CN114573704B

CN114573704B - PD-1/CTLA-4 binding protein and medical application thereof

Info

Publication number: CN114573704B
Application number: CN202210223194.6A
Authority: CN
Inventors: 王雷; 刘钰莹; 王宇; 陈思萌; 张伟; 张满; 廖成
Original assignee: Beijing Tuojie Biomedical Technology Co ltd
Current assignee: Beijing Tuojie Biomedical Technology Co ltd
Priority date: 2021-03-10
Filing date: 2022-03-09
Publication date: 2022-11-01
Anticipated expiration: 2042-03-09
Also published as: CN114573704A

Abstract

The present disclosure relates to PD-1/CTLA-4 binding proteins and medical uses thereof. In particular, the disclosure relates to PD-1/CTLA-4 binding proteins comprising a first antigen-binding domain that specifically binds PD-1 and a second antigen-binding domain that specifically binds CTLA-4, and pharmaceutical uses thereof for treating cancer.

Description

PD-1/CTLA-4 binding protein and medical application thereof

Technical Field

The present disclosure is in the field of biomedicine and relates to PD-1/CTLA-4 binding proteins and their use in treating diseases (e.g., cancer).

Background

PD-1 (Programmed Cell death-1) belongs to the CD28 receptor family and is an immunosuppressive receptor. PD-1 is a type I transmembrane protein, expressed predominantly in activated B, T and bone marrow cells (Chen et al 2013, nat. Rev. Immunol.13, 227-42), with two cell surface glycoprotein ligands, PD-L1 and PD-L2, respectively. After PD-1 binds to PD-L1 or PD-L2, the functions of T cells are down-regulated, including reduction of activation, differentiation and proliferation of T cells, secretion of cytokines and the like. Blocking the interaction of PD-1 and PD-L1 can reverse immunosuppression, while inhibiting the effects of PD-1 and PD-L1, PD-L2 can play a synergistic role.

CTLA-4 (cytotoxic T lymphocyte-associated protein 4, also known as CD 152), a receptor protein with immune checkpoint and down-regulation of immune response, is expressed on activated CD4⁺And CD8⁺T cells, which are capable of halting the response of activated T cells and mediating the suppressive function of tregs, therefore CTLA-4 antibodies or CTLA-4 ligands can prevent CTLA-4 from binding its natural ligands, thereby blocking CTLA-4 transduction of T cell negative regulatory signals and enhancing T cell responsiveness to various antigens (Margaret K et al (2010), semin oncol.37 (5): 473-484.4). Currently, the CTLA-4 antibody, ipilimumab (ipilimumab,

) Has been approved for the treatment of melanoma, non-small cell lung cancer, renal cell carcinoma, mesothelioma, colorectal cancer and hepatocellular carcinoma. In addition, CTLA-4 antibodies such as tremelimumab and zalifrelimaab are being studied clinically for the treatment of bladder cancer, small cell lung cancer, cervical cancer, angiosarcoma, soft tissue sarcoma, and other cancers.

The camelid can generate single-domain antibody, the molecular weight is only 12-15kDa, and is one tenth of the traditional antibody. Single domain antibodies contain 3 CDRs, of which CDR3 plays a major role for affinity. Compared with human antibody VH, the CDR3 of the single-domain antibody is longer, can form a bulge loop (loop) structure, and can go deep into the antigen, thereby better combining the antigen. In addition, the hydrophobic residues of FR2 in single domain antibodies are replaced with hydrophilic residues, which are more water soluble and less prone to aggregate formation. Compared with the traditional antibody, the single-domain antibody has the advantages of high binding force, high specificity, high solubility, high stability, high expression level and the like.

At present, single-domain antibodies aiming at PD-1 are all in early development stage in the world, no single-domain antibody medicament targeting PD-1 is on the market, and no double-antibody medicament aiming at PD-1/CTLA-4 is on the market, and all are in early development stage. The present disclosure provides bispecific antibodies that bind both PD-1 and CTLA-4 and block PD-1-and CTLA-4-mediated immune system suppression to promote or continue activation of the immune system, effectively treating cancer and pathogen-related diseases.

Disclosure of Invention

The present disclosure provides a PD-1 binding protein, more specifically, a PD-1/CTLA-4 binding protein or a combination of PD-1 binding protein and CTLA-4 binding protein, and pharmaceutical uses thereof.

PD-1/CTLA-4 binding proteins

The present disclosure provides a PD-1/CTLA-4 binding protein comprising a first antigen-binding domain that specifically binds to PD-1 and a second antigen-binding domain that specifically binds to CTLA-4, the first antigen-binding domain that specifically binds to PD-1 comprising at least one immunoglobulin single variable domain comprising three complementarity determining regions CDR1, CDR2, and CDR3, wherein:

CDR1 comprises a compound as in X₂₂KCMG (SEQ ID NO: 152), wherein X₂₂Selected from N, D, E, F, G, H, I, K, L, M, P, Q, R or S, CDR2 comprises, for example, VVDFGGTIYAX₂₅An amino acid sequence of SVKG (SEQ ID NO: 187), wherein X₂₅Selected from A or D, CDR3 comprises, for example, GSYTSA X₂₃SCQPDAL (SEQ ID NO: 153) wherein X₂₃Selected from N, A, E, F, G, H, K, P, Q, R or S; or

CDR1 comprises SEQ ID NO:62 and CDR2 comprises X₁IDSVGX₂TX₃YX₄X₅SVKG (SEQ ID NO: 115) wherein X₁Selected from S or T, X₂Selected from T or A, X₃Selected from D, N or G, X₄Selected from T or A, X₅Selected from N or D, CDR3 comprises SEQ ID NO: 64; or

CDR1 comprises SEQ ID NO:81, CDR2 comprises VVDRX₂₄GGX₆IYAX₇SVKX₈(SEQ ID NO: 116) wherein X₂₄Selected from Y or F, X₆Selected from I or T, X₇Selected from A or D, X₈Selected from K or D, CDR3 comprises GSYTX₉X₁₀X₁₁SCX₁₂Amino acid represented by PDAL (SEQ ID NO: 117)Sequence of, wherein, X₉Selected from S or D, X₁₀Selected from A or D, X₁₁Selected from N or G, X₁₂Is selected from Q or H; or

CDR1 comprises YNX₁₃MX₁₄(SEQ ID NO: 118) wherein X₁₃Selected from F or Y, X₁₄Selected from S or T, CDR2 comprises SEQ ID NO:66, CDR3 comprises the amino acid sequence of SEQ ID NO: 67; or

CDR1 comprises SEQ ID NO:84, CDR2 comprises VINTGX₁₅NX₁₆TYYADSVKG (SEQ ID NO: 119), wherein X₁₅Selected from A or T, X₁₆Selected from S or T, CDR3 comprises SEQ ID NO: 86; or

CDR1 comprises SEQ ID NO:78, CDR2 comprises X₁₇YPTAGX₁₈TYX₁₉X₂₀DSX₂₁KG (SEQ ID NO: 120) wherein X₁₇Selected from L or I, X₁₈Selected from R or K, X₁₉Selected from Y or F, X₂₀Selected from G or A, X₂₁Selected from M or V, CDR3 comprises SEQ ID NO: 80; or

CDR1, CDR2, CDR3 comprise SEQ ID NO: 59. 60, 61; or

CDR1, CDR2, CDR3 comprise SEQ ID NO: 74. 75, 76; or

CDR1, CDR2, CDR3 comprise SEQ ID NO: 88. 89 and 90;

or, CDR1, CDR2, CDR3 comprise SEQ ID NO: 96. 97, 98.

The PD-1/CTLA-4 binding protein of some embodiments, wherein the immunoglobulin single variable domain in the first antigen-binding domain that specifically binds PD-1 comprises CDR1, CDR2, and CDR3 as follows:

CDR1 comprises the amino acid sequence as set forth in SEQ ID NO:129-141, CDR2 comprises the amino acid sequence as set forth in SEQ ID NO:71 or 82, CDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 83; or

CDR1 comprises the amino acid sequence as set forth in SEQ ID NO:81 and CDR2 comprises the amino acid sequence as set forth in SEQ ID NO:71 or 82, CDR3 comprises the amino acid sequence as set forth in SEQ ID NO:142-151, or a pharmaceutically acceptable salt thereof; or

CDR1, CDR2, CDR3 comprise the amino acid sequences as set forth in SEQ ID NOs: 129. 82, 145; or

CDR1, CDR2, CDR3 comprise the amino acid sequences as set forth in SEQ ID NOs: 132. 82, 145; or

CDR1, CDR2, CDR3 comprise the amino acid sequences as set forth in SEQ ID NOs: 129. 82, 146; or

CDR1, CDR2, CDR3 comprise the amino acid sequences as set forth in SEQ ID NOs: 132. 82, 146; or

CDR1, CDR2, CDR3 comprise the amino acid sequences as set forth in SEQ ID NOs: 129. 71, 145; or

CDR1, CDR2, CDR3 comprise the amino acid sequences as set forth in SEQ ID NOs: 132. 71, 145; or

CDR1, CDR2, CDR3 comprise the amino acid sequences as set forth in SEQ ID NOs: 129. 71, 146; or

CDR1, CDR2, CDR3 comprise the amino acid sequences as set forth in SEQ ID NOs: 132. 71, 146; or

CDR1 comprises SEQ ID NO:62 and CDR2 comprises the amino acid sequence of SEQ ID NO: 63. 68, 69, 70, 72, 77, CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 64; or

CDR1, CDR2, CDR3 comprise the amino acid sequences as set forth in SEQ ID NOs: 62. 63, 73; or

CDR1 comprises SEQ ID NO:81 and CDR2 comprises the amino acid sequence of SEQ ID NO: 71. 82, CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 83; or

CDR1 comprises SEQ ID NO:81 and CDR2 comprises the amino acid sequence of SEQ ID NO: 91. 93, CDR3 comprises the amino acid sequence shown in SEQ ID NO: 92; or

CDR1, CDR2, CDR3 comprise the amino acid sequences as set forth in SEQ ID NOs: 81. 94 and 95; or

CDR1 comprises SEQ ID NO: 65. 113, 114, CDR2 comprises the amino acid sequence set forth in SEQ ID NO:66, CDR3 comprises the amino acid sequence of SEQ ID NO: 67; or

CDR1 comprises SEQ ID NO:84, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 85. 102, CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 86; or

CDR1 comprises SEQ ID NO:78 and CDR2 comprises the amino acid sequence shown in SEQ ID NO: 79. 87, 99, 100, 101, CDR3 comprises the amino acid sequence of SEQ ID NO:80, or a pharmaceutically acceptable salt thereof.

The PD-1/CTLA-4 binding protein of some embodiments, wherein the immunoglobulin single variable domain of the first antigen-binding domain that specifically binds PD-1 comprises an amino acid sequence as set forth in SEQ ID NO:154-157, 7-33, 35-58, 123-128 or an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99% sequence identity thereto.

The PD-1/CTLA-4 binding protein of some embodiments, wherein the second antigen-binding domain that specifically binds CTLA-4 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

VH comprises the amino acid sequence as set forth in SEQ ID NO:158-160 HCDR1, HCDR2, HCDR3, VL comprising the amino acid sequence as set forth in SEQ ID NO: 161. 163, and a LCDR3, and a LCDR2 as shown in the GAF amino acid sequence.

The PD-1/CTLA-4 binding protein of some embodiments, wherein the VH of the second antigen-binding domain that specifically binds CTLA-4 comprises the amino acid sequence as set forth in SEQ ID NO:164 or at least 90%, at least 95%, at least 98%, at least 99% sequence identity thereto, and VL comprises an amino acid sequence as set forth in SEQ ID NO:165 or an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99% sequence identity thereto; in some embodiments, the VH comprises the amino acid sequence as set forth in SEQ ID NO:164 and VL comprises the amino acid sequence as set forth in SEQ ID NO:165, or a pharmaceutically acceptable salt thereof.

The PD-1/CTLA-4 binding protein of some embodiments, wherein the second antigen-binding domain that specifically binds CTLA-4 comprises a full-length Heavy Chain (HC) and a full-length Light Chain (LC); for example, the full-length heavy chain is of the IgG1 or IgG4 isotype and the full-length light chain is of the Kappa isotype; for example, the full-length heavy chain is SEQ ID NO:166 or has at least 90% sequence identity thereto, and the full length light chain is SEQ ID NO:167 or has at least 90% sequence identity thereto; for example, the full-length heavy chain and the full-length light chain are SEQ ID NOs: 166. 167 as shown.

the immunoglobulin single variable domain that specifically binds the first antigen-binding domain of PD-1 is located N-terminal to the heavy chain variable region or the full-length heavy chain of the second antigen-binding domain that specifically binds CTLA-4;

the immunoglobulin single variable domain that specifically binds the first antigen-binding domain of PD-1 is located C-terminal to the heavy chain variable region or the full-length heavy chain of the second antigen-binding domain that specifically binds CTLA-4;

the immunoglobulin single variable domain that specifically binds the first antigen-binding domain of PD-1 is N-terminal to the light chain variable region or the full-length light chain of the second antigen-binding domain that specifically binds CTLA-4; and/or

The immunoglobulin single variable domain that specifically binds the first antigen-binding domain of PD-1 is C-terminal to the light chain variable region or the full-length light chain of the second antigen-binding domain that specifically binds CTLA-4.

The PD-1/CTLA-4 binding protein of some embodiments, wherein an immunoglobulin single variable domain that specifically binds to a first antigen-binding domain of PD-1 is linked, directly or by a linker, to a second antigen-binding domain that specifically binds to CTLA-4; for example, the GGGS (SEQ ID NO: 174), LGGGSG (SEQ ID NO: 175), GGGSGGGSGGG (SEQ ID NO: 176), ASTKG (SEQ ID NO: 177), DKTTCPPCP (SEQ ID NO: 178), EPKSCDKTHTCPPCP (SEQ ID NO: 179), LEPKSS (SEQ ID NO: 180), APSSS (SEQ ID NO: 181), and APSSSPME (SEQ ID NO: 182), LEPKSADKTHTCPPC (SEQ ID NO: 183), GGC and GGG; as another example, the linker can have a structure as (G)₄S)_xThe amino acid sequence shown, wherein x is independently selected from an integer from 1 to 20 (e.g., x is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20); for example, the linker is (G)₄S)₄、(G₄S)₆The amino acid sequence shown.

The PD-1/CTLA-4 binding protein of some embodiments, comprising a first polypeptide chain comprising an amino acid sequence as set forth in SEQ ID NO:168-173 or an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99% sequence identity thereto, and a second polypeptide chain comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 167 or an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99% sequence identity thereto. For example, the first polypeptide chain of the PD-1/CTLA-4 binding protein comprises an amino acid sequence as set forth in SEQ ID NO:168-173 and a second polypeptide chain comprising an amino acid sequence as set forth in any of SEQ ID NOs: 167.

A PD-1/CTLA-4 binding protein in some embodiments, having an activity selected from at least one of:

(a) At most 10^-7K of_DValues bind to human PD-1 or a fragment thereof;

(b) To be less than or equal to 10^-7K of_DValues bind to human CTLA-4 or a fragment thereof;

(c) Inhibit the binding of PD-1 to PD-L1;

(d) Inhibits the binding of PD-1 to PD-L2;

(e) Inhibits binding of CTLA-4 to CD80 and/or CD 86;

(f) Inducing T cells to secrete IFN-gamma and/or IL-2;

(g) Enhanced activation of PBMCs;

(h) Enhancing T cell activation, stimulating T cell response, or stimulating T cell proliferation;

(i) Inhibiting tumor growth and delaying cancer development.

In some embodiments, the PD-1/CTLA-4 binding protein described above is an anti-PD-1/CTLA-4 bispecific antibody.

In some embodiments, the anti-PD-1/CTLA-4 bispecific antibody comprises an immunoglobulin single variable domain in a first antigen-binding domain that specifically binds PD-1, as provided by the foregoing disclosure, and a heavy chain variable region (VH) and a light chain variable region (VL) in a second antigen-binding domain that specifically binds CTLA-4, as provided by the foregoing disclosure.

In some embodiments, the anti-PD-1/CTLA-4 bispecific antibody is one in which:

the first antigen binding domain that specifically binds PD-1 is a first antibody, which is a VHH, having CDR1, CDR2 and CDR3 of any of the immunoglobulin single variable domains provided by the foregoing disclosure; and, the second antigen-binding domain that specifically binds CTLA-4 is a second antibody comprising a Heavy Chain (HC) and a Light Chain (LC).

In some embodiments, the second antibody is any anti-CTLA-4 antibody. For example, exemplary anti-CTLA 4 antibodies include tremelimumab (tremelimumab) (e.g., disclosed in US6682736 and WO 00/37504); and ipilimumab (ipilimumab) (CTLA-4 antibody, also known as MDX-010, cas number 477202-00-9, disclosed in, e.g., US5811097, US7605238, WO00/32231, and WO 97/20574). Other exemplary anti-CTLA-4 antibodies are disclosed in, for example, US5811097, WO2014209804A, WO2017128534A, WO2017106061A, WO2017193032A, WO2017218707A, WO2019090002A, WO2019179391A, WO2019179421A, WO2019094637A, WO2020205516A, WO2020057610A, WO2020057611A, and WO2021023117A.

In some embodiments, the VHH is located as a first antibody at the N-terminus and/or C-terminus of the heavy or light chain of a second antibody.

In some embodiments, the anti-PD-1/CTLA-4 bispecific antibody comprises 1 second antibody and 2 first antibodies of VHH; the second antibody comprises two HCs and two LCs, the VH of one HC of the second antibody forms an antigen-binding site with the VL of one LC, and the VH of the other HC forms an antigen-binding site with the VL of the other LC.

In some embodiments, the first antibody of one VHH of the anti-PD-1/CTLA-4 bispecific antibody is located N-terminal to the heavy or light chain of the second antibody and the first antibody of the other VHH is located C-terminal to the heavy or light chain of the second antibody.

In some embodiments, the first antibody of each VHH in the anti-PD-1/CTLA-4 bispecific antibody is N-terminal to both heavy chains or both light chains of the second antibody, respectively; alternatively, the first antibody of each VHH is C-terminal to both heavy chains or both light chains of the second antibody, respectively.

In some embodiments, the first antibody of each VHH of the anti-PD-1/CTLA-4 bispecific antibody is located N-terminal to the two heavy chains of the first antibody, respectively; alternatively, the first antibody of each VHH is C-terminal to both heavy chains of the first antibody;

in some embodiments, the second antibody of the anti-PD-1/CTLA-4 bispecific antibody can have attached thereto first antibodies of 1, 2, 3, 4, 5, 6, 7, 8 VHHs, which first antibodies of VHH can be the same or different, can all be attached to the N-terminus of the heavy chain of the second antibody, or all be attached to the C-terminus of the heavy chain of the second antibody, or all be attached to the N-terminus of the light chain of the second antibody, or all be attached to the C-terminus of the light chain of the second antibody, or any combination of the N-terminus of the heavy chain, the C-terminus of the heavy chain, the N-terminus of the light chain, and the C-terminus of the light chain.

In some embodiments, the first antibody against VHH in the PD-1/CTLA-4 bispecific antibody is linked to the N-terminus or C-terminus of each heavy chain of the second antibody, either directly or through a linker. The linker is selected from: such as (G)_mS_n)_xThe amino acid sequence shown, wherein m, n are each independently selected from an integer from 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8), and x is independently selected from an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). For example, the linker is G₄S、(G₄S)₂、(G₄S)₃、(G₄S)₄、(G₄S)₅、(G₄S)₆The amino acid sequence shown.

In some embodiments, the second antibody of the anti-PD-1/CTLA-4 bispecific antibody has a heavy chain comprising a heavy chain variable region (VH) and a heavy chain constant region (CH), and a light chain comprising a light chain variable region (VL) and a light chain constant region (CL). The second antibody may be a full length antibody.

In some embodiments, the heavy chain of the second antibody of the anti-PD-1/CTLA-4 bispecific antibody is of an IgG isotype, e.g., igG1, igG2, igG3, or IgG4, e.g., is of an IgG1 isotype; and/or the light chain of the second antibody is of Kappa isotype.

In some embodiments, both HCs of the anti-PD-1/CTLA-4 bispecific antibody comprise the same CDR and/or both LCs comprise the same CDR. In some embodiments, the two HCs of the second antibody comprise the same VH and/or the two LCs comprise the same VL. In some embodiments, the two HCs of the second antibody have the same amino acid sequence and/or the two LCs have the same amino acid sequence.

In some embodiments, the anti-PD-1/CTLA-4 bispecific antibody the first two VHH antibodies have identical or non-identical amino acid sequences. For example, the first antibodies of two of said VHHs have the same amino acid sequence.

In some embodiments, the anti-PD-1/CTLA-4 bispecific antibody comprises two first polypeptide chains and two second polypeptide chains, wherein for each polypeptide chain:

a) The first polypeptide chain comprises each independently a Heavy Chain (HC) of a first antibody and a second antibody of a VHH; and b) the second polypeptide chains each independently comprise a Light Chain (LC) of a second antibody; wherein the VHH is linked to the N-terminus and/or C-terminus of the HC of the first antibody via a linker; alternatively, the first and second electrodes may be,

i) The first polypeptide chains each independently comprise a Heavy Chain (HC) of a second antibody; and ii) a second polypeptide chain each independently comprises a Light Chain (LC) of a first antibody and a second antibody of VHH; wherein the VHH is linked to the N-terminus and/or C-terminus of the LC of the second antibody directly or via a linker.

In some embodiments, the anti-PD-1/CTLA-4 bispecific antibody comprises two identical first polypeptide chains and two identical second polypeptide chains.

In some embodiments, antibodies are provided that compete for binding to the same epitope as the PD-1 binding proteins, PD-1/CTLA-4 binding proteins, anti-PD-1 antibodies, anti-PD-1/CTLA-4 bispecific antibodies of the present disclosure.

In some embodiments, the PD-1 binding protein, CTLA-4 binding protein, PD-1/CTLA-4 binding protein, anti-PD-1/CTLA-4 bispecific antibody of the present disclosure further comprises a human immunoglobulin Fc region; for example, the Fc region is that of human IgG1, igG2, or IgG4. The Fc region may have a mutation. Exemplary mutations are selected from: L234A/L235A on IgG1, V234A/G237A/P238S/H268A/V309L/A330S/P331S on IgG2, F234A/L235A on IgG4, S228P or S228P/F234A/L235A on IgG4, N297A on IgG1, igG2, igG3 or IgG4, V234A/G237A on IgG2, K214T/E233P/L234V/L235A/G236 deletion/A G/P331A/D365E/L358MV309L/A330S/P331S on IgG1, L234F/L235E/D265A on IgG1, L234A/L235A/G237A/P238S/H268A/A330S/P331S on IgG1, S228P/F234A/L235A/G238A/P237S and P237A/P236S 237A/P236S on IgG4. Hybrid IgG2/4Fc domains, e.g., fc having residues 117-260 from IgG2 and residues 261-447 from IgG4, can also be used.

In some specific embodiments, the Fc region of human IgG4 has the S228P, F A, L a and/or K447A mutations. In some specific embodiments, the Fc region of human IgG1 has the L234A/L235A or L234A/L235A/P329G mutations.

Compositions of PD-1 binding proteins and CTLA-4 binding proteins

The present disclosure provides compositions comprising PD-1 binding protein and CTLA-4 binding protein, wherein PD-1 binding protein comprises a first antigen-binding domain that specifically binds PD-1 as provided by the foregoing disclosure, and CTLA-4 binding protein comprises a second antigen-binding domain that specifically binds CTLA-4 as provided by the foregoing disclosure.

In some embodiments, the PD-1 binding protein in the composition comprises at least one immunoglobulin single variable domain comprising three complementarity determining regions CDR1, CDR2, and CDR3, the CDR1, CDR2, CDR3 being as set forth in SEQ ID NO:154-157, 7-33, 35-58, 123-128, wherein the CDRs are defined according to the Kabat, IMGT, chothia, abM or Contact numbering system, and in some embodiments according to the Kabat numbering system.

In some embodiments, the immunoglobulin single variable domain:

CDR1, CDR2, CDR3 comprise the amino acid sequences as set forth in SEQ ID NOs: 152. 187, 153;

CDR1, CDR2, CDR3 comprise the amino acid sequences as set forth in SEQ ID NOs: 62. 115, 64;

CDR1, CDR2, CDR3 comprise the amino acid sequences as set forth in SEQ ID NOs: 81. 116, 117;

CDR1, CDR2, CDR3 comprise the amino acid sequences as set forth in SEQ ID NOs: 118. 66, 67, respectively;

CDR1, CDR2, CDR3 comprise the amino acid sequences as set forth in SEQ ID NOs: 84. 119, 86;

CDR1, CDR2, CDR3 comprise the amino acid sequences as set forth in SEQ ID NOs: 78. 120, 80;

CDR1, CDR2, CDR3 comprise the amino acid sequences as set forth in SEQ ID NOs: 59-61;

CDR1, CDR2, CDR3 comprise the amino acid sequences as set forth in SEQ ID NOs: 74-76;

CDR1, CDR2, CDR3 comprise the amino acid sequences as set forth in SEQ ID NOs: 88-90; or

CDR1, CDR2, CDR3 comprise the amino acid sequences as set forth in SEQ ID NOs: 96-98.

In some embodiments, CDR1 comprises a CDR sequence as set forth in SEQ ID NO:129-141, CDR2 comprises the amino acid sequence as set forth in SEQ ID NO:71 or 82, CDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 83; or

CDR1 comprises SEQ ID NO:62 and CDR2 comprises the amino acid sequence of SEQ ID NO: 63. 68, 69, 70, 72, 77, CDR3 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 64; or

CDR1 comprises SEQ ID NO: 65. 113, 114, CDR2 comprises the amino acid sequence set forth in SEQ ID NO:66, CDR3 comprises the amino acid sequence shown in SEQ ID NO: 67; or

CDR1 comprises SEQ ID NO:84, CDR2 comprises the amino acid sequence of SEQ ID NO: 85. 102, CDR3 comprises the amino acid sequence of SEQ ID NO: 86; or

CDR1 comprises SEQ ID NO:78 and CDR2 comprises the amino acid sequence of SEQ ID NO: 79. 87, 99, 100, 101, CDR3 comprises the amino acid sequence of SEQ ID NO:80, or a pharmaceutically acceptable salt thereof.

In some embodiments, the PD-1 binding protein comprises a sequence as set forth in SEQ ID NO:154-157, 7-33, 35-58, 123-128 or an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99% sequence identity thereto.

In some embodiments, the immunoglobulin single variable domain in the PD-1 binding protein is a VHH, e.g., a VHH that is humanized and/or affinity matured.

In some embodiments, the PD-1 binding protein is an antibody that specifically binds to PD-1 or a fragment thereof; preferably, the antibody is a camelid antibody, a chimeric antibody, a humanized antibody, a fully human antibody. In some embodiments, the PD-1 binding protein further comprises a human immunoglobulin Fc region, e.g., an Fc region of human IgG1 or IgG4, e.g., having a mutation of S228P, F A, L a and/or K447A.

In some embodiments, the CTLA-4 binding protein is an anti-CTLA-4 antibody. For example, tiximumab and ipilimumab, other exemplary anti-CTLA-4 antibodies are disclosed in US5811097, WO2014209804A, WO2017128534A, WO2017106061A, WO2017193032A, WO2017218707A, WO2019090002A, WO2019179391A, WO2019179421A, WO2019094637A, WO2020205516A, WO2020057610A, WO2020057611a, and WO2021023117a.

In some embodiments, the CTLA-4 binding protein comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: VH comprises the amino acid sequence as set forth in SEQ ID NO:158-160 HCDR1, HCDR2, HCDR3, VL comprising the amino acid sequence as set forth in SEQ ID NO: 161. 163, and a LCDR3, and a LCDR2 as shown in the GAF amino acid sequence.

In some embodiments, the CTLA-4 binding protein is a full-length antibody comprising a full-length Heavy Chain (HC), e.g., an IgG1 or IgG4 isotype, and a full-length Light Chain (LC), e.g., a Kappa isotype.

In some embodiments, the VH of the CTLA-4 binding protein comprises the amino acid sequence as set forth in SEQ ID NO:164 or at least 90%, at least 95%, at least 98%, at least 99% sequence identity thereto, and VL comprises an amino acid sequence as set forth in SEQ ID NO:165 or an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99% sequence identity thereto; in some embodiments, the VH comprises the amino acid sequence as set forth in SEQ ID NO:164 and VL comprises the amino acid sequence set forth in SEQ ID NO:165 has at least 95% identity thereto.

In some embodiments, the full length heavy chain of the CTLA-4 binding protein is SEQ ID NO:166 or has at least 80%, at least 90%, at least 95%, at least 98%, at least 99% sequence identity thereto, and the full-length light chain is SEQ ID NO:167 or has at least 80%, at least 90%, at least 95%, at least 98%, at least 99% sequence identity thereto; in some embodiments, the full-length heavy chain and the full-length light chain of the CTLA-4 binding protein are SEQ ID NOs: 166. 167 as shown.

Polynucleotide

The present disclosure provides polynucleotides encoding the PD-1 binding proteins, CTLA-4 binding proteins, PD-1/CTLA-4 binding proteins, anti-PD-1/CTLA-4 bispecific antibodies of the present disclosure. The polynucleotides of the present disclosure may be RNA, DNA, or cDNA. According to some embodiments of the disclosure, the polynucleotide of the disclosure is a substantially isolated nucleic acid.

The polynucleotides of the present disclosure may also be in the form of, may be present in and/or may be part of a vector, such as a plasmid, cosmid, YAC, or viral vector. The vector may especially be an expression vector, i.e. a vector providing for the expression of the PD-1 binding protein in vitro and/or in vivo (i.e. in a suitable host cell, host organism and/or expression system). The expression vector typically comprises at least one nucleic acid of the present disclosure operably linked to one or more suitable expression regulatory elements (e.g., promoters, enhancers, terminators, and the like). The selection of the elements and their sequences for expression in a particular host is within the knowledge of one skilled in the art. Regulatory elements and other elements useful or necessary for expression of the PD-1 binding proteins of the present disclosure are, for example, promoters, enhancers, terminators, integration factors, selection markers, leader sequences, reporter genes.

Polynucleotides of the disclosure may be prepared or obtained by known means (e.g., by automated DNA synthesis and/or recombinant DNA techniques) based on information on the amino acid sequence of the polypeptides of the disclosure, and/or may be isolated from a suitable natural source.

Host cell

The present disclosure provides recombinant host cells that express or are capable of expressing one or more PD-1 binding proteins, CTLA-4 binding proteins, PD-1/CTLA-4 binding proteins, anti-PD-1/CTLA-4 bispecific antibodies of the present disclosure, and/or containing a polynucleotide or vector of the present disclosure. In some embodiments, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

Bacterial cells include, for example, cells of gram-negative bacterial strains, such as Escherichia coli (Escherichia coli) strains, proteus (Proteus) strains and Pseudomonas (Pseudomonas) strains, and gram-positive bacterial strains, such as Bacillus (Bacillus) strains, streptomyces (Streptomyces) strains, staphylococcus (Staphylococcus) strains and Lactococcus (Lactococcus) strains.

Fungal cells include, for example, cells of species of the genera Trichoderma (Trichoderma), neurospora (Neurospora), and Aspergillus (Aspergillus); or cells of species including Saccharomyces (Saccharomyces) such as Saccharomyces cerevisiae, schizosaccharomyces (Schizosaccharomyces pombe), pichia (Pichia) such as Pichia pastoris and Pichia methanolica, and Hansenula.

Mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, heLa cells, COS cells, and the like.

However, the present disclosure may also use amphibian cells, insect cells, plant cells, and any other cells used in the art for expression of heterologous proteins.

The cells of the present disclosure are unable to develop into a finished plant or individual animal.

Production or preparation method

The present disclosure provides methods of making PD-1 binding proteins, CTLA-4 binding proteins, PD-1/CTLA-4 binding proteins, anti-PD-1/CTLA-4 bispecific antibodies of the present disclosure, the methods generally comprising the steps of:

-culturing a host cell of the disclosure under conditions that allow expression of the PD-1 binding protein, CTLA-4 binding protein, PD-1/CTLA-4 binding protein, anti-PD-1/CTLA-4 bispecific antibody of the disclosure; and

-recovering the protein of interest expressed by the host cell from the culture; and

optionally, further purification and/or modification of the protein of interest of the present disclosure.

The PD-1 binding proteins, CTLA-4 binding proteins, PD-1/CTLA-4 binding proteins, anti-PD-1/CTLA-4 bispecific antibodies of the present disclosure can be produced intracellularly (e.g., in the cytoplasm, in the periplasm, or in inclusion bodies) in cells as described above, followed by isolation from the host cell and optionally further purification; or it may be produced extracellularly (e.g. in the medium in which the host cell is cultured), followed by isolation from the medium and optionally further purification.

Methods and reagents for recombinant production of polypeptides, e.g., specifically adapted expression vectors, transformation or transfection methods, selection markers, methods of inducing protein expression, culture conditions, and the like, are known in the art. Similarly, protein isolation and purification techniques suitable for use in methods of making PD-1 binding proteins of the present disclosure are well known to those skilled in the art.

However, the PD-1 binding proteins, CTLA-4 binding proteins, PD-1/CTLA-4 binding proteins, anti-PD-1/CTLA-4 bispecific antibodies of the present disclosure may also be obtained by other methods of protein production known in the art, such as chemical synthesis, including solid phase or liquid phase synthesis.

Pharmaceutical composition

The present disclosure provides pharmaceutical compositions containing a prophylactically or therapeutically effective amount of a PD-1-binding protein, CTLA-4-binding protein, PD-1/CTLA-4-binding protein, anti-PD-1/CTLA-4 bispecific antibody or encoding polynucleotide thereof of the present disclosure, as described above, and one or more pharmaceutically acceptable carriers, diluents, buffers, or excipients.

In some embodiments, the pharmaceutical composition may comprise 0.01 to 99% by weight of PD-1 binding protein, PD-1/CTLA-4 binding protein, or anti-PD-1/CTLA-4 bispecific antibody in a unit dose. In other embodiments, the pharmaceutical composition comprises PD-1 binding protein, PD-1/CTLA-4 binding protein, or an anti-PD-1/CTLA-4 bispecific antibody in an amount of 0.1 to 2000mg; and in some embodiments 1 to 1000mg.

Reagent kit

The present disclosure provides kits comprising PD-1 binding protein, CTLA-4 binding protein, PD-1/CTLA-4 binding protein, anti-PD-1/CTLA-4 bispecific antibody, and/or encoding polynucleotides thereof of the present disclosure. In some embodiments, diagnostic reagents comprising the polynucleotides described above are also provided, as are related diagnostic uses.

Method for treating diseases and pharmaceutical use

The present disclosure provides uses and methods of the PD-1/CTLA-4 binding proteins, anti-PD-1/CTLA-4 bispecific antibodies or antigen-binding fragments thereof, compositions, polynucleotides, pharmaceutical compositions of PD-1 binding proteins and CTLA-4 binding proteins, of the present disclosure in the prevention and/or treatment of diseases, which may or may not be associated with the PD-1 signaling pathway. In some embodiments, the present disclosure provides a method of preventing and/or treating a disease associated with PD-1, the method comprising administering to a subject a prophylactically and/or therapeutically effective amount of a PD-1 binding protein of the present disclosure, or a pharmaceutical composition comprising a PD-1 binding protein of the present disclosure. And also provides application of the PD-1 binding protein in preparing medicaments for preventing and/or diseases related to PD-1.

The PD-1/CTLA-4 binding proteins, anti-PD-1/CTLA-4 bispecific antibodies or antigen-binding fragments thereof, compositions, polynucleotides, pharmaceutical compositions of PD-1 binding proteins and CTLA-4 binding proteins of the present disclosure can be used alone or in combination with other anti-tumor therapeutic approaches (e.g., with other immunogenic agents, standard cancer therapies, or other antibody molecules) to inhibit the growth of cancerous tumors.

In some embodiments, the disclosure provides a method of promoting T cell proliferation, in other embodiments, the disclosure provides a method of benefiting a patient or a subject from an up-regulation of an immune response, and in other embodiments, a method of promoting expression of a cytokine (e.g., INF γ, IL-2) in a subject or patient, each comprising administering to the patient or subject a prophylactically and/or therapeutically effective amount of a PD-1/CTLA-4 binding protein, an anti-PD-1/CTLA-4 bispecific antibody or antigen-binding fragment thereof, a composition of PD-1 binding protein and CTLA-4 binding protein, a polynucleotide, a pharmaceutical composition of the disclosure.

In some embodiments, the present disclosure provides a method of preventing and/or treating cancer, comprising administering to a patient or subject a prophylactically and/or therapeutically effective amount of a PD-1/CTLA-4 binding protein, an anti-PD-1/CTLA-4 bispecific antibody or antigen-binding fragment thereof, a composition of a PD-1 binding protein and a CTLA-4 binding protein, a polynucleotide, a pharmaceutical composition of the present disclosure, inhibiting tumor cell growth in the patient or subject. In some embodiments, the cancer is preferably, but not limited to, a cancer responsive to immunotherapy.

Non-limiting examples of cancers in the above methods include lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma (e.g., metastatic malignant melanoma), renal cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematological malignancies, head and neck cancer, glioma, gastric cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine body tumor, and osteosarcoma. Examples of other cancers that may be treated with the methods of the present disclosure include: bone cancer, pancreatic cancer, skin cancer, prostate cancer, cutaneous or intraocular malignant melanoma, uterine cancer, cancer of the anal region, testicular cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the vagina, carcinoma of the vulva, hodgkin's disease, non-hodgkin's lymphoma, carcinoma of the esophagus, cancer of the small intestine, cancer of the endocrine system, carcinoma of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemias, including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors of childhood, lymphocytic lymphomas, cancer of the bladder, kidney or ureter, carcinoma of the renal pelvis, tumors of the Central Nervous System (CNS), primary CNS lymphoma, tumoral angiogenesis, spinal axis tumors, brain stem glioma, pituitary adenoma, kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, including asbestos-induced cancers, and combinations of said cancers. In some embodiments, the cancer or tumor is metastatic.

In some embodiments, the present disclosure provides a method of a PD-1 and/or CTLA-4 associated condition and disease, including autoimmune diseases, such as Systemic Lupus Erythematosus (SLE), psoriasis, systemic scleroderma, autoimmune diabetes, and the like, comprising administering an effective amount of a PD-1/CTLA-4 binding protein, an anti-PD-1/CTLA-4 bispecific antibody or antigen-binding fragment thereof, a composition of PD-1 binding protein and CTLA-4 binding protein, a polynucleotide, a pharmaceutical composition of the present disclosure.

In addition, the present disclosure also provides a method of preventing and/or treating an infectious disease in a subject or patient, comprising administering to the subject or patient a PD-1/CTLA-4 binding protein, an anti-PD-1/CTLA-4 bispecific antibody or antigen-binding fragment thereof, a composition of PD-1 binding protein and CTLA-4 binding protein, a polynucleotide, a pharmaceutical composition of the present disclosure, such that the infectious disease in the subject is prevented and/or treated. Similar to the use for tumors as described above, PD-1 binding proteins can be used alone, or in combination with vaccines to stimulate immune responses to pathogens, toxins and autoantigens. Examples of pathogens for which this treatment may be particularly applicable include pathogens for which no effective vaccine is currently available, or pathogens for which conventional vaccines are not fully effective. Including but not limited to HIV, hepatitis viruses (a, b, c), influenza, herpes, giardia, malaria, leishmania, staphylococcus aureus, pseudomonas aeruginosa.

The disclosure also provides pharmaceutical uses of PD-1 binding protein, PD-1/CTLA-4 binding protein, and PD-1 binding protein in combination with CTLA-4 binding protein for the above methods.

In some embodiments, there is provided the use of a PD-1 binding protein in combination with a CTLA-4 binding protein for the manufacture of a medicament for treating a tumor (cancer), treating an autoimmune disease, treating an infection, promoting T cell proliferation, benefiting a subject or patient from an upregulation of an immune response, and/or promoting expression of a cytokine (e.g., INF γ, IL-2) in a subject or patient.

The present disclosure includes the following embodiments:

a pd-1/CTLA-4 binding protein comprising:

a first antigen binding domain that specifically binds PD-1, and

a second antigen-binding domain that specifically binds CTLA-4,

the first antigen-binding domain that specifically binds PD-1 comprises at least one immunoglobulin single variable domain comprising three complementarity determining regions CDR1, CDR2, and CDR3, wherein:

1) CDR1, CDR2, and CDR3 comprise the amino acid sequences as set forth in SEQ ID NOs: 152. 187, 153; or

2) CDR1, CDR2 and CDR3 comprise the amino acid sequences as set forth in SEQ ID NOs: 62. 115, 64; or

3) CDR1, CDR2 and CDR3 comprise the amino acid sequences as set forth in SEQ ID NOs: 81. 116, 117; or

4) CDR1, CDR2 and CDR3 comprise the amino acid sequences as set forth in SEQ ID NOs: 118. 66, 67; or

5) CDR1, CDR2, and CDR3 comprise the amino acid sequences as set forth in SEQ ID NOs: 84. 119, 86; or

6) CDR1, CDR2 and CDR3 comprise the amino acid sequences as set forth in SEQ ID NOs: 78. 120, 80; or

7) CDR1, CDR2 and CDR3 comprise the amino acid sequences as set forth in SEQ ID NOs: 59. 60, 61; or

8) CDR1, CDR2 and CDR3 comprise the amino acid sequences as set forth in SEQ ID NOs: 74. 75, 76; or

9) CDR1, CDR2 and CDR3 comprise the amino acid sequences as set forth in SEQ ID NOs: 88. 89 and 90; or

10 CDR1, CDR2 and CDR3 comprise the amino acid sequences as set forth in SEQ ID NOs: 96. 97, 98.

2. The PD-1/CTLA-4 binding protein of claim 1, wherein the immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 as follows:

1) CDR1 comprises the amino acid sequence as set forth in SEQ ID NO:129, CDR2 comprises the amino acid sequence as set forth in SEQ ID NO:71 or 82, CDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 145; or

2) CDR1 comprises the amino acid sequence as set forth in SEQ ID NO:129-141, CDR2 comprises the amino acid sequence set forth in SEQ ID NO:71 or 82, CDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 83; or

3) CDR1 comprises the amino acid sequence as set forth in SEQ ID NO:81 and CDR2 comprises the amino acid sequence as set forth in SEQ ID NO:71 or 82, CDR3 comprises the amino acid sequence as set forth in SEQ ID NO:142-151, or a pharmaceutically acceptable salt thereof; or

4) CDR1 comprises the amino acid sequence as set forth in SEQ ID NO:132, CDR2 comprises the amino acid sequence as set forth in SEQ ID NO:71 or 82, CDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 145; or

5) CDR1 comprises the amino acid sequence as set forth in SEQ ID NO:129, CDR2 comprises the amino acid sequence as set forth in SEQ ID NO:71 or 82, CDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 146; or

6) CDR1 comprises the amino acid sequence as set forth in SEQ ID NO:132, CDR2 comprises the amino acid sequence as set forth in SEQ ID NO:71 or 82, CDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 146; or

7) CDR1 comprises SEQ ID NO:62 and CDR2 comprises the amino acid sequence of SEQ ID NO: 63. 68, 69, 70, 72, 77, CDR3 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 64; or

8) CDR1 comprises SEQ ID NO:62 and CDR2 comprises the amino acid sequence of SEQ ID NO:63 and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 73; or

9) CDR1 comprises SEQ ID NO:81 and CDR2 comprises the amino acid sequence of SEQ ID NO: 71. 82, CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 83; or

10 CDR1 comprises SEQ ID NO:81 and CDR2 comprises the amino acid sequence of SEQ ID NO: 91. 93, CDR3 comprises the amino acid sequence shown in SEQ ID NO: 92; or

11 CDR1 comprises SEQ ID NO:81 and CDR2 comprises the amino acid sequence of SEQ ID NO:94 and CDR3 comprises the amino acid sequence of SEQ ID NO: 95; or

12 CDR1 comprises SEQ ID NO: 65. 113, 114, CDR2 comprises the amino acid sequence set forth in SEQ ID NO:66, CDR3 comprises the amino acid sequence of SEQ ID NO:67, or a pharmaceutically acceptable salt thereof; or

13 CDR1 comprises SEQ ID NO:84, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 85. 102, CDR3 comprises the amino acid sequence of SEQ ID NO: 86; or

14 CDR1 comprises SEQ ID NO:78 and CDR2 comprises the amino acid sequence of SEQ ID NO: 79. 87, 99, 100, 101, CDR3 comprises the amino acid sequence of SEQ ID NO:80, or a pharmaceutically acceptable salt thereof.

3. The PD-1/CTLA-4 binding protein of

claim

1 or 2, the immunoglobulin single variable domain comprises an amino acid sequence as set forth in SEQ ID NO:154-157, 7-33, 35-58, 123-128 or an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99% sequence identity thereto.

4. The PD-1/CTLA-4 binding protein of any one of the preceding claims, wherein the second antigen-binding domain that specifically binds CTLA-4 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

VH comprises the amino acid sequences as set forth in SEQ ID NOs: 158-160 HCDR1, HCDR2, HCDR3, VL comprising the amino acid sequences set forth in SEQ ID NOs: 161. 163, and a LCDR3, and a LCDR2 as shown in the GAF amino acid sequence.

5. The PD-1/CTLA-4 binding protein of item 4, wherein the VH comprises an amino acid sequence as set forth in SEQ ID NO:164 or at least 90%, at least 95%, at least 98%, at least 99% sequence identity thereto, and VL comprises an amino acid sequence as set forth in SEQ ID NO:165 or an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99% sequence identity thereto.

6. The PD-1/CTLA-4 binding protein of any one of

claims

4 or 5, wherein the second antigen-binding domain that specifically binds CTLA-4 comprises a full-length Heavy Chain (HC) and a full-length Light Chain (LC);

preferably, the full length heavy chain is of the IgG1 or IgG4 isotype and the full length light chain is of the Kappa isotype;

more preferably, the full-length heavy chain is SEQ ID NO:166 or has at least 90% sequence identity thereto, and the full-length light chain is SEQ ID NO:167 or has at least 90% sequence identity thereto.

7. The PD-1/CTLA-4 binding protein of any preceding claim, wherein:

the immunoglobulin single variable domain that specifically binds the first antigen-binding domain of PD-1 is N-terminal to the heavy chain variable region that specifically binds the second antigen-binding domain of CTLA-4;

the immunoglobulin single variable domain that specifically binds the first antigen-binding domain of PD-1 is C-terminal to the heavy chain variable region that specifically binds the second antigen-binding domain of CTLA-4;

the immunoglobulin single variable domain that specifically binds the first antigen-binding domain of PD-1 is N-terminal to the light chain variable region that specifically binds the second antigen-binding domain of CTLA-4; and/or

The immunoglobulin single variable domain that specifically binds the first antigen-binding domain of PD-1 is C-terminal to the light chain variable region that specifically binds the second antigen-binding domain of CTLA-4.

8. The PD-1/CTLA-4 binding protein of claim 7, wherein the immunoglobulin single variable domain that specifically binds the first antigen-binding domain of PD-1 is linked, either directly or through a linker, to a second antigen-binding domain that specifically binds CTLA-4;

preferably, the linker is a peptide having an amino acid sequence as shown by (G4S) x, wherein x is independently selected from an integer of 1 to 20;

more preferably, the linker is an amino acid sequence represented by (G4S) 4 or (G4S) 6.

9. The PD-1/CTLA-4 binding protein of any one of the preceding claims, comprising a first polypeptide chain and a second polypeptide chain, wherein:

the first polypeptide chain comprises the amino acid sequence set forth as SEQ ID NO:168-173 or an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99% sequence identity thereto,

the second polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO:167 or an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99% sequence identity thereto.

10. An anti-PD-1/CTLA-4 bispecific antibody comprising:

an immunoglobulin single variable domain as described in any one of items 1 to 3, and

a heavy chain variable region (VH) and a light chain variable region (VL) in the second antigen-binding domain of any one of claims 4-5;

preferably, the anti-PD-1/CTLA-4 bispecific antibody further contains an Fc region of human IgG1 or IgG4.

11. A polynucleotide encoding the PD-1/CTLA-4 binding protein of any one of items 1 to 9 or the anti-PD-1/CTLA-4 bispecific antibody of item 10.

12. A vector comprising the polynucleotide of item 11.

13. A host cell comprising the vector of item 12.

14. A method of making a PD-1/CTLA-4 binding protein, an anti-PD-1/CTLA-4 bispecific antibody, comprising the steps of:

culturing the host cell of item 13;

recovering the PD-1/CTLA-4 binding protein of any one of items 1 to 9 or the anti-PD-1/CTLA-4 bispecific antibody of item 10;

optionally, the PD-1/CTLA-4 binding protein, anti-PD-1/CTLA-4 bispecific antibody is purified and/or modified.

15. A composition or kit comprising a PD-1 binding protein and a CTLA-4 binding protein, wherein the PD-1 binding protein comprises an immunoglobulin single variable domain of any one of claims 1 to 3;

preferably, the CTLA-4 binding protein comprises a heavy chain variable region (VH) and a light chain variable region (VL) in the second antigen-binding domain of any one of items 4-5, or a full length Heavy Chain (HC) and a full length Light Chain (LC) in the second antigen-binding domain of item 6;

preferably, the PD-1 binding protein and CTLA-4 binding protein are in the same or different vessels.

16. The composition or kit of parts according to claim 15, wherein said immunoglobulin single variable domain is a VHH, preferably said VHH is a humanized and/or affinity matured VHH.

17. The composition or kit of any one of claims 15 to 16, wherein the PD-1 binding protein is an antibody that specifically binds PD-1 or a fragment thereof; preferably, the antibody is a camelid antibody, a chimeric antibody, a humanized antibody, a fully human antibody.

18. The composition or kit of any one of claims 15-17, wherein the PD-1 binding protein further comprises a human immunoglobulin Fc region;

preferably, the Fc region is that of human IgG1 or IgG4;

more preferably, the Fc region of human IgG4 has 228P, 234A, 235A and/or 447A mutations.

19. The composition or kit of any one of claims 15 to 18, wherein the CTLA-4 binding protein comprises the heavy chain variable region (VH) and light chain variable region (VL) in the second antigen-binding domain of any one of claims 4 to 5, or the full length Heavy Chain (HC) and full length Light Chain (LC) in the second antigen-binding domain of claim 6.

20. A pharmaceutical composition comprising:

one or more pharmaceutically acceptable carriers, diluents, buffers or excipients, and

any one selected from the group consisting of:

a therapeutically or prophylactically effective amount of the PD-1/CTLA-4 binding protein of any one of claims 1-9, the anti-PD-1/CTLA-4 bispecific antibody of claim 10, the polynucleotide of claim 11, the composition or kit of any one of claims 15-19, or a combination thereof.

21. Use of the PD-1/CTLA-4 binding protein of any one of claims 1-9, the anti-PD-1/CTLA-4 bispecific antibody of claim 10, the composition or kit of any one of claims 15-19, the pharmaceutical composition of claim 20, or a combination thereof, in the manufacture of a medicament for any one selected from the group consisting of:

preventing cancer, treating cancer, inhibiting PD-1 and/or CTLA-4 activity, and promoting T cell proliferation.

Use of a PD-1 binding protein in combination with a CTLA-4 binding protein, the PD-1 binding protein comprising an immunoglobulin single variable domain as described in any one of claims 1 to 3, and the CTLA-4 binding protein comprising a second antigen-binding domain as described in any one of claims 4 to 6, in the manufacture of a medicament for the treatment of cancer;

preferably, the cancer is selected from lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, renal cancer, squamous cell carcinoma, hematological cancer, or a disease or disorder characterized by uncontrolled cell growth.

Drawings

FIG. 1 is a graph showing the binding results of PD-1 antibody to PD-1 on CHO-PD-1 cell line stably and highly expressing PD-1.

FIG. 2 is a graph showing the results of PD-1 antibody blocking the binding of PD-L1 protein to PD-1 on CHO-PD-1 cell line stably expressing PD-1 at high level.

FIG. 3 is a graph showing the results of in vitro immune activation by PD-1 antibody to release the blocking of PD-1/PD-L1.

FIG. 4 is a graph showing the results of in vitro activation of T cells and secretion of IFN γ by PD-1 antibodies numbered 7#, 32# _ hu _3, 106#, and 107 #.

FIG. 5 is a graph showing the results of in vitro activation of T cells and secretion of IFN γ by PD-1 single domain antibodies numbered 32# _ hu _3_IgG4, 7# _ hu _4_hIgG4, 106# _ hu _1_hIgG4, 107# _ hu _4 _hIgG4.

FIGS. 6A-6B are the results of the inhibition of mouse M38 colon cancer tumor growth by the PD-1 antibody and mouse body weight.

Fig. 7A-7B are exemplary dual-impedance structures, PR2001, PR2009, and PR2011 structures are shown in fig. 7A, and PR2004, PR2012, and PR2014 are shown in fig. 7B.

FIG. 8 is a 10% SDS-PAGE gel electrophoresis of the double antibodies.

FIG. 9 is a graph showing the results of binding of antibodies numbered PR2001, PR2004, PR2009, and PR2012 to PD-1 on cell line JurKAT-PD-1 stably highly expressing PD-1.

Fig. 10 is a graph showing the binding results of the antibodies numbered PR2001, PR2004, PR2009, and PR2012 and CTLA4 on the transient cell line CHOK1-CTLA4 highly expressing CTLA 4.

Figure 11 is a graph of the immune activation results of the in vitro release of PD-1/PD-L1blockade by antibody numbered PR 2009.

FIG. 12 is a graph showing the results of in vitro activation of T cells and secretion of IL-2 in an antibody SEB assay numbered PR 2009.

FIG. 13 is a graph showing the results of in vitro activation of T cells and secretion of IL-2 in an antibody MLR assay numbered PR 2009.

FIGS. 14A-14B are the results of PR2009 antibody inhibiting growth of mouse M38 colon cancer tumor and mouse body weight.

Detailed Description

Term(s)

In order that the disclosure may be more readily understood, certain techniques and sciences are now specifically defined. Unless clearly defined otherwise herein, all other techniques and sciences used in the present disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The three letter codes and the one letter codes for amino acids used in this disclosure are as described in j. Diol. Chem,243, p3558 (1968).

"PD-1" refers to the T cell co-inhibitor programmed death-1 protein, also known as CD279, which is used interchangeably with "programmed death 1", "programmed cell death 1", "protein PD-1", "PDCD1", and "hPD-1", and includes variants, isoforms, species homologs of human PD-1, and analogs having at least one common epitope with PD-1. The amino acid sequence of full-length PD-1 is provided in GenBank as accession number NP _ 005009.2. The term "PD-1" includes recombinant PD-1 or fragments thereof. The term also encompasses PD-1 or fragments thereof coupled to, for example, a histidine tag, a mouse or human Fc, or a signal sequence such as ROR 1. For example, the term includes SEQ ID NO: 1-4. The term "PD-1" refers to human PD-1 unless specified as being from a non-human species.

"CTLA-4" refers to cytotoxic T lymphocyte-associated protein 4, an immune checkpoint receptor or T cell co-suppressor, also known as CD152. The amino acid sequence of full-length human CTLA-4 is provided as SEQ ID NO:505 (accession number NP-005205.2). The term "CTLA-4" includes recombinant CTLA-4 or fragments thereof. The term also encompasses CTLA-4 or fragments thereof, coupled to, for example, a histidine tag, a mouse or human Fc, a signal sequence, or the transmembrane and cytoplasmic domains of CD300a (aa 181-299; accession number NP 009192.2). For example, the terms include the sequences discussed in the examples, such as a myc-myc-polyhistidine tag comprising the C-terminus of full-length CTLA-4 or a mouse Fc (mIgG 2 a) comprising the C-terminus of full-length CTLA-4. The term "CTLA-4" means human CTLA-4 unless explicitly stated to be from a non-human species.

"PD-1 binding protein" means any protein or any molecule comprising said protein that is capable of specifically binding PD-1. The PD-1 binding protein can include an antibody, antigen-binding fragment thereof, or conjugate thereof, as defined in the present disclosure, directed to PD-1. PD-1 binding proteins also encompass immunoglobulin superfamily antibodies (IgSF) or CDR grafted molecules. A "PD-1 binding protein" of the present disclosure may comprise at least one immunoglobulin single variable domain (e.g., VHH) that binds PD-1. In some embodiments, a "PD-1 binding protein" may comprise 2, 3, 4, or more immunoglobulin single variable domains (e.g., VHH) that bind PD-1. The PD-1 binding proteins of the present disclosure may also comprise, in addition to binding to an immunoglobulin single variable domain comprising PD-1, a linker and/or a moiety with effector function, for example a half-life extending moiety (such as an immunoglobulin single variable domain that binds serum albumin), and/or a fusion partner (such as serum albumin) and/or a conjugated polymer (such as PEG) and/or an Fc region. In some embodiments, a "PD-1 binding protein" of the present disclosure also encompasses a bi/multispecific antibody containing immunoglobulins that bind to different antigens (e.g., a first antibody that binds to a first antigen (e.g., PD-1) and a second antibody that binds to a second antigen (e.g., CTLA-4), optionally including a third antibody that binds to a third antigen, further optionally including a fourth antibody that binds to a fourth antigen.

By "CTLA-4 binding protein" is meant any protein or any molecule comprising said protein capable of specifically binding CTLA-4. CTLA-4 binding proteins can include antibodies, antigen-binding fragments thereof, or conjugates thereof, as defined in the present disclosure, directed against CTLA-4.

"PD-1/CTLA-4 binding protein" means any protein or any molecule comprising said protein capable of specifically binding to PD-1 and CTLA-4, encompassing the aforementioned definition of "PD-1 binding protein". PD-1/CTLA-4 binding proteins can include antibodies, antigen-binding fragments thereof, or conjugates thereof, as defined in the present disclosure, directed against PD-1 and CTLA-4.

"binding to PD-1" means capable of interacting with PD-1 or a fragment thereof or an epitope thereof, which PD-1 or a fragment thereof or an epitope thereof may be of human origin. "binding to CTLA-4" means capable of interacting with CTLA-4 or a fragment or epitope thereof, and said PD-1 or fragment or epitope thereof may be of human origin.

"antibody" or "immunoglobulin" broadly encompasses conventional antibodies (tetrapeptide chain structured antibodies made from two identical heavy chains and two identical light chains linked by an interchain disulfide bond), as well as Fab, fv, sFv, F (ab') 2, linear antibodies, single chain antibodies, scFv, sdAb, sdFv, nanobodies, peptidic antibodies, peptibodies, domain antibodies (heavy chain (VH) antibodies, light chain (VL) antibodies), and multispecific antibodies (bispecific antibodies, diabodies, triabodies, and tetrabodies, tandem di-scfvs, tandem tri-scfvs) having antigen binding activity, and thus "antibodies" as used in this disclosure include full-length antibodies, individual chains thereof, and any portions, domains, or fragments thereof having antigen binding activity, as well as multispecific antibodies (including, but not limited to, antigen binding domains or fragments, such as VHH domains or VH domains, respectively) comprising individual chains thereof and any portions, domains or fragments thereof having antigen binding activity. Conventional antibodies or immunoglobulins are generally tetrapeptide chain structures made of two identical heavy chains and two identical light chains joined by interchain disulfide bonds. The constant regions of the heavy chains differ in amino acid composition and arrangement, and therefore, in antigenicity. Accordingly, immunoglobulins can be classified into five classes, otherwise known as the isotype of immunoglobulins, i.e., igM, igD, igG, igA, and IgE, with their corresponding heavy chains being the μ, δ, γ, α, and ε chains, respectively. The same class of igs can be divided into different subclasses according to differences in amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain, and for example, iggs can be classified into IgG1, igG2, igG3 and IgG4. Light chains are classified as either kappa or lambda chains by the differences in the constant regions. Each of the five classes of Ig may have a kappa (kappa) chain or a lambda (lambda) chain. In some embodiments, an antibody of the present disclosure specifically or substantially specifically binds to PD-1.

"antibodies" of the present disclosure include, but are not limited to: (i) a Fab fragment consisting of the VL, VH, CL and CH1 domains; (ii) an Fd fragment consisting of VH and CH1 domains; (iii) A F (ab') 2 fragment, a bivalent fragment comprising two linked Fab fragments; (vii) A single chain Fv molecule (scFv), wherein the VH domain and the VL domain are connected by a peptide linker that allows the two domains to bind to form an antigen binding site; (Bird et al, 1988, 242 in Science 242, huston et al, 1988, proc. Natl. Acad. Sci. U.S. 85, 5879-5883), 242, incorporated herein by reference in its entirety; (iv) "bifunctional antibodies" or "trifunctional antibodies," multivalent or multispecific fragments constructed by gene fusion (Tomlinson et al, 2000, methods in enzymology 326, 461-479, wo94/13804, holliger et al, 1993, journal of the american national academy of sciences, 90, 6444-6448, all incorporated herein by reference); (v) "domain antibodies" or "dAbs" (sometimes referred to as "immunoglobulin single variable domains"), including immunoglobulin single variable domains from other species, such as rodents (e.g., as disclosed in WO 00/29004), nurse sharks and camelidae V-HH dAbs; (vi) SMIPs (small molecule immunopharmaceuticals), camel antibodies, nanobodies, and ignars; (vii) the humanized antibody of the above (i) to (vi).

Antibodies of the present disclosure generally use the Kabat numbering system, where not specifically indicated. EU numbering in Kabat is also generally used for constant domains and/or Fc domains.

"Domain" of a polypeptide or protein refers to a folded protein structure that is capable of maintaining its tertiary structure independently of the rest of the protein. In general, a domain is responsible for a single functional property of a protein, and in many cases may be added, removed, or transferred to other proteins without loss of function of the rest of the protein and/or the domain.

"immunoglobulin domain" refers to a globular region of an antibody chain (e.g., a chain of a conventional tetrapeptide chain structured antibody or a chain of a heavy chain antibody), or to a polypeptide consisting essentially of such a globular region. Immunoglobulin domains are characterized by their ability to maintain the immunoglobulin fold characteristics of an antibody molecule, e.g., in conventional tetrapeptide chain structural antibodies, which consist of two beta sheets linked by intrachain disulfide bonds of the heavy and light chains.

"immunoglobulin variable domain" means a region consisting essentially of four "framework regions," referred to in the art and hereinafter as "framework region" and "CDR" region, respectively, containing "framework region 1" or "FR1", "framework region 2" or "FR2", "framework region 3" or "FR3", and "framework region 4" or "FR4", wherein the framework regions are separated by three "complementarity determining regions" or "CDRs" referred to as "complementarity determining region 1" or "CDR1", "complementarity determining region 2" or "CDR2", and "complementarity determining region 3" or "CDR3", respectively. Thus, the general structure or sequence of an immunoglobulin variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Immunoglobulin variable domains confer specificity for antigens by virtue of having an antigen binding site.

"antibody Framework (FR)" refers to a portion of a variable domain that serves as a scaffold for the antigen binding loops (CDRs) of the variable domain.

For the determination or definition of "CDRs", a deterministic delineation of CDRs and identification of residues comprising the binding site of an antibody can be accomplished by distinguishing the structure of the antibody and/or distinguishing the structure of the antibody-ligand complex. This can be achieved by any of a variety of techniques known to those skilled in the art, such as X-ray crystallography. A variety of analytical methods can be used to identify CDRs, including but not limited to Kabat numbering system, chothia numbering system, abM numbering system, IMGT numbering system. The methods used herein may utilize CDRs defined according to any of these methods.

An "immunoglobulin single variable domain" is generally used to refer to an immunoglobulin variable domain (which may be a heavy or light chain domain, including a VH, VHH or VL domain) that can form a functional antigen binding site without interacting with other variable domains (e.g., without VH/VL interaction as is required between the VH and VL domains of conventional four-chain monoclonal antibodies). Examples of "immunoglobulin single variable domains" include nanobodies (including VHH, humanized VHH and/or camelized VH, e.g. camelized human VH), ignars, domains, (single domain) antibodies (such as dAbs) as or derived from VH domains^TM) And (single domain) antibodies (such as dAbs) as or derived from VL domains^TM). Immunoglobulin single variable domains based on and/or derived from heavy chain variable domains (such as VH or VHH domains) are generally preferred. A specific example of an immunoglobulin single variable domain is a "VHH domain" (or simply "VHH") as defined below.

"VHH domains", also known as heavy chain single domain antibodies, VHHs, VHH antibody fragments, VHH antibodies, nanobodies, are variable domains of antigen-binding immunoglobulins known as "heavy chain antibodies" (i.e. "antibodies lacking the light chain") (Hamers-Casterman C, atarhouch T, muydermans S, robinson G, hamers C, songa EB, bendahman N, hamers R.: nature cure antibodies void of light chains "; nature363, 446-448 (1993)). The term "VHH domain" is used to distinguish the variable domain from the heavy chain variable domain (which is referred to in this disclosure as "VH domain") and the light chain variable domain (which is referred to in this disclosure as "VL domain") present in conventional tetrapeptide chain structural antibodies. The VHH domain specifically binds to an epitope without the need for an additional antigen binding domain (as opposed to the VH or VL domain in conventional tetrapeptide chain structural antibodies, in which case the epitope is recognized by the VL domain together with the VH domain). The VHH domain is a small, stable and efficient antigen recognition unit formed from a single immunoglobulin domain. The terms "heavy chain single domain antibody", "VHH domain", "VHH antibody fragment", "VHH antibody" and "domain" ("Nanobody" is a trademark of Ablynx n.v. company, ghent, belgium) are used interchangeably. "VHH domains" include, but are not limited to, natural antibodies produced by camelids, antibodies produced by camelids which are subsequently humanized, or screened by phage display technology. The total number of amino acid residues in the VHH domain will generally range from 110 to 120, often between 112 and 115. It should be noted, however, that smaller and longer sequences may also be suitable for the purposes described in this disclosure.

By "Fc variant" or "variant Fc" is meant a protein comprising amino acid modifications in the Fc domain. The Fc variants of the present disclosure are defined in terms of the amino acid modifications that make up them. Thus, for example, S228P or 228P is an Fc variant having a proline substitution at position 228 relative to a parent Fc polypeptide, wherein numbering is according to the EU index. The identity of the WT amino acid may be unspecified, in which case the aforementioned variant is referred to as 228P.

Examples of "humanization" include that VHH domains derived from camelidae may be "humanized" (also referred to herein as "sequence optimization", which in addition to humanization may also encompass other modifications to the sequence by one or more mutations providing VHH modifying properties, such as removal of potential post-translational modification sites) by replacing one or more amino acid residues in the amino acid sequence of the original VHH sequence with one or more amino acid residues present at corresponding positions in the VH domain of a human conventional tetrapeptide chain structured antibody. The humanized VHH domain may contain one or more fully human framework region sequences, and in some embodiments, may contain the human framework region sequences of IGHV 3. Yet another example of "humanization" includes grafting heterologous CDR sequences to a human antibody variable region framework, i.e., an antibody produced from different types of human germline antibody framework sequences. Can overcome the strong antibody variable antibody reaction induced by the chimeric antibody because of carrying a large amount of heterologous protein components. Humanization methods such as protein surface amino acid humanization (resurfacing) and antibody humanization general framework grafting (CDR grafting to a non-reactive framework) are methods of "grafting" CDRs onto other "scaffolds (including but not limited to human scaffolds or non-immunoglobulin scaffolds). Scaffolds and techniques suitable for such CDR grafting are known in the art. Germline DNA Sequences of genes such as the human heavy and light chain variable regions can be found in the "VBase" human germline sequence database, as well as in Kabat, E.A. et al, 1991Sequences of Proteins of Immunological Interest, 5 th edition. In addition, to avoid reduced immunogenicity and reduced activity, the human antibody variable region framework sequences may be back-mutated or back-mutated minimally to retain activity.

An "affinity matured" PD-1 binding protein or PD-1 antibody has one or more changes in one or more CDRs that result in an increased affinity for an antigen compared to its parent antibody. Affinity matured antibodies can be prepared, for example, by methods known in the art as described below: marks et al, 1992, biotechnology 10:779-783 or Barbas et al, 1994, proc. Nat. Acad. Sci, USA 91: 3809-3813; shier et al, 1995, gene 169:147 to 155; yelton et al, 1995, immunol.155:1994-2004; jackson et al, 1995, J.Immunol.154 (7): 3310-9; and Hawkins et al, 1992, J.MoI.biol.226 (3): 889896; KS Johnson and RE Hawkins, "Affinity mapping of antibodies using phase display", oxford University Press 1996.

Generally, the PD-1 binding proteins of the present disclosure will be measured as preferably 10 as measured in Biacore or KinExA or Fortibio assays^-7To 10^-10Mole/liter (M), more preferably 10^-8To 10^-10Mol/liter, even more preferably 10^-9To 10^-10Or a dissociation constant (KD) of less, and/or at least 10^-7M, preferably at least 10^-8M, more preferably at least 10^-9M, more preferably at least 10^-10The association constant (KA) of M binds to the antigen to be bound (i.e., PD-1). Any greater than 10^-4The KD value of M is generally considered to indicate non-specific binding. Specific binding of an antigen binding protein to an antigen or epitope can be determined in any suitable manner known, including, for example, surface Plasmon Resonance (SPR) assays, scatchard assays, and/or competitive binding assays (e.g., radioimmunoassays (RIA), enzyme Immunoassays (EIA), and sandwich competitive assays, as described in the present disclosure.

"inhibit" or "block" are used interchangeably and encompass both partial and complete inhibition/blocking.

"inhibiting growth" (e.g., reference to a cell) is intended to include any measurable reduction in cell growth.

"specific binding", "selective binding" refers to binding of an antibody to an epitope on a predetermined antigen. Typically, antibodies have a molecular weight of about less than 10 when measured in an instrument by Surface Plasmon Resonance (SPR) techniques using recombinant human PD-1 or an epitope thereof as the analyte and an antibody as the ligand^-7M or even smaller equilibrium dissociation constant (K)_D) Binds to a predetermined antigen or epitope thereof and binds to the predetermined antigen or epitope thereof with at least twice the affinity as it binds to a non-specific antigen other than the predetermined antigen (or epitope thereof) or closely related antigen (e.g., BSA, etc.).

"amino acid mutation" includes amino acid substitutions, deletions, insertions, modifications, and any combination thereof, to achieve the final construct such that the final construct possesses the desired properties, e.g., enhanced stability, increased activity. Amino acid sequence deletions and insertions include amino and/or carboxy-terminal deletions and amino acid insertions. Preferred amino acid mutations are amino acid substitutions. To alter the binding properties of, for example, an anti-PD-1 antibody, non-conservative amino acid substitutions may be made, i.e., one amino acid is replaced with another amino acid having a different structural and/or chemical property. Preferred amino acid substitutions include the substitution of a hydrophilic amino acid for a hydrophobic amino acid. Amino acid substitutions include substitutions with non-naturally occurring amino acids or with naturally occurring amino acid derivatives of the 20 standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods well known in the art, including methods of site-directed mutagenesis, PCR, gene synthesis, chemical modification, and the like. Amino acid mutations can occur in the CDR regions, FR regions, or Fc regions of an antibody.

"Back-mutation" refers to the mutation of amino acid residues in the FR region of a human antibody to the amino acid residues in the corresponding position in the original antibody, usually in order to avoid the reduction in immunogenicity and thus activity of the humanized antibody, and the humanized antibody variable region may be subjected to minimal reverse mutation to maintain the activity of the antibody.

The "PD-1 binding protein", "PD-1 antibody", "PD-1/CTLA-4 binding protein", "anti-PD-1/CTLA-4 antibody" of the present disclosure may comprise one or more effector molecules, for example, in a conjugated manner. Such "effector molecules" include, for example, antineoplastic agents, drugs, toxins, biologically active proteins (e.g., enzymes), other antibodies or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof (e.g., DNA, RNA, and fragments thereof), radionuclides, particularly radioiodinates, radioisotopes, chelated metals, nanoparticles, and reporter groups such as fluorescent compounds or compounds detectable by NMR or ESR spectroscopy.

"sequence" (for example in the terms "immunoglobulin sequence", "antibody sequence", "single variable domain sequence", "VHH sequence" or "protein sequence") should generally be understood as including both the relevant amino acid sequences and the nucleic acid or nucleotide sequences encoding the sequences, unless the disclosure requires a further defined interpretation.

"Polynucleotide" or "nucleic acid" refers to a chain of nucleotides of any length, including DNA and RNA.

"homology" or "identity" refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., 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 matching or homologous positions shared by the two sequences divided by the number of positions compared x 100%. For example, two sequences are 60% homologous if there are 6 matches or homologies at 10 positions in the two sequences when the sequences are optimally aligned. In general, comparisons are made when aligning two sequences to obtain the greatest percentage of homology.

A "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any material that, when combined with an active ingredient, allows the ingredient to retain biological activity and not react with the immune system of a subject. Examples include, but are not limited to, any standard pharmaceutical carrier, such as phosphate buffered saline solution, water, emulsions such as oil/water emulsions, and various types of wetting agents. In some embodiments, the diluent for aerosol or parenteral administration is Phosphate Buffered Saline (PBS) or physiological (0.9%) saline. Compositions comprising such carriers are formulated by well-known conventional methods (see, e.g., remington's Pharmaceutical Sciences, 18 th edition, A.Gennaro, eds., mack Publishing Co., easton, PA,1990; and R Remington, the Science and Practice of Pharmacy, 20 th edition Mack Publishing, 2000).

"cancer" and "cancerous" and "tumor" refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth. In some embodiments, the cancer is selected from: non-small cell lung cancer, glioblastoma, neuroblastoma, melanoma, breast cancer (e.g., triple negative breast cancer), gastric cancer, colorectal cancer (CRC), and hepatocellular carcinoma. Also, in some embodiments, the cancer is selected from: non-small cell lung cancer, colorectal cancer, glioblastoma and breast cancer (e.g., triple negative breast cancer), including metastatic forms of those cancers.

"preventing cancer" refers to delaying, inhibiting or preventing the onset of cancer in a subject in which the onset of cancer or tumorigenesis has not been confirmed, but a susceptibility to cancer has been identified, e.g., by genetic screening or other methods. The method also includes treating a subject having a pre-cancerous condition to stop progression or cause regression of the pre-cancerous condition to the malignant tumor.

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

By "treating" is meant administering an internal or external therapeutic agent, such as a pharmaceutical composition comprising any of the antibodies of the present disclosure as a therapeutic agent, to a subject who has had, is suspected of having, or is predisposed to having, one or more proliferative diseases or symptoms thereof for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in the subject or population being treated in an amount effective to alleviate one or more symptoms of the disease, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measurable degree. The amount of therapeutic agent effective to alleviate any particular disease symptom (also referred to as a "therapeutically effective amount") can vary depending on a variety of factors, such as the disease state, age, and weight of the subject, and the ability of the drug to produce a desired therapeutic effect in the subject. Whether a disease symptom has been reduced can be assessed by any clinical test commonly used by physicians or other health professional to assess the severity or progression of the symptom. Although embodiments of the present disclosure (e.g., methods of treatment or articles of manufacture) may be ineffective in alleviating a symptom of a target disease in a subject, they should alleviate the symptom of the target disease in a statistically significant number of subjects as determined by any statistical test method known in the art, such as Student's t-test, chi-square test, U-test by Mann and Whitney, kruskal-Wallis test (H-test), jonckhere-Terpstra test, and Wilcoxon test.

An "effective amount" comprises an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition. An effective amount also means an amount sufficient to allow or facilitate diagnosis. An effective amount for a subject may vary depending on the following factors: such as the condition to be treated, the general health of the subject, the method and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or dosage regimen that avoids significant side effects or toxic effects. The subject of the present disclosure may be an animal or human subject.

"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.

By "subject", "patient" as used in this disclosure is meant a mammal, particularly a primate, particularly a human.

Detailed description of the preferred embodiments

The following examples are presented to further illustrate the present disclosure, but are not intended to limit the scope of the present disclosure. The experimental methods of the present disclosure, in which specific conditions are not specified, are generally performed according to conventional conditions, such as the antibody technical laboratory manual of cold spring harbor, molecular cloning manual; or according to the conditions recommended by the manufacturer of the raw material or the goods. Reagents of specific sources are not indicated, and conventional reagents are purchased in the market.

Example 1: preparation of PD-1 antigen and protein for detection

PD-1 antigen design:

human PD-1 was used as a PD-1 template to design the amino acid sequences of the PD-1 antigen and the protein for detection (the PD-1 antigen is not specifically described below and refers to human PD-1).

Human PD-1 full-length protein:

( Note that: the double transverse line part is Signal peptide (Signal peptide); the cross-line part is PD-1 Extracellular region (Extracellular domain), wherein 35-144 bits are Ig-like V-type 1domain,70-77 bits are Interaction with CD274; the dotted-dashed portion is a Transmembrane region portion (Transmembrane domain); the oblique part is divided into intracellular domains (Cytoplasmic domains). )

SEQ ID NO：1

Monkey PD-1 full-length amino acid sequence:

( Note that: the double transverse line part is a signal peptide; the cross-line part is the extracellular region of PD-1, wherein, the 38-127 sites are V-Set domains, and the 39-125 sites are Ig-like; the dotted line portion is a Transmembrane domain portion (Transmembrane domain); the oblique portion is divided into intracellular domains (Cytoplasmic domains). )

SEQ ID NO：2

Human PD-1 antigen (a commercial product (Nano Biological Cat.10377-H08H)) is used for screening and detection:

LDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQD CRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQAHHHHHHHHHH (Note: the cross-hatched part is the PD-1 extracellular region; the italic part is the His-tag.)

SEQ ID NO：3

Human PD-1-Fc antigen (a commercial product (Baiying biology: B3789)) for screening and detection:LDSPDRPWNPPT FSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRA RRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

( And (3) annotation: the cross line part is an extracellular region; the italicized portion is the human Fc label. )

SEQ ID NO：4

Human PD-L1 antigen (commercially available product as Nano Biological cat) for detection：10084-H08H-B))：FT VTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNA ALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQV LSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTAHHHHHHHHHH

( Note that: the cross-line part is a PD-L1 extracellular region; the italic part is a His-tag label. )

SEQ ID NO：5

Human PD-L2 antigen (a commercial product (Sino Biological cat: 10292-H08H-B)) for detection:LF TVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQKVENDTSPHRERATLLEEQLPLGKASFHIPQVQVRDE GQYQCIIIYGVAWDYKYLTLKVKASYRKINTHILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGL YQVTSVLRLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHPAHHHHHHHHHH (Note: the cross-hatched part is the PD-L2 extracellular region; the italic part is the His-tag.)

SEQ ID NO：6

Example 2 screening of Positive antibody sequences that specifically bind to human PD-1

Human PD-1 proteins (ACRO, cat # PD-1-H5259 and ACRO, cat # PD-1-H5221) were used to immunize two bactrian camels (Camelus dromedarius) separately, 5mL of camel serum before immunization was taken and serum was separated. Freund's complete adjuvant was mixed with antigen volume 1:1 after mixing, the camels were immunized subcutaneously in multiple spots (immunization dose of 100. Mu.g protein/mouse, freund's complete adjuvant for the first time and Freund's incomplete adjuvant for the remainder). Boosters were performed every two weeks and titers were measured after four immunizations. Plates (100. Mu.L/well) were coated with 5. Mu.g/mL PD-1-his protein overnight at 4 ℃. The next day of washing was followed by addition of 4% skimmed milk powder for blocking at 37 deg.C for 2h. After washing, different dilutions of camel serum were added and incubated at 37 ℃ for 1 h. Negative controls were preimmune serum (1. After incubation, washed three times with PBST, HRP-anti-camelid antibody (1. Finally, alkaline phosphatase developing solution is added for washing, 2M sulfuric acid is used for termination, and the absorption value is read at the wavelength of 450 nm. 1: titers were detected after 25600-fold dilution. And (5) collecting camel peripheral blood for library construction when the potency is qualified.

The camel peripheral blood is used for separating lymphocytes, and the cell count is 1.2 multiplied by 10⁸Resuspend with Trizol reagent (1X 10)⁷Individual cells/mL Trizol) to lyse the cells, left on ice for 5min; centrifuging at 13000rpm for 3min, collecting supernatant, and removing precipitate; adding 1/5 volume of chloroform, shaking vigorously for 30-60s, and standing in ice bath for 2min; centrifuging at 13000rpm for 10min, and sucking the upper aqueous phase layer into a new 1.5mL tube; adding isopropanol with equal volume, mixing, and standing at-20 deg.C for 30min; centrifuging at 13000rpm for 10min, removing supernatant, and retaining precipitate; adding pre-cooled 75% ethanol, washing the precipitate, and standing at room temperature for 5-10min; adding 600 mu L of deionized water removed by RNase, redissolving to obtain RNA, carrying out reverse transcription to obtain cDNA, and constructing a phage library.

Obtaining a single domain antibody with high affinity with PD-1 antigenic protein by screening a phage library, combining 20 mu g of PD-1-avi-biotin protein with 1mg of Dynabeads MyOne streptavidin T1, standing at 37 ℃ for one hour, sealing with 2% skim milk at room temperature for 2 hours, adding a camel heavy chain single domain antibody phage display library, and acting at room temperature for 1 hour. Washing with PBST (0.05% Tween-20) solution 9 times, removing unbound phage. Phages specifically bound to PD-1 were eluted with 1mg/mL trypsin and infected with E.coli TG1, which was grown in log phase, and phages were generated and purified for the next round of screening. The same screening process was repeated for 2-3 rounds. Positive clones were enriched.

From the screening of enriched positive clones, 96 monoclonal colonies were picked and packaged into phage single chain antibodies for phage ELISA testing. The ELISA plates were coated with 2. Mu.g/mL of PD-1-his protein, and the diluted phage supernatant was added and detected with anti-M13 HRP. Clones tested by ELISA binding to an OD450 value greater than 0.5 were sequenced to give 51 specific sequences.

EXAMPLE 3 construction of intact monoclonal antibodies

Complete antibodies are constructed by 51 specific sequences obtained by screening phage libraries in example 2, and then 25 antibodies are determined to have strong binding capacity and can inhibit the interaction between PD-1 and PD-L1 by an ELISA binding experiment and an ELISA competition experiment, and the results are shown in Table 1.

TABLE 1 ELISA test results for PD-1 antibody

The complete VHH sequence is as follows:

>2

QVQLVESGGGSVQAGGSLRLFCSPSGYTYSRDCMGWFRQAPGKEREGVAAICSSGRNTYYTYYADSVKG RFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADLRSSGGDLTYGLAPGPYEYKYWGQGTQVTVSS

SEQ ID NO：7

>4

QVQLVESGGGSVQAGGSLRLSCAVSGFRYSPILMGWFRQGPGKDREGVASIDSVGTTDYTDSVKGRFTISRDNAENTLFLQMNSLKPEDTGMYYCAAALMRTYLPLQPRQYDFWGQGTLVTVSS

SEQ ID NO：8

>6

HVQLVESGGGSVQAGGSLRLSCAVSGFRYSPILMGWFRQGPGKDRGGVASIDSVGTTDYTDSVKGRFTISRDNAENTLFLQMNSLKPEDTGMYYCAAALMRTYLPLQPRQYDFWGQGTLVTVSS

SEQ ID NO：9

>7

HVQLVESGGGSVQAGGSLRLACASSRNTNRYNFMTWFRQAPGKEREGVAAIYTGFGNTYYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYHCAAALRDGSWSSQNYDYWGQGTQVTVSS

SEQ ID NO：10

>11

DVQLVESGGGSVQAGGSLRLSCAVSGFRYSPILMGWFRQGPGKDREGVASIDSVGTTDYADSVKGRFTISRDNAENTLFLQMNSLKPEDTGMYYCAAALMRTYLPLQPRQYDFWGQGTQVTVSS

SEQ ID NO：11

>19

HVQLVESGGGSVQAGGSLRLSCAVSGFRYSPILMGWFRQGPGKDREGVASIDSVGTTNYTNSVKGPFTISLDNAQNTLFLQMNILKPEDTGMYYCAAALMRTYLPLQPRQYDFWGQGTLDIVSS

SEQ ID NO：12

>32

QVQLVESGGGSVQAGGSLRLSCAVSGFRYSPILMGWFRQGPGKDREGVASIDSVGATDYADSVKGRFTISRDNAENTLFLQMNSLKPEDTGMYYCAAALMRTYLPLQPRQYDFWGQGTLVTVSS

SEQ ID NO：13

>41

HVQLVESGGGSVQAGGSLRLSCAVSGFRYSPILMGWFRQGPGKDREGVASIDSVGTTDYADSVKGRFTISRDNAENTLFLQMNSLKPEDTGMYYCAAALMRTYLPLQPRQYDFWGQGTLVTVSS

SEQ ID NO：14

>54

HVQLVESGGGSVQAGGSLRLSCAVSGFRYSPILMGWFRQGPGKDREGVATIDSVGTTDYTDSVKGRFTISRDNAENTLFLQMNSLKPEDTGMYYCAAALMRTYLPLQPRQYDFWGQGTQVTVSS

SEQ ID NO：15

>56

DVQLVESGGGSVQAGGSLRLSCAVSGFRYSPILMGWFRQGPRKDREGVASIDSVGTTDYTDSVKGRFTISRDNAENTLFLQMNSLKPEDTGMYYCAAALMRTYLPLQPRQYDFWGQGTQVTVSS

SEQ ID NO：16

>59

QVQLVESGGGSVQAEGSLRLSCAVSGFRYSPILMGWFRQGPGKDREGVASIDSVGTTDYTDSVKGRFTISRDNAENTLFLQMNSLKPEDTGMYYCAAALMRTYLPLQPRQYDFWGQGTLVTVSS

SEQ ID NO：17

>61

HVQLVESGGGSVQAGGSLRLSCAASGYTYSSLCMGWFRQAPGKEREGVATIYTGDSSTYYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAAAYGRRWCERLYMYDSWGQGTQVTVSS

SEQ ID NO：18

>62

HVQLVESGGGSVQAGGSLRLSCAVSGFRYSPILMGWFRQGPGKDREGVASIDSVGTTDYTDSVKGRFTISRDNAENTLFLQMNSLKPEDTGMYYCAAALMRTYLPLQPRQYDFWGQGTLVTVSS

SEQ ID NO：19

>68

QVQLVESGGGSVQAGGSLRLSCAVSGFRYSPILMGWFRQGPGKDREGVASIDSVGTTGYTDSVKGRFTISRDNAENTLFLQMNSLKPEDTGMYYCAAALMRTYLPLQPRQYDFWGQGTQVTVSS

SEQ ID NO：20

>104

HVQLVESGGGSVQAGGSLRLSCAASGATFSVYSMGWFRQAPGKEREAVVALYPTAGRTYYGDSVKGRFTISQDNAENTVYLQMNSLQPEDTAMYYCAAGLTGRWWLPEADYWGQGTQVTVSS

SEQ ID NO：21

>106

EVQLVESGGGSAQAGGSLRLSCTSSTYTFKNKCMGWFRQAPGKEREGVAVVDRFGGTIYAASVKGRFAISKDDAKNTLDLLMNSLKPEDTAMYYCAAGSYTSANSCQPDALWGQGTQVTVSS

SEQ ID NO：22

>107

DVQLVGSGGGSVQVGGSLRLSCAASGYTGNRRYCMGWFRQAPGNEREGVAVINTGANTTYYADSVKGRF TISQDNAKNTVYLQMNSLKPEDTAMYYCGVGWRALCEVNGYVYDSWGQGTQVTVSS

SEQ ID NO：23

>108

QVQLVESGGGSVQAGGSLRLSCAASGATFSVYSMGWFRQAPGKERESVVALYPTAGRTYYADSVKGRFTVSQDNAENTVYLQMNSLQPEDTAMYYCAAGLTGRWWLPEADYWGQGIQVTVSS

SEQ ID NO：24

>109

HVQLVESGGDSVQAGGSLRLSCVVSGVTYSFRYMGWFRQAPGKERELVADIYTPSGQTYYGDSVKGRFTISHDYAKNTVHLQMNNLQPEDTAIYHCAAAEGVLGRPLTPAQYSYWGQGTLVTVSS

SEQ ID NO：25

>112

EVQLVESGGGSAQAGGSLRLSCTSSTYTFKNKCMGWFRQAPGKEREGVAVVDRYGGIIYAASVKGRFAISKDDAKNTLDLLMNSLKAEDTAMYYCAAGSYTSDGSCQPDALWGQGTLVTVSS

SEQ ID NO：26

>113

DVQLVESGGGSAQAGGSLRLSCTSSTYTFRNKCMGWFRQAPRKEREGVAVVDRFGGTIYAASVKDRFTISKDDTKNTLDLLMNSLKPEDTAMYYCAAGSYTDAGSCHPDALWGQGTLVTVSS

SEQ ID NO：27

>114

DVQLVESGGGSVQAGGSLTLSCTASKGYTYVRNLMAWFRQAPGNEREGVAVIYVGDTTYYADSVKGRFTISEDNAKNTIYLQMNGLKPEDTAMYYCAAKTGIIQVDDALQPNEYNYWGQGTQVTVSS

SEQ ID NO：28

>116

DVQLVESGGGSVQAGGSLTLSCAASGATFSVYSMGWFRQAPGKEREAVAAIYPTAGRTYFADSVKGRFTISQDNAENTVYLQMNSLQAEDTAIYYCAAGLTGRWWLPEADYWGQGTQVTVSS

SEQ ID NO：29

>118

QVQLVESGGGSVQAGGSLRLSCAGSGATFSVYSMGWFRQAPGKEREAVAAIYPTAGKTYYADSMRGRFTISQDNAENTVYLHMNSLQPEDTAMYYCAAGLTGRWWLPEADYWGQGTLVTVSS

SEQ ID NO：30

>119

DVQLVESGGGSVQAGGSLTLSCAGSGATFSVYSMGWFRQAPGKEREAVAAIYPTAGKTYYADSMKGRFTISQDNAENTVYLHMNSLQPEDTAMYYCAAGLTGRWWLPEADYWGQGTQVTVSS

SEQ ID NO：31

>122

EVQLVESGGGSVQVGGSLRLSCAASGYTGNRRYCMGWFRQAPGNEREGVAVINTGTNSTYYADSVKGRF TISQDNAKNTVYLQMNSLKPEDTAMYYCAVGWRALCEVNGYVYDSWGQGTLVTVSS

SEQ ID NO：32

>123

QVQLVESGGGSVQAGGSLRLSCAASGATFSVYSMGWFRQAPGKEREAVTAIYPTAGRTYFADSVKGRFTISQDNAENTVYLQMNSLQPEDTAMYYCAAGLTGRWWLPEADYWGQGTQVTVSS

SEQ ID NO：33

the above sequence SEQ ID NO:7-33, FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the FR sequence in italic, and the CDR1, CDR2 and CDR3 sequences in underlined, respectively. The numbering conventions provided for PD-1 antibodies in this disclosure are all Kabat, and the CDR sequences are summarized in table 2.

TABLE 2 CDR sequences of PD-1 Single Domain antibodies

The antibody sequence is fused with a human IgG1-Fc (CH 2-CH 3) segment sequence and is constructed into a PTT5 expression vector, and the sequence of the connected human IgG1-Fc can be shown as follows:

EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO：103

the following is the whole protein sequence of the antibody sequence fused to the human Fc (CH 2-CH 3) segment, the human IgG1-Fc (CH 2-CH 3) segment sequence (shown in SEQ ID NO: 103) is single underlined, and the linker sequence is double underlined. The protein sequences are as follows (see, for example, nos. 32#, 7#, 106#, and 107#, as well as other PD-1 antibodies):

32#-IgG1：

7#-IgG1：

106#-IgG1:

107#-IgG1:

example 4 humanization of PD-1 antibodies

Three-dimensional structure homology modeling is carried out on selected specific PD-1 single domain antibody molecules, and combined with the results of comparison with a V-base human germline sequence database and an IMGT human antibody heavy chain variable region germline gene database, heavy chain variable region germline genes with high homology with the screened antibodies are selected as templates, CDRs of camelid source single domain antibodies are transplanted into corresponding human templates, and variable region sequences with the sequence of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 are formed. And (3) carrying out three-dimensional structure simulation and analysis on the transplanted single-domain antibody again, and carrying out back mutation on a specific site influencing the structural morphology of the CDR region in the FR region. The humanized specific sequences obtained were as follows:

2#_Hu_1：

EVQLVESGGGLVQPGGSLRLSCAASGYTYSRDCMGWFRQAPGKGLEGVSAICSSGRNTYYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAADLRSSGGDLTYGLAPGPYEYKYWGQGTLVTVSS

SEQ ID NO：35

7#_Hu_1：

EVQLVESGGGLVQPGGSLRLSCAASRNTNRYNFMTWFRQAPGKEREGVSAIYTGFGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAALRDGSWSSQNYDYWGQGTLVTVSS

SEQ ID NO：36

7#_Hu_2：

EVQLVESGGGLVQPGGSLRLSCAASRNTNRYNFMTWFRQAPGKGLEGVSAIYTGFGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAALRDGSWSSQNYDYWGQGTLVTVSS

SEQ ID NO：37

7#_Hu_3

EVQLVESGGGLVQPGGSLRLSCAASRNTNRYNFMTWFRQAPGKGREGVSAIYTGFGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAALRDGSWSSQNYDYWGQGTLVTVSS

SEQ ID NO：38

7#_Hu_4

EVQLVESGGGLVQPGGSLRLSCAASRNTNRYNFMTWFRQAPGKEREGVSAIYTGFGNTYYADSVKGRFTISQDNSKNTLYLQMNSLRAEDTAVYYCAAALRDGSWSSQNYDYWGQGTLVTVSS

SEQ ID NO：39

7#_Hu_5

EVQLVESGGGLVQPGGSLRLSCAASRNTNRYNFMSWFRQAPGKEREGVSAIYTGFGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAALRDGSWSSQNYDYWGQGTLVTVSS

SEQ ID NO：40

7#_Hu_6

EVQLVESGGGLVQPGGSLRLSCAASRNTNRYNYMSWFRQAPGKEREGVSAIYTGFGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAALRDGSWSSQNYDYWGQGTLVTVSS

SEQ ID NO：41

32#_Hu_1

EVQLVESGGGLVQPGGSLRLSCAASGFRYSPILMGWFRQAPGKGLEGVSSIDSVGATDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAALMRTYLPLQPRQYDFWGQGTLVTVSS

SEQ ID NO：42

32#_Hu_2

EVQLVESGGGLVQPGGSLRLSCAASGFRYSPILMGWFRQAPGKGREGVSSIDSVGATDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAALMRTYLPLQPRQYDFWGQGTLVTVSS

SEQ ID NO：43

32#_Hu_3

EVQLVESGGGLVQPGGSLRLSCAASGFRYSPILMGWFRQAPGKDREGVSSIDSVGATDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAALMRTYLPLQPRQYDFWGQGTLVTVSS

SEQ ID NO：44

32#_Hu_4

EVQLVESGGGLVQPGGSLRLSCAASGFRYSPILMGWFRQAPGKGLEGVASIDSVGATDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAALMRTYLPLQPRQYDFWGQGTLVTVSS

SEQ ID NO：45

32#_Hu_5

EVQLVESGGGLVQPGGSLRLSCAVSGFRYSPILMGWFRQAPGKGLEGVSSIDSVGATDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAALMRTYLPLQPRQYDFWGQGTLVTVSS

SEQ ID NO：46

32#_Hu_6

EVQLVESGGGLVQPGGSLRLSCAASGFTVSPILMGWFRQAPGKDREGVSSIDSVGATDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAALMRTYLPLQPRQYDFWGQGTLVTVSS

SEQ ID NO：123

32#_Hu_7

EVQLVESGGGLVQPGGSLRLSCAASGFTYSPILMGWFRQAPGKDREGVSSIDSVGATDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAALMRTYLPLQPRQYDFWGQGTLVTVSS

SEQ ID NO：124

61#_Hu_1

EVQLVESGGGLVQPGGSLRLSCAASGYTYSSLCMGWFRQAPGKGLEGVSTIYTGDSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAAYGRRWCERLYMYDSWGQGTLVTVSS

SEQ ID NO：47

61#_Hu_2

EVQLVESGGGLVQPGGSLRLSCAASGYTYSSLCMGWFRQAPGKGLEGVSTIYTGDSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAAAYGRRWCERLYMYDSWGQGTLVTVSS

SEQ ID NO：48

106#_Hu_1

EVQLVESGGGLVQPGGSLRLSCAASTYTFKNKCMGWFRQAPGKEREGVSVVDRFGGTIYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGSYTSANSCQPDALWGQGTLVTVSS

SEQ ID NO：49

106#_Hu_2

EVQLVESGGGLVQPGGSLRLSCAASTYTFKNKCMGWFRQAPGKGREGVSVVDRFGGTIYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGSYTSANSCQPDALWGQGTLVTVSS

SEQ ID NO：50

106#_Hu_3

EVQLVESGGGLVQPGGSLRLSCAASTYTFKNKCMGWFRQAPGKGLEGVSVVDRFGGTIYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGSYTSANSCQPDALWGQGTLVTVSS

SEQ ID NO：51

106#_Hu_4

EVQLVESGGGLVQPGGSLRLSCAASTYTFKNKCMGWFRQAPGKEREGVSVVDRFGGTIYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCAAGSYTSANSCQPDALWGQGTLVTVSS

SEQ ID NO：52

106#_Hu_5

EVQLVESGGGLVQPGGSLRLSCAASTYTFKNKCMGWFRQAPGKEREGVSVVDRFGGTIYADSVKGRFTISRDNSKNTLDLQMNSLRAEDTAVYYCAAGSYTSANSCQPDALWGQGTLVTVSS

SEQ ID NO：53

106#_Hu_6

EVQLVESGGGLVQPGGSLRLSCAASGFTVSNKCMGWFRQAPGKEREGVSVVDRFGGTIYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGSYTSANSCQPDALWGQGTLVTVSS

SEQ ID NO：125

106#_Hu_7

EVQLVESGGGLVQPGGSLRLSCAASGFTVKNKCMGWFRQAPGKEREGVSVVDRFGGTIYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGSYTSANSCQPDALWGQGTLVTVSS

SEQ ID NO：126

106#_Hu_8

EVQLVESGGGLVQPGGSLRLSCAASGFTFKNKCMGWFRQAPGKEREGVSVVDRFGGTIYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGSYTSANSCQPDALWGQGTLVTVSS

SEQ ID NO：127

106#_Hu_9

EVQLVESGGGLVQPGGSLRLSCAASGYTFKNKCMGWFRQAPGKEREGVSVVDRFGGTIYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGSYTSANSCQPDALWGQGTLVTVSS

SEQ ID NO：128

107#_Hu_1

EVQLVESGGGLVQPGGSLRLSCAASGYTGNRRYCMGWFRQAPGKEREGVSVINTGANTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAVGWRALCEVNGYVYDSWGQGTLVTVSS

SEQ ID NO：54

107#_Hu_2

EVQLVESGGGLVQPGGSLRLSCAASGYTGNRRYCMGWFRQAPGKGREGVSVINTGANTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAVGWRALCEVNGYVYDSWGQGTLVTVSS

SEQ ID NO：55

107#_Hu_3

EVQLVESGGGLVQPGGSLRLSCAASGYTGNRRYCMGWFRQAPGKGLEGVSVINTGANTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAVGWRALCEVNGYVYDSWGQGTLVTVSS

SEQ ID NO：56

107#_Hu_4

EVQLVESGGGLVQPGGSLRLSCAASGYTGNRRYCMGWFRQAPGKEREGVSVINTGANTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGVGWRALCEVNGYVYDSWGQGTLVTVSS

SEQ ID NO：57

112#_Hu_1

EVQLVESGGGLVQPGGSLRLSCAASTYTFKNKCMGWFRQAPGKEREGVSVVDRFGGIIYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGSYTSDGSCQPDALWGQGTLVTVSS

SEQ ID NO：58。

as shown in the above sequence, during humanization and back mutation, the CDRs of a portion of the antibody were changed, such as the T35S mutation in No. 7 Hu _5, the CDR 1sequence shown in YNFMS (SEQ ID NO: 113); 7# _ Hu _6 having F33Y and T35S mutations, the CDR 1sequence shown by YYMS (SEQ ID NO: 114); 106\\ u hu 1 to 6 have A61D mutation, and the obtained VVDRFGGTIYADSVKG (SEQ ID NO: 71) shows a CDR2 sequence; 112\ u hu 1 has mutations in Y54F and A61D, and the CDR2 sequence shown in VVDRFGGIIYADSVKG (SEQ ID NO: 93) was obtained.

The method of example 4 was used to construct a whole protein sequence of a humanized PD-1 single domain antibody fused to the Fc (CH 2-CH 3) segment of hIgG1, the hIgG1-Fc (CH 2-CH 3) segment sequence (shown in SEQ ID NO: 103) being single underlined, and the linker sequence being double underlined. The protein sequence is as follows (taking 32 hu hu_3-IgG1 as an example, as is the other humanized PD-1 single domain antibodies):

32#_hu_3-hIgG1：

the method in example 4 was used to construct the whole protein sequence of the humanized PD-1 single domain antibody fused to the Fc (CH 2-CH 3) segment of hIgG4, the single underlined sequence of the hIgG4-Fc (CH 2-CH 3) segment (shown in SEQ ID NO: 108).

The sequence of the attached human IgG4-Fc is shown below:

ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO：108

the obtained antibody sequences were as follows:

32#_hu_3_hIgG4：

EVQLVESGGGLVQPGGSLRLSCAASGFRYSPILMGWFRQAPGKDREGVSSIDSVGATDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAALMRTYLPLQPRQYDFWGQGTLVTVSSESKYGPPCPPCPAPEFLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO：109

7#_hu_4_hIgG4：

EVQLVESGGGLVQPGGSLRLSCAASRNTNRYNFMTWFRQAPGKEREGVSAIYTGFGNTYYADSVKGRFTISQDNSKNTLYLQMNSLRAEDTAVYYCAAALRDGSWSSQNYDYWGQGTLVTVSSESKYGPPCPPCPAPEFLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO：110

106#_hu_1_hIgG4：

EVQLVESGGGLVQPGGSLRLSCAASTYTFKNKCMGWFRQAPGKEREGVSVVDRFGGTIYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGSYTSANSCQPDALWGQGTLVTVSSESKYGPPCPPCPAPEFLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO：111

107#_hu_4_hIgG4：

EVQLVESGGGLVQPGGSLRLSCAASGYTGNRRYCMGWFRQAPGKEREGVSVINTGANTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGVGWRALCEVNGYVYDSWGQGTLVTVSSESKYGPPCPPCPAPEFLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO：112

the plasmid was transfected into HEK293 cells, expression supernatant was collected after 6 days, high speed centrifuged to remove impurities, and purified using Protein a column. Equilibrate with PBS until a280 reading falls to baseline. Eluting the target protein with acidic eluent of pH3.0-3.5, and neutralizing with 1M Tris-HCl, pH 8.0-9.0. After the eluted sample was appropriately concentrated, it was further purified by gel chromatography Superdex200 (GE) equilibrated with PBS to remove the aggregates, collect the monomer peak, and split for future use. Upon detection, the PD-1 single domain antibodies of the present disclosure are obtained.

Example 5 glycosylation engineering and expression purification of PD-1 antibodies

CDR1 (in 106# hu _1 _hIgG4sequenceNKCMG) and CDR3 (GSYTSA)NSCQPDAL) contains two N-sugar glycosylation sites (NXC), each of which is N₃₁KC and N₁₀₄SC, the ratio of glycosylation of NKC in CDR1 was 11% and the ratio of glycosylation of NSC in CDR3 was 30% as analyzed by mapping (the specific method in example 6 below), and N was adjusted accordingly₃₁The following amino acid mutations were performed: n is a radical of₃₁-D/E/F/G/H/I/K/L/M/P/Q/R/S；N₁₀₄The following amino acid mutations were performed: n is a radical of₁₀₄-A/E/F/G/H/K/P/Q/R/S; the sequences with little change in affinity, each N, were selected by the method of SPR (Biacore T200) (the specific method of example 7 below)₃₁-D/G and N₁₀₄-G/H。

Wherein 106#_hu_1_hIgG4(N₃₁-D，N₁₀₄-G) was named 0076# hIgG4;106# hu _1_hIgG4 (N)₃₁-G，N₁₀₄-G) was named 0077# _ hIgG4;106# hu _1_hIgG4 (N)₃₁-D，N₁₀₄-H) is named 0078# _ hIgG4;106# hu _1_hIgG4 (N)₃₁-G，N₁₀₄-H) was named 0079# _ hIgG4. The sequences of CDR1 and CDR2 are summarized in table 3.

TABLE 3 CDR1 and CDR2 sequences of glycosylation modified PD-1 antibody

(the sequence of CDR2 is VVDRFGGTIYADSVKG (SEQ ID NO: 71))

Accordingly, the present disclosure provides PD-1 antibodies, the CDR1 of which is X₂₂KCMG (SEQ ID NO: 152), wherein X₂₂Selected from N, D, E, F, G, H, I, K, L, M, P, Q, R or S; CDR2 is VVDRFGGTIYADSVKG (SEQ ID NO: 71); CDR3 is GSYTSAX₂₃SCQPDAL (SEQ ID NO: 153) wherein X₂₃Selected from N, A, E, F, G, H, K, P, Q, R or S.

An example of the sequence of the antibody obtained after glycosylation engineering of 106# hu _1 is as follows:

0076#

EVQLVESGGGLVQPGGSLRLSCAASTYTFKDKCMGWFRQAPGKEREGVSVVDRFGGTIYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGSYTSAGSCQPDALWGQGTLVTVSS

SEQ ID NO：154

0077#

EVQLVESGGGLVQPGGSLRLSCAASTYTFKGKCMGWFRQAPGKEREGVSVVDRFGGTIYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGSYTSAGSCQPDALWGQGTLVTVSS

SEQ ID NO：155

0078#

EVQLVESGGGLVQPGGSLRLSCAASTYTFKDKCMGWFRQAPGKEREGVSVVDRFGGTIYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGSYTSAHSCQPDALWGQGTLVTVSS

SEQ ID NO：156

0079#

EVQLVESGGGLVQPGGSLRLSCAASTYTFKGKCMGWFRQAPGKEREGVSVVDRFGGTIYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGSYTSAHSCQPDALWGQGTLVTVSS

SEQ ID NO：157

0076#_hIgG4

EVQLVESGGGLVQPGGSLRLSCAASTYTFKDKCMGWFRQAPGKEREGVSVVDRFGGTIYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGSYTSAGSCQPDALWGQGTLVTVSSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

SEQ ID NO：184

0077#_hIgG4

EVQLVESGGGLVQPGGSLRLSCAASTYTFKGKCMGWFRQAPGKEREGVSVVDRFGGTIYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGSYTSAGSCQPDALWGQGTLVTVSSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

SEQ ID NO：185

0078#_hIgG4

EVQLVESGGGLVQPGGSLRLSCAASTYTFKDKCMGWFRQAPGKEREGVSVVDRFGGTIYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGSYTSAHSCQPDALWGQGTLVTVSSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

SEQ ID NO：186

0079#_hIgG4

EVQLVESGGGLVQPGGSLRLSCAASTYTFKGKCMGWFRQAPGKEREGVSVVDRFGGTIYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGSYTSAHSCQPDALWGQGTLVTVSSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

SEQ ID NO：162

constructing plasmid transfection Expi-CHO cells, culturing for 9 days, collecting expression supernatant, removing impurities by high-speed centrifugation, and purifying by a Protein A column. Equilibrate with PBS until a280 reading falls to baseline. Eluting the target protein with acidic eluent of pH3.0-3.5, and neutralizing with 1M Tris-HCl, pH8.0-9.0. After the eluted sample was appropriately concentrated, it was further purified by gel chromatography Superdex200 (GE) equilibrated with PBS to remove the aggregates, collect the monomer peak, and split for future use. Through detection, the PD-1 antibody of the disclosure is prepared and obtained.

Example 6 Mass Spectrometry and glycosylation analysis of PD-1 antibodies

An instrument device: an American Agilent Q TOF 6530 mass spectrometer equipped with Dual AJS ESI ion source and data analysis Software Agilent MassHunter BioConfirm Software B.08.00, agilent 1290Infinity high performance liquid chromatography System, USA.

Chromatographic conditions are as follows: the chromatographic column is an Agilent advanced Bio C18 (2.1x150mm, 2.7 um) peptide spectrum analysis chromatographic column; mobile phase: phase A as 100% H2O-0.1% FA, phase B as 100% ACN-0.1% FA; chromatographic elution gradient 0-65min 3% -35% >/B; 65-68min 35% -95% of B;68-70min 95%; 70-72min 95% -3%B;72-75min 3% by weight of B; the flow rate is 0.4mL/min; the column temperature is 60 ℃; the sample size is 20ul.

Mass spectrum parameters: the mass spectrum ion source is a Dual AJS ESI electrospray ion source; the ion injection voltage is 3.5KV; a Gas temperature of 250 ℃; a shear Gas temperature of 350 ℃; the shear Gas Flow is 12l/min; drying Gas is 10l/min; nebulizer 35psi; detecting in a positive ion mode; the mass number range is m/z 200-3000; the acquisition rate is 5 mass spectrograms per second; separation peak width: medium (about 4 m/z).

Sample treatment: adding a certain amount of guanidine hydrochloride into each sample, adding a reducing agent DTT to enable the final concentration to be 20mmol/L, and incubating for 1h at 60 ℃; adding alkylating agent IAM to make the final concentration be 40mmol/L, and reacting for 1h in a dark place; then, the samples were diluted respectively until the guanidine hydrochloride concentration was below 2mol/L, according to protein: trypsin mass ratio 25.

Data processing: raw data collected in LC/MS/MS was processed using data analysis Software Agilent MassHunter BioConfirm Software B.08.00. The results were searched in mAb sequences, including various common modifications of alkylation (C), oxidation (M), deamidation (NQ), pyroglutamylation (E), and glycosylation (N); the allowable error of mass spectrum matching is +/-20ppm, and the allowable error of MS/MS matching is +/-50 ppm. Two trypsin nick sites were allowed. The results are shown in Table 4.

The results showed that 106# hu _1 _hIgG4had three glycosylation modifications and the glycosylation ratio of the VHH segment was 11% and 30%, respectively, resulting in heterogeneity of the antibody protein. The reconstructed 0076# antibody only has one glycosylation modification site, and the protein homogeneity is good.

TABLE 4 glycosylation modification ratio of PD-1 antibody

Example 7 affinity assay for binding of PD-1 antibodies to PD-1 protein

To test the in vitro binding capacity of the screened PD-1 single domain antibodies to human PD-1 protein and monkey PD-1, human PD-1 (Sino Biological Cat.10377-H08H) and monkey PD-1 (Sino Biological Cat.90311-C08H) were used for in vitro binding assays by ELISA binding experiments.

The negative control used in this example was PBS, opdivo (available from Shanghai Rui Zhi chemical (chempartner) lot: 180612001) was used as the positive control, and IgG4 type PD-1Ab646 (hereinafter referred to as PD-1Ab 646) from WO2017054646 was used as the positive control in some experiments, and the sequences were as follows:

PD-1 antibody heavy chain:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYMMSWVRQAPGKGLEWVATISGGGANTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQLYYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP PCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

SEQ ID NO：121

PD-1 antibody light chain:

DIQMTQSPSSLSASVGDRVTITCLASQTIGTWLTWYQQKPGKAPKLLIYTATSLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVYSIPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO：122

the protein with the PD-1 antibody was diluted to 2. Mu.g/mL with PBS buffer, pH7.4, added to a 96-well plate (corning, 9018/box 96well clear float bottom plate) in a volume of 100. Mu.L/well, and left overnight at 4 ℃ for 16-20 hours. After discarding the liquid, the plate was washed three times with PBST (pH 7.4,0.05% Tween-20) buffer solution, and then 2% BSA blocking solution (300. Mu.L/well) diluted with PBS buffer solution was added, and the blocking was performed by incubating in an incubator at 37 ℃ for 2 hours. After blocking was completed, the blocking solution was discarded, and after washing the plate 3 times with PBST buffer, PD-1 antigen (Sino Biological Cat.10377-H08H) protein was added at an initial concentration of 30. Mu.g/mL, and the plate was diluted three-fold with PBS buffer for 8 gradients and incubated at 37 ℃ in an incubator for 1 hour. After completion of the incubation, the reaction solution in the microplate was discarded, and the plate was washed 6 times with PBST, 100. Mu.L/HRP-labeled secondary antibody against his (Abcam ab 1187) (1 dilution. Washing the plate with PBST for 6 times, adding 100 μ LTMB chromogenic substrate, incubating at room temperature for 3-5min, adding 100 μ L1M sulfuric acid to stop the reaction, reading the absorbance at 450nm with a SpectraMax M5 microplate reader, and calculating the antibody-antigen binding EC₅₀The value is obtained. EC of partial antibody₅₀The results are shown in Table 3. The results show that the polypeptide has better binding force with human and monkey PD-1 antigens.

TABLE 5 binding of PD-1 antibodies to human and monkey PD-1 antigens EC₅₀(nM)

Antibody numbering	EC that bind to human PD-1₅₀	Monkey PD-1 binding EC ₅₀
			7#	1.86	2.2
32#	1.99	4.8
			32#_hu_1	4.08	6.2
32#_hu_2	3.43	2.3
			32#_hu_3	2.98	1.2
61#	1.85	/
			106#	2.56	0.67
107#	3.14	2.9
			112#	2.51	1.5
Positive control (Opdivo)	1.69	2.88
			Negative control (PBS)	0	0

(Note: "/" indicates no detection)

In addition, dissociation constants of the PD-1 antibody and the PD-1 protein were also determined by a Biacore 8K (GE Healthcare) instrument. Firstly, an anti-human IgG Fc antibody (GE Healthcare, # BR-1008-39) is covalently coupled to a CM 5S series chip, a PD-1 antibody to be detected is captured to the surface of the chip through affinity, then PD-1 proteins (SEQ ID NO: 3) with different concentrations flow through the surface of the chip, a Biacore instrument is utilized to detect reaction signals in real time so as to obtain a binding dissociation curve, and a binding force constant is obtained through fitting. The solution used for the experiment was an HBS-P solution (10mM HEPES,150mM NaCl, 0.005%; P20, pH 7.4). At the end of each experimental cycle, 3M MgCl was used₂The chip is cleaned and regenerated by the solution. The affinity results for the partial antibodies are shown in table 6. The results show that the affinity of the antibodies obtained by the screening of the disclosure to PD-1 is equivalent to that of the positive control.

TABLE 6 affinity of PD-1 antibodies to PD-1

Antibody numbering	Antigens	k_a(1/Ms)	k_d(1/s)	K_D(M)
					7#	PD-1	1.36E+05	2.81E-04	2.06E-09
32#	PD-1	3.25E+05	2.07E-03	6.35E-09
					32#_hu_1	PD-1	1.79E+05	2.91E-03	1.63E-08
32#_hu_2	PD-1	1.67E+05	1.56E-03	9.36E-09
					32#_hu_3	PD-1	2.20E+05	2.01E-03	9.11E-09
32#_hu_4	PD-1	1.75E+05	3.53E-03	2.02E-08
					32#_hu_5	PD-1	1.62E+05	3.19E-03	1.96E-08
61#	PD-1	1.54E+05	8.19E-04	5.33E-09
					61#_hu_1	PD-1	2.26E+05	4.61E-03	2.04E-08
106#	PD-1	7.94E+04	4.77E-04	6.01E-09
					107#	PD-1	9.65E+04	7.82E-04	8.10E-09
Opdivo	PD-1	5.91E+05	1.45E-03	2.45E-09

In addition, dissociation constants of PD-1 antibody and PD-1 protein were also determined using a Biacore T200 (GE Healthcare) instrument. Covalently coupling protein A (elegant RSPA 05) to a CM 5S series chip, capturing an antibody to be detected to the surface of the chip through affinity, then flowing PD-1 protein (Sino Biological Cat.10377-H08H) with different concentrations on the surface of the chip, detecting a reaction signal in real time to obtain a binding dissociation curve, and obtaining a binding force constant through fitting. The solution used for the experiment was HBS-EP solution (10mM HEPES,150mM NaCl,3mM EDTA,0.005% P20, pH 7.4). At the end of each experimental cycle, the chips were washed and regenerated with 10mM glycine, pH =1.5 (GE, BR-1003-54). See tables 7 and 8 for results.

TABLE 7 affinity K of PD-1 antibodies to human PD-1_D

Antibody numbering	k_a(1/Ms)	k_d(1/s)	K_D(M)
				32#_hu_3_hIgG4	1.05E+05	2.01E-03	1.92E-08
7#_hu_4_hIgG4	4.72E+04	5.84E-03	1.24E-07
				106#_hu_1_hIgG4	8.17E+03	7.05E-04	8.63E-08
107#_hu_4_hIgG4	9.40E+03	1.20E-03	1.28E-07
				PD-1Ab646	6.18E+04	4.79E-04	7.75E-09

TABLE 8 affinity K of PD-1 antibody to PD-1_D

( Note: "/" is not shown for detection specific values; the affinity scale "+ +" means <3.00E-07, and "+" means ≧ 3.00E-07 )

Example 8 blocking of the binding of PD-1 and PD-L1, PD-L2 by the PD-1 antibody

The functional assay for PD-1 antibodies was performed by ELISA competition assays that blocked the binding between PD-1 and PD-L2.

The Fc-tagged PD-1 fusion protein was diluted to a concentration of 2. Mu.g/mL with PBS buffer, pH7.4, added to a 96-well microplate at a volume of 100. Mu.L/well, and left overnight at 4 ℃ for 16-20 hours. After discarding the liquid, the plate was washed three times with PBST (pH 7.4,0.05% Tween-20) buffer, and then, 300. Mu.L/well of 2% BSA blocking solution diluted with PBS buffer was added, and the plate was incubated at 37 ℃ for 2 hours for blocking. After blocking was complete, the blocking solution was discarded and the plate was washed 3 times with PBST buffer, added with biotinylated PD-L1 and PD-L2 proteins at a protein concentration of 6. Mu.g/mL, 50. Mu.L per well, followed by PD-1 antibody protein at an initial concentration of 30. Mu.g/mL, diluted three-fold in PBS buffer for 6 gradients, and incubated for 1 hour at 37 ℃. After completion of the incubation, the reaction solution in the microplate was discarded, the plate was washed 6 times with PBST, and 100. Mu.L/well of HRP-labeled secondary antibody against SA (Peroxidase-conj. Mu. Gated Streptavidin, jackson 136861) diluted with PBS (0.5% BSA) (1. Washing the plate 6 times with PBST, adding 100 μ L/well TMB chromogenic substrate, incubating at room temperature for 3-5min, adding 1M sulfuric acid to stop the reaction, reading the absorbance at 450nm with a SpectraMax M5 microplate reader, and calculating the binding IC of the antibody to the antigen₅₀The value is obtained. IC of partial antibody₅₀The results are shown in Table 4. The results show that the antibodies all competed with PD-L1 and PD-L2 for binding to PD-1, PBS was used as a negative control, and Opdivo was used as a positive control (Opdivo used in this disclosure was purchased from Shanghai Ruizi chemical (chempartner) lot: 180612001). The results of the partial antibodies blocking the binding of PD-1 to PD-L1 are shown in tables 9 and 10.

TABLE 9 IC of different PD-1 antibodies competing for PD-1 antigen with PD-L1 and PD-L2₅₀(nM)

TABLE 10 IC of different PD-1 antibodies competing for PD-1 antigen with PD-L1₅₀(nM)

Antibody numbering	IC blocking binding of PD-1 to PD-L1 ₅₀
		32#_hu_3_hIgG4	2.42
7#_hu_4_hIgG4	1.22
		106#_hu_1_hIgG4	3.14
PD-1Ab646	2.79
		Negative control (PBS)	9999

Example 9 in vitro cell binding assay for PD-1 antibodies

Cloning the human PD-1 full-length gene onto a mammalian cell expression vector pTargeT by PCR, taking a linear plasmid to electrically transfect CHO-S cells (preset CHO cell parameters carried by an electrotransformation instrument), screening for 2 weeks by 1mg/ml G418, carrying out 2 times of limited dilution, detecting the PD-1 gene on the cell surface by a flow cytometry analyzer, and selecting a monoclonal cell strain high-expression human PD-1. Was named CHO-PD-1.

Collecting the cell line CHO-PD-1 with stable and high expression PD-1, 5X 10 per well⁵A cell. The PD-1 antibody was diluted in gradient at 16.67. Mu.g/mL, 5.55. Mu.g/mL, 1.85. Mu.g/mL, 0.617. Mu.g/mL, 0.205. Mu.g/mL, 0.069. Mu.g/mL and incubated with CHO-PD-1 on ice for 1 hour in the absence of light. After one-time rinsing with PBS, FITC anti-human IgG (1). After one more PBS rinse, 100. Mu.L PBS per tube was resuspended and fluorescence detected on a BD C6 Plus flow cytometer. The mean fluorescence intensity from each dose treatment of the antibody was curve fitted and plotted using Graphpad Prism9 software to quantify the binding of PD-1 antibody to CHO-PD-1 cells. The results show that the binding strength of PD-1 antibody to CHO-PD-1 cells is antibody dose dependent.

Binding capacity of partial antibodies EC₅₀The results are shown in table 11, table 12 and fig. 1. The results show that the binding force of the antibodies (such as 2#, 32# hu _1, 32# hu _2, 32# hu _3, 61#, 32# hu _3_hIgG4, 7# hu _4_hIgG4, 106# hu _1 _hIgG4and 107# hu _4 _hIgG4) obtained by screening in the disclosure and PD-1 is significantly better than that of the positive control Opdivo. The binding capacity of molecules such as 0076# and 0078# after mutation compared with PD-1 is not reduced by 106# _ hu _1_hIgG4.

Where the negative control of the disclosure is NC, it is an antibody that has the same constant region IgG4 as the PD-1 antibody of the disclosure, but the variable region does not recognize the antigen PD-1. The positive control used Opdivo.

TABLE 11 binding EC of PD-1 antibodies to the cell surface antigen PD-1₅₀

TABLE 12 binding EC of PD-1 antibodies to the cell surface antigen PD-1₅₀

Example 10 blocking of PD-1 binding to PD-L1 on cells by PD-1 antibody

Collecting stable high expression PD-1 cell line CHO-PD-1, adjusting to 5X 10 per hole⁵A cell. Gradient dilution PD-1 antibodies 50. Mu.g/mL, 16.67. Mu.g/mL, 5.55. Mu.g/mL, 1.85. Mu.g/mL, 0.617. Mu.g/mL, 0.205. Mu.g/mL, 0.069. Mu.g/mL, with CHO-PD-1 cells were incubated on ice for 1 hour. After one wash with PBS, each tube was incubated for 1 hour on ice with 1. Mu.g/mL of PD-L1-mIgG2a protein, and washed again with PBS. After washing, each tube was incubated on ice for 1 hour with PE anti-mouse IgG2a (1. The mean fluorescence intensity from each dose treatment of the antibody was curve fitted and plotted using Graphpad Prism9 software to quantify PD-1 binding to PD-L1 on PD-1 antibody-blocked cells.

The result shows that the PD-1 antibody can block the binding of PD-L1 protein to CHO-PD-1 cells, the antibody dose dependence is shown, and the blocking IC of partial antibody₅₀The results are shown in table 13, table 14 and fig. 2. And, antibodies of the disclosure (e.g., 7, 32_hu _1, 32_hu _2, 32_hu _3, 106, 107, 112) have a greater ability to block PD-L1 from binding to PD-1 than control Opdivo. And, the ability of the post-mutation molecules such as 0076# and 0078# to block PD-L1 binding to PD-1 was also not reduced compared to 106# _ hu _1 \/higg4.

TABLE 13 IC of PD-1 antibody blocking PD-L1 protein and cell surface antigen PD-1₅₀(nM)

TABLE 14 IC of PD-1 antibody blocking PD-L1 protein and cell surface antigen PD-1₅₀(nM)

Example 11 immune activation assay of PD-1 antibodies in vitro to Release PD-1/PD-L1blockade

The CHO-PD-L1 aAPC cell line (purchased from Promega, PD-1/PD-L1Block Bioassay, J1252) endogenously stably and highly expressing PD-L1 and TCR-activating molecules was collected, PD-L1 negative CHO cells were used as a control, and the cell density was adjusted to 4X 10 using complete medium⁵mL, 100uL per well, 5% CO at 37 ℃₂The incubator is used for 20-24 hours.

PD-1 antibody was gradient diluted to 1000, 250, 62.5, 15.6, 3.91, 0.98, 0.24, 0.06nM using assay medium on the day of testing, with 2 duplicate wells set at each concentration.

Effector cells Jurkat-PD-1 (purchased from Promega, PD-1/PD-L1Block Bioassay, J1252) stably expressing PD-1 in high endogenous and simultaneously expressing NFAT-activated luciferase reporter gene in stable endogenous are collected, and the cell density is adjusted to 1.25X 10 by using an analysis medium⁶/mL。

Taking out the culture plate inoculated with CHO-PD-L1 aAPC cells one day before, discarding the supernatant, adding the diluted antibody and the effector cells Jurkat-PD-1 with adjusted density into the cell culture plate at one time, adding 40uL of each well, mixing gently, and standing at 37 deg.C for 5% CO₂The incubator of (1) was incubated for 6 hours.

During antibody incubation, bio-Glo^TMThe Reagent (Promega) was removed and the temperature was returned to room temperature. After the mixed culture is completed, the cell culture plate is taken out, is kept stand for 5 to 10 minutes at room temperature, and then 80uL Bio-Glo is added into each hole^TMReagent, mixing gently, reading chemiluminescence value by using a Molecular Device SpectraMax multifunctional microplate reader, and performing curve fitting analysis on the obtained data by using Graphpad Prism9 software and mapping. The partial antibody results are shown in table 15 and fig. 3.

The results show that both 0076# and 106# hu _1 _hIgG4can well relieve the immune activation blocked by PD-1/PD-L1, and the effect of 0076# is better than that of 106# hu _1_hIgG4.

TABLE 15 PD-1 antibody Destruction PD-1/PD-L1 blocked immune activated IC₅₀

Antibody numbering	IC₅₀(nM)
		106#_hu_1_hIgG4	29.18
0076#	14.25

Example 12 PD-1 antibody promotes cytokine secretion by Mixed lymphocytes in vitro

Isolation of CD14 from fresh or resuscitated PBMCs by EasySep human CD14 Positive screening kit (STEMCELL technologies, 17858)⁺A monocyte. Separated CD14⁺Dendritic cell differentiation kit (R) derived from cells according to monocytes&D system, CDK 004) was prepared by adding IL-4 and GM-CSF for 6 days, and then adding TNF-. Alpha.for further 3 days to obtain mature DCs.

Human PBMC CD3 isolation by EasySep human CD3 Positive screening kit (STEMCELL technologies, 18051)⁺T cells (different donor source than DCs). The isolated T and DC cells were plated at 10:1, while adding a low endotoxin-controlled PD-1 antibody, and culturing for 5 days, and then using a human IFN γ quantikine ELISA kit (R)&D system, DIF 50) to detect IFN γ secretion by activated T cells.

The amounts of IFN γ secretion after mixed lymph culture are shown in table 16 and table 17, and fig. 4 and fig. 5. The results show that a plurality of PD-1 antibodies obtained by screening can effectively enhance the activation of T cells and secrete IFN gamma.

TABLE 16 IFN γ secretion promoting amount of PD-1 antibody

Antibody numbering	IFN gamma secretion (pg/mL)
		7#	963.5
32#	555.3
		32#_hu_3	1031.5
106#	1164.2
		107#	1776.6
Negative Control (NC)	49
		Positive control (Opdivo)	1181.5

TABLE 17 IFN γ secretion promoting amount of PD-1 antibody

Example 13 inhibition of tumor growth in a mouse colon cancer model by PD-1 antibody

Animal experiments were performed by Shanghai Ai Fei medicine science and technology, inc., using HuPD-1 humanized transgenic mice, female, 6-8 weeks old, purchased from Nanjing Yinhe biomedical Inc.

PBS resuspended mouse colon carcinoma cell line MC38 cells at 5X 10⁵The seeds were inoculated subcutaneously in the right flank of HuPD-1 humanized mouse at a concentration of 0.1mL and a volume of 0.1 mL. When the average tumor volume reaches 100mm³(70-120mm³) When the method is used, mice with moderate individual tumor volumes are selected and grouped, and the right tumor volume is taken as a grouping basis. The medicine is administered on the grouping day, and the administration dose is 0.3mg/kg; the dosing frequency was once every three days for a total of three weeks; the mode of administration is intravenous injection.

The results of the PD-1 antibody inhibiting the growth of colon cancer tumor in mice are shown in Table 18 and FIGS. 6A and 6B. The results showed that the positive control had a tumor suppression ratio of 47.3% at day 24; the tumor inhibition ratio of 32# _ hu _3 _hIgG4is 50.8%; the tumor inhibition ratio of 7# _ hu _4 \/higg4 is 68.4%; the tumor inhibition ratio of 106# hu _3 _hIgG4is 64.4%, and the tumor growth in mice can be effectively inhibited.

TABLE 18 results of inhibition of Colon cancer tumor growth in mice by PD-1 antibody

Example 14 construction, expression and purification of PD-1/CTLA-4 bispecific antibody

1. Structure of bispecific antibody binding to PD-1/CTLA4

The PD-1 antibodies of the present disclosure are combined with ipilimumab (ipilimumab) to construct bispecific antibodies PR2001, PR2009, and PR2011, PR2004, PR2012, and PR2014 that target PD-1 and CTLA 4. The Fc part of the antibodies introduces two point mutations of L234F/L235E on the basis of IgG1 so as to eliminate Fc-mediated cell killing effect. The CDRs of ipilimumab are shown in table 19.

TABLE 19 Epipilimumab CDR

And (3) ipilimumab VH:

QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSS

SEQ ID NO：164

ipilimumab VL:

EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK

SEQ ID NO：165

full length of ipilimumab heavy chain:

QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO：166

full length of ipilimumab light chain:

EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO：167

the structures of 3 molecules PR2001, PR2009, and PR2011 are shown in fig. 7A, and the structures of 3 molecules PR2004, PR2012, and PR2014 are shown in fig. 7B. PR2001, PR2009 and PR2011, PR2004, PR2012 and PR2014 differ in the antibody sequence of PD-1 used. PR2001 and PR2004, PR2009 and PR2012, PR2011 and PR2014 differ in the anti-reactive groups usedThe bulk form is different. The linker-1 (linker-1) used in the double antibody structure of PR2001, PR2009, PR2011 has a sequence structure of (G)₄S)₄(i.e., GGGGSGGGGSGGSGGGGS). The linker 2 between the Fc of PR2004, PR2012 and PR2014 and the PD-1 single-domain antibody is (G)₄S)₆(i.e., GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS). The light chains used for these 4 bispecific antibodies were identical. Each antibody sequence is as follows:

PR2001 first polypeptide chain (linker underlined):

EVQLVESGGGLVQPGGSLRLSCAASGFRYSPILMGWFRQAPGKDREGVSSIDSVGATDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAALMRTYLPLQPRQYDFWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO：168

PR2004 first polypeptide chain (linker underlined):

QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGG GGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFRYSPILMGWFRQAPGKDREGVSSIDSVGATDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAALMRTYLPLQPRQYDFWGQGTLVTVSS

SEQ ID NO：169

first polypeptide chain of PR2009 (linker underlined):

EVQLVESGGGLVQPGGSLRLSCAASTYTFKNKCMGWFRQAPGKEREGVSVVDRFGGTIYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGSYTSANSCQPDALWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO：170

first polypeptide chain of PR2012 (linker underlined):

QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGG GGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASTYTFKNKCMGWFRQAPGKEREGVSVVDRFGGTIYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGSYTSANSCQPDALWGQGTLVTVSS

SEQ ID NO：171

PR2011 first polypeptide chain (linker underlined):

EVQLVESGGGLVQPGGSLRLSCAASTYTFKDKCMGWFRQAPGKEREGVSVVDRFGGTIYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGSYTSAGSCQPDALWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO：172

first polypeptide chain of PR2014 (linker underlined):

QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGG GGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASTYTFKDKCMGWFRQAPGKEREGVSVVDRFGG

TIYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGSYTSAGSCQPDALWGQGTLVTVSS

SEQ ID NO：173

PR2001, PR2009, and PR2011, PR2004, PR2012, and PR2014 are all SEQ ID NOs: 167.

2. production of bispecific antibody binding to PD-1/CTLA4

CHO-S cells (from Thermo, cat # A29133) in a good growth state in a logarithmic growth phase were used, centrifuged and grown at 6X 10⁶cells/mL were seeded at 250mL. Adding the solution 2 (diluted with culture solution 9.2mL to 800ul of transfection reagent, and mixed) into the solution 1 (diluted with culture solution 10mL to 250 ug of plasmid, and mixed) to obtain a total volume of 20mL, gently mixing, incubating at room temperature for 1-5 min, no more than 5min, adding the mixed transfection solution dropwise into the cell fluid, and adding while shaking. Then placing the flask in 5% CO₂After culturing at 32 ℃ on a shaking table, 16mL of auxiliary material Feed (purchased from Thermo company, product No. A29133) and 0.6mL of Enhancer Enhancer (purchased from Thermo company, product No. A29133) are added for 18-22 hours. Adding adjuvants Feed (from Thermo company, cat. No. A29133) 1695l, 123rpm,5% CO on the fifth day₂Culturing at 32 deg.C, and centrifuging on day 12-14 to collect supernatant. By affinity chromatography (Protein A) and ion exchange in two stepsAnd (5) purifying the recombinant antibody. The PD-1/CTLA4 bispecific antibodies PR2001, PR2009 and PR2011, PR2004, PR2012 and PR2014 are prepared. Detected by 10% SDS-PAGE gel electrophoresis, as shown in FIG. 8.

The results show that the theoretical molecular weight of the double antibody is 174kDa, the first polypeptide chain is 64kDa, the second polypeptide chain is 23kDa, and the sizes of the bands are judged by the size of a non-reducing band and the size of a reducing band of SDS-PAGE gel electrophoresis, and the sizes of the bands are consistent with expectations, which indicates that the recombinant bispecific antibody can be correctly assembled and expressed without obvious aggregation degradation.

Example 15 detection of the affinity of PD-1/CTLA-4 double antibodies to human PD-1 and human CTLA-4

Flow cytometry was used to detect binding of the antibody to Jurkat-PD-1 cells (purchased from Promega, CS 187102). Selecting Jurkat cells in logarithmic growth phase, collecting cells, washing with PBS, centrifuging, and resuspending the cells with 2% FBS PBS. Using 100. Mu.l of 2X 10 per well⁵Cells were plated and centrifuged at 400g for 5min. Adding antibodies to be detected with different concentrations, incubating on ice for 1h, washing with PBS, and centrifuging at 400g for 5min. A secondary antibody Alexa Fluor 647mouse anti-human lgG, fc gamma Fragment Specific (from Jackson) was added with a fluorophore for ice bath staining for 1h, followed by PBS washing and in-machine detection. As shown in FIG. 9, all the anti-PD-1 nanobodies detected were able to bind to Jurkat-PD-1 cells. TABLE 20 bound EC₅₀The value is obtained.

TABLE 20 EC for Biantibody binding to PD-1₅₀(FACS)

Antibody numbering	EC₅₀(nM)
		PR2001	0.198
PR2004	0.755
		PR2009	0.632
PR2012	2.010

Flow cytometry was used to detect binding of the antibody to CHOK1-CTLA4 cells. Selecting CHOK1 cells in logarithmic growth phase, transfecting CTLA4 plasmid (purchased from Yiqian Shenzhou) by Lipo2000 (purchased from Invitrogen), removing culture medium after 48h, washing by PBS, centrifuging, adding a proper amount of 0.25% pancreatin (purchased from Invitrogen) for digestion, adding complete culture medium to terminate digestion, centrifuging to collect cells, and 2, resuspending the cells by FBS PBS. Using 100. Mu.l of 2X 10 per well⁵Cells were plated and centrifuged at 400g for 5min. Adding antibodies to be detected with different concentrations, incubating on ice for 1h, washing with PBS, and centrifuging at 400g for 5min. Adding a secondary antibody Alexa Fluor 647mouse anti-human lgG with a fluorescent group, carrying out ice bath staining for 1h, washing with PBS, and then carrying out on-line detection. As shown in FIG. 10, all antibodies of the present disclosure were detected to bind to CHOK1-CTLA4 cells. TABLE 21 bound EC₅₀The value is obtained.

TABLE 21. EC for combination of diabodies with CTLA4₅₀(FACS)

Antibody numbering	EC₅₀(nM)
		Ipilimumab	2.334
PR2001	2.967
		PR2004	3.047
PR2009	3.856
		PR2012	2.669

Example 16 detection of inhibitory Activity of PD-1/CTLA-4 double antibodies against PD-1/PD-L1 signals

Two cell engineering strains, gloResponse NFAT-luc2/PD1 Jurkat (from Promega, CS 187102) and PD-L1-CHOK1 (from Promega, CS 187108) were used in this experiment. GloResponse NFAT-luc2/PD1 Jurkat cells are Jurkat cells stably expressing PD1 and simultaneously stably transfer NFAT promoter/Luciferase reporter gene system vectors downstream of the TCR signaling pathway. PD-L1-CHOK1 cells are CHO-K1 cell lines stably expressing PD-L1, and can activate TCR (T cell receptor) signals of Jurkat cells. When GloResponse NFAT-luc2/PD1 Jurkat cells and PD-L1-CHOK1 are co-incubated, TCR signals are inhibited due to the binding of PD-1 and PD-L1, and when the PD-1 antibody is added, the binding of PD-1 and PD-L1 can be blocked, the inhibition of the TCR signals is released, and the expression of Luciferase reporter genes is induced.

After washing PD-L1-CHOK1 with PBS, adding appropriate amount of 0.25% pancreatin (purchased from Gibico) for digestion, supplementing F-12 complete culture medium (purchased from Gibico) to terminate digestion, centrifuging to collect cells, and resuspending the cells to 2X 10 in F-12 complete culture medium⁵Perkinelmer 6005680 to 96-well plate (Perkinelmer 5363) was added cell density adjusted PD-L1-CHOK1 cell suspension, 100. Mu.L/well, placed at 37 ℃ and 5% CO₂Culturing in an incubator for 24h. GloResponse NFAT-luc2/PD1 Jurkat cells were harvested, washed with PBS and centrifuged, and RPMI1640 assay medium (purchased from Gibico) resuspended cells. The cell culture plate of PD-L1-CHOK1 was removed, the supernatant was removed, and the diluted antibody was adjustedThe high density GloResponse NFAT-luc2/PD1 Jurkat cells were added to the cell culture plate in order of 40. Mu.L/well at 37 ℃ 5% CO₂Culturing in an incubator. Bright-Glo^TMReagent (from Promega, E-2620) was removed and the temperature was returned to room temperature. The cell culture plate was removed, left at room temperature for 10min, and then 40. Mu.L of Bright-Glo was added to each well^TMAnd (5) reading the Reagent, placing the Reagent in the room temperature and keeping out of the light for 5min, and reading the photometric value by using a multifunctional microplate reader. As shown in fig. 11, the detected antibody PR2009 had inhibitory activity against PD-1/PD-L1 signals.

Example 17 staphylococcal toxin (SEB) assay to detect T cell activating Activity of PD-1/CTLA-4 double antibody

The activity of the antibody molecules on the activation of T cells under the stimulation of SEB is detected by detecting the IL-2 secretion of peripheral blood mononuclear cells. PBMC (purchased from ALLCELLS) were thawed, supplemented with the appropriate amount of RPMI1640 complete medium, and centrifuged at 400g for 5min. Add RPMI1640 complete medium containing 100ng/ml SEB (ex Congboning 2090681) to resuspend the cells to 2X 10⁶and/mL. The cells were added to a 96-well cell culture plate at 100. Mu.L/well, and the diluted antibody of RPMI1640 complete medium at 100. Mu.L/well was added, which was incubated at 37 ℃ for 72 hours in a 5% CO2 incubator. Supernatants were harvested and IL-2 concentration was assayed using Human IL2 kits (purchased from Cisbio,62HIL02 PEG). As shown in fig. 12, the detection antibody PR2009 had activation activity on T cells under SEB stimulation.

Example 18 Mixed Lymphocyte Reaction (MLR) assay to detect T cell activating Activity of PD-1/CTLA-4 double antibody

Fresh PBMC (from ALLCELLS) were washed once by adding the autoMACS Running buffer (from Miltenyi 130-091-221) and centrifuged at 400g for 5 minutes. 1.6ml of buffer was added for resuspension, and 400ul of human CD14 MicroBeads (purchased from Miltenyi # 130-050-201) were added and incubated at 4 ℃ for 15mins. After adding an appropriate amount of buffer, the supernatant was centrifuged off and 2ml of buffer was resuspended. The column (from Miltenyi) was prepared for loading, 2ml buffer for column washing, loading, and 2ml buffer for column washing. The column was removed, 2ml of buffer eluted and CD14 was collected⁺cells,400g, centrifuge for 5 minutes. Complete medium containing 250U/ml IL-4 (from Peprotech 200-04) and 500U/ml GMCSF (from Peprotech 300-03) RPMI1640 was addedCultured in a CO2 incubator at 37 ℃ and 5%for 5 days. Adding LPS (purchased from Sigma # L2880) with a final concentration of 1 μ g/ml, culturing for 48h, collecting cells, washing with RPMI1640 complete medium once, and resuspending the RPMI1640 complete medium to 4X 10⁵and/mL. PBMCs (purchased from ALLCELLS) were thawed, washed once with the addition of an AutoMACS Running buffer, centrifuged at 400g for 5 minutes, and the supernatant removed. Using EasySep^TMHuman CD4⁺Isolation of CD4 from T Cell Isolation Kit (from Stemcell # 17952)⁺T cells, RPMI1640 complete Medium resuspended to 2X 10⁶The volume is/mL. Equal volume mixing of diluted mature DCs and CD4⁺T cells were cultured in 96-well cell culture plates at 100. Mu.L/well, diluted with RPMI1640 complete medium at 50. Mu.L/well, and in a 5-vol CO2 incubator at 37 ℃ for 72 hours. Supernatants were harvested and IL-2 concentration was assayed using Human IL2 kits (purchased from Cisbio,62HIL02 PEG). As shown in FIG. 13, the detection antibody PR2009 was directed against CD4 in MLR⁺Activating activity of T cells.

Example 19 detection of antitumor Activity of PD-1/CTLA-4 double antibody in mouse subcutaneous tumor model

A drug efficacy experiment (Beijing Baioehcet chart) is carried out by using an MC38-hPD-L1 colon cancer animal model of a B-hPD-1/hCTLA4 humanized mouse, and the in vivo anti-tumor activity of an antibody PR2009 is detected. Resuspending PBS on MC38-hPD-L1 colon cancer cells at 5X 10⁵One/0.1 mL concentration, 0.1 mL/volume was inoculated subcutaneously on the right side of B-hPD-1/hCTLA4 humanized mice. When the mean tumor volume reached about 100mm³At this time, 24 mice with appropriate individual tumor volumes were selected and placed into the main experimental group, and animals were randomly assigned to 4 experimental groups by tumor volume, 6 animals per group, namely, G1 hIgG1 (1 mg/kg), G2 SHR1901 (1 mg/kg), G3 SHR1901+ ipilimumab (1 + 0.3mg/kg) and G4 PR2009 (1.3 mg/kg). All groups were administered by intraperitoneal injection 1 time every 3 days, and 1 time a week after 3 consecutive administrations. Tumor volumes were measured in 2 mice weekly during dosing and observation and the measurements were recorded. As shown in fig. 14A and 14B, the detection antibody PR2009 had an in vivo anti-tumor activity.

SEQUENCE LISTING

<110> Beijing Tuo biomedical science and technology Co., ltd

<120> PD-1/CTLA-4 binding protein and medical application thereof

<150> CN202110261228.6

<151> 2021-03-10

<150> CN202110259670.5

<151> 2021-03-10

<160> 187

<170> PatentIn version 3.5

<210> 1

<211> 288

<212> PRT

<213> Homo sapiens

<400> 1

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> 2

<211> 288

<212> PRT

<213> Macaca fascicularis

<400> 2

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 Glu Ser Pro Asp Arg Pro Trp

20 25 30

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

35 40 45

Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Ala 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 Arg 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 Ala 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 Gln Gly Thr Ile Glu 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 Ala Pro

225 230 235 240

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

245 250 255

Leu Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg

260 265 270

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

275 280 285

<210> 3

<211> 154

<212> PRT

<213> Artificial Sequence

<220>

<223> commercialization of human PD-1 antigen

<400> 3

Leu Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr Phe Ser Pro Ala

1 5 10 15

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

20 25 30

Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr Arg Met Ser Pro

35 40 45

Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu Asp Arg Ser Gln

50 55 60

Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu Pro Asn Gly Arg

65 70 75 80

Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn Asp Ser Gly Thr

85 90 95

Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala Gln Ile Lys Glu

100 105 110

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

115 120 125

Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly Gln Phe Gln Ala

130 135 140

His His His His His His His His His His

145 150

<210> 4

<211> 375

<212> PRT

<213> Artificial Sequence

<220>

<223> commercial anti-human PD-1-Fc antigen

<400> 4

Leu Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr Phe Ser Pro Ala

1 5 10 15

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

20 25 30

Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr Arg Met Ser Pro

35 40 45

Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu Asp Arg Ser Gln

50 55 60

Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu Pro Asn Gly Arg

65 70 75 80

Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn Asp Ser Gly Thr

85 90 95

Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala Gln Ile Lys Glu

100 105 110

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

115 120 125

Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly Gln Phe Gln Glu

130 135 140

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

145 150 155 160

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

165 170 175

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

180 185 190

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

195 200 205

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

210 215 220

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

225 230 235 240

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

245 250 255

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

260 265 270

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys

275 280 285

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

290 295 300

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

305 310 315 320

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

325 330 335

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

340 345 350

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

355 360 365

Leu Ser Leu Ser Pro Gly Lys

370 375

<210> 5

<211> 232

<212> PRT

<213> Artificial Sequence

<220>

<223> commercialization of human PD-L1 antigen

<400> 5

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

1 5 10 15

Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu

20 25 30

Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile Ile Gln

35 40 45

Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg

50 55 60

Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala

65 70 75 80

Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Arg Cys

85 90 95

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

100 105 110

Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro

115 120 125

Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys

130 135 140

Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys

145 150 155 160

Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr

165 170 175

Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr

180 185 190

Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile

195 200 205

Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Thr Ala His His

210 215 220

His His His His His His His His

225 230

<210> 6

<211> 211

<212> PRT

<213> Artificial Sequence

<220>

<223> commercial human PD-L2 antigen

<400> 6

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

1 5 10 15

Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser His Val Asn

20 25 30

Leu Gly Ala Ile Thr Ala Ser Leu Gln Lys Val Glu Asn Asp Thr Ser

35 40 45

Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu Pro Leu Gly

50 55 60

Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp Glu Gly Gln

65 70 75 80

Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr Lys Tyr Leu

85 90 95

Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr His Ile Leu

100 105 110

Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln Ala Thr Gly

115 120 125

Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val Pro Ala Asn

130 135 140

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

145 150 155 160

Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys Val Phe Trp

165 170 175

Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp Leu Gln Ser

180 185 190

Gln Met Glu Pro Arg Thr His Pro Ala His His His His His His His

195 200 205

His His His

210

<210> 7

<211> 134

<212> PRT

<213> Camelus dromedarius

<400> 7

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

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

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

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

50 55 60

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn

65 70 75 80

Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met

85 90 95

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

100 105 110

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

115 120 125

Gln Val Thr Val Ser Ser

130

<210> 8

<211> 124

<212> PRT

<213> Camelus dromedarius

<400> 8

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Arg Tyr Ser Pro Ile

20 25 30

Leu Met Gly Trp Phe Arg Gln Gly Pro Gly Lys Asp Arg Glu Gly Val

35 40 45

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

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Asn Thr Leu Phe Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Met Tyr Tyr Cys Ala

85 90 95

Ala Ala Leu Met Arg Thr Tyr Leu Pro Leu Gln Pro Arg Gln Tyr Asp

100 105 110

Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 9

<211> 124

<212> PRT

<213> Camelus dromedarius

<400> 9

His Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Arg Tyr Ser Pro Ile

20 25 30

Leu Met Gly Trp Phe Arg Gln Gly Pro Gly Lys Asp Arg Gly Gly Val

35 40 45

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

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Asn Thr Leu Phe Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Met Tyr Tyr Cys Ala

85 90 95

Ala Ala Leu Met Arg Thr Tyr Leu Pro Leu Gln Pro Arg Gln Tyr Asp

100 105 110

Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 10

<211> 123

<212> PRT

<213> Camelus dromedarius

<400> 10

His Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ala Cys Ala Ser Ser Arg Asn Thr Asn Arg Tyr Asn

20 25 30

Phe Met Thr Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

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

50 55 60

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

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr His Cys

85 90 95

Ala Ala Ala Leu Arg Asp Gly Ser Trp Ser Ser Gln Asn Tyr Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 11

<211> 124

<212> PRT

<213> Camelus dromedarius

<400> 11

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Arg Tyr Ser Pro Ile

20 25 30

Leu Met Gly Trp Phe Arg Gln Gly Pro Gly Lys Asp Arg Glu Gly Val

35 40 45

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

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Asn Thr Leu Phe Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Met Tyr Tyr Cys Ala

85 90 95

Ala Ala Leu Met Arg Thr Tyr Leu Pro Leu Gln Pro Arg Gln Tyr Asp

100 105 110

Phe Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 12

<211> 124

<212> PRT

<213> Camelus dromedarius

<400> 12

His Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Arg Tyr Ser Pro Ile

20 25 30

Leu Met Gly Trp Phe Arg Gln Gly Pro Gly Lys Asp Arg Glu Gly Val

35 40 45

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

50 55 60

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

65 70 75 80

Gln Met Asn Ile Leu Lys Pro Glu Asp Thr Gly Met Tyr Tyr Cys Ala

85 90 95

Ala Ala Leu Met Arg Thr Tyr Leu Pro Leu Gln Pro Arg Gln Tyr Asp

100 105 110

Phe Trp Gly Gln Gly Thr Leu Asp Ile Val Ser Ser

115 120

<210> 13

<211> 124

<212> PRT

<213> Camelus dromedarius

<400> 13

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Arg Tyr Ser Pro Ile

20 25 30

Leu Met Gly Trp Phe Arg Gln Gly Pro Gly Lys Asp Arg Glu Gly Val

35 40 45

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

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Asn Thr Leu Phe Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Met Tyr Tyr Cys Ala

85 90 95

Ala Ala Leu Met Arg Thr Tyr Leu Pro Leu Gln Pro Arg Gln Tyr Asp

100 105 110

Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 14

<211> 124

<212> PRT

<213> Camelus dromedarius

<400> 14

His Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Arg Tyr Ser Pro Ile

20 25 30

Leu Met Gly Trp Phe Arg Gln Gly Pro Gly Lys Asp Arg Glu Gly Val

35 40 45

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

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Asn Thr Leu Phe Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Met Tyr Tyr Cys Ala

85 90 95

Ala Ala Leu Met Arg Thr Tyr Leu Pro Leu Gln Pro Arg Gln Tyr Asp

100 105 110

Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 15

<211> 124

<212> PRT

<213> Camelus dromedarius

<400> 15

His Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Arg Tyr Ser Pro Ile

20 25 30

Leu Met Gly Trp Phe Arg Gln Gly Pro Gly Lys Asp Arg Glu Gly Val

35 40 45

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

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Asn Thr Leu Phe Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Met Tyr Tyr Cys Ala

85 90 95

Ala Ala Leu Met Arg Thr Tyr Leu Pro Leu Gln Pro Arg Gln Tyr Asp

100 105 110

Phe Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 16

<211> 124

<212> PRT

<213> Camelus dromedarius

<400> 16

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Arg Tyr Ser Pro Ile

20 25 30

Leu Met Gly Trp Phe Arg Gln Gly Pro Arg Lys Asp Arg Glu Gly Val

35 40 45

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

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Asn Thr Leu Phe Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Met Tyr Tyr Cys Ala

85 90 95

Ala Ala Leu Met Arg Thr Tyr Leu Pro Leu Gln Pro Arg Gln Tyr Asp

100 105 110

Phe Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 17

<211> 124

<212> PRT

<213> Camelus dromedarius

<400> 17

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Glu Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Arg Tyr Ser Pro Ile

20 25 30

Leu Met Gly Trp Phe Arg Gln Gly Pro Gly Lys Asp Arg Glu Gly Val

35 40 45

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

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Asn Thr Leu Phe Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Met Tyr Tyr Cys Ala

85 90 95

Ala Ala Leu Met Arg Thr Tyr Leu Pro Leu Gln Pro Arg Gln Tyr Asp

100 105 110

Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 18

<211> 124

<212> PRT

<213> Camelus dromedarius

<400> 18

His Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

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

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

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

50 55 60

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

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Ala Ala Tyr Gly Arg Arg Trp Cys Glu Arg Leu Tyr Met Tyr Asp

100 105 110

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

115 120

<210> 19

<211> 124

<212> PRT

<213> Camelus dromedarius

<400> 19

His Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Arg Tyr Ser Pro Ile

20 25 30

Leu Met Gly Trp Phe Arg Gln Gly Pro Gly Lys Asp Arg Glu Gly Val

35 40 45

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

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Asn Thr Leu Phe Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Met Tyr Tyr Cys Ala

85 90 95

Ala Ala Leu Met Arg Thr Tyr Leu Pro Leu Gln Pro Arg Gln Tyr Asp

100 105 110

Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 20

<211> 124

<212> PRT

<213> Camelus dromedarius

<400> 20

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Arg Tyr Ser Pro Ile

20 25 30

Leu Met Gly Trp Phe Arg Gln Gly Pro Gly Lys Asp Arg Glu Gly Val

35 40 45

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

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Asn Thr Leu Phe Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Met Tyr Tyr Cys Ala

85 90 95

Ala Ala Leu Met Arg Thr Tyr Leu Pro Leu Gln Pro Arg Gln Tyr Asp

100 105 110

Phe Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 21

<211> 122

<212> PRT

<213> Camelus dromedarius

<400> 21

His Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ala Gly Leu Thr Gly Arg Trp Trp Leu Pro Glu Ala Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 22

<211> 122

<212> PRT

<213> Camelus dromedarius

<400> 22

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Ala Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ser Ser Thr Tyr Thr Phe Lys Asn Lys

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Gly Ser Tyr Thr Ser Ala Asn Ser Cys Gln Pro Asp Ala Leu Trp

100 105 110

Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 23

<211> 125

<212> PRT

<213> Camelus dromedarius

<400> 23

Asp Val Gln Leu Val Gly Ser Gly Gly Gly Ser Val Gln Val Gly Gly

1 5 10 15

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

20 25 30

Tyr Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Asn Glu Arg Glu Gly

35 40 45

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

50 55 60

Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val

65 70 75 80

Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr

85 90 95

Cys Gly Val Gly Trp Arg Ala Leu Cys Glu Val Asn Gly Tyr Val Tyr

100 105 110

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

115 120 125

<210> 24

<211> 122

<212> PRT

<213> Camelus dromedarius

<400> 24

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Lys Gly Arg Phe Thr Val Ser Gln Asp Asn Ala Glu Asn Thr Val Tyr

65 70 75 80

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

85 90 95

Ala Ala Gly Leu Thr Gly Arg Trp Trp Leu Pro Glu Ala Asp Tyr Trp

100 105 110

Gly Gln Gly Ile Gln Val Thr Val Ser Ser

115 120

<210> 25

<211> 125

<212> PRT

<213> Camelus dromedarius

<400> 25

His Val Gln Leu Val Glu Ser Gly Gly Asp Ser Val Gln Ala Gly Gly

1 5 10 15

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

20 25 30

Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val

35 40 45

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

50 55 60

Lys Gly Arg Phe Thr Ile Ser His Asp Tyr Ala Lys Asn Thr Val His

65 70 75 80

Leu Gln Met Asn Asn Leu Gln Pro Glu Asp Thr Ala Ile Tyr His Cys

85 90 95

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

100 105 110

Ser Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 26

<211> 122

<212> PRT

<213> Camelus dromedarius

<400> 26

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Ala Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ser Ser Thr Tyr Thr Phe Lys Asn Lys

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Gly Ser Tyr Thr Ser Asp Gly Ser Cys Gln Pro Asp Ala Leu Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 27

<211> 122

<212> PRT

<213> Camelus dromedarius

<400> 27

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Ser Ala Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ser Ser Thr Tyr Thr Phe Arg Asn Lys

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Pro Arg Lys Glu Arg Glu Gly Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Gly Ser Tyr Thr Asp Ala Gly Ser Cys His Pro Asp Ala Leu Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 28

<211> 127

<212> PRT

<213> Camelus dromedarius

<400> 28

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

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

20 25 30

Asn Leu Met Ala Trp Phe Arg Gln Ala Pro Gly Asn Glu Arg Glu Gly

35 40 45

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

50 55 60

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

65 70 75 80

Leu Gln Met Asn Gly Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Ala Lys Thr Gly Ile Ile Gln Val Asp Asp Ala Leu Gln Pro Asn

100 105 110

Glu Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 29

<211> 122

<212> PRT

<213> Camelus dromedarius

<400> 29

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

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

20 25 30

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

35 40 45

Ala Ala Ile Tyr Pro Thr Ala Gly Arg Thr Tyr Phe Ala Asp Ser Val

50 55 60

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

65 70 75 80

Leu Gln Met Asn Ser Leu Gln Ala Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Ala Gly Leu Thr Gly Arg Trp Trp Leu Pro Glu Ala Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 30

<211> 122

<212> PRT

<213> Camelus dromedarius

<400> 30

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

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

20 25 30

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

35 40 45

Ala Ala Ile Tyr Pro Thr Ala Gly Lys Thr Tyr Tyr Ala Asp Ser Met

50 55 60

Arg Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Glu Asn Thr Val Tyr

65 70 75 80

Leu His Met Asn Ser Leu Gln Pro Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Ala Gly Leu Thr Gly Arg Trp Trp Leu Pro Glu Ala Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 31

<211> 122

<212> PRT

<213> Camelus dromedarius

<400> 31

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

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

20 25 30

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

35 40 45

Ala Ala Ile Tyr Pro Thr Ala Gly Lys Thr Tyr Tyr Ala Asp Ser Met

50 55 60

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

65 70 75 80

Leu His Met Asn Ser Leu Gln Pro Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Ala Gly Leu Thr Gly Arg Trp Trp Leu Pro Glu Ala Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 32

<211> 125

<212> PRT

<213> Camelus dromedarius

<400> 32

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

1 5 10 15

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

20 25 30

Tyr Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Asn Glu Arg Glu Gly

35 40 45

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

50 55 60

Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val

65 70 75 80

Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr

85 90 95

Cys Ala Val Gly Trp Arg Ala Leu Cys Glu Val Asn Gly Tyr Val Tyr

100 105 110

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

115 120 125

<210> 33

<211> 122

<212> PRT

<213> Camelus dromedarius

<400> 33

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

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

20 25 30

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

35 40 45

Thr Ala Ile Tyr Pro Thr Ala Gly Arg Thr Tyr Phe Ala Asp Ser Val

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ala Gly Leu Thr Gly Arg Trp Trp Leu Pro Glu Ala Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 34

<211> 360

<212> PRT

<213> Artificial Sequence

<220>

<223> 32#-IgG1

<400> 34

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Arg Tyr Ser Pro Ile

20 25 30

Leu Met Gly Trp Phe Arg Gln Gly Pro Gly Lys Asp Arg Glu Gly Val

35 40 45

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

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Asn Thr Leu Phe Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Met Tyr Tyr Cys Ala

85 90 95

Ala Ala Leu Met Arg Thr Tyr Leu Pro Leu Gln Pro Arg Gln Tyr Asp

100 105 110

Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Gly Ser

115 120 125

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

130 135 140

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

145 150 155 160

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

165 170 175

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

180 185 190

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

195 200 205

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

210 215 220

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

225 230 235 240

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

245 250 255

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

260 265 270

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

275 280 285

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

290 295 300

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

305 310 315 320

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

325 330 335

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

340 345 350

Ser Leu Ser Leu Ser Pro Gly Lys

355 360

<210> 35

<211> 134

<212> PRT

<213> Artificial Sequence

<220>

<223> 2#_Hu_1

<400> 35

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 Tyr Ser Arg Asp

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val

35 40 45

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

50 55 60

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Leu Val Thr Val Ser Ser

130

<210> 36

<211> 123

<212> PRT

<213> Artificial Sequence

<220>

<223> 7#_Hu_1

<400> 36

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 Arg Asn Thr Asn Arg Tyr Asn

20 25 30

Phe Met Thr Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

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

50 55 60

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

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 37

<211> 123

<212> PRT

<213> Artificial Sequence

<220>

<223> 7#_Hu_2

<400> 37

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 Arg Asn Thr Asn Arg Tyr Asn

20 25 30

Phe Met Thr Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val

35 40 45

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

50 55 60

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

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 38

<211> 123

<212> PRT

<213> Artificial Sequence

<220>

<223> 7#_Hu_3

<400> 38

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 Arg Asn Thr Asn Arg Tyr Asn

20 25 30

Phe Met Thr Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Gly Val

35 40 45

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

50 55 60

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

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 39

<211> 123

<212> PRT

<213> Artificial Sequence

<220>

<223> 7#_Hu_4

<400> 39

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 Arg Asn Thr Asn Arg Tyr Asn

20 25 30

Phe Met Thr Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

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

50 55 60

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

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 40

<211> 123

<212> PRT

<213> Artificial Sequence

<220>

<223> 7#_Hu_5

<400> 40

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 Arg Asn Thr Asn Arg Tyr Asn

20 25 30

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

35 40 45

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

50 55 60

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

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 41

<211> 123

<212> PRT

<213> Artificial Sequence

<220>

<223> 7#_Hu_6

<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 Arg Asn Thr Asn Arg Tyr Asn

20 25 30

Tyr Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

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

50 55 60

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

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 42

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> 32#_Hu_1

<400> 42

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 Arg Tyr Ser Pro Ile

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ala Leu Met Arg Thr Tyr Leu Pro Leu Gln Pro Arg Gln Tyr Asp

100 105 110

Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 43

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> 32#_Hu_2

<400> 43

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 Arg Tyr Ser Pro Ile

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ala Leu Met Arg Thr Tyr Leu Pro Leu Gln Pro Arg Gln Tyr Asp

100 105 110

Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 44

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> 32#_Hu_3

<400> 44

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 Arg Tyr Ser Pro Ile

20 25 30

Leu Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Asp Arg Glu Gly Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ala Leu Met Arg Thr Tyr Leu Pro Leu Gln Pro Arg Gln Tyr Asp

100 105 110

Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 45

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> 32#_Hu_4

<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 Arg Tyr Ser Pro Ile

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ala Leu Met Arg Thr Tyr Leu Pro Leu Gln Pro Arg Gln Tyr Asp

100 105 110

Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 46

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> 32#_Hu_5

<400> 46

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 Val Ser Gly Phe Arg Tyr Ser Pro Ile

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ala Leu Met Arg Thr Tyr Leu Pro Leu Gln Pro Arg Gln Tyr Asp

100 105 110

Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 47

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> 61#_Hu_1

<400> 47

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 Tyr Ser Ser Leu

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val

35 40 45

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

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 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 Ala Ala Tyr Gly Arg Arg Trp Cys Glu Arg Leu Tyr Met Tyr Asp

100 105 110

Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 48

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> 61#_Hu_2

<400> 48

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 Tyr Ser Ser Leu

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ala Ala Tyr Gly Arg Arg Trp Cys Glu Arg Leu Tyr Met Tyr Asp

100 105 110

Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 49

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> 106#_Hu_1

<400> 49

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 Thr Tyr Thr Phe Lys Asn Lys

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Gly Ser Tyr Thr Ser Ala Asn Ser Cys Gln Pro Asp Ala Leu Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 50

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> 106#_Hu_2

<400> 50

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 Thr Tyr Thr Phe Lys Asn Lys

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Gly Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Gly Ser Tyr Thr Ser Ala Asn Ser Cys Gln Pro Asp Ala Leu Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 51

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> 106#_Hu_3

<400> 51

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 Thr Tyr Thr Phe Lys Asn Lys

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Gly Ser Tyr Thr Ser Ala Asn Ser Cys Gln Pro Asp Ala Leu Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 52

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> 106#_Hu_4

<400> 52

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 Thr Tyr Thr Phe Lys Asn Lys

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Gly Ser Tyr Thr Ser Ala Asn Ser Cys Gln Pro Asp Ala Leu Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 53

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> 106#_Hu_5

<400> 53

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 Thr Tyr Thr Phe Lys Asn Lys

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Gly Ser Tyr Thr Ser Ala Asn Ser Cys Gln Pro Asp Ala Leu Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 54

<211> 125

<212> PRT

<213> Artificial Sequence

<220>

<223> 107#_Hu_1

<400> 54

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 Gly Asn Arg Arg

20 25 30

Tyr Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Cys Ala Val Gly Trp Arg Ala Leu Cys Glu Val Asn Gly Tyr Val Tyr

100 105 110

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

115 120 125

<210> 55

<211> 125

<212> PRT

<213> Artificial Sequence

<220>

<223> 107#_Hu_2

<400> 55

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 Gly Asn Arg Arg

20 25 30

Tyr Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Gly

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Cys Ala Val Gly Trp Arg Ala Leu Cys Glu Val Asn Gly Tyr Val Tyr

100 105 110

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

115 120 125

<210> 56

<211> 125

<212> PRT

<213> Artificial Sequence

<220>

<223> 107#_Hu_3

<400> 56

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 Gly Asn Arg Arg

20 25 30

Tyr Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Cys Ala Val Gly Trp Arg Ala Leu Cys Glu Val Asn Gly Tyr Val Tyr

100 105 110

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

115 120 125

<210> 57

<211> 125

<212> PRT

<213> Artificial Sequence

<220>

<223> 107#_Hu_4

<400> 57

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 Gly Asn Arg Arg

20 25 30

Tyr Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Cys Gly Val Gly Trp Arg Ala Leu Cys Glu Val Asn Gly Tyr Val Tyr

100 105 110

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

115 120 125

<210> 58

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> 112#_Hu_1

<400> 58

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 Thr Tyr Thr Phe Lys Asn Lys

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Gly Ser Tyr Thr Ser Asp Gly Ser Cys Gln Pro Asp Ala Leu Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 59

<211> 5

<212> PRT

<213> Camelus dromedarius

<400> 59

Arg Asp Cys Met Gly

1 5

<210> 60

<211> 20

<212> PRT

<213> Camelus dromedarius

<400> 60

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

1 5 10 15

Ser Val Lys Gly

20

<210> 61

<211> 22

<212> PRT

<213> Camelus dromedarius

<400> 61

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

1 5 10 15

Pro Tyr Glu Tyr Lys Tyr

20

<210> 62

<211> 5

<212> PRT

<213> Camelus dromedarius

<400> 62

Pro Ile Leu Met Gly

1 5

<210> 63

<211> 16

<212> PRT

<213> Camelus dromedarius

<400> 63

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

1 5 10 15

<210> 64

<211> 16

<212> PRT

<213> Camelus dromedarius

<400> 64

Ala Leu Met Arg Thr Tyr Leu Pro Leu Gln Pro Arg Gln Tyr Asp Phe

1 5 10 15

<210> 65

<211> 4

<212> PRT

<213> Camelus dromedarius

<400> 65

Asn Phe Met Thr

1

<210> 66

<211> 17

<212> PRT

<213> Camelus dromedarius

<400> 66

Ala Ile Tyr Thr Gly Phe Gly Asn Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 67

<211> 14

<212> PRT

<213> Camelus dromedarius

<400> 67

Ala Leu Arg Asp Gly Ser Trp Ser Ser Gln Asn Tyr Asp Tyr

1 5 10

<210> 68

<211> 16

<212> PRT

<213> Camelus dromedarius

<400> 68

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

1 5 10 15

<210> 69

<211> 16

<212> PRT

<213> Camelus dromedarius

<400> 69

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

1 5 10 15

<210> 70

<211> 16

<212> PRT

<213> Camelus dromedarius

<400> 70

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

1 5 10 15

<210> 71

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> 106_hu_1 CDR2

<400> 71

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

1 5 10 15

<210> 72

<211> 16

<212> PRT

<213> Camelus dromedarius

<400> 72

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

1 5 10 15

<210> 73

<211> 18

<212> PRT

<213> Camelus dromedarius

<400> 73

Ala Leu Met Arg Thr Tyr Leu Pro Leu Gln Pro Arg Gln Tyr Asp Phe

1 5 10 15

Trp Gly

<210> 74

<211> 5

<212> PRT

<213> Camelus dromedarius

<400> 74

Ser Leu Cys Met Gly

1 5

<210> 75

<211> 17

<212> PRT

<213> Camelus dromedarius

<400> 75

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

1 5 10 15

Gly

<210> 76

<211> 15

<212> PRT

<213> Camelus dromedarius

<400> 76

Ala Tyr Gly Arg Arg Trp Cys Glu Arg Leu Tyr Met Tyr Asp Ser

1 5 10 15

<210> 77

<211> 16

<212> PRT

<213> Camelus dromedarius

<400> 77

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

1 5 10 15

<210> 78

<211> 5

<212> PRT

<213> Camelus dromedarius

<400> 78

Val Tyr Ser Met Gly

1 5

<210> 79

<211> 17

<212> PRT

<213> Camelus dromedarius

<400> 79

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

1 5 10 15

Gly

<210> 80

<211> 13

<212> PRT

<213> Camelus dromedarius

<400> 80

Gly Leu Thr Gly Arg Trp Trp Leu Pro Glu Ala Asp Tyr

1 5 10

<210> 81

<211> 5

<212> PRT

<213> Camelus dromedarius

<400> 81

Asn Lys Cys Met Gly

1 5

<210> 82

<211> 16

<212> PRT

<213> Camelus dromedarius

<400> 82

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

1 5 10 15

<210> 83

<211> 14

<212> PRT

<213> Camelus dromedarius

<400> 83

Gly Ser Tyr Thr Ser Ala Asn Ser Cys Gln Pro Asp Ala Leu

1 5 10

<210> 84

<211> 5

<212> PRT

<213> Camelus dromedarius

<400> 84

Arg Tyr Cys Met Gly

1 5

<210> 85

<211> 17

<212> PRT

<213> Camelus dromedarius

<400> 85

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

1 5 10 15

Gly

<210> 86

<211> 15

<212> PRT

<213> Camelus dromedarius

<400> 86

Gly Trp Arg Ala Leu Cys Glu Val Asn Gly Tyr Val Tyr Asp Ser

1 5 10 15

<210> 87

<211> 16

<212> PRT

<213> Camelus dromedarius

<400> 87

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

1 5 10 15

<210> 88

<211> 5

<212> PRT

<213> Camelus dromedarius

<400> 88

Phe Arg Tyr Met Gly

1 5

<210> 89

<211> 17

<212> PRT

<213> Camelus dromedarius

<400> 89

Asp Ile Tyr Thr Pro Ser Gly Gln Thr Tyr Tyr Gly Asp Ser Val Lys

1 5 10 15

Gly

<210> 90

<211> 16

<212> PRT

<213> Camelus dromedarius

<400> 90

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

1 5 10 15

<210> 91

<211> 16

<212> PRT

<213> Camelus dromedarius

<400> 91

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

1 5 10 15

<210> 92

<211> 14

<212> PRT

<213> Camelus dromedarius

<400> 92

Gly Ser Tyr Thr Ser Asp Gly Ser Cys Gln Pro Asp Ala Leu

1 5 10

<210> 93

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> 112_hu_1 CDR2

<400> 93

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

1 5 10 15

<210> 94

<211> 16

<212> PRT

<213> Camelus dromedarius

<400> 94

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

1 5 10 15

<210> 95

<211> 14

<212> PRT

<213> Camelus dromedarius

<400> 95

Gly Ser Tyr Thr Asp Ala Gly Ser Cys His Pro Asp Ala Leu

1 5 10

<210> 96

<211> 5

<212> PRT

<213> Camelus dromedarius

<400> 96

Arg Asn Leu Met Ala

1 5

<210> 97

<211> 16

<212> PRT

<213> Camelus dromedarius

<400> 97

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

1 5 10 15

<210> 98

<211> 18

<212> PRT

<213> Camelus dromedarius

<400> 98

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

1 5 10 15

Asn Tyr

<210> 99

<211> 17

<212> PRT

<213> Camelus dromedarius

<400> 99

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

1 5 10 15

Gly

<210> 100

<211> 17

<212> PRT

<213> Camelus dromedarius

<400> 100

Ala Ile Tyr Pro Thr Ala Gly Lys Thr Tyr Tyr Ala Asp Ser Met Arg

1 5 10 15

Gly

<210> 101

<211> 17

<212> PRT

<213> Camelus dromedarius

<400> 101

Ala Ile Tyr Pro Thr Ala Gly Lys Thr Tyr Tyr Ala Asp Ser Met Lys

1 5 10 15

Gly

<210> 102

<211> 17

<212> PRT

<213> Camelus dromedarius

<400> 102

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

1 5 10 15

Gly

<210> 103

<211> 232

<212> PRT

<213> Homo sapiens

<400> 103

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

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

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

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

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

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

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

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

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 104

<211> 359

<212> PRT

<213> Artificial Sequence

<220>

<223> 7#-IgG1

<400> 104

His Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ala Cys Ala Ser Ser Arg Asn Thr Asn Arg Tyr Asn

20 25 30

Phe Met Thr Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

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

50 55 60

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

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr His Cys

85 90 95

Ala Ala Ala Leu Arg Asp Gly Ser Trp Ser Ser Gln Asn Tyr Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ser Gly Ser Glu

115 120 125

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

130 135 140

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

145 150 155 160

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

165 170 175

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

180 185 190

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

195 200 205

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

210 215 220

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

225 230 235 240

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

245 250 255

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

260 265 270

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

275 280 285

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

290 295 300

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

305 310 315 320

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

325 330 335

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

340 345 350

Leu Ser Leu Ser Pro Gly Lys

355

<210> 105

<211> 358

<212> PRT

<213> Artificial Sequence

<220>

<223> 106#-IgG1

<400> 105

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Ala Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ser Ser Thr Tyr Thr Phe Lys Asn Lys

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Gly Ser Tyr Thr Ser Ala Asn Ser Cys Gln Pro Asp Ala Leu Trp

100 105 110

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

115 120 125

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

130 135 140

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

145 150 155 160

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

165 170 175

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

180 185 190

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

195 200 205

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

275 280 285

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

290 295 300

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

305 310 315 320

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

325 330 335

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

340 345 350

Ser Leu Ser Pro Gly Lys

355

<210> 106

<211> 361

<212> PRT

<213> Artificial Sequence

<220>

<223> 107#-IgG1

<400> 106

Asp Val Gln Leu Val Gly Ser Gly Gly Gly Ser Val Gln Val Gly Gly

1 5 10 15

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

20 25 30

Tyr Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Asn Glu Arg Glu Gly

35 40 45

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

50 55 60

Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val

65 70 75 80

Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr

85 90 95

Cys Gly Val Gly Trp Arg Ala Leu Cys Glu Val Asn Gly Tyr Val Tyr

100 105 110

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

115 120 125

Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

130 135 140

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

145 150 155 160

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

165 170 175

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

180 185 190

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

195 200 205

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

210 215 220

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

225 230 235 240

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

245 250 255

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

260 265 270

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

275 280 285

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

290 295 300

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

305 310 315 320

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

325 330 335

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

340 345 350

Lys Ser Leu Ser Leu Ser Pro Gly Lys

355 360

<210> 107

<211> 360

<212> PRT

<213> Artificial Sequence

<220>

<223> 32#_hu_3-hIgG1

<400> 107

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 Arg Tyr Ser Pro Ile

20 25 30

Leu Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Asp Arg Glu Gly Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ala Leu Met Arg Thr Tyr Leu Pro Leu Gln Pro Arg Gln Tyr Asp

100 105 110

Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Gly Ser

115 120 125

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

130 135 140

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

145 150 155 160

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

165 170 175

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

180 185 190

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

195 200 205

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

210 215 220

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

225 230 235 240

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

245 250 255

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

260 265 270

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

275 280 285

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

290 295 300

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

305 310 315 320

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

325 330 335

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

340 345 350

Ser Leu Ser Leu Ser Pro Gly Lys

355 360

<210> 108

<211> 229

<212> PRT

<213> Homo sapiens

<400> 108

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe

1 5 10 15

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

20 25 30

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

35 40 45

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

50 55 60

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

65 70 75 80

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

85 90 95

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

195 200 205

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

210 215 220

Leu Ser Leu Gly Lys

225

<210> 109

<211> 353

<212> PRT

<213> Artificial Sequence

<220>

<223> 32#_hu_3_hIgG4

<400> 109

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 Arg Tyr Ser Pro Ile

20 25 30

Leu Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Asp Arg Glu Gly Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ala Leu Met Arg Thr Tyr Leu Pro Leu Gln Pro Arg Gln Tyr Asp

100 105 110

Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Glu Ser Lys Tyr

115 120 125

Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

Lys

<210> 110

<211> 352

<212> PRT

<213> Artificial Sequence

<220>

<223> 7#_hu_4_hIgG4

<400> 110

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 Arg Asn Thr Asn Arg Tyr Asn

20 25 30

Phe Met Thr Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

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

50 55 60

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

100 105 110

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

115 120 125

Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser

130 135 140

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

145 150 155 160

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr

225 230 235 240

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

245 250 255

Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

<210> 111

<211> 351

<212> PRT

<213> Artificial Sequence

<220>

<223> 106#_hu_1_hIgG4

<400> 111

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 Thr Tyr Thr Phe Lys Asn Lys

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Gly Ser Tyr Thr Ser Ala Asn Ser Cys Gln Pro Asp Ala Leu Trp

100 105 110

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

115 120 125

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

130 135 140

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

145 150 155 160

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

165 170 175

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

180 185 190

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

195 200 205

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

210 215 220

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

225 230 235 240

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

245 250 255

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

325 330 335

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

340 345 350

<210> 112

<211> 354

<212> PRT

<213> Artificial Sequence

<220>

<223> 107#_hu_4_hIgG4

<400> 112

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 Gly Asn Arg Arg

20 25 30

Tyr Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Cys Gly Val Gly Trp Arg Ala Leu Cys Glu Val Asn Gly Tyr Val Tyr

100 105 110

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

115 120 125

Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu

340 345 350

Gly Lys

<210> 113

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> 7#_Hu_5 CDR1

<400> 113

Tyr Asn Phe Met Ser

1 5

<210> 114

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> 7#_Hu_6 CDR2

<400> 114

Tyr Asn Tyr Met Ser

1 5

<210> 115

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2 of the formula

<220>

<221> VARIANT

<222> (1)..(1)

<223> Xaa selected from S or T

<220>

<221> VARIANT

<222> (7)..(7)

<223> Xaa selected from T or A

<220>

<221> VARIANT

<222> (9)..(9)

<223> Xaa selected from D, N or G

<220>

<221> VARIANT

<222> (11)..(11)

<223> Xaa selected from T or A

<220>

<221> VARIANT

<222> (12)..(12)

<223> Xaa selected from N or D

<400> 115

Xaa Ile Asp Ser Val Gly Xaa Thr Xaa Tyr Xaa Xaa Ser Val Lys Gly

1 5 10 15

<210> 116

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2 of the formula

<220>

<221> VARIANT

<222> (5)..(5)

<223> Xaa selected from Y or F

<220>

<221> VARIANT

<222> (8)..(8)

<223> Xaa selected from I or T

<220>

<221> VARIANT

<222> (12)..(12)

<223> Xaa selected from A or D

<220>

<221> VARIANT

<222> (16)..(16)

<223> Xaa selected from K or D

<400> 116

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

1 5 10 15

<210> 117

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 general formula

<220>

<221> VARIANT

<222> (5)..(5)

<223> Xaa is selected from S or D

<220>

<221> VARIANT

<222> (6)..(6)

<223> Xaa selected from A or D

<220>

<221> VARIANT

<222> (7)..(7)

<223> Xaa selected from N or G

<220>

<221> VARIANT

<222> (10)..(10)

<223> Xaa is selected from Q or H

<400> 117

Gly Ser Tyr Thr Xaa Xaa Xaa Ser Cys Xaa Pro Asp Ala Leu

1 5 10

<210> 118

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1 of the formula

<220>

<221> VARIANT

<222> (3)..(3)

<223> Xaa selected from F or Y

<220>

<221> VARIANT

<222> (5)..(5)

<223> Xaa selected from S or T

<400> 118

Tyr Asn Xaa Met Xaa

1 5

<210> 119

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2 of the formula

<220>

<221> VARIANT

<222> (6)..(6)

<223> Xaa selected from A or T

<220>

<221> VARIANT

<222> (8)..(8)

<223> Xaa selected from S or T

<400> 119

Val Ile Asn Thr Gly Xaa Asn Xaa Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 120

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2 general formula

<220>

<221> VARIANT

<222> (1)..(1)

<223> Xaa is selected from L or I

<220>

<221> VARIANT

<222> (7)..(7)

<223> Xaa selected from R or K

<220>

<221> VARIANT

<222> (10)..(10)

<223> Xaa selected from Y or F

<220>

<221> VARIANT

<222> (11)..(11)

<223> Xaa selected from G or A

<220>

<221> VARIANT

<222> (14)..(14)

<223> Xaa selected from M or V

<400> 120

Xaa Tyr Pro Thr Ala Gly Xaa Thr Tyr Xaa Xaa Asp Ser Xaa Lys Gly

1 5 10 15

<210> 121

<211> 443

<212> PRT

<213> Artificial Sequence

<220>

<223> heavy chain of PD-1 antibody

<400> 121

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

Met 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 Ala Asn Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys 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 Gln Leu Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val

100 105 110

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

115 120 125

Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro

210 215 220

Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

225 230 235 240

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

245 250 255

Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn

260 265 270

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

275 280 285

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

290 295 300

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

305 310 315 320

Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys

325 330 335

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

340 345 350

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

355 360 365

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

370 375 380

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

385 390 395 400

Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly

405 410 415

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

420 425 430

Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210> 122

<211> 214

<212> PRT

<213> Artificial Sequence

<220>

<223> PD-1 antibody light chain

<400> 122

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 Leu Ala Ser Gln Thr Ile Gly Thr Trp

20 25 30

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

35 40 45

Tyr Thr Ala Thr Ser Leu Ala Asp 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 Val Tyr Ser Ile 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> 123

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> 32#_Hu_6

<400> 123

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 Val Ser Pro Ile

20 25 30

Leu Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Asp Arg Glu Gly Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ala Leu Met Arg Thr Tyr Leu Pro Leu Gln Pro Arg Gln Tyr Asp

100 105 110

Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 124

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> 32#_Hu_7

<400> 124

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 Tyr Ser Pro Ile

20 25 30

Leu Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Asp Arg Glu Gly Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ala Leu Met Arg Thr Tyr Leu Pro Leu Gln Pro Arg Gln Tyr Asp

100 105 110

Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 125

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> 106#_Hu_6

<400> 125

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 Val Ser Asn Lys

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Gly Ser Tyr Thr Ser Ala Asn Ser Cys Gln Pro Asp Ala Leu Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 126

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> 106#_Hu_7

<400> 126

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 Val Lys Asn Lys

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Gly Ser Tyr Thr Ser Ala Asn Ser Cys Gln Pro Asp Ala Leu Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 127

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> 106#_Hu_8

<400> 127

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 Lys Asn Lys

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Gly Ser Tyr Thr Ser Ala Asn Ser Cys Gln Pro Asp Ala Leu Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 128

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> 106#_Hu_9

<400> 128

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 Lys Asn Lys

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Gly Ser Tyr Thr Ser Ala Asn Ser Cys Gln Pro Asp Ala Leu Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 129

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> 106#_hu_1_hIgG4（N31-D） CDR1

<400> 129

Asp Lys Cys Met Gly

1 5

<210> 130

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> 106#_hu_1_hIgG4（N31-E） CDR1

<400> 130

Glu Lys Cys Met Gly

1 5

<210> 131

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> 106#_hu_1_hIgG4（N31-F） CDR1

<400> 131

Phe Lys Cys Met Gly

1 5

<210> 132

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> 106#_hu_1_hIgG4（N31-G） CDR1

<400> 132

Gly Lys Cys Met Gly

1 5

<210> 133

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> 106#_hu_1_hIgG4（N31-H） CDR1

<400> 133

His Lys Cys Met Gly

1 5

<210> 134

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> 106#_hu_1_hIgG4（N31-I） CDR1

<400> 134

Ile Lys Cys Met Gly

1 5

<210> 135

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> 106#_hu_1_hIgG4（N31-K） CDR1

<400> 135

Lys Lys Cys Met Gly

1 5

<210> 136

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> 106#_hu_1_hIgG4（N31-L） CDR1

<400> 136

Leu Lys Cys Met Gly

1 5

<210> 137

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> 106#_hu_1_hIgG4（N31-M） CDR1

<400> 137

Met Lys Cys Met Gly

1 5

<210> 138

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> 106#_hu_1_hIgG4（N31-P） CDR1

<400> 138

Pro Lys Cys Met Gly

1 5

<210> 139

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> 106#_hu_1_hIgG4（N31-Q） CDR1

<400> 139

Gln Lys Cys Met Gly

1 5

<210> 140

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> 106#_hu_1_hIgG4（N31-R） CDR1

<400> 140

Arg Lys Cys Met Gly

1 5

<210> 141

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> 106#_hu_1_hIgG4（N31-S） CDR1

<400> 141

Ser Lys Cys Met Gly

1 5

<210> 142

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> 106#_hu_1_hIgG4（N104-A） CDR3

<400> 142

Gly Ser Tyr Thr Ser Ala Ala Ser Cys Gln Pro Asp Ala Leu

1 5 10

<210> 143

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> 106#_hu_1_hIgG4（N104-E） CDR3

<400> 143

Gly Ser Tyr Thr Ser Ala Glu Ser Cys Gln Pro Asp Ala Leu

1 5 10

<210> 144

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> 106#_hu_1_hIgG4（N104-F） CDR3

<400> 144

Gly Ser Tyr Thr Ser Ala Phe Ser Cys Gln Pro Asp Ala Leu

1 5 10

<210> 145

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> 106#_hu_1_hIgG4（N104-G） CDR3

<400> 145

Gly Ser Tyr Thr Ser Ala Gly Ser Cys Gln Pro Asp Ala Leu

1 5 10

<210> 146

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> 106#_hu_1_hIgG4（N104-H） CDR3

<400> 146

Gly Ser Tyr Thr Ser Ala His Ser Cys Gln Pro Asp Ala Leu

1 5 10

<210> 147

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> 106#_hu_1_hIgG4（N104-K） CDR3

<400> 147

Gly Ser Tyr Thr Ser Ala Lys Ser Cys Gln Pro Asp Ala Leu

1 5 10

<210> 148

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> 106#_hu_1_hIgG4（N104-P） CDR3

<400> 148

Gly Ser Tyr Thr Ser Ala Pro Ser Cys Gln Pro Asp Ala Leu

1 5 10

<210> 149

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> 106#_hu_1_hIgG4（N104-Q）

<400> 149

Gly Ser Tyr Thr Ser Ala Gln Ser Cys Gln Pro Asp Ala Leu

1 5 10

<210> 150

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> 106#_hu_1_hIgG4（N104-R） CDR3

<400> 150

Gly Ser Tyr Thr Ser Ala Arg Ser Cys Gln Pro Asp Ala Leu

1 5 10

<210> 151

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> 106#_hu_1_hIgG4（N104-S） CDR1

<400> 151

Gly Ser Tyr Thr Ser Ala Ser Ser Cys Gln Pro Asp Ala Leu

1 5 10

<210> 152

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1 of the formula

<220>

<221> VARIANT

<222> (1)..(1)

<223> Xaa selected from N, D, E, F, G, H, I, K, L, M, P, Q, R or S

<400> 152

Xaa Lys Cys Met Gly

1 5

<210> 153

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 general formula

<220>

<221> VARIANT

<222> (7)..(7)

<223> Xaa selected from N, A, E, F, G, H, K, P, Q, R or S

<400> 153

Gly Ser Tyr Thr Ser Ala Xaa Ser Cys Gln Pro Asp Ala Leu

1 5 10

<210> 154

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> 0076#

<400> 154

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 Thr Tyr Thr Phe Lys Asp Lys

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Gly Ser Tyr Thr Ser Ala Gly Ser Cys Gln Pro Asp Ala Leu Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 155

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> 0077#

<400> 155

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 Thr Tyr Thr Phe Lys Gly Lys

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Gly Ser Tyr Thr Ser Ala Gly Ser Cys Gln Pro Asp Ala Leu Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 156

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> 0078#

<400> 156

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 Thr Tyr Thr Phe Lys Asp Lys

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Gly Ser Tyr Thr Ser Ala His Ser Cys Gln Pro Asp Ala Leu Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 157

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> 0079#

<400> 157

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 Thr Tyr Thr Phe Lys Gly Lys

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Gly Ser Tyr Thr Ser Ala His Ser Cys Gln Pro Asp Ala Leu Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 158

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> ipilimumab HCDR1

<400> 158

Gly Phe Thr Phe Ser Ser Tyr

1 5

<210> 159

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> ipilimumab HCDR2

<400> 159

Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys

1 5 10

<210> 160

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> ipilimumab HCDR3

<400> 160

Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr

1 5 10

<210> 161

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Ipimumab LCDR1

<400> 161

Gln Ser Val Gly Ser Ser Tyr

1 5

<210> 162

<211> 351

<212> PRT

<213> Artificial Sequence

<220>

<223> 0079#_hIgG4

<400> 162

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 Thr Tyr Thr Phe Lys Gly Lys

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Gly Ser Tyr Thr Ser Ala His Ser Cys Gln Pro Asp Ala Leu Trp

100 105 110

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

115 120 125

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

130 135 140

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

145 150 155 160

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

165 170 175

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

180 185 190

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

195 200 205

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

210 215 220

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

225 230 235 240

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

245 250 255

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

325 330 335

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

340 345 350

<210> 163

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Ipimab LCDR3

<400> 163

Gln Gln Tyr Gly Ser Ser Pro Trp Thr

1 5

<210> 164

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> ipilimumab VH

<400> 164

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

1 5 10 15

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

20 25 30

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

35 40 45

Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 165

<211> 108

<212> PRT

<213> Artificial Sequence

<220>

<223> ipilimumab VL

<400> 165

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

1 5 10 15

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

20 25 30

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

35 40 45

Ile Tyr Gly Ala Phe Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

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

65 70 75 80

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

85 90 95

Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 166

<211> 448

<212> PRT

<213> Artificial Sequence

<220>

<223> ipilimumab heavy chain

<400> 166

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

1 5 10 15

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

20 25 30

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

35 40 45

Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

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

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 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 Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

<210> 167

<211> 215

<212> PRT

<213> Artificial Sequence

<220>

<223> Ipimuzumab light chain

<400> 167

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

1 5 10 15

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

20 25 30

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

35 40 45

Ile Tyr Gly Ala Phe Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

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

65 70 75 80

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

85 90 95

Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

180 185 190

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 168

<211> 592

<212> PRT

<213> Artificial Sequence

<220>

<223> PR2001 first polypeptide chain

<400> 168

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 Arg Tyr Ser Pro Ile

20 25 30

Leu Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Asp Arg Glu Gly Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ala Leu Met Arg Thr Tyr Leu Pro Leu Gln Pro Arg Gln Tyr Asp

100 105 110

Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val

195 200 205

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

210 215 220

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

225 230 235 240

Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr 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 Ser Ser Lys Ser Thr Ser Gly Gly 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 Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

340 345 350

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

355 360 365

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

<210> 169

<211> 601

<212> PRT

<213> Artificial Sequence

<220>

<223> PR2004 first polypeptide chain

<400> 169

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

1 5 10 15

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

20 25 30

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

35 40 45

Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

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

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 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 Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

450 455 460

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln

465 470 475 480

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

485 490 495

Leu Ser Cys Ala Ala Ser Gly Phe Arg Tyr Ser Pro Ile Leu Met Gly

500 505 510

Trp Phe Arg Gln Ala Pro Gly Lys Asp Arg Glu Gly Val Ser Ser Ile

515 520 525

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

530 535 540

Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn

545 550 555 560

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

565 570 575

Met Arg Thr Tyr Leu Pro Leu Gln Pro Arg Gln Tyr Asp Phe Trp Gly

580 585 590

Gln Gly Thr Leu Val Thr Val Ser Ser

595 600

<210> 170

<211> 590

<212> PRT

<213> Artificial Sequence

<220>

<223> first polypeptide chain of PR2009

<400> 170

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 Thr Tyr Thr Phe Lys Asn Lys

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Gly Ser Tyr Thr Ser Ala Asn Ser Cys Gln Pro Asp Ala Leu Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val

130 135 140

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

145 150 155 160

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

165 170 175

His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Thr Phe

180 185 190

Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly

195 200 205

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

210 215 220

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

225 230 235 240

Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val

245 250 255

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

260 265 270

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

275 280 285

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

290 295 300

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

305 310 315 320

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

325 330 335

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

340 345 350

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

355 360 365

Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe

370 375 380

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

385 390 395 400

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

405 410 415

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

420 425 430

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

435 440 445

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

450 455 460

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

465 470 475 480

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

485 490 495

Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

565 570 575

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

580 585 590

<210> 171

<211> 599

<212> PRT

<213> Artificial Sequence

<220>

<223> first polypeptide chain of PR2012

<400> 171

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

1 5 10 15

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

20 25 30

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

35 40 45

Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

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

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 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 Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

450 455 460

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn

545 550 555 560

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

565 570 575

Tyr Thr Ser Ala Asn Ser Cys Gln Pro Asp Ala Leu Trp Gly Gln Gly

580 585 590

Thr Leu Val Thr Val Ser Ser

595

<210> 172

<211> 590

<212> PRT

<213> Artificial Sequence

<220>

<223> first polypeptide chain of PR2011

<400> 172

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 Thr Tyr Thr Phe Lys Asp Lys

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Gly Ser Tyr Thr Ser Ala Gly Ser Cys Gln Pro Asp Ala Leu Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val

130 135 140

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

145 150 155 160

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

165 170 175

His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Thr Phe

180 185 190

Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly

195 200 205

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

210 215 220

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

225 230 235 240

Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val

245 250 255

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

260 265 270

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

275 280 285

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

290 295 300

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

305 310 315 320

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

325 330 335

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

340 345 350

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

355 360 365

Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe

370 375 380

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

385 390 395 400

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

405 410 415

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

420 425 430

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

435 440 445

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

450 455 460

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

465 470 475 480

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

485 490 495

Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

565 570 575

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

580 585 590

<210> 173

<211> 599

<212> PRT

<213> Artificial Sequence

<220>

<223> first polypeptide chain of PR2014

<400> 173

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

1 5 10 15

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

20 25 30

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

35 40 45

Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

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

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 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 Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

450 455 460

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn

545 550 555 560

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

565 570 575

Tyr Thr Ser Ala Gly Ser Cys Gln Pro Asp Ala Leu Trp Gly Gln Gly

580 585 590

Thr Leu Val Thr Val Ser Ser

595

<210> 174

<211> 4

<212> PRT

<213> Artificial Sequence

<220>

<223> linker

<400> 174

Gly Gly Gly Ser

1

<210> 175

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> linker

<400> 175

Leu Gly Gly Gly Ser Gly

1 5

<210> 176

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> linker

<400> 176

Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly

1 5 10

<210> 177

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> linker

<400> 177

Ala Ser Thr Lys Gly

1 5

<210> 178

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> linker

<400> 178

Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10

<210> 179

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> linker

<400> 179

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

<210> 180

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> linker

<400> 180

Leu Glu Pro Lys Ser Ser

1 5

<210> 181

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> linker

<400> 181

Ala Pro Ser Ser Ser

1 5

<210> 182

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> linker

<400> 182

Ala Pro Ser Ser Ser Pro Met Glu

1 5

<210> 183

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> linker

<400> 183

Leu Glu Pro Lys Ser Ala Asp Lys Thr His Thr Cys Pro Pro Cys

1 5 10 15

<210> 184

<211> 351

<212> PRT

<213> Artificial Sequence

<220>

<223> 0076#_hIgG4

<400> 184

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 Thr Tyr Thr Phe Lys Asp Lys

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Gly Ser Tyr Thr Ser Ala Gly Ser Cys Gln Pro Asp Ala Leu Trp

100 105 110

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

115 120 125

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

130 135 140

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

145 150 155 160

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

165 170 175

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

180 185 190

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

195 200 205

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

210 215 220

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

225 230 235 240

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

245 250 255

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

325 330 335

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

340 345 350

<210> 185

<211> 351

<212> PRT

<213> Artificial Sequence

<220>

<223> 0077#_hIgG4

<400> 185

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 Thr Tyr Thr Phe Lys Gly Lys

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Gly Ser Tyr Thr Ser Ala Gly Ser Cys Gln Pro Asp Ala Leu Trp

100 105 110

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

115 120 125

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

130 135 140

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

145 150 155 160

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

165 170 175

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

180 185 190

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

195 200 205

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

210 215 220

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

225 230 235 240

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

245 250 255

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

325 330 335

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

340 345 350

<210> 186

<211> 351

<212> PRT

<213> Artificial Sequence

<220>

<223> 0078#_hIgG4

<400> 186

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 Thr Tyr Thr Phe Lys Asp Lys

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Gly Ser Tyr Thr Ser Ala His Ser Cys Gln Pro Asp Ala Leu Trp

100 105 110

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

115 120 125

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

130 135 140

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

145 150 155 160

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

165 170 175

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

180 185 190

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

195 200 205

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

210 215 220

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

225 230 235 240

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

245 250 255

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

325 330 335

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

340 345 350

<210> 187

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2 of the formula

<220>

<221> VARIANT

<222> (12)..(12)

<223> Xaa selected from A or D

<400> 187

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

1 5 10 15

Claims

A pd-1/CTLA-4 binding protein comprising:

a first antigen-binding domain that specifically binds PD-1, and

a second antigen-binding domain that specifically binds CTLA-4,

the first antigen binding domain that specifically binds PD-1 comprises at least one immunoglobulin single variable domain comprising three complementarity determining regions CDR1, CDR2, and CDR3, wherein: the immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 as follows:

1) The amino acid sequence of CDR1 is shown in SEQ ID NO:129, and the amino acid sequence of CDR2 is shown in SEQ ID NO:71, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 145; or

2) The amino acid sequence of CDR1 is shown in SEQ ID NO:129-141, the amino acid sequence of CDR2 is as set forth in SEQ ID NO:71, and the amino acid sequence of CDR3 is shown in SEQ ID NO:83 is shown; or

3) The amino acid sequence of CDR1 is shown in SEQ ID NO:81, and the amino acid sequence of CDR2 is shown in SEQ ID NO:71, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 142-151; or

4) The amino acid sequence of CDR1 is shown in SEQ ID NO:132, the amino acid sequence of CDR2 is as shown in SEQ ID NO:71, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 145; or

5) The amino acid sequence of CDR1 is shown in SEQ ID NO:129, the amino acid sequence of CDR2 is shown in SEQ ID NO:71, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 146; or

6) The amino acid sequence of CDR1 is shown in SEQ ID NO:132, and the amino acid sequence of CDR2 is shown in SEQ ID NO:71, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 146; or

7) Amino acid sequence of CDR 1SEQ ID NO:81, and the amino acid sequence of CDR2 is shown in SEQ ID NO:71, the amino acid sequence of CDR3 SEQ ID NO:83, respectively.
2. The PD-1/CTLA-4 binding protein of claim 1, the immunoglobulin single variable domain comprises an amino acid sequence as set forth in SEQ ID NO:154-157, 49-53 and 125-128.
3. The PD-1/CTLA-4 binding protein of claim 1, the second antigen-binding domain that specifically binds CTLA-4 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

VH comprises the amino acid sequences as set forth in SEQ ID NOs: 158-160, HCDR1, HCDR2, HCDR3; VL comprises the amino acid sequences set forth in SEQ ID NOs: 161. 163, and the LCDR1, LCDR3 of the amino acid sequence shown in seq id no, and LCDR2 of the amino acid sequence shown in GAF.
4. The PD-1/CTLA-4 binding protein of claim 3, wherein the VH comprises an amino acid sequence as set forth in SEQ ID NO:164 and VL comprises the amino acid sequence as set forth in SEQ ID NO:165, or a pharmaceutically acceptable salt thereof.
5. The PD-1/CTLA-4 binding protein of claim 2, wherein the second antigen-binding domain that specifically binds CTLA-4 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

VH comprises the amino acid sequences as set forth in SEQ ID NOs: 158-160, HCDR1, HCDR2, HCDR3; VL comprises the amino acid sequences set forth in SEQ ID NOs: 161. 163, and the LCDR1, LCDR3 of the amino acid sequence shown in seq id no, and LCDR2 of the amino acid sequence shown in GAF.
6. The PD-1/CTLA-4 binding protein of claim 5, wherein the VH comprises an amino acid sequence as set forth in SEQ ID NO:164 and VL comprises the amino acid sequence as set forth in SEQ ID NO:165, or a pharmaceutically acceptable salt thereof.
7. The PD-1/CTLA-4 binding protein of any one of the preceding claims, comprising a first polypeptide chain and a second polypeptide chain, wherein:

the first polypeptide chain comprises the amino acid sequence set forth as SEQ ID NO:170-173, or a pharmaceutically acceptable salt thereof,

the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:167.
8. A polynucleotide encoding the PD-1/CTLA-4 binding protein of any one of claims 1 to 7.
9. A pharmaceutical composition comprising: one or more pharmaceutically acceptable carriers, diluents, buffers or excipients, and the PD-1/CTLA-4 binding protein of any one of claims 1 to 7.
10. Use of a PD-1/CTLA-4 binding protein according to any one of claims 1 to 7 in the manufacture of a medicament for the treatment of intestinal cancer.
11. The use according to claim 10, wherein the intestinal cancer is selected from colon cancer and rectal cancer.