Liquid biopsy markers, treatment targets and their applications for clear cell renal cell carcinoma

Abstract

The present invention discloses a liquid biopsy marker and a therapeutic target for clear cell renal cell carcinoma and their applications, belonging to the field of clinical diagnosis and treatment. Through bioinformatics analysis, it is found that the mRNA of PD-L2 in clear cell renal cell carcinoma is highly expressed after carcinogenesis, and is higher than the expression level of the mRNA of PD-L1, and is also related to poor staging. Further immunohistochemistry is used to verify the expression and localization of PD-L2 in clear cell renal cell carcinoma and adjacent tissues, and it is found that PD-L2 is highly expressed in the intercellular space after carcinogenesis of clear cell renal cell carcinoma, indicating an increase in its secretion after carcinogenesis. This application reveals the role and mechanism of exosomal PD-L2 in the development of clear cell renal cell carcinoma with or without immunity, indicates its potential as a liquid biopsy detection index for predicting the prognosis of clear cell renal cell carcinoma, and provides a theoretical basis for exosomal PD-L2 to become a therapeutic target for clear cell renal cell carcinoma.

Description

Technical Field

[0001] The present invention relates to the field of clinical diagnosis and treatment, and specifically relates to liquid biopsy markers and treatment targets for clear cell renal carcinoma and their applications. Background Art

[0002] Renal cancer is a general term for malignant tumors originating from the renal system. Among the cancers diagnosed and causing death worldwide every year, renal cancer accounts for approximately 2-3%. The male-to-female ratio is 2:1, indicating a certain gender predisposition. Approximately 90% of renal cancers originate from renal epithelial cells, that is, renal cell carcinoma, which can be further divided into more than 10 subtypes according to tissue and molecular characteristics. Among them, clear cell renal carcinoma accounts for about 85% of renal cell carcinoma and is a common subtype causing death in renal cancer patients.

[0003] Cancer Genome Project research found that among 18 different types of epithelial cancers, clear cell renal carcinoma has the highest angiogenesis score and the highest immune cell infiltration rate, which provides a theoretical basis and application prospect for the clinical application of immune checkpoint blocker antibodies. At the same time, the combination of immune checkpoint blockers and tyrosine kinase inhibitors as the first-line treatment method for patients with metastatic renal cancer has been applied to a certain extent in the clinical treatment of metastatic renal cancer.

[0004] PD-1 is programmed death receptor 1, an important immunosuppressive molecule and a member of the CD28 superfamily. PD-1 belongs to type I transmembrane protein and consists of an extracellular domain, a transmembrane anchoring region, and an intracellular signal transduction region. It is mainly induced and expressed on the surface of activated T lymphocytes and B lymphocytes. The ligands of PD-1 are B7-H1 (PD-L1) and B7-DC (PD-L2). After their binding, they inhibit the proliferation and activation of T cells and play a negative regulatory role in T cell responses, belonging to inhibitory receptors. However, preliminary clinical studies found that the response rate of clear cell renal carcinoma to the PD-1 antibody Nivolumab is only about 25%, and patients did not show tumor shrinkage. At the same time, the expression of PD-L1 has no correlation with the response rate. This indicates that our research on renal cancer immunotherapy has just started, and there is an urgent need to find more effective biomarkers for immune response and solve problems such as how to convert non-responsive patients into responsive ones. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention proposes liquid biopsy markers and treatment targets for clear cell renal carcinoma and their applications.

[0006] The object of the present invention can be achieved by the following technical solutions:

[0007] A tumor detection kit, comprising a reagent for detecting the content of PD-L2.

[0008] An anti-tumor drug, comprising:

[0009] A component that can inhibit the production of PD-L2 or reduce the content of PD-L2, which is used for the treatment of patients except those with primary or secondary severe immunodeficiency of T and B lymphocytes; or

[0010] A component that can promote the production of PD-L2 or increase the content of PD-L2, which is used for the treatment of patients with severe immunodeficiency of T and B lymphocytes.

[0011] Use of an inhibitor of PD-L2 in the preparation of an anti-tumor drug for patients except those with primary or secondary severe immunodeficiency of T and B lymphocytes.

[0012] Use of a promoter of PD-L2 in the preparation of an anti-tumor drug for patients with severe immunodeficiency of T and B lymphocytes.

[0013] Advantages of the present invention:

[0014] 1. Based on the importance of immune checkpoint therapy and tumor microenvironment in the treatment of clear cell renal cell carcinoma, the present invention explores the existence form and proportion of PD-L2 in clear cell renal cell carcinoma, and confirms the existence of exosomal PD-L2 in clear cell renal cell carcinoma, providing a new index for liquid biopsy in the prognostic diagnosis of clear cell renal cell carcinoma.

[0015] 2. The biomarker of the present invention uses a variety of technical means to reveal the influence and mechanism of exosomal PD-L2 on the immune microenvironment of clear cell renal cell carcinoma from the levels of molecules, cells, animals and bioinformatics, providing a theoretical basis for the progression of clear cell renal cell carcinoma and ultimately providing a therapeutic target for the treatment of clear cell renal cell carcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The present invention will be further described below with reference to the accompanying drawings.

[0017] Figure 1 are the expression levels of mRNA and protein of PD-L1 and PD-L2 in TCGA and HPA databases;

[0018] Figure 2 is the relationship between PD-L2 and the stage of clear cell renal cell carcinoma in TCGA database;

[0019] Figure 3 is the PD-L2 immunohistochemistry of clear cell renal cell carcinoma tissue chip;

[0020] Figure 4 is the content of PD-L2 in each component outside clear cell renal cell carcinoma cells;

[0021] Figure 5 is the influence of knocking out the key protein Rab27a in the exosome release process on intracellular PD-L2 and exosome marker CD63;

[0022] Figure 6 It is the quality control of exosome mass spectrometry, and the analysis results are verified by western blot of PD-L2;

[0023] Figure 7 It is the content of PD-L2 in the supernatant of primary cells and tumor tissue fluid exosomes of patients with clear cell renal cell carcinoma;

[0024] Figure 8 It is the effect of PD-L2 on the proliferation and metastasis of clear cell renal cell carcinoma at the in vitro cell level;

[0025] Figure 9 It is the effect of exosomal PD-L2 on the proliferation and metastasis of clear cell renal cell carcinoma in vitro;

[0026] Figure 10 It is the effect of exosomal PD-L2 on the proliferation and metastasis of clear cell renal cell carcinoma in immunodeficient mice;

[0027] Figure 11 It is the effect of exosomal PD-L2 on the proliferation of T lymphocytes;

[0028] Figure 12 It is the effect of exosomal PD-L2 on the production of inflammatory cytokines in T lymphocytes;

[0029] Figure 13 It is the effect of exosomal PD-L2 on the apoptosis of T lymphocytes;

[0030] Figure 14 It is the effect of exosomal PD-L2 on the CD4+ and CD8+ subtyping of T lymphocytes;

[0031] Figure 15 It is the effect of exosomal PD-L2 on the proportion of cytotoxic T lymphocytes in T lymphocytes;

[0032] Figure 16 It is the effect of exosomal PD-L2 on the killing ability of T lymphocytes against clear cell renal cell carcinoma cells;

[0033] Figure 17 It is the binding situation between exosomal PD-L2 and PD-1 on the surface of T lymphocytes;

[0034] Figure 18 It is the effect of exosomal PD-L2 on the proliferation and metastasis of clear cell renal cell carcinoma in immunocompetent mice;

[0035] Figure 19 It is the effect of exosomal PD-L2 on the proportion of Treg and CTL in TIL and spleen in immunocompetent mice;

[0036] Figure 20The effects of PD-L2 antibody treatment on the proliferation and metastasis of clear cell renal cell carcinoma in immunocompetent mice;

[0037] Figure 21 The effects of PD-L2 antibody treatment on the ratios of TIL to Treg and CTL in the spleen in immunocompetent mice. Specific implementation manners

[0038] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0039] A clear cell renal cell carcinoma marker, treatment target and its application. The marker and treatment target are targeted at exosomal PD-L2. Based on the changes in expression level and expression position leading to functional changes, relying on technical means such as immunohistochemistry, tissue immunofluorescence, exosome isolation and purification, exosome mass spectrometry sequencing, flow cytometry, ELISA, and bioinformatics, it reveals the molecular basis of exosomal PD-L2 participating in the remodeling of the immune microenvironment of clear cell renal cell carcinoma and then promoting the metastasis of clear cell renal cell carcinoma from the molecular, cellular, animal, clear cell renal cell carcinoma clinical patient, and bioinformatics levels, and finally provides new liquid biopsy indicators and treatment targets for the treatment of clear cell renal cell carcinoma.

[0040] 1) Construct a PD-L2 knockout cell line:

[0041] Design 2-3 sgRNAs targeting the PD-L2 gene, construct them into a co-expression vector of sgRNA and CRISPR-Cas9 protein, use electroporation technology to transfect the 786-0 cell line with the expression vector, screen the successfully transfected positive cells, extract genomic DNA, perform PCR on the first exon, sequence the product, compare the PD-L2 gene fragment, detect whether there are deletion mutations, and verify the knockout success by western blot;

[0042] 2) Construct PD-L2 overexpression and knockdown cell lines:

[0043] Design primers targeting the full-length coding region of the PD-L2 gene, amplify the full-length PD-L2 gene by PCR, construct a PD-L2 overexpression vector, package the corresponding lentivirus through HEK293T cells, infect renal clear cell carcinoma 786-0 cells and ACHN cells with the overexpressed lentivirus, design shRNA against the PD-L2 gene, and also knock down PD-L2 in renal clear cell carcinoma 786-0 cells and ACHN cells by means of lentivirus packaging and infection;

[0044] 1. Expression of PD-L2 in renal clear cell carcinoma:

[0045] 1) Expression and clinicopathological bioinformatics analysis of PD-L2 in renal clear cell carcinoma

[0046] As Figure 1 shown, by analyzing the TCGA and HPA databases, it was found that the mRNA expression level of PD-L2 in renal clear cell carcinoma was higher than that in adjacent tissues, and significantly higher than the expression level of PD-L1 mRNA. Moreover, the protein level of PD-L2 could be detected in renal clear cell carcinoma, indicating a basis for its role in renal clear cell carcinoma. Further analysis of the relationship between PD-L2 and the stage of renal clear cell carcinoma patients in TCGA, as Figure 2 shown, the mRNA level of PD-L2 was higher in the group with unfavorable staging of renal clear cell carcinoma, indicating that PD-L2 plays a pro-cancer role in renal clear cell carcinoma.

[0047] 2) Immunohistochemical detection of PD-L2 expression and distribution in renal clear cell carcinoma and adjacent tissues

[0048] To verify the expression changes of PD-L2 in renal clear cell carcinoma, immunohistochemical staining of PD-L2 in cancer and adjacent tissues of 165 pairs of renal clear cell carcinoma patients was performed. As Figure 3 shown, it was found that the distribution of PD-L2 changed after carcinogenesis in renal clear cell carcinoma: from mainly expressing in the cytoplasm and cell membrane to highly expressing in the intercellular space of tumor cells, indicating that it is secreted extracellularly to play a role.

[0049] 3) Existence form and content of PD-L2 outside renal clear cell carcinoma cells

[0050] To explore the existence form of PD-L2 outside renal clear cell carcinoma cells, the 786-0 renal clear cell carcinoma cell line was used as a model for research. The free proteins, microvesicles and exosomes in the culture supernatant were separated by ultrafiltration combined with sucrose density gradient centrifugation, and the content of PD-L2 in each component was detected by ELISA. As Figure 4 shown, it was found that PD-L2 was expressed in each component, but mainly in the form of exosomes.

[0051] 4) Verification of the presence of exosomal PD-L2 in clear cell renal cell carcinoma

[0052] By detecting the change in the protein content of PD-L2 in the cell line after knocking down the key gene Rab27a in the exosome secretion process, as Figure 5 shown, when the release of exosomes into the extracellular space is inhibited, the proteins of exosome markers CD63 and PD-L2 accumulate intracellularly, and the co-localization of CD63 and PD-L2 proteins intracellularly increases, indicating that PD-L2 can be released extracellularly in the form of exosomes. As Figure 6 shown, the presence of exosomal PD-L2 was found in the exosome proteomics of the in situ clear cell renal carcinoma cell line 786-0 and the highly metastatic clear cell carcinoma cell line ACHN, and PD-L2 was highly expressed in the exosomes of ACHN. The results of exosome proteomics were verified by western blot experiments, and the expression of PD-L2 protein in the exosomes of the purified 786-0 and ACHN cell lines was detected, which was the same as the results of mass spectrometry sequencing, and the expression level of exosomal PD-L2 was high in highly metastatic clear cell renal carcinoma. Subsequently, the clinical samples of patients were used to test exosomal PD-L2, and ELISA was used to detect the protein expression of PD-L2 in the exosomes extracted from the primary cell culture supernatant and tumor tissue fluid of clear cell renal carcinoma patients, as Figure 7 shown, exosomal PD-L2 could be detected in the primary cell culture supernatant and tumor tissue fluid of patients, and there were individual differences among patients, with the potential to be used as a detection marker. In summary, the above experiments confirmed the real existence of exosomal PD-L2 in clear cell renal cell lines and patients, and it was highly expressed in highly metastatic cell lines.

[0053] 2. Exosomal PD-L2 inhibits immunodeficient clear cell renal cell carcinoma:

[0054] 1) Effects of exosomal PD-L2 on clear cell renal cell carcinoma at the in vitro cell level

[0055] First, we evaluated the effects of PD-L2 on proliferation and metastasis in vitro using CTG and Transwell, as Figure 8 shown, PD-L2 at the cell level inhibits the proliferation and metastasis of renal cancer cells. Subsequently, we extracted exosomes from the PD-L2 knockout, PD-L2 overexpression, and empty control groups, and after purification, NTA was used to count the exosomes. 786-0 cells were stained with CFSE, and then 10 11 exosomes were added. After 96 hours, the cells were analyzed by flow cytometry to analyze the numerical change in the relative fluorescence intensity of FITC. The smaller the relative fluorescence intensity, the faster the cell proliferation. As Figure 9 shown, exosomal PD-L2 inhibits the proliferation of the clear cell renal carcinoma cell line 786-0. The same number of 786-0 cells were seeded into non-adherent 6-well plates, and 1011 Exosomes were used to feed the cells. After 48 hours, the cells were counted and seeded into 24-well Transwell plates to observe the effect of exosomal PD-L2 on the metastasis of 786-0 cells. Similar to the effect of PD-L2 at the cellular level, exosomal PD-L2 also inhibited the metastasis of tumor cells ( Figure 9 ).

[0056] 1) Effect of exosomal PD-L2 on immunodeficient clear cell renal carcinoma in vitro and in vivo

[0057] PD-L2 is relatively conserved in mice. Therefore, we established an orthotopic tissue model of renal cancer in severely immunodeficient SCID-BALB / c mice. On the 7th and 14th days after modeling, 10 12 exosomes extracted from Renca cells overexpressing PD-L2 were injected into the tail veins of each mouse to study the function of exosomal PD-L2. The mice were sacrificed by cervical dislocation 21 days after modeling. The mice were dissected to observe the growth of the orthotopic tumors and the metastasis to various organs. The weight of the left kidney with tumor was subtracted from the weight of the right kidney, and the difference was recorded as the weight of the orthotopic tumor of the mouse. The weights of the orthotopic tumors in the two groups were statistically analyzed. As Figure 10 shown, in the group of mice injected with exosomes overexpressing PD-L2 in immunodeficient mice, the growth of the orthotopic tumors slowed down, indicating that exosomal PD-L2 inhibited the growth of tumor cells. After dissecting and separating the organs of the mice, obvious metastatic foci were only observed in the lungs macroscopically. The lungs, liver, spleen, and the right kidney without tumor injection were paraffin-embedded and examined for metastatic foci by HE staining. No metastatic foci were observed in other organs except the lungs. The tumors in the lungs were weighed and the number of metastatic foci in the lung HE-stained sections was counted. The results showed that the metastasis in the group injected with exosomes overexpressing PD-L2 was reduced, indicating that exosomal PD-L2 inhibited the metastasis of tumors in severely immunodeficient mice. In summary, these experiments confirmed in vivo and in vitro that exosomal PD-L2 inhibited the proliferation and metastasis of renal cancer itself without the participation of the immune system.

[0058] 1. Exosomal PD-L2 inhibits the activity and function of T lymphocytes through PD-1:

[0059] 1) Exosomal PD-L2 inhibits the activity and function of T lymphocytes

[0060] T lymphocytes were fed with exosomes with different PD-L2 expression levels, and the proliferation of lymphocytes, the production of inflammatory cytokines IL-2 and IFN-γ, and the apoptosis of T lymphocytes were detected respectively. Exosomal PD-L2 could inhibit the proliferation of T lymphocytes ( Figure 11 ), inhibit the production of activation cytokines IL-2 and IFN-γ ( Figure 12 ), and promote the apoptosis of T lymphocytes ( Figure 13)。How does exosomal PD-L2 affect the subtype ratio of lymphocytes? The basic CD4+ and CD8+ T cells were distinguished by flow cytometry staining, as Figure 14 shown. We found that compared with the control group of empty exosomes, after feeding exosomes overexpressing PD-L2, the proportion of CD4+ cells increased. The proportion of effector T cells was also evaluated, as Figure 15 shown. The proportion of effector T cells decreased after feeding PD-L2 OE exosomes. Considering the multiple inhibitory effects of exosomal PD-L2 on lymphocytes, it led to a weakened killing ability of lymphocytes in the group fed with exosomes overexpressing PD-L2 against ccRCC cells ( Figure 16 ).

[0061] 2) Exosomal PD-L2 exerts its function by binding to PD-1 on the surface of T lymphocytes

[0062] As Figure 17 shown, exosomes carrying PD-L2-GFP were extracted from 786-0 cells overexpressing PD-L2-GFP, and these exosomes were used to feed T lymphocytes, Jurkat cells. After 48 hours of feeding, Jurkat cells with PD-1 knockout were fixed, and the proportion of GFP+ cells in immune cells was analyzed by flow cytometry. It was found that exosomal PD-L2-GFP could bind to T lymphocytes and Jurkat cells, while exosomal PD-L2-GFP hardly bound to Jurkat cells with PD-1 knockout. This indicates that exosomal PD-L2 exerts its function by binding to PD-1 on the surface of T lymphocytes.

[0063] 2. Exosomal PD-L2 promotes the proliferation and metastasis of immunocompetent clear cell renal cell carcinoma:

[0064] 1) Exosomal PD-L2 remodels the inhibitory immune microenvironment to promote the proliferation and metastasis of immunocompetent clear cell renal cell carcinoma

[0065] Similar to the experimental method for immunodeficient mice described above, the difference is that we replaced SCID mice with immunocompetent healthy BALB / c mice for the experiment. Completely different phenotypes were generated in immunocompetent mice. The weight of the orthotopic tumor and the number of metastatic sites in the lungs and spleens of mice injected with exosomes overexpressing PD-L2 also increased ( Figure 18 ). We further performed flow cytometry detection of tumor-infiltrating T cells and immune cells in the spleen according to surface markers, as Figure 19 shown. It was found that exosomal PD-L2 could inhibit the proportion of cytotoxic T cells and promote the proportion of inhibitory regulatory T cells, comprehensively playing a role in inhibiting the tumor in situ and peripheral secondary immune organs, helping clear cell renal cell carcinoma achieve immune escape and playing a carcinogenic role.

[0066] 2) PD-L2 antibody treatment can rescue the pro-cancer effect of exosomal PD-L2 in clear cell renal cell carcinoma

[0067] To verify whether exosomal PD-L2 can be a therapeutic target for clear cell renal cell carcinoma, we orthotopically injected Renca cells with different PD-L2 expression levels into the left kidney of mice, and starting from the third day after modeling, injected mouse PD-L2 antibody / IgG into the tail vein once every 3 days. We still took the orthotopic tumor, lung, spleen, and liver for observation and experiments. We found that as Figure 20 shown, the volume of the orthotopic tumor in the PD-L2 overexpression group also increased, and PD-L2 antibody treatment could rescue the impact of PD-L2 overexpression on the orthotopic tumor. Similar situations also existed in the lung metastases. However, perhaps due to slightly insufficient amount of PD-L2 antibody, the number of metastases could not be completely rescued, and the group treated with PD-L2 antibody had not yet returned to the same level as the empty vector control group. In tumor-infiltrating T lymphocytes and the spleen, similar to the previous research results, the PD-L2 overexpression group would increase the content of regulatory T cells, while antibody treatment would rescue the phenomenon of elevated regulatory T cells( Figure 21 ).

[0068] In the description of this specification, the descriptions referring to terms such as "one embodiment", "example", "specific example", etc. mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples.

[0069] The above shows and describes the basic principles, main features, and advantages of the present invention. Those skilled in the art of this industry should understand that the present invention is not limited by the above embodiments. The above embodiments and the descriptions in the specification only illustrate the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed.

Claims

1. Use of an inhibitor of PD-L2 in the preparation of an anti-tumor drug for immunocompetent patients without primary or secondary severe immunodeficiency of T and B lymphocytes, characterized in that, The inhibitor of PD-L2 includes a PD-L2 antibody, and the tumor is clear cell renal carcinoma.

2. Use of a promoter of PD-L2 in the preparation of an anti-tumor drug for patients with severe immune deficiency of T and B lymphocytes, characterized in that, The promoter of PD-L2 includes overexpressed PD-L2 exosomes, and the tumor is clear cell renal carcinoma.

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