CN111196852A

CN111196852A - anti-TIGIT antibodies and uses thereof

Info

Publication number: CN111196852A
Application number: CN201811366907.4A
Authority: CN
Inventors: 肖亮; 周利; 刘登念; 孙见宇; 胡江江; 刘雪梅; 喻海旻; 郑云程; 李强; 薛彤彤
Original assignee: Anyuan Pharmaceutical Technology Shanghai Co ltd; Sichuan Kelun Biotech Biopharmaceutical Co Ltd
Current assignee: Anyuan Pharmaceutical Technology Shanghai Co ltd; Sichuan Kelun Biotech Biopharmaceutical Co Ltd
Priority date: 2018-11-16
Filing date: 2018-11-16
Publication date: 2020-05-26
Also published as: WO2020098734A1

Abstract

The present invention relates to the field of disease therapy and immunology, and in particular, the present invention relates to antibodies or antigen binding fragments thereof against TIGIT, nucleic acid molecules encoding them, and methods for making them. The anti-TIGIT antibody or the antigen binding fragment thereof has high specificity and high affinity to TIGIT, can effectively block the binding of the TIGIT and a ligand thereof, and inhibits and/or blocks the intracellular signal transduction mediated by the binding of the TIGIT to the ligand thereof. Thus, the invention further relates to a pharmaceutical composition comprising said antibody or antigen binding fragment thereof, and to the use thereof for the preparation of a medicament for increasing the activity of an immune cell, for enhancing an immune response, or for the prevention and/or treatment of a tumor, an infection or an infectious disease.

Description

anti-TIGIT antibodies and uses thereof

Technical Field

The present invention is in the field of therapeutic monoclonal antibodies, and more particularly, the present invention relates to an antibody directed against a T cell immunoreceptor with Ig and ITIM domains (TIGIT); it also relates to the use of said antibodies in the treatment of diseases, including but not limited to cancer, infections, etc.

Background

TIGIT (a T cell immunoreceptor containing Ig and ITIM domains) belongs to a member of the immunoglobulin superfamily, also known as Wucam, Vstm3 or Vsig9, with an extracellular immunoglobulin domain, a type I transmembrane region, and two Immunoreceptor Tyrosine Inhibitory Motifs (ITIMs). TIGIT is mainly distributed in regulatory T cells (Tregs), activated T cells, natural killer cells (NK) and the like, is a co-inhibitory receptor protein and can form a co-stimulation network with positive protein CD226(Dnam-1) on T cells, ligand CD155(Pvr or Necl-5) expressed on APC and CD112(Pvrl-2 or Nectin 2). Wherein TIGIT competes with CD226 for binding to CD155 and CD112, and TIGIT binds its ligand with higher affinity than CD 226. Ligation of TIGIT to CD155 or CD112 is mediated by its cytoplasmic ITIM or ITT-like motif, recruiting the phosphatase SHIP-1 to the TIGIT tail triggering inhibitory signaling. In addition, the ITIM domain is also responsible for the inhibitory ability of mouse TIGIT. TIGIT/CD155 binding inhibits NK cytotoxicity, attenuates T cell responses and anti-tumor responses, while the CD226/CD155 binding effect is reversed.

TIGIT may "shut down" the immune response of the body by a variety of mechanisms. Initial studies have shown that TIGIT indirectly suppresses immune responses by triggering the CD155 cytoplasmic tail on Dendritic Cells (DCs), thereby blocking DC maturation and inducing the production of the immunosuppressive cytokine IL-10. Further studies have shown that TIGIT directly blocks naive T cell (Tn) activation, proliferation and acquisition of effector function by targeting signaling pathway molecules of T cell antigen receptors (TCRs) and is capable of inhibiting CD4⁺T cell proliferation and production of inflammatory cytokines. Recent studies have shown that TIGIT enhances Tregs stability and its suppressive function on gamma interferon (IFN- γ) -producing T cell proliferation, selectively suppressing helper T cell 1(Th1) and helper T cell 17(Th17) responses-the primary responses driving inflammation in autoimmune tissues. In addition, by inhibiting the cytolytic activity of NK cells, TIGIT reduced the production of NK cell cytokine IFN- γ, reducing its cytotoxicity. Therefore, blocking TIGIT signaling has the effect of enhancing autoimmune responseThe function of (1). Other related studies have also found that enhanced or activated binding to Fc γ can mediate TIGIT through tumor site Antibody Dependent Cellular Cytotoxicity (ADCC) or phagocytosis (ADCP)⁺Tregs selective depletion, and TIGIT blocking antibodies can increase CD8⁺T cells, tumor infiltrating lymphocytes (CD 8)⁺TILs) and cytokine-induced killer Cell (CIK) cell proliferation and cytokine production such as IFN- γ. In many clinical tumor environments, TIGIT is at CD8⁺The high surface expression of T cells, Tregs, TILs or NK cells, including melanoma, non-small cell lung cancer, colon cancer, endometrial cancer, breast cancer, renal clear cell carcinoma and other cancers, indicates that the anti-TIGIT antibody has the potential for treating various tumors.

Multiple researches show that TIGIT is related to various diseases such as cancer, autoimmunity and infection, and is expected to become a specific target for treating various diseases. Blocking of CD 155-highly expressing melanoma CIK cells with Anti-TIGIT antibody significantly increased IFN-. gamma.production and far higher positive cell killing than control (Baofu ZH et al, cancer Immunol. Immunother.,65:305-14, 2016). Experimental Allergic Encephalomyelitis (EAE) series of experiments showed elevated levels of encephalitogenic T cells and proinflammatory cytokines IL-6, IFN- γ and IL-17 in TIGIT deficient mice (Joller N et al, J.Immunol.,186: 1338-1342, 2011) and TIGIT antibody blockade could lead to disease exacerbation by enhanced proinflammatory T cell responses (Levin SD et al, Eur.J.Immunol.,41: 902-915, 2011). Furthermore, TIGIT interacts with effector CD8 during infection by Human Immunodeficiency Virus (HIV) or Simian Immunodeficiency Virus (SIV)⁺Programmed death receptor-1 (PD-1) co-expression on T cells, increased expression of TIGIT associated with disease progression (Chew GM et al, PLoSPathog.,12: e1005349,2016); also, TIGIT co-blocking antibody with PD-1 ligand (PD-L1) was able to restore CD8 depleted induced in chronic lymphocytic choriomeningitis virus (LCMV) infection⁺Production of T cell cytokines (Johnston RJ et al, Cancer cell.,26: 923-. Recent research shows that the high-affinity Anti-TIGIT antibody can reverse the NK cell exhaustion of a T cell-deficient mouse but cannot reverse the T cell exhaustion of the NK cell-deficient mouse, and the result shows that the NK cell can independently kill a targetCells, but T cells, must rely on NK cells for their immune function (Zhang Q et al, nat. Immunol.,2018Jun18.doi:10.1038/s41590-018-0132-0.), which is consistent with the study that NK cells express the specific immune protein FLT3LG, recruit SDCs (DC cells with stimulation) to stimulate dormant T cells and thus exert anti-tumor effect (BarryKC et al, nat. Med.2018Jun 25.doi:10.1038/s 41591-018-0085-8.). The reversal of the T cell depletion of the T cell deficient mice by the TIGIT antibody can effectively inhibit the tumor growth of the mice, so that the mice have nearly lifelong strong anti-tumor immunological memory, and the patients generate stronger response to the PD-1 antibody due to the incapability of reversing the T cell depletion of the NK cell deficient mice, so that the long-term immune response and the higher overall survival rate are caused, which indicates that the anti-TIGIT monoclonal antibody has the potential application of treating various related diseases by being used alone or being used together with an antibody (such as the PD-1 antibody) which plays a therapeutic role depending on the T cell immune effect.

Several TIGIT antibodies are currently in clinical trials including MTIG7192A from Genentech, BMS 986207, MK-7684 from MSD, COM-701 developed by Bayer in cooperation with Comugen, and AB154 from Arcus Biosciences. However, no relevant drugs are yet on the market.

Therefore, the development of anti-TIGIT antibodies with higher specificity, lower toxic and side effects and better clinical efficacy is urgently and necessarily required, which will provide more medication options for patients with cancer or infectious diseases.

Disclosure of Invention

Antibody molecules that bind to TIGIT with high affinity and specificity are disclosed in the present invention. Nucleic acid molecules encoding the antibody molecules, expression vectors, host cells, and methods for making the antibody molecules are also provided. Also provided are multispecific or bispecific antibodies, pharmaceutical compositions, and immunogenic compositions comprising the antibody molecules. Additionally, the use of the anti-TIGIT antibody molecules disclosed herein (alone or in combination with other active agents or therapeutic modalities) in the preparation of a medicament for the treatment and/or prevention of tumors, infections and infectious diseases is also provided.

In one aspect, the invention provides an antibody or antigen-binding fragment thereof capable of specifically binding TIGIT, comprising:

(a) the following CDRs of 3 heavy chain variable regions (VH):

(i) CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 1, or a sequence having substitution, deletion, or addition of one or several amino acids (e.g., substitution, deletion, or addition of 1, 2, or 3 amino acids) as compared with the sequence of CDR-H1 contained in the VH;

(ii) CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 1, or a sequence having substitution, deletion, or addition of one or several amino acids (e.g., substitution, deletion, or addition of 1, 2, or 3 amino acids) as compared with the sequence of CDR-H2 contained in the VH; and

(iii) CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 1, or a sequence having substitution, deletion, or addition of one or several amino acids (e.g., substitution, deletion, or addition of 1, 2, or 3 amino acids) as compared with the sequence of CDR-H3 contained in the VH;

and/or the presence of a gas in the gas,

(b) the CDRs of the following 3 light chain variable regions (VL):

(iv) CDR-L1 having the amino acid sequence as set forth in SEQ ID NO: 2, or a sequence having substitution, deletion, or addition of one or several amino acids (e.g., substitution, deletion, or addition of 1, 2, or 3 amino acids) as compared with the sequence of CDR-L1 contained in the VL;

(v) CDR-L2 having the amino acid sequence as set forth in SEQ ID NO: 2, or a sequence having substitution, deletion, or addition of one or several amino acids (e.g., substitution, deletion, or addition of 1, 2, or 3 amino acids) as compared with the sequence of CDR-L2 contained in the VL; and

(vi) CDR-L3 having the amino acid sequence as set forth in SEQ ID NO: 2, or a sequence having substitution, deletion, or addition of one or several amino acids (e.g., substitution, deletion, or addition of 1, 2, or 3 amino acids) as compared with the sequence of CDR-L3 contained in the VL.

In certain preferred embodiments, the substitution recited in any one of (i) - (vi) is a conservative substitution.

In certain preferred embodiments, the CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH), and/or the CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region (VL) are defined by the Kabat, Chothia or IMGT numbering system.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:

(a) 3 CDRs contained in a heavy chain variable region (VH) selected from:

as shown in SEQ ID NO: 1. SEQ ID NO: 31. SEQ ID NO: 35. SEQ ID NO: 39 and SEQ ID NO: 43, VH set forth in any one of;

and/or

(b) 3 CDRs contained in a light chain variable region (VL) selected from:

as shown in SEQ ID NO: 2. SEQ ID NO: 32. SEQ ID NO: 36. SEQ ID NO: 40 and SEQ ID NO: 44 of any one of VL.

In certain preferred embodiments, the 3 CDRs contained in the heavy chain variable region (VH) and/or the 3 CDRs contained in the light chain variable region (VL) are defined by the Kabat, Chothia or IMGT numbering system.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises: as shown in SEQ ID NO: 1 (VH) comprises 3 CDRs; and/or, as shown in SEQ ID NO: 2 (VL) comprises 3 CDRs; wherein the 3 CDRs contained in the heavy chain variable region (VH) and the 3 CDRs contained in the light chain variable region (VL) are determined by the IMGT numbering system.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises:

(a) the following 3 heavy chain variable region (VH) CDRs:

(i) CDR-H1, consisting of the sequence: SEQ ID NO:5, or a variant of SEQ ID NO:5 compared with a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids),

(ii) CDR-H2, consisting of the sequence: SEQ ID NO: 6, or a variant of SEQ ID NO: 6 to a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids), and

(iii) CDR-H3, consisting of the sequence: SEQ ID NO: 7, or a variant of SEQ ID NO: 7 with one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions);

and/or the presence of a gas in the gas,

(b) the following 3 light chain variable region (VL) CDRs:

(iv) CDR-L1, consisting of the following sequence: SEQ ID NO: 8, or a variant of SEQ ID NO: 8 compared with a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids),

(v) CDR-L2, consisting of the following sequence: SEQ ID NO: 9, or a variant of SEQ ID NO: 9 has a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids), and

(vi) CDR-L3, consisting of the following sequence: SEQ ID NO: 10, or a variant of SEQ ID NO: 10 with one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions);

wherein the heavy chain variable region (VH) CDR and the light chain variable region (VL) CDR are defined by the IMGT numbering system.

In certain preferred embodiments, the VH of an antibody or antigen-binding fragment thereof of the invention comprises: as shown in SEQ ID NO:5 CDR-H1; as shown in SEQ ID NO: 6 CDR-H2; and, as shown in SEQ ID NO: 7 CDR-H3; and, the VL of the antibody or antigen-binding fragment thereof comprises: as shown in SEQ ID NO: 8 CDR-L1; as shown in SEQ ID NO: 9 CDR-L2; and, as shown in SEQ ID NO: 10, CDR-L3.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises: as shown in SEQ ID NO: 1 (VH) comprises 3 CDRs; and/or, as shown in SEQ ID NO: 2 (VL) comprises 3 CDRs;

wherein the 3 CDRs contained in the heavy chain variable region (VH) and the 3 CDRs contained in the light chain variable region (VL) are defined by the Chothia numbering system.

(a) the following 3 heavy chain variable region (VH) CDRs:

(i) CDR-H1, consisting of the sequence: SEQ ID NO: 27, or a variant of SEQ ID NO: 27 compared to a sequence having a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids),

(ii) CDR-H2, consisting of the sequence: SEQ ID NO: 28, or a variant of SEQ ID NO: 28 is compared with a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids), and

(iii) CDR-H3, consisting of the sequence: SEQ ID NO: 19, or a variant of SEQ ID NO: 19 with one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions);

and/or the presence of a gas in the gas,

(b) the following 3 light chain variable region (VL) CDRs:

(iv) CDR-L1, consisting of the following sequence: SEQ ID NO: 20, or a variant of SEQ ID NO: 20 compared with a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids),

(v) CDR-L2, consisting of the following sequence: SEQ ID NO: 21, or a variant of SEQ ID NO: 21 has a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids), and

wherein the heavy chain variable region (VH) CDR and the light chain variable region (VL) CDR are defined by the Chothia numbering system.

In certain preferred embodiments, the VH of an antibody or antigen-binding fragment thereof of the invention comprises: as shown in SEQ ID NO: CDR-H1 shown in FIG. 27; as shown in SEQ ID NO: 28 CDR-H2; and, as shown in SEQ ID NO: 19 CDR-H3; and, the VL of the antibody or antigen-binding fragment thereof comprises: as shown in SEQ ID NO: 20 CDR-L1; as shown in SEQ ID NO: 21 CDR-L2; and, as shown in SEQ ID NO: 10, CDR-L3.

wherein the 3 CDRs contained in the heavy chain variable region (VH) and the 3 CDRs contained in the light chain variable region (VL) are defined by the Kabat numbering system.

(a) the following 3 heavy chain variable region (VH) CDRs:

(i) CDR-H1, consisting of the sequence: SEQ ID NO: 17, or a variant of SEQ ID NO: 17 compared with a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids),

(ii) CDR-H2, consisting of the sequence: SEQ ID NO: 18, or a variant of SEQ ID NO: 18 to a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids), and

and/or the presence of a gas in the gas,

(b) the following 3 light chain variable region (VL) CDRs:

wherein the heavy chain variable region (VH) CDRs and the light chain variable region (VL) CDRs are defined by the Kabat numbering system.

In certain preferred embodiments, the VH of an antibody or antigen-binding fragment thereof of the invention comprises: (a) such as SEQ ID NO: 17 CDR-H1; as shown in SEQ ID NO: 18 CDR-H2; and, as shown in SEQ ID NO: 19 CDR-H3; and, the VL of the antibody or antigen-binding fragment thereof comprises: as shown in SEQ ID NO: 20 CDR-L1; as shown in SEQ ID NO: 21 CDR-L2; and, as shown in SEQ ID NO: 10, CDR-L3.

In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the present invention further comprise a Framework Region (FR) derived from a mammalian (e.g., murine or human) immunoglobulin.

In certain preferred embodiments, the VH of an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) Framework Region (FR) derived from a murine immunoglobulin, and/or the VL of said antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) Framework Region (FR) derived from a murine immunoglobulin.

In certain preferred embodiments, the VH of an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) Framework Region (FR) derived from a human immunoglobulin, and/or the VL of said antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) Framework Region (FR) derived from a human immunoglobulin. In such embodiments, the heavy chain variable region FR and/or the light chain variable region FR of the antibody or antigen binding fragment thereof of the invention may comprise one or more non-human (e.g., murine) amino acid residues, e.g., the heavy chain framework region FR and/or the light chain framework region FR may comprise one or more amino acid back mutations in which there is a corresponding murine amino acid residue.

(a) a heavy chain framework region of a human immunoglobulin or a variant thereof having conservative substitutions of up to 20 amino acids (e.g., conservative substitutions of up to 15, up to 10, or up to 5 amino acids; e.g., conservative substitutions of 1, 2, 3, 4, or 5 amino acids) in comparison to the sequence from which it is derived; and/or

(b) A light chain framework region of a human immunoglobulin or a variant thereof, which variant has conservative substitutions of up to 20 amino acids (e.g., conservative substitutions of up to 15, up to 10, or up to 5 amino acids; e.g., conservative substitutions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence from which it is derived.

Thus, in certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the present invention are humanized. In certain preferred embodiments, the antibodies or antigen binding fragments thereof of the invention are humanized to a degree of at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%.

(a) a heavy chain variable region (VH) comprising an amino acid sequence selected from:

(i) SEQ ID NOs: 1. 31, 35, 39, 43;

(ii) and SEQ ID NOs: 1. 31, 35, 39 or 43 with one or more amino acid substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions); or

(iii) And SEQ ID NOs: 1. 31, 35, 39, 43, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity;

and/or the presence of a gas in the gas,

(b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

(iv) SEQ ID NOs: 2. 32, 36, 40, 44;

(v) and SEQ ID NOs: 2. 32, 36, 40, 44, having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) as compared to the sequence set forth in any one of seq id no; or

(vi) And SEQ ID NOs: 2. 32, 36, 40, 44, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

In certain preferred embodiments, the substitutions recited in (ii) or (v) are conservative substitutions.

(i) SEQ ID NO: 1;

(ii) and SEQ ID NO: 1 compared to a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions); or

(iii) And SEQ ID NO: 1, has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity;

and

(iv) SEQ ID NO: 2;

(v) and SEQ ID NO: 2 compared to a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions); or

(vi) And SEQ ID NO: 2, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

(i) SEQ ID NO: 31;

(ii) and SEQ ID NO: 31 with one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence set forth in seq id no; or

(iii) And SEQ ID NO: 31, having a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%;

and

(iv) SEQ ID NO: 32;

(v) and SEQ ID NO: 32 compared to a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions); or

(vi) And SEQ ID NO: 32, has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

(i) SEQ ID NO: 35;

(ii) and SEQ ID NO: 35 compared to a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions); or

(iii) And SEQ ID NO: 35, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity;

and

(iv) SEQ ID NO: 36;

(v) and SEQ ID NO: 36 with one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in (a); or

(vi) And SEQ ID NO: 36, has a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%.

(i) SEQ ID NO: 39;

(ii) and SEQ ID NO: 39 with one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence set forth in seq id no; or

(iii) And SEQ ID NO: 39, has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity;

and

(iv) SEQ ID NO: 40;

(v) and SEQ ID NO: 40 with one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence set forth in seq id no; or

(vi) And SEQ ID NO: 40, has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

(i) SEQ ID NO: 43;

(ii) and SEQ ID NO: 43 with one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in (b); or

(iii) And SEQ ID NO: 43, has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity;

and

(iv) SEQ ID NO: 44, or a sequence shown in SEQ ID NO;

(v) and SEQ ID NO: 44 compared to a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions); or

(vi) And SEQ ID NO: 44, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

(1) has the sequence shown in SEQ ID NO: 1 and VH having the sequence shown as SEQ ID NO: 2, VL of the sequence set forth in seq id no;

(2) has the sequence shown in SEQ ID NO: 31 and a VH having the sequence shown as SEQ ID NO: 32, VL of the sequence set forth in seq id no;

(3) has the sequence shown in SEQ ID NO: 35 and a VH having the sequence shown as SEQ ID NO: 36, VL of the sequence set forth in seq id no;

(4) has the sequence shown in SEQ ID NO: 39 and VH having the sequence shown as SEQ ID NO: 40, VL of the sequence set forth in seq id no; or

(5) Has the sequence shown in SEQ ID NO: 43 and a VH having the sequence shown as SEQ ID NO: 44, VL of the sequence set forth in seq id no.

In another aspect, the invention provides an antibody or antigen-binding fragment thereof capable of specifically binding TIGIT, comprising:

(a) the following CDRs of 3 heavy chain variable regions (VH):

(i) CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 3, or a sequence having substitution, deletion, or addition of one or several amino acids (e.g., substitution, deletion, or addition of 1, 2, or 3 amino acids) as compared with the sequence of CDR-H1 contained in the VH;

(ii) CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 3, or a sequence having substitution, deletion, or addition of one or several amino acids (e.g., substitution, deletion, or addition of 1, 2, or 3 amino acids) as compared with the sequence of CDR-H2 contained in the VH; and

(iii) CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 3, or a sequence having substitution, deletion, or addition of one or several amino acids (e.g., substitution, deletion, or addition of 1, 2, or 3 amino acids) as compared with the sequence of CDR-H3 contained in the VH;

and/or the presence of a gas in the gas,

(b) the CDRs of the following 3 light chain variable regions (VL):

(iv) CDR-L1 having the amino acid sequence as set forth in SEQ ID NO:4, or a sequence having substitution, deletion, or addition of one or several amino acids (e.g., substitution, deletion, or addition of 1, 2, or 3 amino acids) as compared with the sequence of CDR-L1 contained in the VL;

(v) CDR-L2 having the amino acid sequence as set forth in SEQ ID NO:4, or a sequence having substitution, deletion, or addition of one or several amino acids (e.g., substitution, deletion, or addition of 1, 2, or 3 amino acids) as compared with the sequence of CDR-L2 contained in the VL; and

(vi) CDR-L3 having the amino acid sequence as set forth in SEQ ID NO:4, or a sequence having substitution, deletion, or addition of one or several amino acids (e.g., substitution, deletion, or addition of 1, 2, or 3 amino acids) as compared with the sequence of CDR-L3 contained in the VL.

(a) 3 CDRs contained in a heavy chain variable region (VH) selected from:

as shown in SEQ ID NO: 3. SEQ ID NO: 33. SEQ ID NO: 37 and SEQ ID NO: 41, VH as set forth in any one of claims 41;

and/or the presence of a gas in the gas,

(b) 3 CDRs contained in a light chain variable region (VL) selected from:

as shown in SEQ ID NO: 4. SEQ ID NO: 34. SEQ ID NO: 38 and SEQ ID NO: 42 VL of any one of.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises: as shown in SEQ ID NO: 3 (3) of the 3 CDRs contained in the heavy chain variable region (VH); and/or, as shown in SEQ ID NO:4 (VL) comprising 3 CDRs; wherein the 3 CDRs contained in the heavy chain variable region (VH) and the 3 CDRs contained in the light chain variable region (VL) are determined by the IMGT numbering system.

(a) the following CDRs of 3 heavy chain variable regions (VH):

(i) CDR-H1, consisting of the sequence: SEQ ID NO: 11, or a variant of SEQ ID NO: 11 compared with a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids),

(ii) CDR-H2, consisting of the sequence: SEQ ID NO: 12, or a variant of SEQ ID NO: 12 to a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions), and

(iii) CDR-H3, consisting of the sequence: SEQ ID NO: 13, or a variant of SEQ ID NO: 13 with one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions);

and/or the presence of a gas in the gas,

(b) the CDRs of the following 3 light chain variable regions (VL):

(iv) CDR-L1, consisting of the following sequence: SEQ ID NO: 14, or a sequence identical to SEQ ID NO: 14 with one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions),

(v) CDR-L2, consisting of the following sequence: SEQ ID NO: 15, or a variant of SEQ ID NO: 15 to a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids), and

(vi) CDR-L3, consisting of the following sequence: SEQ ID NO: 16, or a variant of SEQ ID NO: 16 with one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions);

In certain preferred embodiments, the VH of an antibody or antigen-binding fragment thereof of the invention comprises: as shown in SEQ ID NO: 11 CDR-H1; as shown in SEQ ID NO: CDR-H2 shown in FIG. 12; and, as shown in SEQ ID NO: 13 CDR-H3; and, the VL of the antibody or antigen-binding fragment thereof comprises: as shown in SEQ ID NO: 14 CDR-L1; as shown in SEQ ID NO: 15 CDR-L2; and, as shown in SEQ ID NO: 16, CDR-L3.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises: as shown in SEQ ID NO: 3 (3) of the 3 CDRs contained in the heavy chain variable region (VH); and/or, as shown in SEQ ID NO:4 (VL) comprising 3 CDRs;

(a) the following 3 heavy chain variable region (VH) CDRs:

(i) CDR-H1, consisting of the sequence: SEQ ID NO: 29, or a variant of SEQ ID NO: 29 compared to a sequence having a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids),

(ii) CDR-H2, consisting of the sequence: SEQ ID NO: 30, or a variant of SEQ ID NO: 30 to a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids), and

(iii) CDR-H3, consisting of the sequence: SEQ ID NO: 24, or a sequence identical to SEQ ID NO: 24 compared to a sequence having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions);

and/or the presence of a gas in the gas,

(b) the following 3 light chain variable region (VL) CDRs:

(iv) CDR-L1, consisting of the following sequence: SEQ ID NO: 25, or a variant of SEQ ID NO: 25 by comparison with a sequence having a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids),

(v) CDR-L2, consisting of the following sequence: SEQ ID NO: 26, or a variant of SEQ ID NO: 26 to a sequence having a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids), and

In certain preferred embodiments, the VH of an antibody or antigen-binding fragment thereof of the invention comprises: as shown in SEQ ID NO: CDR-H1 shown in FIG. 29; as shown in SEQ ID NO: CDR-H2 shown in FIG. 30; and, as shown in SEQ ID NO: CDR-H3 shown in FIG. 24; and, the VL of the antibody or antigen-binding fragment thereof comprises: as shown in SEQ ID NO: 25 CDR-L1; as shown in SEQ ID NO: CDR-L2 shown in FIG. 26; and, as shown in SEQ ID NO: 16, CDR-L3.

(a) the following 3 heavy chain variable region (VH) CDRs:

(i) CDR-H1, consisting of the sequence: SEQ ID NO: 22, or a sequence identical to SEQ ID NO: 22 compared to a sequence having a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids),

(ii) CDR-H2, consisting of the sequence: SEQ ID NO: 23, or a variant of SEQ ID NO: 23 to a sequence having a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids), and

and/or the presence of a gas in the gas,

(b) the following 3 light chain variable region (VL) CDRs:

In certain preferred embodiments, the VH of an antibody or antigen-binding fragment thereof of the invention comprises: as shown in SEQ ID NO: 22 CDR-H1; as shown in SEQ ID NO: CDR-H2 shown in FIG. 23; and, as shown in SEQ ID NO: CDR-H3 shown in FIG. 24; and, the VL of the antibody or antigen-binding fragment thereof comprises: as shown in SEQ ID NO: 25 CDR-L1; as shown in SEQ ID NO: CDR-L2 shown in FIG. 26; and, as shown in SEQ ID NO: 16, CDR-L3.

(i) SEQ ID NOs: 3. 33, 37 or 41;

(ii) and SEQ ID NOs: 3. 33, 37 or 41 having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids); or

(iii) And SEQ ID NOs: 3. 33, 37, 41, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity;

and/or the presence of a gas in the gas,

(iv) SEQ ID NOs: 4. 34, 38, 42;

(v) and SEQ ID NOs: 4. 34, 38 or 42 with one or more amino acid substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in any one of the above; or

(vi) And SEQ ID NOs: 4. 34, 38, 42, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

(i) SEQ ID NO: 3;

(ii) and SEQ ID NO: 3 compared to a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions); or

(iii) And SEQ ID NO: 3, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity;

and

(iv) SEQ ID NO: 4;

(v) and SEQ ID NO:4 compared to a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions); or

(vi) And SEQ ID NO:4, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

(i) SEQ ID NO: 33;

(ii) and SEQ ID NO: 33 compared to a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions); or

(iii) And SEQ ID NO: 33, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity;

and

(iv) SEQ ID NO: 34;

(v) and SEQ ID NO: 34 compared to a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions); or

(vi) And SEQ ID NO: 34, has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

(i) SEQ ID NO: 37;

(ii) and SEQ ID NO: 37 compared to a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions); or

(iii) And SEQ ID NO: 37, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity;

and

(iv) SEQ ID NO: 38;

(v) and SEQ ID NO: 38 compared to a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions); or

(vi) And SEQ ID NO: 38, or a variant thereof, 38, a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

(i) SEQ ID NO: 41;

(ii) and SEQ ID NO: 41 with one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in (b); or

(iii) And SEQ ID NO: 41, has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity;

and

(iv) SEQ ID NO: 42;

(v) and SEQ ID NO: 42 compared to a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions); or

(vi) And SEQ ID NO: 42, has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

(1) has the sequence shown in SEQ ID NO: 3 and VH having the sequence shown as SEQ ID NO:4, VL of the sequence set forth in seq id no;

(2) has the sequence shown in SEQ ID NO: 33 and a VH having the sequence shown as SEQ ID NO: 34, VL of the sequence set forth in seq id no;

(3) has the sequence shown in SEQ ID NO: 37 and a VH having the sequence shown as SEQ ID NO: 38, VL of the sequence set forth in seq id no; or

(4) Has the sequence shown in SEQ ID NO: 41 and VH having the sequence shown as SEQ ID NO: 42, VL of the sequence set forth in seq id no.

In any of the above aspects, the antibody or antigen-binding fragment thereof of the invention may further comprise a constant region sequence derived from a mammalian (e.g., murine or human) immunoglobulin, or a variant thereof having one or more substitutions, deletions or additions compared to the sequence from which it is derived. In certain preferred embodiments, the variant has conservative substitutions of one or more amino acids compared to the sequence from which it is derived. In certain embodiments, the anti-TIGIT antibody molecule has a heavy chain constant region (Fc) selected from, for example, the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE; in particular from heavy chain constant regions such as IgG1, IgG2, IgG3 and IgG4, more in particular from heavy chain constant regions such as IgG1 or IgG4 (e.g.human IgG1 or IgG 4). In some embodiments, the anti-TIGIT antibody molecule has a light chain constant region selected from light chain constant regions such as kappa or lambda, preferably a kappa light chain constant region (e.g., a human kappa light chain).

In certain preferred embodiments, the heavy chain of an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain constant region (CH) of a human immunoglobulin or a variant thereof having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of up to 20, up to 15, up to 10, or up to 5 amino acids; e.g., substitutions, deletions or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence from which it is derived; and/or the presence of a gas in the gas,

the light chain of the antibody or antigen-binding fragment thereof of the invention comprises a light chain constant region (CL) of a human immunoglobulin or a variant thereof having conservative substitutions of up to 20 amino acids (e.g., conservative substitutions of up to 15, up to 10, or up to 5 amino acids; e.g., conservative substitutions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence from which it is derived.

In certain embodiments, the heavy chain of an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain constant region (CH) of a murine immunoglobulin or a variant thereof having conservative substitutions of up to 20 amino acids (e.g., conservative substitutions of up to 15, up to 10, or up to 5 amino acids; e.g., conservative substitutions of 1, 2, 3, 4, or 5 amino acids) as compared to the sequence from which it is derived; and/or the presence of a gas in the gas,

the light chain of the antibody or antigen-binding fragment thereof of the invention comprises a light chain constant region (CL) of a murine immunoglobulin or a variant thereof having conservative substitutions of up to 20 amino acids (e.g., conservative substitutions of up to 15, up to 10, or up to 5 amino acids; e.g., conservative substitutions of 1, 2, 3, 4, or 5 amino acids) as compared to the sequence from which it is derived.

In some embodiments, the constant region is altered, e.g., mutated, to modify a property of the anti-TIGIT antibody molecule (e.g., to alter one or more of Fc receptor binding, antibody glycosylation, number of cysteine residues, effector cell function, or complement function). A functional change may be produced by replacing at least one amino acid residue in the constant region of an antibody with a different residue, for example, by altering the affinity of the antibody for an effector ligand (e.g., FcR or complement C1q), thereby altering (e.g., enhancing, reducing or eliminating) effector function. Methods of substituting amino acid residues in the Fc region of an antibody to alter its effector function are known in the art (see, e.g., ep388,151a1, US564,8260, US562,4821). The Fc region of an antibody mediates several important effector functions, such as ADCC, phagocytosis, CDC, and the like. In certain cases, these effector functions are required for therapeutic antibodies; in other cases, however, these effector functions may be unnecessary or even detrimental, depending on the intended purpose. Thus, in certain embodiments, an antibody or antigen-binding fragment thereof of the invention has reduced or even eliminated effector function (e.g., ADCC and/or CDC activity). Furthermore, it is also conceivable to introduce amino acid mutations in human IgG4 that stabilize the antibody structure, such as S228P (EU nomenclature, S241P in Kabat nomenclature).

In such embodiments, the antibody or antigen-binding fragment thereof of the invention comprises a variant of a human IgG heavy chain constant region having at least one of the following substitutions compared to the wild-type sequence from which it is derived: ser228Pro, Leu234Ala, Leu235Ala, Gly237Ala, Asp265Ala, Asn297Ala, Pro329Ala, Asp356Glu and Leu358Met (the amino acid positions mentioned above are according to the EU numbering system, Edelman GM et al, Proc Natl Acad USA,63,78-85(1969). PMID: 5257969).

In certain exemplary embodiments, the antibodies or antigen-binding fragments thereof of the invention comprise a human wild-type IgG1 heavy chain constant region. In such embodiments, the antibody or antigen-binding fragment thereof of the invention has ADCC and CDC activity.

In certain exemplary embodiments, the antibodies or antigen-binding fragments thereof of the invention comprise a variant of the human IgG1 heavy chain constant region having the following substitutions as compared to the wild-type sequence from which it is derived: asn297Ala (position according to the EU numbering system). In such embodiments, the antibody or antigen-binding fragment thereof of the invention has abrogated ADCC activity.

In certain exemplary embodiments, the antibodies or antigen-binding fragments thereof of the invention comprise a variant of the human IgG1 heavy chain constant region having the following substitutions as compared to the wild-type sequence from which it is derived: asp265Ala, Pro329Ala (position according to the EU numbering system). In such embodiments, the antibody or antigen-binding fragment thereof of the invention has abrogated ADCC activity.

In certain exemplary embodiments, the antibodies or antigen-binding fragments thereof of the invention comprise a variant of the human IgG4 heavy chain constant region having the following substitutions as compared to the wild-type sequence from which it is derived: ser228Pro (position according to EU numbering system). In such embodiments, the antibodies or antigen binding fragments thereof of the present invention are structurally stable, can reduce Fab-arm exchange, and thus are less prone to half-antibody formation.

In certain preferred embodiments, the heavy chain of an antibody or antigen-binding fragment thereof of the invention comprises a variant of the heavy chain constant region (CH) of a human immunoglobulin that has substantially unchanged effector function compared to the wild-type sequence from which it is derived. In such embodiments, the variant may have conservative substitutions of up to 20 amino acids (e.g., conservative substitutions of up to 15, up to 10, or up to 5 amino acids; e.g., conservative substitutions of 1, 2, 3, 4, or 5 amino acids) as compared to the wild-type sequence from which it is derived.

(a) a heavy chain comprising an amino acid sequence selected from the group consisting of:

(i) SEQ ID NO: 63;

(ii) and SEQ ID NO: 63 compared to a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions); or

(iii) And SEQ ID NO: 63, has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity;

and

(b) a light chain comprising an amino acid sequence selected from the group consisting of:

(iv) SEQ ID NO: 64;

(v) and SEQ ID NO: 64 with one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions); or

(vi) And SEQ ID NO: 64, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

(i) SEQ ID NO: 65;

(ii) and SEQ ID NO: 65 with one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence set forth in seq id no; or

(iii) And SEQ ID NO: 65, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity;

and

(iv) SEQ ID NO: 66;

(v) and SEQ ID NO: 66 with one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in (b); or

(vi) And SEQ ID NO: 66, has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

In certain exemplary embodiments, an antibody or antigen-binding fragment thereof of the invention comprises:

(1) has the sequence shown in SEQ ID NO: 63 and a light chain having the sequence shown as SEQ ID NO: 64; or

(2) Has the sequence shown in SEQ ID NO: 65 and a light chain having the sequence shown as SEQ ID NO: 66, or a light chain of the sequence shown in seq id no.

The anti-TIGIT antibodies or antigen binding fragments thereof disclosed herein may inhibit, reduce, or neutralize one or more activities of TIGIT, e.g., resulting in the blocking or reduction of immune checkpoints on T cells or NK cells, or the reactivation of an immune response by modulation of antigen presenting cells.

In one embodiment, an antibody molecule or antigen-binding fragment thereof of the invention may exhibit at least one of the following properties:

(a) with a K of 100nM or less, preferably 10nM or less, 1nM or 100pM or less_DIn combination with TIGIT (especially human TIGIT), e.g. by means of biofilm interference technique (BLI) (e.g. ForteBio)

) Measured; (b) induce and/or enhance an immune response; (c) increasing effector T cellsActivity; (d) increasing Cytotoxic T Lymphocyte (CTL) activity; (e) increasing NK cell activity; (f) inhibiting TIGIT activation; (g) inhibiting TIGIT-mediated signaling; (h) inhibits or blocks binding of CD155 and/or CD112 to TIGIT; (i) reducing the number of regulatory T cells in the tissue or circulation; (j) inhibiting suppression of effector T cells by regulatory T cells.

In certain preferred embodiments, an antibody or antigen-binding fragment thereof of the invention has any combination of the above biological functions.

In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the present invention are derived from or are monoclonal antibodies produced by the following hybridoma cell lines:

hybridoma cell strain #7, which is deposited in China Center for Type Culture Collection (CCTCC) in No. 10/month 24 of 2018 and has a deposition number of CCTCC NO. C2018210.

Hybridoma cell strain #21, which is deposited in China Center for Type Culture Collection (CCTCC) in No. 10/month 24 of 2018 and has a deposition number of CCTCC NO. C2018209.

In certain preferred embodiments, the antibodies of the invention are chimeric or humanized antibodies. In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention is selected from the group consisting of ScFv, Fab ', (Fab')₂Fv fragments, disulfide-linked Fv (dsfv), diabodies (diabodies), bispecific antibodies, and multispecific antibodies.

In another aspect of the invention, there is provided an isolated nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof of the invention, its heavy and/or light chain, or its heavy chain variable region and/or light chain variable region. In certain embodiments, the nucleotide sequence is codon optimized. In certain preferred embodiments, the isolated nucleic acid molecule comprises first and second nucleic acids encoding the heavy chain variable region and the light chain variable region, respectively, of an antibody of the invention, or an antigen-binding fragment thereof, selected from any one of the following: mab21, AB12V3, AB12V5, AB12V7, and AB12V 9; or a sequence substantially identical to said first and second nucleic acids. In certain preferred embodiments, the isolated nucleic acid molecule comprises first and second nucleic acids encoding the heavy chain variable region and the light chain variable region, respectively, of an antibody of the invention, or an antigen-binding fragment thereof, selected from any one of the following: mab7, AB12V4, AB12V6, AB12V 8; or a sequence substantially identical to said first and second nucleic acids. For example, the isolated nucleic acid molecule can comprise the AB12V8 and AB12V9 nucleotide sequences shown in the sequence listing or sequences substantially identical thereto (e.g., sequences having at least about 85%, 90%, 95%, 99% or more sequence identity thereto or having one or more nucleotide substitutions, or sequences that differ by no more than 3, 6, 15, 30, or 45 nucleotides from the sequences shown in the sequence listing).

In certain preferred embodiments, the present invention provides an isolated nucleic acid molecule comprising a nucleic acid molecule encoding an antibody heavy chain variable region, and/or a nucleic acid molecule encoding an antibody light chain variable region, wherein the nucleic acid molecule encoding an antibody heavy chain variable region has a sequence selected from the group consisting of: (a) 49 or 51, or (b) a sequence substantially identical to the nucleotide sequence of (a) (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity, or a sequence having one or more nucleotide substitutions, as compared to the nucleotide sequence of (a)), or (c) a sequence that differs from the nucleotide sequence of (a) by NO more than 3, 6, 15, 30, or 45 nucleotides; the nucleic acid molecule encoding the variable region of the antibody light chain has a sequence selected from the group consisting of: (a) a nucleotide sequence as set forth in SEQ ID No. 50 or 52, or (b) a sequence substantially identical to the nucleotide sequence set forth in (a) (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity, or a sequence having one or more nucleotide substitutions, as compared to the nucleotide sequence set forth in (a)), or (c) a sequence that differs from the nucleotide sequence set forth in (a) by NO more than 3, 6, 15, 30, or 45 nucleotides.

In certain preferred embodiments, the nucleic acid molecule encoding the variable region of the antibody heavy chain has the nucleotide sequence shown in SEQ ID NO. 49 and the nucleic acid molecule encoding the variable region of the antibody light chain has the nucleotide sequence shown in SEQ ID NO. 50. In certain preferred embodiments, the isolated nucleic acid molecule of the invention comprises a nucleic acid molecule encoding an antibody heavy chain variable region as shown in SEQ ID NO. 49 and/or a nucleic acid molecule encoding an antibody light chain variable region as shown in SEQ ID NO. 50.

In certain preferred embodiments, the nucleic acid molecule encoding the variable region of the antibody heavy chain has the nucleotide sequence shown in SEQ ID NO:51 and the nucleic acid molecule encoding the variable region of the antibody light chain has the nucleotide sequence shown in SEQ ID NO: 52. In certain preferred embodiments, the isolated nucleic acid molecule of the invention comprises a nucleic acid molecule encoding an antibody heavy chain variable region as shown in SEQ ID NO:51 and/or a nucleic acid molecule encoding an antibody light chain variable region as shown in SEQ ID NO: 52.

In certain embodiments, the antibodies or antigen-binding fragments thereof of the invention can be used to construct Chimeric Antigen Receptors (CARs) comprising an extracellular antigen-binding domain (e.g., ScFv) that specifically binds TIGIT, a transmembrane domain, and one or more intracellular T cell signaling domains. In such embodiments, an isolated nucleic acid molecule of the invention can comprise a nucleotide sequence encoding a chimeric antigen receptor that further comprises a nucleotide sequence encoding an antibody of the invention or an antigen-binding fragment thereof (e.g., ScFv). In certain embodiments, an isolated nucleic acid molecule of the invention encodes a chimeric antigen receptor comprising an antigen-binding fragment (e.g., ScFv) of an antibody of the invention.

In another aspect of the invention, the invention provides a vector (e.g., a cloning vector or an expression vector) comprising an isolated nucleic acid molecule of the invention. In certain preferred embodiments, the vectors of the invention are, for example, plasmids, cosmids, phages, lentiviruses, and the like. In certain preferred embodiments, the vector is capable of expressing an antibody or antigen-binding fragment thereof of the invention in a subject (e.g., a mammal, e.g., a human).

In certain embodiments, the antibodies or antigen-binding fragments thereof of the invention can be used to construct chimeric antigen receptors. In such embodiments, the isolated nucleic acid molecule comprised by the vector of the invention may comprise a nucleotide sequence encoding a chimeric antigen receptor that further comprises a nucleotide sequence encoding an antibody of the invention or an antigen-binding fragment thereof (e.g., ScFv). In certain embodiments, the isolated nucleic acid molecule comprised by the vector of the invention encodes a chimeric antigen receptor comprising an antigen-binding fragment (e.g., ScFv) of the antibody of the invention.

In another aspect of the invention, the invention provides a host cell comprising an isolated nucleic acid molecule of the invention or a vector of the invention. The host cell may be a eukaryotic cell (e.g., mammalian cell, insect cell, yeast cell) or a prokaryotic cell (e.g., E.coli). Suitable eukaryotic cells include, but are not limited to, NS0 cells, Vero cells, Hela cells, COS cells, CHO cells, HEK293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells. In certain preferred embodiments, the host cell of the invention is a mammalian cell, such as CHO (e.g., CHO-K1, CHO-S, CHO DXB11, CHO DG 44).

In certain embodiments, the host cell of the invention can be a chimeric antigen receptor T cell (CAR-T). In such embodiments, the isolated nucleic acid molecule comprised by the host cell may comprise a nucleotide sequence encoding a chimeric antigen receptor that further comprises a nucleotide sequence encoding an antibody or antigen-binding fragment thereof (e.g., ScFv) of the present invention. In certain embodiments, the isolated nucleic acid molecule comprised by the host cell encodes a chimeric antigen receptor comprising an antigen-binding fragment (e.g., ScFv) of an antibody of the invention.

In another aspect of the invention, there is provided a method of producing an antibody or antigen-binding fragment thereof of the invention, comprising culturing a host cell of the invention under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

In another aspect of the invention, a pharmaceutical composition is disclosed comprising an antibody or antigen-binding fragment thereof, a vector or a host cell of the invention, and a pharmaceutically acceptable carrier and/or excipient.

In certain preferred embodiments, the pharmaceutical composition of the invention comprises an antibody or antigen-binding fragment thereof of the invention, and a pharmaceutically acceptable carrier and/or excipient.

In certain preferred embodiments, the pharmaceutical compositions of the invention comprise a vector or host cell of the invention, and a pharmaceutically acceptable carrier and/or excipient. In such embodiments, the isolated nucleic acid molecule comprised by the vector comprises a nucleotide sequence encoding a chimeric antigen receptor that further comprises a nucleotide sequence encoding an antibody of the invention or an antigen-binding fragment thereof (e.g., ScFv); the host cell comprises the isolated nucleic acid molecule or vector as described previously. In certain preferred embodiments, the isolated nucleic acid molecule encodes a chimeric antigen receptor comprising an antigen-binding fragment (e.g., ScFv) of an antibody of the invention. In certain preferred embodiments, the host cell is a T cell. In certain preferred embodiments, the host cell is a chimeric antigen receptor T cell (CAR-T).

In certain preferred embodiments, the pharmaceutical composition may further comprise an additional pharmaceutically active agent. In certain preferred embodiments, the additional pharmaceutically active agent is a drug for the treatment of an immune-related disease. In certain preferred embodiments, the additional pharmaceutically active agent is a drug having anti-tumor activity. In certain preferred embodiments, the additional pharmaceutically active agent is a drug for the treatment of an infection or infectious disease.

In certain preferred embodiments, in the pharmaceutical composition, the antibody or antigen-binding fragment thereof of the invention and the additional pharmaceutically active agent are provided as separate components or as components of the same composition. Thus, the antibody or antigen-binding fragment thereof of the invention and the additional pharmaceutically active agent may be administered simultaneously, separately or sequentially.

In another aspect, the antibody or antigen-binding fragment thereof, vector or host cell in the pharmaceutical composition of the invention is sufficient to:

(a) induce and/or enhance an immune response; (b) increasing effector T cell activity; (c) increasing Cytotoxic T Lymphocyte (CTL) activity; (d) increasing NK cell activity; (e) inhibiting TIGIT activation; (f) inhibiting TIGIT-mediated signaling; (g) inhibits or blocks binding of CD155 and/or CD112 to TIGIT; (h) reducing the number of regulatory T cells in the tissue or circulation; (i) inhibiting suppression of effector T cells by regulatory T cells; or (j) any combination of (a) - (i).

In another aspect, the pharmaceutical composition of the invention further comprises a second antibody that specifically binds to or a nucleic acid encoding a receptor or ligand selected from the group consisting of: PD-1, PD-L1, PD-L2, TIM-3, LAG-3, VISTA, CTLA-4, OX40, BTLA, 4-1BB, CD96, CD27, CD28, CD40, LAIR1, CD160, 2B4, TGF-R, KIR, ICOS, GITR, CD3, CD30, BAFFR, HVEM, CD7, LIGHT, SLAMF7, NKp80, B7-H3, and any combination thereof.

In certain specific embodiments, the second antibody is an antibody or antigen-binding fragment thereof that binds human PD-1. In certain preferred embodiments, the pharmaceutical compositions of the invention comprise an antibody or antigen-binding fragment thereof that binds human PD-1.

In certain specific embodiments, the second antibody is an antibody or antigen-binding fragment thereof that binds human PD-L1. In certain preferred embodiments, the pharmaceutical compositions of the invention comprise an antibody or antigen-binding fragment thereof that binds human PD-L1.

In another aspect of the invention, there is provided the use of an antibody or antigen-binding fragment thereof, vector or host cell of the invention in the manufacture of a medicament for:

(1) increasing immune cell activity in vitro or in a subject (e.g., a human);

(2) enhancing an immune response in a subject (e.g., a human);

(3) treating a tumor in a subject (e.g., a human); or

(4) Treating an infection or infectious disease in a subject (e.g., a human).

In certain preferred embodiments, when the vector or host cell of the invention is used in the manufacture of a medicament, the isolated nucleic acid molecule comprised by the vector comprises a nucleotide sequence encoding a chimeric antigen receptor further comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof (e.g., ScFv) of the invention; the host cell comprises the isolated nucleic acid molecule or vector as described previously. In certain preferred embodiments, the isolated nucleic acid molecule encodes a chimeric antigen receptor comprising an antigen-binding fragment (e.g., ScFv) of an antibody of the invention. In certain preferred embodiments, the host cell is a T cell. In certain preferred embodiments, the host cell is a chimeric antigen receptor T cell (CAR-T).

In certain preferred embodiments, when the vector or host cell of the invention is used in the preparation of a medicament, the medicament is for treating a tumor in a subject (e.g., a human).

In certain preferred embodiments, the tumor to which the antibody or antigen-binding fragment thereof, vector or host cell of the invention is directed is selected from the group consisting of a solid tumor, a hematological tumor (e.g., leukemia, lymphoma, myeloma, e.g., multiple myeloma), and a metastatic, refractory or recurrent lesion of cancer; for example, including, but not limited to, esophageal cancer, gastrointestinal cancer, pancreatic cancer, thyroid cancer, colorectal cancer, renal cancer, lung cancer (e.g., non-small cell lung cancer), liver cancer, stomach cancer, head and neck cancer, bladder cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, germ cell cancer, bone cancer, skin cancer, thymus cancer, bile duct cancer, melanoma, mesothelioma, lymphoma, myeloma, sarcoma, glioblastoma, leukemia, or metastatic, refractory, or recurrent lesions of the cancer.

In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the present invention are directed against an infection or infectious disease selected from the group consisting of viral, bacterial, fungal, and parasitic infections, including but not limited to HIV, hepatitis, herpes, CMV, EBV, influenza, or tetanus.

In another aspect of the invention, an immunogenic composition is provided comprising an antibody or antigen-binding fragment thereof of the invention, and an immunogen.

In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the present invention act as adjuvants.

In certain preferred embodiments, the immunogen is selected from the group consisting of a tumor cell, an antigen (e.g., a protein, polypeptide, or carbohydrate molecule) associated with a tumor, a dendritic cell primed by the antigen, and any combination thereof.

In certain preferred embodiments, the immunogen is selected from the group consisting of an inactivated or attenuated pathogen, an antigen (e.g., a protein, polypeptide, or carbohydrate molecule) associated with a pathogen (e.g., a virus), a dendritic cell primed by the antigen, and any combination thereof.

In certain preferred embodiments, the immunogenic composition further comprises a pharmaceutically acceptable carrier and/or excipient.

In certain preferred embodiments, in the immunogenic composition, the antibody or antigen-binding fragment thereof of the invention is provided as a separate component from the immunogen or as a component of the same composition. Thus, the antibody or antigen-binding fragment thereof of the invention and the immunogen may be administered simultaneously, separately or sequentially.

In another aspect, the invention relates to the use of an antibody or antigen-binding fragment thereof of the invention as an adjuvant, or in the preparation of an immunogenic composition for enhancing an immune response in a subject; wherein the immunogenic composition comprises an antibody or antigen-binding fragment thereof of the invention and an immunogen.

In certain preferred embodiments, the immunogen is selected from the group consisting of a tumor-associated antigen (e.g., a protein, polypeptide, or carbohydrate molecule), a tumor cell, a dendritic cell primed with the antigen, and any combination thereof. In such embodiments, the immunogenic composition is used to prevent and/or treat a tumor, delay tumor progression, or reduce or inhibit tumor recurrence in a subject.

In certain preferred embodiments, the tumor to which the antibody or antigen-binding fragment thereof of the present invention is directed is selected from the group consisting of a solid tumor, a hematological tumor (e.g., leukemia, lymphoma, myeloma, e.g., multiple myeloma), and a metastatic, refractory, or recurrent lesion of cancer; for example, including, but not limited to, esophageal cancer, gastrointestinal cancer, pancreatic cancer, thyroid cancer, colorectal cancer, renal cancer, lung cancer (e.g., non-small cell lung cancer), liver cancer, stomach cancer, head and neck cancer, bladder cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, germ cell cancer, bone cancer, skin cancer, thymus cancer, bile duct cancer, melanoma, mesothelioma, lymphoma, myeloma, sarcoma, glioblastoma, leukemia, or metastatic, refractory, or recurrent lesions of the cancer.

In certain preferred embodiments, the immunogen is selected from the group consisting of an antigen (e.g., a protein, polypeptide, or carbohydrate molecule) associated with a pathogen (e.g., a virus), an inactivated or attenuated pathogen, a dendritic cell primed by the antigen, and any combination thereof. In such embodiments, the immunogenic composition is for use in the prevention and/or treatment of an infection or infectious disease in a subject. In such embodiments, the immunogenic composition is used to delay the progression of, or reduce or inhibit the recurrence of, an infection or infectious disease in a subject.

In certain preferred embodiments, the immunogenic composition further comprises a pharmaceutically acceptable carrier and/or excipient. In certain preferred embodiments, the immunogenic composition comprises a stabilizer.

In another aspect, the invention relates to the use of said antibody or antigen-binding fragment thereof or pharmaceutical or immunogenic composition for the manufacture of a medicament for the treatment of an immune-related disease, wherein the immune-related disease involves T cell dysfunction and/or NK cell dysfunction. In certain preferred embodiments, the T cell dysfunction includes, for example, T cell disability, exhaustion, or reduced cytokine secretion; and/or, the NK cell dysfunction includes, for example, NK cell disability, depletion, or decreased cytokine secretion.

In another aspect, the invention provides a method of increasing the activity of an immune cell in vitro, the method comprising the step of contacting the immune cell with an antibody or antigen-binding fragment thereof of the invention.

In certain preferred embodiments, any suitable indicator can be used to measure the activity of the immune cells. Non-limiting examples of such suitable indicators include: in the presence of an antibody or antigen-binding fragment thereof of the invention, an immune cell (e.g., a T cell) has an increased level of cytokine (e.g., IL-2, IFN- γ, etc.) secretion, proliferative activity, and/or expression of an activation marker (e.g., CD25, CD69, etc.).

In certain preferred embodiments, the methods are used to treat tumors. In such embodiments, the immune cells obtained by the above methods can be adoptively transferred into a subject to treat a tumor. Activation in vitro in the presence of an antibody or antigen-binding fragment thereof of the invention is expected to increase the activity of adoptively transferred immune cells, thereby facilitating tumor killing of these adoptively transferred immune cells in a subject. In certain preferred embodiments, the immune cell is a tumor infiltrating lymphocyte.

In certain preferred embodiments, the immune response stimulating agent is selected from the group consisting of IL-1, IL-2, IL-3, IL-7, IL-12, IL-15, IL-18, IL-21, IFN- γ, IL-10, TGF- β, GM-CSF, M-CSF, G-CSF, TNF- α, TNF- β, and any combination thereof.

In another aspect, the invention provides a method of enhancing T cell activation and/or response in a subject. In another aspect, the invention provides a method of enhancing NK cell activation and/or response in a subject. The method comprises the following steps: administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to the invention or a pharmaceutical composition according to the invention or an immunogenic composition according to the invention.

In certain embodiments, the method may further comprise administering to the subject a second therapy selected from the group consisting of surgery, chemotherapy, radiation therapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof.

In another aspect, the invention provides a method of increasing immune cell activity in a subject, comprising: administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to the invention or a pharmaceutical composition according to the invention or an immunogenic composition according to the invention. In certain embodiments, the immune cell is a T cell. In certain embodiments, the immune cell is a cytotoxic T Cell (CTL). In certain embodiments, the immune cell is an NK cell. In certain embodiments, the immune cell is any combination of the above immune cells.

In certain preferred embodiments, the methods are used for the prevention and/or treatment of tumors. In certain preferred embodiments, the method is for delaying tumor progression. In certain preferred embodiments, the methods are used to reduce or inhibit tumor recurrence.

In certain preferred embodiments, the methods are used for the prevention and/or treatment of infection or infectious disease. In certain preferred embodiments, the methods are used to delay the progression of an infection or infectious disease. In certain preferred embodiments, the methods are used to reduce or inhibit infection or infectious disease recurrence.

In another aspect, the invention provides a method of enhancing an immune response in a subject, comprising administering to a subject in need thereof an effective amount of an antibody or antigen-binding fragment thereof of the invention, a pharmaceutical composition of the invention, or an immunogenic composition of the invention. In certain embodiments, the immune response is an antigen-specific T cell response.

In another aspect, the present invention provides a method of preventing and/or treating a tumor in a subject. In another aspect, the invention provides a method of delaying tumor progression in a subject. In another aspect, the invention provides a method of reducing or inhibiting tumor recurrence in a subject. The methods described above comprise administering to a subject in need thereof an effective amount of an antibody or antigen-binding fragment thereof of the invention, a host cell of the invention, or a pharmaceutical composition of the invention, or an immunogenic composition of the invention.

When the host cell of the invention is used in the methods described above, the host cell expresses a chimeric antigen receptor comprising an antigen-binding fragment (e.g., ScFv) of the antibody of the invention. Thus, in certain preferred embodiments, the isolated nucleic acid molecule comprised by the host cell comprises a nucleotide sequence encoding a chimeric antigen receptor that further comprises a nucleotide sequence encoding an antibody or antigen-binding fragment thereof (e.g., ScFv) of the invention. In certain preferred embodiments, the isolated nucleic acid molecule encodes a chimeric antigen receptor comprising an antigen-binding fragment (e.g., ScFv) of an antibody of the invention. In certain preferred embodiments, the host cell is a T cell. In certain preferred embodiments, the host cell is a chimeric antigen receptor T cell (CAR-T).

In another aspect, the methods described above further comprise administering to the subject a second therapy selected from the group consisting of surgery, chemotherapy, radiation therapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof.

In another aspect, the invention provides a method of preventing and/or treating an infection or infectious disease in a subject. In another aspect, the invention provides a method of delaying the progression of an infection or infectious disease in a subject. In another aspect, the invention provides a method of reducing or inhibiting the recurrence of an infection or infectious disease in a subject. The methods described above comprise administering to a subject in need thereof an effective amount of an antibody or antigen-binding fragment thereof of the invention, a pharmaceutical composition of the invention, or an immunogenic composition of the invention.

In another aspect, the invention provides a method of detecting the presence or level of TIGIT in a sample, the method comprising forming a complex between the antibody or antigen-binding fragment thereof and TIGIT, contacting the sample with the antibody or antigen-binding fragment thereof of the invention, and detecting the formation of the complex.

In another aspect, the invention provides a diagnostic or therapeutic kit comprising an antibody or antigen-binding fragment thereof, vector or host cell of the invention, and instructions for use.

In another aspect, the invention provides a diagnostic or therapeutic kit comprising a pharmaceutical composition of the invention and instructions for use.

The antibody of the invention has high binding affinity with TIGIT and extremely strong specificity, and can enhance the activity of immunocytes in vitro/in vivo and stimulate immune response. Accordingly, the antibody of the present invention has a potential for use in the prevention and/or treatment of a tumor, an immune-related disease, an infection, or an infectious disease. The humanized antibody provided by the invention retains the functions and properties of a parent murine antibody, and in vivo anti-tumor research data show that the humanized antibody provided by the invention can obviously inhibit the growth of mouse transplanted tumors, even part of mice completely disappear. Furthermore, the humanized antibodies of the invention have a high degree of humanization and can therefore be safely administered to human subjects without eliciting an immunogenic response. Thus, the antibodies of the invention (particularly humanized antibodies) are of significant clinical value.

Detailed Description

Abbreviations and Definitions

TIGIT cell immune receptor containing Ig and ITIM structural domain

Complementarity determining regions in CDR immunoglobulin variable regions

FR antibody framework regions: amino acid residues other than CDR residues in antibody variable regions

VH antibody heavy chain variable region

VL antibody light chain variable region

IgG immunoglobulin G

Kabat, by Elvin a. Kabat, the immunoglobulin alignment and numbering system (see, e.g., Kabat al, Sequences of Proteins of Immunological Interest,5th ed. public Health service, National Institutes of Health, Bethesda, Md., 1991).

Chothia by Chothia et al proposed immunoglobulin numbering system, which is based on the structural loop region position identification CDR region boundary classic rules (see, for example, Chothia & Lesk (1987) J.mol.biol.196:901 and 917; Chothia et al (1989) Nature 342:878 and 883).

IMGT is based on the International ImmunoGeneTiCs information System (International ImmunoGeneTiCs information) initiated by Lefranc et al

(IMGT)) see Lefranc et al, dev.company.immunol.27: 55-77,2003.

mAb monoclonal antibodies

EC₅₀Concentrations that give 50% efficacy or binding

IC₅₀Concentration giving 50% inhibition

ELISA enzyme-linked immunosorbent assay

PCR polymerase chain reaction

HRP horse radish peroxidase

IL-2 Interleukin 2

IFN interferon

K_DEquilibrium dissociation constant

K_aConstant of binding rate

K_dOff rate constant

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, the procedures of cell culture, biochemistry, nucleic acid chemistry, immunological laboratories and the like used herein are all conventional procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

The term "TIGIT" includes human TIGIT, cynomolgus TIGIT, rat TIGIT, mouse TIGIT, and fragments thereof (such as mature fragments thereof lacking a signal peptide). In one embodiment of the invention, the amino acid sequence of human TIGIT comprises the amino acid sequence disclosed in amino acid residues 25-244 of Genbank accession No. NP _ 776160.2. In some embodiments, the antibody molecules provided herein specifically bind to an epitope (e.g., a linear or conformational epitope) on TIGIT of a mammal (e.g., a human). In some embodiments, the binding epitope is at least a portion of an IgV domain of human TIGIT.

The term "antibody" is used in the broadest sense to broadly refer to any immunobinder or molecule comprising an antigen binding domain, including intact antibodies (e.g., IgG1 or IgG3), monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), chimeric antibodies, various functional fragments (e.g., may include only antigen binding portions, such as Fab, Fab ', F (ab')₂Fd, Fd', Fv, or ScFv fragments) and variants thereof (e.g., humanized, glycosylated, etc.), so long as they exhibit the desired biological activity. The invention also includes anti-TIGIT antibodies with glycosylation modifications. In some applications, modifications are made to remove undesired glycosylation sites, such as defucose modifications on the oligosaccharide chains to enhance Antibody Dependent Cellular Cytotoxicity (ADCC) function; in other applications, galactosylation modifications can be made to alter Complement Dependent Cytotoxicity (CDC) effects.

Generally, naturally occurring immunoglobulins have heavy and light chains interconnected by disulfide bonds, heavy chains can be classified as μ, δ, γ, α, or ε, and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA, and IgE, respectively, based on the amino acid sequence of the constant region and the amino acid sequence of the framework region of the variable region, and "light chains" are divided into two strict classes, kappa (κ) and lambda (λ), the subclasses of IgG isotypes are classified in certain species as IgG1, IgG2, IgG3 and IgG4 in humans, and IgG1, IgG2a, IgG2b and IgG3 in mice, immunoglobulins such as human IgG1, exist as several allotypes that differ from each other in up to several amino acids.

Each heavy and light chain contains a constant region and a variable region (the regions are also referred to as "domains"). ' V_H"or" VH "refers to the variable region of an immunoglobulin heavy chain, including the variable region of an Fv, ScFv, dsFv, or Fab; ' V_L"or" VL "refers to the variable region of an immunoglobulin light chain, including the variable regions of Fv, ScFv, dsFv, or Fab. The light and heavy chain variable regions contain"framework regions" separated by three hypervariable regions (also referred to as "complementarity determining regions" or "CDRs"). "Framework Regions (FR)" are regions other than CDRs within the variable regions of heavy and light chains, and the amino acid composition and arrangement is relatively invariant. The CDRs are primarily responsible for binding to an epitope of the antigen. The CDRs of each chain are generally referred to as CDR-H1, CDR-H2, CDR-H3, numbered sequentially from the N-terminus, and are also generally identified by the chain in which the particular CDR is located. Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. Although the CDRs differ from antibody to antibody, only a limited number of amino acid positions of the CDRs are directly involved in antigen binding.

The variable regions of the heavy and light chains of an antibody contain binding domains that interact with an antigen. While antibodies exhibit binding specificity for a particular antigen, immunoglobulins include antibodies and other antibody-like molecules that lack antigen specificity. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (e.g., C1 q). The Fc region of a naturally occurring antibody binds to elements of the complement system and also to receptors on effector cells, including, for example, effector cells that mediate cytotoxicity (including specific ADCP). It is known in the art that the affinity and/or other binding properties of the Fc region for Fc receptors can be modulated by glycosylation or other modifications.

The humanized antibody or antibody fragment may be naturally occurring or chemically synthesized in whole or in part. The antibodies may thus be derived from any suitable source, for example recombinant sources and/or transgenic animal or plant production, or produced in eggs using IgY technology. Thus, antibody molecules can be produced in vivo or in vitro.

The term "hypervariable region" or "CDR region" or "complementarity determining region" refers to the amino acid residues of an antibody which are responsible for antigen binding. CDR region sequences can be defined by IMGT, Kabat, Chothia and AbM methods or the field known as any CDR region sequence determination method to identify the variable region within the amino acid residues. Antibody CDRs can be identified as hypervariable regions originally defined by Kabat et al, e.g., residues 24-34(L1), 50-56(L2) and 89-97(L3) of the light chain variable domain and residues 31-35(H1), 50-65(H2) and 95-102(H3) of the heavy chain variable domain, see Kabat EA et al, 1991, Sequences of proteins of Immunological Interest,5th edition, public Health Service, National Institutes of Health, Behinda, Md.; the position of the CDRs can also be identified as originally defined by the "hypervariable loop" (HVL) structure described by Chothia et al. IMGT (ImmunoGeneTiCs) also provides a numbering system for immunoglobulin variable regions including CDRs, the CDR regions being defined according to IMGT numbering, e.g., residues 27-32(L1), 50-52(L2) and 89-97(L3) of the light chain variable domain and residues 26-35(H1), 51-57(H2) and 93-102(H3) of the heavy chain variable domain, see, e.g., Dev.Comp.Immunol.,2003,27:55-77, by Lefranc MP et al, which is incorporated herein by reference. Other methods for CDR identification include "AbM definition," which is a compromise between Kabat and Chothia and is obtained using Oxford Molecular's AbM antibody model software; or "contact definition" of CDRs, based on observed antigen contact and described in MacCallum RM et al, 1996, J.mol biol.,262: 732-. In the "configuration definition" approach to CDRs, the position of a CDR can be identified as a residue contributing to antigen binding, see, e.g., Makabe K et al, 2008, J.biol chem.,283: 1156-. The CDRs contained in the antibodies of the invention or antigen-binding fragments thereof can be determined according to various numbering systems known in the art. In certain embodiments, the CDRs contained by the antibodies or antigen binding fragments thereof of the present invention are preferably determined by the Kabat, Chothia, or IMGT numbering system.

The term "epitope" or "antigenic determinant" refers to a particular chemical group or peptide sequence on a molecule that is antigenic (i.e., capable of eliciting a specific immune response), being the site on an antigen (e.g., TIGIT) to which an immunoglobulin or antibody specifically binds. The immunoglobulin or antibody specifically binds to a particular epitope on the polypeptide (e.g., TIGIT). OK epitope-determining regions usually consist of chemically active surface groups of the molecule (e.g. amino acids or glycosyl side chains) and usually have specific three-dimensional structural properties as well as specific charge properties.

The term "Fab fragment" consists of one light chain and one heavy chainChain CH1 and the variable region. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. A "Fab ' fragment" contains the VH and CH1 domains of one heavy and one light chain and the constant region portion between the CH1 and CH2 domains, whereby an interchain disulfide bond can be formed between the two heavy chains of two Fab ' fragments to form F (ab ')₂A molecule. "F (ab')₂A fragment "contains the VH and CH1 domains of the two heavy and two light chains and the constant region portion between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. Thus, F (ab')₂The fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains. The "Fv region" comprises variable regions from both the heavy and light chains, but lacks the constant region. The "Fd fragment" consists of CH1 and the variable domain of one heavy chain, and is the heavy chain portion remaining after the Fab fragment has removed the light chain. The "disulfide-bond stability protein (dsFv)" introduces a cysteine mutation point in each of the VH and VL regions, thereby forming a disulfide bond between the VH and VL to achieve structural stability.

The term "Fc region" or "Fc" refers to the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the hinge region, the CH2 domain, and the CH3 domain, which mediates binding of the immunoglobulin to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or to the first component of the classical complement system (e.g., C1q), including native sequence Fc regions and variant Fc regions. Typically, the human IgG heavy chain Fc region is the carboxy-terminal stretch from the amino acid residue at position Cys226 or Pro230, but the boundaries may vary. The C-terminal lysine of the Fc region (residue 447, according to the EU numbering system) may or may not be present. Fc may also refer to this region of sequestration, or in the case of Fc-containing protein polypeptides, such as "binding proteins comprising an Fc region," also referred to as "Fc fusion proteins" (e.g., antibodies or immunoadhesins). The native sequence Fc region in the antibodies of the invention includes human IgG1, IgG2(IgG2A, IgG2B), IgG3 and IgG 4. In IgG, IgA, and IgD antibody isotypes, the Fc region comprises the CH2 and CH3 constant domains of each of the two heavy chains of an antibody; the IgM and IgEFc regions comprise three heavy chain constant domains (CH domains 2-4) in each polypeptide chain.

The term "Fc receptor" or "FcR" refers to a receptor that binds the Fc region of an immunoglobulin. The FcR may be a native sequence human FcR or an FcR (gamma receptor) that binds IgG antibodies, as well as allelic variants and alternatively spliced forms of these receptors. The Fc γ R family consists of three activating receptors (Fc γ RI, Fc γ RIII and Fc γ RIV in mice; Fc γ RIA, Fc γ RIIA and Fc γ RIIIA in humans) and one inhibitory receptor (Fc γ RIIb or equivalent Fc γ RIIb). Fc γ RII receptors include Fc γ RIIA ("activating receptor") and Fc γ RIIB ("inhibiting receptor"), which have similar amino acid sequences. The cytoplasmic domain of Fc γ RIIA contains an immunoreceptor tyrosine-based activation motif (ITAM). The cytoplasmic domain of Fc γ RIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) (see M.Annu. Rev. Immunol.15:203-234, 1997). Most native effector cell types co-express one or more activating Fc γ rs and inhibitory Fc γ RIIb, while NK cells selectively express one of the activating Fc receptors (Fc γ RIII in mice and Fc γ RIIIA in humans), but do not express inhibitory Fc γ RIIb in mice and humans. Human IgG1 binds to most human Fc receptors and is considered equivalent to murine IgG2a in the type of activating Fc receptor that it binds. The term "FcR" encompasses other fcrs herein, including those that will be identified in the future. Methods for measuring binding to FcRn are known (see, e.g., Ghetie V et al, immunological Today 18:592-8, 1997; Ghetie V et al, Nature Biotechnology,15:637-40, 1997). The in vivo binding and serum half-life of human FcRn high affinity binding polypeptides to FcRn can be determined, for example, in transgenic mice or transfected human cell lines expressing human FcRn. The term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer RL et al, J.Immunol.117:587,1976) and (Kim YJ et al, J.Immunol.24: 249,1994).

The term "single chain Fv antibody (or ScFv antibody)" refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. For a review of ScFv, see International patent application publication No. WO88/01649 and U.S. Pat. Nos. 4946,778 and 5260,203.

The term "single domain antibody" is obtained by genetic engineering methods and has mainly 3 classes, the first of which is the heavy chain variable region obtained from camelid HCAb, as a single folded unit, which retains intact antigen binding activity and is the smallest fragment of a natural antibody. The second type is a heavy chain variable region obtained from IgNAR of cartilaginous fish such as shark, which is denoted by VNAR. The third type is a heavy chain or light chain variable region obtained from a monoclonal antibody of human or murine origin, which retains antigen binding activity but has greatly reduced affinity and solubility.

The term "monoclonal antibody (mAb)" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprised by the population are identical except for the few naturally occurring mutations that may occur, exhibiting a single binding specificity and affinity for a particular epitope. The modifier "monoclonal" indicates the identity of the antibody obtained from a substantially homogeneous population of antibodies, and does not require that the antibody be produced by a particular method. Monoclonal antibodies are produced by methods known to those skilled in the art, such as by fusing myeloma cells and immune spleen cells to produce hybrid antibody producing cells. Synthesized by culturing hybridomas, no contamination with any other antibodies. Monoclonal antibodies can also be obtained recombinantly, e.g., using recombinant techniques, phage display techniques, synthetic techniques, or other known techniques.

The term "multispecific antibody" is an antibody having specificity for at least two different antigens. The multispecific antibody may be a full-length antibody or a fragment of said antibody. For example, bispecific antibodies may recognize two different antigens or may recognize different epitopes of one antigen.

The term "chimeric antibody" refers to a portion of the heavy and/or light chain that is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No.4,816,567; Morrison SL et al, Proc. Natl. Acad. Sci. USA,81: 6851-. For example, the term "chimeric antibody" can include an antibody (e.g., a human murine chimeric antibody) in which the heavy and light chain variable regions of the antibody are from a first antibody (e.g., a murine antibody) and the heavy and light chain constant regions of the antibody are from a second antibody (e.g., a human antibody).

The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) and a light chain variable domain (VL) linked thereto in the same polypeptide chain (VH-VL or VL-VH). By using a linker that is too short to allow pairing between two variable domains on the same chain, the domains are forced to pair with the complementary domains of the other chain and two antigen binding sites are created. Diabodies are more fully described in e.g. EP 404,097; WO 93/11161; and Holliger et al (1993) Proc.Natl.Acad.Sci.USA 90: 6444-. For a review of engineered antibody variants, see generally Holliger and Hudson (2005) nat. Biotechnol.,23: 1126-1136.

The terms "full length antibody", "intact antibody", are used interchangeably and refer to a substantially intact form of an antibody, as opposed to an antibody fragment. In particular, whole antibodies include those having a heavy chain and a light chain (including an Fc region). The constant region may be a native sequence constant region (e.g., a human native sequence constant region) or an amino acid sequence variant thereof. In some cases, an intact antibody may have one or more effector functions.

The term "humanized antibody" refers to a non-human antibody that has been genetically engineered to have an amino acid sequence modified to increase homology to the sequence of a human antibody. Most or all of the amino acids outside the CDR domain of a non-human antibody, e.g., a mouse antibody, are replaced with corresponding amino acids from a human immunoglobulin, while most or all of the amino acids within one or more CDR regions are unchanged. Addition, deletion, insertion, substitution, or modification of small amino acids is permissible as long as they do not abrogate the ability of the antibody to bind to a particular antigen. "humanized" antibodies retain antigen specificity similar to the original antibody. The source of the CDR is not particularly limited and may be derived from any animal. For example, antibodies derived from mouse, rat, rabbit, or non-human primates (e.g., cynomolgus monkey) can be used.

The term "human" antibody (HuMab) refers to an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In addition, if the antibody contains constant regions, the constant regions are also derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

The term "antibody fragment" or "antigen-binding fragment" refers to antigen-binding fragments and antibody analogs of antibodies that retain the ability to specifically bind to an antigen (e.g., TIGIT), which typically include at least a portion of the antigen-binding or variable region of the parent antibody (parent antibody). Antibody fragments retain at least some of the binding specificity of the parent antibody. Typically, when expressed as a mole of activity, an antibody fragment retains at least 10% of the parent binding activity. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% of the binding affinity of the parent antibody to the target. Antibody fragments include, but are not limited to: fab fragment, Fab 'fragment, F (ab')₂Fragments, Fv fragments, Fd fragments, Complementarity Determining Region (CDR) fragments, disulfide bond stability proteins (dsFv), and the like; linear antibodies (Linear antibodies), Single chain antibodies (e.g., ScFv Single antibodies) (technique from Genmab), bivalent Single chain antibodies, Single chain phage antibodies, Single Domain antibodies (e.g., VH Domain antibodies), Domain antibodies (technique from AbIynx); multispecific antibodies formed from antibody fragments (e.g., three-chain antibodies, four-chain antibodies, etc.); and engineered antibodies such as Chimeric antibodies (e.g., humanized murine antibodies), Heteroconjugate antibodies (Heteroconjugate antibodies), and the like. These antibody fragments are obtained by conventional techniques known to those skilled in the art and are screened for utility in the same manner as are intact antibodies.

The term "domain antibody" is an immunologically functional immunoglobulin fragment that contains only the variable region of a heavy chain or the variable region of a light chain. In some cases, two or more VH regions are covalently joined with a peptide linker to produce a bivalent domain antibody. The two VH regions of the bivalent domain antibody may target the same or different antigens.

The term "functional fragment" encompasses a portion of an intact antibody, typically including the antigen binding or variable region of an intact antibody or the Fc region of an antibody that retains or has the ability to improve FcR binding. Examples of functional fragments of antibodies include linear antibodies, single chain antibody molecules, and multispecific antibodies formed from antibody fragments.

The terms "conjugate", "linked" and "linking" refer to the association of two or more molecules. The linkage may also be genetic (i.e., recombinant fusion). In a specific context, the term includes reference to linking a ligand (e.g., an antibody moiety) to an effector molecule. Such attachment can be accomplished using a variety of art-recognized techniques, such as by chemical or recombinant means. By "chemical means" is meant a reaction between the antibody moiety and an effector molecule such that a covalent bond is formed between the two molecules to form one molecule.

The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of a cell and/or causes destruction of a cell. The term is intended to include radioisotopes (e.g., I)¹³¹、I¹²⁵、Y⁹⁰、Re¹⁸⁶) Chemotherapeutic agents, and toxins (such as enzymatically active toxins of bacterial, fungal, plant or animal origin), or fragments thereof.

Examples of such cytokines include lymphokines, monokines, interleukins ("IL"), such as IL-2, IL-6, IL-17A-F, tumor necrosis factors, such as TNF- α or TNF- β, and other polypeptide factors, such as leukemia inhibitory factor ("LIF").

The term "inflammatory cell" refers to a cell that enhances an inflammatory response, such as monocytes, macrophages and polymorphonuclear neutrophils (PMNs).

The term "immune-related disorder" refers to an immune-related disorder in a mammal that is caused, mediated, or otherwise contributed to by components of the mammal's immune system, and also includes disorders in which stimulation or intervention of an immune response has an ameliorating effect on the development of the disorder. The term includes immune-mediated inflammatory diseases, non-immune-mediated inflammatory diseases, infectious diseases, immunodeficiency diseases, tumors, and the like.

The term "depletion" refers to the depletion of T cells as a state of T cell dysfunction resulting from sustained TCR signaling or sustained NK receptor (such as NCR) signaling that occurs during many chronic infections and cancers, due to sustained signaling. Depletion may result from both extrinsic negative regulatory pathways (e.g., immunoregulatory cytokines) and cell intrinsic negative regulatory (co-stimulatory) pathways.

The term "enhancing NK cell activity" means inducing, causing or stimulating NK cells to have sustained or amplified biological activity, or restoring or reactivating exhausted or inactive NK cells. Examples of enhancing NK cell activity include: increased IFN- γ secretion, increased proliferation, increased antigen responsiveness (e.g., clearance of a virus, pathogen, or tumor) relative to such levels prior to intervention. In one embodiment, the level of enhancement is at least 50%, e.g., 60%, 70%, 80%, 90%, 100%, 120%, 150%, or 200%. The manner of measuring this level of enhancement is known to those of ordinary skill in the art.

The term "tumor immunity" refers to the process by which a tumor evades immune recognition and clearance. As such, as a therapeutic concept, tumor immunity is "treated" when such evasion is diminished, and the tumor is recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage and tumor clearance.

The term "immunogenicity" refers to the ability of a particular substance to elicit an immune response. Tumors are immunogenic and enhancing tumor immunogenicity aids in the elimination of tumor cells by immune response. Examples of enhancing tumor immunogenicity include, but are not limited to, treatment with TIGIT inhibitors (e.g., anti-TIGIT antibodies).

The term "immune response" is the effect of soluble macromolecules produced by cells of the immune system (e.g., T lymphocytes, B lymphocytes, NK cells, antigen presenting cells, macrophages, eosinophils, mast cells, DC cells or neutrophils) and by immune cells or the liver, by any of these cells or liver (including antibodies, cytokines and complements), which results in selective damage to, destruction of, or clearance from the human body of invading pathogens, cells or tissues infected by pathogens, cancer cells, or normal human cells or tissues in the case of pathological inflammation. Immune responses include, for example, T cells (e.g., effector T cells or Th cells, such as CD)⁸⁺Or CD⁴⁺T cells), or inhibits or depletes NK cells or Treg cells.

The term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and the antigen against which it is directed. The term "immunological binding" refers to a specific binding reaction that occurs between an antibody molecule and an antigen for which the antibody is specific. The strength or affinity of an immunological binding interaction may be the equilibrium dissociation constant (K) of the interaction_D) Is represented by the formula, wherein K_DSmaller values indicate higher affinity. The immunological binding properties between the two molecules can be quantified using methods well known in the art. One method involves measuring the rate of antigen binding site/antigen complex formation and dissociation. Refers to the "binding rate constant" (K) of a particular antibody-antigen interaction_aOr K_on) And "dissociation rate constant" (K)_dOr K_off) Both can be calculated from the concentration and the actual rate of association and dissociation, see Malmqvist M,1993, Nature,361: 186-187. K_d/K_aIs equal to the dissociation constant K_DSee Davies DR et al, 1990, Annual Rev biochem, 59: 439-. K can be measured by any effective method_D、K_aAnd K_dThe value is obtained. In a preferred embodiment, bioluminescence interferometry (e.g., the ForteBio Octet method described in example 4) is used to measure dissociation constantsAnd (4) counting. In other preferred embodiments, the dissociation constant can be measured using surface plasmon resonance techniques (e.g., Biacore) or KinExa. An antibody that "specifically binds to human TIGIT" refers to K that binds to human TIGIT that is soluble or binds cells_DIs 10^-7M or less, e.g. less than about 10^-8M，10^-9M,10^-10M or 10^-11M even lower antibodies, but do not bind unrelated antigens with high affinity.

The term "cross-reactive" refers to the ability of an antibody described herein to bind TIGIT from a different species. For example, an antibody that binds to human TIGIT described herein may also bind to TIGIT from other species (e.g., cynomolgus TIGIT). Cross-reactivity can be measured by detecting specific reactivity with purified antigens in binding assays (e.g., SPR, ELISA), or binding to or otherwise interacting with the function of cells physiologically expressing TIGIT. Examples of assays known in the art to determine binding affinity include surface plasmon resonance (e.g., Biacore) or similar techniques (e.g., KinExa or OCTET).

The term "nucleic acid molecule" is intended to include both DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, and may be a cDNA.

The term "polypeptide" refers to a chain comprising at least two amino acid residues linked in series, with no upper limit on the length of the chain. One or more amino acid residues in a protein may contain modifications such as, but not limited to, glycosylation, phosphorylation or disulfide bonds. A "protein" may comprise one or more polypeptides.

The term "host cell" refers to a cell in which a vector can be propagated and the DNA expressed, which cell may be a prokaryotic cell or a eukaryotic cell. The term also includes any progeny of the subject host cell. It is understood that not all progeny may be identical to the parent cell, since mutations may occur during replication and such progeny are included. In certain embodiments, the host cell of the invention may be a chimeric antigen receptor T cell.

The term "identity" is used to refer to the degree of sequence similarity between two polypeptides or between two nucleic acids. When a position in both of the sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 of 10 positions of two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 of the total 6 positions match). Typically, the comparison is made when the two sequences are aligned to yield maximum identity. Such alignment can be achieved using, for example, the method of Needleman et al (1970) J.mol.biol.,48: 443-. The algorithm of E.Meyers and W.Miller (Compout. appl. biosci., 4:11-17,1988), which has been incorporated into the ALIGN program (version 2.0), can also be used to determine percent identity between two amino acid sequences using a PAM120 weight residue table (weight residue table), a gap length penalty of 12, and a gap penalty of 4. Furthermore, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J.MoI.biol.,48: 444-.

The term "conservative substitutions" means amino acid substitutions that do not adversely affect or alter the intended properties of the Protein/polypeptide comprising the amino acid sequence.conservative substitutions may be introduced, for example, by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis.conservative substitutions include substitutions in which an amino acid residue is replaced with an amino acid residue having a similar side chain, for example, with a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., having a similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds, etc.. families of amino acid residues having similar side chains have been defined in the art, including those having basic side chains (e.g., lysine, arginine and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), branched side chains of β (e.g., threonine, valine, phenylalanine, tyrosine, tryptophan, etc.), the corresponding side chains (e.g., alanine, phenylalanine, serine, tryptophan, phenylalanine, tyrosine, phenylalanine, tryptophan, phenylalanine, tryptophan, phenylalanine, tryptophan, phenylalanine, tryptophan.

The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and an episomal mammalian vector). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are usually present in the form of plasmids. However, other forms of expression vectors are also included, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

The term "Chimeric Antigen Receptor (CAR)" refers to an engineered T cell receptor having an antibody-derived extracellular targeting domain (e.g., ScFv) linked to one or more intracellular signaling domains of the T cell receptor. In the present invention, the term "chimeric antigen receptor T cell" is a T cell that expresses a CAR and has antigen specificity determined by the targeting domain of the CAR (e.g., ScFv). Methods of making CARs (e.g., for cancer therapy) are known in the art, and can be found, for example, in Park TS et al, Trends biotechnol, 29:550-557, 2011; grupps SA et al, N.Engl.J.Med.,368: 1509-; han EQ et al, j.hematol oncol, 6:47,2013, which are all incorporated herein by reference in their entirety.

The term "pharmaceutically acceptable carrier and/or excipient" refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and active ingredient, and which is non-toxic to the cells or mammal to which it is exposed at the dosages and concentrations employed. Including but not limited to: pH adjusting agents, surfactants, adjuvants, ionic strength enhancers, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives. For example, pH adjusting agents include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate salts and gelatin. Diluents include, but are not limited to, water, aqueous buffers (e.g., buffered saline), alcohols and polyols (e.g., glycerol), and the like. Preservatives include, but are not limited to, various antibacterial and antifungal agents, for example, thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like. Stabilizers have the meaning generally understood by those skilled in the art to be capable of stabilizing the desired activity of the active ingredient in a medicament, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or degradation products thereof (such as lactalbumin hydrolysate), and the like.

The term "effective amount" refers to an amount sufficient to obtain, or at least partially obtain, the desired effect. For example, a prophylactically effective amount (e.g., tumor or infection) refers to an amount sufficient to prevent, or delay the onset of a disease (e.g., tumor or infection); a therapeutically effective amount for a disease is an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. It is well within the ability of those skilled in the art to determine such effective amounts. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient, e.g., age, weight and sex, the mode of administration of the drug, and other treatments administered concurrently, and the like.

The term "prevention" refers to a method performed in order to prevent or delay the onset of a disease or disorder or condition (e.g., tumor or infection) in a subject or if its effects are minimized. The term "treatment" refers to a method performed in order to obtain a beneficial or desired clinical result. Beneficial or desired clinical results include, but are not limited to, decreasing the rate of disease progression, ameliorating or palliating the disease state, and regression or improved prognosis, whether detectable or undetectable. The amount of therapeutic agent effective to alleviate any particular disease symptom may vary depending on factors such as the disease state, age and weight of the patient, and the ability of the drug to elicit a desired response in the subject. Whether a symptom of a disease is alleviated can be assessed by any clinical measure that is typically used by a physician or other skilled healthcare provider to assess the severity or progression of the symptom.

The term "delaying the progression of a disease" means delaying, hindering, slowing, delaying, stabilizing, and/or delaying the development of a disease (such as cancer or tumor immunity). This delay can be of varying lengths of time depending on the disease history and/or the individual being treated. As will be apparent to those skilled in the art, a sufficient or significant delay may essentially encompass prevention, as the individual does not develop disease. For example, the formation of late stage cancer, such as metastasis, may be delayed.

The term "reducing or inhibiting cancer recurrence" means reducing or inhibiting tumor or cancer recurrence or tumor or cancer progression. As disclosed herein, cancer recurrence and/or cancer progression includes, but is not limited to, cancer metastasis.

The terms "inhibit" or "block" (e.g., refer to inhibiting/blocking the binding of CD155 or CD112 to TIGIT on a cell) are used interchangeably and include both partial and complete inhibition/blocking to, for example, at least about 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%.

The term "cytotoxicity" refers to, for example, the toxicity of an immunotoxin to the cell intended to be targeted, but not to other cells of the organism.

The term "chemotherapy" or "chemotherapy" refers to any chemical agent administered for therapeutic use in the treatment of a disease characterized by abnormal cell growth. Such diseases include diseases characterized by proliferative growth, such as tumors. More than one agent is administered in combination with chemotherapy to treat cancer.

The terms "patient" and "subject", "individual" and "subject" refer to any human or non-human animal, particularly a human, undergoing prophylactic or therapeutic treatment. For example, the antibodies, methods, and compositions described herein can be used to treat a subject having cancer. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cattle, chickens, amphibians, reptiles, and the like.

The term "immune cell" includes cells having hematopoietic origin and playing a role in the immune response, such as lymphocytes, e.g., B cells and T cells; a natural killer cell; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils and granulocytes.

The term "immunotherapy" refers to the treatment of a subject suffering from or at risk of developing a disease or the recurrence of an infectious disease by a method that includes inducing, enhancing, suppressing or otherwise modifying an immune response.

The term "effector functions" refers to those biological activities attributable to the Fc region of an antibody (either the native sequence Fc region or the amino acid sequence variant Fc region), and which vary with the antibody isotype. Examples of antibody effector functions include, but are not limited to: fc receptor binding affinity, antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), down-regulation of cell surface receptors (e.g., B cell receptors), B cell activation, cytokine secretion, half-life/clearance of antibodies and antigen-antibody complexes, and the like. Methods of altering effector functions of antibodies are known in the art, for example by introducing mutations in the Fc region.

The term "antibody-dependent cell-mediated cytotoxicity (ADCC)" refers to a cytotoxic form in which Ig binds specifically to antigen-attached target cells by binding to fcrs present on cytotoxic cells (e.g., NK cells, neutrophils, or macrophages) and then kills the target cells by secretion of cytotoxins. Methods for detecting ADCC activity of an antibody are known in the art and can be assessed, for example, by determining the binding activity between the antibody to be tested and an FcR (e.g., CD16 a).

The term "Complement Dependent Cytotoxicity (CDC)" refers to the cytotoxic form of the complement cascade activated by binding complement component C1q to antibody Fc. Methods for detecting CDC activity of an antibody are known in the art and can be assessed, for example, by measuring binding activity between the antibody to be tested and an Fc receptor (e.g., C1 q).

The term "autoimmunity" refers to the state of immune tolerance in which certain amounts of autoreactive T cells and autoantibodies are ubiquitous in the peripheral immune system of all individuals, helping to eliminate the senescent and degenerative components of the body, and having important physiological significance in maintaining the stability of the immune system's autoimmunity.

The term "immune tolerance" refers to the ability of the immune system of the normal body to distinguish between "self" and "non-self", to be able to mount an immune response to non-self antigens, and to be in a state of no or weak response to the principle of self-antibodies.

The terms "cancer," "tumor," and "tumors" are used interchangeably and refer to a broad class of diseases characterized by the uncontrolled growth of abnormal cells in vivo. Unregulated cell division may lead to the formation of malignant tumors or cells that invade adjacent tissues and may migrate to remote sites in the body through the lymphatic system or the bloodstream. Cancer includes benign and malignant cancers as well as dormant tumors or micrometastases. Cancer also includes hematologic malignancies.

The term "hematological malignancy" includes lymphoma, leukemia, myeloma or lymphoid malignancies, as well as spleen cancer and lymph node tumors. Exemplary lymphomas include B-cell lymphomas and T-cell lymphomas. B cell lymphomas, including, for example, hodgkin lymphoma. T cell lymphomas, including, for example, cutaneous T cell lymphomas. Hematological malignancies also include leukemias, such as secondary leukemia or acute lymphocytic leukemia. Hematological malignancies also include myelomas (e.g., multiple myeloma) and other hematological and/or B-cell or T-cell related cancers.

The term "dysfunction" in the context of immune dysfunction refers to a state of reduced immune responsiveness to antigenic stimulation. The term includes the common elements of both depletion and/or anergy that antigen recognition can occur, but the subsequent immune response is ineffective to control infection or tumor growth. The term "dysfunction" also includes a lack of perception or response to antigen recognition, in particular, an impaired ability to translate antigen recognition into downstream T cell effector functions, such as proliferation, cytokine production (e.g., IL-2) and/or target cell killing.

Homologous antibodies

The present invention also relates to variants comprising heavy and light chain variable regions comprising amino acid sequences that are homologous to the amino acid sequences of the antibodies or antigen-binding fragments thereof provided by any of the above aspects, and wherein the variants retain the desired functional properties of the anti-TIGIT antibodies of the invention.

For example, the invention provides a humanized TIGIT-binding antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein: (a) the heavy chain variable region comprises a heavy chain variable region substantially identical to a light chain variable region selected from the group consisting of SEQ ID NOs: 31. 33, 35, 37, 39, 41 and 43, or an amino acid sequence that is at least 70% homologous; more preferably, the heavy chain variable region comprises a heavy chain variable region comprising a heavy chain variable region substituted with a heavy chain variable region selected from the group consisting of SEQ ID NOs: 31. 33, 35, 37, 39, 41 and 43, at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous; (b) the light chain variable region comprises a light chain variable region substantially identical to a light chain variable region selected from the group consisting of SEQ ID NOs: 32. 34, 36, 38, 40, 42, and 44, amino acid sequences that are at least 70% homologous; more preferably, the light chain variable region comprises a heavy chain variable region substantially identical to a light chain variable region selected from SEQ ID NOs: 32. 34, 36, 38, 40, 42, and 44, at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous.

Methods of sequence alignment for comparison are well known in the art. Various programs and alignment algorithms are described in: SmithTF and Waterman MS, adv.appl.math, 2:482,1981; higgins DG and Sharp PM, CABIOS5:151,1989. Altschul SF et al, Nature Genet, 6:119,1994 provide detailed ideas for sequence alignment methods and homology calculations.

Improvement of stability of anti-TIGIT antibodies

Preferably, the stability of the anti-TIGIT antibody or antigen-binding fragment thereof of the present invention is improved. In general, the thermal melting temperature (Tm) value of an antibody can be used as an excellent index of thermal stability of an antibody, and can also be used as an index of the storage period of an antibody. Lower Tm value indicates lower association/stability; higher Tm value indicates less association formation and higher stability. Therefore, it is preferable to provide an antibody having a high Tm value. In one non-limiting embodiment of the invention, antibodies with higher Tm values are selected. In some embodiments, antibodies are selected that have a Tm value of at least 50 ℃, 55 ℃, 60 ℃,65 ℃, 70 ℃,75 ℃, 80 ℃,85 ℃, 90 ℃, 95 ℃, or more than 100 ℃.

Stability of an antibody can be determined using any method known in the art, e.g., differential scanning fluorescence analysis (DSF), Circular Dichroism (CD), intrinsic protein fluorescence, differential scanning calorimetry, spectroscopy, light scattering (e.g., Dynamic Light Scattering (DLS) and Static Light Scattering (SLS) and self-interaction chromatography (SIC).

DSF assays monitor thermal unfolding of proteins in the presence of fluorescent dyes and are typically performed by using real-time PCR instruments. DSF can be applied to a variety of proteins, including antibody light and heavy chains. Fluorescent dyes used for DSF have high fluorescence in a non-polar environment, such as hydrophobic sites on unfolded proteins, compared to aqueous solutions where fluorescence is quenched. The various dyes that have been used differ in the fluorescence quantum yield caused by binding to denatured proteins. When protein stability analysis was performed using DSF, the fluorescence intensity was plotted as a function of temperature, resulting in a sigmoidal curve that can be described by a two-state transition. The inflection point (Tm) of the transition curve is calculated using a simple equation, such as Boltzmann's equation (Boltzmann's equation) (see, e.g., Nissen F.H., Bergren's H., and Viddy M.; Nature's laboratory Manual 2:2212-21(2007)) using differential scanning fluorometry to detect ligand interactions that promote protein stability.

The stability of the anti-TIGIT antibody and the fragment thereof of the present invention can be changed by randomly changing each amino acid residue constituting the antibody represented by the humanized anti-TIGIT antibody. In addition, the stability may be changed by substituting a part of the amino acid sequence constituting the humanized antibody represented by the humanized anti TIGIT antibody used for the change with an amino acid sequence constituting a known antibody having a high Tm value, and a part of the amino acid sequence of the humanized antibody represented by the humanized TIGIT antibody used for the change with a view point of the steric structure of the antibody. The position of the amino acid residue to be substituted is not limited, but it may be preferable to change the amino acid residue in FR. The amino acid residues in the CDRs may be changed as appropriate as long as the binding activity to the antigen is not reduced. The number of amino acid residues to be modified is not particularly limited, and may be carried out by substituting a specific fragment of FR with a desired fragment. The substitution of the fragment may be carried out by changing each fragment of FR1, FR2, FR3, FR4 in all FRs, or by combining changes of one or more of each fragment.

Immune cell activation

In some embodiments, an antibody or antigen binding fragment of the invention increases the activity of an immune cell. The increase in immune cell activity can be detected using any method known in the art. An increase in immune cell activity can be detected by measuring the proliferation of immune cells. For example, an increase in NK cell activity can be detected by measuring NK cell proliferation or promotion of cytokine secretion or ADCC enhancement. An increase in the activity of an immune cell can be detected by measuring the increase in CTL or NK cell ADCC effect on a specific target cell or IFN- γ cytokines associated with the stimulation of anti-tumor immunity.

In one embodiment, the increase in NK cell killing activity can be determined by measuring the production of one or more proinflammatory cytokines selected from the group consisting of, but not limited to, IL-1, TNF α, IL-5, IFN- γ, GM-CSF, IL-8, and IL-10.

Use of anti-TIGIT antibodies

The anti-TIGIT antibodies of the invention or antigen binding fragments thereof can be used as therapeutic agents to enhance immune responses to treat diseases such as cancer or infection. In such embodiments, the antibodies described herein are preferably humanized antibodies. For example, an anti-TIGIT antibody or antigen-binding fragment thereof described herein can be administered to cells cultured in vitro or ex vivo, or to a human subject, to enhance immunogenicity of various diseases. Preferred subjects include human patients in need of immune response enhancement. The treatment population includes human patients having a disorder that can be treated by enhancing an immune response (e.g., an NK cell-mediated immune response).

The anti-TIGIT antibodies of the invention, or antigen-binding fragments thereof, can be used to prepare pharmaceutical compositions comprising pharmaceutically acceptable carriers and/or excipients. In some preferred embodiments, the pharmaceutical composition further comprises an additional pharmaceutically active agent. In some preferred embodiments, the pharmaceutically active agent is a drug for the treatment of immune-related diseases. In some preferred embodiments, the pharmaceutically active agent is a drug having anti-tumor activity. In some preferred embodiments, the pharmaceutically active agent is a medicament for the treatment of an infection or infectious disease.

In some embodiments, the amount of anti-TIGIT antibody or antigen-binding fragment thereof in the pharmaceutical compositions of the invention is sufficient to elicit one of the following responses in a subject: (a) induce and/or enhance an immune response; (b) increasing effector T cell activity; (c) increasing Cytotoxic T Lymphocyte (CTL) activity; (d) increasing NK cell activity; (e) inhibiting TIGIT activation; (f) inhibiting TIGIT-mediated signaling; (g) inhibits or blocks binding of CD155 and/or CD112 to TIGIT; (h) reducing the number of regulatory T cells in the tissue or circulation; (i) inhibiting suppression of effector T cells by regulatory T cells; or (j) any combination of (a) - (i).

In some embodiments, the pharmaceutical compositions of the invention further comprise a second antibody or a nucleic acid encoding said second antibody that specifically binds to a receptor or ligand selected from the group consisting of: PD-1, PD-L1, PD-L2, TIM-3, LAG-3, VISTA, CTLA-4, OX40, BTLA, 4-1BB, CD96, CD27, CD28, CD40, LAIR1, CD160, 2B4, TGF-R, KIR, ICOS, GITR, CD3, CD30, BAFFR, HVEM, CD7, LIGHT, SLAMF7, NKp80, B7-H3, and any combination thereof.

The anti-TIGIT antibodies of the invention, or antigen binding fragments thereof, can be used to increase immune cell activity in vitro or in vivo in a subject (e.g., a human), and/or to enhance an immune response in a subject (e.g., a human).

The anti-TIGIT antibodies of the invention, or antigen-binding fragments thereof, can be used to treat a tumor in a subject (e.g., a human). In some preferred embodiments, the tumor is selected from the group consisting of a solid tumor, a hematological tumor (e.g., leukemia, lymphoma, myeloma, e.g., multiple myeloma), and a metastatic, refractory, or recurrent lesion of the cancer; for example, including, but not limited to, esophageal cancer, gastrointestinal cancer, pancreatic cancer, thyroid cancer, colorectal cancer, renal cancer, lung cancer (e.g., non-small cell lung cancer), liver cancer, stomach cancer, head and neck cancer, bladder cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, germ cell cancer, bone cancer, skin cancer, thymus cancer, bile duct cancer, melanoma, mesothelioma, lymphoma, myeloma, sarcoma, glioblastoma, leukemia, or metastatic, refractory, or recurrent lesions of the cancer.

The anti-TIGIT antibodies of the invention, or antigen binding fragments thereof, are useful for treating an infection or infectious disease in a subject (e.g., a human). In some preferred embodiments, the infection or infectious disease is selected from the group consisting of viral, bacterial, fungal, and parasitic infections, including but not limited to HIV, hepatitis, herpes, CMV, EBV, influenza, or tetanus.

The anti-TIGIT antibodies of the invention, or antigen binding fragments thereof, can be used to prepare immunogenic compositions for increasing immune cell activity and/or enhancing an immune response in a subject. Wherein the immunogenic composition comprises the antibody or antigen-binding fragment thereof and an immunogen. In such embodiments, the antibody or antigen-binding fragment thereof of the invention may act as an adjuvant.

In some preferred embodiments, the immunogen is selected from the group consisting of a tumor cell, an antigen (e.g., a protein, polypeptide, or carbohydrate molecule) associated with a tumor, a dendritic cell primed by the antigen, and any combination thereof. In some preferred embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof antigen-binding fragment is for preventing and/or treating a tumor in a subject. In some preferred embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof is used to delay tumor progression in a subject. In some preferred embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof is used to reduce or inhibit tumor recurrence in a subject. In certain preferred embodiments, the tumor to which the antibody or antigen-binding fragment thereof of the present invention is directed is selected from the group consisting of a solid tumor, a hematological tumor (e.g., leukemia, lymphoma, myeloma, e.g., multiple myeloma), and a metastatic, refractory, or recurrent lesion of cancer; for example, including, but not limited to, esophageal cancer, gastrointestinal cancer, pancreatic cancer, thyroid cancer, colorectal cancer, renal cancer, lung cancer (e.g., non-small cell lung cancer), liver cancer, stomach cancer, head and neck cancer, bladder cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, germ cell cancer, bone cancer, skin cancer, thymus cancer, bile duct cancer, melanoma, mesothelioma, lymphoma, myeloma, sarcoma, glioblastoma, leukemia, or metastatic, refractory, or recurrent lesions of the cancer.

In some preferred embodiments, the immunogen is selected from the group consisting of an inactivated or attenuated pathogen, an antigen (e.g., a protein, polypeptide, or carbohydrate molecule) associated with a pathogen, a dendritic cell primed by the antigen, and any combination thereof. In some preferred embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof is for use in the prevention and/or treatment of an infection or infectious disease in a subject. In some preferred embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof is used to delay infection or infectious disease progression in a subject. In some preferred embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof is used to reduce or inhibit infection or infectious disease relapse in a subject. In some preferred embodiments, the infection or infectious disease is selected from the group consisting of viral, bacterial, fungal, and parasitic infections, including but not limited to HIV, hepatitis, herpes, CMV, EBV, influenza, or tetanus.

In some preferred embodiments, the immunogenic composition further comprises a pharmaceutically acceptable carrier and/or excipient. In certain preferred embodiments, in the immunogenic composition, the antibody or antigen-binding fragment thereof of the invention is provided as a separate component from the immunogen or as a component of the same composition. Thus, the antibody or antigen-binding fragment thereof of the invention and the immunogen may be administered simultaneously, separately or sequentially.

The anti-TIGIT antibodies or antigen binding fragments thereof of the invention are useful for the preparation of a medicament for the treatment of an immune related disease, wherein the immune related disease involves T cell dysfunction and/or NK cell dysfunction. In certain preferred embodiments, the T cell dysfunction includes, for example, T cell disability, depletion, or reduced cytokine secretion and the NK cell dysfunction includes, for example, NK cell disability, depletion, or reduced cytokine secretion.

In some preferred embodiments, the method further comprises the step of contacting the immune cell with an additional pharmaceutically active agent in some preferred embodiments, the additional pharmaceutically active agent is an immune response stimulator selected from the group consisting of IL-1, IL-2, IL-3, IL-7, IL-12, IL-15, IL-18, IL-21, IFN- γ, IL-10, TGF- β, GM-CSF, M-CSF, G-CSF, TNF- α, and TNF- β, and any combination thereof.

In some embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof of the invention can enhance T cell activity in a subject. In some embodiments, the anti-TIGIT antibodies or antigen-binding fragments thereof of the invention can enhance cytotoxic T Cell (CTL) activity in a subject. In some embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof of the invention can enhance NK cell activity in a subject.

In some embodiments, the anti-TIGIT antibodies or antigen-binding fragments thereof of the invention can enhance T cell activation and/or response in a subject. In some embodiments, the anti-TIGIT antibodies or antigen-binding fragments thereof of the invention can enhance NK cell activation and/or response in a subject.

The anti-TIGIT antibodies of the invention or antigen binding fragments thereof are used to increase immune cell activity in a subject. In some embodiments, the anti-TIGIT antibodies of the invention or antigen binding fragments thereof are used to enhance an immune response in a subject.

The anti-TIGIT antibodies or antigen-binding fragments thereof of the invention are useful for preventing and/or treating a tumor, delaying tumor progression, and/or reducing or inhibiting tumor recurrence in a subject. The invention also relates to methods of depleting Treg cells from the tumor microenvironment of a subject having a tumor, the methods comprising administering to the subject a therapeutically effective amount of an anti-TIGIT antibody and antigen binding fragments thereof described herein, wherein the anti-TIGIT antibody and antigen binding fragments thereof comprise an Fc capable of stimulating depletion of Treg cells in the tumor microenvironment. The Fc can, for example, be an Fc having effector function or enhanced effector function, e.g., an Fc that binds to or has enhanced binding to one or more activated Fc receptors.

The anti-TIGIT antibodies of the invention, or antigen-binding fragments thereof, are used to prevent and/or treat infection or infectious disease, delay infection or infectious disease progression, and/or reduce or inhibit infection or infectious disease relapse in a subject.

In any of the above uses, the anti-TIGIT antibody or antigen-binding fragment thereof of the present invention may be used in combination. Thus, in some embodiments, an effective amount of any one or more (e.g., 1, 2, 3, 4 or more) of the anti-TIGIT antibodies of the invention, or antigen-binding fragments thereof, is administered to an individual to treat or delay progression of the immune-related disease (e.g., tumor) in the individual or to increase, enhance or stimulate an immune response or function in the individual.

The use as described above further comprises the combination with a second therapy selected from the group consisting of surgery, chemotherapy, radiation therapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy and any combination. Administration of the antibodies, antigen-binding fragments, pharmaceutical compositions disclosed herein may be accompanied by administration of other anti-cancer agents or other treatments (e.g., surgical resection of a tumor). Any suitable anti-cancer agent can be administered in combination with the antibodies, antibody fragments, pharmaceutical compositions, immunogenic compositions disclosed herein. Exemplary anticancer agents can include chemotherapeutic agents (e.g., mitotic inhibitors), alkylating agents (e.g., nitrogen mustards), antimetabolites (e.g., folic acid analogs), natural products (e.g., Vinca alkaloids, e.g., platinum coordination complexes), hormones and antagonists (e.g., adrenal corticosteroids), immunomodulators (e.g., bripirimimine, Upjohn), and the like. Other anti-cancer therapies include other antibodies that specifically target cancer cells.

A therapeutically effective dose of an anti-TIGIT antibody or antigen-binding fragment thereof of the invention will depend on the severity of the disease and the health of the patient. In addition, the amount required for administration will depend in part on the binding affinity of the antibody to the antigen and the pharmacokinetic properties of the antibody in the subject. A typical range for administration of a therapeutically effective dose of an anti-TIGIT antibody or antigen-binding fragment thereof of the invention (by way of non-limiting example) may be from about 0.1mg/kg body weight to about 50mg/kg body weight. The usual frequency of administration may range, for example, from twice daily to once a week. The dose and method of administration may vary depending on the body weight, age and symptoms of the patient, and are appropriately selected by those skilled in the art.

The anti-TIGIT antibodies of the invention, or antigen binding fragments thereof, can be used to detect the presence of TIGIT in a sample, the method comprising contacting the sample with an antibody of the invention, or antigen binding fragment thereof, under conditions that allow formation of a complex between the antibody, or antigen binding fragment thereof, and TIGIT, and detecting the formation of the complex. In certain embodiments, the method comprises the steps of: i. incubating the sample with an anti-TIGIT antibody of the invention, or an antigen-binding fragment thereof; detecting bound TIGIT using a detectable probe; comparing the amount of ii to a standard curve obtained from a reference sample containing a known amount of TIGIT; calculating the amount of TIGIT in the sample from the standard curve.

The anti-TIGIT antibodies of the invention or antigen binding fragments thereof can be used to prepare diagnostic or therapeutic kits comprising the antibodies of the invention or antigen binding fragments thereof, and optionally instructions for use. The pharmaceutical compositions of the invention may be used to prepare diagnostic or therapeutic kits comprising the pharmaceutical compositions of the invention, and optionally instructions for use.

Cancer treatment

The anti-TIGIT antibodies of the invention, or antigen-binding fragments, vectors, or host cells thereof, can be used to prepare pharmaceutical compositions comprising pharmaceutically acceptable carriers and/or excipients. In certain preferred embodiments, the pharmaceutical composition comprises an anti-TIGIT antibody or antigen-binding fragment thereof of the invention. In certain preferred embodiments, the pharmaceutical composition comprises a vector or host cell of the invention. In such embodiments, the isolated nucleic acid molecule comprised by the vector comprises a nucleotide sequence encoding a chimeric antigen receptor that further comprises a nucleotide sequence encoding an antibody of the invention or an antigen-binding fragment thereof (e.g., ScFv); the host cell comprises the isolated nucleic acid molecule or vector as described previously. In certain preferred embodiments, the isolated nucleic acid molecule encodes a chimeric antigen receptor comprising an antigen-binding fragment (e.g., ScFv) of an antibody of the invention. In certain preferred embodiments, the host cell is a T cell. In certain preferred embodiments, the host cell is a chimeric antigen receptor T cell (CAR-T).

In some preferred embodiments, the pharmaceutical composition further comprises an additional pharmaceutically active agent. In some preferred embodiments, the pharmaceutical composition is a medicament for the treatment of an immune-related disorder. In some preferred embodiments, the pharmaceutical composition is a drug with anti-tumor activity.

In some embodiments, a therapeutically effective amount of an anti-TIGIT antibody, or antigen-binding fragment, vector, or host cell thereof, in a pharmaceutical composition of the invention is sufficient to elicit in a subject one of the following responses: (a) induce and/or enhance an immune response; (b) increasing effector T cell activity; (c) increasing Cytotoxic T Lymphocyte (CTL) activity; (d) increasing NK cell activity; (e) inhibiting TIGIT activation; (f) inhibiting TIGIT-mediated signaling; (g) inhibits or blocks binding of CD155 and/or CD112 to TIGIT; (h) reducing the number of regulatory T cells in the tissue or circulation; (i) inhibiting suppression of effector T cells by regulatory T cells; or (j) any combination of (a) - (i). In some preferred embodiments, the subject has a tumor or cancer-related disease.

In some embodiments, an anti-TIGIT antibody or antigen-binding fragment thereof of the invention may be combined with an immunogen to form an immunogenic complex. In some preferred embodiments, the immunogen is selected from the group consisting of a tumor-associated antigen (e.g., a protein, polypeptide, or carbohydrate molecule), a tumor cell, a dendritic cell primed by the antigen, and any combination thereof.

In some preferred embodiments, the antibodies and antigen binding fragments, vectors or host cells thereof described above are used for preventing and/or treating tumors in a subject. In some preferred embodiments, the antibodies and antigen binding fragments, vectors or host cells thereof described above are used to delay tumor progression in a subject. In some preferred embodiments, the antibodies and antigen-binding fragments, vectors, or host cells thereof described above are used to reduce or inhibit tumor recurrence in a subject. In some preferred embodiments, the antibody or antigen-binding fragment thereof described above may be provided simultaneously with or prior to the immunogen.

The anti-TIGIT antibodies of the invention or antigen binding fragments thereof are useful for treating or delaying progression of an immune related disease. In some embodiments, the immune-related disease involves T cell dysfunction. In some embodiments, the T cell dysfunction includes, for example, T cell disability, depletion, or reduced cytokine secretion. In some embodiments, the immune-related disease involves NK cell dysfunction. In some embodiments, the NK cell dysfunction comprises NK cell disability, depletion, or reduced cytokine secretion. In some embodiments, the immune-related disease comprises a tumor. For example, such that the growth of cancerous tumors is inhibited or reduced, and/or tumors regress. In some embodiments, the anti-TIGIT antibodies of the invention, or antigen binding fragments thereof, can be used to increase, enhance, or stimulate an immune response or function thereof in an individual in need thereof.

In some embodiments, the anti-TIGIT antibodies or antigen-binding fragments thereof, pharmaceutical compositions, and immunogenic compositions of the invention can enhance T cell activity in a subject. In some embodiments, the antibodies and antigen-binding fragments thereof, pharmaceutical compositions, and immunogenic compositions of the invention can enhance cytotoxic T Cell (CTL) activity in a subject. In some embodiments, the antibodies and antigen-binding fragments thereof, pharmaceutical compositions, and immunogenic compositions of the invention can enhance NK cell activity in a subject. In some embodiments, the cellular activity described above is associated with tumor immunity.

In some embodiments, the anti-TIGIT antibodies or antigen-binding fragments thereof, pharmaceutical compositions, and immunogenic compositions of the invention can enhance T cell activation and/or response in a subject. In some embodiments, the antibodies and antigen binding fragments thereof, pharmaceutical compositions, and immunogenic compositions of the invention can enhance NK cell activation and/or response in a subject. In some embodiments, the cell activation and/or response described above is associated with tumor immunity.

In some embodiments, the anti-TIGIT antibodies or antigen-binding fragments thereof, pharmaceutical compositions, and immunogenic compositions of the invention can enhance immune cell activity and/or enhance an immune response in a subject. In some embodiments, the cellular activity or immune response described above is associated with tumor immunity.

In some preferred embodiments, the anti-TIGIT antibodies or antigen-binding fragments thereof, vectors, host cells, pharmaceutical compositions, and immunogenic compositions described above are used to prevent and/or treat a tumor in a subject. In some preferred embodiments, the antibodies and antigen binding fragments thereof, vectors, host cells, pharmaceutical compositions, and immunogenic compositions described above are used to delay tumor progression in a subject. In some preferred embodiments, the antibodies and antigen binding fragments thereof, vectors, host cells, pharmaceutical compositions, and immunogenic compositions described above are used to reduce or inhibit tumor recurrence in a subject.

In some embodiments, an effective amount of any one or more (e.g., 1, 2, 3, 4 or more) of the anti-TIGIT antibodies of the invention, or antigen-binding fragments thereof, is administered to the subject.

Alternatively, the anti-TIGIT antibody or antigen-binding fragment thereof can be used in combination with another agent (e.g., other antibody, other immunogenic agent, or standard cancer therapy). the anti-TIGIT antibody or antigen-binding fragment thereof can be used in combination with other antibodies that activate the immune response of the host, including but not limited to T cell co-stimulatory molecules such as inhibitors of PD-1, PD-L1, and CTLA-4 (e.g., U.S. Pat. No. 5,811,097), OX-40 (Weing berAD et al, Immunol.,164: 2160-.

The combination of anti-TIGIT antibodies or antigen binding fragments thereof of the present invention uses a nucleic acid comprising or encoding a second antibody that specifically binds to a receptor or ligand selected from the group consisting of: PD-1, PD-L1, PD-L2, TIM-3, LAG-3, VISTA, CTLA-4, OX40, BTLA, 4-1BB, CD96, CD27, CD28, CD40, LAIR1, CD160, 2B4, TGF-R, KIR, ICOS, GITR, CD3, CD30, BAFFR, HVEM, CD7, LIGHT, SLAMF7, NKp80, B7-H3, and any combination thereof.

The anti-TIGIT antibodies of the invention or antigen binding fragments thereof may be administered in combination with an immunogenic agent, such as cancer cells, purified tumor antigens (including recombinant proteins, peptides and carbohydrate molecules) or cells transfected with genes encoding immunostimulatory cytokines (He YF et al j. immunol.173: 4919-. The anti-TIGIT antibodies or antigen binding fragments thereof of the invention can be co-administered with DC cells to effectively enhance anti-tumor responses. The anti-TIGIT antibodies of the invention, or antigen binding fragments or combinations thereof, can also be administered in combination with standard cancer therapies (e.g., surgery, radiation, and chemotherapy).

In some preferred embodiments, the tumors described above include, but are not limited to, solid tumors, hematological tumors (e.g., leukemia, lymphoma, myeloma, e.g., multiple myeloma), and metastatic, refractory, or recurrent lesions of cancer; for example, including, but not limited to, esophageal cancer, gastrointestinal cancer, pancreatic cancer, thyroid cancer, colorectal cancer, renal cancer, lung cancer (e.g., non-small cell lung cancer), liver cancer, stomach cancer, head and neck cancer, bladder cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, germ cell cancer, bone cancer, skin cancer, thymus cancer, bile duct cancer, melanoma, mesothelioma, lymphoma, myeloma, sarcoma, glioblastoma, leukemia, or metastatic, refractory, or recurrent lesions of the cancer.

The use described above may further comprise a second therapy selected from the group consisting of surgery, chemotherapy, radiation therapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof.

The antibodies or antigen binding fragments thereof, pharmaceutical compositions or immunogenic compositions of the invention may be administered by any suitable method known in the art, including, but not limited to, subcutaneous, intravenous, intramuscular, topical, oral, transdermal, intraperitoneal, intraorbital, by implantation, by inhalation, intrathecal, intraventricular or intranasal administration. However, for many therapeutic uses, the preferred route/mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). The skilled artisan will appreciate that the route and/or mode of administration will vary depending on the intended purpose. In a preferred embodiment, the antibody or antigen-binding fragment thereof, pharmaceutical composition or immunogenic composition of the invention is administered by intravenous infusion or injection. In some embodiments, the individual or subject is a human.

A therapeutically effective dose of an antibody or antigen-binding fragment thereof of the invention will depend on the severity of the disease and the health of the patient. In addition, the amount required for administration will depend in part on the binding affinity of the antibody to the antigen and the pharmacokinetic properties of the antibody in the subject. A typical range for administering a therapeutically effective dose of an antibody or antigen-binding fragment thereof of the invention may be, by way of non-limiting example, from about 0.1mg/kg body weight to about 50mg/kg body weight. The usual frequency of administration may range, for example, from twice daily to once a week. The dose and method of administration may vary depending on the body weight, age and symptoms of the patient, and are appropriately selected by those skilled in the art.

Infectious diseases

The anti-TIGIT antibodies of the invention, or antigen binding fragments thereof, can be used to prepare pharmaceutical compositions comprising pharmaceutically acceptable carriers and/or excipients. In some preferred embodiments, the pharmaceutical composition further comprises an additional pharmaceutically active agent. In some preferred embodiments, the pharmaceutical composition is a medicament for the treatment of an immune-related disorder. In some preferred embodiments, the pharmaceutical composition is a medicament for the treatment of an infection or infectious disease.

In some embodiments, a therapeutically effective amount of an anti-TIGIT antibody or antigen-binding fragment thereof in a pharmaceutical composition of the invention is sufficient to elicit one of the following responses in a subject: (a) induce and/or enhance an immune response; (b) increasing effector T cell activity; (c) increasing Cytotoxic T Lymphocyte (CTL) activity; (d) increasing NK cell activity; (e) inhibiting TIGIT activation; (f) inhibiting TIGIT-mediated signaling; (g) inhibits or blocks binding of CD155 and/or CD112 to TIGIT; (h) reducing the number of regulatory T cells in the tissue or circulation; (i) inhibiting suppression of effector T cells by regulatory T cells; or (j) any combination of (a) - (i). In some preferred embodiments, the subject has an infection or infectious disease.

In some embodiments, an anti-TIGIT antibody or antigen-binding fragment thereof of the invention may be combined with an immunogen to form an immunogenic complex. In some preferred embodiments, the immunogen is selected from the group consisting of an antigen (e.g., a protein, polypeptide, or carbohydrate molecule) associated with a pathogen, an inactivated or attenuated pathogen, a dendritic cell primed with the antigen, and any combination thereof.

In some preferred embodiments, the antibodies and antigen binding fragments thereof are used to prevent and/or treat infection or infectious disease in a subject. In some preferred embodiments, the antibodies and antigen binding fragments thereof are used to delay infection or infectious disease progression in a subject. In some preferred embodiments, the antibodies and antigen binding fragments thereof are used to reduce or inhibit infection or infectious disease recurrence in a subject. In some preferred embodiments, the antibody or antigen-binding fragment thereof may be provided simultaneously with or prior to the immunogen.

The anti-TIGIT antibodies of the invention or antigen binding fragments thereof are useful for treating or delaying progression of an immune related disease. In some embodiments, the immune-related disease involves T cell dysfunction. In some embodiments, the T cell dysfunction includes, for example, T cell disability, depletion, or reduced cytokine secretion. In some embodiments, the immune-related disease involves NK cell dysfunction. In some embodiments, the NK cell dysfunction includes, for example, NK cell disability, depletion, or reduced cytokine secretion. In some embodiments, the immune-related disease comprises an infection or an infectious disease.

In some embodiments, the anti-TIGIT antibodies or antigen-binding fragments thereof, pharmaceutical compositions, and immunogenic compositions of the invention can enhance T cell activity in a subject. In some embodiments, the anti-TIGIT antibodies or antigen-binding fragments thereof, pharmaceutical compositions, and immunogenic compositions of the invention can enhance cytotoxic T Cell (CTL) activity in a subject. In some embodiments, the anti-TIGIT antibodies or antigen-binding fragments thereof, pharmaceutical compositions, and immunogenic compositions of the invention can enhance NK cell activity in a subject. In some embodiments, the cellular activity described above is immunologically correlated with an infection or infectious disease.

In some embodiments, the anti-TIGIT antibodies or antigen-binding fragments thereof, pharmaceutical compositions, and immunogenic compositions of the invention can enhance T cell activation and/or response in a subject. In some embodiments, the antibodies and antigen binding fragments thereof, pharmaceutical compositions, and immunogenic compositions of the invention can enhance NK cell activation and/or response in a subject. In some embodiments, the cell activation and/or response described above is immunologically correlated with an infection or infectious disease.

In some preferred embodiments, the anti-TIGIT antibodies or antigen-binding fragments thereof, pharmaceutical compositions, and immunogenic compositions are used to prevent and/or treat infection or infectious disease in a subject. In some preferred embodiments, the anti-TIGIT antibodies or antigen-binding fragments thereof, pharmaceutical compositions, and immunogenic compositions are used to delay infection or infectious disease progression in a subject. In some preferred embodiments, the anti-TIGIT antibodies or antigen-binding fragments thereof, pharmaceutical compositions, and immunogenic compositions are used to reduce or inhibit infection or infectious disease relapse in a subject.

In some embodiments, an effective amount of any one or more (e.g., 1, 2, 3, 4, or more) of the anti-TIGIT antibodies of the invention, or antigen-binding fragments thereof, can be administered to a subject.

The anti-TIGIT antibodies of the invention or antigen binding fragments thereof can be used alone to treat infection or infectious disease. Alternatively, the anti-TIGIT antibodies of the invention or antigen binding fragments thereof can be used in combination with other regimens (e.g., other antibodies, standard therapy, or chemoradiotherapy). The combination of anti-TIGIT antibodies or antigen binding fragments thereof of the present invention uses a nucleic acid comprising or encoding a second antibody that specifically binds to a receptor or ligand selected from the group consisting of: PD-1, PD-L1, PD-L2, TIM-3, LAG-3, VISTA, CTLA-4, OX40, BTLA, 4-1BB, CD96, CD27, CD28, CD40, LAIR1, CD160, 2B4, TGF-R, KIR, ICOS, GITR, CD3, CD30, BAFFR, HVEM, CD7, LIGHT, SLAMF7, NKp80, B7-H3, and any combination thereof.

Infections are broadly classified as bacterial, viral, fungal or parasitic based on the type of infectious organism or substance involved. Other less common types of infections include, for example, infectious diseases involving rickettsia, mycoplasma, and matter. Examples of bacteria, viruses, fungi and parasites that cause infections are well known in the art. The infection may be acute, subacute, chronic or latent, and it may be local or systemic. In some embodiments, the immune-related disorder comprises acute and chronic infections of unknown cause. In some embodiments, the infection or infectious disease includes, but is not limited to, viral infection, bacterial infection, fungal infection, and parasitic infection.

Infectious diseases are caused by infectious bacteria. Non-limiting examples of infectious bacteria include: helicobacter pylori, Borrelia burgdorferi, Mycobacterium species (e.g. Mycobacterium tuberculosis), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Streptococcus pyogenes (group A streptococci), Streptococcus pneumoniae, Campylobacter species, enterococcus species, Haemophilus influenzae, Bacillus anthracis, Corynebacterium diphtheriae, Erythrinum erythrosepticum, Clostridium tetani, Clostridium nucleatum, Streptococcus moniliformis, Treponema pallidum. Other infectious organisms (e.g., protists) include: plasmodium falciparum and toxoplasma. The compositions and methods described herein are expected to be useful in the treatment of infections caused by these pathogens.

Infectious diseases are caused by infectious viruses. The viral antigen may be any antigen that is present in an infectious virus and induces an immune response in a subject. Non-limiting examples of infectious viruses include: HIV, poliovirus, yellow fever virus, rotavirus, coronavirus, mumps virus, ebola virus, echovirus, measles virus, influenza virus, dengue virus, hemorrhagic fever virus, coxsackie virus, reovirus, hepatitis b virus, parvovirus, papilloma virus, adenoviridae, herpes virus, smallpox virus, rabies virus, and unclassified viruses (e.g., the causative agent of spongiform encephalopathy). The compositions and methods described herein are expected to be useful in treating infections caused by these viral pathogens.

Infectious diseases are caused by infectious fungi. Non-limiting examples of infectious pathogenic fungi include Candida albicans, Candida krusei, Cryptococcus, Histoplasma, Aspergillus niger, Rhizopus, Sporothrix schenckii, Coccidioides immaturus, and Histoplasma capsulatum.

Infectious diseases are caused by infectious parasites. Non-limiting examples of infectious pathogenic parasites include entomorphus dysenteriae, dactylopilus coli, proteus forbesii, acanthamoeba species, giardia lamblia, cryptosporidium species, pneumocystis carinii, plasmodium vivax, babesia parvum, trypanosoma brucei, trypanosoma cruzi, leishmania donovani, toxoplasma gondii and teres brazilianae.

Vaccine adjuvant

The anti-TIGIT antibodies of the invention, or antigen-binding fragments thereof, can be administered in combination with an immunogen (e.g., a vaccine) to enhance an antigen-specific immune response. Accordingly, the present invention also relates to a method of increasing an immune response to an immunogen in a subject comprising administering to the subject: (i) an immunogen; (ii) the anti-TIGIT antibodies, or antigen binding fragments thereof, of the invention result in an enhanced immune response to the immunogen in a subject. The invention also relates to the use of an anti-TIGIT antibody or antigen-binding fragment thereof of the invention as an adjuvant or for the preparation of an immunogenic composition, wherein the immunogenic composition comprises an antibody or antigen-binding fragment thereof of the invention and an immunogen.

In some preferred embodiments, the immunogen is selected from the group consisting of a tumor-associated antigen (e.g., a protein, polypeptide, or carbohydrate molecule), a tumor cell, a dendritic cell primed by the antigen, and any combination thereof. In such embodiments, the methods can further be used to prevent and/or treat a tumor, delay tumor progression, and/or reduce or inhibit tumor recurrence in a subject.

In some preferred embodiments, the immunogen is selected from the group consisting of an antigen (e.g., a protein, polypeptide, or carbohydrate molecule) associated with a pathogen, an inactivated or attenuated pathogen, a dendritic cell primed by the antigen, and any combination thereof. In such embodiments, the methods can further be used to prevent and/or treat infection or infectious disease, delay progression of infection or infectious disease, and/or reduce or inhibit relapse of infection or infectious disease in a subject.

Pharmaceutical composition

The anti-TIGIT antibody (preferably a humanized antibody) or antigen-binding fragment thereof of the present invention can be used to prepare a pharmaceutical or sterile composition by mixing the anti-TIGIT antibody or antigen-binding fragment thereof with a pharmaceutically acceptable carrier and/or excipient. The pharmaceutical composition can include one or a combination (e.g., two or more different) of the anti-TIGIT antibodies of the invention or antigen-binding fragments thereof. For example, a pharmaceutical composition of the invention may comprise a combination of antibodies or antibody fragments with complementary activity that bind to different epitopes on a target antigen.

The vectors or host cells of the invention may also be used in the preparation of pharmaceutical or sterile compositions by mixing them with pharmaceutically acceptable carriers and/or excipients. In such embodiments, the isolated nucleic acid molecule comprised by the vector comprises a nucleotide sequence encoding a chimeric antigen receptor that further comprises a nucleotide sequence encoding an antibody of the invention or an antigen-binding fragment thereof (e.g., ScFv); the host cell comprises the isolated nucleic acid molecule or vector as described previously. In certain preferred embodiments, the isolated nucleic acid molecule encodes a chimeric antigen receptor comprising an antigen-binding fragment (e.g., ScFv) of an antibody of the invention. In certain preferred embodiments, the host cell is a T cell. In certain preferred embodiments, the host cell is a chimeric antigen receptor T cell (CAR-T).

The pharmaceutical composition of the present invention may be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, troches, suppositories, injections (including injections, sterile powders for injections and concentrated solutions for injections), inhalants, sprays and the like. The preferred dosage form depends on the intended mode of administration and therapeutic use. In certain embodiments, the formulation of the pharmaceutical composition may be prepared by mixing with a pharmaceutically acceptable carrier and/or excipient, for example, in the form of a lyophilized powder, slurry, aqueous solution or suspension.

The term "pharmaceutically acceptable" means that the molecular entity, molecular fragment, or composition does not produce an adverse, allergic, or other untoward reaction when properly administered to an animal or human. Specific examples of some substances that may serve as pharmaceutically acceptable carriers or components thereof include sugars (e.g., lactose), starch, cellulose and its derivatives, vegetable oils, gelatin, polyols (e.g., propylene glycol), alginic acid, and the like.

In some embodiments, a therapeutically effective amount of an anti-TIGIT antibody, or antigen-binding fragment, vector, or host cell thereof, in a pharmaceutical composition of the invention is sufficient to elicit in a subject one of the following responses: (a) induce and/or enhance an immune response; (b) increasing effector T cell activity; (c) increasing Cytotoxic T Lymphocyte (CTL) activity; (d) increasing NK cell activity; (e) inhibiting TIGIT activation; (f) inhibiting TIGIT-mediated signaling; (g) inhibits or blocks binding of CD155 and/or CD112 to TIGIT; (h) reducing the number of regulatory T cells in the tissue or circulation; (i) inhibiting suppression of effector T cells by regulatory T cells; or (j) any combination of (a) - (i). In some preferred embodiments, the subject has an infection or infectious disease. In some preferred embodiments, the subject has a tumor.

In some preferred embodiments, the pharmaceutical compositions of the invention are used to treat immune-related diseases. Wherein the immune related disease involves T cell dysfunction including, for example, T cell disability, depletion or reduced cytokine secretion and/or NK cell dysfunction including, for example, NK cell disability, depletion or reduced cytokine secretion. In some preferred embodiments, the pharmaceutical composition of the invention is used for the treatment of tumors. In some preferred embodiments, the pharmaceutical compositions of the invention are used to treat infections or infectious diseases.

In some embodiments, the pharmaceutical compositions of the invention can enhance T cell activity in a subject. In some embodiments, the pharmaceutical compositions of the present invention can enhance cytotoxic T Cell (CTL) activity in a subject. In some embodiments, the pharmaceutical compositions of the invention can enhance NK cell activity in a subject.

In some embodiments, the pharmaceutical compositions of the invention can enhance T cell activation and/or response in a subject. In some embodiments, the pharmaceutical compositions of the invention can enhance NK cell activation and/or response in a subject.

In some preferred embodiments, the pharmaceutical composition of the present invention is for use in the prevention and/or treatment of a tumor in a subject. In some preferred embodiments, the pharmaceutical composition is for delaying tumor progression in a subject. In some preferred embodiments, the pharmaceutical composition is for use in reducing or inhibiting tumor recurrence in a subject.

In some preferred embodiments, the pharmaceutical composition of the present invention is for use in the prevention and/or treatment of an infection or infectious disease in a subject. In some preferred embodiments, the pharmaceutical composition is for delaying infection or infectious disease progression in a subject. In some preferred embodiments, the pharmaceutical composition is for use in reducing or inhibiting infection or infectious disease recurrence in a subject.

In some embodiments, the pharmaceutical compositions of the invention comprise a second antibody or a nucleic acid encoding the second antibody that specifically binds to a receptor or ligand selected from the group consisting of: PD-1, PD-L1, PD-L2, TIM-3, LAG-3, VISTA, CTLA-4, OX40, BTLA, 4-1BB, CD96, CD27, CD28, CD40, LAIR1, CD160, 2B4, TGF-R, KIR, ICOS, GITR, CD3, CD30, BAFFR, HVEM, CD7, LIGHT, SLAMF7, NKp80, B7-H3, and any combination thereof.

The anti-TIGIT antibodies or antigen binding fragments thereof of the present invention may be used in conjunction with one or more other therapeutic agents, such as a toxin, cytotoxic agent, radioisotope, immunosuppressive agent or vaccine. The anti-TIGIT antibody or antigen binding fragment thereof can be linked to the therapeutic agent (as an immune complex) or administered separately from the therapeutic agent. In the latter case, the anti-TIGIT antibody or antigen-binding fragment thereof may be administered before, after, or in conjunction with the therapeutic agent, or may be administered in conjunction with other known therapies (e.g., anti-cancer therapies, such as radiation).

Therapeutic agents include, but are not limited to, antineoplastic agents, such as doxorubicin (adriamycin), cisplatin bleomycin sulfate, nitrosourea nitrogen mustard, chlorambucil, and cyclophosphamide hydroxyurea, which are themselves effective only at toxic or sub-toxic levels to the patient. Target-specific effector cells, such as effector cells linked to an anti-TIGIT antibody or antigen-binding fragment thereof of the invention, can also be used as therapeutic agents. The targeted effector cells may be human leukocytes such as macrophages, neutrophils or monocytes. Other cells include eosinophils, NK cells and other cells bearing IgG or IgA receptors. If desired, effector cells can be obtained from the subject to be treated. Target-specific effector cells may be administered as a suspension of cells in a physiologically acceptable solution. The number of cells administered may be in the range of 10⁸-10⁹Within an order of magnitude, but may vary depending on the therapeutic purpose. Generally, the amount is sufficient to achieve localization in a target cell (e.g., a cell of a TIGIT-expressing tumor) and cell killing by, e.g., phagocytosis. Routes of administration also vary, including oral, rectal, transmucosal, enteral, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, dermal, transdermal or intraarterial.

Compositions of the invention having a complement binding site, such as a portion from IgG1, IgG2, or IgG4, or IgM that binds to complement, can also be used in the presence of complement. The compositions of the invention may also be administered with complement, e.g., in combination with C1 q.

Therapy with administration of target-specific effector cells can be performed in conjunction with other techniques for clearing target cells. For example, anti-tumor therapies using the compositions of the present invention and/or effector cells equipped with these compositions are used in combination with chemotherapy. Non-limiting examples of antibody combination therapies of the invention include surgery, chemotherapy, radiation therapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof.

Determination of the appropriate dosage is made by a clinician, for example, using parameters or factors known or suspected in the art to affect treatment. Typically, a dose is started in an amount slightly less than the optimal dose, and it is then increased in smaller increments until the desired or optimal effect is achieved relative to any negative side effects. Important diagnostic measures include, for example, a measure of the symptoms of inflammation or the levels of inflammatory cytokines produced.

The invention provides a container (e.g., a plastic or glass vial, e.g., with a cap or chromatography column, a hollow bore needle, or a syringe barrel) comprising any of the antibodies or antigen-binding fragments of the invention or a pharmaceutical composition thereof. The invention also provides an injection device comprising any of the antibodies or antigen-binding fragments of the invention or a pharmaceutical composition thereof.

Combination therapy

The invention encompasses the use of an anti-TIGIT antibody or antigen-binding fragment thereof in combination with one or more additional active therapeutic agents (e.g., chemotherapeutic agents) or other prophylactic or therapeutic modalities (e.g., radiation therapy). In such combination therapies, the various active agents often have different complementary mechanisms of action, which may result in synergistic effects. Combination therapy includes therapeutic agents that affect an immune response (e.g., enhance or activate a response) and therapeutic agents that affect (e.g., inhibit or kill) tumor/cancer cells. Combination therapy can reduce the likelihood of drug resistant cancer cells. Combination therapy may allow for a reduction in the dosage of one or more of the agents to reduce or eliminate adverse effects associated with one or more of the agents. Such combination therapies may have a synergistic therapeutic or prophylactic effect on the underlying disease, disorder or condition.

"combination" includes therapies that can be administered separately, e.g., formulated separately for individual administration (e.g., as can be provided in a kit), and therapies that can be administered together in a single formulation (i.e., "co-formulation"). In certain embodiments, the anti-TIGIT antibodies or antigen-binding fragments thereof of the invention may be administered sequentially. In other embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof may be administered simultaneously. The anti-TIGIT antibodies of the invention or antigen binding fragments thereof can be used in any combination with at least one other (active) agent.

The anti-TIGIT antibody or antigen binding fragment thereof treatment of the present invention may be combined with other treatments that may be effective against the condition to be treated. Non-limiting examples of antibody combination therapies of the invention include surgery, chemotherapy, radiation therapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof.

Detection method and kit

The present invention provides methods of detecting the presence or level of TIGIT in a sample (e.g., a biological sample, e.g., blood, serum, semen, or urine, or a tissue biopsy sample (e.g., from a hyperproliferative or cancerous lesion)) e.g., in vitro or in vivo. The method may be used for diagnostic purposes, or for non-diagnostic purposes (e.g., the sample is a cell sample, not a sample from a patient). The methods can be used to assess (e.g., monitor the treatment or progression of, diagnosis and/or stage of) a disease described herein (e.g., an immune-related disorder, cancer, or infectious disease) in a subject. The method can comprise the following steps: i. incubating the sample with an antibody or antigen-binding fragment thereof of the invention; detecting a TIGIT complex using a detectable probe; comparing the amount of ii to a standard curve obtained from a reference sample containing a known amount of TIGIT; calculating the amount of TIGIT in the sample from the standard curve. The formation of a complex indicates the presence of TIGIT and may indicate the suitability or need of treatment as described herein. The method may involve, for example, immunohistochemistry, immunocytochemistry, flow cytometry, magnetic beads complexed with antibody molecules, ELISA assays, PCR techniques (e.g., RT-PCR). Generally, anti-TIGIT antibody molecules used in vivo and in vitro diagnostic methods are labeled, directly or indirectly, with a detectable substance to output a detection signal. Suitable detectable substances include, but are not limited to, various biologically active enzymes, prosthetic groups, fluorescent substances, luminescent substances, and radioactive substances.

Also provided are reagents and kits for diagnosis or detection comprising one or more anti-TIGIT antibodies of the invention or antigen binding fragments thereof, optionally comprising instructions for performing the detection methods described above. In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the present invention are detectably labeled. In a preferred embodiment, the kit further comprises a second antibody that specifically recognizes the antibody of the invention or an antigen-binding fragment thereof. Preferably, the second antibody further comprises a detectable label.

In the present invention, the detectable label can be any substance detectable by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics or chemical means.³H、¹²⁵I、³⁵S、¹⁴C or³²P), fluorescent dyes (e.g., Fluorescein Isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), Phycoerythrin (PE), texas red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., Cy7, Alexa 750)), acridine ester compounds, magnetic beads (e.g.,

) A calorimetric label such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to the label-modified avidin (e.g., streptavidin) described above. Patents that teach the use of such markers include, but are not limited to, U.S. Pat. nos. 3,817,837; 3,850,752, respectively; 3,939,350, respectively; 3,996,345; 4,277,437; 4,275,149; and 4,366,241 (all incorporated herein by reference). The markers encompassed by the present invention can be detected by methods known in the art. For example, the radioactive label can be detected using photographic film or scintillation calculators, fluorescent labelsThe object may be detected using a light detector to detect the emitted light. Enzyme labels are generally detected by providing a substrate for the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, and calorimetric labels are detected by simply visualizing the colored label. In certain embodiments, detectable labels as described above can be attached to recombinant proteins of the invention via linkers of different lengths to reduce potential steric hindrance.

In some embodiments, the kit may be used in a variety of detection assays, including, for example, immunoassays, such as ELISA (sandwich or competitive format). The kit may include other additives such as stabilizers, buffers (e.g., blocking buffer or lysis buffer), and the like. In particular, the reagents in the kit may be provided as a dry powder, which is typically lyophilized, including excipients, which, upon dissolution, will provide a reagent solution having the appropriate concentration.

The components of the kit may be pre-attached to the solid support, or may be applied to the surface of the solid support when the kit is used. In some embodiments of the invention, the signal-generating device may be pre-associated with an antibody or antigen-binding fragment thereof of the invention, or it may be desirable to combine one or more components, such as buffers, antibody-enzyme conjugates, enzyme substrates, and the like, prior to use. In particular aspects, enzymes that catalyze the formation of a chemiluminescent or chromogenic product or the reduction of a chemiluminescent or chromogenic substrate are components of the signal producing device. Enzyme labels are generally detected by providing a substrate for the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, and calorimetric labels are detected by simply visualizing the colored label. In certain embodiments, detectable labels as described above can be attached to recombinant proteins of the invention via linkers of different lengths to reduce potential steric hindrance. Such enzymes are well known in the art.

The kit may also include additional reagents such as blocking reagents for reducing non-specific binding to the solid phase surface, washing reagents, enzyme substrates, and the like. The solid phase surface may be in the form of a tube, bead, microtiter plate, microsphere, or other material suitable for immobilizing proteins, peptides, or polypeptides.

The kit also includes a device or apparatus for performing the detection or monitoring methods described herein.

The kit components may be packaged together or in two or more containers. In some embodiments, the container may be a vial containing a sterile lyophilized formulation of a composition suitable for reconstitution.

Also provided are therapeutic kits comprising one or more anti-TIGIT antibodies or antigen-binding fragments thereof of the invention, optionally comprising instructions for use of the anti-TIGIT antibodies or antigen-binding fragments thereof of the invention to prevent and/or treat one or more disease states (e.g., tumor, infection, or infectious disease) as described herein.

Also provided are therapeutic kits comprising the vectors or host cells of the invention, optionally comprising instructions for use of the vectors or host cells of the invention in the prevention and/or treatment of a tumor as described herein. In such embodiments, the isolated nucleic acid molecule comprised by the vector comprises a nucleotide sequence encoding a chimeric antigen receptor that further comprises a nucleotide sequence encoding an antibody of the invention or an antigen-binding fragment thereof (e.g., ScFv); the host cell comprises the isolated nucleic acid molecule or vector as described previously. In certain preferred embodiments, the isolated nucleic acid molecule encodes a chimeric antigen receptor comprising an antigen-binding fragment (e.g., ScFv) of an antibody of the invention. In certain preferred embodiments, the host cell is a T cell. In certain preferred embodiments, the host cell is a chimeric antigen receptor T cell (CAR-T).

In one embodiment, the kit comprises an anti-TIGIT antibody (e.g., a humanized antibody) or antigen-binding fragment thereof, or a vector or host cell as described above, packaged in a container.

In one embodiment, the kit comprises an anti-TIGIT antibody (e.g., a humanized antibody) or antigen-binding fragment thereof or a vector or host cell as described above, and one or more pharmaceutically acceptable carriers packaged in a container.

In one embodiment, the kit comprises a combination of the invention comprising an anti-TIGIT antibody or antigen-binding fragment thereof of the invention or a vector or host cell as described above in a single common container together with one or more pharmaceutically acceptable carriers, optionally in combination with one or more therapeutic agents, optionally formulated together in a pharmaceutical composition.

In one embodiment, the kit comprises an anti-TIGIT antibody or antigen-binding fragment thereof or vector or host cell as described above or a pharmaceutical composition thereof of the invention in one container and a pharmaceutical composition and/or therapeutic agent thereof in another container.

Simultaneous administration of two therapeutic agents does not require that the agents be administered simultaneously or by the same route, so long as there is an overlap in the time periods during which the agents exert their therapeutic effects.

In another aspect, the above kits comprise a label attached to or packaged with a container, the label describing the contents of the container and providing instructions for the indication and/or methods for using the contents of the container, instructions useful for treating and/or preventing one or more disease states as described herein.

The kit may optionally further comprise a syringe for parenteral, e.g. intravenous, administration. The kit also includes means for closely containing the vial for commercial sale and/or for convenient packaging and delivery.

Derivatized antibodies

The antibodies or antigen-binding fragments thereof of the invention can be derivatized, e.g., linked to another molecule (e.g., another polypeptide or protein). In general, derivatization (e.g., labeling) of an antibody or antigen-binding fragment thereof does not adversely affect its binding to TIGIT, particularly human TIGIT. Thus, the antibodies or antigen-binding fragments thereof of the present invention are also intended to include such derivatized forms. For example, an antibody or antigen-binding fragment thereof of the invention can be functionally linked (by chemical coupling, genetic fusion, non-covalent linkage, or other means) to one or more other molecular moieties, such as another antibody (e.g., to form a bispecific antibody), a detection reagent, a pharmaceutical agent, and/or a protein or polypeptide (e.g., avidin or polyhistidine tag) capable of mediating binding of the antibody or antigen-binding fragment to another molecule.

One type of derivatized antibody (e.g., a bispecific antibody) is produced by cross-linking 2 or more antibodies (of the same type or of different types). Methods for obtaining bispecific antibodies are well known in the art, and examples include, but are not limited to, chemical cross-linking methods, cell engineering methods (hybridoma methods), or genetic engineering methods.

Another type of derivatized antibody is an antibody linked to a therapeutic moiety. The therapeutic moiety of the present invention may be a bacterial toxin, cytotoxic drug or radiotoxin, examples of which include, but are not limited to, taxol, cytochalasin b, mitomycin, etoposide, vincristine or other antimetabolites, alkylating agents, antibiotics or antimitotic drugs.

Another type of derivatized antibody is a labeled antibody, for example, an antibody of the invention or antigen-binding fragment thereof can be linked to a detectable label such labels are well known in the art and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β -galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C, or 32P), fluorescent dyes (e.g., Fluorescein Isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), Phycoerythrin (PE), Texas red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., Cy7, Alexa 750)), acridinium ester compounds, magnetic beads (e.g.,

) Calorimetric labels such as gold colloids or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and modified for binding such labelsAvidin (e.g., streptavidin) biotin. In certain embodiments, a detectable label as described above can be attached to an antibody or antigen-binding fragment thereof of the invention via a linker of varying length to reduce potential steric hindrance.

In addition, the antibodies or antigen-binding fragments thereof of the present invention may also be derivatized with chemical groups, such as polyethylene glycol (PEG), methyl or ethyl, or glycosyl groups. These groups can be used to improve the biological properties of the antibody, for example to increase serum half-life.

Preparation of monoclonal antibodies

The mAbs of the invention can be prepared by a variety of techniques, including conventional mAb methodologies, such as standard somatic hybridization techniques as described in Kohler G and Milstein C, Nature,256:49,1975. In the hybridoma method, a mouse, hamster, or other suitable host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Although somatic hybridization protocols are preferred, in principle other methods of making mabs may be used, such as viral or oncogenic transformation of B lymphocytes.

The animal used for preparing the hybridoma is not particularly limited, but is preferably selected in consideration of its compatibility with the parent cell for cell fusion. Generally transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Preferably, the animal system is a murine system. The preparation of hybridomas in mice is a very well established protocol. Immunization protocols and techniques for isolating immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion protocols are also known.

Methods for obtaining mabs from hybridomas include culturing hybridomas and obtaining mabs from culture supernatants according to standard methods. Another method involves administering the hybridoma to a mammal compatible with the hybridoma to proliferate the same, and obtaining the mAb from ascites fluid. The former method is suitable for obtaining high purity antibodies, and the latter method is suitable for mass production of antibodies.

The culture medium containing the hybridoma cells can then be assayed for the presence of monoclonal antibodies. Such techniques and assays are known in the art. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay or enzyme-linked immunosorbent assay. The binding affinity of monoclonal antibodies can be determined by, for example, Scatchard analysis of Munson and Pollard, anal. biochem.,107:220,1980.

The antibody thus obtained can be isolated from the inside or outside of the host cell (medium, etc.), and then can be purified to a substantially pure and homogeneous antibody. The separation and purification of the antibody is carried out by separation and purification methods generally used in the purification of polypeptides. For example, the separation and purification of the polypeptide is carried out by any method including chromatography column, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, and a combination thereof.

To express the antibody or antibody fragment thereof, DNA encoding partial or full-length light and heavy chains can be obtained by standard molecular biology techniques (e.g., PCR amplification or cDNA cloning using hybridomas that express the antibody of interest), and the DNA can be inserted into an expression vector, thereby operably linking the gene of interest to transcriptional and translational regulatory sequences, transfecting a host cell for expression, preferably a eukaryotic expression vector, more preferably a mammalian cell, such as CHO and derived cell lines thereof.

The humanized antibody of the present invention can be prepared based on the murine antibody sequence prepared as described above. DNA encoding the heavy and light chain immunoglobulins can be obtained from a murine hybridoma of interest and engineered to contain non-murine (e.g., human) immunoglobulin sequences using standard molecular biology techniques. For example, to create humanized antibodies, murine CDR regions can be inserted into human framework sequences using methods known in the art, see U.S. Pat. No.5,225,539 to Winter and U.S. Pat. nos.5,530,101 to Queen et al; 5,585,089; 5,693,762 and 6,180,370. Transgenic animals, such as HuMAb mice (Metarex, Inc.) containing a human immunoglobulin gene minilocus (minioci) encoding unrearranged human heavy (mu and gamma) and kappa light chain immunoglobulin sequences, in addition to targeted mutations that inactivate endogenous mu and kappa chain loci, can also be used (see, e.g., Lonberg N et al, Nature,368: 856-; or "KM mouse TM" carrying human heavy chain transgene and human light chain transchromosome (see WO 02/43478) for antibody humanization. Other methods of humanizing antibodies include, for example, phage display techniques.

The epitope on the TIGIT molecule recognized by the anti-TIGIT antibody or antigen-binding fragment thereof of the present invention is not limited to a specific epitope. The anti-TIGIT antibody or antigen binding fragment thereof may recognize any epitope as long as the epitope is present on the TIGIT molecule. Thus, any fragment may also be used as an antigen for producing the anti-TIGIT antibody of the present invention as long as it contains an epitope present on the TIGIT molecule.

The invention is further illustrated by the following examples, which should not be construed as further limiting. The contents of all figures and all references, patents and published patent applications cited throughout this application are hereby expressly incorporated herein by reference in their entirety.

Drawings

Fig. 1, ELISA test anti-TIGIT murine antibodies Mab21 and Mab7 for their ability to bind to human TIGIT antigen.

Fig. 2, ELISA test the ability of anti-TIGIT humanized antibody to bind to human TIGIT antigen.

FIG. 3-1 ELISA detects cross-reactivity of anti-TIGIT humanized antibody with mouse or rat TIGIT antigen.

Fig. 3-2, ELISA detect cross-reactivity of anti-TIGIT humanized antibody with cynomolgus monkey TIGIT antigen.

Fig. 4, DSF detects Tm values of anti-TIGIT humanized antibodies.

Figure 5-1, ELISA test anti-TIGIT humanized antibody blocks the ability of human TIGIT to bind to CD 155.

Fig. 5-2, ELISA test anti-TIGIT humanized antibody for its ability to block human TIGIT and CD112 binding.

FIG. 6 flow cytometry detection of the affinity of AB12V8 and AB12V9 antibodies to Jurkat-TIGIT cells.

FIG. 7, flow cytometry to detect the ability of AB12V8 and AB12V9 antibodies to block Jurkat-TIGIT binding to CD 155.

Fig. 8, flow cytometry detects AB12V8 and AB12V9 antibody binding to CIK cells.

FIG. 9, microplate reader detection of CDC activity of AB12V8 and AB12V9 antibodies.

Figure 10 microplate reader assay AB12V7, AB12V8 and AB12V9 ability to bind FcRn.

FIG. 11, microplate reader assay for ADCC activity of AB12V8 and AB12V 9.

FIG. 12-1, AB12V1 and PD-1 antibody AB12M4 were administered in combination to test for the ability to promote IFN-. gamma.secretion.

FIG. 12-2, AB12V4 and PD-1 antibody AB12M4 were administered in combination to test for the ability to promote IFN-. gamma.secretion.

FIG. 12-3, AB12V8 and PD-1 antibody AB12M4 were administered in combination to test for the ability to promote IFN-. gamma.secretion.

FIG. 12-4, AB12V9 and PD-1 antibody AB12M4 were administered in combination to test for the ability to promote IFN-. gamma.secretion.

Figure 13, AB12V8, and AB12V9 improve the detection of NK cells and T cells' ability to kill HCC827 cells.

FIG. 14, AB12V8 effect on inhibiting growth of tumor cell MC38 in TIGIT transgenic mice.

Description of biological Material preservation

The present invention relates to the following biological materials which have been preserved in the China center for type culture Collection (CCTCC, university of Wuhan, China):

Detailed Description

The invention will now be described with reference to the following examples, which are intended to illustrate the invention, but not to limit it.

Unless otherwise indicated, the molecular biological experimental methods and immunoassay methods used in the present invention are essentially described by reference to j.sambrook et al, molecular cloning: a laboratory manual, 2 nd edition, cold spring harbor laboratory Press, 1989, and F.M. Ausubel et al, eds. molecular biology laboratory Manual, 3 rd edition, John Wiley & Sons, Inc., 1995; the use of restriction enzymes follows the conditions recommended by the product manufacturer. The examples are given by way of illustration and are not intended to limit the scope of the invention as claimed.

Example 1 preparation of mouse-derived monoclonal antibody against human TIGIT

50 mu g of human TIGIT ectodomain antigen (Uniport entry: Q495A1, manufactured by Anyuan pharmaceutical technology (Shanghai) Co., Ltd.) was fully emulsified with Freund's complete adjuvant, and then a multi-point immunization method was used to immunize male Balb/C mice with an immunization cycle of three weeks. On day 10 after the 3 rd immunization, blood was taken through the tail vein, and plasma anti-human TIGIT antibody titers were tested by ELISA to monitor the degree of immune response of the mice, and then the mice producing the highest anti-human TIGIT antibody titer were boosted once 3 days before the fusion. After 3 days, the mice were sacrificed and their spleens were removed and fused with a mouse myeloma Sp 2/0 cell line. Mixing 2X 10⁸ Sp 2/0 cells and 2X 10 cells⁸Individual splenocytes were fused in 50% polyethylene glycol (molecular weight 1450) and 5% Dimethylsulfoxide (DMSO) solution. Spleen cell counts were adjusted to 5X 10 using HAT selection medium (DMEM medium containing 10% fetal bovine serum, 100U/mL penicillin, 100. mu.g/mL streptomycin, 0.1mM hypoxanthine, 0.4. mu.M aminopterin and 16. mu.g thymidine)⁵0.3 mL/mL, added to wells of a 96-well plate and placed at 37 ℃ in 5% CO₂In the incubator. After culturing for 10 days, high-throughput ELISA method is adopted to detect clones of the antibody in the supernatant which are combined with TIGIT-His with high affinity respectively. And subcloning the fusion cells in the hole of the monoclonal antibody. Then, positive wells that compete with human CD155/hFc (purchased from SinoBiological Co., Ltd., product No. 10109-H02H) for binding to TIGIT were selected by the competitive ELISA method (see example 6.4), and hybridoma cell lines #21 and #7 were obtained. Specificity of production by culture in RPMI1640 Medium supplemented with 10% FBSCloning of the antibody. When the cell density reaches about 5X 10⁵At individual cells/mL, the medium was replaced with serum-free medium. After 2 to 4 days, the cultured medium was centrifuged to collect a culture supernatant. Protein G columns were used to purify the antibodies. The monoclonal antibody eluate was dialyzed against 150mM NaCl. The dialyzed solution was filter-sterilized through a 0.2 μm filter to obtain mouse monoclonal antibodies Mab21, Mab7 corresponding to the purified hybridoma cell lines #21, #7 to be tested.

Example 2 measurement of binding Capacity of murine antibody to human TIGIT antigen by ELISA method

The elisa plate was coated with human TIGIT overnight at room temperature. The coating solution was discarded, each well was blocked with skim milk dissolved in Phosphate Buffered Saline (PBS) for 1 hour, and the wells were washed with PBS containing 0.05% Tween 20 (Tween-20). Then 50. mu.L of purified anti-human TIGIT murine antibodies Mab21, Mab7 were added to each well, murine IgG (prepared by purification of mouse serum using ProteinG) was used as a negative control, PBS was used as a blank, incubation was carried out at room temperature for 1 hour, the wells were washed with PBS containing 0.05% Tween 20(Tween-20), and then 50. mu.L of HRP-labeled goat anti-mouse IgG polyclonal antibody (purchased from Jackson laboratory Co., Ltd., cat # 115-.

The results are shown in FIG. 1, murine antibodies Mab21 and Mab7 have higher affinity with human TIGIT, and both EC₅₀Are all about 18 pM.

Example 3 subtype identification and variable region amplification of anti-TIGIT murine mAb

And (3) antibody subtype identification: taking hybridoma cell culture supernatant, and adopting IsoTrip^TMThe subtype of the mouse monoclonal antibody was identified using a mouse monoclonal antibody subtype identification kit (Santa Cruz Biotechnology, cat # sc-24958). The murine Mab21 subtype was identified as the IgG2b (Kappa) type. The murine Mab7 subtype was identified as the IgG1(Kappa) type.

Antibody variable region amplification: candidate hybridoma cells #21, #7 were cultured to a total number of 10⁷The cells were centrifuged at 1000rpm for 10min to collect total RNA and extracted with mRNA extraction kit (from NEB, cat # S1550S), and synthesized with reverse transcription kit SMARTER RACE (from TAKARA, cat # 634858)First strand cDNA, and using the first strand cDNA as a subsequent template to amplify the antibody variable region DNA sequence corresponding to the hybridoma cell. According to the subtype identification result, obtaining the heavy chain and light chain constant region sequences of the antibody subtype, designing a specific nested PCR primer, and the primer sequence used in the amplification reaction is complementary with the first framework region and the constant region of the antibody variable region. Amplifying a target gene by adopting a conventional PCR method, sequencing an amplification product to obtain a heavy chain variable region sequence SEQ ID NO: 1 and light chain variable region sequences SEQ ID NO: 2. the heavy chain variable region sequence of hybridoma clone #7 secretory antibody Mab7 SEQ ID NO: 3 and the light chain variable region sequence SEQ ID NO: 4.

the amino acid sequences of the heavy chain CDRs (CDR-H1, CDR-H2 and CDR-H3) and light chain CDRs (CDR-L1, CDR-L2 and CDR-L3) of antibodies Mab21 and Mab7, respectively, are as follows:

tables 1 to 1: heavy and light chain CDRs of murine antibody Mab21

Tables 1 to 2: heavy and light chain CDRs of murine antibody Mab7

The above CDR region sequences are defined by the Chothia, Kabat and IMGT methods, respectively, and any other art-recognized CDR region sequence determination method can be used to identify the amino acid residues in the CDR region of the variable region.

Example 4 affinity detection of anti-human TIGIT murine antibody

The binding affinity constants of the purified murine monoclonal antibodies Mab21, Mab7 and antigen were determined by the biofilm interference technique (BLI) using the ForteBio Octet QK system from PALL. Multichannel parallel quantitative analysis concentration gradients were set as: 3.1256.25, 12.5, 25, 50 and 100nM, Human TIGIT (His tag) coupled Ni-NTA sensors (available from Fortebio, Inc., cat # 18-5101). The results of the affinity measurements are shown in table 2, and show that murine monoclonal antibodies Mab21 and Mab7 have very high binding affinity for human TIGIT, reaching 10^-11And 10^-12Of the order of M.

Table 2: affinity assay results for murine mAbs

Example 5 humanization of anti-TIGIT murine antibodies

The humanized transformation of the murine antibodies Mab21 and Mab7 was performed using a CDR-grafted antibody humanized transformation method. Briefly, humanization engineering involves the following steps: comparing the amino acid sequence of the mouse-derived monoclonal antibody with the amino acid sequence of the human embryonic system antibody to find out a sequence with high homology and excellent physicochemical properties as a human embryonic system framework sequence; analyzing and investigating HLA-DR affinity, and selecting a human embryonic line framework sequence with low affinity; six CDRs of murine antibody were grafted onto selected heavy and light chain framework sequences, respectively.

Specifically, the heavy and light chain CDR regions of murine antibodies Mab21, Mab7, respectively, were grafted onto the FR framework of the corresponding humanized template. The humanized templates for heavy and light chains of Mab21 were the human germline gene sequences IGHV1-69-2 a 01 (see IMGT accession No. KF698734) and IGKV6-21 a 02 (see IMGT accession No. KM455568), respectively, and the humanized templates for heavy and light chains of Mab7 were the human germline gene sequences IGHV1-2 a 02 (see IMGT accession No. X62106) and IGKV1-39 a 01 (see IMGT accession No. X59315), respectively.

Further, the computer simulation technology is utilized, the molecular docking is applied to analyze the variable region and the framework amino acid sequence around the variable region, and the spatial and stereo combination mode of the variable region and the framework amino acid sequence is investigated. Through calculating electrostatic force, van der Waals force, hydrophilicity and hydrophobicity and entropy, the amino acid sequence of the murine antibody is analyzed for key amino acids which can act with TIGIT and maintain space structure, and the murine amino acids are retained in the transplanted antibody. That is, a series of back mutations were made to the amino acid residues in the FR region of the above humanized template so that the humanized antibody retains the antigen-binding ability of the murine antibody as much as possible.

According to the method, 2 humanized antibodies are constructed on the basis of the CDR of the murine antibody Mab21 and are respectively named as AB12V7 and AB12V 9; wherein the heavy chain constant region of each antibody is a human wild-type IgG1 heavy chain constant region (SEQ ID NO: 47), and the amino acid sequence of each light chain constant region is as shown in SEQ ID NO: shown at 48. Meanwhile, 1 strain of humanized antibody is constructed on the basis of the CDR of the murine antibody Mab7 and is named AB12V 8; wherein the heavy chain constant region of the antibody is a human wild-type IgG1 heavy chain constant region (SEQ ID NO: 47), and the amino acid sequence of the light chain constant region is as shown in SEQ ID NO: shown at 48.

Furthermore, the variable region sequences of the antibody secreted from the hybridoma cells obtained as described above were also humanized by a resurfacing method. The surface remodeling method refers to the humanized modification of heterologous antibody surface amino acid residues, and the method only replaces the region with obvious difference with human antibody surface amino acids, and selects the amino acid replacement similar to the human antibody surface residues on the basis of maintaining the antibody activity and reducing the heterogeneity. Specifically, the resurfacing humanization transformation process involves the following steps: firstly, comparing the amino acid sequence of the antibody secreted by each hybridoma cell with the amino acid sequence of the human embryonic antibody to find out a sequence with high homology; the exposed surface amino acids are then replaced with human embryonic antibody amino acids using computer modeling techniques when the solubility (solvent accessibility) is selected to be greater than 30%. Residues that affect side chain size, charge, hydrophobicity, or the potential for hydrogen bonding to affect the conformation of the complementarity determining regions of an antibody are minimally replaced.

According to the above method, humanized antibodies AB12V3 and AB12V5 were constructed based on the CDRs of murine antibody Mab21 and using the human IGHV1-69-2 x 01 heavy chain variable region and human IGKV6-21 x 02 light chain variable region as template sequences; humanized antibodies AB12V4 and AB12V6 were constructed based on the CDRs of murine antibody Mab7 and using the human IGHV1-2 x 02 heavy chain variable region and the human IGKV1-39 x 01 light chain variable region as template sequences.

Wherein, the heavy chain constant regions of AB12V3 and AB12V4 are human IgG4 heavy chain constant region variants (IgG4V1, SEQ ID NO: 46), are human IgG4 heavy chain constant regions containing Ser228Pro mutation, have stable structures, can reduce Fab-arm exchange, thereby half antibodies are not easy to form, and the amino acid sequences of the light chain constant regions are all shown as SEQ ID NO: 48 is shown; the heavy chain constant regions of AB12V5 and AB12V6 are human wild type IgG1 heavy chain constant regions (IgG 1; SEQ ID NO: 47), and the amino acid sequences of the light chain constant regions are shown in SEQ ID NO: shown at 48.

The amino acid sequences of the heavy and light chain variable regions and the heavy and light chain constant regions of the humanized antibodies AB12V3, AB12V4, AB12V5, AB12V6, AB12V7, AB12V8 and AB12V9 are shown in Table 3.

Table 3: humanized antibody variable and constant region amino acid sequences

Based on the heavy and light chain sequences obtained in the above examples, the coding cDNA was designed to be inserted into the pCMAB2M eukaryotic expression vector (Anyuan pharmaceutical technology (Shanghai) Co., Ltd.). The expression vector plasmid contains the cytomegalovirus early gene promoter-enhancer required for high level expression in mammalian cells. Meanwhile, the vector plasmid contains a selectable marker gene to confer ampicillin resistance in bacteria and G418 resistance in mammalian cells. In addition, the vector plasmid contains a dihydrofolate reductase (DHFR) gene, and in a suitable host cell, the antibody gene and the DHFR gene can be co-amplified with Methotrexate (MTX).

The above-constructed recombinant expression vector plasmid is transfected into a mammalian host cell line to express a humanized antibody. For stable high level expression, the preferred host cell line is DHFR-deficient Chinese Hamster Ovary (CHO) cells (see U.S. patent No.4,818,679). The preferred method of transfection is electroporation, although other methods, includingIncluding calcium phosphate co-precipitation, lipofection, protoplast fusion, and the like. In electroporation, 2X 10 cells were placed in a cuvette using a GenePulser (Bio-Rad Laboratories) set at 300V electric field and 1050. mu. Fd capacitance⁷The cells were suspended in 0.8ml of PBS and contained 20. mu.g of the expression vector plasmid. After 2 days of transfection, a solution containing 0.2mg/ml G418 and 200nM MTX (Sigma) was added. To achieve higher levels of expression, the transfected antibody genes were co-amplified with DHFR gene suppressed by MTX drug. The secretion rate of each cell line was measured by limiting dilution subcloned transfectants and ELISA method, and cell lines expressing high levels of antibody were selected. The conditioned medium of the antibody was collected for determination of its biological activity in vitro and in vivo.

Example 6 screening and identification of anti-TIGIT humanized antibodies

6.1 measurement of binding Capacity of humanized TIGIT antibody to human TIGIT

Human TIGIT/mFc (manufactured by Anyuan pharmaceutical technology (Shanghai) Co., Ltd.) was diluted to 1. mu.g/ml with PBS buffer, added to a 96-well plate in a volume of 100. mu.l/well, and left at 4 ℃ for 16 to 20 hours. Discarding the coating solution, adding PBS/2% BSA solution, and blocking at 37 deg.C for 2 h; removing blocking solution, washing the plate 3 times with PBST (PBS with pH 7.4 and 0.05% Tween 20) buffer, diluting the anti-TIGIT humanized antibody to be tested AB12V4, AB12V5, AB12V7, AB12V8, AB12V9 and reference antibody AB12V1 (variable region sequence is from anti-TIGIT antibody 22G2 in Chinese patent CN107207594, and heavy chain constant region and light chain constant region are respectively shown in SEQ ID NO:46 and 48), AB12V2 (variable region sequence is from anti-TIGIT antibody 10A7 in US patent US20150216970A1, and heavy chain constant region and light chain constant region are respectively shown in SEQ ID NO:47 and 48), adding a series of concentration gradients of TIGIT antibody to be tested, incubating at 37 ℃ for 2 h; the reaction system was removed, and after washing the plate 3 times with PBST, HRP-anti human IgG (H + L) (purchased from Jackson ImmunoResearch, cat # 109-035-003) was diluted with PBS/2% BSA solution at 100. mu.l/well and incubated at 37 ℃ for 1H; adding 100 μ l/hole TMB solution into corresponding hole, and developing at room temperature for 10 min; 50 μ l of 1M H was added₂SO₄The solution is stopped to develop color, and the absorbance is read by a microplate reader at 450 nm. The results are shown in fig. 2 and table 4, and the affinity between TIGIT and most antibodies isAt 10^-10M scale, with very high binding affinity, with AB12V5, AB12V7, and AB12V8 having the strongest ability to bind antigen.

Table 4: measurement result of binding ability of anti-TIGIT humanized antibody to human TIGIT

Name of antibody	EC50(nM)
		AB12V1	0.07357
AB12V2	0.1679
		AB12V4	0.07094
AB12V5	0.04737
		AB12V7	0.05387
AB12V8	0.05421
		AB12V9	0.1671

6.2 species Cross-reactivity assay of humanized antibodies

To examine the differences in specific binding of the anti-TIGIT humanized antibody to different species other than human, TIGIT of mouse, cynomolgus monkey and rat was used for the binding assay.

Mice TIGIT/His and cynomolgus monkey TIGIT/His (both purchased from Acro Biosystems, and having TIT-M52E6 and TIT-C5223 as a product number, respectively) were diluted to 0.1. mu.g/ml with PBS buffer, added to a 96-well plate in a volume of 100. mu.l/well, and left at 4 ℃ for 16 to 20 hours. The coating solution in the 96-well plate was aspirated, the plate was washed 3 times with PBST buffer, 200. mu.l/well PBST/1% skim milk powder was added, and the blocking was performed by incubation at room temperature for 1 h. The blocking solution was removed, the plates were washed 3 times with PBST buffer, a series of concentration gradients of TIGIT antibody to be tested were added, 100. mu.l/well, and incubated at room temperature for 2 h. The reaction system was removed and after 3 PBST washes, 100. mu.l/well of HRP-labeled secondary goat anti-human IgG antibody (purchased from Jackson immuno Research, cat # 109-. PBST washing plate 3 times, adding 100u l/hole TMB, room temperature incubation 5-10 min. 50. mu.l/well of 0.2M H was added₂SO₄The reaction was terminated. The microplate reader reads the absorbance at a dual wavelength of 450/620 nm.

Since rat TIGIT antigen is a human Fc tag (purchased from Acro Biosystems, Cat.: TIT-R5258), in order to examine the ability of anti-TIGIT antibody to specifically bind to rat TIGIT antigen, EZ-Link was used as the anti-TIGIT humanized antibody to be examined^TMThe Sulfo-NHS-Biotinylation kit (available from Thermo Scientific, cat # 21425) was biotinylated using HRP-labeled streptavidin (available from BD Biosciences, cat # 554066) as the detection antibody, in the same manner as described above.

Results as shown in fig. 3-1, 3-2, all anti-TIGIT humanized antibodies did not cross-react with mouse TIGIT antigen species, and all anti-TIGIT humanized antibodies specifically bound to cynomolgus TIGIT antigen; in addition, all anti-TIGIT humanized antibodies did not cross-react with the rat TIGIT antigen species, but the reference antibody AB12V1 bound to the rat TIGIT antigen, and the anti-TIGIT humanized antibody of the invention had a different binding region than AB12V 1.

6.3 measurement of Tm value of humanized antibody

The Tm value of the anti-TIGIT antibody was measured by the DSF (differential fluorescence scanning technique) method.The specific experimental procedure was as follows, 12.5. mu.l of 40 XSSYPRO Orange dye (from Life technologies, cat # 56651), 5. mu.l of 1mg/ml TIGIT antibody to be tested (PBS dilution) and 7.5. mu.l of deionised H₂O in an EP tube, the above sample mixture was added to a Q-PCR system (AB Applied Biosystems ABI, 7500) for reaction, Q-PCR parameters set: target (ROX), program (25 ℃,3 min; 1% rate, 95 ℃,2 min). As a result, Tm values were calculated by introducing Graph Prism software, and as shown in fig. 4 and table 5, Tm values of all anti-TIGIT humanized antibodies were higher than 65 ℃, better than those of AB12V1 and AB12V2, and had excellent thermostability. The Tm values of AB12V6, AB12V8, and AB12V9 reached about 70 ℃, indicating that AB12V6, AB12V8, and AB12V9 have significantly superior thermal stability.

Table 5: results of measurement of Tm value of humanized antibody

Name of antibody	Tm(℃)
		AB12V1	63.53
AB12V2	64.03
		AB12V4	66.87
AB12V5	68.13
		AB12V6	73.86
AB12V7	65.8
		AB12V8	71.32
AB12V9	69.79

6.4 humanized antibody blocks TIGIT and CD155 or TIGIT and CD112 binding experiment

To detect blocking of the TIGIT/CD155 interaction by the humanized TIGIT antibody, the CD155-mFc protein was labeled according to the protocol of the HRP labeling kit (available from BigjingO-Tokyo Co., Ltd., cat # BF06095-1000), and the blocking activity of the humanized antibody was measured by the competitive Elisa method.

Human TIGIT was diluted to 1. mu.g/ml with PBS buffer, added to a 96-well plate in a volume of 100. mu.l/well, and left at 4 ℃ for 16-20 h. The coating solution was discarded, 200. mu.l/well PBS/2% BSA solution was added and incubated at 37 ℃ for 2h with blocking. After removing the blocking solution and washing the plate 3 times with PBST buffer, a series of concentration gradients of 50. mu.l/well of TIGIT antibody to be tested and 50. mu.l/well of HRP-CD155 (25. mu.g/ml) were added and incubated at 37 ℃ for 2 h. The reaction was removed and the plates were washed 5 times with PBST, then 100. mu.l/well TMB was added and developed for 5min at room temperature. 50. mu.l/well 1M H was added₂SO₄The reaction was terminated. The microplate reader reads the absorbance at a wavelength of 450 nm.

To detect blocking of the TIGIT/CD112 interaction by the humanized TIGIT antibody, biotinylated CD112-hFc protein (from Acro Biosystems, Cat.: CD2-H82F8) was purchased for competitive Elisa to detect the blocking activity of the humanized TIGIT antibody.

TIGIT-mFc was diluted to 2. mu.g/ml with PBS buffer, added to a 96-well plate in a volume of 100. mu.l/well, and left at 4 ℃ for 16-20 hours. The coating solution was discarded, the plates were washed 3 times with PBST, 200. mu.l/well of PBST/1% skim milk powder was added, and the plates were incubated at room temperature for 1h for blocking. After removing the blocking solution and washing the plate 3 times with PBST buffer, a series of concentration gradients of 50. mu.l/well of TIGIT antibody to be tested and 50. mu.l/well of 2. mu.g/ml biotinylated human CD112 (purchased from Acro Biosy)stems corporation, cat number: CD2-H82F8), incubated for 2H at room temperature. The reaction system was removed and the plates were washed 3 times with PBST, 100. mu.l/well diluted with PBST/1% skim milk powder (dilution ratio 1:500) and HRP-labeled streptavidin (purchased from BD Biosciences, cat.: 554066) was added and incubated at room temperature for 1 h. After PBST washing 3 times, 100. mu.l/well TMB was added and incubated at room temperature for 10-15 min. 50. mu.l/well of 0.2M H was added₂SO₄The reaction was terminated. The microplate reader reads the absorbance at a dual wavelength of 450/620 nm.

As shown in FIGS. 5-1, 5-2 and Table 6, all of the humanized TIGIT antibodies tested specifically blocked the binding of human TIGIT to CD155 (FIG. 5-1) or to human CD112 (FIG. 5-2), and had excellent blocking activity in nM. The AB12V4 and AB12V8 antibodies have stronger capability of blocking TIGIT/CD155 combination than AB12V1 and AB12V 2; the ability of the AB12V9 antibody to block TIGIT/CD155 binding was substantially comparable to AB12V1 and AB12V 2. The AB12V4 antibody has stronger capability of blocking TIGIT/CD112 binding than AB12V 1; the ability of the AB12V8 antibody to block TIGIT/CD112 binding was substantially comparable to AB12V 1.

Table 6: detection of blocking TIGIT/CD155 and blocking TIGIT/CD112 binding by anti-TIGIT humanized antibody

6.5 affinity determination and kinetic analysis of anti-TIGIT humanized antibody

The characterized affinity and binding kinetics of the purified anti-TIGIT humanized antibody were determined using biofilm interference technique (BLI). The measurement was carried out according to the standard procedure of an Octet molecular interaction apparatus (ForteBio Octet QK System, PALL). Multichannel parallel quantitative analysis concentration gradients were set as: 3.125, 6.25, 12.5, 25, 50 and 100nM, human TIGIT-His coupled Ni-NTA sensor. Antigen-antibody binding kinetics and dissociation kinetics were followed. Analysis of the data obtained, K determined in this way_a、K_dAnd K_DThe values are shown in Table 7.All humanized monoclonal antibodies showed 10^-11Affinity of the M class, where the equilibrium dissociation constant K of AB12V4, AB12V8 and AB12V9_DThe values are 1.56X 10 respectively^-11M，1.79×10^- ¹¹M and 2.52X 10^-11M, showing good antigen binding activity.

Table 7: humanized TIGIT antibody affinity assay results

6.6 detection of affinity of anti-TIGIT humanized antibody and Jurkat-TIGIT cell

To examine the cell affinity of the anti-TIGIT humanized antibody, first, a full-length TIGIT Gene (Gene ID:201633) was transferred into Jurkat cells (purchased from Beijing Korea Biotech Co., Ltd.) by a lentivirus method, and pressure-screening was performed with puromycin to construct a Jurkat-TIGIT stable cell line. The anti-TIGIT humanized antibody to be tested was labeled with NHS-Fluro (available from Thermo, cat # 46610), diluted with PBS buffer to the labeled antibody, 1. mu.M initial, 4-fold gradient dilution, 3X 10⁵Jurkat-TIGIT cells were resuspended in 50. mu.l of diluted antibody, incubated at 4 ℃ for 0.5h, washed twice with PBS and then detected using a flow cytometer (Beckman, model Cytoflex). The results of fluorescence signal values were introduced into Graph Prism software to calculate IC50 values, and the results are shown in FIG. 6 and Table 8, which indicate that both the AB12V8 and AB12V9 antibodies can specifically bind to TIGIT on the cell surface, and that the affinity of the AB12V8 for Jurkat-TIGIT cells is stronger than that of the reference antibody AB12V 1.

Table 8: detection result of affinity of anti-TIGIT humanized and Jurkat-TIGIT cells

Name of antibody	EC₅₀(nM)
		AB12V1	0.744
AB12V8	0.682
		AB12V9	1.12

6.7 anti-TIGIT humanized antibody blocks Jurkat-TIGIT/CD155 binding detection

CD155 was labeled with NHS-Fluro (available from Thermo Co.) as a reagent. Diluting the anti-TIGIT humanized antibody to be detected to 200 mu g/ml by PBS buffer solution, diluting by 4-time gradient, setting 10 concentration gradients, diluting FITC-CD155 to 600nM by PBS buffer solution, mixing the antibody to be detected with a series of concentration gradients with FITC-CD 1551: 1 uniformly, and multiplying by 3 to 10⁵Jurkat-TIGIT cells (see example 6.6 for preparation) were resuspended in 50. mu.l of a mixture containing the antibody to be tested and FITC-CD155, incubated at 4 ℃ for 0.5h, washed twice with PBS and then detected using a flow cytometer (Beckman, model Cytoflex). Fluorescence signal value results are imported into Graph Prism software for calculating IC₅₀The results, shown in figure 7 and table 9, indicate that both AB12V8 and AB12V9 antibodies blocked Jurkat-TIGIT/CD155 binding and were significantly stronger than the reference antibody AB12V 1.

Table 9: detection result of Jurakt-TIGIT/CD155 combination blocking of anti-TIGIT humanized antibody

Name of antibody	IC₅₀(nM)
		AB12V1	44.77
AB12V8	14.6
		AB12V9	29.02

6.8 detection of anti-TIGIT humanized antibody combined with CIK cells

To examine the affinity of the anti-TIGIT humanized antibody to human CIK (cytokine-induced killer) cells, PBMC were isolated from healthy human fresh blood and cultured with anti-CD3 and anti-CD28 antibodies. NHS-Fluro labeling the anti-TIGIT humanized antibody to be detected, diluting with PBS buffer solution to the labeled antibody, 0.4 mu M initial, 5 times gradient dilution, 3X 10⁵CIK cells cultured for 15 days were resuspended with 50. mu.l of diluted antibody, incubated at 4 ℃ for 0.5h, washed twice with PBS and then detected using a flow cytometer (Beckman, model CytoFlex). The fluorescence signal value results are introduced into Graph Prism software, the results are shown in FIG. 8, and the results show that the AB12V8 and AB12V9 antibodies have stronger affinity with CIK cells, wherein the affinity of the AB12V8 antibody with the CIK cells is stronger than that of the reference antibody AB12V 1.

6.9 CDC Activity assay of anti-TIGIT humanized antibody

In this example, CDC activity of humanized antibodies was evaluated by measuring the binding activity of the antibody to C1q and the activity of the antibody to complement in direct killing of target cells.

To determine the binding activity of the antibody to C1q, the anti-TIGIT humanized antibody to be tested was diluted to 600 μ g/ml with a carbonate buffer pH 9.6, diluted in a 3-fold gradient, set at 11 concentration points, and a series of antibodies to be tested were added to a 96-well plate at a volume of 100 μ l/well overnight at 4 ℃; discarding the coating solution, and washing the plate for 1 time by PBS; adding PBS/2% BSA solution, and blocking at 37 ℃ for 2 h; the blocking solution was removed, the plates were washed 3 times with PBST buffer, C1q (from Prospec) was diluted to 5. mu.g/ml with PBS/2% BSA solution, added to a 96-well plate at 100. mu.l/well, and incubated at 37 ℃ for 2 h; PBST plate washing 3 times; 100. mu.l/well of Shp pAb to C1q (HRP) (from A) diluted with PBS/2% BSA (dilution ratio 1: 300) was addedbcam corporation), incubated at 37 ℃ for 1 h; washing the plate with PBST buffer solution for 5 times, adding 100 μ l/well TMB solution into corresponding well, and developing at room temperature for 5 min; 50 μ l of 1M H was added₂SO₄The solution is stopped to develop color, and the absorbance is read by a microplate reader at 450 nm.

The results show that AB12V7, AB12V8 and AB12V9 all bind to C1q, whereas the reference antibody AB12V1 is of the IgG4 subtype and does not bind to C1 q.

For determination of the activity of the antibody and complement for direct killing of target cells, the anti-TIGIT humanized antibody was diluted in RPMI1640 medium containing 20% guinea pig serum, 100. mu.g/ml of initial, 5-fold dilution, 100. mu.l/well; Jurkat-TIGIT cells (see example 6.6 for preparation method) were harvested, centrifuged, and adjusted to 5 × 10 density in RPMI1640 medium containing 20% guinea pig serum⁵And/ml, adding 100 mu l/hole into the corresponding hole, uniformly mixing, culturing at 37 ℃ for 3h, culturing at 20 mu l/hole, continuously incubating at 37 ℃ for 5h, and reading the absorbance value of 450nm by using an enzyme-labeling instrument. The results are shown in fig. 9 and table 10, and indicate that AB12V1 has no CDC activity, while AB12V8 and AB12V9 have similar activities, both reaching nM level.

TABLE 10 CDC Activity assay results for anti-TIGIT humanized antibody

Name of antibody	EC₅₀(nM)
		AB12V1	Is inactive
AB12V8	0.84
		AB12V9	0.82

6.10 determination of FcRn binding Activity of anti-TIGIT humanized antibody

Diluting the anti-TIGIT humanized antibody to be detected to 5 mu g/ml by using a carbonic acid buffer solution with the pH of 9.6, adding the diluted antibody to a 96-well plate in a volume of 100 mu l/well, and standing overnight at the temperature of 4 ℃; discarding the coating solution, and washing the plate for 1 time by PBS; adding Triethanolamine Buffer Salt (TBS) solution containing 0.5% BSA, and blocking at 37 deg.C for 2 hr; the blocking solution was removed, and FcRn (purchased from Acro biosystems, Cat.: FCM-H82W4) was diluted with TBS (pH 6.0)/0.5% BSA solution to 10. mu.g/ml, 3-fold gradient diluted, 11 concentration points, added to a 96-well plate in a volume of 100. mu.l/well, and incubated at 37 ℃ for 2 hours; washing the plate for 3 times by TBST; 100. mu.l/well of HRP-labeled streptavidin (purchased from Proteitech, Inc., cat. No.: SA00001-0) diluted with TBS/0.5% BSA (dilution ratio 1:500) was added and incubated at 37 ℃ for 1 h. Washing the plate for 5 times with TBST buffer solution, adding 100 μ l/hole TMB solution into corresponding hole, and developing at room temperature for 5 min; adding 50 μ l of 1MH₂SO₄The solution stopped the color development, and the microplate reader read the absorbance at 450nm, and the results are shown in FIG. 10 and Table 11, in which the FcRn binding activity of AB12V8 is the strongest (EC)₅₀1.474nM) and the affinity of the antibody for FcRn is positively correlated with its half-life in vivo, presumably AB12V8 will have a longer in vivo half-life than the reference antibody AB12V 1.

Table 11: results of determination of FcRn binding Activity of anti-TIGIT humanized antibody

Name of antibody	EC₅₀(nM)
		AB12V1	2.727
AB12V7	3.35
		AB12V8	1.474
AB12V9	3.276

6.11 measurement of ADCC Activity of anti-TIGIT humanized antibody

In order to detect ADCC activity on anti-TIGIT humanized antibody cells, a full-length CD16a Gene (Gene ID:2214) and a luciferase Gene are transferred into Jurkat cells (purchased from Beijing Korea Biotech, Ltd.) by a lentivirus mode, and a Jurkat-NFAT/luciferase-CD16a stable cell line is constructed by pressure screening of puromycin and bleomycin to serve as effector cells; similarly, the full-length TIGIT Gene (Gene ID:201633) was lentivirally transferred into CHO-S cells (purchased from Thermo, Inc.), and a CHO-S-TIGIT stable cell line was constructed as a target cell by pressure screening with puromycin and bleomycin. When the effector cells, the antibody and the target cells are present at the same time, the expression of luciferase is activated, and then the ONE-Glo reagent (available from Promega, cat.: PRE-6120) is used as a substrate, and a fluorescence signal is detected by an enzyme-linked immunosorbent assay. 400g, 5min centrifugation is carried out to collect Jurkat-NFAT/luciferase-CD16a and CHO-S-TIGIT cells, RPMI1640 + 1% FBS culture medium is used for resuspension, and the density is adjusted to 2.5 multiplied by 10⁶A mixture of 1.25X 10 and/ml⁶Add/ml, 40. mu.L/well to 96-well plate; RPMI1640 + 1% FBS medium diluted antibodies (100. mu.g/mL, 3 fold dilution, 11 concentration points), 20. mu.L/well, 37 ℃ incubation for 5 h; adding 20 mu L of One-glo detection reagent, shaking and uniformly mixing, reading a fluorescence signal value by using a microplate reader, and introducing the result into Graph Prism software to calculate EC₅₀As shown in fig. 11 and table 12, the results indicated that both AB12V8 and AB12V9 had good ADCC activity, whereas the reference antibody AB12V1 had no ADCC activity.

Table 12: ADCC Activity measurement results of anti-TIGIT humanized antibody

Name of antibody	EC50(nM)
		AB12V1	Is inactive
AB12V8	0.3826
		AB12V9	0.3902

Example 7 functional characterization of anti-TIGIT humanized antibody

7.1 functional experiment of anti-TIGIT humanized antibody for improving secretion of IFN-gamma by CEFT activated PBMC

This assay was used to evaluate the effect of anti-TIGIT humanized antibodies administered alone or in combination with anti-PD-1 antibodies on IFN- γ production by PBMCs stimulated with antigen peptide mixture CEFT (from CMV, EBV, influenza or tetanus).

By density gradient centrifugation (Lymphoprep)^TMHuman lymphocyte isolate, from Stemcell corporation) fresh PBMCs were obtained from human peripheral blood and purified using CEFT (from jp peptide Technologies, cat #: PM-CEFT-4) RPMI1640 medium (containing 10% inactivated FBS) resuspended PBMC at a cell density of 10⁶One/ml, inoculated in a 96-well plate, 100. mu.l/well; 20 μ g/ml of AB12V1, AB12V4, AB12V8, AB12V9, AB12M4, isotype IgG control antibody or a combination of the above anti-TIGIT antibody and anti-PD-1 antibody AB12M4 (which is disclosed in Chinese patent application CN106519034A) was added to a 96-well plate at 100 μ l/well in triplicate wells; at 37 ℃ with 5% CO₂After 6 days of culture in the incubator, the supernatant was subjected to IFN-. gamma.concentration measurement using OptEIA ELISA kit (purchased from BD Bioscience, cat. No.: 555142), and the results were shown in FIGS. 12-1, 12-2, 12-3, and 12-4, respectively.

The results show that AB12V4, AB12V8 or AB12V9 can promote the T cells to secrete IFN-gamma, wherein the IFN-gamma secretion promoting capability of AB12V8 is strongest and is obviously better than that of a reference antibody AB12V 1; in the aspect of combined administration with AB12M4, the combination of each antibody and AB12M4 can promote the T cells to secrete IFN-gamma, wherein the combination of AB12V8 or AB12V9 and anti-PD-1 antibody AB12M4 can obviously promote factor secretion, and is obviously better than the combination effect of a reference antibody AB12V1 and AB12M 4.

7.2 function experiment of humanized antibody for improving killing of HCC827 cells by human NK cells and T cells

To examine the effect of anti-TIGIT humanized antibody on the killing of HCC827 cells (human non-small cell lung carcinoma cells, highly expressing CD155, purchased from cell banks of Chinese academy of sciences) by human NK and T cells (Zhang Q et al. nat. Immunol.,19:723-732, 2018). This example uses a density gradient centrifugation method (Lymphoprep)^TMHuman lymphocyte isolate, purchased from Stemcell, cat # stock: #70851) fresh PBMCs were obtained from human peripheral blood using CD56⁺The selection kit (purchased from America, Tian, and whirly, Biotech Co., Ltd., Germany, Cat. No.: 130-⁶Each ml of the cells was supplemented with 5. mu.l/10 of beads (purchased from America, Eddy, Biotech, Ltd., Cat. No.: 130-094-483) for activating NK cells⁶Expanding and culturing the cells for 12 days; t cells after sorting NK cells were additionally expanded and cultured in 1640 medium containing 1. mu.g/ml OKT3, 500IU/ml IL-2 and 10% FBS, and the T cells were frozen on day 9.

Resuscitating the cryopreserved T cells one day before killing experiment, and stimulating the T cells one day with 1640 medium containing 1. mu.g/ml OKT3, 500IU/ml IL-2 and 10% FBS; the next day, HCC827 cells were digested with 0.25% pancreatin, microscopically observed to be rounded, the digestion was stopped, the cells were blasted, and the cells were counted to adjust the HCC827 cell density to 2 × 10⁵One/ml, 50 μ l/well, seeded in 96-well plates, two sets of wells with only HCC827 cells were set up as target cell maximum release LDH set and target cell self release LDH set; t cells and NK cells after restimulationMixing at a ratio of 1:1, resuspending the mixed cells, and using them as effector cells with a density adjusted to 10⁶One/ml, inoculated in the 96-well plate, 50. mu.l/well; 30 mu g/ml, 6 mu g/ml, 1.2 mu g/ml of antibodies to be tested AB12V1, AB12V8, AB12V9 or isotype IgG control antibody are added into the 96-well plate, 50 mu l/well is formed, three wells are formed, a group of groups with effector cells and target cells is formed, a group with only effective effector cells is formed, the plate is placed at 37 ℃ and 5% CO₂Culturing for 5-6h in an incubator; 1h before collection of the supernatant, 16. mu.l Lysis solution was added to the target cell only wells; cells 250 Xg, centrifuge for 5min, take 35. mu.l of supernatant in a new 96-well flat bottom plate; adding 35. mu.l/well CytoTox96Reagent (purchased from Promega corporation, Cat. No: G1780), and incubating at room temperature in the dark for 30 min; add 35. mu.l/well StopSolution, read at 492nm, and calculate the specific killing rate of the antibody based on OD 492 values as follows:

the results show that in fig. 13, AB12V1, AB12V8, or AB12V9 can significantly improve the specific killing ability of NK cells and T cells, wherein the specific killing effect of AB12V8 and AB12V9 is significantly better than that of the reference antibody AB12V 1.

Example 8 anti-TIGIT humanized antibody inhibits Colon cancer cell growth in hTIGIT transgenic mice

Using 10% fetal bovine serum in RPMI1640 medium at 37 deg.C and 5% CO₂MC38 cells (mouse colon cancer cells, purchased from tokyo bai biotechnology limited). Collecting MC38 cells in exponential growth phase, resuspending PBS to proper concentration, and inoculating to female B-hTIGIT mice to establish colon cancer model subcutaneously. The average volume of the tumor to be detected is about 91.56mm³At this time, they were randomly divided according to the tumor size, and were intraperitoneally administered 2 times a week for 3 weeks, in a Human IgG (negative control) group, AB12V8 monotherapy group, and AB12V8 combined with anti-mouse PD-1 antibody (purchased from BioxCell, Inc., Cat. No.: BE0146), respectively. The tumor volumes of the mice were observed and measured periodically after the administration, and the results are shown in FIG. 14.

The test results show that the AB12V8 single drug group has obvious inhibition effect on the tumor growth of the MC38 colon cancer transplantation tumor model, and simultaneously, the combined group of the AB12V8 and anti-mouse PD-1(0.5mg/kg) has obvious synergistic effect relative to each antibody single drug group. All treatment groups showed no animal death and significant weight loss during the observation period, showed no significant drug toxicity, and mice were well tolerated by the AB12V8 antibody during the treatment period.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

SEQUENCE LISTING

<110> Sichuan Kolun Bordete biomedical corporation

Anyuan pharmaceutical technology (Shanghai) Co., Ltd

<120> anti-TIGIT antibody and use thereof

<130>IDC180239

<160>66

<170>PatentIn version 3.5

<210>1

<211>119

<212>PRT

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<223> murine antibody Mab21 heavy chain variable region

<400>1

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

1 5 10 15

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

20 25 30

Tyr Ile Asn Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Pro Lys Ser Gly Asp Tyr Gly Trp Tyr Phe Asp Val Trp Gly Ala Gly

100 105110

Thr Thr Val Thr Val Ser Ser

115

<210>2

<211>108

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<223> murine antibody Mab21 light chain variable region

<400>2

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

1 5 10 15

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

20 25 30

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

35 40 45

Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Tyr Phe Thr Leu Ser Ile Asn Ser Val Glu Thr

65 70 75 80

Glu Asp Phe Gly Met Tyr Phe Cys Gln Gln Ser Asn Ser Trp Leu Thr

85 90 95

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Ala

100 105

<210>3

<211>119

<212>PRT

<213> Artificial sequence

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<223> murine antibody Mab7 heavy chain variable region

<400>3

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Gly Ser Tyr Gly Ser Ser Tyr Thr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser

115

<210>4

<211>109

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<223> murine antibody Mab7 light chain variable region

<400>4

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

1 5 10 15

Asp Ser Val Ser Ile Thr Cys Lys Ala Ser Gln His Val Ser Thr Ala

20 25 30

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

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala

65 70 75 80

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

85 90 95

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

100 105

<210>5

<211>8

<212>PRT

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<223>IMGT Mab21 CDR-H1

<400>5

Gly Phe Asn Ile Lys Asp Thr Tyr

1 5

<210>6

<211>8

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<223>IMGT Mab21 CDR-H2

<400>6

Ile Asp Pro Ala Asn Gly Asp Thr

1 5

<210>7

<211>12

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<223>IMGT Mab21 CDR-H3

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Pro Lys Ser Gly Asp Tyr Gly Trp Tyr Phe Asp Val

1 5 10

<210>8

<211>6

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<220>

<223>IMGT Mab21 CDR-L1

<400>8

Gln Ser Ile Ser Asn Asn

1 5

<210>9

<211>3

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<220>

<223>IMGT Mab21 CDR-L2

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Tyr Ala Ser

1

<210>10

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<220>

<223>IMGT/Kabat/Chothia Mab21 CDR-L3

<400>10

Gln Gln Ser Asn Ser Trp Leu Thr

1 5

<210>11

<211>8

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<223>IMGT Mab7 CDR-H1

<400>11

Gly Tyr Ala Phe Thr Asn Tyr Leu

1 5

<210>12

<211>8

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<213> Artificial sequence

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<223>IMGT Mab7 CDR-H2

<400>12

Ile Asn Pro Gly Ser Gly Gly Thr

1 5

<210>13

<211>12

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<223>IMGT Mab7 CDR-H3

<400>13

Ala Gly Ser Tyr Gly Ser Ser Tyr Thr Phe Asp Tyr

1 5 10

<210>14

<211>6

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<223>IMGT Mab7 CDR-L1

<400>14

Gln His Val Ser Thr Ala

1 5

<210>15

<211>3

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<220>

<223>IMGT Mab7 CDR-L2

<400>15

Ser Pro Ser

1

<210>16

<211>9

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<220>

<223>IMGT/Kabat/Chothia Mab7 CDR-L3

<400>16

His Gln His Tyr Ser Thr Pro Trp Thr

1 5

<210>17

<211>5

<212>PRT

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<220>

<223>Kabat Mab21 CDR-H1

<400>17

Asp Thr Tyr Ile Asn

1 5

<210>18

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<220>

<223>Kabat Mab21 CDR-H2

<400>18

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

1 5 10 15

Gly

<210>19

<211>10

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<223>Kabat/Chothia Mab21 CDR-H3

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Ser Gly Asp Tyr Gly Trp Tyr Phe Asp Val

1 5 10

<210>20

<211>11

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<223>Kabat/Chothia Mab21 CDR-L1

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Arg Ala Ser Gln Ser Ile Ser Asn Asn Leu His

1 5 10

<210>21

<211>7

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<223>Kabat/Chothia Mab21 CDR-L2

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Tyr Ala Ser Gln Ser Ile Ser

1 5

<210>22

<211>5

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<223>Kabat Mab7 CDR-H1

<400>22

Asn Tyr Leu Ile Glu

1 5

<210>23

<211>17

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<220>

<223>Kabat Mab7 CDR-H2

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

1 5 10 15

Gly

<210>24

<211>10

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<223>Kabat/Chothia Mab7 CDR-H3

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Ser Tyr Gly Ser Ser Tyr Thr Phe Asp Tyr

1 5 10

<210>25

<211>11

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<213> Artificial sequence

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<223>Kabat/Chothia Mab7 CDR-L1

<400>25

Lys Ala Ser Gln His Val Ser Thr Ala Val Val

1 5 10

<210>26

<211>7

<212>PRT

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<223>Kabat/Chothia Mab7 CDR-L2

<400>26

Ser Pro Ser Tyr Arg Tyr Thr

1 5

<210>27

<211>7

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<220>

<223>Chothia Mab21 CDR-H1

<400>27

Gly Phe Asn Ile Lys Asp Thr

1 5

<210>28

<211>6

<212>PRT

<213> Artificial sequence

<220>

<223>Chothia Mab21 CDR-H2

<400>28

Asp Pro Ala Asn Gly Asp

1 5

<210>29

<211>7

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<213> Artificial sequence

<220>

<223>Chothia Mab7 CDR-H1

<400>29

Gly Tyr Ala Phe Thr Asn Tyr

1 5

<210>30

<211>6

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<213> Artificial sequence

<220>

<223>Chothia Mab7 CDR-H2

<400>30

Asn Pro Gly Ser Gly Gly

1 5

<210>31

<211>119

<212>PRT

<213> Artificial sequence

<220>

<223> humanized antibody AB12V3 heavy chain variable region

<400>31

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

1 5 10 15

Thr Val Lys Leu Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile Asn Trp Val Lys Gln Arg Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Pro Lys Ser Gly Asp Tyr Gly Trp Tyr Phe Asp Val Trp Gly Gln Gly

100 105 110

Thr Met Val Thr Val Ser Ser

115

<210>32

<211>106

<212>PRT

<213> Artificial sequence

<220>

<223> humanized antibody AB12V3 light chain variable region

<400>32

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

1 5 10 15

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

20 25 30

Leu His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Tyr Phe Thr Leu Ser Ile Asn Ser Val Glu Ala

65 70 75 80

Glu Asp Phe Gly Met Tyr Phe Cys Gln Gln Ser Asn Ser Trp Leu Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>33

<211>119

<212>PRT

<213> Artificial sequence

<220>

<223> humanized antibody AB12V4 heavy chain variable region

<400>33

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Val Ile Asn Pro Gly Ser Gly Gly Thr Ile Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Gly Ser Tyr Gly Ser Ser Tyr Thr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Leu Thr Val Ser Ser

115

<210>34

<211>107

<212>PRT

<213> Artificial sequence

<220>

<223> humanized antibody AB12V4 light chain variable region

<400>34

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr Ser Pro Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys His Gln His Tyr Ser Thr Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210>35

<211>119

<212>PRT

<213> Artificial sequence

<220>

<223> humanized antibody AB12V5 heavy chain variable region

<400>35

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

1 5 10 15

Thr Val Lys Leu Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile Asn Trp Val Lys Gln Arg Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Pro Lys Ser Gly Asp Tyr Gly Trp Tyr Phe Asp Val Trp Gly Gln Gly

100 105 110

Thr Met Val Thr Val Ser Ser

115

<210>36

<211>106

<212>PRT

<213> Artificial sequence

<220>

<223> humanized antibody AB12V5 light chain variable region

<400>36

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

1 5 10 15

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

20 25 30

Leu His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Tyr Phe Thr Leu Ser Ile Asn Ser Val Glu Ala

65 70 75 80

Glu Asp Phe Gly Met Tyr Phe Cys Gln Gln Ser Asn Ser Trp Leu Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>37

<211>119

<212>PRT

<213> Artificial sequence

<220>

<223> humanized antibody AB12V6 heavy chain variable region

<400>37

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Val Ile Asn Pro Gly Ser Gly Gly Thr Ile Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Gly Ser Tyr Gly Ser Ser Tyr Thr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Leu Thr Val Ser Ser

115

<210>38

<211>107

<212>PRT

<213> Artificial sequence

<220>

<223> humanized antibody AB12V6 light chain variable region

<400>38

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr Ser Pro Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys His Gln His Tyr Ser Thr Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210>39

<211>119

<212>PRT

<213> Artificial sequence

<220>

<223> humanized antibody AB12V7 heavy chain variable region

<400>39

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

Pro Lys Ser Gly Asp Tyr Gly Trp Tyr Phe Asp Val Trp Gly Gln Gly

100 105 110

Thr Met Val Thr Val Ser Ser

115

<210>40

<211>106

<212>PRT

<213> Artificial sequence

<220>

<223> humanized antibody AB12V7 light chain variable region

<400>40

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

1 5 10 15

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

20 25 30

Leu His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Arg Leu Leu Ile

35 4045

Lys Tyr Ala Ser Gln Ser Ile Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Tyr Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala

65 70 75 80

Glu Asp Ala Ala Thr Tyr Phe Cys Gln Gln Ser Asn Ser Trp Leu Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>41

<211>119

<212>PRT

<213> Artificial sequence

<220>

<223> humanized antibody AB12V8 heavy chain variable region

<400>41

Gln 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 Ala Phe Thr Asn Tyr

20 25 30

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

35 40 45

Gly Val Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Ala Gln Lys Leu

50 55 60

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

65 70 75 80

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

85 90 95

Ala Gly Ser Tyr Gly Ser Ser Tyr Thr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210>42

<211>107

<212>PRT

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<220>

<223> humanized antibody AB12V8 light chain variable region

<400>42

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys His Gln His Tyr Ser Thr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>43

<211>119

<212>PRT

<213> Artificial sequence

<220>

<223> humanized antibody AB12V9 heavy chain variable region

<400>43

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu AspThr Ala Val Tyr Tyr Cys

85 90 95

Pro Lys Ser Gly Asp Tyr Gly Trp Tyr Phe Asp Val Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210>44

<211>106

<212>PRT

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<220>

<223> humanized antibody AB12V9 light chain variable region

<400>44

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

1 5 10 15

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

20 25 30

Leu His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Ile Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Ala Ala Thr Tyr Phe Cys Gln Gln Ser Asn Ser Trp Leu Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>45

<211>327

<212>PRT

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<220>

<223> human IgG4 heavy chain constant region

<400>45

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 Ser Cys Pro Ala Pro

100105 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

260265 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>46

<211>327

<212>PRT

<213> Artificial sequence

<220>

<223> human IgG4 heavy chain constant region variant (IgG4V 1)

<400>46

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>47

<211>330

<212>PRT

<213> Artificial sequence

<220>

<223> human IgG1 heavy chain constant region

<400>47

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

1 5 10 15

Ser Thr Ser Gly Gly 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 Gln Thr

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210>48

<211>107

<212>PRT

<213> Artificial sequence

<220>

<223> human kappa light chain constant region

<400>48

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>49

<211>1347

<212>DNA

<213> Artificial sequence

<220>

<223> humanized antibody AB12V8 heavy chain variable region nucleotide sequence

<400>49

caggtgcagc tggtgcagtc tggcgccgag gtgaagaagc caggcgcttc tgtgaaggtg 60

tcctgcaagg ccagcggcta tgctttcacc aactacctga tcgagtggat cagacaggct 120

cctggacagg gactggagtg gatgggcgtg atcaacccag gaagcggagg aacaaattac 180

gctcagaagc tgcagggaag ggtgaccctg acagctgaca agtctaccaa tacagcttat 240

atggagctga gaagcctgcg ctctgacgat accgccgtgt acttttgcgc tggctcttac 300

ggctccagct atacattcga ctactggggc cagggcaccc tggtgacagt gtcttccgct 360

tctaccaagg gcccatccgt gtttcccctg gcccctagct ctaagtctac ctccggagga 420

acagccgctc tgggatgtct ggtgaaggat tacttccccg agcctgtgac cgtgtcttgg 480

aactccggcg ctctgacctc tggcgtgcac acatttcctg ccgtgctgca gtccagcggc 540

ctgtattccc tgtcttccgt ggtgaccgtg ccaagctctt ccctgggcac ccagacatac 600

atctgcaacg tgaatcacaa gccctctaat acaaaggtgg acaagaaggt ggagcctaag 660

tcctgtgata agacccatac atgcccccct tgtccagctc cagagctgct gggaggacca 720

tccgtgttcc tgtttccacc caagcccaag gacaccctga tgatctctcg gacccctgag 780

gtgacatgcg tggtggtgga cgtgtcccac gaggacccag aggtgaagtt caactggtac 840

gtggatggcg tggaggtgca taatgccaag accaagccta gagaggagca gtataactct 900

acataccgcg tggtgtccgt gctgaccgtg ctgcaccagg actggctgaa cggcaaggag 960

tacaagtgca aggtgagcaa taaggctctg cctgccccaa tcgagaagac aatctctaag 1020

gctaagggcc agccaaggga gccccaggtgtataccctgc ctccaagccg ggacgagctg 1080

acaaagaacc aggtgtctct gacctgtctg gtgaagggct tctacccatc tgatatcgcc 1140

gtggagtggg agtccaatgg ccagcccgag aacaattata agaccacacc ccctgtgctg 1200

gacagcgatg gatccttctt tctgtactcc aagctgaccg tggacaagag caggtggcag 1260

cagggcaacg tgtttagctg ctctgtgatg catgaggctc tgcacaatca ttacacacag 1320

aagtccctga gcctgtctcc tggcaag 1347

<210>50

<211>642

<212>DNA

<213> Artificial sequence

<220>

<223> humanized antibody AB12V8 light chain variable region nucleotide sequence

<400>50

gatatccaga tgacccagtc ccctagctct ctgagcgcct ctgtgggcga cagagtgacc 60

atcacatgtc gcgcctctca gcacgtgtcc acagctgtgg tgtggtatca gcagaagcca 120

ggcaaggctc ccaagctgct gatctactcc cctagctaca ggtataccgg cgtgccaagc 180

cggttttctg gctccggcag cggcacagac ttcaccctga caatctccag cctgcagcca 240

gaggattttg ccacctacta ttgccaccag cattactcta ccccctggac attcggccag 300

ggcacaaagc tggagatcaa gcgcaccgtg gctgcccctt ccgtgttcat ctttcctcca 360

tccgacgagc agctgaagtc tggcacagcc tccgtggtgt gcctgctgaa caatttctat 420

ccaagagagg ctaaggtgca gtggaaggtg gataacgccc tgcagtctgg caattcccag 480

gagagcgtga cagagcagga ctctaaggat tccacctaca gcctgtccag caccctgaca 540

ctgagcaagg ctgattatga gaagcacaag gtgtacgcct gcgaggtgac acatcagggc 600

ctgtcttccc ccgtgaccaa gagcttcaac cgcggcgagt gt 642

<210>51

<211>1347

<212>DNA

<213> Artificial sequence

<220>

<223> humanized antibody AB12V9 heavy chain variable region nucleotide sequence

<400>51

caggtgcagc tggtgcagag cggagctgag gtgaagaagc ctggctccag cgtgaaggtg 60

tcctgcaagg ctagcggctt taacatcaag gacacctaca tcaattgggt gagacaggct 120

ccaggacagg gactggagtg gatgggaagg atcgaccctg ctagcggcga taccaagtac 180

gctcagaagt tccagggcag agtgacaatc accacagaca cctctacaaa caccgcctat 240

atggagctgt cttccctgcg cagcgaggat accgccgtgt actattgccc aaagtctggc 300

gactacggct ggtattttga cgtgtggggc cagggcacac tggtgaccgt gagctccgct 360

tctaccaagg gcccatccgt gtttcccctg gcccctagct ctaagtctac ctccggagga 420

acagccgctc tgggatgtct ggtgaaggat tacttccccg agcctgtgac cgtgtcttgg 480

aactccggcg ctctgacctc tggcgtgcac acatttcctg ccgtgctgca gtccagcggc 540

ctgtattccc tgtcttccgt ggtgaccgtg ccaagctctt ccctgggcac ccagacatac 600

atctgcaacg tgaatcacaa gccctctaat acaaaggtgg acaagaaggt ggagcctaag 660

tcctgtgata agacccatac atgcccccct tgtccagctc cagagctgct gggaggacca 720

tccgtgttcc tgtttccacc caagcccaag gacaccctga tgatctctcg gacccctgag 780

gtgacatgcg tggtggtgga cgtgtcccac gaggacccag aggtgaagtt caactggtac 840

gtggatggcg tggaggtgca taatgccaag accaagccta gagaggagca gtataactct 900

acataccgcg tggtgtccgt gctgaccgtg ctgcaccagg actggctgaa cggcaaggag 960

tacaagtgca aggtgagcaa taaggctctg cctgccccaa tcgagaagac aatctctaag 1020

gctaagggcc agccaaggga gccccaggtg tataccctgc ctccaagccg ggacgagctg 1080

acaaagaacc aggtgtctct gacctgtctg gtgaagggct tctacccatc tgatatcgcc 1140

gtggagtggg agtccaatgg ccagcccgag aacaattata agaccacacc ccctgtgctg 1200

gacagcgatg gatccttctt tctgtactcc aagctgaccg tggacaagag caggtggcag 1260

cagggcaacg tgtttagctg ctctgtgatg catgaggctc tgcacaatca ttacacacag 1320

aagtccctga gcctgtctcc tggcaag 1347

<210>52

<211>639

<212>DNA

<213> Artificial sequence

<220>

<223> humanized antibody AB12V9 light chain variable region nucleotide sequence

<400>52

gagatcgtgc tgacccagtc tccagacttc cagtccgtga cacccaagga gaaggtgaca 60

atcacctgta gggcctccca gagcatctcc agcaacctgc actggtacca gcagaagcca 120

gatcagtccc ccaagctgct gatcaagtat gcttctcagt ccatcagcgg agtgccaagc 180

cggttctctg gatccggaag cggaaccgac tttacactga ccatctcttc cctggaggcc240

gaggatgccg ctacatattt ctgccagcag tctaattcct ggctgacctt tggccagggc 300

acaaagctgg agatcaagcg caccgtggct gccccttccg tgttcatctt tcctccatcc 360

gacgagcagc tgaagtctgg cacagcctcc gtggtgtgcc tgctgaacaa tttctatcca 420

agagaggcta aggtgcagtg gaaggtggat aacgccctgc agtctggcaa ttcccaggag 480

agcgtgacag agcaggactc taaggattcc acctacagcc tgtccagcac cctgacactg 540

agcaaggctg attatgagaa gcacaaggtg tacgcctgcg aggtgacaca tcagggcctg 600

tcttcccccg tgaccaagag cttcaaccgc ggcgagtgt 639

<210>53

<211>446

<212>PRT

<213> Artificial sequence

<220>

<223> AB12V3 heavy chain

<400>53

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

1 5 10 15

Thr Val Lys Leu Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile Asn Trp Val Lys Gln Arg Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

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

50 55 60

Gln Gly Arg Ala ThrIle Thr Thr Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

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

85 90 95

Pro Lys Ser Gly Asp Tyr Gly Trp Tyr Phe Asp Val Trp Gly Gln Gly

100 105 110

Thr Met 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>54

<211>213

<212>PRT

<213> Artificial sequence

<220>

<223> AB12V3 light chain

<400>54

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

1 5 10 15

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

20 25 30

Leu His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Tyr Phe Thr Leu Ser Ile AsnSer Val Glu Ala

65 70 75 80

Glu Asp Phe Gly Met Tyr Phe Cys Gln Gln Ser Asn Ser Trp Leu Thr

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Asn Arg Gly Glu Cys

210

<210>55

<211>446

<212>PRT

<213> Artificial sequence

<220>

<223> AB12V4 heavy chain

<400>55

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Val Ile Asn Pro Gly Ser Gly Gly Thr Ile Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Gly Ser Tyr Gly Ser Ser Tyr Thr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Leu 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 135140

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>56

<211>214

<212>PRT

<213> Artificial sequence

<220>

<223> AB12V4 light chain

<400>56

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr Ser Pro Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys His Gln His Tyr Ser Thr Pro Trp

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210>57

<211>449

<212>PRT

<213> Artificial sequence

<220>

<223> AB12V5 heavy chain

<400>57

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

1 5 10 15

Thr Val Lys Leu Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile Asn Trp Val Lys Gln Arg Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Pro Lys Ser Gly Asp Tyr Gly Trp Tyr Phe Asp Val Trp Gly Gln Gly

100 105 110

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

115 120 125

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

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

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

260 265 270

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

275 280 285

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

405 410 415

Ser Arg Trp Gln Gln 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 Pro Gly

435 440 445

Lys

<210>58

<211>213

<212>PRT

<213> Artificial sequence

<220>

<223> AB12V5 light chain

<400>58

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

1 5 10 15

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

20 25 30

Leu His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Tyr Phe Thr Leu Ser Ile Asn Ser Val Glu Ala

65 70 75 80

Glu Asp Phe Gly Met Tyr Phe Cys Gln Gln Ser Asn Ser Trp Leu Thr

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Asn Arg Gly Glu Cys

210

<210>59

<211>449

<212>PRT

<213> Artificial sequence

<220>

<223> AB12V6 heavy chain

<400>59

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Val Ile Asn Pro Gly Ser Gly Gly Thr Ile Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Gly Ser Tyr Gly Ser Ser Tyr Thr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

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

115120 125

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

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

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

260 265 270

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

275 280285

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

405 410 415

Ser Arg Trp Gln Gln 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 Pro Gly

435 440445

Lys

<210>60

<211>214

<212>PRT

<213> Artificial sequence

<220>

<223> AB12V6 light chain

<400>60

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr Ser Pro Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys His Gln His Tyr Ser Thr Pro Trp

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210>61

<211>449

<212>PRT

<213> Artificial sequence

<220>

<223> AB12V7 heavy chain

<400>61

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

Pro Lys Ser Gly Asp Tyr Gly Trp Tyr Phe Asp Val Trp Gly Gln Gly

100 105 110

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

115 120 125

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

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

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

260 265 270

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

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 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 Ala Leu Pro Ala Pro 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 ProPro Ser Arg Asp Glu Leu 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 Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln 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 Pro Gly

435 440 445

Lys

<210>62

<211>213

<212>PRT

<213> Artificial sequence

<220>

<223> AB12V7 light chain

<400>62

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

1 5 10 15

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

20 25 30

Leu His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Ile Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Tyr Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala

65 70 75 80

Glu Asp Ala Ala Thr Tyr Phe Cys Gln Gln Ser Asn Ser Trp Leu Thr

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Asn Arg Gly Glu Cys

210

<210>63

<211>449

<212>PRT

<213> Artificial sequence

<220>

<223> AB12V8 heavy chain

<400>63

Gln 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 Ala Phe Thr Asn Tyr

20 25 30

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

35 40 45

Gly Val Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Ala Gln Lys Leu

50 55 60

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

65 70 75 80

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

85 90 95

Ala Gly Ser Tyr Gly Ser Ser Tyr Thr 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 Ser Ser Lys Ser Thr Ser Gly Gly 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 Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

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

260 265 270

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

275 280 285

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

405 410 415

Ser Arg Trp Gln Gln 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 Pro Gly

435 440 445

Lys

<210>64

<211>214

<212>PRT

<213> Artificial sequence

<220>

<223> AB12V8 light chain

<400>64

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr Ser Pro Ser Tyr 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 His Gln His Tyr Ser Thr Pro Trp

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>65

<211>449

<212>PRT

<213> Artificial sequence

<220>

<223> AB12V9 heavy chain

<400>65

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

1 510 15

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

20 25 30

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

35 40 45

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

50 55 60

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

Pro Lys Ser Gly Asp Tyr Gly Trp Tyr Phe Asp Val 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 Ser Ser Lys Ser Thr Ser Gly Gly 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 170175

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

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

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

260 265 270

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

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 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 Ala Leu Pro Ala Pro Ile Glu Lys

325 330335

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

340 345 350

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

405 410 415

Ser Arg Trp Gln Gln 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 Pro Gly

435 440 445

Lys

<210>66

<211>213

<212>PRT

<213> Artificial sequence

<220>

<223> AB12V9 light chain

<400>66

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

1 5 10 15

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

20 25 30

Leu His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Ile Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Ala Ala Thr Tyr Phe Cys Gln Gln Ser Asn Ser Trp Leu Thr

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Asn Arg Gly Glu Cys

210

Claims

1. An antibody or antigen-binding fragment thereof capable of specifically binding TIGIT, the antibody or antigen-binding fragment thereof comprising Complementarity Determining Regions (CDRs) selected from the group consisting of:

(a) the following CDRs of 3 heavy chain variable regions (VH):

(iii) CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 1, or a sequence having substitution, deletion, or addition of one or several amino acids (e.g., substitution, deletion, or addition of 1, 2, or 3 amino acids) as compared with the sequence of CDR-H3 contained in the VH; and/or the presence of a gas in the gas,

the CDRs of the following 3 light chain variable regions (VL):

(vi) CDR-L3 having the amino acid sequence as set forth in SEQ ID NO: 2, or a sequence having substitution, deletion, or addition of one or several amino acids (e.g., substitution, deletion, or addition of 1, 2, or 3 amino acids) as compared with the sequence of CDR-L3 contained in the VL;

or,

(b) the following CDRs of 3 heavy chain variable regions (VH):

(iii) CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 3, or a sequence having substitution, deletion, or addition of one or several amino acids (e.g., substitution, deletion, or addition of 1, 2, or 3 amino acids) as compared with the sequence of CDR-H3 contained in the VH; and/or the presence of a gas in the gas,

the CDRs of the following 3 light chain variable regions (VL):

(vi) CDR-L3 having the amino acid sequence as set forth in SEQ ID NO:4, or a sequence having substitution, deletion, or addition of one or several amino acids (e.g., substitution, deletion, or addition of 1, 2, or 3 amino acids) as compared with the sequence of CDR-L3 contained in the VL;

preferably, the substitution recited in any one of (i) - (vi) is a conservative substitution;

preferably, the CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) and/or the CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region (VL) are defined by the Kabat, Chothia or IMGT numbering system.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises:

(a) 3 CDRs contained in a heavy chain variable region (VH) selected from:

as shown in SEQ ID NO: 1. SEQ ID NO: 31. SEQ ID NO: 35. SEQ ID NO: 39 and SEQ ID NO: 43 or VH set forth in any one of items;

and/or the presence of a gas in the gas,

3 CDRs contained in a light chain variable region (VL) selected from:

as shown in SEQ ID NO: 2. SEQ ID NO: 32. SEQ ID NO: 36. SEQ ID NO: 40 and SEQ ID NO: 44 VL of any one of;

or

(b) 3 CDRs contained in a heavy chain variable region (VH) selected from:

and/or the presence of a gas in the gas,

3 CDRs contained in a light chain variable region (VL) selected from:

as shown in SEQ ID NO: 4. SEQ ID NO: 34. SEQ ID NO: 38 and SEQ ID NO: 42 VL of any one of;

preferably, the 3 CDRs contained in the heavy chain variable region (VH) and/or the 3 CDRs contained in the light chain variable region (VL) are defined by the Kabat, Chothia or IMGT numbering system.

3. The antibody or antigen-binding fragment thereof of claim 2, wherein the antibody or antigen-binding fragment thereof comprises:

(1) (a) as set forth in SEQ ID NO: 1 (VH) comprises 3 CDRs; and/or, as shown in SEQ ID NO: 2 (VL) comprises 3 CDRs; or (b) as set forth in SEQ ID NO: 3 (3) of the 3 CDRs contained in the heavy chain variable region (VH); and/or, as shown in SEQ ID NO:4 (VL) comprising 3 CDRs;

wherein the 3 CDRs contained in the heavy chain variable region (VH) and the 3 CDRs contained in the light chain variable region (VL) are defined by the IMGT numbering system;

(2) (a) as set forth in SEQ ID NO: 1 (VH) comprises 3 CDRs; and/or, as shown in SEQ ID NO: 2 (VL) comprises 3 CDRs; or (b) as set forth in SEQ ID NO: 3 (3) of the 3 CDRs contained in the heavy chain variable region (VH); and/or, as shown in SEQ ID NO:4 (VL) comprising 3 CDRs; or (c) as shown in SEQ ID NO: 41 (VH) comprising 3 CDRs; and/or, as shown in SEQ ID NO: 42 in the light chain variable region (VL) comprising 3 CDRs; or (d) as shown in SEQ ID NO: 43, 3 CDRs contained in the heavy chain variable region (VH); and/or, as shown in SEQ ID NO: 44 in the light chain variable region (VL) comprising 3 CDRs;

wherein the 3 CDRs contained in the heavy chain variable region (VH) and the 3 CDRs contained in the light chain variable region (VL) are defined by the Kabat numbering system; or

(3) As shown in SEQ ID NO: 1 (VH) comprises 3 CDRs; and/or, as shown in SEQ ID NO: 2 (VL) comprises 3 CDRs; or (b) as set forth in SEQ ID NO: 3 (3) of the 3 CDRs contained in the heavy chain variable region (VH); and/or, as shown in SEQ ID NO:4 (VL) comprising 3 CDRs; or (c) as shown in SEQ ID NO: 41 (VH) comprising 3 CDRs; and/or, as shown in SEQ ID NO: 42 in the light chain variable region (VL) comprising 3 CDRs; or (d) as shown in SEQ ID NO: 43, 3 CDRs contained in the heavy chain variable region (VH); and/or, as shown in SEQ ID NO: 44 in the light chain variable region (VL) comprising 3 CDRs;

4. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises:

(1) CDRs defined by IMGT numbering system:

(a) the following CDRs of 3 heavy chain variable regions (VH):

and/or

The CDRs of the following 3 light chain variable regions (VL):

(vi) CDR-L3, consisting of the following sequence: SEQ ID NO: 10, or a variant of SEQ ID NO: 10 with one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions); or

(b) The following CDRs of 3 heavy chain variable regions (VH):

and/or

The CDRs of the following 3 light chain variable regions (VL):

or,

(2) CDRs defined by Chothia numbering system:

(a) the following 3 heavy chain variable region (VH) CDRs:

and/or the presence of a gas in the gas,

the following 3 light chain variable region (VL) CDRs:

(b) The following CDRs of 3 heavy chain variable regions (VH):

the following 3 heavy chain variable region (VH) CDRs:

and/or the presence of a gas in the gas,

the following 3 light chain variable region (VL) CDRs:

or,

(3) CDRs defined by Kabat numbering system:

(a) the following 3 heavy chain variable region (VH) CDRs:

and/or the presence of a gas in the gas,

the following 3 light chain variable region (VL) CDRs:

(b) The following CDRs of 3 heavy chain variable regions (VH):

and/or the presence of a gas in the gas,

the following 3 light chain variable region (VL) CDRs:

preferably, the substitution recited in any one of (i) - (vi) is a conservative substitution.

5. The antibody or antigen-binding fragment thereof of claim 4, wherein:

(a) the VH of the antibody or antigen-binding fragment thereof comprises: as shown in SEQ ID NO:5 CDR-H1; as shown in SEQ ID NO: 6 CDR-H2; and, as shown in SEQ ID NO: 7 CDR-H3;

and, the VL of the antibody or antigen-binding fragment thereof comprises: as shown in SEQ ID NO: 8 CDR-L1; as shown in SEQ ID NO: 9 CDR-L2; and, as shown in SEQ ID NO: 10 CDR-L3; or

(b) The VH of the antibody or antigen-binding fragment thereof comprises: as shown in SEQ ID NO: 11 CDR-H1; as shown in SEQ ID NO: CDR-H2 shown in FIG. 12; and, as shown in SEQ ID NO: 13 CDR-H3;

and, the VL of the antibody or antigen-binding fragment thereof comprises: as shown in SEQ ID NO: 14 CDR-L1; as shown in SEQ ID NO: 15 CDR-L2; and, as shown in SEQ ID NO: 16 CDR-L3;

wherein, each CDR is defined by an IMGT numbering system.

6. The antibody or antigen-binding fragment thereof of claim 4, wherein:

(a) the VH of the antibody or antigen-binding fragment thereof comprises: as shown in SEQ ID NO: CDR-H1 shown in FIG. 27; as shown in SEQ ID NO: 28 CDR-H2; and, as shown in SEQ ID NO: 19 CDR-H3; and, the VL of the antibody or antigen-binding fragment thereof comprises: as shown in SEQ ID NO: 20 CDR-L1; as shown in SEQ ID NO: 21 CDR-L2; and, as shown in seq id NO: 10 CDR-L3; or

(b) The VH of the antibody or antigen-binding fragment thereof comprises: as shown in SEQ ID NO: CDR-H1 shown in FIG. 29; as shown in SEQ ID NO: CDR-H2 shown in FIG. 30; and, as shown in SEQ ID NO: CDR-H3 shown in FIG. 24; and, the VL of the antibody or antigen-binding fragment thereof comprises: as shown in SEQ ID NO: 25 CDR-L1; as shown in SEQ ID NO: CDR-L2 shown in FIG. 26; and, as shown in seq id NO: 16 CDR-L3;

wherein, each CDR is defined by Chothia numbering system.

7. The antibody or antigen-binding fragment thereof of claim 4, wherein:

(a) the VH of the antibody or antigen-binding fragment thereof comprises: as shown in SEQ ID NO: 17 CDR-H1; as shown in SEQ ID NO: 18 CDR-H2; and, as shown in SEQ ID NO: 19 CDR-H3; and, the VL of the antibody or antigen-binding fragment thereof comprises: as shown in SEQ ID NO: 20 CDR-L1; as shown in SEQ ID NO: 21 CDR-L2; and, as shown in seq id NO: 10 CDR-L3; or

(b) The VH of the antibody or antigen-binding fragment thereof comprises: as shown in SEQ ID NO: 22 CDR-H1; as shown in SEQ ID NO: CDR-H2 shown in FIG. 23; and, as shown in SEQ ID NO: CDR-H3 shown in FIG. 24; and, the VL of the antibody or antigen-binding fragment thereof comprises: as shown in SEQ ID NO: 25 CDR-L1; as shown in SEQ ID NO: CDR-L2 shown in FIG. 26; and, as shown in seq id NO: 16 CDR-L3;

wherein each of the above CDRs is defined by the Kabat numbering system.

8. The antibody or antigen-binding fragment thereof of any one of claims 1-7, wherein the antibody or antigen-binding fragment thereof comprises:

(1) (a) a heavy chain variable region (VH) comprising an amino acid sequence selected from:

(i) SEQ ID NOs: 1. 31, 35, 39, 43;

and/or

(iv) SEQ ID NOs: 2. 32, 36, 40, 44;

(vi) And SEQ ID NOs: 2. 32, 36, 40, 44, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity;

or

(2) (a) a heavy chain variable region (VH) comprising an amino acid sequence selected from:

(i) SEQ ID NOs: 3. 33, 37 or 41;

and/or

(iv) SEQ ID NOs: 4. 34, 38, 42;

(vi) And SEQ ID NOs: 4. 34, 38, 42, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity;

preferably, the substitutions described in (ii) or (v) are conservative substitutions.

9. The antibody or antigen-binding fragment thereof of any one of claims 1-8, wherein the antibody or antigen-binding fragment thereof comprises:

(a) a heavy chain variable region (VH) and a light chain variable region (VL) comprising:

(i) has the sequence shown in SEQ ID NO: 1 and VH having the sequence shown as SEQ ID NO: 2, VL of the sequence set forth in seq id no;

(ii) has the sequence shown in SEQ ID NO: 31 and a VH having the sequence shown as SEQ ID NO: 32, VL of the sequence set forth in seq id no;

(iii) has the sequence shown in SEQ ID NO: 35 and a VH having the sequence shown as SEQ ID NO: 36, VL of the sequence set forth in seq id no;

(iv) has the sequence shown in SEQ ID NO: 39 and VH having the sequence shown as SEQ ID NO: 40, VL of the sequence set forth in seq id no; or

(v) Has the sequence shown in SEQ ID NO: 43 and a VH having the sequence shown as SEQ ID NO: 44, VL of the sequence set forth in seq id no;

or

(b) A heavy chain variable region (VH) and a light chain variable region (VL) comprising:

(i) has the sequence shown in SEQ ID NO: 3 and VH having the sequence shown as SEQ ID NO:4, VL of the sequence set forth in seq id no;

(ii) has the sequence shown in SEQ ID NO: 33 and a VH having the sequence shown as SEQ ID NO: 34, VL of the sequence set forth in seq id no;

(iii) has the sequence shown in SEQ ID NO: 37 and a VH having the sequence shown as SEQ ID NO: 38, VL of the sequence set forth in seq id no; or

(iv) Has the sequence shown in SEQ ID NO: 41 and VH having the sequence shown as SEQ ID NO: 42, VL of the sequence set forth in seq id no.

10. The antibody or antigen-binding fragment thereof of any one of claims 1-9, wherein the antibody or antigen-binding fragment thereof further comprises:

(a) a heavy chain constant region (CH) of a human immunoglobulin or a variant thereof having conservative substitutions of up to 20 amino acids (e.g., conservative substitutions of up to 15, up to 10, or up to 5 amino acids; e.g., conservative substitutions of 1, 2, 3, 4, or 5 amino acids) as compared to the sequence from which it is derived; and

(b) a light chain constant region (CL) of a human immunoglobulin or a variant thereof having conservative substitutions of up to 20 amino acids (e.g., conservative substitutions of up to 15, up to 10, or up to 5 amino acids; e.g., conservative substitutions of 1, 2, 3, 4, or 5 amino acids) as compared to the sequence from which it is derived;

preferably, the heavy chain constant region is an IgG heavy chain constant region, e.g., an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region;

preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region selected from the group consisting of:

(1) human IgG1 heavy chain constant region;

(2) human IgG4 heavy chain constant region;

(3) a variant of the human IgG4 heavy chain constant region having the following substitutions in comparison to the wild type sequence from which it is derived: ser228 Pro;

wherein the above-mentioned amino acid positions are positions according to the EU numbering system;

preferably, the antibody or antigen binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID NOs: 45-47 of any one of the heavy chain constant regions (CH);

preferably, the light chain constant region is a kappa light chain constant region;

preferably, the antibody or antigen binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID NO: 48 (CL) as shown.

11. The antibody or antigen-binding fragment thereof of any one of claims 1-10, wherein the antibody is a chimeric antibody or a humanized antibody.

12. The antibody or antigen-binding fragment thereof of any one of claims 1-11, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of ScFv, Fab ', (Fab')₂Fv fragments, disulfide-linked Fv (dsfv), diabodies (diabodies), bispecific antibodies and multispecific antibodies.

13. The antibody or antigen-binding fragment thereof of any one of claims 1-12, wherein the antibody or antigen-binding fragment thereof is labeled; preferably, the antibody or antigen-binding fragment thereof carries a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin.

14. The antibody or antigen-binding fragment thereof of any one of claims 1-13, wherein the antibody or antigen-binding fragment thereof is at about 100 x 10^-10K of M or less_DIn conjunction with TIGIT (e.g., human TIGIT);

preferably, the antibody or antigen-binding fragment thereof is present at about 1 × 10^-10K of M or less_DIn conjunction with TIGIT (e.g., human TIGIT).

15. An isolated nucleic acid molecule encoding the antibody or antigen binding fragment thereof of any one of claims 1-14, a heavy chain and/or light chain thereof, or a heavy chain variable region and/or light chain variable region thereof.

16. The isolated nucleic acid molecule of claim 15, comprising a nucleic acid molecule encoding an antibody heavy chain variable region, and/or a nucleic acid molecule encoding an antibody light chain variable region, wherein,

the nucleic acid molecule encoding the variable region of the antibody heavy chain has a sequence selected from the group consisting of: (a) 49 or 51, or (b) a sequence substantially identical to the nucleotide sequence of (a) (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity, or a sequence having one or more nucleotide substitutions, as compared to the nucleotide sequence of (a)), or (c) a sequence that differs from the nucleotide sequence of (a) by NO more than 3, 6, 15, 30, or 45 nucleotides, and,

the nucleic acid molecule encoding the variable region of the antibody light chain has a sequence selected from the group consisting of: (a) 50 or 52, or (b) a sequence substantially identical to the nucleotide sequence of (a) (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity, or a sequence having one or more nucleotide substitutions, as compared to the nucleotide sequence of (a)), or (c) a sequence that differs from the nucleotide sequence of (a) by NO more than 3, 6, 15, 30, or 45 nucleotides;

preferably, the nucleic acid molecule encoding the heavy chain variable region of the antibody has the nucleotide sequence shown as SEQ ID NO. 49, and the nucleic acid molecule encoding the light chain variable region of the antibody has the nucleotide sequence shown as SEQ ID NO. 50;

preferably, the nucleic acid molecule encoding the heavy chain variable region of the antibody has the nucleotide sequence shown as SEQ ID NO:51, and the nucleic acid molecule encoding the light chain variable region of the antibody has the nucleotide sequence shown as SEQ ID NO: 52.

17. A vector comprising the nucleic acid molecule of claim 15 or 16; preferably, the vector is a cloning vector or an expression vector.

18. A host cell comprising the nucleic acid molecule of claim 15 or 16 or the vector of claim 17.

19. A method of making the antibody or antigen-binding fragment thereof of any one of claims 1-14, comprising culturing the host cell of claim 18 under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

20. A hybridoma cell strain which is: hybridoma cell strain #7, which is preserved in China Center for Type Culture Collection (CCTCC) and has a preservation number of CCTCC NO. C2018210.

21. A hybridoma cell strain which is: hybridoma cell strain #21, which is preserved in China Center for Type Culture Collection (CCTCC) and has a preservation number of CCTCC NO. C2018209.

22. A monoclonal antibody produced by the hybridoma cell line of claim 20 or 21.

23. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-14, or the vector of claim 17, or the host cell of claim 18, and a pharmaceutically acceptable carrier and/or excipient;

preferably, the pharmaceutical composition further comprises an additional pharmaceutically active agent;

preferably, the additional pharmaceutically active agent is a drug for the treatment of an immune related disease;

preferably, the additional pharmaceutically active agent is a drug with anti-tumor activity;

preferably, the additional pharmaceutically active agent is a medicament for the treatment of an infection or infectious disease;

preferably, the antibody or antigen-binding fragment thereof and the additional pharmaceutically active agent are provided as separate components or as components of the same composition.

24. The pharmaceutical composition of claim 23, wherein the amount of antibody or antigen-binding fragment thereof in the pharmaceutical composition is sufficient to:

(a) induce and/or enhance an immune response;

(b) increasing effector T cell activity;

(c) increasing Cytotoxic T Lymphocyte (CTL) activity;

(d) increasing NK cell activity;

(e) inhibiting TIGIT activation;

(f) inhibiting TIGIT-mediated signaling;

(g) inhibits or blocks binding of CD155 and/or CD112 to TIGIT;

(h) reducing the number of regulatory T cells in the tissue or circulation;

(i) inhibiting suppression of effector T cells by regulatory T cells; or,

(j) any combination of (a) - (i).

25. The pharmaceutical composition of claim 23 or 24, further comprising a second antibody that specifically binds to or encodes a second antibody selected from the group consisting of a receptor or ligand selected from the group consisting of: PD-1, PD-L1, PD-L2, TIM-3, LAG-3, VISTA, CTLA-4, OX40, BTLA, 4-1BB, CD96, CD27, CD28, CD40, LAIR1, CD160, 2B4, TGF-R, KIR, ICOS, GITR, CD3, CD30, BAFFR, HVEM, CD7, LIGHT, SLAMF7, NKp80, B7-H3, and any combination thereof;

preferably, the second antibody is an antibody or antigen-binding fragment thereof that binds human PD-1;

preferably, the second antibody is an antibody or antigen-binding fragment thereof that binds human PD-L1.

26. An immunogenic composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-14, and an immunogen;

preferably, the antibody or antigen-binding fragment thereof is used as an adjuvant;

preferably, the immunogen is selected from the group consisting of a tumor cell, an antigen associated with a tumor (e.g., a protein, polypeptide, or carbohydrate molecule), a dendritic cell primed by the antigen, and any combination thereof;

preferably, the immunogen is selected from the group consisting of an inactivated or attenuated pathogen, an antigen (e.g., a protein, polypeptide, or carbohydrate molecule) associated with a pathogen, a dendritic cell primed by the antigen, and any combination thereof;

preferably, the antibody or antigen-binding fragment thereof is provided as a separate component from the immunogen or as a component of the same composition.

27. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-14, or the vector of claim 17, or the host cell of claim 18, in the preparation of a medicament for:

(1) increasing immune cell activity in vitro or in a subject (e.g., a human);

(2) enhancing an immune response in a subject (e.g., a human);

(3) treating a tumor in a subject (e.g., a human); or

(4) Treating an infection or infectious disease in a subject (e.g., a human);

preferably, the tumor is selected from the group consisting of a solid tumor, a hematological tumor (e.g., leukemia, lymphoma, myeloma, e.g., multiple myeloma), and a metastatic, refractory or recurrent lesion of the cancer;

preferably, the infection or infectious disease is selected from the group consisting of viral, bacterial, fungal and parasitic infections, including but not limited to HIV, hepatitis, herpes virus, CMV, EBV, influenza or tetanus.

28. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-14 in the preparation of an immunogenic composition for increasing immune cell activity and/or enhancing an immune response in a subject; wherein the immunogenic composition comprises the antibody or antigen-binding fragment thereof and an immunogen;

preferably, the immunogen is selected from the group consisting of a tumor cell, an antigen associated with a tumor (e.g., a protein, polypeptide, or carbohydrate molecule), a dendritic cell primed by the antigen, and any combination thereof; the immunogenic composition is for use in the prevention and/or treatment of a tumor and/or delay of tumor progression and/or reduction or inhibition of tumor recurrence in a subject;

preferably, the immunogen is selected from the group consisting of an inactivated or attenuated pathogen, an antigen (e.g., a protein, polypeptide, or carbohydrate molecule) associated with a pathogen, a dendritic cell primed by the antigen, and any combination thereof; the immunogenic composition is used for preventing and/or treating infection or infectious disease and/or delaying infection or infectious disease progression and/or reducing or inhibiting infection or infectious disease recurrence in a subject.

29. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 14 or a pharmaceutical composition according to any one of claims 23 to 25 or an immunogenic composition according to claim 26 in the manufacture of a medicament for the treatment of an immune-related disease involving T cell dysfunction and/or NK cell dysfunction;

preferably, the T cell dysfunction includes, for example, T cell disability, exhaustion, or reduced cytokine secretion; and/or, the NK cell dysfunction includes, for example, NK cell disability, depletion, or decreased cytokine secretion;

preferably, the immune-related disease is selected from the group consisting of a tumor and an infection or infectious disease.

30. A method of increasing the activity of an immune cell in vitro, the method comprising the step of contacting the immune cell with the antibody or antigen-binding fragment thereof of any one of claims 1-14;

preferably, the method further comprises the step of contacting the immune cell with an additional pharmaceutically active agent;

preferably, the additional pharmaceutically active agent is an immune response stimulating agent;

preferably, the immune response stimulating agent is selected from the group consisting of IL-1, IL-2, IL-3, IL-7, IL-12, IL-15, IL-18, IL-21, IFN- γ, IL-10, TGF- β, GM-CSF, M-CSF, G-CSF, TNF- α, and TNF- β, and any combination thereof.

31. A method of enhancing immune cell activity in a subject, the method comprising: administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1-14 or the pharmaceutical composition of any one of claims 23-25 or the immunogenic composition of claim 26;

preferably, the immune cell is selected from the group consisting of a T cell, a cytotoxic T Cell (CTL), an NK cell, and any combination thereof;

preferably, the method is for: (a) preventing and/or treating a tumor and/or delaying tumor progression and/or reducing or inhibiting tumor recurrence; or (b) preventing and/or treating infection or infectious disease and/or delaying progression of infection or infectious disease and/or reducing or inhibiting recurrence of infection or infectious disease.

32. A method of enhancing the activation and/or response of T cells and/or NK cells in a subject, the method comprising: administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1-14 or the pharmaceutical composition of any one of claims 23-25 or the immunogenic composition of claim 26.

33. A method of enhancing an immune response in a subject, the method comprising administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1-14, the pharmaceutical composition of any one of claims 23-25, or the immunogenic composition of claim 26;

preferably, the immune response is an antigen-specific T cell response;

34. A method for preventing and/or treating a tumor, and/or delaying tumor progression, and/or reducing or inhibiting tumor recurrence in a subject, the method comprising administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1-14, the pharmaceutical composition of any one of claims 23-25, or the immunogenic composition of claim 26.

35. A method for preventing and/or treating, and/or delaying progression, and/or reducing or inhibiting relapse of, an infection or an infectious disease in a subject, the method comprising administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1-14, the pharmaceutical composition of any one of claims 23-25, or the immunogenic composition of claim 26.

36. The method of any one of claims 30-35, further comprising administering to the subject a second therapy selected from the group consisting of surgery, chemotherapy, radiation therapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof.

37. The use of claim 27 or 28, or the method of claim 33 or 34, wherein the tumour is selected from oesophageal cancer, gastrointestinal cancer, pancreatic cancer, thyroid cancer, colorectal cancer, renal cancer, lung cancer (e.g. non-small cell lung cancer), liver cancer, gastric cancer, head and neck cancer, bladder cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, germ cell cancer, bone cancer, skin cancer, thymus cancer, bile duct cancer, melanoma, mesothelioma, lymphoma, myeloma, sarcoma, glioblastoma, leukaemia or metastatic, refractory or recurrent lesions of cancer.

38. A method of detecting the presence or level of TIGIT in a sample comprising contacting the sample with the antibody or antigen-binding fragment thereof of any one of claims 1-14 under conditions that allow formation of a complex between the antibody or antigen-binding fragment thereof and TIGIT, and detecting formation of the complex.

39. A diagnostic or therapeutic kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1-14 or comprising the pharmaceutical composition of any one of claims 23-25, and optionally instructions for use.