 655

His Glu Lys

<210> 104

<211> 214

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> CHAIN

<223> light chain of By24.3

<400> 104

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

1 5 10 15

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

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

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

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 105

<211> 583

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> CHAIN

<223> heavy chain of DVD-GS8

<400> 105

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asp Tyr

20 25 30

Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu

130 135 140

Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr

145 150 155 160

Asp Phe Thr His Tyr Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys

165 170 175

Gly Leu Glu Trp Val Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr

180 185 190

Tyr Ala Ala Asp Phe Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser

195 200 205

Lys Ser Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

210 215 220

Ala Val Tyr Tyr Cys Ala Lys Tyr Pro Tyr Tyr Tyr Gly Thr Ser His

225 230 235 240

Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

245 250 255

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

260 265 270

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

275 280 285

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

290 295 300

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

305 310 315 320

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

325 330 335

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

340 345 350

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

355 360 365

Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

370 375 380

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

385 390 395 400

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

405 410 415

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

420 425 430

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

435 440 445

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

450 455 460

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

465 470 475 480

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

485 490 495

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

500 505 510

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

515 520 525

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

530 535 540

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

545 550 555 560

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

565 570 575

Leu Ser Leu Ser Leu Gly Lys

580

<210> 106

<211> 334

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> CHAIN

<223> light chain of DVD-GS8

<400> 106

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Thr Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

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

115 120 125

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

130 135 140

Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro

145 150 155 160

Gly Lys Ala Pro Lys Val Leu Ile Tyr Phe Thr Ser Ser Leu His Ser

165 170 175

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

180 185 190

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

195 200 205

Gln Gln Tyr Ser Thr Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val

210 215 220

Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro

225 230 235 240

Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu

245 250 255

Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn

260 265 270

Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser

275 280 285

Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala

290 295 300

Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly

305 310 315 320

Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

325 330

<210> 107

<211> 585

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> CHAIN

<223> heavy chain of DVD-GS10

<400> 107

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asp Tyr

20 25 30

Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly

130 135 140

Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

145 150 155 160

Gly Tyr Asp Phe Thr His Tyr Gly Met Asn Trp Val Arg Gln Ala Pro

165 170 175

Gly Lys Gly Leu Glu Trp Val Gly Trp Ile Asn Thr Tyr Thr Gly Glu

180 185 190

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

195 200 205

Thr Ser Lys Ser Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

210 215 220

Asp Thr Ala Val Tyr Tyr Cys Ala Lys Tyr Pro Tyr Tyr Tyr Gly Thr

225 230 235 240

Ser His Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val

245 250 255

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

260 265 270

Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys

275 280 285

Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu

290 295 300

Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu

305 310 315 320

Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr

325 330 335

Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val

340 345 350

Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro

355 360 365

Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

370 375 380

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

385 390 395 400

Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr

405 410 415

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

420 425 430

Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

435 440 445

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

450 455 460

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

465 470 475 480

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met

485 490 495

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

500 505 510

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

515 520 525

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

530 535 540

Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val

545 550 555 560

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

565 570 575

Lys Ser Leu Ser Leu Ser Leu Gly Lys

580 585

<210> 108

<211> 336

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> CHAIN

<223> light chain of DVD-GS10

<400> 108

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Thr Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln

115 120 125

Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr

130 135 140

Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp Thr Phe Gly Gln Gly Thr

210 215 220

Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe

225 230 235 240

Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys

245 250 255

Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val

260 265 270

Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln

275 280 285

Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser

290 295 300

Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His

305 310 315 320

Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

325 330 335

<210> 109

<211> 594

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> CHAIN

<223> heavy chain of DVD-H19L14

<400> 109

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asp Tyr

20 25 30

Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

130 135 140

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

145 150 155 160

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Asp Phe Thr His Tyr

165 170 175

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

180 185 190

Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe

195 200 205

Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr

210 215 220

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

225 230 235 240

Ala Lys Tyr Pro Tyr Tyr Tyr Gly Thr Ser His Trp Tyr Phe Asp Val

245 250 255

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

260 265 270

Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser

275 280 285

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

290 295 300

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

305 310 315 320

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

325 330 335

Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val

340 345 350

Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys

355 360 365

Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

370 375 380

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

385 390 395 400

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu

405 410 415

Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

420 425 430

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg

435 440 445

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

450 455 460

Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu

465 470 475 480

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

485 490 495

Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu

500 505 510

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

515 520 525

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

530 535 540

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp

545 550 555 560

Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His

565 570 575

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu

580 585 590

Gly Lys

<210> 110

<211> 340

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> CHAIN

<223> light chain of DVD-H19L14

<400> 110

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Thr Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp Ile

115 120 125

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

130 135 140

Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn

145 150 155 160

Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile Tyr Phe

165 170 175

Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly

180 185 190

Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp

195 200 205

Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp Thr Phe

210 215 220

Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser

225 230 235 240

Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala

245 250 255

Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val

260 265 270

Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser

275 280 285

Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr

290 295 300

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

305 310 315 320

Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn

325 330 335

Arg Gly Glu Cys

340

<210> 111

<211> 597

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> CHAIN

<223> heavy chain of DVD-H21L14

<400> 111

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asp Tyr

20 25 30

Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

130 135 140

Gly Ser Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

145 150 155 160

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Asp Phe

165 170 175

Thr His Tyr Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

180 185 190

Glu Trp Val Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala

195 200 205

Ala Asp Phe Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser

210 215 220

Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

225 230 235 240

Tyr Tyr Cys Ala Lys Tyr Pro Tyr Tyr Tyr Gly Thr Ser His Trp Tyr

245 250 255

Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser

260 265 270

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

275 280 285

Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro

290 295 300

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

305 310 315 320

His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

325 330 335

Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr

340 345 350

Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val

355 360 365

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala

370 375 380

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

385 390 395 400

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

405 410 415

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

420 425 430

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

435 440 445

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

450 455 460

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

465 470 475 480

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

485 490 495

Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

500 505 510

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

515 520 525

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

530 535 540

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

545 550 555 560

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

565 570 575

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

580 585 590

Leu Ser Leu Gly Lys

595

<210> 112

<211> 589

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> CHAIN

<223> heavy chain of DVD-GS14

<400> 112

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asp Tyr

20 25 30

Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Val

130 135 140

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

145 150 155 160

Cys Ala Ala Ser Gly Tyr Asp Phe Thr His Tyr Gly Met Asn Trp Val

165 170 175

Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Trp Ile Asn Thr

180 185 190

Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe Lys Arg Arg Phe Thr

195 200 205

Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr Leu Gln Met Asn Ser

210 215 220

Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Tyr Pro Tyr

225 230 235 240

Tyr Tyr Gly Thr Ser His Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr

245 250 255

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

260 265 270

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

275 280 285

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

290 295 300

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

305 310 315 320

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

325 330 335

Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

340 345 350

Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

355 360 365

Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu

370 375 380

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

385 390 395 400

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

405 410 415

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

420 425 430

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

435 440 445

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

515 520 525

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

530 535 540

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

545 550 555 560

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

565 570 575

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

580 585

<210> 113

<211> 345

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> CHAIN

<223> light chain of DVD-GS19

<400> 113

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Thr Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Val Leu Ile Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg

180 185 190

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser

195 200 205

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

210 215 220

Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr

225 230 235 240

Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu

245 250 255

Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro

260 265 270

Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly

275 280 285

Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr

290 295 300

Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His

305 310 315 320

Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val

325 330 335

Thr Lys Ser Phe Asn Arg Gly Glu Cys

340 345

<210> 114

<211> 599

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> CHAIN

<223> heavy chain of DVD-H24L19

<400> 114

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asp Tyr

20 25 30

Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

130 135 140

Ser Gly Gly Gly Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu

145 150 155 160

Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr

165 170 175

Asp Phe Thr His Tyr Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys

180 185 190

Gly Leu Glu Trp Val Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr

195 200 205

Tyr Ala Ala Asp Phe Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser

210 215 220

Lys Ser Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

225 230 235 240

Ala Val Tyr Tyr Cys Ala Lys Tyr Pro Tyr Tyr Tyr Gly Thr Ser His

245 250 255

Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

260 265 270

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

275 280 285

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

290 295 300

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

305 310 315 320

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

325 330 335

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

340 345 350

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

355 360 365

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

370 375 380

Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

385 390 395 400

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

405 410 415

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

420 425 430

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

435 440 445

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

450 455 460

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

465 470 475 480

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

485 490 495

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

500 505 510

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

515 520 525

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

530 535 540

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

545 550 555 560

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

565 570 575

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

580 585 590

Leu Ser Leu Ser Leu Gly Lys

595

<210> 115

<211> 706

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> CHAIN

<223> heavy chain of IgG-GS10-scFv

<400> 115

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asp Tyr

20 25 30

Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

115 120 125

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

130 135 140

Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys

195 200 205

Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu

210 215 220

Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

225 230 235 240

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

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

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

405 410 415

Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln

450 455 460

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

465 470 475 480

Leu Ser Cys Ala Ala Ser Gly Tyr Asp Phe Thr His Tyr Gly Met Asn

485 490 495

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

500 505 510

Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe Lys Arg Arg

515 520 525

Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr Leu Gln Met

530 535 540

Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Tyr

545 550 555 560

Pro Tyr Tyr Tyr Gly Thr Ser His Trp Tyr Phe Asp Val Trp Gly Gln

565 570 575

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

Lys Ala Pro Lys Val Leu Ile Tyr Phe Thr Ser Ser Leu His Ser Gly

645 650 655

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

660 665 670

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

675 680 685

Gln Tyr Ser Thr Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu

690 695 700

Ile Lys

705

<210> 116

<211> 709

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> CHAIN

<223> heavy chain of IgG-GS13-scFv

<400> 116

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asp Tyr

20 25 30

Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

115 120 125

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

130 135 140

Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys

195 200 205

Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu

210 215 220

Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

225 230 235 240

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

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

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

405 410 415

Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Leu Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

450 455 460

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

465 470 475 480

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Asp Phe Thr His Tyr

485 490 495

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

500 505 510

Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe

515 520 525

Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr

530 535 540

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

545 550 555 560

Ala Lys Tyr Pro Tyr Tyr Tyr Gly Thr Ser His Trp Tyr Phe Asp Val

565 570 575

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

580 585 590

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln

595 600 605

Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr

610 615 620

Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp Thr Phe Gly Gln Gly Thr

690 695 700

Lys Val Glu Ile Lys

705

<210> 117

<211> 715

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> CHAIN

<223> heavy chain of IgG-GS19-scFv

<400> 117

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asp Tyr

20 25 30

Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

115 120 125

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

130 135 140

Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys

195 200 205

Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu

210 215 220

Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

225 230 235 240

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

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

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

405 410 415

Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

450 455 460

Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly

465 470 475 480

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

485 490 495

Tyr Asp Phe Thr His Tyr Gly Met Asn Trp Val Arg Gln Ala Pro Gly

500 505 510

Lys Gly Leu Glu Trp Val Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro

515 520 525

Thr Tyr Ala Ala Asp Phe Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr

530 535 540

Ser Lys Ser Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

545 550 555 560

Thr Ala Val Tyr Tyr Cys Ala Lys Tyr Pro Tyr Tyr Tyr Gly Thr Ser

565 570 575

His Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser

580 585 590

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

660 665 670

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

675 680 685

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp

690 695 700

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

705 710 715

<210> 118

<211> 675

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> CHAIN

<223> heavy chain of Hu23-2TE

<400> 118

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asp Tyr

20 25 30

Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

115 120 125

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

130 135 140

Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys

195 200 205

Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu

210 215 220

Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

225 230 235 240

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

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

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

405 410 415

Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

450 455 460

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

465 470 475 480

Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg Glu Leu

485 490 495

Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr Leu Lys

500 505 510

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

515 520 525

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

530 535 540

Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr

545 550 555 560

Asn Tyr Leu Thr His Arg Gln Thr Asn Cys Ile Ile Asp Val Val Leu

565 570 575

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

580 585 590

Asn Cys Thr Ala Arg Thr Cys Leu Asn Val Gly Ile Asp Phe Asn Trp

595 600 605

Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn Arg Asp

610 615 620

Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser Thr Leu

625 630 635 640

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

645 650 655

Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val Arg Val

660 665 670

His Glu Lys

675

<210> 119

<211> 675

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> CHAIN

<223> heavy chain of Hu23-3ST

<400> 119

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asp Tyr

20 25 30

Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

115 120 125

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

130 135 140

Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys

195 200 205

Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu

210 215 220

Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

225 230 235 240

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

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

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

405 410 415

Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

450 455 460

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

465 470 475 480

Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg Glu Leu

485 490 495

Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr Leu Lys

500 505 510

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

515 520 525

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

530 535 540

Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr

545 550 555 560

Asn Tyr Leu Thr His Arg Gln Thr Asn Thr Ile Ile Asp Val Val Leu

565 570 575

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

580 585 590

Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe Asn Trp

595 600 605

Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn Arg Asp

610 615 620

Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser Thr Leu

625 630 635 640

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

645 650 655

Ala Cys Ser Gly Leu Met Cys Lys Lys Asn Ser Thr Phe Val Arg Val

660 665 670

His Glu Lys

675

<210> 120

<211> 674

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> CHAIN

<223> heavy chain of Hu23-5LK

<400> 120

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

1 5 10 15

Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asp Tyr Glu

20 25 30

Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly

35 40 45

Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe Lys

50 55 60

Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met

65 70 75 80

Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe Asp

100 105 110

Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu

130 135 140

Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn

195 200 205

Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser

210 215 220

Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln

260 265 270

Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

450 455 460

Ser Gly Gly Gly Gly Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr

465 470 475 480

Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg Glu Leu Val

485 490 495

Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys

500 505 510

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

515 520 525

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

530 535 540

Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn

545 550 555 560

Tyr Leu Thr His Arg Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser

565 570 575

Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn

580 585 590

Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu

595 600 605

Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn Arg Asp Cys

610 615 620

Lys Thr Gln Ser Gly Ser Glu Met Lys Cys Phe Leu Ser Thr Leu Thr

625 630 635 640

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

645 650 655

Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val Arg Val His

660 665 670

Glu Lys

<210> 121

<211> 678

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> CHAIN

<223> heavy chain of 3-6

<400> 121

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asp Tyr

20 25 30

Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

115 120 125

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

130 135 140

Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys

195 200 205

Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu

210 215 220

Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

225 230 235 240

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

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

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

405 410 415

Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Leu Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

450 455 460

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

465 470 475 480

Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly

485 490 495

Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val

500 505 510

Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg

515 520 525

Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr

530 535 540

Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu

545 550 555 560

Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn Thr Thr Ile Asp

565 570 575

Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Ala Gly Glu Lys

580 585 590

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

595 600 605

Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val

610 615 620

Asn Arg Asp Leu Lys Thr Gln Ser Gln Ser Thr Met Asn Lys Tyr Leu

625 630 635 640

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

645 650 655

Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe

660 665 670

Val Arg Val His Glu Lys

675

<210> 122

<211> 678

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> CHAIN

<223> heavy chain of 3-12

<400> 122

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asp Tyr

20 25 30

Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

115 120 125

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

130 135 140

Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys

195 200 205

Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu

210 215 220

Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

225 230 235 240

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

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

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

405 410 415

Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Leu Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

450 455 460

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

465 470 475 480

Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly

485 490 495

Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val

500 505 510

Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg

515 520 525

Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr

530 535 540

Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu

545 550 555 560

Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn Thr Ile Ile Asp

565 570 575

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

580 585 590

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

595 600 605

Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val

610 615 620

Asn Arg Asp Leu Lys Thr Gln Ser Thr Ser Asp Met Ala Lys Arg Leu

625 630 635 640

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

645 650 655

Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe

660 665 670

Val Arg Val His Glu Lys

675

<210> 123

<211> 678

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> CHAIN

<223> heavy chain of 6-37

<400> 123

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asp Tyr

20 25 30

Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

115 120 125

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

130 135 140

Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys

195 200 205

Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu

210 215 220

Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

225 230 235 240

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

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

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

405 410 415

Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Leu Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

450 455 460

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

465 470 475 480

Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly

485 490 495

Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val

500 505 510

Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg

515 520 525

Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr

530 535 540

Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu

545 550 555 560

Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn Thr Ile Ile Asp

565 570 575

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

580 585 590

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

595 600 605

Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val

610 615 620

Asn Arg Asp Leu Lys Thr Gln Ser Pro Ser Ser Met Glu Lys Ala Leu

625 630 635 640

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

645 650 655

Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe

660 665 670

Val Arg Val His Glu Lys

675

<210> 124

<211> 676

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> CHAIN

Heavy chain of <223> #2

<400> 124

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg

85 90 95

Gln Thr Asn Cys Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr

115 120 125

Cys Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys

130 135 140

His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly

145 150 155 160

Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr

165 170 175

Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met

180 185 190

Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Gly Gly Gly

195 200 205

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

210 215 220

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

225 230 235 240

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asp Tyr

245 250 255

Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

260 265 270

Gly Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe

275 280 285

Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

290 295 300

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

305 310 315 320

Ala Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe

325 330 335

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

340 345 350

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

355 360 365

Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

370 375 380

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

385 390 395 400

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

405 410 415

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys

420 425 430

Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu

435 440 445

Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

450 455 460

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

465 470 475 480

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

485 490 495

Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

500 505 510

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

515 520 525

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

530 535 540

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser

545 550 555 560

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

565 570 575

Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val

580 585 590

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

595 600 605

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

610 615 620

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

625 630 635 640

Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val

645 650 655

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

660 665 670

Ser Leu Gly Lys

675

<210> 125

<211> 676

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> CHAIN

Heavy chain of <223> #3

<400> 125

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg

85 90 95

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr

115 120 125

Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys

130 135 140

His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly

145 150 155 160

Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr

165 170 175

Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Cys Ser Gly Leu Met

180 185 190

Cys Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Gly Gly Gly

195 200 205

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

210 215 220

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

225 230 235 240

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asp Tyr

245 250 255

Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

260 265 270

Gly Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe

275 280 285

Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

290 295 300

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

305 310 315 320

Ala Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe

325 330 335

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

340 345 350

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

355 360 365

Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

370 375 380

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

385 390 395 400

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

405 410 415

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys

420 425 430

Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu

435 440 445

Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

450 455 460

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

465 470 475 480

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

485 490 495

Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

500 505 510

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

515 520 525

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

530 535 540

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser

545 550 555 560

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

565 570 575

Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val

580 585 590

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

595 600 605

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

610 615 620

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

625 630 635 640

Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val

645 650 655

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

660 665 670

Ser Leu Gly Lys

675

<210> 126

<211> 676

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> CHAIN

Heavy chain of <223> #5

<400> 126

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg

85 90 95

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr

115 120 125

Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys

130 135 140

His Gln His Lys Lys Leu Val Asn Arg Asp Cys Lys Thr Gln Ser Gly

145 150 155 160

Ser Glu Met Lys Cys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr

165 170 175

Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met

180 185 190

Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Gly Gly Gly

195 200 205

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

210 215 220

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

225 230 235 240

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asp Tyr

245 250 255

Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

260 265 270

Gly Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe

275 280 285

Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

290 295 300

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

305 310 315 320

Ala Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe

325 330 335

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

340 345 350

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

355 360 365

Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

370 375 380

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

385 390 395 400

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

405 410 415

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys

420 425 430

Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu

435 440 445

Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

450 455 460

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

465 470 475 480

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

485 490 495

Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

500 505 510

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

515 520 525

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

530 535 540

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser

545 550 555 560

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

565 570 575

Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val

580 585 590

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

595 600 605

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

610 615 620

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

625 630 635 640

Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val

645 650 655

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

660 665 670

Ser Leu Gly Lys

675

<210> 127

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<223> peptide linker

<400> 127

Gly Ser Gly Gly Gly Gly Ser Gly

1 5

<210> 128

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<223> peptide linker

<400> 128

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 129

<211> 19

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<223> peptide linker

<400> 129

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly

<210> 130

<211> 14

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<223> peptide linker

<400> 130

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

1 5 10

<210> 131

<211> 22

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<223> peptide linker

<400> 131

Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

Gly Gly Gly Gly Ser Gly

20

<210> 132

<211> 24

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<223> peptide linker

<400> 132

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly

20

<210> 133

<211> 13

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<223> peptide linker

<400> 133

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 134

<211> 20

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<223> peptide linker

<400> 134

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

20

<210> 135

<211> 582

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> CHAIN

<223> heavy chain

<400> 135

Glu Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Pro Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ala Gln Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr

85 90 95

Tyr Cys Thr Gly Ser Tyr Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val

130 135 140

Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Asp

145 150 155 160

Phe Thr His Tyr Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly

165 170 175

Leu Glu Trp Val Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr

180 185 190

Ala Ala Asp Phe Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys

195 200 205

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

210 215 220

Val Tyr Tyr Cys Ala Lys Tyr Pro Tyr Tyr Tyr Gly Thr Ser His Trp

225 230 235 240

Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala

245 250 255

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

260 265 270

Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe

275 280 285

Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly

290 295 300

Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu

305 310 315 320

Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr

325 330 335

Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg

340 345 350

Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu

355 360 365

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

370 375 380

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

385 390 395 400

Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

405 410 415

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

420 425 430

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

435 440 445

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

450 455 460

Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

465 470 475 480

Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn

485 490 495

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

500 505 510

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

515 520 525

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

530 535 540

Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys

545 550 555 560

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

565 570 575

Ser Leu Ser Leu Gly Lys

580

<210> 136

<211> 340

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> CHAIN

<223> light chain

<400> 136

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

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Arg Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Val Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser

85 90 95

Thr His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp Ile

115 120 125

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

130 135 140

Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn

145 150 155 160

Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile Tyr Phe

165 170 175

Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly

180 185 190

Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp

195 200 205

Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp Thr Phe

210 215 220

Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser

225 230 235 240

Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala

245 250 255

Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val

260 265 270

Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser

275 280 285

Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr

290 295 300

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

305 310 315 320

Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn

325 330 335

Arg Gly Glu Cys

340

<210> 137

<211> 563

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> CHAIN

<223> heavy chain of Hu33-V6

<400> 137

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

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

35 40 45

Ala Thr Ile Ser Gly Gly Gly Val Asp Thr Tyr Tyr Gln Asp Asn Val

50 55 60

Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

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

85 90 95

Ala Ser Pro Tyr Gly His Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly

435 440 445

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

450 455 460

Asp Thr Gly Arg Pro Phe Val Glu Met Cys Ser Glu Ile Pro Glu Ile

465 470 475 480

Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr

485 490 495

Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu

500 505 510

Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile

515 520 525

Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala

530 535 540

Thr Val Asn Gly His Leu Tyr Lys Cys Asn Tyr Leu Thr His Arg Gln

545 550 555 560

Thr Asn Thr

<210> 138

<211> 570

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> CHAIN

<223> heavy chain of Hu23-V6

<400> 138

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asp Tyr

20 25 30

Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

115 120 125

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

130 135 140

Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys

195 200 205

Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu

210 215 220

Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

225 230 235 240

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

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

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

405 410 415

Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

450 455 460

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

465 470 475 480

Cys Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg Glu Leu

485 490 495

Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr Leu Lys

500 505 510

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

515 520 525

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

530 535 540

Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys Cys

545 550 555 560

Asn Tyr Leu Thr His Arg Gln Thr Asn Thr

565 570

<210> 139

<211> 563

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> CHAIN

<223> heavy chain of Hu33-WT

<400> 139

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

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

35 40 45

Ala Thr Ile Ser Gly Gly Gly Val Asp Thr Tyr Tyr Gln Asp Asn Val

50 55 60

Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

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

85 90 95

Ala Ser Pro Tyr Gly His Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly

435 440 445

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

450 455 460

Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile

465 470 475 480

Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr

485 490 495

Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu

500 505 510

Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile

515 520 525

Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala

530 535 540

Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln

545 550 555 560

Thr Asn Thr

<210> 140

<211> 570

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> CHAIN

<223> heavy chain of Hu23-WT

<400> 140

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asp Tyr

20 25 30

Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Leu Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Glu Arg Phe Ser Tyr Tyr Gly Ser Thr Ser Asp Trp Tyr Phe

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

115 120 125

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

130 135 140

Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys

195 200 205

Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu

210 215 220

Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

225 230 235 240

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

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

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

405 410 415

Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

450 455 460

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

465 470 475 480

Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg Glu Leu

485 490 495

Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr Leu Lys

500 505 510

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

515 520 525

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

530 535 540

Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr

545 550 555 560

Asn Tyr Leu Thr His Arg Gln Thr Asn Thr

565 570

<210> 141

<211> 100

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> WT

<400> 141

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg

85 90 95

Gln Thr Asn Thr

100

<210> 142

<211> 100

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> V6

<400> 142

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Cys Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Cys Asn Tyr Leu Thr His Arg

85 90 95

Gln Thr Asn Thr

100

Claims

1. A bispecific antigen binding molecule comprising at least one first antigen binding domain capable of specifically binding to PD-1, and at least one second antigen binding domain capable of specifically binding to VEGF;

the PD-1-VH has: SEQ ID NO:8, PD-1-HCDR1 of the amino acid sequence of SEQ ID NO:9, and PD-1-HCDR2 of the amino acid sequence of SEQ ID NO:10, PD-1-HCDR3 of the amino acid sequence of seq id no; and is also provided with

The PD-1-VL has: SEQ ID NO:49, PD-1-LCDR1 of the amino acid sequence of SEQ ID NO:12, and PD-1-LCDR2 of the amino acid sequence of SEQ ID NO:13, PD-1-LCDR3 of the amino acid sequence of 13; and

wherein the second antigen-binding domain capable of specifically binding VEGF comprises a heavy chain variable region VEGF-VH and a light chain variable region VEGF-VL, wherein:

the VEGF-VH has: SEQ ID NO:87, VEGF-HCDR1 of the amino acid sequence of SEQ ID NO:88, and VEGF-HCDR2 of the amino acid sequence of SEQ ID NO:89, VEGF-HCDR3 of the amino acid sequence; and is also provided with

The VEGF-VL has: SEQ ID NO:90, and VEGF-LCDR1 of the amino acid sequence of SEQ ID NO:91, and VEGF-LCDR2 of the amino acid sequence of SEQ ID NO:92, VEGF-LCDR3 of the amino acid sequence; and

wherein the PD-1-VH is linked to the VEGF-VH by a linker and the PD-1-VL is linked to the VEGF-VL by a linker.

2. The bispecific antigen-binding molecule of claim 1, wherein the PD-1-VH comprises SEQ ID NO:27, and the PD-1-VL comprises the amino acid sequence of SEQ ID NO: 55.

3. The bispecific antigen binding molecule of claim 1, wherein the bispecific antigen binding molecule further comprises a heavy chain constant region and a light chain constant region.

4. The bispecific antigen binding molecule of claim 3, wherein the heavy chain constant region comprises SEQ ID NO:72, and the light chain constant region comprises the amino acid sequence of SEQ ID NO:73, and an amino acid sequence of 73.

5. The bispecific antigen binding molecule according to claim 1,

the VEGF-VH comprises the sequence of SEQ ID NO:85, and said VEGF-VL comprises the amino acid sequence of SEQ ID NO:86, and a sequence of amino acids.

6. The bispecific antigen binding molecule of claim 1, having a first chain of the structure of formula (i) and a second chain of the structure of formula (ii),

(i) [ PD-1-VH ] - [ linker ] - [ VEGF-VH ] - [ heavy chain constant region ];

(ii) [ PD-1-VL ] - [ linker ] - [ VEGF-VL ] - [ light chain constant region ];

7. The bispecific antigen-binding molecule of claim 6, wherein the PD-1-VH comprises SEQ ID NO:27, and the PD-1-VL comprises the amino acid sequence of SEQ ID NO:55, an amino acid sequence of seq id no; and

the second antigen-binding domain capable of specifically binding to VEGF comprises a heavy chain variable region VEGF-VH and a light chain variable region VEGF-VL; wherein the VEGF-VH comprises the sequence of SEQ ID NO:85, and said VEGF-VL comprises the amino acid sequence of SEQ ID NO:86, and a sequence of amino acids.

8. The bispecific antigen binding molecule of claim 7, further comprising a heavy chain constant region and a light chain constant region; wherein the heavy chain constant region comprises SEQ ID NO:72, and the light chain constant region comprises the amino acid sequence of SEQ ID NO:73, and an amino acid sequence of 73.

9. The bispecific antigen binding molecule of claim 1, wherein the linker is a peptide linker.

10. The bispecific antigen binding molecule of claim 9, wherein each of the peptide linkers independently has L ₁ -(GGGGS) _n -L ₂ Wherein L is ₁ Is a bond, A, GS, GGS or GGGS, n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, L ₂ Is a bond, G, GG, GGG or GGGGGG.

11. The bispecific antigen binding molecule of claim 9, wherein the peptide linkers each independently have a sequence selected from the group consisting of SEQ ID NOs: 127 to SEQ ID NO:134, and a fragment thereof.

12. The bispecific antigen binding molecule of claim 1, wherein the bispecific antigen binding molecule has:

comprising a sequence selected from the group consisting of SEQ ID NOs: 105. SEQ ID NO: 107. SEQ ID NO: 109. SEQ ID NO: 111. SEQ ID NO:112 and SEQ ID NO:114 and a first strand comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 106. SEQ ID NO: 108. SEQ ID NO:110 and SEQ ID NO:113, and a second strand of the amino acid sequence of the group consisting of 113.

13. The bispecific antigen binding molecule of claim 12, having:

Comprising SEQ ID NO:114, and a first strand comprising the amino acid sequence of SEQ ID NO:113, and a second strand of an amino acid sequence of 113.

14. The bispecific antigen binding molecule of claim 13, having:

comprising SEQ ID NO:112, and a first strand comprising the amino acid sequence of SEQ ID NO:110, and a second strand of an amino acid sequence of seq id no.

15. A pharmaceutical composition comprising:

a therapeutically effective amount of the bispecific antigen binding molecule of any one of claims 1 to 14, and one or more pharmaceutically acceptable carriers, diluents, buffers or excipients.

16. A nucleic acid molecule encoding the bispecific antigen binding molecule of any one of claims 1 to 14.

17. A host cell comprising the nucleic acid molecule of claim 16.

18. A method for immunodetection or assay of PD-1, the method comprising the step of contacting the bispecific antigen binding molecule of any one of claims 1 to 14 with a subject or a sample from a subject, the method being for non-disease diagnostic purposes.

19. A kit comprising the bispecific antigen binding molecule of any one of claims 1 to 14.

20. Use of a bispecific antigen binding molecule of any one of claims 1 to 14, or a pharmaceutical composition of claim 15, or a nucleic acid molecule of claim 16, in the manufacture of a medicament for the treatment of a tumor.

21. The use of claim 20, wherein the tumor is selected from the group consisting of: head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumor, laryngeal cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatobiliary cancer, pancreatic cancer, gastrointestinal cancer, colorectal cancer, renal cancer, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, bone cancer, chondrosarcoma, myeloma, squamous cell carcinoma, ewing's sarcoma, systemic light chain amyloidosis, and meckerr cell carcinoma.

22. The use of claim 21, wherein the lymphoma is selected from the group consisting of: hodgkin's lymphoma and non-hodgkin's lymphoma,

the lung cancer is selected from: non-small cell lung cancer and small cell lung cancer,

the leukemia is selected from: lymphoblastic leukemia, and myeloid leukemia.

23. The use of claim 20, wherein the tumor is selected from the group consisting of: PD-L1 positive melanoma, non-small cell lung cancer, breast cancer, gastric cancer, kidney cancer, bladder cancer, bowel cancer, colon cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, clear cell renal cell carcinoma, multiple myeloma, myelodysplastic syndrome, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, acute myeloid leukemia, and chronic lymphocytic leukemia.