WO2020098734A1

WO2020098734A1 - Anti-tigit antibody and use thereof

Info

Publication number: WO2020098734A1
Application number: PCT/CN2019/118354
Authority: WO
Inventors: 肖亮; 周利; 刘登念; 孙见宇; 胡江江; 刘雪梅; 喻海旻; 郑云程; 李强; 薛彤彤
Original assignee: 安源医药科技（上海）有限公司; 四川科伦博泰生物医药股份有限公司
Priority date: 2018-11-16
Filing date: 2019-11-14
Publication date: 2020-05-22
Also published as: CN111196852A

Abstract

The present invention relates to the field of disease treatment and immunology. Specifically, the present invention relates to an anti-TIGIT antibody or an antigen-binding fragment thereof, a nucleic acid molecule for encoding same, and a method for preparing same. The anti-TIGIT antibody or the antigen-binding fragment thereof in the present invention has a high specificity and a high affinity with a TIGIT, and can efficiently block the binding of the TIGIT with a ligand thereof, and inhibit and/or block the intracellular signal transduction mediated by the ligand thereof bound with the TIGIT. Therefore, the present invention also relates to a pharmaceutical composition comprising the antibody or the antigen-binding fragment thereof, and the use thereof in preparing a drug for improving the immune cell activity, enhancing an immune response or preventing and/or treating tumors, infections or infectious diseases.

Description

Anti-TIGIT antibody and its use

Related application

This application requires the priority of the Chinese patent application CN201811366907.4 filed on November 16, 2018. The content of the priority application cited above is incorporated herein by reference.

Technical field

The present invention belongs to the field of therapeutic monoclonal antibodies. More specifically, the present invention relates to an antibody against T cell immunoreceptors (TIGIT) having Ig and ITIM domains; it also relates to the treatment of diseases (including but not Limited to use in cancer, infection, etc.).

Background technique

TIGIT (T-cell immunoreceptor containing Ig and ITIM domains) belongs to the immunoglobulin superfamily, also known as Wucam, Vstm3 or Vsig9, has an extracellular immunoglobulin domain, type I transmembrane domain and two Immune receptor tyrosine inhibition motif (ITIM). TIGIT is mainly distributed in regulatory T cells (Tregs), activated T cells and natural killer cells (NK), etc. It is a co-suppressive receptor protein, which can be combined with the positive proteins CD226 (Dnam-1) and APC on T cells The expressed ligands CD155 (Pvr or Necl-5) and CD112 (Pvrl-2 or Nectin2) constitute a costimulatory network. Among them, TIGIT competes with CD226 to bind CD155 and CD112, and TIGIT binds its ligand with a higher affinity than CD226. The connection between TIGIT and CD155 or CD112 is mediated by its cytoplasmic ITIM or ITT-like motif, recruiting phosphatase SHIP-1 to the tail of TIGIT to trigger inhibitory signaling. In addition, the ITIM domain is also responsible for the inhibitory ability of mouse TIGIT. TIGIT / CD155 combination can inhibit NK cell toxicity, weaken T cell response and anti-tumor response, while the binding effect of CD226 / CD155 is just the opposite.

TIGIT can "turn off" the body's immune response through various mechanisms. Initial research showed that TIGIT indirectly suppresses the immune response by triggering the CD155 cytoplasmic tail on dendritic cells (DC), thereby blocking DC maturation and inducing the production of the immunosuppressive cytokine IL-10. Further research shows that by targeting T cell antigen receptor (TCR) signaling pathway molecules, TIGIT directly blocks the activation, proliferation, and effector function of initial T cells (Tn), and can inhibit CD4 ⁺ T cell cell proliferation and inflammation Cytokine production. Recent studies have pointed out that TIGIT can enhance the stability of Tregs and its inhibitory function on the proliferation of T cells producing IFN-γ, and selectively inhibits helper T cells 1 (Th1) and helper T cells 17 ( Th17) response-this is the main response that drives inflammation of autoimmune tissues. In addition, by inhibiting the cytolytic activity of NK cells, TIGIT reduces the production of NK cell cytokine IFN-γ and reduces its cytotoxicity. Therefore, blocking TIGIT signal transduction has the effect of enhancing autoimmune response. Other relevant studies have also found that enhancing or activating the binding to Fcγ can mediate selective depletion of TIGIT ⁺ Tregs by antibody-dependent cytotoxicity (ADCC) or phagocytosis (ADCP) at the tumor site, and TIGIT blocking antibodies can increase CD8 ⁺ Cell proliferation of T cells, tumor infiltrating lymphocytes (CD8 ⁺ TILs) and cytokine-induced killer cells (CIK) and production of cytokines such as IFN-γ. In many clinical tumor environments, TIGIT is highly expressed on the surface of CD8 ⁺ T cells, Tregs, TILs or NK cells, including melanoma, non-small cell lung cancer, colon cancer, endometrial cancer, breast cancer and renal clear cell carcinoma, etc. A variety of cancers, indicating that anti-TIGIT antibodies have the potential to be used to treat a variety of tumors.

Multiple studies have shown that TIGIT is associated with various diseases such as cancer, autoimmunity, and infection, and is expected to become a specific target for the treatment of various diseases. Using Anti-TIGIT antibody to block CD155 melanoma CIK cells, the IFN-γ production was significantly increased and the positive cell killing rate was much higher than the control group (Baofu ZH et al., Cancer Immunol. Immunother., 65: 305-14 , 2016). Experimental allergic encephalomyelitis (EAE) series of experiments showed that the levels of encephalitis T cells and proinflammatory cytokines IL-6, IFN-γ and IL-17 in TIGIT-deficient mice were increased (Joller N Et al., J. Immunol., 186: 1338–1342, 2011), and TIGIT antibody blockade can lead to aggravation of disease caused by enhanced proinflammatory T cell responses (Levin SD et al., Eur. J. Immunol., 41: 902 –915,2011). In addition, during human immunodeficiency virus (HIV) or monkey immunodeficiency virus (SIV) infection, TIGIT is co-expressed with programmed death receptor-1 (PD-1) on effector CD8 ⁺ T cells, and TIGIT is associated with disease progression Increased expression (Chew GM et al., PLoS Pathog., 12: e1005349, 2016); and, TIGIT and PD-1 ligand (PD-L1) co-blocking antibody can restore chronic lymphocytic choriomeningitis virus (LCMV ) The production of depleted CD8 ⁺ T cell cytokines induced during infection (Johnston RJ et al., Cancer cell., 26: 923–937, 2014). Recent studies have shown that high-affinity Anti-TIGIT antibodies can reverse NK cell depletion in T cell-deficient mice, but cannot reverse T cell depletion in NK cell-deficient mice, indicating that NK cells can independently kill target cells, but T cells must Rely on NK cells to exert immune function (Zhang Q et al., Nat. Immunol., 2018Jun 18.doi: 10.1038 / s41590-018-0132-0.), Which is related to the ability of NK cells to express the specific immune protein FLT3LG and recruit SDCs (to stimulate The effect of DC cells) stimulated dormant T cells to play an anti-tumor effect is consistent (Barry KC et al., Nat. Med. 2018Jun 25.doi: 10.1038 / s41591-018-0085-8.). The reversal of TIGIT antibody on NK cell depletion in T-cell deficient mice can effectively inhibit the growth of tumors in mice, making it possess a nearly life-long strong anti-tumor immune memory. Because of the inability to reverse the T cell depletion in NK-deficient mice, the patient's PD -1 antibody produces a stronger response, leading to a long-term immune response and higher overall survival, which indicates that anti-TIGIT monoclonal antibodies have antibodies that can exert therapeutic effects alone or with T-cell immune effects (such as PD-1 antibodies ) Potential use of combined treatment of multiple related diseases.

Currently, several TIGIT antibodies are in clinical trials, including MTIG7192A of Genentech, 986207 of BMS, MK-7684 of MSD, COM-701 developed by Bayer and Compugen, and AB154 of Arcus Biosciences. However, there are no related drugs on the market.

Therefore, it is urgent and necessary to develop anti-TIGIT antibodies with higher specificity, lower toxic side effects, and better clinical efficacy, which will provide patients with cancer or infectious diseases with more choices of medication.

Summary of the invention

The present invention discloses antibody molecules that bind to TIGIT with high affinity and specificity. Also provided are nucleic acid molecules encoding antibody molecules, expression vectors, host cells, and methods for making antibody molecules. Also provided are multispecific antibodies or bispecific antibodies containing antibody molecules, pharmaceutical compositions, and immunogenic compositions. In addition, the application of the anti-TIGIT antibody molecule disclosed by the present invention (alone or in combination with other active agents or therapeutic methods) in the preparation of drugs for treating and / or preventing tumors, infections and infectious diseases is also provided.

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

(a) The following three CDRs of the heavy chain variable region (VH):

(i) CDR-H1, which has the sequence of CDR-H1 contained in VH as shown in SEQ ID NO: 1, or one or several amino acids compared to the sequence of CDR-H1 contained in the VH Sequences of substitutions, deletions or additions (for example, substitutions, deletions or additions of 1, 2 or 3 amino acids);

(ii) CDR-H2, which has the sequence of CDR-H2 contained in VH shown in SEQ ID NO: 1, or one or several amino acids compared to the sequence of CDR-H2 contained in the VH Sequences of substitutions, deletions or additions (for example, substitutions, deletions or additions of 1, 2 or 3 amino acids); and

(iii) CDR-H3, which has the sequence of CDR-H3 contained in VH shown in SEQ ID NO: 1, or one or several amino acids compared to the sequence of CDR-H3 contained in the VH Sequences of substitutions, deletions or additions (for example, substitutions, deletions or additions of 1, 2 or 3 amino acids);

and / or,

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

(iv) CDR-L1, which has the sequence of CDR-L1 contained in the VL shown in SEQ ID NO: 2, or one or several amino acids compared to the sequence of CDR-L1 contained in the VL Sequences of substitutions, deletions or additions (for example, substitutions, deletions or additions of 1, 2 or 3 amino acids);

(v) CDR-L2, which has the sequence of CDR-L2 contained in the VL shown in SEQ ID NO: 2, or one or several amino acids compared to the sequence of CDR-L2 contained in the VL Sequences of substitutions, deletions or additions (for example, substitutions, deletions or additions of 1, 2 or 3 amino acids); and

(vi) CDR-L3, which has the sequence of CDR-L3 contained in the VL shown in SEQ ID NO: 2, or one or several amino acids compared to the sequence of CDR-L3 contained in the VL Sequences of substitutions, deletions or additions (for example, substitutions, deletions or additions of 1, 2 or 3 amino acids).

In certain preferred embodiments, the substitutions described in any one of (i)-(vi) are conservative substitutions.

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

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

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

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

and / or

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

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

In certain preferred embodiments, the three CDRs contained in the heavy chain variable region (VH) and / or the three CDRs contained in the light chain variable region (VL) are composed of Kabat, Chothia or IMGT Numbering system definition.

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

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

(a) The following three heavy chain variable region (VH) CDRs:

(i) CDR-H1, which consists of the following sequence: SEQ ID NO: 5, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 5 (

eg

1, 2, or 3) Amino acid substitutions, deletions or additions),

(ii) CDR-H2, which consists of the following sequence: SEQ ID NO: 6, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 6 (

eg

1, 2, or 3) Amino acid substitutions, deletions or additions), and

(iii) CDR-H3, which consists of the following sequence: SEQ ID NO: 7, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 7 (

eg

1, 2, or 3) Amino acid substitutions, deletions or additions);

and / or,

(b) The following three light chain variable region (VL) CDRs:

(iv) CDR-L1, which consists of the following sequence: SEQ ID NO: 8, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 8 (

eg

1, 2, or 3) Amino acid substitutions, deletions or additions),

(v) CDR-L2, which consists of the following sequence: SEQ ID NO: 9, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 9 (

eg

1, 2, or 3) Amino acid substitutions, deletions or additions), and

(vi) CDR-L3, which consists of the following sequence: SEQ ID NO: 10, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 10 (

eg

1, 2, or 3) Amino acid substitutions, deletions or additions);

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

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

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

Among them, the three CDRs contained in the heavy chain variable region (VH) and the three CDRs contained in the light chain variable region (VL) are defined by the Chothia numbering system.

(a) The following three heavy chain variable region (VH) CDRs:

(i) CDR-H1, which consists of the following sequence: SEQ ID NO: 27, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 27 (

eg

1, 2, or 3) Amino acid substitutions, deletions or additions),

(ii) CDR-H2, which consists of the following sequence: SEQ ID NO: 28, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 28 (

eg

1, 2, or 3) Amino acid substitutions, deletions or additions), and

(iii) CDR-H3, which is composed of the following sequence: SEQ ID NO: 19, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 19 (

eg

1, 2, or 3) Amino acid substitutions, deletions or additions);

and / or,

(b) The following three light chain variable region (VL) CDRs:

(iv) CDR-L1, which consists of the following sequence: SEQ ID NO: 20, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 20 (

eg

1, 2, or 3) Amino acid substitutions, deletions or additions),

(v) CDR-L2, which consists of the following sequence: SEQ ID NO: 21, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 21 (

eg

1, 2, or 3) Amino acid substitutions, deletions or additions), and

eg

1, 2, or 3) Amino acid substitutions, deletions or additions);

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

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

Among them, the three CDRs contained in the heavy chain variable region (VH) and the three CDRs contained in the light chain variable region (VL) are defined by the Kabat numbering system.

(a) The following three heavy chain variable region (VH) CDRs:

(i) CDR-H1, which consists of the following sequence: SEQ ID NO: 17, or has one or several amino acid substitutions, deletions, or additions compared to SEQ ID NO: 17 (

eg

1, 2, or 3) Amino acid substitutions, deletions or additions),

(ii) CDR-H2, which consists of the following sequence: SEQ ID NO: 18, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 18 (

eg

1, 2, or 3) Amino acid substitutions, deletions or additions), and

eg

1, 2, or 3) Amino acid substitutions, deletions or additions);

and / or,

(b) The following three light chain variable region (VL) CDRs:

eg

1, 2, or 3) Amino acid substitutions, deletions or additions),

eg

1, 2, or 3) Amino acid substitutions, deletions or additions), and

eg

1, 2, or 3) Amino acid substitutions, deletions or additions);

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

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

In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the present invention further comprises a framework region (FR) derived from a mammalian (e.g., murine or human) immunoglobulin.

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

In certain preferred embodiments, the VH of the antibody or antigen-binding fragment of the invention comprises a heavy chain variable region (VH) framework region (FR) derived from human immunoglobulin, and / or the antibody or The VL of the antigen-binding fragment contains a light chain variable region (VL) framework region (FR) derived from human immunoglobulin. In such embodiments, the heavy chain variable region FR and / or light chain variable region FR of the antibody or antigen-binding fragment of the invention may contain one or more non-human (eg, murine) amino acid residues For example, the heavy chain framework region FR and / or the light chain framework region FR may contain one or more amino acid back mutations in which there are corresponding murine amino acid residues.

(a) The heavy chain framework region of human immunoglobulin or a variant thereof, said variant having a conservative substitution of at most 20 amino acids (eg, at most 15, at most 10, or at most 5 Conservative substitutions of 1 amino acid; for example, conservative substitutions of 1, 2, 3, 4 or 5 amino acids); and / or

(b) The light chain framework region of human immunoglobulin or a variant thereof, said variant having a conservative substitution of at most 20 amino acids (e.g. at most 15, at most 10, or at most 5 Conservative substitutions of 1 amino acid; for example, conservative substitutions of 1, 2, 3, 4 or 5 amino acids).

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

(a) Heavy chain variable region (VH), which comprises an amino acid sequence selected from the following:

(i) SEQ ID NOs: the sequence shown in any of 1, 31, 35, 39, 43;

(ii) One or several amino acid substitutions, deletions, or additions compared to the sequence shown in any of SEQ ID NOs: 1, 31, 35, 39, 43 (

eg

1, 2, 3, 4 Sequence of one or five amino acid substitutions, deletions or additions); or

(iii) The sequence shown in any one of SEQ ID NOs: 1, 31, 35, 39, 43 has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least A sequence of 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity;

and / or,

(b) Light chain variable region (VL), which contains an amino acid sequence selected from the following:

(iv) SEQ ID NOs: the sequence shown in any one of 2, 32, 36, 40, 44;

(v) One or several amino acid substitutions, deletions, or additions compared to the sequence shown in any of SEQ ID NOs: 2, 32, 36, 40, 44 (

eg

(vi) The sequence shown in any one of SEQ ID NOs: 2, 32, 36, 40, 44 has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least A sequence of 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

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

(i) The sequence shown in SEQ ID NO: 1;

(ii) One or several amino acid substitutions, deletions or additions compared to the sequence shown in SEQ ID NO: 1 (for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions ) Sequence; or

(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% with the sequence shown in SEQ ID NO: 1, A sequence of at least 97%, at least 98%, at least 99%, or 100% sequence identity;

with

(iv) The sequence shown in SEQ ID NO: 2;

(v) One or several amino acid substitutions, deletions or additions compared to the sequence shown in SEQ ID NO: 2 (

eg

1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions ) Sequence; or

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

(i) SEQ ID NO: the sequence shown in 31;

(ii) One or several amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO: 31 (for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions, or additions ) Sequence; or

(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% with the sequence shown in SEQ ID NO: A sequence of at least 97%, at least 98%, at least 99%, or 100% sequence identity;

with

(iv) SEQ ID NO: the sequence shown in 32;

(v) One or several amino acid substitutions, deletions or additions compared to the sequence shown in SEQ ID NO: 32 (

eg

1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions ) Sequence; or

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

(i) SEQ ID NO: the sequence shown in 35;

(ii) One or several amino acid substitutions, deletions or additions compared to the sequence shown in SEQ ID NO: 35 (for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions ) Sequence; or

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

with

(iv) SEQ ID NO: the sequence shown in 36;

(v) Compared with the sequence shown in SEQ ID NO: 36, it has one or several amino acid substitutions, deletions or additions (

eg

1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) ) Sequence; or

(vi) The sequence shown in SEQ ID NO: 36 has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, Sequences with at least 97%, at least 98%, at least 99%, or 100% sequence identity.

(i) SEQ ID NO: the sequence shown in 39;

(ii) One or several amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO: 39 (for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions, or additions ) Sequence; or

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

with

(iv) SEQ ID NO: the sequence shown in 40;

(v) One or several amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO: 40 (for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions, or additions ) Sequence; or

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

(i) SEQ ID NO: the sequence shown in 43;

(ii) One or several amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO: 43 (for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions, or additions ) Sequence; or

(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% with the sequence shown in SEQ ID NO: 43, A sequence of at least 97%, at least 98%, at least 99%, or 100% sequence identity;

with

(iv) SEQ ID NO: the sequence shown in 44;

(v) One or several amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO: 44 (for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions, or additions ) Sequence; or

(vi) The sequence shown in SEQ ID NO: 44 has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, Sequences with at least 97%, at least 98%, at least 99%, or 100% sequence identity.

(1) VH with the sequence shown in SEQ ID NO: 1 and VL with the sequence shown in SEQ ID NO: 2;

(2) VH with the sequence shown in SEQ ID NO: 31 and VL with the sequence shown in SEQ ID NO: 32;

(3) VH with the sequence shown in SEQ ID NO: 35 and VL with the sequence shown in SEQ ID NO: 36;

(4) VH having the sequence shown in SEQ ID NO: 39 and VL having the sequence shown in SEQ ID NO: 40; or

(5) VH having the sequence shown in SEQ ID NO: 43 and VL having the sequence shown in SEQ ID NO: 44.

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

(a) The following three CDRs of the heavy chain variable region (VH):

(i) CDR-H1, which has the sequence of CDR-H1 contained in VH shown in SEQ ID NO: 3, or one or several amino acids compared to the sequence of CDR-H1 contained in the VH Sequences of substitutions, deletions or additions (for example, substitutions, deletions or additions of 1, 2 or 3 amino acids);

(ii) CDR-H2, which has the sequence of CDR-H2 contained in the VH shown in SEQ ID NO: 3, or one or several amino acids compared to the sequence of CDR-H2 contained in the VH Sequences of substitutions, deletions or additions (for example, substitutions, deletions or additions of 1, 2 or 3 amino acids); and

(iii) CDR-H3, which has the sequence of CDR-H3 contained in the VH shown in SEQ ID NO: 3, or one or several amino acids compared to the sequence of CDR-H3 contained in the VH Sequences of substitutions, deletions or additions (for example, substitutions, deletions or additions of 1, 2 or 3 amino acids);

and / or,

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

(iv) CDR-L1, which has the sequence of CDR-L1 contained in the VL shown in SEQ ID NO: 4, or one or several amino acids compared to the sequence of CDR-L1 contained in the VL Sequences of substitutions, deletions or additions (for example, substitutions, deletions or additions of 1, 2 or 3 amino acids);

(v) CDR-L2, which has the sequence of CDR-L2 contained in the VL shown in SEQ ID NO: 4, or one or several amino acids compared to the sequence of CDR-L2 contained in the VL Sequences of substitutions, deletions or additions (for example, substitutions, deletions or additions of 1, 2 or 3 amino acids); and

(vi) CDR-L3, which has the sequence of CDR-L3 contained in the VL shown in SEQ ID NO: 4, or one or several amino acids compared to the sequence of CDR-L3 contained in the VL Sequences of substitutions, deletions or additions (for example, substitutions, deletions or additions of 1, 2 or 3 amino acids).

VH as shown in any of SEQ ID NO: 3, SEQ ID NO: 33, SEQ ID NO: 37 and SEQ ID NO: 41;

and / or,

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

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

(a) The following three CDRs of the heavy chain variable region (VH):

(i) CDR-H1, which consists of the following sequence: SEQ ID NO: 11, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 11 (

eg

1, 2, or 3) Amino acid substitutions, deletions or additions),

(ii) CDR-H2, which consists of the following sequence: SEQ ID NO: 12, or has one or several amino acid substitutions, deletions or additions compared to SEQ ID NO: 12 (

eg

1, 2, or 3) Amino acid substitutions, deletions or additions), and

(iii) CDR-H3, which consists of the following sequence: SEQ ID NO: 13, or has one or several amino acid substitutions, deletions or additions compared to SEQ ID NO: 13 (

eg

1, 2, or 3 Amino acid substitutions, deletions or additions);

and / or,

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

(iv) CDR-L1, which consists of the following sequence: SEQ ID NO: 14, or a substitution, deletion, or addition of one or a few amino acids compared to SEQ ID NO: 14 (

eg

1, 2, or 3) Amino acid substitutions, deletions or additions),

(v) CDR-L2, which consists of the following sequence: SEQ ID NO: 15, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 15 (

eg

1, 2, or 3) Amino acid substitutions, deletions or additions), and

(vi) CDR-L3, which consists of the following sequence: SEQ ID NO: 16, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 16 (

eg

1, 2, or 3) Amino acid substitutions, deletions or additions);

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

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

(a) The following three heavy chain variable region (VH) CDRs:

(i) CDR-H1, which is composed of the following sequence: SEQ ID NO: 29, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 29 (

eg

1, 2, or 3) Amino acid substitutions, deletions or additions),

(ii) CDR-H2, which consists of the following sequence: SEQ ID NO: 30, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 30 (

eg

1, 2, or 3) Amino acid substitutions, deletions or additions), and

(iii) CDR-H3, which consists of the following sequence: SEQ ID NO: 24, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 24 (

eg

1, 2, or 3) Amino acid substitutions, deletions or additions);

and / or,

(b) The following three light chain variable region (VL) CDRs:

(iv) CDR-L1, which consists of the following sequence: SEQ ID NO: 25, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 25 (

eg

1, 2, or 3) Amino acid substitutions, deletions or additions),

(v) CDR-L2, which consists of the following sequence: SEQ ID NO: 26, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 26 (

eg

1, 2, or 3) Amino acid substitutions, deletions or additions), and

eg

1, 2, or 3) Amino acid substitutions, deletions or additions);

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

(a) The following three heavy chain variable region (VH) CDRs:

(i) CDR-H1, which consists of the following sequence: SEQ ID NO: 22, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 22 (

eg

1, 2, or 3) Amino acid substitutions, deletions or additions),

(ii) CDR-H2, which consists of the following sequence: SEQ ID NO: 23, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 23 (

eg

1, 2, or 3) Amino acid substitutions, deletions or additions), and

eg

1, 2, or 3) Amino acid substitutions, deletions or additions);

and / or,

(b) The following three light chain variable region (VL) CDRs:

eg

1, 2, or 3) Amino acid substitutions, deletions or additions),

eg

1, 2, or 3) Amino acid substitutions, deletions or additions), and

eg

1, 2, or 3) Amino acid substitutions, deletions or additions);

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

In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the present invention further comprise framework regions (FR) derived from mammalian (eg, murine or human) immunoglobulins.

(i) SEQ ID NOs: the sequence shown in any of 3, 33, 37, 41;

(ii) One or several amino acid substitutions, deletions, or additions compared to the sequence shown in any of SEQ ID NOs: 3, 33, 37, 41 (for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions); or

(iii) The sequence shown in any one of SEQ ID NOs: 3, 33, 37, 41 has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% , At least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity;

and / or,

(iv) SEQ ID NOs: the sequence shown in any of 4, 34, 38, 42;

(v) One or several amino acid substitutions, deletions, or additions compared to the sequence shown in any of SEQ ID NOs: 4, 34, 38, 42 (

eg

1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions); or

(vi) The sequence shown in any one of SEQ ID NOs: 4, 34, 38, 42 has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% , At least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

(i) SEQ ID NO: 3 shows the sequence;

(ii) One or several amino acid substitutions, deletions or additions compared to the sequence shown in SEQ ID NO: 3 (for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions ) Sequence; or

(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% with the sequence shown in SEQ ID NO: 3, A sequence of at least 97%, at least 98%, at least 99%, or 100% sequence identity;

with

(iv) SEQ ID NO: 4 shows the sequence;

(v) One or several amino acid substitutions, deletions or additions compared to the sequence shown in SEQ ID NO: 4 (for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions ) Sequence; or

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

(i) SEQ ID NO: the sequence shown in 33;

(ii) One or several amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO: 33 (for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions, or additions ) Sequence; or

(iii) with the sequence shown in SEQ ID NO: 33 having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, A sequence of at least 97%, at least 98%, at least 99%, or 100% sequence identity;

with

(iv) SEQ ID NO: the sequence shown in 34;

(v) Compared with the sequence shown in SEQ ID NO: 34, it has one or several amino acid substitutions, deletions or additions (for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) ) Sequence; or

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

(i) SEQ ID NO: the sequence shown in 37;

(ii) One or several amino acid substitutions, deletions or additions compared to the sequence shown in SEQ ID NO: 37 (for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions ) Sequence; or

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

with

(iv) SEQ ID NO: the sequence shown in 38;

(v) Compared with the sequence shown in SEQ ID NO: 38, it has one or several amino acid substitutions, deletions or additions (for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) ) Sequence; or

(vi) the sequence shown in SEQ ID NO: 38 has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, Sequences with at least 97%, at least 98%, at least 99%, or 100% sequence identity.

(i) SEQ ID NO: the sequence shown in 41;

(ii) One or several amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO: 41 (eg, 1, 2, 3, 4 or 5 amino acid substitutions, deletions, or additions ) Sequence; or

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

with

(iv) SEQ ID NO: the sequence shown in 42;

(v) One or several amino acid substitutions, deletions or additions compared to the sequence shown in SEQ ID NO: 42 (

eg

1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions ) Sequence; or

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

(1) VH with the sequence shown in SEQ ID NO: 3 and VL with the sequence shown in SEQ ID NO: 4;

(2) VH with the sequence shown in SEQ ID NO: 33 and VL with the sequence shown in SEQ ID NO: 34;

(3) VH having the sequence shown in SEQ ID NO: 37 and VL having the sequence shown in SEQ ID NO: 38; or

(4) VH having the sequence shown in SEQ ID NO: 41 and VL having the sequence shown in SEQ ID NO: 42.

In any of the above aspects, the antibody or antigen-binding fragment thereof of the present invention may further comprise a constant region sequence derived from a mammalian (eg, murine or human) immunoglobulin or a variant thereof, which variant is derived from Of sequences have one or more substitutions, deletions or additions. In certain preferred embodiments, the variant has a conservative substitution of one or more amino acids compared to the sequence from which it was derived. In certain embodiments, the anti-TIGIT antibody molecule has a heavy chain constant region (Fc) selected from heavy chain constant regions of, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE; specifically selected The heavy chain constant regions from, for example, IgG1, IgG2, IgG3, and IgG4 are more particularly selected from the heavy chain constant regions of IgG1 or IgG4 (eg, human IgG1 or IgG4). In some embodiments, the anti-TIGIT antibody molecule has a light chain constant region selected from, for example, kappa or lambda light chain constant regions, preferably a kappa light chain constant region (eg, human kappa light chain).

In certain preferred embodiments, the heavy chain of the antibody or antigen-binding fragment thereof of the present invention comprises the heavy chain constant region (CH) of human immunoglobulin or a variant thereof compared to the sequence from which it is derived Substitution, deletion or addition of one or more amino acids (for example, substitution, deletion or addition of at most 20, at most 15, at most 10, or at most 5 amino acids; for example 1, 2, 3, 4 Substitutions, deletions or additions of 5 or 5 amino acids); and / or,

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

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

The light chain of the antibody or antigen-binding fragment thereof of the present invention comprises the light chain constant region (CL) of murine immunoglobulin or a variant thereof, which has a conservative substitution of up to 20 amino acids compared to the sequence from which it is derived (E.g. conservative substitutions of at most 15, at most 10, or at most 5 amino acids; for example, conservative substitutions of 1, 2, 3, 4 or 5 amino acids).

In some embodiments, the constant region is altered, eg, mutated, to modify the properties of the anti-TIGIT antibody molecule (eg, alter one or more of the following characteristics: Fc receptor binding, antibody glycosylation, cysteine Number of residues, effector cell function or complement function). Functional changes can be produced by replacing at least one amino acid residue in the antibody constant region with a different residue, for example, by changing the affinity of the antibody for an effector ligand (such as FcR or complement C1q), thereby changing the effector function (for example, enhancing , Reduce or eliminate). Methods for replacing amino acid residues in the Fc region of an antibody to change its effector function are known in the art (see, for example, EP388,151A1, US564,8260, US562,4821). The Fc region of antibodies mediates several important effector functions, such as ADCC, phagocytosis, and CDC. In some cases, these effector functions are needed for therapeutic antibodies; in other cases, these effector functions may be unnecessary or even harmful, depending on the intended purpose. Thus, in certain embodiments, the antibodies or antigen-binding fragments of the invention have reduced or even eliminated effector functions (eg, ADCC and / or CDC activity). In addition, it is also possible to introduce amino acid mutations stabilizing the antibody structure into human IgG4, for example, S228P (EU nomenclature, S241P in Kabat nomenclature).

In such embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises a variant of the constant region of a human IgG heavy chain, which variant has at least one of the following substitutions compared to the wild-type sequence from which it was derived: Ser228Pro , Leu234Ala, Leu235Ala, Gly237Ala, Asp265Ala, Asn297Ala, Pro329Ala, Asp356Glu and Leu358Met (the above-mentioned amino acid positions are based on the position of the EU numbering system, Edelman GM, etc., Proc Natl Acad USA, 63, 78-85 (1969) .PMID : 5257969).

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

In certain exemplary embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises a variant of the human IgG1 heavy chain constant region, which variant has the following substitutions compared to the wild-type sequence from which it was derived: Asn297Ala (according to Position of the EU numbering system). In such embodiments, the antibody or antigen-binding fragment of the invention has abolished ADCC activity.

In certain exemplary embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises a variant of the human IgG1 heavy chain constant region, which variant has the following substitutions compared to the wild-type sequence from which it is derived: Asp265Ala, Pro329Ala (According to the position of the EU numbering system). In such embodiments, the antibody or antigen-binding fragment of the invention has abolished ADCC activity.

In certain exemplary embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises a variant of a human IgG4 heavy chain constant region, which variant has the following substitutions compared to the wild-type sequence from which it was derived: Ser228Pro (based on Position of the EU numbering system). In such an embodiment, the antibody or antigen-binding fragment of the present invention is structurally stable and can reduce Fab-arm exchange, making it difficult to form a half-antibody.

In certain preferred embodiments, the heavy chain of the antibody or antigen-binding fragment thereof of the present invention comprises a variant of the heavy chain constant region (CH) of human immunoglobulin, which variant and the wild-type sequence from which it is derived Compared with the effector function is basically unchanged. In such embodiments, the variant may have a conservative substitution of at most 20 amino acids (e.g., at most 15, at most 10, or at most 5 amino acids, compared to the wild-type sequence from which it was derived; for example (1, 2, 3, 4 or 5 amino acid conservative substitutions).

(a) A heavy chain comprising an amino acid sequence selected from the following:

(i) SEQ ID NO: the sequence shown in 63;

(ii) One or several amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO: 63 (for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions, or additions ) Sequence; or

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

with

(b) A light chain comprising an amino acid sequence selected from the following:

(iv) SEQ ID NO: the sequence shown in 64;

(v) Compared with the sequence shown in SEQ ID NO: 64, it has one or several amino acid substitutions, deletions or additions (

eg

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

(i) SEQ ID NO: the sequence shown in 65;

(ii) One or several amino acid substitutions, deletions or additions compared to the sequence shown in SEQ ID NO: 65 (

eg

1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions ) Sequence; or

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

with

(iv) SEQ ID NO: the sequence shown in 66;

(v) Compared with the sequence shown in SEQ ID NO: 66, it has one or several amino acid substitutions, deletions or additions (for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) ) Sequence; or

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

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

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

(2) A heavy chain having the sequence shown in SEQ ID NO: 65 and a light chain having the sequence shown in SEQ ID NO: 66.

The anti-TIGIT antibody or antigen-binding fragment disclosed herein can inhibit, reduce, or neutralize one or more activities of TIGIT, for example, result in the blocking or reduction of immune checkpoints on T cells or NK cells, or The immune response is reactivated by adjusting antigen presenting cells.

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

(a) Combined with TIGIT (especially human TIGIT) with K _{D of} 100 nM or less, preferably ≤10 nM, ≤1 nM, or ≤100 pM, such as through biological thin film interference technology (BLI) (eg, ForteBio

) Measured; (b) induce and / or enhance immune response; (c) increase effector T cell activity; (d) increase cytotoxic T lymphocyte (CTL) activity; (e) increase NK cell activity; (f) Inhibit TIGIT activation; (g) inhibit TIGIT-mediated signaling; (h) inhibit or block the binding of CD155 and / or CD112 to TIGIT; (i) reduce the number of regulatory T cells in tissues or circulation; (j) inhibit The suppression of effector T cells by regulatory T cells.

In certain preferred embodiments, the antibodies or antigen-binding fragments of the invention possess any combination of the above biological functions.

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

Hybridoma cell line # 7, which was deposited on October 24, 2018 at the China Type Culture Collection (CCTCC), and has the deposit number CCTCC NO.C2018210.

Hybridoma cell line # 21, which was deposited on October 24, 2018 in the Chinese Type Culture Collection (CCTCC), and has the deposit number CCTCC NO.C2018209.

In certain preferred embodiments, the antibodies of the invention are chimeric antibodies or humanized antibodies. In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the present invention is selected from ScFv, Fab, Fab ', (Fab') ₂ , Fv fragment, disulfide-linked Fv (dsFv), diabody ), Bispecific antibodies, and multispecific antibodies.

In another aspect of the invention, there is provided an isolated nucleic acid molecule comprising an antibody encoding the invention or an antigen-binding fragment thereof, its heavy chain and / or light chain, or its heavy chain variable region and / or light chain The nucleotide sequence of the variable region. In certain embodiments, the nucleotide sequence is codon optimized. In certain preferred embodiments, the isolated nucleic acid molecule comprises first and second nucleic acids encoding the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment thereof of the invention, respectively. From any one of the group: Mab21, AB12V3, AB12V5, AB12V7 and AB12V9; or a sequence substantially the same as the first and second nucleic acids. In certain preferred embodiments, the isolated nucleic acid molecule comprises first and second nucleic acids encoding the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment thereof of the invention, respectively. From any one of the group: Mab7, AB12V4, AB12V6, AB12V8; or a sequence substantially the same as the first and second nucleic acids. For example, the isolated nucleic acid molecule may comprise the AB12V8 and AB12V9 nucleotide sequences shown in the sequence listing or a sequence substantially identical thereto (eg, having at least about 85%, 90%, 95%, 99% compared to it Or a sequence with a higher sequence identity or a sequence with one or more nucleotide substitutions, or a sequence no more than 3, 6, 15, 30, or 45 nucleotides from the sequence shown in the sequence listing) .

In certain preferred embodiments, the present invention provides an isolated nucleic acid molecule comprising a nucleic acid molecule encoding an antibody heavy chain variable region, and / or a nucleic acid molecule encoding an antibody light chain variable region, wherein, The nucleic acid molecule encoding the variable region of the antibody heavy chain has a sequence selected from the following: (a) the nucleotide sequence shown in SEQ ID NO: 49 or 51, or the nucleus described in (b) and (a) A sequence of substantially the same nucleotide sequence (for example, a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to the nucleotide sequence described in (a), or having A sequence of one or more nucleotide substitutions), or (c) the sequence of nucleotides described in (a) does not differ by more than 3, 6, 15, 30, or 45 nucleotides; the encoding The nucleic acid molecule of the variable region of the antibody light chain has a sequence selected from the following: (a) the nucleotide sequence shown in SEQ ID NO: 50 or 52, or the nucleotide sequences described in (b) and (a) Substantially the same sequence (for example, a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to the nucleotide sequence described in (a), or having one or more A sequence of multiple nucleotide substitutions), or a sequence of (c) no more than 3, 6, 15, 30, or 45 nucleotides from the nucleotide sequence described in (a).

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

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

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

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

In certain embodiments, the antibodies or antigen-binding fragments of the invention can be used to construct chimeric antigen receptors. In such embodiments, the isolated nucleic acid molecule contained in the vector of the invention may comprise a nucleotide sequence encoding a chimeric antigen receptor, the nucleotide sequence encoding the chimeric antigen receptor further comprising the invention Nucleotide sequence of antibodies or antigen-binding fragments (eg ScFv). In certain embodiments, the isolated nucleic acid molecule contained in the vector of the present invention encodes a chimeric antigen receptor comprising an antigen-binding fragment (eg, ScFv) of the antibody of the present invention.

In another aspect of the invention, the invention provides a host cell comprising the isolated nucleic acid molecule of the invention or the vector of the invention. The host cell may be a eukaryotic cell (eg mammalian cell, insect cell, yeast cell) or a prokaryotic cell (eg E. coli). Suitable eukaryotic cells include, but are not limited to, NSO cells, Vero cells, Hela cells, COS cells, CHO cells, HEK293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to Sf9 cells. In certain preferred embodiments, the host cell of the invention is a mammalian cell, such as CHO (eg CHO-K1, CHO-S, CHO DXB11, CHO DG44).

In certain embodiments, the host cell of the invention may be a chimeric antigen receptor T cell (CAR-T). In such embodiments, the isolated nucleic acid molecule contained in the host cell may comprise a nucleotide sequence encoding a chimeric antigen receptor, the nucleotide sequence encoding the chimeric antigen receptor further comprising the invention Nucleotide sequence of antibodies or antigen-binding fragments (eg ScFv). In certain embodiments, the isolated nucleic acid molecule contained in the host cell encodes a chimeric antigen receptor comprising an antigen-binding fragment (eg, ScFv) of the antibody of the invention.

In another aspect of the present invention, there is provided a method of preparing the antibody or antigen-binding fragment thereof of the present invention, which comprises culturing the host cell of the present invention under conditions permitting expression of the antibody or antigen-binding fragment thereof, and The antibody or antigen-binding fragment thereof is recovered from the host cell culture.

In another aspect of the present invention, a pharmaceutical composition is disclosed, which comprises the antibody or antigen-binding fragment of the present invention, a carrier or a host cell, and a pharmaceutically acceptable carrier and / or excipient.

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

In certain preferred embodiments, the pharmaceutical composition of the invention comprises the vector or host cell of the invention, and a pharmaceutically acceptable carrier and / or excipient. In such embodiments, the isolated nucleic acid molecule contained in the vector comprises a nucleotide sequence encoding a chimeric antigen receptor, and the nucleotide sequence encoding the chimeric antigen receptor further comprises an antibody encoding the invention Or the nucleotide sequence of its antigen-binding fragment (eg ScFv); the host cell contains the isolated nucleic acid molecule or vector as described above. In certain preferred embodiments, the isolated nucleic acid molecule encodes a chimeric antigen receptor comprising an antigen-binding fragment (eg, ScFv) of the antibody of the invention. In certain preferred embodiments, the host cell is a T cell. In certain preferred embodiments, the host cell is a chimeric antigen receptor T cell (CAR-T).

In certain preferred embodiments, the pharmaceutical composition may further comprise additional pharmaceutically active agents. In certain preferred embodiments, the additional pharmaceutically active agent is a drug used to treat immune-related diseases. In certain preferred embodiments, the additional pharmaceutically active agent is a drug with anti-tumor activity. In certain preferred embodiments, the additional pharmaceutically active agent is a drug used to treat an infection or infectious disease.

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

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

(a) induce and / or enhance immune response; (b) increase effector T cell activity; (c) increase cytotoxic T lymphocyte (CTL) activity; (d) increase NK cell activity; (e) inhibit TIGIT activation; f) inhibit TIGIT-mediated signaling; (g) inhibit or block the binding of CD155 and / or CD112 to TIGIT; (h) reduce the number of regulatory T cells in tissues or circulation; (i) inhibit the regulatory T cell pair Inhibition of effector T cells; or any combination of (j) (a)-(i).

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

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

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

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

(1) Improving the activity of immune cells in vitro or in a subject (such as a human);

(2) Enhance immune response in subjects (such as humans);

(3) Treatment of tumors in subjects (eg humans); or

(4) Treatment of infections or infectious diseases in subjects (eg humans).

In certain preferred embodiments, when the vector or host cell of the present invention is used to prepare a drug, the isolated nucleic acid molecule contained in the vector includes a nucleotide sequence encoding a chimeric antigen receptor, and the The nucleotide sequence of the antigen-receptor further includes a nucleotide sequence encoding the antibody of the present invention or an antigen-binding fragment thereof (eg, ScFv); the host cell contains the isolated nucleic acid molecule or vector as described above. In certain preferred embodiments, the isolated nucleic acid molecule encodes a chimeric antigen receptor comprising an antigen-binding fragment (eg, ScFv) of the antibody of the invention. In certain preferred embodiments, the host cell is a T cell. In certain preferred embodiments, the host cell is a chimeric antigen receptor T cell (CAR-T).

In certain preferred embodiments, when the vector or host cell of the present invention is used to prepare a drug, the drug is used to treat a tumor in a subject (eg, a human).

In certain preferred embodiments, the tumors involved in the antibodies or antigen-binding fragments, vectors, or host cells of the present invention are selected from solid tumors, hematological tumors (eg, leukemia, lymphoma, myeloma, eg, multiple Myeloma) and metastatic, refractory, or recurrent lesions of cancer; for example, including but not limited to esophageal cancer, gastrointestinal cancer, pancreatic cancer, thyroid cancer, colorectal cancer, renal cancer, lung cancer (such as non-small cell lung cancer) ), Liver cancer, stomach cancer, head and neck cancer, bladder cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, germ cell cancer, bone cancer, skin cancer, thymus cancer, cholangiocarcinoma, melanoma, Metastatic, refractory, or recurrent lesions of mesothelioma, lymphoma, myeloma, sarcoma, glioblastoma, glioblastoma, leukemia, or cancer.

In certain preferred embodiments, the infections or infectious diseases involved in the antibodies or antigen-binding fragments of the present invention are selected from viral infections, bacterial infections, fungal infections and parasitic infections, including but not limited to HIV, hepatitis virus , Herpes virus, CMV, EBV, flu or tetanus.

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

In certain preferred embodiments, the antibodies or antigen-binding fragments of the invention are used as adjuvants.

In certain preferred embodiments, the immunogen is selected from tumor cells, tumor-associated antigens (such as proteins, polypeptides, or carbohydrate molecules), dendritic cells sensitized by the antigens, and any combination thereof .

In certain preferred embodiments, the immunogen is selected from inactivated or attenuated pathogens, antigens (eg proteins, polypeptides or carbohydrate molecules) associated with the pathogens (eg viruses), sensitized by the antigens Dendritic cells, and any combination thereof.

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

In certain preferred embodiments, in the immunogenic composition, the antibody or antigen-binding fragment of the invention and the immunogen are provided as separate components or as components of the same composition. Therefore, the antibody or antigen-binding fragment of the present invention and the immunogen can be administered simultaneously, separately, or sequentially.

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

In certain preferred embodiments, the immunogen is selected from tumor-associated antigens (eg, proteins, polypeptides, or carbohydrate molecules), tumor cells, dendritic cells sensitized by the antigen, and any combination thereof . In such embodiments, the immunogenic composition is used to prevent and / or treat tumors, delay tumor progression, or reduce or inhibit tumor recurrence in a subject.

In certain preferred embodiments, the tumors involved in the antibodies or antigen-binding fragments thereof of the present invention are selected from solid tumors, hematological tumors (eg, leukemia, lymphoma, myeloma, eg, multiple myeloma), and cancer Metastatic, refractory, or recurrent lesions; for example, including but not limited to esophageal cancer, gastrointestinal cancer, pancreatic cancer, thyroid cancer, colorectal cancer, renal cancer, lung cancer (such as non-small cell lung cancer), liver cancer, gastric cancer , Head and neck cancer, bladder cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, germ cell cancer, bone cancer, skin cancer, thymus cancer, cholangiocarcinoma, melanoma, mesothelioma, lymph Metastatic, refractory, or recurrent lesions of tumors, myeloma, sarcoma, glioblastoma, glioblastoma, leukemia, or cancer.

In certain preferred embodiments, the immunogen is selected from antigens (eg proteins, polypeptides or carbohydrate molecules) associated with pathogens (eg viruses), inactivated or attenuated pathogens, sensitized by the antigens Dendritic cells, and any combination thereof. In such embodiments, the immunogenic composition is used to prevent and / or treat infections or infectious diseases in a subject. In such embodiments, the immunogenic composition is used to delay the progression of infection or infectious disease, or reduce or suppress the recurrence of infection or infectious disease in a subject.

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

In certain preferred embodiments, in the immunogenic composition, the antibody or antigen-binding fragment of the present invention and the immunogen are provided as separate components or as components of the same composition. Therefore, the antibody or antigen-binding fragment of the present invention and the immunogen can be administered simultaneously, separately, or sequentially.

In another aspect, the invention relates to the use of said antibody or antigen-binding fragment or pharmaceutical composition or immunogenic composition thereof in the preparation of a medicament for the treatment of immune-related diseases, wherein the immune-related diseases involve T cell dysfunction and / Or NK cell dysfunction. In certain preferred embodiments, the T cell dysfunction includes, for example, T cell incapacitation, depletion, or reduced cytokine secretion; and / or, the NK cell dysfunction includes, for example, NK cell incapacitation, depletion, or reduced cytokine secretion .

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

In certain preferred embodiments, any suitable indicator can be used to measure the activity of immune cells. Non-limiting examples of such suitable indicators include: cytokines (eg, IL-2, IFN-γ, etc.) of immune cells (eg, T cells) in the presence of the antibody or antigen-binding fragment thereof of the present invention Secretory levels, proliferation activity, and / or activation marker (eg, CD25, CD69, etc.) expression levels are increased.

In certain preferred embodiments, the method is used to treat tumors. In such embodiments, the immune cells obtained by the above method can be adoptively transferred into the subject to treat the tumor. In vitro activation in the presence of the antibody or antigen-binding fragment of the present invention can be expected to increase the activity of adoptively transferred immune cells, thereby facilitating the tumor killing effect of these adoptively transferred immune cells in the subject. In certain preferred embodiments, the immune cells are tumor infiltrating lymphocytes.

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

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

In certain embodiments, the method may further include administering to the subject a second therapy selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nano Therapy, viral therapy, adjuvant therapy and any combination thereof.

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

In certain preferred embodiments, the method is used to prevent and / or treat tumors. In certain preferred embodiments, the method is used to delay tumor progression. In certain preferred embodiments, the method is used to reduce or inhibit tumor recurrence.

In certain preferred embodiments, the method is used to prevent and / or treat infections or infectious diseases. In certain preferred embodiments, the method is used to delay the progression of infection or infectious disease. In certain preferred embodiments, the method is used to reduce or suppress the recurrence of infection or infectious disease.

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

In certain preferred embodiments, the method is used to prevent and / or treat infections or infectious diseases. In certain preferred embodiments, the method is used to delay the progression of infection or infectious disease. In certain preferred embodiments, the method is used to reduce or inhibit infection or recurrence of infectious diseases.

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

When the host cell of the present invention is used in the method described above, the host cell expresses a chimeric antigen receptor containing an antigen-binding fragment (eg, ScFv) of the antibody of the present invention. Therefore, in certain preferred embodiments, the isolated nucleic acid molecule contained in the host cell comprises a nucleotide sequence encoding a chimeric antigen receptor, and the nucleotide sequence encoding the chimeric antigen receptor further comprises The nucleotide sequence encoding the antibody of the present invention or an antigen-binding fragment thereof (for example, ScFv). In certain preferred embodiments, the isolated nucleic acid molecule encodes a chimeric antigen receptor comprising an antigen-binding fragment (eg, ScFv) of the antibody of the invention. In certain preferred embodiments, the host cell is a T cell. In certain preferred embodiments, the host cell is a chimeric antigen receptor T cell (CAR-T).

In another aspect, the method described above further includes administering to the subject a second therapy selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, Viral therapy, adjuvant therapy and any combination thereof.

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

In another aspect, the present invention provides a method for detecting the presence or level of TIGIT in a sample, the method comprising forming a complex between the antibody or antigen-binding fragment thereof and TIGIT, so that the sample and the present invention The antibody or antigen-binding fragment thereof is contacted, and the formation of the complex is detected.

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

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

The antibody of the present invention has high binding affinity with TIGIT, and has extremely strong specificity, and can enhance the activity of immune cells in vitro / in vivo and stimulate the immune response. Therefore, the antibodies of the present invention have the potential for preventing and / or treating tumors, immune-related diseases, infections or infectious diseases. The humanized antibody of the present invention retains the functions and properties of the parent mouse antibody. The in vivo anti-tumor research data shows that the humanized antibody provided by the present invention can significantly inhibit the growth of transplanted tumors in mice, and even some mice The tumor completely disappeared. Moreover, the humanized antibody of the present invention has a high degree of humanization, so that it can be safely administered to human subjects without inducing an immunogenic reaction. Therefore, the antibodies of the present invention (particularly humanized antibodies) have great clinical value.

Detailed description of the invention

Abbreviations and definitions

TIGIT T cell immune receptors containing Ig and ITIM domains

CDR Complementarity determining region in immunoglobulin variable region

FR antibody framework region: amino acid residues other than CDR residues in the variable region of the antibody

VH antibody heavy chain variable region

VL antibody light chain variable region

IgG immunoglobulin G

Kabat The immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, for example, Kabat et al.,

Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service,

National Institutes of Health, Bethesda, Md., 1991).

Chothia, an immunoglobulin numbering system proposed by Chothia et al., Is based on the identification of the location of structural loop regions

Classic rules for the boundaries of CDR regions (see, for example, Chothia & Lesk (1987) J. Mol. Biol.

196: 901-917; Chothia et al. (1989) Nature 342: 878-883).

IMGT is based on the International Immunogenetics Information System (The International) initiated by Lefranc et al.

ImMunoGeneTics information

(IMGT)), please refer to Lefranc et

"Al., Dev. Comparat. Immunol. 27: 55-77, 2003."

mAb monoclonal antibody

EC ₅₀ concentration that produces 50% efficacy or binding

IC ₅₀ concentration that produces 50% inhibition

ELISA Enzyme-linked immunosorbent assay

PCR polymerase chain reaction

HRP horseradish peroxidase

IL-2 Interleukin 2

IFN interferon

K _D equilibrium dissociation constant

K _a binding rate constant

K ^d dissociation rate constant

In the present invention, unless otherwise stated, the scientific and technical terms used herein have the meaning commonly understood by those skilled in the art. In addition, the cell culture, biochemistry, nucleic acid chemistry, immunology laboratory and other procedures used in this article are all routine procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

The term "TIGIT" includes human TIGIT, cynomolgus monkey TIGIT, rat TIGIT, mouse TIGIT, and fragments thereof (such as mature fragments which lack a signal peptide). In one embodiment of the invention, the amino acid sequence of human TIGIT comprises the amino acid sequence disclosed in amino acid residues 25-244 of Genbank accession number NP_776160.2. In some embodiments, the antibody molecules provided by the present invention specifically bind to epitopes (eg, linear or conformational epitopes) on TIGIT of mammals (eg, humans). In some embodiments, the binding epitope is at least part of the IgV domain of human TIGIT.

The term "antibody" is used in the broadest sense and generally refers to any immunobinder or molecule that contains an antigen-binding domain, including intact antibodies (eg, IgG1 or IgG3), monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies , Multispecific antibodies (eg, bispecific antibodies), chimeric antibodies, various functional fragments (eg, may only include antigen-binding portions, such as Fab, Fab ', F (ab') ₂ , Fd, Fd ', Fv or ScFv fragments) and variants thereof (eg, humanization, glycosylation, etc.), as long as they exhibit the desired biological activity. The invention also includes anti-TIGIT antibodies with glycosylation modifications. In some applications, modifications are made to remove undesired glycosylation sites, such as defucose modification on the oligosaccharide chain to enhance antibody-dependent cytotoxicity (ADCC) function; in other applications, galactose can be performed Modification to alter complement-dependent cytotoxicity (CDC) effects.

Generally, naturally occurring immunoglobulins have heavy and light chains connected to each other by disulfide bonds. Based on the amino acid sequence of the constant region and the amino acid sequence of the backbone region of the variable region, the heavy chain can be classified as μ, δ, γ, α, or ε, and the antibody isotypes are defined as IgM, IgD, IgG, IgA and IgE, "light chain" is divided into two strict types: kappa (κ) and lambda (λ). IgG isotypes are divided into subclasses of certain species: IgG1, IgG2, IgG3 and IgG4 in humans and IgG1, IgG2a, IgG2b and IgG3 in mice. Immunoglobulins such as human IgG1 exist in several allotypes, which differ from each other in up to several amino acids. Unless otherwise indicated, the immunoglobulin in the present invention may be from any known antibody class.

Each heavy and light chain contains constant and variable regions (the regions are also referred to as "domains"). "V _H " or "VH" refers to the variable region of the immunoglobulin heavy chain, including the variable region of Fv, ScFv, dsFv or Fab; "V _L " or "VL" refers to the immunoglobulin light chain Variable regions, including Fv, ScFv, dsFv or Fab variable regions. The light and heavy chain variable regions contain "framework regions" separated by three hypervariable regions (also called "complementarity determining regions" or "CDRs"). The "framework region (FR)" is a region other than CDRs in the variable regions of the heavy and light chains, and the amino acid composition and arrangement are relatively difficult to change. CDR is mainly responsible for binding to epitopes. The CDRs of each chain are generally called CDR-H1, CDR-H2, CDR-H3, and are numbered sequentially from the N-terminus, and are generally identified by the chain where the specific CDR is located. Antibodies with different specificities (ie, different binding sites for different antigens) have different CDRs. Although CDRs differ from antibody to antibody, only a limited number of amino acid positions of the CDR are directly involved in antigen binding.

The variable regions of the heavy and light chains of antibodies contain binding domains that interact with the antigen. Although antibodies show binding specificity for specific antigens, immunoglobulins include antibodies and other antibody-like molecules that lack antigen specificity. The constant region of the antibody may mediate the binding of immunoglobulins to host tissues or factors, including the binding of various cells of the immune system (eg, effector cells) and the first component of the classical complement system (eg, Clq). The Fc region of naturally occurring antibodies binds to elements of the complement system, and also to receptors on effector cells, including, for example, effector cells that mediate cytotoxicity (including specific ADCP). It is known in the art that the affinity and / or other binding properties of the Fc region for Fc receptors can be adjusted by glycosylation or other modified forms.

Humanized antibodies or antibody fragments can be produced naturally or chemically synthesized in whole or in part. The antibody can therefore be derived from any suitable source, such as recombinant sources and / or transgenic animal or plant production, or produced in eggs using IgY technology. Therefore, antibody molecules can be produced in vivo or in vitro.

The term "hypervariable region" or "CDR region" or "complementarity determining region" refers to antibody amino acid residues responsible for antigen binding. The CDR region sequence can be defined by the IMGT, Kabat, Chothia, and AbM methods or the amino acid residues in the variable region identified by any CDR region sequence determination method well known in the art. Antibody CDRs can be identified as hypervariable regions originally defined by Kabat et al., For example, residues and weights at positions 24-34 (L1), 50-56 (L2), and 89-97 (L3) of the light chain variable domain 31-35 (H1), 50-65 (H2) and 95-102 (H3) residues of the chain variable domain, see Kabat EA et al., 1991, Sequences of Proteins of Immunological Interest (protein sequence of immunological target) ), 5th edition, Public Health, National Institutes of Health, Bethesda, Md .; the position of the CDR can also be identified as initially defined by the "hypervariable loop" (HVL) structure described by Chothia et al. IMGT (ImMunoGeneTics) also provides a numbering system for immunoglobulin variable regions that includes CDRs. The CDR regions are defined according to IMGT numbering, for example, 27-32 (L1), 50-52 (L2) of light chain variable domains and Residues 89-97 (L3) and residues 26-35 (H1), 51-57 (H2) and 93-102 (H3) of the heavy chain variable domain, see Dev.Comp such as Lefranc MP, etc. . Immunol., 2003, 27: 55-77, which is incorporated herein by reference. Other methods for CDR identification include "AbM definition", which is a compromise between Kabat and Chothia and is obtained using Oxford's Molecular's AbM antibody model software; or CDR's "contact definition", which is based on the observed antigen contact and Elaborated in MacCallum RM et al., 1996, J. Mol Biol., 262: 732-745. In the "configuration definition" method of the CDR, the position of the CDR can be identified as a residue that contributes to antigen binding, see, for example, Makbe K et al., 2008, J. Biol Chem., 283: 1156-1166. The CDR contained in the antibody or antigen-binding fragment of the present invention can be determined according to various numbering systems known in the art. In certain embodiments, the CDRs contained in the antibody or antigen-binding fragment of the invention are preferably determined by the Kabat, Chothia, or IMGT numbering system.

The term "epitope" or "antigenic determinant" refers to a specific chemical group or peptide sequence on a molecule that has antigenicity (that is, to induce a specific immune response), and is an antigen to which immunoglobulins or antibodies specifically bind (eg TIGIT ). An immunoglobulin or antibody specifically binds to a specific epitope on a polypeptide (eg, TIGIT). The OK epitope determining region is usually composed of chemically active surface groups of molecules (such as amino acids or sugar side chains) and usually has specific three-dimensional structural properties and specific charge properties.

The term "Fab fragment" consists of a light chain and a heavy chain CH1 and variable regions. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. "Fab 'fragment" contains a heavy chain and a light chain VH domain and CH1 domain and the constant region part between CH1 and CH2 domain, which can be between two heavy chains of two Fab' fragments Interchain disulfide bonds are formed to form F (ab ') ₂ molecules. The "F (ab ') ₂ fragment" contains two heavy chains and two light chains, the VH domain and the CH1 domain, and the constant region portion between the CH1 and CH2 domains, thereby forming a chain between the two heavy chains Disulfide bond. Therefore, the F (ab ') ₂ fragment consists of two Fab' fragments held together by disulfide bonds between the two heavy chains. The "Fv region" contains variable regions from both heavy and light chains, but lacks constant regions. "Fd fragment" is composed of CH1 and variable region of a heavy chain, and is the heavy chain part remaining after the light chain is removed from the Fab fragment. "Disulfide bond stable protein (dsFv)" introduces a cysteine mutation point in the VH and VL regions, respectively, thereby forming a disulfide bond between VH and VL to achieve structural stability.

The term "Fc region" or "Fc" refers to the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the hinge region, the CH2 domain and the CH3 domain, which mediate the binding of the immunoglobulin to host tissues or factors , Including binding to Fc receptors located on various cells of the immune system (eg, effector cells) or binding to the first component of the classical complement system (eg, Clq), including native sequence Fc regions and variant Fc regions. Generally, the Fc region of the human IgG heavy chain is the segment from the amino acid residue at the position of Cys226 or Pro230 to the carboxyl terminal, but the boundary may vary. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may or may not be present. Fc can also refer to this isolated region, or in the case of Fc-containing protein polypeptides, such as "binding proteins containing Fc regions", also known as "Fc fusion proteins" (eg, antibodies or immunoadhesins). The natural sequence Fc region of the antibody of the present invention includes human IgG1, IgG2 (IgG2A, IgG2B), IgG3 and IgG4. In IgG, IgA and IgD antibody isotypes, the Fc region contains the CH2 and CH3 constant domains of each of the two heavy chains of the antibody; the IgM and IgE Fc regions contain the three heavy chain constants in each polypeptide chain Domain (CH domain 2-4).

The term "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an immunoglobulin. The FcR may be a natural sequence human FcR, or it may be an FcR (gamma receptor) that binds an IgG antibody, as well as allelic variants and alternative splicing forms of these receptors. The FcyR family consists of three activated receptors (FcyRI, FcyRIII and FcyRIV in mice; FcyRIA, FcyRIIA and FcyRIIIA in humans) and an inhibitory receptor (FcyRIIb or equivalent FcyRIIB). FcyRII receptors include FcyRIIA ("activated receptor") and FcyRIIB ("inhibited receptor"), which have similar amino acid sequences. The cytoplasmic domain of FcyRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM). The cytoplasmic domain of FcyRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) (see M. Annu. Rev. Immunol. 15: 203-234, 1997). Most natural effector cell types co-express one or more activated FcγR and inhibitory FcγRIIb, while NK cells selectively express an activated Fc receptor (FcγRIII in mice and FcγRIIIA in humans), but mice and The inhibitory FcγRIIb is not expressed in humans. Human IgG1 binds to most human Fc receptors and is considered equivalent to murine IgG2a in terms of the type of activated Fc receptors it binds. The term "FcR" encompasses other FcRs herein, including those that will be identified in the future. Methods of measuring binding to FcRn are known (see, for example, Ghetie V et al., Immunol Today 18: 592-8, 1997; Ghetie V et al., Nature Biotechnology, 15: 637-40, 1997). The in vivo binding and serum half-life of the human FcRn high affinity binding polypeptide to FcRn can be determined, for example, in transgenic mice expressing human FcRn or transfected human cell lines. The term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for transferring maternal IgG to the fetus (Guyer RL et al., J. Immunol. 117: 587, 1976) and (Kim YJ et al., J. Immunol ., 24: 249, 1994).

The term "single chain Fv antibody (or ScFv antibody)" refers to an antibody fragment comprising the VH and VL domains of an antibody, where these domains are present in a single polypeptide chain. For an overview of ScFv, see International Patent Application Publication No. WO 88/01649 and US Patent Nos. 4946,778 and 5260,203.

The term "single domain antibody" is obtained by genetic engineering methods. There are three main types. The first type is the heavy chain variable region obtained from the camel HCAb. It is a single folding unit that retains complete antigen-binding activity and is the smallest. Natural antibody fragments. The second type is the heavy chain variable region obtained from IgNAR, a cartilage fish such as shark, which is represented by VNAR. The third type is the heavy chain or light chain variable region obtained from human or murine monoclonal antibodies, which retains antigen binding activity, but the affinity and solubility are greatly reduced.

The term "monoclonal antibody (mAb)" refers to an antibody obtained from a substantially homogeneous population of antibodies, ie, except for a few occurrences of possible naturally occurring mutations, the individual antibodies contained in the population are the same, showing a single binding specificity and affinity for a specific epitope . The modifier "monoclonal" indicates the nature of the antibody obtained from a substantially identical group of antibodies, and no specific method is required to produce the antibody. Monoclonal antibodies are produced by methods known to those skilled in the art, for example, by preparing myeloma cells and immune spleen cells to produce hybrid antibody-producing cells. Synthesized by culturing hybridomas, it will not contaminate any other antibodies. Monoclonal antibodies can also be obtained by recombination, such as recombinant technology, phage display technology, synthetic technology, or other existing technologies.

The term "chimeric antibody" means that a portion of the heavy chain and / or light chain is the same as or homologous to the corresponding sequence in an antibody derived from a specific species or belongs to a specific antibody class or subclass, while the remaining portion of the chain is derived from another The corresponding sequences in antibodies of one species or of another antibody class or subclass are the same or homologous, and fragments of such antibodies as long as they exhibit the desired biological activity (US Patent No. 4,816,567; Morrison SL et al., Proc . Natl. Acad. Sci. USA, 81: 6851-6855, 1984). For example, the term "chimeric antibody" may include antibodies (eg, human and murine chimeric antibodies) in which the heavy and light chain variable regions of the antibody are derived from the first antibody (eg, murine antibody), and the heavy chain and The light chain constant region is from a second antibody (eg, human antibody).

The term "diabodies" refers to small antibody fragments with two antigen binding sites, which fragments comprise a heavy chain variable domain (VH) and a light chain variable domain (VL) connected to it in the same polypeptide chain (VH-VL or VL-VH). By using a linker that is too short to allow pairing between the two variable domains on the same chain, the domain is forced to pair with the complementary domain of the other chain, and two antigen binding sites are created. Diabodies are more fully described in, for example, EP 404,097; WO 93/11161; and Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448. For a review of engineered antibody variants, see Holliger and Hudson (2005) Nat. Biotechnol., 23: 1126-1136 in general.

The term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase the sequence homology with the human antibody. Most or all amino acids outside the CDR domain of non-human antibodies, such as mouse antibodies, are replaced with corresponding amino acids from human immunoglobulins, while most or all amino acids in one or more CDR regions are unchanged. The addition, deletion, insertion, substitution or modification of small molecule amino acids is allowed, as long as they do not eliminate the antibody's ability to bind a specific antigen. "Humanized" antibodies retain antigen specificity similar to the original antibody. The source of the CDR is not particularly limited, and can be derived from any animal. For example, antibodies derived from mouse antibodies, rat antibodies, rabbit antibodies, or non-human primates (eg, cynomolgus monkeys) can be used.

The term "antibody fragment" or "antigen-binding fragment" refers to an antigen-binding fragment and antibody analog of an antibody that retains specific binding ability to an antigen (eg, TIGIT), which usually includes at least part of the antigen binding of the parental antibody (Parental Antibody) Zone or variable zone. Antibody fragments retain at least some of the binding specificity of the parent antibody. Generally, when the activity is expressed in moles, antibody fragments retain at least 10% of the maternal binding activity. Preferably, the antibody fragments retain at least 20%, 50%, 70%, 80%, 90%, 95% or 100% of the binding affinity of the parent antibody to the target. Antibody fragments include, but are not limited to: Fab fragments, Fab 'fragments, F (ab') ₂ fragments, Fv fragments, Fd fragments, complementarity determining region (CDR) fragments, disulfide bond stable proteins (dsFv), etc .; linear antibodies ( Linear Antibody), single chain antibody (e.g. ScFv monoclonal antibody) (technique from Genmab), bivalent single chain antibody, single chain phage antibody, single domain antibody (Single Domain Antibody) (e.g. VH domain antibody), domain antibody ( Technology from AbIynx); multispecific antibodies formed by antibody fragments (eg, three-chain antibodies, tetra-chain antibodies, etc.); and engineered antibodies such as chimeric antibodies (eg, humanized murine antibodies), heteroconjugation Antibodies (Heteroconjugate Antibody) and so on. These antibody fragments are obtained by conventional techniques known to those skilled in the art, and these fragments are screened for utility by the same method as intact antibodies.

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

The term "functional fragment" encompasses a part of an intact antibody and generally includes the antigen-binding region or variable region of the intact antibody or the Fc region in which the antibody retains or has improved FcR binding capacity. Examples of functional fragments of antibodies include linear antibodies, single chain antibody molecules, and multispecific antibodies formed from antibody fragments.

The terms "conjugated" and "linked" refer to the association of two or more molecules. The connection can also be genetic (ie recombinant fusion). In a specific context, the term includes references to linking ligands (eg, antibody moieties) to effector molecules. The connection can be achieved using a variety of art-recognized techniques, for example, by chemical or recombinant means. "Chemical way" refers to the reaction between the antibody portion and the effector molecule, so that a covalent bond is formed between the two molecules to form a molecule.

The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and / or causes destruction of cells. The term is intended to include radioisotopes (eg, I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ ), chemotherapeutic agents, and toxins (such as enzymatically active toxins of bacterial, fungal, plant, or animal origin), or fragments thereof.

The term "cytokine" generally refers to a protein released by a cell population that acts as an intercellular medium on another cell or has autocrine effects on cells that produce the protein. Examples of such cytokines include lymphokines, mononuclear factors; interleukins ("IL"), such as IL-2, IL-6, IL-17A-F; tumor necrosis factors, such as TNF-α or TNF-β; and Other polypeptide factors, such as leukemia inhibitory factor ("LIF").

The term "immune-related diseases" refers to immune-related diseases in mammals that are caused by, mediated by, or otherwise contributing to the onset of components of the mammalian immune system, and also include diseases that stimulate or intervene in the immune response to improve the development of the disease. The term includes immune-mediated inflammatory diseases, non-immune-mediated inflammatory diseases, infectious diseases, immunodeficiency diseases, tumors and the like.

The term "depletion" refers to a state of T cell depletion that results from a T cell dysfunction state that persists in TCR signaling during many chronic infections and cancers or a state of NK cell dysfunction caused by sustained NK receptor (eg, NCR) signaling. NK cell depletion occurs due to continuous signaling. Depletion can result from both extrinsic negative regulatory pathways (e.g. immunomodulatory cytokines) and cell-intrinsic negative regulatory (costimulatory) pathways.

The term "enhancement of NK cell activity" means to induce, cause or stimulate NK cells to have sustained or amplified biological activity, or to restore or reactivate depleted or inactive NK cells. Examples of enhanced NK cell activity include: increased levels of IFN-γ secretion, increased proliferation, and increased antigen responsiveness (eg, clearance of viruses, pathogens, or tumors) relative to such levels before intervention. In one embodiment, the level of enhancement is at least 50%, such as 60%, 70%, 80%, 90%, 100%, 120%, 150%, or 200%. Ways to measure this level of enhancement are known to those of ordinary skill in the art.

The term "immunogenicity" refers to the ability of a particular substance to elicit an immune response. Tumors are immunogenic, and enhancing tumor immunogenicity helps clear tumor cells through an immune response. Examples of enhancing tumor immunogenicity include, but are not limited to treatment with TIGIT inhibitors (eg, anti-TIGIT antibodies).

The term "immune response" is composed of cells of the immune system (eg T lymphocytes, B lymphocytes, NK cells, antigen presenting cells, macrophages, eosinophils, mast cells, DC cells or neutrophils) and The role of soluble macromolecules produced by immune cells or the liver is produced by any of these cells or liver (including antibodies, cytokines and complements). This effect leads to the invasion of invasive pathogens, cells or tissues infected by pathogens, cancer Cells, or normal human cells or tissues in the case of pathological inflammation, selectively damage, destroy, or remove them from the body. The immune response includes, for example, activation or suppression of T cells (eg, effector T cells or Th cells, such as CD ⁸⁺ or CD ⁴⁺ T cells), or suppression or depletion of NK cells or Treg cells.

The term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen it targets. The term "immunobinding" refers to a specific binding reaction that occurs between an antibody molecule and an antigen to which the antibody is specific. The strength or affinity of the immune binding interaction can be expressed by the equilibrium dissociation constant (K _D ) of the interaction, where the smaller the K _D value, the higher the affinity. The immunological binding properties between two molecules can be quantified using methods well known in the art. One method involves measuring the rate of antigen binding site / antigen complex formation and dissociation. Both the "association rate constant" (K _a or K _on ) and the "dissociation rate constant" (K _d or K _off ) of a specific antibody-antigen interaction can be calculated from the concentration and the actual rate of association and dissociation It is concluded that see Malmqvist M, 1993, Nature, 361: 186-187. The ratio of K _d / K _a is equal to the dissociation constant K _D , see Davies DR et al., 1990, Annual Rev Biochem., 59: 439-473. By any effective method of measuring K _D, K _a and K _d values. In a preferred embodiment, a bioluminescence interferometry method (eg, the ForteBio Octet method described in Example 4) is used to measure the dissociation constant. In other preferred embodiments, surface plasmon resonance techniques (eg Biacore) or KinExa can be used to measure the dissociation constant. An "antibody that specifically binds to human TIGIT" refers to a K _D that binds to human TIGIT that is soluble or binds to a cell of 10 ^-7 M or less, for example, less than about 10 ^-8 M, 10 ^-9 M, 10 ^-10 M or 10 ^-11 M or even lower antibodies, but do not bind unrelated antigens with high affinity.

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

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

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

The term "host cell" is a cell in which a vector can proliferate and its DNA can be expressed, and the cell may be a prokaryotic cell or a eukaryotic cell. The term also includes any progeny of the test host cell. It should be understood that not all progeny are the same as the parent cell because mutations may occur during the replication process and such progeny are included. In certain embodiments, the host cell of the invention may be a chimeric antigen receptor T cell.

The term "identity" is used to refer to the degree of sequence similarity between two polypeptides or between two nucleic acids. When a position in two compared sequences is occupied by the same base or amino acid monomer subunit (for example, a position in each of two DNA molecules is occupied by adenine, or two A certain position in each of the polypeptides is occupied by lysine), then each molecule is the same at this position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions for comparison x 100. For example, if 6 of the 10 positions of the two sequences match, the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions match). Generally, comparisons are made when two sequences are aligned to produce maximum identity. Such an alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol., 48: 443-453, which is conveniently performed by a computer program such as the Align program (DNAstar, Inc.). You can also use the algorithms of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17, 1988) integrated into the ALIGN program (version 2.0), using the PAM120 weight residue table (weight residue table) , A gap length penalty of 12, and a gap penalty of 4 to determine the percent identity between two amino acid sequences. In addition, the Needleman and Wunsch (J. MoI. Biol., 48: 444-453, 1970) algorithm in the GAP program that has been integrated into the GCG software package (available at www.gcg.com) can be used, using Blossum Matrix or PAM250 matrix and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6 to determine the percentage between two amino acid sequences Identity.

The term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can 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 amino acid residues with amino acid residues that have similar side chains, such as those that are physically or functionally similar to the corresponding amino acid residues (eg, have similar size, shape, charge, chemical properties, including Substitution of residues such as the ability to form covalent bonds or hydrogen bonds. A family of amino acid residues with similar side chains has been defined in the art. These families include basic side chains (eg, lysine, arginine, and histidine), acidic side chains (eg, aspartic acid, glutamic acid), and uncharged polar side chains (eg, glycine) , Asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (eg alanine, valine, leucine, isole Amino acid, proline, phenylalanine, methionine), beta branched side chains (eg, threonine, valine, isoleucine) and aromatic side chains (eg, tyrosine, Phenylalanine, tryptophan, histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, for example, Brummell DA et al., Biochem., 32: 1180-1187, 1993; Kobayashi H et al., Protein Eng., 12: 879-884, 1999; and Burks EA et al., Proc. Natl. Acad. Set USA, 94: 412-417, 1997, which is incorporated herein by reference).

The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA segments can be joined. Another type of vector is a viral vector, where additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors with bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell after introduction into the host cell, and thereby replicate together with the host genome. In addition, certain vectors can direct the expression of genes to which they are effectively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Generally, expression vectors useful in recombinant DNA technology usually exist in the form of plasmids. However, other forms of expression vectors are also included, such as viral vectors (eg, replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions.

The term "chimeric antigen receptor (CAR)" refers to an engineered, extracellular targeting domain (eg, ScFv) derived from an antibody that is linked to one or more intracellular signaling domains of a T cell receptor T cell receptor. In the present invention, the term "chimeric antigen receptor T cell" is a T cell that expresses CAR and has antigen specificity determined by the targeting domain (eg, ScFv) of the CAR. Methods for manufacturing CARs (for example, for cancer treatment) are known in the art, see, for example, Park TS and others, Trends Biotechnol., 29: 550-557, 2011; Grupps SA and others, N. Engl. J. Med ., 368: 1509-1518, 2013; Han EQ et al., J. Hematol Oncol., 6:47, 2013, all of which are incorporated by reference in their entirety.

The term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, at the dosage employed And concentration is not toxic to cells or mammals exposed to it. Including, but not limited to: pH adjusting agents, surfactants, adjuvants, ionic strength enhancers, diluents, agents for maintaining osmotic pressure, agents for delaying absorption, preservatives. For example, pH adjusting agents include but are not limited to phosphate buffer. 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 for maintaining osmotic pressure include but are not limited to sugar, NaCl and the like. Agents that delay absorption include but are not limited to monostearate and gelatin. Diluents include but are not limited to water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerin), 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. A stabilizer has a meaning commonly understood by those skilled in the art, and it can stabilize the desired activity of active ingredients in medicines, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose , Lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin or casein) or their degradation products (such as lactalbumin hydrolysate) and so on.

The term "effective amount" refers to an amount sufficient to obtain or at least partially achieve the desired effect. For example, an effective amount to prevent a disease (eg, a tumor or an infection) refers to an amount sufficient to prevent, prevent, or delay the occurrence of a disease (eg, a tumor or an infection); an effective amount to treat a disease refers to an amount sufficient to cure or at least partially prevent a disease The amount of disease and its complications in patients with the disease. It is well within the ability of those skilled in the art to determine such an effective amount. For example, the amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the general condition of the patient such as age, weight and gender, the mode of administration of the drug, and other treatments administered simultaneously and many more.

The term "prevention" refers to a method implemented to prevent or delay the occurrence of a disease or condition or symptom (eg, tumor or infection) in a subject or if its effect is minimized. The term "treatment" refers to a method performed in order to obtain beneficial or desired clinical results. Beneficial or desired clinical outcomes include, but are not limited to, reducing the rate of disease progression, improving or alleviating the disease state, and regressing or improving the prognosis, whether detectable or undetectable. The amount of therapeutic agent effective to relieve the symptoms of any particular disease may vary depending on factors such as the patient's disease state, age and weight, and the ability of the drug to elicit the desired response in the subject. Whether a symptom of the disease is alleviated can be assessed by any clinical measurement, which is usually used by a doctor or other skilled healthcare provider to assess the severity or progress of the symptom.

The term "delaying the progression of a disease" means delaying, hindering, slowing, delaying, stabilizing, and / or delaying the formation of a disease (such as cancer or tumor immunity). Depending on the history of the disease and / or the individual being treated, this delay can be of varying lengths of time. As is obvious to the person skilled in the art, a sufficient or significant delay can substantially cover prevention, because the individual does not develop a disease. For example, late stage cancers, such as the formation of metastases, can be delayed.

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

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

The term "cytotoxicity" refers to, for example, the toxicity of immunotoxins to cells intended to be targeted, rather than to other cells of the organism.

The term "chemotherapy" or "chemotherapy" refers to the administration of any chemical agent for therapeutic use in the treatment of diseases characterized by abnormal cell growth. Such diseases include diseases characterized by proliferative growth, such as tumors. Combination chemotherapy gives more than one agent to treat cancer.

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

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

The term "effector function" refers to those biological activities that can be attributed to the Fc region of an antibody (native sequence Fc region or amino acid sequence variant Fc region), and it varies with the antibody isotype. Examples of antibody effector functions include, but are not limited to: Fc receptor binding affinity, antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP) , Down regulation of cell surface receptors (such as B cell receptors), B cell activation, cytokine secretion, half-life / clearance of antibodies and antigen-antibody complexes, etc. Methods for changing the effector function of antibodies are known in the art, for example, by introducing mutations in the Fc region.

The term "antibody-dependent cell-mediated cytotoxicity (ADCC)" refers to a form of cytotoxicity in which Ig binds to FcR present on cytotoxic cells (such as NK cells, neutrophils, or macrophages), These cytotoxic effector cells are specifically bound to the target cells to which the antigen is attached, and then the target cells are killed by secreting cytotoxin. Methods for detecting ADCC activity of antibodies are known in the art, and can be evaluated, for example, by measuring the binding activity between the antibody to be tested and FcR (eg, CD16a).

The term "complement dependent cytotoxicity (CDC)" refers to a form of cytotoxicity that activates the complement cascade by binding the complement component C1q to the antibody Fc. Methods for detecting the CDC activity of antibodies are known in the art, and can be evaluated, for example, by measuring the binding activity between the antibody to be tested and the Fc receptor (eg, Clq).

The term "autoimmune" in the state of immune tolerance, a certain amount of self-reactive T cells and autoantibodies are ubiquitous in the peripheral immune system of all individuals, which is helpful to help clear the aging and degeneration of its own components, which is Immune stability has important physiological significance.

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

The term "hematological malignancies" includes lymphoma, leukemia, myeloma or lymphoid malignancies, as well as splenic cancer and lymph node tumors. Exemplary lymphomas include B-cell lymphoma and T-cell lymphoma. B-cell lymphomas include, for example, Hodgkin lymphoma. T-cell lymphomas include, for example, cutaneous T-cell lymphomas. Hematological malignancies also include leukemia, such as secondary leukemia or acute lymphocytic leukemia. Hematological malignancies also include myeloma (eg, multiple myeloma) and other hematological and / or B cell or T cell related cancers.

The term "dysfunction" in the context of immune dysfunction refers to a state of reduced immune responsiveness to antigenic stimuli. The term includes the common elements of both depletion and / or non-responsiveness where antigen recognition can occur but the subsequent immune response is not effective in controlling infection or tumor growth. The term "dysfunction" also includes insensitivity or non-response to antigen recognition, in particular, converting antigen recognition into downstream T cell effector functions such as proliferation, cytokine production (eg IL-2) and / or target cell killing Is impaired.

Homologous antibody

The present invention also relates to variants that contain amino acid sequences contained in the heavy and light chain variable regions that are homologous to the amino acid sequences of the antibody or antigen-binding fragment thereof provided in any of the above aspects, and wherein the variant The body retains the desired functional properties of the anti-TIGIT antibody of the present invention.

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

Methods of sequence alignment for comparison are well known in the art. Various procedures and alignment algorithms are described in: Smith TF and Waterman MS, Adv. Appl. Math., 2: 482, 1981; Higgins DG and Sharp PM, CABIOS 5: 151, 1989. Altschul SF et al., Nature Genet., 6: 119, 1994 provide detailed ideas for sequence alignment methods and homology calculation.

Improvement of anti-TIGIT antibody stability

The anti-TIGIT antibody or antigen-binding fragment of the present invention preferably has improved stability. In general, the thermal melting temperature (Tm) value of an antibody can be used as an excellent indicator of the thermal stability of the antibody, and can also be used as an indicator of the storage period of the antibody. The lower the Tm value, the lower the association / stability is shown; the higher the Tm value, the lower the association / stability is. Therefore, it is preferable to provide an antibody with a high Tm value. In a non-limiting aspect of the present invention, antibodies with higher Tm values are selected. In some embodiments, antibodies having a Tm value of at least 50 ° C, 55 ° C, 60 ° C, 65 ° C, 70 ° C, 75 ° C, 80 ° C, 85 ° C, 90 ° C, 95 ° C, or 100 ° C or more are selected.

The stability of antibodies can be determined using any method known in the art, such as differential scanning fluorescence analysis (DSF), circular dichroism (CD), intrinsic protein fluorescence, differential scanning calorimetry, spectroscopy Science, light scattering (eg, dynamic light scattering (DLS) and static light scattering (SLS) and self-interaction chromatography (SIC).

The DSF assay monitors the thermal unfolding of proteins in the presence of fluorescent dyes and is usually performed by using real-time PCR instruments. DSF can be applied to various proteins, including antibody light and heavy chains. Compared to aqueous solutions where fluorescence is quenched, the fluorescent dyes used for DSF have high fluorescence in non-polar environments, such as hydrophobic sites on unfolded proteins. The various dyes that have been used differ in the fluorescence quantum yield caused by binding to denatured proteins. When using DSF for protein stability analysis, plot the fluorescence intensity as a function of temperature, producing an S-shaped curve that can be described by a two-state transition. Use simple equations, such as Boltzmannequation, to calculate the inflection point (Tm) of the transition curve (see, for example, Nissen FH, Berglund H., and Vidadi M .; use differential scanning fluorometry to detect promoting proteins Stable ligand interaction. "Natural Experiment Manual" 2: 2212-21 (2007)).

In the present invention, the stability of the anti-TIGIT antibody and its fragments can be changed by randomly changing the amino acid residues of the antibody constituting the antibody represented by the humanized anti-TIGIT antibody. In addition, stability can also be changed by replacing a part of the amino acid sequence of the humanized antibody represented by the humanized anti-TIGIT antibody with the following amino acid sequence, which has a high composition The amino acid sequence of the known antibody of the Tm value, and is considered to be the corresponding sequence from the viewpoint that a part of the amino acid sequence of the humanized antibody represented by the humanized TIGIT antibody used for the change is related to the three-dimensional structure of the antibody . The position of the substituted amino acid residue is not limited, but the amino acid residue in the FR can be preferably changed. In addition, it may be an amino acid residue in the CDR as long as it is not accompanied by a decrease in the binding activity to the antigen, and can be appropriately changed. In addition, the number of amino acid residues to be changed is not particularly limited, and can also be performed by substituting a specific fragment of FR with a desired fragment. The substitution of the fragment can be performed by changing the fragments of FR1, FR2, FR3, and FR4 in all FRs, or combining the changes of one or more fragments.

Immune cell activation

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

Increased immune cell activity can also be detected by measuring NK cell and / or T cell killing activity ex vivo in samples derived from individuals. In one embodiment, the increase in NK cell killing activity can be determined by measuring the production of one or more proinflammatory cytokines selected from the group including but not limited to: IL-1, TNFα, IL-5, IFN -γ, GM-CSF, IL-8 and IL-10. In one embodiment, the increase in NK cell killing activity can be achieved by enhancing the action of ADCC. In one embodiment, the increase in T cell killing activity can be detected by measuring T cell proliferation. In one embodiment, the increase in T cell killing activity can be detected by measuring the production of one or more proinflammatory cytokines.

Use of anti-TIGIT antibody

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

The anti-TIGIT antibody or antigen-binding fragment thereof of the present invention can be used to prepare a pharmaceutical composition, which includes a pharmaceutically acceptable carrier and / or excipient. In some preferred embodiments, the pharmaceutical composition further comprises an additional pharmaceutically active agent. In some preferred embodiments, the pharmaceutically active agent is a drug used to treat immune-related diseases. In some preferred embodiments, the pharmaceutically active agent is a drug with anti-tumor activity. In some preferred embodiments, the pharmaceutically active agent is used for the treatment of infections or infectious diseases.

In some embodiments, the amount of anti-TIGIT antibody or antigen-binding fragment thereof in the pharmaceutical composition of the invention is sufficient to elicit one of the following reactions in the subject: (a) induce and / or enhance the immune response; (b) increase Effector T cell activity; (c) increase cytotoxic T lymphocyte (CTL) activity; (d) increase NK cell activity; (e) inhibit TIGIT activation; (f) inhibit TIGIT-mediated signaling; (g) inhibit or Block the binding of CD155 and / or CD112 to TIGIT; (h) reduce the number of regulatory T cells in the tissue or circulation; (i) inhibit the suppression of regulatory T cells against effector T cells; or (j) (a)- (i) any combination.

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

The anti-TIGIT antibody or antigen-binding fragment thereof of the present invention can be used to increase immune cell activity in vitro or in a subject (eg, a human), and / or to enhance an immune response in a subject (eg, a human).

The anti-TIGIT antibody or antigen-binding fragment thereof of the present invention can be used to treat tumors in a subject (eg, human). In some preferred embodiments, the tumor is selected from solid tumors, hematological tumors (eg, leukemia, lymphoma, myeloma, eg, multiple myeloma), and metastatic, refractory, or recurrent lesions of cancer; for example , Including but not limited to esophageal cancer, gastrointestinal cancer, pancreatic cancer, thyroid cancer, colorectal cancer, kidney cancer, lung cancer (such as non-small cell lung cancer), liver cancer, stomach cancer, head and neck cancer, bladder cancer, breast cancer, uterine cancer, Cervical cancer, ovarian cancer, prostate cancer, testicular cancer, germ cell cancer, bone cancer, skin cancer, thymus cancer, cholangiocarcinoma, melanoma, mesothelioma, lymphoma, myeloma, sarcoma, glioblastoma, Metastatic, refractory, or recurrent lesions of glioblastoma, leukemia, or cancer.

The anti-TIGIT antibody or antigen-binding fragment thereof of the present invention can be used to treat infections or infectious diseases in a subject (such as a human). In some preferred embodiments, the infection or infectious disease is selected from viral infections, bacterial infections, fungal infections and parasitic infections, including but not limited to HIV, hepatitis virus, herpes virus, CMV, EBV, influenza or tetanus.

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

In some preferred embodiments, the immunogen is selected from tumor cells, tumor-associated antigens (eg, proteins, polypeptides, or carbohydrate molecules), dendritic cells sensitized by the antigens, and any combination thereof. In some preferred embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof is used to prevent and / or treat tumors in a subject. In some preferred embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof is used to delay tumor progression in a subject. In some preferred embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof is used to reduce or inhibit tumor recurrence in a subject. In certain preferred embodiments, the tumors involved in the antibodies or antigen-binding fragments thereof of the present invention are selected from solid tumors, hematological tumors (eg, leukemia, lymphoma, myeloma, eg, multiple myeloma), and cancer Metastatic, refractory, or recurrent lesions; for example, including but not limited to esophageal cancer, gastrointestinal cancer, pancreatic cancer, thyroid cancer, colorectal cancer, renal cancer, lung cancer (such as non-small cell lung cancer), liver cancer, gastric cancer , Head and neck cancer, bladder cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, germ cell cancer, bone cancer, skin cancer, thymus cancer, cholangiocarcinoma, melanoma, mesothelioma, lymph Metastatic, refractory, or recurrent lesions of tumors, myeloma, sarcoma, glioblastoma, glioblastoma, leukemia, or cancer.

In some preferred embodiments, the immunogen is selected from inactivated or attenuated pathogens, antigens associated with the pathogens (such as proteins, polypeptides, or carbohydrate molecules), dendritic cells sensitized by the antigens, and Any combination. In some preferred embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof is used to prevent and / or treat an infection or infectious disease in a subject. In some preferred embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof is used to delay the progression of infection or infectious disease in a subject. In some preferred embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof is used to reduce or inhibit infection or relapse of an infectious disease in a subject. In some preferred embodiments, the infection or infectious disease is selected from viral infections, bacterial infections, fungal infections and parasitic infections, including but not limited to HIV, hepatitis virus, herpes virus, CMV, EBV, influenza or tetanus.

In some preferred embodiments, the immunogenic composition further comprises a pharmaceutically acceptable carrier and / or excipient. In certain preferred embodiments, in the immunogenic composition, the antibody or antigen-binding fragment of the present invention and the immunogen are provided as separate components or as components of the same composition. Therefore, the antibody or antigen-binding fragment of the present invention and the immunogen can be administered simultaneously, separately, or sequentially.

The anti-TIGIT antibody or antigen-binding fragment thereof of the present invention can be used to prepare a medicament for treating immune-related diseases, wherein the immune-related diseases involve T cell dysfunction and / or NK cell dysfunction. In certain preferred embodiments, the T cell dysfunction includes, for example, T cell disability, depletion, or reduced cytokine secretion, and the NK cell dysfunction includes, for example, NK cell disability, depletion, or reduced cytokine secretion.

The anti-TIGIT antibody or antigen-binding fragment thereof of the present invention can be used to increase the activity of immune cells in vitro. Therefore, the present invention also relates to a method for increasing the activity of immune cells in vitro, which method includes the step of contacting the immune cells with the antibody or antigen-binding fragment thereof of the present invention. In some preferred embodiments, the method further includes the step of contacting the immune cell with an additional pharmaceutically active agent. In some preferred embodiments, the additional pharmaceutically active agent is an immune response stimulant. In some preferred embodiments, the immune response stimulator is selected from IL-1, IL-2, IL-3, IL-7, IL-12, IL-15, IL-18, IL-21, IFN- γ, IL-10, TGF-β, GM-CSF, M-CSF, G-CSF, TNF-α and TNF-β and any combination thereof.

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

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

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

The anti-TIGIT antibody or antigen-binding fragment thereof of the present invention can be used to prevent and / or treat tumors, delay tumor progression, and / or reduce or inhibit tumor recurrence in a subject. The invention also relates to a method of depleting Treg cells from the tumor microenvironment of a subject with a tumor, the method comprising administering to the subject a therapeutically effective amount of an anti-TIGIT antibody and antigen-binding fragment thereof described herein, wherein The anti-TIGIT antibody and its antigen-binding fragments contain Fc that can stimulate the depletion of Treg cells in the tumor microenvironment. The Fc may be, for example, an Fc having an effector function or an enhanced effector function, for example, an Fc that binds to one or more activated Fc receptors or has enhanced binding to one or more activated Fc receptors.

The anti-TIGIT antibody or antigen-binding fragment thereof of the present invention is used to prevent and / or treat an infection or infectious disease, delay the progression of infection or infectious disease, and / or reduce or inhibit the recurrence of infection or infectious disease in a subject.

In any of the above uses, the anti-TIGIT antibody of the present invention or an antigen-binding fragment thereof can be used in combination. Therefore, in some embodiments, an effective amount of any one or more (

eg

1, 2, 3, 4 or more) anti-TIGIT antibodies or antigen-binding fragments of the invention are administered to an individual Treating or delaying the progression of the immune-related disease (eg, tumor) in an individual or increasing, enhancing, or stimulating an immune response or function in the individual.

The uses described above also include the use in combination with a second therapy selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination. Administration of the antibodies, antigen-binding fragments, and pharmaceutical compositions disclosed herein may be accompanied by administration of other anti-cancer agents or other treatments (eg, surgical resection of tumors). Any suitable anti-cancer agent can be administered in combination with the antibodies, antibody fragments, pharmaceutical compositions, immunogenic compositions disclosed herein. Exemplary anticancer agents may include chemotherapeutic agents (e.g. mitotic inhibitors), alkylating agents (e.g. Nitrogen Mustard), antimetabolites (e.g. folic acid analogs), natural products (e.g. Vinca alkaloid , A variety of reagents (such as platinum coordination complexes), hormones and antagonists (such as adrenal corticosteroids), immunomodulators (such as bropilamine Bropirimine, Upjohn) and so on. Other anti-cancer treatments include other antibodies that specifically target cancer cells.

The therapeutically effective dose of the anti-TIGIT antibody or antigen-binding fragment of the invention will depend on the severity of the disease and the patient's health. In addition, the amount required for administration also depends in part on the binding affinity of the antibody to the antigen and the pharmacokinetic properties of the antibody in the subject. The usual range of therapeutically effective dose administration of the anti-TIGIT antibody or antigen-binding fragment of the invention (by way of non-limiting example) may be from about 0.1 mg / kg body weight to about 50 mg / kg body weight. Commonly used dosing frequencies can range, for example, from twice a day to once a week. The dosage and method of administration may vary according to the patient's weight, age, and symptoms, and are appropriately selected by those skilled in the art.

The anti-TIGIT antibody or antigen-binding fragment thereof of the present invention can be used to detect the presence of TIGIT in a sample. The method includes subjecting the sample or antibody of the present invention to a complex of the antibody or antigen-binding fragment thereof and TIGIT under conditions permitting formation of a complex. The antigen-binding fragments are contacted and the formation of the complex is detected. In certain embodiments, the method includes the steps of: i. Incubating the sample with an anti-TIGIT antibody or antigen-binding fragment thereof of the invention; ii. Detecting bound TIGIT using a detectable probe; iii. Ii The amount of is compared with a standard curve obtained from a reference sample containing a known amount of TIGIT; and iv. The amount of TIGIT in the sample is calculated from the standard curve.

The anti-TIGIT antibody or antigen-binding fragment of the present invention can be used to prepare a diagnostic or therapeutic kit, which includes the antibody or antigen-binding fragment of the present invention, and optional instructions for use. The pharmaceutical composition of the present invention can be used to prepare a diagnostic or therapeutic kit, which includes the pharmaceutical composition of the present invention and optional instructions for use.

cancer

The anti-TIGIT antibody or antigen-binding fragment, carrier or host cell of the present invention can be used to prepare a pharmaceutical composition, which includes a pharmaceutically acceptable carrier and / or excipient. In certain preferred embodiments, the pharmaceutical composition comprises an anti-TIGIT antibody or antigen-binding fragment thereof of the invention. In certain preferred embodiments, the pharmaceutical composition comprises the vector or host cell of the invention. In such embodiments, the isolated nucleic acid molecule contained in the vector comprises a nucleotide sequence encoding a chimeric antigen receptor, and the nucleotide sequence encoding the chimeric antigen receptor further comprises an antibody encoding the invention Or the nucleotide sequence of its antigen-binding fragment (eg ScFv); the host cell contains the isolated nucleic acid molecule or vector as described above. In certain preferred embodiments, the isolated nucleic acid molecule encodes a chimeric antigen receptor comprising an antigen-binding fragment (eg, ScFv) of the antibody of the invention. In certain preferred embodiments, the host cell is a T cell. In certain preferred embodiments, the host cell is a chimeric antigen receptor T cell (CAR-T).

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

In some embodiments, the therapeutically effective amount of the anti-TIGIT antibody or antigen-binding fragment, vector, or host cell in the pharmaceutical composition of the invention is sufficient to elicit one of the following reactions in the subject: (a) induce and / or enhance Immune response; (b) increase effector T cell activity; (c) increase cytotoxic T lymphocyte (CTL) activity; (d) increase NK cell activity; (e) inhibit TIGIT activation; (f) inhibit TIGIT-mediated signaling Conduction; (g) inhibit or block the binding of CD155 and / or CD112 to TIGIT; (h) reduce the number of regulatory T cells in the tissue or circulation; (i) inhibit the suppression of regulatory T cells on effector T cells; or (j) Any combination of (a)-(i). In some preferred embodiments, the subject has a tumor or cancer-related disease.

In some embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof of the present invention may form an immunogenic complex with an immunogen. In some preferred embodiments, the immunogen is selected from tumor-associated antigens (eg, proteins, polypeptides, or carbohydrate molecules), tumor cells, dendritic cells sensitized by the antigen, and any combination thereof.

In some preferred embodiments, the antibodies and antigen-binding fragments, vectors or host cells described above are used to prevent and / or treat tumors in a subject. In some preferred embodiments, the antibodies and antigen-binding fragments, vectors, or host cells described above are used to delay tumor progression in a subject. In some preferred embodiments, the antibodies and antigen-binding fragments, vectors, or host cells described above are used to reduce or inhibit tumor recurrence in a subject. In some preferred embodiments, the above antibodies or antigen-binding fragments thereof may be provided together with or before the immunogen.

The anti-TIGIT antibody or antigen-binding fragment thereof of the present invention is used to treat or delay the progression of immune-related diseases. In some embodiments, the immune-related disease involves T cell dysfunction. In some embodiments, the T cell dysfunction includes, for example, T cell disability, depletion, or reduced cytokine secretion. In some embodiments, the immune-related disease involves NK cell dysfunction. In some embodiments, the NK cell dysfunction includes NK cell incapacitation, depletion, or reduced cytokine secretion. In some embodiments, the immune-related disease includes a tumor. For example, the growth of cancerous tumors is suppressed or reduced, and / or the tumors regress. In some embodiments, the anti-TIGIT antibodies or antigen-binding fragments thereof of the invention can be used to increase, enhance or stimulate their immune response or function in individuals in need.

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

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

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

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

In some embodiments, the subject is administered an effective amount of any one or more (

eg

1, 2, 3, 4 or more) anti-TIGIT antibodies or antigen-binding fragments thereof of the invention.

The anti-TIGIT antibody or antigen-binding fragment thereof of the present invention can be used alone to inhibit the growth of cancer tumors. Alternatively, the anti-TIGIT antibody or antigen-binding fragment thereof can be used in combination with another agent (eg, other antibody, other immunogenic agent, or standard cancer treatment). Anti-TIGIT antibodies or antigen-binding fragments can be used in combination with other antibodies that activate the host immune response. Other antibodies include but are not limited to T cell costimulatory molecules, such as PD-1, PD-L1, and CTLA-4 (e.g., U.S. Patent No. 5,811,097), OX-40 (Weinberg AD, etc., Immunol., 164: 2160-2169, 2000) and 4-1BB (Melero I, Nature, Medicine, 3: 682-685, 1997), DC cell surface Molecules, molecules that activate T cells (Hutloff A et al., Nature, 397: 262-266, 1999). Anti-TIGIT antibodies can be combined with immune checkpoint inhibitors to enhance the body's anti-tumor response. Anti-TIGIT antibodies can also be used in combination with bispecific antibodies that target effector cells expressing Fca or Fcy receptors to tumor cells (see, eg, US Patent Nos. 5,922,845 and 5,837,243).

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

The anti-TIGIT antibody or antigen-binding fragment of the present invention can be transfected with immunogenic agents, such as cancer cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules) or with genes encoding immunostimulatory cytokines Combined application (He YF et al. J. Immunol. 173: 4919-28, 2004). The anti-TIGIT antibody or antigen-binding fragment of the present invention can be co-administered with DC cells to effectively enhance the anti-tumor response. The anti-TIGIT antibody or antigen-binding fragment or combination of the invention can also be administered in combination with standard cancer treatments (eg, surgery, radiation, and chemotherapy).

In some preferred embodiments, the above-mentioned tumors include but are not limited to solid tumors, hematological tumors (eg, leukemia, lymphoma, myeloma, eg, multiple myeloma), and metastatic, refractory, or recurrent cancer Lesions; for example, including but not limited to esophageal cancer, gastrointestinal cancer, pancreatic cancer, thyroid cancer, colorectal cancer, kidney cancer, lung cancer (such as non-small cell lung cancer), liver cancer, stomach cancer, head and neck cancer, bladder cancer, breast cancer, Uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, germ cell cancer, bone cancer, skin cancer, thymus cancer, cholangiocarcinoma, melanoma, mesothelioma, lymphoma, myeloma, sarcoma, glioblastoma Metastatic, refractory, or recurrent lesions of cell tumors, glioblastoma, leukemia, or cancer.

The uses described above may also include a second therapy selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof.

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

The therapeutically effective dose of the antibody or antigen-binding fragment of the invention will depend on the severity of the disease and the patient's health. In addition, the amount required for administration also depends in part on the binding affinity of the antibody to the antigen and the pharmacokinetic properties of the antibody in the subject. The usual range of therapeutically effective dose administration of the antibodies or antigen-binding fragments of the invention (by way of non-limiting examples) may be from about 0.1 mg / kg body weight to about 50 mg / kg body weight. Commonly used dosing frequencies can range, for example, from twice a day to once a week. The dosage and method of administration may vary according to the patient's weight, age, and symptoms, and are appropriately selected by those skilled in the art.

Infectious disease

The anti-TIGIT antibody or antigen-binding fragment thereof of the present invention can be used to prepare a pharmaceutical composition, which includes a pharmaceutically acceptable carrier and / or excipient. In some preferred embodiments, the pharmaceutical composition further comprises an additional pharmaceutically active agent. In some preferred embodiments, the pharmaceutical composition is a medicament for treating immune-related diseases. In some preferred embodiments, the pharmaceutical composition is a medicament for treating an infection or infectious disease.

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

In some embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof of the present invention may form an immunogenic complex with an immunogen. In some preferred embodiments, the immunogen is selected from antigens (eg proteins, polypeptides or carbohydrate molecules) associated with pathogens, inactivated or attenuated pathogens, dendritic cells sensitized by the antigens, And any combination thereof.

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

The anti-TIGIT antibody or antigen-binding fragment thereof of the present invention is used to treat or delay the progression of immune-related diseases. In some embodiments, the immune-related disease involves T cell dysfunction. In some embodiments, the T cell dysfunction includes, for example, T cell disability, depletion, or reduced cytokine secretion. In some embodiments, the immune-related disease involves NK cell dysfunction. In some embodiments, the NK cell dysfunction includes, for example, NK cell incapacitation, depletion, or reduced cytokine secretion. In some embodiments, the immune-related diseases include infections or infectious diseases.

In some embodiments, the anti-TIGIT antibodies or antigen-binding fragments, pharmaceutical compositions, and immunogenic compositions of the present invention can enhance T cell activity in a subject. In some embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof, pharmaceutical composition, and immunogenic composition of the present invention can enhance cytotoxic T cell (CTL) activity in a subject. In some embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof, pharmaceutical composition, and immunogenic composition of the present invention can enhance NK cell activity in a subject. In some embodiments, the cell activity described above is immunologically related to infection or infectious disease.

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

In some preferred embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof, pharmaceutical composition and immunogenic composition are used to prevent and / or treat infections or infectious diseases in a subject. In some preferred embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof, pharmaceutical composition, and immunogenic composition are used to delay the progression of infection or infectious disease in a subject. In some preferred embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof, pharmaceutical composition, and immunogenic composition are used to reduce or inhibit infection or recurrence of an infectious disease in a subject.

In some embodiments, an effective amount of any one or more (

eg

1, 2, 3, 4 or more) anti-TIGIT antibodies or antigen-binding fragments of the invention can be administered to a subject.

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

Based on the types of infectious organisms or substances involved, infections are broadly classified as bacterial, viral, fungal or parasitic. Other less common types of infections include, for example, infectious diseases involving Rickettsia, Mycoplasma and substances. Examples of bacteria, viruses, fungi and parasites that cause infections are well known in the art. The infection can be acute, subacute, chronic, or latent, and it can be local or systemic. In some embodiments, the immune-related diseases include acute infection of unknown cause and chronic infection. In some embodiments, the infection or infectious disease includes, but is not limited to, viral infection, bacterial infection, fungal infection, and parasitic infection.

Infectious diseases are caused by infectious bacteria. Non-limiting examples of infectious bacteria include: Helicobacter pylori, Borrelia burgdorferi, Mycobacterium species (such as Mycobacterium tuberculosis), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Streptococcus pyogenes ( Group A Streptococcus), Streptococcus pneumoniae, pathogenic Campylobacter spp, Enterococcus spp, Haemophilus influenzae, Bacillus anthracis, Corynebacterium diphtheriae, Erysipelothrix erysipelas, Clostridium tetani, Fusobacterium Candida streptococci, Treponema pallidum. Other infectious organisms (such as protists) include: Plasmodium falciparum and Toxoplasma gondii. The compositions and methods described herein are expected to be used to treat infections caused by these pathogens.

Infectious diseases are caused by infectious viruses. The viral antigen may be any antigen that is present in an infectious virus and induces an immune response in a subject. Non-limiting examples of infectious viruses include: HIV, poliovirus, yellow fever virus, rotavirus, coronavirus, mumps virus, Ebola virus, Eco virus, measles virus, influenza virus, dengue virus , Hemorrhagic fever virus, Coxsackie virus, Reovirus, Hepatitis B virus, Parvovirus, Papilloma virus, Adenoviridae, Herpes virus, Smallpox virus, Rabies virus, and unclassified viruses (eg, sponge The causative agent of craniocerebral disease). The compositions and methods described herein are expected to be used to treat infections caused by these viral pathogens.

Infectious diseases are caused by infectious fungi. Non-limiting examples of infectious pathogenic fungi include Candida albicans, Candida krusei, Cryptococcus, histoplasma, Aspergillus niger, Rhizopus, S. schenckii, Coccidioides spp Histoplasma.

Infectious diseases are caused by infectious parasites. Non-limiting examples of infectious pathogenic parasites include dysentery amoeba, colonic pouch ciliate, Gree-resistant Amphiprion, Amoeba species, Acanthopanax blueis, Cryptosporidium, Cryptosporidium species, Pneumocystis carinii, Plasmodium vivax, Babesia microcystis, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, and Japanese yenella

Vaccine adjuvant

The anti-TIGIT antibody or antigen-binding fragment of the present invention can be administered in combination with an immunogen (for example, a vaccine) to enhance an antigen-specific immune response. Therefore, the present invention also relates to a method of increasing the immune response to an immunogen in a subject, comprising administering to the subject: (i) the immunogen; (ii) the anti-TIGIT antibody or antigen-binding fragment thereof of the present invention , So that the immune response to the immunogen in the subject is enhanced. The invention also relates to the use of the anti-TIGIT antibody or antigen-binding fragment of the invention as an adjuvant, or for the preparation of an immunogenic composition, wherein the immunogenic composition includes the antibody or antigen of the invention Binding fragments and immunogens.

In some preferred embodiments, the immunogen is selected from tumor-associated antigens (eg, proteins, polypeptides, or carbohydrate molecules), tumor cells, dendritic cells sensitized by the antigen, and any combination thereof. In such embodiments, the method can be further used to prevent and / or treat tumors, delay tumor progression, and / or reduce or inhibit tumor recurrence in a subject.

In some preferred embodiments, the immunogen is selected from antigens (eg proteins, polypeptides or carbohydrate molecules) associated with pathogens, inactivated or attenuated pathogens, dendritic cells sensitized by the antigens, and Any combination. In such embodiments, the method can be further used to prevent and / or treat infections or infectious diseases, delay the progression of infections or infectious diseases, and / or reduce or inhibit the recurrence of infections or infectious diseases in a subject .

Pharmaceutical composition

The anti-TIGIT antibody (preferably humanized antibody) or antigen-binding fragment thereof of the present invention can be used to prepare a pharmaceutical composition or a sterile composition, the anti-TIGIT antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier and / or The excipients are mixed. The pharmaceutical composition may include one or a combination (such as two or more different) anti-TIGIT antibodies or antigen-binding fragments thereof of the present invention. For example, the pharmaceutical composition of the present invention may comprise a combination of antibodies or antibody fragments having complementary activities that bind to different epitopes on the target antigen.

The vector or host cell of the present invention can also be used to prepare a pharmaceutical composition or a sterile composition, and the vector or host cell is mixed with a pharmaceutically acceptable carrier and / or excipient. In such embodiments, the isolated nucleic acid molecule contained in the vector comprises a nucleotide sequence encoding a chimeric antigen receptor, and the nucleotide sequence encoding the chimeric antigen receptor further comprises an antibody encoding the invention Or the nucleotide sequence of its antigen-binding fragment (eg ScFv); the host cell contains the isolated nucleic acid molecule or vector as described above. In certain preferred embodiments, the isolated nucleic acid molecule encodes a chimeric antigen receptor comprising an antigen-binding fragment (eg, ScFv) of the antibody of the invention. In certain preferred embodiments, the host cell is a T cell. In certain preferred embodiments, the host cell is a chimeric antigen receptor T cell (CAR-T).

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

The term "pharmaceutically acceptable" means that when the molecular body, molecular fragment, or composition is properly administered to an animal or human, they will not produce adverse, allergic, or other adverse reactions. Specific examples of some substances that can be used as pharmaceutically acceptable carriers or components include sugars (such as lactose), starch, cellulose and derivatives thereof, vegetable oils, gelatin, polyols (such as propylene glycol), alginic acid, and the like.

In some embodiments, the therapeutically effective amount of the anti-TIGIT antibody or antigen-binding fragment, vector, or host cell in the pharmaceutical composition of the invention is sufficient to elicit one of the following reactions in the subject: (a) induce and / or enhance Immune response; (b) increase effector T cell activity; (c) increase cytotoxic T lymphocyte (CTL) activity; (d) increase NK cell activity; (e) inhibit TIGIT activation; (f) inhibit TIGIT-mediated signaling Conduction; (g) inhibit or block the binding of CD155 and / or CD112 to TIGIT; (h) reduce the number of regulatory T cells in the tissue or circulation; (i) inhibit the suppression of regulatory T cells on effector T cells; or (j) Any combination of (a)-(i). In some preferred embodiments, the subject has an infection or infectious disease. In some preferred embodiments, the subject has a tumor.

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

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

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

In some preferred embodiments, the pharmaceutical composition of the present invention is used to prevent and / or treat tumors in a subject. In some preferred embodiments, the pharmaceutical composition is used to delay tumor progression in a subject. In some preferred embodiments, the pharmaceutical composition is used to reduce or inhibit tumor recurrence in a subject.

In some preferred embodiments, the pharmaceutical composition of the present invention is used to prevent and / or treat infections or infectious diseases in a subject. In some preferred embodiments, the pharmaceutical composition is used to delay the progression of infection or infectious disease in a subject. In some preferred embodiments, the pharmaceutical composition is used to reduce or inhibit infection or relapse of infectious disease in a subject.

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

The anti-TIGIT antibody or antigen-binding fragment thereof of the present invention can be used together with one or more other therapeutic agents, such as toxins, cytotoxic agents, radioisotopes, immunosuppressants or vaccines. The anti-TIGIT antibody or antigen-binding fragment thereof may be linked to the therapeutic agent (as an immune complex) or administered separately from the therapeutic agent. In the latter case, the anti-TIGIT antibody or antigen-binding fragment thereof may be administered before, after, or co-administered with the therapeutic agent, or may be co-administered with other known therapies (eg, anti-cancer therapy, such as radiation) Apply.

Therapeutic agents include but are not limited to anti-tumor agents such as doxorubicin (doxorubicin), cisplatin bleomycin sulfate, nitrosourea mustard, chlorambucil and cyclophosphamide hydroxyurea, these The therapeutic agent itself is only effective when it has a toxic or sub-toxic level to the patient. Target-specific effector cells, such as effector cells linked to the anti-TIGIT antibody or antigen-binding fragment of the invention, can also be used as therapeutic agents. The targeted effector cells may be human leukocytes, such as macrophages, neutrophils or monocytes. Other cells include eosinophils, NK cells and other cells with IgG or IgA receptors. If necessary, effector cells can be obtained from the subject to be treated. Target-specific effector cells can be administered as a suspension of cells in a physiologically acceptable solution. The number of cells administered can be in the order of 10 ⁸ -10 ⁹ but may vary according to the purpose of treatment. In general, this amount is sufficient to achieve localization on target cells (such as TIGIT-expressing tumor cells) and cell killing by, for example, phagocytosis. The route of administration also varies, including oral, rectal, transmucosal, enteral, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular , Inhalation, insufflation, topical, skin, percutaneous or intraarterial.

In the presence of complement, the composition of the present invention having a complement binding site such as a portion from IgG1, IgG2, or IgG4 or IgM that binds to complement may also be used. The composition of the invention can also be administered together with complement, for example in combination with CIq.

Therapies that administer target-specific effector cells can be performed in conjunction with other techniques to eliminate target cells. For example, anti-tumor therapy using the compositions of the present invention and / or effector cells equipped with these compositions is used in combination with chemotherapy. Non-limiting examples of antibody combination therapy of the present invention include surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof.

The determination of the appropriate dose is performed by a clinician, for example using parameters or factors known or suspected in the art to affect the treatment. Generally, the dose starts with an amount slightly less than the optimal dose, and it is then increased in small increments until the desired or optimal effect is achieved relative to any negative side effects. Important diagnostic measures include, for example, symptoms of inflammation or measures of the level of inflammatory cytokines produced.

The present invention provides a container (such as a plastic or glass vial, such as a lid or a chromatographic column, a hollow hole needle, or a syringe cylinder), which contains any antibody or antigen-binding fragment of the present invention or a pharmaceutical composition thereof. The invention also provides an injection device comprising any antibody or antigen-binding fragment of the invention or a pharmaceutical composition thereof.

Combination therapy

The invention encompasses the use of an anti-TIGIT antibody or antigen-binding fragment thereof in combination with one or more additional active therapeutic agents (eg, chemotherapeutic agents) or other preventive or therapeutic modalities (eg, radiotherapy). In such combination therapies, various active agents often have different complementary mechanisms of action, and combination therapies may lead to synergistic effects. Combination therapies include therapeutic agents that affect the immune response (eg, enhance or activate the response) and therapeutic agents that affect (eg, inhibit or kill) tumors / cancer cells. Combination therapy can reduce the possibility of drug-resistant cancer cells. Combination therapy may allow the dose of one or more of the agents to be reduced to reduce or eliminate the adverse effects associated with one or more of the agents. Such combination therapy may have a synergistic treatment or prevention effect on the underlying disease, disorder or condition.

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

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

Detection method and kit

The present invention provides (eg, in vitro or in vivo) to detect the presence or presence of TIGIT in samples (eg, biological samples, eg, blood, serum, semen, or urine or tissue biopsy samples (eg, from hyperproliferative or cancerous lesions)) Its level approach. The method can be used for diagnostic purposes or non-diagnostic purposes (eg, the sample is a cell sample, not a sample from a patient). This method can be used to evaluate (e.g., monitor the treatment or progress of the disease (e.g., immune-related disorders, cancer, or infectious diseases) of the present invention, its diagnosis, and / or staging) in the subject. The method may include: i. Incubating the sample with the antibody or antigen-binding fragment thereof of the present invention; ii. Detecting the TIGIT complex using a detectable probe; iii. Combining the amount of ii with a known amount of TIGIT The standard curve obtained from the reference sample is compared; and iv. The amount of TIGIT in the sample is calculated from the standard curve. The formation of the complex indicates the presence of TIGIT and may indicate the suitability or need of the treatment described herein. The method may involve, for example, immunohistochemistry, immunocytochemistry, flow cytometry, magnetic beads complexed with antibody molecules, ELISA assay, PCR technology (eg, RT-PCR). Generally, anti-TIGIT antibody molecules used in in vivo and in vitro diagnostic methods are directly or indirectly labeled with a detectable substance to output a detection signal. Suitable detectable substances include but are not limited to various biologically active enzymes, prosthetic groups, fluorescent substances, luminescent substances and radioactive substances.

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

In the present invention, the detectable label may be any substance that can be detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical, or chemical means. It is particularly preferred that such labels can be suitable for immunological detection (eg, enzyme-linked immunoassay, radioimmunoassay, fluorescent immunoassay, chemiluminescence immunoassay, etc.). Such labels are well known in the art and include, but are not limited to, enzymes (eg, horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides ( For example, ³ H, ¹²⁵ I, ³⁵ S, ¹⁴ C, or ³² P), fluorescent dyes (eg, fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), algae Hemoglobin (PE), Texas Red, Rhodamine, quantum dots or cyanine dye derivatives (eg Cy7, Alexa 750)), acridine ester compounds, magnetic beads (eg,

), Calorimetric markers such as colloidal gold or colored glass or plastic (eg, polystyrene, polypropylene, latex, etc.) beads, and avidin (eg, streptavidin) modified to bind the above markers ) Of biotin. Patents that teach the use of this marker include, but are not limited to, US Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241 (all incorporated herein by reference). The markers covered in the present invention can be detected by methods known in the art. For example, radioactive labels can be detected using photographic film or scintillation calculators, and fluorescent labels can be detected using photodetectors to detect the emitted light. Enzyme labels are generally detected by providing a substrate for the enzyme and detecting the reaction products generated by the action of the enzyme on the substrate, and calorimetric labels are detected by simply visualizing the colored labels. In certain embodiments, a detectable label as described above can be attached to the recombinant protein of the present invention through linkers of different lengths to reduce potential steric hindrance.

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

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

The kit may also include additional reagents, such as blocking reagents, washing reagents, enzyme substrates, etc. for reducing non-specific binding to the solid phase surface. The solid surface can be in the form of tubes, beads, microtiter plates, microspheres, or other materials suitable for immobilizing proteins, peptides, or polypeptides.

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

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

There is also provided a therapeutic kit comprising one or more anti-TIGIT antibodies of the invention or antigen-binding fragments thereof, the kit optionally comprising a prophylaxis and / or treatment using the anti-TIGIT antibodies of the invention or antigen-binding fragments thereof Instructions for use for one or more disease states (eg, tumor, infection, or infectious disease) as described herein.

There is also provided a therapeutic kit comprising the vector or host cell of the present invention, the kit optionally comprising instructions for use of the vector or host cell of the present invention to prevent and / or treat a tumor as described herein. In such embodiments, the isolated nucleic acid molecule contained in the vector comprises a nucleotide sequence encoding a chimeric antigen receptor, and the nucleotide sequence encoding the chimeric antigen receptor further comprises an antibody encoding the invention Or the nucleotide sequence of its antigen-binding fragment (eg ScFv); the host cell contains the isolated nucleic acid molecule or vector as described above. In certain preferred embodiments, the isolated nucleic acid molecule encodes a chimeric antigen receptor comprising an antigen-binding fragment (eg, ScFv) of the antibody of the invention. In certain preferred embodiments, the host cell is a T cell. In certain preferred embodiments, the host cell is a chimeric antigen receptor T cell (CAR-T).

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

In one embodiment, the kit contains an anti-TIGIT antibody (eg, a humanized antibody) or antigen-binding fragment thereof or a carrier or host cell as described above, and one or more pharmaceutically acceptable Carrier.

In one embodiment, the kit contains the combination of the present invention, which includes the anti-TIGIT antibody or antigen-binding fragment of the present invention or the vector or host cell as described above together with one or more pharmaceutically acceptable in a single common container The accepted carrier, optionally combined with one or more therapeutic agents, is optionally formulated together in a pharmaceutical composition.

In one embodiment, the kit contains the anti-TIGIT antibody or antigen-binding fragment thereof of the present invention in one container or the vector or host cell or pharmaceutical composition thereof as described above and the pharmaceutical composition and / or its composition in another container and / or Or therapeutic agents.

Simultaneous administration of the two therapeutic agents does not require the agents to be administered at the same time or through the same route, as long as there is overlap in the time period over which the agents exert their therapeutic effects.

In another aspect, the above kit contains a label attached to or packaged with the container, the label describing the contents of the container and providing indications and / or methods for using the contents of the container, which are useful for treatment And / or instructions for preventing one or more disease states as described herein.

The kit may optionally further include a syringe for parenteral, such as intravenous administration. The kit also includes a device for tightly containing the vial for commercial sale and / or packaging and delivery.

Derived antibody

The antibody or antigen-binding fragment of the present invention can be derivatized, for example, linked to another molecule (eg, another polypeptide or protein). Generally, derivatization (eg, labeling) of an antibody or antigen-binding fragment thereof does not adversely affect its binding to TIGIT (particularly human TIGIT). Therefore, the antibodies or antigen-binding fragments thereof of the present invention are also intended to include such derivatized forms. For example, the antibody or antigen-binding fragment of the invention can be functionally linked (by chemical coupling, gene fusion, non-covalent linkage, or other means) to one or more other molecular groups, such as another antibody (eg, formed Bispecific antibodies), detection reagents, pharmaceutical reagents, and / or proteins or polypeptides (eg, avidin or polyhistidine tags) that can mediate the binding of the antibody or antigen-binding fragment to another molecule.

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

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

Another type of derivatized antibody is a labeled antibody. For example, the antibody or antigen-binding fragment of the present invention can be linked to a detectable label. The detectable label according to the present invention may be any substance that can be detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical or chemical means. Such labels are well known in the art, and examples include, but are not limited to, enzymes (eg, horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radioactive nuclei (E.g., 3H, 125I, 35S, 14C, or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin ( PE), Texas Red, Rhodamine, quantum dots or cyanine dye derivatives (eg Cy7, Alexa 750)), acridine ester compounds, magnetic beads (eg,

), Calorimetric markers such as colloidal gold or colored glass or plastic (eg, polystyrene, polypropylene, latex, etc.) beads, and avidin (eg, streptavidin) modified to bind the above markers ) Of biotin. In certain embodiments, a detectable label as described above can be linked to the antibody or antigen-binding fragment of the present invention through linkers of different lengths to reduce potential steric hindrance.

In addition, the antibody or antigen-binding fragment of the present invention can also be derivatized with chemical groups, such as polyethylene glycol (PEG), methyl or ethyl, or sugar groups. These groups can be used to improve the biological properties of antibodies, such as increasing serum half-life.

Monoclonal antibody preparation

The mAb of the present invention can be prepared by a variety of techniques, including conventional mAb methodology, such as the standard somatic cell hybridization technique described in Kohler G and Milstein C, Nature, 256: 49, 1975. In the hybridoma method, mice, hamsters, or other suitable host animals are usually immunized with an immunological agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind the immunological agent. Although somatic cell hybridization protocols are preferred, in principle other methods of preparing mAb, such as viral or oncogenic transformation of B lymphocytes, can also be used.

The animal used for preparing the hybridoma is not particularly limited, but it is preferably selected considering its compatibility with the parent cell used for cell fusion. Usually transformed mammalian cells, especially myeloma cells of rodent, bovine and human origin. The preferred animal system is the murine system. The preparation of hybridomas in mice is a very complete procedure. Immunization protocols and techniques for isolating immunized spleen cells for fusion are known in the art. Fusion partners (eg, murine myeloma cells) and fusion protocols are also known.

Methods for obtaining mAbs from hybridomas include culturing hybridomas and obtaining mAbs from the culture supernatant according to standard methods. Another method involves applying hybridomas to hybridoma compatible mammals to proliferate them, and obtaining mAbs from ascites fluid. The former method is suitable for obtaining high-purity antibodies, and the latter method is suitable for mass production of antibodies.

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

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

To express antibodies or antibody fragments thereof, DNA encoding partial or full-length light and heavy chains can be obtained by standard molecular biology techniques (eg, PCR amplification or cDNA cloning using hybridomas expressing the target antibody), and the DNA It is inserted into an expression vector so that the gene of interest is operably linked to transcriptional and translational regulatory sequences to transfect the host cell for expression. The expression host is preferably a eukaryotic expression vector, and more preferably a mammalian cell, such as CHO and its derived cell lines.

The humanized antibody of the present invention can be prepared according to the mouse anti-sequence prepared above. DNA encoding heavy and light chain immunoglobulins can be obtained from target murine hybridomas and engineered using standard molecular biology techniques to include non-mouse (eg, human) immunoglobulin sequences. For example, to create humanized antibodies, mouse CDR regions can be inserted into human framework sequences using methods known in the art, see Winter US Patent No. 5,225,539 and Queen et al. US Patent Nos. 5,530,101; 5,585,089; 6,180,370. Transgenic animals can also be used, for example, HuMAb mice (Medarex, Inc.) containing human immunoglobulin gene miniloci (miniloci) encoding unrearranged human heavy chain (μ and γ) and κ light chain immunoglobulin sequences ), Plus targeted mutations that inactivate endogenous μ and κ chain loci (see, for example, Lonberg N et al., Nature, 368: 856-859, 1994); or those carrying human heavy chain transgenes and human light chain transchromosomes " "KM mouse TM" (see patent WO 02/43478) for antibody humanization. Other methods of humanized antibody modification include, for example, phage display technology.

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

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

BRIEF DESCRIPTION

Figure 1. The ability of anti-TIGIT murine antibodies Mab21 and Mab7 to bind to human TIGIT antigen by ELISA.

Figure 2. ELISA detects the ability of anti-TIGIT humanized antibodies to bind to human TIGIT antigen.

Figure 3-1. ELISA detects the cross-reaction of anti-TIGIT humanized antibody with mouse or rat TIGIT antigen.

Figure 3-2. ELISA detects the cross-reaction of anti-TIGIT humanized antibody with cynomolgus monkey TIGIT antigen.

Figure 4. TSF value of anti-TIGIT humanized antibody detected by DSF.

Figure 5-1. ELISA detects the ability of anti-TIGIT humanized antibodies to block the binding of human TIGIT to CD155.

Figure 5-2. ELISA detects the ability of anti-TIGIT humanized antibodies to block the binding of human TIGIT to CD112.

Figure 6. Flow cytometry to detect the affinity of AB12V8 and AB12V9 antibodies to Jurkat-TIGIT cells.

Figure 7. Flow cytometry to detect the ability of AB12V8 and AB12V9 antibodies to block the binding of Jurkat-TIGIT to CD155.

Figure 8. Flow cytometry detection of AB12V8 and AB12V9 antibodies binding to CIK cells.

Figure 9. Microplate reader detects the CDC activity of AB12V8 and AB12V9 antibodies.

Figure 10. Microplate reader detects the ability of AB12V7, AB12V8 and AB12V9 to bind to FcRn.

Figure 11. The microplate reader detects the ADCC activity of AB12V8 and AB12V9.

Figure 12-1. The detection of the ability of AB12V1 and PD-1 antibody AB12M4 to promote IFN-γ secretion.

Figure 12-2. The detection of the ability of AB12V4 combined with PD-1 antibody AB12M4 to promote IFN-γ secretion.

Figure 12-3. The detection of the ability of AB12V8 and PD-1 antibody AB12M4 to promote IFN-γ secretion.

Figure 12-4. The detection of the ability of AB12V9 combined with PD-1 antibody AB12M4 to promote IFN-γ secretion.

Figure 13. Detection of AB12V8 and AB12V9 enhancing the ability of NK cells and T cells to kill HCC827 cells.

Figure 14. AB12V8 and AB12V9 increase luciferase expression in the TIGIT / CD155 luciferase reporter gene system.

Figure 15. AB12V8 inhibits the growth of tumor cell MC38 cells in TIGIT transgenic mice.

Notes on the preservation of biological materials

The present invention relates to the following biological materials that have been deposited in the Chinese Type Culture Collection (CCTCC, Wuhan University, Wuhan, China):

detailed description

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

Unless otherwise specified, the molecular biology experimental methods and immunoassays used in the present invention basically refer to J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989, and FMAusubel et al., Compilation of Experimental Guidelines for Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995. The use of restriction enzymes is in accordance with the conditions recommended by the product manufacturer. Those skilled in the art know that the embodiments describe the present invention by way of example, and are not intended to limit the scope of the claimed invention.

Example 1. Preparation of anti-human TIGIT mouse monoclonal antibody

After 50μg of human TIGIT extracellular domain antigen (Uniport entry: Q495A1, prepared by Anyuan Pharmaceutical Technology (Shanghai) Co., Ltd.) was fully emulsified with complete Freund's adjuvant, male Balb / C mice were immunized with multi-point immunization, and the immunization cycle was Once every three weeks. On the 10th day after the third immunization, blood was drawn through the tail vein and the plasma anti-human TIGIT antibody titer was tested by ELISA to monitor the immune response of the mice. Then, 3 days before fusion, the highest anti-human TIGIT antibody titer Mice are boosted once. After 3 days, the mouse was sacrificed and the spleen of the mouse was removed and fused with the mouse myeloma Sp 2/0 cell line. 2 × 10 ⁸ Sp 2/0 cells were mixed with 2 × 10 ⁸ splenocytes in 50% polyethylene glycol (molecular weight 1450) and 5% dimethyl sulfoxide (DMSO) solution for fusion. Use HAT selection medium (DMEM medium, containing 10% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, 0.1 mM hypoxanthine, 0.4 μM aminopterin and 16 μg thymidine) to adjust the number of spleen cells To 5 × 10 ⁵ / mL, 0.3mL was added to the well of 96-well culture plate, and placed in 37 ° C, 5% CO ₂ incubator. After culturing for 10 days, high-throughput ELISA method was used to detect clones with high affinity binding of antibody and TIGIT-His in the supernatant. The fusion cells in the wells of the above monoclonal antibodies are then subcloned. Then, by a competitive ELISA method, the positive wells that compete with human CD155 / hFc (purchased from Sino Biological Company, article number: 10109-H02H) to bind TIGIT (see Example 6.4 for the method) are obtained, and hybridoma cell line # 21 is obtained , # 7. Specific antibody-producing clones were cultured in RPMI 1640 medium supplemented with 10% FBS. When the cell density reached approximately 5 × 10 ⁵ cells / mL, the medium was replaced with serum-free medium. After 2 to 4 days, the cultured medium was centrifuged to collect the culture supernatant. The protein G column was used for antibody purification. The monoclonal antibody eluate was dialyzed against 150 mM NaCl. The dialysis solution was filtered and sterilized through a 0.2 μm filter to obtain purified mouse monoclonal antibodies Mab21 and Mab7 corresponding to the hybridoma cell lines # 21 and # 7 to be tested.

Example 2. ELISA method was used to determine the binding ability of murine antibody to human TIGIT antigen

Enzyme plates were coated with human TIGIT and kept at room temperature overnight. The coating solution was discarded, and each well was closed with skim milk dissolved in phosphate buffered saline (PBS) for 1 hour, and the well was washed with PBS containing 0.05% Tween-20 (Tween-20). Then add 50 μL of purified anti-human TIGIT murine antibody Mab21, Mab7 per well, using mouse IgG (prepared by mouse serum with ProteinG purification) as a negative control, and PBS as a blank control, incubate at room temperature for 1 hour, containing Tween 20 (Tween-20) was washed with PBS, and then 50 μL of HRP-labeled goat anti-mouse IgG polyclonal antibody (purchased from Jackson Laboratory, catalog number: 115-035-071) was added as a detection antibody.

The results are shown in Figure 1. The murine antibodies Mab21 and Mab7 have a high affinity with human TIGIT, both of which have an EC ₅₀ of about 18 pM.

Example 3. Subtype identification and variable region amplification of anti-TIGIT murine monoclonal antibody

Antibody subtype identification: The supernatant of the hybridoma cell culture was taken, and the antibody subtype was identified using the IsoStrip ^™ mouse monoclonal antibody subtype identification kit (Santa Cruz Biotechnology, article number: sc-24958). The murine monoclonal antibody Mab21 subtype was identified as IgG2b (Kappa) type. The mouse monoclonal antibody Mab7 subtype was identified as IgG1 (Kappa) type.

Antibody variable region amplification: the candidate hybridoma # 21, # 7 culture to a total number of ¹⁰⁷ cells, cells were collected by centrifugation 1000rpm 10min, and using mRNA extraction kit (purchased from NEB, NO: S1550S) Extraction of Total For RNA, first-strand cDNA was synthesized using the reverse transcription kit SMARTer RACE (purchased from TAKARA, article number: 634858), and the first-strand cDNA was used as the subsequent template to amplify the DNA sequence of the antibody variable region corresponding to the hybridoma cells. According to the subtype identification results, the heavy and light chain constant region sequences of the antibody subtype are obtained, and specific nested PCR primers are designed. The primer sequences used in the amplification reaction and the first framework region of the antibody variable region and The constant regions are complementary. Amplify the target gene by conventional PCR method and sequence the amplified product to obtain the heavy chain variable region sequence SEQ ID NO: 1 and the light chain variable region sequence SEQ ID NO: 2 of hybridoma clone # 21 secreted antibody Mab21. The heavy chain variable region sequence SEQ ID NO: 3 and the light chain variable region sequence SEQ ID NO: 4 of the hybridoma clone # 7 secreted antibody Mab7 were obtained.

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

Table 1-1: Heavy chain CDR and light chain CDR of murine antibody Mab21

Table 1-2: Heavy chain CDR and light chain CDR of murine antibody Mab7

The above CDR region sequences are defined by Chothia, Kabat, and IMGT methods, respectively, and any other CDR region sequence determination methods known in the art may also be used to identify the amino acid residues of the CDR regions within the variable region.

Example 4. Affinity detection of anti-human TIGIT murine antibody

The binding affinity constants of the purified mouse monoclonal antibodies Mab21 and Mab7 to the antigen were measured using biofilm interference technology (BLI). The instrument was ForteBio Octet QK system of PALL. Multi-channel parallel quantitative analysis concentration gradients were set to: 3.125, 6.25, 12.5, 25, 50, and 100 nM. Human TIGIT (His tag) coupled Ni-NTA sensor (purchased from Fortebio, article number: 18-5101). The results of the affinity determination are shown in Table 2. The results show that the mouse monoclonal antibodies Mab21 and Mab7 have extremely high binding affinity to human TIGIT, reaching the order of 10 ^-11 and 10 ^-12 M, respectively.

Table 2: Results of affinity determination of murine monoclonal antibody

Example 5. Humanization of anti-TIGIT murine antibody

The humanized modification of murine antibodies Mab21 and Mab7 was carried out by the humanized modification method of CDR grafted antibody. In short, the humanization transformation involves the following steps: the amino acid sequence of the mouse monoclonal antibody is compared with the amino acid sequence of the human germline antibody to find the sequence with high homology and better physical and chemical properties, as the human embryonic line Framework sequence; analyze and investigate the affinity of HLA-DR, select the human embryonic framework sequence with low affinity; then transplant the six CDRs of murine antibody to the selected heavy chain and light chain framework sequences.

Specifically, the heavy chain and light chain CDR regions of the murine antibodies Mab21 and Mab7 were respectively transplanted into the FR framework of the corresponding humanized template. The heavy chain and light chain humanized templates of Mab21 are the human germline gene sequences IGHV1-69-2 * 01 (see IMGT accession number KF698734) and IGKV6-21 * 02 (see IMGT accession number KM455568), the heavy chain of Mab7, The humanized light chain templates are the human germline gene sequences IGHV1-2 * 02 (see IMGT accession number X62106) and IGKV1-39 * 01 (see IMGT accession number X59315).

Furthermore, using computer simulation technology, molecular docking was used to analyze the framework amino acid sequence of the variable region and its surroundings, and to examine the spatial three-dimensional binding mode. By calculating electrostatic force, van der Waals force, hydrophobicity and entropy value, the amino acid sequence of the mouse antibody can be analyzed to interact with TIGIT and maintain the key amino acids of the space framework, and retain these mouse amino acids in the antibody after transplantation. That is, a series of back mutations were made to the amino acid residues of the FR region of the humanized template, so that the humanized antibody retains the antigen-binding ability of the murine antibody as much as possible.

According to the above method, based on the CDR of the mouse antibody Mab21, a total of 2 humanized antibodies were constructed, named AB12V7 and AB12V9; wherein, the heavy chain constant regions of each antibody were human wild-type IgG1 heavy chain constant regions ( SEQ ID NO: 47), the amino acid sequence of each light chain constant region is shown in SEQ ID NO: 48. At the same time, based on the CDR of the mouse antibody Mab7, a humanized antibody was constructed, named AB12V8; of which, the heavy chain constant region of the antibody was the human wild type IgG1 heavy chain constant region (SEQ ID NO: 47), light chain The amino acid sequence of the constant region is shown in SEQ ID NO: 48.

In addition, the variable region sequence of the antibody secreted by the hybridoma cells obtained above is also humanized by surface remodeling. The surface remodeling method refers to the humanized modification of the amino acid residues on the surface of the heterologous antibody. This method only replaces the areas that are significantly different from the amino acid on the surface of the human antibody. Amino acid substitutions with similar residues on the antibody surface. Specifically, the process of surface remodeling and humanization involves the following steps: First, the amino acid sequence of the antibody secreted by each hybridoma cell is compared with the amino acid sequence of the human embryonic antibody to find a sequence with high homology; then use a computer Simulation technology, when the solubility (solvent) is greater than 30%, the exposed surface amino acid is substituted for the amino acid of the adult embryonic antibody. Residues that affect the size, charge, hydrophobicity of the side chain, or may form hydrogen bonds, which affect the conformation of the complementarity determining region of the antibody, are not replaced as much as possible.

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

Among them, the heavy chain constant region of AB12V3 and AB12V4 is a human IgG4 heavy chain constant region variant (IgG4V1, SEQ ID NO: 46), which is a human IgG4 heavy chain constant region containing Ser228Pro mutation, which has a stable structure and can reduce Fab-arm exchange , So that it is not easy to form a half antibody, the amino acid sequence of the light chain constant region is shown in SEQ ID NO: 48; the heavy chain constant region of AB12V5, AB12V6 is the human wild type IgG1 heavy chain constant region (IgG1; SEQ ID NO: 47), The amino acid sequence of the light chain constant region is shown in SEQ ID NO: 48.

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

Table 3: Amino acid sequences of variable and constant regions of humanized antibodies

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

The recombinant expression vector plasmid constructed above is transfected into a mammalian host cell line to express humanized antibodies. To stabilize high levels of expression, the preferred host cell line is DHFR deficient Chinese hamster ovary (CHO) cells (see US Patent No. 4,818,679). The preferred transfection method is electroporation, and other methods can also be used, including calcium phosphate co-sedimentation, lipofection, and protoplast fusion. In electroporation, a GenePulser (Bio-Rad Laboratories) set to an electric field of 300 V and a capacitance of 1050 μFd was added to the cuvette, 2 × 10 ⁷ cells were suspended in 0.8 ml of PBS, and contained 20 μg of the expression vector plasmid. Two days after transfection, M418 (Sigma) containing 0.2 mg / ml G418 and 200 nM was added. In order to achieve higher levels of expression, the transfected antibody gene was co-amplified with the DHFR gene inhibited by MTX drugs. The secretion rate of each cell line was measured by limiting dilution subcloning transfectants and ELISA method, and cell lines expressing antibodies at high levels were selected. The conditioned medium of the antibody was collected and used to determine its biological activity in vitro and in vivo.

Example 6. Screening and identification of anti-TIGIT humanized antibodies

6.1. Determination of the binding ability of humanized TIGIT antibody to human TIGIT

Human TIGIT / mFc (manufactured by Anyuan Pharmaceutical Technology (Shanghai) Co., Ltd.) was diluted to 1 μg / ml with PBS buffer, added to a 96-well plate at a volume of 100 μl / well, and placed at 4 ° C for 16-20 h. Discard the coating solution, add PBS / 2% BSA solution, block at 37 ° C for 2h; remove the blocking solution, wash the plate 3 times with PBST (pH 7.4, PBS containing 0.05% Tween 20) buffer, and use PBS / 2% The anti-TIGIT humanized antibodies AB12V4, AB12V5, AB12V7, AB12V8, AB12V9 and the reference antibody AB12V1 diluted in the BSA solution were tested. As shown in SEQ ID NOs: 67 and 68 herein), AB12V2 (the variable region sequence is from anti-TIGIT antibody 10A7 in US Patent No. US 20150216970A1, and its heavy and light chains are shown in SEQ ID NOs: 69 and 70, respectively), Add a series of concentration gradients of the tested TIGIT antibody, 100μl / well, and incubate at 37 ° C for 2h; remove the reaction system, wash the plate 3 times with PBST, and dilute HRP-anti human IgG (H + L) with PBS / 2% BSA solution (Purchased from Jackson ImmunoResearch, article number: 109-035-003), 100 μl / well, incubated at 37 ° C for 1 h; add 100 μl / well TMB solution to the corresponding well, and develop at room temperature for 10 min; add 50 μl 1M H ₂ SO ₄ solution The color development was terminated, and the absorbance was read at 450 nm using a microplate reader. The results are shown in Figure 2 and Table 4. Most antibodies have a TIGIT affinity of 10 ^-10 M, which has a very high binding affinity. Among them, AB12V5, AB12V7 and AB12V8 have the strongest antigen binding ability.

Table 4: Measurement results of the binding ability of anti-TIGIT humanized antibody to human TIGIT

抗体名称Antibody name	EC50(nM)EC50 (nM)
AB12V1AB12V1	0.073570.07357
AB12V2AB12V2	0.16790.1679
AB12V4AB12V4	0.070940.07094
AB12V5AB12V5	0.047370.04737
AB12V7AB12V7	0.053870.05387
AB12V8AB12V8	0.054210.05421
AB12V9AB12V9	0.16710.1671

6.2. Species cross-reaction determination of humanized antibodies

In order to detect the differences in specific binding of anti-TIGIT humanized antibodies to different species other than humans, mouse, cynomolgus monkey, and rat TIGIT were used for binding detection.

Dilute mouse TIGIT / His and cynomolgus monkey TIGIT / His (both purchased from Acro Biosystems, article numbers are TIT-M52E6, TIT-C5223) with PBS buffer to 0.1 μg / ml at a volume of 100 μl / well Add to 96-well plate, and place at 4 ℃ for 16-20h. Aspirate the coating solution from the 96-well plate, wash the plate 3 times with PBST buffer, add 200 μl / well PBST / 1% skim milk powder, and incubate at room temperature for 1 hour to block. Remove the blocking solution, wash the plate 3 times with PBST buffer, add a series of concentration gradients of the tested TIGIT antibody, 100μl / well, and incubate at room temperature for 2h. After removing the reaction system and washing the plate 3 times with PBST, add 100 μl / well of HRP-labeled goat anti-human IgG secondary antibody (purchased from Jackson Immuno Research Corporation, diluted with PBST / 1% skim milk powder (dilution ratio 1: 5000) : 109-035-003), incubated at room temperature for 1h. Wash the plate 3 times with PBST, add 100 μl / well TMB, and incubate at room temperature for 5-10 min. Add 50 μl / well of 0.2M H ₂ SO _{4 to} stop the reaction. The microplate reader reads the absorbance at a dual wavelength of 450 / 620nm.

Since the rat TIGIT antigen is a human Fc tag (purchased from Acro Biosystems, article number: TIT-R5258), in order to test the ability of anti-TIGIT antibodies to specifically bind to rat TIGIT antigen, the anti-TIGIT humanized antibody to be tested uses EZ-Link ^TM Sulfo-NHS-Biotinylation kit (purchased from Thermo Scientific, catalog number: 21425) was biotin labeled, using HRP-labeled streptavidin (purchased from BD Biosciences, catalog number: 554066) as the detection antibody, the specific method is the same as above Said.

The results are shown in Figures 3-1 and 3-2. All anti-TIGIT humanized antibodies have no species cross-reactivity with the mouse TIGIT antigen, and all anti-TIGIT humanized antibodies can specifically bind to the cynomolgus monkey TIGIT Antigen; in addition, all anti-TIGIT humanized antibodies do not have species cross-reactivity with rat TIGIT antigens, but the reference antibody AB12V1 can bind to rat TIGIT antigen. The anti-TIGIT humanized antibody of the present invention has a different binding than AB12V1 region.

6.3 Determination of Tm value of humanized antibody

The Tm value of anti-TIGIT antibody was determined by DSF (differential fluorescence scanning technique) method. The specific experimental steps are as follows. 12.5 μl 40 × SYPRO Orange dye (purchased from Life technologies, Catalog No .: 56651), 5 μl 1 mg / ml TIGIT antibody to be tested (diluted in PBS), and 7.5 μl deionized H ₂ O are mixed in the EP tube , The above sample mixture was added to the Q-PCR system (AB Applied Biosystems ABI, 7500) to react, Q-PCR parameter settings: Target (ROX), program (25 ℃, 3min; 1% rate, 95 ℃; 95 ℃, 2min ). The results were imported into Graph Prism software to calculate the Tm value. As shown in Figure 4 and Table 5, the Tm values of all anti-TIGIT humanized antibodies were higher than 65 ° C, better than the Tm values of AB12V1 and AB12V2, and had excellent thermal stability. The Tm value of AB12V6, AB12V8 and AB12V9 reaches about 70 ° C, indicating that AB12V6, AB12V8 and AB12V9 have significantly excellent thermal stability.

Table 5: Measurement results of humanized antibody Tm value

抗体名称Antibody name	Tm(℃)Tm (℃)
AB12V1AB12V1	63.5363.53
AB12V2AB12V2	64.0364.03
AB12V4AB12V4	66.8766.87

AB12V5AB12V5	68.1368.13
AB12V6AB12V6	73.8673.86
AB12V7AB12V7	65.865.8
AB12V8AB12V8	71.3271.32
AB12V9AB12V9	69.7969.79

6.4. Humanized antibody blocking TIGIT binding to CD155 or TIGIT binding to CD112

In order to detect the humanized TIGIT antibody and block the interaction between TIGIT / CD155, label the CD155-mFc protein according to the instructions of the HRP labeling kit (purchased from Beijing Boaolong Company, article number: BF06095-1000), and determine the human source by the competitive Elisa method Blocking activity of anti-body.

Dilute human TIGIT to 1 μg / ml with PBS buffer, add it to a 96-well plate at a volume of 100 μl / well, and place it at 4 ° C for 16-20 h. Discard the coating solution, add 200 μl / well PBS / 2% BSA solution, and incubate at 37 ° C for 2h. Remove the blocking solution, wash the plate 3 times with PBST buffer, add 50μl / well of a series of concentration gradient test TIGIT antibody and 50μl / well HRP-CD155 (25μg / ml), and incubate at 37 ℃ for 2h. Remove the reaction system, wash the plate 5 times with PBST, add 100 μl / well TMB, and develop at room temperature for 5 min. Add 50 μl / well 1M H ₂ SO _{4 to} stop the reaction. The microplate reader reads the absorbance at a wavelength of 450 nm.

In order to detect the humanized TIGIT antibody and block the interaction between TIGIT / CD112, the biotinylated CD112-hFc protein (purchased from Acro Biosystems, catalog number: CD2-H82F8) was used to compete with the Elisa method for the detection of humanized TIGIT Blocking activity.

Dilute TIGIT-mFc to 2μg / ml with PBS buffer, add it to a 96-well plate at a volume of 100μl / well, and place at 4 ° C for 16-20h. Discard the coating solution, wash the plate 3 times with PBST, add 200 μl / well PBST / 1% skimmed milk powder, and incubate at room temperature for 1 h to block. Remove the blocking solution, wash the plate 3 times with PBST buffer, add 50μl / well of a series of concentration gradients of the test TIGIT antibody and 50μl / well of 2μg / ml biotinylated human CD112 (purchased from Acro Biosystems, article number: CD2 -H82F8), incubate at room temperature for 2h. Remove the reaction system, wash the plate 3 times with PBST, add 100 μl / well and dilute with PBST / 1% skimmed milk powder (dilution ratio 1: 500) HRP-labeled streptavidin (purchased from BD Biosciences, article number: 554066) , Incubate at room temperature for 1h. After washing the plate 3 times with PBST, add 100 μl / well TMB and incubate at room temperature for 10-15 min. Add 50 μl / well of 0.2M H ₂ SO _{4 to} stop the reaction. The microplate reader reads the absorbance at a dual wavelength of 450 / 620nm.

The results are shown in Figures 5-1, 5-2, and Table 6. All humanized TIGIT antibodies tested specifically blocked human TIGIT and CD155 (Figure 5-1) or human CD112 (Figure 5-2) ) Has excellent blocking activity of nM level. The ability of AB12V4 and AB12V8 antibodies to block TIGIT / CD155 binding is stronger than that of AB12V1 and AB12V2; the ability of AB12V9 antibody to block TIGIT / CD155 binding is basically equivalent to AB12V1 and AB12V2. The ability of AB12V4 antibody to block TIGIT / CD112 binding is stronger than AB12V1; the ability of AB12V8 antibody to block TIGIT / CD112 binding is basically equivalent to AB12V1.

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

Antibody name

IC ₅₀ (pM)

A	TIGIT/CD155TIGIT / CD155	TIGIT/CD112TIGIT / CD112
AB12V1AB12V1	475.7475.7	762.11762.11
AB12V2AB12V2	492.1492.1	未检测Not detected
AB12V4AB12V4	351.1351.1	583.7583.7
AB12V5AB12V5	872.9872.9	未检测Not detected
AB12V6AB12V6	未检测Not detected	10431043
AB12V7AB12V7	11551155	未检测Not detected
AB12V8AB12V8	340.5340.5	774774
AB12V9AB12V9	501.3501.3	932932

6.5 Affinity determination and kinetic analysis of anti-TIGIT humanized antibodies

Biofilm interference technology (BLI) was used to determine the affinity and binding kinetics of the purified anti-TIGIT humanized antibody. The measurement was carried out according to the standard operating method of Octet Molecular Interaction Apparatus (ForteBio Octet QK System, PALL Corporation). Multi-channel parallel quantitative analysis concentration gradients were set to: 3.125, 6.25, 12.5, 25, 50 and 100 nM, and human TIGIT-His was coupled to Ni-NTA sensor. Track antigen-antibody binding kinetics and dissociation kinetics. The resulting analytical data, K _a, K _d and K _D values measured by this method are shown in Table 7. All humanized monoclonal antibodies showed affinities of 10 ^-11 M level. Among them, the equilibrium dissociation constant K _D values of AB12V4, AB12V8 and AB12V9 were 1.56 × 10 ^-11 M, 1.79 × 10 ^-11 M and 2.52, respectively. × 10 ^-11 M, showing good antigen binding activity.

Table 7: Humanized TIGIT antibody affinity determination results

6.6 、 Affinity test of anti-TIGIT humanized antibody and Jurkat-TIGIT cells

In order to detect the cell affinity of anti-TIGIT humanized antibodies, the full-length TIGIT gene (Gene ID: 201633) was first transferred into Jurkat cells (purchased from Nanjing Kebai Biotechnology Co., Ltd.) by lentiviral method. Perform pressure screening to construct Jurkat-TIGIT stable cell line. The anti-TIGIT humanized antibody to be tested was labeled with NHS-Fluro (purchased from Thermo Company, Catalog No. 46610), diluted with PBS buffer to the labeled antibody, starting at 1 μM, 4-fold gradient dilution, 3 × 10 ⁵ Jurkat- TIGIT cells were resuspended with 50 μl of diluted antibody, incubated at 4 ° C for 0.5 h, washed twice with PBS, and detected by flow cytometry (Beckman, model CytoFlex). The result of the fluorescence signal value is imported into Graph Prism software to calculate the IC ₅₀ value. The results are shown in Figure 6 and Table 8. The results show that AB12V8 and AB12V9 antibodies can specifically bind to TIGIT on the cell surface, and AB12V8 and Jurkat-TIGIT cell affinity is stronger than the reference Antibody AB12V1.

Table 8: Anti-TIGIT humanization and Jurkat-TIGIT cell affinity test results

抗体名称Antibody name	EC ₅₀(nM) EC ₅₀ (nM)
AB12V1AB12V1	0.7440.744
AB12V8AB12V8	0.6820.682
AB12V9AB12V9	1.121.12

6.7. Anti-TIGIT humanized antibody blocks Jurkat-TIGIT / CD155 binding detection

N155-Fluro (purchased from Thermo Company) labeled CD155 was used as a reagent. Dilute the anti-TIGIT humanized antibody to be tested to 200 μg / ml in PBS buffer, dilute in a 4-fold gradient, and set up 10 concentration gradients. In addition, dilute FITC-CD155 to 600 nM with PBS buffer. The test antibody and FITC-CD155 were mixed 1: 1, and 3 × 10 ⁵ Jurkat-TIGIT cells (for the preparation method see Example 6.6) were resuspended with 50 μl of the mixture containing the test antibody and FITC-CD155, and incubated at 4 ° C for 0.5 h. After washing twice with PBS, it was detected using a flow cytometer (Beckman, model CytoFlex). The result of the fluorescence signal value is imported into Graph Prism software to calculate the IC ₅₀ value. The results are shown in Figure 7 and Table 9. The results show that both AB12V8 and AB12V9 antibodies can block the Jurkat-TIGIT / CD155 binding and are significantly stronger than the reference antibody AB12V1.

Table 9: Detection results of anti-TIGIT humanized antibody blocking Jurakt-TIGIT / CD155 binding

抗体名称Antibody name	IC ₅₀(nM) IC ₅₀ (nM)
AB12V1AB12V1	44.7744.77
AB12V8AB12V8	14.614.6
AB12V9AB12V9	29.0229.02

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

In order to detect the affinity of anti-TIGIT humanized antibodies and human CIK (cytokine-induced killer) cells, PBMCs were isolated from fresh blood of healthy people and cultured with anti-CD3 and anti-CD28 antibodies. Label the anti-TIGIT humanized antibody to be tested with NHS-Fluro, dilute the labeled antibody with PBS buffer, start with 0.4 μM, 5-fold gradient dilution, 3 × 10 ⁵ CIK cells cultured for 15 days with 50 μl after dilution The antibody was resuspended, incubated at 4 ° C for 0.5h, washed twice with PBS, and detected using a flow cytometer (Beckman, model CytoFlex). The results of the fluorescence signal values were imported into Graph Prism software. The results are shown in Figure 8. The results show that AB12V8 and AB12V9 antibodies have a strong affinity with CIK cells, of which AB12V8 antibody has a stronger affinity with CIK cells than reference antibody AB12V1.

6.9. Anti-TIGIT humanized antibody CDC activity determination

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

To determine the binding activity of the antibody to C1q, the anti-TIGIT humanized antibody to be tested was diluted to 600 μg / ml in carbonate buffer pH 9.6, diluted in a 3-fold gradient, 11 concentration points were set, and a series of tested antibodies were tested at 100 μl / Well volume was added to 96-well plate at 4 ° C overnight; discard the coating solution and wash the plate once with PBS; add PBS / 2% BSA solution and block at 37 ° C for 2h; remove the blocking solution and wash the plate with PBST buffer 3 times, dilute C1q (purchased from Prospec) with PBS / 2% BSA solution to 5 μg / ml, add 100 μl / well to 96-well plate, incubate at 37 ° C for 2 h; wash the plate 3 times with PBST; add 100 μl / well PBS / 2% BSA solution diluted (dilution ratio 1: 300) Shp pAb to C1q (HRP) (purchased from Abcam), incubate at 37 ℃ for 1h; wash the plate 5 times with PBST buffer, add 100μl / well TMB solution to the corresponding In the wells, the color was developed at room temperature for 5 min; 50 μl of 1M H ₂ SO ₄ solution was added to stop the color development, and the absorbance was read by a microplate reader at 450 nm.

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

To determine the activity of antibody and complement to directly kill target cells, anti-TIGIT humanized antibody was diluted in RPMI 1640 medium containing 20% guinea pig serum, starting at 100 μg / ml, 5-fold dilution, 100 μl / well; Jurkat-TIGIT cells were taken ( For the preparation method, see Example 6.6), centrifugation, adjust the density to 5 × 10 ⁵ / ml in RPMI 1640 medium containing 20% guinea pig serum, add 100 μl / well to the corresponding well, mix and incubate at 37 ° C for 3 h, 20 μl / Well, the microplate reader reads the absorbance at 450nm after incubating at 37 ℃ for 5h. The results are shown in Figure 9 and Table 10. The results show that AB12V1 has no CDC activity, while AB12V8 and AB12V9 have similar activities and can reach nM level.

Table 10: Test results of anti-TIGIT humanized antibody CDC activity

抗体名称Antibody name	EC ₅₀(nM) EC ₅₀ (nM)
AB12V1AB12V1	无活性Inactive
AB12V8AB12V8	0.840.84
AB12V9AB12V9	0.820.82

6.10. Anti-TIGIT humanized antibody FcRn binding activity determination

The anti-TIGIT humanized antibody to be tested was diluted to 5 μg / ml with carbonate buffer pH 9.6, and added to a 96-well plate at a volume of 100 μl / well at 4 ° C overnight; the coating solution was discarded and the plate was washed once ; Add triethanolamine buffer salt (TBS) solution containing 0.5% BSA, block at 37 ℃ for 2h; remove the blocking solution, dilute FcRn with TBS (pH 6.0) /0.5% BSA solution (purchased from Acro Biosystems, article number: FCM- H82W4) to 10μg / ml, 3-fold gradient dilution, 11 concentration points, add 100μl / well to 96-well plate, incubate at 37 ° C for 2h; wash plate 3 times with TBST; add 100μl / well with TBS / 0.5% Dilute BSA (dilution ratio 1: 500) HRP-labeled streptavidin (purchased from Proteintech, catalog number: SA00001-0), and incubate at 37 ° C for 1 h. Wash the plate 5 times with TBST buffer, add 100μl / well TMB solution to the corresponding well, and develop the color at room temperature for 5min; add 50μl 1M H ₂ SO ₄ solution to stop the color development. The absorbance is read at 450nm with a microplate reader. The results are shown in Figure 10 and As shown in Table 11, AB12V8 has the strongest FcRn-binding activity (EC ₅₀ = 1.474 nM), and the affinity of antibody to FcRn has a positive correlation with its half-life in vivo. It is speculated that the half-life of AB12V8 in vivo will be longer than that of reference antibody AB12V1.

Table 11: Results of measurement of anti-TIGIT humanized antibody FcRn binding activity

抗体名称Antibody name	EC ₅₀(nM) EC ₅₀ (nM)
AB12V1AB12V1	2.7272.727
AB12V7AB12V7	3.353.35
AB12V8AB12V8	1.4741.474
AB12V9AB12V9	3.2763.276

6.11. Determination of ADCC activity of anti-TIGIT humanized antibody

To detect the ADCC activity on anti-TIGIT humanized antibody cells, the full-length CD16a gene (Gene ID: 2214) and luciferase gene were transferred into Jurkat cells (purchased from Nanjing Kebai Biotechnology Co., Ltd.) by lentivirus ), And pressurized screening with puromycin and bleomycin to construct Jurkat-NFAT / luciferase-CD16a stable cell line as effector cells; similarly, the full-length TIGIT gene (Gene ID: 201633) was lentivirused It was transferred into CHO-S cells (purchased from Thermo Company), and subjected to pressurized screening with puromycin and bleomycin to construct a CHO-S-TIGIT stable cell line as a target cell. When the effector cells, antibodies and target cells coexist, it will activate the expression of luciferase, and then use ONE-Glo reagent (purchased from Promega, article number: PRE-6120) as a substrate, and the microplate reader detects the fluorescent signal . Collect Jurkat-NFAT / luciferase-CD16a and CHO-S-TIGIT cells by centrifugation at 400g for 5min, resuspend in RPMI 1640 + 1% FBS medium, adjust the density to 2.5 × 10 ⁶ / ml and 1.25 × 10 ⁶ / ml, 40 μL / Well added to 96-well plate; RPMI 1640 + 1% FBS medium diluted antibody (100μg / mL, 3-fold dilution, 11 concentration points), 20μL / well, incubated at 37 ℃ for 5h; added 20μL One-glo detection reagent After mixing and shaking, use a microplate reader to read the fluorescence signal value. The result is imported into Graph Prism software to calculate the EC ₅₀ value. The results are shown in Figure 11 and Table 12. The results show that AB12V8 and AB12V9 have good ADCC activity. Specific antibody AB12V1 does not have ADCC activity.

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

抗体名称Antibody name	EC50(nM)EC50 (nM)
AB12V1AB12V1	无活性Inactive
AB12V8AB12V8	0.38260.3826
AB12V9AB12V9	0.39020.3902

Example 7. Functional identification of anti-TIGIT humanized antibody

7.1. Anti-TIGIT humanized antibody improves the function of CEFT activated PBMC to secrete IFN-γ

This test is used to evaluate the effect of anti-TIGIT humanized antibody alone or in combination with anti-PD-1 antibody on IFN-γ produced by PBMC stimulated with antigen peptide mixture CEFT (from CMV, EBV, influenza or tetanus).

Fresh PBMC was obtained from human peripheral blood by density gradient centrifugation (Lymphoprep ^™ , human lymphocyte separation liquid, purchased from Stemcell), and CEFT (purchased from JPT Peptide) containing 100 IU / ml IL-2 and 4 μg / ml antigen peptide mixture was used. Technologies Inc., Cat: PM-CEFT-4) RPMI 1640 medium (containing 10% inactivated FBS) are resuspended PBMCs, cell density was adjusted to ¹⁰⁶ / ml, and seeded in 96-well plate, 100μl / hole; and 20μg / ml of AB12V1, AB12V4, AB12V8, AB12V9, AB12M4, isotype IgG control antibody or a combination of the above anti-TIGIT antibody and anti-PD-1 antibody AB12M4 (which is disclosed in Chinese patent application CN 106519034A), added to a 96-well plate, 100 μl / Well, set up three wells; after 6 days incubation at 37 ° C, 5% CO ₂ incubator, take the supernatant and measure the concentration of IFN-γ using OptEIA ELISA kit (purchased from BD Bioscience, Catalog No. 555142). As shown in Figure 12-1, 12-2, 12-3, 12-4.

The results show that AB12V4, AB12V8 or AB12V9 can promote T cells to secrete IFN-γ, of which AB12V8 has the strongest ability to promote IFN-γ secretion, and is significantly better than the reference antibody AB12V1; in combination with AB12M4, each antibody and AB12M4 The combination of both can promote the secretion of IFN-γ by T cells. Among them, the combination of AB12V8 or AB12V9 and the anti-PD-1 antibody AB12M4 can significantly promote the secretion of factors, and is significantly better than the combination effect of the reference antibody AB12V1 and AB12M4.

7.2 、 Humanized antibody enhances the function of human NK cells and T cells to kill HCC827 cells

In order to detect the effect of anti-TIGIT humanized antibodies on human NK and T cells killing HCC827 cells (human non-small cell lung cancer cells, high expression CD155, purchased from the Chinese Academy of Sciences cell bank) (Zhang Q et al. Nat. Immunol., 19: 723-732,2018). In this example, density gradient centrifugation (Lymphoprep ^™ , human lymphocyte separation solution, purchased from Stemcell, article number: # 70851) was used to obtain fresh PBMC from human peripheral blood, using CD56 ⁺ positive selection kit (purchased from Meitian, Germany) Ni Biotechnology Co., Ltd., Catalog No .: 130-050-401) Sort human NK cells, resuspend NK cells in Cell Genix GMP SCGM medium containing 500IU / ml IL-2 and 5% human AB serum, and adjust the cell density to 10 ⁶ cells / ml, adding beads of activated NK cells (purchased from Germany Meitani Biotechnology Co., Ltd., article number: 130-094-483), 5 μl / 10 ⁶ cells, expanded culture for 12 days; additionally sort NK After the cells, the T cells were expanded and cultured in 1640 medium containing 1 μg / ml OKT3, 500 IU / ml IL-2 and 10% FBS, and the T cells were frozen on the 9th day.

One day before the killing experiment, the above frozen T cells were recovered, and the T cells were restimulated with 1640 medium containing 1 μg / ml OKT3, 500 IU / ml IL-2 and 10% FBS for one day; the next day, they were digested with 0.25% trypsin HCC827 cells, observed under a microscope until the cells are round, stop digestion, pipette the cells, and count the cells, adjust the HCC827 cell density to 2 × 10 ⁵ cells / ml, 50 μl / well, inoculate in a 96-well plate, set up two groups Only the holes of HCC827 cells are used as the target cell maximum release LDH group and the target cell itself release LDH group; the restimulated T cells and NK cells are mixed in a 1: 1 ratio, and the mixed cells are resuspended to use them as effector cells. density was adjusted to 10 ⁶ effector cells / ml, seeded in 96-well plates in the above, 50μl / hole; 30μg / ml, 6μg / ml , test antibody AB12V1 1.2μg / ml of, AB12V8, AB12V9 or IgG isotype control antibody was added Go to the above 96-well plate, 50μl / well, set three multiple wells, set a group with effector cells and target cell groups, a group with only effector cells group, put them in a 37 ℃, 5% CO ₂ incubator 5- 6h; 1h before collecting the supernatant, add 16μl Lysis Solution to the wells with only target cells; cells 250 × g, centrifuge for 5min, take 35μl supernatant in a new 96-well flat bottom plate; add 35μl / well CytoTox 96Reagent ( (Purchased from Promega, article number: G1780), incubated at room temperature in the dark for 30 min; add 35 μl / well Stop Solution, read at 492 nm, and then calculate the specific kill rate of the antibody according to the OD 492 value, the formula is as follows:

The results show in Figure 13, AB12V1, AB12V8 or AB12V9 can significantly improve the specific killing ability of NK cells and T cells, of which AB12V8 and AB12V9 specific killing effect is significantly better than the reference antibody AB12V1.

7.3. Luciferase reporter gene system to detect the action intensity of anti-TIGIT humanized antibody

To test the effect of anti-TIGIT humanized antibodies on the TIGIT / CD155 luciferase reporter gene system, we used TIGIT / NFAT Reporter-Jurkat cell line and CD155 / TCR Activator-CHO cell line as two cell lines in the system.

CD155 / TCR Activator-CHO cells (purchased from BPS Bioscience, catalog number: # 60548) were collected by centrifugation, containing 10% FBS (purchased from Life Technologies, catalog number: # 10100139), 1% Penn-Strep (purchased from Life Technologies, catalog number) : # 15140122), 500 μg / ml Hygromycin B (purchased from Life Technologies, catalog number: # 10687010) and 5 μg / ml Puromycin (purchased from Invivogen, catalog number: # ant-pr-1) of Ham's F12 medium (purchased from Life Technologies , Article number: # 21127022) Adjust the cell density to 4 × 10 ⁵ cells / ml, inoculate 100 μl / well in a 96-well opaque whiteboard; the next day, dilute the test antibodies AB12V1, AB12V8, AB12V9 and the positive control antibody Anti -TIGIT neutralizing antibody (purchased from BPS Bioscience, Catalog No .: # 71340), with a maximum concentration of 20 μg / ml, diluted 11 gradients at a 3-fold ratio; TIGIT / NFAT Reporter-Jurkat cells (purchased from BPS Bioscience, Catalog No .: # 60538), using RPMI 1640 medium (purchased from Life Technologies, catalog number: 10% FBS, 1% Penn-Strep, 1 mg / ml Geneticin (purchased from Life Technologies, catalog number: # 11811031) and 200 μg / ml Hygromycin B # A10491-01) Adjust the cell density to 4 × 10 ⁵ cells / ml, mix the above-mentioned TIGIT antibody with different concentration gradients and TIGIT / NFAT Reporter-Jurkat cells at a volume ratio of 1: 1, and place at 37 ℃, 5% CO ₂ Pre-incubate in the incubator for 15 minutes; after incubation, remove the medium in CD155 / TCR Activator-CHO cells, and add 100 μl of TIGIT / NFAT Reporter-Jurkat with different concentrations of anti-TIGIT antibodies to the corresponding wells in the 96-well whiteboard The cells were placed in a 37 ° C, 5% CO ₂ incubator for 5 hours; then, 100-l / well was added to One-Step Luciferase reagent (purchased from BPS Bioscience, catalog number: # 60690) and mixed, incubated at room temperature for 30 minutes, using spectra The analyzer measures the luminosity value, and then calculates the fold of the luciferase activity after the antibody action according to the luminosity value. The formula is as follows:

The results are shown in Figure 14 and Table 13. AB12V1, AB12V8, AB12V9 or positive control antibody can significantly increase the expression of luciferase in the TIGIT / CD155 luciferase reporter gene system. weaker, but with reference AB12V9 AB12V8 and ₅₀ by less than one order of magnitude than the EC AB12V1 antibody, the effect is quite basic.

Table 13: Measurement results of the effect of anti-TIGIT humanized antibody on TIGIT / CD155 luciferase reporter gene system

抗体名称Antibody name	EC ₅₀(nM) EC ₅₀ (nM)
Anti-TIGIT中和抗体Anti-TIGIT	3.2323.232
AB12V1AB12V1	0.81050.8105
AB12V8AB12V8	1.3701.370
AB12V9AB12V9	5.3575.357

Example 8. Anti-TIGIT humanized antibody inhibits the growth of colon cancer cells in hTIGIT transgenic mice

MC38 cells (mouse colon cancer cells, purchased from Nanjing Kebai Biotechnology Co., Ltd.) were cultured with RPMI 1640 medium containing 10% fetal bovine serum under the conditions of 37 ° C and 5% CO ₂ . Collect MC38 cells in the exponential growth phase, resuspend in PBS to a suitable concentration, and inoculate female B-hTIGIT mice subcutaneously to establish a colon cancer model. When the average tumor volume is about 91.56mm ³ , they are randomly grouped according to the size of the tumor, which are the Human IgG (negative control) group, the AB12V8 single drug group, and the AB12V8 and Anti-mouse PD-1 antibodies (purchased from BioxCell, article number: BE0146) The combination group was injected intraperitoneally twice a week for 3 weeks. Observe and regularly measure the tumor volume of mice after administration. The specific results are shown in Figure 15.

It can be seen from the test results that the AB12V8 single-drug group has a significant inhibitory effect on the tumor growth of the MC38 colon cancer xenograft model. There is a clear synergistic effect. During the observation period, there was no animal death and significant weight loss in the treatment group, and no obvious drug toxicity was shown. During the treatment period, the mice tolerated AB12V8 antibody well.

All documents mentioned in the present invention are cited as references in this application, just as each document is individually cited as a reference. In addition, it should be understood that, after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

An antibody or antigen-binding fragment thereof capable of specifically binding TIGIT, the antibody or antigen-binding fragment thereof comprising a complementarity determining region (CDR) selected from the group consisting of:

(a) The following three CDRs of the heavy chain variable region (VH):

(i) CDR-H1, which has the sequence of CDR-H1 contained in VH as shown in SEQ ID NO: 1, or one or several amino acids compared to the sequence of CDR-H1 contained in the VH Sequences of substitutions, deletions or additions (for example, substitutions, deletions or additions of 1, 2 or 3 amino acids);

(ii) CDR-H2, which has the sequence of CDR-H2 contained in VH shown in SEQ ID NO: 1, or one or several amino acids compared to the sequence of CDR-H2 contained in the VH Sequences of substitutions, deletions or additions (for example, substitutions, deletions or additions of 1, 2 or 3 amino acids); and

(iii) CDR-H3, which has the sequence of CDR-H3 contained in VH shown in SEQ ID NO: 1, or one or several amino acids compared to the sequence of CDR-H3 contained in the VH Sequences of substitutions, deletions or additions (for example, substitutions, deletions or additions of 1, 2 or 3 amino acids); and / or,

The CDRs of the following three light chain variable regions (VL):

(iv) CDR-L1, which has the sequence of CDR-L1 contained in the VL shown in SEQ ID NO: 2, or one or several amino acids compared to the sequence of CDR-L1 contained in the VL Sequences of substitutions, deletions or additions (for example, substitutions, deletions or additions of 1, 2 or 3 amino acids);

(v) CDR-L2, which has the sequence of CDR-L2 contained in the VL shown in SEQ ID NO: 2, or one or several amino acids compared to the sequence of CDR-L2 contained in the VL Sequences of substitutions, deletions or additions (for example, substitutions, deletions or additions of 1, 2 or 3 amino acids); and

(vi) CDR-L3, which has the sequence of CDR-L3 contained in the VL shown in SEQ ID NO: 2, or one or several amino acids compared to the sequence of CDR-L3 contained in the VL Sequences of substitutions, deletions or additions (for example, substitutions, deletions or additions of 1, 2 or 3 amino acids);

or,

(b) The CDRs of the following three heavy chain variable regions (VH):

(i) CDR-H1, which has the sequence of CDR-H1 contained in VH shown in SEQ ID NO: 3, or one or several amino acids compared to the sequence of CDR-H1 contained in the VH Sequences of substitutions, deletions or additions (for example, substitutions, deletions or additions of 1, 2 or 3 amino acids);

(ii) CDR-H2, which has the sequence of CDR-H2 contained in the VH shown in SEQ ID NO: 3, or one or several amino acids compared to the sequence of CDR-H2 contained in the VH Sequences of substitutions, deletions or additions (for example, substitutions, deletions or additions of 1, 2 or 3 amino acids); and

(iii) CDR-H3, which has the sequence of CDR-H3 contained in the VH shown in SEQ ID NO: 3, or one or several amino acids compared to the sequence of CDR-H3 contained in the VH Sequences of substitutions, deletions or additions (for example, substitutions, deletions or additions of 1, 2 or 3 amino acids); and / or,

The CDRs of the following three light chain variable regions (VL):

(iv) CDR-L1, which has the sequence of CDR-L1 contained in the VL shown in SEQ ID NO: 4, or one or several amino acids compared to the sequence of CDR-L1 contained in the VL Sequences of substitutions, deletions or additions (for example, substitutions, deletions or additions of 1, 2 or 3 amino acids);

(v) CDR-L2, which has the sequence of CDR-L2 contained in the VL shown in SEQ ID NO: 4, or one or several amino acids compared to the sequence of CDR-L2 contained in the VL Sequences of substitutions, deletions or additions (for example, substitutions, deletions or additions of 1, 2 or 3 amino acids); and

(vi) CDR-L3, which has the sequence of CDR-L3 contained in the VL shown in SEQ ID NO: 4, or one or several amino acids compared to the sequence of CDR-L3 contained in the VL Sequences of substitutions, deletions or additions (for example, substitutions, deletions or additions of 1, 2 or 3 amino acids);

Preferably, the substitution described in any one of (i)-(vi) is a conservative substitution;

Preferably, the CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH), and / or the CDR-L1, CDR- contained in the light chain variable region (VL) L2 and CDR-L3 are defined by Kabat, Chothia or IMGT numbering system.
The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises:

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

VH as shown in any of SEQ ID NO: 1, SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 39 and SEQ ID NO: 43; and / or,

3 CDRs contained in the light chain variable region (VL) selected from the following:

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

or

(b) 3 CDRs contained in the heavy chain variable region (VH) selected from the following:

VH as shown in any of SEQ ID NO: 3, SEQ ID NO: 33, SEQ ID NO: 37 and SEQ ID NO: 41;

and / or,

3 CDRs contained in the light chain variable region (VL) selected from the following:

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

Preferably, the three CDRs contained in the heavy chain variable region (VH) and / or the three CDRs contained in the light chain variable region (VL) are defined by the Kabat, Chothia, or IMGT numbering system.
The antibody or antigen-binding fragment thereof of claim 2, wherein the antibody or antigen-binding fragment thereof comprises:

(1) (a) 3 CDRs contained in the heavy chain variable region (VH) shown in SEQ ID NO: 1; and / or, the light chain variable region (VL shown in SEQ ID NO: 2 ) Contains 3 CDRs; or (b) 3 CDRs contained in the heavy chain variable region (VH) shown in SEQ ID NO: 3; and / or light CDRs shown in SEQ ID NO: 4 3 CDRs contained in the chain variable region (VL);

Wherein, the three CDRs contained in the heavy chain variable region (VH) and the three CDRs contained in the light chain variable region (VL) are defined by the IMGT numbering system;

(2) (a) 3 CDRs contained in the heavy chain variable region (VH) shown in SEQ ID NO: 1; and / or the light chain variable region (VL shown in SEQ ID NO: 2 ) Contains 3 CDRs; or (b) 3 CDRs contained in the heavy chain variable region (VH) shown in SEQ ID NO: 3; and / or light CDRs shown in SEQ ID NO: 4 3 CDRs contained in the chain variable region (VL); or (c) 3 CDRs contained in the heavy chain variable region (VH) shown in SEQ ID NO: 41; and / or, such as SEQ ID NO : 3 CDRs contained in the light chain variable region (VL) shown in 42; or (d) 3 CDRs contained in the heavy chain variable region (VH) shown in SEQ ID NO: 43; and / Or, the three CDRs contained in the light chain variable region (VL) shown in SEQ ID NO: 44;

Wherein, the three CDRs contained in the heavy chain variable region (VH) and the three CDRs contained in the light chain variable region (VL) are defined by the Kabat numbering system; or

(3) 3 CDRs contained in the heavy chain variable region (VH) shown in SEQ ID NO: 1; and / or, contained in the light chain variable region (VL) shown in SEQ ID NO: 2 3 CDRs; or (b) 3 CDRs contained in the heavy chain variable region (VH) shown in SEQ ID NO: 3; and / or, the light chain variable shown in SEQ ID NO: 4 3 CDRs contained in the region (VL); or (c) 3 CDRs contained in the heavy chain variable region (VH) shown in SEQ ID NO: 41; and / or, as shown in SEQ ID NO: 42 3 CDRs contained in the light chain variable region (VL) shown; or (d) 3 CDRs contained in the heavy chain variable region (VH) shown in SEQ ID NO: 43; and / or, such as SEQ ID NO: 3 CDRs contained in the light chain variable region (VL) shown in 44;

Among them, the three CDRs contained in the heavy chain variable region (VH) and the three CDRs contained in the light chain variable region (VL) are defined by the Chothia numbering system.
The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises:

(1) CDR defined by IMGT numbering system:

(a) The following three CDRs of the heavy chain variable region (VH):

(i) CDR-H1, which consists of the following sequence: SEQ ID NO: 5, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 5 (eg 1, 2, or 3) Amino acid substitutions, deletions or additions),

(ii) CDR-H2, which consists of the following sequence: SEQ ID NO: 6, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 6 (eg 1, 2, or 3) Amino acid substitutions, deletions or additions), and

(iii) CDR-H3, which consists of the following sequence: SEQ ID NO: 7, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 7 (eg 1, 2, or 3) Amino acid substitutions, deletions or additions);

and / or

The CDRs of the following three light chain variable regions (VL):

(iv) CDR-L1, which consists of the following sequence: SEQ ID NO: 8, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 8 (eg 1, 2, or 3) Amino acid substitutions, deletions or additions),

(v) CDR-L2, which consists of the following sequence: SEQ ID NO: 9, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 9 (eg 1, 2, or 3) Amino acid substitutions, deletions or additions), and

(vi) CDR-L3, which consists of the following sequence: SEQ ID NO: 10, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 10 (eg 1, 2, or 3) Amino acid substitutions, deletions or additions); or

(b) The CDRs of the following three heavy chain variable regions (VH):

(i) CDR-H1, which consists of the following sequence: SEQ ID NO: 11, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 11 (eg 1, 2, or 3) Amino acid substitutions, deletions or additions),

(ii) CDR-H2, which consists of the following sequence: SEQ ID NO: 12, or has one or several amino acid substitutions, deletions or additions compared to SEQ ID NO: 12 (eg 1, 2, or 3) Amino acid substitutions, deletions or additions), and

(iii) CDR-H3, which consists of the following sequence: SEQ ID NO: 13, or has one or several amino acid substitutions, deletions or additions compared to SEQ ID NO: 13 (eg 1, 2, or 3 Amino acid substitutions, deletions or additions);

and / or

The CDRs of the following three light chain variable regions (VL):

(iv) CDR-L1, which consists of the following sequence: SEQ ID NO: 14, or a substitution, deletion, or addition of one or a few amino acids compared to SEQ ID NO: 14 (eg 1, 2, or 3) Amino acid substitutions, deletions or additions),

(v) CDR-L2, which consists of the following sequence: SEQ ID NO: 15, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 15 (eg 1, 2, or 3) Amino acid substitutions, deletions or additions), and

(vi) CDR-L3, which consists of the following sequence: SEQ ID NO: 16, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 16 (eg 1, 2, or 3) Amino acid substitutions, deletions or additions);

or,

(2) CDR defined by Chothia numbering system:

(a) The following three heavy chain variable region (VH) CDRs:

(i) CDR-H1, which consists of the following sequence: SEQ ID NO: 27, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 27 (eg 1, 2, or 3) Amino acid substitutions, deletions or additions),

(ii) CDR-H2, which consists of the following sequence: SEQ ID NO: 28, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 28 (eg 1, 2, or 3) Amino acid substitutions, deletions or additions), and

(iii) CDR-H3, which is composed of the following sequence: SEQ ID NO: 19, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 19 (eg 1, 2, or 3) Amino acid substitutions, deletions or additions);

and / or,

The following three light chain variable region (VL) CDRs:

(iv) CDR-L1, which consists of the following sequence: SEQ ID NO: 20, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 20 (eg 1, 2, or 3) Amino acid substitutions, deletions or additions),

(v) CDR-L2, which consists of the following sequence: SEQ ID NO: 21, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 21 (eg 1, 2, or 3) Amino acid substitutions, deletions or additions), and

(vi) CDR-L3, which consists of the following sequence: SEQ ID NO: 10, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 10 (eg 1, 2, or 3) Amino acid substitutions, deletions or additions); or

(b) The CDRs of the following three heavy chain variable regions (VH):

The following three heavy chain variable region (VH) CDRs:

(i) CDR-H1, which is composed of the following sequence: SEQ ID NO: 29, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 29 (eg 1, 2, or 3) Amino acid substitutions, deletions or additions),

(ii) CDR-H2, which consists of the following sequence: SEQ ID NO: 30, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 30 (eg 1, 2, or 3) Amino acid substitutions, deletions or additions), and

(iii) CDR-H3, which consists of the following sequence: SEQ ID NO: 24, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 24 (eg 1, 2, or 3) Amino acid substitutions, deletions or additions);

and / or,

The following three light chain variable region (VL) CDRs:

(iv) CDR-L1, which consists of the following sequence: SEQ ID NO: 25, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 25 (eg 1, 2, or 3) Amino acid substitutions, deletions or additions),

(v) CDR-L2, which consists of the following sequence: SEQ ID NO: 26, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 26 (eg 1, 2, or 3) Amino acid substitutions, deletions or additions), and

(vi) CDR-L3, which consists of the following sequence: SEQ ID NO: 16, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 16 (eg 1, 2, or 3) Amino acid substitutions, deletions or additions);

or,

(3) CDR defined by Kabat numbering system:

(a) The following three heavy chain variable region (VH) CDRs:

(i) CDR-H1, which consists of the following sequence: SEQ ID NO: 17, or has one or several amino acid substitutions, deletions, or additions compared to SEQ ID NO: 17 (eg 1, 2, or 3) Amino acid substitutions, deletions or additions),

(ii) CDR-H2, which consists of the following sequence: SEQ ID NO: 18, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 18 (eg 1, 2, or 3) Amino acid substitutions, deletions or additions), and

(iii) CDR-H3, which is composed of the following sequence: SEQ ID NO: 19, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 19 (eg 1, 2, or 3) Amino acid substitutions, deletions or additions);

and / or,

The following three light chain variable region (VL) CDRs:

(iv) CDR-L1, which consists of the following sequence: SEQ ID NO: 20, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 20 (eg 1, 2, or 3) Amino acid substitutions, deletions or additions),

(v) CDR-L2, which consists of the following sequence: SEQ ID NO: 21, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 21 (eg 1, 2, or 3) Amino acid substitutions, deletions or additions), and

(vi) CDR-L3, which consists of the following sequence: SEQ ID NO: 10, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 10 (eg 1, 2, or 3) Amino acid substitutions, deletions or additions); or

(b) The CDRs of the following three heavy chain variable regions (VH):

(i) CDR-H1, which consists of the following sequence: SEQ ID NO: 22, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 22 (eg 1, 2, or 3) Amino acid substitutions, deletions or additions),

(ii) CDR-H2, which consists of the following sequence: SEQ ID NO: 23, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 23 (eg 1, 2, or 3) Amino acid substitutions, deletions or additions), and

(iii) CDR-H3, which consists of the following sequence: SEQ ID NO: 24, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 24 (eg 1, 2, or 3) Amino acid substitutions, deletions or additions);

and / or,

The following three light chain variable region (VL) CDRs:

(iv) CDR-L1, which consists of the following sequence: SEQ ID NO: 25, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 25 (eg 1, 2, or 3) Amino acid substitutions, deletions or additions),

(v) CDR-L2, which consists of the following sequence: SEQ ID NO: 26, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 26 (eg 1, 2, or 3) Amino acid substitutions, deletions or additions), and

(vi) CDR-L3, which consists of the following sequence: SEQ ID NO: 16, or a substitution, deletion, or addition of one or several amino acids compared to SEQ ID NO: 16 (eg 1, 2, or 3) Amino acid substitutions, deletions or additions);

Preferably, the substitution described in any one of (i)-(vi) is a conservative substitution.
The antibody or antigen-binding fragment thereof according to claim 4, wherein:

(a) The VH of the antibody or antigen-binding fragment thereof includes: CDR-H1 shown in SEQ ID NO: 5; CDR-H2 shown in SEQ ID NO: 6; and, as shown in SEQ ID NO: 7. CDR-H3 shown;

Moreover, the VL of the antibody or antigen-binding fragment thereof includes: CDR-L1 shown in SEQ ID NO: 8; CDR-L2 shown in SEQ ID NO: 9; and, shown in SEQ ID NO: 10 CDR-L3; or

(b) The VH of the antibody or antigen-binding fragment thereof includes: CDR-H1 shown in SEQ ID NO: 11; CDR-H2 shown in SEQ ID NO: 12; and, as shown in SEQ ID NO: 13 CDR-H3 shown;

Furthermore, the VL of the antibody or antigen-binding fragment thereof includes: CDR-L1 shown in SEQ ID NO: 14; CDR-L2 shown in SEQ ID NO: 15; and, shown in SEQ ID NO: 16 CDR-L3;

Among them, the above CDRs are defined by the IMGT numbering system.
The antibody or antigen-binding fragment thereof according to claim 4, wherein:

(a) The VH of the antibody or antigen-binding fragment thereof includes: CDR-H1 shown in SEQ ID NO: 27; CDR-H2 shown in SEQ ID NO: 28; and, as shown in SEQ ID NO: 19 CDR-H3 shown; and, the VL of the antibody or antigen-binding fragment thereof includes: CDR-L1 shown in SEQ ID NO: 20; CDR-L2 shown in SEQ ID NO: 21; and, SEQ ID NO: CDR-L3 shown in 10; or

(b) The VH of the antibody or antigen-binding fragment thereof includes: CDR-H1 shown in SEQ ID NO: 29; CDR-H2 shown in SEQ ID NO: 30; and, as shown in SEQ ID NO: 24 CDR-H3 shown; and, the VL of the antibody or antigen-binding fragment thereof includes: CDR-L1 shown in SEQ ID NO: 25; CDR-L2 shown in SEQ ID NO: 26; and, as SEQ ID NO: CDR-L3 shown in 16;

Among them, the above CDRs are defined by Chothia numbering system.
The antibody or antigen-binding fragment thereof according to claim 4, wherein:

(a) The VH of the antibody or antigen-binding fragment thereof includes: CDR-H1 shown in SEQ ID NO: 17; CDR-H2 shown in SEQ ID NO: 18; and, as shown in SEQ ID NO: 19 CDR-H3 shown; and, the VL of the antibody or antigen-binding fragment thereof includes: CDR-L1 shown in SEQ ID NO: 20; CDR-L2 shown in SEQ ID NO: 21; and, SEQ ID NO: CDR-L3 shown in 10; or

(b) The VH of the antibody or antigen-binding fragment thereof includes: CDR-H1 as shown in SEQ ID NO: 22; CDR-H2 as shown in SEQ ID NO: 23; and, as shown in SEQ ID NO: 24 CDR-H3 shown; and, the VL of the antibody or antigen-binding fragment thereof includes: CDR-L1 shown in SEQ ID NO: 25; CDR-L2 shown in SEQ ID NO: 26; and, as SEQ ID NO: CDR-L3 shown in 16;

Among them, the above CDRs are defined by the Kabat numbering system.
The antibody or antigen-binding fragment thereof according to any one of claims 1-7, wherein the antibody or antigen-binding fragment thereof comprises:

(1) (a) Heavy chain variable region (VH), which comprises an amino acid sequence selected from the following:

(i) SEQ ID NOs: the sequence shown in any of 1, 31, 35, 39, 43;

(ii) One or several amino acid substitutions, deletions, or additions compared to the sequence shown in any of SEQ ID NOs: 1, 31, 35, 39, 43 (eg 1, 2, 3, 4 Sequence of one or five amino acid substitutions, deletions or additions); or

(iii) The sequence shown in any one of SEQ ID NOs: 1, 31, 35, 39, 43 has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least A sequence of 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity;

and / or

(b) Light chain variable region (VL), which contains an amino acid sequence selected from the following:

(iv) SEQ ID NOs: the sequence shown in any one of 2, 32, 36, 40, 44;

(v) One or several amino acid substitutions, deletions, or additions compared to the sequence shown in any of SEQ ID NOs: 2, 32, 36, 40, 44 (eg 1, 2, 3, 4 Sequence of one or five amino acid substitutions, deletions or additions); or

(vi) The sequence shown in any one of SEQ ID NOs: 2, 32, 36, 40, 44 has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least A sequence of 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity;

or

(2) (a) Heavy chain variable region (VH), which contains an amino acid sequence selected from the following:

(i) SEQ ID NOs: the sequence shown in any of 3, 33, 37, 41;

(ii) One or several amino acid substitutions, deletions, or additions compared to the sequence shown in any of SEQ ID NOs: 3, 33, 37, 41 (for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions); or

(iii) The sequence shown in any one of SEQ ID NOs: 3, 33, 37, 41 has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% , At least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity;

and / or

(b) Light chain variable region (VL), which contains an amino acid sequence selected from the following:

(iv) SEQ ID NOs: the sequence shown in any of 4, 34, 38, 42;

(v) One or several amino acid substitutions, deletions, or additions compared to the sequence shown in any of SEQ ID NOs: 4, 34, 38, 42 (eg 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions); or

(vi) The sequence shown in any of SEQ ID NOs: 4, 34, 38, 42 has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% , At least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity;

Preferably, the substitutions described in (ii) or (v) are conservative substitutions.
The antibody or antigen-binding fragment thereof according to any one of claims 1-8, wherein the antibody or antigen-binding fragment thereof comprises:

(a) Heavy chain variable region (VH) and light chain variable region (VL), which include:

(i) VH with the sequence shown in SEQ ID NO: 1 and VL with the sequence shown in SEQ ID NO: 2;

(ii) VH having the sequence shown in SEQ ID NO: 31 and VL having the sequence shown in SEQ ID NO: 32;

(iii) VH having the sequence shown in SEQ ID NO: 35 and VL having the sequence shown in SEQ ID NO: 36;

(iv) VH having the sequence shown in SEQ ID NO: 39 and VL having the sequence shown in SEQ ID NO: 40; or

(v) VH with the sequence shown in SEQ ID NO: 43 and VL with the sequence shown in SEQ ID NO: 44;

or

(b) Heavy chain variable region (VH) and light chain variable region (VL), which include:

(i) VH with the sequence shown in SEQ ID NO: 3 and VL with the sequence shown in SEQ ID NO: 4;

(ii) VH having the sequence shown in SEQ ID NO: 33 and VL having the sequence shown in SEQ ID NO: 34;

(iii) VH having the sequence shown in SEQ ID NO: 37 and VL having the sequence shown in SEQ ID NO: 38; or

(iv) VH having the sequence shown in SEQ ID NO: 41 and VL having the sequence shown in SEQ ID NO: 42.
The antibody or antigen-binding fragment thereof according to any one of claims 1-9, wherein the antibody or antigen-binding fragment further comprises:

(a) The heavy chain constant region (CH) of a human immunoglobulin or a variant thereof, which variant has conservative substitutions of up to 20 amino acids (e.g. up to 15, up to 10, Or a conservative substitution of up to 5 amino acids; for example, a conservative substitution of 1, 2, 3, 4 or 5 amino acids); and

(b) The light chain constant region (CL) of human immunoglobulin or a variant thereof, said variant having a conservative substitution of up to 20 amino acids (e.g. up to 15, up to 10, Or a conservative substitution of up to 5 amino acids; for example, a conservative substitution of 1, 2, 3, 4 or 5 amino acids);

Preferably, the heavy chain constant region is an IgG heavy chain constant region, such as an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region;

Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region selected from the following:

(1) Human IgG1 heavy chain constant region;

(2) Human IgG4 heavy chain constant region;

(3) A variant of the human IgG4 heavy chain constant region, which has the following substitutions compared to the wild-type sequence from which it was derived: Ser228Pro;

Among them, the above-mentioned amino acid position is based on the position of the EU numbering system;

Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as shown in any one of SEQ ID NOs: 45-47;

Preferably, the light chain constant region is a kappa light chain constant region;

Preferably, the antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) as shown in SEQ ID NO: 48.
The antibody or antigen-binding fragment thereof according to any one of claims 1-10, wherein the antibody is a chimeric antibody or a humanized antibody.
The antibody or antigen-binding fragment thereof according to any one of claims 1-11, wherein the antibody or antigen-binding fragment is selected from ScFv, Fab, Fab ', (Fab') 2 , Fv fragment, disulfide-linked Fv (dsFv), diabody, bispecific antibody and multispecific antibody.
The antibody or antigen-binding fragment thereof according to any one of claims 1-12, wherein the antibody or antigen-binding fragment thereof has a label; preferably, the antibody or antigen-binding fragment thereof has a detectable label, Examples are enzymes (eg horseradish peroxidase), radionuclides, fluorescent dyes, luminescent substances (eg chemiluminescent substances) or biotin.
The antibody or antigen-binding fragment thereof of any one of claims 1-13, wherein the antibody or antigen-binding fragment thereof binds TIGIT (eg, human TIGIT) with a K D of about 100 × 10 -10 M or less;

Preferably, the antibody or antigen-binding fragment thereof binds TIGIT (eg, human TIGIT) with a K D of about 1 × 10 -10 M or less.
An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1-14, its heavy chain and / or light chain, or its heavy chain variable region and / or light chain variable region.
The isolated nucleic acid molecule of claim 15, comprising a nucleic acid molecule encoding an antibody heavy chain variable region, and / or a nucleic acid molecule encoding an antibody light chain variable region, wherein,

The nucleic acid molecule encoding the variable region of the antibody heavy chain has a sequence selected from the following: (a) the nucleotide sequence shown in SEQ ID NO: 49 or 51, or the nucleus described in (b) and (a) A sequence of substantially the same nucleotide sequence (for example, a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to the nucleotide sequence described in (a), or having One or more nucleotide substitutions), or (c) with

(a) the nucleotide sequence differs by no more than 3, 6, 15, 30 or 45 nucleotide sequences, and,

The nucleic acid molecule encoding the variable region of the antibody light chain has a sequence selected from: (a) the nucleotide sequence shown in SEQ ID NO: 50 or 52, or the nucleus described in (b) and (a) A sequence of substantially the same nucleotide sequence (for example, a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to the nucleotide sequence described in (a), or having A sequence of one or more nucleotide substitutions), or (c) a sequence that does not differ from the nucleotide sequence described in (a) by more than 3, 6, 15, 30, or 45 nucleotides;

Preferably, the nucleic acid molecule encoding the variable region of the antibody heavy chain has a nucleotide sequence shown in SEQ ID NO: 49, and the nucleic acid molecule encoding the variable region of the antibody light chain has a SEQ ID NO: 50 The nucleotide sequence shown;

Preferably, the nucleic acid molecule encoding the variable region of the antibody heavy chain has a nucleotide sequence shown in SEQ ID NO: 51, and the nucleic acid molecule encoding the variable region of the antibody light chain has a SEQ ID NO: 52 The nucleotide sequence shown.
A vector comprising the nucleic acid molecule according to claim 15 or 16; preferably, the vector is a cloning vector or an expression vector.
A host cell comprising the nucleic acid molecule according to claim 15 or 16 or the vector according to claim 17.
A method of preparing the antibody or antigen-binding fragment thereof according to any one of claims 1-14, which comprises culturing the host cell of claim 18 under conditions that allow expression of the antibody or antigen-binding fragment thereof, and The antibody or antigen-binding fragment thereof is recovered from the cultured host cell culture.
A hybridoma cell strain is: hybridoma cell strain # 7, which is deposited at the China Type Culture Collection (CCTCC) and has the deposit number CCTCC NO.C2018210.
A hybridoma cell strain is: hybridoma cell strain # 21, which is deposited at the China Type Culture Collection (CCTCC) and has the deposit number CCTCC NO.C2018209.
A monoclonal antibody produced by the hybridoma cell line according to claim 20 or 21.
A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1-14, or the carrier of claim 17, or the host cell of claim 18, and a pharmaceutically acceptable carrier And / or excipients;

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

Preferably, the additional pharmaceutically active agent is a drug used to treat immune-related diseases;

Preferably, the additional pharmaceutically active agent is a drug with anti-tumor activity;

Preferably, the additional pharmaceutically active agent is a drug used to treat infections or infectious diseases;

Preferably, the antibody or antigen-binding fragment thereof and the additional pharmaceutically active agent are provided as separate components or as components of the same composition.
The pharmaceutical composition of claim 23, wherein the amount of antibody or antigen-binding fragment thereof in the pharmaceutical composition is sufficient in the subject:

(a) Induce and / or enhance immune response;

(b) Increase the activity of effector T cells;

(c) Increase cytotoxic T lymphocyte (CTL) activity;

(d) Increase NK cell activity;

(e) Inhibit TIGIT activation;

(f) inhibit TIGIT-mediated signaling;

(g) inhibit or block the binding of CD155 and / or CD112 to TIGIT;

(h) Reduce the number of regulatory T cells in tissues or circulation;

(i) inhibiting the suppression of effector T cells by regulatory T cells; or,

(j) Any combination of (a)-(i).
The pharmaceutical composition according to claim 23 or 24, further comprising a second antibody specifically binding to a receptor or ligand selected from the following or a nucleic acid encoding the second antibody, wherein the receptor or ligand Selected from: PD-1, PD-L1, PD-L2, TIM-3, LAG-3, VISTA, CTLA-4, OX40, BTLA, 4-1BB, CD96, CD27, CD28, CD40, LAIR1, CD160, 2B4 , TGF-R, KIR, ICOS, GITR, CD3, CD30, BAFFR, HVEM, CD7, LIGHT, SLAMF7, NKp80, B7-H3 and any combination thereof;

Preferably, the second antibody is an antibody that binds to human PD-1 or an antigen-binding fragment thereof;

Preferably, the second antibody is an antibody that binds to human PD-L1 or an antigen-binding fragment thereof.
An immunogenic composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-14, and an immunogen;

Preferably, the antibody or antigen-binding fragment thereof is used as an adjuvant;

Preferably, the immunogen is selected from tumor cells, tumor-associated antigens (such as proteins, polypeptides or carbohydrate molecules), dendritic cells sensitized by the antigens, and any combination thereof;

Preferably, the immunogen is selected from inactivated or attenuated pathogens, antigens (eg proteins, polypeptides or carbohydrate molecules) associated with the pathogens, dendritic cells sensitized by the antigens, and any combination thereof;

Preferably, the antibody or antigen-binding fragment thereof and the immunogen are provided as separate components or as components of the same composition.
Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-14, or the vector according to claim 17, or the host cell according to claim 18, in the preparation of a medicament, the medicament being used for:

(1) Improving the activity of immune cells in vitro or in a subject (such as a human);

(2) Enhance immune response in subjects (such as humans);

(3) Treatment of tumors in subjects (eg humans); or

(4) Treatment of infections or infectious diseases in subjects (such as humans);

Preferably, the tumor is selected from solid tumors, hematological tumors (eg, leukemia, lymphoma, myeloma, eg, multiple myeloma), and metastatic, refractory, or recurrent lesions of cancer;

Preferably, the infection or infectious disease is selected from viral infections, bacterial infections, fungal infections and parasitic infections, including but not limited to HIV, hepatitis virus, herpes virus, CMV, EBV, influenza or tetanus.
Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14 in the preparation of an immunogenic composition for improving the activity and / or enhancement of immune cells in a subject An immune response; wherein the immunogenic composition comprises the antibody or antigen-binding fragment thereof and an immunogen;

Preferably, the immunogen is selected from tumor cells, tumor-associated antigens (such as proteins, polypeptides, or carbohydrate molecules), dendritic cells sensitized by the antigen, and any combination thereof; the immunogenicity The composition is used to prevent and / or treat tumors and / or delay tumor progression and / or reduce or inhibit tumor recurrence in a subject;

Preferably, the immunogen is selected from inactivated or attenuated pathogens, antigens (eg proteins, polypeptides or carbohydrate molecules) associated with the pathogens, dendritic cells sensitized by the antigens, and any combination thereof; The immunogenic composition is used to prevent and / or treat an infection or infectious disease and / or delay the progression of infection or infectious disease and / or reduce or inhibit the recurrence of infection or infectious disease in a subject.
The antibody or antigen-binding fragment thereof according to any one of claims 1-14 or the pharmaceutical composition according to any one of claims 23-25 or the immunogenic composition according to claim 26 is prepared for the treatment of immune-related diseases Use in medicine, wherein the immune-related disease involves T cell dysfunction and / or NK cell dysfunction;

Preferably, the T cell dysfunction includes, for example, T cell incapacitation, depletion, or reduced cytokine secretion; and / or, the NK cell dysfunction includes, for example, NK cell incapacitation, depletion, or reduced cytokine secretion;

Preferably, the immune-related diseases are selected from tumors and infections or infectious diseases.
A method for increasing the activity of immune cells in vitro, the method comprising the step of contacting the immune cells with the antibody or antigen-binding fragment thereof according to any one of claims 1-14;

Preferably, the method further includes the step of contacting the immune cells with another pharmaceutically active agent;

Preferably, the additional pharmaceutically active agent is an immune response stimulant;

Preferably, the immune response stimulating agent is selected from IL-1, IL-2, IL-3, IL-7, IL-12, IL-15, IL-18, IL-21, IFN-γ, IL-10 , TGF-β, GM-CSF, M-CSF, G-CSF, TNF-α and TNF-β and any combination thereof.
A method for enhancing the activity of immune cells in a subject, the method comprising: administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14 or claim 23 The pharmaceutical composition according to any one of -25 or the immunogenic composition according to claim 26;

Preferably, the immune cells are selected from T cells, cytotoxic T cells (CTL), NK cells, and any combination thereof;

Preferably, the method is used to: (a) prevent and / or treat tumors and / or delay tumor progression and / or reduce or inhibit tumor recurrence; or (b) prevent and / or treat infections or infectious diseases and / or Delay the progression of infection or infectious disease and / or reduce or inhibit the recurrence of infection or infectious disease.
A method for enhancing the activation and / or response of T cells and / or NK cells in a subject, the method comprising: administering to the subject a therapeutically effective amount of the antibody according to any one of claims 1-14 Or an antigen-binding fragment thereof or the pharmaceutical composition according to any one of claims 23-25 or the immunogenic composition according to claim 26.
A method of enhancing an immune response in a subject, the method comprising administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1-14, claim 23 The pharmaceutical composition according to any one of -25 or the immunogenic composition according to claim 26;

Preferably, the immune response is an antigen-specific T cell response;

Preferably, the method is used to: (a) prevent and / or treat tumors and / or delay tumor progression and / or reduce or inhibit tumor recurrence; or (b) prevent and / or treat infections or infectious diseases and / or Delay the progression of infection or infectious disease and / or reduce or inhibit the recurrence of infection or infectious disease.
A method for preventing and / or treating tumors in a subject, and / or delaying tumor progression, and / or reducing or inhibiting tumor recurrence, the method comprising administering to a subject in need thereof an effective amount of The antibody or antigen-binding fragment thereof according to any one of claims 1-14, the pharmaceutical composition according to any one of claims 23-25, or the immunogenic composition according to claim 26.
A method for preventing and / or treating an infection or infectious disease in a subject, and / or delaying the progression of the infection or infectious disease, and / or reducing or inhibiting the recurrence of the infection or infectious disease, the method comprising Administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment of any one of claims 1-14, the pharmaceutical composition of any one of claims 23-25, or the claim 26 Of the immunogenic composition.
The method of any one of claims 30-35, further comprising administering a second therapy to the subject, the second therapy selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA Therapy, nanotherapy, viral therapy, adjuvant therapy and any combination thereof.
The use of claim 27 or 28, or the method of claim 33 or 34, wherein the tumor is selected from esophageal cancer, gastrointestinal cancer, pancreatic cancer, thyroid cancer, colorectal cancer, renal cancer, lung cancer (eg non-small cells Lung cancer), liver cancer, stomach cancer, head and neck cancer, bladder cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, germ cell cancer, bone cancer, skin cancer, thymus cancer, cholangiocarcinoma, melanoma , Mesothelioma, lymphoma, myeloma, sarcoma, glioblastoma, glioblastoma, leukemia or cancer metastatic, refractory or recurrent lesions.
A method for detecting the presence or level of TIGIT in a sample, which comprises subjecting the sample to any one of claims 1 to 14 under conditions permitting the formation of a complex between the antibody or antigen-binding fragment thereof and TIGIT The antibody or antigen-binding fragment thereof is contacted, and the formation of the complex is detected.
A diagnostic or therapeutic kit comprising the antibody or antigen-binding fragment of any one of claims 1-14 or the pharmaceutical composition of any one of claims 23-25, and optionally user's Guide.