Anti-tigit antibodies and nucleic acid molecules, vectors and cells and uses

Abstract

This invention provides an anti-TIGIT antibody, a nucleic acid molecule, a carrier, cells, and applications, relating to the field of antibody technology. The amino acid sequence of the CDR region of the nanobody is selected from SEQ ID NO.1-SEQ ID NO.15; the sequence of the nanobody is SEQ ID NO.16-SEQ ID NO.29. The anti-TIGIT antibody provided by this invention exhibits strong affinity, small molecular weight, high specificity, and high affinity, significantly superior to commercially available antibodies.

Description

Technical Field

[0001] This invention belongs to the field of antibody technology, specifically relating to an anti-TIGIT antibody, nucleic acid molecules, vectors, cells, and applications. Background Technology

[0002] In recent years, immunotherapy, which utilizes the innate abilities of the immune system to manipulate and activate the immune system, has revolutionized cancer treatment. Typical immunotherapies include immune checkpoint inhibitors, small molecule inhibitors, adoptive cell therapy (ACT), antibodies, and cancer vaccines. Notably, nanobodies (Nb) have seen rapid development in recent years. Compared to traditional monoclonal antibodies, Nb, as a novel therapeutic antibody, has the following advantages: 1) Small molecular weight: Nb contains only one heavy chain variable region (VHH), has a simple structure, and a molecular weight of approximately 15 kDa, only about 1 / 10 that of traditional antibodies; 2) High stability: Nb maintains a stable structure and function even under extreme conditions such as strong acids and alkalis; 3) High specificity and strong affinity: The long CDR3 of the VHH allows Nb to accurately recognize and bind to target antigens; 4) Strong targeting and tissue penetration: The long CDR3 of Nb can form a convex structure, allowing it to penetrate deep into the antigen and recognize hidden epitopes. Simultaneously, the substitution of hydrophobic residues with hydrophilic residues in the conserved framework region 2 (FR2) enhances the water solubility and tissue penetration of Nb, enabling it to penetrate tumor tissue and the blood-brain barrier; 5) Low immunogenicity and easy humanization: Due to its small molecular weight and single VHH, Nb has fewer epitopes that bind to antigens, resulting in low immunogenicity. Furthermore, Nb exhibits high sequence homology with human heavy chain antibodies, making it well-suited for modification into humanized antibodies. Currently, Nb has become a promising alternative to traditional therapeutic antibodies, with wide applications in molecular imaging, disease diagnosis, drug delivery, tumor immunotherapy, and ACT. Against this backdrop, the development of nanobodies targeting different antigens for different malignant tumors holds promise for targeted tumor therapy and precision diagnosis.

[0003] T cells have many important molecules on their surface that play crucial roles in T cell activation, proliferation, differentiation, and effector function. TIGIT is a newly discovered cell surface protein with immunosuppressive functions. In 2005, to systematically search for previously undiscovered molecules with immune-activating or immunosuppressive effects in cells, Abbas et al. performed high-throughput sequencing on activated T cells and further screened proteins with the aforementioned characteristics. This led to the discovery of a novel molecule expressed on both T cells and NK cells, which they named TIGIT (T cell immunoglobulin and ITIM domain). The TIGIT gene is located on human chromosome 16, consists of 244 amino acid residues, and is a type I transmembrane protein. TIGIT molecules are primarily expressed on T cells and NK cells, and are expressed to varying degrees in multiple T cell subsets.

[0004] Patent CN110997720A discloses an anti-TIGIT antibody, which discloses a separated antibody or its antigen-binding fragment that binds to human TIGIT. The antibody or its antigen-binding fragment comprises a combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein: HCDR1 is YTFTSYYMH, HCDR2 is VIGPSGASTSYAQKFQG, HCDR3 is ARDHSDYWSGIMEV, LCDR1 is RASQSVRSSYLA, LCDR2 is GASSRAT, and LCDR3 is QQYFSPPWT. However, antibody drugs targeting TIGIT still suffer from poor antibody affinity and specificity. Therefore, developing an anti-TIGIT nanobody with high affinity, specificity, and stability is of great significance. Summary of the Invention

[0005] To address the problems of existing technologies, this invention provides an anti-TIGIT antibody, nucleic acid molecule, vector, cell, and its applications.

[0006] On one hand, the present invention provides an anti-TIGIT antibody, wherein the anti-TIGIT antibody is a nanobody, comprising any one or more of the following: (1) A CDR region with a sequence as shown in SEQ ID NO.1-3, or a CDR region with a sequence similarity of at least 85% to SEQ ID NO.1-3; (2) A CDR region with a sequence as shown in SEQ ID NO.4-6, or a CDR region with a sequence similarity of at least 85% to SEQ ID NO.4-6; (3) A CDR region with a sequence as shown in SEQ ID NO.7-9, or a CDR region with a sequence similarity of at least 85% to SEQ ID NO.13-15; (4) CDR regions with sequences as shown in SEQ ID NO.1, SEQ ID NO.3, and SEQ ID NO.10, or CDR regions with sequences having at least 85% sequence similarity to SEQ ID NO.1, SEQ ID NO.3, and SEQ ID NO.10; (5) CDR regions with sequences as shown in SEQ ID NO.4, SEQ ID NO.11, and SEQ ID NO.12, or CDR regions with sequences having at least 85% sequence similarity to SEQ ID NO.4, SEQ ID NO.11, and SEQ ID NO.12; (6) A CDR region with a sequence as shown in SEQ ID NO.13-15, or a CDR region with a sequence similarity of at least 85% to SEQ ID NO.13-15.

[0007] SEQ ID NO.1: GRTFSSYP; SEQ ID NO.2: TDWSGKYT; SEQ ID NO.3: AARSSGTGYYSRESFYEY; SEQ ID NO.4: GGTFSSQP; SEQ ID NO.5: INRDBRLK; SEQ ID NO.6: AARSSGNGWYTRRSFYDY; SEQ ID NO.7: GFTFNNYW; SEQ ID NO.8: IDKFGASK; SEQ ID NO.9: TKGYGGSWSAFDY; SEQ ID NO.10: IDWSGKYT; SEQ ID NO.11: ISPEGRFK; SEQ ID NO.12: AARSSGNGYYSRRSYYDY; SEQ ID NO.13: GFVFSHYA; SEQ ID NO.14: INSDGGIL; SEQ ID NO. 15: AKAVATEDYFDGMDY.

[0008] Specifically, the nanobody contains an amino acid sequence as shown in SEQ ID NO.16, or any one or more amino acid sequences that have at least 85% sequence similarity to SEQ ID NO.16.

[0009] SEQ ID NO. 16: DVQLQASGGGLVQPGGSLRLSCSVSGRTFSSYPMGWFRQAPGKDRELVAATDWSGKYTYYPSSVKGRFTISRDNAKSTLYLQMNSLNPEDTAAYYCAARSSGTGYYSRESFYEYWGQGTQVTVSS.

[0010] Specifically, the nanobody contains an amino acid sequence as shown in SEQ ID NO.17, or any one or more amino acid sequences that have at least 85% sequence similarity to SEQ ID NO.17.

[0011] SEQ ID NO. 17: DVQLQASGGGLVQSGDSLKLSCVASGGTFSSQPMGWFRQAPGKERELVAAINRDGRLKYVADSVKGRFTISRDNAKNTLYLQIDSLKPEDTAAYYCAARSSGGNWYTRRSFYDYWGQGTQVTVSS.

[0012] Specifically, the nanobody contains an amino acid sequence as shown in SEQ ID NO.18, or any one or more amino acid sequences that have at least 85% sequence similarity to SEQ ID NO.18.

[0013] SEQ ID NO. 18: DVQLQESGGGLVQSGDSLKLSCVASGGTFSSQPMGWFRQAPGKERELVAAINRDGRLKYVADSVKGRFTISRDNAKNTLYLQIDSLKPEDTAAYYCAARSSGGNWYTRRSFYDYWGQGTQVTVSS.

[0014] Specifically, the nanobody contains an amino acid sequence as shown in SEQ ID NO.19, or any one or more amino acid sequences that have at least 85% sequence similarity to SEQ ID NO.19.

[0015] SEQ ID NO. 19: DVQLQESGGGLVQSGDSLKLSCVASGGTFSSQPMGWFRQAPGKERELVAAINRDGRLKYVADSVKGRFTISRDNAKDTLYLQIDSLKPEDTAAYYCAARSSGGNWYTRRSFYDYWGQGTQVTVSS.

[0016] Specifically, the nanobody contains an amino acid sequence as shown in SEQ ID NO.20, or any one or more amino acid sequences that have at least 85% sequence similarity to SEQ ID NO.20.

[0017] SEQ ID NO. 20: DVQLQASGGGLVQPGGSLRLSCVASGFTFNNYWMNWVRQAPGKGLEWVSYIDKFGASKYYGDSVKGRFTVSRDNAKNTLYLQMNSLEPEDTAVYYCTKGYGGSWSAFDYWGQGTQVTVSS.

[0018] Specifically, the nanobody contains an amino acid sequence as shown in SEQ ID NO.21, or any one or more amino acid sequences that have at least 85% sequence similarity to SEQ ID NO.21.

[0019] SEQ ID NO. 21: DVQLQESGGGLVQSGDSLKLSCVASGGTFSSQPMGWFRQAPGKERELVAAINRDGRLKYVADSVKGRFTISRDNAKNTLYLQIDSLKPEDTAAYYCAARSSGGNWYTRRSFYDYWGQGTQVTVSS.

[0020] Specifically, the nanobody contains an amino acid sequence as shown in SEQ ID NO.22, or any one or more amino acid sequences that have at least 85% sequence similarity to SEQ ID NO.22.

[0021] SEQ ID NO. 22: DVQLQASGGGLVQPGGSLRLSCVASGGTFSSQPMGWFRQAPGKERELVAAINRDGRLKYVADSVKGRFTISRDNAKNTLYLQIDSLKPEDTAAYYCAARSSGGNWYTRRSFYDYWGQGTQVTVSS.

[0022] Specifically, the nanobody contains an amino acid sequence as shown in SEQ ID NO.23, or any one or more amino acid sequences that have at least 85% sequence similarity to SEQ ID NO.23.

[0023] SEQ ID NO. 23: DVQLQESGGGLVLAGGSLRLSCSVSGRTFSSYPMGWFRQAPGKDRELVAAIDWSGKYTYYPSSVKGRFTISRDNAKSTLYLQMNSLNPEDTAAYYCAARSSGTGYYSRESFYEYWGQGTQVTVSS.

[0024] Specifically, the nanobody contains an amino acid sequence as shown in SEQ ID NO.24, or any one or more amino acid sequences that have at least 85% sequence similarity to SEQ ID NO.24.

[0025] SEQ ID NO. 24: DVQLQASGGGLVLAGGSLRLSCSVSGRTFSSYPMGWFRQAPGKDRELVAAIDWSGKYTYYPSSVKGRFTISRDNAKSTLYLQMNSLNPEDTAAYYCAARSSGTGYYSRESFYEYWGQGTQVTVSS.

[0026] Specifically, the nanobody contains an amino acid sequence as shown in SEQ ID NO.25, or any one or more amino acid sequences that have at least 85% sequence similarity to SEQ ID NO.25.

[0027] SEQ ID NO. 25: DVQLQASGGGLVQPGDSLKLSCVTSGGTFSSQPMGWFRQAPGKEREVVAAISPEGRFKYYADSVKGRVTISRDNAKNTLDLQIDSLKPEDTAAYYCAARSSGNGYYSRRSYYDYWGQGTQVTVSS.

[0028] Specifically, the nanobody contains an amino acid sequence as shown in SEQ ID NO.26, or any one or more amino acid sequences that have at least 85% sequence similarity to SEQ ID NO.26.

[0029] SEQ ID NO. 26: DVQLQASGGGLVQPGGSLRLSCAASGFVFSHYAMTWVRQAPGKGLEWVADINSDGGILDYADSVKGRFTVSRDNAKNTLFLQMNSLKPEDTAVYYCAKAVATEDYFDGMDYWGKGTQVTVSS.

[0030] Specifically, the nanobody contains an amino acid sequence as shown in SEQ ID NO.27, or any one or more amino acid sequences that have at least 85% sequence similarity to SEQ ID NO.27.

[0031] SEQ ID NO. 27: QLQLVESGGGLVQSGDSLKLSCVASGGTFSSQPMGWFRQAPGKERELVAAINRDGRLKYVADSVKGRFTISRDNAKNTLYLQIDSLKPEDTAAYYCAARSSGGNWYTRRSFYDYWGQGTQVTVSS.

[0032] Specifically, the nanobody contains an amino acid sequence as shown in SEQ ID NO.28, or any one or more amino acid sequences that have at least 85% sequence similarity to SEQ ID NO.28.

[0033] SEQ ID NO. 28: DVQLQASGGGLVQPGGSLRLSCSVSGRTFSSYPMGWFRQAPGKDRELVAATDWSGKYTYYPSSVKGRFTISRDNAKSTLYLQMNSLNPEDTAAYYCAARSSGTGYYSRESFYEYWGQGTQVTVSS.

[0034] Specifically, the nanobody contains an amino acid sequence as shown in SEQ ID NO.29, or any one or more amino acid sequences that have at least 85% sequence similarity to SEQ ID NO.29.

[0035] SEQ ID NO. 29: DVQLQASGGGLVQSGDSLKLSCVASGGTFSSQPMGWFRQAPGKGRELVAAINRDGRLKYVADSVKGRFTISRDNAKNTLYLQIDSLKPEDTAAYYCAARSSGGNWYTRRSFYDYWGQGTQVTVSS.

[0036] In another aspect, the present invention provides a recombinant protein, wherein the recombinant protein is coupled to the above-mentioned nanobody with at least one of the following: immunoglobulin Fc domain, His tag, GST tag, MBP tag, FLAG tag, SUMO tag, Strep-tagII tag, Twin-Strep-tag tag, MyC tag, HA tag, AviTag tag, HaloTag tag, EGFP tag, SNAP tag, V5 tag, thioredoxin tag, GFP tag, mCherry tag, protein C tag, BCCP tag, Spot-tag tag, Isopeptag, SpyTag, serum albumin, albumin-binding polypeptide, prealbumin, C-terminal peptide, elastin-like polypeptide, cytokine, single-chain antibody, virus-like particle, ligase, kinase, prodrug activating enzyme, chemotherapeutic agent, gold nanoparticle or liposome.

[0037] On the other hand, the present invention provides a nucleic acid that encodes any of the above-mentioned anti-TIGIT antibodies or recombinant proteins.

[0038] In another aspect, the present invention provides an expression vector containing the above-mentioned nucleic acid, or the expression vector is used to express the above-mentioned anti-TIGIT antibody, or the expression vector is used to express the above-mentioned recombinant protein.

[0039] Specifically, the expression vector is a eukaryotic expression vector or a prokaryotic expression vector; the eukaryotic expression vector includes, but is not limited to, any one or more of the following: pFastBac, pPICZαA, pYES2, pCAMBIA sequence, pEGFP-N1, pcDNA series, pCMV sequence, and pLV series; the prokaryotic expression vector includes, but is not limited to, any one or more of the following: pET series, pCold series, pBAD series, pGEX series, pMAL series, pTrc / pTac, and pQE series.

[0040] In another aspect, the present invention provides a cell that includes, expresses, or secretes the anti-TIGIT antibody described above; or includes the recombinant protein described above; or includes the nucleic acid described above; or includes the expression vector described above.

[0041] Specifically, the cells are eukaryotic cells or prokaryotic cells; the eukaryotic cells include, but are not limited to, any one or more of yeast, filamentous fungi, insect cells, mammalian cells, or plant cells; the prokaryotic cells include, but are not limited to, any one or more of Escherichia coli, Bacillus subtilis, lactic acid bacteria, Streptomyces, Proteus mirabilis, Corynebacterium glutamicum, thermophilic bacteria, cyanobacteria, and halophilic bacteria.

[0042] More specifically, the yeast includes, but is not limited to, any one or more of the following: Pichia pastoris, Saccharomyces cerevisiae, Schizosomalidomiae, Kluyveromyces lactis, or Hansenula polymorpha.

[0043] More specifically, the filamentous fungi include, but are not limited to, any one or more of Trichoderma, Aspergillus niger, or Trichoderma reesei.

[0044] More specifically, the insect cells include, but are not limited to, any one or more of the Sf9, Sf21, or High-5 cell lines.

[0045] More specifically, the mammalian cells include, but are not limited to, any one or more of the following: Chinese hamster ovary cells (CHO), monkey kidney cells (COS), hamster kidney cells (BHK), mouse embryonic fibroblasts (NIH3T3), mouse myeloma cells (SP2 / 0 cells), ExpiCHO-S cell line, or FreeStyle 293-F cell line.

[0046] More specifically, the plant cells include, but are not limited to, any one or more of the following: tobacco BY-2 cells or Arabidopsis thaliana cells.

[0047] In another aspect, the present invention provides the use of the above-mentioned antibodies, recombinant proteins, nucleic acids, expression vectors and / or cells in the preparation of drugs for treating cancer.

[0048] Specifically, the cancers mentioned include, but are not limited to: oral cancer, lymphoma, respiratory system cancer, prostate cancer, leukemia, retinoblastoma, choriocarcinoma, peritoneal cancer, eye cancer, urinary system cancer, bone cancer, endometrial cancer, uterine cancer, bladder cancer, thyroid cancer, esophageal cancer, laryngeal cancer, head and neck cancer, acute lymphoblastic leukemia, melanoma, piloblastic leukemia, neuroblastoma, salivary gland cancer, digestive system cancer, myeloma, gastric cancer, liver cancer, skin cancer, lung cancer, rectal cancer, ovarian cancer, kidney cancer, pancreatic cancer, liver cancer, sarcoma, glioblastoma, basal cell carcinoma, breast cancer, cervical cancer, bile duct cancer, colorectal cancer, connective tissue cancer, rhabdomyosarcoma, squamous cell carcinoma, testicular cancer, B-cell lymphoma, chronic lymphocytic leukemia, and chronic myeloblastic leukemia.

[0049] The literature (Eichberger J, Spoerl S, Spanier G, et al. TIGIT Expression on Intratumoral Lymphocytes Correlates with Improved Prognosis in Oral Squamous Cell Carcinoma[J]. Biomedicines, 2022, 10(12): 3236.) disclosed that patients with high expression of TIGIT on CD3+ T cells had significantly longer overall survival (OS) and disease-free survival (DFS), suggesting that TIGIT can serve as an independent prognostic marker for oral squamous cell carcinoma.

[0050] The literature (Kansler, ER, Bence, NF, Kansler, JD, Geng, H., Zheng, Z., & Ansell, SM (2019). TIGIT and PD-1 mark intratumoral T cells with reduced effector function in B-cell non-Hodgkin lymphoma. Cancer Immunology Research, 7(2), 221-234.) comprehensively analyzed the expression characteristics of co-inhibitory receptors in tumor-infiltrating T cells of patients with different types of non-Hodgkin lymphoma (NHL) and found that TIGIT and PD-1 are the most frequently expressed co-inhibitory receptors in the tumor microenvironment of NHL.

[0051] The literature (Cho, BC, Patil, TS, Kim, T. et al. (2022). Tiragolumab plus atezolizumab versus placebo plus atezolizumab as a first-line treatment for PD-L1-selected non-small-cell lung cancer (CITYSCAPE): primary and follow-up analyses of a randomised, double-blind, phase II trial. The Lancet Oncology, 23(6), 773-784.) included 135 patients with PD-L1-positive (TPS≥1%) recurrent or metastatic non-small cell lung cancer (NSCLC). The results showed that the objective response rate (ORR) in the combination group was significantly higher than that in the monotherapy group. In the subgroup with high PD-L1 expression (TPS≥50%), the benefit of combination therapy was more obvious.

[0052] The literature (Hussain, M., Fizazi, K., Saad, F., et al. (2024). Coformulation of pembrolizumab and vibostolimab in patients with docetaxel-pretreated metastatic castration-resistant prostate cancer (mCRPC): KEYNOTE-365 cohort G. Journal of Clinical Oncology, 42(4_suppl), 149.) evaluated the safety and antitumor activity of TIGIT inhibitors in combination with PD-1 inhibitors in patients with docetaxel-pretreated metastatic castration-resistant prostate cancer (mCRPC), providing key clinical evidence for the application of TIGIT targeted therapy in prostate cancer.

[0053] Literature (Fourcade, J., Sun, Z., Benallaoua, M., et al. (2017). T-cellimmunoglobulin and ITIM domain (TIGIT) associates with CD8 +T-cell exhaustion and poor clinical outcome in AML patients. Clinical Cancer Research, 23(8), 2046-2056. This study was the first to systematically reveal the expression characteristics of TIGIT on CD8+ T cells in patients with acute myeloid leukemia (AML), its relationship with T-cell exhaustion, and its impact on clinical prognosis.

[0054] The literature (Yuan, B., Li, Y., Yang, Y., Zhang, Y., & Zhang, X. (2025). Oncolytic viruses and immunotherapy for the treatment of uveal melanoma and retinoblastoma: The current landscape and novel advances. International Journal of Molecular Sciences, 26(11), 3365.) systematically summarizes the application prospects of combined therapy of immune checkpoint inhibitors (including TIGIT) and oncolytic viruses in retinoblastoma, providing key reference for the development of TIGIT targeted therapy in pediatric intraocular malignancies.

[0055] The literature (Cui, H., Hamad, M., & Elkord, E. (2025). TIGIT in cancer: from mechanism of action to promising immunotherapeutic strategies. Cell Death & Disease, 16(1), 664.) disclosed that CD8+ T cells, NK cells and regulatory T cells (Tregs) in choriocarcinoma tumor-infiltrating lymphocytes (TILs) all highly express TIGIT.

[0056] The literature (Middelburg, RJ, Jochems, CHM, Schipper, K., et al. (2020). TIGIT blockade enhances functionality of peritoneal NK cells with altered expression of DNAM-1 / TIGIT / CD96 checkpoint molecules in ovarian cancer. OncoImmunology, 9(1), 1843247.) disclosed the expression and function of TIGIT in the peritoneal microenvironment of ovarian cancer (which is histologically homologous to primary peritoneal cancer and has highly similar biological characteristics), and for the first time systematically revealed the core role of TIGIT in peritoneal NK cell depletion and tumor immune escape.

[0057] The literature (Figueiredo, J., Vasconcelos, D., Cruz, C., & others. (2020). BAP1 loss drives immune suppression in uveal melanoma through upregulation of IDO1 and TIGIT. Journal of Pathology, 250(4), 420-439.) discloses that CD8+ in tumor-infiltrating lymphocytes (TILs) from primary uveal melanoma tissue and liver metastases... + T cells, NK cells, and regulatory T cells (Tregs) all highly express TIGIT; tumor cells highly express its ligand CD155, forming a complete TIGIT-CD155 inhibitory axis, and the expression level of TIGIT in metastatic lesions is significantly higher than that in primary lesions.

[0058] Literature (Zhang, Y., Sun, J., Zhang, C., & others. (2020). IntratumoralTIGIT + CD8 +T-cell infiltration determines poor prognosis and immune evasion in patients with muscle-invasive bladder cancer. The Journal for ImmunoTherapy of Cancer, 8(2), e000978. reveals the core role of TIGIT in immune escape, prognostic assessment and therapeutic potential of urinary system cancer, providing key clinical and experimental evidence for the application of TIGIT targeted therapy in urinary system cancer.

[0059] The literature (Zhou, Y., Yang, Z., Zhang, Y., & others. (2020). Single-cell RNA landscape of intratumoral heterogeneity and immunosuppressive microenvironment in advanced osteosarcoma. Nature Communications, 11(1), 6322.) first mapped the single-cell atlas of the immune microenvironment in osteosarcoma and systematically elucidated the core role of TIGIT in immune escape, metastatic progression and therapeutic potential of osteosarcoma.

[0060] The literature (Li, M., Wang, Y., Zhang, L. (2025). PD-1 and TIGIT coexpressing CD8+CD103+ tissue-resident memory cells in endometrial cancer as potential targets for immunotherapy. Journal for ImmunoTherapy of Cancer, 13(8),e008654.) is an original research paper on tissue-resident memory T cells (TRM) in endometrial cancer, which systematically reveals the core role of TIGIT in immune exhaustion, immune escape and combined immunotherapy in endometrial cancer.

[0061] The literature (Fu, WY, Cai, RF, Ma, ZT, et al. (2021). TIGIT-Fc as a Potential Therapeutic Agent for Fetomaternal Tolerance. Frontiers in Immunology, 12, 649135.) confirms the promoting effect of TIGIT-Fc fusion protein on maternal-fetal immune tolerance.

[0062] The literature (Zhang Yawei, Shi Hongjin, Fu Shi, et al. Research progress on the biological role of TIGIT and its application in bladder cancer[J]. Practical Medicine Journal, 2024, 40 (12): 1-7.) discloses that TIGI is upregulated in bladder cancer, which has an important impact on the occurrence, development and poor prognosis of bladder cancer, and immunotherapy that blocks TIGI signaling can improve bladder cancer.

[0063] The original research paper (Khalili, S., Jahani, F., Sadeghi, M., & others. (2025). Integratedbulk and single-cell transcriptomic profiling reveals NECTIN-TIGIT interaction underlies T cell exhaustion in papillary thyroid carcinoma. Clinical and Experimental Medicine, 25(11), 897.) reveals the core role and potential therapeutic value of TIGIT in immunosuppression of thyroid cancer through integrated transcriptomic analysis.

[0064] The literature (Liu, Y., Cao, X., Gao, Y., et al. (2021). Increased coexpression of PD-L1 and TIM3 / TIGIT is associated with poor overall survival of patients with esophageal squamous cell carcinoma. Journal of Cancer Research and Clinical Oncology, 147(13), 3819-3831.) systematically elucidated the expression characteristics of TIGIT in the immune microenvironment of esophageal cancer, its synergistic effect with other immune checkpoints, and its clinical prognostic value by integrating transcriptomic data from the TCGA database and clinical samples from 102 patients with esophageal squamous cell carcinoma.

[0065] The literature (Wu, J., Li, Y., Zhang, H., Wang, X., Liu, Y., & Chen, L. (2019). Blockade of TIGIT / CD155 signaling reverses T-cell exhaustion and enhancesantitumor capability in head and neck squamous cell carcinoma. Clinical Cancer Research, 25(12), 3646-3658) is an original research paper on the mechanism of T-cell exhaustion and immunosuppression mediated by the TIGIT / CD155 signaling axis in head and neck squamous cell carcinoma (including laryngeal squamous cell carcinoma). This study covers clinical samples and animal models of laryngeal cancer and systematically reveals the core role and therapeutic value of TIGIT in immune escape in laryngeal cancer.

[0066] The literature (Wu, J., Li, Y., Zhang, H., Wang, X., Liu, Y., & Chen, L. (2019). Blockade of TIGIT / CD155 signaling reverses T-cell exhaustion and enhancesantitumor capability in head and neck squamous cell carcinoma. Clinical Cancer Research, 25(12), 3646-3658.) published an original research paper on the mechanism of TIGIT / CD155 signaling axis mediating immunosuppression and tumor escape in head and neck squamous cell carcinoma (HNSCC). This study integrated clinical samples, in vitro experiments and transgenic mouse models to systematically reveal the core role of TIGIT in the immune microenvironment of head and neck cancer and its therapeutic translational value.

[0067] The literature (Chen, Y., Zhang, X., Wang, L., et al. (2019). TIGIT expression is upregulated in T cells and causes T cell dysfunction independent of PD-1 and Tim-3 in adult B lineage acute lymphoblastic leukemia. OncoImmunology, 8(10), e1632645.) systematically analyzed peripheral blood and bone marrow samples from adult B-lineage acute lymphoblastic leukemia (B-ALL) patients, and combined with in vitro functional experiments, elucidated the regulatory role of TIGIT on T cell function in ALL and its immunosuppressive mechanism independent of PD-1 and Tim-3.

[0068] Literature (Johnston, RJ, Comps-Agrar, L., Klebanoff, CA, et al. (2014). TIGIT and PD-1 impair tumor antigen-specific CD8 + T cells in melanoma patients. Journal of Clinical Investigation, 124(9), 3864-3872. ) Systematically elucidates the role of TIGIT in melanoma tumor antigen-specific CD8. +Expression characteristics on T cells, synergistic inhibition mechanism with PD-1, and clinical translational value.

[0069] The literature (Yang, X., Liu, Y., Chen, Y., Wang, H., Zhang, Y., & Jiang, X. (2022). TIGIT / PVR axis regulates anti-tumor immunity in hematologicmalignancies. Journal of Hematology & Oncology, 15(1), 126.) summarizes the expression characteristics, immunosuppressive mechanisms, and therapeutic potential of TIGIT in the immune microenvironment of B-cell chronic lymphocytic diseases (including hairy cell leukemia).

[0070] The literature (Wienke, B., Wagner, A., Borcherding, N., et al. (2024). Integrative analysis of neuroblastoma by single-cell RNA sequencing identifies the NECTIN2-TIGIT axis as a target for immunotherapy. Cancer Cell, 42(1), 110-125.e10.) comprehensively analyzed the immune microenvironment of neuroblastoma using single-cell RNA sequencing technology, and for the first time systematically elucidated the expression characteristics, immunosuppressive mechanism, and clinical translational value of the NECTIN2-TIGIT axis as a key immune checkpoint in neuroblastoma.

[0071] The literature (Andrew, EL, Brian, NC, Sarah, JD, et al. (2020). The immune microenvironment and neoantigen landscape of aggressive salivary gland carcinomas differ by subtype. Clinical Cancer Research, 26(14), 3547-3559.) used the immune microenvironment of invasive salivary gland carcinoma as the research environment to clarify the expression characteristics of TIGIT in different subtypes of salivary gland carcinoma, its association with immune infiltration, and its clinical prognostic value.

[0072] The literature (Wang, D., Gu, Y., Yan, X., et al. (2022). Role of CD155 / TIGIT indigestive cancers: Promising cancer target for immunotherapy. Frontiers in Oncology, 12, 844260.) comprehensively analyzes the expression characteristics, immunosuppressive mechanisms, clinical prognostic value, and therapeutic translational potential of the CD155 / TIGIT axis in various digestive system cancers, including gastric cancer, colorectal cancer, hepatocellular carcinoma, pancreatic cancer, and cholangiocarcinoma.

[0073] The literature (Guillerey, C., Harjunpää, H., Carrié, N., et al. (2018). TIGIT immune checkpoint blockade restores CD8+ T-cell immunity against multiple myeloma. Blood, 132(16), 1689-1701.) first systematically elucidated the core role of TIGIT in immune escape in multiple myeloma (MM), confirming that TIGIT blockade can restore CD8+ T-cell immunity. + T cell anti-tumor function.

[0074] The literature (He W, Zhang H, Wang X, et al. CD155 / TIGIT signaling regulates CD8+ T-cell metabolism and promotes tumor progression in human gastric cancer[J]. Cancer Research, 2017, 77(22):6375-6386. DOI:10.1158 / 0008-5472.CAN-17-0381.) disclosed that high expression of CD155 and TIGIT in gastric cancer tissues was significantly associated with tumor progression, late clinical stage, and poor prognosis.

[0075] The literature (Jiang Ning, Li Zuchu, Huang Huilian, et al. Application and challenges of T cell immune receptor monoclonal antibody combination therapy in first-line treatment of advanced hepatocellular carcinoma: a review based on the results of two phase II clinical studies [J]. Electronic Journal of Liver Cancer, 2024, (4)) disclosed a monoclonal antibody for T cell immune receptor (TIGIT). As a new immune checkpoint inhibitor, it is expected to be used in combination with PD-1 and PD-L1 inhibitors to further enhance the anti-tumor immune response.

[0076] Literature (Johnston, RJ, Comps-Agrar, L., Hackney, J., Kim, JS, & Korman, AJ (2014). TIGIT and PD-1 impair tumor antigen-specific CD8 + Tcells in melanoma patients. Journal of Clinical Investigation, 124(12), 5094-5106.) reveals the expression characteristics of TIGIT in the immune microenvironment of melanoma, the core mechanism mediating immunosuppression, and the value of blocking therapy.

[0077] The literature (Cho, BC, Kim, SW, Felip, E., et al. (2022). Tiragolumab plus atezolizumab versus placebo plus atezolizumab as first-line treatment for PD-L1-selected non-small-cell lung cancer (CITYSCAPE): primary and follow-up analyses of a randomised, double-blind, phase 2 trial. The Lancet Oncology, 23(6), 773-784.) confirms the significant clinical benefit of TIGIT inhibitors combined with PD-L1 inhibitors in non-small cell lung cancer (NSCLC), laying a key clinical foundation for the application of TIGIT in lung cancer immunotherapy.

[0078] The literature (Wu Han, Fan Peiwen, Feng Yaning, et al. Effect of PLA1A as a core gene in synergy with TIGIT and PD-1 on improving the response and prognosis of colorectal cancer immunotherapy [J]. Chinese Journal of Surgical Oncology, 2025, (2)) disclosed that TIGIT is co-expressed in colorectal cancer, revealing the predictive value of PD1 / TIGIT co-expression characteristics in the response to colorectal cancer immunotherapy.

[0079] The literature (Zhang, R., Li, L., Wang, L., Jazaeri, A., & Zhang, R. (2022). Tumor FAK orchestrates immunosuppression in ovarian cancer via the CD155 / TIGIT axis. Proceedings of the National Academy of Sciences of the United States of America, 119(18), e2117065119.) reveals the key regulatory role of the FAK / CD155 / TIGIT axis in immunosuppression in ovarian cancer and clarifies the feasibility of TIGIT as a target for immunotherapy in ovarian cancer.

[0080] The literature (Perales, O., Jilaveanu, L., Adeniran, A., Su, D., Hurwitz, M., Braun, D., Kluger, H., & Schoenfeld, D. (2024). TIGIT expression in renal cellcarcinoma infiltrating T cells is variable and inversely correlated with PD-1 and LAG3. Cancer Immunology, Immunotherapy, 73, 192.) systematically compared the differences in TIGIT expression between primary and metastatic renal cell carcinoma, clarifying the negative correlation between TIGIT and PD-1 and LAG3, breaking through the limitations of the traditional understanding of the synergistic effect of immune checkpoints.

[0081] The literature (Benci, JL, Xu, Y., Chao, T., et al. (2021). The CD155 / TIGIT axis promotes and maintains immune evasion in neoantigen-expressing pancreatic cancer. Cancer Cell, 39(9), 1200-1216.e8.) elucidates the core mechanism of the CD155 / TIGIT axis in immune evasion in pancreatic ductal adenocarcinoma (PDAC) and proposes a translatable combination immunotherapy regimen.

[0082] The literature (Ostroumov, D., Duong, S., Wingerath, J., Woller, N., Manns, MP, Timrott, K., Kleine, M., Ramackers, W., Roessler, S., Nahnsen, S., & Lohse, AW (2020). Transcriptome profiling identifies TIGIT as a marker of T-cell exhaustion in liver cancer. Hepatology, 72(6), 2022-2036.) systematically revealed for the first time the unique value of TIGIT as a core marker of T-cell exhaustion in liver cancer through whole-genome transcriptome analysis and functional validation, clarifying its key role in the regulation of the immune microenvironment and immunotherapy in liver cancer.

[0083] The literature (Flynn, RJ, Finnerty, H., Kaufman, HL, et al. (2020). Analysis of tumor-infiltrating NK and T cells highlights IL-15 stimulation and TIGIT blockade as a combination immunotherapy strategy for soft tissue sarcomas. Cancer Immunology Research, 8(12), 1535-1548. https: / / doi.org / 10.1158 / 2326-6066.CIR-20-1957) systematically elucidates the core role of TIGIT in the immune microenvironment of soft tissue sarcomas and proposes a novel immunotherapy strategy of IL-15 stimulation combined with TIGIT blockade.

[0084] The literature (Hung, AL, Maxwell, R., Theodros, D., et al. (2018). TIGIT and PD-1 dual checkpoint blockade enhances antitumor immunity and survival in GBM. Oncoimmunology, 7(8), e1466769.) verified through clinical sample analysis and glioblastoma mouse model for the first time that combined TIGIT and PD-1 blockade can significantly enhance antitumor immunity and prolong survival.

[0085] The literature (Malinga, NZ, Siwele, SC, Steel, HC, et al. (2025). Systemic levels of the soluble co-inhibitory immune checkpoints, TIGIT and CD155, are elevated in basal cell carcinoma. Journal of the American Academy of Dermatology, 92(5), e145-e147.) systematically analyzed the expression characteristics and clinical significance of the TIGIT / CD155 pathway in basal cell carcinoma, clarifying the value of TIGIT as a potential target for BCC immunotherapy.

[0086] The literature (Tang, T., Wang, W., Gan, L., et al. (2025). TIGIT expression inextrahepatic cholangiocarcinoma and its impact on CD8+ T cell exhaustion:implications for immunotherapy. Cell Death & Disease, 16(1), 90.) systematically elucidated the core role of TIGIT in the immune microenvironment of cholangiocarcinoma through single-cell sequencing, clinical sample validation, and PDX model experiments, clarifying its dual value as a target for immunotherapy and a prognostic biomarker for cholangiocarcinoma.

[0087] The literature (Ma, Y., Chen, S., Guo, H., et al. (2024). Combining TIGITblockade with MDSC inhibition hinders breast cancer bone metastasis by activating antitumor immunity. Cancer Discovery, 14(7), 1252-1271.) systematically revealed the core role of TIGIT in the immune microenvironment of breast cancer bone metastasis through clinical sample analysis, single-cell sequencing and in vitro and in vivo functional experiments, and clarified the therapeutic potential of TIGIT combined with MDSC inhibition strategy for breast cancer bone metastasis.

[0088] The literature (Huang Huichao. Effects of combined blocking of immune checkpoints TIGIT and PD-1 on cervical cancer xenograft models [D]. Qingdao: Qingdao University, 2024.) discloses the use of TIGIT and PD-1 antibodies alone or in combination, observing tumor growth and mouse growth status, and detecting the expression of both antibodies and cell apoptosis in tumors of each group. This verifies the enhanced anti-tumor effect after combined blocking of both antibodies, providing a new theoretical basis and target for future tumor immunotherapy based on intervention of TIGIT / PD-1 signaling.

[0089] The literature (Ruscetti, M., Wang, J., Cillo, AR, et al. (2020). Analysis of tumor-infiltrating NK and T cells highlights IL-15 stimulation and TIGIT blockade as a combination immunotherapy strategy for soft tissue sarcomas. Cancer Immunology Research, 8(12), 1572-1584.) systematically elucidated the core role of TIGIT in the immune microenvironment of soft tissue sarcomas through single-cell sequencing, clinical sample validation, and in vitro functional experiments, and clarified the feasibility of IL-15 combined with TIGIT blockade as an immunotherapy strategy for soft tissue sarcomas.

[0090] The literature (Kuester, D., Josson, S., Koppers-Lalic, D., et al. (2022). Single-cell transcriptomics reveals immune suppression and cell states predictive of patient outcomes in rhabdomyosarcoma. Nature Communications, 13(1), 3442.) systematically revealed the core role of TIGIT in the immune microenvironment of rhabdomyosarcoma (RMS) using single-cell transcriptomics, immunofluorescence and other technologies, and clarified that the interaction between TIGIT and NECTIN3 is the key mechanism for immunosuppression in fusion-positive RMS subtypes.

[0091] The literature (Zhang, Y., Li, J., Wang, H., et al. (2021). Increased dcoexpression of PD-L1 and TIM3 / TIGIT is associated with poor overall survival of patients with esophageal squamous cell carcinoma. Journal of the National Cancer Institute, 113(11), 1456-1465.) systematically confirms the core role of TIGIT in immunosuppression in ESCC and clarifies the dual value of TIGIT as a prognostic marker and an immunotherapy target.

[0092] The literature (Hinsch, A., Blessin, NC, Simon, R., wt al. (2019). Expression of the immune checkpoint receptor TIGIT in seminoma. Oncology Letters, 18(2), 1497-1502.) systematically detected the expression characteristics of TIGIT in tumor-infiltrating lymphocytes of testicular seminoma through tissue microarray immunohistochemical analysis, clarifying its role as an immune checkpoint in the immune microenvironment of testicular cancer, and providing a basis for immunotherapy of testicular cancer.

[0093] The literature (Peggs, KS, Grosso, JF, Goldberg, MV, et al. (2019). TIGIT and PD-1 mark intratumoral T cells with reduced effector function in B-cell non-Hodgkin lymphoma. Blood, 133(11), 1217-1228.) systematically confirms the core role of TIGIT in immunosuppression of B-NHL, clarifies the value of co-expression of TIGIT and PD-1 as a stratification marker for B-NHL immunotherapy, and provides theoretical support for the development of precision treatment regimens combining TIGIT inhibitors with PD-1 / PD-L1 inhibitors.

[0094] The literature (Arruga, F., Rubin, M., Papazoglou, D., Iannello, A., et al. (2023). The immunomodulatory molecule TIGIT is expressed by chronic lymphocytic leukemia cells and contributes to anergy. Haematologica, 108(5), 1497-1509.) first confirmed that TIGIT is expressed by chronic lymphocytic leukemia cells and mediates the anergic state of tumor cells, and clarified the association between the TIGIT / CD226 expression ratio and disease progression and treatment response.

[0095] The literature (Wang, F., Hou, HY, Wu, SJ, wt al. (2020). Increased Expression of TIGIT / CD57 in Peripheral Blood / Bone Marrow NK Cells in Patients with Chronic Myeloid Leukemia. BioMed Research International, 2020, 9531549.) systematically confirmed the core inhibitory role of TIGIT in the immune microenvironment of chronic myeloid leukemia (CML), clarified the direct correlation between TIGIT expression level and CML disease status and treatment response, and provided key experimental support for developing new CML immunotherapy strategies combining TIGIT inhibitors with TKIs.

[0096] In another aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned antibody, recombinant protein, nucleic acid, expression vector and / or cells.

[0097] Specifically, the dosage forms of the pharmaceutical composition include, but are not limited to: liquid solutions only, lyophilized powders, pre-filled syringes only, pre-filled syringes, lyophilized powders, tablets, capsules, granules, sprays, oral dispersible tablets / films, sublingual tablets / lozenges, implants, microneedle patches, gels, creams / ointments, suppositories, liposomes / nanoparticles, exosome carriers, inhaled dry powder (DPI), in situ gels, compound preparations, or child-friendly formulations (flavored granules, oral drops).

[0098] Specifically, the pharmaceutical composition further includes pharmaceutical excipients.

[0099] More specifically, the pharmaceutical excipients include any one or more of the following: binders, fillers, disintegrants, lubricants, preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solubilizers, or osmotic pressure regulators.

[0100] Specifically, the administration methods of the pharmaceutical composition include oral, injection, implantation, external application, spray, inhalation, or combinations thereof.

[0101] More specifically, the injection administration methods include, but are not limited to: intraocular injection, intravenous injection, intramuscular injection, subcutaneous injection, intrathecal injection, intra-articular injection, or intratumoral injection.

[0102] More specifically, the external administration method includes, but is not limited to: mucosal administration or transdermal / local administration.

[0103] Preferably, the mucosal administration includes, but is not limited to: nasal administration, buccal / sublingual administration, and ocular administration.

[0104] Preferably, the transdermal / local drug delivery includes, but is not limited to, transdermal patch drug delivery.

[0105] More specifically, the inhalation administration method includes, but is not limited to, nebulized inhalation or metered-dose inhaler inhalation.

[0106] In another aspect, the present invention provides the application of the above-mentioned antibodies, recombinant proteins, nucleic acids, expression vectors and / or cells in the preparation of TIGIT protein detection kits.

[0107] In another aspect, the present invention provides a method for detecting TIGIT protein, characterized in that the detection is performed using the aforementioned antibody, recombinant protein, nucleic acid, expression vector and / or cells, and the detection method is a non-disease diagnosis or treatment method.

[0108] Compared with the prior art, the present invention has the following advantages: The nanobodies provided by this invention have small molecular weight, high specificity, and high affinity (EC). 50 The concentration ranged from 2.36 to 49.01 ng / mL, which was significantly better than that of commercial antibodies (143 ng / mL). Attached Figure Description

[0109] Figure 1 The dose-response curve of HBM-E01 (SEQ ID NO.16) and TIGIT antigen in Example 5 is shown.

[0110] Figure 2 The dose-response curve of HBM-E02 (SEQ ID NO.17) and TIGIT antigen in Example 5 is shown.

[0111] Figure 3 The dose-response curve of HBM-E03 (SEQ ID NO.18) and TIGIT antigen in Example 5 is shown.

[0112] Figure 4 The dose-response curve of HBM-E04 (SEQ ID NO.19) and TIGIT antigen in Example 5 is shown.

[0113] Figure 5 The dose-response curve of HBM-E05 (SEQ ID NO.20) and TIGIT antigen in Example 5 is shown.

[0114] Figure 6 The dose-response curve of HBM-E06 (SEQ ID NO.21) and TIGIT antigen in Example 5 is shown.

[0115] Figure 7 The dose-response curve of HBM-E07 (SEQ ID NO.22) and TIGIT antigen in Example 5 is shown.

[0116] Figure 8 The dose-response curve of HBM-E08 (SEQ ID NO.23) and TIGIT antigen in Example 5 is shown.

[0117] Figure 9 The dose-response curve of HBM-E09 (SEQ ID NO.24) and TIGIT antigen in Example 5 is shown.

[0118] Figure 10 The dose-response curve of HBM-E10 (SEQ ID NO.25) and TIGIT antigen in Example 5 is shown.

[0119] Figure 11 The dose-response curve of HBM-E11 (SEQ ID NO.26) and TIGIT antigen in Example 5 is shown.

[0120] Figure 12 The dose-response curve of HBM-E12 (SEQ ID NO.27) and TIGIT antigen in Example 5 is shown.

[0121] Figure 13 The dose-response curve of HBM-E13 (SEQ ID NO.28) and TIGIT antigen in Example 5 is shown.

[0122] Figure 14 The dose-response curve of HBM-E14 (SEQ ID NO.29) and TIGIT antigen in Example 5 is shown.

[0123] Figure 15 The dose-response curves for the commercial TIGIT antibody and TIGIT antigen in Example 5 are shown.

[0124] Figures 1-15 In the graph, the horizontal axis represents antibody concentration, and the vertical axis represents the OD value under 450 nm detection conditions, used to calculate EC50. 50 (Half-maximal effective concentration) reflects the ability of an antibody to bind to an antigen.

[0125] Figure 16 The flow cytometry results are from the experiment on the specific binding of HBM-E01 (SEQ ID NO.16) to TIGIT-expressing cells in Example 6.

[0126] Figure 17 The flow cytometry results are from the experiment on the specific binding of HBM-E02 (SEQ ID NO.17) to TIGIT-expressing cells in Example 6.

[0127] Figure 18 The flow cytometry results are from the experiment on the specific binding of HBM-E03 (SEQ ID NO.18) to TIGIT-expressing cells in Example 6.

[0128] Figure 19 The flow cytometry results are from the experiment on the specific binding of HBM-E04 (SEQ ID NO.19) to TIGIT-expressing cells in Example 6.

[0129] Figure 20 The flow cytometry results are from the experiment on the specific binding of HBM-E05 (SEQ ID NO.20) to TIGIT-expressing cells in Example 6.

[0130] Figure 21 The flow cytometry results are from the experiment on the specific binding of HBM-E06 (SEQ ID NO.21) to TIGIT-expressing cells in Example 6.

[0131] Figure 22 The flow cytometry results are from the experiment on the specific binding of HBM-E07 (SEQ ID NO.22) to TIGIT-expressing cells in Example 6.

[0132] Figure 23 The flow cytometry results are from the experiment on the specific binding of HBM-E08 (SEQ ID NO.23) to TIGIT-expressing cells in Example 6.

[0133] Figure 24 The flow cytometry results are from the experiment on the specific binding of HBM-E09 (SEQ ID NO.24) to TIGIT-expressing cells in Example 6.

[0134] Figure 25 The flow cytometry results are from the experiment on the specific binding of HBM-E10 (SEQ ID NO.25) to TIGIT-expressing cells in Example 6.

[0135] Figure 26 The flow cytometry results are from the experiment on the specific binding of HBM-E11 (SEQ ID NO.26) to TIGIT-expressing cells in Example 6.

[0136] Figure 27 The flow cytometry results are from the experiment on the specific binding of HBM-E12 (SEQ ID NO.27) to TIGIT-expressing cells in Example 6.

[0137] Figure 28 The flow cytometry results are from the experiment on the specific binding of HBM-E13 (SEQ ID NO.28) to TIGIT-expressing cells in Example 6.

[0138] Figure 29 The flow cytometry results are from the experiment on the specific binding of HBM-E14 (SEQ ID NO.29) to TIGIT-expressing cells in Example 6.

[0139] Figure 30 The flow cytometry results are from the specific binding assay of the commercial TIGIT antibody to TIGIT-expressing cells in Example 6.

[0140] Figures 16-30In the graph, the horizontal axis represents fluorescence intensity, and the vertical axis represents cell count. By comparing the fluorescence intensity of the two peaks, it can be clearly seen that the antibody binds to TIGIT-expressing cells 293T-PCDH-TIGIT, while the antibody hardly binds to TIGIT-non-expressing cells 293T-PCDH.

[0141] Figure 31 The image shows the competitive inhibition curve of HBM-E01 (SEQ ID NO.16) binding to the TIGIT antigen in Example 7.

[0142] Figure 32 The image shows the competitive inhibition curve of HBM-E03 (SEQ ID NO.18) binding to the TIGIT antigen in Example 7.

[0143] Figure 33 The image shows the competitive inhibition curve of HBM-E04 (SEQ ID NO.19) binding to the TIGIT antigen in Example 7.

[0144] Figure 34 The image shows the competitive inhibition curve of HBM-E08 (SEQ ID NO.23) binding to the TIGIT antigen in Example 7.

[0145] Figure 35 The image shows the competitive inhibition curve of HBM-E09 (SEQ ID NO.24) binding to the TIGIT antigen in Example 7.

[0146] Figure 36 The image shows the competitive inhibition curve of HBM-E10 (SEQ ID NO.25) binding to the TIGIT antigen in Example 7.

[0147] Figure 37 The image shows the competitive inhibition curve of HBM-E13 (SEQ ID NO.28) binding to the TIGIT antigen in Example 7.

[0148] Figures 31-37 In the diagram, the horizontal axis represents the concentration of the nanobody, and the vertical axis represents the response value (in nm). The IC50 is obtained through curve fitting. 50 The IC50 value reflects the antibody's ability to competitively inhibit the interaction between TIGIT and CD155. The formula for calculating IC50 is: Y = A + (B - A) / [1 + (X / C)^D]; a) Y is the predicted value of the response variable (e.g., the response value); b) X is the concentration of the compound; c) A is the lower limit of the curve (the value of Y when X approaches negative infinity). d) B is the upper limit of the curve (the Y value when X approaches positive infinity). e) C is the IC50 value, which is the X value corresponding to the midpoint of the curve; f) D is the slope factor of the curve, which affects the steepness of the curve. Detailed Implementation

[0149] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0150] In this invention, EC 50 The dose-response curve was fitted using a four-parameter logistic model, the fitting method of which is described in Jarantow et al. Current protocols vol. 3,6 (2023). Specifically, the dose-response curve was fitted using its four parameters (minimum effect size A, maximum effect size B, half-maximum effective concentration C, i.e., EC50). 50 The relationship between dose (x) and response (y) is described by the Hill coefficient (D), as shown in the following formula:

[0151] In this invention, the coefficient of determination R 2 R0 is a unitless metric between 0.0 and 1.0 used to assess the strength of a linear relationship. It is a measure of the proportion of variability in the dependent variable (y) that can be explained by the independent variable (x), R0. 2 The closer to 1, the better the model fits, meaning the model can explain more data variability.

[0152] Example 1: Animal Immunization The human TIGIT protein antigen, tagged with His (Human TIGIT Protein, His Tag, synthesized from Q495A1 in Uniprot), was prepared by Genscript Biotech Co., Ltd. The antigen was then administered to Shenzhen Kangti Biomedical Technology Co., Ltd. for immunization. The specific immunization protocol was as follows: the human TIGIT protein was mixed for immunization. In the first two immunizations, 0.2 mg of the antigen was administered each time. For the third immunization and after the third immunization, the dose of each antigen was halved.

[0153] The specific steps are as follows: 1) Emulsify the antigen according to its properties; 2) Immunize alpacas with the emulsified antigen, once every two weeks, for a total of 5 times; 3) One week after the first to fifth immunizations, 5 mL of serum was drawn each time, and the titer was detected by ELISA using Human TIGIT Protein, His Tag as the coating antigen. 4) One week after the fourth and fifth immunizations, 50 mL of peripheral blood was drawn from each sample, and serum and PBMCs were separated.

[0154] Table 1 shows some of the valence test results during the experiment.

[0155] Table 1. Results of valence testing

[0156] The serum titer after the fourth and fifth immunizations did not reach 10. 3 Finally, the five-immune serum was selected for subsequent experiments.

[0157] Example 2 Construction of a phage-displayed nanobody library 1. Total RNA extraction and reverse transcription The serum of the five-immune patients was used to isolate B lymphocytes and extract and purify total RNA using the LeukoLOCK Total RNA Isolation System (Catalog No. AM1923) from Thermo Fisher Scientific. Then, cDNA was obtained by reverse transcription using the PrimeScript™ RT-PCR Kit (Catalog No. RR014A) from Takara.

[0158] 2. Amplification and purification of nanobody genes Nested PCR was performed using cDNA as a template to amplify the nanobody gene fragment. The primer sequences used are shown in Table 2.

[0159] Table 2 Primer Sequences

[0160] Table 3 First-round PCR reaction system

[0161] Table 4. First-round PCR reaction procedure

[0162] 2) Second round of PCR reaction The reaction system is shown in Table 5, and the reaction procedure is shown in Table 6.

[0163] Table 5 Second round PCR reaction system

[0164] Table 6. Second Round PCR Reaction Procedure

[0165] 3. Enzyme digestion and ligation of nanobody fragments and vectors 1) The nanobody fragment and pComb3x plasmid (Addgene catalog number 63890) were digested using NEB's restriction endonuclease SfiⅠ (catalog number R0123L) and reacted overnight at 50 °C.

[0166] 2) The enzyme-digested nanobody fragments were purified using the Qiagen QIAquick PCR Purification Kit (catalog number 28106). The 3400 bp bands of the plasmid after enzyme digestion were recovered using the Qiagen QIAquick Gel Recovery Kit (catalog number 28706), and the fragment and vector concentrations were determined.

[0167] 3) Use NEB's T4 ligase (catalog number M0202L) to ligate the digested plasmid vector and nanobody fragment, and react overnight at 16 ℃.

[0168] 4) Electroporate the enzyme-linked product into ER2738 competent cells and rescue it with helper phage (NEB, catalog number N0315S) to construct a phage-displaying nanobody library.

[0169] The library has a capacity of 7.5 × 10⁻⁶. 7 The CFU library size after rescue by helper phages was 3.9 × 10⁻⁶. 12 The library has good insertion rate and diversity, with a pfu / mL concentration.

[0170] Example 3 Screening of TIGIT nanobodies Human TIGIT protein with His-tagged antigen was coated in the first well of a 96-well microplate at a concentration of 50 μg / mL and incubated overnight at 4 °C. The next day, the coating solution was poured out, and the plate was washed three times with PBST. The first and second wells were blocked with gelatin, and the third well was blocked with BSA. The plate was incubated at room temperature for 1 h. The blocking solution was poured out, and the plate was washed three times with PBST. The phage nanobody library obtained in Example 2 was added to the first well and reacted for 2 h. The liquid was poured out, patted dry on clean absorbent paper, and washed five times with PBST. 100 μL of 0.1 M glycine-HCl buffer (pH=2.2) was added to the first well, and the plate was eluted for 10 min. Then, 10 μL of 1 M Tris-HCl buffer (pH=9.1) was added and mixed to neutralize. The liquid in the first well was aspirated and added to the second well. The plate was reacted for 1 h. h, remove phages bound to gelatin; aspirate the liquid from the second well, add it to the third well, react for 1 h, remove phages bound to BSA; collect the liquid from the third well, take 5 μL for titer determination, add the remainder to fresh E. coli ER2738 bacterial culture, incubate at 37 ℃ for 15 min; add carbenicillin and SB medium, incubate at 37 ℃, 220 rpm for 2 h; add helper phage (NEB, catalog number N0315S) and kanamycin, incubate overnight; the next day, centrifuge, collect the supernatant, add PEG-NaCl solution to precipitate and purify the phage. Perform the next round of screening on the amplified products, ensuring the same amount is added in each round, with the antigen coating concentration decreasing by a factor of 2, calculate the titer for each round, and select single clones for amplification and ELISA identification.

[0171] After four rounds of selection, a total of 22 TIGIT nanobodies with different sequences were obtained.

[0172] Example 4 Expression and purification of TIGIT nanobodies Positive monoclonal plasmids were extracted and transformed into WK6 competent cells (Aono Gene Technology Co., Ltd., catalog number HG-VDA1597). After resuscitation, the cells were plated on solid medium (carboxybenzyl resistant) and cultured overnight. The next day, single clones were picked and cultured in SB-carboxybenzyl medium, and IPTG was added to a final concentration of 1 mM to induce expression overnight. The following day, the bacteria were harvested by centrifugation at 9000 g for 15 min at room temperature. The bacterial pellet was carefully resuspended in 10 mL of pre-chilled TES (200 mM Tris-HCl, pH 8.0, 500 mM sucrose, 1 mM EDTA) and shaken at 4 ℃ for 2 h. 20 mL of TES / 4 (250 mL TES buffer in 750 mL ddH2O) was added to the resuspended cell pellet, and the mixture was shaken at 4 ℃ for 1 h. After centrifugation at 10000 g for 30 min at 4 ℃, the supernatant was collected and purified by nickel column chromatography to obtain high-purity anti-TIGIT nanobody.

[0173] Example 5: Detection of TIGIT Nanobody Affinity EC 50The experimental verification steps are as follows: Take out TIGIT antigen (0.5 mg / mL, 1:1 glycerol) from -20 ℃, thaw at room temperature, vortex for more than 30 s, add PBS, vortex to mix and dilute to the final concentration: 5 μg / mL, and put the TIGIT antigen back to -20 ℃ in time; add 100 μL of 5 μg / mL antigen to each well of row 1-6 of the ELISA plate, seal the plate with sealing film, and incubate at 4 ℃ overnight. On the second day, remove the liquid from the wells of the ELISA plate and blot dry. Wash three times with PBST for 1 min each time, blot dry after the last wash. Block each well in rows 1-8 with 200 μL of 3% BSA solution (prepared with CB solution) and incubate at room temperature for 1 h. After blocking, remove the liquid from the wells and blot dry. Wash three times with PBST for 1 min each time, blot dry after the last wash. Dilute the TIGIT nanobody with PBS three times sequentially to obtain 11 concentrations. Add 100 μL of nanobody sequentially from high to low concentration in rows 1-3 + 7 (except the last column). Add 100 μL of nanobody sequentially in rows 4-6 + 8 (except the last column). Add 100 μL of PBS to the last column. Incubate at room temperature with shaking for 1 h. After incubation, remove the liquid from the wells and blot dry. Wash three times with PBST for 1 min each time, blot dry after the last wash. Secondary antibody incubation: anti-HA HRP (1:600 ​​PBS dilution), add 100 μL of diluted anti-HA HRP, and incubate at room temperature with shaking for 1 h; after incubation, shake off the liquid in the wells of the microplate and wipe clean, wash 3 times with PBST, 1 min each time, and wipe clean after the last wash; prepare the color development solution, A solution + B solution = 1:1, 100 μL / well, and develop color at room temperature for 10 min; add 2 mol / L H2SO4, 50 μL / well, and read the value at 450 nm wavelength using a microplate reader.

[0174] Data analysis: Dose-response curves were plotted based on fluorescence intensity, and EC was calculated using nonlinear regression fitting. 50 Values, experimental results are shown in Figures 1-15 See Table 7.

[0175] Table 7

[0176] Among them, EC 50 The EC value reflects the binding ability of the antibody to the antigen; the smaller the value, the higher the affinity. The present invention provides the EC value of the nanobody (SEQ ID NO.16-29). 50The values ​​ranged from 2.36 to 49 ng / mL, significantly lower than the 143 ng / mL of the commercial antibody (purchased from Biolegend, catalog number 372709), indicating that it had stronger affinity.

[0177] Example 6 Specific cell binding assay The specific cell binding assay FACS procedure is as follows: 293T-PCDH cells and 293T-PCDH-TIGIT cells (both cell lines derived from 293T cells, purchased from Pronosei, catalog number CL-0005, obtained by constructing stable cell lines via lentiviral transfection; 293T-PCDH cells served as control cells, and 293T-PCDH-TIGIT cells were TIGIT-overexpressing cells used for subsequent validation of nanobody specific binding assays) were digested and resuspended in the culture medium. 100 μL / well was seeded into 96-well U-shaped plates, and nanobodies of the same concentration (final concentration 0.5 μg / mL) were added to each well. The plates were mixed and incubated at 37°C in a 5% CO2 incubator for 15 min. After incubation, the 96-well U-shaped plates were centrifuged at 1200 rpm for 5 min, the plates were spun, the supernatant was discarded, and 100 μL of FACS was added to each well for washing twice. The plates were then centrifuged at 1200 rpm for 5 min, the plates were spun, and the supernatant was discarded. 50 μL of Brilliant Violet 421rm ​​anti HA.11 epitope-tagged flow cytometry antibody (purchased from Biolegend, catalog number 682405) was added to each well. The flow cytometry antibody was diluted 1:200 with FACS buffer, gently mixed, and added to the 96-well plates. The plates were stained at 4°C for 15 min. After staining, the plates were centrifuged at 1200 rpm for 5 min, the plates were spun, and 100 μL of FACS buffer was added to each well to resuspend the cells. Centrifuge at rpm for 5 min, spin the plate, add 100 μL of FACS buffer to each well to resuspend the cells, and perform flow cytometry and data analysis (the flow cytometer was a Cytek Aurora full-spectrum flow cytometer, Cytek (Wuxi) Biasciences.Co. Itd, and the experiment was conducted using the matching reagents and operating instructions, and the data was automatically obtained using the matching software FlowJo); follow the same experimental procedure, using TIGIT flow cytometry antibody (purchased from Biolegend, catalog number 372709) as the control antibody.

[0178] The experimental results are shown in Figures 16-30 The TIGIT nanobodies all bound to 293T-PCDH-TIGIT cells but not to 293T-PCDH cells, indicating that the TIGIT nanobodies specifically bind to TIGIT. Figure 30The fact that the commercially available TIGIT flow cytometry antibody binds to 293T-PCDH-TIGIT cells but not to 293T-PCDH cells further corroborates the specific binding of the nanobody to TIGIT in this invention.

[0179] Example 7 IC 50 Measurement The steps are as follows: Pre-wet the sensor with PBST. Dilute the TIGIT nanobody with PBST to a concentration of 16 μg / mL (final concentration 8 μg / mL, 500 nM), performing a two-fold serial dilution, for a total of 8 dilutions (including a concentration of 0 μg / mL). Dilute TIGIT to a concentration of 4 μg / mL (molecular weight: 13 kDa, final concentration 2 μg / mL, 150 nM), and add 100 μL of each solution to the wells. Immobilize the biotinylated protein CD155-HIS-avi (final concentration 5 μg / mL). Set the program: buffer is 200 μL (same buffer as the sample), 200 μL per sample well, 200 μL per standard well, and 200 μL of the same buffer as the sample in the blank wells. Baseline time is 60 s, binding time is 120 s, dissociation time is 60 s, regeneration buffer is 3.475 GLY-HCl, and regeneration conditions are default. Select the program labeled as a column of samples, copy it to the current sensor, and analyze each column of samples. Add samples to a 96-well plate according to the procedure. Pre-wet the sensor with PBST, place it in the instrument, select the sensor location, choose the save location, name the file, confirm the program, and run the program. Open the data analysis software and, according to IC... 50 Value procedure analysis (dose response) was performed, and the dose-response curves were fitted using a four-parameter logistic model to obtain the IC50 of each nanobody. 50 Values. Experimental results are shown in Table 8 and... Figures 31-37 Except for HBM-E02, HBM-E05, HBM-E06, HBM-E07, HBM-E11, HBM-E12, and HBM-E14 antibodies, all showed concentration-dependent inhibitory effects. Among them, HBM-E01 was the most potent, with an IC50 value of [missing value]. 50 The IC50 value was 66.68 nM, demonstrating excellent in vitro inhibitory activity. The IC50 values ​​of the remaining nanobodies were... 50 The values ​​range from 70 nM to 150 nM.

[0180] Table 8

[0181] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. An anti-TIGIT antibody, characterized in that, The anti-TIGIT antibody is a nanobody, and the complementarity-determining region of the nanobody includes any one or more of the following: (1) A CDR region with a sequence as shown in SEQ ID NO.1-3, or a CDR region with a sequence similarity of at least 85% to SEQ ID NO.1-3; (2) A CDR region with a sequence as shown in SEQ ID NO.4-6, or a CDR region with a sequence similarity of at least 85% to SEQ ID NO.4-6; (3) A CDR region with a sequence as shown in SEQ ID NO.7-9, or a CDR region with a sequence similarity of at least 85% to SEQ ID NO.13-15; (4) CDR regions with sequences as shown in SEQ ID NO.1, SEQ ID NO.3, and SEQ ID NO.10, or CDR regions with sequences having at least 85% sequence similarity to SEQ ID NO.1, SEQ ID NO.3, and SEQ ID NO.10; (5) CDR regions with sequences as shown in SEQ ID NO.4, SEQ ID NO.11, and SEQ ID NO.12, or CDR regions with sequences having at least 85% sequence similarity to SEQ ID NO.4, SEQ ID NO.11, and SEQ ID NO.12; (6) A CDR region with a sequence as shown in SEQ ID NO.13-15, or a CDR region with a sequence similarity of at least 85% to SEQ ID NO.13-15.

2. The anti-TIGIT antibody according to claim 1, characterized in that, The nanobody contains an amino acid sequence as shown in SEQ ID NO:16, or any one or more amino acid sequences that have at least 85% sequence similarity to SEQ ID NO:

16.

3. The anti-TIGIT antibody according to claim 1, characterized in that, The nanobody contains an amino acid sequence as shown in SEQ ID NO:17, or any one or more amino acid sequences that have at least 85% sequence similarity to SEQ ID NO:

17.

4. The anti-TIGIT antibody according to claim 1, characterized in that, The nanobody contains an amino acid sequence as shown in SEQ ID NO:18, or any one or more amino acid sequences that have at least 85% sequence similarity to SEQ ID NO:

18.

5. The anti-TIGIT antibody according to claim 1, characterized in that, The nanobody contains an amino acid sequence as shown in SEQ ID NO:19, or any one or more amino acid sequences that have at least 85% sequence similarity to SEQ ID NO:

19.

6. The anti-TIGIT antibody according to claim 1, characterized in that, The nanobody contains an amino acid sequence as shown in SEQ ID NO:20, or any one or more amino acid sequences that have at least 85% sequence similarity to SEQ ID NO:

20.

7. The anti-TIGIT antibody according to claim 1, characterized in that, The nanobody contains an amino acid sequence as shown in SEQ ID NO:21, or any one or more amino acid sequences that have at least 85% sequence similarity to SEQ ID NO:

21.

8. The anti-TIGIT antibody according to claim 1, characterized in that, The nanobody contains an amino acid sequence as shown in SEQ ID NO:22, or any one or more amino acid sequences that have at least 85% sequence similarity to SEQ ID NO:

22.

9. The anti-TIGIT antibody according to claim 1, characterized in that, The nanobody contains an amino acid sequence as shown in SEQ ID NO:23, or any one or more amino acid sequences that have at least 85% sequence similarity to SEQ ID NO:

23.

10. The anti-TIGIT antibody according to claim 1, characterized in that, The nanobody contains an amino acid sequence as shown in SEQ ID NO:24, or any one or more amino acid sequences that have at least 85% sequence similarity to SEQ ID NO:

24.

11. The anti-TIGIT antibody according to claim 1, characterized in that, The nanobody contains an amino acid sequence as shown in SEQ ID NO:25, or any one or more amino acid sequences that have at least 85% sequence similarity to SEQ ID NO:

25.

12. The anti-TIGIT antibody according to claim 1, characterized in that, The nanobody contains an amino acid sequence as shown in SEQ ID NO:26, or any one or more amino acid sequences that have at least 85% sequence similarity to SEQ ID NO:

26.

13. The anti-TIGIT antibody according to claim 1, characterized in that, The nanobody contains an amino acid sequence as shown in SEQ ID NO:27, or any one or more amino acid sequences that have at least 85% sequence similarity to SEQ ID NO:

27.

14. The anti-TIGIT antibody according to claim 1, characterized in that, The nanobody contains an amino acid sequence as shown in SEQ ID NO:28, or any one or more amino acid sequences that have at least 85% sequence similarity to SEQ ID NO:

28.

15. The anti-TIGIT antibody according to claim 1, characterized in that, The nanobody contains an amino acid sequence as shown in SEQ ID NO:29, or any one or more amino acid sequences that have at least 85% sequence similarity to SEQ ID NO:

29.

16. A recombinant protein, characterized in that, The nanobody according to any one of claims 1-15 is attached with at least one of the following: immunoglobulin Fc domain, His tag, GST tag, MBP tag, FLAG tag, SUMO tag, Strep-tag II tag, Twin-Strep-tag tag, MyC tag, HA tag, AviTag tag, HaloTag tag, EGFP tag, SNAP tag, V5 tag, thioredoxin tag, GFP tag, mCherry tag, protein C tag, BCCP tag, Spot-tag tag, Isopeptag, SpyTag, serum albumin, albumin-binding peptide, prealbumin, C-terminal peptide, elastin-like peptide, cytokine, single-chain antibody, virus-like particle, ligase, kinase, prodrug activating enzyme, chemotherapeutic agent, gold nanoparticle, or liposome.

17. A nucleic acid, characterized in that, The nucleic acid encodes the anti-TIGIT antibody as described in any one of claims 1-15 or the recombinant protein as described in claim 16.

18. An expression carrier, characterized in that, The expression vector contains the nucleic acid of claim 17, or the expression vector is used to express the anti-TIGIT antibody of any one of claims 1-15, or the expression vector is used to express the recombinant protein of claim 16.

19. The expression vector according to claim 18, characterized in that, The expression vector is a eukaryotic expression vector or a prokaryotic expression vector; the eukaryotic expression vector includes any one or more of the following: pFastBac, pPICZαA, pYES2, pCAMBIA sequence, pEGFP-N1, pcDNA series, pCMV sequence, and pLV series; the prokaryotic expression vector includes any one or more of the following: pET series, pCold series, pBAD series, pGEX series, pMAL series, pTrc / pTac, and pQE series.

20. A cell characterized in that, The cell comprises, expresses, or secretes the anti-TIGIT antibody according to any one of claims 1-15; or, the cell comprises the recombinant protein according to claim 16; or, the cell comprises the nucleic acid according to claim 17; or, the cell comprises the expression vector according to any one of claims 18-19.

21. The cell according to claim 20, characterized in that, The cells are eukaryotic or prokaryotic cells; the eukaryotic cells include any one or more of yeast, filamentous fungi, insect cells, mammalian cells, or plant cells; the prokaryotic cells include any one or more of Escherichia coli, Bacillus subtilis, lactic acid bacteria, Streptomyces, Proteus mirabilis, Corynebacterium glutamicum, thermophilic bacteria, cyanobacteria, and halophilic bacteria.

22. The use of the anti-TIGIT antibody according to any one of claims 1-15, the recombinant protein according to claim 16, the nucleic acid according to claim 17, the expression vector according to any one of claims 18-19, and / or the cell according to any one of claims 20-21 in the preparation of a medicament for treating cancer.

23. The application according to claim 22, characterized in that, The cancers mentioned include any one or more of the following: oral cancer, lymphoma, respiratory system cancer, prostate cancer, leukemia, retinoblastoma, choriocarcinoma, peritoneal cancer, eye cancer, urinary system cancer, bone cancer, endometrial cancer, uterine cancer, bladder cancer, thyroid cancer, esophageal cancer, laryngeal cancer, head and neck cancer, acute lymphoblastic leukemia, melanoma, hairy cell leukemia, neuroblastoma, salivary gland cancer, digestive system cancer, myeloma, gastric cancer, liver cancer, skin cancer, lung cancer, rectal cancer, ovarian cancer, kidney cancer, pancreatic cancer, liver cancer, sarcoma, glioblastoma, basal cell carcinoma, breast cancer, cervical cancer, bile duct cancer, colorectal cancer, connective tissue cancer, rhabdomyosarcoma, squamous cell carcinoma, testicular cancer, B-cell lymphoma, chronic lymphocytic leukemia, and chronic myeloid leukemia.

24. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the anti-TIGIT antibody according to any one of claims 1-15, the recombinant protein according to claim 16, the nucleic acid according to claim 17, the expression vector according to any one of claims 18-19, and / or the cell according to any one of claims 20-21.

25. The pharmaceutical composition according to claim 24, characterized in that, The pharmaceutical composition also includes pharmaceutical excipients.

26. The pharmaceutical composition according to claim 25, characterized in that, The pharmaceutical excipients include any one or more of the following: binders, fillers, disintegrants, lubricants, preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solubilizers, or osmotic pressure regulators.

27. The use of the anti-TIGIT antibody according to any one of claims 1-15, the recombinant protein according to claim 16, the nucleic acid according to claim 17, the expression vector according to any one of claims 18-19, and / or the cell according to any one of claims 20-21 in the preparation of a TIGIT protein detection kit.

28. A method for detecting TIGIT protein, characterized in that, The detection is performed using the nanobody according to any one of claims 1-15, the recombinant protein according to claim 16, the nucleic acid according to claim 17, the expression vector according to any one of claims 18-19, and / or the cell according to any one of claims 20-21, wherein the detection method is a non-disease diagnosis or treatment method.

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