WO2020132789A1 - 突变的人2Ig-B7-H3蛋白编码基因、重组载体、包含其的宿主细胞、药物组合物及其应用 - Google Patents

突变的人2Ig-B7-H3蛋白编码基因、重组载体、包含其的宿主细胞、药物组合物及其应用 Download PDF

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WO2020132789A1
WO2020132789A1 PCT/CN2018/123049 CN2018123049W WO2020132789A1 WO 2020132789 A1 WO2020132789 A1 WO 2020132789A1 CN 2018123049 W CN2018123049 W CN 2018123049W WO 2020132789 A1 WO2020132789 A1 WO 2020132789A1
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protein
vector
human
gene
cancer
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WO2020132789A8 (zh
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黄海东
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Chen Hanqiang
Zhou Xiaoyi
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Chen Hanqiang
Zhou Xiaoyi
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Priority to SG11202106952UA priority Critical patent/SG11202106952UA/en
Priority to NZ777659A priority patent/NZ777659B2/en
Priority to PCT/CN2018/123049 priority patent/WO2020132789A1/zh
Priority to AU2018455720A priority patent/AU2018455720B2/en
Priority to EP18944354.2A priority patent/EP3889263A4/en
Priority to CN201880099908.9A priority patent/CN113260703A/zh
Priority to CA3124721A priority patent/CA3124721A1/en
Priority to US17/417,991 priority patent/US12415846B2/en
Application filed by Chen Hanqiang, Zhou Xiaoyi filed Critical Chen Hanqiang
Priority to JP2021560752A priority patent/JP7315703B2/ja
Publication of WO2020132789A1 publication Critical patent/WO2020132789A1/zh
Publication of WO2020132789A8 publication Critical patent/WO2020132789A8/zh
Priority to IL284294A priority patent/IL284294A/en
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Priority to ZA2021/04520A priority patent/ZA202104520B/en
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/10—Seeds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70532—B7 molecules, e.g. CD80, CD86
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present invention relates to the field of genetic engineering, and in particular to a human 2Ig-B7-H3 protein coding gene, its encoded protein, a recombinant vector, a host cell containing the same, a pharmaceutical composition, and applications.
  • T cell activation requires two different signals.
  • the first signal comes from the interaction between the TCR and the antigen peptide-MHC complex, and the second signal comes from the synergistic stimulation signal generated by the B7 family molecule on APC and its ligand on the T cell CD28 family molecule, such as B7-1B7-2 Combined with CD28 and CTLA-4, this pathway is called the classic B7 pathway.
  • B7-H3 The human B7-H3 gene was first discovered by Chapoval et al. in the cDNA library of human dendritic cells. Because its structure is similar to the genes of the B7 family, it was named B7Homolog 3, abbreviated as B7-H3. It is a type I transmembrane glycoprotein and belongs to the immunoglobulin superfamily. It has 20-27% homology with other members of the B7 family in the amino acid sequence outside the cell.
  • B7-H3 has a wide range of expression: B7-H3 is expressed in most tissues at the transcription level, and only in a few tissues such as human liver, lung, bladder, testis, prostate, breast, placenta, and lymphoid organs at the protein level; The difference in the expression of B7-H3 at the gene (mRNA) level and protein level may be related to the post-transcriptional regulation of the molecule.
  • B7-H3 is an immunomodulatory molecule. In recent years, it has also been found to have important clinical significance in many tumor cells: that is, it may be a tumor resistance Regulatory factor.
  • the human B7-H3 gene is located on chromosome 15 and the protein has two different forms of spliceosomes in the body: 2IgB7-H3 and 4IgB7-H3.
  • the extracellular segment of 2IgB7-H3 consists of two immunoglobulin domains, IgV-IgC composition. The applicant hopes to find through research that the correlation between 2IgB7-H3 gene mutation and tumor resistance.
  • the technical problem to be solved by the present invention is to provide a human 2Ig-B7-H3 protein coding gene, its encoded protein, a recombinant vector, a host cell containing the same and a pharmaceutical composition and application thereof, said human 2Ig-B7-H3 protein
  • the coding gene provides a new way to treat cancer.
  • the invention discloses a mutated human 2Ig-B7-H3 protein coding gene, which has the nucleotide sequence shown in SEQ ID NO:1.
  • the invention discloses a human 2Ig-B7-H3 protein.
  • the human 2Ig-B7-H3 protein coding gene is the mutated human 2Ig-B7-H3 protein coding gene.
  • the invention discloses a recombinant vector.
  • the recombinant vector includes a vector and a target gene carried by the vector.
  • the target gene is a mutated human 2Ig-B7-H3 protein coding gene described in the above technical solution.
  • the vector is selected from the group consisting of cloning vector, eukaryotic expression vector, prokaryotic expression vector and shuttle vector.
  • the invention discloses a pharmaceutical composition, which comprises an excipient and one or more of the human 2Ig-B7-H3 protein and the recombinant vector described in the above technical solution.
  • the pharmaceutical composition is an injection solution
  • the injection solution includes a pharmaceutically acceptable excipient and one or more selected from the recombinant carriers described in the above technical solution.
  • the invention discloses the application of the mutated human 2Ig-B7-H3 protein coding gene described in the above technical scheme in the preparation of drugs for preventing and treating cancer.
  • the present invention provides a mutated human 2Ig-B7-H3 protein coding gene, the protein encoded by it, a recombinant vector, and a pharmaceutical composition including the gene or protein.
  • the protein encoded by the gene of the present invention its regulatory expression, interaction and signal transmission play an extremely important role in the process of tumor immune response, and in particular provide a favorable new way for the prevention and treatment of cancer.
  • Figure 1 shows the results of flow cytometric analysis of 293T cells.
  • Figure 2 shows the results of flow cytometric analysis of SHG44 cells.
  • the embodiment of the present invention provides a mutated human 2Ig-B7-H3 protein coding gene, the gene is:
  • the mutated human 2Ig-B7-H3 protein encoding gene contains a total of 2765 bases, located at the 1'position at the 5'end, a C>T heterozygous mutation occurs, so that the C and T bases at the 1488 position are each half position.
  • "y" at position 1488 in SEQ ID NO: 1 indicates that a C>T heterozygous mutation has occurred, and at position 1488, the C and T bases each occupy half of the position.
  • the invention discloses a human 2Ig-B7-H3 protein.
  • the human 2Ig-B7-H3 protein coding gene is the mutated human 2Ig-B7-H3 protein coding gene.
  • a person skilled in the art obtains the nucleoside from a nucleotide sequence shown in SEQ ID NO: 1 disclosed in the present invention according to a well-known codon table through biological methods (such as PCR method, point mutation method) or chemical synthesis method
  • the acid sequence is used in recombinant technology and gene therapy, so this part of the nucleotide sequence should be included in the scope of the present invention.
  • using the DNA sequences disclosed herein can also be performed by methods known in the art, such as the method of Sambrook et al. (Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, Second Edition, 1989), by modifying the invention The nucleic acid sequence provided.
  • the embodiment of the present invention discloses a recombinant vector.
  • the recombinant vector includes a vector and a target gene carried by the vector.
  • the target gene is a mutated human 2Ig-B7-H3 protein coding gene described in the above technical solution.
  • the target gene may further include a regulatory sequence, such as a promoter, terminator and enhancer for expression of the one or more target genes.
  • the target gene may also include a marker gene (for example, a gene encoding ⁇ -galactosidase, green fluorescent protein, or other fluorescent protein) or a gene whose product regulates the expression of other genes.
  • the target gene may also be mRNA, tRNA, or rRNA, and may also include related transcriptional regulatory sequences generally associated with transcribed sequences, such as transcription termination signals, polyadenylation sites, and downstream enhancer elements.
  • the vector may be various vectors commonly used in the art that can carry the gene of interest, and various vectors that can carry the gene of interest that are available for technological development and improvement.
  • Such vectors are, for example, plasmids (naked DNA), liposomes, molecular couplings, polymers and viruses.
  • the plasmid can carry the gene of interest, and the plasmid carrying the gene of interest can be directly injected into the tissue cells by gene gun, electroporation and electrofusion technology.
  • ultrasound helps improve the efficiency of plasmid transfer.
  • Ultrasonic wave combined with microbubble echogenic agent can improve the permeability of cell membrane, thus significantly improve the transfer and expression efficiency of naked DNA. This membrane penetration technique can instantly create small pores on the surface of the cell membrane, and DNA takes the opportunity to enter the cell.
  • the liposome is a particle composed of a lipid bilayer, which can mediate the gene of interest to pass through the cell membrane.
  • the lipid may be a natural phospholipid mainly derived from egg yolk and soybeans, which is mainly lecithin (phosphatidylcholine, PC); it may also be dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylethanolamine (DPPC). DPPE), distearoylphosphatidylcholine (DSPC) and other synthetic phospholipids; may also contain cholesterol.
  • the preferred liposomes are cationic liposomes, which are mainly composed of positively charged lipids and neutral auxiliary lipids mixed in equimolar. The positively-charged liposomes and negatively-charged DNA can effectively form a complex and migrate into the cell through endocytosis.
  • the polymer uses cationic polymers, such as the positive charge on poly-L-lysine combined with the negative charge on DNA to electrically neutralize guanidine to form a stable polymer/DNA complex.
  • cationic polymers such as the positive charge on poly-L-lysine combined with the negative charge on DNA to electrically neutralize guanidine to form a stable polymer/DNA complex.
  • the resulting complex of cationic polymer and DNA is still positively charged and can bind to a negatively charged receptor on the surface of the cell and be infiltrated into the cell.
  • the molecular coupler is to covalently bind the exogenous DNA of the target gene to the ligand of the specific receptor on the cell surface or the monoclonal antibody or viral membrane protein, and use the specific binding characteristics to mediate the introduction of the exogenous gene into the Specific types of cells.
  • Viruses can usually enter specific cells with high efficiency, express their own proteins, and generate new virus particles. Therefore, the transformed virus first becomes a vector for gene therapy.
  • lentiviral vectors retroviral vectors, adenovirus vectors, adeno-associated virus vectors, and herpes simplex virus vectors. .
  • expression vector refers to a vector containing a recombinant polynucleotide containing an expression control sequence operably linked to the nucleotide sequence to be expressed.
  • Expression vectors include all expression vectors known in the art, including cosmids, plasmids (eg, naked or contained in liposomes) introduced into recombinant polynucleotides, and viruses (eg, lentiviruses, retroviruses) , Adenovirus and adeno-associated virus).
  • lentivirus belongs to the family of retroviruses. Lentiviruses can infect dividing and non-dividing cells. After lentivirus infection, a large amount of genetic information can be delivered to the host cell, and it can be stably expressed for a long time, and it can be inherited stably along with cell division. Therefore, lentivirus is one of the most effective tools for introducing foreign genes. Examples of lentiviruses include human immunodeficiency virus (HIV), monkey immunodeficiency virus (SIV), equine infectious anemia (EIA), feline immunodeficiency virus (FIV).
  • HCV human immunodeficiency virus
  • SIV monkey immunodeficiency virus
  • EIA equine infectious anemia
  • FFIV feline immunodeficiency virus
  • Lentiviral vectors can effectively integrate foreign genes into the host chromosome, thereby achieving persistent expression. In terms of infectivity, it can effectively infect various types of cells such as neuronal cells, liver cells, cardiomyocytes, tumor cells, endothelial cells, stem cells, etc., so as to achieve a good gene therapy effect.
  • the present invention uses lentiviral vectors.
  • the present invention also provides a host cell, wherein the host contains the recombinant vector of the present invention.
  • the recombinant vector containing the gene encoding the mutant human 2Ig-B7-H3 protein of the present invention can be transformed into a host body and can be used to study its relationship with tumor cell expression.
  • the host is selected from one or more of E. coli, 239 cells and SHG44 cells. Among them, E.
  • coli as a genetically engineered bacterium, may contain the recombinant cloning vector of the present invention to realize the amplification of the gene encoding the mutant human 2Ig-B7-H3 protein of the present inventors, or may contain the recombinant expression vector of the present invention to achieve the present invention. Extensive expression of human mutant human 2Ig-B7-H3 protein coding genes.
  • the recombinant vector is a recombinant adenovirus vector
  • the vector can be amplified in SHG44 and 239 cells.
  • the embodiments of the present invention disclose a pharmaceutical composition, which includes an excipient and one or more of the human 2Ig-B7-H3 protein and the recombinant vector described in the above technical solution.
  • the pharmaceutical-acceptable excipients refer to non-toxic solid, semi-solid or liquid fillers, diluents, encapsulating materials or other preparation auxiliary materials, such as, but not limited to, saline, buffered saline, glucose, water, glycerin, ethanol And their mixtures.
  • the pharmaceutical composition is suitable for parenteral, sublingual, intracisternal, intravaginal, intraperitoneal, intrarectal, buccal, or epidermal administration.
  • Parenteral administration includes intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, intraarticular injection and infusion.
  • Pharmaceutical compositions suitable for parenteral administration include sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and powders for formulation in sterile injectable solutions or dispersions immediately before use.
  • Suitable aqueous or non-aqueous carriers, diluents, solvents or excipients include water, ethanol, glycerin, propylene glycol, polyethylene glycol, carboxymethyl cellulose, vegetable oils and injectable organic esters such as ethyl oleate.
  • compositions may also contain preservatives, wetting agents, emulsifiers, protective agents, and dispersant adjuvants, such as inositol, sorbitol, and sucrose.
  • preservatives such as sugar, sodium chloride, potassium chloride is added.
  • Epidermal administration includes administration on the skin, mucous membranes, and lung and ocular surfaces.
  • Such pharmaceutical compositions include powders, ointments, drops, transdermal patches, iontophoresis devices, inhalants, and the like.
  • the composition for rectal or vaginal administration is preferably a suppository, which can be prepared by mixing the recombinant carrier of the present invention with a suitable non-irritating excipient such as cocoa butter, polyethylene glycol, or suppository wax.
  • the carrier is solid at room temperature and liquid at body temperature, so it melts in the rectum or vaginal cavity and releases the active compound.
  • the pharmaceutical composition is an injection solution
  • the injection solution includes a pharmaceutically acceptable excipient and a gene selected from the gene encoding the human glucokinase mutant of the present invention and the recombinant vector of the present invention.
  • a pharmaceutically acceptable excipient and a gene selected from the gene encoding the human glucokinase mutant of the present invention and the recombinant vector of the present invention.
  • the pharmaceutical composition is an injection solution
  • the injection solution includes a pharmaceutically acceptable excipient and one or more selected from the recombinant carriers described in the above technical solution.
  • the embodiment of the present invention discloses the application of the mutated human 2Ig-B7-H3 protein coding gene described in the above technical solution in the preparation of drugs for preventing and treating cancer.
  • mice Ectopic expression of several tumor cell lines in mice can induce the activation of tumor-specific cytotoxic T lymphocytes, thereby delaying the growth of cancer cells or even completely eliminating the tumor.
  • Transfected cancer cell lines can be significantly extended after implantation in mice The survival time of mice.
  • the mutated human 2Ig-B7-H3 protein-encoding gene contains a total of 2765 bases, located at the 1488th position of the 5'end, and a C>T heterozygous mutation occurs, so that the C and T bases at the 1488th position each occupy half of the position.
  • the nucleotide sequence of the gene encoding the mutant human 2Ig-B7-H3 protein is shown in SEQ ID NO:1. As shown in the sequence table, "y" at position 1488 in SEQ ID NO: 1 indicates that a C>T heterozygous mutation has occurred, and at position 1488, the C and T bases each occupy half of the position.
  • the nucleotide sequence shown in SEQ ID NO: 1 of the sequence table was inserted between the NHeI and NotI cleavage sites of the pIRES2-EGFP vector to obtain the recombinant plasmid pIRES2-EGFP/2Ig-B7-H3.
  • Adopt LipofectamineTM2000 cationic liposome transfection kit and operate according to the kit instructions, and introduce the recombinant plasmid pIRES2-EGFP/2Ig-B7-H3 into 239T cells to obtain recombinant cells.
  • step 2 Inoculate the recombinant cells obtained in step 1 into DMEM/F12 medium containing 5% (volume ratio) newborn bovine serum, and then cultivate in a 37°C, 5% CO2 incubator for 48 hours, and then collect the supernatant.
  • step 3 Take the supernatant obtained in step 2, filter with 0.45 ⁇ m filter membrane and collect the filtrate, then adjust the pH to 7.4.
  • Equilibrium buffer Tris-HCl buffer containing 0.5M NaCl at pH7.4 and 0.5M;
  • A280nm detects the ultraviolet absorption peak.
  • a collection tube was used to collect the target peak, and then the solution in the collection tube was transferred to a dialysis bag for dialysis in pH 7.4, 0.01M PBS buffer to obtain human 2Ig-B7-H3 protein.
  • SHG44 and 293T cells were infected with the vector obtained in Example 2 and the negative control lentivirus. The cells were harvested after infection and the expression of 2IgB7-H3 on the cell surface was detected by flow cytometry.
  • Target cells SHG44, 293T.
  • Flow cytometry reagent 2IgB7-H3 flow cytometry antibody (human).
  • the SHG44 and 293T cells infected with the vector obtained in Example 2 and the negative control lentivirus were cultured in a 5% CO2 and 37°C carbon dioxide incubator.
  • mice Mouse liver cancer H22 cells frozen in liquid nitrogen were quickly thawed in a 37°C water bath, the cell density was adjusted to 1 ⁇ 10 7 /mL, and 2 BALB/c mice were inoculated intraperitoneally, 0.2 mL each. After the mouse's abdomen was swollen, the mice were sacrificed by cervical dislocation, the abdomen was disinfected, the ascites was extracted and combined, and the cell density was adjusted to 1 ⁇ 10 7 /m L with PBS. 20 BALB/c mice were inoculated subcutaneously. The mice were divided into two groups of 10 mice, and were treated as follows:
  • the first group no treatment, no vaccination;
  • the second group subcutaneous injection of human 2Ig-B7-H3 protein in the abdomen, immunization 3 times (0.2mL each time), single immunization dose is 20ug/only;
  • the first immunization was performed on the 12th day: the second immunization was performed on the 15th day: the third immunization was performed on the 18th day.
  • V ab 2 /2 (V-volume, a-tumor long diameter, b-tumor short diameter). See Table 1 for changes in tumor volume.
  • the human 2Ig-B7-H3 protein has a significant therapeutic effect on the induced liver cancer model subcutaneously inoculated. After treatment with human 2Ig-B7-H3 protein, the growth rate of subcutaneous tumors was significantly slowed, and the tumor volume became smaller.
  • DMEM medium containing 100 mL/L fetal bovine serum
  • the subject of the experiment was a patient with lung adenocarcinoma.
  • the synthetic polypeptide was dissolved in a pH 7.4 PBS (Hyclone) solution, the concentration was adjusted to 2.5 mg/ml, and 5% Aldaracream was covered after each 200 mg subcutaneous injection of the upper arm. iNova Pharmaceuticals Australia Pty Ltd.), once a week, 12 weeks as a cycle.
  • the secretion of IFN- ⁇ before administration, 3 weeks, 7 weeks, and 11 weeks of administration was measured by ELISA.
  • the synthetic polypeptides used in the specific examples and the test results before and after administration are shown in Table 3.
  • the level of IFN- ⁇ secreted by T cells has a significantly increased trend, and even shows an exponential increase, indicating that the use of the protein of the present invention can increase the peripheral blood of patients with lung adenocarcinoma
  • the tumor killing capacity further proves the effect of the present invention.
  • the protein provided by the present invention has a significant effect on the treatment of lung adenocarcinoma.
  • the cancer cells are induced to produce dendritic cells to form antigen presenting cells, thereby stimulating killer T cells in the body The realization of the treatment of lung adenocarcinoma.
  • the synthetic polypeptide of the present invention has no effect on stimulating the secretion of IFN- ⁇ to the body without cancer cells, and has less toxic and side effects.

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Abstract

涉及基因工程领域，特别涉及一种突变的人2Ig-B7-H3蛋白编码基因，蛋白，重组载体，以及包括所述基因或蛋白的药物组合物。涉及的基因所编码的蛋白，其调节性表达、相互作用以及信号传递在肿瘤免疫应答过程中起着极其重要的作用，尤其为预防和治疗癌症提供了有利的途径。

Description

突变的人2Ig-B7-H3蛋白编码基因、重组载体、包含其的宿主细胞、药物组合物及其应用 技术领域
本发明涉及基因工程领域，特别涉及一种人2Ig-B7-H3蛋白编码基因、其编码的蛋白、重组载体、包含其的宿主细胞、药物组合物及应用。
背景技术
T细胞的活化需要两个不同的信号。第一信号来自TCR与抗原肽-MHC复合物相互作用，第二信号来自APC上的B7家族分子与其在T细胞上的配体CD28家族分子相结合产生的协同刺激信号，如B7-1B7-2与CD28，CTLA-4结合，该途径被称为经典的B7途径。
人B7-H3基因首先是由Chapoval等在人类树突状细胞的cDNA文库中发现的，由于其结构类似B7家族的基因，因此命名为B7Homolog 3，简称B7-H3。它是一种Ⅰ型跨膜糖蛋白，属于免疫球蛋白超家族，在氨基酸序列上与B7家族的其他成员在细胞外有20-27％的同源性。
B7-H3有着广泛的表达：在转录水平B7-H3表达于大多数组织，在蛋白水平上仅表达于少数组织如人体肝、肺、膀胱、睾丸、前列腺、乳房、胎盘和淋巴样器官等；B7-H3在基因(mRNA)水平和蛋白水平上表达的差异可能与分子的转录后调控有关。
B7-H3除了在抗原特异性的体液免疫过程中能够调控淋巴细胞增殖，是一个免疫调控分子，近年来又发现它在许多肿瘤细胞中也有着重要的临床意义：即它可能是一个肿瘤抗性的调控因子。
人B7-H3基因定位在15号染色体上，蛋白在体内有两种不同形式的剪切体：2IgB7-H3和4IgB7-H3。2IgB7-H3胞外段由IgV-IgC两个免疫球蛋白结构域组成。申请人希望通过研究发现，2IgB7-H3的基因突变与肿瘤抗性的相关性。
发明内容
本发明要解决的技术问题是提供一种人2Ig-B7-H3蛋白编码基因、其编码的蛋白、重组载体、包含其的宿主细胞及药物组合物及应用，所述人2Ig-B7-H3蛋白编码基因对癌症的治疗提供了新的途径。
本发明公开了一种突变的人2Ig-B7-H3蛋白编码基因，所述基因具有SEQ ID NO:1所示的核苷酸序列。
本发明公开了一种人2Ig-B7-H3蛋白，所述人2Ig-B7-H3蛋白的编码基因是所述突变的人2Ig-B7-H3蛋白编码基因。
本发明公开了一种重组载体，所述重组载体包括载体及其携带的目的基因，所述目的基因是上述技术方案所述突变的人2Ig-B7-H3蛋白编码基因。
优选的，所述载体选自由克隆载体、真核表达载体、原核表达载体和穿梭载体。
本发明公开了一种药物组合物，所述药物组合物包括赋形剂以及选择上述技术方案所述人2Ig-B7-H3蛋白和所述的重组载体中的一种或几种。
优选的，所述药物组合物为注射液，所述注射液包括药学上可接受的赋形剂以及选自上述技术方案所述的重组载体中的一种或几种。
本发明公开了上述技术方案所述突变的人2Ig-B7-H3蛋白编码基因在制备预防和治疗癌症的药物中的应用。
与现有技术相比，本发明提供了一种突变的人2Ig-B7-H3蛋白编码基因，由其编码的蛋白，重组载体，以及包括所述基因或蛋白的药物组合物。本发明的基因所编码的蛋白，其调节性表达、相互作用以及信号传递在肿瘤免疫应答过程中起着极其重要的作用，尤其为预防和治疗癌症提供了有利的新途径。
附图说明
图1示出了293T细胞的流式数据分析结果。
图2示出了SHG44细胞的流式数据分析结果。
具体实施方式
下面将参照附图更详细地描述本公开的示例性实施例。虽然附图中显示 了本公开的示例性实施例，然而应当理解，可以以各种形式实现本公开而不应被这里阐述的实施例所限制。相反，提供这些实施例是为了能够更透彻地理解本公开，并且能够将本公开的范围完整的传达给本领域的技术人员。
本发明的实施例提供了一种突变的人2Ig-B7-H3蛋白编码基因，所述基因是：
SEQ ID NO:1所示的核苷酸序列。
所述突变的人2Ig-B7-H3蛋白编码基因共包含2765个碱基，位于5’端第1488位，发生C＞T杂合突变，使得在第1488位的C和T碱基各占一半位置。如序列表中所示的SEQ ID NO:1中第1488位的“y”表示发生C＞T杂合突变，在第1488位处C和T碱基各占一半位置。
本发明公开了一种人2Ig-B7-H3蛋白，所述人2Ig-B7-H3蛋白的编码基因是所述突变的人2Ig-B7-H3蛋白编码基因。
本领域公知，组成蛋白质的20种不同的氨基酸中，除Met(ATG)或Trp(TGG)分别为单一密码子编码外，其他18种氨基酸分别由2-6个密码子编码(Sambrook等，分子克隆，冷泉港实验室出版社，纽约，美国，第二版，1989，见950页附录D)。即由于遗传密码子的简并性，决定一个氨基酸的密码子大多不止一个，三联体密码子中第三个核苷酸的置换，往往不会改变氨基酸的组成，因此编码相同氨基酸序列的蛋白的核苷酸序列可以不同。本领域人员根据公知的密码子表，从本发明公开的SEQ ID NO：1所示的核苷酸序列，通过生物学方法(如PCR方法、点突变方法)或化学合成方法得到所述核苷酸序列，应用到重组技术以及基因治疗当中，因此该部分核苷酸序列都应该包括在本发明范围内。相反，利用本文公开的DNA序列，也可以通过本领域公知的方法，例如Sambrook等的方法(分子克隆，冷泉港实验室出版社，纽约，美国，第二版，1989)进行，通过修改本发明提供的核酸序列。
本发明的实施例公开了一种重组载体，所述重组载体包括载体及其携带的目的基因，所述目的基因是上述技术方案所述突变的人2Ig-B7-H3蛋白编码基因。
其中，所述目的基因还可以包括调控序列，例如所述一种或几种目的基因表达的启动子、终止子和增强子。所述目的基因也可以包括标记基因(例如， 编码β-半乳糖苷酶、绿色荧光蛋白或其它荧光蛋白的基因)或其产物调节其它基因表达的基因。所述目的基因除可以是DNA外，还可以是mRNA、tRNA或rRNA，还可以包括通常与转录序列相关的相关转录调控序列，例如转录终止信号、聚腺苷酸化位点和下游增强子元件。
所述载体可以是本领域常用的各种能携带目的基因的载体以及技术发展改进的可用的各种能携带目的基因的载体。所述载体例如，质粒(裸DNA)、脂质体、分子耦联体、多聚物和病毒。
所述质粒(裸DNA)可以携带目的基因，该携带有目的基因的质粒可以直接注射或通过基因枪、电穿孔及电融合技术导入到组织细胞中。此外，超声波有助于提高质粒的转移效率。超声波配合微泡回声比差剂可提高细胞膜的通透性，从而显著提高裸DNA的转移和表达效率。这项胞膜渗透技术可在细胞膜表面瞬间制造小孔，DNA则趁机进入细胞内。
所述脂质体是由脂质双分子层组成的颗粒，可介导目的基因穿过细胞膜。所述脂质可以是来源于蛋黄和大豆的以卵磷脂(磷脂酰胆碱，PC)为主的天然磷脂；也可以是二棕榈酰磷脂酰胆碱(DPPC)、二棕榈酰磷脂酰乙醇胺(DPPE)、二硬脂酰磷脂酰胆碱(DSPC)等合成磷脂；还可以含有胆固醇。优选的脂质体为阳离子脂质体，其主要由带正电荷的脂类及中性辅助脂类以等摩尔混合而成。该带正电荷的脂质体与带负电荷的DNA之间可有效地形成复合物，并通过内吞作用移入细胞内。
所述多聚物，即利用阳离子多聚体，如多聚左旋赖氨酸上的正电荷与DNA上的负电荷结合发生电性中和胍，而形成稳定的多聚物/DNA复合物。所得阳离子多聚体与DNA的复合物仍带正电荷，可与细胞表面带负电荷的受体结合，而被渗入至细胞内。
所述分子耦联体是将目的基因外源性DNA共价结合到细胞表面特异受体的配基或单克隆抗体或病毒胞膜蛋白上，利用特异的结合特性介导外源性基因导入至特定类型的细胞中。
病毒通常可以高效率地进入特定的细胞，表达自身蛋白，产生新的病毒粒子，因此被改造的病毒首先成为了基因治疗的载体。例如，慢病毒载体、反转录病毒载体、腺病毒载体、腺相关病毒载体及单纯疱疹病毒载体等。。
术语“表达载体”是指包含重组多核苷酸的载体，该重组核苷酸含有可操作地连接到待表达核苷酸序列的表达控制序列。表达载体包括本领域已知的所有表达载体，包括引入重组多核苷酸中的粘粒、质粒(例如，裸露的或包含在脂质体中的质粒)和病毒(例如，慢病毒、逆转录病毒、腺病毒和腺相关病毒)。
术语“慢病毒”属于逆转录病毒科。慢病毒能够感染分裂期细胞和非分裂期细胞。慢病毒感染后，可以将大量的遗传信息递送到宿主细胞，并长时间持续稳定地表达，同时能够随细胞分裂稳定遗传下去。因此慢病毒是导入外源基因的最有效工具之一。慢病毒的实例包括人类免疫缺陷病毒(HIV)、猴免疫缺陷病毒(SIV)、马传染性贫血(EIA)、猫免疫缺陷病毒(FIV)。
慢病毒载体可以将外源基因有效地整合到宿主染色体上，从而达到持久性表达。在感染能力方面可有效地感染神经元细胞、肝细胞、心肌细胞、肿瘤细胞、内皮细胞、干细胞等多种类型的细胞，从而达到良好的基因治疗效果。
优选地，本发明采用慢病毒载体。
本发明还提供了一种宿主细胞，其中，所述宿主含有本发明所述的重组载体。将含有本发明人突变的人2Ig-B7-H3蛋白编码基因的重组载体转化到宿主体内，可以用于研究其与肿瘤细胞表达的关系。优选所述宿主选自大肠杆菌、239细胞和SHG44细胞中的一种或几种。其中，大肠杆菌作为基因工程菌，既可以含有本发明重组克隆载体以实现对本发明人的突变的人2Ig-B7-H3蛋白编码基因的扩增，也可以含有本发明重组表达载体以实现对本发明人的突变的人2Ig-B7-H3蛋白编码基因的大量表达。当所述重组载体为重组腺病毒载体时，该载体可以在SHG44和239细胞中得到扩增。
本发明的实施例公开了一种药物组合物，所述药物组合物包括赋形剂以及选择上述技术方案所述人2Ig-B7-H3蛋白和所述的重组载体中的一种或几种。
所述药物接受的赋形剂指无毒固态、半固态或液态填充剂、稀释剂、包囊材料或其他制剂辅料，例如，包括但不限于盐水、缓冲盐液、葡萄糖、水、甘油、乙醇及其混合物。所述药物组合物适于胃肠外、舌下、脑池内、阴道 内、腹膜内、直肠内、颊内或表皮给药。
胃肠外给药包括静脉内、肌内、腹膜内、胸骨内、皮下、关节内注射和输注。适于胃肠外给药的药物组合物包括无菌水溶液或非水溶液、分散液、悬浮液或乳液，以及用于在临使用前在无菌可注射溶液或分散液中配制的粉末。适宜的水性或非水性载体、稀释剂、溶剂或赋形剂包括水、乙醇、甘油、丙二醇、聚乙二醇、羧甲基纤维素、植物油和可注射的有机酯如油酸乙酯。这些组合物还可以含有防腐剂、润湿剂、乳化剂、保护剂和分散剂佐剂，例如肌醇、山梨醇和蔗糖。优选加入渗透压调节剂如糖类、氯化钠、氯化钾。
表皮给药包括在皮肤、黏膜上以及在肺和眼表面给药。这样的药物组合物包括粉剂、软膏、滴剂、透皮贴剂、离子电渗疗法装置以及吸入剂等。直肠或阴道给药的组合物优选为栓剂，它可以通过将本发明的重组载体与适宜的非刺激性赋形剂如可可脂、聚乙二醇或栓剂蜡混合制备，所述赋形剂或载体在室温为固态，在体温下为液态，因此在直肠或阴道腔内熔化并释放出活性化合物。
优选地，所述药物组合物为注射液，所述注射液包括药学上可接受的赋形剂以及选自本发明所述的人葡萄糖激酶突变体编码基因和本发明所述的重组载体中的一种或几种。
优选的，所述药物组合物为注射液，所述注射液包括药学上可接受的赋形剂以及选自上述技术方案所述的重组载体中的一种或几种。
本发明的实施例公开了上述技术方案所述突变的人2Ig-B7-H3蛋白编码基因在制备预防和治疗癌症的药物中的应用。
在鼠的几种肿瘤细胞株异位表达后可以诱导肿瘤特异性细胞毒性T淋巴细胞活化，从而延缓癌细胞的生长甚至完全消除肿瘤,转染的癌细胞株植入小鼠后，可以明显延长小鼠的生存期。
下面通过实施例来进一步说明本公开的技术方案。本领域技术人员应该明了，所述实施例仅仅是帮助理解本公开，不应视为对本公开的具体限制。
为使本领域具有普通知识的人员可了解本公开的特点及效果，以下谨就说明书及申请专利范围中提及的术语及用语进行一般性的说明及定义。除非另有指明，否则文中使用的所有技术及科学上的字词，皆具有本领域技术人 员对于本公开所了解的通常意义，当有冲突情形时，应以本说明书的定义为准。
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。
实施例1
人2Ig-B7-H3蛋白编码基因
突变的人2Ig-B7-H3蛋白编码基因共包含2765个碱基，位于5’端第1488位，发生C＞T杂合突变，使得在第1488位的C和T碱基各占一半位置。该突变的人2Ig-B7-H3蛋白编码基因的核苷酸序列如SEQ ID NO:1所示。如序列表中所示的SEQ ID NO:1中第1488位的“y”表示发生C＞T杂合突变，在第1488位处C和T碱基各占一半位置。
实施例2
重组载体构建
在pIRES2-EGFP载体的NHeI和NotI酶切位点之间插入序列表的SEQ ID NO:1所示的核苷酸序列，得到重组质粒pIRES2-EGFP/2Ig-B7-H3。
1、采用LipofectamineTM2000阳离子脂质体转染试剂盒并按试剂盒说明书操作，将重组质粒pIRES2-EGFP/2Ig-B7-H3导入239T细胞，得到重组细胞。
2、将步骤1得到的重组细胞接种至含5％(体积比)新生牛血清的DMEM/F12培养基，然后在37℃、5％CO2的培养箱中培养48小时，然后收集上清液。
3、取步骤2得到的上清液，用0.45μm滤膜过滤并收集滤液，然后调pH至7.4。
4、将步骤3得到的滤液进行亲和层析纯化。
平衡缓冲液：含0.5M NaCl的pH7.4、0.5M的Tris-HCl缓冲液；
洗脱液：pH3.0、0.1M的Gly-HCl缓冲液。
先用平衡缓冲液洗涤3个柱体积，然后用洗脱液洗涤目标物，流速均为5mL/min。
A280nm检测紫外吸收峰。
采用收集管收集目标峰，然后将收集管中的溶液转移至透析袋中，在pH7.4、0.01M PBS缓冲液中进行透析，得到人2Ig-B7-H3蛋白。
实施例3
人2Ig-B7-H3蛋白编码基因在细胞表面的表达
为了检测2IgB7-H3在细胞表面的表达，用实施例2得到的载体及阴性对照慢病毒感染SHG44、293T细胞。在感染后收细胞，流式检测细胞表面2IgB7-H3的表达。
1、材料和仪器
目的细胞：SHG44、293T。
培养基：DMEM+10％FBS+1％P/S。
流式检测试剂：2IgB7-H3流式抗体(人)。
流式仪器：BD FASAria Cell Sorter。
2、实验步骤1)细胞培养：
将用实施例2得到的载体及阴性对照慢病毒感染的SHG44、293T细胞在5％CO2，37℃二氧化碳培养箱中培养。
2)感染细胞：
(1)将处于对数生长期的SHG44、293T细胞进行胰酶消化，制成细胞悬液。
(2)将SHG44、293T细胞悬液接种于6孔板中，5％CO2，37℃二氧化碳培养箱中培养过夜。
(3)根据病毒滴度各孔中分别加入适量的实施例2所制备的载体和阴性对照病毒，SHG44的MOI为100；293T的MOI值为2，实验分组如下：
Figure PCTCN2018123049-appb-000001
感染48小时后，拍照记录。
3)细胞染色及流式检测
i)去除培养基，用PBS洗两次，用胰酶消化底面贴壁的细胞，收集细胞，1000rpm离心5分钟。
ii)去上清，1ml PBS重悬细胞，1000rpm离心5分钟。
iii)去上清，每个样品用500ul PBS重悬细胞，轻轻吹匀。
iv)每组分别加入2IgB7-H3流式抗体及同型对照，轻轻混匀。
v)避光4℃孵育30分钟。
vi)1000rpm离心5分钟，去上清，1ml PBS重悬细胞。
vii)1000rpm离心5分钟，去上清，500ul PBS重悬细胞。
viii)上机流式检测。
4、实验结果
流式数据分析
293T细胞的流式数据分析结果如图1所示。
SHG44细胞的流式数据分析结果如图2所示。
5、结论
由结果可知，抗原提呈细胞293T和SHG44细胞的WT+MT组的CD276的平均荧光强度均高于WT组和MT组，即WT和MT组的CD276的表达量较高。
实施例4
在抗癌方面的应用
将液氮冻存的小鼠肝癌H22细胞于37 0C水浴锅中快速解冻，细胞密度调至1X 10 7/mL，腹腔接种2只BALB/c小鼠，每只0.2mL。待小鼠腹部胀大后，将小鼠颈椎脱臼处死，消毒腹部，抽取腹水合并，用PBS调整细胞密度至1X10 7/m L，皮下接种20只BALB/c小鼠，每只0.2mL12天后将小鼠分成两组，每组10只，进行如下处理:
第一组：不做处理，不接种任何治疗药物；
第二组：腹部皮下注射人2Ig-B7-H3蛋白，免疫3次(每次0.2mL)，单次免疫剂量为20ug/只；
按种肿瘤细胞后第12天进行第一次免疫:第15天进行第二次免疫:第18 天进行第三次免疫。从第二天免疫开始，每天观察肿瘤生长情况，记录肿瘤大小，按以下公式计算肿瘤体积:V＝ab 2/2(V-体积，a-肿瘤长径，b-肿瘤短径)。肿瘤体积变化见表1。
Figure PCTCN2018123049-appb-000002
从结果可以看出，人2Ig-B7-H3蛋白对皮下接种的诱导型肝癌模型有明显的治疗作用。人2Ig-B7-H3蛋白治疗后皮下肿瘤的生长速度明显减慢，肿瘤体积变小。
实施例5
将人肺腺癌细胞PC-9培养于含100mL/L胎牛血清的DMEM培养基中，以1×10 5/mL的细胞浓度植入圆形玻底培养皿(Φ＝35mm)，36℃，5％CO2细胞培养箱中培养22h后，弃去培养液，平均分成10份，加入用DMEM培养基溶解的人2Ig-B7-H3蛋白(12μM，冰浴预冷)(以下简称“用药”)；冰浴避光孵育2h后，分别弃去多肽溶液，并用预冷PBS洗涤2次。
在操作过程中，观察并记录微管蛋白厚度(即微管壁厚度)的变化，观察结果显示，在所有10份试验中，人肺腺癌细胞PC-9的微管蛋白厚度均出现变薄的现象，具体结果如表2所示。从表2可知，用药后的微管蛋白厚度均为用药之前的80％±2％，说明本发明的蛋白都能破坏癌细胞的微管动力学，起到阻止癌细胞增殖的抗有丝分裂剂的作用(减慢或阻止癌细胞的有丝分裂)。
表2
Figure PCTCN2018123049-appb-000003
实施例6
实验对象为肺腺癌患者，临床IV期，将所述合成多肽溶解于PH值为 7.4PBS(Hyclone)溶液中，浓度调至2.5mg/ml，每次上臂皮下注射200ug后覆盖5％Aldaracream(iNova Pharmaceuticals Australia Pty Ltd.)，每周一次，12周为一个周期。分别用ELISA检测用药前、用药3周、7周、11周的IFN-γ的分泌情况，具体实施例所用合成多肽以及用药前后的测试结果如表3所示。
从表3可以看出，在用药后，T细胞分泌的IFN-γ水平具有明显升高的趋势，甚至出现了指数型增长的情况，说明使用了本发明的蛋白能增加肺腺癌患者外周血的肿瘤杀伤能力，进一步证明了本发明的效果。推测可能是本发明提供的蛋白对于治疗肺腺癌具有明显的疗效，通过与肺腺癌细胞的特异性结合，诱导癌细胞产生树突状细胞，形成抗原呈递细胞，进而刺激体内杀伤性T细胞的产生，实现对于肺腺癌的治疗。
随后，用体内无癌细胞的志愿者替代肺腺癌患者重复上述试验，并用ELISA检测用药前、用药3周、7周、11周的IFN-γ的分泌情况，发现并无明显的波动，说明本发明的合成多肽对于无癌细胞的机体没有刺激分泌IFN-γ的作用，毒副作用较小。
表3
Figure PCTCN2018123049-appb-000004
以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域的普通技术人员来说，在不脱离本明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。
对所公开的实施例的上述说明，使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下，在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。

Claims (11)

  1. 一种突变的人2Ig-B7-H3蛋白编码基因，其特征在于，所述基因具有SEQ ID NO:1所示的核苷酸序列。
  2. 一种人2Ig-B7-H3蛋白，其特征在于，所述人2Ig-B7-H3蛋白的编码基因是权利要求1所述突变的人2Ig-B7-H3蛋白编码基因。
  3. 一种重组载体，所述重组载体包括载体及其携带的目的基因，其特征在于，所述目的基因是权利要求1所述突变的人2Ig-B7-H3蛋白编码基因。
  4. 根据权利要求3所述的重组载体，其特征在于，所述载体选自由克隆载体、真核表达载体、原核表达载体和穿梭载体所组成的组。
  5. 根据权利要求3所述的重组载体，其特征在于，所述载体选自由pIRES2-EGFP、pCMVp-NEO.BAN、pEGFT-Actin、慢病毒载体和腺病毒载体所组成的组。
  6. 一种宿主细胞，其含有权利要求3-5中任意一项所述的重组载体。
  7. 根据权利要求6所述的宿主细胞，其特征在于，所述宿主细胞选自239T细胞、SHG44细胞中的一种或几种。
  8. 一种药物组合物，其特征在于，所述药物组合物包含药学上可接受的赋形剂以及选自权利要求1所述的突变的人2Ig-B7-H3蛋白编码基因、权利要求2所述的人2Ig-B7-H3蛋白、权利要求3～5中任一项所述的重组载体和权利要求6～7中任一项所述的宿主细胞中的一种或几种。
  9. 根据权利要求8所述的药物组合物，其特征在于，所述药物组合物为注射液。
  10. 权利要求1所述突变的人2Ig-B7-H3蛋白编码基因、权利要求2所述的人2Ig-B7-H3蛋白、权利要求3～5中任一项所述的重组载体和权利要求6～7中任一项所述的宿主细胞在制备预防或治疗癌症的药物中的应用。
  11. 权利要求11所述的应用，其特征在于，所述癌症为肝癌、肺癌、前列腺癌、胰腺癌、小肠癌、结肠癌以及宫颈癌。
PCT/CN2018/123049 2018-12-24 2018-12-24 突变的人2Ig-B7-H3蛋白编码基因、重组载体、包含其的宿主细胞、药物组合物及其应用 Ceased WO2020132789A1 (zh)

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