CN117586398A

CN117586398A - anti-TIGIT antibodies and uses thereof

Info

Publication number: CN117586398A
Application number: CN202310994050.5A
Authority: CN
Inventors: 郑轩; 陈艳; 李春霞; 唐雨蕊; 韦涛; 丁兆
Original assignee: Sichuan Huiyu Haiyue Pharmaceutical Technology Co ltd; SICHUAN HUIYU PHARMACEUTICAL CO Ltd
Current assignee: Sichuan Huiyu Haiyue Pharmaceutical Technology Co ltd; SICHUAN HUIYU PHARMACEUTICAL CO Ltd
Priority date: 2022-08-10
Filing date: 2023-08-09
Publication date: 2024-02-23

Abstract

The present invention is in the field of therapeutic monoclonal antibodies, and in particular, relates to antibodies against TIGIT or antigen binding fragments thereof, nucleic acid molecules encoding them, and methods of making them. The anti-TIGIT antibody or the antigen binding fragment thereof has good specificity and affinity to the TIGIT, and can effectively promote secretion of IFN-gamma and IL-2. Thus, further relates to pharmaceutical compositions comprising said antibodies or antigen binding fragments thereof, and to the use thereof in the manufacture of a medicament for the prevention and/or treatment of TIGIT-related diseases.

Description

anti-TIGIT antibodies and uses thereof

The present application claims priority from China patent application 2022109593346 with application date 2022/8/10. The present application refers to the entirety of the above-mentioned chinese patent application.

Technical Field

The present invention is in the field of therapeutic monoclonal antibodies, more specifically, the invention relates to an antibody directed against TIGIT; also relates to the use of said antibodies in the treatment of diseases.

Background

The immune system is closely related to the occurrence of various diseases, including some autoimmune diseases, cancers and the like, and more results indicate that inhibitory receptors play a very important role in regulating innate immunity and adaptive immunity.

Inhibitory receptors include PD1 (programmed death receptor 1), TIGIT (T cell immune receptor containing Ig and ITIM domains). TIGIT, also known as Wucam, vstm3 or Vsig9, belongs to an immunoglobulin superfamily member. It is mainly distributed in T cells and NK cells and plays an important role in regulating innate and adaptive immunity. TIGIT is composed of mainly one extracellular immunoglobulin domain, a type I transmembrane region, two suppressor immune receptor-tyrosine suppressor motifs (ITIMs) and immunoglobulin tyrosine-like (ITT) suppressor motifs. TIGIT can constitute a costimulatory network with the positive protein CD226 (Dnam-1) on T cells and the ligands CD155 (Pvr or Necl-5) and CD112 (Pvrl-2 or Nectin 2) expressed on CD96 and APC. CD226 and TIGIT share a common ligand (CD 112 and CD 155), although binding of CD226, TIGIT to the ligand results in two opposite outcomes, CD226 binding to the ligand activating NK, T cell function; TIGIT binding to the ligand inhibits NK and T cell function. Interestingly, TIGIT binds to CD155 with much greater affinity than CD226 to CD155, which would further limit activation of CD 226.

TIGIT's inhibition of immune function is mainly manifested in several aspects: 1. TIGIT can inhibit the function of DC cells to indirectly influence T cell activation, and the TIGIT is combined with CD155 on the surface of the DC cells to inhibit DC maturation, so that the secretion of IL-12 is reduced, and the inhibitory cytokine IL-10 is induced to be produced; 2. studies have shown that TIGIT can affect TCR signaling pathways to directly inhibit T cell activation; meanwhile, the killing effect of NK cells on the tumor with high expression of CD155 can be weakened, the immune escape of the tumor is increased, and the NK cells can be combined with CD155 on the surface of MDSC to inhibit ZAP70/Syk and ERK1/2 signal paths so as to further inhibit the activation of NK cell functions; 3. TIGIT can increase the immunosuppressive function of tregs. Treg cells highly expressing TIGIT exhibit stronger immunosuppression, inhibit the response of TH17 and TH1 to T cells, and further promote the production of IL-10 by tregs and inhibit T cell activation by fibrin 2; 4. TIGIT inhibits activation of the CD226 signaling pathway and thus T cell function.

In recent years, immunotherapy of cancer has attracted more and more attention, TIGIT is an important immunosuppressive molecule, and related to the occurrence of various cancers, and many reports have demonstrated the importance of TIGIT in the process of cancer occurrence, and TIGIT blocking or knockout in various tumor models in vitro and in vivo can promote NK cell anti-tumor effect. Researchers also find that in a CT26 colon cancer and B16 melanoma mouse lung metastasis model, the response of CD8+ T cells can be promoted after TIGIT is blocked, and the effect of killing tumors is remarkably improved. It has been found that TIGIT in combination with other immune checkpoint antibody molecules or cytokines may further enhance the therapeutic effect, for example in malignant melanoma, TIGIT in combination with PD1 may significantly enhance the therapeutic effect. In some patients with non-small cell lung cancer, the combined administration of PDL1 and TIGIT can also significantly improve the treatment effect. In a melanoma mouse model, IL-15 and TIGIT combined increase the killing effect of NK on tumors, and simultaneously effectively reduce metastasis of tumors.

As can be seen from the existing reports, TIGIT is a very potential tumor immunotherapy target, but no related drugs are marketed at present, so that the development of anti-TIGIT antibodies is necessary, and the development of anti-TIGIT antibodies can bring hopes to more cancer patients.

Disclosure of Invention

Antibodies of the invention

Accordingly, in one aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds TIGIT, wherein:

the antibody or antigen-binding fragment thereof comprises 3 CDRs contained in a heavy chain variable region (VH) and 3 CDRs contained in a light chain variable region (VL) selected from any one of the following groups:

(1) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 97; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO. 125; or (b)

(2) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 87; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO 115; or (b)

(3) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 86; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO 114; or (b)

(4) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO. 85; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO 113; or (b)

(5) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 83; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO. 132; or (b)

(6) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 84; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO 112; or (b)

(7) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 88; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO 116; or (b)

(8) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 89; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO 117; or (b)

(9) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 90; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO. 118; or (b)

(10) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO. 91; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO 119; or (b)

(11) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 92; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO. 120; or (b)

(12) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO. 93; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO. 121; or (b)

(13) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO. 94; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO. 122; or (b)

(14) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 95; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO. 123; or (b)

(15) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO. 96; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO 124; or (b)

(16) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 98; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO. 126; or (b)

(17) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 99; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO 127; or (b)

(18) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO. 100; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO 128; or (b)

(19) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 101; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO. 129; or (b)

(20) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 102; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO. 130; or (b)

(21) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 103; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO. 131; or (b)

(22) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 107; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO 134; or (b)

(23) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 107; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO. 135; or (b)

(24) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 109; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO 137; or (b)

(25) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO. 110; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO 138; or (b)

(26) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 104; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO. 140; or (b)

(27) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 104; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO 133; or (b)

(28) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 105; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO 133; or (b)

(29) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 106; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO 133; or (b)

(30) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO. 108; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO 136; or (b)

(31) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 111; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO 139; or (b)

(32) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 111; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO 138;

in certain preferred embodiments, the CDRs are defined according to the numbering system of Kabat, IMGT, chothia, contact, abM or a combination thereof.

In certain embodiments, the antibody or antigen-binding fragment thereof that specifically binds TIGIT, wherein the antibody or antigen-binding fragment thereof comprises the following heavy chain variable region (VH) and light chain variable region (VL), wherein CDRs are defined by the IMGT numbering system:

(1) VH comprising 3 CDRs: comprising SEQ ID NO:11, comprising the CDR-H1 of the sequence set forth in SEQ ID NO:27, comprising the sequence set forth in SEQ ID NO:43, CDR-H3 of the sequence shown; and

VL comprising 3 CDRs: comprising SEQ ID NO:56, comprising the sequence set forth in SEQ ID NO:69, comprising the CDR-L2 of the sequence set forth in SEQ ID NO:80, CDR-L3 of the sequence shown;

or (b)

(2) VH comprising 3 CDRs: comprising SEQ ID NO:5, comprising the CDR-H1 of the sequence set forth in SEQ ID NO:18, comprising the CDR-H2 of the sequence set forth in SEQ ID NO:35, CDR-H3 of the sequence shown; and

VL comprising 3 CDRs: comprising SEQ ID NO:52, comprising the CDR-L1 of the sequence set forth in SEQ ID NO:64, comprising the sequence set forth in SEQ ID NO:76, CDR-L3 of the sequence shown;

or (b)

(3) VH comprising 3 CDRs: comprising SEQ ID NO:4, comprising the CDR-H1 of the sequence set forth in SEQ ID NO:17, comprising the CDR-H2 of the sequence set forth in SEQ ID NO:34, CDR-H3 of the sequence shown in seq id no; and

VL comprising 3 CDRs: comprising SEQ ID NO:51, comprising the CDR-L1 of the sequence set forth in SEQ ID NO:63, comprising the sequence set forth in SEQ ID NO:75, CDR-L3 of the sequence shown;

or (b)

(4) VH comprising 3 CDRs: comprising SEQ ID NO:3, comprising the CDR-H1 of the sequence set forth in SEQ ID NO:16, comprising the sequence set forth in SEQ ID NO:33, CDR-H3 of the sequence shown in seq id no; and

VL comprising 3 CDRs: comprising SEQ ID NO:50, comprising the CDR-L1 of the sequence set forth in SEQ ID NO:62, comprising the CDR-L2 of the sequence set forth in SEQ ID NO:74 to CDR-L3 of the sequence shown;

Or (b)

(5) VH comprising 3 CDRs: comprising SEQ ID NO:1, comprising the CDR-H1 of the sequence set forth in SEQ ID NO:14, comprising the sequence set forth in SEQ ID NO:31, CDR-H3 of the sequence shown; and

VL comprising 3 CDRs: comprising SEQ ID NO:48, comprising the sequence set forth in SEQ ID NO:60, comprising the sequence set forth in SEQ ID NO:72, CDR-L3 of the sequence shown;

or (b)

(6) VH comprising 3 CDRs: comprising SEQ ID NO:2, comprising the CDR-H1 of the sequence set forth in SEQ ID NO:15, comprising the CDR-H2 of the sequence set forth in SEQ ID NO:32, CDR-H3 of the sequence shown; and

VL comprising 3 CDRs: comprising SEQ ID NO:49, comprising the CDR-L1 of the sequence set forth in SEQ ID NO:61, comprising the sequence set forth in SEQ ID NO:73, CDR-L3 of the sequence shown;

or (b)

(7) VH comprising 3 CDRs: comprising SEQ ID NO:6, comprising the CDR-H1 of the sequence set forth in SEQ ID NO:19, comprising the sequence set forth in SEQ ID NO:36, CDR-H3 of the sequence shown in seq id no; and

VL comprising 3 CDRs: comprising SEQ ID NO:53, comprising the sequence set forth in SEQ ID NO:65, comprising the sequence set forth in SEQ ID NO:77, CDR-L3 of the sequence shown;

or (b)

(8) VH comprising 3 CDRs: comprising SEQ ID NO:7, comprising the CDR-H1 of the sequence set forth in SEQ ID NO:20, comprising the sequence set forth in SEQ ID NO:37, CDR-H3 of the sequence shown; and

VL comprising 3 CDRs: comprising SEQ ID NO:48, comprising the sequence set forth in SEQ ID NO:66, comprising the sequence set forth in SEQ ID NO:77, CDR-L3 of the sequence shown;

or (b)

(9) VH comprising 3 CDRs: comprising SEQ ID NO:8, comprising the CDR-H1 of the sequence set forth in SEQ ID NO:20, comprising the sequence set forth in SEQ ID NO:37, CDR-H3 of the sequence shown; and

VL comprising 3 CDRs: comprising SEQ ID NO:53, comprising the sequence set forth in SEQ ID NO:66, comprising the sequence set forth in SEQ ID NO:77, CDR-L3 of the sequence shown;

or (b)

(10) VH comprising 3 CDRs: comprising SEQ ID NO:9, comprising the CDR-H1 of the sequence set forth in SEQ ID NO:21, comprising the sequence set forth in SEQ ID NO:38 to CDR-H3 of the sequence shown; and

VL comprising 3 CDRs: comprising SEQ ID NO:48, comprising the sequence set forth in SEQ ID NO:67, comprising the sequence set forth in SEQ ID NO:77, CDR-L3 of the sequence shown;

or (b)

(11) VH comprising 3 CDRs: comprising SEQ ID NO:7, comprising the CDR-H1 of the sequence set forth in SEQ ID NO:22, comprising the sequence set forth in SEQ ID NO:39, CDR-H3 of the sequence shown; and

Or (b)

(12) VH comprising 3 CDRs: comprising SEQ ID NO:7, comprising the CDR-H1 of the sequence set forth in SEQ ID NO:23, comprising the CDR-H2 of the sequence set forth in SEQ ID NO:40, CDR-H3 of the sequence shown; and

VL comprising 3 CDRs: comprising SEQ ID NO:54, comprising the sequence set forth in SEQ ID NO:68, comprising the sequence set forth in SEQ ID NO:78, CDR-L3 of the sequence shown;

or (b)

(13) VH comprising 3 CDRs: comprising SEQ ID NO:10, comprising the CDR-H1 of the sequence set forth in SEQ ID NO:24, comprising the CDR-H2 of the sequence set forth in SEQ ID NO:41, CDR-H3 of the sequence shown; and

VL comprising 3 CDRs: comprising SEQ ID NO:55, comprising the sequence set forth in SEQ ID NO:62, comprising the CDR-L2 of the sequence set forth in SEQ ID NO:79, CDR-L3 of the sequence shown;

or (b)

(14) VH comprising 3 CDRs: comprising SEQ ID NO:8, comprising the CDR-H1 of the sequence set forth in SEQ ID NO:25, comprising the CDR-H2 of the sequence set forth in SEQ ID NO:42, CDR-H3 of the sequence shown; and

or (b)

(15) VH comprising 3 CDRs: comprising SEQ ID NO:7, comprising the CDR-H1 of the sequence set forth in SEQ ID NO:26, comprising the sequence set forth in SEQ ID NO:37, CDR-H3 of the sequence shown; and

VL comprising 3 CDRs: comprising SEQ ID NO:48, comprising the sequence set forth in SEQ ID NO:65, comprising the sequence set forth in SEQ ID NO:77, CDR-L3 of the sequence shown;

or (b)

(16) VH comprising 3 CDRs: comprising SEQ ID NO:11, comprising the CDR-H1 of the sequence set forth in SEQ ID NO:27, comprising the sequence set forth in SEQ ID NO:43, CDR-H3 of the sequence shown; and

VL comprising 3 CDRs: comprising SEQ ID NO:57, comprising the sequence set forth in SEQ ID NO:69, comprising the CDR-L2 of the sequence set forth in SEQ ID NO:80, CDR-L3 of the sequence shown;

or (b)

(17) VH comprising 3 CDRs: comprising SEQ ID NO:12, comprising the CDR-H1 of the sequence set forth in SEQ ID NO:28, comprising the sequence set forth in SEQ ID NO:44 to CDR-H3 of the sequence shown; and

VL comprising 3 CDRs: comprising SEQ ID NO:58, comprising the CDR-L1 of the sequence set forth in SEQ ID NO:70, comprising the sequence set forth in SEQ ID NO:82 to CDR-L3 of the sequence shown;

or (b)

(18) VH comprising 3 CDRs: comprising SEQ ID NO:12, comprising the CDR-H1 of the sequence set forth in SEQ ID NO:29, comprising the sequence set forth in SEQ ID NO:44 to CDR-H3 of the sequence shown; and

VL comprising 3 CDRs: comprising SEQ ID NO:58, comprising the CDR-L1 of the sequence set forth in SEQ ID NO:71, comprising the sequence set forth in SEQ ID NO:82 to CDR-L3 of the sequence shown;

Or (b)

(19) VH comprising 3 CDRs: comprising SEQ ID NO:13, comprising the CDR-H1 of the sequence set forth in SEQ ID NO:27, comprising the sequence set forth in SEQ ID NO:45 to CDR-H3 of the sequence shown; and

or (b)

(20) VH comprising 3 CDRs: comprising SEQ ID NO:13, comprising the CDR-H1 of the sequence set forth in SEQ ID NO:30, comprising the sequence set forth in SEQ ID NO:46, CDR-H3 of the sequence shown; and

or (b)

(21) VH comprising 3 CDRs: comprising SEQ ID NO:13, comprising the CDR-H1 of the sequence set forth in SEQ ID NO:27, comprising the sequence set forth in SEQ ID NO:47, CDR-H3 of the sequence shown in seq id no; and

VL comprising 3 CDRs: comprising SEQ ID NO:59 comprising the sequence set forth in SEQ ID NO:71, comprising the sequence set forth in SEQ ID NO:81 to CDR-L3 of the sequence shown;

in certain alternative embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL) wherein the CDRs are defined according to the Kabat numbering system:

(A1) VH comprising 3 CDRs: CDR-H1 comprising the sequence shown in SEQ ID NO. 190, CDR-H2 comprising the sequence shown in SEQ ID NO. 191, and CDR-H3 comprising the sequence shown in SEQ ID NO. 192; and

VL comprising 3 CDRs: CDR-L1 comprising the sequence shown as SEQ ID NO. 193, CDR-L2 comprising the sequence shown as SEQ ID NO. 194, and CDR-L3 comprising the sequence shown as SEQ ID NO. 80;

or (b)

(A2) VH comprising 3 CDRs: CDR-H1 comprising the sequence shown in SEQ ID NO. 175, CDR-H2 comprising the sequence shown in SEQ ID NO. 179, and CDR-H3 comprising the sequence shown in SEQ ID NO. 183; and

VL comprising 3 CDRs: CDR-L1 comprising the sequence shown as SEQ ID NO. 185, CDR-L2 comprising the sequence shown as SEQ ID NO. 187, and CDR-L3 comprising the sequence shown as SEQ ID NO. 76;

or (b)

(A3) VH comprising 3 CDRs: CDR-H1 comprising the sequence shown in SEQ ID NO. 145, CDR-H2 comprising the sequence shown in SEQ ID NO. 149, and CDR-H3 comprising the sequence shown in SEQ ID NO. 153; and

VL comprising 3 CDRs: CDR-L1 comprising the sequence shown in SEQ ID NO. 155, CDR-L2 comprising the sequence shown in SEQ ID NO. 157, and CDR-L3 comprising the sequence shown in SEQ ID NO. 75;

or (b)

(A4) VH comprising 3 CDRs: CDR-H1 comprising the sequence shown as SEQ ID NO. 160, CDR-H2 comprising the sequence shown as SEQ ID NO. 164, and CDR-H3 comprising the sequence shown as SEQ ID NO. 168; and

VL comprising 3 CDRs: CDR-L1 comprising the sequence shown in SEQ ID NO. 170, CDR-L2 comprising the sequence shown in SEQ ID NO. 172, and CDR-L3 comprising the sequence shown in SEQ ID NO. 74;

in certain alternative embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL) as follows, wherein the CDRs are defined by the AbM numbering system:

(B1) VH comprising 3 CDRs: CDR-H1 comprising the sequence shown as SEQ ID NO. 195, CDR-H2 comprising the sequence shown as SEQ ID NO. 196, and CDR-H3 comprising the sequence shown as SEQ ID NO. 192; and

or (b)

(B2) VH comprising 3 CDRs: CDR-H1 comprising the sequence shown in SEQ ID NO. 176, CDR-H2 comprising the sequence shown in SEQ ID NO. 180, and CDR-H3 comprising the sequence shown in SEQ ID NO. 183; and

or (b)

(B3) VH comprising 3 CDRs: CDR-H1 comprising the sequence shown in SEQ ID NO. 146, CDR-H2 comprising the sequence shown in SEQ ID NO. 150, and CDR-H3 comprising the sequence shown in SEQ ID NO. 153; and

or (b)

(B4) VH comprising 3 CDRs: CDR-H1 comprising the sequence shown as SEQ ID NO. 161, CDR-H2 comprising the sequence shown as SEQ ID NO. 165, and CDR-H3 comprising the sequence shown as SEQ ID NO. 168; and

in certain alternative embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL) as follows, wherein the CDRs are defined by the Chothia numbering system:

(C1) VH comprising 3 CDRs: CDR-H1 comprising the sequence shown in SEQ ID NO. 197, CDR-H2 comprising the sequence shown in SEQ ID NO. 198, and CDR-H3 comprising the sequence shown in SEQ ID NO. 192; and

or (b)

(C2) VH comprising 3 CDRs: CDR-H1 comprising the sequence shown as SEQ ID NO. 177, CDR-H2 comprising the sequence shown as SEQ ID NO. 181, and CDR-H3 comprising the sequence shown as SEQ ID NO. 183; and

or (b)

(C3) VH comprising 3 CDRs: CDR-H1 comprising the sequence shown in SEQ ID NO. 147, CDR-H2 comprising the sequence shown in SEQ ID NO. 151, and CDR-H3 comprising the sequence shown in SEQ ID NO. 153; and

or (b)

(C4) VH comprising 3 CDRs: CDR-H1 comprising the sequence shown as SEQ ID NO. 162, CDR-H2 comprising the sequence shown as SEQ ID NO. 166, and CDR-H3 comprising the sequence shown as SEQ ID NO. 168; and

in certain alternative embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL) as follows, wherein the CDRs are defined by the Contact numbering system:

(D1) VH comprising 3 CDRs: CDR-H1 comprising the sequence shown as SEQ ID NO:199, CDR-H2 comprising the sequence shown as SEQ ID NO:200, and CDR-H3 comprising the sequence shown as SEQ ID NO: 201; and, a step of, in the first embodiment,

VL comprising 3 CDRs: CDR-L1 comprising the sequence shown in SEQ ID NO. 202, CDR-L2 comprising the sequence shown in SEQ ID NO. 203, and CDR-L3 comprising the sequence shown in SEQ ID NO. 204.

Or (b)

(D2) VH comprising 3 CDRs: CDR-H1 comprising the sequence shown in SEQ ID NO. 178, CDR-H2 comprising the sequence shown in SEQ ID NO. 182, and CDR-H3 comprising the sequence shown in SEQ ID NO. 184; and

VL comprising 3 CDRs: CDR-L1 comprising the sequence shown in SEQ ID NO. 186, CDR-L2 comprising the sequence shown in SEQ ID NO. 188, and CDR-L3 comprising the sequence shown in SEQ ID NO. 189;

or (b)

(D3) VH comprising 3 CDRs: CDR-H1 comprising the sequence shown in SEQ ID NO. 148, CDR-H2 comprising the sequence shown in SEQ ID NO. 152, and CDR-H3 comprising the sequence shown in SEQ ID NO. 154; and

VL comprising 3 CDRs: CDR-L1 comprising the sequence shown in SEQ ID NO. 156, CDR-L2 comprising the sequence shown in SEQ ID NO. 158, and CDR-L3 comprising the sequence shown in SEQ ID NO. 159;

or (b)

(D4) VH comprising 3 CDRs: CDR-H1 comprising the sequence shown as SEQ ID NO. 163, CDR-H2 comprising the sequence shown as SEQ ID NO. 167, and CDR-H3 comprising the sequence shown as SEQ ID NO. 169; and

VL comprising 3 CDRs: CDR-L1 comprising the sequence shown as SEQ ID NO. 171, CDR-L2 comprising the sequence shown as SEQ ID NO. 173, and CDR-L3 comprising the sequence shown as SEQ ID NO. 174;

In certain alternative embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL) wherein the CDRs are defined by the numbering system of Kabat, IMGT, chothia, contact or a combination of abms:

(E1) VH comprising 3 CDRs: comprising CDR-H1 as shown in SEQ ID NO 195, comprising CDR-H2 as shown in SEQ ID NO 191, and comprising CDR-H3 as shown in SEQ ID NOs 43; and

VL comprising 3 CDRs: comprising CDR-L1 as shown in SEQ ID NO:193, comprising CDR-L2 as shown in SEQ ID NO:194, and comprising CDR-L3 as shown in SEQ ID NOs: 80;

or (b)

(E2) VH comprising 3 CDRs: comprising CDR-H1 as shown in SEQ ID NO. 176, comprising CDR-H2 as shown in SEQ ID NO. 179, and comprising CDR-H3 as shown in SEQ ID NOs.35; and

VL comprising 3 CDRs: comprising CDR-L1 as shown in SEQ ID NO:185, comprising CDR-L2 as shown in SEQ ID NO:187, and comprising CDR-L3 as shown in SEQ ID NOs: 76;

or (b)

(E3) VH comprising 3 CDRs: comprising CDR-H1 as shown in SEQ ID NO. 146, comprising CDR-H2 as shown in SEQ ID NO. 149, and comprising CDR-H3 as shown in SEQ ID NOs:34; and

VL comprising 3 CDRs: comprising CDR-L1 as shown in SEQ ID NO:155, comprising CDR-L2 as shown in SEQ ID NO:157, and comprising CDR-L3 as shown in SEQ ID NOs: 75.

In certain embodiments, the antibody or antigen-binding fragment thereof that specifically binds TIGIT, wherein,

the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL) selected from any one of the following:

(1) SEQ ID NO:97 and VH and SEQ ID NO:125, VL of the sequence shown in seq id no;

(2) SEQ ID NO:87 and VH and SEQ ID NO:115, VL of the sequence shown in seq id no;

(3) SEQ ID NO:86 and VH and SEQ ID NO:114, VL of the sequence shown in seq id no;

(4) SEQ ID NO:85 and VH and SEQ ID NO:113, VL of the sequence shown in seq id no;

(5) SEQ ID NO:83 and VH and SEQ ID NO:132, VL of the sequence shown in seq id no;

(6) SEQ ID NO:84 and VH and SEQ ID NO:112, VL of the sequence shown in seq id no;

(7) SEQ ID NO:88 and VH and SEQ ID NO:116, VL of the sequence shown in seq id no;

(8) SEQ ID NO:89 and VH and SEQ ID NO: 117;

(9) SEQ ID NO:90 and VH and SEQ ID NO:118, VL of the sequence shown in seq id no;

(10) SEQ ID NO:91 and VH and SEQ ID NO:119, VL of the sequence shown in seq id no;

(11) SEQ ID NO:92 and VH and SEQ ID NO:120, VL of the sequence shown in seq id no;

(12) SEQ ID NO:93 and VH and SEQ ID NO:121, VL of the sequence shown in seq id no;

(13) SEQ ID NO:94 and VH and SEQ ID NO:122, VL of the sequence shown in seq id no;

(14) SEQ ID NO:95 and VH and SEQ ID NO:123 VL of the sequence shown in seq id no;

(15) SEQ ID NO:96 and VH and SEQ ID NO:124, VL of the sequence shown;

(16) SEQ ID NO:98 and VH and SEQ ID NO:126, VL of the sequence shown;

(17) SEQ ID NO:99 and VH and SEQ ID NO:127, VL of the sequence shown in seq id no;

(18) SEQ ID NO:100 and VH and SEQ ID NO:128, VL of the sequence shown in seq id no;

(19) SEQ ID NO:101 and VH and SEQ ID NO:129, VL of the sequence shown in seq id no;

(20) SEQ ID NO:102 and VH and SEQ ID NO:130, VL of the sequence shown in seq id no;

(21) SEQ ID NO:103 and SEQ ID NO:131, VL of the sequence shown in seq id no;

(22) SEQ ID NO:104 and SEQ ID NO:140, VL of the sequence shown in seq id no;

(23) SEQ ID NO:104 and SEQ ID NO:133, VL of the sequence shown;

(24) SEQ ID NO:105 and the sequence shown in SEQ ID NO:133, VL of the sequence shown;

(25) SEQ ID NO:106 and the sequence set forth in SEQ ID NO:133, VL of the sequence shown;

(26) SEQ ID NO:107 and VH and SEQ ID NO:134, VL of the sequence shown;

(27) SEQ ID NO:107 and VH and SEQ ID NO:135, VL of the sequence shown in seq id no;

(28) SEQ ID NO:108 and VH and SEQ ID NO:136, VL of the sequence shown in seq id no;

(29) SEQ ID NO:109 and VH and SEQ ID NO:137, VL of the sequence shown in seq id no;

(30) SEQ ID NO:110 and SEQ ID NO:138, VL of the sequence shown in seq id no;

(31) SEQ ID NO:111 and VH and SEQ ID NO:139, VL of the sequence shown in seq id no;

(32) SEQ ID NO:111 and VH and SEQ ID NO:138, VL of the sequence shown in seq id no;

in certain alternative embodiments, the VH and VL have at least 70%, 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 compared to any of (1) to (32); and, VL has at least 70%, 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%, or at least 99% sequence identity;

in certain alternative embodiments, the VH and VL have one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) as compared to any of groups (1) to (32); and VL has a 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); in certain preferred embodiments, the substitutions are conservative substitutions.

In certain embodiments, the antibody or antigen-binding fragment thereof that specifically binds TIGIT is selected from the group consisting of Fab fragments, fab 'fragments, F (ab)' ₂ Fragments, single chain antibodies or disulfide stabilized Fv proteins (dsFv);

in certain preferred embodiments, the single chain antibody is selected from scFv, di-scFv or (scFv) ₂ 。

In certain embodiments, the antibody or antigen-binding fragment thereof that specifically binds TIGIT, wherein the antibody or antigen-binding fragment thereof is a murine antibody, chimeric antibody, humanized antibody, or other species-derived antibody (e.g., rabbit, camel, or shark).

the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region (CH) and a light chain constant region (CL);

in certain preferred embodiments, the heavy chain constant region is selected from the group consisting of a heavy chain constant region of IgG, igM, igE, igD or IgA or a variant thereof; and/or the number of the groups of groups,

the light chain constant region is selected from a kappa or lambda light chain constant region or variant thereof;

the variant has one or more amino acid substitutions, deletions or additions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions or additions) as compared to the wild-type sequence from which it was derived;

In certain more preferred embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain constant region and a light chain constant region selected from the group consisting of:

(1) A human IgG1 heavy chain constant region (e.g., a human IgG1 heavy chain constant region as set forth in SEQ ID NO: 141) or a variant thereof (e.g., a variant of a human IgG1 heavy chain constant region wherein the variant has one or more amino acid substitutions, deletions, or additions at position 233, 234, 235, 236, 239, 327, 330, 331, or 332 (e.g., substitutions of E233P, L234V, L235A, A327G, A330S, P S, S35239D, A L or I332E; e.g., deletions of deltaG 236) as compared to the human IgG1 heavy chain constant region sequence from which it is derived according to the EU numbering system; in certain more preferred embodiments, the sequence of the variant is selected from SEQ ID NO:142 or 143);

(2) A human IgG1 light chain constant region (e.g., a human kappa light chain constant region as set forth in SEQ ID NO: 144) or variant thereof;

in certain further preferred embodiments, the antibody or antigen binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID NO:141 and a heavy chain constant region (CH) as set forth in SEQ ID NO:144, a light chain constant region (CL);

Or comprises the amino acid sequence as set forth in SEQ ID NO:142 and a heavy chain constant region (CH) as set forth in SEQ ID NO:144, a light chain constant region (CL);

or comprises the amino acid sequence as set forth in SEQ ID NO:143 and a heavy chain constant region (CH) as set forth in SEQ ID NO:144, and a light chain constant region (CL).

the antibody or antigen binding fragment thereof has:

binding TIGIT (e.g., human TIGIT) with a KD of less than 500nM, such as less than 100nM, 50nM, 40nM, 20nM, 10nM, 1nM, 0.1nM, 0.01nM or less; and/or;

with an EC of less than 500nM, e.g., less than 100nM, 10nM, 1nM, 0.9nM, 0.8nM, 0.7nM, 0.6nM, 0.5nM, 0.4nM, 0.3nM, 0.2nM, 0.1nM, 0.01nM or less ₅₀ Binding to tumor cells expressing TIGIT; in certain preferred embodiments, the EC ₅₀ Measured by flow cytometry or cell competition ELISA.

Multispecific antibodies

In another aspect, the invention provides a multispecific antibody comprising an antibody or antigen-binding fragment thereof of the invention; in certain preferred embodiments, the multispecific antibody is a bispecific antibody or a trispecific antibody or a tetraspecific antibody.

Derived antibodies

The antibodies or antigen binding fragments thereof of the invention may be derivatized, e.g., linked to another molecule (e.g., another polypeptide or protein). In general, derivatization (e.g., labeling) of the 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 invention are also intended to include such derivatized forms. For example, an antibody or antigen-binding fragment thereof of the invention may be functionally linked (by chemical coupling, gene fusion, non-covalent linkage, or otherwise) to one or more other molecular groups, such as another antibody (e.g., forming a bispecific antibody), a detection reagent, a pharmaceutical reagent, 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., bispecific antibody) is produced by cross-linking 2 or more antibodies (of the same type or different types). Methods for obtaining bispecific antibodies are well known in the art, examples of which 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, a cytotoxic drug or a radioactive toxin, examples of which include, but are not limited to, taxol, cytochalasin B (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 or antigen binding fragment thereof of the invention may be linked to a detectable label. The detectable label according to the present invention may be any substance that is detectable by fluorescence, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. Such labels are well known in the art, examples of which include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., ³ H、 ¹² 5I、 ³⁵ S、 ¹⁴ c or ³² P), 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 esters, magnetic beads, calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.), and biotin for binding to the label-modified avidin (e.g., streptavidin) described above. Patents teaching the use of such markers include, but are not limited to, U.S. Pat. nos. 3,817,837;3,850,752;3,939,350;3,996,345;4,277,437;4,275,149; and 4,366,241 (incorporated by reference in its entirety). The detectable labels described above can be detected by methods known in the art. For example, the radiolabel may be detected using a photographic film or scintillation calculator, and the fluorescent label may be detected using a photodetector to detect the emitted light. Enzyme labels are typically prepared by providing an enzyme with a substrate and detecting the reaction generated by the action of the enzyme on the substrate The products should be detected and the calorimetric markers detected by simple visualization of the colored markers. In certain embodiments, such labels can be suitable for immunological detection (e.g., enzyme-linked immunoassay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, etc.). In certain embodiments, a detectable label as described above may be attached to an antibody or antigen binding fragment thereof of the invention by linkers of different lengths to reduce potential steric hindrance.

Furthermore, the antibodies of the invention or antigen binding fragments thereof 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 antibodies, such as increasing serum half-life.

Isolated nucleic acid molecules

In another aspect, the invention provides a nucleic acid molecule encoding an antibody or antigen-binding fragment thereof of the invention, or a multispecific antibody of the invention.

Carrier body

In another aspect, the invention provides a vector comprising a nucleic acid molecule of the invention; in certain preferred embodiments, the vector is a cloning vector or an expression vector; in certain more preferred embodiments, the expression vector is selected from the group consisting of a pt 5 vector, a pJex vector, or a pD912 vector.

Host cells

In another aspect, the invention provides a host cell comprising a nucleic acid molecule of the invention or a vector of the invention. In certain preferred embodiments, the host cell of the invention is a CHO cell (e.g., a CHO-K1, CHO-S, CHO DG44 or an Expi-CHOs cell). In certain more preferred embodiments, the CHO cells are Expi-CHOs cells.

Preparation of antibodies

In another aspect, the invention provides a method of making an antibody or antigen-binding fragment thereof, or a multispecific antibody that specifically binds TIGIT, comprising culturing a host cell of the invention under conditions that allow expression of the antibody or antigen-binding fragment thereof, or the multispecific antibody, and recovering the antibody or antigen-binding fragment thereof, or the multispecific antibody, from the cultured host cell culture.

Conjugate(s)

In another aspect, the invention provides a conjugate comprising an antibody or antigen-binding fragment thereof of the invention, or a multispecific antibody of the invention, or a nucleic acid molecule of the invention, and a coupling moiety; the antibody or antigen-binding fragment thereof, or the multispecific antibody, or the nucleic acid molecule is linked to the conjugate moiety directly or via a linker;

In certain preferred embodiments, the coupling moiety is selected from the group consisting of: a detectable label (e.g., a radioisotope, fluorescent substance, luminescent substance, colored substance, or enzyme) or therapeutic agent (e.g., a nuclide, an immune agonist, an immunosuppressant, a cytokine, a toxin, and other active substances that inhibit tumor cell growth, promote tumor cell apoptosis, or necrosis).

Pharmaceutical composition

In another aspect, the invention provides a pharmaceutical composition comprising an antibody of the invention or an antigen-binding fragment thereof, or a multispecific antibody of the invention, or a nucleic acid molecule of the invention, or a vector of the invention, or a host cell of the invention, or a conjugate of the invention, and a pharmaceutically acceptable carrier and/or excipient;

in certain preferred embodiments, the pharmaceutical composition further comprises an additional pharmaceutically active agent;

in certain more preferred embodiments, the additional pharmaceutically active agent is a drug having anti-tumor activity, such as a chemotherapeutic (e.g., platinum-based) or small molecule inhibitor; and/or the number of the groups of groups,

the additional pharmaceutically active agent is an immunotherapeutic-related agent, such as an immunodetection point inhibitor (e.g., a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, or any combination thereof), an agent that enhances immunity (e.g., an interferon, an interleukin), or an immune system activator (e.g., TLR, ONDs, cpG, LPS, SEB, SEA, STING or SEC activator); and/or the number of the groups of groups,

The additional pharmaceutically active agent is an oncolytic virus, an immune cell, or an engineered immune cell or immune cell of an engineered immune cell; and/or the number of the groups of groups,

the additional pharmaceutically active agent is a radioactive substance or a color-developing agent.

Kit for detecting a substance in a sample

In another aspect, the invention provides a kit comprising an antibody of the invention or an antigen-binding fragment thereof, or a multispecific antibody of the invention, or a nucleic acid molecule of the invention, or a vector of the invention, or a host cell of the invention, or a conjugate of the invention, or a pharmaceutical composition of the invention.

Use of the same

In another aspect, the invention provides the use of an antibody of the invention or an antigen binding fragment thereof, or a multispecific antibody of the invention, or a nucleic acid molecule of the invention, or a vector of the invention, or a host cell of the invention, or a conjugate of the invention, or a pharmaceutical composition of the invention, in the manufacture of a medicament for the prevention and/or treatment and/or adjuvant treatment and/or neoadjuvant treatment of a TIGIT-related disease;

in certain preferred embodiments, the TIGIT-related disorder is a tumor;

in certain preferred embodiments, the medicament is for: (a) inducing apoptosis of tumor cells; (b) inhibiting tumor cell proliferation; (c) inducing and/or increasing T cell infiltration; (d) inducing and/or enhancing an immune response; (e) increasing NK cell activity; (f) inhibiting TIGIT expression and activation; (g) inhibiting TIGIT-mediated signaling; (h) Induce and/or promote secretion of cytokines (e.g., IFN-gamma or IL-2); or any combination of (a) - (h);

In certain preferred embodiments, the antibody or antigen-binding fragment thereof, multispecific antibody, nucleic acid molecule, vector, host cell, conjugate, or pharmaceutical composition is further administered separately, in combination, simultaneously or sequentially with another pharmaceutically active agent; in certain further preferred embodiments, the additional pharmaceutically active agent is a drug having anti-tumor activity, such as a chemotherapeutic (e.g., platinum-based) or small molecule inhibitor; and/or the additional pharmaceutically active agent is an immunotherapeutic-related agent, such as an immunodetection point inhibitor (e.g., PD-1 inhibitor, PD-L1 inhibitor, CTLA-4 inhibitor, or any combination thereof), an agent that enhances immunity (e.g., an interferon, an interleukin), or an immune system activator (e.g., TLR, ONDs, cpG, LPS, SEB, SEA, STING or SEC activator); and/or the additional pharmaceutically active agent is an oncolytic virus, an immune cell, or an engineered immune cell or an immune cell engineered by an engineered immune cell; and/or the additional pharmaceutically active agent is a radioactive material or a color-developing agent.

In certain embodiments, according to the use as described above, wherein the tumor is selected from a solid tumor or a hematological tumor;

In certain preferred embodiments, the solid tumor is selected from melanoma, lung cancer (e.g., non-small cell lung cancer), kidney cancer, bladder cancer, head and neck tumor (e.g., head and neck squamous carcinoma), stomach cancer, esophagus cancer, liver cancer, skin cancer (e.g., mercker skin cancer), colon cancer, ureter cancer, or a microsatellite highly unstable solid tumor; the hematological neoplasm is selected from lymphomas (e.g., hodgkin's lymphoma).

Detection method

In another aspect, the invention provides a method of detecting the presence or level of TIGIT in a sample comprising contacting the sample with an antibody of the invention or an antigen binding fragment thereof, or a multispecific antibody of the invention, or a nucleic acid molecule of the invention, or a vector of the invention, or a host cell of the invention, or a conjugate of the invention, or a pharmaceutical composition of the invention, under conditions that allow for the formation of an antibody-antigen immune complex, and detecting the formation of the complex.

Use of a diagnostic kit

In another aspect, the invention provides the use of an antibody of the invention or an antigen-binding fragment thereof, or a multispecific antibody of the invention, or a nucleic acid molecule of the invention, or a vector of the invention, or a host cell of the invention, or a conjugate of the invention, or a pharmaceutical composition of the invention, in the manufacture of a diagnostic kit for the diagnosis or differential diagnosis of a disease associated with TIGIT; in certain preferred embodiments, the TIGIT-related disorder is a tumor (e.g., the tumor is selected from a solid tumor or a hematological tumor);

In certain more preferred embodiments, the solid tumor is selected from melanoma, lung cancer (e.g., non-small cell lung cancer), kidney cancer, bladder cancer, head and neck tumor (e.g., head and neck squamous carcinoma), stomach cancer, esophagus cancer, liver cancer, skin cancer (e.g., mercker skin cancer), colon cancer, ureter cancer, or a microsatellite highly unstable solid tumor; the hematological neoplasm is selected from lymphomas (e.g., hodgkin's lymphoma).

Definition of terms

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Moreover, the virology, biochemistry, immunology laboratory procedures used herein are all conventional procedures widely used in the corresponding field. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

When used herein, the terms "for example," such as, "" including, "" comprising, "or variations thereof, are not to be construed as limiting terms, but rather as meaning" but not limited to "or" not limited to.

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

As used herein, the term "antibody" refers to an immunoglobulin molecule that is typically composed of two pairs of polypeptide chains, each pair having one Light Chain (LC) and one Heavy Chain (HC). Antibody light chains can be classified as kappa (kappa) and lambda (lambda) light chains. 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. Within the light and heavy chains, the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain also comprises a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH 1, CH2 and CH 3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The constant domains are not directly involved in binding of antibodies to antigens, but exhibit a variety of effector functions, such as may mediate binding of immunoglobulins 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 (C1 q). VH and VL regions can also be subdivided into regions of high variability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FR). Each VH and VL is prepared from the following sequence: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 consist of 3 CDRs and 4 FRs arranged from amino-terminus to carboxy-terminus. The variable regions (VH and VL) of each heavy chain, light chain pair, respectively, form antigen binding sites. The assignment of amino acids to regions or domains can be carried out by Kabat, sequences of Proteins of Immunological Interest (National Institutes of Health, bethesda, md. (1987 and 1991)), or Chothia & Lesk (1987) J.mol. Biol.196:901-917; chothia et al (1989) Nature 342:878-883.

In this context, unless the context clearly indicates otherwise, when referring to the term "antibody" it includes not only whole antibodies, but also antigen-binding fragments of antibodies.

As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in an antibody variable region that are responsible for antigen binding. Three CDRs, designated CDR1, CDR2 and CDR3, are contained in each of the variable regions of the heavy and light chains. The precise boundaries of these CDRs may be defined according to various numbering systems known in the art, such as those defined in the Kabat numbering system (Kabat et al, sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md, 1991), the Chothia numbering system (Chothia & Lesk (1987) J.mol. Biol.196:901-917; chothia et al (1989) Nature 342:878-883), the IMGT numbering system (Lefranc et al, dev. Comparat. Immunol.27:55-77,2003), the AbM numbering system (Martin ACR, cheethane JC, rees (1989) Modelling Antibody hypervariable loops:A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272) or the Contact numbering system (MacCallum, R.M., martin, A.C.R., thorton, J.M. (1996) anti-antigen Interactions: contact Analysis and Binding Site. Of Molecular Biology, 375). For a given antibody, one skilled in the art will readily identify the CDRs defined by each numbering system. Also, the correspondence between the different numbering systems is well known to those skilled in the art (see, for example, lefranc et al, dev. Comparat. Immunol.27:55-77,2003).

In the present invention, the CDRs contained in the antibodies or antigen binding fragments thereof of the present invention can be determined according to various numbering systems known in the art, for example by the Kabat, chothia, IMGT, abM or Contact numbering system. In certain embodiments, the antibodies of the invention, or antigen binding fragments thereof, comprise CDRs that are determined by the Kabat, chothia, IMGT, abM or Contact numbering system.

As used herein, the term "framework region" or "FR" residues refer to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

The term "antibody" is not limited by any particular method of producing an antibody. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibodies may be of different isotypes, for example, igG (e.g., igG1, igG2, igG3, or IgG4 subclasses), igA1, igA2, igD, igE, or IgM antibodies.

As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains a specific junctionThe ability to bind the same antigen to which the full-length antibody binds, and/or to compete with the full-length antibody for specific binding to the antigen, also referred to as an "antigen-binding portion". See generally Fundamental Immunology, ch.7 (Paul, W., ed., 2 nd edition, raven Press, N.Y. (1989), which is incorporated herein by reference in its entirety for all purposes, antigen binding fragments of antibodies may be generated by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies non-limiting examples of antigen binding fragments include Fab, fab ', F (ab') ₂ Fd, fv, complementarity Determining Region (CDR) fragments, scFv, diabodies (diabodies), single domain antibodies (singledomain antibody), chimeric antibodies, linear antibodies (linear antibodies), nanobodies (technology from Dommantis), probody and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen binding capacity to the polypeptide.

As used herein, the term "full length antibody" means an antibody consisting of two "full length heavy chains" and two "full length light chains". Wherein "full length heavy chain" refers to a polypeptide chain consisting of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a Hinge Region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain in the N-to C-terminal direction; and, when the full length antibody is an IgE isotype, optionally further comprises a heavy chain constant region CH4 domain. In certain preferred embodiments, a "full length heavy chain" is a polypeptide chain consisting of VH, CH1, HR, CH2, and CH3 in the N-to C-terminal direction. A "full length light chain" is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-to C-terminal direction. The two pairs of full length antibody chains are linked together by a disulfide bond between CL and CH1 and a disulfide bond between HR of the two full length heavy chains. Full length antibodies comprise two antigen binding sites formed by VH and VL pairs, respectively, that specifically recognize, and bind the same antigen.

As used herein, the term "Fab fragment" means an antibody fragment consisting of VL, VH, CL and CH1 domains; the term "F (ab') ₂ Fragment "means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; surgery (operation)The term "Fab 'fragment" means a reduction-linked F (ab') ₂ The resulting fragment after disulfide bonding of the two heavy chain fragments in the fragment consists of one complete light and heavy chain Fd fragment (consisting of VH and CH1 domains).

As used herein, the term "Fv" means an antibody fragment consisting of VL and VH domains of a single arm of an antibody. Fv fragments are generally considered to be the smallest antibody fragment that forms the complete antigen binding site. It is believed that the six CDRs confer antigen binding specificity to the antibody. However, even one variable region (e.g., fd fragment, which contains only three CDRs specific for an antigen) is able to recognize and bind antigen, although its affinity may be lower than the complete binding site.

As used herein, the term "Fc domain" or "Fc region" means a portion of the heavy chain constant region comprising CH2 and CH3. The Fc fragment of an antibody has a number of different functions, but does not participate in antigen binding. "effector functions" mediated by the Fc region include Fc receptor binding; clq binding and Complement Dependent Cytotoxicity (CDC); antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors); and B cell activation, etc. In some embodiments, the Fc region comprises a hinge, CH2, and CH3. When the Fc region comprises a hinge, the hinge modulates dimerization between two Fc-containing polypeptides. The Fc region can be any antibody heavy chain constant region isotype, e.g., igG1, igG2, igG3, or IgG4.

The Fc domain may include both a native Fc region and a variant Fc region. The native Fc region comprises an amino acid sequence that corresponds to the amino acid sequence of an Fc region found in nature, e.g., the native sequence human Fc region includes the native sequence human IgG1 Fc region (non-a and a allotypes); a native sequence human IgG2 Fc region; a native sequence human IgG3 Fc region; and the native sequence human IgG4 Fc region, and naturally occurring variants thereof. The variant Fc region comprises an amino acid sequence that differs from the amino acid sequence of the native sequence Fc region by at least one amino acid modification. In some embodiments, the variant Fc region may possess altered effector functions (e.g., fc receptor binding, antibody glycosylation, number of cysteine residues, effector cell function, or complement function) compared to the native Fc region.

As used herein, the term "scFv" refers to a single polypeptide chain comprising VL and VH domains, wherein the VL and VH are connected by a linker (linker). Such scFv molecules may have the general structure: NH (NH) ₂ -VL-linker-VH-COOH or NH ₂ -VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a polypeptide having an amino acid sequence (GGGGS) can be used ₄ But variants thereof may also be used. In some cases, disulfide bonds may also exist between VH and VL of scFv.

As used herein, the term "diabody" means that its VH and VL domains are expressed on a single polypeptide chain, but uses a linker that is too short to allow pairing between two domains of the same chain, forcing the domains to pair with complementary domains of the other chain and creating two antigen binding sites (see, e.g., holliger p. Et al, proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak R.J. Et al, structures 2:1121-1123 (1994)).

Each of the above antibody fragments retains the ability to specifically bind to the same antigen to which the full-length antibody binds and/or competes with the full-length antibody for specific binding to the antigen.

As used herein, "bispecific antibody" refers to a conjugate formed by a first antibody (fragment) and a second antibody (fragment) or antibody analog via a conjugate arm, by means including, but not limited to, chemical reaction, gene fusion, protein fusion, polypeptide fusion, and enzymatic. "multispecific antibodies" include, for example: trispecific antibodies, which are antibodies with three different antigen binding specificities, and tetraspecific antibodies, which are antibodies with four different antigen binding specificities.

In this context, techniques for obtaining antibodies may use conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods) to obtain antigen-binding fragments of antibodies (e.g., the antibody fragments described above) from a given antibody (e.g., an antibody provided by the present invention), and specifically screen the antigen-binding fragments of antibodies in the same manner as for intact antibodies.

As used herein, the terms "monoclonal antibody," "mAb," and "mAb" have the same meaning and are used interchangeably to refer to an antibody or a fragment of an antibody from a population of highly homologous antibody molecules, i.e., a population of identical antibody molecules except for natural mutations that may occur spontaneously. Monoclonal antibodies have a high specificity for a single epitope on an antigen. Polyclonal antibodies are relative to monoclonal antibodies, which typically comprise at least 2 or more different antibodies, which typically recognize different epitopes on an antigen. Furthermore, the modifier "monoclonal" merely indicates the character of the antibody as being obtained from a population of highly homologous antibodies, and is not to be construed as requiring preparation of the antibody by any particular method.

As used herein, the term "murine antibody" refers to B cells derived from immunized mice fused with myeloma cells, followed by screening for murine hybrid fusion cells that can both immortalize and secrete antibodies, and further screening, antibody production, and antibody purification. Or the plasma cells can produce secretory antibodies due to the formation of plasma cells by the differentiation and proliferation of B cells after the invasion of the antigen into the mouse body. The antigen is stimulated to produce, and the antibody is produced through the interaction of various immune cells after the antigen invades human body, so that B cells in lymphocyte are differentiated and proliferated to form plasma cells, and the plasma cells can produce secreted antibody.

As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence is modified to increase homology with the sequence of a human antibody. Typically, all or part of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody) and all or part of the non-CDR regions (e.g., variable region FR and/or constant regions) are derived from a human immunoglobulin (acceptor antibody). Humanized antibodies generally retain the desired properties of the donor antibody including, but not limited to, antigen specificity, affinity, reactivity, ability to enhance immune cell activity, ability to enhance immune responses, and the like. The donor antibody can be a mouse, rat, rabbit, or non-human primate (e.g., cynomolgus monkey) antibody having the desired properties (e.g., antigen specificity, affinity, reactivity, ability to enhance immune cell activity, and/or ability to enhance an immune response).

Humanized antibodies are particularly advantageous because they are capable of retaining the desired properties of non-human donor antibodies (e.g., murine antibodies) and are also capable of effectively reducing the immunogenicity of non-human donor antibodies (e.g., murine antibodies) in human subjects. However, due to matching problems between CDRs of a donor antibody and FRs of a recipient antibody, the desired properties of a humanized antibody (e.g., antigen specificity, affinity, reactivity, ability to enhance immune cell activity, and/or ability to enhance immune response) are typically lower than non-human donor antibodies (e.g., murine antibodies).

Thus, although researchers in the field have developed intensive research into the humanization of antibodies and made some progress (see, e.g., jones et al, nature,321:522 525 (1986), reichmann et al, nature,332:323 329 (1988), presta, curr. Op. Structure. Biol.,2:593 596 (1992), and Clark, immunol. Today21:397 402 (2000)), how to adequately humanize a donor antibody so that the resulting humanized antibody has as high a degree of humanization as possible while retaining as much as possible the intended properties of the donor antibody, the prior art does not provide exhaustive guidance. The skilled artisan needs to fumbly, explore and engineer specific donor antibodies, with the great effort of inventiveness being possible to obtain, humanized antibodies that have both a high degree of humanization (e.g., 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% degree of humanization) and retain the desired properties of the specific donor antibody.

In the present invention, in order for the humanized antibody to retain the properties of the donor antibody (including, for example, antigen specificity, affinity, reactivity, ability to enhance immune cell activity, and/or ability to enhance immune response) as much as possible, the Framework Region (FR) in the humanized antibody of the present invention may contain both amino acid residues of the human acceptor antibody and amino acid residues of the corresponding non-human donor antibody.

As used herein, the term "degree of humanization" is an indicator for evaluating the number of non-human amino acid residues in a humanized antibody. The degree of humanization of a humanized antibody can be predicted, for example, by the IMGT website DomainGapAlign, to predict the homology of the variable region sequence to the human V domain.

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against which it is directed. The strength or affinity of a specific binding interaction can be determined by the equilibrium dissociation constant (KD) or half maximal Effect Concentration (EC) ₅₀ ) And (3) representing.

The specific binding properties between two molecules can be determined using methods well known in the art. One method involves measuring the rate of antigen binding site or antigen complex formation and dissociation. Both the "binding rate constant" (ka or kon) and the "dissociation rate constant" (kdis or koff) can be calculated from the concentration and the actual rate of association and dissociation (see Malmqvist M, nature,1993, 361:186-187). The ratio of kdis or kon is equal to the dissociation constant KD (see Davies et al, annual Rev Biochem,1990; 59:439-473). KD, kon and kdis values can be measured by any effective method. In certain embodiments, the dissociation constant may be measured using bioluminescence interferometry (e.g., forteBio oct method). In addition to this, the dissociation constants can be measured using surface plasmon resonance techniques (e.g., biacore) or Kineca.

As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes, such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC), or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. Animal viruses that may be used as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus, papilloma vacuolation virus (e.g., SV 40). A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication origin.

Expression and cloning vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells. Typically, in cloning vectors, this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and which includes origins of replication or autonomously replicating sequences. The term "expression vector" as used herein refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or by an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, a prokaryotic cell such as e.g. escherichia coli or bacillus subtilis, a fungal cell such as e.g. yeast cells or aspergillus, an insect cell such as e.g. S2 drosophila cells or Sf9, or an animal cell such as e.g. fibroblasts, CHO cells, COS cells, NSO cells, heLa cells, BHK cells, HEK 293 cells or human cells.

As used herein, the term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the first amino acid sequence or nucleic acid sequence for optimal alignment with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When a position in a first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in a second sequence, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical overlapping positions/total number of positions x 100%). In certain embodiments, the two sequences are the same length.

Determination of percent identity between two sequences can also be accomplished using mathematical algorithms. One non-limiting example of a mathematical algorithm for comparison of two sequences is the algorithm of Karlin and Altschul, 1990, proc.Natl. Acad. Sci.U.S. A.87:2264-2268, as modified in Karlin and Altschul,1993, proc.Natl. Acad. Sci.U.S. A.90:5873-5877. Such an algorithm was integrated into the NBLAST and XBLAST programs of Altschul et al, 1990, J.mol. Biol. 215:403.

As used herein, the term "variant", in the context of polypeptides (including polypeptides), also refers to polypeptides or peptides comprising an amino acid sequence that has been altered by the introduction of amino acid residue substitutions, deletions or additions. In some instances, the term "variant" also refers to a polypeptide or peptide that has been modified (i.e., by covalently linking any type of molecule to the polypeptide or peptide). For example, but not by way of limitation, the polypeptide may be modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting groups, proteolytic cleavage, attachment to a cell ligand or other protein, and the like. The derivatized polypeptide or peptide may be produced by chemical modification using techniques known to those skilled in the art, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In addition, the variants have similar, identical or improved functions as the polypeptide or peptide from which they are derived.

As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the intended properties of a protein, polypeptide comprising the amino acid sequence. For example, conservative substitutions may be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions that replace an amino acid residue with an amino acid residue having a similar side chain, such as substitutions with residues that are physically or functionally similar (e.g., of similar size, shape, charge, chemical nature, including the ability to form covalent or hydrogen bonds, etc.) to the corresponding amino acid residue. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with 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), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Furthermore, amino acid residues can also be divided into categories defined by alternative physical and functional properties. For example, alcohol group-containing residues (S and T), aliphatic residues (I, L, V and M), cycloalkenyl-related residues (F, H, W and Y), hydrophobic residues (A, C, F, G, H, I, L, M, R, T, V, W and Y), negatively charged residues (D and E), polar residues (C, D, E, H, K, N, Q, R, S and T), positively charged residues (H, K and R), small residues (A, C, D, G, N, P, S, T and V), very small residues (A, G and S), residues involving corner formation (A, C, D, E, G, H, K, N, Q, R, S, P and T), flexible residues (Q, T, K, S, G, P, D, E and R). Methods for identifying conservative substitutions of amino acids are well known in the art (see, e.g., brummell et al, biochem.32:1180-1187 (1993); kobayashi et al Protein Eng.12 (10): 879-884 (1999); and Burks et al Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

The twenty conventional amino acids referred to herein are written following conventional usage. See, e.g., immunology-a Synthesis (2nd Edition,E.S.Golub and D.R.Gren,Eds, sinauer Associates, sundland, mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in the present invention, amino acids are generally indicated by single-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

As used herein, the term "linker" refers to a molecular moiety for linking an antibody or antigen binding fragment thereof that targets a specific antigen to a coupling moiety to form a conjugate. In some cases, the linker further comprises a spacer to alter the steric hindrance of the coupling moiety. In some conjugates, a linker is necessary; in other conjugates, a linker is not necessary. The linkers are divided into non-cleavable linkers and cleavable linkers. Conjugates containing such non-cleavable linkers require degradation of the antibody or antigen binding fragment thereof by internalization to release the conjugated moiety; conjugates containing such cleavable linkers may release the coupling moiety by internalizing the degrading antibody or antigen binding fragment thereof, or may release the coupling moiety by cleavage of the structure of the linker by enzymatic or chemical catalytic cleavage occurring at the cleavage site without internalization. Examples of the non-cleavable linker include N-succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC). Examples of such cleavable linkers include linkers with disulfide linkages as cleavage sites, hydrazone linkers with acyl groups as cleavage sites, linkers with enzymatic sites as cleavage sites (e.g., dipeptide linkers Val-Cit, val-Ala; tetrapeptide linkers Gly-Gly-Phe-Gly; linkers containing phosphoric acid and pyrophosphoric acid).

The linker may link the antibody or antigen binding fragment thereof to the coupling moiety by covalent attachment through chemical functional group reaction by coupling techniques known in the art. The coupling technique may be based on chemical specific in situ antibody modification, e.g. ligation by reaction of lysine residues with electrophilic groups, ligation by reaction of disulfide bonds, bridging again by sulfhydryl groups, and ligation by glycosylation of glycans. The coupling technique may also be based on site-specific bioconjugation of engineered antibodies, e.g., by adding enzyme-specific recognition natural or unnatural amino acids to the antibody for ligation by enzyme catalysis.

As used herein, 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, which is well known in the art (see, e.g., remington's Pharmaceutical sciences. Mediated by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995), and includes, but is not limited to: pH modifiers, surfactants, adjuvants, ionic strength enhancers, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives. For example, pH adjusters 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, sugar, naCl, and the like. Agents that delay absorption include, but are not limited to, monostearates 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, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like. Stabilizers have the meaning commonly understood by those skilled in the art and are capable of stabilizing the desired activity of the active ingredient in a medicament, including but not limited to sodium glutamate, gelatin, SPGA, saccharides (e.g., sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (e.g., glutamic acid, glycine), proteins (e.g., dried whey, albumin or casein) or degradation products thereof (e.g., lactalbumin hydrolysate), and the like.

As used herein, the term "preventing" refers to a method that is performed in order to prevent or delay the occurrence of a disease or disorder or symptom in a subject. As used herein, the term "treatment" refers to a method that is performed in order to obtain beneficial or desired clinical results. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., no longer worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment or inhibition of disease recurrence, and remission (whether partial or total), whether detectable or undetectable. Furthermore, "treatment" may also refer to an extension of survival compared to the expected survival (if not treated).

As used herein, the term "subject" refers to a mammal, such as a primate mammal, e.g., a human. In certain embodiments, the subject (e.g., a human) has, or is at risk of having, a tumor (e.g., a tumor that expresses MSLN), an inflammatory disease, or an autoimmune disease.

As used herein, 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 of a disease (e.g., a tumor, an inflammatory disease, or an autoimmune disease) refers to an amount sufficient to prevent, arrest, or delay the onset of the disease (e.g., a tumor, an inflammatory disease, or an autoimmune disease); a therapeutically effective amount refers to an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Determination of such effective amounts is well within the ability of those skilled in the art. For example, the 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 such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, and the like.

The terms "cancer" and "tumor" are used interchangeably and refer to a broad class of diseases characterized by uncontrolled growth of abnormal cells in the body. Unregulated cell division may result in the formation of malignant tumors or cells that invade adjacent tissues and may metastasize to distant sites of the body through the lymphatic system or blood flow. Cancers include benign and malignant cancers, dormant tumors or micrometastases. Cancers also include hematological malignancies.

As used herein, the term "pharmaceutically acceptable" refers to a molecule that does not produce adverse, allergic or other untoward reactions when the molecule entity, fragment or composition is properly administered to an animal or human. Specific examples of some substances that may be pharmaceutically acceptable carriers or components thereof include saccharides (e.g., lactose), starches, celluloses and derivatives thereof, vegetable oils, gelatins, polyols (e.g., propylene glycol), alginic acid and the like.

Herein, combination therapy includes the use of an anti-TIGIT antibody or antigen-binding fragment thereof contemplated by the present invention in combination with one or more additional active therapeutic agents of a second therapy (e.g., chemotherapeutic agents) or other prophylactic or therapeutic modalities (e.g., radiation therapy).

Exemplary anti-cancer agents for the second therapy may include chemotherapeutic agents (e.g., mitotic inhibitors), alkylating agents (e.g., nitrogen Mustard Nitrogen mustards), antimetabolites (e.g., folic acid analogs), natural products (e.g., vinca alkaloids), various agents (e.g., platinum coordination complexes), hormones and antagonists (e.g., adrenocorticosteroids), immunomodulators (e.g., bromopirimine, upjohn), and the like. Other anti-cancer treatments include other antibodies that specifically target cancer cells.

In such combination therapies, the various active agents often have different complementary mechanisms of action, which may lead to synergistic effects. Combination therapies include therapeutic agents that affect an immune response (e.g., enhance or activate a response) and therapeutic agents that affect (e.g., inhibit or kill) a tumor or cancer cell. Combination therapy may reduce the likelihood of drug-resistant cancer cell development. Combination therapy may allow for a reduced 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 a potential disease, disorder, or condition.

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

Advantageous effects of the invention

In the present application, the inventors first developed murine antibodies with superior properties that are capable of specifically recognizing or binding TIGIT. Based on this, the inventors have made intensive studies and alterations on the murine antibody, thereby developing a humanized antibody of the murine antibody. The humanized antibodies of the present invention not only have a higher degree of humanization, but also have substantially the same (or even superior) biological function as murine antibodies.

Thus, the antibodies of the invention (particularly humanized antibodies) are highly advantageous, not only in retaining the functions and properties of the parent murine antibody, e.g., binding TIGIT with good affinity and specificity, effectively promoting secretion of IFN- γ and IL-2, and can be used for preventing and treating TIGIT-related diseases (e.g., tumors); the humanized antibodies of the present invention have a high degree of humanization and thus can be safely administered to human subjects without eliciting an immunogenic response. The antibodies of the invention are of great clinical value.

Embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are only for illustrating the present invention and are not to be construed as limiting the scope of the present invention. Various objects and advantageous aspects of the invention will become apparent to those skilled in the art from the following detailed description of the preferred embodiments and the accompanying drawings.

Drawings

Fig. 1A: anti-TIGIT chimeric antibodies 7TI-024, 7TI-025, 7TI-026, 7TI-027 increased the detection of SEA activated PBMC (donor 007) secretion of IFN- γ;

fig. 1B: anti-TIGIT chimeric antibody 7TI-026, 7TI-027 enhances the detection of SEA activated PBMC (donor 095) secreting IFN- γ;

fig. 1C: anti-TIGIT chimeric antibody 7TI-035, 7TI-036 improved the detection of SEA activated PBMC (donor 095) secreting IFN- γ;

fig. 1D: anti-TIGIT chimeric antibodies 7TI-038, 7TI-041, 7TI-042, 7TI-046 improved the detection of SEA activated PBMC (donor 095) secretion of IFN- γ;

fig. 1E: anti-TIGIT chimeric antibody 7TI-042, 7TI-046 improved the detection of SEA activated PBMC (donor 007) secretion of IFN- γ;

fig. 2A: anti-TIGIT chimeric antibodies 7TI-024, 7TI-025, 7TI-026, 7TI-027 increased the detection of SEA activated PBMC (donor 007) secreting IL-2;

fig. 2B: anti-TIGIT chimeric antibodies 7TI-042, 7TI-046 improved the detection of SEA activated PBMC (donor 007) secreting IL-2.

The information of the sequences to which the invention relates is described in the following table:

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remarks: the VH and VL sequences in the table above are bolded and underlined are CDR regions.

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, molecular biology experimental methods and immunoassays used in the present invention are basically described in j.sambrook et al, molecular cloning: laboratory Manual, 2 nd edition, cold spring harbor laboratory Press, 1989, and F.M. Ausubel et al, fine-compiled guidelines for molecular biology experiments, 3 rd edition, john Wiley & Sons, inc., 1995; the use of restriction enzymes was in accordance with the conditions recommended by the manufacturer of the product. Those skilled in the art will appreciate that the examples describe the invention by way of example and are not intended to limit the scope of the invention as claimed.

Example 1: preparation of anti-TIGIT murine monoclonal antibodies

1.1 mouse immunization and hybridoma fusion

100. Mu.g of human TIGIT ectodomain huTIGIT-msIgG2a-Fc-tag antigen (purchased by Arco; TIT-H5253) was fully emulsified with complete Freund's adjuvant at an equal volume of 1:1 and immunized by tail vein injection and intraperitoneal injection for 5 to 6 weeks of BALB/c mice. The immunization period was 1/2 weeks, and the jugular vein was collected after the end of each immunization period, and the serum titers of the mice were evaluated by ELISA detection. Two weeks after immunization 3, the mice serum anti-human and cynomolgus TIGIT antigen antibody titers were tested by ELISA to monitor the extent of the mouse immune response. And the mice with highest anti-human and cynomolgus TIGIT antibody titer are boosted once, and spleen cells of the mice are isolated after 3 days to obtain spleen cell single cell suspension. Will be 1.0X10 ⁸ Sp2/0 cells (Nanjac Bai, CBP 60881) and 3.0X10 ⁸ Single cell suspension of spleen cells by electrofusionThe fusion is carried out in a mode. Adjustment of the number of fused cells to 2.5X10 with HAT Medium Screen ⁵ 0.2mL of each of the above-mentioned substances was added to a 96-well culture plate well and the well was placed at 37℃in 5% CO ₂ Culturing in an incubator for 10 days to obtain hybridoma culture solution.

1.2ELISA, FACS high throughput screening of clones with affinity

1.2.1ELISA high throughput screening

The coated antigens were human TIGIT-His (huTIGIT-His, organism, Q495A 1-1) and monkey TIGIT-His (cynotiGIT-His, acro; TIT-C5223). The antigens huTIGIT-His and cynotiit-His were diluted to 1 μg/ml with PBS in 96-well plates, respectively, followed by 100 μl of diluted antigen per well for incubation, 3 times daily PBST wash at 4 ℃ overnight, then 3% bsa was added, blocked for 1 hour at 37 ℃ and washed 3 times with PBST. Then, 50ul of the supernatant of the hybridoma culture solution obtained in example 1.1 was added to each well, incubated at 37℃for 1 hour, and washed 3 times with PBST; anti-mouse-IgG-HRP secondary antibody (Thermo Fisher, 32460) was added, diluted 1:5000, incubated at 4℃for 1 hour, washed 3 times with PBST, the substrate was added for color development, quenched with 2mM hydrochloric acid after completion of the reaction, and finally plates were read with an ELISA reader at 450 nm. Positive clones binding to both huTIGIT and cyno-TIGIT were selected based on the plate reading results and further screened by FACS.

1.2.2FACS high throughput screening

On a 6-well plate, 10. Mu.l of lentivirus (Hantao custom made) of human TIGIT (huTIGIT) target gene (SEQ ID NO: 205) was added to 60% density Jurkat cells (Nanjac Bai, CBP 60520), and after 24 hours of culture, a new medium was changed and puromycin was added to give a final working concentration of 10. Mu.g/ml for resistance screening, and after 4 days positive polyclonal cells obtained by the screening were subjected to expansion culture and seed preservation to obtain Jurkat-huTIGIT cells. 293T cells (CBP 60439, nanjac Bai) were infected with lentivirus (Hemsl) packed with cynomolgus TIGIT (cynoTIGIT) target gene (SEQ ID NO: 206) according to the above preparation method to obtain 293T-cynotiGIT cells.

The Jurkat-huTIGIT cells and 293T-cynotiit cells obtained by the screening were added to 96-well plates with 1X 10 per well ⁵ Cells were then added to 50. Mu.l of positive hybridoma supernatant screened in experiment 1.2.1, incubated for 1 hour at 37℃and washed twice with FACS Buffer (PBS+2% FBS), followed by 50. Mu.l/well of anti-mouse-IgG-APC secondary antibody (Biolegend, 405308), incubated at 4℃for 30min in the absence of light, eluted twice with FACS Buffer (PBS+2% FBS), and 200. Mu.l of FACS Buffer was added per well and detected with a flow meter. According to the result, positive clone strains with strong binding effect to Jurkat-huTIGIT and 293T-cynotiit cells are selected for further analysis and detection.

1.3CD155 competitive binding and reporter gene activation screening activation function mother clone strain

ELISA and FACS are used for dual screening of positive clone strains with binding to further detect the NF- κB signal path activating effect, so that the functional anti-TIGIT positive clone strains are further screened.

After preliminary screening by ELISA, FACS and competitive binding and reporter gene activation function assays, 21 candidate anti-TIGIT murine monoclonal antibodies were obtained, designated 1BT4-10A3, 2BT1-11A6-A10, 2BT3-4A6-H12, 2IT1-18B2-C3, 2IT1-10A3-2H6, 3IT2-4E1-G6, 3IT2-4G9-B11, 3IT2-7B9-C2, 3IT2-15B1-G8, 3IT2-16F2-C2, 3IT2-18G2-E7, 3IT2-19F6-G8, 3IT2-26G7-B7, 3IT2-28E5-G3, 3A03A03, 6B01A10, 16F10A06, 33F12A12, 41A07A03, 50F02C 03H04, and further expanded antibodies were cultured and analyzed for further characterization.

EXAMPLE 2 variable region amplification and sequence analysis of anti-TIGIT murine monoclonal antibody

The candidate monoclonal obtained in example 1 was further amplified, a part of the cells was taken for seed preservation, the other part was used for total RNA extraction using RNA kit (KaTaRa), and reverse transcribed into cDNA, PCR amplified with primers for heavy chain variable region (VH) and light chain variable region (VL) of murine monoclonal antibody, respectively, ligated to a vector, and then clones were picked up for DNA sequencing. Analysis of the sequencing results and definition of CDR regions. The variable region and CDR sequences of the obtained anti-TIGIT murine monoclonal antibody are shown in Table 1:

TABLE 1 variable region and CDR amino acid sequences of anti-TIGIT murine antibodies

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EXAMPLE 3 humanization of murine antibodies to TIGIT

The murine antibodies in table 1 were humanized. Specifically, the amino acid sequence of the light chain variable region of the murine antibody was linked to the amino acid sequence of the light chain constant region (SEQ ID NO: 144), and the amino acid sequence of the heavy chain variable region was linked to the amino acid sequence of the IgG1 heavy chain constant region (SEQ ID NO: 141) or the amino acid sequence of the heavy chain constant region variant (SEQ ID NO: 142), respectively, to construct a chimeric antibody, and the variable region and constant region amino acid sequences of the obtained anti-TIGIT chimeric antibody were as shown in Table 2:

TABLE 2 amino acid sequences of variable and constant regions of anti-TIGIT chimeric antibodies

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Example 4 detection of cell binding of anti-TIGIT chimeric antibodies to human or monkey TIGIT cells

The chimeric antibody was detected by flow cytometry fluorescence sorting (FACS) and cell binding detected with Jurkat-huTIGIT cells and 293T-cynotiGIT cells, respectively. The method comprises the following steps:

1) Jurkat-huTIGIT or 293T-cynoTIGIT cells were added to the U-bottom plates of 96-well plates, respectively, 1X 10 cells per well ⁵ Individual cells.

2) Antibody preparation, the antibody to be detected was diluted starting from 30. Mu.g/ml for the first concentration gradient, 3-fold gradient dilution, 11 spots in total, and 50. Mu.l per well.

3) The samples were placed in a 37 ℃ incubator for 1 hour incubation.

4) 350g, centrifuged for 5min, cells were collected and washed twice with flow staining buffer (FACS buffer).

5) The secondary antibody was prepared, and the fluorescent conjugated secondary antibody anti-mouse-IgG-APC (1:100 dilution) (Biolegend, 405308) was added at 50 μl per well and reacted at 4deg.C for 30min in the absence of light.

6) 350g, centrifuged for 5min, and cells were collected and washed twice with FACS Buffer, 200. Mu.l FACS Buffer was added to each well and detected by flow-through.

Analyzing the flow result by FlowJo software, sorting the fluorescence value and importing the fluorescence value into Graphpad Prism to calculate EC ₅₀ The analysis results are shown in Table 3 and Table 4, and from the results, the target antibodies are shown: b60 (IMGT No. INN (International Nonproprietary Name): tiragolumab), 7TI-002 (VH/VL sequence alignment antibody B60, heavy chain constant region variant amino acid sequence shown as SEQ ID NO:142, light chain constant region amino acid sequence shown as SEQ ID NO: 144), or 7TI-003 (VH/VL sequence alignment antibody B60, heavy chain constant region variant amino acid sequence shown as SEQ ID NO:143, light chain constant region amino acid sequence shown as SEQ ID NO: 144); as shown by the results of TIGIT cross-binding experiments of human and monkey, the antibody of the invention has good cell binding activity with TIGIT cells expressing human or monkey.

Table 3: anti-TIGIT chimeric antibodies bind to Jurkat-huTIGIT cells

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Remarks: a. b, c, d represent different experimental detection batches;

table 4: binding of anti-TIGIT chimeric antibodies to 293T-cynotiit cells

Remarks: a. the b and c batches represent different experimental detection batches.

Example 5 anti-TIGIT chimeric antibodies compete with CD155 for binding and reporter activating ability

The in vitro activity of the antibodies was evaluated using Jurkat-NFAT-LUC-huTIGIT effector cells (Nanjac Bai, CBP 74020) and CHO-TCR-CD155 (Nanjac Bai, CBP 74073) target cells. The specific operation steps are as follows:

1) Taking logarithmic growth CHO-TCR-CD155 cells, re-suspending by pancreatin digestion in newly prepared F12K medium containing 10% FBS, and adjusting the density of the re-suspended cells to 4.0X10% ⁵ And each ml.

2) Resuspended cells were inoculated into white-wall clear-bottomed 96-well cell culture plates, 100 μl/well of cell suspension was added, and incubated overnight in an incubator at 37 ℃.

3) The next day, F12K medium in 96-well plates inoculated with CHO-TCR-CD155 cells was blotted dry, samples were further diluted in gradient with 10% serum RPMI1640 medium, gradient diluted concentration chimeric antibody (50 μl/well) was added to 96-well plates inoculated with cells, samples were 3-fold diluted 11 concentration gradients starting from the highest concentration, and blank medium control wells were additionally set.

4) The supernatant was removed by centrifugation from Jurkat-NFAT-LUC-huTIGIT cells grown in log phase, and the resuspended cells were adjusted to 4.0X10 by resuspension in fresh 10% FBS RPMI1640 medium ⁵ Cells were then added to the 96-well plate of step 3) at 50 μl per well and incubated in an incubator at 37℃for 5 to 6 hours.

5) The 96-well plate was removed from the incubator and 100. Mu.l/well Bright-Glo was added ^TM The luciferase detection reagent is placed for 3 to 5 minutes and put into an enzyme-labeled instrument to read the value.

6) Fitting a gradient curve of the sample to cell activation using Graphpad Prism software according to the read value corresponding to each gradient concentration well, and calculating EC of the sample ₅₀ 。

The specific analysis results are shown in Table 5. The results show that the antibodies of the invention in Table 5 are effective in activating the reporter gene.

Table 5: anti-TIGIT chimeric antibodies compete with CD155 for binding and reporter-activated EC ₅₀

Remarks: a. b, c, d, e batches represent different experimental detection batches.

EXAMPLE 6 physicochemical Property detection of anti-TIGIT chimeric antibody

Physicochemical property evaluation is carried out on the anti-TIGIT chimeric antibody molecules, wherein the physicochemical property evaluation comprises three indexes of TM, tagg and HIC, and the detection methods of the four detection indexes are as follows:

1) TM detection: a DSF (differential fluorescence scanning technique) method is employed. 50ul of the anti-TIGIT chimeric antibody to be tested, 1mg/ml, was diluted to 17.5. Mu.l with PBS, 2.5. Mu.l of 1000X protein Thermal shift dye (Thermo, 4461146) was added and mixed in the EP tube, and the above sample mixture was added to the Q-PCR system (Quant Studio) for reaction, and the Q-PCR parameters were set: target (ROX), procedure (25 ℃,3min;0.05 ℃ C./s temperature rise to 99 ℃, 99 ℃ C., 2 min). Result importing Graph Prism software to calculate T _m Values.

2) HIC hydrophobicity detection: the sample to be tested was prepared by 10ug (1 mg/ml) and loaded onto an HPLC liquid chromatograph (SHIMADZU, shimadzu LC 2030C), a corresponding detection program was selected, and after the detection was completed, analysis was performed by using analysis software (Lab solution) built in the instrument.

3) Tagg detection: 9ul (1 mg/ml) of each sample to be tested is prepared, the sample to be tested is put into a detection instrument (Uncle-0734), and after detection, the analysis is carried out by software.

The detection results of these three detection indexes are summarized in table 6. The results show that the antibodies of the invention have stable physicochemical properties.

Table 6: physicochemical Properties of anti-TIGIT chimeric antibodies

Remarks: N/A represents undetected.

EXAMPLE 7SPR (surface plasmon resonance) detection of affinity of anti-TIGIT chimeric antibodies to human or monkey antigens

The binding kinetics of anti-TIGIT chimeric antibodies to human (huTIGIT-his) or monkey (cynotiit-his) were characterized by SPR assay using BIAcore. The anti-TIGIT chimeric antibody to be detected was added to the protein a amino-coupled CM5 biosensor chip (cytova, BR 100839) at a final concentration of 5 ug/ml. The gradient diluted huTIGIT-his ( organism, Q495 A1-1) and cynotigitis-his (Acro; TIT-C5223) antigens (gradient dilution starting at 100nM, 3-fold dilution, 9 spots diluted) were then added separately from low to high concentrations, following the procedure Kinetics: start up (60 s) -Capture (60 s) -Association (200 s) -Association (600 s) -Regeneration (40 s), and after the program is finished, the result file is read by Insight Evaluation Software software for analysis, and the specific analysis results are shown in table 7:

Table 7: SPR detection of chimeric antibodies affinity to human and monkey TIGIT

Example 8 functional assay of anti-TIGIT chimeric antibodies to increase secretion of IFN-gamma and IL-2 by SEA activated PBMCs

Human PBMC donor 095 (cat No. SC 12095) and donor 007 (SC 12007) were purchased from Chimaphila organisms to resuspend PBMC with RIPM-1640 medium and inactivated 10% FBS, and cell density was adjusted to 1.0X10 after counting ⁶ Per mL, 100ng/mL SEA (staphylococcal enterotoxin A, purchased from Toxin Technology, AT 101) was added to stimulate T cell activation, and cells were seeded in 96-well plates with 100. Mu.l per well. Chimeric antibody was used at six concentrations (100. Mu.g/ml),50. Mu.g/ml, 25. Mu.g/ml, 10. Mu.g/ml, 1. Mu.g/ml, 0.1. Mu.g/ml) were serially diluted, added to 96-well plates, 100. Mu.l/well, and three wells were made; at 37℃with 5% CO ₂ After 3 days of incubation in the incubator, the supernatants were assayed for IL-2 concentration using an HTRF kit (from PerkinElmer Cisbo, human IL-2 kit). After 4 days of incubation, the supernatant was assayed for IFN-gamma concentration using HTRF kit (from PerkinElmer Cisbo, human IFN gamma kit) and the results of IFN-gamma assay are shown in FIGS. 1A, 1B, 1C, 1D, and 1E, respectively. IL-2 detection results are shown in FIGS. 2A and 2B. As can be seen from the figure, the antibody of the present invention is effective in promoting secretion of IFN-gamma and IL-2.

EXAMPLE 9 humanization of murine antibodies to TIGIT

The murine antibody 2IT1-18B2-C3,2IT1-10A3-2H6,3A03A03 was humanized. Specifically, the amino acid sequence of the murine antibody is compared with the amino acid sequence of the human germ line antibody, and a sequence with high homology and better physicochemical property is found out to be used as a humanized antibody framework sequence; defining a murine antibody CDR region according to IMGT+Kabat, and transplanting the murine antibody CDR region on a human antibody framework sequence; based on sequence analysis and 3D modeling, the amino acids of the CDR and framework regions, and the amino acids that may interact with the antibody surface, are recovered; further, 11 mutants of 7TI-152, 7TI-153, 7TI-161, 7TI-165, 7TI-172, 7TI-174, 7TI-177, 7TI-202, 7TI-208, 7TI-215 and 7TI-218 are obtained through computer calculation, primary affinity detection is carried out, antibodies with high affinity and humanization degree are further expressed, and various antigens, cell binding capacity and endocytosis detection are carried out. Wherein, the primary humanized antibody generated by the murine antibody 2IT1-18B2-C3 is: 7TI-152, 7TI-153, 7TI-161, 7TI-165; the primary humanized antibody generated by the murine antibody 2IT1-10A3-2H6 is: 7TI-172, 7TI-174, 7TI-177; the primary human antibody produced by the murine antibody 3a03 is: 7TI-202, 7TI-208, 7TI-215, 7TI-218.

The amino acid sequences of the variable region and the constant region of the humanized antibody are shown in Table 8:

TABLE 8 amino acid sequences of variable and constant regions of humanized anti-TIGIT antibodies

EXAMPLE 10 detection of cell binding of humanized anti-TIGIT antibodies to human or monkey TIGIT cells

The humanized antibodies of tables 9 and 10 were examined by FACS for cell binding with Jurkat-huTIGIT cells and 293T-cynotiGIT cells, respectively, and the specific assay method was as described in example 4. EC of humanized antibody binding to Jurkat-huTIGIT cells ₅₀ See Table 9, EC of humanized antibodies binding to 293T-cynoTIGIT cells ₅₀ See table 10. The results show that the humanized antibodies of the present invention have good cell binding activity to TIGIT cells expressing human or monkey.

Table 9: binding of humanized antibody TIGIT to Jurkat-huTIGIT cells

Remarks: a. b represents different experimental detection batches;

table 10: EC of humanized antibody TIGIT binding to 293T-cynotiGIT cells ₅₀

Remarks: a. batch b represents different experimental detection batches.

Example 11 humanized anti-TIGIT antibodies compete with CD155 for binding and reporter activating ability

Humanized antibodies in table 11 were tested for competitive binding to CD155 and reporter gene activation, and specific test methods are described in example 5. Humanized antibody CD155 competing for binding and reporter gene activating EC ₅₀ See table 11 below. The results show that the antibodies of the invention in Table 11 are effective in activating the reporter gene.

TABLE 11 humanized anti-TIGIT antibodies and CD155 bidContention binding and reporter-activated EC ₅₀

Remarks: a. batch b represents different experimental detection batches.

EXAMPLE 12 detection of physicochemical Properties of humanized antibody against TIGIT

Physical and chemical properties TM and Tagg values of the humanized antibodies in table 12 were measured, and specific measurement methods are described in example 6, and the measurement results are shown in table 12 below. It can be seen that the chimeric antibody of the present invention is stable in physicochemical properties after humanization.

TABLE 12 physicochemical Properties of humanized antibody TIGIT

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Remarks: N/A represents undetected.

EXAMPLE 13SPR (surface plasmon resonance) detection of affinity of anti-TIGIT humanized antibodies to human antigen

BIAcore was used to characterize the binding kinetics of humanized antibodies to humans, and specific detection methods are described in example 7, with the results shown in Table 13.

TABLE 13SPR (surface plasmon resonance) detection of affinity of humanized antibody TIGIT to human TIGIT antigen

Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate that: many modifications and variations of details may be made to adapt to a particular situation and the invention is intended to be within the scope of the invention. The full scope of the invention is given by the appended claims together with any equivalents thereof.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds TIGIT, wherein:

(1) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 97; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO. 125;

or (b)

(2) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 87; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO 115;

or (b)

(3) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 86; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO 114;

or (b)

(4) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO. 85; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO 113;

or (b)

(5) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 83; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO. 132;

or (b)

(6) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 84; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO 112;

Or (b)

(7) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 88; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO 116;

or (b)

(8) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 89; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO 117;

or (b)

(9) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 90; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO. 118;

or (b)

(10) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO. 91; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO 119;

or (b)

(11) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 92; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO. 120;

or (b)

(12) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO. 93; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO. 121;

or (b)

(13) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO. 94; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO. 122;

or (b)

(14) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 95; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO. 123;

or (b)

(15) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO. 96; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO 124;

or (b)

(16) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 98; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO. 126;

or (b)

(17) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 99; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO 127;

or (b)

(18) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO. 100; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO 128;

or (b)

(19) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 101; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO. 129;

or (b)

(20) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 102; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO. 130;

or (b)

(21) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 103; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO. 131;

Or (b)

(22) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 107; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO 134;

or (b)

(23) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 107; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO. 135;

or (b)

(24) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 109; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO 137;

or (b)

(25) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO. 110; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO 138;

or (b)

(26) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 104; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO. 140;

or (b)

(27) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 104; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO 133;

or (b)

(28) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 105; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO 133;

or (b)

(29) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 106; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO 133;

or (b)

(30) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO. 108; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO 136;

or (b)

(31) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 111; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO 139;

or (b)

(32) CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO 111; and

CDR-L1, CDR-L2 and CDR-L3 contained in VL represented by SEQ ID NO 138;

preferably, the CDRs are defined according to the numbering system of Kabat, IMGT, chothia, contact, abM or a combination thereof.

2. An antibody or antigen-binding fragment thereof that specifically binds TIGIT according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises the following heavy chain variable region (VH) and light chain variable region (VL), wherein CDRs are defined by the IMGT numbering system:

Or (b)

or (b)

Or (b)

or (b)

Or (b)

or (b)

alternatively, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL) wherein the CDRs are defined according to the Kabat numbering system:

or (b)

Alternatively, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL) wherein the CDRs are defined by the AbM numbering system:

or (b)

Or (b)

alternatively, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL) wherein the CDRs are defined by the Chothia numbering system:

or (b)

Or (b)

or (b)

alternatively, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL) wherein the CDRs are defined by the Contact numbering system:

Or (b)

or (b)

alternatively, the antibody or antigen-binding fragment thereof comprises the following heavy chain variable region (VH) and light chain variable region (VL), wherein the CDRs are defined by the numbering system of Kabat, IMGT, chothia, contact or a combination of abms:

or (b)

3. The antibody or antigen-binding fragment thereof that specifically binds TIGIT according to any one of claims 1 or 2, wherein:

(8) SEQ ID NO:89 and VH and SEQ ID NO: 117;

(15) SEQ ID NO:96 and VH and SEQ ID NO:124, VL of the sequence shown;

(16) SEQ ID NO:98 and VH and SEQ ID NO:126, VL of the sequence shown;

(21) SEQ ID NO:103 and SEQ ID NO:131, VL of the sequence shown in seq id no;

(22) SEQ ID NO:104 and SEQ ID NO:140, VL of the sequence shown in seq id no;

(23) SEQ ID NO:104 and SEQ ID NO:133, VL of the sequence shown;

(26) SEQ ID NO:107 and VH and SEQ ID NO:134, VL of the sequence shown;

(30) SEQ ID NO:110 and SEQ ID NO:138, VL of the sequence shown in seq id no;

alternatively, the VH and VL have at least 70%, 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 compared to any of the groups (1) to (32); and, VL has at least 70%, 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%, or at least 99% sequence identity;

alternatively, the VH and VL have one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) as compared to any of the groups (1) to (32); and VL has a 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); preferably, the substitutions are conservative substitutions.

4. An antibody or antigen-binding fragment thereof that specifically binds TIGIT according to any one of claims 1-3, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of Fab fragments, fab 'fragments, F (ab)' ₂ Fragments, single chain antibodies or disulfide stabilized Fv proteins (dsFv);

preferably, the single chain antibody is selected from scFv, di-scFv or (scFv) ₂ 。

5. An antibody or antigen-binding fragment thereof that specifically binds TIGIT according to any one of claims 1-4, wherein the antibody or antigen-binding fragment thereof is a murine antibody, chimeric antibody, humanized antibody or other species derived antibody (e.g. rabbit, camel or shark).

6. An antibody or antigen-binding fragment thereof that specifically binds TIGIT according to any one of claims 1-5, wherein,

the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and a light chain constant region;

preferably, the heavy chain constant region is selected from the group consisting of a heavy chain constant region of IgG, igM, igE, igD or IgA or a variant thereof; and/or the number of the groups of groups,

wherein the variant has a substitution, deletion, or addition of one or more amino acids (e.g., a substitution, deletion, or addition of up to 20, up to 15, up to 10, or up to 5 amino acids; e.g., a substitution, deletion, or addition of 1, 2, 3, 4, or 5 amino acids) as compared to the wild-type sequence from which it is derived;

More preferably, the antibody or antigen binding fragment thereof comprises a heavy chain constant region and a light chain constant region selected from the group consisting of:

(1) A human IgG1 heavy chain constant region (e.g., a human IgG1 heavy chain constant region as set forth in SEQ ID NO: 141) or a variant thereof (e.g., a variant of a human IgG1 heavy chain constant region wherein the variant has one or more amino acid substitutions, deletions, or additions at positions 233, 234, 235, 236, 239, 327, 330, 331, or 332 (e.g., substitutions of E233P, L, 234V, L, 235A, A327G, A330S, P S, S, D, A L or I332E; e.g., deletions of deltaG 236) as compared to the human IgG1 heavy chain constant region sequence from which it is derived according to the EU numbering system), more preferably the sequence of the variant is selected from SEQ ID NO:142 or 143;

further preferred, the antibody or antigen binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID NO:141 and a heavy chain constant region as set forth in SEQ ID NO: 144.

Or comprises the amino acid sequence as set forth in SEQ ID NO:142 and a heavy chain constant region as set forth in SEQ ID NO: 144.

Or comprises the amino acid sequence as set forth in SEQ ID NO:143 and a heavy chain constant region as set forth in SEQ ID NO: 144.

7. A multispecific antibody comprising the antibody or antigen-binding fragment thereof of any one of claims 1-6; preferably, the multispecific antibody is a bispecific or trispecific or tetraspecific antibody.

8. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-6, or the multispecific antibody of claim 7.

9. A vector comprising the nucleic acid molecule of claim 8; preferably, the vector is a cloning vector or an expression vector; more preferably, the expression vector is selected from the group consisting of pTT5 vector, pJex vector or pD912 vector.

10. A host cell comprising the nucleic acid molecule of claim 8 or the vector of claim 9; preferably, the host cell is a CHO cell; more preferably, the CHO cells are Expi-CHOs cells.

11. A method of making an antibody or antigen-binding fragment thereof that specifically binds TIGIT, or a multispecific antibody, comprising culturing the host cell of claim 10 under conditions that allow expression of the antibody or antigen-binding fragment thereof, or the multispecific antibody, and recovering the antibody or antigen-binding fragment thereof, or the multispecific antibody from the cultured host cell culture.

12. A conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 6, or the multispecific antibody of claim 7, or the nucleic acid molecule of claim 8, and a coupling moiety; the antibody or antigen-binding fragment thereof, or the multispecific antibody, or the nucleic acid molecule is linked to the conjugate moiety directly or via a linker;

preferably, the coupling moiety is selected from: a detectable label (e.g., a radioisotope, fluorescent substance, luminescent substance, colored substance, or enzyme) or therapeutic agent (e.g., a nuclide, an immune agonist, an immunosuppressant, a cytokine, a toxin, and other active substances that inhibit tumor cell growth, promote tumor cell apoptosis, or necrosis).

13. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, or a multispecific antibody according to claim 7, or a nucleic acid molecule according to claim 8, or a vector according to claim 9, or a host cell according to claim 10, or a conjugate according to claim 12, and a pharmaceutically acceptable carrier and/or excipient;

Preferably, the pharmaceutical composition further comprises an additional pharmaceutically active agent;

more preferably, the additional pharmaceutically active agent is a drug having anti-tumor activity, such as a chemotherapeutic (e.g. platinum-based) or a small molecule inhibitor; and/or the number of the groups of groups,

14. A kit comprising an antibody or antigen binding fragment thereof according to any one of claims 1 to 6, or a multispecific antibody according to claim 7, or a nucleic acid molecule according to claim 8, or a vector according to claim 9, or a host cell according to claim 10, or a conjugate according to claim 12, or a pharmaceutical composition according to claim 13.

15. Use of an antibody or antigen binding fragment thereof according to any one of claims 1 to 6, or a multispecific antibody according to claim 7, or a nucleic acid molecule according to claim 8, or a vector according to claim 9, or a host cell according to claim 10, or a conjugate according to claim 12, or a pharmaceutical composition according to claim 13, for the manufacture of a medicament for the prevention and/or treatment and/or adjuvant treatment and/or neoadjuvant treatment of TIGIT-related diseases;

preferably, the TIGIT-related disease is a tumor;

preferably, the medicament is for: (a) inducing apoptosis of tumor cells; (b) inhibiting tumor cell proliferation; (c) inducing and/or increasing T cell infiltration; (d) inducing and/or enhancing an immune response; (e) increasing NK cell activity; (f) inhibiting TIGIT expression and activation; (g) inhibiting TIGIT-mediated signaling; (h) Induce and/or promote secretion of cytokines (e.g., IFN-gamma or IL-2); or any combination of (a) - (h);

preferably, the antibody or antigen-binding fragment thereof, multispecific antibody, nucleic acid molecule, vector, host cell, conjugate, or pharmaceutical composition is further administered separately, in combination, simultaneously or sequentially with another pharmaceutically active agent; further preferably, the additional pharmaceutically active agent is a drug having anti-tumor activity, such as a chemotherapeutic (e.g. platinum-based) or a small molecule inhibitor; and/or the additional pharmaceutically active agent is an immunotherapeutic-related agent, such as an immunodetection point inhibitor (e.g., PD-1 inhibitor, PD-L1 inhibitor, CTLA-4 inhibitor, or any combination thereof), an agent that enhances immunity (e.g., an interferon, an interleukin), or an immune system activator (e.g., TLR, ONDs, cpG, LPS, SEB, SEA, STING or SEC activator); and/or the additional pharmaceutically active agent is an oncolytic virus, an immune cell, or an engineered immune cell or an immune cell engineered by an engineered immune cell; and/or the additional pharmaceutically active agent is a radioactive material or a color-developing agent.

16. The use according to claim 15, wherein the tumor is selected from a solid tumor or a hematological tumor;

preferably, the solid tumor is selected from melanoma, lung cancer (e.g., non-small cell lung cancer), kidney cancer, bladder cancer, head and neck tumor (e.g., head and neck squamous carcinoma), stomach cancer, esophageal cancer, liver cancer, skin cancer (e.g., merck skin cancer), colon cancer, ureter cancer, or microsatellite highly unstable solid tumor; the hematological neoplasm is selected from lymphomas (e.g., hodgkin's lymphoma).

17. 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 1-6, or the multispecific antibody of claim 7, or the nucleic acid molecule of claim 8, or the vector of claim 9, or the host cell of claim 10, or the conjugate of claim 12, or the pharmaceutical composition of claim 13, under conditions that allow formation of an antibody-antigen immune complex, and detecting formation of the complex.

18. Use of an antibody or antigen binding fragment thereof according to any one of claims 1 to 6, or a multispecific antibody according to claim 7, or a nucleic acid molecule according to claim 8, or a vector according to claim 9, or a host cell according to claim 10, or a conjugate according to claim 12, or a pharmaceutical composition according to claim 13, in the manufacture of a diagnostic kit for the diagnosis or differential diagnosis of a TIGIT-related disease; preferably, the TIGIT-related disorder is a tumor (e.g., the tumor is selected from a solid tumor or a hematological tumor);

More preferably, the solid tumor is selected from melanoma, lung cancer (e.g., non-small cell lung cancer), kidney cancer, bladder cancer, head and neck tumor (e.g., head and neck squamous carcinoma), stomach cancer, esophageal cancer, liver cancer, skin cancer (e.g., merck skin cancer), colon cancer, ureter cancer, or a microsatellite highly unstable solid tumor; the hematological neoplasm is selected from lymphomas (e.g., hodgkin's lymphoma).