Small molecule polypeptide NEAT1-31 and its applications and products

Abstract

The present invention provides a small molecule polypeptide NEAT1-31, its applications and products, relating to the field of biotechnology. The small molecule polypeptide NEAT1-31 provided by the present invention has an amino acid sequence as shown in SEQ ID NO.1. Through research by the inventors, it has been found that Neat1-31 has the effect of promoting phagocytosis both in vivo and in vitro. And it has been confirmed that Neat1-31 and anti-CD47 antibody have a synergistic effect, and the two jointly inhibit tumor progression. This provides a new direction for tumor treatment and brings new hope to patients resistant to immune checkpoint blockade (ICB) therapy.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a small molecule polypeptide NEAT1-31 and its applications and products. Background Art

[0002] Advances in single-cell multi-omics have provided a deeper understanding of the complex interactions between different immune cell subsets and tumor cells in the tumor microenvironment, and the results have provided promising ideas for overcoming these problems. In addition to playing an important role in innate immunity, macrophages are also indispensably involved in adaptive immunity by phagocytosing and presenting antigens, which is a multi-step cellular process involving target cell recognition. Phagocytosis and lysosomal digestion are regulated by receptor-ligand interactions between target cells and phagocytes, which is called the "eat me" signal. Once the inherent "eat me" signal is activated by immunoreceptor tyrosine-based activation motifs (ITAMs), the cytoskeleton of phagocytes is remodeled for phagocytosis. In contrast, cancer cells evade immune clearance with the help of anti-phagocytic molecules, including CD47, CD24, PD-L1, MHC-I, STC-1, and GD2, which are called the "don't eat me" signals. Most phagocytosis-related ICB therapies focus on blocking the "don't eat me" signals, such as CD47 and PD-L1.

[0003] Although immune checkpoint blockade (ICB) therapies have achieved unprecedented success in suppressing cancer by enhancing T cell responses, drug resistance prevents most patients from benefiting from this therapy. Moreover, there are few reports on phagocytosis activators currently.

[0004] In view of this, the present invention is specifically proposed. Summary of the Invention

[0005] The first object of the present invention is to provide a small molecule polypeptide NEAT1-31, which has the function of improving the phagocytic ability of macrophages and treating cancer, so as to solve the above problems.

[0006] The second object of the present invention is to provide the application of the above small molecule polypeptide NEAT1-31 in improving the phagocytic ability of macrophages.

[0007] The third object of the present invention is to provide the application of the above small molecule polypeptide NEAT1-31 in the preparation of drugs for treating cancer.

[0008] The fourth object of the present invention is to provide the application of the above small molecule polypeptide NEAT1-31 combined with anti-CD47 antibody in the preparation of drugs for treating cancer.

[0009] The fifth object of the present invention is to provide a phagocyte activator.

[0010] The sixth object of the present invention is to provide a drug for treating cancer.

[0011] In order to achieve the above object, the following technical solutions are specifically adopted:

[0012] In the first aspect, the present invention provides a small molecule polypeptide NEAT1-31, and the amino acid sequence of the small molecule polypeptide NEAT1-31 is shown in SEQ ID NO.1.

[0013] In the second aspect, the present invention provides the application of the above small molecule polypeptide NEAT1-31 in improving the phagocytic ability of macrophages.

[0014] In the third aspect, the present invention provides the application of the above small molecule polypeptide NEAT1-31 in the preparation of a drug for treating cancer.

[0015] As a further technical solution, the cancer includes breast cancer, gastric cancer, colon cancer, low-grade glioma, glioblastoma, bladder urothelial carcinoma, liver cancer, lung cancer, pancreatic ductal adenocarcinoma and ovarian cancer.

[0016] In the fourth aspect, the present invention provides the application of the above small molecule polypeptide NEAT1-31 combined with an anti-CD47 antibody in the preparation of a drug for treating cancer.

[0017] As a further technical solution, the cancer includes breast cancer, gastric cancer, colon cancer, low-grade glioma, glioblastoma, bladder urothelial carcinoma, liver cancer, lung cancer, pancreatic ductal adenocarcinoma and ovarian cancer.

[0018] In the fifth aspect, the present invention provides a phagocyte activator, and the phagocyte activator includes the above small molecule polypeptide NEAT1-31.

[0019] In the sixth aspect, the present invention provides a drug for treating cancer, and the drug includes the above phagocyte activator.

[0020] As a further technical solution, the drug further includes an anti-CD47 antibody.

[0021] As a further technical solution, the cancer includes breast cancer, gastric cancer, colon cancer, low-grade glioma, glioblastoma, bladder urothelial carcinoma, liver cancer, lung cancer, pancreatic ductal adenocarcinoma and ovarian cancer.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The small molecule polypeptide NEAT1-31 provided by the present invention, as discovered by the inventors through research, has the effect of promoting phagocytosis both in vivo and in vitro. It has also been confirmed that Neat1-31 and anti-CD47 antibody have a synergistic effect, and the two jointly inhibit tumor progression. This provides a new direction for tumor treatment and brings new hope to patients resistant to immune checkpoint blockade (ICB) therapy. Description of the Drawings

[0024] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0025] Figure 1 Schematic diagram for ribosome profiling analysis of phagocytes, macrophages and cancer cells;

[0026] Figure 2 Ribosome profiling analysis of phagocytes, wherein, B. Venn diagram of dysregulated mRNA, LincRNA, 3'UTR and 5'UTR; C. Relative expression distribution of mRNA, LincRNA, 3’UTR, 5’UTR; D. Pie chart distribution of mRNA, LincRNA, 3'UTR, 5'UTR; E. Mapping of ribosome protected fragments (RPF) to transcripts, percentage of RPF mapping for each ORF; F. Length distribution of lincRNA; G. Phagocytic expression of each ORF and ORF score, x-axis, ORF score, y-axis, relative phagocyte expression;

[0027] Figure 3NEAT1-31 encoded by LincNeat1 is enriched in anti-tumor macrophages, predicting a better ICB treatment response; A. RFP density of LincNEAT1 from ribosome profiling; B. Left: Flag knock-in and LincNeat1 knockout cell lines were established in HEK293T cells, and Flag and NEAT1-31 were detected by immunoblotting; Right: THP1-induced macrophages (M0 / M1 / M2), NEAT1-31 was detected; C. Flow cytometry was used to detect the distribution of NEAT1-31 in anti- / pro-tumor macrophages; D. Macrophages were derived from different sample types, divided into anti- / pro-tumor macrophages, and the expression of NEAT1-31 was detected; E. Percentage of NEAT1-31-positive macrophages from different cancer types; F. Percentage of NEAT1-31-positive macrophages in anti- / pro-tumor macrophages from different tumor types; G. The ratio of NEAT1-31+ macrophages to CD45 cells was calculated by flow cytometry, and patients were divided into NEAT1-31 low / high groups, with the average ratio of NEAT1-31+ macrophages / CD45 cells as the cut-off value. The effective rate (RR) of ICB treatment was calculated for each cohort;

[0028] Figure 4 NEAT1-31 and anti-CD47 antibody inhibit tumor progression through synergistic effects;

[0029] Figure 5 To verify that NEAT1-31 and anti-CD47 antibody inhibit tumor progression through synergistic effects. Detailed implementation manners

[0030] The implementation schemes of the present invention will be described in detail below in combination with the implementation manners and examples. However, those skilled in the art will understand that the following implementation manners and examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. Based on the examples in the present invention, all other examples obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention. Those not specifying specific conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. Those reagents or instruments not indicating the manufacturer can be obtained as conventional products through commercial purchase.

[0031] In a first aspect, the present invention provides a small molecule polypeptide NEAT1-31, and the amino acid sequence of the small molecule polypeptide NEAT1-31 is shown as SEQ ID NO.1:

[0032] MGIVGREWARCLYYMCDLKTLLGSELRLLNW (SEQ ID NO.1).

[0033] The small molecule polypeptide NEAT1-31 has the effect of enhancing the phagocytic ability of macrophages and treating cancer.

[0034] In a second aspect, the present invention provides the use of the above-mentioned small molecule polypeptide NEAT1-31 in enhancing the phagocytic ability of macrophages.

[0035] The small molecule polypeptide NEAT1-31 provided by the present invention has the effect of enhancing the phagocytic ability of macrophages, and therefore can be used to enhance the phagocytic ability of macrophages.

[0036] In a third aspect, the present invention provides the use of the above-mentioned small molecule polypeptide NEAT1-31 in the preparation of a drug for treating cancer.

[0037] The small molecule polypeptide NEAT1-31 provided by the present invention has the effect of treating cancer, and therefore can be used to prepare a drug for treating cancer.

[0038] In some alternative embodiments, the cancer includes but is not limited to breast cancer, gastric cancer, colon cancer, low-grade glioma, glioblastoma, bladder urothelial carcinoma, liver cancer, lung cancer, pancreatic ductal adenocarcinoma, and ovarian cancer.

[0039] In a fourth aspect, the present invention provides the use of the above-mentioned small molecule polypeptide NEAT1-31 in combination with an anti-CD47 antibody in the preparation of a drug for treating cancer.

[0040] Through the research of the inventors, it is found that the small molecule polypeptide NEAT1-31 and the anti-CD47 antibody have a synergistic effect in cancer treatment, and the combination of the two can further improve the effect of treating cancer.

[0041] In some alternative embodiments, the cancer includes but is not limited to breast cancer, gastric cancer, colon cancer, low-grade glioma, glioblastoma, bladder urothelial carcinoma, liver cancer, lung cancer, pancreatic ductal adenocarcinoma, and ovarian cancer.

[0042] In a fifth aspect, the present invention provides a phagocyte activator, which includes the above-mentioned small molecule polypeptide NEAT1-31.

[0043] This phagocyte activator has the effect of promoting macrophage phagocytosis.

[0044] In a sixth aspect, the present invention provides a drug for treating cancer, which includes the above-mentioned phagocyte activator.

[0045] This drug can be used for the treatment of cancer.

[0046] In some alternative embodiments, the drug further includes an anti-CD47 antibody.

[0047] The small molecule polypeptide NEAT1-31 and the anti-CD47 antibody have a synergistic effect, and through this cooperation, the therapeutic effect of cancer can be further improved.

[0048] In some alternative embodiments, the cancers include, but are not limited to, breast cancer, gastric cancer, colon cancer, low-grade glioma, glioblastoma, bladder urothelial carcinoma, liver cancer, lung cancer, pancreatic ductal adenocarcinoma, and ovarian cancer.

[0049] The present invention will be further illustrated by specific examples below. However, it should be understood that these examples are only for more detailed illustration and should not be construed as limiting the present invention in any form.

[0050] Example 1

[0051] To identify potential phagocyte activators, the inventors first established an in vitro phagocytosis assay as follows.

[0052] Peripheral blood was collected and peripheral blood mononuclear cells (PBMCs) were isolated. Subsequently, macrophages were induced and cultured in ImmunoCult TM -SF macrophage medium and then labeled with CellTraceTM Blue (Thermo Fisher, cat# C34568). Similarly, MDA-MB-231 breast cancer cells were labeled with 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFSE). The macrophages were incubated with MDA-MB-231 cells for 2 hours and fluorescence-activated cell sorting (FACS) analysis was performed. Cells labeled with the blue cell tracker and CFSE were considered phagocytes. In addition to conventional FACS, the inventors also applied mild trypsinization to avoid cell adhesion. Cells with only the blue cell tracker were identified as macrophages without phagocytic ability. Meanwhile, equal amounts of phagocytes, macrophages, and MDA-MB231 cells were collected from 100 donors for ribosome profiling (as Figure 1as shown). Ribosome protected fragments (RPFs) were collected and subjected to transcriptional mapping. The open reading frames mapped to the transcriptional regions showed a distinct 3-nt periodicity, indicating reliable quality control. Further applying principal component analysis (PCA), the RPF signatures could distinguish cancer cells, phagocytes, and macrophages. To further confirm, relative FPKM and RPF counts were applied to analyze the well-studied phagocytic checkpoints. The expression of "don't eat me" markers CLEC-1, Siglec-10, SIRPα, LILRB1, and PD1 in macrophages was significantly higher than that in phagocytes, and the above data all had reliable specificity and sensitivity. Therefore, the inventors adopted a separate analysis procedure. However, phagocytosis actually involves two cell types, and phagocytes "eat" cancer cells. To determine the reliability of using phagocytic activators, the inventors adopted a rigorous analysis process. First, phagocyte-specific genes were determined by comparing phagocytes with cancer cells (FPKM of MDA-MB-231 cells = 0, FPKM of phagocytes > 0). Next, the expression of phagocyte-specific genes in phagocytes and macrophages was compared, and finally, phagocytic activators were determined. Accordingly, Venn diagrams of mRNAs, lincRNAs, 3'UTRs, and 5'UTRs were constructed. A total of 2,606 potential phagocytic activators were identified, including 7 mRNAs, 750 lincRNAs, 85 3'UTRs, and 1,764 5'UTRs (as shown in B of Figure 2 ). Summarizing the expression levels of each group, the open reading frame expression level in 5'UTR was the highest (as shown in C of Figure 2 ). Most RPFs originated from 3'UTRs, accounting for 67.69% of all RPFs (as shown in D of Figure 2 ). Approximately 67.69% of RPFs were mapped to 3'UTRs, 31.2% of RPFs were mapped to lincRNAs, and less than 10% of RPFs were mapped to mRNAs and 5'UTRs (as shown in E of Figure 2 ). The distribution and length of lincORFs were analyzed, and the peak number of lincORFs at 80 - 130 bp was determined (as shown in F of Figure 2 ). ORF scores were calculated to determine the degree of active translation, and expression levels were measured to verify the biological importance of each group. The putative lincRNA ORF (lincNEAT1-31aa) that met the ORF score and phagocyte expression criteria was significantly upregulated in phagocytes (phagocyte FPKM = 11.62, cancer FPKM = 0, macrophage FPKM = 0.65, P = 3.01E-06, FC = 4.16, where the P value is a numerical value in statistics representing the significance of the difference, and FC refers to the fold change) (as shown in G of Figure 2 ).

[0053] The linNEAT1 ORF is located in the chromosomal region 2833-2928 of the ENST00000645023 transcript. Ribosome profiling predicts that this ORF encodes a 31-amino acid micropeptide (hereinafter referred to as NEAT1-31) (as shown in A of Figure 3 ). To confirm the translational function of NEAT1-31, the inventors established a flag knock-in and knock-out system in HEK293T cells. Since there is no effective strategy to induce phagocytes, the inventors treated THP1 cells (human monocytes) with phorbol 12-myristate 13-acetate (PMA), a phorbol ester that is an activator of protein kinase C and SphK, lipopolysaccharide (LPS), or IL4 to induce them into macrophages. Immunoblotting was used to detect NEAT1-31 with an anti-flag antibody, and a specific monoclonal antibody (anti-NEAT1-31 antibody) was generated to directly detect NEAT1-31 (as shown in B of Figure 3 ). Figure 3 Left panel in B of Figure 3 : WT is HEK293T cells without treatment, KI is HEK293T cells with flag knock-in, and K.O. is HEK293T cells with LincNeat1 knockout. Flag and NEAT1-31 were detected by immunoblotting. Right panel: THP1 was induced into macrophages (M0 / M1 / M2, where M0, M1, and M2 are different subtypes of macrophages), and NEAT1-31 was detected. To further confirm, the inventors used FACS to determine the proportion of NEAT1-31+ in different macrophages. Next, macrophages were collected from healthy donors and detected with NEAT1-31, iNOS, and CD206 antibodies. NEAT1-31 is abundantly expressed in iNOS+ macrophages and hardly expressed in CD206+ macrophages. iNOS and CD206 are two widely used classical markers for anti-tumor / pro-tumor (AT / PT) macrophages (as shown in C of Figure 3 ​as shown in D and E). Subsequently, macrophages were divided into anti-tumor macrophages and pro-tumor macrophages (anti-tumor macrophages refer to M1 macrophages, which usually express iNOS; pro-tumor cells refer to M2 macrophages, which usually express CD206). NEAT1-31+ macrophages in each group were detected. The results showed that NEAT1-31 was specifically enriched in anti-tumor macrophages of different tumors (as shown in F in Figure 3 ). Patients receiving immunotherapy were included, tumor samples were collected, and infiltrating lymphocytes were isolated. The ratio of NEAT1-31+ macrophages to CD45 cells was calculated by flow cytometry, and the patients were divided into NEAT1-31 low / high groups, with the average ratio of NEAT1-31+ macrophages / CD45 cells as the cut-off value. The effective rate (RR) of ICB treatment in each cohort was calculated, and the effective rate in the NEAT1-31 high group was greater than that in the NEAT1-31 low group. Taken together, these findings revealed that lincNEAT1 encodes NEAT1-31, which is specifically enriched in anti-tumor macrophages and predicts a good ICB response in multiple cancers (as shown in G in Figure 2 ).

[0054] CD47 is one of the most important phagocytic immune checkpoints, and specific blockade of CD47 by antibodies can promote phagocytosis. To verify the clinical application of NEAT1-31 in promoting phagocytosis, we treated donor-derived macrophages (DDMs) with NEAT1-31, with or without anti-CD47 antibody. A phagocytosis experiment was performed on the cells: First, sufficient macrophages and tumor cells were cultured, then the tumor cells (MDA-MB-231 breast cancer cells or human glioma cells U251) were labeled with CFSE, and the macrophages were labeled with blue. These labeled macrophages and tumor cells were co-cultured, and after sufficient phagocytosis, they were analyzed by flow cytometry and the phagocytosis rate was calculated. The results showed that NEAT1-31 and anti-CD47 antibody promoted cell phagocytosis as expected, and NEAT1-31 plus anti-CD47 antibody had a synergistic effect ( Figure 4 in A-C). Figure 4 A and B in are the treatment of DDM1 and DDM2 (DDM1 and DDM2 are different donor-derived macrophages) with NEAT 1-31, anti-CD 47 antibody or combination therapy. An in vitro phagocytosis experiment was performed, and phagocytic cells were detected by flow cytometry. Figure 4 C in is Figure 4Statistical graphs of A and B in []. Next, we established a quasi-in situ (implanted into the renal capsule) xenograft model. The specific experimental steps included implanting 1×10^6 MDA-MB-231-Luc or 1×10^6 U251 tumor cells into the renal capsule. Mice carrying tumor cells were treated with IgG or anti-CD47 antibody. Then, monocytes were extracted from the blood of the mice and sorted, leaving macrophages, which were transfected with the Neat-31 overexpression plasmid and then infused back into the mice via the tail vein. A total of 400 μg anti-CD47 (clone B6.H12, Bio X Cell) was injected intraperitoneally into the mice every other day. Bioluminescence imaging (BLI) and an IVIS instrument (PhotoSound PAFT / 256) were used to detect the growth of tumor xenografts (as shown in E in Figure 4 ), and the relative flux intensity was measured and statistically analyzed (as shown in F in Figure 4 . In the figure, in the BLI technique, the luciferase gene is inserted into the cell chromosomal DNA, enabling the cells to express luciferase. When luciferin is exogenously administered, luciferase catalyzes the oxidation reaction of luciferin in the presence of ATP and oxygen, producing a luminescence phenomenon. This luminescence only occurs within living cells, and the intensity of the light is linearly related to the number of labeled cells. Therefore, by measuring the light flux intensity, the size and growth of the tumor can be quantitatively evaluated.). The total survival time was measured and statistically analyzed (as shown in G in Figure 4 ). The results showed that both NEAT1-31 and anti-CD47 could promote the elimination of cancer cells in breast cancer and GBM mice and extend the total survival time (as shown in D-G in Figure 4 ). Figure 4 Figure D in [] is an illustration of the combination treatment strategy, where mice were treated with macrophages treated with anti-CD47 antibody or NEAT1-31. These findings were further confirmed in the C57 model (the experiment was conducted according to the above experimental procedure, with the difference that normal mice were replaced with C57 mice). NEAT1-31 and anti-CD47 antibody inhibited tumor progression through a synergistic effect (as shown in A-C in Figure 5 ).

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A small molecule polypeptide NEAT1-31, characterized in that: The amino acid sequence of the small molecule polypeptide NEAT1-31 is shown in SEQ ID NO.

1.

2. Use of the small molecule polypeptide NEAT1-31 according to claim 1 in the preparation of drugs for treating cancer.

3. The use according to claim 2, characterized in that: The cancers include breast cancer, gastric cancer, colon cancer, low-grade glioma, glioblastoma, bladder urothelial carcinoma, liver cancer, lung cancer, pancreatic ductal adenocarcinoma, and ovarian cancer.

4. Use of the small molecule polypeptide NEAT1-31 according to claim 1 in combination with an anti-CD47 antibody in the preparation of a drug for treating cancer.

5. The use according to claim 4, characterized in that: The cancers include breast cancer, gastric cancer, colon cancer, low-grade glioma, glioblastoma, bladder urothelial carcinoma, liver cancer, lung cancer, pancreatic ductal adenocarcinoma, and ovarian cancer.

6. A drug for treating cancer, characterized in that: The drug comprises the small molecule polypeptide NEAT1-31 according to claim 1.

7. The drug according to claim 6, characterized in that The medicament also includes anti-CD47 antibodies.

8. The drug according to claim 6, characterized in that The cancers include breast cancer, gastric cancer, colon cancer, low-grade glioma, glioblastoma, bladder urothelial carcinoma, liver cancer, lung cancer, pancreatic ductal adenocarcinoma, and ovarian cancer.

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