WO2018082590A1 - 肿瘤免疫治疗靶标及其应用 - Google Patents

肿瘤免疫治疗靶标及其应用 Download PDF

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WO2018082590A1
WO2018082590A1 PCT/CN2017/109031 CN2017109031W WO2018082590A1 WO 2018082590 A1 WO2018082590 A1 WO 2018082590A1 CN 2017109031 W CN2017109031 W CN 2017109031W WO 2018082590 A1 WO2018082590 A1 WO 2018082590A1
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btn3a3
tumor
sequence
lsectin
cells
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PCT/CN2017/109031
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French (fr)
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唐丽
贺福初
柳迪
陆倩
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北京蛋白质组研究中心
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Priority claimed from CN201610953057.2A external-priority patent/CN108014327B/zh
Priority claimed from CN201610952945.2A external-priority patent/CN107998396B/zh
Application filed by 北京蛋白质组研究中心 filed Critical 北京蛋白质组研究中心
Priority to US16/346,316 priority Critical patent/US11634493B2/en
Publication of WO2018082590A1 publication Critical patent/WO2018082590A1/zh

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Definitions

  • the invention belongs to the field of biotechnology, and particularly relates to a tumor immunotherapy target for tumor-associated macrophages.
  • the global incidence of cancer has been on the rise since the late 1970s.
  • the treatment of tumors mainly includes surgery, radiotherapy, chemotherapy, endocrine therapy, targeted therapy and adjuvant treatment of traditional Chinese medicine.
  • the root cause of cancer treatment is that the tumor has certain drug resistance and recurrence. Studies have shown that the root cause of tumor resistance and recurrence is the maintenance and enhancement of tumor cell dryness. Therefore, the targeted treatment of tumor cell stem has become a research hotspot.
  • the dryness of tumor cells is regulated by three factors: gene diversity, epigenetic and tumor microenvironment. Due to the high degree of heterogeneity of tumor cells, it is difficult to propose effective treatment options from the perspective of genetic diversity and epigenetics. In contrast, immune cells infiltrated by tumor microenvironment maintain and promote the dryness of tumor cells through interaction, which has become a research hotspot because of its strong application prospects. Based on the basic principles of tumor immunotherapy, targeted drugs for the interaction of immune cells on tumor cells, it is expected to apply immunological principles and methods to improve the immunogenicity of tumor cells and sensitivity to effector cell killing, and stimulate and enhance the body resistance. Tumor immune response, synergistic with the body's immune system, thereby inhibiting tumor growth, metastasis and recurrence.
  • Tumor microenvironment factors can promote the dry cell-dependent interaction of tumor cells.
  • stromal cells and infiltrating immune cells in the tumor microenvironment. By blocking the interaction between these cells and tumor cells, it is expected to reduce the dryness of tumor cells. Thereby inhibiting the occurrence and development of tumors.
  • tumor-associated macrophages are a general term for macrophages present in the tumor microenvironment and an important component of infiltration of immune cells in the tumor microenvironment.
  • Studies have shown that tumor-associated macrophages can promote tumor progression through a variety of ways, including inhibition of tumor immune response, promotion of tumor immune tolerance environment, and promotion of tumor tissue angiogenesis. In view of the fact that tumor-associated macrophages promote tumor progression, clearing tumor-associated macrophages and promoting the transformation of tumor-associated macrophages into immune activation has become a potential tumor treatment.
  • the application is based on the infiltration of tumor-associated macrophage-expressing LSECtin to interact with BTN3A3 expressed on the surface of tumor cells, thereby promoting the dryness of tumor cells and promoting the occurrence and development of tumors, using two targets of LSECtin and BTN3A3. Development or design of tumor immunotherapy drugs.
  • the tumor is a tumor in which tumor-associated macrophages express LSECtin and tumor cells express BTN3A3, including but not limited to breast cancer, myeloma, liver cancer, gastric cancer, colon cancer, lung cancer, giant cell tumor of bone, kidney cancer, and larynx. Cancer and salivary gland cancer.
  • Another object of the present invention is to provide an application of LSECtin or tumor-expressing BTN3A3 in invasive tumor-associated macrophage expression as a target in tumor immunotherapy.
  • the application is based on the expression of LSECtin infiltrating tumor-associated macrophage cells and the expression of BTN3A3 on the surface of tumor cells, each of which has the characteristics of promoting tumor cell dryness and promoting tumor occurrence and development, and developing or designing using LSECtin or BTN3A3 as a target. Inhibitors thus inhibit the occurrence and development of tumors, and screen for tumor immunotherapy drugs.
  • the tumor in this application is one of: c1) a tumor infiltrating tumor-associated macrophage cells expressing LSECtin; c2) a tumor expressing BTN3A2 and/or BTN3A3; and c3) a tumor satisfying both c1) and c2).
  • Such tumors include, but are not limited to, breast cancer, myeloma, liver cancer, gastric cancer, colon cancer, lung cancer, giant cell tumor of bone, kidney cancer, laryngeal cancer, and salivary gland cancer.
  • the drug has at least one of the following functions (1) to (5): (1) treating and/or preventing tumor; (2) inhibiting growth of tumor cells; and (3) inhibiting tumor Maintenance or promotion of cell dryness; (4) inhibition of expression of stem cell-related characteristic molecules; and (5) inhibition of STAT3 phosphorylation in tumor cells.
  • the tumor cell dry correlation characteristic molecule is an Oct4 gene and/or a Nanog gene and/or a Sox gene; the growth of the tumor suppressing cell is reduced in reducing the tumor formation rate of the tumor cell and/or reducing the volume of the tumor cell.
  • Still another object of the present invention is to provide a substance which inhibits LSECtin activity in infiltrating tumor-associated macrophage-derived cells, inhibits tumor-expressing BTN3A3 activity, or inhibits interaction of said LSECtin with BTN3A3, including but not limited to: interference with BTN3A2 and BTN3A3 Expressed RNA molecule, anti-LSECtin antibody, LSECtin small molecule inhibitor, LSECtin soluble protein, RNA molecule that interferes with LSECtin expression, anti-BTN3A2 antibody, BTN3A2 small molecule inhibitor, BTN3A2 soluble protein, RNA molecule that interferes with BTN3A2 expression, anti-BTN3A3 antibody , BTN3A3 small molecule inhibitor, BTN3A3 small molecule inhibitor, BTN3A3 soluble protein and RNA molecules that interfere with BTN3A3 expression.
  • a specific first class of substances are RNA molecules that interfere with the expression of BTN3A2 and BTN3A3 or RNA molecules that interfere with the expression of BTN3A3, which are any of b1)-b4): b1) shRNA molecules shown in SEQ ID NO: 4; b2) sequences 4 deleting or adding or changing one or several nucleotides, and nucleotides having the same function as sequence 4; b3) shRNA molecules shown in sequence 5; and b4) deleting or increasing or changing sequence 5 by one or several Nucleotide, and nucleotides that have the same function as sequence 5.
  • a specific second type of substance is a soluble protein, which is a fusion protein having an activity of inhibiting tumor progression, which can block
  • BTN3A3-Ig The interaction between LSECtin and BTN3A3, named BTN3A3-Ig, is a recombinant protein obtained by linking human BTN3A3 and human IgG1 via a linker peptide.
  • the fusion protein BTN3A3-Ig is one of the following amino acid residue sequences: 1) the sequence 9 in the sequence listing; 2) the amino acid residue sequence of the sequence 9 in the sequence listing is substituted by one or several amino acid residues, a protein that is deleted or added and has activity to inhibit tumor progression; 3) a protein in which the amino acid residue sequence of SEQ ID NO: 9 is substituted, deleted or added by an amino acid residue and has a tumor suppressing activity, and the new protein is the same as the sequence 9
  • the source is 80% or higher.
  • Sequence 9 in the sequence listing consists of 490 amino acid residues, starting from the amino acid (N) end to the start codon, from the amino terminus to the 2nd to the signal peptide, and from the amino terminus to the 16th to the 262th position.
  • BTN3A3 is a linker from positions 263 to 266 of the amino terminus and human IgG1 from the amino terminus at positions 267 to 490.
  • the gene BTN3A3-Ig encoding the fusion protein BTN3A3-Ig is also within the scope of the present invention.
  • the gene BTN3A3-Ig encoding the fusion protein BTN3A3-Ig is one of the following nucleotide sequences: 1) the DNA sequence of SEQ ID NO: 10 in the sequence listing; 2) the DNA sequence encoding SEQ ID NO: 9 in the sequence listing; 3) and the coding sequence
  • the DNA sequence of SEQ ID NO:9 in the list has one or several base changes and has a nucleotide sequence which inhibits tumor process activity; 4) 80% or more of the encoded sequence is homologous to sequence 10 in the sequence listing and has tumor suppressing progress.
  • the sequence 10 in the sequence listing consists of 1470 bases, the coding sequence of which is 1-1470 bases from the 5' end, encoding a protein having the sequence of the amino acid residue shown by 1 in the sequence listing, from the 5' end to the 46th.
  • the -786 base encodes human BTN3A3, which encodes a linker from the 5'-end 787-798 base and encodes human IgG1 from the 5' end of positions 797-1470.
  • An expression vector, a transgenic cell line or a host strain containing the fusion gene BTN3A3-Ig is also included in the present invention.
  • a primer pair that amplifies any of the fusion gene BTN3A3-Ig is also in the present invention.
  • the method for expressing the fusion protein BTN3A3-Ig of the present invention comprises the following steps:
  • fusion protein BTN3A3-Ig The recombinant expression vector pIRES2-EGFP-BTN3A3-Ig containing the fusion gene BTN3A3-Ig was transformed or transfected into host cells and their progeny cells, and the recombinant host cells were cultured to obtain the fusion gene BTN3A3-Ig. expression;
  • the fusion gene BTN3A3-Ig was ligated into the NheI and SalI restriction sites in the vector pIRES2-EGFP to obtain a recombinant expression vector, which was named pIRES2-EGFP-BTN3A3-Ig.
  • the host cell in the step 2) is a cell capable of expressing a foreign gene, including 293T cells, 293 cells, CHO-S cells, and the like; and the culture of the recombinant host cell containing the fusion gene BTN3A3-Ig is cultured in the step 2).
  • step 2) containing the recombinant fusion gene is BTN3A3-Ig
  • the culture conditions of the host cells are culture conditions suitable for growth of the host cells, and culture is carried out at 36.5 to 37.5 ° C for 24-120 hours, preferably at 37 ° C for 96 hours.
  • a Protein G Sepharose column for example, a Protein G Sepharose column (Protein G Sepharose column), a Protein A/G Sepharose column (Protein A/G Sepharose column), or a Protein A Sepharose column (Protein A agar) can be used.
  • the recombinant expressed protein is purified by a sugar gel column or the like, preferably a Protein G Sepharose column, and the purification method is: adding the culture supernatant of the cell (recombinant host cell containing the fusion gene BTN3A3-Ig) to an equilibration buffer (20 mM PBS, 150 mM).
  • the fusion protein BTN3A3-Ig of the present invention is prepared into a pharmaceutical composition by a pharmaceutically acceptable pharmaceutical carrier suitable for administration, and suitable pharmaceutical carriers are well known to those skilled in the art, including but not limited to physiological saline, phosphate buffer, Water, liposomes, nanocarriers, and the like.
  • suitable pharmaceutical carriers are well known to those skilled in the art, including but not limited to physiological saline, phosphate buffer, Water, liposomes, nanocarriers, and the like.
  • a pharmaceutical carrier containing the fusion protein BTN3A3-Ig can be produced by a conventional method.
  • the pharmaceutical composition of the fusion protein BTN3A3-Ig of the present invention can be administered to humans or other mammals by various routes of administration including, but not limited to, intravenous (iv), intravenous infusion, intramuscular injection. (im), subcutaneous injection (sc) and oral (po). Different routes of administration can be chosen for different diseases.
  • the fusion protein of the present invention BTN3A3-Ig is generally administered at a dose of 0.5-2.5 ⁇ g/g based on a mouse model, and the course of treatment is generally 15-30 days.
  • the dosage and course of treatment in the actual application can be adjusted according to the actual situation.
  • a specific third class of substances is a monoclonal antibody having activity to inhibit tumor progression, which blocks the interaction between LSECtin and BTN3A3.
  • the heavy chain variable region of the monoclonal antibody is a polypeptide represented by a sequence of 13 amino acid residues in the sequence listing, and a sequence of amino acid residues of sequence 13 in the sequence listing is substituted, deleted or substituted with one to ten amino acid residues.
  • the light chain variable region of the monoclonal antibody is: from the sequence listing a polypeptide represented by a sequence of 14 amino acid residues, a sequence of amino acid residues of sequence 14 in the sequence listing, substituted or deleted by one to ten amino acid residues
  • Sequence 13 in the sequence listing consists of 141 amino acid residues, and sequence 14 in the sequence listing consists of 130 amino acid residues.
  • the gene encoding the monoclonal antibody has a heavy chain variable region encoding gene: a DNA sequence having the sequence 15 in the sequence listing, a DNA sequence encoding the sequence 13 in the sequence listing, and a DNA sequence having the sequence 13 in the sequence of the encoded sequence.
  • One or more base-changing sequences, 80% or more of the encoded sequences are homologous to the sequence of SEQ ID NO: 15 in the sequence listing, and nucleosides which hybridize under high stringency conditions to the DNA sequence defined by SEQ ID NO: 15 in the Sequence Listing Acid sequence
  • the gene encoding the monoclonal antibody has a light chain variable region encoding gene: a DNA sequence having the sequence 16 in the sequence listing, a DNA sequence encoding the sequence 14 in the sequence listing, and a DNA sequence having the sequence 14 in the sequence of the encoded sequence.
  • a DNA sequence having the sequence 16 in the sequence listing a DNA sequence encoding the sequence 14 in the sequence listing
  • a DNA sequence having the sequence 14 in the sequence of the encoded sequence One or more base-changing sequences, 80% or more of the encoded sequence homologous to the sequence of sequence 16 in the sequence listing, and a nucleoside which hybridizes under high stringency conditions to the DNA sequence defined by SEQ ID NO: 16 in the Sequence Listing Acid sequence.
  • the high stringency conditions were hybridization and washing of the membrane in a solution of 0.1 x SSPE (or 0.1 x SSC), 0.1% SDS at 65 °C.
  • sequence 15 in the sequence listing consists of 423 bases, encoding a protein having the sequence of the amino acid residue of SEQ ID NO: 13 in the sequence listing, and the sequence 16 in the sequence listing consists of 390 bases, the sequence having the sequence 14 in the sequence listing.
  • the protein of the amino acid residue sequence consists of 423 bases, encoding a protein having the sequence of the amino acid residue of SEQ ID NO: 13 in the sequence listing, and the sequence 16 in the sequence listing consists of 390 bases, the sequence having the sequence 14 in the sequence listing.
  • An expression vector, a transgenic cell line or a host strain containing the gene 5E08 of the present invention belongs to the present invention.
  • Primer pairs that amplify any of the fragments of the gene 5E08 of the present invention are also within the scope of the invention.
  • a hybridoma cell strain capable of secreting a monoclonal antibody that blocks the interaction between LSECtin and BTN3A3, which is a fusion protein obtained by linking human BTN3A3 and mouse IgG2a via a linker peptide (designated BTN3A3) is also included in the present invention.
  • BTN3A3 a linker peptide
  • the fusion protein BTN3A3-mIg is one of the following amino acid residue sequences: 1) the sequence 11 in the sequence listing; 2) the amino acid residue sequence of the sequence 11 in the sequence listing is substituted by one or several amino acid residues, a protein that is deleted or added and has anti-BTN3A3 immunogenicity; 3) a protein that has been substituted, deleted or added to the amino acid residue of sequence 11 in the sequence listing and has anti-BTN3A3 immunogenicity, new protein and sequence 1 homology reaches 80% or higher.
  • sequence 11 in the sequence listing consists of 499 amino acid residues, the first position from the amino (N) end is the start codon, the second to the signal peptide, and the 16th to the 262th are the human BTN3A3, from the amino terminus.
  • the gene encoding the above fusion protein BTN3A3-mIg also belongs to the present invention.
  • the gene BTN3A3-mIg encoding the above fusion protein is one of the following nucleotide sequences: 1) the DNA sequence of SEQ ID NO: 12 in the sequence listing; 2) the DNA sequence of SEQ ID NO: 11 in the sequence listing; 3) The DNA sequence of SEQ ID NO: 11 in the sequence listing has a nucleotide sequence of one or several bases and has anti-BTN3A3 immunogenicity; 4) 80% or more of the encoded sequence is homologous to sequence 12 in the sequence listing And having a nucleotide sequence which is anti-BTN3A3 immunogenic; 5) a nucleotide sequence which hybridizes under high stringency conditions to a DNA sequence defined by SEQ ID NO: 12 in the Sequence Listing.
  • the high stringency conditions were after washing, the membrane was washed at 65 ° C with a solution containing 0.1 x SSPE (or 0.1 x SSC), 0.1% SDS.
  • the sequence 12 in the sequence listing consists of 1497 bases, the coding sequence of which is from the 1st to the 5th end of the 1-5497 base, encoding a protein having the sequence of the amino acid residue shown by 11 in the sequence listing, from the 5' end
  • the 1-3 position is the start codon
  • the base 4-48 signal peptide from the 5' end encodes the human BTN3A3 from the 5' end 46-786, and the base 787-798 from the 5' end.
  • the base encodes a linker, which encodes mouse IgG2a from the 5' end of positions 799-1494, and the stop codon from the 5' end of positions 1495-1497.
  • An expression vector, a transgenic cell line or a host strain containing the fusion gene BTN3A3-mIg belongs to the present invention.
  • Primer pairs that amplify any of the fusion gene BTN3A3-mIg are also within the scope of the invention.
  • a method of expressing the above fusion protein BTN3A3-mIg is also in the present invention.
  • the method for expressing the fusion protein BTN3A3-mIg provided by the invention may comprise the following steps: (1) constructing a recombinant expression vector: ligating the fusion gene BTN3A3-mIg into an expression vector to obtain a recombinant expression vector containing the fusion gene BTN3A3-mIg (2) expressing the fusion protein BTN3A3-mIg: transforming or transfecting the recombinant expression vector containing the fusion gene BTN3A3-mIg into the host cell and its progeny cells, culturing the recombinant host cell, and obtaining the fusion gene BTN3A3-mIg; (3) Purification: Purification of the recombinantly expressed protein to obtain the fusion protein BTN3A3-mIg.
  • the hybridoma cell line was immunized with the fusion protein BTN3A3-mIg as an immunogen to obtain a hybridoma cell line with a specific monoclonal antibody which inhibits the tumor process activity, and the name is anti-P3 (5E08).
  • the cell line was in 2017. On September 26th, the company was deposited at the General Microbiology Center of the China General Microorganisms Collection and Management Committee at No. 3, Beichen West Road, Chaoyang District, Beijing, China. The deposit number is CGMCC No.14723.
  • the monoclonal antibody secreted by the hybridoma cell line anti-P3 was named 5E08 and was derived from mouse mouse (Mus musculus), which blocked the interaction between LSECtin and BTN3A3 and inhibited tumor process activity.
  • the monoclonal antibody 5E08 belongs to the present invention.
  • the present invention also provides hybridomas capable of secreting monoclonal antibodies that block the interaction between LSECtin and BTN3A3.
  • the method for obtaining a cell strain may include the following steps: 1) immunizing an animal with the fusion protein BTN3A3-mIg as an immunogen; 2) isolating the spleen cells of the immunized animal, and fusing it with the myeloma cells to form a hybridoma; 3) screening hybridization On the tumor cells, a hybridoma cell line anti-P3 (5E08) was obtained.
  • the concentration of the fusion protein BTN3A3-mIg in the step 1) is 100-400 ⁇ g/mL, preferably 400 ⁇ g/mL; and the immunization for preparing the monoclonal antibody
  • the animal may be a mammal such as mouse, rat, rabbit, goat, sheep, pig, donkey, horse, etc., preferably a mouse.
  • the serum antibody level of the immunized animal reaches a peak, the spleen cells of the animal can be isolated and prepared as a single cell suspension.
  • the spleen cells can be screened using an immunoadsorption method and fused with myeloma cells (preferably mouse myeloma cells SP2/0) under the induction of a suitable fusing agent such as polyethylene glycol to form a hybridoma.
  • the step 3) can be cultured in a selective medium (such as HAT medium) to screen the fused hybridoma cells, and further, the method can be identified by flow cytometry, Western blotting, immunoprecipitation, or the like. Positive resistant cell line.
  • the hybridoma cell strain anti-specific monoclonal antibody 5E08 secreting the activity of inhibiting tumor progression can be cultured in vitro (such as in a tissue culture flask or a porous fiber reactor) or in vivo (mouse ascites).
  • P3 (5E08) and monoclonal antibody 5E08 was collected and purified from cell culture fluid or mouse ascites fluid.
  • the method for obtaining the monoclonal antibody 5E08 of the present invention adds the following steps based on the above steps:
  • the monoclonal antibody 5E08 was isolated and purified from the culture solution of the hybridoma cell line anti-P3 (5E08) or the ascites fluid of the animal inoculated with the hybridoma cell line anti-P3 (5E08).
  • the expression vector of monoclonal antibody 5E08 is constructed by using the heavy chain variable region and the light chain variable region amino acid sequence or DNA sequence of the monoclonal antibody 5E08 mentioned, and a single agent having a tumor suppressing activity is obtained by conventional protein expression. Cloning antibodies.
  • a further object of the present invention is to provide an expression vector, a transgenic cell line or a host strain, a monoclonal antibody 5E08, and an immunogenic fusion protein BTN3A3 of the RNA molecule, the fusion protein BTN3A3-Ig, the fusion gene BTN3A3-mIg, the fusion gene BTN3A3-Ig.
  • -mIg and its coding gene BTN3A3-mIg in the preparation of a product having at least one of the following functions (b1)-(b5): (b1) treating and/or preventing tumor; (b2) inhibiting tumor progression; (b3 Inhibiting the maintenance or promotion of stem cell dryness; (b4) inhibiting the expression of tumor cell-associated characteristic molecules; and (b5) inhibiting STAT3 phosphorylation in tumor cells.
  • the tumor is a tumor in which the tumor-associated macrophage expresses LSECtin and the tumor cell expresses BTN3A3, and the tumor-associated macrophage expresses LSECtin and the tumor cell expresses BTN3A3 tumor including but not limited to breast cancer, myeloma, liver cancer, gastric cancer, colon Cancer, lung cancer, giant cell tumor of bone, kidney cancer, laryngeal cancer and salivary gland cancer.
  • the present invention proposes infiltration of tumor-associated macrophage-derived LSECtin and tumor-expressing BTN3A3 alone or in combination
  • the RNA molecule, the fusion protein BTN3A3-Ig and the monoclonal antibody 5E08 were used as active ingredients to prepare tumor immunotherapy drugs.
  • LSECtin, BTN3A2 and BNT3A3 promote tumor progression by promoting the maintenance of tumor cell dryness, which is embodied in promoting tumor cell ball formation, expression of dry transcription factors, and promotion of tumor progression in mouse tumor models; inhibition of LSECtin and The interaction between BTN3A2 and BTN3A3 can effectively slow down the tumor progression, which is reflected in reducing tumor incidence and slowing tumor volume growth.
  • the experiment also proved that the fusion protein BTN3A3-Ig has binding activity to LSECtin protein, and the fusion protein BTN3A3-Ig can block the interaction between LSECtin and cell surface expressed BTN3A3.
  • the fusion protein BTN3A3-Ig can block LSECtin to promote tumor cells.
  • the dry, fusion protein BTN3A3-Ig inhibits tumor progression, and the fusion protein BTN3A3-Ig inhibits tumor progression dependent on LSECtin.
  • the experiment also proved that the hybridoma cell line anti-P3(5E08)CGMCC No.14723 can secrete monoclonal antibody 5E08, monoclonal antibody 5E08 has binding activity to BTN3A3 protein, and monoclonal antibody 5E08 can block mutual interaction between LSECtin and BTN3A3.
  • Role, monoclonal antibody 5E08 can block LSECtin to promote the dryness of tumor cells, monoclonal antibody 5E08 can inhibit tumor growth.
  • the invention provides a new idea for the immunotherapy of tumors, and has broad application prospects.
  • Figure 1 shows that LSECtin in Example 1 promotes tumor formation. among them:
  • Figure 1 (a) is a test result curve of tumor volume in spontaneous breast cancer model mice, showing the tumor-forming volume of LSECtin + / + PyMT and LSECtin - / - PyMT mice;
  • Figure 1(b) is a graph showing the number of tumor foci in spontaneous breast cancer model mice, showing LSECtin +/+ PyMT and LSECtin -/- PyMT breast cancer tumors in each mouse model of MMTV-PyMT spontaneous breast cancer. Number comparison
  • Figure 1(c) is a bar graph showing the results of the number of lung tumor metastases in spontaneous breast cancer model mice, showing LSECtin +/+ PyMT and LSECtin -/- PyMT lung tumors in each mouse model of MMTV-PyMT spontaneous breast cancer. Comparison of the number of metastases;
  • Figure 1 (d) is a graph showing the detection of tumor volume in a human-nude mouse breast cancer transplantation model, showing tumor volume comparisons in the LSECtin +/+ Nude -/- group and the LSECtin -/- Nude -/- group.
  • Figure 2 shows the detection of LSECtin expression levels in the tumor microenvironment in Example 2. among them:
  • Figure 2 (a) is a histogram of mRNA level gene expression levels, indicating qPCR detection of LSECtin expression levels in the tumor microenvironment of MMTV-PyMT spontaneous breast cancer model mice, wherein CD11b - MHCII - represents lymphocytes and TAM represents tumor-associated macrophages Cells, TAN represents tumor-associated neutrophils, and Mo represents monocytes;
  • Figure 2(b) is a histogram of gene expression levels at the mRNA level, indicating qPCR detection of LSECtin expression levels in the tumor microenvironment of a human-nude mouse breast cancer transplantation model;
  • Figure 2 (c) is a graph showing the results of in situ immunofluorescence staining of mouse tumors, indicating that LSECtin colocalizes with macrophage marker molecule F4/80 in the spontaneous breast cancer microenvironment of MMTV-PyMT mice;
  • Figure 2 (d) is a flow-type detection of LSECtin expression level peak map, indicating the expression level of LSECtin on the surface of human breast cancer tumor-associated macrophages;
  • Figure 2 (e) is a flow pattern detection of LSECtin expression level peak map, indicating the expression level of LSECtin on the surface of tumor-associated macrophages in patients with myeloma;
  • Figure 2(f) is a flow pattern detection of the expression level of LSECtin expression level, indicating the expression level of LSECtin on the surface of tumor-associated macrophages in lung cancer patients;
  • Figure 2 (g) is a flow pattern detection of LSECtin expression level peak map, indicating the expression level of LSECtin on the surface of tumor-associated macrophages in colon cancer patients;
  • Figure 2 (h) is a flow-type detection of LSECtin expression level peak map, indicating the expression level of LSECtin on the surface of tumor-associated macrophages in patients with giant cell tumor of bone;
  • Figure 2 (i) is a flow pattern detection of LSECtin expression level peak map, indicating the expression level of LSECtin on the surface of tumor-associated macrophages in renal cancer patients;
  • Figure 2 (j) is a flow pattern detection of LSECtin expression level peak map, indicating the expression level of LSECtin on the surface of tumor-associated macrophages in patients with laryngeal cancer;
  • Figure 2(k) is a flow chart for the detection of LSECtin expression level scatter plots showing the expression of LSECtin on the surface of tumor-associated macrophages in patients with salivary gland cancer.
  • Figure 3 shows the clinical sample tumor cells expressing BTN3A3 in Example 4. among them
  • Figure 3 (a) is a peak pattern of BTN3A expression level in breast cancer patients, showing the expression of BTN3A on the surface of tumor cells in tumor tissues;
  • Figure 3 (b) is a scatter plot of the expression level of BTN3A3 in flow cytometry, indicating the expression of BTN3A on the surface of tumor cells in lung cancer patients;
  • Figure 3 (c) is a scatter plot of flow cytometric expression of BTN3A3, indicating the expression of BTN3A on the surface of tumor cells in colon cancer patients;
  • Figure 3 (d) is a scatter plot of the expression level of BTN3A3 in flow cytometry, indicating the expression of BTN3A on the surface of tumor cells in the tumor tissue of patients with giant cell tumor of bone;
  • Figure 3 (e) is a scatter plot of flow cytometry to detect BTN3A3 expression level, indicating tumor cell surface in tumor tissues of patients with renal cancer Express BTN3A levels;
  • Figure 3 (f) is a peak-shaped plot of the expression level of BTN3A3 in flow cytometry, indicating the expression of BTN3A on the surface of tumor cells in tumor tissues of patients with salivary gland cancer.
  • FIG. 4 shows that various tumor cell lines in Example 5 express BTN3A3. among them:
  • Figure 4 (a) is a picture of the results of Western Blot detection of BTN3A3 expressed by various breast cancer cell lines
  • Figure 4 (b) is a picture showing the results of Western Blot detection of BTN3A3 expressed by various liver cancer cell lines
  • Figure 4 (c) is a picture showing the results of Western Blot detection of human melanoma cell line expressing BTN3A3;
  • Figure 4 (d) is a picture of the results of Western Blot detection of BTN3A3 expressed by various gastric cancer cell lines;
  • Figure 4 (e) is a photograph of the results of Western Blot detection of BTN3A3 expressed by colon cancer cell lines.
  • FIG. 5 shows the expression of BTN3A2 and BTN3A3 on breast cancer cells in Example 6. among them:
  • Figure 5 (a) is a histogram of qPCR detection of BTN3A2 and BTN3A3 results, indicating that breast cancer cell lines express BTN3A2 and BTN3A3;
  • Figure 5 (b) is a peak map showing the expression of BTN3A in the breast cancer cell line, indicating that BTN3A is expressed on the surface of breast cancer cell lines;
  • Figure 5 (c) is a graph showing the results of immunofluorescence staining of cells showing the localization of BTN3A3 on the surface membrane of breast cancer cells.
  • FIG. 6 shows that BTN3A3 expressed by tumor cells in Example 7 promotes tumor formation. among them:
  • Figure 6 (a) is a histogram of qPCR detection of BTN3A2 and BTN3A3 results, indicating shRNA low knockdown of BTN3A2 and BTN3A3 expression;
  • Figure 6(b) is a photograph showing the results of Western Blot of protein levels of BTN3A3, showing the effect of shRNA knockdown on the expression of BTN3A3 protein;
  • Figure 6 (c) is a test result curve of tumor volume in a human-nude mouse breast cancer transplantation model, showing a tumor volume comparison of 231-NC and 231-sh4;
  • Figure 7 is a scatter plot of the BTN3A3 adhesion expressed by the flow-through detection of LSECtin recombinant protein and tumor cells in Example 8, indicating direct and specific interaction between human and murine LSECtin and BTN3A3. among them:
  • Figure 7 (a) is a scatter plot of BTN3A3 adhesion expressed by human-derived LSECtin and tumor cells by flow cytometry, indicating that there is a direct and specific interaction between human LSECtin recombinant protein and BTN3A3;
  • Figure 7 (b) is a scatter plot of BTN3A3 adhesion expressed by murine LSECtin and tumor cells by flow cytometry, showing direct and specific interaction between murine LSECtin and BTN3A3.
  • Figure 8 shows that LSECtin promotes tumor cells by interacting with BTN3A3 expressed by tumor cells in Example 9. Dry. among them:
  • Fig. 8(a) and Fig. 8(b) are bar graphs showing the number of spheres, indicating that LSECtin/BTN3A3 promotes the ability of tumor cells to form MDA-MB-231;
  • Figure 8 (c) is a histogram of qPCR detection of dry transcription factor expression levels, indicating that LSECtin interacts with BTN3A3 to promote upregulation of tumor cell dry transcription factors.
  • Figure 9 shows the interaction of LSECtin with BTN3A3 expressed by tumor cells in Example 10 to promote STAT3 phosphorylation in tumor cells to promote tumor cell dry maintenance. among them:
  • Figure 9 (a) is a photograph of the results of the Wethern Blot assay for LSECtin/BTN3A3 interaction to promote STAT3 phosphorylation in tumor cells, indicating no activation of other STAT molecules;
  • Figure 9(b) is a histogram of the number of spheres in which LSECtin/BTN3A3 promotes tumor cell formation, indicating that STAT3 inhibitors block LSECtin/BTN3A3 promoting tumor cell glomeration.
  • Figure 10 is a photograph showing the results of Coomassie blue staining of the recombinantly expressed fusion protein BTN3A3-Ig;
  • Figure 11 is a photograph showing the results of Western Blot detection of the recombinantly expressed fusion protein BTN3A3-Ig;
  • Figure 12 is a graph showing the Elisa detection result of the binding activity of the fusion protein BTN3A3-Ig to the LSECtin protein;
  • Figure 13 is a scattergram of the adhesion test results of the fusion protein BTN3A3-Ig blocking the interaction between the LSECtin protein and the membrane form BTN3A3;
  • Figure 14 is a graph showing the detection results of tumor volume of macrophage-specific knockout LSECtin spontaneous breast cancer model mice
  • Figure 15 is a graph showing the results of detection of the number of tumor foci in macrophage-specific knockout LSECtin spontaneous breast cancer model mice;
  • Figure 16 is a bar graph showing the results of detection of the number of lung tumor metastases in macrophage-specific knockout LSECtin spontaneous breast cancer model mice;
  • Figure 17 is a bar graph showing the results of detection of the fusion protein BTN3A3-Ig blocking LSECtin to promote tumor cell dryness;
  • Figure 18 is a graph showing the results of inhibition of tumor progression by the fusion protein BTN3A3-Ig;
  • Figure 19 is a graph showing the results of inhibition of tumor progression-dependent LSECtin by the fusion protein BTN3A3-Ig;
  • Fig. 20 is a diagram showing the sections of heart, liver, spleen, lung and kidney after the action of the experimental drug of Example 16, showing the results of the detection of the non-toxic side effects of the fusion protein BTN3A3-Ig.
  • Figure 21 is a photograph showing the results of Coomassie blue staining of the recombinantly expressed fusion protein BTN3A3-mIg;
  • Figure 22 is a photograph showing the results of Western Blot detection of recombinantly expressed fusion protein BTN3A3-mIg;
  • Figure 23 is a photograph showing the results of flow cytometry of the ratio of binding of monoclonal antibody 5E08 to BTN3A3 secreted by hybridoma cells;
  • Figure 24 is a photograph showing the results of an adhesion test of the monoclonal antibody 5E08 blocking the interaction between the LSECtin protein and the membrane form BTN3A3;
  • Figure 25 is a bar graph showing the results of detection of monoclonal antibody 5E08 blocking LSECtin to promote tumor cell dryness
  • Figure 26 is a graph showing the results of a mouse prophylactic model test for the inhibitory effect of monoclonal antibody 5E08 on tumor progression;
  • Figure 27 is a graph showing the results of a mouse therapeutic model test for the inhibitory effect of monoclonal antibody 5E08 on tumor progression;
  • Figure 28 is a diagram showing the sections of heart, liver, spleen, lung and kidney after the action of the experimental drug of Example 26, showing that the monoclonal antibody 5E08 has no toxic and side effects on the mouse prevention model;
  • Figure 29 is a sectional view of the heart, liver, spleen, lung and kidney tissues of the Example 26 after the action of the experimental drug, showing that the monoclonal antibody 5E08 has no toxic side effects on the mouse treatment model.
  • LSECtin protein function includes negative regulation of T cell immune response in melanoma, promotion of inflammatory reaction induced by Ebola virus, inhibition of CTL-dependent HBV virus clearance, promotion of colon cancer tumor cell liver Shift, the inventors He Fuchu, Tang Li et al found that LSECtin may be used as a target for melanoma immunotherapy (CN104906575A, 2014100898325), and also found that LSECtin or a fusion protein containing LSECtin can be used to prepare drugs that inhibit cancer cells from metastasis to the liver, LSECtin Proteins and their fusion proteins can adhere to colon cancer cells and inhibit the homing migration of colon cancer cells to the liver, and become a new target for anti-adhesion treatment of tumor liver metastasis (CN101732715A, 2008102257147).
  • the functions of the LSECtin protein present in tumor cells or in metastatic organs disclosed in these prior art do not give an indication of whether LSECtin affects the formation and development of tumors in the case where the tumor cells themselves do not express LSECtin.
  • the inventors unexpectedly discovered that tumor-associated macrophages also express LSECtin protein, and that the LSECtin protein expressed by the tumor-associated macrophage has characteristics of promoting tumor formation and development.
  • the mechanism by which LSECtin protein exerts this property is to mediate the direct contact of tumor-associated macrophages with tumor cells and to promote the maintenance of tumor cell dryness.
  • the present invention first proposes that LSECtin proteins expressed by tumor-associated macrophages can be used as targets for immunotherapy of such tumors.
  • LSECtin Liver Sinusoidal Endothelial Cells lectin
  • LSECtin Liver Sinusoidal Endothelial Cells lectin
  • the inventors further explored the interaction membrane protein of LSECtin and screened and inhibited LSECtin and This kind of membrane protein interaction material provides a new idea for the targeted treatment of tumor cell stem.
  • LSECtin can interact with BTN3A3 (Butyrophilin subfamily 3 member A3) expressed on the surface of tumor cells, thereby promoting the dryness of tumor cells and promoting the occurrence and development of tumors.
  • BTN3A3 Butyrophilin subfamily 3 member A3
  • BTN3A3 (Butyrophilin subfamily 3 member A3) is a type I transmembrane glycoprotein located in human 6p22.2 and is a member of the B7 superfamily. It is known that BTN3A3 protein may negatively regulate lymphocyte activity, and gene mutations encoding BTN3A3 protein are associated with tumor susceptibility (Peedicayil, A., et al. Risk of ovarian cancer and inherited variants in relapse-associated genes. PLoS One 5, E8884 (2010).).
  • the inventors have experimentally verified that it can be used as a target in tumor immunotherapy in the present invention.
  • LSECtin based on the interaction of LSECtin with BTN3A3 expressed on the surface of tumor cells to promote the discovery of dryness of tumor cells, the inventors screened and excavated substances capable of blocking the interaction between LSECtin and BTN3A3, and developed them to inhibit tumor-associated giants.
  • a targeted drug that promotes the development of tumor dryness by phagocytes.
  • a substance capable of blocking the interaction between LSECtin and BTN3A3 is proposed, which is used to inhibit the occurrence and development of tumors and inhibit tumor progression.
  • the substance provided by the present invention is an RNA molecule that interferes with the expression of BTN3A2 and BTN3A3 or an RNA molecule that interferes with the expression of BTN3A3.
  • the substance provided by the present invention is a fusion protein mainly composed of two functional parts of human BTN3A3 and human IgG1, and can block the interaction between LSECtin and cell surface expressed BTN3A3, and the fusion protein is named BTN3A3. -Ig.
  • the substance provided by the present invention is a monoclonal antibody having an activity of inhibiting tumor progression, and is named 5E08.
  • the present invention also proposes a hybridoma cell line anti-P3 (5E08) capable of secreting 5E08.
  • the percentage concentration is mass/mass (W/W, unit g/100g) percentage concentration, mass/volume (W/V, unit g/100mL) percentage concentration or volume/volume (V/V, unless otherwise specified). Percent concentration in units of mL/100 mL).
  • RNA extraction kit cDNA reverse transcription kit (A3500) and Mix (A6001) in the following examples are all products of Promega.
  • the cell counting plate (145-0011) in the following examples is a product of Biorad Corporation.
  • the CCK8 Cell Counting Kit (CK04) in the following examples is a product of Dongren Chemical Technology Co., Ltd.
  • qPCR primers of LSECtin (QT01034446), BTN3A2 (QT00060039) and BTN3A3 (QT00060039) in the following examples were synthesized by Qiagen.
  • mice The 4-6 week female nude mice in the following examples are products of the company.
  • the collagenase IV (C5138) in the following examples is a product of Sigma.
  • the DNase I in the following examples is a product of Ximeijie Company.
  • mice MHCII Percp-cy5.5 in the following examples is a product of Biolegend.
  • mice Ly6G APC-cy7 (560600), human CD14 V500 (561392) and human CD15 PE-CF594 (562372) in the following examples are all products of BD Corporation.
  • the blocking solution in the following examples the solvent is water, the solute is Na 2 HPO 4 , KH 2 PO 4 , NaCl and skim milk; the solute Na 2 HPO 4 , KH 2 PO 4 , NaCl and skim milk in the blocking solution
  • concentrations were 0.02 M, 0.0015 M, 0.14 M and 3% (mass percentage), respectively.
  • the human breast cancer cells MDA-MB-231 in the following examples were purchased from the National Experimental Cell Resource Sharing Platform and cultured according to the culture provided by the platform.
  • the human breast cancer cell line MDA-MB-231 was cultured in RPMI 1640 medium (Thermofish, Cat. No. SH30809.01B) containing 10% fetal calf serum (Gibico, Cat. No. 10100-147-FBS) at 37 °C. Incubate with 5% CO 2 .
  • the MMTV-PyMT in the following examples are spontaneous breast cancer model mice, LSECtin +/+ PyMT is a wild-type spontaneous breast cancer model mouse, and LSECtin -/- PyMT is an LSECtin knock-out spontaneous breast cancer model mouse.
  • the specific methods were as follows: C57Bl/6J background wild type MMTV-PyMT spontaneous breast cancer model male rats were mated with C57Bl/6J background LSECtin knockout female mice, and LSECtin was obtained as a heterozygous spontaneous breast cancer model male mouse.
  • genotype identification can obtain LSECtin + / + PyMT (LSEC-expressing wild-type spontaneous breast cancer model mice) and LSECtin -/- PyMT (LSECtin -/- PyMT is LSECtin) Knock out spontaneous breast cancer model mice).
  • PyMT represents a spontaneous breast cancer model transgene
  • +/+ represents wild-type homozygotes
  • -/- represents knockout homozygotes.
  • the above C57Bl/6J background LSECtin knockout mice are described in the literature "Tang L, Yang J, Liu W, et al.
  • Gastroenterology, 2009, 137 (4): published in 1498-1508.e5. the public can obtain from the Academy of Military Medical Sciences.
  • the above C57B1/6J background wild type MMTV-PyMT spontaneous breast cancer model mouse is in the literature "Davie SA, Maglione JE, Manner CK, et al. Effects of FVB/NJ and C57Bl/6J strain backgrounds on mammary tumor phenotype in inducible nitric oxide Synthase deficient mice [J].
  • Transgenic research, 2007, 16 (2): 193-201. published in the public, the public can be obtained from the Academy of Military Medical Sciences.
  • LSECtin +/+ Nude -/- was a wild type nude mouse
  • LSECtin -/- Nude -/- was an LSECtin knockout nude mouse.
  • the specific methods were as follows: BALB/c background male nude mouse LSECtin +/+ Nude -/- (purchased from Vitaliv) was mated with BALB/c background female LSECtin -/- Nude +/+ mice to obtain LSECtin + /- Nude +/- mice.
  • LSECtin +/- Nude +/- mice Male LSECtin +/- Nude +/- mice were mated with female LSECtin +/- Nude +/- mice, and female nude mice in their offspring were genotyped to obtain LSECtin +/+ Nude -/- (LSECtin Expression of wild-type nude mice) and LSECtin -/- Nude -/- (LSECtin knockout nude mice).
  • the mouse anti-human LSECtin antibody CCB059 in the following examples is in the literature "Zhao D, Han X, Zheng X, et al. Correction: The Myeloid LSECtin Is a DAP12-Coupled Receptor That Is Crucial for Inflammatory Response Induced by Ebola Virus Glycoprotein [J]. PLoS pathogens, 2016, 12 (3).” disclosed in the public can be obtained from the Academy of Military Medical Sciences.
  • the rabbit anti-mouse LSECtin polyclonal antibody in the following examples is described in the literature "Xu F, Liu J, Liu D, et al. LSECtin expressed on melanoma cells promotes tumor progression by inhibiting antitumor T-cell responses [J]. Cancer research, Published in 2014, 74(13): 3418-3428., the public is available from the Academy of Military Medical Sciences.
  • the murine source LSECtin-Fc in the following examples is in the literature "Tang L, Yang J, Tang X, et al.
  • the DC-SIGN family member LSECtin is a novel ligand of CD44on activated T cells [J]. European journal of immunology, 2010, 40(4): 1185-1191.” The public is available from the Academy of Military Medical Sciences.
  • Example 1 LSECtin promotes tumor formation
  • LSECtin promotes tumor formation and tumor progression in spontaneous breast cancer in mice
  • the spontaneous breast cancer model mouse LSECtin +/+ PyMT and the spontaneous breast cancer model mouse LSECtin -/- PyMT were separately raised and propagated in the experimental animal platform of the Academy of Military Medical Sciences. Tumor volume was measured starting at 13 weeks. Then, once a week, the long diameter a and the short diameter b of the mouse tumor were measured using a vernier caliper, and the tumor volume was calculated. The calculation formula of the tumor volume was 0.5*ab 2 . The mice were sacrificed until 22 weeks.
  • Fig. 1(a) The results of the detection of tumor volume in spontaneous breast cancer model mice are shown in Fig. 1(a), at the 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, and 22th week, LSECtin + /
  • the tumor volumes of the + -PyMT group were 0.000 ⁇ 0.000, 0.000 ⁇ 0.000, 0.000 ⁇ 0.000, 1.553 ⁇ 2.763, 3.566 ⁇ 4.912, 7.049 ⁇ 12.477, 13.867 ⁇ 18.089, 27.189 ⁇ 25.164, 90.020 ⁇ 53.954, and 176.631 ⁇ 80.076, respectively.
  • the tumor volume of the mice was 0.000 ⁇ 0.000, 0.000 ⁇ 0.000, 0.000 ⁇ 0.000, 0.658 ⁇ 1.612, 0.927 ⁇ 2.269, 1.795 ⁇ 4.397, 7.136 ⁇ 7.950, 12.340 ⁇ 13.105, 23.115 ⁇ 27.922, 36.883 ⁇ 41.680, 103.638 ⁇ 91.174, 173.123 ⁇ 112.662 (in mm 3 ).
  • mice in the LSECtin -/-- PyMT group were significantly smaller than that in the LSECtin +/+- PyMT group, indicating that LSECtin promoted tumorigenesis and tumor progression in spontaneous breast cancer in mice.
  • a single spontaneous breast cancer model mouse LSECtin +/+ PyMT and a single spontaneous breast cancer model mouse LSECtin -/- PyMT were separately raised and propagated in the experimental animal platform of the Academy of Military Medical Sciences. The number of single tumor foci was counted starting at 14 weeks, and then observed every two weeks until 20 weeks, and the mice were sacrificed.
  • Fig. 1(b) The results of the number of tumor foci in spontaneous breast cancer model mice are shown in Fig. 1(b). It can be seen from the figure that the number of tumor lesions in LSECtin -/-- PyMT single mice is significantly less than that of LSECtin +/+. -PyMT mice. This indicates that LSECtin promotes tumorigenesis and tumor progression in spontaneous breast cancer in mice.
  • a single spontaneous breast cancer model mouse LSECtin +/+ PyMT and a single spontaneous breast cancer model mouse LSECtin -/- PyMT were separately raised and propagated in the experimental animal platform of the Academy of Military Medical Sciences.
  • the mice were sacrificed, the lungs were removed, HE staining results were obtained by paraffin embedding, tissue sectioning and staining, and the number of lung metastases per section was obtained by counting.
  • human breast cancer cells MDA-MB-231, Matrigel (BD, 354230) and PBS (Hyclone, SH30256.01) were mixed to obtain a mixture; the mixture was planted for 5 weeks, female LSECtin +/+ Nude -/- nude
  • the human-nude mouse breast cancer transplantation model was established in the lower mammary gland of the mouse (LSECtin +/+ Nude -/- group) and LSECtin -/- Nude -/- nude mouse (LSECtin -/- Nude -/- group).
  • the long diameter a and the short diameter b of the mouse tumor were measured using vernier calipers, and the tumor volume and tumor formation rate were calculated.
  • the calculation formula of tumor volume was 0.5*ab 2
  • the calculation formula of the tumor formation rate was Tumor formation (only) / total number of models (only).
  • Fig. 1(d) The results of the tumor volume detection of LSECtin +/+ Nude -/- group and LSECtin -/- Nude -/- group nude mice are shown in Fig. 1(d) at 0, 1, 2, 3, 4, 5, and 6.
  • the tumor volumes of the LSECtin +/+ -Nude -/- group were 0.000 ⁇ 0.000, 0.000 ⁇ 0.000, 23.820 ⁇ 9.802, 49.392 ⁇ 23.256, 73.482 ⁇ 38.720, 129.332 ⁇ 86.165, 228.424 ⁇ 170.106, 329.700, respectively.
  • the tumor volumes of the LSECtin -/- Nude -/- group were 0.000 ⁇ 0.000, 0.000, respectively. ⁇ 0.000, 5.953 ⁇ 6.052, 13.096 ⁇ 10.624, 25.466 ⁇ 26.931, 37.257 ⁇ 40.210, 65.645 ⁇ 53.518, and 91.430 ⁇ 59.608 (in mm 3 ).
  • the tumor volume of LSECtin -/- Nude -/- mice was significantly smaller than that of LSECtin +/+ -Nude -/- mice, indicating that LSECtin can promote the formation of breast cancer in mice.
  • the results of the tumor formation rate of LSECtin +/+ Nude -/- group and LSECtin -/- Nude -/- group nude mice are shown in Table 1.
  • the tumor formation rate of LSECtin -/- Nude -/- mice was significantly lower than that of LSECtin. +/+ Nude -/- mice. This indicates that LSECtin can promote the formation of tumors in mouse breast cancer.
  • MMTV-PyMT spontaneous breast cancer mouse model human-nude mouse breast cancer transplantation model and clinical samples of tumor infiltrating myeloid cells
  • the tumor digest was filtered through a 70 ⁇ m sieve and centrifuged at 250 g for 10 min.
  • tumor-associated macrophage TAM (CD45 + CD11b + CD11c + MHCII + Ly6C - Ly6G - ); monocyte Mo (CD45 + CD11b + CD11c - MHC - Ly6C + Ly6G - ); tumor-associated neutrophil TAN (CD45 + CD11b + CD11c +/- MHCII - Ly6C + Ly6G + ); other myeloid cells CD11b - MHCII - (CD45 + CD11b - MHCII - ).
  • step 1 (1) infiltrating the myeloid cells of each mouse model obtained in step 1, in accordance with the method of RNA extraction kit, extracting RNA; synthesizing cDNA according to the method of cDNA synthesis kit;
  • the LSECtin and GAPDH were amplified using a real-time fluorescence quantitative nucleic acid amplification detection system, and the relative expression of LSECtin was analyzed by software.
  • the primer sequences are as follows:
  • LSECtin upstream primer GGTGCCCATCTGGTGATTGT;
  • LSECtin downstream primer CAGTGGCTGAAGTTGAGTGAGG;
  • GAPDH upstream primer AGGTCGGTGTGAACGGATTTG;
  • GAPDH downstream primer TGTAGACCATGTAGTTGAGGTCA.
  • Fig. 2(a) shows qPCR detection of LSECtin expression in the spontaneous breast cancer microenvironment of MMTV-PyMT mice
  • Fig. 2(b) shows qPCR detection of human-nude mouse breast cancer transplantation model tumor microenvironment
  • TAM breast cancer tumor-associated macrophages
  • TAN granulocytes
  • the specimen was added with a small amount of OCT embedding agent, placed in a cryostat, sliced to a thickness of 4-5 ⁇ m, and attached to a glass slide for use. Dry at room temperature for 30 min.
  • Tritonx-100 First 3% Tritonx-100 10 ml (0.3 ml Trixtonx-100 stock solution. 9.7 ml PBS), and then diluted to 0.3% tritonx-100.
  • Tissue cells were isolated from fresh clinical patient tumor samples and tumor infiltrating immune cell subsets were obtained by flow cytometry.
  • the specific steps are as follows: the living cell population is sequentially removed by SSC-H/FSC-W and SSC-W/FSC-H to remove adhesion cells, SSC-A and FSC-A to remove cell debris, CD45 + to obtain immune cell subsets, CD3 - CD19 - CD56 - Obtained myeloid cell enrichment subgroup, CD14, CD11b and CD15 define TAM (CD11b + CD14 + CD15 - ), and obtain CD45 + CD3 - CD15 - CD19 - CD56 - CD11b + CD14 + tumor-associated macrophages
  • the test group was labeled with LSECtin flow antibody; the isotype control antibody was used as the control group.
  • LSECtin is highly expressed in tumor-associated macrophages of breast cancer CD45 + CD3 - CD15 - CD19 - CD56 - CD11b + CD14 + .
  • Tissue cells were isolated from tumor samples from fresh clinical myeloma patients, and tumor infiltrating immune cell subsets were obtained by flow cytometry.
  • the specific steps are as follows: the living cell population is sequentially removed by SSC-H/FSC-W and SSC-W/FSC-H to remove adhesion cells, SSC-A and FSC-A to remove cell debris, CD45 + to obtain immune cell subsets, CD3 - CD19 - CD56 - Obtained myeloid cell enrichment subgroup, CD14, CD11b and CD15 define TAM (CD11b + CD14 + CD15 - ), and obtain CD45 + CD3 - CD15 - CD19 - CD56 - CD11b + CD14 + tumor-associated macrophages
  • the test group was labeled with LSECtin flow antibody; the isotype control antibody was used as the control group.
  • LSECtin is highly expressed in myeloma CD45 + CD3 - CD15 - CD19 - CD56 - CD11b + CD14 + tumor-associated macrophages.
  • Tissue cells were isolated from tumor samples of fresh clinical lung cancer patients, and tumor infiltrating immune cell subsets were obtained by flow cytometry.
  • the specific steps are as follows: the living cell population is sequentially removed by SSC-H/FSC-W and SSC-W/FSC-H to remove adhesion cells, SSC-A and FSC-A to remove cell debris, CD45 + to obtain immune cell subsets, CD3 - CD19 - CD56 - Obtained myeloid cell enrichment subgroup, CD14, CD11b and CD15 define TAM (CD11b + CD14 + CD15 - ), and obtain CD45 + CD3 - CD15 - CD19 - CD56 - CD11b + CD14 + tumor-associated macrophages
  • the test group was labeled with LSECtin flow antibody; the isotype control antibody was used as the control group.
  • LSECtin is highly expressed in lung cancer CD45 + CD3 - CD15 - CD19 - CD56 - CD11b + CD14 + tumor-associated macrophages.
  • Tissue cells were isolated from tumor samples from fresh clinical colon cancer patients, and tumor infiltrating immune cell subsets were obtained by flow cytometry.
  • the specific steps are as follows: the living cell population is sequentially removed by SSC-H/FSC-W and SSC-W/FSC-H to remove adhesion cells, SSC-A and FSC-A to remove cell debris, CD45 + to obtain immune cell subsets, CD3 - CD19 - CD56 - Obtained myeloid cell enrichment subgroup, CD14, CD11b and CD15 define TAM (CD11b + CD14 + CD15 - ), and obtain CD45 + CD3 - CD15 - CD19 - CD56 - CD11b + CD14 + tumor-associated macrophages
  • the test group was labeled with LSECtin flow antibody; the isotype control antibody was used as the control group.
  • LSECtin is highly expressed in colon cancer-associated macrophages of colon cancer CD45 + CD3 - CD15 - CD19 - CD56 - CD11b + CD14 + .
  • Tissue cells were isolated from tumor samples from patients with fresh clinical giant cell tumors, and tumor infiltrating immune cell subsets were obtained by flow cytometry.
  • the specific steps are as follows: the living cell population is sequentially removed by SSC-H/FSC-W and SSC-W/FSC-H to remove adherent cells, SSC-A and FSC-A to remove cell debris, CD45 + to obtain immune cell subsets, CD3 - CD19 - CD56 - Obtained myeloid cell enrichment subgroup, CD14, CD11b and CD15 define TAM (CD11b + CD14 + CD15 - ), and obtain CD45 + CD3 - CD15 - CD19 - CD56 - CD11b + CD14 + tumor-associated macrophages
  • the test group was labeled with LSECtin flow antibody; the isotype control antibody was used as the control group.
  • LSECtin is highly expressed in tumor-associated macrophages of giant cell tumor CD45 + CD3 - CD15 - CD19 - CD56 - CD11b + CD14 + .
  • Tissue cells were isolated from tumor samples of fresh clinical kidney cancer patients, and tumor infiltrating immune cell subsets were obtained by flow cytometry.
  • the specific steps are as follows: the living cell population is sequentially removed by SSC-H/FSC-W and SSC-W/FSC-H to remove adhesion cells, SSC-A and FSC-A to remove cell debris, CD45 + to obtain immune cell subsets, CD3 - CD19 - CD56 - Obtained myeloid cell enrichment subgroup, CD14, CD11b and CD15 define TAM (CD11b + CD14 + CD15 - ), and obtain CD45 + CD3 - CD15 - CD19 - CD56 - CD11b + CD14 + tumor-associated macrophages
  • the test group was labeled with LSECtin flow antibody; the isotype control antibody was used as the control group.
  • LSECtin is highly expressed in tumor-associated macrophages of the renal cell carcinoma CD45 + CD3 - CD15 - CD19 - CD56 - CD11b + CD14 + .
  • Tissue cells were isolated from tumor samples from fresh clinical laryngeal cancer patients, and tumor infiltrating immune cell subsets were obtained by flow cytometry.
  • the specific steps are as follows: the living cell population is sequentially removed by SSC-H/FSC-W and SSC-W/FSC-H to remove adhesion cells, SSC-A and FSC-A to remove cell debris, CD45 + to obtain immune cell subsets, CD3 - CD19 - CD56 - Obtained myeloid cell enrichment subgroup, CD14, CD11b and CD15 define TAM (CD11b + CD14 + CD15 - ), and obtain CD45 + CD3 - CD15 - CD19 - CD56 - CD11b + CD14 + tumor-associated macrophages
  • the test group was labeled with LSECtin flow antibody; the isotype control antibody was used as the control group.
  • LSECtin is highly expressed in tumor-associated macrophages of laryngeal carcinoma CD45 + CD3 - CD15 - CD19 - CD56 - CD11b + CD14 + .
  • Tissue cells were isolated from tumor samples from fresh clinical salivary gland cancer patients, and tumor infiltrating immune cell subsets were obtained by flow cytometry.
  • the specific steps are as follows: the living cell population is sequentially removed by SSC-H/FSC-W and SSC-W/FSC-H to remove adhesion cells, SSC-A and FSC-A to remove cell debris, CD45 + to obtain immune cell subsets, CD3 - CD19 - CD56 - CD15 - obtains a non-granulocyte-rich subpopulation of myeloid cells, in which CD11b+ defines TAM, and CD45 + CD3 - CD15 - CD19 - CD56 - CD11b + tumor-associated macrophages (TAM group); CD45 + CD3 - CD15 + CD19 - CD56 - CD11b + is a non-TAM group.
  • LSECtin is highly expressed in tumor-associated macrophages of the salivary gland cancer CD45 + CD3 - CD15 - CD19 - CD56 - CD11b + .
  • the MMTV-PyMT in the following examples are spontaneous breast cancer model mice; LyZ2 Cre is a LyZ2 gene Cre enzyme knock-in mouse; LSECtin fl/fl is a LoxP knock-in mouse; WT-PyMT-LyZ2 is wild-type spontaneous In a breast cancer model mouse, KO-PyMT-LyZ2 is a macrophage LSECtin knockout spontaneous breast cancer model mouse.
  • the specific methods were as follows: C57Bl/6J background wild type MMTV-PyMT spontaneous breast cancer model male rats were mated with C57Bl/6J background LyZ2 Cre female mice, and genotype identification, male clones of spontaneous breast cancer model with Cre enzyme knock-in were obtained.
  • the mouse was mated with C57Bl/6J background LSECtin fl/fl female, and the male model of spontaneous breast cancer model with Cre enzyme knock-in and LoxP gene heterozygous was obtained by genotype identification.
  • the mouse was mated with LoxP knock-in mice to obtain WT-PyMT-LyZ2 mice (MMTV + LyZ2 Cre- LSECtin fl/fl ) and KO-PyMT-LyZ2 mice (MMTV + LyZ2 Cre+ LSECtin fl/fl ).
  • MMTV represents a spontaneous breast cancer model transgene
  • Cre- represents Cre enzyme not knocked in
  • Cre+ represents Cre enzyme knock-in
  • fl/- represents LoxP gene knock-in heterozygote
  • fl/fl represents LoxP gene knock-in homozygote.
  • the above-mentioned Cre knock-in mice can be purchased from Jackson, and LoxP knock-in mice can be purchased from TACONIC, and the public can also obtain from the Beijing Proteome Research Center.
  • the above C57B1/6J background wild type MMTV-PyMT spontaneous breast cancer model mouse is in the literature "Davie SA, Maglione JE, Manner CK, et al.
  • mice WT-PyMT-LyZ2 and the experimental group KO-PyMT-LyZ2, which were knocked out of macrophage knockout LSECtin, were housed and propagated in the animal platform of the National Proteome Research Center. Tumor volume was measured starting at 13 weeks. Then, once a week, the long diameter a and the short diameter b of the mouse tumor were measured using a vernier caliper, and the tumor volume was calculated. The calculation formula of the tumor volume was 0.5*ab 2 . The mice were sacrificed until 21 weeks.
  • Spontaneous breast cancer model control mice WT-PyMT-LyZ2 and experimental group KO-PyMT-LyZ2, which were knocked out of macrophage knockout LSECtin, were separately raised and propagated in the National Proteome Research Center experimental animal platform. The number of single tumor foci was counted starting at 14 weeks, and then observed every two weeks until 20 weeks, and the mice were sacrificed.
  • Fig. 15 The results of the detection of the number of tumor foci in spontaneous breast cancer model mice are shown in Fig. 15. As can be seen from the figure, the number of tumors in KO-PyMT-LyZ2 single mice was significantly less than that in WT-PyMT-LyZ2 mice. It indicated that knockout macrophage LSECtin can significantly inhibit tumor formation and tumor progression in mouse breast cancer.
  • a single spontaneous breast cancer model mouse WT-PyMT-LyZ2 and a single spontaneous breast cancer model mouse KO-PyMT-LyZ2 were separately raised and propagated in the animal platform of the National Proteome Research Center. At 25 weeks, the mice were sacrificed, the lungs were removed, HE staining results were obtained by paraffin embedding, tissue sectioning and staining, and the number of lung metastases per section was obtained by counting.
  • Example 4 multiple clinical samples of tumor cells expressing BTN3A2 and BTN3A3
  • step (2) Take the tumor tissue of the fresh clinical patient separately, cut it, and put it into the digestive juice prepared in step (1) to obtain the tumor digestive juice, and digest it at 37 ° C for 40 min.
  • the tumor digest was filtered through a 70 ⁇ m sieve and centrifuged at 250 g for 10 min.
  • the expression level of tumor cell BTN3A in tumor tissues of breast cancer patients was detected.
  • BTN3A flow cytometry results are shown in the expression level in breast cancer patient 3 (a) below: in CD45 - breast cancer tumor cells defined by expression of anti-CD277 antibody is labeled cells BTN3A experimental group, control group labeled isotype control antibody Cell.
  • the expression level of tumor cell BTN3A in tumor tissues of lung cancer patients was detected.
  • BTN3A flow cytometry results are shown in the expression level 3 (b) below: in CD45 - cell lung cancer defined, expressed as the experimental group BTN3A with anti-CD277 antibody labeled cells in the control group isotype control antibody labeled cells.
  • tumor cell BTN3A The expression level of tumor cell BTN3A in tumor tissues of colon cancer patients was detected as described in the above.
  • BTN3A flow cytometry results are shown in the expression level 3 (c) below: in CD45 - colon tumor cells defined by expression BTN3A anti-CD277 antibody labeled cells as the experimental group, control group isotype control antibody labeled cells.
  • tumor cell BTN3A The expression level of tumor cell BTN3A in tumor tissue of patients with giant cell tumor of bone was detected as described in the above.
  • BTN3A flow cytometry results are shown in the expression level 3 (d) below: in CD45 - Giant cell tumor of bone tumors defined, expressed as the experimental group BTN3A with anti-CD277 antibody labeled cells in the control group isotype control antibody-labeled cells .
  • tumor cell BTN3A The expression level of tumor cell BTN3A in tumor tissues of renal cancer patients was detected as described in the above.
  • BTN3A flow cytometry results are shown in the expression level 3 (e) below: in CD45 - renal tumor cells defined by expression of anti-CD277 antibody BTN3A labeled cells as the experimental group, control group isotype control antibody labeled cells.
  • tumor cell BTN3A in tumor tissues of patients with salivary gland cancer was detected.
  • BTN3A flow cytometry results are shown in the expression level 3 (f) below: in CD45 - cells defined parotid gland tumor, expression of the experimental group BTN3A portion with anti-CD277 antibody labeled cells in the control group isotype control antibody-labeled cells .
  • Example 5 multiple tumor cell lines express BTN3A3
  • MCF7 (3111C0001CCC000013), ZR75-1 (3111C0001CCC000090), BT474 (3111C0001CCC000129), T47D (3111C0001CCC000265), MDA-MB-453 (3111C0001CCC000016), SKBR3 (3111C0001CCC000085), MDA-MB-468 (3111C0001CCC000249) ), MDA-MB-436 (3111C0001CCC000352); MDA-MB-231 (3111C0001CCC000013); liver cancer cell lines: BEL-7402 (3131C0001000700010), HepG2 (3111C0001CCC000035), HCC-LM3 (3142C0001000000316), HHCC (3111C0002000000069), Hep3B ( 3111C0001CCC000376), QGY7701 (3131C0001000700042), SMCC7721 (3111C0001CCC000087), Huh7 (3131C0001000700
  • the above cell lines were lysed by RIPA lysate (Thermofisher, 89901), respectively, and the cell lysate was obtained, and the cell lysate was detected by Western Blot, and the expression of BTN3A3 was detected by anti-BTN3A3 antibody (Sigma, HPA007904). Level.
  • RNA extract kit was used to extract the following breast cancer cells: MCF7, ZR75-1, BT474, T47D, SKBR3, MDA-MB-468, MDA-MB-231, MDA-MB-436 RNA; according to the cDNA synthesis kit Method Synthesis of cDNA.
  • BTN3A2, BTN3A3 and GAPDH were amplified using a real-time fluorescent quantitative nucleic acid amplification detection system (qPCR), and the relative expression levels of BTN3A2 and BTN3A3 were analyzed by software.
  • qPCR real-time fluorescent quantitative nucleic acid amplification detection system
  • the anti-CD277 antibody (which simultaneously recognizes BTN3A1, BTN3A2, BTN3A3 in membrane form) against breast cancer cell lines: MCF7, ZR75-1, BT474, MDA-MB-468, MDA-MB-231, MDA-MB- 436 for flow detection.
  • the specific steps were as follows: the experimental group diluted anti-CD277 antibody (eBioscience, 14-2779) in a volume ratio of 1:50 with 1 ⁇ PBS, and the control group diluted the isotype control antibody with a volume of 1:50 in 1 ⁇ PBS (eBioscience, 14- 4714-82), incubate for 30 min at 4 °C. After washing the cells 3 times with 1 x PBS, the supernatant was discarded.
  • the goat anti-mouse PE-labeled fluorescent secondary antibody (Biolegend, 405307) was diluted at a ratio of 1:50 and incubated for 30 min at 4 °C. After washing the cells three times with 1 x PBS, the supernatant was discarded, resuspended in 300 ⁇ l of PBS, and subjected to flow detection.
  • MDA-MB-231 cells in the cultured state were collected, washed three times with PBS, and excess serum was washed away. Dilute a 10 ⁇ permeabilizing solution (Dakko, 421002) into a 1 ⁇ working solution. The cells were resuspended in a permeabilized solution, centrifuged at 350 g for 10 min, the supernatant was discarded, and the procedure was repeated once. The ruptured cells were resuspended in 100 ul permeabilized liquid, and incubated with anti-BTN3A3 antibody (Sigma, HPA007904) diluted 1:200 in a permeabilizing solution, and incubated at 4 ° C for 30 min.
  • anti-BTN3A3 antibody Sigma, HPA007904
  • Both 231-sh3 cells and 231-sh4 cells are human breast cancer cells MDA-MB-231 stably expressing green fluorescent protein and knocking down BTN3A3.
  • 231-NC cells are human breast cancer cells MDA-MB- stably expressing green fluorescent protein. 231. The specific construction steps are as follows
  • sh3 GCCACAGATGGATCTCATATC (SEQ ID NO: 4); sh4: CCCTTCTGCAACAACCAATCA (SEQ ID NO: 5); NC (negative control sequence): TTCTCCGAACGTGTCACGTTTC.
  • the constructed sh3 expression plasmid was transfected into the target cell (human breast cancer cell MDA-MB-231) by lentivirus, and screened by fluorescent protein to obtain a stable cell line 231-sh3 knockdown of BTN3A3.
  • the constructed sh4 expression plasmid was transfected into the target cell (human breast cancer cell MDA-MB-231) by lentivirus, and screened by fluorescent protein to obtain a stable cell line 231-sh4 knocking down BTN3A3.
  • the constructed NC expression plasmid was transfected into the target cells (human breast cancer cells MDA-MB-231) by lentivirus, and screened by fluorescent protein to obtain a control cell line 231-NC.
  • the specific procedure for the above transfection was as follows: 18-24 hours before virus transfection, adherent cells were plated at 1 x 10 5 cells/well into 24-well plates. The number of cells when transfected with lentivirus was about 2 x 10 5 MOI/well. On the next day, the original medium was replaced with 2 ml of fresh medium containing 6 ⁇ g/ml of polybrene (Suzhou Jima), and an appropriate amount of virus suspension was added. The culture was continued for 24 hours, and the virus-containing medium was replaced with fresh medium. Significant fluorescence was observed after 48 hours of viral infection, which was more pronounced after 72 hours. The culture was expanded for one week, and cells with GFP were obtained by flow sorting.
  • RNA extraction and reverse transcription according to the method described in the kit instructions, and the obtained cDNA is subjected to qPCR using qPCR primers.
  • the RNA Mini Kit (74034) was purchased from Qiagen.
  • the RNA Reverse Transcription Kit (Promega) was purchased from Promega.
  • BTN3A1 (QF00264803), BTN3A2 (QT00060039), BTN3A3 (QF00264803) gene qPCR primers were purchased from Qiagen.
  • BTN3A3 protein level expression 10 5 231-sh3 cells, 231-sh4 cells and 231-NC control cells were separately collected, and BTN3A3 protein level expression was detected in 231-sh3 cells, 231-sh4 cells and 231-NC control cells.
  • the specific steps are as follows: VIIPA lysate (Thermofisher, 89901) was used to lyse each cell line, respectively, to obtain cell lysate, and the cell lysate was detected by Western Blot, and detected by anti-BTN3A3 antibody (Sigma, HPA007904).
  • BTN3A3 expression level the specific method refers to the antibody specification.
  • the stable cell line 231-sh3 of BTN3A3 was knocked down as compared with the control cell 231-NC.
  • the expression level of BTN3A3 in the stable cell line 231-sh4 of BTN3A3 was significantly decreased.
  • the control cells 231-NC and 231-sh4 cells were used to implant the mammary fat pad of nude mice in 1000 cells/in situ. After inoculation of the cells, observation was started on the 8th day, and then observed once a week, and the long diameter a and the short diameter b of the mouse tumor were respectively measured using a vernier caliper, and the tumor volume and the tumor formation rate were calculated. The tumor volume calculation formula is 0.5*ab 2 . The mice were sacrificed until 6 weeks.
  • mice at 0, 1, 2, 3, 4, 5, and 6 weeks after inoculation of 231-NC cells were 0.000 ⁇ 0.000, 3.398 ⁇ 5.829, respectively. 10.627 ⁇ 10.152, 32.637 ⁇ 20.258, 60.116 ⁇ 24.670, 158.550 ⁇ 59.933 and 331.362 ⁇ 100.745 (unit: mm 3 ); mice 0, 1, 2, 3, 4, 5, 6 weeks after inoculation of 231-sh4 cells
  • the tumor volumes were 0.000 ⁇ 0.000, 0.000 ⁇ 0.000, 4.068 ⁇ 6.957, 14.460 ⁇ 20.532, 13.208 ⁇ 17.609, 36.183 ⁇ 48.657 and 96.543 ⁇ 127.005 (unit: mm 3 );
  • the results of the tumor formation rate are shown in Table 2.
  • the tumor formation rate of the control cells in the 231-NC group was significantly higher than that in the 231-sh4 group.
  • the BTN3A1 sequence (sequence 6), BTN3A2 sequence (sequence 7), and BTN3A3 sequence (sequence 8) were substituted for the DNA fragments between the NdeI and XhoI cleavage sites of the pIRES2-EGFP vector (Clotech, 6029-1), respectively.
  • step 3 Digestion and collection of each cell obtained in step 2, respectively, using human LSECtin protein (R&D, 2947-CL) and murine LSECtin-Fc protein to adhere to each of the above cells, and detecting human-derived LSECtin adhesion with mouse anti-human LSECtin antibody CCB059
  • the adhesion rate of the murine LSECtin was measured by anti-IgG (Biolegend, 405307), and the adhesion ratio was detected by flow cytometry.
  • the specific steps of the adhesion experiment refer to the literature "Tang L, Yang J, Tang X, et al.
  • the DC-SIGN family member LSECtin is a novel ligand of CD44on activated T cells [J]. European journal of immunology, 2010, 40 (4 ): The method in 1185-1191.”
  • Example 9 LSECtin interacts with BTN3A2 and BTN3A3 to promote the maintenance of tumor cell dryness
  • the spheres were collected by centrifugation at 800 rpm, digested with trypsin (Gibico, 25300120), filtered through a 40 ⁇ m sieve, and subjected to a secondary sphere formation experiment.
  • the LSECitn-stimulated tumor cells were added to each of the above culture systems at a concentration of 100 ng/ml, and each of the cells stimulated by LSECitn was obtained.
  • LSECtin can promote the formation of 231-NC cell spheres in control cells; however, it does not promote the knockdown of 231-sh3 cells of BTN3A2 and BTN3A3.
  • the 231-sh4 cells formed a sphere.
  • LSECtin-stimulated concentration LSECtin failed to promote globular formation of BT474-EGFP overexpressing empty vector; however, it promoted globular formation of BT474-BTN3A2 overexpressing BTN3A2 and BT474-BTN3A3 overexpressing BTN3A3.
  • the 231-sh4 cells and 231-NC cells in the ball formation experiment obtained in step 4 of the first step were collected, and the expression changes of the dry tumor-related characteristic molecules such as OCT4, NANOG, and SOX2 of the breast tumor were detected by qPCR.
  • DC-SIGN represents a negative control
  • Control is a non-LSECtin-stimulated group
  • 231-sh4 is a cell that knocks down BTN3A3
  • 231-NC is a control cell.
  • the primer sequences were as follows: OCT4: Up-GCTCGAGAAGGATGTGGTCC; Down-GTTGTGCATAGTCGCTGCT; NANOG: Up-TCTGGACACTGGCTGAATCCT; Down-CGCTGATTAGGCTCCAACCAT; SOX2: Up-GCTCGCAGACCTACATGAAC; Down-GGGAGGAAGAGGTAACCACA.
  • LSECtin can promote the expression of the dry key transcription factors Oct4, Nanog and Sox at a stimulation concentration of 100 ng of LSECtin; however, this promotion effect cannot be obtained after knocking out BTN3A3.
  • Example 10 LSECtin interacts with BTN3A2 and BTN3A3 to promote tumor progression dependent on activation of STAT3 phosphorylation in tumor cells
  • LSECtin stimulates STAT3 phosphorylation in breast cancer cells expressing BTN3A3
  • the LSECtin-stimulated spheres obtained in Example 4 of Example 8 were separately collected to form a sample: 231-sh3 cells knocking down BTN3A3 (LSECitn-231-sh3), and 231-sh4 cells knocking down BTN3A3 (LSECitn-231-sh4) , cell 231-NC (LSECitn-231-NC) and the LSEC-stimulated sphere obtained in step 3 of Example 8 to form a sample: knock down 231-sh3 cells of BTN3A3 (231-sh3), knock down BTN3A3 231-sh4 cells (231-sh4), cells 231-NC (231-NC).
  • Fig. 9(a) The results are shown in Fig. 9(a).
  • the p-STAT3 levels of 231-NC cells expressing BTN3A2 and BTN3A3 were significantly up-regulated, but the 231-sh3 cells of BTN3A2 and BTN3A3 were knocked down (LSECitn-231-sh3).
  • LSECitn-231-sh4 cells LSECitn-231-sh4 cells
  • LSECtin could not stimulate the up-regulation of p-STAT3 levels; but whether or not the BTN3A3 was knocked down, the phosphorylation levels of STAT1, STAT5, and STAT6 in other STAT families No upregulation.
  • LSECtin promotes the formation of BT3 phosphorylation in breast cancer cells expressing BTN3A2 and BTN3A3.
  • the pellet-forming samples obtained in the fourth step of Example 8 were separately collected: knockdown of BTN3A3 cells 231-sh3, knockdown of BTN3A3 cells 231-sh4, and knockdown of BTN3A3 cells 231-NC, and after collecting samples
  • the STAT3 inhibitor (selleck, S1155) was added for three days and the number of sphere formation was measured on the tenth day.
  • LSECtin promoted the globular formation ability of 231-NC cells expressing BTN3A2 and BTN3A3 normally.
  • the 231-sh3 and 231-sh4 cells of BTN3A2 and BTN3A3 were knocked down.
  • the addition of a STAT3 inhibitor completely prevented the LSECtin from promoting the formation of the ball.
  • Example 1 - Example 10 LSECtin expressed by tumor-associated macrophages and BTN3A2 and/or BNT3A3 expressed by tumor cells promote tumor progression by promoting the maintenance of tumor cell dryness, which is embodied in promoting tumor cell ball formation.
  • the LSECtin is a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in the Sequence Listing; or the amino acid sequence of SEQ ID NO: 1 is substituted and/or deleted and/or added by one or several amino acid residues and has the same Functional protein derived from SEQ ID NO: 1.
  • BTN3A2 is a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 in the Sequence Listing; or the sequence 2 is substituted and/or deleted and/or added by the amino acid sequence of SEQ ID NO: 2 and having the same function. Derived protein.
  • BTN3A3 is the amino acid sequence shown by SEQ ID NO:3 in the Sequence Listing a protein consisting of; or a protein derived from sequence 3 having the amino acid sequence of SEQ ID NO: 3 substituted and/or deleted and/or added by one or several amino acid residues and having the same function.
  • LSECtin and BTN3A3 can be used together as immunotherapy.
  • the present invention further proposes a class of substances which inhibit the interaction of LSECtin with BTN3A2 or BTN3A3, and which can prepare at least one of the following functions (b1)-(b5)
  • the product :
  • the substance which inhibits the interaction of LSECtin with BTN3A2 and BTN3A3 is any one of the following: an RNA molecule which interferes with the expression of BTN3A2 and BTN3A3, an anti-LSECtin antibody, a small molecule inhibitor of LSECtin, a soluble protein of LSECtin, an RNA molecule which interferes with the expression of LSECtin, and an anti-BTN3A2 Antibody, BTN3A2 small molecule inhibitor, BTN3A2 soluble protein, RNA molecule that interferes with BTN3A2 expression, anti-BTN3A3 antibody, BTN3A3 small molecule inhibitor, BTN3A3 soluble protein and RNA molecule that interferes with BTN3A3 expression.
  • RNA molecule which interferes with the expression of BTN3A2 and BTN3A3 or the RNA molecule which interferes with the expression of BTN3A3 is: the shRNA molecule shown in SEQ ID NO: 4 or the sequence 4 is deleted or increased or changed by one or several nucleotides, and the sequence a nucleotide of the same function; or a shRNA molecule of SEQ ID NO: 5 or a nucleotide which deletes or adds or changes one or more nucleotides of sequence 5 and has the same function as that of SEQ ID NO:5.
  • Example 11 - Example 18 is described in detail for the fusion protein BTN3A3-Ig which blocks the interaction of LSECtin with BTN3A3.
  • the substance that blocks the interaction between LSECtin and BTN3A3 is the fusion protein BTN3A3-Ig, and the expression method thereof comprises the following steps:
  • BTN3A3-Ig Construction of the fusion gene BTN3A3-Ig: According to the human BTN3A3 gene sequence searched in the gene bank (GenBank No.: BT007251.1) and human IgG1 gene sequence (GenBank No.: AY623427.1), select BTN3A3 extracellular domain protein (this example considers the interaction of LSECtin and BTN3A3 extracellular domain, blocking the interaction of the protein needs and membrane form BTN3A competes with LSECtin, so BTN3A3 extracellular domain protein is selected, and then the appropriate linker coding sequence is selected, and the fusion gene BTN3A3-Ig is obtained by artificial synthesis.
  • the nucleotide sequence is shown in sequence 10 in the sequence listing.
  • the coding sequence is 1-1470 bases from the 5' end, encoding a protein having the sequence of the amino acid residue shown by 1 in the sequence listing, and the base BTN3A3 from the 5' end of the 46-786 base, from the 5' end 787-798 base-coding linker, encoding human IgG1 from base 57-1470 of the 5' end;
  • the recombinant expression vector pIRES2-EGFP-BTN3A3-Ig containing the fusion gene BTN3A3-Ig was transfected into 293T cells (derived from the national experimental cell resource sharing platform), and the serum-free medium M293TI was purchased.
  • Recombinant 293T cells were cultured at 37 ° C ( ⁇ 0.5 ° C) from Beijing Yiqiao Shenzhou Technology Co., Ltd., filtered through 0.45 ⁇ m filter (PN4614, Pall), and the supernatant was collected and replaced with medium every 24 hours. , until 96 hours (24-120 hours), the end of the culture, the fusion gene BTN3A3-Ig obtained expression;
  • Protein G Sepharose column purchased from Kangwei Century Biotechnology Co., Ltd.
  • Protein G protein G agarose gel column is a cell surface isolated from G or C type Streptococcus The protein, which interacts with the Fc region of immunoglobulin (Ig), binds to most mammalian IgG.
  • Native protein G has albumin and cell surface binding domains, and recombinant Protein G removes albumin and cell surface binding domains.
  • the protein can be used to purify IgG after coupling with Sepharose.
  • Purification of the recombinantly expressed protein by adding the culture supernatant of the cell (recombinant 293T cells containing the fusion gene BTN3A3-Ig) to the equilibrium Buffer (20 mM PBS, 150 mM NaCl, pH 8.0) to pH 8.0, add the cell supernatant to a Protein G Sepharose column that has been equilibrated with equilibration buffer, wash the column with equilibration buffer until no impurities are detected in the effluent The protein was eluted with elution buffer (0.1 M glycine, pH 3.0), and the effluent was collected, neutralized immediately with neutralizing buffer (1 M Tris ⁇ HCl, pH 9.0), and dialyzed against pH 7.2 0.01 mol/L PBS.
  • elution buffer 0.1 M glycine, pH 3.0
  • the fusion protein BTN3A3-Ig was obtained, which was consistent with the expected results.
  • the amino acid sequence of the fusion protein BTN3A3-Ig is shown in SEQ ID NO:9 in the sequence listing, and the sequence 9 is composed of 490 amino acid residues, the first position from the amino (N) end is the start codon, and the amino terminal is from the amino end 2-15.
  • the position is a signal peptide, which is human BTN3A3 from the 16th to the 262th position of the amino terminus, a linker from the amino terminus at positions 263 to 266, and a human IgG1 from the amino terminus at positions 267 to 490.
  • the samples were measured for OD260 and OD280 on a violet spectrophotometer, and the protein content was calculated using a BCA protein quantification kit (purchased from Kangwei Century Biotechnology Co., Ltd.), and the result was 1 mg/ml, and stored at -80 ° C after dispensing.
  • Example 12 Coomassie blue staining and Western Blot detection of fusion protein BTN3A3-Ig
  • the recombinantly expressed BTN3A3-Ig fusion protein sample of Example 11 was collected, and a 10% SDS-PAGE gel was prepared and electrophoresed for Coomassie blue staining.
  • the control was a lysate of cells (recombinant 293T cells containing the fusion gene BTN3A3-Ig).
  • the recombinantly expressed BTN3A3-Ig fusion protein sample of Example 10 was collected, and a 10% SDS-PAGE gel was prepared, electrophoresed, transferred, and blocked with a TBST solution containing 5% skim milk powder, using anti-anti-CD277 (the antibody specification indicates that BTN3A3) (purchased from Thermo Scientific) was used for primary antibody incubation, and secondary antibody incubation was performed with Mouse IgG HRP-conjugated Antibody (purchased from R&D, HAF007).
  • the experimental control protein was human IgG (purchased from R&D).
  • Example 13 Elisa detects the binding activity of fusion protein BTN3A3-Ig to LSECtin protein
  • the LSECtin protein (2947-CL, R&D) was coated at a concentration of 1 ⁇ g/mL overnight at 4 °C.
  • the blocking solution was prepared with PBST at a concentration of 5% skim milk powder. After washing the plate, 300 ⁇ L per well was blocked at room temperature for 2 h. After washing the plate, the fusion protein BTN3A3-Ig was diluted at a concentration of 2 ⁇ g/mL, and incubated for 3 h at room temperature. After washing the plate, the anti-anti-CD277 (the antibody specification indicates that BTN3A3 can be recognized) (purchased from Thermo Scientific) was incubated for 40 min.
  • the secondary antibody Mouse IgG HRP-conjugated Antibody purchased from R&D, HAF007 was incubated for 30 min. Wash plate, develop color, terminate, OD450 reading.
  • the experimental control protein was human IgG.
  • the ELSE assay protein can directly interact with the fusion protein BTN3A3-Ig, while the control protein human IgG cannot be recognized by the anti-CD277 antibody, while the BTN3A3-Ig antibody exhibits a gradient indicating that the above system is purified.
  • the purity of the BTN3A3-Ig protein is good.
  • Example 14 Adhesion assay to detect the interaction between the fusion protein BTN3A3-Ig and the membrane form BTN3A3
  • the purified fusion protein BTN3A3-Ig and LSECtin protein were mixed at a ratio of 1:1, and BT474 overexpressed BTN3A3 cells BT474-BTN3A3, and then adhered to the adhesion test.
  • the DC-SIGN family member LSECtin is a novel ligand of CD44on activated T cells [J]. European journal of immunology, 2010, 40 (4) :1185-1191.”.
  • the experimental control protein was human IgG.
  • the fusion protein BTN3A3-Ig blocked the interaction between LSECtin and BTN3A3.
  • the LSECtin adhesion rate was calculated as LSECtin adhesion-positive cells/ZSG-positive cells.
  • the adhesion rate of BTN3A3 cells was 33.9%, and the formula was 11.5/(11.5+22.4).
  • the adhesion rate of LSECtin to overexpressing BTN3A3 cells was only 3.2%, and the formula was 0.969/( 0.969+29.3).
  • the results showed that BTN3A3-Ig blocked the interaction between LSECtin and BTN3A3.
  • Example 15 Fusion protein BTN3A3-Ig blocks LSECtin to promote tumor cell dryness
  • the B27 (purchased from Life), bFGF (purchased from Sigma), EGF (purchased from Sigma), insulin (purchased from Sigma), heparin (purchased from Sigma), and DMEM/F12 serum-free medium were mixed to obtain a culture system.
  • the concentrations of the respective components in the culture system were: B27 (10 ng/mL), bFGF (20 ng/mL), EGF (20 ng/mL), insulin (5 ⁇ g/mL), and heparin (4 ⁇ g/mL).
  • the breast cancer cell MDA-MB-231 (derived from the National Experimental Cell Resource Sharing Platform) was made into a single cell suspension, and plated at 20,000/mL, and 100 ng of BTN3A3-Ig, LSECtin+IgG, and LSECtin+BTN3A3-Ig were added respectively. After blanking for 7-10 days, the number of spheres larger than 75 ⁇ m in diameter was calculated and photographed.
  • IgG control group intraperitoneal injection of IgG (10 ⁇ g / only)
  • BTN3A3-Ig group intraperitoneal injection of BTN3A3-Ig (10 ⁇ g/only)
  • MDA-MB-231 cells were inoculated separately into each of the above wild type nude mice. After modeling, the protein was injected intraperitoneally every 3 days, and the inhibitory effect of BTN3A3-Ig on tumor progression was observed by measuring the tumor volume.
  • the tumor volumes at 1, 2, 3, 4, 5, 6, 7, and 8 weeks after modeling were as follows:
  • the IgG group was 0 ⁇ 0.35, 9.83 ⁇ 9.22, 18.13 ⁇ 12.68, 41.54 ⁇ 26.64, 134.08 ⁇ 66.72, 362.16 ⁇ 186.56, 661.32 ⁇ 359.54 and 1089.43 ⁇ 584.70 (unit: mm 3 );
  • the BTN3A3-Ig group was 0.00 ⁇ 0.00, 3.72 ⁇ 5.15, 4.25 ⁇ 6.01, 6.23 ⁇ 8.53, 30.45 ⁇ 9.44, 71.80 ⁇ 48.32, 125.24 ⁇ 106.49 and 240.17 ⁇ 255.17 (unit: mm 3 ).
  • the tumor volume of the injected BTN3A3-Ig group was significantly smaller than that of the injected IgG group, indicating that the injection of the fusion protein BTN3A3-Ig can inhibit tumor growth and can be used for the preparation of antitumor drugs and tumors.
  • Example 17 Detection of fusion protein BTN3A3-Ig inhibits tumor progression dependent on LSECtin
  • LSECtin +/+ Nude -/- was a wild type nude mouse
  • LSECtin -/- Nude -/- was an LSECtin knockout nude mouse.
  • the specific methods were as follows: BALB/c background male nude mouse LSECtin +/+ Nude -/- (purchased from Vitaliv) was mated with BALB/c background female LSECtin -/- Nude +/+ mice to obtain LSECtin + /- Nude +/- mice.
  • LSECtin +/- Nude +/- mice Male LSECtin +/- Nude +/- mice were mated with female LSECtin +/- Nude +/- mice, and female nude mice in their offspring were genotyped to obtain LSECtin +/+ Nude -/- (LSECtin Expression of wild-type nude mice) and LSECtin -/- Nude -/- (LSECtin knockout nude mice).
  • the above-mentioned BALB/c background female LSECtin -/- Nude +/+ mouse information is in the literature "Zuo Y, Ren S, Wang M, et al. Novel roles of liver sinusoidal endothelial cell lectin in colon carcinoma cell adhesion, migration[J] Published in .Gut, 2013, 62(8): 1169-1178., the public is available from the National Proteome Research Center.
  • MDA-MB-231 cells were each inoculated into each of the above-mentioned groups of LSECtin knockout nude mice according to the method of Example 16. After modeling, the protein was injected intraperitoneally every 3 days, and the inhibitory effect of BTN3A3-Ig on tumor progression was observed by measuring the tumor volume.
  • the tumor volumes at 1, 2, 3, 4, 5, 6, and 7 weeks after modeling were as follows:
  • the IgG group was 0.00 ⁇ 0.00, 14.08 ⁇ 11.03, 23.89 ⁇ 21.31, 66.90 ⁇ 8.90, 143.47 ⁇ 34.76, 240.21 ⁇ 42.42 and 400.47 ⁇ 28.29 (unit: mm 3 );
  • the BTN3A3-Ig group was 0.00 ⁇ 0.00, 9.93 ⁇ 11.47, 28.19 ⁇ 32.63, 52.12 ⁇ 33.41, 100.68 ⁇ 63.98, 199.67 ⁇ 78.36, and 350.12 ⁇ 45.83 (unit: mm 3 ).
  • Example 18 the fusion protein BTN3A3-Ig inhibits tumors without toxic side effects
  • Example 16 The model in Example 16 was used for the experiment. After treatment with the experimental drug IgG or the fusion protein BTN3A3-Ig, the heart, liver, spleen, lung and kidney tissues of the mice were placed in formalin solution and sent to Beijing Jiasi Jiayang Company for tissue embedding and sectioning. And HE staining.
  • the tissue sections were as shown in Fig. 20.
  • the heart, liver, spleen, lung and kidney tissues of the mice were not damaged, and no obvious inflammatory cell infiltration occurred. , indicating that the intraperitoneal injection of the fusion protein BTN3A3-Ig has no toxic side effects.
  • Example 19 - Example 26 further provides a substance capable of blocking the interaction between LSECtin and BTN3A3, thereby inhibiting the occurrence and development of tumors (inhibiting tumor progression).
  • This substance is a monoclonal antibody capable of blocking the interaction between LSECtin and BTN3A3 and having an activity of inhibiting tumor progression.
  • the present embodiment first obtains an immunogen for preparing the antibody, which is a specially designed fusion protein, named BTN3A3-mIg, which is a small human BTN3A3.
  • BTN3A3-mIg a specially designed fusion protein
  • a recombinant protein obtained by ligating mouse IgG2a by a ligation peptide.
  • the expression method of the fusion protein BTN3A3-mIg comprises the following steps:
  • BTN3A3-mIg Construction of the fusion gene BTN3A3-mIg: The BTN3A3 extracellular domain protein was selected based on the human BTN3A3 gene sequence (GenBank number: BT007251.1) and the mouse IgG2a gene sequence (GenBank number: BC018535.1) searched in the gene bank ( The present invention contemplates that LSECtin interacts with the extracellular domain of BTN3A3, and the protein that blocks its interaction needs to compete with the membrane form BTN3A for binding to LSECtin, thus selecting the BTN3A3 extracellular domain protein), selecting a suitable linker coding sequence, and obtaining it by artificial synthesis.
  • the fusion gene BTN3A3-mIg has a nucleotide sequence as shown in SEQ ID NO: 12 in the sequence listing, and its coding sequence is from the 1st to the 497th base of the 5' end, and encodes a protein having the sequence of the amino acid residue shown by 1 in the sequence listing. From the 5' end, the 1-3th position is the start codon, the base 4-45 signal peptide from the 5' end, from the 5' end 46-786 base encoding human BTN3A3, from the 5' end
  • the 787-798 base encodes a linker, encoding mouse IgG2a from the 5' end of the 799-1494 base, and the stop codon from the 5' end of the 1495-1497;
  • the recombinant expression vector pIRES2-EGFP-BTN3A3-mIg containing the fusion gene BTN3A3-mIg was transfected into 293T cells (derived from the national experimental cell resource sharing platform), and the serum-free medium M293TI was purchased.
  • Recombinant 293T cells were cultured at 37 ° C ( ⁇ 0.5 ° C) from Beijing Yiqiao Shenzhou Technology Co., Ltd., filtered through 0.45 ⁇ m filter (PN4614, Pall), and the supernatant was collected and replaced with medium every 24 hours. To the end of 96 hours (24-120 hours), the fusion gene BTN3A3-mIg was expressed;
  • Protein G Sepharose column purchased from Kangwei Century Biotechnology Co., Ltd.
  • Protein G protein G agarose gel column is a cell surface isolated from G or C type Streptococcus The protein, which interacts with the Fc region of immunoglobulin (Ig), binds to most mammalian IgG.
  • Native protein G has albumin and cell surface binding domains, and recombinant Protein G removes albumin and cell surface binding domains.
  • the protein can be used to purify IgG after coupling with Sepharose.
  • Purification of the recombinantly expressed protein by adding the culture supernatant of the cell (recombinant 293T cells containing the fusion gene BTN3A3-mIg) to the equilibrium Buffer (20 mM PBS, 150 mM NaCl, pH 8.0) to pH 8.0, add the cell supernatant to a Protein G Sepharose column that has been equilibrated with equilibration buffer, wash the column with equilibration buffer until no impurities are detected in the effluent The protein was eluted with an elution buffer (0.1 M glycine, pH 3.0), and the effluent was collected, neutralized immediately with a neutralization buffer (1 M Tris ⁇ HCl, pH 9.0), and PBS was used at pH 7.2 0.01 mol/L.
  • the fusion protein BTN3A3-mIg was obtained, which was consistent with the expected result.
  • the amino acid sequence of the fusion protein BTN3A3-mIg is shown in SEQ ID NO: 11 in the sequence listing, and the sequence 11 in the sequence listing consists of 499 amino acid residues from the amino group (N).
  • the first position of the end is the start codon
  • the second is the signal peptide
  • the 16th to the 262th is the human BTN3A3
  • the 263-266 from the amino terminus is the linker
  • the 267-498 from the amino terminus Mouse IgG2a position 499 is the stop codon.
  • the samples were measured for OD260 and OD280 on a violet spectrophotometer, and the protein content was calculated using a BCA protein quantification kit (purchased from Kangwei Century Biotechnology Co., Ltd.), and the result was 1 mg/ml, and stored at -80 ° C after dispensing.
  • Example 20 Coomassie blue staining and Western Blot detection of fusion protein BTN3A3-mIg
  • the BTN3A3-mIg fusion protein sample recombinantly expressed in Example 19 was collected, and a 10% SDS-PAGE gel was prepared and electrophoresed for Coomassie blue staining.
  • the control was a lysate of cells (recombinant 293T cells containing the fusion gene BTN3A3-mIg).
  • the recombinantly expressed BTN3A3-mIg fusion protein sample of Example 19 was collected, and a 10% SDS-PAGE gel was prepared, electrophoresed, transferred, blocked with TBST solution containing 5% skim milk powder, and anti-human BTN3A3 antibody (purchased from Thermo Fisher)
  • the primary antibody was incubated and the secondary antibody was incubated with Mouse IgG HRP-conjugated Antibody (purchased from R&D, HAF007).
  • the experimental control protein was mouse IgG (purchased from Abcam, ab37355).
  • the results of Western Blot assay are shown in Figure 22. It can be seen that the fusion protein recombinantly expressed in Example 1 can specifically bind to the anti-human BTN3A3 antibody, indicating that the BTN3A3-mIg protein can be obtained from the above protein expression and purification system.
  • the fusion protein BTN3A3-mIg can be used as an immunizing antigen for the preparation of BTN3A3 antibody.
  • Example 21 a hybridoma cell line anti-P3 (5E08) which continuously and stably secretes monoclonal antibody 5E08 which inhibits tumor process activity is obtained.
  • the method for obtaining the hybridoma cell line anti-P3 (5E08) comprises the following steps:
  • BTN3A3-mIg 100 ⁇ g (50-100 ⁇ g of the fusion protein) BTN3A3-mIg was diluted with physiological saline to 0.25 mL (0.25-0.50 mL), and mixed and stirred with an equal volume of Freund's complete adjuvant (purchased from Sigma). ,emulsification.
  • mice were immunized with the fusion protein BTN3A3-mIg as the antigen at a concentration of 400 ⁇ g/mL (200-400 ⁇ g/mL), the immunization dose was 0.25 mL (0.25-0.50 mL), and the bladder was injected subcutaneously into the back of the neck.
  • c healthy female mice (6-8 weeks old, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.), the other intraperitoneal injection;
  • the ratio of the serum of the immunized mouse to the unimmunized mouse serum is greater than 2 (ie, the OD 450 nm value is greater than the negative control 2 times as a positive judgment basis), and can be used to prepare hybridoma cells.
  • mice were dissected from the neck and immersed in 75% alcohol for 5 minutes, then placed in a clean bench, placed abdomen in a dish or fixed on a anatomical plate.
  • spleen cell suspension (cell concentration: 2.5 ⁇ 10 6 -5.0 ⁇ 10 6 /ML), and transferred to In a 50 mL centrifuge tube, add RPMI1640 medium to 30 mL, centrifuge at 1500-2000 rpm for 5 minutes, discard the supernatant, count, and take 1 ⁇ 10 8 cells for use.
  • 2.2 Spleen cells Myeloma cells were mixed at a ratio of 10:1 cells and centrifuged at 2000 rpm for 3 minutes. The supernatant was poured out, and the cell pellet was blanched and placed in a 37 ° C water bath. Add 1 mL of fusion agent in 1 minute (purchased from Sigma) Division), the cells were stirred, 37 ° C water bath for 45 seconds, 1 mL of RPMI1640 medium was added in 1 minute and the cells were stirred, 5 mL of RPMI1640 medium was added every 2 minutes and the cells were stirred.
  • HAT medium purchased from Sigma
  • 80-100 ⁇ L was added dropwise to the gun, and cultured at 37 ° C in a CO 2 incubator.
  • the cells were carefully observed, and the growth state of the cells, the number of hybridoma cells per well, the number of blocks, the presence or absence of contamination of the culture solution, and the nourishment of cells were recorded.
  • the HAT medium was changed, and after 10 days, the HT medium (purchased from Sigma) was cultured for 20 days, and the RPMI1640 medium was further cultured for 48 hours, and the supernatant was collected and arranged in the order of arrangement on the cloning plate. Number, for example, 5E08, etc.
  • the cell supernatant was subjected to Elisa detection according to the method in the first step, and the clone with positive value greater than 1.5 (5E08, etc.) was selected for flow detection, and the specific method is as follows:
  • the BTN3A3 gene sequence (GenBank No.: BC018535.1, BTN3A3 cDNA full length) was replaced with the DNA fragment between the NheI and SalI cleavage sites in the pIRES2-EGFP vector (purchased from Clotech) to obtain the BTN3A3 overexpression vector, named pIRES2. -EGFP-BTN3A3, the nucleotide sequence of which is shown in SEQ ID NO:8 in the Sequence Listing.
  • BTN3A3 overexpression vector pIRES2-EGFP-BTN3A3 and pIRES2-EGFP empty vector were transfected into BT474 cells (from the national experimental cell resource sharing platform). After transfection for 36h, recombinant cells were obtained: BT474-BTN3A3 overexpressing BTN3A3. And BT474-EGFP overexpressing the pIRES2-EGFP empty vector.
  • step 3.3 Digestion Collect the recombinant cells obtained in step 3.2, using mouse IgG (purchased from Thermo Fisher), commercial BTN3A3 flow antibody (purchased from Thermo Scientific) and monoclonal antibodies secreted by hybridoma cells (step 2.3
  • the collected antibody markers were labeled with a goat anti-mouse PE-labeled fluorescent secondary antibody (purchased from Biolegend) at a volume ratio of 1:50 and incubated at 4 ° C for 30 minutes. After washing the cells 3 times with 1 x PBS, the supernatant was discarded. Incubate at 4 ° C for 30 minutes. After washing the cells three times with 1 x PBS, the supernatant was discarded, resuspended in 300 ⁇ L of PBS, and subjected to flow cytometry.
  • the hybridoma cell strain is subcloned by making the hybridoma cells into a single cell suspension and diluting them into 96-well plates plated with trophoblast cells, so that the number of hybridoma cells per well is not more than one. After 10 days of normal culture, the supernatant was taken for Elisa detection, and the cells in the 5 wells with the highest positive value were subcloned again until five subclones were completed, thereby obtaining a hybridoma cell which continuously and stably secreted the antibody.
  • Example 22 Detection and purification of monoclonal antibody 5E08 secreted by hybridoma cell line anti-P3 (5E08)
  • mice were intraperitoneally injected with 0.5 mL of liquid paraffin, and the pretreated mice were used for 2-3 months.
  • the hybridoma cell anti-P3 (5E08) with good culture state was blown down, centrifuged at 1000 rpm for 5 minutes at room temperature, the supernatant was discarded, and the hybridoma cell anti-P3 (5E08) was resuspended and mixed with serum-free RPMI1640 medium, and the cells were incubated. The concentration was adjusted to 2 ⁇ 10 6 /mL, and each mouse was intraperitoneally injected with 0.5 mL.
  • Monoclonal antibody 5E08 in mouse ascites collected in step 1 was purified using the IgM monoclonal antibody purification kit (purchased from Beijing Boaolong Immunotechnology Co., Ltd.).
  • the purified monoclonal antibody 5E08 was subjected to flow cytometry as in Example 3.
  • the BTN3A3-his recombinant protein (purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.) was coated with 8 microplate wells in PBS buffer, placed at 4 ° C for 12 hours, and then washed once with a washing machine PBST. Next, 100 ⁇ L of hybridoma cell anti-P3 (5E08) culture supernatant was added to each well, incubated at 37 ° C for 30 minutes, and then washed 5 times with a plate washer PBST.
  • the subtype test results showed that the heavy chain of monoclonal antibody 5E08 secreted by hybridoma cell anti-P3 (5E08) was IgM.
  • the light chain is Kappa.
  • the Kingsray Company was commissioned to sequence the variable region of the monoclonal antibody 5E08 secreted by the hybridoma cell anti-P3 (5E08).
  • the heavy chain variable region encoding gene of 5E08 has the DNA sequence of SEQ ID NO: 15 in the sequence listing, encoding the amino acid residue sequence shown in SEQ ID NO: 13 in the sequence listing; the light chain variable region encoding gene has the DNA sequence of SEQ ID NO: 16 in the sequence listing. And encodes the amino acid residue sequence shown in SEQ ID NO: 14 in the Sequence Listing.
  • Sequence 13 in the sequence listing consists of 141 amino acid residues
  • sequence 14 in the sequence listing consists of 130 amino acid residues
  • sequence 15 in the sequence listing consists of 423 bases, encoding an amino acid having sequence 13 in the sequence listing
  • the protein of the residue sequence which consists of 390 bases in the sequence listing, encodes a protein having the sequence of the amino acid residue of SEQ ID NO: 14 in the Sequence Listing.
  • Example 23 adhesion assay to detect the activity of monoclonal antibody 5E08 blocking the interaction of LSECtin protein with membrane form BTN3A3
  • the purified monoclonal antibody 5E08 and LSECtin protein were mixed at a ratio of 1:1 according to the amount of the substance, and the BT474 cell line BT474-BTN3A3 expressing BTN3A3 was incubated, and then the adhesion test was performed.
  • the specific steps of the adhesion test refer to the literature "Tang L, Yang J, Tang X, et al.
  • the DC-SIGN family member LSECtin is a novel ligand of CD44on activated T cells [J]. European journal of immunology, 2010, 40 (4 ): The method described in 1185-1191.”
  • the experimental control protein was human IgG.
  • the adhesion test results of monoclonal antibody 5E08 blocking the interaction between LSECtin protein and membrane form BTN3A3 are shown in Fig. 24.
  • the calculation method of LSECtin adhesion rate is LSECtin adhesion positive cells and ZSG positive cells/ZSG positive cells.
  • the adhesion rate of LSECtin to overexpressing BTN3A3 cells was 16.1%, and the formula was 9.72/(9.72+50.6).
  • the monoclonal antibody 5E08 was added, the adhesion rate of LSECtin to overexpressing BTN3A3 cells was 1.4. %, the formula is 0.828/(0.828+58.8).
  • B27 (purchased from Life), bFGF (purchased from Sigma), EGF (purchased from Sigma), insulin (purchased from Sigma), heparin (purchased from Sigma), and DMEM/F12 serum-free medium. After that, a culture system was obtained, and the concentration of each component in the culture system was: B27 (10 ng/mL), bFGF (20 ng/mL), EGF (20 ng/mL), insulin (5 ⁇ g/mL), and heparin (4 ⁇ g/mL). ).
  • the breast cancer cells MDA-MB-231 (derived from the National Experimental Cell Resource Sharing Platform) were prepared as single cell suspensions and plated at 20,000/mL. After adding 100 ng of LSECtin, add 0 ⁇ g/mL, 12.5 ⁇ g/mL, 25 ⁇ g/mL, 50 ⁇ g/mL, 100 ⁇ g/mL monoclonal antibody 5E08, and use human IgG as control. After 7-10 days of culture, calculate spheres larger than 75 ⁇ m in diameter. Quantity and take pictures.
  • IgG control group intraperitoneal injection of human IgG (50 ⁇ g / only);
  • Group 5E08 intraperitoneal injection of monoclonal antibody 5E08 (50 ⁇ g/head).
  • MDA-MB-231 cells were inoculated separately into each of the above groups of mice. After modeling, the protein was injected intraperitoneally every 3 days, and the inhibitory effect of monoclonal antibody 5E08 on tumor progression was observed by measuring the tumor volume.
  • the tumor volume at 1, 2, 3, 4, 5, 6, and 7 weeks after modeling was as follows:
  • the IgG group is 0.00 ⁇ 0.00, 0.00 ⁇ 0.00, 0.00 ⁇ 0.00, 0.00 ⁇ 0.00, 20.67 ⁇ 18.82, 64.98 ⁇ 34.16, 124.03 ⁇ 47.41, 350.71 ⁇ 165.45 and 848.36 ⁇ 243.67 units are mm3);
  • the 5E08 group was 0.00 ⁇ 0.00, 0.00 ⁇ 0.00, 0.00 ⁇ 0.00, 0.00 ⁇ 0.00, 4.38 ⁇ 12.37, 24.49 ⁇ 24.97, 35.57 ⁇ 37.09, 110.98 ⁇ 86.41 and 325.89 ⁇ 233.46 (unit: mm 3 ).
  • the results of the assay are shown in Figure 26.
  • the tumor volume of the injected monoclonal antibody 5E08 group was significantly smaller than that of the injected human IgG group.
  • the test results show that monoclonal antibody 5E08 can inhibit tumor growth and can be used for the preparation of anti-tumor drugs and tumors.
  • the mouse treatment model was used to detect the inhibitory effect of monoclonal antibody 5E08 on tumor progression.
  • MDA-MB-231 cells were modeled for 1 month, they were divided into IgG control group according to the equal mean tumor volume, ie intratumoral injection of human IgG (50 ⁇ g/mouse); 5E08 group, ie intratumoral injection of monoclonal antibody 5E08 (50 ⁇ g/only). The inhibitory effect of monoclonal antibody 5E08 on tumor progression was observed by measuring tumor volume.
  • the tumor volume after treatment is as follows:
  • the IgG group was 197.40 ⁇ 47.98, 270.52 ⁇ 28.76, 389.69 ⁇ 17.26, 571.45 ⁇ 59.02, 933.05 ⁇ 158.71 and 1202.53 ⁇ 60.31 (unit: mm3);
  • the 5E08 group was 179.87 ⁇ 42.91, 269.22 ⁇ 54.97, 276.68 ⁇ 58.52, 331.02 ⁇ 94.42, 443.16 ⁇ 48.39 and 492.66 ⁇ 71.39 (unit: mm 3 ).
  • the results of the assay are shown in Figure 27.
  • the tumor volume of the injected monoclonal antibody 5E08 group was significantly smaller than that of the injected human IgG group.
  • the test results show that monoclonal antibody 5E08 can inhibit tumor growth and can be used for the preparation of anti-tumor drugs and tumors.
  • Example 26 monoclonal antibody 5E08 inhibits tumors without toxic side effects
  • Monoclonal antibody 5E08 inhibits tumor toxic side effects in mouse prevention model
  • Example 25 The prevention model in Example 25 was used for the experiment. After taking the experimental drug IgG or 5E08, the heart, liver, spleen, lung and kidney tissues of the mice were placed in formalin solution and sent to Beijing Ji Si Jia Yang Company for tissue embedding, sectioning and HE staining.
  • the monoclonal antibody 5E08 inhibits tumor toxic side effects in the mouse treatment model
  • Example 25 The treatment model in Example 25 was used for the experiment. After taking the experimental drug IgG or 5E08, the heart, liver, spleen, lung and kidney tissues of the mice were placed in formalin solution and sent to Beijing Ji Si Jia Yang Company for tissue embedding, sectioning and HE staining.
  • the present invention provides an application of inducing tumor-associated macrophage-expressing LSECtin and tumor-expressing BTN3A3 alone or in combination as a target in tumor immunotherapy, and further proposes an LSECtin activity capable of inhibiting infiltration of tumor-associated macrophage-derived cells,
  • a substance that inhibits tumor-expressing BTN3A3 activity or inhibits the interaction of the LSECtin with BTN3A3, including an RNA molecule, a fusion protein BTN3A3-Ig, and a monoclonal antibody 5E08, can be used as an active ingredient to prepare a tumor immunotherapy drug, and is suitable for industrial applications. .

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Abstract

本发明提供了肿瘤免疫治疗靶标及其应用，具体地，提供了浸润肿瘤相关巨噬细细胞表达的LSECtin和肿瘤表达BTN3A3单独或共同作为靶点在肿瘤免疫治疗中的应用，并进一步提供了能抑制浸润肿瘤相关巨噬细细胞表达的LSECtin活性、抑制肿瘤表达的BTN3A3活性、或抑制所述LSECtin与BTN3A3相互作用的物质，包括RNA分子、融合蛋白BTN3A3-Ig和单克隆抗体5E08，以其作为活性成分能制备肿瘤免疫治疗药物。

Description

肿瘤免疫治疗靶标及其应用 技术领域
本发明属于生物技术领域，具体涉及针对肿瘤相关巨噬细胞的肿瘤免疫治疗靶标。
背景技术
全球肿瘤发病率自20世纪70年代末开始一直呈上升趋势。目前，对于肿瘤的治疗手段主要包括手术、放疗、化疗、内分泌治疗、靶向治疗及中医药辅助治疗等。困扰肿瘤治疗的根本原因是肿瘤具备一定的耐药性及复发能力。研究表明，导致肿瘤具有耐药性以及复发能力的根本原因在于肿瘤细胞干性的维持与提升，因此，肿瘤细胞干性的靶向性治疗日益成为研究的热点。
肿瘤细胞的干性受基因多样性、表观遗传以及肿瘤微环境三个因素调控。由于肿瘤细胞存在高度异质性，因此，从基因多样性及表观遗传角度，很难提出行之有效的治疗方案。与之相比，肿瘤微环境浸润的免疫细胞通过交互作用对肿瘤细胞干性的维持与促进作用，因其具有较强的应用前景日益成为研究的热点。基于肿瘤免疫治疗的基本原理，针对免疫细胞对肿瘤细胞交互作用的靶向药物，有望应用免疫学原理和方法，提高肿瘤细胞的免疫原性和对效应细胞杀伤的敏感性，激发和增强机体抗肿瘤免疫应答，协同机体免疫系统，从而抑制肿瘤的生长、转移与复发。
肿瘤微环境因素能促进肿瘤细胞干性依赖细胞间交互作用，肿瘤微环境中存在有基质细胞、浸润免疫细胞等，通过阻断这些细胞与肿瘤细胞间的交互作用，有望降低肿瘤细胞干性，从而抑制肿瘤的发生与发展。其中，肿瘤相关巨噬细胞是肿瘤微环境中存在的巨噬细胞的总称，是肿瘤微环境中浸润免疫细胞的重要组成部分。已有研究显示，肿瘤相关巨噬细胞能够通过多种途径促进肿瘤进程，包括抑制肿瘤免疫反应、促进肿瘤免疫耐受环境的形成、促进肿瘤组织血管生成等。鉴于肿瘤相关巨噬细胞促进肿瘤进程现象明确，清除肿瘤相关巨噬细胞、促使肿瘤相关巨噬细胞向免疫激活方向转化已成为潜在的肿瘤治疗方法。
发明内容
本发明的一个目的是提供浸润肿瘤相关巨噬细细胞表达的LSECtin与肿瘤表达BTN3A3共同作为靶点在肿瘤免疫治疗中的应用。
所述应用基于浸润肿瘤相关巨噬细细胞表达的LSECtin能够与肿瘤细胞表面表达的BTN3A3相互作用，从而促进肿瘤细胞的干性，并且促进肿瘤的发生与发展，利用LSECtin和BTN3A3两个靶点来开发或设计肿瘤免疫治疗药物。
这里，所述肿瘤为与肿瘤相关巨噬细胞表达LSECtin且肿瘤细胞表达BTN3A3的肿瘤，包括但不限于乳腺癌、骨髓瘤、肝癌、胃癌、结肠癌、肺癌、骨巨细胞瘤、肾癌、喉癌和腮腺癌。
本发明的另一目的是提供浸润肿瘤相关巨噬细细胞表达的LSECtin或肿瘤表达BTN3A3单独作为靶点在肿瘤免疫治疗中的应用。
所述应用基于浸润肿瘤相关巨噬细细胞表达的LSECtin和肿瘤细胞表面表达的BTN3A3均各自具有促进肿瘤细胞干性，促进肿瘤的发生与发展的特性，利用LSECtin或BTN3A3作为靶点来开发或设计抑制剂从而抑制肿瘤的发生与发展，筛选获得肿瘤免疫治疗药物。
该应用中所述肿瘤为如下一种：c1)浸润肿瘤相关巨噬细细胞表达LSECtin的肿瘤；c2)表达BTN3A2和/或BTN3A3的肿瘤；和c3)同时满足c1)和c2)的肿瘤。所述肿瘤包括但不限于乳腺癌、骨髓瘤、肝癌、胃癌、结肠癌、肺癌、骨巨细胞瘤、肾癌、喉癌和腮腺癌。
作为开发或设计药物的应用，所述药物具备如下(1)-(5)中至少一种功能：(1)治疗和/或预防肿瘤；(2)抑制肿瘤细胞的生长；(3)抑制肿瘤细胞干性的维持或促进；(4)抑制肿瘤细胞干性相关特征分子的表达；和(5)抑制肿瘤细胞内部STAT3磷酸化。
这里，所述肿瘤细胞干性相关特征分子为Oct4基因和/或Nanog基因和/或Sox基因；所述抑制肿瘤细胞的生长体现在降低肿瘤细胞的成瘤率和/或减小肿瘤细胞的体积。
本发明还一目的在于提供抑制浸润肿瘤相关巨噬细细胞表达的LSECtin活性、抑制肿瘤表达的BTN3A3活性、或抑制所述LSECtin与BTN3A3相互作用的物质，这些物质包括但不限于：干扰BTN3A2和BTN3A3表达的RNA分子、抗LSECtin抗体、LSECtin小分子抑制剂、LSECtin可溶性蛋白、干扰LSECtin表达的RNA分子、抗BTN3A2抗体、BTN3A2小分子抑制剂、BTN3A2可溶性蛋白、干扰BTN3A2表达的RNA分子、抗BTN3A3抗体、BTN3A3小分子抑制剂、BTN3A3可溶性蛋白和干扰BTN3A3表达的RNA分子。
具体的第一类物质为干扰BTN3A2和BTN3A3表达的RNA分子或干扰BTN3A3表达的RNA分子，其为如下b1)-b4)中任一种：b1)序列4所示的shRNA分子；b2)将序列4删除或增加或改变一个或几个核苷酸，且与序列4相同功能的核苷酸；b3)序列5所示的shRNA分子；和b4)将序列5删除或增加或改变一个或几个核苷酸，且与序列5相同功能的核苷酸。
具体的第二类物质为可溶性蛋白，其为具有抑制肿瘤进程活性的融合蛋白，其能阻断 LSECtin与BTN3A3之间相互作用，命名为BTN3A3-Ig，是将人BTN3A3与人IgG1通过连接肽连接后获得的重组蛋白。
所述融合蛋白BTN3A3-Ig是下述氨基酸残基序列之一：1)序列表中的序列9；2)将序列表中序列9的氨基酸残基序列经过一个或几个氨基酸残基的取代、缺失或添加且具有抑制肿瘤进程活性的蛋白质；3)将序列表中序列9的氨基酸残基序列经过氨基酸残基的取代、缺失或添加且具有抑制肿瘤进程活性的蛋白质，新蛋白质与序列9同源性达到80％或更高。
序列表中的序列9由490个氨基酸残基组成，自氨基(N)端第1位为起始密码子，自氨基端第2-15位为信号肽，自氨基端第16-262位为人BTN3A3，自氨基端第263-266位为连接肽(linker)，自氨基端第267-490位为人IgG1。
编码所述融合蛋白BTN3A3-Ig的基因BTN3A3-Ig也属于本发明内容。编码融合蛋白BTN3A3-Ig的基因BTN3A3-Ig是下述核苷酸序列之一：1)序列表中序列10的DNA序列；2)编码序列表中序列9的DNA序列；3)与所编码序列表中序列9的DNA序列有一个或几个碱基变化且具有抑制肿瘤进程活性的核苷酸序列；4)所编码的序列80％或以上同源于序列表中序列10且具有抑制肿瘤进程活性的核苷酸序列；和5)在高严谨条件下可与序列表中序列10限定的DNA序列杂交的核苷酸序列。
序列表中的序列10由1470个碱基组成，其编码序列为自5’端第1-1470位碱基，编码具有序列表中1所示氨基酸残基序列的蛋白质，自5’端第46-786位碱基编码人BTN3A3，自5’端第787-798位碱基编码连接肽(linker)，自5’端第799-1470位碱基编码人IgG1。
含有所述融合基因BTN3A3-Ig的表达载体、转基因细胞系或宿主菌也属于本发明。
扩增融合基因BTN3A3-Ig中任一片段的引物对也属于本发明。
本发明表达所述融合蛋白BTN3A3-Ig的方法，包括以下步骤：
1)构建重组表达载体：将所述的融合基因BTN3A3-Ig连接入表达载体中，得到含有融合基因BTN3A3-Ig的重组表达载体；
2)表达融合蛋白BTN3A3-Ig：将含有融合基因BTN3A3-Ig的重组表达载体pIRES2-EGFP-BTN3A3-Ig转化或转染宿主细胞及其后代细胞，培养重组宿主细胞，使融合基因BTN3A3-Ig获得表达；
3)纯化：对重组表达蛋白进行纯化，得到融合蛋白BTN3A3-Ig。
其中：所述步骤1)中将融合基因BTN3A3-Ig连接入载体pIRES2-EGFP中的NheⅠ和 SalⅠ酶切位点之间，得到重组表达载体，命名为pIRES2-EGFP-BTN3A3-Ig。所述步骤2)中的宿主细胞为可表达外源基因的细胞，包括293T细胞、293细胞和CHO-S细胞等；所述步骤2)中培养含有融合基因BTN3A3-Ig的重组宿主细胞的培养基为适于宿主细胞生长的培养基，如无血清培养基M293TI、M293TI、CD CHO或CD OptiCHOTM等，优选为无血清培养基M293TI；所述步骤2)中含有融合基因BTN3A3-Ig的重组宿主细胞的培养条件为适于宿主细胞生长的培养条件，36.5-37.5℃培养24-120小时，优选为37℃培养96小时。
所述步骤3)中可使用如Protein G Sepharose柱(蛋白G琼脂糖凝胶柱)、Protein A/G Sepharose柱(蛋白A/G琼脂糖凝胶柱)、或Protein A Sepharose柱(蛋白A琼脂糖凝胶柱)等对重组表达蛋白进行纯化，优选为Protein G Sepharose柱，纯化方法为：将细胞(含有融合基因BTN3A3-Ig的重组宿主细胞)培养上清加入平衡缓冲液(20mM PBS，150mM NaCl，pH 8.0)至pH 8.0，将细胞上清加入已经用平衡缓冲液平衡好的Protein G Sepharose柱中，用平衡缓冲液洗柱，直到流出液中检测不到杂蛋白为止，用洗脱缓冲液(0.1M甘氨酸，pH 3.0)洗脱，收集流出液，立即用中和缓冲液(1M Tris·HCl，pH 9.0)中和，用pH 7.2 0.01mol/L PBS透析72h，得到融合蛋白BTN3A3-Ig。
本发明的融合蛋白BTN3A3-Ig通过药学可接受的、适合于给药的药物载体制备成药物组合物，合适的药物载体为本领域技术人员熟知的，包括但不限于生理盐水、磷酸缓冲液、水、脂质体、纳米载体等。含有融合蛋白BTN3A3-Ig的药物载体可通过常规方法制备。
本发明融合蛋白BTN3A3-Ig的药物组合物可以通过各种给药途径施用有效剂量于人类或其它哺乳动物，给药途径包括但不限于静脉注射(iv)、静脉滴注(infusion)、肌肉注射(im)、皮下注射(sc)和口服(po)等。对于不同的疾病，可以选择不同的给药途径。
本发明融合蛋白BTN3A3-Ig基于小鼠模型的给药剂量一般为0.5-2.5μg/g，疗程一般为15-30天。实际应用中的剂量和疗程可以根据实际情况进行调整。
具体的第三类物质为具有抑制肿瘤进程活性的单克隆抗体，其能阻断LSECtin与BTN3A3之间相互作用。
所述单克隆抗体的重链可变区为：由序列表中序列13氨基酸残基序列表示的多肽、将序列表中序列13的氨基酸残基序列经过一至十个氨基酸残基的取代、缺失或添加且可与人BTN3A3特异结合的多肽、或与序列表序列13有80％或以上同源性且可与人BTN3A3特异结合的多肽；单克隆抗体的轻链可变区为：由序列表中序列14氨基酸残基序列表示的多肽、将序列表中序列14的氨基酸残基序列经过一至十个氨基酸残基的取代、缺失或 添加且可与人BTN3A3特异结合的多肽、或与序列表序列14有80％或以上同源性且可与人BTN3A3特异结合的多肽。
序列表中的序列13由141个氨基酸残基组成，序列表中的序列14由130个氨基酸残基组成。
编码单克隆抗体的基因重链可变区编码基因为以下之一：具有序列表中序列15的DNA序列、编码序列表中序列13的DNA序列、与所编码序列表中序列13的DNA序列有一个或多个碱基变化的序列、所编码的序列80％或以上同源于序列表中序列15的序列、和在高严谨条件下可与序列表中序列15限定的DNA序列杂交的核苷酸序列；
编码单克隆抗体的基因轻链可变区编码基因为以下之一：具有序列表中序列16的DNA序列、编码序列表中序列14的DNA序列、与所编码序列表中序列14的DNA序列有一个或多个碱基变化的序列、所编码的序列80％或以上同源于序列表中序列16的序列、和在高严谨条件下可与序列表中序列16限定的DNA序列杂交的核苷酸序列。
所述高严谨条件为在0.1×SSPE(或0.1×SSC)、0.1％SDS的溶液中，65℃条件下杂交并洗膜。
其中：序列表中的序列15由423个碱基组成，编码具有序列表中序列13的氨基酸残基序列的蛋白质，序列表中的序列16由390个碱基组成，编码具有序列表中序列14的氨基酸残基序列的蛋白质。
含有本发明基因5E08的表达载体、转基因细胞系或宿主菌均属于本发明。
扩增本发明基因5E08中任一片段的引物对也在本发明的范围之内。
能分泌阻断LSECtin与BTN3A3之间相互作用单克隆抗体的杂交瘤细胞株也属于本发明，该杂交瘤细胞株是以将人BTN3A3与小鼠IgG2a通过连接肽连接得到的融合蛋白(命名为BTN3A3-mIg)为免疫原免疫小鼠，获得持续、稳定分泌具有抑制肿瘤进程活性的单克隆抗体的杂交瘤细胞株。
所述融合蛋白BTN3A3-mIg是下述氨基酸残基序列之一：1)序列表中的序列11；2)将序列表中序列11的氨基酸残基序列经过一个或几个氨基酸残基的取代、缺失或添加且具有抗BTN3A3免疫原性的蛋白质；3)将序列表中序列11的氨基酸残基序列经过氨基酸残基的取代、缺失或添加且具有抗BTN3A3免疫原性的蛋白质，新蛋白质与序列1同源性达到80％或更高。
其中：序列表中的序列11由499个氨基酸残基组成，自氨基(N)端第1位为起始密码子，第2-15为信号肽，第16-262位为人BTN3A3，自氨基端第263-266位为连接肽 (linker)，自氨基端第267-498位为小鼠IgG2a，第499位为终止密码子。
编码上述融合蛋白BTN3A3-mIg的基因，命名为BTN3A3-mIg，也属于本发明。具体来讲，编码上述融合蛋白的基因BTN3A3-mIg，是下述核苷酸序列之一：1)序列表中序列12的DNA序列；2)编码序列表中序列11的DNA序列；3)与所编码序列表中序列11的DNA序列有一个或几个碱基变化且具有抗BTN3A3免疫原性的的核苷酸序列；4)所编码的序列80％或以上同源于序列表中序列12且具有抗BTN3A3免疫原性的的核苷酸序列；5)在高严谨条件下可与序列表中序列12限定的DNA序列杂交的核苷酸序列。
所述高严谨条件为杂交后用含0.1×SSPE(或0.1×SSC)、0.1％SDS的溶液在65℃下洗膜。
其中：序列表中的序列12由1497个碱基组成，其编码序列为自5’端第1-1497位碱基，编码具有序列表中11所示氨基酸残基序列的蛋白质，自5’端1-3位为起始密码子，自5’端第4-45位碱基编码信号肽，自5’端第46-786位碱基编码人BTN3A3，自5’端第787-798位碱基编码连接肽(linker)，自5’端第799-1494位碱基编码小鼠IgG2a，自5’端第1495-1497位为终止密码子。
含有融合基因BTN3A3-mIg的表达载体、转基因细胞系或宿主菌均属于本发明。
扩增融合基因BTN3A3-mIg中任一片段的引物对也属于本发明。
表达上述融合蛋白BTN3A3-mIg的方法也属于本发明。本发明所提供的融合蛋白BTN3A3-mIg的表达方法，可包括以下步骤：(1)构建重组表达载体：将融合基因BTN3A3-mIg连接入表达载体中，得到含有融合基因BTN3A3-mIg的重组表达载体；(2)表达融合蛋白BTN3A3-mIg：将含有融合基因BTN3A3-mIg的重组表达载体转化或转染宿主细胞及其后代细胞，培养重组宿主细胞，使融合基因BTN3A3-mIg获得表达；(3)纯化：对重组表达蛋白进行纯化，得到融合蛋白BTN3A3-mIg。
以融合蛋白BTN3A3-mIg为免疫原免疫小鼠，获得持续、稳定分泌具有抑制肿瘤进程活性的特异性单克隆抗体的杂交瘤细胞株，名称为anti-P3(5E08)，该细胞株已于2017年9月26日保藏于位于中国北京市朝阳区北辰西路1号院3号的中国普通微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.14723。
由杂交瘤细胞株anti-P3(5E08)分泌的单克隆抗体命名为5E08，来源于小鼠属小鼠(Mus musculus)，其能阻断LSECtin与BTN3A3之间相互作用而具有抑制肿瘤进程活性，单克隆抗体5E08属于本发明。
本发明还提供了能分泌阻断LSECtin与BTN3A3之间相互作用单克隆抗体的杂交瘤细 胞株的获取方法，可包括以下步骤：1)用融合蛋白BTN3A3-mIg作为免疫原免疫动物；2)分离免疫动物的脾细胞，将其与骨髓瘤细胞融合，形成杂交瘤；3)筛选杂交瘤细胞，得到杂交瘤细胞株anti-P3(5E08)。
该杂交瘤细胞株anti-P3(5E08)的获得方法中，所述步骤1)中融合蛋白BTN3A3-mIg的浓度为100-400μg/mL，优选为400μg/mL；用于制备单克隆抗体的免疫动物可为小鼠、大鼠、兔、山羊、绵羊、猪、驴、马等哺乳动物，优选为小鼠。所述步骤2)中当被免疫动物的血清抗体水平达到峰值时，可分离动物的脾细胞并制备成单细胞悬液。必要时，可使用免疫吸附方法筛选脾细胞，并在适当的融合剂(如聚乙二醇)的诱导下与骨髓瘤细胞(优选为小鼠骨髓瘤细胞SP2/0)融合以形成杂交瘤。所述步骤3)中可以在选择性培养基(如HAT培养基)中培养以筛选融合的杂交瘤细胞，并进一步可使用流式细胞术、Western印迹法、免疫沉淀法等方法鉴定所需的阳性抗性细胞株。所述步骤4)中可于体外(如在组织培养瓶或多孔纤维反应器中)或体内(小鼠腹水)培养分泌具有抑制肿瘤进程活性的特异性单克隆抗体5E08的杂交瘤细胞株anti-P3(5E08)，并从细胞培养液或小鼠腹水液中收集和纯化出单克隆抗体5E08。
获得本发明单克隆抗体5E08的方法，在上述步骤的基础上增加以下步骤：
4)从杂交瘤细胞株anti-P3(5E08)的培养液或接种杂交瘤细胞株anti-P3(5E08)的动物的腹水液中分离并纯化出单克隆抗体5E08。
或者，利用所提及的单克隆抗体5E08的重链可变区和轻链可变区氨基酸序列或DNA序列构建单克隆抗体5E08的表达载体，通过常规蛋白表达方式得到具有抑制肿瘤进程活性的单克隆抗体。
本发明再一目的在于提出所述RNA分子、融合蛋白BTN3A3-Ig、融合基因BTN3A3-mIg、融合基因BTN3A3-Ig的表达载体、转基因细胞系或宿主菌、单克隆抗体5E08、免疫原融合蛋白BTN3A3-mIg以及其编码基因BTN3A3-mIg在制备具有如下(b1)-(b5)中至少一种功能的产品中的应用：(b1)治疗和/或预防肿瘤；(b2)抑制肿瘤进程；(b3)抑制肿瘤细胞干性的维持或促进；(b4)抑制肿瘤细胞干性相关特征分子的表达；和(b5)抑制肿瘤细胞内部STAT3磷酸化。所述肿瘤为肿瘤相关巨噬细胞表达LSECtin且肿瘤细胞表达BTN3A3的肿瘤，所述肿瘤相关巨噬细胞表达LSECtin且肿瘤细胞表达BTN3A3的肿瘤包括但不限于乳腺癌、骨髓瘤、肝癌、胃癌、结肠癌、肺癌、骨巨细胞瘤、肾癌、喉癌和腮腺癌。
本发明提出了浸润肿瘤相关巨噬细细胞表达的LSECtin和肿瘤表达BTN3A3单独或共 同作为靶点在肿瘤免疫治疗中的应用，并进一步提出了能抑制浸润肿瘤相关巨噬细细胞表达的LSECtin活性、抑制肿瘤表达的BTN3A3活性、或抑制所述LSECtin与BTN3A3相互作用的物质，包括RNA分子、融合蛋白BTN3A3-Ig和单克隆抗体5E08，以其作为活性成分制备肿瘤免疫治疗药物。实验证明：LSECtin、BTN3A2及BNT3A3通过促进肿瘤细胞干性的维持促进肿瘤进程，具体体现在促进肿瘤细胞球形成、干性转录因子的表达以及小鼠肿瘤模型中对肿瘤进程的促进；抑制LSECtin与BTN3A2及BTN3A3的相互作用能够有效减缓肿瘤进程，具体体现在降低肿瘤发生率和减缓肿瘤体积增长。实验还证明：融合蛋白BTN3A3-Ig与LSECtin蛋白具有结合活性，融合蛋白BTN3A3-Ig能够阻断LSECtin与细胞表面表达的BTN3A3之间的相互作用，融合蛋白BTN3A3-Ig能够阻断LSECtin促进肿瘤细胞的干性，融合蛋白BTN3A3-Ig可以抑制肿瘤进程，融合蛋白BTN3A3-Ig抑制肿瘤进程依赖LSECtin。实验还证明：杂交瘤细胞株anti-P3(5E08)CGMCC No.14723能分泌单克隆抗体5E08，单克隆抗体5E08与BTN3A3蛋白具有结合活性，单克隆抗体5E08能够阻断LSECtin与BTN3A3之间的相互作用，单克隆抗体5E08能够阻断LSECtin促进肿瘤细胞的干性，单克隆抗体5E08能够抑制肿瘤生长。本发明为肿瘤的免疫治疗提供了一个新的思路，应用前景广阔。
附图说明
图1显示实施例1中LSECtin能促进肿瘤形成。其中：
图1(a)为自发乳腺癌模型小鼠肿瘤体积的检测结果曲线，表示LSECtin+/+PyMT与LSECtin-/-PyMT小鼠乳腺癌成瘤体积对比；
图1(b)为自发乳腺癌模型小鼠肿瘤灶个数的检测结果曲线，表示MMTV-PyMT自发乳腺癌模型中LSECtin+/+PyMT与LSECtin-/-PyMT每只小鼠乳腺癌肿瘤灶个数对比；
图1(c)为自发乳腺癌模型小鼠肺脏肿瘤转移灶个数的检测结果柱状图，表示MMTV-PyMT自发乳腺癌模型中LSECtin+/+PyMT与LSECtin-/-PyMT每只小鼠肺脏肿瘤转移灶个数对比；
图1(d)为人-裸鼠乳腺癌移植模型肿瘤体积的检测结果曲线，表示LSECtin+/+Nude-/-组和LSECtin-/-Nude-/-组小鼠肿瘤体积对比。
图2显示实施例2中肿瘤微环境中LSECtin表达水平的检测。其中：
图2(a)为mRNA水平基因表达水平柱状图，表示qPCR检测MMTV-PyMT自发乳腺癌模型小鼠肿瘤微环境中LSECtin表达水平，其中，CD11b-MHCⅡ-代表淋巴细胞，TAM代表肿瘤相关巨噬细胞，TAN代表肿瘤相关中性粒细胞，Mo代表单核细胞；
图2(b)为mRNA水平基因表达水平柱状图，表示qPCR检测人-裸鼠乳腺癌移植模型肿瘤微环境中LSECtin表达水平；
图2(c)为小鼠肿瘤原位免疫荧光染色结果图，表示MMTV-PyMT小鼠自发乳腺癌微环境中LSECtin与巨噬细胞标志分子F4/80共定位；
图2(d)为流式检测LSECtin表达水平峰型图，表示人乳腺癌肿瘤相关巨噬细胞表面LSECtin表达水平；
图2(e)为流式检测LSECtin表达水平峰型图，表示骨髓瘤病人肿瘤相关巨噬细胞表面LSECtin表达水平；
图2(f)为流式检测LSECtin表达水平峰型图，表示肺癌病人肿瘤相关巨噬细胞表面LSECtin表达水平；
图2(g)为流式检测LSECtin表达水平峰型图，表示结肠癌病人肿瘤相关巨噬细胞表面LSECtin表达水平；
图2(h)为流式检测LSECtin表达水平峰型图，表示骨巨细胞瘤病人肿瘤相关巨噬细胞表面LSECtin表达水平；
图2(i)为流式检测LSECtin表达水平峰型图，表示肾癌病人肿瘤相关巨噬细胞表面LSECtin表达水平；
图2(j)为流式检测LSECtin表达水平峰型图，表示喉癌病人肿瘤相关巨噬细胞表面LSECtin表达水平；
图2(k)为流式检测LSECtin表达水平散点图，表示腮腺癌病人肿瘤相关巨噬细胞表面LSECtin表达水平。
图3显示实施例4中临床样本肿瘤细胞表达BTN3A3。其中
图3(a)为流式检测乳腺癌病人BTN3A表达水平峰型图，表示肿瘤组织中肿瘤细胞表面表达BTN3A水平；
图3(b)为流式检测BTN3A3表达水平散点图，表示肺癌病人肿瘤组织中肿瘤细胞表面表达BTN3A水平；
图3(c)为流式检测BTN3A3表达水平散点图，表示结肠癌病人肿瘤组织中肿瘤细胞表面表达BTN3A水平；
图3(d)为流式检测BTN3A3表达水平散点图，表示骨巨细胞瘤病人肿瘤组织中肿瘤细胞表面表达BTN3A水平；
图3(e)为流式检测BTN3A3表达水平散点图，表示肾癌病人肿瘤组织中肿瘤细胞表 面表达BTN3A水平；
图3(f)为流式检测BTN3A3表达水平峰形图，表示腮腺癌病人肿瘤组织中肿瘤细胞表面表达BTN3A水平。
图4显示实施例5中多种肿瘤细胞系表达BTN3A3。其中：
图4(a)为多种乳腺癌细胞系表达BTN3A3的Western Blot检测结果图片；
图4(b)为多种肝癌细胞系表达BTN3A3的Western Blot检测结果图片；
图4(c)为人黑色素瘤细胞系表达BTN3A3的Western Blot检测结果图片；
图4(d)为多种胃癌细胞系表达BTN3A3的Western Blot检测结果图片；
图4(e)为结肠癌细胞系表达BTN3A3的Western Blot检测结果图片。
图5显示实施例6中BTN3A2及BTN3A3在乳腺癌细胞上的表达。其中：
图5(a)为qPCR检测BTN3A2及BTN3A3结果柱状图，表明乳腺癌细胞系表达BTN3A2与BTN3A3；
图5(b)为乳腺癌细胞系流式检测BTN3A表达峰型图，表示BTN3A表达于乳腺癌细胞系表面；
图5(c)为细胞免疫荧光染色结果图，表示BTN3A3于乳腺癌细胞表面膜定位。
图6显示实施例7中肿瘤细胞表达的BTN3A3促进肿瘤形成。其中：
图6(a)为qPCR检测BTN3A2及BTN3A3结果柱状图，表示shRNA对BTN3A2及BTN3A3表达的敲低；
图6(b)为BTN3A3的蛋白水平的Western Blot检测结果图片，表示shRNA敲低对BTN3A3蛋白水平表达的影响；
图6(c)为人-裸鼠乳腺癌移植模型肿瘤体积的检测结果曲线，表示231-NC与231-sh4成瘤体积对比；
图7为实施例8中流式检测LSECtin重组蛋白与肿瘤细胞表达的BTN3A3粘附散点图，表示人源及鼠源LSECtin与BTN3A3存在直接且特异性的相互作用。其中：
图7(a)为流式检测人源LSECtin与肿瘤细胞表达的BTN3A3粘附散点图，表示人源LSECtin重组蛋白与BTN3A3存在直接且特异性的相互作用；
图7(b)为流式检测鼠源LSECtin与肿瘤细胞表达的BTN3A3粘附散点图，表示鼠源LSECtin与BTN3A3存在直接且特异性的相互作用。
图8显示实施例9中LSECtin通过与肿瘤细胞表达的BTN3A3相互作用促进肿瘤细胞 干性。其中：
图8(a)和图8(b)为球体数量统计柱状图，表示LSECtin/BTN3A3促进肿瘤细胞MDA-MB-231球形成能力；
图8(c)为qPCR检测干性转录因子表达水平柱状图，表示LSECtin与BTN3A3相互作用促进肿瘤细胞干性转录因子上调。
图9显示实施例10中LSECtin与肿瘤细胞表达的BTN3A3相互作用促进肿瘤细胞内STAT3磷酸化进而促进肿瘤细胞干性维持结果。其中：
图9(a)为LSECtin/BTN3A3相互作用促进肿瘤细胞STAT3磷酸化的Weatern Blot检测结果图片，表明对其它STAT分子无激活作用；
图9(b)为LSECtin/BTN3A3促进肿瘤细胞球形成的球体数量统计柱状图，表示STAT3抑制剂可阻断LSECtin/BTN3A3促进肿瘤细胞成球效应。
图10为重组表达的融合蛋白BTN3A3-Ig的考马斯亮蓝染色结果图片；
图11为重组表达的融合蛋白BTN3A3-Ig的Western Blot检测结果图片；
图12为融合蛋白BTN3A3-Ig与LSECtin蛋白结合活性的Elisa检测结果曲线；
图13为融合蛋白BTN3A3-Ig阻断LSECtin蛋白与膜形式BTN3A3之间相互作用的粘附实验检测结果散点图；
图14为巨噬细胞特异性敲除LSECtin自发乳腺癌模型小鼠肿瘤体积的检测结果曲线；
图15为巨噬细胞特异性敲除LSECtin自发乳腺癌模型小鼠肿瘤灶个数的检测结果曲线；
图16为巨噬细胞特异性敲除LSECtin自发乳腺癌模型小鼠肺脏肿瘤转移灶个数的检测结果柱状图；
图17为融合蛋白BTN3A3-Ig阻断LSECtin促进肿瘤细胞干性的检测结果柱状图；
图18为融合蛋白BTN3A3-Ig抑制肿瘤进程的检测结果曲线；
图19为融合蛋白BTN3A3-Ig抑制肿瘤进程依赖LSECtin的检测结果曲线；
图20为实施例16实验药物作用后心脏、肝脏、脾脏、肺脏以及肾脏组织切片图，显示融合蛋白BTN3A3-Ig抑制肿瘤无毒副作用检测结果图片。
图21为重组表达的融合蛋白BTN3A3-mIg的考马斯亮蓝染色结果图片；
图22为重组表达的融合蛋白BTN3A3-mIg的Western Blot检测结果图片；
图23为杂交瘤细胞分泌的单克隆抗体5E08与BTN3A3结合比例的流式细胞检测结果图片；
图24为单克隆抗体5E08阻断LSECtin蛋白与膜形式BTN3A3相互作用活性的粘附实验检测结果图片；
图25为单克隆抗体5E08阻断LSECtin促进肿瘤细胞干性的检测结果柱图；
图26为单克隆抗体5E08对肿瘤进程的抑制效果的小鼠预防模型检测结果曲线；
图27为单克隆抗体5E08对肿瘤进程的抑制效果的小鼠治疗模型检测结果曲线；
图28为实施例26实验药物作用后心脏、肝脏、脾脏、肺脏以及肾脏组织切片图，显示单克隆抗体5E08对小鼠预防模型无毒副作用；
图29为实施例26实验药物作用后心脏、肝脏、脾脏、肺脏以及肾脏组织切片图，显示单克隆抗体5E08对小鼠治疗模型无毒副作用。
具体实施方式
在肿瘤免疫治疗的研究中，已知LSECtin蛋白功能包括在黑色素瘤中负调节T细胞免疫反应，促进埃博拉病毒引发的炎性反应，抑制CTL依赖的HBV病毒清除，促进结肠癌肿瘤细胞肝转移，发明人贺福初、唐丽等之前发现LSECtin可能作为黑色素瘤免疫治疗的靶点(CN104906575A，2014100898325)，还发现LSECtin或含有LSECtin的融合蛋白可应用于制备抑制癌细胞向肝转移的药物，LSECtin蛋白及其融合蛋白能粘附结肠癌细胞、抑制结肠癌细胞往肝脏的归巢性迁移，成为抗粘附治疗肿瘤肝转移的一个新靶标(CN101732715A，2008102257147)。
然而，这些现有技术中披露的为肿瘤细胞中或转移器官中存在的LSECtin蛋白功能，并未对肿瘤细胞自身不表达LSECtin的情况下LSECtin是否影响肿瘤的形成与发展给出启示。在近期研究中，发明人意外地发现肿瘤相关巨噬细胞也表达LSECtin蛋白，且该肿瘤相关巨噬细胞表达的LSECtin蛋白具有促进肿瘤形成与发展的特性。LSECtin蛋白发挥此种特性的机制是介导肿瘤相关巨噬细胞与肿瘤细胞直接接触，并促进肿瘤细胞干性的维持。鉴于此，通过实验验证，本发明首先提出了肿瘤相关巨噬细胞表达的LSECtin蛋白能够作为该类肿瘤进行免疫治疗的靶点。
LSECtin(Liver Sinusoidal Endothelial Cells lectin)是Ⅱ型跨膜糖蛋白，位于人类19p13.3，是C型凝集素家族的新成员。
基于以上研究成果，鉴于LSECtin蛋白发挥促进肿瘤发生与发展功能依赖于与肿瘤细胞的直接接触及对肿瘤细胞干性的维持作用，因此，发明人进一步挖掘LSECtin的相互作用膜蛋白，筛选抑制LSECtin与该类膜蛋白间相互作用的物质，为肿瘤细胞干性的靶向性治疗提供新思路。
经过深入研究，发明人首次发现LSECtin能够与肿瘤细胞表面表达的BTN3A3(Butyrophilin subfamily 3 member A3)相互作用，从而促进肿瘤细胞的干性，并且促进肿瘤的发生与发展。
BTN3A3(嗜乳脂蛋白3-A3，Butyrophilin subfamily 3 member A3)为Ⅰ型跨膜糖蛋白，位于人类6p22.2，是B7超家族的成员。已知BTN3A3蛋白有可能负调节淋巴细胞活性，并且编码BTN3A3蛋白的基因突变与肿瘤易感性相关(Peedicayil,A.,et al.Risk of ovarian cancer and inherited variants in relapse-associated genes.PLoS One 5,e8884(2010).)。
针对肿瘤表达的BTN3A3蛋白，发明人通过实验验证，在本发明中提出其能够作为靶点在肿瘤免疫治疗中应用。
进一步，基于LSECtin能与肿瘤细胞表面表达的BTN3A3相互作用促进肿瘤细胞的干性的发现，发明人针对能阻断LSECtin与BTN3A3相互作用的物质进行筛选与挖掘，并将其开发为抑制肿瘤相关巨噬细胞促进肿瘤干性发展的靶向性药物。
因此，在本发明中提出一种能阻断LSECtin与BTN3A3之间的相互作用的物质，利用该物质抑制肿瘤的发生与发展，抑制肿瘤进程。
具体的，本发明提供的该物质为干扰BTN3A2和BTN3A3表达的RNA分子或干扰BTN3A3表达的RNA分子。
具体的，本发明提供的该物质为一种融合蛋白，主要由人BTN3A3和人IgG1两个功能部分组成，能够阻断LSECtin与细胞表面表达的BTN3A3之间的相互作用，该融合蛋白命名为BTN3A3-Ig。
具体的，本发明提供的该物质为具有抑制肿瘤进程活性的单克隆抗体，命名为5E08，本发明同时提出能分泌5E08的杂交瘤细胞株anti-P3(5E08)。
下面结合具体实施例对本发明做进一步详细说明。实施例中所用方法如无特别说明均为常规方法，具体步骤可参见：《分子克隆实验指南》(《Molecular Cloning:A Laboratory Manual》Sambrook，J.，Russell，David W.，Molecular Cloning:A Laboratory Manual，3rd edition，2001，NY，Cold Spring Harbor)。
所述百分比浓度如无特别说明均为质量/质量(W/W，单位g/100g)百分比浓度、质量/体积(W/V，单位g/100mL)百分比浓度或体积/体积(V/V，单位mL/100mL)百分比浓度。
实施例中描述到的各种生物材料的取得途径仅是提供一种实验获取的途径以达到具体公开的目的，不应成为对本发明生物材料来源的限制。事实上，所用到的生物材料的来 源是广泛的，任何不违反法律和道德伦理能够获取的生物材料都可以按照实施例中的提示替换使用。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
下述实施例中的定量试验，均设置三次重复实验，结果取平均值。
下述实施例中的RNA提取试剂盒、cDNA反转录试剂盒(A3500)和Mix(A6001)均为Promega公司的产品。
下述实施例中的细胞计数板(145-0011)是Biorad公司的产品。
下述实施例中的CCK8细胞计数试剂盒(CK04)是东仁化学科技公司的产品。
下述实施例中的LSECtin(QT01034446)、BTN3A2(QT00060039)和BTN3A3(QT00060039)的qPCR引物均是Qiagen公司合成。
下述实施例中的4-6周雌性裸鼠是维通利华公司的产品。
下述实施例中的胶原酶Ⅳ(C5138)是Sigma公司的产品。
下述实施例中的DNase Ⅰ是西美杰公司的产品。
下述实施例中的小鼠MHCⅡ Percp-cy5.5是Biolegend公司的产品。
下述实施例中的小鼠Ly6G APC-cy7(560600)、人CD14 V500(561392)和人CD15 PE-CF594(562372)均是BD公司的产品。
下述实施例中的小鼠Ly6C APC(17-5932)、CD11b PE-cy7(25-0112)、人CD3 FITC(11-0038)、CD19 FITC(11-0199)、CD56 FITC(11-0566)和CD11b PE-cy7(25-0118)均是eBioscience公司的产品。
下述实施例中的封闭液：溶剂为水，溶质为Na2HPO4、KH2PO4、NaCl和脱脂牛奶；溶质Na2HPO4、KH2PO4、NaCl和脱脂牛奶在封闭液中的浓度分别为0.02M、0.0015M、0.14M和3％(质量百分含量)。
下述实施例中的人乳腺癌细胞MDA-MB-231购自国家实验细胞资源共享平台，并按照该平台提供的培养方式培养。人乳腺癌细胞系MDA-MB-231的培养条件为在含有10％的胎牛血清(Gibico，货号：10100-147-FBS)的RPMI 1640培养基(Thermofish，货号：SH30809.01B)中37℃，5％CO2孵育。
下述实施例中的MMTV-PyMT为自发乳腺癌模型小鼠，LSECtin+/+PyMT为野生型自发乳腺癌模型小鼠，LSECtin-/-PyMT为LSECtin敲除自发乳腺癌模型小鼠。具体获得方法如下：C57Bl/6J背景野生型MMTV-PyMT自发乳腺癌模型雄鼠与C57Bl/6J背景LSECtin敲除雌鼠 交配，获得LSECtin为杂合的自发乳腺癌模型雄鼠，将该小鼠与C57Bl/6J背景LSECtin杂合雌鼠交配，经基因型鉴定可分别获得LSECtin+/+PyMT(LSECtin表达的野生型自发乳腺癌模型小鼠)和LSECtin-/-PyMT(LSECtin-/-PyMT为LSECtin敲除自发乳腺癌模型小鼠)。上述标注中，PyMT代表自发乳腺癌模型转基因，+/+代表野生型纯合子，-/-代表敲除纯合子。上述C57Bl/6J背景LSECtin敲除小鼠在文献“Tang L,Yang J,Liu W,et al.Liver sinusoidal endothelial cell lectin,LSECtin,negatively regulates hepatic T-cell immune response[J].Gastroenterology,2009,137(4):1498-1508.e5.”中公开过，公众可从军事医学科学院获得。上述C57Bl/6J背景野生型MMTV-PyMT自发乳腺癌模型小鼠在文献“Davie S A,Maglione J E,Manner C K,et al.Effects of FVB/NJ and C57Bl/6J strain backgrounds on mammary tumor phenotype in inducible nitric oxide synthase deficient mice[J].Transgenic research,2007,16(2):193-201.”中公布开过，公众可从军事医学科学院获得。
下述实施例中的LSECtin+/+Nude-/-为野生型裸鼠，LSECtin-/-Nude-/-为LSECtin敲除裸鼠。具体获得方法如下：将BALB/c背景雄性裸鼠LSECtin+/+Nude-/-(购自维通利华)与BALB/c背景雌性LSECtin-/-Nude+/+小鼠交配，获得LSECtin+/-Nude+/-小鼠。雄性LSECtin+/-Nude+/-小鼠与雌性LSECtin+/-Nude+/-小鼠交配，其子代中的雌性裸鼠，经基因型鉴定可获得LSECtin+/+Nude-/-(LSECtin表达的野生型裸鼠)和LSECtin-/-Nude-/-(LSECtin敲除裸鼠)。上述BALB/c背景雌性LSECtin-/-Nude+/+小鼠信息在文献“Liu B,Wang M,Wang X,et al.Liver sinusoidal endothelial cell lectin inhibits CTL-dependent virus clearance in mouse models of viral hepatitis[J].The Journal of Immunology,2013,190(8):4185-4195.”中公布开过，公众可从军事医学科学院获得。
下述实施例中的小鼠抗人LSECtin抗体CCB059在文献“Zhao D,Han X,Zheng X,et al.Correction:The Myeloid LSECtin Is a DAP12-Coupled Receptor That Is Crucial for Inflammatory Response Induced by Ebola Virus Glycoprotein[J].PLoS pathogens,2016,12(3).”中公开过，公众可从军事医学科学院获得。
下述实施例中的兔抗小鼠LSECtin多抗在文献“Xu F,Liu J,Liu D,et al.LSECtin expressed on melanoma cells promotes tumor progression by inhibiting antitumor T-cell responses[J].Cancer research,2014,74(13):3418-3428.”中公开过，公众可从军事医学科学院获得。
下述实施例中的鼠源LSECtin-Fc在文献“Tang L,Yang J,Tang X,et al.The  DC-SIGN family member LSECtin is a novel ligand of CD44on activated T cells[J].European journal of immunology,2010,40(4):1185-1191.”中公开过，公众可从军事医学科学院获得。
实施例1、LSECtin促进肿瘤形成
一、LSECtin促进小鼠自发乳腺癌的肿瘤成瘤及肿瘤进程
1、自发乳腺癌模型小鼠肿瘤体积的检测
分别将自发乳腺癌模型小鼠LSECtin+/+PyMT和自发乳腺癌模型小鼠LSECtin-/-PyMT饲养并繁殖于军事医学科学院实验动物平台。于13周开始测量肿瘤体积。之后每周观察一次，使用游标卡尺分别测量小鼠肿瘤的长径a和短径b，计算肿瘤体积，肿瘤体积计算公式为0.5*ab2。直到22周，处死小鼠。
自发乳腺癌模型小鼠肿瘤体积的检测结果如图1(a)所示，在第11、12、13、14、15、16、17、18、19、20、21、22周，LSECtin+/+-PyMT组小鼠的肿瘤体积分别为0.000±0.000、0.000±0.000、0.000±0.000、1.553±2.763、3.566±4.912、7.049±12.477、13.867±18.089、27.189±25.164、90.020±53.954、176.631±80.076、709.085±334.051、1017.960±434.164(单位为mm3)；在第11、12、13、14、15、16、17、18、19、20、21、22周，LSECtin-/--PyMT组小鼠的肿瘤体积分别为0.000±0.000、0.000±0.000、0.000±0.000、0.658±1.612、0.927±2.269、1.795±4.397、7.136±7.950、12.340±13.105、23.115±27.922、36.883±41.680、103.638±91.174、173.123±112.662(单位为mm3)。上述结果表明：LSECtin-/--PyMT组小鼠肿瘤体积明显小于LSECtin+/+-PyMT组小鼠，说明LSECtin促进小鼠自发乳腺癌的肿瘤成瘤及肿瘤进程。
2、自发乳腺癌模型小鼠肿瘤灶个数的检测
分别将单只自发乳腺癌模型小鼠LSECtin+/+PyMT和单只自发乳腺癌模型小鼠LSECtin-/-PyMT饲养并繁殖于军事医学科学院实验动物平台。于14周开始统计单只肿瘤灶个数，之后每隔两周观察一次，直到20周，处死小鼠。
自发乳腺癌模型小鼠肿瘤灶个数的检测结果如图1(b)所示，从图中可以看出，LSECtin-/--PyMT单只小鼠肿瘤灶个数明显少于LSECtin+/+-PyMT小鼠。说明LSECtin促进小鼠自发乳腺癌的肿瘤成瘤及肿瘤进程。
3、自发乳腺癌模型小鼠肺脏肿瘤转移灶个数的检测
分别将单只自发乳腺癌模型小鼠LSECtin+/+PyMT和单只自发乳腺癌模型小鼠LSECtin-/-PyMT饲养并繁殖于军事医学科学院实验动物平台。于20周，处死小鼠，取出肺 脏，通过石蜡包埋、组织切片及染色获得HE染色结果，并通过计数获得每张切片肺脏转移灶个数的统计结果。
自发乳腺癌模型小鼠肺脏肿瘤转移灶个数的检测结果如图1(c)所示，从图中可以看出，LSECtin-/--PyMT单只小鼠肺脏肿瘤转移灶个数明显少于LSECtin+/+-PyMT小鼠。说明LSECtin促进小鼠自发乳腺癌向肺部的转移。
二、人-裸鼠乳腺癌移植模型的建立与肿瘤体积观测
将1000个人乳腺癌细胞MDA-MB-231、基质胶(BD，354230)及PBS(Hyclone，SH30256.01)混匀，得到混合物；将混合物分别种植5周雌性LSECtin+/+Nude-/-裸鼠(LSECtin+/+Nude-/-组)和LSECtin-/-Nude-/-裸鼠(LSECtin-/-Nude-/-组)的下乳腺，建立人-裸鼠乳腺癌移植模型。两个月中每隔一周观测一次，使用游标卡尺分别测量小鼠肿瘤的长径a和短径b，计算肿瘤体积和成瘤率，肿瘤体积计算公式为0.5*ab2，成瘤率计算公式为成瘤(只)/建模总数(只)。
1、人-裸鼠乳腺癌移植模型小鼠肿瘤体积的检测
LSECtin+/+Nude-/-组和LSECtin-/-Nude-/-组裸鼠肿瘤体积的检测结果如图1(d)所示，在第0、1、2、3、4、5、6、7周，LSECtin+/+-Nude-/-组小鼠的肿瘤体积分别为0.000±0.000、0.000±0.000、23.820±9.802、49.392±23.256、73.482±38.720、129.332±86.165、228.424±170.106、329.700±229.062(单位为mm3)；而在第0、1、2、3、4、5、6、7周，LSECtin-/-Nude-/-组小鼠的肿瘤体积分别为0.000±0.000、0.000±0.000、5.953±6.052、13.096±10.624、25.466±26.931、37.257±40.210、65.645±53.518和91.430±59.608(单位为mm3)。说明LSECtin-/-Nude-/-小鼠的肿瘤体积明显小于LSECtin+/+-Nude-/-小鼠，说明LSECtin能够促进小鼠乳腺癌肿瘤的形成。
2、人-裸鼠乳腺癌移植模型小鼠成瘤率的检测
LSECtin+/+Nude-/-组和LSECtin-/-Nude-/-组裸鼠成瘤率的检测结果如表1所示，LSECtin-/-Nude-/-小鼠成瘤率显著低于LSECtin+/+Nude-/-小鼠。说明LSECtin能够促进小鼠乳腺癌肿瘤的形成。
表1：裸鼠成瘤率的检测结果
组别 成瘤(只)/建模总数(只) 成瘤率(％)
LSECtin+/+Nude-/- 5/11 46
LSECtin-/-Nude-/- 1/8 12
实施例2、肿瘤微环境中LSECtin表达水平的检测
一、小鼠乳腺癌微环境中LSECtin表达水平的检测
1、MMTV-PyMT自发乳腺癌小鼠模型、人-裸鼠乳腺癌移植模型以及临床样本肿瘤浸润髓系细胞的分离
(1)消化液的配制：将20ml的1640培养基(Hyclone公司，SH30809)、20mg胶原酶Ⅳ及1mg DNaseⅠ混匀，得到消化液，0.45μm滤膜过滤。
(2)分别从MMTV-PyMT自发乳腺癌小鼠和人-裸鼠乳腺癌移植模型的小鼠体内剥离种植了8周的乳腺肿瘤组织，或者新鲜临床肿瘤样本，剪碎后，放入步骤(1)配制的消化液中，得到肿瘤消化液，37℃消化40min。
(3)用70μm的滤网过滤肿瘤消化液，250g离心10min。
(4)用1640培养基清洗肿瘤消化液中的肿瘤细胞3次，通过流式分选分别获得如下各个小鼠模型肿瘤浸润髓系细胞(括号内为流式标记)：肿瘤相关巨噬细胞TAM(CD45+CD11b+CD11c+MHCⅡ+Ly6C-Ly6G-)；单核细胞Mo(CD45+CD11b+CD11c-MHC-Ly6C+Ly6G-)；肿瘤相关中性粒细胞TAN(CD45+CD11b+CD11c+/-MHCⅡ-Ly6C+Ly6G+)；其它髓系细胞CD11b-MHCⅡ-(CD45+CD11b-MHCⅡ-)。
2、qPCR检测MMTV-PyMT小鼠自发乳腺癌微环境和人-裸鼠乳腺癌移植微环境中LSECtin表达水平
(1)将步骤1获得的各个小鼠模型肿瘤浸润髓系细胞，按照RNA提取试剂盒的方法，提取RNA；按照cDNA合成试剂盒的方法合成cDNA；
(2)以步骤(1)获得的cDNA为模板，使用实时荧光定量核酸扩增检测系统扩增LSECtin及GAPDH，通过软件分析LSECtin相对表达量。引物序列如下：
LSECtin上游引物：GGTGCCCATCTGGTGATTGT；
LSECtin下游引物：CAGTGGCTGAAGTTGAGTGAGG；
GAPDH上游引物：AGGTCGGTGTGAACGGATTTG；
GAPDH下游引物：TGTAGACCATGTAGTTGAGGTCA。
结果如图2所示，图2(a)为qPCR检测MMTV-PyMT小鼠自发乳腺癌微环境中LSECtin的表达情况；图2(b)为qPCR检测人-裸鼠乳腺癌移植模型肿瘤微环境中LSECtin的表达情况。从图中可以看出：LSECtin在MMTV-PyMT及人-裸鼠移植模型的乳腺癌肿瘤相关巨噬细胞(TAM)中高表达，而在单核浸润细胞(Mo)与粒细胞(TAN)中低表达。
3、免疫荧光检测MMTV-PyMT小鼠自发乳腺癌微环境中LSECtin表达水平
(1)标本加少许OCT包埋剂，置于恒冷切片机中切片，厚度为4-5μm，贴附于载玻片上备用。室温干燥30min。
(2)PBS漂洗，5min×3次。
(3)加入0.01M PBS中漂洗，3min×3次。
(4)0.3％Tritonx-100作用30min(不用洗片，吸出0.3％Tritonx-100即可)。
0.3％Tritonx-100：先3％Tritonx-100 10ml(0.3ml Trixtonx-100原液.9.7ml PBS)，再稀释成0.3％tritonx-100。
(5)5％山羊血清37℃封闭30min(不用洗片，吸出血清即可)，加入以抗体稀释液(3％Tritonx-100 0.4ml、BSA 0.04g、PBS 3.6ml)稀释至工作浓度的兔抗小鼠LSECtin多抗，阴性对照一抗用0.01M PBS替代，4℃置湿盒中过夜。
(6)0.01M PBS中漂洗，3min×3次，除去未结合的兔抗小鼠LSECtin多抗。
(7)滴加稀释好的荧光素标记的抗体，可用0.01M的PBS(PH7.4)对荧光抗体进行稀释，室温孵育1-2hour或37℃孵育30min。
(8)PBS洗，5min×3次，洗去未与组织结合的抗体。
(9)复染细胞核：Hoechst33258室温孵育15min。
(10)0.01M PBS中漂洗，3min×3次。
(11)室温下轻度风干切片。
(12)共聚焦显微镜下观察，采集图像。
小鼠肿瘤原位免疫荧光染色结果如图2(c)所示：表示MMTV-PyMT小鼠自发乳腺癌微环境中LSECtin与巨噬细胞标志分子F4/80共定位，表明MMTV-PyMT小鼠自发乳腺癌微环境中，LSECtin高表达于TAM。
二、乳腺癌病人肿瘤组织中LSECtin表达水平的检测
从新鲜临床病人肿瘤样本中分离组织细胞，并通过流式分析获得肿瘤浸润免疫细胞亚群。具体步骤如下：将活细胞群依次通过SSC-H/FSC-W及SSC-W/FSC-H去除粘连细胞、SSC-A与FSC-A去除细胞碎片、CD45+获得免疫细胞亚群、CD3-CD19-CD56-获得髓系细胞富集亚群、CD14、CD11b和CD15定义TAM(CD11b+CD14+CD15-)，得到CD45+CD3-CD15-CD19-CD56-CD11b+CD14+的肿瘤相关巨噬细胞，用LSECtin流式抗体标记为实验组；以同型对照抗体标记作为对照组。
流式检测LSECtin表达水平结果如图2(d)所示：LSECtin高表达于乳腺癌CD45+CD3-CD15-CD19-CD56-CD11b+CD14+的肿瘤相关巨噬细胞。
三、骨髓瘤病人肿瘤组织中LSECtin表达水平的检测
从新鲜临床骨髓瘤病人肿瘤样本中分离组织细胞，并通过流式分析获得肿瘤浸润免疫细胞亚群。具体步骤如下：将活细胞群依次通过SSC-H/FSC-W及SSC-W/FSC-H去除粘连细胞、SSC-A与FSC-A去除细胞碎片、CD45+获得免疫细胞亚群、CD3-CD19-CD56-获得髓系细胞富集亚群、CD14、CD11b和CD15定义TAM(CD11b+CD14+CD15-)，得到CD45+CD3-CD15-CD19-CD56-CD11b+CD14+的肿瘤相关巨噬细胞，用LSECtin流式抗体标记为实验组；以同型对照抗体标记作为对照组。
流式检测LSECtin表达水平结果如图2(e)所示：LSECtin高表达于骨髓瘤CD45+CD3-CD15-CD19-CD56-CD11b+CD14+的肿瘤相关巨噬细胞。
四、肺癌病人肿瘤组织中LSECtin表达水平的检测
从新鲜临床肺癌病人肿瘤样本中分离组织细胞，并通过流式分析获得肿瘤浸润免疫细胞亚群。具体步骤如下：将活细胞群依次通过SSC-H/FSC-W及SSC-W/FSC-H去除粘连细胞、SSC-A与FSC-A去除细胞碎片、CD45+获得免疫细胞亚群、CD3-CD19-CD56-获得髓系细胞富集亚群、CD14、CD11b和CD15定义TAM(CD11b+CD14+CD15-)，得到CD45+CD3-CD15-CD19-CD56-CD11b+CD14+的肿瘤相关巨噬细胞，用LSECtin流式抗体标记为实验组；以同型对照抗体标记作为对照组。
流式检测LSECtin表达水平结果如图2(f)所示：LSECtin高表达于肺癌CD45+CD3-CD15-CD19-CD56-CD11b+CD14+的肿瘤相关巨噬细胞。
五、结肠癌病人肿瘤组织中LSECtin表达水平的检测
从新鲜临床结肠癌病人肿瘤样本中分离组织细胞，并通过流式分析获得肿瘤浸润免疫细胞亚群。具体步骤如下：将活细胞群依次通过SSC-H/FSC-W及SSC-W/FSC-H去除粘连细胞、SSC-A与FSC-A去除细胞碎片、CD45+获得免疫细胞亚群、CD3-CD19-CD56-获得髓系细胞富集亚群、CD14、CD11b和CD15定义TAM(CD11b+CD14+CD15-)，得到CD45+CD3-CD15-CD19-CD56-CD11b+CD14+的肿瘤相关巨噬细胞，用LSECtin流式抗体标记为实验组；以同型对照抗体标记作为对照组。
流式检测LSECtin表达水平结果如图2(g)所示：LSECtin高表达于结肠癌CD45+CD3-CD15-CD19-CD56-CD11b+CD14+的肿瘤相关巨噬细胞。
六、骨巨细胞瘤病人肿瘤组织中LSECtin表达水平的检测
从新鲜临床骨巨细胞瘤病人肿瘤样本中分离组织细胞，并通过流式分析获得肿瘤浸润免疫细胞亚群。具体步骤如下：将活细胞群依次通过SSC-H/FSC-W及SSC-W/FSC-H去除粘 连细胞、SSC-A与FSC-A去除细胞碎片、CD45+获得免疫细胞亚群、CD3-CD19-CD56-获得髓系细胞富集亚群、CD14、CD11b和CD15定义TAM(CD11b+CD14+CD15-)，得到CD45+CD3-CD15-CD19-CD56-CD11b+CD14+的肿瘤相关巨噬细胞，用LSECtin流式抗体标记为实验组；以同型对照抗体标记作为对照组。
流式检测LSECtin表达水平结果如图2(h)所示：LSECtin高表达于骨巨细胞瘤CD45+CD3-CD15-CD19-CD56-CD11b+CD14+的肿瘤相关巨噬细胞。
七、肾癌病人肿瘤组织中LSECtin表达水平的检测
从新鲜临床肾癌病人肿瘤样本中分离组织细胞，并通过流式分析获得肿瘤浸润免疫细胞亚群。具体步骤如下：将活细胞群依次通过SSC-H/FSC-W及SSC-W/FSC-H去除粘连细胞、SSC-A与FSC-A去除细胞碎片、CD45+获得免疫细胞亚群、CD3-CD19-CD56-获得髓系细胞富集亚群、CD14、CD11b和CD15定义TAM(CD11b+CD14+CD15-)，得到CD45+CD3-CD15-CD19-CD56-CD11b+CD14+的肿瘤相关巨噬细胞，用LSECtin流式抗体标记为实验组；以同型对照抗体标记作为对照组。
流式检测LSECtin表达水平结果如图2(i)所示：LSECtin高表达于肾癌CD45+CD3-CD15-CD19-CD56-CD11b+CD14+的肿瘤相关巨噬细胞。
八、喉癌病人肿瘤组织中LSECtin表达水平的检测
从新鲜临床喉癌病人肿瘤样本中分离组织细胞，并通过流式分析获得肿瘤浸润免疫细胞亚群。具体步骤如下：将活细胞群依次通过SSC-H/FSC-W及SSC-W/FSC-H去除粘连细胞、SSC-A与FSC-A去除细胞碎片、CD45+获得免疫细胞亚群、CD3-CD19-CD56-获得髓系细胞富集亚群、CD14、CD11b和CD15定义TAM(CD11b+CD14+CD15-)，得到CD45+CD3-CD15-CD19-CD56-CD11b+CD14+的肿瘤相关巨噬细胞，用LSECtin流式抗体标记为实验组；以同型对照抗体标记作为对照组。
流式检测LSECtin表达水平结果如图2(j)所示：LSECtin高表达于喉癌CD45+CD3-CD15-CD19-CD56-CD11b+CD14+的肿瘤相关巨噬细胞。
九、腮腺癌病人肿瘤组织中LSECtin表达水平的检测
从新鲜临床腮腺癌病人肿瘤样本中分离组织细胞，并通过流式分析获得肿瘤浸润免疫细胞亚群。具体步骤如下：将活细胞群依次通过SSC-H/FSC-W及SSC-W/FSC-H去除粘连细胞、SSC-A与FSC-A去除细胞碎片、CD45+获得免疫细胞亚群、CD3-CD19-CD56-CD15-获得髓系细胞非粒细胞富集亚群，其中CD11b+定义TAM，得到CD45+CD3-CD15-CD19-CD56-CD11b+的肿瘤相关巨噬细胞(TAM组)；CD45+CD3-CD15+CD19-CD56-CD11b+为非TAM组。
流式检测LSECtin表达水平结果如图2(k)所示：LSECtin高表达于腮腺癌CD45+CD3-CD15-CD19-CD56-CD11b+的肿瘤相关巨噬细胞。
实施例3、巨噬细胞特异性敲除LSECtin抑制肿瘤进程
下述实施例中的MMTV-PyMT为自发乳腺癌模型小鼠；LyZ2Cre为LyZ2基因Cre酶敲入小鼠；LSECtinfl/fl为LoxP基因敲入小鼠；WT-PyMT-LyZ2为野生型自发乳腺癌模型小鼠，KO-PyMT-LyZ2为巨噬细胞LSECtin敲除自发乳腺癌模型小鼠。具体获得方法如下：C57Bl/6J背景野生型MMTV-PyMT自发乳腺癌模型雄鼠与C57Bl/6J背景LyZ2Cre雌鼠交配，经基因型鉴定，获得Cre酶敲入的自发乳腺癌模型雄鼠。将该小鼠与C57Bl/6J背景LSECtinfl/fl雌鼠交配，经基因型鉴定可获得获得Cre酶敲入且LoxP基因为杂合的自发乳腺癌模型雄鼠。将该小鼠与LoxP基因敲入小鼠交配，可获得WT-PyMT-LyZ2小鼠(MMTV+LyZ2Cre-LSECtinfl/fl)和KO-PyMT-LyZ2小鼠(MMTV+LyZ2Cre+LSECtinfl/fl)。上述标注中，MMTV代表自发乳腺癌模型转基因，Cre-代表Cre酶未敲入，Cre+代表Cre酶敲入，fl/-代表LoxP基因敲入杂合子，fl/fl代表LoxP基因敲入纯合子。上述Cre酶敲入小鼠可从Jackson公司购买，LoxP基因敲入小鼠可从TACONIC购买，公众也可从北京蛋白质组研究中心获得。上述C57Bl/6J背景野生型MMTV-PyMT自发乳腺癌模型小鼠在文献“Davie S A,Maglione J E,Manner C K,et al.Effects of FVB/NJ and C57Bl/6J strain backgrounds on mammary tumor phenotype in inducible nitric oxide synthase deficient mice[J].Transgenic research,2007,16(2):193-201.”中公开过，公众可从北京蛋白质组研究中心获得。
一、巨噬细胞特异性敲除LSECtin自发乳腺癌模型小鼠肿瘤体积的检测
分别将巨噬细胞敲除LSECtin的自发乳腺癌模型对照组小鼠WT-PyMT-LyZ2和实验组KO-PyMT-LyZ2饲养并繁殖于国家蛋白质组研究中心动物平台。于13周开始测量肿瘤体积。之后每周观察1次，使用游标卡尺分别测量小鼠肿瘤的长径a和短径b，计算肿瘤体积，肿瘤体积计算公式为0.5*ab2。直到21周，处死小鼠。
自发乳腺癌模型小鼠肿瘤体积的检测结果如图14所示，在第11、12、13、14、15、16、17、18、19、20、21周，WT-PyMT-LyZ2组小鼠的肿瘤体积分别为0.00±0.000、3.94±7.63、17.16±23.96、34.56±37.33、81.59±79.46、106.29±93.45、188.42±127.41、396.37±181.39、599.75±224.66、814.60±336.65、1227.49±516.97(单位为mm3)；在第11、12、13、14、15、16、17、18、19、20、21周，KO-PyMT-LyZ2 组小鼠的肿瘤体积分别为0.000±0.000、0.000±0.000、3.00±11.25、9.32±15.89、11.01±19.49、34.71±43.83、96.71±98.74、203.46±196.01、272.47±254.20、440.47±315.91、653.70±419.70(单位为mm3)。上述结果表明：KO-PyMT-LyZ2组小鼠肿瘤体积明显小于WT-PyMT-LyZ2组小鼠，说明敲除巨噬细胞LSECtin可显著抑制小鼠乳腺癌的肿瘤成瘤及肿瘤进程。
二、巨噬细胞特异性敲除LSECtin自发乳腺癌模型小鼠肿瘤灶个数的检测
分别将将巨噬细胞敲除LSECtin的自发乳腺癌模型对照组小鼠WT-PyMT-LyZ2和实验组KO-PyMT-LyZ2饲养并繁殖于国家蛋白质组研究中心实验动物平台。于14周开始统计单只肿瘤灶个数，之后每隔两周观察1次，直到20周，处死小鼠。
自发乳腺癌模型小鼠肿瘤灶个数的检测结果如图15所示，从图中可以看出，KO-PyMT-LyZ2单只小鼠肿瘤灶个数明显少于WT-PyMT-LyZ2小鼠，说明敲除巨噬细胞LSECtin可显著抑制小鼠乳腺癌的肿瘤成瘤及肿瘤进程。
三、巨噬细胞特异性敲除LSECtin自发乳腺癌模型小鼠肺脏肿瘤转移灶个数的检测
分别将单只自发乳腺癌模型小鼠WT-PyMT-LyZ2和单只自发乳腺癌模型小鼠KO-PyMT-LyZ2饲养并繁殖于国家蛋白质组研究中心动物平台。于25周，处死小鼠，取出肺脏，通过石蜡包埋、组织切片及染色获得HE染色结果，并通过计数获得每张切片肺脏转移灶个数的统计结果。
自发乳腺癌模型小鼠肺脏肿瘤转移灶个数的检测结果如图16所示，从图中可以看出，KO-PyMT-LyZ2单只小鼠肺脏肿瘤转移灶个数明显少于WT-PyMT-LyZ2小鼠，说明敲除巨噬细胞LSECtin可显著抑制小鼠自发乳腺癌向肺部的转移。
实施例4、多种临床样本肿瘤细胞表达BTN3A2及BTN3A3
一、临床样本肿瘤细胞的分离
(1)消化液的配制：将20ml的1640培养基(Hyclone公司，SH30809)、20mg胶原酶Ⅳ及1mg DNaseⅠ混匀，得到消化液，0.45μm滤膜过滤。
(2)分别取新鲜临床病人肿瘤组织，剪碎后，放入步骤(1)配制的消化液中，得到肿瘤消化液，37℃消化40min。
(3)用70μm的滤网过滤肿瘤消化液，250g离心10min。
(4)用1640培养基清洗肿瘤消化液中的肿瘤细胞3次，通过流式分选分别获得肿瘤细胞富集亚群：具体步骤如下：将活细胞群依次通过SSC-H/FSC-W及SSC-W/FSC-H去除粘连细胞、SSC-A与FSC-A去除细胞碎片、CD45-获得肿瘤细胞富集亚群，并通过用anti-CD277 抗体(该抗体同时识别膜形式的BTN3A1、BTN3A2、BTN3A3)进行BTN3A表达检测。
二、乳腺癌病人肿瘤组织中肿瘤细胞BTN3A表达水平的检测
按照上述一中的描述，对乳腺癌病人肿瘤组织中肿瘤细胞BTN3A表达水平进行检测。
流式检测乳腺癌病人BTN3A表达水平结果如图3(a)所示：以CD45-定义的乳腺癌肿瘤细胞，用anti-CD277抗体标记的细胞为实验组表达BTN3A，对照组为同型对照抗体标记的细胞。
三、肺癌病人肿瘤组织中肿瘤细胞BTN3A表达水平的检测
按照上述一中的描述，对肺癌病人肿瘤组织中肿瘤细胞BTN3A表达水平进行检测。
流式检测BTN3A表达水平结果如图3(b)所示：以CD45-定义的肺癌肿瘤细胞，用anti-CD277抗体标记的细胞为实验组表达BTN3A，对照组为同型对照抗体标记的细胞。
四、结肠癌病人肿瘤组织中肿瘤细胞BTN3A表达水平的检测
按照上述一中的描述，对结肠癌病人肿瘤组织中肿瘤细胞BTN3A表达水平进行检测。
流式检测BTN3A表达水平结果如图3(c)所示：以CD45-定义的结肠癌肿瘤细胞，用anti-CD277抗体标记的细胞为实验组表达BTN3A，对照组为同型对照抗体标记的细胞。
五、骨巨细胞瘤病人肿瘤组织中肿瘤细胞BTN3A表达水平的检测
按照上述一中的描述，对骨巨细胞瘤病人肿瘤组织中肿瘤细胞BTN3A表达水平进行检测。
流式检测BTN3A表达水平结果如图3(d)所示：以CD45-定义的骨巨细胞瘤肿瘤，用anti-CD277抗体标记的细胞为实验组表达BTN3A，对照组为同型对照抗体标记的细胞。
六、肾癌病人肿瘤组织中肿瘤细胞BTN3A表达水平的检测
按照上述一中的描述，对肾癌病人肿瘤组织中肿瘤细胞BTN3A表达水平进行检测。
流式检测BTN3A表达水平结果如图3(e)所示：以CD45-定义的肾癌肿瘤细胞，用anti-CD277抗体标记的细胞为实验组表达BTN3A，对照组为同型对照抗体标记的细胞。
七、腮腺癌病人肿瘤组织中肿瘤细胞BTN3A表达水平的检测
按照上述一中的描述，对腮腺癌病人肿瘤组织中肿瘤细胞BTN3A表达水平进行检测。
流式检测BTN3A表达水平结果如图3(f)所示：以CD45-定义的腮腺癌肿瘤细胞，用anti-CD277抗体标记的细胞为实验组部分表达BTN3A，对照组为同型对照抗体标记的细胞。
实施例5、多种肿瘤细胞系表达BTN3A3
1、肿瘤细胞系的培养
培养如下乳腺癌细胞系：MCF7(3111C0001CCC000013)、ZR75-1(3111C0001CCC000090)、BT474(3111C0001CCC000129)、T47D(3111C0001CCC000265)、MDA-MB-453(3111C0001CCC000016)、SKBR3(3111C0001CCC000085)、MDA-MB-468(3111C0001CCC000249)、MDA-MB-436(3111C0001CCC000352)；MDA-MB-231(3111C0001CCC000013)；肝癌细胞系：BEL-7402(3131C0001000700010)、HepG2(3111C0001CCC000035)、HCC-LM3(3142C0001000000316)、HHCC(3111C0002000000069)、Hep3B(3111C0001CCC000376)、QGY7701(3131C0001000700042)、SMCC7721(3111C0001CCC000087)、Huh7(3131C0001000700182)；黑色素瘤细胞系：A875(3111C0001CCC000094)、A375(3131C0001000700004)；胃癌细胞系：MKN28(3111C0001CCC000482)、NCI-N87(3111C0001CCC000481)、MGC-803(3111C0001CCC000227)、SGC-7901(3131C0001000700046)；结肠癌细胞系：LOVO(3111C0001CCC000164)、SW480(3142C0001000000064)、LS174T(3111C0001CCC000248)、DLD-1(3131C0001000700134)。上述细胞均购自国家实验细胞资源共享平台。上述细胞培养条件按“国家实验细胞资源共享平台”网站查询的方法培养。
2、用RIPA裂解液(Thermofisher，89901)分别将上述各个细胞系进行裂解，分别获得细胞裂解液，并分别对细胞裂解液进行Western Blot检测，通过anti-BTN3A3抗体(Sigma,HPA007904)检测BTN3A3表达水平。
Western Blot检测结果如图4(a)-(e)所示：从图中可以看出，多种肿瘤细胞系表达BTN3A3。
实施例6、BTN3A2和BTN3A3在乳腺癌细胞上的表达
一、qPCR检测乳腺癌细胞系上BTN3A2和BTN3A3的表达水平
1、RNA的提取及cDNA的反转录
采用RNA提取试剂盒提取如下各乳腺癌细胞：MCF7、ZR75-1、BT474、T47D、SKBR3、MDA-MB-468、MDA-MB-231、MDA-MB-436的RNA；按照cDNA合成试剂盒的方法合成cDNA。
2、qPCR检测乳腺癌细胞系上BTN3A3表达水平
以步骤1获得的cDNA为模板，使用实时荧光定量核酸扩增检测系统(qPCR)扩增BTN3A2，BTN3A3及GAPDH，并通过软件分析BTN3A2，BTN3A3相对表达量。上述BTN3A2，BTN3A3以及GAPDH引物均购自Qiagen公司。
结果如图5(a)所示。qPCR检测结果表明：乳腺癌细胞MDA-MB-231、MDA-MB-468、MDA-MB-436上高表达BTN3A2和BTN3A3。
二、流式细胞显示BTN3A分子表达于乳腺癌细胞表面
用anti-CD277抗体(该抗体同时识别膜形式的BTN3A1、BTN3A2、BTN3A3)对如下乳腺癌细胞系：MCF7、ZR75-1、BT474、MDA-MB-468、MDA-MB-231、MDA-MB-436进行流式检测。具体步骤如下：实验组用1×PBS以1:50体积比稀释anti-CD277抗体(eBioscience，14-2779)，对照组用1×PBS以1:50体积比稀释同型对照抗体(eBioscience，14-4714-82)，4℃孵育30min。用1×PBS洗细胞3次后，弃上清。以1:50体积比稀释羊抗小鼠PE标记荧光二抗(Biolegend，405307)标记细胞，4℃孵育30min。用1×PBS洗细胞3次后，弃上清，用300μl PBS重悬后进行流式检测。
结果如图5(b)所示，从图中可以看出：BTN3A表达于乳腺癌细胞MDA-MB-468、MDA-MB-231和MDA-MB-436的表面。
三、细胞免疫荧光检测BTN3A3于乳腺癌细胞表面膜定位
收集培养状态下的MDA-MB-231细胞，用PBS洗三遍，洗去多余血清。稀释10×的透膜液(达科为，421002)成1×的工作液。用透膜液重悬固定细胞，350g离心10min，弃上清，重复该步骤一次。100ul透膜液重悬固定破膜后的细胞，用透膜液以1:200体积比稀释的anti-BTN3A3抗体(Sigma，HPA007904)，4℃孵育30min。用透膜液洗细胞3次后，弃上清。加入以体积比1:200稀释的兔TRITC荧光抗体(中杉金桥，ZF-0318)，4℃避光孵育30min。用透膜液洗细胞三次后，将细胞放入载玻片，荧光显微镜下观察BTN3A3表达情况。
结果如图5(c)所示，从图中可以看出：MDA-MB-231细胞表达BTN3A3，并主要定位于细胞膜。
实施例7、肿瘤细胞表达的BTN3A2和BTN3A3促进肿瘤形成
利用231-NC细胞及231-sh4细胞验证肿瘤表达的BTN3A3对肿瘤进程的影响。具体步骤如下：
1、敲低BTN3A3的人乳腺癌细胞MDA-MB-231的构建
231-sh3细胞和231-sh4细胞均为稳定表达绿色荧光蛋白且敲低BTN3A3的人乳腺癌细胞MDA-MB-231，231-NC细胞是稳定表达绿色荧光蛋白的人乳腺癌细胞MDA-MB-231。具体构建步骤如下
(1)委托苏州吉玛公司完成敲低BTN3A3的sh表达质粒的构建及慢病毒的包装与纯 化的步骤。sh RNA序列如下：sh3：GCCACAGATGGATCTCATATC(序列4)；sh4：CCCTTCTGCAACAACCAATCA(序列5)；NC(阴性对照序列)：TTCTCCGAACGTGTCACGTTTC。
(2)利用慢病毒分别将构建的sh3表达质粒转染目的细胞(人乳腺癌细胞MDA-MB-231)，并通过荧光蛋白进行筛选，得到敲低BTN3A3的稳定细胞系231-sh3。
利用慢病毒分别将构建的sh4表达质粒转染目的细胞(人乳腺癌细胞MDA-MB-231)，并通过荧光蛋白进行筛选，得到敲低BTN3A3的稳定细胞系231-sh4。
利用慢病毒分别将构建的NC表达质粒转染目的细胞(人乳腺癌细胞MDA-MB-231)，并通过荧光蛋白进行筛选，得到对照细胞系231-NC。
上述转染的具体步骤如下：病毒转染前18-24小时，将贴壁细胞以1×105个/孔铺到24孔板中。使细胞在慢病毒转染时的数量为2×105MOI/孔左右。第二天，用含有6μg/ml polybrene(苏州吉玛公司)的2ml新鲜培养基替换原培养基，加入适量病毒悬液。继续培养24小时，用新鲜培养基替换含有病毒的培养基。病毒感染48小时后可见明显荧光表达，72小时后更加明显。扩大培养一周，通过流式分选获得带有GFP的细胞。
(3)BTN3A3和BTN3A2的mRNA水平表达量检测
分别收集105个231-sh4细胞和231-NC对照细胞，利用qPCR分别对231-sh4细胞和231-NC对照细胞的BTN3A1，BTN3A2，BTN3A3mRNA水平表达进行检测。具体步骤如下：按照试剂盒说明书所示方法进行RNA提取及反转录，获得的cDNA，再利用qPCR引物进行qPCR。其中，RNA小提试剂盒(74034)购自Qiagen公司。RNA反转录试剂盒(Promega)购自Promega公司。BTN3A1(QF00264803)，BTN3A2(QT00060039)，BTN3A3(QF00264803)基因qPCR引物均购自Qiagen公司。
检测结果如图6(a)所示，与对照细胞231-NC相比，敲低BTN3A3的稳定细胞系231-sh4中，BTN3A2及BTN3A3的相对表达量均低于0.4，说明敲低BTN3A3的稳定细胞系231-sh4中BTN3A2及BTN3A3表达水平均明显降低。
(4)BTN3A3的蛋白水平表达量检测
分别收集105个231-sh3细胞、231-sh4细胞和231-NC对照细胞，对231-sh3细胞、231-sh4细胞和231-NC对照细胞的BTN3A3蛋白水平表达进行检测。具体步骤如下：用RIPA裂解液(Thermofisher，89901)分别将上述各个细胞系进行裂解，分别获得细胞裂解液，并分别对细胞裂解液进行Western Blot检测，通过anti-BTN3A3抗体(Sigma,HPA007904)检测BTN3A3表达水平，具体方法参照抗体说明书。
结果如图6(b)所示，与对照细胞231-NC相比，敲低BTN3A3的稳定细胞系231-sh3 和敲低BTN3A3的稳定细胞系231-sh4中BTN3A3表达水平明显降低。
2、分别将对照细胞231-NC和231-sh4细胞以1000个细胞/只原位种植裸鼠乳腺脂肪垫。在接种细胞后，第8天开始观察，之后每周观察一次，使用游标卡尺分别测量小鼠肿瘤的长径a和短径b，并计算肿瘤体积和成瘤率。肿瘤体积计算公式为0.5*ab2。直到6周时，处死小鼠。
肿瘤体积的检测结果如图6(c)所示，小鼠在接种231-NC细胞后第0、1、2、3、4、5、6周肿瘤体积分别为0.000±0.000、3.398±5.829、10.627±10.152、32.637±20.258、60.116±24.670、158.550±59.933和331.362±100.745(单位为mm3)；小鼠在接种231-sh4细胞后第0、1、2、3、4、5、6周肿瘤体积分别为0.000±0.000、0.000±0.000、4.068±6.957、14.460±20.532、13.208±17.609、36.183±48.657和96.543±127.005(单位为mm3)；
成瘤率的检测结果如表2所示，对照细胞231-NC组小鼠的成瘤率明显高于231-sh4组。
表2：成瘤率的检测结果
组别 成瘤(只)/建模总数(只) 成瘤率(％)
231-NC 5/10 50
231-sh4 2/10 20
上述结果均表明：敲低BTN3A2和BTN3A3后，乳腺癌细胞在裸鼠体内的成瘤率、成瘤体积均明显下降。说明BTN3A2和BTN3A3促进肿瘤形成。
实施例8、LSECtin与BTN3A2及BTN3A3存在直接且特异性的相互作用
粘附实验验证LSECtin与BTN3A2及BTN3A3存在直接且特异的相互作用
1、过表达BTN3A1，BTN3A2，BTN3A3的载体的构建
分别将BTN3A1序列(序列6)、BTN3A2序列(序列7)、BTN3A3序列(序列8)替换pIRES2-EGFP载体(Clotech，6029-1)的NdeⅠ和XhoⅠ酶切位点间的DNA片段，分别获得表达BTN3A1的载体pIRES2-EGFP-BTN3A1、表达BTN3A2的pIRES2-EGFP-BTN3A2、表达BTN3A3的pIRES2-EGFP-BTN3A3。
2、分别将表达BTN3A1的载体pIRES2-EGFP-BTN3A1、表达BTN3A2的pIRES2-EGFP-BTN3A2、表达BTN3A3的pIRES2-EGFP-BTN3A3及空载体pIRES2-EGFP转染BT474细胞(国家实验细胞资源共享平台，3111C0001CCC000129)，转染36h后，分别得到过表达BTN3A1的细胞BT474-BTN3A1、过表达BTN3A2的细胞BT474-BTN3A2、过表达 BTN3A3的细胞BT474-BTN3A3和过表达空载体的细胞BT474-EGFP。
3、消化收集步骤2获得的各个细胞，分别用人源LSECtin蛋白(R&D,2947-CL)和鼠源LSECtin-Fc蛋白与上述各个细胞粘附，并用小鼠抗人LSECtin抗体CCB059检测人源LSECtin粘附率，用anti-IgG(Biolegend，405307)检测鼠源LSECtin粘附率，通过流式检测粘附比例。粘附实验具体步骤参照文献“Tang L,Yang J,Tang X,et al.The DC-SIGN family member LSECtin is a novel ligand of CD44on activated T cells[J].European journal of immunology,2010,40(4):1185-1191.”中的方法。
粘附实验验证LSECtin与BTN3A3存在直接且特异性的相互作用的结果如图7(a)和图7(b)所示。其中，人源LSECtin粘附结果如图7(a)所示，LSECtin与过表达空载体的细胞BT474-EGFP不粘附(粘附率为3.579％)，与过表达BTN3A1的细胞BT474-BTN3A1不粘附(粘附率为0.775％)，与过表达BTN3A2的细胞BT474-BTN3A2弱粘附(粘附率为42.33％)，与过表达BTN3A3的细胞BT474-BTN3A3强粘附(粘附率为57.587％)。鼠源LSECtin-Fc粘附结果如图7(b)所示，LSECtin与过表达空载体的细胞BT474-EGFP不粘附(粘附率为0.967％)、与过表达BTN3A1的细胞BT474-BTN3A1不粘附(粘附率为1.598％)，与过表达BTN3A2的细胞BT474-BTN3A2不粘附(粘附率为6.61％)，与BT474-BTN3A3粘附(粘附率为27.345％)。
上述结果说明，人源及鼠源LSECtin与人肿瘤细胞表面表达的BTN3A3存在直接且特异的强相互作用，人源LSECtin与人肿瘤细胞表面表达的BTN3A3存在强相互作用。
实施例9、LSECtin与BTN3A2及BTN3A3相互作用促进肿瘤细胞干性的维持
一、球形成实验证明LSECtin与BTN3A2及BTN3A3相互作用促进肿瘤细胞干性的维持
本实施例通过球形成实验证明LSECtin与BTN3A2及BTN3A3相互作用促进肿瘤细胞干性的维持。具体步骤如下：
1、分别将231-sh3细胞、231-sh4细胞和231-NC细胞，以及实施例7步骤二中的2获得的过表达BTN3A1的细胞BT474-BTN3A1、过表达BTN3A2的细胞BT474-BTN3A2、过表达BTN3A3的细胞BT474-BTN3A3和过表达空载体的细胞BT474-EGFP制成单细胞悬液，第一代细胞以20,000个/mL铺板，传代以1000个/mL铺板。
2、分别将上述各个细胞、B27(Life,17504044)、bFGF(Sigam,SRP2092)、EGF(Sigma,E9644)、胰岛素(Sigma,I3536)、肝素(sigma,1235853)和DMEM/F12无血清培养基混匀后，分别得到培养体系，各个组分在培养体系中的浓度为：B27(10ng/ml)、bFGF(20ng/ml)、EGF(20ng/ml)、胰岛素(5μg/ml)、肝素(4μg/ml)，培养7-10天后，计 算直径大于75μm的球体数量并拍照。
3、800rpm离心收集球体，胰酶(Gibico,25300120)消化，40μm筛网过滤，进行二次球体形成实验。
4、分别向上述各个培养体系中加入浓度为100ng/ml的LSECitn刺激肿瘤细胞，分别得到LSECitn刺激后的各个细胞。
结果如图8(a)和图8(b)所示，在100ng的LSECtin刺激浓度下，LSECtin能够促进对照细胞231-NC细胞球形成；但不能促进敲低BTN3A2及BTN3A3的231-sh3细胞和231-sh4细胞的球形成。在100ng的LSECtin刺激浓度下，LSECtin不能促进过表达空载体的细胞BT474-EGFP的球形成；但可以促进过表达BTN3A2的细胞BT474-BTN3A2、过表达BTN3A3的细胞BT474-BTN3A3的球形成。
二、干性特征分子表达水平的检测
收集步骤一中的4获得的球形成实验中的231-sh4细胞和231-NC细胞，通过qPCR检测乳腺肿瘤干性相关特征分子如OCT4、NANOG、SOX2的表达变化。DC-SIGN代表阴性对照，Control为未用LSECtin刺激组，231-sh4为敲低BTN3A3的细胞，231-NC为对照细胞。引物序列如下：OCT4：Up-GCTCGAGAAGGATGTGGTCC；Down-GTTGTGCATAGTCGCTGCT；NANOG：Up-TCTGGACACTGGCTGAATCCT；Down-CGCTGATTAGGCTCCAACCAT；SOX2：Up-GCTCGCAGACCTACATGAAC；Down-GGGAGGAAGAGGTAACCACA。
结果如图8(c)所示，在100ng的LSECtin刺激浓度下，LSECtin能够促进干性关键转录因子Oct4，Nanog和Sox的表达；但该促进效应在敲除BTN3A3后，则不能。
上述结果说明，LSECtin与BTN3A2及BTN3A3相互作用促进肿瘤进程依赖与促进肿瘤细胞干性的维持。
实施例10、LSECtin与BTN3A2及BTN3A3相互作用促进肿瘤进程依赖激活肿瘤细胞内STAT3磷酸化
一、LSECtin刺激表达BTN3A3的乳腺癌细胞STAT3磷酸化
分别收集实施例8中步骤一的4获得的经LSECtin刺激的球形成样本：敲低BTN3A3的231-sh3细胞(LSECitn-231-sh3)、敲低BTN3A3的231-sh4细胞(LSECitn-231-sh4)、细胞231-NC(LSECitn-231-NC)和实施例8步骤一的3中获得的未经LSECtin刺激的球形成样本：敲低BTN3A3的231-sh3细胞(231-sh3)、敲低BTN3A3的231-sh4细胞(231-sh4)、细胞231-NC(231-NC)。利用胰酶进行消化，4℃，1000rmp离心后，弃上清，收集细胞，并通过PBS洗细胞三次。通过RIPA裂解液冰上裂解30min，12,000rmp离心，取上清，获 得细胞裂解液。通过Stat Antibody Sampler Kit试剂盒(Cell signaling technology，9939)Western Blot检测裂解液中STAT1、STAT3、STAT5、STAT6的本底水平，通过Phospho-Stat Antibody Sampler Kit试剂盒(Cell signaling technology，9914)检测裂解液中STAT1、STAT3、STAT5、STAT6的磷酸化水平。具体检测方法参照试剂盒中的说明书。
结果如图9(a)所示，LSECtin刺激下，正常表达BTN3A2及BTN3A3的231-NC细胞的p-STAT3水平明显上调；但在敲低BTN3A2及BTN3A3的231-sh3细胞(LSECitn-231-sh3)、敲低BTN3A2及BTN3A3的231-sh4细胞(LSECitn-231-sh4)中，LSECtin不能刺激p-STAT3水平上调；但无论是否敲低BTN3A3，STAT家族的其它分子STAT1、STAT5、STAT6磷酸化水平无上调作用。
上述结果说明，LSECtin通过与BTN3A3相互作用，能够刺激p-STAT3水平上调。
二、LSECtin促进表达BTN3A2及BTN3A3的乳腺癌细胞球形成依赖STAT3磷酸化
分别收集实施例8步骤一的4中获得的球形成样本：敲低BTN3A3的细胞231-sh3、敲低BTN3A3的细胞231-sh4和敲低BTN3A3的细胞231-NC，并在收集样本后的第三天加入STAT3抑制剂(selleck，S1155)，第十天检测球形成数量。
结果如图9(b)所示，加入STAT3抑制剂后，LSECtin促进正常表达BTN3A2及BTN3A3的231-NC细胞的球形成能力明显降低；在敲低BTN3A2及BTN3A3的231-sh3及231-sh4细胞中，加入STAT3抑制剂使得LSECtin促进球形成作用完全消失。
上述结果说明，LSECtin与乳腺癌细胞表达的BTN3A2及BTN3A3相互作用促进肿瘤进程依赖于细胞内部的STAT3磷酸化水平。
通过以上实施例1-实施例10实验证明：肿瘤相关巨噬细胞表达的LSECtin、肿瘤细胞表达的BTN3A2和/或BNT3A3通过促进肿瘤细胞干性的维持促进肿瘤进程，具体体现在促进肿瘤细胞球形成、干性转录因子的表达以及小鼠肿瘤模型中对肿瘤进程的促进，由此表明肿瘤相关巨噬细胞表达的LSECtin和肿瘤表达的BNT3A3可分别作为肿瘤免疫治疗的靶点，这类肿瘤共同的特点为能浸润肿瘤相关巨噬细细胞表达LSECtin。
以上实施例中，LSECtin为由序列表中序列1所示的氨基酸序列组成的蛋白质；或将序列1的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的由序列1衍生的蛋白质。BTN3A2为由序列表中序列2所示的氨基酸序列组成的蛋白质；或将序列2的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的由序列2衍生的蛋白质。BTN3A3为由序列表中序列3所示的氨基酸序列 组成的蛋白质；或将序列3的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的由序列3衍生的蛋白质。
以上实施例实验还证明：抑制LSECtin与BTN3A2和/或BTN3A3的相互作用能够有效减缓肿瘤进程，具体体现在降低肿瘤发生率和减缓肿瘤体积增长，由此也提示出LSECtin与BTN3A3可共同作为免疫治疗与肿瘤相关巨噬细胞表达LSECtin且肿瘤细胞表达BTN3A3的肿瘤的靶点，这类肿瘤包括但不限于乳腺癌、骨髓瘤、肝癌、胃癌、结肠癌、肺癌、骨巨细胞瘤、肾癌、喉癌或腮腺癌。
基于LSECtin与BTN3A3或BTN3A2相互作用促进肿瘤进程的实验结果，本发明进一步提出一类抑制LSECtin与BTN3A2或BTN3A3相互作用的物质，这些物质能制备具有如下(b1)-(b5)中至少一种功能的产品：
(b1)治疗和/或预防肿瘤；
(b2)抑制肿瘤进程；
(b3)抑制肿瘤细胞干性的维持或促进；
(b4)抑制肿瘤细胞干性相关特征分子的表达；和
(b5)抑制肿瘤细胞内部STAT3磷酸化。
这里，抑制LSECtin与BTN3A2及BTN3A3相互作用的物质为如下任一种：干扰BTN3A2和BTN3A3表达的RNA分子、抗LSECtin抗体、LSECtin小分子抑制剂、LSECtin可溶性蛋白、干扰LSECtin表达的RNA分子、抗BTN3A2抗体、BTN3A2小分子抑制剂、BTN3A2可溶性蛋白、干扰BTN3A2表达的RNA分子、抗BTN3A3抗体、BTN3A3小分子抑制剂、BTN3A3可溶性蛋白和干扰BTN3A3表达的RNA分子。其中，所述干扰BTN3A2和BTN3A3表达的RNA分子或所述干扰BTN3A3表达的RNA分子为：序列4所示的shRNA分子或将序列4删除或增加或改变一个或几个核苷酸，且与序列4相同功能的核苷酸；或，序列5所示的shRNA分子或将序列5删除或增加或改变一个或几个核苷酸，且与序列5相同功能的核苷酸。
以下继续以实施例具体说明能抑制LSECtin与BTN3A3相互作用的物质。实施例11-实施例18针对能阻断LSECtin与BTN3A3相互作用的融合蛋白BTN3A3-Ig进行详细介绍。
实施例11、融合蛋白BTN3A3-Ig的表达
阻断LSECtin与BTN3A3相互作用的物质为融合蛋白BTN3A3-Ig，其表达方法包括以下步骤：
1)构建融合基因BTN3A3-Ig：根据基因库中搜索到的人BTN3A3基因序列(GenBank 号：BT007251.1)和人IgG1基因序列(GenBank号：AY623427.1)，选择BTN3A3胞外区蛋白(本实施例考虑LSECtin与BTN3A3胞外区相互作用，封闭其相互作用的蛋白需要与膜形式BTN3A竞争结合LSECtin，因此选择BTN3A3胞外区蛋白)，再选择合适的连接肽编码序列，用人工合成的方法获得融合基因BTN3A3-Ig，其核苷酸序列如序列表中序列10所示，其编码序列为自5’端第1-1470位碱基，编码具有序列表中1所示氨基酸残基序列的蛋白质，自5’端第46-786位碱基编码人BTN3A3，自5’端第787-798位碱基编码连接肽(linker)，自5’端第799-1470位碱基编码人IgG1；
2)构建重组表达载体：将融合基因BTN3A3-Ig连接入载体pIRES2-EGFP(购自Clotech公司)中的NheⅠ和SalⅠ酶切位点之间，得到重组表达载体pIRES2-EGFP-BTN3A3-Ig；
3)表达融合蛋白BTN3A3-Ig：将含有融合基因BTN3A3-Ig的重组表达载体pIRES2-EGFP-BTN3A3-Ig转染293T细胞(来源于国家实验细胞资源共享平台)，用无血清培养基M293TI(购自北京义翘神州科技有限公司，用0.45μm滤膜(PN4614，Pall)过滤，冰上保存)在37℃(±0.5℃)下培养重组293T细胞，每24小时收集细胞上清并更换培养基，至96小时(24-120小时均可)培养结束，使融合基因BTN3A3-Ig获得表达；
4)纯化：用Protein G Sepharose柱(购自康为世纪生物科技公司)(蛋白G琼脂糖凝胶柱天然蛋白G(Protein G)是一种分离自G型或C型链球菌属的细胞表面蛋白，主要通过与免疫球蛋白(Ig)的Fc区相互作用，可结合大多数哺乳动物的IgG。天然蛋白G具有白蛋白和细胞表面结合域，重组Protein G去除了白蛋白和细胞表面结合域，以减少非特异性结合，该蛋白与Sepharose偶联后可用于纯化IgG。)对重组表达蛋白进行纯化，纯化方法为：将细胞(含有融合基因BTN3A3-Ig的重组293T细胞)培养上清加入平衡缓冲液(20mM PBS，150mM NaCl，pH 8.0)至pH 8.0，将细胞上清加入已经用平衡缓冲液平衡好的Protein G Sepharose柱中，用平衡缓冲液洗柱，直到流出液中检测不到杂蛋白为止，用洗脱缓冲液(0.1M甘氨酸，pH 3.0)洗脱，收集流出液，立即用中和缓冲液(1M Tris·HCl，pH 9.0)中和，用pH 7.2 0.01mol/L PBS透析72h，得到融合蛋白BTN3A3-Ig，与预期结果相符。该融合蛋白BTN3A3-Ig氨基酸序列如序列表中的序列9所示，序列9由490个氨基酸残基组成，自氨基(N)端第1位为起始密码子，自氨基端第2-15位为信号肽，自氨基端第16-262位为人BTN3A3，自氨基端第263-266位为连接肽(linker)，自氨基端第267-490位为人IgG1。取样在紫分光光度计上测OD260、OD280，用BCA蛋白定量试剂盒(购自康为世纪生物科技公司)计算蛋白质含量，结果为1mg/ml，分装后于-80℃保存。
实施例12、融合蛋白BTN3A3-Ig的考马斯亮蓝染色及Western Blot检测
一、融合蛋白BTN3A3-Ig的考马斯亮蓝染色
收集实施例11重组表达的BTN3A3-Ig融合蛋白样品，配制10％SDS-PAGE凝胶，电泳，进行考马斯亮蓝染色。对照为细胞(含有融合基因BTN3A3-Ig的重组293T细胞)裂解液。
考马斯亮蓝染色结果如图10所示，经表达获得了分子量约55kD的蛋白，与预期结果相符。
二、融合蛋白BTN3A3-Ig的Western Blot检测
收集实施例10重组表达的BTN3A3-Ig融合蛋白样品，配制10％SDS-PAGE凝胶，电泳，转膜，用含有5％脱脂奶粉的TBST溶液封闭，用抗anti-CD277(该抗体说明书指明能够识别BTN3A3)(购自赛默飞)进行一抗孵育，用Mouse IgG HRP-conjugated Antibody(购自R&D，HAF007)进行二抗孵育。实验对照蛋白为人IgG(购自R&D)。
Western Blot检测结果如图11所示，可以看出，实施例11重组表达的融合蛋白BTN3A3-Ig可以与抗人BTN3A3抗体特异结合，而对照蛋白人IgG则无法识别，表明BTN3A3-Ig蛋白能够从上述蛋白表达及纯化体系中获得。
实施例13、Elisa检测融合蛋白BTN3A3-Ig与LSECtin蛋白的结合活性
用1μg/mL浓度包被LSECtin蛋白(2947-CL，R&D)，4℃过夜。用PBST以5％脱脂奶粉浓度配制封闭液。洗板后，每孔300μL室温封闭2h。洗板后，以2μg/mL浓度为起始浓度倍比稀释融合蛋白BTN3A3-Ig并室温孵育3h。洗板后，孵育一抗anti-CD277(该抗体说明书指明能够识别BTN3A3)(购自赛默飞)40min。洗板后，孵育二抗Mouse IgG HRP-conjugated Antibody(购自R&D，HAF007)30min。洗板，显色，终止，OD450读数。实验对照蛋白为人IgG。
Elisa检测结果如图12所示，LSECtin蛋白能够与融合蛋白BTN3A3-Ig发生直接相互作用，而对照蛋白人IgG无法被anti-CD277抗体识别，而BTN3A3-Ig抗体呈现梯度，表明上述体系纯化出的BTN3A3-Ig蛋白的纯度较好。
实施例14、粘附实验检测融合蛋白BTN3A3-Ig阻断LSECtin蛋白与膜形式BTN3A3之间的相互作用
粘附实验过程中，将纯化后的融合蛋白BTN3A3-Ig与LSECtin蛋白按照物质的量比为1:1的比例混合，孵育BT474过表达BTN3A3细胞BT474-BTN3A3，然后进行粘附实验，粘 附实验方法参照文献：“Tang L,Yang J,Tang X,et al.The DC‐SIGN family member LSECtin is a novel ligand of CD44on activated T cells[J].European journal of immunology,2010,40(4):1185-1191.”。实验对照蛋白为人IgG。
融合蛋白BTN3A3-Ig阻断LSECtin与BTN3A3之间相互作用的检测结果如图13所示，LSECtin粘附率的计算方法为LSECtin粘附阳性细胞/ZSG阳性细胞，加入对照IgG时，LSECtin与过表达BTN3A3细胞的粘附率为33.9％,计算公式为11.5/(11.5+22.4)，加入融合蛋白BTN3A3-Ig后，LSECtin与过表达BTN3A3细胞的粘附率仅为3.2％，计算公式为0.969/(0.969+29.3)。检测结果表明BTN3A3-Ig能够阻断LSECtin与BTN3A3之间的相互作用。
实施例15、融合蛋白BTN3A3-Ig阻断LSECtin促进肿瘤细胞干性
将B27(购自Life)、bFGF(购自Sigma)、EGF(购自Sigma)、胰岛素(购自Sigma)、肝素(购自Sigma)和DMEM/F12无血清培养基混匀后，得到培养体系，各个组分在培养体系中的浓度为：B27(10ng/mL)、bFGF(20ng/mL)、EGF(20ng/mL)、胰岛素(5μg/mL)、肝素(4μg/mL)。
将乳腺癌细胞MDA-MB-231(来源于国家实验细胞资源共享平台)制成单细胞悬液，以20,000个/mL铺板，分别加入100ng的BTN3A3-Ig、LSECtin+IgG和LSECtin+BTN3A3-Ig，以空白为对照，培养7-10天后，计算直径大于75μm的球体数量并拍照。
检测结果如图17所示，在100ng的LSECtin刺激浓度下，LSECtin能够促进MDA-MB-231细胞球形成，但不能促进加入融合蛋白BTN3A3-Ig后MDA-MB-231细胞球形成，表明融合蛋白BTN3A3-Ig可阻断LSECtin促进肿瘤细胞干性。
实施例16、融合蛋白BTN3A3-Ig抑制肿瘤进程
将10000个人乳腺癌细胞MDA-MB-231、基质胶(BD，354230)及PBS(Hyclone，SH30256.01)混匀，得到混合物；将混合物分别种植5周雌性裸鼠的下乳腺，建立人-裸鼠乳腺癌移植模型。两个月中每隔一周观测一次，使用游标卡尺分别测量小鼠肿瘤的长径a和短径b，计算肿瘤体积和成瘤率，肿瘤体积计算公式为0.5*ab2，成瘤率计算公式为成瘤(只)/建模总数(只)。
在细胞种植裸鼠建模前1天，按照注射药剂的不同分为如下组：
IgG对照组：腹腔注射IgG(10μg/只)
BTN3A3-Ig组：腹腔注射BTN3A3-Ig(10μg/只)
将MDA-MB-231细胞分别接种于上述各组野生型裸鼠中。在建模后，每3天腹腔注射蛋白，通过测量肿瘤体积观测BTN3A3-Ig对肿瘤进程的抑制效果。
建模后第1、2、3、4、5、6、7、8周肿瘤体积分别如下：
IgG组为0±0.35、9.83±9.22、18.13±12.68、41.54±26.64、134.08±66.72、362.16±186.56、661.32±359.54和1089.43±584.70(单位为mm3)；
BTN3A3-Ig组为0.00±0.00、3.72±5.15、4.25±6.01、6.23±8.53、30.45±9.44、71.80±48.32、125.24±106.49和240.17±255.17(单位为mm3)。
检测结果如图18所示，注射BTN3A3-Ig组肿瘤体积明显小于注射IgG组,表明注射融合蛋白BTN3A3-Ig能够抑制肿瘤生长，可用于制备抗肿瘤药物及肿瘤的治疗。
实施例17、检测融合蛋白BTN3A3-Ig抑制肿瘤进程依赖LSECtin
下述实施例中的LSECtin+/+Nude-/-为野生型裸鼠，LSECtin-/-Nude-/-为LSECtin敲除裸鼠。具体获得方法如下：将BALB/c背景雄性裸鼠LSECtin+/+Nude-/-(购自维通利华)与BALB/c背景雌性LSECtin-/-Nude+/+小鼠交配，获得LSECtin+/-Nude+/-小鼠。雄性LSECtin+/-Nude+/-小鼠与雌性LSECtin+/-Nude+/-小鼠交配，其子代中的雌性裸鼠，经基因型鉴定可获得LSECtin+/+Nude-/-(LSECtin表达的野生型裸鼠)和LSECtin-/-Nude-/-(LSECtin敲除裸鼠)。上述BALB/c背景雌性LSECtin-/-Nude+/+小鼠信息在文献“Zuo Y,Ren S,Wang M,et al.Novel roles of liver sinusoidal endothelial cell lectin in colon carcinoma cell adhesion,migration[J].Gut,2013,62(8):1169-1178.”中公开过，公众可从国家蛋白质组研究中心获得。
按照实施例16中的方法，将MDA-MB-231细胞分别接种于上述各组LSECtin敲除裸鼠中。在建模后，每3天腹腔注射蛋白，通过测量肿瘤体积观测BTN3A3-Ig对肿瘤进程的抑制效果。
建模后第1、2、3、4、5、6、7周肿瘤体积分别如下：
IgG组为0.00±0.00、14.08±11.03、23.89±21.31、66.90±8.90、143.47±34.76、240.21±42.42和400.47±28.29(单位为mm3)；
BTN3A3-Ig组为0.00±0.00、9.93±11.47、28.19±32.63、52.12±33.41、100.68±63.98、199.67±78.36和350.12±45.83(单位为mm3)。
检测结果如图19所示，在缺失LSECtin的情况下，融合蛋白BTN3A3-Ig不能发挥抑 制肿瘤生长的作用，因此BTN3A3-Ig发挥抑制肿瘤生长作用依赖机体中的LSECtin。
实施例18、融合蛋白BTN3A3-Ig抑制肿瘤无毒副作用
实验使用实施例16中的模型。取实验药物IgG或融合蛋白BTN3A3-Ig治疗后，小鼠中的心脏、肝脏、脾脏、肺脏以及肾脏组织，放入福尔马林溶液后，送北京集思佳阳公司进行组织包埋、切片及HE染色。
实验药物作用后各组织切片如图20所示，腹腔注射IgG或融合蛋白BTN3A3-Ig后，小鼠中的心脏、肝脏、脾脏、肺脏以及肾脏组织无损伤，亦无出现明显的炎性细胞浸润，说明腹腔注射融合蛋白BTN3A3-Ig无毒副作用。
实施例19-实施例26进一步提供能够阻断LSECtin与BTN3A3之间相互作用的物质，从而抑制肿瘤的发生与发展(抑制肿瘤进程)。该物质为能够阻断LSECtin与BTN3A3之间的相互作用，具有抑制肿瘤进程活性的单克隆抗体。
实施例19、融合蛋白BTN3A3-mIg的表达
为得到所述具有抑制肿瘤进程活性的单克隆抗体，本实施例先得到用于制备该抗体的免疫原，其为一种特别设计的融合蛋白，命名为BTN3A3-mIg，是将人BTN3A3与小鼠IgG2a通过连接肽连接后获得的重组蛋白。
融合蛋白BTN3A3-mIg的表达方法，包括以下步骤：
1)构建融合基因BTN3A3-mIg：根据基因库中搜索到的人BTN3A3基因序列(GenBank号：BT007251.1)和小鼠IgG2a基因序列(GenBank号：BC018535.1)，选择BTN3A3胞外区蛋白(本发明考虑LSECtin与BTN3A3胞外区相互作用，封闭其相互作用的蛋白需要与膜形式BTN3A竞争结合LSECtin，因此选择BTN3A3胞外区蛋白)，选择合适的连接肽编码序列，用人工合成的方法获得融合基因BTN3A3-mIg，其核苷酸序列如序列表中序列12所示，其编码序列为自5’端第1-1497位碱基，编码具有序列表中1所示氨基酸残基序列的蛋白质，自5’端第1-3位为起始密码子，自5’端第4-45位碱基编码信号肽，自5’端第46-786位碱基编码人BTN3A3，自5’端第787-798位碱基编码连接肽(linker)，自5’端第799-1494位碱基编码小鼠IgG2a，自5’端第1495-1497位为终止密码子；
2)构建重组表达载体：将融合基因BTN3A3-mIg连接入载体pIRES2-EGFP(购自Clotech公司)中的NheⅠ和SalⅠ酶切位点之间，得到重组表达载体，命名为pIRES2-EGFP-BTN3A3-mIg；
3)表达融合蛋白BTN3A3-mIg：将含有融合基因BTN3A3-mIg的重组表达载体pIRES2-EGFP-BTN3A3-mIg转染293T细胞(来源于国家实验细胞资源共享平台)，用无血清培养基M293TI(购自北京义翘神州科技有限公司，用0.45μm滤膜(PN4614，Pall)过滤，冰上保存)在37℃(±0.5℃)下培养重组293T细胞，每24小时收集细胞上清并更换培养基，至96小时(24-120小时均可)培养结束，使融合基因BTN3A3-mIg获得表达；
4)纯化：用Protein G Sepharose柱(购自康为世纪生物科技公司)(蛋白G琼脂糖凝胶柱天然蛋白G(Protein G)是一种分离自G型或C型链球菌属的细胞表面蛋白，主要通过与免疫球蛋白(Ig)的Fc区相互作用，可结合大多数哺乳动物的IgG。天然蛋白G具有白蛋白和细胞表面结合域，重组Protein G去除了白蛋白和细胞表面结合域，以减少非特异性结合，该蛋白与Sepharose偶联后可用于纯化IgG。)对重组表达蛋白进行纯化，纯化方法为：将细胞(含有融合基因BTN3A3-mIg的重组293T细胞)培养上清加入平衡缓冲液(20mM PBS，150mM NaCl，pH 8.0)至pH 8.0，将细胞上清加入已经用平衡缓冲液平衡好的Protein G Sepharose柱中，用平衡缓冲液洗柱，直到流出液中检测不到杂蛋白为止，用洗脱缓冲液(0.1M甘氨酸，pH 3.0)洗脱，收集流出液，立即用中和缓冲液(1M Tris·HCl，pH 9.0)中和，用pH 7.2 0.01mol/L PBS透析72h，得到融合蛋白BTN3A3-mIg，与预期结果相符，融合蛋白BTN3A3-mIg氨基酸序列如序列表中的序列11所示，序列表中的序列11由499个氨基酸残基组成，自氨基(N)端第1位为起始密码子，第2-15为信号肽，第16-262位为人BTN3A3，自氨基端第263-266位为连接肽(linker)，自氨基端第267-498位为小鼠IgG2a，第499位为终止密码子。
取样在紫分光光度计上测OD260、OD280，用BCA蛋白定量试剂盒(购自康为世纪生物科技公司)计算蛋白质含量，结果为1mg/ml，分装后于-80℃保存。
实施例20、融合蛋白BTN3A3-mIg的考马斯亮蓝染色及Western Blot检测
一、融合蛋白BTN3A3-mIg的考马斯亮蓝染色
收集实施例19重组表达的BTN3A3-mIg融合蛋白样品，配制10％SDS-PAGE凝胶，电泳，进行考马斯亮蓝染色。对照为细胞(含有融合基因BTN3A3-mIg的重组293T细胞)裂解液。
考马斯亮蓝染色结果如图21所示，经表达获得了分子量约55kD的蛋白，与预期结果相符。
二、融合蛋白BTN3A3-mIg的Western Blot检测
收集实施例19重组表达的BTN3A3-mIg融合蛋白样品，配制10％SDS-PAGE凝胶，电泳，转膜，用含有5％脱脂奶粉的TBST溶液封闭，用抗人BTN3A3抗体(购自赛默飞)进行一抗孵育，用Mouse IgG HRP-conjugated Antibody(购自R&D，HAF007)进行二抗孵育。实验对照蛋白为小鼠IgG(购自Abcam，ab37355)。
Western Blot检测结果如图22所示，可以看出，实施例1重组表达的融合蛋白可以与抗人BTN3A3抗体特异结合，表明BTN3A3-mIg蛋白能够从上述蛋白表达及纯化体系中获得。融合蛋白BTN3A3-mIg能作为制备BTN3A3抗体的免疫抗原。
实施例21、获得持续、稳定分泌具有抑制肿瘤进程活性的单克隆抗体5E08的杂交瘤细胞株anti-P3(5E08)
杂交瘤细胞株anti-P3(5E08)的获得方法，包括以下步骤：
一、动物免疫
1、抗原制备方法
取100μg(50-100μg均可)融合蛋白BTN3A3-mIg，用生理盐水稀释为0.25mL(0.25-0.50mL均可)，与等体积的弗氏完全佐剂(购自Sigma公司)充分混合、搅拌、乳化。
2、免疫方案
首次免疫：以融合蛋白BTN3A3-mIg为抗原免疫小鼠，浓度为400μg/mL(200-400μg/mL均可)，免疫剂量为0.25mL(0.25-0.50mL均可)，颈背部皮下注射Balb/c健康雌性小鼠(6-8周龄，购自北京维通利华实验动物技术有限公司)，其余腹腔注射；
二次免疫：首次免疫30天后，免疫方法与首次免疫相同；
测定血清效价：二次免疫7天后，尾静脉采血，ELISA测定血清效价；
加强免疫：在融合前3-4天，加强免疫，不加免疫佐剂，采用腹腔注射或尾静脉注射，免疫浓度和免疫剂量与首次免疫相同。
3、免疫小鼠外周血以及腹水效价的Elisa检测
3.1用1μg/mL浓度BTN3A3-his重组蛋白(购自北京义翘神州科技有限公司)包被酶标板，200μl/孔，4℃过夜(12-16小时)后，PBST洗液洗涤3次；
3.2每孔加150μL封闭液，4℃过夜(12-16小时)，洗涤3次，拍干，置4℃冰箱保存备用；
3.3检测效价时，取免疫小鼠外周血血清或腹水进行四倍比稀释(稀释度依次为500， 2000，8000，32000，128000，512000,2048000,8192000)，以100μl/孔加样到包被好的酶标板中，同时，每板分别选取未免疫小鼠血清及正常IgG作为阴性对照，37℃孵育30分钟，洗板4次，拍干；
3.4加二抗Mouse IgG HRP-conjugated Antibody(购自R&D公司，HAF007)，37℃孵育40分钟，洗板8次，拍干；
3.5加显色液(购自赛默飞公司)10-20分钟，显色；
3.6加终止液(浓度为2mol/L浓度的硫酸水溶液)，终止显色反应；
3.7测定OD450nm值，获得检测结果。
用目测法比较，比阴性对照颜色深的腹水最高稀释度作为腹水效价。
免疫小鼠血清与未免疫小鼠血清读数之比大于2(即以OD450nm值大于阴性对照2倍为阳性判断依据)，可用于制备杂交瘤细胞。
二、杂交瘤细胞的融合和筛选
1、制备滋养细胞
将Balb/c小鼠拉颈脱臼处死，浸泡于75％酒精5分钟，随即放入超净工作台内，腹部朝上放于平皿内或固定于解剖板上。用镊子夹起小鼠腹部皮肤，用剪刀剪一小口，注意切勿剪破腹膜，以免腹腔液外流。然后，用剪刀上下两侧做钝性分离，充分暴露腹膜，用酒精棉球擦拭腹膜消毒。用注射器吸取5mL RPMI1640基础培养液(购自Hyclone公司)，注入小鼠腹腔，注射器停留不动，晃动小鼠或反复抽吸几次，用原注射器抽回腹腔内液体，注入离心管，如此反复操作3-4次。1000rpm离心10分钟，弃上清，留下部细胞备用。用20-50mL完全培养液(含10％FBS的RPMI1640培养基，FBS购自Gibico公司)重悬细胞，100μL/孔滴加到培养板，置37℃培养箱作为滋养细胞备用。
2、脾细胞与骨髓瘤细胞融合
2.1取加强免疫小鼠1只，眼眶采血后脱臼处死，在75％酒精中消毒后取脾脏，制备脾细胞悬液(细胞浓度为2.5×106-5.0×106个/ML)，转移到50mL离心管中，加RPMI1640培养基至30mL，1500-2000rpm离心5分钟，弃上清，计数，取1×108个细胞待用。取2瓶生长状态良好的(活细胞数>95％)骨髓瘤细胞(来源于国家实验细胞资源共享平台)，将骨髓瘤细胞完全吹下，转移到50mL离心管中，加RPMI1640培养基至30mL，1500-2000rpm离心5分钟，弃上清，加RPMI1640培养基至30mL，计数，取1×107个细胞待用。
2.2将脾细胞：骨髓瘤细胞以10:1细胞数比例混合，2000rpm离心3分钟。将上清倒干，把细胞沉淀弹成糊状，置37℃水浴。在1分钟内加入1mL融合剂(购自Sigma公 司)，搅拌细胞，37℃水浴45秒，在1分钟内加入1mL RPMI1640培养基并搅拌细胞，每2分钟内加入5mL RPMI1640培养基并搅拌细胞。
2.3轻轻弹匀细胞，缓缓加入HAT培养液(购自Sigma公司)至体积为40ml-50ml，将细胞重悬，轻轻地混匀，加到步骤1预先准备好的滋养细胞板中。排枪滴加80-100μL(配10mL/板)，37℃、CO2培养箱培养、观察。
从细胞融合后第1天开始，对细胞进行仔细观察，记录细胞的生长状态、每孔杂交瘤细胞个数、块数、培养液有无污染、滋养细胞等情况。培养3-5天用HAT培养液换液，10天后换HT培养液(购自Sigma公司)培养至20天，换RPMI1640培养基继续培养48小时，收集上清并按照克隆板上排列的顺序进行编号，例如，5E08等。
3、筛选分泌与BTN3A3特异结合的单克隆抗体5E08的杂交瘤细胞
将细胞上清按步骤一中的方法进行Elisa检测，选择阳性值大于1.5的克隆(5E08等)进行流式检测，具体方法如下：
3.1构建BTN3A3过表达载体
将BTN3A3基因序列(GenBank号：BC018535.1，BTN3A3cDNA全长)替换pIRES2-EGFP载体(购自Clotech公司)中NheⅠ和SalⅠ酶切位点之间的DNA片段，获得BTN3A3过表达载体，命名为pIRES2-EGFP-BTN3A3，其核苷酸序列如序列表中序列8所示。
3.2将BTN3A3过表达载体pIRES2-EGFP-BTN3A3及pIRES2-EGFP空载体分别转染BT474细胞(来源于国家实验细胞资源共享平台)，转染36h后，得到重组细胞：过表达BTN3A3的细胞BT474-BTN3A3和过表达pIRES2-EGFP空载体的细胞BT474-EGFP。
3.3消化收集步骤3.2获得的重组细胞，分别用小鼠IgG(购自赛默飞公司)、商业化BTN3A3流式抗体(购自赛默飞公司)以及杂交瘤细胞分泌的单克隆抗体(步骤2.3收集的)抗体标记，以1:50体积比稀释羊抗小鼠PE标记荧光二抗(购自Biolegend公司)标记细胞，4℃孵育30分钟。用1×PBS洗细胞3次后，弃上清。4℃孵育30分钟。用1×PBS洗细胞3次后，弃上清，用300μL PBS重悬后进行流式细胞检测。
3.4将杂交瘤细胞株进行亚克隆，方法为，将杂交瘤细胞制成单细胞悬液并稀释，滴入铺有滋养细胞的96孔板中，使得每孔杂交瘤细胞数量不超过1个。正常培养10天后，取上清进行Elisa检测，阳性值最高的5个孔中的细胞，进行再次亚克隆，直至完成五次亚克隆，从而获得持续、稳定分泌抗体的杂交瘤细胞。检测结果如图23所示，其中筛选出的杂交瘤细胞分泌的单克隆抗体5E08能够识别人肿瘤细胞表面表达的BTN3A3，表明获得了分泌与BTN3A3特异结合的单克隆抗体5E08的杂交瘤细胞，命名为anti-P3(5E08)， 该细胞株已于2017年9月26日保藏于位于中国北京市朝阳区北辰西路1号院3号的中国普通微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.14723。
实施例22、杂交瘤细胞株anti-P3(5E08)分泌的单克隆抗体5E08的特征检测及纯化
一、腹水的制备及单克隆抗体5E08的纯化
1、制备腹水
在接种杂交瘤细胞anti-P3(5E08)前1-2周，先给小鼠腹腔注射0.5mL液体石蜡，预处理过的小鼠在2-3个月内均可使用。将培养状态良好的杂交瘤细胞anti-P3(5E08)吹打下来，室温1000rpm离心5分钟，弃上清，用无血清RPMI1640培养液将杂交瘤细胞anti-P3(5E08)重悬混匀，将细胞浓度调整至2×106个/mL，每只小鼠腹腔注射0.5mL。接种杂交瘤细胞anti-P3(5E08)后的7-12天，可见小鼠腹部明显膨大，腹部膨大一定程度后，颈椎脱臼处死，将腹腔剪开一个小口，用1mL移液器将腹水收集起来，抽取的腹水经3000rpm离心20分钟，收集上清，-20℃冻存备用。
2、纯化单克隆抗体5E08
使用IgM类单克隆抗体纯化试剂盒(购自北京博奥龙免疫技术有限公司)纯化步骤1收集的小鼠腹水中的单克隆抗体5E08。
3、鉴定纯化的单克隆抗体5E08
纯化后的单克隆抗体5E08按实施例3中的方法进行流式细胞检测。
检测结果表明单克隆抗体5E08能够识别人肿瘤细胞表面表达的BTN3A3，表明获得了纯化的单克隆抗体5E08。
二、单克隆抗体5E08的特征检测
1、亚型检测
将BTN3A3-his重组蛋白(购自北京义翘神州科技有限公司)用PBS缓冲液包被酶标8个微孔板孔，4℃放置12小时，随后用洗板机PBST洗1遍。接下来，每孔加入100μL杂交瘤细胞anti-P3(5E08)培养上清，37℃温育30分钟，而后用洗板机PBST洗5遍。此后，每孔添加100μL Goat Anti-Mouse Ig(G1\G2a\G2b\G3\M\A\κ\λ)-HRP二抗(购自北京博奥龙免疫技术有限公司)，37℃温育30分钟后继续用洗板机PBST洗5遍，随后加入TMB显色液(购自赛默飞公司)，37℃避光显色，20分钟即可判定结果，肉眼观察蓝色孔即为阳性。
亚型检测结果显示杂交瘤细胞anti-P3(5E08)分泌的单克隆抗体5E08的重链为IgM， 轻链为Kappa。
2、测定单克隆抗体5E08的可变区序列
委托金斯瑞公司对杂交瘤细胞anti-P3(5E08)分泌的单克隆抗体5E08的可变区进行测序。结果5E08的重链可变区编码基因具有序列表中序列15的DNA序列，编码序列表中序列13所示的氨基酸残基序列；轻链可变区编码基因具有序列表中序列16的DNA序列，编码序列表中序列14所示的氨基酸残基序列。
序列表中的序列13由141个氨基酸残基组成，序列表中的序列14由130个氨基酸残基组成；序列表中的序列15由423个碱基组成，编码具有序列表中序列13的氨基酸残基序列的蛋白质，序列表中的序列16由390个碱基组成，编码具有序列表中序列14的氨基酸残基序列的蛋白质。
可以了解，本领域技术人员利用以上所述单克隆抗体5E08的重链可变区和轻链可变区氨基酸序列或DNA序列通过常规分子克隆手段可获得单克隆抗体5E08的表达载体，利用常规蛋白表达方法能够更便捷获取具有抑制肿瘤进程活性的单克隆抗体，利用该方式制备具有抑制肿瘤进程活性的单克隆抗体属于本实施例公开内容。
实施例23、粘附实验检测单克隆抗体5E08阻断LSECtin蛋白与膜形式BTN3A3相互作用的活性
粘附实验过程中，将纯化后的单克隆抗体5E08与LSECtin蛋白按照物质的量比为1:1的比例混合，孵育过表达BTN3A3的BT474细胞系BT474-BTN3A3细胞，然后进行粘附实验。粘附实验具体步骤参照文献“Tang L,Yang J,Tang X,et al.The DC‐SIGN family member LSECtin is a novel ligand of CD44on activated T cells[J].European journal of immunology,2010,40(4):1185-1191.”中记载的方法。实验对照蛋白为人IgG。
单克隆抗体5E08阻断LSECtin蛋白与膜形式BTN3A3相互作用活性的粘附实验检测结果如图24所示，LSECtin粘附率的计算方法为LSECtin粘附阳性细胞且ZSG阳性细胞/ZSG阳性细胞，结果加入对照人IgG时，LSECtin与过表达BTN3A3细胞的粘附率为16.1％，计算公式为9.72/(9.72+50.6)，但加入单克隆抗体5E08，LSECtin与过表达BTN3A3细胞的粘附率为1.4％，计算公式为0.828/(0.828+58.8)。
检测结果表明单克隆抗体5E08能够阻断LSECtin与BTN3A3之间的相互作用。
实施例24、检测单克隆抗体5E08阻断LSECtin促进肿瘤细胞干性
将B27(购自Life公司)、bFGF(购自Sigma公司)、EGF(购自Sigma公司)、胰岛素(购自Sigma公司)、肝素(购自Sigma公司)和DMEM/F12无血清培养基混匀后，得到培养体系，各个组分在培养体系中的浓度为：B27(10ng/mL)、bFGF(20ng/mL)、EGF(20ng/mL)、胰岛素(5μg/mL)、肝素(4μg/mL)。
将乳腺癌细胞MDA-MB-231(来源于国家实验细胞资源共享平台)制成单细胞悬液，以20,000个/mL铺板。加入100ng LSECtin后，分别加入0μg/mL、12.5μg/mL、25μg/mL、50μg/mL、100μg/mL单克隆抗体5E08，以人IgG为对照，培养7-10天后，计算直径大于75μm的球体数量并拍照。
检测结果如图25所示，在刺激浓度下，LSECtin能够促进MDA-MB-231细胞球形成，但不能促进加入了50μg/mL浓度以及100μg/mL浓度单克隆抗体5E08后MDA-MB-231细胞球形成。
检测结果表明单克隆抗体5E08可阻断LSECtin促进肿瘤细胞干性。
实施例25、检测单克隆抗体5E08抑制肿瘤进程
一、用小鼠预防模型检测单克隆抗体5E08对肿瘤进程的抑制效果
将10000个人乳腺癌细胞MDA-MB-231、基质胶(BD，354230)及PBS(Hyclone，SH30256.01)混匀，得到混合物；将混合物分别种植5周雌性裸鼠的下乳腺，建立人-裸鼠乳腺癌移植模型。两个月中每隔一周观测一次，使用游标卡尺分别测量小鼠肿瘤的长径a和短径b，计算肿瘤体积和成瘤率，肿瘤体积计算公式为0.5*ab2，成瘤率计算公式为成瘤(只)/建模总数(只)。
在细胞种植小鼠建模前一天，按照注射药剂的不同分为如下组：
IgG对照组：腹腔注射人IgG(50μg/只)；
5E08组：腹腔注射单克隆抗体5E08(50μg/只)。
将MDA-MB-231细胞分别接种于上述各组小鼠中。建模后，每3天腹腔注射蛋白，通过测量肿瘤体积观测单克隆抗体5E08对肿瘤进程的抑制效果。
建模后第1、2、3、4、5、6、7周肿瘤体积如下：
IgG组为0.00±0.00、0.00±0.00、0.00±0.00、0.00±0.00、20.67±18.82、64.98±34.16、124.03±47.41、350.71±165.45和848.36±243.67单位为mm3)；
5E08组为0.00±0.00、0.00±0.00、0.00±0.00、0.00±0.00、4.38±12.37、 24.49±24.97、35.57±37.09、110.98±86.41和325.89±233.46(单位为mm3)。
检测结果如图26所示，注射单克隆抗体5E08组肿瘤体积明显小于注射人IgG组。
检测结果表明单克隆抗体5E08能够抑制肿瘤生长，可用于制备抗肿瘤药物及肿瘤的治疗。
二、用小鼠治疗模型检测单克隆抗体5E08对肿瘤进程的抑制效果
在MDA-MB-231细胞种植小鼠建模1个月时，按照等平均肿瘤体积分为IgG对照组，即肿瘤内注射人IgG(50μg/只)；5E08组，即肿瘤内注射单克隆抗体5E08(50μg/只)。通过测量肿瘤体积观测单克隆抗体5E08对肿瘤进程的抑制效果。
治疗后第肿瘤体积如下：
IgG组为197.40±47.98、270.52±28.76、389.69±17.26、571.45±59.02、933.05±158.71和1202.53±60.31(单位为mm3)；
5E08组为179.87±42.91、269.22±54.97、276.68±58.52、331.02±94.42、443.16±48.39和492.66±71.39(单位为mm3)。
检测结果如图27所示，注射单克隆抗体5E08组肿瘤体积明显小于注射人IgG组。检测结果表明单克隆抗体5E08能够抑制肿瘤生长，可用于制备抗肿瘤药物及肿瘤的治疗。
实施例26、单克隆抗体5E08抑制肿瘤无毒副作用
一、小鼠预防模型中单克隆抗体5E08抑制肿瘤无毒副作用
实验使用实施例25中的预防模型。取实验药物IgG或5E08治疗后，小鼠中的心脏、肝脏、脾脏、肺脏以及肾脏组织，放入福尔马林溶液后，送北京集思佳阳公司进行组织包埋、切片及HE染色。
实验药物作用后各组织切片如图28所示，腹腔注射IgG或单克隆抗体5E08后，小鼠中的心脏、肝脏、脾脏、肺脏以及肾脏组织无损伤，亦无出现明显的炎性细胞浸润，说明腹腔注射单克隆抗体5E08无毒副作用。
二、小鼠治疗模型中单克隆抗体5E08抑制肿瘤无毒副作用
实验使用实施例25中的治疗模型。取实验药物IgG或5E08治疗后，小鼠中的心脏、肝脏、脾脏、肺脏以及肾脏组织，放入福尔马林溶液后，送北京集思佳阳公司进行组织包埋、切片及HE染色。
实验药物作用后各组织切片如图29所示，肿瘤内注射IgG或单克隆抗体5E08后，小鼠中的心脏、肝脏、脾脏、肺脏以及肾脏组织无损伤，亦无出现明显的炎性细胞浸润，说 明肿瘤内注射单克隆抗体5E08无毒副作用。
工业应用性
本发明提出了浸润肿瘤相关巨噬细细胞表达的LSECtin和肿瘤表达BTN3A3单独或共同作为靶点在肿瘤免疫治疗中的应用，并进一步提出了能抑制浸润肿瘤相关巨噬细细胞表达的LSECtin活性、抑制肿瘤表达的BTN3A3活性、或抑制所述LSECtin与BTN3A3相互作用的物质，包括RNA分子、融合蛋白BTN3A3-Ig和单克隆抗体5E08，以其作为活性成分能制备肿瘤免疫治疗药物，适于工业应用。

Claims (36)

  1. 浸润肿瘤相关巨噬细细胞表达的LSECtin与肿瘤表达BTN3A3共同作为靶点在肿瘤免疫治疗中的应用。
  2. 根据权利要求1所述应用，为在开发或设计肿瘤免疫治疗药物中的应用。
  3. 根据权利要求1或2所述应用，所述肿瘤为与肿瘤相关巨噬细胞表达LSECtin且肿瘤细胞表达BTN3A3的肿瘤。
  4. 根据权利要求3所述应用，所述肿瘤包括但不限于乳腺癌、骨髓瘤、肝癌、胃癌、结肠癌、肺癌、骨巨细胞瘤、肾癌、喉癌和腮腺癌。
  5. 根据权利要求2或3或4所述应用，所述药物具备如下(1)-(5)中至少一种功能：
    (1)治疗和/或预防肿瘤；
    (2)抑制肿瘤细胞的生长；
    (3)抑制肿瘤细胞干性的维持或促进；
    (4)抑制肿瘤细胞干性相关特征分子的表达；和
    (5)抑制肿瘤细胞内部STAT3磷酸化。
  6. 浸润肿瘤相关巨噬细细胞表达的LSECtin或肿瘤表达BTN3A3单独作为靶点在肿瘤免疫治疗中的应用。
  7. 根据权利要求6所述应用，为在开发或设计肿瘤免疫治疗药物中的应用。
  8. 根据权利要求6或7所述应用，所述肿瘤为如下一种：
    c1)浸润肿瘤相关巨噬细细胞表达LSECtin的肿瘤；
    c2)表达BTN3A2和/或BTN3A3的肿瘤；和
    c3)同时满足c1)和c2)的肿瘤。
  9. 根据权利要求8所述应用，所述肿瘤包括但不限于乳腺癌、骨髓瘤、肝癌、胃癌、结肠癌、肺癌、骨巨细胞瘤、肾癌、喉癌和腮腺癌。
  10. 根据权利要求7或8或9所述应用，所述药物具备如下(1)-(5)中至少一种功能：
    (1)治疗和/或预防肿瘤；
    (2)抑制肿瘤细胞的生长；
    (3)抑制肿瘤细胞干性的维持或促进；
    (4)抑制肿瘤细胞干性相关特征分子的表达；和
    (5)抑制肿瘤细胞内部STAT3磷酸化。
  11. 根据权利要求5或10所述应用，所述肿瘤细胞干性相关特征分子为Oct4基因和/或Nanog基因和/或Sox基因；
    所述抑制肿瘤细胞的生长体现在降低肿瘤细胞的成瘤率和/或减小肿瘤细胞的体积。
  12. 抑制浸润肿瘤相关巨噬细细胞表达的LSECtin或肿瘤表达的BTN3A3活性或抑制所述LSECtin与BTN3A3相互作用的物质，包括但不限于：干扰BTN3A2和BTN3A3表达的RNA分子、抗LSECtin抗体、LSECtin小分子抑制剂、LSECtin可溶性蛋白、干扰LSECtin表达的RNA分子、抗BTN3A2抗体、BTN3A2小分子抑制剂、BTN3A2可溶性蛋白、干扰BTN3A2表达的RNA分子、抗BTN3A3抗体、BTN3A3小分子抑制剂、BTN3A3可溶性蛋白和干扰BTN3A3表达的RNA分子。
  13. 根据权利要求12所述物质，为干扰BTN3A2和BTN3A3表达的RNA分子或干扰BTN3A3表达的RNA分子，其为如下b1)-b4)中任一种：
    b1)序列4所示的shRNA分子；
    b2)将序列4删除或增加或改变一个或几个核苷酸，且与序列4相同功能的核苷酸；
    b3)序列5所示的shRNA分子；和
    b4)将序列5删除或增加或改变一个或几个核苷酸，且与序列5相同功能的核苷酸。
  14. 根据权利要求12所述物质，为具有抑制肿瘤进程活性的融合蛋白，能阻断LSECtin与BTN3A3之间相互作用，命名为BTN3A3-Ig，是将人BTN3A3与人IgG1通过连接肽连接后获得的重组蛋白。
  15. 根据权利要求14所述物质，所述融合蛋白BTN3A3-Ig是下述氨基酸残基序列之一：
    1)序列表中的序列9；
    2)将序列表中序列9的氨基酸残基序列经过一个或几个氨基酸残基的取代、缺失或添加且具有抑制肿瘤进程活性的蛋白质；
    3)将序列表中序列9的氨基酸残基序列经过氨基酸残基的取代、缺失或添加且具有抑制肿瘤进程活性的蛋白质，新蛋白质与序列9同源性达到80％或更高。
  16. 编码权利要求14或15所述的融合蛋白BTN3A3-Ig的基因BTN3A3-Ig。
  17. 根据权利要求16所述的基因，编码融合蛋白BTN3A3-Ig的基因BTN3A3-Ig是下述核苷酸序列之一：
    1)序列表中序列10的DNA序列；
    2)编码序列表中序列9的DNA序列；
    3)与所编码序列表中序列9的DNA序列有一个或几个碱基变化且具有抑制肿瘤进程活性的核苷酸序列；
    4)所编码的序列80％或以上同源于序列表中序列10且具有抑制肿瘤进程活性的核苷酸序列；
    5)在高严谨条件下可与序列表中序列10限定的DNA序列杂交的核苷酸序列。
  18. 含有权利要求16或17所述融合基因BTN3A3-Ig的表达载体、转基因细胞系或宿主菌。
  19. 一种表达权利要求14或15所述融合蛋白BTN3A3-Ig的方法，包括以下步骤：
    1)构建重组表达载体：将权利要求16或17所述的融合基因BTN3A3-Ig连接入表达载体中，得到含有融合基因BTN3A3-Ig的重组表达载体；
    2)表达融合蛋白BTN3A3-Ig：将含有融合基因BTN3A3-Ig的重组表达载体pIRES2-EGFP-BTN3A3-Ig转化或转染宿主细胞及其后代细胞，培养重组宿主细胞，使融合基因BTN3A3-Ig获得表达；
    3)纯化：对重组表达蛋白进行纯化，得到融合蛋白BTN3A3-Ig。
  20. 根据权利要求19所述的表达方法，所述步骤1)中将融合基因BTN3A3-Ig连接入载体pIRES2-EGFP中的NheⅠ和SalⅠ酶切位点之间，得到重组表达载体，命名为pIRES2-EGFP-BTN3A3-Ig。
  21. 根据权利要求19或20所述的表达方法，所述步骤2)中的宿主细胞为可表达外源基因的细胞，包括293T细胞、293细胞和CHO-S细胞等；所述步骤2)中培养含有融合基因BTN3A3-Ig的重组宿主细胞的培养基为适于宿主细胞生长的培养基，如无血清培养基M293TI、M293TI、CD CHO或CD OptiCHOTM等，优选为无血清培养基M293TI；所述步骤2)中含有融合基因BTN3A3-Ig的重组宿主细胞的培养条件为适于宿主细胞生长的培养条件，36.5-37.5℃培养24-120小时，优选为37℃培养96小时。
  22. 根据权利要求19或20或21所述的表达方法，所述步骤3)中可使用如Protein  G Sepharose柱(蛋白G琼脂糖凝胶柱)、Protein A/G Sepharose柱(蛋白A/G琼脂糖凝胶柱)、或Protein A Sepharose柱(蛋白A琼脂糖凝胶柱)等对重组表达蛋白进行纯化，优选为Protein G Sepharose柱，纯化方法为：将细胞(含有融合基因BTN3A3-Ig的重组宿主细胞)培养上清加入平衡缓冲液(20mM PBS，150mM NaCl，pH 8.0)至pH 8.0，将细胞上清加入已经用平衡缓冲液平衡好的Protein G Sepharose柱中，用平衡缓冲液洗柱，直到流出液中检测不到杂蛋白为止，用洗脱缓冲液(0.1M甘氨酸，pH 3.0)洗脱，收集流出液，立即用中和缓冲液(1M Tris·HCl，pH 9.0)中和，用pH 7.2 0.01mol/L PBS透析72h，得到融合蛋白BTN3A3-Ig。
  23. 根据权利要求12所述的物质，为具有抑制肿瘤进程活性的单克隆抗体，能阻断LSECtin与BTN3A3之间相互作用。
  24. 根据权利要求23所述的物质，所述单克隆抗体的重链可变区为：由序列表中序列13氨基酸残基序列表示的多肽、将序列表中序列13的氨基酸残基序列经过一至十个氨基酸残基的取代、缺失或添加且可与人BTN3A3特异结合的多肽、或与序列表序列13有80％或以上同源性且可与人BTN3A3特异结合的多肽；
    单克隆抗体的轻链可变区为：由序列表中序列14氨基酸残基序列表示的多肽、将序列表中序列14的氨基酸残基序列经过一至十个氨基酸残基的取代、缺失或添加且可与人BTN3A3特异结合的多肽、或与序列表序列14有80％或以上同源性且可与人BTN3A3特异结合的多肽。
  25. 根据权利要求24所述的物质，编码单克隆抗体的基因重链可变区编码基因为以下之一：具有序列表中序列15的DNA序列、编码序列表中序列13的DNA序列、与所编码序列表中序列13的DNA序列有一个或多个碱基变化的序列、所编码的序列80％或以上同源于序列表中序列15的序列、和在高严谨条件下可与序列表中序列15限定的DNA序列杂交的核苷酸序列；
    编码单克隆抗体的基因轻链可变区编码基因为以下之一：具有序列表中序列16的DNA序列、编码序列表中序列14的DNA序列、与所编码序列表中序列14的DNA序列有一个或多个碱基变化的序列、所编码的序列80％或以上同源于序列表中序列16的序列、和在高严谨条件下可与序列表中序列16限定的DNA序列杂交的核苷酸序列。
  26. 能分泌阻断LSECtin与BTN3A3之间相互作用单克隆抗体的杂交瘤细胞株，是以将人BTN3A3与小鼠IgG2a通过连接肽连接得到的融合蛋白(命名为BTN3A3-mIg)为 免疫原免疫小鼠，获得持续、稳定分泌具有抑制肿瘤进程活性的单克隆抗体的杂交瘤细胞株。
  27. 根据权利要求26所述杂交瘤细胞株，所述融合蛋白BTN3A3-mIg是下述氨基酸残基序列之一：
    1)序列表中的序列11；
    2)将序列表中序列11的氨基酸残基序列经过一个或几个氨基酸残基的取代、缺失或添加且具有抗BTN3A3免疫原性的蛋白质；
    3)将序列表中序列11的氨基酸残基序列经过氨基酸残基的取代、缺失或添加且具有抗BTN3A3免疫原性的蛋白质，新蛋白质与序列1同源性达到80％或更高。
  28. 根据权利要求26或27所述杂交瘤细胞株，其名称为anti-P3(5E08)，保藏编号为CGMCC No.14723。
  29. 能阻断LSECtin与BTN3A3之间相互作用具有抑制肿瘤进程活性的单克隆抗体，由权利要求26或27或28所述的杂交瘤细胞株分泌的单克隆抗体，命名为5E08。
  30. 能分泌阻断LSECtin与BTN3A3之间相互作用单克隆抗体的杂交瘤细胞株的获取方法，可包括以下步骤：
    1)用融合蛋白BTN3A3-mIg作为免疫原免疫动物；
    2)分离免疫动物的脾细胞，将其与骨髓瘤细胞融合，形成杂交瘤；
    3)筛选杂交瘤细胞，得到杂交瘤细胞株anti-P3(5E08)。
  31. 能阻断LSECtin与BTN3A3之间相互作用具有抑制肿瘤进程活性的单克隆抗体的制备方法，采用：
    方式一：从权利要求26或27或28所述能分泌阻断LSECtin与BTN3A3之间相互作用单克隆抗体的杂交瘤细胞株的培养液或接种所述杂交瘤细胞株的动物的腹水液中分离并纯化得到具有抑制肿瘤进程活性的单克隆抗体，或
    方式二：利用权利要求24或25中提及的单克隆抗体5E08的重链可变区和轻链可变区氨基酸序列或DNA序列构建单克隆抗体5E08的表达载体，通过常规蛋白表达方式得到具有抑制肿瘤进程活性的单克隆抗体。
  32. 用于制备具有抑制肿瘤进程活性的单克隆抗体中的免疫原，为将人BTN3A3与小鼠IgG2a通过连接肽连接得到的融合蛋白，命名为BTN3A3-mIg。
  33. 根据权利要求32所述免疫原，所述融合蛋白BTN3A3-mIg是下述氨基酸残基 序列之一：
    1)序列表中的序列11；
    2)将序列表中序列11的氨基酸残基序列经过一个或几个氨基酸残基的取代、缺失或添加且具有抗BTN3A3免疫原性的蛋白质；和
    3)将序列表中序列11的氨基酸残基序列经过氨基酸残基的取代、缺失或添加且具有抗BTN3A3免疫原性的蛋白质，新蛋白质与序列11同源性达到80％或更高。
  34. 编码权利要求32或33所述融合蛋白的基因BTN3A3-mIg，是下述核苷酸序列之一：
    1)序列表中序列12的DNA序列；
    2)编码序列表中序列11的DNA序列；
    3)与所编码序列表中序列11的DNA序列有一个或几个碱基变化且具有抗BTN3A3免疫原性的的核苷酸序列；
    4)所编码的序列80％或以上同源于序列表中序列12且具有抗BTN3A3免疫原性的的核苷酸序列；和
    5)在高严谨条件下可与序列表中序列12限定的DNA序列杂交的核苷酸序列。
  35. 权利要求13所述RNA分子、权利要求14或15所述融合蛋白BTN3A3-Ig、权利要求16或17所述基因BTN3A3-mIg、权利要求18所述融合基因BTN3A3-Ig的表达载体、转基因细胞系或宿主菌、权利要求23或24或29提及的单克隆抗体5E08、权利要求32或33所述免疫原以及权利要求34所述基因BTN3A3-mIg在制备具有如下(b1)-(b5)中至少一种功能的产品中的应用：
    (b1)治疗和/或预防肿瘤；
    (b2)抑制肿瘤进程；
    (b3)抑制肿瘤细胞干性的维持或促进；
    (b4)抑制肿瘤细胞干性相关特征分子的表达；和
    (b5)抑制肿瘤细胞内部STAT3磷酸化；
    所述肿瘤为肿瘤相关巨噬细胞表达LSECtin且肿瘤细胞表达BTN3A3的肿瘤。
  36. 根据权利要求35所述应用，所述肿瘤相关巨噬细胞表达LSECtin且肿瘤细胞表达BTN3A3的肿瘤包括但不限于乳腺癌、骨髓瘤、肝癌、胃癌、结肠癌、肺癌、骨巨细胞瘤、肾癌、喉癌和腮腺癌。
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