CN111744013B

CN111744013B - Methods and pharmaceutical combinations for treating diseases using anti-TIGIT antibodies in combination with PD-1 inhibitors

Info

Publication number: CN111744013B
Application number: CN202010228904.5A
Authority: CN
Inventors: 付雅媛; 许志宾; 陶维康
Original assignee: Jiangsu Hengrui Medicine Co Ltd; Shanghai Hengrui Pharmaceutical Co Ltd
Current assignee: Jiangsu Hengrui Medicine Co Ltd; Shanghai Hengrui Pharmaceutical Co Ltd
Priority date: 2019-03-29
Filing date: 2020-03-27
Publication date: 2022-07-26
Anticipated expiration: 2040-03-27
Also published as: CN111744013A

Abstract

The present disclosure relates to methods and pharmaceutical combinations of an anti-TIGIT antibody in combination with a PD-1 inhibitor for treating disease. Further, the disclosure relates to medicaments of anti-TIGIT antibodies or antigen-binding fragments thereof in combination with PD-1 inhibitors (e.g., anti-PD-1 antibodies) for the treatment of human TIGIT-associated diseases (e.g., tumors).

Description

Methods and pharmaceutical combinations for treating diseases using anti-TIGIT antibodies in combination with PD-1 inhibitors

Technical Field

The present disclosure relates to methods and pharmaceutical combinations of an anti-TIGIT antibody in combination with a PD-1 inhibitor for treating disease.

Background

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

In recent years, immune checkpoint therapy against immune cell co-inhibitory receptors has made tremendous progress in tumor immunotherapy, and the discovery and validation of new co-inhibitory receptors has become a global competitive hotspot. T cells are key mediators of the immune response, and activation of T cells is dependent on TCR and costimulatory signals. The costimulatory signal is then a limiting signal for T cell activation, the dysfunction of which is involved in the development of autoimmune diseases (Immunol Rev,2012,248: 122-139; Autoimmun Rev,2013,12: 1171-1176). Tigit (T cell immunoglobulin and ITIM domain) is a newly discovered co-inhibitory signal molecule located on the surface of NK cells and T cells, and is closely related to the functional regulation of T cells, NK cells, dendritic cell DCs, and the like.

The TIGIT gene is located on human chromosome 16 and encodes a type I transmembrane protein consisting of 244 amino acids. The human TIGIT molecule has 141 amino acids in the ectodomain and 1 immunoglobulin V-like domain; 23 amino acids in the transmembrane region; the cytoplasmic region is short, 80 amino acids, with 1 PDZ binding domain and 1 ITIM motif. TIGIT molecules belong to a member of IgSF (immunoglobulin superfamily IgSF), the structures of which are relatively conservative, homologous molecules of which are found in various mammals, and human TIGIT molecules have 88%, 67% and 58% homology with TIGIT molecules of monkeys, dogs and mice, respectively (Nat Immunol,2009,10 (1): 48-57).

The TIGIT molecule is predominantly expressed on the surface of T cells and NK cells (Nat Immunol,2009,10: 48-57).

Both T cells and resting memory T cells expressed low expression of TIGIT and were up-regulated upon in vitro activation (J Immunol,2012,188: 3869-3875). NK cell surface TIGIT has higher expression level (Proc Natl Acad Sci USA, 2009, 106(42): 17858-17863). TIGIT is a novel potential immunotherapy target. Monoclonal antibodies that specifically block TIGIT have been shown to exhibit significant anti-tumor effects in animal models (Martinet and Smyth 2015). TIGIT antibodies have been reported in WO2009126688, WO2014089113, WO2015009856, WO2015143343, WO2015174439, WO2017053748, WO2017030823, WO2016106302, US20160176963, US20130251720 and other patents.

PD-l (programmed death molecule 1) is a protein receptor expressed on the surface of T cells discovered in 1992 and is involved in the process of apoptosis. PD-l belongs to the CD28 family, has 23% amino acid homology with cytotoxic T lymphocyte antigen 4 (CTLA-4), but is expressed differently from CTLA, mainly on activated T cells, B cells and myeloid cells. PD-1 has two ligands, PD-L1 and PD-L2. PD-L1 is expressed predominantly on T cells, B cells, macrophages and Dendritic Cells (DCs), and expression on activated cells can be upregulated. While expression of PD-L2 is relatively restricted, it is predominantly expressed on antigen presenting cells, such as activated macrophages and dendritic cells.

PD-L1 inhibits the immune system by binding to PD-1 and B7-1, and many tumor cells and immune cells in the microenvironment of tumor tissues express PD-L1. The new research finds that the expression of high PD-L1 protein is detected in human tumor tissues such as breast cancer, lung cancer, gastric cancer, intestinal cancer, renal cancer, melanoma, non-small cell lung cancer, colon cancer, bladder cancer, ovarian cancer, pancreatic cancer, liver cancer and the like, and the expression level of PD-L1 is closely related to the clinic and prognosis of patients.

The anti-PD-L1/PD-1 monoclonal antibody can block the combination between PD-L1/PD-1, and improve the immune response of a patient to a tumor to the maximum extent, thereby achieving the aim of killing tumor cells. Currently, a variety of PD-1 inhibitors are internationally approved for the market, including anti-PD-1 antibodies, such as: nivolumab (trade name Opdivo), Pembrolizumab (trade name Keytruda); anti-PD-L1 antibodies, for example: atezolizumab (trade name tecentiq), Avelumab (trade name Bavencio), and Durvalumab (trade name Imfinzi) for the treatment of tumors such as melanoma, hodgkin lymphoma patients, non-small cell lung cancer, kidney cancer, bladder cancer, head and neck squamous cell carcinoma, gastric cancer, liver cancer, mercker skin cancer, and all microsatellite-unstable solid tumors. Domestic anti-PD-1 antibodies: tereprinizumab (Junchenyibio) and Cedilizumab (Xindar bio) have also been approved for marketing in China, and Carrilizumab (SHR1210, Henrie medicine) and Terrilizumab (Baiji State) have also been filed for drug marketing. In addition, WO2017084495a1 discloses anti-PDL-1 antibodies: HRP00052 and HRP00049, other patent documents WO 013978, WO2013173223, WO2014195852, WO2013181634, WO2015048520, WO2015036511, US2014335093, WO2014100079, WO2014055897, US6803192B1, WO2014022758, US8617546B2, WO2010089411a2, WO2015085847, EP3378871a1 and the like also disclose various anti-PD-L1/PD-1 monoclonal antibodies.

In addition, WO2015009856 discloses the combination of a PD-1 axis binding antagonist and an agent that decreases TIGIT expression or activity to treat cancer. However, the Chew et al study found that the combination of TIGIT and PD-1 antibodies promoted the killing of HIV and melanoma by CD8T cells, but this effect disappeared with the blockade of CD226 (Chew, Fujita et al.2016).

Disclosure of Invention

The present disclosure provides anti-TIGIT antibodies in combination with PD-1 inhibitors for the treatment of disease.

In one aspect, the present disclosure provides a method of treating a disease associated with human TIGIT, the method comprising administering to a subject a pharmaceutically effective amount of a monoclonal antibody, or antigen-binding fragment thereof, that specifically binds human TIGIT, wherein the monoclonal antibody, or antigen-binding fragment thereof, that specifically binds human TIGIT comprises a heavy chain variable region and a light chain variable region, and a pharmaceutically effective amount of a PD-1 inhibitor, wherein:

i) the heavy chain variable region comprises a sequence identical to SEQ ID NO:5 HCDR1, HCDR2 and HCDR3 have the same sequences as HCDR1, HCDR2 and HCDR3 respectively, and the light chain variable region comprises a light chain variable region having the same sequence as SEQ ID NO: LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in FIG. 6 have LCDR1, LCDR2 and LCDR3 having the same sequence;

ii) the heavy chain variable region comprises a sequence identical to the sequence of SEQ ID NO: HCDR1, HCDR2 and HCDR3 of the heavy chain variable region depicted in fig. 7 have HCDR1, HCDR2 and HCDR3 of the same sequence, and the light chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in fig. 8 have LCDR1, LCDR2 and LCDR3 of the same sequence;

iii) the variable region of the heavy chain comprises a sequence identical to the sequence of SEQ ID NO: HCDR1, HCDR2 and HCDR3 of the heavy chain variable region shown in fig. 9 have HCDR1, HCDR2 and HCDR3 of the same sequence, and the light chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in fig. 10 have LCDR1, LCDR2 and LCDR3 of the same sequence;

iv) the heavy chain variable region comprises a sequence identical to SEQ ID NO: 11 and HCDR1, HCDR2 and HCDR3 having the same sequences as HCDR1, HCDR2 and HCDR3 of the heavy chain variable region, the light chain variable region comprising a light chain variable region having the sequence of SEQ ID NO: LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in fig. 12 have LCDR1, LCDR2 and LCDR3 of the same sequence; or

v) the heavy chain variable region comprises a sequence identical to SEQ ID NO: 13, and HCDR1, HCDR2 and HCDR3 having the same sequences as HCDR1, HCDR2 and HCDR3 of the heavy chain variable region, and the light chain variable region comprises a light chain variable region having the same sequence as SEQ ID NO: LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in fig. 14 have the same sequence as LCDR1, LCDR2 and LCDR 3. In some embodiments, the monoclonal antibody or antigen binding fragment thereof that specifically binds to human TIGIT and the PD-1 inhibitor are administered simultaneously, or sequentially.

In one aspect, the present disclosure provides use of a monoclonal antibody or antigen-binding fragment thereof that specifically binds to human TIGIT in combination with a PD-1 inhibitor in the manufacture of a medicament for treating a disease associated with human TIGIT, wherein the monoclonal antibody or antigen-binding fragment thereof that specifically binds to human TIGIT comprises a heavy chain variable region and a light chain variable region, wherein:

i) the heavy chain variable region comprises a heavy chain variable region corresponding to the amino acid sequence set forth in SEQ ID NO:5, and a light chain variable region comprising HCDR1, HCDR2 and HCDR3 having the same sequence as the heavy chain variable region shown in SEQ ID NO:6, LCDR1, LCDR2, and LCDR3 having the same sequence as the light chain variable region shown in SEQ ID NO;

ii) the heavy chain variable region comprises a sequence identical to the sequence set forth in SEQ ID NO:7, and a light chain variable region comprising HCDR1, HCDR2, and HCDR3 having the same sequence as the heavy chain variable region shown in SEQ ID NO: LCDR1, LCDR2 and LCDR3 having the same sequence as the light chain variable region set forth in fig. 8;

iii) the heavy chain variable region comprises a sequence identical to the sequence set forth in SEQ ID NO:9, and a light chain variable region comprising HCDR1, HCDR2, and HCDR3 having the same sequence as the heavy chain variable region shown in SEQ ID NO: 10, LCDR1, LCDR2, and LCDR3 having the same sequence as the light chain variable region set forth in seq id No. 10;

iv) the heavy chain variable region comprises a sequence identical to the sequence set forth in SEQ ID NO: 11, and a light chain variable region comprising HCDR1, HCDR2, and HCDR3 having the same sequence as the heavy chain variable region set forth in SEQ ID NO: 12, LCDR1, LCDR2 and LCDR3 having the same sequence as the light chain variable region shown in fig. 12; or

v) the heavy chain variable region comprises a sequence identical to the sequence set forth in SEQ ID NO: 13, and a light chain variable region comprising HCDR1, HCDR2 and HCDR3 having the same sequence as the heavy chain variable region shown in SEQ ID NO: 14, LCDR1, LCDR2 and LCDR 3. In some embodiments, the monoclonal antibody or antigen binding fragment thereof that specifically binds to human TIGIT and the PD-1 inhibitor are administered simultaneously, or sequentially.

In another aspect, the present disclosure provides a monoclonal antibody or antigen-binding fragment thereof that specifically binds to human TIGIT and a PD-1 inhibitor as medicaments, wherein the monoclonal antibody or antigen-binding fragment thereof that specifically binds to human TIGIT comprises a heavy chain variable region and a light chain variable region, wherein:

ii) the heavy chain variable region comprises a sequence identical to SEQ ID NO:7 and HCDR1, HCDR2 and HCDR3 having the same sequences as HCDR1, HCDR2 and HCDR3 of the heavy chain variable region, the light chain variable region comprising a light chain variable region having the sequence of SEQ ID NO: LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in fig. 8 have LCDR1, LCDR2 and LCDR3 of the same sequence;

iii) the heavy chain variable region comprises a sequence identical to SEQ ID NO:9 HCDR1, HCDR2 and HCDR3 have the same sequences as HCDR1, HCDR2 and HCDR3 respectively, and the light chain variable region comprises a light chain variable region having the same sequence as SEQ ID NO: LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in fig. 10 have LCDR1, LCDR2 and LCDR3 of the same sequence;

iv) the heavy chain variable region comprises a sequence identical to SEQ ID NO: 11 and HCDR1, HCDR2 and HCDR3 having the same sequences as HCDR1, HCDR2 and HCDR3, respectively, and the light chain variable region comprises a light chain variable region having the same sequence as SEQ ID NO: LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in fig. 12 have LCDR1, LCDR2 and LCDR3 of the same sequence; or

v) the heavy chain variable region comprises a sequence identical to SEQ ID NO: 13, and HCDR1, HCDR2 and HCDR3 having the same sequences as HCDR1, HCDR2 and HCDR3 of the heavy chain variable region, and the light chain variable region comprises a light chain variable region having the same sequence as SEQ ID NO: LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in fig. 14 have LCDR1, LCDR2 and LCDR3 with the same sequence. In some embodiments, the medicament is for treating a disease associated with human TIGIT. In some embodiments, the monoclonal antibody or antigen-binding fragment thereof that specifically binds to human TIGIT and the PD-1 inhibitor are administered simultaneously or sequentially with the treatment.

In some embodiments, wherein the monoclonal antibody or antigen-binding fragment thereof that specifically binds to human TIGIT of any of the foregoing comprises a heavy chain variable region and a light chain variable region, wherein:

vi) the heavy chain variable region comprises the amino acid sequences shown in SEQ ID NOs: 15. 16 and 17, and a light chain variable region comprising the amino acid sequences of HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOs: 18. 19 and 20 amino acid sequences shown as LCDR1, LCDR2, and LCDR 3;

vii) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 21. 22 and 23, and a light chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 24. LCDR1, LCDR2 and LCDR3 as shown in amino acid sequences 25 and 26;

viii) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 27. 28 and 29, and a light chain variable region comprising the amino acid sequences HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NOs: 30. LCDR1, LCDR2 and LCDR3 as shown in amino acid sequences 31 and 32;

ix) the heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 33. 34 and 35, and a light chain variable region comprising the amino acid sequences shown in SEQ ID NOs: 36. LCDR1, LCDR2 and LCDR3 as shown in amino acid sequences 37 and 38; or

x) the heavy chain variable region comprises the amino acid sequences as set forth in SEQ ID NOs: 39. 40 and 41, and a light chain variable region comprising the amino acid sequences HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOs: 42. LCDR1, LCDR2 and LCDR3 as shown in amino acid sequences 43 and 44.

In some embodiments, wherein any one of the monoclonal antibodies or antigen binding fragments thereof that specifically bind to human TIGIT comprises:

a) the heavy chain variable region as set forth in SEQ ID NO 45 or 50 and the light chain variable region as set forth in SEQ ID NO 46, 47, 48 or 49;

b) the heavy chain variable region as set forth in SEQ ID NO 51, 54 or 55 and the light chain variable region as set forth in SEQ ID NO 52 or 53;

c) the heavy chain variable region as set forth in SEQ ID NO 56, 61, 62 or 63 and the light chain variable region as set forth in SEQ ID NO 57, 58, 59 or 60;

d) the heavy chain variable region as set forth in SEQ ID NO 64, 67, 68, 69 or 70 and the light chain variable region as set forth in SEQ ID NO 65 or 66; or

e) The heavy chain variable region as set forth in SEQ ID NO 71, 75, 76 or 77 and the light chain variable region as set forth in SEQ ID NO 72, 73 or 74.

Preferably, the monoclonal antibody or antigen-binding fragment thereof that specifically binds to human TIGIT comprises an amino acid sequence set forth as SEQ ID NO:51 and the heavy chain variable region as set forth in SEQ ID NO: a light chain variable region shown at 53; or as shown in SEQ ID NO:64 and the heavy chain variable region as set forth in SEQ ID NO:65, light chain variable region shown.

In some embodiments, wherein any of the monoclonal antibodies described above that specifically binds to human TIGIT is a full length antibody comprising human antibody constant regions; in some embodiments, the monoclonal antibody that specifically binds to human TIGIT comprises a human antibody heavy chain constant region as set forth in SEQ ID NO. 78 and/or a human antibody light chain constant region as set forth in SEQ ID NO. 79.

In some embodiments, wherein the monoclonal antibody that specifically binds to human TIGIT comprises:

f) a heavy chain as shown in SEQ ID NO. 83, and a light chain as shown in SEQ ID NO. 82;

g) a heavy chain as set forth in SEQ ID NO. 85 and a light chain as set forth in SEQ ID NO. 84;

h) the heavy chain as shown in SEQ ID NO. 87 and the light chain as shown in SEQ ID NO. 86;

i) a heavy chain as set forth in SEQ ID NO. 89, and a light chain as set forth in SEQ ID NO. 88; or

j) The heavy chain shown as SEQ ID NO. 91, and the light chain shown as SEQ ID NO. 90.

In some embodiments, wherein the PD-1 inhibitor is selected from: anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-PD-L2 antibodies.

In some embodiments, wherein the PD-1 inhibitor is selected from: nivolumab, Pembrolizumab, Atezolizumab, Avelumab, Durvalumab, tereprinimab, sediluzumab, tiraleuzumab, caprolixizumab, HRP00052, and HRP 00049.

In some embodiments, wherein the PD-1 inhibitor is selected from the group consisting of cabrayleigh mab and the monoclonal antibody that specifically binds to human TIGIT is selected from the group consisting of: h1707-02 (heavy chain as SEQ ID NO:83, light chain as SEQ ID NO: 82), h1711-04 (heavy chain as SEQ ID NO:91, light chain as SEQ ID NO: 90), h1709-10 (heavy chain as SEQ ID NO:87, light chain as SEQ ID NO: 86), h1708-04 (heavy chain as SEQ ID NO:85, light chain as SEQ ID NO: 84) and h1710-01 (heavy chain as SEQ ID NO:89, light chain as SEQ ID NO: 88).

In some embodiments, the disease of any of the foregoing, wherein the disease is a T cell dysfunctional disorder; in some embodiments, wherein the disease is: a tumor, an immune disease, or an infectious disorder; in some embodiments, wherein the disease is a CD 155-positive or PVR-positive tumor, an immunological disease, or an infectious disorder; in some embodiments, wherein the tumor is selected from the group consisting of: head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumor, throat cancer, nasopharyngeal carcinoma, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, hepatic carcinoma, hepatobiliary cancer, pancreatic cancer, gastric cancer, gastrointestinal cancer, intestinal cancer, colon cancer, colorectal cancer, renal cancer, clear cell renal cell cancer, 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 tumors, squamous cell carcinoma, ewing's sarcoma, systemic light chain amyloidosis, and merkel cell carcinoma; in some 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 in T-cells/histiocytes, and lymphoplasmacytic lymphoma, said lung cancer being selected from the group consisting of: non-small cell lung cancer and small cell lung cancer, said leukemia being selected from: chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and myeloid leukemia.

In other embodiments, the present disclosure provides a pharmaceutical composition comprising a monoclonal antibody or antigen-binding fragment thereof that specifically binds to human TIGIT, and a PD-1 inhibitor, wherein the monoclonal antibody or antigen-binding fragment thereof that specifically binds to human TIGIT comprises a heavy chain variable region and a light chain variable region, wherein:

ii) the heavy chain variable region comprises a sequence identical to the sequence set forth in SEQ ID NO:7, and a light chain variable region comprising HCDR1, HCDR2 and HCDR3 having the same sequence as the heavy chain variable region shown in SEQ ID NO: LCDR1, LCDR2 and LCDR3 having the same sequence as the light chain variable region shown in fig. 8;

iii) the heavy chain variable region comprises a sequence identical to the sequence set forth in SEQ ID NO:9, and a light chain variable region comprising HCDR1, HCDR2 and HCDR3 having the same sequence as the heavy chain variable region shown in SEQ ID NO: 10, LCDR1, LCDR2, and LCDR3 having the same sequence as the light chain variable region set forth in fig. 10;

iv) the heavy chain variable region comprises a sequence identical to the sequence set forth in SEQ ID NO: 11, and a light chain variable region comprising HCDR1, HCDR2 and HCDR3 having the same sequence as the heavy chain variable region shown in SEQ ID NO: 12, LCDR1, LCDR2, and LCDR3 having the same sequence as the light chain variable region set forth in fig. 12; or

v) the heavy chain variable region comprises a sequence substantially identical to the sequence set forth in SEQ ID NO: 13, and a light chain variable region comprising HCDR1, HCDR2, and HCDR3 having the same sequence as the heavy chain variable region set forth in SEQ ID NO: 14, LCDR1, LCDR2 and LCDR3 having the same sequence as the light chain variable region shown in fig. 14. In some embodiments, the monoclonal antibody or antigen binding fragment thereof that specifically binds to human TIGIT and the PD-1 inhibitor are mixed directly, mixed prior to use, or mixed in a subject at the time of administration. In some embodiments, the pharmaceutical composition is for treating a tumor. In other embodiments, the present disclosure provides a pharmaceutical combination comprising a package insert further comprising a monoclonal antibody or antigen-binding fragment thereof that specifically binds to human TIGIT and/or a PD-1 inhibitor, the package insert comprising instructions for using the monoclonal antibody or antigen-binding fragment thereof that specifically binds to human TIGIT in combination with the PD-1 inhibitor to treat a tumor, wherein the monoclonal antibody or antigen-binding fragment thereof that specifically binds to human TIGIT comprises a heavy chain variable region and a light chain variable region, wherein:

i) the heavy chain variable region comprises a sequence identical to SEQ ID NO:5, and HCDR1, HCDR2 and HCDR3 having the same sequences as HCDR1, HCDR2 and HCDR3, respectively, and a light chain variable region comprising a heavy chain variable region having the same sequence as SEQ ID NO: LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in FIG. 6 have LCDR1, LCDR2 and LCDR3 having the same sequence;

ii) the heavy chain variable region comprises a sequence identical to SEQ ID NO: HCDR1, HCDR2 and HCDR3 of the heavy chain variable region depicted in fig. 7 have HCDR1, HCDR2 and HCDR3 of the same sequence, and the light chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: LCDR1, LCDR2 and LCDR3 of the light chain variable region set forth in fig. 8 have LCDR1, LCDR2 and LCDR3 of the same sequence;

iii) the variable region of the heavy chain comprises a sequence identical to the sequence of SEQ ID NO:9 HCDR1, HCDR2 and HCDR3 have the same sequences as HCDR1, HCDR2 and HCDR3 respectively, and the light chain variable region comprises a light chain variable region having the same sequence as SEQ ID NO: LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in fig. 10 have LCDR1, LCDR2 and LCDR3 of the same sequence;

iv) the heavy chain variable region comprises a sequence identical to SEQ ID NO: 11 and HCDR1, HCDR2 and HCDR3 having the same sequences as HCDR1, HCDR2 and HCDR3, respectively, and the light chain variable region comprises a light chain variable region having the same sequence as SEQ ID NO: 12, wherein the light chain variable region shown in fig. 12 has LCDR1, LCDR2 and LCDR3 with the same sequence as LCDR1, LCDR2 and LCDR 3; or

v) the heavy chain variable region comprises a sequence identical to SEQ ID NO: 13, and HCDR1, HCDR2 and HCDR3 having the same sequences as HCDR1, HCDR2 and HCDR3 of the heavy chain variable region, and the light chain variable region comprises a light chain variable region having the same sequence as SEQ ID NO: LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in fig. 14 have LCDR1, LCDR2 and LCDR3 with the same sequence. In some embodiments, the pharmaceutical composition is for treating a tumor.

In other embodiments, the present disclosure provides a kit comprising a package insert further comprising a monoclonal antibody or antigen-binding fragment thereof that specifically binds to human TIGIT and/or a PD-1 inhibitor, the package insert comprising instructions for using the monoclonal antibody or antigen-binding fragment thereof that specifically binds to human TIGIT in combination with the PD-1 inhibitor to treat a tumor, wherein the monoclonal antibody or antigen-binding fragment thereof that specifically binds to human TIGIT comprises a heavy chain variable region and a light chain variable region, wherein:

i) the heavy chain variable region comprises a heavy chain variable region corresponding to the amino acid sequence set forth in SEQ ID NO:5, and a light chain variable region comprising HCDR1, HCDR2, and HCDR3 having the same sequence as the heavy chain variable region shown in SEQ ID NO:6, LCDR1, LCDR2, and LCDR3 having the same sequence as the light chain variable region shown in SEQ ID NO;

ii) the heavy chain variable region comprises a sequence substantially identical to the sequence set forth in SEQ ID NO:7, and a light chain variable region comprising HCDR1, HCDR2 and HCDR3 having the same sequence as the heavy chain variable region shown in SEQ ID NO: LCDR1, LCDR2 and LCDR3 having the same sequence as the light chain variable region shown in fig. 8;

iii) the heavy chain variable region comprises a sequence substantially identical to the sequence set forth in SEQ ID NO:9, and a light chain variable region comprising HCDR1, HCDR2, and HCDR3 having the same sequence as the heavy chain variable region shown in SEQ ID NO: 10, LCDR1, LCDR2, and LCDR3 having the same sequence as the light chain variable region set forth in fig. 10;

iv) the heavy chain variable region comprises a sequence substantially identical to the sequence set forth in SEQ ID NO: 11, and a light chain variable region comprising HCDR1, HCDR2 and HCDR3 having the same sequence as the heavy chain variable region shown in SEQ ID NO: 12, LCDR1, LCDR2 and LCDR3 having the same sequence as the light chain variable region shown in fig. 12; or

v) the heavy chain variable region comprises a sequence identical to the sequence set forth in SEQ ID NO: 13, and a light chain variable region comprising HCDR1, HCDR2 and HCDR3 having the same sequence as the heavy chain variable region shown in SEQ ID NO: 14, LCDR1, LCDR2 and LCDR 3. In some embodiments, the kit is for treating a tumor.

In some embodiments, 0.01 to 99% by weight of a monoclonal antibody or antigen-binding fragment thereof that specifically binds to human TIGIT and a PD-1 inhibitor can be contained in the pharmaceutical composition or kit unit dose of any of the foregoing; in some embodiments, the pharmaceutical composition contains a monoclonal antibody or antigen-binding fragment thereof that specifically binds to human TIGIT and/or a PD-1 inhibitor in an amount of 0.1-2000 mg; in still other embodiments, the pharmaceutical composition contains a monoclonal antibody or antigen-binding fragment thereof that specifically binds to human TIGIT and/or the PD-1 inhibitor in an amount of 1-1000mg per unit dose.

In some embodiments, the pharmaceutical composition or kit of any one of the preceding, wherein the monoclonal antibody or antigen-binding fragment thereof that specifically binds to human TIGIT comprises a heavy chain variable region and a light chain variable region, wherein:

k) the heavy chain variable region comprises the amino acid sequences shown as SEQ ID NO: 15. 16 and 17, and a light chain variable region comprising the amino acid sequences of HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOs: 18. 19 and 20 amino acid sequences shown as LCDR1, LCDR2, and LCDR 3;

l) the heavy chain variable region comprises the amino acid sequences shown in SEQ ID NOs: 21. 22 and 23, and a light chain variable region comprising the amino acid sequences of HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NOs: 24. LCDR1, LCDR2 and LCDR3 as shown in amino acid sequences 25 and 26;

m) the heavy chain variable region comprises the amino acid sequences shown in SEQ ID NO: 27. 28 and 29, and a light chain variable region comprising the amino acid sequences HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOs: 30. LCDR1, LCDR2 and LCDR3 as shown in amino acid sequences 31 and 32;

n) the heavy chain variable region comprises the amino acid sequences shown as SEQ ID NO: 33. 34 and 35, and a light chain variable region comprising the amino acid sequences shown in SEQ ID NOs: 36. LCDR1, LCDR2 and LCDR3 as shown in amino acid sequences 37 and 38; or

o) the heavy chain variable region comprises the amino acid sequences shown in SEQ ID NOs: 39. 40 and 41, and a light chain variable region comprising the amino acid sequences HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOs: 42. LCDR1, LCDR2 and LCDR3 as shown in amino acid sequences 43 and 44.

In some embodiments, the pharmaceutical composition or kit of any one of the preceding, wherein the monoclonal antibody or antigen-binding fragment thereof that specifically binds to human TIGIT comprises:

p) the heavy chain variable region as set forth in SEQ ID NO 45 or 50 and the light chain variable region as set forth in SEQ ID NO 46, 47, 48 or 49;

q) the heavy chain variable region as set forth in SEQ ID NO 51, 54 or 55 and the light chain variable region as set forth in SEQ ID NO 52 or 53;

r) the heavy chain variable region as set forth in SEQ ID NO 56, 61, 62 or 63 and the light chain variable region as set forth in SEQ ID NO 57, 58, 59 or 60;

s) the heavy chain variable region as set forth in SEQ ID NO 64, 67, 68, 69 or 70 and the light chain variable region as set forth in SEQ ID NO 65 or 66; or

t) the heavy chain variable region as set forth in SEQ ID NO 71, 75, 76 or 77 and the light chain variable region as set forth in SEQ ID NO 72, 73 or 74.

In some embodiments, the pharmaceutical composition or kit of any one of the preceding, wherein the monoclonal antibody that specifically binds to human TIGIT comprises:

u) a heavy chain as set forth in SEQ ID NO 83 and a light chain as set forth in SEQ ID NO 82;

v) a heavy chain as set forth in SEQ ID NO:85 and a light chain as set forth in SEQ ID NO: 84;

w) a heavy chain as set forth in SEQ ID NO:87 and a light chain as set forth in SEQ ID NO: 86;

x) a heavy chain as set forth in SEQ ID NO. 89, and a light chain as set forth in SEQ ID NO. 88; or

y) a heavy chain as set forth in SEQ ID NO. 91 and a light chain as set forth in SEQ ID NO. 90.

In some embodiments, the pharmaceutical composition or kit of any one of the preceding, wherein the PD-1 inhibitor is selected from the group consisting of: anti-PD-1, anti-PD-L1, and anti-PD-L2 antibodies; preferably, the PD-1 inhibitor is selected from: nivolumab, Pembrolizumab, Atezolizumab, Avelumab, Durvalumab, Terepril mab, Cedilizumab, Terralizumab, Cerralizumab, HRP00052, and HRP 00049.

In some embodiments, the pharmaceutical composition or kit of any one of the preceding claims, wherein the tumor is selected from the group consisting of: head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumor, laryngeal cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatoma, hepatobiliary cancer, pancreatic cancer, gastric cancer, gastrointestinal cancer, intestinal cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell cancer, 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 tumors, squamous cell carcinoma, ewing's sarcoma, systemic light chain amyloidosis, and merkel cell carcinoma; in some 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 in T-cells/histiocytes, and lymphoplasmacytic lymphoma, said lung cancer being selected from the group consisting of: non-small cell lung cancer and small cell lung cancer, said leukemia being selected from: chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and myeloid leukemia. In some embodiments, the tumor is selected from a CD 155-positive or PVR-positive tumor. In some embodiments, the tumor is selected from a CD 155-positive or PVR-positive tumor, and in some embodiments, the tumor is associated with human TIGIT.

Drawings

FIG. 1: the experimental results of the influence of the anti-TIGIT antibody and the anti-PD-1 antibody on the activation of human T lymphocyte leukemia cell lines.

FIG. 2 is a schematic diagram: the effect of anti-TIGIT antibodies in combination with anti-PD-1 antibodies on the engraftment of MC38 tumor-bearing mice; in FIG. 2, vs IgG (D22) indicates the P value of each group relative to the blank IgG group at day 22, where:indicatesp < 0.05; p < 0.01; denotes p < 0.001; (ii) a Column for vs anti-murine PD-1 antibody (D22) shows the P-values at day 22 for each group relative to the anti-murine PD-1 antibody group, where # denotes P < 0.05; # indicates p < 0.01; # # denotes p < 0.001).

Detailed Description

Term(s)

In order that the disclosure may be more readily understood, certain technical and scientific terms are specifically defined below. Unless otherwise specifically defined herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

The three letter and one letter codes for amino acids used in this disclosure are as described in j. biol. chem,243, p3558 (1968).

The term "antibody" as used in this disclosure refers to an immunoglobulin, an intact antibody is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The constant regions of immunoglobulin heavy chains differ in their amino acid composition and arrangement, and thus, their antigenicity. Accordingly, immunoglobulins can be classified into five classes, otherwise known as the isotype of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, with their corresponding heavy chains being the μ chain, the δ chain, the γ chain, the α chain, and the ε chain, respectively. The same class of Ig can be divided into different subclasses according to the differences of amino acid composition of the hinge region and the number and position of disulfide bonds of heavy chains, for example, IgG can be divided into IgG1, IgG2, IgG3 and IgG 4. Light chains are classified as either kappa or lambda chains by differences in the constant regions. Each of the five classes of Ig may have either a kappa chain or a lambda chain.

In the present disclosure, the antibody light chain of the present disclosure may further comprise a light chain constant region comprising a human or murine kappa, lambda chain or variant thereof.

In the present disclosure, the antibody heavy chains of the present disclosure may further comprise a heavy chain constant region comprising human or murine IgG1, IgG2, IgG3, IgG4, or variants thereof.

The sequences of the antibody heavy and light chains, near the N-terminus, are widely varied by about 110 amino acids, the variable region (Fv region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region. The variable regions include 3 hypervariable regions (HVRs) and 4 Framework Regions (FRs) which are relatively sequence conserved. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each of the light chain variable region (LCVR or VL) and heavy chain variable region (HCVR or VH) consists of 3 CDR regions and 4 FR regions, arranged sequentially from amino terminus to carboxy terminus in the order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR 3; the 3 CDR regions of the heavy chain are referred to as HCDR1, HCDR2 and HCDR 3. In some examples, the amino acid residues of the CDRs of the LCVR and HCVR regions of the antibody or antigen-binding fragment are in numbers and positions conforming to known Kabat numbering conventions (LCDR1-3, HCDR 1-3).

"monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies (e.g., antibodies containing naturally occurring mutations or mutations generated during the manufacture of monoclonal antibody preparations, which variants are typically present in minor amounts). Unlike polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation (formulation) is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the identity of the antibody as obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies of the disclosure can be prepared by a variety of techniques including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods that utilize transgenic animals containing all or part of a human immunoglobulin locus, such methods, as well as other exemplary methods for preparing monoclonal antibodies, are described herein.

Antibodies of the present disclosure include murine, chimeric, humanized antibodies.

The term "murine antibody" is used in this disclosure to refer to monoclonal antibodies to an antigen (e.g., human TIGIT) prepared according to the knowledge and skill in the art. The preparation is carried out by injecting a test subject with TIGIT antigen and then isolating hybridomas expressing antibodies with the desired sequence or functional properties. In a preferred embodiment of the present disclosure, the murine TIGIT antibody or antigen binding fragment thereof may further comprise a light chain constant region of a murine kappa, lambda chain or variant thereof, or further comprise a heavy chain constant region of a murine IgG1, IgG2, IgG3, IgG4 or variant thereof.

The term "chimeric antibody" refers to an antibody obtained by fusing a variable region of a murine antibody to a constant region of a human antibody, and can reduce an immune response induced by the murine antibody. Establishing chimeric antibody, firstly establishing hybridoma secreting mouse-derived specific monoclonal antibody, then cloning variable region gene from mouse hybridoma cell, cloning constant region gene of human antibody according to the need, connecting mouse variable region gene and human constant region gene into chimeric gene, inserting into expression vector, and finally expressing chimeric antibody molecule in eukaryotic system or prokaryotic system. In a preferred embodiment of the present disclosure, the antibody light chain of the TIGIT chimeric antibody further comprises a light chain constant region of a human kappa, lambda chain or variant thereof. The antibody heavy chain of the TIGIT chimeric antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4, or a variant thereof, preferably comprises a human IgG1, IgG2, or IgG4 heavy chain constant region, or an IgG1, IgG2, or IgG4 variant using amino acid mutations (e.g., YTE mutation or back mutation, S228P).

The term "humanized antibody", including CDR-grafted antibodies, refers to antibodies produced by grafting murine CDR sequences into human antibody variable region frameworks, i.e., different types of human germline antibody framework sequences. Can overcome the heterogeneous reaction induced by the chimeric antibody because of carrying a large amount of mouse protein components. Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. Germline DNA sequences for genes such as the human heavy and light chain variable regions may be at "VBase "race series database (on the Internet)www.mrccpe.com.ac.uk/vbaseAvailable), and is found in Kabat, e.a. et al, 1991Sequences of Proteins of Immunological Interest, 5 th edition. To avoid decreased immunogenicity and, at the same time, decreased activity, the human antibody variable region framework sequences may be minimally back-mutated or back-mutated to retain activity. The humanized antibodies of the present disclosure also include humanized antibodies after further affinity maturation of the CDRs by phage display. In a preferred embodiment of the present disclosure, the murine CDR sequences of the TIGIT humanized antibody are selected from the group consisting of SEQ ID NO: 15-44; the human antibody variable region framework is designed and selected, wherein the heavy chain FR region sequence on the antibody heavy chain variable region, the human germline heavy chain is selected from the group consisting of: (IGHV3-7 x 01 and hjh2), (IGHV1-46 x 01 and hjh 4.1.1) and (IGHV1-69 x 02 and hjh 4.1.1), and the human germline light chain is selected from the group consisting of: (IGKV1-39 × 02 and hjk2.1), (IGKV1-39 × 01 and hjk4.1) and (IGKV4-1 × 01 and hjk 4.1). In order to avoid the decrease in immunogenicity and the resulting decrease in activity, the variable region of a human antibody may be subjected to minimal reverse mutation (back mutation, i.e., mutation of amino acid residues in the FR region of a human antibody to amino acid residues at positions corresponding to those of the original antibody) to retain the activity. In one embodiment of the disclosure, wherein the light/heavy chain variable region of the TIGIT humanized antibody is back-mutated to obtain the optimized light/heavy chain variable region combination of h1707, h1708, h1709, h17010, h17011 humanized antibody, specifically as shown in tables 1-5 below,

table 1: h1707 humanized antibody light/heavy chain variable region combinatorial table:

note: in the table, "H1707-02V" indicates the H1707-H2 heavy chain variable region (shown in SEQ ID NO: 50) and the light/heavy chain variable region pair with the H1707-L1 light chain variable region (shown in SEQ ID NO: 46), and so on.

Table 2: h1708 humanized antibody light/heavy chain variable region combination table:

note: in the table, "H1708-04V" represents the light/heavy chain variable region pair consisting of the heavy chain variable region H1708-H1 (shown in SEQ ID NO: 51) and the light chain variable region H1708-L2 (shown in SEQ ID NO: 53), and so on.

Table 3: h1709 humanized antibody light/heavy chain variable region combinatorial table:

note: in the table, "H1709-10V" represents the light/heavy chain variable region pair consisting of the H1709-H2 heavy chain variable region (shown in SEQ ID NO: 61) and the H1709-L3 light chain variable region (shown in SEQ ID NO: 59), and so on.

Table 4: h1710 humanized antibody light/heavy chain variable region combinatorial table:

note: in the table, "H1710-01V" represents a light/heavy chain variable region pair consisting of H1710-H1 heavy chain variable region (shown in SEQ ID NO: 64) and H1710-L1 light chain variable region (shown in SEQ ID NO: 65), and so on.

Table 5: h1711 humanized antibody light/heavy chain variable region combinatorial table:

note: in the table, "H1711-04V" represents the light/heavy chain variable region pair consisting of H1711-H4 heavy chain variable region (shown as SEQ ID NO: 77) and H1711-L1 light chain variable region (shown as SEQ ID NO: 72), and so on.

The light/heavy chain variable region combinations of the humanized antibodies of the foregoing tables 1-5, whose heavy chain variable region can be linked to the amino terminus of a heavy chain constant region of IgG1, IgG2, IgG3, IgG4 or variants thereof of a human antibody (e.g., a variant of IgG1, IgG2 or IgG4 comprising a human IgG1, IgG2 or IgG4 heavy chain constant region, or using an amino acid mutation such as one or more of a YTE mutation or a back mutation, a S228P mutation, a F234A mutation and an L235A mutation) can be linked to a full-length antibody heavy chain, and whose light chain variable region can be linked to the amino terminus of a light chain constant region of a kappa, lambda chain or variants thereof of a human antibody to a full-length antibody light chain. For example, the light/heavy chain variable region combination H1707-02V, whose heavy chain variable region H1707-H2(SEQ ID NO: 50) is linked to the amino terminus of IgG1, IgG2, IgG3, IgG4 or a variant thereof (e.g., one or more of YTE mutation or reversion mutation, S228P mutation, F234A mutation, and L235A mutation) of a human antibody to form a heavy chain of the antibody, and whose light chain variable region H1707-L1(SEQ ID NO: 46) is linked to the amino terminus of kappa, lambda chain constant region, or a variant thereof of a human antibody to form a light chain of the antibody, and then the heavy chain is linked to the light chain to form a full-length humanized antibody; in one embodiment, the light/heavy chain variable region is combined with H1707-02V and the heavy chain variable region H1707-H2(SEQ ID NO: 50) is combined with a light chain variable region as set forth in SEQ ID NO:78 are linked at the amino terminus of the constant region of the IgG4 variant to form the heavy chain of the antibody (as shown in SEQ ID NO: 83), and the light chain variable region h1707-L1(SEQ ID NO: 46) is linked at the amino terminus of the kappa light chain constant region of the human antibody to form the light chain of the antibody (as shown in SEQ ID NO: 82).

Grafting of CDRs can result in reduced affinity of the TIGIT antibody or antigen-binding fragment thereof for the antigen due to framework residues in contact with the antigen. Such interactions may be the result of somatic hypermutations. Thus, there may still be a need to graft such donor framework amino acids to the framework of a humanized antibody. Amino acid residues from the non-human TIGIT antibody or antigen-binding fragment thereof that are involved in antigen binding can be identified by examining the murine monoclonal antibody variable region sequence and structure. Residues in the CDR donor framework that differ from the germline can be considered related. If the closest germline cannot be determined, the sequence can be compared to a subgroup consensus sequence or a consensus sequence of murine sequences with a high percentage of similarity. Rare framework residues are thought to be likely the result of somatic hypermutation and thus play an important role in binding.

"amino acid difference" in the analogous "variants having 3, 2 or 1 amino acid differences" means that there is an amino acid change or mutation in the variant protein or polypeptide compared to the original protein or polypeptide, including the occurrence of 1 or several amino acid insertions, deletions or substitutions on the basis of the original protein or polypeptide.

The term "antigen-binding fragment" or "functional fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen (e.g., TIGIT). It has been shown that fragments of full-length antibodies can be used to achieve the antigen-binding function of an antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include (i) Fab fragments, monovalent fragments consisting of the VL, VH, CL and CH1 domains; (ii) f (ab') ₂ A fragment, a bivalent fragment comprising two Fab fragments connected by a disulfide bridge at the hinge region, (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) (ii) an Fv fragment consisting of the VH and VL domains of a single arm of an antibody; (v) a single domain or dAb fragment (Ward et al, (1989) Nature 341: 544-546) which consists of a VH domain; and (vi) an isolated Complementarity Determining Region (CDR) or (vii) a combination of two or more isolated CDRs which may optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined by a synthetic linker using recombinant methods, such that it is capable of generating a single protein chain in which the VL and VH regions pair to form monovalent molecules (referred to as single chain Fv (scFv); see, e.g., Bird et al (1988) Science242: 423-. Such single chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as for intact antibodies. Antigen binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins. The antibodies can be of different isotypes, e.g., IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

Antigen binding fragments of the present disclosure include Fab, F (ab ')2, Fab', single chain antibodies (scFv), dimerized V regions (diabodies), disulfide stabilized V regions (dsFv), peptides comprising CDRs, and the like.

Fab is an antibody fragment having a molecular weight of about 50,000 and having an antigen binding activity among fragments obtained by treating an IgG antibody molecule with protease papain (which cleaves the amino acid residue at position 224 of the H chain), in which about half of the N-terminal side of the H chain and the entire L chain are bonded together by a disulfide bond.

The Fab of the present disclosure may be produced by treating a monoclonal antibody of the present disclosure, which specifically recognizes human TIGIT and binds to an amino acid sequence of an extracellular region or a three-dimensional structure thereof, with papain. In addition, the Fab may be produced by inserting DNA encoding the Fab of the antibody into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryote or a eukaryote to express the Fab.

F (ab')2 is an antibody fragment having a molecular weight of about 100,000 and having antigen binding activity and comprising two Fab regions joined at the hinge position obtained by digestion of the lower part of the two disulfide bonds in the IgG hinge region with the enzyme pepsin.

The F (ab')2 of the present disclosure can be produced by treating a monoclonal antibody of the present disclosure, which specifically recognizes human TIGIT and binds to the amino acid sequence of the extracellular region or its three-dimensional structure, with pepsin. Further, the F (ab ')2 can be produced 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 an antigen-binding activity, which is obtained by cleaving the disulfide bond in the hinge region of the above-mentioned F (ab') 2. Fab's of the present disclosure can be produced by treating F (ab')2 of the present disclosure that specifically recognizes TIGIT and binds to the amino acid sequence of the extracellular region or its three-dimensional structure with a reducing agent such as dithiothreitol.

In addition, the Fab ' may be produced by inserting DNA encoding the Fab ' fragment of the antibody into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryote or a eukaryote to express the Fab '.

The term "single chain antibody", "single chain Fv" or "scFv" means a molecule comprising an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) joined by a linker. Such scFv molecules can have the general structure: NH ₂ -VL-linker-VH-COOH or NH ₂ -VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof, for example using variants of 1-4 repeats (Holliger et al (1993), Proc. Natl. Acad. Sci. USA90: 6444-. Other linkers useful in the present disclosure are described by Alfthan et al (1995), Protein Eng.8: 725-.

The scFv of the present disclosure can be produced by the following steps: obtaining cDNA encoding VH and VL of the monoclonal antibody of the present disclosure, which specifically recognizes human TIGIT and binds to the amino acid sequence of the extracellular region or the three-dimensional structure thereof, constructing DNA encoding scFv, inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector, and then introducing the expression vector into a prokaryote or a eukaryote to express scFv.

Diabodies are antibody fragments in which an scFv is dimerized, and are antibody fragments that have bivalent antigen binding activity. In the divalent antigen binding activity, the two antigens may be the same or different.

Diabodies of the present disclosure can be produced by the following steps: obtaining cdnas encoding VH and VL of the monoclonal antibody of the present disclosure that specifically recognizes human TIGIT and binds to an amino acid sequence of an extracellular region or a three-dimensional structure thereof, constructing DNA encoding scFv such that the amino acid sequence of a peptide linker is 8 residues or less in length, inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector, and then introducing the expression vector into a prokaryote or a eukaryote to express a diabody.

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

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

The CDR-containing peptide is constituted by one or more regions in a CDR containing VH or VL. Peptides comprising multiple CDRs may be linked directly or via a suitable peptide linker.

The peptides of the present disclosure comprising CDRs may be produced by: the DNA encoding CDRs of VH and VL of the monoclonal antibody of the present disclosure, which specifically recognizes human TIGIT and binds to the amino acid sequence of the extracellular region or a three-dimensional structure thereof, is constructed, inserted into a prokaryotic expression vector or a eukaryotic expression vector, and then the expression vector is introduced into a prokaryote or a eukaryote to express the peptide. The CDR-containing peptides may also be produced by chemical synthesis methods such as Fmoc method or tBoc method.

The term "antibody framework" as used herein refers to a portion of a variable domain, VL or VH, that serves as a scaffold for the antigen binding loops (CDRs) of that variable domain. It is essentially a variable domain without CDRs.

The term "complementarity determining region", "CDR" or "hypervariable region" refers to one of the 6 hypervariable regions within the variable domain of an antibody which primarily contributes to antigen binding. Typically, there are three CDRs (HCDR1, HCDR2, HCDR3) per heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) per light chain variable region. The amino acid sequence boundaries of the CDRs may be determined using any of a variety of well known protocols, including the "Kabat" numbering convention (see Kabat et Al (1991), "Sequences of Proteins of Immunological Interest", 5 th edition, Public Health Service, National Institutes of Health, Bethesda, MD), "Chothia" numbering convention (see Al-Lazikani et Al, (1997) JMB 273: 927-948) and ImmunoGenTics IMGT (Lefranc M.P., Methylogist, 7, 132-136 (1999); Lefranc, M.P. et Al, Dev.Comp., 27, 55-77(2003), (2003) et Al, for example, following the classical Kabat convention, the numbering convention for the variable domains of the heavy chain (VH) is that of amino acid residues 35-31), the variable domains of the light chain (VH) is that of CDR 35-31), the variable domains of the light chain (CDR 34-3650) (LCDR 3-3634) and the variable domains of the light chain (LCDR 3-11) (35-35) (CDDR 54) and the LCDR 3-11 (35-11) (LCDR 3-11) are followed by the numbering convention for the classical (LCbat numbering convention, CDR amino acid numbers in VH were 26-32(HCDR1), 52-56(HCDR2) and 95-102(HCDR 3); and amino acid residues in VL are numbered 26-32(LCDR1), 50-52(LCDR2) and 91-96(LCDR 3). By combining the CDR definitions of both Kabat and Chothia, the CDRs are made up of amino acid residues 26-35(HCDR1), 50-65(HCDR2) and 95-102(HCDR3) in the human VH and amino acid residues 24-34(LCDR1), 50-56(LCDR2) and 89-97(LCDR3) in the human VL. Following the rules of IMGT, the CDR amino acid residue numbers in VH are roughly 26-35(CDR1), 51-57(CDR2) and 93-102(CDR3), and the CDR amino acid residue numbers in VL are roughly 27-32(CDR1), 50-52(CDR2) and 89-97(CDR 3). Following the IMGT rules, the CDR regions of the antibody can be determined using the program IMGT/DomainGap Align.

The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds (e.g., a specific site on a TIGIT molecule). Epitopes typically comprise at least 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14 or 15 contiguous or non-contiguous amino acids in a unique spatial conformation. See, e.g., Epitope Mapping Protocols in Methods in Molecular B biology, volume 66, g.e. morris, Ed. (1996).

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

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

The term "PD-1 inhibitor" refers to a molecule that reduces, blocks, inhibits, eliminates, or interferes with the signal transduction of the interaction of PD-1 with its ligand (e.g., PD-L1, PD-L2). For example, in some embodiments, PD-1 inhibitors include anti-PD-1 antibodies or antigen-binding fragments thereof, or immunoadhesins thereof, or fusion proteins thereof, or oligopeptides thereof and other molecules that reduce, block, inhibit, eliminate, or interfere with the signaling of PD-1 interaction with PD-L1 and/or PD-L2; in some embodiments, the PD-1 inhibitor comprises an anti-PD-L1 antibody or antigen-binding fragment thereof, or an immunoadhesin thereof, or a fusion protein thereof, or an oligopeptide thereof and other molecules that reduce, block, inhibit, eliminate, or interfere with the signaling of the interaction of PD-1 with PD-L1; in some embodiments, the PD-1 inhibitor comprises an anti-PD-L2 antibody or antigen-binding fragment thereof, or an immunoadhesin thereof, or a fusion protein thereof, or an oligopeptide thereof and other molecules that reduce, block, inhibit, eliminate, or interfere with the signaling of the interaction of PD-1 with PD-L2. In some embodiments, the PD-1 inhibitor is selected from known anti-PD-1 and/or PD-L1 antibodies: for example, antibodies disclosed in Nivolumab (trade name optodivo), Pembrolizumab (trade name Keytruda), Atezolizumab (trade name Tecentriq), Avelumab (trade name Bavencio), Durvalumab (trade name Imfinzi), tereprinimab (juniper bioscience), fiducilizumab (fidaxania), charyleigh mab (SHR1210, constant ruit medicine), tiraleizumab (state of great relief), HRP00052 (see WO 3838 2017084495a1), and HRP00049 (see WO2017084495a1), as well as other patent documents WO013972, WO2013173223, WO2014195852, WO2013181634, WO 048520, WO2015036511, US2014335093, WO2014100079, WO2014055897, US 387 6803192B1, WO 2014758, US8617546B2, WO 36 2, WO 5808580847, WO 084624 a, 20140824 a, 20144647, and/3652, or anti-PD antibodies disclosed in EP 013976, and the like.

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

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

Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art, such as the Cold spring harbor antibody protocols, chapters 5-8 and 15. For example, mice can be immunized with human TIGIT or a fragment thereof, and the resulting antibodies can be renatured, purified, and amino acid sequenced using conventional methods. Antigen-binding fragments can likewise be prepared by conventional methods. The antibody or antigen-binding fragment of the invention is genetically engineered to add one or more human FR regions to the CDR regions of non-human origin. Human FR germline sequences can be obtained from the website http:// IMGT. cities.fr of ImmunoGeneTiCs (IMGT) or from the immunoglobulin journal, 2001ISBN012441351, by aligning the IMGT human antibody variable region germline gene database with the MOE software.

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

Engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified using conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into a GS expression vector. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As a more recommended prior art, mammalian expression systems result in glycosylation of antibodies, particularly at the highly conserved N-terminal site of the Fc region. Stable clones were obtained by expression of antibodies that specifically bind to human TIGIT. Positive clones were expanded in bioreactor serum-free medium to produce antibodies. The antibody-secreting culture medium can be purified by conventional techniques. For example, purification is carried out on an A or G Sepharose FF column containing the adjusted buffer. Non-specifically bound fractions were washed away. And eluting the bound antibody by using a pH gradient method, detecting antibody fragments by using SDS-PAGE, and collecting. The antibody can be concentrated by filtration by a conventional method. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange. The resulting product is immediately frozen, e.g., -70 ℃, or lyophilized.

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

By "treating" is meant administering a therapeutic agent, e.g., a composition comprising any one of the binding compounds of the present disclosure, either internally or externally to a patient who has one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the patient or population being treated to induce regression of such symptoms or to inhibit the development of such symptoms to any clinically measured degree. The amount of therapeutic agent effective to alleviate any particular disease symptom (also referred to as a "therapeutically effective amount") can vary depending on a variety of factors, such as the disease state, age, and weight of the patient, and the ability of the drug to produce a desired therapeutic effect in the patient. Whether a symptom of a disease has been alleviated can be assessed by any clinical test commonly used by physicians or other health care professionals to assess the severity or progression of the symptom. Although embodiments of the present disclosure (e.g., methods of treatment or articles of manufacture) may be ineffective in alleviating the symptoms of each disease of interest, it should alleviate the symptoms of the disease of interest in a statistically significant number of patients as determined by any statistical test known in the art, such as the Student's t-test, chi-square test, U-test by Mann and Whitney, Kruskal-Wallis test (H-test), jonckhere-Terpstra test, and Wilcoxon test.

"conservative modifications" or "conservative substitutions or replacements" refer 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.) such that changes may be made frequently without altering the biological activity of the protein. It is known to The person skilled in The art that, in general, a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter The biological activity (see, for example, Watson et al (1987) Molecular Biology of The Gene, The Benjamin/Cummings pub. Co., page 224, (4 th edition)). In addition, substitution of structurally or functionally similar amino acids is unlikely to abolish biological activity.

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

"exogenous" refers to a substance produced outside an organism, cell or human body as the case may be. "endogenous" refers to a substance produced in a cell, organism, or human body as the case may be.

"homology" refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared x 100. For example, if there are 6 matches or homologies at 10 positions in two sequences when the sequences are optimally aligned, then the two sequences are 60% homologous; two sequences are 95% homologous if there are 95 matches or homologies at 100 positions in the two sequences. In general, comparisons are made when aligning two sequences to obtain the greatest percentage of homology.

As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the words "transformant" and "transformed cell" include the primary test cell and cultures derived therefrom, regardless of the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where different names are intended, they are clearly visible from the context.

As used herein, "polymerase chain reaction" or "PCR" refers to a procedure or technique in which minute amounts of a particular portion of nucleic acid, RNA, and/or DNA are amplified as described, for example, in U.S. patent No. 4,683,195. In general, it is desirable to obtain sequence information from the ends of or beyond the target region so that oligonucleotide primers can be designed; these primers are identical or similar in sequence to the corresponding strands of the template to be amplified. The 5' terminal nucleotide of the 2 primers may coincide with the end of the material to be amplified. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA, phage or plasmid sequences transcribed from total cellular RNA, and the like. See generally Mullis et al (1987) Cold Spring Harbor Symp. Ouant. biol.51: 263; erlich editors, (1989) PCR TECHNOLOGY (Stockton Press, N.Y.). PCR, as used herein, is considered to be one example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample that includes using a known nucleic acid as a primer and a nucleic acid polymerase to amplify or generate a specific portion of the nucleic acid.

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

The kit is a container comprising an agent (e.g., a monoclonal antibody or antigen-binding fragment thereof that specifically binds to human TIGIT and/or a PD-1 inhibitor). Suitable containers include, for example: bottles, vials (e.g., dual chamber vials), syringes (e.g., single or dual chamber syringes), and test tubes. The container may be made of a variety of materials, such as glass or plastic. The kit may comprise a label or package insert. The label or package insert may indicate instructions for use of the medicament within the kit. The kit may further comprise other materials, such as other buffers, diluents, filters, needles, syringes, and the like. The medicament or pharmaceutical composition of the present disclosure may be placed in a kit.

"pharmaceutical composition" or "pharmaceutical combination" means a mixture comprising one or more compounds or physiologically/pharmaceutically acceptable salts or prodrugs thereof together with other chemical components, such as physiologically/pharmaceutically acceptable carriers and excipients. In some embodiments, a pharmaceutical combination of the present disclosure comprises a monoclonal antibody or antigen-binding fragment thereof that specifically binds to human TIGIT, and a PD-1 inhibitor; in other embodiments, a pharmaceutical combination of the disclosure comprises a package insert further comprising a monoclonal antibody or antigen-binding fragment thereof that specifically binds to human TIGIT and/or a PD-1 inhibitor, the package insert comprising instructions for using the monoclonal antibody or antigen-binding fragment thereof that specifically binds to human TIGIT in combination with the PD-1 inhibitor to treat a tumor. In some embodiments, the pharmaceutical combination of the present disclosure, a monoclonal antibody or antigen-binding fragment thereof that specifically binds to human TIGIT and a PD-1 inhibitor are directly mixed to form a pharmaceutical composition, which can further comprise a physiological/pharmaceutically acceptable carrier and excipient, which, when administered, administers the pharmaceutical composition to a subject; in some embodiments, the pharmaceutical combination of the present disclosure, the PD-1 inhibitor and the monoclonal antibody or antigen-binding fragment thereof that specifically binds to human TIGIT are mixed prior to use, i.e., the PD-1 inhibitor and the monoclonal antibody or antigen-binding fragment thereof that specifically binds to human TIGIT are kept separately until they are mixed just prior to use, and the mixture is then administered to the subject; in other embodiments, the pharmaceutical combination of the disclosure, the PD-1 inhibitor and the monoclonal antibody or antigen-binding fragment thereof that specifically binds to human TIGIT are combined in the subject, i.e., the PD-1 inhibitor and the monoclonal antibody or antigen-binding fragment thereof that specifically binds to human TIGIT are administered to the subject separately, and the PD-1 inhibitor and the monoclonal antibody or antigen-binding fragment thereof that specifically binds to human TIGIT are combined in the subject.

The present disclosure "combination" is a mode of administration and may include "simultaneous administration" or "sequential administration". By "concurrently administering" is meant that 1 or more drugs are administered to a subject concurrently; "sequential administration" means that 1 or more drugs are administered to a subject separately, and the order of administration is not particularly limited. For example, in some embodiments of the disclosure, a monoclonal antibody or antigen-binding fragment thereof that specifically binds to human TIGIT and a PD-1 inhibitor are administered to a subject concurrently to treat a disease. In other embodiments, a monoclonal antibody or antigen-binding fragment thereof that specifically binds to human TIGIT and a PD-1 inhibitor are administered sequentially to a subject to treat a disease, for example: administering a monoclonal antibody or antigen-binding fragment thereof that specifically binds to human TIGIT to the subject, followed by administering a PD-1 inhibitor to the subject; or administering a PD-1 inhibitor to the subject prior to administering a monoclonal antibody or antigen binding fragment thereof that specifically binds to human TIGIT to the subject; there may also be sequential administration of the PD-1 inhibitor or the monoclonal antibody or antigen-binding fragment thereof that specifically binds to human TIGIT in any order, e.g., the monoclonal antibody or antigen-binding fragment thereof that specifically binds to human TIGIT is administered to the subject first, followed by administration of the monoclonal antibody or antigen-binding fragment thereof that specifically binds to human TIGIT to the subject again, followed by administration of the PD-1 inhibitor to the subject to treat the disease.

The TIGIT-associated disease is not limited so long as it is a TIGIT-associated disease, e.g., a therapeutic response induced using the molecules of the present disclosure can suppress or inhibit a T cell dysfunctional disorder by binding to human TIGIT, in some embodiments a malignancy, cancer, or infectious disorder, in some embodiments a clinically responsive tumor or cancer type, e.g., a CD 155-positive, or PVR-positive tumor, immunological disease, or infectious disorder, observed in a clinical trial of an immunotherapeutic agent targeting an immunotherapeutic checkpoint. In some embodiments, wherein the tumor is selected from the group consisting of: head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumor, laryngeal cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatoma, hepatobiliary cancer, pancreatic cancer, gastric cancer, gastrointestinal cancer, intestinal cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell cancer, 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 tumors, squamous cell carcinoma, ewing's sarcoma, systemic light chain amyloidosis, and merkel cell carcinoma; in some 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 enriched in T-cells/histiocytes, and lymphoplasmacytic lymphoma, said lung cancer being selected from the group consisting of: non-small cell lung cancer and small cell lung cancer, said leukemia being selected from: chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and myeloid leukemia.

The TIGIT-associated disease described above can be diagnosed by detecting or measuring TIGIT-expressing cells with the monoclonal antibody or antibody fragment of the present disclosure.

In the present disclosure, the method for detecting or determining the amount of TIGIT may be any known method. For example, it includes immunodetection or assay methods.

The immunoassay or measuring method is a method of detecting or measuring the amount of an antibody or the amount of an antigen using a labeled antigen or antibody. Examples of the immunological detection or measurement method include a radioactive substance-labeled immune antibody method (RIA), an enzyme immunoassay (EIA or ELISA), a Fluorescence Immunoassay (FIA), a luminescence immunoassay, a western immunoblotting method, a physicochemical method, and the like.

For detecting cells expressing the polypeptide, a known immunoassay method can be used, and immunoprecipitation, fluorescent cell staining, immunohistological staining, or the like is preferably used. In addition, a fluorescent antibody staining method using FMAT8100HTS system (Applied Biosystem) or the like can be used.

In the present disclosure, the living sample for detecting or measuring TIGIT is not particularly limited as long as it has a possibility of containing cells expressing TIGIT, such as tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid, or culture fluid.

The diagnostic agent containing the monoclonal antibody or antibody fragment thereof of the present disclosure may further contain a reagent for performing an antigen-antibody reaction or a reagent for detecting a reaction, depending on the desired diagnostic method. Reagents for performing antigen-antibody reactions include buffers, salts, and the like. The reagent for detection includes reagents generally used in immunodetection or assay methods, such as a labeled secondary antibody recognizing the monoclonal antibody, an antibody fragment thereof or a binding substance thereof, a substrate corresponding to the label, and the like.

The TIGIT monoclonal antibody or the antigen binding fragment provided by the embodiment of the disclosure has very high specificity to TIGIT and high affinity with TIGIT, wherein the immunogenicity of the humanized antibody is greatly reduced, and simultaneously the specificity, high affinity and excellent in vitro and in vivo activity of the murine antibody are completely retained. In some implementations, the antibodies or antigen-binding fragments that specifically bind to human TIGIT of the present disclosure are described in international patent application PCT/CN2018/108246(WO2019062832a1), which is incorporated herein by reference in its entirety.

Detailed Description

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

Example 1 preparation of TIGIT antigen antibody

1.1 protein design and expression

Human TIGIT protein (Uniprot No. Q495A1) is used as a template of the TIGIT of the disclosure, the amino acid sequences of the antigen and the protein for detection of the disclosure are designed, different labels are fused on the basis of the TIGIT protein optionally, the antigen and the protein for detection of the disclosure are respectively cloned on pHr carrier (self-produced) or pXC-17.4 carrier (LONZA), and the antigen and the protein for detection of the disclosure are obtained by transient expression in 293 cells or stable expression and purification in CHO cells. In the following, TIGIT antigens are not specifically indicated as human TIGIT.

Fusion protein of TIGIT extracellular region and mouse IgG2aFc segment: TIGIT-mFc for immunization and detection

MEFGLSWLFLVAILKGVQCMMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIPEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

SEQ ID NO：1

Note that: the crosshatched portion is the signal peptide and the italicized portion is the mFc.

Fusion protein of TIGIT extracellular region and human IgG1 Fc segment: TIGIT-Fc for detection

MEFGLSWLFLVAILKGVQCMMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIPEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO：2

Note that: the signal peptide is in the cross-hatched portion and Fc in the italic portion.

And (3) full-length TIGIT: used for constructing a TIGIT over-expression cell strain and detecting

SEQ ID NO：3Signal peptide (Single underline)+ extracellular region +

+ intracellular region (italic part)

Fusion protein of cynoTIGIT extracellular region and mouse IgG2aFc segment: CynoTIGIT-mFc for detection

MEFGLSWLFLVAILKGVQCMMTGTIETTGNISAKKGGSVILQCHLSSTMAQVTQVNWEQHDHSLLAIRNAELGWHIYPAFKDRVAPGPGLGLTLQSLTMNDTGEYFCTYHTYPDGTYRGRIFLEVLESSVAEHSARFQIPEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

SEQ ID NO：4

1.2 purification of TIGIT-related recombinant protein, and purification of hybridoma antibody and recombinant antibody

(1) Hybridoma supernatant isolation purification/ProteinG affinity chromatography:

for purification of mouse hybridoma supernatant, preferred protein G was subjected to affinity chromatography, cultured hybridoma cells were centrifuged to obtain supernatant, and 1M Tris-HCl (pH8.0-8.5) was added in an amount of 10-15% by volume based on the volume of the supernatant to adjust the pH of the supernatant. Washing the ProteinG column by using 6M guanidine hydrochloride for 3-5 times of column volume, and then washing the ProteinG column by using pure water for 3-5 times of column volume; equilibrating the column for 3-5 column volumes using, for example, 1 XPBS (pH7.4) buffer as equilibration buffer; the cell supernatant is combined by low flow rate sample loading, and the flow rate is controlled to keep the retention time for about 1min or more; washing the column with 1 × PBS (pH7.4) for 3-5 times of column volume until the UV absorption falls to baseline; eluting with 0.1M acetic acid/sodium acetate (pH3.0) buffer solution, collecting eluate peak according to ultraviolet detection, and rapidly adjusting pH of the eluate product to 5-6 with 1M Tris-HCl (pH8.0) for temporary storage. The eluted product may be subjected to solution displacement by methods well known to those skilled in the art, such as ultrafiltration concentration using an ultrafiltration tube and solution displacement to the desired buffer system, or desalting using size exclusion such as G-25 to replace the desired buffer system, or removing the polymer component from the eluted product using a high resolution size exclusion column such as Superdex 200 to increase the purity of the sample.

(2) Protein A affinity chromatography extraction of Fc-tagged fusion Protein or antibody:

first, cell culture supernatants expressing Fc fusion proteins or antibodies are subjected to high speed centrifugation to collect the supernatants. The ProteinA affinity column was washed 3-5 column volumes with 6M guanidine hydrochloride, then 3-5 column volumes with pure water. The column is equilibrated by 3-5 column volumes using, for example, 1 XPBS (pH7.4) buffer system as the equilibration buffer. The cell supernatant was combined by low flow rate loading, the flow rate was controlled to allow retention time of about 1min or more, and after the combination was completed, the column was washed 3-5 times the column volume with 1 × PBS (ph7.4) until the uv absorbance fell back to baseline. Eluting with 0.1M acetic acid/sodium acetate (pH3.0-3.5) buffer solution, collecting eluate peak according to ultraviolet detection, and rapidly adjusting pH of the eluate to 5-6 with 1M Tris-HCl (pH8.0) for temporary storage. The eluted product may be subjected to solution displacement by methods well known to those skilled in the art, such as ultrafiltration concentration using an ultrafiltration tube and solution displacement to the desired buffer system, or by size exclusion such as desalting with G-25 to replace the desired buffer system, or by removing the polymer component from the eluted product using a high resolution size exclusion column such as Superdex 200 to increase the purity of the sample.

Example 2 preparation of anti-human TIGIT hybridoma monoclonal antibody

2.1 immunization

Anti-human TIGIT monoclonal antibodies were generated by immunization of mice. The experimental SJL white mice, female, 6-8 weeks old (Beijing Witongliwa laboratory animal technology, Inc., animal production license number: SCXK (Jing) 2012-0001). A breeding environment: SPF grade. After the mice are purchased, the mice are raised in a laboratory environment for 1 week, and the light/dark period is regulated for 12/12 hours at the temperature of 20-25 ℃; the humidity is 40-60%. Mice that had been acclimatized were immunized according to the following protocol. The immunizing antigen is human TIGIT extracellular region with mFc (SEQ ID NO: 1).

Immunization protocol: by using

Gold Adjuvant (Sigma Cat No. T2684) and Thermo

Alum (Thermo Cat No.77161) adjuvant cross-immunization. Antigen and adjuvant: (

Gold adjuvants) ratio 1:1, antigen to Adjuvant (Thermo)

Alum) ratio of 3:1, 50. mu.g/mouse (prime), 25. mu.g/mouse (boost). The antigen was emulsified and inoculated for 0, 14, 28, 42, and 56 days. Day 0 Intraperitoneal (IP) injection of 50. mu.g/pellet of post-emulsification antigen. On day 14, 25. mu.g/mouse were injected subcutaneously (sc) at multiple sites (typically 6-8 sites on the back). On days 28 and 42, dorsal or intraperitoneal injections of antigen were selected based on the presence of dorsal lumps and abdominal swelling. Blood was collected on days 21, 35, 49 and 63, and the antibody titer in mouse serum was determined by ELISA. After 4-5 immunizations, mice with high antibody titers in serum and titers approaching a plateau were selected for splenocyte fusion. The booster immunization was performed 3 days before the splenocyte fusion, and 50. mu.g/tube of antigen solution in physiological saline was Intraperitoneally (IP) injected.

2.2 spleen cell fusion

Spleen lymphocytes and myeloma cells Sp2/0 cells Using an optimized PEG-mediated fusion procedure (

CRL-8287 ^TM ) And fusing to obtain the hybridoma cells. The fused hybridoma cells are cultured in a complete medium (containing 20% FBS, 0.5-1 × 10^6/ml,DMEM medium 1 XHAT, 1 XPI) resuspended, seeded in 96-well plates 100. mu.l/well, 37 ℃ 5% CO ₂ After 3-4 days of incubation, 100. mu.l/well of HAT complete medium was supplemented, and culture was continued for 3-4 days until needlepoint-like colonies were formed. The supernatant was removed and 200. mu.l/well of HT complete medium (RPMI-1640 medium containing 20% FBS, 1 XHT and 1 XPI), 37 ℃ C., 5% CO ₂ ELISA detection was performed after 3 days of culture.

2.3 hybridoma cell selection

According to the growth density of the hybridoma cells, the detection of the hybridoma culture supernatant is carried out by combining an ELISA method. And cell binding experiments and cell blocking experiments are carried out on the positive hole cell supernatant combined with ELISA detection. And (3) combining and blocking the positive cell, and timely performing amplification, freezing, seed preservation and two to three times of subcloning until obtaining single cell clone.

TIGIT binding ELISA, HTRF blocking experiments, cell binding experiments and cell blocking experiments are required to detect each subcloned cell. Hybridoma clones obtained by the above experimental screening were further prepared by serum-free cell culture, and the antibodies were purified according to the purification examples for use in the test examples.

2.4 sequencing of hybridoma Positive clones

The cloning of sequences from positive hybridomas is as follows. Hybridoma cells in logarithmic growth phase were harvested, RNA was extracted using Trizol (Invitrogen, Cat No.15596-018) according to the kit instructions, and PrimeScript was used ^TM Reverse transcription using the Reverse Transcriptase kit (Takara, Cat No. 2680A). The cDNA obtained by reverse transcription was amplified by PCR using mouse Ig-Primer Set (Novagen, TB326 Rev. B0503) and then sequenced. The amino acid sequences of the antibody variable regions corresponding to the positive clones m1707, m1708, m1709, m1710 and m1711 obtained by screening were as follows:

m1707-HCVR

EVKLVESGGGLVQPGGSLKLSCAASGFIFSDYHMYWVRQTPEKRLEWVAYISKGGISTYYPDTVKGRFTISRDNAKHTLYLQMSRLKSEDTAMYYCARQSSYDFAMDYWGRGTSVTVSS

SEQ ID NO：5

m1707-LCVR

DIVMTQSHKFMSTSVGVRVSITCKASQDVGTSVAWYQQKPGQSPKLLIYWASARHTGVPDRFTGSGSGTDFTLTITNVQSEDLADYFCQQYSSYPLTFGAGTKLELK

SEQ ID NO：6

m1708-HCVR

QVQLQQPGAELVKPGSSVKLSCKASGYTFTNYWMHWVKQGPGRGLEWIGRIDPDSTGSKYNEKFKTKASLTVDTVSGTAYMQLSSLTSEDSAVYFCAREGAYGYYFDYWGQGTTLTVSS

SEQ ID NO：7

m1708-LCVR

DIQMTQSPASLSASVGETVTITCRASENIYSYLAWYQQKQGKSPQLLVYNARTLAESVPSRFSGSGSGTQFSLKINSLQPEDFGSYYCQYHSGSPLPFGAGTKLALK

SEQ ID NO：8

m1709-HCVR

EVQLQQSGPVLVKPGPSVKISCKASGFTFTDYYMHWVKQSLGKSLEWIGLVYPYNDNTGYNRKFKGKATLTVDTSSSTAYIELNSLTSEDSAVYYCARGGPSNWNYFDYWGQGTTLTVSS

SEQ ID NO：9

m1709-LCVR

DIVMTQSQKFMSTTVGDRVSITCKASQNVVTAVAWYQQKPGQSPKLLIYSASNRYTGVPDRFTGSGSGTDFTLTINNVQSEDLADYFCQQYTLYPLTFGAGTKLELK

SEQ ID NO：10

m1710-HCVR

QVQLQQPGAELVKFGASVKLSCKASGYTFTNYYMHWVKQRPGRGLEWIGRIDPTSGATKYNDNFKGKATLTVDKPSTTAYMQLSSLTSEDSAVYYCAREGGFGYYFDYWGQGTTLTVSS

SEQ ID NO：11

m1710-LCVR

DIQMTQSPASLSASVGETVTITCRTSENIFTYLAWYQQKQGKSPQLLVYNAKTFAEGVPSRFSGSGSGTQFSLKISSLQPEDFGIYYCQHHYGIPLPFGAGTKLELK

SEQ ID NO：12

m1711-HCVR

QVQLQQSGTELVRPGTSVKMSCKASGYTFTNYWIGWAKQRPGHGLEWIGDIYPGGAYTNYNEKFKDKATLTADKSSSTAYMQFSSLTSEDSAIYYCTRGDYYDSSGRAMDYWGQGTSVTVSS

SEQ ID NO：13

m1711-LCVR

DIVMSQSPSSLAVSVGEKVSMSCKSSQSLLYSRNQMNYLAWYQQKPGQSPKLLIYWTSTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPYTFGGGTKLEIK

SEQ ID NO：14

wherein the CDR sequences in each antibody light and heavy chain are as shown in table 6 (CDR sequences determined and annotated by Kabat numbering system).

Table 6: antibody heavy and light chain CDR sequences

Example 3 humanization of murine anti-human TIGIT antibodies

By comparing an IMGT human antibody heavy chain and light chain variable region germline gene database with MOE software, respectively selecting heavy chain and light chain variable region germline genes with high homology with a murine antibody as templates, respectively transplanting CDRs of the murine antibody into corresponding human templates to form variable region sequences in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4. If necessary, amino acids in the FR region are subjected to back mutation to obtain the humanized anti-TIGIT monoclonal antibody. In the following exemplary embodiments, wherein CDR region amino acid residues are determined and annotated by Kabat numbering system.

The light chain and heavy chain variable region of the murine antibody is connected with the light chain and heavy chain constant region of the human antibody to form a chimeric antibody, the chimeric antibody corresponding to the m1707 antibody is named as ch1707, and the rest antibodies are analogized.

3.1 humanization of hybridoma clone m1707

(1) m1707 humanized framework selection

The humanized light chain templates of the murine antibody m1707 are IGKV1-39 x 02 and hjk2.1, the humanized heavy chain templates are IGHV3-7 x 01 and hjh2, and the humanized antibody h1707 is obtained after humanization, and the humanized variable region sequence is as follows:

h1707 VH-CDR graft

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYHMYWVRQAPGKGLEWVAYISKGGISTYYPDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQSSYDFAMDYWGRGTLVTVSS

SEQ ID NO：45

h1707VL-CDR graft

DIQMTQSPSFLSASVGDRVTITCKASQDVGTSVAWYQQKPGKAPKLLIYWASARHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSYPLTFGQGTKLEIK

SEQ ID NO：46

note: the sequence FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the FR sequences being in italics and the CDR sequences being underlined.

(2) The h1707 back-mutations were designed as follows:

table 7: h1707 reverse mutation design

Note: as shown in S60D, the sequence number is determined in accordance with the natural sequence of the amino acid sequence, and the position 60S is changed back to D. Grafted stands for murine antibody CDR-Grafted human germline FR region sequences.

The sequence of the h1707 humanized antibody light/heavy chain variable region after back mutation design is as follows:

h1707-L1 (same as h1707 VL-CDR gradient)

SEQ ID NO：46

>h1707-L2

DIQMTQSPSFLSASVGDRVTITCKASQDVGTSVAWYQQKPGKAPKLLIYWASARHTGVPDRFSGSGSGTDFTLTISSLQPEDFADYYCQQYSSYPLTFGQGTKLEIK

SEQ ID NO：47

>h1707-L3

DIQMTQSPSFLSASVGDRVTITCKASQDVGTSVAWYQQKPGKSPKLLIYWASARHTGVPDRFSGSGSGTDFTLTISSLQPEDFADYYCQQYSSYPLTFGQGTKLEIK

SEQ ID NO：48

>h1707-L4

DIQMTQSPSFLSASVGDRVTITCKASQDVGTSVAWYQQKPGKSPKLLIYWASARHTGVPDRFTGSGSGTDFTLTISSLQPEDFADYYCQQYSSYPLTFGQGTKLEIK

SEQ ID NO：49

H1707-H1 (same as H1707 VH-CDR graft)

SEQ ID NO：45

>h1707-H2

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYHMYWVRQAPGKGLEWVAYISKGGISTYYPDTVKGRFTISRDNAKNSLYLQMSRLRAEDTAVYYCARQSSYDFAMDYWGRGTLVTVSS

SEQ ID NO：50

3.2 humanization of hybridoma clone m1708

(1) m1708 selection of humanized frameworks

The humanized light chain templates of the murine antibody m1708 are IGKV1-39 x 01 and hjk4.1, and the humanized heavy chain templates are IGHV1-46 x 01 and hjh 4.1.1, and the humanized antibody h1708 is obtained after humanization, and the humanized variable region sequences are as follows:

h1708VH-CDR graft

EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQAPGQGLEWMGRIDPDSTGSKYNEKFKTRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREGAYGYYFDYWGQGTLVTVSS

SEQ ID NO：51

h1708VL-CDR graft

DIQMTQSPSSLSASVGDRVTITCRASENIYSYLAWYQQKPGKAPKLLIYNARTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQYHSGSPLPFGGGTKVEIK

SEQ ID NO：52

note: the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, with the FR sequences in italics and the CDR sequences underlined.

(2) The h1708 back mutations were designed as follows:

table 8: h1708 Back-mutation design

Note: A43A was mutated back to S as indicated by A43S, numbering according to the natural sequence of the amino acid sequence. Grafted stands for murine antibody CDR-implanted human germline FR region sequences.

The sequence of the h1708 humanized antibody light/heavy chain variable region after back mutation design is as follows:

h1708-L1 (same as h1708 VL-CDR gradient)

SEQ ID NO：52

>h1708-L2

DIQMTQSPSSLSASVGDRVTITCRASENIYSYLAWYQQKPGKSPKLLIYNARTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQYHSGSPLPFGGGTKVEIK

SEQ ID NO：53

H1708-H1 (same as H1708 VH-CDR graft)

SEQ ID NO：51

>h1708-H2

EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQAPGQGLEWMGRIDPDSTGSKYNEKFKTRVTMTVDTSTSTVYMELSSLRSEDTAVYYCAREGAYGYYFDYWGQGTLVTVSS

SEQ ID NO：54

>h1708-H3

EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQAPGQGLEWIGRIDPDSTGSKYNEKFKTRVTMTVDTSTSTAYMELSSLRSEDTAVYYCAREGAYGYYFDYWGQGTLVTVSS

SEQ ID NO：55

3.3 humanization of hybridoma clone m1709

(1) m1709 selection of humanized frameworks

The humanized light chain templates of the murine antibody m1709 are IGKV1-39 x 01 and hjk4.1, the humanized heavy chain templates are IGHV1-46 x 01 and hjh 4.1.1, and the humanized antibody h1709 is obtained after humanization, and the humanized variable region sequences are as follows:

h1709VH-CDR graft

EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQAPGQGLEWMGLVYPYNDNTGYNRKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGPSNWNYFDYWGQGTLVTVSS

SEQ ID NO：56

h1709VL-CDR graftDIQMTQSPSSLSASVGDRVTITCKASQNVVTAVAWYQQKPGKAPKLLIYSA SNRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYTLYPLTFGGGTKVEIK

SEQ ID NO：57

(2) The h1709 back-mutations were designed as follows:

table 9: h1709 Back-mutation design

Note: as shown in S60D, the sequence number is determined according to the natural sequence of the amino acid sequence, and the position 60S is changed back to A. Grafted stands for murine antibody CDR-implanted human germline FR region sequences.

The h1709 humanized antibody light/heavy chain variable region sequence after back mutation design is as follows:

h1709-L1 (same as h1709 VL-CDR gradient)

DIQMTQSPSSLSASVGDRVTITCKASQNVVTAVAWYQQKPGKAPKLLIYSASNRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYTLYPLTFGGGTKVEIK

SEQ ID NO：57

>h1709-L2

DIQMTQSPSSLSASVGDRVTITCKASQNVVTAVAWYQQKPGKAPKLLIYSASNRYTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQQYTLYPLTFGGGTKVEIK

SEQ ID NO：58

>h1709L3

DIQMTQSPSSLSASVGDRVTITCKASQNVVTAVAWYQQKPGKSPKLLIYSASNRYTGVPDRFSGSGSGTDFTLTISSLQPEDFATYFCQQYTLYPLTFGGGTKVEIK

SEQ ID NO：59

>h1709-L4

DIVMTQSPSSLSASVGDRVTITCKASQNVVTAVAWYQQKPGKSPKLLIYSASNRYTGVPDRFSGSGSGTDFTLTISSLQPEDFATYFCQQYTLYPLTFGGGTKVEIK

SEQ ID NO：60

H1709-H1 (same as H1709 VH-CDR graft)

SEQ ID NO：56

>h1709-H2

EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQAPGQGLEWMGLVYPYNDNTGYNRKFKGRVTMTVDTSTSTVYMELNSLRSEDTAVYYCARGGPSNWNYFDYWGQGTLVTVSS

SEQ ID NO：61

>h1709-H3

EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQAPGQGLEWMGLVYPYNDNTGYNRKFKGRVTMTVDTSTSTAYMELNSLRSEDTAVYYCARGGPSNWNYFDYWGQGTLVTVSS

SEQ ID NO：62

>h1709-H4

EVQLVQSGAEVKKPGASVKVSCKASGFTFTDYYMHWVRQAPGQGLEWIGLVYPYNDNTGYNRKFKGRVTMTVDTSTSTAYMELNSLRSEDTAVYYCARGGPSNWNYFDYWGQGTLVTVSS

SEQ ID NO：63

3.4 humanization of hybridoma clone m1710

(1) m1710 humanized framework selection

The humanized light chain templates of the murine antibody m1710 are IGKV1-39 x 01 and hjk4.1, the humanized heavy chain templates are IGHV1-46 x 01 and hjh 4.1.1, and the humanized antibody h1710 is obtained after humanization, and the humanized variable region sequence is as follows:

h1710VH-CDR graft

EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGQGLEWMGRIDPTSGATKYNDNFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREGGFGYYFDYWGQGTTVTVSS

SEQ ID NO：64

h1710VL-CDR graft

DIQMTQSPSSLSASVGDRVTITCRTSENIFTYLAWYQQKPGKAPKLLIYNAKTFAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYGIPLPFGQGTKLEIK

SEQ ID NO：65

(2) The h1710 back-mutation design is as follows:

table 10: h1710 reverse mutation design

Note: if A43S indicates the numbering according to the natural sequence of the amino acid sequence, the A at position 43 is mutated back to S. Grafted stands for murine antibody CDR-implanted human germline FR region sequences.

The sequence of the light/heavy chain variable region of the h1710 humanized antibody after back mutation design is as follows:

h1710-L1 (h 1710 VL-CDR gradient)

SEQ ID NO：65

>h1710-L2

DIQMTQSPSSLSASVGDRVTITCRTSENIFTYLAWYQQKPGKSPKLLVYNAKTFAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYGIPLPFGQGTKLEIK

SEQ ID NO：66

H1710-H1 (same H1710 VH-CDR gradient)

SEQ ID NO：64

>h1710-H2

EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGQGLEWMGRIDPTSGATKYNDNFKGRVTMTVDKSTSTVYMELSSLRSEDTAVYYCAREGGFGYYFDYWGQGTTVTVSS

SEQ ID NO：67

>h1710-H3

EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGQGLEWMGRIDPTSGATKYNDNFKGRVTLTVDKSTSTAYMELSSLRSEDTAVYYCAREGGFGYYFDYWGQGTTVTVSS

SEQ ID NO：68

>h1710-H4

EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGQGLEWIGRIDPTSGATKYNDNFKGRATLTVDKSTSTAYMELSSLRSEDTAVYYCAREGGFGYYFDYWGQGTTVTVSS

SEQ ID NO：69

>h1710-H5

EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVKQAPGQGLEWIGRIDPTSGATKYNDNFKGKATLTVDKSTSTAYMELSSLRSEDTAVYYCAREGGFGYYFDYWGQGTTVTVSS

SEQ ID NO：70

3.5 humanization of hybridoma clone m1711

(1) m1711 selection of humanized frameworks

The humanized light chain templates of the murine antibody m1711 are IGKV4-1 × 01 and hjk4.1, the humanized heavy chain templates are IGHV1-69 × 02 and hjh 4.1.1, and the humanized antibody h1711 is obtained after humanization, and the humanized variable region sequences are as follows:

h1711VH-CDR graft

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWIGWVRQAPGQGLEWMGDIYPGGAYTNYNEKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARGDYYDSSGRAMDYWGQGTLVTV

SEQ ID NO：71

h1711VL-CDR graft

DIVMTQSPDSLAVSLGERATINCKSSQSLLYSRNQMNYLAWYQQKPGQPPKLLIYWTSTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPYTFGGGTKVEIK

SEQ ID NO：72

(2) The design of the h1711 back mutation is as follows:

table 11: h1711 reverse mutation design

Note: p49 is mutated back to S as indicated by P49S, numbering according to the natural order of the amino acid sequence. Grafted stands for murine antibody CDR-implanted human germline FR region sequences.

The sequence of the light/heavy chain variable region of the h1711 humanized antibody after back mutation design is as follows:

h1711-L1 (h 1711 VL-CDR gradient)

SEQ ID NO：72

>h1711-L2

DIVMTQSPDSLAVSLGERATINCKSSQSLLYSRNQMNYLAWYQQKPGQSPKLLIYWTSTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPYTFGGGTKVEIK

SEQ ID NO：73

>h1711-L3

DIVMTQSPDSLAVSLGERATISCKSSQSLLYSRNQMNYLAWYQQKPGQSPKLLIYWTSTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPYTFGGGTKVEIK

SEQ ID NO：74

H1711-H1 (H1711 VH-CDR graft)

SS

SEQ ID NO：71

>h1711-H2

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWIGWVRQAPGQGLEWIGDIYPGGAYTNYNEKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARGDYYDSSGRAMDYWGQGTLVTVSS

SEQ ID NO：75

>h1711-H3

EVQLVQSGAEVKKPGSSVKVSCKASGYTFSNYWIGWVRQAPGQGLEWIGDIYPGGAYTNYNEKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARGDYYDSSGRAMDYWGQGTLVTVSS

SEQ ID NO：76

>h1711-H4

EVQLVQSGAEVKKPGSSVKVSCKASGYTFSNYWIGWVRQAPGQGLEWIGDIYPGGAYTNYNEKFKDRVTITADKSTSTAYMEFSSLRSEDTAVYYCTRGDYYDSSGRAMDYWGQGTLVTVSS

SEQ ID NO：77

3.6 construction and expression of humanized antibodies

Connecting the heavy chain variable region of the humanized antibody obtained after the back mutation with the amino terminal of the constant region of the heavy chain of the human antibody to form an antibody heavy chain, and connecting the light chain variable region with the amino terminal of the constant region of the light chain of the human antibody to form an antibody light chain; designing primers, carrying out PCR (polymerase chain reaction) to build each humanized antibody VH/VK gene fragment, carrying out homologous recombination with an expression vector pHr (with signal peptide and a constant region gene (CH1-Fc/CL) fragment), constructing an antibody full-length expression vector VH-CH1-Fc-pHr/VK-CL-pHr, and carrying out expression of humanized antibodies. Illustratively, the antibody heavy chain variable region is attached to the amino terminus of the IgG4 heavy chain constant region with a mutation at S228P (corresponding to position 108 of SEQ ID NO: 78) and the light chain variable region is attached to the amino terminus of the kappa light chain constant region, resulting in h1707, h1708, h1709, h1710, h1711 humanized antibodies, as shown in tables 12-16 below:

table 12: h1707 humanized antibodies

Note: in the table, "H1707-02" indicates the amino acid sequence represented by the heavy chain variable region of H1707-H2 (shown as SEQ ID NO: 50) and the amino acid sequence shown as SEQ ID NO:78 to the variable region of h1707-L1 light chain (as shown in SEQ ID NO: 46) and the amino terminus of the heavy chain constant region as shown in SEQ ID NO:79, and so on, in the form of a full-length antibody comprising a light chain linked to the amino terminus of a light chain constant region.

Table 13: h1708 humanized antibodies

Note: in the table, "H1708-04" indicates the heavy chain variable region consisting of H1708-H1 (shown in SEQ ID NO: 51) and the amino acid sequence shown in SEQ ID NO:78 to the variable region of h1708-L2 light chain (as shown in SEQ ID NO: 53) and the amino terminus of the heavy chain constant region as shown in SEQ ID NO:79, and so on, in the form of a full-length antibody comprising a light chain linked to the amino terminus of a light chain constant region.

Table 14: h1709 humanized antibodies

Note: in the table, "H1709-10" indicates the amino acid sequence represented by the heavy chain variable region of H1709-H2 (shown in SEQ ID NO: 61) and the amino acid sequence shown in SEQ ID NO:78 to the variable region of h1709-L3 light chain (as shown in SEQ ID NO: 59) and the amino terminus of the heavy chain constant region as shown in SEQ ID NO:79, and so on, in the form of a full-length antibody comprising a light chain linked to the amino terminus of a light chain constant region.

Table 15: h1710 humanized antibody

Note: in the table, "H1710-01" indicates the amino acid sequence represented by the heavy chain variable region of H1710-H1 (shown as SEQ ID NO: 64) and the amino acid sequence shown as SEQ ID NO:78 to the variable region of h1710-L1 (shown as SEQ ID NO: 65) and the variable region of SEQ ID NO:79, and so on, in the form of a full-length antibody formed by a light chain linked at the amino terminus of a light chain constant region.

Table 16: h1711 humanized antibodies

Note: in the table, "H1711-04" indicates the heavy chain variable region represented by H1711-H4 (shown in SEQ ID NO: 77) and the amino acid sequence shown in SEQ ID NO:78 to the variable region of h1711-L1 (shown in SEQ ID NO: 72) and the amino terminus of the heavy chain constant region shown in SEQ ID NO:79, and so on, in the form of a full-length antibody comprising a light chain linked to the amino terminus of a light chain constant region.

Exemplary antibody constant region sequences are shown below:

IgG4 heavy chain constant region with S228P mutation:

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

SEQ ID NO：78

kappa light chain constant region:

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO：79

the positive control antibody 22G2-H3Q, whose VH and VL sequences (SEQ ID NO:8 and 9 of US20160176963A1, respectively) were identical to the sequences set forth above for SEQ ID NO:78 and SEQ ID NO:79 to form a complete full-length antibody. Specific sequences of 22G2-H3Q for VH and VL are as follows:

22G2-H3Q VH：

QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGIYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDYYVSGNYYNVDYYFFGVDVWGQGTTVTVSS

SEQ ID NO：80

22G2-H3Q VL：

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPLFTFGPGTKVDIK

SEQ ID NO：81

exemplary anti-human TIGIT antibody light/heavy chain full length sequences are as follows:

h1707-02 light chain full-length sequence

DIQMTQSPSFLSASVGDRVTITCKASQDVGTSVAWYQQKPGKAPKLLIYWASARHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO：82

H1707-02 heavy chain full-length sequence

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYHMYWVRQAPGKGLEWVAYISKGGISTYYPDTVKGRFTISRDNAKNSLYLQMSRLRAEDTAVYYCARQSSYDFAMDYWGRGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

SEQ ID NO：83

H1708-04 full-length light chain sequence

DIQMTQSPSSLSASVGDRVTITCRASENIYSYLAWYQQKPGKSPKLLIYNARTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQYHSGSPLPFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO：84

H1708-04 heavy chain full-length sequence

EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQAPGQGLEWMGRIDPDSTGSKYNEKFKTRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREGAYGYYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

SEQ ID NO：85

H1709-10 full-length light chain sequence

DIQMTQSPSSLSASVGDRVTITCKASQNVVTAVAWYQQKPGKSPKLLIYSASNRYTGVPDRFSGSGSGTDFTLTISSLQPEDFATYFCQQYTLYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO：86

H1709-10 heavy chain full-length sequence

EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQAPGQGLEWMGLVYPYNDNTGYNRKFKGRVTMTVDTSTSTVYMELNSLRSEDTAVYYCARGGPSNWNYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

SEQ ID NO：87

H1710-01 full-length light chain sequence

DIQMTQSPSSLSASVGDRVTITCRTSENIFTYLAWYQQKPGKAPKLLIYNAKTFAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYGIPLPFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO：88

H1710-01 heavy chain full-length sequence

EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGQGLEWMGRIDPTSGATKYNDNFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREGGFGYYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

SEQ ID NO：89

H1711-04 light chain full-length sequence

DIVMTQSPDSLAVSLGERATINCKSSQSLLYSRNQMNYLAWYQQKPGQPPKLLIYWTSTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO：90

H1711-04 heavy chain full-length sequence

EVQLVQSGAEVKKPGSSVKVSCKASGYTFSNYWIGWVRQAPGQGLEWIGDIYPGGAYTNYNEKFKDRVTITADKSTSTAYMEFSSLRSEDTAVYYCTRGDYYDSSGRAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

SEQ ID NO：91

The following biochemical test methods were used to verify the activity and efficacy of the antibodies of the present disclosure

Test example 1: biacore assay

And (3) measuring the affinity of the anti-TIGIT antibody to be measured with a Biacore and GE instrument to human and monkey TIGIT.

Human anti-capture antibodies were affinity-captured on a Protein a biosensor chip (Cat. #29127556, GE) or covalently coupled to a biosensor chip (Cat. #28-9538-28, GE) according to the methods described in the specifications of the human anti-capture kit (Cat. #28-9538-28, GE) to affinity-capture a quantity of the antibody to be detected, followed by flowing a series of concentration-graded human and monkey TIGIT antigens on the chip surface, wherein the human TIGIT was selected from TIGIT from the group consisting of kyush bio (cat.10917-H08-H, sino.biol), which was purified by expression in examples 1 and 2, and the binding and dissociation curves were obtained by detecting the reaction signals in real time using a Biacore instrument (Biacore T200, GE). After dissociation of each cycle was completed, the biochip was washed and regenerated with a regeneration solution prepared in a human anti-capture kit or a glycine-hydrochloric acid regeneration solution (Cat. # BR-1003-54, GE) of ph 1.5. The amino coupling kit used in the experiment was purchased from GE (Cat. # BR-1000-50, GE) and the buffer was HBS-EP +10 Xbuffer solution (Cat. # BR-1006-69, GE) diluted to 1X (pH7.4) with D.I.Water.

The data obtained from the experiment were fitted using BIAevaluation version 4.1, GE software using a (1:1) Langmuir model to obtain affinity values, the results of which are shown in tables 17-19.

TABLE 17 reaction affinities of molecules to be tested and human TIGIT protein

TABLE 18 reaction affinities of molecules to be tested and cynoTIGIT protein

TABLE 19 reaction affinities of ch1711 and its humanized antibodies to human TIGIT protein

Test example 2: blocking experiment of binding of CD155 protein and CHO cell over-expressing TIGIT by TIGIT antibody

Blocking ability of anti-TIGIT antibodies was detected by FACS experiments in which the antibodies blocked binding of CD155 to TIGIT overexpressing CHO cells. After transfecting TIGIT full-length plasmid into CHO cells by an electrotransfection method and screening for two weeks under pressure, the expression level of TIGIT is detected. After pre-incubating the over-expressed cells with anti-TIGIT antibodies with different concentrations and a negative control (human IgG4 antibody not binding to TIGIT (hIgG4)), the over-expressed cells are added with fluorescence labeled CD155-Fc for incubation, and the capability of the antibodies for blocking the binding of the CD155 and the CHO cells over-expressing TIGIT is judged by the strength of signals, wherein the specific experimental method comprises the following steps:

first labeling CF 155-Fc (Sino Biological, Cat No.10109-H02H) with CD155-Fc ^TM 633(Sigma Aldrich, Cat No. MX633S100). Dissolving CD155-Fc with PBS until the concentration is 0.5-1mg/ml, adding 10 xMix-n-Stain Reaction Buffer with 9 times of sample volume, and mixing uniformly; then adding CF ^TM 633 fluorescent dye, and incubating for 30 minutes at room temperature in the dark to finish the fluorescent labeling.

TIGIT-CHO cells at 5X 10 ⁶ Density of 100. mu.l/well in a 96-well round bottom plate (Corning, Cat No.32915001), Centrifuge (Beckman Coulter, Allegra X-15R Centrifuge) for 5 minutes at 1500rpm, discard the supernatant; the cells were resuspended in 200. mu.l PBS, centrifuged, and the supernatant discarded and repeated. The cells were resuspended by adding 100. mu.l/well of a test antibody solution which had been diluted in a sample dilution (pH7.4 PBS containing 1% BSA) and incubated for 1 hour at 4 ℃. After the incubation was completed, centrifugation was carried out at 1500rpm for 5 minutes, the supernatant was discarded, and after washing the cells twice with the sample diluent, 100. mu.l of 2. mu.g/ml CF was added ^TM 633 fluorescently labeled CD155-Fc solution resuspended cells and incubated at 4 ℃ for 1 hour. After the incubation is finished, centrifuging at 1500rpm for 5 minutes, discarding the supernatant, washing the cells twice with a sample diluent, finally resuspending the cells with a 200. mu.l/well sample diluent, detecting the intensity of a fluorescence signal on a flow cytometer (BD FACS Canto II), analyzing data with GraphPad Prism5, and calculating the blocking capacity of the TIGIT antibody on the binding of CD155 and TIGIT-CHO cells. The results are given in Table 20 below:

TABLE 20 blocking experiment results of binding of CD155 protein to TIGIT-overexpressing CHO cells by TIGIT antibody

In the test example, the antibodies h1707-02, h1708-04, h1709-10, h1710-01 and h1711-04 can well block the combination of CD155 and TIGIT over-expressed CHO cells.

Test example 3: effect of anti-TIGIT antibodies in combination with PD-1 inhibitors on activation of human T-lymphocytic leukemia cell lines

To investigate the effect of antibodies on T cell function, Jurkat cell lines with high expression of PD-1, TIGIT and CD226 carrying NFAT reporter (GloResponse) ^TM ) And co-culturing with Chinese hamster ovary cells (CHO-K1) highly expressing T cell activating molecules, PD-L1 and CD155 for 18 hours, adding luciferase substrate to detect a luminescence signal value, and judging the activation activity of the antibody on the T cells. The specific experimental process is as follows:

GloResponse NFAT-luc2/PD-1/TIGIT/CD226 Jurkat cells were cultured in RPMI 1640 medium supplemented with 10% (v/v) Fetal Bovine Serum (FBS), 100. mu.g/ml Hygromycin B (Hygromycin B), 1mg/ml G418, 2. mu.g/ml puromycin (puromycin), 37 ℃, 5% CO ₂ Culturing under the condition. CHO-K1/PD-L1/CD155 cells cultured in F-12Nutrient Mixture (purchased from Ham) medium supplemented with 10% (v/v) FBS, 200. mu.g/ml Hygromycin B, 250. mu.g/ml G418, 37 ℃, 5% CO ₂ Culturing under the condition.

Experiment day one CHO-K1/PD-L1/CD155 cells were seeded in 96-well plates at a density of 40,000 cells per well at 37 ℃ with 5% CO ₂ Incubated under conditions for 24 hours. The next day, the medium in the cell culture plate was discarded, washed with 2% FBS-containing RPMI 1640 medium, and the GloResponse NFAT-luc2/PD-1/TIGIT/CD226 Jurkat cells were resuspended and seeded into a 96-well plate at a cell density of about 50,000 cells/well, while a gradient diluted antibody sample was added (dilution of 2% FBS-containing RPMI 1640, starting concentrations of anti-PD-1 antibody and anti-TIGIT antibody were 30ug/ml, 3-fold each, 9 dilutions in total), and an equal amount of isotype IgG was used as a blank. 37 ℃ and 5% CO ₂ After 18h incubation in the incubator, 40. mu.L of the prepared Luciferase substrate Bio-GloTM Luciferase Assay system (purchased from Promega, G7940) was added to each well, left for 10 minutes at room temperature in the absence of light, and then usedThe luminescence signal values were read on a Victor3 multifunctional microplate reader (from Perkin Elmer). IC50 values for the test samples were calculated using the data processing software Graphpad prism 5.0.

As shown in FIG. 1, TIGIT humanized antibodies h1707-02, h1708-04, h1709-10, h1710-01 and h1711-04 can enhance the luminescent signal value generated by Jurkat cell activation to different degrees (wherein the signal values of h1707-02, h1708-04 and h1709-10 antibodies are very similar, and 3 curves thereof are almost overlapped and cannot be distinguished in FIG. 1), and have drug concentration dose effect. Furthermore, when the anti-TIGIT humanized antibody (h1707-02, h1708-04, h1709-10, h1710-01 and h1711-04) and the anti-PD-1 antibody (Carrayleigh monoclonal antibody (SHR1210, constant Ri medicine)) are used in combination, the intensity of the optical signal generated by activation of Jurkat cells can be greatly enhanced, and the activation intensity is higher than that when the TIGIT antibody or the PD-1 antibody is used alone.

Test example 4: in vivo efficacy test of mice combined by anti-TIGIT antibody and PD-1 inhibitor

MC38 mouse colon cancer cells are inoculated subcutaneously in TIGIT transgenic C57 mice, TIGIT antibodies h1708-04, h1710-01 and an anti-mouse PD-1 antibody are given, and the TIGIT antibody is combined with the anti-mouse PD-1 antibody (clone No. J43, available on the market), the change of the size of the tumor volume of the mice is detected, and the influence of the tested antibody on the tumor growth of the tumor-bearing mice is evaluated.

The TIGIT transgenic C57 mice, 100, male, were purchased from Shanghai south China Modular Biotechnology, Inc. at 6 weeks of age, and were bred in SPF environment at 20-25 deg.C, with a weight of 20-28g and 5 mice/cage; humidity is 40-60%, license number SCXK (Shanghai) 2014 and 0002. The environment is adapted for about 12 days.

MC38 cells (purchased from Nanjing Galaxy Biomedicine Co., Ltd.) (5X 10) ⁵ One) 100 mul of the cells were inoculated subcutaneously into the right flank of 100 TIGIT transgenic C57 mice to form a tumor (130 mm) ³ ) Thereafter, the over-and under-sized tumors were removed and mice were randomly divided into 8 groups of 7-9 mice per group based on tumor volume. The day the injection of antibody by intraperitoneal injection was started, 2 times per week for 3 weeks. Tumor volume was measured 2 times per week, body weight was weighed, and data was recorded. The grouping and dosing are shown in table 21.

TABLE 21 test grouping and dosing

At the end of the experiment, tumor tissue was taken from the mice and stored at-80 ℃. Using Excel statistical software: the average is calculated as avg; the SD value is calculated as STDEV; SEM values were calculated as STDEV/SQRT (number of animals per group); the P values for differences between groups were analyzed as Two-way Anova using GraphPad Prism software for plotting.

Tumor volume (V) was calculated as: v1/2 xl _{Long and long} ×L _{Short length} ²

Relative volume (RTV) ═ V _T /V ₀

Tumor inhibition rate (%) ═ C _RTV -T _RTV )/C _RTV (％)

Wherein V ₀ 、V _T Tumor volumes at the beginning and end of the experiment, respectively. C _RTV 、T _RTV Relative tumor volumes of Vehicle (blank control group) and experimental group at the end of experiment, respectively.

The test result is shown in figure 2, and compared with a blank control (IgG) group, the anti-mouse PD-1 antibody single-use group has the tumor inhibition rate of 6 percent; the tumor inhibition rate of the h1708-04-10mg/kg single use group is 4%, the tumor inhibition rate of the h1708-04-10mg/kg and the anti-mouse PD-1 antibody 0.125mg/kg combined use group is 40% (p is less than 0.001), the combined use group can obviously inhibit the growth of MC38 tumor, and the drug effect of the combined use group is obviously better than that of the single use group (p is less than 0.001); the tumor inhibition rate of the single-use group of h 1708-0425 mg/kg is 23% (p is less than 0.001), the tumor inhibition rate of the combined group of h 1708-0425 mg/kg and the anti-mouse PD-1 antibody 0.125mg/kg is 31% (p is less than 0.001), the growth of the MC38 tumor can be obviously inhibited, and the tumor inhibition rate of the combined group is higher than that of the single-use group; the tumor inhibition rate of the h 1710-0125 mg/kg single use group is 8%, the tumor inhibition rate of the h 1710-0125 mg/kg and the anti-mouse PD-1 antibody 0.125mg/kg combined use is 34% (p is less than 0.001), the growth of MC38 tumor can be obviously inhibited (p is less than 0.001), and the drug effect of the combined use group is obviously superior to that of the single use group (p is less than 0.01).

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

Sequence listing

<110> Hengrui pharmaceuticals, Inc. of Jiangsu and Hengrui pharmaceuticals, Inc. of Shanghai

<120> method and pharmaceutical combination for treating diseases using anti-TIGIT antibody in combination with PD-1 inhibitor

<130> 2019

<150> 201910247537.0

<151> 2019-03-29

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

1 5 10 15

Val Gln Cys Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn Ile Ser

20 25 30

Ala Glu Lys Gly Gly Ser Ile Ile Leu Gln Cys His Leu Ser Ser Thr

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Thr Ala Gln Val Thr Gln Val Asn Trp Glu Gln Gln Asp Gln Leu Leu

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Ala Ile Cys Asn Ala Asp Leu Gly Trp His Ile Ser Pro Ser Phe Lys

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

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Thr Val Asn Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His Thr Tyr Pro

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

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Val Ala Glu His Gly Ala Arg Phe Gln Ile Pro Glu Pro Arg Gly Pro

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Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser

225 230 235 240

Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro

245 250 255

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

260 265 270

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

275 280 285

Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val

290 295 300

Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu

305 310 315 320

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

325 330 335

Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val

340 345 350

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

355 360 365

Thr Pro Gly Lys

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<223> fusion protein of TIGIT extracellular region and human IgG1 Fc fragment: TIGIT-Fc

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

1 5 10 15

Val Gln Cys Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn Ile Ser

20 25 30

Ala Glu Lys Gly Gly Ser Ile Ile Leu Gln Cys His Leu Ser Ser Thr

35 40 45

Thr Ala Gln Val Thr Gln Val Asn Trp Glu Gln Gln Asp Gln Leu Leu

50 55 60

Ala Ile Cys Asn Ala Asp Leu Gly Trp His Ile Ser Pro Ser Phe Lys

65 70 75 80

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

85 90 95

Thr Val Asn Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His Thr Tyr Pro

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Pro Gly Lys

370

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<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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<221> DOMAIN

<223> full-length TIGIT

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Met Arg Trp Cys Leu Leu Leu Ile Trp Ala Gln Gly Leu Arg Gln Ala

1 5 10 15

Pro Leu Ala Ser Gly Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn

20 25 30

Ile Ser Ala Glu Lys Gly Gly Ser Ile Ile Leu Gln Cys His Leu Ser

35 40 45

Ser Thr Thr Ala Gln Val Thr Gln Val Asn Trp Glu Gln Gln Asp Gln

50 55 60

Leu Leu Ala Ile Cys Asn Ala Asp Leu Gly Trp His Ile Ser Pro Ser

65 70 75 80

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

85 90 95

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

100 105 110

Tyr Pro Asp Gly Thr Tyr Thr Gly Arg Ile Phe Leu Glu Val Leu Glu

115 120 125

Ser Ser Val Ala Glu His Gly Ala Arg Phe Gln Ile Pro Leu Leu Gly

130 135 140

Ala Met Ala Ala Thr Leu Val Val Ile Cys Thr Ala Val Ile Val Val

145 150 155 160

Val Ala Leu Thr Arg Lys Lys Lys Ala Leu Arg Ile His Ser Val Glu

165 170 175

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

180 185 190

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

195 200 205

Gly Leu Cys Gly Glu Gln Arg Gly Glu Asp Cys Ala Glu Leu His Asp

210 215 220

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

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Thr Glu Thr Gly

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<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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<223> fusion protein of extracellular region of cynoTIGIT and IgG2aFc fragment of mouse: cynoTIGIT-mFc

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

1 5 10 15

Val Gln Cys Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn Ile Ser

20 25 30

Ala Lys Lys Gly Gly Ser Val Ile Leu Gln Cys His Leu Ser Ser Thr

35 40 45

Met Ala Gln Val Thr Gln Val Asn Trp Glu Gln His Asp His Ser Leu

50 55 60

Leu Ala Ile Arg Asn Ala Glu Leu Gly Trp His Ile Tyr Pro Ala Phe

65 70 75 80

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

85 90 95

Leu Thr Met Asn Asp Thr Gly Glu Tyr Phe Cys Thr Tyr His Thr Tyr

100 105 110

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

115 120 125

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

130 135 140

Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu

145 150 155 160

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

165 170 175

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

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

195 200 205

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

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Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met

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Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala

245 250 255

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

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

275 280 285

Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr

290 295 300

Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr

305 310 315 320

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

325 330 335

Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser

340 345 350

Val Val His Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser

355 360 365

Arg Thr Pro Gly Lys

370

<210> 5

<211> 119

<212> PRT

<213> mouse (Mus musculus)

<220>

<221> DOMAIN

<223> m1707-HCVR sequence

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

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Asp Tyr

20 25 30

His Met Tyr Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gln Ser Ser Tyr Asp Phe Ala Met Asp Tyr Trp Gly Arg Gly

100 105 110

Thr Ser Val Thr Val Ser Ser

115

<210> 6

<211> 107

<212> PRT

<213> mouse (Mus musculus)

<220>

<221> DOMAIN

<223> m1707-LCVR sequence

<400> 6

Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Val Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ser

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 7

<211> 119

<212> PRT

<213> mouse (Mus musculus)

<220>

<221> DOMAIN

<223> m1708-HCVR sequence

<400> 7

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Thr Leu Thr Val Ser Ser

115

<210> 8

<211> 107

<212> PRT

<213> mouse (Mus musculus)

<220>

<221> DOMAIN

<223> m1708-LCVR

<400> 8

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

1 5 10 15

Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr

20 25 30

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

35 40 45

Tyr Asn Ala Arg Thr Leu Ala Glu Ser Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Gly Ser Tyr Tyr Cys Gln Tyr His Ser Gly Ser Pro Leu

85 90 95

Pro Phe Gly Ala Gly Thr Lys Leu Ala Leu Lys

100 105

<210> 9

<211> 120

<212> PRT

<213> mouse (Mus musculus)

<220>

<221> DOMAIN

<223> m1709-HCVR sequence

<400> 9

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

1 5 10 15

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

20 25 30

Tyr Met His Trp Val Lys Gln Ser Leu Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Leu Val Tyr Pro Tyr Asn Asp Asn Thr Gly Tyr Asn Arg Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gly Gly Pro Ser Asn Trp Asn Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Leu Thr Val Ser Ser

115 120

<210> 10

<211> 107

<212> PRT

<213> mouse (Mus musculus)

<220>

<221> DOMAIN

<223> m1709-LCVR sequence

<400> 10

Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Thr Val Gly

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Thr Leu Tyr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 11

<211> 119

<212> PRT

<213> mouse (Mus musculus)

<220>

<221> DOMAIN

<223> m1710-HCVR sequence

<400> 11

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

1 5 10 15

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

20 25 30

Tyr Met His Trp Val Lys Gln Arg Pro Gly Arg Gly Leu Glu Trp Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Thr Leu Thr Val Ser Ser

115

<210> 12

<211> 107

<212> PRT

<213> mouse (Mus musculus)

<220>

<221> DOMAIN

<223> m1710-LCVR sequence

<400> 12

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr Asn Ala Lys Thr Phe Ala Glu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Gly Ile Tyr Tyr Cys Gln His His Tyr Gly Ile Pro Leu

85 90 95

Pro Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 13

<211> 122

<212> PRT

<213> mouse (Mus musculus)

<220>

<221> DOMAIN

<223> m1711-HCVR sequence

<400> 13

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

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

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

35 40 45

Gly Asp Ile Tyr Pro Gly Gly Ala Tyr Thr Asn Tyr Asn Glu Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Thr Arg Gly Asp Tyr Tyr Asp Ser Ser Gly Arg Ala Met Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 14

<211> 113

<212> PRT

<213> mouse (Mus musculus)

<220>

<221> DOMAIN

<223> m1711-LCVR sequence

<400> 14

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Lys

<210> 15

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1707-HCDR 1sequence

<400> 15

Asp Tyr His Met Tyr

1 5

<210> 16

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1707 HCDR2 sequence

<400> 16

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

1 5 10 15

Gly

<210> 17

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1707 HCDR3 sequence

<400> 17

Gln Ser Ser Tyr Asp Phe Ala Met Asp Tyr

1 5 10

<210> 18

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1707 LCDR 1sequence

<400> 18

Lys Ala Ser Gln Asp Val Gly Thr Ser Val Ala

1 5 10

<210> 19

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1707 LCDR2 sequence

<400> 19

Trp Ala Ser Ala Arg His Thr

1 5

<210> 20

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1707 LCDR3 sequence

<400> 20

Gln Gln Tyr Ser Ser Tyr Pro Leu Thr

1 5

<210> 21

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1708 HCDR 1sequence

<400> 21

Asn Tyr Trp Met His

1 5

<210> 22

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1708 HCDR2 sequence

<400> 22

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

1 5 10 15

Thr

<210> 23

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1708 HCDR3 sequence

<400> 23

Glu Gly Ala Tyr Gly Tyr Tyr Phe Asp Tyr

1 5 10

<210> 24

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1708 LCDR 1sequence

<400> 24

Arg Ala Ser Glu Asn Ile Tyr Ser Tyr Leu Ala

1 5 10

<210> 25

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1708 LCDR2 sequence

<400> 25

Asn Ala Arg Thr Leu Ala Glu

1 5

<210> 26

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1708 LCDR3 sequence

<400> 26

Gln Tyr His Ser Gly Ser Pro Leu Pro

1 5

<210> 27

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1709 HCDR 1sequence

<400> 27

Asp Tyr Tyr Met His

1 5

<210> 28

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1709 HCDR2 sequence

<400> 28

Leu Val Tyr Pro Tyr Asn Asp Asn Thr Gly Tyr Asn Arg Lys Phe Lys

1 5 10 15

Gly

<210> 29

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1709 HCDR3 sequence

<400> 29

Gly Gly Pro Ser Asn Trp Asn Tyr Phe Asp Tyr

1 5 10

<210> 30

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1709 LCDR 1sequence

<400> 30

Lys Ala Ser Gln Asn Val Val Thr Ala Val Ala

1 5 10

<210> 31

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1709-LCDR2 sequence

<400> 31

Ser Ala Ser Asn Arg Tyr Thr

1 5

<210> 32

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1709-LCDR3 sequence

<400> 32

Gln Gln Tyr Thr Leu Tyr Pro Leu Thr

1 5

<210> 33

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1710-HCDR 1sequence

<400> 33

Asn Tyr Tyr Met His

1 5

<210> 34

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1710-HCDR2 sequence

<400> 34

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

1 5 10 15

Gly

<210> 35

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1710-HCDR3 sequence

<400> 35

Glu Gly Gly Phe Gly Tyr Tyr Phe Asp Tyr

1 5 10

<210> 36

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1710-LCDR 1sequence

<400> 36

Arg Thr Ser Glu Asn Ile Phe Thr Tyr Leu Ala

1 5 10

<210> 37

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1710-LCDR2 sequence

<400> 37

Asn Ala Lys Thr Phe Ala Glu

1 5

<210> 38

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1710-LCDR3 sequence

<400> 38

Gln His His Tyr Gly Ile Pro Leu Pro

1 5

<210> 39

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1711-HCDR 1sequence

<400> 39

Asn Tyr Trp Ile Gly

1 5

<210> 40

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1711-HCDR2 sequence

<400> 40

Asp Ile Tyr Pro Gly Gly Ala Tyr Thr Asn Tyr Asn Glu Lys Phe Lys

1 5 10 15

Asp

<210> 41

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1711-HCDR3 sequence

<400> 41

Gly Asp Tyr Tyr Asp Ser Ser Gly Arg Ala Met Asp Tyr

1 5 10

<210> 42

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1711-LCDR 1sequence

<400> 42

Lys Ser Ser Gln Ser Leu Leu Tyr Ser Arg Asn Gln Met Asn Tyr Leu

1 5 10 15

Ala

<210> 43

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1711-LCDR2 sequence

<400> 43

Trp Thr Ser Thr Arg Glu Ser

1 5

<210> 44

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1711-LCDR3 sequence

<400> 44

Gln Gln Tyr Tyr Ser Tyr Pro Tyr Thr

1 5

<210> 45

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> H1707 VH-CDR graft, H1707-H1 sequence

<400> 45

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gln Ser Ser Tyr Asp Phe Ala Met Asp Tyr Trp Gly Arg Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 46

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> h1707 VL-CDR graft, h1707-L1 sequence

<400> 46

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ser

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 47

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> h1707-L2 sequence

<400> 47

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ser

20 25 30

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

35 40 45

Tyr Trp Ala Ser Ala Arg His Thr Gly Val Pro Asp 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 Asp Tyr Tyr Cys Gln Gln Tyr Ser Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 48

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> h1707-L3 sequence

<400> 48

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ser

20 25 30

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

35 40 45

Tyr Trp Ala Ser Ala Arg His Thr Gly Val Pro Asp 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 Asp Tyr Tyr Cys Gln Gln Tyr Ser Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 49

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> h1707-L4 sequence

<400> 49

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ser

20 25 30

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

35 40 45

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

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 50

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> H1707-H2 sequence

<400> 50

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gln Ser Ser Tyr Asp Phe Ala Met Asp Tyr Trp Gly Arg Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 51

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> H1708 VH-CDR graft, H1708-H1 sequence

<400> 51

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Lys Thr Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val 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 Ala Tyr Gly Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 52

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> h1708 VL-CDR graft, h1708-L1 sequences

<400> 52

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 Glu Asn Ile Tyr Ser Tyr

20 25 30

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

35 40 45

Tyr Asn Ala Arg Thr Leu Ala Glu 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 Tyr His Ser Gly Ser Pro Leu

85 90 95

Pro Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 53

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> h1708-L2 sequence

<400> 53

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 Glu Asn Ile Tyr Ser Tyr

20 25 30

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

35 40 45

Tyr Asn Ala Arg Thr Leu Ala Glu 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 Tyr His Ser Gly Ser Pro Leu

85 90 95

Pro Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 54

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> H1708-H2 sequence

<400> 54

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Lys Thr Arg Val Thr Met Thr Val Asp Thr Ser Thr Ser Thr Val 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 Ala Tyr Gly Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 55

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> H1708-H3 sequence

<400> 55

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Lys Thr Arg Val Thr Met Thr Val Asp Thr 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 Ala Tyr Gly Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 56

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> H1709 VH-CDR graft, H1709-H1 sequence

<400> 56

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Leu Val Tyr Pro Tyr Asn Asp Asn Thr Gly Tyr Asn Arg Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val 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 Gly Gly Pro Ser Asn Trp Asn Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 57

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> h1709 VL-CDR graft, h1709-L1 sequences

<400> 57

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 Lys Ala Ser Gln Asn Val Val Thr Ala

20 25 30

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

35 40 45

Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Thr Leu Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 58

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> h1709-L2 sequence

<400> 58

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 Lys Ala Ser Gln Asn Val Val Thr Ala

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Thr Leu Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 59

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> h1709-L3 sequence

<400> 59

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 Lys Ala Ser Gln Asn Val Val Thr Ala

20 25 30

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

35 40 45

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 60

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> h1709-L4 sequence

<400> 60

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

1 5 10 15

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

20 25 30

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

35 40 45

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 61

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> H1709-H2 sequence

<400> 61

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Leu Val Tyr Pro Tyr Asn Asp Asn Thr Gly Tyr Asn Arg Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gly Gly Pro Ser Asn Trp Asn Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 62

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> H1709-H3 sequence

<400> 62

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Leu Val Tyr Pro Tyr Asn Asp Asn Thr Gly Tyr Asn Arg Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gly Gly Pro Ser Asn Trp Asn Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 63

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> H1709-H4 sequence

<400> 63

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Leu Val Tyr Pro Tyr Asn Asp Asn Thr Gly Tyr Asn Arg Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gly Gly Pro Ser Asn Trp Asn Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 64

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> H1710 VH-CDR graft, H1710-H1 sequence

<400> 64

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val 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 Gly Phe Gly Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 65

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> h1710 VL-CDR graft, h1710-L1 sequence

<400> 65

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 Thr Ser Glu Asn Ile Phe Thr Tyr

20 25 30

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

35 40 45

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

85 90 95

Pro Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 66

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> h1710-L2 sequence

<400> 66

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 Thr Ser Glu Asn Ile Phe Thr Tyr

20 25 30

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

35 40 45

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

85 90 95

Pro Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 67

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> H1710-H2 sequence

<400> 67

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Lys Gly Arg Val Thr Met Thr Val Asp Lys Ser Thr Ser Thr Val 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 Gly Phe Gly Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 68

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> H1710-H3 sequence

<400> 68

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Lys Gly Arg Val Thr Leu Thr Val 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 Gly Phe Gly Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 69

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> H1710-H4 sequence

<400> 69

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Lys Gly Arg Ala Thr Leu Thr Val 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 Gly Phe Gly Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 70

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> H1710-H5 sequence

<400> 70

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val 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 Gly Phe Gly Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 71

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> H1711 VH-CDR graft, H1711-H1 sequence

<400> 71

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 Asn Tyr

20 25 30

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

35 40 45

Gly Asp Ile Tyr Pro Gly Gly Ala Tyr Thr Asn Tyr Asn Glu 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 Gly Asp Tyr Tyr Asp Ser Ser Gly Arg Ala Met Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val

115 120

<210> 72

<211> 113

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> h1711 VL-CDR graft, h1711-L1 sequence

<400> 72

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

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Lys

<210> 73

<211> 113

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> h1711-L2 sequence

<400> 73

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

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Lys

<210> 74

<211> 113

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> h1711-L3 sequence

<400> 74

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

1 5 10 15

Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Lys

<210> 75

<211> 122

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> H1711-H2 sequence

<400> 75

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 Asn Tyr

20 25 30

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

35 40 45

Gly Asp Ile Tyr Pro Gly Gly Ala Tyr Thr Asn Tyr Asn Glu 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 Gly Asp Tyr Tyr Asp Ser Ser Gly Arg Ala Met Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 76

<211> 122

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> H1711-H3 sequence

<400> 76

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Asp Ile Tyr Pro Gly Gly Ala Tyr Thr Asn Tyr Asn Glu 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 Gly Asp Tyr Tyr Asp Ser Ser Gly Arg Ala Met Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 77

<211> 122

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> H1711-H4 sequence

<400> 77

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 Asn Tyr

20 25 30

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

35 40 45

Gly Asp Ile Tyr Pro Gly Gly Ala Tyr Thr Asn Tyr Asn Glu 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 Phe Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Asp Tyr Tyr Asp Ser Ser Gly Arg Ala Met Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 78

<211> 327

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> human IgG4 heavy chain constant region sequence with S228P mutation

<400> 78

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> 79

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> kappa light chain constant region

<400> 79

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> 80

<211> 130

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 22G2-H3Q VH sequence

<400> 80

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Val Ser Ser Gly

20 25 30

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

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser

50 55 60

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

65 70 75 80

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

85 90 95

Cys Ala Arg Asp Tyr Tyr Val Ser Gly Asn Tyr Tyr Asn Val Asp Tyr

100 105 110

Tyr Phe Phe Gly Val Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val

115 120 125

Ser Ser

130

<210> 81

<211> 109

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 22G2-H3Q VL sequence

<400> 81

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro

85 90 95

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

100 105

<210> 82

<211> 214

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> h1707-02 full-length light chain sequence

<400> 82

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ser

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu 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> 83

<211> 446

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> h1707-02 heavy chain full-length sequence

<400> 83

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gln Ser Ser Tyr Asp Phe Ala Met Asp Tyr Trp Gly Arg Gly

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

<210> 84

<211> 214

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> h1708-04 full-length light chain sequence

<400> 84

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 Glu Asn Ile Tyr Ser Tyr

20 25 30

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

35 40 45

Tyr Asn Ala Arg Thr Leu Ala Glu 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 Tyr His Ser Gly Ser Pro Leu

85 90 95

Pro 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> 85

<211> 446

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> h1708-04 heavy chain full-length sequence

<400> 85

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Lys Thr Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val 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 Ala Tyr Gly Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

<210> 86

<211> 214

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> h1709-10 full-length light chain sequence

<400> 86

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 Lys Ala Ser Gln Asn Val Val Thr Ala

20 25 30

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

35 40 45

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

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> 87

<211> 447

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> h1709-10 heavy chain full-length sequence

<400> 87

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Leu Val Tyr Pro Tyr Asn Asp Asn Thr Gly Tyr Asn Arg Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gly Gly Pro Ser Asn Trp Asn Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu 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> 88

<211> 214

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> h1710-01 full-length light chain sequence

<400> 88

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 Thr Ser Glu Asn Ile Phe Thr Tyr

20 25 30

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

35 40 45

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

85 90 95

Pro Phe Gly Gln Gly Thr Lys Leu 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> 89

<211> 446

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> h1710-01 heavy chain full-length sequence

<400> 89

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val 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 Gly Phe Gly Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

<210> 90

<211> 220

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> h1711-04 light chain full-length sequence

<400> 90

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

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 91

<211> 449

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> h1711-04 heavy chain full-length sequence

<400> 91

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 Asn Tyr

20 25 30

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

35 40 45

Gly Asp Ile Tyr Pro Gly Gly Ala Tyr Thr Asn Tyr Asn Glu 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 Phe Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Asp Tyr Tyr Asp Ser Ser Gly Arg Ala Met Asp Tyr Trp

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Lys

Claims

1. Use of a monoclonal antibody or antigen binding fragment thereof that specifically binds to human TIGIT in combination with a PD-1 inhibitor for the manufacture of a medicament for treating a tumor, wherein the monoclonal antibody or antigen binding fragment that specifically binds to human TIGIT comprises a heavy chain variable region and a light chain variable region, wherein:

the heavy chain variable region comprises the amino acid sequences shown as SEQ ID NO: 21. 22 and 23, and a light chain variable region comprising the amino acid sequences of HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NOs: 24. LCDR1, LCDR2 and LCDR3 as shown in amino acid sequences 25 and 26.

2. The use of claim 1, wherein the monoclonal antibody or antigen-binding fragment thereof that specifically binds to human TIGIT comprises:

the heavy chain variable region as set forth in SEQ ID NO 51, 54 or 55 and the light chain variable region as set forth in SEQ ID NO 52 or 53.

3. The use of claim 1, wherein the monoclonal antibody that specifically binds to human TIGIT is a full length antibody comprising human antibody constant regions.

4. The use of claim 1, wherein the monoclonal antibody that specifically binds to human TIGIT is a full length antibody comprising a human antibody heavy chain constant region as set forth in SEQ ID NO:78 and/or a human antibody light chain constant region as set forth in SEQ ID NO: 79.

5. The use of claim 1, wherein the monoclonal antibody that specifically binds to human TIGIT comprises: the heavy chain shown as SEQ ID NO. 85, and the light chain shown as SEQ ID NO. 84.

6. The use of claim 1, wherein the PD-1 inhibitor is selected from: anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-PD-L2 antibodies.

7. The use of claim 1, wherein the PD-1 inhibitor is selected from: nivolumab, Pembrolizumab, Atezolizumab, Avelumab, Durvalumab, tereprinimab, sediluzumab, tiraleuzumab, caprolixizumab, HRP00052, and HRP 00049.

8. The use of claim 1, wherein the tumor is selected from the group consisting of: head and neck cancer, central nervous system cancer, neuroendocrine tumor, esophageal cancer, malignant pleural mesothelioma, lung cancer, breast cancer, hepatobiliary cancer, pancreatic cancer, gastrointestinal cancer, kidney cancer, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, leukemia, lymphoma, bone cancer, squamous cell carcinoma, and ewing's sarcoma.

9. The use of claim 8, wherein the tumor is selected from the group consisting of: head and neck squamous cell carcinoma, brain cancer, glioma, neuroblastoma, neuroendocrine tumor, laryngeal cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatoma, pancreatic cancer, gastric cancer, intestinal cancer, clear cell renal cell carcinoma, melanoma, chondrosarcoma, myeloma, and myeloproliferative tumors.

10. The use of claim 8, wherein 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 enriched in T-cells/histiocytes, and lymphoplasmacytic lymphoma, said lung cancer being selected from the group consisting of: non-small cell lung cancer and small cell lung cancer, said leukemia being selected from: lymphocytic leukemia and myeloid leukemia.

11. A pharmaceutical composition comprising a monoclonal antibody or antigen-binding fragment thereof that specifically binds to human TIGIT, and a PD-1 inhibitor; wherein the monoclonal antibody or antigen binding fragment thereof that specifically binds to human TIGIT comprises a heavy chain variable region and a light chain variable region, wherein:

12. A kit comprising a package insert further comprising a monoclonal antibody or antigen-binding fragment thereof that specifically binds to human TIGIT and a PD-1 inhibitor, the package insert comprising instructions for using the monoclonal antibody or antigen-binding fragment thereof that specifically binds to human TIGIT in combination with the PD-1 inhibitor to treat a tumor; wherein the monoclonal antibody or antigen binding fragment thereof that specifically binds to human TIGIT comprises a heavy chain variable region and a light chain variable region, wherein:

13. The pharmaceutical composition of claim 11 or the kit of claim 12, wherein the monoclonal antibody or antigen-binding fragment thereof that specifically binds to human TIGIT comprises:

the heavy chain variable region shown in SEQ ID NO 51, 54 or 55 and the light chain variable region shown in SEQ ID NO 52 or 53.

14. The pharmaceutical composition of claim 11 or the kit of claim 12, wherein the monoclonal antibody that specifically binds to human TIGIT comprises: the heavy chain shown as SEQ ID NO. 85, and the light chain shown as SEQ ID NO. 84.

15. The pharmaceutical composition of claim 11 or the kit of claim 12, wherein the PD-1 inhibitor is selected from the group consisting of: anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-PD-L2 antibodies.

16. The pharmaceutical composition of claim 11 or the kit of claim 12, wherein the PD-1 inhibitor is selected from the group consisting of: nivolumab, Pembrolizumab, Atezolizumab, Avelumab, Durvalumab, tereprinimab, sediluzumab, tiraleuzumab, caprolixizumab, HRP00052, and HRP 00049.

17. The pharmaceutical composition according to claim 11 or the kit according to claim 12, for use in the treatment of a tumor selected from the group consisting of: head and neck cancer, central nervous system cancer, neuroendocrine tumor, esophageal cancer, malignant pleural mesothelioma, lung cancer, breast cancer, hepatobiliary cancer, pancreatic cancer, gastrointestinal cancer, kidney cancer, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, leukemia, lymphoma, bone cancer, squamous cell carcinoma, and ewing's sarcoma.

18. Use of the pharmaceutical composition or kit according to claim 17 for the treatment of a tumor, wherein said tumor is selected from the group consisting of: head and neck squamous cell carcinoma, brain cancer, glioma, neuroblastoma, neuroendocrine tumor, laryngeal cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatoma, pancreatic cancer, gastric cancer, intestinal cancer, clear cell renal cell carcinoma, melanoma, chondrosarcoma, myeloma, myeloproliferative tumor, and ewing's sarcoma.

19. Use of the pharmaceutical composition or kit according to claim 17 for the treatment of tumors, wherein said lymphoma is selected from the group consisting of: 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 in T-cells/histiocytes, and lymphoplasmacytic lymphoma, said lung cancer being selected from the group consisting of: non-small cell lung cancer and small cell lung cancer, said leukemia being selected from: lymphocytic leukemia and myeloid leukemia.