RU2807484C2 - Antibody to pd-1, its antigen-binding fragment and its pharmaceutical use - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: antibody to PD-1, its antigen-binding fragment, a pharmaceutical composition containing an antibody to PD-1 and its antigen-binding fragment, a nucleic acid molecule encoding an antibody to PD-1 or its antigen-binding fragment. In particular, a host cell for producing an antibody to PD-1 or an antigen-binding fragment thereof and a method for treating diseases associated with PD-1 are proposed.

EFFECT: used for the treatment of cancer.

20 cl, 3 dwg, 21 tbl, 7 ex

Description

TECHNICAL FIELD

The present invention relates to the field of biotechnology. More specifically, the present invention relates to anti-PD-1 antibodies and uses thereof.

BACKGROUND OF THE ART

Statements in this section relate only to general information associated with the present invention and do not necessarily constitute prior art.

Tumor immunotherapy is a treatment method that fully utilizes and mobilizes killer T cells in cancer patients to destroy the tumor. At the same time, tumor cell evasion is a huge obstacle for tumor immunotherapy. Tumor cells use their own inhibitory effects on the immune system to promote the rapid growth of tumors. There is a very complex relationship between the mechanism of tumor evasion from the immune response and the body's immune response to tumors. At the early stage of tumor immunotherapy, tumor-specific killer T cells have their biological activity, but as the tumor grows, they lose their killing function at a later stage.

To activate T cells in the human body, two systems of signaling pathways are used. In addition to the first signal transmitted to T cells by the presentation of MHC antigen peptides by antigen-presenting cells, a second signal transmitted by a series of co-stimulatory molecules is also required for T cells to mount normal immune responses. This dual signaling system plays a vital role in the balance of the immune system in the body. It strictly regulates the body's various immune responses to its own and foreign antigens. If the second signal conveyed by the co-stimulatory molecule is missing, it will result in no response or a sustained T cell-specific immune response, leading to tolerance. Therefore, the second signaling pathway plays a very important regulatory role in the entire process of the body's immune response.

Programmed death-1 (PD-1) is a protein receptor expressed on the surface of T cells, discovered in 1992, that is involved in the process of cell apoptosis. PD-1 belongs to the CD28 family. It shares 23% amino acid homology with cytotoxic T lymphocyte antigen-4 (CTLA-4), but its expression is different from that of CTLA and it is mainly expressed on activated T cells, B cells and myeloid cells. PD-1 has two ligands, PD-L1 and PD-L2, respectively. PD-L1 is mainly expressed on T cells, B cells, macrophages, and dendritic cells (DCs), and cell expression can be upregulated upon activation. PD-L2 expression is relatively limited and primarily occurs on antigen-presenting cells such as activated macrophages and dendritic cells.

PD-L1 suppresses the immune system by binding to PD-1 and B7-1. Many tumor cells and immune cells in the tumor tissue microenvironment express PD-L1. New studies have shown that high expression of PD-L1 protein has been found in breast cancer, lung cancer (eg non-small cell lung cancer), stomach cancer, colorectal cancer, kidney cancer, melanoma, colon cancer, bladder cancer, ovarian cancer, pancreatic cancer and liver cancer, and in other human tumor tissues, and the expression level of PD-L1 was closely associated with the clinical manifestations and prognosis of patients.

An anti-PD-1 monoclonal antibody can maximize the patient's own immune system response to tumors by blocking PD-L1/PD-1 binding, thereby achieving the goal of killing tumor cells. Currently available anti-PD-1 antibodies are pembrolizumab (also known as Merck keytruda, keytruda, Merck-Pemb, Merck-keytruda, Merck-PD-1, pembrolizumab) and nivolumab (also known as BMS Opdivo, Opdivo, BMS-Nivolumab, Nivolumab ) has been approved by the US Food and Drug Administration for the treatment of melanoma, patients with Hodgkin's lymphoma, non-small cell lung cancer and other tumors. In addition, patent documents such as WO200139722, WO2006121168, WO2010036959, WO2010089411, WO2011110604, WO2013173223, WO2013181634, US2014335093, US6803192B1, US8617546B2 and WO2015085847 also disclose a range of anti-PD-1 monoclonal antibodies.

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to a novel anti-PD-1 antibody, its antigen binding fragment and its medical use.

In some alternative embodiments, the present invention provides an anti-PD-1 antibody or an antigen-binding fragment thereof selected from any of the following i) - iii):

i) an anti-PD-1 antibody or antigen-binding fragment thereof, the heavy chain variable region of which contains HCDR1 represented in SEQ ID NO: 8 or having no more than 3, 2 or 1 amino acid mutation relative to it, HCDR2 represented in SEQ ID NO: 9 or having no more than 3, 2 or 1 amino acid mutation relative to it, and HCDR3 represented in SEQ ID NO: 10 or having no more than 8, 3, 2 or 1 amino acid mutation relative to it; the light chain variable region of which contains LCDR1, represented in SEQ ID NO: 11 or having no more than 4, 3, 2 or 1 amino acid mutation relative to it, LCDR2, presented in SEQ ID NO: 12 or having no more than 3, 2 or 1 amino acid mutation mutations relative thereto, and LCDR3 represented in SEQ ID NO: 13 or having no more than 3, 2 or 1 amino acid mutation relative to it;

ii) an anti-PD-1 antibody or antigen-binding fragment thereof, the heavy chain variable region of which contains HCDR1, represented in SEQ ID NO: 14, or having no more than 3, 2, or 1 amino acid mutation relative to it, HCDR2, represented in SEQ ID NO: 15 or having no more than 3, 2 or 1 amino acid mutation relative to it, and HCDR3 represented in SEQ ID NO: 16 or having no more than 8, 3, 2 or 1 amino acid mutation relative to it; the light chain variable region of which contains LCDR1, represented in SEQ ID NO: 17 or having no more than 4, 3, 2 or 1 amino acid mutation relative to it, LCDR2, presented in SEQ ID NO: 12 or having no more than 3, 2 or 1 amino acid mutation mutations relative thereto, and LCDR3 represented in SEQ ID NO: 18 or having no more than 3, 2 or 1 amino acid mutation relative to it; And

iii) an anti-PD-1 antibody or antigen-binding fragment thereof, the heavy chain variable region of which contains HCDR1 represented in SEQ ID NO: 21 or having no more than 3, 2 or 1 amino acid mutation relative to it, HCDR2 represented in SEQ ID NO: 22 or having no more than 3, 2 or 1 amino acid mutation relative to it, and HCDR3 represented in SEQ ID NO: 23 or having no more than 3, 2 or 1 amino acid mutation relative to it; the light chain variable region of which contains LCDR1, represented in SEQ ID NO: 24 or having no more than 3, 2 or 1 amino acid mutation relative to it, LCDR2, presented in SEQ ID NO: 25 or having no more than 3, 2 or 1 amino acid mutation relative to it it, and LCDR3 represented in SEQ ID NO: 26 or having no more than 3, 2 or 1 amino acid mutation relative to it.

In some embodiments, the above-mentioned anti-PD-1 antibody or antigen binding fragment thereof of the present invention binds to human PD-1 with an equilibrium dissociation constant of 10 ^-7 M or less. In some embodiments, it binds to human PD-1 with an equilibrium dissociation constant equal to or less than 10 ^-8 M, 10 ^-9 M, 10 ^-10 M, or 10 ^-11 M.

In some alternative embodiments, the present invention provides an anti-PD-1 antibody or antigen-binding fragment thereof, the heavy chain variable region of which comprises: HCDR1, represented by SEQ ID NO: 65, HCDR2, represented by SEQ ID NO: 66, and HCDR3, represented by in SEQ ID NO: 67; the light chain variable region of which contains: LCDR1, represented in SEQ ID NO: 68, LCDR2, presented in SEQ ID NO: 12, and LCDR3, presented in SEQ ID NO: 69; The sequences are presented in Table 1 below:

Table 1

Heavy chain Light chain HCDR1 DYE X ₁ H, X ₁ selected from I or M;
(SEQ ID NO: 65) LCDR1 RSSQSX ₁₃ VHSX ₁₄ X ₁₅ X ₁₆ TYLE where X ₁₃ is selected from I or L, X ₁₄ is selected from N, Q, L, T or D, X ₁₅ is selected from G, A or V and X ₁₆ is selected from N or K ;
(SEQ ID NO: 68) HCDR2 LX ₂ DPETGGX ₃ VYNQKFKX ₄ , X ₂ selected from F or I, X ₃ selected from I/T and X ₄ selected from G or D;
(SEQ ID NO: 66) LCDR2 KVSNRFS;
(SEQ ID NO: 12) HCDR3 EX ₅ X ₆ X ₇ X ₈ YX ₉ X ₁₀ X ₁₁ X ₁₂ DWYFDV, X ₅ is selected from G or R, X ₆ is selected from F or missing, X ₇ is S or missing, X ₈ is Y or missing, X ₉ is G or absent, X ₁₀ is S or absent, X ₁₁ is selected from N or T, and X ₁₂ is selected from R or S;
(SEQ ID NO: 67) LCDR3 FQGSHVPYX ₁₇ , X ₁₇ selected from A or T;
(SEQ ID NO: 69)

In some alternative embodiments, in the above-mentioned anti-PD-1 antibody or antigen-binding fragment thereof, the heavy chain variable region comprises HCDR1, shown in SEQ ID NO: 8, HCDR2, shown in SEQ ID NO: 9, and HCDR3, shown in SEQ ID NO: 10; the light chain variable region contains LCDR2, represented by SEQ ID NO: 12, LCDR3, represented by SEQ ID NO: 13, and LCDR1, represented by the general formula RSSQSX ₁₃ VHSX ₁₄ X ₁₅ X ₁₆ TYLE (SEQ ID NO: 68), where X ₁₃ is selected from L, X ₁₄ is selected from N, Q, L, T or D, X ₁₅ is selected from G, A or V, and X ₁₆ is selected from N.

In some alternative embodiments, the anti-PD-1 antibody or antigen binding fragment thereof is selected from any of the following (a) - (e):

(a) an anti-PD-1 antibody or antigen binding fragment thereof comprising HCDR1, HCDR2 and HCDR3 set forth in SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, respectively, and LCDR2 and LCDR3 set forth in SEQ ID NO: 12 and SEQ ID NO: 13, respectively, and LCDR1 represented in SEQ ID NO: 11, 47, 48, 49, 50, 51 or 52;

(b) an anti-PD-1 antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2 and HCDR3 represented by SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively, and LCDR1, LCDR2 and LCDR3 represented by in SEQ ID NO: 17, SEQ ID NO: 12 and SEQ ID NO: 18, respectively;

(c) an anti-PD-1 antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2 and HCDR3 represented by SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23, respectively, and LCDR1, LCDR2 and LCDR3 represented by in SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, respectively;

(d) an anti-PD-1 antibody or antigen binding fragment thereof comprising HCDR1, HCDR2 and HCDR3 set forth in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively, and LCDR2 and LCDR3 set forth in SEQ ID NO: 12 and SEQ ID NO: 13, respectively, and LCDR1 represented in SEQ ID NO: 11, 47, 48, 49, 50, 51 or 52; And

(e) the heavy chain variable region contains HCDR1, HCDR2 and HCDR3 presented in SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, respectively, and the light chain variable region contains LCDR1, LCDR2 and LCDR3 presented in SEQ ID NO: 17, SEQ ID NO: 12 and SEQ ID NO: 18, respectively.

In some embodiments, an anti-PD-1 antibody or antigen binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, as set forth in SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, respectively, and the light chain variable region contains LCDR1, LCDR2 and LCDR3, shown in SEQ ID NO: 49, SEQ ID NO: 12 and SEQ ID NO: 13, respectively.

In some embodiments, an anti-PD-1 antibody or antigen binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, as set forth in SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23, respectively, and the light chain variable region contains LCDR1, LCDR2 and LCDR3, shown in SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, respectively.

In some alternative embodiments, the present invention provides an anti-PD-1 antibody or an antigen-binding fragment thereof selected from any of the following iv) - vi):

iv) an anti-PD-1 antibody or antigen binding fragment thereof, the heavy chain variable region of which contains HCDR1, HCDR2 and HCDR3 with the same sequences as in the heavy chain variable region shown in SEQ ID NO: 4, and the light chain variable region contains LCDR1, LCDR2 and LCDR3 with the same sequences as in the light chain variable region shown in SEQ ID NO: 5;

v) an anti-PD-1 antibody or an antigen-binding fragment thereof, the heavy chain variable region of which contains HCDR1, HCDR2 and HCDR3 with the same sequences as in the heavy chain variable region shown in SEQ ID NO: 6, and the light chain variable region contains LCDR1, LCDR2 and LCDR3 with the same sequences as in the light chain variable region shown in SEQ ID NO: 7; And

vi) an anti-PD-1 antibody or antigen binding fragment thereof, the heavy chain variable region of which contains HCDR1, HCDR2 and HCDR3 with the same sequences as the heavy chain variable region shown in SEQ ID NO: 19, and the light chain variable region contains LCDR1, LCDR2 and LCDR3 with the same sequences as in the light chain variable region shown in SEQ ID NO: 20.

In some embodiments of the above-mentioned anti-PD-1 antibody or antigen-binding fragment thereof, the anti-PD-1 antibody or antigen-binding fragment thereof is a murine antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a fully human antibody or antigen-binding fragment thereof, or a humanized antibody or an antigen-binding fragment thereof.

In some embodiments of the above-mentioned anti-PD-1 antibody or antigen binding fragment thereof, the anti-PD-1 antibody or antigen binding fragment thereof is a humanized antibody or antigen binding fragment thereof.

In some embodiments, the humanized antibody comprises a framework region derived from a human antibody, or a variant of a framework region thereof.

In some embodiments, the framework region variant has no more than 11 reverse amino acid mutations in each of the light chain framework region and/or heavy chain framework region of the human antibody.

In some embodiments, the framework region variant comprises mutation(s) selected from any of the following (f) - (h):

(f) a reverse amino acid mutation 2G contained in the light chain variable region and/or one or more reverse amino acid mutations selected from 27Y, 48I, 67T, 69L, 82F and 93T contained in the heavy chain variable region;

(g) a reverse amino acid mutation 2V contained in the light chain variable region and/or one or more reverse amino acid mutations selected from 26D, 27F, 30T, 38K, 43H, 48I, 66K, 67A, 69L, 82F and 93T contained in the variable region of the heavy chain; And

(h) one or more reverse amino acid mutations selected from 42G, 44V and 71Y contained in the light chain variable region, and/or reverse amino acid mutations 1K and/or 94S contained in the heavy chain variable region.

In some embodiments of the above-mentioned anti-PD-1 antibody or antigen-binding fragment thereof, the anti-PD-1 antibody or antigen-binding fragment thereof comprises antibody variable regions selected from the group consisting of:

(a2) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, respectively, and one or more reverse amino acid mutations of 27Y, 48I, 67T , 69L, 82F and 93T contained in the heavy chain framework region, and

a light chain variable region comprising LCDR2 and LCDR3 shown in SEQ ID NO: 12 and SEQ ID NO: 13, respectively, and LCDR1 shown in SEQ ID NO: 11, 47, 48, 49, 50, 51 or 52, and reverse amino acid mutation 2G contained in the framework region of the light chain;

(b2) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively, and one or more reverse amino acid mutations selected from 26D, 27F , 30T, 38K, 43H, 48I, 66K, 67A, 69L, 82F and 93T contained in the heavy chain variable region; And

a light chain variable region comprising LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 17, SEQ ID NO: 12 and SEQ ID NO: 18, respectively, and a reverse amino acid mutation 2V contained in the light chain framework region;

(c2) a heavy chain variable region containing HCDR1, HCDR2 and HCDR3 represented in SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23, respectively, and reverse amino acid mutations 1K and/or 94S contained in the framework heavy chain regions, and

a light chain variable region comprising LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, respectively, and one or more reverse amino acid mutations selected from 42G, 44V and 71Y, contained in the light chain framework region.

In some embodiments of the above-mentioned anti-PD-1 antibody or antigen-binding fragment thereof, the anti-PD-1 antibody or antigen-binding fragment thereof comprises antibody variable regions selected from any of the following (i) - (o):

(i) a heavy chain variable region the sequence of which corresponds to that shown in SEQ ID NO: 4 or has at least 90% sequence identity to SEQ ID NO: 4; and/or

a light chain variable region the sequence of which corresponds to that shown in SEQ ID NO: 5 or has at least 90% sequence identity to SEQ ID NO: 5;

(j) a heavy chain variable region the sequence of which corresponds to that shown in SEQ ID NO: 6 or has at least 90% sequence identity to SEQ ID NO: 6; and/or

a light chain variable region the sequence of which corresponds to that shown in SEQ ID NO: 7 or has at least 90% sequence identity to SEQ ID NO: 7;

(k) a heavy chain variable region the sequence of which corresponds to that shown in SEQ ID NO: 19 or has at least 90% sequence identity to SEQ ID NO: 19; and/or

a light chain variable region the sequence of which corresponds to that shown in SEQ ID NO: 20 or has at least 90% sequence identity to SEQ ID NO: 20;

(l) a heavy chain variable region the sequence of which corresponds to SEQ ID NO: 27, 30, 31 or 32, or has at least 90% sequence identity to SEQ ID NO: 27, 30, 31 or 32, respectively; and/or

a light chain variable region having the same sequence as set forth in SEQ ID NO: 28, 29, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, or 64, or having at least 90% identity sequences SEQ ID NO: 28, 29, 34, 35, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 or 64, respectively;

(m) a heavy chain variable region the sequence of which corresponds to SEQ ID NO: 33, 36, 37, 38, 39 or 40, or has at least 90% sequence identity to SEQ ID NO: 33, 36, 37, 38, 39 or 40, respectively; and/or

a light chain variable region the sequence of which corresponds to SEQ ID NO: 34, 35, 28, 29, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 or 64, or has at least at least 90% sequence identity to SEQ ID NO: 34, 35, 28, 29, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 or 64, respectively;

(n) a heavy chain variable region the sequence of which corresponds to SEQ ID NO: 41, 45 or 46, or has at least 90% sequence identity to SEQ ID NO: 41, 45 or 46, respectively; and/or

a light chain variable region having a sequence corresponding to SEQ ID NO: 42, 43 or 44, or having at least 90% sequence identity to SEQ ID NO: 42, 43 or 44, respectively;

(o) a heavy chain variable region the sequence of which corresponds to that shown in SEQ ID NO: 70 or has at least 90% sequence identity to SEQ ID NO: 70; and/or

a light chain variable region the sequence of which corresponds to that shown in SEQ ID NO: 71 or has at least 90% sequence identity to SEQ ID NO: 71; And

(p) a heavy chain variable region the sequence of which corresponds to SEQ ID NO: 27, 30, 31 or 32, or has at least 90% sequence identity to SEQ ID NO: 27, 30, 31 or 32, respectively; and/or

a light chain variable region the sequence of which corresponds to SEQ ID NO: 34 or 35 or has at least 90% sequence identity to SEQ ID NO: 34 or 35, respectively;

wherein the sequences of SEQ ID NO: 70 and SEQ ID NO: 71 are sequences of the general formula as shown in Table 2:

table 2

In some embodiments, the anti-PD-1 antibody heavy chain variable region sequence is as set forth in SEQ ID NO: 27 or has at least 90% identity to SEQ ID NO: 27, and the anti-PD-1 antibody light chain variable region sequence is as set forth in SEQ ID NO: 27 ID NO: 55 or has at least 90% sequence identity to SEQ ID NO: 55.

In some embodiments, the anti-PD-1 antibody heavy chain variable region is as set forth in SEQ ID NO: 46 or has at least 90% identity to SEQ ID NO: 46, and the anti-PD-1 antibody light chain variable region sequence is as set forth in SEQ ID NO: 46 ID NO: 43 or has at least 90% sequence identity to SEQ ID NO: 43.

The above-mentioned “at least 90% identity” includes at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

In some other embodiments of the above-mentioned anti-PD-1 antibody or antigen binding fragment thereof, the antibody further comprises antibody constant regions; in some other embodiments, the heavy chain constant region of the antibody constant region is selected from the human IgG1, IgG2, IgG3, and IgG4 constant regions and conventional variants thereof, the light chain constant region of the antibody constant region is selected from the human antibody κ and λ chain constant regions and their traditional options; in some other embodiments, the antibody constant regions comprise an IgG4 heavy chain constant region into which one or more mutations of S228P, F234A and L235A are introduced, for example, comprising an IgG4 heavy chain constant region having three amino acid mutations of S228P, F234A and L235A; in certain other embodiments, the antibody comprises the heavy chain constant region set forth in SEQ ID NO: 72 or SEQ ID NO: 79 and the light chain constant region set forth in SEQ ID NO: 73.

In some embodiments of the above-mentioned anti-PD-1 antibody or antigen binding fragment thereof, the anti-PD-1 antibody comprises a light chain set forth in SEQ ID NO: 78 and a heavy chain set forth in SEQ ID NO: 77 or 82; or

a light chain represented by SEQ ID NO: 75; and a heavy chain represented by SEQ ID NO: 74, 76, 80 or 81.

In some embodiments, the anti-PD-1 antibody comprises:

the heavy chain shown in SEQ ID NO: 74 and the light chain shown in SEQ ID NO: 75; or

the heavy chain shown in SEQ ID NO: 77, and the light chain shown in SEQ ID NO: 78.

In some embodiments, an anti-PD-1 antibody or antigen-binding fragment thereof is provided, wherein the antibody competes with any of the above-mentioned anti-PD-1 antibodies or antigen-binding fragments thereof for binding to human PD-1, or binds to the same epitope of human PD-1, as any of the above-mentioned anti-PD-1 antibodies or antigen-binding fragments thereof.

In some embodiments of the above-mentioned anti-PD-1 antibody or antigen-binding fragment thereof, the antibody is a bispecific antibody or a multispecific antibody.

In some embodiments of an anti-PD-1 antibody or antigen binding fragment thereof, the antigen binding fragment is selected from the group consisting of Fab, Fab', F(ab') ₂ , single chain antibody (scFv), dimerized V region (diabody), and disulfide bond stabilized V-regions (dsFv).

In some embodiments, an isolated monoclonal antibody or antigen binding fragment thereof is disclosed, wherein said antibody competes with an anti-PD-1 antibody or antigen binding fragment thereof for binding to human PD-1 according to any of the above.

Some embodiments of the present invention also provide a pharmaceutical composition comprising a therapeutically effective amount of an anti-PD-1 antibody or an antigen binding fragment thereof according to any of the above, or a therapeutically effective amount of the above isolated monoclonal antibody or an antigen binding fragment thereof, and one or more pharmaceutically acceptable carriers, diluents, buffers or excipients. In some embodiments, a therapeutically effective amount refers to a single dose of a composition containing from 0.1 to 3000 mg of the above-mentioned anti-PD-1 antibody or antigen-binding fragment thereof.

In some embodiments, the present invention also provides a nucleic acid molecule encoding an anti-PD-1 antibody or an antigen binding fragment thereof according to any of the above, or encoding the above isolated monoclonal antibody or an antigen binding fragment thereof.

In some embodiments, the present invention also relates to a host cell containing the aforementioned nucleic acid molecule.

In some embodiments, the present invention also provides a method for immunodetection or determination of PD-1, which includes the step of using an anti-PD-1 antibody or an antigen binding fragment thereof according to any of the above, or the step of using the above isolated monoclonal antibody or an antigen binding fragment thereof.

In some embodiments, the present invention also provides a kit comprising the above-mentioned anti-PD-1 antibody or antigen-binding fragment thereof, or the above-mentioned isolated monoclonal antibody or antigen-binding fragment thereof.

Some embodiments provide the use of the above-mentioned anti-PD-1 antibody or antigen-binding fragment thereof to produce diagnostic agents for PD-1-related diseases.

In some embodiments, the present invention also provides a method for treating diseases, comprising administering to a subject a therapeutically effective amount of an anti-PD-1 antibody or an antigen binding fragment thereof according to any of the above, or the above isolated monoclonal antibody or an antigen binding fragment thereof, or the above pharmaceutical composition, or the aforementioned nucleic acid molecule.

In some embodiments, the disease is a tumor.

In some other embodiments, the disease is selected from: head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumor, pharyngeal cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer gland, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatoma, hepatocellular carcinoma, hepatobiliary cancer, pancreatic cancer, stomach cancer, gastrointestinal cancer, intestinal cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, bone cancer, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, myeloproliferative neoplasms, squamous cell carcinoma, Ewing's sarcoma, systemic light chain amyloidosis and Merkel cell carcinoma; in some of these embodiments, the lymphoma is selected from: Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, large B-cell lymphoma, rich T-cell/histiocyte, and lymphoplasmatic lymphoma, lung cancer selected from: non-small cell lung cancer and small cell lung cancer, leukemia selected from: chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic ary leukemia and myeloid leukemia; in some other embodiments, the disease is selected from: PD-L1 positive melanoma, lung cancer, non-small cell lung cancer, breast cancer, stomach cancer, kidney cancer, bladder cancer, bowel cancer, and colon cancer.

In some embodiments, the present invention also provides the use of an anti-PD-1 antibody or antigen binding fragment thereof according to any of the above, or the above isolated monoclonal antibody or antigen binding fragment thereof, or the above pharmaceutical composition, or the above nucleic acid molecule for the production of drugs for the treatment or prevention of diseases.

In some embodiments, the disease is a tumor.

In some other embodiments, the disease is selected from: head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumor, pharyngeal cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer gland, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatoma, hepatocellular carcinoma, hepatobiliary cancer, pancreatic cancer, stomach cancer, gastrointestinal cancer, intestinal cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, bone cancer, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, myeloproliferative neoplasms, squamous cell carcinoma, Ewing's sarcoma, systemic light chain amyloidosis and Merkel cell carcinoma; in some of these embodiments, the lymphoma is selected from: Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, large B-cell lymphoma, rich T-cell/histiocyte, and lymphoplasmatic lymphoma, lung cancer selected from: non-small cell lung cancer and small cell lung cancer, leukemia selected from: chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic ary leukemia and myeloid leukemia; in some other embodiments, the disease is selected from: PD-L1 positive melanoma, lung cancer, non-small cell lung cancer, breast cancer, stomach cancer, kidney cancer, bladder cancer, bowel cancer, and colon cancer.

In some embodiments, the present invention also provides an anti-PD-1 antibody or an antigen-binding fragment thereof according to any of the above, or the above isolated monoclonal antibody or an antigen-binding fragment thereof, or the above-mentioned nucleic acid molecule, or the above-mentioned pharmaceutical composition for use as medicines.

In some embodiments, the drug is used to treat or prevent diseases associated with PD-1.

In some embodiments, the disease is a tumor.

In some other embodiments, the disease is selected from: head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumor, pharyngeal cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer gland, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatoma, hepatocellular carcinoma, hepatobiliary cancer, pancreatic cancer, stomach cancer, gastrointestinal cancer, intestinal cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, bone cancer, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, myeloproliferative neoplasms, squamous cell carcinoma, Ewing's sarcoma, systemic light chain amyloidosis and Merkel cell carcinoma; in some of these embodiments, the lymphoma is selected from: Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, large B-cell lymphoma, rich T-cell/histiocyte, and lymphoplasmatic lymphoma, lung cancer selected from: non-small cell lung cancer and small cell lung cancer, leukemia selected from: chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic ary leukemia and myeloid leukemia; in some other embodiments, the disease is selected from: PD-L1 positive melanoma, lung cancer, non-small cell lung cancer, breast cancer, stomach cancer, kidney cancer, bladder cancer, bowel cancer, and colon cancer.

DESCRIPTION OF GRAPHIC MATERIALS

Fig. 1: results of a test for blocking the binding of PD-1 to its ligand by antibodies to PD-1;

Fig. 2: the effect of antibodies to PD-1 on the secretion of IFNγ from PBMC cells;

Fig. 3: Efficacy of anti-PD-1 antibodies against MC38 colon cancer tumor xenograft in mice;

Fig. 4: Effect of anti-PD-1 antibodies on tumor volume of MC38 colon cancer in mice.

DETAILED DESCRIPTION OF THE INVENTION

Review

To facilitate understanding of the present invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention pertains.

The terms "programmed death 1", "programmed cell death 1", "PD-1 protein", "PD-1", "PDCD1" and "hPD-1" are used interchangeably and include variants, isotypes, species homologues and analogues of human PD -1, which share at least one epitope with PD-1. The complete sequence of PD-1 can be found under GenBank accession number U64863.

The term “programmed death ligand-1 (PD-L1)” is one of two surface glycoprotein ligands of PD-1 (the other is called PD-L2) that inhibits T cell activation and cytokine secretion when bound to PD-1. As used herein, the term “PD-L1” includes human PD-L1 (hPD-L1), hPD-L1 variants, isotypes, and cross-species homologues, as well as 5 analogs that share at least one epitope with hPD-1. The complete sequence of hPD-L1 can be found under GenBank accession number Q9NZQ7.

The term "cytokine" is a general term for proteins released by a population of cells that act on other cells as mediators of cell-cell interactions. Examples of such cytokines include lymphokines, monokines, chemokines and traditional polypeptide hormones. Examples of cytokines include: human IL-2, IFN-γ, IL-6, TNFα, IL-17 and IL-5.

The three-letter codes and one-letter codes for amino acids used herein are those described in J. biol. chem, 243, p3558 (1968).

An “antibody” described herein generally refers to an immunoglobulin. A natural intact antibody has a tetrapeptide chain structure consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant regions of the immunoglobulin heavy chain differ, so their antigenicity also differs. Thus, immunoglobulins can be divided into five classes, otherwise called immunoglobulin isotypes, namely, IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are μ-chain, δ-chain, γ-chain, α- chain and ε-chain, respectively. The same Ig class can be divided into different subclasses according to differences in the amino acid composition of the hinge region and the number and position of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3 and IgG4. The light chain is classified into κ chain or λ chain according to the difference in constant region. Each of the five Ig classes can have a κ chain or a λ chain. Antibodies referred to herein include antibodies or antigen-binding fragments thereof, including antibodies or antigen-binding fragments thereof that have been modified from an immunoglobulin while retaining the ability to bind antigen; including monospecific antibodies, bispecific antibodies or multispecific antibodies; also including monovalent antibodies, divalent antibodies or multivalent antibodies. For example, the antigen binding fragments of antibodies may be antigen binding fragments comprising at least one VH-CH1 structure and at least one VL-CL structure, wherein the VH and VL structures can approach each other on an interchain basis and retain antigen binding ability. In some embodiments, the antigen binding fragment of the antibody is a monovalent Fab fragment (Fab1 fragment), a divalent Fab fragment (F(ab)2), a trivalent Fab fragment (F(ab)3), a polyvalent (two or more) Fab fragment, and may also be other monospecific, bispecific or multispecific antigen-binding fragments containing at least one Fab fragment.

"Bispecific antibody" refers to an antibody (including an antibody or an antigen-binding fragment thereof, such as a single chain antibody) that can specifically bind to two different antigens or two different epitopes of the same antigen. The prior art discloses bispecific antibodies with various structures. Based on the integrity of the IgG molecule, they can be divided into IgG-like bispecific antibodies and bispecific antibodies similar to antibody fragments. Based on the number and configuration of antigen-binding regions, they can be divided into divalent, trivalent, tetravalent, or bispecific antibodies having a higher valency. Depending on whether the structure is symmetrical, they can be divided into bispecific antibodies with a symmetrical structure and bispecific antibodies with an asymmetrical structure. Among them, bispecific antibodies based on antibody fragments, for example, Fab fragments lacking an Fc fragment form bispecific antibodies by linking two or more Fab fragments in one molecule. These antibodies have lower immunogenicity, lower molecular weight and higher permeability into tumor tissue. Typical antibody structures of this type are bispecific antibodies such as F(ab)2, scFv-Fab, (scFv)2-Fab, etc. As for IgG-like bispecific antibodies (for example, with an Fc fragment), this type of antibody has a large molecular weight. The Fc fragment helps purify the antibody at later stages and improves its solubility and stability. The Fc moiety may also bind to the FcRn receptor and increase the serum half-life of the antibody. Typical bispecific antibody structure models are KiH, CrossMAb, Triomab quad hybridoma, FcΔAdp, ART-Ig, BiMAb, Biclonics, BEAT, DuoBody, Azymetric, XmAb, 2:1 TCBs, 1Fab-IgG TDB, FynomAb, two-in-one/DAF , scFv-Fab-IgG, DART-Fc, LP-DART, CODV-Fab-TL, HLE-BiTE, F(ab)2-CrossMAb, IgG-(scFv)2, Bs4Ab, DVD-Ig, tetravalent-DART- Fc, (scFv)4-Fc, CODV-Ig, mAb2, F(ab)4-CrossMAb and other bispecific antibodies (see Aran F. Labrijn et al., Nature Reviews Drug Discovery volume 18, pages 585-608 (2019 ); Chen S1 et al., J Immunol Res. 2019 Feb 11; 2019:4516041).

The term "monivalent", "divalent", "trivalent" or "multivalent" refers to the presence of a specified number of antigen binding sites in an antibody or polypeptide complex. For example, "monovalent antibody" means that the antibody has one antigen-binding site, "monovalent polypeptide complex" means that the polypeptide complex has one antigen-binding site; "divalent antibody" means that there are two antigen binding sites in the antibody, "divalent polypeptide complex" means that there are two antigen binding sites in the polypeptide complex; "trivalent antibody" means that there are three antigen binding sites in the antibody, "trivalent polypeptide complex" means that there are three antigen binding sites in the polypeptide complex; "multivalent antibody" means that there are multiple (three or more) antigen binding sites in the antibody, and "multivalent polypeptide complex" means that there are multiple (two or more) antigen binding sites in the polypeptide complex.

The term "antibody fusion protein" refers to a biologically active fusion protein formed by binding a protein of interest (polypeptide) to an immunoglobulin. The fusion protein has the biological activity of both the bound protein and the immunoglobulin.

The sequence of approximately 110 amino acids near the N-terminus of the heavy and light chains of an antibody varies widely and constitutes the variable region (Fv region); the remaining amino acid sequence near the C-terminus is relatively stable and constitutes a constant region. The variable region includes 3 hypervariable regions (HVR) and 4 framework regions (FR) with relatively conserved sequences. The 3 hypervariable regions that determine the specificity of an antibody are also known as complementarity determining regions (CDRs). Each of the light chain variable region (VL) and the heavy chain variable region (VH) consists of 3 CDR regions and 4 FR regions. The order from amino terminus to carboxyl terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 light chain CDR regions are LCDR1, LCDR2 and LCDR3; and 3 heavy chain CDR regions are HCDR1, HCDR2 and HCDR3.

Antibodies of the present invention include murine antibodies, chimeric antibodies, humanized antibodies and fully human antibodies, preferably humanized antibodies.

The term “mouse antibody” as used herein means a monoclonal antibody to human PD-1 prepared in accordance with knowledge and skill in the art. During preparation, the test subject is injected with PD-1 antigen, and then hybridomas expressing antibodies with the desired sequence or functional properties are isolated. In a preferred embodiment of the present invention, the mouse anti-PD-1 antibody or antigen binding fragment thereof may further comprise a mouse κ, λ light chain constant region, or a variant thereof, or further comprise a mouse IgG1, IgG2, IgG3 heavy chain constant region, or a variant thereof.

The term "chimeric antibody" means an antibody formed by fusing the variable region of a mouse antibody with the constant region of a human antibody, which can reduce the immune response induced by the mouse antibody. Creation of a chimeric antibody requires first creating a hybridoma secreting mouse-specific monoclonal antibodies, then cloning the variable region gene from mouse hybridoma cells, and then cloning the human antibody constant region gene, if necessary, linking the mouse variable region gene with the human constant region gene to form a chimeric gene , inserting the chimeric gene into an expression vector, and finally expressing the chimeric antibody molecule in a eukaryotic system or prokaryotic system. In a preferred embodiment of the present invention, the antibody light chain of the chimeric anti-PD-L1 antibody further comprises a human κ-, λ-chain light chain constant region, or a variant thereof. The antibody heavy chain of the chimeric anti-PD-1 antibody further comprises a human IgG1, IgG2, IgG3, IgG4 heavy chain constant region or a variant thereof, preferably contains a human IgG1, IgG2 or IgG4 heavy chain constant region, or contains IgG1, IgG2 or IgG4 variants with amino acids mutations (eg, L234A and/or L235A mutations, and/or S228P mutations).

The term “humanized antibody,” also known as a CDR-grafted antibody, refers to an antibody produced by grafting murine CDR sequences onto a human antibody variable region framework, that is, it refers to an antibody derived from various types of human germline antibody framework sequences. It can overcome the heterogeneous response induced by the chimeric antibody because it carries a large number of murine protein components. Such framework sequences can be obtained from publicly available DNA databases or published references that include germline antibody gene sequences. For example, germline DNA sequences of the human heavy and light chain variable region genes can be found in the human germline sequence database "VBase" (available at www.mrccpe.com.ac.uk/vbase) and also in Kabat, E. A., et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition. To avoid decreased activity concomitantly caused by decreased immunogenicity, the human antibody variable region framework sequence may be subject to minimal reverse mutations or back mutations to maintain activity. The humanized antibodies of the present invention also include humanized antibodies that have been further subjected to CDR affinity maturation using yeast display.

Because the residues are in contact with the antigen, grafting with a CDR may result in a decrease in the affinity of the resulting antibody or antigen binding fragment thereof for that antigen due to the framework residues being in contact with the antigen. Such interactions may result from hypermutation of somatic cells. Therefore, it may still be necessary to graft such donor framework amino acids onto the humanized antibody framework. Amino acid residues involved in antigen binding and derived from non-human antibodies or antigen-binding fragments thereof can be identified by examining the sequence and structure of the variable region of an animal monoclonal antibody. Residues in the donor CDR scaffold that differ from the germline can be considered related. If the closest germline cannot be determined, the sequence can be compared to a subclass consensus sequence or an antibody sequence from an animal with a high percentage of similarity. Rare scaffold residues are thought to result from hypermutation in somatic cells and therefore play an important role in binding.

In one embodiment of the present invention, the antibody or antigen binding fragment thereof may further comprise a light chain constant region of a human or murine κ, λ chain or variant thereof, or further comprise a heavy chain constant region of a human or murine IgG1, IgG2, IgG3, IgG4 or her version; preferably contains the heavy chain constant region of human IgG1, IgG2 or IgG4, or a variant of IgG1, IgG2 or IgG4 with amino acid mutations (eg, L234A and/or L235A mutation, and/or S228P mutation).

A "conventional variant" of a human antibody heavy chain constant region and a human antibody light chain constant region described herein refers to a variant of the heavy chain constant region or light chain constant region disclosed in the prior art that does not alter the structure and function of the antibody variable region . Examples of variants include heavy chain constant region variants of IgG1, IgG2, IgG3, or IgG4 with site-directed modifications and amino acid substitutions in the heavy chain constant region. Specific substitutions are YTE mutations, L234A and/or L235A mutations, S228P mutations, and/or knob-to-valve mutations (which converts the antibody heavy chain to a knob-Fc and trench-Fc combination), known in the art. These mutations have been shown to impart new properties to the antibody without changing the function of the antibody variable region.

"HuMAb", "human antibody", "whole human antibody" and "whole human antibody" may be used interchangeably and may refer to antibodies derived from a human or antibodies derived from a genetically modified organism that has been "engineered" to produce certain human antibodies in response to antigen stimulation, and can be obtained by any method known in the art. In some technologies, elements of the human heavy and light chain loci are introduced into cell lines of organisms derived from embryonic stem cell lines in which the endogenous heavy chain and light chain loci have been specifically disrupted by agents targeting the endogenous heavy chain and light chain loci contained in these cell lines. Transgenic organisms can synthesize human antibodies specific for human antigens, and these organisms can be used to produce hybridomas that secrete human antibodies. A human antibody may also be an antibody in which the heavy and light chains are encoded by nucleotide sequences derived from one or more human DNA sources. Fully human antibodies can also be constructed by gene or chromosome transfection techniques and phage display technology, or constructed from in vitro activated B cells, all of which are known in the art.

The terms “full-length antibody,” “intact antibody,” “whole antibody,” and “full antibody” are used interchangeably herein and refer to an antibody in substantially intact form, as opposed to antigen-binding fragments as defined below. These terms refer exclusively to an antibody in which the heavy chain includes an Fc region.

The term "antigen binding fragment" or "functional fragment" of an antibody refers to one or more antibody fragments that retain the ability to specifically bind an antigen (eg, PD-1). It has been shown that fragments of full-length antibodies can be used to perform the antigen-binding function of antibodies. Examples of the binding fragment included in the term “antigen binding fragment” of an antibody include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) F(ab') ₂ fragments, including divalent fragments of two Fab fragments linked by a disulfide bridge in the hinge region, (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of the VH and VL domains of one arm of the antibody; (V) single domains or dAb fragments (Ward et al., (1989) Nature 341:544-546), which consist of a VH domain; and (vi) isolated complementarity determining regions (CDRs) or (vii) combinations of two or more isolated CDRs, optionally linked by synthetic linkers. In addition, although the two VL and VH domains of the Fv fragment are encoded by separate genes, recombination techniques can be used to link them using synthetic linkers so that it can be produced as a single protein chain in which the VL and VH regions are paired with formation of a monovalent molecule (called single chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85: 5879- 5883). Such single chain antibodies are also intended to be encompassed by the term “antigen binding fragment” of an antibody. Such antibody fragments are prepared using conventional methods known to those skilled in the art, and the functions of the fragments are screened in the same manner as the functions of intact antibodies. The antigen-binding fragment can be obtained using recombinant DNA technology or enzymatic or chemical cleavage of intact immunoglobulin. Antibodies can be antibodies of different isotypes, for example, IgG antibodies (eg, IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM.

The antigen binding fragments of the present invention include Fab, F(ab') ₂ , Fab', single chain antibody (scFv), dimerized V region (diabody), disulfide stabilized V region (dsFv), CDR containing peptide and the like.

Fab is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity, and is produced by treating IgG antibody molecules with the protease papain (cleaves the amino acid residue at position 224 of the H chain), in which approximately half of the N-terminal side of the H chain and all The L-chain is linked together by disulfide bond(s).

The Fab of the present invention can be produced using papain to treat a monoclonal antibody of the present invention that specifically recognizes human PD-1 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure. In addition, a Fab can be produced by inserting DNA encoding a Fab antibody into a prokaryotic expression vector or eukaryotic expression vector and introducing the vector into a prokaryotic organism or eukaryotic organism to express the Fab.

F(ab') ₂ is an antibody fragment that has a molecular weight of about 100,000, has antigen-binding activity and contains two Fab regions linked at a hinge position, and is prepared by cleaving the bottom of the two disulfide bonds in the IgG hinge region with the enzyme pepsin.

F(ab') ₂ according to the present invention can be obtained using pepsin to treat a monoclonal antibody according to the present invention, which specifically recognizes human PD-1 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure. In addition, F(ab') ₂ can be obtained by linking Fab', described below, with a thioether bond or a disulfide bond.

Fab' is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity, obtained by cleavage of the disulfide bond in the F(ab') ₂ hinge region. Fab' according to the present invention can be obtained using reducing agents, for example, dithiothreitol, to treat F(ab') ₂ according to the present invention, which specifically recognizes PD-1 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure.

In addition, a Fab can be produced by inserting DNA encoding a Fab' fragment of an antibody into a prokaryotic expression vector or eukaryotic expression vector, and then introducing the vector into a prokaryotic organism or eukaryotic organism to express the Fab'.

The term "single chain antibody", "single chain Fv" or "scFv" refers to molecules containing an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) linked by a linker. Such scFv molecules may have the general structure: NH ₂ -VL-linker-VH-COOH or NH ₂ -VH-linker-VL-COOH. Suitable linkers in the art consist of repeated amino acid sequences GGGGS or variants thereof, for example, a variant with 1-4 sequence repeats (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers that can be used in the present invention are described in Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol.

The ScFv of the present invention can be produced by the following steps: obtaining a cDNA encoding the VH and VL of a monoclonal antibody of the present invention that specifically recognizes human PD-1 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure, constructing the DNA encoding the scFv, inserting DNA into a prokaryotic expression vector or eukaryotic expression vector, and then introducing the expression vector into a prokaryotic organism or eukaryotic organism to express the scFv.

The diabody is an antibody fragment in which the scFv is dimerized and is an antibody fragment with bivalent antigen-binding activity. In bivalent antigen-binding activity, the two antigens may be the same or different.

The diabody of the present invention can be produced by the following steps: obtaining a cDNA encoding the VH and VL of a monoclonal antibody of the present invention that specifically recognizes human PD-1 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure, constructing a DNA encoding the scFv, so that the peptide linker is 8 or less amino acid residues in length, inserting the DNA into a prokaryotic expression vector or eukaryotic expression vector, and then introducing the expression vector into a prokaryotic organism or eukaryotic organism to express the diabody.

dsFv is produced by linking polypeptides in which one amino acid residue in each of VH and VL is replaced by a cysteine residue via a disulfide bond between the cysteine residues. Amino acid residues substituted by cysteine residues can be selected according to a known method (Protein Engineering, 7, 697 (1994)) based on the prediction of the three-dimensional structure of the antibody.

The dsFv of the present invention can be produced by the following steps: obtaining a cDNA encoding the VH and VL of a monoclonal antibody of the present invention that specifically recognizes human PD-1 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure, constructing the DNA encoding the dsFv, inserting DNA into a prokaryotic expression vector or eukaryotic expression vector, and then introducing the expression vector into a prokaryotic organism or eukaryotic organism to express dsFv.

The CDR-containing peptide consists of one or more regions including the VH or VL CDR region(s). Peptides containing multiple CDRs can be linked directly or through a suitable peptide linker.

The CDR-containing peptide of the present invention can be obtained by the following steps: constructing cDNA sequence(s) encoding the CDR VH and VL region(s) of a monoclonal antibody of the present invention that specifically recognizes human PD-1 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure, inserting the DNA sequence(s) into a prokaryotic expression vector or eukaryotic expression vector, and then introducing the expression vector into a prokaryotic organism or eukaryotic organism to express the peptide. The CDR-containing peptide can also be obtained by chemical synthesis methods, for example, the Fmoc method or the tBoc method.

The term "amino acid difference" or "amino acid mutation" refers to the presence of amino acid changes or mutations in a variant protein or polypeptide compared to the parent protein or polypeptide, including the presence of 1, 2, 3 or more amino acid insertions, deletions or substitutions relative to the parent protein or polypeptide .

The term “antibody framework” or “FR region” refers to a fragment of the VL or VH variable domain that serves as a framework for the antigen binding loop (CDR) of the variable domain. In fact, it is a variable domain without a CDR.

The term “complementarity determining region,” “CDR,” or “hypervariable region” refers to one of the six hypervariable regions in the variable domain of an antibody that primarily contribute to antigen binding. Typically there are three CDRs (HCDR1, HCDR2 and HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2 and LCDR3) in each light chain variable region. Any of the well-known schemes, including the "Kabat" numbering system, can be used to delineate CDR amino acid sequences (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering system (see Al-Lazikani et al., (1997) JMB 273:927-948) and the ImmunoGenTics (IMGT) numbering system (Lefranc M. P., Immunologist, 7, 132- 136 (1999); Lefranc, M. P., et al., Dev. Comp. Immunol., 27, 55-77 (2003)), etc. For example, for the classical format according to the Kabat system, the CDR amino acid residue numbering in the heavy chain variable domain (VH) is 31-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3); The numbering of the CDR amino acid residues in the light chain variable domain (VL) is 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3). According to the Chothia system, the numbering of the CDR amino acid residues in VH is 26-32 (HCDR1), 52-56 (HCDR2) and 95-102 (HCDR3); and the numbering of amino acid residues in VL is 26-32 (LCDR1), 50-52 (LCDR2) and 91-96 (LCDR3). According to a combination of the CDR definitions according to Kabat and Chothia, CDRs consist of amino acid residues 26–35 (HCDR1), 50–65 (HCDR2) and 95–102 (HCDR3) in human VH and amino acid residues 24–34 (LCDR1), 50 -56 (LCDR2) and 89-97 (LCDR3) in human VL. According to the IMGT system, the CDR amino acid residue numbering in VH is approximately 26-35 (CDR1), 51-57 (CDR2) and 93-102 (CDR3), and the CDR amino acid residue numbering in VL is approximately 27-32 (CDR1 ), 50-52 (CDR2) and 89-97 (CDR3). According to the IMGT system, antibody CDR regions can be determined using the IMGT/DomainGap Align program.

The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds (eg, a specific site on PD-L1 molecules). Epitopes typically include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-sequential amino acids in a unique spatial conformation. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996).

The terms “specifically binds,” “selectively binds,” “binds selectively,” and “binds specifically” refer to the binding of an antibody to an epitope on a selected antigen. Typically, antibodies bind with an affinity (KD) of less than about 10 ^-8 M, such as less than about 10 ^-9 M, 10 ^-10 M, 10 ^-11 M or less.

The term "KD" or "Kd" refers to the equilibrium dissociation constant for a particular antibody-antigen interaction. Typically, antibodies of the present invention bind to PD-1 with an equilibrium dissociation constant (KD) of less than about 10 ^-7 M, for example, less than about 10 ^-8 M or 10 ^-9 M, for example, when measured on a BIACORE instrument using surface plasma technology resonance (SPR).

When the term "competition" is used in the context of antigen-binding proteins that compete for the same epitope (e.g., neutralizing antigen-binding proteins or neutralizing antibodies), it refers to the competition between antigen-binding proteins, which can be determined using the following assay: in the assay subject test, an antigen binding protein (eg, an antibody or an immunologically functional fragment thereof) prevents or inhibits (eg, reduces) the specific binding of a reference antigen binding protein (eg, a ligand or a reference antibody) to a common antigen (eg, PD-1 or a fragment thereof). To determine whether one antigen-binding protein competes with another, many types of competitive binding assays can be used, for example: direct or indirect radioimmunoassay (RIA), direct or indirect enzyme-linked immunosorbent assay (EIA), competitive sandwich assay (see e.g. , Stahli et al, 1983, Methods in Enzymology 9: 242-253); solid phase direct biotin-avidin EIA (see, for example, Kirkland et al, 1986, J. Immunol. 137: 3614-3619), solid phase direct labeling assay, solid phase direct labeling sandwich assay (see, for example, Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase RIA with direct labeling with I-125 labels (see, for example, Morel et al, 1988, Molec. Immunol. 25: 7-15); solid phase direct biotin-avidin EIA (see, for example, Cheung, et al, 1990, Virology 176: 546-552); and direct labeling RIA (Moldenhauer et al, 1990, Scand. J. Immunol. 32: 77-82). Typically, the assay involves the use of purified antigen bound to a solid surface or cell bearing either an unlabeled test antigen-binding protein or a labeled reference antigen-binding protein. Competitive inhibition is measured by measuring the amount of label bound to a solid surface or cell in the presence of a test antigen binding protein. Typically, the antigen binding protein being tested is present in excess. Antigen-binding proteins identified by competition analysis (competing antigen-binding proteins) include: antigen-binding proteins that bind to the same epitope as the reference antigen-binding protein; and antigen-binding proteins that bind to adjacent epitopes located sufficiently close to the epitope to which the reference antigen-binding protein binds, where the two epitopes spatially hinder each other's binding. Additional details regarding methods for determining binding competition are provided in the examples provided herein. Typically, when a competing antigen binding protein is present in excess, it will inhibit (e.g., reduce) the specific binding of the reference antigen binding protein to a common antigen by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70%-75% or 75%, or even more. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97%, or more.

The term "nucleic acid molecule" as used herein refers to DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded and is preferably double-stranded DNA or single-stranded mRNA or modified mRNA. When a nucleic acid is operably linked to another nucleic acid sequence, the nucleic acid is "operably linked". For example, if a promoter or enhancer influences transcription of a coding sequence, then the promoter or enhancer is operably linked to the coding sequence.

The term "vector" refers to a nucleic acid molecule capable of carrying another nucleic acid to which it has been linked. In one embodiment, the vector is a "plasmid", which refers to a circular double-stranded loop of DNA to which additional DNA segments can be linked. In yet another embodiment, the vector is a viral vector, in which additional DNA segments can be linked to the viral genome. The vectors disclosed herein may autonomously replicate in the host cell into which they have been introduced (e.g., a bacterial vector with a bacterial origin of replication and a mammalian episomal vector), or may be integrated into the genome of the host cell after introduction into the host cell. to replicate along with the host genome (eg, a non-episomal mammalian vector).

Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art, for example, Antibodies: A Laboratory Manual, Cold Spring Harbor, chapters 5-8 and 15. For example, mice can be immunized with human PD-1 or fragments thereof, and the resulting antibodies can be renatured, purified, and amino acid sequencing can be performed using conventional methods. Antigen binding fragments can also be prepared by conventional methods. One or more human FR regions are added to non-human CDR regions of antibodies or antigen binding fragments of the invention using genetic engineering techniques. Human germline FR sequences can be obtained from the ImmunoGeneTics (IMGT) website, http://imgt.cines.fr, by comparing the IMGT database of human antibody variable region germline genes and MOE software, or obtained from The Immunoglobulin FactsBook, 2001ISBN012441351.

The term "host cell" refers to the cell into which the expression vector has been introduced. Host cells may include bacteria, microorganisms, plant or animal cells. Bacteria that can be easily transformed include members of the Enterobacteriaceae, such as Escherichia coli or Salmonella strains; Bacillaceae, for example Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO cells (a Chinese hamster ovary cell line) and NS0.

Genetically modified antibodies or antigen binding fragments according to the present invention can be obtained and purified by conventional methods. For example, the cDNA sequences encoding the heavy and light chains can be cloned and recombined into a GS expression vector. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. More preferably, mammalian expression systems can result in glycosylation of antibodies, especially at the highly conserved N-terminal sites of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human PD-1. Positive clones are propagated in a serum-free bioreactor medium to obtain antibodies. The culture medium into which the antibodies are secreted can be purified by conventional methods. For example, using a Sepharose FF A or G column with adjusted buffer for purification. Nonspecifically binding components are washed away. Binding antibodies are then eluted using a pH gradient, and antibody fragments are detected by SDS-PAGE and collected. Antibodies can be filtered and concentrated using conventional methods. Soluble mixtures and polymers can also be removed by conventional methods such as molecular sieves and ion exchange. The resulting product must be immediately frozen, for example at -70 °C, or lyophilized.

"Administration", "administration", "treatment" and "treatment" when applied to animals, humans, experimental subjects, cells, tissues, organs or body fluids refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent or composition with animals, humans , subjects, cells, tissues, organs or biological fluids. "Administration", "administration", "treatment" and "treatment" may refer to, for example, treatment, pharmacokinetics, diagnostics, research and experimental methods. Cell processing involves contact of reagents with cells and contact of reagents with liquids, where liquids contact cells. “Administration,” “administration,” and “treatment” also refer to the treatment, for example, of cells with reagents, diagnostic, binding compositions, or other cells in vitro and ex vivo. “Treatment,” as applied to a human patient, animal patient or test subject, refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.

“Treatment” refers to the administration of an internal or external therapeutic agent, for example, a composition containing any of the binding compounds of the present invention, to a patient who has one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Generally, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of a disease in a patient or population being treated to cause regression of such symptoms or suppress the development of such symptoms to any clinically measurable extent. The amount of a therapeutic agent effective to relieve any particular symptom of a disease (also referred to as a "therapeutically effective amount") may vary depending on various factors, such as the stage of the disease, the age and weight of the patient, and the ability of the drug to provide the desired therapeutic benefit. effect in the patient. Whether symptoms of a disease have been alleviated can be assessed using any of the clinical testing methods commonly used by doctors or other health care professionals to assess the severity or progression of symptoms. Although embodiments of the present invention (e.g., treatments or products) may not be effective in alleviating every symptom of the target disease, they should alleviate the symptom of the target disease in a statistically significant number of patients, as determined by any statistical test known in the art, such as as Student's t-test, chi-square test, Mann-Whitney U-test, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test and Wilcoxon test.

"Conservative modification" or "conservative substitution or substitution" refers to the replacement of amino acids in a protein with other amino acids having similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.) so that it was common to make changes without changing the biological activity of the protein. It is known to those skilled in the art that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co. ., Page 224, (4th edition)). In addition, substitution with amino acids of similar structure or function is unlikely to affect biological activity. Exemplary conservative amino acid substitutions are shown in the table “Illustrative Conservative Amino Acid Substitutions” below.

Table 3. Illustrative Conservative Amino Acid Substitutions

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

"Effective amount" or "effective dose" refers to the amount of a drug, compound or pharmaceutical composition required to obtain any one or more beneficial or desired therapeutic results. For prophylactic purposes, beneficial or desired outcomes include eliminating or reducing the risk, reducing the severity or delaying the onset of disease, including the biochemical, histological and behavioral manifestations of the disease, its complications and intermediate pathological phenotypes that occur during the development of the disease process. For therapeutic purposes, beneficial or desired results include clinical results such as reducing the incidence of various disorders of the present invention associated with the target antigen, or improving one or more symptoms of the disorder, reducing the dose of other agents needed to treat the disorder, increasing the therapeutic efficacy of another agent and/or delaying the progression of the disorder of the present invention associated with the target antigen in the patient.

"Exogenous" refers to substances produced outside of organisms, cells, or human bodies, as the case may be. "Endogenous" refers to substances produced within cells, organisms, or human bodies, as the case may be.

"Homology" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When positions in two sequences being compared are occupied by the same base or subunit of an amino acid monomer, for example, if a position in each of two DNA molecules is occupied by an adenine, then the molecules are homologous at that position. The percentage of 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 being compared, and then multiplied by 100. For example, when two sequences are optimally aligned, if 6 of the 10 positions in the two sequences are matched or homologous, then the two sequences are 60% homologous; if 95 out of 100 positions in two sequences are the same or homologous, then the two sequences are 95% homologous. Typically, when two sequences are aligned, comparisons are made to obtain the maximum percentage of homology. For example, the comparison may be performed by the BLAST algorithm, in which the parameters of the algorithm are chosen to provide the maximum match for each sequence over the entire length of each reference sequence. The following references refer to the BLAST algorithm, which is often used for sequence analysis: BLAST ALGORITHMS: Altschul, S. F. et al., (1990) J. Mol. Biol. 215:403-410; Gish, W. et al., (1993) Nature Genet. 3:266-272; Madden, T. L. et al., (1996) Meth. Enzymol. 266:131-141; Altschul, S. F. et al., (1997) Nucleic Acids Res. 25:3389-3402; Zhang, J. et al., (1997) Genome Res. 7:649-656. Other traditional BLAST algorithms, such as those available from NCBI BLAST, are also well known to those skilled in the art.

The expressions "cell", "cell line" and "cell culture" as used herein may be used interchangeably, and all such names include progeny. Therefore, the words “transformant” and “transformed cell” include the primary test cells and cultures derived from them, regardless of the number of passages. It should also be kept in mind that due to intentional or accidental mutations, all offspring may not be exactly the same in terms of DNA content. Included are mutant progeny with the same function or biological activity as found in the screening of the original transformed cells. When another concept is meant, it will be clear from the context.

"Polymerase chain reaction" or "PCR" as used herein refers to a procedure or technique in which a trace amount of a specific portion of nucleic acid, RNA and/or DNA, is amplified, as described, for example, in US Pat. No. 4,683,195. In general, sequence information at the end or outside the target region must be obtained so that oligonucleotide primers can be designed; these primers are the same or similar in sequence to the corresponding strands of the template to be amplified. The 5'-terminal nucleotides of the two primers may be identical to the ends of the substance to be amplified. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, as well as cDNA, phage or plasmid sequences transcribed from total cellular RNA, etc. See generally Mullis et al. (1987) Cold Spring Harbor, Symp. Ouant. Biol. 51:263; Erlich ed., (1989) PCR TECHNOLOGY (Stockton Press, N.Y.). PCR as used herein is considered as an example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a test nucleic acid sample, and this method involves the use of known nucleic acids as primers and nucleic acid polymerases to amplify or produce a specific portion of the nucleic acid.

“Isolated” refers to a purified state and in this case means that the designated molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates or other substances, such as cellular debris and growth media. In general, the term “isolated” does not imply the complete absence of these substances or the absence of water, buffers or salts, unless they are present in an amount that significantly interferes with the experimental or therapeutic use of the compound as described herein.

“Optional” or “optional” means that the event or environment described below may, but does not necessarily have to, occur, and the description includes cases in which the event or environment does or does not occur. For example, “optionally containing from 1 to 3 antibody heavy chain variable regions” means that antibody heavy chain variable regions with certain sequences may, but need not necessarily, be present.

"Pharmaceutical composition" means a mixture containing one or more compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, for example, physiological/pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate the administration of a compound into the body, which facilitates the absorption of the active ingredient, causing it to exhibit biological activity.

The term "pharmaceutically acceptable carrier" refers to any inactive substance suitable for use in a formulation for the delivery of antibodies or antigen-binding moieties. The carrier may be an antiadhesive agent, a binder, a coating agent, a disintegrant, a filler or diluent, a preservative (such as an antioxidant, antibacterial or antifungal agent), a sweetener, an absorption retarding agent, a wetting agent, an emulsifier, a buffer, etc. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyols (eg, glycerin, propylene glycol, polyethylene glycol, etc.), dextrose, vegetable oils (eg, olive oil), saline, buffer, buffered saline, and isotonic agents. for example, sugars, polyhydric alcohols, sorbitol and sodium chloride.

In addition, the present invention includes agents for treating diseases associated with cells positive for a target antigen (eg, PD-1), wherein the agents contain an anti-PD-1 antibody or an antigen-binding fragment thereof according to the present invention as an active ingredient.

The PD-1 related disease is not limited in the present invention as long as it is a PD-1 related disease. For example, the therapeutic response induced by a molecule of the present invention can be achieved by binding to human PD-1 and then inhibiting the binding of PD-1 and its ligands PD-L1 and PD-L2, or killing tumor cells overexpressing PD-1. Therefore, when included in preparations and formulations suitable for therapeutic use, the molecules of the present invention are very effective for people having a tumor or cancer, preferably melanoma, colon cancer, breast cancer, lung cancer, stomach cancer, colon cancer, kidney cancer , non-small cell lung cancer, bladder cancer, etc.

In addition, the present invention relates to methods for immunodetection or determination of target antigens (for example, PD-1), reagents for immunodetection or determination of target antigens (for example, PD-1), methods for immunodetection or determination of cells expressing target antigens (for example , PD-1), and diagnostic agents for diagnosing diseases associated with cells positive for a target antigen (e.g., PD-1), which include an antibody or antibody fragment of the present invention that specifically recognizes a target antigen (e.g., human PD-1) and bind to the amino acid sequence of the extracellular region or its three-dimensional structure, being used as an active ingredient.

In the present invention, the method used to detect or measure the amount of a target antigen (eg, PD-1) may be any known method. For example, it includes immunodetection or measurement methods.

Immunodetection or measurement methods are methods for detecting or measuring the amount of antibody or antigen using labeled antigens or antibodies. Examples of immunodetection or measurement methods include radioimmunoassay (RIA), enzyme-linked immunosorbent assay (EIA or ELISA), fluorescence immunoassay (FIA), luminescent immunoassay, Western blotting, physicochemical methods, etc.

The above diseases associated with PD-1 positive target cells can be diagnosed by detecting or measuring cells expressing PD-1 using antibodies or antibody fragments according to the present invention.

Known immunodetection methods, preferably using immunoprecipitation, fluorescent cell staining, immunohistochemical staining, etc., can be used to detect cells expressing the polypeptide. In addition, a fluorescent antibody staining method can be used using the FMAT8100HTS system (Applied Biosystem).

In the present invention, the in vivo sample used to detect or measure a target antigen (eg, PD-1) is not particularly limited as long as it may contain cells expressing the target antigen (eg, PD-1), such as histocytes , blood, plasma, serum, pancreatic juice, urine, feces, interstitial fluid or culture fluid.

Depending on the required diagnostic method, the diagnostic agent containing the monoclonal antibody or fragment thereof according to the present invention may also contain reagents for conducting an antigen-antibody reaction or reagents for detecting such a reaction. Reagents used to carry out the antigen-antibody reaction include buffers, salts, etc. Reagents used for detection include agents commonly used in immunodetection measurement methods, for example, labeled secondary antibodies recognizing a monoclonal antibody, fragment or conjugate thereof, and a substrate corresponding to the label, etc.

Obtaining antigens

1. Construction of antigens:

The human PD-1-IgG1Fc fusion protein is designed and synthesized with the 150 amino acids of the extracellular region of human PD-1 at the N-terminus and the Fc fragment of human IgG1 (hIgG1Fc) at the C-terminus. High purity recombinant PD-1-Fc protein can be obtained after protein A affinity column purification and used to detect anti-PD-1 antibody binding to antigen.

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

Note: The underlined portion represents the signal peptide, the unhighlighted portion represents the extracellular region of human PD-1, and the italicized portion represents hIgG1Fc (signal peptide + extracellular region + hIgG1Fc).

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

MEFGLSWLFLVAILKGVQCPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVGSSDYKDDDDKHHHHHH.

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

MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGA RRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL.

Obtaining antibodies

Antibodies to human PD-1 can be obtained by immunizing mice, and can also be obtained from an anti-human PD-1 phage library obtained by immunizing mice.

The method for producing anti-human PD-1 antibody by immunizing mice is as follows:

1. Immunization: SJL albino mice, female, 6-8 weeks old, and Balb/c albino mice, female, 6-8 weeks old. Conditions of detention: SPF level (free from pathogenic microflora). After acquisition, mice were kept in laboratory conditions for 1 week for adaptation, on a 12/12 hour light/dark cycle at 20–25°C; humidity 40-60%. Mice adapted to the environment were immunized according to different protocols, 6-10 mice in each group. The immunoantigen could be purified recombinant PD-1-IgG1Fc protein (see SEQ ID NO: 1), PD-1-his (see SEQ ID NO: 2) or Jurkat/CHO-PD-1 cells transfected with PD-1 , as an antigen (see SEQ ID NO: 3). One antigen in combination with different immune adjuvants or different types of immunogens could be used for cross-immunization. The immunization site could be the abdominal cavity or the area under the skin on the back, or immunization could be administered alternately at the two sites. TiterMax ^® Gold Adjuvant (hereinafter referred to as Titermax, purchased from Sigma, cat. no. T2684) and Imject Alum Adjuvant (hereinafter referred to as Alum, purchased from Pierce, cat. no. 77161) were used for cross-immunization. Antigen to adjuvant ratio (Titermax) was 1:1, antigen to adjuvant ratio (Alum) was 3:1, 25-50 μg/animal (first immunization), 50 μg/animal (booster immunization) or 1×10 ⁷ cells Jurkat/CHO-PD-1/animal. On day 0, 25-50 μg/animal of emulsified antigen was administered intraperitoneally, once a week or every two weeks after the first immunization, Titermax and Alum were used alternately, for a total of 5-8 times.

2. Cell fusion: Mice with high serum antibody titers were selected for spleen cell fusion. Blood was collected from the eyes of mice 72 hours after booster immunization. Mice were sacrificed by neck dislocation and placed in 75% ethanol for disinfection. Spleen lymphocytes were fused with Sp2/0 myeloma cells (Chinese Academy of Sciences) to generate hybridoma cells using an optimized PEG-mediated fusion procedure. Confluent hybridoma cells were resuspended in complete HAT medium (RPMI-1640 medium containing 20% FBS, 1xHAT and 1xOPI) and aliquoted into 96-well cell culture plates ( ^1x105 /150 μl/well) , incubated at 37 °C, 5% CO ₂ and plated in a total of approximately 10-30 plates. On day 5 after confluence, complete HAT medium was added at 50 μl/well and incubated at 37 °C, 5% CO ₂ . From 7-8 days after confluence, depending on cell growth density, the medium was completely changed at 200 μl/well and incubated at 37 °C, 5% CO ₂ .

3. Screening of hybridoma cells: 7-9 days after fusion, depending on cell growth density, ELISA was performed to detect antibody binding to PD-1, and cells in positive wells were further detected by ELISA to detect blocking of PD-1 binding. 1/PDL1. The media in the positive wells was changed, and the cells were transferred to 24-well plates after reaching the appropriate cell density. Cell lines transferred to 24-well plates were retested and then maintained and subcloned for the first time. Cells positive in the first subcloning screen were retained and a second or third subcloning was performed until single cell progeny clones were obtained. Through multiple fusions, hybridoma cells were obtained that had the effect of blocking the binding of PD-1 and PDL1.

The method for obtaining antibodies to human PD-1 using a phage mouse immune library to human PD-1 was as follows:

1. Construction of a phage mouse immune library to human PD-1: spleens from mice with high serum antibody titers were selected and total RNA was extracted from the tissue using Trizol (Invitrogen, cat. no. 15596-018). cDNA was prepared using the PrimeScript™ II 1st Strand cDNA Synthesis Kit (Takara, cat. no. 6210A) for reverse transcription. Primers for library construction were designed and synthesized according to the IMGT database. Single-chain antibody fragments were generated using three rounds of PCR reactions. Single-chain antibody fragments and modified library construction vector pCantab5E (Amersham Biosciences/GE, cat. no. 27-9400-01) were digested with Sfi1 (NEB, cat. no. R0123L), and purified and isolated using the EZNA ^® Gel Extraction Kit (Omega, cat. no. D2500-02) after electrophoresis. Then T4 DNA ligase (NEB Cat. No. M0202L) was used for ligation at 16 °C for 16-18 hours, and the above kit was used for purification and isolation, and finally elution was carried out with deionized water. 1 μg of ligation product was taken and mixed with 1 vial of competent TG1 (Lucigen, Cat. No. 60502-2) for electrotransformation, and electrotransformation was carried out using an electroporator (Bio Rad Micropulser) with parameters set to 2.5 kV, 200 ohms and 25 µF. The transformation was repeated 10 times. The product was distributed into plates and cultured inverted at 37 °C for 16-18 hours. All colonies were then separated and mixed together, glycerol was added to a final concentration of 15%, and stored at −80 °C for future use.

2. Mouse phage immune library screening for human PD-1: packaged phage immune library for human PD-1 (1×10 ¹² - 1×10 ¹³ ) and 100 μl of streptomycin microbeads (Miltenyi Biotec, Auburn, CA) were added to 1 ml of sodium phosphate buffer containing 2% skim milk (MPBS) and incubated at room temperature for 1 h, placed on a magnetic stand and the supernatant was collected. 10 μg/ml biotinylated human PD-1-ECD-his protein (purchased from Sino Biological) was added to the supernatant and incubated for 1 h at room temperature. Then, 100 μl of streptavidin-coated magnetic beads (pre-incubated with 1 ml of MPBS) was added and incubated for 1 h at room temperature. The product was loaded onto a magnetic sorting rack system and the supernatant was aspirated. Add 1 ml PBST (phosphate buffer containing 0.1% Tween-20) and invert several times. After complete aspiration, fresh wash solution was added, repeated 11 times to remove unbound antibody fragments, and 0.5 mL of elution solution (50 μL of 10 mg/mL trypsin stock solution added in 450 μL PBS) was added. It was shaken for 15 min at room temperature, placed on a magnetic stand, and the supernatant was aspirated into a new Eppendorf tube. TG1 was sown in 2YT medium and propagated to a bacterial culture density of OD600 = 0.4. 1.75 ml of TG1 (OD600 = 0.4) was added to each tube and 250 µl of eluted phage (phage) was added, incubated in a water bath at 37 °C for 30 minutes and distributed onto gradient dilution plates to check the titer. The remaining TG1 solution was centrifuged, distributed into plates, and incubated overnight at 37 °C.

The murine phage immune library was subjected to 2-3 rounds of screening using biotinylated human PD-1-ECD-his antigen, with MACS screening (magnetic streptomycin beads, Invitrogen), and finally monoclonal antibodies capable of binding to PD-1 and capable of blocking the binding of PD-1 to PD-L1. The sequences of the variable region of these antibodies were obtained.

Purification of recombinant antigenic proteins/antibodies

1. Separation and purification of hybridoma supernatant/Protein G affinity chromatography:

Protein G affinity chromatography was the preferred method for purification of mouse hybridoma supernatant. The hybridoma culture was centrifuged to collect the supernatant and, based on the volume of the supernatant, 10-15% volume of 1 M Tris-HCl (pH 8.0-8.5) was added to adjust Supernatant pH. For the protein G column, 6 M guanidine hydrochloride was used to wash 3-5 column volumes, and then pure water was used to wash 3-5 column volumes; a buffer system such as 1×PBS (pH 7.4) was used as an equilibration buffer to equilibrate the column with 3-5 column volumes. The cell supernatant was loaded and bound using a low flow rate, which was controlled so that the retention time was about 1 minute or more; 1×PBS (pH 7.4) was used to wash the chromatography column with 3-5 column volumes until the UV absorbance decreased to the original level. A 0.1 M acetic acid/sodium acetate buffer (pH 3.0) was used to elute the sample, the elution peaks were collected according to UV detection, and 1 M Tris-HCl (pH 8.0) was used to quickly adjust the pH of the eluted product up to 5-6 for temporary storage. For the eluted product, solution replacement can be accomplished by methods well known to those skilled in the art, such as using ultrafiltration tubes and concentrating and replacing the solution with the required buffer system, or using a molecular exclusion agent such as G-25 to desalt and replace with required buffer system, or the use of high-resolution molecular exclusion columns such as Superdex 200 to remove components aggregated in the eluting product to improve sample purity.

2. Purification of proteins or antibodies using protein A affinity chromatography:

First, the supernatant of a cell culture expressing an antigenic protein or antibody was centrifuged at high speed to collect the supernatant. For the Protein A affinity column, 6 M guanidine hydrochloride was used to wash 3-5 column volumes, and then pure water was used to wash 3-5 column volumes. A buffer system such as 1×PBS (pH 7.4) was used as an equilibration buffer to equilibrate the chromatography column in 3-5 column volumes. The cell supernatant was loaded and bound using a low flow rate, which was controlled so that the retention time was about 1 minute or more. Once binding was complete, 1×PBS (pH 7.4) was used to wash the chromatography column with 3-5 column volumes until the UV absorbance decreased to baseline levels. A 0.1 M acetic acid/sodium acetate buffer (pH 3.0-3.5) was used to elute the sample, the elution peaks were collected according to UV detection, and 1 M Tris-HCl (pH 8.0) was used to quickly bringing the pH of the eluted product to 5-6 for temporary storage. For the eluted product, solution replacement can be accomplished by methods well known to those skilled in the art, such as using ultrafiltration tubes and concentrating and replacing the solution with the required buffer system, or using a molecular exclusion agent, such as G-25, to desalt and replace with required buffer system, or the use of high-resolution molecular exclusion columns such as Superdex 200 to remove components aggregated in the eluting product to improve sample purity.

EXAMPLES

The present invention is further described below in conjunction with examples, but these examples do not limit the scope of the present invention. Experimental methods for which conditions are not specifically mentioned in the examples of the present invention were generally carried out under normal conditions, such as those described in Antibodies: A Laboratory Manual and Molecular Cloning: A Laboratory Manual, Cold Spring Harbor; or under conditions recommended by the manufacturer of the raw material or product. Reagents whose sources are not specifically stated are common reagents available on the market.

Example 1: Production of Mouse Anti-Human PD-1 Antibodies

Mouse anti-human PD-1 antibodies obtained by the above method were subjected to antigen binding experiments and screened to obtain several antibody strains with good activity, which included M23, M32 and M33. Single cell progeny clones were expanded and cultured, RNA was extracted, and degenerate mouse Ig primers were used to perform reverse transcription-amplification (RT-PCR) to obtain the antibody variable region sequence. The variable region sequence of the mouse antibody was linked to the constant region sequence of the human antibody. The chimeric antibody from the mouse monoclonal antibody was cloned and recombinantly expressed, and in vitro activity experiments were performed to confirm that the resulting monoclonal antibody variable region sequence was correct.

The sequences of the variable regions of murine antibodies M23, M32 and M33 are determined as follows:

Mouse Antibody M23 Heavy Chain Variable Region (SEQ ID NO: 4):

Mouse Antibody M23 Light Chain Variable Region (SEQ ID NO: 5):

Mouse Antibody Heavy Chain Variable Region M32 (SEQ ID NO: 6):

Mouse Antibody M32 Light Chain Variable Region (SEQ ID NO: 7):

Mouse Antibody Heavy Chain Variable Region M33 (SEQ ID NO: 19):

Mouse Antibody M33 Light Chain Variable Region (SEQ ID NO: 20):

Note: In the variable heavy chain and variable light chain sequences of the above antibodies, the CDR sequences defined according to the Kabat numbering system are underlined in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

Table 4. Sequences of the heavy and light chain CDR regions of mouse antibodies M23, M32 and M33

Antibody name Heavy chain Light chain M23 HCDR1 DYEMH
(SEQ ID NO: 8) LCDR1 RSSQSLVHSNGNTYLE
(SEQ ID NO: 11) HCDR2 LIDPETGGTVYNQKFKD
(SEQ ID NO: 9) LCDR2 KVSNRFS
(SEQ ID NO: 12) HCDR3 ERFSYYGSTSDWYFDV
(SEQ ID NO: 10) LCDR3 FQGSHVPYT
(SEQ ID NO: 13) M32 HCDR1 DYEIH
(SEQ ID NO: 14) LCDR1 RSSQSIVHSNGNTYLE
(SEQ ID NO: 17) HCDR2 LFDPETGGIVYNQKFKG
(SEQ ID NO: 15) LCDR2 KVSNRFS
(SEQ ID NO: 12) HCDR3 EGYNRDWYFDV
(SEQ ID NO: 16) LCDR3 FQGSHVPYA
(SEQ ID NO: 18) M33 HCDR1 SYAMS
(SEQ ID NO: 21) LCDR1 RASQDINNFLN
(SEQ ID NO: 24) HCDR2 TISGGGVDTYYQDNVQG
(SEQ ID NO: 22) LCDR2 YTSSLHS
(SEQ ID NO: 25) HCDR3 PYGHGYFDV
(SEQ ID NO: 23) LCDR3 QQGNTLPWT
(SEQ ID NO: 26)

Note: Antibody CDR sequences in the table are defined according to the Kabat numbering system.

Example 2: Humanization of Anti-Human PD-1 Monoclonal Antibodies

By alignment with the IMGT database of human antibody heavy and light chain variable region germline genes and analysis in MOE software, human germline heavy and light chain variable region genes with high identity to the light and heavy chain sequences M23, M32 and M33. The CDRs of these 3 mouse antibodies were grafted onto the corresponding human antibody scaffolds to construct corresponding humanized antibodies from them, respectively.

1. Humanization of mouse M23 antibody

1.1 Selection of scaffold for humanization of murine antibody M23

The light chain humanization frameworks for the murine M23 antibody are IGKV2-40*01 and IGKJ4*01, and the heavy chain humanization frameworks are IGHV1-69*02 and IGHJ6*01. The sequences of the variable regions after humanization were as follows (CDR sequences are underlined):

Hu23VH-CDR-grafted: (SEQ ID NO: 27)

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

1.2 Humanization scaffold selection and design of murine M23 antibody back mutations

Table 5. Back mutations of humanized antibodies based on the murine M23 antibody

VL VH Hu23VL1 Grafted Hu23VH1 Grafted Hu23VL2 I2G Hu23VH2 G27Y I69L Hu23VH3 G27Y M48I V67T I69L L82F Hu23VH4 G27Y M48I V67T I69L L82F A93T

Note: “grafted” means that the murine antibody CDRs are grafted into the human germline FR region sequence. Amino acid residues are defined and annotated according to the Kabat numbering system, for example, I2G means that the I at position 2 of the Kabat numbering is mutated back to G according to the Kabat numbering system.

The light/heavy chain variable region sequences of the M23-based humanized antibodies are as follows:

> Hu23VL1 (same as Hu23VL-CDR-grafted): (SEQ ID NO: 28)

DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIK.

> Hu23VL2 (SEQ ID NO: 29)

DGVMTQTPLSLPVTPGEPASISCRSSQSLVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIK.

> Hu23VH1 (same as Hu23VH-CDR-grafted): (SEQ ID NO: 27)

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSDYEMHWVRQAPGQGLEWMGLIDPETGGTVYNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARERFSYYGSTSDWYFDVWGQGTTVTVSS.

> Hu23VH2 (SEQ ID NO: 30)

EVQLVQSGAEVKKPGSSVKVSCKASGYTFSDYEMHWVRQAPGQGLEWMGLIDPETGGTVYNQKFKDRVTLTADKSTSTAYMELSSLRSEDTAVYYCARERFSYYGSTSDWYFDVWGQGTTVTVSS.

> Hu23VH3 (SEQ ID NO: 31)

EVQLVQSGAEVKKPGSSVKVSCKASGYTFSDYEMHWVRQAPGQGLEWIGLIDPETGGTVYNQKFKDRTTLTADKSTSTAYMEFSSLRSEDTAVYYCARERFSYYGSTSDWYFDVWGQGTTVTVSS.

> Hu23VH4 (SEQ ID NO: 32)

EVQLVQSGAEVKKPGSSVKVSCKASGYTFSDYEMHWVRQAPGQGLEWIGLIDPETGGTVYNQKFKDRTTLTADKSTSTAYMEFSSLRSEDTAVYYCTRERFSYYGSTSDWYFDVWGQGTTVTVSS.

1.3 Combination of humanized murine antibody M23 sequences

Antibodies and combinations of their variable regions obtained by humanization of the murine M23 antibody are shown in the table below.

Table 6. Humanized Antibody Hu23 Variable Region Combinations

VL
VH Hu23VL1 Hu23VL2 Hu23VH1 Hu23-1 Hu23-5 Hu23VH2 Hu23-2 Hu23-6 Hu23VH3 Hu23-3 Hu23-7 Hu23VH4 Hu23-4 Hu23-8

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

Combinations of antibody light/heavy chain variable regions (eg, Hu23-1) shown in the table above can be linked to antibody light/heavy chain constant regions to form full-length antibodies, respectively; Unless otherwise specified herein, when a full-length antibody is formed, the light chain variable region binds to the kappa chain constant region set forth in SEQ ID NO: 73 to form the antibody light chain, and the heavy chain variable region binds to the IgG4 heavy chain constant region. -AA represented in SEQ ID NO: 72, or with the heavy chain constant region of IgG4-P presented in SEQ ID NO: 79 to form an antibody heavy chain, and a table name referring to the combination of the antibody light/heavy chain variable regions (e.g. Hu23-1), with the suffix ". IgG4AA" means a full-length antibody formed by ligation with the heavy chain constant region of IgG4AA, with the suffix ". IgG4P" is a full-length antibody formed by ligation with the constant region of the heavy chain of IgG4-P. For example, "Hu23-1.IgG4AA" means a full-length antibody formed by binding a heavy chain formed by binding the heavy chain variable region of Hu23VH1 and the heavy chain constant region of IgG4-AA shown in SEQ ID NO: 72, and a light chain formed by binding the Hu23VL1 light chain variable region and the kappa chain constant region shown in SEQ ID NO: 73. “Hu23-1.IgG4P” means a full-length antibody formed by binding a heavy chain formed by binding a Hu23VH1 heavy chain variable region and a heavy chain constant region an IgG4-P chain shown in SEQ ID NO: 79, and a light chain formed by linking the Hu23VL1 light chain variable region and the kappa chain constant region shown in SEQ ID NO: 73.

2. Humanization of mouse antibody M32

2.1 Selection of scaffold for humanization of murine antibody M32

The light chain backbones for humanization of the murine antibody M32 are IGKV2-40*01 and IGKJ4*01, and the heavy chain backbones for humanization are IGHV1-69*02 and IGHJ6*01. The sequences of the humanized variable regions were as follows (CDR sequences are underlined):

Hu32VH-CDR-grafted: (SEQ ID NO: 33) IGHV1-69*02 and IGHJ6*01

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

2.2 Humanization scaffold selection and design of murine M32 antibody back mutations

Table 7. Back mutations of humanized antibodies based on the murine M32 antibody

VL VH Hu32VL1 Grafted Hu32VH1 Grafted Hu32VL2 I2V Hu32VH2 G27F, I69L, A93T Hu32VH3 G26D, G27F, I69L, A93T Hu32VH4 G27F, M48I, V67A, I69L, L82F, A93T Hu32VH5 G26D, G27F, S30T, M48I, V67A, I69L, L82F, A93T Hu32VH6 G26D, G27F, S30T, R38K, Q43H, M48I, R66K, V67A, I69L, L82F, A93T

Note: “grafted” means that the murine antibody CDRs are grafted into the human germline FR region sequence. Amino acid residues are defined and annotated according to the Kabat numbering system, for example, I2V means that the I at position 2 of the Kabat numbering is mutated back to V according to the Kabat numbering system.

The sequences of the light and heavy chain variable regions of the humanized antibodies based on the murine M32 antibody are as follows:

> Hu32VL1 (same as Hu32VL-CDR-grafted): (SEQ ID NO: 34)

DIVMTQTPLSLPVTPGEPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYAFGGGTKVEIK.

> Hu32VL2 (SEQ ID NO: 35)

DVVMTQTPLSLPVTPGEPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYAFGGGTKVEIK.

> Hu32VH1 (same as Hu23VH-CDR-grafted): (SEQ ID NO: 33)

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSDYEIHWVRQAPGQGLEWMGLFDPETGGIVYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREGYNRDWYFDVWGQGTTVTVSS.

> Hu32VH2 (SEQ ID NO: 36)

EVQLVQSGAEVKKPGSSVKVSCKASGFTFSDYEIHWVRQAPGQGLEWMGLFDPETGGIVYNQKFKGRVTLTADKSTSTAYMELSSLRSEDTAVYYCTREGYNRDWYFDVWGQGTTVTVSS.

> Hu32VH3 (SEQ ID NO: 37)

EVQLVQSGAEVKKPGSSVKVSCKASDFTFSDYEIHWVRQAPGQGLEWMGLFDPETGGIVYNQKFKGRVTLTADKSTSTAYMELSSLRSEDTAVYYCTREGYNRDWYFDVWGQGTTVTVSS.

> Hu32VH4 (SEQ ID NO: 38)

EVQLVQSGAEVKKPGSSVKVSCKASGFTFSDYEIHWVRQAPGQGLEWIGLFDPETGGIVYNQKFKGRATLTADKSTSTAYMEFSSLRSEDTAVYYCTREGYNRDWYFDVWGQGTTVTVSS.

> Hu32VH5 (SEQ ID NO: 39)

EVQLVQSGAEVKKPGSSVKVSCKASDFTFTDYEIHWVRQAPGQGLEWIGLFDPETGGIVYNQKFKGRATLTADKSTSTAYMEFSSLRSEDTAVYYCTREGYNRDWYFDVWGQGTTVTVSS.

> Hu32VH6 (SEQ ID NO: 40)

EVQLVQSGAEVKKPGSSVKVSCKASDFTFTDYEIHWVKQAPGHGLEWIGLFDPETGGIVYNQKFKGKATLTADKSTSTAYMEFSSLRSEDTAVYYCTREGYNRDWYFDVWGQGTTVTVSS.

2.3 Combination of humanized murine antibody M32 sequences

Antibodies and combinations of their variable regions obtained by humanization of the murine M32 antibody.

Table 8. Humanized Antibody Hu32 Light/Heavy Chain Variable Region Combinations

VL
VH Hu32VL1 Hu32VL2 Hu32VH1 Hu32-1 Hu32-7 Hu32VH2 Hu32-2 Hu32-8 Hu32VH3 Hu32-3 Hu32-9 Hu32VH4 Hu32-4 Hu32-10 Hu32VH5 Hu32-5 Hu32-11 Hu32VH6 Hu32-6 Hu32-12

Note: For example, in the table, "Hu32-1" refers to an antibody with the combination of the Hu32VL1 light chain variable region and the Hu32VH1 heavy chain variable region, and so on.

Combinations of antibody light/heavy chain variable regions (eg, Hu32-1) shown in the table above can be linked to antibody light/heavy chain constant regions to form full-length antibodies, respectively; Unless otherwise specified herein, when a full-length antibody is formed, the light chain variable region binds to the kappa chain constant region set forth in SEQ ID NO: 73 to form the antibody light chain, and the heavy chain variable region binds to the IgG4 heavy chain constant region. -AA represented in SEQ ID NO: 72, or with the heavy chain constant region of IgG4-P presented in SEQ ID NO: 79 to form an antibody heavy chain, and a table name referring to the combination of the antibody light/heavy chain variable regions (e.g. Hu32-1), with the suffix ". IgG4AA" means a full-length antibody formed by ligation with the heavy chain constant region of IgG4AA, with the suffix ". IgG4P" is a full-length antibody formed by ligation with the constant region of the heavy chain of IgG4-P. For example, "Hu32-1.IgG4AA" means a full-length antibody formed by binding a heavy chain formed by binding the heavy chain variable region of Hu32VH1 and the heavy chain constant region of IgG4-AA shown in SEQ ID NO: 72, and a light chain formed by binding the Hu32VL1 light chain variable region and the kappa chain constant region shown in SEQ ID NO: 73. "Hu32-1.IgG4P" means a full-length antibody formed by binding a heavy chain formed by binding the Hu32VH1 heavy chain variable region and a heavy chain constant region the IgG4-P chain shown in SEQ ID NO: 79, and a light chain formed by linking the Hu32VL1 light chain variable region and the kappa chain constant region shown in SEQ ID NO: 73.

3. Humanization of mouse M33 antibody

3.1 Selection of scaffold for humanization of murine antibody M33

The light chain backbones for humanization of the murine antibody M33 are IGKV1-39*01 and IGKJ4*01, and the heavy chain backbones for humanization are IGHV3-7 and IGHJ6*01. The sequences of the humanized variable regions were as follows (CDR sequences are underlined):

Hu33VH-CDR-grafted (SEQ ID NO: 41):

Hu33VL-CDR-grafted (SEQ ID NO: 42):

3.2 Humanization scaffold selection and design of murine M33 antibody back mutations

Table 9. Back mutations of humanized antibodies based on the murine M33 antibody

VL VH Hu33VL1 Grafted Hu33VH1 Grafted Hu33VL2 F71Y Hu33VH2 R94S Hu33VL3 K42G P44V F71Y Hu33VH3 E1K R94S

Note: “grafted” means that the murine antibody CDRs are grafted into the human germline FR region sequence. Amino acid residues are defined and annotated according to the Kabat numbering system, for example, F71Y means that the F at position 71 of the Kabat numbering is mutated back to Y according to the Kabat numbering system.

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

> Hu33VL1 (same as Hu33VL-CDR-grafted): (SEQ ID NO: 42)

DIQMTQSPSSLSASVGDRVTITCRASQDINNFLNWYQQKPGKAPKLLIYYTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTFGGGTKVEIK.

> Hu33VL2 (SEQ ID NO: 43)

DIQMTQSPSSLSASVGDRVTITCRASQDINNFLNWYQQKPGKAPKLLIYYTSSLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGGGTKVEIK.

> Hu33VL3 (SEQ ID NO: 44)

DIQMTQSPSSLSASVGDRVTITCRASQDINNFLNWYQQKPGGAVKLLIYYTSSLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGGGTKVEIK.

> Hu33VH1 (same as Hu33VH-CDR-grafted): (SEQ ID NO: 41)

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVATISGGGVDTYYQDNVQGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPYGHGYFDVWGQGTTVTVSS.

> Hu33VH2 (SEQ ID NO: 45)

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVATISGGGVDTYYQDNVQGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASPYGHGYFDVWGQGTTVTVSS.

> Hu33VH3 (SEQ ID NO: 46)

KVQLVESGGGLVQPGGSLRLSCAASGFFSSYAMSWVRQAPGKGLEWVATISGGGVDTYYQDNVQGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASPYGHGYFDVWGQGTTVTVSS.

3.3 Combination of humanized murine M33 antibody sequences

Table 10. Humanized Antibody Light and Heavy Chain Variable Region Combinations

VL
VH Hu33VL1 Hu33VL2 Hu33VL3 Hu33VH1 Hu33-1 Hu33-4 Hu33-7 Hu33VH2 Hu33-2 Hu33-5 Hu33-8 Hu33VH3 Hu33-3 Hu33-6 Hu33-9

Note: For example, in the table, "Hu33-6" refers to an antibody with the combination of the light chain variable region Hu33VL2 and the heavy chain variable region Hu33VH3, and so on.

Combinations of antibody light/heavy chain variable regions (eg, Hu33-6) presented in the table above can be linked to antibody light/heavy chain constant regions to form full-length antibodies, respectively; Unless otherwise specified herein, when a full-length antibody is formed, the light chain variable region binds to the kappa chain constant region set forth in SEQ ID NO: 73 to form the antibody light chain, and the heavy chain variable region binds to the IgG4 heavy chain constant region. -AA represented in SEQ ID NO: 72, or with the heavy chain constant region of IgG4-P presented in SEQ ID NO: 79 to form an antibody heavy chain, and a table name referring to the combination of the antibody light/heavy chain variable regions (e.g. Hu33-6), with the suffix ". "IgG4AA" means a full-length antibody formed by ligation with the heavy chain constant region of IgG4AA, with the suffix ".IgG4P" means a full-length antibody formed by ligation with the heavy chain constant region of IgG4-P. For example, "Hu32-6.IgG4AA" means a full-length antibody formed by binding a heavy chain formed by binding the heavy chain variable region of Hu33VH3 and the heavy chain constant region of IgG4-AA shown in SEQ ID NO: 72, and a light chain formed by binding the Hu33VL2 light chain variable region and the kappa chain constant region shown in SEQ ID NO: 73. “Hu33-6.IgG4P” means a full-length antibody formed by binding a heavy chain formed by binding a Hu33VH3 heavy chain variable region and a heavy chain constant region the IgG4-P chain shown in SEQ ID NO: 79, and a light chain formed by linking the Hu33VL2 light chain variable region and the kappa chain constant region shown in SEQ ID NO: 73.

4. Humanized antibody mutants

4.1 Mutant antibodies based on the humanized antibody Hu23

Through computer modeling, amino acids at specific light chain LCDR1 sites (SEQ ID NO: 11) of the humanized Hu23 antibody were subjected to site-directed mutations, and the specific mutations are listed in Table 11:

Table 11. Hu23 light chain LCDR1 mutant sequences:

Name Sequence (SEQ ID NO:) Hu23LCDR1(N28Q) RSSQSLVHSQGNTYLE
(SEQ ID NO: 47) Hu23LCDR1(N28L) RSSQSLVHSLGNTYLE
(SEQ ID NO: 48) Hu23LCDR1(N28T) RSSQSLVHSTGNTYLE
(SEQ ID NO: 49) Hu23LCDR1(N28D) RSSQSLVHSDGNTYLE
(SEQ ID NO: 50) Hu23LCDR1(G29A) RSSQSLVHSNANTYLE
(SEQ ID NO: 51) Hu23LCDR1(G29V) RSSQSLVHSNVNTYLE
(SEQ ID NO: 52)

Note: Hu23LCDR1 (N28Q) means an LCDR1 mutation sequence in which the N at position 28 according to the Kabat numbering system of the light chain variable region of Hu23VL1 or Hu23VL2 of the humanized antibody Hu23 is mutated to Q, Hu23LCDR1 (G29A) means an LCDR1 mutation sequence in which The G at position 29 according to the Kabat numbering system of the Hu23VL1 or Hu23VL2 light chain variable region of the humanized antibody Hu23 is mutated to A (CDRs defined using the Kabat numbering system).

The light chain variable region sequences of the humanized antibody Hu23 after introducing mutations in LCDR1 are as follows:

> The sequence of Hu23VL1(N28Q) is:

DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSQGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIK (SEQ ID NO: 53)

> The sequence of Hu23VL1(N28L) is:

DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSLGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIK (SEQ ID NO: 54)

> The sequence of Hu23VL1(N28T) is:

DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSTGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIK (SEQ ID NO: 55)

> The sequence of Hu23VL1(N28D) is:

DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSDGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIK (SEQ ID NO: 56)

> The sequence of Hu23VL1(G29A) is:

DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSNANTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIK (SEQ ID NO: 57)

> The sequence Hu23VL1(G29V) is:

DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSNVNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIK (SEQ ID NO: 58)

> The sequence Hu23VL2(N28Q) is:

DGVMTQTPLSLPVTPGEPASISCRSSQSLVHSQGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIK (SEQ ID NO: 59)

> The sequence Hu23VL2(N28L) is:

DGVMTQTPLSLPVTPGEPASISCRSSQSLVHSLGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIK (SEQ ID NO: 60)

> The sequence Hu23VL2(N28T) is:

DGVMTQTPLSLPVTPGEPASISCRSSQSLVHSTGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIK (SEQ ID NO: 61)

> The sequence Hu23VL2(N28D) is:

DGVMTQTPLSLPVTPGEPASISCRSSQSLVHSDGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIK (SEQ ID NO: 62)

> The sequence Hu23VL2(G29A) is:

DGVMTQTPLSLPVTPGEPASISCRSSQSLVHSNANTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIK (SEQ ID NO: 63)

> The sequence Hu23VL2(G29V) is:

DGVMTQTPLSLPVTPGEPASISCRSSQSLVHSNVNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIK (SEQ ID NO: 64)

Table 12. Humanized Antibody Hu23 Light and Heavy Chain Variable Region Combinations

VH
VL Hu23VH1 Hu23VH2 Hu23VH3 Hu23VH4 Hu23VL1(N28Q) Hu23-9 Hu23-21 Hu23-33 Hu23-45 Hu23VL1(N28L) Hu23-10 Hu23-22 Hu23-34 Hu23-46 Hu23VL1(N28T) Hu23-11 Hu23-23 Hu23-35 Hu23-47 Hu23VL1(N28D) Hu23-12 Hu23-24 Hu23-36 Hu23-48 Hu23VL1(G29A) Hu23-13 Hu23-25 Hu23-37 Hu23-49 Hu23VL1(G29V) Hu23-14 Hu23-26 Hu23-38 Hu23-50 Hu23VL2(N28Q) Hu23-15 Hu23-27 Hu23-39 Hu23-51 Hu23VL2(N28L) Hu23-16 Hu23-28 Hu23-40 Hu23-52 Hu23VL2(N28T) Hu23-17 Hu23-29 Hu23-41 Hu23-53 Hu23VL2(N28D) Hu23-18 Hu23-30 Hu23-42 Hu23-54 Hu23VL2(G29A) Hu23-19 Hu23-31 Hu23-43 Hu23-55 Hu23VL2(G29V) Hu23-20 Hu23-32 Hu23-44 Hu23-56

Note: For example, in the table, "Hu23-11" refers to an antibody with the combination of the light chain variable region Hu23VL1(N28T) and the heavy chain variable region Hu23VH1, and so on.

Combinations of antibody light/heavy chain variable regions (eg, Hu23-11) shown in the table above can be linked to antibody light/heavy chain constant regions to form full-length antibodies, respectively; Unless otherwise specified herein, when a full-length antibody is formed, the light chain variable region binds to the kappa chain constant region set forth in SEQ ID NO: 73 to form the antibody light chain, and the heavy chain variable region binds to the IgG4 heavy chain constant region. -AA represented in SEQ ID NO: 72, or with the heavy chain constant region of IgG4-P presented in SEQ ID NO: 79 to form an antibody heavy chain, and a table name referring to the combination of the antibody light/heavy chain variable regions (e.g. Hu23-11), with the suffix ". IgG4AA" means a full-length antibody formed by ligation with the heavy chain constant region of IgG4AA, with the suffix ". IgG4P" is a full-length antibody formed by ligation with the constant region of the heavy chain of IgG4-P. For example, "Hu23-11.IgG4AA" means a full-length antibody formed by binding a heavy chain formed by binding the heavy chain variable region of Hu23VH1 and the heavy chain constant region of IgG4-AA shown in SEQ ID NO: 72, and a light chain formed by binding the light chain variable region of Hu23VL1(N28T) and the kappa chain constant region shown in SEQ ID NO: 73. “Hu23-11.IgG4P” means the full-length antibody formed by binding the heavy chain formed by binding the heavy chain variable region of Hu23VH1 and an IgG4-P heavy chain constant region shown in SEQ ID NO: 79, and a light chain formed by linking the light chain variable region of Hu23VL1(N28T) and the kappa chain constant region shown in SEQ ID NO: 73.

The experimental results showed that after site-directed mutations, all humanized antibodies Hu23LCDR1 (N28Q), Hu23LCDR1 (N28L), Hu23LCDR1 (N28T), Hu23LCDR1 (N28D), Hu23LCDR1 (G29A) and Hu23LCDR1 (G29V) retained the ability to bind to PD-1 ( table 16).

4.2 Mutant antibodies based on the humanized Hu32 antibody

The M23-derived Hu23 humanized antibody series and the M32-derived Hu32 humanized antibody series had high sequence identity in sequence analysis. The Hu23 light chain variable region and the Hu32 heavy chain variable region were combined into new combinations of light and heavy chain variable regions. The experimental results showed that humanized antibodies containing new combinations of light and heavy chain variable regions retain the ability to bind to the PD-1 antigen (Table 16).

Table 13. General formulas of consensus sequences of the variable region of antibodies Hu32 and Hu23

Table 14. Combinations of Hu32 heavy chain variable regions and Hu23 light chain variable regions

VH
VL Hu32VH1 Hu32VH2 Hu32VH3 Hu32VH4 Hu32VH5 Hu32VH6 Hu23VL1(N28Q) Hu32a-13 Hu32a-27 Hu32a-41 Hu32a-55 Hu32a-69 Hu32a-83 Hu23VL1(N28L) Hu32a-14 Hu32a-28 Hu32a-42 Hu32a-56 Hu32a-70 Hu32a-84 Hu23VL1(N28T) Hu32a-15 Hu32a-29 Hu32a-43 Hu32a-57 Hu32a-71 Hu32a-85 Hu23VL1(N28D) Hu32a-16 Hu32a-30 Hu32a-44 Hu32a-58 Hu32a-72 Hu32a-86 Hu23VL1(G29A) Hu32a-17 Hu32a-31 Hu32a-45 Hu32a-59 Hu32a-73 Hu32a-87 Hu23VL1(G29V) Hu32a-18 Hu32a-32 Hu32a-46 Hu32a-60 Hu32a-74 Hu32a-88 Hu23VL2(N28Q) Hu32a-19 Hu32a-33 Hu32a-47 Hu32a-61 Hu32a-75 Hu32a-89 Hu23VL2(N28L) Hu32a-20 Hu32a-34 Hu32a-48 Hu32a-62 Hu32a-76 Hu32a-90 Hu23VL2(N28T) Hu32a-21 Hu32a-35 Hu32a-49 Hu32a-63 Hu32a-77 Hu32a-91 Hu23VL2(N28D) Hu32a-22 Hu32a-36 Hu32a-50 Hu32a-64 Hu32a-78 Hu32a-92 Hu23VL2(G29A) Hu32a-23 Hu32a-37 Hu32a-51 Hu32a-65 Hu32a-79 Hu32a-93 Hu23VL2(G29V) Hu32a-24 Hu32a-38 Hu32a-52 Hu32a-66 Hu32a-80 Hu32a-94 Hu23VL1 Hu32a-25 Hu32a-39 Hu32a-53 Hu32a-67 Hu32a-81 Hu32a-95 Hu23VL2 Hu32a-26 Hu32a-40 Hu32a-54 Hu32a-68 Hu32a-82 Hu32a-96

Note: For example, in the table, "Hu32a-85" refers to the light chain variable region antibody Hu23VL1(N28T) and the heavy chain variable region Hu32VH6, and so on.

Combinations of antibody light/heavy chain variable regions (eg, Hu32a-85) shown in the table above can be linked to antibody light/heavy chain constant regions to form full-length antibodies, respectively; Unless otherwise specified herein, when a full-length antibody is formed, the light chain variable region binds to the kappa chain constant region set forth in SEQ ID NO: 73 to form the antibody light chain, and the heavy chain variable region binds to the IgG4 heavy chain constant region. -AA represented in SEQ ID NO: 72, or with the heavy chain constant region of IgG4-P presented in SEQ ID NO: 79 to form an antibody heavy chain, and a table name referring to the combination of the antibody light/heavy chain variable regions (e.g. Hu32a-85), with the suffix ". IgG4AA" means a full-length antibody formed by ligation with the heavy chain constant region of IgG4AA, with the suffix ". IgG4P" is a full-length antibody formed by ligation with the constant region of the heavy chain of IgG4-P. For example, "Hu32a-85.IgG4AA" means a full-length antibody formed by linking a heavy chain formed by linking the heavy chain variable region of Hu32VH6 and the heavy chain constant region of IgG4-AA shown in SEQ ID NO: 72, and a light chain formed by binding the light chain variable region of Hu23VL1(N28T) and the kappa chain constant region shown in SEQ ID NO: 73. “Hu32a-85.IgG4P” means the full-length antibody formed by binding the heavy chain formed by binding the heavy chain variable region of Hu32VH6 and an IgG4-P heavy chain constant region shown in SEQ ID NO: 79, and a light chain formed by linking the light chain variable region of Hu23VL1(N28T) and the kappa chain constant region shown in SEQ ID NO: 73.

Table 15. Combinations of Hu32 heavy chain variable regions and Hu23 light chain variable regions

VH
VL Hu23VH1 Hu23VH2 Hu23VH3 Hu32VL1 Hu23a-57 Hu23a-59 Hu23a-61 Hu32VL2 Hu23a-58 Hu23a-60 Hu23a-62

Note: For example, in the table, "Hu23a-57" refers to an antibody with the combination of the light chain variable region Hu32VL1 and the heavy chain variable region Hu23VH1, and so on.

Combinations of antibody light/heavy chain variable regions (eg, Hu23a-57) shown in the table above can be linked to antibody light/heavy chain constant regions to form full-length antibodies, respectively; Unless otherwise specified herein, when a full-length antibody is formed, the light chain variable region binds to the kappa chain constant region set forth in SEQ ID NO: 73 to form the antibody light chain, and the heavy chain variable region binds to the IgG4 heavy chain constant region. -AA represented in SEQ ID NO: 72, or with the IgG4-P heavy chain constant region presented in SEQ ID NO: 79 to form an antibody heavy chain, and a table name referring to the combination of the antibody light/heavy chain variable regions (e.g. Hu32a-85), with the suffix ". IgG4AA" means a full-length antibody formed by ligation with the heavy chain constant region of IgG4AA, with the suffix ". IgG4P" is a full-length antibody formed by ligation with the constant region of the heavy chain of IgG4-P. For example, "Hu23a-57.IgG4AA" means a full-length antibody formed by linking a heavy chain formed by linking the heavy chain variable region of Hu23VH1 and the heavy chain constant region of IgG4-AA shown in SEQ ID NO: 72, and a light chain formed by binding the Hu32VL1 light chain variable region and the kappa chain constant region shown in SEQ ID NO: 73. “Hu23a-57.IgG4P” means a full-length antibody formed by binding a heavy chain formed by binding a Hu23VH1 heavy chain variable region and a heavy chain constant region an IgG4-P chain shown in SEQ ID NO: 79, and a light chain formed by linking the Hu32VL1 light chain variable region and the kappa chain constant region shown in SEQ ID NO: 73.

5. Screening for humanized antibodies

Determination of the affinity of various humanized antibodies was performed using Biacore (see method in Test Example 3), and the results are shown in Table 16. The results showed that various humanized antibodies retain the ability to bind to PD-1, and some humanized antibodies have an affinity even substantially similar to the affinity of the mouse antibodies from which they are derived.

Table 16. Affinity of humanized antibody Hu23 to human PD-1

Antibody ka (1/Ms) kd (1/s) KD(M) M23 4.57E+04 1.32E-04 2.89E-09 Hu23-1.IgG4AA 3.43E+04 1.10E-04 3.20E-09 Hu23-5.IgG4AA 2.99E+04 1.14E-04 3.82E-09 Hu23-2.IgG4AA 2.74E+04 1.61E-04 5.86E-09 Hu23-6.IgG4AA 2.79E+04 1.55E-04 5.57E-09 Hu23-3.IgG4AA 2.93E+04 1.60E-04 5.46E-09 Hu23-7.IgG4AA 2.77E+04 1.66E-04 5.97E-09 Hu23-4.IgG4AA 4.24E+04 1.44E-04 3.39E-09 Hu23-8.IgG4AA 4.11E+04 1.47E-04 3.56E-09 Hu23-9.IgG4AA 6.16E+04 1.32E-04 2.15E-09 Hu23-10.IgG4AA 5.51E+04 1.53E-04 2.77E-09 Hu23-11.IgG4AA 4.22E+04 1.14E-04 2.71E-09 Hu23-12.IgG4AA 5.45E+04 1.10E-04 2.02E-09 Hu23-13.IgG4AA 4.24E+04 1.22E-04 2.88E-09 Hu23-14.IgG4AA 7.23E+04 1.61E-04 2.22E-09 M32 7.83E+04 4.15E-04 5.3E-09 Hu32a-15.IgG4AA 4.89E+04 2.52E-03 5.14E-08 Hu32a-29.IgG4AA 7.89E+04 6.20E-04 7.86E-09 Hu32a-43.IgG4AA 8.39E+04 6.85E-04 8.16E-09 Hu32a-57.IgG4AA 7.94E+04 6.24E-04 7.85E-09 Hu32a-71.IgG4AA 8.60E+04 3.96E-04 4.61E-09 Hu32a-85.IgG4AA 9.90E+04 3.15E-04 3.18E-09 M33 3.08E+05 2.27E-04 7.37E-10 Hu33-1.IgG4AA 6.61E+04 1.28E-03 1.93E-08 Hu33-4.IgG4AA 8.11E+04 2.55E-03 3.14E-08 Hu33-7.IgG4AA 7.69E+04 2.60E-03 3.38E-08 Hu33-2.IgG4AA 1.35E+05 1.43E-04 1.06E-09 Hu33-5.IgG4AA 1.46E+05 1.35E-04 9.26E-10 Hu33-8.IgG4AA 1.53E+05 1.62E-04 1.06E-09 Hu33-3.IgG4AA 1.25E+05 1.36E-04 1.09E-09 Hu33-6.IgG4AA 1.30E+05 1.40E-04 1.08E-09 Hu33-9.IgG4AA 1.40E+05 1.53E-04 1.09E-09

Example 3: Construction and Expression of Humanized Anti-PD-1 Antibodies

Each humanized antibody VH/VK gene fragment was constructed using designed primers by PCR and then used to construct the VH-CH1-Fc-pHr/VK-CL-pHr vector expressing the full-length antibody by homologous recombination with the pHr expression vector (with signal peptide and constant region gene fragment (CH1-Fc/CL)). IgG4-P is the S228P mutation (corresponding to position 108 of SEQ ID NO: 72 or SEQ ID NO: 79), and IgG4-AA is the F234A mutation (corresponding to position 114 of SEQ ID NO: 72 or SEQ ID NO: 79), L235A (corresponding to provision 115 of SEQ ID NO: 72 or SEQ ID NO: 79) and S228P (corresponding to provision 108 of SEQ ID NO: 72 or SEQ ID NO: 79). The IgG4-AA and IgG4-P antibody formats can be generated by simple point mutations in the IgG4 antibody format.

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

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

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

Examples of engineered full-length antibody sequences of the IgG4AA form are as follows:

Antibody heavy chain Hu23-11.IgG4AA (SEQ ID NO: 74):

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSDYEMHWVRQAPGQGLEWMGLIDPETGGTVYNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARERFSYYGSTSDWYFDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

Hu23-11.IgG4AA light chain (SEQ ID NO: 75):

DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSTGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC.

Heavy chain Hu32a-85.IgG4AA (SEQ ID NO: 76):

EVQLVQSGAEVKKPGSSVKVSCKASDFTFTDYEIHWVKQAPGHGLEWIGLFDPETGGIVYNQKFKGKATLTADKSTSTAYMEFSSLRSEDTAVYYCTREGYNRDWYFDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

Hu32a-85.IgG4AA light chain (same as Hu23-11.IgG4AA light chain, SEQ ID NO: 75):

DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSTGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC.

Heavy chain Hu33-6.IgG4AA (SEQ ID NO: 77):

KVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVATISGGGVDTYYQDNVQGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASPYGHGYFDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC NVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

Hu33-6.IgG4AA light chain (SEQ ID NO: 78):

DIQMTQSPSSLSASVGDRVTITCRASQDINNFLNWYQQKPGKAPKLLIYYTSSLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGEC.

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

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP P CPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

Examples of engineered full-length IgG4-P antibody sequences are as follows:

Hu23-11.IgG4P antibody heavy chain (SEQ ID NO: 80):

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSDYEMHWVRQAPGQGLEWMGLIDPETGGTVYNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARERFSYYGSTSDWYFDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

Hu23-11.IgG4P light chain (same as Hu23-11.IgG4AA light chain, SEQ ID NO: 75):

DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSTGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC.

Heavy chain Hu32a-85.IgG4P (SEQ ID NO: 81):

EVQLVQSGAEVKKPGSSVKVSCKASDFTFTDYEIHWVKQAPGHGLEWIGLFDPETGGIVYNQKFKGKATLTADKSTSTAYMEFSSLRSEDTAVYYCTREGYNRDWYFDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

Hu32a-85.IgG4P light chain (same as Hu23-11.IgG4AA light chain, SEQ ID NO: 75):

DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSTGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC.

Hu33-6.IgG4P Heavy Chain (SEQ ID NO: 82):

KVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVATISGGGVDTYYQDNVQGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASPYGHGYFDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC NVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

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

DIQMTQSPSSLSASVGDRVTITCRASQDINNFLNWYQQKPGKAPKLLIYYTSSLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGEC.

Test cases

Test Example 1: ELISA study of anti-PD-1 antibody binding to PD-1 ligand and blocking binding in vitro

PD-L1 on the surface of tumor cells binds to PD-1 on the surface of T cells, thereby inhibiting T cell proliferation. Anti-PD-1 antibodies can block the PD-L1/PD-1 signaling pathway by binding to PD-1, thereby stimulating T cell proliferation. A PD-1/PD-L1 binding blocking experiment was used to detect the blocking activity of anti-PD-1 antibodies on the signaling pathway.

In this experiment, after coating 96-well plates with PD-1-His protein (Cat. No. 10377H08H, Sino Biological), the tested anti-PD-1 antibodies (including antibodies: Hu23-11.IgG4AA, Hu32a-85. IgG4AA and Hu33-6.IgG4AA, positive control antibody: H005-1 (see antibody H005-1 in WO2015085847)) and the reaction mixture was incubated; then HRP-labeled goat anti-human IgG (H+L) (cat. no. 109-035-003, Jackson ImmunoResearch) was added and incubated. After washing the plates, the binding amounts of HRP-labeled goat anti-human IgG (H+L) were determined and the EC ₅₀ values of anti-PD-1 antibody binding to the PD-1 ligand were calculated.

In this experiment, after coating 96-well plates with PD-1 protein fused to an Fc in the extracellular region (PD-1-Fc, see SEQ ID NO: 1), the test anti-PD-1 antibodies were individually added. 1 (including antibodies: Hu23-11.IgG4AA, Hu32a-85.IgG4AA and Hu33-6.IgG4AA, positive control antibody: H005-1 (see antibody H005-1 in WO2015085847)) and incubated; then biotin-labeled PD-L1/PD-L2 was added and incubated. After washing the plates, binding amounts of biotin-labeled PD-L1/PD-L2 were determined and IC ₅₀ values of anti-PD-1 antibodies for blocking PD-L1/PD-L2 ligand binding were calculated.

CB buffer with pH 9.6 (1.59 g Na ₂ CO ₃ and 2.93 g NaHCO ₃ dissolved in 1 L of distilled water) was used to dilute PD-1-Fc to 1 μg/ml, which was added to 96- well plates in a volume of 100 μl/well and kept at 4 °C for 16-20 hours. PBS buffer was aspirated from the 96-well plates, and the plates were washed once with PBST buffer (pH 7.4, PBS containing 0.05% tween 20). 120 μl/well PBST/1% milk was added and incubated at room temperature for 1 h to block. The blocking solution was removed and the plates were washed once with PBST buffer. 90 μl of test anti-PD-1 antibody diluted to the appropriate concentration in sample diluent (pH 7.4 PBS containing 5% BSA, 0.05% Tween20) was added and preincubated at 4°C for 1 hour. A 10-fold concentration of biotin-labeled PD-L1/PD-L2 (Beijing Sino Biological Co., Ltd.) (10 μg/ml) was added in a volume of 10 μl/well, vortexed and mixed well on a shaker, and then incubated at 37° C for 1 hour. The reaction system was removed and the plates were washed 6 times with PBST. 100 μl/well of streptavidin peroxidase polymer diluted 1:400 with PBST buffer was added and incubated with shaking at room temperature for 50 min. The plates were washed 6 times with PBST. 100 μl/well of TMB was added and incubated at room temperature for 5-10 min. 100 µl/well of 1M H ₂ SO ₄ was added to stop the reaction. Absorbance values at 450 nm were read using a microplate reader and IC ₅₀ values of anti-PD-1 antibodies for blocking PD-L1/PD-L2 ligand binding were calculated. The data is detailed below in Table 17.

Table 17. ELISA assay of anti-PD-1 antibodies of the present invention for binding to PD-1 and blocking PD-L1/PD-L2 ligand binding

Antibody Antigen binding Blocking PD-1 and PD-L1/PD-L2 binding Maximum OD value for human PD-1-his EC50 for human PD-1-his (ng/ml) IC50 for human PD-1-Fc/PD-L1 (ng/ml) IC50 for human PD-1-Fc/PD-L2 (ng/ml) Hu23-11.IgG4AA 1.46 237.3 92.35 245.1 Hu32a-85.IgG4AA 1.265 135.9 78.63 205.9 Hu33-6.IgG4AA 1,706 205.6 103.5 207.2 H005-1 1.21 113.1 109.6 225.3

The exemplary anti-PD-1 antibodies Hu23-11.IgG4AA, Hu32a-85.IgG4AA and Hu33-6.IgG4AA of the present invention are all capable of effectively blocking the binding of PD-1 to PD-L1/PD-L2, and their blocking activity is similar to that of the antibody positive control.

Test Example 2: Ligand Blocking Study with Exemplary Antibodies

The blocking effect of antibodies on the binding of PD-1 and PD-L1 was studied. The experimental process is briefly described as follows:

CHOK1/PD-L1 cells (Promega) were digested, added to 96-well plates at 100 μl/well, and placed in a 37 °C, 5% CO ₂ incubator for 24 h. Controls and samples were diluted to desired concentrations using PBS. Jurkat/PD-1 cells (Jurkat cells stably transfected with PD-1) were counted and seeded at a specific proportion (90 μL/well) into cell culture plates containing CHOK1/PD-L1 cells, and 10 μL/well of diluted antibody was added ( antibody: Hu23-11.IgG4AA, Hu32a-85.IgG4AA and Hu33-6.IgG4AA, positive control antibody: H005-1, negative control: IgG4 protein, antibody gradient dilution concentration: 0.3 mg/ml, 3 mg/ml , 30 mg/ml) and placed in an incubator at 37 °C, 5% CO ₂ for incubation for 5 h. Cell culture plates were removed and kept at room temperature for 5 min. 50 µl of Bio-Glo™ reagent was then added to each well and incubated at room temperature for 5 minutes before plate reading. The results of the experiment are shown in Fig. 1.

The results showed that exemplary anti-PD-1 antibodies Hu23-11.IgG4AA, Hu32a-85.IgG4AA and Hu33-6.IgG4AA according to the present invention can effectively block the binding of PD-1 and PD-L1.

Test Example 3: Antibody Affinity Experiment Using BIAcore on Illustrative Antibodies

IgG affinity capture was performed using a Protein A biosensor chip (Cat. No. 29127556, GE). Human PD-1 antigen (Cat. No. 10377H08H, Sino Biological) and cynomolgus PD-1 antigen (purchased from Sino Biological) were passed over the surface of the chip, and binding and dissociation curves were obtained by real-time detection of anti-PD antibody response signals. 1 and PD-1 antigen using the Biacore T200 device. After the dissociation of each experimental run was completed, the biosensor chip was washed and regenerated with 10 mM glycine-HCl buffer pH 1.5. The experimental buffer system was 1×HBS-EP buffer solution (Cat. No. BR-1001-88, GE). After the experiment, GE Biacore T200 Evaluation software version 3.0 was used to fit the data with a Langmuir model (1:1), and the affinity value was obtained. The results are shown in Table 18.

Table 18. Affinity of anti-PD-1 antibodies to human PD1 and cynomolgus PD-1

Antibody Antigen ka (1/Ms) kd (1/s) KD(M) Hu23-11.IgG4AA Human PD-1-His 6.06E+04 1.13E-04 1.86E-09 Cynomolgus macaque PD-1 4.79E+04 1.26E-04 2.62E-09 Hu32a-85.IgG4AA Human PD-1-His 8.56E+04 2.78E-04 3.25E-09 Cynomolgus macaque PD-1 1.16E+05 4.75E-04 4.11E-09 Hu33-6.IgG4AA Human PD-1-His 1.24E+05 1.14E-04 9.18E-10 Cynomolgus macaque PD-1 2.51E+05 2.41E-04 9.60E-10

The results showed that the exemplary anti-PD-1 antibodies Hu23-11.IgG4AA, Hu32a-85.IgG4AA and Hu33-6.IgG4AA of the present invention can all bind to human PD-1 and cynomolgus PD-1.

Test example 4. Effect of antibodies on the secretion of IFNγ by cells in an experiment on activation of PBMC-T lymphocytes

To study the effect of anti-PD-1 antibodies on the function of primary human T lymphocytes, peripheral blood mononuclear cells (PBMCs) were collected and purified, stimulated with tuberculin (TB) for 5 days, and the level of secretion of the cytokine IFNγ was determined. The experimental process is briefly described as follows:

PBMCs were obtained from fresh blood using Ficoll-Hypaque (17-5442-02, GE) for density gradient centrifugation (Stem Cell Technologies), cultured in RPMI 1640 medium (SH30809.01, GE) supplemented with 10% (v/v) vol.) FBS (10099-141, Gibco) at 37 °C and 5% CO ₂ .

Freshly isolated and purified PBMCs were brought to a density of ^2x106 cells/ml using RPMI 1640 medium. 40 µl tuberculin (97-8800, Synbiotics) was added to 20 ml cell suspension and cultured in an incubator at 37 °C, 5% CO ₂ in within 5 days. On day 5, the above cultured cells were collected by centrifugation, resuspended in fresh RPMI 1640 medium, adjusted to a density of 1.1×10 ⁶ cells/ml and seeded into 96-well cell culture plates at 90 µl per well. At the same time, antibody samples (including antibodies according to the present invention: Hu23-11.IgG4AA, Hu32a-85.IgG4AA and Hu33-6.IgG4AA, positive control antibody H005-1 and negative control IgG4 protein, antibody gradient dilution concentration 0.3 mg/ ml, 3 mg/ml and 30 mg/ml), the gradient was diluted in PBS (B320, Shanghai BasalMedia Technologies Co., Ltd.), 10 μl/well. Cell culture plates were placed in an incubator at 37 °C, 5% CO ₂ for incubation for 3 days. The cell culture plates were removed and the cell culture supernatant was collected by centrifugation (4000 rpm, 10 min). IFN-γ levels were determined using an ELISA method (human IFN-γ kit (EHC102g.96, Neobioscience)). For specific operations, the instructions supplied with the reagents were used.

The test results are shown in Fig. 2. The results showed that all the anti-PD-1 antibodies Hu23-11.IgG4AA, Hu32a-85.IgG4AA and Hu33-6.IgG4AA according to the present invention can effectively activate the secretion of IFN-γ.

Test Example 5: Effect of Anti-PD-1 Antibodies on MC38 Colon Cancer Model in PD-1 Transgenic Mice

MC38 cells were inoculated at a dose of 5×10 ⁵ cells/mouse/100 μl into 90 hPD-1 TG mice (Biocytogen) subcutaneously in the region of the right ribs. After 10 days, animals with tumors that were too large or too small were excluded. Based on the average tumor volume of approximately 120 mm ^{3 ,} mice were randomly divided into: vehicle blank control (PBS), positive control H005-1 3 mg/kg, Hu32a-85.IgG4AA 1 mg/kg, Hu32a-85.IgG4AA 3 mg /kg, Hu23-11.IgG4AA 1 mg/kg, Hu23-11.IgG4AA 3 mg/kg and Hu33-6.IgG4AA 3 mg/kg, for a total of 7 groups of 8 animals each. Antibodies from each group were administered intraperitoneally three times per week starting on day 0. After the first week of administration, tumors were found to be significantly inhibited. In the second and third weeks, the frequency of administration was adjusted to once a week, for a total of 5 administrations. The tumor volume and weight of the animals were monitored twice a week and the data were recorded. When tumor volume exceeded 2000 mm ³ , or the majority of tumors showed ulceration, or body weight decreased by 20%, animals with tumors were sacrificed as the end point of the experiment.

Tumor volume (TV) = 1/2 × L _long × L _short ²

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

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

Where T and T0 represent the tumor volumes at the end of the test and the beginning of the test in the antibody administration group, respectively, and C and C0 represent the tumor volumes at the end of the test and the beginning of the test in the blank control group, respectively.

The test results are shown in Table 19 and FIG. 3. The test results showed that, compared with the blank control, all the antibodies of the present invention were able to significantly inhibit the growth of MC38 colon cancer xenograft tumors in mice. Among them, the Hu32a-85.IgG4AA-3 mg/kg group had the highest tumor inhibition rate, and the tumor inhibition rate was 77.64% at the last measurement. When the dosing frequency was three times a week for 3 administrations, when measured on the seventh day, the results showed that all tumor inhibition rates of the antibodies of the present invention were significantly better than those of the positive control antibody H005-1; thereafter, the dosing frequency was reduced to once a week, after two administrations (day 21), the difference in the effectiveness of the antibodies according to the present invention gradually increased and appeared in a dose-dependent manner, among which Hu32a-85.IgG4AA was significantly better than the same dose of H005- 1 (p < 0.05). In addition, tumor-bearing mice could tolerate all anti-PD-1 antibodies well, with body weight continuously increasing throughout the administration process and no apparent drug-induced weight loss or other symptoms observed.

Table 19. Effect of antibodies to PD-1 on the degree of inhibition of MC38 colon tumor growth in mice (mm ³ )

Days after first administration H005-1 Hu32a-85.IgG4AA Hu23-11.IgG4AA Hu33-6.IgG4AA 3 mg/kg 1 mg/kg 3 mg/kg 1 mg/kg 3 mg/kg 3 mg/kg 21 46.82% 67.12% 77.64% 45.12% 75.32% 59.31%

Test Example 6: Effect of Anti-PD-1 Antibodies on MC38 Colon Cancer Model in PD-1 Transgenic Mouse

PD-1 transgenic mice were purchased from ISIS INNOVATION LIMITED, University Offices, Wellington Square, Oxford OX1 2JD, England, and bred at Cephrim Biosciences, Inc. to obtain fifth generation mice. MC38 cells were inoculated at a dose of 5×10 ⁵ cells/100 μl/animal into hPD-1 transgenic mice (half female and half male) subcutaneously into the posterior region of the right ribs. When the average tumor volume in mice reached 80-100 mm ³ , animals with too much or too little body weight or tumors were excluded. According to tumor volume, tumor-bearing mice were randomly divided into 5 groups (8 animals each): negative control hIgG 30 mg/kg, H005-1 10 mg/kg, H005-1 30 mg/kg, Hu33-6. IgG4AA 10 mg/kg and Hu33-6.IgG4AA 30 mg/kg. The date of group division and administration was set as day 0. After group division, each drug was administered intraperitoneally for 22 days, once every two days, for a total of 11 times. Tumor volume was measured twice a week, body weight was measured, and data were recorded. Animal body weights and tumor volumes in each group were presented as mean ± standard deviation (mean ± SEM), and Graphpad Prism 5 and Excel software were used for plotting, and Student's t test was used for statistical analysis.

Tumor volume (TV) = 0.5236 × L _long × L _short ²

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

Degree of tumor growth inhibition %TGI = 1- T/C %

Where T and T0 represent the tumor volumes at the end of the test and the beginning of the test in the antibody administration group, respectively, and C and C0 represent the tumor volumes at the end of the test and the beginning of the test in the blank control group, respectively.

The test results are shown in Table 20 and FIG. 4. The test results showed that, compared with the control group, the antibodies of the present invention were able to significantly inhibit the growth of MC38 colon cancer xenograft tumors in mice. Among them, the Hu33-6.IgG4AA-30 mg/kg group had the highest tumor inhibition rate, and the tumor inhibition rate was 80.4% at 20 days. In the low dose group (10 mg/kg), the efficacy of Hu33-6.IgG4AA-10 mg/kg was better than that of the positive control group H005-1-10 mg/kg.

Table 20. Effect of anti-PD-1 antibodies on MC38 colon tumor volume in mice

Days after first administration hIgG control group Group H005-1 10 mg/kg Group H005-1 30 mg/kg Group Hu33-6.IgG4AA 10 mg/kg Group Hu33-6.IgG4AA 30 mg/kg 0 96.2 96.1 95.3 94.7 94.5 20 1797.2 794.0 434.9 550.1 352.8 23 1034.2 534.1 632.4 387.6

Note: The unit of mean tumor volume for each group in the table is mm ³ .

Test case 7: Pharmacokinetic test of anti-PD-1 antibodies in cynomolgus monkeys

The cynomolgus macaques used in the experiment were 6 males, 2–5 years old, 2–5 kg, purchased from Guangdong Frontier Biotechnology Co., Ltd., license number: SCXK (Guangdong) 2015-0037, animal certificate number: 44613900000219.

Housing conditions: The room temperature was maintained at 18°C-26°C, relative humidity was 40%-70%, and lighting was alternately light and dark for 12 hours. Mice were given free access to food and water unless when fasting was necessary.

Animals were weighed before administration and weighed between 2.81 and 3.52 kg. Administration was carried out using an injection pump for subcutaneous intravenous infusion into the forelimbs or hindlimbs. The dose for each group was 1 mg/kg, administered by a single intravenous injection at a rate of 0.1 ml/kg/min over an administration period of approximately 30 minutes. Before administration and after 5 minutes, 0.25 hour, 0.5 hour (immediately after completion of administration), 1 hour, 2 hours, 4 hours, 8 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 7 days, 10 days, 13 days, 14 days, 21 days and 28 days after the start of the intravenous infusion, whole blood of the animal was collected from the veins of the hind limbs and the serum was separated. Of these, approximately 2 ml of whole blood was collected before administration and 14 days, 21 days and 28 days after the start of the intravenous infusion, and approximately 1 ml of whole blood was collected at the remaining blood collection points. Serum blood drug concentrations were determined by ELISA and PK analysis was performed. The results are shown in Table 21.

Table 21. Pharmacokinetics of humanized anti-PD1 antibodies in cynomolgus monkeys

Hu23-11.IgG4AA Hu33-6.IgG4AA t1/2 (days) 5.5±0.7 4.6±1.3 Cmax (µg/ml) 23.75±2.29 21.47±2.13 AUC (h⋅µg/ml) 2775±241 2319±518 CL (ml/day/kg) 8.7±0.7 10.7±2.3 Vz (ml/kg) 69±3.6 67.9±3.4

The results showed that Hu23-11.IgG4AA and Hu33-6.IgG4AA had good pharmacokinetic activities.

Although the above invention has been described in detail with the help of figures and examples for a clear understanding, the descriptions and examples should not be interpreted as limiting the scope of the present invention. The disclosure of all patents and scientific documents cited herein is expressly incorporated by reference in its entirety.

--->

LIST OF SEQUENCES

<110> JIANGSU HENGRUI MEDICINE CO., LTD.

SHANGHAI HENGRUI PHARMACEUTICAL CO., LTD.

<120> ANTIBODY TO PD-1, ITS ANTIGEN-BINDING FRAGMENT AND ITS PHARMACEUTICAL APPLICATION

<130> 2019

<160> 82

<170> SIPOSequenceListing 1.0

<210> 1

<211> 401

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Human PD-1-IgG1Fc sequence

<400> 1

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Lys

<210> 2

<211> 186

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Sequence of human PD-1-his

<400> 2

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Asp Asp Asp Lys His His His His His

180 185

<210> 3

<211> 288

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> PD-1 antigen sequence for cell transfection

<400> 3

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

<210> 4

<211> 125

<212> Protein

<213> Mus musculus

<220>

<221> DOMAIN

<223> M23 heavy chain variable region sequence

<400> 4

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

<210> 5

<211> 112

<212> Protein

<213> Mus musculus

<220>

<221> DOMAIN

<223> M23 light chain variable region sequence

<400> 5

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 6

<211> 120

<212> Protein

<213> Mus musculus

<220>

<221> DOMAIN

<223> M32 heavy chain variable region sequence

<400> 6

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 7

<211> 112

<212> Protein

<213> Mus musculus

<220>

<221> DOMAIN

<223> M32 light chain variable region sequence

<400> 7

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 8

<211> 5

<212> Protein

<213> Mus musculus

<220>

<221> DOMAIN

<223> HCDR1 M23 sequence

<400> 8

Asp Tyr Glu Met His

15

<210> 9

<211> 17

<212> Protein

<213> Mus musculus

<220>

<221> DOMAIN

<223> HCDR2 M23 sequence

<400> 9

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

1 5 10 15

Asp

<210> 10

<211> 16

<212> Protein

<213> Mus musculus

<220>

<221> DOMAIN

<223> HCDR3 M23 sequence

<400> 10

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

1 5 10 15

<210> 11

<211> 16

<212> Protein

<213> Mus musculus

<220>

<221> DOMAIN

<223> Sequence LCDR1 M23

<400> 11

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

1 5 10 15

<210> 12

<211> 7

<212> Protein

<213> Mus musculus

<220>

<221> DOMAIN

<223> LCDR2 sequence M23/M32

<400> 12

Lys Val Ser Asn Arg Phe Ser

15

<210> 13

<211> 9

<212> Protein

<213> Mus musculus

<220>

<221> DOMAIN

<223> LCDR3 sequence M23

<400> 13

Phe Gln Gly Ser His Val Pro Tyr Thr

15

<210> 14

<211> 5

<212> Protein

<213> Mus musculus

<220>

<221> DOMAIN

<223> HCDR1 M32 sequence

<400> 14

Asp Tyr Glu Ile His

15

<210> 15

<211> 17

<212> Protein

<213> Mus musculus

<220>

<221> DOMAIN

<223> HCDR2 M32 sequence

<400> 15

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

1 5 10 15

Gly

<210> 16

<211> 11

<212> Protein

<213> Mus musculus

<220>

<221> DOMAIN

<223> HCDR3 M32 sequence

<400> 16

Glu Gly Tyr Asn Arg Asp Trp Tyr Phe Asp Val

1 5 10

<210> 17

<211> 16

<212> Protein

<213> Mus musculus

<220>

<221> DOMAIN

<223> LCDR1 M32 sequence

<400> 17

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

1 5 10 15

<210> 18

<211> 9

<212> Protein

<213> Mus musculus

<220>

<221> DOMAIN

<223> LCDR3 M32 sequence

<400> 18

Phe Gln Gly Ser His Val Pro Tyr Ala

15

<210> 19

<211> 118

<212> Protein

<213> Mus musculus

<220>

<221> DOMAIN

<223> M33 heavy chain variable region sequence

<400> 19

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 20

<211> 107

<212> Protein

<213> Mus musculus

<220>

<221> DOMAIN

<223> M33 light chain variable region sequence

<400> 20

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 21

<211> 5

<212> Protein

<213> Mus musculus

<220>

<221> DOMAIN

<223> HCDR1 M33 sequence

<400> 21

Ser Tyr Ala Met Ser

15

<210> 22

<211> 17

<212> Protein

<213> Mus musculus

<220>

<221> DOMAIN

<223> HCDR2 M33 sequence

<400> 22

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

1 5 10 15

Gly

<210> 23

<211> 9

<212> Protein

<213> Mus musculus

<220>

<221> DOMAIN

<223> HCDR3 M33 sequence

<400> 23

Pro Tyr Gly His Gly Tyr Phe Asp Val

15

<210> 24

<211> 11

<212> Protein

<213> Mus musculus

<220>

<221> DOMAIN

<223> Sequence LCDR1 M33

<400> 24

Arg Ala Ser Gln Asp Ile Asn Asn Phe Leu Asn

1 5 10

<210> 25

<211> 7

<212> Protein

<213> Mus musculus

<220>

<221> DOMAIN

<223> LCDR2 M33 sequence

<400> 25

Tyr Thr Ser Ser Leu His Ser

15

<210> 26

<211> 9

<212> Protein

<213> Mus musculus

<220>

<221> DOMAIN

<223> LCDR3 M33 sequence

<400> 26

Gln Gln Gly Asn Thr Leu Pro Trp Thr

15

<210> 27

<211> 125

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Hu23VH1 /Hu23VH-CDR-grafted sequence

<400> 27

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

<210> 28

<211> 112

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Hu23VL1 /Hu23VL-CDR-grafted sequence

<400> 28

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 29

<211> 112

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Sequence Hu23VL2

<400> 29

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 30

<211> 125

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Sequence Hu23VH2

<400> 30

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

<210> 31

<211> 125

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Sequence Hu23VH3

<400> 31

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

<210> 32

<211> 125

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Sequence Hu23VH4

<400> 32

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

<210> 33

<211> 120

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Hu32VH1 /Hu32VH-CDR-grafted sequence

<400> 33

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 34

<211> 112

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Hu32VL1 /Hu32VL-CDR-grafted sequence

<400> 34

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 35

<211> 112

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Sequence Hu32VL2

<400> 35

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 36

<211> 120

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Sequence Hu32VH2

<400> 36

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 37

<211> 120

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Sequence Hu32VH3

<400> 37

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 38

<211> 120

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Sequence Hu32VH4

<400> 38

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 39

<211> 120

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Sequence Hu32VH5

<400> 39

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 40

<211> 120

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Sequence Hu32VH6

<400> 40

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 41

<211> 118

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Hu33VH1/Hu33VH-CDR-grafted sequence

<400> 41

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 42

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Hu33VL1/Hu33VL-CDR-grafted sequence

<400> 42

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 43

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Hu33VL2 sequence

<400> 43

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 44

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Hu33VL3 sequence

<400> 44

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 45

<211> 118

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Sequence Hu33VH2

<400> 45

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 46

<211> 118

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Sequence Hu33VH3

<400> 46

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 47

<211> 16

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Sequence Hu23LCDR1(N33Q)

<400> 47

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

1 5 10 15

<210> 48

<211> 16

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Sequence Hu23LCDR1(N33L)

<400> 48

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

1 5 10 15

<210> 49

<211> 16

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Sequence Hu23LCDR1(N33T)

<400> 49

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

1 5 10 15

<210> 50

<211> 16

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Sequence Hu23LCDR1(N33D)

<400> 50

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

1 5 10 15

<210> 51

<211> 16

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Sequence Hu23LCDR1(G34A)

<400> 51

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

1 5 10 15

<210> 52

<211> 16

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Sequence Hu23LCDR1(G34V)

<400> 52

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

1 5 10 15

<210> 53

<211> 112

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Sequence Hu23VL1(N33Q)

<400> 53

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 54

<211> 112

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Sequence Hu23VL1(N33L)

<400> 54

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 55

<211> 112

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Sequence Hu23VL1(N33T)

<400> 55

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 56

<211> 112

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Sequence Hu23VL1(N33D)

<400> 56

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 57

<211> 112

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Sequence Hu23VL1(G34A)

<400> 57

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 58

<211> 112

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Sequence Hu23VL1(G34V)

<400> 58

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 59

<211> 112

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Sequence Hu23VL2(N33Q)

<400> 59

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 60

<211> 112

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Sequence Hu23VL2(N33L)

<400> 60

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 61

<211> 112

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Sequence Hu23VL2(N33T)

<400> 61

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 62

<211> 112

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Sequence Hu23VL2(N33D)

<400> 62

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 63

<211> 112

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Sequence Hu23VL2(G34A)

<400> 63

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 64

<211> 112

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Sequence Hu23VL2(G34V)

<400> 64

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 65

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> General sequence formula of HCDR1 antibodies Hu32 and Hu23

<220>

<221> DOMAIN

<222>(4)..(4)

<223> Xaa selected from Ile or Met

<400> 65

Asp Tyr Glu Xaa His

15

<210> 66

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> General sequence formula of HCDR2 antibodies Hu32 and Hu23

<220>

<221> DOMAIN

<222>(2)..(2)

<223> Xaa selected from Phe or Ile

<220>

<221> DOMAIN

<222>(9)..(9)

<223> Xaa selected from Ile or Thr

<220>

<221> DOMAIN

<222>(17)..(17)

<223> Xaa selected from Gly or Asp

<400> 66

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

1 5 10 15

Xaa

<210> 67

<211> 16

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> General sequence formula of HCDR3 antibodies Hu32 and Hu23

<220>

<221> DOMAIN

<222>(2)..(2)

<223> Xaa selected from Gly or Arg

<220>

<221> DOMAIN

<222>(3)..(3)

<223> Xaa selected from Phe or missing

<220>

<221> DOMAIN

<222>(4)..(4)

<223> Xaa selected from Ser or missing

<220>

<221> DOMAIN

<222>(5)..(5)

<223> Xaa selected from Tyr or missing

<220>

<221> DOMAIN

<222>(7)..(7)

<223> Xaa selected from Gly or missing

<220>

<221> DOMAIN

<222>(8)..(8)

<223> Xaa selected from Ser or missing

<220>

<221> DOMAIN

<222>(9)..(9)

<223> Xaa selected from Asn or Thr

<220>

<221> DOMAIN

<222>(10)..(10)

<223> Xaa selected from Arg or Ser

<400> 67

Glu Xaa Xaa Xaa Xaa Tyr Xaa Xaa Xaa Xaa Asp Trp Tyr Phe Asp Val

1 5 10 15

<210> 68

<211> 16

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> General sequence formula LCDR1 antibodies Hu32 and Hu23

<220>

<221> DOMAIN

<222>(6)..(6)

<223> Xaa selected from Ile or Leu

<220>

<221> DOMAIN

<222>(10)..(10)

<223> Xaa selected from Asn, Gln, Leu, Thr or Asp

<220>

<221> DOMAIN

<222>(11)..(11)

<223> Xaa selected from Gly, Ala or Val

<220>

<221> DOMAIN

<222>(12)..(12)

<223> Xaa selected from Asn or Lys

<400> 68

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

1 5 10 15

<210> 69

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> General sequence formula of LCDR3 antibodies Hu32 and Hu23

<220>

<221> DOMAIN

<222>(9)..(9)

<223> Xaa selected from Ala or Thr

<400> 69

Phe Gln Gly Ser His Val Pro Tyr Xaa

15

<210> 70

<211> 125

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> General formula of the variable region sequence of the antibody heavy chain Hu32 and Hu23

<220>

<221> DOMAIN

<222>(26)..(26)

<223> Xaa selected from Gly or Asp

<220>

<221> DOMAIN

<222>(27)..(27)

<223> Xaa selected from Gly, Phe or Tyr

<220>

<221> DOMAIN

<222>(30)..(30)

<223> Xaa selected from Ser or Thr

<220>

<221> DOMAIN

<222>(34)..(34)

<223> Xaa selected from Ile or Met

<220>

<221> DOMAIN

<222>(38)..(38)

<223> Xaa selected from Arg or Lys

<220>

<221> DOMAIN

<222>(43)..(43)

<223> Xaa selected from Gln or His

<220>

<221> DOMAIN

<222>(48)..(48)

<223> Xaa selected from Ile or Met

<220>

<221> DOMAIN

<222>(51)..(51)

<223> Xaa selected from Phe or Ile

<220>

<221> DOMAIN

<222>(58)..(58)

<223> Xaa selected from Ile or Thr

<220>

<221> DOMAIN

<222>(66)..(66)

<223> Xaa selected from Gly or Asp

<220>

<221> DOMAIN

<222>(67)..(67)

<223> Xaa selected from Arg or Lys

<220>

<221> DOMAIN

<222>(68)..(68)

<223> Xaa selected from Val, Ala or Thr

<220>

<221> DOMAIN

<222>(70)..(70)

<223> Xaa selected from Arg or Lys

<220>

<221> DOMAIN

<222>(83)..(83)

<223> Xaa selected from Leu or Phe

<220>

<221> DOMAIN

<222>(97)..(97)

<223> Xaa selected from Ala or Thr

<220>

<221> DOMAIN

<222>(100)..(100)

<223> Xaa selected from Gly or Arg

<220>

<221> DOMAIN

<222>(101)..(101)

<223> Xaa selected from Phe or missing

<220>

<221> DOMAIN

<222>(102)..(102)

<223> Xaa selected from Ser or missing

<220>

<221> DOMAIN

<222>(103)..(103)

<223> Xaa selected from Tyr or missing

<220>

<221> DOMAIN

<222>(105)..(105)

<223> Xaa selected from Gly or missing

<220>

<221> DOMAIN

<222>(106)..(106)

<223> Xaa selected from Ser or missing

<220>

<221> DOMAIN

<222>(107)..(107)

<223> Xaa selected from Asn or Thr

<220>

<221> DOMAIN

<222>(108)..(108)

<223> Xaa selected from Arg or Ser

<400> 70

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

1 5 10 15

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

20 25 30

Glu Xaa His Trp Val Xaa Gln Ala Pro Gly Xaa Gly Leu Glu Trp Xaa

35 40 45

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

50 55 60

Lys Xaa Xaa Xaa Thr Xaa Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Xaa Arg Glu Xaa Xaa Xaa Xaa Tyr Xaa Xaa Xaa Xaa Asp Trp Tyr Phe

100 105 110

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

115 120 125

<210> 71

<211> 112

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> General formula of the antibody light chain variable region sequence of Hu32 and Hu23

<220>

<221> DOMAIN

<222>(2)..(2)

<223> Xaa selected from Ile, Val or Gly

<220>

<221> DOMAIN

<222>(29)..(29)

<223> Xaa selected from Ile or Leu

<220>

<221> DOMAIN

<222>(33)..(33)

<223> Xaa selected from Asn, Gln, Leu, Thr or Asp

<220>

<221> DOMAIN

<222>(34)..(34)

<223> Xaa selected from Gly, Ala or Val

<220>

<221> DOMAIN

<222>(35)..(35)

<223> Xaa selected from Asn or Lys

<220>

<221> DOMAIN

<222>(102)..(102)

<223> Xaa selected from Ala or Thr

<400> 71

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 72

<211> 327

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> IgG4-AA heavy chain constant region sequence

<400> 72

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

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

115 120 125

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

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210> 73

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Sequence of the antibody kappa light chain constant region

<400> 73

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

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

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 74

<211> 452

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Sequence of the heavy chain of the antibody Hu23-11.IgG4AA

<400> 74

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

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

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys

195 200 205

Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu

210 215 220

Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

225 230 235 240

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

245 250 255

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

260 265 270

Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

290 295 300

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

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val

420 425 430

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

435 440 445

Ser Leu Gly Lys

450

<210> 75

<211> 219

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Light chain sequence Hu23-11.IgG4AA/Hu32a-85.IgG4AA/Hu23-11.IgG4P/Hu32a-85.IgG4P

<400> 75

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

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

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 76

<211> 447

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Heavy chain sequence Hu32a-85.IgG4AA

<400> 76

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

130 135 140

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

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

210 215 220

Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val

225 230 235 240

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

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

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

290 295 300

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

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

325 330 335

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

340 345 350

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

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

435 440 445

<210> 77

<211> 445

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Heavy chain sequence Hu33-6.IgG4AA

<400> 77

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 78

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Light chain sequence Hu33-6.IgG4AA/Hu33-6.IgG4P

<400> 78

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

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

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

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

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 79

<211> 327

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> IgG4-P heavy chain constant region sequence

<400> 79

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

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

115 120 125

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

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210> 80

<211> 452

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Heavy chain sequence of antibody Hu23-11. IgG4P

<400> 80

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

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

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys

195 200 205

Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

290 295 300

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

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val

420 425 430

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

435 440 445

Ser Leu Gly Lys

450

<210> 81

<211> 447

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Hu32a-85. Heavy chain sequence of Hu32a-85. IgG4P

<400> 81

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

130 135 140

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

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

210 215 220

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

225 230 235 240

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

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

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

290 295 300

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

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

325 330 335

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

340 345 350

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

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

435 440 445

<210> 82

<211> 445

<212> Protein

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Heavy chain sequence of Hu33-6. IgG4P

<400> 82

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

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Claims (39)

1. An anti-PD-1 antibody or an antigen binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the combination of the heavy chain variable region and the light chain variable region is as follows: a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, respectively, and a light chain variable region comprising LCDR2 and LCDR3, shown in SEQ ID NO: 12 and SEQ ID NO: 13, respectively, and LCDR1, shown in SEQ ID NO: 11, 47, 48, 49, 50, 51 or 52. 2. Antibody to PD-1 or antigen-binding fragment thereof according to claim 1, containing a combination of a heavy chain variable region and a light chain variable region as follows: (a1) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, respectively, and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 49, SEQ ID NO: 12 and SEQ ID NO: 13, respectively. 3. An anti-PD-1 antibody or an antigen-binding fragment thereof according to claim 1 or 2, wherein the anti-PD-1 antibody or an antigen-binding fragment thereof is a murine antibody or an antigen-binding fragment thereof, a chimeric antibody or an antigen-binding fragment thereof, a fully human antibody or an antigen-binding fragment thereof fragment, or a humanized antibody or antigen-binding fragment thereof. 4. An anti-PD-1 antibody or an antigen-binding fragment thereof according to claim 3, wherein the anti-PD-1 antibody is a humanized antibody comprising a framework region derived from a human antibody, or a variant thereof, wherein: The framework region variant contains mutation(s) as follows: reverse amino acid mutation 2G contained in the light chain variable region, and/or reverse amino acid mutations contained in the heavy chain variable region, as indicated by one of the following: (f1) reverse amino acid mutations 27Y and 69L contained in the heavy chain variable region; or (f2) reverse amino acid mutations 27Y, 48I, 67T, 69L and 82F contained in the heavy chain variable region; or (f3) reverse amino acid mutations 27Y, 48I, 67T, 69L, 82F and 93T contained in the heavy chain variable region; where amino acid positions are numbered and defined according to the Kabat system. 5. An anti-PD-1 antibody or an antigen-binding fragment thereof according to claim 3, wherein the combination of the heavy chain variable region and the light chain variable region of the antibody is selected from (i) or (l): (i) a heavy chain variable region, the sequence of which corresponds to that shown in SEQ ID NO: 4; and/or a light chain variable region, the sequence of which corresponds to that presented in SEQ ID NO: 5; (l) a heavy chain variable region the sequence of which corresponds to SEQ ID NO: 27, 30, 31 or 32, and/or a light chain variable region whose sequence corresponds to SEQ ID NO: 28, 29, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 or 64. 6. An anti-PD-1 antibody or an antigen-binding fragment thereof according to claim 5, wherein the combination of the heavy chain variable region and the light chain variable region is selected from: (p) a heavy chain variable region, the sequence of which corresponds to that shown in SEQ ID NO: 27; and/or light chain variable region, the sequence of which corresponds to that presented in SEQ ID NO: 55. 7. An antibody to PD-1 or an antigen-binding fragment thereof according to any one of paragraphs. 1-6, wherein the antibody comprises an antibody constant region. 8. The anti-PD-1 antibody or antigen-binding fragment thereof according to claim 7, wherein the heavy chain constant region of the antibody constant region is selected from the group consisting of human IgG1, IgG2, IgG3 and IgG4 constant regions and conventional variants thereof, the constant light chain constant region region of the antibody is selected from the group consisting of human antibody κ and λ chain constant regions and conventional variants thereof. 9. The anti-PD-1 antibody or antigen binding fragment thereof according to claim 8, wherein the antibody comprises a heavy chain constant region set forth in SEQ ID NO: 72 or 79 and a light chain constant region set forth in SEQ ID NO: 73. 10. An antibody to PD-1 or an antigen-binding fragment thereof according to any one of paragraphs. 1-9, wherein the anti-PD-1 antibody comprises a light chain set forth in SEQ ID NO: 75 and a heavy chain set forth in SEQ ID NO: 74 or 80. 11. Antibody to PD-1 or an antigen-binding fragment thereof according to claim 10, where the antibody to PD-1 contains: the heavy chain shown in SEQ ID NO: 74, and the light chain shown in SEQ ID NO: 75. 12. An antibody to PD-1 or an antigen-binding fragment thereof according to any one of paragraphs. 1-11, wherein the antigen binding fragment is selected from the group consisting of Fab, Fab', F(ab') ₂ , single chain antibody (scFv), dimerized V region (diabody) and disulfide stabilized V region (dsFv). 13. Pharmaceutical composition for the treatment of diseases associated with PD-1, containing: a therapeutically effective amount of an anti-PD-1 antibody or an antigen-binding fragment thereof according to any one of claims. 1-12 and one or more pharmaceutically acceptable carriers, diluents, buffers or excipients. 14. A nucleic acid molecule encoding an antibody to PD-1 or an antigen-binding fragment thereof according to any one of claims. 1-12. 15. Host cell for producing an antibody to PD-1 or an antigen-binding fragment thereof according to any one of paragraphs. 1-12, containing a nucleic acid molecule according to claim 14. 16. A method of treating diseases associated with PD-1, comprising administering to a subject a therapeutically effective amount of an anti-PD-1 antibody or an antigen-binding fragment thereof according to any one of claims. 1-12 or a pharmaceutical composition according to claim 13, or a nucleic acid molecule according to claim 14. 17. The method according to claim 16, where the disease is a tumor. 18. The method according to claim 17, where the tumor is selected from the group consisting of: squamous cell carcinoma of the head and neck, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, cancer of the central nervous system, neuroendocrine tumor, pharyngeal cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatoma, hepatocellular carcinoma, hepatobiliary cancer, pancreatic cancer, stomach cancer, gastrointestinal cancer, intestinal cancer, colon cancer colon, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, bone cancer, chondrosarcoma, myeloma , multiple myeloma, myelodysplastic syndrome, myeloproliferative neoplasm, squamous cell carcinoma, Ewing's sarcoma, systemic light chain amyloidosis and Merkel cell carcinoma. 19. The method according to claim 18, where the lymphoma is selected from the group consisting of: Hodgkin lymphoma, non-Hodgkin lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, large cell T-cell/histiocyte-rich B-cell lymphoma and lymphoplasmacytic lymphoma, lung cancer selected from the group consisting of: non-small cell lung cancer and small cell lung cancer, leukemia selected from the group consisting of: chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia and myeloid leukemia. 20. The method of claim 17, wherein the tumor is selected from the group consisting of: PD-L1-positive melanoma, lung cancer, non-small cell lung cancer, breast cancer, stomach cancer, kidney cancer, bladder cancer, bowel cancer and colorectal cancer intestines.