Anti-triggering receptor-1 antibody or antigen-binding fragment thereof, and use thereof

Abstract

Provided in the present application is an anti-triggering receptor-1 antibody or an antigen-binding fragment thereof. The anti-triggering receptor-1 antibody with high affinity is developed in the present application. The antibody is screened and prepared from a humanized recombinant antibody phage display library. Moreover, the prepared antibody is subjected to protein-level binding assay, cell-level binding assay, affinity kinetic characterization, epitope binning, binding activity assay on human monocytes, assay for the effect on the expression of a surface marker of human monocytes, assay for the promotion of cytokine secretion by human monocytes, assay for the promotion of pro-inflammatory cytokine secretion by human primary PBMCs, internalization assay, anti-tumor activity assay, etc. The antibody provides a new tool and a new concept for the diagnosis, prevention or treatment of tumors expressing a triggering receptor-1 and diseases involving the triggering receptor-1.

Description

Anti-triggering receptor-1 antibody or antigen-binding fragment thereof and use thereof Technical Field

[0001] The present application belongs to the field of biotechnology, and specifically relates to an anti-triggering receptor-1 antibody or an antigen-binding fragment thereof and applications thereof. Background Art

[0002] Myeloid cells are a major component of the tumor microenvironment (TME). They are a heterogeneous mixed cell population composed of monocytes, neutrophils, tumor-associated macrophages (TAMs), and dendritic cells (DCs). These myeloid cell populations exist in two states: immunostimulatory and immunosuppressive, which can stimulate or inhibit anti-tumor T cell-mediated responses, respectively. Immunosuppressive myeloid cells are an important cause of T cell dysfunction and resistance to anticancer therapy in tumors, and studies have gradually investigated them as targets for tumor treatment. Strategies targeting myeloid cells include inhibiting myeloid cell survival or recruitment to tumors, depleting immunosuppressive cells, blocking immunosuppression, and reprogramming immunosuppressive cells into proinflammatory cells.

[0003] Triggering receptor expressed on myeloid cells-1 (TREM1, also known as CD354) is a member of the immunoglobulin variable domain receptor superfamily and is primarily expressed on neutrophils, monocyte subsets, and tissue macrophages. TREM-1 consists of an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain contains an Ig V region, while the intracellular domain of TREM1 lacks a signaling motif and transmits intracellular signals by recruiting DNAX activating protein 12 kDa (DAP12), a transmembrane adaptor protein containing an immunoreceptor tyrosine-based activation motif (ITAM). The TREM1-DAP12 signaling pathway amplifies toll-like receptor (TLR)-mediated inflammatory responses, including the induction of cytokines such as IL-8 and TNFα and chemokines such as MCP-1, as well as neutrophil degranulation and phagocytosis. TREM1 is expressed on myeloid cell populations with an immunosuppressive phenotype within the TME and is upregulated by immunosuppressive signals within the TME, such as hypoxia. TREM1 is highly expressed in a variety of tumors, including hepatocellular carcinoma (HCC), non-small cell lung cancer (NSCLC), and breast cancer (BC), and is a predictor of progression and poor prognosis. The tumor microenvironment is complex, and although the exact mechanism by which TREM1 inhibits anti-tumor immune responses remains unclear, stimulating TREM1 signaling in the TME to activate myeloid cells provides a strategy for tumor immunotherapy. By leveraging the pro-inflammatory function of TREM1, the suppressive myeloid cell phenotype in the TME is converted to a pro-inflammatory phenotype, thereby generating an anti-tumor response. Furthermore, TREM1 mediates innate immune responses to microbial pathogens and participates in the development of various diseases. Studies have shown that in patients with sepsis, elevated TREM-1 expression on immune cells and elevated circulating levels of sTREM-1 are associated with increased mortality. TREM1 is highly expressed in intestinal macrophages, promoting the development of inflammatory bowel disease (IBD).

[0004] In summary, targeting TREM1 and developing antibodies against TREM1 is of great significance in the field of new drug research and development. Summary of the Invention

[0005] The present application provides an anti-trigger receptor-1 antibody or its antigen-binding fragment and its application, develops a new anti-trigger receptor-1 antibody or its antigen-binding fragment, and provides new tools and new ideas for the diagnosis, prevention or treatment of tumors expressing trigger receptor-1 and diseases involving trigger receptor-1.

[0006] In a first aspect, the present application provides an anti-triggering receptor-1 antibody or an antigen-binding fragment thereof, wherein the anti-triggering receptor-1 antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3;

[0007] The amino acid sequence of the HCDR1 is selected from any one of the following sequences: the sequence shown in SEQ ID NO: 19, 25, 31, 37 or 43, and sequences having at least 90% homology thereto and function identical or similar thereto; the amino acid sequence of the HCDR2 is selected from any one of the following sequences: the sequence shown in SEQ ID NO: 20, 26, 32, 38 or 44, and sequences having at least 90% homology thereto and function identical or similar thereto; the amino acid sequence of the HCDR3 is selected from any one of the following sequences: the sequence shown in SEQ ID NO: 21, 27, 33 or 39, and sequences having at least 90% homology thereto and function identical or similar thereto;

[0008] The amino acid sequence of the LCDR1 is selected from any one of the following sequences: the sequence shown in SEQ ID NO: 22, 28, 34, 40 or 45, and sequences with at least 90% homology to each other and functionally identical or similar; the amino acid sequence of the LCDR2 is selected from any one of the following sequences: SEQ ID NO: 23, 29, 35 or 41, and sequences with at least 90% homology to each other and functionally identical or similar; the amino acid sequence of the LCDR3 is selected from any one of the following sequences: the sequence shown in SEQ ID NO: 24, 30, 36, 42 or 46, and sequences with at least 90% homology to each other and functionally identical or similar.

[0009] This application develops high-affinity anti-trigger receptor-1 antibodies (anti-TREM1 antibodies) targeting trigger receptor-1, screens and prepares a variety of anti-TREM1 antibodies from a humanized recombinant antibody phage display library, and conducts protein level binding assays, cell level binding assays, affinity kinetic characterization, epitope grouping, binding activity assays on human monocytes, assays on the effects on the expression of human monocyte surface markers, assays on the promotion of cytokine secretion by human monocytes, assays on the promotion of pro-inflammatory cytokines secretion by human primary PBMCs, internalization assays, and anti-tumor activity assays on the prepared anti-TREM1 antibodies, fully analyzing each anti-TREM1 antibody and providing a basis for its precise application.

[0010] In this application, the homology (or identity) refers to a sequence that can be evaluated by the naked eye or by computer software (such as the software program described in Current Protocols in Molecular Biology by Ausubel et al. eds. (2007) or the Basic Local Alignment Search Tool (BLAST) tool, etc.). When a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The homology between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the homology between related sequences. A polynucleotide sequence or amino acid sequence that has a certain percentage (e.g., 90%, 95%, 98% or 99%) of "sequence homology" with another sequence means that when the sequences are aligned, that percentage of bases or amino acids in the two compared sequences are the same.

[0011] It is understood that the present application has discovered anti-trigger receptor-1 antibodies or antigen-binding fragments thereof in specific CDR regions. Based on the present application, those skilled in the art can use the common technical means in the art to replace, delete, or add one or at least two amino acid residues (such as conservative mutations) to obtain other sequences with the same or similar functions. In addition, the sequences of antibody constant regions known in the art can also be used to further construct antigen-binding fragments in the form of Fab, Fab', F(ab')2, scFv, (scFv)2, etc. There are no technical barriers, and therefore, they should be within the scope of protection of this application.

[0012] In this application, all limitations on "at least 90% homology" can be freely selected from 90% to 100%, for example, it can be 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, etc., preferably at least 95%, more preferably at least 98%, and even more preferably at least 99%.

[0013] Preferably, the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are any one of the following sequences:

[0014] The sequences shown in SEQ ID NO: 19, 20 and 21 or sequences having at least 90% homology thereto and having the same or similar functions,

[0015] The sequences shown in SEQ ID NO: 25, 26 and 27 or sequences having at least 90% homology thereto and having the same or similar functions,

[0016] The sequences shown in SEQ ID NO: 31, 32 and 33, or sequences having at least 90% homology thereto and having the same or similar functions,

[0017] The sequences shown in SEQ ID NO: 37, 38 and 39 or sequences having at least 90% homology thereto and having the same or similar functions, or,

[0018] The sequences shown in SEQ ID NO: 43, 44 and 21 or sequences having at least 90% homology thereto and having the same or similar functions.

[0019] Preferably, the amino acid sequences of LCDR1, LCDR2 and LCDR3 are any one of the following sequences:

[0020] The sequences shown in SEQ ID NO: 22, 23 and 24, or sequences having at least 90% homology thereto and having the same or similar functions,

[0021] The sequences shown in SEQ ID NO: 28, 29 and 30 or sequences having at least 90% homology thereto and having the same or similar functions,

[0022] The sequences shown in SEQ ID NO: 34, 35 and 36, or sequences having at least 90% homology thereto and having the same or similar functions,

[0023] The sequences shown in SEQ ID NO: 40, 41 and 42 or sequences having at least 90% homology thereto and having the same or similar functions, or,

[0024] The sequences shown in SEQ ID NO: 45, 23 and 46 or sequences having at least 90% homology thereto and having the same or similar functions.

[0025] Preferably, the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the anti-trigger receptor-1 antibody or antigen-binding fragment thereof are SEQ ID NOs: 19, 20 and 21, respectively, or sequences that have at least 90% homology to each other and are functionally identical or similar; the amino acid sequences of LCDR1, LCDR2 and LCDR3 are SEQ ID NOs: 22, 23 and 24, respectively, or sequences that have at least 90% homology to each other and are functionally identical or similar; or,

[0026] The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the anti-trigger receptor-1 antibody or antigen-binding fragment thereof are SEQ ID NOs: 25, 26 and 27, respectively, or sequences that have at least 90% homology to each other and function identically or similarly; the amino acid sequences of LCDR1, LCDR2 and LCDR3 are SEQ ID NOs: 28, 29 and 30, respectively, or sequences that have at least 90% homology to each other and function identically or similarly; or

[0027] The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the anti-trigger receptor-1 antibody or antigen-binding fragment thereof are SEQ ID NOs: 31, 32 and 33, respectively, or sequences that have at least 90% homology to each other and function identically or similarly; the amino acid sequences of LCDR1, LCDR2 and LCDR3 are SEQ ID NOs: 34, 35 and 36, respectively, or sequences that have at least 90% homology to each other and function identically or similarly; or

[0028] The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the anti-trigger receptor-1 antibody or antigen-binding fragment thereof are SEQ ID NOs: 37, 38 and 39, respectively, or sequences that have at least 90% homology to each other and function identically or similarly; the amino acid sequences of LCDR1, LCDR2 and LCDR3 are SEQ ID NOs: 40, 41 and 42, respectively, or sequences that have at least 90% homology to each other and function identically or similarly; or

[0029] The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the anti-trigger receptor-1 antibody or antigen-binding fragment thereof are respectively the sequences shown in SEQ ID NOs: 43, 44 and 21, or sequences that have at least 90% homology to each other and are functionally identical or similar; the amino acid sequences of LCDR1, LCDR2 and LCDR3 are respectively the sequences shown in SEQ ID NOs: 45, 23 and 46, or sequences that have at least 90% homology to each other and are functionally identical or similar.

[0030] Preferably, the amino acid sequence of the heavy chain variable region of the anti-trigger receptor-1 antibody or antigen-binding fragment thereof is the sequence shown in SEQ ID NO: 9 or a sequence thereof with at least 90% homology and the same or similar functions; the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO: 10 or a sequence thereof with at least 90% homology and the same or similar functions; or,

[0031] The amino acid sequence of the heavy chain variable region of the anti-trigger receptor-1 antibody or antigen-binding fragment thereof is the sequence shown in SEQ ID NO: 11 or a sequence thereof with at least 90% homology and the same or similar functions; the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO: 12 or a sequence thereof with at least 90% homology and the same or similar functions; or

[0032] The amino acid sequence of the heavy chain variable region of the anti-trigger receptor-1 antibody or antigen-binding fragment thereof is the sequence shown in SEQ ID NO: 13 or a sequence thereof with at least 90% homology and the same or similar functions; the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO: 14 or a sequence thereof with at least 90% homology and the same or similar functions; or

[0033] The amino acid sequence of the heavy chain variable region of the anti-trigger receptor-1 antibody or antigen-binding fragment thereof is the sequence shown in SEQ ID NO: 15 or a sequence thereof with at least 90% homology and the same or similar functions; the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO: 16 or a sequence thereof with at least 90% homology and the same or similar functions; or

[0034] The amino acid sequence of the heavy chain variable region of the anti-trigger receptor-1 antibody or its antigen-binding fragment is the sequence shown in SEQ ID NO: 17 or a sequence thereof with at least 90% homology and the same or similar functions; the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO: 18 or a sequence thereof with at least 90% homology and the same or similar functions.

[0035] Preferably, the anti-triggering receptor-1 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and a light chain constant region.

[0036] Preferably, the heavy chain constant region comprises a human IgG1, IgG2 or IgG4 constant region, and the light chain constant region comprises a human Ig kappa (κ) chain or lambda (λ) chain constant region;.

[0037] Preferably, the amino acid sequence of the heavy chain constant region includes the sequence shown in SEQ ID NO:4, and the light chain constant region includes the sequence shown in SEQ ID NO:5.

[0038] In a second aspect, the present application provides a nucleic acid molecule encoding the anti-triggering receptor-1 antibody or its antigen-binding fragment as described in the first aspect.

[0039] In a third aspect, the present application provides an expression vector comprising the nucleic acid molecule described in the second aspect.

[0040] In a fourth aspect, the present application provides a host cell, wherein the host cell contains at least one copy of the expression vector as described in the third aspect; or the nucleic acid molecule as described in the second aspect is integrated into the chromosome of the host cell.

[0041] In a fifth aspect, the present application provides a conjugate or fusion protein, which comprises: the anti-triggering receptor-1 antibody or its antigen-binding fragment described in the first aspect.

[0042] Preferably, the conjugate or fusion protein further comprises other parts directly or indirectly connected to the anti-triggering receptor-1 antibody or its antigen-binding fragment, including: any one or a combination of at least two of cell surface receptors, small molecule compounds, small molecule polymers, active proteins or polypeptides.

[0043] In a sixth aspect, the present application provides a pharmaceutical composition, comprising any one or a combination of at least two of the anti-trigger receptor-1 antibody or its antigen-binding fragment described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the host cell described in the fourth aspect, or the conjugate or fusion protein described in the fifth aspect.

[0044] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0045] In the seventh aspect, the present application provides a kit comprising: the anti-trigger receptor-1 antibody or its antigen-binding fragment described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the host cell described in the fourth aspect, the conjugate or fusion protein described in the fifth aspect, or any one or a combination of at least two of the pharmaceutical composition described in the sixth aspect.

[0046] In the eighth aspect, the present application provides the use of any one or a combination of at least two of the anti-trigger receptor-1 antibody or its antigen-binding fragment described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the host cell described in the fourth aspect, the conjugate or fusion protein described in the fifth aspect, or the pharmaceutical composition described in the sixth aspect in the preparation of a drug targeting trigger receptor-1, characterized in that the drug is used to prevent and / or treat tumors expressing trigger receptor-1 and / or diseases in which trigger receptor-1 is involved.

[0047] Preferably, the diseases in which triggering receptor-1 is involved include inflammatory diseases and / or autoimmune diseases.

[0048] Preferably, the tumor expressing triggering receptor-1 includes solid tumors such as non-small cell lung cancer (NSCLC), breast cancer, ovarian cancer, colorectal cancer, head and neck squamous cell carcinoma, etc.; blood tumors such as acute myeloid leukemia (AML), etc.

[0049] In the ninth aspect, the present application provides the use of any one or a combination of at least two of the anti-trigger receptor-1 antibody or its antigen-binding fragment as described in the first aspect, the nucleic acid molecule as described in the second aspect, the expression vector as described in the third aspect, the host cell as described in the fourth aspect, the conjugate or fusion protein as described in the fifth aspect, or the pharmaceutical composition as described in the sixth aspect in combination with other anti-tumor drugs or other drugs for treating autoimmune diseases in the preparation of drugs, wherein the drugs are used to prevent and / or treat tumors expressing trigger receptor-1 and / or diseases in which trigger receptor-1 is involved.

[0050] Preferably, the diseases in which triggering receptor-1 is involved include inflammatory diseases and / or autoimmune diseases.

[0051] Preferably, the other anti-tumor drugs include any one or a combination of at least two of immunomodulators, cytotoxic chemotherapy drugs or small molecule targeted drugs.

[0052] Preferably, the immunomodulator comprises any one of anti-PD1 antibodies, anti-PDL1 antibodies or anti-CTLA4 antibodies, or a combination of at least two of them.

[0053] Preferably, the cytotoxic chemotherapy drug comprises any one or a combination of at least two of a topoisomerase inhibitor, an alkylating agent or an antimetabolite drug.

[0054] In the tenth aspect, the present application provides a method for preventing or treating tumors expressing trigger receptor-1 and / or diseases in which trigger receptor-1 is involved, the method comprising administering to a subject in need thereof an anti-trigger receptor-1 antibody or antigen-binding fragment thereof as described in the first aspect, the nucleic acid molecule as described in the second aspect, the expression vector as described in the third aspect, the host cell as described in the fourth aspect, the conjugate or fusion protein as described in the fifth aspect, or the pharmaceutical composition as described in the sixth aspect, any one or a combination of at least two thereof.

[0055] In the eleventh aspect, the present application provides a method for detecting triggering receptor-1, which comprises contacting the anti-triggering receptor-1 antibody or its antigen-binding fragment described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the host cell described in the fourth aspect, the conjugate or fusion protein described in the fifth aspect, or the pharmaceutical composition described in the sixth aspect with any one or a combination of at least two of them with a sample to be detected.

[0056] Compared with the prior art, this application has the following beneficial effects:

[0057] This application targets trigger receptor-1, develops high-affinity anti-trigger receptor-1 antibodies (anti-TREM1 antibodies), and fully analyzes each anti-TREM1 antibody to provide a basis for its precise application, providing new tools and new ideas for the diagnosis, prevention or treatment of tumors expressing trigger receptor-1 and diseases involving trigger receptor-1. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 is a binding curve graph of anti-TREM1 antibodies on cells overexpressing human TREM1.

[0059] Figure 2 is a binding curve of anti-TREM1 antibodies on monkey TREM1 overexpressing cells.

[0060] FIG3 is a schematic diagram of the BLI detection antibody epitope competition experiment.

[0061] Figure 4 is a graph showing the binding activity test results of anti-TREM1 antibodies on human monocytes.

[0062] Figure 5 shows the detection results of anti-TREM1 antibodies promoting HLA-DR expression on the surface of monocytes, where Figure A shows the detection results of NL-A109 and B133 antibodies; Figure B shows the detection results of B192 and B210 antibodies.

[0063] Figure 6 shows the detection results of anti-TREM1 antibodies activating monocytes to produce the cytokine IL-8, where Figure A shows the detection results of NL_A109 and B133 antibodies; Figure B shows the detection results of B192 and B210 antibodies.

[0064] Figure 7 shows the detection results of anti-TREM1 antibodies activating monocytes to produce the cytokine TNFα, where Figure A shows the detection results of NL_A109 and N_PR_B109 antibodies; Figure B shows the detection results of B192 and B210 antibodies.

[0065] Figure 8 shows the detection results of anti-TREM1 antibody activating PBMC to secrete cytokine IL-8.

[0066] Figure 9 shows the detection results of anti-TREM1 antibody activating PBMC to secrete cytokine TNFα.

[0067] Figure 10 shows the detection results of anti-TREM1 antibody activating PBMC to secrete cytokine IL-6.

[0068] Figure 11 shows the results of Fab-ZAP detection of anti-TREM1 antibody endocytosis, where Figure A shows the detection results of NL_A109, B133, and B192 antibodies; Figure B shows the detection results of B210 and B265 antibodies.

[0069] Figure 12 shows the results of the anti-TREM1 antibody B210's inhibition test on MC38 mouse colon cancer growth. DETAILED DESCRIPTION

[0070] To further illustrate the technical means and effects of this application, the following further describes this application in conjunction with examples and drawings. It should be understood that the specific implementation methods described herein are only used to explain this application, rather than to limit this application.

[0071] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0072] Example 1

[0073] In this example, anti-TREM1 antibodies were prepared.

[0074] Screening of anti-TREM1 antibodies from a humanized recombinant antibody phage display library

[0075] Using immunotubes and magnetic bead screening, a humanized recombinant antibody phage display library was screened by solid-phase and liquid-phase cross-screening. Specific binding antibodies against the extracellular region of recombinant human TREM1 (SEQ ID NO: 1) (hu-TREM1-ECD) and recombinant cynomolgus monkey TREM1 (SEQ ID NO: 3) (Cyno-TREM1-ECD) were enriched by trypsin elution and rounds of panning. Clones with binding activity were selected by ELISA and sequenced. The sequence of the human anti-TREM1 antibody variable region obtained by sequencing was combined with the human IgG1 heavy chain constant region (SEQ ID NO: 4) and the kappa light chain constant region sequence (SEQ ID NO: 5) to construct a human full-length IgG1 antibody. The activity of 5 preferred antibodies was studied.

[0076] The specific sequences involved are shown below, and the underlined sequences in each heavy chain and light chain of the antibody are CDR1-3, respectively.

[0077] Example 2

[0078] This example performs protein level binding detection of anti-TREM1 antibodies.

[0079] The ELISA method was used to screen anti-TREM1 antibodies that bind to the extracellular domains of TREM1 from humans, crab-eating macaques, and mice (hu-TREM1-ECD, cyno-TREM1-ECD, and mu-TREM1-ECD). Recombinant hu-TREM1-ECD, recombinant cyno-TREM1-ECD protein, or mu-TREM1-ECD protein was coated in a 96-well ELISA plate and blocked with blocking solution (5% PBSM) at 25°C for 2 hours after overnight at 4°C. Gradually diluted anti-TREM1 antibodies were added to the plate for incubation, and HRP-labeled goat anti-human IgG Fc (goat-anti-human IgG Fc-HRP) was used as the secondary antibody. GraphPa software was used to fit the binding curve and calculate the EC 50 Table 1 shows the EC values ​​of anti-TREM1 antibodies from different sources binding to human, cynomolgus monkey and mouse TREM1 extracellular domain protein (SEQ ID NO: 2). 50 The antibody was shown to bind to both the human and monkey TREM1 extracellular regions, but not to the mouse TREM1 extracellular region.

[0080] Table 1 Anti-TREM1 antibody ELISA binding to EC 50 (μg / mL)

[0081] Note: N stands for not combined

[0082] Example 3

[0083] This example performs cell-level binding detection of anti-TREM1 antibodies.

[0084] The binding activity of anti-TREM1 antibodies on cells overexpressing TREM1 was detected by flow cytometry (FACS). CHOS cells overexpressing human TREM1 in logarithmic phase were collected from the culture, washed, and plated at 1×10 5 Cells / well were plated in a 96-well round-bottom plate. Gradient concentrations of anti-TREM1 antibody, positive control antibody (PC) or isotype control IgG1 were added to the cells and incubated at 4°C for 1 hour. PE fluorescently labeled anti-human IgG Fc secondary antibody diluted in FACS buffer was added to the corresponding wells at 100 μL / well and incubated at 4°C for 30 minutes. The plate was washed twice with FACS buffer. Fluorescence signals were detected by flow cytometry and EC values ​​were calculated using GraphPa software. 50 HEK293 cells overexpressing cynomolgus monkey TREM1 in logarithmic phase were collected from culture, washed, and centrifuged at 1×10 5Cells / well were plated in a 96-well round-bottom plate. Gradient concentrations of anti-TREM1 antibody or isotype control IgG1 were added to the cells and incubated at 4°C for 1 hour. PE fluorescently labeled anti-human IgG Fc secondary antibody diluted in FACS buffer was added to the corresponding wells at 100 μL / well and incubated at 4°C for 30 minutes. The plate was washed twice with FACS buffer. Fluorescence signals were detected by flow cytometry and EC was calculated using GraphPa software. 50 value.

[0085] The positive control antibody (PC) used in this experiment was self-expressed according to the sequence (SEQ ID NO: 34 and 35) in the patent (US2020 / 0255529A1) and served as a system control. Human IgG1 Isotype control (purchased from Yiqiao) was used as a negative control. Figure 1 is a flow cytometry binding curve of anti-TREM1 antibodies in human TREM1 overexpressing cell lines. Figure 2 is a flow cytometry binding curve of anti-TREM1 antibodies in monkey TREM1 overexpressing cell lines. From the above results, it can be seen that some of the four anti-TREM1 antibodies bind to both human TREM1 and monkey TREM1 at the cellular level, and another antibody B265 binds weakly to monkey TREM1. Table 2 shows the binding of anti-TREM1 antibodies to EC on cells overexpressing human TREM1 and monkey TREM1. 50 value.

[0086] Table 2 Cellular binding of anti-TREM1 antibody molecules

[0087] Note: W stands for weak binding

[0088] Example 4

[0089] This example characterizes the affinity kinetics of anti-TREM1 antibodies.

[0090] The affinity kinetics of anti-TREM1 antibodies for human TREM1 and cynomolgus monkey TREM1 were detected using surface plasmon resonance (SPR) on a Biacore T200 (Cytiva) instrument. All data were collected at 25°C. Antibodies were prepared using HBS-EP+ (pH 7.4) buffer, and anti-TREM1 antibodies were immobilized on channels 2, 3, and 4 on a protein A chip, with channel 1 serving as a reference control. 0.58 to 150 nM of human or cynomolgus monkey TREM1 extracellular domain protein was injected into the flow cell at a flow rate of 30 μL / min. Kinetic data were collected and the equilibrium dissociation constant (K) was calculated. DThe affinity kinetic results of anti-TREM1 antibodies to human TREM1 antigen are shown in Table 3. B192 has the strongest affinity to human TREM1 antigen, with K D was 3.89 nM, and the K D The affinity kinetics of anti-TREM1 antibodies to monkey TREM1 antigen were then tested. As shown in Table 4, the K D The values ​​range from 12.8nM to 263nM.

[0091] Table 3 Kinetic affinity K of an-TREM1 antibodies for human TREM1 antigen D value

[0092] Table 4 Kinetic affinity K of anti-TREM1 antibodies to monkey TREM1 antigen D value

[0093] Example 5

[0094] This example performs epitope grouping of anti-TREM1 antibodies.

[0095] Biolayer interferometry (BLI) was used to detect epitope competition experiments and group anti-TREM1 antibodies into epitope groups. Using a GATOR (ProbeLife) instrument, the hu-TREM1-ECD-his antigen was captured with a probe, and two antibodies were added sequentially. Figure 3 shows the principle diagram of the tandem method for detecting antibody epitope competition experiments. The experimental results are shown in Table 5. Anti-TREM1 antibody molecules can be divided into two epitope groups, of which bin 1 epitopes do not compete with bin 2 epitopes.

[0096] Table 5 Anti-TREM1 antibody epitope grouping

[0097] Example 6

[0098] This example analyzes the binding activity of anti-TREM1 antibodies on human monocytes.

[0099] The frozen human monocytes (purchased from Allcells) were quickly thawed in a 37°C water bath and transferred to pre-warmed culture medium. They were centrifuged at 1500 rpm for 10 min and 1×10 6 After resuspending in 1×10 cells / mL, place in CO2 incubator for 2 hours to recover. 5Cells / well were plated in a 96-well round-bottom plate. Gradient concentrations of anti-TREM1 antibodies NL-A109, B133, and B210 were added to the cells and incubated at 4°C for 1 hour. PE fluorescently labeled anti-human IgG Fc secondary antibody diluted in FACS buffer was added to the corresponding wells at 100 μL / well and incubated at 4°C for 30 minutes. The plate was washed twice with FACS buffer. Fluorescence signals were detected by flow cytometry, and EC was calculated using GraphPad software. 50 value.

[0100] The experimental results showed that antibodies NL-A109, B133 and B210 all bind to monocytes isolated from human peripheral blood. The binding curve is shown in Figure 4, where cells + sec refers to the addition of only fluorescently labeled secondary antibodies as background control, and EC 50 The values ​​are shown in Table 6.

[0101] Table 6 Anti-TREM1 antibody binding to EC on primary human monocytes 50

[0102] Example 7

[0103] This example analyzes the effect of anti-TREM1 antibodies on the expression of human monocyte surface markers.

[0104] Anti-TREM1 was diluted with PBS to 4-5 different concentrations. 70 μL of the antibody dilution was added to a 96-well plate using a 100 μL 12-channel pipette. The plate was placed in a 4°C refrigerator overnight. Frozen mononuclear cells were rapidly thawed in a 37°C water bath and transferred to pre-warmed culture medium. The cells were centrifuged at 1500 rpm for 10 min and 1×10 6 After resuspension, the cells were placed in a CO2 incubator for 1 hour. The recovered monocytes were tested for quality using APC-labeled anti-CD14 monoclonal antibody (Thermo Fisher). The qualified monocytes were adjusted to a cell density of 5 × 10 cells / mL using 1640 complete medium. 5 cells / mL, using a 200μL 12-channel pipette, add the dilution to a 96-well cell plate coated with anti-TREM1 antibody and incubate in a 37°C, 5% CO2 incubator for 24 hours. Lipopolysaccharide (LPS) was used as a positive control, and isotype control IgG1 (Isotype IgG1) antibody was used as a negative control. After incubation, centrifuge at 500g for 5 minutes, resuspend the cells in flow cytometry buffer, and incubate with APC-labeled anti-HLA-DR antibody for 30 minutes. Fluorescence intensity was measured by flow cytometry.

[0105] Coating anti-TREM1 antibodies in 96-well plates enhances antibody cross-linking and produces receptor agonist activity. As shown in Figure 5, anti-TREM1 antibodies increase HLA-DR expression on the surface of monocytes to varying degrees, with antibodies NL-A109 and B192 exhibiting the strongest HLA-DR activity.

[0106] Example 8

[0107] This example analyzes how anti-TREM1 antibodies promote cytokine secretion in human monocytes.

[0108] Anti-TREM1 was diluted with PBS to 4-5 different concentrations. 70 μL of the antibody dilution was added to a 96-well cell plate using a 100 μL 12-channel pipette. The plate was placed in a 4°C refrigerator overnight. Frozen mononuclear cells were rapidly thawed in a 37°C water bath and transferred to pre-warmed culture medium. The cells were centrifuged at 1500 rpm for 10 min and 1×10 6 After resuspension, the cells were placed in a CO2 incubator for 1 hour. The recovered monocytes were tested for quality using APC-labeled anti-CD14 monoclonal antibody (Thermo Fisher). The qualified monocytes were adjusted to a cell density of 5 × 10 cells / mL using 1640 complete medium. 5 cells / mL, using a 200μL 12-channel pipette, add the dilution to a 96-well cell plate coated with anti-TREM1 antibody and incubate in a 37°C, 5% CO2 incubator for 24 hours. Lipopolysaccharide (LPS) was used as a positive control, and isotype control IgG1 antibody was used as a negative control. After incubation, the cells were centrifuged at 500g for 5 minutes, and the supernatant was collected. IL-8 and TNFα secretion were detected using IL-8 and TNFα ELISA kits (both purchased from BD Biosciences).

[0109] Coating anti-TREM1 antibodies in 96-well plates can enhance antibody cross-linking, activate TREM1 receptors on the surface of monocytes, and promote monocyte cytokine secretion. As shown in Figure 6, compared with the isotype control antibody IgG1, NL-A109, B133, B192, and B210 antibodies can significantly activate monocytes to secrete the cytokine IL-8. As shown in Figure 7, all anti-TREM1 antibody molecules activate monocytes to secrete the cytokine TNFα.

[0110] Example 9

[0111] This application analyzes how anti-TREM1 antibodies promote the secretion of pro-inflammatory factors by human primary PBMCs.

[0112] The anti-TREM1 antibody was prepared into 7 dilutions with PBS. The antibody dilutions were added to a 96-well cell plate using a 100 μL 12-channel pipette, 100 μL per well. PBMCs were revived in advance, added to RPMI1640 complete medium, and cultured in a 37°C cell culture incubator for 18 hours. The cells were then transferred to a centrifuge tube, centrifuged at 1600 rpm for 10 minutes, and the cell density was adjusted to 5×10 cells / well using RPMI1640 medium containing 10% FBS. 6 cells / mL. 100 μL of cells were added to a cell plate containing antibody diluent and incubated in a 37°C, 5% CO2 incubator for 24 hours. After incubation, the cells were centrifuged at 500 g for 5 minutes, and the supernatant was collected and assayed for IL-8, TNFα, and IL-6 secretion using ELISA kits.

[0113] The anti-TREM1 antibody solution was incubated with human peripheral blood mononuclear cells (PBMC) for 24 hours, activating the TREM1 receptor on the immune cells and secreting the pro-inflammatory cytokines IL-8, TNFα, and IL-6 (as shown in Figures 8 to 10). Among them, the anti-TREM1 antibodies NL-A109, B265, and B210 significantly enhanced the release of IL-8, TNFα, and IL-6 cytokines, while the concentrations of IL-8, TNFα, and IL-6 produced by antibody B133 in solution and incubated with PBMC were comparable to those of the isotype control at the same concentration, and the agonist activity was weak.

[0114] Example 10

[0115] In this example, anti-TREM1 antibody internalization detection was performed.

[0116] Antibody internalization was assessed using the Fab-ZAP assay on hu-TREM1-HEK293 cells overexpressing human TREM1 using candidate antibodies. The principle is that the antibody-coupled Fab-ZAP enters the cells and kills them, and the cell viability is then measured to calculate antibody internalization.

[0117] The cultured hu-TREM1-HEK293 cells were resuspended in DMEM medium containing 10% FBS and counted, and the cell density was adjusted to 6×10 4cells / mL. Use a 100μL pipette to add cells to a 96-well plate, adding 50μL to each well. Place the cell culture plate in a 37°C, 5% CO2 incubator and culture for 13h. Fab-ZAP (purchased from Atsbio) was diluted to a 2.00nM dilution with DMEM culture medium containing 10% FBS. The anti-TREM1 candidate antibody was diluted with the prepared Fab-ZAP dilution into 8 concentration gradient dilutions. After incubation at 25°C for 30min, a 100μL 12-channel pipette was used to add the antibody dilution to the cell plate, 50μL per well, gently tap to mix, and place the cell plate in a 37°C, 5% CO2 incubator for 72h. Before detection, remove the cell plate from the incubator and equilibrate it at 25°C for 10min. Add 7.5 μL TritonX-100 solution to the wells of the complete lysis group (Cell+Triton) in the cell plate equilibrated to 25°C, gently tap to mix, and place the cell culture plate in a 37°C cell culture incubator for 30 minutes. Using a 12-channel gun, add 20 μL MTS (purchased from Promega) to each well and incubate at 37°C for 3 hours. Use a microplate reader to read the OD value at a detection wavelength of 492 nm. Calculate the cell killing IC compared with the TritonX-100 complete lysis group. 50 value.

[0118] The experimental results showed that when the antibody was coupled with Fab-ZAP and incubated with human TREM1 overexpressing cells for 72 h, the results were shown in Figure 11. Among them, antibodies NL-A109, B133, B192, and B265 could bring Fab-ZAP into the cells and cause cell killing in a concentration-dependent manner. IC 50 The IC values ​​of each antibody were 0.00552~0.02581nM, indicating that these antibodies can be internalized. However, the cell killing effect of B210 antibody was weak, indicating that the internalization rate of this antibody was low. 50 See table for values.

[0119] Table 7 Fab-ZAP assay for antibody endocytosis efficiency IC 50 value

[0120] Example 11

[0121] This example tests the anti-tumor activity of anti-TREM1 in the mouse colon cancer MC38 model.

[0122] MC38 mouse colon cancer cells were subcutaneously inoculated on the right side of the back of humanized TREM1 (B-hTREM1) mice (purchased from Biocytogen). Each animal was inoculated with 1×10 6 Tumor cells, 0.1 mL / mouse. Wait until the tumor grows to an average volume of approximately 95 mm3 The animals were randomly divided into two groups, with 5 animals in each group. Group 2 was given B210-mIgG2a chimeric antibody (mouse IgG2a-Fc replaced human IgG1-Fc) by intraperitoneal injection at 10 mg / kg twice a week, while Group 1 was injected with only an equal amount of solvent (PBS) as a control. Tumor volume was measured regularly, and when the tumor volume reached 2000 mm 3 The experiment was terminated at 4 pm. The tumor growth inhibition rate (TGI) was calculated to evaluate the anti-tumor activity of the antibody.

[0123] The experimental results (Table 8 and Figure 12) showed that antibody B210 exerted anti-tumor activity in humanized TREM1 mice by activating TREM1 and delaying tumor growth.

[0124] Table 8 In vivo antitumor efficacy test results of MC38 mouse colon cancer *p<0.05.

[0125] In summary, the present application develops high-affinity anti-trigger receptor-1 antibodies (anti-TREM1 antibodies) for trigger receptor-1, screens and prepares a variety of anti-TREM1 antibodies from a humanized recombinant antibody phage display library, and performs protein level binding assays, cell level binding assays, affinity kinetic characterization, epitope grouping, binding activity assays on human monocytes, effects on human monocyte surface marker expression, cytokine secretion promotion in human monocytes, pro-inflammatory cytokine secretion promotion in human primary PBMCs, internalization assays, and anti-tumor activity assays on the prepared anti-TREM1 antibodies. This fully analyzes each anti-TREM1 antibody, provides a basis for its precise application, and provides new tools and new ideas for the diagnosis, prevention or treatment of tumors expressing trigger receptor-1 and diseases in which trigger receptor-1 is involved.

[0126] The applicant declares that while the above-mentioned embodiments are used to illustrate the detailed methods of the present application, the present application is not limited to the above-mentioned detailed methods, which does not mean that the present application must rely on the above-mentioned detailed methods in order to be implemented. Those skilled in the art should understand that any improvements to the present application, equivalent replacements for the raw materials of the present application's products, addition of auxiliary ingredients, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present application.

Claims

1. An anti-triggering receptor-1 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region; in, The heavy chain variable region includes HCDR1, HCDR2 and HCDR3, and the light chain variable region includes LCDR1, LCDR2 and LCDR3; The amino acid sequence of the HCDR1 is selected from any one of the following sequences: the sequence shown in SEQ ID NO: 19, 25, 31, 37 or 43, and sequences having at least 90% homology thereto and function identical or similar thereto; the amino acid sequence of the HCDR2 is selected from any one of the following sequences: the sequence shown in SEQ ID NO: 20, 26, 32, 38 or 44, and sequences having at least 90% homology thereto and function identical or similar thereto; the amino acid sequence of the HCDR3 is selected from any one of the following sequences: the sequence shown in SEQ ID NO: 21, 27, 33 or 39, and sequences having at least 90% homology thereto and function identical or similar thereto; The amino acid sequence of the LCDR1 is selected from any one of the following sequences: the sequence shown in SEQ ID NO: 22, 28, 34, 40 or 45, and sequences with at least 90% homology to each other and functionally identical or similar; the amino acid sequence of the LCDR2 is selected from any one of the following sequences: SEQ ID NO: 23, 29, 35 or 41, and sequences with at least 90% homology to each other and functionally identical or similar; the amino acid sequence of the LCDR3 is selected from any one of the following sequences: the sequence shown in SEQ ID NO: 24, 30, 36, 42 or 46, and sequences with at least 90% homology to each other and functionally identical or similar.

2. The anti-triggering receptor-1 antibody or antigen-binding fragment thereof according to claim 1, wherein The amino acid sequences of the HCDR1, HCDR2 and HCDR3 are any one of the following sequences: The sequences shown in SEQ ID NO: 19, 20 and 21 or sequences having at least 90% homology thereto and having the same or similar functions, The sequences shown in SEQ ID NO: 25, 26 and 27 or sequences having at least 90% homology thereto and having the same or similar functions, The sequences shown in SEQ ID NO: 31, 32 and 33, or sequences having at least 90% homology thereto and having the same or similar functions, The sequences shown in SEQ ID NO: 37, 38 and 39 or sequences having at least 90% homology thereto and having the same or similar functions, or, The sequences shown in SEQ ID NO: 43, 44 and 21 or sequences having at least 90% homology thereto and having the same or similar functions.

3. The anti-triggering receptor-1 antibody or antigen-binding fragment thereof according to claim 1, wherein The amino acid sequences of the LCDR1, LCDR2 and LCDR3 are any one of the following sequences: The sequences shown in SEQ ID NO: 22, 23 and 24, or sequences having at least 90% homology thereto and having the same or similar functions, The sequences shown in SEQ ID NO: 28, 29 and 30 or sequences having at least 90% homology thereto and having the same or similar functions, The sequences shown in SEQ ID NO: 34, 35 and 36, or sequences having at least 90% homology thereto and having the same or similar functions, The sequences shown in SEQ ID NO: 40, 41 and 42 or sequences having at least 90% homology thereto and having the same or similar functions, or, The sequences shown in SEQ ID NO: 45, 23 and 46 or sequences having at least 90% homology thereto and having the same or similar functions.

4. The anti-triggering receptor-1 antibody or antigen-binding fragment thereof according to claim 1, wherein The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the anti-trigger receptor-1 antibody or antigen-binding fragment thereof are SEQ ID NOs: 19, 20 and 21, respectively, or sequences that have at least 90% homology to each other and function identically or similarly; the amino acid sequences of LCDR1, LCDR2 and LCDR3 are SEQ ID NOs: 22, 23 and 24, respectively, or sequences that have at least 90% homology to each other and function identically or similarly; or The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the anti-trigger receptor-1 antibody or antigen-binding fragment thereof are SEQ ID NOs: 25, 26 and 27, respectively, or sequences that have at least 90% homology to each other and function identically or similarly; the amino acid sequences of LCDR1, LCDR2 and LCDR3 are SEQ ID NOs: 28, 29 and 30, respectively, or sequences that have at least 90% homology to each other and function identically or similarly; or The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the anti-trigger receptor-1 antibody or antigen-binding fragment thereof are SEQ ID NOs: 31, 32 and 33, respectively, or sequences that have at least 90% homology to each other and function identically or similarly; the amino acid sequences of LCDR1, LCDR2 and LCDR3 are SEQ ID NOs: 34, 35 and 36, respectively, or sequences that have at least 90% homology to each other and function identically or similarly; or The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the anti-trigger receptor-1 antibody or antigen-binding fragment thereof are SEQ ID NOs: 37, 38 and 39, respectively, or sequences that have at least 90% homology to each other and function identically or similarly; the amino acid sequences of LCDR1, LCDR2 and LCDR3 are SEQ ID NOs: 40, 41 and 42, respectively, or sequences that have at least 90% homology to each other and function identically or similarly; or The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the anti-trigger receptor-1 antibody or antigen-binding fragment thereof are respectively the sequences shown in SEQ ID NOs: 43, 44 and 21, or sequences that have at least 90% homology to each other and are functionally identical or similar; the amino acid sequences of LCDR1, LCDR2 and LCDR3 are respectively the sequences shown in SEQ ID NOs: 45, 23 and 46, or sequences that have at least 90% homology to each other and are functionally identical or similar.

5. The anti-triggering receptor-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein The amino acid sequence of the heavy chain variable region of the anti-trigger receptor-1 antibody or antigen-binding fragment thereof is the sequence shown in SEQ ID NO: 9 or a sequence thereof with at least 90% homology and the same or similar functions; the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO: 10 or a sequence thereof with at least 90% homology and the same or similar functions; or The amino acid sequence of the heavy chain variable region of the anti-trigger receptor-1 antibody or antigen-binding fragment thereof is the sequence shown in SEQ ID NO: 11 or a sequence thereof with at least 90% homology and the same or similar functions; the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO: 12 or a sequence thereof with at least 90% homology and the same or similar functions; or The amino acid sequence of the heavy chain variable region of the anti-trigger receptor-1 antibody or antigen-binding fragment thereof is the sequence shown in SEQ ID NO: 13 or a sequence thereof with at least 90% homology and the same or similar functions; the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO: 14 or a sequence thereof with at least 90% homology and the same or similar functions; or The amino acid sequence of the heavy chain variable region of the anti-trigger receptor-1 antibody or antigen-binding fragment thereof is the sequence shown in SEQ ID NO: 15 or a sequence thereof with at least 90% homology and the same or similar functions; the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO: 16 or a sequence thereof with at least 90% homology and the same or similar functions; or The amino acid sequence of the heavy chain variable region of the anti-trigger receptor-1 antibody or its antigen-binding fragment is the sequence shown in SEQ ID NO: 17 or a sequence thereof with at least 90% homology and the same or similar functions; the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO: 18 or a sequence thereof with at least 90% homology and the same or similar functions.

6. The anti-triggering receptor-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein: The anti-triggering receptor-1 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and a light chain constant region; Preferably, the heavy chain constant region comprises a human IgG1, IgG2 or IgG4 constant region, and the light chain constant region comprises a human Ig kappa chain or lambda chain constant region; Preferably, the amino acid sequence of the heavy chain constant region includes the sequence shown in SEQ ID NO:4, and the light chain constant region includes the sequence shown in SEQ ID NO:

5.

7. A nucleic acid molecule encoding the anti-triggering receptor-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6. An expression vector comprising the nucleic acid molecule according to claim 7 .

9. A host cell containing at least one copy of the expression vector according to claim 8; or the nucleic acid molecule according to claim 7 is integrated into the chromosome of the host cell.

10. A conjugate or fusion protein comprising: The anti-triggering receptor-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6; Preferably, the conjugate or fusion protein further comprises other parts directly or indirectly connected to the anti-triggering receptor-1 antibody or its antigen-binding fragment, including: any one or a combination of at least two of cell surface receptors, small molecule compounds, small molecule polymers, active proteins or polypeptides.

11. A pharmaceutical composition comprising: Any one or a combination of at least two of the anti-triggering receptor-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, the nucleic acid molecule according to claim 7, the expression vector according to claim 8, the host cell according to claim 9, or the conjugate or fusion protein according to claim 10; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

12. A kit comprising: Any one or a combination of at least two of the anti-trigger receptor-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, the nucleic acid molecule according to claim 7, the expression vector according to claim 8, the host cell according to claim 9, the conjugate or fusion protein according to claim 10, or the pharmaceutical composition according to claim 11.

13. Use of any one or a combination of at least two of the anti-triggering receptor 1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, the nucleic acid molecule according to claim 7, the expression vector according to claim 8, the host cell according to claim 9, the conjugate or fusion protein according to claim 10, or the pharmaceutical composition according to claim 11 in the preparation of a drug targeting triggering receptor 1; in, The drug is used for preventing and / or treating tumors expressing triggering receptor-1 and / or diseases in which triggering receptor-1 is involved; Preferably, the diseases in which triggering receptor-1 is involved include inflammatory diseases and / or autoimmune diseases.

14. Use of any one or a combination of at least two of the anti-triggering receptor-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, the nucleic acid molecule according to claim 7, the expression vector according to claim 8, the host cell according to claim 9, the conjugate or fusion protein according to claim 10, or the pharmaceutical composition according to claim 11 in combination with other anti-tumor drugs or other drugs for treating autoimmune diseases in the preparation of a medicament; in, The drug is used for preventing and / or treating tumors expressing triggering receptor-1 and / or diseases in which triggering receptor-1 is involved; Preferably, the diseases in which triggering receptor-1 is involved include inflammatory diseases and / or autoimmune diseases; Preferably, the other anti-tumor drugs include any one or a combination of at least two of immunomodulators, cytotoxic chemotherapy drugs or small molecule targeted drugs; Preferably, the immunomodulator comprises any one or a combination of at least two of anti-PD1 antibodies, anti-PDL1 antibodies or anti-CTLA4 antibodies; Preferably, the cytotoxic chemotherapy drug comprises any one or a combination of at least two of a topoisomerase inhibitor, an alkylating agent or an antimetabolite drug.

15. A method for preventing or treating tumors expressing triggering receptor 1 and / or diseases in which triggering receptor 1 is involved, comprising administering to a subject in need thereof any one or a combination of at least two of the anti-triggering receptor 1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, the nucleic acid molecule according to claim 7, the expression vector according to claim 8, the host cell according to claim 9, the conjugate or fusion protein according to claim 10, or the pharmaceutical composition according to claim 11.

16. A method for detecting triggering receptor 1, comprising contacting a sample to be detected with any one or a combination of at least two of the anti-triggering receptor 1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, the nucleic acid molecule according to claim 7, the expression vector according to claim 8, the host cell according to claim 9, the conjugate or fusion protein according to claim 10, or the pharmaceutical composition according to claim 11.

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