Immune checkpoint molecule and pharmaceutical application thereof

Abstract

According to the immune checkpoint molecule and the pharmaceutical application thereof, a kit for detecting ILDR2 comprises an antibody specifically combined with ILDR2 protein or a primer pair for detecting ILDR2 mRNA, and the kit is used for detecting the expression level of ILDR2 in a liver cancer tissue sample in vitro. The invention discloses application of detection of B7 family protein ILDR2 expression quantity in liver cancer diagnosis and treatment. It is found for the first time that ILDR2 is highly expressed in liver cancer, and the highly expressed ILDR2 is poorly related to prognosis of patients. Liver cancer cells express ILDR2 to inhibit the anti-tumor function of CD4 + T cells, promote formation of regulatory T cells and inhibit polarization of the CD4 + T cells to Th1. The ILDR2 is knocked down, so that liver cancer cell proliferation can be obviously inhibited. Detection of ILDR2 expression can better guide treatment of liver cancer patients with high ILDR2 expression, and targeted ILDR2 can screen candidate immune checkpoint drugs for treating liver cancer.

Description

Technical Field

[0001] The present invention belongs to the field of immunotechnology, and specifically relates to an immune checkpoint molecule and its pharmaceutical use in liver cancer treatment drugs. Background Art

[0002] Liver cancer, a digestive system tumor with high malignancy, insidious disease progression, and a short overall survival rate, poses a serious threat to human health. Current mainstream clinical treatment options include surgical resection, transarterial chemoembolization, and local ablation, but these have significant limitations. According to statistics, the five-year survival rate for patients with primary liver cancer who undergo radical resection is only 37%-57%, suggesting that the current treatment system is in urgent need of reform.

[0003] Breakthroughs have been made in the field of tumor immunotherapy in recent years. Immune checkpoint inhibitors, by blocking key signaling pathways such as CTLA-4 and PD-1 / PD-L1, effectively restore T cell anti-tumor activity, demonstrating unique advantages in the treatment of solid tumors such as melanoma and non-small cell lung cancer, resulting in sustained remissions. However, the unique immunosuppressive microenvironment of liver cancer significantly limits the response rate of existing immunotherapies: the objective response rate (ORR) of FDA-approved PD-1 / PD-L1 inhibitors (such as nivolumab and atezolizumab) alone is only 14%-18%, and the ORR of combination therapy is only increased to 20%-35%. This dilemma highlights the important clinical value of in-depth analysis of the immune escape mechanism of liver cancer and the development of novel immune checkpoints.

[0004] Immunoglobulin-like domain receptor 2 (ILDR2), a recently discovered B7 family transmembrane protein, plays a key role in the regulation of autoimmune diseases. Preclinical studies have shown that ILDR2 participates in the pathological process of diseases such as rheumatoid arthritis and multiple sclerosis by inhibiting T cell activation. Notably, researchers have found that in mouse melanoma, bladder cancer and other transplant tumor models, ILDR2 monoclonal antibodies (such as BAY 1905254) exhibit significant anti-tumor activity as a single agent, and have a synergistic effect when combined with PD-L1 inhibitors. Mechanistic studies have revealed that ILDR2 may regulate immune responses through novel signaling pathways distinct from traditional checkpoints, suggesting that it has unique potential in overcoming resistance to existing immunotherapies.

[0005] However, the biological function of ILDR2 in the development and progression of liver cancer remains largely unknown. In particular, (1) the expression profile of ILDR2 in liver cancer tissue and its clinical relevance are not yet clear; (2) whether ILDR2 is involved in regulating the formation of the liver cancer immune microenvironment is still lacking in systematic studies; and (3) the synergistic effect of ILDR2-targeted therapy combined with existing immune checkpoint inhibitors needs to be verified. Answers to these key scientific questions will provide important theoretical basis for the development of new immunotherapy strategies for liver cancer. Summary of the Invention

[0006] Technical problem to be solved: The present invention provides an immune checkpoint molecule and its pharmaceutical use, and uses ILDR2 as a new immune checkpoint molecule for detecting tumors such as liver cancer with high ILDR2 expression; the present invention also provides the use of ILDR2 as a new target for liver cancer immunotherapy in the preparation or screening of treatments for liver cancer and other tumors.

[0007] Technical solution: A kit for detecting ILDR2, comprising an antibody that specifically binds to ILDR2 protein or a primer pair for detecting ILDR2 mRNA. The kit is used to detect the expression level of ILDR2 in liver cancer tissue samples in vitro.

[0008] The heavy chain variable region sequence of the above antibody is shown in SEQ ID NO: 35, and the light chain variable region sequence is shown in SEQ ID NO: 36.

[0009] VH (SEQ ID NO: 35):

[0010] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVAVIWYDGGSETYYADSV KGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVGYGSWFAYWGQGTLVTVSA

[0011] VL (SEQ ID NO: 36):

[0012] DIQMTQSPASSLSASVGDRVTITCRASQSISSYLHWYQQKPGKAPKLLIYDTNNLASGVPPRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYSTPYTFGQGTKLEIK

[0013] The sequences of the primer pair are shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0014] A use of an inhibitor targeting ILDR2 in the preparation of a drug for treating liver cancer, wherein the inhibitor is selected from at least one of a monoclonal antibody, a bispecific antibody, a small molecule compound or shRNA.

[0015] The sequence of the shRNA comprises the nucleotide sequences shown in SEQ ID NOs: 37-46 as follows:

[0016] sh-ILDR2-1F:CCGGGCGGACTCTATTACTGTATTACTCGAGTAATACAGTAATAGAGTCCGCTTTTTG;

[0017] sh-ILDR2-1R:AATTCAAAAAGCGGACTCTATTACTGTATTACTCGAGTAATACAGTAATAGAGTCCGC;

[0018] sh-ILDR2-2F:CCGGCTACATGAGGAGGACAGCAATCTCGAGATTGCTGTCCTCCTCATGTAGTTTTTG;

[0019] sh-ILDR2-2R:AATTCAAAAACTACATGAGGAGGACAGCAATCTCGAGATTGCTGTCCTCCTCATGTAG;

[0020] sh-ILDR2-3F:CCGGGCAGATATAACAACACCATCTCTCGAGAGATGGTGTTGTTATATCTGCTTTTTG;

[0021] sh-ILDR2-3R:AATTCAAAAAGCAGATATAACAACACCATCTCTCGAGAGATGGTGTTGTTATATCTGC;

[0022] sh-ILDR2-4F:CCGGGCTTACCAAGAAAGCAAGAATCTCGAGATTCTTGCTTTCTTGGTAAGCTTTTTG;

[0023] sh-ILDR2-4R:AATTCAAAAAGCTTACCAAGAAAGCAAGAATCTCGAGATTCTTGCTTTCTTGGTAAGC;

[0024] sh-ILDR2-5F:CCGGCTCAGCAAGAGAAACCTGGAACTCGAGTTCCAGGTTTCCTTGCTGAGTTTTTG;

[0025] sh-ILDR2-5R:AATTCAAAAACTCAGCAAGAGAAACCTGGAACTCGAGTTCCAGGTTTCCTTGCTGAG。

[0026] A pharmaceutical composition comprises a therapeutically effective amount of an ILDR2 inhibitor and a pharmaceutically acceptable carrier, wherein the ILDR2 inhibitor is the above-mentioned inhibitor.

[0027] A method for screening ILDR2 inhibitors in vitro comprises the following steps: a) contacting a candidate compound with an ILDR2-expressing liver cancer cell line in vitro; b) detecting the inhibitory effect of the candidate compound on the ILDR2 expression level or activity; and c) selecting a compound that can significantly reduce ILDR2 expression or activity as a candidate drug.

[0028] A use of ILDR2 as a biomarker in preparing a kit for evaluating the prognosis of liver cancer.

[0029] A method for constructing a model for studying the immune microenvironment of liver cancer in vitro, comprising: a) constructing an ILDR2-knockdown liver cancer cell line; b) co-culturing the cell line with immune cells; and c) detecting changes in immune cell activation or function.

[0030] Beneficial effects: The present invention discloses the application of detecting the expression of B7 family protein ILDR2 in the diagnosis and treatment of liver cancer. It is the first time to find that ILDR2 is highly expressed in liver cancer and that high expression of ILDR2 is associated with a worse prognosis for patients. + T cell anti-tumor function, promoting the formation of regulatory T cells and inhibiting CD4 + T cells polarize toward Th1. Knockdown of ILDR2 significantly inhibits liver cancer cell proliferation. Detecting ILDR2 expression can better guide the treatment of patients with liver cancer who have high ILDR2 expression. Targeting ILDR2 can screen candidate immune checkpoint drugs for liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Expression of ILDR2 in liver cancer tissues. A: The Cancer Genome Atlas (TCGA) database analyzed ILDR2 expression in liver cancer tissues and adjacent tissues; B: The applicant collected ILDR2 expression in 80 pairs of liver cancer tissues and adjacent tissues; C: TCGA database analysis of the correlation between ILDR2 and overall survival (OS) of liver cancer patients; D: TCGA database analysis of the correlation between ILDR2 and disease-free survival (DFS) of liver cancer patients; E: qRT-PCR analysis of ILDR2 expression in normal liver cell lines and liver cancer cell lines; F: Western Blot analysis of ILDR2 expression in normal liver cell lines and liver cancer cell lines.

[0032] Figure 2Knockdown of ILDR2 in liver cancer cells inhibits liver cancer cell proliferation. A: Western blot analysis of shRNA knockdown of ILDR2 in Hepa1-6 cells; B: qRT-PCR analysis of shRNA knockdown of ILDR2 in Hepa1-6 cells; C: CCK8 and colony formation analysis of Hepa1-6 proliferation after shRNA knockdown of ILDR2; D: Western blot analysis of shRNA knockdown of ILDR2 in HCCLM3 cells; E: qRT-PCR analysis of shRNA knockdown of ILDR2 in HCCLM3 cells; F: CCK8 and colony formation analysis of HCCLM3 proliferation after shRNA knockdown of ILDR2.

[0033] Figure 3 Knockdown of ILDR2 inhibits the tumorigenicity of liver cancer cells. A: Tumor volume and growth rate were measured in mice bearing subcutaneous tumors containing control and ILDR2-knockdown Hepa1-6 cells. B: Liver weight / body weight ratio and tumor volume were measured in mice bearing orthotopic tumors containing control and ILDR2-knockdown Hepa1-6 cells.

[0034] Figure 4 Knockdown of ILDR2 promotes CD4 + T cell activation ELISA was used to detect the IFN-γ levels in the supernatant of mouse spleen cells after co-culture with control and ILDR2 knockdown Hepa1-6 cells under different conditions; A: Flow cytometry was used to detect the proportion of effector T cells after co-culture of mouse spleen cells with control and ILDR2 knockdown Hepa1-6 cells; B: ELISA was used to detect the IFN-γ levels in the supernatant of mouse spleen cells after co-culture with control and ILDR2 knockdown Hepa1-6 cells; C: Flow cytometry was used to detect the expression of T cell activation markers after co-culture of mouse spleen cells with control and ILDR2 knockdown Hepa1-6 cells.

[0035] Figure 5 To knock down ILDR2 to inhibit Treg formation and promote the increase of Th1 ratio; A: Schematic diagram of experimental process; B: Flow cytometry detection of CD4 + T cell sorting efficiency; C: qRT-PCR detection of mouse CD4 + The expression of main functional genes of T cells after co-culture with control and ILDR2 knockdown Hepa1-6 cells; D: Flow cytometry detection of mouse CD4 + The ratio of Treg and Th1 after co-culture of T cells with control and ILDR2 knockdown Hepa1-6 cells. DETAILED DESCRIPTION

[0036] The following examples may enable those skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way.

[0037] Example 1

[0038] Detection of ILDR2 expression level in liver cancer tissue

[0039] Main reagents

[0040] ILDR2 primers (Sanggong), ILDR2 antibody (Invitrogen), qRT-PCR related reagents (Novizan), Western Blot related reagents (Beyotime)

[0041] Main instruments

[0042] qRT-PCR instrument, Western Blot electrophoresis instrument, chemiluminescence imaging analyzer

[0043] Main Methods

[0044] qRT-PCR

[0045] After cells were lysed with Trizol, RNA was extracted using chloroform and isopropanol, and then reverse transcribed into cDNA for qRT-PCR analysis, using GAPDH as an internal control.

[0046] The ILDR2 forward primer sequence is 5′-TGTGGAGATTATGCCAGAGTGG-3′; (SEQ ID NO: 1)

[0047] The ILDR2 reverse primer sequence is 5′-GACATTTCTCTCGATCGCTCTGT-3′; (SEQ ID NO: 2)

[0048] The GAPDH forward primer sequence is 5′-AGATCCCTCCAAAATCAAGTGG-3′; (SEQ ID NO: 3)

[0049] The GAPDH reverse primer sequence is 5′-GGCAGAGATGATGACCCTTTT-3′; (SEQ ID NO: 4)

[0050] Western Blot

[0051] Samples were lysed with RIPA protein lysis buffer and quantified. Loading buffer was added and the protein was heated at 100°C for 10 minutes. Protein samples were run on a gel, transferred to a membrane, incubated with the corresponding primary antibody overnight, and then incubated with the corresponding secondary antibody before exposure and imaging.

[0052] result

[0053] The present inventors analyzed the liver cancer tissues and adjacent tissues in the TCGA database ( Figure 1:A), and 80 pairs of liver cancer tissues and adjacent tissues collected ( Figure 1 :B) The expression of ILDR2 gene was found to be highly expressed in liver cancer tissues. ILDR2 was negatively correlated with the overall survival (OS) and disease-free survival (DFS) of patients ( Figure 1 :C, D). qRT-PCR and Western Blot were then used to detect the levels of ILDR2 in liver cancer cell lines. It was found that ILDR2 was differentially expressed in liver cancer cell lines ( Figure 1 :E, F). This suggests that ILDR2 plays an important role in the occurrence and development of liver cancer.

[0054] Example 2

[0055] Knockdown of ILDR2 inhibits the proliferation of liver cancer cells

[0056] Main reagents

[0057] ILDR2 antibody (Invitrogen), CCK8 (Yisun), crystal violet (Sangong),

[0058] shRNA:

[0059] sh-ILDR2-3F:CCGGGCAGATATAACAACACCATCTCTCGAGAGATGGTGTTGTTATATCTGCTTTTTG;

[0060] sh-ILDR2-3R:AATTCAAAAAGCAGATATAACAACACCATCTCTCGAGAGATGGTGTTGTTATATCTGC.

[0061] Main instruments

[0062] microplate reader

[0063] Main Methods

[0064] CCK8

[0065] Tumor cells were seeded into 96-well plates (2,000 / well), and CCK8 (10 μL) was added at the time indicated in the figure. The reaction was continued for 1.5 hours and the reading was taken at 450 nm.

[0066] Colony formation

[0067] Tumor cells were seeded into 6-well plates (2,000 / well), and fresh culture medium was replaced every 3 days. After 15 days, the cells were stained with crystal violet and photographed.

[0068] result

[0069] To explore the role of ILDR2 in the occurrence and development of liver cancer, the present inventors first constructed a stable liver cancer cell line with shRNA knockdown of ILDR2. qRT-PCR and Western Blot were used to verify the knockdown efficiency. CCK8 and clone formation experiments showed that ILDR2 knockdown significantly inhibited the growth of mouse liver cancer cells Hepa1-6 ( Figure 2 :C), and human hepatocellular carcinoma cell line HCCLM3 ( Figure 2 :F) proliferation ability.

[0070] Example 3

[0071] Knockdown of ILDR2 inhibits the tumorigenicity of liver cancer cells

[0072] Main reagents

[0073] Sodium pentobarbital

[0074] Main Methods

[0075] After anesthesia for 6-8 weeks, C57 mice were anesthetized and then subjected to subcutaneous tumor loading (5 million micrograms / mouse) and orthotopic hepatic tumor loading (1 million micrograms / mouse). Tumor growth rates were measured. Mice were sacrificed after 6 weeks for subcutaneous models and 3 weeks for orthotopic models. Tumors were then excised, weighed, and volume measured.

[0076] result

[0077] To further explore the role of ILDR2 in the occurrence and development of liver cancer, the present inventors used liver cancer cells with ILDR2 knockdown to load mice with tumors. It was found that knockdown of ILDR2 significantly inhibited the formation of liver cancer tumors ( Figure 3 ), suggesting the important role of ILDR2 in regulating the growth of liver cancer tumors.

[0078] Example 4

[0079] Knockdown of ILDR2 promotes CD4 + T cell activation

[0080] Main reagents

[0081] Mouse IFN-γ ELISA kit (Dayou), T cell-related flow cytometry antibodies: CD45, CD3, CD4, CD8, CD25, CD69, IFN-γ, Foxp3 (Biolegend), CD3, CD28 antibodies (Biolegend), IL-2 (Abclonal).

[0082] Main instruments

[0083] Microplate reader, flow cytometer

[0084] Main Methods

[0085] Co-culture of mouse spleen cells and liver cancer cells

[0086] After killing the mice, the spleen was removed, ground and filtered, and the red blood cells were lysed. After the cells were counted, they were co-cultured with a certain number of liver cancer cells after counting. CD3 and CD28 antibodies were added to activate T cells, and the cytokine IL-2 was added to maintain T cells.

[0087] ELISA

[0088] The cell culture supernatant was collected and centrifuged at 1500 rpm for 5 minutes. The supernatant was aspirated and tested according to the kit instructions.

[0089] Flow cytometry

[0090] Collect the cell culture medium, centrifuge at 1500rpm for 5 minutes, collect the cell pellet, add staining buffer for washing, centrifuge at 1500rpm for 5 minutes, perform cell surface staining, protect from light for 15 minutes, add buffer for washing, fix the cells and break the membrane for intracellular staining, protect from light for 30 minutes, add buffer for washing, reselect with appropriate amount of buffer and then detect on the machine.

[0091] result

[0092] IFN-γ is a key cytokine for the anti-tumor effect of T cells. The inventors found that after knocking down ILDR2, the effect of liver cancer cells on inhibiting the secretion of IFN-γ by mouse cells was weakened ( Figure 4 :A). Flow cytometry analysis showed that knockdown of ILDR2 reduced the expression of IFN-γ + CD4 + The proportion of T cells increased significantly ( Figure 4 :B). Further detection of T cell activation markers CD25 and CD69 revealed that after knocking down ILDR2, liver cancer cells induced CD4 + Increased levels of CD25 and CD69 on the surface of T cells, as well as CD8 + Increased levels of CD69 on the surface of T cells ( Figure 4 : D). It revealed that ILDR2 is involved in regulating the anti-tumor effect of T cells, and T cells, especially CD4 + Activation of T cells.

[0093] Example 5

[0094] Knockdown of ILDR2 inhibits Treg formation and promotes the increase of Th1 proportion

[0095] Main reagents

[0096] Mouse CD4 + T cell separation magnetic beads (Biolegend)

[0097] Main instruments

[0098] Sorting poles

[0099] Main Methods

[0100] Mouse CD4 + T cell sorting

[0101] The mice were sacrificed and the spleens were taken out. The red blood cells were lysed and made into single cell suspension, and then sorted according to the instructions.

[0102] qRT-PCR

[0103] Mouse Il1b forward primer sequence is 5′-TCTTTGAAGTTGACGGACCC-3′ (SEQ ID NO: 5)

[0104] Mouse Il1b reverse primer sequence: 5′-TGAGTGATACTGCCTGCCTG-3′; (SEQ ID NO: 6)

[0105] Mouse Il9 forward primer sequence is 5′-ATGTTGGTGACATACATCCTTGC-3′ (SEQ ID NO: 7)

[0106] Mouse Il9 reverse primer sequence: 5′-TGACGGTGGATCATCCTTCAG-3′; (SEQ ID NO: 8)

[0107] Mouse Il8 forward primer sequence is 5′-TTCACCCATGGAGCATCAGG-3′, (SEQ ID NO: 9)

[0108] Mouse Il8 reverse primer sequence: 5′-CTAGGCATCTTCGTCCGTCC-3′; (SEQ ID NO: 10)

[0109] Mouse Il21 forward primer sequence is 5′-AGGTCCAATGTGTTCCCACC-3′ (SEQ ID NO: 11)

[0110] Mouse Il21 reverse primer sequence: 5′-TGCACAGCAGTCTTGAACCT-3′; (SEQ ID NO: 12)

[0111] Mouse Il22 forward primer sequence is 5′-ACGGCATGGATCTCAAAGAC-3′ (SEQ ID NO: 13)

[0112] Mouse Il22 reverse primer sequence: 5′-AACTGTTGACACTTGTGCGA-3′; (SEQ ID NO: 14)

[0113] Mouse Tnf forward primer sequence is 5′-TGTGGAGATTATGCCAGAGTGG-3′, (SEQ ID NO: 15)

[0114] Mouse Tnf reverse primer sequence: 5′-AGATAGCAAATCGGCTGACG-3′; (SEQ ID NO: 16)

[0115] Mouse Il17f forward primer sequence is 5′-AGCCAACTTTTAGGAGCA′ (SEQ ID NO: 17)

[0116] Mouse Il17f reverse primer sequence: 5′-GTCAGGAAGACAGCACCA-3′; (SEQ ID NO: 18)

[0117] Mouse Il2 forward primer sequence is 5′-CCTTGCTAATCACTCCTCAC-3′ (SEQ ID NO: 19)

[0118] Mouse Il2 reverse primer sequence: 5′-CTGTGCTTCCGCTGTAGA-3′; (SEQ ID NO: 20)

[0119] Mouse Il6 forward primer sequence: 5′-ACAAAGCCAGAGTCCTTCAGAGAG-3′ (SEQ ID NO: 21)

[0120] Mouse Il6 reverse primer sequence: 5′-TTGGATGGTCTTGGTCCTTAGCCA-3′; (SEQ ID NO: 22)

[0121] Mouse Tgfb1 forward primer sequence: 5′-GAGCCCGAAGCGGACTACTA-3′ (SEQ ID NO: 23)

[0122] Mouse Tgfb1 reverse primer sequence: 5′-TGGTTTTCTCATAGATGGCGTTG-3′; (SEQ ID NO: 24)

[0123] Mouse Il10 forward primer sequence is 5′-CGCAGCTCTAGGAGCATGTG-3′ (SEQ ID NO: 25)

[0124] Mouse Il10 reverse primer sequence: 5′-GCTCTTACTGACTGGCATGAG-3′; (SEQ ID NO: 26)

[0125] Mouse Il4 forward primer sequence: 5′-CTCTCTGTGGTGTTCTTCGT-3′ (SEQ ID NO: 27)

[0126] Mouse Il4 reverse primer sequence: 5′-TCATCCTGCTCTTCTTTCTC-3′; (SEQ ID NO: 28)

[0127] Mouse Ifng forward primer sequence: 5′-CTCTCTGTGGTGTTCTTCGT-3′ (SEQ ID NO: 29)

[0128] Mouse Ifng reverse primer sequence: 5′-CACACCTGATTACTACCTTCTTC-3′; (SEQ ID NO: 30)

[0129] Mouse Il17a forward primer sequence: 5′-GTTGACCTTCACATTCTGG-3′ (SEQ ID NO: 31)

[0130] Mouse Il17a reverse primer sequence: 5′-TGTCTCTGATGCTGTTGCT-3′; (SEQ ID NO: 32)

[0131] Mouse Foxp3 forward primer sequence is 5′-CCTTCTCGCTCTCCACTC-3′, (SEQ ID NO: 33)

[0132] Mouse Foxp3 reverse primer sequence is 5′-CACCTATGCCACCCTTATC-3′. (SEQ ID NO: 34)

[0133] result

[0134] CD4 + CD25 + Foxp3 + Regulatory T cells (Treg) are a group of immune cells that negatively regulate T cell activity. In tumor immunity, Treg often become the "accomplice" of tumor escape, and CD4 + IFN-γ + Helper T cells 1 (Th1) play an anti-tumor role by activating and maintaining tumor-killing T cells. The inventors first sorted out CD4 T cells with a purity of about 95% from mouse spleen cells.+ T cells ( Figure 5 :B), we then explored the effect of knockdown of ILDR2 on the CD4 + The results showed that ILDR2 was associated with the decreased expression of Il6, Il8, and Foxp3 and the upregulation of Ifng ( Figure 5 :C). Flow cytometry analysis showed that after knocking down ILDR2, the proportion of Treg induced by liver cancer cells decreased ( Figure 5 :D), and the Th1 ratio increased ( Figure 5 :E). This indicates that ILDR2 expression can promote Treg formation and inhibit Th1 differentiation, suggesting that ILDR2 expression in liver cancer cells is one of the potential mechanisms of liver cancer immune escape and ILDR2 is a potential target for liver cancer treatment.

Claims

1. A kit for detecting ILDR2, characterized in that: The kit comprises an antibody that specifically binds to ILDR2 protein or a primer pair that detects ILDR2 mRNA. The kit is used for in vitro detection of the expression level of ILDR2 in liver cancer tissue samples.

2. The kit according to claim 1, wherein The heavy chain variable region sequence of the antibody is shown in SEQ ID NO: 35, and the light chain variable region sequence is shown in SEQ ID NO:

36.

3. The kit according to claim 1, wherein: The sequences of the primer pair are shown in SEQ ID NO: 1 and SEQ ID NO:

2.

4. Use of an inhibitor targeting ILDR2 in the preparation of a drug for treating liver cancer, wherein the inhibitor is selected from at least one of a monoclonal antibody, a bispecific antibody, a small molecule compound or shRNA.

5. The use according to claim 3, characterized in that: The sequence of the shRNA comprises the nucleotide sequence shown in SEQ ID NO: 37-46.

6. A pharmaceutical composition, characterized in that: The invention comprises a therapeutically effective amount of an ILDR2 inhibitor and a pharmaceutically acceptable carrier, wherein the ILDR2 inhibitor is the inhibitor according to claim 3 or 4.

7. A method for screening ILDR2 inhibitors in vitro, characterized in that The following steps are involved: a) contacting a candidate compound with an ILDR2-expressing liver cancer cell line in vitro; b) detecting the inhibitory effect of the candidate compound on the ILDR2 expression level or activity; c) selecting a compound that can significantly reduce the ILDR2 expression or activity as a candidate drug.

8. Use of ILDR2 as a biomarker in preparing a kit for evaluating the prognosis of liver cancer.

9. A method for constructing a model for studying the immune microenvironment of liver cancer in vitro, characterized in that include: a) Construction of ILDR2 knockdown liver cancer cell line; b) co-culturing the cell line with immune cells; c) Detect changes in immune cell activation or function.

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