Biomarker for predicting treatment effectiveness of OX40 agonist antibody as well as screening method and application of biomarker

Abstract

The invention relates to the technical field of medicines, in particular to a biomarker for predicting treatment effectiveness of an OX40 agonist antibody and a screening method and application of the biomarker. The biomarker for predicting the treatment effectiveness of the OX40 agonist antibody comprises at least one of a Clec4d gene, a Clec4e gene and a Ccrl2 gene. In an OX40 agonist antibody treatment response group, the expression quantities of Clec4d, Clec4e and Ccrl2 genes on myeloid cells have a rising trend, the curative effect of the OX40 agonist antibody can be predicted through the expression change of the Ccrl2, Clec4d and Clec4e genes before and after the treatment of the OX40 agonist antibody on the same mouse, and a reference is provided for realizing accurate immunotherapy.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to biomarkers for predicting the effectiveness of OX40 agonist antibody therapy and screening methods and applications thereof. Background Art

[0002] At present, the mechanism of action of OX40 agonist antibodies is not completely clear. Existing research results show that OX40 agonist antibodies have an effect on T cells. In multiple preclinical models, OX40 agonists or OX40 agonist antibodies induce T cell activation in in vitro and in vivo models, inhibit Treg-mediated immunosuppression, and show tumor inhibitory efficacy in tumor-bearing mouse models. However, there is currently no clinical biomarker that can predict the effectiveness of OX40 agonist antibody treatment. Summary of the invention

[0003] The present invention aims to solve at least one of the technical problems existing in the related art. To this end, the present invention provides a biomarker for predicting the effectiveness of OX40 agonist antibody treatment. Based on experimental data, it was found that in the OX40 agonist antibody treatment response group, the expression of Clec4d, Clec4e, and Ccrl2 genes on myeloid cells was significantly increased (P value less than 0.05, T test). By comparing the changes in Ccrl2, Clec4d and Clec4e gene expression before and after OX40 agonist antibody treatment in the same mouse, the efficacy of OX40 agonist antibody can be predicted, providing a reference for achieving precise immunotherapy.

[0004] In one aspect of the present invention, the present invention provides a biomarker for predicting the effectiveness of OX40 agonist antibody treatment, wherein the biomarker comprises at least one of Clec4d gene, Clec4e gene and Ccrl2 gene. Furthermore, before treatment with OX40 agonist antibodies, When at least one of the Clec4d gene, the Clec4e gene, and the Ccrl2 gene is highly expressed in myeloid cells, it is predicted that OX40 agonist antibody treatment is effective;

[0005] When Clec4d, Clec4e and Ccrl2 genes are all lowly expressed in myeloid cells, it is predicted that OX40 agonist antibody treatment is ineffective.

[0006] Furthermore, the myeloid cells include monocytes, macrophages and / or neutrophils.

[0007] Furthermore, the biomarker is Clec4d.

[0008] Furthermore, the OX40 agonist antibody includes an OX40 humanized agonist antibody (2-2-1). In another aspect of the present invention, the present invention provides an application of the aforementioned biomarker for predicting the effectiveness of OX40 agonist antibody therapy in predicting the effectiveness of OX40 agonist antibody therapy, the application comprising: before using OX40 agonist antibody therapy, When at least one of the Clec4d gene, Clec4e gene, and Ccrl2 gene is highly expressed in myeloid cells, it is predicted that OX40 agonist antibody treatment is effective;

[0009] When Clec4d, Clec4e and Ccrl2 genes are all lowly expressed in myeloid cells, it is predicted that OX40 agonist antibody treatment is ineffective.

[0010] Furthermore, when the OX40 agonist antibody treatment is effective, the expression levels of the Clec4d gene, the Clec4e gene and / or the Ccrl2 gene are monitored. Compared with before the OX40 agonist antibody treatment, the expression of any one of the Clec4d gene, the Clec4e gene and the Ccrl2 gene in the myeloid cells is significantly increased after the OX40 agonist antibody treatment. Further, when the OX40 agonist antibody treatment is effective, the expression level of the Clec4d gene in the myeloid cells is significantly higher than the expression level of the Clec4d gene when the OX40 agonist antibody treatment is ineffective; or, When the OX40 agonist antibody treatment is effective, the expression level of the Clec4e gene in the myeloid cells is significantly higher than the expression level of the Clec4e gene when the OX40 agonist antibody treatment is ineffective; or,

[0011] When OX40 agonist antibody treatment is effective, the expression level of Ccrl2 gene in myeloid cells is higher than that when OX40 agonist antibody treatment is ineffective.

[0012] In another aspect of the present invention, the present invention provides a method for screening biomarkers for predicting the effectiveness of OX40 agonist antibody treatment as described above, characterized in that it comprises the following steps: S1. Establish a bilateral tumor-grafting model in OX40 humanized mice with intact immune systems and determine the phenotypes of the responding and non-responding groups. S2. Analyze and compare the precise composition, functional status and / or gene expression of tumor-infiltrating immune cells in mice that respond to OX40 agonist antibody treatment and those that do not respond; S3. Screen at least one of the target genes Clec4d, Clec4e and Ccrl2 that are related to myeloid cells and highly expressed in the OX40 agonist antibody treatment response group as a biomarker for predicting the effectiveness of OX40 agonist antibody treatment. Further, in step S3, the effect of knocking out at least one of the Clec4d gene and the Ccrl2 gene on the efficacy of the OX40 agonist antibody is analyzed to determine a biomarker for predicting the effectiveness of the OX40 agonist antibody treatment;

[0013] And / or, knocking out the Clec4d gene has no effect on the efficacy of OX40 agonist antibody, and determining that the biomarker for predicting the effectiveness of OX40 agonist antibody treatment is Clec4d.

[0014] The above one or more technical solutions in the embodiments of the present invention have at least one of the following technical effects: In the OX40 agonist antibody treatment response group, the expression levels of Clec4d, Clec4e, and Ccrl2 genes on myeloid cells tended to increase. By observing the changes in Ccrl2, Clec4d, and Clec4e gene expression before and after OX40 agonist antibody treatment in the same mice, the efficacy of OX40 agonist antibodies can be predicted, providing a reference for achieving precise immunotherapy.

[0015] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 This is a cluster diagram of differentially expressed genes in transcriptome sequencing of the OX40 agonist antibody treatment response group and the non-response group in Example 1 of the present invention.

[0018] Figure 2 It is a volcano plot of differentially expressed genes in transcriptome sequencing in Example 1 of the present invention.

[0019] Figure 3 It is the GO (Gene Ontology database) pathway enrichment analysis of differentially expressed genes in transcriptome sequencing in Example 1 of the present invention.

[0020] Figures 4 to 9The expressions of genes such as Gzmf, Gzmg, Nos2, Clec4d, Clec4e and Ccrl2 in the response group and the non-response group were detected by qRT-PCR in Example 1 of the present invention.

[0021] Fig.10 It is the display result of the unsupervised clustering UMAP (Uniform Manifold Approximation and Projection) of the single-cell sequencing results of the response group and the non-response group in Example 2 of the present invention.

[0022] Fig.11 It is a comparison of the distribution of immune cell clusters in the responding and non-responding groups in Example 2 of the present invention.

[0023] Fig.12 It is a bar graph of the cell ratios of different immune cell populations in the response group and the non-response group in Example 2 of the present invention.

[0024] Figures 13 to 18 They are CD8 T cells, CD8 T cells in the multicolor flow cytometry response group and the non-response group in Example 2 of the present invention. + IFNγ + , CD4 T cells, macrophages, neutrophils, and changes in the proportions of M1 (F4 / 80+Nos2+) and M2 (F4 / 80+Cd206+) macrophages.

[0025] Figures 19 to 21 They are respectively the cell populations expressing the Ccrl2, Clec4d and Clec4e genes shown in the UMAP map in Example 2 of the present invention.

[0026] Fig. 22 It is a violin plot of the expression of Ccrl2, Clec4d and Clec4e genes in all cell subsets of mononuclear macrophages (M_0-M_11) and neutrophils (G5a, G5b) in Example 2 of the present invention.

[0027] Fig.23 This is the Ccrl2 gene knockout strategy in Example 3 of the present invention.

[0028] Fig.24 This is the Clec4d gene knockout strategy in Example 3 of the present invention.

[0029] Fig.25 This is the electrophoresis result of Ccrl2 genotype identification in Example 3 of the present invention.

[0030] Fig.26 This is the electrophoresis result of Clec4d genotype identification in Example 3 of the present invention.

[0031] Fig. 27 This is the mouse tumor growth curve after OX40 agonist antibody treatment in Ccrl2 gene knockout mice in Example 3 of the present invention.

[0032] Fig.28 This is the survival curve of mice after OX40 agonist antibody treatment in Ccrl2 gene knockout mice in Example 3 of the present invention.

[0033] Figure 29 to Figure 31 They are respectively the flow cytometry analysis of MHCII expression of macrophages (CD11b+F4 / 80+), monocytes (CD11b+Ly6C+) and neutrophils (CD11b+Ly6G+) in the OX40 agonist antibody treated and untreated groups in Ccrl2 knockout and non-knockout mice in Example 3 of the present invention.

[0034] Fig.32 This is the mouse tumor growth curve after OX40 agonist antibody treatment in Clec4d gene knockout mice in Example 3 of the present invention.

[0035] Fig.33 This is the mouse survival curve after OX40 agonist antibody treatment in Clec4d gene knockout mice in Example 3 of the present invention.

[0036] Fig.34 This is the expression of Clec4d in the untreated tumor on the left side of the same mouse in the OX40 agonist antibody treatment response group and the non-response group in Example 4 of the present invention.

[0037] Fig.35 This is the expression of Clec4e in the untreated tumor on the left side of the same mouse in the OX40 agonist antibody treatment response group and the non-response group in Example 4 of the present invention.

[0038] Fig.36 This is the expression of Ccrl2 in the untreated tumor on the left side of the same mouse in the OX40 agonist antibody treatment response group and the non-response group in Example 4 of the present invention. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme of the present invention will be clearly and completely described below in conjunction with the drawings in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. The following embodiments are used to illustrate the present invention, but cannot be used to limit the scope of the present invention.

[0040] In the description of the embodiments of the present invention, it should be noted that the terms "center", "longitudinal", "lateral", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside" and the like indicate positions or positional relationships based on the positions or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the embodiments of the present invention and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a specific orientation, be constructed and operate in a specific orientation, and therefore cannot be understood as limitations on the embodiments of the present invention. In addition, the terms "first", "second", and "third" are used for descriptive purposes only and cannot be understood as indicating or implying relative importance.

[0041] In the description of the embodiments of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "connected" and "connection" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium. For ordinary technicians in this field, the specific meanings of the above terms in the embodiments of the present invention can be understood according to specific circumstances.

[0042] In the embodiments of the present invention, unless otherwise clearly specified and limited, the first feature being "above" or "below" the second feature may mean that the first and second features are in direct contact, or the first and second features are in indirect contact through an intermediate medium. Moreover, the first feature being "above", "above" and "above" the second feature may mean that the first feature is directly above or obliquely above the second feature, or simply means that the first feature is higher in level than the second feature. The first feature being "below", "below" and "below" the second feature may mean that the first feature is directly below or obliquely below the second feature, or simply means that the first feature is lower in level than the second feature.

[0043] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the embodiment of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0044] In one aspect of the present invention, the present invention provides a biomarker for predicting the effectiveness of OX40 agonist antibody treatment, wherein the biomarker comprises at least one of Clec4d gene, Clec4e gene and Ccrl2 gene.

[0045] It should be noted that Ccrl2 binds chemokines at the N-terminus, allowing the C-terminal peptide sequence to interact with cmklr1 and promote the recruitment of cmklr1-expressing cells, such as monocytes / macrophages, dendritic cells, plasmacytoid dendritic cells, and NK cells; Ccrl2-CXCR2 mediates neutrophil recruitment at inflammatory sites and is also expressed in various myeloid immune cell subsets (functional role of CCRL2 in activating immunostimulatory macrophages, enhancing anti-tumor T cell responses and tumor rejection). Macrophage C-type lectins MCL (Clec4d) and Mincle (Clec4e) are mainly expressed in mononuclear macrophages and neutrophils. The surface expression of Clec4d requires binding to the signaling adapter FcRγ, which also mediates downstream signaling through the Syk / CARD9 pathway. Clec4d is able to form a functional receptor complex with Mincle, and Clec4d is required for the induction and surface expression of Clec4e. Clec4d and Mincle play a regulatory role in a mutually dependent manner during inflammation and infection. Clec4e acts as an activating pattern recognition receptor that helps recognize pathogens and enhances adaptive immune responses, releasing pro-inflammatory cytokines.

[0046] After treatment with OX40 agonist antibodies, the expression levels of Clec4d, Clec4e and Ccrl2 genes on myeloid cells in the response group were significantly increased compared with the non-response group (P value less than 0.05, T test). By analyzing the changes in Ccrl2, Clec4d and Clec4e gene expression before and after treatment with OX40 agonist antibodies in the same mice, the efficacy of OX40 agonist antibodies can be predicted, providing a reference for achieving precise immunotherapy.

[0047] In some embodiments of the present invention, before using OX40 agonist antibody treatment, when at least one of the Clec4d gene, the Clec4e gene and the Ccrl2 gene is highly expressed in myeloid cells, it is predicted that the OX40 agonist antibody treatment is effective; when the Clec4d gene, the Clec4e gene and the Ccrl2 gene are all lowly expressed in myeloid cells, it is predicted that the OX40 agonist antibody treatment is ineffective.

[0048] In some embodiments of the present invention, the myeloid cells include monocytes, macrophages and / or neutrophils.

[0049] In some preferred embodiments of the present invention, the biomarker is Clec4d.

[0050] In some embodiments of the present invention, the OX40 agonist antibody comprises an OX40 humanized agonist antibody (2-2-1).

[0051] In some specific embodiments of the present invention, the OX40 humanized agonist antibody (2-2-1) is prepared in the following manner: first, an antibody library binding to the OX40 target is screened by phage display technology, and then several active OX40 agonist antibodies are screened from the OX40 antibody binding library by high-throughput screening based on microfluidics technology, and the activities of these OX40 agonist antibodies are evaluated in vitro and in vivo, and finally the OX40 agonist antibody with the highest activity is screened and named 2-2-1.

[0052] In another aspect of the present invention, the present invention provides an application of the aforementioned biomarker for predicting the effectiveness of OX40 agonist antibody therapy in predicting the effectiveness of OX40 agonist antibody therapy, comprising: before using OX40 agonist antibody therapy, when at least one of the Clec4d gene, the Clec4e gene and the Ccrl2 gene is highly expressed in myeloid cells, it is predicted that the OX40 agonist antibody therapy is effective; when the Clec4d gene, the Clec4e gene and the Ccrl2 gene are all lowly expressed in myeloid cells, it is predicted that the OX40 agonist antibody therapy is ineffective.

[0053] In some embodiments of the present invention, when OX40 agonist antibody treatment is effective, the expression levels of Clec4d gene, Clec4e gene and / or Ccrl2 gene are monitored, and compared with before treatment with OX40 agonist antibody, the expression of any one of Clec4d gene, Clec4e gene and Ccrl2 gene in myeloid cells is significantly increased after treatment with OX40 agonist antibody (significant increase means: P value is less than 0.05, T test).

[0054] In some embodiments of the present invention, when OX40 agonist antibody treatment is effective, the expression level of Clec4d gene in myeloid cells is higher than the expression level of Clec4d gene when OX40 agonist antibody treatment is ineffective; or, when OX40 agonist antibody treatment is effective, the expression level of Clec4e gene in myeloid cells is significantly higher (significantly higher means: P value is less than 0.05, T test) than the expression level of Clec4e gene when OX40 agonist antibody treatment is ineffective; or, when OX40 agonist antibody treatment is effective, the expression level of Ccrl2 gene in myeloid cells is significantly higher (significantly higher means: P value is less than 0.05, T test) than the expression level of Ccrl2 gene when OX40 agonist antibody treatment is ineffective.

[0055] In another aspect of the present invention, the present invention provides a method for screening biomarkers for predicting the effectiveness of OX40 agonist antibody treatment as described above, the screening method comprising the following steps: S1. Establish bilateral tumor grafting model in OX40 humanized mice with intact immune system and determine the phenotypes of responding and non-responding groups. S2. Analyze and compare the precise composition, functional status and / or gene expression of tumor-infiltrating immune cells in mice that respond to OX40 agonist antibody treatment and those that do not respond; S3. Screen target genes related to myeloid cells, including at least one of Clec4d, Clec4e and Ccrl2 as biomarkers for predicting the effectiveness of OX40 agonist antibody treatment.

[0056] In some embodiments of the present invention, in step S2, the analysis and comparison is performed by multi-color flow cytometry, transcriptome sequencing analysis and / or single-cell transcriptome sequencing analysis.

[0057] In some embodiments of the present invention, in step S3, the effect of knocking out the Clec4d gene and / or the Ccrl2 gene on the efficacy of the OX40 agonist antibody is analyzed to determine a biomarker for predicting the therapeutic effectiveness of the OX40 agonist antibody.

[0058] It should be noted that the function of the Clec4e gene will be affected after knocking out the Clec4d gene.

[0059] In some embodiments of the present invention, knocking out the Clec4d gene has no effect on the efficacy of the OX40 agonist antibody, and the biomarker for predicting the effectiveness of the OX40 agonist antibody treatment is determined to be Clec4d.

[0060] In some specific embodiments of the present invention, a bilateral tumor grafting model is established in OX40 humanized mice with intact immune systems. The tumor on one side is removed and the wound is sutured in the early stage of treatment, and the growth of the tumor on the other side is continuously monitored to determine whether the treatment is effective or not, and they are named as the response group and the non-response group, respectively. The tumors of the response group and the non-response group of mice are subjected to multicolor flow cytometry analysis, transcriptome and single-cell transcriptome sequencing analysis, and then the precise composition, functional state and gene expression of tumor-infiltrating immune cells in the mice in the response group and the non-response group of mice treated with OX40 agonist antibodies are compared based on various bioinformatics analyses, and target genes related to myeloid cells are mined, including at least one of Clec4d, Clec4e and Ccrl2 as biomarkers for determining the effectiveness of OX40 agonist antibody treatment.

[0061] The inventors of the present invention have found that the mechanism of action of OX40 agonist antibodies is not completely clear at present. Most of the published results focus on the effects of OX40 agonist antibodies on T cells, while the effects of myeloid cells on T cells after OX40 agonist antibody treatment are not studied in depth. Based on this, the present invention firstly performed transcriptome sequencing and single-cell transcriptome sequencing analysis on the responding and non-responding mice after OX40 agonist antibody treatment, and found that compared with the non-responding group, the target genes Ccrl2, Clec4d and Clec4e related to myeloid cells in the responding group were significantly upregulated, so these three genes were taken as the research focus, and then RNA was extracted from the left untreated tumor of the same mouse that responded to OX40 agonist antibody treatment and qRT-PCR detection was performed. It was found that before OX40 agonist antibody treatment, compared with the untreated non-responding group, the expression levels of Clec4d, Clec4e and Ccrl2 genes in the untreated responding group showed an increasing trend, while in the responding group after OX40 agonist antibody treatment, the expression levels of Clec4d, Clec4e and Ccrl2 genes were significantly increased (P value less than 0.05, T test), thereby confirming that Clec4d, Clec4e and Ccrl2 can be used as predictive markers for OX40 agonist antibody treatment response. Currently, there is no biomarker in clinical practice that can predict the effectiveness of OX40 agonist antibody therapy. Ccrl2, Clec4d and Clec4e can predict the efficacy of OX40 agonist antibodies and provide a reference for achieving precise immunotherapy.

[0062] The present invention is further described below in conjunction with specific examples. It should be noted that the following examples are only used to explain the present invention and are not to be construed as limiting the present invention. Example

[0063] The specific steps of the division of the response group and the non-response group, the single-cell sequencing analysis of the mouse tumor-infiltrating immune cells in the mouse tumor model treated with OX40 agonist antibodies, and the multicolor flow cytometry analysis of the changes in the mouse tumor-infiltrating immune cells used in the following examples are as follows: 1. Construction of bilateral subcutaneous tumor-bearing model in mice and division into response group and non-response group The specific operation steps are as follows: revive MC38 (mouse colon cancer) cells, remove the cells from the incubator, observe the cell status under a microscope, and determine whether the cell density that can be subcultured has been reached. Sterilize the clean bench and required consumables by ultraviolet irradiation for 30 minutes. Preheat PBS buffer and complete culture medium in a 37°C water bath. After the UV ends, place the cells in the clean bench, aspirate the culture medium, add 5ml PBS to rinse the cells, gently add the rinse solution from one side of the culture dish to avoid damaging the cell layer, gently shake the culture dish to rinse the cells, and discard the PBS buffer. Add 2ml 0.25% trypsin, gently shake the culture bottle to infiltrate all cells, and discard the trypsin. Place the culture dish in a 37°C incubator for incubation. The cell digestion time is about 1min; tap the outer wall of the culture container so that the cells can detach from the bottle wall. Resuspend the cells in PBS buffer and count them, and adjust the cell density to 1*10 7 / mL and place on ice for later use. Use a syringe to draw 100ul of cell suspension, grab the fixed mouse with your left hand, and inject subcutaneously on both sides of the mouse. When inoculating, the needle should be inserted a little deeper, about 1cm deep, to reduce the overflow of cell suspension from the needle eye after injection.

[0064] Tumor size reaches 50-100mm 3 The mice were enrolled at 1:10 pm. On the second day after three treatments with OX40 agonist antibodies, the mice were anesthetized and the left tumor tissue was removed under sterile conditions. After quick freezing with liquid nitrogen, it was stored at -80°C. The incision was sutured, and the right tumor was observed to determine the treatment effect. By observing the response results of the right tumor, the treatment response and non-response groups were named R and NR, respectively. The tumor tissues of the five mice with the best treatment effect in the response group and the five mice with the worst treatment effect in the non-response group were selected and sent to Jinweizhi Company for transcriptome sequencing analysis. Subsequently, RNA was extracted and reverse transcribed from the samples of the R and NR groups, and the expression of Clec4d, Clec4e, and Ccrl2 genes in the OX40 agonist antibody treatment response and non-response groups was detected by qRT-PCR.

[0065] The tissue extraction steps before OX40 agonist antibody treatment are as follows: tumor volume reaches 50-100mm 3At 1:10 pm, the mice were ear-tagged (1-30), the left tumor tissue was removed under sterile conditions after anesthesia, frozen in liquid nitrogen and stored at -80℃, the incision was sutured, and 5mg / kg OX40 agonist antibody was treated the next day. The drug was given every two days for a total of 3 times. The tumor growth curve of the mice was then monitored, and the treatment response group and non-response group were determined according to the tumor volume. The left tumor of the response group before treatment was named the untreated response group (UR, n=5), and the left tumor of the non-response group before treatment was named the untreated non-response group (UNR, n=5) by corresponding ear tag numbers. RNA was then extracted and reverse transcribed from the tumors of UR and UNR, and the expression of Clec4d, Clec4e, and Ccrl2 genes was detected by qRT-PCR. 2. Analysis of changes in immune cells and differential gene expression in tumor tissues of mice in the response group and non-response group

[0066] MC38 bilateral tumor-bearing mice were treated with 4% paracentesis and 2% paracentesis when the tumor volume grew to 50-100 mm. 3 The OX40 agonist antibody was used to treat the tumor every two days. After three treatments, one side of the tumor was removed in a sterile environment and digested and ground into a single cell suspension. CD45 positive magnetic beads were used to detect the CD45 in the tumor tissue. + Immune cells were isolated, and their cell number and cell activity were measured, and the cell viability was ensured to be above 90%. After labeling the immune cells of each mouse with hashtag antibodies, single-cell transcriptome sequencing samples were prepared using the 10×Genomics single-cell transcriptome sequencing platform. Master Mix (mainly including poly-dT primers and enzymes, etc.), cell suspension, barcode-containing gel beads and oil droplets were added to different chambers of Chromium Chip B, and the oil droplet-encapsulated gel beads (GelBead-In-EMulsion, GEM) were formed through the microfluidic "double cross" cross system. GEMs were collected and mixed evenly. The gel beads automatically dissolved in each oil droplet to release a large number of primers carrying barcodes, and the cells were lysed to release mRNA. The mRNA was then reversely transcribed under the action of reverse transcriptase to produce cDNA carrying barcode and UMI (specific molecular label) information. After preparing the cDNA library according to the above standard process, it was sent to Beijing Yimeitongde Company for sequencing using an Illumina sequencer. After sequencing is completed, data analysis is performed to determine the changes in immune cells and differential gene expression in the tumor tissues of mice in the response and non-response groups. 3. Study the therapeutic effect of OX40 agonist antibody on Clec4d gene and Ccrl2 gene knockout mice

[0067] CRISPR / Cas9 technology was used to design gRNA and Cas9 expression plasmids for Ccrl2 exon 2, Clec4d exons 2, 3, 4, and 5, resulting in the knockout of the functional regions of Ccrl2 and Clec4d, and obtaining a mouse model in which the Ccrl2 gene or Clec4d gene was not expressed in all tissues and cells of the body (labeled as Ccrl2- / hOX40 mice and Clec4d- / hOX40 mice, respectively). Ccrl2 knockout OX40 humanized mice (labeled as Ccrl2- / hOX40) and Ccrl2 non-knockout OX40 humanized mice (labeled as Ccrl2+ / hOX40) were subcutaneously inoculated with 1x10 6 MC38 tumor cells, and wait until the tumor volume grows to 50-100 mm 3 At the same time, Ccrl2- / hOX40 mice and Ccrl2+ / hOX40 mice were divided into two groups, each with 6 mice, and treated with isotype control antibody IgG and OX40 agonist antibody, respectively. The antibody was intraperitoneally administered at 5 mg / kg for a total of three times. After the OX40 agonist antibody was intraperitoneally administered three times, the tumor volume of the MC38 tumor-bearing Ccrl2- / hOX40 mice used for drug efficacy testing was monitored, and the tumor growth curve of the mice was plotted by Graphpad. For flow cytometry analysis of immune cell changes in MC38 tumor-bearing Ccrl2- / hOX40 mice, after three doses, the mice were anesthetized and killed by cervical dislocation, the tumor was removed, mechanically chopped up, treated with enzymatic solution for 20 minutes, and then filtered through a 70μm filter. After red blood cell lysis, a single tumor cell suspension was prepared and added to a V-bottom 96-well plate. A series of flow cytometry analysis schemes (panels) were designed. After cell blocking, the single cell suspension was stained with live and dead dyes, surface flow antibodies, and intracellular flow antibodies. The samples were analyzed using a BD LSRFortessa flow cytometer, and FlowJo software was used to analyze the expression of macrophages (CD11b + F4 / 80 + ), monocytes (CD11b + Ly6C + ) and neutrophils (CD11b + 6G + ) MHCII expression. Example 1 Gene expression in mice responding and non-responding to OX40 agonist antibody treatment

[0068] A mouse model with bilateral colon cancer cells (MC38) was used to analyze the differences in the composition of tumor-infiltrating immune cells and gene expression between the responding and non-responding groups of mice. After three treatments with OX40 agonist antibodies, the left tumor was removed, and the mice were divided into a treatment response group and a non-responding group by continuously monitoring the growth of the right tumor. The removed tumors of the responding and non-responding groups were analyzed. The clustering results of differentially expressed genes in transcriptome sequencing between the responding and non-responding groups are shown in Figure 1. Compared with the non-responding group, granzyme G (Gzmg), granzyme F (Gzmf), interleukin 1β (Il1b) and nitric oxide synthase 2 (NOS2), and other genes related to immune activation and pro-inflammation were highly expressed in the responding group (results are shown in Figure 1). Figure 2 As shown in Figure 2, qRT-PCR also further verified the reliability of the transcriptome sequencing results. Based on differentially expressed gene analysis, the genes of interest Ccrl2, Clec4d, and Clec4e that were significantly upregulated in the response group and related to myeloid cells were selected as the research focus. Compared with the non-response group, they were all highly expressed in the OX40 agonist antibody treatment response group (the results are shown in Figure 2). Figures 4 to 9 As shown, Figure 4 p = 0.0238, Figure 5 p=0.0278, Figure 6 p = 0.0298, Figure 7 p = 0.0048, Figure 8 p=0.0152, Fig. 9 p=0.04). Differentially expressed genes and immune-related enrichment pathways include the response to lipopolysaccharide signal (response to lipopolysaccharide), and most of the differentially expressed genes related to this signal pathway were upregulated in the response group (results as shown in Figure 3 As shown, Figure 3 The biological process-related pathways in which differentially expressed genes are enriched are displayed. The response group-VS-non-response group refers to the response group-versus (control)-non-response group).

[0069] The above results showed that the transcriptome sequencing and qRT-PCR results of mice in the OX40 agonist antibody treatment response and non-response groups showed that the Clec4d, Clec4e and Ccrl2 genes were highly expressed in the OX40 agonist antibody treatment response group. Example 2 Single-cell transcriptome sequencing further analyzed the expression of genes such as Ccrl2, Clec4d and Clec4e in monocytes, macrophages and neutrophils

[0070] After OX40 agonist antibody treatment of MC38 tumor-bearing mice three times, one side of the tumor was removed on the second day, tumor-infiltrating immune cells were isolated and subjected to single-cell transcriptome sequencing, and the other side of the tumor was used to determine whether it responded to OX40 agonist antibody treatment. All cells were divided into groups such as T cells, NK cells, monocytes, macrophages, neutrophils, DC cells and mast cells by unsupervised cluster analysis (results are shown in Fig.10 By comparing the number of various immune cells in the response group and the non-response group, it was found that the proportion of lymphoid T and NK cells in the response group increased significantly, while the proportion of myeloid cells including monocytes, macrophages and DC cells decreased significantly, and the proportion of neutrophils increased slightly (the results are shown in Fig.11 and Fig.12 Flow cytometry analysis confirmed the results of single-cell sequencing. Flow cytometry data showed that macrophages in the OX40 agonist antibody treatment response group changed from the immunosuppressive M2 type to the pro-inflammatory and anti-tumor M1 type. At the same time, the proportion of IFNγ released by CD8T cells in the response group increased (results shown in Figures 13 to 18 as shown).

[0071] Single-cell sequencing data showed that genes such as Ccrl2, Clec4d, and Clec4e were highly expressed in monocytes, macrophages, and neutrophils (results shown in Figures 19 to 21 The expression of Ccrl2 in the monocyte-macrophage M_2, M_3 and M_11 subpopulations was significantly upregulated in the response group; the expression of Clec4d in the M_0, M_2, M_4, M_6, M_7, M_8 and M_11 and the neutrophil G5b subpopulation was significantly upregulated in the response group; the expression of Clec4e in the M_1, M_7, M_9 and M_11 and the neutrophil G5b subpopulation was significantly upregulated in the response group (the results are shown in Fig. 22 As shown, Fig. 22 The horizontal axis in the middle represents the different subpopulations of monocytes, macrophages and neutrophils corresponding to the response group and the non-response group). Example 3 Study on the effect of Ccrl2 gene and Clec4d gene knockout on the efficacy of OX4 agonist antibody

[0072] CRISPR / Cas9 technology was used to design gRNA and Cas9 expression plasmids for Ccrl2 exon 2 and Clec4d exons 2, 3, 4, and 5, resulting in the knockout of the functional regions of Ccrl2 and Clec4d, thereby obtaining a mouse model in which the gene was not expressed in all tissues and cells of the body (the results are shown in Figure 2). Fig.23 and Fig.24 As shown, Fig.23 In the figure, gRNA was designed upstream and downstream of exon 2 for knockout. Fig.24The upstream and downstream gRNAs were designed for knockout, as shown in the figure. The upstream primer F1 was located upstream of the knockout fragment, the downstream primer R2 was located in the knockout fragment, and the downstream primer R1 was located downstream of the knockout fragment. The electrophoresis results showed that the single lane F1+R1 indicated the knockout homozygote, the single lane F1+R2 indicated the wild type, and the presence of bands in both lanes indicated the heterozygote. We have obtained homozygous Ccrl2 and Clec4d gene knockout mice (the results are shown in Fig.25 and Fig.26 As shown, Fig.25 R1 means F1+R1, R2 means F1+R2; Fig.26 Here, R1 represents F1+R1, and R2 represents F1+R2).

[0073] To further investigate the effects of Ccrl2 and Clec4d on the efficacy of OX40 agonist antibodies, Ccrl2 knockout mice, Clec4d knockout mice, and OX40 humanized mice were mated to obtain Ccrl2 knockout OX40 humanized mice (Ccrl2- / hOX40) and Clec4d knockout OX40 humanized mice (Clec4d- / hOX40). The OX40 humanized mice without gene knockout were named Ccrl2+ / hOX40 mice or Clec4d+ / hOX40 mice.

[0074] Ccrl2- / hOX40 mice and Ccrl2+ / hOX40 mice were inoculated with MC38 tumor cells and the tumor volume grew to 50-100 mm. 3 The mice were divided into two groups, Ccrl2- / hOX40 mice and Ccrl2+ / hOX40 mice, with six mice in each group. They were treated with isotype control antibody IgG and OX40 agonist antibody, respectively. The antibody was intraperitoneally administered at 5 mg / kg for a total of three times. The tumor volume of the mice was monitored, and the tumor growth curve and survival curve of the mice were plotted using Graphpad. The results are shown in the figure below. Figures 27 to 31 As shown ( Figures 27 to 31In the data, WT-isotype control irrelevant antibody IgG refers to mice in which the Ccrl2 gene was not knocked out and treated with isotype control antibody IgG, WT-OX40 agonist antibody refers to mice in which the Ccrl2 gene was not knocked out and treated with OX40 agonist antibody, Ccrl2 KO-isotype control irrelevant antibody IgG refers to mice in which the Ccrl2 gene was knocked out and treated with isotype control antibody IgG, and Ccrl2 KO-OX40 agonist antibody refers to mice in which the Ccrl2 gene was knocked out and treated with OX40 agonist antibody). The results showed that the efficacy of OX40 agonist antibody was partially affected after the Ccrl2 gene was knocked out; the flow cytometry results showed that the expression of MHCII on the surface of monocytes, macrophages and neutrophils was significantly downregulated, which indicated that the antigen presentation function of myeloid cells was affected after the Ccrl2 gene was knocked out.

[0075] Clec4d- / hOX40 mice and Clec4d+ / hOX40 mice were inoculated with MC38 tumor cells and the tumors were grown to 50-100 mm 3 At the time of the experiment, the mice were divided into two groups according to Clec4d- / hOX40 mice and Clec4d+ / hOX40 mice, with 6 mice in each group. Each group was treated with isotype control antibody IgG and OX40 agonist antibody, respectively. The antibody was intraperitoneally administered at 5 mg / kg for a total of three times. The tumor volume of the mice was monitored, and the tumor growth curve and survival curve of the mice were plotted using Graphpad. The results are shown in the figure below. Fig.32 and Fig.33 As shown ( Fig.32 and Fig.33 In the data, WT-isotype control irrelevant antibody IgG refers to mice in which the Clec4d gene was not knocked out and treated with isotype control antibody IgG, WT-OX40 agonist antibody refers to mice in which the Clec4d gene was not knocked out and treated with OX40 agonist antibody, Clec4d KO-isotype control irrelevant antibody IgG refers to mice in which the Clec4d gene was knocked out and treated with isotype control antibody IgG, and Clec4d KO-OX40 agonist antibody refers to mice in which the Clec4d gene was knocked out and treated with OX40 agonist antibody). The results indicate that knocking out the Clec4d gene did not affect the therapeutic effect of OX40 agonist antibody on tumor-bearing mice. In addition, the expression of this gene was significantly upregulated in the OX40 agonist antibody treatment response group. We speculate that this gene is more likely to serve as a marker for OX40 agonist antibody treatment response. Example 4 Ccrl2, Clec4d and Clec4e genes are used to predict the effectiveness of OX40 agonist antibody therapy

[0076] OX40 humanized mice were inoculated with MC38 tumor cells on both sides, and the tumor volume reached 50-100mm 3 At 14:00, the mice were ear-tagged (1-30), and the left tumor tissue was removed under sterile conditions after anesthesia. The tumor tissue was quickly frozen in liquid nitrogen and stored at -80°C. The incision was sutured, and 5 mg / kg OX40 agonist antibody was treated the next day. The drug was given every two days for a total of 3 times. The tumor growth curve of the mice was then monitored, and the treatment response group and non-response group were determined according to the tumor volume. The tumor removed from the left side of the same mouse in the response group before treatment was named the untreated response group (UR, n=5), and the tumor on the left side of the same mouse in the non-response group before treatment was named the untreated non-response group (UNR, n=5) according to the corresponding ear tag number. RNA was then extracted and reverse transcribed from the tumors of UR and UNR, and the expression of Clec4d, Clec4e, and Ccrl2 genes was detected by qRT-PCR. The results are shown in Figure 2. Figure 34 to Figure 36 As shown ( Fig.34 p=0.1942, Fig.35 p=0.1138, Fig.36 Compared with the untreated non-responder group, the expression of Clec4d, Clec4e and Ccrl2 genes in the untreated corresponding responder group tended to increase. The changes in the expression of Clec4d, Clec4e and Ccrl2 in the responder group and the non-responder group before and after OX40 agonist antibody treatment and the results of the drug efficacy test in gene knockout mice indicate that Clec4d, Clec4e and Ccrl2 can be used as markers to predict the response to OX40 agonist antibody treatment.

[0077] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A biomarker for predicting the effectiveness of OX40 agonist antibody therapy, characterized in that The biomarker includes at least one of the Clec4d gene, the Clec4e gene and the Ccrl2 gene.

2. The biomarker for predicting the effectiveness of OX40 agonist antibody treatment according to claim 1, characterized in that Before treatment with OX40 agonist antibodies, When at least one of the Clec4d gene, the Clec4e gene, and the Ccrl2 gene is highly expressed in myeloid cells, it is predicted that OX40 agonist antibody treatment is effective; When Clec4d, Clec4e and Ccrl2 genes are all lowly expressed in myeloid cells, it is predicted that OX40 agonist antibody treatment is ineffective.

3. The biomarker for predicting the effectiveness of OX40 agonist antibody treatment according to claim 2, characterized in that The myeloid cells include monocytes, macrophages and / or neutrophils.

4. The biomarker for predicting the effectiveness of OX40 agonist antibody treatment according to claim 2 or 3, characterized in that The biomarker is Clec4d.

5. The biomarker for predicting the effectiveness of OX40 agonist antibody treatment according to claim 1, characterized in that The OX40 agonist antibody includes an OX40 humanized agonist antibody (2-2-1).

6. Use of a biomarker for predicting the effectiveness of OX40 agonist antibody therapy according to any one of claims 1 to 5 in predicting the effectiveness of OX40 agonist antibody therapy, characterized in that: include: Before treatment with OX40 agonist antibodies, When at least one of the Clec4d gene, Clec4e gene, and Ccrl2 gene is highly expressed in myeloid cells, it is predicted that OX40 agonist antibody treatment is effective; When Clec4d, Clec4e and Ccrl2 genes are all lowly expressed in myeloid cells, it is predicted that OX40 agonist antibody treatment is ineffective.

7. The use according to claim 6, characterized in that: When OX40 agonist antibody treatment is effective, the expression levels of Clec4d gene, Clec4e gene and / or Ccrl2 gene are monitored. Compared with before treatment with OX40 agonist antibody, the expression of any one of Clec4d gene, Clec4e gene and Ccrl2 gene in myeloid cells is significantly increased after treatment with OX40 agonist antibody.

8. The use according to claim 6 or 7, characterized in that: When the OX40 agonist antibody treatment is effective, the expression level of the Clec4d gene in the myeloid cells is higher than the expression level of the Clec4d gene when the OX40 agonist antibody treatment is ineffective; or, When the OX40 agonist antibody treatment is effective, the expression level of the Clec4e gene in the myeloid cells is significantly higher than the expression level of the Clec4e gene when the OX40 agonist antibody treatment is ineffective; or, When OX40 agonist antibody treatment is effective, the expression level of Ccrl2 gene in myeloid cells is significantly higher than that when OX40 agonist antibody treatment is ineffective.

9. A method for screening biomarkers for predicting the effectiveness of OX40 agonist antibody therapy according to any one of claims 1 to 5, characterized in that: The following steps are involved: S1. Establish a bilateral tumor-grafting model in OX40 humanized mice with intact immune systems and determine the phenotypes of the responding and non-responding groups. S2. Analyze and compare the precise composition, functional status and / or gene expression of tumor-infiltrating immune cells in mice that respond to OX40 agonist antibody treatment and those that do not respond; S3. Screen at least one of the target genes Clec4d, Clec4e and Ccrl2 that are related to myeloid cells and highly expressed in the OX40 agonist antibody treatment response group as a biomarker for predicting the effectiveness of OX40 agonist antibody treatment.

10. The method for screening biomarkers for predicting the effectiveness of OX40 agonist antibody therapy according to claim 9, characterized in that: In step S3, analyzing the effect of knocking out the Clec4d gene and / or the Ccrl2 gene on the efficacy of the OX40 agonist antibody to determine a biomarker predicting the effectiveness of the OX40 agonist antibody treatment; And / or, knocking out the Clec4d gene has no effect on the efficacy of OX40 agonist antibody, and determining that the biomarker for predicting the effectiveness of OX40 agonist antibody treatment is Clec4d.

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