CN111744007B

CN111744007B - anti-TIGIT antibody pharmaceutical composition and application thereof

Info

Publication number: CN111744007B
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: 2023-05-12
Anticipated expiration: 2040-03-27
Also published as: CN111744007A

Abstract

The present disclosure relates to an anti-TIGIT antibody pharmaceutical composition and uses thereof. In particular, the present 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 anti-TIGIT antibodies in combination with PD-1 inhibitors for treating diseases.

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 immune responses, and activation of T cells depends on TCR signaling and costimulatory signaling. The costimulatory signal is then a limiting signal for T cell activation, whose dysfunction is involved in the development of autoimmune disease (Immunol Rev,2012,248:122-139;Autoimmun Rev,2013,12:1171-1176). TIGIT (T cell immunoglobulin and ITIM domain) is a newly discovered co-inhibitory signal molecule 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. Human TIGIT molecules are 141 amino acids long in the extracellular domain, with 1 immunoglobulin V-like domain; 23 amino acids in the transmembrane region; the cytoplasmic region is short, 80 amino acids, with 1 PDZ binding domain and 1 ITIM motif. TIGIT molecules belong to a member of the immunoglobulin superfamily IgSF, whose structure is conserved, and homologous molecules are found in a variety of mammals, human TIGIT molecules having 88%, 67% and 58% homology with TIGIT molecules of monkeys, dogs and mice, respectively (Nat Immunol,2009,10 (1): 48-57).

TIGIT molecules are predominantly expressed on T cells and NK cell surfaces (Nat Immunol,2009, 10:48-57).

Both T cells and resting memory T cells expressed TIGIT down and up-regulated after in vitro activation (J Immunol,2012, 188:3869-3875). NK cell surface TIGIT has higher level expression (Proc Natl Acad Sci USA,2009, 106 (42): 17858-17863). TIGIT is a new target for immunotherapy with great potential. Monoclonal antibodies that specifically block TIGIT have been shown to exhibit significant anti-tumor effects in animal models (Martinet and Smyth 2015). Antibodies to TIGIT and related applications have been reported in the patents WO2009126688, WO2014089113, WO2015009856, WO2015143343, WO2015174439, WO2017053748, WO2017030823, WO2016106302, US20160176963, US20130251720, etc. WO2018204405A1 also discloses an anti-TIGIT antibody formulation comprising an antioxidant. However, TIGIT antibodies have not been used clinically so far.

Disclosure of Invention

The present 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 histidine salt buffer, succinate buffer, acetate buffer, and phosphate buffer, the pH of the buffer being 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 buffer has a pH of 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 the group consisting of: 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 histidine-acetic acid buffer having a pH of about 5.0 to 5.8; in some embodiments, the buffer is histidine-acetate buffer at a pH of about 5.5. In other embodiments, the pharmaceutical composition of the present disclosure, wherein the buffer concentration is about 5mM to 30mM, about 5mM to 15mM, or about 10mM.

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

In some embodiments, the pharmaceutical composition of any of the preceding, further comprising a sugar. Without limitation, the sugar may be of conventional composition (CH ₂ O) 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, stachyose, maltose, lactulose, maltulose, sorbitol, maltitol, lactitol, isomalt, 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 100mg/ml; in some embodiments, the sugar concentration is about 75mg/ml to 85mg/ml; in some embodiments, the sugar concentration is about 65mg/ml, 70mg/ml, 75mg/ml, 80mg/ml, or 85mg/ml. In some embodiments, the sugar concentration is about 80mg/ml.

In some embodiments, the pharmaceutical composition of any of the preceding, further comprising a surfactant. The surfactant may be selected from, without limitation, polysorbate 20, polysorbate 80, polyhydroxyen, triton, sodium dodecyl sulfonate, 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, oleamidopropyl-betaine, myristamidopropyl-betaine, palmitoamidopropyl-betaine, isostearamidopropyl-betaine, myristamidopropyl-dimethylamine, palmitoamidopropyl-dimethylamine, isostearamidopropyl-dimethylamine, sodium methyl cocoa acyl, sodium methyl oleyl 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 1.5mg/ml; in other embodiments, the concentration of the surfactant is about 0.1mg/ml to about 0.8mg/ml, and in other embodiments, the concentration of the surfactant is about 0.4mg/ml.

In some embodiments, the pharmaceutical composition of any of the foregoing comprises: a) an anti-TIGIT antibody or antigen-binding fragment thereof at a concentration of about 1mg/ml to about 100mg/ml, b) a histidine salt buffer, succinate buffer, acetate buffer or phosphate buffer at a pH of about 5.0 to about 7.0, c) sucrose at a concentration of about 65mg/ml to about 100mg/ml, and d) polysorbate 80 at a concentration of about 0.05mg/ml to about 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 about 60mg/ml, b 1) sodium acetate-acetate at a pH of about 5.0 to about 5.7, sodium succinate-succinate at a pH of about 5.0 to about 6.0, histidine-histidine hydrochloride at a pH of about 5.5 to about 6.0, or histidine-acetic acid at a pH of about 5.0 to about 6.0, c 1) sucrose at a concentration of about 75mg/ml to about 85mg/ml, and d 1) polysorbate 80 at a concentration of about 0.1mg/ml to about 0.8 mg/ml.

In other embodiments, the pharmaceutical composition comprises: 10mM histidine-acetic buffer at pH of about 5.5, 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 of the preceding, wherein the antibody or antigen-binding fragment thereof that specifically binds 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 of the preceding, wherein the antibody or antigen-binding fragment thereof that specifically binds 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 the sequence set forth in SEQ ID NO:5, and HCDR1, HCDR2 and HCDR3 of the same sequence of a heavy chain variable region comprising a sequence identical as set forth in SEQ ID NO:6, LCDR1, LCDR2 and LCDR3 of the same sequence as the light chain variable region shown in figure 6;

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

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

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

v) the heavy chain variable region comprises a sequence identical to the sequence set forth in SEQ ID NO:13, and HCDR1, HCDR2 and HCDR3 of the same sequence of a heavy chain variable region comprising a sequence identical as set forth in SEQ ID NO:14, LCDR1, LCDR2 and LCDR3 of the same sequence as the light chain variable region shown in figure 14.

In some embodiments, the pharmaceutical composition of any one of the preceding, 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 sequence set forth in SEQ ID NO: 15. 16 and 17, and HCDR1, HCDR2 and HCDR3, the light chain variable region comprising amino acid sequences as set forth in SEQ ID NOs: 18. 19 and 20 amino acid sequences LCDR1, LCDR2 and LCDR3;

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

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

ix) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 33. 34 and 35, and HCDR1, HCDR2 and HCDR3, the light chain variable region comprising amino acid sequences as set forth in SEQ ID NOs: 36. 37 and 38 amino acid sequences LCDR1, LCDR2 and LCDR3; or (b)

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

In some embodiments, the pharmaceutical composition of any 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 having 1-10 amino acid changes in the heavy chain variable region sequence as set forth in SEQ ID No. 45, 51, 56, 64, or 71; and/or a light chain variable region as shown in SEQ ID NO. 46, 52, 57, 65 or 72, or a light chain variable region variant having 1-10 amino acid changes in the light chain variable region as shown 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 FR region position of the heavy chain variable region shown in SEQ ID NO. 45, 51, 56, 64 or 71; preferably, the back mutation is selected from:

An amino acid back mutation having one or more selected from the group consisting of N84S and S85R on the heavy chain variable region shown in SEQ ID NO. 45; or (b)

An amino acid back mutation in the heavy chain variable region shown in SEQ ID NO. 51 selected from one or more of M48I, R V and V79A; or (b)

An amino acid back mutation having one or more amino acids selected from the group consisting of Y27F, M I, R V, V79A and S84N and on the heavy chain variable region shown in SEQ ID NO. 56; or (b)

An amino acid back mutation having one or more of the amino acid back mutations selected from the group consisting of R38K, R67K, R V, T74K, M48I, V68A, M L and V79A and on the heavy chain variable region shown in SEQ ID NO. 64; or (b)

The heavy chain variable region shown in SEQ ID NO. 71 has an amino acid back mutation selected from one or more of G27Y, M48I, L83F and A97T.

In other embodiments, the light chain variable region variant is a back mutation of 1 to 10 amino acids at the FR region position of the light chain variable region shown in SEQ ID NO. 46, 52, 57, 65 or 72; preferably, the back mutation is selected from:

an amino acid back mutation on the light chain variable region shown in SEQ ID NO. 46 having one or more of S60D, T85D, A S and S63T; or (b)

An amino acid back mutation in the light chain variable region shown in SEQ ID NO. 52 selected from the group consisting of A43S; or (b)

An amino acid back mutation on the light chain variable region shown in SEQ ID NO. 57 having one or more selected from the group consisting of Q3V, A43S, S D and Y87F; or (b)

An amino acid back mutation on the light chain variable region shown in SEQ ID NO. 65 having one or more of the amino acids selected from the group consisting of A43S and I48V; or (b)

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

In some embodiments, the pharmaceutical composition of any of the preceding, wherein the antibody that specifically binds 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) A heavy chain variable region as set forth in SEQ ID NO. 64, 67, 68, 69 or 70, and/or a light chain variable region as set forth in SEQ ID NO. 65 or 66; or (b)

e) A heavy chain variable region as set forth in SEQ ID NO. 71, 75, 76 or 77, and/or a 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 the amino acid sequence as set forth in SEQ ID NO:51 and a heavy chain variable region as set forth in SEQ ID NO: 53.

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

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 shown in SEQ ID NO. 85, and/or a light chain as shown 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 shown in SEQ ID NO. 89, and/or a light chain as shown in SEQ ID NO. 88; or (b)

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

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

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

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

In another aspect, in some embodiments, the present disclosure provides a lyophilized formulation comprising an anti-TIGIT antibody or antigen-binding fragment thereof that is reconstituted to form the pharmaceutical composition described previously.

In another aspect, in some embodiments, the present disclosure provides a reconstituted solution comprising an anti-TIGIT antibody or antigen-binding fragment thereof obtained by reconstitution of a lyophilized formulation comprising an anti-TIGIT antibody or antigen-binding fragment thereof as described previously.

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

In another aspect, in some embodiments, the present disclosure provides a pharmaceutical composition, lyophilized formulation, reconstituted solution or article of manufacture as any one of the preceding claims as a medicament for treating a disease associated with human TIGIT. In some embodiments, wherein the disease associated with human TIGIT is a tumor; in some embodiments, the tumor is selected from: head and neck squamous cell carcinoma, head and neck carcinoma, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system carcinoma, neuroendocrine tumor, laryngeal carcinoma, nasopharyngeal carcinoma, esophageal carcinoma, thyroid carcinoma, malignant pleural mesothelioma, lung carcinoma, breast carcinoma, liver carcinoma, hepatoma, hepatobiliary carcinoma, pancreatic carcinoma, stomach carcinoma, gastrointestinal carcinoma, intestinal carcinoma, colon carcinoma, colorectal carcinoma, renal carcinoma, clear cell renal cell carcinoma, ovarian carcinoma, endometrial carcinoma, cervical carcinoma, bladder carcinoma, prostate carcinoma, testicular carcinoma, skin carcinoma, melanoma, leukemia, lymphoma, bone carcinoma, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, myeloproliferative neoplasm, squamous cell carcinoma, ewing's sarcoma, systemic light chain amyloidosis, and merkel cell carcinoma; in some embodiments, the lymphoma is selected from the group consisting of: hodgkin's lymphoma, non-hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, T-cell/tissue cell enriched large B-cell lymphoma, and lymphoplasmacytic lymphoma, the lung cancer being selected from the group consisting of: non-small cell lung cancer and small cell lung cancer, said leukemia being selected from the group consisting of: chronic myeloid leukemia, acute myeloid leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, and myeloid leukemia; in some embodiments, the tumor is selected from CD155 positive or PVR positive tumors.

In another aspect, in some embodiments, the present disclosure provides the use of a pharmaceutical composition, lyophilized formulation, reconstituted solution or article of manufacture as described in any one of the preceding claims in the manufacture of a medicament for treating a disease associated with human TIGIT. In some embodiments, wherein the disease associated with human TIGIT is a tumor; in some embodiments, the tumor is selected from: head and neck squamous cell carcinoma, head and neck carcinoma, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system carcinoma, neuroendocrine tumor, laryngeal carcinoma, nasopharyngeal carcinoma, esophageal carcinoma, thyroid carcinoma, malignant pleural mesothelioma, lung carcinoma, breast carcinoma, liver carcinoma, hepatoma, hepatobiliary carcinoma, pancreatic carcinoma, stomach carcinoma, gastrointestinal carcinoma, intestinal carcinoma, colon carcinoma, colorectal carcinoma, renal carcinoma, clear cell renal cell carcinoma, ovarian carcinoma, endometrial carcinoma, cervical carcinoma, bladder carcinoma, prostate carcinoma, testicular carcinoma, skin carcinoma, melanoma, leukemia, lymphoma, bone carcinoma, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, myeloproliferative neoplasm, squamous cell carcinoma, ewing's sarcoma, systemic light chain amyloidosis, and merkel cell carcinoma; in some embodiments, the lymphoma is selected from the group consisting of: hodgkin's lymphoma, non-hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, T-cell/tissue cell enriched large B-cell lymphoma, and lymphoplasmacytic lymphoma, the lung cancer being selected from the group consisting of: non-small cell lung cancer and small cell lung cancer, said leukemia being selected from the group consisting of: chronic myeloid leukemia, acute myeloid leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, and myeloid leukemia; in some embodiments, the tumor is selected from CD155 positive or PVR positive tumors.

In another aspect, in some embodiments, the present disclosure provides a method of treating or preventing TIGIT-related disorders, the method comprising administering to a subject a therapeutically effective amount of a pharmaceutical composition, lyophilized formulation, reconstituted solution, or article of manufacture of any one of the preceding claims. In some embodiments, wherein the disease associated with human TIGIT is a tumor; in some embodiments, the tumor is selected from: head and neck squamous cell carcinoma, head and neck carcinoma, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system carcinoma, neuroendocrine tumor, laryngeal carcinoma, nasopharyngeal carcinoma, esophageal carcinoma, thyroid carcinoma, malignant pleural mesothelioma, lung carcinoma, breast carcinoma, liver carcinoma, hepatoma, hepatobiliary carcinoma, pancreatic carcinoma, stomach carcinoma, gastrointestinal carcinoma, intestinal carcinoma, colon carcinoma, colorectal carcinoma, renal carcinoma, clear cell renal cell carcinoma, ovarian carcinoma, endometrial carcinoma, cervical carcinoma, bladder carcinoma, prostate carcinoma, testicular carcinoma, skin carcinoma, melanoma, leukemia, lymphoma, bone carcinoma, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, myeloproliferative neoplasm, squamous cell carcinoma, ewing's sarcoma, systemic light chain amyloidosis, and merkel cell carcinoma; in some embodiments, the lymphoma is selected from the group consisting of: hodgkin's lymphoma, non-hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, T-cell/tissue cell enriched large B-cell lymphoma, and lymphoplasmacytic lymphoma, the lung cancer being selected from the group consisting of: non-small cell lung cancer and small cell lung cancer, said leukemia being selected from the group consisting of: chronic myeloid leukemia, acute myeloid leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, and myeloid leukemia; in some embodiments, the tumor is selected from CD155 positive or PVR positive tumors.

Drawings

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

Fig. 2: ELISA detection of anti-TIGIT antibodies binding to monkey TIGIT protein.

Fig. 3: binding assays of anti-TIGIT antibodies to CHO cells overexpressing human TIGIT.

Fig. 4: binding affinity assay of anti-TIGIT antibodies to human PBMC.

Fig. 5: FIG. 5A is a block assay of binding of human TIGIT to CD155 by ch1708 and its humanized antibodies; FIG. 5B is a block effect assay of anti-TIGIT humanized antibodies on binding of human TIGIT to CD 155.

Fig. 6: blocking experiments of TIGIT antigen binding to CHO cells overexpressing CD155 by anti-TIGIT antibodies.

Fig. 7: blocking experiments of CD155 binding to CHO cells overexpressing TIGIT 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 assay of anti-TIGIT antibodies in TIGIT overexpressing CHO cells, wherein endocytosis is for 1 hour.

Fig. 10: FIG. 10A is a cell killing experiment of an anti-TIGIT antibody to promote Natural Killer (NK) cells; fig. 10B: anti-TIGIT antibodies facilitate Natural Killer (NK) cell killing experiments.

Fig. 11: anti-TIGIT antibodies were tested for PBMC-T lymphocyte activation.

Fig. 12: pharmacokinetic detection of anti-TIGIT humanized antibodies in rats.

Fig. 13: anti TIGIT antibody prescription DOE screening of histidine-acetic acid (His-AA) buffer contour plots, each contour plot parameter in fig. 13 is given in the following table:

fig. 14: anti TIGIT antibody prescription DOE screening of histidine-HCl (His-HCl) buffer contour plots, with individual contour plot parameters in fig. 14 as follows:

fig. 15: anti TIGIT antibody prescription DOE screening of succinic acid-sodium Succinate (SA) buffer contour plots, each contour plot parameter in fig. 15 is as follows:

Detailed Description

Terminology

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

"buffer" or "buffering agent" refers to a buffer that is resistant to changes in pH by the action of its acid-base conjugate 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.

A "histidine salt buffer" or "histidine salt buffer" is a buffer comprising histidine ions. Examples of histidine buffers 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-hcl, histidine-acetic acid. The histidine-acetic acid buffer is prepared from histidine and acetic acid or histidine and histidine-acetate.

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

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

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

"pharmaceutical composition" means a mixture comprising one or more of the 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, and facilitates the absorption of the active ingredients so as to exert biological activity. As used herein, the terms "pharmaceutical composition" and "formulation" are not intended to be mutually exclusive. In some embodiments, a pharmaceutical combination of the present disclosure comprises a monoclonal antibody or antigen-binding fragment thereof that specifically binds human TIGIT. In some embodiments, the pharmaceutical compositions or formulations of the present disclosure are 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: an amount of purified antibody solution is taken, solvent displaced (preferably ultrafiltration) with a buffer free of antibody (e.g., 10mM histidine-acetic acid buffer, pH 5.5), displaced by at least 6 volumes through the ultrafiltration membrane, and the antibody is concentrated to about 70mg/mL. Adding a certain volume of sucrose mother liquor, and uniformly mixing to obtain the final sucrose concentration of 80mg/mL. A volume of polysorbate 80 stock solution was added and mixed to give a final polysorbate 80 concentration of 0.4mg/mL. The volume was fixed by adding 10mM histidine-acetic buffer, pH5.5, to a concentration of 50mg/mL antibody (other formulations to be tested or stable formulations may be formulated with reference to similar procedures). The product is filtered, sampled and detected asepsis by central control. The stock solution was filtered through a 0.22 μm PVDF filter cartridge and the filtrate was collected. And a second step of: the filling amount is regulated to 2.15ml, the filtrate is filled in a 2ml penicillin bottle, a plug is added, and the filling amount difference is detected by sampling central control respectively at the beginning, the middle and the end of filling. And a third step of: and opening the capping machine, adding an aluminum cap, and capping. Fourth step: and (5) visual inspection, namely confirming that the product has no defects of inaccurate loading and the like. Printing and pasting a penicillin bottle label; printing paper box labels, folding paper boxes, boxing and sticking paper box labels.

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

The pharmaceutical compositions described in this disclosure are in the form of solutions, wherein the solvent is water unless otherwise specified.

By "lyophilized formulation" is meant a pharmaceutical composition in liquid or solution form 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 pharmaceutical compositions or liquid or solution formulations. Lyophilization is performed by freezing the formulation and subsequently sublimating the water at a temperature suitable for primary drying. Under these conditions, the product temperature is below the eutectic or decomposition temperature of the formulation. Typically, the storage temperature of the primary drying is in the range of about-30 to 25 c (assuming the product remains frozen during the primary drying) at a suitable pressure, typically in the range of about 50-250 mtorr. The size and type of formulation, the container (e.g., glass vial) containing the sample, and the volume of liquid determine the time required for drying, which may range from a few hours to a few 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 humidity level in the product and typically takes at least about 5 hours. Typically, the water content of the lyophilized formulation is less than about 5%, preferably less than about 3%. The pressure may be the same as the pressure applied in the primary drying step, preferably the pressure of the secondary drying is lower than the primary drying. The lyophilization conditions may vary with formulation and vial size. In some embodiments of the present disclosure, the lyophilized formulation is prepared by filling an anti-TIGIT antibody formulation into a 6mL vial at 2.15 mL/vial or into a 15mL vial at 12.3 mL/vial, filling into a lyophilization tank, and lyophilizing; the freeze-drying procedure is prefreezing, primary drying and secondary drying; after the lyophilization procedure was completed, vacuum stoppered.

The terms "about," "approximately" or "substantially comprise," as used herein, mean that the value is within an acceptable error range for the particular value being determined by one of ordinary skill in the art, which value depends in part on how the measurement or determination is made (i.e., the limits of the measurement system). For example, "about" in each implementation in the art may mean within 1 or exceeding a standard deviation of 1. Alternatively, "about," "about," or "substantially comprising" may mean a range of up to 20%. Furthermore, the term may mean at most one order of magnitude or at most 5 times the value, especially for biological systems or processes. Unless otherwise indicated, when a particular value is found in this application and in the claims, the meaning of "about", "about" or "substantially comprising" should be assumed to be within the acceptable error of that particular value.

The pharmaceutical composition disclosed by the disclosure can achieve a stable effect: a pharmaceutical composition wherein the antibody substantially retains its physical and/or chemical stability and/or biological activity after storage, preferably the pharmaceutical composition substantially retains its physical and chemical stability and its biological activity after storage. The shelf life is generally selected based on the predetermined shelf life of the pharmaceutical composition. There are a number of analytical techniques for measuring protein stability that 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 in the following cases: the storage is at refrigeration temperature (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 those that: which exhibits desired characteristics after storage at temperatures including 25 ℃ and 40 ℃ for periods including 1 month, 3 months, 6 months. Stable formulations such as: the pharmaceutical antibody formulation was colorless, or clear to slightly milky, by visual analysis. The concentration, pH and osmolality of the formulation have a variation of no more than + -10%. Typically no more than about 10%, preferably no more than about 5% truncation is observed. Usually no more than about 10%, preferably no more than about 5% of aggregates are formed. In some embodiments, the pharmaceutical compositions or lyophilized formulations in the present disclosure are 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; stable for at least 3 months, at least 6 months at 25 ℃.

An antibody "retains its physical stability" in a pharmaceutical formulation if it does not exhibit a 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). The change in protein conformation can be assessed by fluorescence spectroscopy (which determines the tertiary structure of the protein) and by FTIR spectroscopy (which determines the secondary structure of the protein).

An antibody "retains its chemical stability" in a pharmaceutical formulation if it does not exhibit a significant chemical change. Chemical stability can be assessed by detecting and quantifying chemically altered forms 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 in combination with mass spectrometry or MALDI/TOF/MS), deamidation (assessed by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide spectroscopy, isoaspartic acid measurement, etc.), 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 biological activities exhibited when the pharmaceutical formulation is prepared. The biological activity of an antibody may be determined, for example, by an antigen binding assay.

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

The "antibody" in the present disclosure refers to an immunoglobulin, and the complete antibody is a tetrapeptide chain structure formed by connecting two identical heavy chains and two identical light chains through inter-chain disulfide bonds. The immunoglobulin heavy chain constant region differs in amino acid composition and sequence, and thus, in antigenicity. Accordingly, immunoglobulins can be assigned to five classes, or isotypes of immunoglobulins, igM, igD, igG, igA and IgE, with their respective heavy chains being the μ, δ, γ, α, and epsilon chains, respectively. The same class of Ig can be further classified into different subclasses according to the amino acid composition of the hinge region and the number and position of disulfide bonds of the heavy chain, e.g., igG can be classified into IgG1, igG2, igG3, and IgG4. Light chains are classified by the difference in constant regions as either kappa chains or lambda chains. 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 kappa, lambda chain of human or murine origin, or variants thereof.

In the present disclosure, the antibody heavy chain of the present disclosure may further comprise a heavy chain constant region comprising IgG1, igG2, igG3, igG4, or variants thereof, of human or murine origin.

The sequences of the heavy and light chains of antibodies, near the N-terminus, vary widely, being the variable region (Fv region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region. The variable region includes 3 hypervariable regions (HVRs) and 4 Framework Regions (FR) that are relatively conserved in sequence. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each 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 in order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain are referred to as HCDR1, HCDR2 and HCDR3. In some exemplary embodiments, the CDR amino acid residues of the LCVR region and HCVR region of the antibody or antigen-binding fragment conform in number and position to the known Kabat numbering convention (LCDR 1-3, 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 to the same epitope except for possible variant antibodies (e.g., comprising naturally occurring mutations or mutations generated during manufacture of monoclonal antibody preparations, which are typically present in minor amounts). Unlike polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation (formulation) is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being 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 present disclosure can be prepared by a variety of techniques including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods, and other exemplary methods for preparing monoclonal antibodies are described herein.

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

The term "murine antibody" is in this disclosure a monoclonal antibody to an antigen (e.g., human TIGIT) prepared according to the knowledge and skill in the art. The preparation is performed by injecting the test subjects with TIGIT antigen and then isolating hybridomas expressing antibodies having 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 murine kappa, lambda chain or variant light chain constant region or further comprise a murine IgG1, igG2, igG3, igG4 or variant heavy chain constant region.

The term "chimeric antibody (chimeric antibody)" refers to an antibody in which a variable region of a murine antibody is fused to a constant region of a human antibody, and which can reduce an immune response induced by the murine antibody. The chimeric antibody is established by firstly establishing a hybridoma secreting the murine specific monoclonal antibody, cloning a variable region gene from a mouse hybridoma cell, cloning a constant region gene of a human antibody according to requirements, connecting the mouse variable region gene and the human constant region gene into a chimeric gene, inserting the chimeric gene into an expression vector, and finally expressing the chimeric antibody molecule in a eukaryotic system or a 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 a human IgG1, igG2, igG3, igG4 or variant thereof, preferably comprises a human IgG1, igG2 or IgG4 heavy chain constant region, or an IgG1, igG2 or IgG4 variant using amino acid mutation (e.g., YTE mutation or back mutation, S228P).

The term "humanized antibody (humanized antibody)", including CDR-grafted antibodies, refers to a human antibody variable region framework, i.e., a different type of human germline, grafted with murine CDR sequencesAntibodies produced in the antibody framework sequences. The heterologous reaction induced by chimeric antibodies due to the large amount of murine protein components can be overcome. Such framework sequences may be obtained from public DNA databases including germline antibody gene sequences or published references. The germline DNA sequences of, for example, human heavy and light chain variable region genes, can be found in the "VBase" human germline sequence database (in the Internetwww.mrccpe.com.ac.uk/vbaseAvailable) and found in Kabat, e.a. et al, 1991, sequences of Proteins of Immunological Interest, 5 th edition. To avoid a decrease in immunogenicity while at the same time causing a decrease in activity, the human antibody variable region framework sequences may be subjected to minimal reverse or back-mutations to maintain activity. Humanized antibodies of the present disclosure also include humanized antibodies that are further affinity matured for 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 selected from the group consisting of the heavy chain FR region sequences on the antibody heavy chain variable region, and the human germline heavy chain is selected from the group consisting of: the combined sequences of (IGHV 3-7 x 01 and hjh), (IGHV 1-46 x 01 and hjh 4.1) and (IGHV 1-69 x 02 and hjh 4.1), and the human germline light chain are selected from: (IGKV 1-39 x 02 and hjk 2.1), (IGKV 1-39 x 01 and hjk 4.1) and (IGKV 4-1 x 01 and hjk 4.1). To avoid a decrease in immunogenicity while at the same time causing a decrease in activity, the human antibody variable region may be subjected to minimal reverse mutation (back mutation, i.e., mutation of amino acid residues in the FR region of human antibody to amino acid residues at positions corresponding to the original source antibody) to maintain activity. In one embodiment of the present disclosure, wherein the light/heavy chain variable region of the anti-TIGIT humanized antibody is back mutated, an optimized combination of light/heavy chain variable regions of h1707, h1708, h1709, h17010, h17011 humanized antibodies is obtained, see in particular tables 1-5 below,

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

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

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

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

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

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

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

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

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

And (3) injection: "H1711-04V" in the table represents the light/heavy chain variable region pair consisting of the H1711-H4 heavy chain variable region (shown as SEQ ID NO: 77) and the 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 tables 1-5 described above, the heavy chain variable region thereof may be joined to the heavy chain constant region of a human antibody (e.g., a heavy chain constant region comprising a human IgG1, igG2, or IgG4 heavy chain constant region, or a variant of IgG1, igG2, or IgG4 using an amino acid mutation (e.g., one or more of a YTE mutation or back mutation, an F234A mutation, and an L235A mutation) to form a full length antibody heavy chain, and the light chain variable region thereof may be joined to the amino terminus of the light chain constant region of a kappa, lambda, or variant thereof of a human antibody to form a full length antibody light chain. For example, the light/heavy chain variable region H1707-02V, the heavy chain variable region H1707-H2 (SEQ ID NO: 50) thereof is linked to the amino terminus of IgG1, igG2, igG3, igG4, or a variant thereof (e.g., one or more of the YTE mutation or back mutation, the S228P mutation, the F234A mutation, and the L235A mutation) of a human antibody to form the heavy chain of the antibody, the light chain variable region H1707-L1 (SEQ ID NO: 46) is linked to the kappa, lambda chain constant region of the human antibody, or a variant amino terminus thereof to form the antibody light chain, and the heavy chain is then linked to the light chain to form the full length humanized antibody; in one embodiment, wherein the light/heavy chain variable region comprises region H1707-02V, the heavy chain variable region H1707-H2 (SEQ ID NO: 50) thereof is substantially identical to the sequence set forth in SEQ ID NO:78 to form an antibody heavy chain (shown as SEQ ID NO: 83), and a light chain variable region h1707-L1 (SEQ ID NO: 46) to the amino terminus of the kappa light chain constant region of a human antibody (shown as SEQ ID NO: 82).

Grafting of CDRs may result in reduced affinity of the resulting TIGIT antibody or antigen binding fragment thereof to the antigen due to framework residues that contact the antigen. Such interactions may be the result of somatic hypermutations. Thus, it may still be desirable to graft such donor framework amino acids to the framework of a humanized antibody. Amino acid residues involved in antigen binding from a non-human TIGIT antibody or antigen binding fragment thereof can be identified by examining the sequence and structure of the murine monoclonal antibody variable region. Residues in the CDR donor framework that differ from the germline can be considered relevant. If the closest germline cannot be determined, the sequences can be compared to subtype consensus sequences or consensus sequences of murine sequences with a high percentage of similarity. Rare framework residues are thought to be the result of highly mutated somatic cells, thereby playing 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 on the basis of the original protein or polypeptide. In one embodiment of the present disclosure, wherein the light/heavy chain variable region of the anti-TIGIT humanized antibody is back mutated, in some embodiments, the present 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 an antigen (e.g., TIGIT). Fragments of full length antibodies have been shown to be useful in achieving antigen binding functions of antibodies. Examples of binding fragments comprised in the term "antigen-binding fragment" of an antibody include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) F (ab') ₂ A fragment comprising a bivalent fragment of two Fab fragments linked by a disulfide bridge at the hinge region, (iii) an Fd fragment consisting of VH and CH1 domains; (iv) Fv fragments consisting of the VH and VL domains of the single arm of the antibody; (v) Single domain or dAb fragments (Ward et al, (1989) Nature 341:544-546) consisting of VH domains; and (vi) an isolated Complementarity Determining Region (CDR) or (vii) a combination of two or more isolated CDRs, optionally linked 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, using recombinant methods, by a synthetic linker, allowing for the production of a construct in which the VL and VH regions are pairedSingle protein chains forming monovalent molecules (known as single chain Fv (scFv); see, e.g., bird et al (1988) Science242:423-426; and Huston et al (1988) Proc. Natl. Acad. Sci USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed by the term "antigen-binding fragment" of an antibody. Such antibody fragments are 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. The antigen binding portion may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins. The antibodies may be of different isotypes, for example, igG (e.g., igG1, igG2, igG3, or IgG4 subclasses), igA1, igA2, igD, igE, or IgM antibodies.

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

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

The Fab of the present disclosure may be produced by treating a monoclonal antibody of the present disclosure that specifically recognizes human TIGIT and binds to the amino acid sequence of the extracellular region or its three-dimensional structure with papain. In addition, the Fab may be produced by inserting DNA encoding the Fab of the antibody into a prokaryotic or eukaryotic expression vector and introducing the vector into a prokaryote or 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 linked at hinge positions, 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 may be produced by treating a monoclonal antibody of the present disclosure that specifically recognizes human TIGIT and binds to the amino acid sequence of the extracellular region or its three-dimensional structure with pepsin. In addition, the F (ab ') 2 may 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 antigen binding activity obtained by cleavage of disulfide bonds in the hinge region of the above F (ab') 2. Fab '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 a Fab ' fragment of an antibody into a prokaryotic or eukaryotic expression vector and introducing the vector into a prokaryote or eukaryotic organism 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) connected by a linker. Such scFv molecules may have the general structure: NH (NH) ₂ -VL-linker-VH-COOH or NH ₂ -VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof, e.g.using 1-4 repeated variants (Holliger et al (1993), proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present disclosure are described by Alfthan et al (1995), protein Eng.8:725-731, choi et al (2001), eur.J.Immunol.31:94-106, hu et al (1996), cancer Res.56:3055-3061, kipriyanov et al (1999), J.mol.biol.293:41-56 and Roovers et al (2001), cancer Immunol.

The scFv of the present disclosure can be produced by the following steps: coding cdnas of VH and VL of a monoclonal antibody specifically recognizing human TIGIT and binding to an amino acid sequence of an extracellular region or a three-dimensional structure thereof of the present disclosure are obtained, DNA encoding scFv is constructed, the DNA is inserted into a prokaryotic or eukaryotic expression vector, and then the expression vector is introduced into a prokaryote or eukaryotic organism to express scFv.

Diabodies are antibody fragments in which 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 may be produced by the steps of: coding cdnas of VH and VL of a monoclonal antibody specifically recognizing human TIGIT and binding to the amino acid sequence of extracellular region or three-dimensional structure thereof of the present disclosure are obtained, DNA encoding scFv is constructed so that the amino acid sequence of peptide linker is 8 residues or less in length, the DNA is inserted into a prokaryotic or eukaryotic expression vector, and then the expression vector is introduced into a prokaryote or eukaryotic organism to express diabodies.

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

The dsFv of the present disclosure may be produced by the steps of: coding cdnas of VH and VL of a monoclonal antibody specifically recognizing human TIGIT and binding to an amino acid sequence of an extracellular region or a three-dimensional structure thereof of the present disclosure are obtained, DNA encoding dsFv is constructed, the DNA is inserted into a prokaryotic or eukaryotic expression vector, and then the expression vector is introduced into a prokaryote or eukaryotic organism to express the dsFv.

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

The CDR-containing peptides of the present disclosure can be produced by: constructing a DNA encoding CDRs of VH and VL of a monoclonal antibody specifically recognizing human TIGIT and binding to an amino acid sequence of an extracellular region or a three-dimensional structure thereof of the present disclosure, inserting the DNA into a prokaryotic or eukaryotic expression vector, and then introducing the expression vector into a prokaryote or eukaryotic organism to express the peptide. The CDR-containing peptide may also be produced by a chemical synthesis method 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 that serves as a scaffold for the antigen binding loops (CDRs) of the variable domain. Essentially, it is a variable domain that does not have 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 that contribute primarily to antigen binding. Typically, there are three CDRs (HCDR 1, HCDR2, HCDR 3) in each heavy chain variable region, and three CDRs (LCDR 1, LCDR2, LCDR 3) in each light chain variable region. The amino acid sequence boundaries of the CDRs can be determined using any of a variety of well-known schemes, including "Kabat" numbering convention (see Kabat et Al (1991), "Sequences of Proteins of Immunological Interest", 5 th edition, public Health Service, national Institutes of Health, bethesda, MD), "Chothia" numbering convention (see Al-Lazikani et Al, (1997) JMB 273:927-948) and ImMunoGenTics (IMGT) numbering convention (Lefranc M.P.), immunolist, 7, 132-136 (1999); lefranc, M.P. et Al, dev. Comp. Immunol.,27, 55-77 (2003) et Al, for example, for classical format, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR 1), 50-65 (HCDR 2) and 95-102 (HCDR 3), the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-35 (HCDR 1), 50-65 (VL 2) and 95 (HCDR 3), the amino acid residues in the light chain variable domain (VL) are numbered 24-35 (VL) and 50-35 (VL 2), the amino acid residues in the light chain variable domain (VL) are numbered 26-35 (VL) and 95-35 (VL) and the amino acid residues in the amino acid variable domain (VL) are numbered 26-35 (VL) and 95-35 (HCDR 2) and 95 (VL) are combined by amino acid numbers 50-35, VL 2 and VL 2-35 (VL 1) and VL 2 (VL 1) are defined by amino acid (HCDR 1) and HCDR2 50-56 (LCDR 2) and 89-97 (LCDR 3). Following the IMGT rules, the CDR amino acid residues in VH are approximately 26-35 (CDR 1), 51-57 (CDR 2) and 93-102 (CDR 3), and the CDR amino acid residues in VL are approximately 27-32 (CDR 1), 50-52 (CDR 2) and 89-97 (CDR 3). Following IMGT rules, CDR regions of antibodies can be determined using the procedure IMGT/DomainGap alignment.

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 generally comprise at least 3,4,5,6,7,8,9, 10, 11, 12, 13, 14 or 15 contiguous or non-contiguous amino acids in a unique spatial conformation. See, e.g., epitope Mapping Protocols in Methods in Molecular B iology, volume 66, g.e.Morris, ed. (1996).

The terms "specific binding," "selective binding," "selectively binding," and "specifically binding" refer to binding of an antibody to an epitope on a predetermined antigen. Typically, the antibody is present at about less than 10 ^-7 M, e.g. less than about 10 ^-8 M、10 ^- ⁹ M、10 ^-10 M、10 ^-11 Affinity (KD) binding of M or less.

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 ^-8 M、10 ^-9 M or 10 ^-10 The dissociation equilibrium constant (KD) of M or less binds TIGIT, e.g., as determined in a BIACORE instrument using Surface Plasmon Resonance (SPR) techniques.

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

Methods for producing and purifying antibodies and antigen binding fragments are well known in the art, such as the guidelines for antibody experimentation in Cold spring harbor, chapters 5-8 and 15. For example, the mice may be immunized with human TIGIT or fragments thereof, the resulting antibodies may be renatured, purified, and amino acid sequenced using conventional procedures. Antigen binding fragments can likewise be prepared by conventional methods. The antibodies or antigen binding fragments of the invention are engineered to incorporate one or more human FR regions into the non-human CDR regions. Human FR germline sequences can be obtained from the website http:// IMGT. Cines. FR of ImMunoGeneTics (IMGT), or from the journal of immunoglobulins, 2001ISBN012441351 by aligning IMGT human antibody variable region germline gene databases with 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 that are susceptible to transformation include members of the Enterobacteriaceae family, such as strains of Escherichia coli (Escherichia coli) or Salmonella (Salmonella); the family of bacillus (bacillus) such as bacillus subtilis (Bacillus subtilis); pneumococci (pneumococci); streptococcus (Streptococcus) and haemophilus influenzae (Haemophilus influenzae). Suitable microorganisms include Saccharomyces cerevisiae (Saccharomyces cerevisiae) and Pichia pastoris (Pichia pastoris). Suitable animal host cell lines include CHO (chinese hamster ovary cell line) and NS0 cells.

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

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

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

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

An "effective amount" comprises an amount sufficient to ameliorate or prevent a symptom or condition of a medical disorder. An effective amount is also meant to be an amount sufficient to permit 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 route and dosage of administration, and the severity of the side effects. An effective amount may be the maximum dose or regimen that avoids significant side effects or toxic effects.

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

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

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

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

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

The TIGIT-related disease is not limited so long as it is a TIGIT-related disease, e.g., a therapeutic response induced with a molecule of the present disclosure may be through binding to human TIGIT followed by suppression or inhibition of a T cell dysfunctional disorder, in some embodiments a malignancy, cancer, or an infectious disorder, in some embodiments a clinically responsive tumor or cancer type, e.g., a CD155 positive, or PVR positive tumor, immune disease, or infectious disorder, observed in clinical trials of immunotherapeutic agents 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 carcinoma, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system carcinoma, neuroendocrine tumor, laryngeal carcinoma, nasopharyngeal carcinoma, esophageal carcinoma, thyroid carcinoma, malignant pleural mesothelioma, lung carcinoma, breast carcinoma, liver carcinoma, hepatoma, hepatobiliary carcinoma, pancreatic carcinoma, stomach carcinoma, gastrointestinal carcinoma, intestinal carcinoma, colon carcinoma, colorectal carcinoma, renal carcinoma, clear cell renal cell carcinoma, ovarian carcinoma, endometrial carcinoma, cervical carcinoma, bladder carcinoma, prostate carcinoma, testicular carcinoma, skin carcinoma, melanoma, leukemia, lymphoma, bone carcinoma, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, myeloproliferative neoplasm, squamous cell carcinoma, ewing's sarcoma, systemic light chain amyloidosis, and merkel cell carcinoma; in some embodiments, the lymphoma is selected from the group consisting of: hodgkin's lymphoma, non-hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, T-cell/tissue cell enriched large B-cell lymphoma, and lymphoplasmacytic lymphoma, the lung cancer being selected from the group consisting of: non-small cell lung cancer and small cell lung cancer, said leukemia being selected from the group consisting of: chronic myeloid leukemia, acute myeloid leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, and myeloid leukemia.

The above-described TIGIT-related diseases may be diagnosed by detecting or assaying TIGIT-expressing cells with the monoclonal antibodies or antibody fragments 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 immunological detection or assay methods.

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

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

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

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

The TIGIT monoclonal antibody or antigen binding fragment provided by the embodiment of the disclosure has high specificity to the TIGIT and high affinity to the TIGIT, wherein the immunogenicity of the humanized antibody is greatly reduced, and meanwhile, the specificity of the murine antibody is completely reserved, and the higher affinity and the excellent in-vitro and in-vivo activity are realized. In some implementations, the antibodies or antigen-binding fragments of the present disclosure that specifically bind to human TIGIT are those described in international patent application PCT/CN2018/108246 (WO 2019062832 A1), which is incorporated herein by reference in its entirety.

Detailed Description

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

Example 1 preparation of anti-TIGIT antibodies

1. Preparation of antigen antibodies

1.1 protein design and expression

The antigen of the present disclosure and the amino acid sequence of the protein for detection are designed by taking human TIGIT protein (Uniprot number: Q495A 1) as a template of the TIGIT of the present disclosure, and different tags are optionally fused on the basis of the TIGIT protein and cloned on a pHr vector (self-produced) or a pXC-17.4 vector (LONZA) respectively, and the antigen for encoding the present disclosure and the protein for detection are obtained by transient expression in 293 cells or stable expression and purification in CHO cells. The TIGIT antigens are not specifically described below and refer to human TIGIT.

Fusion protein of TIGIT extracellular domain and mouse IgG2 fc fragment: TIGIT-mFc for immunization and detection

Annotation: the cross-hatched portion is the signal peptide and the italic portion is mFc.

Fusion protein of TIGIT extracellular domain and human IgG1 Fc fragment: TIGIT-Fc for detection of

Annotation: the cross-hatched portion is the signal peptide and the italic portion is Fc.

Full length TIGIT: for constructing TIGIT over-expression cell strain and for detecting

Fusion protein of the extracellular domain of (italic part) cynoTIGIT with the mouse IgG2 fc fragment: cynogit-mFc for detection

1.2 purification of TIGIT-related recombinant proteins, hybridoma antibodies, and purification of recombinant antibodies

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

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

(2) Protein a affinity chromatography extracts Fc-tagged fusion proteins or antibodies:

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

2. Preparation of anti-human TIGIT hybridoma monoclonal antibodies

2.1 immunization

Anti-human TIGIT monoclonal antibodies were generated by immunizing mice. Experimental SJL white mice, females, 6-8 weeks old (animal production license number SCXK 2012-0001, experimental animal technologies limited, beijing verritun). Feeding environment: SPF stage. After the mice are purchased, the mice are fed in a laboratory environment for 1 week, the light/dark period is regulated for 12/12 hours, and the temperature is 20-25 ℃; humidity is 40-60%. The acclimatized mice were immunized as follows. The immune antigen is the extracellular region of human TIGIT with mFc (SEQ ID NO: 1).

Immunization scheme: by using

Gold Adjuvant (Sigma Cat No. T2684) and Thermo +.>

Alum (Thermo Cat No. 77161) adjuvant. Antigen and adjuvant ()>

Gold Adjuvant) ratio of 1:1, antigen to Adjuvant (Thermo +.>

Alum) ratio was 3:1, 50 μg/dose (primary immunization), 25 μg/dose (booster immunization). The antigen was emulsified and inoculated for days 0, 14, 28, 42, 56. Day 0 Intraperitoneal (IP) injection of 50 μg/dose of post-emulsification antigen. Subcutaneous (sc) multipoint (typically 6-8 points on the back) injections at day 14 were 25 μg/dose. Day 28, 42, back or intraperitoneal injections of antigen were selected based on back caking and abdominal swelling. On days 21, 35, 49, 63, the antibody titer in the mouse serum was determined by ELISA. After 4-5 immunizations, mice with high and plateau titers in serum were selected for spleen cell fusion. Immunization was boosted 3 days before spleen cell fusion, and an antigen solution prepared by 50. Mu.g/saline was injected Intraperitoneally (IP).

2.2 spleen cell fusion

Spleen lymphocytes and myeloma Sp2/0 cells are fused by adopting an optimized PEG-mediated fusion step

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

2.3 hybridoma cell screening

Hybridoma culture supernatants were assayed by binding ELISA, based on hybridoma cell growth density. And cell binding experiments and cell blocking experiments were performed on positive well cell supernatants that were detected in combination with ELISA. And (3) timely amplifying, freezing, preserving and subcloning the hole cells with positive binding and blocking until obtaining single cell clones.

TIGIT binding ELISA, HTRF blocking assays, cell binding assays, and cell blocking assays were also performed for each subclone of cells. Hybridoma clones were obtained by the above experimental screening, antibodies were further prepared by serum-free cell culture, and purified according to the purification examples for use in the test cases.

2.4 sequence determination of hybridoma Positive clones

Cloning of sequences from positive hybridomas proceeds as follows. Collecting hybridoma cells in logarithmic growth phase, extracting RNA with Trizol (Invitrogen, cat No. 15596-018) according to the procedure of kit instruction, and extracting RNA with PrimeScript ^TM Reverse Transcriptase kit reverse transcription (Takara, cat No. 2680A). The cDNA obtained by reverse transcription was subjected to PCR amplification using a mouse Ig-Primer Set (Novagen, TB326 Rev.B 0503), and then sequenced. Obtaining selected positive clones from the obtained DNA sequenceThe amino acid sequences of the antibody variable regions corresponding to m1707, m1708, m1709, m1710 and m1711 are 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 the light and heavy chains of each antibody are as shown in table 6 (CDR sequences are determined and annotated by the Kabat numbering system).

Table 6: antibody heavy and light chain CDR region sequences

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3. Humanization of murine anti-human TIGIT antibodies

By comparing the germline gene database of the heavy and light chain variable regions of the IMGT human antibody with MOE software, heavy and light chain variable region germline genes with high homology with the murine antibody are respectively selected as templates, and CDRs of the murine antibody are respectively transplanted into corresponding human templates to form variable region sequences with the sequence of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4. And (3) carrying out back mutation on the amino acid in the FR region according to the requirement to obtain the humanized anti-TIGIT antibody. Illustratively, in the following specific examples wherein the determination of CDR region amino acid residues is determined and annotated by the Kabat numbering system.

The light and heavy chain variable region of the murine antibody is linked to the light and heavy chain constant region of the human antibody to form a chimeric antibody, and the chimeric antibody corresponding to the m1707 antibody is designated ch1707, and the other antibodies are analogized.

3.1 humanization of hybridoma clone m1707

(1) m1707 humanized framework selection

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

h1707 VH-CDR graft

h1707VL-CDR graft

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

(2) The h1707 back mutation was designed as follows:

table 7: h1707 back mutation design

Note that: if S60D is shown, the S at position 60 is mutated back to D according to the natural sequence numbering of the amino acid sequences. Grafted represents 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 h1707 VL-CDR graft)

>h1707-L2

>h1707-L3

>h1707-L4

H1707-H1 (same H1707 VH-CDR graft)

>h1707-H2

/>

3.2 humanization of hybridoma clone m1708

(1) m1708 humanized framework selection

Humanized light chain templates of the murine antibody m1708 are IGKV1-39 x 01 and hjk4.1, humanized heavy chain templates are IGHV1-46 x 01 and hjh4.1, and humanized antibody h1708 is obtained after humanization, and the humanized variable region sequence is as follows:

h1708VH-CDR graft

h1708VL-CDR graft

And (3) injection: the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, italics in sequence is FR sequence and underlined is CDR sequence.

(2) The h1708 back mutation was designed as follows:

table 8: h1708 back mutation design

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

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

h1708-L1 (same h1708 VL-CDR graft)

>h1708-L2

H1708-H1 (same H1708 VH-CDR graft)

>h1708-H2

>h1708-H3

3.3 humanization of hybridoma clone m1709

(1) m1709 humanized framework selection

Humanized light chain templates of the murine antibody m1709 are IGKV1-39 x 01 and hjk4.1, humanized heavy chain templates are IGHV1-46 x 01 and hjh 4.1.1, and 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 mutation was designed as follows:

table 9: h1709 back mutation design

Note that: if S60D is shown, the S at position 60 is mutated back to A according to the natural sequence numbering of the amino acid sequences. Grafted represents murine antibody CDR-implanted human germline FR region sequences.

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

H1709-L1 (same h1709 VL-CDR graft)

>h1709-L2

>h1709L3

>h1709-L4

H1709-H1 (same H1709 VH-CDR graft)

>h1709-H2

>h1709-H3

>h1709-H4

3.4 humanization of hybridoma clone m1710

(1) m1710 humanized framework selection

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

h1710VH-CDR graft

h1710VL-CDR graft

(2) h1710 back mutations were designed as follows:

table 10: h1710 reverse mutation design

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

h1710-L1 (same as h1710 VL-CDR graft)

>h1710-L2

H1710-H1 (same H1710 VH-CDR graft)

>h1710-H2

>h1710-H3

/>

>h1710-H4

>h1710-H5

3.5 humanization of hybridoma clone m1711

(1) m1711 humanized framework selection

Humanized light chain templates of the murine antibody m1711 are IGKV4-1 x 01 and hjk4.1, humanized heavy chain templates are IGHV1-69 x 02 and hjh4.1, and humanized antibody h1711 is obtained after humanization, and the humanized variable region sequence is as follows:

h1711VH-CDR graft

h1711VL-CDR graft

(2) The h1711 back mutation was designed as follows:

table 11: h1711 back mutation design

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Note that: if P49S indicates numbering according to the natural sequence of the amino acid sequence, the P at position 49 is mutated back to S. Grafted represents murine antibody CDR-implanted human germline FR region sequences.

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

h1711-L1 (same h1711 VL-CDR graft)

>h1711-L2

>h1711-L3

H1711-H1 (same H1711 VH-CDR graft)

>h1711-H2

>h1711-H3

>h1711-H4

3.6 construction and expression of humanized antibodies

Connecting the humanized antibody heavy chain variable region obtained by the reverse mutation design with the amino terminal of the human antibody heavy chain constant region to form an antibody heavy chain, and connecting the light chain variable region with the amino terminal of the human antibody light chain constant region to form an antibody light chain; designing primer PCR to build each humanized antibody VH/VK gene fragment, carrying out homologous recombination with an expression vector pHr (with signal peptide and constant region gene (CH 1-Fc/CL) fragment), constructing an antibody full-length expression vector VH-CH1-Fc-pHr/VK-CL-pHr, and carrying out humanized antibody expression. Illustratively, the antibody heavy chain variable region was linked to the amino terminus of the IgG4 heavy chain constant region with the mutation of S228P (corresponding to position 108 of sequence SEQ ID NO: 78), and the light chain variable region was linked to the amino terminus of the kappa light chain constant region, resulting in the humanized antibodies of h1707, h1708, h1709, h1710, h1711, see specifically tables 12-16 below:

Table 12: h1707 humanized antibodies

Note that: "H1707-02" in the table represents the variable region of the heavy chain consisting of H1707-H2 (as shown in SEQ ID NO: 50) and the sequence shown in SEQ ID NO:78, and a heavy chain comprising an h1707-L1 light chain variable region (as shown in SEQ ID NO: 46) and a heavy chain comprising an amino-terminal linkage of a heavy chain constant region as shown in SEQ ID NO:79, and so forth.

Table 13: h1708 humanized antibodies

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Note that: "H1708-04" in the table represents the variable region of the heavy chain consisting of H1708-H1 (as shown in SEQ ID NO: 51) and the sequence shown in SEQ ID NO:78, and a heavy chain comprising an h1708-L2 light chain variable region (as shown in SEQ ID NO: 53) and a heavy chain comprising an amino-terminal linkage of a heavy chain constant region as shown in SEQ ID NO:79, and so forth.

Table 14: h1709 humanized antibodies

Note that: "H1709-10" in the table represents the variable region of the heavy chain consisting of H1709-H2 (as shown in SEQ ID NO: 61) and the sequence shown in SEQ ID NO:78, and a heavy chain comprising an h1709-L3 light chain variable region (as shown in SEQ ID NO: 59) and a heavy chain comprising an amino-terminal linkage of a heavy chain constant region as shown in SEQ ID NO:79, and so forth.

Table 15: h1710 humanized antibodies

Note that: in the table, "H1710-01" represents a heavy chain variable region consisting of H1710-H1 (as shown in SEQ ID NO: 64) and a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO:78, and a heavy chain comprising an h1710-L1 light chain variable region (as shown in SEQ ID NO: 65) and a heavy chain comprising a heavy chain constant region as shown in SEQ ID NO:79, and so forth.

Table 16: h1711 humanized antibodies

Note that: in the table, "H1711-04" represents the variable region of the heavy chain consisting of H1711-H4 (as shown in SEQ ID NO: 77) and as set forth in SEQ ID NO:78, and a heavy chain comprising an h1711-L1 light chain variable region (as shown in SEQ ID NO: 72) and a heavy chain comprising an amino-terminal linkage of a heavy chain constant region as shown in SEQ ID NO:79, and so forth.

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

positive control antibody 22G2-H3Q, the VH and VL sequences (SEQ ID NOs 8 and 9, respectively, of US20160176963 A1) of which are identical to the above-described SEQ ID NOs: 78 and SEQ ID NO:79 and a light chain constant region linked 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

Exemplary anti-human TIGIT antibodies light/heavy chain full length sequences are as follows:

h1707-02 full-Length sequence of the light chain

H1707-02 heavy chain full-Length sequence

H1708-04 full-length sequence of light chain

H1708-04 heavy chain full-length sequence

H1709-10 full-length sequence of light chain

H1709-10 heavy chain full-Length sequence

H1710-01 full-length light chain sequence

H1710-01 heavy chain full-length sequence

H1711-04 full-length sequence of light chain

H1711-04 heavy chain full-Length sequence

Example 2 anti-TIGIT antibody Activity test experiments

And (3) a step of: ELISA experiments of TIGIT antibody binding to human TIGIT protein

The binding of anti-TIGIT antibodies was detected by ELISA experiments of antibodies with human TIGIT protein. The strength of the signal after antibody addition was used to determine the binding activity of the antibodies to TIGIT by binding to the anti-Fc or mFc antibody coated on the elisa plate using Fc or mFc tagged TIGIT fusion proteins, the positive control molecules 22G2-H3Q and 10a7 hIgG4 (wherein the light and heavy chain variable region sequences of 10a7 hIgG4 are from SEQ ID nos. 21 and 22 in US20130251720A1, and the light and heavy chain constant regions linked to the light and heavy chain variable regions thereof to form a full length antibody are SEQ ID nos. 79 and 78, respectively), were as follows:

sheep anti-human Fc antibody (Jackson Immuno Research, cat No. 109-005-008) or sheep anti-mouse Fc antibody (Sigma, cat No. M3534-1 ML) was diluted to a concentration of 2. Mu.g/ML with PBS (Shanghai source culture, cat No. B320) buffer at pH7.4, and added at a concentration of 50. Mu.l The volume of the wells was added to a 96-well ELISA plate (Corning, cat No. CLS3590-100 EA) and placed in an incubator at 37℃for 2 hours. After discarding the liquid, 200. Mu.l/well of a blocking solution of 5% skim milk (BD ski milk, cat No. 232100) diluted with PBS was added, and incubated in an incubator at 37℃for 3 hours or left overnight (16-18 hours) at 4℃for blocking. After blocking was completed, blocking solution was discarded, and after washing the plate 5 times with PBST buffer (pH 7.4 PBS containing 0.05% tween-20), 50. Mu.l/well of sample dilution (pH 7.4 PBS containing 1% BSA) was added to dilute to 0.5. Mu.g/ml of TIGIT-Fc fusion protein (produced internally) or TIGIT-mFc fusion protein (produced internally), and incubated in an incubator at 37℃for 1 hour or overnight at 4 ℃. After the incubation, the reaction solution in the ELISA plate was discarded, and after washing the plate 5 times with PBST, 50. Mu.l/well of the antibodies to be tested (hybridoma purified antibodies or humanized antibodies) at different concentrations diluted with the sample dilution were added, and incubated in an incubator at 37℃for 1 hour. After the incubation, the plates were washed 5 times with PBST, and 50. Mu.l/well of HRP-labeled goat anti-mouse secondary antibody (Jackson Immuno Research, cat No. 115-035-003) or goat anti-human secondary antibody (Jackson Immuno Research, cat No. 109-035-003) diluted with the sample dilution was added, followed by incubation at 37℃for 1 hour. After washing the plate 5 times with PBST, 50. Mu.l/well TMB chromogenic substrate (KPL, cat No. 52-00-03) was added, incubated at room temperature for 5-10min, 50. Mu.l/well 1M H was added ₂ SO ₄ The reaction was stopped, the absorbance was read at 450nm using a microplate reader (Thermo scientific Multiskan, MK 3), the data was analyzed using GraphPad Prism 5, and the binding EC50 value of the TIGIT antibody to human TIGIT protein was calculated. The results are shown in FIG. 1.

And II: ELISA experiments of TIGIT antibody binding to cynomolgus monkey TIGIT protein

The monkey cross-binding capacity of anti-TIGIT antibodies was detected by ELISA experiments of antibodies with cynomolgus TIGIT protein. The cynomolgus TIGIT fusion protein with the Fc or mFc tag is fixed in the 96-well ELISA plate by combining with the anti-Fc or mFc antibody coated in the ELISA plate, and the intensity of the signal after the antibody is added is used for judging the binding activity of the antibody and the cynomolgus TIGIT, and the specific experimental method is as follows:

sheep anti-human Fc antibody (Jackson Immuno Research, cat No. 109-005-008) or sheep anti-mouse were buffered with PBS (Shanghai source culture, cat No. B320) pH7.4Fc antibody (Sigma, cat No. M3534-1 ML) was diluted to a concentration of 2. Mu.g/ML, added to a 96-well ELISA plate (Corning, cat No. CLS3590-100 EA) in a volume of 50. Mu.l/well, and placed in an incubator at 37℃for 2 hours. After discarding the liquid, 200. Mu.l/well of a blocking solution of 5% skim milk (BD ski milk, cat No. 232100) diluted with PBS was added, and incubated in an incubator at 37℃for 3 hours or left overnight (16-18 hours) at 4℃for blocking. After blocking was completed, blocking solution was discarded, and after washing the plate 5 times with PBST buffer (pH 7.4 PBS containing 0.05% tween-20), 50. Mu.l/well of sample dilution (pH 7.4 PBS containing 1% BSA) was added to dilute to 0.5. Mu.g/ml of cynomolgus TIGIT-Fc fusion protein (produced internally) or cynomolgus TIGIT-mFc fusion protein (produced internally), and incubated in a 37℃incubator for 1 hour or overnight at 4 ℃. After the incubation, the reaction solution in the ELISA plate was discarded, and after washing the plate 5 times with PBST, 50. Mu.l/well of the antibodies to be tested (hybridoma purified antibodies or humanized antibodies) at different concentrations diluted with the sample dilution were added, and incubated in an incubator at 37℃for 1 hour. After the incubation, the plates were washed 5 times with PBST, and 50. Mu.l/well of HRP-labeled goat anti-mouse secondary antibody (Jackson Immuno Research, cat No. 115-035-003) or goat anti-human secondary antibody (Jackson Immuno Research, cat No. 109-035-003) diluted with the sample dilution was added, followed by incubation at 37℃for 1 hour. After washing the plate 5 times with PBST, 50. Mu.l/well TMB chromogenic substrate (KPL, cat No. 52-00-03) was added, incubated at room temperature for 5-10min, 50. Mu.l/well 1M H was added ₂ SO ₄ The reaction was terminated, absorbance was read at a wavelength of 450nm using a microplate reader (Thermo scientific Multiskan MK 3), data was analyzed using GraphPad Prism5, and the binding EC50 value of TIGIT antibody to monkey TIGIT was calculated. The results are shown in FIG. 2.

Thirdly,: binding experiments of TIGIT antibodies with human TIGIT overexpressing CHO cells

Binding of anti-TIGIT antibodies was detected by binding experiments of antibodies to CHO cells overexpressing TIGIT proteins. After transferring the full-length TIGIT plasmid into CHO cells by electrotransfection and screening for two weeks, the expression level of TIGIT was detected. After the over-expression cells are fixed on the bottom of the 96-well plate, the intensity of the signal after the antibody is added is used for judging the binding activity of the antibody and the 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. After washing the supernatant three times with PBS, 100. Mu.l/well of cell immunofixative (Beyotime, cat No. P0098) was added and the mixture was fixed at room temperature for half an hour, and the washing with PBS was performed four times. After discarding the liquid, 200. Mu.l/well of a blocking solution of 5% skim milk (BD ski milk, cat No. 232100) diluted with PBS was added, and the mixture was incubated in an incubator at 37℃for 3 hours for blocking. After blocking, the blocking solution was discarded, and after washing the plate 5 times with PBST buffer (pH 7.4 PBS containing 0.05% tween-20), 50. Mu.l/well of the antibody to be tested (hybridoma purified antibody or humanized antibody) at different concentrations diluted with the sample dilution was added, and incubated in an incubator at 37℃for 1 hour. After the incubation, the plates were washed 5 times with PBST, and 50. Mu.l/well of HRP-labeled goat anti-mouse secondary antibody (Jackson Immuno Research, cat No. 115-035-003) or goat anti-human secondary antibody (Jackson Immuno Research, cat No. 109-035-003) diluted with the sample dilution was added, followed by incubation at 37℃for 1 hour. After washing the plate 5 times with PBST, 50. Mu.l/well TMB chromogenic substrate (KPL, cat No. 52-00-03) was added, incubated at room temperature for 5-15min, 50. Mu.l/well 1M H2SO4 was added to terminate the reaction, absorbance was read at wavelength 450nm using an microplate reader (Thermo scientific Multiskan MK 3), data was 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.

Fourth, the method comprises the following steps: binding experiments of TIGIT antibodies to human PBMC

Binding of anti-TIGIT antibodies was detected by binding experiments of antibodies to in vitro activated human PBMCs. Human PBMC were activated by the method of superantigen staphylococcus aureus enterotoxin B (SEB) stimulation, and the intensity of the fluorescent signal after antibody addition was used to determine the binding activity of the antibody to activated human PBMC, as follows:

fresh blood was centrifuged (Stem Cell Technologies) using Ficoll-Hypaque density gradient to obtain PBMC, and cultured in RPMI 1640 medium supplemented with 10% (v/v) FBS and 500ng/ml superantigen Staphylococcus aureus enterotoxin B (SEB), 37℃and 5% CO ₂ Culturing under the condition for 4 days.

Activated PBMC cells were seeded at a density of 5X 10≡6/ml, 100 μl/well in 96-well round bottom plates (Corning, cat No. 32915001), centrifuged at 1500rpm for 5 min with a Centrifuge (Beckman Coulter, allegra X-15R Centrifuge) and the supernatant discarded; cells were resuspended in 200 μl PBS, centrifuged, and the supernatant discarded and repeated. Cells were resuspended by adding 100. Mu.l/well of the antibody solution to be assayed which had been diluted in a gradient with sample dilution (pH 7.4 PBS containing 1% BSA), and incubated at 4℃for 1 hour. After the incubation was completed, the cells were resuspended in 100. Mu.l of a dilution of PE-goat anti-human IgG (Jackson ImmunoResearch, 109-115-098) after centrifugation at 1500rpm for 5 minutes, the supernatant was discarded, and the cells were washed twice with the sample dilution, and incubated at 4℃for 1 hour. After the incubation was completed, centrifugation was performed at 1500rpm for 5 minutes, the supernatant was discarded, the cells were washed twice with the sample diluent, and finally the cells were resuspended with 200. Mu.l/well of the sample diluent, the fluorescence signal intensity was detected on a flow cytometer (BD FACS Canto II), the data was analyzed by GraphPad Prism 5, and the EC50 value of TIGIT antibody binding to human PBMC cells was calculated. The results are shown in FIG. 4.

Fifth step: biacore assay

The affinity of the anti-TIGIT antibody to be tested to human and monkey TIGIT was determined with Biacore, GE instrument.

Human anti-capture antibodies were either affinity captured using a Protein A biosensing chip (Cat. # 29119556, GE) or covalently coupled to a biosensing chip (Cat. #28-9538-28, GE) according to the methods described in the instructions of human anti-capture kit (Cat. #28-9538-28, GE), thereby affinity capturing a certain amount of the antibodies to be tested, and then passed over the chip surface through human, monkey TIGIT antigen under a series of concentration gradients, which was selected from Yiqiao Shenzhou biological company (Cat.10917-H08-H, sino.biol), which was purified by expression of example 1 and example 2, and the reaction signals were detected in real time using a Biacore instrument (Biacore T200, GE) to obtain binding and dissociation curves. After completion of each cycle of dissociation, the biochip was washed and regenerated with a regeneration solution prepared in a human anti-capture kit or glycine-hydrochloric acid regeneration solution (Cat. #BR-1003-54, GE) at pH 1.5. The amino coupling kit used in the experiments was purchased from GE company (Cat. #BR-1000-50, GE), and the buffer was HBS-EP+10Xbuffer (Cat. #BR-1006-69, GE) diluted to 1X (pH 7.4) with D.I. water.

The experimental data were fitted with BIAevaluation version.4.1 using GE software using the (1:1) Langmuir model to give affinity values, the results are shown in tables 17-19.

TABLE 17 reaction affinity of test molecules with human TIGIT protein

Table 18, reaction affinity of test molecules with cynoTIGIT protein

Table 19, ch1711 and reaction affinity of humanized antibodies to human TIGIT protein

Sixth,: blocking assay of TIGIT antigen binding to CD155 protein by TIGIT antibodies

The blocking ability of anti-TIGIT antibodies was detected by HTRF experiments in which the antibodies blocked TIGIT binding to CD155 protein. The pair of donor and acceptor bound to TIGIT-Fc and biotylinylated CD155 (R & D, cat No. 2530-CD-050/CF) with Pab Anti-Human IgG-Tb (Cisbio, cat No. 61HFCTAA) and strepavidin-XL 665 (Cisbio, cat No. 610SAXLA), or the intensity of the signal after hybridoma purification or humanized antibody addition was used to determine the activity of antibody blocking TIGIT and CD155 by binding to TIGIT-Fc and biotylinylated CD155 (R & D, cat No. 2530-CD-050/CF), or by binding to Pab Anti-mouse-IgG-XL665 (Cisbio, cat No. 61PAMXLA) and strepavidin-Tb (Cisbio, cat No. 610SATLA), as follows:

10. Mu.l/Kong Jiaru 384 well assay plates (Corning, cat No. 3706) of different concentrations of antibodies to be tested (hybridoma purified antibodies or humanized antibodies) diluted in diluent (pH 7.4 PBS containing 1% BSA) were centrifuged at 1000rpm for 1min, 2.5. Mu.l/well of TIGIT-Fc or TIGIT-mFc diluted with sample diluent to 2. Mu.g/ml was added, centrifuged at 1000rpm for 1min, 2.5. Mu.l/well of biotin-CD155 diluted to 4. Mu.g/ml was added, centrifuged at 1000rpm for 1min, pre-incubated at room temperature for 10min, then 2.5. Mu.l/well of sample diluent was added to 3.2. Mu.g/ml of Pab Anti-Human IgG-Tb (Cisbio, cat No. 61HFCTAA) and 2.5. Mu.l/well of 0.08. Mu.g/ml strepavidin-XL 665 (Cisbio, cat No. 610SAXLA), or 2.5. Mu.l/well of sample dilutions were diluted to 3.2. Mu.g/ml of Pab Anti-mouse-IgG-XL665 (Cisbio, cat No. 61PAMXLA) and 2.5. Mu.l/well of 0.08. Mu.g/ml strepavidin-Tb (Cisbio, cat No. 610SATLA), were left at room temperature for 1 hour, the emission light values at 665nm and 620nm were detected with a PHEARstar FS enzyme-labeled instrument (BMG LABETECH), the data were analyzed with GraphPad Prism 5, and the inhibitory activity of TIGIT antibodies on Human TIGIT and CD155 proteins was calculated. The results are shown in FIGS. 5A and 5B.

7. Blocking experiments of TIGIT antibodies on TIGIT antigen binding to CHO cells overexpressing CD155

The blocking ability of anti-TIGIT antibodies was detected by ELISA experiments where antibodies blocked TIGIT binding to CHO cells overexpressing CD 155. After transfection of CD155 full-length plasmid into CHO cells by electrotransfection and pressure screening for two weeks, the expression level of CD155 was detected. After the over-expression cells are fixed on the bottom of a 96-well plate, the TIGIT and diluted anti-TIGIT antibodies with different concentrations are preincubated, then the well plate is added, and the strength of a signal is used for judging the capability of the antibody to block the binding of the TIGIT and the CHO cells over-expressing CD155 after the secondary antibody is added, and the specific experimental method is as follows:

CD155-CHO cells were cultured overnight in 96 well plates at a density of 5X 10≡5/ml and 100. Mu.l/well. After washing the supernatant three times with PBS, 100. Mu.l/well of cell immunofixative (Beyotidme, cat No. P0098) was added and the mixture was fixed at room temperature for half an hour, and the washing with PBS was performed four times. After discarding the liquid, 200. Mu.l/well of a blocking solution of 5% skim milk (BD ski milk, cat No. 232100) diluted with PBS was added, and the mixture was incubated in an incubator at 37℃for 3 hours for blocking. After blocking was completed, the blocking solution was discarded, and after washing the plate 5 times with PBST buffer (pH 7.4 PBS containing 0.05% tween-20), 50. Mu.l/well of a pre-mixed incubation for 1 hour with a sample dilution (pH 7.4 PBS containing 1% BSA) was added, and the mixture was incubated in an incubator at 37℃for 1 hour with a final concentration of 0.2. Mu.g/ml of human TIGIT-hFc (produced internally) or a mixture of human TIGIT-mFc (produced internally) and an antigen-antibody of the antibody to be tested in a gradient concentration. After the incubation, the reaction solution in the ELISA plate was discarded, and after washing the plate 5 times with PBST, 50. Mu.l/well of HRP-labeled goat anti-human secondary antibody (Jackson Immuno Research, cat No. 109-035-003) or goat anti-mouse secondary antibody (Jackson Immuno Research, cat No. 115-035-003) diluted with the sample dilution was added, and incubated at 37℃for 1 hour. After washing the plates 5 times with PBST, 50. Mu.l/well TMB chromogenic substrate (KPL, cat No. 52-00-03) was added, the reaction was stopped by incubation at room temperature for 5-15 minutes, 50. Mu.l/well 1M H2SO4 solution was added, the absorbance was read at a wavelength of 450nm using a microplate reader (Thermo scientific Multiskan, MK 3), the data were analyzed using GraphPad Prism 5, and the blocking effect of TIGIT antibodies on antigen binding to CHO cells overexpressing CD155 was calculated. The results are shown in FIG. 6.

8. Blocking experiments of binding of TIGIT antibodies to CD155 protein and CHO cells overexpressing TIGIT

The blocking ability of anti-TIGIT antibodies was detected by FACS experiments in which the antibodies blocked the binding of CD155 to TIGIT overexpressing CHO cells. After transfection of the TIGIT full-length plasmid into CHO cells by electrotransfection for two weeks, the expression level of TIGIT was detected. After pre-incubating the over-expression cells with anti-TIGIT antibodies with different concentrations, adding fluorescence labeled CD155-Fc for incubation, and judging the ability of the antibody to block the combination of CD155 and the CHO cells over-expressing TIGIT through the strength of signals, wherein the specific experimental method is as follows:

CD155-Fc (Sino Biological, cat No. 10109-H02H) was first labeled with CF ^TM 633 (Sigma Aldrich, cat No. MX 633S100). Dissolving CD155-Fc with PBS to a concentration of 0.5-1mg/ml, adding 10 XMix-n-Stain Reaction Buffer with a sample volume of 9 times, and mixing; CF is then added ^TM 633 fluorescent dye, after incubation for 30 minutes at room temperature in the dark, the fluorescent labeling is completed.

TIGIT-CHO cells were seeded at a density of 5×10≡6/ml, 100 μl/well in 96-well round bottom plates (Corning, cat No. 32915001), centrifuged at 1500rpm for 5 min with a Centrifuge (Beckman Coulter, allegra X-15R Centrifuge) and the supernatant discarded; cells were resuspended in 200 μl PBS, centrifuged, and the supernatant discarded and repeated. Cells were resuspended by adding 100. Mu.l/well of the antibody solution to be assayed which had been diluted in a gradient with sample dilution (pH 7.4 PBS containing 1% BSA), and incubated at 4℃for 1 hour. After completion of incubation, centrifugation at 1500rpm for 5 minutes, the supernatant was discarded, and after washing the cells twice with the sample diluent, 100. Mu.l of 2. Mu.g/ml of CF was added ^TM 633 fluorescence-labeled CD155-Fc solution resuspended cells and incubated at 4℃for 1 hour. After the incubation was completed, centrifugation was performed at 1500rpm for 5 minutes, the supernatant was discarded, the cells were washed twice with the sample diluent, and finally the cells were resuspended with 200. Mu.l/well of the sample diluent, the fluorescence signal intensity was detected on a flow cytometer (BD FACS Canto II), the data was analyzed by GraphPad Prism 5, and the blocking ability of the TIGIT antibody against CD155 binding to TIGIT-CHO cells was calculated. The results are shown in FIG. 7.

9. Blocking experiments of TIGIT antibodies on TIGIT antigen binding to CHO cells overexpressing CD112

The 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 CD112 plasmid into CHO cells by electrotransfection and screening for two weeks under pressure, the expression level of CD112 was detected. Pre-incubating TIGIT-mFc protein with diluted anti-TIGIT antibodies with different concentrations, adding the pre-incubated anti-TIGIT antibodies into a CD112 over-expressed CHO cell for incubation, and detecting the intensity of TIGIT signals by an antibody added with PE label is used for judging the ability of the antibody to block the binding of TIGIT and the over-expressed CD112 CHO cell, wherein the specific experimental method is as follows:

diluting the humanized antibody sample by 1% BSA, diluting nine concentration points from the initial concentration of 20 mug/mL by two times, simultaneously diluting TIGIT-mFc to 2 mug/mL, uniformly mixing the antigen and the antibodies with different concentrations according to the volume ratio of 1:1, and pre-incubating for 30 minutes at 37 ℃; CD112-CHOs cells were collected, washed once with PBS, and distributed at 0.5 x 10≡6/test; the cells were resuspended in 150. Mu.l of antigen-antibody mixture, incubated at 4℃for 60 min, and washed 3 times with 1% BSA; PE-goat anti-mouse IgG (Biolegend, 405307) dilutions were used to resuspend cells 100. Mu.l, incubated at 4℃for 40 min, washed 3 times with 1% BSA, resuspended cells with 200. Mu.l 1% BSA, and the MFI of each sample was read on the flow cytometer BD FACSCanto II, graphPad Prism 5 assay data to determine the blocking effect of TIGIT antibodies on antigen binding to CD112-CHO cells. The results are shown in FIG. 8.

10. Binding endocytic assay of TIGIT antibodies in TIGIT overexpressing CHO cells

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

TIGIT-CHO cells were seeded at a density of 2X 10X 6/ml, 100 μl/well in 96-well round bottom plates (Corning, cat No. 32915001), centrifuged at 1500rpm for 5 min with a Centrifuge (Beckman Coulter, allegra X-15R Centrifuge) and the supernatant discarded; cells were resuspended with 200 μl 1% BSA, centrifuged, and the supernatant discarded and repeated. The humanized antibody sample was diluted to a concentration of 4. Mu.g/mL with 1% BSA, 100. Mu.l/well was added to the cells to resuspend the cells, and incubated on ice for 1 hour. After the end of incubation, centrifugation at 1500rpm for 5 minutes, the supernatant was discarded, after washing the cells three times with 1% BSA, the cells were resuspended with 10% FBS-DMEM/F-12 medium and divided into two parts, one part was incubated in a 37℃incubator for 1 hour (endocytic group, internalization group) and the other part was further incubated on ice for 1 hour (binding affinity group ). After the incubation was completed, cells were washed once with 1% BSA, PE-anti-Fc antibody (Jackson, 109-115-098) diluted with 1% BSA was added, and after incubation on ice for one hour, 1% BSA was washed 3 times, cells were resuspended with 1% BSA, and each sample MFI was read on a flow cytometer BD FACSCanto II, and the endocytosis rate of the antibody was calculated as follows. And (3) injection: black is incubated without anti TIGIT antibody, with PE-anti Fc antibody diluted with 1% BSA alone on ice for one hour followed by 3 washes to read MFI in resuspension. The results are shown in FIG. 9.

Endocytic ratio (Internalization Ratio)% = (binding affinity group-endocytic group) ×100/(binding affinity group-blank group)

11. Cell killing experiments of Natural killer cells (NK)

To investigate the effect of TIGIT antibodies on NK cell killing function, human Peripheral Blood Mononuclear Cells (PBMC) 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 detected. The specific experimental process is as follows:

human colorectal cancer cell line WiDr was cultured in MEM medium supplemented with 10% (v/v) Fetal Bovine Serum (FBS), 37℃and 5% CO ₂ Culturing under the condition. Fresh blood was centrifuged using Ficoll-Hypaque density gradient (Stem Cell Technologies) to obtain PBMC, and human primary NK cells were extracted from freshly isolated PBMC (Milenyi, CAT#130-092-657) in RPMI 1640 medium supplemented with 10% (v/v) FBS,37℃and 5% CO ₂ Culturing under the condition.

Human primary NK cells were inoculated into 6-well cell culture plates at a cell density of about 2X 10-6/mL, cultured overnight with 100U/mL human IL-2, washed with phenol red-free RPMI 1640 medium, resuspended, and inoculated into 96-well U-bottom plates at a cell density of about 3X 10-5/well, while gradient diluted antibody samples (diluted with PBS) or an equivalent amount of isotype IgG was added as a blank. 37 ℃,5% CO ₂ After incubation in incubator for 1h, target cells WiDr were co-cultured with human primary NK cells at 1:1 ratio, 37℃at 5% CO ₂ After 4 hours of culture in the incubator, the cell culture supernatant was collected. By CytoTox

The cell culture supernatant was assayed for the level of LDH secretion by the Non-Radioactive Cytotoxicity Assay (Promega, CAT#G1780) protocol. The percentage of specific cell lysis is determined by the following formula: % dissolution = 100× (ER-SR 1-SR 2)/(MR-SR 1), where ER, SR (1)&2) And MR represent experimental, spontaneous (1 as target cell, 2 as human primary NK cell) and maximal LDH release, respectively. Spontaneous release of LDH released by target cells or human primary NK cells cultured alone in a medium, maximum release of LDH measured when all target cells are lysed with lysate. As shown in FIG. 10A or FIG. 10B, the antibodies h1707-02, h1708-04, h1710-01 and the like of the TIGIT humanized candidate antibodies can enhance the killing of the primary NK cells to target cells to different degrees, and have a drug concentration dose effect.

12. PBMC-T lymphocyte activation assay

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

Fresh blood was centrifuged (Stem Cell Technologies) using Ficoll-Hypaque density gradient to obtain PBMC, and cultured in RPMI 1640 medium supplemented with 10% (v/v))FBS，37℃、5％CO ₂ Culturing under the condition.

Fresh isolated and purified PBMC were conditioned to a density of 2X 10≡6/mL in RPMI 1640 medium, 25. Mu.l tuberculin was added to 20mL cell suspension, 37℃and 5% CO ₂ The incubator was cultured for 5 days. On day 5, CD155 (recombinant CD155/PVR Protein, R) was added to 96-well cell culture plates&D, 2530-CD-050/CF), 0.25 μg per well, coated overnight at 4 ℃. On day 6, the cultured cells were collected, centrifuged once with PBS, resuspended in fresh RPMI 1640 medium, adjusted to a density of 1X 10≡6/ml, and plated onto CD155 coated 96 well cell culture plates at 90. Mu.l per well. A gradient diluted antibody sample (diluted with PBS) or an equivalent of isotype IgG was added as a blank, 10 μl per well. The cell culture plate was placed at 37℃with 5% CO ₂ Incubators were incubated for 3 days. The cell culture plates were removed, centrifuged (4000 rpm,10 min) to collect the cell culture supernatant, and the IFN-gamma levels were detected by ELISA (human IFN-gamma detection kit, xinbo, EHC102 g.96). For specific operation reference is made to the reagent instructions. As shown in FIG. 11, antibodies such as TIGIT humanized candidate antibodies h1708-04, h1710-01 can enhance the secretion of cytokine IFN-gamma by activated primary T lymphocytes to different degrees, and have a drug concentration dose effect.

13. Humanized TIGIT antibody rat pharmacokinetic evaluation

SD male rats weighing 180-240g purchased from Sipuler-BiKai laboratory animal Co. The feed and water are freely taken during the feeding period, the laboratory environment adaptability 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, randomly grouped, 3 per group. On the day of the experiment, five groups of rats are respectively injected with the tested drugs H1707-02, H1708-04, H1710-01, H1711-04 and H1709-10 by intravenous injection or subcutaneous injection, and the dosage of the drugs is 3mg/kg; the injection volumes were 5ml/kg.

Intravenous administration blood was collected at each time point of 5min,8h,1d,2d,4d,7d,10d,14d,21d,28d before administration. Subcutaneous injection was used to collect blood at each time point of 1h,4h,8h,1d,2d,4d,8d,11d,14d,21d,28d before and after administration. 0.2ml of whole blood was taken from each animal, without anticoagulant, placed at 4℃for 30min, centrifuged at 1000g for 15min, and the supernatant was placed in an EP tube and stored at-80 ℃.

The concentration of antibodies in the serum was measured by ELISA (see example 2. One, supra) and the pharmacokinetic parameters of the test drug were calculated using Winnolin software. The principal pharmacokinetic results of the obtained fractions are shown in FIG. 12.

After detection, the SD rat is given 3mg/kg of anti-TIGIT antibody h1707-02, h1708-04, h1710-01, h1709-10 and h1711-04 by intravenous injection, the exposure in the rat is similar; the bioavailability of subcutaneous administration is high and approaches 100%; the elimination half-life of each antibody is longer and better than that of the 22G2-H3Q antibody.

The following stabilized formulations of anti-TIGIT antibodies were formulated by experimentation (exemplary anti-TIGIT antibodies in examples 3-11 below were the h1708-04 antibodies described previously)

EXAMPLE 3 determination of Tm value of tigit antibody

Preparing 10mM disodium hydrogen phosphate-citric acid (PB-CA) buffer solutions (see below 1) to 6) with different pH values, and preparing an antibody preparation with an anti-TIGIT antibody concentration 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 thermostability of the anti-TIGIT antibodies in each group of formulations was determined by differential scanning calorimetry (differential scanning calorimetry, DSC). The test results are shown in Table 20, and the results show that the anti-TIGIT antibody is stable at pH 5.0-7.0.

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 the buffer System for anti-TIGIT antibody formulations for pH

Different buffers (see groups 1) to 12) were prepared and antibody preparations were prepared with anti-TIGIT antibody concentrations of 50 mg/ml:

1) 10mM acetic acid-sodium acetate, pH5.0;

2) 10mM acetic acid-sodium acetate, pH5.5;

3) 10mM acetic acid-sodium acetate, pH5.7;

4) 10mM sodium succinate, pH5.0;

5) 10mM sodium succinate, pH5.5;

6) 10mM sodium succinate, pH6.0;

7) 10mM histidine-histidine hydrochloride, pH5.5;

8) 10mM histidine-histidine hydrochloride, pH6.0;

9) 10mM histidine-histidine hydrochloride, pH6.5;

10 10mM histidine-acetic acid, pH5.0;

11 10mM histidine-acetic acid, pH5.5;

12 10mM histidine-acetic acid, pH6.0.

Samples of each group of anti-TIGIT antibody formulations were taken for high temperature (40 ℃) stability studies and the test results are shown in table 21. The results show that: the pH is in the range of 5.0-6.0, the difference of the purity values of the SEC monomers of the 4 buffer systems is not large, the main peak change of CE-SDS (NR) is within 2%, the neutral peak change of ICE is within the range of 13.1-17.5%, and the pH is more stable relative to the buffer solution with the pH of 6.5.

TABLE 21 pH screening results of tigit antibody preparation buffer System

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Note that: m represents a month, e.g., M0 represents 0 month of storage, M1 represents 1 month of storage at 40℃C

Example 5 screening of surfactant species in anti-TIGIT antibody preparations

A buffer system of 10mM histidine-histidine hydrochloride pH6.0, 80mg/ml sucrose, and a group of different types of surfactants (see below 1) to 2) were selected to prepare a preparation having an anti-TIGIT antibody concentration of 50 mg/ml:

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

TABLE 22 stability results of TIGIT antibody surfactant class screening

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Note that: m represents a month, D represents a day, e.g., M0 represents 0 month of storage, D20 represents 20 days of storage at 40℃and M6 represents 6 months of storage at 25℃and M6 represents 2 to 8℃for 6 months.

Example 6 screening of surfactant concentration in tigit antibody formulations

An antibody preparation with an anti-TIGIT antibody concentration of 50mg/ml was prepared by selecting a buffer system of 10mM histidine-histidine hydrochloride ph5.5, 80mg/ml sucrose, and different concentrations of polysorbate 80 (see groups 1) to 6) below:

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.

The preparation without polysorbate 80 is obviously deteriorated in appearance after being shaken for 7 days, which indicates that the addition of the polysorbate 80 serving as a surfactant has a stabilizing effect on the preparation, and the formulations of each group of polysorbate 80 containing 0.1mg/ml to 0.8mg/ml have no obvious difference and are stable.

TABLE 23 results of the TIGIT antibody Polysorbate 80 concentration screening shaking experiments

Note that: shakingD7 represents shaking for 7 days, and 0h represents shaking for 0 hour.

EXAMPLE 7 screening of sugar concentration in anti-TIGIT antibody preparations

An antibody preparation with an anti-TIGIT antibody concentration of 50mg/ml was prepared by selecting a buffer system of 10mM histidine-histidine hydrochloride ph5.5,0.4mg/ml polysorbate 80 and different concentrations of sucrose (see groups 1) to 5):

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 results are shown in Table 24, and the experimental results show that the preparation is isotonic when the concentration of sucrose is 80 mg/ml.

TABLE 24 osmotic pressure results of anti-TIGIT antibodies at various sucrose concentrations

Group of	Sucrose concentration	Osmotic pressure value (mOsm)
			1	65mg/ml	244
2	70mg/ml	264
			3	75mg/ml	286
4	80mg/ml	304
			5	85mg/ml	319

Example 8 different buffer System stability of anti-TIGIT antibodies

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

1) 10mM histidine-histidine hydrochloride pH5.5;

2) 10mM histidine-acetic acid pH5.5;

3) 10mM sodium succinate pH5.5.

The stability of the anti-TIGIT antibody preparation in different buffer systems at 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 in the buffer systems of 10mM sodium acetate-10 mM histidine-histidine hydrochloride and 10mM succinic acid has good stability after being placed at 25 ℃ for 6 months and 2-8 ℃ for 6 months, and no obvious difference is seen between the anti-TIGIT antibody preparation and the anti-TIGIT antibody preparation.

TABLE 25 results of stability at 25℃for different buffer systems

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

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

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

Example 9 comprehensive screening of anti-TIGIT antibody formulation Components

For further comprehensive screening of antibody concentrations (40-60 mg/ml), pH (5.0-6.0) and buffer systems (His-HCl, his-AA, SA), DOE design was performed using JMP software, and a series of prescriptions (see below 1) to 13) were obtained using RSM model), in different buffer systems, anti-TIGIT antibody preparations containing 0.4mg/ml polysorbate 80 and 80mg/ml sucrose were prepared:

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 repeat test was designed for group 6 of median values);

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 sodium succinate, pH5.0, 50mg/ml anti-TIGIT antibody;

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

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

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

the stability analysis was performed by storing each group of antibody preparation samples at 40℃or under light (5.+ -. 3 ℃ C., 4500 lx), and the experimental 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 5.0-5.8 is optional; histidine-histidine hydrochloride (His-HCl) with an optional pH in the range of 5.5 to 6.0; the pH range of succinic acid-sodium Succinate (SA) is chosen to be relatively narrow. In addition, the decline in SA purity data was large relative to His-AA and His-AA. Thus, the buffer 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

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Note that: 40℃ D25 means preservation at 40℃ for 25 days, 0h means preservation for 0 hour, and light D10 means illumination (5.+ -. 3 ℃ C., 4500 lx) means preservation for 10 days.

EXAMPLE 10 lyophilization of anti-TIGIT antibody formulations

An antibody preparation containing 80mg/ml sucrose, 0.4mg/ml polysorbate 80 and having an anti-TIGIT antibody concentration of 50mg/ml was prepared with a buffer containing 10mM histidine-acetic acid at pH 5.5. The antibody was filled into 6mL penicillin bottles at 2.15 mL/bottle, filled into lyophilization boxes, and lyophilized. The lyophilization procedure is prefreezing, primary drying and secondary drying. After the lyophilization procedure was completed, vacuum stoppered. The reconstituted samples were subjected to lyophilization before and after comparison. The results show that the reconstituted solution can maintain good performance of the liquid formulation.

TABLE 28 lyophilization step of the formulations

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

Example 11 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-acetate buffer ph5.6;

(2) 1mg/ml anti-TIGIT antibody, 100mg/ml sucrose, 0.05mg/ml polysorbate 80, and 5mM histidine-acetate buffer pH5.9;

(3) 20mg/ml anti-TIGIT antibody, 90mg/ml sucrose, 1.0mg/ml polysorbate 80, and 15mM histidine-acetate buffer pH5.7;

(4) 80mg/ml anti-TIGIT antibody, 81mg/ml sucrose, 0.6mg/ml polysorbate 80, and 20mM histidine-acetate buffer pH5.6;

(5) 50mg/ml anti-TIGIT antibody, 83mg/ml sucrose, 1.2mg/ml polysorbate 80, and 25mM histidine-acetate buffer pH5.5;

(6) 55mg/ml anti-TIGIT antibody, 78mg/ml sucrose, 0.4mg/ml polysorbate 80, and 9mM histidine-acetate buffer pH5.4;

(7) 30mg/ml anti-TIGIT antibody, 75mg/ml sucrose, 1.4mg/ml polysorbate 80, and 8mM histidine-acetate buffer pH5.3;

(8) 70mg/ml anti-TIGIT antibody, 82mg/ml sucrose, 0.8mg/ml polysorbate 80, and 11mM histidine-acetate buffer ph5.2;

(9) 50mg/ml anti-TIGIT antibody, 77mg/ml sucrose, 1.3mg/ml polysorbate 80, and 12mM histidine-acetate buffer pH5.1;

(10) 50mg/ml anti-TIGIT antibody, 80mg/ml sucrose, 0.5mg/ml polysorbate 80, and 13mM histidine-acetate buffer pH5.2;

(11) 52mg/ml anti-TIGIT antibody, 84mg/ml sucrose, 1.1mg/ml polysorbate 80, and 14mM histidine-acetate buffer ph5.5;

(12) 48mg/ml anti-TIGIT antibody, 79mg/ml sucrose, 0.7mg/ml polysorbate 80, and 12mM histidine-hcl buffer pH6.1;

(13) 49mg/ml anti-TIGIT antibody, 74mg/ml sucrose, 0.3mg/ml polysorbate 80, and 15mM histidine-histidine hydrochloride buffer pH6.2;

(14) 45mg/ml anti-TIGIT antibody, 80mg/ml sucrose, 0.9mg/ml polysorbate 80, and 10mM histidine-histidine hydrochloride buffer pH6.3;

(15) 65mg/ml anti-TIGIT antibody, 80mg/ml sucrose, 0.4mg/ml polysorbate 80, and 10mM histidine-histidine hydrochloride buffer pH6.4;

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.

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Arg Thr Pro Gly Lys

370

<210> 5

<211> 119

<212> PRT

<213> mice (Mus musculus)

<220>

<221> DOMAIN

<223> m1707-HCVR sequence

<400> 5

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Ser Val Thr Val Ser Ser

115

<210> 6

<211> 107

<212> PRT

<213> mice (Mus musculus)

<220>

<221> DOMAIN

<223> m1707-LCVR sequence

<400> 6

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 7

<211> 119

<212> PRT

<213> mice (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> mice (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> mice (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> mice (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> mice (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> mice (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> mice (Mus musculus)

<220>

<221> DOMAIN

<223> m1711-HCVR sequence

<400> 13

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gly Gln Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 14

<211> 113

<212> PRT

<213> mice (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-HCDR1 sequence

<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 LCDR1 sequence

<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 HCDR1 sequence

<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 LCDR1 sequence

<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 HCDR1 sequence

<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 LCDR1 sequence

<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-HCDR1 sequence

<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-LCDR1 sequence

<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-HCDR1 sequence

<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-LCDR1 sequence

<400> 42

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

1 5 10 15

Ala

<210> 43

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1711-LCDR2 sequence

<400> 43

Trp Thr Ser Thr Arg Glu Ser

1 5

<210> 44

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1711-LCDR3 sequence

<400> 44

Gln Gln Tyr Tyr Ser Tyr Pro Tyr Thr

1 5

<210> 45

<211> 119

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

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

<400> 45

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 46

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

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

<400> 46

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 47

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> h1707-L2 sequence

<400> 47

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 48

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> h1707-L3 sequence

<400> 48

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 49

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> h1707-L4 sequence

<400> 49

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 50

<211> 119

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> H1707-H2 sequence

<400> 50

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 51

<211> 119

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

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

<400> 51

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 52

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

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

<400> 52

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Pro Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 53

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> h1708-L2 sequence

<400> 53

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Pro Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 54

<211> 119

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> H1708-H2 sequence

<400> 54

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 55

<211> 119

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> H1708-H3 sequence

<400> 55

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 56

<211> 120

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

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

<400> 56

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 57

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

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

<400> 57

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 58

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> h1709-L2 sequence

<400> 58

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 59

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> h1709-L3 sequence

<400> 59

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 60

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> h1709-L4 sequence

<400> 60

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 61

<211> 120

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> H1709-H2 sequence

<400> 61

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 62

<211> 120

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> H1709-H3 sequence

<400> 62

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 63

<211> 120

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> H1709-H4 sequence

<400> 63

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 64

<211> 119

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

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

<400> 64

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 65

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

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

<400> 65

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Pro Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 66

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> h1710-L2 sequence

<400> 66

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Pro Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 67

<211> 119

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> H1710-H2 sequence

<400> 67

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 68

<211> 119

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> H1710-H3 sequence

<400> 68

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 69

<211> 119

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> H1710-H4 sequence

<400> 69

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 70

<211> 119

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> H1710-H5 sequence

<400> 70

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 71

<211> 120

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

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

<400> 71

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gly Gln Gly Thr Leu Val Thr Val

115 120

<210> 72

<211> 113

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

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

<400> 72

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Lys

<210> 73

<211> 113

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> h1711-L2 sequence

<400> 73

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Lys

<210> 74

<211> 113

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> h1711-L3 sequence

<400> 74

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Lys

<210> 75

<211> 122

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> H1711-H2 sequence

<400> 75

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 76

<211> 122

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> H1711-H3 sequence

<400> 76

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 77

<211> 122

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> H1711-H4 sequence

<400> 77

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 78

<211> 327

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

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

<400> 78

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210> 79

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> kappa light chain constant region

<400> 79

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 80

<211> 130

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 22G2-H3Q VH sequence

<400> 80

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Ser Ser

130

<210> 81

<211> 109

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 22G2-H3Q VL sequence

<400> 81

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105

<210> 82

<211> 214

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

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

<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 sequence of light chain

<400> 84

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 85

<211> 446

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

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

<400> 85

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

<210> 86

<211> 214

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

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

<400> 86

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

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

115 120 125

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

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 87

<211> 447

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

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

<400> 87

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

<210> 88

<211> 214

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

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

<400> 88

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Pro Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

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

115 120 125

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

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 89

<211> 446

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

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

<400> 89

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

<210> 90

<211> 220

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

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

<400> 90

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

<210> 91

<211> 449

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> DOMAIN

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

<400> 91

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Lys

Claims

1. 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 histidine salt buffer, succinate buffer, acetate buffer, and phosphate buffer, the pH of the buffer being 5.0 to 7.0;

the anti-TIGIT antibody or antigen-binding fragment thereof that specifically binds to human TIGIT comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 21. 22 and 23, and HCDR1, HCDR2, and HCDR3, the light chain variable region comprising amino acid sequences as set forth in SEQ ID NOs: 24. 25 and 26 amino acid sequences of LCDR1, LCDR2 and LCDR3.

2. The pharmaceutical composition of claim 1, wherein the pH of the buffer is 5.0 to 6.0.

3. The pharmaceutical composition of claim 1, 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.

4. The pharmaceutical composition of claim 3, wherein the buffer is selected from the group consisting of an acetic acid-sodium acetate buffer at a pH of 5.0 to 5.7, a succinic acid-sodium succinate buffer at a pH of 5.0 to 6.0, a histidine-hydrochloric acid buffer at a pH of 5.5 to 6.0, or a histidine-acetic acid buffer at a pH of 5.0 to 6.0.

5. The pharmaceutical composition of claim 3, wherein the buffer is histidine-acetate buffer at pH 5.5.

6. The pharmaceutical composition of claim 1, wherein the buffer concentration is 5mM to 30mM.

7. The pharmaceutical composition of claim 1, wherein the buffer concentration is 5mM to 15mM.

8. The pharmaceutical composition of claim 1, wherein the buffer concentration is 10mM.

9. The pharmaceutical composition of claim 1, wherein the anti-TIGIT antibody or antigen-binding fragment thereof is at a concentration of 1mg/ml to 100mg/ml.

10. The pharmaceutical composition of claim 1, wherein the anti-TIGIT antibody or antigen-binding fragment thereof is at a concentration of 40mg/ml to 60mg/ml.

11. The pharmaceutical composition of claim 1, wherein the anti-TIGIT antibody or antigen-binding fragment thereof is at a concentration of 50mg/ml.

12. The pharmaceutical composition of claim 1, further comprising a sugar.

13. The pharmaceutical composition of claim 12, wherein the sugar is a disaccharide.

14. The pharmaceutical composition of claim 12, wherein the sugar is sucrose.

15. The pharmaceutical composition of claim 12, wherein the sugar concentration is 65mg/ml to 100mg/ml.

16. The pharmaceutical composition of claim 12, wherein the sugar concentration is 75mg/ml to 85mg/ml.

17. The pharmaceutical composition of claim 12, wherein the sugar concentration is 80mg/ml.

18. The pharmaceutical composition of claim 1, further comprising a surfactant.

19. The pharmaceutical composition of claim 18, wherein the surfactant is a polysorbate.

20. The pharmaceutical composition of claim 18, wherein the surfactant is polysorbate 80.

21. The pharmaceutical composition of claim 18, wherein the concentration of the surfactant is 0.05mg/ml to 1.5mg/ml.

22. The pharmaceutical composition of claim 18, wherein the concentration of the surfactant is 0.1mg/ml to 0.8mg/ml.

23. The pharmaceutical composition of claim 18, wherein the concentration of the surfactant is 0.4mg/ml.

24. The pharmaceutical composition according to claim 1, comprising:

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

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

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

d) Polysorbate 80 at a concentration of 0.05mg/ml to 1.5 mg/ml.

25. The pharmaceutical composition according to claim 1, comprising:

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

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

c1 Sucrose at a concentration of 75mg/ml to 85mg/ml, and

d1 Polysorbate 80 at a concentration of 0.1mg/ml to 0.8 mg/ml.

26. The pharmaceutical composition according to claim 1, comprising:

10mM histidine-acetic acid buffer pH 5.5, at a concentration of 50mg/ml anti-TIGIT antibody or antigen binding fragment thereof specifically binding to human TIGIT, at a concentration of 80mg/ml sucrose, and at a concentration of 0.4mg/ml polysorbate 80.

27. The pharmaceutical composition of claim 1, wherein the antibody or antigen-binding fragment thereof that specifically binds human TIGIT comprises a heavy chain variable region as set forth in SEQ ID NOs 51, 54, or 55, and/or a light chain variable region as set forth in SEQ ID NOs 52 or 53.

28. The pharmaceutical composition of claim 1, wherein the antibody or antigen-binding fragment thereof that specifically binds human TIGIT comprises the amino acid sequence as set forth in SEQ ID NO:51 and a heavy chain variable region as set forth in SEQ ID NO: 53.

29. The pharmaceutical composition of claim 1, wherein the antibody that specifically binds human TIGIT is a full length antibody comprising a human antibody constant region.

30. The pharmaceutical composition of claim 29, wherein the antibody that specifically binds human TIGIT is a full length antibody comprising a human antibody heavy chain constant region as set forth in SEQ ID No. 78 and/or a human antibody light chain constant region as set forth in SEQ ID No. 79.

31. The pharmaceutical composition of claim 1, wherein the antibody that specifically binds human TIGIT is a full length antibody comprising the heavy chain as set forth in SEQ ID No. 85 and/or the light chain as set forth in SEQ ID No. 84.

32. A method of preparing the pharmaceutical composition of any one of claims 1 to 31, the method comprising the step of mixing an anti-TIGIT antibody or antigen-binding fragment thereof with a buffer; the buffer is selected from histidine salt buffer, succinate buffer, acetate buffer and phosphate buffer, and the pH of the buffer is 5.0 to 7.0.

33. The method of claim 32, wherein the pH of the buffer is 5.0 to 6.0.

34. A lyophilized formulation comprising an anti-TIGIT antibody or antigen-binding fragment thereof, the lyophilized formulation obtained by freeze-drying the pharmaceutical composition of any one of claims 1-31.

35. A lyophilized formulation comprising an anti-TIGIT antibody or antigen-binding fragment thereof, which is reconstituted to form the pharmaceutical composition of any one of claims 1-31.

36. A reconstituted solution comprising an anti-TIGIT antibody or antigen-binding fragment thereof, the reconstituted solution being obtained by reconstitution of the lyophilized formulation of claim 34 or 35.

37. An article of manufacture comprising a container containing a pharmaceutical composition according to any one of claims 1 to 31, or a lyophilized formulation according to claim 34 or 35, or a reconstituted solution according to claim 36.

38. Use of a pharmaceutical composition according to any one of claims 1 to 31, or a lyophilized formulation according to claim 34 or 35, or a reconstituted solution according to claim 36, or an article according to claim 37, for the manufacture of a medicament for the treatment of tumors associated with human TIGIT.

39. The use of claim 38, wherein the tumor is selected from the group consisting of: head and neck cancer, central nervous system cancer, neuroendocrine tumors, malignant pleural mesothelioma, lung cancer, breast cancer, liver and gall cancer, pancreatic cancer, gastrointestinal cancer, kidney cancer, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, leukemia, lymphoma, bone cancer, myeloma, squamous cell carcinoma, and systemic light chain amyloidosis.

40. The use according to claim 39, wherein the lymphoma is selected from the group consisting of: hodgkin's lymphoma, non-hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, T-cell/tissue cell enriched large B-cell lymphoma, and lymphoplasmacytic lymphoma, the lung cancer being selected from the group consisting of: non-small cell lung cancer and small cell lung cancer, said leukemia being selected from the group consisting of: chronic myeloid leukemia, acute myeloid leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, and myeloid leukemia.

41. The use according to claim 40, wherein the lymphoma is selected from CD155 positive or PVR positive tumors.

42. The use of claim 38, wherein the tumor is selected from the group consisting of: squamous cell carcinoma of the head and neck, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, laryngeal carcinoma, nasopharyngeal carcinoma, esophageal carcinoma, thyroid carcinoma, liver cancer, stomach cancer, intestinal cancer, colorectal cancer, clear cell renal cell carcinoma, melanoma, chondrosarcoma, myelodysplastic syndrome, myeloproliferative neoplasm, ewing's sarcoma, and meckerr cell carcinoma.

43. The use according to claim 42, wherein the liver cancer is hepatoma; the colorectal cancer is colon cancer; the myeloproliferative neoplasm is multiple myeloma.