CN116139275A - Use of CD300LD inhibitors in the preparation of a product for the prevention, diagnosis or treatment of tumors - Google Patents

Abstract

The invention relates to the field of biotechnology, in particular to application of a CD300LD inhibitor in preparing a tumor product for preventing, diagnosing or treating, wherein the CD300LD inhibitor regulates migration and functions of PMN-MDSC cells by regulating S100A8/A9 in a downstream signal path of the CD300LD inhibitor, and plays an important role in establishing a tumor microenvironment; the CD300LD inhibitor can effectively inhibit the development of various solid tumor mouse models, and has obvious anti-tumor synergistic effect with PD1 antibodies; the administration of CD300LD inhibitors can significantly inhibit tumor progression at the stage of tumor establishment. The invention provides a new target spot and thought for tumor immunotherapy.

Description

Use of CD300LD inhibitors in the preparation of products for preventing, diagnosing or treating tumors

technical field

The invention relates to the field of biotechnology, in particular to the use of CD300LD inhibitors in the preparation of products for preventing, diagnosing or treating tumors.

Background technique

Immune checkpoint blockade therapies such as PD1 and CTLA4 only have good therapeutic effects in some patients, so finding new immune checkpoints to further promote tumor immunotherapy is a major challenge in the field. Polymorphic myeloid-derived suppressor cells (PMN-MDSCs) are a type of pathologically induced neutrophils that are widely present in various tumors. They can inhibit the function of effector cells such as T cells and NK cells, and promote tumor development, invasion and Metastasis plays a key role in the regulation of tumor immunity. Specific targeted drugs against PMN-MDSC can further advance the immunotherapy of tumors, but the specific immune checkpoint molecules are still to be discovered.

Contents of the invention

In view of the shortcomings of the prior art described above, the purpose of the present invention is to provide the use of CD300LD inhibitors in the preparation of products for preventing, diagnosing or treating tumors, so as to solve the problems in the prior art.

To achieve the above and other related purposes, the present invention firstly provides the use of CD300LD gene as a target in the preparation of tumor prevention, diagnosis or treatment drugs.

The present invention also provides the use of CD300LD gene and PD1 as targets in the preparation of tumor prevention, diagnosis or treatment drugs.

The present invention also provides the use of CD300LD inhibitors in the preparation of products with at least one of the following effects:

1) Prevention, diagnosis and treatment of tumors;

2) regulating the activity and/or migration of myeloid cells;

3) Regulating T cell activity;

4) Regulating the downstream signaling pathway of CD300LD.

The present invention also provides a composition, the composition includes a CD300LD inhibitor, and the composition is used for any one or more of the following purposes:

1) Prevention, diagnosis and treatment of tumors;

2) regulating the activity and/or migration of myeloid cells;

3) Regulating T cell activity;

4) Regulating the downstream signaling pathway of CD300LD.

As mentioned above, the use of the CD300LD inhibitor of the present invention in the preparation of products for preventing, diagnosing or treating tumors has the following beneficial effects: a PMN-MDSC-specific new immune checkpoint molecule——CD300LD is provided, so that it is specific, Efficiently target and regulate PMN-MDSC, and provide new targets and ideas for solving targeted regulation of PMN-MDSC for tumor immunotherapy.

Description of drawings

(CD45⁺CD11b⁺F4/80⁺)，单核髓来源抑制细胞M-MDSC(CD45⁺CD11b⁺Ly6c⁺ly6G^-)，多型核髓来源抑制细胞PMN-MDSC(CD45⁺CD11b⁺Ly6c^lowly6G⁺)，结果显示CD300LD在PMN-MDSC中特异性高表达。F，qRT-PCR显示CD300LD在荷瘤小鼠PMN-MDSC与正常小鼠中性粒细胞中的mRNA水平。G，Human Protein Atlas数据库分析显示CD300LD基因主要表达在血液/免疫组织，在细胞亚型上主要表达在中性粒细胞上。H，qRT-PCR对健康人与结肠癌病人外周血粒细胞的CD300LD基因定量。双尾t检验，*p<0.05,**p<0.01,***p<0.001。Figure 1 shows the discovery of the CD300LD gene and its gene expression profile in immune cells in Example 1 of the present invention. A, Differentially expressed genes between PMN-MDSCs and neutrophils. B and C, Schematic diagram of the screening process for in vivo screening of myeloid cell surface receptors using Crispr-Cas9 and B16-F10 tumor models (B); screening enriched genes (C), compared with the bone marrow of tumor-bearing mice, CD300LD gRNA is specifically deleted in tumors. D, Flow cytometry analysis shows that in normal mouse immune cells, CD300LD is specifically expressed in myeloid cells in white blood cells, especially in neutrophils (CD45 ⁺ CD11b ⁺ Ly6G ⁺ ) (n=3 ). E, Flow cytometric analysis of CD300LD expression in intratumoral myeloid cells: macrophages

(CD45 ⁺ CD11b ⁺ F4/80 ⁺ ), mononuclear myeloid-derived suppressor cells M-MDSC (CD45 ⁺ CD11b ⁺ Ly6c ⁺ ly6G ^- ), polymorphic nuclear myeloid-derived suppressor cells PMN-MDSC (CD45 ⁺ CD11b ⁺ Ly6c ^low ly6G ⁺ ), the results showed that CD300LD was specifically highly expressed in PMN-MDSC. F, qRT-PCR shows the mRNA level of CD300LD in PMN-MDSC of tumor-bearing mice and neutrophils of normal mice. G, Analysis of Human Protein Atlas database shows that CD300LD gene is mainly expressed in blood/immune tissues, and mainly expressed in neutrophils in terms of cell subtypes. H, Quantification of CD300LD gene in peripheral blood granulocytes of healthy people and colon cancer patients by qRT-PCR. Two-tailed t-test, *p<0.05, **p<0.01, ***p<0.001.

Figure 2 shows that CD300LD knockout in Example 2 of the present invention inhibits the occurrence and development of various tumors. A&B, Schematic diagram of the construction of CD300LDKO mice (A) and genotyping results (B). C, Flow cytometric analysis of the expression of CD300LD in neutrophils of CD300LD WT and KO mice. D-F, Tumor growth curve (D), 20-day tumor size (E), and mouse survival curve (F) of CD300LD WT and KO mouse melanoma (B16-F10) tumor models (n=11). G, Tumor growth curves of TC-1 cervical cancer model in CD300LD WT and KO mice (n=6). H, Tumor growth curves of MC-38 colon cancer model in CD300LD WT and KO mice (n=6). I, Tumor growth curves of LLC lung cancer model in CD300LD WT and KO mice (n=6). Two-tailed t-test, *p<0.05, **p<0.01, ***p<0.001.

Figure 3 shows that CD300LD knockout inhibits tumor development through PMN-MDSC in Example 3 of the present invention. A, Knocking out CD300LD in myeloid cells by LyzCre can significantly inhibit the development of B16-F10 tumors (n=6). B, CD300LD of PMN-MDSC in myeloid cells can be knocked out by S100A8Cre conditionally, B16-F10 can obviously inhibit the development of tumor (n=9). C, Depletion of PMN-MDSCs in mice by Ly6g antibody significantly inhibited the development of B16-F10 tumors in wild-type mice, but had no significant effect in the B16-F10 tumor model of CD300LD knockout mice. D-F, Isolation of macrophages (D), mononuclear myeloid-derived suppressor cells (E), and PMN-MDSC (F) cells in B16-F10 tumors of wild-type mice or CD300LD KO mice, 1:1 The proportion of B16-F10 cells mixed with B16-F10 cells were inoculated subcutaneously in wild mice. The growth curves of the mice are shown in the figure. Only the presence or absence of CD300LD of multinucleated myeloid-derived suppressor cells leads to differences in tumor growth. Two-tailed t-test, *p<0.05, **p<0.01, ***p<0.001.

Figure 4 shows that CD300LD knockout in Example 4 of the present invention improves the tumor immune microenvironment (flow cytometric analysis results). A, Flow cytometric analysis of the ratio of white blood cells (CD45 ⁺ ) and major myeloid cell subsets in B16-F10 tumors. B, Flow cytometric analysis of the changes in the proportion of lymphocyte subsets in B16-F10 tumors. C&D, immunofluorescence analysis of intratumoral PMN-MDSC (Ly6g ⁺ ) and CD8 ⁺ T cells in B16-F10 tumors, where C is the result of immunofluorescence (Bar=200um), and D is the statistics of immunofluorescence analysis. Two-tailed t-test, *p<0.05, **p<0.01, ***p<0.001.

Figure 5 shows that CD300LD knockout in Example 4 of the present invention improves the tumor immune microenvironment (single-cell sequencing results). A&B, using single-cell sequencing to group immune cells in the B16-F10 tumor immune microenvironment, where A is the tSNE map, B&C, the distribution of immune cells in the B16-F10 tumor microenvironment of CD300LD WT and KO mice, in which the KO group PMN-MDSCs were significantly reduced, and CD8 ⁺ T cells were significantly increased; B is the tSNE map, and C is the ratio map of WT and KO corresponding cells. D, The expression levels of PMN-MDSC function-related genes, T cell recruitment and function-related genes in immune cells of WT and KO groups, the results showed that PMN-MDSC function-related genes were significantly down-regulated in the KO group, T cell infiltration and function-related Gene expression was significantly increased. Two-tailed t-test, *p<0.05, **p<0.01, ***p<0.001.

FIG. 6 shows that CD300LD-KO significantly reduces the migration and function of PMN-MDSCs in Example 5 of the present invention. A, RNAseq analysis of gene expression profiles of CD300LD WT and KO PMN-MDSCs, and GO analysis revealed significant differences in granulocyte migration signaling pathways. B, GSEA analysis found that granulocyte migration-related genes were significantly enriched in WT PMN-MDSCs. C, PMN-MDSCs were isolated from tumor-bearing CD300LD WT and KO mice for in vitro migration experiments, where the left side is a schematic diagram, and the right side is the experimental statistical results (n=3). D-F, PMN-MDSCs were isolated from tumor-bearing CD300LD WT and KO mice for in vivo migration experiments: D is a schematic diagram of the process. E&F, Flow cytometric analysis (E) and statistical results (F) of WT and KO PMN-MDSCs in bone marrow and tumor before and after injection. G&H, analysis of the ability of CD300LD WT and KO PMN-MDSC to inhibit T cell proliferation, where G is the flow diagram, and H is the statistical analysis (n=3). Two-tailed t-test, *p<0.05, **p<0.01, ***p<0.001.

Figure 7 shows that CD300LD regulates the migration and function of PMN-MDSC through S100A8/A9 in Example 6 of the present invention. A, RNAseq analysis of differential genes between CD300LD WT and KO PMN-MDSC, in which the expression of PMN-MDSC-related gene S100A8/A9 was significantly down-regulated in the KO group. B, ELISA analysis of the levels of S100A8/A9 in serum of WT and KO tumor-bearing mice. C, qRT-PCR analysis of S100A8/A9 expression in CD300LD WT and KO PMN-MDSCs (n=3). D, Effect of S100A8/A9 protein on the migration ability of PMN-MDSC in vitro (n=3). E, Effect of S100A8/A9 inhibitor Tasquinimod on WT and KO PMN-MDSCs inhibiting T cell proliferation (n=3). F&G, Tasquinimod, an inhibitor of S100A8/A9, was injected intraperitoneally during the tumor-bearing process of mice, and its effect on the tumor growth curve (F) and the ratio of PMN-MDSC in the tumor (G) were analyzed (n=4-5). Two-tailed t-test, *p<0.05, **p<0.01, ***p<0.001.

Figure 8 shows that CD300LD knockout and PD antibody in Example 7 of the present invention have anti-tumor synergistic effect. A, The ratio of PD1 ⁺ CD8 ⁺ cells, PD-L1 ⁺ PMN-MDSC cells, and PD-L1 ⁺ tumor cells in tumors of CD300LDWT and KO mice. B, After CD300LD WT and KO mice were inoculated with B16-F10 tumor cells, they were treated with PD1 antibody on the 7th day, 200 μg/mouse, once every 2 days, a total of 3 times; tumor growth curve (B) and mouse survival curve (C ) (n=11-15). Two-tailed t-test, *p<0.05, **p<0.01, ***p<0.001.

Fig. 9 shows the result that CD300LD knockout in Example 8 of the present invention has no obvious effect on the development of the immune and hematopoietic system. A, T (CD3 ⁺ ), B (B220 ⁺ ), myeloid (Mac1 ⁺ ) and neutrophils (Mac1 ⁺ Gr1 ⁺ ) and other immune cell ratio flow cytometric analysis statistics (n=3). B&C, flow cytometric analysis (B) and statistics (C) of HSCs and various precursor cells in the bone marrow of CD300LDWT and KO mice (n=3). D, Clonogenic ability of CD300LD WT and KO mouse HSCs (n=3). Two-tailed t-test, *p<0.05, **p<0.01, ***p<0.001.

FIG. 10 shows that the induced knockout of CD300LD in Example 9 of the present invention can inhibit the development of established tumors and can be used as an effective target for tumor therapy. A, B16-F10 tumor model was established in CD300LD ^(f/f) /Mx1-Cre and CD300LD ^(f/f) mice, and PolyI:C was injected on the 7th day of tumor development to induce knockout of CD300LD. Tumor growth curves showed that tumor development was significantly inhibited in CD300LD ^(f/f) /Mx1-Cre mice (n=6). B, the control of group A, the mice were the same as group A, if PolyI:C was not injected, there was no significant difference in tumor growth between the two groups of mice (n=6). Two-tailed t-test, *p<0.05, **p<0.01, ***p<0.001.

Detailed ways

The present invention focuses on finding key checkpoint molecules that regulate PMN-MDSCs. Because PMN-MDSC and neutrophils are similar in molecular typing but have opposite functions, the present invention firstly performs transcriptome sequencing on the two to analyze the difference in expression of membrane proteins; meanwhile, an sgRNA library for PMN-MDSC membrane proteins is also constructed, In vivo CRISPR-Cas9 screening in combination with mouse tumor models. Both strategies simultaneously point to a novel membrane protein CD300LD. Both human and mouse CD300LD were specifically expressed on neutrophils/PMN-MDSCs, and the expression was significantly up-regulated in the latter after tumor bearing. Knockout of CD300LD significantly inhibits tumor development in various models including melanoma, colon, breast and lung cancer. Using CD300LD conditional knockout or Ly6G antibody to clear PMN-MDSCs in vivo, and to isolate different myeloid cell subsets for mixed tumorigenesis experiments, etc., proved that PMN-MDSCs specifically regulate tumor immunity through CD300LD. Thus, CD300LD is a novel PMN-MDSC-specific tumor immunosuppressive receptor, a potent immune checkpoint molecule.

Mechanistic studies have found that knockout of CD300LD leads to a significant improvement in the tumor immunosuppressive microenvironment: PMN-MDSC sharply decreases, CD8 ⁺ T cell infiltration significantly increases; PMN-MDSC migration and function-related signaling pathways are significantly down-regulated; T cell migration and killing-related signals pathways were markedly upregulated. Further studies found that CD300LD regulates PMN-MDSC tumor migration and inhibits T cell function through downstream S100A8/A9. In summary, CD300LD knockout regulates the recruitment and function of PMN-MDSCs through S100A8/A9, thereby significantly improving the tumor immunosuppressive microenvironment and producing a broad-spectrum anti-tumor effect.

In terms of safety, unlike the currently widespread concern that CSF1R knockout, which is widely expressed in myeloid cells, is lethal to the body, and CD47 has strong side effects, CD300LD knockout does not affect the normal development of the body and the immune system. In terms of effectiveness, CD300LD inhibits the development of various tumor models, and exhibits a significant anti-tumor synergistic effect with PD1 antibodies; more importantly, inducing the knockout of CD300LD can also significantly inhibit tumor development when the tumor has been established. In conclusion, CD300LD, as a new target, has good safety and anti-tumor efficacy.

Based on the above studies, the present invention first provides the use of the CD300LD gene as a target in the preparation of tumor prevention, diagnosis or treatment drugs.

In the present invention, the term "prophylaxis" includes prophylactic treatment that results in the desired pharmaceutical and/or physiological effect. The better effect means that medically it can block, delay the occurrence of the disease and/or reduce the risk of the development or deterioration of the disease.

In the present invention, the term "diagnosis" can refer to judging whether there is a certain disease based on the expression level of CD300LD.

As used herein, the term "treatment" includes curative or palliative treatments that result in a desired pharmaceutical and/or physiological effect. The effect preferably means that one or more symptoms of the disease can be reduced medically or the disease can be completely eliminated.

Unless otherwise specified, the tumor described in the present invention is a tumor overexpressing CD300LD. The overexpression of CD300LD usually means that the expression level of CD300LD is higher than a certain standard, and this expression can be at the molecular level or at the protein level. For example, CD300LD positivity may be overexpression of CD300LD mRNA in tumor tissue. For another example, positive CD300LD may be the overexpression of CD300LD-detectable protein in the tumor tissue. For another example, positive CD300LD may mean that the mRNA expression level of CD300LD in tumor tissue is higher than that in surrounding healthy tissue. For another example, positive CD300LD may mean that the expression level of CD300LD protein in tumor tissue is higher than that in surrounding healthy tissue. The tumor may be a solid tumor (such as a digestive tract tumor, a respiratory tract tumor) or a blood tumor, and more specifically may be colon cancer, lung cancer, liver cancer, breast cancer, esophageal cancer, head and neck cancer, skin cancer, kidney cancer, leukemia, cervical cancer , coad (colon cancer), lihc (hepatocellular carcinoma), ov (ovarian serous cystadenocarcinoma), ucec (endometrial carcinoma), thca (thyroid carcinoma), skcm (cutaneous melanoma), luad (lung adenocarcinoma) ), hnsc (head and neck squamous cell carcinoma), gbm (glioblastoma multiforme), prad (prostate cancer), thym (thymus carcinoma), lgg (low-grade glioma of the brain), read (rectal adenocarcinoma) , pcpg (pheochromocytoma and paraganglioma), esca (esophageal carcinoma), kirc (clear cell renal cell carcinoma), blca (urothelial carcinoma of the bladder), kirp (papillary cell carcinoma of the kidney), paad (pancreatic carcinoma ), stad (stomach cancer), kich (chromophobe renal cell carcinoma), brca (breast invasive carcinoma), lusc (lung squamous cell carcinoma), sarc (sarcoma), LAML (acute myeloid leukemia), etc.

The CD300LD gene as a target in the preparation of preventive, diagnostic or tumor treatment drugs specifically refers to: using the CD300LD gene as an action object, screening drugs or preparations to find a drug that can inhibit the expression of the CD300LD gene as a candidate drug for tumor treatment. For example, small molecule interfering RNA (siRNA) is obtained by screening the human CD300LD gene as an action object, and can be used as a drug capable of inhibiting tumor cell proliferation. In addition, such as antibody drugs, small molecule drugs, etc. can also target the CD300LD gene.

The use of the human CD300LD gene as a target in the preparation of tumor diagnostic drugs specifically refers to: using the CD300LD gene expression product as a tumor diagnostic index in the preparation of tumor diagnostic drugs.

In one embodiment, the tumor treatment drug is a molecule that can specifically inhibit the transcription or translation of the CD300LD gene, or can specifically inhibit the expression or activity of the CD300LD protein, thereby reducing the expression level of the CD300LD gene in tumor cells, or It can competitively inhibit CD300LD and achieve the goal of significantly improving the tumor immunosuppressive microenvironment. The significant improvement in the tumor immunosuppressive microenvironment is manifested in a sharp decrease in PMN-MDSC and a significant increase in infiltration of CD8 ⁺ T cells; PMN-MDSC migration and functional correlation The signaling pathways were significantly down-regulated; the signaling pathways related to T cell migration and killing were significantly up-regulated. The inventors found through mechanism research that CD300LD in tumor therapeutic drugs regulates the tumor migration of PMN-MDSCs and inhibits the function of T cells through downstream S100A8/A9, resulting in a broad-spectrum anti-tumor effect.

The tumor therapeutic drug or tumor diagnostic drug obtained through CD300LD gene preparation includes but not limited to: nucleic acid molecules, carbohydrates, lipids, small molecule chemical drugs, antibody drugs, polypeptides, proteins or interfering lentiviruses.

The nucleic acid includes but is not limited to: antisense oligonucleotide, double-stranded RNA (dsRNA), ribozyme, small interfering RNA prepared by endoribonuclease III or short hairpin RNA (shRNA).

In one embodiment, the dosage of the drug for treating tumor is a dosage sufficient to reduce the transcription or translation of human CD300LD gene, or the dosage sufficient to reduce the expression or activity of CD300LD protein. Such that the expression or activity of the human CD300LD gene is at least reduced by 50%, 80%, 90%, 95% or 99%.

In another embodiment, the dose sufficient to reduce the activity of the CD300LD protein is a dose sufficient to competitively bind with the CD300LD protein or its receptor, so that the CD300LD protein cannot function.

The method of treating tumors with the aforementioned tumor therapeutic drugs mainly achieves the purpose of treatment by reducing the expression or activity level of human CD300LD gene and inhibiting the proliferation of tumor cells. Specifically, during treatment, the substance that can effectively reduce the expression or activity level of human CD300LD gene is administered to the patient.

In one embodiment, the target of the CD300LD gene is Exon2.

The present invention also provides the use of CD300LD gene and PD1 as targets in the preparation of tumor prevention, diagnosis or treatment drugs.

The present invention also provides the use of CD300LD inhibitors in the preparation of products with at least one of the following effects:

1) Prevention, diagnosis and treatment of tumors;

2) regulating the activity and/or migration of myeloid cells;

3) Regulating T cell activity;

4) Regulating the downstream signaling pathway of CD300LD.

The inventors of the present invention have found that CD300LD inhibitors can modulate the activity of myeloid cells. The regulation of the activity of myeloid cells may be, for example, reducing the number of myeloid cells, or reducing the expression of genes related to immune suppression on myeloid cells.

The regulation of migration of myeloid cells may be achieved by significantly down-regulating signaling pathways related to migration of myeloid cells.

The myeloid cells are selected from macrophages, M-MDSCs or PMN-MDSCs.

Regulation of T cell activity can be, for example, a significant increase in infiltrating immune cells in the tumor microenvironment, a significant increase in the number of CD4 ⁺ and CD8 ⁺ T cells, a significant decrease in Treg cells, a significant increase in the expression of genes related to T cell killing function, or a significant increase in T cell recruitment related genes Significantly increased. CD300LD inhibitors can regulate T cell proliferation by inhibiting the T cell inhibitory function of myeloid cells.

The regulation of the downstream signaling pathway of CD300LD may be through positive regulation of a certain gene in the downstream signaling pathway, or through negative regulation of a certain gene in the downstream signaling pathway. The genes of the downstream signaling pathway are, for example, S100A8, S100A9, Trim10, Ms4a6d, Apol11b.

In the products provided by the present invention, CD300LD inhibitors are generally used as immunotherapeutic drugs. Immunotherapy drugs used to treat tumors usually restore the body's normal anti-tumor immune response by restarting and maintaining the tumor-immune cycle, thereby changing the tumor microenvironment to achieve the effect of controlling and eliminating tumors.

The product necessarily includes a CD300LD inhibitor, and uses the CD300LD inhibitor as an active ingredient for the aforementioned effects.

In the product, the active ingredient that exerts the aforementioned functions may only be a CD300LD inhibitor, or may contain other molecules that can exert the aforementioned functions.

That is, the CD300LD inhibitor is the only active ingredient or one of the active ingredients of the product.

The product may be a single-ingredient substance or a multi-ingredient substance.

The form of the product is not particularly limited, and can be in various forms such as solid, liquid, gel, semi-fluid, and aerosol.

The main targets of the products are mammals. The mammal is preferably a rodent, an artiodactyla, a perissodactyla, a lagomorpha, a primate or the like. The primate is preferably a monkey, ape or human.

The products include but are not limited to medicines, health care products, food, kits, etc.

The CD300LD inhibitor can be a nucleic acid molecule, an antibody, or a small molecular compound.

As listed in the embodiments of the present invention, the CD300LD inhibitor may be a nucleic acid molecule that reduces the expression of CD300LD gene in tumor cells.

In the present invention, a CD300LD inhibitor generally refers to a substance that can inhibit the expression and/or function of CD300LD. For example, the CD300LD inhibitor can partially inhibit, that is, reduce the expression and/or function of CD300LD, for example, reduce the expression and/or function of CD300LD by 50%, 60%, 70%, 80%, 90%, 95%, 99%; It can also be completely inhibited, ie the expression of CD300LD and/or its function is substantially completely eliminated. The class of suitable substances capable of acting as CD300LD inhibitors will be known to those skilled in the art. For example, a CD300LD inhibitor can be an antagonist, a blocker, and the like. For another example, the inhibitory function of the CD300LD inhibitor can be the inhibition of CD300LD gene expression level at the nucleic acid molecule level (eg, mRNA level, DNA level) and/or protein molecule level. For another example, the CD300LD inhibitor may also be a substance that competes with CD300LD for binding to its ligand, or a substance that competes with the ligand of CD300LD for binding to CD300LD. More specifically, CD300LD inhibitors can be nucleic acid molecules, protein molecules or compounds, etc. For example, the nucleic acid molecule can be selected from interfering RNA against CD300LD, antisense oligonucleotides against CD300LD, substances for knocking out or knocking down the expression of CD300LD, more specifically siRNA, miRNA, shRNA, gene knockout vector, Gene expression vectors (for example, capable of expressing siRNA, shRNA, interfering RNA) and the like. In a specific embodiment of the present invention, the target sequence of the nucleic acid molecule may be exon 2 of CD300LD. For example, protein molecules can be selected from anti-CD300LD antibodies, and these antibodies can be monoclonal antibodies, polyclonal antibodies, and the like. For another example, the CD300LD inhibitor can be CD300LD-ECD (CD300LD extracellular region), and the amino acid sequence of CD300LD-ECD can be derived from rat, mouse, human, etc.

The present invention also provides a composition, the composition includes a CD300LD inhibitor, and the composition is used for any one or more of the following purposes:

1) Treat tumors;

2) regulating the activity and/or migration of myeloid cells;

3) Regulating T cell activity;

4) Regulating the downstream signaling pathway of CD300LD.

The CD300LD inhibitor can be various CD300LD inhibitors as described above.

In the present invention, the above-mentioned products or compositions may be used for diagnostic or therapeutic purposes, or may be used for non-diagnostic or therapeutic purposes.

The present invention also provides an in vitro drug screening method, which includes the following steps: after mixing the cells overexpressing CD300LD with the drug to be screened, detecting changes in the cells.

In one embodiment, the drug to be screened is an antineoplastic drug. The anti-tumor drug is, for example, a CD300LD inhibitor.

In one embodiment, the cells overexpressing CD300LD are myeloid immune cells. More specifically, the myeloid immune cells are selected from macrophages, M-MDSCs and PMN-MDSCs.

The changes in the cells include changes in cell activity, migration, expression of CD300LD, changes in downstream signaling pathways of CD300D, and the like.

The present invention also provides a regulation method, which includes administering to an individual an effective amount of a CD300LD inhibitor, or the composition provided above to regulate the activity of myeloid cells, or regulate the activity of T cells, or regulate the downstream signal of CD300LD path.

The present invention also provides a treatment method comprising: administering to an individual a therapeutically effective dose of a CD300LD inhibitor, or the composition provided above in the present invention.

The treatment method provided by the present invention can be used to treat indications including but not limited to tumors and the like.

In the present invention, "individual" generally includes humans and non-human primates, such as mammals, dogs, cats, horses, sheep, pigs, cows, etc., which can use the above-mentioned drugs, compositions, preparations, kits, etc. Or combined preparations to benefit from treatment.

In the present invention, "therapeutically effective dose" generally refers to an amount that can achieve the effect of treating the diseases listed above after an appropriate administration period. Specifically, when the CD300LD inhibitor or composition is administered to a subject, the dosage varies according to the age and weight of the patient, the characteristics and severity of the disease, and the route of administration. The results of animal experiments and In various cases, the total dosage should not exceed a certain range.

In some embodiments, the CD300LD inhibitor described herein, or the composition, is administered in an amount of about 0.001 mg/Kg to about 500 mg/Kg (eg, about 0.001 mg/Kg to about 200 mg/Kg; about 0.01 mg/Kg Kg to about 200 mg/Kg; about 0.01 mg/Kg to about 150 mg/Kg; about 0.01 mg/Kg to about 100 mg/Kg; about 0.01 mg/Kg to about 50 mg/Kg; about 0.01 mg/Kg to about 10 mg/Kg about 0.01 mg/Kg to about 5 mg/Kg; about 0.01 mg/Kg to about 1 mg/Kg; about 0.01 mg/Kg to about 0.5 mg/Kg; about 0.01 mg/Kg to about 0.1 mg/Kg; about 0.1 mg /Kg to about 200 mg/Kg; about 0.1 mg/Kg to about 150 mg/Kg; about 0.1 mg/Kg to about 100 mg/Kg; about 0.1 mg/Kg to about 50 mg/Kg; about 0.1 mg/Kg to about 10 mg/Kg Kg; about 0.1 mg/Kg to about 5 mg/Kg; about 0.1 mg/Kg to about 1 mg/Kg; about 0.1 mg/Kg to about 0.5 mg/Kg). The total daily dosage may be administered in divided and divided doses throughout the day or by providing continuous delivery. Can be daily (e.g., as a single dose or as two or more divided doses) or non-daily (e.g., every other day, every two days, every three days, weekly, twice a week, every two weeks , once a month) administration. In some embodiments, the CD300LD inhibitor described herein, or the administration time of the composition is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days , 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months , 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months or longer. In another embodiment, the administration stop time is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days , 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months or more. In one embodiment, a subject is administered a CD300LD inhibitor, or composition, for treatment for a period of time, followed by separate periods of time. In another embodiment, the therapeutic CD300LD inhibitor, or composition, is administered for a first period, discontinued for a second period of time after the first period, and then restarted for a third period of time with the CD300LD inhibitor , or the composition, and then discontinue dosing for a fourth period after the third period. In one embodiment, the administration time is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months months, 9 months, 10 months, 11 months, 12 months or more. In another embodiment, the time to stop administration is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months , 8 months, 9 months, 10 months, 11 months, 12 months or longer.

In some embodiments, the methods described herein may further comprise administering one or more additional therapies (e.g., one or more additional therapeutic agents and/or one or more treatment regimens) in combination with the compounds described herein ).

In some embodiments, the methods described herein can further comprise administering one or more additional cancer therapies. One or more additional cancer therapies may include, but are not limited to: surgery, radiation therapy, chemotherapy, toxin therapy, immunotherapy, cryotherapy, cancer vaccines (e.g., HPV vaccine, hepatitis B vaccine, Oncophage, Provenge) and Gene therapy, and combinations thereof. Immunotherapy, including but not limited to adoptive cell therapy, derivation of stem cells and/or dendritic cells, blood transfusion, lavage and/or other therapies including but not limited to freezing tumors.

In some embodiments, the one or more additional cancer therapies are chemotherapy, which may comprise administration of one or more additional chemotherapy agents.

In some embodiments, the other chemotherapeutic agent is an immunomodulatory molecule, such as an immune checkpoint inhibitor. In some of these embodiments, the immune checkpoint inhibitor targets an immune checkpoint receptor selected from the group consisting of CTLA-4, PD-1, PD-L1, PD-1–PD-L1, PD-1–PD -L2.

In some of these embodiments, the immune checkpoint inhibitor is selected from the group consisting of: Pembrolizumab (PD1), Nivolumab (PD1), Atezolizumab (formerly known as MPDL3280A) (PDL1), MEDI4736 (PD-L1), Avelumab (Avelumab) (PD-L1), PDR001 (PD1), BMS-986016, MGA271, Lirilumab (Lirilumab), IPH2201, Yin Ma Ketuzumab Emactuzumab, INCB024360, Galunisertib, Ulocuplumab, BKT140, Bavituximab, CC-90002, Bevacizumab, MNRP1685A, and MGA271.

Embodiments of the present invention are described below through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation modes, and various modifications or changes can be made to the details in this specification based on different viewpoints and applications without departing from the spirit of the present invention.

Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the following specific specific embodiments; it should also be understood that the terms used in the examples of the present invention are to describe specific specific embodiments, It is not intended to limit the protection scope of the present invention; in the description and claims of the present invention, unless the context clearly indicates otherwise, the singular forms "a", "an" and "the" include plural forms.

When the examples give numerical ranges, it should be understood that, unless otherwise stated in the present invention, the two endpoints of each numerical range and any value between the two endpoints can be selected. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, equipment, and materials used in the examples, according to those skilled in the art's grasp of the prior art and the description of the present invention, the methods, equipment, and materials described in the examples of the present invention can also be used Any methods, apparatus and materials of the prior art similar or equivalent to the practice of the present invention.

Example 1 Key function receptor screening of PMN-MDSC

The immunosuppressive microenvironment of tumors is an important factor for tumor immune escape. In the tumor microenvironment, polymorphic myeloid-derived suppressor cells (PMN-MDSCs) are an important cell population leading to immunosuppression. Because PMN-MDSC and neutrophils are similar in molecular typing but have opposite functions, this example firstly performs transcriptome sequencing on the two, and analyzes the differences in expression of membrane proteins; at the same time, constructs an sgRNA library for PMN-MDSC membrane proteins, combining Mouse tumor models for CRISPR-Cas9 in vivo screening. The key functional receptors of PMN-MDSCs were screened using these two strategies, and expression profiling was performed on the obtained candidate molecules.

Materials and Methods:

1-1) Transcriptome analysis of PMN-MDSC and neutrophils: Neutrophils (CD11b ⁺ Ly6G ⁺ ) in bone marrow of normal C57 mice and spleen of B16-F10 tumor-bearing mice were separated by AutoMACS magnetic sorting From the PMN-MDSC (CD11b ⁺ Ly6G ⁺ ), total cellular RNA was extracted according to methods well known in the art for RNAseq sequencing analysis.

1-2) Construction and packaging of an sgRNA library targeting myeloid cell-specific surface receptors: using the RNAseq data of immune cells, for the membrane protein genes in them, calculate their expression ratio in myeloid cells/lymphoid cells, According to the ratio >2, 300 receptor genes specifically expressed in myeloid cells were selected. For each gene, 10 sgRNA oligos were designed and synthesized according to methods well known in the art, and constructed into the Syn003-modified lentiviral expression plasmid to obtain an sgRNA lentiviral expression library targeting myeloid cell surface receptors. The library plasmid and the lentiviral packaging plasmid were co-transfected into 293T cells using Lipofectin, and the virus supernatant was collected after 48 hrs, filtered through a 0.45 μm filter membrane, and used for infection of target cells.

1-3) Screening of key receptors related to myeloid cells in the tumor microenvironment: use magnetic sorting to separate hematopoietic precursor cells (Lineage-negative cells) in Cas9 mouse fetal liver, and then use the aforementioned sgRNA library virus supernatant to infect these After the precursor cells were transplanted into irradiated mice (9G) to reconstitute the immune system of the mice; the immune cells of these mice would express different sgRNAs. After 4 weeks of transplantation, 1.5×10 ⁵ B16 cells were inoculated subcutaneously in the mice for tumor formation, and the myeloid cells in the mouse bone marrow and tumor were isolated 2 weeks later, and the gRNA in them was amplified according to methods well known in the art for deep sequencing and comparison analyze.

1-4) Analysis of the expression of CD300LD in various types of immune cells in mice: Isolate the bone marrow and tumors of normal mice or B16 tumor-bearing mice, prepare single cell suspensions, and use CD300LD antibodies and antibodies for various immune cells to label Afterwards, flow cytometry was performed to analyze the protein expression levels of CD300LD on the surface of various types of immune cells. The surface markers of each immune cell were: macrophages (CD45 ⁺ /CD11b ⁺ //F4/80 ⁺ ), monocytes/ M-MDSC (CD45 ⁺ /CD11b ⁺ /Ly6c ⁺ ), neutrophils/PMN-MDSC (CD45 ⁺ /CD11b ⁺ /Ly6G ⁺ ), DC (CD45 ⁺ /CD11b ^- /CD11c ⁺ ), T cells (CD45 ⁺ /CD3 ⁺ ), CD4 ⁺ T (CD45 ⁺ /CD3 ⁺ /CD4 ⁺ ), CD8 ⁺ T (CD45 ⁺ /CD3 ⁺ /CD8 ⁺ ), B cells (CD45 ⁺ /B220 ⁺ ), NK cells (CD45 ⁺ /NKG2D ⁺ ). In order to analyze the RNA expression level of CD300LD, the present invention uses the above molecular markers to sort out normal or tumor-bearing mouse bone marrow or immune cells in the tumor by flow cytometry. ) reverse transcription, and then perform qRT-PCR detection according to the primers and conditions as described below.

mLD-For: 5'ACATTGACCAAGCCCCAAAAA3' (SEQ ID NO.1)

mLD-Rev: 5'AGCAGGGGTAACTCCACAAAG 3' (SEQ ID NO.2)

mGAPDH-For: 5'GTCTACATGTTCCAGTATGACTCC 3' (SEQ ID NO.3)

mGDPDH-Rev: 5'AGTGAGTTGTCATATTTCTCGTGGT 3' (SEQ ID NO.4)

qPCR conditions: 95°C for 5min, (95°C for 30s, 58°C for 30s, 72°C for 30s, 40cycles), 72°C for 10min, 4°C

10min

1-5) Analysis of the expression of CD300LD in various types of human immune cells: using the Human Protein Atlas database, the mRNA expression levels of CD300LD in different types of human immune cells were analyzed. In order to analyze the expression level of CD300LD in clinical tumor patients, the present invention collects the peripheral blood of normal people and colorectal cancer patients, extracts leukocyte mRNA, and performs qRT-PCR detection according to the following primers and conditions:

huLD-For: 5'TCCCAGGTTACTCCATTGCC 3' (SEQ ID NO.5)

huLD-Rev: 5'GCCTGAGCCATAAGCACACT 3' (SEQ ID NO.6)

huGAPDH-For: 5'TGTGGGCATCAATGGATTTGG 3' (SEQ ID NO.7)

huGDPDH-Rev: 5'ACACCATGTATTCCGGGTCAAT 3' (SEQ ID NO.8)

qPCR conditions: 95°C for 5min, (95°C for 30s, 58°C for 30s, 72°C for 30s, 40cycles), 72°C for 10min, 4°C for 10min)

result:

Transcriptome analysis results showed that CD300LD was significantly upregulated in PMN-MDSCs compared with normal neutrophils (Fig. 1A). At the same time, Crispr-Cas9 was used to screen the membrane proteins specifically expressed by myeloid cells in vivo (Fig. 1B). The results showed that the abundance of CD300LD-gRNA in myeloid cells in the tumor microenvironment of tumor-bearing mice was significantly lower than that of bone marrow. Myeloid cells (Fig. 1C), suggesting that the tumor microenvironment rejects CD300LD knockdown. The above two screens suggested that CD300LD may be the key receptor of PMN-MDSC.

Cells expressing CD300LD were analyzed. CD300LD was specifically and highly expressed in normal neutrophils, as well as tumor PMN-MDSCs (Fig. 1D and E). Compared with normal neutrophils, the expression level of CD300LD was significantly increased in PMN-MDSCs (Fig. 1F). In human immune cell populations, CD300LD is also specifically highly expressed on neutrophils (Fig. 1G); and in PMN-MDSCs derived from tumor patients, the expression level of CD300LD is significantly higher than that of normal neutrophils (Fig. 1H) .

The above results show that CD300LD is specifically highly expressed on neutrophils/PMN-MDSCs, and its expression on PMN-MDSCs is significantly increased during tumorigenesis; combined with the results of Crispr-Cas9 screening, that is, the tumor microenvironment excludes CD300LD knockout cells. In addition, it is suggested that CD300LD is likely to be the key receptor required for the recruitment, differentiation and function of PMN-MDSC in tumors.

Example 2 CD300LD knockout significantly inhibits the development of various types of tumors

In order to evaluate the function of CD300LD in tumor development, CD300LD knockout mice were constructed, and various tumor models were used to analyze the effect of CD300LD knockout on tumor development.

Materials and Methods:

2-1) Construction of CD300LD knockout mice (entrusted by a third-party company, Jizui Yaokang Co., Ltd. to construct): using Cas9/sgRNA (gRNA1 sequence is shown in SEQ ID NO.9; gRNA2 sequence is shown in SEQ ID NO.10) , construct a mouse that knocks out CD300LDExon2 (nucleotide sequence such as SEQ ID NO.11);

2-2) B16-F10 melanoma tumor mouse model: inoculate B16 cells (according to 1.5×10 ⁵ cells/mouse) into mice subcutaneously, measure tumor size every 2-3 days, and draw tumor growth curve; tumor volume It was judged as dead at ^1000cm3 , and the survival curve of the mice was drawn;

2-3) TC-1 cervical cancer mouse model: inoculate TC1 cells (according to 1.5×10 ⁵ cells/mouse) into mice subcutaneously, measure the size of the tumor every 2-3 days, and draw the tumor growth curve; tumor volume It was judged as dead at ^1000cm3 , and the survival curve of the mice was drawn;

2-4) MC-38 colon cancer mouse model: inoculate MC38 cells (according to 1.0×10 ⁶ cells/mouse) into mice subcutaneously, measure the size of the tumor every 2-3 days, and draw the tumor growth curve; tumor volume It was judged as dead at ^1000cm3 , and the survival curve of the mice was drawn;

2-5) LLC lung cancer mouse model: Inoculate LLC cells (5×10 ⁵ cells/mouse) into mice subcutaneously, measure the size of the tumor every 2-3 days, and draw the tumor growth curve; the tumor volume is 1000 cm ³ Determined as dead, draw the survival curve of the mouse

result:

CD300LD knockout mice were constructed, and CD300LD was knocked out at the genome level and protein level (Fig. 2A-C). Using the B16-F10 melanoma model, it was found that in CD300LD knockout mice, tumor growth was significantly lower than that in wild-type mice (Figure 2D and E), and the survival time of mice was also significantly prolonged (Figure 2F). In the other three tumor models including TC-1 cervical cancer, MC-38 colon cancer, and LLC lung cancer, the tumor growth of CD300LD knockout mice was significantly lower than that of wild-type mice (Fig. 2G-I). The above results show that knocking out host CD300LD can significantly inhibit the development of various types of tumors.

Example 3 CD300LD knockout inhibits tumor development through PMN-MDSC

CD300LD knockout mice can significantly inhibit the development of various types of tumors, therefore, it is extremely critical to determine which cell type CD300LD plays a role in it. The main cell population expressing CD300LD was myeloid immune cells, including macrophages, M-MDSCs, and PMN-MDSCs; the expression of CD300LD on PMN-MDSCs was much higher than that on other types of cells (Figure 1D and E). Therefore, this example analyzes which type of myeloid cells whose CD300LD is knocked out inhibits the development of tumors.

Materials and Methods:

3-1) The effect of conditional knockout of CD300LD in different cell populations on tumor development: Construction of CD300LD fl/fl conditional knockout mice (entrusted to a third-party company, Jizui Yaokang Co., Ltd. to construct; gRNA1 sequence as shown in SEQ ID NO.12 shown, the gRNA2 sequence is shown in SEQ ID NO.13), and the mouse was bred with different Cre mice, including LyzCre (macrophages, neutrophils/PMN-MDSC and other myeloid cells specifically express Cre) and S100A8Cre (neutrophils/PMN-MDSCs specifically express Cre), so as to obtain mice with conditional knockout of CD300LD in different types of myeloid cells. The above mice were used to carry out B16-F10 tumor experiment to detect tumor growth (see 2-2 in Example 2 for the steps).

3-2) The effect on tumor development after using Ly6G antibody to eliminate PMN-MDSC: B16-F10 tumor cells were inoculated subcutaneously in mice, and Ly6G antibody or Isotype control antibody (200 μg /mouse/time) to detect tumor growth.

3-3) Detection of the effect of co-transplantation of different myeloid cells and tumor cells on tumor growth: Isolate macrophages, M-MDSC and PMN-MDSC cells from WT or CD300LD-KO tumor-bearing mice, and use a ratio of 1:1 Ratio mixed with B16-F10 cells and inoculated subcutaneously into wild-type mice for tumor formation experiments to detect tumor growth.

result:

The results showed that using LyzCre to conditionally knock out CD300LD in various myeloid cells (including macrophages/neutrophils/PMN-MDSCs) significantly inhibited the development of B16 tumors (Fig. 3A); Conditional knockout of CD300LD in PMN-MDSCs also significantly inhibited tumor development (Fig. 3B); indicating that CD300LD expressed by PMN-MDSCs plays an important role in tumor development.

In order to further prove that CD300LD plays a role in tumor through PMN-MDSC, this example uses Ly6G antibody to eliminate neutrophils/PMN-MDSC in wild-type and CD300LD-KO tumor mice, and the results show that in wild-type mice Tumor development was significantly slowed down, while Ly6G antibody had no effect in CD300LD-KO mice (Fig. 3C), indicating that the anti-tumor effect caused by CD300LD knockout was mainly accomplished by neutrophils/PMN-MDSCs.

In order to further prove that CD300LD regulates tumor development through PMN-MDSC, macrophages, M-MDSC and PMN-MDSC cells were isolated from CD300LD WT and KO tumor-bearing mice, and then co-injection was used to analyze the effect of different cell populations on tumor development. Impact. The results showed that only CD300LD-KO PMN-MDSCs significantly inhibited tumor development (Fig. 3D-F).

Based on the above results, CD300LD plays an important role in tumor development mainly through PMN-MDSC cells.

Example 4 CD300LD knockout reverses tumor immunosuppressive microenvironment

In order to study how CD300LD knockout inhibits the development of tumors, this example uses flow cytometry analysis and immunohistochemistry to analyze the changes of various immune cell populations in the tumor microenvironment of WT and CD300LD knockout mice, and at the same time, the immune cells in the tumor microenvironment cells for further single-cell sequencing analysis.

Materials and Methods:

4-1) B16-F10 melanoma tumor mouse model: B16-F10 cells (1.5×10 ⁵ cells/mouse) were subcutaneously inoculated into mice, and the tumor size was measured every 2-3 days.

4-2) Tumor microenvironment analysis: Take mouse tumors to prepare single-cell suspension, and use the following two groups of antibodies for labeling: Antibody group 1 (Anti-Ly6C FITC, Anti-MHCII PE, Anti-F4/80PE-Cy7, Anti- -Cd11b PB, Anti-CD45BV510, Anti-Cd11c APC, Anti-Ly6G APC-Cy7) and Antibody Group 2 (Anti-CD3 FITC, Anti-CD8 PE, Anti-B220 PE-Cy7, Anti-CD45 BV510, Anti-CD49b APC, Anti-CD4 APC-Cy7), the proportion of each immune cell population was analyzed by flow cytometry.

4-3) Immunofluorescence: Isolate mouse tumors for frozen sections, stain with CD8-FITC antibody or Ly6G-PE antibody at 4 degrees overnight, wash 3 times, seal with DAPI-containing mounting solution, and examine under a fluorescent microscope observe.

4-4) Single-cell sequencing of the tumor immune microenvironment: take the tumor tissue of B16 tumor mice to prepare single-cell suspension, flow cytometrically sort CD45+ immune cells, perform 10× single-cell sequencing, and use Sereut for dimensionality reduction and grouping, etc. analyze

result:

The results are shown in Figure 4. Compared with the tumor microenvironment of WT mice, CD300LD knockout led to a significant increase in infiltrating immune cells and a significant decrease in PMN-MDSC cells in the tumor microenvironment, accompanied by a significant increase in CD4 ⁺ and CD8 ⁺ T cells, As well as a significant reduction in Treg cells (Figure 4A and B). Immunofluorescence results also showed that in the tumors of CD300LD knockout mice, PMN-MDSCs were significantly reduced and CD8 ⁺ T cells were significantly increased (Fig. 4C and D).

This example further analyzes the impact of CD300LD knockout on the tumor immune microenvironment from the single-cell transcriptome level. The results also showed that CD300LD knockdown led to a significant reduction of PMN-MDSCs and a significant increase in the infiltration of CD8 ⁺ T cells in the tumor microenvironment (Fig. 5A-C). At the same time, PMN-MDSC inhibited the expression of many important genes related to immune suppression, such as S100a8, S100a9, Cxcr2, Cxcl2, Cxcl3, etc.; while the expression of many important genes related to T cell killing function increased significantly, such as Gzma, Gzmb, Ifng , Prf1 and Klrk1, etc.; T cell recruitment-related genes were also significantly increased, such as Cxcl10, Cxcl9, Ccl5 and H2-Ab1, etc. (Fig. 5D).

The above results proved that CD300LD knockout led to a significant decrease in the immunosuppressive cell population PMN-MDSC in the tumor microenvironment, while a significant increase in immune effector T cells, indicating that CD300LD knockout significantly improved the immunosuppressive microenvironment in the tumor.

Example 5 CD300LD Knockout Inhibits the Migration and Function of PMN-MDSC

Since CD300LD knockout inhibits the development of tumors by regulating PMN-MDSC, and the PMN-MDSC in the tumor microenvironment after knockout decreases sharply, therefore, this example further verifies whether CD300LD knockout affects the migration process of PMN-MDSC. At the same time, as an immunosuppressive cell, PMN-MDSC can significantly inhibit the activity of T cells. Therefore, this embodiment will also analyze whether CD300LD knockout affects the immunosuppressive function of PMN-MDSC.

Materials and Methods:

5-1) RNAseq: Separation of spleens from WT and CD300LD-KO B16 tumor-bearing mice, preparation of single cell suspension and antibody labeling, flow cytometry sorting of PMN-MDSC cells (CD45+/CD11b+/Ly6G+), extraction of total cell RNA, reverse-transcribed into cDNA for transcriptome sequencing and analysis.

5-2) PMN-MDSC in vitro cell migration assay (Transwell): the spleen of tumor-bearing mice was isolated to prepare a single cell suspension, which was labeled with antibodies, and PMN-MDSC cells (CD45+/CD11b+/Ly6G+) were sorted by flow cytometry. Using Transwell wells, add 10 ⁵ PMN-MDSC cells to the upper layer and culture in a 37°C incubator for 2 hours, analyze the number of PMN-MDSC cells in the lower layer by flow cytometry, and calculate the migration ratio of the cells.

5-3) PMN-MDSC in vivo cell migration experiment: Spleen of B16 tumor-bearing mice was isolated to prepare a single cell suspension and labeled with antibodies, and PMN-MDSC cells (CD45+/CD11b+/Ly6G+) were sorted by flow cytometry. Using CTV staining, WT PMN-MDSCs and CD300LD-KO PMN-MDSCs were stained for high and low CTV markers, respectively. Mix WT/KO PMN-MDSC at a ratio of 1:1, inject intravenously into tumor-bearing WT mice, isolate the mouse bone marrow, spleen and tumor after 24 hours, and analyze the ratio of WT and CD300LD-KO PMN-MDSC in it by flow cytometry .

5-4) T cell proliferation experiment: isolate the spleen of OT-1 mice, prepare a single cell suspension and suspend it in PBS (5×10 ⁷ cells/ml), add CTV dye at a final concentration of 5 μM, and incubate in a water bath at 37°C for 20 Minutes, add 5 times the volume of ice-cold RPMI medium (RPMI-1640+10% FBS), invert and mix well, place at room temperature for 5 minutes, centrifuge at 400g for 5 minutes, and resuspend the cells in RPMI medium (1×10 ⁶ cells/ml). Take 80 μl of CTV labeled cells, add 40 μl of OVA peptide with a concentration of 100ng/ml, add 80ul of RPMI medium, add to the wells of a 96-well U-bottom plate, culture in a carbon dioxide incubator at 37°C, and carry out antibody labeling and flow after 48-72 hours formula analysis

5-5) Experiment of PMN-MDSC inhibiting T cell proliferation: Isolate the spleen of B16 tumor-bearing mice to prepare a single cell suspension and perform antibody labeling, and flow sorting to obtain PMN-MDSC cells (CD45 ^+/ CD11b ⁺ /Lying6G ⁺ ) , resuspended in RPMI medium (1×10 ⁶ cells/ml). Take 80 μl of CTV-labeled cells in 8-1), add 40ul of OVA peptide with a concentration of 100ng/ml, and PMN-MDSC cells, medium, into the wells of a 96-well U-bottom plate, culture in a 37°C carbon dioxide incubator, 48- Antibody labeling and flow analysis were performed 72 hr later. During the above T cell proliferation experiments, PMN-MDSC cells were added in different proportions, and antibody labeling and flow analysis were performed 48-72 hours later

result:

In this example, RNAseq analysis was performed on CD300LD WT and KO PMN-MDSCs, and the results showed that after CD300LD knockout, neutrophil migration-related signaling pathways were significantly down-regulated (Figure 6A and B). In vitro cell migration experiments demonstrated that the migration ability of CD300LD-KO PMN-MDSCs was significantly lower than that of WT PMN-MDSCs (Fig. 6C). CD300LD WT and KO PMN-MDSCs were mixed at a ratio of 1:1 and injected into tumor-bearing mice for migration competition experiments, and the results showed that the recruitment of WT PMN-MDSCs in tumors was significantly higher than that of CD300LD-KO PMN-MDSCs (Figure 6D-F ). The above results indicated that CD300LD knockdown inhibited the migratory ability of PMN-MDSCs, leading to a significant reduction in tumor recruitment of PMN-MDSCs.

The effect of CD300LD on PMN-MDSC function was further analyzed. The results of T cell proliferation experiments showed that WT PMN-MDSC could efficiently inhibit T cell proliferation, which was significantly decreased in CD300LD-KO PMN-MDSC (Figure 6G and H), indicating that the T cell inhibitory function of PMN-MDSC was affected by CD300LD Positive regulation. The above results indicated that CD300LD knockout also suppressed the T cell suppressive function of PMN-MDSCs.

Example 6 CD300LD regulates the migration and function of PMN-MDSC through S100A8/A9

In order to study the molecular mechanism of how CD300LD regulates PMN-MDSC, this example performs transcriptome sequencing on WT and KO PMN-MDSC, and analyzes the significantly different genes in order to study which downstream gene CD300LD regulates the cell migration and function of PMN-MDSC .

Materials and Methods:

6-1) PMN-MDSC in vitro cell migration assay (Transwell): Spleen of B16 tumor-bearing mice was isolated to prepare a single cell suspension and labeled with antibodies, and PMN-MDSC cells (CD45+/CD11b+/Lying6G+) were sorted by flow cytometry. Using Transwell wells, add 10 ⁵ PMN-MDSC cells to the upper layer and place them in a 37°C incubator for culture. Add 1 μg/ml final concentration of S100A8/A9 inhibitor (Tasquinimod) or DMSO control to the culture medium; 2 hours later flow analysis The number of PMN-MDSC cells in the lower layer was used to calculate the migration ratio of the cells.

6-2) Tumor model: B16 cells (1.5×10 ⁵ cells/mouse) were inoculated subcutaneously into mice, and 20 μg Tasquinimod/mouse were injected intraperitoneally on day 2, and the tumor size was measured every 2-3 days, Draw tumor growth curves. On the 19th day, the mouse tumors were taken, and the proportion of immune cells was analyzed for loss.

result:

To further explore the molecular mechanism by which CD300LD regulates PMN-MDSCs, PMN-MDSCs from WT and KO mice were isolated for RNA-seq differential analysis, in which S100A8/A9 was the most significantly down-regulated gene in KO PMN-MDSCs (Fig. 7A), and Reported to be functionally related to PMN-MDSC. The level of S100A8/A9 in serum of WT tumor-bearing mice was significantly higher than that of normal mice, while the level of S100A8/A9 in KO tumor-bearing mice was not significantly different from that of normal mice (Fig. 7B). Meanwhile, the expression levels of S100A8 and S100A9 in CD300LD-KO PMN-MDSCs were also significantly lower than those in WT PMN-MDSCs (Fig. 7C). Migration experiments in vitro proved that S100A8/A9 proteins could compensate for the weakened migration ability of KO PMN-MDSCs (Fig. 7D). T cell inhibition experiments showed that S100A8/A9 inhibitors could significantly reduce the inhibitory effect of WT PMN-MDSCs, but had no significant effect on KO PMN-MDSCs (Fig. 7E). Further tests were performed using tumor models, and the results showed that in vivo injection of the S100A8/A9 inhibitor Tasquinimod could inhibit tumor growth in WT mice, but could not further inhibit tumor growth in KO mice, and the ratio of intratumoral PMN-MDSC was also consistent with tumor changes ( Figure 7F and G). The above results prove that CD300LD regulates the migration and function of PMN-MDSCs through S100A8/A9, and S100A8/A9 is an important effector molecule downstream of CD300LD.

Example 7 CD300LD knockout and PD1 antibody have significant anti-tumor synergistic effect

Since CD300LD is mainly expressed on myeloid cells PMN-MDSC, while PD1 is mainly expressed on T cells, theoretically these two targets will have a synergistic effect on anti-tumor effects. Therefore, this example analyzes the anti-tumor effect of CD300LD knockout combined with PD1 antibody.

Materials and Methods:

7-1) Expression analysis of PD1 and its ligand PD-L1: Isolate B16 mouse tumors to prepare single cell suspension, stain with PD1 antibody and PD-L1 antibody, and analyze PD1 and PD on the surface of tumor cells and immune cells by flow cytometry - Expression of L1.

7-2) Tumor model: B16-F10 cells (according to 1.5×10 ⁵ cells/mouse) were inoculated subcutaneously into WT and CD300LD KO mice, and on the 7th day after inoculation, PD1 antibody was injected intravenously, 200 μg/mouse, 3 times in total. The size of the tumor was measured every 2-3 days, and the tumor growth curve was drawn.

result:

The results of flow cytometry showed that compared with WT mice, the proportion of PD1 ⁺ CD8 ⁺ T cells in tumors of KO mice was increased, and the expression ratio of PD-L1 in tumor cells and PMN-MDSC cells was also increased (Fig. 8A) , further suggesting that this target will have a synergistic effect with PD1. We tested the effect of PD1 antibody in the B16-F10 tumor model of WT and CD300LD-KO mice. The results showed that the treatment of WT mice with PD-1 antibody alone had the same effect as CD300LD KO, and both significantly inhibited the development of tumors. In the context of CD300LD KO, PD-1 antibody could further inhibit tumor development and prolong the survival time of mice (Fig. 8B and C). The above results prove that CD300LD knockout and PD-1 immune checkpoint have obvious anti-tumor synergistic effects.

Example 8 CD300LD knockout does not affect the normal development of mice

In order to explore the systemic risks that may be caused by targeting CD300LD, this example analyzes the impact of CD300LD knockout on body development, and conducts a preliminary assessment of the safety of the CD300LD target.

Materials and Methods:

8-1) Immune cell population analysis: separate mouse bone marrow, spleen and peripheral blood, prepare single cell suspension, and use flow cytometry antibody to analyze the proportion of different immune cell populations, including T cells (CD3 ⁺ ), B cells ( B220 ⁺ ), myeloid cells (CD11b(Mac1) ⁺ ), granulocytes (CD11b ⁺ Gr1 ⁺ ); LSK hematopoietic precursor cells (Lin ^- Sca1 ⁺ cKit ⁺ ), LT-HSC (Lin ^- Sca1 ⁺ cKit ⁺ CD34 ^- Flt3 ^- ), MMP (Lin ^- Sca1 ⁺ cKit ⁺ CD34 ⁺ Flt3 ⁺ ), GMP (Lin ^- cKit ⁺ Sca1 ^- CD34 ^High FcyR ^High ), CMP (Lin ^- cKit ⁺ Sca1 ^- CD34 ^High FcyR ^Mid ), MEP (Lin ^- cKit ⁺ Sca1 ^- CD34 ^Low FcyR ^Low ), CLP (Lin ^- cKit ^Mid Sca1 ^Mid Flt3 ^High IL7R ^High ). A single cell suspension was prepared from the tumor, stained with PD1 antibody and PD-L1 antibody, and the expression of PD1 and PD-L1 on the surface of tumor cells and immune cells was analyzed by flow cytometry.

8-2) CFU experiment: Isolate mouse bone marrow, prepare single cell suspension, take 10 ⁵ cells and culture them in cellulose semi-solid medium (MethoCult GF M3434, Stem Cell Company), after 7 days of culture, BFU- E. CFU-GM and CFU-GEMM clones were counted; or bone marrow cells were cultured in cellulose semi-solid medium (MethoCult GFM3630, Stem Cell Company), and after 7 days of culture, Pre-B clones were counted under a microscope.

result:

CD300LD KO and WT mice grew and developed normally without visible behavioral abnormalities. The results of flow cytometry analysis showed that there was no significant difference in the proportions of T cells, B cells and myeloid cells in the immune and hematopoietic organs of KO and WT mice (Figure 9A); long-term and short-term hematopoietic stem cells (LT-HSC, ST-HSC ), multipotent hematopoietic progenitor cells (MPP), and other precursor cells, including myeloid precursor cells CMP, GMP, MEP, and lymphoid precursor cells CLP (Fig. 9B, C). Further analysis of the clonogenic ability of hematopoietic stem cells in KO and WT mice showed that there was no significant difference between them ( FIG. 9D ). The above results show that the knockout of CD300LD does not affect the normal development of mice and the development of the hematopoietic/immune system, and targeting CD300LD will have certain safety.

Example 9 CD300LD Knockout Inhibits the Development of Established Tumors

This example further analyzes whether the induced knockout of CD300LD can inhibit the development of tumors in the case of established tumors.

Materials and Methods:

Induced CD300LD knockout tumor model: Using CD300LD ^fl/fl Mx1CRE mice, B16 cells (according to 1.5×10 ⁵ cells/mouse) were inoculated subcutaneously into mice, and on the 7th day after inoculation, PolyIC was injected to induce gene knockout , once every 2 days, a total of 4 times. The size of the tumor was measured every 2-3 days, and the tumor growth curve was drawn.

result:

CD300LD ^fl/fl Mx1CRE mice can be bred by CD300LD ^fl/fl mice and Mx1CRE mice, which can knock out CD300LD under the induction of PolyIC drugs. The mice were inoculated with B16-F10 cells for subcutaneous tumor formation, and PolyIC treatment was performed when the tumor grew to ~100 mm ³ . The results showed that induced knockout of CD300LD significantly inhibited the development of established tumors (Fig. 9A), while there was no difference in tumor growth in the same two groups of mice without administration (Fig. 9B). The above results indicated that inducing knockout of CD300LD could significantly inhibit the development of tumors at the established stage of tumors, which further indicated that CD300LD could be used as an effective target for tumor immunotherapy.

The above examples are intended to illustrate the disclosed embodiments of the present invention, and should not be construed as limiting the present invention. In addition, various modifications set forth herein, as well as changes in the method of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in connection with various specific preferred embodiments of the invention, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as mentioned above which are obvious to those skilled in the art to obtain the invention should be included in the scope of the present invention.

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<110> Fudan University

Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences

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Claims (11)

1. Use of a cd300ld gene as a target in the preparation of a medicament for tumor prevention, diagnosis or treatment.
2. Use of a cd300ld gene and PD1 together as targets in the manufacture of a medicament for tumor prevention, diagnosis or treatment.
3. Use of a cd300ld inhibitor in the manufacture of a product having at least one of the following effects:

   1) Preventing, diagnosing or treating a tumor;

   2) Modulating the activity and/or migration of myeloid lineage cells;

   3) Modulating T cell activity;

   4) Downstream signal paths of CD300LD are regulated.
4. 4. The use according to claim 3, wherein the target of the CD300LD inhibitor is CD300LDExon2 and/or the CD300LD inhibitor is selected from a nucleic acid molecule, a protein molecule or a small molecule compound.
5. 5. The use according to claim 3, wherein the tumor is a CD300LD overexpressing tumor; preferably, the tumor is selected from the group consisting of bowel cancer, lung cancer, liver cancer, breast cancer, esophageal cancer, head and neck cancer, skin cancer, kidney cancer, leukemia, cervical cancer, colon cancer, hepatocellular carcinoma, ovarian serous cyst adenocarcinoma, endometrial cancer, thyroid cancer, skin melanoma, lung adenocarcinoma, head and neck squamous cell carcinoma, glioblastoma multiforme, prostate cancer, thymus cancer, brain low-grade glioma, rectal adenocarcinoma, pheochromocytoma and paraganglioma, esophageal cancer, renal clear cell carcinoma, bladder urothelial carcinoma, renal papillary cell carcinoma, pancreatic cancer, gastric cancer, renal chromophobe carcinoma, breast infiltration carcinoma, lung squamous carcinoma, sarcoma, or acute myeloid leukemia.
6. 6. The use according to claim 3, wherein the myeloid cells are selected from macrophages, M-MDSCs or PMN-MDSCs.
7. 7. The use according to claim 3, wherein said modulating the activity of cells of the myeloid lineage is selected from reducing the cell number of cells of the myeloid lineage or lowering the expression of genes on cells of the myeloid lineage that suppress immunity.
8. 8. The use according to claim 3, wherein modulating T cell activity is selected from any one or more of the following: increasing CD4 ⁺ And CD8 ⁺ Cell number of T cells; reducing the number of cells of tregs; increasing the gene expression related to the T cell killing function; resulting in increased expression of the gene associated with T cell recruitment.
9. 9. The use of claim 3, wherein modulating the downstream signaling pathway of CD300LD comprises modulating the S100A8/A9 gene in the downstream signaling pathway of CD300 LD.
10. 10. A composition comprising a CD300LD inhibitor therein for any one or more of the following uses:

    1) Preventing, diagnosing and treating tumors;

    2) Modulating the activity and/or migration of myeloid lineage cells;

    3) Modulating T cell activity;

    4) Downstream signal paths of CD300LD are regulated.
11. 11. An in vitro drug screening method, comprising the steps of: after mixing the cells over-expressing CD300LD with the drug to be screened, the change of the cells is detected.

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