CN111744007A

CN111744007A - anti-TIGIT antibody pharmaceutical composition and application thereof

Info

Publication number: CN111744007A
Application number: CN202010228589.6A
Authority: CN
Inventors: 杨健健; 李皓; 刘洵; 付雅媛
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: 2020-10-09
Anticipated expiration: 2040-03-27
Also published as: CN111744007B

Abstract

The disclosure relates to an anti-TIGIT antibody pharmaceutical composition and use thereof. In particular, the disclosure relates to a pharmaceutical composition comprising an anti-TIGIT antibody or antigen-binding fragment thereof and a buffer. Further, the pharmaceutical composition further comprises a sugar and a surfactant. The pharmaceutical compositions of the present disclosure are useful for treating T cell dysfunctional disorders.

Description

anti-TIGIT antibody pharmaceutical composition and application thereof

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 T cell activation is dependent on both 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 (ImmunRev, 2012,248: 122-139; AutoimmunRev,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 ectodomain of human TIGIT molecule is 141 amino acids long and has 1 immunoglobulin V-like domain; 23 amino acids in the transmembrane region; the cytoplasmic region is shorter, 80 amino acids, with 1 PDZ binding domain and 1 ITIM motif. TIGIT molecules belong to a member of the immunoglobulin superfamily IgSF, are structurally conserved, homologous molecules are found in many mammals, and human TIGIT molecules have 88%, 67%, and 58% homology with monkey, dog, and mouse TIGIT molecules, 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). At present, patents such as WO2009126688, WO2014089113, WO2015009856, WO2015143343, WO2015174439, WO2017053748, WO2017030823, WO2016106302, US20160176963, US20130251720 and the like report antibodies of TIGIT and related applications. WO2018204405a1 also discloses an anti-TIGIT antibody formulation comprising an antioxidant. However, no TIGIT antibody has been clinically used so far.

Disclosure of Invention

The disclosure provides an anti-TIGIT antibody pharmaceutical composition and uses thereof.

In one aspect, the present disclosure provides a pharmaceutical composition comprising an anti-TIGIT antibody or antigen-binding fragment thereof that specifically binds to human TIGIT, and a buffer selected from the group consisting of a histidine salt buffer, a succinate buffer, an acetate buffer, and a phosphate buffer, the buffer having a pH of about 5.0 to 7.0; in some alternative embodiments, the pH of the buffer is about 5.0 to 6.5, about 5.0 to 6.0, about 5.5 to 6.5, about 5.0 to 5.7, or about 5.0 to 5.8; in other embodiments, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0; in other embodiments, the pH of the buffer is about 5.0, about 5.5, about 5.7, about 5.8, about 6.0, about 6.5, or about 7.0; in other embodiments, the buffer has a pH of about 5.5.

In some embodiments, the pharmaceutical composition of the present disclosure, wherein the buffer is selected from the group consisting of: acetic acid-sodium acetate buffer, succinic acid-sodium succinate buffer, histidine-hydrochloric acid buffer and histidine-acetic acid buffer. In some embodiments, the buffer is selected from: an acetic acid-sodium acetate buffer at a pH of about 5.0 to 5.7, a succinic acid-sodium succinate buffer at a pH of about 5.0 to 6.0, a histidine-hydrochloric acid buffer at a pH of about 5.5 to 6.0, or a histidine-acetic acid buffer at a pH of about 5.0 to 6.0; in some embodiments, the buffer is a histidine-acetic acid buffer at a pH of about 5.0 to 5.8; in some embodiments, the buffer is a histidine-acetic acid buffer at a pH of about 5.5. In other embodiments, the pharmaceutical composition of the disclosure, wherein the buffer concentration is about 5mM to 30mM, about 5mM to 15mM, or about 10 mM.

In some embodiments, the pharmaceutical composition of any one of the foregoing, wherein the anti-TIGIT antibody or antigen-binding fragment thereof is at a concentration of about 1mg/ml to 100 mg/ml; in some embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof is present at a concentration of about 40mg/ml to 60 mg/ml; in other embodiments, the concentration of the anti-TIGIT antibody or antigen-binding fragment thereof is about 50 mg/ml.

In some embodiments, the pharmaceutical composition of any of the preceding, further comprising a sugar. Without limitation, the sugar may be a conventional Composition (CH)₂O) n or derivatives thereof, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, and the like. In some embodiments, the sugar is selected from glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerol, erythritol, glycerol, arabitol, xylitol, sorbitol, mannitol, melibiose, melezitose, raffinose, mannotriose, stachyose, maltose, lactulose, maltulose, sorbitol, maltitol, lactitol, iso-maltulose, and the like. In some embodiments, the sugar is a disaccharide. In other embodiments, the sugar is sucrose. In some embodiments, the sugar concentration is about 65mg/ml to 100 mg/ml; in some embodiments, the sugar concentration is about 75mg/ml to 85 mg/ml; in some embodiments, the sugar concentration is about 65mg/ml, 70mg/ml, 75mg/ml, 80mg/ml, or 85 mg/ml. In some embodiments, the sugar concentration is about 80 mg/ml.

In some embodiments, the pharmaceutical composition of any of the preceding, further comprising a surfactant. Without limitation, the surfactant may be selected from polysorbate 20, polysorbate 80, polyhydroxylated hydrocarbon, Triton, sodium dodecylsulfonate, sodium lauryl sulfonate, sodium octyl glucoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-betaine, myristyl-betaine, cetyl-betaine, lauramidopropyl-betaine, cocamidopropyl-betaine, linoleamidopropyl-betaine, myrimidopropyl-betaine, palmamidopropyl-betaine, isosteamidopropyl-betaine, sodium lauryl sulfate, sodium lauryl sarcosinate, sodium myrimidopropyl, Myristamidopropyl-dimethylamine, palmitoamidopropyl-dimethylamine, isostearamidopropyl-dimethylamine, sodium methylcoconoyl, sodium methyloleyl taurate, polyethylene glycol, polypropylene glycol, and copolymers of ethylene and propylene glycol. In some embodiments, the surfactant is a polysorbate; in some embodiments, the surfactant is polysorbate 80. In some embodiments, wherein the concentration of surfactant is about 0.05mg/ml to 1.5 mg/ml; in other embodiments, the surfactant is present at a concentration of about 0.1mg/ml to about 0.8mg/ml, and in other embodiments, the surfactant is present at a concentration of about 0.4 mg/ml.

In some embodiments, the pharmaceutical composition of any of the preceding claims, comprising: a) an anti-TIGIT antibody or antigen-binding fragment thereof at a concentration of about 1mg/ml to 100mg/ml, b) a histidine buffer, a succinate buffer, an acetate buffer, or a phosphate buffer at a pH of about 5.0 to 7.0, c) sucrose at a concentration of about 65mg/ml to 100mg/ml, and d) polysorbate 80 at a concentration of about 0.05mg/ml to 1.5 mg/ml.

In some embodiments, the pharmaceutical composition comprises: a1) an anti-TIGIT antibody or antigen-binding fragment thereof at a concentration of about 40mg/ml to 60mg/ml, b1) sodium acetate-acetate at a pH of about 5.0 to 5.7, sodium succinate at a pH of about 5.0 to 6.0, histidine-histidine hydrochloride at a pH of about 5.5 to 6.0, or histidine-acetic acid at a pH of about 5.0 to 6.0, c1) sucrose at a concentration of about 75mg/ml to 85mg/ml, and d1) polysorbate 80 at a concentration of about 0.1mg/ml to 0.8 mg/ml.

In other embodiments, the pharmaceutical composition comprises: a 10mM histidine-acetic acid buffer at a pH of about 5.5, an anti-TIGIT antibody at a concentration of about 50mg/ml, sucrose at a concentration of about 80mg/ml, and polysorbate 80 at a concentration of about 0.4 mg/ml.

In some embodiments, the pharmaceutical composition of any one of the foregoing, wherein the antibody or antigen-binding fragment thereof that specifically binds to human TIGIT is a murine antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, or a humanized antibody or antigen-binding fragment thereof.

In some embodiments, the pharmaceutical composition of any one of the foregoing, wherein the 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 and a heavy chain variable region as 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 set forth in SEQ ID NO: LCDR1, LCDR2 and LCDR3 having the same sequence as the light chain variable region shown in FIG. 6;

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 set forth in SEQ ID NO: LCDR1, LCDR2 and LCDR3 of the same sequence as the light chain variable region set forth in fig. 8;

iii) the heavy chain variable region comprises a heavy chain variable region substantially identical to the heavy chain variable region as 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 set forth in SEQ ID NO: 10, LCDR1, LCDR2 and LCDR3 of the same sequence as the light chain variable region;

iv) the heavy chain variable region comprises a heavy chain variable region substantially identical to the heavy chain variable region as 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; 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 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 pharmaceutical composition of any one of the foregoing, the 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:

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 sequences as 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 sequences set forth in SEQ ID NOs: 27. 28 and 29, and a light chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown 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 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

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 shown in SEQ ID NOs: 42. LCDR1, LCDR2 and LCDR3 shown in amino acid sequences 43 and 44.

In some embodiments, the pharmaceutical composition of any one of the preceding, wherein the antibody that specifically binds to human TIGIT is a humanized antibody, wherein the antibody or antigen-binding fragment thereof that specifically binds to human TIGIT comprises a heavy chain variable region as set forth in SEQ ID No. 45, 51, 56, 64, or 71, or a heavy chain variable region variant comprising 1-10 amino acid changes in the heavy chain variable region sequence set forth in SEQ ID No. 45, 51, 56, 64, or 71; and/or a light chain variable region variant comprising a light chain variable region as set forth in SEQ ID NO 46, 52, 57, 65 or 72, or comprising 1-10 amino acid changes in the light chain variable region as set forth in SEQ ID NO 46, 52, 57, 65 or 72.

In some embodiments, the heavy chain variable region variant is a back mutation having 1 to 10 amino acids at the position of the FR region of the heavy chain variable region represented by SEQ ID NO 45, 51, 56, 64 or 71; preferably, the back-mutation is selected from:

(ii) a back mutation of an amino acid selected from one or more of N84S and S85R in the heavy chain variable region set forth in SEQ ID NO: 45; or

51 has one or more amino acid back mutations selected from M48I, R72V and V79A in the heavy chain variable region; or

56, having one or more amino acid back mutations selected from the group consisting of Y27F, M48I, R72V, V79A, and S84N; or

64, and one or more amino acid back mutations selected from the group consisting of R38K, R67K, R72V, T74K, M48I, V68A, M70L, and V79A; or

71 has one or more amino acid back mutations selected from G27Y, M48I, L83F and a 97T.

In other embodiments, the light chain variable region variant is a back mutation having 1 to 10 amino acids at the position of the FR region of the light chain variable region represented by SEQ ID NOs 46, 52, 57, 65, or 72; preferably, the back-mutation is selected from:

46 with one or more amino acid back mutations selected from the group consisting of S60D, T85D, A43S and S63T; or

52 having an amino acid back mutation selected from A43S in the light chain variable region; or

A back mutation of an amino acid having one or more selected from the group consisting of Q3V, A43S, S60D and Y87F in the variable region of the light chain represented by SEQ ID NO. 57; or

A back mutation of an amino acid having one or more selected from the group consisting of A43S and I48V in the light chain variable region represented by SEQ ID NO. 65; or

72 has one or more amino acid back mutations selected from the group consisting of N22S and P49S in the light chain variable region shown in SEQ ID NO.

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

a) a heavy chain variable region as set forth in

SEQ ID NO

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

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

c) a heavy chain variable region as set forth in SEQ ID NO 56, 61, 62 or 63 and/or a 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/or 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/or 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 as set forth in SEQ ID NO:51 and the heavy chain variable region set forth in SEQ ID NO: 53, or a light chain variable region as shown in fig.

In some embodiments, the pharmaceutical composition of any one of the preceding, wherein the antibody that specifically binds to human TIGIT is a full length antibody comprising a human antibody constant region; preferably, the antibody comprises a human antibody heavy chain constant region as shown in SEQ ID NO:78, and/or a human antibody light chain constant region as shown in SEQ ID NO: 79.

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

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

h) a heavy chain as shown in SEQ ID NO. 87, and/or a light chain as shown in SEQ ID NO. 86;

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

j) The heavy chain shown as SEQ ID NO. 91 and/or the light chain shown as SEQ ID NO. 90. Preferably, the monoclonal antibody or antigen binding fragment thereof that specifically binds to human TIGIT comprises a heavy chain as set forth in SEQ ID NO. 85 and a light chain as set forth in SEQ ID NO. 84;

in some embodiments, the pharmaceutical composition of any one of the foregoing, can contain in a unit dose amount from 0.01 to 99% by weight of an antibody or antigen-binding fragment thereof that specifically binds to human TIGIT; in some embodiments, the amount of antibody or antigen-binding fragment thereof that specifically binds to human TIGIT in a unit dose of the pharmaceutical composition is 0.1-2000 mg; in still other embodiments, the amount of antibody or antigen-binding fragment thereof that specifically binds to human TIGIT in a unit dose of the pharmaceutical composition is 1-1000 mg.

In another aspect, the present disclosure provides, in some embodiments, a method of making the pharmaceutical composition of any one of the foregoing, the method comprising the step of mixing an anti-TIGIT antibody or antigen-binding fragment thereof with a pharmaceutically acceptable excipient.

In another aspect, the present disclosure provides a lyophilized formulation comprising an anti-TIGIT antibody or antigen-binding fragment thereof obtained by freeze-drying the pharmaceutical composition of any of the foregoing. In some embodiments, the freeze-drying comprises the steps of prefreezing, primary drying, and secondary drying in sequence.

In another aspect, the present disclosure provides, in some embodiments, a lyophilized formulation comprising an anti-TIGIT antibody or antigen-binding fragment thereof, which upon reconstitution can form the pharmaceutical composition described previously.

In another aspect, the present disclosure provides a reconstitution solution comprising an anti-TIGIT antibody or antigen binding fragment thereof prepared by reconstituting a lyophilized formulation comprising an anti-TIGIT antibody or antigen binding fragment thereof as previously described.

In another aspect, in some embodiments, the present disclosure provides an article of manufacture comprising a container having the pharmaceutical composition, lyophilized formulation, or reconstituted solution of any one of the foregoing therein.

In another aspect, the present disclosure provides the pharmaceutical composition, lyophilized formulation, reconstituted solution or article of manufacture of any one of the foregoing as a medicament for treating a TIGIT-related disease in a human. In some embodiments, wherein the TIGIT-associated disease in humans is a tumor; in some embodiments, the tumor is selected from: head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumor, 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; in some embodiments, the tumor is selected from a CD 155-positive or PVR-positive tumor.

In another aspect, the present disclosure provides a use of the pharmaceutical composition, lyophilized formulation, reconstituted solution or article of manufacture of any one of the foregoing in the manufacture of a medicament for treating a TIGIT-related disease in a human. In some embodiments, wherein the TIGIT-associated disease in humans is a tumor; in some embodiments, the tumor is selected from: head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumor, 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; in some embodiments, the tumor is selected from a CD 155-positive or PVR-positive tumor.

In another aspect, in some embodiments, the present disclosure provides a method of treating or preventing a TIGIT-associated disease, the method comprising administering to a subject a therapeutically effective amount of the pharmaceutical composition, lyophilized formulation, reconstituted solution, or article of manufacture of any one of the foregoing. In some embodiments, wherein the TIGIT-associated disease in humans is a tumor; in some embodiments, the tumor is selected from: head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumor, 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; in some embodiments, the tumor is selected from a CD 155-positive or PVR-positive tumor.

Drawings

FIG. 1: ELISA detection of anti-TIGIT antibodies binding to human TIGIT protein.

FIG. 2: ELISA detection of anti-TIGIT antibody binding to monkey TIGIT protein.

FIG. 3: binding of anti-TIGIT antibodies to CHO cells overexpressing human TIGIT was detected.

FIG. 4: binding affinity of anti-TIGIT antibodies to human PBMC was detected.

FIG. 5: FIG. 5A is a graph of the blocking effect of ch1708 and its humanized antibodies on human TIGIT binding to CD 155; fig. 5B is a graph of the blocking effect of anti-TIGIT humanized antibody on human TIGIT binding to CD 155.

FIG. 6: blocking experiment of TIGIT antigen binding to CHO cells overexpressing CD155 by anti-TIGIT antibody.

FIG. 7: blocking of CD155 binding to TIGIT overexpressing CHO cells by anti-TIGIT antibodies.

FIG. 8: blocking experiments of TIGIT antigen binding to CHO cells overexpressing CD112 by anti-TIGIT antibodies.

FIG. 9: binding endocytosis experiments of anti-TIGIT antibodies in TIGIT overexpressed CHO cells, wherein endocytosis was 1 hour.

FIG. 10: FIG. 10A is a cell killing experiment of anti-TIGIT antibodies promoting natural killer cells (NK); FIG. 10B: cell killing experiments of Natural Killer (NK) cells were facilitated by anti-TIGIT antibodies.

FIG. 11: PBMC-T lymphocyte activation experiments with anti-TIGIT antibodies.

FIG. 12: pharmacokinetic assays of anti-TIGIT humanized antibodies in rats.

FIG. 13: DOE screening histidine-acetic acid (His-AA) buffer contours for anti-TIGIT antibody formulations, and the various contour delineation parameters in fig. 13 are shown in the following table:

FIG. 14: DOE screening histidine-histidine hydrochloride (His-HCl) buffer contours for anti-TIGIT antibody formulations, with the various contour scriber parameters in fig. 14 shown in the table below:

FIG. 15: the contour plots for the DOE screen succinic acid-sodium Succinate (SA) buffer for anti-TIGIT antibody formulations, with the various contour plot parameters in fig. 15 given in the table below:

Detailed Description

Term(s) for

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.

"buffer" or "buffering agent" refers to a buffer that is resistant to pH changes by the action of its acid-base conjugated components. Examples of buffers to control the pH in the appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine and other organic acid buffers.

"histidine salt buffer" or "histidine salt buffer" is a buffer comprising histidine ions. Examples of the histidine buffer include histidine-hydrochloric acid, histidine-acetic acid, histidine-phosphate, histidine-sulfate and the like. In some embodiments of the present disclosure, the buffer is selected from histidine-hydrochloric acid, histidine-acetic acid. The histidine-acetic acid buffer solution is prepared by histidine and acetic acid or histidine and histidine-acetate.

"succinate buffer" or "succinate buffer" is a buffer comprising succinate ions. Examples of succinate buffers include succinic acid-sodium succinate, succinic acid-potassium succinate, succinic acid-calcium succinate, and the like. In some embodiments of the present disclosure, the succinic acid buffer is succinic acid-sodium succinate.

An "acetate buffer" or "acetate buffer" is a buffer that includes acetate ions. Examples of the acetate buffer include acetic acid-sodium acetate, acetic acid histidine salt, acetic acid-potassium acetate, acetic acid calcium acetate, acetic acid magnesium acetate, and the like. In some embodiments of the present disclosure, the acetate buffer is acetic acid-sodium acetate.

A "phosphate buffer" or "phosphate buffer" is a buffer that includes phosphate ions. Examples of the phosphate buffer include disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, disodium hydrogen phosphate-citric acid, and the like. The present disclosure, in some embodiments, disodium hydrogen phosphate-citric acid.

"pharmaceutical composition" means a mixture containing one or more compounds described herein or a physiologically/pharmaceutically acceptable salt or prodrug thereof and other chemical components, such as physiologically/pharmaceutically acceptable carriers and excipients. The pharmaceutical composition maintains the stability of the active ingredients of the antibody, promotes the administration to organisms, is beneficial to the absorption of the active ingredients and further exerts the biological activity. Herein, "pharmaceutical composition" and "formulation" are not mutually exclusive. 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. In some embodiments, the pharmaceutical composition or formulation of the present disclosure is in the form of a solution, wherein the solvent is water.

The pharmaceutical compositions or formulations of the present disclosure may be prepared by methods well known in the art. Exemplary, preparation of antibody pharmaceutical compositions or formulations: the first step is as follows: a quantity of the purified antibody solution is subjected to solvent displacement (preferably ultrafiltration) with a buffer free of antibody (e.g., 10mM histidine-acetic acid buffer, pH 5.5), and the antibody is concentrated to about 70mg/mL by at least 6 volumes through an ultrafiltration membrane. Adding a certain volume of sucrose mother liquor, and uniformly mixing to make the final sucrose concentration be 80 mg/mL. A volume of polysorbate 80 stock solution was added and mixed well to give a final polysorbate 80 concentration of 0.4 mg/mL. 10mM histidine-acetic acid buffer pH5.5 was added to the solution to a volume of 50mg/mL antibody (other formulations to be tested or stability formulations were prepared by similar procedures). The product is filtered, and then is subjected to central control sampling detection and is aseptic. The stock solution was passed through a 0.22 μm PVDF filter cartridge and the filtrate was collected. The second step is that: adjusting the filling amount to 2.15ml, filling the filtrate into a 2ml penicillin bottle, plugging, and controlling and detecting the filling amount difference in sampling at the beginning of filling, in the middle of filling and at the end of filling respectively. The third step: and opening the capping machine, adding an aluminum cap, and capping. The fourth step: visual inspection is carried out to confirm that the product has no defects such as inaccurate loading and the like. Printing and sticking a penicillin bottle label; printing paper box labels, folding paper boxes, boxing and pasting paper box labels.

"Tm value" refers to the temperature at which a half of a protein is thermally denatured, i.e., the temperature at which the protein is unfolded, and the spatial structure of the protein is broken, so that the higher the Tm value, the higher the thermal stability of the protein.

In the solution form of the pharmaceutical composition described in the present disclosure, the solvent is water unless otherwise specified.

"lyophilized formulation" means a pharmaceutical composition in the form of a liquid or solution or a formulation or pharmaceutical composition obtained after a vacuum freeze-drying step of a liquid or solution formulation. Lyophilized formulations can be obtained by freeze-drying the pharmaceutical composition or liquid or solution formulation. Lyophilization is carried out by freezing the formulation and subsequently subliming the water at a temperature suitable for primary drying. Under these conditions, the product temperature is below the eutectic point or decomposition temperature of the formulation. At suitable pressures, typically in the range of about 50-250 millitorr, the primary drying is typically stored at a temperature in the range of about-30 to 25 ℃ (assuming the product remains frozen during the primary drying). The formulation, the size and type of container (e.g., glass vial) holding the sample, and the volume of liquid determine the time required for drying, which can range from a few hours to several days (e.g., 40-60 hours). The secondary drying stage may be carried out at about 0-40 c, depending primarily on the type and size of the container and the type of protein employed. The secondary drying time is determined by the desired residual moisture level in the product and typically takes at least about 5 hours. Typically, the lyophilized formulation has a water content of less than about 5%, preferably less than about 3%. The pressure may be the same as that applied in the primary drying step, and preferably, the pressure of the secondary drying is lower than that of the primary drying. The freeze-drying conditions may vary with formulation and vial size. In some embodiments of the present disclosure, the lyophilized formulation is prepared by filling the anti-TIGIT antibody formulation into 6mL _ vial at 2.15 mL/vial or into 15mL _ vial at 12.3 mL/vial, filling into a lyophilizer, and lyophilizing; the freeze-drying procedure comprises pre-freezing, primary drying and secondary drying; after the lyophilization procedure was completed, the stopper was vacuum stoppered.

The terms "about," "about," or "substantially comprising," as used herein, mean that a numerical value is within an acceptable error range for the particular value determined by one of ordinary skill in the art, with the numerical value depending in part on how the value is measured or determined (i.e., the limits of the measurement system). For example, "about" in each practice in the art may mean within 1 or a standard deviation of more than 1. Alternatively, "about" or "consisting essentially of" may mean a range of up to 20%. Furthermore, particularly for biological systems or processes, the term may mean at most an order of magnitude or at most 5 times the value. Unless otherwise indicated, when a particular value appears in the application and claims, the meaning of "about", "about" or "substantially comprising" should be assumed to be within an acceptable error range for that particular value.

The pharmaceutical composition of the present disclosure can achieve a stable effect: a pharmaceutical composition wherein the antibody substantially retains its physical and/or chemical stability and/or biological activity upon storage, preferably the pharmaceutical composition substantially retains its physical and chemical stability and its biological activity upon storage. The shelf life is generally selected based on a predetermined shelf life of the pharmaceutical composition. There are a number of analytical techniques currently available for measuring protein stability, which can measure stability after storage at a selected temperature for a selected period of time.

A stable pharmaceutical antibody formulation is one in which no significant change is observed under the following conditions: stored at refrigeration temperatures (2-8 ℃) for at least 3 months, preferably 6 months, more preferably 1 year, and even more preferably up to 2 years. In addition, stable liquid formulations include liquid formulations that: which exhibits desirable characteristics after storage at temperatures including 25 ℃ and 40 ℃ for periods of time including 1 month, 3 months, 6 months. Stable formulations such as: the pharmaceutical antibody formulation was colorless, or clear to slightly opalescent, by visual analysis. The concentration, pH and osmolality of the formulation have no more than ± 10% variation. Typically, no more than about 10%, preferably no more than about 5% truncation is observed. Typically no more than about 10%, preferably no more than about 5% aggregates are formed. In some embodiments, the pharmaceutical composition or lyophilized formulation of the present disclosure is stable at 2-8 ℃ for at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months; can be stably stored at 25 deg.C for at least 3 months and at least 6 months.

An antibody "retains its physical stability" in a pharmaceutical formulation if it does not exhibit significant increase in aggregation, precipitation and/or denaturation after visual inspection of color and/or clarity, or as measured by UV light scattering, Size Exclusion Chromatography (SEC) and Dynamic Light Scattering (DLS). Changes in protein conformation can be assessed by fluorescence spectroscopy (which determines the protein tertiary structure) and by FTIR spectroscopy (which determines the protein secondary structure).

An antibody "retains its chemical stability" in a pharmaceutical formulation if it does not exhibit significant chemical changes. Chemical stability can be assessed by detecting and quantifying the chemically altered form of the protein. Degradation processes that often alter the chemical structure of proteins include hydrolysis or truncation (assessed by methods such as size exclusion chromatography and SDS-PAGE), oxidation (assessed by methods such as peptide spectroscopy coupled to mass spectrometry or MALDI/TOF/MS), deamidation (assessed by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide spectroscopy, isoaspartic acid measurement) and isomerization (assessed by measuring isoaspartic acid content, peptide spectroscopy, etc.).

An antibody "retains its biological activity" in a pharmaceutical formulation if the biological activity of the antibody at a given time is within a predetermined range of the biological activity exhibited at the time of preparation of the pharmaceutical formulation. The biological activity of an antibody can be determined, for example, by an antigen binding assay.

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

An "antibody" as described in the present disclosure refers to an immunoglobulin, a complete antibody being a tetrapeptide chain structure formed by 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, i.e., IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being the μ, γ, α, and IgE chains, respectively. The same class of igs can be divided into different subclasses according to differences in amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain, and for example, iggs can be classified into IgG1, IgG2, IgG3 and 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 chain 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 exemplary embodiments, 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 the known Kabat numbering convention (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 (preparation) 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 a monoclonal antibody 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 an antibody produced by grafting murine CDR sequences into a human antibody variable region framework, i.e., a different type of human germline antibody framework sequence. Can overcome the heterogenous reaction induced by the chimeric antibody carrying a large amount of murine protein components. Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. Germline DNA sequences of, for example, human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (at the Internet)www.mrccpe.com.ac.uk/vbaseAvailable), and found in Kabat, e.a. et al, 1991Sequences of proteins of Immunological Interest, 5 th edition. To avoid reduced immunogenicity and reduced activity, the human antibody variable region framework sequences may be minimally back-mutated or back-mutated to retain activity. 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 nos 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 × 01 and hjh2), (IGHV1-46 × 01 and hjh 4.1.1) and (IGHV1-69 × 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 hjk4.1)) The combined sequence of (1). In order to avoid the decrease in immunogenicity and the resulting decrease in activity, the variable region of the human antibody may be subjected to minimal reverse mutation (back mutation, i.e., mutation of amino acid residues in the FR region from which the human antibody is derived to amino acid residues at positions corresponding to those in the original antibody from which it was derived) to retain activity. In one embodiment of the disclosure, wherein the light/heavy chain variable region of the anti-TIGIT humanized antibody is subjected to back mutation design, optimized light/heavy chain variable region combinations of h1707, h1708, h1709, h17010 and h17011 humanized antibodies are obtained, 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 heavy chain variable region of H1707-H2 (shown in SEQ ID NO: 50) and the light/heavy chain variable region pair consisting of the light chain variable region of H1707-L1 (shown in SEQ ID NO: 46), and so on.

Table 2: h1708 humanized antibody light/heavy chain variable region combinatorial 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 heavy chain variable region H1709-H2 (shown in SEQ ID NO: 61) and the light chain variable region H1709-L3 (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 a 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 variants thereof (e.g., one or more of YTE mutation or back 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 the kappa, lambda chain constant region or variants 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 in combination H1707-02V, and the heavy chain variable region H1707-H2(SEQ ID NO: 50) is substantially identical to the 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 the CDRs may 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 a high degree of somatic mutation. Thus, there may still be a need to graft such donor framework amino acids to the framework of humanized antibodies. 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 subtype 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.

By "amino acid change" or "amino acid difference" is meant the presence of an amino acid change or mutation in a variant protein or polypeptide as compared to the original protein or polypeptide, including the insertion, deletion or substitution of 1 or several amino acids based on the original protein or polypeptide. In one embodiment of the disclosure, wherein the light/heavy chain variable region of the anti-TIGIT humanized antibody is back-mutated in design, in some embodiments, of the disclosure

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 to 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')₂A 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) single domain or dAb fragments (Ward et al, (1989) Nature 341: 544-546) consisting 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 possible to generate a single protein chain in which the VL and VH regions pair to form monovalent molecules (known 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 antibody may be of a different isotype, for example, an IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM antibody.

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 of 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 (NH)₂-VL-linker-VH-COOH or NH₂-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof, e.g.using 1-4 repeated variants (Holliger et al (1993), Proc. Natl. Acad. Sci. USA90: 6444-. Other linkers useful in the present disclosure are made by Alfthan et al (1995), Protein Eng.8:725-Human (1999), J.mol.biol.293:41-56 and Rovers et al (2001), Cancer Immunol.

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 having 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 cDNA 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 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 coding cDNA of VH and VL of the monoclonal antibody which specifically recognizes human TIGIT and is combined with the amino acid sequence of the extracellular region or the three-dimensional structure thereof, constructing DNA coding dsFv, 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 dsFv.

A CDR-containing peptide is composed of one or more regions in a CDR that contains 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 an amino acid sequence of an 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 can also be produced by chemical synthesis methods such as the Fmoc method or the tBoc method.

The term "antibody framework" as used herein refers to a portion of a variable domain, VL or VH, which 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-, 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 approximately 26-35(CDR1), 51-57(CDR2) and 93-102(CDR3), and the CDR amino acid residue numbers in VL are approximately 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., epipope 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^-7M, e.g. less than about 10^-8M、10^- ⁹M、10^-10M、10^-11M or less affinity (KD) binding.

The term "KD" or "KD" refers to the dissociation equilibrium constant of 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^-8M、10^-9M or 10^-10A 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 "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 so as to be 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 fragments 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 may 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 using an A or G Sepharose FF column containing a buffer adjusted. Non-specifically bound fractions are washed away. And eluting the bound antibody by using a pH gradient method, detecting the antibody fragment 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 either 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" may 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 the subject patient or population in an amount effective to alleviate one or more symptoms of the disease, to induce regression of such symptoms or to inhibit development of such symptoms to any clinically measurable degree. The amount of therapeutic agent effective to alleviate any particular disease symptom (also referred to as a "therapeutically effective amount") can vary depending on a variety of factors, such as the disease state, age, and weight of the patient, and the ability of the drug to produce a desired therapeutic effect in the patient. Whether a disease symptom has been reduced can be assessed by any clinical test commonly used by physicians or other health professional to assess the severity or progression of the symptom. Although embodiments of the present disclosure (e.g., methods of treatment or articles of manufacture) may be ineffective in alleviating the symptoms of each target disease, they should alleviate the symptoms of the target disease in a statistically significant number of patients as determined according to any statistical test method known in the art, such as Student's t-test, chi-square test, U-test by Mann and Whitney, Kruskal-Wallis test (H-test), Jonckhere-Terpstra test, and Wilcoxon test.

"conservative modification" or "conservative substitution" refers to the replacement of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation, and rigidity, etc.) so that changes can be made frequently without changing 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, He Gene, The Benjamin/Cummings pub. Co., p. 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 general health of the patient, the method and dosage of administration, and the severity of side effects. An effective amount may 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, two sequences are 60% homologous if there are 6 matches or homologies at 10 positions in the two sequences when the sequences are optimally aligned; 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 harborSymp. 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, which includes the use of known nucleic acids and nucleic acid polymerases as primers to amplify or generate specific portions 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 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 for 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 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 according to conventional conditions, such as the antibody technical laboratory manual of cold spring harbor, molecular cloning manual; or according to the conditions recommended by the manufacturer of the raw material or the goods. Reagents of specific sources are not indicated, and conventional reagents are purchased in the market.

Example 1 preparation of anti-TIGIT antibody

First, preparation of 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

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

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

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

(italic part) fusion protein of cynoTIGIT extracellular region with mouse IgG2aFc fragment: CynoTIGIT-mFc for detection

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 cell supernatant, preferably, protein G is subjected to affinity chromatography, cultured hybridoma cells are centrifuged to obtain supernatant, and 10-15% by volume of 1M Tris-HCl (pH8.0-8.5) is added according to 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 system 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 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.

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

firstly, cell culture supernatant expressing Fc fusion protein or antibody is subjected to high-speed centrifugation to collect the supernatant. The ProteinA affinity column was washed 3-5 column volumes with 6M guanidine hydrochloride and then 3-5 column volumes with pure water. The column is equilibrated 3-5 column volumes using, for example, a1 XPBS (pH7.4) buffer system as the equilibration buffer. The cell supernatant was combined by low flow 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 with 1 × PBS (ph7.4) for 3-5 column volumes 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.

Preparation of anti-human TIGIT hybridoma monoclonal antibody

2.1 immunization

Anti-human TIGIT monoclonal antibodies were produced by immunizing 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 a 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-immunizes. Antigen and adjuvant: (

Golddjuvant) ratio of 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. First, the50 μ g/pellet of post-emulsification antigen was injected Intraperitoneally (IP) 0 days. 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 tending to plateau were selected for splenocyte fusion. 3 days before splenocyte fusion, the immunization was boosted by Intraperitoneal (IP) injection of 50. mu.g/mouse of antigen solution in physiological saline.

2.2 spleen cell fusion

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

CRL-8287^TM) The fused hybridoma cells were resuspended in complete medium (DMEM medium containing 20% FBS, 1 × HAT, 1 × OPI) at a density of 0.5-1 × 10^6/ml, seeded in 96-well plates at 37 ℃ and 5% CO at 100. mu.l/well₂After 3-4 days of incubation, 100. mu.l/well HAT complete medium was supplemented, and incubation was continued for 3-4 days until pinpoint colonies formed, the supernatant was removed, and 200. mu.l/well of HT complete medium (RPMI-1640 medium containing 20% FBS, 1 × HT and 1 × OPI) was added at 37 ℃ and 5% CO₂ELISA was performed after 3 days of culture.

2.3 hybridoma cell selection

Hybridoma culture supernatant detection was performed by a combination of ELISA method according to the growth density of hybridoma cells. And cell binding experiments and cell blocking experiments are carried out on the cell supernatants of the positive wells combined with ELISA detection. And combining and blocking the positive well cells, and performing amplification and cryopreservation and two to three times of subcloning in time until single cell clone is obtained.

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

2.4 sequencing of hybridoma Positive clones

The sequence cloning from positive hybridomas is as follows. The logarithmic growth phase hybridoma cells were harvested, RNA was extracted using Trizol (Invitrogen, Cat No.15596-018) according to the kit instructions, and PrimeScript was used^TMReverse transcription using the Reverse Transcriptase kit (Takara, Cat No. 2680A). The cDNA obtained by reverse transcription was subjected to PCR amplification 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 region sequences

Humanized anti-human TIGIT antibody of mouse source

By comparing an IMGT human antibody heavy-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. And (3) carrying out back mutation on the amino acid in the FR region as required to obtain the humanized anti-TIGIT antibody. Exemplarily, the following embodiments wherein the determination of the amino acid residues in the CDR regions is determined and annotated by the Kabat numbering system.

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

3.1 humanization of hybridoma clone m1707

(1) m1707 selection of humanized frameworks

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

h1707VL-CDR graft

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

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

table 7: h1707 Back-mutation design

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

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

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

>h1707-L2

>h1707-L3

>h1707-L4

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

>h1707-H2

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, the humanized heavy chain templates are IGHV1-46 x 01 and hjh4.1, and the humanized antibody h1708 is obtained after humanization, and the humanized variable region sequence is as follows:

h1708VH-CDR graft

h1708VL-CDR graft

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

table 8: h1708 Back-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 h1708 humanized antibody light/heavy chain variable region after back mutation design is as follows:

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

>h1708-L2

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

>h1708-H2

>h1708-H3

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 hjh4.1, and the humanized antibody h1709 is obtained after humanization, and the humanized variable region sequence is as follows:

h1709VH-CDR graft

h1709VL-CDR graft

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

table 9: h1709 Back-mutation design

Note: as shown in S60D, the position 60S is mutated back to A according to the natural sequence numbering of the amino acid sequence. 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 (h 1709 VL-CDR graft)

>h1709-L2

>h1709L3

>h1709-L4

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

>h1709-H2

>h1709-H3

>h1709-H4

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

h1710VL-CDR graft

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

table 10: h1710 reverse mutation design

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

h1710-L1 (h 1710 VL-CDR gradient)

>h1710-L2

H1710-H1 (same H1710 VH-CDR gradient)

>h1710-H2

>h1710-H3

>h1710-H4

>h1710-H5

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, wherein the humanized variable region sequences are as follows:

h1711VH-CDR graft

h1711VL-CDR graft

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

table 11: h1711 reverse mutation design

Note: p49 is mutated back to S as indicated by P49S, which is numbered 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 h1711 humanized antibody light/heavy chain variable region after back mutation design is as follows:

h1711-L1 (h 1711 VL-CDR gradient)

>h1711-L2

>h1711-L3

H1711-H1 (H1711 VH-CDR graft)

>h1711-H2

>h1711-H3

>h1711-H4

3.6 construction and expression of humanized antibodies

Connecting the heavy chain variable region of the humanized antibody obtained by the design of 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 formed by a light chain linked at the amino terminus of a light chain constant region.

Table 13: h1708 humanized antibodies

Note: in the table, "H1708-04" indicates the amino acid sequence represented by the heavy chain variable region of H1708-H1 (shown as SEQ ID NO: 51) and the amino acid sequence shown as SEQ ID NO:78 to the variable region of h1708-L2 light chain (shown as SEQ ID NO: 53) and the amino terminus of the heavy chain constant region shown as 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 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 as SEQ ID NO: 61) and the amino acid sequence shown as 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 formed by a light chain linked at 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 in 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 antibody

Note: in the table, "H1711-04" indicates the amino acid sequence represented by the heavy chain variable region of H1711-H4 (shown as SEQ ID NO: 77) and the amino acid sequence shown as SEQ ID NO:78 to the variable region h1711-L1 (shown as SEQ ID NO: 72) and the amino terminus of the heavy chain constant region shown as 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.

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 in SEQ ID NO:78 and SEQ ID NO:79 to form a full length antibody. The specific sequences of VH and VL of 22G2-H3Q are as follows:

22G2-H3Q VH：

QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGIYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDYYVSGNYYNVDYYFFGVDVWGQGTTVTVSS

SEQ ID NO：80

22G2-H3Q VL：

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPLFTFGPGTKVDIK

SEQ ID NO：81

an exemplary anti-human TIGIT antibody light/heavy chain full length sequence is as follows:

h1707-02 light chain full-length sequence

H1707-02 heavy chain full-length sequence

H1708-04 full-length light chain sequence

H1708-04 heavy chain full-length sequence

H1709-10 full-length light chain sequence

H1709-10 heavy chain full-length sequence

H1710-01 full-length light chain sequence

H1710-01 heavy chain full-length sequence

H1711-04 light chain full-length sequence

H1711-04 heavy chain full-length sequence

Example 2 anti-TIGIT antibody Activity test experiment

Firstly, the method comprises the following steps: ELISA experiment of TIGIT antibody combined with human TIGIT protein

The binding capacity of the anti-TIGIT antibody was detected by ELISA assay of the antibody with human TIGIT protein. The Fc or mFc labeled TIGIT fusion protein is fixed in a 96-well enzyme label plate by binding with an anti-Fc or mFc antibody coated in the enzyme label plate, the strength of a signal after the addition of the antibody is used for judging the binding activity of the antibody and TIGIT, and the positive control molecules are 22G2-H3Q and 10A7 hIgG4 (wherein the light and heavy chain variable region sequence of the 10A7 hIgG4 is from SEQ ID NO: 21 and 22 in US20130251720A1, and the light and heavy chain constant regions connected with the light and heavy chain variable regions thereof to form a full-length antibody are respectively SEQ ID NO:79 and 78 of the application) are specifically tested as follows:

goat anti-human Fc antibody (Jackson ImmunoResearch, Cat No. 109-. After discarding the liquid, 200. mu.l/well of 5% skim milk (BD ski mil, Cat No.232100) diluted with PBS was added and blocked by incubation in an incubator at 37 ℃ for 3 hours or overnight (16-18 hours) at 4 ℃. After blocking, the blocking solution was discarded, and after washing the plate 5 times with PBST buffer (pH7.4 PBS containing 0.05% tween-20), 50. mu.l/well of TIGIT-Fc fusion protein (in-house production) or TIGIT-mFc fusion protein (in-house production) diluted to 0.5. mu.g/ml with a sample diluent (pH7.4 PBS containing 1% BSA) was added, and the mixture was incubated in an incubator at 37 ℃ for 1 hour or left overnight at 4 ℃. After the incubation, the reaction solution in the microplate was discarded, and after washing the plate 5 times with PBST, 50. mu.l/well of the antibody to be tested (hybridoma purified antibody or humanized antibody) diluted with the sample diluent was added thereto and incubated in an incubator at 37 ℃ for 1 hour. After completion of incubation, the plate was washed 5 times with PBST, and 50. mu.l/well of HRP-labeled secondary goat anti-mouse antibody (Jackson ImmunoResearch, Cat No. 115-. Washing the plate with PBST for 5 times, adding 50 μ l/well TMB chromogenic substrate (KPL, Cat No.52-00-03), incubating at room temperature for 5-10min, addingInto 50. mu.l/well 1M H₂SO₄The reaction was stopped, absorbance was read at a wavelength of 450nm using a microplate reader (thermal Multiskan, MK3), and data were analyzed using GraphPad Prism5 to calculate the binding EC50 value of TIGIT antibody to human TIGIT protein. The results are shown in FIG. 1.

II, secondly: ELISA experiment of TIGIT antibody combined with TIGIT protein of cynomolgus monkey

Monkey cross-binding of anti-TIGIT antibodies was detected by ELISA of the antibodies with cynomolgus monkey (cynomolgus) TIGIT protein. The cynomolgus monkey TIGIT fusion protein with Fc or mFc label is combined with an anti-Fc or mFc antibody coated in an enzyme label plate so as to be fixed in a 96-hole enzyme label plate, the intensity of a signal after the addition of the antibody is used for judging the combination activity of the antibody and the cynomolgus monkey TIGIT, and the specific experimental method is as follows:

goat anti-human Fc antibody (Jackson ImmunoResearch, Cat No. 109-. After discarding the liquid, 200. mu.l/well of 5% skim milk (BD ski mil, Cat No.232100) diluted with PBS was added and blocked by incubation in an incubator at 37 ℃ for 3 hours or overnight (16-18 hours) at 4 ℃. After blocking, the blocking solution was discarded, and the plate was washed with PBST buffer (pH7.4 PBS containing 0.05% tween-20) for 5 times, then 50. mu.l/well of a sample diluent (pH7.4 PBS containing 1% BSA) was added to dilute the solution to 0.5. mu.g/ml of cynomolgus monkey TIGIT-Fc fusion protein (in-house production) or cynomolgus monkey TIGIT-mFc fusion protein (in-house production), and the mixture was incubated in an incubator at 37 ℃ for 1 hour or left overnight at 4 ℃. After the incubation, the reaction solution in the microplate was discarded, and after washing the plate 5 times with PBST, 50. mu.l/well of the antibody to be tested (hybridoma purified antibody or humanized antibody) diluted with the sample diluent was added thereto and incubated in an incubator at 37 ℃ for 1 hour. After completion of incubation, the plate was washed 5 times with PBST, and 50. mu.l/well of HRP-labeled secondary goat anti-mouse antibody (Jackson Immuno Research, Cat No. 115-. After washing the plates 5 times with PBST, addAdding 50 μ l/well TMB chromogenic substrate (KPL, Cat No.52-00-03), incubating at room temperature for 5-10min, adding 50 μ l/well 1M H₂SO₄The reaction was stopped, absorbance was read at a wavelength of 450nm using a microplate reader (Thermo scientific Multiskan MK3), and data were analyzed using GraphPad Prism5 to calculate the binding EC50 value of TIGIT antibody to monkey TIGIT. The results are shown in FIG. 2.

Thirdly, the method comprises the following steps: binding experiment of TIGIT antibody and human TIGIT overexpression CHO cell

The binding capacity of the anti-TIGIT antibody was detected by binding experiments of the antibody to CHO cells overexpressing TIGIT protein. Transferring the TIGIT full-length plasmid into CHO cells by an electrotransfection method, pressurizing and screening for two weeks, and then detecting the expression level of the TIGIT. After the over-expression cells are fixed at the bottom of a 96-well plate, the strength of signals after the addition of the antibody is used for judging the binding activity of the antibody and TIGIT over-expression CHO cells, and the specific experimental method is as follows:

cells were seeded at a density of 5X 10^5/ml, 100. mu.l/well in 96-well plates for overnight culture. The supernatant was discarded, washed three times with PBS, and then fixed with 100. mu.l/well of cell-mediated immunity fixative (Beyotime, Cat No. P0098) at room temperature for half an hour, and washed four times with PBS. After discarding the liquid, 200. mu.l/well of a 5% skim milk (BD ski milk, Cat No.232100) blocking solution diluted with PBS was added, and the mixture was blocked by incubation in an incubator at 37 ℃ for 3 hours. After blocking was complete, the blocking solution was discarded, and after washing the plate 5 times with PBST buffer (pH7.4 PBS containing 0.05% tween-20), 50. mu.l/well of an antibody to be tested (hybridoma purified antibody or humanized antibody) diluted with a sample diluent at various concentrations was added and incubated in an incubator at 37 ℃ for 1 hour. After completion of incubation, the plate was washed 5 times with PBST, and 50. mu.l/well of HRP-labeled secondary goat anti-mouse antibody (Jackson ImmunoResearch, Cat No. 115-. After washing the plate 5 times with PBST, 50. mu.l/well of TMB chromogenic substrate (KPL, Cat No.52-00-03) was added, the reaction was incubated at room temperature for 5-15min, 50. mu.l/well of 1M H2SO4 was added to stop the reaction, absorbance was read at a wavelength of 450nm using a microplate reader (Thermosific Multiskan MK3), data were analyzed using GraphPad Prism5, and the binding EC50 value of TIGIT antibody to TIGIT overexpressing CHO cells was calculated. The results are shown in FIG. 3.

Fourthly, the method comprises the following steps: binding experiment of TIGIT antibody and human PBMC

Binding of anti-TIGIT antibodies was detected by antibody binding experiments with in vitro activated human PBMCs. Human PBMC are activated by a superantigen staphylococcus aureus enterotoxin B (SEB) stimulation method, and the strength of a fluorescence signal after the antibody is added is used for judging the binding activity of the antibody and the activated human PBMC, wherein the specific experimental method is as follows:

fresh blood PBMC were obtained by Ficoll-Hypaque density gradient centrifugation (Stem Cell Technologies) and cultured in RPMI 1640 medium supplemented with 10% (v/v) FBS and 500ng/ml of superantigen Staphylococcus aureus enterotoxin B (SEB), 37 ℃ and 5% CO₂Cultured under the conditions for 4 days.

The activated PBMC cells were seeded at a density of 5X 10^6/ml, 100. mu.l/well in a 96-well round bottom plate (Corning, Cat No.32915001), centrifuged at 1500rpm for 5 minutes in a Centrifuge (Beckman Coulter, Allegra X-15R Centrifuge), and the supernatant was discarded; 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, the cells were centrifuged at 1500rpm for 5 minutes, the supernatant was discarded, and after washing the cells twice with the sample diluent, 100. mu.l of PE-goat anti-human IgG (Jackson ImmunoResearch, 109-. After incubation, centrifugation was carried out at 1500rpm for 5 minutes, the supernatant was discarded, the cells were washed twice with a sample diluent, and finally the cells were resuspended in a 200. mu.l/well sample diluent, the intensity of the fluorescence signal was detected on a flow cytometer (BD FACS Canto II), and the data was analyzed with GraphPad Prism5 to calculate the EC50 value of the TIGIT antibody binding to human PBMC cells. The results are shown in FIG. 4.

Fifthly: 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 covalently coupled to a biosensor chip (Cat. #28-9538-28, GE) using Protein a biosensor chip (Cat. #29127556, GE) for affinity capture or following the methods described in the specification of the human anti-capture kit (Cat. #28-9538-28, GE) to affinity capture an amount of the antibody to be detected, followed by flowing human and monkey TIGIT antigens under a series of concentration gradients on the surface of the chip, the human TIGIT being selected from the group consisting of kyushania bio (cat.10917-H08-H, sino.biol), the monkey TIGIT being purified from the expression of examples 1 and 2, and the binding and dissociation curves being obtained by detecting the reaction signals in real time using a Biacore instrument (Biacore t200, GE). After each cycle of dissociation was completed, the biochip was washed and regenerated with a regeneration solution prepared in a human 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 corporation (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 the (1:1) Langmuir model to give affinity values, and the results 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 with cynoTIGIT protein

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

Sixthly, the method comprises the following steps: blocking test of TIGIT antibody on binding of TIGIT antigen and CD155 protein

Blocking ability of anti-TIGIT antibodies was tested by HTRF experiments in which antibodies block TIGIT binding to CD155 protein. Using Pab Anti-Human IgG-Tb (Cisbio, Cat No.61HFCTAA) and Streptavidin-XL665(Cisbio, Cat No.610SAXLA) pairs of donors and recipients bound to TIGIT-Fc and biotinylated CD155(R & D, Cat No.2530-CD 050/CF), or Pab Anti-mouse-IgG-XL665(Cisbio, Cat No.61PAMXLA) and Streptavidin-Tb (Cisbio, Cat No.610SATLA) bound to TIGIT-mFc and biotinylated CD155(R & D, Cat No.2530-CD-050/CF), the intensity of signal after hybridoma purified antibody or humanized antibody addition was used to determine whether the antibody blocked TIGIT and CD155 activity, as follows:

mu.l/well of an antibody to be tested (hybridoma purified antibody or humanized antibody) at different concentrations diluted with a diluent (pH7.4 PBS containing 1% BSA) was added to a 384-well test plate (Corning, Cat No.3706), 2.5. mu.l/well of TIGIT-Fc or TIGIT-mFc diluted to 2. mu.g/ml with a sample diluent was added after centrifugation at 1000rpm for 1min, 2.5. mu.l/well of biotin-CD155 diluted to 4. mu.g/ml was added after centrifugation at 1000rpm for 1min, preincubation was performed at room temperature for 10min after centrifugation at 1000rpm for 1min, and then 2.5. mu.l/well of a sample diluent was added to 3.2. mu.g/ml of Pab Anti-Human IgG-Tb (Cisbio, Cat No.61HFCTAA) and 2.5. mu.08. mu.g/ml of Streptavidin-XL (Cisbio, Cat No.610SAXLA) diluted to 2.5. mu.5. mu.l/well of sample diluent of Pastevi-XL, cat No.61PAMXLA) and 2.5. mu.l/well 0.08. mu.g/ml Streptavidin-Tb (Cisbio, Cat No.610SATLA), left at room temperature for 1 hour, detected by PHEARstar FS microplate reader (BMG LABTECH) at 665nm and 620nm, analyzed by GraphPad Prism5, and calculated the inhibitory activity of TIGIT antibody against human TIGIT and CD155 proteins. The results are shown in FIGS. 5A and 5B.

Seventhly, blocking experiment of TIGIT antibody on binding of TIGIT antigen and CHO cell over-expressing CD155

Blocking ability of anti-TIGIT antibody was tested by ELISA experiments with antibodies blocking TIGIT binding to CHO cells overexpressing CD 155. After transfection of the full-length CD155 plasmid into CHO cells by electrotransfection method and two weeks of pressurized screening, the expression level of CD155 was determined. After the over-expression cells are fixed at the bottom of a 96-well plate, the TIGIT is preincubated with anti-TIGIT antibodies with different diluted concentrations and then added into the plate, and the strength of a signal after the addition of the secondary antibody is used for judging the capacity of the antibody for blocking the combination of the TIGIT and CHO cells over-expressing CD155, wherein the specific experimental method comprises the following steps:

CD155-CHO cells were seeded in 96-well plates at a density of 5X 10^5/ml, 100. mu.l/well overnight. The supernatant was discarded, washed three times with PBS, and then fixed with 100. mu.l/well of a cell immobilizer (Beyotime, Cat No. P0098) at room temperature for half an hour, and washed four times with PBS. After discarding the liquid, 200. mu.l/well of 5% skim milk (BD ski milk, Cat No.232100) diluted with PBS was added and the mixture was incubated at 37 ℃ for 3 hours for blocking. After blocking, the blocking solution was discarded, and the plate was washed 5 times with PBST buffer (pH7.4 PBS containing 0.05% tween-20), and then 50. mu.l/well of a premixed antigen-antibody mixture of human TIGIT-hFc (in-house) diluted with a sample diluent (pH7.4 PBS containing 1% BSA) and human TIGIT-mFc (in-house) having a final concentration of 0.2. mu.g/ml and an antibody to be detected having a gradient concentration was added thereto and incubated at 37 ℃ for 1 hour in an incubator. After the incubation was completed, the reaction solution in the microplate was discarded, and after washing the plate 5 times with PBST, 50. mu.l/well of HRP-labeled secondary goat anti-human antibody (Jackson Immuno Research, Cat No. 109-. After washing the plate 5 times with PBST, 50. mu.l/well of TMB chromogenic substrate (KPL, Cat No.52-00-03) was added, the reaction was stopped by incubating at room temperature for 5-15 minutes, 50. mu.l/well of 1M H2SO4 solution was added, absorbance was read at a wavelength of 450nm using a microplate reader (Thermo scientific Multiskan, MK3), and the blocking effect of the TIGIT antibody on the binding of antigen to CD 155-overexpressing CHO cells was calculated by analyzing data with GraphPad Prism 5. The results are shown in FIG. 6.

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 and anti-TIGIT antibodies with different concentrations, adding fluorescence labeled CD155-Fc for incubation, and judging the capacity of the antibodies for blocking the combination of the CD155 and CHO cells over-expressing TIGIT according to the strength of signals, wherein the specific experimental method comprises the following steps:

firstly, labeling CD155-Fc (Sino Biological, Cat No.10109-H02H)CF^TM633(SigmaAldrich, Cat No. MX633S100) CD155-Fc was dissolved in PBS to a concentration of 0.5-1mg/ml, 10 × Mix-n-Stain Reaction Buffer in a volume 9 times the volume of the sample was added and mixed, and CF was added^TM633 fluorescent dye, and after incubation for 30 minutes at room temperature in the dark, the fluorescent labeling is completed.

TIGIT-CHO cells were seeded at 5 × 10^6/ml, 100. mu.l/well in a 96-well round bottom plate (Corning, Cat No.32915001), centrifuged at 1500rpm for 5 minutes in a Centrifuge (Beckman Coulter, Allegra X-15R Centrifuge), the supernatant was discarded, the cells were resuspended in 200. mu.l PBS, centrifuged, the supernatant was discarded, and the procedure was repeated, 100. mu.l/well of a test antibody solution which had been diluted in a gradient with a sample diluent (pH7.4 PBS containing 1% BSA) was added to the resuspended cells, the incubation was carried out at 4 ℃ for 1 hour, after completion of the incubation, 1500rpm was centrifuged for 5 minutes, the supernatant was discarded, the cells were washed twice with the sample diluent, and 100. mu.l of CF 2. mu.g/ml was added^TM633 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 FACSCAnto II), analyzing data with GraphPad Prism5, and calculating the blocking capacity of the TIGIT antibody on the combination of CD155 and TIGIT-CHO cells. The results are shown in FIG. 7.

Blocking experiment of TIGIT antibody on binding of TIGIT antigen and CHO cell over-expressing CD112

Blocking ability of anti-TIGIT antibodies was detected by FACS experiments in which the antibodies blocked TIGIT binding to CD112 overexpressing CHO cells. After transfection of the full-length plasmid of CD112 into CHO cells by the electrotransfection method, the expression level of CD112 was measured after two weeks of pressurized selection. The method comprises the following steps of pre-incubating TIGIT-mFc protein and diluted anti-TIGIT antibodies with different concentrations, adding the pre-incubated TIGIT-mFc protein into CHO cells over-expressed by CD112 for incubation, and adding an antibody of PE label to detect the intensity of TIGIT signals so as to judge the capacity of the antibodies for blocking the combination of TIGIT and the CHO cells over-expressed by CD112, wherein the specific experimental method comprises the following steps:

diluting a humanized antibody sample by 1% BSA, diluting nine concentration points from 20 mu g/mL of initial concentration twice, simultaneously diluting TIGIT-mFc to 2 mu g/mL, uniformly mixing an antigen and antibodies with different concentrations according to a volume ratio of 1:1, and pre-incubating for 30 minutes at 37 ℃; collecting CD112-CHOs cells, washing once with PBS, and distributing according to 0.5 x 10^ 6/test; resuspend cells with 150. mu.l antigen-antibody mixture, incubate for 60 min at 4 ℃ and wash 3 times with 1% BSA; PE-goat anti-mouse IgG (Biolegend,405307) dilution 100u l heavy suspension cells, 4 degrees C were incubated for 40 minutes, 1% BSA 3 times, using 200 u l 1% BSA heavy suspension cells, flow cytometry BDFACSCAnto II reading each sample MFI, GraphPad Prism5 analysis data, judge TIGIT antibody antigen and CD112-CHO cell binding blocking effect. The results are shown in FIG. 8.

Ten, TIGIT antibody in TIGIT over-expression CHO cell combined endocytosis experiment

To investigate the ability of TIGIT antibodies to endocytose upon binding to cell surface antigens, FACS experiments for TIGIT antibody endocytosis were performed with CHO cells overexpressing full-length TIGIT. The specific experimental method is as follows:

TIGIT-CHO cells were seeded at a density of 2X 10^6/ml in 100. mu.l/well in a 96-well round bottom plate (Corning, Cat No.32915001), centrifuged at 1500rpm for 5 minutes in a Centrifuge (Beckman Coulter, Allegra X-15R Centrifuge), and the supernatant was discarded; cells were resuspended in 200. mu.l of 1% BSA, centrifuged, and the supernatant discarded and repeated. Humanized antibody samples were diluted to a concentration of 4. mu.g/mL with 1% BSA, resuspended cells were added to 100. mu.l/well, and incubated on ice for 1 hour. After the incubation was completed, centrifugation was carried out at 1500rpm for 5 minutes, the supernatant was discarded, and after washing the cells with 1% BSA for three times, the cells were resuspended in 10% FBS-DMEM/F-12 medium and divided into two portions, one portion was placed in an incubator at 37 ℃ and incubated for 1 hour (endocytosis group), and the other portion was further incubated on ice for 1 hour (Binding affinity group). After the incubation was completed, after washing the cells once with 1% BSA and adding PE-anti-Fc antibody (Jackson, 109-. Note: black was incubated without anti-TIGIT antibody, and the MFI was read by washing 3 resuspensions after incubation on ice for one hour with PE-anti-Fc antibody diluted with 1% BSA. The results are shown in FIG. 9.

Endocytosis Ratio (Internalization Ratio)% (binding affinity group-endocytosis group) × 100/(binding affinity group-blank group)

Eleventh, cell killing experiment of Natural killer cells (NK)

To investigate the effect of TIGIT antibody on NK cell killing function, human Peripheral Blood Mononuclear Cells (PBMCs) were collected and purified, natural killer cells (NK) were extracted, co-cultured with human colorectal cancer cells WiDr for 4h, and the secretion level of Lactate Dehydrogenase (LDH) was measured. The specific experimental process is as follows:

the human large intestine cancer cell line WiDr was cultured in MEM medium supplemented with 10% (v/v) Fetal Bovine Serum (FBS), 5% CO at 37 ℃₂Culturing under the condition. Fresh blood PBMC were obtained by Ficoll-Hypaque density gradient centrifugation (Stem cell technologies), and human primary NK cells were extracted from freshly isolated PBMC (Miltenyi, CAT #130-₂Culturing under the condition.

Inoculating human primary NK cells to a 6-well cell culture plate with a cell density of about 2 × 10^6/mL, adding 100U/mL human IL-2 for overnight culture, washing with phenol red-free RPMI 1640 medium, resuspending, inoculating to a 96-well U-plate with a cell density of about 3 × 10^ 5/well, and simultaneously adding a gradient diluted antibody sample (diluted with PBS) or an equivalent amount of isotype IgG as a blank control, 37 ℃ and 5% CO₂After incubation in an incubator for 1h, target cells WiDr were CO-cultured with human primary NK cells at a ratio of 1:1, 37 ℃ with 5% CO₂After 4h of incubator culture, cell culture supernatant was collected. Using CytoTox

The method of Non-radioactive cytotoxin Assay (Promega, CAT # G1780) specification detects LDH secretion levels in cell culture supernatants the percentage of specific cytolysis is determined by the following formula:% lysis 100 × (ER-SR1-SR2)/(MR-SR1), where ER and SR (1)&2) And MR for experimental, spontaneous (1 as target cells, 2 as human primary NK cells) and maximal LDH release, respectively. The spontaneous release line is released by target cells or human primary NK cells cultured in a medium aloneLDH (b) as determined by lysis of all target cells with lysis solution, maximum release. As shown in FIG. 10A or FIG. 10B, the antibodies H1707-02, H1708-04, and H1710-01, which are TIGIT humanized candidate antibodies, all enhanced the killing of human primary NK cells on target cells to different degrees, and had drug concentration dose effect.

Twelve, PBMC-T lymphocyte activation experiment

To study the effect of TIGIT antibodies on human primary T lymphocyte function, human Peripheral Blood Mononuclear Cells (PBMCs) were collected and purified and cytokine IFN γ secretion levels were measured after 5 days of in vitro stimulation with Tuberculin (TB). The specific experimental process is as follows:

fresh blood PBMC were obtained by Ficoll-Hypaque density gradient centrifugation (Stem Cell Technologies) and cultured in RPMI 1640 medium supplemented with 10% (v/v) FBS at 37 ℃ and 5% CO₂Culturing under the condition.

Freshly isolated and purified PBMC were adjusted to a density of 2 × 10^6/mL in RPMI 1640 medium, 25. mu.l tuberculin was added to 20mL of cell suspension at 37 ℃ with 5% CO₂The incubator is used for 5 days. Day 5, CD155 (recombinant CD155/PVR Protein, R) was added to 96-well cell culture plates&D, 2530-CD-050/CF), 0.25 mu g per well, coating overnight at 4 ℃, day 6, collecting the cultured cells, centrifuging, washing once with PBS, suspending into fresh RPMI 1640 medium, adjusting the density to 1 × 10^6/ml, inoculating to a CD 155-coated 96-well cell culture plate, adding 90 mu l per well, simultaneously adding a gradient diluted antibody sample (diluted with PBS) or an equivalent amount of isotype IgG as a blank control, and placing 10 mu l per well, wherein the cell culture plate is placed at 37 ℃ and 5% CO₂Incubate for 3 days. The cell culture plate was removed, centrifuged (4000rpm, 10min) to collect the cell culture supernatant, and the IFN-. gamma.level was measured by ELISA (human IFN-. gamma.assay kit, Xinbo Sheng, EHC102g.96). The specific operation refers to the reagent specification. The results are shown in FIG. 11, and the antibodies such as TIGIT humanized candidate antibodies h1708-04, h1710-01 and the like can enhance the secretion of cytokine IFN-gamma by activated primary T lymphocytes to different degrees and have the effect of drug concentration dose.

Thirteen, humanized TIGIT antibody rat pharmacokinetic evaluation

SD male rats weighing 180-. The feed and water are freely taken during the feeding period, the laboratory environment adaptive feeding is not less than 3 days, the light/dark period is regulated for 12/12 hours, the temperature is 16-26 ℃, and the relative humidity is 40-70%. The day before the start of the experiment, SD rats were numbered and randomly grouped into 3 animals per group. On the day of experiment, five groups of rats are injected with test drugs H1707-02, H1708-04, H1710-01, H1711-04 and H1709-10 by intravenous injection or subcutaneous injection respectively, 22G2-H3Q are positive controls, and the administration dose is 3 mg/kg; the injection volume was 5 ml/kg.

The intravenous administration mode comprises blood collection at each time point of 5min,8h,1d,2d,4d,7d,10d,14d,21d and 28d before and after administration. The subcutaneous injection mode of administration comprises blood collection at each time point of 1h,4h,8h,1d,2d,4d,8d,11d,14d,21d and 28d before and after administration. Taking 0.2ml of whole blood from each animal, adding no anticoagulant, standing at 4 deg.C for 30min, centrifuging at 1000g for 15min, collecting supernatant, and storing at-80 deg.C in EP tube.

The concentration of antibody in serum was measured by ELISA (see example 2. I above) and pharmacokinetic parameters of the test drug were calculated using Winnolin software. The partial primary pharmacokinetic results are shown in FIG. 12.

Through detection, after SD rats are administrated with 3mg/kg of anti-TIGIT antibody h1707-02, h1708-04, h1710-01, h1709-10 and h1711-04 by intravenous injection, the exposure amount in the rats is similar; the subcutaneous administration has high bioavailability which is close to 100 percent; the elimination half-life of each antibody is longer and better than that of the 22G2-H3Q antibody.

An anti-TIGIT antibody stabilizing preparation (exemplary, the anti-TIGIT antibody in examples 3 to 11 described below is the aforementioned h1708-04 antibody) was prepared by the following experiment

Example 3 determination of Tm value of TIGIT antibody

Preparing 10mM disodium hydrogen phosphate-citric acid (PB-CA) buffers (see the following 1) to 6) at different pH values), and preparing an antibody preparation with the concentration of the anti-TIGIT antibody of 1 mg/ml:

1)10mM disodium hydrogen phosphate-citric acid, pH4.5

2)10mM disodium hydrogen phosphate-citric acid, pH5.0

3)10mM disodium hydrogen phosphate-citric acid, pH5.5

4)10mM disodium hydrogen phosphate-citric acid, pH6.0

5)10mM disodium hydrogen phosphate-citric acid, pH6.5

6)10mM disodium hydrogen phosphate-citric acid, pH7.0

The thermal stability of the anti-TIGIT antibodies in each formulation was determined by Differential Scanning Calorimetry (DSC). The test results are shown in Table 20, and the results show that the anti-TIGIT antibody is stable at pH5.0-7.0.

TABLE 20 Tm values of different pH formulations of anti-TIGIT antibodies

Group of	pH	Tm Onset℃	Tm℃
					1	4.5	51.16	75.49
2	5.0	54.68	76.86
				3	5.5	58.06	77.73
4	6.0	62.56	77.74
				5	6.5	63.29	77.62
6	7.0	63.69	77.20

Example 4 screening of pH value of buffer System for anti-TIGIT antibody preparation

Preparing different buffer solutions (see 1) to 12) below), preparing an antibody preparation with an anti-TIGIT antibody concentration of 50 mg/ml:

1)10mM acetic acid-sodium acetate, pH 5.0;

2)10mM acetic acid-sodium acetate, pH 5.5;

3)10mM acetic acid-sodium acetate, pH 5.7;

4)10mM succinic acid-sodium succinate, pH 5.0;

5)10mM succinic acid-sodium succinate, pH 5.5;

6)10mM succinic acid-sodium succinate, pH 6.0;

7)10mM histidine-histidine hydrochloride, pH 5.5;

8)10mM histidine-histidine hydrochloride, pH 6.0;

9)10mM histidine-histidine hydrochloride, pH 6.5;

10)10mM histidine-acetic acid, pH 5.0;

11)10mM histidine-acetic acid, pH 5.5;

12)10mM histidine-acetic acid, pH 6.0.

Samples of each anti-TIGIT antibody preparation were taken for high temperature (40 ℃) stability studies, and the test results are shown in Table 21. The results show that: the pH value is within the range of 5.0-6.0, the SEC monomer purity values of 4 buffer systems are not greatly different, the main peak change of CE-SDS (NR) is within 2%, the ICE neutral peak change is within the range of 13.1% -17.5%, and the SEC monomer purity values are more stable compared with a buffer solution with the pH value of 6.5.

TABLE 21 pH value screening results of TIGIT antibody preparation buffer system

Note: m represents a month, for example, M0 represents 0 month of storage, M1 represents 40 ℃ for 1 month

Example 5 screening of surfactant classes in anti-TIGIT antibody formulations

Selecting a 10mM histidine-histidine hydrochloride pH6.0 buffer system, 80mg/ml sucrose and different surfactant (see the following 1) to 2)), a formulation with an anti-TIGIT antibody concentration of 50mg/ml was prepared:

1)10mM histidine-histidine hydrochloride pH6.0, 0.4mg/ml polysorbate 80, 80mg/ml sucrose;

2)10mM histidine-histidine hydrochloride pH6.0, 0.4mg/ml polysorbate 20, 80mg/ml sucrose.

The appearance stability of the anti-TIGIT antibody preparations of different surfactants at 40 ℃, 25 ℃ and 2-8 ℃ was observed, and the experimental results are shown in table 22. The results show that the anti-TIGIT antibody preparation containing polysorbate 80 is stable at 40 ℃, 25 ℃ and 2-8 ℃.

TABLE 22 stability results of TIGIT antibody surfactant species screening

Note: m represents month, D represents day, for example, M0 represents 0 month, D20 represents 20 days at 40 deg.C, M6 at 25 deg.C represents 6 months at 25 deg.C, and M6 represents 2-8 deg.C for 6 months.

Example 6 screening of surfactant concentration in TIGIT antibody formulations

Selecting a buffer system of 10mM histidine-histidine hydrochloride pH5.5, 80mg/ml sucrose and polysorbate 80 (see 1) to 6) groups at different concentrations), an antibody preparation with an anti-TIGIT antibody concentration of 50mg/ml was prepared:

1)10mM histidine-histidine hydrochloride ph5.5, without polysorbate 80;

2)10mM histidine-histidine hydrochloride pH5.5, 0.1mg/ml polysorbate 80;

3)10mM histidine-histidine hydrochloride pH5.5, 0.2mg/ml polysorbate 80;

4)10mM histidine-histidine hydrochloride pH5.5, 0.4mg/ml polysorbate 80;

5)10mM histidine-histidine hydrochloride pH5.5, 0.6mg/ml polysorbate 80;

6)10mM histidine-histidine hydrochloride pH5.5, 0.8mg/ml polysorbate 80.

Placing and shaking for a shaking stability test (25 ℃, 300rpm) for 7 days, wherein the test results are shown in Table 23, and the results show that the appearance of the preparation without adding polysorbate 80 is obviously poor after shaking for 7 days, which indicates that the addition of the polysorbate 80 as a surfactant has a stabilizing effect on the preparation, and the preparations of all groups of the polysorbate 80 containing 0.1 mg/ml-0.8 mg/ml have no obvious difference, and the preparations of all groups are stable.

TABLE 23 TIGIT antibody polysorbate 80 concentration screening shaking test results

Note: ShakingD7 indicates shaking for 7 days, and 0h indicates shaking for 0 hour.

Example 7 screening of sugar concentration in anti-TIGIT antibody formulations

Selecting 10mM histidine-histidine hydrochloride ph5.5 buffer system, 0.4mg/ml polysorbate 80 and different concentrations of sucrose (see below 1-5), an antibody formulation was prepared with an anti-TIGIT antibody concentration of 50 mg/ml:

1)10mM histidine-histidine hydrochloride pH5.5, 65mg/ml sucrose, 0.4mg/ml polysorbate 80;

2)10mM histidine-histidine hydrochloride pH5.5, 70mg/ml sucrose, 0.4mg/ml polysorbate 80;

3)10mM histidine-histidine hydrochloride pH5.5, 75mg/ml sucrose, 0.4mg/ml polysorbate 80;

4)10mM histidine-histidine hydrochloride pH5.5, 80mg/ml sucrose, 0.4mg/ml polysorbate 80;

5)10mM histidine-histidine hydrochloride pH5.5, 85mg/ml sucrose, 0.4mg/ml polysorbate 80;

the osmotic pressure of each group of samples is detected, the experimental result is shown in table 24, and the experimental result shows that the preparation is isotonic when the sucrose concentration is 80 mg/ml.

TABLE 24 osmotic pressure results for different sucrose concentrations of anti-TIGIT antibody

Group of	Concentration of sucrose	Osmolarity value (mOsm)
			1	65mg/ml	244
2	70mg/ml	264
			3	75mg/ml	286
4	80mg/ml	304
			5	85mg/ml	319

Example 8 stability of different buffer systems for anti-TIGIT antibodies

Antibody formulations containing 50mg/ml anti-TIGIT antibody, 80mg/ml sucrose, 0.4mg/ml polysorbate 80 were prepared using the following buffer systems (see groups 1) to 3) below):

1)10mM histidine-histidine hydrochloride pH 5.5;

2)10mM histidine-acetic acid pH 5.5;

3)10mM succinic acid-sodium succinate pH 5.5.

The stability of the anti-TIGIT antibody preparation in different buffer systems of 25 ℃ and 2-8 ℃ is tested, the experimental results are shown in tables 25 and 26, and the results show that the anti-TIGIT antibody preparation with 10mM acetic acid-sodium acetate, 10mM histidine-histidine hydrochloride and 10mM succinic acid buffer systems has good stability and has no obvious difference with each other after being placed at 25 ℃ for 6 months and 2-8 ℃ for 6 months.

TABLE 25 stability results at 25 ℃ for different buffer systems

Note: m represents a month, for example M6 represents 6 months.

TABLE 26 stability results at 2-8 ℃ for different buffer systems

Note: m represents a month, for example M6 represents 6 months.

Example 9 Integrated screening of anti-TIGIT antibody formulation Components

In order to further comprehensively screen the antibody concentration (40-60 mg/ml), the pH (5.0-6.0) and a buffer system (His-HCl, His-AA and SA), JMP software is used for DOE design, an RSM model is used for obtaining a series of prescription (see the following 1) -13) groups), and anti-TIGIT antibody preparations containing 0.4mg/ml polysorbate 80 and 80mg/ml sucrose are prepared in different buffer systems:

1)10mM histidine-histidine hydrochloride, pH5.5, 43mg/ml anti-TIGIT antibody;

2)10mM histidine-histidine hydrochloride, pH5.55, 58mg/ml anti-TIGIT antibody;

3)10mM histidine-histidine hydrochloride, pH6.0, 47mg/ml anti-TIGIT antibody;

4)10mM histidine-acetic acid, pH5.0, 40mg/ml anti-TIGIT antibody;

5)10mM histidine-acetic acid, pH5.0, 60mg/ml anti-TIGIT antibody;

6)10mM histidine-acetic acid, pH5.5, 50mg/ml anti-TIGIT antibody;

7)10mM histidine-acetic acid, ph5.5, 50mg/ml anti-TIGIT antibody (note: one replicate was designed for group 6 of medians);

8)10mM histidine-acetic acid, pH6.0, 40mg/ml anti-TIGIT antibody;

9)10mM histidine-acetic acid, pH6.0, 60mg/ml anti-TIGIT antibody;

10)10mM succinic acid-sodium succinate, pH5.0, 50mg/ml anti-TIGIT antibody;

11)10mM succinic acid-sodium succinate, pH5.5, 40mg/ml anti-TIGIT antibody;

12)10mM succinic acid-sodium succinate, pH5.5, 60mg/ml anti-TIGIT antibody;

13)10mM succinic acid-sodium succinate, pH6.0, 50mg/ml anti-TIGIT antibody;

the antibody preparation samples of each group were stored at 40 ℃ or under illumination (5. + -. 3 ℃ C., 4500lx) for stability analysis, and the results are shown in Table 27 and FIGS. 13-15. The result shows that the optional pH range of histidine-acetic acid (His-AA) is the largest, and the pH range can be 5.0-5.8; the optional pH range of histidine-histidine hydrochloride (His-HCl) is 5.5-6.0; the pH range of choice for succinic acid-sodium Succinate (SA) is relatively narrow. In addition, the decrease in SA purity data was greater relative to His-AA and His-AA. Therefore, the buffer solution of the anti-TIGIT antibody preparation can be His-AA, the pH range is 5.0-5.8, and the concentration of the anti-TIGIT antibody is 40-60 mg/ml.

TABLE 27 DOE screening test results

Note: 40 ℃ D25 indicates storage at 40 ℃ for 25 days, 0h indicates storage for 0 hour, and LightD10 indicates storage under light (5. + -. 3 ℃ C., 4500lx) for 10 days.

Example 10 lyophilization of anti-TIGIT antibody formulations

An antibody preparation containing sucrose at a concentration of 80mg/ml and polysorbate 80 at a concentration of 50mg/ml for the anti-TIGIT antibody was prepared using a buffer containing histidine-acetic acid at 10mM, pH 5.5. Filling the antibody into a 6mL penicillin bottle at a concentration of 2.15 mL/bottle, filling the penicillin bottle into a freeze-drying box, and freeze-drying. The freeze-drying procedure is pre-freezing, primary drying and secondary drying. After the lyophilization procedure was completed, the stopper was vacuum stoppered. Reconstituted samples were compared before and after lyophilization. The results show that the redissolution can maintain good performance of the liquid formulation.

TABLE 28 lyophilization step of formulations

Note: N/A (not applicable) indicates that the table is not applicable to it

Example 11 other alternative formulation formulations

In addition, the present disclosure also provides anti-TIGIT antibody pharmaceutical formulations of other formulation formulations, including but not limited to:

(1)100mg/ml anti-TIGIT antibody, 95mg/ml sucrose, 1.5mg/ml polysorbate 80, and 30mM histidine-acetic acid buffer pH 5.6;

(2)1mg/ml anti-TIGIT antibody, 100mg/ml sucrose, 0.05mg/ml polysorbate 80, and 5mM histidine-acetic acid buffer ph 5.9;

(3)20mg/ml anti-TIGIT antibody, 90mg/ml sucrose, 1.0mg/ml polysorbate 80, and 15mM histidine-acetic acid buffer ph 5.7;

(4)80mg/ml anti-TIGIT antibody, 81mg/ml sucrose, 0.6mg/ml polysorbate 80, and 20mM histidine-acetic acid buffer pH 5.6;

(5)50mg/ml anti-TIGIT antibody, 83mg/ml sucrose, 1.2mg/ml polysorbate 80, and 25mM histidine-acetic acid buffer pH 5.5;

(6)55mg/ml anti-TIGIT antibody, 78mg/ml sucrose, 0.4mg/ml polysorbate 80, and 9mM histidine-acetic acid buffer ph 5.4;

(7)30mg/ml anti-TIGIT antibody, 75mg/ml sucrose, 1.4mg/ml polysorbate 80, and 8mM histidine-acetic acid buffer ph 5.3;

(8)70mg/ml anti-TIGIT antibody, 82mg/ml sucrose, 0.8mg/ml polysorbate 80, and 11mM histidine-acetic acid buffer ph 5.2;

(9)50mg/ml anti-TIGIT antibody, 77mg/ml sucrose, 1.3mg/ml polysorbate 80, and 12mM histidine-acetic acid buffer pH 5.1;

(10)50mg/ml anti-TIGIT antibody, 80mg/ml sucrose, 0.5mg/ml polysorbate 80, and 13mM histidine-acetic acid buffer pH 5.2;

(11)52mg/ml anti-TIGIT antibody, 84mg/ml sucrose, 1.1mg/ml polysorbate 80, and 14mM histidine-acetic acid buffer pH 5.5;

(12)48mg/ml anti-TIGIT antibody, 79mg/ml sucrose, 0.7mg/ml polysorbate 80, and 12mM histidine-histidine hydrochloride buffer pH 6.1;

(13)49mg/ml anti-TIGIT antibody, 74mg/ml sucrose, 0.3mg/ml polysorbate 80, and 15mM histidine-histidine hydrochloride buffer pH 6.2;

(14)45mg/ml anti-TIGIT antibody, 80mg/ml sucrose, 0.9mg/ml polysorbate 80, and 10mM histidine-histidine hydrochloride buffer ph 6.3;

(15)65mg/ml anti-TIGIT antibody, 80mg/ml sucrose, 0.4mg/ml polysorbate 80, and 10mM histidine-histidine hydrochloride buffer pH 6.4;

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> anti-TIGIT antibody pharmaceutical composition and application thereof

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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 sequence

<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 LeuGln 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 ValTyr 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 105110

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

100105 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 GlyAla

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

1 510 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

SerVal 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

1 510 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

180185 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

245250 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

405410 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 LeuTyr 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

195200 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. A pharmaceutical composition comprising an anti-TIGIT antibody or antigen-binding fragment thereof that specifically binds to human TIGIT, and a buffer selected from the group consisting of a histidine buffer, a succinate buffer, an acetate buffer, and a phosphate buffer, the buffer having a pH of about 5.0 to 7.0, preferably a pH of about 5.0 to 6.0.

2. The pharmaceutical composition of claim 1, wherein the buffer is selected from the group consisting of an acetate-sodium acetate buffer, a succinate-sodium succinate buffer, a histidine-hydrochloric acid buffer, and a histidine-acetate buffer; preferably an acetic acid-sodium acetate buffer at a pH of about 5.0 to 5.7, a succinic acid-sodium succinate buffer at a pH of about 5.0 to 6.0, a histidine-hydrochloric acid buffer at a pH of about 5.5 to 6.0, or a histidine-acetic acid buffer at a pH of about 5.0 to 6.0; most preferred is a histidine-acetic acid buffer at a pH of about 5.5.

3. The pharmaceutical composition according to claim 1 or 2, wherein the buffer concentration is about 5mM to 30mM, preferably about 5mM to 15mM, more preferably about 10 mM.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the anti-TIGIT antibody or antigen-binding fragment thereof is at a concentration of about 1 to 100mg/ml, preferably about 40 to 60mg/ml, more preferably about 50 mg/ml.

5. The pharmaceutical composition according to any one of claims 1 to 4, further comprising a sugar, preferably a disaccharide, most preferably sucrose; the sugar concentration is preferably from about 65mg/ml to 100mg/ml, more preferably from about 75mg/ml to 85mg/ml, most preferably about 80 mg/ml.

6. The pharmaceutical composition according to any one of claims 1 to 5, further comprising a surfactant, preferably a polysorbate, more preferably polysorbate 80; the concentration of the surfactant is preferably about 0.05mg/ml to 1.5mg/ml, preferably about 0.1mg/ml to 0.8mg/ml, more preferably about 0.4 mg/ml.

7. The pharmaceutical composition according to any one of claims 1 to 6, comprising:

a) an anti-TIGIT antibody or antigen-binding fragment thereof at a concentration of about 1mg/ml to 100mg/ml,

b) a histidine salt buffer, a succinate buffer, an acetate buffer or a phosphate buffer at a pH of about 5.0 to 7.0,

c) sucrose at a concentration of about 65mg/ml to 100mg/ml, and

d) polysorbate 80 at a concentration of about 0.05mg/ml to 1.5 mg/ml;

preferably, the pharmaceutical composition comprises:

a1) an anti-TIGIT antibody or antigen-binding fragment thereof at a concentration of about 40mg/ml to 60mg/ml,

b1) acetic acid-sodium acetate at a pH of about 5.0 to 5.7, succinic acid-sodium succinate at a pH of about 5.0 to 6.0, histidine-histidine hydrochloride at a pH of about 5.5 to 6.0, or histidine-acetic acid at a pH of about 5.0 to 6.0,

c1) sucrose at a concentration of about 75mg/ml to 85mg/ml, and

d1) polysorbate 80 at a concentration of about 0.1mg/ml to 0.8 mg/ml.

More preferably, the pharmaceutical composition comprises: about 10mM histidine-acetic acid buffer at pH about 5.5, an anti-TIGIT antibody or antigen-binding fragment thereof that specifically binds to human TIGIT at a concentration of about 50mg/ml, sucrose at a concentration of about 80mg/ml, and polysorbate 80 at a concentration of about 0.4 mg/ml.

8. The pharmaceutical composition of any one of claims 1-7, wherein the 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 comprising HCDR1, HCDR2 and HCDR3 having the same sequence as the heavy chain variable region set forth in sequence SEQ ID NO: LCDR1, LCDR2 and LCDR3 having the same sequence as the light chain variable region shown in FIG. 6;

ii) the heavy chain variable region comprises a sequence identical to 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 set forth in sequence SEQ ID NO: LCDR1, LCDR2 and LCDR3 of 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 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 of the same sequence as the light chain variable region;

iv) the heavy chain variable region comprises a sequence identical to 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 sequence SEQ ID NO: 12, LCDR1, LCDR2 and LCDR3 having the same sequence as the light chain variable region; or

v) the heavy chain variable region comprises a sequence identical to 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 sequence SEQ ID NO: LCDR1, LCDR2 and LCDR3 of the same sequence as the light chain variable region set forth in fig. 14;

preferably, the 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:

vi) the heavy chain variable region comprises the amino acid sequences as set forth in SEQ ID NOs: 15. 16 and 17, and a light chain variable region comprising the amino acid sequences 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 sequences as set forth in SEQ ID NOs: 21. 22 and 23, and a light chain variable region comprising the amino acid sequences 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;

viii) the heavy chain variable region comprises the amino acid sequences set forth in SEQ ID NOs: 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 sequences shown as SEQ ID NO: 33. 34 and 35, and the light chain variable region comprises HCDR1, HCDR2 and HCDR3 as 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 shown as SEQ ID NO: 39. 40 and 41, and the light chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 42. LCDR1, LCDR2 and LCDR3 shown in amino acid sequences 43 and 44.

9. The pharmaceutical composition of claim 8, wherein the antibody or antigen-binding fragment thereof that specifically binds to human TIGIT comprises a heavy chain variable region as set forth in SEQ ID NOs 45, 51, 56, 64, or 71, or a heavy chain variable region variant comprising 1-10 amino acid changes in the heavy chain variable region sequence set forth in SEQ ID NOs 45, 51, 56, 64, or 71; and/or

A light chain variable region variant comprising the light chain variable region of SEQ ID NO 46, 52, 57, 65 or 72 or comprising 1-10 amino acid changes in the light chain variable region of SEQ ID NO 46, 52, 57, 65 or 72;

preferably, wherein the antibody or antigen-binding fragment thereof that specifically binds to human TIGIT comprises:

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

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

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

more preferably, the monoclonal antibody or antigen binding fragment thereof that specifically binds to human TIGIT comprises an amino acid sequence as set forth in SEQ id no:51 and the heavy chain variable region set forth in SEQ ID NO: 53, or a light chain variable region as shown in fig.

10. The pharmaceutical composition of claim 8 or 9, wherein the antibody that specifically binds to human TIGIT is a full-length antibody comprising human antibody constant regions; preferably comprising a human antibody heavy chain constant region as shown in SEQ ID NO. 78, and/or a human antibody light chain constant region as shown in SEQ ID NO. 79;

more preferably, wherein the antibody that specifically binds to human TIGIT comprises:

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

11. A method of making the pharmaceutical composition of any one of claims 1 to 10, the method comprising the step of mixing an anti-TIGIT antibody or antigen-binding fragment thereof with a buffer; the buffer is selected from the group consisting of histidine buffer, succinate buffer, acetate buffer and phosphate buffer, said buffer having a pH of about 5.0 to 7.0, preferably a pH of about 5.0 to 6.0.

12. A lyophilized formulation comprising an anti-TIGIT antibody or antigen-binding fragment thereof obtained by lyophilizing the pharmaceutical composition of any one of claims 1-10.

13. A lyophilized formulation comprising an anti-TIGIT antibody or antigen-binding fragment thereof, which upon reconstitution can form the pharmaceutical composition of any one of claims 1-10.

14. A reconstituted solution containing an anti-TIGIT antibody or antigen-binding fragment thereof prepared by reconstituting the lyophilized formulation of claim 12 or 13.

15. An article of manufacture comprising a container containing a pharmaceutical composition according to any one of claims 1 to 10, or a lyophilized formulation according to claim 12 or 13, or a reconstituted solution according to claim 14.

16. Use of a pharmaceutical composition according to any one of claims 1 to 10, or a lyophilized formulation according to claim 12 or 13, or a reconstituted solution according to claim 14, or an article of manufacture according to claim 15, in the manufacture of a medicament for the treatment of TIGIT-related disease in humans; preferably, the disease is a tumor; preferably, the tumor is selected from: head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumor, 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; preferably, 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; more preferably, the tumor is selected from a CD 155-positive or PVR-positive tumor.